An expanded CAG repeat is the underlying genetic defect in Huntington disease, a disorder characterized by motor, psychiatric and cognitive deficits and striatal atrophy associated with neuronal loss. An accurate animal model of this disease is crucial for elucidation of the underlying natural history of the illness and also for testing experimental therapeutics. We established a new yeast artificial chromosome (YAC) mouse model of HD with the entire human HD gene containing 128 CAG repeats (YAC128) which develops motor abnormalities and age-dependent brain atrophy including cortical and striatal atrophy associated with striatal neuronal loss. YAC128 mice exhibit initial hyperactivity, followed by the onset of a motor deficit and finally hypokinesis. The motor deficit in the YAC128 mice is highly correlated with striatal neuronal loss, providing a structural correlate for the behavioral changes. The natural history of HD-related changes in the YAC128 mice has been defined, demonstrating the presence of huntingtin inclusions after the onset of behavior and neuropathological changes. The HD-related phenotypes of the YAC128 mice show phenotypic uniformity with low inter-animal variability present, which together with the age-dependent striatal neurodegeneration make it an ideal mouse model for the assessment of neuroprotective and other therapeutic interventions.

Cleavage of huntingtin (htt) has been characterized in vitro, and accumulation of caspase cleavage fragments represents an early pathological change in brains of Huntington's disease (HD) patients. However, the relationship between htt proteolysis and the pathogenesis of HD is unknown. To determine whether caspase cleavage of htt is a key event in the neuronal dysfunction and selective neurodegeneration in HD, we generated YAC mice expressing caspase-3- and caspase-6-resistant mutant htt. Mice expressing mutant htt, resistant to cleavage by caspase-6 but not caspase-3, maintain normal neuronal function and do not develop striatal neurodegeneration. Furthermore, caspase-6-resistant mutant htt mice are protected against neurotoxicity induced by multiple stressors including NMDA, quinolinic acid (QA), and staurosporine. These results are consistent with proteolysis of htt at the caspase-6 cleavage site being an important event in mediating neuronal dysfunction and neurodegeneration and highlight the significant role of htt proteolysis and excitotoxicity in HD.

We have produced yeast artificial chromosome (YAC) transgenic mice expressing normal (YAC18) and mutant (YAC46 and YAC72) huntingtin (htt) in a developmental and tissue-specific manner identical to that observed in Huntington's disease (HD). YAC46 and YAC72 mice show early electrophysiological abnormalities, indicating cytoplasmic dysfunction prior to observed nuclear inclusions or neurodegeneration. By 12 months of age, YAC72 mice have a selective degeneration of medium spiny neurons in the lateral striatum associated with the translocation of N-terminal htt fragments to the nucleus. Neurodegeneration can be present in the absence of macro- or microaggregates, clearly showing that aggregates are not essential to initiation of neuronal death. These mice demonstrate that initial neuronal cytoplasmic toxicity is followed by cleavage of htt, nuclear translocation of htt N-terminal fragments, and selective neurodegeneration.

Plasma HDL cholesterol levels are inversely related to risk for atherosclerosis. The ATP-binding cassette, subfamily A, member 1 (ABCA1) mediates the rate-controlling step in HDL particle formation, the assembly of free cholesterol and phospholipids with apoA-I. ABCA1 is expressed in many tissues; however, the physiological functions of ABCA1 in specific tissues and organs are still elusive. The liver is known to be the major source of plasma HDL, but it is likely that there are other important sites of HDL biogenesis. To assess the contribution of intestinal ABCA1 to plasma HDL levels in vivo, we generated mice that specifically lack ABCA1 in the intestine. Our results indicate that approximately 30% of the steady-state plasma HDL pool is contributed by intestinal ABCA1 in mice. In addition, our data suggest that HDL derived from intestinal ABCA1 is secreted directly into the circulation and that HDL in lymph is predominantly derived from the plasma compartment. These data establish a critical role for intestinal ABCA1 in plasma HDL biogenesis in vivo.Introduction HDL particles mediate the transport of cholesterol from peripheral tissues to the liver in a process termed reverse cholesterol transport (1, 2), which is postulated to explain, at least in part, their ability to protect against foam cell formation and atherosclerosis. Despite the widespread interest in HDL as a potential therapeutic target (3), the origins of plasma HDL are still elusive. The ATP-binding cassette, subfamily A, member 1 (ABCA1) mediates the rate-controlling step in HDL particle formation by promoting the efflux of cholesterol and phospholipids to apoA-I (4, 5). Mutations in ABCA1 cause Tangier disease (6-8), characterized by near absence of HDL cholesterol and increased risk for atherosclerosis (9-11). ABCA1 is widely expressed throughout the body (12, 13); however, the contributions of ABCA1 in specific tissues to HDL levels and reverse cholesterol transport are still being unraveled, and only recently the role of hepatic ABCA1 in homeostasis of HDL levels was elucidated.Overexpression of hepatic ABCA1 raises HDL cholesterol levels (14, 15), and liver-specific deletion of ABCA1 results in a substantial (∼80%) decrease in plasma HDL cholesterol in chow-fed mice (16). Similarly, a 50% knockdown of hepatic ABCA1 expression by adenovirus-mediated RNA interference in mice is associated with a 40% decrease in HDL cholesterol (17). These results indicate that the liver is the single most important source of plasma HDL in vivo but also suggest the existence of additional, extrahepatic sites of HDL biogenesis.The intestine, along with the liver, is an important site for the synthesis and secretion of apoA-I, the principal apoprotein of HDL,

