In search of a cure: PACS1 Research Foundation as a model of rare disease therapy development.

Rare diseases affect nearly 400 million people worldwide and have a devastating impact on patients and families. Although these diseases are collectively common, they are often overlooked by the research community. We present the ongoing work of the PACS1 Syndrome Research Foundation as a paradigm for approaching rare disease research.

Pathogenic MAST3 Variants in the STK Domain Are Associated with Epilepsy.

OBJECTIVE: The MAST family of microtubule-associated serine-threonine kinases (STKs) have distinct expression patterns in the developing and mature human and mouse brain. To date, only MAST1 has been conclusively associated with neurological disease, with de novo variants in individuals with a neurodevelopmental disorder, including a mega corpus callosum. METHODS: Using exome sequencing, we identify MAST3 missense variants in individuals with epilepsy. We also assess the effect of these variants on the ability of MAST3 to phosphorylate the target gene product ARPP-16 in HEK293T cells. RESULTS: We identify de novo missense variants in the STK domain in 11 individuals, including 2 recurrent variants p.G510S (n = 5) and p.G515S (n = 3). All 11 individuals had developmental and epileptic encephalopathy, with 8 having normal development prior to seizure onset at <2 years of age. All patients developed multiple seizure types, 9 of 11 patients had seizures triggered by fever and 9 of 11 patients had drug-resistant seizures. In vitro analysis of HEK293T cells transfected with MAST3 cDNA carrying a subset of these patient-specific missense variants demonstrated variable but generally lower expression, with concomitant increased phosphorylation of the MAST3 target, ARPP-16, compared to wild-type. These findings suggest the patient-specific variants may confer MAST3 gain-of-function. Moreover, single-nuclei RNA sequencing and immunohistochemistry shows that MAST3 expression is restricted to excitatory neurons in the cortex late in prenatal development and postnatally. INTERPRETATION: In summary, we describe MAST3 as a novel epilepsy-associated gene with a potential gain-of-function pathogenic mechanism that may be primarily restricted to excitatory neurons in the cortex. ANN NEUROL 2021;90:274-284.

Homozygous Mutations in TBC1D23 Lead to a Non-degenerative Form of Pontocerebellar Hypoplasia.

Pontocerebellar hypoplasia (PCH) represents a group of recessive developmental disorders characterized by impaired growth of the pons and cerebellum, which frequently follows a degenerative course. Currently, there are 10 partially overlapping clinical subtypes and 13 genes known mutated in PCH. Here, we report biallelic TBC1D23 mutations in six individuals from four unrelated families manifesting a non-degenerative form of PCH. In addition to reduced volume of pons and cerebellum, affected individuals had microcephaly, psychomotor delay, and ataxia. In zebrafish, tbc1d23 morphants replicated the human phenotype showing hindbrain volume loss. TBC1D23 localized at the trans-Golgi and was regulated by the small GTPases Arl1 and Arl8, suggesting a role in trans-Golgi membrane trafficking. Altogether, this study provides a causative link between TBC1D23 mutations and PCH and suggests a less severe clinical course than other PCH subtypes.

Characterization of seizure susceptibility in Pcdh19 mice.

OBJECTIVE: PCDH19-related epilepsy is characterized by a distinctive pattern of X-linked inheritance, where heterozygous females exhibit seizures and hemizygous males are asymptomatic. A cellular interference mechanism resulting from the presence of both wild-type and mutant PCDH19 neurons in heterozygous patients or mosaic carriers of PCDH19 variants has been hypothesized. We aim to investigate seizure susceptibility and progression in the Pchd19 mouse model. METHODS: We assessed seizure susceptibility and progression in the Pcdh19 mouse model using three acute seizure induction paradigms. We first induced focal, clonic seizures using the 6-Hz psychomotor test. Mice were stimulated with increasing current intensities and graded according to a modified Racine scale. We next induced generalized seizures using flurothyl or pentylenetetrazol (PTZ), both γ-aminobutyric acid type A receptor function inhibitors, and recorded latencies to myoclonic and generalized tonic-clonic seizures. RESULTS: Pcdh19 knockout and heterozygous females displayed increased seizure susceptibility across all current intensities in the 6-Hz psychomotor test, and increased severity overall. They also exhibited shorter latencies to generalized seizures following flurothyl, but not PTZ, seizure induction. Hemizygous males showed comparable seizure incidence and severity to their wild-type male littermates across all paradigms tested. SIGNIFICANCE: The heightened susceptibility observed in Pcdh19 knockout females suggests additional mechanisms other than cellular interference are at play in PCDH19-related epilepsy. Further experiments are needed to understand the variability in seizure susceptibility so that this model can be best utilized toward development of future therapeutic strategies for PCDH19-related epilepsy.

