HD Mice Research Articles

Msh3 and Pms1 Set Neuronal CAG-repeat Migration Rate to Drive Selective Striatal and Cortical Pathogenesis in HD Mice.

Msh3 and Pms1 are genetic drivers of sequential striatal and cortical pathogenesis in Q140 mice by mediating selective CAG-repeat migration in HD vulnerable neurons.

Longitudinal alterations in brain perfusion and vascular reactivity in the zQ175DN mouse model of Huntington’s disease

BackgroundHuntington’s disease (HD) is marked by a CAG-repeat expansion in the huntingtin gene that causes neuronal dysfunction and loss, affecting mainly the striatum and the cortex. Alterations in the neurovascular coupling system have been shown to lead to dysregulated energy supply to brain regions in several neurological diseases, including HD, which could potentially trigger the process of neurodegeneration. In particular, it has been observed in cross-sectional human HD studies that vascular alterations are associated to impaired cerebral blood flow (CBF). To assess whether whole-brain changes in CBF are present and follow a pattern of progression, we investigated both resting-state brain perfusion and vascular reactivity longitudinally in the zQ175DN mouse model of HD.MethodsUsing pseudo-continuous arterial spin labelling (pCASL) MRI in the zQ175DN model of HD and age-matched wild-type (WT) mice, we assessed whole-brain, resting-state perfusion at 3, 6 and 9 and 13 months of age, and assessed hypercapnia-induced cerebrovascular reactivity (CVR), at 4.5, 6, 9 and 15 months of age.ResultsWe found increased perfusion in cortical regions of zQ175DN HET mice at 3 months of age, and a reduction of this anomaly at 6 and 9 months, ages at which behavioural deficits have been reported. On the other hand, under hypercapnia, CBF was reduced in zQ175DN HET mice as compared to the WT: for multiple brain regions at 6 months of age, for only somatosensory and retrosplenial cortices at 9 months of age, and brain-wide by 15 months. CVR impairments in cortical regions, the thalamus and globus pallidus were observed in zQ175DN HET mice at 9 months, with whole brain reactivity diminished at 15 months of age. Interestingly, blood vessel density was increased in the motor cortex at 3 months, while average vessel length was reduced in the lateral portion of the caudate putamen at 6 months of age.ConclusionOur findings reveal early cortical resting-state hyperperfusion and impaired CVR at ages that present motor anomalies in this HD model, suggesting that further characterization of brain perfusion alterations in animal models is warranted as a potential therapeutic target in HD.

Unprecedented effect of vitamin D3 on T-cell receptor beta subunit and alpha7 nicotinic acetylcholine receptor expression in a 3-nitropropionic acid induced mouse model of Huntington’s disease

Introduction3-NP induction in rodent models has been shown to induce selective neurodegeneration in the striatum followed by the cortex (Brouillet, 2014). However, it remains unclear whether, under such a neurotoxic condition, characterized by neuroinflammation and oxidative stress, the gene expression of the immune resident protein, T-cell receptor beta subunit (TCR-β), α7 nicotinic acetylcholine receptor (α7 nAChRs), the nuclear factor kappa B (NF-κB), inflammatory cytokines (TNF-α and IL-6), and antioxidants (Cat and GpX4) get modulated on Vitamin D3 (VD) supplementation in the central nervous system. MethodsIn the present study, real-time polymerase chain reaction (RT-PCR) was performed to study the expression of respective genes. Male C57BL/6 mice (8–12 weeks) were divided into four groups namely, Group I: Control (saline); Group II: 3-NP induction via i.p (HD); Group III: Vitamin D3 (VD) and Group IV: (HD + VD) (Manjari et al., 2022). ResultsOn administration of 500IU/kg/day of VD, HD mice showed a significant reduction in the gene expression of the immune receptor, TCR-β subunit, nuclear factor kappa B (NF-κB), inflammatory cytokines, and key antioxidants, followed by a decrease in the acetylcholinesterase activity. ConclusionA novel neuroprotective effect of VD in HD is demonstrated by combating the immune receptor, TCR-β gene expression, antioxidant markers, and inflammatory cytokines. In addition, HD mice on VD administration for 0–15 days showed an enhancement in cholinergic signaling with restoration in α7 nAChRs mRNA and protein expression in the striatum and cortex.

