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Gaucher disease (GD) is caused by insufficient activity of acid $\$-glucosidase (GCase) resulting from mutations in GBA1. To understand the pathogenesis of the neuronopathic GD,induced pluripotent stem cells (iPSCs) were generated from fibroblasts isolated from three GD type 2 (GD2) and 2 unaffected (normal and GD carrier) individuals. The iPSCs were converted to neural precursor cells (NPCs) which were further differentiated into neurons. Parental GD2 fibroblasts as well as iPSCs,NPCs,and neurons had similar degrees of GCase deficiency. Lipid analyses showed increases of glucosylsphingosine and glucosylceramide in the GD2 cells. In addition,GD2 neurons showed increased $\$-synuclein protein compared to control neurons. Whole cell patch-clamping of the GD2 and control iPSCs-derived neurons demonstrated excitation characteristics of neurons,but intriguingly,those from GD2 exhibited consistently less negative resting membrane potentials with various degree of reduction in action potential amplitudes,sodium and potassium currents. Culture of control neurons in the presence of the GCase inhibitor (conduritol B epoxide) recapitulated these findings,providing a functional link between decreased GCase activity in GD and abnormal neuronal electrophysiological properties. To our knowledge,this study is first to report abnormal electrophysiological properties in GD2 iPSC-derived neurons that may underlie the neuropathic phenotype in Gaucher disease. View Publication

Insight into the normal function of PrP(C),and how it can be subverted to produce neurotoxic effects,is provided by PrP molecules carrying deletions encompassing the conserved central region. The most neurotoxic of these mutants,Δ105-125 (called ΔCR),produces a spontaneous neurodegenerative illness when expressed in transgenic mice,and this phenotype can be dose-dependently suppressed by co-expression of wild-type PrP. Whether the toxic activity of ΔCR PrP and the protective activity or wild-type PrP are cell-autonomous,or can be exerted on neighboring cells,is unknown. To investigate this question,we have utilized co-cultures of differentiated neural stem cells derived from mice expressing ΔCR or wild-type PrP. Cells from the two kinds of mice,which are marked by the presence or absence of GFP,are differentiated together to yield neurons,astrocytes,and oligodendrocytes. As a surrogate read-out of ΔCR PrP toxicity,we assayed sensitivity of the cells to the cationic antibiotic,Zeocin. In a previous study,we reported that cells expressing ΔCR PrP are hypersensitive to the toxic effects of several cationic antibiotics,an effect that is suppressed by co-expression of wild type PrP,similar to the rescue of the neurodegenerative phenotype observed in transgenic mice. Using this system,we find that while ΔCR-dependent toxicity is cell-autonomous,the rescuing activity of wild-type PrP can be exerted in trans from nearby cells. These results provide important insights into how ΔCR PrP subverts a normal physiological function of PrP(C),and the cellular mechanisms underlying the rescuing process. View Publication

Reactive oxygen species (ROS) damage brain lipids,carbohydrates,proteins,as well as DNA and may contribute to neurodegeneration. We previously reported that ER- and oxidative stress cause neuronal apoptosis in infantile neuronal ceroid lipofuscinosis (INCL),a lethal neurodegenerative storage disease,caused by palmitoyl-protein thioesterase-1 (PPT1) deficiency. Polyunsaturated fatty acids (PUFA) are essential components of cell membrane phospholipids in the brain and excessive ROS may cause oxidative damage of PUFA leading to neuronal death. Using cultured neurons and neuroprogenitor cells from mice lacking Ppt1,which mimic INCL,we demonstrate that Ppt1-deficient neurons and neuroprogenitor cells contain high levels of ROS,which may cause peroxidation of PUFA and render them incapable of providing protection against oxidative stress. We tested whether treatment of these cells with omega-3 or omega-6 PUFA protects the neurons and neuroprogenitor cells from oxidative stress and suppress apoptosis. We report here that both omega-3 and omega-6 fatty acids protect the Ppt1-deficient cells from ER- as well as oxidative stress and suppress apoptosis. Our results suggest that PUFA supplementation may have neuroprotective effects in INCL. View Publication

