Endogenous mouse huntingtin is highly abundant in cranial nerve nuclei, co-aggregates to Abeta plaques and is induced in reactive astrocytes in a transgenic mouse model of Alzheimer’s disease

The expression of endogenous HTT in brains of 3-month-old wild type animals was analyzed in the inbred mouse lines C57BL/6 N (N = 3) and BALB/c (N = 3) and in the outbred line CD1 (N = 3) as well as in Wistar rats (N = 3) and hamster (N = 1). In order to reveal a potential influence of amyloid pathology on endogenous HTT expression in brain, human APP-transgenic Tg2576 mice and wild type littermates at postnatal ages of 15, 18, 24, 28 and 32 months were analyzed. Tg2576 mice are on C57BL/6xSJL background and express human APP695 carrying the Swedish mutation KM670/671NL driven by the hamster prion protein promoter [39]. Founder mice were provided by K. Hsiao-Ashe. Animals were housed at 12 h day/12 h night cycles with food and water ad libitum in cages that contained red plastic houses (Tecniplast, Hohenpeißenberg, Germany) and shredded paper flakes to allow nest building. All experimental protocols were approved by Landesdirektion Sachsen, license numbers T11/16 (rat), T14/16 (hamster) and T28/16 (mouse) and all methods were carried out in accordance with the relevant guidelines and regulations.

Primary neuronal and astroglial cultures were established from APP-transgenic Tg2576 mice and wild type littermates and grown under standard conditions [35]. Briefly, primary neuronal cell cultures were derived from fetal mouse brain at gestation day 16. Astrocyte-rich primary cell cultures were derived from brains of newborn mice. Cells were grown in 24 well plates on poly-L-lysine-coated coverslips and maintained in DMEM-based medium at 37 °C in a humidified atmosphere with 95% air/5% CO₂. The purity of primary cultures was verified by RT-PCR and by immunocytochemistry against neuronal and astrocytic markers (Additional file 1: Figure S2).

A real time quantitative polymerase chain reaction (RT-qPCR) with RNA from primary neurons and astrocytes derived from wild type and Tg2576 mouse brains, respectively, was performed to analyze cell type-specific HTT expression. RNA of cultivated wild type and Tg2576 neurons and astrocytes was isolated using the Trizol RNA isolation protocol [10]. Quality and concentration of RNA was analyzed with the photometer NanoDrop 2000 at wavelengths 260 nm and 280 nm. Based on the results obtained from primer testing (Additional file 1: Figure S3a), two primer pairs (#1 and #4) were chosen to analyze the HTT expression in cultivated primary astrocytes and neurons. Primer pair #1 is directed against a region spanning HTT exon 1 and 2, whereas primer pair #4 recognizes a region located more carboxy-terminally corresponding to the epitope of HTT antibody used in this study. A one-step RT-qPCR was performed to analyze expression of HTT. All working steps were performed on ice and Qiagen RT-PCR Kit was used. RNA of three wild type and three Tg2576 cell preparations were diluted in RNase free water to obtain a 20 ng/μl solution. All diluted RNA samples were denatured in the Rotor GeneTM 6000 Cycler at 70 °C for 5 min to dissolve possible secondary structures and were stored on ice until further usage. Cyclophilin A (CycA) was used as reference gene. Primers for CycA [81] and HTT (50 μM each) were diluted in aqua dest. to obtain a concentration of 10 μM. Of all samples 0.5 μl RNA were added to 9.5 μl of master mix and the RT-qPCR was performed with a Rotor GeneTM 6000 Cycler according to Additional file 1: Table S1. Specificity of PCR products was verified by electrophoresis and by analysis of the obtained C_T values. A detailed analysis of threshold cycle (CT) results obtained with RT-qPCR was performed with the ΔΔCT method for relative quantification. The linearity of results obtained was validated in a standard curve (Additional file 1: Figure S3b) and PCR control experiments without RT and after DNAse treatment are shown in Additional file 1: Figure S3c.

To determine the cell type-specific expression of endogenous HTT in primary neurons and astrocytes double immunofluorescent labellings of HTT and cell type-specific markers were performed as described for immunohistochemistry on brain sections. Finally, coverslips were air-dried, embedded in entellan/toluene on microscopic slides and stored at 4 °C in the dark. Additionally, double labelling with neuron- and astrocyte-specific antibodies was performed on neuronal and astroglial primary cultures, to examine the identity and purity of the respective cell cultures.

