Antonio Falace

SLC35A2 encodes the Golgi uridine diphosphate galactose transporter, which is essential for glycosylation of glycoproteins and glycolipids. Variants in this gene, either germline or somatic, have emerged as causes of diverse neurological disorders ranging from congenital disorders of glycosylation (SLC35A2-CDG) to focal cortical malformations such as mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy (MOGHE). This review summarizes the molecular function of SLC35A2, clinical phenotypes of congenital and somatic variants, insights from functional assays and animal models, and therapeutic perspectives including galactose supplementation and precision medicine. We aim to provide an integrative synthesis of human genetics, neuropathology, glycomics, and translational approaches.

Brain somatic variants in the SLC35A2 gene, encoding for a Golgi galactose transporter, represent the major cause of mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy (MOGHE). Clinical features associated with MOGHE include early-onset epileptic encephalopathy, drug-resistant focal epilepsy with developmental delay, and intellectual disability. Half of somatic SLC35A2 variants identified in MOGHE patients are predicted to encode full-length SLC35A2 protein or stable protein products. We investigated the pathophysiological basis of MOGHE by analyzing the functional consequences of SLC35A2 pathogenetic variants in vitro and in vivo models. We assessed how different SLC35A2 variants impact protein stability and expression in transfected cellular models. We used in utero electroporation in the rat brain to model mosaic expression of SLC35A2 pathogenetic variants in the cerebral cortex and assessed their effect on neurons migration and morphology. We found that SLC35A2 variants identified in MOGHE patients variably impact on SLC35A2 protein expression. In utero expression of a SLC35A2 missense (p.G282A) or frameshift (p.F280Tfs*10) variants resulted in neuronal heterotopia in the white matter and impaired dendritogenesis at postnatal stages, suggesting a cell autonomous role for SLC35A2 in neuronal development. These phenotypes were recapitulated by in utero silencing of rat Slc35a2 gene. We successfully developed an in vivo mosaic model for the characterization of SLC35A2 variants identified in MOGHE patients and demonstrated that the expression of single SLC35A2 variants triggers the pathophysiological cascade associated with SLC35A2 dysfunction in neurons.

Purpose of the Review Preclinical and clinical evidence support the notion that in Developmental and Epileptic Encephalopathies (DEEs) both neurodevelopmental and progressive alterations coexist. Several studies have contributed to the identification of pathophysiological mechanisms underlying DEEs, allowing novel interpretative meanings regarding the mechanisms involved in the progression of DEE symptomatology. The purpose of this review is to critically discuss emerging evidence linking DEEs, genetics, inflammation, and neurodegeneration. Recent Findings In the last two decades, genetic findings have highlighted the role of cellular processes involved in both early neurodevelopment and neurodegeneration. In this scenario, evidence linking both autophagic and synaptic dysfunction to DEE provides the needed bridge between such pivotal processes for neuronal homeostasis and neurodegeneration. Furthermore, genetic defects affecting ion currents and cellular osmolarity, cytoskeletal structure and integrity of myelinated axons, neuronal morphology, and purine metabolism have been found to underlie DEE with progressive course. Recent evidence also demonstrates that neuroinflammation during gestation and in the immature brain can induce immune priming and increase the risk of developing DEEs. Summary The phenotypic evolution of many DEEs beyond early childhood is still poorly understood and hardly preventable. Recent studies showed that DEEs, at least some of them, may be the consequence of early inflammatory and neurodegenerative dysfunctions with a variable severity determined by specific genetic and epigenetic factors. These findings open new avenues for uncovering the underpinnings of DEEs progression and symptomatology.

Vacuolar-type ATPase (v-ATPase) is a multimeric protein complex that regulates H⁺ transport across membranes and intra-cellular organelle acidification. Catabolic processes, such as endocytic degradation and autophagy, strictly rely on v-ATPase-dependent luminal acidification in lysosomes. The v-ATPase complex is expressed at high levels in the brain and its impairment triggers neuronal dysfunction and neurodegeneration. Due to their post-mitotic nature and highly specialized function and morphology, neurons display a unique vulnerability to lysosomal dyshomeostasis. Alterations in genes encoding subunits composing v-ATPase or v-ATPase-related proteins impair brain development and synaptic function in animal models and underlie genetic diseases in humans, such as encephalopathies, epilepsy, as well as neurodevelopmental, and degenerative disorders. This review presents the genetic and functional evidence linking v-ATPase subunits and accessory proteins to various brain disorders, from early-onset developmental epileptic encephalopathy to neurodegenerative diseases. We highlight the latest emerging therapeutic strategies aimed at mitigating lysosomal defects associated with v-ATPase dysfunction.