Loss of Protocadherin-12 Leads to Diencephalic-Mesencephalic Junction Dysplasia Syndrome.

OBJECTIVE: To identify causes of the autosomal-recessive malformation, diencephalic-mesencephalic junction dysplasia (DMJD) syndrome. METHODS: Eight families with DMJD were studied by whole-exome or targeted sequencing, with detailed clinical and radiological characterization. Patient-derived induced pluripotent stem cells were derived into neural precursor and endothelial cells to study gene expression. RESULTS: All patients showed biallelic mutations in the nonclustered protocadherin-12 (PCDH12) gene. The characteristic clinical presentation included progressive microcephaly, craniofacial dysmorphism, psychomotor disability, epilepsy, and axial hypotonia with variable appendicular spasticity. Brain imaging showed brainstem malformations and with frequent thinned corpus callosum with punctate brain calcifications, reflecting expression of PCDH12 in neural and endothelial cells. These cells showed lack of PCDH12 expression and impaired neurite outgrowth. INTERPRETATION: DMJD patients have biallelic mutations in PCDH12 and lack of protein expression. These patients present with characteristic microcephaly and abnormalities of white matter tracts. Such pathogenic variants predict a poor outcome as a result of brainstem malformation and evidence of white matter tract defects, and should be added to the phenotypic spectrum associated with PCDH12-related conditions. Ann Neurol 2018;84:646-655.

Mutations in CSPP1 lead to classical Joubert syndrome.

Joubert syndrome and related disorders (JSRDs) are genetically heterogeneous and characterized by a distinctive mid-hindbrain malformation. Causative mutations lead to primary cilia dysfunction, which often results in variable involvement of other organs such as the liver, retina, and kidney. We identified predicted null mutations in CSPP1 in six individuals affected by classical JSRDs. CSPP1 encodes a protein localized to centrosomes and spindle poles, as well as to the primary cilium. Despite the known interaction between CSPP1 and nephronophthisis-associated proteins, none of the affected individuals in our cohort presented with kidney disease, and further, screening of a large cohort of individuals with nephronophthisis demonstrated no mutations. CSPP1 is broadly expressed in neural tissue, and its encoded protein localizes to the primary cilium in an in vitro model of human neurogenesis. Here, we show abrogated protein levels and ciliogenesis in affected fibroblasts. Our data thus suggest that CSPP1 is involved in neural-specific functions of primary cilia.

iPSC-derived models of PACS1 syndrome reveal transcriptional and functional deficits in neuron activity.

PACS1 syndrome is a neurodevelopmental disorder characterized by intellectual disability and distinct craniofacial abnormalities resulting from a de novo p.R203W variant in phosphofurin acidic cluster sorting protein 1 (PACS1). PACS1 is known to have functions in the endosomal pathway and nucleus, but how the p.R203W variant affects developing neurons is not fully understood. Here we differentiated stem cells towards neuronal models including cortical organoids to investigate the impact of the PACS1 syndrome-causing variant on neurodevelopment. While few deleterious effects were detected in PACS1(+/R203W) neural precursors, mature PACS1(+/R203W) glutamatergic neurons exhibited impaired expression of genes involved in synaptic signaling processes. Subsequent characterization of neural activity using calcium imaging and multielectrode arrays revealed the p.R203W PACS1 variant leads to a prolonged neuronal network burst duration mediated by an increased interspike interval. These findings demonstrate the impact of the PACS1 p.R203W variant on developing human neural tissue and uncover putative electrophysiological underpinnings of disease.

