Genotype-phenotype correlation in contactin-associated protein-like 2 (CNTNAP-2) developmental disorder.

Contactin-associated protein-like 2 (CNTNAP2) gene encodes for CASPR2, a presynaptic type 1 transmembrane protein, involved in cell-cell adhesion and synaptic interactions. Biallelic CNTNAP2 loss has been associated with "Pitt-Hopkins-like syndrome-1" (MIM#610042), while the pathogenic role of heterozygous variants remains controversial. We report 22 novel patients harboring mono- (n = 2) and bi-allelic (n = 20) CNTNAP2 variants and carried out a literature review to characterize the genotype-phenotype correlation. Patients (M:F 14:8) were aged between 3 and 19 years and affected by global developmental delay (GDD) (n = 21), moderate to profound intellectual disability (n = 17) and epilepsy (n = 21). Seizures mainly started in the first two years of life (median 22.5 months). Antiseizure medications were successful in controlling the seizures in about two-thirds of the patients. Autism spectrum disorder (ASD) and/or other neuropsychiatric comorbidities were present in nine patients (40.9%). Nonspecific midline brain anomalies were noted in most patients while focal signal abnormalities in the temporal lobes were noted in three subjects. Genotype-phenotype correlation was performed by also including 50 previously published patients (15 mono- and 35 bi-allelic variants). Overall, GDD (p < 0.0001), epilepsy (p < 0.0001), hyporeflexia (p = 0.012), ASD (p = 0.009), language impairment (p = 0.020) and severe cognitive impairment (p = 0.031) were significantly associated with the presence of biallelic versus monoallelic variants. We have defined the main features associated with biallelic CNTNAP2 variants, as severe cognitive impairment, epilepsy and behavioral abnormalities. We propose CASPR2-deficiency neurodevelopmental disorder as an exclusively recessive disease while the contribution of heterozygous variants is less likely to follow an autosomal dominant inheritance pattern.

Crucial neuroprotective roles of the metabolite BH4 in dopaminergic neurons.

Dopa-responsive dystonia (DRD) and Parkinson's disease (PD) are movement disorders caused by the dysfunction of nigrostriatal dopaminergic neurons. Identifying druggable pathways and biomarkers for guiding therapies is crucial due to the debilitating nature of these disorders. Recent genetic studies have identified variants of GTP cyclohydrolase-1 (GCH1), the rate-limiting enzyme in tetrahydrobiopterin (BH4) synthesis, as causative for these movement disorders. Here, we show that genetic and pharmacological inhibition of BH4 synthesis in mice and human midbrain-like organoids accurately recapitulates motor, behavioral and biochemical characteristics of these human diseases, with severity of the phenotype correlating with extent of BH4 deficiency. We also show that BH4 deficiency increases sensitivities to several PD-related stressors in mice and PD human cells, resulting in worse behavioral and physiological outcomes. Conversely, genetic and pharmacological augmentation of BH4 protects mice from genetically- and chemically induced PD-related stressors. Importantly, increasing BH4 levels also protects primary cells from PD-affected individuals and human midbrain-like organoids (hMLOs) from these stressors. Mechanistically, BH4 not only serves as an essential cofactor for dopamine synthesis, but also independently regulates tyrosine hydroxylase levels, protects against ferroptosis, scavenges mitochondrial ROS, maintains neuronal excitability and promotes mitochondrial ATP production, thereby enhancing mitochondrial fitness and cellular respiration in multiple preclinical PD animal models, human dopaminergic midbrain-like organoids and primary cells from PD-affected individuals. Our findings pinpoint the BH4 pathway as a key metabolic program at the intersection of multiple protective mechanisms for the health and function of midbrain dopaminergic neurons, identifying it as a potential therapeutic target for PD.

FIBCD1 is an endocytic GAG receptor associated with a novel neurodevelopmental disorder.

