An IGFBP2-derived peptide promotes neuroplasticity and rescues deficits in a mouse model of Phelan-McDermid syndrome.

We developed an IGFBP2-mimetic peptide fragment, JB2, and showed that it promotes basal synaptic structural and functional plasticity in cultured neurons and mice. We demonstrate that JB2 directly binds to dendrites and synapses, and its biological activity involves NMDA receptor activation, gene transcription and translation, and IGF2 receptors. It is not IGF1 receptor-dependent. In neurons, JB2 induced extensive remodeling of the membrane phosphoproteome. Synapse and cytoskeletal regulation, autism spectrum disorder (ASD) risk factors, and a Shank3-associated protein network were significantly enriched among phosphorylated and dephosphorylated proteins. Haploinsufficiency of the SHANK3 gene on chromosome 22q13.3 often causes Phelan-McDermid Syndrome (PMS), a genetically defined form of autism with profound deficits in motor behavior, sensory processing, language, and cognitive function. We identified multiple disease-relevant phenotypes in a Shank3 heterozygous mouse and showed that JB2 rescued deficits in synaptic function and plasticity, learning and memory, ultrasonic vocalizations, and motor function; it also normalized neuronal excitability and seizure susceptibility. Notably, JB2 rescued deficits in the auditory evoked response latency, alpha peak frequency, and steady-state electroencephalography response, measures with direct translational value to human subjects. These data demonstrate that JB2 is a potent modulator of neuroplasticity with therapeutic potential for the treatment of PMS and ASD.

Genetic and metabolic profiling of individuals with Phelan-McDermid syndrome presenting with seizures.

Phelan-McDermid syndrome (PMS) (OMIM*606232) is a rare genetic disorder characterized by intellectual disability, autistic features, speech delay, minor dysmorphia, and seizures. This study was conducted to investigate the prevalence of seizures and the association with genetic and metabolic features since there has been little research related to seizures in PMS. For 57 individuals, seizure data was collected from caregiver interviews, genetic data from existing cytogenetic records and Sanger sequencing for nine 22q13 genes, and metabolic profiling from the Phenotype Mammalian MicroArray (PM-M) developed by Biolog. Results showed that 46% of individuals had seizures with the most common type being absence and grand-mal seizures. Seizures were most prevalent in individuals with pathogenic SHANK3 mutations (70%), those with deletion sizes >4 Mb (16%), and those with deletion sizes <4 Mb (71%) suggesting involvement of genes in addition to SHANK3. Additionally, a 3 Mb genomic region on 22q13.31 containing the gene TBC1D22A, was found to be significantly associated with seizure prevalence. A distinct metabolic profile was identified for individuals with PMS with seizures and suggested among other features a disrupted utilization of main energy sources using Biolog plates. The results of this study will be helpful for clinicians and families in anticipating seizures in these children and for researchers to identify candidate genes for the seizure phenotype.

Restoring Shank3 in the rostral brainstem of shank3ab-/- zebrafish autism models rescues sensory deficits.

People with Phelan-McDermid Syndrome, caused by mutations in the SHANK3 gene, commonly exhibit reduced responses to sensory stimuli; yet the changes in brain-wide activity that link these symptoms to mutations in the shank3 gene remain unknown. Here we quantify movement in response to sudden darkness in larvae of two shank3 zebrafish mutant models and show that both models exhibit dampened responses to this stimulus. Using brain-wide activity mapping, we find that shank3-/- light-sensing brain regions show normal levels of activity while sensorimotor integration and motor regions are less active. Specifically restoring Shank3 function in a sensorimotor nucleus of the rostral brainstem enables the shank3-/- model to respond like wild-type. In sum, we find that reduced sensory responsiveness in shank3-/- models is associated with reduced activity in sensory processing brain regions and can be rescued by restoring Shank3 function in the rostral brainstem. These studies highlight the importance of Shank3 function in the rostral brainstem for integrating sensory inputs to generate behavioral adaptations to changing sensory stimuli.

Homer1a regulates Shank3 expression and underlies behavioral vulnerability to stress in a model of Phelan-McDermid syndrome.

