Viral delivery of C9orf72 hexanucleotide repeat expansions in mice leads to repeat-length-dependent neuropathology and behavioural deficits

ABSTRACT Intronic GGGGCC repeat expansions in C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Two major pathologies stemming from the hexanucleotide RNA expansions (HREs) have been identified in postmortem tissue: intracellular RNA foci and repeat-associated non-ATG dependent (RAN) dipeptides, although it is unclear how these and other hallmarks of disease contribute to the pathophysiology of neuronal injury. Here, we describe two novel lines of mice that overexpress either 10 pure or 102 interrupted GGGGCC repeats mediated by adeno-associated virus (AAV) and recapitulate the relevant human pathology and disease-related behavioural phenotypes. Similar levels of intracellular RNA foci developed in both lines of mice, but only mice expressing 102 repeats generated C9orf72 RAN pathology, neuromuscular junction (NMJ) abnormalities, dispersal of the hippocampal CA1, enhanced apoptosis, and deficits in gait and cognition. Neither line of mice, however, showed extensive TAR DNA-binding protein 43 (TDP-43) pathology or neurodegeneration. Our data suggest that RNA foci pathology is not a good predictor of C9orf72 RAN dipeptide formation, and that RAN dipeptides and NMJ dysfunction are drivers of C9orf72 disease pathogenesis. These AAV-mediated models of C9orf72-associated ALS/FTD will be useful tools for studying disease pathophysiology and developing new therapeutic approaches. Summary: C9orf72-linked motor neuron disease models with viral-mediated expression of GGGGCC repeat expansion in mice show neuropathology and behavioural deficits.

[1]  Hannah A. Pliner,et al.  C9ORF72 hexanucleotide repeat exerts toxicity in a stable, inducible motor neuronal cell model, which is rescued by partial depletion of Pten , 2017, Human molecular genetics.

[2]  Diana M. Mitrea,et al.  C9orf72 Dipeptide Repeats Impair the Assembly, Dynamics, and Function of Membrane-Less Organelles , 2016, Cell.

[3]  L. Petrucelli,et al.  Poly(GR) in C9ORF72-Related ALS/FTD Compromises Mitochondrial Function and Increases Oxidative Stress and DNA Damage in iPSC-Derived Motor Neurons , 2016, Neuron.

[4]  N. Shneider,et al.  The C9ORF72 GGGGCC expansion forms RNA G-quadruplex inclusions and sequesters hnRNP H to disrupt splicing in ALS brains , 2016, eLife.

[5]  Rachael A. Powis,et al.  Systemic restoration of UBA1 ameliorates disease in spinal muscular atrophy , 2016, JCI insight.

[6]  D. Borchelt,et al.  C9orf72 BAC Mouse Model with Motor Deficits and Neurodegenerative Features of ALS/FTD , 2016, Neuron.

[7]  C. Heyser,et al.  Gain of Toxicity from ALS/FTD-Linked Repeat Expansions in C9ORF72 Is Alleviated by Antisense Oligonucleotides Targeting GGGGCC-Containing RNAs , 2016, Neuron.

[8]  L. Petrucelli,et al.  C9ORF72 poly(GA) aggregates sequester and impair HR23 and nucleocytoplasmic transport proteins , 2016, Nature Neuroscience.

[9]  D. Underhill,et al.  C9orf72 is required for proper macrophage and microglial function in mice , 2016, Science.

[10]  A. Grierson,et al.  AAV9-mediated central nervous system–targeted gene delivery via cisterna magna route in mice , 2016, Molecular therapy. Methods & clinical development.

[11]  L. Petrucelli,et al.  C9orf72 BAC Transgenic Mice Display Typical Pathologic Features of ALS/FTD , 2015, Neuron.

[12]  H. Horvitz,et al.  Human C9ORF72 Hexanucleotide Expansion Reproduces RNA Foci and Dipeptide Repeat Proteins but Not Neurodegeneration in BAC Transgenic Mice , 2015, Neuron.

[13]  Z. Weng,et al.  Differential Toxicity of Nuclear RNA Foci versus Dipeptide Repeat Proteins in a Drosophila Model of C9ORF72 FTD/ALS , 2015, Neuron.

[14]  Bruce L. Miller,et al.  GGGGCC repeat expansion in C9orf72 compromises nucleocytoplasmic transport , 2015, Nature.

[15]  Sean J. Miller,et al.  The C9orf72 repeat expansion disrupts nucleocytoplasmic transport , 2015, Nature.

