Histone deacetylases (HDACs) in frontotemporal lobar degeneration

1 This article is protected by copyright. All rights reserved.

Histone deacetylases (HDACs) in Frontotemporal Lobar Degeneration 1

Amy Whitehouse1, Klara Doherty

1, Hsin Hsien Yeh

1, Andrew C. Robinson

1, Sara

Rollinson1,2

, Stuart Pickering-Brown1,2

, Julie Snowden1, Jennifer C Thompson

1, Yvonne

S. Davidson1, David M. A. Mann

1

1,2 Clinical and Cognitive Sciences Research Group, Institute of Brain, Behaviour and

Mental Health, Faculty of Medical and Human Sciences, University of Manchester, 1Salford Royal Hospital, Salford, M6 8HD and

2A.V Hill Building, Oxford Rd,

Manchester, M13 9PL, UK

Correspondence to:

Prof. David M.A. Mann,

Clinical and Cognitive Neuroscience Research Group,

University of Manchester,

Salford Royal Foundation NHS Trust,

Salford M6 8HD, UK.

Email: [email protected]

Tel. +44 (0) 161-206-2580

Fax. +44 (0) 161-206-0388

This article has been accepted for publication and undergone full peer review but has not been through the

copyediting, typesetting, pagination and proofreading process, which may lead to differences between this

version and the Version of Record. Please cite this article as doi: 10.1111/nan.12153

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Abstract

Aims: Frontotemporal lobar degeneration (FTLD) is clinically and pathologically

heterogeneous. Although associated with variations in MAPT, GRN and C9ORF72, the

pathogenesis of these, and of other non-genetic, forms of FTLD, remains unknown.

Epigenetic factors such as histone regulation by histone deacetylases (HDAC) may play a

role in the dysregulation of transcriptional activity, thought to underpin the

neurodegenerative process.

Methods: The distribution and intensity of HDACs 4, 5 and 6 was assessed semi-

quantitatively in immunostained sections of temporal cortex with hippocampus, and

cerebellum, from 33 pathologically confirmed cases of FTLD and 27 controls.

Results: We found a significantly greater intensity of cytoplasmic immunostaining for

HDAC4 and HDAC6 in granule cells of the dentate gyrus in cases of FTLD overall

compared to controls, and specifically in cases of FTLD tau-Picks compared to FTLD

tau-MAPT and controls. No differences were noted between FTLD-TDP subtypes, or

between the different genetic and non-genetic forms of FTLD. No changes were seen in

HDAC5 in any FTLD or control cases.

Conclusions: Dysregulation of HDAC4 and/or HDAC6 could play a role in the

pathogenesis of FTLD-tau associated with Pick bodies, though their lack of

immunostaining implies that such changes do not contribute directly to the formation of

Pick bodies. Acc

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Introduction

Frontotemporal Lobar Degeneration (FTLD), after Alzheimer’s disease, is the second

most common cause of dementia in the under 65s, accounting for 20% of cases [1]. It is a

primary neurodegenerative disorder characterized by circumscribed neurodegeneration of

the frontal and anterior temporal lobes. It is clinically, pathologically and genetically,

heterogeneous, and may in fact represent several distinct disorders united under this

umbrella [1]. In pathological terms, about half of cases can be defined by the presence of

tau-immunoreactive changes in neurones, and sometimes glial cells, of the frontal and

temporal cortex, and hippocampus, characterised by either neurofibrillary tangle-like

structures or Pick bodies; some of these cases are associated with mutations in the tau

gene, MAPT and have been classified as FTLD-tau [2]. Most of the remaining cases of

FTLD are associated with inclusion bodies (neuronal cytoplasmic inclusions, NCI and

neuronal intranuclear inclusions, NII) containing the transactive response (TAR) DNA

binding protein with Mw 43kDa, known as TDP-43, within neurones of the same regions

of cerebral cortex [3-5]. Such cases are termed FTLD-TDP [2], and have been associated

with mutations in various genes, but chiefly in progranulin (GRN) [6] and most recently

in C9ORF72 [7,8] where an expansion of a hexanucleotide repeat within the first intron

has now been identified as responsible for disease. Lastly, around 5% of cases are

characterized by NCI containing fused in sarcoma protein (FUS) [9], and are termed

FTLD-FUS [2].

Both TDP-43 and FUS are normally found in the nucleus of neurons, shuttling between

nucleus and cytoplasm. However, in FTLD-TDP and FTLD-FUS they become Acc

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translocated to the cytoplasm and accumulate as the characterizing inclusion bodies.

TDP-43 is part of the heterogeneous nuclear ribonucleoprotein (hnRNP) family and thus

regulates the transcription and splicing of RNA and the transport and translation of

mRNA. In this role it can act both as a suppressive or enhancing factor. Physiologically

the role of FUS, like TDP-43, involves regulation of DNA expression, RNA splicing,

mRNA transport into the nucleus, and mRNA translation, including local translation at

specific dendritic spines, thereby implying a role in synaptic plasticity [10].