First, the expression of endogenous HTT in C57BL/6 N mouse brain was analyzed by immunohistochemistry. HTT immunoreactivity was present at low levels throughout the brain but was highly enriched in a number of morphologically clearly defined neuronal nuclei. The identities of these nuclei brought out by HTT labelling were determined according to their morphology as well as to their rostro-caudal and dorso-ventral position using a mouse brain atlas [22].

At a high concentration of the primary antibody, a wide-spread HTT immunoreactivity was detected throughout the mouse brain. When diluting the HTT primary antibody from 1:500 to 1:4000, only neuronal populations with highly abundant HTT expression were detected (Additional file 1: Figure S1a). Applying a high HTT antibody dilution to horizontal mouse brain sections, a significant number of neuronal populations in caudal brain regions were still strongly HTT immunoreactive while ubiquitous neuronal staining was abolished (Fig. 1).

The screening of every 6th coronal section identified HTT immunoreactive neurons at different coronal levels. These populations include a distinct subpopulation of striatal neurons, neurons in the basal forebrain including medial septum (MS), vertical and horizontal diagonal band (VDB/HDB), ventral pallidum (VP), olfactory tubercle (Tu), nucleus basalis magnocellularis (Nbm) as well as amygdala (Amy) (Fig. 2). At more caudal coronal brain levels, significant HTT immunoreactivity was detected in Edinger-Westphal nucleus (EWN), pedunculopontine tegmental nucleus (PPT), laterodorsal tegmental nucleus (LDT), motor trigeminal nucleus (Mo5), nucleus abducens (6), nucleus facialis (7) and the perifacial zone (P7), the parvicellular medial vestibular nucleus (MVePC), nucleus ambiguus (Amb), nucleus vagus (10) and the hypoglossal nucleus (12) (Fig. 2).

Interestingly, EP867Y detected HTT immunoreactivity is strongly enriched in cranial nerve nuclei and also significantly present in basal forebrain and in a subset of striatal neurons. In order to validate the identity of the HTT-positive neuronal nuclei and subpopulations, respectively, double labellings with ChAT, Ucn-1, DARPP-32, a marker for medium spiny neurons, and TH were performed. As a result, almost all heavily stained HTT-positive nuclei were found to be cholinergic which was evident by ChAT co-expression. This demonstrates HTT expression by neurons of the respective cranial nerve nuclei and by cholinergic neurons of the basal forebrain (MS, VDB/HDB and Nbm) as well as cholinergic interneurons of the striatum (Fig. 2). In contrast, HTT immunoreactivity was absent from DARPP-32-positive striatal medium spiny neurons and from TH-positive locus coeruleus neurons neighboring the LDT (not shown). The only non-cholinergic neuronal population showing high HTT immunoreactivity in mouse brain was the Ucn-1-positive neuronal subregion of EWN (Fig. 2). For a general overview, the HTT immunoreactive neuronal populations along the rostro-caudal extension of the brain and their attribution to cranial nerve nuclei are summarized in Table 2.

Since this is the first detailed analysis of endogenous HTT expression in mouse brain, we considered it important to validate our observations made in the C57BL/6 N strain in brains of two other mouse lines (CD1 and BALB/c). In the brains of these mouse lines very similar HTT expression patterns were observed (not shown). Also in Wistar rat (Fig. 3) and in hamster (Additional file 1: Figure S4) as different rodent species there was an enrichment of HTT immunoreactivity in cranial nerve nuclei and in the cholinergic basal forebrain and caudate putamen structures. A distinct difference to the HTT immunoreactivity in mouse brain, however, was the labelling of neocortical neurons, in particular in layer III pyramidal neurons, CA1 pyramidal neurons and dentate gyrus granule neurons (Fig. 3; Additional file 1: Figure S4), which was not depicted in mouse brain using the same HTT antibody dilution. However, the cortical and hippocampal neurons immunoreactive for HTT in rat brain did not display a distinct cytoplasmic labelling typical for basal forebrain and cranial nerve nuclei, but rather a nuclear HTT localization. The presence of HTT in neuronal nuclei is exemplarily shown for dentate gyrus granule cells (Fig. 3). However, most neuronal populations in basal forebrain and in cranial nerve nuclei displayed cytoplasmic/neuritic labelling (Fig. 3).

