An RNA-binding protein, Qki5, regulates embryonic neural stem cells through pre-mRNA processing in cell adhesion signaling.

Cell type-specific transcriptomes are enabled by the action of multiple regulators, which are frequently expressed within restricted tissue regions. In the present study, we identify one such regulator, Quaking 5 (Qki5), as an RNA-binding protein (RNABP) that is expressed in early embryonic neural stem cells and subsequently down-regulated during neurogenesis. mRNA sequencing analysis in neural stem cell culture indicates that Qki proteins play supporting roles in the neural stem cell transcriptome and various forms of mRNA processing that may result from regionally restricted expression and subcellular localization. Also, our in utero electroporation gain-of-function study suggests that the nuclear-type Qki isoform Qki5 supports the neural stem cell state. We next performed in vivo transcriptome-wide protein-RNA interaction mapping to search for direct targets of Qki5 and elucidate how Qki5 regulates neural stem cell function. Combined with our transcriptome analysis, this mapping analysis yielded a bona fide map of Qki5-RNA interaction at single-nucleotide resolution, the identification of 892 Qki5 direct target genes, and an accurate Qki5-dependent alternative splicing rule in the developing brain. Last, our target gene list provides the first compelling evidence that Qki5 is associated with specific biological events; namely, cell-cell adhesion. This prediction was confirmed by histological analysis of mice in which Qki proteins were genetically ablated, which revealed disruption of the apical surface of the lateral wall in the developing brain. These data collectively indicate that Qki5 regulates communication between neural stem cells by mediating numerous RNA processing events and suggest new links between splicing regulation and neural stem cell states.

Stage-dependent immunity orchestrates AQP4 antibody-guided NMOSD pathology: a role for netting neutrophils with resident memory T cells in situ.

Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune disease of the CNS characterized by the production of disease-specific autoantibodies against aquaporin-4 (AQP4) water channels. Animal model studies suggest that anti-AQP4 antibodies cause a loss of AQP4-expressing astrocytes, primarily via complement-dependent cytotoxicity. Nonetheless, several aspects of the disease remain unclear, including: how anti-AQP4 antibodies cross the blood-brain barrier from the periphery to the CNS; how NMOSD expands into longitudinally extensive transverse myelitis or optic neuritis; how multiphasic courses occur; and how to prevent attacks without depleting circulating anti-AQP4 antibodies, especially when employing B-cell-depleting therapies. To address these knowledge gaps, we conducted a comprehensive 'stage-dependent' investigation of immune cell elements in situ in human NMOSD lesions, based on neuropathological techniques for autopsied/biopsied CNS materials. The present study provided three major findings. First, activated or netting neutrophils and melanoma cell adhesion molecule-positive (MCAM+) helper T (TH) 17/cytotoxic T (TC) 17 cells are prominent, and the numbers of these correlate with the size of NMOSD lesions in the initial or early-active stages. Second, forkhead box P3-positive (FOXP3+) regulatory T (Treg) cells are recruited to NMOSD lesions during the initial, early-active or late-active stages, suggesting rapid suppression of proinflammatory autoimmune events in the active stages of NMOSD. Third, compartmentalized resident memory immune cells, including CD103+ tissue-resident memory T (TRM) cells with long-lasting inflammatory potential, are detected under "standby" conditions in all stages. Furthermore, CD103+ TRM cells express high levels of granzyme B/perforin-1 in the initial or early-active stages of NMOSD in situ. We infer that stage-dependent compartmentalized immune traits orchestrate the pathology of anti-AQP4 antibody-guided NMOSD in situ. Our work further suggests that targeting activated/netting neutrophils, MCAM+ TH17/TC17 cells, and CD103+ TRM cells, as well as promoting the expansion of FOXP3+ Treg cells, may be effective in treating and preventing relapses of NMOSD.

Brain-specific glycosylation of protein tyrosine phosphatase receptor type Z (PTPRZ) marks a demyelination-associated astrocyte subtype.