Mutations in TBC1D24 are described in patients with a spectrum of neurological diseases, including mild and severe epilepsies and complex syndromic phenotypes such as Deafness, Onycodystrophy, Osteodystrophy, Mental Retardation and Seizure (DOORS) syndrome. The product of TBC1D24 is a multifunctional protein involved in neuronal development, regulation of synaptic vesicle trafficking, and protection from oxidative stress. Although pathogenic mutations in TBC1D24 span the entire coding sequence, no clear genotype/phenotype correlations have emerged. However most patients bearing predicted loss of function mutations exhibit a severe neurodevelopmental disorder. Aim of the study is to investigate the impact of TBC1D24 knockdown during the first stages of neuronal differentiation when axonal specification and outgrowth take place. In rat cortical primary neurons silenced for TBC1D24, we found defects in axonal specification, the maturation of axonal initial segment and action potential firing. The axonal phenotype was accompanied by an impairment of endocytosis at the growth cone and an altered activation of the TBC1D24 molecular partner ADP ribosylation factor 6. Accordingly, acute knockdown of TBC1D24 in cerebrocortical neurons in vivo analogously impairs callosal projections. The axonal defect was also investigated in human induced pluripotent stem cell-derived neurons from patients carrying TBC1D24 mutations. Reprogrammed neurons from a patient with severe developmental encephalopathy show significant axon formation defect that were absent from reprogrammed neurons of a patient with mild early onset epilepsy. Our data reveal that alterations of membrane trafficking at the growth cone induced by TBC1D24 loss of function cause axonal and excitability defects. The axonal phenotype correlates with the disease severity and highlight an important role for TBC1D24 in connectivity during brain development.

The development of the cerebral cortex requires complex sequential processes that have to be precisely orchestrated. The localization and timing of neuronal progenitor proliferation and of neuronal migration define the identity, laminar positioning, and specific connectivity of each single cortical neuron. Alterations at any step of this organized series of events-due to genetic mutations or environmental factors-lead to defined brain pathologies collectively known as malformations of cortical development (MCDs), which are now recognized as a leading cause of drug-resistant epilepsy and intellectual disability. In this heterogeneous group of disorders, macroscopic alterations of brain structure (eg, heterotopic nodules, small or absent gyri, double cortex) can be recognized and probably subtend a general reorganization of neuronal circuits. In this review, we provide an overview of the molecular mechanisms that are implicated in the generation of genetic MCDs associated with aberrations at various steps of neurogenesis and cortical development.

Birth defects that involve the cerebral cortex — also known as malformations of cortical development (MCD) — are important causes of intellectual disability and account for 20-40% of drug-resistant epilepsy in childhood. High-resolution brain imaging has facilitated in vivo identification of a large group of MCD phenotypes. Despite the advances in brain imaging, genomic analysis and generation of animal models, a straightforward workflow to systematically prioritize candidate genes and to test functional effects of putative mutations is missing. To overcome this problem, an experimental strategy enabling the identification of novel causative genes for MCD was developed and validated. This strategy is based on identifying candidate genomic regions or genes via array-CGH or whole-exome sequencing and characterizing the effects of their inactivation or of overexpression of specific mutations in developing rodent brains via in utero electroporation. This approach led to the identification of the C6orf70 gene, encoding for a putative vesicular protein, to the pathogenesis of periventricular nodular heterotopia, a MCD caused by defective neuronal migration.

Objective: To evaluate the phenotypic spectrum associated with mutations in TBC1D24. Methods: We acquired new clinical, EEG, and neuroimaging data of 11 previously unreported and 37 published patients. TBC1D24 mutations, identified through various sequencing methods, can be found online (http://lovd.nl/TBC1D24). Results: Forty-eight patients were included (28 men, 20 women, average age 21 years) from 30 independent families. Eighteen patients (38%) had myoclonic epilepsies. The other patients carried diagnoses of focal (25%), multifocal (2%), generalized (4%), and unclassified epilepsy (6%), and early-onset epileptic encephalopathy (25%). Most patients had drug-resistant epilepsy. We detail EEG, neuroimaging, developmental, and cognitive features, treatment responsiveness, and physical examination. In silico evaluation revealed 7 different highly conserved motifs, with the most common pathogenic mutation located in the first. Neuronal outgrowth assays showed that some TBC1D24 mutations, associated with the most severe TBC1D24-associated disorders, are not necessarily the most disruptive to this gene function. Conclusions: TBC1D24-related epilepsy syndromes show marked phenotypic pleiotropy, with multisystem involvement and severity spectrum ranging from isolated deafness (not studied here), benign myoclonic epilepsy restricted to childhood with complete seizure control and normal intellect, to early-onset epileptic encephalopathy with severe developmental delay and early death. There is no distinct correlation with mutation type or location yet, but patterns are emerging. Given the phenotypic breadth observed, TBC1D24 mutation screening is indicated in a wide variety of epilepsies. A TBC1D24 consortium was formed to develop further research on this gene and its associated phenotypes.

Recycling of synaptic vesicles (SVs) is a fundamental step in the process of neurotransmission. Endocytosed SV can travel directly into the recycling pool or recycle through endosomes but little is known about the molecular actors regulating the switch between these SV recycling routes. ADP ribosylation factor 6 (Arf6) is a small GTPase known to participate in constitutive trafficking between plasma membrane and early endosomes. Here, we have morphologically and functionally investigated Arf6-silenced hippocampal synapses and found an activity dependent accumulation of synaptic endosome-like organelles and increased release-competent docked SVs. These features were phenocopied by pharmacological blockage of Arf6 activation. The data reveal an unexpected role for this small GTPase in reducing the size of the readily releasable pool of SVs and in channeling retrieved SVs toward direct recycling rather than endosomal sorting. We propose that Arf6 acts at the presynapse to define the fate of an endocytosed SV.