Calcium/calmodulin-dependent Serine Protein Kinase Research Articles

363 A CRISPR/dCas9 Epigenetic Therapuetic Approach for CASK-Related MICPCH

OBJECTIVES/GOALS: CASK-related microcephaly with pontine and cerebellar hypoplasia (MICPCH) is a rare X-linked neurodevelopmental disorder caused by mutations in calcium/calmodulin-dependent serine protein kinase (CASK). We aim to rescue CASK expression via an CRISPR/dCas9 epigenetic therapeutic and create iPSC-based CASK relevant in vitro model systems. METHODS/STUDY POPULATION: As females have two X-chromosomes, disease causing mutations present with a 50/50 expression of mutant and wildtype, due to the mosaicism caused by random X-chromosome inactivation (XCI). This project will adapt an established CRISPR/dCas9 epigenetic approach to rescue expression from the silenced, wild-type CASK allele. We aim to accomplish this through testing different dCas9 orthologues and a guide RNA screen targeting the CASK promoter. Constructs will be tested for optimal targeting efficacy in vitro and assessed via RT-qPCR. Additionally, epigenetic modifications from our approach will be analyzed through bisulfite sequencing. We also aim to apply this epigenetic rescue technology in disease relevant cell lines and eventually in engineered patient mutation iPSC-derived neurons. RESULTS/ANTICIPATED RESULTS: Our results show the ability to target CASK and assess gene expression changes with CRISPR/dCas9 paired with an epigenetic modifier and transcriptional activator. Additionally, our fibroblast model with nonpathogenic single nucleotide polymorphisms within CASKallow for allele specific analysis of our targeted reactivation. We anticipate that following an increase of CASK expression, there would be a decrease in region specific promoter methylation. Further, with the identification of clinically described disease-causing point mutations that result in a loss of function of CASK protein, induction of the mutant sequence onto a healthy cell background will result in a similar reduction of CASK protein in our cell model. DISCUSSION/SIGNIFICANCE: This project will demonstrate the first therapeutic avenue for CASK-related MICPCH, and the potential to utilize targeted X-reactivation as a platform approach for X-linked disorders. Further, investigation of smaller dCas9 orthologues prepares our approach for future translational applications such as packaging into AAV for delivery.

CASK Mediates Oxidative Stress-Induced Microglial Apoptosis-Inducing Factor-Independent Parthanatos Cell Death via Promoting PARP-1 Hyperactivation and Mitochondrial Dysfunction.

Calcium/calmodulin-dependent serine protein kinase (CASK) is a scaffold protein and plays critical roles in neuronal synaptic formation and brain development. Previously, CASK was shown to associate with EGFR to maintain the vulval cell differentiation in C. elegans. In this study, we explored the role of CASK in CHME3 microglial cells. We found that CASK silencing protects cells from H2O2-induced cell death by attenuating PARP-1 activation, mitochondrial membrane potential loss, reactive oxygen species production, and mitochondrial fission, but it increases oxidative phosphorylation. The PARP-1 inhibitor olaparib blocks H2O2-induced cell death, suggesting the death mode of parthanatos. CASK silencing also increases AKT activation but decreases AMPK activation under H2O2 treatment. Pharmacological data further indicate that both signaling changes contribute to cell protection. Different from the canonical parthanatos pathway, we did not observe the AIF translocation from mitochondria into the nucleus, suggesting a non-canonical AIF-independent parthanatos in H2O2-treated CHME3 cells. Moreover, we found that CASK silencing upregulates the EGFR gene and protein expression and increases H2O2-induced EGFR phosphorylation in CHME3 microglia. However, EGFR activation does not contribute to cell protection caused by CASK silencing. In conclusion, CASK plays a crucial role in microglial parthanatos upon H2O2 treatment via stimulation of PARP-1 and AMPK but the inhibition of AKT. These findings suggest that CASK might be an ideal therapeutic target for CNS disorders.