The role of epigenetic regulators in the control of adult neurogenesis is largely undefined. We show that the histone demethylase enzyme Kdm5b (Jarid1b) negatively regulates neurogenesis from adult subventricular zone (SVZ) neural stem cells (NSCs) in culture. shRNA-mediated depletion of Kdm5b in proliferating adult NSCs decreased proliferation rates and reduced neurosphere formation in culture. When transferred to differentiation culture conditions,Kdm5b-depleted adult NSCs migrated from neurospheres with increased velocity. Whole-genome expression screening revealed widespread transcriptional changes with Kdm5b depletion,notably the up-regulation of reelin (Reln),the inhibition of steroid biosynthetic pathway component genes and the activation of genes with intracellular transport functions in cultured adult NSCs. Kdm5b depletion increased extracellular reelin concentration in the culture medium and increased phosphorylation of the downstream reelin signaling target Disabled-1 (Dab1). Sequestration of extracellular reelin with CR-50 reelin-blocking antibodies suppressed the increase in migratory velocity of Kdm5b-depleted adult NSCs. Chromatin immunoprecipitation revealed that Kdm5b is present at the proximal promoter of Reln,and H3K4me3 methylation was increased at this locus with Kdm5b depletion in differentiating adult NSCs. Combined the data suggest Kdm5b negatively regulates neurogenesis and represses Reln in neural stem cells from the adult SVZ. View Publication

Pediatric-onset ataxias often present clinically as developmental delay and intellectual disability,with prominent cerebellar atrophy as a key neuroradiographic finding. Here we describe a new clinically distinguishable recessive syndrome in 12 families with cerebellar atrophy together with ataxia,coarsened facial features and intellectual disability,due to truncating mutations in the sorting nexin gene SNX14,encoding a ubiquitously expressed modular PX domain-containing sorting factor. We found SNX14 localized to lysosomes and associated with phosphatidylinositol (3,5)-bisphosphate,a key component of late endosomes/lysosomes. Patient-derived cells showed engorged lysosomes and a slower autophagosome clearance rate upon autophagy induction by starvation. Zebrafish morphants for snx14 showed dramatic loss of cerebellar parenchyma,accumulation of autophagosomes and activation of apoptosis. Our results characterize a unique ataxia syndrome due to biallelic SNX14 mutations leading to lysosome-autophagosome dysfunction. View Publication

BACKGROUND Disruptive mutation in the CHD8 gene is one of the top genetic risk factors in autism spectrum disorders (ASDs). Previous analyses of genome-wide CHD8 occupancy and reduced expression of CHD8 by shRNA knockdown in committed neural cells showed that CHD8 regulates multiple cell processes critical for neural functions,and its targets are enriched with ASD-associated genes. METHODS To further understand the molecular links between CHD8 functions and ASD,we have applied the CRISPR/Cas9 technology to knockout one copy of CHD8 in induced pluripotent stem cells (iPSCs) to better mimic the loss-of-function status that would exist in the developing human embryo prior to neuronal differentiation. We then carried out transcriptomic and bioinformatic analyses of neural progenitors and neurons derived from the CHD8 mutant iPSCs. RESULTS Transcriptome profiling revealed that CHD8 hemizygosity (CHD8 (+/-)) affected the expression of several thousands of genes in neural progenitors and early differentiating neurons. The differentially expressed genes were enriched for functions of neural development,$$-catenin/Wnt signaling,extracellular matrix,and skeletal system development. They also exhibited significant overlap with genes previously associated with autism and schizophrenia,as well as the downstream transcriptional targets of multiple genes implicated in autism. Providing important insight into how CHD8 mutations might give rise to macrocephaly,we found that seven of the twelve genes associated with human brain volume or head size by genome-wide association studies (e.g.,HGMA2) were dysregulated in CHD8 (+/-) neural progenitors or neurons. CONCLUSIONS We have established a renewable source of CHD8 (+/-) iPSC lines that would be valuable for investigating the molecular and cellular functions of CHD8. Transcriptomic profiling showed that CHD8 regulates multiple genes implicated in ASD pathogenesis and genes associated with brain volume. View Publication

