A Parkinson's disease gene regulatory network identifies the signaling protein RGS2 as a modulator of LRRK2 activity and neuronal toxicity.

Mutations in LRRK2 are one of the primary genetic causes of Parkinson's disease (PD). LRRK2 contains a kinase and a GTPase domain, and familial PD mutations affect both enzymatic activities. However, the signaling mechanisms regulating LRRK2 and the pathogenic effects of familial mutations remain unknown. Identifying the signaling proteins that regulate LRRK2 function and toxicity remains a critical goal for the development of effective therapeutic strategies. In this study, we apply systems biology tools to human PD brain and blood transcriptomes to reverse-engineer a LRRK2-centered gene regulatory network. This network identifies several putative master regulators of LRRK2 function. In particular, the signaling gene RGS2, which encodes for a GTPase-activating protein (GAP), is a key regulatory hub connecting the familial PD-associated genes DJ-1 and PINK1 with LRRK2 in the network. RGS2 expression levels are reduced in the striata of LRRK2 and sporadic PD patients. We identify RGS2 as a novel interacting partner of LRRK2 in vivo. RGS2 regulates both the GTPase and kinase activities of LRRK2. We show in mammalian neurons that RGS2 regulates LRRK2 function in the control of neuronal process length. RGS2 is also protective against neuronal toxicity of the most prevalent mutation in LRRK2, G2019S. We find that RGS2 regulates LRRK2 function and neuronal toxicity through its effects on kinase activity and independently of GTPase activity, which reveals a novel mode of action for GAP proteins. This work identifies RGS2 as a promising target for interfering with neurodegeneration due to LRRK2 mutations in PD patients.

Transcription Factor IIA tau is associated with undifferentiated cells and its gene expression is repressed in primary neurons at the chromatin level in vivo.

The levels of General Transcription Factor (TF) IIA were examined during mammalian brain development and in rat embryo fibroblasts and transformed cell lines. The large TFIIA subunit paralogues alphabeta and tau are largely produced in unsynchronized cell lines, yet only TFIIA alphabeta is observed in a number of differentiated tissue extracts. Steady-state protein levels of the TFIIA tau, alphabeta, and gamma subunits were significantly reduced when human embryonal (ec) and hepatic carcinoma cell lines were stimulated to differentiate with either all-trans-retinoic acid (ATRA) or sodium butyrate. ATRA-treated NT2-ec cells required replating to induce a neuronal phenotype and loss of detectable TFIIA tau and gamma proteins. High levels of TFIIA tau, alphabeta, and gamma and Sp factors were identified in extracts from human fetal and rat embryonic day-18 brains, but not in human and rat adult brain extracts. A high histone H3 Lys9/Lys4 methylation ratio was observed in the TFIIA tau promoter of primary hippocampal neurons from day-18 rat embryos, suggesting that repressive epigenetic marks of chromatin prevent TFIIA tau from being transcribed in neurons. We conclude that TFIIA tau is associated with undifferentiated cells during development, yet is down-regulated at the chromatin level upon cellular differentiation.

Similar patterns of mitochondrial vulnerability and rescue induced by genetic modification of alpha-synuclein, parkin, and DJ-1 in Caenorhabditis elegans.

How genetic and environmental factors interact in Parkinson disease is poorly understood. We have now compared the patterns of vulnerability and rescue of Caenorhabditis elegans with genetic modifications of three different genetic factors implicated in Parkinson disease (PD). We observed that expressing alpha-synuclein, deleting parkin (K08E3.7), or knocking down DJ-1 (B0432.2) or parkin produces similar patterns of pharmacological vulnerability and rescue. C. elegans lines with these genetic changes were more vulnerable than nontransgenic nematodes to mitochondrial complex I inhibitors, including rotenone, fenperoximate, pyridaben, or stigmatellin. In contrast, the genetic manipulations did not increase sensitivity to paraquat, sodium azide, divalent metal ions (Fe(II) or Cu(II)), or etoposide compared with the nontransgenic nematodes. Each of the PD-related lines was also partially rescued by the antioxidant probucol, the mitochondrial complex II activator, D-beta-hydroxybutyrate, or the anti-apoptotic bile acid tauroursodeoxycholic acid. Complete protection in all lines was achieved by combining d-beta-hydroxybutyrate with tauroursodeoxycholic acid but not with probucol. These results show that diverse PD-related genetic modifications disrupt the mitochondrial function in C. elegans, and they raise the possibility that mitochondrial disruption is a pathway shared in common by many types of familial PD.

Up-regulation of NMDAR1 subunit gene expression in cortical neurons via a PKA-dependent pathway.

Transcription mediated by protein kinase A and the cAMP response element binding protein (CREB) has been linked to the establishment of long-term memory and cell survival. However, all of the major targets for activated CREB have yet to be identified. Given the fact that CREB-mediated transcription is intimately involved in cellular processes of learning and memory and that CREB activity can be regulated by synaptic N-methyl-d-aspartate receptors (NMDARs) and metabotropic GABA receptors, we have studied the role of the cAMP-dependent signaling pathway in the regulation of the NMDA receptor subunit 1 (NMDAR1), a subunit required for functional receptor formation. We now report that levels of NMDAR1 subunit protein in primary neocortical cultures are increased 66% in response to forskolin, an activator of adenylyl cyclase. Up-regulation of NMDAR1 is paralleled by a twofold increase in mRNA levels and an 83% increase in NMDAR1 promoter/luciferase reporter activity that is dependent on protein kinase A. Three cAMP regulatory elements (CREs) in the rat NMDAR1 promoter (- 228, - 67, and - 39) bind CREB in vitro and forskolin increases binding to two of the sites (- 228 and - 67). Chromatin immunoprecipitation of neuronal rat genomic DNA reveals that CREB is bound in vivo to the endogenous NMDAR1 gene. Increased presence of the activated Ser133 phosphorylated form is dependent on the length of exposure to forskolin. Taken together with the results of mutational analysis, the findings strongly suggest that transcription of NMDAR1 is regulated by the c-AMP signaling pathway, most likely through the binding of CREB and its activation by signal-dependent phosphorylation.