Certain heterozygous variants in the kinase domain of the serine/threonine kinase NEK8 can cause an autosomal dominant form of polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) resulting from pathogenic variants in PKD1 and PKD2 is the most common form of PKD, but other genetic causes tied to primary cilia function have been identified. Biallelic pathogenic variants in the serine/threonine kinase NEK8 cause a syndromic ciliopathy with extra-kidney manifestations. Here we identify NEK8 as a disease gene for ADPKD in 12 families. Clinical evaluation was combined with functional studies using fibroblasts and tubuloids from affected individuals. Nek8 knockout mouse kidney epithelial (IMCD3) cells transfected with wild type or variant NEK8 were further used to study ciliogenesis, ciliary trafficking, kinase function, and DNA damage responses. Twenty-one affected monoallelic individuals uniformly exhibited cystic kidney disease (mostly neonatal) without consistent extra-kidney manifestations. Recurrent de novo mutations of the NEK8 missense variant p.Arg45Trp, including mosaicism, were seen in ten families. Missense variants elsewhere within the kinase domain (p.Ile150Met and p.Lys157Gln) were also identified. Functional studies demonstrated normal localization of the NEK8 protein to the proximal cilium and no consistent cilia formation defects in patient-derived cells. NEK8-wild type protein and all variant forms of the protein expressed in Nek8 knockout IMCD3 cells were localized to cilia and supported ciliogenesis. However, Nek8 knockout IMCD3 cells expressing NEK8-p.Arg45Trp and NEK8-p.Lys157Gln showed significantly decreased polycystin-2 but normal ANKS6 localization in cilia. Moreover, p.Arg45Trp NEK8 exhibited reduced kinase activity in vitro. In patient derived tubuloids and IMCD3 cells expressing NEK8-p.Arg45Trp, DNA damage signaling was increased compared to healthy passage-matched controls. Thus, we propose a dominant-negative effect for specific heterozygous missense variants in the NEK8 kinase domain as a new cause of PKD.

Biallelic variants in FLII cause pediatric cardiomyopathy by disrupting cardiomyocyte cell adhesion and myofibril organization.

Pediatric cardiomyopathy (CM) represents a group of rare, severe disorders that affect the myocardium. To date, the etiology and mechanisms underlying pediatric CM are incompletely understood, hampering accurate diagnosis and individualized therapy development. Here, we identified biallelic variants in the highly conserved flightless-I (FLII) gene in 3 families with idiopathic, early-onset dilated CM. We demonstrated that patient-specific FLII variants, when brought into the zebrafish genome using CRISPR/Cas9 genome editing, resulted in the manifestation of key aspects of morphological and functional abnormalities of the heart, as observed in our patients. Importantly, using these genetic animal models, complemented with in-depth loss-of-function studies, we provided insights into the function of Flii during ventricular chamber morphogenesis in vivo, including myofibril organization and cardiomyocyte cell adhesion, as well as trabeculation. In addition, we identified Flii function to be important for the regulation of Notch and Hippo signaling, crucial pathways associated with cardiac morphogenesis and function. Taken together, our data provide experimental evidence for a role for FLII in the pathogenesis of pediatric CM and report biallelic variants as a genetic cause of pediatric CM.

AMFR dysfunction causes autosomal recessive spastic paraplegia in human that is amenable to statin treatment in a preclinical model.

Hereditary spastic paraplegias (HSP) are rare, inherited neurodegenerative or neurodevelopmental disorders that mainly present with lower limb spasticity and muscle weakness due to motor neuron dysfunction. Whole genome sequencing identified bi-allelic truncating variants in AMFR, encoding a RING-H2 finger E3 ubiquitin ligase anchored at the membrane of the endoplasmic reticulum (ER), in two previously genetically unexplained HSP-affected siblings. Subsequently, international collaboration recognized additional HSP-affected individuals with similar bi-allelic truncating AMFR variants, resulting in a cohort of 20 individuals from 8 unrelated, consanguineous families. Variants segregated with a phenotype of mainly pure but also complex HSP consisting of global developmental delay, mild intellectual disability, motor dysfunction, and progressive spasticity. Patient-derived fibroblasts, neural stem cells (NSCs), and in vivo zebrafish modeling were used to investigate pathomechanisms, including initial preclinical therapy assessment. The absence of AMFR disturbs lipid homeostasis, causing lipid droplet accumulation in NSCs and patient-derived fibroblasts which is rescued upon AMFR re-expression. Electron microscopy indicates ER morphology alterations in the absence of AMFR. Similar findings are seen in amfra-/- zebrafish larvae, in addition to altered touch-evoked escape response and defects in motor neuron branching, phenocopying the HSP observed in patients. Interestingly, administration of FDA-approved statins improves touch-evoked escape response and motor neuron branching defects in amfra-/- zebrafish larvae, suggesting potential therapeutic implications. Our genetic and functional studies identify bi-allelic truncating variants in AMFR as a cause of a novel autosomal recessive HSP by altering lipid metabolism, which may potentially be therapeutically modulated using precision medicine with statins.

