Universitätsklinikum Ulm Institut für Pathologie

Universitätsklinikum Ulm

Institut für Pathologie

Ärztlicher Direktor: Prof. Dr. med. Peter Möller

Mitochondrial alterations in different Alzheimers d isease

mouse models

Dissertation

zur Erlangung des Doktorgrades der Medizin

der Medizinischen Fakultät

der Universität Ulm

vorgelegt von

Frederik Scheibe

Aachen

2014

Amtierender Dekan: Prof. Dr. Thomas Wirth

1. Berichterstatter: Prof. Dr. Dietmar Thal

2. Berichterstatter: Prof. Dr. Christine von Arnim

Tag der Promotion: 24.04.2015

Contents

I

Contents

Contents........................................... ...................................................................... I

Abbreviations ...................................... ................................................................ III

1 Introduction ....................................... ..................................................... 1

1.1 Alzheimers disease ....................................................................... 1

1.2 Neuropathology ............................................................................. 1

1.2.1 APP and Aβ ........................................................................... 2

1.2.2 The amyloid hypothesis ......................................................... 3

1.2.3 Intraneuronal Abeta ............................................................... 4

1.3 Mitochondria.................................................................................. 5

1.3.1 Mitochondrial function in AD .................................................. 6

1.4 Mouse models ............................................................................... 7

1.5 Aims of the study......................................................................... 10

2 Material and Methods ............................... ........................................... 12

2.1 Material and Equipments used.................................................... 12

2.2 Mouse models ............................................................................. 15

2.3 Tissue praparation and DiI tracing............................................... 15

2.4 Microscopic and quantitative analysis ......................................... 16

2.5 Immunohistochemistry ................................................................ 17

2.6 Protein extraction from brain tissue ............................................. 18

2.7 SDS-PAGE and Western blot analysis........................................ 18

2.8 Aβ Enzyme Linked Immunosorbent Assay (ELISA) .................... 19

2.9 Stereology ................................................................................... 19

2.10 Electron microscopy, immuno electron microscopy..................... 19

2.10.1 Semiquantitative analysis of synapse densities ................. 20

2.10.2 Semiquantitative analysis of dystrophic dendrites ............. 20

2.10.3 Analysis of mitochondria .................................................... 21

2.11 Statistical analysis ....................................................................... 23

3 Results ............................................ ...................................................... 25

3.1 Different patterns of Aβ-pathology in APP23 and APP48 mice ... 25

3.2 Degeneration of neurites and asymetric synapses in APP23 but

not in APP48 mice.................................................................................. 28

II

3.3 Neuron loss in the CA1 subfield but not in the frontocentral cortex

of APP48 and APP23 mice .................................................................... 34

3.4 Increased mitochondrial alterations in APP48 in neuronal somata

of APP48 but not in APP23 .................................................................... 34

4 Discussion......................................... ................................................... 39

4.1 Aβ effects on mitochondrial integrity............................................ 40

4.2 Neurite and synapse degeneration in APP23 and APP48 mice .. 42

4.3 Two types of Aβ-induced neurodegeneration in the frontocentral

cortex: somatic type and neuritic type.................................................... 43

4.4 Conclusions................................................................................. 45

5 Summary............................................ ................................................... 47

6 References......................................... ................................................... 49

Supplementary Material ............................. ........................................................ 64

Supplementary Tab. 1............................................................................ 64

Acknowledgements................................... ......................................................... 77

Curriculum vitae ................................... .............................................................. 78

Abbreviations

III

Abbreviations

ABAD Aβ-binding alcohol dehydrogenase

ABC complex avidin-biotin-peroxidase complex

Acetyl-CoA acetyl coenzyme A

AD Alzheimers disease

ADP Adenosine diphosphate

AICD Amyloid precursor protein intracellular domain

ANOVA Analysis of variance

APH-1 anterior pharynx defective protein 1

APOE Apolipoprotein E

APP Amyloid precursor protein

ATP Adenosine triphosphate

Aβ Amyloid beta protein

CA1 Cornu ammonis

CAA Cerebral amyloid angiopathy

CTF C-terminal fragment

CypD Cyclophilin D

DAB 3,3'-Diaminobenzidine

DNA Deoxyribonucleic acid

ELISA Enzyme Linked Immunosorbent Assay

ETC electron transfer chain

FADH flavin adenine dinucleotide

IgG Immunoglobulin G

IHC Immunohistochemistry

kDa kilo Dalton

LDS lithium dodecyl sulfate

M Mol

M1 primary motor cortex

M2 secondary motor cortex

MW molecular weight

NADH Nicotinamide adenine dinucleotide

NFTs Neurofibrillary tangles

Abbreviations

IV

P1 pellet containing the membrane-associated and the

insoluble, plaque-associated fraction after

homogenization

P2 dispersible fraction after ultracentrifugation

P3 pellet after SDS-resuspension and centrifugation

pAβ phosphorylated Amyloid beta protein

PBS Phosphate buffer saline

PEN2 Presenilin enhancer 2

PFA Phosphate buffer saline

PSEN1 Presenilin 1

PSEN2 Presenilin 2

RNA Ribonucleic acid

ROS reactive oxygen species

S1 supernatant with the soluble and dispersible fraction in

homogenization

S2 supernatant after ultracentrifugation

S3 supernatant after SDS-resuspension and centrifugation

sAPPα soluble amyloid precursor protein N-terminal fragment α

sAPPβ soluble amyloid precursor protein N-terminal fragment β

SC1 primary somatosensory cortex

SDS sodium dodecyl sulfate

SDS-PAGE sodium dodecyl sulfate polyacrylamide gel

electrophoresis

TBS Tris-Buffer Saline

TIM Translocase of the inner mitochondrial membrane

TOM Translocase of the outer mitochondrial membrane

τ protein Tau protein

Introduction

1

1 Introduction

1.1 Alzheimers disease

Alzheimers disease (AD) is the most common form of dementia in the elderly. It

was first described by Alois Alzheimer in 1907. He reported the case of a 51 year

old woman with progressive mental disorders and histologic brain pathology, i.e.

senile plaques and neurofibrillary tangles (Alzheimer, 1907). Accordingly, the

typical clinical symptoms of AD are difficulties in learning, memory loss,

disorientation in time and/or place, changes in mood and personality. In 2000 the

prevalence of AD in the U.S. population was estimated to be as high as 4.5 million

persons with an expected increase up to 13.2 million by 2050 (Hebert et al., 2003).

The prevalence of AD in the age group of people ≥ 65 years is 13 % while it

increases in the group of people ≥ 85 years up to 43% (Thies and Bleiler, 2011).

Most AD cases are sporadic. Most sporadic AD cases are late-onset AD cases,

i.e. dementia manifests at the age of 65 years or older. Here, age and the

prevalence of the Apolipoprotein E (APOE) ε4 allele are the most important risk

factors. When AD manifests at age < 65 years, the cases are referred to as “early-

onset” AD cases. Early onset cases frequently have a familial history and carry

mutations in the APP, PS1 or PS2 gene. Other established risk factors are mild

cognitive impairment, cardiovascular disease risk factors, traumatic brain injury

and female gender (Corder et al., 2004).

1.2 Neuropathology

In patients with typical AD symptoms there are degenerative changes in various

brain regions. Macroscopic examination of the brain shows cortical atrophy

especially in frontal, temporal and parietal lobes. Accompanied with a widening of

the cerebral sulci and with enlargement of the central ventricel. Microscopically,

the hallmarks of the AD neuropathology are:

1) Extracellular senile plaques, consisting of Amyloid-β (Aβ) which is generated

from the amyloid precursor protein (APP) and that forms aggregates in a

hierarchical manner in the brain (Masters et al., 1985; Thal et al., 2002). The major

component of these amyloid plaques consist of the 40-42 amino acid amyloid beta

protein which can also be shown in different Alzheimers disease mouse models.

Introduction

2

2) Intracellular neurofibrillary tangles (NFTs), consisting of the protein tau (τ) which

is physiologically involved in stabilization and assembly of microtubules in nerve

cells (Goedert and Spillantini, 2006). Its pathologic hyperphosphorylated

appearance leads to the aggregation and formation of NFTs. Tau is a major

hallmark of different kinds of dementia including AD in the class of tauopathies

(Duyckaerts et al., 2009).

3) Cerebral amyloid angiopathy (CAA), showing vascular Aβ deposits in brain

vessels similar to senile plaques and being capable of causing hemorrhage and

infarction (Ghiso et al., 2010). In almost all persons with dementia CAA was found

(Arvanitakis et al., 2011).

Although these changes follow a typical regional progression in AD brain they are

not specific (Montine et al., 2012) and can also be seen in non-demented elderly.

Additionaly neuron loss, dendritic and synaptic degeneration are seen in AD

brains.

1.2.1 APP and A β

Aβ protein was identified as the major component in cerebrovascular amyloid

deposits (Glenner and Wong, 1984a) and as the main core protein in AD typical

extracellular plaques (Masters et al., 1985). It is a 36-43 amino acid peptide of 4

kDa molecular weight with its most common monomeric forms Aβ1-40 and Aβ1-42.

Aβ is derived from a larger protein, the APP, by proteolytic cleavage. APP is a

highly conserved single-pass transmembrane protein with a large extracellular

domain (O'Brien and Wong, 2011) and its 695 amino acids form is the most

common isoform in the central nervous system (Bayer et al., 1999). It is cleaved

by proteases, forming different proteolytic fragments.

Two different pathways for APP cleavage exist, the non-amyloidogenic pathway by

the α-secretase without pathological consequences and the amyloidogenic

pathway driven by the β-secretase leading to the formation of toxic Aβ.

The α-secretase cleaves APP within the Aβ region, leading to generation of the

secreted N-terminal APP processing product, (sAPPα) and retending a C-terminal

membrane-bound fragment (CTF) which can be cleaved by the γ-secretase

releasing the p3 fragment (Selkoe, 2001) and the APP intracellular domain (AICD).

It was shown that sAPPα is an actor in many physiological processes, i.e.

neuroprotection (Mattson et al., 1993; Furukawa et al., 1996; Kogel et al., 2005),

Introduction

3

modulation of neuronal excitability (Mattson et al., 1993; Mattson and Furukawa,

1998), synaptic plasticity (Turner et al., 2003; Ring et al., 2007), axonal growth and

branching (Moya et al., 1994; Ikin et al., 2007), dendritic outgrowth (Mattson,

1997) and in synaptogenesis (Mattson and Furukawa, 1998; Wang et al., 2011).

Also the α-secretase derived C-terminal fragment p3 and AICD are discussed to

have neuroprotective and neurotrophic properties. This non-amyloidogenic

pathway precludes the formation of toxic Aβ by cutting APP within the Aβ region

(Esch et al., 1990; Sinha and Lieberburg, 1999).

Alternatively to the non-amyloidic pathway, APP can be cleaved by β-and γ-

secretase, releasing a smaller fragment, sAPPβ, Aβ and AICD. This

amyloidogenic pathway leads to Aβ-formation when γ-secretase processes the β-

secretase derived C-terminal fragment (APP-CTFβ) fragment and leads to the

formation of different Aβ species, especially Aβ1-40 and Aβ1-42 (Seubert et al.,

1993) and the C-terminal fragment.

The γ-secretase is a protein complex consisting of Presenilin 1 (PSEN1) or

Presenilin 2 (PSEN2) and Nicastrin, Presenilin enhancer 2 (PEN2) and the

anterior pharynx defective protein (APH-1) (De Strooper, 2003). The presenilins

form the active center of the γ-secretase. Presenilin 1 and 2 knockout mice and

cells did not produce Aβ (De Strooper et al., 1998; Herreman et al., 2000).

Mutations causing familial Alzheimer disease occur in the PSEN1, PSEN2 and

APP genes (Goate et al., 1991; Levy-Lahad et al., 1995; Sherrington et al., 1995).

A pathological role of APP and Aβ in AD was considered because mutations on

the APP gene locus were shown to cause familial early-onset AD (Goate et al.,

1991; Mullan et al., 1992) by an elevated production of Aβ (Citron et al., 1992; Cai

et al., 1993). Interestingely nearly all patients with trisomy 21 beyond 45 years of

age develop an AD and plaque pathology (Glenner and Wong, 1984b) while the

APP encoding gene resides on chromosome 21 (Robakis et al., 1987). Recently, a

protective APP mutation was found in an icelandic cohort that decreases formation

of amyloid peptides, protects against cognitive decline in the elderly and is

discussed to reduce β–cleavage of APP (Jonsson et al., 2012).

1.2.2 The amyloid hypothesis

Because neuritic plaques contain Aβ, the so called “amyloid hypothesis” was

instandly suggested (Hardy and Higgins, 1992) and later on modified in

Introduction

4

accordance to new findings (Hardy and Selkoe, 2002). It proposes that altered

APP processing drives Aβ production, Aβ results in plaque formation, plaques or

soluble Aβ aggregates induce neurodegeneration, and the resultant neuronal loss

results in the clinical dementia syndrome typical of AD (Hardy and Allsop, 1991;

Hardy, 1997; Swerdlow, 2007b). The presenilin mutations lead to an increased

production and aggregation of Aβ42 in total but also in relation to Aβ40 (Scheuner et

al., 1996). The Aβ42 protein is more prone to aggregate than Aβ40, is predominant

in senile plaques (Roher et al., 1993) and is toxic to cells in culture (Younkin,

1995; Swerdlow, 2007b).

The amyloid hypothesis has evolved since it was first postulated. Two major

modifications are 1) Neuritic plaques are no longer considered as the toxic

aggregates but as sinks for Aβ. Soluble Aβ aggregates such as oligomers not

sequestered in plaques are today suggested to represent toxic agents in AD

(Hardy and Selkoe, 2002; Lesne and Kotilinek, 2005; Walsh et al., 2005). 2) Aβ42

has a major importance for toxicity instead of total Aβ (Younkin, 1995).

Extracellular deposits of Aβ are typical for AD brains but nevertheless are also

found in cognitively normal elderly people. The number of Aβ deposits found do

not allow a conclusion to the degree of cognitive impairment (Hardy and Selkoe,

2002). However, amyloid plaque deposition in the AD brain follows a hierarchichal

sequence in which many brain areas are subsequently affected (Thal et al., 2002).

1.2.3 Intraneuronal Abeta

In addition to extracellular Aβ-deposition in plaques, intracellular Aβ occurs in

nerve cells in the AD brain (Gouras et al., 2000; D'Andrea et al., 2002) and in

mouse models for AD (Tomiyama et al, 2010; Wirths et al., 2001; Takahashi et al.,

2002; Abramowski et al., 2012b). The role of intracellular Aβ for

neurodegeneration and the development of AD has been discussed

controversially. Mutant intracellular Aβ has been shown to induce hippocampal cell

loss associated with endoplasmatic reticulum stress and mitochondrial alterations

in AD mouse models (Umeda et al., 2011), suggesting that intracellular Aβ might

be an early event in AD pathology (LaFerla et al., 2007). In young patients with

trisomy 21 it has been shown that high intracellular Aβ levels declined when

extracellular plaques accumulated (Mori et al., 2002). Within the neuron Aβ

accumulates in multivesicular bodies in pre- and postsynaptic compartments while

Introduction

5

abnormal synaptic morpholgy is present in AD mouse models Tg2576 with the

human APP Swedish mutation 670/671 (Takahashi et al., 2002). In this mouse

model mitochondria were found to be a site of Aβ accumulation, also showing a

decrease of cytochrome c activity suggesting that Aβ could affect mitochondrial

function. Here, an increase of hydrogen peroxide was directly correlated to levels

of soluble Aβ suggesting that soluble Aβ may be responsible for hydrogen

peroxide production leading to oxidative stress (Manczak et al., 2006).

In the following paragraph I will give a short introduction to general mitochondrial

function and its role in AD pathology.

1.3 Mitochondria

Mitochondria are essential parts of the eucaryotic cell and are often localized close

to structures with a high metabolic rate of ATP, e.g. in muscles near the actin and

myosin filaments. The average diameter of mitochondria is about 0,5 – 1 μm but

studies have shown that size and plasticity of mitochondria are in a state of

permanent change and that they have the ability to fuse with other mitochondria

(fusion) and to seperate again (fission) (Dimmer and Scorrano, 2006). Whithin the

cell mitochondria are transported along microtubules which determine direction of

internal transport and distribution in distant cell types.

Mitochondria are enclosed by two membranes, the inner and the outer

mitochondrial membrane. These membranes divide the inner mitochondrial space

in two seperate compartments; the inner matrix and the intermembranal space.

There are more than 1000 mitochondrial proteins which are encoded in the

nucleus and imported to the different mitochondrial compartments by translocases

of the outer and inner membrane (TOM/TIM). The outer membrane also contains

transport proteins like voltage-dependant anion channels making it permeable to

molecules and proteins of the size of 5000 daltons or less (Wohlrab, 2009). The

inner membrane is highly specialised and is characterised by cristae which

increase the inner membrane surface. In contrast to the outer membrane it is

impermeable to ions by its lipid bilayer membrane but different transport proteins

make it selectively permeable to metabolized molecules or those which are

requiered by enzymes in the matrix for Krebs cycle and the electron transport

chain. In the Krebs cycle pyruvate and fatty acids are converted to acetyl CoA.

Great amounts of NADH (and FADH2) are created by this oxidative reaction. This

Introduction

6

energy carrier looses its electrons on its way through the respiratory chain in the

inner mitochondrial membrane converting O2 to H20. In so doing, its energy is

stored as an electrochemical proton gradient which enables the membranebound

enzyme ATP synthase to convert ADP to ATP via oxidative phosphorylation. 15

times more ATP can be produced this way than by glycolysis alone. This

demonstrates the importance of mitochondria to the cell for its energy generation.

