YAC128 Huntington's Disease Mouse Research Articles

Differential changes in thalamic and cortical excitatory synapses onto striatal spiny projection neurons in a Huntington disease mouse model

Huntington disease (HD), a neurodegenerative disorder caused by CAG repeat expansion in the gene encoding huntingtin, predominantly affects the striatum, especially the spiny projection neurons (SPN). The striatum receives excitatory input from cortex and thalamus, and the role of the former has been well-studied in HD. Here, we report that mutated huntingtin alters function of thalamostriatal connections. We used a novel thalamostriatal (T-S) coculture and an established corticostriatal (C-S) coculture, generated from YAC128 HD and WT (FVB/NJ background strain) mice, to investigate excitatory neurotransmission onto striatal SPN. SPN in T-S coculture from WT mice showed similar mini-excitatory postsynaptic current (mEPSC) frequency and amplitude as in C-S coculture; however, both the frequency and amplitude were significantly reduced in YAC128 T-S coculture. Further investigation in T-S coculture showed similar excitatory synapse density in WT and YAC128 SPN dendrites by immunostaining, suggesting changes in total dendritic length or probability of release as possible explanations for mEPSC frequency changes. Synaptic N-methyl-d-aspartate receptor (NMDAR) current was similar, but extrasynaptic current, associated with cell death signaling, was enhanced in YAC128 SPN in T-S coculture. Employing optical stimulation of cortical versus thalamic afferents and recording from striatal SPN in brain slice, we found increased glutamate release probability and reduced AMPAR/NMDAR current ratios in thalamostriatal synapses, most prominently in YAC128. Enhanced extrasynaptic NMDAR current in YAC128 SPN was apparent with both cortical and thalamic stimulation. We conclude that thalamic afferents to the striatum are affected early, prior to an overt HD phenotype; however, changes in NMDAR localization in SPN are independent of the source of glutamatergic input.

Protection by dietary restriction in the YAC128 mouse model of Huntington's disease: Relation to genes regulating histone acetylation and HTT

Huntington's disease (HD) is a fatal neurodegenerative disease characterized by metabolic, cognitive, and motor deficits. HD is caused by an expanded CAG repeat in the first exon of the HTT gene, resulting in an expanded polyglutamine section. Dietary restriction (DR) increases lifespan and ameliorates age-related pathologies, including in a model of HD, but the mechanisms mediating these protective effects are unknown. We report metabolic and behavioral effects of DR in the full-length YAC128 HD mouse model, and associated transcriptional changes in hypothalamus and striatum. DR corrected many effects of the transgene including increased body weight, decreased blood glucose, and impaired motor function. These changes were associated with reduced striatal human (but not mouse) HTT expression, as well as alteration in gene expression regulating histone acetylation modifications, particularly Hdac2. Other mRNAs related to Huntington's pathology in striatal tissue showed significant modulation by the transgene, dietary restriction or both. These results establish a protective role of DR in a transgenic model that contains the complete human HTT gene and for the first time suggest a role for DR in lowering HTT level, which correlates with severity of symptoms.

Adult neural progenitor cells from Huntington's disease mouse brain exhibit increased proliferation and migration due to enhanced calcium and ROS signals.

Huntington's disease (HD) is an inherited human neurodegenerative disorder characterized by uncontrollable movement, psychiatric disturbance and cognitive decline. Impaired proliferative/differentiational potentials of adult neural progenitor cells (ANPCs) have been thought to be a pathogenic mechanism involved in it. In this study, we aimed to elucidate intrinsic properties of ANPCs subjected to neurodegenerative condition in YAC128 HD mice. ANPCs were isolated from the SVZ regions of 4-month-old WT and YAC128 mice. Cell proliferation, migration and neuronal differentiation invitro were compared between these two genotypes with/without Ca(2+) inhibitors or ROS scavenger treatments. Differences in ANPC proliferation and differentiation capabilities invivo between the two genotypes were evaluated using Ki-67 and Doublecortin (DCX) immunofluorescence respectively. Compared to WT ANPCs, YAC128 ANPCs had significantly enhanced cell proliferation, migration and neuronal differentiation invitro, accompanied by increased Ca(2+) and ROS signals. Raised proliferation and migration in YAC128 ANPCs were abolished by Ca(2+) signalling antagonists and ROS scavenging. However, invivo, HD ANPCs failed to show any elevated proliferation or differentiation. Increased Ca(2+) signalling and higher level of ROS conferred HD ANPC enhancement of proliferation and migration potentials. However, the invivo micro-environment did not support endogenous ANPCs to respond appropriately to neuronal loss in these YAC128 mouse brains.

