NCAM induces CaMKIIα-mediated RPTPα phosphorylation to enhance its catalytic activity and neurite outgrowth

Receptor protein tyrosine phosphatase α (RPTPα) phosphatase activity is required for intracellular signaling cascades that are activated in motile cells and growing neurites. Little is known, however, about mechanisms that coordinate RPTPα activity with cell behavior. We show that clustering of neural cell adhesion molecule (NCAM) at the cell surface is coupled to an increase in serine phosphorylation and phosphatase activity of RPTPα. NCAM associates with T- and L-type voltage-dependent Ca2+ channels, and NCAM clustering at the cell surface results in Ca2+ influx via these channels and activation of NCAM-associated calmodulin-dependent protein kinase IIα (CaMKIIα). Clustering of NCAM promotes its redistribution to lipid rafts and the formation of a NCAM–RPTPα–CaMKIIα complex, resulting in serine phosphorylation of RPTPα by CaMKIIα. Overexpression of RPTPα with mutated Ser180 and Ser204 interferes with NCAM-induced neurite outgrowth, which indicates that neurite extension depends on NCAM-induced up-regulation of RPTPα activity. Thus, we reveal a novel function for a cell adhesion molecule in coordination of cell behavior with intracellular phosphatase activity

Bovine PrPC directly interacts with αB-crystalline

AbstractWe used a bovine brain cDNA library to perform a yeast two-hybrid assay with bovine mature PrPC as bait. The screening result showed that αB-crystalline interacted with PrPC. The interaction was further evaluated both in vivo and in vitro with different methods, such as immunofluorescent colocalization, native polyacrylamide-gel electrophoresis, and IAsys biosensor assays. The results suggested that αB-crystalline may have the ability to refold denatured prion proteins, and provided first evidence that αB-crystalline is directly associated with prion protein

Transcriptional Mechanisms Involved in Long-term Potentiation of Hippocampal Neurons

Long-term potentiation (LTP), the persistent strengthening of synaptic connections following high frequency stimulation, is a form of synaptic plasticity proposed to underlie memory formation and consolidation. Despite decades of extensive study our understanding of the molecular mechanisms underpinning LTP remains incomplete. Whilst many of the protein components involved in the mechanisms of LTP have been extensively described the long non-protein-coding RNAs (lncRNAs) remain largely unexplored. Expression of lncRNAs is particularly enriched in the mammalian brain where they potentially impact LTP through regulation of epigentetic processes, transcription, mRNA splicing and translation. Characterisation of all the proteins and lncRNAs involved in LTP may elucidate the molecular mechanisms underlying memory formation and consolidation as well as provide greater insight into perturbation of these processes in developmental and neurodegenerative diseases. This study aimed to comprehensively analyse the transcriptome of primary hippocampal neurons, from neonatal mice, undergoing LTP induction in order to identify and quantify the protein-coding genes and ncRNAs expressed during LTP induction. Furthermore, this project aimed to investigate the transcriptomic changes that result from inhibition of LTP through disruption of synaptic adhesion. Analysis of transcriptome sequencing data led to identification of 64 differentially expressed genes, including four unannotated noncoding lincRNAs, across four distinct LTP conditions. Among those genes four distinct expression patterns could be identified. Gene Ontology (GO) analysis identified numerous enriched GO terms including those associated with intracellular signalling, transcriptional and translational regulation, as well as numerous clusters associated with immune response. The novel unannotated transcripts identified in this study were characterised as putative long intervening non-coding RNAs (lincRNAs), two of which demonstrated potential micropeptide expression. Future studies will determine the role and function of these putative lincRNAs in the induction of LTP. Meta-analysis comparing the results of the present study with those of a recent study on LTP induction in rat hippocampal neurons found no common differentially expressed genes

The long-term effects of maternal deprivation on the number and size of inhibitory interneurons in the rat amygdala and nucleus accumbens