Astrocytes are the most abundant glial cell type in the brain, where they participate in various homeostatic functions. Transcriptomically, diverse astrocyte subpopulations play distinct roles during development and disease progression. However, the biochemical identification of astrocyte subtypes, especially by membrane surface protein glycosylation, remains poorly investigated. Protein tyrosine phosphatase receptor type zeta (PTPRZ) is a highly expressed membrane protein in CNS glia cells that can be modified with diverse glycosylation, including the unique HNK-1 capped O-mannosyl (O-Man) core M2 glycan mediated by brain-specific branching enzyme GnT-IX. Although PTPRZ modified with HNK-1 capped O-Man glycans (HNK-1-O-Man+ PTPRZ) is increased in reactive astrocytes of demyelination model mice, whether such astrocytes emerge in a broad range of disease-associated conditions or are limited to conditions associated with demyelination remains unclear. Here, we show that HNK-1-O-Man+ PTPRZ localizes in hypertrophic astrocytes of damaged brain areas in patients with multiple sclerosis. Furthermore, we show that astrocytes expressing HNK-1-O-Man+ PTPRZ are present in two demyelination mouse models (cuprizone-fed mice and a vanishing white matter disease model), while traumatic brain injury does not induce glycosylation. Administration of cuprizone to Aldh1l1-eGFP and Olig2KICreER/+ ;Rosa26eGFP mice revealed that cells expressing HNK-1-O-Man+ PTPRZ are derived from cells in the astrocyte lineage. Notably, GnT-IX but not PTPRZ mRNA was up-regulated in astrocytes isolated from the corpus callosum of cuprizone model mice. These results suggest that the unique PTPRZ glycosylation plays a key role in the patterning of demyelination-associated astrocytes.

Disruption of the anterior commissure in Olig2 deficient mice.

In the present study, we examined neural circuit formation in the forebrain of the Olig2 knockout (Olig2-KO) mouse model and found disruption of the anterior commissure at the late foetal stage. Axon bundles of the anterior commissure encountered the wall of the third ventricle and ceased axonal extension. L1-CAM immunohistochemistry showed that Olig2-KO mice lose decussation formation in the basal forebrain. DiI tracing revealed that the thin bundles of the anterior commissure axons crossed the midline but ceased further extension into the deep part of the contralateral side. Furthermore, some fractions of DiI-labelled axons were oriented dorsolaterally, which was not observed in the control mouse forebrain. The rostral part of the third ventricle was much wider in the Olig2-KO mice than in wild-type mice, which likely resulted in the delay of midline fusion and subsequent delay and malformation of the anterior commissure. We analysed gene expression alterations in the Olig2-KO mice using a public database and found multiple genes, which are related to axon guidance and epithelial-mesenchymal transition, showing subtle expression changes. These results suggest that Olig2 is essential for anterior commissure formation, likely by regulating multiple biological processes.

CCP1, a Regulator of Tubulin Post-Translational Modifications, Potentially Plays an Essential Role in Cerebellar Development.

The cytosolic carboxypeptidase (CCP) 1 protein, encoded by CCP1, is expressed in cerebellar Purkinje cells (PCs). The dysfunction of CCP1 protein (caused by CCP1 point mutation) and the deletion of CCP1 protein (caused by CCP1 gene knockout) all lead to the degeneration of cerebellar PCs, which leads to cerebellar ataxia. Thus, two CCP1 mutants (i.e., Ataxia and Male Sterility [AMS] mice and Nna1 knockout [KO] mice) are used as disease models. We investigated the cerebellar CCP1 distribution in wild-type (WT), AMS and Nna1 KO mice on postnatal days (P) 7-28 to investigate the differential effects of CCP protein deficiency and disorder on cerebellar development. Immunohistochemical and immunofluorescence studies revealed significant differences in the cerebellar CCP1 expression in WT and mutant mice of P7 and P15, but no significant difference between AMS and Nna1 KO mice. Electron microscopy showed slight abnormality in the nuclear membrane structure of PCs in the AMS and Nna1 KO mice at P15 and significant abnormality with depolymerization and fragmentation of microtubule structure at P21. Using two CCP1 mutant mice strains, we revealed the morphological changes of PCs at postnatal stages and indicated that CCP1 played an important role in cerebellar development, most likely via polyglutamylation.

