Loss of the Intellectual Disability and Autism Gene Cc2d1a and Its Homolog Cc2d1b Differentially Affect Spatial Memory, Anxiety, and Hyperactivity

Hundreds of genes are mutated in non-syndromic intellectual disability (ID) and autism spectrum disorder (ASD), with each gene often involved in only a handful of cases. Such heterogeneity can be daunting, but rare recessive loss of function (LOF) mutations can be a good starting point to provide insight into the mechanisms of neurodevelopmental disease. Biallelic LOF mutations in the signaling scaffold CC2D1Acause a rare form of autosomal recessive ID, sometimes associated with ASD and seizures. In parallel, we recently reported that Cc2d1a-deficient mice present with cognitive and social deficits, hyperactivity and anxiety. In Drosophila, loss of the only ortholog of Cc2d1a, lgd, is embryonically lethal, while in vertebrates, Cc2d1a has a homolog Cc2d1b which appears to be compensating, indicating that Cc2d1a and Cc2d1b have a redundant function in humans and mice. Here, we generate an allelic series of Cc2d1a and Cc2d1b LOF to determine the relative role of these genes during behavioral development. We generated Cc2d1b knockout (KO), Cc2d1a/1b double heterozygous and double KO mice, then performed behavioral studies to analyze learning and memory, social interactions, anxiety, and hyperactivity. We found that Cc2d1a and Cc2d1b have partially overlapping roles. Overall, loss of Cc2d1b is less severe than loss of Cc2d1a, only leading to cognitive deficits, while Cc2d1a/1b double heterozygous animals are similar to Cc2d1a-deficient mice. These results will help us better understand the deficits in individuals with CC2D1A mutations, suggesting that recessive CC2D1B mutations and trans-heterozygous CC2D1A and CC2D1B mutations could also contribute to the genetics of ID

Translating genetic and preclinical findings into autism therapies

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder characterized by social deficits and repetitive/restrictive interests. ASD is associated with multiple comorbidities, including intellectual disability, anxiety, and epilepsy. Evidence that ASD is highly heritable has spurred major efforts to unravel its genetics, revealing possible contributions from hundreds of genes through rare and common variation and through copy-number changes. In this perspective, we provide an overview of the current state of ASD genetics and of how genetic research has spurred the development of in vivo and in vitro models using animals and patient cells to evaluate the impact of genetic mutations on cellular function leading to disease. Efforts to translate these findings into successful therapies have yet to bear fruit. We discuss how the valuable insight into the disorder provided by these new models can be used to better understand ASD and develop future clinical trials

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the cerebellar neuronal types (granule cells, Purkinje cells and inhibitory interneurons), granule neurons are by far the most numerous and are the most abundant type of neurons in the mammalian brain. In rodents, cerebellar granule neurons are generated during the first two post-natal weeks from progenitor cells in the outermost layer of the cerebellar cortex, the external granule layer (EGL). The protocol presented here describes techniques to enrich and culture granule neurons and their progenitor cells from post-natal mouse cerebellum. We will describe procedures to obtain cultures of increasing purity3,4, which can be used to study the differentiation of proliferating progenitor cells into granule neurons5,6. Once the progenitor cells differentiate, the cultures also provide a homogenous population of granule neurons for experimental manipulation and characterization of phenomena such as synaptogenesis, glutamate receptor function7, interaction with other purified cerebellar cells8,9 or cell death7

Structure of the polyisoprenyl-phosphate glycosyltransferase GtrB and insights into the mechanism of catalysis

The attachment of a sugar to a hydrophobic polyisoprenyl carrier is the first step for all extracellular glycosylation processes. The enzymes that perform these reactions, polyisoprenyl-glycosyltransferases (PI-GTs) include dolichol phosphate mannose synthase (DPMS), which generates the mannose donor for glycosylation in the endoplasmic reticulum. Here we report the 3.0Å resolution crystal structure of GtrB, a glucose-specific PI-GT from Synechocystis, showing a tetramer in which each protomer contributes two helices to a membrane-spanning bundle. The active site is 15 Å from the membrane, raising the question of how water-soluble and membrane-embedded substrates are brought into apposition for catalysis. A conserved juxtamembrane domain harbours disease mutations, which compromised activity in GtrB in vitro and in humanDPM1 tested in zebrafish. We hypothesize a role of this domain in shielding the polyisoprenyl-phosphate for transport to the active site. Our results reveal the basis of PI-GT function, and provide a potential molecular explanation for DPM1-related disease

CC2D1A regulates human intellectual and social function as well as NF-κB signaling homeostasis.

