Microglial proliferation and astrocytic protein alterations in the human Huntington's disease cortex

Huntington’s disease (HD) is a neurodegenerative disorder that severely affects the basal ganglia and regions of the cerebral cortex. While astrocytosis and microgliosis both contribute to basal ganglia pathology , the contribution of gliosis and potential factors driving glial activity in the human HD cerebral cortex is less understood. Our study aims to identify nuanced indicators of gliosis in HD which is challenging to identify in the severely degenerated basal ganglia, by investigating the middle temporal gyrus (MTG), a cortical region previously documented to demonstrate milder neuronal loss. Immunohistochemistry was conducted on MTG paraffin-embedded tissue microarrays (TMAs) comprising 29 HD and 35 neurologically normal cases to compare the immunoreactivity patterns of key astrocytic proteins (glial fibrillary acidic protein, GFAP; inwardly rectifying potassium channel 4.1, Kir4.1; glutamate transporter-1, GLT-1; aquaporin-4, AQP4), key microglial proteins (ionised calcium-binding adapter molecule-1, IBA-1; human leukocyte antigen (HLA)-DR; transmembrane protein 119, TMEM119; purinergic receptor P2RY12, P2RY12), and indicators of proliferation (Ki-67; proliferative cell nuclear antigen, PCNA). Our findings demonstrate an upregulation of GFAP + protein expression attributed to the presence of more GFAP + expressing cells in HD, which correlated with greater cortical mutant huntingtin (mHTT) deposition. In contrast, Kir4.1, GLT-1, and AQP4 immunoreactivity levels were unchanged in HD. We also demonstrate an increased number of IBA-1 + and TMEM119 + microglia with somal enlargement. IBA-1 + , TMEM119 + , and P2RY12 + reactive microglia immunophenotypes were also identified in HD, evidenced by the presence of rod-shaped, hypertrophic, and dystrophic microglia. In HD cases, IBA-1 + cells contained either Ki-67 or PCNA, whereas GFAP + astrocytes were devoid of proliferative nuclei. These findings suggest cortical microgliosis may be driven by proliferation in HD, supporting the hypothesis of microglial proliferation as a feature of HD pathophysiology. In contrast, astrocytes in HD demonstrate an altered GFAP expression profile that is associated with the degree of mHTT deposition.


Introduction
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder which stems from a cytosine-adenine-guanine (CAG) trinucleotide repeat expansion, resulting in the production of a mutant form of the huntingtin (HTT) protein [31].Consequently, there is an aberrant accumulation of mutant HTT (mHTT) protein accompanied by degeneration of select neuronal populations throughout the human brain.While the basal ganglia has been a major area of focus in HD, analysis of cortical neuronal loss has critically influenced the understanding of HD symptom heterogeneity, which our laboratory and other studies have well documented [27,35,37,44,45,52,66].However the contribution of non-neuronal cells, including glia, to disease pathogenesis and heterogeneity is not as well understood.While glial cells play a significant role in maintaining healthy brain function, there is still a lack of understanding of cortical gliosis in HD.
Astrocytes and microglia are vital to maintaining homeostasis and regulating neuronal activity in the brain.In response to damage or disease, these cells enter a reactive state characterised by functional and morphological changes [57,60].Entering a reactive state allows glial cells to maintain homeostasis, chronic activation is thought to result in detrimental effects on the brain, as proffered in other neurodegenerative diseases such as Alzheimer's disease (AD) [30,39,60].Gliosis within the human HD striatum is substantiated with prior studies documenting (1) astrocytic increases in glial fibrillary acidic protein (GFAP) immunoreactivity, (2) astrocyte and microglial cell somal enlargement, (3) thickened tortuous glial processes, and (4) greater presence of immunohistochemically-and PETdetected reactive microglial phenotypes [12,13,33,38,47,50,55,63,70,71].Furthermore, as the structural GFAP + changes have been implicated in HD striatal dysfunction, subsequent studies have served to investigate the mechanisms underlying HD astrogliosis.
In the post-mortem human striatum, glutamate transporter-1 (GLT-1) has been reported to progressively reduce with advancing neuropathological grade [13].Subsequent HD animal model studies also theorise that astrocytic impairment is not limited to glutamate transport but may, in fact, include the aberrant buffering of extracellular K + through deficits in the expression of the inwardly rectifying K + channel 4.1 (Kir4.1)[68].In addition to the altered buffering of glutamate and K + , the aquaporin-4 (AQP4) channels, involved in the clearance of extracellular waste and mediation of K + buffering, have been implicated in the clearance of extracellular mHTT protein [8].

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Journal Pre-proof On the other hand, while reactive microglia have been reported in the striatum and cortex of both living HD patients and post-mortem human tissue [38,41,43,47,63], it remains unclear as to when microglia enter the reactive phenotypes and the biological context surrounding this shift.Therefore, to provide further clarity surrounding the immunophenotypes of microglia in HD, the following markers were examined: IBA-1, HLA-DR, TMEM119, and P2RY12.IBA-1 is a widely utilised immunohistochemical microglia marker that has been reported to be upregulated in the human HD striatum [12,50].Similarly, HLA-DR is an antigen-presentation molecule commonly utilised to identify and quantify reactive microglia in the post-mortem human brain [22,51,61].While IBA-1 is a commonly utilised marker, it is also known to immunolabel perivascular macrophages [18,61].As such, this study also investigates the expression of two microglia-specific markers, TMEM119 and P2RY12, which are reportedly altered in reactive microglia of the post-mortem Alzheimer's disease human brain [1,36,48,62].
This study aims to utilise a well-established human brain tissue microarray (TMA) and highcontent analysis platform to examine neuropathological and glial molecular changes in the human HD temporal cortex [53].We hypothesise that profiling the temporal cortex in HD will aid in the discovery of more subtle HD-related molecular changes in glia which would be more challenging to detect in severely degenerated regions, due to the lesser degree of neuronal loss we have quantified in this region [37].We have recently confirmed the presence of various species of HD aggregates within TMAs constructed from the middle temporal gyrus (MTG), with the degree of N-terminal HTT deposition correlating with the length of the CAG repeat expansion and symptom onset, but not neuronal loss [28].
Therefore, extending our investigation to non-neuronal cells in the MTG, commencing with glia, provides an opportunity to identify more subtle HD-related molecular changes and elucidate potential drivers of HD pathogenesis that may underpin HTT deposition and neuropathology.Fixation and dissection protocols for human brain tissue preparation were previously described [28,53,73].In brief, donated brains were perfused with formalin and the MTG was divided into four equal sized blocks (MTG0-3, Supplementary Fig. 1).