Single-Cell Suspension Methodology Favors Survival and Vascularization of Fetal Striatal Grafts in the YAC128 Mouse Model of Huntington's Disease

Cell replacement therapies have yielded variable and short-lived benefits in Huntington's disease (HD) patients. This suboptimal outcome is likely due to the fact that graft survival is compromised long term because grafts are subjected to a host's microglial inflammatory response, to a lack of adequate trophic support, and possibly to cortical excitotoxicity. However, graft demise may also relate to more straightforward issues such as cell preparation methodology (solid grafts vs. cell suspension). Indeed, we recently reported that solid grafts are poorly revascularized in HD patients transplanted 9 and 12 years previously. To evaluate whether methodological issues relating to cell preparation may have an impact on graft viability, we implanted green fluorescent protein (GFP(+)) single-cell suspensions of fetal striatal neuronal cells into the striatum of YAC128 HD mice. Postmortem evaluation yielded comparable graft survival in YAC128 mice and their wild-type littermates (noncarrier) at 1 and 3 months posttransplantation. Additionally, the degrees of graft revascularization in the YAC128 and noncarrier mice were similar, with both capillaries and large-caliber vessels observable within the grafted tissue. Furthermore, GFP(+) cells interacted well with host blood vessels, indicating integration of the donor cells within the recipient brain. These observations, combined with our recent report of poor revascularization of solid grafts in the HD-transplanted patients, suggest that the success of cell transplantation can be improved by optimizing methodological aspects relating to cell preparation.

M04 Preventing Formation Of Toxic N-terminal Huntingtin Fragments Through Antisense Oligonucleotide-mediated Protein Modification

Several studies have implicated the importance of proteolytic cleavage of the mutant huntingtin protein in Huntington disease (HD) pathogenesis and it is generally accepted that N-terminal polyglutamine-containing huntingtin fragments are more toxic than full-length huntingtin protein. While recent publications have shown promising results reducing mutant huntingtin toxicity by lowering huntingtin levels using antisense oligonucleotides, there is some concern regarding unwanted side effects caused by lowering huntingtin protein levels too much. In our study we reduce mutant huntingtin toxicity by modifying the huntingtin protein to prevent proteolytic cleavage and subsequent formation of toxic N-terminal huntingtin fragments. Using antisense oligonucleotides, we induce skipping of huntingtin exon 12 that encodes important cleavage sites. In vitro studies showed successful exon skipping and appearance of a shorter huntingtin protein, that is resistant to caspase 6 cleavage. Pilot studies in the YAC128 transgenic HD mouse model revealed skipping of human huntingtin exon 12 after a single intracerebroventricular injection of antisense oligonucleotides. We have recently started a follow-up study to assess the longitudinal phenotypical effect of antisense oligonucleotide-mediated exon skipping in YAC128 HD mice.

Alterations in STriatal‐Enriched protein tyrosine Phosphatase expression, activation, and downstream signaling in early and late stages of the YAC128 Huntington's disease mouse model

