USP8 Deubiquitinates SHANK3 to Control Synapse Density and SHANK3 Activity-Dependent Protein Levels

Mutations or altered protein levels of SHANK3 are implicated in neurodevelopmental disorders such as Phelan–McDermid syndrome, autism spectrum disorders, and schizophrenia (Guilmatre et al., 2014). Loss of SHANK3 in mouse models results in decreased synapse density and reduction in the levels of multiple synaptic proteins (Jiang and Ehlers, 2013). The family of SHANK scaffolding molecules are among the most heavily ubiquitinated proteins at the postsynaptic density. The ubiquitin-dependent proteasome degradation of SHANK is regulated by synaptic activity and may contribute to activity-dependent synaptic remodeling (Ehlers, 2003; Shin et al., 2012). However, the identity of the specific deubiquitinating enzymes and E3 ligases that regulate SHANK ubiquitination at synapses are unknown. Here we identify USP8/UBPY as a deubiquitinating enzyme that regulates SHANK3 and SHANK1 ubiquitination and protein levels. In primary rat neurons, USP8 enhances SHANK3 and SHANK1 protein levels via deubiquitination and increases dendritic spine density. Additionally, USP8 is essential for changes in SHANK3 protein levels following synaptic activity modulation. These data identify USP8 as a key modulator of SHANK3 downstream of synaptic activity. SIGNIFICANCE STATEMENT Precise regulation of the protein levels of the postsynaptic scaffolding protein SHANK3 is essential for proper neurodevelopment. Mutations of SHANK3 have been identified in Phelan–McDermid syndrome, autism spectrum disorders, and schizophrenia (Guilmatre et al., 2014). In this research, we identify USP8 as a key enzyme that regulates SHANK3 protein levels in neurons. USP8 acts to deubiquitinate SHANK3, which prevents its proteasomal-mediated degradation and enhances overall dendritic spine stability. In the future, the modulation of USP8 deubiquitinating activity could potentially be used to titrate the protein levels of SHANK3 to ameliorate disease.

[1]  Ute Moog,et al.  Mutations in the SHANK2 synaptic scaffolding gene in autism spectrum disorder and mental retardation , 2010, Nature Genetics.

[2]  David Komander,et al.  Ubiquitin modifications , 2016, Cell Research.

[3]  M. Sheng,et al.  Degradation of Postsynaptic Scaffold GKAP and Regulation of Dendritic Spine Morphology by the TRIM3 Ubiquitin Ligase in Rat Hippocampal Neurons , 2010, PloS one.

[4]  A. Matouschek,et al.  Regulated protein turnover: snapshots of the proteasome in action , 2014, Nature Reviews Molecular Cell Biology.

[5]  L. Schwarz,et al.  Synaptic Strength Is Bidirectionally Controlled by Opposing Activity-Dependent Regulation of Nedd4-1 and USP8 , 2014, The Journal of Neuroscience.

[6]  E. Blennow,et al.  FISH-mapping of a 100-kb terminal 22q13 deletion , 2002, Human Genetics.

[7]  P. Worley,et al.  Shank, a Novel Family of Postsynaptic Density Proteins that Binds to the NMDA Receptor/PSD-95/GKAP Complex and Cortactin , 1999, Neuron.

[8]  M. Sheng,et al.  The Shank family of scaffold proteins. , 2000, Journal of cell science.

[9]  M. Sheng,et al.  GKAP/SAPAP orchestrates activity-dependent postsynaptic protein remodeling and homeostatic scaling , 2012, Nature Neuroscience.

[10]  K. Miyazawa,et al.  A Deubiquitinating Enzyme UBPY Interacts with the Src Homology 3 Domain of Hrs-binding Protein via a Novel Binding Motif PX(V/I)(D/N)RXXKP* , 2000, The Journal of Biological Chemistry.

[11]  Mark J. Harris,et al.  Haploinsufficiency of the autism-associated Shank3 gene leads to deficits in synaptic function, social interaction, and social communication , 2010, Molecular autism.

[12]  M. Komada,et al.  Regulation of epidermal growth factor receptor down-regulation by UBPY-mediated deubiquitination at endosomes. , 2005, Molecular biology of the cell.

