ALS-linked FUS exerts a gain of toxic function involving aberrant p38 MAPK activation

© The Author(s), 2017. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Scientific Reports 7 (2017): 115, doi:10.1038/s41598-017-00091-1.Mutations in Fused in Sarcoma/Translocated in Liposarcoma (FUS) cause familial forms of amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by progressive axonal degeneration mainly affecting motor neurons. Evidence from transgenic mouse models suggests mutant forms of FUS exert an unknown gain-of-toxic function in motor neurons, but mechanisms underlying this effect remain unknown. Towards this end, we studied the effect of wild type FUS (FUS WT) and three ALS-linked variants (G230C, R521G and R495X) on fast axonal transport (FAT), a cellular process critical for appropriate maintenance of axonal connectivity. All ALS-FUS variants impaired anterograde and retrograde FAT in squid axoplasm, whereas FUS WT had no effect. Misfolding of mutant FUS is implicated in this process, as the molecular chaperone Hsp110 mitigated these toxic effects. Interestingly, mutant FUS-induced impairment of FAT in squid axoplasm and of axonal outgrowth in mammalian primary motor neurons involved aberrant activation of the p38 MAPK pathway, as also reported for ALS-linked forms of Cu, Zn superoxide dismutase (SOD1). Accordingly, increased levels of active p38 MAPK were detected in post-mortem human ALS-FUS brain tissues. These data provide evidence for a novel gain-of-toxic function for ALS-linked FUS involving p38 MAPK activation.We are grateful for funding from NIH/NINDS (R01 NS078145, R01 NS090352, and R21 NS091860 to D.A.B., R01 NS066942A and R21 NS096642 to G.M., R01NS023868 and R01NS041170 to S.T.B.), the ALS Therapy Alliance/CVS Pharmacy (to D.A.B. and G.M.) and the ALS Association (to C.F. and J.M.)

High-efficiency transfection of cultured primary motor neurons to study protein localization, trafficking, and function

<p>Abstract</p> <p>Background</p> <p>Cultured spinal motor neurons are a valuable tool to study basic mechanisms of development, axon growth and pathfinding, and, importantly, to analyze the pathomechanisms underlying motor neuron diseases. However, the application of this cell culture model is limited by the lack of efficient gene transfer techniques which are available for other neurons. To address this problem, we have established magnetofection as a novel method for the simple and efficient transfection of mouse embryonic motor neurons. This technique allows for the study of the effects of gene expression and silencing on the development and survival of motor neurons.</p> <p>Results</p> <p>We found that magnetofection, a novel transfection technology based on the delivery of DNA-coated magnetic nanobeads, can be used to transfect primary motor neurons. Therefore, in order to use this method as a new tool for studying the localization and transport of axonal proteins, we optimized conditions and determined parameters for efficient transfection rates of >45% while minimizing toxic effects on survival and morphology. To demonstrate the potential of this method, we have used transfection with plasmids encoding fluorescent fusion-proteins to show for the first time that the spinal muscular atrophy-disease protein Smn is actively transported along axons of live primary motor neurons, supporting an axon-specific role for Smn that is different from its canonical function in mRNA splicing. We were also able to show the suitability of magnetofection for gene knockdown with shRNA-based constructs by significantly reducing Smn levels in both cell bodies and axons, opening new opportunities for the study of the function of axonal proteins in motor neurons.</p> <p>Conclusions</p> <p>In this study we have established an optimized magnetofection protocol as a novel transfection method for primary motor neurons that is simple, efficient and non-toxic. We anticipate that this novel approach will have a broad applicability in the study of motor neuron development, axonal trafficking, and molecular mechanisms of motor neuron diseases.</p

Bioenergetic status modulates motor neuron vulnerability and pathogenesis in a zebrafish model of spinal muscular atrophy

