- Open Access
X-linked intellectual disability gene CASK regulates postnatal brain growth in a non-cell autonomous manner
© Srivastava et al. 2016
Received: 4 March 2016
Accepted: 7 March 2016
Published: 31 March 2016
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.
CASK is an evolutionarily conserved gene which encodes for a member of the membrane-associated guanylate kinase (MAGUK) protein family . In mammals CASK was discovered due to its ability to bind to the cytosolic tail of neuronal adhesion molecules neurexins and therefore is primarily identified as a scaffolding protein at the neuronal synapse . However, CASK deletion does not alter synapse formation in C. elegans , Drosophila  or mouse . CASK ortholog lin-2 was identified in C. elegans as early as 1980 and was found in screens for cell lineage specificity rather than synaptic function [16, 24]. Although CASK is essential for survival in mouse, detailed electrophysiological analysis on CASK null mice failed to uncover changes in core neuronal functions such as membrane excitability, calcium-dependent pre-synaptic release or post-synaptic receptor organization . In fact, CASK evolved before the emergence of the nervous system [31, 43] and is present in tissues from all three germ layers in mammals [21, 60]. Within the brain, CASK is expressed by neurons and non-neuronal cells such as oligodendrocytes  and astrocytes . The phylogenetic data and tissue distribution of CASK, together with the phenotypes of CASK-mutated animal models, strongly suggest that CASK may have an important but currently ill-defined non-synaptic function(s). In fact, CASK has been noted to play a role in wide variety of cellular functions including transcription regulation , insulin signaling and secretion [67, 72], and cancer biology .
Subjects with mutations in CASK exhibit autistic traits, intellectual disability , Ohtahara syndrome , infantile spasms , FG syndrome , mental retardation and microcephaly with pontine and cerebellar hypoplasia (MICPCH) [45, 47, 64]. In addition, CASK mutations are associated with growth retardation, optic nerve hypoplasia/ atrophy, epilepsy, sensorineural deafness and hypotonia often resulting in scoliosis [8, 42]. This symptomatology is strikingly similar to that of metabolic diseases . Mitochondrial respiratory chain defects and mutations in mitochondrial proteins are often associated with cerebellar hypoplasia [14, 34]. Mitochondrial diseases frequently affect the optic nerve and auditory sensory circuits (reviewed in [9, 55]). Moreover, epilepsy and hypotonia are primary manifestations in mitochondrial encephalopathies [30, 61]. Syndromes associated with CASK mutations, e.g. Ohtahara syndrome, can also arise from respiratory chain defects [10, 69]. Recently, we found that CASK interacts with mitochondrial proteins in Drosophila melanogaster, pointing to a potential role of CASK in the regulation of metabolism .
A large component of CASK-associated pathology develops postnatally, which has been reported by some authors as postnatal microcephaly or even atrophy . Consistent with this idea, the brain of CASK null mice is normal at birth and properly laminated, and no defect in synapse formation is detectable ; deletion of CASK does, however, lead to an increased apoptotic rate in the mouse brain by 3-folds , which might easily explain the postnatal microcephaly. Since CASK constitutive knockout mice die within a few hours after birth, further analysis has not been possible.
Here, we demonstrate that the cerebellar hypoplasia caused by CASK heterozygous deletion mutation is not due to the loss of CASK gene specifically in cerebellar neurons. In fact, some of the MICPCH phenotypes may be primarily due to a non-neuronal function of CASK. Mice that are heterozygous for CASK deletion in neurons appear normal, but the constitutive whole-body heterozygous knockout (CASK (+/-)) female mice phenocopy the human disease state, indicating that MICPCH occurs due to an overall haploinsufficiency of the CASK gene. Surprisingly, the observed microcephaly in CASK (+/-) mice is not simply due to loss of only the CASK null cells i.e. it occurs in a non-cell autonomous manner. We further demonstrate that CASK interacts with mitochondrial proteins and that CASK (+/-) mice exhibit metabolic defects. Knocking down CASK expression in a human cell line recapitulates these defects. These findings indicate that mammalian CASK has a novel role in metabolic regulation which is crucial for postnatal brain growth in mammals.
Materials and methods
Generation of CASK (+/-) heterozygous and CASK neuronal knockout mice
CASK (+/floxed) female mice were crossed with male mice carrying a transgenic Cre recombinase controlled by the zona pellucida 3 promoter . The F1 generation offsprings were genotyped to identify female mice carrying the Cre recombinase and the floxed CASK gene. These female mice were crossed with wild-type C57Bl6 male mice to obtain female CASK (+/-) mice. Crossings were continued with wild-type male mice to eliminate the Cre transgene. Colonies were maintained as CASK (+/-) females. CASK neuronal knockout mice were generated by crossing the CASK (+/floxed) female mice with a mouse line expressing Cre recombinase under the control of synapsin 1 promoter . Male mice from the F1 generation bearing the Cre transgene and floxed CASK were analyzed. A table providing information on all mice used is shown in Additional file 1: Figure S1A.
Generation of CASK knockdown cells
HEK 293 cells were transduced with PLKO lentiviral particles carrying CASK shRNA or empty lentiviral particles. Experiments were performed 96 h post-transduction. Cell homogenates were analyzed for CASK expression using quantitative immunoblotting.
Mitochondrial respiration measurements
Mitochondrial oxygen consumption was measured in whole brain homogenates using the conventional Clark electrode assay as previously described . Briefly, total respiration was measured in a buffer containing 0.3 M mannitol, 10 mM KCl, 5 mM MgCl2, 10 mM KH2PO4 and 1 mg/ml BSA (pH 7.4) in a water-jacketed cell magnetically stirred at 37 °C (Hansatech instruments, Norfolk, UK). Oxygen consumption rates were measured both in the presence and absence of potassium cyanide (KCN) to assess the rate of KCN sensitive respiration.Oxygen consumption measurements from HEK 293 cells were done as previously described [58, 59]. Briefly, HEK 293 cells were trypsinized and harvested in a sucrose-containing buffer (25 mM Tris–HCl, 10 mM K2HPO4 and 150 mM sucrose (pH 7.4)). Oxygen consumption in cell suspensions were measured in a water-jacketed cell magnetically stirred at 37 °C (Hansatech instruments, Norfolk, UK).
