There is considerable interest in targeting dopaminergic neurons in the substantia nigra compacta (SNc) and ventral tegmental area (VTA) to monitor or modulate their activity in normal and diseased states. The limited availability of selective promoters has hampered progress in non-transgenic animals. Thus, we designed MiniPromoters to target TH-expressing neurons in the midbrain of mice and non-human primates. After screening eight recombinant AAVs encoding MiniPromoters driving EmGFP in mice by intravenous (IV) and intracerebroventricular (ICV) injections, we identified Ple384 (TH) and Ple388 (PITX3), which resulted in robust selective expression in SNc and VTA. These rAAVs were further characterized in rhesus macaques using ICV where both MiniPromoters expressed throughout the SNc, with Ple384 (TH) more widespread than Ple388 (PITX3) in VTA neurons. Both MiniPromoters will be useful for expression of research and gene therapy molecules in midbrain dopaminergic neurons in mice and non-human primates.

Compact cell type-specific promoters are important tools for basic and preclinical research and clinical delivery of gene therapy. In this work, we designed novel MiniPromoters to target D1 and D2 type dopaminoceptive medium spiny neurons in the striatum by manually identifying candidate regulatory regions or employing the OnTarget webserver. We then empirically tested the designs in rAAV-PHP.B for specificity and robustness in three systems: intravenous injection in mice, intracerebroventricular injection in mice, and intracerebroventricular injection in non-human primates. Twelve MiniPromoters were designed from eight genes: seven manually and five using OnTarget. When delivered intravenously in mice, three MiniPromoters demonstrated highly selective expression in the striatum, with Ple389 (ADORA2A) showing high levels of dopamine D2-receptor cell co-localization. The same three MiniPromoters also displayed enriched expression in the striatum when delivered intracerebroventricularly in mice with high levels of DARPP32 co-localization. Finally, Ple389 (ADORA2A) was intracerebroventricularly injected in non-human primates and showed enriched expression in the striatum as in the mouse. Ple389 (ADORA2A) demonstrated expression in the medium spiny neurons in all three systems tested and exhibited the highest level of D2-MSNs and DARPP32 co-labeling in mice, demonstrating its potential as a tool for gene therapy approaches for Parkinson and Huntington disease treatment.

Huntington disease (HD) is an adult-onset neurodegenerative disorder that is caused by a trinucleotide CAG repeat expansion in the HTT gene that codes for the protein huntingtin (HTT in humans or Htt in mice). HTT is a multi-functional, ubiquitously expressed protein that is essential for embryonic survival, normal neurodevelopment, and adult brain function. The ability of wild-type HTT to protect neurons against various forms of death raises the possibility that loss of normal HTT function may worsen disease progression in HD. Huntingtin-lowering therapeutics are being evaluated in clinical trials for HD, but concerns have been raised that decreasing wild-type HTT levels may have adverse effects. Here we show that Htt levels modulate the occurrence of an idiopathic seizure disorder that spontaneously occurs in approximately 28% of FVB/N mice, which we have called FVB/N Seizure Disorder with SUDEP (FSDS). These abnormal FVB/N mice demonstrate the cardinal features of mouse models of epilepsy including spontaneous seizures, astrocytosis, neuronal hypertrophy, upregulation of brain-derived neurotrophic factor (BDNF), and sudden seizure-related death. Interestingly, mice heterozygous for the targeted inactivation of Htt (Htt+/- mice) exhibit an increased frequency of this disorder (71% FSDS phenotype), while over-expression of either full length wild-type HTT in YAC18 mice or full length mutant HTT in YAC128 mice completely prevents it (0% FSDS phenotype). Examination of the mechanism underlying huntingtin's ability to modulate the frequency of this seizure disorder indicated that overexpression of full length HTT can promote neuronal survival following seizures. Overall, our results demonstrate a protective role for huntingtin in this form of epilepsy and provide a plausible explanation for the observation of seizures in the juvenile form of HD, Lopes-Maciel-Rodan syndrome, and Wolf-Hirschhorn syndrome. Adverse effects caused by decreasing huntingtin levels have ramifications for huntingtin-lowering therapies that are being developed to treat HD.

A reduced incidence of various forms of cancer has been reported in Huntington's disease patients and may be due to pro-apoptotic effects of mutant huntingtin. We tested this hypothesis by assessing the effects of huntingtin protein overexpression on survival in two murine cancer models. We generatedYACHDmice containing human huntingtin transgenes with various CAG tract lengths (YAC18, YAC72, YAC128) on either an Msh2 or p53 null background which have increased cancer incidence. In both mouse models of cancer, the overexpression of either mutant or wild-type huntingtin had no significant effect on overall survival. These results do not support the hypothesis that mutant huntingtin expression is protective against cancer.