[13]  T. Reese,et al.  Activity induced changes in the distribution of Shanks at hippocampal synapses , 2010, Neuroscience.

[14]  Laurent Mottron,et al.  Novel de novo SHANK3 mutation in autistic patients , 2009, American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics.

[15]  M. Schmeisser.  Translational neurobiology in Shank mutant mice--model systems for neuropsychiatric disorders. , 2015, Annals of anatomy = Anatomischer Anzeiger : official organ of the Anatomische Gesellschaft.

[16]  A. Breman,et al.  SHANK3 overexpression causes manic-like behavior with unique pharmacogenetic properties , 2013, Nature.

[17]  S. Scherer,et al.  Inherited and de novo SHANK2 variants associated with autism spectrum disorder impair neuronal morphogenesis and physiology , 2011, Human molecular genetics.

[18]  Karl Deisseroth,et al.  Modulation of prefrontal cortex excitation/inhibition balance rescues social behavior in CNTNAP2-deficient mice , 2017, Science Translational Medicine.

[19]  Guoping Feng,et al.  Adult Restoration of Shank3 Expression Rescues Selective Autistic-Like Phenotypes , 2016, Nature.

[20]  Elodie Ey,et al.  Meta-analysis of SHANK Mutations in Autism Spectrum Disorders: A Gradient of Severity in Cognitive Impairments , 2014, PLoS genetics.

[21]  Nicholas A. Frost,et al.  Shank–cortactin interactions control actin dynamics to maintain flexibility of neuronal spines and synapses , 2016, The European journal of neuroscience.

[22]  T. Bourgeron,et al.  SHANK3 mutations identified in autism lead to modification of dendritic spine morphology via an actin-dependent mechanism , 2011, Molecular Psychiatry.

[23]  Xiuli Wu,et al.  Stabilization of the E3 Ubiquitin Ligase Nrdp1 by the Deubiquitinating Enzyme USP8 , 2004, Molecular and Cellular Biology.

[24]  P. Worley,et al.  Shank Expression Is Sufficient to Induce Functional Dendritic Spine Synapses in Aspiny Neurons , 2005, The Journal of Neuroscience.

[25]  M. Ehlers,et al.  Modeling Autism by SHANK Gene Mutations in Mice , 2013, Neuron.

[26]  G. Ellis‐Davies,et al.  Structural basis of long-term potentiation in single dendritic spines , 2004, Nature.

[27]  S. Buono,et al.  Schizophrenia in a patient with subtelomeric duplication of chromosome 22q , 2007, Clinical genetics.

[28]  P. Nash,et al.  Regulation of Endocytic Sorting by ESCRT–DUB-Mediated Deubiquitination , 2011, Cell Biochemistry and Biophysics.

[29]  Post-transcriptional regulation of SHANK3 expression by microRNAs related to multiple neuropsychiatric disorders , 2015, Molecular Brain.

[30]  D. Ledbetter,et al.  Molecular characterization of a 130-kb terminal microdeletion at 22q in a child with mild mental retardation. , 1997, American journal of human genetics.

[31]  M. Bear,et al.  Ubiquitination Regulates PSD-95 Degradation and AMPA Receptor Surface Expression , 2003, Neuron.

[32]  E. Schuman,et al.  Synaptic protein degradation by the ubiquitin proteasome system , 2005, Current Opinion in Neurobiology.

[33]  Thomas Bourgeron,et al.  The emerging role of SHANK genes in neuropsychiatric disorders , 2014, Developmental neurobiology.

[34]  René Bernards,et al.  A Genomic and Functional Inventory of Deubiquitinating Enzymes , 2005, Cell.

[35]  Morgan Sheng,et al.  Deconstruction for Reconstruction: The Role of Proteolysis in Neural Plasticity and Disease , 2011, Neuron.

[36]  Tony Pawson,et al.  A High-Affinity Arg-X-X-Lys SH3 Binding Motif Confers Specificity for the Interaction between Gads and SLP-76 in T Cell Signaling , 2002, Current Biology.

