Abstract
Recent advances in the genetics of neurodevelopmental disorders (NDDs) have identified the transcription factor
FOXP2
as one of numerous risk genes, e.g. in autism spectrum disorders (ASD) ...and attention-deficit/hyperactivity disorder (ADHD). FOXP2 function is suggested to be involved in GABAergic signalling and numerous studies demonstrate that GABAergic function is altered in NDDs, thus disrupting the excitation/inhibition balance. Interestingly, GABAergic signalling components, including glutamate-decarboxylase 1 (Gad1) and GABA receptors, are putative transcriptional targets of FOXP2. However, the specific role of FOXP2 in the pathomechanism of NDDs remains elusive. Here we test the hypothesis that Foxp2 affects behavioural dimensions via GABAergic signalling using zebrafish as model organism. We demonstrate that
foxp2
is expressed by a subset of GABAergic neurons located in brain regions involved in motor functions, including the subpallium, posterior tuberculum, thalamus and medulla oblongata. Using CRISPR/Cas9 gene-editing we generated a novel
foxp2
zebrafish loss-of-function mutant that exhibits increased locomotor activity. Further, genetic and/or pharmacological disruption of Gad1 or GABA-A receptors causes increased locomotor activity, resembling the phenotype of
foxp2
mutants. Application of muscimol, a GABA-A receptor agonist, rescues the hyperactive phenotype induced by the
foxp2
loss-of-function. By reverse translation of the therapeutic effect on hyperactive behaviour exerted by methylphenidate, we note that application of methylphenidate evokes different responses in wildtype compared to
foxp2
or
gad1b
loss-of-function animals. Together, our findings support the hypothesis that
foxp2
regulates locomotor activity via GABAergic signalling. This provides one targetable mechanism, which may contribute to behavioural phenotypes commonly observed in NDDs.
The fatal neurodegenerative disorders amyotrophic lateral sclerosis and spinal muscular atrophy are, respectively, the most common motoneuron disease and genetic cause of infant death. Various in ...vitro model systems have been established to investigate motoneuron disease mechanisms, in particular immortalized cell lines and primary neurons. Using quantitative mass-spectrometry-based proteomics, we compared the proteomes of primary motoneurons to motoneuron-like cell lines NSC-34 and N2a, as well as to non-neuronal control cells, at a depth of 10,000 proteins. We used this resource to evaluate the suitability of murine in vitro model systems for cell biological and biochemical analysis of motoneuron disease mechanisms. Individual protein and pathway analysis indicated substantial differences between motoneuron-like cell lines and primary motoneurons, especially for proteins involved in differentiation, cytoskeleton, and receptor signaling, whereas common metabolic pathways were more similar. The proteins associated with amyotrophic lateral sclerosis also showed distinct differences between cell lines and primary motoneurons, providing a molecular basis for understanding fundamental alterations between cell lines and neurons with respect to neuronal pathways with relevance for disease mechanisms. Our study provides a proteomics resource for motoneuron research and presents a paradigm of how mass-spectrometry-based proteomics can be used to evaluate disease model systems.
Abstract Amyotrophic lateral sclerosis (ALS), the major form of motor neuron disease in the adult occurs as a sporadic disease in more than 95% of all cases. Analysis of familial forms is considered ...as a key to understand the pathophysiology of the disease. It is expected that mutations responsible for familial forms are also found in sporadic ALS. During the past years, several loci and genes have been identified in which disease associated mutations have been discovered. We report here on the screening of 596 sporadic ALS patients, 41 familial ALS cases and other motor neuron disease patients from Germany for mutations in the FUS/TLS gene. Sequencing of the last two exons in all patients revealed the C1561T transversion, which leads to the amino acid substitution at R521C, in one familial and one sporadic ALS patient. In addition three patients with a synonymous mutation at codon 522 were identified. None of these variants were present in the control population. Our results indicate that mutations in FUS/TLS are not a major cause of sporadic ALS in the German population.
