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Lost in Translation: Cul3-Dependent Pathological Mechanisms in Psychiatric Disorders
Neuron
Previews Lost in Translation: Cul3-Dependent Pathological Mechanisms in Psychiatric Disorders Huei-Ying Chen1 and Brady J. Maher1,2,3,* 1Lieber
Institute for Brain Development, Johns Hopkins Medical Campus, Baltimore, MD, USA of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, USA 3The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, USA *Correspondence: [email protected] https://doi.org/10.1016/j.neuron.2020.01.010 2Department
In this issue of Neuron, Dong et al. (2020) finds that deficiency of the psychiatric risk gene Cul3, which encodes an E3 ubiquitin ligase, leads to an upregulation of Cap-dependent protein translation. The resulting imbalance in protein synthesis and degradation is found to disrupt glutamatergic transmission and excitability in networks that underlie sociability and anxiety. Progress in psychiatric genetics has led to the identification of hundreds of genes and genetic variants associated with risk. One such gene is Cullin 3 (Cul3), which was initially identified as an autism spectrum disorder (ASD) risk gene by analysis of whole-exome sequencing of parent-child trios exhibiting sporadic ASD (Kong et al., 2012; O’Roak et al., 2012). In addition, genome-wide association studies (GWASs) of schizophrenia (SCZ) have identified more than 100 common variants that each contribute a small amount of risk. One of these common risk variants is located in a genomic region containing CUL3 and is associated with reduced CUL3 expression in postmortem brain (Collado-Torres et al., 2019), thus implicating CUL3 in risk for SCZ. Despite abundant expression of CUL3 in the brain and its disease relevance, understanding of its role in brain development and function is limited. By studying Cul3 deficiency in a mouse model, Dong and colleagues have potentially identified shared pathophysiology between ASD and SCZ (Dong et al., 2020). The addition of ubiquitin to proteins is the main signal for proteasomal degradation. The process of ubiquitination is tightly regulated and is involved in many cellular processes, including cell-cycle regulation, transcription, signaling, trafficking, and protein quality control. CUL3 encodes an E3 ubiquitin ligase that forms a complex with RING-box protein and BTB protein that plays a critical role in the ligation of ubiquitin
to substrate proteins (Ande´rica-Romero et al., 2013). Dysfunction of proteasomal degradation has been previously associated with several human diseases. For instance, the accumulation of misfolded proteins is common to neurodegenerative disorders, while proteasome inhibitors are used to treat some forms of cancers. In this issue of Neuron, Dong et al. (2020) demonstrate that deficiencies in protein degradation pathways can lead to physiological and behavioral phenotypes that may underlie neuropsychiatric disorders. To understand how Cul3 deficiency leads to ASD, Dong et al. (2020) primarily focused on Cul3 heterozygous mice by crossing a Cul3 floxed animal to either GFAP-cre or NEX-cre mice. These mouse models produce Cul3 deficiency primarily in excitatory neurons, with GFAP-cre also targeting glial cells, and both models showed social deficits on the three-chamber social interaction tests and increased anxiety-like phenotype in the open field arena. With these behavioral deficits in hand, the authors then set out to determine whether morphological and physiological phenotypes were present within the hippocampus. They observed that in both GFAP-cre and NEX-cre lines, Cul3 deficiency in CA1 neurons had profound effects on neuron and network excitability. CA1 neurons showed increased spine density, increased miniature excitatory postsynaptic current (mEPSC) frequency, increased action potential output, enhanced presynaptic glutamate
398 Neuron 105, February 5, 2020 Crown Copyright ª 2020 Published by Elsevier Inc.
release, and a developmental increase in GABAergic miniature inhibitory postsynaptic current frequency, which equated to an overall excitation-inhibition (E/I) imbalance within the CA1 network. To link CUL3-dependent behavioral deficits to morphological and physiological phenotypes, Dong et al. (2020) turned to tandem mass tagging (TMT)-based quantitative proteomics to help identify associated biochemical mechanisms. Using this method, they identified a total of 5,720 proteins, of which 335 were differentially expressed (DE). Gene ontology analysis of increased DE proteins was consistent with increased synaptic transmission, showing enrichment for neuronal excitability, SNARE complex disassembly, synaptic vesicle localization, synaptic vesicle cycle, and presynaptic assembly. Although these DE proteins failed to survive false discovery rate correction, western blot analysis confirmed that many of these proteins were significantly upregulated. Dong et al. (2020) then focused their attention on eIF4G1, which was one of 14 upregulated DE proteins in common between Cul3 homozygous and heterozygous knockout samples and was previously shown to regulate Cap-dependent translation and synthesis of synaptic proteins (Marcotrigiano et al., 1999). They demonstrated that eIF4G1 expression is regulated by CUL3-dependent ubiquitination and that upregulation of eIF4G1 increased protein translation in Cul3-deficient animals.
