Complete Loss of CASK Causes Severe Ataxia Through Cerebellar Degeneration

Heterozygous loss of X-linked genes like CASK and MeCP2 (Rett syndrome) causes neurodevelopmental disorders (NDD) in girls, while in boys loss of the only allele of these genes leads to profound encephalopathy. The cellular basis for these disorders remains unknown. CASK is presumed to work through the Tbr1-reelin pathway in neuronal migration. Here we report clinical and histopathological analysis of a deceased 2-month-old boy with a CASK-null mutation. Although smaller in size, the CASK-null human brain exhibits normal lamination without defective neuronal differentiation, migration, or axonal guidance, excluding the role of reelin. The hypoplastic cerebellum instead displayed astrogliosis, a marker for neuronal loss. We therefore hypothesized that cerebellar hypoplasia with CASK loss is a result of early neurodegeneration. We generated a mouse line where CASK is completely deleted (hemizygous and homozygous) from post-migratory neurons in the cerebellum. Data conrm that a small cerebellum in CASK-loss results from post-developmental degeneration of cerebellar granule neurons. We further demonstrate that at least in cerebellum the functional loss with CASK deletion results secondary to degeneration of granule cells rather that any acute molecular functional loss of CASK. Intriguingly, female mice with heterozygous deletion of CASK in the cerebellum did not display any neurodegeneration. We suggest that NDDs like CASK mutation and Rett syndrome are pathologically neurodegenerative; however, random X-chromosome inactivation in the heterozygous mutant girls results in 50% of cells expressing the functional gene, resulting in a non-progressive pathology, whereas complete loss of the only allele in boys leads to unconstrained degeneration and encephalopathy.