IntroductionThere is an increasing evidence supporting the hypothesis that traumatic experiences during early developmental periods might be associated with psychopathology later in life. Maternal deprivation (MD) in rodents has been proposed as an animal model for certain aspects of neuropsychiatric disorders.MethodsTo determine whether early-life stress leads to changes in GABAergic, inhibitory interneurons in the limbic system structures, specifically the amygdala and nucleus accumbens, 9-day-old Wistar rats were exposed to a 24 h MD. On postnatal day 60 (P60), the rats were sacrificed for morphometric analysis and their brains were compared to the control group.ResultsResults show that MD affect GABAergic interneurons, leading to the decrease in density and size of the calcium-binding proteins parvalbumin-, calbindin-, and calretinin-expressing interneurons in the amygdala and nucleus accumbens.DiscussionThis study indicates that early stress in life leads to changes in the number and morphology of the GABAergic, inhibitory interneurons in the amygdala and nucleus accumbens, most probably due to the loss of neurons during postnatal development and it further contributes to understanding the effects of maternal deprivation on brain development

SUMOylation of the MAGUK protein CASK regulates dendritic spinogenesis

Membrane-associated guanylate kinase (MAGUK) proteins interact with several synaptogenesis-triggering adhesion molecules. However, direct evidence for the involvement of MAGUK proteins in synapse formation is lacking. In this study, we investigate the function of calcium/calmodulin-dependent serine protein kinase (CASK), a MAGUK protein, in dendritic spine formation by RNA interference. Knockdown of CASK in cultured hippocampal neurons reduces spine density and shrinks dendritic spines. Our analysis of the time course of RNA interference and CASK overexpression experiments further suggests that CASK stabilizes or maintains spine morphology. Experiments using only the CASK PDZ domain or a mutant lacking the protein 4.1–binding site indicate an involvement of CASK in linking transmembrane adhesion molecules and the actin cytoskeleton. We also find that CASK is SUMOylated. Conjugation of small ubiquitin-like modifier 1 (SUMO1) to CASK reduces the interaction between CASK and protein 4.1. Overexpression of a CASK–SUMO1 fusion construct, which mimicks CASK SUMOylation, impairs spine formation. Our study suggests that CASK contributes to spinogenesis and that this is controlled by SUMOylation

Asociación de S25p-MARCKS con microdominios de membrana e interacción con el citoesqueleto de actina en neuroblastos y neuronas del embrión de pollo

Orientador: Prof. Dra. Cristina Arruti.Tribunal: Dr. Omar Macadar, Dra. Silvia Chifflet, Dra. Adriana Esteve