SMPD4 regulates mitotic nuclear envelope dynamics and its loss causes microcephaly and diabetes.

Biallelic loss-of-function variants in SMPD4 cause a rare and severe neurodevelopmental disorder with progressive congenital microcephaly and early death. SMPD4 encodes a sphingomyelinase that hydrolyses sphingomyelin into ceramide at neutral pH and can thereby affect membrane lipid homeostasis. SMPD4 localizes to the membranes of the endoplasmic reticulum and nuclear envelope and interacts with nuclear pore complexes (NPC). We refine the clinical phenotype of loss-of-function SMPD4 variants by describing five individuals from three unrelated families with longitudinal data due to prolonged survival. All individuals surviving beyond infancy developed insulin-dependent diabetes, besides presenting with a severe neurodevelopmental disorder and microcephaly, making diabetes one of the most frequent age-dependent non-cerebral abnormalities. We studied the function of SMPD4 at the cellular and organ levels. Knock-down of SMPD4 in human neural stem cells causes reduced proliferation rates and prolonged mitosis. Moreover, SMPD4 depletion results in abnormal nuclear envelope breakdown and reassembly during mitosis and decreased post-mitotic NPC insertion. Fibroblasts from affected individuals show deficient SMPD4-specific neutral sphingomyelinase activity, without changing (sub)cellular lipidome fractions, which suggests a local function of SMPD4 on the nuclear envelope. In embryonic mouse brain, knockdown of Smpd4 impairs cortical progenitor proliferation and induces premature differentiation by altering the balance between neurogenic and proliferative progenitor cell divisions. We hypothesize that, in individuals with SMPD4-related disease, nuclear envelope bending, which is needed to insert NPCs in the nuclear envelope, is impaired in the absence of SMPD4 and interferes with cerebral corticogenesis and survival of pancreatic beta cells.

Myelination generates aberrant ultrastructure that is resolved by microglia.

To enable rapid propagation of action potentials, axons are ensheathed by myelin, a multilayered insulating membrane formed by oligodendrocytes. Most of the myelin is generated early in development, resulting in the generation of long-lasting stable membrane structures. Here, we explored structural and dynamic changes in central nervous system myelin during development. To achieve this, we performed an ultrastructural analysis of mouse optic nerves by serial block face scanning electron microscopy (SBF-SEM) and confocal time-lapse imaging in the zebrafish spinal cord. We found that myelin undergoes extensive ultrastructural changes during early postnatal development. Myelin degeneration profiles were engulfed and phagocytosed by microglia using exposed phosphatidylserine as one "eat me" signal. In contrast, retractions of entire myelin sheaths occurred independently of microglia and involved uptake of myelin by the oligodendrocyte itself. Our findings show that the generation of myelin early in development is an inaccurate process associated with aberrant ultrastructural features that require substantial refinement.

Unexplained mismatch repair deficiency: Case closed.

