LRRK2 and RAB7L1 coordinately regulate axonal morphology and lysosome integrity in diverse cellular contexts.

Leucine-rich repeat kinase 2 (LRRK2) has been linked to several clinical disorders including Parkinson's disease (PD), Crohn's disease, and leprosy. Furthermore in rodents, LRRK2 deficiency or inhibition leads to lysosomal pathology in kidney and lung. Here we provide evidence that LRRK2 functions together with a second PD-associated gene, RAB7L1, within an evolutionarily conserved genetic module in diverse cellular contexts. In C. elegans neurons, orthologues of LRRK2 and RAB7L1 act coordinately in an ordered genetic pathway to regulate axonal elongation. Further genetic studies implicated the AP-3 complex, which is a known regulator of axonal morphology as well as of intracellular protein trafficking to the lysosome compartment, as a physiological downstream effector of LRRK2 and RAB7L1. Additional cell-based studies implicated LRRK2 in the AP-3 complex-related intracellular trafficking of lysosomal membrane proteins. In mice, deficiency of either RAB7L1 or LRRK2 leads to prominent age-associated lysosomal defects in kidney proximal tubule cells, in the absence of frank CNS pathology. We hypothesize that defects in this evolutionarily conserved genetic pathway underlie the diverse pathologies associated with LRRK2 in humans and in animal models.

Mutations in LRRK2 potentiate age-related impairment of autophagic flux.

Autophagy is thought to play a pivotal role in the pathophysiology of Parkinson's disease, but little is known about how genes linked to PD affect autophagy in the context of aging. We generated lines of C. elegans expressing reporters for the autophagosome and lysosome expressed only in dopaminergic neurons, and examined autophagy throughout the lifespan in nematode lines expressing LRRK2 and α-synuclein. Dopamine neurons exhibit a progressive loss of autophagic function with aging. G2019S LRRK2 inhibited autophagy and accelerated the age-related loss of autophagic function, while WT LRRK2 improved autophagy throughout the life-span. Expressing α-synuclein with G2019S or WT LRRK2 caused age-related synergistic inhibition of autophagy and increase in degeneration of dopaminergic neurons. The presence of α-synuclein particularly accentuated age-related inhibition of autophagy by G2019S LRRK2. This work indicates that LRRK2 exhibits a selective, age-linked deleterious interaction with α-synuclein that promotes neurodegeneration.

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.

Regulation of autophagy by LRRK2 in Caenorhabditis elegans.

BACKGROUND: Mutations in LRRK2 (leucine-rich repeat kinase 2) are a common cause of familial Parkinson's disease. However, the mechanisms through which LRRK2 mutations contribute to neurodegeneration are poorly understood. OBJECTIVE: We investigated the effects of WT, G2019S (GS), R1441C (RC) and kinase dead LRRK2 across multiple different cellular compartments in order to gain insight into the breadth of LRRK2 effects on cellular function. METHODS: Nematodes expressing lgg-1::RFP, hsp1::GFP, hsp4::GFP and hsp6::GFP were crossed to nematode lines expressing WT, GS, RC or kinase dead LRRK2. RESULTS: We observed that GS and RC LRRK2 inhibited autophagy, while WT, GS and RC LRRK2 increased the response of the mitochondrial hsp6 reporter to stress. The response of the hsp reporters under basal conditions was more nuanced. CONCLUSION: These results support a putative role of LRRK2 in the autophagic and mitochondrial systems.

Polycomblike protein PHF1b: a transcriptional sensor for GABA receptor activity.

BACKGROUND: The γ-aminobutyric acid (GABA) type A receptor (GABA(A)R) contains the recognition sites for a variety of agents used in the treatment of brain disorders, including anxiety and epilepsy. A better understanding of how receptor expression is regulated in individual neurons may provide novel opportunities for therapeutic intervention. Towards this goal we have studied transcription of a GABA(A)R subunit gene (GABRB1) whose activity is autologously regulated by GABA via a 10 base pair initiator-like element (ß(1)-INR). METHODS: By screening a human cDNA brain library with a yeast one-hybrid assay, the Polycomblike (PCL) gene product PHD finger protein transcript b (PHF1b) was identified as a ß(1)-INR associated protein. Promoter/reporter assays in primary rat cortical cells demonstrate that PHF1b is an activator at GABRB1, and chromatin immunoprecipitation assays reveal that presence of PHF1 at endogenous Gabrb1 is regulated by GABA(A)R activation. RESULTS: PCL is a member of the Polycomb group required for correct spatial expression of homeotic genes in Drosophila. We now show that PHF1b recognition of ß(1)-INR is dependent on a plant homeodomain, an adjacent helix-loop-helix, and short glycine rich motif. In neurons, it co-immunoprecipitates with SUZ12, a key component of the Polycomb Repressive Complex 2 (PRC2) that regulates a number of important cellular processes, including gene silencing via histone H3 lysine 27 trimethylation (H3K27me3). CONCLUSIONS: The observation that chronic exposure to GABA reduces PHF1 binding and H3K27 monomethylation, which is associated with transcriptional activation, strongly suggests that PHF1b may be a molecular transducer of GABA(A)R function and thus GABA-mediated neurotransmission in the central nervous system.

