Over 95% of the neurons that make up the striatum (caudate and nucleus accumbens) are medium size spiny neurons (MSNs). All MSNs are dopaminoceptive; i.e. they receive dopamine input and express one or more of the dopamine receptor subtypes. Dysregulation of gene expression in MSNs is implicated in the pathophysiology and treatment of many neuropsychiatric diseases, including Huntington's disease, Parkinson's disease, drug addiction, affective disorders, attention deficit hyperactivity disorder, and schizophrenia; despite this, few details are available regarding how the differentiated MSN phenotype is specified at the molecular level. The dopamine and cyclic AMP-regulated phosphoprotein, 32 kDa (DARPP- 32) is the most widely used marker of the differentiated MSN, and DARPP-32 is a key modulator of MSN functions, especially those mediated by dopamine. DARPP-32 levels are highly influenced in vivo and in vitro by levels of brain-derived neurotrophic factor (BDNF'), and BDNF regulates DARPP-32 expression via the phosphatidylinositoi 3-kinase (PI3K) and p35/cdk5 pathways. The objectives of this proposal are:
SPECIFIC AIM 1 To identify the cis-acting sequence(s) within the 9Kb DARPP-32 genomic fragment identified in this laboratory that direct(s) transgene expression to the MSNs, using transgenesis, transient transfection, electrophoretic mobility shift assays and DNase footprinting, with particular attention to distinctions between striatal patch and matrix compartments.
SPECIFIC AIM 2 To determine in vitro: A) whether the DNA cis-acting regions mediating MSN-specific transcription also mediate regulation by BDNF and PI3-K within these neurons; B) the relationship between PI3-K, Akt and cdk5 in the regulation of DARPP-32 expression utilizing primary neuronal cultures, transfection, antisense oligonucleotides and viral transduction.
