Glutamate excitotoxicity leads to fragmented mitochondria in neurodegenerative diseases, mediated by nitric oxide and S-nitrosylation of dynamin-related protein 1, a mitochondrial outer membrane ...fission protein. Optic atrophy gene 1 (OPA1) is an inner membrane protein important for mitochondrial fusion. Autosomal dominant optic atrophy (ADOA), caused by mutations in OPA1, is a neurodegenerative disease affecting mainly retinal ganglion cells (RGCs). Here, we showed that OPA1 deficiency in an ADOA model influences N-methyl-D-aspartate (NMDA) receptor expression, which is involved in glutamate excitotoxicity and oxidative stress. Opa1(enu/+) mice show a slow progressive loss of RGCs, activation of astroglia and microglia, and pronounced mitochondrial fission in optic nerve heads as found by electron tomography. Expression of NMDA receptors (NR1, 2A, and 2B) in the retina of Opa1(enu/+) mice was significantly increased as determined by western blot and immunohistochemistry. Superoxide dismutase 2 (SOD2) expression was significantly decreased, the apoptotic pathway was activated as Bax was increased, and phosphorylated Bad and BcL-xL were decreased. Our results conclusively demonstrate that not only glutamate excitotoxicity and/or oxidative stress alters mitochondrial fission/fusion, but that an imbalance in mitochondrial fission/fusion in turn leads to NMDA receptor upregulation and oxidative stress. Therefore, we propose a new vicious cycle involved in neurodegeneration that includes glutamate excitotoxicity, oxidative stress, and mitochondrial dynamics.
Autosomal dominant optic atrophy (adOA) is a juvenile onset, progressive ocular disorder characterized by bilateral loss of vision, central visual field defects, colour vision disturbances, and optic ...disc pallor. adOA is most frequently associated with mutations in OPA1 encoding a dynamin-related large GTPase that localizes to mitochondria. Histopathological studies in adOA patients have shown a degeneration of retinal ganglion cells (RGCs) and a loss of axons in the optic nerve. However little is known about the molecular mechanism and pathophysiology of adOA due to the lack of appropriate in vivo models. Here we report a first mouse model carrying a splice site mutation (c.1065 + 5G → A) in the Opa1 gene. The mutation induces a skipping of exon 10 during transcript processing and leads to an in-frame deletion of 27 amino acid residues in the GTPase domain. Western blot analysis showed no evidence of a shortened mutant protein but a ∼50% reduced OPA1 protein level supporting haploinsufficiency as a major disease mechanism in adOA. Homozygous mutant mice die in utero during embryogenesis with first notable developmental delay at E8.5 as detected by magnetic resonance imaging (MRI). Heterozygous mutants are viable and of normal habitus but exhibit an age-dependent loss of RGCs that eventually progresses to a severe degeneration of the ganglion cell and nerve fibre layer. In addition optic nerves of mutant mice showed a reduced number of axons, and a swelling and abnormal shape of the remaining axons. Mitochondria in these axons showed disorganized cristae structures. All these defects recapitulate crucial features of adOA in humans and therefore document the validity and importance of this model for future research.
Purpose
Hereditary retinal dystrophies (RD) are a group of heterogeneous disorders caused by mutations in over 200 genes. RDs can be subdivided into different groups based on the primary degeneration ...of rod or cone photoreceptor cells. This study was conducted to investigate the underlying RD genes and mutation in consanguineous families from Pakistan.
Methods
Families were recruited after informed consent. Peripheral blood was collected and genomic DNA was extracted according to standard procedures. Homozygosity mapping was performed using Affymetrix Gene Chip Human Mapping 250 K‐NspI arrays. The data were analyzed using Homozygosity Mapper software. Primers for amplifying all exons and intron boundaries of all genes were designed with Primer3plus software followed by PCR and Sanger sequencing. Minigene splicing assay and DNA walking were performed on respective samples.
Results
Homozygosity mapping identified a novel locus in family A, one novel gene (C8ORF37) in family B and a large novel genomic deletion of the (LCA5) gene in family C.
Conclusions
Our study indicates the heterogeneous nature of retinal dystrophies in Pakistan. Although earlier studies have explored a number of RD families, but underlying genes are still unknown for significant proportion of Pakistani families. Theoretically the traditional screening methods (homozygosity mapping based candidate gene sequencing) were successful, however SNP based genome wide scan were further implemented to improve the detection of underlying variations responsible for Retinal Dystrophies. Therefore the amalgamation of traditional and modern molecular techniques is required for accurate identification of mutations. It is anticipated that these findings will contribute to future genetic testing in Pakistani families to minimize the risk of recessive disorders.