Degeneration and loss of lower motor neurons is the major pathological hallmark of spinal muscular atrophy (SMA), resulting from low levels of ubiquitously-expressed survival motor neuron (SMN) protein. One remarkable, yet unresolved, feature of SMA is that not all motor neurons are equally affected, with some populations displaying a robust resistance to the disease. Here, we demonstrate that selective vulnerability of distinct motor neuron pools arises from fundamental modifications to their basal molecular profiles. Comparative gene expression profiling of motor neurons innervating the extensor digitorum longus (disease-resistant), gastrocnemius (intermediate vulnerability), and tibialis anterior (vulnerable) muscles in mice revealed that disease susceptibility correlates strongly with a modified bioenergetic profile. Targeting of identified bioenergetic pathways by enhancing mitochondrial biogenesis rescued motor axon defects in SMA zebrafish. Moreover, targeting of a single bioenergetic protein, phosphoglycerate kinase 1 (Pgk1), was found to modulate motor neuron vulnerability in vivo. Knockdown of pgk1 alone was sufficient to partially mimic the SMA phenotype in wild-type zebrafish. Conversely, Pgk1 overexpression, or treatment with terazosin (an FDA-approved small molecule that binds and activates Pgk1), rescued motor axon phenotypes in SMA zebrafish. We conclude that global bioenergetics pathways can be therapeutically manipulated to ameliorate SMA motor neuron phenotypes in vivo

Sistemi a pompa di calore elioassistita : modello di simulazione in ambiente TRNSYS e confronto energetico di configurazioni impiantistiche

LAUREA SPECIALISTICAIl presente lavoro di tesi è volto ad effettuare un’analisi energetica di diverse configurazioni impiantistiche con pompa di calore elioassistita accoppiata indirettamente ai collettori solari mediante circuito ad acqua o ad aria per il riscaldamento invernale di un edificio. Mediante simulazione in ambiente TRNSYS si sono sviluppati modelli sia per pompe di calore ad acqua che ad aria, con i quali, variando parametri come la tipologia e la superficie del campo collettori, si è giunti ad un confronto energetico.This work is about an energetic analysis of different plant solutions with solar assisted indirect heat pump coupled with water or air circuit to heat a building during winter season. Models both water and air heat pumps have been developed by simulations with TRNSYS and an energetic comparison has been made and changing parameters such as type and area of the solar collectors

The 3' untranslated region of human Cyclin-Dependent Kinase 5 Regulatory subunit 1 contains regulatory elements affecting transcript stability

<p>Abstract</p> <p>Background</p> <p><it>CDK5R1 </it>plays a central role in neuronal migration and differentiation during central nervous system development. <it>CDK5R1 </it>has been implicated in neurodegenerative disorders and proposed as a candidate gene for mental retardation. The remarkable size of <it>CDK5R1 </it>3'-untranslated region (3'-UTR) suggests a role in post-transcriptional regulation of <it>CDK5R1 </it>expression.</p> <p>Results</p> <p>The bioinformatic study shows a high conservation degree in mammals and predicts several AU-Rich Elements (AREs). The insertion of <it>CDK5R1 </it>3'-UTR into luciferase 3'-UTR causes a decreased luciferase activity in four transfected cell lines. We identified 3'-UTR subregions which tend to reduce the reporter gene expression, sometimes in a cell line-dependent manner. In most cases the quantitative analysis of luciferase mRNA suggests that CDK5R1 3'-UTR affects mRNA stability. A region, leading to a very strong mRNA destabilization, showed a significantly low half-life, indicating an accelerated mRNA degradation. The 3' end of the transcript, containing a class I ARE, specifically displays a stabilizing effect in neuroblastoma cell lines. We also observed the interaction of the stabilizing neuronal RNA-binding proteins ELAV with the CDK5R1 transcript in SH-SY5Y cells and identified three 3'-UTR sub-regions showing affinity for ELAV proteins.</p> <p>Conclusion</p> <p>Our findings evince the presence of both destabilizing and stabilizing regulatory elements in <it>CDK5R1 </it>3'-UTR and support the hypothesis that <it>CDK5R1 </it>gene expression is post-transcriptionally controlled in neurons by ELAV-mediated mechanisms. This is the first evidence of the involvement of 3'-UTR in the modulation of <it>CDK5R1 </it>expression. The fine tuning of <it>CDK5R1 </it>expression by 3'-UTR may have a role in central nervous system development and functioning, with potential implications in neurodegenerative and cognitive disorders.</p

Modulation of actin polymerization affects nucleocytoplasmic transport in multiple forms of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of unknown etiology. Although defects in nucleocytoplasmic transport (NCT) may be central to the pathogenesis of ALS and other neurodegenerative diseases, the molecular mechanisms modulating the nuclear pore function are still largely unknown. Here we show that genetic and pharmacological modulation of actin polymerization disrupts nuclear pore integrity, nuclear import, and downstream pathways such as mRNA post-transcriptional regulation. Importantly, we demonstrate that modulation of actin homeostasis can rescue nuclear pore instability and dysfunction caused by mutant PFN1 as well as by C9ORF72 repeat expansion, the most common mutation in ALS patients. Collectively, our data link NCT defects to ALS-associated cellular pathology and propose the regulation of actin homeostasis as a novel therapeutic strategy for ALS and other neurodegenerative diseases