Whole-exome sequencing of two patients with idiopathic complex neurodevelopmental disorder (NDD) identified biallelic variants of unknown significance within FIBCD1, encoding an endocytic acetyl group-binding transmembrane receptor with no known function in the central nervous system. We found that FIBCD1 preferentially binds and endocytoses glycosaminoglycan (GAG) chondroitin sulphate-4S (CS-4S) and regulates GAG content of the brain extracellular matrix (ECM). In silico molecular simulation studies and GAG binding analyses of patient variants determined that such variants are loss-of-function by disrupting FIBCD1-CS-4S association. Gene knockdown in flies resulted in morphological disruption of the neuromuscular junction and motor-related behavioural deficits. In humans and mice, FIBCD1 is expressed in discrete brain regions, including the hippocampus. Fibcd1 KO mice exhibited normal hippocampal neuronal morphology but impaired hippocampal-dependent learning. Further, hippocampal synaptic remodelling in acute slices from Fibcd1 KO mice was deficient but restored upon enzymatically modulating the ECM. Together, we identified FIBCD1 as an endocytic receptor for GAGs in the brain ECM and a novel gene associated with an NDD, revealing a critical role in nervous system structure, function and plasticity.

Leu226Trp CACNA1A variant associated with juvenile myoclonic epilepsy with and without intellectual disability.

OBJECTIVE: Epilepsy is a disease of Central Nervous System (CNS) characterized by abnormal brain activity and recurrent seizures and is considered a clinically and genetically heterogeneous disease. Here, we investigated pathogenic genetic alteration and described the clinical characteristics of three Iranian family members affected by Idiopathic Generalized Epilepsy (IGE) with and without intellectual disability. METHODS: A non-consanguineous Iranian family with juvenile myoclonic epilepsy was enrolled in the study. The comprehensive neurological evaluation included motor and sensory skills, vision, hearing, speech, coordination, and mood. Whole-exome Sequencing (WES) was performed on the proband to detect probable pathogenic variant, and after the filtering process, probable variants were evaluated with familial segregation analysis using Sanger sequencing. RESULTS: Using WES, we identified a heterozygous missense substitution (NM_023035.3:c.T677G:p.Leu226Trp) in CACNA1A gene in the studied family with juvenile myoclonic epilepsy with and without intellectual disability and psychiatric phenotype. Considering the patients' clinical synopsis, familial segregation analysis, and literature review, we postulated this variant to be causative of the disease. Indeed, the resulting missense mutation of Leu226Trp affects a highly conserved residue supporting our hypothesis that this mutation is potentially pathogenic. CONCLUSION: To the best of our knowledge, this is the first report of juvenile myoclonic epilepsy related to CACNA1A gene. Our results provide evidence for expanding the clinical and molecular findings related to the CACNA1A gene.

Transcription factor mesenchyme homeobox protein 2 (MEOX2) modulates nociceptor function.

Mesenchyme homeobox protein 2 (MEOX2) is a transcription factor involved in mesoderm differentiation, including development of bones, muscles, vasculature and dermatomes. We have previously identified dysregulation of MEOX2 in fibroblasts from Congenital Insensitivity to Pain patients, and confirmed that btn, the Drosophila homologue of MEOX2, plays a role in nocifensive responses to noxious heat stimuli. To determine the importance of MEOX2 in the mammalian peripheral nervous system, we used a Meox2 heterozygous (Meox2+/- ) mouse model to characterise its function in the sensory nervous system, and more specifically, in nociception. MEOX2 is expressed in the mouse dorsal root ganglia (DRG) and spinal cord, and localises in the nuclei of a subset of sensory neurons. Functional studies of the mouse model, including behavioural, cellular and electrophysiological analyses, showed altered nociception encompassing impaired action potential initiation upon depolarisation. Mechanistically, we noted decreased expression of Scn9a and Scn11a genes encoding Nav 1.7 and Nav 1.9 voltage-gated sodium channels respectively, that are crucial in subthreshold amplification and action potential initiation in nociceptors. Further transcriptomic analyses of Meox2+/- DRG revealed downregulation of a specific subset of genes including those previously associated with pain perception, such as PENK and NPY. Based on these observations, we propose a novel role of MEOX2 in primary afferent nociceptor neurons for the maintenance of a transcriptional programme required for proper perception of acute and inflammatory noxious stimuli.

