Usher syndrome type 3 (USH3) is an autosomal recessive disorder characterized by progressive hearing loss, severe retinal degeneration, and variably present vestibular dysfunction, assigned to 3q21-q25. Here, we report on the positional cloning of the USH3 gene. By haplotype and linkage-disequilibrium analyses in Finnish carriers of a putative founder mutation, the critical region was narrowed to 250 kb, of which we sequenced, assembled, and annotated 207 kb. Two novel genes-NOPAR and UCRP-and one previously identified gene-H963-were excluded as USH3, on the basis of mutational analysis. USH3, the candidate gene that we identified, encodes a 120-amino-acid protein. Fifty-two Finnish patients were homozygous for a termination mutation, Y100X; patients in two Finnish families were compound heterozygous for Y100X and for a missense mutation, M44K, whereas patients in an Italian family were homozygous for a 3-bp deletion leading to an amino acid deletion and substitution. USH3 has two predicted transmembrane domains, and it shows no homology to known genes. As revealed by northern blotting and reverse-transcriptase PCR, it is expressed in many tissues, including the retina.

The progressive myoclonus epilepsies, featuring the triad of myoclonus, seizures, and ataxia, comprise a large group of inherited neurodegenerative diseases that remain poorly understood and refractory to treatment. The Cystatin B gene is mutated in one of the most common forms of progressive myoclonus epilepsy, Unverricht-Lundborg disease (EPM1). Cystatin B knockout in a mouse model of EPM1 triggers progressive degeneration of cerebellar granule neurons. Here, we report impaired redox homeostasis as a key mechanism by which Cystatin B deficiency triggers neurodegeneration. Oxidative stress induces the expression of Cystatin B in cerebellar granule neurons, and EPM1 patient-linked mutation of the Cystatin B gene promoter impairs oxidative stress induction of Cystatin B transcription. Importantly, Cystatin B knockout or knockdown sensitizes cerebellar granule neurons to oxidative stress-induced cell death. The Cystatin B deficiency-induced predisposition to oxidative stress in neurons is mediated by the lysosomal protease Cathepsin B. We uncover evidence of oxidative damage, reflected by depletion of antioxidants and increased lipid peroxidation, in the cerebellum of Cystatin B knock-out mice in vivo. Collectively, our findings define a pathophysiological mechanism in EPM1, whereby Cystatin B deficiency couples oxidative stress to neuronal death and degeneration, and may thus provide the basis for novel treatment approaches for the progressive myoclonus epilepsies.

Alternative oxidase (AOX) is a non‐mammalian enzyme that can bypass blockade of the complex III‐IV segment of the respiratory chain (RC). We crossed a Ciona intestinalis AOX transgene into RC complex III (cIII)‐deficient Bcs1l p.S78G knock‐in mice, displaying multiple visceral manifestations and premature death. The homozygotes expressing AOX were viable, and their median survival was extended from 210 to 590 days due to permanent prevention of lethal cardiomyopathy. AOX also prevented renal tubular atrophy and cerebral astrogliosis, but not liver disease, growth restriction, or lipodystrophy, suggesting distinct tissue‐specific pathogenetic mechanisms. Assessment of reactive oxygen species (ROS) production and damage suggested that ROS were not instrumental in the rescue. Cardiac mitochondrial ultrastructure, mitochondrial respiration, and pathological transcriptome and metabolome alterations were essentially normalized by AOX, showing that the restored electron flow upstream of cIII was sufficient to prevent cardiac energetic crisis and detrimental decompensation. These findings demonstrate the value of AOX, both as a mechanistic tool and a potential therapeutic strategy, for cIII deficiencies.

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) is a hereditary neurodegenerative disorder caused by mutations in the cystatin B (CSTB) gene encoding an inhibitor of cysteine proteases. Here, we provide the first detailed description of the onset and progression of pathologic changes in the CNS of Cstb-deficient (Cstb) mice. Our data reveal early and localized glial activation in brain regions where neuron loss subsequently occurs. These changes are most pronounced in the thalamocortical system, with neuron loss occurring first within the cortex and only subsequently in the corresponding thalamic relay nucleus. Microglial activation precedes the emergence of myoclonia and is followed by successive astrocytosis and selective neuron loss. Neuron loss was not detected in thalamic relay nuclei that displayed no glial activation. Microglia showed morphologic changes during disease progression from that of phagocytic brain macrophages in young animals to having thickened branched processes in older animals. These novel data on the timing of pathologic events in the CSTB-deficient brain highlight the potential role of glial activation at the initial stages of the disease. Determining the precise sequence of the neurodegenerative events in Cstb mouse brains will lay the basis for understanding the pathophysiology of EPM1.

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) is an autosomal recessive neurodegenerative disorder caused by mutations in the cystatin B gene (CSTB) that encodes an inhibitor of several lysosomal cathepsins. An unstable expansion of a dodecamer repeat in the CSTB promoter accounts for the majority of EPM1 disease alleles worldwide. We here describe a novel PCR protocol for detection of the dodecamer repeat expansion. We describe two novel EPM1-associated mutations, c.149G4A leading to the p.G50E missense change and an intronic 18-bp deletion (c.168 þ 1_18del), which affects splicing of CSTB. The p.G50E mutation that affects the conserved QVVAG amino acid sequence critical for cathepsin binding fails to associate with lysosomes. This further supports the previously implicated physiological importance of the CSTB-lysosome association. Expression of CSTB mRNA and protein was markedly reduced in lymphoblastoid cells of the patients irrespective of the mutation type. Patients homozygous for the dodecamer expansion mutation showed 5-10% expression compared to controls. By combining database searches with RT-PCR we identified several alternatively spliced CSTB isoforms. One of these, CSTB2, was also present in mouse and was analyzed in more detail. In real-time PCR quantification, CSTB2 expression was less than 5% of total CSTB expression in all human adult and fetal tissues analyzed. In patients homozygous for the minisatellite mutation, the level of CSTB2 was reduced similarly to that of CSTB implicating regulation from the same promoter. The physiological significance of CSTB2 remains to be determined.