Huntington disease (HD) is an adult-onset neurodegenerative disorder that is caused by a trinucleotide CAG repeat expansion in the HTT gene that codes for the protein huntingtin (HTT or Htt in mice). HTT is a multi-functional, ubiquitously expressed protein that is essential for embryonic survival, normal neurodevelopment, and adult brain function. The ability of wild-type HTT to protect neurons against various forms of death raises the possibility that loss of normal HTT function may worsen disease progression in HD. Huntingtin-lowering therapeutics are being evaluated in clinical trials for HD, but concerns have been raised that decreasing wild-type HTT levels may have adverse effects. Here we show that Htt levels modulate the occurrence of an idiopathic seizure disorder that spontaneously occurs in FVB/N mice. These abnormal FVB/N mice demonstrate various cardinal features of mouse models of epilepsy including spontaneous seizures, astrocytosis, neuronal hypertrophy, upregulation of brain-derived neurotrophic factor (BDNF), and sudden seizure-related death. Interestingly, decreasing wild-type Htt levels increased the frequency of this disorder, while over-expression of HTT completely prevented it. Examination of the mechanism underlying huntingtin’s ability to modulate the frequency of this seizure disorder indicated that over-expression of full length HTT can promote neuronal survival following seizures. Overall, our results demonstrate a protective role for huntingtin in this form of epilepsy and provide a plausible explanation for the observation of seizures in the juvenile form of HD, Lopes-Maciel-Rodan syndrome, and Wolf-Hirschhorn syndrome. Adverse effects caused by altering huntingtin levels has ramifications related to Huntingtin-lowering therapies in development to treat HD.

The granulin protein (also known as, and hereafter referred to as, progranulin) is a secreted glycoprotein that contributes to overall brain health. Heterozygous loss-of-function mutations in the gene encoding the progranulin protein (Granulin Precursor, GRN) are a common cause of familial frontotemporal dementia (FTD). Gene therapy approaches that aim to increase progranulin expression from a single wild-type allele, an area of active investigation for the potential treatment of GRN-dependent FTD, will benefit from the availability of a mouse model that expresses a genomic copy of the human GRN gene. Here we report the development and characterization of a novel mouse model that expresses the entire human GRN gene in its native genomic context as a single copy inserted into a defined locus (Hprt) in the mouse genome. We show that human and mouse progranulin are expressed in a similar tissue-specific pattern, suggesting that the two genes are regulated by similar mechanisms. Human progranulin rescues a phenotype characteristic of progranulin-null mice, the exaggerated and early deposition of the aging pigment lipofuscin in the brain, indicating that the two proteins are functionally similar. Longitudinal behavioural and neuropathological analyses revealed no significant differences between wild-type and human progranulin-overexpressing mice up to 18 months of age, providing evidence that long-term increase of progranulin levels is well tolerated in mice. Finally, we demonstrate that human progranulin expression can be increased in the brain using an antisense oligonucleotide that inhibits a known GRN-regulating micro-RNA, demonstrating that the transgene is responsive to potential gene therapy drugs. Human progranulin-expressing mice represent a novel and valuable tool to expedite the development of progranulin-modulating therapeutics.

Viral vectors made from adeno-associated virus (AAV) have emerged as preferred tools in basic and translational neuroscience research to introduce or modify genetic material in cells of interest. The use of viral vectors is particularly attractive in nontransgenic species, such as nonhuman primates. Injection of AAV solutions into the cerebrospinal fluid is an effective method to achieve a broad distribution of a transgene in the central nervous system. In this study, we conducted injections of AAV9-PHP.B, a recently described AAV capsid mutant, in the lateral ventricle of mice and rhesus macaques. To enhance the expression of the transgene (the tag protein emerald green fluorescent protein [EmGFP]), we used a gene promoter that confers high neuron-specific expression of the transgene, the human synapsin 1 (SYN1) promoter. The efficacy of the viral vector was first tested in mice. Our results show that intracerebroventricular injections of AAV9-PHP.B SYN1-EmGFP-woodchuck hepatitis virus posttranscriptional regulatory element resulted in neuronal EmGFP expression throughout the mice and monkey brains. We have provided a thorough characterization of the brain regions expressing EmGFP in both species. EmGFP was observed in neuronal cell bodies over the whole cerebral cortex and in the cerebellum, as well as in some subcortical regions, including the striatum and hippocampus. We also observed densely labeled neuropil in areas known to receive projections from these regions. Double fluorescence studies demonstrated that EmGFP was expressed by several types of neurons throughout the mouse and monkey brain. Our results demonstrate that a single injection in the lateral ventricle is an efficient method to obtain transgene expression in many cortical and subcortical regions, obviating the need of multiple intraparenchymal injections to cover large brain areas. The use of intraventricular injections of AAV9-PHP.B SYN1-EmGFP could provide a powerful approach to transduce widespread areas of the brain and may contribute to further development of methods to genetically target-specific populations of neurons.