Human tissue collection and processing
A total of 35 normal cases with no history of neurological abnormalities (Table 1) and 29 HD cases (Table 2) were utilised in the construction of MTG TMAs.The normal and HD cases were matched closely for age and post-mortem delay.In addition to the usage of TMAs, larger MTG sections from either block zero or one were utilised for single and double-label paraffin immunohistochemistry to validate indicators of microglial proliferation in those cases identified by TMA to exhibit proliferation (Table 3).
Table 1 Normal cases for the middle temporal gyrus tissue microarrays.Details include the case number, sex, age at death (years), post-mortem delay (PMD; hours), and cause of death.The normal cases included 11 females and 24 males, aged 32-98 years with a post-mortem delay of 5.5-48 hours.F = female; M = male; SD = standard deviation.

TMA design and production
The preparation, construction, and analysis protocols of the TMAs utilised in this study have been described in Singh-Bains et al [53].Briefly, 7 μm thick sections were obtained from donor paraffin-embedded MTG blocks of 35 normal and 29 HD cases (Table 1 and Table 2) and mounted onto positively charged slides (überFrost Plus 3000) for antigenicity detection and neuroanatomical localisation studies.Neuroanatomical identification of cortical grey matter layers II-VI was identified by labelling a section from each donor block with cresyl violet, followed by subsequent inspection and identification of candidate coring sites by a neuroanatomist (AYST or MKSB).Once coring sites have been identified, a 2 mm tissue core was extracted from the donor blocks using the Advanced Tissue Arrayer (VTA-100, Veridiam).The extracted cores were inserted into a blank recipient TMA paraffin block, forming an array of cores.The recipient block was serially sectioned into 7 μm thick slices using the paraffin microtome in preparation for immunohistochemistry.

Single-label paraffin immunohistochemistry
Standard paraffin immunohistochemistry protocols were carried out on the sections, as described previously [28,53,74].A summary of all antibodies and respective antigen retrieval protocols can be found in Table 4.The TMA sections were placed on a hot plate at 60°C for 1 hour to allow the tissue to anneal to the glass slides and aid in paraffin dewaxing.
The sections were subsequently dewaxed in two xylene immersions (1 hour and 10 minutes, respectively), and rehydrated through two 5-minute 100% ethanol immersions, followed by 95%, 80%, and 75% ethanol immersion (2-minutes each), and three 5-minute washes in J o u r n a l P r e -p r o o f Journal Pre-proof milliQ water (mQH 2 O).After deparaffinisation, the sections underwent heat-induced antigen retrieval (AR) at 121°C for 20 minutes (Table 4; Model 2100-retriever, Pick Cell Laboratories), followed by a 2-hour cool-down period.Following AR, the sections were washed three times in mQH 2 O for 5 minutes each.Endogenous peroxidases were blocked for 20 minutes at room temperature in a blocking solution (50% methanol, 1% H 2 O 2 , diluted in mQH 2 O).The sections were rinsed in mQH 2 O followed by three 5-minute washes in PBS.
The sections were exposed to a blocking buffer (10% normal goat or donkey serum diluted in PBS) for 1 hour at room temperature.After blocking, the sections were incubated overnight with the primary antibodies at 4°C.
The following day, the sections were washed in PBS with 0.2% Triton X-100 (PBS-T) for 5minutes, followed by two 5-minute PBS washes, before incubation in secondary antibody at room temperature for 3 hours.The sections were washed in PBS-T and PBS before ExtrAvidin®-Peroxidase incubation at room temperature for 1 hour.Washing steps were conducted again prior to slide incubation in the peroxidase substrate (0.5% 3,3'diaminobenzidine (DAB), 0.01% H 2 O 2 , intensified with 0.04% Ni(NH 4 ) 2 SO 4 ).DAB was washed off with three PBS and three mQH 2 O washes (5-minutes each) and the sections were subsequently dehydrated (2-minutes each in 75%, 80%, and 95% ethanol, twice for 5-minutes in 100% ethanol, and three times for 10-minutes in xylene) before a coverslip was applied using DPX mounting medium.All antibodies were diluted in 1% normal goat or donkey serum (diluted in PBS).No-primary controls, where the primary antibody was omitted, were included to assess non-specific secondary antibody labelling.The presence of Ki-67 and proliferative cell nuclear antigen (PCNA) immunoreactivity within HD tissue was validated by assessing antigenicity on formalin-fixed paraffin-embedded glioblastoma (GBM) tissue, which served as a positive control due to the known presence of highly proliferative cells [10].

Double-label chromogen paraffin immunohistochemistry
Detection of a second antigen on top of tissue subjected to one round of single-label DAB immunohistochemistry was achieved using VinaGreen (BRR807AS, BioCare Medical) amplification in a second round of immunohistochemistry.After DAB exposure and subsequent PBS and mQH2O washes were completed for detection of the first immunolabel, the sections underwent a second AR round for the second immunolabel.Following AR for the second antigen, endogenous peroxidases were blocked for 20 minutes and subsequently

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Journal Pre-proof exposed to 10% normal goat/donkey serum for 1 hour before incubation with the second primary antibody overnight.The following day, the sections were incubated in secondary antibody and ExtrAvidin®-Peroxidase. Washes were conducted before incubation in VinaGreen (prepared as per manufacturer's directions).The sections were briefly washed in PBS and mQH2O before being dehydrated and coverslipped.

