Axon guidance genes modulate neurotoxicity of ALS-associated UBQLN2

Mutations in the ubiquitin (Ub) chaperone Ubiquilin 2 (UBQLN2) cause X-linked forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) through unknown mechanisms. Here, we show that aggregation-prone, ALS-associated mutants of UBQLN2 (UBQLN2ALS) trigger heat stress-dependent neurodegeneration in Drosophila. A genetic modifier screen implicated endolysosomal and axon guidance genes, including the netrin receptor, Unc-5, as key modulators of UBQLN2 toxicity. Reduced gene dosage of Unc-5 or its coreceptor Dcc/frazzled diminished neurodegenerative phenotypes, including motor dysfunction, neuromuscular junction defects, and shortened lifespan, in flies expressing UBQLN2ALS alleles. Induced pluripotent stem cells (iPSCs) harboring UBQLN2ALS knockin mutations exhibited lysosomal defects while inducible motor neurons (iMNs) expressing UBQLN2ALS alleles exhibited cytosolic UBQLN2 inclusions, reduced neurite complexity, and growth cone defects that were partially reversed by silencing of UNC5B and DCC. The combined findings suggest that altered growth cone dynamics are a conserved pathomechanism in UBQLN2-associated ALS/FTD.

[1]  S. Almeida,et al.  How Villains are Made: The Translation of Dipeptide Repeat Proteins in C9ORF72-ALS/FTD. , 2023, Gene.

[2]  Anna L. Brown,et al.  TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A , 2022, Nature.

[3]  Caitlin M. Rodriguez,et al.  TDP-43 represses cryptic exon inclusion in the FTD–ALS gene UNC13A , 2021, Nature.

[4]  C. Ghigna,et al.  A ligand-insensitive UNC5B splicing isoform regulates angiogenesis by promoting apoptosis , 2021, Nature Communications.

[5]  K. Ye,et al.  Netrin-1 receptor UNC5C cleavage by active δ-secretase enhances neurodegeneration, promoting Alzheimer’s disease pathologies , 2021, Science Advances.

[6]  Shyam M. Saladi,et al.  The STI1‐domain is a flexible alpha‐helical fold with a hydrophobic groove , 2021, Protein science : a publication of the Protein Society.

[7]  M. Saarma,et al.  Netrin‐1 and its receptor DCC modulate survival and death of dopamine neurons and Parkinson’s disease features , 2020, The EMBO journal.

[8]  L. Van Den Bosch,et al.  Role and therapeutic potential of liquid–liquid phase separation in amyotrophic lateral sclerosis , 2020, Journal of molecular cell biology.

[9]  C. Castañeda,et al.  Structure, dynamics and functions of UBQLNs: at the crossroads of protein quality control machinery. , 2020, The Biochemical journal.

[10]  K. Ye,et al.  Netrin1 deficiency activates MST1 via UNC5B receptor, promoting dopaminergic apoptosis in Parkinson’s disease , 2020, Proceedings of the National Academy of Sciences.

[11]  H. Paulson,et al.  Modeling UBQLN2-mediated neurodegenerative disease in mice: Shared and divergent properties of wild type and mutant UBQLN2 in phase separation, subcellular localization, altered proteostasis pathways, and selective cytotoxicity , 2020, Neurobiology of Disease.

[12]  Robert J Millikin,et al.  A Bayesian Null Interval Hypothesis Test Controls False Discovery Rates and Improves Sensitivity in Label-Free Quantitative Proteomics. , 2020, Journal of proteome research.

[13]  James E. Allen,et al.  Ensembl Genomes 2020—enabling non-vertebrate genomic research , 2019, Nucleic Acids Res..

[14]  Fen-Biao Gao,et al.  Transcription elongation factor AFF2/FMR2 regulates expression of expanded GGGGCC repeat-containing C9ORF72 allele in ALS/FTD , 2019, Nature Communications.

[15]  J. Trojanowski,et al.  Drosophila Ref1/ALYREF regulates transcription and toxicity associated with ALS/FTD disease etiologies , 2019, Acta neuropathologica communications.

[16]  Alireza Hadj Khodabakhshi,et al.  Metascape provides a biologist-oriented resource for the analysis of systems-level datasets , 2019, Nature Communications.

[17]  A. Mikos,et al.  Ubiquilins Regulate Autophagic Flux through mTOR Signaling and Lysosomal Acidification , 2019, Nature Cell Biology.