BioProfiling.jl: profiling biological perturbations with high-content imaging in single cells and heterogeneous populations.

MOTIVATION: High-content imaging screens provide a cost-effective and scalable way to assess cell states across diverse experimental conditions. The analysis of the acquired microscopy images involves assembling and curating raw cellular measurements into morphological profiles suitable for testing biological hypotheses. Despite being a critical step, general-purpose and adaptable tools for morphological profiling are lacking and no solution is available for the high-performance Julia programming language. RESULTS: Here, we introduce BioProfiling.jl, an efficient end-to-end solution for compiling and filtering informative morphological profiles in Julia. The package contains all the necessary data structures to curate morphological measurements and helper functions to transform, normalize and visualize profiles. Robust statistical distances and permutation tests enable quantification of the significance of the observed changes despite the high fraction of outliers inherent to high-content screens. This package also simplifies visual artifact diagnostics, thus streamlining a bottleneck of morphological analyses. We showcase the features of the package by analyzing a chemical imaging screen, in which the morphological profiles prove to be informative about the compounds' mechanisms of action and can be conveniently integrated with the network localization of molecular targets. AVAILABILITY AND IMPLEMENTATION: The Julia package is available on GitHub: https://github.com/menchelab/BioProfiling.jl. We also provide Jupyter notebooks reproducing our analyses: https://github.com/menchelab/BioProfilingNotebooks. The data underlying this article are available from FigShare, at https://doi.org/10.6084/m9.figshare.14784678.v2. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Network analysis reveals rare disease signatures across multiple levels of biological organization.

Rare genetic diseases are typically caused by a single gene defect. Despite this clear causal relationship between genotype and phenotype, identifying the pathobiological mechanisms at various levels of biological organization remains a practical and conceptual challenge. Here, we introduce a network approach for evaluating the impact of rare gene defects across biological scales. We construct a multiplex network consisting of over 20 million gene relationships that are organized into 46 network layers spanning six major biological scales between genotype and phenotype. A comprehensive analysis of 3,771 rare diseases reveals distinct phenotypic modules within individual layers. These modules can be exploited to mechanistically dissect the impact of gene defects and accurately predict rare disease gene candidates. Our results show that the disease module formalism can be applied to rare diseases and generalized beyond physical interaction networks. These findings open up new venues to apply network-based tools for cross-scale data integration.

PRDM12 Is Transcriptionally Active and Required for Nociceptor Function Throughout Life.

PR domain-containing member 12 (PRDM12) is a key developmental transcription factor in sensory neuronal specification and survival. Patients with rare deleterious variants in PRDM12 are born with congenital insensitivity to pain (CIP) due to the complete absence of a subtype of peripheral neurons that detect pain. In this paper, we report two additional CIP cases with a novel homozygous PRDM12 variant. To elucidate the function of PRDM12 during mammalian development and adulthood, we generated temporal and spatial conditional mouse models. We find that PRDM12 is expressed throughout the adult nervous system. We observed that loss of PRDM12 during mid-sensory neurogenesis but not in the adult leads to reduced survival. Comparing cellular biophysical nociceptive properties in developmental and adult-onset PRDM12 deletion mouse models, we find that PRDM12 is necessary for proper nociceptive responses throughout life. However, we find that PRDM12 regulates distinct age-dependent transcriptional programs. Together, our results implicate PRDM12 as a viable therapeutic target for specific pain therapies even in adults.

Expanding the known phenotype of Mullegama-Klein-Martinez syndrome in male patients.

Here, we report a novel case of a male patient with a hemizygous missense variant in STAG2 (p.Tyr159His) resulting in Mullegama-Klein-Martinez syndrome (MKMS), a rare X-linked cohesinopathy. He shares distinct clinical features with a previously reported male patient carrying the STAG2 variant p.Tyr159Cys, suggesting that this phenotype is determined by the position of the mutation. Additionally, our patient exhibits symptoms not previously associated with MKMS, expanding the known clinical phenotype of this rare disease.

