Complete CASK loss in humans causes profound neurodevastation and cerebellar atrophy
MICPCH subjects with heterozygous CASK mutations are known to live past their 30s. Cask+/− female mice are fertile beyond 6 months. We have allowed four Cask+/− mice to age more than two years, considered to be old for mice. All four mice survived to that age without adverse events. We did not observe any obvious phenotypes in these aged mice compared to wild-type littermates. The cerebellum displayed the typical layers and configuration without severe deterioration, indicating that the disorder is non-progressive (Supplemental Fig. 1).
Null mutation of CASK in mice is, however, lethal and in boys, produces progressive encephalopathy. Due to the early lethality of Cask null mice, the postnatal pathology of complete Cask loss has been difficult to study to date. Here we describe detailed clinical findings and autopsy results from a 2-month-old boy with a CASK null mutation who expired due to hypoventilation and neurogenic respiratory failure. A copy number variation study was unremarkable, but next generation sequencing of genes revealed a c.79C > T (p.Arginine27Ter) CASK mutation in exon 2 (Fig. 1A). This CASK mutation introduces a stop codon in the very N-terminus of the CASK protein, precluding expression of any splice variant of CASK (Supplemental Fig. 2). Magnetic resonance imaging (MRI) indicated normal lateral and third ventricles with an elongated fourth ventricle. The cerebellum appeared markedly hypoplastic without a vermis. The small posterior fossa was filled with fluid (Fig. 1B). The corpus callosum was thin but present without any midline shift, and myelination was delayed for age. The cavum septum pellucidum seemed to be more prominent. No heterotopic cells were noted in any area, but there was some degree of smoothening, particularly of the frontal cortex. The brain stem appeared to be extremely thin.
Video electroencephalographic (vEEG) monitoring was done both during awake and sleeping states. Awake-state background EEG displayed a burst-suppression pattern with variable amounts of bursts and suppressions (Fig. 1C and Supplemental Fig. 3). This EEG pattern is typical of Ohtahara syndrome, a devastating epileptic encephalopathy, that usually co-occurs with CASK-null mutations (4, 15, 16). The burst phase was dominated by a mixture of theta and delta waves. Overall, the EEG retained its symmetry in both hemispheres but was discontinuous. No electroclinical seizures were observed during the period of recording, although intermittent and independent sharp waves were observed, predominantly in the right temporal and occipital region. The sleep EEG was similar to the waking EEG and included burst-suppression signals. A spectral analysis of the entire epoch revealed skewing towards lower frequency with delta and alpha power dominating the spectra (Fig. 1D-G).
At autopsy, head circumference was 32.7 cm, with a 37.0 cm crown-rump length and crown-heel length of 51.0 cm. The decedent was small for his age, and the brain weight was 300.8 grams, which is 60% of what is expected at this age (Fig. 2A). Except for lung, heart, and spleen, most other organs were smaller than expected but had an overall normal gross appearance (Fig. 2A). The brain was well formed with normal gyri formations in the cerebral hemispheres. Tertiary gyri were present, and there was no evidence of polymicrogyria or other abnormal configuration. The Sylvian fissure was well formed, and the leptomeninges were clear (Fig. 2B). Vascularization, including the circle of Willis, was normally formed. The central part of the cerebral hemispheres was edematous, and the septum cavum pellucidum was present (0.9 cm in vertical length). The basal ganglia displayed a normal architecture bilaterally. The left hippocampus was also architecturally normal with a serpiginous appearance. The right hippocampus had a blurred appearance (Supplemental Fig. 4). The thalamus was normally formed and firm. The lateral ventricles were not dilated; the midbrain was very small with a patent but pinpoint cerebral aqueduct, and the fourth ventricle was slit-like. The cerebellum and the pons were markedly hypoplastic (Fig. 2C, Supplemental Fig. 4). The cerebellum, despite hypoplasia, had a normal configuration but did not exhibit the usual folia. There was no evidence for heterotopia of cells. The anterior vermis was not identifiable and appeared to be membrane-like; cerebellar hemispheres were thin, flattened and firm. The spinal cord was of uniform caliber and had no obvious pathology.