Calcium/Calmodulin-Dependent Serine Protein Kinase (CASK) Gene Polymorphisms in Pigeons

Calcium/calmodulin-dependent serine protein kinase (CASK) is an multidomain protein involved in tissue development and cell signalling. In skeletal muscle, it is involved in the development of neuromuscular junctions. The participation of a pigeon in racing is a great physical effort that causes many changes in the skeletal muscles. Thus, the purpose of the study was to detect the nucleotide sequence variability in the calcium/calmodulin-dependent serine kinase (CASK) gene in domestic pigeons (Columba livia domestica) and assess the potential impact of DNA polymorphisms on the flight performance of pigeons. The research included a total of 517 individuals. DNA was extracted from the blood. A DNA fragment from nucleotides 8689 to 9049 of the CASK (NW_004973256.1 sequence) of six unrelated pigeons were sequenced. One of the detected polymorphic sites (g.8893G > A), located a very close to the start codon, was selected for genotyping in all individuals. The association studies included a total of 311 young homing pigeons that participated in racing competitions. The homing pigeons showed higher frequencies of the AA genotype than non-homing ones (p < 0.05). In rock pigeons only the GG genotype was found. Further research could confirm the functionality of the CASK g.8893G > A SNP in shaping the racing phenotype of pigeons, and the AA genotype could be useful as a selection criterion in pigeon breeding.

CNTNAP2 intracellular domain (CICD) generated by γ-secretase cleavage improves autism-related behaviors

As the most prevalent neurodevelopmental disorders in children, autism spectrum disorders (ASD) are characterized by deficits in language development, social interaction, and repetitive behaviors or inflexible interests. Contactin associated protein like 2 (CNTNAP2), encoding a single transmembrane protein (CNTNAP2) with 1331 amino acid residues, is a widely validated ASD-susceptible gene. Cntnap2-deficient mice also show core autism-relevant behaviors, including the social deficits and repetitive behavior. However, the cellular mechanisms underlying dysfunction CNTNAP2 and ASD remain elusive. In this study, we found a motif within the transmembrane domain of CNTNAP2 was highly homologous to the γ-secretase cleavage site of amyloid-β precursor protein (APP), suggesting that CNTNAP2 may undergo proteolytic cleavage. Further biochemical analysis indicated that CNTNAP2 is cleaved by γ-secretase to produce the CNTNAP2 intracellular domain (CICD). Virally delivery of CICD to the medial prefrontal cortex (mPFC) in Cntnap2-deficient (Cntnap2−/−) mice normalized the deficit in the ASD-related behaviors, including social deficit and repetitive behaviors. Furthermore, CICD promoted the nuclear translocation of calcium/calmodulin-dependent serine protein kinase (CASK) to regulate the transcription of genes, such as Prader Willi syndrome gene Necdin. Whereas Necdin deficiency led to reduced social interaction in mice, virally expression of Necdin in the mPFC normalized the deficit in social preference of Cntnap2−/− mice. Our results thus reveal a critical function of CICD and highlight a role of the CNTNAP2-CASK-Necdin signaling pathway in ASD.

A novel de novo variant in CASK causes a severe neurodevelopmental disorder that masks the phenotype of a novel de novo variant in EEF2.

We report a 9-year-old Spanish boy with severe psychomotor developmental delay, short stature, microcephaly and abnormalities of the brain morphology, including cerebellar atrophy. Whole-exome sequencing (WES) uncovered two novel de novo variants, a hemizygous variant in CASK (Calcium/Calmodulin Dependent Serine Protein Kinase) and a heterozygous variant in EEF2 (Eukaryotic Translation Elongation Factor 2). CASK gene encodes the peripheral plasma membrane protein CASK that is a scaffold protein located at the synapses in the brain. The c.2506-6 A > G CASK variant induced two alternative splicing events that account for the 80% of the total transcripts, which are likely to be degraded by NMD. Pathogenic variants in CASK have been associated with severe neurological disorders such as mental retardation with or without nystagmus also called FG syndrome 4 (FGS4), and intellectual developmental disorder with microcephaly and pontine and cerebellar hypoplasia (MICPCH). Heterozygous variants in EEF2, which encodes the elongation factor 2 (eEF2), have been associated to Spinocerebellar ataxia 26 (SCA26) and more recently to a childhood-onset neurodevelopmental disorder with benign external hydrocephalus. The yeast model system used to investigate the functional consequences of the c.34 A > G EEF2 variant supported its pathogenicity by demonstrating it affects translational fidelity. In conclusion, the phenotype associated with the CASK variant is more severe and masks the milder phenotype of EEF2 variant.