The inner matrix contains the mitochondrial genome, ribosomes, transfer RNA and

a variety of enzymes necessary for expression of mitochondrial genes. The

mitochondrial genome is present as a double stranded, circular DNA molecule

which provides the mitochondrion with only a part of the requiered proteins. Some

of the catalytically most important subunits of the cytochrome c oxidase complex

are encoded in the mitochondrial genome (Swerdlow et al., 2010). However, most

proteins of the mitochondrial matrix are encoded in the cellular DNA and are

imported from the cytosol. The mitochondrial DNA, which is especially important to

the electron chain and ATP generation is highly susceptible to reactive oxygen

species (ROS). When mitochondrial DNA is damaged by accumulation of ROS a

threshold can be reached leading to a point of no return that results in the

induction of apoptosis. This intrinsic pathway of apoptosis can also be induced by

other mechanisms of cell injury, for example DNA damage, oxidative stress,

accumulation of misfolded proteins and the lack of oxygen or nutrients. It is started

by the release of cytochrome c, a part of the electron transport chain, to the

cytosol what is followed by the activation of the caspase cascade, ultimately

leading to nuclear fragmentation and cell death.

1.3.1 Mitochondrial function in AD

An alternative hypothesis for the development of AD is that oxidative stress leads

to mitochondrial dysfunction as one of the primary events in AD related

neurodegeneration. This mitochondrial cascade hypohesis was proposed in order

to understand the correlation of advancing age and AD and the cause of Aβ

amyloidosis in late-onset AD (Swerdlow and Khan, 2004). The main assertions of

the hypothesis are that

1. mitochondrial function and durability is determined by inheritance,

2. the structural and functional changes of mitochondria going along with age are

determined by mitochondrial durability and

Introduction

7

3. mitochondrial changes with age pass a threshold causing AD pathology and

symptoms e.g. via apoptosis of nerve cells (Swerdlow et al., 2010).

Mitochondrial dysfunction has been described in several studies of AD brains, in

APP mouse models and cell lines (Gibson et al., 1998; Keil et al., 2004; Lustbader

et al., 2004; Manczak et al., 2004; Reddy et al., 2004; Caspersen et al., 2005; Devi

et al., 2006; Manczak et al., 2006; Eckert et al., 2008; Hauptmann et al., 2009).

Structural changes and a reduction of neuronal mitochondria were shown in

morphological studies (Hirai et al., 2001). Also early defects in glucose metabolism

of AD patients suggest alterations in mitochondrial functions (Hoyer, 2000).

Important findings were also the deficiency of enzymes of the Krebs cycle, such as

pyruvate dehydrogenase complex, ketoglutarate dehydrogenase complex, and the

reduction of cytochrome c oxidase activity. Here, it was shown that Aβ directly

disrupts mitochondrial function by inhibiting key enzymes and may thereby

contribute to the deficiency of energy metabolism seen in AD (Casley et al., 2002).

The reduction of cytochrome c oxidase activity was also shown in so called cybrid

cell lines with AD mitochondria compared to cell lines with control subject

mitochondria. Other findings in these cybrid cells were altered homeostasis,

reduction of mitochondrial membrane potential, activation of apoptosis pathways

and increased Aβ42 production (Swerdlow, 2007a). Some of these studies suggest

that mitochondrial changes preceed AD pathology like Aβ accumulation, others do

not. It is tempting to hypothesize that there are several events of mitochondrial

damage affecting each other leading to a vicious cycle of neurodegeneration.

1.4 Mouse models

Transgenic mouse models for AD pathology have been created to understand

disease mechanisms. They are also important for drug tests and clinical trials. A

great number of mouse models has been developed, most of them overexpressing

wildtype or mutant APP, PSEN 1, PSEN 2 or tau protein or expressing two or

more of the gene combination in multiple transgenic mice (Sturchler-Pierrat et al.,

1997; Mineur et al., 2005). However, it is difficult to create a mouse model

imitating human AD pathology. Even the coexpression of Aβ and tau phenotype

does not allow to answer the questions why this pathology develops in the human

brain without overproduction of Aβ and tau as it is necessary in the mouse model

to create the pathology.

Introduction

8

The APP23 mouse overexpresses mutant APP with the Swedish double mutation

(Sturchler-Pierrat et al., 1997). This mutation results in an increased production

and secretion of Aβ due to increased β-secretase activity in the secretory pathway

in patients developing early onset AD (Haass et al., 1995). In APP23 mice this

double mutation is driven by a murine Thy-1 promoter which leads to an

increased, selective expression of APP in neuronal cells. Neuropathologically, Aβ

is highly overexpressed and mice present Aβ deposits from the age of 6 month in

neocortex and hippocampus. Aβ-deposits increase with age in size and number,

affecting following brain regions such as thalamus, olfactory nucleus and caudate

putamen. Other findings were neuron loss especially found in hippocampal region

CA1 (Calhoun et al., 1998), what is also seen in AD patients (West et al., 1994),

furthermore dystrophic neurites, hyperphosphorylated tau-protein, inflammatory

response and synapse loss. Also cerebral amyloid angiopathy was shown (Winkler

et al., 2001). In 2006 a retrograde DiI tracing study demonstrated that especially

layer III commissural neurons with highly ramified dendritic trees, i.e. type I

commissural neurons of the frontocentral neocortex (Tab. 1) were reduced in

number, these neurons also presented alterations in the dendritic tree (Capetillo-

Zarate et al., 2006).

Introduction

9

Table 1 : Types of commissural neurons in layer III of the frontocentral cortex

identified by DiI-tracing (Capetillo-Zarate et al., 2006)

Type I commissural

neurons

Pyramidal neurons with a highly ramified dendritic

tree showing multiple secondary and tertiary

branches

Type II commissural

neurons

Pyramidal neurons with a dendritic tree that

branches distant from the cell soma, secondary and

tertiary branches are usually not observed in 100µm

thick sections

Type III commissural

neurons

Non-pyramidal commissural neurons

Not only neuropathologically but also in behavioral tests APP23 mice showed

cognitive decline compared to wildtype littermates from the age of 3 months

onwards, preceeding amyloid plaque depositioning (Van Dam et al., 2003).

Because of these findings APP23 mice were considered to be a useful model to

analyze the contribution of APP and Aβ for the pathogenesis of AD (Sturchler-

Pierrat and Staufenbiel, 2000; Kuo et al., 2001). However, other authors presume

that this mouse model is a limited model of AD because only low tau

phosphorylation without NFT formation and low complement and microglial

response was seen compared to AD patients (Schwab et al., 2004). Therefore,

other transgenic mouse models some of which combine two or more mutations

were created in order to mimic AD pathology more exactly. Accordingly, PSEN1/2

and/or tau-protein mutations were combined with APP mutation. Here amyloid

pathology was combined with neuron loss described earlier and the generation of

NFTs (Lewis et al., 2001; Perez et al., 2005). In another 3x transgenic mouse

models (APP, PSEN1 and Tau transgenes) the mutations also led to these

pathologies but showed also intracellular τ accumulation (Oddo et al., 2003; Oddo

et al., 2006). Combining these features in a single mouse model made it an

interesting model in AD research. A problem in investigating AD pathology with

these multi-mutant mouse models is the interaction of the different proteins.

However, to clarify which protein leads to a specific pathology these models are

Introduction

10

suboptimal in comparison to single trangenic ones. Here, the APP23 mouse model

combines a single mutation and some of the typical AD pathology features.

The APP48 transgenic mouse model expresses intracellular Aβ42 and shows

intracellular Aβ lesions. Neurodegeneration is represented by the reduction of

hippocampal CA1 neurons and white matter reduction going along with an

impaired motor function in the Rotarod test (Abramowski et al., 2012b) In

comparison to the APP23 mice, APP48 mice do not show extracellular amyloid

plaques. In so doing, they can serve as an artificial model for intraneuronal Aβ

toxicity.

In so doing, the APP23 and APP48 mouse models are ideally suited to study the

influence of APP, intra- and extracellular Aβ on mitochondrial structure in vivo.

APP23 mice show intra- and extracellular Aβ pathology and an overexpression of

APP whereas intracellular Aβ is studied in APP48 mice. In contrast to other

transgenic mouse models these mouse models permit the analysis of Aβ effects

under distinct conditions without interference of a second transgenic manipulation

that could explain pathologies as well:

APP23 mice are used for the analysis of extra- and intraneuronal Aβ production by

APP cleavage.

APP48 mice are used for the analysis of APP independent intraneuronal Aβ

toxicity.

1.5 Aims of the study

To address the question whether 1. intra- or 2. extracellular Aβ leads to

mitochondrial and/or dendritic alterations and to identify the role of APP-derived

Aβ production for Aβ-induced mitochondrial and/or dendritic changes I studied the

mitochondrial morphology and distribution in neurons of the frontocentral cortex of

APP23 and APP48 transgenic mice in comparison to that of wildtype mice.

In detail my specific aim was to answer the following question:

1. does intracellular overexpression of Aβ independent from APP cleavage in

APP48 mice affect mitochondrial integrity and/or distribution in the cell and

dendritic morphology?

Introduction

11

2. does extracellular Aβ accumulation in APP23 mice affect intracellular

mitochondrial structure and/or distribution and/or dendritic morphology?

By addressing these questions I will be able to elucidate whether beta- and

gamma-secretase directed APP processing including Aβ formation or APP

independent Aβ formation leads to intra- and extracellular AD pathology. Although

the mouse models are artificial and can not be considered as good models for

human AD pathology, this study will lead to a better understanding of the cellular

and subcellular effects induced by Aβ in the brain under different conditions.

Material and Methods

12

2 Material and Methods

2.1 Material and Equipments used

Table 2: Buffer and Reagents

A. Tris-Buffer Saline (TBS), pH 7.4

Components Concentration/Amount Company Tris-HCl 20 mM Merck, Darmstadt, Germany NaCl 150 mM Merck, Darmstadt, Germany

ddH2O To 1 liter Millipore GmbH, Schwalbach, Germany

B. Phosphate buffer saline (PBS), pH 7.6

KCl 2.7 Mm Merck, Darmstadt, Germany KH2PO4 1.46 Mm Merck, Darmstadt, Germany NaCl 137 mM Merck, Darmstadt, Germany Na2HPO4.2H2O 8.1 mM Merck, Darmstadt, Germany

ddH2O To 1 liter Millipore GmbH, Schwalbach, Germany

Tween 0.05 % Bio-Rad, Hercules, CA, USA C. 2.6% Phosphate buffer paraformaldehyde solution (PFA), pH 7.6

PFA 2.6% Sigma, Taufkirchen, Germany PBS 0.2M, pH 7.6 500 ml See PBS solution

ddH2O 500 ml Millipore GmbH, Schwalbach, Germany

D. Reducing Solution (IHC)

Methanol 10% Merck, Darmstadt, Germany H2O2 30% Sigma, Taufkirchen, Germany in TBS, pH 7.6 0.05 M See TBS solution E. Blocking Solution (IHC)

DL-Lysine 0.1 M Sigma, Taufkirchen, Germany Triton-X 0.25% Sigma, Taufkirchen, Germany in BSA 10% Sigma, Taufkirchen, Germany F. Perfusion solution

Heparin 0,5% Roche, Mannheim, Germany In TBS 0,05 M pH 7,3

To 1 liter See TBS solution


13

G. Tracing solution in PFA Iodocetic acid 0,8% Sigma, Taufkirchen, Germany Sodium periodate 0.8% Sigma, Taufkirchen, Germany DL-Lysine 0.1 M Sigma, Taufkirchen, Germany In PFA To 1 liter See PFA solution H. Other Reagents

3,3'-Diaminobenzidine (DAB) Merck, Darmstadt, Germany

Alcohol Sigma-Aldrich, Steinheim, Germany

Avidin-Biotin Complex Kit (ABC-Kit) Vectastain, Vector laboratories, Burlingame, CA. USA

Aβ42 ELISA plates ELISA from Innogenetics, Ghent, Belgium

BCA Protein Assay Bio-Rad, Hercules, CA, USA BSA (10%) Sigma, Taufkirchen, Germany BSAc Aurion, Wageningen, Netherlands

Carbocyanine DiI Molecular Probes, Eugene, OR, USA

cold washed fish gelatine (CWFG) BBInternational, Cardiff, UK

ECL detection system

Supersignal Pico Western system, ThermoScientific-Pierce, Waltham, MA, USA

ECL Hyperfilm GE Healthcare, Buckinghamshire, UK

Epon Sigma-Aldrich, Steinheim, Germany

Eukitt© Kindler, Freiburg, Germany Formic acid Applichem, Darmstadt, Germany Glutaraldehyd Plano, Wetzlar, Germany

Ketamin 25% Bela-Pharma GmbH, Vechta, Germany

lithium dodecyl sulfate (LDS) sample buffer Invitrogen, Carlsbad, CA, USA

LR-white London Resins Co. Ltd, Birkshire, England

Micropestle Eppendorf, Hamburg, Germany Na-Borhydrit Merck, Darmstadt, Germany NGS (normal goat serum) Aurion, Wageningen, Netherlands NuPAGE 4-12% Bis-Tris gel system Invitrogen, Carlsbad, CA, USA Osmium tetroxide Chempur, Karlsruhe, Germany

Ponceau S staining or β-actin Santa Cruz Biotechnology, CA, USA

Propanol Sigma-Aldrich, Steinheim, Germany

Propylenoxid Merck, Darmstadt, Germany protease and phosphatase inhibitor-cocktail (Complete and PhosphoSTOP)

Roche, Mannheim, Germany

synthetic Aβ1-42 Bachem, Bubendorf, Switzerland Uranyl acetate Merck, Darmstadt, Germany


14

Xylazin 2% Riemser Arzneimttel AG, Greifswald, Germany

Table 3: List of Antibodies used for Immunoelectron microscopy

Primary antibody Antigen Source of primary antibody

4G8 Aβ17-24 Covance, Dedham, USA

MBC42 Aβ37-42 Gift of H. Yamaguchi, Gunma, Japan

GAM IgG Aurion, Wageningen, Netherlands IgG polyclonal goat,

MSIgG Biomeda, Foster City, CA, USA

6E10 anti-Aβ1-17 Covance, Dedham, USA

anti-AβN3pE AβN3pE IBL International GmbH, Hamburg, Germany

Phosphorylated Aβ (pAβ) Phosphorylated Aβ

Girft of J. Walter, Department of Neurology, University of Bonn, Bonn, Germany

Table 4: List of Equipments used

Table centrifuge

Mikro 200 Hettich Zentrifugen, Tuttlingen, Germany

vacuum centrifuge

Vacufuge Eppendorf, Hamburg, Germany

Magnetic Stirrer

MR Hei Standard Heidolph Instruments, Solingen, Germany

Microscopes Fluorescence Microscope Leica DMLB

Leica, Bensheim, Germany

Scanning Confocal Microscope Leica TSC NT

Leica, Bensheim, Germany

Electron microscope

JEM 1400 Jeol, Tokyo, Japan

EM400T 120KV Philips, Eindhoven, The Netherlands

EM 10 Zeiss, Oberkochen, Germany

pH Meter pH-meter 210 Hanna Intruments, Kehl am Rhein, Germany

Vortex VM 3000 Mini vortexer Henry Troemner LLC, NJ, USA Vibratome Leica VT 1000S Leica, Bensheim, Germany Grids for EM Plano, Wetzlar, Germany Diamont knive Diatomeknives Hatfield, PA, USA Embedding film

TED Pella Inc., Redding, CA, USA

Microtome Ultracut Reichert Jung, Depew, NY, USA


15

Table 5: List of Softwares used

CorelDRAW Graphics Suite 12 CorelDRAW, Unterschleissheim, Germany

EndNote 8.0 Thomson ISI ResearchSoft, Philadelphia, PA, USA

ImageJ, Imaging Processing and Analysis Software

NIH, Bethesda, MD, USA

Leica FireCam Software Leica, Bersheim, Germany Leica TCS Software Leica, Bersheim, Germany

Open Office Apache Software Foundation, DE, USA

SPSS software Chicago, IL, USA Microsoft Office Microsoft, CA, USA

2.2 Mouse models

Table 6: Mouse models used

Model 3 month 15 - 18 month APP23 n = 12 n = 17 APP48 n = 12 n = 17 Wildtype n = 14 n = 20

APP23 and APP48 mice were generated as described previously (Sturchler-

Pierrat et al., 1997; Herzig et al., 2004; Abramowski et al., 2012b) and

continuously back-crossed to C57BL/6. An expression construct containing a

murine Thy-1 promoter was used to drive neuron-specific expression of human

mutant APP751 with the Swedish mutation 670/671 KM→NL in APP23 mice. In

APP48 mice an expression construct encoding a rat proenkephalin signal

sequence followed by Aβ1-42 driven by the neuron specific Thy-1 promoter was

used to drive neuron-specific expression of human wildtype Aβ1-42.

For this study female APP23 mice (3 months, n = 12; 15 months, n = 17) and

female and male APP48 mice (3 months, n = 12; 18 months, n = 17) were

analyzed. As control, respective female and male wildtype littermates of 3 (n = 14),

15 (n = 10), and 18 months (n = 10) were used (Rijal Upadhaya et al., 2013).