Neuron
Previews To confirm that eIF4G1 upregulation was responsible for cellular and behavioral phenotypes associated with Cul3 deficiency, Dong and colleagues performed intracerebral ventricular injections of 4EGI-1, an inhibitor of Cap-dependent translation. This treatment rescued social deficits in Cul3-deficient mice but had little effect on anxiety-like behaviors. When they looked at 4EGI-1 rescue of neuronal morphology and physiology, they observed that spine density, mEPSC frequency, expression of synaptic vesicle proteins, and glutamate release were all normalized; however, 4EGI-1 failed to rescue increased intrinsic excitability. To specifically reduce intrinsic excitability of CA1 neurons, they relied on a chemogenic approach by virally injecting hM4Di, an inhibitory DREADD construct, into the hippocampus. Treatment with clozapine N-oxide (CNO) to activate the hM4Di receptor led to a reduction in CA1 action potential output and abolished the anxiety-like behaviors. Together, these two independent rescue experiments identified unique cellular phenotypes that underlie specific behavioral deficits. The results by Dong et al. (2020) are somewhat consistent with previous phenotypes observed in several mouse models of syndromic ASD risk genes. For instance, mutations in the UBE3A gene, which also encodes an E3 ubiquitin ligase and causes Angelman syndrome, leads to similar defects in synaptic vesicle cycling and E/I imbalance (Wallace et al., 2012). Together, these two studies demonstrate that ubiquitination is a critical process for the regulation of synaptic vesicle function, further implicating presynaptic terminals and the regulation of neurotransmitter release as a potential hotspot for ASD pathophysiology. How-
ever, one important distinction between these two reports is that the mouse model used in Wallace et al. (2012) had a germline mutation in the maternal Ube3a allele, which preferentially disrupted GABAergic terminals, leaving excitatory terminals intact despite Ube3a expression in all neurons. It will be interesting to see whether Cul3 deficiency in GABAergic neurons has any effect on inhibitory transmission by using a GABAergic-specific cre line or, even better, mutating Cul3 in the germline to match the human ASD condition. Dong et al. (2020) also demonstrated that Cul3 deficiency leads to increased intrinsic excitability, which is consistent with excitability phenotypes reported in mouse models of fragile X syndrome (Fmr1) and Rett’s syndrome (Mecp2) (Gibson et al., 2008; Wu et al., 2016) and suggests that dysregulation of ion channel expression and function may be essential to our understanding of ASD pathophysiology. Dong et al. (2020) showed that chemogenic knockdown of excitability in the hippocampus rescued hyperexcitability and anxiety-like behaviors, providing a physiological mechanism. However, the molecular mechanism remains unknown. Thus, an exciting next step would be to identify the ion channels involved and demonstrate pharmacological rescue, which could potentially pave the way to developing a therapeutic target. Overall, Dong et al. (2020) provide good evidence that Cul3 deficiency disrupts glutamatergic presynaptic transmission due to an imbalance of protein synthesis and degradation, thus building on evidence that the dysregulation of presynaptic transmitter release may play a prominent role in psychiatric disorders.