Mutations of SHANK3 cause Phelan-McDermid syndrome (PMS), and these individuals can exhibit sensitivity to stress, resulting in behavioral deterioration. Here, we examine the interaction of stress with genotype using a mouse model with face validity to PMS. In Shank3ΔC/+ mice, swim stress produces an altered transcriptomic response in pyramidal neurons that impacts genes and pathways involved in synaptic function, signaling, and protein turnover. Homer1a, which is part of the Shank3-mGluR-N-methyl-D-aspartate (NMDA) receptor complex, is super-induced and is implicated in the stress response because stress-induced social deficits in Shank3ΔC/+ mice are mitigated in Shank3ΔC/+;Homer1a-/- mice. Several lines of evidence demonstrate that Shank3 expression is regulated by Homer1a in competition with crosslinking forms of Homer, and consistent with this model, Shank3 expression and function that are reduced in Shank3ΔC/+ mice are rescued in Shank3ΔC/+;Homer1a-/- mice. Studies highlight the interaction between stress and genetics and focus attention on activity-dependent changes that may contribute to pathogenesis.

Cecr2 mutant mice as a model for human cat eye syndrome.

Cat eye syndrome (CES), a human genetic disorder caused by the inverted duplication of a region on chromosome 22, has been known since the late 1890s. Despite the significant impact this disorder has on affected individuals, models for CES have not been produced due to the difficulty of effectively duplicating the corresponding chromosome region in an animal model. However, the study of phenotypes associated with individual genes in this region such as CECR2 may shed light on the etiology of CES. In this study we have shown that deleterious loss of function mutations in mouse Cecr2 effectively demonstrate many of the abnormal features present in human patients with CES, including coloboma and specific skeletal, kidney and heart defects. Beyond phenotypic analyses we have demonstrated the importance of utilizing multiple genetic backgrounds to study disease models, as we see major differences in penetrance of Cecr2-related abnormal phenotype between mouse strains, reminiscent of the variability in the human syndrome. These findings suggest that Cecr2 is involved in the abnormal features of CES and that Cecr2 mice can be used as a model system to study the wide range of phenotypes present in CES.

The Mitochondrial Citrate Carrier SLC25A1/CIC and the Fundamental Role of Citrate in Cancer, Inflammation and Beyond.

The mitochondrial citrate/isocitrate carrier, CIC, has been shown to play an important role in a growing list of human diseases. CIC belongs to a large family of nuclear-encoded mitochondrial transporters that serve the fundamental function of allowing the transit of ions and metabolites through the impermeable mitochondrial membrane. Citrate is central to mitochondrial metabolism and respiration and plays fundamental activities in the cytosol, serving as a metabolic substrate, an allosteric enzymatic regulator and, as the source of Acetyl-Coenzyme A, also as an epigenetic modifier. In this review, we highlight the complexity of the mechanisms of action of this transporter, describing its involvement in human diseases and the therapeutic opportunities for targeting its activity in several pathological conditions.

Chromosome 22q11.2 deletion causes PERK-dependent vulnerability in dopaminergic neurons.

BACKGROUND: The chromosome 22q11.2 deletion is an extremely high risk genetic factor for various neuropsychiatric disorders; however, the 22q11.2 deletion-related brain pathology in humans at the cellular and molecular levels remains unclear. METHODS: We generated iPS cells from healthy controls (control group) and patients with 22q11.2 deletion (22DS group), and differentiated them into dopaminergic neurons. Semiquantitative proteomic analysis was performed to compare the two groups. Next, we conducted molecular, cell biological and pharmacological assays. FINDINGS: Semiquantitative proteomic analysis identified 'protein processing in the endoplasmic reticulum (ER)' as the most altered pathway in the 22DS group. In particular, we found a severe defect in protein kinase R-like endoplasmic reticulum kinase (PERK) expression and its activity in the 22DS group. The decreased PERK expression was also shown in the midbrain of a 22q11.2 deletion mouse model. The 22DS group showed characteristic phenotypes, including poor tolerance to ER stress, abnormal F-actin dynamics, and decrease in protein synthesis. Some of phenotypes were rescued by the pharmacological manipulation of PERK activity and phenocopied in PERK-deficient dopaminergic neurons. We lastly showed that DGCR14 was associated with reduction in PERK expression. INTERPRETATION: Our findings led us to conclude that the 22q11.2 deletion causes various vulnerabilities in dopaminergic neurons, dependent on PERK dysfunction. FUNDING: This study was supported by the AMED under grant nos JP20dm0107087, JP20dm0207075, JP20ak0101113, JP20dk0307081, and JP18dm0207004h0005; the MEXT KAKENHI under grant nos. 16K19760, 19K08015, 18H04040, and 18K19511; the Uehara Memorial Foundation under grant no. 201810122; and 2019 iPS Academia Japan Grant.