Elevated levels of mosaic aneuploidy in brain cells and fibroblast cell lines from human Huntington’s disease donors and in brain cells from Huntington’s disease mouse models

AbstractBackgroundHuntington’s disease (HD) is a progressive neurodegenerative disorder that presents with motor and cognitive deficits and psychiatric disturbances. HD is associated with autosomal dominant inheritance of a CAG trinucleotide repeat expansion mutation in the huntingtin (HTT) gene. The huntingtin protein localizes to spindle poles and is required for proper mitotic spindle orientation, chromosome segregation, and cell cycle regulation. The mature mutant huntingtin protein has an expanded polyglutamine region (>35 repeats) near its N terminus, which leads to neuronal dysfunction and death. Mouse models of HD exhibit defects in microtubule function and cell cycle regulation. Studies from our laboratory and others have shown elevated levels of aneuploidy in many neurodegenerative disorders characterized by cognitive deficits, including Alzheimer’s disease and frontotemporal lobar degeneration. Here, we investigated whether chromosome segregation defects that lead to mosaic aneuploidy and consequent apoptosis may also contribute to neuronal loss in HD.MethodsSingle‐cell suspensions from cortex and cerebellum from human HD donors were analyzed using FISH for chromosome 21, NeuN immunostaining, and/or TUNEL staining. Fibroblast cell lines from human HD donors were also analyzed using FISH. Single‐cell suspensions from cortex and cerebellum from HD mouse models were analyzed using FISH for chromosomes 5 and 16. Cell cycle arrest, mitotic spindle defects, ploidy, and apoptosis profiles in the HD fibroblast cell lines are being analyzed by flow cytometry and immunofluorescence microscopy.ResultsIncreased levels of mosaic aneuploidy and apoptosis were observed in brain cells and in fibroblast cell lines from HD donors compared to age‐matched healthy control donors. Increased levels of mosaic aneuploidy were also observed in brain cells from HD mice compared to age‐matched wild‐type control mice. Results of ongoing studies of cell cycle and mitotic defects will also be presented.ConclusionsTaken together with the results of our previous studies, these data provide evidence that chromosome segregation defects that lead to genomic instability may serve as a shared mechanism underlying many neurodegenerative disorders. Our findings highlight the need to further investigate the biological mechanism(s) that contribute to HD pathology and set the stage for novel therapeutic approaches to HD that may also be applied to other neurodegenerative disorders.

Striatal spatial heterogeneity, clustering, and white matter association of GFAP+ astrocytes in a mouse model of Huntington's disease.

Huntington's disease (HD) is a neurodegenerative disease that primarily affects the striatum, a brain region that controls movement and some forms of cognition. Neuronal dysfunction and loss in HD is accompanied by increased astrocyte density and astrocyte pathology. Astrocytes are a heterogeneous population classified into multiple subtypes depending on the expression of different gene markers. Studying whether mutant Huntingtin (HTT) alters specific subtypes of astrocytes is necessary to understand their relative contribution to HD. Here, we studied whether astrocytes expressing two different markers; glial fibrillary acidic protein (GFAP), associated with astrocyte activation, and S100 calcium-binding protein B (S100B), a marker of matured astrocytes and inflammation, were differentially altered in HD. First, we found three distinct populations in the striatum of WT and symptomatic zQ175 mice: GFAP+, S100B+, and dual GFAP+S100B+. The number of GFAP+ and S100B+ astrocytes throughout the striatum was increased in HD mice compared to WT, coinciding with an increase in HTT aggregation. Overlap between GFAP and S100B staining was expected, but dual GFAP+S100B+ astrocytes only accounted for less than 10% of all tested astrocytes and the number of GFAP+S100B+ astrocytes did not differ between WT and HD, suggesting that GFAP+ astrocytes and S100B+ astrocytes are distinct types of astrocytes. Interestingly, a spatial characterization of these astrocyte subtypes in HD mice showed that while S100B+ were homogeneously distributed throughout the striatum, GFAP+ preferentially accumulated in "patches" in the dorsomedial (dm) striatum, a region associated with goal-directed behaviors. In addition, GFAP+ astrocytes in the dm striatum of zQ175 mice showed increased clustering and association with white matter fascicles and were preferentially located in areas with low HTT aggregate load. In summary, we showed that GFAP+ and S100B+ astrocyte subtypes are distinctly affected in HD and exist in distinct spatial arrangements that may offer new insights to the function of these specific astrocytes subtypes and their potential implications in HD pathology.