In eukaryotic cells, DNA lies in a protein complex known as chromatin, the individual

units of which, called nucleosomes, consist of 146 base pairs of DNA wound around 8

histone proteins (2 each of H2A, H2B, H3 and H4). Epigenetic modifications involve

regulating how easily transcription machinery can access the DNA; setting up obstacles

or opening up the DNA respectively and can include (i) modifications of the nucleotides

i.e. methylation of the DNA or synthesis of non-coding RNAs which interferes with gene

transcription and translation; (ii) nucleosome remodelling where energy is required to

move the DNA along the histone octomers, thus changing what DNA is accessible for

transcription and (iii) ‘post-translational modifications’ of histones involving acetylation,

methylation, phosphorylation, ribosylation and ubiquitination. In acetylation, an acetyl

group is transferred onto the ε-amino group of a specific lysine residue located on the tail

of the histone, reducing affinity between neighbouring histones and facilitating DNA

translation [11]. Without its acetyl group (ie on deacetylation) the histone tail has a

positive charge and high affinity for the negative DNA, resulting in tightly packed DNA

which cannot be accessed by transcription machinery. Acetylation and deacetylation is Acc

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mediated by two groups of enzymes; histone acetyltransferases (HAT) and histone

deacetylases (HDAC), respectively. The human genome encodes 11 HDAC proteins,

divided into 5 classes according to structure; class I, IIa, IIb, III and IV. Class IIa

(HDAC4, 5, 7 and 9) and class IIb (HDAC6 and 10) are differentially expressed in brain

tissue [12,13], and some HDACs have been implicated in neurodegenerative disease. For

example, studies have shown HDAC4 to have a protective role against oxidative stress

and neurotoxicity [14], whereas others conclude that HDAC4 promotes cell death [15],

especially when it undergoes accumulation in the nucleus. Quinti et al [16] demonstrated

significant increases in HDAC1 and decreases in HDAC4, 5 and 6 in R6/2 Huntington

mice, though others have shown increases in HDAC4 and 5 to occur in human patients

with Huntington’s disease [17].

HDAC6 is present mainly in the cytoplasm where it deacetylates α-tubulin and

colocalises with microtubules [18], and therefore plays a role in the regulation of

intracellular transport. It has been shown to interact with tau and promote

phosphorylation at residue Thr231, which is believed to encourage hyperphosphorylation

of tau at other residues [19], thereby reducing the binding and stabilisation of

microtubules and promoting inclusion formation [20,21]. It also controls the fusion of

autophagosomes and lysosomes, and therefore may play a part in the sequestration and

elimination of intracellular aggregates such as huntingtin in HD [22] and α-synuclein in

Parkinson’s disease [23]. Importantly, it has been suggested that both FUS and TDP-43

regulate expression of HDAC6 [24,25]. The proteins were found to form a complex

which associates with HDAC6 mRNA at endogenous expression levels in mammalian Acc

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cell culture [24]. Expression of HDAC6 was reduced by RNAi silencing of either TDP-

43 or FUS [24]. Additionally, depletion of TDP-43 in HEK-293E cells has been shown to

reduce levels of HDAC6 [25,26]. Moreover, work showing that parkin ubiquitinates

TDP-43 and promotes its cytosolic accumulation via interactions with HDAC6 [27]

emphasise the potential role changes in HDAC might play in these forms of FTLD.

Thus, as with FTLD-tau, dysregulation of HDACs may play a role in FTLD-TDP and

FTLD-FUS. Indeed, Odagira et al [28] reported increased HDAC6 immunoreactivity in

the temporal cortex in patients with FTLD-TDP, but not in patients with AD, relative to

controls, and this was also raised in glial cells in the cerebellar white matter in Multiple

System Atrophy. In a preliminary study [17], we noted increased levels of HDAC4 and

HDAC5 in the cerebellum of FTLD cases, especially those with FTLD-TDP. Therefore,

the aim of the present study was to follow up these observations in more cases of FTLD,

and to expand the study to include neocortical and hippocampal regions, and to include

HDAC6 in the analysis. Temporal cortex and hippocampus were chosen as sites for

analysis because the principal changes of all histological forms of FTLD (ie Pick bodies

in FTLD-tau, TDP-43 and p62 inclusions in FTLD-TDP) fall on this region. We included

cerebellum as an area of interest because it is known that this region is affected in FTLD

(p62 immunoreactive inclusions in granule cells) associated with an expansion in

C9ORF72, at least [7,8]. We also compared cases of FTLD-tau with those of FTLD-TDP,

both overall and as separate histological and/or genetic subtypes, in order to determine

whether any changes in HDAC might be preferentially associated with any of these

particular variants of FTLD. Acc

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Patients and Methods

Patients

The patient groups comprised 33 patients with FTLD and 27 controls (Table 1). The

mean age of disease onset in the whole FTLD group was 57 years (range 43-73 years),

the mean duration of disease was 7.7 years (range 2 – 18 years) and the mean age at death

was 64 years (range 45-77 years). The latter was not significantly different from that of

the control group, where mean age at death was 64 years (range 26-92 years). Sections

from FTLD cases were obtained from the Manchester Brain Bank through appropriate

consenting procedures for the collection and use of the human brain tissues. Sections

from control cases were from Manchester Brain Bank (12 cases) or Thomas Willis Brain

Bank (15 cases) these, again, having been obtained through appropriate consenting

procedures.