At sites of endogenous HTT expression, the formation of cytotoxic HTT aggregates is likely when HTT variants with expanded CAG repeats are expressed. However, in different clinical conditions associated with pathogenic protein aggregation in brain, the formation of protein co-aggregates generated in cross-seeding events is reported.

In order to investigate potential protein cross seeding of endogenous mouse HTT by Abeta aggregates, we analyzed the HTT immunoreactivity in brains of 18-month-old APP-transgenic Tg2576 mice with amyloid pathology. Basically, the general HTT staining pattern in Tg2576 mouse brain reflected the labelling detected in wild type mice (not shown). However, neocortical and hippocampal HTT immunoreactivity in aged Tg2576 mice appeared to be additionally associated with amyloid plaque pathology. Therefore, a series of double labellings of HTT with (i) ThS, (ii) the Abeta antibody 4G8 and (iii) the astrocyte marker GFAP were performed and analyzed by confocal laser scanning microscopy (Fig. 4a). These double immunofluorescent stainings demonstrated a punctate HTT immunoreactivity in the near periphery of fibrillary (ThS-positive) and Abeta-immunoreactive extracellular plaques. This labelling pattern was consistently detected irrespective of the brain region (hippocampus and neocortex) and of plaque size. In addition, marked HTT immunoreactivity was detected in amyloid plaque-associated reactive astrocytes (Fig. 4a) but not in microglial cells (not shown). In brains of wild type mice neither punctate nor astrocytic HTT immunoreactivity was observed (Fig. 4a).

In order to reveal whether HTT association with amyloid plaques directly reflects its stimulated aggregation by Abeta peptides, HTT aggregation assays in the absence or presence of 1 μM Abeta (1–42) were performed. These assays revealed stimulated HTT aggregation kinetics in the presence of Abeta (Fig. 4b). While the HTT aggregation rate increased significantly in the presence of Abeta peptides, no shortening of the lag phase was observed (Fig. 4b). This indicates that Abeta peptides increase the efficiency of the polymerization event while they do not accelerate the nucleation process. The co-aggregation of HTT with Abeta in fibrils was also demonstrated by transmission electron microscopy (Fig. 4c).

Next, we sought to investigate whether the accumulation of endogenous HTT near Abeta plaques and its induction in reactive astrocytes are age-dependent and increase along with plaque load. Therefore, coronal brain sections of APP-transgenic mice at the postnatal age of 15, 18, 20, 24 and 32 months, respectively, were used for immunocytochemical triple labelling of HTT, fibrillary Abeta plaques (ThS) and reactive astrocytes (GFAP). We observed an age-dependent increase in hippocampal HTT immunoreactivity, which was paralleled by increases in plaque load and GFAP immunoreactivity (Fig. 5). In negative control experiments without primary antibodies, no labelling for HTT and GFAP was generated (not shown). Additionally, in wild type mice no ThS-positive deposits were detected and HTT and GFAP labellings were comparable to young Tg2576 mice (Fig. 5).

The presence of astrocytic HTT immunoreactivity in hippocampus of Tg2576 mice is particularly interesting and may result from astrocytic HTT expression and/or uptake of HTT released from neurons. In order to investigate the capability of astrocytes to express HTT, Htt mRNA and HTT protein expression were analyzed by RT-qPCR and by immunocytochemistry in primary mouse neuronal and astrocytic cultures. The purity of neuronal and astrocytic cultures was demonstrated by the detection of the neuronal marker NeuN and the astrocytic marker GFAP by RT-PCR and by immunocytochemistry (Additional file 1: Figure S2). These data clearly show the absence of neurons in astrocytic cultures and vice versa.

The analysis of RT-qPCR products by gel electrophoresis demonstrated the expected product size and similar HTT expression levels in primary neurons and astrocytes of both, wild type and Tg2576 origin (Fig. 6a). C_T mean values for each cell type, genotype and applied primer pair were calculated and are shown in Table 3 with the corresponding ΔC_T value. The calculated ΔC_T values were further taken to perform relative quantification by comparing the values among each other, obtaining ΔΔC_T. Subsequently, based on ΔΔC_T values, the factor of difference between the two investigated samples was calculated (Table 4). The relative quantification revealed that both cell types investigated express Htt mRNA at comparable levels with small fluctuations. In conclusion, by means of Htt RT-qPCR we were able to validate that primary astrocytes of wild type and Tg2576 background do express Htt mRNA in addition to primary neurons.