Image acquisition
Images for the immunolabelled TMA sections were acquired using a VSlide Automated Slide Scanning Microscope (Metasystems) running Metafer4 software (v3.12.133), utilising a previously described TMA imaging protocol [53].An automated pre-scan image of the TMA acquired with a 2.5x objective lens was used to localise the immunolabeled cores through threshold-based segmentation with the 'Microarray Tool' function.A 6x10 grid of interconnected dots was overlaid on the thresholded cores, and each dot was manually adjusted to align with the centre of each core of interest.Once finalised, a subsequent automated re-scan was carried out for each core using a 10x objective lens across the entire TMA.Four images were acquired around the centre of each core to capture the entire 2 mm core with sufficient detail for analysis.The re-scan images were inspected for folded, missing, or torn cores, which were excluded from subsequent analysis.

Image analysis using high-content screening
Images acquired were analysed using MetaMorph (v7.8.10,Molecular Devices).Image analysis was conducted using the 'Count Nuclei' module as previously described [53].For each immunolabel, the Count Nuclei measurements included: total nuclei (number of cells), integrated intensity (immunolabel expression), and total area (of the immunolabel).In addition, for glial morphology measurements, the 'Neurite Outgrowth' module was used to generate marker-specific segmentation masks to log the mean cell body size [53].
Furthermore, the total area of the core was measured using the 'Threshold Image' command that isolated the region of interest above a pre-defined greyscale value, in this case, the whole core [53].Data for each immunolabel was presented as the mean across four images acquired per core.

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Image analysis using manual quantification
Manual quantification methods were performed on Ki-67 and PCNA immunolabelled TMAs using a Nikon Ni-E microscope.Using the 'Large Image Grabbing' function within the NiS Elements Advanced Research Software (v4.50), the tissue was scanned in the X, Y and Z plane with a 63x oil objective for Ki-67 immunoreactivity and 40x water objective for PCNA immunoreactivity.If positive punctate immunoreactivity was detected, it would be verified with a Z-plane scan before being quantified and imaged.If no immunoreactivity was detected, the field of view moved to the adjacent sampling site located 0.1 mm (63x objective) or 0.4 mm (40x objective) apart until the entire core or tissue section was quantified.For TMA cases with identified Ki-67 and PCNA immunoreactivity, larger tissue sections from the donor MTG block (Table 3) were immunolabeled and quantified manually to validate the presence of proliferative nuclei in a larger area.All data points pertaining to the number of Ki-67 + and PCNA + nuclei are presented as a sum of all immunopositive nuclei within the TMA core or large MTG tissue section.

Statistical analysis
All data was statistically analysed using GraphPad Prism (v8.0) and presented as mean ± SD.
The normal and HD TMA data were screened for departures from normality.The TMA data for each immunolabel was not represented by a Gaussian population.Therefore, the comparisons between normal and HD groups were conducted using a non-parametric 2-tailed Mann-Whitney test.HD subgroup comparisons between early and advanced Vonsattel grade were investigated using a Mann-Whitney test.A p-value < 0.05 was considered statistically significant.Additionally, the TMA data were also correlated with various clinicopathological parameters including the number of SMI-32 + (Neurofilament H non-phosphorylated; Covance; SMI-32R; 1:500) pyramidal neurons, the number of 1C2 + (Millipore; MAB1574; 1:5000) and B4-S830 + (Gillian Bate's Laboratory, 1:100) mHTT aggregates, CAG repeat length, symptom onset, age at death, and post-mortem delay using Spearman's test.The correlation strengths were interpreted according to the r-values [28] with statistical significance set at p<0.05.

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Further correlations were conducted to investigate the potential relationship between the number of GFAP + astrocytes and indicators of HD clinicopathology for each case (Supplementary Table 2 and Supplementary Fig. 2).Subgrouping the HD cohort into early (Grade 1-2) versus advanced (Grade 3-4) Vonsattel neuropathological grade did not demonstrate a greater number of cortical astrocytes with advanced striatal pathology (Supplementary Table 2).Furthermore, the relationship between GFAP + cell number with respect to the degree of SMI-32 + neuronal loss, 1C2 + HTT aggregate deposition, and B4-S830 + HTT aggregate deposition was also investigated (Supplementary Table 2).A moderate correlation (r=0.58,p=0.016) was observed between the number of GFAP + cells and the number of B4-S830 + aggregates, which suggests a potential link between astrocytic changes and the degree of mHTT deposition (Supplementary Fig. 2).The number of GFAP + cells did not correlate with CAG repeat length, symptom onset, age at death, or post-mortem delay (Supplementary Table 2), nor were there any gender-related differences across the HD and normal cohorts (Supplementary Table 3).Journal Pre-proof significant strong positive correlation (r=0.82,p<0.00010) was identified between AQP4 + and GLT-1 + immunolabel coverage in the pooled cohort (n=42), which can be attributed to the significant strong positive correlation identified within the separate normal (r=0.88,p<0.00010, n=21) and HD (r=0.72,p=0.00030, n=21) cohorts.An unpaired Mann-Whitney test was conducted for comparisons between the HD and normal cohorts.A Spearman's correlation test was used to obtain the r-and p-values for the relationship investigated.Data presented as mean ± SD. *p < 0.05.AQP4 = aquaporin-4, GFAP = glial fibrillary acidic protein, GLT-1 = glutamate transporter-1, HD = Huntington's disease, Kir4.1 = inwardly rectifying potassium channel 4.1, TMA = tissue microarray.

Preservation of Kir4.1, GLT-1, and AQP4 expression in the human HD temporal cortex
Kir4.1 immunoreactivity, in both normal and HD cores, presented with distinct astrocyte cell bodies and fine processes (Fig. 1F-G).TMA analysis revealed no significant differences in Kir4.1 + integrated intensity, coverage, and cell number between the HD and normal cohorts (Fig. 1H-J).
In both normal and HD cores, GLT-1 immunoreactivity presented with two distinct staining patterns: domain-like patches of staining (Fig. 1K-L) and diffuse immunoreactivity (Fig. 1M-N).However, these staining differences were not specific to a particular subgroup of cases.
TMA analysis revealed no significant differences in GLT-1 + integrated intensity and coverage between the HD and normal cohorts, which is likely to be attributable to the heterogenous staining patterns (Fig. 1O-P).
Similar to GLT-1, AQP4 presented with distinct domain-like patches of staining (Fig. 1Q-R) and diffuse immunoreactivity (Fig. 1S-T) which were widely present throughout the HD and normal cohorts.TMA analysis of AQP4 + immunoreactivity revealed no significant differences in integrated intensity and coverage between the HD and normal cohorts (Fig. 1U-V).As the "patch" and "diffuse" staining patterns of AQP4 and GLT-1 presented similarly, a correlation was conducted to investigate if the staining patterns were present in the same cases.A significant strong positive correlation was identified between GLT-1 + and AQP4 + coverage (Fig. 1W, r=0.82, p<0.00010).To determine whether the observed relationship is a disease-specific event, correlations between GLT-1 + and AQP4 + coverage was conducted for the HD and normal cohorts independently.Significant strong positive correlations were identified in both the normal (r=0.88,p<0.00010) and HD (r=0.72,p=0.00030) cohorts suggesting that this relationship is not influenced by disease and can likely be attributed to overall case variation.