Quantifying differentiation of progenitor populations using cerebral organoid models for neurodevelopmental disorders.

Neurodevelopmental disorders are characterized by complex phenotypes that often result from concomitant dysregulation of cell proliferation, differentiation, or other crucial developmental processes. Here, we present a protocol to quantify differentiation of progenitor populations during early stages of neurogenesis in induced pluripotent stem cell (iPSC)-derived cerebral organoids. We describe steps for organoid differentiation and maturation, sample preparation, immunofluorescence, and imaging and analysis using epifluorescence microscopy. This protocol can be used to compare cerebral organoids from control and patient-derived iPSCs. For complete details on the use and execution of this protocol, please refer to Rakotomamonjy et al. (2023).1.

Impaired migration and premature differentiation underlie the neurological phenotype associated with PCDH12 loss of function.

Protocadherins (PCDHs) are cell adhesion molecules that regulate many essential neurodevelopmental processes related to neuronal maturation, dendritic arbor formation, axon pathfinding, and synaptic plasticity. Bi-allelic loss-of-function variants in PCDH12 are associated with several neurodevelopmental disorders (NDDs) such as diencephalic-mesencephalic dysplasia syndrome, cerebral palsy, cerebellar ataxia, and microcephaly. Despite the highly deleterious outcome resulting from loss of PCDH12, little is known about its role during brain development and disease. Here, we show that PCDH12 loss severely impairs cerebral organoid development with reduced proliferative areas and disrupted laminar organization. 2D models further show that neural progenitor cells lacking PCDH12 prematurely exit cell cycle and differentiate earlier when compared to wildtype. Furthermore, we show that PCDH12 regulates neuronal migration through a mechanism requiring ADAM10-mediated ectodomain shedding and membrane recruitment of cytoskeleton regulators. Our data demonstrate a critical and broad involvement of PCDH12 in cortical development, revealing the pathogenic mechanisms underlying PCDH12-related NDDs.

PCDH12 loss results in premature neuronal differentiation and impeded migration in a cortical organoid model.

Protocadherins (PCDHs) are cell adhesion molecules that regulate many essential neurodevelopmental processes related to neuronal maturation, dendritic arbor formation, axon pathfinding, and synaptic plasticity. Biallelic loss-of-function variants in PCDH12 are associated with several neurodevelopmental disorders (NDDs). Despite the highly deleterious outcome resulting from loss of PCDH12, little is known about its role during brain development and disease. Here, we show that PCDH12 loss severely impairs cerebral organoid development, with reduced proliferative areas and disrupted laminar organization. 2D models further show that neural progenitor cells lacking PCDH12 prematurely exit the cell cycle and differentiate earlier when compared with wild type. Furthermore, we show that PCDH12 regulates neuronal migration and suggest that this could be through a mechanism requiring ADAM10-mediated ectodomain shedding and/or membrane recruitment of cytoskeleton regulators. Our results demonstrate a critical involvement of PCDH12 in cortical organoid development, suggesting a potential cause for the pathogenic mechanisms underlying PCDH12-related NDDs.

Reactive oxygen species are related to ionic fluxes and volume decrease in apoptotic cerebellar granule neurons: role of NOX enzymes.

Reactive oxygen species (ROS) are produced early during apoptosis of cerebellar granule neurons induced by low potassium (K5) and staurosporine (Sts). In addition, K5 and Sts activate NADPH oxidases (NOX). Recently, we described that K5 and Sts induce apoptotic volume decrease (AVD) at a time when ROS generation and NOX activity occur. In the present study, we evaluated the relationship between ROS generation and ionic fluxes during AVD. Here, we showed that K5- and Sts-induced AVD was inhibited by antioxidants and that direct ROS production induced AVD. Moreover, NOX inhibitors eliminated AVD induced by both K5 and Sts. Sts, but not K5, failed to induce AVD in cerebellar granule neurons from NOX2 knockout mice. These findings suggest that K5- and Sts-induced AVD is largely mediated by ROS produced by NOX. On the other hand, we also found that the blockage of ionic fluxes involved in AVD inhibited both ROS generation and NOX activity. These findings suggest that ROS generation and NOX activity are involved in ionic fluxes activation, which in turn could maintain ROS generation by activating NOX, leading to a self-amplifying cycle.