To identify Lynch syndrome (LS) carriers, DNA mismatch repair (MMR) immunohistochemistry (IHC) is performed on colorectal cancers (CRCs). Upon subsequent LS diagnostics, MMR deficiency (MMRd) sometimes remains unexplained (UMMRd). Recently, the importance of complete LS diagnostics to explain UMMRd, involving MMR methylation, germline, and somatic analyses, was stressed. To explore why some MMRd CRCs remain unsolved, we performed a systematic review of the literature and mapped patients with UMMRd diagnosed in our center. A systematic literature search was performed in Ovid Medline, Embase, Web of Science, Cochrane CENTRAL, and Google Scholar for articles on UMMRd CRCs after complete LS diagnostics published until December 15, 2021. Additionally, UMMRd CRCs diagnosed in our center since 1993 were mapped. Of 754 identified articles, 17 were included, covering 74 patients with UMMRd. Five CRCs were microsatellite stable. Upon complete diagnostics, 39 patients had single somatic MMR hits, and six an MMR germline variant of unknown significance (VUS). Ten had somatic pathogenic variants (PVs) in POLD1, MLH3, MSH3, and APC. The remaining 14 patients were the only identifiable cases in the literature without a plausible identified cause of the UMMRd. Of those, nine were suspected to have LS. In our center, complete LS diagnostics in approximately 5,000 CRCs left seven MMRd CRCs unexplained. All had a somatic MMR hit or MMR germline VUS, indicative of a missed second MMR hit. In vitually all patients with UMMRd, complete LS diagnostics suggest MMR gene involvement. Optimizing detection of currently undetectable PVs and VUS interpretation might explain all UMMRd CRCs, considering UMMRd a case closed.

Web-accessible application for identifying pathogenic transcripts with RNA-seq: Increased sensitivity in diagnosis of neurodevelopmental disorders.

For neurodevelopmental disorders (NDDs), a molecular diagnosis is key for management, predicting outcome, and counseling. Often, routine DNA-based tests fail to establish a genetic diagnosis in NDDs. Transcriptome analysis (RNA sequencing [RNA-seq]) promises to improve the diagnostic yield but has not been applied to NDDs in routine diagnostics. Here, we explored the diagnostic potential of RNA-seq in 96 individuals including 67 undiagnosed subjects with NDDs. We performed RNA-seq on single individuals' cultured skin fibroblasts, with and without cycloheximide treatment, and used modified OUTRIDER Z scores to detect gene expression outliers and mis-splicing by exonic and intronic outliers. Analysis was performed by a user-friendly web application, and candidate pathogenic transcriptional events were confirmed by secondary assays. We identified intragenic deletions, monoallelic expression, and pseudoexonic insertions but also synonymous and non-synonymous variants with deleterious effects on transcription, increasing the diagnostic yield for NDDs by 13%. We found that cycloheximide treatment and exonic/intronic Z score analysis increased detection and resolution of aberrant splicing. Importantly, in one individual mis-splicing was found in a candidate gene nearly matching the individual's specific phenotype. However, pathogenic splicing occurred in another neuronal-expressed gene and provided a molecular diagnosis, stressing the need to customize RNA-seq. Lastly, our web browser application allowed custom analysis settings that facilitate diagnostic application and ranked pathogenic transcripts as top candidates. Our results demonstrate that RNA-seq is a complementary method in the genomic diagnosis of NDDs and, by providing accessible analysis with improved sensitivity, our transcriptome analysis approach facilitates wider implementation of RNA-seq in routine genome diagnostics.

CLEC16A interacts with retromer and TRIM27, and its loss impairs endosomal trafficking and neurodevelopment.

CLEC16A is a membrane-associated C-type lectin protein that functions as a E3-ubiquitin ligase. CLEC16A regulates autophagy and mitophagy, and reportedly localizes to late endosomes. GWAS studies have associated CLEC16A SNPs to various auto-immune and neurological disorders, including multiple sclerosis and Parkinson disease. Studies in mouse models imply a role for CLEC16A in neurodegeneration. We identified bi-allelic CLEC16A truncating variants in siblings from unrelated families presenting with a severe neurodevelopmental disorder including microcephaly, brain atrophy, corpus callosum dysgenesis, and growth retardation. To understand the function of CLEC16A in neurodevelopment we used in vitro models and zebrafish embryos. We observed CLEC16A localization to early endosomes in HEK293T cells. Mass spectrometry of human CLEC16A showed interaction with endosomal retromer complex subunits and the endosomal ubiquitin ligase TRIM27. Expression of the human variant leading to C-terminal truncated CLEC16A, abolishes both its endosomal localization and interaction with TRIM27, suggesting a loss-of-function effect. CLEC16A knockdown increased TRIM27 adhesion to early endosomes and abnormal accumulation of endosomal F-actin, a sign of disrupted vesicle sorting. Mutagenesis of clec16a by CRISPR-Cas9 in zebrafish embryos resulted in accumulated acidic/phagolysosome compartments, in neurons and microglia, and dysregulated mitophagy. The autophagocytic phenotype was rescued by wild-type human CLEC16A but not the C-terminal truncated CLEC16A. Our results demonstrate that CLEC16A closely interacts with retromer components and regulates endosomal fate by fine-tuning levels of TRIM27 and polymerized F-actin on the endosome surface. Dysregulation of CLEC16A-mediated endosomal sorting is associated with neurodegeneration, but it also causes accumulation of autophagosomes and unhealthy mitochondria during brain development.