BACKGROUND:TheCre/loxP system allows for the temporal and spatial investigation of the expression of a single gene in the nervous system. Current methods of validating conditional knock-out mouse models rely on heterogeneous brain tissue or primary culture. These methods may assess the extent of genetic knockdown in the brain but do not provide age-appropriate, cell-type specific information.NEW METHOD:We isolated specific cell types from adult murine brain using FACS to assess cell type-specific gene expression in conditional mouse models.RESULTS:We identified robust but incomplete genetic knockdown in microglia isolated from two separate microglia-specific knockout models.COMPARISONWITH EXISTING METHODS(S):Genetic knockdown in isolated adult microglia differed significantly from cultured primary microglia.CONCLUSIONS:Differences observed in primary cultured microglia compared to isolated adult microglia suggest that current methods used to validate microglia-specific gene deletion over-estimate deletion efficiency. Assessment of gene expression in isolated adult microglia provides a more accurate assessment of Cre-mediated gene deletion.

Huntington's disease (HD) is caused by a CAG repeat expansion in the HTT gene and is characterized by early and selective striatal neurodegeneration. The huntingtin (HTT) protein is ubiquitously expressed in many tissues and the cellular pathogenesis of the disease is not fully understood. Immune cell dysfunction due to mutant HTT (mHTT) expression and aberrant immune system activation in HD patients suggests that inflammatory processes may contribute to HD pathogenesis. Here we used the BACHD mouse model of HD, which carries a conditional transgene expressing full-length human mHTT, to selectively deplete mHTT expression in myeloid lineage cells, including microglia, and evaluated the effects on HD-related behavior and neuropathology. In the converse experiment, we depleted mHTT expression in the majority of cells in the brain but specifically excluding microglia and again evaluated behavior and neuropathology. In mice with myeloid-specific mHTT-depletion, we observed no significant rescue of any behavioral or neuropathological outcome measures, while neural-specific knockout mice showed significant rescue of body weight, rotarod performance and striatal volume. We conclude that mHTT expression in microglia, though clearly affecting specific aspects of microglia function, does not alter disease pathogenesis in the BACHD mouse model. This may have implications for current or future therapeutic trials testing immune-modulating drugs in HD patients.

Huntington's disease (HD) is a hereditary neurodegenerative disorder resulting from N-terminal polyglutamine expansion in the huntingtin protein. A relatively selective and early loss of medium spiny neurons in the striatum is a hallmark of HD neuropathology. Although the exact mechanism of mutant huntingtin-mediated neurodegeneration is unclear, recent evidence suggests that NMDA-receptor-mediated excitotoxicity is involved. Our previously published findings show that decreasing levels of the cdk5 activators, p35 and p25, reduces NMDA receptor-mediated excitotoxicity in striatal neurons in vivo. In this study we directly examined the effect of reducing levels of p35 and p25 in the context of mutant huntingtin toxicity, using the B6 YAC128 mouse model of HD. Our findings demonstrate that deletion of a single allele of p35 in the B6 YAC128 mice results in an upregulation of Akt activity, and increases phosphorylation of mutant huntingtin at Ser421. Longitudinal behavioral analysis showed that this 50% reduction in p35 and p25 levels did not improve accelerating Rotarod performance in these YAC128 mice. However, a complete deletion of p35 normalized the accelerating Rotarod performance relative to their non-transgenic littermates at four months of age.