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Journal Pre-proof Additional analyses were conducted to investigate the potential impact that gender may have on Kir4.1 + , GLT-1 + , and AQP4 + integrated intensity (Supplementary Table 3).We can conclude that gender has no impact on our reported integrated intensity data in the MTG.As Kir4.1, GLT-1, and AQP4 immunoreactivity was preserved between the HD and normal cohorts, no further analysis was conducted to investigate the relationships between Kir4.1, GLT-1, and AQP4 immunoreactivity and clinicopathological variables.

Increased number of IBA-1 + microglia with altered morphology in HD temporal cortex
To further understand the degree of gliosis within the HD TMA, the immunoreactivity patterns of classical microglial markers, IBA-1 and HLA-DR, were investigated.IBA-1 immunolabelled small, round cell bodies and fine, highly branched processes, indicating the presence of ramified microglia within both HD and normal cohorts (Fig. 2A-B).Additionally, within the HD cores, more rod-shaped, hypertrophic, and dystrophic microglia were identified (Fig. 2C-E).Analysis of IBA-1 + microglia revealed a significant 48% increase in cell number (p=0.0027) in HD compared to normal cases, with no differences in IBA-1 + integrated intensity and coverage (Fig. 2F-H).Morphology quantification of IBA-1 + microglia revealed a significant 14% increase (p=0.0017) in the cell body size in HD cases relative to the normal cases (Fig. 2I).However, HD and normal IBA-1 + microglia presented with a comparable degree of outgrowths, processes, and branches per cell (Supplementary Figure 3 A-C).
To elucidate the relationship between the observed increase in IBA-1 + cell number and mean cell body size with various measures of clinicopathology, further analyses were conducted, yielding no significant relationships (Supplementary Table 2 and 3).Subgrouping the HD cohort into early (Grade 1-2) versus advanced (Grade 3-4) Vonsattel neuropathological grade did not demonstrate greater cortical microgliosis with advanced striatal pathology.
Closer examination of HLA-DR + microglia (Fig. 2J-N) demonstrated similar morphologies in both HD and normal cases.Quantitative analysis revealed no significant changes in HLA-DR integrated intensity, coverage, cell number, soma size, outgrowths per cell, processes per cell, or branches per cell between normal and HD cores (Fig. 2O-R and Supplementary Figure 3 D-F).No significant gender-related HLA-DR + integrated intensity differences were identified across the HD and normal cohort.As HLA-DR + immunoreactivity was preserved between the HD and normal cohorts, no further analysis was conducted to investigate the relationship between HLA-DR + immunoreactivity and clinicopathological variables.Journal Pre-proof p=0.072) and immunolabel coverage (G, p=0.069) between the HD (n=22) and normal cohort (n=22).(H) A significant 48% increase in the number of IBA-1 + microglia (p=0.0027) were identified in the HD cohort compared to the normal cohort.(I) TMA analysis of morphological parameters revealed a significant 14% increase in IBA-1 + mean cell body area (p=0.0017) in the HD cohort compared to the normal cohort.(J-N) Representative photomicrographs of HLA-DR immunoreactivity from a (J) normal and an (K) HD case, with black arrowheads denoting HLA-DR + microglia.(O-R) No significant differences were identified in integrated intensity (O, p=0.34), immunolabel coverage (P, p=0.32), cell number (Q, p=0.15), and cell body area (R, p=0.43) between the HD (n=19) and normal cohorts (n=21).Omission of the outlier case H245 (circled in red) does not influence the mean comparisons between HD and normal cohorts and was left in the dataset due to no biologically relevant reasons to exclude this case (post-mortem delay, cause of death, age at death within typical range).An unpaired Mann-Whitney test was used to obtain the p-value, with the data presented as mean ± SD. ** p < 0.010.HD = Huntington's disease, HLA-DR = human leukocyte antigen-DR isotype, TMA = tissue microarray, IBA-1 = ionised calcium-binding adapter protein 1.

Increased number and soma size of TMEM119 + microglia contrasted with the preservation of P2RY12 expression
As IBA-1 expression is expressed by both microglia and border-associated macrophage populations in the human brain[18], TMEM119 and P2RY12 were subsequently utilised to specifically elucidate the degree of microgliosis within the HD human brain.Similar to IBA-1, TMEM119 immunolabelled distinct ramified microglia within both HD and normal cohorts (Fig. 3A-B).Furthermore, TMEM119 + microglia presented with a range of morphologies, including rod-shaped, hypertrophic, and dystrophic microglia in the HD cohort (Fig. 3C-E).Analysis of TMEM119 + microglia revealed a significant 47% increase in the number of microglia (p=0.010) in the HD cohort compared to the normal cohort, with no significant differences in TMEM119 + integrated intensity or coverage (Fig. 3F-H).
Subsequent morphometric analysis of TMEM119 + microglia revealed an 8% increase in soma size in HD (Fig. 3I, p=0.033).Similar to IBA-1 + microglia, TMEM119 + microglia in the HD cohort did not present with a significantly altered length of outgrowths per cell, or the number of processes and branches per cell (Supplementary Figure 3 G-I).
To investigate the relationship between the observed increase in TMEM119 + cell number and mean cell body size with various clinicopathological variables in HD, further analyses were conducted, yielding no significant relationships (Supplementary Table 2 and 3).Subgrouping the HD cohort into early (Grade 1-2) versus advanced (Grade 3-4) Vonsattel grade did not demonstrate greater cortical microgliosis with advanced striatal pathology.(H) A significant 47% increase in the number of TMEM119 + microglia (Q, p=0.010) with a significant 8% increase in mean soma size (R, p=0.033) was identified in the HD cohort compared to the normal cohort.(J-N) Representative photomicrographs of P2RY12 immunoreactivity from a (J) normal and an (K) HD case, with arrowheads denoting the presences of ramified (black arrowheads) and rod-shaped (white arrowhead) P2RY12 + microglia.The presence of (L) rod-shaped, (M) hypertrophic, and (N) dystrophic P2RY12 + microglia was identified in the HD cohort.(O-R) No significant differences were identified in integrated intensity (O, p=0.91), immunolabel coverage (P, p=0.91), cell number (Q, p>0.99), and mean cell body area (R, p=0.16) between the HD (n=20) and normal cohorts (n=21).An unpaired Mann-Whitney test was used to obtain the p-value, with the data presented as mean ± SD. * p < 0.050, HD = Huntington's disease, P2RY12 = purinergic receptor P2RY12, TMA = tissue microarray, TMEM119 = transmembrane protein 119.