High-yield identification of pathogenic NF1 variants by skin fibroblast transcriptome screening after apparently normal diagnostic DNA testing.

Neurofibromatosis type 1 (NF1) is caused by inactivating mutations in NF1. Due to the size, complexity, and high mutation rate at the NF1 locus, the identification of causative variants can be challenging. To obtain a molecular diagnosis in 15 individuals meeting diagnostic criteria for NF1, we performed transcriptome analysis (RNA-seq) on RNA obtained from cultured skin fibroblasts. In each case, routine molecular DNA diagnostics had failed to identify a disease-causing variant in NF1. A pathogenic variant or abnormal mRNA splicing was identified in 13 cases: 6 deep intronic variants and 2 transposon insertions causing noncanonical splicing, 3 postzygotic changes, 1 branch point mutation and, in 1 case, abnormal splicing for which the responsible DNA change remains to be identified. These findings helped resolve the molecular findings for an additional 17 individuals in multiple families with NF1, demonstrating the utility of skin-fibroblast-based transcriptome analysis for molecular diagnostics. RNA-seq improves mutation detection in NF1 and provides a powerful complementary approach to DNA-based methods. Importantly, our approach is applicable to other genetic disorders, particularly those caused by a wide variety of variants in a limited number of genes and specifically for individuals in whom routine molecular DNA diagnostics did not identify the causative variant.

Dominant-acting CSF1R variants cause microglial depletion and altered astrocytic phenotype in zebrafish and adult-onset leukodystrophy.

Tissue-resident macrophages of the brain, including microglia, are implicated in the pathogenesis of various CNS disorders and are possible therapeutic targets by their chemical depletion or replenishment by hematopoietic stem cell therapy. Nevertheless, a comprehensive understanding of microglial function and the consequences of microglial depletion in the human brain is lacking. In human disease, heterozygous variants in CSF1R, encoding the Colony-stimulating factor 1 receptor, can lead to adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) possibly caused by microglial depletion. Here, we investigate the effects of ALSP-causing CSF1R variants on microglia and explore the consequences of microglial depletion in the brain. In intermediate- and late-stage ALSP post-mortem brain, we establish that there is an overall loss of homeostatic microglia and that this is predominantly seen in the white matter. By introducing ALSP-causing missense variants into the zebrafish genomic csf1ra locus, we show that these variants act dominant negatively on the number of microglia in vertebrate brain development. Transcriptomics and proteomics on relatively spared ALSP brain tissue validated a downregulation of microglia-associated genes and revealed elevated astrocytic proteins, possibly suggesting involvement of astrocytes in early pathogenesis. Indeed, neuropathological analysis and in vivo imaging of csf1r zebrafish models showed an astrocytic phenotype associated with enhanced, possibly compensatory, endocytosis. Together, our findings indicate that microglial depletion in zebrafish and human disease, likely as a consequence of dominant-acting pathogenic CSF1R variants, correlates with altered astrocytes. These findings underscore the unique opportunity CSF1R variants provide to gain insight into the roles of microglia in the human brain, and the need to further investigate how microglia, astrocytes, and their interactions contribute to white matter homeostasis.

M-CSFR/CSF1R signaling regulates myeloid fates in zebrafish via distinct action of its receptors and ligands.