All FTLD cases fulfilled Lund-Manchester clinical diagnostic criteria for FTLD [29] and

the more recent consensus criteria [30]. All had been longitudinally assessed within the

Cerebral Function Unit, Salford Royal Hospital, employing the Manchester

Neuropsychological Test Battery. Data collected on each patient included gender, age at

diagnosis and at death, and clinical diagnosis. All cases were from the Manchester Brain

Bank. Pathological diagnoses were made by an experienced neuropathologist (Professor

David Mann) and genetic analyses (for C9ORF72, MAPT and GRN) were performed in

the laboratory of Professor Stuart Pickering-Brown.

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The FTLD group was composed of 14 males and 19 females. There were 23 patients with

bvFTD (67%), 5 with FTD+MND (15%), 2 with SD (6%) and 3 with PNFA (9%).

Pathological examination of the FTLD group showed 20 patients with FTLD-TDP (61%),

of which 10 had type A pathology (50%), 8 had type B pathology (40%) and 2 had type

C pathology (10%) [2]. Thirteen cases had FTLD-tau (39%), of which 7 had Pick’s

disease (54%) and 6 had tau pathology consistent with exon 10 +16 MAPT mutation. The

FTLD-TDP type A group comprised 8 patients with bvFTD and 2 with PNFA. The

FTLD-TDP type B group comprised 5 patients with FTD+MND and 3 with bvFTD. Both

of the patients with FTLD-TDP C had SD. One patient with PNFA had FTLD-tau (Pick’s

disease type), whereas the other 2 patients had FTLD-TDP type A pathology. Of the 22

patients with bvFTD, 12 showed FTLD-tau pathology (6 with Pick’s disease and 6 with

exon 10 +16 MAPT mutation) and 10 showed FTLD-TDP pathology (8 type A and 2 type

B).

Nineteen of the FTLD cases (58%) had a positive first-degree family history, of which 17

bore a mutation on the MAPT, GRN or C9ORF72 gene. Of these 17 familial cases, 7 bore

a mutation on GRN gene, one with exon 1 C31LfsX34, one with exon 4 Q130SfsX124,

two with exon 10 V452WfsX38 mutation, one with exon 10 Q468X, and one with exon

11 R493X. These all had FTLD-TDP type A pathology, 4 with bvFTD and 2 with PNFA.

6 patients bore MAPT mutation (+16 splice site mutation on intron to exon 10) and all

had a family history of a FTLD-type disorder. The other 4 patients bore a hexanucleotide

repeat expansion on C9ORF72 gene. One of these had FTLD-TDP type A based Acc

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pathology, with a diagnosis of bvFTD but three had a pathological diagnosis of FTLD-

TDP type B, 2 with FTD+MND and one with bvFTD.

The mean duration of disease for each mutation type was similar (8-10 years), though the

mean age at onset of disease varied significantly (59 years for GRN; 48 years for MAPT

and 58 years for C9ORF72; p=0.028 between GRN and MAPT).

The 27 controls (11 males and 16 females) were judged to be clinically normal. Twelve

of these were obtained from the Manchester Brain Bank whereas the other 15 were

obtained from the Thomas Willis Brain Bank, University of Oxford. None of the 27

control cases showed any pathology beyond that which might be anticipated for age.

Consequently, 10 cases (over 65 years of age) showed mild deposition of amyloid beta

protein, mostly in the form of diffuse amyloid plaques, and 6 of these showed mild

neurofibrillary pathology (Braak stages I-II), confined essentially to amygdala and

hippocampal formation. Two cases showed moderate cerebrovascular disease and 1

showed mild cerebrovascular disease post-mortem: the remaining 13 cases were

apparently histological abnormality.

Methods

Immunohistochemistry

Sections of temporal cortex with hippocampus (Brodmann areas 21/22) and cerebellar

cortex were cut at 6µm thickness from formalin fixed, paraffin embedded blocks and

mounted on to glass slides. Acc

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The same immunostaining protocol was performed on each of the HDAC antibodies for

all of the 33 FTLD cases and 27 control cases. A single antibody for HDAC4, HDAC 5

and HDAC6 was employed. All 3 antibodies were obtained from Cell Signaling

Technology. The HDAC4 antibody (#2072) was produced by immunizing rabbit with a

synthetic KLH-coupled peptide corresponding to residues surrounding amino acid 10 of

human HDAC4. In mouse brain it detects a single band at the predicted 140kDa level.