Absence of CASK does not affect neuronal migration, axonal guidance, or lamination in humans but may promote neuronal loss
Histologically, the cerebellum itself displayed proper cellular organization, with a defined external granular layer (EGL), molecular layer, and internal granular layer (IGL). There was a uniform single layer of Purkinje cells between the molecular layer and the internal granular layer (Fig. 2D, E). A proper migratory pattern of granule cells was visible and appropriate for age. The white matter was poorly organized, and the dentate nucleus was absent. The midbrain consisted of astrocytic cells with pink cytoplasm and some neuronal cells, however no organized substantia nigra was noted (Fig. 2F). Sections of the cortex indicated orderly and proper neuronal migration; the germinal matrix was appropriately thinned for this age. The white matter tracts were discreet and adequate for this age (Supplemental Fig. 4). The basal ganglia displayed normal numbers of neurons. The hippocampi were properly organized with uniform neuronal populations in all CA (cornu ammonis) zones. The midbrain and pons displayed corticospinal tracts. The cerebral aqueduct was patent and dilated. Within the pons, the pontine decussation was seen and the locus coeruleus properly formed. The medullary olives were poorly formed, and the fourth ventricle was widely patent. The spinal cord was unremarkable with adequate anterior horn cells and uniform radiating column. The central canal was patent throughout (Supplemental Fig. 4).
Histologically, almost all organs including the bone marrow, heart, and intestines were unremarkable. The endocrine glands also appeared normal, except for the adrenal cortex, which was thinned out. The kidneys had appropriate and orderly glomerular and tubular development (Supplemental Fig. 5C, D). The heart rate varied between 90 and 150 beats per minute and displayed a sinus rhythm (Supplemental Fig. 6).
Data clearly indicate that although CASK loss affects the size of the brain globally, the cerebellum and brainstem are disproportionately affected. Both in murine models and the human subject, early lethality is likely linked with the dysfunction of the brainstem leading to respiratory failure. Cask null mice display hypoventilation and die within hours of birth although the brain is of normal size and properly laminated at death (18).
The normal lamination and configuration of the brain in both CASK null humans and mice suggests that the histological pathology related to CASK loss is likely to be neurodegenerative, with neuronal loss. One of the most common hallmarks of neuronal damage and neuronal loss is reactive gliosis. We therefore next evaluated the cerebellum of the decedent for the ability to form synapses and for evidence of astrogliosis (Fig. 3). Previous studies in the murine model have shown that CASK loss-of-function does not negatively impact synapse formation (18, 54), and in the human cerebellum evaluated here, immunostaining revealed that levels of the synaptic marker synaptophysin in the decedent’s cerebellum were similar to levels observed in an age-matched control cerebellum (Fig. 3A, B). GFAP (glial fibrillary acidic protein) staining of the cerebellum to detect astrogliosis, however, indicates that, compared to the control, the decedent exhibits ~ 5-fold higher amounts of GFAP immunoreactivity, specifically in the IGL (Fig. 3A, C, D). In fact, large reactive astrocytes in the IGL were readily observed (Fig. 3C). Together these data suggest that loss of CASK produces delayed neurodegenerative changes, causing the CASK-linked phenotype to typically manifest postnatally. We finally investigated myelination within the cerebellar cortex using FluoroMyelin lipid staining (Fig. 3E) and observed that the myelination pattern exhibited disturbed arrangement within the IGL of the R27Ter subject, with discrete myelinated tracts, in contrast to the diffuse mesh-like myelin staining observed in the control subject. The histological features thus indicate that the disorganized white matter described earlier may be secondary to ongoing cerebellar grey matter degeneration. Thus our observations support the notion that loss of CASK induces cerebellar cortical degeneration, specifically in the IGL. To test this idea, we next employed murine genetic experiments, where CASK is deleted in a temporally and spatially specified manner using Cre-LoxP-mediated gene excision.