Huntington's Disease (HD) is an inherited neurodegenerative disease caused by a polyglutamine expansion in the huntingtin protein. The YAC128 mouse model of HD expresses the full-length human huntingtin protein with 128 CAG repeats and replicates the phenotype and neurodegeneration that occur in HD. Several studies have implicated a role for neuroinflammation in HD pathogenesis. Studies on presymptomatic HD patients have illustrated microgliosis (activated microglia) in brain regions affected in HD. Mutant huntingtin expressing isolated primary monocytes (human HD patients) and primary macrophages (YAC128) are overactive in response to lipopolysaccharide (LPS) stimulation. In this study we demonstrate that cultured primary microglia (the resident immune cells of the brain cells) from YAC128 mice differentially express a wide number of cytokines compared to wildtype microglia cultures in response to LPS. Furthermore, this study outlines a direct interaction between mutant huntingtin and cytokine secretion in HD microglia. Increased cytokine release in YAC128 microglia can be blocked by cannabinoid activation or by mutant huntingtin knockdown with anti-sense oligonucleotide treatment. Matrix metalloprotease 3 (MMP3), an endogenous neuronal activator of microglia, also induces increased cytokine release from YAC128 microglia compared to wildtype microglia. We found elevated MMP levels in HD CSF, and MMP levels correlate with disease severity in HD. These data support a novel role for MMPs and microglial activation in HD pathogenesis. With an improved understanding of the specific cellular processes involved in HD neuroinflammation, novel therapeutic agents targeting these processes can be developed and hold great promise in the treatment of HD.

OBJECTIVE: Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative condition, characterized by the preferential destruction of motor neurons innervating fast-twitch muscle. The goal of this study is to promote skeletal muscle fiber type conversion from fast to slow phenotype using exercise. We hypothesise that this conversion would delay neurodegeneration in the G93A human SOD1 transgenic mouse model of ALS. BACKGROUND: Is exercise neuroprotective, or does high-intensity activity influence ALS development? This is an ongoing debate in the ALS literature. Due to degeneration of fast-twitch muscle motor units, conversion of motor units to a slow-oxidative phenotype should promote neuroprotection in ALS. Muscle regulatory factors, myogenin and MyoD have been used to alter muscle fiber type composition in mice; myogenin gene transfer increases oxidative phenotype, whereas MyoD gene transfer reduces oxidative phenotype and hastens disease process. This study aims to demonstrate a relationship between exercise and altered muscle expression of myogenin and MyoD. DESIGN/METHODS: Transgenic mice overexpressing the G93A human SOD1 mutation (n=9) and wild type controls (n=6) were randomized into exercise and sedentary groups. Mice engaged in a treadmill-based interval program or remained sedentary three days weekly for eight weeks. Mice were weighed weekly. Spinal cord and muscle samples were collected and frozen following the intervention; half were used for mRNA extraction and cDNA generation for qPCR analysis using standard techniques. RESULTS: Mice showed improvement on the running task over the study duration. Real-time quantitative polymerase chain reaction (PCR) of muscle regulatory factors (myogenin and MyoD), myosin heavy-chain type (Myh7, Myh2, Myh4), and IGF-1 will be completed on hindlimb and spinal cord samples. A succinate dehydrogenase enzyme assay will be performed to assess muscle oxidative capacity. CONCLUSIONS: These results will inform efforts to develop specific muscle-directed exercise therapies for motor neuron diseases. Study Supported by: AAN Student Research Scholarship, UBC, and CMMT

Loss-of-function mutations in the progranulin (GRN) gene are a common cause of autosomal dominant frontotemporal lobar degeneration, a fatal and progressive neurodegenerative disorder common in people less than 65 years of age. In the brain, progranulin is expressed in multiple regions at varying levels, and has been hypothesized to play a neuroprotective or neurotrophic role. Four neurotoxic agents were injected in vivo into constitutive progranulin knockout (Grn(-/-)) mice and their wild-type (Grn(+/+)) counterparts to assess neuronal sensitivity to toxic stress. Administration of 3-nitropropionic acid, quinolinic acid, kainic acid, and pilocarpine induced robust and measurable neuronal cell death in affected brain regions, but no differential cell death was observed between Grn(+/+) and Grn(-/-) mice. Thus, constitutive progranulin knockout mice do not have increased sensitivity to neuronal cell death induced by the acute chemical models of neuronal injury used in this study. (C) 2013 Published by Elsevier Inc.