Samples were separated by 10 % SDS-PAGE, transferred to nitrocellulose, blocked in 5 % skimmed milk for 2 h and incubated with primary antibody for 1 h followed by incubation with secondary antibody (1:5000 dilution) for 30 min at room temperature. The chemiluminescent signal was detected via the enzymatic reaction using ECL detection reagents (Amersham) and visualized on ChemiDoc (Biorad). For quantitative immunodetection, the blots were incubated with a fluorescent secondary antibody (Alexa 488) for 30 min at room temperature. The primary antibodies used included anti-ATP synthase subunit β (MitoSciences MS503, 1:1000), anti-synaptophysin (Sigma, 1:1000), anti-tubulin (DSHB, 1:1000), anti-CASK (Neuromab, 1:1000), and anti IDH (AssayBiotech, 1:1000).
Energy expenditure and respiratory exchange ratio measurements
The energy expenditure and respiratory exchange ratios were measured over 48 h using the indirect calorimetry (TSE Systems, Chesterfield, MO). Energy expenditure data were expressed relative to free fat mass and presented as mean ± SEM. The TSE system is equipped with beams pointing in the horizontal (X,Y) and vertical (Z) directions. As mice break these beams, the LabMaster software sums this movement, which is translated into cage activity expressed as meters/hr.
All protein quantitations were done using the Coomassie Bradford reagent from Biorad, following the manufacturer’s instruction.
Brain sectioning and immunofluorescent staining
Animals were deeply anesthetized to avoid any pain and sacrificed mechanically either by decapitation or exsanguination. All animal procedures were performed in accordance with the Virginia Tech guidelines for use and care of laboratory animals. Three-month-old mature adult mice were deeply anaesthetized using isoflurane, and mice hearts were cannulated and perfused with PBS (exsanguination) followed by 4 % paraformaldehyde (PFA). The perfused mice were decapitated and brains were dissected out and fixed in 4 % PFA overnight. Brains were cryopreserved by incubation in 30 % sucrose solution for 48 h. For sectioning, brains were embedded in CryoTek™ and 20 μm-thick cortical sections were generated using a cryostat (IEC). The cortical sections were immunostained as floating sections. Sections were permeabilized with 0.025 % Triton X-100 followed by blocking with 5 % goat serum. Sections were stained with primary antibodies for two hours followed by secondary antibody (1:500) incubation for 30 min. Finally, sections were mounted on slides using PermaFluor™ mounting medium (Thermo Scientific) and coverslips were sealed using nail polish.
Fatty-acid and glucose oxidation assays
Fatty-acid oxidation was assessed in the mouse brain, red and white gastrocnemius muscle and quadriceps femoris muscle by measuring and summing 14CO2 production and 14C-labeled acid-soluble metabolites from the oxidation of [1-14C]-palmitic acid (American Radiolabeled Chemicals, St. Louis MO) respectively. Briefly, samples were incubated in 0.5 μCi/ml of [1-14C]-palmitic acid for 3 h. Media was then removed and exposed to 45 % perchloric acid for 1 h to liberate 14CO2, which was trapped in a tube containing 1 M sodium hydroxide (NaOH). The NaOH was then placed into a scintillation vial with 5 ml scintillation fluid added. The vial was then placed on a scintillation counter (LS 4500, Beckman Coulter) and counted for the presence of 14C. Acid soluble metabolites were determined by collecting the acidified media and measuring 14C content. Glucose oxidation was assessed by measuring 14CO2 production in a similar manner to fatty acid oxidation with the exception that [U-14C]-glucose was substituted for [1-14C]-palmitic acid.
Body composition measurement
Body composition was measured using Burker mini spec LF90, as previously described .
Glutathione-S-transferase (GST) pull down assay
GST-fusion CASK and GST were expressed in the BL21-DE3 strain of E. coli and purified by affinity chromatography on a glutathione–Sepharose column (Amersham) . GST-pulldowns from rat brain were performed essentially as described .
Triton X-114 phase separation of homogenized mouse brain
Triton X-114 phase separation protocol was based on a previously published method . Briefly, 4 % Triton X-114 (Sigma) in PBS containing protease inhibitors was mixed 1:1 with brain homogenate. The mixture was first incubated on ice for 10 min and then incubated at 37 °C for 10 min to promote separation of two phases. The sample was then centrifuged at 25 °C for 10 min at 13,000 g for separation into 1) aqueous phase on top, 2) a detergent phase in the middle, and 3) a pellet at the bottom.
Linear glycerol gradient centrifugation
A continuous glycerol gradient (10 % - 40 %) was generated using a Gradient Master™ (BioComp Instruments, Inc.) containing 25 mM HEPES-NaOH, pH 7.2, 150 mM NaCl, 5 mM DTT, 2 mM EDTA and protease inhibitors On a 10 mL continuous glycerol gradient buffer in ultracentrifuge tubes used with SW41 Ti Rotor (Beckman Coulter), 0.5 mL of the aqueous phase from the Triton X-114 phase separation of homogenized brain was carefully layered and then centrifuged for 20 h, at 35,000 rpm at 4 °C. A total of 9 fractions (1.1 mL each) were collected using the Piston Gradient Fractionator (BioComp Instruments, Inc.). Separate tubes were used for recombinant CASK protein and protein standards in the same experiment.
Brain subcellular fractionation and solubilization
Subcellular fractionation was performed similar to previous publication . Briefly, dissected brains were rapidly transferred to ice cold homogenization buffer (i.e. 0.32 M sucrose and 20 mM HEPES pH 7.4 with protease inhibitors). The brain was homogenized in a motorized homogenizer (~20 strokes). Brain homogenates were centrifuged at 1000 g for 10 min to generate post nuclear supernatant (PNS). The PNS was centrifuged at 17,000 g for 15 min to obtain a pellet, which was subsequently washed once with homogenization buffer and then resuspended in homogenization buffer to obtain crude synaptosomes. Crude synaptosomes were subjected to ultracentrifugation (120,000 g). The supernatant was filtered through a 200 μm filter to obtain cytosolic fraction. The pellet was first solubilized in PBS containing 1 % Triton X-100 and protease inhibitors for two hours to obtain a Triton X-100-soluble fraction. The remaining pellet was further solubilized in PBS containing 1 % deoxycholic acid and protease inhibitors at 4 °C.