An expanded hexanucleotide repeat in a noncoding region of the C9orf72 gene is a major cause of amyotrophic lateral sclerosis (ALS),accounting for up to 40% of familial cases and 7% of sporadic ALS in European populations. We have generated induced pluripotent stem cells (iPSCs) from fibroblasts of patients carrying C9orf72 hexanucleotide expansions,differentiated these to functional motor and cortical neurons and performed an extensive phenotypic characterization. In C9orf72 iPSC-derived motor neurons,decreased cell survival is correlated with dysfunction in Ca(2+) homeostasis,reduced levels of the anti-apoptotic protein Bcl-2,increased endoplasmic reticulum (ER) stress and reduced mitochondrial membrane potential. Furthermore,C9orf72 motor neurons,and also cortical neurons,show evidence of abnormal protein aggregation and stress granule formation. This study is an extensive characterization of iPSC-derived motor neurons as cellular models of ALS carrying C9orf72 hexanucleotide repeats,which describes a novel pathogenic link between C9orf72 mutations,dysregulation of calcium signalling and altered proteostasis and provides a potential pharmacological target for the treatment of ALS and the related neurodegenerative disease frontotemporal dementia (FTD). This article is protected by copyright. All rights reserved. View Publication

FBW7 is a crucial component of an SCF-type E3 ubiquitin ligase,which mediates degradation of an array of different target proteins. The Fbw7 locus comprises three different isoforms,each with its own promoter and each suspected to have a distinct set of substrates. Most FBW7 targets have important functions in developmental processes and oncogenesis,including Notch proteins,which are functionally important substrates of SCF(Fbw7). Notch signalling controls a plethora of cell differentiation decisions in a wide range of species. A prominent role of this signalling pathway is that of mediating lateral inhibition,a process where exchange of signals that repress Notch ligand production amplifies initial differences in Notch activation levels between neighbouring cells,resulting in unequal cell differentiation decisions. Here we show that the downstream Notch signalling effector HES5 directly represses transcription of the E3 ligase Fbw7β,thereby directly bearing on the process of lateral inhibition. Fbw7(Δ/+) heterozygous mice showed haploinsufficiency for Notch degradation causing impaired intestinal progenitor cell and neural stem cell differentiation. Notably,concomitant inactivation of Hes5 rescued both phenotypes and restored normal stem cell differentiation potential. In silico modelling suggests that the NICD/HES5/FBW7β positive feedback loop underlies Fbw7 haploinsufficiency. Thus repression of Fbw7β transcription by Notch signalling is an essential mechanism that is coupled to and required for the correct specification of cell fates induced by lateral inhibition. View Publication

Synaptic cadherin adhesion complexes are known to be key regulators of synapse plasticity. However,the molecular mechanisms that coordinate activity-induced modifications in cadherin localization and adhesion and the subsequent changes in synapse morphology and efficacy remain unknown. We demonstrate that the intracellular cadherin binding protein δ-catenin is transiently palmitoylated by DHHC5 after enhanced synaptic activity and that palmitoylation increases δ-catenin-cadherin interactions at synapses. Both the palmitoylation of δ-catenin and its binding to cadherin are required for activity-induced stabilization of N-cadherin at synapses and the enlargement of postsynaptic spines,as well as the insertion of GluA1 and GluA2 subunits into the synaptic membrane and the concomitant increase in miniature excitatory postsynaptic current amplitude. Notably,context-dependent fear conditioning in mice resulted in increased δ-catenin palmitoylation,as well as increased δ-catenin-cadherin associations at hippocampal synapses. Together these findings suggest a role for palmitoylated δ-catenin in coordinating activity-dependent changes in synaptic adhesion molecules,synapse structure and receptor localization that are involved in memory formation. View Publication