Cellular Models and High-Throughput Screening for Genetic Causality of Intellectual Disability.

Intellectual disabilities (ID) are a type of neurodevelopmental disorder (NDD). They can have a genetic cause, including an emerging class of ID centring around Rho GTPases, such as Ras-related C3 botulinum toxin substrate 1 (RAC1). Guidelines for establishing genetic causality include the use of cellular models, which often have morphological aberrations, a long-standing hallmark of ID. Disease cellular models can facilitate high-throughput screening (HTS) of chemical or genetic perturbations, which can provide translatable biological insight. Here, we discuss a class of IDs centring around RAC1. We review novel and established cellular models of ID, including mouse and human primary cells and reprogrammed or induced neurons. Finally, we review progress and remaining challenges in the adoption of HTS methodologies by the community studying neurological disorders.

PRDM12: New Opportunity in Pain Research.

PRDM12 is a newly identified causative gene for a type of congenital insensitivity to pain disorder, which is characterized by the inability to perceive pain. Here, we discuss the (patho)physiology of PRDM12 function and the opportunities and challenges those data provide for novel therapeutic approaches in various pain disorders.

Identification of ALK in Thinness.

There is considerable inter-individual variability in susceptibility to weight gain despite an equally obesogenic environment in large parts of the world. Whereas many studies have focused on identifying the genetic susceptibility to obesity, we performed a GWAS on metabolically healthy thin individuals (lowest 6th percentile of the population-wide BMI spectrum) in a uniquely phenotyped Estonian cohort. We discovered anaplastic lymphoma kinase (ALK) as a candidate thinness gene. In Drosophila, RNAi mediated knockdown of Alk led to decreased triglyceride levels. In mice, genetic deletion of Alk resulted in thin animals with marked resistance to diet- and leptin-mutation-induced obesity. Mechanistically, we found that ALK expression in hypothalamic neurons controls energy expenditure via sympathetic control of adipose tissue lipolysis. Our genetic and mechanistic experiments identify ALK as a thinness gene, which is involved in the resistance to weight gain.

HACE1 deficiency leads to structural and functional neurodevelopmental defects.

OBJECTIVE: We aim to characterize the causality and molecular and functional underpinnings of HACE1 deficiency in a mouse model of a recessive neurodevelopmental syndrome called spastic paraplegia and psychomotor retardation with or without seizures (SPPRS). METHODS: By exome sequencing, we identified 2 novel homozygous truncating mutations in HACE1 in 3 patients from 2 families, p.Q209* and p.R332*. Furthermore, we performed detailed molecular and phenotypic analyses of Hace1 knock-out (KO) mice and SPPRS patient fibroblasts. RESULTS: We show that Hace1 KO mice display many clinical features of SPPRS including enlarged ventricles, hypoplastic corpus callosum, as well as locomotion and learning deficiencies. Mechanistically, loss of HACE1 results in altered levels and activity of the small guanosine triphosphate (GTP)ase, RAC1. In addition, HACE1 deficiency results in reduction in synaptic puncta number and long-term potentiation in the hippocampus. Similarly, in SPPRS patient-derived fibroblasts, carrying a disruptive HACE1 mutation resembling loss of HACE1 in KO mice, we observed marked upregulation of the total and active, GTP-bound, form of RAC1, along with an induction of RAC1-regulated downstream pathways. CONCLUSIONS: Our results provide a first animal model to dissect this complex human disease syndrome, establishing the first causal proof that a HACE1 deficiency results in decreased synapse number and structural and behavioral neuropathologic features that resemble SPPRS patients.

Prdm12 Directs Nociceptive Sensory Neuron Development by Regulating the Expression of the NGF Receptor TrkA.