We previously showed that the expression and activity of indoleamine 2,3-dioxygenase (Ido1) are chronically elevated in the striatum of YAC128 mouse model of HD. This was followed by increased production of neurotoxic metabolite hydroxykynurenine (3-HK) in the striatum of symptomatic mice. We therefore hypothesized that the chronic Ido1 induction in the striatum of YAC128 mice leads to increased neurotoxicity in this mouse model; based on this hypothesis, we predicted that the absence of Ido1 expression would result in decreased sensitivity to neurotoxicity in mice. The work described in this brief communication will include the characterization of Ido(-/-) striatum in terms of enzymatic expression and activity in the first step of the pathway. Additionally, we assessed the sensitivity of the striatum to excitotoxic insult in the absence of Ido1 expression in the striatum of constitutive Ido1 null mice (Ido(-/-)) and demonstrated that Ido(-/-) mice are less sensitive to QA-induced striatal neurotoxicity. Finally, through measurement of kynurenine pathway (KP) metabolites in Ido(-/-) mice, we showed decreased levels of 3-HK in the striatum of these mice. This study suggests that the inhibition of the first step in the KP may be neuroprotective and should be considered as a potential therapeutic target in HD and other neurodegenerative diseases.

Indoleamine 2,3 dioxygenase (Ido1), the first and rate-limiting enzyme of the kynurenine pathway (KP), is a striatally enriched gene with increased expression levels in the YAC128 mouse model of Huntington disease (HD). Our objective in this study was to delineate age-related KP alterations in this model. Three enzymes potentially catalyze the first step of the KP; Ido1 and Indoleamine 2,3 dioxygenase-2 were highly expressed in the striatum and Tryptophan 2,3 dioxygenase (Tdo2) in the cerebellum. During development, Ido1 mRNA expression is dynamically regulated and chronically up-regulated in YAC128 mice. Kynurenine (Kyn) to tryptophan (Trp) ratio, a measure of activity in the first step of the KP, was elevated in YAC128 striatum, but no change in Tdo2 mRNA levels or Kyn to Trp ratio was detected in the cerebellum. Ido1 induction was coincident with Trp depletion at 3months and Kyn accumulation at 12months of age in striatum. Changes in downstream KP metabolites of YAC128 mice generally followed a biphasic pattern with neurotoxic metabolites reduced at 3months and increased at 12months of age. Striatally specific induction of Ido1 and downstream KP alterations suggest involvement in HD pathogenesis, and should be taken into account in future therapeutic developments for HD.

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by an expanded CAG trinucleotide repeat sequence in the huntingtin gene. The resulting poly-glutamine expansion in the huntingtin protein imparts a novel toxic gain of function causing selective loss of medium spiny neurons (MSNs) in the striatum. Although the exact mechanism of cell death is unclear, recent evidence suggests involvement of NMDA-receptor mediated excitotoxicity and aberrant cyclin dependent kinase 5 (cdk5) activity in striatal cells undergoing neurodegeneration. In this study we directly tested the effect of reduced levels of p25 and p35, two proteins required for cdk5 activation, on striatal neurodegeneration using mice with targeted deletion of p35. Quinolinic acid (QA) injected into the striatum of mice causes NMDA-receptor mediated cell death, and these QA-induced striatal lesions were examined in p35 hemizygous null (p35+/-) and wildtype (WT) mice. Striatal QA lesion volumes were 30% smaller in p35+/- mice than in WT mice. Furthermore, primary neuronal cultures of MSNs from P0 p35+/- pups displayed 33% less apoptotic neurons following NMDA treatment than those from WT pups. Examination of YAC128 mouse model of HD showed elevated p25 levels in striatum following intrastriatal QA injection. Our findings provide direct evidence for p25 and p35 involvement in excitotoxic neurodegeneration of MSNs and suggest a role for the cdk5 pathway in HD striatal neurodegeneration.

Progranulin haploinsufficiency is a common cause of familial frontotemporal dementia (FTD), but the role of progranulin in the brain is poorly understood. To investigate the role of murine progranulin (Grn) in the CNS in vivo, we generated mice targeted at the progranulin locus (Grn) using a gene-trap vector. Constitutive progranulin knockout mice (GrnKO) show moderate abnormalities in anxiety-related behaviors, social interactions, motor coordination, and novel object recognition at 8months of age, many of which differ between males and females. Analysis of synaptic transmission in 10–12month old GrnKO male mice indicates altered synaptic connectivity and impaired synaptic plasticity. Additionally, apical dendrites in pyramidal cells in the CA1 region of the hippocampus in GrnKO males display an altered morphology and have significantly decreased spine density compared to wild-type (WT) mice. The observed changes in behavior, synaptic transmission, and neuronal morphology in GrnKO mice occur prior to neuropathological abnormalities, most of which are apparent at 18 but not at 8months of age. We conclude that progranulin deficiency leads to reduced synaptic connectivity and impaired plasticity, which may contribute to FTD pathology in human patients.