Treatment with the Glycosphingolipid Modulator THI Rescues Myelin Integrity in the Striatum of R6/2 HD Mice

Huntington's disease is one of the most common dominantly inherited neurodegenerative disorders caused by an expansion of a polyglutamine (polyQ) stretch in the N-terminal region of huntingtin (Htt). Among all the molecular mechanisms, affected by the mutation, emerging evidence proposes glycosphingolipid dysfunction as one of the major determinants. High levels of sphingolipids have been found to localize in the myelin sheaths of oligodendrocytes, where they play an important role in myelination stability and functions. In this study, we investigated any potential existing link between sphingolipid modulation and myelin structure by performing both ultrastructural and biochemical analyses. Our findings demonstrated that the treatment with the glycosphingolipid modulator THI preserved myelin thickness and the overall structure and reduced both area and diameter of pathologically giant axons in the striatum of HD mice. These ultrastructural findings were associated with restoration of different myelin marker protein, such as myelin-associated glycoprotein (MAG), myelin basic protein (MBP) and 2', 3' Cyclic Nucleotide 3'-Phosphodiesterase (CNP). Interestingly, the compound modulated the expression of glycosphingolipid biosynthetic enzymes and increased levels of GM1, whose elevation has been extensively reported to be associated with reduced toxicity of mutant Htt in different HD pre-clinical models. Our study further supports the evidence that the metabolism of glycosphingolipids may represent an effective therapeutic target for the disease.

S-07-6: NEUTROPHIL EXTRACELLULAR TRAPS AS MEDIATORS OF ENDOTHELIAL DAMAGE IN HYPERTENSION AND DIABETES

Objective: Endothelial dysfunction (ED) plays a key role in the pathogenesis of hypertension and diabetes, yet its molecular determinants are poorly understood. Concomitant hypertension and diabetes can synergistically increase damage to the vasculature, with increasing evidence suggesting that resulting immune dysregulation may contribute to endothelial injury. Neutrophil extracellular traps (NETs), networks of chromatin and protein-containing extracellular fibers released to fight infection, are generated from neutrophils following activation of the citrullinating enzyme peptidylarginine deiminase 4 (PAD4). PAD4 has been shown to be upregulated in atherosclerosis, vasculitis, and various inflammatory autoimmune diseases, leading to NET-induced endothelial injury. Whether NETs could contribute to hypertension- and diabetes-induced ED has not been established. The objective of the present study is to investigate the impact of hypertension and diabetes on PAD4 activity and the contribution of NETs to resulting vascular injury. Design and Methods: Promyelocytic HL-60 cells were differentiated to neutrophil-like cells and treated with angiotensin II (10−7 M) and/or high glucose (25 mM) for 24 hours, with or without subsequent NET-inducing PMA stimulation (500 nM). NETs were isolated from the culture medium by differential centrifugation and assessed by Western blot analysis of citrullinated histone H3 (citH3, a primary NET component). Isolated NETs were used to treat Human Umbilical Vein Endothelial Cells (HUVECs) at 0.5–500 ng/ml for 24 hours, after which cell viability (XTT cell assay) and proliferation (BrdU assay) were measured. We also assessed NETosis in mice expressing human renin in the liver (TTRhRen) intercrossed with OVE26 diabetic mice [i.e. hypertensive-diabetic, HD mice], compared with wild-type (WT) FVBN/J mice. We examined kidney levels of citH3, neutrophil elastase (NE), and PAD4 expression by immunohistochemistry. Results: Treatment with high glucose, but not angiotensin II, increased citH3 levels in neutrophil-like cells. NETs isolated from PMA-stimulated cells decreased HUVEC viability by ∼2-fold at 5 ng/ml (P = < 0.0001), and dose dependently decreased proliferation from 0.5–500 ng/ml, with a ∼80% decrease at 500 ng/ml. In HD mice, we observed a significant increase in kidney levels of citH3 (P < 0.0001), NE (P < 0.0001), and PAD4 (P < 0.0001), which was associated with the presence of albuminuria and increased blood pressure levels. Conclusion: These results suggest that NETs are upregulated in hypertension and diabetes and may contribute to endothelial damage and microvascular injury.

Treatment of GM2 Gangliosidosis in Adult Sandhoff Mice Using an Intravenous Self-Complementary Hexosaminidase Vector.