The HDAC5 antibody (#2082) was produced by immunizing rabbits with a synthetic

peptide corresponding to the carboxy-terminal sequence of human HDAC5. In cell lines

it detects a single band at the predicted 124kDa level. The HDAC6 antibody (#7558) is a

monoclonal antibody (clone D2E5) produced by immunizing rabbits with a recombinant

protein specific to the carboxy-terminal sequence of human HDAC6. In cell lines it

detects a single band at the predicted 160kDa level. A second series of HDAC antibodies

were purchased from Santa Cruz Biotechnology, and employed on a subseries of sections

from both FTLD and control groups. Of these, the HDAC4 antibody (H-92: sc-11418) is

a rabbit polyclonal antibody raised against residues 530-561 of human HDAC4. In

NIH/3T3 cells it detects a single band at the predicted 140kDa level. The HDAC5

antibody (B-11; sc-133106) is a mouse monoclonal antibody raised against residues 371-

443 of human HDAC5 and in cell lines detects a single band at 140-150kDa level. The

HDAC6 antibody (D11: sc-28326) is a mouse monoclonal antibody raised against

residues 916-1215 of human HDAC6. In cell lines it detects a single band at the predicted

160kDa level.

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Sections were firstly hydrated through successive baths of xylene, alcohols of decreasing

concentration and distilled water. Antigen unmasking was performed by pressure cooking

in citrate buffer (pH 6 10mM) for 30 minutes, reaching 120 degrees Celsius and >15 kPa

pressure. Sections were incubated for 30 minutes at room temperature in 0.3% peroxide

in methanol to quench endogenous peroxidise activity, and then for a further 30 minutes

at room temperature in Vectastain Elite PK-6101 goat serum as blocking buffer. Sections

were then incubated for one hour at room temperature in the appropriate Cell Signalling

or Santa Cruz antibody, at a concentration of 1 in 50 of blocking buffer for both sets of

antibodies. The sections were incubated for 30 minutes in a biotinylated secondary

antibody followed by 30 minutes in Avidin Biotin Complex (ABC) reagent (both

Vectastain Elite PK-6101 Rabbit IgG), both at room temperature.

Sites of immunoreactions were visualised by incubating in DAB (3,3′-diaminobenzidine

tetrahydrochloride) for 5 minutes, followed by light counterstaining with haematoxylin

(Vector H-3401). Sections were dehydrated and mounted for analysis under the

microscope.

Microscopic analysis

Sections of hippocampus and temporal cortex, immunostained using the Cell Signaling

antibodies, were scored for the degree of staining of granular neurons in the dentate gyrus

(DG) of the hippocampus and pyramidal cells of the inferior temporal gyrus (ITG).

Because not all cells in DG or ITG showed nuclear and/or cytoplasmic staining, it was

decided to assess these separately. Staining was rated on a scale of 0-3 both for the Acc

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proportion of cells stained, and for the intensity of staining, in both the cytoplasm and

nucleus (where appropriate).

The proportion of cells/nuclei staining was scored according to:

0 = no cells staining

1 = a few cells staining

2 = a moderate number of cells staining

3 = most/all cells staining

The relative strength of cytoplasmic/nuclear staining was scored according to:

0 = no staining

1 = mild staining; some light brown or slightly denser granules present

2 = moderate staining; moderate number of medium brown or denser staining granules

present

3 = strong staining; many dark brown granules present

After scoring, the proportion and strength ratings in the nucleus and cytoplasm were

summated separately to give a total score for each (maximum score of 6) in each region.

The cerebellar sections were scored for immunostaining of Purkinje cells (PC), again

when using the Cell Signaling antibodies. Because the PC nucleus was unstained in all

instances, each PC was given a score according to the strength of immunostaining in the

cytoplasm alone (see scoring system for hippocampus and ITG) each cell thereby

attaining a potential maximum score of 3. In order to account for significant variation in Acc

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the strength of cytoplasmic immunostaining between PC in the same section, it was

decided to examine 30 consecutively located PC per section.

Each ‘set’ of stained sections (ie all section from each area stained by each antibody) was

evaluated at x20 magnification at one sitting by the same assessor. Assessments were

repeated twice on a subset of sections, and any cases where there was disagreement from

the original staining assessment were subject to reconciliation with a second observer

(DMAM).

Statistical Analysis

Statistical analysis was performed using IBM® SPSS® Statistics Version 20. Differences

in HDAC staining in each area of the brain between all FTLD cases and controls,

between all FTLD-tau and all FTLD-TDP cases, and between the histological subtypes of

FTLD-tau (tau-MAPT and tau-Pi) were analysed using Mann-Whitney U tests. Kruskal-

Wallis analysis with post-hoc Dunn’s test (if significant) was used to test for differences

in staining between the histological subtypes of FTLD-TDP (TDP-A, TDP-B and TDP-

C) as well as between cases of FTLD-TDP with different genetic backgrounds (mutations

in MAPT, C9ORF72 and GRN with cases with no known mutation).