Perturbation of monoamine metabolism and enhanced fear responses in mice defective in the regeneration of tetrahydrobiopterin.

Increasing evidence suggests the involvement of peripheral amino acid metabolism in the pathophysiology of neuropsychiatric disorders, whereas the molecular mechanisms are largely unknown. Tetrahydrobiopterin (BH4) is a cofactor for enzymes that catalyze phenylalanine metabolism, monoamine synthesis, nitric oxide production, and lipid metabolism. BH4 is synthesized from guanosine triphosphate and regenerated by quinonoid dihydropteridine reductase (QDPR), which catalyzes the reduction of quinonoid dihydrobiopterin. We analyzed Qdpr-/- mice to elucidate the physiological significance of the regeneration of BH4. We found that the Qdpr-/- mice exhibited mild hyperphenylalaninemia and monoamine deficiency in the brain, despite the presence of substantial amounts of BH4 in the liver and brain. Hyperphenylalaninemia was ameliorated by exogenously administered BH4, and dietary phenylalanine restriction was effective for restoring the decreased monoamine contents in the brain of the Qdpr-/- mice, suggesting that monoamine deficiency was caused by the secondary effect of hyperphenylalaninemia. Immunohistochemical analysis showed that QDPR was primarily distributed in oligodendrocytes but hardly detectable in monoaminergic neurons in the brain. Finally, we performed a behavioral assessment using a test battery. The Qdpr-/- mice exhibited enhanced fear responses after electrical foot shock. Taken together, our data suggest that the perturbation of BH4 metabolism should affect brain monoamine levels through alterations in peripheral amino acid metabolism, and might contribute to the development of anxiety-related psychiatric disorders. Cover Image for this issue: https://doi.org/10.1111/jnc.15398.

Nna1, Essential for Purkinje Cell Survival, Is also Associated with Emotion and Memory.

Nna1/CCP1 is generally known as a causative gene for a spontaneous autosomal recessive mouse mutation, Purkinje cell degeneration (pcd). There is enough evidence that the cytosolic function of the zinc carboxypeptidase (CP) domain at the C-terminus of the Nna1 protein is associated with cell death. On the other hand, this molecule's two nuclear localization signals (NLSs) suggest some other functions exist. We generated exon 3-deficient mice (Nna1N KO), which encode a portion of the N-terminal NLS. Despite the frameshift occurring in these mice, there was an expression of the Nna1 protein lacking the N-terminal side. Surprisingly, the pcd phenotype did not occur in the Nna1N KO mouse. Behavioral analysis revealed that they were less anxious when assessed by the elevated plus maze and the light/dark box tests compared to the control. Furthermore, they showed impairments in context-dependent and sound stimulus-dependent learning. Biochemical analysis of Nna1N KO mice revealed a reduced level of the AMPA-type glutamine receptor GluA2 in the hippocampal synaptosomal fraction. In addition, the motor protein kinesin-1, which transports GluA2 to dendrites, was also decreased. These results indicate that Nna1 is also involved in emotion and memory learning, presumably through the trafficking and expression of synaptic signaling molecules, besides a known role in cell survival.

Ddx20, DEAD box helicase 20, is essential for the differentiation of oligodendrocyte and maintenance of myelin gene expression.

