Holdase activity of secreted Hsp70 masks amyloid-ß42 neurotoxicity in Drosophila.

Alzheimer's disease (AD) is the most prevalent of a large group of related proteinopathies for which there is currently no cure. Here, we used Drosophila to explore a strategy to block Aß42 neurotoxicity through engineering of the Heat shock protein 70 (Hsp70), a chaperone that has demonstrated neuroprotective activity against several intracellular amyloids. To target its protective activity against extracellular Aß42, we added a signal peptide to Hsp70. This secreted form of Hsp70 (secHsp70) suppresses Aß42 neurotoxicity in adult eyes, reduces cell death, protects the structural integrity of adult neurons, alleviates locomotor dysfunction, and extends lifespan. SecHsp70 binding to Aß42 through its holdase domain is neuroprotective, but its ATPase activity is not required in the extracellular space. Thus, the holdase activity of secHsp70 masks Aß42 neurotoxicity by promoting the accumulation of nontoxic aggregates. Combined with other approaches, this strategy may contribute to reduce the burden of AD and other extracellular proteinopathies.

C-terminal proline deletions in KCNC3 cause delayed channel inactivation and an adult-onset progressive SCA13 with spasticity.

Mutations in the potassium channel gene KCNC3 (Kv3.3) cause the autosomal dominant neurological disease, spinocerebellar ataxia 13 (SCA13). In this study, we expand the genotype-phenotype repertoire of SCA13 by describing the novel KCNC3 deletion p.Pro583_Pro585del highlighting the allelic heterogeneity observed in SCA13 patients. We characterize adult-onset, progressive clinical symptoms of two afflicted kindred and introduce the symptom of profound spasticity not previously associated with the SCA13 phenotype. We also present molecular and electrophysiological characterizations of the mutant protein in mammalian cell culture. Mechanistically, the p.Pro583_Pro585del protein showed normal membrane trafficking with an altered electrophysiological profile, including slower inactivation and decreased sensitivity to the inactivation-accelerating effects of the actin depolymerizer latrunculin B. Taken together, our results highlight the clinical importance of the intracellular C-terminal portion of Kv3.3 and its association with ion channel function.

Anti-Aß single-chain variable fragment antibodies exert synergistic neuroprotective activities in Drosophila models of Alzheimer's disease.

Both active and passive immunotherapy protocols decrease insoluble amyloid-ß42 (Aß42) peptide in animal models, suggesting potential therapeutic applications against the main pathological trigger in Alzheimer's disease (AD). However, recent clinical trials have reported no significant benefits from humanized anti-Aß42 antibodies. Engineered single-chain variable fragment antibodies (scFv) are much smaller and can easily penetrate the brain, but identifying the most effective scFvs in murine AD models is slow and costly. We show here that scFvs against the N- and C-terminus of Aß42 (scFv9 and scFV42.2, respectively) that decrease insoluble Aß42 in CRND mice are neuroprotective in Drosophila models of Aß42 and amyloid precursor protein neurotoxicity. Both scFv9 and scFv42.2 suppress eye toxicity, reduce cell death in brain neurons, protect the structural integrity of dendritic terminals in brain neurons and delay locomotor dysfunction. Additionally, we show for the first time that co-expression of both anti-Aß scFvs display synergistic neuroprotective activities, suggesting that combined therapies targeting distinct Aß42 epitopes can be more effective than targeting a single epitope. Overall, we demonstrate the feasibility of using Drosophila as a first step for characterizing neuroprotective anti-Aß scFvs in vivo and identifying scFv combinations with synergistic neuroprotective activities.

KCNC3(R420H), a K(+) channel mutation causative in spinocerebellar ataxia 13 displays aberrant intracellular trafficking.

Spinocerebellar ataxia 13 (SCA13) is an autosomal dominant disease resulting from mutations in KCNC3 (Kv3.3), a voltage-gated potassium channel. The KCNC3(R420H) mutation was first identified as causative for SCA13 in a four-generation Filipino kindred with over 20 affected individuals. Electrophysiological analyses in oocytes previously showed that this mutation did not lead to a functional channel and displayed a dominant negative phenotype. In an effort to identify the molecular basis of this allelic form of SCA13, we first determined that human KCNC3(WT) and KCNC3(R420H) display disparate post-translational modifications, and the mutant protein has reduced complex glycan adducts. Immunohistochemical analyses demonstrated that KCNC3(R420H) was not properly trafficking to the plasma membrane and surface biotinylation demonstrated that KCNC3(R420H) exhibited only 24% as much surface expression as KCNC3(WT). KCNC3(R420H) trafficked through the ER but was retained in the Golgi. KCNC3(R420H) expression results in altered Golgi and cellular morphology. Electron microscopy of KCNC3(R420H) localization further supports retention in the Golgi. These results are specific to the KCNC3(R420H) allele and provide new insight into the molecular basis of disease manifestation in SCA13.

Subchronic Toxicity Assessment of Orally Administered Methanol (70%) Seed Extract of Abrus precatorius L. in Wistar Albino Rats.