Force plate actometry and rotarod treadmill experiments
For force plate analyses, the adult littermate mice per genotype performed on a force plate (Bioseb) for 5 min as described previously . Ataxic index was calculated as the area traveled over the distance covered on the force plate. We also tested the ability of mice to balance on a fixed speed rotarod test (10RPM) (Ugobasile). Each mouse was given three runs each day and an average was calculated.
Cerebellar neuron-specific deletion of CASK does not produce cerebellar hypoplasia
Heterozygous deletion mutations in CASK produce cerebellar hypoplasia [8, 45]. It has also been demonstrated that mice carrying a floxed CASK gene (CASK floxed) express ~ 33 % CASK compared to the wildtype littermates, and exhibit hypoplasia of cerebellar vermis [3, 45]. These data indicate that CASK may play a crucial role in the cerebellum. We therefore decided to use the Cre-loxP method to specifically delete CASK from cerebellar neurons. In order to ascertain the validity of this methodology, we first produced neuron-glia mixed cultures from P1.5 (postnatal day 1.5) CASK floxed mice and transduced them with Cre-expressing lentivirus. Immunoblotting revealed that Cre expression specifically and completely eliminated CASK expression in these cultures within 96 h of virus addition. (Additional file 1: Figure S1B,C).
We next deleted CASK from the cerebellar granule cells by crossing the CASK floxed mouse line to a Math5-Cre transgenic line. Math5-Cre is expressed in many different areas of brain including the hippocampus, the fourth layer of the cortex and the cerebellar cortex. In the cerebellum, cells expressing Math5-Cre give rise to the tightly packed cells in the granular layer . We confirmed this distribution by crossing the Math5-Cre mouse line to an indicator mouse line that expresses tdTomato in a Cre-dependent manner . Our experimental results show that Math5-Cre is expressed in granular cells as well as in a few cells in the molecular layer (Fig. 1h, middle panels). Granular cells are formed in the external granular layer during cerebellar genesis and subsequently migrate inwards. The CASK null granular cells are capable of this migration as indicated by their proper localization in the adult mouse cerebellum (Fig. 1h, lower row). Furthermore, we found that deleting CASK from the cerebellar granule cells did not alter the size of the cerebellum or layering of neurons compared to the CASK floxed mice (Fig. 1i, j, k). No specific motor deficits were observed in any of these mice (data not shown). Our data therefore imply that although CASK may play an important role in cell survival in the postnatal brain, deleting it from specific neuronal subtypes is compatible with their survival. Furthermore, our data indicates that the cerebellar hypoplasia phenotype observed in CASK mutation subjects may not be directly related to the loss of CASK function in cerebellar neurons.
MICPCH pathology is not purely neuronal in origin
CASK (+/-) mice mimic the human MICPCH phenotype
CASK deletion affects cell number in a non-cell-autonomous manner
CASK is an intracellular cytosolic protein and interacts with mitochondrial proteins
CASK is thought to be either completely or mainly membrane-anchored protein [21, 28]. Deletion of a signaling molecule at the membrane may explain a non-cell autonomous effect. We therefore decided to re-examine the cellular localization of CASK to confirm its presence at the membrane. We fractionated the wild-type mouse brain and looked for the solubility of CASK compared to PSD95, another MAGUK protein which is known to be membrane anchored [28, 63]. As expected, we found that PSD95 is completely membrane-anchored and is solubilized only with deoxycholic acid treatment, whereas a significant amount of CASK is also present in the cytosolic fraction isolated from the mouse brain, indicating that CASK is a soluble cytosolic protein (Fig. 5c). In this regard our finding is consistent with a previous report . To further explore CASK’s cellular localization, we performed a Triton X-114 phase separation experiment. Triton X-114 solution is homogenous at 0 °C but separates into a detergent and aqueous phase at 20 °C, allowing for separation of hydrophilic cytosolic proteins from amphiphilic transmembrane proteins . Since Triton X-114 is a mild non-ionic detergent, it is unlikely to disrupt strong interactions . Interestingly, most of the CASK protein partitioned in the aqueous phase indicating that CASK is not tightly membrane-anchored and predominantly a cytosolic protein (Fig. 5d). In contrast to CASK, only a minor fraction of PSD95 is present in the aqueous phase (Fig. 5d). These results suggest that a large fraction of CASK protein may be cytosolic in the mouse brain, and in this aspect CASK differs substantially from other MAGUK proteins such as PSD95, which is tethered to the membrane to orchestrate cell-to-cell signaling.
In Drosophila, it is known that CASK interacts with different protein complexes in different cell types . We therefore sought to determine if in the mammalian brain, CASK is also part of large protein complexes. We first expressed and purified full-length recombinant rat CASK (rCASK). CASK is a monomeric protein based on the gel exclusion chromatography experiments (data not shown). After glycerol gradient centrifugation, the rCASK (MW 120 kDa) protein was present in the second fraction along with a 150 kDa standard marker, as expected. Since rCASK migrates at ~ 120 kDa on SDS-PAGE, this confirms that rCASK is a monomer. In contrast, a large portion of the mouse brain cytosolic CASK sedimented in the fourth fraction along with a 460 kDa marker, indicating that when in cytosol, CASK is likely a part of a multi-protein complex (Fig. 5e). Surprisingly, small amounts of CASK were found to sediment in all fractions unlike the other well-known scaffolding protein e.g. liprin-α3 which displayed a well-defined sedimentation fraction indicating that the mouse brain CASK may be present in many complexes with molecular weights larger than 500 kDa (Fig. 5e).
