[Show abstract][Hide abstract] ABSTRACT: SHANK proteins are crucial for the formation and plasticity of excitatory synapses. Although mutations in all three SHANK genes are associated with autism spectrum disorder (ASD), SHANK3 appears to be the major ASD gene with a prevalence of approximately 0.5% for SHANK3 mutations in ASD, with higher rates in individuals with ASD and intellectual disability (ID). Interestingly, the most relevant mutations are typically de novo and often are frameshift or nonsense mutations resulting in a premature stop and a truncation of SHANK3 protein.
We analyzed three different SHANK3 stop mutations that we identified in individuals with ASD and/or ID, one novel (c.5008A > T) and two that we recently described (c.1527G > A, c.2497delG). The mutations were inserted into the human SHANK3a sequence and analyzed for effects on subcellular localization and neuronal morphology when overexpressed in rat primary hippocampal neurons.
Clinically, all three individuals harboring these mutations had global developmental delays and ID. In our in vitro assay, c.1527G > A and c.2497delG both result in proteins that lack most of the SHANK3a C-terminus and accumulate in the nucleus of transfected cells. Cells expressing these mutants exhibit converging morphological phenotypes including reduced complexity of the dendritic tree, less spines, and less excitatory, but not inhibitory synapses. In contrast, the truncated protein based on c.5008A > T, which lacks only a short part of the sterile alpha motif (SAM) domain in the very SHANK3a C-terminus, does not accumulate in the nucleus and has minor effects on neuronal morphology.
In spite of the prevalence of SHANK3 disruptions in ASD and ID, only a few human mutations have been functionally characterized; here we characterize three additional mutations. Considering the transcriptional and functional complexity of SHANK3 in healthy neurons, we propose that any heterozygous stop mutation in SHANK3 will lead to a dysequilibrium of SHANK3 isoform expression and alterations in the stoichiometry of SHANK3 protein complexes, resulting in a distinct perturbation of neuronal morphology. This could explain why the clinical phenotype in all three individuals included in this study remains quite severe - regardless of whether there are disruptions in one or more SHANK3 interaction domains.
[Show abstract][Hide abstract] ABSTRACT: Rap GTPase-activating proteins (RapGAPs) are essential for synaptic function as they tightly regulate synaptic Rap signaling. Among the most abundant synaptic RapGAPs in brain are the Spine-associated RapGAPs (SPARs) Sipa1l1/SPAR and Sipa1l2/SPAR2, while nothing has been reported on Sipa1l3/SPAR3. In this study, we show that Sipa1l3/SPAR3 is conserved across species, has a distinct expression pattern in the developing rat brain and is localized at excitatory postsynapses. We further demonstrate that the Sipa1l3/SPAR3 C-terminus is required for postsynaptic targeting and represents an interaction module for Fezzins such as ProSAPiP1/Lzts3, a binding partner of the postsynaptic scaffold protein Shank3. Taken together, our data imply that Sipa1l3/SPAR3 is a hitherto unknown synaptic RapGAP, which is targeted to postsynaptic specializations and interacts with Fezzins. This article is protected by copyright. All rights reserved.
Journal of Neurochemistry 09/2015; DOI:10.1111/jnc.13353 · 4.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A dyshomeostasis of zinc ions has been reported for many psychiatric and neurodegenerative disorders, including schizophrenia, attention deficit hyperactivity disorder, depression, autism, Parkinson's and Alzheimer's disease. Furthermore, alterations in zinc-levels have been associated with seizures and traumatic brain injury. Thus, altering zinc-levels within the brain is emerging as a new target for the prevention and treatment of psychiatric and neurological diseases. However, given the restriction of zinc uptake into the brain by the blood-brain barrier, methods for controlled regulation and manipulation of zinc concentrations within the brain are rare. Here, we performed in vivo studies investigating the possibility of brain targeted zinc delivery using zinc-loaded nanoparticles which are able to cross the blood-brain barrier. After injecting these nanoparticles, we analyzed the regional and time-dependent distribution of zinc and nanoparticles within the brain. Moreover, we evaluated whether the presence of zinc-loaded nanoparticles alters the expression of zinc sensitive genes and proteins such as metallothioneins and zinc transporters and quantified possible toxic effects. Our results show that zinc loaded g7 nanoparticles offer a promising approach as a novel non - invasive method to selectively enrich Zn2+ in the brain within a small amount of time.