[16]  Raymond D. Schellevis,et al.  C9orf72 ablation in mice does not cause motor neuron degeneration or motor deficits , 2015, Annals of neurology.

[17]  E. Kremmer,et al.  Distribution of dipeptide repeat proteins in cellular models and C9orf72 mutation cases suggests link to transcriptional silencing , 2015, Acta Neuropathologica.

[18]  Kevin F. Bieniek,et al.  C9ORF72 repeat expansions in mice cause TDP-43 pathology, neuronal loss, and behavioral deficits , 2015, Science.

[19]  M. Aittaleb,et al.  The Knockdown of αkap Alters the Postsynaptic Apparatus of Neuromuscular Junctions in Living Mice , 2015, The Journal of Neuroscience.

[20]  P. Rabins,et al.  Hippocampal sclerosis dementia with the C9ORF72 hexanucleotide repeat expansion , 2014, Neurobiology of Aging.

[21]  A. Grierson,et al.  Early Detection of Motor Dysfunction in the SOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis (ALS) Using Home Cage Running Wheels , 2014, PloS one.

[22]  O. Hendrich,et al.  C9orf72 repeat expansions cause neurodegeneration in Drosophila through arginine-rich proteins , 2014, Science.

[23]  Stuart A. Wilson,et al.  Sequestration of multiple RNA recognition motif-containing proteins by C9orf72 repeat expansions , 2014, Brain : a journal of neurology.

[24]  E. Giralt,et al.  Reelin delays amyloid-beta fibril formation and rescues cognitive deficits in a model of Alzheimer’s disease , 2014, Nature Communications.

[25]  Patrick G. Shaw,et al.  C9orf72 Nucleotide Repeat Structures Initiate Molecular Cascades of Disease , 2014, Nature.

[26]  Adriano Chiò,et al.  State of play in amyotrophic lateral sclerosis genetics , 2013, Nature Neuroscience.

[27]  C. E. Pearson,et al.  TMPyP4 Porphyrin Distorts RNA G-quadruplex Structures of the Disease-associated r(GGGGCC)n Repeat of the C9orf72 Gene and Blocks Interaction of RNA-binding Proteins* , 2013, The Journal of Biological Chemistry.

[28]  Michel Boulouard,et al.  Object recognition test in mice , 2013, Nature Protocols.

[29]  L. Petrucelli,et al.  Reduced C9orf72 gene expression in c9FTD/ALS is caused by histone trimethylation, an epigenetic event detectable in blood , 2013, Acta Neuropathologica.

[30]  E. Kremmer,et al.  Bidirectional transcripts of the expanded C9orf72 hexanucleotide repeat are translated into aggregating dipeptide repeat proteins , 2013, Acta Neuropathologica.

[31]  Nipun A. Mistry,et al.  RNA Toxicity from the ALS/FTD C9ORF72 Expansion Is Mitigated by Antisense Intervention , 2013, Neuron.

[32]  S. Lorenzl,et al.  Dipeptide repeat protein pathology in C9ORF72 mutation cases: clinico-pathological correlations , 2013, Acta Neuropathologica.

[33]  A. Pastore,et al.  The Role of Interruptions in polyQ in the Pathology of SCA1 , 2013, PLoS genetics.

[34]  E. Kremmer,et al.  The C9orf72 GGGGCC Repeat Is Translated into Aggregating Dipeptide-Repeat Proteins in FTLD/ALS , 2013, Science.

[35]  G. Sobue,et al.  Loss of TDP-43 causes age-dependent progressive motor neuron degeneration. , 2013, Brain : a journal of neurology.

[36]  C. E. Pearson,et al.  The Disease-associated r(GGGGCC)n Repeat from the C9orf72 Gene Forms Tract Length-dependent Uni- and Multimolecular RNA G-quadruplex Structures* , 2013, The Journal of Biological Chemistry.

[37]  M. Koch,et al.  Impairment of cognitive performance after reelin knockdown in the medial prefrontal cortex of pubertal or adult rats , 2011, Neurobiology of Disease.

[38]  Bruce L. Miller,et al.  Expanded GGGGCC Hexanucleotide Repeat in Noncoding Region of C9ORF72 Causes Chromosome 9p-Linked FTD and ALS , 2011, Neuron.

[39]  David Heckerman,et al.  A Hexanucleotide Repeat Expansion in C9ORF72 Is the Cause of Chromosome 9p21-Linked ALS-FTD , 2011, Neuron.