OBJECTIVES: Abrus precatorius L. is a famous medicinal plant of the family Fabaceae and is widely used in traditional medicine for the treatment of various ailments. However, there are limited toxicological data available regarding its safety following repeated exposure; therefore, the present study was designed to evaluate the 28-day subchronic toxicity of methanol (70%) crude extract of A. precatorius seeds in adult Wistar albino rats. MATERIALS AND METHODS: A subchronic toxicity experiment was conducted by oral administration of graded doses (200 mg/kg and 400 mg/kg) of test extract daily for 28 days. Signs of toxicity, food and water consumption, body weight, and gross pathology as well as relative organ weight were evaluated. The toxic effects were also assessed using hematological and biochemical data followed by histopathological examination of various internal organs. All data collected were expressed as mean ± standard deviation. ANOVA followed by the Bonferroni test was used for data interpretation and p<0.05 was considered significant. RESULTS: No deaths or evident toxic signs were found during the experimental period. There were no significant differences in body weight, gross pathology, organ weight, or food and water consumption between the control and the treated groups. There were no treatment-related differences in hematological or biochemical indices. Moreover, no gross abnormalities or histological alterations were observed. CONCLUSION: The methanol extract of A. precatorius seeds was nontoxic in subchronic intake at the dosages tested. Thus, this study is expected to be beneficial for clinical and traditional applications for safe consumption and to utilize A. precatorius as a remedy at a recommended dosage.

Prothrombotic state in HIV: A study on protein C, protein S, homocysteine and correlation with CD4 counts.

Introduction: Human immunodeficiency virus (HIV) may result in variable haematological manifestations. Thrombotic events are more common among HIV-infected persons than the general population, possibly due to the increased inflammatory/hypercoagulable state and presence of concurrent comorbidities. Aims and Objectives: (1) Screen for coagulation abnormalities in HIV-infected patients. (2) Detect certain prothrombotic factors such as deficiency of protein C and protein S and elevation of homocysteine as possible precursors of coagulation defects in HIV patients. (3) Correlation of coagulation abnormalities with CD4 counts. Methods: A pilot study of 1-year duration conducted in the Department of Pathology in collaboration with ART centre, KGMU Lucknow. All diagnosed HIV-seropositive patients (n = 30) who were not taking Vitamin K, antithrombotic and antiplatelet drugs including aspirin, oral contraceptives and not having known protein C/S deficiency were included in the present study as cases. Apart from this, 30 age- and sex-matched healthy individuals were also included in the present study. Assessment of the bleeding time, prothrombin time and activated partial thromboplastin time, complete blood count was done. Protein C and S were measured by calorimetric assay. Serum homocysteine was measured by the semi-automated method. CD4 count was done by flow cytometry. Results: The findings of the present study suggest a relationship between HIV, its complications and thrombosis. The HIV-seropositive patients have reduced levels of haemoglobin, CD4 counts, platelet counts, mean platelet volume, protein C and S activity as compared to the healthy individuals. Thrombophilic abnormality in the form of hyperhomocysteinaemia is more frequent in HIV-infected patients. All these parameters have a definite correlation with CD4 count.

A KCNC3 mutation causes a neurodevelopmental, non-progressive SCA13 subtype associated with dominant negative effects and aberrant EGFR trafficking.

The autosomal dominant spinocerebellar ataxias (SCAs) are a diverse group of neurological disorders anchored by the phenotypes of motor incoordination and cerebellar atrophy. Disease heterogeneity is appreciated through varying comorbidities: dysarthria, dysphagia, oculomotor and/or retinal abnormalities, motor neuron pathology, epilepsy, cognitive impairment, autonomic dysfunction, and psychiatric manifestations. Our study focuses on SCA13, which is caused by several allelic variants in the voltage-gated potassium channel KCNC3 (Kv3.3). We detail the clinical phenotype of four SCA13 kindreds that confirm causation of the KCNC3R423H allele. The heralding features demonstrate congenital onset with non-progressive, neurodevelopmental cerebellar hypoplasia and lifetime improvement in motor and cognitive function that implicate compensatory neural mechanisms. Targeted expression of human KCNC3R423H in Drosophila triggers aberrant wing veins, maldeveloped eyes, and fused ommatidia consistent with the neurodevelopmental presentation of patients. Furthermore, human KCNC3R423H expression in mammalian cells results in altered glycosylation and aberrant retention of the channel in anterograde and/or endosomal vesicles. Confirmation of the absence of plasma membrane targeting was based on the loss of current conductance in cells expressing the mutant channel. Mechanistically, genetic studies in Drosophila, along with cellular and biophysical studies in mammalian systems, demonstrate the dominant negative effect exerted by the mutant on the wild-type (WT) protein, which explains dominant inheritance. We demonstrate that ocular co-expression of KCNC3R423H with Drosophila epidermal growth factor receptor (dEgfr) results in striking rescue of the eye phenotype, whereas KCNC3R423H expression in mammalian cells results in aberrant intracellular retention of human epidermal growth factor receptor (EGFR). Together, these results indicate that the neurodevelopmental consequences of KCNC3R423H may be mediated through indirect effects on EGFR signaling in the developing cerebellum. Our results therefore confirm the KCNC3R423H allele as causative for SCA13, through a dominant negative effect on KCNC3WT and links with EGFR that account for dominant inheritance, congenital onset, and disease pathology.

Purification of transcripts and metabolites from Drosophila heads.

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.