[Show abstract][Hide abstract] ABSTRACT: Proteolytic processing of amyloid-β precursor protein (APP) by beta-site APP cleaving enzyme 1 (BACE1) is the initial step in the production of amyloid beta (Aβ), which accumulates in senile plaques in Alzheimer's disease (AD). Essential for this cleavage is the transport and sorting of both proteins through endosomal/Golgi compartments. Golgi-localized γ-ear-containing ARF-binding (GGA) proteins have striking cargo-sorting functions in these pathways. Recently, GGA1 and GGA3 were shown to interact with BACE1, to be expressed in neurons, and to be decreased in AD brain, whereas little is known about GGA2. Since GGA1 impacts Aβ generation by confining APP to the Golgi and perinuclear compartments, we tested whether all GGAs modulate BACE1 and APP transport and processing. We observed decreased levels of secreted APP alpha (sAPPα), sAPPβ, and Aβ upon GGA overexpression, which could be reverted by knockdown. GGA-BACE1 co-immunoprecipitation was impaired upon GGA-GAE but not VHS domain deletion. Autoinhibition of the GGA1-VHS domain was irrelevant for BACE1 interaction. Our data suggest that all three GGAs affect APP processing via the GGA-GAE domain.
PLoS ONE 06/2015; 10(6). DOI:10.1371/journal.pone.0129047 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Amyotrophic lateral sclerosis (ALS) is a genetically heterogeneous neurodegenerative syndrome hallmarked by adult-onset loss of motor neurons. We performed exome sequencing of 252 familial ALS (fALS) and 827 control individuals. Gene-based rare variant analysis identified an exome-wide significant enrichment of eight loss-of-function (LoF) mutations in TBK1 (encoding TANK-binding kinase 1) in 13 fALS pedigrees. No enrichment of LoF mutations was observed in a targeted mutation screen of 1,010 sporadic ALS and 650 additional control individuals. Linkage analysis in four families gave an aggregate LOD score of 4.6. In vitro experiments confirmed the loss of expression of TBK1 LoF mutant alleles, or loss of interaction of the C-terminal TBK1 coiled-coil domain (CCD2) mutants with the TBK1 adaptor protein optineurin, which has been shown to be involved in ALS pathogenesis. We conclude that haploinsufficiency of TBK1 causes ALS and fronto-temporal dementia.
[Show abstract][Hide abstract] ABSTRACT: Various recent studies revealed that biometal dyshomeostasis plays a crucial role in the pathogenesis of neurological disorders such as autism spectrum disorders (ASD). Substantial evidence indicates that disrupted neuronal homeostasis of different metal ions such as Fe, Cu, Pb, Hg, Se, and Zn may mediate synaptic dysfunction and impair synapse formation and maturation. Here, we performed in vitro studies investigating the consequences of an imbalance of transition metals on glutamatergic synapses of hippocampal neurons. We analyzed whether an imbalance of any one metal ion alters cell health and synapse numbers. Moreover, we evaluated whether a biometal profile characteristic for ASD patients influences synapse formation, maturation, and composition regarding NMDA receptor subunits and Shank proteins. Our results show that an ASD like biometal profile leads to a reduction of NMDAR (NR/Grin/GluN) subunit 1 and 2a, as well as Shank gene expression along with a reduction of synapse density. Additionally, synaptic protein levels of GluN2a and Shanks are reduced. Although Zn supplementation is able to rescue the aforementioned alterations, Zn deficiency is not solely responsible as causative factor. Thus, we conclude that balancing Zn levels in ASD might be a prime target to normalize synaptic alterations caused by biometal dyshomeostasis.