Macrophage colony-stimulating factor receptor (M-CSFR/CSF1R) signaling is crucial for the differentiation, proliferation, and survival of myeloid cells. The CSF1R pathway is a promising therapeutic target in many human diseases, including neurological disorders and cancer. Zebrafish are commonly used for human disease modeling and preclinical therapeutic screening. Therefore, it is necessary to understand the proper function of cytokine signaling in zebrafish to reliably model human-related diseases. Here, we investigate the roles of zebrafish Csf1rs and their ligands (Csf1a, Csf1b, and Il34) in embryonic and adult myelopoiesis. The proliferative effect of exogenous Csf1a on embryonic macrophages is connected to both receptors, Csf1ra and Csf1rb, however there is no evident effect of Csf1b in zebrafish embryonic myelopoiesis. Furthermore, we uncover an unknown role of Csf1rb in zebrafish granulopoiesis. Deregulation of Csf1rb signaling leads to failure in myeloid differentiation, resulting in neutropenia throughout the whole lifespan. Surprisingly, Il34 signaling through Csf1rb seems to be of high importance as both csf1rbΔ4bp-deficient and il34Δ5bp-deficient zebrafish larvae lack granulocytes. Our single-cell RNA sequencing analysis of adult whole kidney marrow (WKM) hematopoietic cells suggests that csf1rb is expressed mainly by blood and myeloid progenitors, and the expression of csf1ra and csf1rb is nonoverlapping. We point out differentially expressed genes important in hematopoietic cell differentiation and immune response in selected WKM populations. Our findings could improve the understanding of myeloid cell function and lead to the further study of CSF1R pathway deregulation in disease, mostly in cancerogenesis.

Transcriptome analysis reveals the contribution of oligodendrocyte and radial glia-derived cues for maintenance of microglia identity.

Microglia are increasingly being recognized as druggable targets in neurodegenerative disorders, and good in vitro models are crucial to address cell biological questions. Major challenges are to recapitulate the complex microglial morphology and their in vivo transcriptome. We have therefore exposed primary microglia from adult rhesus macaques to a variety of different culture conditions including exposure to soluble factors as M-CSF, IL-34, and TGF-ß as well as serum replacement approaches, and compared their morphologies and transcriptomes to those of mature, homeostatic in vivo microglia. This enabled us to develop a new, partially serum-free, monoculture protocol, that yields high numbers of ramified cells. We also demonstrate that exposure of adult microglia to M-CSF or IL-34 induces similar transcriptomes, and that exposure to TGF-ß has much less pronounced effects than it does on rodent microglia. However, regardless of culture conditions, the transcriptomes of in vitro and in vivo microglia remained substantially different. Analysis of differentially expressed genes inspired us to perform 3D-spherical coculture experiments of microglia with oligodendrocytes and radial glia. In such spheres, microglia signature genes were strongly induced, even in the absence of neurons and astrocytes. These data reveal a novel role for oligodendrocyte and radial glia-derived cues in the maintenance of microglial identity, providing new anchor points to study microglia in health and disease.

Comprehensive multi-omics integration identifies differentially active enhancers during human brain development with clinical relevance.

BACKGROUND: Non-coding regulatory elements (NCREs), such as enhancers, play a crucial role in gene regulation, and genetic aberrations in NCREs can lead to human disease, including brain disorders. The human brain is a complex organ that is susceptible to numerous disorders; many of these are caused by genetic changes, but a multitude remain currently unexplained. Understanding NCREs acting during brain development has the potential to shed light on previously unrecognized genetic causes of human brain disease. Despite immense community-wide efforts to understand the role of the non-coding genome and NCREs, annotating functional NCREs remains challenging. METHODS: Here we performed an integrative computational analysis of virtually all currently available epigenome data sets related to human fetal brain. RESULTS: Our in-depth analysis unravels 39,709 differentially active enhancers (DAEs) that show dynamic epigenomic rearrangement during early stages of human brain development, indicating likely biological function. Many of these DAEs are linked to clinically relevant genes, and functional validation of selected DAEs in cell models and zebrafish confirms their role in gene regulation. Compared to enhancers without dynamic epigenomic rearrangement, DAEs are subjected to higher sequence constraints in humans, have distinct sequence characteristics and are bound by a distinct transcription factor landscape. DAEs are enriched for GWAS loci for brain-related traits and for genetic variation found in individuals with neurodevelopmental disorders, including autism. CONCLUSION: This compendium of high-confidence enhancers will assist in deciphering the mechanism behind developmental genetics of human brain and will be relevant to uncover missing heritability in human genetic brain disorders.

Reduction of oxidative stress suppresses poly-GR-mediated toxicity in zebrafish embryos.