GM2 gangliosidosis is a neurodegenerative, lysosomal storage disease caused by the deficiency of β-hexosaminidase A enzyme (Hex A), an α/β-subunit heterodimer. A novel variant of the human hexosaminidase α-subunit, coded by HEX M, has previously been shown to form a stable homodimer, Hex M, that hydrolyzes GM2 gangliosides (GM2) in vivo. The current study assessed the efficacy of intravenous (IV) delivery of a self-complementary adeno-associated virus serotype 9 (scAAV9) vector incorporating the HEXM transgene, scAAV9/HEXM, including the outcomes based on the dosages provided to the Sandhoff (SD) mice. Six-week-old SD mice were injected with either 2.5E+12 vector genomes (low dose, LD) or 1.0E+13 vg (high dose, HD). We hypothesized that when examining the dosage comparison for scAAV9/HEXM in adult SD mice, the HD group would have more beneficial outcomes than the LD cohort. Assessments included survival, behavioral outcomes, vector biodistribution, and enzyme activity within the central nervous system. Toxicity was observed in the HD cohort, with 8 of 14 mice dying within one month of the injection. As compared to untreated SD mice, which have typical survival of 16 weeks, the LD cohort and the remaining HD mice had a significant survival benefit with an average/median survival of 40.6/34.5 and 55.9/56.7 weeks, respectively. Significant behavioral, biochemical and molecular benefits were also observed. The second aim of the study was to investigate the effects of IV mannitol infusions on the biodistribution of the LD scAAV9/HEXM vector and the survival of the SD mice. Increases in both the biodistribution of the vector as well as the survival benefit (average/median of 41.6/49.3 weeks) were observed. These results demonstrate the potential benefit and critical limitations of the treatment of GM2 gangliosidosis using IV delivered AAV vectors.

DNAJB6b-enriched small extracellular vesicles decrease polyglutamine aggregation in in vitro and in vivo models of Huntington disease

SummaryHuntington disease (HD) is a devastating neurodegenerative disorder characterized by aggregation of huntingtin (HTT) protein containing expanded polyglutamine (polyQ) tracts. DNAJB6, a member of the DNAJ chaperone family, was reported to efficiently inhibit polyQ aggregation in vitro, in cell models, and in vivo in flies, xenopus, and mice. For the delivery of exogenous DNAJB6 to the brain, the DNAJB6 needs to be protected against (enzymatic) degradation and show good penetration into brain tissue. Here, we tested the potential of small extracellular vesicles (sEVs) derived from neural stem cells (NSCs) for delivery of DNAJB6 as anti-amyloidogenic cargo. Administration of sEVs isolated from DNAJB6-overexpressing cells to cells expressing expanded polyQ tracts suppressed HTT aggregation. Furthermore, intrathecal injection of DNAJB6-enriched sEVs into R6/2 transgenic HD mice significantly reduced mutant HTT aggregation in the brain. Taken together, our data suggest that sEV-mediated molecular chaperone delivery may hold potential to delay disease onset in HD.

Small-molecule suppression of calpastatin degradation reduces neuropathology in models of Huntington\u2019s disease

Mitochondrial dysfunction is a common hallmark of neurological disorders, and reducing mitochondrial damage is considered a promising neuroprotective therapeutic strategy. Here, we used high-throughput small molecule screening to identify CHIR99021 as a potent enhancer of mitochondrial function. CHIR99021 improved mitochondrial phenotypes and enhanced cell viability in several models of Huntington’s disease (HD), a fatal inherited neurodegenerative disorder. Notably, CHIR99201 treatment reduced HD-associated neuropathology and behavioral defects in HD mice and improved mitochondrial function and cell survival in HD patient-derived neurons. Independent of its known inhibitory activity against glycogen synthase kinase 3 (GSK3), CHIR99021 treatment in HD models suppressed the proteasomal degradation of calpastatin (CAST), and subsequently inhibited calpain activation, a well-established effector of neural death, and Drp1, a driver of mitochondrial fragmentation. Our results established CAST-Drp1 as a druggable signaling axis in HD pathogenesis and highlighted CHIR99021 as a mitochondrial function enhancer and a potential lead for developing HD therapies.

Isoform-Specific Reduction of the Basic Helix-Loop-Helix Transcription Factor TCF4 Levels in Huntington's Disease.