Expanding the natural history of CASK-related disorders to the prenatal period.

To assess whether microcephaly with pontine and cerebellar hypoplasia (MICPCH) could manifest in the prenatal period in patients with calcium/calmodulin-dependent serine protein kinase (CASK) gene disorders. In this international multicentre retrospective study, we contacted a CASK parents' social media group and colleagues with expertise in cerebellar malformations and asked them to supply clinical and imaging information. Centiles and standard deviations (SD) were calculated according to age by nomograms. The study consisted of 49 patients (44 females and 5 males). Information regarding prenatal head circumference was available in 19 patients; 11 out of 19 had a fetal head circumference below -2SD (range -4.1SD to -2.02SD, mean gestational age at diagnosis 20 weeks). Progressive prenatal deceleration of head circumference growth rate was observed in 15 out of 19. At birth, 20 out of 42 had a head circumference below -2SD. A total of 6 out of 15fetuses had a TCD z-score below -2 (range -5.88 to -2.02). This study expands the natural history of CASK-related disorders to the prenatal period, showing evidence of progressive deceleration of head circumference growth rate, head circumference below -2SD, or small TCD. Most cases will not be diagnosed according to current recommendations for fetal central nervous system routine assessment. Consecutive measurements and genetic studies are advised in the presence of progressive deceleration of head circumference growth rates or small TCD. Progressive deceleration of fetal head circumference growth rate can be observed. A small transcerebellar diameter is an additional important manifestation. Most cases will not be diagnosed according to current recommendations for fetal central nervous system routine assessment. Consecutive measurements are advised when measurements are within the low range of norm.

Involvement of membrane palmitoylated protein 2 (MPP2) in the synaptic molecular complex at the mouse cerebellar glomerulus.

We previously reported that the membrane skeletal protein 4.1G in the peripheral nervous system transports membrane palmitoylated protein 6 (MPP6), which interacts with the synaptic scaffolding protein Lin7 and cell adhesion molecule 4 (CADM4) in Schwann cells that form myelin. In the present study, we investigated the localization of and proteins related to MPP2, a highly homologous family protein of MPP6, in the cerebellum of the mouse central nervous system, in which neurons are well organized. Immunostaining for MPP2 was observed at cerebellar glomeruli (CG) in the granular layer after postnatal day 14. Using the high-resolution Airyscan mode of a confocal laser-scanning microscope, MPP2 was detected as a dot pattern and colocalized with CADM1 and Lin7, recognized as small ring/line patterns, as well as with calcium/calmodulin-dependent serine protein kinase (CASK), NMDA glutamate receptor 1 (GluN1), and M-cadherin, recognized as dot patterns, indicating the localization of MPP2 in the excitatory postsynaptic region and adherens junctions of granule cells. An immunoprecipitation analysis revealed that MPP2 formed a molecular complex with CADM1, CASK, M-cadherin, and Lin7. Furthermore, the Lin7 staining pattern showed small rings surrounding mossy fibers in wild-type CG, while it changed to the dot/spot pattern inside small rings detected with CADM1 staining in MPP2-deficient CG. These results indicate that MPP2 influences the distribution of Lin7 to synaptic cell membranes at postsynaptic regions in granule cells at CG, at which electric signals enter the cerebellum.

The Non-Linear Path from Gene Dysfunction to Genetic Disease: Lessons from the MICPCH Mouse Model.

Most human disease manifests as a result of tissue pathology, due to an underlying disease process (pathogenesis), rather than the acute loss of specific molecular function(s). Successful therapeutic strategies thus may either target the correction of a specific molecular function or halt the disease process. For the vast majority of brain diseases, clear etiologic and pathogenic mechanisms are still elusive, impeding the discovery or design of effective disease-modifying drugs. The development of valid animal models and their proper characterization is thus critical for uncovering the molecular basis of the underlying pathobiological processes of brain disorders. MICPCH (microcephaly and pontocerebellar hypoplasia) is a monogenic condition that results from variants of an X-linked gene, CASK (calcium/calmodulin-dependent serine protein kinase). CASK variants are associated with a wide range of clinical presentations, from lethality and epileptic encephalopathies to intellectual disabilities, microcephaly, and autistic traits. We have examined CASK loss-of-function mutations in model organisms to simultaneously understand the pathogenesis of MICPCH and the molecular function/s of CASK. Our studies point to a highly complex relationship between the potential molecular function/s of CASK and the phenotypes observed in model organisms and humans. Here we discuss the implications of our observations from the pathogenesis of MICPCH as a cautionary narrative against oversimplifying molecular interpretations of data obtained from genetically modified animal models of human diseases.