Genetic Analysis of Dog Congenital Deafness and Herding Behavior

Strong artificial selections of canine morphological and behavioral traits lead to the formation of more than 400 modern dog (Canis familiaris, CFA) breeds within the past 300 years. Most dog breeds are derived from small numbers of founders, and this closed genetic pool within each breed results in the high frequency of occurrence of canine congenital disorders. The majority of these heredopathies share common clinical signs with corresponding human diseases. Therefore, dogs are appropriate spontaneous models for studying human diseases. Congenital deafness can cause both health and welfare problems in dogs, and it is quite prevalent among several dog breeds such as Dalmatian, Australian Cattle Dog, English Setter and Australian Stumpy Tail Cattle Dog (ASCD). However, hearing loss causative or associated genes in these dog breeds are not yet identified. The purpose of the study in Chapter 2 was to identify congenital deafness related genes in ASCD. Three bilateral deaf and one normal hearing ASCDs were whole genome sequenced. The publicly available 722 canine whole genome sequences were also used to investigate potential causative mutations in this study. A case-control genome-wide association study (GWAS) was conducted by setting three deafness affected ASCDs as cases, and one unaffected ASCD and 43 additional herding group dogs were used as controls. The GWAS identified several loci on six chromosomes with potential canine deafness association (CFA3, 8, 17, 23, 28 and 37), and most (7 out of 13) of the significantly associated loci were located within CFA37. The private variants unique to three deaf ASCD were filtered by comparison to 722 canine controls of over 144 modern breeds. Subsequent annotation of these variants was performed, only potentially functional variants were filtered resulting in four remaining missense mutations. A missense mutation in the Kruppel-like factor 7 (KLF7) gene (NC_006619.3: g.15562684G>A; XP_022270984.1: p.Leu173Phe) on CFA37 could be emphasized to be associated considering the variant effect prediction and gene function. KLF7 inner ear expression and a corresponding functional impact in development of inner ear and sensory neurons is known. Further genotyping of the KLF7 variant in 28 affected and 27 normal hearing ASCDs still supported its association with ASCD congenital deafness. Dogs have been selectively bred to intensify the performance abilities in regard to diverse tasks such as herding, hunting or companionship. Finally, modern dog breeds vary diversely in not only morphological but also behavioral traits. GWAS analysis of dog morphological traits using breed standard values have been well studied, and many auspicious genes were identified. However, due to the complexity of dog behavior traits, research progress on this topic is still limited. The study of Chapter 3 was intended to elucidate the candidate genes underlying dog behavior traits including herding, predation, temperament and trainability. The phenotype information of these behavioral traits was obtained from American Kennel Club, which classified dog breeds into seven groups (Herding, Hound, Working, Terrier, Toy, Sporting and Non-sporting) based on the behavior, heritage and historical roles. 268 publicly available dog whole genome sequences of 130 modern breeds were used in this study. Four GWASs were performed to investigate potential candidate genes. Dogs with herding behavior were compared with the other dog categories by GWAS. Candidate neurological genes such as THOC1, ASIC2, MSRB3, LLPH, RFX8 and CHL1 were detected within or nearest to the significant loci of herding GWAS. Regarding dog predation behavior, herding behavior is the modified predatory behavior like repression of killing instinct, while hound dogs were selectively bred to enhance predation behaviors. We then use hound and herding group dogs in GWAS to analyze the dog predation behavior. Three neural genes JAK2, MEIS1 and LRRTM4 that were nearest to the significant loci of predation GWAS were revealed as candidates. In temperament GWAS, candidate neurological gene ACSS3 was significantly associated with dog temperament trait. Dog behaviors were reported to be associated with body mass, so we repeated the four GWASs with incorporating dog breed standard body size as covariates. Similar results except for the significant associations of ASIC2, JAK2 and MEIS1 were observed, while these three candidate genes could contribute to dog behaviors through their effects on dog brain architecture. Linkage disequilibrium (LD) analysis of the herding GWAS significant associated signals were also conducted. Promising neurological processes or cellular components were disclosed in GO analysis of potentially functional private genes of herding dogs. In the study described in Chapter 4, one loss of function mutation in ABHD16B was identified to be associated with bull infertility. However, the exact gene function of ABHD16B remains unknown. Western blot was applied to locate ABHD16B protein expression, uncovering its occurrence in bull testis tissue but not in sperm cells. ABHD16B protein owns a function domain of α/β-hydrolase (ABHD) and several ABHD members are involved in lipid metabolism. It is assumed that ABHD16B could play roles in biosynthesis of sperm membrane lipids. Lipidomes of heterozygous and homozygous wild-type bull sperms were analyzed to explore potential aberrations. Several lipid components including PC, DAG, Cer, SM and PC were found significantly altered which verified our hypothesis. Therefore, the imbalanced lipid homeostasis of sperm membrane could be responsible for the bull infertility problem subjected in this study.2021-10-1

Identification of novel cytosolic binding partners of the neural cell adhesion molecule NCAM and functional analysis of these interactions