A novel aberration of COL1A1-PDGFB fusion as an insertion in chromosome 15 in one case of dermatofibrosarcoma protuberans involving a rare location.

Dermatofibrosarcoma protuberans (DFSP) is a rare sarcoma of the skin arising from the dermis. Its location is most commonly presented on the trunk of middle-aged adults and rarely on the face. The characteristic genetic aberration in the form of a reciprocal translocation t(17;22)(q21;q13) or a ring fusing the COL1A1 and PDGFB genes is found in 90% of DFSP. We present a case of a 42-year-old man who presented with a DFSP on the left cheek with foci of myxoid-fibrosarcomatous transformation. A conventional chromosomal analysis revealed a complex karyotype without a supernumerary ring chromosome or a linear translocation t(17;22). Comparative genome hybridization and fluorescence in-situ hybridization revealed the fusion of COL1A1 and PDGFB probes inserted in chromosome 15. This is a unique case of DFSP characterized by a rare body location, unique histopathological features, and novel chromosome COL1A1-PDGFB insertion, and may help guide future diagnostic and patient care modalities.

Increased rates of cerebral protein synthesis in Shank3 knockout mice: Implications for a link between synaptic protein deficit and dysregulated protein synthesis in autism spectrum disorder/intellectual disability.

SHANK3 is a postsynaptic scaffolding protein that plays a critical role in synaptic development and brain function. Mutations in SHANK3 are implicated in Phelan-McDermid syndrome (PMS), a neurodevelopmental disorder characterized by autistic-like behavior, delayed speech, hypotonia, and intellectual disability (ID). Moreover, mutations in SHANK3 occur in 1-2% of cases of idiopathic autism spectrum disorder (ASD). In fragile X syndrome (FXS), a syndromic form of autism, SHANK3 is one of the 842 targets of fragile X mental retardation protein (FMRP), the protein product of the silenced FMR1 gene. FXS is likely a primary disorder of the regulation of translation, whereas other syndromic forms of ASD/ID, e.g. PMS, appear to be primary disorders of synaptic structure. In this study, we asked if a knockout of the synaptic protein, Shank3, is linked to an effect on translation. Specifically, we measured the effect of Shank3 loss on rates of cerebral protein synthesis (rCPS) in vivo by means of the L-[1-14C]leucine quantitative autoradiographic method. We found that Shank3 knockout mice had significantly increased rCPS in every brain region examined. Our results suggest a link in ASD/ID between synaptic structure and regulation of translation.

22q11.2 duplication: a review of neuropsychiatric correlates and a newly observed case of prototypic sociopathy.

Callous-unemotional (CU) traits are highly disabling behavioral characteristics, common predictors of delinquency and criminality, and pathognomonic for antisocial personality disorder. They are highly heritable, but their specific molecular genetic causes are unknown. Here, we briefly review the literature on neuropsychiatric correlates of 22q11.2 duplication and describe a newly identified case of a 737-kb microduplication within the low copy repeat (LCR) B-D region, involving a 13-yr-old early adoptee with mild developmental delay and severe, chronic antisocial behavior of early childhood onset. When psychiatric symptoms have been reported in relation to duplications in this specific region, 19% of the reports feature aggression-but never previously CU traits-as a component of the phenotype. We discuss the potential implications of gain of function in this chromosomal region for heritable origins of sociopathy and their possible relation to genetic influences on aggression.

Further support linking the 22q11.2 microduplication to an increased risk of bladder exstrophy and highlighting LZTR1 as a candidate gene.