Oligodendrocytes form myelin sheaths that surround axons, contributing to saltatory conduction and proper central nervous system (CNS) function. Oligodendrocyte progenitor cells (OPCs) are generated during the embryonic stage and differentiate into myelinating oligodendrocytes postnatally. Ddx20 is a multifunctional, DEAD-box helicase involved in multiple cellular processes, including transcription, splicing, microRNA biogenesis, and translation. Although defects in each of these processes result in abnormal oligodendrocyte differentiation and myelination, the involvement of Ddx20 in oligodendrocyte terminal differentiation remains unknown. To address this question, we used Mbp-Cre mice to generate Ddx20 conditional knockout (cKO) mice to allow for the deletion of Ddx20 from mature oligodendrocytes. Mbp-Cre;Ddx20 cKO mice demonstrated small body sizes, behavioral abnormalities, muscle weakness, and short lifespans, with mortality by the age of 2 months old. Histological analyses demonstrated significant reductions in the number of mature oligodendrocytes and drastic reductions in the expression levels of myelin-associated mRNAs, such as Mbp and Plp at postnatal day 42. The number of OPCs did not change. A thin myelin layer was observed for large-diameter axons in Ddx20 cKO mice, based on electron microscopic analysis. A bromodeoxyuridine (BrdU) labeling experiment demonstrated that terminal differentiation was perturbed from ages 2 weeks to 7 weeks in the CNS of Mbp-Cre;Ddx20 cKO mice. The activation of mitogen-activated protein (MAP) kinase, which promotes myelination, was downregulated in the Ddx20 cKO mice based on immunohistochemical detection. These results indicate that Ddx20 is an essential factor for terminal differentiation of oligodendrocytes and maintenance of myelin gene expression.

Olig2-Induced Semaphorin Expression Drives Corticospinal Axon Retraction After Spinal Cord Injury.

Axon regeneration is limited in the central nervous system, which hinders the reconstruction of functional circuits following spinal cord injury (SCI). Although various extrinsic molecules to repel axons following SCI have been identified, the role of semaphorins, a major class of axon guidance molecules, has not been thoroughly explored. Here we show that expression of semaphorins, including Sema5a and Sema6d, is elevated after SCI, and genetic deletion of either molecule or their receptors (neuropilin1 and plexinA1, respectively) suppresses axon retraction or dieback in injured corticospinal neurons. We further show that Olig2+ cells are essential for SCI-induced semaphorin expression, and that Olig2 binds to putative enhancer regions of the semaphorin genes. Finally, conditional deletion of Olig2 in the spinal cord reduces the expression of semaphorins, alleviating the axon retraction. These results demonstrate that semaphorins function as axon repellents following SCI, and reveal a novel transcriptional mechanism for controlling semaphorin levels around injured neurons to create zones hostile to axon regrowth.

Olig2 regulates terminal differentiation and maturation of peripheral olfactory sensory neurons.

The bHLH transcription factor Olig2 is required for sequential cell fate determination of both motor neurons and oligodendrocytes and for progenitor proliferation in the central nervous system. However, the role of Olig2 in peripheral sensory neurogenesis remains unknown. We report that Olig2 is transiently expressed in the newly differentiated olfactory sensory neurons (OSNs) and is down-regulated in the mature OSNs in mice from early gestation to adulthood. Genetic fate mapping demonstrates that Olig2-expressing cells solely give rise to OSNs in the peripheral olfactory system. Olig2 depletion does not affect the proliferation of peripheral olfactory progenitors and the fate determination of OSNs, sustentacular cells, and the olfactory ensheathing cells. However, the terminal differentiation and maturation of OSNs are compromised in either Olig2 single or Olig1/Olig2 double knockout mice, associated with significantly diminished expression of multiple OSN maturation and odorant signaling genes, including Omp, Gnal, Adcy3, and Olfr15. We further demonstrate that Olig2 binds to the E-box in the Omp promoter region to regulate its expression. Taken together, our results reveal a distinctly novel function of Olig2 in the periphery nervous system to regulate the terminal differentiation and maturation of olfactory sensory neurons.

Cofilin Signaling in the CNS Physiology and Neurodegeneration.

