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Abstract

Huntington's disease is an autosomal dominantly inherited neurodegenerative disorder characterized by progressive motor dysfunction, dementia, psychiatric symptoms, and weight loss, eventually leading to death. Postmortem analysis of the brains of HD patients reveals marked striatal and cortical atrophy, and decreased levels of key neuronal signaling proteins including BNDF. The HD-causing mutation was identified in 1993 as an expanded trinucleotide CAG repeat in the gene encoding the huntingtin protein (htt). Sixteen years have now passed since the identification of the HD-causing gene mutation and the achievements in understanding HD pathology have been numerous. However, there is still no consensus as to which molecular or cellular pathway impairments are the principal drivers of neuronal dysfunction. The need for effective therapies for Huntington's disease motivated us to combine studies of disease etiology with unbiased molecular screens in order to widen the possibilities for identifying new therapeutic agents. We employed a relevant in vitro HD model comprising lentiviral vector (LV)-mediated overexpression of mutant htt in primary striatal cultures. The molecular features of this model reproduce many of the pathological mechanisms previously implicated in HD. Our group has been centrally involved in the discovery of HD-related changes in gene expression; the transcriptiomic signature of HD contains candidate targets for potential therapeutic strategies. Thus, we tested the benefits of restoring the expression levels of HD-downregulated mRNAs. The results, of these studies, provide evidence that the transcriptomic HD signature contains compensatory RNA expression responses that protect neurons against mutant htt's effects. Additional tests of in vitro neuroprotection in primary striatal models of HD included previously identified and novel neuroprotectants. Such compounds not only serve as candidates for drug development but also provide opportunities for gaining improved insights into neuroprotective and neurodegenerative mechanisms in HD. Changes in cerebral cortical function and cortical neuron survival are also major clinical and pathologic aspects of HD. However, there are few models available to assess the specific effects of mutant htt on cortical neuron function, activity, connectivity and viability. We therefore established an in vitro cortical model of HD and assessed the effect of mutant htt expression on neuronal network activity using multielectrode arrays (MEAs). This enabled us to non-invasively monitor the electrical activity from multiple independent sites and to characterize spontaneous firing activity in both HD model and control cells. Our results show that mutant htt causes severe cortical network dysfunction consisting of a decreased occurrence of synchronized burst firing attributable to synaptic impairment. The neurotrophic factor BDNF is believed to be an important regulator of striatal neuron survival, differentiation, and plasticity. Moreover, reduction of BDNF delivery to the striatum has been implicated in the pathophysiology of HD. Nevertheless, many essential aspects of BDNF responses in striatal neurons remain to be elucidated. Therefore, we assessed the relative contributions of multipartite intracellular signaling pathways to the short-term induction of striatal gene expression by BDNF. Striatal genes regulated by BDNF were first identified by DNA microarray analysis, followed by subsequent dissection of signal transduction pathways underlying gene induction using pharmacological agents and quantitative real-time PCR. Our studies have shown that mutant htt may wield striatal effects at multiple levels of the BDNF signaling pathway. These include decreased basal TrkB mRNA expression, decreased Akt activation and complex changes in BDNF-induced gene expression. Increasing Akt activation by expression of a constitutively active form of Akt1 (Myr-Akt1) in striatal neurons not only prevented mutant htt toxicity, but also increased neuronal counts in control cultures, supporting a critical role of Akt1 in neuronal survival. Mutant htt expression has been previously reported to alter Bdnf production in cortical neurons. Here we demonstrated that cortical cultures expressing mutant htt have lower BDNF protein levels and show impaired activity-dependent regulation of Bdnf transcription. Our results indicate that mutant htt can impair signaling pathways controlling both cortical Bndf production and striatal BDNF effects. These altered signaling pathways provide potential therapeutic targets for HD and suggest new mechanisms for decreased neurotrophic support in HD.

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