Since CASK interacts with mitochondrial proteins in Drosophila , we next sought to determine if CASK also interacts with mitochondrial proteins in mammals. Using immobilized full-length recombinant CASK protein covalently attached to AminoLink™ agarose resin, we performed an affinity chromatography experiment using rat brain lysate and identified several CASK-interacting mitochondrial proteins from rat brain (unpublished data). To test the specificity of these interactions, we performed a pull-down assay with a GST (glutathione-S-transferase)-tagged CASK and found that CASK interacts with the mitochondrial ATP synthase β subunit and isocitrate dehydrogenase (IDH) (Fig. 5f).
CASK (+/-) heterozygous mice show aberrant metabolism
Because some studies indicate that CASK plays a role in endocrine function such as insulin release , it is important to consider the fact that an overall measure of organism or organ metabolism may not be indicative of CASK’s direct involvement in cellular metabolism. We therefore examined the rate of metabolic fuel oxidation in skeletal muscle homogenates isolated from CASK (+/-) mice compared to the sex-matched CASK (+/+) littermate controls. Interestingly, we observed that the skeletal muscles from CASK (+/-) mice oxidize significantly lower amounts of fatty acids (Fig. 6g) and glucose (Fig. 6h) compared to the CASK (+/+) mice, suggesting that CASK is involved in the regulation of cellular substrate metabolism.
CASK regulates mitochondrial respiration and oxidative metabolism
Since the neonatal brain is highly susceptible to defects in metabolism, we next examined the effects of CASK deletion on oxidative metabolism in the brain. We adopted the same strategy as in muscles by using the brain homogenates to rule out any indirect effect/s of CASK deletion. Whole brain homogenates have been shown to exhibit a more tightly coupled respiration than isolated mitochondria ; and we also recently found that respiration can be measured more effectively from whole brain homogenates . The rate of endogenous respiration in whole brain homogenates from CASK (+/-) mice was significantly reduced (~25 % decrease) compared to the CASK (+/+) brain homogenates (Fig. 7f), further suggesting that CASK is a regulator of mitochondrial respiration in the brain. Since glucose is the major source of fuel in the brain, we next asked whether glucose oxidation was altered in the brain of CASK (+/-) mice. We found a significant reduction (~20 % decrease) in the rate of glucose oxidation in CASK (+/-) mice brain compared to the CASK (+/+) brain (Fig. 7g), suggesting that CASK regulates oxidative metabolism in the brain.
XLID affects males at a significantly higher rate than females because males have a single X-chromosome . In females, X-linked mutations create a mosaic brain due to random X-inactivation, with ~ 50 % of brain cells expressing the mutant gene. This results in a relatively milder phenotype, as exemplified by the doublecortin mutations . Most CASK deletion mutation patients are females, indicating that CASK expression is essential in the majority of cells for adequate brain function.
CASK heterozygous deletion mutation patient phenotypes have been classified as MICPCH or pontocerebellar hypoplasia. Here, we demonstrated that cerebellar hypoplasia associated with CASK mutations may not be a local neuronal pathology. One possible explanation for the disproportionate cerebellar hypoplasia could be the time of onset of this disorder; CASK heterozygous deletion mutations lead to aberrant postnatal brain growth and since cerebellar growth is largely postnatal, it may be affected disproportionately. In fact, it is known that although nutritional deprivation during postnatal brain development inhibits overall brain growth, it affects cerebellum disproportionately .
Consistent with the notion that CASK is not involved in core neuronal functions , we demonstrate that neuronal-specific CASK deletion does not alter neuronal migration or survival. Although the neuron-specific CASK heterozygous knockout mice did not exhibit any major phenotype, the complete neuronal CASK knockout mice exhibited pronounced growth retardation and epilepsy, and both are symptoms of CASK-related syndromes. Since, the CASK neuronal-null mutants are made on a hypomorphic background, at present it is not possible to specifically point out the consequences of deleting CASK only from neurons. However, our data suggests that the disruption of neuronal CASK function does contribute to the overall CASK mutation-related phenotypes.
Phenotypes associated with CASK heterozygous deletion mutation in humans may represent a cumulative loss of CASK function in different brain cell types. Since CASK interacts with multiple synaptic proteins, it is presumed to possess a synaptic function . Our recent proteomic screening experiments in Drosophila melanogaster confirms the synaptic protein interactions of CASK, but also revealed a hitherto unknown interaction of CASK with mitochondrial proteins . In agreement with our previous findings, we demonstrate here that mammalian CASK also interacts with certain metabolic/mitochondrial proteins. Since a significant amount of CASK was found to be cytosolic in the mouse brain, we speculate that these interactions may be transient and taking place in the cytosol. Interestingly, the cytosolic interaction and phosphorylation of nuclear encoded mitochondrial proteins with other kinases have been shown to affect their import into mitochondria . Reducing CASK expression in a cellular or animal model alters mitochondrial function, including oxygen consumption and fuel metabolism. Since the cell biological role of neuroglial cells and neurons are considerably different, specific deletion of CASK in each cell type is likely to produce different phenotypes. A metabolic function of CASK could also explain the phenotypic differences observed in multiple animal models of CASK deficiency, including the vulval phenotype in worms and a defective locomotion phenotype in flies, both of which are known to be associated with abnormal metabolism [12, 29]. In mammals, the high metabolic demand of the growing neonatal brain could explain the lethality and postnatal onset of microcephaly and cerebellar hypoplasia in cases of CASK deficiency. It is possible that the species-specific phenotypic differences represent a difference in the metabolic requirements of each specific nervous system rather than an evolutionary difference in the molecular function of CASK.