REFERENCES Ande´rica-Romero, A.C., Gonza´lez-Herrera, I.G., Santamarı´a, A., and Pedraza-Chaverri, J. (2013). Cullin 3 as a novel target in diverse pathologies. Redox Biol. 1, 366–372. Collado-Torres, L., Burke, E.E., Peterson, A., Shin, J., Straub, R.E., Rajpurohit, A., Semick, S.A., Ulrich, W.S., Price, A.J., Valencia, C., et al.; BrainSeq Consortium (2019). Regional Heterogeneity in Gene Expression, Regulation, and Coherence in the Frontal Cortex and Hippocampus across Development and Schizophrenia. Neuron 103, 203–216.e8. Dong, Z., Chen, W., Chen, C., Wang, H., Cui, W., Tan, Z., Robinson, H., Gao, N., Luo, B., Zhang, L., et al. (2020). CUL3 Deficiency Causes Social Deficits and Anxiety-like Behaviors by Impairing Excitation-Inhibition Balance through the Promotion of Cap-Dependent Translation. Neuron 105, this issue, 475–490. Gibson, J.R., Bartley, A.F., Hays, S.A., and Huber, K.M. (2008). Imbalance of neocortical excitation and inhibition and altered UP states reflect network hyperexcitability in the mouse model of fragile X syndrome. J. Neurophysiol. 100, 2615–2626. Kong, S.W., Collins, C.D., Shimizu-Motohashi, Y., Holm, I.A., Campbell, M.G., Lee, I.-H., Brewster, S.J., Hanson, E., Harris, H.K., Lowe, K.R., et al. (2012). Characteristics and predictive value of blood transcriptome signature in males with autism spectrum disorders. PLoS ONE 7, e49475. Marcotrigiano, J., Gingras, A.C., Sonenberg, N., and Burley, S.K. (1999). Cap-dependent translation initiation in eukaryotes is regulated by a molecular mimic of eIF4G. Mol. Cell 3, 707–716. O’Roak, B.J., Vives, L., Girirajan, S., Karakoc, E., Krumm, N., Coe, B.P., Levy, R., Ko, A., Lee, C., Smith, J.D., et al. (2012). Sporadic autism exomes reveal a highly interconnected protein network of de novo mutations. Nature 485, 246–250. Wallace, M.L., Burette, A.C., Weinberg, R.J., and Philpot, B.D. (2012). Maternal loss of Ube3a produces an excitatory/inhibitory imbalance through neuron type-specific synaptic defects. Neuron 74, 793–800. Wu, Y., Zhong, W., Cui, N., Johnson, C.M., Xing, H., Zhang, S., and Jiang, C. (2016). Characterization of Rett Syndrome-like phenotypes in Mecp2-knockout rats. J. Neurodev. Disord. 8, 23.
Neuron 105, February 5, 2020 399
Previews Lost in Translation: Cul3-Dependent Pathological Mechanisms in Psychiatric Disorders Huei-Ying Chen1 and Brady J. Maher1,2,3,* 1Lieber
Institute for Brain Development, Johns Hopkins Medical Campus, Baltimore, MD, USA of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, USA 3The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, USA *Correspondence: [email protected] https://doi.org/10.1016/j.neuron.2020.01.010 2Department
In this issue of Neuron, Dong et al. (2020) finds that deficiency of the psychiatric risk gene Cul3, which encodes an E3 ubiquitin ligase, leads to an upregulation of Cap-dependent protein translation. The resulting imbalance in protein synthesis and degradation is found to disrupt glutamatergic transmission and excitability in networks that underlie sociability and anxiety. Progress in psychiatric genetics has led to the identification of hundreds of genes and genetic variants associated with risk. One such gene is Cullin 3 (Cul3), which was initially identified as an autism spectrum disorder (ASD) risk gene by analysis of whole-exome sequencing of parent-child trios exhibiting sporadic ASD (Kong et al., 2012; O’Roak et al., 2012). In addition, genome-wide association studies (GWASs) of schizophrenia (SCZ) have identified more than 100 common variants that each contribute a small amount of risk. One of these common risk variants is located in a genomic region containing CUL3 and is associated with reduced CUL3 expression in postmortem brain (Collado-Torres et al., 2019), thus implicating CUL3 in risk for SCZ. Despite abundant expression of CUL3 in the brain and its disease relevance, understanding of its role in brain development and function is limited. By studying Cul3 deficiency in a mouse model, Dong and colleagues have potentially identified shared pathophysiology between ASD and SCZ (Dong et al., 2020). The addition of ubiquitin to proteins is the main signal for proteasomal degradation. The process of ubiquitination is tightly regulated and is involved in many cellular processes, including cell-cycle regulation, transcription, signaling, trafficking, and protein quality control. CUL3 encodes an E3 ubiquitin ligase that forms a complex with RING-box protein and BTB protein that plays a critical role in the ligation of ubiquitin