2.3 Tissue praparation and DiI tracing

The commissural neurons in the frontocentral cortex were stained using DiI as a

tracer. The dye is a lipophilic tracer and is absorbed through the cell membranes

being in contact to the dye and diffuses along the cell membrane labelling the cell

with all of its processes. In neurons this results in an antero- and retrograde


16

tracing which leads to the staining of the complete cell including synapses and

dendrites. This dye allows precise Golgi-like tracing of neurons in postmortem

fixed tissue in a quality similar to in vivo tracing methods using rhodamine tracers

even in only weakly traced neurons (Galuske and Singer, 1996; Capetillo-Zarate et

al., 2006).

For DiI tracing the brains of 3 and 15-18-months-old APP23, APP48 and wildtype

mice were studied. Animals were treated in agreement with the German laws on

the use of laboratory animals. Mice were anesthetized. Perfusion was performed

transcardially with Tris-buffered saline (TBS) with heparin (pH 7.4) followed by the

injection of 0.1 M PBS (pH 7.4) containing 2.6% paraformaldehyde (PFA), 0.8%

iodoacetic acid, 0.8% sodium periodate and 0.1 M D-L lysine. The brains were

removed in total and post-fixed in 2.6% phosphate-buffered PFA (pH 7.4)

containing 0.8% iodoacetic acid, 0.8% sodium periodate and 0.1 M D-L lysine

(Galuske et al., 2000). Three days later a single crystal (~0.3 mm3) of the

carbocyanine dye DiI was implanted into the left frontocentral cortex, 1 mm

rostrally from the central sulcus, 2 mm laterally from the middle line and 1 mm

deep in the cortex as reported earlier (Capetillo-Zarate et al., 2006). After

incubation in 2.6% phosphate-buffered PFA for at least 3 months at 37°C, 100-µm

thick coronal vibratome sections were cut. All sections of a given mouse brain

were separately stored and continuously numbered. Sections were temporarily

mounted in TBS for microscopic analysis (Rijal Upadhaya et al., 2013).

2.4 Microscopic and quantitative analysis

In layer III of the frontocentral cortex of the right hemisphere, contralateral to the

implantation site of the tracer, the morphology of traced commissural neurons was

examined. The traced neurons were assigned to different types according to their

morphology (Capetillo-Zarate et al., 2006) (Tab. 1). Then the number of traced

commissural neurons of each type in wildtype mice was counted and compared

with that in APP23 and APP48 mice. For qualitative and quantitative analysis 10

consecutive sections (100-mm thickness each) representing a tissue block of 1

mm thickness were studied for each mouse. Analysis started at the anterior

commissure setting the caudal limit of the investigated tissue block. For each

coronal section, the medial boundary of the region investigated was set as the

vertical line at the cingulum that separated the cingulate cortex from secondary


17

motor cortex (M2). The horizontal boundary was set as the horizontal line

separating the primary somatosensory cortex (SC1) from the insular cortex.

For the qualitative analysis a laser scanning confocal microscope was used.

Stacks of 2D images were superimposed digitally using the Image J Image

Processing and Analysis software, and 3D data sets were generated for the

visualization of neurons with their entire dendritic tree. For quantification, traced

neurons in layer III were counted in the region of interest in 10 consecutive

sections of the tissue block taken for qualitative and quantitative analysis using a

fluorescence microscope. In so doing, we analyzed a cortex volume of 5–6 mm³ in

each mouse. Mean and median values of the number of traced neurons were

calculated and compared between wildtype, APP23 and APP48 mice (Rijal

Upadhaya et al., 2013).

2.5 Immunohistochemistry

Immunohistochemistry was performed for the visualization of Aβ pathology as well

as dendritic morphology in APP48 and APP23 mice. After formic acid pretreatment

free-floating sections were incubated in goat anti-mouse immunoglobulin (IgG) to

block cross-reactions with intrinsic mouse IgG as previously described (Thal et al.,

2007). To detect Aβ-positive material the sections were stained with monoclonal

antibodies specifically detecting the C-terminus of Aβ42 (MBC42) (Yamaguchi et

al., 1998). In APP23 mice anti-Aβ17-24 (4G8) was used to stain Aβ-deposits

regardless of Aβ40 or Aβ42. C-terminus-specific MBC40 (Yamaguchi et al., 1998),

antibodies detecting exclusively the C-terminus of Aβ40 were used in APP23 mice

as well. N-terminal-truncated and pyroglutamate modified AβN3pE was detected

with anti-AβN3pE (Saido et al., 1995). Phosphorylation of serine 8 of Aβ was

detected with antibodies against phosphorylated Aβ (pAβ) (Kumar et al., 2011;

Kumar et al., 2013).

The primary antibodies were detected with a biotinylated secondary antibody and

the avidin-biotin-peroxidase complex (ABC complex), and visualized with

Diaminobenzidine (DAB) (Hsu et al., 1981). Sections were mounted in Eukitt©.

The immunostained sections were analyzed with a Leica DMLB fluorescence

microscope. Positive and negative controls were performed (Rijal Upadhaya et al.,

2013).


18

2.6 Protein extraction from brain tissue

Protein extraction was carried out from female APP23 (n = 4), APP48 (n = 4), and

wildtype littermates (n = 4) mouse brains, aged 9–11 months. Mice of this age

were taken to demonstrate the differences in the biochemical distribution of Aβ in

APP23 and APP48 mice.

Fresh frozen forebrain (0.4 g) was homogenized in 2 ml of 0.32 M sucrose

dissolved in TBS containing a protease and phosphatase inhibitor-cocktail with

Micropestle followed by sonication. The homogenate was centrifuged for 30 min at

14.000 × g at 4°C. The supernatant (S1) with the soluble and dispersible fraction

not separated from one another was kept. The pellet (P1) containing the

membrane-associated and the insoluble, plaque-associated fraction was

resuspended in 2% sodium dodecyl sulfate (SDS).

Ultracentrifugation of the supernatant S1 at 175.000 × g was used to separate the

soluble, i.e. the supernatant after ultracentrifugation (S2), from the dispersible

fraction, i.e. the resulting pellet (P2). The pellet P2 with the dispersible fraction was

resuspended in TBS.

The SDS-resuspended pellet P1 was centrifuged at 14.000 × g. The supernatant

(S3) was kept as membrane-associated SDS-soluble fraction. The pellet (P3) that

remained was dissolved in 70% formic acid and dried in a vacuum centrifuge and

reconstituted in 100 µl of 2X lithium dodecyl sulfate (LDS) sample buffer followed

by heating at 70°C for 5 min. The resultant sample was considered as insoluble,

plaque-associated fraction (Mc Donald et al., 2010). The total protein amounts of

soluble, dispersible, and membrane-associated fractions were determined using

BCA Protein Assay (Rijal Upadhaya et al., 2013).

2.7 SDS-PAGE and Western blot analysis

For sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE,)

soluble (S2), dispersible (P2), membrane-associated (SDS-sobluble, S3), and

insoluble, plaque-associated (formic acid soluble, P3) fractions (50 µg total

protein) were electrophoretically resolved in a precast NuPAGE 4-12% Bis-Tris gel

system. The protein load was controlled either by Ponceau S staining or β-actin

immunoblotting. Aβ was detected by western blotting with anti-Aβ1-17. Blots were

developed with an ECL detection system and illuminated in ECL Hyperfilm (Rijal



19

2.8 Aβ Enzyme Linked Immunosorbent Assay (ELISA)

For analysis of Aβ by ELISA, forebrain homogenates from APP23 and APP48

mice of each age group (2-3 months: n = 6 (APP23), 6 (APP48); 15-18 months: n

= 7 (APP23), (8 APP48)) were homogenized, centrifuged and loaded on sandwich

ELISA plates for quantification of Aβ peptides as previously described (Capetillo-

Zarate et al., 2006). Standard curves were prepared with synthetic Aβ1-42 and

diluted in extracts of non-transgenic mouse forebrain prepared in parallel as

described above. Each sample was analyzed in duplicate (Rijal Upadhaya et al.,

2013).

2.9 Stereology

Six APP23, six APP48, and six wildtype mice at the age of 3 and 15-18-months,

respectively, were chosen randomly for stereology. One brain section of the

frontocentral cortex already quantified for the number of DiI-traced neurons was

selected by chance and stained with aldehyde fuchsin-Darrow red. Quantification

of neurons was performed according to the principles of unbiased stereology

(Schmitz and Hof, 2000). The frontocentral cortex volume was defined as the

volume of the subfields M2, primary motor cortex (M1), SC1 starting at the level of

the anterior commissure as described previously (Capetillo-Zarate et al., 2006).

The CA1 volume was measured in serial 100µm thick sections of the entire mouse

brain at 5x magnification. Neurons were counted in three different, randomly

chosen microscopic fields (40 x magnification) of an aldehyde fuchsin-Darrow red

stained section of the frontocentral cortex and CA1, respectively. For optical

dissection, stacks of 10 images in 2 µm focus distance were generated for each

microscopic field. Only those neurons having nuclei with dark and round nucleoli

visible in the center of soma in one of the stack-images were considered for

quantification using the ImageJ software. The number of neurons in the

frontocentral cortex and CA1 was calculated on the basis of the respective

reference volumes and neuron densities (Rijal Upadhaya et al., 2013).

2.10 Electron microscopy, immuno electron microsco py

100 µm thick vibratome sections of the frontocentral cortex and of the

hippocampus from six wildtype, six APP23 and six APP48 mice, aged 3 and 15-18

month respectively, were flat-embedded in Epon. A second vibratom section from


20

each animal and region was flat embedded in LR-White-Resin. A part of the

frontocentral cortex covering all six cortical layers was dissected under

microscopic control and pasted on Epon blocks with a drop of Epon. Likewise, a

part of CA1 subfield of the hippocampus with adjacent stratum oriens and

radiatum was cut and pasted on a second Epon block. Ultrathin sections were cut

at 70 nm. Epon sections were block stained with uranyl acetate and lead citrate,

and viewed with a Philips EM400T 120KV, a Zeiss EM10, or a JEM-1400 electron

microscope. LR-White sections were immunostained with anti-Aβ42 (MBC42) and

anti-Aβ1-17 (6E10) antibodies and visualized with anti-mouse secondary antibodies

labeled with 10 nm nanogold particles. Digital pictures were taken (Rijal Upadhaya

et al., 2013).

2.10.1 Semiquantitative analysis of synapse densiti es

Digital pictures from Epon embedded sections were taken from 20 soma- and

plaque-free neuropil areas located in layers II-VI at 4600-times magnification. The

numbers of the symmetric and asymmetric synapses were counted and the length

of the synapses was determined with the ImageJ software. The synaptic density

was determined separately for symmetric and asymmetric synapses according to

DeFelipe et al. (DeFelipe et al., 1999) (synaptic density = Number of synapse-

profiles in a given area/length of synaptic profiles). These semiquantitative data

were used to compare the synaptic densities between the different mouse lines.

Asymmetric and symmetric synapses were distinguished according to published

criteria (Colonnier, 1968; Miranda et al., 2009).

Synaptic densities in the CA1 regions were measured in 10 randomly taken

pictures of the stratum oriens and in 10 randomly taken pictures of the stratum

radiatum at 4600-times magnification (Rijal Upadhaya et al., 2013).

2.10.2 Semiquantitative analysis of dystrophic dend rites

The frequency of dystrophic dendrites was observed by counting the number of

dystrophic neurites in the 20 pictures taken for the determination of the synapse

densities. The criteria for the identification of dystrophic dendrites at the

ultrastructural level were: neurite profiles with a disorganized cytoplasm,

occurrence of multilamellar, whirl-like structures in the absence of ultrastructurally

intact cell organelles in the area of the lesion, and an enlarged size compared with

neighboring neuritic profiles (Fig. 1) (Rijal Upadhaya et al., 2012). This number


21

was determined in six APP23, six APP48 and six wildtype mice and used as a

semiquantitative score for structural dendritic alterations (Rijal Upadhaya et al.,

2013).

Fig. 1 Identification of dystrophic neurites. Electron microscopy was used to identify dystrophic neurites (arrows ) as previously described (Rijal Upadhaya et al., 2012) and shown here in the frontocentral cortex of 15-month-old APP23 mice. Such neurites are characterized by neuritic swelling and contain vesicles with electron dense bodies (black arrowheads ) probably representing autophagic vacuoles. Mitochondria (M) in these neurites appear morphologically intact. Few multivesicular bodies are seen in these neurites as well (white arrowhead ). The calibration bar corresponds to: 250 nm (Rijal Upadhaya et al., 2013).

2.10.3 Analysis of mitochondria

To clarify whether APP-independant production of intraneuronal Aβ or APP-

derived intra- and extracellular Aβ accumulation are critical for mitochondrial

changes in neurons, mitochondrial alterations in frontocentral and CA1 neurons of

six APP23, six APP48 and six wildtype mice were compared at the electron-

microscopic level. For this purpose I compared the area profiles from neuritic and

somatic neuronal mitochondria as well as from the respective somata and


22

peripheral neurites in plaque-free areas. Mitochondria in peripheral dendrites and

axons were analyzed in the 20 pictures taken from soma- and plaque-free areas of

the frontocentral cortex and in 20 pictures of the stratum oriens and radiatum of

the CA1 hippocampal subfield also used for the assessment of synapse densities.

The mitochondria in neuronal somata were studied in 40 pictures from 10 different

randomly taken of layer II – layer VI neurons of the frontocentral cortex and in 40

pictures from 10 randomly taken CA1 neurons with a Philips EM400T 120 KV

electron microscope at 6000-times magnification (4 pictures per neuron) in each

individual mouse. The area profiles from morphologically intact mitochondria and

from those with an altered ultrastructure, i.e. degeneration of christae as

previously described in prion disease (Siskova et al., 2010), were separately

obtained for somatic and peripheral neuritic mitochondria. Volume densities in

percent of soma and neurite profiles were calculated according to the criteria for

unbiased stereology (Weibel, 1969) following the determinations provided in (Tab.

7).

Those mitochondria which exhibited th typical ultrastructural pattern with outer and

inner membranes as well as christa membranes and altered mitochondria with

clear structural changes of the christa membranes like rarefication, loss, abnormal

structures were considered for quantification (Fig. 2). Assessments were

performed without previous knowledge of the genotypes of the animals.

Two APP23, two APP48, and two wildtype mice at the age of 15-18-months were

chosen randomly for immunoelectron microscopy analysis of Aβ deposition in

nerve cell somata and soma-free neuropil. LR-White sections were immunostained

with anti-Aβ42 (MBC42) antibodies and visualized with anti-mouse secondary

antibodies labelled with 15 nm nanogold particels. Digital pictures were taken with

a JEM 1400 electron microscope and a Zeiss EM 10 from 20 soma-free neuropil

areas located in layers II-VI and of 10 neuron somata at various magnification

levels. The different Aβ-deposition characteristics seen as the 15 nm gold particels

in the neurite, soma or mitochondria were compared to relate them with

stereological findings (Rijal Upadhaya et al., 2013).


23

Fig. 2: Electromicroscopical images for several steps of the assesment of stereological data of neuronal mitochondria in the frontocentral cortex of an APP48 mouse. A, various cell compartements are shown, nucleus (N), mitochondria (M), rough endoplasmic reticulum (RER), axon (Ax ). B, mitochondria measured and labelled black. C, alterated mnitochondria labelled red, normal mitochondria labelled black. D, intracellular, perinuclear area labelled in black. Pictures were taken with a Philips EM400T 120KV at 6000 times magnification.

2.11 Statistical analysis

SPSS 19.0 (SPSS, Chicago, IL, USA) software was used to calculate statistical

tests. Non-parametric tests were used to compare wild type, APP23, and APP48

mice. p-values were corrected for multiple testing using the Bonferroni method.

Parametric data were analyzed by ANOVA with subsequent Games-Howell post-

hoc test to correct for multiple testing or using the Welch test. The results of the

statistical analysis are summarized in Supplementary Tab. 1 (Rijal Upadhaya et

al., 2013).


24

Table 7 : Determinations of the parameters employed for quantification of mitochondrial alterations

in neurites and nerve cell somata (Rijal Upadhaya et al., 2013)

Percentage of altered mitochondria in nerve cell somata =

∑ altered mitochondrial area within nerve cell somata

∑ complete mitochondrial area within nerve cell somatax100

Percentage of altered mitochondria in neurites (axons and dendrites) =

∑ altered mitochondrial area within neurites

∑ complete mitochondrial area within neuritesx 100

Mitochondrial volume density in the nerve cell somata =

∑ complete mitochondrial area within nerve cell somata

∑ area of nerve cell somatax100

Mitochondrial volume density in neurites =

∑ complete area of mitochondria within neurites

∑ area of neuritesx100

Volume density of altered mitochondria in the nerve cell somata =

∑ altered mitochondrial area within nerve cell somata

∑ area of nerve cell somatax100

Volume density of altered mitochondria in neurites =

∑ altered mitochondrial area within neurites

∑ area of neuritesx 100

Results

25

3 Results

3.1 Different patterns of A β-pathology in APP23 and APP48 mice

As previously published, 15 month-old APP23 mice exhibited a high number of

extracellular Aβ-plaques in the cerebral cortex (Fig. 3a – indicated by arrows) as

well as cerebral amyloid angiopathy as previously described in male and female

animals (Sturchler-Pierrat and Staufenbiel, 2000). Intracellular Aβ42 detectable

with MBC42 was not abundant at the light microscopic level in this region neither

in neurons nor in glial cells. MBC40-positive Aβ40 was observed in the perikarya of

pyramidal neurons. At 3 month of age APP23 mice did not exhibit Aβ plaques or

vascular Aβ deposits as previously described in male and female animals

(Abramowski et al., 2012a; Rijal Upadhaya et al., 2013). APP48 mice, on the other

hand, did not show Aβ plaques but intracellular accumulation of Aβ in dendritic

threads, somatic granules in neurons and in microglial Aβ-grains at 3 and 18

months of age (Fig. 3b). This pathology was seen in male and female animals

(Abramowski et al., 2012a).