How does a cell-autonomous metabolic function of CASK translate into the reduction of cell numbers in a non-cell-autonomous manner? Although it may be assumed that a significant reduction in the mitochondrial function would be deleterious to the survival of neurons, the extent to which the energetic function is compromised may further determine whether neurons may survive abnormally or undergo cell death. In human mitochondrial encephalopathies, the symptoms typically do not manifest until two years of age, indicating adequate survival of neurons during development . Importantly, the central nervous system pathology like stroke episodes in mitochondrial encephalopathies may also be related to an aberrant extracellular signaling e.g. nitric oxide mediated signaling . The non-cell autonomous systemic manifestations of mitochondrial stress are gradually getting recognized . It is possible that CASK deficiency modulates one or more metabolic pathway/s which are critical for the secretion of a factor(s) essential for cellular survival. Conversely, it is also possible that CASK deficiency leads to the release of toxic intermediates, resulting in the loss of cells in a non-cell autonomous manner. Mitochondrial function in the brain is not only critical to ATP production but also in regulating amino-acid metabolism, which governs neurotransmitter production. An altered milieu of different neurotransmitters in the brain may have profound implications on neurodevelopment. It can therefore be hypothesized that the postnatal microcephaly associated with CASK heterozygous deletion mutation may be caused by altered levels of one or more extracellular factors. Although much remains to be done to identify the specific players involved, the most exciting aspect of this study is the possibility that a metabolic intervention may potentially prevent some of the postnatal consequences associated with CASK deletion mutation.
Taken together our results suggest that lack of neuronal CASK function contributes to the phenotypic spectrum of CASK linked pathologies, however MICPCH is not purely neuronal in origin. Therefore, constitutive heterozygous CASK knockout females are better animal model to investigate MICPCH. Our data further indicates that although CASK is a cytosolic protein and may perform cell-autonomous function in regulating metabolism, CASK-linked microcephaly occurs in a non-cell autonomous manner which involves loss of both CASK positive and CASK null cells. Finally, the observed cerebellar hypoplasia is not a local neuronal pathology; disproportionate cerebellar hypoplasia may simply represent timing of the disorder which has a large postnatal component. It is known that nutritional deprivation during the postnatal brain growth spurt specifically affects the cerebellum. Therefore, our findings also provide a valid framework for the investigation of non-CASK related cerebellar hypoplasia.
All procedures performed in studies involving animals were in accordance with the ethical standards of the VirginiaTech institutional animal care and use committee.
We gratefully acknowledge Prof. Thomas Südhof for kindly providing us with the mouse carrying the floxed CASK gene. We also thank Dr. Michael Fox for providing us with the Math5-Cre mice, Dr. Alexei Morozov for providing us with LSL-tdt-Tomato mice. We thank Dr. Leslie LaConte for careful reading of the manuscript. This work was partially supported by NIH award 1R01EY024712-01A1 to KM.
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- Aigner L, Fluegel D, Dietrich J, Ploetz S, Winkler J. Isolated lissencephaly sequence and double-cortex syndrome in a German family with a novel doublecortin mutation. Neuropediatrics. 2000;31:195–8. doi:10.1055/s-2000-7452.View ArticlePubMedGoogle Scholar
- Anitei M, Ifrim M, Ewart MA, Cowan AE, Carson JH, Bansal R, Pfeiffer SE. A role for Sec8 in oligodendrocyte morphological differentiation. J Cell Sci. 2006;119:807–18. doi:10.1242/jcs.02785.View ArticlePubMedGoogle Scholar
- Atasoy D, Schoch S, Ho A, Nadasy KA, Liu X, Zhang W, Mukherjee K, Nosyreva ED, Fernandez-Chacon R, Missler M, et al. Deletion of CASK in mice is lethal and impairs synaptic function. Proc Natl Acad Sci U S A. 2007;104:2525–30. doi:10.1073/pnas.0611003104.View ArticlePubMedPubMed CentralGoogle Scholar
- Barski JJ, Dethleffsen K, Meyer M. Cre recombinase expression in cerebellar Purkinje cells. Genesis. 2000;28:93–8.View ArticlePubMedGoogle Scholar
- Birkebaek NH, Patel L, Wright NB, Grigg JR, Sinha S, Hall CM, Price DA, Lloyd IC, Clayton PE. Endocrine status in patients with optic nerve hypoplasia: relationship to midline central nervous system abnormalities and appearance of the hypothalamic-pituitary axis on magnetic resonance imaging. J Clin Endocrinol Metab. 2003;88:5281–6. doi:10.1210/jc.2003-030527.View ArticlePubMedGoogle Scholar
- Bordier C. Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem. 1981;256:1604–7.PubMedGoogle Scholar
- Brusca JS, Radolf JD. Isolation of integral membrane proteins by phase partitioning with Triton X-114. Methods Enzymol. 1994;228:182–93.View ArticlePubMedGoogle Scholar
- Burglen L, Chantot-Bastaraud S, Garel C, Milh M, Touraine R, Zanni G, Petit F, Afenjar A, Goizet C, Barresi S, et al. Spectrum of pontocerebellar hypoplasia in 13 girls and boys with CASK mutations: confirmation of a recognizable phenotype and first description of a male mosaic patient. Orphanet J Rare Dis. 2012;7:18. 10.1186/1750-1172-7-18.