The neural cell adhesion molecule (NCAM) plays an important role during brain development and in adult brain. NCAM functions through interactions with several proteins leading to intracellular signal transduction pathways ultimately causing cellular proliferation, differentiation, migration, survival, and neuritogenesis. This thesis aimed for the identification of novel, yet unknown intracellular interaction partners of NCAM to further understand the mechanisms underlying NCAM’s role in the brain. Purified intracellular domains of human NCAM180 or NCAM140 were applied onto a protein macroarray containing 24000 expression clones of human fetal brain. Using this approach, several novel potential interaction partners were detected, including ubiquitin carboxyl-terminal hydrolase isozyme L1, ubiquitin-fold modifier-conjugating enzyme 1, and kinesin light chain 1 (KLC1). KLC1 is part of kinesin-1, a motor protein that transports cargoes towards the plus end of microtubules in axons and dendrites. As the transport mechanism of NCAM in neurons is still unknown, the potential role of kinesin-1 in NCAM trafficking was specifically interesting and analyzed in detail herein. The interaction of NCAM and KLC1 was verified in mouse brain tissue by co-immunoprecipitation. Co-localization studies in Chinese Hamster Ovary (CHO) cells overexpressing NCAM and kinesin-1 and in primary hippocampal neurons revealed an overlap of NCAM with subunits of kinesin-1. Functional studies showed that significantly more NCAM was delivered to the cell surface in NCAM and kinesin-1 overexpressing CHO cells. This effect was inhibited by excess of free full-length intracellular domain of NCAM as well as by several shorter peptides thereof. This showed that the intracellular domain of NCAM is required for the transport of NCAM to the cell surface. Further studies were carried out in primary cortical neurons. Whereas the kinesin-1 dependent transport of NCAM seemed to be mediated constitutively in CHO cells, the amount of cell surface NCAM significantly increased only after antibody-stimulated NCAM endocytosis in primary cortical neurons. In agreement, co-localization of internalized NCAM and KLC1 was observed in these neurons. Finally, an 8 amino acid sequence within the intracellular domain of NCAM was identified in an ELISA to be sufficient to directly interact with KLC1. The KLC1-binding region within NCAM overlaps with the domain responsible for binding to p21-activated kinase 1 (PAK1) which was shown to compete with KLC1 for binding to NCAM in a pull-down assay. This competition may provide a regulatory mechanism for the interaction between NCAM and KLC1 and could potentially be involved in the detachment of NCAM from KLC1 after delivery to the cell surface. Knowledge of the exact transport mechanism of NCAM will contribute to an advanced understanding of the underlying mechanisms of its functions during brain development and in adult brain

Activation of translocator protein by XBD173 ameliorates cognitive deficits and neuropathology in an Alzheimer’s mouse model