MKK6 binds and regulates expression of Parkinson's disease-related protein LRRK2.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are prevalent causes of late-onset Parkinson's disease. Here, we show that LRRK2 binds to MAPK kinases (MKK) 3, 6, and 7, and that LRRK2 is able to phosphorylate MKK3, 6 and 7. Over-expression of LRRK2 and MKK6 increased the steady state levels of each protein beyond that observed with over-expression of either protein alone. Co-expression increased levels of MKK6 in the membrane more than in the cytoplasm. The increased expression of LRRK2 and MKK6 requires MKK6 activity. The disease-linked LRRK2 mutations, G2019S, R1441C and I2020T, enhance binding of LRRK2 to MKK6. This interaction was further supported by in vivo studies in C. elegans. RNAi knockdown in C. elegans of the endogenous orthologs for MKK6 or p38, sek-1 and pmk-1, abolishes LRRK2-mediated protection against mitochondrial stress. These results were confirmed by deletion of sek-1 in C. elegans. These data demonstrate that MKKs and LRRK2 function in similar biological pathways, and support a role for LRRK2 in modulating the cellular stress response.

LRRK2 modulates vulnerability to mitochondrial dysfunction in Caenorhabditis elegans.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal-dominant familial Parkinson's disease. We generated lines of Caenorhabditis elegans expressing neuronally directed human LRRK2. Expressing human LRRK2 increased nematode survival in response to rotenone or paraquat, which are agents that cause mitochondrial dysfunction. Protection by G2019S, R1441C, or kinase-dead LRRK2 was less than protection by wild-type LRRK2. Knockdown of lrk-1, the endogenous ortholog of LRRK2 in C. elegans, reduced survival associated with mitochondrial dysfunction. C. elegans expressing LRRK2 showed rapid loss of dopaminergic markers (DAT::GFP fluorescence and dopamine levels) beginning in early adulthood. Loss of dopaminergic markers was greater for the G2019S LRRK2 line than for the wild-type line. Rotenone treatment induced a larger loss of dopamine markers in C. elegans expressing G2019S LRRK2 than in C. elegans expressing wild-type LRRK2; however, loss of dopaminergic markers in the G2019S LRRK2 nematode lines was not statistically different from that in the control line. These data suggest that LRRK2 plays an important role in modulating the response to mitochondrial inhibition and raises the possibility that mutations in LRRK2 selectively enhance the vulnerability of dopaminergic neurons to a stressor associated with Parkinson's disease.

Investigating convergent actions of genes linked to familial Parkinson's disease.

BACKGROUND: Mutations in LRRK2 are among the most frequent genetic changes identified in Parkinson's disease (PD), but how LRRK2 contributes to the pathophysiology of PD is not known. OBJECTIVES: To investigate how expressing wild-type or G2019S LRRK2 modifies cellular responses to rotenone, a mitochondrial toxin. METHODS: We investigated the vulnerability to mitochondrial toxins in Caenorhabditis elegans expressing wild-type or G2019S LRRK2. RESULTS: We observed a powerful role for LRRK2 in mitochondrial biology. Overexpressing LRRK2 strongly protects C. elegans against rotenone toxicity. The G2019S LRRK2 construct also protected LRRK2 against rotenone, but to a lesser degree than wild-type LRRK2. Knockdown of lrk-1 potentiated rotenone toxicity. CONCLUSIONS: These data suggest that LRRK1/2 regulate mitochondrial physiology.

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.

RNA sequences that work as transcriptional activating regions.

We describe a set of RNA molecules that work as transcriptional activators when tethered to DNA. These RNA activating regions were found amongst a randomized set of molecules bearing variants of a 10 nt loop attached to an RNA stem. The various RNA activating regions all bear an identical five- residue sequence with an interspersed sixth residue. The result shows that although all natural activating regions characterized thus far are peptidic, this function can be served by other kinds of moieties as well.