Members of the family of metabotropic glutamate receptors are involved in the pathomechanism of several disorders of the nervous system. Besides the well-investigated function of dysfunctional ...glutamate receptor signaling in neurodegenerative diseases, neurodevelopmental disorders (NDD), like autism spectrum disorders (ASD) and attention-deficit and hyperactivity disorder (ADHD) might also be partly caused by disturbed glutamate signaling during development. However, the underlying mechanism of the type III metabotropic glutamate receptor 8 (mGluR8 or GRM8) involvement in neurodevelopment and disease mechanism is largely unknown. Here we show that the expression pattern of the two orthologs of human
GRM8
,
grm8a
and
grm8b
, have evolved partially distinct expression patterns in the brain of zebrafish (
Danio rerio
), especially at adult stages, suggesting sub-functionalization of these two genes during evolution. Using double
in situ
hybridization staining in the developing brain we demonstrate that
grm8a
is expressed in a subset of
gad1a
-positive cells, pointing towards glutamatergic modulation of GABAergic signaling. Building on this result we generated loss-of-function models of both genes using CRISPR/Cas9. Both mutant lines are viable and display no obvious gross morphological phenotypes making them suitable for further analysis. Initial behavioral characterization revealed distinct phenotypes in larvae. Whereas
grm8a
mutant animals display reduced swimming velocity,
grm8b
mutant animals show increased thigmotaxis behavior, suggesting an anxiety-like phenotype. We anticipate that our two novel metabotropic glutamate receptor 8 zebrafish models may contribute to a deeper understanding of its function in normal development and its role in the pathomechanism of disorders of the central nervous system.
Cultured spinal motoneurons are a valuable tool for studying the basic mechanisms of axon and dendrite growth and also for analyses of pathomechanisms underlying diseases like amyotrophic lateral ...sclerosis (ALS) and spinal muscular atrophy (SMA). As motoneurons in the developing spinal cord of mice constitute only a minor population of neurons, these cells need to be enriched in order to study them in the absence of contaminating neuronal and non-neuronal cells. Here, we describe a protocol for the isolation and in vitro cultivation of embryonic primary motoneurons from individual mouse embryos. Tissue dissection, cell isolation and a p75(NTR)-antibody-based panning technique, which highly enriches motoneurons within <8 h are described. This protocol is aimed to provide an alternative to the established FACS-based protocols describing p75(NTR)-based enrichments of neurons. This protocol will help in facilitating the research on molecular mechanisms underlying motoneuron development, survival and disease mechanisms.
Alterations in fear learning/generalization are considered to be relevant mechanisms engendering the development of anxiety disorders being the most prevalent mental disorders. Although anxiety ...disorders almost exclusively have their first onset in childhood and adolescence, etiological research focuses on adult individuals. In this study, we evaluated findings of a recent meta-analysis of genome-wide association studies in adult anxiety disorders with significant associations of four single nucleotide polymorphisms (SNPs) in a large cohort of 347 healthy children (8–12 years) characterized for dimensional anxiety. We investigated the modulation of anxiety parameters by these SNPs in a discriminative fear conditioning and generalization paradigm in the to-date largest sample of children. Results extended findings of the meta-analysis showing a genomic locus on 2p21 to modulate anxious personality traits and arousal ratings. These SNPs might, thus, serve as susceptibility markers for a shared risk across pathological anxiety, presumably mediated by alterations in arousal.
Vacuolar-vesicular protein sorting (Vps) factors are involved in vesicular trafficking in eukaryotic cells. We identified the missense mutation L967Q in Vps54 in the wobbler mouse, an animal model of ...amyotrophic lateral sclerosis, and also characterized a lethal allele, Vps54β-geo. Motoneuron survival and spermiogenesis are severely compromised in the wobbler mouse, indicating that Vps54 has an essential role in these processes.