The data on Htt mRNA expression are supported by immunocytochemical labelling of HTT in mouse primary neurons and astrocytes demonstrating neuronal and astrocytic HTT protein expression (Fig. 6b). Interestingly, primary astrocytes from wild type mice expressed Htt mRNA and HTT protein whereas in brain of adult mice astrocytic HTT immunoreactivity was only observed in transgenic mice and associated with amyloid pathology (Fig. 4).

In HD, the number of CAG repeats of the HTT gene determines the age of disease onset in a fully dominant fashion [47]. When assessing the neuronal populations affected by pathogenic HTT protein aggregation, it is very likely that neurons with abundant HTT expression are more affected than neurons expressing low levels of HTT or no HTT at all. Thus, the defined cellular HTT expression levels might be decisive for the neuron type-specific pathology. From that point of view it is surprising that no detailed analyses on the neuron type-specific endogenous HTT expression patterns in brains of frequently used experimental animal species exist. There is only an early report showing HTT immunoreactivity in the cytoplasm of neurons in rat neocortex and in human cerebellar Purkinje and neocortical neurons as well as neurons of caudate nucleus [17].

Therefore, we here comprehensively analyzed the brain region- and neuron type-specific expression of endogenous HTT in brains of three mouse lines, Wistar rat and hamster using a monoclonal rabbit antibody (EP867Y) that binds to the HTT mid-region. The specificity of this antibody is validated by Western blot analysis of HTT knock-out cells analyzed along with wild type cells (Product information sheet: https://​www.​abcam.​com/​huntingtin-antibody-ep867y-ab45169.​html). Furthermore, in the transgenic BACHD HTT mouse model we demonstrate much stronger EP867Y labelling than in wild type mouse brain tissue (Additional file 1: Figure S1b). In addition, the specificity of the EP867Y antibody was demonstrated in a number of recent publications [32, 56, 75].

Interestingly, in the striatum of all mouse lines tested, as well as in rat and hamster, strong HTT immunoreactivity stood out in cholinergic interneurons that are spared in HD [20]. In contrast, in DARPP-32-positive striatal medium-spiny neurons, which are known to be affected in HD [29], significant HTT-labelling with the high threshold selected here was missing. However, also in human brain large striatal neurons that are spared in HD express HTT [64] and deficits in cholinergic markers such as acetylcholinesterase and vesicular acetylcholine transporter without accompanying cell loss have been reported in HD affected subjects [1, 73].

We detected ubiquitous low level HTT immunoreactivity throughout the brain but a highly selective enrichment of HTT in cholinergic neurons of the basal forebrain and amygdala, and in particular in cholinergic neurons of cranial nerve nuclei. In addition, prominent HTT expression was revealed in Ucn-1-positive EWN neurons and represented the only non-cholinergic neuronal population with abundant HTT expression identified in the present study. These observations indicate that not only neurons in striatum, but also defined neuronal populations in midbrain and brain stem may be affected by the pathogenic mechanisms resulting from polyQ extension of the HTT protein in HD. Indeed, in HD patients HTT protein inclusions were demonstrated in cranial nerve nuclei III (oculomotor nerve), IV (trochlear nerve), V (trigeminal nerve), VI (abducens nerve), VII (facial nerve), VIII (vestibulocochlear nerve), IX (glossopharyngeal nerve), X (vagal nerve) and XII (hypoglossal nerve) [63]. This is in good accordance with our observation of prominent HTT expression in mouse, rat and hamster brain and would explain some clinical symptoms, (i.e. oculomotor and vestibular deficits) that cannot solely be attributed to degeneration of striatal neurons. Interestingly, HTT-rich vestibular projection neurons predominantly terminate on HTT-negative medium spiny neurons in dorsolateral striatum [78], where significant neurodegeneration in HD occurs. Thus, in addition to local HTT expression, an indirect pathogenic HTT effect in striatum via innervating fibers should be considered based on the evidence presented here.