- Cacace AT, Pinheiro JM. The mitochondrial connection in auditory neuropathy. Audiol Neurootol. 2011;16:398–413. doi:10.1159/000323276.View ArticlePubMedGoogle Scholar
- Castro-Gago M, Blanco-Barca MO, Gomez-Lado C, Eiris-Punal J, Campos-Gonzalez Y, Arenas-Barbero J. Respiratory chain complex I deficiency in an infant with Ohtahara syndrome. Brain Dev. 2009;31:322–5. doi:10.1016/j.braindev.2008.05.009.View ArticlePubMedGoogle Scholar
- Chavan V, Willis J, Walker SK, Clark HR, Liu X, Fox MA, Srivastava S, Mukherjee K. Central presynaptic terminals are enriched in ATP but the majority lack mitochondria. PLoS One. 2015;10:e0125185. doi:10.1371/journal.pone.0125185.View ArticlePubMedPubMed CentralGoogle Scholar
- Cheng Z, Tsuda M, Kishita Y, Sato Y, Aigaki T. Impaired energy metabolism in a Drosophila model of mitochondrial aconitase deficiency. Biochem Biophys Res Commun. 2013;433:145–50. doi:10.1016/j.bbrc.2013.02.040.View ArticlePubMedGoogle Scholar
- Dobbing J. Vulnerable periods of brain development. In: lipids, malnutrition & the developing brain. Ciba Found Symp. 1971: 9-29. PMID: 4949882.Google Scholar
- Edvardson S, Shaag A, Kolesnikova O, Gomori JM, Tarassov I, Einbinder T, Saada A, Elpeleg O. Deleterious mutation in the mitochondrial arginyl-transfer RNA synthetase gene is associated with pontocerebellar hypoplasia. Am J Hum Genet. 2007;81:857–62. doi:10.1086/521227.View ArticlePubMedPubMed CentralGoogle Scholar
- El-Hattab AW, Adesina AM, Jones J, Scaglia F. MELAS syndrome: Clinical manifestations, pathogenesis, and treatment options. Mol Genet Metab. 2015;116:4–12. doi:10.1016/j.ymgme.2015.06.004.View ArticlePubMedGoogle Scholar
- Ferguson EL, Horvitz HR. Identification and characterization of 22 genes that affect the vulval cell lineages of the nematode Caenorhabditis elegans. Genetics. 1985;110:17–72.PubMedPubMed CentralGoogle Scholar
- Goldowitz D, Cushing RC, Laywell E, D'Arcangelo G, Sheldon M, Sweet HO, Davisson M, Steindler D, Curran T. Cerebellar disorganization characteristic of reeler in scrambler mutant mice despite presence of reelin. J Neurosci. 1997;17:8767–77.PubMedGoogle Scholar
- Guan KL, Dixon JE. Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal Biochem. 1991;192:262–7.View ArticlePubMedGoogle Scholar
- Guyenet SJ, Furrer SA, Damian VM, Baughan TD, La Spada AR, Garden GA. A simple composite phenotype scoring system for evaluating mouse models of cerebellar ataxia. J Vis Exp. 2010: doi10.3791/1787.
- Hackett A, Tarpey PS, Licata A, Cox J, Whibley A, Boyle J, Rogers C, Grigg J, Partington M, Stevenson RE, et al. CASK mutations are frequent in males and cause X-linked nystagmus and variable XLMR phenotypes. Eur J Hum Genet. 2010;18:544–52. doi:10.1038/ejhg.2009.220.View ArticlePubMedPubMed CentralGoogle Scholar
- Hata Y, Butz S, Sudhof TC. CASK: a novel dlg/PSD95 homolog with an N-terminal calmodulin-dependent protein kinase domain identified by interaction with neurexins. J Neurosci. 1996;16:2488–94.PubMedGoogle Scholar
- Ho A, Morishita W, Atasoy D, Liu X, Tabuchi K, Hammer RE, Malenka RC, Sudhof TC. Genetic analysis of Mint/X11 proteins: essential presynaptic functions of a neuronal adaptor protein family. J Neurosci. 2006;26:13089–101. doi:10.1523/JNEUROSCI.2855-06.2006.View ArticlePubMedGoogle Scholar
- Hong ST, Mah W. A critical role of GIT1 in vertebrate and invertebrate brain development. Exp Neurobiol. 2015;24:8–16. doi:10.5607/en.2015.24.1.8.View ArticlePubMedPubMed CentralGoogle Scholar
- Horvitz HR, Sulston JE. Isolation and genetic characterization of cell-lineage mutants of the nematode Caenorhabditis elegans. Genetics. 1980;96:435–54.PubMedPubMed CentralGoogle Scholar
- Hoskins R, Hajnal AF, Harp SA, Kim SK. The C. elegans vulval induction gene lin-2 encodes a member of the MAGUK family of cell junction proteins. Development. 1996;122:97–111.PubMedGoogle Scholar
- Hsueh YP. The role of the MAGUK protein CASK in neural development and synaptic function. Curr Med Chem. 2006;13:1915–27.View ArticlePubMedGoogle Scholar
- Hsueh YP, Wang TF, Yang FC, Sheng M. Nuclear translocation and transcription regulation by the membrane-associated guanylate kinase CASK/LIN-2. Nature. 2000;404:298–302. doi:10.1038/35005118.View ArticlePubMedGoogle Scholar
- Hsueh YP, Yang FC, Kharazia V, Naisbitt S, Cohen AR, Weinberg RJ, Sheng M. Direct interaction of CASK/LIN-2 and syndecan heparan sulfate proteoglycan and their overlapping distribution in neuronal synapses. J Cell Biol. 1998;142:139–51.View ArticlePubMedPubMed CentralGoogle Scholar
- Jia K, Albert PS, Riddle DL. DAF-9, a cytochrome P450 regulating C. elegans larval development and adult longevity. Development. 2002;129:221–31.PubMedGoogle Scholar
- Kang HC, Lee YM, Kim HD. Mitochondrial disease and epilepsy. Brain Dev.2013: Doi 10.1016/j.braindev.2013.01.006.
