YY1 mutations disrupt corticogenesis through a cell-type specific rewiring of cell-autonomous and non-cell-autonomous transcriptional programs.

Germline mutations of YY1 cause Gabriele-de Vries syndrome (GADEVS), a neurodevelopmental disorder featuring intellectual disability and a wide range of systemic manifestations. To dissect the cellular and molecular mechanisms underlying GADEVS, we combined large-scale imaging, single-cell multiomics and gene regulatory network reconstruction in 2D and 3D patient-derived physiopathologically relevant cell lineages. YY1 haploinsufficiency causes a pervasive alteration of cell type specific transcriptional networks, disrupting corticogenesis at the level of neural progenitors and terminally differentiated neurons, including cytoarchitectural defects reminiscent of GADEVS clinical features. Transcriptional alterations in neurons propagated to neighboring astrocytes through a major non-cell autonomous pro-inflammatory effect that grounds the rationale for modulatory interventions. Together, neurodevelopmental trajectories, synaptic formation and neuronal-astrocyte cross talk emerged as salient domains of YY1 dosage-dependent vulnerability. Mechanistically, cell-type resolved reconstruction of gene regulatory networks uncovered the regulatory interplay between YY1, NEUROG2 and ETV5 and its aberrant rewiring in GADEVS. Our findings underscore the reach of advanced in vitro models in capturing developmental antecedents of clinical features and exposing their underlying mechanisms to guide the search for targeted interventions.

Neuroinflammatory Pathways in the ALS-FTD Continuum: A Focus on Genetic Variants.

Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal dementia (FDT) are progressive neurodegenerative disorders that, in several cases, overlap in clinical presentation, and genetic and pathological disease mechanisms. About 10-15% of ALS cases and up to 40% of FTD are familial, usually with dominant traits. ALS and FTD, in several cases, share common gene mutations, such as in C9ORF72, TARDBP, SQSTM-1, FUS, VCP, CHCHD10, and TBK-1. Also, several mechanisms are involved in ALS and FTD pathogenesis, such as protein misfolding, oxidative stress, and impaired axonal transport. In addition, neuroinflammation and neuroinflammatory cells, such as astrocytes, oligodendrocytes, microglia, and lymphocytes and, overall, the cellular microenvironment, have been proposed as pivotal players in the pathogenesis the ALS-FTD spectrum disorders. This review overviews the current evidence regarding neuroinflammatory markers in the ALS/FTD continuum, focusing on the neuroinflammatory pathways involved in the genetic cases, moving from post-mortem reports to in vivo biofluid and neuroimaging data. We further discuss the potential link between genetic and autoimmune disorders and potential therapeutic implications.

Investigating the Prognostic Role of Peripheral Inflammatory Markers in Mild Cognitive Impairment.

Growing evidence suggests that neuroinflammation plays a critical role in the pathogenesis of neurodegenerative diseases. Peripheral markers of inflammation, including blood cell counts and their ratios, such as the neutrophil-to-lymphocyte ratio (NLR), have been reported as an easily accessible and reliable proxy of central nervous system inflammation. However, the role of peripheral inflammation in dementia and Mild Cognitive Impairment (MCI) still needs to be clarified. In the current study, we aimed to assess the prognostic role of the NLR and other peripheral markers of inflammation in a sample of 130 amnestic MCI, followed up for two to five years. The Mini-Mental state examination (MMSE) score at baseline and follow-up visits was used to assess global cognitive status at each visit and the degree of cognitive decline over time. Baseline peripheral markers of inflammation included blood cell counts and ratios, specifically the NLR, the platelet-to-lymphocyte ratio (PLR), the monocyte-to-lymphocyte ratio (MLR), and the systemic immune inflammation index (SII). After classifying subjects into CONVERTERS and non-CONVERTERS (respectively, patients converting to dementia and subjects showing stability at the last available follow-up), we compared peripheral markers of inflammation among groups ed correlated them with cognitive measures, testing the ability of significant factors to predict conversion to dementia. In our cohort, CONVERTERS showed lower baseline MMSE scores (p-value = 0.004) than non-CONVERTERS. In addition, CONVERTERS had statistically elevated NLR (p-value = 0.005), PLR (p-value = 0.002), and SII levels (p-value = 0.015), besides a lower number of lymphocytes (p-value = 0.004) compared with non-CONVERTERS. In a logistic regression analysis, baseline MMSE scores and NLR predicted conversion to dementia. Tertiles analysis showed that MCI with the highest NLR values had a higher conversion risk. Our study supports the hypothesis that a dysregulation of peripheral inflammation involving both lymphocytes and neutrophils may play a role in the pathogenesis of dementia, even at the early stages of neurodegeneration, as in the MCI condition.