Hypoxic stressors contribute to neuronal death in many brain diseases. Astrocyte processes surround most neurons and are therefore anatomically well-positioned to shield them from hypoxic injury. Excitatory amino acid transporters (EAATs),represent the sole mechanism of active reuptake of glutamate into the astrocytes and neurons and are essential to dampen neuronal excitation following glutamate release at synapses. Glutamate clearance impairment from any factors is bound to result in an increase in hypoxic neuronal injury. The brain energy metabolism under hypoxic conditions depends on monocarboxylate transporters (MCTs) that are expressed by neurons and glia. Previous co-immunoprecipitation experiments revealed that MCT4 directly modulate EAAT1 in astrocytes. The reduction in both surface proteins may act synergistically to induce neuronal hyperexcitability and excitotoxicity. Therefore we hypothesized that astrocytes would respond to hypoxic conditions by enhancing their expression of MCT4 and EAAT1,which,in turn,would enable them to better support neurons to survive lethal hypoxia injury. An oxygen deprivation (OD) protocol was used in primary cultures of neurons,astrocytes,and astrocytes-neurons derived from rat hippocampus,with or without MCT4-targeted short hairpin RNA (shRNA) transfection. Cell survival,expression of MCT4,EAAT1,glial fibrillary acidic protein and neuronal nuclear antigen were evaluated. OD resulted in significant cell death in neuronal cultures and up-regulation of MCT4,EAAT1 expression respectively in primary cell cultures,but no injury in neuron-astrocyte co-cultures and astrocyte cultures. However,neuronal cell death in co-cultures was increased exposure to shRNA-MCT4 prior to OD. These findings demonstrate that the MCT4-mediated expression of EAAT1 is involved in the resistance to hypoxia injury in astrocyte-neuron co-cultures. View Publication

Alzheimer's disease (AD) is emerging as a synaptopathology driven by metaplasticity. Indeed,reminiscent of metaplasticity,oligomeric forms of the amyloid-beta$ peptide (oAbeta$) prevent induction of long-term potentiation (LTP) via the prior activation of GluN2B-containing NMDA receptors (NMDARs). However,the downstream Ca2+-dependent signaling molecules that mediate aberrant metaplasticity are unknown. In this study,we show that oAbeta$ promotes the activation of Ca2+/calmodulin-dependent kinase II (CaMKII) via GluN2B-containing NMDARs. Importantly,we find that CaMKII inhibition rescues both the LTP impairment and the dendritic spine loss mediated by oAbeta$. Mechanistically resembling metaplasticity,oAbeta$ prevents subsequent rounds of plasticity from inducing CaMKII T286 autophosphorylation,as well as the associated anchoring and accumulation of synaptic AMPA receptors (AMPARs). Finally,prolonged oAbeta$ treatment-induced CaMKII misactivation leads to dendritic spine loss via the destabilization of surface AMPARs. Thus,our study demonstrates that oAbeta$ engages synaptic metaplasticity via aberrant CaMKII activation. View Publication

Glioblastoma multiforme (GBM) is the most common and aggressive brain cancer,and despite treatment advances,patient prognosis remains poor. During routine animal studies,we serendipitously observed that fenbendazole,a benzimidazole antihelminthic used to treat pinworm infection,inhibited brain tumor engraftment. Subsequent in vitro and in vivo experiments with benzimidazoles identified mebendazole as the more promising drug for GBM therapy. In GBM cell lines,mebendazole displayed cytotoxicity,with half-maximal inhibitory concentrations ranging from 0.1 to 0.3 µM. Mebendazole disrupted microtubule formation in GBM cells,and in vitro activity was correlated with reduced tubulin polymerization. Subsequently,we showed that mebendazole significantly extended mean survival up to 63% in syngeneic and xenograft orthotopic mouse glioma models. Mebendazole has been approved by the US Food and Drug Administration for parasitic infections,has a long track-record of safe human use,and was effective in our animal models with doses documented as safe in humans. Our findings indicate that mebendazole is a possible novel anti-brain tumor therapeutic that could be further tested in clinical trials. View Publication