Introduction
Heterozygous mutations in certain X-linked genes (e.g., CDKL5, MeCP2 in Rett syndrome, and CASK in MICPCH (mental retardation and microcephaly with pontine and cerebellar hypoplasia (OMIM: 300749)) are linked to postnatal microcephaly in girls (Seltzer and Paciorkowski 2014). Hemizygous mutations in these same genes give rise to progressive epileptic encephalopathy and lethality in boys (Jakimiec et al. 2020;Kankirawatana et al. 2006;Saitsu et al. 2012). Rett syndrome was the rst such disorder to be reported; it was described as a cerebral atrophic syndrome by Andreas Rett in 1966(Rett 1966. Until the 1990s, Rett syndrome was considered a neurodegenerative disorder (FitzGerald et al. 1990). With the discovery, however, of the MeCP2 gene association, postmortem autopsy observations, and the development of preclinical models, focus shifted to dendritic morphology and synapse development and dysfunction, resulting in the re-classi cation of Rett syndrome as a neurodevelopmental disorder (Neul and Zoghbi 2004) (Zoghbi 2003). Studies on the cellular pathology associated with MeCP2 loss in boys with epileptic encephalopathies have, however, been limited (Schule et al. 2008).
MICPCH is also considered to be a neurodevelopmental disorder that occurs due to heterozygous mutations in the X-linked gene CASK (calcium/calmodulin-dependent serine protein kinase) in girls.
Despite the microcephaly associated with CASK mutation being described as postnatal and progressive, females with MICPCH grow into adulthood, often with an intellectual disability that is non-progressive (Burglen et al. 2012;LaConte et al. 2018; Moog et al. 2011;Najm et al. 2008;Takanashi et al. 2012). Such mutations in hemizygous males are, however, lethal. These boys exhibit epileptic encephalopathy with pronounced cerebellar hypoplasia and progressive supratentorial atrophy (Moog et al. 2015;Saitsu et al. 2012). Regression of motor skills has also been noted in a girl with MICPCH in adolescence (Nishio et al. 2020). The cellular pathology of CASK-linked disorders remains uncertain. This problem is exacerbated by the fact that CASK-null mice die within hours of birth and do not exhibit a difference in brain size or morphology from their wild type littermates at birth (Atasoy et al. 2007). Based on the standard Theiler developmental staging of mice (Xue et al. 2013), the immediate postnatal period of mice best parallels the third trimester of human embryonic development (Carnegie staging; (O'Rahilly and Muller 2010)), making any interpretation of postnatal brain pathology di cult.
Although often considered to be a component of presynaptic terminals, CASK in fact is ubiquitously expressed in the body and has been implicated in a variety of functions (Hata et al. 1996;Stevenson et al. 2000). Outside of the brain, CASK has been shown to participate in cell proliferation (Ojeh et al. 2008), cell polarization (Caruana 2002), gap junctions and wound healing (Marquez-Rosado et al. 2012), insulin secretion and signaling (Wang et al. 2006;Zhu et al. 2014), hypoxia response Weigand et al. 2012), renal development and disease (Ahn et al. 2013;Beaudreuil et al. 2019), spermatogenesis and sperm motility (Aravindan et al. 2012;Burkin et al. 2004), and cardiac conductivity (Beuriot et al. 2020;Eichel et al. 2016), to name a few examples. In the brain, CASK has been examined as both a pre-and post-synaptic molecule (Butz et al. 1998;Hsueh et al. 1998). It has also been suggested that CASK is involved in protein tra cking via its interaction with SAP97 (Jeyifous et al. 2009;Lin et al. 2013). CASK is proposed to be involved in axonal branching (Kuo et al. 2010), dendritic arborization (Gao et al. 2018), dendrite spinogenesis (Chao et al. 2008) and synaptogenesis (Samuels et al. 2007). Thus, many hypotheses as to why loss of CASK leads to defects in brain development can be proposed.
CASK also has a function in regulating gene transcription (Hsueh et al. 2000;Wang et al. 2004a;Wang et al. 2004b). It has been suggested that CASK translocates to the nucleus, where it regulates the function of T-box transcription factor (Tbr-1) (Bredt 2000;Hsueh et al. 2000). It is proposed that CASK forms a ternary complex together with CINAP (CASK-interacting nucleosome assembly protein) and Tbr-1 to induce expression of molecules such as reelin that play a crucial role in brain development (Wang et al. 2004a). Reelin is a secreted extracellular molecule critical for neuronal migration (Hirotsune et al. 1995).
Indeed, both in the reeler mice and Tbr-1 knockout mice, defects in proper lamination of cortex are seen (Hamburgh 1963;Hevner et al. 2001). In addition to the cortex, reeler mice also display a hypoplastic disorganized cerebellum with defects in neuronal migration and suppressed neurogenesis (Hamburgh 1963). The neurodevelopmental function of CASK has been speci cally attributed to the CASK's interaction with Tbr-1and presumed regulation of reelin expression (Najm et al. 2008;Namavar et al. 2012;Takanashi et al. 2010).
Here, we report a detailed clinical description and autopsy ndings from a 2-month-old boy harboring the CASK null mutation R27*. Although the brain is small, the clear presence of tertiary gyri that form near term, as well as proper cortical and cerebellar lamination, argue against defects in neuronal migration; instead we uncover evidence of neurodegeneration suggested by reactive astrogliosis in the cerebellum.
We then design and execute a genetic experiment in mice that provides conclusive evidence that loss of CASK indeed produces neurodegeneration in the cerebellum. Most pontocerebellar hypoplasias (PCH) are progressive, but based on the postnatal brain growth pattern, it has been hypothesized that MICPCH has a distinct pathogenic mechanism (van Dijk et al. 2020). We instead provide evidence that mechanistically, MICPCH in girls with heterozygous CASK mutations is also degenerative, and the non-progressive course of MICPCH is dictated by uniqueness of the X-linked inheritance pattern in which 50% of brain cells express the normal gene.

Statement of ethics
All studies described herein were approved by the Virginia Tech Institutional Animal Care and Use Committee and Institutional Review Board.

Statistics
A two-tailed Student's t-test was used as a comparison between two genotypes in each experiment to compute signi cance with an alpha of 0.05.