Huntington’s disease (HD) is an inherited neurodegenerative disorder with onset of characteristic motor symptoms at midlife, preceded by subtle cognitive and behavioral disturbances. Transcriptional dysregulation emerges early in the disease course and is considered central to HD pathogenesis. Using wild-type (wt) and HD knock-in mouse striatal cell lines we observed a HD genotype-dependent reduction in the protein levels of transcription factor 4 (TCF4), a member of the basic helix-loop-helix (bHLH) family with critical roles in brain development and function. We characterized mouse Tcf4 gene structure and expression of alternative mRNAs and protein isoforms in cell-based models of HD, and in four different brain regions of male transgenic HD mice (R6/1) from young to mature adulthood. The largest decrease in the levels of TCF4 at mRNA and specific protein isoforms were detected in the R6/1 mouse hippocampus. Translating this finding to human disease, we found reduced expression of long TCF4 isoforms in the postmortem hippocampal CA1 area and in the cerebral cortex of HD patients. Additionally, TCF4 protein isoforms showed differential synergism with the proneural transcription factor ASCL1 in activating reporter gene transcription in hippocampal and cortical cultured neurons. Induction of neuronal activity increased these synergistic effects in hippocampal but not in cortical neurons, suggesting brain region-dependent differences in TCF4 functions. Collectively, this study demonstrates isoform-specific changes in TCF4 expression in HD that could contribute to the progressive impairment of transcriptional regulation and neuronal function in this disease.

Accumulation of Endogenous Mutant Huntingtin in Astrocytes Exacerbates Neuropathology of Huntington Disease in Mice.

Selective neuronal accumulation of misfolded proteins is a key step toward neurodegeneration in a wide range of neurodegenerative diseases, including Huntington's (HD) diseases. Our recent studies suggest that Hsp70-binding protein 1 (HspBP1), an Hsp70/CHIP inhibitor that reduces protein folding, is highly expressed in neuronal cells and accounts for the accumulation of the HD protein huntingtin (HTT) in neuronal cells. To further determine the role of HspBP1 in regulation of mutant protein accumulation, we investigated whether increasing expression of HspBP1 in glial cells can also induce the accumulation of endogenous mutant HTT in glial cells and yield non-cell-autonomous toxic effects. We performed stereotaxic injection of AAV to selectively express HspBP1 in astrocytes in the brains of HD140Q knock-in (KI) mice that express mutant HTT ubiquitously but do not display obvious neurodegeneration. However, HspBP1 expression in HD140Q astrocytes led to the increased accumulation of endogenous mutant HTT and robust neuronal loss in the striatum of HD140Q KI mice. In transgenic HD mice that selectively express mutant HTT in astrocytes, increased accumulation of mutant HTT in astrocytes via HspBP1 expression did not elicit neurodegeneration but could exacerbate neurological symptoms. Consistently, suppressing the expression of endogenous HspBp1 in the striatum of HD140Q KI mice via CRISPR/Cas9 led to a significant reduction of mutant HTT accumulation. Our findings suggest that although endogenous mutant HTT in astrocytes can exacerbate neurological symptoms, it mediates neurodegeneration only when mutant HTT is also accumulated in neuronal cells.

Exosomes as a potential messenger unit during heterochronic parabiosis for amelioration of Huntington's disease

BackgroundHuntington's disease (HD) starts its pathology long before clinical manifestation, however, there is no therapy to cure it completely and only a few studies have been reported for delaying the progression of HD. Recently, it has been shown that heterochronic parabiosis can modulate the neurodegenerative diseases. Despite the importance of the transportation process of positive factors during heterochronic parabiosis, there were limited understandings because the transportation process is nanoscale, which makes it difficult to identify the messenger unit. We demonstrated that heterochronic parabiosis could modulate HD in R6/2 mice model, and identified the messenger unit for transferring positive factors in the young blood serum. MethodsR6/2 mice were surgically connected with young wild-type mice (n = 13), old wild-type mice (n = 8), or R6/2 mice (n = 6) to examine the effect of heterochronic parabiosis. Parabionts composed of 5- to 6-week-old transgenic and wild-type mice were observed for 6 weeks in a single cage. The in vitro cellular model of HD cells were treated by the blood serum of the young or old mice, and by the exosomes isolated from thereof. The in vitro cellular model of HD were developed by differentiating neural stem cells cultured from SVZ of the brain. ResultsAfter the heterochronic parabiosis, the weight loss and survival of HD mice was improved. Also, mutant Huntingtin aggregation (EM48 p < 0.005), improvement of mitochondria dysfunction (PGC-1a p < 0.05, p-CREB/CREB p < 0.005), cell death (p53 p < 0.05, Bax p < 0.05, Cleaved-caspase3 p < 0.05), and cognition (DCX p < 0.5) showed a near complete restoration. In addition, treating in vitro cellular model of HD by the exosomes from young blood serum improved mutant Huntingtin aggregation (EM48 p < 0.05), mitochondria biogenesis (p-CREB/CREB p < 0.005), cell death (p53 p < 0.05, Bax p < 0.005, Cleaved-caspase3 p < 0.05, Bcl-2 p < 0.05), and cell proliferation (WST-1 p < 0.005). ConclusionsWe found that the overall pathology of HD could be improved by the shared blood circulation through heterochronic parabiosis, furthermore, we demonstrated that the exosomes could be messengers for transferring positive factors, showing the potential of exosomes from young blood for the amelioration of HD.

