Transgenic mice expressing caspase-6-derived N-terminal fragments of mutant huntingtin develop neurologic abnormalities with predominant cytoplasmic inclusion pathology composed largely of a smaller proteolytic derivative.

Recent studies have implicated an N-terminal caspase-6 cleavage product of mutant huntingtin (htt) as an important mediator of toxicity in Huntington's disease (HD). To directly assess the consequences of such fragments on neurologic function, we produced transgenic mice that express a caspase-6 length N-terminal fragment of mutant htt (N586) with both normal (23Q) and disease (82Q) length glutamine repeats. In contrast to mice expressing N586-23Q, mice expressing N586-82Q accumulate large cytoplasmic inclusion bodies that can be visualized with antibodies to epitopes throughout the N586 protein. However, biochemical analyses of aggregated mutant huntingtin in these mice demonstrated that the inclusion bodies are composed largely of a much smaller htt fragment (terminating before residue 115), with lesser amounts of full-length N586-82Q fragments. Mice expressing the N586-82Q fragment show symptoms typical of previously generated mice expressing mutant huntingtin fragments, including failure to maintain weight, small brain weight and reductions in specific mRNAs in the striatum. Uniquely, these N586-82Q mice develop a progressive movement disorder that includes dramatic deficits in motor performance on the rotarod and ataxia. Our findings suggest that caspase-6-derived fragments of mutant htt are capable of inducing novel HD-related phenotypes, but these fragments are not terminal cleavage products as they are subject to further proteolysis. In this scenario, mutant htt fragments derived from caspase 6, or possibly other proteases, could mediate HD pathogenesis via a 'hit and run' type of mechanism in which caspase-6, or other larger N-terminal fragments, mediate a neurotoxic process before being cleaved to a smaller fragment that accumulates pathologically.

Longitudinal behavioral, cross-sectional transcriptional and histopathological characterization of a knock-in mouse model of Huntington's disease with 140 CAG repeats.

The discovery of the gene mutation responsible for Huntington's disease (HD), huntingtin, in 1993 allowed for a better understanding of the pathology of and enabled the development of animal models. HD is caused by the expansion of a polyglutamine repeat region in the N-terminal of the huntingtin protein. Here we examine the behavioral, transcriptional, histopathological and anatomical characteristics of a knock-in HD mouse model with a 140 polyglutamine expansion in the huntingtin protein. This CAG 140 model contains a portion of the human exon 1 with 140 CAG repeats knocked into the mouse huntingtin gene. We have longitudinally examined the rearing behavior, accelerating rotarod, constant speed rotarod and gait for age-matched heterozygote, homozygote and non-transgenic mice and have found a significant difference in the afflicted mice. However, while there were significant differences between the non-transgenic and the knock-in mice, these behaviors were not progressive. As in HD, we show that the CAG 140 mice also have a significant decrease in striatally enriched mRNA transcripts. In addition, striatal neuronal intranuclear inclusion density increases with age. Lastly these CAG 140 mice show slight cortical thinning compared to non-transgenic mice, similarly to the cortical thinning recently reported in HD.

Tight Long-term dynamic doxycycline responsive nigrostriatal GDNF using a single rAAV vector.

Glial cell line-derived neurotrophic factor (GDNF) gene transfer is being developed as a treatment for Parkinson's disease (PD). Due to the potential for side effects, external transgene regulation should enhance this strategy's safety profile. Here, we demonstrate dynamic control during long-term expression of GDNF using a recombinant adeno-associated virus (rAAV)-based bicistronic tetracycline (tet)-off construct. Nigrostriatal GDNF overexpression induces body weight alterations in rodents, enabling longitudinal in vivo tracking of GDNF expression after nigral vector delivery. Regulated GDNF expression was highly sensitive to dietary doxycycline (DOX), displaying undetectable striatal GDNF levels at serum DOX levels below those required for antimicrobial activity. However, in the absence of DOX, striatal GDNF levels exceeded levels required for efficacy in PD models. We also demonstrate the absence of a series of known GDNF-associated side effects when using direct intrastriatal vector delivery. Therefore, this single rAAV vector system meets most of the requirements for an experimental reagent for treatment of PD.

Nigrostriatal rAAV-mediated GDNF overexpression induces robust weight loss in a rat model of age-related obesity.

Intraventricular administration of glial cell line-derived neurotrophic factor (GDNF) in primate and humans to study Parkinson's disease (PD) has revealed the potential for GDNF to induce weight loss. Our previous data indicate that bilateral continuous hypothalamic GDNF overexpression via recombinant adeno-associated virus (rAAV) results in significant failure to gain weight in young rats and weight loss in aged rats. Based on these previous results, we hypothesized that because the nigrostriatal tract passes through the lateral hypothalamus, motor hyperactivity mediated by nigrostriatal dopamine (DA) may have been responsible for the previously observed effect on body weight. In this study, we compared bilateral injections of rAAV2/5-GDNF in hypothalamus versus substantia nigra (SN) in aged Brown-Norway X Fisher 344 rats. Nigrostriatal GDNF overexpression resulted in significantly greater weight loss than rats treated in hypothalamus. The nigral or hypothalamic GDNF-induced weight loss was unrelated to motor activity levels of the rats, though some of the weight loss could be attributed to a transient reduction in food intake. Forebrain DA levels did not account for the observed effects on body weight, although GDNF-induced increases in nucleus accumbens DA may have partially contributed to this effect in the hypothalamic GDNF-treated group. However, only nigrostriatal GDNF overexpression induced activation of phosphorylated extracellular signal-regulated kinase (p-ERK) in a small population of corticotrophin-releasing factor [corticotrophin-releasing hormone (CRH)] neurons located specifically in the medial parvocellullar division (MPD) of the paraventricular nucleus of the hypothalamus. Activation of these hypothalamic CRH neurons likely accounted for the observed metabolic effects leading to weight loss in obese rats.