Modified Aβ such as AβN3pE was detected in a large number of plaques in 15-

month-old APP23 mice (Figure 3c). In APP48 mice only few dendritic threads

exhibited AβN3pE at 3 months of age (Figure 3d) whereas at 18 months of age a

significant number of AβN3pE-positive inclusions was observed as shown previously

(Abramowski et al., 2012a). pAβ in APP23 mice was detected in plaques of 15

months old APP23 mice (Figure 3e). Intraneuronal pAβ in APP23 mice was

apparent as previously reported in APP-PS1 transgenic animals (Kumar et al.,

2013). Only single pAβ-positive threads were stained in 3-month-old APP48 mice

(Figure 3f) whereas a few more pAβ-positive threads, grains and somatic granules

were observed at 18 months of age (Rijal Upadhaya et al., 2013).

Results

26

Fig. 3 Amyloid beta protein (Aβ) -pathology in 15-month-old APP23 (a, c, e) and 3-month-old-APP48 mice (b, d, f). a: The APP23 mouse showed a high number of extracellular Aβ plaques detectable with an antibody raised against Aβ17-24 (arrows ). Intracellular Aβ was negligible. b: In the 3-month-old APP48 mouse no extracellular Aβ -pathology was apparent. These animals showed intraneuronal dendritic threads (arrowheads) and somatic granules (lucent arrow ) as well as intramicroglial Aβ–grains (arrows ) detectable with anti-Aβ17-24 as previously published (Abramowski et al., 2012a). c: Amyloid plaques in APP23 mice did also contain N-terminal truncated and pyroglutamate modified AβN3pE (arrows ). d: AβN3pE was also found in even 3-month-old APP48 mice in some neuritic threads (arrowheads ). e: phosphorylated Aβ (pAβ) was detected in amyloid plaques in 15-month-old APP23 mice. f: In 3-month-old APP48 mice only single threads showed labeling with anti-pAβ . Calibration bar in b corresponds to: a, b = 30 μm; c = 80 μm; e = 60 μm; d, f = 20 μm (Rijal Upadhaya et al., 2013).

Biochemical analysis revealed that 3-month-old APP48 mice contained ~70 times

more total Aβ42 detected by ELISA than APP23 mice whereas at 15–18 months of

Results

27

age APP23 mice contained Aβ42 in a concentration ~17 fold higher than in APP48

mice (Figure 4a, Supplementary Tab. 1a) (Rijal Upadhaya et al., 2013).

Fig. 4 Biochemical analysis of Amyloid beta protein (Aβ) in APP23 and APP48 mice. a: Total Aβ42 levels detected by ELISA in forebrain hemispheres of 2–3 and 15-18-month-old APP23 and APP48 mice. At 2–3 months APP23 mice exhibited low amounts of Aβ42 whereas APP48 mice displayed significantly more Aβ42 in the brain. At 15–18 months APP48 mice showed more Aβ than at 2–3 months of age but APP23 mice exhibited several times more Aβ in the forebrain. b: For demonstration of the types of Aβ aggregates in APP23 and APP48 mice brain homogenates of 9-11-month-old animals were analyzed by SDS-PAGE and western blotting after preparation of the soluble, dispersible, membrane-associated and insoluble (plaque-associated) fraction. Soluble Aβ as detected with antibodies raised against Aβ1-17 (6E10) was restricted to APP23 mice. Dispersible, membrane-associated, and insoluble (plaque-associated, formic acid soluble) Aβ aggregates were found in both transgenic mouse lines. The Aβ detected in the insoluble fraction of the forebrain homogenates of APP48 mice represents Aβ aggregates that require formic acid pretreatment before analysis similar to plaque-associated Aβ in APP23 mice. Since APP48 mice did not develop Aβ plaques this insoluble Aβ presumably represented intracellular fibrillar aggregates, such as dendritic threads. Wildtype controls did not exhibit detectable amounts of Aβ in all four fractions. The original western blots are depicted in Supplementary Fig. 1 (ELISA data from APP23 mice were previously published in a different context (Abramowski et al., 2012a). ***p < 0.001 Welch-test (Rijal Upadhaya et al., 2013).

Results

28

To document the distribution of Aβ aggregates brain homogenates of 9-11-month-

old APP23 and APP48 mice for Aβ in the soluble (S2), dispersible (P2),

membrane-associated (S3) and insoluble fraction (P3) were analyzed. APP23

mice exhibited soluble, dispersible, membrane-associated and insoluble, plaque-

associated Aβ (Figure 4b) as reported previously in detail (Rijal Upadhaya et al.,

2012). In contrast, APP48 mice only exhibited dispersible, membrane-associated

and insoluble, aggregated Aβ whereas soluble Aβ was not detectable (Figure 4b)

(Rijal Upadhaya et al., 2013).

3.2 Degeneration of neurites and asymetric synapses in APP23

but not in APP48 mice

Using retrograde tracing with DiI three types of commissural neurons were

subclassified as previously published (Capetillo-Zarate et al., 2006) in APP23,

APP48, and wildtype mice (Tab. 1). Type I and type II commissural neurons

exhibited alterations in the dendritic tree as well as a decrease in number in

APP23 mice compared to wildtype littermates as previously reported (Capetillo-

Zarate et al., 2006) (Fig. 5). Type III commissural neurons did not exhibit

differences in their morphological appearance among APP48, APP23, and

wildtype littermates. There were no significant differences in the numbers of type I,

II, and III commissural neurons in APP48 mice compared to the respective

wildtype littermates (Fig. 5 c-i, Supplementary Tab. 1). Thus, 15 month-old APP23

mice exhibited dendritic degeneration of commissural neurons whereas APP48

mice did not (Rijal Upadhaya et al., 2013).

Results

29

Fig. 5 Dendritic degeneration in frontocentral commissural neurons of APP23 and APP48 mice. a: A type I neuron in an 18 month-old wildtype animal exhibits a symmetric dendritic tree with prominent secondary and tertiary branches. b: In contrast, the dendritic tree of a representative type I commissural neuron (I) in a 15-month-old APP23 mouse is degenerated. Most basal dendrites were shrunken and had a reduced caliber (arrows ). The degenerated dendrites showed some branches (arrows ) that distinguished the degenerated type I neuron (I) from type II neurons without ramifications near the soma (II). c: Such a degeneration of the dendritic tree was not seen in APP48 mice. d-f : Numbers of DiI-traced type I, type II and type III commissural neurons in 15-18-month-old mice. d: APP23 mice at 15 months of age showed a decrease by more than 50% of the type I commissural neurons compared with 18-month-old wild type and APP48 mice. e: There was a significant reduction of type II commissural neurons in APP23 mice at 15 months of age compared with wild type littermates and APP48 mice at 18 months of age. f: Although APP23 mice had higher numbers of type III commissural neurons there was no significant difference from wild type littermates. g-h : No significant differences among the frequencies of DiI-traced type I, type II, and type III commissural neurons were observed at 3 months of age. ** p < 0.01 (Further statistical analysis: Supplementary Tab. 1). Means and standard errors are depicted in d-i. (Quantitative data from APP23 mice and their respective wild type littermates were previously published in a different context (Abramowski et al., 2012a; Rijal Upadhaya et al., 2013). Calibration bar in c corresponds to: a-c = 30 μ m. To confirm neuritic degeneration transmission electron microscopy was used to

compare the presence of dystrophic neurites among APP23, APP48 and wildtype

mice. The frequency of dystrophic neurites was higher in the frontocentral cortex

of 15 month-old APP23 mice when compared to 15-18 month-old APP48 and

Results

30

wildtype mice (Fig. 6, Supplementary Tab. 1c). There were no differences in the

frequency of dystrophic neurites between APP48 and wildtype mice or between 3

month-old animals of each genotype (Fig. 6) (Rijal Upadhaya et al., 2013).

Fig. 6 Frequencies of dystrophic neurites in wild type, APP23, and APP48 mice. The semiquantitatively assessed frequency of dystrophic neurites in the soma- and plaque-free frontocentral neuropil at the electron microscopic level was higher in 15-month-old APP23 mice than in wild type and APP48 mice of the same age group. In APP48 mice there was no increase in the frequency of dystrophic neurites in comparison to wild type mice. 3-month-old mice did not exhibit significant differences in the frequency of dystrophic neurites among the 3 genotypes nor were differences found in neuropil of the stratum radiatum and oriens of the CA1 region. (Data from APP23 mice and their respective wild type littermates were previously published in a different context (Rijal Upadhaya et al., 2012). *p < 0.05 (Further statistical analysis: Supplementary Tab. 1). Means and standard errors are depicted (Rijal Upadhaya et al., 2013). Qualitative changes in synapse morphology other than the generation of

dystrophic neurites as in 15 month-old APP23 mice were not observed.

Semiquantitative analysis of the densities of symmetric and asymmetric synapses

showed a reduction of the density of asymmetric synapses in the frontocentral

cortex of 3 and 15 month-oldAPP23 mice in comparison to wildtype mice. In the

stratum radiatum and oriens of CA1 a similar trend was observed but did not reach

significance. Such a reduction of asymmetric synapses was not observed in

APP48 mice in comparison to wildtype littermates (Fig. 7a). There were no

significant differences in the numbers of symmetric synapses among APP23,

Results

31

APP48, and WT mice (Fig. 7b, Supplementary Tab. 1d) (Rijal Upadhaya et al.,

2013).

Figure 7 Synapse densities in wild type, APP23 and APP48 mic e. a: Loss of asymmetric synapses in the frontocentral cortex of 3- and 15-month-old APP23 mice in comparison to wild type mice. 18-month-old APP48 and wild type mice did not differ significantly in the density of asymmetric synapses. At 3 months of age APP48 mice had even more asymmetric synapses than wild type animals. In CA1 there were also slightly less asymmetric synapses in APP23 mice than in wild type controls and APP48 mice. However, these differences were not significant. b: There were no significant differences in the number of symmetric synapses in the frontocentral cortex and in CA1 in 3- and 15-18-month-old animals. c: APP23 and APP48 mice of both age groups exhibited reduced numbers of CA1 neurons compared to wild type mice whereby CA1 neuron loss was most pronounced in APP23 mice. d: The number of neurons in the frontocentral cortex did not vary significantly among 15-18-month-old wild type, APP23, and APP48 mice. Therefore, younger animals were not studied for the number of neurons in the frontocentral cortex. *p < 0.05, **p < 0.01, ***p < 0.001 (Further statistical analysis: Supplementary Tab. 1). Means and standard errors are depicted. (Some data were previously published in a different context (Abramowski et al., 2012a; Rijal Upadhaya et al., 2012; Rijal Upadhaya et al., 2013). The number of asymmetric synapses increased with age in the frontocentral

neocortex of wild type, APP23, and APP48 mice (Figure 7a, Supplementary Tab.

1d). Such an increase in the number of asymmetric synapses with age was not

seen in the stratum radiatum and oriens of CA1 (Figure 7a, Supplementary Tab.

1d). The number of symmetric synapses did not differ between 3 and 15-18-

month-old mice of each genotype (Figure 7b, Supplementary Tab. 1d)(Rijal

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Immunoelectron microscopy showed Aβ within dendrites of 15-18 month-old

APP23 and APP48 mice. In 15 month-old APP23 mice extracellular Aβ plaques

contained fibrillar Aβ that could easily be labelled with Anti-Aβ1-17 (Fig.8a, b).

Plaque-associated dystrophic neurites were seen in the middle of bundles of

extracellular Aβ fibrils. No fibrillar Aβ was found within neurites of APP23 mice.

However, intracellular Aβ was detected in a few of these dystrophic neurites near

the membrane in electron dense spherical particles, which may represent non-

fibrillar Aβ oligomers or protofibrils (Figure 8c-e). In APP48 mice fibrillar Aβ

aggregates presumably representing the ultrastructural correlate of dendritic

threads were found in the dendrites as seen morphologically in Epon-embedded

tissue. These dendrites were not enlarged (Figure 8f). Immunoelectron microscopy

with anti-Aβ1-17 indicated that the fibrillar structures identified in the Epon-

embedded sections contain Aβ (Figure 8g) (Abramowski et al., 2012a). Organelles

near dendritic threads in APP48 mice, thereby, did not differ from that elsewhere in

APP48 mouse neurons as demonstrated for a mitochondrium in Figure 8g (m)

(Rijal Upadhaya et al., 2013).

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Fig. 8 Electron microscopy and immunogold labeling of Amyloid beta protein (Aβ) in APP23 and APP48 mice. a, b: Immunogold particles specifically labeled fibrillar Aβ (arrowheads ) of a plaque in a 15-month-old APP23 mouse. At high magnification small amyloid fibrils were identified (arrowheads in b ). They were located in the extracellular space. c, d: Dystrophic neurites (outlined structures ) were associated with extracellular bundles of plaque-associated Aβ fibrils (arrowheads ) in an 15-month-old APP23 mouse. Within the neurite, Aβ was localized in electron

Results

34

dense material near the surface as well as in the center of the neurite (arrows in c ). d: A second dendrite without signs of dystrophy such as multilamellar bodies was also located near extracellular Aβ fibrils (outlined structure labeled with e ). e: Higher magnification of this dendrite showed a dendrite cross section with an intact mitochondrium (m) and with condensed Aβ–positive material in the cytoplasm (arrows ). Similar Aβ -positive material was found in the neighboring extracellular space (arrowhead ). Both, intra- and extracellular Aβ aggregates did not exhibit fibrillar morphology. As such it is quite likely that these Aβ aggregates represent non-fibrillar oligomers and/or protofibrils occurring in the neighborhood of extracellular, plaque-associated Aβ fibrils. f: Fibrillar material (arrows ) was observed in some dendrites of a 3-month-old APP48 mouse in an Epon-embedded, not immunostained section presumably representing the ultrastructural correlative for dendritic threads. g: Immunoelectron microscopy confirmed Aβ –positive material in fibrillar aggregates within dendritic threads labeled by gold particles (arrows ) in APP48 mice. No Aβ was observed in the neighboring, non-altered mitochondrium (m). Calibration bar in g is valid for: a = 570 nm, b = 200 nm, c = 750 nm, d = 1000 nm, e = 275 nm, f, g = 350 nm (Rijal Upadhaya et al., 2013).

3.3 Neuron loss in the CA1 subfield but not in the frontocentral

cortex of APP48 and APP23 mice

The number of CA1 neurons was lower in 3 and 18 month-old APP48 mice

compared to wildtype animals indicating CA1 neuron loss in APP48 mice

(Abramowski et al., 2012a). A decrease in the number of CA1 neurons was also

observed in 3 and 15-18 month-old APP23 mice in comparison to wildtype animals

(Fig. 7c, Supplementary Tab. 1e) (Rijal Upadhaya et al., 2012).

In contrast, the number of neurons in the frontocentral cortex of 15-18 month-old

APP23 and APP48 mice was not significantly different from that in wildtype mice

(Fig. 7d, Supplementary Tab. 1e) (Abramowski et al., 2012a). Therefore, 3 month-

old animals were not analyzed for the numbers of frontocentral neurons (Rijal


3.4 Increased mitochondrial alterations in APP48 in neuronal

somata of APP48 but not in APP23

The analysis of mitochondrial alterations in neurites and the somata of

frontocentral neurons showed differences between 15-18 month-old wildtype,

APP23, and APP48 mice (Fig. 9 a-f). Increased percentages and volume densities

of altered mitochondria with destruction and rarefaction of the christa membranes

as depicted in Figure 9c were observed in somata of neurons from APP48 mice in

contrast to predominantly ultrastructually intact mitochondria in wildtype and

APP23 mice (Figure 9 a, b, d, e, Supplementary Tab. 1 f, g). However, few altered

mitochondria were also observed in wildtype and APP23 mice. Such an increase

in the percentage and volume density of altered mitochondria in APP48 mice was

Results

35

not observed in the hippocampal subfield CA1 and in the frontocentral neocortex

of 3-month-old animals (Figure 9 d, e, g, h, Supplementary Tab. 1 f, g). Neurites

studied distant from nerve cell somata exhibited a small number of altered

mitochondria in all mice but did not show differences in the percentage and

volume densities of altered mitochondria among the three mouse lines at both

ages and locations Supplementary Tab. 1i, j, Supplementary Tab. 1a-c). The

volume densities of somatic and neuritic mitochondria in general, i.e. unaltered

and altered mitochondria together, did not significantly differ in APP23, APP48 and

wild type mice (Figure 9 f, i, Supplementary Tab. 1h, k, Supplementary Tab. 1c, f).

In the neuronal somata, the percentages and volume densities of altered

mitochondria in frontocentral and CA1 neurons increased with age (Figure 9 d, e,

g, h, Supplementary Tab. 1 f, g). The volume densities of all, altered and healthy-

looking mitochondria increased in APP23 and APP48 mice with age whereas in

wildtype animals such an increase was not observed (Figure 9 f, i, Supplementary

Tab. 1h).

In neuritic processes, an increase or at least an increasing trend with age in the

percentages and volume densities of altered mitochondria was observed in the

frontocentral cortex of APP23, APP48 and wildtype mice (Supplementary Tab. 1 k,

Supplementary Tab. 1 a, b). In the stratum radiatum and oriens neurites of CA1,

the percentages and volume densities of altered mitochondria in 15-month-old

APP23 mice were lower than in wildtype mice whereas no differences were

observed between 3-month-old APP23 and wildtype mice as well as between

APP48 mice of any age and wildtype mice (Supplementary Tab. 1 k,

Supplementary Tab. 1 d, e). In neurites, the volume densities of all, healthy-

looking and altered mitochondria did not increase with age (Supplementary Tab. 1

c, f, k) (Rijal Upadhaya et al., 2013).