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Results

HDAC4

Using the Cell Signalling antibody, a moderate to strong, granular cytoplasmic staining

for HDAC4 was observed in most, if not all, nerve cells of both the DG (Figure 1a, b)

and ITG (not shown) in most of the control cases (Figure 1a), as well as in most cases of

FTLD cases (Figure 1b). To the eye, there were no unambiguous group differences

between the FTLD cases and controls (compare Figure 1a and 1b), as regards either the

proportion of cells showing cytoplasmic staining, or the intensity of that staining, in

either region. In the cerebellum, most cases of FTLD and controls showed no

immunostaining of PC cytoplasm, though occasional cases in both groups showed a weak

immunostaining of isolated cells. There was usually no nuclear staining in DG (Figure

1a,b), ITG or PC in any case, though an occasional cell in all regions showed some weak

immunoreactivity. The Santa Cruz HDAC4 antibody showed a similar pattern of

immunostaining (on line supplementary figure 1a, b).

Nonetheless, the total cytoplasmic staining score (ie the sum of scores of proportion of

cells staining plus that of the relative strength of staining) was significantly higher in cells

of the DG (p=0.013) in FTLD overall compared to controls. This was due to both to an

increase in the proportion of cells showing cytoplasmic staining (p=0.016), as well as an

increase in the intensity of staining (p=0.050). Otherwise, there were no significant

differences in nuclear staining between FTLD and controls in DG, or between FTLD and

controls for both cytoplasmic and nuclear staining in ITG or in PC. There were no Acc

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differences in the degree of staining of nuclei or cytoplasm between all FTLD-tau and all

FTLD-TDP cases, in DG, ITG or PC. However, comparison of FTLD-Picks and FTLD-

MAPT cases with controls showed a highly significant (p=0.007) difference in total

cytoplasmic (but not nuclear) staining in DG between the groups. Post-hoc testing

showed that this was due to higher staining scores in FTLD-Picks than in FTLD-MAPT

(p=0.038) and controls (p=0.022), which did not differ significantly from each other

(p=0.058). Furthermore, the higher staining score in DG cells in FTLD-Picks was due to

a higher level of cytoplasmic staining (p=0.018), rather than a greater proportion of cells

staining (p=0.332). There were no significant differences in either nuclear or cytoplasmic

staining between the different subtypes of FTLD-TDP, or the different genetic types of

FTLD overall.

HDAC5

Cytoplasmic immunostaining for HDAC5 in the DG was generally absent or weak, with

only a small to moderate proportion of cells showing slight nuclear staining in both

individual control or FTLD cases (Figure 1c, d). In the ITG, more cells showed a granular

cytoplasmic staining for HDAC5, though this was generally weak (not shown). However,

nuclear immunostaining for HDAC5 in nerve cells of the ITG was more intense, though

again the number of cells that stained varied widely between cases, but usually affecting

only a small proportion of cells. The Santa Cruz HDAC5 antibody showed a similar

pattern of immunostaining (on line supplementary figure 1c, d).

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Statistically, there were no significant differences in any measure of HDAC5

immunostaining in cells of DG or ITG, or in PC, between all FTLD and control cases,

between FTLD-tau and FTLD-TDP cases, between the different histological subtypes of

FTLD-TDP, or the different genetic types of FTLD overall, Also, there were no

significant differences in staining between FTLD-MAPT and FTLD-Picks.

HDAC6

Generally, in control cases, no cells showed any cytoplasmic immunostaining for

HDAC6 (Figure 1e), though in some instances a few cells did show a very weak

immunostaining. However, the proportion of cells staining, and the intensity of staining,

was greater in some FTLD cases, especially those with FTLD-tau associated with Pick

bodies (Figure 1f). In the ITG, the intensity of cytoplasmic staining, and the proportion of

cells staining, again varied greatly, though there were no obvious differences between

FTLD cases and controls. Nuclear staining was present only in a small proportion of

cells, whether they were in FTLD or control cases, and was weak when present (Figure

1e, f). Little, or no, staining was observed in PC in control cases, with only a few FTLD

cases showing a weak, diffuse cytoplasmic staining of isolated cells. The Santa Cruz

HDAC6 antibody showed a similar pattern of immunostaining (on line supplementary

figure 1e, f).

The total cytoplasmic staining score was significantly higher in cells of the DG (p=0.013)

in FTLD overall compared to controls. Again, this was due to both to an increase in the

proportion of cells showing cytoplasmic staining (p=0.023), as well as an increase in the Acc

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intensity of staining (p=0.039). Otherwise, there were no significant differences in

nuclear staining between FTLD and controls in DG, or between FTLD and controls for

both cytoplasmic and nuclear staining in ITG or in PC.

There were no differences in the degree of staining of nuclei or cytoplasm between all

FTLD-tau and all FTLD-TDP cases, in DG, ITG or PC. However, comparison of FTLD-

Picks and FTLD-MAPT cases with controls showed a significantly higher total

cytoplasmic (but not nuclear) score in DG in FTLD-Picks (p=0.01) compared to FTLD-

MAPT (p=0.01) and controls (p=0.008). This was due to an increase in the intensity of

cytoplasmic staining (p=0.033), as well as an increase in proportion of cells staining

(p=0.018). There were no significant differences in either nuclear or cytoplasmic staining

between the different subtypes of FTLD-TDP, or the different genetic types of FTLD

overall.