The hexanucleotide (G4C2)-repeat expansion in the C9ORF72 gene is the most common pathogenic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). This repeat expansion can be translated into dipeptide repeat proteins (DPRs), and distribution of the poly-GR DPR correlates with neurodegeneration in postmortem C9FTD/ALS brains. Here, we assessed poly-GR toxicity in zebrafish embryos, using an annexin A5-based fluorescent transgenic line (secA5) that allows for detection and quantification of apoptosis in vivo. Microinjection of RNA encoding poly-GR into fertilized oocytes evoked apoptosis in the brain and abnormal motor neuron morphology in the trunk of 1-4-days postfertilization embryos. Poly-GR can be specifically detected in protein homogenates from injected zebrafish and in the frontal cortexes of C9FTD/ALS cases. Poly-GR expression further elevated MitoSOX levels in zebrafish embryos, indicating oxidative stress. Inhibition of reactive oxygen species using Trolox showed full suppression of poly-GR toxicity. Our study indicates that poly-GR can exert its toxicity via oxidative stress. This zebrafish model can be used to find suppressors of poly-GR toxicity and identify its molecular targets underlying neurodegeneration observed in C9FTD/ALS.

Multidisciplinary interaction and MCD gene discovery. The perspective of the clinical geneticist.

The increasing pace of gene discovery in the last decade has brought a major change in the way the genetic causes of brain malformations are being diagnosed. Unbiased genomic screening has gained the first place in the diagnostic protocol of a child with congenital (brain) anomalies and the detected variants are matched with the phenotypic presentation afterwards. This process is defined as "reverse phenotyping". Screening of DNA, through copy number variant analysis of microarrays and analysis of exome data on different platforms, obtained from the index patient and both parents has become a routine approach in many centers worldwide. Clinicians are used to multidisciplinary team interaction in patient care and disease management and this explains why the majority of research that has led to the discovery of new genetic disorders nowadays proceeds from clinical observations to genomic analysis and to data exchange facilitated by open access sharing databases. However, the relevance of multidisciplinary team interaction has not been object of systematic research in the field of brain malformations. This review will illustrate some examples of how diagnostically driven questions through multidisciplinary interaction, among clinical and preclinical disciplines, can be successful in the discovery of new genes related to brain malformations. The first example illustrates the setting of interaction among neurologists, geneticists and neuro-radiologists. The second illustrates the importance of interaction among clinical dysmorphologists for pattern recognition of syndromes with multiple congenital anomalies. The third example shows how fruitful it can be to step out of the "clinical comfort zone", and interact with basic scientists in applying emerging technologies to solve the diagnostic puzzles.

The multicellular interplay of microglia in health and disease: lessons from leukodystrophy.

Microglia are highly dynamic cells crucial for developing and maintaining lifelong brain function and health through their many interactions with essentially all cellular components of the central nervous system. The frequent connection of microglia to leukodystrophies, genetic disorders of the white matter, has highlighted their involvement in the maintenance of white matter integrity. However, the mechanisms that underlie their putative roles in these processes remain largely uncharacterized. Microglia have also been gaining attention as possible therapeutic targets for many neurological conditions, increasing the demand to understand their broad spectrum of functions and the impact of their dysregulation. In this Review, we compare the pathological features of two groups of genetic leukodystrophies: those in which microglial dysfunction holds a central role, termed 'microgliopathies', and those in which lysosomal or peroxisomal defects are considered to be the primary driver. The latter are suspected to have notable microglia involvement, as some affected individuals benefit from microglia-replenishing therapy. Based on overlapping pathology, we discuss multiple ways through which aberrant microglia could lead to white matter defects and brain dysfunction. We propose that the study of leukodystrophies, and their extensively multicellular pathology, will benefit from complementing analyses of human patient material with the examination of cellular dynamics in vivo using animal models, such as zebrafish. Together, this will yield important insight into the cell biological mechanisms of microglial impact in the central nervous system, particularly in the development and maintenance of myelin, that will facilitate the development of new, and refinement of existing, therapeutic options for a range of brain diseases.

Bi-allelic VPS16 variants limit HOPS/CORVET levels and cause a mucopolysaccharidosis-like disease.