Apoptosis and autophagy in rat cerebellar granule neuron death: Role of reactive oxygen species.

Programmed cell death (PCD) has been defined as an active, controlled process in which cells participate in their own demise. Apoptosis, or type I PCD, has been widely characterized, both morphologically and biochemically. More recently, autophagy, the self-digesting mechanism involved in the removal of cytoplasmic long-lived proteins, has been involved in cell death, and type II PCD is defined as cell death occurring with autophagic features. Neurons can undergo more than one type of PCD as a backup mechanism when the traditional death pathway is inhibited or in response to a particular death-inducing stimulus. Reactive oxygen species (ROS) have been shown to be important signaling molecules in the execution of apoptosis and, more recently, in the autophagic pathway. In this work, we characterize apoptotic and autophagic cell death in rat cerebellar granule neuron (CGN) culture, a widespread model for the study of neuronal death. Potassium deprivation (K5) and staurosporine (STS) were used for death induction. We found apoptotic and autophagic features under both conditions. Caspase inhibition as well as autophagy inhibition by 3-methyl adenine decreased cell death. Moreover, CGN can undergo the alternative type of cell death when the other one is inhibited. An antioxidant or NADPH oxidase inhibitors delayed apoptosis and had no effect in autophagic features. Thus, we found that autophagy plays a role in cell death of CGN and that, when cells are treated with K5 or STS, both autophagy and ROS seem to promote apoptosis by independent mechanisms.

PYRC2-Related Hypomyelinating Leukodystrophy: More to This Than Meets the Eye.

Loss-of-function variants in the PYRC2 gene cause hypomyelinating leukodystrophy 10 (HLD10), but the associated pathogenic mechanisms are unknown. In this issue of Neuron, Escande-Beillard et al. (2020) reveal that PYRC2 is a key enzyme for proper brain development and a regulator of glycine homeostasis, uncovering hyperglycinemia as a driver of HLD10 pathogenesis.

NOX2 mediates apoptotic death induced by staurosporine but not by potassium deprivation in cerebellar granule neurons.

Neuronal apoptotic death involves the participation of reactive oxygen species (ROS), but their sources have not been completely elucidated. Previous studies have demonstrated that the ROS-producing enzyme NADPH oxidase is present in neuronal cells and that this enzyme could participate in the apoptotic neuronal death. Cerebellar granule neurons (CGN) undergo apoptosis when cells are transferred from a medium with 25 mM KCl (K25) to a 5 mM KCl (K5) medium or when they are treated with staurosporine (ST). Under these conditions, apoptotic death of CGN is dependent on ROS production. In this study, we evaluated the role of NOX2, an NADPH oxidase homolog, in the apoptotic death of CGN induced by two different conditions. In CGN from NOX2-deficient (ko) mice, a significantly lower rate of apoptotic death occurs compared with wild-type (wt) CGN. Also, caspase-3 activation, NADPH oxidase activity, and superoxide anion production induced by ST were markedly lower in ko neurons than in wt CGN. In contrast to the case with ST, when CGN were treated with K5, no differences were observed between ko and wt cells in any of the parameters measured. However, all NADPH oxidase inhibitors tested noticeably reduced cell death and apoptotic parameters induced by K5 in both wt and ko CGN. These results suggest that NOX2 could be necessary for apoptotic death induced by ST, but not by K5, which could require other member of the NOX family in the apoptotic process.

Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures.

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early postnatal development, cerebellar Purkinje cells are known to go through a vulnerable period, during which they undergo programmed cell death. Here, we provide a detailed protocol to perform mouse organotypic cerebellar slice culture during this critical time. The slices are further labeled to assess Purkinje cell survival and the efficacy of neuroprotective treatments. This method can be extremely valuable to screen for new neuroactive molecules.

Genetic Causes and Modifiers of Autism Spectrum Disorder.