BACKGROUND: The bladder exstrophy-epispadias complex (BEEC) is a congenital malformation of the bladder and urethra. The underlying causes of this malformation are still largely unknown; however, aside from environment, genetics is thought to play an essential role. The recurrent 22q11.2 microduplication is the most persistently detected genetic aberration found in BEEC cases. METHODS: We performed array comparative genomic hybridization (array-CGH) analysis of 76 Swedish BEEC patients. Statistical analysis was performed on current dataset pooled with previously published data on the 22q11.2 microduplication in BEEC patients. We performed massive parallel sequencing (MPS) of the 22q11.2 region in 20 BEEC patients without the 22q11.2 microduplication followed by functional studies. RESULTS: We identified three additional cases with the 22q11.2 microduplication. Pooling data from this study with previously published reports showed a statistically significant enrichment of the 22q11.2 microduplication in BEEC patients (2.61% in cases vs. 0.08% in controls; OR = 32.6; p = 8.7 × 10-4 ). MPS of the 22q11.2 region in 20 BEEC patients without the 22q11.2 microduplication identified a novel variant in LZTR1 (p.Ser698Phe) in one patient. Functional evaluation of the LZTR1 p.Ser698Phe variant in live NIH 3T3 cells showed that the concentration and cytoplasmic mobility differ between the Lztr1wt and Lztr1mut , indicating a potential functional effect of the LZTR1mut . CONCLUSION: Our study further emphasizes the involvement of the 22q11.2 region in BEEC development and highlights LZTR1 as a candidate gene underlying the urogenital malformation.

Systems Analysis of the 22q11.2 Microdeletion Syndrome Converges on a Mitochondrial Interactome Necessary for Synapse Function and Behavior.

Neurodevelopmental disorders offer insight into synaptic mechanisms. To unbiasedly uncover these mechanisms, we studied the 22q11.2 syndrome, a recurrent copy number variant, which is the highest schizophrenia genetic risk factor. We quantified the proteomes of 22q11.2 mutant human fibroblasts from both sexes and mouse brains carrying a 22q11.2-like defect, Df(16)A+/- Molecular ontologies defined mitochondrial compartments and pathways as some of top ranked categories. In particular, we identified perturbations in the SLC25A1-SLC25A4 mitochondrial transporter interactome as associated with the 22q11.2 genetic defect. Expression of SLC25A1-SLC25A4 interactome components was affected in neuronal cells from schizophrenia patients. Furthermore, hemideficiency of the Drosophila SLC25A1 or SLC25A4 orthologues, dSLC25A1-sea and dSLC25A4-sesB, affected synapse morphology, neurotransmission, plasticity, and sleep patterns. Our findings indicate that synapses are sensitive to partial loss of function of mitochondrial solute transporters. We propose that mitoproteomes regulate synapse development and function in normal and pathological conditions in a cell-specific manner.SIGNIFICANCE STATEMENT We address the central question of how to comprehensively define molecular mechanisms of the most prevalent and penetrant microdeletion associated with neurodevelopmental disorders, the 22q11.2 microdeletion syndrome. This complex mutation reduces gene dosage of ∼63 genes in humans. We describe a disruption of the mitoproteome in 22q11.2 patients and brains of a 22q11.2 mouse model. In particular, we identify a network of inner mitochondrial membrane transporters as a hub required for synapse function. Our findings suggest that mitochondrial composition and function modulate the risk of neurodevelopmental disorders, such as schizophrenia.

X-ray Structures and Feasibility Assessment of CLK2 Inhibitors for Phelan-McDermid Syndrome.

CLK2 inhibition has been proposed as a potential mechanism to improve autism and neuronal functions in Phelan-McDermid syndrome (PMDS). Herein, the discovery of a very potent indazole CLK inhibitor series and the CLK2 X-ray structure of the most potent analogue are reported. This new indazole series was identified through a biochemical CLK2 Caliper assay screen with 30k compounds selected by an in silico approach. Novel high-resolution X-ray structures of all CLKs, including the first CLK4 X-ray structure, bound to known CLK2 inhibitor tool compounds (e.g., TG003, CX-4945), are also shown and yield insight into inhibitor selectivity in the CLK family. The efficacy of the new CLK2 inhibitors from the indazole series was demonstrated in the mouse brain slice assay, and potential safety concerns were investigated. Genotoxicity findings in the human lymphocyte micronucleus test (MNT) assay are shown by using two structurally different CLK inhibitors to reveal a major concern for pan-CLK inhibition in PMDS.

Murine model indicates 22q11.2 signaling adaptor CRKL is a dosage-sensitive regulator of genitourinary development.