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36

Figure 9 Electron microscopy of mitochondria in wil d type, APP23, and APP48 mice. a-b: Electron microscopy showed predominantly non-altered mitochondria in nerve cell somata of wildtype (WT) (a) and APP23 mice (b). c: More altered mitochondria in the nerve cell somata were observed in frontocentral neurons of 18-month-old APP48 mice. Mitochondrial alteration was characterized by a loss of mitochondrial christae although the double membrane architecture and at least single christa structures (arrow ) were preserved. d-f : The percentage of altered mitochondria in the nerve cell somata of 18-month-old APP48 mice was higher than in 15-18-month-old wild type and APP23 mice (d). The volume density of altered mitochondria in the frontocentral nerve cell somata of 18-month-old APP48 mice was increased in comparison to 15-18-month-old wild type and APP23 mice (e). At 3 months of age such differences in the presence of altered mitochondria were not observed. The total mitochondrial volume density, i.e. altered and non-altered mitochondria together, did not differ among the investigated mouse lines in both age groups (f). g-i : In CA1 there were no obvious changes in the percentage and volume density of altered mitochondria in the nerve cell somata. *p < 0.05, **p < 0.01 (Further statistical analysis: Supplementary Tab. 1). Means and standard errors are depicted in d-f. Calibration bar in c is valid for: a-c = 150 nm (Rijal Upadhaya et al., 2013).

At the immuno electron microscopic level Aβ was detected in lipofuscin granules

or in association with other cytoplasmic material in neurons of APP23 (Figure 8 c-

e, Figure 10 a, b) and APP48 mice as previously reported (Huang et al., 2006;

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37

Abramowski et al., 2012a; Rijal Upadhaya et al., 2012) with or without

mitochondrial alterations. Even neurons of wildtype mice without detectable Aβ

contained single altered mitochondria. In 15-month-old APP23 mice Aβ was seen

only in few mitochondria (Figure 10 c) whereas most somatic and neuritic

mitochondria did not contain Aβ-positive material (Figure 10 b). In contrast, altered

and non-altered mitochondria labeled for Aβ showing goldparticles in association

with mitochondrial membranes were often found in the nerve cell somata of 18-

month-old APP48 mice (Figure 10d, e) (Rijal Upadhaya et al., 2013).

Results

38

Figure 10 Immunoelectron microscopy of mitochondria in wild type, APP23, and APP48 mice. a : Immunoelectron microscopy revealed gold-labeled Amyloid beta protein (Aβ) (arrows ) within lipofuscin-like granules in the cytoplasm of frontocentral pyramidal neurons in 15-month-old APP23 mice. b. Most mitochondria in the somata of frontocentral pyramidal neurons in 15-month-old APP23 mice did not contain immunogold labeled Aβ–positive material (arrowheads ). c: Only few neuronal mitochondria exhibited single gold particles in a 15-month-old APP23 mouse indicating anti-Aβ-positive material within these mitochondria (arrow ). d: In a few mitochondria within frontocentral pyramidal nerve cell somata of 18-month-old APP48 mice we found immunogold labeled Aβ -positive material associated with the membranes of healthy-looking mitochondria (arrow ). Neighboring non-altered mitochondria often did not contain Aβ (arrowheads ). Aβ fibrils were not seen inside the mitochondria. e: Altered mitochondria within pyramidal neurons in the frontocentral cortex of a 18-month-old APP48 mouse also exhibited single gold particles in association with their membranes indicating the presence of Aβ (arrows ) although Aβ fibrils were not observed inside the mitochondria. Due to the use of LR-white embedded tissue required for post-embedding immunoelectron microscopy (a,b) the tissue preservation was less good than in the Epon embedded sections used for the morphological analysis of the mitochondria (Figure 8a-c). Calibration bar in e is valid for: a, b = 540 nm, c = 325 nm, d = 288 nm, e = 180 nm (Rijal Upadhaya et al., 2013).

Discussion

39

4 Discussion Here we employed two different mouse models for Aβ pathology to clarify

mechanisms of Aβ-related neurodegeneration. In APP48 mice, where exclusively

intracellular Aβ is exhibited, increased numbers of mitochondria exhibiting

alterations in frontocentral pyramidal cells and neuron loss in CA1 neurons were

found. Synaptic and dendritic degeneration was not seen in APP48 in contrast to

APP23 mice. The latter mouse model overexpresses human mutant APP and

shows extra- and intracellular Aβ pathology and neurodegeneration. Here

mitochondrial alterations did not exceed the levels seen in wildtype animals. These

results lead to the suggestion that different mechanisms are causative for neuron

loss in the two AD mouse models. The following paragraphs will discuss causes

and consequences of Aβ accumulation in the brain of the described mouse models

as well as its significance for the AD pathology.

APP23 and APP48 mice differ considerably with respect to the mechanism of

transgenic, human Aβ generation. Cleavage of Aβ from APP with the Swedish

mutation as in APP23 mice occurs after APP internalization from the membrane in

the early endosomal compartment (Rajendran et al., 2006; Rajendran and

Annaert, 2012). Once generated Aβ is rapidly secreted to a large extent. In APP48

mice, on the other hand, expression of Aβ42 with a cleaved signal sequence

targets the released Aβ to the endoplasmic reticulum. Intracellular accumulation

occurs in neurites and lysosomes whereas only very little Aβ42 is secreted into the

extracellular space (Abramowski et al., 2012a). APP48 mice lack human APP and

its metabolites except for Aβ42 whereas APP23 mice produce more Aβ40 than Aβ42

and, in addition to Aβ, N- and C-terminal fragments of APP. A transgenic mouse

expressing only Aβ40 with a similar construct as used in APP48 mice for Aβ42 did

not show Aβ aggregation and neuronal degeneration (Abramowski et al., 2012a).

In double transgenic animals expressing both Aβ42 and Aβ40 constructs a similar

pattern of pathological lesions was observed as in APP48 mice (Abramowski et

al., 2012a) indicating that Aβ40 has only a marginal effect in these animals.

Both, APP23 and APP48 mice exhibited intracellular Aβ in the lysosomal

compartment and in mitochondria whereas early endosomal Aβ was not seen in

APP48 mice. The distribution of the transgene mRNA in the neocortex and the

hippocampus as well as the expression levels were quite similar in APP23 and

APP48 mice (Sturchler-Pierrat et al., 1997; Abramowski et al., 2012a).

Discussion

40

Accordingly, neocortical and hippocampal neurons did not vary much in the

transgene expression levels in APP23 and APP48 mice. Both mouse models were

used to determine the effects of Aβ under given artificial conditions. None of these

two mouse models reflects human AD pathology because none showed significant

neurofibrillary tangle pathology and both overexpressed a transgenic construct

made to produce large amounts of Aβ (Sturchler-Pierrat et al., 1997; Abramowski

et al., 2012a; Rijal Upadhaya et al., 2013).

4.1 Aβ effects on mitochondrial integrity

The results obtained in this study show that APP-independant intraneuronal

production and accumulation of Aβ in 18 month-old APP48 mice is associated with

increased numbers of mitochondria exhibiting alterations in frontocentral nerve

cells compared to APP23 and wildtype mice. The questions which need to be

discussed are a) how does Aβ affect mitochondrial structure and b) how does

mitochondrial damage affect the neuron?

Structural changes were found, i.e. increased levels of mitochondria with distorted

christae in APP48 mice similar to those recently reported in prion disease (Siskova

et al., 2010). The increase of mitochondrial alterations in APP48 mice was accom-

panied by the detection of Aβ in a moderate number of altered and non-altered

neuronal somatic mitochondria whereas only few somatic mitochondria in APP23

mice exhibited Aβ and none in wild type animals. Several arguments suggest that

the increase of mitochondrial alterations in APP48 mice was related to the

presence of Aβ and may have functional consequences:

1) Aβ is capable to induce apoptosis through the mitochondria-caspase-3 pathway

(Costa et al., 2010; Wang et al., 2010), and

2) mitochondrial Aβ levels are associated with the extent of mitochondrial

dysfunction, cognitive impairment and oxidative stress in AD mouse models and

AD (Dragicevic et al., 2010),

3) histologically altered mitochondria showed a reduced number of christa

membranes presumably providing the morphological correlative for an impairment

of mitochondrial function, they were found most frequently in APP48 mice which

also contained more mitochondrial Aβ than APP23 mice.

Oxidative stress with consecutive mitochondrial dysfunction and cell death may

result from Aβ interaction with Aβ-binding alcohol-dehydrogenase (ABAD) as a Aβ

Discussion

41

binding site in the mitochondrion, (Lustbader et al., 2004; Takuma et al., 2005), Aβ

interaction with Cyclophillin D (CypD) (Du et al., 2008) or Aβ induced inhibition of

complex IV of the ETC (Canevari et al., 1999; Crouch et al., 2005; Manczak et al.,

2006; Wang et al., 2008).

As shown, there are a number of mechanisms by which intracellular Aβ in

mitochondria could lead to mitochondrial damage. It is proposed that Aβ-related

mitochondrial pathology is compensated by various mechanisms until

mitochondria reach a functional threshold which leads to neurodegeneration

(Swerdlow et al., 2010).

For APP23 mice, however, we cannot rule out that trophic effects reported for APP

expression (Mattson, 1997) compensate the Aβ toxicity to mitochondria.

It is noteworthy that only mitochondria in the nerve cell somata showed increased

alterations in APP48 mice whereas mitochondria in distal dendrites and axons did

not exhibit differences in volume density as well as in the percentage of alterations

among APP23, APP48 and wildtype mice. Thus, intraneuronal Aβ42 exhibited its

major toxic effects on mitochondria close to its production site in APP48 mice, i.e.

close to the endoplasmic reticulum. Biochemical assessment of respiratory chain

complex I and complex IV, α-ketoglutarate dehydrogenase, and tricarboxylic acid

cycle enzyme activity did not show significant differences in forebrain

homogenates (data not shown, received from Staufenbiel M). However, the local

effect in the soma is probably not sufficient to reduce the overall activities in a

brain homogenate.

In the AD brain and in in-vitro studies, APP was found to accumulate in

mitochondria in neurons of AD brains (Anandatheerthavarada et al., 2003; Keil et

al., 2004; Park et al., 2006), leading to mitochondrial dysfunction. APP was found

to be imported into mitochondria via mitochondrial import channels and formed

complexes with them. These complexes were suggested to lead to to the blockade

of further import of nuclear encoded proteins with the result of decreased

cytochrome c oxidase activity and increased reactive oxygen species

(Anandatheerthavarada et al., 2003; Devi et al., 2006; Rijal Upadhaya et al.,

2013).

Discussion

42

4.2 Neurite and synapse degeneration in APP23 and A PP48 mice

APP23 mice showed dendritic degeneration in DiI-traced commissural neurons,

loss of asymmetric synapses and dystrophic neurites whereas none of these

pathologies was observed in APP48 mice. In this context, it is important to note

that fibrillar Aβ was present in dendrites of APP48 mice indicating that the mere

presence of intracellular Aβ was not sufficient to induce this pathology.

Accordingly, neuritic degeneration and loss of asymmetric synapses may either

require high concentrations of extracellular Aβ40/42 and/or the presence of APP and

its metabolites including APP-derived Aβ as in APP23 mice. The amount of total

Aβ42 determined by ELISA did not explain neuritic degeneration because APP48

mice contained more Aβ42 in the brain than 3–5 months old APP23 mice (see also

(Capetillo-Zarate et al., 2006)) although 5-month-old APP23 mice did already

exhibit dendrite pathology as previously reported (Capetillo-Zarate et al., 2006).

Consistent with our observations, the prevalence of high levels of extracellular Aβ

aggregates, e.g. soluble Aβ oligomers and protofibrils and Aβ plaques have been

shown to be associated with altered synapse function (Wang et al., 2002; Lacor et

al., 2004; Shankar et al., 2008). Neuritic alterations were frequently seen near

amyloid plaques (Tsai et al., 2004; Spires et al., 2005). The electron microscopic

detection of extracellular non-fibrillar and fibrillar Aβ-positive aggregates in

association with dystrophic neurites in APP23 but not in APP48 mice further

argues in favor of a critical role of extracellular Aβ in neuritic degeneration.

Electrophysiological changes in response to extracellularly administered Aβ–

aggregates were mainly reported for excitatory synapses (Wang et al., 2002;

Shankar et al., 2008). Since asymmetric synapses represent mainly excitatory

(glutamatergic) synapses (Miranda et al., 2009) the loss of these synapses likely

represented the result of toxic interactions with Aβ aggregates. We have detected

Aβ within few dystrophic neurites in APP23 mice indicating that we cannot exclude

a contribution of intraneuronal Aβ to neuritic degeneration.

The Bri-Aβ42 mouse, that produces only extracellular Aβ42 derived from an ABri-

Aβ42 construct did not show behavioral changes but Aβ plaques (McGowan et al.,

2005). Bri-Aβ40 mice expressing an ABri-Aβ40 construct did not even develop

plaques. These findings may argue against a contribution of extracellular Aβ to Aβ

toxicity. However, it is not yet clear whether the ABri-derived Aβ42 aggregates

contain similar amounts of AβN3pE and pAβ as Aβ aggregates in APP23 or APP48

Discussion

43

mice, whether APP expression or the presence of both, Aβ42 and Aβ40 , is required

for extracellular Aβ toxicity, or whether this mouse model did not produce enough

Aβ42 to cause symptoms.

APPE693Δ transgenic mice produce Aβ lacking glutamate-22 (E22Δ). This mouse

model does not develop amyloid plaques but APP-derived intracellular Aβ

aggregates and synapse loss (Tomiyama et al., 2010). Exogenous synthetic

AβE22Δ also lead to synaptic alterations in hippocampal slice culture experiments or

after intraventricular injection (Takuma et al., 2008; Tomiyama et al., 2008)

[46,47]. As such, neuritic changes and/or synapse pathology can be explained by

APP-derived Aβ production regardless of the presence of Aβ plaques whereas

such changes were not reported in APP48, Bri-Aβ42 and Bri-Aβ40 mice, which

produce non-APP derived Aβ. A further argument for a role of APP in Aβ toxicity in

APP23 mice is the recent finding of a coaggregation of Aβ with C-terminal APP

fragments in dispersible Aβ aggregates (Rijal Upadhaya et al., 2012) supporting

the hypothesis that APP is a molecular target of Aβ toxicity (Bignante et al., 2013;

Rijal Upadhaya et al., 2013).

4.3 Two types of A β-induced neurodegeneration in the

frontocentral cortex: somatic type and neuritic typ e

In the frontocentral cortex of APP23 mice neuritic degeneration and asymmetric

synapse loss was found in the absence of neuron loss suggesting that both events

indicate a neuritic type of nerve cell degeneration with neuritic degeneration

preceding nerve cell death. APP48 mice, on the other hand, showed a different

type of nerve cell damage characterized by morphologically altered mitochondria

in the cell soma and thread-like Aβ aggregates in dendrites. Although the

appearance of the dendrites and axons was, except for the Aβ threads,

morphologically normal and no synapse loss was observed, mitochondrial

changes in nerve cell somata represented early signs of a somatic type of

neurodegeneration. Since mitochondrial alterations caused by Aβ have been

demonstrated to induce apoptosis (Costa et al., 2010; Wang et al., 2010) it is

tempting to speculate that this type of somatic neurodegeneration with increased

amounts of morphologically altered mitochondria finally results in apoptosis

without the development of dystrophic neurites and dendrite degeneration before

cell death. Hence, APP23 mice and APP48 mice develop two different types of

Discussion

44

nerve cell degeneration in the frontocentral cortex (Figure 11):

1) APP23 mice showed a neuritic type of neurodegeneration with early neuritic

and synaptic degeneration but without increased numbers of altered mitochondria;

2) APP48 mice exhibited a somatic type of neurodegeneration with increased

somatic mitochondrial degeneration but morphologically intact dendrites and

axons. APP48 and APP23 mice both showed neuron loss in the CA1 region as

previously described (Calhoun et al., 1998; Abramowski et al., 2012a; Rijal


It was not accompanied by a reduction in synaptic density suggesting replacement

of lost synapses by surviving neurons. Except for Aβ plaques in APP23 mice and

intracellular Aβ in both transgenic animals, significant reduction of synapse

densities and mitochondrial alterations could not be identified in this brain region

possibly because the more vulnerable CA1 neurons die faster than altered

neurons in the frontocentral cortex and do not accumulate at early stages of nerve

cell degeneration. Since 3-month-old animals have more CA1 neurons than 15-18-

month-old mice (Figure 7c) and the decrease in number was higher than the age-

related loss of CA1 neurons in wild type mice it appears likely that the age-related

loss of CA1 neurons is enhanced by Aβ toxicity in APP23 and APP48 mice.

Although both types of Aβ-related neurodegeneration were observed in artificial

mouse models that were made to produce large amounts of Aβ there are

arguments for the hypothesis that both mechanisms of Aβ-related

neurodegeneration are relevant in AD: 1) synapse loss and dystrophic neurites

especially around neuritic plaques are well known features of AD pathology

(DeKosky and Scheff, 1990; Wang and Munoz, 1995; Dickson, 1997) that might

be explained in part by the neuritic type of Aβ-related neurodegeneration and 2)

mitochondrial alterations are well known in AD cases as well (Lustbader et al.,

2004) presumably indicative for somatic type neurodegeneration in the presence

of intracellular Aβ (Gouras et al., 2000; Rijal Upadhaya et al., 2013).