Proliferation of IBA-1 + microglia within the HD temporal cortex
To further understand the basis of gliosis in the HD TMAs, a screen of routinely used antibodies to proliferation markers, Ki-67 and PCNA, was conducted to address the potential contribution of cellular proliferation (Fig. 4).The experiments undertaken on HD TMAs were also conducted in tandem on GBM tissue known to contain highly proliferative cells that are positive for PCNA and Ki-67 (Fig. 4A,E).To determine if the increase in the number of IBA-1 + microglia in HD may be attributable to proliferation, chromogen co-labelling was first conducted with IBA-1 and Ki-67 (Fig. 4A-D), and IBA-1 and PCNA (Fig. 4E-H).We were able to detect both PCNA and Ki-67 immunoreactivity within IBA-1 + microglia, indicative of microglial proliferation in the HD cerebral cortex (Fig. 4G-H).Subsequent co-labelling with GFAP and PCNA was conducted to determine if the increase in GFAP + astrocyte cell number in HD was attributable to proliferation (Fig. 4I-J).No co-labelling was identified, indicating that the increase in GFAP + cell number in HD cannot be attributed to astrocytic proliferation.

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To quantify the presence of proliferative nuclei in the HD MTG, TMA analysis was conducted and revealed a statistically significant subset of HD cases that demonstrated an increase in the number of both p=0.046) and PCNA (Fig. 5B,p=0.0046)immunopositive nuclei within the HD cohort, with 25% (4/16 cases) and 31% (8/26 cases) of HD cases in the TMA immunolabelling for Ki-67 and PCNA, respectively.No normal cases contained any signs of PCNA or Ki-67 immunoreactivity.To determine if the degree of proliferation within the 2 mm HD TMA cores recapitulates proliferation in the wider MTG, PCNA immunoreactivity was quantified in larger MTG tissue sections obtained from the HD TMA cases with known PCNA immunoreactivity.A strong significant correlation (Fig. 5C, r=0.87, p=0.0091) was identified between the number of PCNA + nuclei within the larger tissue sections versus the number of PCNA + nuclei in the TMA cores, thereby validating that the TMA recapitulates proliferation seen in larger areas of tissue sampled.As the TMA immunoreactivity was validated, subsequent correlations were conducted to compare PCNA immunoreactivity with various HD clinicopathological variables.
A very strong negative correlation (Fig. 5D, r=-0.90, p=0.083) was identified between the number of PCNA + nuclei and number of SMI-32 + pyramidal cells, however, this association did not reach statistical significance.A strong significant positive correlation (Fig. 5E, r=0.80, p=0.024) was observed between the number of PCNA + nuclei and CAG repeat length.Furthermore, a strong significant negative correlation was observed between the number of PCNA + nuclei and age at death (Fig. 5F, r=-0.88,p=0.0065) suggesting HD cases with longer CAG repeat length and earlier age at death have more PCNA + nuclei.

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Journal Pre-proof the few studies that have been conducted to quantify glial involvement within the human HD cerebral cortex, the findings have been restricted to predominantly microglia, rather than the contribution of astrocytosis [38,41,47,58,63,76].Therefore, this study serves to be the first of its kind to utilise cortical human brain TMAs to survey the contributions of both cortical astrocytes and microglia to HD clinicopathology.By investigating the MTG, a region with a milder degree of neuronal loss, our study was able to elucidate nuanced disease-related indicators, including evidence of microglial proliferation in the HD cortex, which is difficult to uncover in more severely degenerated regions of the HD human brain.Our results confirm astrocytic and microglial reactivity in the HD human cortex, which reinforces the need to consider these cell types as potential targets and modulators for HD therapies.

Contribution of astrogliosis to HD cortical pathology
The degree of astrogliosis can be typically characterised into (1) mild to moderate, (2) severe diffuse, and (3) severe with compact scar formation [57].Phenotypically, as the severity of astrogliosis progresses, astrocytes demonstrate a parallel upregulation of GFAP expression and hypertrophy.Within the post-mortem human HD striatum, reactive astrocytes have been immunohistochemically documented, with studies reporting increases in GFAP staining intensity, soma size, and thickening of tortuous processes [12,13,56,70].Indeed, the progression of astrogliosis from mild to severe corresponds to the overall degree of striatal HD neurodegeneration [72].In contrast, while cortical astrocytic dysfunction has been alluded to by previous studies, a comprehensive immunohistochemical characterisation of astrocytes using multiple antibodies and quantitative analysis approaches has not been undertaken in human brain tissue.Astrocytes in prior studies were either examined qualitatively, semi-quantitatively through western blots or quantitively through a cresyl violet stain which is not astrocyte-specific [19,42,49,58,76].Our MTG TMA data revealed clear indicators of astrocytic alterations through significant increases in the number of GFAP + cells and overall protein expression and coverage in the HD cases.Importantly, TMA analysis revealed no co-labelling of proliferative markers and GFAP in HD, suggesting that the increase in the number of GFAP + astrocytes within the HD cortex may be largely driven by protein expression and morphology changes, rather than astrocytic proliferation.While future studies would be required to determine the degree and subsequent consequence of astrogliosis in the HD MTG, this study proposes that astrocytic changes within this region presents in a mild state.