Specific Deletion of CASK in Pancreatic β Cells Affects Glucose Homeostasis and Improves Insulin Sensitivity in Obese Mice by Reducing Hyperinsulinemia Running Title: β Cell CASK Deletion Reduces Hyperinsulinemia.

Calcium/calmodulin-dependent serine protein kinase (CASK) is involved in the secretion of insulin vesicles in pancreatic β-cells. The present study revealed a new in vivo role of CASK in glucose homeostasis during the progression of type 2 diabetes mellitus (T2DM). A Cre-loxP system was used to specifically delete the Cask gene in mouse β-cells (βCASKKO), and the glucose metabolism was evaluated in βCASKKO mice fed a normal chow diet (ND) or a high-fat diet (HFD). ND-fed mice exhibited impaired insulin secretion in response to glucose stimulation. Transmission electron microscopy showed significantly reduced numbers of insulin granules at or near the cell membrane in the islets of βCASKKO mice. By contrast, HFD-fed βCASKKO mice showed reduced blood glucose and a partial relief of hyperinsulinemia and insulin resistance when compared to HFD-fed wildtype mice. The IRS1/PI3K/AKT signaling pathway was upregulated in the adipose tissue of HFD-βCASKKO mice. These results indicated that knockout of the Cask gene in β cells had a diverse effect on glucose homeostasis: reduced insulin secretion in ND-fed mice, but improves insulin sensitivity in HFD-fed mice. Therefore, CASK appears to function in the insulin secretion and contributes to hyperinsulinemia and insulin resistance during the development of obesity-related T2DM.

CASK regulates Notch pathway and functions as a tumor promoter in pancreatic cancer

Calcium/calmodulin-dependent serine protein kinase (CASK), a member of membrane-associated guanylate kinase (MAGUK) super-family, is implicated in regulating cell proliferation, cytoskeletal remodeling, and cell metastasis. Our study aimed to investigate the effect of CASK on the malignant behaviors of pancreatic cancer cells and to determine the signaling pathway involved. CASK expression in pancreatic cancer tissues based on the TCGA database was analyzed using GEPIA online tool. The overall survival (OS) and disease-free survival (DFS) in patients with pancreatic cancer based on CASK expression was also analyzed using GEPIA. KEGG pathway enrichment analysis was used to show the association of 1522 CASK-related genes and signaling pathways. The expression of CASK, Notch1 and Hey1 was detected by Western blot. Cell proliferation, colony number, invasion, and apoptosis were detected by CCK-8, colony formation assay, Transwell invasion assay, and flow cytometry analysis, respectively. Results showed that CASK was upregulated in pancreatic cancer tissues and cells. Pancreatic cancer patients with high CASK expression showed shorter OS and DFS than patients with low CASK expression. KEGG pathway enrichment analysis proved that CASK and 1522 CASK-associated genes were primarily associated with the Notch pathway. CASK silencing inhibited cell proliferation, colony formation ability, and invasion and elicited apoptosis in pancreatic cancer cells. Additionally, we confirmed that CASK silencing inhibited the Notch pathway in pancreatic cancer cells. Overexpression of Notch1 resisted the anti-tumor functions of CASK knockdown in pancreatic cancer cells. In conclusion, CASK knockdown suppressed the malignant behaviors of pancreatic cancer cells by inactivating the Notch pathway.

Solution NMR Structure of the SH3 Domain of Human Caskin1 Validates the Lack of a Typical Peptide Binding Groove and Supports a Role in Lipid Mediator Binding.