In humans, many cases of congenital insensitivity to pain (CIP) are caused by mutations of components of the NGF/TrkA signaling pathway, which is required for survival and specification of nociceptors and plays a major role in pain processing. Mutations in PRDM12 have been identified in CIP patients that indicate a putative role for this transcriptional regulator in pain sensing. Here, we show that Prdm12 expression is restricted to developing and adult nociceptors and that its genetic ablation compromises their viability and maturation. Mechanistically, we find that Prdm12 is required for the initiation and maintenance of the expression of TrkA by acting as a modulator of Neurogenin1/2 transcription factor activity, in frogs, mice, and humans. Altogether, our results identify Prdm12 as an evolutionarily conserved key regulator of nociceptor specification and as an actionable target for new pain therapeutics.

The evolutionarily conserved transcription factor PRDM12 controls sensory neuron development and pain perception.

PR homology domain-containing member 12 (PRDM12) belongs to a family of conserved transcription factors implicated in cell fate decisions. Here we show that PRDM12 is a key regulator of sensory neuronal specification in Xenopus. Modeling of human PRDM12 mutations that cause hereditary sensory and autonomic neuropathy (HSAN) revealed remarkable conservation of the mutated residues in evolution. Expression of wild-type human PRDM12 in Xenopus induced the expression of sensory neuronal markers, which was reduced using various human PRDM12 mutants. In Drosophila, we identified Hamlet as the functional PRDM12 homolog that controls nociceptive behavior in sensory neurons. Furthermore, expression analysis of human patient fibroblasts with PRDM12 mutations uncovered possible downstream target genes. Knockdown of several of these target genes including thyrotropin-releasing hormone degrading enzyme (TRHDE) in Drosophila sensory neurons resulted in altered cellular morphology and impaired nociception. These data show that PRDM12 and its functional fly homolog Hamlet are evolutionary conserved master regulators of sensory neuronal specification and play a critical role in pain perception. Our data also uncover novel pathways in multiple species that regulate evolutionary conserved nociception.

The RANKL-RANK Story.

Receptor activator of nuclear factor x03BA;B (RANK) and its ligand (RANKL) have originally been described for their key roles in bone metabolism and the immune system. Subsequently, it has been shown that the RANKL-RANK system is critical in the formation of mammary epithelia in lactating females and the thermoregulation of the central nervous system. RANKL and RANK are under the tight control of the female sex hormones estradiol and progesterone. A reduction of the circulating female sex hormones leading to an increase in RANKL-RANK signaling is the leading cause of osteoporosis in postmenopausal women. Denosumab, a human monoclonal anti-RANKL antibody, has been approved for the treatment of postmenopausal osteoporosis, where it is showing great promise. In addition, RANKL-RANK signaling also plays a critical role in other bone pathologies, bone metastasis or hormone-driven breast cancer. This review will highlight some of the functions of RANKL-RANK in bone turnover, the immune system and brain with a focus on the regulatory role of the female sex hormones.

SHARPIN forms a linear ubiquitin ligase complex regulating NF-κB activity and apoptosis.

SHARPIN is a ubiquitin-binding and ubiquitin-like-domain-containing protein which, when mutated in mice, results in immune system disorders and multi-organ inflammation. Here we report that SHARPIN functions as a novel component of the linear ubiquitin chain assembly complex (LUBAC) and that the absence of SHARPIN causes dysregulation of NF-κB and apoptotic signalling pathways, explaining the severe phenotypes displayed by chronic proliferative dermatitis (cpdm) in SHARPIN-deficient mice. Upon binding to the LUBAC subunit HOIP (also known as RNF31), SHARPIN stimulates the formation of linear ubiquitin chains in vitro and in vivo. Coexpression of SHARPIN and HOIP promotes linear ubiquitination of NEMO (also known as IKBKG), an adaptor of the IκB kinases (IKKs) and subsequent activation of NF-κB signalling, whereas SHARPIN deficiency in mice causes an impaired activation of the IKK complex and NF-κB in B cells, macrophages and mouse embryonic fibroblasts (MEFs). This effect is further enhanced upon concurrent downregulation of HOIL-1L (also known as RBCK1), another HOIP-binding component of LUBAC. In addition, SHARPIN deficiency leads to rapid cell death upon tumour-necrosis factor α (TNF-α) stimulation via FADD- and caspase-8-dependent pathways. SHARPIN thus activates NF-κB and inhibits apoptosis via distinct pathways in vivo.