Discussion

45

Figure 11 Schematic representation of the somatic a nd neuritic type of Amyloid beta protein (Aβ) -related neurodegeneration in frontocentral neuro ns of APP23 and APP48 mice. In APP48 mice Aβ accumulates within neurons and in microglial cells as previously reported (Abramowski et al., 2012a). Extracellular Aβ is not detectable suggesting that Amyloid precursor protein (APP) independently produced intracellular Aβ leads to functional impairment of neurons as indicated by motor deficits (Abramowski et al., 2012a). Mitochondrial alterations occur more frequently in the nerve cell somata of APP48 mice than in wildtype and APP23 mice and are proposed to lead to apoptotic cell death as suggested previously (Umeda et al., 2011) without preceding neuritic alteration. This type of somatic neurodegeneration in APP48 mice is different from that seen in APP23 mice, which contains less intracellular Aβ but significant amounts of extracellular Aβ aggregates including plaques. We propose that extra- and intracellular APP-derived Aβ causes synapse loss, dendrite degeneration and often plaque-associated, dystrophic neurites in APP23 mice indicative for a second neuritic type of neurodegeneration. Intracellular Aβ in APP23 mice may be produced within the nerve cell or may be taken up from the extracellular space (Wirths et al., 2001; Tampellini et al., 2007; Ovsepian et al., 2013; Ovsepian and Herms, 2013; Rijal Upadhaya et al., 2013).

4.4 Conclusions

The data suggest two independent mechanisms by which Aβ causes

neurodegeneration (Fig. 11): a somatic type and a neuritic type. The neuritic type

of neurodegeneration is characterized by a loss of asymmetric synapses,

degeneration of dendrites, occurrence of dystrophic neurites and is associated

with the occurrence of extracellular Aβ aggregates in APP23 mice. The somatic

type of neurodegeneration shows mitochondrial alterations in the neuronal somata

but no changes in neurite morphology. It is linked to intraneuronal accumulation of

Aβ and functional changes in APP48 mice (Abramowski et al., 2012a). Both

Discussion

46

mechanisms may finally lead to neuron loss as observed in the hippocampal

sector CA1 in APP23 and APP48 mice (Abramowski et al., 2012a) (Calhoun et al.,

1998) and both mechanisms may account for neurodegeneration in AD. As such,

for the development of therapeutic strategies aimed at protecting neurons from

AD-related degeneration it appears important to consider both types of Aβ-related

neurodegeneration (Rijal Upadhaya et al., 2013).

Summary

47

5 Summary

The deposition of the amyloid beta protein (Aβ) peptide in the brain is one of the

hallmarks of Alzheimer’s disease. It is not yet clear whether Aβ always leads to

similar changes or whether it induces different features of neurodegeneration in

relation to its intra- and/or extracellular localization. Two transgenic mouse models

APP48 and APP23 mice were analyzed. The APP48 mouse expresses Aβ1-42 with

a signal sequence in neurons. These animals produce intracellular Aβ42

independent of amyloid precursor protein (APP) but do not develop extracellular

Aβ plaques. The APP23 mouse overexpresses human APP with the Swedish

mutation (KM670/671NL) in neurons and produces APP-derived extracellular Aβ

plaques and intracellular Aβ aggregates. Tracing of commissural neurons in layer

III of the frontocentral cortex with the DiI tracer showed no morphological signs of

dendritic degeneration in APP48 mice compared to littermate controls. In contrast,

the dendritic tree of highly ramified commissural frontocentral neurons was altered

in 15-month-old APP23 mice. The density of asymmetric synapses in the

frontocentral cortex was reduced in 3- and 15-month-old APP23 but not in 3- and

18-month-old APP48 mice. Frontocentral neurons of 18-month-old APP48 mice

showed an increased percentage of altered mitochondria in the soma compared to

wildtype and APP23 mice. Aβ was often seen in the membrane of neuronal

mitochondria in APP48 mice at the electron microscopic level. These results

indicate that APP-independent intracellular Aβ accumulation in APP48 mice is not

associated with neuritic degeneration but with mitochondrial alterations whereas

APP-derived intra- and extracellular Aβ pathology in APP23 mice is linked to

dendrite degeneration and synapse loss independent of obvious mitochondrial

alterations. Thus, Aβ aggregates in APP23 and APP48 mice induce

neurodegeneration presumably by a somatic and a neuritic mechanism. The

neuritic type of neurodegeneration is characterized by a loss of asymmetric

synapses, degeneration of dendrites, occurrence of dystrophic neurites and is

associated with the occurrence of extracellular Aβ aggregates in APP23 mice. The

somatic type of neurodegeneration shows mitochondrial alterations in the neuronal

somata but no changes in neurite morphology. It is linked to intraneuronal

accumulation of Aβ and functional changes in APP48 mice. Both mechanisms may

Summary

48

finally lead to neuron loss as observed in the hippocampal sector CA1 in APP23

and APP48 mice and both mechanisms may account for neurodegeneration in AD.

References

49

6 References

1) Abramowski D, Rabe S, Rijal-Upadhaya A, Reichwald J, Danner S, Staab

D, Capetillo-Zarate E, Yamaguchi H, Saido TC, Wiederhold KH, Thal DR,

Staufenbiel M (2012a) Transgenic Expression of Intraneuronal Abeta42 But

Not Abeta40 Leads to Cellular Abeta Lesions, Degeneration, and

Functional Impairment without Typical Alzheimer's Disease Pathology. J

Neurosci 32:1273-1283.

2) Abramowski D, Rabe S, Upadhaya AR, Reichwald J, Danner S, Staab D,

Capetillo-Zarate E, Yamaguchi H, Saido TC, Wiederhold KH, Thal DR,

Staufenbiel M (2012b) Transgenic Expression of Intraneuronal Abeta42 But

Not Abeta40 Leads to Cellular Abeta Lesions, Degeneration, and

Functional Impairment without Typical Alzheimer's Disease Pathology. J

Neurosci 32:1273-1283.

3) Alzheimer A (1907) Über eine eigenartige Erkrankung der Hirnrinde. Allg

Zschr Psych 64:146-148.

4) Anandatheerthavarada HK, Biswas G, Robin MA, Avadhani NG (2003)

Mitochondrial targeting and a novel transmembrane arrest of Alzheimer's

amyloid precursor protein impairs mitochondrial function in neuronal cells. J

Cell Biol 161:41-54.

5) Arvanitakis Z, Leurgans SE, Wang Z, Wilson RS, Bennett DA, Schneider JA

(2011) Cerebral amyloid angiopathy pathology and cognitive domains in

older persons. Ann Neurol 69:320-327.

6) Bayer TA, Cappai R, Masters CL, Beyreuther K, Multhaup G (1999) It all

sticks together--the APP-related family of proteins and Alzheimer's disease.

Mol Psychiatry 4:524-528.

7) Bignante EA, Heredia F, Morfini G, Lorenzo A (2013) Amyloid beta

precursor protein as a molecular target for amyloid beta--induced neuronal

degeneration in Alzheimer's disease. Neurobiol Aging 34:2525-2537.

8) Cai XD, Golde TE, Younkin SG (1993) Release of excess amyloid beta

protein from a mutant amyloid beta protein precursor. Science 259:514-516.

9) Calhoun ME, Wiederhold KH, Abramowski D, Phinney AL, Probst A,

Sturchler-Pierrat C, Staufenbiel M, Sommer B, Jucker M (1998) Neuron

loss in APP transgenic mice. Nature 395:755-756.

References

50

10) Canevari L, Clark JB, Bates TE (1999) beta-Amyloid fragment 25-35

selectively decreases complex IV activity in isolated mitochondria. FEBS

Lett 457:131-134.

11) Capetillo-Zarate E, Staufenbiel M, Abramowski D, Haass C, Escher A,

Stadelmann C, Yamaguchi H, Wiestler OD, Thal DR (2006) Selective

vulnerability of different types of commissural neurons for amyloid beta-

protein-induced neurodegeneration in APP23 mice correlates with dendritic

tree morphology. Brain 129:2992-3005.

12) Casley CS, Canevari L, Land JM, Clark JB, Sharpe MA (2002) Beta-

amyloid inhibits integrated mitochondrial respiration and key enzyme

activities. J Neurochem 80:91-100.

13) Caspersen C, Wang N, Yao J, Sosunov A, Chen X, Lustbader JW, Xu HW,

Stern D, McKhann G, Yan SD (2005) Mitochondrial Abeta: a potential focal

point for neuronal metabolic dysfunction in Alzheimer's disease. Faseb J

19:2040-2041.

14) Citron M, Oltersdorf T, Haass C, McConlogue L, Hung AY, Seubert P, Vigo-

Pelfrey C, Lieberburg I, Selkoe DJ (1992) Mutation of the beta-amyloid

precursor protein in familial Alzheimer's disease increases beta-protein

production. Nature 360:672-674.

15) Colonnier M (1968) Synaptic patterns on different cell types in the different

laminae of the cat visual cortex. An electron microscope study. Brain Res

9:268-287.

16) Corder EH, Ghebremedhin E, Taylor MG, Thal DR, Ohm TG, Braak H

(2004) The biphasic relationship between regional brain senile plaque and

neurofibrillary tangle distributions: modification by age, sex, and APOE

polymorphism. Ann N Y Acad Sci 1019:24-28.

17) Costa RO, Ferreiro E, Cardoso SM, Oliveira CR, Pereira CM (2010) ER

stress-mediated apoptotic pathway induced by Abeta peptide requires the

presence of functional mitochondria. J Alzheimers Dis 20:625-636.

18) Crouch PJ, Blake R, Duce JA, Ciccotosto GD, Li QX, Barnham KJ, Curtain

CC, Cherny RA, Cappai R, Dyrks T, Masters CL, Trounce IA (2005)

Copper-dependent inhibition of human cytochrome c oxidase by a dimeric

conformer of amyloid-beta1-42. J Neurosci 25:672-679.

References

51

19) D'Andrea MR, Nagele RG, Wang HY, Lee DH (2002) Consistent

immunohistochemical detection of intracellular beta-amyloid42 in pyramidal

neurons of Alzheimer's disease entorhinal cortex. Neurosci Lett 333:163-

166.

20) De Strooper B (2003) Aph-1, Pen-2, and Nicastrin with Presenilin generate

an active gamma-Secretase complex. Neuron 38:9-12.

21) De Strooper B, Saftig P, Craessaerts K, Vanderstichele H, Guhde G,

Annaert W, Von Figura K, Van Leuven F (1998) Deficiency of presenilin-1

inhibits the normal cleavage of amyloid precursor protein. Nature 391:387-

390.

22) DeFelipe J, Marco P, Busturia I, Merchan-Perez A (1999) Estimation of the

number of synapses in the cerebral cortex: methodological considerations.

Cereb Cortex 9:722-732.

23) DeKosky ST, Scheff SW (1990) Synapse loss in frontal cortex biopsies in

Alzheimer's disease: correlation with cognitive severity. Ann Neurol 27:457-

464.

24) Devi L, Prabhu BM, Galati DF, Avadhani NG, Anandatheerthavarada HK

(2006) Accumulation of amyloid precursor protein in the mitochondrial

import channels of human Alzheimer's disease brain is associated with

mitochondrial dysfunction. J Neurosci 26:9057-9068.

25) Dickson DW (1997) The pathogenesis of senile plaques. J Neuropathol Exp

Neurol 56:321-339.

26) Dimmer KS, Scorrano L (2006) (De)constructing mitochondria: what for?

Physiology (Bethesda) 21:233-241.

27) Dragicevic N, Mamcarz M, Zhu Y, Buzzeo R, Tan J, Arendash GW,

Bradshaw PC (2010) Mitochondrial amyloid-beta levels are associated with

the extent of mitochondrial dysfunction in different brain regions and the

degree of cognitive impairment in Alzheimer's transgenic mice. J

Alzheimers Dis 20 Suppl 2:S535-550.

28) Du H, Guo L, Fang F, Chen D, Sosunov AA, McKhann GM, Yan Y, Wang

C, Zhang H, Molkentin JD, Gunn-Moore FJ, Vonsattel JP, Arancio O, Chen

JX, Yan SD (2008) Cyclophilin D deficiency attenuates mitochondrial and

neuronal perturbation and ameliorates learning and memory in Alzheimer's

disease. Nat Med 14:1097-1105.

References

52

29) Duyckaerts C, Delatour B, Potier MC (2009) Classification and basic

pathology of Alzheimer disease. Acta Neuropathol 118:5-36.

30) Eckert A, Hauptmann S, Scherping I, Rhein V, Muller-Spahn F, Gotz J,

Muller WE (2008) Soluble beta-amyloid leads to mitochondrial defects in

amyloid precursor protein and tau transgenic mice. Neurodegener Dis

5:157-159.

31) Esch FS, Keim PS, Beattie EC, Blacher RW, Culwell AR, Oltersdorf T,

McClure D, Ward PJ (1990) Cleavage of amyloid beta peptide during

constitutive processing of its precursor. Science 248:1122-1124.

32) Furukawa K, Barger SW, Blalock EM, Mattson MP (1996) Activation of K+

channels and suppression of neuronal activity by secreted beta-amyloid-

precursor protein. Nature 379:74-78.

33) Galuske RA, Singer W (1996) The origin and topography of long-range

intrinsic projections in cat visual cortex: a developmental study. Cereb

Cortex 6:417-430.

34) Galuske RA, Schlote W, Bratzke H, Singer W (2000) Interhemispheric

asymmetries of the modular structure in human temporal cortex. Science

289:1946-1949.

35) Ghiso J, Tomidokoro Y, Revesz T, Frangione B, Rostagno A (2010)

Cerebral Amyloid Angiopathy And Alzheimer's Disease. Hirosaki Igaku

61:S111-S124.

36) Gibson GE, Sheu KF, Blass JP (1998) Abnormalities of mitochondrial

enzymes in Alzheimer disease. J Neural Transm 105:855-870.

37) Glenner GG, Wong CW (1984a) Alzheimer's disease: initial report of the

purification and characterization of a novel cerebrovascular amyloid protein.

Biochem Biophys Res Commun 120:885-890.

38) Glenner GG, Wong CW (1984b) Alzheimer's disease and Down's

syndrome: sharing of a unique cerebrovascular amyloid fibril protein.

Biochem Biophys Res Commun 122:1131-1135.

39) Goate A, Chartier-Harlin MC, Mullan M, Brown J, Crawford F, Fidani L,

Giuffra L, Haynes A, Irving N, James L, et al. (1991) Segregation of a

missense mutation in the amyloid precursor protein gene with familial

Alzheimer's disease. Nature 349:704-706.

References

53

40) Goedert M, Spillantini MG (2006) A century of Alzheimer's disease. Science

314:777-781.

41) Gouras GK, Tsai J, Naslund J, Vincent B, Edgar M, Checler F, Greenfield

JP, Haroutunian V, Buxbaum JD, Xu H, Greengard P, Relkin NR (2000)

Intraneuronal Abeta42 accumulation in human brain. Am J Pathol 156:15-

20.

42) Haass C, Lemere CA, Capell A, Citron M, Seubert P, Schenk D, Lannfelt L,

Selkoe DJ (1995) The Swedish mutation causes early-onset Alzheimer's

disease by beta-secretase cleavage within the secretory pathway. Nat Med

1:1291-1296.

43) Hardy J (1997) Amyloid, the presenilins and Alzheimer's disease. Trends

Neurosci 20:154-159.

44) Hardy J, Allsop D (1991) Amyloid deposition as the central event in the

aetiology of Alzheimer's disease. Trends Pharmacol Sci 12:383-388.

45) Hardy J, Selkoe DJ (2002) The amyloid hypothesis of Alzheimer's disease:

progress and problems on the road to therapeutics. Science 297:353-356.

46) Hardy JA, Higgins GA (1992) Alzheimer's disease: the amyloid cascade

hypothesis. Science 256:184-185.

47) Hauptmann S, Scherping I, Drose S, Brandt U, Schulz KL, Jendrach M,

Leuner K, Eckert A, Muller WE (2009) Mitochondrial dysfunction: an early

event in Alzheimer pathology accumulates with age in AD transgenic mice.

Neurobiol Aging 30:1574-1586.

48) Hebert LE, Scherr PA, Bienias JL, Bennett DA, Evans DA (2003) Alzheimer

disease in the US population: prevalence estimates using the 2000 census.

Arch Neurol 60:1119-1122.

49) Herreman A, Serneels L, Annaert W, Collen D, Schoonjans L, De Strooper

B (2000) Total inactivation of gamma-secretase activity in presenilin-

deficient embryonic stem cells. Nat Cell Biol 2:461-462.

50) Herzig MC, Winkler DT, Burgermeister P, Pfeifer M, Kohler E, Schmidt SD,

Danner S, Abramowski D, Sturchler-Pierrat C, Burki K, van Duinen SG,

Maat-Schieman ML, Staufenbiel M, Mathews PM, Jucker M (2004) Abeta is

targeted to the vasculature in a mouse model of hereditary cerebral

hemorrhage with amyloidosis. Nat Neurosci 7:954-960.

References

54

51) Hirai K, Aliev G, Nunomura A, Fujioka H, Russell RL, Atwood CS, Johnson

AB, Kress Y, Vinters HV, Tabaton M, Shimohama S, Cash AD, Siedlak SL,

Harris PL, Jones PK, Petersen RB, Perry G, Smith MA (2001) Mitochondrial

abnormalities in Alzheimer's disease. J Neurosci 21:3017-3023.