[Show abstract][Hide abstract] ABSTRACT: Located at neuronal terminals, the postsynaptic density (PSD) is a highly complex network of cytoskeletal scaffolding and signaling proteins responsible for the transduction and modulation of glutamatergic signaling between neurons. Using ion-mobility enhanced data-independent label-free LC-MS/MS, we established a reference proteome of crude synaptosomes, synaptic junctions and PSD derived from mouse hippocampus including TOP3-based absolute quantification values for identified proteins. The final dataset across all fractions comprised 49491 peptides corresponding to 4558 protein groups. Of these, 2102 protein groups were identified in highly purified PSD in at least two biological replicates. Identified proteins play pivotal roles in neurological and synaptic processes providing a rich resource for studies on hippocampal PSD function as well as on the pathogenesis of neuropsychiatric disorders. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: SHANK genes code for scaffold proteins located at the post-synaptic density of glutamatergic synapses. In neurons, SHANK2 and SHANK3 have a positive effect on the induction and maturation of dendritic spines, whereas SHANK1 induces the enlargement of spine heads. Mutations in SHANK genes have been associated with autism spectrum disorders (ASD), but their prevalence and clinical relevance remain to be determined. Here, we performed a new screen and a meta-analysis of SHANK copy-number and coding-sequence variants in ASD. Copy-number variants were analyzed in 5,657 patients and 19,163 controls, coding-sequence variants were ascertained in 760 to 2,147 patients and 492 to 1,090 controls (depending on the gene), and, individuals carrying de novo or truncating SHANK mutations underwent an extensive clinical investigation. Copy-number variants and truncating mutations in SHANK genes were present in ∼1% of patients with ASD: mutations in SHANK1 were rare (0.04%) and present in males with normal IQ and autism; mutations in SHANK2 were present in 0.17% of patients with ASD and mild intellectual disability; mutations in SHANK3 were present in 0.69% of patients with ASD and up to 2.12% of the cases with moderate to profound intellectual disability. In summary, mutations of the SHANK genes were detected in the whole spectrum of autism with a gradient of severity in cognitive impairment. Given the rare frequency of SHANK1 and SHANK2 deleterious mutations, the clinical relevance of these genes remains to be ascertained. In contrast, the frequency and the penetrance of SHANK3 mutations in individuals with ASD and intellectual disability-more than 1 in 50-warrant its consideration for mutation screening in clinical practice.
[Show abstract][Hide abstract] ABSTRACT: Recent studies suggest that synaptic pathology in autism spectrum disorder (ASD) might be caused by the disruption of a signaling pathway at excitatory glutamatergic synapses, which can be influenced by environmental factors. Some factors, such as prenatal zinc deficiency, dysfunction of metallothioneins as well as deletion of COMMD1, all affect brain metal-ion homeostasis and have been associated with ASD. Given that COMMD1 regulates copper levels and that copper and zinc have antagonistic properties, here, we followed the idea that copper overload might induce a local zinc deficiency affecting key players of a putative ASD pathway such as ProSAP/Shank proteins as reported before. Our results show that increased copper levels indeed interfere with intracellular zinc concentrations and affect synaptic ProSAP/Shank levels, which similarly are altered by manipulation of copper and zinc levels through overexpression and knockdown of COMMD1. In line with this, acute and prenatal copper overload lead to local zinc deficiencies in mice. Pups exposed to prenatal copper overload furthermore show a reduction in ProSAP/Shank protein levels in the brain as well as a decreased NMDAR subunit 1 concentration. Thus, it might be likely that brain metal ion status influences a distinct pathway in excitatory synapses associated with genetic forms of ASD.
[Show abstract][Hide abstract] ABSTRACT: Polymeric nanoparticles (NPs) offer a promising approach for therapeutic intracellular delivery of proteins, conventionally hampered by short half-lives, instability and immunogenicity. Remarkably, NPs uptake occurs via endocytic internalization leading to NPs content's release within lysosomes. To overcome lysosomal degradation and achieve NPs and/or loaded proteins release into cytosol, we propose the formulation of hybrid NPs by adding 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as pH sensitive component in the formulation of poly-lactide-co-glycolide (PLGA) NPs. Hybrid NPs, featured by different DOPE/PLGA ratios, were characterized in terms of structure, stability and lipid organization within the polymeric matrix. Experiments on neuronal cells and rat primary cultures highlighted the safety profile of hybrid NPs. Moreover, after internalization, NPs are able to transiently destabilize the integrity of lysosomes in which they are taken up, speeding their escape and favoring cytoplasmatic localization. Thus, these DOPE/PLGA-NPs configure themselves as promising carriers for intracellular protein delivery.
