An N-terminal heterozygous missense CASK mutation is associated with microcephaly and bilateral retinal dystrophy plus optic nerve atrophy.

Heterozygous loss-of-function mutations in the X-linked gene CASK are associated with mental retardation and microcephaly with pontine and cerebellar hypoplasia (MICPCH) and ophthalmological disorders including optic nerve atrophy (ONA) and optic nerve hypoplasia (ONH). Recently, we have demonstrated that CASK(+/-) mice display ONH with 100% penetrance but exhibit no change in retinal lamination or structure. It is not clear if CASK loss-of-function predominantly affects retinal ganglion cells, or if other retinal cells like photoreceptors are also involved. Here, we report a heterozygous missense mutation in the N-terminal calcium/calmodulin-dependent kinase (CaMK) domain of the CASK protein in which a highly conserved leucine is mutated to the cyclic amino acid proline. In silico analysis suggests that the mutation may produce destabilizing structural changes. Experimentally, we observe pronounced misfolding and insolubility of the CASKL209P protein. Interestingly, the remaining soluble mutant protein fails to interact with Mint1, which specifically binds to CASK's CaMK domain, suggesting a mechanism for the phenotypes observed with the CASKL209P mutation. In addition to microcephaly, cerebellar hypoplasia and delayed development, the subject with the L209P mutation also presented with bilateral retinal dystrophy and ONA. Electroretinography indicated that rod photoreceptors are the most prominently affected cells. Our data suggest that the CASK interactions mediated by the CaMK domain may play a crucial role in retinal function, and thus, in addition to ONH, individuals with mutations in the CASK gene may exhibit other retinal disorders, depending on the nature of mutation.

Two microcephaly-associated novel missense mutations in CASK specifically disrupt the CASK-neurexin interaction.

Deletion and truncation mutations in the X-linked gene CASK are associated with severe intellectual disability (ID), microcephaly and pontine and cerebellar hypoplasia in girls (MICPCH). The molecular origin of CASK-linked MICPCH is presumed to be due to disruption of the CASK-Tbr-1 interaction. This hypothesis, however, has not been directly tested. Missense variants in CASK are typically asymptomatic in girls. We report three severely affected girls with heterozygous CASK missense mutations (M519T (2), G659D (1)) who exhibit ID, microcephaly, and hindbrain hypoplasia. The mutation M519T results in the replacement of an evolutionarily invariant methionine located in the PDZ signaling domain known to be critical for the CASK-neurexin interaction. CASKM519T is incapable of binding to neurexin, suggesting a critically important role for the CASK-neurexin interaction. The mutation G659D is in the SH3 (Src homology 3) domain of CASK, replacing a semi-conserved glycine with aspartate. We demonstrate that the CASKG659D mutation affects the CASK protein in two independent ways: (1) it increases the protein's propensity to aggregate; and (2) it disrupts the interface between CASK's PDZ (PSD95, Dlg, ZO-1) and SH3 domains, inhibiting the CASK-neurexin interaction despite residing outside of the domain deemed critical for neurexin interaction. Since heterozygosity of other aggregation-inducing mutations (e.g., CASKW919R) does not produce MICPCH, we suggest that the G659D mutation produces microcephaly by disrupting the CASK-neurexin interaction. Our results suggest that disruption of the CASK-neurexin interaction, not the CASK-Tbr-1 interaction, produces microcephaly and cerebellar hypoplasia. These findings underscore the importance of functional validation for variant classification.

CASK stabilizes neurexin and links it to liprin-α in a neuronal activity-dependent manner.