Ca2+ binding to synapsin I regulates resting Ca2+ and recovery from synaptic depression in nerve terminals.

Synapsin I (SynI) is a synaptic vesicle (SV)-associated phosphoprotein that modulates neurotransmission by controlling SV trafficking. The SynI C-domain contains a highly conserved ATP binding site mediating SynI oligomerization and SV clustering and an adjacent main Ca2+ binding site, whose physiological role is unexplored. Molecular dynamics simulations revealed that the E373K point mutation irreversibly deletes Ca2+ binding to SynI, still allowing ATP binding, but inducing a destabilization of the SynI oligomerization interface. Here, we analyzed the effects of this mutation on neurotransmitter release and short-term plasticity in excitatory and inhibitory synapses from primary hippocampal neurons. Patch-clamp recordings showed an increase in the frequency of miniature excitatory postsynaptic currents (EPSCs) that was totally occluded by exogenous Ca2+ chelators and associated with a constitutive increase in resting terminal Ca2+ concentrations. Evoked EPSC amplitude was also reduced, due to a decreased readily releasable pool (RRP) size. Moreover, in both excitatory and inhibitory synapses, we observed a marked impaired recovery from synaptic depression, associated with impaired RRP refilling and depletion of the recycling pool of SVs. Our study identifies SynI as a novel Ca2+ buffer in excitatory terminals. Blocking Ca2+ binding to SynI results in higher constitutive Ca2+ levels that increase the probability of spontaneous release and disperse SVs. This causes a decreased size of the RRP and an impaired recovery from depression due to the failure of SV reclustering after sustained high-frequency stimulation. The results indicate a physiological role of Ca2+ binding to SynI in the regulation of SV clustering and trafficking in nerve terminals.

Increased Prevalence of Neuropsychiatric Disorders during COVID-19 Pandemic in People Needing a Non-Deferrable Neurological Evaluation.

BACKGROUND: The novel coronavirus disease of 2019 (COVID-19) outbreak provoked a profound healthcare system reorganization. This study aimed to compare the reasons for requesting a non-deferrable neurological evaluation before the COVID-19 pandemic and during the lockdown. METHODS: Retrospective observational study including non-deferrable neurological outpatients before the pandemic (pre-COVID-19 group, n = 223) and during the Italian second wave of the COVID-19 pandemic (LOCKDOWN group, n = 318). RESULTS: The number of patients sent for cerebrovascular disorders, headache, and vertigo significantly dropped between the pre-COVID-19 era and the lockdown period. While in the pre-COVID-19 group, the most frequent diagnosis was cerebrovascular disorder; neuropsychiatric disorders ranked first in the LOCKDOWN group. Moreover, the percentage of appropriate non-deferrable neurological evaluations significantly increased in the LOCKDOWN group compared with the pre-COVID-19 group. DISCUSSION: Our study shows a significant increase of neuropsychiatric disorders in non-deferrable neurologic evaluations during the Italian second wave of the COVID-19. Overall, cases were more severe and required a more complex management during the lockdown compared with the pre-COVID era. These findings confirm that a careful approach to prevent the psychological consequences of the pandemic is needed, and long-term rearrangements of the healthcare system are desirable to guarantee appropriate management.

An interaction between PRRT2 and Na+/K+ ATPase contributes to the control of neuronal excitability.

Mutations in PRoline Rich Transmembrane protein 2 (PRRT2) cause pleiotropic syndromes including benign infantile epilepsy, paroxysmal kinesigenic dyskinesia, episodic ataxia, that share the paroxysmal character of the clinical manifestations. PRRT2 is a neuronal protein that plays multiple roles in the regulation of neuronal development, excitability, and neurotransmitter release. To better understand the physiopathology of these clinical phenotypes, we investigated PRRT2 interactome in mouse brain by a pulldown-based proteomic approach and identified α1 and α3 Na+/K+ ATPase (NKA) pumps as major PRRT2-binding proteins. We confirmed PRRT2 and NKA interaction by biochemical approaches and showed their colocalization at neuronal plasma membrane. The acute or constitutive inactivation of PRRT2 had a functional impact on NKA. While PRRT2-deficiency did not modify NKA expression and surface exposure, it caused an increased clustering of α3-NKA on the plasma membrane. Electrophysiological recordings showed that PRRT2-deficiency in primary neurons impaired NKA function during neuronal stimulation without affecting pump activity under resting conditions. Both phenotypes were fully normalized by re-expression of PRRT2 in PRRT2-deficient neurons. In addition, the NKA-dependent afterhyperpolarization that follows high-frequency firing was also reduced in PRRT2-silenced neurons. Taken together, these results demonstrate that PRRT2 is a physiological modulator of NKA function and suggest that an impaired NKA activity contributes to the hyperexcitability phenotype caused by PRRT2 deficiency.