- LaConte L, Mukherjee K. Structural constraints and functional divergences in CASK evolution. Biochem Soc Trans. 2013;41:1017–22. doi:10.1042/BST20130061.View ArticlePubMedGoogle Scholar
- Lee MS. Effect of mitochondrial stress on systemic metabolism. Ann N Y Acad Sci. 2015;1350:61–5. doi:10.1111/nyas.12822.View ArticlePubMedGoogle Scholar
- Lewandoski M, Wassarman KM, Martin GR. Zp3-cre, a transgenic mouse line for the activation or inactivation of loxP-flanked target genes specifically in the female germ line. Curr Biol. 1997;7:148–51.View ArticlePubMedGoogle Scholar
- Lincke CR, van den Bogert C, Nijtmans LG, Wanders RJ, Tamminga P, Barth PG. Cerebellar hypoplasia in respiratory chain dysfunction. Neuropediatrics. 1996;27:216–8. doi:10.1055/s-2007-973792.View ArticlePubMedGoogle Scholar
- Madisen L, Zwingman TA, Sunkin SM, Oh SW, Zariwala HA, Gu H, Ng LL, Palmiter RD, Hawrylycz MJ, Jones AR, et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat Neurosci. 2010;13:133–40. doi:10.1038/nn.2467.View ArticlePubMedPubMed CentralGoogle Scholar
- Marquez-Rosado L, Singh D, Rincon-Arano H, Solan JL, Lampe PD. CASK (LIN2) interacts with Cx43 in wounded skin and their coexpression affects cell migration. J Cell Sci. 2012;125:695–702. doi:10.1242/jcs.084400.View ArticlePubMedPubMed CentralGoogle Scholar
- Matos MF, Mukherjee K, Chen X, Rizo J, Sudhof TC. Evidence for SNARE zippering during Ca2 + -triggered exocytosis in PC12 cells. Neuropharmacology. 2003;45:777–86.View ArticlePubMedGoogle Scholar
- Mattman A, Sirrs S, Mezei MM, Salvarinova-Zivkovic R, Lillquist Y. Mitochondrial disease clinical manifestations: an overview. BC Med J. 2011;53:183–7.Google Scholar
- McMillan RP, Wu Y, Voelker K, Fundaro G, Kavanaugh J, Stevens JR, Shabrokh E, Ali M, Harvey M, Anderson AS, et al. Selective overexpression of Toll-like receptor-4 in skeletal muscle impairs metabolic adaptation to high-fat feeding. Am J Physiol Regul Integr Comp Physiol. 2015;309:R304–313. doi:10.1152/ajpregu.00139.2015.View ArticlePubMedGoogle Scholar
- Michaud JL, Lachance M, Hamdan FF, Carmant L, Lortie A, Diadori P, Major P, Meijer IA, Lemyre E, Cossette P, et al. The genetic landscape of infantile spasms. Hum Mol Genet. 2014;23:4846–58. doi:10.1093/hmg/ddu199.View ArticlePubMedGoogle Scholar
- Moog U, Bierhals T, Brand K, Bautsch J, Biskup S, Brune T, Denecke J, de Die-Smulders CE, Evers C, Hempel M, et al. Phenotypic and molecular insights into CASK-related disorders in males. Orphanet J Rare Dis. 2015;10:44. doi:10.1186/s13023-015-0256-3.View ArticlePubMedPubMed CentralGoogle Scholar
- Moog U, Kutsche K, Kortum F, Chilian B, Bierhals T, Apeshiotis N, Balg S, Chassaing N, Coubes C, Das S, et al. Phenotypic spectrum associated with CASK loss-of-function mutations. J Med Genet. 2011;48:741–51. doi:10.1136/jmedgenet-2011-100218.View ArticlePubMedGoogle Scholar
- Mukherjee K, Sharma M, Jahn R, Wahl MC, Sudhof TC. Evolution of CASK into a Mg2 + -sensitive kinase. Sci Signal. 2010;3:ra33. doi:10.1126/scisignal.2000800.View ArticlePubMedPubMed CentralGoogle Scholar
- Mukherjee K, Slawson JB, Christmann BL, Griffith LC. Neuron-specific protein interactions of Drosophila CASK-beta are revealed by mass spectrometry. Front Mol Neurosci. 2014;7:58. doi:10.3389/fnmol.2014.00058.View ArticlePubMedPubMed CentralGoogle Scholar
- Najm J, Horn D, Wimplinger I, Golden JA, Chizhikov VV, Sudi J, Christian SL, Ullmann R, Kuechler A, Haas CA, et al. Mutations of CASK cause an X-linked brain malformation phenotype with microcephaly and hypoplasia of the brainstem and cerebellum. Nat Genet. 2008;40:1065–7. doi:10.1038/ng.194.View ArticlePubMedGoogle Scholar
- Nakajiri T, Kobayashi K, Okamoto N, Oka M, Miya F, Kosaki K, Yoshinaga H. Late-onset epileptic spasms in a female patient with a CASK mutation. Brain Dev. 2015;37:919–23. doi:10.1016/j.braindev.2015.02.007.View ArticlePubMedGoogle Scholar
- Nakamura K, Nishiyama K, Kodera H, Nakashima M, Tsurusaki Y, Miyake N, Matsumoto N, Saitsu H, Jinnou H, Ohki S, et al. A de novo CASK mutation in pontocerebellar hypoplasia type 3 with early myoclonic epilepsy and tetralogy of Fallot. Brain Dev. 2014;36:272–3. doi:10.1016/j.braindev.2013.03.007.View ArticlePubMedGoogle Scholar
- Piluso G, D'Amico F, Saccone V, Bismuto E, Rotundo IL, Di Domenico M, Aurino S, Schwartz CE, Neri G, Nigro V. A missense mutation in CASK causes FG syndrome in an Italian family. Am J Hum Genet. 2009;84:162–77. doi:10.1016/j.ajhg.2008.12.018.View ArticlePubMedPubMed CentralGoogle Scholar
- Ramos-Jimenez A, Hernandez-Torres RP, Torres-Duran PV, Romero-Gonzalez J, Mascher D, Posadas-Romero C, Juarez-Oropeza MA. The respiratory exchange ratio is associated with fitness indicators both in trained and untrained men: a possible application for people with reduced exercise tolerance. Clin Med Circ Respir Pulm Med. 2008;2:1–9.Google Scholar
- Raymond FL. X linked mental retardation: a clinical guide. J Med Genet. 2006;43:193–200. doi:10.1136/jmg.2005.033043.View ArticlePubMedPubMed CentralGoogle Scholar
- Saitsu H, Kato M, Osaka H, Moriyama N, Horita H, Nishiyama K, Yoneda Y, Kondo Y, Tsurusaki Y, Doi H, et al. CASK aberrations in male patients with Ohtahara syndrome and cerebellar hypoplasia. Epilepsia. 2012;53:1441–9. doi:10.1111/j.1528-1167.2012.03548.x.View ArticlePubMedGoogle Scholar