SH3 domains constitute an important class of protein modules involved in a variety of cellular functions. They participate in protein-protein interactions via their canonical ligand binding interfaces composed of several evolutionarily conserved aromatic residues forming binding grooves for typical (PxxP) and atypical (PxxxPR, RxxK, RKxxY) binding motifs. The calcium/calmodulin-dependent serine protein kinase (CASK)-interacting protein 1, or Caskin1, a multidomain scaffold protein regulating the cortical actin filaments, is enriched in neural synapses in mammals. Based on its known interaction partners and knock-out animal studies, Caskin1 may play various roles in neural function and it is thought to participate in several pathological processes of the brain. Caskin1 has a single, atypical SH3 domain in which key aromatic residues are missing from the canonical binding groove. No protein interacting partner for this SH3 domain has been identified yet. Nevertheless, we have recently demonstrated the specific binding of this SH3 domain to the signaling lipid mediator lysophospatidic acid (LPA) in vitro. Here we report the solution NMR structure of the human Caskin1 SH3 domain and analyze its structural features in comparison with other SH3 domains exemplifying different strategies in target selectivity. The key differences revealed by our structural study show that the canonical binding groove found in typical SH3 domains accommodating proline-rich motifs is missing in Caskin1 SH3, most likely excluding a bona fide protein target for the domain. The LPA binding site is distinct from the altered protein binding groove. We conclude that the SH3 domain of Caskin1 might mediate the association of Caskin1 with membrane surfaces with locally elevated LPA content.

Cask methylation involved in the injury of insulin secretion function caused by interleukin1-β.

Islet inflammation severely impairs pancreatic β‐cell function, but the specific mechanisms are still unclear. Interleukin1‐β (IL‐1β), an essential inflammatory factor, exerts a vital role in multiple physio‐pathologic processes, including diabetes. Calcium/calmodulin‐dependent serine protein kinase (CASK) is an important regulator especially in insulin secretion process. This study aims to unveil the function of CASK in IL‐1β–induced insulin secretion dysfunction and the possible mechanism thereof. Islets of Sprague‐Dawley (SD) rats and INS‐1 cells stimulated with IL‐1β were utilized as models of chronic inflammation. Insulin secretion function associated with Cask and DNA methyltransferases (DNMT) expression were assessed. The possible mechanisms of IL‐1β‐induced pancreatic β‐cell dysfunction were also explored. In this study, CASK overexpression effectively improved IL‐1β‐induced islet β‐cells dysfunction, increased insulin secretion. DNA methyltransferases and the level of methylation in the promoter region of Cask were elevated after IL‐1β administration. Methyltransferase inhibitor 5‐Aza‐2’‐deoxycytidine (5‐Aza‐dC) and si‐DNMTs partially up‐regulated CASK expression and reversed potassium stimulated insulin secretion (KSIS) and glucose‐stimulated insulin secretion (GSIS) function under IL‐1β treatment in INS‐1 and rat islets. These results reveal a previously unknown effect of IL‐1β on insulin secretion dysfunction and demonstrate a novel pathway for Cask silencing based on activation of DNA methyltransferases via inducible nitric oxide synthase (iNOS) and modification of gene promoter methylation.

CASK, APBA1, and STXBP1 collaborate during insulin secretion

Calcium/calmodulin-dependent serine protein kinase (CASK) knockdown reduces insulin vesicle docking to cell membranes. Here, we explored CASK interactions with other proteins during insulin secretion. Using co-immunoprecipitation, liquid chromatography-mass spectrometry and bioinformatic analysis, we identified that CASK, Adapter protein X11 alpha (APBA1), and Syntaxin binding protein 1 (STXBP1) formed tripartite complex during insulin secretion. CASK enhanced APBA1–STXBP1 interaction and mediated their traffic from cytoplasm to plasma membrane during insulin release. High fatty acid stimulation decreased insulin secretion along with CASK, APBA1, and STXBP1 expression; Cask overexpression enhanced CASK/APBA1/STXBP1 tripartite complex function, and may thereby rescue lipotoxicity-induced insulin-release defects. Collectively, our results illustrated the function of CASK in insulin granules exocytosis, which broadens the underlying mechanism of insulin secretion and highlights the clinical potential of CASK as a drug target of type 2 Diabetes Mellitus (T2DM).