CASK, a MAGUK family protein, is an essential protein present in the presynaptic compartment. CASK's cellular role is unknown, but it interacts with multiple proteins important for synapse formation and function, including neurexin, liprin-α, and Mint1. CASK phosphorylates neurexin in a divalent ion-sensitive manner, although the functional relevance of this activity is unclear. Here we find that liprin-α and Mint1 compete for direct binding to CASK, but neurexin1ß eliminates this competition, and all four proteins form a complex. We describe a novel mode of interaction between liprin-α and CASK when CASK is bound to neurexin1ß. We show that CASK phosphorylates neurexin, modulating the interaction of liprin-α with the CASK-neurexin1ß-Mint1 complex. Thus, CASK creates a regulatory and structural link between the presynaptic adhesion molecule neurexin and active zone organizer, liprin-α. In neuronal culture, CASK appears to regulate the stability of neurexin by linking it with this multi-protein presynaptic active zone complex.

Type I interferon-sensitive recombinant newcastle disease virus for oncolytic virotherapy.

Newcastle disease virus (NDV), an avian paramyxovirus, is tumor selective and intrinsically oncolytic because of its potent ability to induce apoptosis. Several studies have demonstrated that NDV is selectively cytotoxic to tumor cells but not normal cells due to defects in the interferon (IFN) antiviral responses of tumor cells. Many naturally occurring strains of NDV have an intact IFN-antagonistic function and can still replicate in normal human cells. To avoid potential toxicity issues with NDV, especially in cancer patients with immunosuppression, safe NDV-oncolytic vectors are needed. We compared the cell killing abilities of (i) a recombinant NDV (rNDV) strain, Beaudette C, containing an IFN-antagonistic, wild-type V protein (rBC), (ii) an isogenic recombinant virus with a mutant V protein (rBC-Edit virus) that induces increased IFN in infected cells and whose replication is restricted in normal human cells, and (iii) a recombinant LaSota virus with a virulent F protein cleavage site that is as interferon sensitive as rBC-Edit virus (LaSota V.F. virus). Our results indicated that the tumor-selective replication of rNDV is determined by the differential regulation of IFN-alpha and downstream antiviral genes induced by IFN-alpha, especially through the IRF-7 pathway. In a nude mouse model of human fibrosarcoma, we show that the IFN-sensitive NDV variants are as effective as IFN-resistant rBC virus in clearing the tumor burden. In addition, mice treated with rNDV exhibited no signs of toxicity to the viruses. These findings indicate that augmentation of innate immune responses by NDV results in selective oncolysis and offer a novel and safe virotherapy platform.

Significance of heme oxygenase-1(HMOX1) gene on fetal hemoglobin induction in sickle cell anemia patients.

Though the patients with sickle cell anemia (SCA) inherit same genetic mutation, they show considerable phenotypic heterogeneity. It has been observed that patients with elevated fetal hemoglobin (HbF) levels have a relatively mild clinical course. There is sparse literature on the association of higher HbF levels leading to reduction in the oxidative stress in SCA patients. Hence in this study, the significance between the HMOX1 gene polymorphisms and the HbF levels has been studied. Preliminary screening was carried out. Genotyping of 3 variants in the HMOX1 gene was performed in 90 SCA patients and 50 healthy controls by PCR-RFLP, GeneScan and direct DNA sequencing. It was observed that SCA patients with higher HbF levels, showed improved hematological indices with an inverse effect on HbS levels. The TT genotype of rs2071746 (A→T) polymorphism was found to be associated with elevated HbF levels (P: 0.012). Also, the long form (> 25 GT repeats) of rs3074372 (GT)n repeats was found to be linked with increased HbF levels. We could not find any association of rs2071749 (A→G) polymorphism with the HbF levels. As, the sickle cell anemia patients show significant oxidative stress due to hemolysis, the study of polymorphisms in the HMOX1 gene may act as a potential independent marker for elevated HbF levels.

Internalization of scavenger receptor ligands by cortical neurons.

