Neuronal intermediate filaments and ALS: A new look at an old question

AbstractOne of the pathological hallmarks of ALS is the presence of axonal spheroids and perikaryal accumulations/aggregations comprised of the neuronal intermediate filament proteins, neurofilaments and peripherin. These abnormalities represent a point of convergence of both familial and sporadic forms of the disease and understanding their formation may reveal shared pathways in what is otherwise considered a highly heterogeneous disorder. Here we provide a review of the basic biology of neurofilaments and peripherin and the evidence linking them with ALS disease pathogenesis

Influence of occupation type on the association between sleep duration and impaired fasting glucose: results from a Chinese population-based study

Objectives Systematic evaluation of the influence of occupation type on the association between sleep–glucose metabolismDesign A cross-sectional study.Setting The Nantong Metabolic Syndrome Study is a Chinese population-based study.Participants 20 502 participants aged 18–74 years old.Intervention No intervention.Primary and secondary outcome measures Impaired fasting glucose (IFG).Results A total of 1503 participants (7.33%) with a slightly longer sleep duration had IFG. After being stratified according to occupation, a sleep duration of ≥10 hours daily corresponded to a 1.321-fold risk of IFG (95% CI 1.071 to 1.628, p=0.0092) among moderate and heavy physical workers compared with those with a daily sleep duration of 7–9 hours. There was no significant relationship between sleep and IFG among other types of workers. Moreover, we discovered a gender difference in the influence of occupation on the sleep–IFG. A positive association among moderate and heavy physical men and a negative association among light or sedentary men were established, but not in unemployed men. However, a positive association was evident only in unemployed women; there was no significant association among other occupations.Conclusion This study highlights the role of occupation in the relationship of sleep–glucose metabolism. A gender difference was found to have been influenced by occupational types on the sleep–metabolic association

Gene targeting of mouse Tardbp negatively affects Masp2 expression.

Amyotrophic Lateral Sclerosis (ALS) is a devastating adult onset neurodegenerative disease affecting both upper and lower motor neurons. TDP-43, encoded by the TARDBP gene, was identified as a component of motor neuron cytoplasmic inclusions in both familial and sporadic ALS and has become a pathological signature of the disease. TDP-43 is a nuclear protein involved in RNA metabolism, however in ALS, TDP-43 is mislocalized to the cytoplasm of affected motor neurons, suggesting that disease might be caused by TDP-43 loss of function. To investigate this hypothesis, we attempted to generate a mouse conditional knockout of the Tardbp gene using the classical Cre-loxP technology. Even though heterozygote mice for the targeted allele were successfully generated, we were unable to obtain homozygotes. Here we show that although the targeting vector was specifically designed to not overlap with Tardbp adjacent genes, the homologous recombination event affected the expression of a downstream gene, Masp2. This may explain the inability to obtain homozygote mice with targeted Tardbp

Synaptic localization of C9orf72 regulates post-synaptic glutamate receptor 1 levels

Abstract A hexanucleotide repeat expansion in a noncoding region of C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Reduction of select or total C9orf72 transcript and protein levels is observed in postmortem C9-ALS/FTD tissue, and loss of C9orf72 orthologues in zebrafish and C. elegans results in motor deficits. However, how the reduction in C9orf72 in ALS and FTD might contribute to the disease process remains poorly understood. It has been shown that C9orf72 interacts and forms a complex with SMCR8 and WDR41, acting as a guanine exchange factor for Rab GTPases. Given the known synaptosomal compartmentalization of C9orf72-interacting Rab GTPases, we hypothesized that C9orf72 localization to synaptosomes would be required for the regulation of Rab GTPases and receptor trafficking. This study combined synaptosomal and post-synaptic density preparations together with a knockout-confirmed monoclonal antibody for C9orf72 to assess the localization and role of C9orf72 in the synaptosomes of mouse forebrains. Here, we found C9orf72 to be localized to both the pre- and post-synaptic compartment, as confirmed by both post-synaptic immunoprecipitation and immunofluorescence labelling. In C9orf72 knockout (C9-KO) mice, we demonstrated that pre-synaptic Rab3a, Rab5, and Rab11 protein levels remained stable compared with wild-type littermates (C9-WT). Strikingly, post-synaptic preparations from C9-KO mouse forebrains demonstrated a complete loss of Smcr8 protein levels, together with a significant downregulation of Rab39b and a concomitant upregulation of GluR1 compared with C9-WT mice. We confirmed the localization of Rab39b downregulation and GluR1 upregulation to the dorsal hippocampus of C9-KO mice by immunofluorescence. These results indicate that C9orf72 is essential for the regulation of post-synaptic receptor levels, and implicates loss of C9orf72 in contributing to synaptic dysfunction and related excitotoxicity in ALS and FTD