We would like to note that this pattern of HTT expression was not recapitulated by other HTT antibodies such as 2B7 and 2166. One possible explanation for this is HTT cleavage by different proteases to generate numerous fragments [44, 57] and the adoption of different conformational states of the HTT protein [65, 69]. The epitope of the EP867Y antibody is within amino acids 550 and 650 of HTT and corresponds to residues specific to the apopain (caspase-3) cleavage site [27, 87]. Thus, this antibody appears to be unique and will allow monitoring distinct HTT expression related to cholinergic nuclei.

From the pattern of endogenous HTT expression in mouse, rat and hamster brain, one would predict that under conditions of pathogenic HTT expression the corresponding neuronal functions such as the reward system (VP, CPu, Tu, PPT, Amy), memory processing and decision making (Amy), arousal and attention (PPT), generation of REM sleep (LDT), pupil constriction and eye movement (EWN, oculomotor nerve nucleus, nucleus abducens), parasympathetic vagal functions (dorsal motor nucleus vagus) and general sensory and motor functions (facial nucleus, Pr5, Mo5, PPT, MVePC, ambiguus nucleus, hypoglossal nucleus) are affected. Indeed, the clinical features of HD such as progressive motor dysfunction, cognitive decline and psychiatric disturbance [86] could be – at least partially – related to dysfunction or death of these neuronal populations. Typical motor dysfunctions in HD include chorea, dystonia, progressive bradykinesia, rigidity and incoordination. In addition, many patients have substantial cognitive or behavioral disturbances before onset of diagnostic motor signs [54]. Interestingly, deep brain stimulation of the PPT might ameliorate gait and postural difficulties in PD subjects [3, 79]. In addition, a significant cholinergic innervation of the striatum and nucleus accumbens arises from brainstem LDT and PPT nuclei [14]. Thus, when affected during HD pathogenesis, dysfunction and/or degeneration of these neurons may contribute to striatal cholinergic deficits. Indeed, targeting the cholinergic system has been proposed as novel HD therapy [18] and may compensate deficits in numerous cholinergic nerve nuclei with high HTT expression levels. This may also apply to the hypoglossal nerve nucleus which provides innervation of extrinsic and intrinsic muscles of the tongue and, therefore, accounts for the prominent dysphagia and disturbance of speech in HD patients [48, 53, 62].

Neurons with highly abundant HTT immunoreactivity displayed cytoplasmic localization of HTT. This is consistent with biochemical and electron microscopic analyses which demonstrated the presence of HTT in neuronal cytoplasm and an association with vesicle membrane proteins [17]. In the cytoplasm, full length HTT seems to be particularly involved in intracellular transport processes and vesicle trafficking, as it interacts with microtubules and clathrin-coated vesicles [24, 36, 82]. In neurons with lower endogenous HTT expression levels, we also observed faint immunoreactivity in the nucleus. This is in line with reported co-immunoprecipitates with the carboxy-terminal binding protein, known to be a transcriptional co-repressor in the nucleus [43]. An amino-terminal HTT fragment, however, has also been shown to interact with a variety of proteins important for nuclear function, like p53 [77] and nuclear receptor co-repressor protein [5].

In several transgenic mouse models of HD, mutant HTT is randomly inserted within the genome and its expression is typically driven by neuron-specific promoters other than the endogenous HTT promoter. Most of these mice develop ubiquitinated nuclear HTT inclusions but do not exhibit the full neuropathological phenotype of human HD brains [83]. In particular, brain regions affected by neuronal loss differ from that in HD and the effects on oligodendrocytes and astrocytes in the human brain are not recapitulated in transgenic mice [83]. On the other hand, in knock in mouse models of HD, transgene expression is driven by the endogenous mouse HTT promoter and should lead to pathology in brain regions identified by us as highly HTT expressing. Interestingly, the manifestation of HD-typical pathology including brain atrophy, striatal neuronal loss, severe motor phenotype, weight loss and premature death depends on the expression of N-terminal HTT fragments as present in the R6/2 model expressing exon 1 of the HTT protein under the control of human HTT promoter [8, 49]. Unfortunately, the more caudal brain regions containing cholinergic cranial nerve nuclei identified by us as strongly immunoreactive for endogenous HTT were not included in the analyses. Thus, we strongly recommend the analyses of these structures and would predict the detection of pathological alterations like HTT protein aggregates and astrogliosis in these clearly defined nuclei.