Summary
Mutations in OPA1 are the main cause of dominant optic atrophy (DOA) and have also been implicated in a variety of syndromic neuropathies such as DOAplus or Behr‐like syndrome. We have ...recently discovered a disease‐causing deep intronic mutation (DIM) in OPA1 that induces a constitutive inclusion of a cryptic exon into OPA1 transcripts. As a potential therapeutic strategy we sought to correct mis‐splicing of the mutant pre‐mRNA by using antisense oligonucleotides targeting the cryptic acceptor splice site and the novel branch point created by the DIM, respectively. Transfection of patient‐derived primary dermal fibroblasts with AONs resulted in splice correction of the mutant pre‐mRNA in a time and concentration dependent manner. Maximal rescue efficacy of up ~55% was obtained with the cryptic acceptor splice site targeting AON. Peak efficacy of splice correction was observed at 4 days after treatment. However, significant effects were still seen 14 days post‐transfection in the proliferating cell culture. Western blot analysis revealed increased amounts of OPA1 protein with maximum amounts at ~3 days post‐treatment. In summary, we provide the first mutation‐specific rescue strategy for OPA1 deficiency using synthetic AONs.
Purpose
Mutations in the
RS1
gene are known to cause retinoschisis, an X-linked hereditary retinal degeneration. Here, we present a case of atypical retinoschisis with clinical findings of ...retinoschisis and retinitis pigmentosa.
Methods
This report is an observational case report. The detailed ophthalmological examinations included visual field determination, multimodal imaging and electrophysiological recordings. Targeted next-generation sequencing of a retinal disease gene panel was performed.
Results
The 55-year-old male, highly hyperopic patient, presented with a best-corrected Snellen visual acuity of 20/100 in the right eye and 20/400 in the left eye. In the kinetic visual field, there was a superior scotoma, as well as a ring scotoma in the inferior hemisphere in the right eye and a concentric visual field constriction to 10° in the left eye. Funduscopy revealed marked pigmentary changes (i.e. bone spicules) in the mid-periphery bilaterally and symmetrically, as well as two small intra-retinal haemorrhages in the left eye. Full-field electroretinography recordings showed extinguished rod and cone responses. Diagnostic–genetic testing revealed a hemizygous missense mutation in the
RS1
gene (c.305G > A; p.Arg102Gln) was identified.
Conclusion
We present a case of atypical retinoschisis with clinical findings of retinitis pigmentosa.
Peters anomaly and Axenfeld–Rieger syndrome (ARS) belong to the overlapping spectrum of disorders summarized as anterior segment dysgenesis (ASD). Five patients from a family with Peters’ anomaly and ...ARS were screened for mutations in the PITX2, CYP1B1 and FOXC1 genes by direct sequencing. All affected family members examined were heterozygous for a single nucleotide substitution, resulting in a nonsense mutation (Q120X) at a highly conserved residue of the FOXC1 gene that is essential for DNA binding. In this pedigree, all affected family members were diagnosed with ARS except for one who shows bilateral Peters’ anomaly. Our findings support the role of FOXC1 mutations in the spectrum of ASD.
Summary
Mutations in OPA1 are a common cause of dominant optic neuropathy (DOA). Recent studies suggest that ~20% of patients carrying OPA1 mutations have additional neurological deficits (DOAplus ...phenotype). Such patients frequently carry missense mutations in the GTPase domain of OPA1 suggesting a gain‐of‐function effect as a major mechanism. We and others recently reported a series of DOAplus patients with biallelic OPA1 mutations as an alternative disease mechanism. Notably most cases were compound heterozygous for a null allele and the Ile382Met variant. The latter is not per se pathogenic but rather acts as a hypomorphic modifier allele that reinforces phenotypic expression in patients with null mutations on the opposite allele. In one biallelic DOAplus family we identified a deep intronic mutation (DIM) that causes a constitutive activation and inclusion of a cryptic frameshift‐inducing exon into OPA1 mRNA. Consistent with the DIM representing a null allele we observed reduced OPA1 protein amounts to about 50% of normal. Applying antisense oligonucleotides targeting the splice acceptor site of the DIM in patient fibroblasts we could efficiently rescue splicing of the mutant mRNA and re‐establish intermediate OPA1 protein levels.