[40]  Aneurin J. Kennerley,et al.  Optimised and Rapid Pre-clinical Screening in the SOD1G93A Transgenic Mouse Model of Amyotrophic Lateral Sclerosis (ALS) , 2011, PloS one.

[41]  Brian B. Gibbens,et al.  Non-ATG–initiated translation directed by microsatellite expansions , 2010, Proceedings of the National Academy of Sciences.

[42]  T. Gillingwater,et al.  Review: Neuromuscular synaptic vulnerability in motor neurone disease: amyotrophic lateral sclerosis and spinal muscular atrophy , 2010, Neuropathology and applied neurobiology.

[43]  Radim Mazanec,et al.  Highly unstable sequence interruptions of the CTG repeat in the myotonic dystrophy gene , 2009, American journal of medical genetics. Part A.

[44]  M. Rich,et al.  Impaired Synaptic Vesicle Release and Immaturity of Neuromuscular Junctions in Spinal Muscular Atrophy Mice , 2009, The Journal of Neuroscience.

[45]  E. Frackelton,et al.  Novel, complex interruptions of the GAA repeat in small, expanded alleles of two affected siblings with late‐onset Friedreich ataxia , 2008, Movement disorders : official journal of the Movement Disorder Society.

[46]  Masaaki Komatsu,et al.  Homeostatic Levels of p62 Control Cytoplasmic Inclusion Body Formation in Autophagy-Deficient Mice , 2007, Cell.

[47]  A. Destée,et al.  Are interrupted SCA2 CAG repeat expansions responsible for parkinsonism? , 2007, Neurology.

[48]  S. Bione,et al.  Influence of intermediate and uninterrupted FMR1 CGG expansions in premature ovarian failure manifestation. , 2006, Human reproduction.

[49]  G. Bjørkøy,et al.  p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death , 2005, The Journal of cell biology.

[50]  A. Vincent,et al.  Increased expression of rapsyn in muscles prevents acetylcholine receptor loss in experimental autoimmune myasthenia gravis. , 2005, Brain : a journal of neurology.

[51]  D. McCarty,et al.  Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis , 2001, Gene Therapy.

[52]  P. Caroni,et al.  Early and Selective Loss of Neuromuscular Synapse Subtypes with Low Sprouting Competence in Motoneuron Diseases , 2000, The Journal of Neuroscience.

[53]  J. Lichtman,et al.  Rapid and reversible effects of activity on acetylcholine receptor density at the neuromuscular junction in vivo. , 1999, Science.

[54]  John Shelton,et al.  Reeler/Disabled-like Disruption of Neuronal Migration in Knockout Mice Lacking the VLDL Receptor and ApoE Receptor 2 , 1999, Cell.

[55]  E. Eichler,et al.  Interruptions in the triplet repeats of SCA1 and FRAXA reduce the propensity and complexity of slipped strand DNA (S-DNA) formation. , 1998, Biochemistry.

[56]  R. Sinden,et al.  Structural analysis of slipped-strand DNA (S-DNA) formed in (CTG)n. (CAG)n repeats from the myotonic dystrophy locus. , 1998, Nucleic acids research.

[57]  Jonathan A. Cooper,et al.  Neuronal position in the developing brain is regulated by mouse disabled-1 , 1997, Nature.

[58]  Jeff W. Lichtman,et al.  A quantitative fluorescence-imaging technique for studying acetylcholine receptor turnover at neuromuscular junctions in living animals , 1996, Journal of Neuroscience Methods.

[59]  E. Eichler,et al.  Length of uninterrupted CGG repeats determines instability in the FMR1 gene , 1994, Nature Genetics.

[60]  A. Joshi,et al.  Edinburgh Research Explorer Dysregulation of ubiquitin homeostasis and -catenin signaling promote spinal muscular atrophy , 2022 .

[61]  R. Reisin.  Frequency of the C9orf72 hexanucleotide repeat expansion in patients with amyotrophic lateral sclerosis and frontotemporal dementia: a cross-sectional study , 2012 .

[62]  D. Nelson,et al.  Interruptions in the expanded ATTCT repeat of spinocerebellar ataxia type 10: repeat purity as a disease modifier? , 2006, American journal of human genetics.

[63]  B. Keats,et al.  Replication-mediated instability of the GAA triplet repeat mutation in Friedreich ataxia. , 2004, Nucleic acids research.

[64]  H. Zoghbi,et al.  Evidence for a mechanism predisposing to intergenerational CAG repeat instability in spinocerebellar ataxia type I , 1993, Nature Genetics.