Clinical History
The decedent was a male born via vaginal delivery at 36.1 weeks of gestation to a 34-year-old woman, G3, P3, A1 (Gravida, para, abortus). He was conceived through in vitro fertilization with a sperm donor. He was born as a monochorionic, diamniotic twin. Ultrasound at the third trimester indicated the presence of slight microcephaly and smaller cerebellum which raised some concerns. He was also small for gestational age with a weight of 2.4 kg (0% Percentile, Z score -7.37), length of 43 cm (4 th Percentile, Z score -1.71) and head circumference of 30 cm (4 th percentile, Z score-1.79). The Apgar scores at 1 and 5 minutes after birth were recorded as 8 and 9. The decedent was discharged from the hospital 2 days after birth. At home he became apneic with hypoventilation and was readmitted to a hospital 4 days later. Despite positive airway pressure ventilation, the apneic spells continued which led to neurological and genetic investigations. He was then diagnosed with microcephaly and pontocerebellar hypoplasia with CASK mutation. He displayed poor feeding, profound hypotonia, microcephaly, micrognathia, bilateral clubfoot, and vertical chordee with penile torsion. Oral-pharyngeal motility studies revealed mild to moderate oral motor dysphagia; there were episodes of silent aspirations with very limited re ux. A gastrostomy tube placement was performed. Fluctuations in body temperature with hypothermia and heart rate were also noted. Within 3 weeks after his birth, torso exions were noted occurring 2-3 times a day. He also displayed tics in the hands, feet and neck which lasted for several seconds to several minutes. The decedent developed irritability and intolerance to feeds, hypothermia and acute respiratory failure with apnea. A surface, 25-channel video electroencephalography (vEEG) was performed using an international 10-20 system. A diagnosis of Ohtahara syndrome was established due to the presence of a typical burst suppression pattern. He was started on keppra and a ketogenic diet. Possibility of long-term palliative care including tracheostomy was discussed, but a decision was made against aggressive continued therapy. He passed away 2 months and 6 days after birth.

EEG Spectral Analysis
Raw data were trimmed for artifacts by a trained observer in the clinic. Data were analyzed in MATLAB 2017a using the EEGLab toolbox. After ltering from 0.01-50Hz, bad channels were removed based on spectral power. Spectral power was plotted for each channel independently using the spectopo() function with a window length of 256 samples, FFT length of 256, and 0 overlap in the entire 0.01-50Hz frequency band. Channels covering each of a given lobe (frontal, parietal, temporal, occipital, central) were then grouped and mean power spectral density was calculated within each biologically relevant frequency band: delta, alpha, beta, theta, and low gamma. Time-frequency plots were generated for a representative 1 minute of the recording using a divisive baseline.

Generation of Mouse Lines
Calb2-Cre mice (strain 010774) was obtained from Jackson Laboratory, Cask oxed mice (strain 006382) was a kind gift from Prof. Thomas Südhof. Cask oxed females were bred with Calb2-Cre positive males to generate the F1 cross Cask oxed ::Calb2-Cre. F1 mice were bred to Ai14-LSL-tdTomato-positive males obtained from Jackson Laboratory (strain 007914) to generate uorescent reporter mice. F1 mice were genotyped by PCR using primers targeted at either LoxP elements, a sequence within the Cre gene, or a sequence within the tdTomato gene. All lines were from a C57BL/6J background backcrossed for at least 25 generations.

Antibodies and Material Reagents
Bassoon monoclonal antibodies were obtained from Enzo Lifescience, GFAP monoclonal antibodies were obtained from Invitrogen, calbindin polyclonal antibody from Invitrogen, synaptophysin antibody from Sigma and secondary antibodies conjugated with AlexaFluor 488, 550 and 633 were obtained from Thermo sher. Hardset Vectashield TM with DAPI was obtained from Vector Laboratories.