Autism Spectrum Disorder (ASD) is one of the most prevalent neurodevelopmental disorders, affecting an estimated 1 in 59 children. ASD is highly genetically heterogeneous and may be caused by both inheritable and de novo gene variations. In the past decade, hundreds of genes have been identified that contribute to the serious deficits in communication, social cognition, and behavior that patients often experience. However, these only account for 10-20% of ASD cases, and patients with similar pathogenic variants may be diagnosed on very different levels of the spectrum. In this review, we will describe the genetic landscape of ASD and discuss how genetic modifiers such as copy number variation, single nucleotide polymorphisms, and epigenetic alterations likely play a key role in modulating the phenotypic spectrum of ASD patients. We also consider how genetic modifiers can alter convergent signaling pathways and lead to impaired neural circuitry formation. Lastly, we review sex-linked modifiers and clinical implications. Further understanding of these mechanisms is crucial for both comprehending ASD and for developing novel therapies.

Zika Virus Protease Cleavage of Host Protein Septin-2 Mediates Mitotic Defects in Neural Progenitors.

Zika virus (ZIKV) targets neural progenitor cells in the brain, attenuates cell proliferation, and leads to cell death. Here, we describe a role for the ZIKV protease NS2B-NS3 heterodimer in mediating neurotoxicity through cleavage of a host protein required for neurogenesis. Similar to ZIKV infection, NS2B-NS3 expression led to cytokinesis defects and cell death in a protease activity-dependent fashion. Among binding partners, NS2B-NS3 cleaved Septin-2, a cytoskeletal factor involved in cytokinesis. Cleavage of Septin-2 occurred at residue 306 and forced expression of a non-cleavable Septin-2 restored cytokinesis, suggesting a direct mechanism of ZIKV-induced neural toxicity. VIDEO ABSTRACT.

Role of NADPH oxidase in the apoptotic death of cultured cerebellar granule neurons.

Cerebellar granule neurons (CGN) cultured in a medium containing 25 mM KCl and treated with staurosporine (ST) or transferred to a medium with 5 mM KCl (K5) die apoptotically. CGN death is mediated by an increase in reactive oxygen species (ROS) production. When CGN are treated with antioxidants all apoptotic parameters and cell death are markedly diminished, showing a central role for ROS in this process. Recently, it has been suggested that a possible ROS source involved in cell death is a NADPH oxidase. In that regard, we found expression in CGN of the components of NADPH proteins, p40phox, p47phox and p67phox, and p22phox, as well as three homologues of the catalytic subunit of this complex, NOX1, 2, and 4. The inhibition of NADPH oxidase with diphenylene iodonium or 4-(2-aminoethyl)benzenesulfonyl fluoride significantly reduced ROS production, NADPH oxidase activity, all the apoptotic events, and cell death induced by both K5 and ST. We conclude that ROS could be an early signal of apoptotic neuronal death and that NADPH oxidase, including NOX1, 2, and/or 4, could have a central role in apoptotic death induced by different conditions in these neurons.

Biallelic mutations in the 3' exonuclease TOE1 cause pontocerebellar hypoplasia and uncover a role in snRNA processing.

Deadenylases are best known for degrading the poly(A) tail during mRNA decay. The deadenylase family has expanded throughout evolution and, in mammals, consists of 12 Mg2+-dependent 3'-end RNases with substrate specificity that is mostly unknown. Pontocerebellar hypoplasia type 7 (PCH7) is a unique recessive syndrome characterized by neurodegeneration and ambiguous genitalia. We studied 12 human families with PCH7, uncovering biallelic, loss-of-function mutations in TOE1, which encodes an unconventional deadenylase. toe1-morphant zebrafish displayed midbrain and hindbrain degeneration, modeling PCH-like structural defects in vivo. Surprisingly, we found that TOE1 associated with small nuclear RNAs (snRNAs) incompletely processed spliceosomal. These pre-snRNAs contained 3' genome-encoded tails often followed by post-transcriptionally added adenosines. Human cells with reduced levels of TOE1 accumulated 3'-end-extended pre-snRNAs, and the immunoisolated TOE1 complex was sufficient for 3'-end maturation of snRNAs. Our findings identify the cause of a neurodegenerative syndrome linked to snRNA maturation and uncover a key factor involved in the processing of snRNA 3' ends.