Neuropathology and Applied Neurobiology (2015), 41, 245–257

doi: 10.1111/nan.12153

Histone deacetylases (HDACs) in frontotemporal lobar degeneration A. Whitehouse*, K. Doherty*, H. H. Yeh*, A. C. Robinson*, S. Rollinson†, S. Pickering-Brown†, J. Snowden*, J. C. Thompson*, Y. S. Davidson* and D. M. A. Mann* *Clinical and Cognitive Sciences Research Group, Institute of Brain, Behaviour and Mental Health, Faculty of Medical and Human Sciences, University of Manchester, Salford Royal Hospital, Salford, and †Clinical and Cognitive Sciences Research Group, Institute of Brain, Behaviour and Mental Health, Faculty of Medical and Human Sciences, University of Manchester, A.V Hill Building, Manchester, UK

A. Whitehouse, K. Doherty, H. H. Yeh, A. C. Robinson, S. Rollinson, S. Pickering-Brown, J. Snowden, J. C. Thompson, Y. S. Davidson and D. M. A. Mann (2015) Neuropathology and Applied Neurobiology 41, 245–257 Histone deacetylases (HDACs) in frontotemporal lobar degeneration 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 nongenetic, 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 with controls, and specifically in cases of FTLD tau-Picks compared with FTLD tau-MAPT and controls. No differences were noted between FTLDTDP subtypes, or between the different genetic and nongenetic 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, although their lack of immunostaining implies that such changes do not contribute directly to the formation of Pick bodies.

Keywords: frontotemporal lobar degeneration, histone deacetylases, immunohistochemistry.

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 Correspondence: David M. A. Mann, Clinical and Cognitive Neuroscience Research Group, University of Manchester, Salford Royal Foundation NHS Trust, Salford M6 8HD, UK. Tel: +44 (0) 161 206 2580; Fax: +44 (0) 161 206 0388; E-mail: david.mann@manchester .ac.uk © 2014 British Neuropathological Society

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, characterized 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 245

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43 kDa, 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 neurones, shuttling between nucleus and cytoplasm. However, in FTLD-TDP and FTLD-FUS they become 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 as an 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 eight histone proteins (two 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, that is, methylation of the DNA or synthesis of noncoding 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 (that is, 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 mediated by two groups of enzymes; histone acetyltransferases (HAT) © 2014 British Neuropathological Society

and histone deacetylases (HDAC) respectively. The human genome encodes 11 HDAC proteins, divided into five 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, although 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 colocalizes 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 stabilization 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 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] emphasize 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, Odagiri 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 (MSA). In a preliminary study [17], we noted increased levels of HDAC4 and HDAC5 in the NAN 2015; 41: 245–257

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cerebellum of FTLD cases, especially those with FTLDTDP. 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 (that is, 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.

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 © 2014 British Neuropathological Society

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C9ORF72, MAPT and GRN) were performed in the laboratory of Professor Stuart Pickering-Brown. The FTLD group was composed of 14 men and 19 women. There were 23 patients with behavioural variant frontotemporal dementia (bvFTD) (67%), five with frontotemporal dementia (FTD) + motor neurone disease (MND) (15%), two with semantic dementia (SD) (6%) and three with progressive nonfluent aphasia (PNFA) (9%). Pathological examination of the FTLD group showed 20 patients with FTLD-TDP (61%), of which 10 had type A pathology (50%), eight had type B pathology (40%) and two had type C pathology (10%) [2]. Thirteen cases had FTLD-tau (39%), of which seven had Pick’s disease (54%) and six had tau pathology consistent with exon 10 +16 MAPT mutation. The FTLD-TDP type A group comprised eight patients with bvFTD and two with PNFA. The FTLDTDP type B group comprised five patients with FTD + MND and three 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 two patients had FTLD-TDP type A pathology. Of the 22 patients with bvFTD, 12 showed FTLD-tau pathology (six with Pick’s disease and six with exon 10 +16 MAPT mutation) and 10 showed FTLD-TDP pathology (eight type A and two type B). Nineteen of the FTLD cases (58%) had a positive firstdegree family history, of which 17 bore a mutation on the MAPT, GRN or C9ORF72 gene. Of these 17 familial cases, seven 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, four with bvFTD and two with PNFA. Six 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 four patients bore a hexanucleotide repeat expansion on C9ORF72 gene. One of these had FTLD-TDP type A-based pathology, with a diagnosis of bvFTD but three had a pathological diagnosis of FTLD-TDP type B, two with FTD + MND and one with bvFTD. The mean duration of disease for each mutation type was similar (8–10 years), although 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 men and 16 women) were judged to be clinically normal. Twelve of these were obtained NAN 2015; 41: 245–257