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Journal Pre-proof Investigations to elucidate the process and timeline of astrocytic activation in HD has predominantly focused on the study of subcortical regions.With the progression of pathological disease severity from Vonsattel Grade 0 to 4, characterised by progressive neuronal loss, there is a paralleled increase in astrogliosis visualised through an increase in GFAP, progressive cellular and process hypertrophy, as well as receptor changes in the basal ganglia [12,13,72].Furthermore, mHTT protein has been found within astrocytes in both the striatum of HD human tissue and HD animal models, with the suggestion that mHTT alters astrocytic function and exacerbates neuronal dysfunction and symptomology in animal and in vitro studies [6,7,13,49].Our data demonstrates the observed increase in the number of GFAP + cells in the HD temporal cortex does not appear to be dependent on advancing striatal neuropathological grade, signifying that upregulation of cortical GFAP expression does not parallel the degree of striatal neuropathology.Further correlations with neuropathological parameters suggest that the increase in GFAP + cells does not correlate with greater SMI-32 + pyramidal neuronal loss.However, it may be influenced by mHTT accumulation as evidenced by the moderate correlation between increasing numbers of GFAP + cells and the presence of more B4-S830 + mHTT aggregates.Indeed, it is hypothesised that astrogliosis within the HD striatum may be partially driven by mHTT protein which impairs astrocytic function and perpetuates further cellular and phenotypic dysfunction [3,6,7,49].In line with this hypothesis, the presence of mHTT in astrocytes has been reported in both the frontal cortex and striatum of HD post-mortem human brains and animal models [25].Therefore, our data suggests that alterations in GFAP expression may be linked to the accumulation and deposition of mHTT aggregates within the human HD MTG.Interestingly, our TMA data revealed a correlative relationship between GFAP + cell number and only B4-S830 + aggregate deposition, but no relationship with the presence of 1C2 + aggregates.These results would lend itself to the hypothesis that different conformations of mHTT protein may confer distinct forms of astrocytic dysfunction [2].As B4-S830 was raised against exon 1 of mHTT protein and 1C2 was raised against the polyglutamine tract of TATA-binding protein, it could be hypothesised that increases in GFAP + cell number, driven by an upregulation of protein expression, may be more closely associated with exon 1 of mHTT protein.Collectively, while our findings advance the idea that a form of astrocytic response occurs within the human HD cortex, further research is required to determine the exact influence of mHTT aggregates on astrocytes.

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Preservation of functional astrocytic proteins in the human HD temporal cortex
Astrocytes are crucial to the maintenance of the CNS homeostatic environment.It is proposed that astrogliosis in HD mouse models, influenced by mHTT protein expression, presents with an early intrinsic disruption of spatial K + buffering and glutamate reuptake with a loss of key proteins, including the Kir4.1 channel and GLT-1 transporter, which may contribute to neuronal deficits in the HD striatum [68].Indeed, in HD cases of Vonsattel Grade 0, the HD striatum is characterised by a 30-40% loss of neurons with no overt signs of reactive astrocytes, despite reports of a significant reduction in GLT-1 expression [13,71].Together, these studies would indicate that loss of astrocytic proteins detrimentally contributes to striatal neuronal dysfunction and may precede reactive astrocytic phenotypes.Contrasting these findings, TMA analysis of the HD cerebral cortex revealed no alterations in overall Kir4.1, GLT-1, and AQP4 expression, despite an upregulation of GFAP immunoreactivity.Therefore, our findings suggest that the astrocytic response within the HD temporal cortex may not be governed by overt disruption in the expression of these functional astrocytic proteins.