Scavenger receptors (SRs) are a family of receptors displaying affinity for a wide variety of ligands including modified lipoproteins. SRs may play a range of physiological functions including intracellular transport, lipid transport and pathogen clearance. The role of SRs has been documented in pathologies such as atherosclerosis and Alzheimer's disease. Although most studies on SRs have focused on macrophages, they are also present in other cells like endothelium, smooth muscles and brain tissue. Within brain, due to its functional similarity, SRs have been studied mostly in microglia. However, in situ images from Allen's brain atlas suggest SRs are abundant in neurons. In this study we have used two fluorophore labeled well characterized SR ligand, maleylated-BSA (MBSA) and polyguanylic acid (poloyG) to probe acute cortical slices. Our data indicate that within cortex, neurons avidly endocytose both ligands. Thus in cerebral cortex neurons may have higher number of functional SRs on the surface than other cell-types.

Correction: Central Presynaptic Terminals Are Enriched in ATP but the Majority Lack Mitochondria.

[This corrects the article DOI: 10.1371/journal.pone.0125185.].

Analysis of Brain Mitochondria Using Serial Block-Face Scanning Electron Microscopy.

Human brain is a high energy consuming organ that mainly relies on glucose as a fuel source. Glucose is catabolized by brain mitochondria via glycolysis, tri-carboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) pathways to produce cellular energy in the form of adenosine triphosphate (ATP). Impairment of mitochondrial ATP production causes mitochondrial disorders, which present clinically with prominent neurological and myopathic symptoms. Mitochondrial defects are also present in neurodevelopmental disorders (e.g. autism spectrum disorder) and neurodegenerative disorders (e.g. amyotrophic lateral sclerosis, Alzheimer's and Parkinson's diseases). Thus, there is an increased interest in the field for performing 3D analysis of mitochondrial morphology, structure and distribution under both healthy and disease states. The brain mitochondrial morphology is extremely diverse, with some mitochondria especially those in the synaptic region being in the range of <200 nm diameter, which is below the resolution limit of traditional light microscopy. Expressing a mitochondrially-targeted green fluorescent protein (GFP) in the brain significantly enhances the organellar detection by confocal microscopy. However, it does not overcome the constraints on the sensitivity of detection of relatively small sized mitochondria without oversaturating the images of large sized mitochondria. While serial transmission electron microscopy has been successfully used to characterize mitochondria at the neuronal synapse, this technique is extremely time-consuming especially when comparing multiple samples. The serial block-face scanning electron microscopy (SBFSEM) technique involves an automated process of sectioning, imaging blocks of tissue and data acquisition. Here, we provide a protocol to perform SBFSEM of a defined region from rodent brain to rapidly reconstruct and visualize mitochondrial morphology. This technique could also be used to provide accurate information on mitochondrial number, volume, size and distribution in a defined brain region. Since the obtained image resolution is high (typically under 10 nm) any gross mitochondrial morphological defects may also be detected.

X-linked intellectual disability gene CASK regulates postnatal brain growth in a non-cell autonomous manner.

The phenotypic spectrum among girls with heterozygous mutations in the X-linked intellectual disability (XLID) gene CASK (calcium/calmodulin-dependent serine protein kinase) includes postnatal microcephaly, ponto-cerebellar hypoplasia, seizures, optic nerve hypoplasia, growth retardation and hypotonia. Although CASK knockout mice were previously reported to exhibit perinatal lethality and a 3-fold increased apoptotic rate in the brain, CASK deletion was not found to affect neuronal physiology and their electrical properties. The pathogenesis of CASK associated disorders and the potential function of CASK therefore remains unknown. Here, using Cre-LoxP mediated gene excision experiments; we demonstrate that deleting CASK specifically from mouse cerebellar neurons does not alter the cerebellar architecture or function. We demonstrate that the neuron-specific deletion of CASK in mice does not cause perinatal lethality but induces severe recurrent epileptic seizures and growth retardation before the onset of adulthood. Furthermore, we demonstrate that although neuron-specific haploinsufficiency of CASK is inconsequential, the CASK mutation associated human phenotypes are replicated with high fidelity in CASK heterozygous knockout female mice (CASK ((+/-))). These data suggest that CASK-related phenotypes are not purely neuronal in origin. Surprisingly, the observed microcephaly in CASK ((+/-)) animals is not associated with a specific loss of CASK null brain cells indicating that CASK regulates postnatal brain growth in a non-cell autonomous manner. Using biochemical assay, we also demonstrate that CASK can interact with metabolic proteins. CASK knockdown in human cell lines cause reduced cellular respiration and CASK ((+/-)) mice display abnormalities in muscle and brain oxidative metabolism, suggesting a novel function of CASK in metabolism. Our data implies that some phenotypic components of CASK heterozygous deletion mutation associated disorders represent systemic manifestation of metabolic stress and therefore amenable to therapeutic intervention.