Calb2 -Cre targets post-migratory granule cells and a subset of Purkinje cells in the cerebellum
Previous neuroimaging data and the comprehensive CASK null brain histological autopsy results presented here clearly indicate that within the brain, loss of CASK is likely to disproportionately affect the hindbrain including the brainstem and cerebellum. In particular, CASK-linked lethality most likely results from effects on the brain stem. Our focus, therefore, in the study presented here is to evaluate the long-term effect of CASK loss in the cerebellum. We have previously demonstrated that CASK loss likely does not affect cerebellar development. We reached this conclusion by examining three different mouse constructs: 1) pan-neuronal Cask knockout mice, which die before P24 (postnatal day 24) but exhibit normal cerebellar formation and lamination (54); 2) Purkinje cell-specific knockout mice, which display normal development and motor function (54); and finally, 3) mice with Cask deletion in a distributed subpopulation of granule cells, which do not exhibit altered cell migration or survival (54). There are two critical reasons that conclusions about cerebellar development from these previous experiments must be tempered: 1) for each of these mouse types, Cask was deleted only in small subset of cells in the cerebellum (55–57); and 2) we did not study the long-term effect of Cask deletion in the cerebellum. To address these gaps and examine the role of CASK in the cerebellum over longer time periods, we have devised a method to delete CASK from most cerebellar cells in a manner that does not produce lethality in mice. To do so, we chose a mouse line in which Cre-recombinase is driven by an endogenous promoter of the signaling molecule Calb2 (calretinin/calbindin2), reported earlier (58). The choice of Calb2-Cre was made instead of a promoter such as Math1, a transcription factor, because Math1 turns on earlier and is also expressed in the brain stem, which could contribute to lethality. It has been shown that Calb2 expresses in nearly all granule cells in the cerebellum (59, 60), but the exact timing of initiation of Calb2-Cre gene recombination in granule cells was not known. There have also been conflicting reports about the expression of Calb2 in the Purkinje cells within the cerebellum (59, 60). We therefore first tested the recombination specificity of Calb2-Cre in mice at ages when the cerebellum is still developing and displays both the EGL and IGL (P8 and P15). We crossed Calb2-Cre mice with Cre-recombination indicator mice (LSL-tdTomato) (Fig. 4A). The distribution of the tdTomato-expressing neurons serves as a proxy for CASK deletion when Calb2-Cre mice are crossed with Caskfloxed mice in parallel (Fig. 4B). Our data indicate that Calb2-Cre is active in the cerebellum as early as P8. By P8, recombination was observed in granule cells but only after migration into the IGL; recombination was also observed in many Purkinje cells (Fig. 4C). By P15, Calb2-Cre already exhibited robust recombination in many parts of the brain and in the entirety of post-migratory granule cells. Dense cellular distribution with recombination was seen in the cerebellum, hippocampus, striatum and olfactory bulb. Sparsely distributed cells were observed throughout the brain, including the cortex (Fig. 4D, E). The brainstem displayed minimal recombination, with sparsely tdTomato-labeled cells. Within the cerebellum, all granular cells in the IGL and a subset of Purkinje cells were positive for recombination at P15. Cells in the EGL, however, did not display any recombination, indicating that Calb2-Cre-driven recombination occurs only after migration of granule cells (Fig. 4E). Our data thus indicate that Calb2-Cre specifically leads to deletion of CASK both in a subset of Purkinje cells and in granule cells within the IGL by P15 and is not likely to affect the brainstem or its function.
Deletion of CASK from cerebellar neurons results in later-onset progressive degeneration of the cerebellum and severe ataxia
We next examined mice from crosses of the Calb2-Cre and Caskfloxed lines. It has been shown previously that the Caskfloxed mouse is a hypomorph that expresses ~ 40% CASK, likely due to a phenomenon known as selection cassette interference (18). Caskfloxed mice are smaller than wild type mice and exhibit cerebellar hypoplasia (13, 18, 54). Caskfloxed;Calb2-Cre F1 mice were genotyped by PCR.