Emerging Role of the Autophagy/Lysosomal Degradative Pathway in Neurodevelopmental Disorders With Epilepsy.

Autophagy is a highly conserved degradative process that conveys dysfunctional proteins, lipids, and organelles to lysosomes for degradation. The post-mitotic nature, complex and highly polarized morphology, and high degree of specialization of neurons make an efficient autophagy essential for their homeostasis and survival. Dysfunctional autophagy occurs in aging and neurodegenerative diseases, and autophagy at synaptic sites seems to play a crucial role in neurodegeneration. Moreover, a role of autophagy is emerging for neural development, synaptogenesis, and the establishment of a correct connectivity. Thus, it is not surprising that defective autophagy has been demonstrated in a spectrum of neurodevelopmental disorders, often associated with early-onset epilepsy. Here, we discuss the multiple roles of autophagy in neurons and the recent experimental evidence linking neurodevelopmental disorders with epilepsy to genes coding for autophagic/lysosomal system-related proteins and envisage possible pathophysiological mechanisms ranging from synaptic dysfunction to neuronal death.

Biallelic DMXL2 mutations impair autophagy and cause Ohtahara syndrome with progressive course.

Ohtahara syndrome, early infantile epileptic encephalopathy with a suppression burst EEG pattern, is an aetiologically heterogeneous condition starting in the first weeks or months of life with intractable seizures and profound developmental disability. Using whole exome sequencing, we identified biallelic DMXL2 mutations in three sibling pairs with Ohtahara syndrome, belonging to three unrelated families. Siblings in Family 1 were compound heterozygous for the c.5135C>T (p.Ala1712Val) missense substitution and the c.4478C>G (p.Ser1493*) nonsense substitution; in Family 2 were homozygous for the c.4478C>A (p.Ser1493*) nonsense substitution and in Family 3 were homozygous for the c.7518-1G>A (p.Trp2507Argfs*4) substitution. The severe developmental and epileptic encephalopathy manifested from the first day of life and was associated with deafness, mild peripheral polyneuropathy and dysmorphic features. Early brain MRI investigations in the first months of life revealed thin corpus callosum with brain hypomyelination in all. Follow-up MRI scans in three patients revealed progressive moderate brain shrinkage with leukoencephalopathy. Five patients died within the first 9 years of life and none achieved developmental, communicative or motor skills following birth. These clinical findings are consistent with a developmental brain disorder that begins in the prenatal brain, prevents neural connections from reaching the expected stages at birth, and follows a progressive course. DMXL2 is highly expressed in the brain and at synaptic terminals, regulates v-ATPase assembly and activity and participates in intracellular signalling pathways; however, its functional role is far from complete elucidation. Expression analysis in patient-derived skin fibroblasts demonstrated absence of the DMXL2 protein, revealing a loss of function phenotype. Patients' fibroblasts also exhibited an increased LysoTracker® signal associated with decreased endolysosomal markers and degradative processes. Defective endolysosomal homeostasis was accompanied by impaired autophagy, revealed by lower LC3II signal, accumulation of polyubiquitinated proteins, and autophagy receptor p62, with morphological alterations of the autolysosomal structures on electron microscopy. Altered lysosomal homeostasis and defective autophagy were recapitulated in Dmxl2-silenced mouse hippocampal neurons, which exhibited impaired neurite elongation and synaptic loss. Impaired lysosomal function and autophagy caused by biallelic DMXL2 mutations affect neuronal development and synapse formation and result in Ohtahara syndrome with profound developmental impairment and reduced life expectancy.

TBC1D24 regulates axonal outgrowth and membrane trafficking at the growth cone in rodent and human neurons.

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 epilepsy-associated protein TBC1D24 is required for normal development, survival and vesicle trafficking in mammalian neurons.