Striatal neurodegeneration and synaptic dysfunction in Huntington's disease are mediated by the mutant huntingtin (mHtt) protein. MHtt disrupts calcium homeostasis and facilitates excitotoxicity, in part by altering NMDA receptor (NMDAR) trafficking and function. Pre-symptomatic (excitotoxin-sensitive) transgenic mice expressing full-length human mHtt with 128 polyglutamine repeats (YAC128 Huntington's disease mice) show increased calpain activity and extrasynaptic NMDAR (Ex-NMDAR) localization and signaling. Furthermore, Ex-NMDAR stimulation facilitates excitotoxicity in wild-type cortical neurons via calpain-mediated cleavage of STriatal-Enriched protein tyrosine Phosphatase 61 (STEP61). The cleavage product, STEP33, cannot dephosphorylate p38 mitogen-activated protein kinase (MAPK), thereby augmenting apoptotic signaling. Here, we show elevated extrasynaptic calpain-mediated cleavage of STEP61 and p38 phosphorylation, as well as STEP61 inactivation and reduced extracellular signal-regulated protein kinase 1/2 phosphorylation (ERK1/2) in the striatum of 6-week-old, excitotoxin-sensitive YAC128 mice. Calpain inhibition reduced basal and NMDA-induced STEP61 cleavage. However, basal p38 phosphorylation was normalized by a peptide disrupting NMDAR-post-synaptic density protein-95 (PSD-95) binding but not by calpain inhibition. In 1-year-old excitotoxin-resistant YAC128 mice, STEP33 levels were not elevated, but STEP61 inactivation and p38 and ERK 1/2 phosphorylation levels were increased. These results show that in YAC128 striatal tissue, enhanced NMDAR-PSD-95 interactions contributes to elevated p38 signaling in early, excitotoxin-sensitive stages, and suggest that STEP61 inactivation enhances MAPK signaling at late, excitotoxin-resistant stages. The YAC128 Huntington's disease mouse model shows early, enhanced susceptibility to NMDA receptor-mediated striatal apoptosis, progressing to late-stage excitotoxicity resistance. This study shows that elevated NMDA receptor-PSD-95 interactions as well as decreased extrasynaptic STriatal-Enriched protein tyrosine Phosphatase 61 (STEP61) activation may contribute to early enhanced apoptotic signaling. In late-stage YAC128 mice, reduced STEP61 levels and activity correlate with elevated MAPK signaling, consistent with excitotoxicity resistance. Solid and dotted arrows indicate conclusions drawn from the current study and other literature, respectively.

Suppressing aberrant GluN3A expression rescues synaptic and behavioral impairments in Huntington's disease models

Huntington's disease is caused by an expanded polyglutamine repeat in huntingtin (Htt), but the pathophysiological sequence of events that trigger synaptic failure and neuronal loss are not fully understood. Alterations in NMDA-type glutamate receptors (NMDARs) have been implicated, yet it remains unclear how the Htt mutation impacts NMDAR function and direct evidence for a causative role is missing. Here we show that mutant Htt re-directs an intracellular store of juvenile NMDARs to the surface of striatal neurons by sequestering and disrupting the subcellular localization of the GluN3A subunit-specific endocytic adaptor PACSIN1. Overexpressing GluN3A in wild-type striatum mimicked the synapse loss observed in Huntington's disease mouse models, whereas genetic deletion of GluN3A prevented synapse degeneration, ameliorated motor and cognitive decline, and reduced striatal atrophy and neuronal loss in the YAC128 model. Furthermore, GluN3A deletion corrected the abnormally enhanced NMDAR currents, which have been linked to cell death in Huntington's disease and other neurodegenerative conditions. Our findings reveal an early pathogenic role of GluN3A dysregulation in Huntington's disease, and suggest that therapies targeting GluN3A or pathogenic Htt-PACSIN1 interactions might prevent or delay disease progression.

B08 Differential sensitivity of aggregate markers in HdhQ150 and YAC128 HD mouse models

Huntington's disease (HD), an inherited neurodegenerative disorder that is characterised histologically by the presence of intranuclear inclusions (NIIs) and the loss of neurons in the striatum and cortex. Protein aggregates are a hallmark of several neurodegenerative disorders including HD. These aggregates contain a variety of other proteins including ubiquitin. The present study was designed to determine the sensitivity of the different antibody in inclusion formations in knock-in HdhQ150 and transgenic YAC128 mouse lines. In order to fully assess the different protein deposition patterns in these mouse models, brains were assessed using S830 or ubiquitin immunohistochemistry at 8 and 18 months of age. In 8 months old HdhQ150 and YAC128 carrier mice, ubiquitin antibody showed more diffuse staining with minimum NIIs in the olfactory tubercle, striatum, piriform cortex, cortex and hippocampus. However, at the same age, the S830 antibody showed more specific binding to NIIs in the HdhQ150 but not in YAC128 mice. As would be predicted the deposition of aggregated protein becomes more widespread at later ages. At 18 months of age the HdhQ150 mice showed increased ubiquitin immunoactivity, however there was little difference found in the YAC128 mice. NIIs were visible with the S830 antibody in the HdhQ150 and YAC128 mice at this stage. The present study demonstrates that the differential sensitivity of different antibodies in identifying NIIs between mouse lines, which may reflect confirmatinal changes in protein aggregates.