- Samuels BA, Hsueh YP, Shu T, Liang H, Tseng HC, Hong CJ, Su SC, Volker J, Neve RL, Yue DT, et al. Cdk5 promotes synaptogenesis by regulating the subcellular distribution of the MAGUK family member CASK. Neuron. 2007;56:823–37. doi:10.1016/j.neuron.2007.09.035.View ArticlePubMedPubMed CentralGoogle Scholar
- Schmidt O, Harbauer AB, Rao S, Eyrich B, Zahedi RP, Stojanovski D, Schonfisch B, Guiard B, Sickmann A, Pfanner N, et al. Regulation of mitochondrial protein import by cytosolic kinases. Cell. 2011;144:227–39. doi:10.1016/j.cell.2010.12.015.View ArticlePubMedGoogle Scholar
- Schoch S, Cibelli G, Thiel G. Neuron-specific gene expression of synapsin I. Major role of a negative regulatory mechanism. J Biol Chem. 1996;271:3317–23.View ArticlePubMedGoogle Scholar
- Sitarz KS, Chinnery PF, Yu-Wai-Man P. Disorders of the optic nerve in mitochondrial cytopathies: new ideas on pathogenesis and therapeutic targets. Curr Neurol Neurosci Rep. 2012;12:308–17. doi:10.1007/s11910-012-0260-0.View ArticlePubMedPubMed CentralGoogle Scholar
- Slawson JB, Kuklin EA, Ejima A, Mukherjee K, Ostrovsky L, Griffith LC. Central regulation of locomotor behavior of Drosophila melanogaster depends on a CASK isoform containing CaMK-like and L27 domains. Genetics. 2011;187:171–84. doi:10.1534/genetics.110.123406.View ArticlePubMedPubMed CentralGoogle Scholar
- Smart JL, Dobbing J, Adlard BP, Lynch A, Sands J. Vulnerability of developing brain: relative effects of growth restriction during the fetal and suckling periods on behavior and brain composition of adult rats. J Nutr. 1973;103:1327–38.PubMedGoogle Scholar
- Srivastava S, Barrett JN, Moraes CT. PGC-1alpha/beta upregulation is associated with improved oxidative phosphorylation in cells harboring nonsense mtDNA mutations. Hum Mol Genet. 2007;16:993–1005. doi:10.1093/hmg/ddm045.View ArticlePubMedPubMed CentralGoogle Scholar
- Srivastava S, Diaz F, Iommarini L, Aure K, Lombes A, Moraes CT. PGC-1alpha/beta induced expression partially compensates for respiratory chain defects in cells from patients with mitochondrial disorders. Hum Mol Genet. 2009;18:1805–12. doi:10.1093/hmg/ddp093.View ArticlePubMedPubMed CentralGoogle Scholar
- Stevenson D, Laverty HG, Wenwieser S, Douglas M, Wilson JB. Mapping and expression analysis of the human CASK gene. Mamm Genome. 2000;11:934–7. doi:10.1007/s003350010170.View ArticlePubMedGoogle Scholar
- Szklarczyk R, Wanschers BF, Nijtmans LG, Rodenburg RJ, Zschocke J, Dikow N, van den Brand MA, Hendriks-Franssen MG, Gilissen C, Veltman JA, et al. A mutation in the FAM36A gene, the human ortholog of COX20, impairs cytochrome c oxidase assembly and is associated with ataxia and muscle hypotonia. Hum Mol Genet. 2013;22:656–67. doi:10.1093/hmg/dds473dds473.View ArticlePubMedGoogle Scholar
- Tialowska B, Kaczor J, Stempak W, Grybos D, Popinigis J. Tightly coupled respiration in rat brain homogenates. Acta Biochim Pol. 1991;38:165–7.PubMedGoogle Scholar
- Topinka JR, Bredt DS. N-terminal palmitoylation of PSD-95 regulates association with cell membranes and interaction with K+ channel Kv1.4. Neuron. 1998;20:125–34.View ArticlePubMedGoogle Scholar
- Valayannopoulos V, Michot C, Rodriguez D, Hubert L, Saillour Y, Labrune P, de Laveaucoupet J, Brunelle F, Amiel J, Lyonnet Set al (2012) Mutations of TSEN and CASK genes are prevalent in pontocerebellar hypoplasias type 2 and 4. Brain. 2012;135: e199; author reply e200 Doi 10.1093/brain/awr108.
- Wagner N, Wagner KD, Scholz H, Kirschner KM, Schedl A. Intermediate filament protein nestin is expressed in developing kidney and heart and might be regulated by the Wilms’ tumor suppressor Wt1. Am J Physiol Regul Integr Comp Physiol. 2006;291:R779–787. doi:10.1152/ajpregu.00219.2006.View ArticlePubMedGoogle Scholar
- Walker AS, Goings GE, Kim Y, Miller RJ, Chenn A, Szele FG. Nestin reporter transgene labels multiple central nervous system precursor cells. Neural Plast. 2010;2010:894374. doi:10.1155/2010/894374.PubMedPubMed CentralGoogle Scholar
- Wang Y, Li R, Du D, Zhang C, Yuan H, Zeng R, Chen Z. Proteomic analysis reveals novel molecules involved in insulin signaling pathway. J Proteome Res. 2006;5:846–55. doi:10.1021/pr050391m.View ArticlePubMedGoogle Scholar
- Wei JL, Fu ZX, Fang M, Zhou QY, Zhao QN, Guo JB, Lu WD, Wang H. High expression of CASK correlates with progression and poor prognosis of colorectal cancer. Tumour Biol. 2014;35:9185–94. doi:10.1007/s13277-014-2179-3.View ArticlePubMedGoogle Scholar
- Williams AN, Gray RG, Poulton K, Ramani P, Whitehouse WP. A case of Ohtahara syndrome with cytochrome oxidase deficiency. Dev Med Child Neurol. 1998;40:568–70.View ArticlePubMedGoogle Scholar
- Yang Z, Ding K, Pan L, Deng M, Gan L. Math5 determines the competence state of retinal ganglion cell progenitors. Dev Biol. 2003;264:240–54.View ArticlePubMedGoogle Scholar
- Zhu Y, Romero MI, Ghosh P, Ye Z, Charnay P, Rushing EJ, Marth JD, Parada LF. Ablation of NF1 function in neurons induces abnormal development of cerebral cortex and reactive gliosis in the brain. Genes Dev. 2001;15:859–76. doi:10.1101/gad.862101.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhu ZQ, Wang D, Xiang D, Yuan YX, Wang Y. Calcium/calmodulin-dependent serine protein kinase is involved in exendin-4-induced insulin secretion in INS-1 cells. Metabolism. 2014;63:120–6. doi:10.1016/j.metabol.2013.09.009.View ArticlePubMedGoogle Scholar