Autism spectrum disorder (ASD) and intellectual disability (ID) are often comorbid, but the extent to which they share common genetic causes remains controversial. Here, we present two autosomal-recessive founder mutations in the CC2D1A gene causing fully penetrant cognitive phenotypes, including mild-to-severe ID, ASD, as well as seizures, suggesting shared developmental mechanisms. CC2D1A regulates multiple intracellular signaling pathways, and we found its strongest effect to be on the transcription factor nuclear factor κB (NF-κB). Cc2d1a gain and loss of function both increase activation of NF-κB, revealing a critical role of Cc2d1a in homeostatic control of intracellular signaling. Cc2d1a knockdown in neurons reduces dendritic complexity and increases NF-κB activity, and the effects of Cc2d1a depletion can be rescued by inhibiting NF-κB activity. Homeostatic regulation of neuronal signaling pathways provides a mechanism whereby common founder mutations could manifest diverse symptoms in different patients

Does salinity modulates the response of Mytilus galloprovincialis exposed to triclosan and diclofenac?

In the present study Mytilus galloprovincialis mussels were exposed for 28 days to three salinities: 30 (control), 25 and 35. Simultaneously, organisms at each salinity were exposed to either the antimicrobial agent Triclosan (TCS) or the pharmaceutical drug Diclofenac (DIC) at 1 μg/L. Salinity alone and exposure to PPCPs changed mussel's metabolic capacity and oxidative status, but no additive or synergetic effects resulting from the combined exposures were observed. Overall, the metabolic capacity of mussels was decreased when exposed to TCS and DIC under control salinity, which was less pronounced at salinities out of the control level. TCS had a notorious effect over glutathione peroxidase activity while DIC exposure enhanced catalase response. Such defence mechanisms were able to prevent cellular damage but still a clear reduction in GSH/GSSG ratio after PPCPs exposures indicates oxidative stress which could compromise bivalve's performance to further stressing events.publishe

Anticancer effects of novel resveratrol analogues on human ovarian cancer cells

Resveratrol, a naturally occurring phytoalexin, has long been known to play an important regulatory role in key functions in cell physiology. This multifunctional role of resveratrol is explained by its ability to interact with several targets of various cell pathways. In the recent past, synthetic chemical modifications have been made in an attempt to enhance the biological effects of resveratrol, including its anti-cancer properties. In this study, we investigated the molecular mechanisms of action of novel trans-restricted analogues of resveratrol in which the C-C double bond of the natural derivative has been replaced by diaryl-substituted imidazole analogues. In ovarian cancer models, the results of in vitro screening revealed that the resveratrol analogues exhibited enhanced anti-proliferative properties compared with resveratrol. We found that the resveratrol analogues also significantly inhibited Akt and MAPK signalling and reduced the migration of IL-6 and EGF-treated cells. Finally, in ascite-derived cancer cells, we demonstrated that the resveratrol analogues reduced the expression of epithelial mesenchymal transition (EMT) markers. Collectively, these findings indicate the enhanced anti-cancer properties of the resveratrol analogues