Analysis of <i>Tardbp</i> and Masp2 expression in Tardbp^2Lox/+mice.

<p>A) Quantification of mRNA in mouse lumbar spinal cord by Real-Time PCR. No significant difference was found between <i>Tardbp</i> transcripts between WT and Tardbp^+/2lox mice (n = 4). B) Immunoblot analysis of TDP-43 protein expression in mouse brain, spinal cord, and liver shows no reduction in expression in <i>Tardbp</i>^+/2lox mice compared to wildtype littermates. Actin was used to normalize the amount of protein loaded. C) Quantification of <i>Masp</i>2 splicing variants mRNA in mouse liver by quantitative Real-Time PCR. <i>Masp2</i> long variant was significantly reduced in <i>Tardbp</i>^+/2lox compared to WT (p = 0.03, n = 3). No significant difference was found for <i>MAp19</i> transcript levels between WT and Tardbp^+/2lox mice. D) Analysis of total protein extracts from mouse liver by Western blot. Masp2 is clearly reduced in Tardbp^+/2lox mice livers when compared to WT, while MAp19 expression is marginally reduced.</p

Attenuation of Sepsis-Induced Acute Kidney Injury by Exogenous H₂S via Inhibition of Ferroptosis

Sepsis-associated acute kidney injury (SA-AKI) results in significant morbidity and mortality, and ferroptosis may play a role in its pathogenesis. Our aim was to examine the effect of exogenous H2S (GYY4137) on ferroptosis and AKI in in vivo and in vitro models of sepsis and explore the possible mechanism involved. Sepsis was induced by cecal ligation and puncture (CLP) in male C57BL/6 mice, which were randomly divided into the sham, CLP, and CLP + GYY4137 group. The indicators of SA-AKI were most prominent at 24 h after CLP, and analysis of the protein expression of ferroptosis indicators showed that ferroptosis was also exacerbated at 24 h after CLP. Moreover, the level of the endogenous H2S synthase CSE (Cystathionine-γ-lyase) and endogenous H2S significantly decreased after CLP. Treatment with GYY4137 reversed or attenuated all these changes. In the in vitro experiments, LPS was used to simulate SA-AKI in mouse renal glomerular endothelial cells (MRGECs). Measurement of ferroptosis-related markers and products of mitochondrial oxidative stress showed that GYY4137 could attenuate ferroptosis and regulate mitochondrial oxidative stress. These findings imply that GYY4137 alleviates SA-AKI by inhibiting ferroptosis triggered by excessive mitochondrial oxidative stress. Thus, GYY4137 may be an effective drug for the clinical treatment of SA-AKI

Strategy and validation of the conditional deletion of <i>Tardbp</i> gene.

<p>A) To delete exons 2 and 3 in the endogenous <i>Tardbp</i> we used a targeting vector with 6 Kb and 4 Kb homology arms, shown as black bars. The neomycin resistance was used for positive selection in embryonic stem cells and was flanked by two FRT sites to allow its removal upon FLP mediated recombination. A DTA cassette allowed negative selection of ES cells bearing random integration of the targeting vector. Upon homologous recombination in ES cells (Xs) the endogenous gene was replaced with the targeted cassette. FLP mediated recombination generated a conditional knockout where exons 2 and 3 were flanked by loxP sites. B) Southern Blot analysis of control (+/+) and targeted ES clones. A HindIII digest produced fragments of 7.3 Kb for the WT and 9.1 Kb for the targeted allele. C) Confirmation of neomycin cassette excision by PCR amplification.</p