Contribution of microgliosis to HD cortical pathology
Our study is the first to conduct an in-depth quantification of key molecular markers of human microglia using human brain cortical TMAs.Studies investigating the relationship between microgliosis and HD severity in the human brain have predominantly focused on the striatum.PET studies have suggested reactive microgliosis occurs within the striatum of HD patients, which appears to be closely associated with disease severity [38,40].Furthermore, IBA-1 + microglia was reported to be predominantly ramified in the HD human striatum before shifting into either a hypertrophic or dystrophic phenotype by Vonsattel Grades 3 and 4 [12,50].Within the human frontal cortex, the presence of reactive microglia was noted to be associated with disease severity and the degree of neuronal loss [47].While PET and immunohistochemical studies have noted the involvement of microglia in HD [12,38,41,42,47,50,55,58,63], the degree of microglial involvement has not be extensively quantified in the human cortex until this current study.
We commenced our investigation with analysis of pan-microglial marker IBA-1, expressed throughout the cytoplasm and processes, where its expression is thought to increase with J o u r n a l P r e -p r o o f Journal Pre-proof microglial reactivity [14,24,50].Our quantitative analysis of IBA-1 immunoreactivity in the HD MTG revealed a significant increase in the number of IBA-1 + cells with hypertrophic morphologies.Furthermore, we identified TMEM119, P2RY12 and IBA-1 positive rodshaped, hypertrophic, and dystrophic microglial morphologies in the HD cortex which was widely absent from our normal cases.Generally, hypertrophic microglia are classically considered the quintessential 'reactive' microglia [59].In contrast, dystrophic microglia, are considered to be damaged microglia in the senescence phase [59].Finally, the functions of rod-shaped microglia are still unknown, but hypothesised to be involved in a neuroprotective response for injured or diseased neurons [47].These data suggest a reactive microglial response occurring in the HD cortex, driven by a shift from a ramified microglial morphology to more reactive phenotypes [11,47,60,71,72].
Based on the changes observed in IBA-1 + microglia in the HD cortex, we extended our investigation to another known microglial marker, HLA-DR.HLA-DR is a major histocompatibility complex (MHC) class II protein expressed on the surface of antigenpresenting cells and upregulated following inflammation and injury [59].HLA-DR immunoreactivity has been reported in the human HD basal ganglia and frontal cortex, however, these studies have been chiefly descriptive and qualitative [33,34,47,55].Our TMA data suggests HLA-DR + microglia are widely preserved in the HD MTG.In a prior study, Sapp et al [47].reported a greater presence of anti-CR3/43 + immunoreactivity, which is an antibody that recognises HLA-DR reactive microglia within the human HD frontal cortex.Based on our previous stereological investigations, the frontal cortex is a region which traditionally undergoes more neuronal loss in HD than the temporal cortex [37].Therefore, it is possible the preservation of HLA-DR immunoreactivity in the HD MTG may be linked to the lesser degree of neuronal loss occurring in this region [37].Taken together, the similar HLA-DR immunoreactive profiles between normal and HD MTG samples suggests a milder reactive response of microglia within the HD temporal cortex compared to the severely degenerated basal ganglia and more vulnerable cortical regions.
With its recent discovery, TMEM119 and P2RY12 are two markers that are becoming increasingly popular in microglial research due to the widely accepted view that IBA-1 is not a microglia-exclusive marker as it is also known to immunolabel border-associated macrophages [1,26,36,61].The function of TMEM119 in homeostatic and reactive microglia is still unknown.However, expression of TMEM119 has been demonstrated to be reduced in the post-mortem AD human brain [26].Due to the novelty of TMEM119 as a J o u r n a l P r e -p r o o f Journal Pre-proof microglial marker, no studies have yet been conducted to investigate expression patterns in HD pathogenesis.As such, this TMA study served as the first to conduct a quantitative investigation of TMEM119 immunoreactivity in HD.TMA analysis of TMEM119 revealed an increase in both the number and somatic size of microglia in HD, reaffirming the IBA-1 data (Fig. 5E-F).Furthermore, similar to IBA-1, TMEM119 microgliosis was not associated with the degree of striatal degeneration.Thus, our findings implicate TMEM119, a 'microglial-specific' marker, in HD cortical pathogenesis, warranting further investigations into additional microglia markers.P2RY12, a purinergic receptor, is commonly considered a homeostatic microglial marker and is expressed by ramified microglia [16].It is suggested that in response to an insult, P2RY12 triggers the extension of microglial processes towards the site of injury.Subsequently, P2RY12 is downregulated, facilitating the retraction of microglial processes [16,20].It has been demonstrated that impairment of P2RY12 resulted in the suppression of the microglial chemotaxis response [29].Our data demonstrates widely preserved P2RY12 immunoreactivity in the HD MTG, with the presence of ramified P2RY12 microglia in both HD and normal cases.However, rod-shaped, hypertrophic, and dystrophic microglia immunoreactive for P2RY12 were identified in the HD cortex exclusively.Therefore, the preservation of P2RY12 in the HD cortex relative to normal, combined with observed P2RY12 microglial morphology differences in HD suggests that P2RY12-expressing microglia may not be migrating to a stimulus but responding to disease through presentation of more reactive microglial phenotypes, which aligns with our IBA1 and TMEM119 results.

Proliferation of HD microglia but not astrocytes in HD cerebral cortex
While our presented data reaffirms the general consensus that glial activation occurs within the HD human brain, [38,47] the contribution of reactive glia to disease pathophysiology has yet to be clearly elucidated.Both reactive microglia and astrocytes have the ability to undergo enhanced proliferation which has been investigated in the context of AD, Amyotrophic Lateral Sclerosis, and prion disease pathogenesis [4,15,17,32].In human brain tissue, a significantly higher expression of microglial proliferation regulatory proteins in the temporal cortex of prion disease and AD human brains has been reported [15].Furthermore, blocking microglial proliferation in prion disease mice resulted in a reduction of J o u r n a l P r e -p r o o f Journal Pre-proof neuronal death [15].In the AD human hippocampus, PCNA was observed to co-label with IBA-1 + microglia but not GFAP + astrocytes, therefore suggesting that microglia are selectively proliferating [32].Within the context of HD, PCNA has been previously reported to co-label with neurons and astrocytes within the subependymal layer of the HD human brain [9].However, investigations into microglial proliferation in HD have been limited, with no evidence of investigations being conducted in the HD cerebral cortex prior to our study.
Based on the observed increase in the number of IBA-1 + microglia and GFAP + astrocytes in our HD MTG TMA cores, we investigated whether the increase could be attributed to alterations in cellular protein expression, or to proliferation of the respective cell types.TMA quantification of Ki-67 and PCNA, revealed a significant subset of HD cases immunopositive for both proliferative markers, with no immunopositive cases within the normal cohort, which reinforces the presence of these indicators of proliferation in response to disease.Our studies revealed PCNA and Ki-67 both co-labelled with IBA-1 but not with GFAP in HD cores.Our data, therefore, suggests microglial proliferation may be a contributor to the reactive glial response within the HD cerebral cortex.On the other hand, this study demonstrates that the upregulation of astrocyte-specific proteins in the HD MTG occurs without astrocytic proliferation.Furthermore, HD cases with indicators of proliferation tended to have longer CAG repeats and an earlier age at death, suggesting that cellular proliferation is a significant event which may be influenced by the severity of the HD genetic mutation.
Microglial dysfunction is believed to contribute towards HD pathogenesis.However, therapeutics inhibiting or reducing microglial response or inflammation have yielded mixed results [5,23,46].In part, the lack of therapeutic efficacy highlights the complexity of inflammation in the human HD brain.Earlier studies have documented the presence of reactive microglia and provided compelling evidence for its involvement in HD pathogenesis that precedes severe neuronal loss [12,38,40,47].However, it is unknown whether the presence of reactive microglia in HD is solely beneficial or detrimental.Instead, it is likely that microglia subpopulations confer different consequences in disease [38,43].For instance, it is hypothesised that the proliferation of rod-shaped microglia is a crucial step in minimising damage and facilitating repair in response to neuronal degeneration [64,65,77].Indeed, it has been reported that rod-shaped microglia were closely associated with cortical neurons in post-mortem human HD brains [47].Coupled with our findings that revealed an association between the number of PCNA + nuclei and the number of SMI-32 + pyramidal neurons, it could be inferred that neuronal dysfunction could contribute to the microglia response by J o u r n a l P r e -p r o o f Journal Pre-proof inducing a phenotypical shift into the rod-shaped phenotype.Extending further, our study reveals that proliferation within the subset of HD cases was significantly associated with CAG repeat length and lifespan, suggesting that proliferation may be a significant event in HD pathophysiology that is influenced by the genetic mutation.Therefore, elucidating the contributions that microglia subpopulations may confer in HD pathogenesis may aid in the development of predictive markers for disease severity or targeted therapeutics.