There were no changes in any of the HDAC scores, in any region, with age within the

control cases, either when all 27 cases were examined by regression analysis, or when

comparing elderly cases with minimal pathology with younger cases without pathology.

Other observations

None of the HDACs, from either source, immunostained the inclusion bodies in any of

the FTLD histological subtypes including Pick bodies in FTLD tau-Pi, neurofibrillary

tangle-like structures and glial cell tangles in FTLD tau-MAPT, neuronal cytoplasmic

inclusions and/or neurites in FTLD-TDP, neuronal intranuclear inclusions in FTLD Acc

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associated with GRN mutations or p62 positive inclusions in hippocampus and

cerebellum in FTLD associated with C9ORF72 hexanucleotide expansions.

Discussion

The main findings from the present study are that certain HDACs may be dysregulated in

particular groups of patients with FTLD compared either to control cases, or when

compared as ‘internal’ subgroups. Although some of the more elderly control cases

showed minimal amounts of tau and/or Aβ pathology, especially, in hippocampus and

temporal lobe, there was no association between scores for any of the HDACs and age at

death for either of these regions, or cerebellum. We therefore conclude that such minor

amounts of pathology are unlikely to have influenced overall control values, and

therefore unlikely to have affected outcomes when comparing FTLD cases and controls.

Firstly, we found a significantly stronger cytoplasmic immunostaining for HDAC4 in

cells of DG in FTLD tau-Picks compared to FTLD tau-MAPT, suggesting that

dysregulation of HDAC4 could play a role in the pathogenesis of this form of FTLD-tau.

No differences in nuclear staining were seen between FTLD tau-Picks and FTLD tau-

MAPT, nor were any similar changes seen in ITG or cerebellum. Likewise, no changes in

HDAC4 were seen in other forms of FTLD (ie FTLD-TDP), or to be associated with any

of the other mutations which cause FTLD (ie GRN or C9ORF72). The increase in

cytoplasmic staining in cells of DG could be due to an increased expression of HDAC4,

or alterations in the cellular localisation of HDAC4, or a combination of the two. Since

HDAC4 is able to translocate between nucleus and cytoplasm, a process regulated in part Acc

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by calcium/calmodulin-dependent kinase, tau-Pi pathology may involve increased

translocation of HDAC4 from the nucleus to the cytoplasm [15]. Downstream effects of

this could induce changes in gene expression or cell viability since nuclear accumulation

of HDAC4 has been linked to neuronal apoptosis [15]. However, since the only

significant difference in immunostaining that was observed was in cytoplasmic, rather

than nuclear, staining, and because HDAC4 is normally localised to the cytoplasm of

neurons [15], it is suggested that dysregulation of HDAC4 in FTLD-tau-Picks may exert

its pathological effects through a cytoplasmic substrate or substrates, rather than changes

in histone acetylation or promotion of apoptosis through nuclear accumulation.

Nevertheless, the differences in cytoplasmic staining observed here may represent subtle

alterations in the balance between nuclear and cytoplasmic levels of HDAC4, thus

affecting histone acetylation or cell viability.

There were no significant differences in immunostaining for HDAC5 in DG, ITG or

cerebellum between any of the groups. This conflicts with previous observations, made

by Yeh et al of increases in HDAC4 and HDAC5 in the caudate and cerebellum (Purkinje

cells (PC)) of FTLD-TDP type B cases compared to controls, with corresponding

reductions in the levels of histone acetylation [17]. However, in this latter study such

observations were based on only a few cases, and no statistical comparison of the results

was performed. Such findings might simply reflect small sample variability [17].

Interestingly, no obvious changes in any HDAC, compared to other FTLD cases, were

seen in the hippocampus or PC of the cerebellum of FTLD-TDP cases bearing

expansions in C9ORF72. In such cases there are p62 immunoreactive neuronal Acc

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cytoplasmic inclusions in granule cells of the DG and cerebellum, and also occasionally

in PC – changes which are characteristic of this form of FTLD [31,32]. Consequently, it

might have been anticipated that cells in DG or PC would show some changes HDAC

activity given that the expansion might interfere with transcriptional activity [7,8].

Significant variations in immunostaining for HDAC6 in the DG, also suggest

dysregulation in FTLD. As with HDAC4, the intensity of immunostaining of the

cytoplasm was stronger, but also staining was present in a greater proportion of cells, in

FTLD tau-Pi than in tau-MAPT, once more suggesting that dysregulation of HDAC6, like

HDAC4, may play a greater role in FTLD tau-Pi than tau-MAPT. Differences in

cytoplasmic levels of HDAC6 could be caused by changes in localisation, expression, or

both and it will be important to determine the mechanism behind the changes in order to

understand the pathogenesis of FTLD more fully. Dysregulation of HDAC6 localisation

could contribute to disease pathology by inducing abnormally high or abnormally low

acetylation levels of normal cellular substrates of HDAC6, or by allowing HDAC6 to act

on novel substrates. Since HDAC6 acts on histones to repress transcription, changes in

localisation or expression could alter the gene expression profile, which may contribute

to the pathology of FTLD.