[37]  C. Hoogenraad,et al.  The postsynaptic architecture of excitatory synapses: a more quantitative view. , 2007, Annual review of biochemistry.

[38]  Thomas Bourgeron,et al.  Mutations in the gene encoding the synaptic scaffolding protein SHANK3 are associated with autism spectrum disorders , 2007, Nature Genetics.

[39]  K. Knobeloch,et al.  Essential Role of Ubiquitin-Specific Protease 8 for Receptor Tyrosine Kinase Stability and Endocytic Trafficking In Vivo , 2007, Molecular and Cellular Biology.

[40]  D. Kirkpatrick,et al.  The mitochondrial deubiquitinase USP30 opposes parkin-mediated mitophagy , 2014, Nature.

[41]  Guosong Liu,et al.  Regulation of Dendritic Spine Morphology and Synaptic Function by Shank and Homer , 2001, Neuron.

[42]  J. Harper,et al.  Altered social behavior and neuronal development in mice lacking the Uba6-Use1 ubiquitin transfer system. , 2013, Molecular cell.

[43]  Yanji Xu,et al.  Synaptic protein ubiquitination in rat brain revealed by antibody-based ubiquitome analysis. , 2012, Journal of proteome research.

[44]  M. Ehlers.  Activity level controls postsynaptic composition and signaling via the ubiquitin-proteasome system , 2003, Nature Neuroscience.

[45]  R. Giorda,et al.  Identification of a recurrent breakpoint within the SHANK3 gene in the 22q13.3 deletion syndrome , 2005, Journal of Medical Genetics.

[46]  A. Beaudet,et al.  Synaptic dysfunction and abnormal behaviors in mice lacking major isoforms of Shank3. , 2011, Human molecular genetics.

[47]  Erin M. Schuman,et al.  Activity-dependent dynamics and sequestration of proteasomes in dendritic spines , 2006, Nature.

[48]  Paola Visconti,et al.  Prevalence of SHANK3 variants in patients with different subtypes of autism spectrum disorders , 2012, European Journal of Human Genetics.

[49]  James Y. Zhang,et al.  Reduced Excitatory Neurotransmission and Mild Autism-Relevant Phenotypes in Adolescent Shank3 Null Mutant Mice , 2012, The Journal of Neuroscience.

[50]  D. Rujescu,et al.  Investigation of SHANK3 in schizophrenia , 2017, American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics.

[51]  F. Mackenzie,et al.  Amino-terminal Dimerization, NRDP1-Rhodanese Interaction, and Inhibited Catalytic Domain Conformation of the Ubiquitin-specific Protease 8 (USP8)* , 2006, Journal of Biological Chemistry.

[52]  T. Bonhoeffer,et al.  Bidirectional Activity-Dependent Morphological Plasticity in Hippocampal Neurons , 2004, Neuron.

[53]  P. Worley,et al.  Coupling of mGluR/Homer and PSD-95 Complexes by the Shank Family of Postsynaptic Density Proteins , 1999, Neuron.

[54]  D. Pinto,et al.  SHANK1 Deletions in Males with Autism Spectrum Disorder. , 2012, American journal of human genetics.

[55]  S. Tenzer,et al.  Proteomic Analysis of Post-synaptic Density Fractions from Shank3 Mutant Mice Reveals Brain Region Specific Changes Relevant to Autism Spectrum Disorder , 2017, Front. Mol. Neurosci..

[56]  J. J. Siegel,et al.  Prefrontal Cortex Dysfunction in Fragile X Mice Depends on the Continued Absence of Fragile X Mental Retardation Protein in the Adult Brain , 2017, The Journal of Neuroscience.

[57]  G. Feng,et al.  Shank3 mutant mice display autistic-like behaviours and striatal dysfunction , 2011, Nature.

[58]  H. McDermid,et al.  Molecular characterisation of the 22q13 deletion syndrome supports the role of haploinsufficiency of SHANK3/PROSAP2 in the major neurological symptoms , 2003, Journal of medical genetics.

[59]  Richard L. Huganir,et al.  Rapid Dispersion of SynGAP from Synaptic Spines Triggers AMPA Receptor Insertion and Spine Enlargement during LTP , 2015, Neuron.