One of the most prevalent forms of dementia among elderly patients, Alzheimer's disease (AD) affects millions of people worldwide and brings a huge burden to the individual as well as the global economy. Accumulation of β-amyloid peptide (Aβ) is a major characteristic feature of AD. Previous clinical studies suggest that depression is a common antecedent of AD and may be an early manifestation of dementia, suggesting biological mechanisms that are partly similar in both these disorders. Targeting the molecular mechanism behind these connected disorders can be an excellent therapeutic strategy. The mitochondrial translocator protein (18 kDa) (TSPO) plays an essential role in neurosteroidogenesis and TSPO ligands are neuroprotective in several neurodisorders. We hypothesized that XBD173 since it induces rapid anxiolysis, may have early (neuro) protective effects in AD pathophysiology. Additionally, previous studies concerning XBD173 show a lower side-effect profile compared to benzodiazepines. Both in-vitro electrophysiological recordings, as well as the cognitive performance of the mice, were accessed to unravel the effect of XBD173 on the pathophysiology of AD. First from the CA1-Long Term Potentiation (CA1-LTP) recordings, we observed that 90 min incubation of Aβ1-42 (50 nM) to murine hippocampal slices, prevented the CA1-LTP development after tetanic stimulation of the Schaffer collaterals. Additionally, there was a reduction in the total spine density of CA1 pyramidal neurons. XBD173 (300 nM) restored LTP deficit as well as spine density in the presence of Aβ1-42. XBD173 incubation recovered mushroom and thin spines, as well as overall spine density, as reflected by Imaris dendritic spine rendering. Interestingly, the incubation of XBD173 did not restore the LTP deficit resulting from Aβ1-42 incubation in a global TSPO knockout (KO) mouse model, suggesting a TSPO-mediated action for XBD173. Chronic administration of TSPO-dependent XBD173 (1mg/kg every second day for 3 months) improves the cognitive performance in 9 months ArcAβ (transgenic AD) mice accessed by the water cross maze. Analyzing the brains of these mice showed that chronic XBD173 treatment reduced plaque load (Methoxy-04 staining) and total Aβ1-42 levels (ELISA) in the cortex. Additionally, we found that chronic treatment with XBD173 reduces astrocytic synaptic pruning in the hippocampus and cortex, which in AD mice was exacerbated. Given, the important role of complement proteins in advancing the pathophysiology of AD, we focused on complement protein C1q which acts as an “eat-me” tag for the neurons to be destroyed. We found that astrocytes in transgenic AD mice contain more C1q engulfment compared to the wild-type. Chronic XBD173 treatment reduces this aberrant astrocytic engulfment of C1q tags. It was interesting to observe that amyloid plaques colocalize with C1q aggregates and these C1q aggregates are reduced in XBD173-treated mice compared to their transgenic counterparts. Additionally, we observed that the activation of TSPO by XBD173 in a chronic-treatment model elevates the levels of the neurosteroids including allopregnanolone, dihydrodeoxycorticosterone (DHDOC), and 3ꞵ5α THDOC in the cortex and hippocampus. We further studied whether neurosteroids could potentially be the main players behind the effectiveness of XBD173 treatment in AD. From the CA1-LTP experiment, we found that 3α5α THDOC (100 nM) and 3ꞵ5α THDOC (100 nM), similar to XBD173 (300 nM), restored the LTP deficit in Aβ1-42 treated slices. However, both XBD (300 nM) and 3α5α THDOC (100 nM), could not prevent the CA1-LTP impairments in GABA delta KO mice. These findings highlight a TSPO-mediated increase in neurosteroidogenesis by XBD173, which, upon release, elevates GABAA receptor activity containing the GABA delta subunit. Allopregnanolone (100 nM) prevents the LTP deficits resulting from Aβ1-40 incubation but not Aβ1-42 incubation. Taken together, the present study highlights the beneficial effects of XBD173 against Aβ-derived pathophysiology. Chronic XBD173 treatment improves cognition and appears to have a disease-modifying effect when applied early in the course of AD. This hypothesis is supported by the reduction of soluble Aβ levels, plaque load, and synaptic pruning by XBD173. In conclusion, our work shows that XBD173, in a TSPO-dependent manner provides neuroprotective benefits in a rodent Alzheimer model evident from both the in vitro as well as in vivo behavioral studies. This study paves the way for further advancements in AD treatments and research and provides a possible effective intervention for AD pathophysiology

Bergmann Glia and the Recognition Molecule CHL1 Organize GABAergic Axons and Direct Innervation of Purkinje Cell Dendrites

The geometric and subcellular organization of axon arbors distributes and regulates electrical signaling in neurons and networks, but the underlying mechanisms have remained elusive. In rodent cerebellar cortex, stellate interneurons elaborate characteristic axon arbors that selectively innervate Purkinje cell dendrites and likely regulate dendritic integration. We used GFP BAC transgenic reporter mice to examine the cellular processes and molecular mechanisms underlying the development of stellate cell axons and their innervation pattern. We show that stellate axons are organized and guided towards Purkinje cell dendrites by an intermediate scaffold of Bergmann glial (BG) fibers. The L1 family immunoglobulin protein Close Homologue of L1 (CHL1) is localized to apical BG fibers and stellate cells during the development of stellate axon arbors. In the absence of CHL1, stellate axons deviate from BG fibers and show aberrant branching and orientation. Furthermore, synapse formation between aberrant stellate axons and Purkinje dendrites is reduced and cannot be maintained, leading to progressive atrophy of axon terminals. These results establish BG fibers as a guiding scaffold and CHL1 a molecular signal in the organization of stellate axon arbors and in directing their dendritic innervation