Ubiquitin ligase complexes: from substrate selectivity to conjugational specificity.

Localization, activity and lifespan of a protein are signaled by a small, 8 kDa protein, ubiquitin (Ub). Ub conjugation is a post-translational modification orchestrated by the sequential action of activating (E1), conjugating (E2), and ligating (E3) enzymes. Although a simple combination of an E2 and an E3 enzyme can be sufficient for an active complex, in other cases ubiquitination can occur in the context of large multimeric complexes with enhanced molecular abilities. Here, we review several Ub ligase complexes to highlight strategies governing conjugational specificity, the gained adaptability in substrate specificity, and modulatory flexibility encoded in regulatory components of these diverse multimers.

Cadherin-8 and N-cadherin differentially regulate pre- and postsynaptic development of the hippocampal mossy fiber pathway.

Cells sort into regions and groups in part by their selective surface expression of particular classic cadherins during development. In the nervous system, cadherin-based sorting can define axon tracts, restrict axonal and dendritic arbors to particular regions or layers, and may encode certain aspects of synapse specificity. The underlying model has been that afferents and their targets hold in common the expression of a particular cadherin, thereby providing a recognition code of homophilic cadherin binding. However, most neurons express multiple cadherins, and it is not clear whether multiple cadherins all act similarly in shaping neural circuitry. Here we asked how two such cadherins, cadherin-8 and N-cadherin, influence the guidance and differentiation of hippocampal mossy fibers. Using organotypic hippocampal cultures, we find that cadherin-8 regulates mossy fiber fasciculation and targeting, but has little effect on CA3 dendrites. In contrast, N-cadherin regulates mossy fiber fasciculation, but has little impact on axonal growth and targeting. However, N-cadherin is essential for CA3 dendrite arborization. Both cadherins are required for formation of proper numbers of presynaptic terminals. Mechanistically, such differential actions of these two cadherins could, in theory, reflect coupling to distinct intracellular binding partners. However, we find that both cadherins bind beta-catenin in dentate gyrus (DG). This suggests that cadherins may engage different intracellular signaling cascades downstream of beta-catenin, coopt different extracellular binding partners, or target distinct subcellular domains. Together our findings demonstrate that cadherin-8 and N-cadherin are critical for generating the mossy fiber pathway, but that each contributes differentially to afferent and target differentiation, thereby complementing one another in the assembly of a synaptic circuit.

The extracellular protease matrix metalloproteinase-9 is activated by inhibitory avoidance learning and required for long-term memory.

Matrix metalloproteinases (MMPs) are a family of extracellularly acting proteolytic enzymes with well-recognized roles in plasticity and remodeling of synaptic circuits during brain development and following brain injury. However, it is now becoming increasingly apparent that MMPs also function in normal, nonpathological synaptic plasticity of the kind that may underlie learning and memory. Here, we extend this idea by investigating the role and regulation of MMP-9 in an inhibitory avoidance (IA) learning and memory task. We demonstrate that following IA training, protein levels and proteolytic activity of MMP-9 become elevated in hippocampus by 6 h, peak at 12-24 h, then decline to baseline values by approximately 72 h. When MMP function is abrogated by intrahippocampal infusion of a potent gelatinase (MMP-2 and MMP-9) inhibitor 3.5 h following IA training, a time prior to the onset of training-induced elevation in levels, IA memory retention is significantly diminished when tested 1-3 d later. Animals impaired at 3 d exhibit robust IA memory when retrained, suggesting that such impairment is not likely attributed to toxic or other deleterious effects that permanently disrupt hippocampal function. In anesthetized adult rats, the effective distance over which synaptic plasticity is impaired by a single intrahippocampal infusion of the MMP inhibitor of the kind that blocks IA memory is approximately 1200 microm. Taken together, these data suggest that IA training induces a slowly emerging, but subsequently protracted period of MMP-mediated proteolysis critical for enabling long-lasting synaptic modification that underlies long-term memory consolidation.