A role for the ELAV RNA-binding proteins in neural stem cells : stabilization of Msi1 mRNA

Post-transcriptional regulation exerted by neural-specific RNA-binding proteins plays a pivotal role in the development and maintenance of the nervous system. Neural ELAV proteins are key inducers of neuronal differentiation through the stabilization and/or translational enhancement of target transcripts bearing the AU-rich elements (AREs), whereas Musashi-1 maintains the stem cell proliferation state by acting as a translational repressor. Since the gene encoding Musashi-1 (Msi1) contains a conserved ARE in its 3' untranslated region, the authors focused on the possibility of a mechanistic relation between ELAV proteins and Musashi-1 in cell fate commitment. Colocalization of neural ELAV proteins with Musashi-1 clearly shows that ELAV proteins are expressed at early stages of neural commitment, whereas interaction studies demonstrate that neural ELAV proteins exert an ARE-dependent binding activity on the Msi1 mRNA. This binding activity has functional effects, since the ELAV protein family member HuD is able to stabilize the Msi1 ARE-contg. mRNA in a sequence-dependent way in a deadenylation/degrdn. assay. Furthermore activation of the neural ELAV proteins by phorbol esters in human SH-SY5Y cells is assocd. with an increase of Musashi-1 protein content in the cytoskeleton. The authors propose that ELAV RNA-binding proteins exert an important post-transcriptional control on Musashi-1 expression in the transition from proliferation to neural differentiation of stem/progenitor cells

ALS-associated KIF5A mutations abolish autoinhibition resulting in a toxic gain of function

Understandingthepathogenicmechanismsof diseasemutations is critical toadvancingtreatments.ALS-associated mutations in the gene encoding the microtubulemotor KIF5A result in skipping of exon 27 (KIF5ADExon27) and the encoding of a protein with a novel 39 amino acid residue C-terminal sequence. Here, we report that expression of ALS-linked mutant KIF5A results in dysregulated motor activity, cellular mislocalization, altered axonal transport, and decreased neuronal survival. Single-molecule analysis revealed that the altered C terminus of mutant KIF5A results in a constitutively active state. Furthermore,mutant KIF5A possesses altered protein and RNA interactions and its expression results in altered gene expression/splicing. Taken together, our data support the hypothesis that causative ALS mutations result in a toxic gain of function in the intracellular motor KIF5A that disrupts intracellular trafficking and neuronal homeostasis

Coaggregation of RNA-Binding Proteins in a Model of TDP-43 Proteinopathy with Selective RGG Motif Methylation and a Role for RRM1 Ubiquitination

TAR DNA-binding protein 43 (TDP-43) is a major component within ubiquitin-positive inclusions of a number of neurodegenerative diseases that increasingly are considered as TDP-43 proteinopathies. Identities of other inclusion proteins associated with TDP-43 aggregation remain poorly defined. In this study, we identify and quantitate 35 co-aggregating proteins in the detergent-resistant fraction of HEK-293 cells in which TDP-43 or a particularly aggregate prone variant, TDP-S6, were enriched following overexpression, using stable isotope-labeled (SILAC) internal standards and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). We also searched for differential post-translational modification (PTM) sites of ubiquitination. Four sites of ubiquitin conjugation to TDP-43 or TDP-S6 were confirmed by dialkylated GST-TDP-43 external reference peptides, occurring on or near RNA binding motif (RRM) 1. RRM-containing proteins co-enriched in cytoplasmic granular structures in HEK-293 cells and primary motor neurons with insoluble TDP-S6, including cytoplasmic stress granule associated proteins G3BP, PABPC1, and eIF4A1. Proteomic evidence for TDP-43 co-aggregation with paraspeckle markers RBM14, PSF and NonO was also validated by western blot and by immunocytochemistry in HEK-293 cells. An increase in peptides from methylated arginine-glycine-glycine (RGG) RNA-binding motifs of FUS/TLS and hnRNPs was found in the detergent-insoluble fraction of TDP-overexpressing cells. Finally, TDP-43 and TDP-S6 detergent-insoluble species were reduced by mutagenesis of the identified ubiquitination sites, even following oxidative or proteolytic stress. Together, these findings define some of the aggregation partners of TDP-43, and suggest that TDP-43 ubiquitination influences TDP-43 oligomerization