ANGIOTENSIN II INCREASES FORMATION OF NEUTROPHIL EXTRACELLULAR TRAPS: ROLE IN HYPERTENSION AND DIABETES

Objective: Endothelial dysfunction plays a central mechanistic role in the pathogenesis of hypertension and diabetes. However, the molecular determinants of endothelial dysfunction are not fully understood. Peptidylarginine deiminases (PADs) irreversibly convert arginine to citrulline in protein, generating neoantigens that are unrecognizable to the immune system. PAD4, an isoform predominantly expressed in neutrophils is upregulated in autoimmune and coronary artery disease. PAD4 activation, in turn, promotes vascular injury through the formation of neutrophil extracellular traps (NETs), networks of chromatin and protein-containing extracellular fibres released to fight infection. Whether PAD4 activity is altered in hypertension and diabetes and whether this contributes to disease pathogenesis is not known. The objective of the present study was to examine the effect of angiotensin II on formation of neutrophil extracellular traps in vitro and in an animal model of hypertension and diabetes. Design and method: Neutrophil-like cells were generated from differentiation of the HL-60 cell line and then treated with angiotensin II (10-7 M) for 24 hours. NET formation was assessed by Western blot analysis of citrullinated histone H3 (a primary component of NETs). We further assessed NET formation in mice expressing human renin under the control of the transthyretin promoter (TTRhRen) intercrossed with OVE26 diabetic mice [i.e. hypertensive-diabetic, HD mice] and compared with wild-type (WT) FVBN/J mice. We examined levels of citrullinated histone H3, neutrophil elastase, and PAD4 expression in the kidney of HD and WT mice by immunohistochemistry. Results: Treatment of neutrophil-like cells with angiotensin-II increased levels of citrullinated histone H3 by ∼2-fold. Similarly, in HD mice we observed a significant increase in citrullinated histone H3 (P &lt; 0.0001), in neutrophil elastase (P &lt; 0.0001), and in PAD4 protein levels (P &lt; 0.0001) compared with WT. Increased PAD4 activity was associated with the presence of albuminuria, renal interstitial fibrosis and reduced glomerular filtration rate. Conclusions: Taken together these data suggest that angiotensin II induces NET formation leading to renal accumulation in hypertension and diabetes. PAD4 may therefore play a role in target organ injury in hypertension and diabetes.

Exercise reduces hyperlipidemia-induced kidney damage in apolipoprotein E-deficient mice.

Hyperlipidemia is a risk factor of kidney damage that can lead to chronic kidney disease. Studies have shown that exercise reduces kidney damage; however, the specific mechanisms underlying the protective effects of exercise remain unclear. For 12 weeks, 8-week-old male apolipoprotein E-deficient (ApoE-/-) mice were randomly divided into four treatment groups (n=7/group) as follows: Mice fed a normal diet (ND group); mice fed a ND and exercised (ND + E group); mice fed a high-fat diet (HD group); and mice fed a HD and exercised (HD + E group). Exercise training consisted of swimming for 40 min, 5 days/week. Metabolic parameters, such as low-density lipoprotein-cholesterol, total cholesterol and creatinine levels were higher in the ApoE-/- HD mice compared with those in the ApoE-/- HD + E mice. Serum levels of glutathione peroxidase and superoxide dismutase were significantly decreased in the HD group compared with those in the HD + E group. Significant pathological changes were observed in the HD + E group compared with in the HD group. Immunohistochemistry and immunoblotting revealed increased levels of oxidative stress (nuclear factor erythroid-2-related factor 2) and fibrosis (Smad3 and TGF-β) markers in the ApoE-/- HD group; however, the expression levels of these markers were significantly decreased in the ApoE-/- HD + E group. Furthermore, NF-κB expression in the HD + E group was significantly lower compared with that in the HD group. These results suggested that exercise may exert protective effects against kidney damage caused by hyperlipidemia.