Mutations in the Tre2/Bub2/Cdc16 (TBC)1 domain family member 24 (TBC1D24) gene are associated with a range of inherited neurological disorders, from drug-refractory lethal epileptic encephalopathy and DOORS syndrome (deafness, onychodystrophy, osteodystrophy, mental retardation, seizures) to non-syndromic hearing loss. TBC1D24 has been implicated in neuronal transmission and maturation, although the molecular function of the gene and the cause of the apparently complex disease spectrum remain unclear. Importantly, heterozygous TBC1D24 mutation carriers have also been reported with seizures, suggesting that haploinsufficiency for TBC1D24 is significant clinically. Here we have systematically investigated an allelic series of disease-associated mutations in neurons alongside a new mouse model to investigate the consequences of TBC1D24 haploinsufficiency to mammalian neurodevelopment and synaptic physiology. The cellular studies reveal that disease-causing mutations that disrupt either of the conserved protein domains in TBC1D24 are implicated in neuronal development and survival and are likely acting as loss-of-function alleles. We then further investigated TBC1D24 haploinsufficiency in vivo and demonstrate that TBC1D24 is also crucial for normal presynaptic function: genetic disruption of Tbc1d24 expression in the mouse leads to an impairment of endocytosis and an enlarged endosomal compartment in neurons with a decrease in spontaneous neurotransmission. These data reveal the essential role for TBC1D24 at the mammalian synapse and help to define common synaptic mechanisms that could underlie the varied effects of TBC1D24 mutations in neurological disease.

APache Is an AP2-Interacting Protein Involved in Synaptic Vesicle Trafficking and Neuronal Development.

Synaptic transmission is critically dependent on synaptic vesicle (SV) recycling. Although the precise mechanisms of SV retrieval are still debated, it is widely accepted that a fundamental role is played by clathrin-mediated endocytosis, a form of endocytosis that capitalizes on the clathrin/adaptor protein complex 2 (AP2) coat and several accessory factors. Here, we show that the previously uncharacterized protein KIAA1107, predicted by bioinformatics analysis to be involved in the SV cycle, is an AP2-interacting clathrin-endocytosis protein (APache). We found that APache is highly enriched in the CNS and is associated with clathrin-coated vesicles via interaction with AP2. APache-silenced neurons exhibit a severe impairment of maturation at early developmental stages, reduced SV density, enlarged endosome-like structures, and defects in synaptic transmission, consistent with an impaired clathrin/AP2-mediated SV recycling. Our data implicate APache as an actor in the complex regulation of SV trafficking, neuronal development, and synaptic plasticity.

Clinical intrafamilial variability in lethal familial neonatal seizure disorder caused by TBC1D24 mutations.

TBC1D24-related disorders include a wide phenotypic ranging from mild to lethal seizure disorders, non-syndromic deafness, and composite syndromes such as DOORS (deafness, onychodystrophy, osteodystrophy, mental retardation, and seizures). The TBC1D24 gene has a role in cerebral cortex development and in presynaptic neurotransmission. Here, we present a familial case of a lethal early-onset epileptic encephalopathy, associated with two novel compound heterozygous missense variants on the TBC1D24 gene, which were detected by exome sequencing. The detailed clinical data of the three siblings is summarized in order to support the variability of the phenotype, severity, and progression of this disorder among these family members. Functional studies demonstrated that the identified novel missense mutations result in a loss of expression of the protein, suggesting a correlation between residual expression, and the disease severity. This indicates that protein expression analysis is important for interpreting genetic results when novel variants are found, as well as for complementing clinical assessment by predicting the functional impact. Further analysis is necessary to delineate the clinical presentation of individuals with TBC1D24 pathogenic variants, as well as to develop markers for diagnosis, prognosis, and potential targeted treatments. © 2016 Wiley Periodicals, Inc.

Establishment of a radial glia-like mouse fetal hypothalamic neural stem cell line (AC1) able to differentiate into neuroendocrine cells.

The present study describes the generation and the characterization of a stable cell line of neural stem cells derived from embryonic mouse hypothalamus. These cells (AC1) grow as an adherent culture in defined serum-free medium and express typical markers of neurogenic radial glia and of hypothalamic precursors. After prolonged expansion, AC1 cells may be efficiently induced to differentiate into neurons and astroglial cells in vitro and start to express some hormonal neuropeptides, like TRH, CRH, and POMC. Based on the capabilities of AC1 cells to be stably expanded and to develop neuroendocrine lineages in vitro, these cells might represent a novel tool to elucidate the mechanisms involved in the development of the hypothalamus and in the specific differentiation of neuroendocrine neurons.