Immunostaining of Mouse Tissue
For all immunostaining, mice were sacri ced by trans-cardiac perfusion rst with phosphate buffered saline (PBS) for exsanguination and subsequently with 4% paraformaldehyde for xation of tissues. Brains were dissected and post-xed for at least 24 hours in 4% paraformaldehyde. After post-xation, brains were hemisected along the longitudinal ssure and 50µm sagittal sections were cut using a ThermoScienti c™ Microm HM650V Vibratome. Sections were submerged in permeabilization/blocking solution composed of 10% fetal bovine serum and 1% Triton-X 100 in PBS overnight at +4°C.
Rabbit anti-calbindin was diluted at 1:50 in blocking solution and mouse anti-bassoon was diluted at 1:200 in blocking solution. For GFAP immunostaining, mouse anti-GFAP was diluted at 1:200 in blocking solution. After blocking/permeabilization overnight, free-oating sections were incubated for 3 hours in dilute primary antibody at room temperature. After incubation in primary antibody, sections were washed 3 times for 5 minutes in PBS before being incubated in secondary antibody for the respective host species for 1 hour at room temperature. Sections were again washed and mounted on slides using VECTASHIELD® anti-fade medium. Quanti cation of synapse density was conducted using the SynQuant algorithm 80 .

Immunostaining of Human Tissue
Human tissue obtained during autopsy was post-xed in 10% formalin overnight, embedded in para n, and subsequently sectioned into 20µm sections onto charged slides. Slides were depara nized with 3 changes of poly-xylenes for 10 minutes each time and rehydrated using an ethanol gradient from 100%-95%-70%-50%-H 2 O for 5 minutes in each condition. Antigen retrieval was conducted by boiling slides in 10mM sodium citrate buffer with 0.1% Tween-20 for 10 minutes in a domestic microwave followed by running the slides under cold tap water for 10 minutes. Immunostaining for GFAP, calbindin and synaptophysin was then conducted using the same procedure described for mouse tissue.

Motor Behavioral Assays
Accelerating Rotarod experiments were conducted by placing 4 Cask ( oxed) ::Calb2-Cre mice at P100 (postataxia onset), at P48 (pre-ataxia onset), and age-matched Cask ( oxed) control mice on an accelerating Rotarod, beginning at 2 cycles/minute and accelerating at a rate of 5 cycles/minute until mice fell off the platform. Three trials were conducted in succession for each mouse, with 5 minutes of rest between trials.