All eukaryotic cells are composed of the cytoskeleton, which plays crucial roles in coordinating diverse cellular functions such as cell division, morphology, migration, macromolecular stabilization, and protein trafficking. The cytoskeleton consists of microtubules, intermediate filaments, and actin filaments. Cofilin, an actin-depolymerizing protein, is indispensable for regulating actin dynamics in the central nervous system (CNS) development and function. Cofilin activities are spatiotemporally orchestrated by numerous extra- and intra-cellular factors. Phosphorylation at Ser-3 by kinases attenuate cofilin's actin-binding activity. In contrast, dephosphorylation at Ser-3 enhances cofilin-induced actin depolymerization. Cofilin functions are also modulated by various binding partners or reactive oxygen species. Although the mechanism of cofilin-mediated actin dynamics has been known for decades, recent research works are unveiling the profound impacts of cofilin dysregulation in neurodegenerative pathophysiology. For instance, oxidative stress-induced increase in cofilin dephosphorylation is linked to the accumulation of tau tangles and amyloid-beta plaques in Alzheimer's disease. In Parkinson's disease, cofilin activation by silencing its upstream kinases increases α-synuclein-fibril entry into the cell. This review describes the molecular mechanism of cofilin-mediated actin dynamics and provides an overview of cofilin's importance in CNS physiology and pathophysiology.

Disruption of dystonin in Schwann cells results in late-onset neuropathy and sensory ataxia.

Dystonin (Dst) is a causative gene for Dystonia musculorum (dt) mice, which is an inherited disorder exhibiting dystonia-like movement and ataxia with sensory degeneration. Dst is expressed in a variety of tissues, including the central nervous system and the peripheral nervous system (PNS), muscles, and skin. However, the Dst-expressing cell type(s) for dt phenotypes have not been well characterized. To address the questions whether the disruption of Dst in Schwann cells induces movement disorders and how much impact does it have on dt phenotypes, we generated Dst conditional knockout (cKO) mice using P0-Cre transgenic mice and Dst gene trap mice. First, we assessed the P0-Cre transgene-dependent Cre recombination using tdTomato reporter mice and then confirmed the preferential tdTomato expression in Schwann cells. In the Dst cKO mice, Dst mRNA expression was significantly decreased in Schwann cells, but it was intact in most of the sensory neurons in the dorsal root ganglion. Next, we analyzed the phenotype of Dst cKO mice. They exhibited a normal motor phenotype during juvenile periods, and thereafter, started exhibiting an ataxia. Behavioral tests and electrophysiological analyses demonstrated impaired motor abilities and slowed motor nerve conduction velocity in Dst cKO mice, but these mice did not manifest dystonic movements. Electron microscopic observation of the PNS of Dst cKO mice revealed significant numbers of hypomyelinated axons and numerous infiltrating macrophages engulfing myelin debris. These results indicate that Dst is important for normal PNS myelin organization and Dst disruption in Schwann cells induces late-onset neuropathy and sensory ataxia. MAIN POINTS: Dystonin (Dst) disruption in Schwann cells results in late-onset neuropathy and sensory ataxia. Dst in Schwann cells is important for normal myelin organization in the peripheral nervous system.

Glial pathology in a novel spontaneous mutant mouse of the Eif2b5 gene: a vanishing white matter disease model.

