Presence of Extracellular Alpha-Synuclein Aggregates Trigger Astrocytic Degeneration Through Enhanced Membrane Rigidity and Deregulation of Store-Operated Calcium Entry (SOCE) into the Endoplasmic Reticulum.

α-Synuclein has a critical role in Parkinson's disease, but the mechanism of how extracellular α-synuclein aggregates lead to astrocytic degeneration remains unknown. Our recent study in astrocytes highlighted that α-synuclein aggregates undergo lower endocytosis than the monomeric-form, even while displaying a higher impact on glutathione-machinery and glutamate-metabolism under sublethal conditions. As optimal intracellular calcium levels are essential for these functions, we aimed to study the effect of extracellular α-synuclein aggregates on ER calcium entry. We assessed the association of extracellular aggregated-α-synuclein (WT and A30P/A53T double-mutant) with the astrocytic membrane (lipid rafts) and studied its effects on membrane fluidity, ER stress, and ER calcium refilling in three systems-purified rat primary midbrain astrocyte culture, human iPSC-derived astrocytes, and U87 cells. The corresponding timeline effect on mitochondrial membrane potential was also evaluated. Post-24 h exposure to extracellular WT and mutant α-synuclein aggregates, fluorescence-based studies showed a significant increase in astrocyte membrane rigidity over control, with membrane association being significantly higher for the double mutant aggregates. α-Synuclein aggregates also showed preferentially higher association with lipid rafts of astrocytic membrane. A simultaneous increase in ER stress markers (phosphorylated PERK and CHOP) with significantly higher SOCE was also observed in aggregate-treated astrocytes, with higher levels for double mutant variant. These observations correlate with increased expression of SOCE markers, especially Orai3, on plasma membrane. Alterations in mitochondrial membrane potential were only noted post-48 h of exposure to α-synuclein aggregates. We therefore suggest that in astrocytes, α-synuclein-aggregates preferentially associate with lipid rafts of membrane, altering membrane fluidity and consequently inducing ER stress mediated by interaction with membrane SOCE proteins, resulting in higher Ca2+ entry. A distinct cascade of events of sequential impairment of ER followed by mitochondrial alteration is observed. The study provides novel evidence elucidating relationships between extracellular α-synuclein aggregates and organellar stress in astrocytes and indicates the therapeutic potential in targeting the association of α-synuclein aggregates with astrocytic membrane.

A rapid and simple resazurin assay to detect minimum inhibitory concentrations of first-line drugs for Mycobacterium tuberculosis isolated from cerebrospinal fluid.

OBJECTIVES: Central nervous system tuberculosis (CNS-TB) is a devastating manifestation of TB. The most common form of CNS-TB is tuberculous meningitis. Drug-resistant TB poses a major threat to the control of TB worldwide. Timely treatment dramatically improves the outcome. Colorimetric techniques for drug susceptibility testing based on the oxidation-reduction principle give results quick and are less expensive. The objectives of this study were to compare the susceptibility of Mycobacterium tuberculosis isolated from cerebrospinal fluid to four first-line drugs using the MGIT automated mycobacterial detection system and the resazurin assay (RA) as well as to estimate the minimum inhibitory concentrations (MICs) by RA. METHODS: A total of 42 M. tuberculosis isolates were analysed for their susceptibilities by MGIT and RA. RESULTS: Of the 42 isolates, 35 gave concordant results with both methods. Agreement between the two tests for streptomycin and rifampicin was 100% with a Fleiss' kappa (κ) value of 1, whereas for isoniazid and ethambutol agreement was 92.86% and 90.48%, respectively, with κ values of 0.853 and 0.738. CONCLUSION: The RA appears to be a good alternative to the automated MGIT technique in resource-limited settings.

Mitochondrial functions of RECQL4 are required for the prevention of aerobic glycolysis-dependent cell invasion.

Germline mutations in RECQL4 helicase are associated with Rothmund-Thomson syndrome, which is characterized by a predisposition to cancer. RECQL4 localizes to the mitochondria, where it acts as an accessory factor during mitochondrial DNA replication. To understand the specific mitochondrial functions of RECQL4, we created isogenic cell lines, in which the mitochondrial localization of the helicase was either retained or abolished. The mitochondrial integrity was affected due to the absence of RECQL4 in mitochondria, leading to a decrease in F1F0-ATP synthase activity. In cells where RECQL4 does not localize to mitochondria, the membrane potential was decreased, whereas ROS levels increased due to the presence of high levels of catalytically inactive SOD2. Inactive SOD2 accumulated owing to diminished SIRT3 activity. Lack of the mitochondrial functions of RECQL4 led to aerobic glycolysis that, in turn, led to an increased invasive capability within these cells. Together, this study demonstrates for the first time that, owing to its mitochondrial functions, the accessory mitochondrial replication helicase RECQL4 prevents the invasive step in the neoplastic transformation process.

Protective effect of curcuminoids on age-related mitochondrial impairment in female Wistar rat brain.

The present study demonstrated the neuroprotective effect of curcuminoids, the active polyphenols of Curcuma longa (L.) rhizomes on mitochondrial dysfunctioning in middle aged and aged female Wistar rat brain. Rats were orally treated with curcuminoids (100 mg/kg) for 3 months and their brain was collected for evaluation of mitochondrial enzymes and complexes activity, ultra structural changes in mitochondria, neuronal nitric oxide synthase (nNOS) protein expression, adenosine triphosphate (ATP) and lipofuscin content. Significant alterations were observed in all the tested parameters in highly aged rat brain when compared with young control. Long term curcuminoids administration prevented this age associated loss of mitochondrial enzymes and complexes activity in middle aged rat brain except for malate dehydrogenase, Complex II and IV activity when compared with young control. Among aged rats, curcuminoids treatment specifically elevated isocitrate and NADH dehydrogenase, cytochrome c oxidase, Complex I and total ATP content. A significant down-regulation of nNOS protein expression along with reduced lipofuscin content was also observed in curucminoids treated middle aged and aged rats. Thus, it was suggested that curcuminoids may act as a putative drug candidate for the prevention of deleterious effects of ageing and age associated neurodegenerative disorders through amelioration of aberrant mitochondrial functioning.

Expression and localization of insulin receptors in dissociated primary cultures of rat Schwann cells.

The objective of the present study was to examine for the presence of the IRs (insulin receptors) in the primary dissociated culture preparation of SCs (Schwann cells). This was achieved using immunological techniques using a rabbit polyclonal anti-IR antibody and at molecular level by RT (reverse transcription)-PCR. Light microscopic immune cytochemistry revealed that almost all SCs in cluster and associated neuritis exhibited positive immune reaction with the antibody, confirming the presence of IRs in them. Immunoblotting detected a prominent protein band of 90 kDa, which is consistent with those reported by the manufacturer. Like the peripheral nerve, primary SC cultures showed a predominantly high affinity IR mRNA lacking exon 11. Ultrastructural immune localization confined the presence of the IRs in the basal lamina, plasma membrane and the cytoplasmic processes of the SCs.