Results
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 con guration without severe deterioration, indicating that the disorder is non-progressive (Supplemental Figure 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 di cult to study to date. Here we describe detailed clinical ndings 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 ( Figure 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 Figure 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 lled with uid ( Figure 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 ( Figure 1C and Supplemental Figure 3). This EEG pattern is typical of Ohtahara syndrome, a devastating epileptic encephalopathy, that usually co-occurs with CASK-null mutations (Moog et al. 2015;Mukherjee et al. 2020;Saitsu et al. 2012). 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 ( Figure 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 ( Figure 2A). Except for lung, heart, and spleen, most other organs were smaller than expected but had an overall normal gross appearance ( Figure 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 con guration. The Sylvian ssure was well formed, and the leptomeninges were clear ( Figure 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 Figure 4). The thalamus was normally formed and rm. 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 ( Figure 2C, Supplemental Figure 4). The cerebellum, despite hypoplasia, had a normal con guration but did not exhibit the usual folia. There was no evidence for heterotopia of cells. The anterior vermis was not identi able and appeared to be membrane-like; cerebellar hemispheres were thin, attened and rm. 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 de ned 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 ( Figure 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 ( Figure 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 Figure 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 Figure 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 Figure 5C, D). The heart rate varied between 90 and 150 beats per minute and displayed a sinus rhythm (Supplemental Figure 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 (Atasoy et al. 2007).
The normal lamination and con guration 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 ( Figure 3). Previous studies in the murine model have shown that CASK loss-of-function does not negatively impact synapse formation (Atasoy et al. 2007;Srivastava et al. 2016), 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 ( Figure  3A, B). GFAP staining of the cerebellum to detect astrogliosis, however, indicates that, compared to the control, the decedent exhibits ~5-fold higher amounts of GFAP immunoreactivity, speci cally in the IGL ( Figure 3A, C, D). In fact, large reactive astrocytes in the IGL were readily observed ( Figure 3C). Together these data suggest that loss of CASK produces delayed neurodegenerative changes, causing the CASKlinked phenotype to typically manifest postnatally. We nally investigated myelination within the cerebellar cortex using FluoroMyelin lipid staining ( Figure 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, speci cally in the IGL. To test this idea, we next employed murine genetic experiments, where CASK is deleted in a temporally and spatially speci ed manner using Cre-LoxPmediated 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 longterm 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 (Srivastava et al. 2016); 2) Purkinje cell-speci c knockout mice, which display normal development and motor function (Srivastava et al. 2016); and nally, 3) mice with Cask deletion in a distributed subpopulation of granule cells, which do not exhibit altered cell migration or survival (Srivastava et al. 2016). 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 (Barski et al. 2000;Saul et al. 2008;Zhu et al. 2001); 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 (Kerr et al. 2019). 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 (Bearzatto et al. 2006;Schiffmann et al. 1999), but the exact timing of initiation of Calb2-Cre gene recombination in granule cells was not known. There have also been con icting reports about the expression of Calb2 in the Purkinje cells within the cerebellum (Bearzatto et al. 2006;Schiffmann et al. 1999). We therefore rst tested the recombination speci city 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) ( Figure 4A). The distribution of the tdTomato-expressing neurons serves as a proxy for CASK deletion when Calb2-Cre mice are crossed with Cask oxed mice in parallel ( Figure 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 ( Figure 4C). By P15, Calb2-Cre already exhibited robust recombination in many parts of the brain and in the entirety of postmigratory 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 ( Figure 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 ( Figure 4E). Our data thus indicate that Calb2-Cre speci cally 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 Cask oxed lines. It has been shown previously that the Cask oxed mouse is a hypomorph that expresses ~40% CASK, likely due to a phenomenon known as selection cassette interference (Atasoy et al. 2007). Cask oxed mice are smaller than wild type mice and exhibit cerebellar hypoplasia (Atasoy et al. 2007;Najm et al. 2008;Srivastava et al. 2016).
Cask oxed ;Calb2-Cre F1 mice were genotyped by PCR. Cask oxed ;Calb2-Cre mice remain indistinguishable from the Cask oxed mice well into adulthood (~40 days), indicating that acute deletion of Cask does not have signi cant effects on cerebellar development, motor learning, or locomotor function. Past two months of age, however, Cask oxed ;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 de cits, the Cask oxed ;Calb2-Cre mice are otherwise healthy and display a slick coat, good body condition score, and are bright, alert and responsive. Compared to littermate Cask oxed controls, the cerebellum of the Cask oxed ;Calb2-Cre mouse is extremely diminished in volume at P100 when the motor phenotype has plateaued ( Figure 5A, B). Comparing the histology of the Cask oxed ;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 Cask oxed ;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 ( Figure 5C). The molecular layer of the cerebellum is thin and collapsed, most likely due to loss of parallel bers arising from the granular cells and loss of synaptic connections between granule cells and Purkinje cells ( Figure 5D-G). We therefore next quanti ed synaptic connections within the cerebellar layers using bassoon as a pre-synaptic marker. As seen ( Figure 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 bers. 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 (Kerr et al. 2019). Our data here thus indicate that loss of CASK results in the disproportionate degeneration of a speci c 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 ( Figure 3E). We therefore quanti ed myelin in the Cask oxed ;Calb2-Cre mice using FluoroMyelin TM staining. As seen in Figure 6A, the myelin appears to be disorganized in the white matter of folia from the Cask oxed ;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 ( Figure 6A). Quanti cation of pixels displayed a strong trend towards a decrease in myelinated bers which did not reach statistical signi cance ( Figure 6B). The degeneration of cerebellar grey matter thus is also associated with disorganization of the white matter in the Cask oxed ;Calb2-Cre mouse cerebellum, and the broadened white matter layer is likely to be lled only with acellular matrix. Because the Cask oxed ;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 con rms 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 brillary protein (GFAP) ( Figure 6C). Although Cask oxed mice display some GFAP positivity, Cask oxed ;Calb2-Cre mice displayed an almost 2-fold higher level of astrogliosis compared to age-matched control Cask oxed mice ( Figure  6D). Overall, this nding 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 Cask oxed ;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 ( Figure 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 ( Figure 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 ( Figure 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.

