THE POSSIBILITY OF A DEMENTIA-FREE FUTURE: FANTASY OR REALITY?

THE POSSIBILITY OF A DEMENTIA-FREE FUTURE: FANTASY OR REALITY?

Developing a Mind…

Newborn 3months 15months 2years

Connections Give Ever Deeper MEANING over

time…

THE BIOLOGICAL BASIS OF THE

MIND IS THE

PERSONALISATION OF THE

BRAIN THROUGH UNIQUE

DYNAMIC CONFIGURATIONS OF

NEURONAL CONNECTIONS,

DRIVEN BY UNIQUE

EXPERIENCES

Developing And Losing One’s Mind…

…Reflected In Brain Cell Branching (Connections)

CURRENT APPROACHES

CURRENT APPROACHES

INVASIVE

APPROACHES

CURRENT APPROACHES

INVASIVE

APPROACHES

NON-INVASIVE

APPROACHES

CURRENT APPROACHES

INVASIVE

APPROACHES

NON-INVASIVE

APPROACHES

THE ‘ANTICIPATORY’

APPROACH

But degeneration can start 20-30 years before the onset of visible symptoms!

THE ANTICIPATORY APPROACH

(A) Marker for in blood detects degeneration before symptoms appear

+(B) Medication for arresting further

neuronal death

=PERMANENT PREVENTION OF

SYMPTOM ONSET!

AN EVENTUAL ‘CURE’?

What is the Basic Mechanism of Neurodegeneration?

Clue 1: Co-pathology: Alzheimer's, Parkinson’s, and ALS

In search of the Basic Mechanism

of Neurodegeneration

‘Global Neurons’

SERIAL Primary sensory,

relays, cerebellum, thalamus, hippocampus, cortex

Amino acids Alar plate Electrically silent Lose plasticity Insensitive to trophic

factors

GLOBAL Locus C., Raphe N.,

A9, A10, basal forebrain motorneurons

Ach, DA, NA, 5-HT Basal plate Spontaneously

active Robust plasticity Sensitive to trophic

factors

‘SERIAL VS GLOBAL’ NEURONS (Woolf, 1996)

SERIAL Primary sensory,

relays, cerebellum, thalamus, hippocampus, cortex

Amino acids Alar plate Electrically silent Lose plasticity Insensitive to trophic

factors

GLOBAL Locus C., Raphe N.,

A9, A10, basal forebrain motorneurons

Ach, DA, NA, 5-HT Basal plate Spontaneously

active Robust plasticity Sensitive to trophic

factors

‘SERIAL VS GLOBAL’ NEURONS (Woolf, 1996)

Cholinergic Striatal Interneurons

Cholinergic Basal Forebrain

Histamine Hypothalamus

Dopaminergic Substantia Nigra

Cholinergic Pontomesencephalon

Noradrenergic Cerulear Region

Serotonergic Raphe

Cholinergic Motor Neurons

Woolf 1996

The ‘Isodendritic Core’: Rossor 1981

NORADRENALINE ACETYLCHOLINESTERASE (AChE)

THE LOCUS COERULEUS Degree of cell death correlates with disease

duration (Albanese & Butcher 1980)

Clue 1: Co-pathology: Alzheimer's, Parkinson’s, and ALS

Clue 2: Presence of key protein (AChE) in all Global neurons/Isodendritic

core

Clue 3: AChE is released

In search of the Basic Mechanism

of Neurodegeneration

Release of AChE from the Substantia Nigra:

(Llinás & Greenfield 1987)

Clue 1: Co-pathology: Alzheimer's, Parkinson’s, and ALS

Clue 2: Presence of key protein (AChE) in all Global neurons/Isodendritic

core

Clue 3: AChE is released

Clue 4: AChE promotes cell growth

In search of the Basic Mechanism

of Neurodegeneration

Trophic action of AChE in Hippocampus

(Day & Greenfield 2002)

Control

AChE

Trophic-Toxic Consequences of Increasing Levels of Calcium:

(Dickie et al., 1996)

Trophic or Toxic? Another Key Factor:

AGE!

Neonatal

Growth factors (e.g. AChE)

Brain develops normal structure

and function

Adult

AChE cells retain plasticity, sensitivity to growth factors and regenerative capacity

Cell Death

Attempt to repopulate in response to growth factors,

harmful ‘developmental’ response

Neurodegenerative Disorders

Non-AChE cells stabilise, lose plasticity and proliferative responses

Cell Death

Compensation by extant cells, no

proliferation

Adult

INSULT

Neurodegeneration: An Aberrant Form of

Development

We have identified the molecular fragment of

AChE that can operate

independent of enzyme function:

‘AChE-Peptide’

AChE C-Terminal Peptides

APP ..VKMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA.. AChE ..RQWKAEFHRWSSYMVHWKNQFDHYSKQDRCSDL

T14

T30

AB42

Control AChE Peptide

Administration of AChE-Peptide can be Toxic

(Greenfield et al 2004)

NEURODEGENERATION IS AN ABERRANT

FORM OF DEVELOPMENT:

AChE-PEPTIDE IS THE PIVOTAL TOXIC

MOLECULEI. BiomarkerII. In Vitro and In Vivo

ModelsIII. Therapeutic Intervention

NEURODEGENERATION IS AN ABERRANT

FORM OF DEVELOPMENT:

AChE-PEPTIDE IS THE PIVOTAL TOXIC

MOLECULEI. Biomarker

Discovery of Biomarkers:Mass Spectrometry

Sampl

e selection

• Selection of Alzheimer patients sample (Diagnosed for 1 to 3 years)

• Selection of age matched control patients

Sampl

e processing

• Filter the proteins with a 10kDa cut-off membrane• Desalting samples for mass spectrometry analysis

Sampl

e analysis

• All samples are analysed by Liquid chromatography coupled with a mass spectrometer

• The data are then manually processed

The AD-index:

Higher AD-index at the early stage of the disease

83% of the values are above the median of the control

50% of the values are above the upper quartile of the control

60 samples (30 AD and 30 control)R2 = 0.1297; P = 0.0040

Time after diagnosis (years)

AD

-ind

ex

NEXT STEPS

• Determine how early the AD-index can diagnose Alzheimer’s disease

• Confirm the specificity of this index (comparison with non-neurodegenerative disorders)

• Produce a diagnostic feature for Alzheimer’s disease (ELISA kit, etc.)