© 2014 British Neuropathological Society FTLD-TDP C 10 normal 6 AD 7 ARC 3 CVD 1 CAA

FTLD-TDP B (C9ORF72)

FTLD-TDP A (C9ORF72) FTLD-TDP B

0:2 (0:100) 11:16 (41:59)

1:2 (33:67)

1:0 (100:0) 3:2 (60:40)

2:5 (29:71)

2:4 (33:67) 1:1 (50:50)

FTLD-tau (MAPT +16) FTLD-TDP A FTLD-TDP A (GRN)

4:3 (57:43)

M : F (%)

FTLD-tau Pi

Pathological diagnosis

58 60 ± 12 67 ± 7 69 ± 4 64 ± 22

59 ± 8 63 ± 11 na

69 ± 4

59 ± 6 49 57 ± 10

58 ± 5 66 ± 3

65 ± 8

57 ± 10 48 ± 2 60 ± 1

Age at death (years)

Age at onset (years)

7±6 na

8±9

9 4±2

10 ± 5

10 ± 4 6±2

8±2

Duration (years)

6 1 1 6 1 1 3 2 2 1 2 27

Y Y N Y N Y Y N Y N N N

7N

Family history

1279 ± 344 1248 ± 34

1287 ± 108

1050 1251 ± 185

1050 ± 168

1132 ± 103 1175 ± 50

1019 ± 126

Brain weight (g)

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 applicable; PNFA, progressive nonfluent aphasia; SD, semantic dementia; Y, yes; N, no.

32–33 34–60

29–31

23 24–28

16–22

5 FTD 2 PNFA 1 FTD 2 FTD 3 FTD + MND 1 FTD 2 FTD + MND 2 SD 24 Normal 3 MCI

6 FTD 1 PNFA 6 FTD 2 FTD

1–7

8–13 14–15

Clinical diagnosis

Case number

Table 1. Selected case details

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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 six of these showed mild neurofibrillary pathology (Braak stages I–II), confined essentially to amygdala and hippocampal formation. Two cases showed moderate cerebrovascular disease and one 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 6 μm thickness from formalin fixed, paraffin embedded blocks and mounted on to glass slides. 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, HDAC5 and HDAC6 was employed. All three antibodies were obtained from Cell Signalling 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 140 kDa 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 124 kDa 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 160 kDa 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 140 kDa level. The HDAC5 antibody (B-11; sc-133106) is a mouse monoclonal antibody raised against residues 371–443 of human HDAC5 and in cell © 2014 British Neuropathological Society

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lines detects a single band at 140–150 kDa 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 160 kDa level. 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, 10 mM) for 30 min, reaching 120 degrees Celsius and >15 kPa pressure. Sections were incubated for 30 min at room temperature in 0.3% peroxide in methanol to quench endogenous peroxidise activity, and then for a further 30 min at room temperature in Vectastain Elite PK-6101 goat serum as blocking buffer. Sections were then incubated for 1 h at room temperature in the appropriate Cell Signalling or Santa Cruz antibody, at a concentration of one in 50 of blocking buffer for both sets of antibodies. The sections were incubated for 30 min in a biotinylated secondary antibody followed by 30 min in Avidin Biotin Complex (ABC) reagent (both Vectastain Elite PK-6101 Rabbit IgG), both at room temperature. Sites of immunoreactions were visualized by incubating in DAB (3,3′-diaminobenzidine tetrahydrochloride) for 5 min, 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 Signalling antibodies, were scored for the degree of staining of granular neurones 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 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 NAN 2015; 41: 245–257

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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 Signalling 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 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 (that is, all section from each area stained by each antibody) was evaluated at ×20 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 FTLDTDP 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).