Discussion
Developmental disorders are de ned based on their clinical course rather than cellular pathology. Presentation of a severe, chronic disability (mental and/or physical impairment) in three or more areas of major life activity by the age of 22 that is likely to continue through the individual's lifetime is classi ed as a developmental disorder (Developmental Disabilities Assistance and Bill of Rights Act of 2000). Strategies for molecular therapeutic intervention, however, are more likely to be dependent on cellular pathology rather than the clinical course of a given disorder. Conditions associated with mutations in the human CASK gene have been described as developmental disorders (Burglen et al. 2012;Moog et al. 2011). CASK is a ubiquitously expressed gene and has been proposed to have a function in a variety of organs including the intestine, kidney, heart and brain (Hata et al. 1996;Stevenson et al. 2000), and mutations in CASK produce microcephaly as well as somatic growth retardation (Burglen et al. 2012;Moog et al. 2011). In boys who do not express CASK, a clear picture has emerged consisting of neurodevastation, microcephaly, pontocerebellar hypoplasia (PCH), and a consistently abnormal EEG pattern characterized by disorganization, low frequency, attenuation and discontinuity. CASK null boys are thus likely to be diagnosed with epileptic encephalopathies such as Ohtahara syndrome and West syndrome. Despite uniform neurological ndings in these subjects, ndings involving other organ systems remain inconsistent and often unremarkable. Our analysis of CASK null mutations in boys indicates that the function/s of CASK that are critical for survival are brain-speci c; all other organ systems can function within the normal range without CASK 16 . In fact, we have previously demonstrated that deletion of CASK in neurons is su cient to produce somatic and brain size reduction in mice (Srivastava et al. 2016). The thinning and dysfunction of the brain stem manifests as aberrant respiratory, deglutition and cardiovascular re exes, and it is this dysfunction that underlies the lethality associated with CASK loss in mammals (Atasoy et al. 2007;Mukherjee et al. 2020).
Neurodevelopment could be stalled at several steps of brain development. This includes cell proliferation, neuronal differentiation and polarization, neuronal migration and nal neuronal maturation including axonal and dendritic growth and synaptogenesis. At a molecular level CASK has been proposed to play a role in all of these processes. Evidence exists that suggests CASK participates in mitotic spindle orientation and cell proliferation (Porter et al. 2019), in cell polarization (Caruana 2002), in axonal and dendritic maturation and synaptogenesis (Gao et al. 2018;Kuo et al. 2010;Samuels et al. 2007). Within the synapse CASK can be found in molecular complexes that include other important molecules like Mint1, Caskin, liprin-α and the adhesion molecule neurexin (Butz et al. 1998;Hata et al. 1996;LaConte et al. 2016;Tabuchi et al. 2002). CASK is a kinase that phosphorylates neurexin and is likely to regulate this complex formation (Cortese et al. 2016;LaConte et al. 2016;Mukherjee et al. 2010;Mukherjee et al. 2008). The most accepted notion of CASK in neurodevelopment has, however, been the role of CASK in neuronal migration via its interaction with CINAP and Tbr-1 resulting in the upregulation of reelin transcription (Hsueh et al. 2000;Mori et al. 2019;Najm et al. 2008;Wang et al. 2004a). Our analysis of the CASK-null human brain is, however, unable to support any of these putative roles of CASK in neurodevelopment.
Genetic manipulation of Cask in murine models has demonstrated that CASK-linked murine brain pathology is postnatal and not likely to be a developmental defect (Kerr et al. 2019;Srivastava et al. 2016). Although the delivery of the decedent described here was late preterm, Apgar scores were normal, and the infant was released from the hospital without concern. Rapid regression within days to weeks has been noted in this and other boys with CASK-null mutations, indicating that although degeneration may begin in the third trimester, it continues rapidly throughout infancy. Despite a smaller size, the gross and histological ndings in a brain without CASK are minimal. Overall, the brain con guration, vasculature, ventricular system, meninges, brain lamination and neuronal migration all remained unaltered. This indicates that in the presence of CASK mutation, embryonic brain development appears unchanged, with no defect in neuronal differentiation and migration. The ndings from neuroimaging and histological studies of human cases are consistent with the ndings of CASK knockout mice where the brain size, lamination and synapse formation are all normal at birth (Atasoy et al. 2007). In fact, absence of an acute locomotor effect in Cask oxed ;Calb2-Cre mice excludes a synaptic role of CASK in cerebellar function. Overall our study here indicates that CASK is not likely to work in neurodevelopment via the purported Tbr-1-reelin pathway. This interpretation is in line with the observation that in the mouse model, abrogation of the CASK-Tbr-1 interaction does not affect brain size or lamination (Huang and Hsueh 2017). A number of CASK missense mutations have been identi ed that are associated with intellectual disability and MICPCH. Recent studies on these missense mutations have also indicated that the CASK-Tbr-1 interaction is an unlikely mechanism of MICPCH 11, 74 .