Distinct calcium/calmodulin-dependent serine protein kinase domains control cardiac sodium channel membrane expression and focal adhesion anchoring

Membrane-associated guanylate kinase proteins function as adaptor proteins to mediate the recruitment and scaffolding of ion channels in the plasma membrane in various cell types. In the heart, the protein calcium/calmodulin-dependent serine protein kinase (CASK) negatively regulates the main cardiac sodium channel NaV1.5, which carries the sodium current (INa) by preventing its anterograde trafficking. CASK is also a new member of the dystrophin-glycoprotein complex and, like syntrophin, binds to the C-terminal domain of the channel. The purpose of this study was to unravel the mechanisms of CASK-mediated negative INa regulation and interaction with the dystrophin-glycoprotein complex in cardiac myocytes. CASK adenoviral truncated constructs with sequential single functional domain deletions were designed for overexpression in cardiac myocytes: CASKΔCAMKII, CASKΔL27A, CASKΔL27B, CASKΔPDZ, CASKΔSH3, CASKΔHOOK, and CASKΔGUK. A combination of whole-cell patch-clamp recording, total internal reflection fluorescence microscopy, and biochemistry experiments was conducted in cardiac myocytes to study the functional consequences of domain deletions. We show that both L27B and GUK domains are required for the negative regulatory effect of CASK on INa and NaV1.5 surface expression and that the HOOK domain is essential for interaction with the cell adhesion dystrophin-glycoprotein complex. This study demonstrates that the multimodular structure of CASK confers an ability to simultaneously interact with several targets within cardiomyocytes. Through its L27B, GUK, and HOOK domains, CASK potentially provides the ability to control channel delivery at adhesion points in cardiomyocytes.

Circulating CASK is associated with recurrent focal segmental glomerulosclerosis after transplantation.

IntroductionFocal and Segmental GlomeruloSclerosis (FSGS) can cause nephrotic syndrome with a risk of progression to end-stage renal disease. The idiopathic form has a high rate of recurrence after transplantation, suggesting the presence of a systemic circulating factor that causes glomerular permeability and can be removed by plasmapheresis or protein-A immunoadsorption.ResultsTo identify this circulating factor, the eluate proteins bound on therapeutic immunoadsorption with protein-A columns were analyzed by comparative electrophoresis and mass spectrometry. A soluble form of calcium/calmodulin-dependent serine protein kinase (CASK) was identified. CASK was immunoprecipitated only in the sera of patients with recurrent FSGS after transplantation and not in control patients. Recombinant-CASK (rCASK) induced the reorganization of the actin cytoskeleton in immortalized podocytes, a redistribution of synaptopodin, ZO-1,vinculin and ENA. rCASK also induced alterations in the permeability of a monolayer of podocytes and increased the motility of pdodocytes in vitro. The extracellular domain of CD98, a transmembrane receptor expressed on renal epithelial cells, has been found to co-immunoprecipitated with rCASK. The invalidation of CD98 with siRNA avoided the structural changes of rCask treated cells suggesting its involvement in physiopathology of the disease. In mice, recombinant CASK induced proteinuria and foot process effacement in podocytes.ConclusionOur results suggest that CASK can induce the recurrence of FSGS after renal transplantation.

PPAR-γ activation increases insulin secretion independent of CASK in INS-1 cells.

Peroxisome proliferator-activated receptor-γ (PPAR-γ) is expressed in pancreatic β cells and is involved in insulin secretion. However, the precise mechanisms remain unclear. Calcium/calmodulin-dependent serine protein kinase (CASK), which plays a vital role in the anchoring of insulin granules on pancreatic β cell membrane, is probably a downstream of the transcription factor PPAR-γ. The aim of the present study was to investigate the correlation among PPAR-γ, CASK and insulin secretion. We found that rosiglitazone (RSG) had a positive effect on the expression of CASK and PPAR-γ in INS-1 cells as shown by real-time polymerase chain reaction (PCR) and western blot analysis, but did not change the cellular location of CASK as shown by immunofluorescence assay. Knockdown of PPAR-γ significantly attenuated the mRNA and protein expression levels of CASK. ChIP-qPCR and luciferase assays showed that PPAR-γ bound with the Cask promoter, and promoter activity of Cask was elevated by RSG. RSG significantly enhanced the insulin secretion with potassium stimulation, but did not alter the insulin content as shown by potassium-stimulated insulin secretion assay. In addition, with RSG pretreatment, knockdown of Cask did not significantly affect the PPAR-γ activation-mediated insulin secretion. Moreover, electron microscopy demonstrated that with RSG pretreatment, silence of Cask did not change the number of vesicles anchored on the cell membranes compared with those in siCask-treated cells. Overall, the present study identifies that CASK is one of the PPAR-γ downstream targets and PPAR-γ exerts a positive effect on the expression of CASK in INS-1 cells. PPAR-γ activation increases insulin secretion independent of the upregulation of CASK.