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,b), 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, although 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, although an occasional cell in all regions showed some weak immunoreactivity. The Santa Cruz HDAC4 antibody showed a similar pattern of immunostaining (online supplementary Figure S1a,b). Nonetheless, the total cytoplasmic staining score (that is, 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 with controls. This was due to both to an increase in the proportion of cells showing cytoplasmic staining (P = 0.016), and to an increase in the intensity of staining (P = 0.050). Otherwise, there were no significant

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 with controls, due to both an increase in the proportion of cells showing cytoplasmic staining (P = 0.016), and 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 with controls, due to both an increase in the proportion of cells showing cytoplasmic staining (P = 0.023), and an increase in the intensity of staining (P = 0.039). Immunoperoxidase-haematoxylin; all at ×40 microscope objective magnification. © 2014 British Neuropathological Society

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(a)

(b)

(c)

(d)

(e)

(f)

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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, although this was generally weak (not shown). However, nuclear immunostaining for HDAC5 in nerve cells of the ITG was more intense, although 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 (online supplementary Figure S1c,d). 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.

ing, 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, although 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 (online supplementary Figure S1e,f). The total cytoplasmic staining score was significantly higher in cells of the DG (P = 0.013) in FTLD overall compared with controls. Again, this was due to both to an increase in the proportion of cells showing cytoplasmic staining (P = 0.023), and to an increase in the 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 FTLDTDP 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 with 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.

HDAC6

Other observations

Generally, in control cases, no cells showed any cytoplasmic immunostaining for HDAC6 (Figure 1e), although in some instances a few cells did show a very weak immunostaining. However, the proportion of cells stain-

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-

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 FTLDMAPT 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

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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.

Discussion The main findings from the present study are that certain HDACs may be dysregulated in particular groups of patients with FTLD either when compared with control cases, or when compared with ‘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 tauPicks compared with 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 tauMAPT, nor were any similar changes seen in ITG or cerebellum. Likewise, no changes in HDAC4 were seen in other forms of FTLD (that is, FTLD-TDP), or to be associated with any of the other mutations which cause FTLD (that is, 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 localization of HDAC4, or a combination of the two. As HDAC4 is able to translocate between nucleus and cytoplasm, a process regulated in part 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 as nuclear accumulation of HDAC4 has been linked to neuronal apoptosis [15]. However, as the only significant difference in immunostaining that was observed was in cytoplasmic, rather than nuclear, staining, and because HDAC4 is normally localized to the cytoplasm of neurones [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 © 2014 British Neuropathological Society

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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 (PC) of FTLDTDP type B cases compared with 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 with 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 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 tauMAPT, 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 localization, 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 localization 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. As HDAC6 acts on histones to repress transcription, changes in localization or expression could alter the gene expression profile, which may contribute to the pathology of FTLD. NAN 2015; 41: 245–257

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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 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 [19–21]. This interaction may promote tau inclusion (Pick) body formation as HDAC6 has been shown to promote phosphorylation of tau at residue Thr231, which promotes hyperphosphorylation of tau [19] and reduces binding and stabilization 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 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 with controls, whereas no significant difference in HDAC6 immunostaining was observed between FTLDTDP 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 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, as Odagiri 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 localization or expression, may have a range of different effects. According to present data, both HDAC4 and HDAC6 © 2014 British Neuropathological Society

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 tauPi, 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 two antibodies for HDAC4, HDAC5 and HDAC6 were 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]. 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.

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.

Author contributions 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. NAN 2015; 41: 245–257

Histone deacetylases (HDACs) in frontotemporal lobar degeneration

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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Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Figure S1. 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

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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 ×40 microscope objective magnification. Received 21 August 2013 Accepted after revision 15 April 2014 Published online Article Accepted on 27 May 2014

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