In girls with heterozygous CASK mutations, the predominant manifestations are also brain-related (Burglen et al. 2012;Moog et al. 2011). Our data presented here demonstrate that although Cask +/mice have cerebellar hypoplasia, they do not signi cantly degenerate further into old age, which agrees with the clinical de nition of a neurodevelopmental disease. In girls with MICPCH and in Cask +/mice, however, ~50% of cells still express CASK (Mori et al. 2019;Srivastava et al. 2016), confounding the study of neuropathology and making it di cult to draw rm conclusions. Conditional genetic animal model experimentation allows us to overcome the di culties presented by this X-linked condition and also helps separate the neurodegenerative pathology of CASK loss from the developmental phase.
CASK deletion in mice is lethal, but by generating otherwise healthy mice that do not express Cask in many parts of the brain including the cerebellum, we are able to clearly demonstrate that lack of CASK produces cerebellar atrophy and degeneration. CASK has previously been identi ed as a biomarker for several neurodegenerative disorders George et al. 2018;Morello et al. 2019;Strand et al. 2005); our study thus explains these previous unbiased ndings. CASK loss, however, does not uniformly produce neurodegeneration; for example, loss of CASK is also associated with optic nerve hypoplasia, but unexpectedly, CASK deletion from retinal ganglion cells (whose axons form the optic nerve) does not negatively affect optic nerve pathology in mice of the same genotype (Kerr et al. 2019).
Similarly, Calb2 is present in many cortical interneurons and neurons of the olfactory bulb, hippocampus and striatum, but we do not observe death of these cells in the Cask oxed ;Calb2-Cre mice. In fact, a closer look within the cerebellum suggests that the cerebellar degeneration may be primarily due to granule cell death, making this condition most similar to Norman-type cerebellar atrophy (OMIM: 213200). Many Purkinje cells, which are among the rst cerebellar cells to express Calb2-Cre, do not die off but rather persist throughout the lifespan, even following the appearance of ataxia. Thus CASK loss may affect neurons differentially.
PCH pathologies are typically thought to be neurodegenerative in their origin beginning antenatally (van Dijk et al. 2020). Defects in both energy production and protein metabolism (speci cally, protein synthesis) are known to disproportionately affect the cerebellum and are likely causes of PCH (Kasher et al. 2011). In the case of CASK mutation, PCH has been described as neurodevelopmental, mostly due to the non-progressive course seen in females (van Dijk et al. 2018). In contrast to hemizygous and homozygous Cask oxed ;Calb2-Cre mice, the heterozygous Cask oxed ;Calb2-Cre mice fail to exhibit cerebellar degeneration ( Figure 7C-E). Our ndings here thus suggest that CASK-linked PCH is also neurodegenerative and the arrest of neurodegeneration in girls most likely arises from mosaicism of the defective X-linked gene, which guarantees that ~50% of brain cells retain a normally functioning CASK gene ( Figure 8). Mechanistically, our ndings demonstrating that CASK is also likely to function in pathways associated with energy production and protein metabolism Srivastava et al. 2016), con rming that MICPCH shares not only the pathogenic mechanism, but also a common molecular pathway with other types of PCH.
Heterozygous mutations in X-linked genes other than CASK, such as MeCP2 and CDKL5, are associated with postnatal microcephaly in girls (Seltzer and Paciorkowski 2014). Intriguingly, subjects with mutations in these genes show an initial normal developmental trajectory followed by developmental arrest and delay (regression) (Fehr et al. 2013;Hagberg et al. 1983). Mutational analysis of orthologous genes in murine models also produces phenotypes which are clearly post-developmental, presenting only in adulthood (Guy et al. 2001). Incidentally, just like CASK mutations, CDKL5 and MeCP2 mutations in males are associated with epileptic encephalopathy (Jakimiec et al. 2020;Kankirawatana et al. 2006). These data raise the possibility that even in disorders such as Rett syndrome (MeCP2 mutation) and CDKL5 de ciency, the pathology may be neurodegenerative, as originally suggested by Andreas Rett (Rett 1966), but the clinical course may not be progressive in females due to the mosaic expression of the normal gene under heterozygous conditions.