Role of mosaic aneuploidy in the development and progression of Huntington's disease

AbstractBackgroundHuntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG trinucleotide repeat expansion mutation in the huntingtin (HTT) gene. HTT encodes the huntingtin protein, which has been shown to localize to spindle poles and is required for proper mitotic spindle orientation, chromosome segregation, and cell cycle regulation. Mutations in HTT lead to the production of a mature huntingtin protein with an expanded polyglutamine region (&gt;35 repeats) at its N terminus. Previous studies have shown that mouse models of HD exhibit defects in microtubule function and in cell cycle regulation. HD is a progressive condition characterized by decline in cognitive and executive functions. Over the years, studies from our laboratory and others have shown elevated levels of aneuploidy in many neurodegenerative disorders that are characterized by cognitive deficits, including Alzheimer’s disease (AD) and frontotemporal lobar degeneration (FTLD). Here, we investigated whether chromosome segregation defects that lead to mosaic aneuploidy and consequent apoptosis may also contribute to neuronal loss in HD.MethodSingle cell suspensions were prepared from human brain samples (cortex and cerebellum) and fibroblast cell lines from HD donors and age‐matched controls and were processed for FISH analysis of human chromosome 21, NeuN immunostaining, and/or TUNEL staining. Single cell suspensions from cerebellum were prepared from mouse models of HD and age‐matched controls for FISH analysis of mouse chromosome 5 or 16, which is syntenic to human chromosome 21.ResultWe observed increased levels of mosaic aneuploidy and associated apoptosis in brain tissues and in cultured fibroblasts from HD donors compared to age‐matched controls. We also observed increased levels of mosaic aneuploidy in brain tissue from HD mice compared to age‐matched controls.ConclusionTogether with our previous studies of AD and FTLD, these data provide evidence that chromosome segregation defects that lead to genomic instability may serve as a shared mechanism underlying many neurodegenerative disorders. Our findings highlight the need to further investigate the biological mechanism(s) underlying HD pathology and set the stage for novel therapeutic approaches to HD that may also be applied to other neurodegenerative disorders.

Disruption of zinc transporter ZnT3 transcriptional activity and synaptic vesicular zinc in the brain of Huntington\u2019s disease transgenic mouse

BackgroundHuntington’s disease (HD) is a neurodegenerative disease that involves a complex combination of psychiatric, cognitive and motor impairments. Synaptic dysfunction has been implicated in HD pathogenesis. However, the mechanisms have not been clearly delineated. Synaptic vesicular zinc is closely linked to modulating synaptic transmission and maintaining cognitive ability. It is significant to assess zinc homeostasis for further revealing the pathogenesis of synaptic dysfunction and cognitive impairment in HD.ResultsHistochemical staining by autometallography indicated that synaptic vesicular zinc was decreased in the hippocampus, cortex and striatum of N171-82Q HD transgenic mice. Analyses by immunohistochemistry, Western blot and RT-PCR found that the expression of zinc transporter 3 (ZnT3) required for transport of zinc into synaptic vesicles was obviously reduced in these three brain regions of the HD mice aged from 14 to 20 weeks and BHK cells expressing mutant huntingtin. Significantly, dual-luciferase reporter gene and chromatin immunoprecipitation assays demonstrated that transcription factor Sp1 could activate ZnT3 transcription via its binding to the GC boxes in ZnT3 promoter. Moreover, mutant huntingtin was found to inhibit the binding of Sp1 to the promoter of ZnT3 and down-regulate ZnT3 expression, and the decline in ZnT3 expression could be ameliorated through overexpression of Sp1.ConclusionsThis is first study to reveal a significant loss of synaptic vesicular zinc and a decline in ZnT3 transcriptional activity in the HD transgenic mice. Our work sheds a novel mechanistic insight into pathogenesis of HD that mutant huntingtin down-regulates expression of ZnT3 through inhibiting binding of Sp1 to the promoter of ZnT3 gene, causing disruption of synaptic vesicular zinc homeostasis. Disrupted vesicular zinc ultimately leads to early synaptic dysfunction and cognitive deficits in HD. It is also suggested that maintaining normal synaptic vesicular zinc concentration is a potential therapeutic strategy for HD.