International Journal of Pharmaceutics 05/2014; DOI:10.1016/j.ijpharm.2014.05.054 · 3.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background: A number of the genes identified as anomalous in genetic screens of the autism spectrum disorders (ASD) population code for proteins that regulate synaptic plasticity, including the Shank family of genes. The link between mutation of these ubiquitously expressed genes and the selective social impairments and repetitive behaviors featured in ASD, though, is still unclear. Transgenic introduction of mutations identified in human ASD subjects into rodent models enables the association of specific genotypes to phenotypes. Rat models present several advantages over mouse models for characterizing ASD mutations due to their rich social behavioral repertoire, metabolic similarities to humans and amenability to complex electrophysiological manipulations.
Objectives: To investigate the relationship between decreased synaptic function and social impairment, we have characterized the behavioral, molecular and electrophysiological phenotype of rats expressing a mutation of the Shank2 gene associated with ASD. The characterization of neural activity associated with aberrant behavior has the potential to identify reliable biomarkers of both disease and drug response.
Methods: Transgenic Sprague-Dawley rats containing a targeted deletion of exon 31 of the SHANK2 gene were generated using zinc finger nuclease technology. The subsequent animals were characterized through a behavioral battery, molecularly, through western blot protein quantification of regional synaptosomal brain homogenates, and electrophysiologically, through in vivo, chronically implanted, surface and depth electrodes.
Results: Mutation of the Shank2 gene results in alterations in social behavior seen throughout development. Homozygous Shank2 mutant rats (HOs) engage in less species typical juvenile play than wild type rats (WTs). This deficit is maintained into adulthood as evidenced by decreased social investigation of conspecifics and transmission of food preference. However, Shank2 mutation in the rat, unlike in the mouse, does not result in decreased social approach. HOs also exhibited several forms of restricted and repetitive behaviors analogous to those observed in ASD, including increased locomotion and abnormal circling and checking behaviors. Uniquely, HOs also show increased motivation in a progressive ratio task that is independent of hyperactivity. The behavioral deficits associated with Shank2 mutation are accompanied by upregulation of Shank3 and mGluR1 in the striatum and downregulation SHANK1 and Homer1 in the hippocampus. Local field potential recordings in multiple nodes of the social brain circuit, including the amygdala, entorhinal cortex and hippocampus were collected to assess differences in network activity resulting from Shank2 mutation.
Conclusions: Shank2 mutation in the rat model recapitulates many of the behavioral features of ASD and has broad effects on the expression of synaptic proteins. Exploration of electrophysiological phenotypes associated with these behaviors in this model may shed light on the neural underpinnings of the symptomatology of the disorder.
2014 International Meeting for Autism Research; 05/2014
[Show abstract][Hide abstract] ABSTRACT: The genetic heterogeneity of autism spectrum disorders (ASDs) is enormous, and the neurobiology of proteins encoded by genes associated with ASD is very diverse. Revealing the mechanisms on which different neurobiological pathways in ASD pathogenesis converge may lead to the identification of drug targets.
The main objective is firstly to outline the main molecular networks and neuronal mechanisms in which ASD gene products participate and secondly to answer the question how these converge. Finally, we aim to pinpoint drug targets within these mechanisms.
Literature review of the neurobiological properties of ASD gene products with a special focus on the developmental consequences of genetic defects and the possibility to reverse these by genetic or pharmacological interventions.