[18]  Michael J. Steinbaugh,et al.  ALS IMPLICATED PROTEIN TDP-43 SUSTAINS LEVELS OF STMN2 A MEDIATOR OF MOTOR NEURON GROWTH AND REPAIR , 2019, Nature Neuroscience.

[19]  Yingnan Yin,et al.  Generation of seven induced pluripotent stem cell lines from neonates of different ethnic backgrounds. , 2019, Stem cell research.

[20]  S. Subramani,et al.  The Roles of Ubiquitin-Binding Protein Shuttles in the Degradative Fate of Ubiquitinated Proteins in the Ubiquitin-Proteasome System and Autophagy , 2019, Cells.

[21]  A. Al-Chalabi,et al.  Striking phenotypic variation in a family with the P506S UBQLN2 mutation including amyotrophic lateral sclerosis, spastic paraplegia, and frontotemporal dementia , 2019, Neurobiology of Aging.

[22]  F. Rigo,et al.  Premature polyadenylation-mediated loss of stathmin-2 is a hallmark of TDP-43-dependent neurodegeneration , 2018, Nature Neuroscience.

[23]  Juan-Pablo Labrador,et al.  Motor axon guidance in Drosophila. , 2017, Seminars in cell & developmental biology.

[24]  L. Tan,et al.  The role of UNC5C in Alzheimer's disease. , 2018, Annals of translational medicine.

[25]  J. Julien,et al.  Neuronal Expression of UBQLN2P497H Exacerbates TDP-43 Pathology in TDP-43G348C Mice through Interaction with Ubiquitin , 2018, Molecular Neurobiology.

[26]  A. Shilatifard,et al.  Aberrant activation of non-coding RNA targets of transcriptional elongation complexes contributes to TDP-43 toxicity , 2018, Nature Communications.

[27]  M. Ivanova,et al.  Mutant UBQLN2 promotes toxicity by modulating intrinsic self-assembly , 2018, Proceedings of the National Academy of Sciences.

[28]  S. Gupton,et al.  Revisiting Netrin-1: One Who Guides (Axons) , 2018, Front. Cell. Neurosci..

[29]  Michael R Shortreed,et al.  Enhanced Global Post-translational Modification Discovery with MetaMorpheus. , 2018, Journal of proteome research.

[30]  J. Taylor,et al.  Ubiquitin Modulates Liquid-Liquid Phase Separation of UBQLN2 via Disruption of Multivalent Interactions. , 2018, Molecules and Cells.

[31]  Jia Gu,et al.  fastp: an ultra-fast all-in-one FASTQ preprocessor , 2018, bioRxiv.

[32]  F. Zoulim,et al.  Hepatocellular carcinoma-associated depletion of the netrin-1 receptor Uncoordinated Phenotype-5A (UNC5A) skews the hepatic unfolded protein response towards prosurvival outcomes. , 2018, Biochemical and biophysical research communications.

[33]  Lloyd M. Smith,et al.  Mutation-dependent aggregation and toxicity in a Drosophila model for UBQLN2-associated ALS , 2018, Human molecular genetics.

[34]  Michael R. Shortreed,et al.  Ultrafast Peptide Label-Free Quantification with FlashLFQ. , 2018, Journal of proteome research.

[35]  M. Zerial,et al.  Rab5 and Alsin regulate stress-activated cytoprotective signaling on mitochondria , 2017, bioRxiv.

[36]  Jost Vielmetter,et al.  Deconstruction of the beaten Path-Sidestep interaction network provides insights into neuromuscular system development , 2017, eLife.

[37]  Machelle D. Wilson,et al.  Valid statistical approaches for analyzing sholl data: Mixed effects versus simple linear models , 2017, Journal of Neuroscience Methods.

[38]  Cao Huang,et al.  Increased Ubqln2 expression causes neuron death in transgenic rats , 2016, Journal of neurochemistry.

[39]  R. Hegde,et al.  Ubiquilins Chaperone and Triage Mitochondrial Membrane Proteins for Degradation , 2016, Molecular cell.

[40]  Brian L. Frey,et al.  Global Identification of Protein Post-translational Modifications in a Single-Pass Database Search , 2015, Journal of proteome research.

[41]  O. Korvatska,et al.  R47H Variant of TREM2 Associated With Alzheimer Disease in a Large Late-Onset Family: Clinical, Genetic, and Neuropathological Study. , 2015, JAMA neurology.

[42]  Frank Fan,et al.  Generation and Expansion of highly-pure Motor Neuron Progenitors from Human Pluripotent Stem Cells , 2015, Nature Communications.