Wake-Promoting and EEG Spectral Effects of Modafinil After Acute or Chronic Administration in the R6/2 Mouse Model of Huntington\u2019s Disease

Huntington’s disease (HD) is characterised by progressive symptoms including cognitive deficits and sleep/wake disturbances reflected in an abnormal electroencephalography (EEG). Modafinil, a wake-promoting and cognitive-enhancing drug, has been considered as a treatment for HD. We used HD (R6/2) mice to investigate the potential for using modafinil to treat sleep-wake disturbance in HD. R6/2 mice show sleep-wake and EEG changes similar to those seen in HD patients, with increased rapid eye movement sleep (REMS), decreased wakefulness/increased non-REMS (NREMS), and pathological changes in EEG spectra, particularly an increase in gamma power. We recorded EEG from R6/2 and wild-type mice treated with modafinil acutely (with single doses between 25 and 100 mg/kg; at 12 and 16 weeks of age), or chronically (64 mg/kg modafinil/day from 6 to 15 weeks). Acutely, modafinil increased wakefulness in R6/2 mice and restored NREMS to wild-type levels at 12 weeks. It also suppressed the pathologically increased REMS. This was accompanied by decreased delta power, increased peak frequency of theta, and increased gamma power. At 16 weeks, acute modafinil also restored wakefulness and NREMS to wild-type levels. However, whilst REMS decreased, it did not return to normal levels. By contrast, in the chronic treatment group, modafinil-induced wakefulness was maintained at 15 weeks (after 9 weeks of treatment). Interestingly, chronic modafinil also caused widespread suppression of power across the EEG spectra, including a reduction in gamma that increases pathologically in R6/2 mice. The complex EEG effects of modafinil in R6/2 mice should provide a baseline for further studies to investigate the translatability of these result to clinical practice.

Nemo-like kinase reduces mutant huntingtin levels and mitigates Huntington's disease.

Nemo-like kinase (NLK), an evolutionarily conserved serine/threonine kinase, is highly expressed in the brain, but its function in the adult brain remains not well understood. In this study, we identify NLK as an interactor of huntingtin protein (HTT). We report that NLK levels are significantly decreased in HD human brain and HD models. Importantly, overexpression of NLK in the striatum attenuates brain atrophy, preserves striatal DARPP32 levels and reduces mutant HTT (mHTT) aggregation in HD mice. In contrast, genetic reduction of NLK exacerbates brain atrophy and loss of DARPP32 in HD mice. Moreover, we demonstrate that NLK lowers mHTT levels in a kinase activity-dependent manner, while having no significant effect on normal HTT protein levels in mouse striatal cells, human cells and HD mouse models. The NLK-mediated lowering of mHTT is associated with enhanced phosphorylation of mHTT. Phosphorylation defective mutation of serine at amino acid 120 (S120) abolishes the mHTT-lowering effect of NLK, suggesting that S120 phosphorylation is an important step in the NLK-mediated lowering of mHTT. A further mechanistic study suggests that NLK promotes mHTT ubiquitination and degradation via the proteasome pathway. Taken together, our results indicate a protective role of NLK in HD and reveal a new molecular target to reduce mHTT levels.

The interplay between redox signalling and proteostasis in neurodegeneration: In vivo effects of a mitochondria-targeted antioxidant in Huntington's disease mice

Abnormal protein homeostasis (proteostasis), dysfunctional mitochondria, and aberrant redox signalling are often associated in neurodegenerative disorders, such as Huntington's (HD), Alzheimer's and Parkinson's diseases. It remains incompletely understood, however, how changes in redox signalling affect proteostasis mechanisms, including protein degradation pathways and unfolded protein responses (UPR). Here we address this open question by investigating the interplay between redox signalling and proteostasis in a mouse model of HD, and by examining the in vivo effects of the mitochondria-targeted antioxidant MitoQ. We performed behavioural tests in wild-type and R6/2 HD mice, examined markers of oxidative stress, UPR activation, and the status of key protein degradation pathways in brain and peripheral tissues. We show that R6/2 mice present widespread markers of oxidative stress, with tissue-specific changes in proteostasis that were more pronounced in the brain and muscle than in the liver. R6/2 mice presented increased levels of cytosolic and mitochondrial chaperones, particularly in muscle, indicating UPR activation. Treatment with MitoQ significantly ameliorated fine motor control of R6/2 mice, and reduced markers of oxidative damage in muscle. Additionally, MitoQ attenuated overactive autophagy induction in the R6/2 muscle, which has been associated with muscle wasting. Treatment with MitoQ did not alter autophagy markers in the brain, in agreement with its low brain bioavailability, which limits the risk of impairing neuronal protein clearance mechanisms. This study supports the hypotheses that abnormal redox signalling in muscle contributes to altered proteostasis and motor impairment in HD, and that redox interventions can improve muscle performance, highlighting the importance of peripheral therapeutics in HD.