52) Hoyer S (2000) Brain glucose and energy metabolism abnormalities in

sporadic Alzheimer disease. Causes and consequences: an update. Exp

Gerontol 35:1363-1372.

53) Hsu SM, Raine L, Fanger H (1981) Use of avidin-biotin-peroxidase complex

(ABC) in immunoperoxidase techniques: a comparison between ABC and

unlabeled antibody (PAP) procedures. J Histochem Cytochem 29:577-580.

54) Huang SM, Mouri A, Kokubo H, Nakajima R, Suemoto T, Higuchi M,

Staufenbiel M, Noda Y, Yamaguchi H, Nabeshima T, Saido TC, Iwata N

(2006) Neprilysin-sensitive synapse-associated amyloid-beta peptide

oligomers impair neuronal plasticity and cognitive function. J Biol Chem

281:17941-17951.

55) Ikin AF, Sabo SL, Lanier LM, Buxbaum JD (2007) A macromolecular

complex involving the amyloid precursor protein (APP) and the cytosolic

adapter FE65 is a negative regulator of axon branching. Mol Cell Neurosci

35:57-63.

56) Jonsson T, Atwal JK, Steinberg S, Snaedal J, Jonsson PV, Bjornsson S,

Stefansson H, Sulem P, Gudbjartsson D, Maloney J, Hoyte K, Gustafson A,

Liu Y, Lu Y, Bhangale T, Graham RR, Huttenlocher J, Bjornsdottir G,

Andreassen OA, Jonsson EG, Palotie A, Behrens TW, Magnusson OT,

Kong A, Thorsteinsdottir U, Watts RJ, Stefansson K (2012) A mutation in

APP protects against Alzheimer's disease and age-related cognitive

decline. Nature 488:96-99.

57) Keil U, Bonert A, Marques CA, Scherping I, Weyermann J, Strosznajder JB,

Muller-Spahn F, Haass C, Czech C, Pradier L, Muller WE, Eckert A (2004)

Amyloid beta-induced changes in nitric oxide production and mitochondrial

activity lead to apoptosis. J Biol Chem 279:50310-50320.

58) Kogel D, Schomburg R, Copanaki E, Prehn JH (2005) Regulation of gene

expression by the amyloid precursor protein: inhibition of the JNK/c-Jun

pathway. Cell Death Differ 12:1-9.

References

55

59) Kumar S, Wirths O, Theil S, Gerth J, Bayer TA, Walter J (2013) Early

intraneuronal accumulation and increased aggregation of phosphorylated

Abeta in a mouse model of Alzheimer's disease. Acta Neuropathol 125:699-

709.

60) Kumar S, Rezaei-Ghaleh N, Terwel D, Thal DR, Richard M, Hoch M, Mc

Donald JM, Wullner U, Glebov K, Heneka MT, Walsh DM, Zweckstetter M,

Walter J (2011) Extracellular phosphorylation of the amyloid beta-peptide

promotes formation of toxic aggregates during the pathogenesis of

Alzheimer's disease. Embo J 30:2255-2265.

61) Kuo YM, Beach TG, Sue LI, Scott S, Layne KJ, Kokjohn TA, Kalback WM,

Luehrs DC, Vishnivetskaya TA, Abramowski D, Sturchler-Pierrat C,

Staufenbiel M, Weller RO, Roher AE (2001) The evolution of A beta peptide

burden in the APP23 transgenic mice: implications for A beta deposition in

Alzheimer disease. Mol Med 7:609-618.

62) Lacor PN, Buniel MC, Chang L, Fernandez SJ, Gong Y, Viola KL, Lambert

MP, Velasco PT, Bigio EH, Finch CE, Krafft GA, Klein WL (2004) Synaptic

targeting by Alzheimer's-related amyloid beta oligomers. J Neurosci

24:10191-10200.

63) LaFerla FM, Green KN, Oddo S (2007) Intracellular amyloid-beta in

Alzheimer's disease. Nat Rev Neurosci 8:499-509.

64) Lesne S, Kotilinek L (2005) Amyloid plaques and amyloid-beta oligomers:

an ongoing debate. J Neurosci 25:9319-9320.

65) Levy-Lahad E, Wasco W, Poorkaj P, Romano DM, Oshima J, Pettingell

WH, Yu CE, Jondro PD, Schmidt SD, Wang K, et al. (1995) Candidate gene

for the chromosome 1 familial Alzheimer's disease locus. Science 269:973-

977.

66) Lewis J, Dickson DW, Lin WL, Chisholm L, Corral A, Jones G, Yen SH,

Sahara N, Skipper L, Yager D, Eckman C, Hardy J, Hutton M, McGowan E

(2001) Enhanced neurofibrillary degeneration in transgenic mice expressing

mutant tau and APP. Science 293:1487-1491.

67) Lustbader JW, Cirilli M, Lin C, Xu HW, Takuma K, Wang N, Caspersen C,

Chen X, Pollak S, Chaney M, Trinchese F, Liu S, Gunn-Moore F, Lue LF,

Walker DG, Kuppusamy P, Zewier ZL, Arancio O, Stern D, Yan SS, Wu H

References

56

(2004) ABAD directly links Abeta to mitochondrial toxicity in Alzheimer's

disease. Science 304:448-452.

68) Manczak M, Park BS, Jung Y, Reddy PH (2004) Differential expression of

oxidative phosphorylation genes in patients with Alzheimer's disease:

implications for early mitochondrial dysfunction and oxidative damage.

Neuromolecular Med 5:147-162.

69) Manczak M, Anekonda TS, Henson E, Park BS, Quinn J, Reddy PH (2006)

Mitochondria are a direct site of A beta accumulation in Alzheimer's disease

neurons: implications for free radical generation and oxidative damage in

disease progression. Hum Mol Genet 15:1437-1449.

70) Masters CL, Simms G, Weinman NA, Multhaup G, McDonald BL,

Beyreuther K (1985) Amyloid plaque core protein in Alzheimer disease and

Down syndrome. Proc Natl Acad Sci U S A 82:4245-4249.

71) Mattson MP (1997) Cellular actions of beta-amyloid precursor protein and

its soluble and fibrillogenic derivatives. Physiol Rev 77:1081-1132.

72) Mattson MP, Furukawa K (1998) Signaling events regulating the

neurodevelopmental triad. Glutamate and secreted forms of beta-amyloid

precursor protein as examples. Perspect Dev Neurobiol 5:337-352.

73) Mattson MP, Cheng B, Culwell AR, Esch FS, Lieberburg I, Rydel RE (1993)

Evidence for excitoprotective and intraneuronal calcium-regulating roles for

secreted forms of the beta-amyloid precursor protein. Neuron 10:243-254.

74) Mc Donald JM, Savva GM, Brayne C, Welzel AT, Forster G, Shankar GM,

Selkoe DJ, Ince PG, Walsh DM (2010) The presence of sodium dodecyl

sulphate-stable Abeta dimers is strongly associated with Alzheimer-type

dementia. Brain 133:1328-1341.

75) McGowan E, Pickford F, Kim J, Onstead L, Eriksen J, Yu C, Skipper L,

Murphy MP, Beard J, Das P, Jansen K, Delucia M, Lin WL, Dolios G, Wang

R, Eckman CB, Dickson DW, Hutton M, Hardy J, Golde T (2005) Abeta42 is

essential for parenchymal and vascular amyloid deposition in mice. Neuron

47:191-199.

76) Mineur YS, McLoughlin D, Crusio WE, Sluyter F (2005) Genetic mouse

models of Alzheimer's disease. Neural Plast 12:299-310.

77) Miranda R, Sebrie C, Degrouard J, Gillet B, Jaillard D, Laroche S, Vaillend

C (2009) Reorganization of inhibitory synapses and increased PSD length

References

57

of perforated excitatory synapses in hippocampal area CA1 of dystrophin-

deficient mdx mice. Cereb Cortex 19:876-888.

78) Montine TJ, Phelps CH, Beach TG, Bigio EH, Cairns NJ, Dickson DW,

Duyckaerts C, Frosch MP, Masliah E, Mirra SS, Nelson PT, Schneider JA,

Thal DR, Trojanowski JQ, Vinters HV, Hyman BT (2012) National Institute

on Aging-Alzheimer's Association guidelines for the neuropathologic

assessment of Alzheimer's disease: a practical approach. Acta Neuropathol

123:1-11.

79) Mori C, Spooner ET, Wisniewsk KE, Wisniewski TM, Yamaguch H, Saido

TC, Tolan DR, Selkoe DJ, Lemere CA (2002) Intraneuronal Abeta42

accumulation in Down syndrome brain. Amyloid 9:88-102.

80) Moya KL, Benowitz LI, Schneider GE, Allinquant B (1994) The amyloid

precursor protein is developmentally regulated and correlated with

synaptogenesis. Dev Biol 161:597-603.

81) Mullan M, Crawford F, Axelman K, Houlden H, Lilius L, Winblad B, Lannfelt

L (1992) A pathogenic mutation for probable Alzheimer's disease in the

APP gene at the N-terminus of beta-amyloid. Nat Genet 1:345-347.

82) O'Brien RJ, Wong PC (2011) Amyloid precursor protein processing and

Alzheimer's disease. Annu Rev Neurosci 34:185-204.

83) Oddo S, Caccamo A, Tran L, Lambert MP, Glabe CG, Klein WL, LaFerla

FM (2006) Temporal profile of amyloid-beta (Abeta) oligomerization in an in

vivo model of Alzheimer disease. A link between Abeta and tau pathology. J

Biol Chem 281:1599-1604.

84) Oddo S, Caccamo A, Shepherd JD, Murphy MP, Golde TE, Kayed R,

Metherate R, Mattson MP, Akbari Y, LaFerla FM (2003) Triple-transgenic

model of Alzheimer's disease with plaques and tangles: intracellular Abeta

and synaptic dysfunction. Neuron 39:409-421.

85) Ovsepian SV, Herms J (2013) Drain of the brain: low-affinity p75

neurotrophin receptor affords a molecular sink for clearance of cortical

amyloid beta by the cholinergic modulator system. Neurobiol Aging

34:2517-2524.

86) Ovsepian SV, Antyborzec I, O'Leary VB, Zaborszky L, Herms J, Oliver Dolly

J (2013) Neurotrophin receptor p75 mediates the uptake of the amyloid

References

58

beta (Abeta) peptide, guiding it to lysosomes for degradation in basal

forebrain cholinergic neurons. Brain Struct Funct.

87) Park HJ, Kim SS, Seong YM, Kim KH, Goo HG, Yoon EJ, Min do S, Kang

S, Rhim H (2006) Beta-amyloid precursor protein is a direct cleavage target

of HtrA2 serine protease. Implications for the physiological function of HtrA2

in the mitochondria. J Biol Chem 281:34277-34287.

88) Perez M, Ribe E, Rubio A, Lim F, Moran MA, Ramos PG, Ferrer I, Isla MT,

Avila J (2005) Characterization of a double (amyloid precursor protein-tau)

transgenic: tau phosphorylation and aggregation. Neuroscience 130:339-

347.

89) Rajendran L, Annaert W (2012) Membrane trafficking pathways in

Alzheimer's disease. Traffic 13:759-770.

90) Rajendran L, Honsho M, Zahn TR, Keller P, Geiger KD, Verkade P, Simons

K (2006) Alzheimer's disease beta-amyloid peptides are released in

association with exosomes. Proc Natl Acad Sci U S A 103:11172-11177.

91) Reddy PH, McWeeney S, Park BS, Manczak M, Gutala RV, Partovi D, Jung

Y, Yau V, Searles R, Mori M, Quinn J (2004) Gene expression profiles of

transcripts in amyloid precursor protein transgenic mice: up-regulation of

mitochondrial metabolism and apoptotic genes is an early cellular change in

Alzheimer's disease. Hum Mol Genet 13:1225-1240.

92) Rijal Upadhaya A, Capetillo-Zarate E, Kosterin I, Abramowski D, Kumar S,

Yamaguchi H, Walter J, Fandrich M, Staufenbiel M, Thal DR (2012)

Dispersible amyloid beta-protein oligomers, protofibrils, and fibrils represent

diffusible but not soluble aggregates: their role in neurodegeneration in

amyloid precursor protein (APP) transgenic mice. Neurobiol Aging.

93) Rijal Upadhaya A, Scheibe F, Kosterin I, Abramowski D, Gerth J, Kumar S,

Liebau S, Yamaguchi H, Walter J, Staufenbiel M, Thal DR (2013) The type

of Abeta-related neuronal degeneration differs between amyloid precursor

protein (APP23) and amyloid beta-peptide (APP48) transgenic mice. Acta

Neuropathol Commun 1:77.

94) Ring S, Weyer SW, Kilian SB, Waldron E, Pietrzik CU, Filippov MA, Herms

J, Buchholz C, Eckman CB, Korte M, Wolfer DP, Muller UC (2007) The

secreted beta-amyloid precursor protein ectodomain APPs alpha is

References

59

sufficient to rescue the anatomical, behavioral, and electrophysiological

abnormalities of APP-deficient mice. J Neurosci 27:7817-7826.

95) Robakis NK, Wisniewski HM, Jenkins EC, Devine-Gage EA, Houck GE,

Yao XL, Ramakrishna N, Wolfe G, Silverman WP, Brown WT (1987)

Chromosome 21q21 sublocalisation of gene encoding beta-amyloid peptide

in cerebral vessels and neuritic (senile) plaques of people with Alzheimer

disease and Down syndrome. Lancet 1:384-385.

96) Roher AE, Lowenson JD, Clarke S, Woods AS, Cotter RJ, Gowing E, Ball

MJ (1993) beta-Amyloid-(1-42) is a major component of cerebrovascular

amyloid deposits: implications for the pathology of Alzheimer disease. Proc

Natl Acad Sci U S A 90:10836-10840.

97) Saido TC, Iwatsubo T, Mann DM, Shimada H, Ihara Y, Kawashima S

(1995) Dominant and differential deposition of distinct beta-amyloid peptide

species, A beta N3(pE), in senile plaques. Neuron 14:457-466.

98) Scheuner D, Eckman C, Jensen M, Song X, Citron M, Suzuki N, Bird TD,

Hardy J, Hutton M, Kukull W, Larson E, Levy-Lahad E, Viitanen M, Peskind

E, Poorkaj P, Schellenberg G, Tanzi R, Wasco W, Lannfelt L, Selkoe D,

Younkin S (1996) Secreted amyloid beta-protein similar to that in the senile

plaques of Alzheimer's disease is increased in vivo by the presenilin 1 and

2 and APP mutations linked to familial Alzheimer's disease. Nat Med 2:864-

870.

99) Schmitz C, Hof PR (2000) Recommendations for straightforward and

rigorous methods of counting neurons based on a computer simulation

approach. J Chem Neuroanat 20:93-114.

100) Schwab C, Hosokawa M, McGeer PL (2004) Transgenic mice

overexpressing amyloid beta protein are an incomplete model of Alzheimer

disease. Exp Neurol 188:52-64.

101) Selkoe DJ (2001) Alzheimer's disease: genes, proteins, and therapy.

Physiol Rev 81:741-766.

102) Seubert P, Oltersdorf T, Lee MG, Barbour R, Blomquist C, Davis DL,

Bryant K, Fritz LC, Galasko D, Thal LJ, et al. (1993) Secretion of beta-

amyloid precursor protein cleaved at the amino terminus of the beta-

amyloid peptide. Nature 361:260-263.

References

60

103) Shankar GM, Li S, Mehta TH, Garcia-Munoz A, Shepardson NE,

Smith I, Brett FM, Farrell MA, Rowan MJ, Lemere CA, Regan CM, Walsh

DM, Sabatini BL, Selkoe DJ (2008) Amyloid-beta protein dimers isolated

directly from Alzheimer's brains impair synaptic plasticity and memory. Nat

Med 14:837-842.

104) Sherrington R, Rogaev EI, Liang Y, Rogaeva EA, Levesque G, Ikeda

M, Chi H, Lin C, Li G, Holman K, Tsuda T, Mar L, Foncin JF, Bruni AC,

Montesi MP, Sorbi S, Rainero I, Pinessi L, Nee L, Chumakov I, Pollen D,

Brookes A, Sanseau P, Polinsky RJ, Wasco W, Da Silva HA, Haines JL,

Perkicak-Vance MA, Tanzi RE, Roses AD, Fraser PE, Rommens JM, St

George-Hyslop PH (1995) Cloning of a gene bearing missense mutations in

early-onset familial Alzheimer's disease. Nature 375:754-760.

105) Sinha S, Lieberburg I (1999) Cellular mechanisms of beta-amyloid

production and secretion. Proc Natl Acad Sci U S A 96:11049-11053.

106) Siskova Z, Mahad DJ, Pudney C, Campbell G, Cadogan M, Asuni A,

O'Connor V, Perry VH (2010) Morphological and functional abnormalities in

mitochondria associated with synaptic degeneration in prion disease. Am J

Pathol 177:1411-1421.

107) Spires TL, Meyer-Luehmann M, Stern EA, McLean PJ, Skoch J,

Nguyen PT, Bacskai BJ, Hyman BT (2005) Dendritic spine abnormalities in

amyloid precursor protein transgenic mice demonstrated by gene transfer

and intravital multiphoton microscopy. J Neurosci 25:7278-7287.

108) Sturchler-Pierrat C, Staufenbiel M (2000) Pathogenic mechanisms of

Alzheimer's disease analyzed in the APP23 transgenic mouse model. Ann

N Y Acad Sci 920:134-139.