[43]  Cao Huang,et al.  Pathogenic Ubqln2 gains toxic properties to induce neuron death , 2015, Acta Neuropathologica.

[44]  E. Stoeckli,et al.  The SynCAM synaptic cell adhesion molecules are involved in sensory axon pathfinding by regulating axon–axon contacts , 2014, Journal of Cell Science.

[45]  J. Haines,et al.  A rare mutation in UNC5C predisposes to late-onset Alzheimer's disease and increases neuronal cell death , 2014, Nature Medicine.

[46]  W. Huber,et al.  Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 , 2014, Genome Biology.

[47]  J. Disterhoft,et al.  Dendritic spinopathy in transgenic mice expressing ALS/dementia-linked mutant UBQLN2 , 2014, Proceedings of the National Academy of Sciences.

[48]  Wei Shi,et al.  featureCounts: an efficient general purpose program for assigning sequence reads to genomic features , 2013, Bioinform..

[49]  J. Haines,et al.  A rare mutation in UNC 5 C predisposes to late-onset Alzheimer ’ s disease and increases neuronal cell death , 2014 .

[50]  D. Cleveland,et al.  Converging Mechanisms in ALS and FTD: Disrupted RNA and Protein Homeostasis , 2013, Neuron.

[51]  F. Hartl,et al.  Structure and function of Hip, an attenuator of the Hsp70 chaperone cycle , 2013, Nature Structural &Molecular Biology.

[52]  D. Arnott,et al.  Ubiquilin4 is an adaptor protein that recruits Ubiquilin1 to the autophagy machinery , 2013, EMBO reports.

[53]  R. Tibbetts,et al.  Identification of Genetic Modifiers of TDP-43 Neurotoxicity in Drosophila , 2013, PloS one.

[54]  Thomas R. Gingeras,et al.  STAR: ultrafast universal RNA-seq aligner , 2013, Bioinform..

[55]  L. Schöls,et al.  Screening in ALS and FTD patients reveals 3 novel UBQLN2 mutations outside the PXX domain and a pure FTD phenotype , 2012, Neurobiology of Aging.

[56]  G. Comi,et al.  Ubiquilin 2 mutations in Italian patients with amyotrophic lateral sclerosis and frontotemporal dementia , 2012, Journal of Neurology, Neurosurgery & Psychiatry.

[57]  V. Meininger,et al.  UBQLN2 mutations are rare in French and French–Canadian amyotrophic lateral sclerosis , 2012, Neurobiology of Aging.

[58]  A. Shilatifard,et al.  The super elongation complex (SEC) family in transcriptional control , 2012, Nature Reviews Molecular Cell Biology.

[59]  J. Buchner,et al.  The architecture of functional modules in the Hsp90 co‐chaperone Sti1/Hop , 2012, The EMBO journal.

[60]  John L. Robinson,et al.  Pattern of ubiquilin pathology in ALS and FTLD indicates presence of C9ORF72 hexanucleotide expansion , 2012, Acta Neuropathologica.

[61]  J. Haines,et al.  Mutations in UBQLN2 cause dominant X-linked juvenile and adult onset ALS and ALS/dementia , 2011, Nature.

[62]  Ji Han Kim,et al.  A Drosophila model of FUS-related neurodegeneration reveals genetic interaction between FUS and TDP-43. , 2011, Human molecular genetics.

[63]  A. Kimchi,et al.  The dependence receptor UNC5H2/B triggers apoptosis via PP2A-mediated dephosphorylation of DAP kinase. , 2010, Molecular cell.

[64]  M. Monteiro,et al.  Ubiquilin at a crossroads in protein degradation pathways , 2010, Autophagy.

[65]  Deepak Srinivasan,et al.  Ubiquilin functions in autophagy and is degraded by chaperone-mediated autophagy. , 2010, Human molecular genetics.

[66]  H. Baba,et al.  Identification of UNC5A as a novel transcriptional target of tumor suppressor p53 and a regulator of apoptosis. , 2010, International journal of oncology.

[67]  H. Aberle.  Searching for guidance cues: Follow the Sidestep trail , 2009, Fly.

[68]  Deepak Srinivasan,et al.  Ubiquilin and p97/VCP bind erasin, forming a complex involved in ERAD , 2009, The Journal of cell biology.

[69]  M. Siebert,et al.  Drosophila motor axons recognize and follow a Sidestep-labeled substrate pathway to reach their target fields. , 2009, Genes & development.