to substrate proteins (Ande´rica-Romero et al., 2013). Dysfunction of proteasomal degradation has been previously associated with several human diseases. For instance, the accumulation of misfolded proteins is common to neurodegenerative disorders, while proteasome inhibitors are used to treat some forms of cancers. In this issue of Neuron, Dong et al. (2020) demonstrate that deficiencies in protein degradation pathways can lead to physiological and behavioral phenotypes that may underlie neuropsychiatric disorders. To understand how Cul3 deficiency leads to ASD, Dong et al. (2020) primarily focused on Cul3 heterozygous mice by crossing a Cul3 floxed animal to either GFAP-cre or NEX-cre mice. These mouse models produce Cul3 deficiency primarily in excitatory neurons, with GFAP-cre also targeting glial cells, and both models showed social deficits on the three-chamber social interaction tests and increased anxiety-like phenotype in the open field arena. With these behavioral deficits in hand, the authors then set out to determine whether morphological and physiological phenotypes were present within the hippocampus. They observed that in both GFAP-cre and NEX-cre lines, Cul3 deficiency in CA1 neurons had profound effects on neuron and network excitability. CA1 neurons showed increased spine density, increased miniature excitatory postsynaptic current (mEPSC) frequency, increased action potential output, enhanced presynaptic glutamate
398 Neuron 105, February 5, 2020 Crown Copyright ª 2020 Published by Elsevier Inc.
release, and a developmental increase in GABAergic miniature inhibitory postsynaptic current frequency, which equated to an overall excitation-inhibition (E/I) imbalance within the CA1 network. To link CUL3-dependent behavioral deficits to morphological and physiological phenotypes, Dong et al. (2020) turned to tandem mass tagging (TMT)-based quantitative proteomics to help identify associated biochemical mechanisms. Using this method, they identified a total of 5,720 proteins, of which 335 were differentially expressed (DE). Gene ontology analysis of increased DE proteins was consistent with increased synaptic transmission, showing enrichment for neuronal excitability, SNARE complex disassembly, synaptic vesicle localization, synaptic vesicle cycle, and presynaptic assembly. Although these DE proteins failed to survive false discovery rate correction, western blot analysis confirmed that many of these proteins were significantly upregulated. Dong et al. (2020) then focused their attention on eIF4G1, which was one of 14 upregulated DE proteins in common between Cul3 homozygous and heterozygous knockout samples and was previously shown to regulate Cap-dependent translation and synthesis of synaptic proteins (Marcotrigiano et al., 1999). They demonstrated that eIF4G1 expression is regulated by CUL3-dependent ubiquitination and that upregulation of eIF4G1 increased protein translation in Cul3-deficient animals.
Neuron
Previews To confirm that eIF4G1 upregulation was responsible for cellular and behavioral phenotypes associated with Cul3 deficiency, Dong and colleagues performed intracerebral ventricular injections of 4EGI-1, an inhibitor of Cap-dependent translation. This treatment rescued social deficits in Cul3-deficient mice but had little effect on anxiety-like behaviors. When they looked at 4EGI-1 rescue of neuronal morphology and physiology, they observed that spine density, mEPSC frequency, expression of synaptic vesicle proteins, and glutamate release were all normalized; however, 4EGI-1 failed to rescue increased intrinsic excitability. To specifically reduce intrinsic excitability of CA1 neurons, they relied on a chemogenic approach by virally injecting hM4Di, an inhibitory DREADD construct, into the hippocampus. Treatment with clozapine N-oxide (CNO) to activate the hM4Di receptor led to a reduction in CA1 action potential output and abolished the anxiety-like behaviors. Together, these two independent rescue experiments identified unique cellular phenotypes that underlie specific behavioral deficits. The results by Dong et al. (2020) are somewhat consistent with previous phenotypes observed in several mouse models of syndromic ASD risk genes. For instance, mutations in the UBE3A gene, which also encodes an E3 ubiquitin ligase and causes Angelman syndrome, leads to similar defects in synaptic vesicle cycling and E/I imbalance (Wallace et al., 2012). Together, these two studies demonstrate that ubiquitination is a critical process for the regulation of synaptic vesicle function, further implicating presynaptic terminals and the regulation of neurotransmitter release as a potential hotspot for ASD pathophysiology. How-