We have serendipitously established a mouse that expresses an N-terminal human huntingtin (htt) fragment with an expanded polyglutamine repeat (Ϸ120) under the control of the endogenous human promoter (shortstop). Frequent and widespread htt inclusions occur early in shortstop mice. Despite these inclusions, shortstop mice display no clinical evidence of neuronal dysfunction and no neuronal degeneration as determined by brain weight, striatal volume, and striatal neuronal count. These results indicate that htt inclusions are not pathogenic in vivo. In contrast, the full-length yeast artificial chromosome (YAC) 128 model with the identical polyglutamine length and same level of transgenic protein expression as the shortstop demonstrates significant neuronal dysfunction and loss. In contrast to the YAC128 mouse, which demonstrates enhanced susceptibility to excitotoxic death, the shortstop mouse is protected from excitotoxicity, providing in vivo evidence suggesting that neurodegeneration in Huntington disease is mediated by excitotoxic mechanisms.Huntington disease ͉ mouse models ͉ excitotoxicity ͉ aggregates ͉ fragment H untingtin (htt), the protein product encoded by the gene mutated in Huntington disease (HD), forms aggregates and inclusion bodies in the presence of a pathogenic expanded polyglutamine (polyQ) repeat. Htt protein inclusions are a hallmark of HD and are present in brains of human patients (1), in HD mouse models (2, 3), and in cell culture models of HD (4). It is still controversial whether htt inclusions are pathogenic (2), benign biomarkers (5), or neuroprotective (4, 6). The distinction between these hypotheses is clinically relevant, because much therapeutic research has focused on screening compounds for their ability to inhibit inclusion formation (7,8). A decrease in inclusion formation has been interpreted as a positive outcome in preclinical therapeutic trials with mouse models (9, 10).Increasing evidence in vitro in cell culture models supports the hypothesis that htt inclusions are not pathogenic (5, 11). In a recent study, Arrasate et al. (4) discovered that in their cell culture system, neurons with inclusions had an increased likelihood of survival compared with neurons without inclusions. However, because these results were obtained in a cell culture system, the question of whether htt inclusions are toxic in vivo during the lifespan of an organism and therefore clinically relevant for patients with HD remains unanswered.Examinations of inclusions in brains from HD patients are limited due to the inability to sample inclusions over the natural history of the disease, and, therefore, studies of mouse models of HD can be useful in determining the role of htt inclusions in vivo. The yeast artificial chromosome (YAC) 128 model of HD, which expresses full-length mutant htt, forms intranuclear inclusions 12 months after the onset of behavioral changes measured by rotarod and 6 months after striatal neuronal degeneration (3).During the development of the full-length YAC mouse models, a m...

Mutations in ABCA1 cause the allelic disorders familial hypolipoproteinemia and Tangier Disease. To identify where ABCA1 was likely to have a functional role, we determined the cellular and tissue-specific patterns of murine ABCA1 expression. RT-PCR and Western blot analysis on dissected murine tissues demonstrated broad expression of ABCA1 mRNA and protein in many tissues with prominent protein expression in liver, testis, and adrenal tissue. In situ hybridization and immunohistochemistry experiments demonstrated specific patterns of ABCA1 expression at the cellular level, with hepatocytes, the epithelial lining of the digestive system and bladder, the proximal convoluted tubule of the kidney, and Purkinje and cortical pyramidal neurons containing abundant ABCA1 protein. Significant discordance between relative mRNA and protein expression patterns suggests the possibility of post-transcriptional regulation of ABCA1 expression in selected cells or tissues. We also show that ABCA1 protein levels are up-regulated specifically in the liver after exposure to an atherogenic diet for 7 days, supporting a major role for the liver in dietary modulation of HDL-C levels. Our observations show that ABCA1 is expressed in a pattern consistent with its role in HDL-C metabolism. Additionally, ABCA1 may have important functional roles in other cell types independent of HDL-C regulation.

By using BAC transgenic mice, we have shown that increased human ABCA1 protein expression results in a significant increase in cholesterol efflux in different tissues and marked elevation in high density lipoprotein (HDL)-cholesterol levels associated with increases in apoAI and apoAII. Three novel ABCA1 transcripts containing three different transcription initiation sites that utilize sequences in intron 1 have been identified. In BAC transgenic mice there is an increased expression of ABCA1 protein, but the distribution of the ABCA1 product in different cells remains similar to wild type mice. An internal promoter in human intron 1 containing liver X response elements is functional in vivo and directly contributes to regulation of the human ABCA1 gene in multiple tissues and to raised HDL cholesterol, apoAI, and apoAII levels. A highly significant relationship between raised protein levels, increased efflux, and level of HDL elevation is evident. These data provide proof of the principle that increased human ABCA1 efflux activity is associated with an increase in HDL levels in vivo.

Huntingtin interacting protein 14 (HIP14, ZDHHC17) is a huntingtin (HTT) interacting protein with palmitoyl transferase activity. In order to interrogate the function of Hip14, we generated mice with disruption in their Hip14 gene. Hip14-/- mice displayed behavioral, biochemical and neuropathological defects that are reminiscent of Huntington disease (HD). Palmitoylation of other HIP14 substrates, but not Htt, was reduced in the Hip14-/- mice. Hip14 is dysfunctional in the presence of mutant htt in the YAC128 mouse model of HD, suggesting that altered palmitoylation mediated by HIP14 may contribute to HD.