Developmental and degenerative features in a complicated spastic paraplegia

ObjectiveWe sought to explore the genetic and molecular causes of Troyer syndrome, one of several complicated hereditary spastic paraplegias (HSPs). Troyer syndrome had been thought to be restricted to the Amish; however, we identified 2 Omani families with HSP, short stature, dysarthria and developmental delay—core features of Troyer syndrome—and a novel mutation in the SPG20 gene, which is also mutated in the Amish. In addition, we analyzed SPG20 expression throughout development to infer how disruption of this gene might generate the constellation of developmental and degenerative Troyer syndrome phenotypes.MethodsClinical characterization of 2 non-Amish families with Troyer syndrome was followed by linkage and sequencing analysis. Quantitative polymerase chain reaction and in situ hybridization analysis of SPG20 expression were carried out in embryonic and adult human and mouse tissue.ResultsTwo Omani families carrying a novel SPG20 mutation displayed clinical features remarkably similar to the Amish patients with Troyer syndrome. SPG20 mRNA is expressed broadly but at low relative levels in the adult brain; however, it is robustly and specifically expressed in the limbs, face, and brain during early morphogenesis.InterpretationNull mutations in SPG20 cause Troyer syndrome, a specific clinical entity with developmental and degenerative features. Maximal expression of SPG20 in the limb buds and forebrain during embryogenesis may explain the developmental origin of the skeletal and cognitive defects observed in this disorder. ANN NEUROL 2010;67:516–52

Mutations in INPP5K Cause a Form of Congenital Muscular Dystrophy Overlapping Marinesco-Sjögren Syndrome and Dystroglycanopathy.

Congenital muscular dystrophies display a wide phenotypic and genetic heterogeneity. The combination of clinical, biochemical, and molecular genetic findings must be considered to obtain the precise diagnosis and provide appropriate genetic counselling. Here we report five individuals from four families presenting with variable clinical features including muscular dystrophy with a reduction in dystroglycan glycosylation, short stature, intellectual disability, and cataracts, overlapping both the dystroglycanopathies and Marinesco-Sjögren syndrome. Whole-exome sequencing revealed homozygous missense and compound heterozygous mutations in INPP5K in the affected members of each family. INPP5K encodes the inositol polyphosphate-5-phosphatase K, also known as SKIP (skeletal muscle and kidney enriched inositol phosphatase), which is highly expressed in the brain and muscle. INPP5K localizes to both the endoplasmic reticulum and to actin ruffles in the cytoplasm. It has been shown to regulate myoblast differentiation and has also been implicated in protein processing through its interaction with the ER chaperone HSPA5/BiP. We show that morpholino-mediated inpp5k loss of function in the zebrafish results in shortened body axis, microphthalmia with disorganized lens, microcephaly, reduced touch-evoked motility, and highly disorganized myofibers. Altogether these data demonstrate that mutations in INPP5K cause a congenital muscular dystrophy syndrome with short stature, cataracts, and intellectual disability

COL4A1 Mutations Cause Ocular Dysgenesis, Neuronal Localization Defects, and Myopathy in Mice and Walker-Warburg Syndrome in Humans

Muscle-eye-brain disease (MEB) and Walker Warburg Syndrome (WWS) belong to a spectrum of autosomal recessive diseases characterized by ocular dysgenesis, neuronal migration defects, and congenital muscular dystrophy. Until now, the pathophysiology of MEB/WWS has been attributed to alteration in dystroglycan post-translational modification. Here, we provide evidence that mutations in a gene coding for a major basement membrane protein, collagen IV alpha 1 (COL4A1), are a novel cause of MEB/WWS. Using a combination of histological, molecular, and biochemical approaches, we show that heterozygous Col4a1 mutant mice have ocular dysgenesis, neuronal localization defects, and myopathy characteristic of MEB/WWS. Importantly, we identified putative heterozygous mutations in COL4A1 in two MEB/WWS patients. Both mutations occur within conserved amino acids of the triple-helix-forming domain of the protein, and at least one mutation interferes with secretion of the mutant proteins, resulting instead in intracellular accumulation. Expression and posttranslational modification of dystroglycan is unaltered in Col4a1 mutant mice indicating that COL4A1 mutations represent a distinct pathogenic mechanism underlying MEB/WWS. These findings implicate a novel gene and a novel mechanism in the etiology of MEB/WWS and expand the clinical spectrum of COL4A1-associated disorders