The spectrum of congenital anomalies affecting either the upper tract (kidneys and ureters) or lower tract (reproductive organs) of the genitourinary (GU) system are fundamentally linked by the developmental origin of multiple GU tissues, including the kidneys, gonads, and reproductive ductal systems: the intermediate mesoderm. Although ∼31% of DiGeorge/del22q11.2 syndrome patients exhibit GU defects, little focus has been placed on the molecular etiology of GU defects in this syndrome. Among del22q11.2 patients exhibiting GU anomalies, we have mapped the smallest relevant region to only five genes, including CRKLCRKL encodes a src-homology adaptor protein implicated in mediating tyrosine kinase signaling, and is expressed in the developing GU-tract in mice and humans. Here we show that Crkl mutant embryos exhibit gene dosage-dependent growth restriction, and homozygous mutants exhibit upper GU defects at a microdissection-detectable rate of 23%. RNA-sequencing revealed that 52 genes are differentially regulated in response to uncoupling Crkl from its signaling pathways in the developing kidney, including a fivefold up-regulation of Foxd1, a known regulator of nephron progenitor differentiation. Additionally, Crkl heterozygous adult males exhibit cryptorchidism, lower testis weight, lower sperm count, and subfertility. Together, these data indicate that CRKL is intimately involved in normal development of both the upper and lower GU tracts, and disruption of CRKL contributes to the high incidence of GU defects associated with deletion at 22q11.2.

Zinc deficiency and low enterocyte zinc transporter expression in human patients with autism related mutations in SHANK3.

Phelan McDermid Syndrome (PMDS) is a genetic disorder characterized by features of Autism spectrum disorders. Similar to reports of Zn deficiency in autistic children, we have previously reported high incidence of Zn deficiency in PMDS. However, the underlying mechanisms are currently not well understood. Here, using inductively coupled plasma mass-spectrometry to measure the concentration of Zinc (Zn) and Copper (Cu) in hair samples from individuals with PMDS with 22q13.3 deletion including SHANK3 (SH3 and multiple ankyrin repeat domains 3), we report a high rate of abnormally low Zn/Cu ratios. To investigate possible underlying mechanisms, we generated enterocytes from PMDS patient-derived induced pluripotent stem cells and used Caco-2 cells with knockdown of SHANK3. We detected decreased expression of Zn uptake transporters ZIP2 and ZIP4 on mRNA and protein level correlating with SHANK3 expression levels, and found reduced levels of ZIP4 protein co-localizing with SHANK3 at the plasma membrane. We demonstrated that especially ZIP4 exists in a complex with SHANK3. Furthermore, we performed immunohistochemistry on gut sections from Shank3αß knockout mice and confirmed a link between enterocytic SHANK3, ZIP2 and ZIP4. We conclude that apart from its well-known role in the CNS, SHANK3 might play a specific role in the GI tract.

Pharmacological enhancement of mGlu5 receptors rescues behavioral deficits in SHANK3 knock-out mice.

SHANK3 (also called PROSAP2) genetic haploinsufficiency is thought to be the major cause of neuropsychiatric symptoms in Phelan-McDermid syndrome (PMS). PMS is a rare genetic disorder that causes a severe form of intellectual disability (ID), expressive language delays and other autistic features. Furthermore, a significant number of SHANK3 mutations have been identified in patients with autism spectrum disorders (ASD), and SHANK3 truncating mutations are associated with moderate to profound ID. The Shank3 protein is a scaffold protein that is located in the postsynaptic density (PSD) of excitatory synapses and is crucial for synapse development and plasticity. In this study, we investigated the molecular mechanisms associated with the ASD-like behaviors observed in Shank3Δ11-/- mice, in which exon 11 has been deleted. Our results indicate that Shank3 is essential to mediating metabotropic glutamate receptor 5 (mGlu5)-receptor signaling by recruiting Homer1b/c to the PSD, specifically in the striatum and cortex. Moreover, augmenting mGlu5-receptor activity by administering 3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide ameliorated the functional and behavioral defects that were observed in Shank3Δ11-/- mice, suggesting that pharmaceutical treatments that increase mGlu5 activity may represent a new approach for treating patients that are affected by PMS and SHANK3 mutations.