Vanishing white matter disease (VWM) is an autosomal recessive neurological disorder caused by mutation(s) in any subunit of eukaryotic translation initiation factor 2B (eIF2B), an activator of translation initiation factor eIF2. VWM occurs with mutation of the genes encoding eIF2B subunits (EIF2B1, EIF2B2, EIF2B3, EIF2B4, and EIF2B5). However, little is known regarding the underlying pathogenetic mechanisms or how to treat patients with VWM. Here we describe the identification and detailed analysis of a new spontaneous mutant mouse harboring a point mutation in the Eif2b5 gene (p.Ile98Met). Homozygous Eif2b5I98M mutant mice exhibited a small body, abnormal gait, male and female infertility, epileptic seizures, and a shortened lifespan. Biochemical analyses indicated that the mutant eIF2B protein with the Eif2b5I98M mutation decreased guanine nucleotide exchange activity on eIF2, and the level of the endoplasmic reticulum stress marker activating transcription factor 4 was elevated in the 1-month-old Eif2b5I98M brain. Histological analyses indicated up-regulated glial fibrillary acidic protein immunoreactivity in the astrocytes of the Eif2b5I98M forebrain and translocation of Bergmann glia in the Eif2b5I98M cerebellum, as well as increased mRNA expression of an endoplasmic reticulum stress marker, C/EBP homologous protein. Disruption of myelin and clustering of oligodendrocyte progenitor cells were also indicated in the white matter of the Eif2b5I98M spinal cord at 8 months old. Our data show that Eif2b5I98M mutants are a good model for understanding VWM pathogenesis and therapy development. Cover Image for this issue: doi: 10.1111/jnc.14751.

BPAG1 in muscles: Structure and function in skeletal, cardiac and smooth muscle.

BPAG1, also known as Dystonin or BP230, belongs to the plakin family of proteins, which has multiple cytoskeleton-binding domains. Several BPAG1 isoforms are produced by a single BPAG1 genomic locus using different promoters and exons. For example, BPAG1a, BPAG1b, and BPAG1e are predominantly expressed in the nervous system, muscle, and skin, respectively. Among BPAG1 isoforms, BPAG1e is well studied because it was first identified as an autoantigen in patients with bullous pemphigoid, an autoimmune skin disease. BPAG1e is a component of hemidesmosomes, the adhesion complexes that promote dermal-epidermal cohesion. In the nervous system, the role of BPAG1a is also well studied because disruption of BPAG1a results in a phenotype identical to that of Dystonia musculorum (dt) mutants, which show progressive motor disorder. However, the expression and function of BPAG1 in muscles is not well studied. The aim of this review is to provide an overview of and highlight some recent findings on the expression and function of BPAG1 in muscles, which can assist future studies designed to delineate the role and regulation of BPAG1 in the dt mouse phenotype and in human hereditary sensory and autonomic neuropathy type 6 (HSAN6).

Acute spatial spread of NO-mediated potentiation during hindpaw ischaemia in mice.

KEY POINTS: Neuropathic pain spreads spatially beyond the injured sites, and the mechanism underlying the spread has been attributed to inflammation occurring in the spinal cord. However, the spatial spread of spinal/cortical potentiation induced by conduction block of the peripheral nerves can be observed prior to inflammation. In the present study, we found that spreading potentiation and hypersensitivity acutely induced by unilateral hindpaw ischaemia are nitric oxide (NO)-dependent and that NO is produced by ischaemia and quickly diffuses within the spinal cord. We also found that NO production induced by ischaemia is not observed in the presence of an antagonist for group II metabotropic glutamate receptors (mGluRs) and that neuronal NO synthase-positive dorsal horn neurons express group II mGluRs. These results suggest strongly that NO-mediated spreading potentiation in the spinal cord is one of the trigger mechanisms for neuropathic pain. ABSTRACT: Cortical/spinal responses to hindpaw stimulation are bilaterally potentiated by unilateral hindpaw ischaemia in mice. We tested the hypothesis that hindpaw ischaemia produces nitric oxide (NO), which diffuses in the spinal cord to induce spatially spreading potentiation. Using flavoprotein fluorescence imaging, we confirmed that the spreading potentiation in hindpaw responses was induced during ischaemia in the non-stimulated hindpaw. This spreading potentiation was blocked by spinal application of l-NAME, an inhibitor of NO synthase (NOS). Furthermore, no spreading potentiation was observed in neural NOS (nNOS) knockout mice. Spinal application of an NO donor was enough to induce cortical potentiation and mechanical hypersensitivity. The spatial distribution of NO during unilateral hindpaw ischaemia was visualized using 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM). An increase in fluorescence derived from the complex of DAF-FM with NO was observed on the ischaemic side of the spinal cord. A similar but smaller increase was also observed on the contralateral side. Somatosensory potentiation after hindpaw ischaemia is known to be inhibited by spinal application of LY354740, an agonist of group II metabotropic glutamate receptors (mGluRs). We confirmed that the spinal DAF-FM fluorescence increases during hindpaw ischaemia were not observed in the presence of LY354740. We also confirmed that approximately half of the nNOS-positive neurons in the superficial laminae of the dorsal horn expressed mGluR2 mRNA. These results suggest that disinhibition of mGluR2 produces NO which in turn induces a spreading potentiation in a wide area of the spinal cord. Such spreading, along with the consequent non-specific potentiation in the spinal cord, may trigger neuropathic pain.