Central presynaptic terminals are enriched in ATP but the majority lack mitochondria.

Synaptic neurotransmission is known to be an energy demanding process. At the presynapse, ATP is required for loading neurotransmitters into synaptic vesicles, for priming synaptic vesicles before release, and as a substrate for various kinases and ATPases. Although it is assumed that presynaptic sites usually harbor local mitochondria, which may serve as energy powerhouse to generate ATP as well as a presynaptic calcium depot, a clear role of presynaptic mitochondria in biochemical functioning of the presynapse is not well-defined. Besides a few synaptic subtypes like the mossy fibers and the Calyx of Held, most central presynaptic sites are either en passant or tiny axonal terminals that have little space to accommodate a large mitochondrion. Here, we have used imaging studies to demonstrate that mitochondrial antigens poorly co-localize with the synaptic vesicle clusters and active zone marker in the cerebral cortex, hippocampus and the cerebellum. Confocal imaging analysis on neuronal cultures revealed that most neuronal mitochondria are either somatic or distributed in the proximal part of major dendrites. A large number of synapses in culture are devoid of any mitochondria. Electron micrographs from neuronal cultures further confirm our finding that the majority of presynapses may not harbor resident mitochondria. We corroborated our ultrastructural findings using serial block face scanning electron microscopy (SBFSEM) and found that more than 60% of the presynaptic terminals lacked discernible mitochondria in the wild-type mice hippocampus. Biochemical fractionation of crude synaptosomes into mitochondria and pure synaptosomes also revealed a sparse presence of mitochondrial antigen at the presynaptic boutons. Despite a low abundance of mitochondria, the synaptosomal membranes were found to be highly enriched in ATP suggesting that the presynapse may possess alternative mechanism/s for concentrating ATP for its function. The potential mechanisms including local glycolysis and the possible roles of ATP-binding synaptic proteins such as synapsins, are discussed.

Identification and glycerol-induced correction of misfolding mutations in the X-linked mental retardation gene CASK.

The overwhelming amount of available genomic sequence variation information demands a streamlined approach to examine known pathogenic mutations of any given protein. Here we seek to outline a strategy to easily classify pathogenic missense mutations that cause protein misfolding and are thus good candidates for chaperone-based therapeutic strategies, using previously identified mutations in the gene CASK. We applied a battery of bioinformatics algorithms designed to predict potential impact on protein structure to five pathogenic missense mutations in the protein CASK that have been shown to underlie pathologies ranging from X-linked mental retardation to autism spectrum disorder. A successful classification of the mutations as damaging was not consistently achieved despite the known pathogenicity. In addition to the bioinformatics analyses, we performed molecular modeling and phylogenetic comparisons. Finally, we developed a simple high-throughput imaging assay to measure the misfolding propensity of the CASK mutants in situ. Our data suggests that a phylogenetic analysis may be a robust method for predicting structurally damaging mutations in CASK. Mutations in two evolutionarily invariant residues (Y728C and W919R) exhibited a strong propensity to misfold and form visible aggregates in the cytosolic milieu. The remaining mutations (R28L, Y268H, and P396S) showed no evidence of aggregation and maintained their interactions with known CASK binding partners liprin-α3 Mint-1, and Veli, indicating an intact structure. Intriguingly, the protein aggregation caused by the Y728C and W919R mutations was reversed by treating the cells with a chemical chaperone (glycerol), providing a possible therapeutic strategy for treating structural mutations in CASK in the future.