Limitations and Future Directions
The present study focuses on the MTG, a region that is mildly implicated in HD pathogenesis [37].Our investigation into this region has provided valuable insight into cortical glial pathology.However, extending our current study to investigating other regions typically associated with HD symptomology would allow us to explore potential associations between an altered glial profile and the clinical phenotype of HD.Furthermore, by comparing the degree of gliosis across the various brain regions, we can aim to provide a multi-faceted understanding of gliosis in a clinicopathological context.
In this study, evidence of glial protein expression changes were identified in the HD MTG through chromogenic immunohistochemical techniques.Extending these findings, future studies could uncover the multi-faceted event of inflammation in the HD human brain through evaluations of a combination of molecular markers, such as functional channels, cytokine or interleukin receptors, and cytoskeletal proteins.Indeed, future studies exploring (1) the spatial relationships between gliotic events and neuronal dysfunction or HTT inclusions, (2) the immunohistochemical phenotype of not only astrocytic and microglial populations, but also perivascular macrophages and other immune cells, or (3) proliferative microglia would serve to provide a larger and holistic understanding of HD pathology.

Conclusion
In conclusion, the present study is the first to quantify astrocytic changes occurring in the human HD MTG and revealed an increase in GFAP expression, coverage, and cell number in HD.Our results also revealed a potential link between astrocytic alterations and the accumulation of B4-S830 + aggregates, but not pyramidal neuronal loss.We demonstrate an increase in the number of IBA-1 and TMEM119 cells accompanied by the presence of rodshaped, hypertrophic, and dystrophic morphologies in HD.Subsequent investigations found J o u r n a l P r e -p r o o f Journal Pre-proof indicators of cell proliferation to be present within HD microglia, but not astrocytes.Taken together, our data confirms that profiling the extent of gliosis within the temporal cortex in HD has enabled the identification of potential drivers of microgliosis and astrocytosis.Within the HD cerebral cortex, astrocytic alterations is driven by GFAP upregulation that may be influenced by the accumulation of mHTT aggregates, whereas microgliosis may be driven by phenotypic alterations and proliferation.Thus, we have identified novel HD-related molecular changes to microglia and astrocytes in the HD temporal cortex, which provides an opportunity for future studies to elucidate the contribution of other non-neuronal cells disease pathogenesis.
All post-mortem human brain tissue used in this study was obtained from the Neurological Foundation Human Brain Bank in the Centre for Brain Research at the University of J o u r n a l P r e -p r o o f Journal Pre-proof Auckland, New Zealand.Protocols in this study were approved by the University of Auckland Human Participation Ethics Committee (Ref.14/NTA/208), and all families provided informed consent.All cases were examined by a neuropathologist (CPT) and were classified based on neurological abnormalities, or lack thereof, in neurologically normal cases.Neuropathological assessments were carried out on the HD cases, which were graded according to Vonsattel striatal neuropathological grade [72].The number of CAG repeats in both alleles of the HTT gene was determined for each HD case by PCR amplification of DNA isolated from cerebellar brain tissue, as previously described [75].Age of clinical symptom onset was based on well-defined criteria outlined by Tippett et al [67].

Fig 2
Fig 2 Alterations in IBA-1 + immunoreactivity indicative of microglial reactivity in the human HD MTG.(A-E) Representative photomicrographs of IBA-1 immunoreactivity from a (A) normal and an (B) HD case with denoting the presence of ramified (black arrowhead) and rod (white arrowhead) IBA-1 + microglia.Higher power images from HD cases demonstrate the presence of (C) rod-shaped, (D) hypertrophic, and (E) dystrophic IBA-1 + microglia.(F-G) TMA analysis revealed no significant differences in integrated intensity (F,

Figure 3
Figure 3 Evidence of TMEM119 + and P2RY12 + reactive microglia in the human HD MTG.(A-E) Representative photomicrographs of TMEM119 immunoreactivity from a (A) normal and an (B) HD case with denoting the presence of ramified (black arrowhead) and rod (white arrowhead) TMEM119 + microglia.Higher power images from HD cases demonstrate the presence of (C) rod-shaped, (D) hypertrophic, and (E) dystrophic TMEM119 + microglia.(F-G)TMA analysis revealed no significant differences in integrated intensity(O, p=0.53)

Figure 5
Figure 5 Proliferation within a subset of HD cases correlates with neuronal loss, CAG repeat length, and age at death.(A) TMA analysis revealed a significant increase (p=0.046) in Ki-67 + immunoreactivity in HD cases (n=16) relative to the normal cohort (n=23).(B) Unpaired Mann-Whitney comparisons of TMA data from HD and normal cohorts revealed a significant increase (p=0.0046) in PCNA + immunoreactivity in HD cases (n=26) relative to normal cases (n=23).(C) Spearman correlation analysis of HD cases containing indicators of proliferation revealed a significant strong positive correlation (r=0.87,p=0.0091) between the number of PCNA + nuclei in TMA compared with larger tissue sections from the MTG.(D-F) Subsequent Spearman correlation analysis revealed a strong negative correlation between the number of PCNA + nuclei and number of SMI-32 + cells (D, r=-0.90, p=0.083), a significant strong positive correlation between number of PCNA + nuclei and increasing CAG repeat length (E, r=0.80; p=0.024), and a significant negative correlation between number of PCNA + nuclei and age at death (F, r=-0.88;p=0.0065).*p < 0.05, **p < 0.01.CAG = cytosine-adenine-guanine, GBM = Glioblastoma multiforme, HD = Huntington's disease, PCNA = proliferative cell nuclear antigen, SMI-32 = Neurofilament H nonphosphorylated.

Table 2
HD cases for the middle temporal gyrus tissue microarrays.Details include case number, sex, age at death (years), post-mortem delay (PMD; hours), symptom onset (years),