Expression of CASK Antibody in Non-Mucinous Colorectal Adenocarcinoma

AbstractBackground: Colorectal cancers are one of the leading causes of cancer related morbidity and mortality worldwide. In Egypt was 4% in the year 2012 ranking as the sixth cancer in Egypt and representing 53% of GI malignancy. It is obvious that there is no single determining factor for the process of carcinogenesis or spread of colorectal carcinoma. There are many proposed contributing factors, including disturbances in Epithelial-Mesenchymal Transition (EMT) and loss of regulation of adhesion molecules and polarity proteins. Cal-cium/calmodulin-dependent serine protein kinase (CASK) belongs to the Membrane-Associated Guanylate Kinase (MAGUK) family. The role of CASK in Colorectal cancer hasn't been yet fully understood.Aim of Study: To evaluate the expression of Calci-um/calmodulin-dependent serine protein kinase (CASK) in non-mucinous colorectal adenocarcinoma and to evaluate its correlation with variable clinicopathological factors.Material and Methods: It is retrospective descriptive study is conducted in the Department of Pathology, Faculty of Medicine, Suez Canal University Teaching Hospital. It include 42 archived paraffin blocks of non-mucinous colorectal adenocarcinoma of patients who underwent surgical excision without previous chemotherapy or radiation, 15 adenomatous polyps and 5 normal mucosa samples. The clinical and path-ological data of studied patients are taken from medical records, pathology referral reports and pathology reports. Patients with incomplete data excluded from the study. Re-viewing the data of survival from the archived registry files at department of Oncology, Suez Canal University Teaching Hospital. Sections cut into 5um thick sections and mounted on positive charged slides for immunohistochemical staining using CASK Antibody (S56A-50): Mouse Monoclonal, Novus Biologicals, Catalogue no. NBP1-47648. Staining intensity graded according to the Allred score on a 0-3 scale.Data Analysis: The relation between CASK antibody staining intensity in the studied biopsies and other clinico-pathological parameters including (age, gender, histopatho-logical tumor features) was evaluated using Pearson's x2 test. The level of statistical significance (p-value) was set at Results: The study revealed that CASK protein had mainly cytoplasmic and cytoplasmic-membranous pattern of expres-sion. The CASK protein was overexpressed in the majority of CRC samples with 85.7% of cases showing moderate to strong expression, while only 14.3% of cases displayed minimal faint expression. On the other hand, CASK expression was low in the majority (53.3%) of adenoma samples. Addi-tionally, adenomas with high grade dysplasia showed stronger staining intensity for CASK protein and higher percentage of positive cases than the ones with low grade dysplasia by IHC. As regard to the association between CASK protein overex-pression and clinicopathological prognostic factors we found that CASK demonstrated significantly higher expression in tumor samples with early stages (I/II) rather than advanced stage (III/IV). Low grade tumors showed higher percentage of positive cases and stronger intensity of staining for CASK protein than high grade tumors. Expression of CASK protein showed no statistically significant correlation with patients' gender or age respectively. There was also no significant correlation with tumor site, gross tumor size, histologic tumor border configuration, lymphovascular invasion or distant metastasis.Conclusion: CASK protein was overexpressed in the majority of non-mucinous colorectal adenocarcinoma (CRC) cases with mainly cytoplasmic and cytoplasmic-membranous localization. However, its expression was significantly less in adenomas with a possible association with the grade of dysplasia; giving positive pattern in highly dysplastic cases suggesting a role in the progression of adenomas into carci-nomas. CASK overexpression also associated with both TNM stage of tumors and their histologic grade. CASK was signif-icantly overexpressed in early stage and low grade tumors rather than tumors with advanced stage and higher histological grades. This suggests that CASK protein is a good prognostic factor and might contribute in tumor confinement and local-ization.