ever, one important distinction between these two reports is that the mouse model used in Wallace et al. (2012) had a germline mutation in the maternal Ube3a allele, which preferentially disrupted GABAergic terminals, leaving excitatory terminals intact despite Ube3a expression in all neurons. It will be interesting to see whether Cul3 deficiency in GABAergic neurons has any effect on inhibitory transmission by using a GABAergic-specific cre line or, even better, mutating Cul3 in the germline to match the human ASD condition. Dong et al. (2020) also demonstrated that Cul3 deficiency leads to increased intrinsic excitability, which is consistent with excitability phenotypes reported in mouse models of fragile X syndrome (Fmr1) and Rett’s syndrome (Mecp2) (Gibson et al., 2008; Wu et al., 2016) and suggests that dysregulation of ion channel expression and function may be essential to our understanding of ASD pathophysiology. Dong et al. (2020) showed that chemogenic knockdown of excitability in the hippocampus rescued hyperexcitability and anxiety-like behaviors, providing a physiological mechanism. However, the molecular mechanism remains unknown. Thus, an exciting next step would be to identify the ion channels involved and demonstrate pharmacological rescue, which could potentially pave the way to developing a therapeutic target. Overall, Dong et al. (2020) provide good evidence that Cul3 deficiency disrupts glutamatergic presynaptic transmission due to an imbalance of protein synthesis and degradation, thus building on evidence that the dysregulation of presynaptic transmitter release may play a prominent role in psychiatric disorders.
REFERENCES Ande´rica-Romero, A.C., Gonza´lez-Herrera, I.G., Santamarı´a, A., and Pedraza-Chaverri, J. (2013). Cullin 3 as a novel target in diverse pathologies. Redox Biol. 1, 366–372. Collado-Torres, L., Burke, E.E., Peterson, A., Shin, J., Straub, R.E., Rajpurohit, A., Semick, S.A., Ulrich, W.S., Price, A.J., Valencia, C., et al.; BrainSeq Consortium (2019). Regional Heterogeneity in Gene Expression, Regulation, and Coherence in the Frontal Cortex and Hippocampus across Development and Schizophrenia. Neuron 103, 203–216.e8. Dong, Z., Chen, W., Chen, C., Wang, H., Cui, W., Tan, Z., Robinson, H., Gao, N., Luo, B., Zhang, L., et al. (2020). CUL3 Deficiency Causes Social Deficits and Anxiety-like Behaviors by Impairing Excitation-Inhibition Balance through the Promotion of Cap-Dependent Translation. Neuron 105, this issue, 475–490. Gibson, J.R., Bartley, A.F., Hays, S.A., and Huber, K.M. (2008). Imbalance of neocortical excitation and inhibition and altered UP states reflect network hyperexcitability in the mouse model of fragile X syndrome. J. Neurophysiol. 100, 2615–2626. Kong, S.W., Collins, C.D., Shimizu-Motohashi, Y., Holm, I.A., Campbell, M.G., Lee, I.-H., Brewster, S.J., Hanson, E., Harris, H.K., Lowe, K.R., et al. (2012). Characteristics and predictive value of blood transcriptome signature in males with autism spectrum disorders. PLoS ONE 7, e49475. Marcotrigiano, J., Gingras, A.C., Sonenberg, N., and Burley, S.K. (1999). Cap-dependent translation initiation in eukaryotes is regulated by a molecular mimic of eIF4G. Mol. Cell 3, 707–716. O’Roak, B.J., Vives, L., Girirajan, S., Karakoc, E., Krumm, N., Coe, B.P., Levy, R., Ko, A., Lee, C., Smith, J.D., et al. (2012). Sporadic autism exomes reveal a highly interconnected protein network of de novo mutations. Nature 485, 246–250. Wallace, M.L., Burette, A.C., Weinberg, R.J., and Philpot, B.D. (2012). Maternal loss of Ube3a produces an excitatory/inhibitory imbalance through neuron type-specific synaptic defects. Neuron 74, 793–800. Wu, Y., Zhong, W., Cui, N., Johnson, C.M., Xing, H., Zhang, S., and Jiang, C. (2016). Characterization of Rett Syndrome-like phenotypes in Mecp2-knockout rats. J. Neurodev. Disord. 8, 23.
Neuron 105, February 5, 2020 399