Caspase-6-Resistant Mutant Huntingtin Does not Rescue the Toxic Effects of Caspase-Cleavable Mutant Huntingtin in vivo.

The amelioration of behavioral and neuropathological deficits in mice expressing caspase-6-resistant (C6R) mutant huntingtin (mhtt), despite the presence of an expanded polyglutamine tract, highlights proteolysis of htt at the 586aa caspase-6 (casp6) site may be an important mechanism in the pathogenesis of Huntington disease (HD). One possible explanation of these effects is that C6R mhtt could act as a dominant negative on mhtt. To determine if the neuroprotective effect observed in the C6R mice is due to dominant negative effects, we crossed the C6R mice to the YAC128 HD mouse model to generate mice expressing both caspase-cleavable and C6R mhtt (YAC/C6R) concurrently and assessed previously defined behavioral and neuropathological endpoints. Our results demonstrate that YAC/C6R animals exhibit similar motor abnormalities and learning deficits as the YAC128 mice. Neuropathological analysis reveals a significant decrease in brain weight and striatal volume in the YAC/C6R mice comparable to the YAC128 mice. In contrast, and similar to previous findings, C6R mice demonstrate preserved brain weight and striatal volume. As expected, body weight is significantly increased in the YAC/C6R mice due to the increased levels of htt. The results of this study suggest that the lack of an HD phenotype in the C6R mice is most likely due to the absence of cleavage of htt and not due to suppression of expression of mhtt.

Longitudinal analyses of operant performance on the serial implicit learning task (SILT) in the YAC128 Huntington's disease mouse line

Huntington's disease is a genetic disorder characterised by progressive striatal and cortical neurodegeneration, resulting in a broad range of motor, cognitive and behavioural abnormalities. The disease is caused by a single mutation in the gene responsible for the protein huntingtin, increasing the number of polyQ repeats and conferring a toxic gain of function to the mutant protein, which ultimately induces cell death. Several mouse models of HD are available. The YAC128 mouse model carries 128 CAG repeats and is known to develop several HD-like symptoms. This model has been well characterised on the FVB/N background strain, a strain that develops severe retinal degeneration. We have therefore sought to characterise YAC128 deficit in mice backcrossed onto the C57BL/6j background strain which is free of visual deficits and therefore more amenable to behavioural testing. In a parallel study (this special issue) we have provided a longitudinal characterisation of the emergence of a motor phenotype in the YAC128/C57BL mice. In the present paper, we have undertaken a more detailed characterisation of cognitive impairment in this mouse line at 6, 12, and 18 months of age using the operant serial implicit learning task (SILT), a task that was first designed to assess impairments in mice similar to the implicit serial learning impairments in HD patients task, and which has subsequently been shown to be highly sensitive to cortico-striatal disruption in mice. On the SILT task, the mouse must attain rewards by correctly nose-poking to 2 stimulus lights (S1 and S2) presented randomly and sequentially in 5 holes (deemed A-E) on a light array. Performance is measured by accuracy and speed of response to the S1 and S2 stimuli. Embedded within the random sequences, was a predictable sequence whereby an S1 in hole B is always followed by the S2 in hole D, which constitutes an implicit learning probe. The YAC128 carriers were less accurate in their responses to both S1 and S2 stimuli in the absence of response latency deficits. The deficits in accuracy to the S2 stimuli were present from 6 months of age and were progressive. There was no difference between the wildtype and the YAC128 carriers in the benefits gained from identifying the predictable B-D sequence. The results suggest that the YAC128 mice have a motor-learning deficit that may reflect impulsive responding and/or impaired visuo-spatial attention consistent with a model of HD.

Selective cognitive impairment in the YAC128 Huntington's disease mouse

People with HD have a demonstrated early extra-dimensional set-shifting deficit. In the present study, we use a novel water T-maze set-shifting procedure and demonstrate its validity as a set-shifting task in a mouse model of Huntington's disease. Three groups of YAC128 mice of different ages (27, 69 and 117 weeks) were run on the task, which incorporated six distinct stages in which the mice must learn a rule and then switch to a different rule. The six stages were: directional learning, directional learning reversal, light discrimination, light discrimination reversal, return to place learning and a maze rotation spatial learning test. Rule changes from place learning to light discrimination and back constitute extra-dimensional shifts. The results of the study demonstrate robust light/dark discrimination reversal learning deficits in transgenic mice from 27 weeks of age, and a directional learning to light discrimination extra-dimensional set-shifting deficit from 69 weeks of age. The extra-dimensional shift deficit was confirmed with control trials demonstrating the validity of the deficit and the task. The onset of reversal learning and extra-dimensional shift deficits corresponded with the development of mutant huntingtin N-terminal fragment aggregates in neurons of relevant forebrain regions.