The transport of glucose across the cell plasma membrane is vital to most mammalian cells. The glucose transporter (GLUT; also called SLC2A) family of transmembrane solute carriers is responsible for ...this function
. GLUT proteins encompass 14 different isoforms in humans with different cell type-specific expression patterns and activities. Central to glucose utilization and delivery in the brain is the neuronally expressed GLUT3. Recent research has shown an involvement of GLUT3 genetic variation or altered expression in several different brain disorders, including Huntington's and Alzheimer's diseases. Furthermore,
was identified as a potential risk gene for multiple psychiatric disorders. To study the role of GLUT3 in brain function and disease a more detailed knowledge of its expression in model organisms is needed. Zebrafish (
) has in recent years gained popularity as a model organism for brain research and is now well-established for modeling psychiatric disorders. Here, we have analyzed the sequence of GLUT3 orthologs and identified two paralogous genes in the zebrafish,
and
. Interestingly, the Glut3b protein sequence contains a unique stretch of amino acids, which may be important for functional regulation. The
transcript is detectable in the central nervous system including distinct cellular populations in telencephalon, diencephalon, mesencephalon and rhombencephalon at embryonic and larval stages. Conversely, the
transcript shows a rather diffuse expression pattern at different embryonic stages and brain regions. Expression of
is maintained in the adult brain and is found in the telencephalon, diencephalon, mesencephalon, cerebellum and medulla oblongata. The
transcripts are present in overlapping as well as distinct regions compared to
. Double
hybridizations were used to demonstrate that
is expressed by some GABAergic neurons at embryonic stages. This detailed description of zebrafish
and
expression at developmental and adult stages paves the way for further investigations of normal GLUT3 function and its role in brain disorders.
Intronic hexanucleotide expansions in C9ORF72 are common in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia, but it is unknown whether loss of function, toxicity by the expanded RNA ...or dipeptides from non-ATG-initiated translation are responsible for the pathophysiology. We determined the interactome of C9ORF72 in motor neurons and found that C9ORF72 was present in a complex with cofilin and other actin binding proteins. Phosphorylation of cofilin was enhanced in C9ORF72-depleted motor neurons, in patient-derived lymphoblastoid cells, induced pluripotent stem cell-derived motor neurons and post-mortem brain samples from ALS patients. C9ORF72 modulates the activity of the small GTPases Arf6 and Rac1, resulting in enhanced activity of LIM-kinases 1 and 2 (LIMK1/2). This results in reduced axonal actin dynamics in C9ORF72-depleted motor neurons. Dominant negative Arf6 rescues this defect, suggesting that C9ORF72 acts as a modulator of small GTPases in a pathway that regulates axonal actin dynamics.
The chromosome 9p21 amyotrophic lateral sclerosis-frontotemporal dementia (ALS-FTD) locus contains one of the last major unidentified autosomal-dominant genes underlying these common ...neurodegenerative diseases. We have previously shown that a founder haplotype, covering the MOBKL2b, IFNK, and C9ORF72 genes, is present in the majority of cases linked to this region. Here we show that there is a large hexanucleotide (GGGGCC) repeat expansion in the first intron of C9ORF72 on the affected haplotype. This repeat expansion segregates perfectly with disease in the Finnish population, underlying 46.0% of familial ALS and 21.1% of sporadic ALS in that population. Taken together with the D90A SOD1 mutation, 87% of familial ALS in Finland is now explained by a simple monogenic cause. The repeat expansion is also present in one-third of familial ALS cases of outbred European descent, making it the most common genetic cause of these fatal neurodegenerative diseases identified to date.
► A GGGGCC hexanucleotide repeat expansion causes chromosome 9p21 FTD/ALS ► The GGGGCC repeat expansion accounts for nearly half of familial ALS in Finland ► The GGGGCC repeat expansion accounts for more than 1/3 of familial ALS in Europe ► The repeat expansion is the most common cause of ALS and FTD identified to date
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