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) is an autosomal recessively inherited neurodegenerative disease, manifesting with myoclonus, seizures and ataxia, caused by mutations in the cystatin B (CSTB) gene. With the aim of understanding the molecular basis of pathogenetic events in EPM1 we characterized gene expression changes in the cerebella of pre-symptomatic postnatal day 7 (P7) and symptomatic P30 cystatin B -deficient (Cstb−/−) mice, a model for the disease, and in cultured Cstb−/− cerebellar granule cells using a pathway-based approach. Differentially expressed genes in P7 cerebella were connected to synaptic function and plasticity, and in cultured cerebellar granule cells, to cell cycle, cytoskeleton, and intracellular transport. In particular, the gene expression data pinpointed alterations in GABAergic pathway. Electrophysiological recordings from Cstb−/− cerebellar Purkinje cells revealed a shift of the balance towards decreased inhibition, yet the amount of inhibitory interneurons was not declined in young animals. Instead, we found diminished number of GABAergic terminals and reduced ligand binding to GABAA receptors in Cstb−/− cerebellum. These results suggest that alterations in GABAergic signaling could result in reduced inhibition in Cstb−/− cerebellum leading to the hyperexcitable phenotype of Cstb−/− mice. At P30, the microarray data revealed a marked upregulation of immune and defense response genes, compatible with the previously reported early glial activation that precedes neuronal degeneration. This further implies the role of early-onset neuroinflammation in the pathogenesis of EPM1.

Childhood‐onset neuronal ceroid lipofuscinoses (NCL) are a group of autosomal recessive progressive encephalopathies characterized by the accumulation of autofluorescent material in various tissues, notably in neurons. Based on clinical features, the country of origin of patients, and the molecular genetic background of the disorder, at least seven different forms are thought to exist. Northern epilepsy is a novel form of NCL so far described only in Finland, where all patients are homozygous for a missense mutation in the CLN8 gene. A variant form of late infantile NCL (vLINCL) present in Turkish patients has been considered a distinct clinical and genetic entity among the NCL, the underlying gene (CLN7) being unknown. Recently, we reported homozygosity over the Northern epilepsy CLN8 gene region on 8p23 in four out of five Turkish vLINCL families studied. However, no common mutation in CLN8 was found in these families. We have now extended the Turkish vLINCL family panel to 18 families, of which only one is nonconsanguineous. Nine families were excluded from CLN8 by lack of homozygosity. In the remaining families, four CLN8 gene mutations were identified indicating that in a subset of patients with Turkish vLINCL, the disorder is allelic to Northern epilepsy. There is no apparent genotype‐phenotype correlation among the Turkish patients with CLN8 mutations, although their phenotype is distinct from that of Finnish Northern epilepsy patients. The molecular genetic background of the Turkish vLINCL families not linked to CLN8 remains to be clarified. Hum Mutat 23:300–305, 2004 © 2004 Wiley‐Liss, Inc.

Progressive encephalopathy with oedema, hypsarrhythmia, and optic atrophy (PEHO) syndrome is an early childhood onset, severe autosomal recessive encephalopathy characterized by extreme cerebellar atrophy due to almost total granule neuron loss. By combining homozygosity mapping in Finnish families with Sanger sequencing of positional candidate genes and with exome sequencing a homozygous missense substitution of leucine for serine at codon 31 in ZNHIT3 was identified as the primary cause of PEHO syndrome. ZNHIT3 encodes a nuclear zinc finger protein previously implicated in transcriptional regulation and in small nucleolar ribonucleoprotein particle assembly and thus possibly to pre-ribosomal RNA processing. The identified mutation affects a highly conserved amino acid residue in the zinc finger domain of ZNHIT3. Both knockdown and genome editing of znhit3 in zebrafish embryos recapitulate the patients' cerebellar defects, microcephaly and oedema. These phenotypes are rescued by wild-type, but not mutant human ZNHIT3 mRNA, suggesting that the patient missense substitution causes disease through a loss-of-function mechanism. Transfection of cell lines with ZNHIT3 expression vectors showed that the PEHO syndrome mutant protein is unstable. Immunohistochemical analysis of mouse cerebellar tissue demonstrated ZNHIT3 to be expressed in proliferating granule cell precursors, in proliferating and post-mitotic granule cells, and in Purkinje cells. Knockdown of Znhit3 in cultured mouse granule neurons and ex vivo cerebellar slices indicate that ZNHIT3 is indispensable for granule neuron survival and migration, consistent with the zebrafish findings and patient neuropathology. These results suggest that loss-of-function of a nuclear regulator protein underlies PEHO syndrome and imply that establishment of its spatiotemporal interaction targets will be the basis for developing therapeutic approaches and for improved understanding of cerebellar development.