Regulation of gene expression is critical for neuronal development, survival, differentiation, and axon/dendritic arborization. While several transcription factors have been shown to be important for the development of mesolimbic dopaminergic neurons, it is unclear how such mechanism can be exploited as effective therapeutic means. The goal of this project is to investigate if transcription cofactor homeodomain interacting protein kinase 2 (HIPK2) can be a therapeutic target to enhance the development and/or survival of mesolimbic dopaminergic neurons. Recent results from my lab indicate that HIPK2 is expressed in high abundance in the substantia nigra and about 40-50% of dopaminergic neurons are lost in the substantia nigra of homozygous HIPK2 (HIPK2-/-) mutants. As a consequence, HIPK2-/- mutants are hypoactive and show spontaneous tremor, shuffling gaits, bradykinesia, clasping of hindlimbs (dystonia) and poor motor coordination. These results underscore the important role of HIPK2 for midbrain dopaminergic neurons and lead us to hypothesize that HIPK2-regulated gene expression is essential for the survival and differentiation of neurons in the substantia nigra. Given the recent evidence that the kinase activity of HIPK2 is important for its function, we hypothesize that targeting HIPK2 kinase activity will provide an innovative approach to promote survival and differentiation of midbrain dopaminergic neurons. To test this hypothesis, we propose to use the chemical genetic approach to alter the highly conserved amino acids in HIPK2 to engineer a functionally silent HIPK2 protein that contains a 'bump' in the catalytic domain. Such modified pocket can interact with ATP analogues in a monospecific fashion and selectively transfer radioactive P32 onto the substrates. We will introduce the 'analogue-sensitive' HIPK2 mutant protein into cultured cell lines or dopaminergic neurons to identify the endogenous substrates of HIPK2. Our long-term goal is to use the 'analogue-sensitive' HIPK2 mutant protein and mouse strain to screen for cell-permeable compounds that can activate HIPK2 and thereby enhance survival and differentiation of dopaminergic neurons.