We investigated the effects of nitrogen deposition and precipitation on Messor pergandei (Mayr) harvester ants and plants to identify alterations in the desert food web in California. We measured ant ...colony attributes and shrub fruit densities, as well as nitrogen and carbon concentrations and stable isotopes, at 18 sites along a nitrogen deposition gradient. Ant nest density increased from low to high deposition sites; however, ant nest mound size and the density of abandoned nests decreased as deposition increased. Nest mound size was positively correlated with the size and age of the colony; therefore, these results suggest that colonization has been more frequent with increased inter-colony competition in areas of high deposition. Nitrogen and carbon isotope values of perennial plant leaves and seeds, annual plant seeds, and ants were significantly enriched in the heavy isotopes from low to high nitrogen deposition regions, indicating the possibility of plants assimilating different sources of both elements, including anthropogenically-produced compounds. Plant carbon isotope discrimination also differed with the decrease in precipitation across the gradient. Considering that deserts are limited by both nitrogen and precipitation, our results suggest that altered nitrogen inputs in conjunction with precipitation may result in cascading effects through trophic levels and drive arid ecosystem change.
•We examined the effects of nitrogen deposition on plants and harvester ants.•Ant nest mound size decreased in high nitrogen and precipitation regions.•Ant nest density increased in high nitrogen and precipitation regions.•N and C isotopes of plants and ants were enriched from high to low nitrogen regions.•Plant and ant N and C content did not vary across the nitrogen gradient.
Purpose
To establish the normal range of values for rod-isolated b-wave amplitudes in achromatopsia and cone dystrophies.
Methods
We reviewed charts of 112 patients with various types of cone ...dystrophy, and compared their standardized electroretinographic rod b-wave amplitudes with age-matched normal controls. Twenty-six patients had known mutations in achromatopsia and cone dystrophy genes, while 53 were characterized by their inheritance pattern since they had yet to have their gene identified. Visual acuity information and scotomata were documented.
Results
We found that patients with achromatopsia and cone dystrophy had rod b-wave amplitudes that were significantly lower than age-matched controls, but found no evidence of rod amplitude progression nor loss of peripheral visual fields in the study group.
Conclusions
We found that cone dystrophy patients of all types had depressed rod-isolated ERGs across the board. If typical diagnostic criteria are used, these patients might be considered to have “abnormal” rod-isolated electroretinographic values, and might be called “cone-rod dystrophy”, even though the waveforms are stable for years. Patients with cone-rod dysfunction patterns on ERG can be better understood by also performing kinetic (Goldmann) visual fields, which will help to distinguish cone dystrophies from progressive cone-rod dystrophies by central scotomata size and progression over time in many forms of cone-rod dystrophy.
Autosomal dominant optic atrophy (ADOA) is considered as the most common form of hereditary optic neuropathy. Although genetic linkage studies point to the OPA1 locus on chromosome 3q28-q29 as by far ...the most common gene locus, previous screening studies-based on sequencing of the coding exons-detected OPA1 mutations in only 32-70% of ADOA patients. We therefore hypothesised that larger deletions or duplications that remained undetected in previous screening approaches may substantially contribute to the prevalence of OPA1 mutations in ADOA.
42 independent ADOA patients were analysed for the presence of genomic rearrangements in OPA1 by means of multiplex ligation probe amplification (MLPA). Deletions or duplications were confirmed either by long distance polymerase chain reaction (PCR) and breakpoint sequencing or loss of heterozygosity analyses with flanking microsatellite markers. Patients underwent ophthalmological examination including visual acuity, colour vision testings, perimetry and funduscopy.
We identified genomic rearrangements in 8 of 42 patients, including single exon deletions of exon 9 and exon 24, respectively, a deletion of exons 1-5, two different deletions of the complete OPA1 gene as well as a duplication of the exons 7-9, with the latter being present in three unrelated families. Patients' phenotypes were highly variable, similar to patients with point mutation in OPA1.
Our findings show that gross genomic aberrations at the OPA1 gene locus are frequent in ADOA and substantially contribute to the spectrum and prevalence of OPA1 mutations in ADOA patients. They further strengthen the hypothesis that haploinsufficiency is a major pathomechanism in OPA1 associated ADOA.
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