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Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of motor neurons. ALS can be modeled in zebrafish (Danio rerio) through the expression of human ALS-causing genes, such as superoxide dismutase 1 (SOD1). Overexpression of mutated human SOD1 protein causes aberrant branching and shortening of spinal motor axons. Despite this, the functional relevance of this axon morphology remains elusive. Our aim was to determine whether this motor axonopathy is correlated with impaired movement in mutant (MT) SOD1-expressing zebrafish. Transgenic zebrafish embryos that express blue fluorescent protein (mTagBFP) in motor neurons were injected with either wild-type (WT) or MT (A4V) human SOD1 messenger ribonucleic acid (mRNA). At 48 hours post-fertilization, larvae movement (distance traveled during behavioral testing) was examined, followed by quantification of motor axon length. Larvae injected with MT SOD1 mRNA had significantly shorter and more aberrantly branched motor axons (p

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive, fatal neurodegenerative disease characterised by the death of upper and lower motor neurons. Approximately 10% of cases have a known family history of ALS and disease-linked mutations in multiple genes have been identified. ALS-linked mutations in CCNF were recently reported, however the pathogenic mechanisms associated with these mutations are yet to be established. To investigate possible disease mechanisms, we developed in vitro and in vivo models based on an ALS-linked missense mutation in CCNF. Proteomic analysis of the in vitro models identified the disruption of several cellular pathways in the mutant model, including caspase-3 mediated cell death. Transient overexpression of human CCNF in zebrafish embryos supported this finding, with fish expressing the mutant protein found to have increased levels of cleaved (activated) caspase-3 and increased cell death in the spinal cord. The mutant CCNF fish also developed a motor neuron axonopathy consisting of shortened primary motor axons and increased frequency of aberrant axonal branching. Importantly, we demonstrated a significant correlation between the severity of the CCNF-induced axonopathy and a reduced motor response to a light stimulus (photomotor response). This is the first report of an ALS-linked CCNF mutation in vivo and taken together with the in vitro model identifies the disruption of cell death pathways as a significant consequence of this mutation. Additionally, this study presents a valuable new tool for use in ongoing studies investigating the pathobiology of ALS-linked CCNF mutations.

We describe a protocol for culturing neurons from transgenic zebrafish embryos to investigate the subcellular distribution and protein aggregation status of neurodegenerative disease-causing proteins. The utility of the protocol was demonstrated on cell cultures from zebrafish that transgenically express disease-causing variants of human fused in sarcoma (FUS) and ataxin-3 proteins, in order to study amyotrophic lateral sclerosis (ALS) and spinocerebellar ataxia type-3 (SCA3), respectively. A mixture of neuronal subtypes, including motor neurons, exhibited differentiation and neurite outgrowth in the cultures. As reported previously, mutant human FUS was found to be mislocalized from nuclei to the cytosol, mimicking the pathology seen in human ALS and the zebrafish FUS model. In contrast, neurons cultured from zebrafish expressing human ataxin-3 with disease-associated expanded polyQ repeats did not accumulate within nuclei in a manner often reported to occur in SCA3. Despite this, the subcellular localization of the human ataxin-3 protein seen in cell cultures was similar to that found in the SCA3 zebrafish themselves. The finding of similar protein localization and aggregation status in the neuronal cultures and corresponding transgenic zebrafish models confirms that this cell culture model is a useful tool for investigating the cell biology and proteinopathy signatures of mutant proteins for the study of neurodegenerative disease., Summary: This article describes the optimization and validation of a protocol for culturing of neurons from transgenic zebrafish for the study of neurodegenerative diseases.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are overlapping, fatal neurodegenerative disorders in which the molecular and pathogenic basis remains poorly understood. Ubiquitinated protein aggregates, of which TDP-43 is a major component, are a characteristic pathological feature of most ALS and FTD patients. Here we use genome-wide linkage analysis in a large ALS/FTD kindred to identify a novel disease locus on chromosome 16p13.3. Whole-exome sequencing identified a CCNF missense mutation at this locus. Interrogation of international cohorts identified additional novel CCNF variants in familial and sporadic ALS and FTD. Enrichment of rare protein-altering CCNF variants was evident in a large sporadic ALS replication cohort. CCNF encodes cyclin F, a component of an E3 ubiquitin–protein ligase complex (SCFCyclin F). Expression of mutant CCNF in neuronal cells caused abnormal ubiquitination and accumulation of ubiquitinated proteins, including TDP-43 and a SCFCyclin F substrate. This implicates common mechanisms, linked to protein homeostasis, underlying neuronal degeneration., Ian Blair and colleagues use genome-wide linkage analysis and whole exome sequencing to identify mutations in the CCNF gene in large cohorts of amyotrophic lateral sclerosis and frontotemporal dementia patients. In addition to validating the mutations in international cohorts, the authors also show that mutant CCNF gene product affects ubiquitination and protein degradation in cultured cells.

Using light and scanning electron microscopy, the internal male and female reproductive anatomy of the praying mantid Ciulfina klassi is identified and described. This is the first detailed study to investigate the internal reproductive morphology of any Mantodea. The female structures identified were (1) paired ovaries with primitive panoistic type ovarioles, (2) a single blind-ended spermatheca with secretory gland cells and surrounding layer of striated muscle, and (3) female accessory glands associated with the production of the ootheca (the egg casing). The male structures identified were (1) paired multi-tubular testes, in which different stages of spermatogenesis were observed, (2) tubular vasa deferentia, (3) seminal vesicles, (4) male accessory glands and (5) a single muscular ejaculatory duct. Knowledge of basic reproductive morphology can be used to infer function and so provide key information for future research into reproductive behavior and physiology in the Mantodea.