RasGRP1 promotes amphetamine-induced motor behavior through a Rhes interaction network (“Rhesactome”) in the striatum

Shahani, Neelam; Swarnkar, Supriya; Giovinazzo, Vincenzo; Morgenweck, Jenny; Bohn, Laura M.; Scharager-Tapia, Catherina; Pascal, Bruce D.; Martínez-Acedo, Pablo; Khare, Kshitij; Subramaniam, Srinivasa

doi:10.1126/scisignal.aaf6670

Cited by 38 publications

(77 citation statements)

References 102 publications

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“…Briefly the cells were . Open field was conducted as described in our previous study (68). The inverted grip test was performed based on the Treat-NMD guidelines.…”

Section: Ribosome Isolation and In Vitro Binding Assaymentioning

confidence: 99%

Global ribosome profiling reveals that mutant huntingtin stalls ribosomes and represses protein synthesis independent of fragile X mental retardation protein

Eshraghi

Karunadharma

Blin

et al. 2019

Preprint

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The regulators that stall ribosome translocation are poorly understood. We find that polyglutamine-expanded mutant Huntingtin (mHtt), the Huntington's disease (HD) causing protein, promotes ribosome stalling and physiologically suppresses protein synthesis. A comprehensive, genome-wide analysis of ribosome footprint profiling (Ribo-Seq) revealed widespread ribosome stalling on mRNA transcripts and a shift in the distribution of ribosomes toward the 5' end, with single-codon unique pauses on selected mRNAs in HD cells. In Ribo-Seq, we found fragile X mental retardation protein (FMRP), a known regulator of ribosome stalling, translationally upregulated and it coimmunoprecipitated with mHtt in HD cells and postmortem brain. Depletion of FMRP gene, Fmr1, however, did not affect the mHtt-mediated suppression of protein synthesis or ribosome stalling in HD cells. Consistent with this, heterozygous deletion of Fmr1 in Q175FDN-Het mouse model, Q175FDN-Het; Fmr1 +/-, showed no discernable phenotype, but a subtle deficit in motor skill learning. On the other hand, depletion of mHtt, which binds directly to ribosomes in an RNase-sensitive manner, enhanced global protein synthesis, increased ribosome translocation and decreased stalling. This mechanistic knowledge advances our understanding of the inhibitory role of mHtt in ribosome translocation and may lead to novel target(s) identification and therapeutic approaches that modulate ribosome stalling in HD. One Sentence Summary:Huntington's disease (HD) protein, mHtt, binds to ribosomes and affects their translocation and promotes stalling independent of the fragile X mental retardation protein.Main Text:

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“…Briefly the cells were . Open field was conducted as described in our previous study (68). The inverted grip test was performed based on the Treat-NMD guidelines.…”

Section: Ribosome Isolation and In Vitro Binding Assaymentioning

confidence: 99%

Global ribosome profiling reveals that mutant huntingtin stalls ribosomes and represses protein synthesis independent of fragile X mental retardation protein

Eshraghi

Karunadharma

Blin

et al. 2019

Preprint

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“…Moreover, RasGRP1 KO mice displayed no significant changes in basal motor behavior or coordination 16 , as shown in Supplementary Figs 3-5, which would not likely contribute much to the altered L-DOPA responses we observed.…”

Section: Introductionmentioning

confidence: 64%

“…Despite its high expression in brain regions, its role in neuronal functions remains unclear. Previously, we found that RasGRP1 interacts and acts as a GEF for Rhes and promotes amphetamine-induced hyperactivity via the striatal protein-protein complex known as Rhesactome 16 . Here, we have reported a major role for RasGRP1 in L-DOPA-induced dyskinesia and demonstrated that RasGRP1 is causally linked to L-DOPAinduced dyskinesia and robustly activates ERK and mTORC1 pathways in the striatum.…”

Section: Introductionmentioning

confidence: 99%

RasGRP1 (CalDAG-GEF-II) Mediates L-DOPA-induced Dyskinesia in a Mouse Model of Parkinson Disease