Feedback inhibition derived from the posterior parietal cortex regulates the neural properties of the mouse visual cortex.

Feedback regulation from the higher association areas is thought to control the primary sensory cortex, contribute to the cortical processing of sensory information, and work for higher cognitive functions such as multimodal integration and attentional control. However, little is known about the underlying neural mechanisms. Here, we show that the posterior parietal cortex (PPC) persistently inhibits the activity of the primary visual cortex (V1) in mice. Activation of the PPC causes the suppression of visual responses in V1 and induces the short-term depression, which is specific to visual stimuli. In contrast, pharmacological inactivation of the PPC or disconnection of cortical pathways from the PPC to V1 results in an effect of transient enhancement of visual responses in V1. Two-photon calcium imaging demonstrated that the cortical disconnection caused V1 excitatory neurons an enhancement of visual responses and a reduction of orientation selectivity index (OSI). These results show that the PPC regulates the response properties of V1 excitatory neurons. Our findings reveal one of the functions of the PPC, which may contribute to higher brain functions in mice.

Biallelic Variants in UBA5 Link Dysfunctional UFM1 Ubiquitin-like Modifier Pathway to Severe Infantile-Onset Encephalopathy.

The ubiquitin fold modifier 1 (UFM1) cascade is a recently identified evolutionarily conserved ubiquitin-like modification system whose function and link to human disease have remained largely uncharacterized. By using exome sequencing in Finnish individuals with severe epileptic syndromes, we identified pathogenic compound heterozygous variants in UBA5, encoding an activating enzyme for UFM1, in two unrelated families. Two additional individuals with biallelic UBA5 variants were identified from the UK-based Deciphering Developmental Disorders study and one from the Northern Finland Intellectual Disability cohort. The affected individuals (n = 9) presented in early infancy with severe irritability, followed by dystonia and stagnation of development. Furthermore, the majority of individuals display postnatal microcephaly and epilepsy and develop spasticity. The affected individuals were compound heterozygous for a missense substitution, c.1111G>A (p.Ala371Thr; allele frequency of 0.28% in Europeans), and a nonsense variant or c.164G>A that encodes an amino acid substitution p.Arg55His, but also affects splicing by facilitating exon 2 skipping, thus also being in effect a loss-of-function allele. Using an in vitro thioester formation assay and cellular analyses, we show that the p.Ala371Thr variant is hypomorphic with attenuated ability to transfer the activated UFM1 to UFC1. Finally, we show that the CNS-specific knockout of Ufm1 in mice causes neonatal death accompanied by microcephaly and apoptosis in specific neurons, further suggesting that the UFM1 system is essential for CNS development and function. Taken together, our data imply that the combination of a hypomorphic p.Ala371Thr variant in trans with a loss-of-function allele in UBA5 underlies a severe infantile-onset encephalopathy.

Direct Relay Pathways from Lemniscal Auditory Thalamus to Secondary Auditory Field in Mice.