Frontotemporal lobar degeneration (FTLD) is a neurodegenerative condition characterized by focal degeneration of the frontal and temporal lobes of the brain. Autosomal dominantly inherited mutations of the progranulin gene (GRN) have been identified as the cause of a subset of cases of familial FTLD. In order to better understand the function of progranulin in the central nervous system (CNS), we have assessed the spatiotemporal expression pattern of both the murine progranulin gene (Grn) and the protein (Grn) by using transgenic knock‐in mice expressing a reporter gene from the Grn locus and by immunohistochemistry, respectively. We compared Grn expression with a panel of established markers for distinct neuronal developmental stages and specific cell lineages at time points ranging from embryonic day 13.5 through to the mature adult. We find that Grn is expressed in both neurons and microglia within the CNS, but that it shows a different developmental expression pattern in each cell type. Grn expression in neurons increases as the cells mature, whereas expression in microglia varies with the cells' state of activation, being specifically upregulated in microglia in response to excitotoxic injury. Our results suggest that progranulin plays distinct roles in neurons and microglia, both of which likely contribute to overall neuronal health and function. J. Comp. Neurol. 518:3931–3947, 2010. © 2010 Wiley‐Liss, Inc.

YAC transgenic mice expressing poly(Q)-expanded full-length huntingtin (mhtt) recapitulate many behavioral and neuropathological features of Huntington disease (HD). We have previously observed a reduction in phosphorylation of mhtt at S421 in the presence of the mutation for HD. In addition, phosphorylation of normal S421-htt is reduced after excitotoxic stimulation of NMDA receptors (NMDARs). To test whether NMDAR stimulation contributes to reduced pS421-htt levels in HD, we determined phosphorylation of htt at Ser421 after NMDA-induced excitotoxicity in neurons from YAC128 mice. Here, we report that the total level of pS421-htt is reduced in YAC128 primary neurons after excitotoxic NMDAR stimulation. Similarly, the total level of pS421-htt is reduced in YAC128 transgenic mice after quinolinic acid injection into the striatum. In contrast, loss of phosphorylation of pS421-htt is prevented in YAC mice that never develop clinical or neuropathological features of HD [the caspase 6-resistant YAC128 transgene (C6R)]. To gain insight into the mechanisms underlying these findings, we determined that the Ser/Thr protein phosphatases PP1 and PP2A dephosphorylate pS421-htt in situ and after excitotoxic stimulation of NMDARs in neurons. Furthermore, increasing the phosphorylation of htt at S421 by blocking PP1 and PP2A activity protects YAC128 striatal neurons from NMDA-induced cell death. These results, together with the observed modulation of pS421-htt levels by dopamine, the reduced expression of PP1 inhibitor Darpp-32 in the striatum of YAC128 mice, and the reduced phosphorylation of PP1 substrate CreB, point to altered regulation of phosphatase activity in HD and highlight enhancing phosphorylation of htt at S421 as a therapeutic target.

Huntington disease (HD) is a neurodegenerative disorder caused by an expanded CAG tract in the HD gene. Polyglutamine expansion of huntingtin (htt) results in early, progressive loss of medium spiny striatal neurons, as well as cortical neurons that project to the striatum. Excitotoxicity has been postulated to play a key role in the selective vulnerability of striatal neurons in HD. Early excitotoxic neuropathological changes observed in human HD brain include increased quinolinate (QUIN) concurrent with proliferative changes such as increased spine density and dendritic length. In later stages of the disease, degenerative-type changes are apparent, such as loss of dendritic arborization, a reduction in spine density and reduced levels of 3-hydroxykynurenine and QUIN. It is currently unknown whether sensitivity to excitotoxic stress varies between initiation and progression of disease. Here, we have assessed the excitotoxic phenotype in the YAC128 mouse model of HD by examining the response to excitotoxic stress at different stages of disease. Our results demonstrate that YAC128 mice display enhanced sensitivity to NMDA ex vivo and QUIN in vivo before obvious phenotypic changes. In contrast, 10-month-old symptomatic YAC128 mice are resistant to QUIN-induced neurotoxicity. These findings are paralleled by a significant increase in NMDAR-mediated membrane currents in presymptomatic YAC128 dissociated medium spiny neurons progressing to reduced NMDAR-mediated membrane currents with disease progression. These data highlight the dynamic nature of the mutant htt-mediated excitotoxic phenotype and suggests that therapeutic approaches to HD may need to be altered, depending on the stage and development of the disease.