leupeptins and Spinocerebellar-Ataxias

Activation of G-protein coupled receptors elevates cAMP levels promoting dissociation of protein kinase A (PKA) holoenzymes and release of catalytic subunits (PKAc). This results in PKAc-mediated phosphorylation of compartmentalized substrates that control central aspects of cell physiology. The mechanism of PKAc activation and signaling have been largely characterized. However, the modes of PKAc inactivation by regulated proteolysis were unknown. Here, we identify a regulatory mechanism that precisely tunes PKAc stability and downstream signaling. Following agonist stimulation, the recruitment of the chaperone-bound E3 ligase CHIP promotes ubiquitylation and proteolysis of PKAc, thus attenuating cAMP signaling. Genetic inactivation of CHIP or pharmacological inhibition of HSP70 enhances PKAc signaling and sustains hippocampal long-term potentiation. Interestingly, primary fibroblasts from autosomal recessive spinocerebellar ataxia 16 (SCAR16) patients carrying germline inactivating mutations of CHIP show a dramatic dysregulation of PKA signaling. This suggests the existence of a negative feedback mechanism for restricting hormonally controlled PKA activities.

Topics: Animals; Cyclic AMP; Cyclic AMP-Dependent Protein Kinase Catalytic Subunits; Feedback, Physiological; Fibroblasts; HEK293 Cells; Hippocampus; Holoenzymes; HSP70 Heat-Shock Proteins; Humans; Leupeptins; Mice; Mice, Inbred C57BL; Molecular Chaperones; Phosphorylation; Primary Cell Culture; Protein Binding; Proteolysis; Purine Nucleosides; Signal Transduction; Spinocerebellar Ataxias; Ubiquitin-Protein Ligases; Ubiquitination

Spinocerebellar ataxia type 8 (SCA8) involves bidirectional expression of CUG (ATXN8OS) and CAG (ATXN8) expansion transcripts. The pathogenesis of SCA8 is complex and the spectrum of clinical presentations is broad. In the present study, we assessed the SCA8 repeat size ranges in Taiwanese Parkinson's disease, Alzheimer's disease and atypical parkinsonism and investigated the genetic variation modulating ATXN8 expression. Thirteen large SCA8 alleles and a novel ATXN8 -62 G/A promoter SNP were found. There is a significant difference in the proportion of the individuals carrying SCA8 larger alleles in atypical parkinsonism (P = 0.044) as compared to that in the control subjects. In lymphoblastoid cells carrying SCA8 large alleles, treatment of MG-132 or staurosporine significantly increases the cell death or caspase 3 activity. Although expressed at low steady-state, ATXN8 expression level is significantly higher (P = 0.012) in cells with SCA8 large alleles than that of the control cells. The ATXN8 transcriptional activity was significantly higher in the luciferase reporter construct containing the -62G allele than that containing the -62A allele in both neuroblastoma and embryonic kidney cells. Therefore, our preliminary results suggest that ATXN8 gene -62 G/A polymorphism may be functional in modulating ATXN8 expression.

Topics: Adult; Aged; Aged, 80 and over; Alzheimer Disease; Base Sequence; Case-Control Studies; Cell Line; Central Nervous System Diseases; DNA Primers; Female; Gene Expression; Humans; Leupeptins; Lymphocytes; Male; Microfilament Proteins; Middle Aged; Nerve Tissue Proteins; Parkinson Disease; Parkinsonian Disorders; Polymorphism, Single Nucleotide; Pyrimidines; RNA, Long Noncoding; RNA, Untranslated; Spinocerebellar Ataxias; Staurosporine; Sulfonamides; Trinucleotide Repeat Expansion