[70]  Hao Wu,et al.  Autoinhibition of UNC5b revealed by the cytoplasmic domain structure of the receptor. , 2009, Molecular cell.

[71]  Xun Hu,et al.  Mutations in FUS, an RNA Processing Protein, Cause Familial Amyotrophic Lateral Sclerosis Type 6 , 2009, Science.

[72]  J L Haines,et al.  Supporting Online Material Materials and Methods Figs. S1 to S7 Tables S1 to S4 References Mutations in the Fus/tls Gene on Chromosome 16 Cause Familial Amyotrophic Lateral Sclerosis , 2022 .

[73]  J. Debnath,et al.  PLIC proteins or ubiquilins regulate autophagy‐dependent cell survival during nutrient starvation , 2009, EMBO reports.

[74]  R. Morimoto,et al.  Integrating the Stress Response: Lessons for Neurodegenerative Diseases from C. Elegans , 2022 .

[75]  C. Eyers.  Universal sample preparation method for proteome analysis , 2009 .

[76]  R. Morimoto,et al.  Proteotoxic stress and inducible chaperone networks in neurodegenerative disease and aging. , 2008, Genes & development.

[77]  P. Mehlen,et al.  Netrin-1 mediates neuronal survival through PIKE-L interaction with the dependence receptor UNC5B , 2008, Nature Cell Biology.

[78]  S. Hadano,et al.  The Rab5 Activator ALS2/alsin Acts as a Novel Rac1 Effector through Rac1-activated Endocytosis* , 2007, Journal of Biological Chemistry.

[79]  M. Tessier-Lavigne,et al.  UNC5A promotes neuronal apoptosis during spinal cord development independent of netrin-1 , 2006, Nature Neuroscience.

[80]  D. Maier,et al.  Genetic Modifier Screens on Hairless Gain-of-Function Phenotypes Reveal Genes Involved in Cell Differentiation, Cell Growth and Apoptosis in Drosophila melanogaster , 2005, Genetics.

[81]  John B. Thomas,et al.  The Homeobox Transcription Factor Even-skipped Regulates Netrin-Receptor Expression to Control Dorsal Motor-Axon Projections in Drosophila , 2005, Current Biology.

[82]  H. Sink,et al.  Target recognition and synaptogenesis by motor axons: responses to the sidestep protein , 2005, International Journal of Developmental Neuroscience.

[83]  A. Kimchi,et al.  The dependence receptor UNC5H2 mediates apoptosis through DAP‐kinase , 2005, The EMBO journal.

[84]  Yusuke Nakamura,et al.  p53RDL1 regulates p53-dependent apoptosis , 2003, Nature Cell Biology.

[85]  T. Südhof,et al.  SynCAM, a Synaptic Adhesion Molecule That Drives Synapse Assembly , 2002, Science.

[86]  F. Llambi,et al.  Netrin‐1 acts as a survival factor via its receptors UNC5H and DCC , 2001, The EMBO journal.

[87]  C. Goodman,et al.  sidestep Encodes a Target-Derived Attractant Essential for Motor Axon Guidance in Drosophila , 2001, Cell.

[88]  D. Bredesen,et al.  The dependence receptor DCC (deleted in colorectal cancer) defines an alternative mechanism for caspase activation , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[89]  Stefan A. Przyborski,et al.  The mouse rostral cerebellar malformation gene encodes an UNC-5-like protein , 1997, Nature.

[90]  M. Masu,et al.  Vertebrate homologues of C. elegans UNC-5 are candidate netrin receptors , 1997, Nature.

[91]  C. Goodman,et al.  The Drosophila beaten path Gene Encodes a Novel Secreted Protein That Regulates Defasciculation at Motor Axon Choice Points , 1996, Cell.

[92]  J. Culotti,et al.  UNC-40, a C. elegans Homolog of DCC (Deleted in Colorectal Cancer), Is Required in Motile Cells Responding to UNC-6 Netrin Cues , 1996, Cell.

[93]  M. Masu,et al.  Deleted in Colorectal Cancer (DCC) Encodes a Netrin Receptor , 1996, Cell.

[94]  Y. Jan,et al.  frazzled Encodes a Drosophila Member of the DCC Immunoglobulin Subfamily and Is Required for CNS and Motor Axon Guidance , 1996, Cell.

[95]  C. Goodman,et al.  Genes that control neuromuscular specificity in Drosophila , 1993, Cell.

[96]  E. Hafen,et al.  Lilliputian : an AF 4 / FMR 2-related protein that controls cell identity and cell growth , 2022 .