Tonotopy is an essential functional organization in the mammalian auditory cortex, and its source in the primary auditory cortex (A1) is the incoming frequency-related topographical projections from the ventral division of the medial geniculate body (MGv). However, circuits that relay this functional organization to higher-order regions such as the secondary auditory field (A2) have yet to be identified. Here, we discovered a new pathway that projects directly from MGv to A2 in mice. Tonotopy was established in A2 even when primary fields including A1 were removed, which indicates that tonotopy in A2 can be established solely by thalamic input. Moreover, the structural nature of differing thalamocortical connections was consistent with the functional organization of the target regions in the auditory cortex. Retrograde tracing revealed that the region of MGv input to a local area in A2 was broader than the region of MGv input to A1. Consistent with this anatomy, two-photon calcium imaging revealed that neuronal responses in the thalamocortical recipient layer of A2 showed wider bandwidth and greater heterogeneity of the best frequency distribution than those of A1. The current study demonstrates a new thalamocortical pathway that relays frequency information to A2 on the basis of the MGv compartmentalization.

Deletion of exons encoding carboxypeptidase domain of Nna1 results in Purkinje cell degeneration (pcd) phenotype.

Purkinje cell degeneration (pcd) was first identified in a spontaneous mouse mutant showing cerebellar ataxia. In addition to cerebellar Purkinje cells (PCs), retinal photoreceptors, mitral cells in the olfactory bulb, and a discrete subpopulation of thalamic neurons also degenerate in the mutant brains. The gene responsible for the pcd mutant is Nna1, also known as ATP/GTP binding protein 1 or cytosolic carboxypeptidase-like 1, which encodes a zinc carboxypeptidase protein. To investigate pathogenesis of the pcd mutation in detail, we generated a conditional Nna1 allele targeting the carboxypeptidase domain at C-terminus. After Cre recombination and heterozygous crossing, we generated Nna1 knockout (KO) mice and found that the Nna1 KO mice began to show cerebellar ataxia at postnatal day 20 (P20). Most PCs degenerated until 4-week-old, except lobule X. Activated microglia and astrocytes were also observed in the Nna1 KO cerebellum. In the mutant brain, the Nna1 mRNA level was dramatically reduced, suggesting that nonsense-mediated mRNA decay occurs in it. Since the Nna1 protein acts as a de-glutamatase on the C-terminus of α-tubulin and ß-tubulin, increased polyglutamylated tubulin was detected in the Nna1 KO cerebellum. In addition, the endoplasmic reticulum stress marker, C/EBP homologous protein (CHOP), was up-regulated in the mutant PCs. We report the generation of a functional Nna1 conditional allele and possible mechanisms of PC death in the Nna1 KO in the cerebellum. OPEN PRACTICES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Developmental defects and aberrant accumulation of endogenous psychosine in oligodendrocytes in a murine model of Krabbe disease.

Krabbe disease (KD), or globoid cell leukodystrophy, is an inherited lysosomal storage disease with leukodystrophy caused by a mutation in the galactosylceramidase (GALC) gene. The majority of patients show the early onset form of KD dominated by cerebral demyelination with apoptotic oligodendrocyte (OL) death. However, the initial pathophysiological changes in developing OLs remain poorly understood. Here, we show that OLs of twitcher mice, an authentic mouse model of KD, exhibited developmental defects and impaired myelin formation in vivo and in vitro. In twitcher mouse brain, abnormal myelination and reduced expression of myelin genes during the period of most active OL differentiation and myelination preceded subsequent progressive OL death and demyelination. Importantly, twitcher mouse OL precursor cells proliferated normally, but their differentiation and survival were intrinsically defective. These defects were associated with aberrant accumulation of endogenous psychosine (galactosylsphingosine) and reduced activation of the Erk1/2 and Akt/mTOR pathways before apoptotic cell death. Collectively, our results demonstrate that GALC deficiency in developing KD OLs profoundly affects their differentiation and maturation, indicating the critical contribution of OL dysfunction to KD pathogenesis.