Cask floxed;Calb2-Cre mice remain indistinguishable from the Caskfloxed mice well into adulthood (~ 40 days), indicating that acute deletion of Cask does not have significant effects on cerebellar development, motor learning, or locomotor function. Past two months of age, however, Caskfloxed;Calb2-Cre mice begin displaying obvious locomotor incoordination and ataxia which are rapidly progressive. By approximately P100, these mice are profoundly ataxic, are unable to keep their balance and repeatedly fall over with an inability to walk forward (supplemental video). Despite profound motor coordination deficits, the Caskfloxed;Calb2-Cre mice are otherwise healthy and display a slick coat, good body condition score, and are bright, alert and responsive. Compared to littermate Caskfloxed controls, the cerebellum of the Caskfloxed;Calb2-Cre mouse is extremely diminished in volume at P100 when the motor phenotype has plateaued (Fig. 5A, B). Comparing the histology of the Caskfloxed;Calb2-Cre cerebellum at P30 (well before onset of ataxia) and P100 (after onset of ataxia), our results indicate that at P30, the cerebellum of Caskfloxed;Calb2-Cre mice is populated with well-placed granule and Purkinje cells. At P100, however, we observe profound loss of granule cells, whereas Purkinje cells remain visible as a standard single layer of cells (Fig. 5C). The molecular layer of the cerebellum is thin and collapsed, most likely due to loss of parallel fibers arising from the granular cells and loss of synaptic connections between granule cells and Purkinje cells (Fig. 5D-G). We therefore next quantified synaptic connections within the cerebellar layers using bassoon as a pre-synaptic marker. As seen (Fig. 5H-K), our data indicate that synapse density is unaltered, although the absolute number of synapses is reduced due to the shrunken volume of the molecular layer. The large number of remaining synapses are likely to be derived from the climbing fibers. Notably, other regions with Calb2-Cre recombination such as the olfactory bulb, hippocampus and striatum do not show the striking hypoplasia observable in the cerebellum. In our previous studies, we did not observe degeneration of retinal ganglion cells, which are also positive for Calb2-Cre (58). Our data here thus indicate that loss of CASK results in the disproportionate degeneration of a specific vulnerable neuronal population, cerebellar granule cells, leading to cerebellar hypoplasia.
A decrease in grey matter creates an impression of increased white matter area. On the other hand, the histopathology in the human cerebellum displayed disorganized white matter (Fig. 3E). We therefore quantified myelin in the Caskfloxed;Calb2-Cre mice using FluoroMyelin™ staining. As seen in Fig. 6A, the myelin appears to be disorganized in the white matter of folia from the Caskfloxed;Calb2-Cre mouse cerebellum, which is most obvious in the region immediately distal to Purkinje cells. We also observed extremely limited myelinated axons in the anterior-most folium (Fig. 6A). Quantification of pixels displayed a strong trend towards a decrease in myelinated fibers which did not reach statistical significance (Fig. 6B). The degeneration of cerebellar grey matter thus is also associated with disorganization of the white matter in the Caskfloxed;Calb2-Cre mouse cerebellum, and the broadened white matter layer is likely to be filled only with acellular matrix. Because the Caskfloxed;Calb2-Cre mouse represents a targeted deletion of CASK in cerebellar neuronal cells, it is reasonable to conclude that the observed disordered white matter is a property of the underlying neuronal pathology rather than an oligodendrocyte-mediated pathology and confirms our observations from the human subject.
Neuronal loss or damage is typically associated with gliosis, as seen in the human subject, so we next immunostained mouse cerebella with a marker for reactive gliosis, glial acidic fibrillary protein (GFAP) (Fig. 6C). Although Caskfloxed mice display some GFAP positivity, Caskfloxed;Calb2-Cre mice displayed an almost 2-fold higher level of astrogliosis compared to age-matched control Caskfloxed mice (Fig. 6D). Overall, this finding suggests that CASK loss-of-function produces protracted neuronal loss in the cerebellum, explaining why MICPCH typically becomes obvious a few months after birth. The cerebellar hypoplasia associated with loss of CASK represents disproportionate neuronal loss in the cerebellum.
Finally, we examined the functional loss associated with the cerebellar degeneration in Caskfloxed;Calb2-Cre mice. By P100, the mouse’s hindlimbs can no longer maintain normal righting, and the mice display hindlimb clasping with no obvious dystonic movement (Fig. 7A). Accelerating rotarod balance experiments suggest that even at P48, the mutant mice have a trend to underperform on a rotarod, indicating that the process of cerebellar degeneration and consequent functional degradation may be ongoing even before obvious locomotor defects are visually noticed within the cage. At P70 the mice are unable to perform on the rotarod at all, demonstrating a rapid degradation of locomotor coordination within a short span of 3 weeks (Fig. 7B). Additionally, the cerebellar degeneration and accompanying motor phenotype only manifest in the homozygous knockout of CASK from cerebellar cells and not in the heterozygous deletion (Fig. 7C-E) indicating that despite CASK being absent from approximately half the cells in the heterozygous deletion due to its X-linked nature, cerebellar degeneration requires total deletion of CASK. Overall, our data indicate that deletion of CASK does not affect brain development and the brain phenotype is unlikely due to defects of reelin function. Further, CASK loss leads to degeneration of cerebellar neurons leading to pronounced cerebellar atrophy that results in a progressive cerebellar ataxia.