The CNTNAP2-CASK complex modulates GluA1 subcellular distribution in interneurons

GABAergic interneurons are emerging as prominent substrates in the pathophysiology of multiple neurodevelopmental disorders, including autism spectrum disorders, schizophrenia, intellectual disability, and epilepsy. Interneuron excitatory activity is influenced by 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl) propanoic acid receptors (AMPARs), which in turn affects excitatory transmission in the central nervous system. Yet how dysregulation of interneuronal AMPARs distinctly contributes to the molecular underpinning of neurobiological disease is drastically underexplored. Contactin-associated protein-like 2 (CNTNAP2) is a neurexin-related adhesion molecule shown to mediate AMPAR subcellular distribution while calcium/calmodulin-dependent serine protein kinase (CASK) is a multi-functional scaffold involved with glutamate receptor trafficking. Mutations in both genes have overlapping disease associations, including autism spectrum disorders, intellectual disability, and epilepsy, thus suggesting converging perturbations of excitatory/inhibitory balance. Our lab has previously shown that CNTNAP2 stabilizes interneuron dendritic arbors through CASK and that CNTNAP2 regulates AMPAR subunit GluA1 trafficking in excitatory neurons. The interaction between these three proteins, however, has not been studied in interneurons. Using biochemical techniques, structured illumination microscopy (SIM) and shRNA technology, we first confirm that these three proteins interact in mouse brain, and then examined relationship between CNTNAP2, CASK and GluA1 in mature interneurons. Using SIM, we ascertain that a large fraction of endogenous CNTNAP2, CASK, and GluA1 molecules collectively colocalize together in a tripartite manner. Finally, individual knockdown of either CNTNAP2 or CASK similarly alter GluA1 levels and localization. These findings offer insight to molecular mechanisms underlying GluA1 regulation in interneurons.

Deficiency of calcium/calmodulin-dependent serine protein kinase disrupts the excitatory-inhibitory balance of synapses by down-regulating GluN2B

Calcium/calmodulin-dependent serine protein kinase (CASK) is a membrane-associated guanylate kinase (MAGUK) protein that is associated with neurodevelopmental disorders. CASK is thought to have both pre- and postsynaptic functions, but the mechanism and consequences of its functions in the brain have yet to be elucidated, because homozygous CASK-knockout (CASK-KO) mice die before brain maturation. Taking advantage of the X-chromosome inactivation (XCI) mechanism, here we examined the synaptic functions of CASK-KO neurons in acute brain slices of heterozygous CASK-KO female mice. We also analyzed CASK-knockdown (KD) neurons in acute brain slices generated by in utero electroporation. Both CASK-KO and CASK-KD neurons showed a disruption of the excitatory and inhibitory (E/I) balance. We further found that the expression level of the N-methyl-d-aspartate receptor subunit GluN2B was decreased in CASK-KD neurons and that overexpressing GluN2B rescued the disrupted E/I balance in CASK-KD neurons. These results suggest that the down-regulation of GluN2B may be involved in the mechanism of the disruption of synaptic E/I balance in CASK-deficient neurons.

Distribution of Caskin1 protein and phenotypic characterization of its knockout mice using a comprehensive behavioral test battery

Calcium/calmodulin-dependent serine protein kinase (CASK)-interacting protein 1 (Caskin1) is a direct binding partner of the synaptic adaptor protein CASK. Because Caskin1 forms homo-multimers and binds not only CASK but also other neuronal proteins in vitro, it is anticipated to have neural functions; but its exact role in mammals remains unclear. Previously, we showed that the concentration of Caskin1 in the spinal dorsal horn increases under chronic pain. To characterize this protein, we generated Caskin1-knockout (Caskin1-KO) mice and specific anti-Caskin1 antibodies. Biochemical and immunohistochemical analyses demonstrated that Caskin1 was broadly distributed in the whole brain and spinal cord, and that it primarily localized at synapses. To elucidate the neural function of Caskin1 in vivo, we subjected Caskin1-KO mice to comprehensive behavioral analysis. The mutant mice exhibited differences in gait, enhanced nociception, and anxiety-like behavior relative to their wild-type littermates. In addition, the knockouts exhibited strong freezing responses, with or without a cue tone, in contextual and cued-fear conditioning tests as well as low memory retention in the Barnes Maze test. Taken together, these results suggest that Caskin1 contributes to a wide spectrum of behavioral phenotypes, including gait, nociception, memory, and stress response, in broad regions of the central nervous system.