However, a number of other roles for HDAC6 have also been described which may be

affected by dysregulation and thus contribute to FTLD pathogenesis. For example, actin

and microtubules (tau) are both substrates of HDAC6 and are involved in aggresome

formation and the delivery of autophagosomal substrates to the lysosome to be removed Acc

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by macroautophagy [21,33]. Therefore, dysregulation of HDAC6 may promote inclusion

formation through abnormal acetylation levels of actin and tubulin proteins [21,33].

Additionally, HDAC6 is able to interact with tau, through its microtubule binding

domain, supporting the idea that dysregulation of HDAC6 may be involved in the

pathogenesis of FTLD-tau [19,20,21]. This interaction may promote tau inclusion (Pick)

body formation since HDAC6 has been shown to promote phosphorylation of tau at

residue Thr231, which promotes hyperphosphorylation of tau [19] and reduces binding

and stabilisation of microtubules [20,21]. In turn, tau also inhibits the activity of HDAC6,

leading to increased microtubule acetylation and reduced autophagy [34,35].

The potential involvement of increased HDAC6 levels in dementia, particularly those

forms with underlying tau pathology, is highlighted by research into other

neurodegenerative diseases, including Alzheimer’s disease and multiple system atrophy

(MSA), where HDAC6 levels have been shown to be increased relative to controls,

particularly in the cortex and hippocampus in Alzheimer’s disease, and the temporal

cortex in MSA [21,28,36].

However, present data conflicts with some previous studies. For example, Odagiri et al

[28] observed significantly increased expression of HDAC6, with increased cytoplasmic

staining, in cases of FTLD-TDP type B compared to controls, whereas no significant

difference in HDAC6 immunostaining was observed between FTLD-TDP and control

cases in this experiment in DG, ITG or cerebellum [28]. It has also been shown that

HDAC6 can form a multi-protein complex with Parkin and TDP-43 to mediate Acc

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translocation of TDP-43 into the cytosol, so it might be expected that dysregulation of

HDAC6 might be involved in FLTD-TDP [27]. These differences in findings could be

due to small sample sizes used or maybe the subjective nature of immunostain analysis,

since Ogadiri et al used immunoblotting to quantify HDAC6 expression [28].

HDACs are able to act on numerous substrates within cells, and so dysregulation, through

changes in localisation or expression, may have a range of different effects. According to

present data, both HDAC4 and HDAC6 appear to be dysregulated in DG in FTLD-tau-

Picks, and changes in these might therefore be associated with Pick body formation.

However, none of the HDACs appeared to immunostain inclusion bodies in any of the

FTLD histological subtypes whether these be Pick bodies in FTLD tau-Pi, neurofibrillary

tangle-like structures and glial cell tangles in FTLD tau-MAPT, neuronal cytoplasmic

inclusions and/or neurites in FTLD-TDP, neuronal intranuclear inclusions in FTLD

associated with GRN mutations or p62 positive inclusions in hippocampus and

cerebellum in FTLD associated with C9ORF72 hexanucleotide expansions. This suggests

that (changes in) HDACs do not play a direct role in inclusion body formation, but

nonetheless could precipitate or promote changes that ultimately lead to the formation of

such structures.

A potential limitation of the present study lies with the fact that only a single antibody for

HDAC, HDAC5 and HDAC was employed. Nonetheless, these antibodies have been well

characterized by the manufacturer, and antibodies against HDACs have been used

successfully in other studies based on human post mortem tissues [12,16,22,28]. Acc

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Nonetheless, it would clearly be important to validate present findings using other

antibodies in order to be able to make more definitive observations. Also, it might be

argued that the number of cases studied was too low to generate sufficient statistical

power, especially with some of the smaller histological or genetic subgroups, and again it

would be important in future to build on the principal findings of the study by recruitment

of further cases.

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Acknowledgements

We acknowledge the support of Alzheimers Research UK and Alzheimer’s Society

through their funding of the Manchester Brain Bank under the Brains for Dementia

Research (BDR) initiative. DMAM also receives funding from MRC and Wellcome Trust

which supported this study in part.

Amy Whitehouse and Klara Doherty did the immunohistochemistry and microscopical

assessments, and helped with paper writing.

Hsin Yeh developed the immunohistochemical staining protocols.

Andrew Robinson prepared sections for staining and immunohistochemistry.

Jennifer Thompson did the data analysis.

Julie Snowden helped with statistical advice and clinical data.

Sara Rollinson and Stuart Pickering-Brown provided genetic data.

Yvonne Davidson provided technical support and training.