Lysosomal storage diseases, including mucopolysaccharidoses, result from genetic defects that impair lysosomal catabolism. Here, we describe two patients from two independent families presenting with progressive psychomotor regression, delayed myelination, brain atrophy, neutropenia, skeletal abnormalities, and mucopolysaccharidosis-like dysmorphic features. Both patients were homozygous for the same intronic variant in VPS16, a gene encoding a subunit of the HOPS and CORVET complexes. The variant impaired normal mRNA splicing and led to an ~85% reduction in VPS16 protein levels in patient-derived fibroblasts. Levels of other HOPS/CORVET subunits, including VPS33A, were similarly reduced, but restored upon re-expression of VPS16. Patient-derived fibroblasts showed defects in the uptake and endosomal trafficking of transferrin as well as accumulation of autophagosomes and lysosomal compartments. Re-expression of VPS16 rescued the cellular phenotypes. Zebrafish with disrupted vps16 expression showed impaired development, reduced myelination, and a similar accumulation of lysosomes and autophagosomes in the brain, particularly in glia cells. This disorder resembles previously reported patients with mutations in VPS33A, thus expanding the family of mucopolysaccharidosis-like diseases that result from mutations in HOPS/CORVET subunits.

Comparative Studies in the A30P and A53T α-Synuclein C. elegans Strains to Investigate the Molecular Origins of Parkinson's Disease.

The aggregation of α-synuclein is a hallmark of Parkinson's disease (PD) and a variety of related neurological disorders. A number of mutations in this protein, including A30P and A53T, are associated with familial forms of the disease. Patients carrying the A30P mutation typically exhibit a similar age of onset and symptoms as sporadic PD, while those carrying the A53T mutation generally have an earlier age of onset and an accelerated progression. We report two C. elegans models of PD (PDA30P and PDA53T), which express these mutational variants in the muscle cells, and probed their behavior relative to animals expressing the wild-type protein (PDWT). PDA30P worms showed a reduced speed of movement and an increased paralysis rate, control worms, but no change in the frequency of body bends. By contrast, in PDA53T worms both speed and frequency of body bends were significantly decreased, and paralysis rate was increased. α-Synuclein was also observed to be less well localized into aggregates in PDA30P worms compared to PDA53T and PDWT worms, and amyloid-like features were evident later in the life of the animals, despite comparable levels of expression of α-synuclein. Furthermore, squalamine, a natural product currently in clinical trials for treating symptomatic aspects of PD, was found to reduce significantly the aggregation of α-synuclein and its associated toxicity in PDA53T and PDWT worms, but had less marked effects in PDA30P. In addition, using an antibody that targets the N-terminal region of α-synuclein, we observed a suppression of toxicity in PDA30P, PDA53T and PDWT worms. These results illustrate the use of these two C. elegans models in fundamental and applied PD research.

Müller glia-myeloid cell crosstalk accelerates optic nerve regeneration in the adult zebrafish.

Neurodegenerative disorders, characterized by progressive neuronal loss, eventually lead to functional impairment in the adult mammalian central nervous system (CNS). Importantly, these deteriorations are irreversible, due to the very limited regenerative potential of these CNS neurons. Stimulating and redirecting neuroinflammation was recently put forward as an important approach to induce axonal regeneration, but it remains elusive how inflammatory processes and CNS repair are intertwined. To gain more insight into these interactions, we investigated how immunomodulation affects the regenerative outcome after optic nerve crush (ONC) in the spontaneously regenerating zebrafish. First, inducing intraocular inflammation using zymosan resulted in an acute inflammatory response, characterized by an increased infiltration and proliferation of innate blood-borne immune cells, reactivation of Müller glia, and altered retinal cytokine expression. Strikingly, inflammatory stimulation also accelerated axonal regrowth after optic nerve injury. Second, we demonstrated that acute depletion of both microglia and macrophages in the retina, using pharmacological treatments with both the CSF1R inhibitor PLX3397 and clodronate liposomes, compromised optic nerve regeneration. Moreover, we observed that csf1ra/b double mutant fish, lacking microglia in both retina and brain, displayed accelerated RGC axonal regrowth after ONC, which was accompanied with unusual Müller glia proliferative gliosis. Altogether, our results highlight the importance of altered glial cell interactions in the axonal regeneration process after ONC in adult zebrafish. Unraveling the relative contribution of the different cell types, as well as the signaling pathways involved, may pinpoint new targets to stimulate repair in the vertebrate CNS.