Conclusions
Overall our data indicate that loss of CASK induces a very early neurodegeneration. Due to the delay in CASK deletion in the current murine model we are conclusively able to temporally separate brain development from the CASK-linked degenerative process. Further, locomotor assessment indicates that the loss-of-function that associates with CASK mutations may be linked to the degenerative process rather than manifestation of loss of CASK molecular function. Thus, our ndings suggest an early detection of CASK mutation after birth may provide us with a window period where therapeutic intervention is likely to halt the disease process.

Declarations
Ethics Approval and Consent to Participate: All studies described herein were approved by the Virginia Tech Institutional Animal Care and Use Committee and Virginia Tech Institutional Review Board.

Consent for Publication:
Informed consent has been taken from parents for this publication.

Availability of Data and Materials:
Raw datasets used in the present study are available upon reasonable request to the corresponding author.
Competing Interests: The authors have declared that no con ict of interest exists.

Funding:
The work was supported with funding from the NIH National Eye Institute (R01EY024712) to KM.  The R27Ter mutation results in early truncation of CASK, pontocerebellar hypoplasia and slowed EEG in a human subject. A) Location of the R27Ter mutation in the male decedent; top row: exons of the human CASK gene; bottom row: corresponding CASK protein domains. B) Brain MRI scan obtained at 2 weeks revealed severely diminished cerebellar and pontine size with otherwise normal formation of cortical gyri; sagittal, coronal, and transverse planes can be seen from left to right, respectively. C) A representative example of discontinuous EEG pattern (arrow and red line in the EEG trace); more examples of burst suppression can be found in Supplemental Figure 3. D) Power spectral density curves in the 0.01-50Hz range for each electrode independently; colors correspond to underlying cortical location of a given electrode. E) Quanti cation of mean power spectral density for each electrode in the entire 0.01-50Hz range. F) Mean power spectral density divided into biologically relevant frequency bands of delta, theta, alpha, beta, and low gamma divided by lobe; error bars represent standard deviation between electrodes within a given lobe. G) Representative time frequency plots of 1 minute of the recording by electrode position.

Figure 2
Absence of CASK leads to smaller organs including brain with normal histology of cerebellum and no defect in lamination or cellular migration. A) Organ weight of the decedent relative to average weight for       Model describing zygosity-based mechanism of a neurodevelopmental versus neurodegenerative clinical course of CASK-linked phenotype based on random X-chromosome inactivation. CASK is an X-linked gene critical for maintenance of cerebellar neurons. Heterozygous mutation in CASK produces CASK lossof-function in only 50% of neurons (red). In the heterozygous condition (red and gray), neurodegeneration thus plateaus (bottom middle), causing an apparent neurodevelopmental disorder, whereas hemizygous CASK mutations (red and purple) in male mice or homozygous CASK mutation (two reds) in female mice produce a progressive phenotype typical of neurodegeneration with severe ataxia (bottom right).

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