The regulation of activity-dependent protein synthesis appears central in the pathogenesis of ASD. Through sequential consequences for axodendritic function, neuronal disabilities arise expressed as behavioral abnormalities and autistic symptoms in ASD patients. Several known ASD gene products have their effect on this central process by affecting protein synthesis intrinsically, e.g., through enhancing the mammalian target of rapamycin (mTOR) signal transduction pathway or through impairing synaptic function in general. These are interrelated processes and can be targeted by compounds from various directions: inhibition of protein synthesis through Lovastatin, mTOR inhibition using rapamycin, or mGluR-related modulation of synaptic activity.
ASD gene products may all feed into a central process of translational control that is important for adequate glutamatergic regulation of dendritic properties. This process can be modulated by available compounds but may also be targeted by yet unexplored routes.
[Show abstract][Hide abstract] ABSTRACT: The application of polymeric nanoparticles (NPs) has a promising future for targeting and delivering drugs into the central nervous system (CNS). However, the fate of NPs once entered in the brain after crossing the blood-brain barrier (BBB) and taken up into neuronal cells is a neglected area of study. Thus, here, we investigate the possible mechanisms of a cell-to-cell transport of poly-lactide-co-glycolide (PLGA) NPs modified with a glycopeptide (g7-NPs), already demonstrated to be able to cross the BBB after in vivo administration in rodents. We also tested antibody (Ab) -modified g7-NPs both in vitro and in vivo to investigate the possibility of specific targeting. Our results show that g7-NPs can be transported intra- and intercellularly within vesicles after vesicular internalization. Moreover, cell-to-cell transport is mediated by tunneling-nanotube (TNT)-like structures in cell lines and most interestingly in glial as well as neuronal cells in vitro. The transport is dependent on F-actin and can be increased by induction of TNT-like structures overexpressing M-Sec, a central factor and inducer of TNT formation. Moreover, cell-to-cell transport occurs independently from NP surface modification with antibodies. These in vitro findings were in part confirmed by in vivo evidence after i.p. administration of NPs in mice.
[Show abstract][Hide abstract] ABSTRACT: Development of dendritic spines is important for synaptic function, and alteration in spine morphogenesis is often associated with mental disorders. Rich2 was an uncharacterized Rho-GAP protein. Here we searched for a role of this protein in spine morphogenesis. We found that it is enriched in dendritic spines of cultured hippocampal pyramidal neurons during early stages of development. Rich2 specifically stimulated the Rac1 GTPase in these neurons. Inhibition of Rac1 by EHT 1864 increased the size and decreased the density of dendritic spines. Similarly, Rich2 overexpression increased the size and decreased the density of dendritic spines, whereas knock-down of the protein by specific si-RNA decreased both size and density of spines. The morphological changes were reflected by the increased amplitude and decreased frequency of miniature EPSCs induced by Rich2 overexpression, while si-RNA treatment decreased both amplitude and frequency of these events. Finally, treatment of neurons with EHT 1864 rescued the phenotype induced by Rich2 knock-down. These results suggested that Rich2 controls dendritic spine morphogenesis and function via inhibition of Rac1.
[Show abstract][Hide abstract] ABSTRACT: Nanocarriers can be useful tools for delivering drugs to the central nervous system (CNS). Their distribution within the brain and their interaction with CNS cells must be assessed accurately before they can be proposed for therapeutic use. In this paper, we investigated these issues by employing poly-lactide-co-glycolide nanoparticles (NPs) specifically engineered with a glycopeptide (g7) conferring to NPs the ability to cross the blood brain barrier (BBB) at a concentration of up to 10% of the injected dose. g7-NPs display increased in vitro uptake in neurons and glial cells. Our results show that in vivo administration of g7-NPs leads to a region- and cell type-specific enrichment of NPs within the brain. We provide evidence that g7-NPs are endocytosed in a clathrin-dependent manner and transported into a specific subset of early endosomes positive for Rab5 in vitro and in vivo. The differential Rab5 expression level is strictly correlated with the amount of g7-NP accumulation. These findings show that g7-NPs can cross the BBB and target specific brain cell populations, suggesting that these NPs can be promising carriers for the treatment of neuropsychiatric and neurodegenerative diseases.