Genetic studies of GRN and IFT74 in amyotrophic lateral sclerosis

There is increasing evidence of a clinical, neuropathological and genetic overlap between frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). We conducted a case–control study using a UK dataset to test the hypothesis that polymorphisms in two FTD-related genes (GRN and FT74) are associated with increased susceptibility to ALS. We evaluated the majority of known genetic variability in IFT74 and GRN. The results revealed that the common variations in IFT74 and GRN neither constitute strong ALS risk factors nor modify the age-at-onset. However, the possibility of a modest risk effect remains to be assessed in large datasets

Immunohistochemical detection of C9orf72 protein in Frontotemporal Lobar Degeneration and Motor Neurone Disease: patterns of immunostaining and an evaluation of commercial antibodies

We have employed as ‘gold standards’ two in-house, well-characterised and validated polyclonal antibodies, C9-L and C9-S, which detect the longer and shorter forms of C9orf72, and have compared seven other commercially available antibodies with these in order to evaluate the utility of the latter as credible tools for the demonstration of C9orf72. C9-L and C9-S antibodies immunostained cytoplasmic ‘speckles’, and the nuclear membrane, respectively, in cerebellar Purkinje cells of the cerebellum in patients with behavioural variant frontotemporal dementia (bvFTD) with amyotrophic lateral sclerosis (ALS), and in patients with ALS alone. Similar staining was seen in Purkinje cells in healthy control tissues and in other neurodegenerative disorders, and in pyramidal cells of CA4 and dentate gyrus of hippocampus. However, in the spinal cord there was little cytoplasmic staining with C9-L antibody. C9-S antibody immunostained the nuclear membrane of anterior horn cells in healthy neurons. In patients with bvFTD + ALS, or ALS alone, this C9-S nuclear staining was redistributed to the plasma membrane. In those patients with bvFTD + ALS or ALS bearing an expansion in C9orf72, none of the commercially available antibodies detected TDP-43 inclusions in anterior horn cells, nor were dipeptide repeat proteins demonstrated. Five of the commercial antibodies provided immunohistochemical staining patterns similar in morphological appearance to the in-house C9-L antibody, but distinct from C9-S antibody. However, only three showed sufficient specificity and intensity of staining for C9orf72 at acceptably low concentrations, to make them of practical value and sufficiently reliable for the detection of at least the longer form of C9orf72 protein.</p

Immunohistochemical detection of C9orf72 protein in frontotemporal lobar degeneration and motor neurone disease: patterns of immunostaining and an evaluation of commercial antibodies

<p>We have employed as ‘gold standards’ two in-house, well-characterised and validated polyclonal antibodies, C9-L and C9-S, which detect the longer and shorter forms of C9orf72, and have compared seven other commercially available antibodies with these in order to evaluate the utility of the latter as credible tools for the demonstration of C9orf72. C9-L and C9-S antibodies immunostained cytoplasmic ‘speckles’, and the nuclear membrane, respectively, in cerebellar Purkinje cells of the cerebellum in patients with behavioural variant frontotemporal dementia (bvFTD) with amyotrophic lateral sclerosis (ALS), and in patients with ALS alone. Similar staining was seen in Purkinje cells in healthy control tissues and in other neurodegenerative disorders, and in pyramidal cells of CA4 and dentate gyrus of hippocampus. However, in the spinal cord there was little cytoplasmic staining with C9-L antibody. C9-S antibody immunostained the nuclear membrane of anterior horn cells in healthy neurons. In patients with bvFTD + ALS, or ALS alone, this C9-S nuclear staining was redistributed to the plasma membrane. In those patients with bvFTD + ALS or ALS bearing an expansion in <i>C9orf72</i>, none of the commercially available antibodies detected TDP-43 inclusions in anterior horn cells, nor were dipeptide repeat proteins demonstrated. Five of the commercial antibodies provided immunohistochemical staining patterns similar in morphological appearance to the in-house C9-L antibody, but distinct from C9-S antibody. However, only three showed sufficient specificity and intensity of staining for C9orf72 at acceptably low concentrations, to make them of practical value and sufficiently reliable for the detection of at least the longer form of C9orf72 protein.</p