Palmitoylation and function of glial glutamate transporter-1 is reduced in the YAC128 mouse model of Huntington disease

Excitotoxicity plays a key role in the selective vulnerability of striatal neurons in Huntington disease (HD). Decreased glutamate uptake by glial cells could account for the excess glutamate at the synapse in patients as well as animal models of HD. The major molecule responsible for clearing glutamate at the synapses is glial glutamate transporter GLT-1. In this study, we show that GLT-1 is palmitoylated at cysteine38 (C38) and further, that this palmitoylation is drastically reduced in HD models both in vitro and in vivo. Palmitoylation is required for normal GLT-1 function. Blocking palmitoylation either with the general palmitoylation inhibitor, 2-bromopalmitate, or with a GLT-1 C38S mutation, severely impairs glutamate uptake activity. In addition, GLT-1-mediated glutamate uptake is indeed impaired in the YAC128 HD mouse brain, with the defect in the striatum evident as early as 3 months prior to obvious neuropathological findings, and in both striatum and cortex at 12 months. These phenotypes are not a result of changes in GLT1 protein expression, suggesting a crucial role of palmitoylation in GLT-1 function. Thus, it appears that impaired GLT-1 palmitoylation is present early in the pathogenesis of HD, and may influence decreased glutamate uptake, excitotoxicity, and ultimately, neuronal cell death in HD.

Mutant Huntingtin N-terminal Fragments of Specific Size Mediate Aggregation and Toxicity in Neuronal Cells

Huntingtin proteolysis is implicated in Huntington disease pathogenesis, yet, the nature of huntingtin toxic fragments remains unclear. Huntingtin undergoes proteolysis by calpains and caspases within an N-terminal region between amino acids 460 and 600. We have focused on proteolytic steps producing shorter N-terminal fragments, which we term cp-1 and cp-2 (distinct from previously described cp-A/cp-B). We used HEK293 cells to express the first 511 residues of huntingtin and further define the cp-1 and cp-2 cleavage sites. Based on epitope mapping with huntingtin-specific antibodies, we found that cp-1 cleavage occurs between residues 81 and 129 of huntingtin. Affinity and size exclusion chromatography were used to further purify huntingtin cleavage products and enrich for the cp-1/cp-2 fragments. Using mass spectrometry, we found that the cp-2 fragment is generated by cleavage of huntingtin at position Arg(167). This site was confirmed by deletion analysis and specific detection with a custom-generated cp-2 site neo-epitope antibody. Furthermore, alterations of this cleavage site resulted in a decrease in toxicity and an increase in aggregation of huntingtin in neuronal cells. These data suggest that cleavage of huntingtin at residue Arg(167) may mediate mutant huntingtin toxicity in Huntington disease.

Ginsenosides protect striatal neurons in a cellular model of Huntington's disease

Ginseng, the root of Panax ginseng C.A. Meyer (Araliaceae), is a widely used herbal medicine. Ginsenosides, the active ingredients of ginseng, are the main components responsible for many beneficial actions of ginseng. In the present study, we tested 10 different ginsenosides in the previously developed in vitro Huntington's disease (HD) assay with primary medium spiny striatal neuronal cultures (MSN) from the YAC128 HD mouse model. We found that nanomolar concentrations of ginsenoside Rb1 and Rc effectively protected YAC128 medium spiny neurons from glutamate-induced apoptosis and that Rg5 was protective at micromolar concentration. The other seven ginsenosides tested were not effective or exerted toxic effects in MSN cultures. From further experiments, we suggested that neuroprotective effects of ginsenosides Rb1, Rc, and Rg5 could correlate with their ability to inhibit glutamate-induced Ca(2+) responses in cultured MSN. From these results we concluded that ginsenosides Rb1, Rc, and Rg5 offer a potential therapeutic choice for the treatment of HD and possibly other neurodegenerative disorders.