109) Sturchler-Pierrat C, Abramowski D, Duke M, Wiederhold KH, Mistl C,

Rothacher S, Ledermann B, Burki K, Frey P, Paganetti PA, Waridel C,

Calhoun ME, Jucker M, Probst A, Staufenbiel M, Sommer B (1997) Two

amyloid precursor protein transgenic mouse models with Alzheimer

disease-like pathology. Proc Natl Acad Sci U S A 94:13287-13292.

110) Swerdlow RH (2007a) Mitochondria in cybrids containing mtDNA

from persons with mitochondriopathies. J Neurosci Res 85:3416-3428.

111) Swerdlow RH (2007b) Pathogenesis of Alzheimer's disease. Clin

Interv Aging 2:347-359.

References

61

112) Swerdlow RH, Khan SM (2004) A "mitochondrial cascade

hypothesis" for sporadic Alzheimer's disease. Med Hypotheses 63:8-20.

113) Swerdlow RH, Burns JM, Khan SM (2010) The Alzheimer's disease

mitochondrial cascade hypothesis. J Alzheimers Dis 20 Suppl 2:S265-279.

114) Takahashi RH, Milner TA, Li F, Nam EE, Edgar MA, Yamaguchi H,

Beal MF, Xu H, Greengard P, Gouras GK (2002) Intraneuronal Alzheimer

abeta42 accumulates in multivesicular bodies and is associated with

synaptic pathology. Am J Pathol 161:1869-1879.

115) Takuma H, Teraoka R, Mori H, Tomiyama T (2008) Amyloid-beta

E22Delta variant induces synaptic alteration in mouse hippocampal slices.

Neuroreport 19:615-619.

116) Takuma K, Yao J, Huang J, Xu H, Chen X, Luddy J, Trillat AC, Stern

DM, Arancio O, Yan SS (2005) ABAD enhances Abeta-induced cell stress

via mitochondrial dysfunction. Faseb J 19:597-598.

117) Tampellini D, Magrane J, Takahashi RH, Li F, Lin MT, Almeida CG,

Gouras GK (2007) Internalized antibodies to the Abeta domain of APP

reduce neuronal Abeta and protect against synaptic alterations. J Biol

Chem 282:18895-18906.

118) Thal DR, Rub U, Orantes M, Braak H (2002) Phases of A beta-

deposition in the human brain and its relevance for the development of AD.

Neurology 58:1791-1800.

119) Thal DR, Larionov S, Abramowski D, Wiederhold KH, Van Dooren T,

Yamaguchi H, Haass C, Van Leuven F, Staufenbiel M, Capetillo-Zarate E

(2007) Occurrence and co-localization of amyloid beta-protein and

apolipoprotein E in perivascular drainage channels of wild-type and APP-

transgenic mice. Neurobiol Aging 28:1221-1230.

120) Thies W, Bleiler L (2011) 2011 Alzheimer's disease facts and figures.

Alzheimers Dement 7:208-244.

121) Tomiyama T, Nagata T, Shimada H, Teraoka R, Fukushima A,

Kanemitsu H, Takuma H, Kuwano R, Imagawa M, Ataka S, Wada Y,

Yoshioka E, Nishizaki T, Watanabe Y, Mori H (2008) A new amyloid beta

variant favoring oligomerization in Alzheimer's-type dementia. Ann Neurol

63:377-387.

References

62

122) Tomiyama T, Matsuyama S, Iso H, Umeda T, Takuma H, Ohnishi K,

Ishibashi K, Teraoka R, Sakama N, Yamashita T, Nishitsuji K, Ito K,

Shimada H, Lambert MP, Klein WL, Mori H (2010) A mouse model of

amyloid beta oligomers: their contribution to synaptic alteration, abnormal

tau phosphorylation, glial activation, and neuronal loss in vivo. J Neurosci

30:4845-4856.

123) Tsai J, Grutzendler J, Duff K, Gan WB (2004) Fibrillar amyloid

deposition leads to local synaptic abnormalities and breakage of neuronal

branches. Nat Neurosci 7:1181-1183.

124) Turner PR, O'Connor K, Tate WP, Abraham WC (2003) Roles of

amyloid precursor protein and its fragments in regulating neural activity,

plasticity and memory. Prog Neurobiol 70:1-32.

125) Umeda T, Tomiyama T, Sakama N, Tanaka S, Lambert MP, Klein

WL, Mori H (2011) Intraneuronal amyloid beta oligomers cause cell death

via endoplasmic reticulum stress, endosomal/lysosomal leakage, and

mitochondrial dysfunction in vivo. J Neurosci Res 89:1031-1042.

126) Van Dam D, D'Hooge R, Staufenbiel M, Van Ginneken C, Van Meir

F, De Deyn PP (2003) Age-dependent cognitive decline in the APP23

model precedes amyloid deposition. Eur J Neurosci 17:388-396.

127) Walsh DM, Klyubin I, Shankar GM, Townsend M, Fadeeva JV, Betts

V, Podlisny MB, Cleary JP, Ashe KH, Rowan MJ, Selkoe DJ (2005) The

role of cell-derived oligomers of Abeta in Alzheimer's disease and avenues

for therapeutic intervention. Biochem Soc Trans 33:1087-1090.

128) Wang D, Munoz DG (1995) Qualitative and quantitative differences in

senile plaque dystrophic neurites of Alzheimer's disease and normal aged

brain. J Neuropathol Exp Neurol 54:548-556.

129) Wang HW, Pasternak JF, Kuo H, Ristic H, Lambert MP, Chromy B,

Viola KL, Klein WL, Stine WB, Krafft GA, Trommer BL (2002) Soluble

oligomers of beta amyloid (1-42) inhibit long-term potentiation but not long-

term depression in rat dentate gyrus. Brain Res 924:133-140.

130) Wang X, Su B, Fujioka H, Zhu X (2008) Dynamin-like protein 1

reduction underlies mitochondrial morphology and distribution abnormalities

in fibroblasts from sporadic Alzheimer's disease patients. Am J Pathol

173:470-482.

References

63

131) Wang Z, Yang L, Zheng H (2011) Role of APP and Abeta in Synaptic

Physiology. Curr Alzheimer Res.

132) Wang ZF, Yin J, Zhang Y, Zhu LQ, Tian Q, Wang XC, Li HL, Wang

JZ (2010) Overexpression of tau proteins antagonizes amyloid-beta-

potentiated apoptosis through mitochondria-caspase-3 pathway in N2a

cells. J Alzheimers Dis 20:145-157.

133) Weibel ER (1969) Stereological principles for morphometry in

electron microscopic cytology. Int Rev Cytol 26:235-302.

134) West MJ, Coleman PD, Flood DG, Troncoso JC (1994) Differences

in the pattern of hippocampal neuronal loss in normal ageing and

Alzheimer's disease. Lancet 344:769-772.

135) Winkler DT, Bondolfi L, Herzig MC, Jann L, Calhoun ME, Wiederhold

KH, Tolnay M, Staufenbiel M, Jucker M (2001) Spontaneous hemorrhagic

stroke in a mouse model of cerebral amyloid angiopathy. J Neurosci

21:1619-1627.

136) Wirths O, Multhaup G, Czech C, Blanchard V, Moussaoui S, Tremp

G, Pradier L, Beyreuther K, Bayer TA (2001) Intraneuronal Abeta

accumulation precedes plaque formation in beta-amyloid precursor protein

and presenilin-1 double-transgenic mice. Neurosci Lett 306:116-120.

137) Wohlrab H (2009) Transport proteins (carriers) of mitochondria.

IUBMB Life 61:40-46.

138) Yamaguchi H, Sugihara S, Ogawa A, Saido TC, Ihara Y (1998)

Diffuse plaques associated with astroglial amyloid beta protein, possibly

showing a disappearing stage of senile plaques. Acta Neuropathol 95:217-

222.

139) Younkin SG (1995) Evidence that A beta 42 is the real culprit in

Alzheimer's disease. Ann Neurol 37:287-288.

Supplementary Material

64


Supplementary Tab. 1

a. Aβ1-42 levels detected by ELISA: Mean, standard devia tion, Welch-test

Mean Aβ1-42 (pmol/g) p-value

APP23 – 2-3 months (n=4) 2.25 +/- 0.36

APP48 – 2-3 months (n=6) 165.00 +/- 6.48 } <0.001

APP23 – 15 months (n=7) 6019.74 +/- 1146.88

APP48 – 18 months (n=8) 352.38 +/- 34.47 } <0.001

(Rijal Upadhaya et al., 2013)

b. DiI tracing of commissural neurons in the fronto central cortex

DiI-tracing of commissural neurons: Mann-Whitney U-test (exact test – two sided)

Comparison between transgenic and wildtype mice

3 months of age Type I neurons Type II

neurons

Type III

neurons

APP23 mice vs wildtype

littermates

p = 0.589 p = 1.000 p = 0.394


littermates

p = 0.573 p = 0.573 p = 0.228

n = 6 (APP23, APP48); n = 5 (wildtype (APP23-littermates)); n = 8 (wildtype

(APP48-littermates))



65


Comparison between transgenic and wildtype mice

15-18 months of age Type I neurons Type II

neurons

Type III

neurons


littermates

p = 0.001 p = 0.004 p = 0.093


littermates

p = 0.422 p = 0.235 p = 0.134

n = 8 (APP23); n = 9 (wildtype (APP23-littermates)); n = 11 (wildtype (APP48-

littermates)); n = 12 (APP48)



Comparison between age groups

3 months vs.

15-18 months

Type I

neurons

Type II

neurons

Type III

neurons

n (3m;

15/18m)

Wildtype

littermates

APP23

p = 0.012 p = 0.689 p < 0.001 * 5; 9

Wildtype

littermates

APP48

p = 0.009 p = 0.152 p = 0.442 8; 11

APP23 p = 0.001 p = 0.001 p = 0.142 6; 8

APP48 p = 0.018 p = 0.018 p = 0.553 6; 12

*not considered different because not confirmed in APP48 wildtype littermates



66

c. Frequency of dystropic neurites at the electron microscopic level

Comparison by genotype: ANOVA with Games-Howell post-hoc test

frontocentral cortex CA1

3 months 15-18

months

3 months 15-18

months

Wildtype vs. APP23 0.445 0.032 0.300 0.970

Wildtype vs. APP48 0.198 0.939 0.829 0.228

n = 6 (each group)


Comparison by age: Welch-test


Wildtype 0.093 0.485

APP23 0.004 0.015

APP48 0.818 0.394

n = 6 (each group)



67

d. Number of synapses: Mann-Whitney-U-test (exact t est-two sided)

Comparison by genotype


Asymmetric synapses 3 months 15-18

months

3 months 15-18

months

Wildtype vs. APP23 0.026 0.041 0.240 0.065

Wildtype vs. APP48 0.041 1.000 0.818 0.240

Symmetric synapses

Wildtype vs. APP23 0.240 0.589 0.093 0.394

Wildtype vs. APP48 0.310 0.310 0.310 0.589

n = 6 (each group)


Comparison by age

Asymmetric synapses frontocentral cortex CA1


APP23 0.002 0.180

APP48 0.009 0.937

Symmetric synapses

wildtype 1.000 0.093

APP23 0.093 0.240

APP48 0.937 0.699

n = 6 (each group)



68

e. Number of hippocampal CA1 and of frontocentral n eurons

Comparison by genotype: ANOVA with Games-Howell post-hoc test

Frontocentral cortex 3 months 15-18 months

Wildtype vs. APP23 n.a. 0.812

Wildtype vs. APP48 n.a. 0.999

CA1

Wildtype vs. APP23 0.004 <0.001

Wildtype vs. APP48 0.004 <0.001

n = 6 (15-18 months wildtype, APP23, APP48 and 3-month-old APP23, APP48); n

= 7 (3-month-old wildtype)


Comparison by age: Welch test

Frontocentral cortex CA1

Wildtype n.a. 0.005

APP23 n.a. <0.001

APP48 n.a. 0.002

n = 6 (15-18 months wildtype, APP23, APP48 and 3-month-old APP23, APP48); n

= 7 (3-month-old wildtype)



69

f. Percentage of altered somatic mitochondria: Mann -Whitney-U-Test (exact

test-two sided)



Wildtype vs. APP23 0.065 0.937


CA1



n = 6 (each group)


comparison by age



APP23 0.002 0.015

APP48 0.002 0.004

n = 6 (each group)



70

g. Volume density of altered somatic mitochondria: Mann-Whitney U-test

(exact test – two sided)





CA1



n = 6 (each group)


Comparison by age



APP23 0.002 0.002

APP48 0.002 0.065

n = 6 (each group)



71

h. Volume density of somatic mitochondria: Mann-Whi tney U-test (exact test

– two sided)





CA1



n = 6 (each group)


Comparison by age



APP23 0.002 0.065

APP48 0.026 0.485

n = 6 (each group)



72

i. Percentage of altered neuritic mitochondria: Man n-Whitney U-test (exact

test – two sided)





CA1



n = 6 (each group)


Comparison by age



APP23 0.002 0.699

APP48 0.180 0.485

n = 6 (each group)



73

j. Volume density of altered neuritic mitochondria: Mann-Whitney U-test

(exact test – two sided)





CA1



n = 6 (each group)


Comparison by age



APP23 0.002 0.699

APP48 0.065 0.065

n = 6 (each group)



74

k. Volume density of neuritic mitochondria: Mann-Wh itney U-test (exact test

– two sided)





CA1



n = 6 (each group)


Comparison by age



APP23 0.394 0.937

APP48 0.240 0.065

n = 6 (each group)



75

Supplementary Fig. 1: Western blot analysis of solu ble, dispersible, membrane-associated, and insoluble (plaque-associated) A β in wildtype, APP48 and APP23 mice. Original blots related to Fig. 4b(Rijal Upadhaya et al., 2013).


76

Supplementary Fig 2: Mitochondrial alterations in peripheral neurites. a-c: Percentage of altered mitochondria (a), volume densities of altered mitochondria (b), and volume densities of all mitochondria (c) in peripheral neurites did not show significant changes among frontocentral neurons in wild type (WT), APP23 and APP48 mice. d-f: No significant differences in the percentage of altered mitochondria (d), volume densities of altered mitochondria (e), and volume densities of all mitochondria (f) in peripheral neurites of the CA1 sector of the Ammon’s horn were found between APP48 mice and wild type controls. 3-month-old APP23 mice had less morphologically altered mitochondria (d, e) than wild type controls. In 15-month-old animals the trend was still visible but did not reach significance (d, e). *p < 0.05 (Further statistical analysis: Supplementary Tab. 1)(Rijal Upadhaya et al., 2013).

Acknowledgements

77

Acknowledgements

I would like to express my gratitude to my supervisor Prof. Dr. med. Dietmar Thal,

for providing me with such an interesting topic, for the excellent supervision and

support during this work as well as for providing immunohistochemical data,

synapse density data and dystrophic neurites frequency data.

I wish to thank all colleagues of the Section of Neuropathology, especially to Dr.

rer. nat. Ajeet Rijal Upadhaya for analysis of DiI tracing data from APP48 and

wildtype mice, for altered mitochondria data of 3 month old mice, stereological

data of hippocampal and frontocentral cortex neuron numbers as well as SDS-

PAGE and Western Blot data and to Irina Kosterin for the help with electron

microscope. I am very grateful to M. Staufenbiel and D. Abramowski at the

Novartis Institute for Biomedical Research (Basel, Switzerland) for providing the

animal models and ELISA data, to Estibaliz Capetillo-Zarate for providing DiI

tracing data for APP23 and wildtype mice (Cornell University, NY, USA), to H.

Yamaguchi at the Gunma University School of Health Sciences (Maebashi,

Gunma, Japan) for the kind gift of antibodies and Stefan Liebau for providing

Laser-Scan Microscopy figures of traced neurons. Thanks to Jochen Walter,

Satish Kumar, Janina Gerth for providing phosphorylated Aβ antibodies and

immunohistochemical data. Thanks to Stephan Harf for technical help on Fig. 1.

This work has been published in Acta Neuropathologica Communications and

contains, in addition to my work, experimental data carried out by coworkers as

described above. The text of this dissertation contains text blocks of its published

version in Acta Neuropathologica Communications.

Special thanks to my parents Helga and Dirk Scheibe for all the love and support

during my studies and this work. I am so thankful for everything you gave me and

all I have learned from you! I know it means a lot to you as it means to me to finish

this work.

Special thanks also to Heike Luise Schwarz for the love and motivation to finish

this work.

Thanks also to my sister and brothers and their families as well as to my friends

and colleagues for the distraction and nice time we had together.

Curriculum vitae

78

Curriculum vitae

Der Lebenslauf ist in der Online-Version aus Gründen des Datenschutzes nicht

enthalten.

Curriculum vitae

79

Parts of this dissertation have already been published in

Ajeet Rijal Upadhaya*, Frederik Scheibe*, Irina Kosterin, Dorothee Abramowski,

Janina Gerth, Sathish Kumar, Stefan Liebau, Haruyasu Yamaguchi, Jochen

Walter, Matthias Staufenbiel and Dietmar Rudolf Thal (2013) The type of

Abeta related neuronal degeneration differs between amyloid precursor

protein (APP23) and amyloid beta-peptide (APP48) transgenic mice. Acta

Neuropathologica Communications 2013, 1:77

* Equal contributors

© 2013 Rijal Upadhaya, Scheibe et al.; licensee BioMed Central Ltd. This is an

Open Access article distributed under the terms of the Creative Commons

Attribution License (http://creativecommons.org/licenses/by/2.0), which permits

unrestricted use, distribution, and reproduction in any medium, provided the

original work is properly cited. The Creative Commons Public Domain Dedication

waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data

made available in this article, unless otherwise stated.

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