Ishragi

Jarquín

Shahani

et al. 2019

Preprint

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The therapeutic benefits of L-3,4-dihydroxyphenylalanine (L-DOPA) in Parkinson disease (PD) patients diminishes with the onset of abnormal involuntary movements (L-DOPA induced dyskinesia), a debilitating motor side effect. L-DOPA induced dyskinesia are due to altered dopaminergic signaling in the striatum, a brain region that controls motor and cognitive functions.However, the molecular mechanisms that promote L-DOPA-induced dyskinesia remain unclear.Here, we have reported that RasGRP1 (also known as CalDAG-GEF-II) physiologically mediated L-DOPA induced dyskinesia in a 6-hydroxy dopamine (6-OHDA) lesioned mouse model of PD. In this study, L-DOPA treatment rapidly upregulated RasGRP1 in the striatum. Our findings showed that RasGRP1 deleted mice (RasGRP1 -/-) had drastically diminished L-DOPAinduced dyskinesia, and RasGRP1 -/mice did not interfere with the therapeutic benefits of L-DOPA. In terms of its mechanism, RasGRP1 mediates L-DOPA-induced extracellular regulated kinase (ERK), the mammalian target of rapamycin kinase (mTOR) and the cAMP/PKA pathway and binds directly with Ras-homolog-enriched in the brain (Rheb), which is a potent activator of mTOR, both in vitro and in the intact striatum. High-resolution tandem mass tag mass spectrometry analysis of striatal tissue revealed significant targets, such as phosphodiesterase (Pde1c), Pde2a, catechol-o-methyltransferase (comt), and glutamate decarboxylase 1 and 2 (Gad1 and Gad2), which are downstream regulators of RasGRP1 and are linked to L-DOPA-induced dyskinesia vulnerability. Collectively, the findings of this study demonstrated that RasGRP1 is a major regulator of L-DOPA-induced dyskinesia in the striatum. Drugs or genedepletion strategies targeting RasGRP1 may offer novel therapeutic opportunities for preventing L-DOPA-induced dyskinesia in PD patients. induced dyskinesia. RESULTS RasGRP1 promoted L-DOPA-induced dyskinesia in a mouse model of Parkinson disease. We hypothesized that RasGRP1 may be an upstream regulator of L-DOPA-induced dyskinesia due to the following reasons: a) L-DOPA treatment of mice with unilateral 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal pathway augmented striatal ERK and mTOR signaling 1-3 ; b) Rhes, a striatal-enriched protein that activates mTOR, is involved in L-DOPA-induced dyskinesia 6, 17, 18 ; and c) RasGRP1 regulated the synaptic localization of Rhes and RasGRP1, and Rhes co-expression strongly activated both ERK and mTORC1 signaling in a cell culture 16 .To test our hypothesis, we subjected WT and RasGRP1 -/-(RasGRP1 KO) mice to a wellestablished 6-OHDA lesion model of L-DOPA-induced dyskinesia as in our earlier study 6 . Fig 1A shows the timeline of the 6-OHDA lesion and L-DOPA-induced dyskinesia analysis. We observed 6-OHDA-induced PD-like symptoms in the drag test, rotarod, and turning test, which were similar between WT and RasGRP1 KO mice (Fig. 1B). The open field test did not show obvious differences (Fig. 1B). Daily treatment of unilaterally-6-OHDA lesioned mice with 5 mg/kg L-DOPA produced dyski...

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“…Mouse STHdh Q7/Q7 (control), STHdh Q7/Q111 (HD-het), and STHdh Q111/Q111 (HD-homo) striatal neuronal cells (37). Striatal cells were cultured in growth medium containing Dulbecco's modified Eagle's medium high glucose (Thermo Fisher Scientific) with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin at 33°C and 5% CO2, as described in our previous works (61,64,67).…”

Section: Cell Culturementioning

confidence: 99%

“…HRP-conjugated secondary antibodies (Jackson ImmunoResearchInc.) were probed to detect bound primary IgG with a chemiluminescence imager (Alpha Innotech) using enhanced chemiluminescence from WesternBright Quantum (Advansta)(67). The band intensities were quantified with ImageJ software (NIH).…”

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confidence: 99%

cGAS, a DNA Sensor, Promotes Inflammatory Responses in Huntington Disease

Sharma

Sumitha

Shahani

et al. 2020

Preprint

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The genetic cause of Huntington disease (HD) is attributed to the N-terminal polyglutamine expansion of huntingtin (mHTT). mHTT, which is a ubiquitously expressed protein, induces noticeable damage to the striatum, which affects motor, psychiatric, and cognitive functions in HD individuals. Although inflammatory responses apparently precede striatal damage and an overall progression of HD, the molecular mechanisms at work remain unclear (1-6). In this study, we found that cyclic GMP-AMP synthase (cGAS), a DNA sensor, which regulates inflammation, autophagy, and cellular senescence (7-9), plays a critical role in the inflammatory responses of HD. Ribosome profiling analysis reveals that cGAS mRNA has a high ribosome occupancy at exon 1 and codon-specific pauses at positions 171 (CCG) and 172 (CGT) in HD cells, compared to the control, indicating an altered cGAS expression. Accordingly, cGAS protein levels and activity, as measured by phosphorylation of stimulator of interferon genes (STING) or TANK-binding kinase 1 (TBK1), are increased in HD striatal cells, mouse Q175HD striatum and human postmortem HD striatum, compared to the healthy control. Furthermore, cGASdependent inflammatory genes such as Cxcl10 and Ccl5 show enhanced ribosome occupancy at exon 3 and exon 1, respectively and are upregulated in HD cells. Depletion of cGAS via CRISPR/Cas-9 diminishes cGAS activity and decreases expression of inflammatory genes while suppressing the autophagy upregulation in HD cells. We additionally detected the presence of numerous micronuclei, a known inducer of cGAS, in the cytoplasm of HD cells. Overall, the data indicates that cGAS is highly upregulated in HD and mediates inflammatory and autophagy responses. Thus, targeting cGAS may offer therapeutic benefits in HD.

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RasGRP1 promotes amphetamine-induced motor behavior through a Rhes interaction network (“Rhesactome”) in the striatum

Cited by 38 publications

References 102 publications

Global ribosome profiling reveals that mutant huntingtin stalls ribosomes and represses protein synthesis independent of fragile X mental retardation protein

Global ribosome profiling reveals that mutant huntingtin stalls ribosomes and represses protein synthesis independent of fragile X mental retardation protein

RasGRP1 (CalDAG-GEF-II) Mediates L-DOPA-induced Dyskinesia in a Mouse Model of Parkinson Disease

cGAS, a DNA Sensor, Promotes Inflammatory Responses in Huntington Disease

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