An important aspect of the present work was the analyses of a potential HTT protein cross seeding by Abeta aggregates in brains of transgenic mice with amyloid pathology. As the amyloid pathology in the Tg2576 mouse model used is restricted to neocortex and hippocampus, we focused on HTT aggregation in proximity to amyloid plaques in these brain regions. We observed an extracellular halo of HTT immunoreactivity around amyloid plaques labelled with the Abeta-specific antibody 4G8 or by the fibril intercalating dye ThS, respectively. Such protein cross seeding events are known to occur for proteins affected by a particular disease, such as Abeta and tau proteins in AD [28], but also for proteins from different clinical entities such as Abeta and α-synuclein proteins affected in AD and PD [23, 46, 52, 55, 80], respectively. We here demonstrate for the first time that also the endogenous HTT proteins can cross-seed with Abeta aggregates. This process has a clear age-related component and Abeta plaque-associated HTT immunoreactivity in hippocampus increases with plaque load. Since Abeta plaques also contain non-Abeta peptides that could be responsible for HTT recruitment, we directly tested the aggregation of HTT by Abeta (1-42) in ThT aggregation assays. We demonstrate that Abeta peptides increase the efficiency of the polymerization event but have no effect on the nucleation process. The nucleation of polyQ HTT was reported to underlie a different mechanism than nucleation of other amyloid peptides [38]. PolyQ fibrils of HTT do not reveal a typical β-sheet structure but rather assemble into α-helical structures and oligomers which interfere with polyQ sequences and facilitate the formation of amyloid nuclei [58]. A similar effect could occur in the presence of Abeta peptides generating conditions that favor β-sheet structure and thus result in higher aggregation rates [74]. The biological significance and pathogenic potential of these co-aggregates is not yet clear but they might represent novel therapeutic targets. It has been recently shown in an AD mouse model that targeting one pathogenic protein by passive immunization also attenuates the deposition of other pathogenic protein aggregates, most likely by a mechanism involving activation of the complement system and microglia [13].

Currently, no data are available on cross seeding of HTT to Abeta plaques in AD brains. However, there are several reports analyzing Abeta pathology in HD brains [15, 40, 76]. Based on morphological amyloid and tau staging in autopsy cases of HD, a rare co-existence of HD and AD was reported, although initial neuropathological stages of AD were found to be present early in HD patients [40]. On the other hand, in elderly HD subjects with dementia, a high proportion of subjects displayed tau and amyloid pathology, suggesting that co-occurrence of AD may contribute to cognitive decline in elderly HD patients [15]. Moreover, a co-occurrence of mixed proteinopathies involving oligomerization and aggregation of Tau, α-synuclein and TDP-43 in late stage HD was demonstrated [76], indicating common mechanisms of pathological protein aggregation in different neurodegenerative diseases.

In brain sections of Tg2576 mice used for the analyses of HTT protein cross seeding to amyloid plaques we surprisingly observed HTT immunoreactivity in reactive astrocytes in proximity to Abeta plaques. This robust astrocytic HTT immunoreactivity raises the question whether HTT produced by and released from neurons is taken up by astrocytes or whether it is expressed by these glial cells. In order to address this issue, primary neuronal and glial cell cultures from APP-transgenic Tg2576 mice were established and analyzed for Htt mRNA and protein expression. Both, primary neurons and astrocytes were shown to express Htt mRNA and protein. This is in agreement with immunohistochemical labellings of brain slices and supportive of de novo synthesis of HTT by astrocytes, rather than uptake of neuron-derived HTT. However, in contrast to the labelling of mouse brain sections, also astrocytes derived from primary cultures of wild type mice expressed HTT indicating that astrocytic HTT expression can generally be induced under conditions of astrocytic activation and independent of Abeta pathology. HTT immunoreactive astrocytes were also detected in the caudate and putamen of HD subjects [72] and in a transgenic HTT mouse model [71]. The intentional and specific mutant HTT expression by astrocytes was demonstrated to cause age-dependent neurological symptoms including body weight loss, motor function deficits, increased susceptibility to glutamate-induced seizures and early death [6, 7]. Moreover, the mutant HTT expression by cultured primary astrocytes was shown to induce cell death of co-cultured wild type neurons [71]. On the other hand, reduction of astrocytic mutant HTT expression slows disease progression in the BACHD conditional HD mouse model and improves motor and psychiatric-like phenotypes [89]. Thus, there is solid evidence to conclude that astrocytic HTT expression contributes to neurodegeneration in HD.