David Mann provided study design, supervision, helped with microscopical assessments

and wrote the paper.

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Case

number

Clinical

Diagnosis

Pathological

Diagnosis

M : F (%) Age at

onset

(years)

Age at

death

(years)

Duration

(years)

Family

history

Brain

weight (g)

1-7 6 FTD

1 PNFA

FTLD-tau Pi 4:3 (57:43) 57 ± 10 65 ± 8 8 ± 2 7 N 1019 ± 126

8-13 6 FTD FTLD-tau

(MAPT +16)

2:4 (33:67) 48 ± 2 58 ± 5 10 ± 4 6 Y 1132± 103

14-15 2 FTD FTLD-TDP A 1:1 (50:50) 60 ± 1 66 ± 3 6 ± 2 1Y

1 N

1175 ± 50

16-22 5 FTD

2 PNFA

FTLD-TDP A

(GRN)

2:5 (29:71) 59 ± 6 69 ± 4 10 ± 5 6 Y

1 N

1050 ± 168

23 1 FTD FTLD-TDP A

(C9ORF72)

1:0 (100:0) 49 58 9 1Y 1050

24-28 2 FTD

3 FTD+MND

FTLD-TDP B 3:2 (60:40) 57± 10 60 ± 12 4 ± 2 3Y

2 N

1251± 185

29-31 1 FTD

2 FTD+MND

FTLD-TDP B

(C9ORF72)

1:2 (33:67) 59± 8 67 ± 7 8 ± 9 2 Y

1 N

1287± 108

32-33 2 SD FTLD-TDP C 0:2 (0:100) 63 ± 11 69± 4 7 ± 6 2 N 1279± 344

34-60 24 Normal

3 MCI

10 normal

6 AD

7 ARC

3 CVD

1 CAA

11:16

(41:59)

NA 64± 22 na 27 N 1248± 34

Table 1: Selected case details. AD = Alzheimer’s disease, ARC = age-related changes,

CAA = cerebral amyloid angiopathy, CVD = cerebrovascular disease, FH = family

history, FTD = frontotemporal dementia, MCI = mild cognitive impairment, MND =

motor neurone disease, na = not available, PNFA = progressive non-fluent aphasia, SD =

semantic dementia, Y = yes, N = no, * = some data unavailable.

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Legends to Figure

Figure 1: HDAC immunostaining in cells of the dentate gyrus of the hippocampus in

control (a,c,e) and FTLD-tau with Pick bodies (b,d,f) for: HDAC4 (a,b), HDAC5 (c,d)

and HDAC6 (e,f) employing Cell Signalling HDAC antibodies. In most control (a) and

FTLD (b) cases, a similarly moderate to strong, granular cytoplasmic staining for

HDAC4 was observed, and no clear group differences could be ascertained on visual

inspection alone. Nonetheless, statistical analysis showed a significantly higher

cytoplasmic staining score (p=0.013) in FTLD overall compared to controls, due to both

to an increase in the proportion of cells showing cytoplasmic staining (p=0.016), as well

as an increase in the intensity of staining (p=0.050). Cytoplasmic immunostaining for

HDAC5 was generally absent or weak, with only a small to moderate proportion of cells

showing slight nuclear staining in either control (c) or FTLD (d) cases. Again no clear

group differences could be seen between control and FTLD cases, and this was borne out

by statistical analysis. Generally, no cells showed any cytoplasmic immunostaining for

HDAC6 in control cases (e), whereas there was strong HDAC6 immunoreactivity in

perikarya and cell processes in FTLD-tau cases associated with Pick bodies (f). Statistical

analysis supported these visual observations inasmuch as the total cytoplasmic staining

score was significantly higher in cells of the DG (p=0.013) in FTLD overall compared to

controls, due to both to an increase in the proportion of cells showing cytoplasmic

staining (p=0.023), as well as an increase in the intensity of staining (p=0.039).

Immunoperoxidase-haematoxylin; all at x40 microscope objective magnification.

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On-line supplementary Figure 1: HDAC immunostaining in cells of the dentate gyrus

of the hippocampus in control (a,c,e) and FTLD-tau with Pick bodies (b,d,f) for: HDAC4

(a,b), HDAC5 (c,d) and HDAC6 (e,f) employing Santa Cruz HDAC antibodies. In most

control (a) and FTLD (b) cases, a similarly moderate to strong, granular cytoplasmic

staining for HDAC4 was observed. Cytoplasmic immunostaining for HDAC5 was

generally absent or weak, with only a small to moderate proportion of cells showing

slight nuclear staining in either control (c) or FTLD (d) cases. Generally, cells showed

weak to moderate cytoplasmic immunostaining for HDAC6 in control cases (e), whereas

there was strong HDAC6 immunoreactivity in perikarya and cell processes in FTLD-tau

cases associated with Pick bodies (f).

Immunoperoxidase-haematoxylin; all at x40 microscope objective magnification.

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Histone deacetylases (HDACs) in frontotemporal lobar degeneration