• Establish the relationship between the AChE-peptide and the current biomarker peptides

• Identify the proteases involved in the cleavage of AChE, and subsequent metabolites of AChE-peptide in current samples

NEURODEGENERATION IS AN ABERRANT

FORM OF DEVELOPMENT:

AChE-PEPTIDE IS THE PIVOTAL TOXIC

MOLECULEI. BiomarkerII. In Vitro and In Vivo

Models

(A) CHROMAFFIN/PC12 CELLS:

(1) Lewy body inclusions (Averback 1983)

(2) Neurofibrilliary tangles & paired helical filaments: (Izumiyama et al., 1990)

(3) Expression of APP (Takeda et al., 1994)

(4) Aberrant secretion of AChE (Appleyard & MacDonald 1991)

(5) A ‘window on the brain’ (Bornstein et al., 2012)

(A) CHROMAFFIN/PC12 CELLS:

AChE-Peptide (T30) is Toxic in PC12 cells....

30 % Reduction on Cell Viability (1 h)

A Specific Compensatory Response?

Could A and T30 peptide share a

final common path of action?

Synergy of A and T30 peptide after 1 hour

Cell viability AChE activity in perfusate

Advantages: Highly reductionistEasy and quick to cultureImplicated already in ADRelease AChE

Disadvantages: Not brain cells

(A) CHROMAFFIN/PC12 CELLS:

(B) OPTICAL IMAGING: BRAIN SLICES

Badin et al., 2013

(B) OPTICAL IMAGING: BRAIN SLICES

Advantages:Brain cellsFunctional circuitry retained (>10m

cells)Time resolution of action potentialsDifferential spatial and temporal

dynamics

Disadvantages: In Vitro

(C) IN VIVO RAT

The Cylinder Test:Sensitive indicator of neurological

damage (Schallert, 2000)

Control

T30 1μM

T30 100μM

7 days post treatmentGFAP IR IN THE BASAL FOREBRAIN

(C) IN VIVO RAT

Advantages: Functional effectsHistological effects

Disadvantages: Indirect actionBasic mechanism not revealed

(A) CHROMAFFIN/PC12 CELLS(B) OPTICAL IMAGING: BRAIN SLICES(C) IN VIVO RAT

In Vitro and In Vivo Models

NEURODEGENERATION IS AN ABERRANT

FORM OF DEVELOPMENT:

AChE-PEPTIDE IS THE PIVOTAL TOXIC

MOLECULEI. BiomarkerII. In Vitro and In Vivo

ModelsIII. Therapeutic Intervention

The 7 Receptor: The Target for AChE-Peptide?

The 7 Nicotinic Receptor• Coexpresses with AChE in developmental brain

(Broide et al,1996)

• Calcium permeability even greater than NMDA

(Séguéla et al,1993)

• Transgenic AD mouse (elevated Ab1-42): upregulation

(Svedverg et al, 2002)

• Binds amyloid peptide (Aβ) (Wang et al, 2000)

• Implicated in AD in conjunction of Aβ (Dineley,2001; Nagele, 2002)

B

10 μM BuChE peptide

10 μM AChE peptide

10 nM AChE peptide

10 μM BuChE peptide

10 μM AChE peptide

120 s

200 nA

α7

α4/β2

A

Log[AChE peptide] M

Per

cen

tag

e A

Ch

res

po

nse

B

C

-14 -12 -8 -10 -6 -4

50

100

150

AChE-Peptides act via 7-nAChR(Greenfield et al., 2004)

Compared with Control (A) & T15 peptide (D) Chronic Treatment with T14 (B) and T30 (C)

Increases α7-nAChR: A FEEDFORWARD MECHANISM!

Bond et al 2009

Normal AChE-Peptide Alpha-7 Blocker

Allosteric Modulator Site

Intracellular Pathways

Ranya Bechara

Cytotoxicity Assays

SRB MTT LDH

Mitochondria

Collagen

MTT

Cell Adhesion to collagen

Mitochondrial Function

Membrane Permeability

Measuring Cytotoxicity

Formazan

Comparison of T30 and Aβ

Cell Adhesion to collagen

Mitochondrial Function

Membrane Permeability

Ca2 +Calpain

Mitochondria

Spectrin

Nucleus

α7nAChR

Caspases

GSK

PFactor

s

Selective Intracellular Signalling Mechanisms of the

AChE Peptide

Ca2 +Calpain

Mitochondria

Spectrin

Nucleus

α7nAChR

Caspases

GSK

P

Factors

Selective Intracellular Signalling Mechanisms of the

AChE Peptide

(A) Marker for AChE PEPTIDE in blood detects degeneration before symptoms appear

+(B) Medication for arresting further

neuronal death by BLOCKING PEPTIDE ACTION

=PERMANENT PREVENTION OF

SYMPTOM ONSET!

Adding the Two Scenarios: An Eventual ‘Cure’?