Differential effect of Ayurvedic nootropics on C. elegans models of Parkinson's disease.

BACKGROUND: Globally, there is increased incidence of Parkinson's Disease (PD), which is the second most common age-related neurodegenerative disease. The currently available PD-therapeutics provide only symptomatic relief. Thus, there is an urgent need to devise an effective and safe treatment strategy for PD. The holistic approach of Ayurveda can be a potential effective strategy for treating PD. The integration of different medicine systems, such as modern bio-medicine and Ayurveda can be an effective strategy for treatment of complex diseases, including PD. OBJECTIVE: This study aimed to evaluate the neuroprotective mechanism of six Ayurvedic nootropics that are commonly used to treat PD. MATERIAL AND METHODS: Six Ayurvedic herbs, namely Mucuna pruriens (MP), Bacopa monnieri (BM), Withania somnifera (WS), Centella asiatica (CA), Sida cordifolia (SC), and Celastrus paniculatus (CP), were selected after consultation with Ayurvedic scholars and physicians. The mode of action of methanolic herbal extracts was evaluated using the Caenorhabditis elegans BZ555 and NL5901 strains, which can be used to model the two main hallmarks of PD, namely degeneration of dopaminergic neurons and aggregation of α-synuclein protein. RESULTS: All six herbal extracts exhibited neuroprotective effect. The extracts of BM and MP exhibited maximum protection against 1-methyl-4-phenylpyridinium iodide (MPP+ iodide)-induced dopaminergic neurodegeneration in the BZ555 strain. Furthermore, the herbal extracts, except CA extract, inhibited the aggregation of heterologously expressed human α-synuclein in the NL5901 strain. CONCLUSION: Ayurvedic herbs used in the treatment of PD exhibited differential neuroprotective and protein aggregation mitigating effects in C. elegans.

Regulation of VDAC trafficking modulates cell death.

The voltage-dependent anion channel (VDAC) and mitochondria-associated hexokinase (HxK) have crucial roles in both cell survival and death. Both the individual abundances and their ratio seem to influence the balance of survival and death and are thus critical in scenarios, such as neurodegeneration and cancer. Elevated levels of both VDAC and HxK have been reported in cancerous cells. Physical interaction is surmised and specific residues or regions involved have been identified, but details of the interaction and the mechanism by which it modulates survival are yet to be elucidated. We and others have shown that heterologous expression of VDAC can induce cell death, which can be mitigated by concomitant overexpression of HxK. We have also observed that upon overexpression, fluorescently tagged VDAC is distributed between the cytosol and mitochondria. In this study, we show that cell death ensues only when the protein, which is synthesized on cytoplasmic ribosomes, migrates to the mitochondrion. Further, coexpression of rat HxK II (rHxKII) can delay the translocation of human VDAC1 (hVDAC1) protein to mitochondria and thereby inhibit VDAC-induced cell death. Variation in the level of HxK protein as seen endogenously in different cell lines, or as experimentally manipulated by silencing and overexpression, can lead to differential VDAC translocation kinetics and related cell death. The N-terminal region of HxK and the Glu73 residue of hVDAC1, which have previously been implicated in a physical interaction, are required for cytosolic retention of VDAC. Finally, we show that, in otherwise unperturbed cells in culture, there is a small but significant amount of soluble VDAC in the cytosol present in a complex with HxK. This complex could well determine how a cell is poised with respect to incoming thanatopic signals, thereby tilting the survival/death balance in pharmacologically interesting situations, such as neurodegeneration and cancer.

Expression and purification of OsVDAC4.

The voltage-dependent anion channel (VDAC), a major component of the mitochondrial outer membrane, has emerged as an important player in cell function, survival, and death signaling. VDAC function is modulated by its interaction with proteins such as hexokinase, adenine nucleotide translocator, and apoptotic proteins like Bax. Monitoring the activity of VDAC and its modulation in the complex cellular milieu is fraught with complications. Minimizing the number of components in the study is one approach to teasing apart various aspects of its function. In this chapter, we have described detailed protocols for the purification of a rice VDAC isoform, OsVDAC4 after overexpression in a bacterial system. The protein is solubilized with LDAO and then reconstituted into liposomes or planar bilayers to verify its competence to fold into a functionally active form.

Mitochondrial VDAC and hexokinase together modulate plant programmed cell death.

The voltage-dependent anion channel (VDAC) and mitochondrially located hexokinase have been implicated both in pathways leading to cell death on the one hand, and immortalization in tumor formation on the other. While both proteins have also been implicated in death processes in plants, their interaction has not been explored. We have examined cell death following heterologous expression of a rice VDAC in the tobacco cell line BY2 and in leaves of tobacco plants and show that it is ameliorated by co-expression of hexokinase. Hexokinase also abrogates death induced by H2O2. We conclude that the ratio of expression of the two proteins and their interaction play a major role in modulating death pathways in plants.

Bacterial expression, purification and characterization of a rice voltage-dependent, anion-selective channel isoform, OsVDAC4.

The voltage-dependent anion-selective channel (VDAC) is the most abundant protein in the mitochondrial outer membrane and forms the major conduit for metabolite transport across this membrane. VDACs from different sources show varied primary sequence but conserved functional properties. Here, we report on the characterization of a rice channel, OsVDAC4, which complements a VDAC1 deficiency in yeast. We present a consensus secondary structure prediction of an N-terminal α-helix and 19 ß-strands. Bacterially expressed OsVDAC4 was purified from inclusion bodies into detergent-containing solution, where it is largely helical. Detergent-solubilized OsVDAC4 inserts spontaneously into artificial membranes of two topologies-spherical liposomes and planar bilayers. Insertion into liposomes results in an increase in ß-structure. Transport of polyethylene glycols was used to estimate a pore diameter of ~2.6 nm in liposomes. Channels formed in planar bilayers exhibit large conductance (4.6 ± 0.3 nS in 1 M KCl), strong voltage dependence and weak anion selectivity. The open state of the channel is shown to be permeable to ATP. These data are consistent with a large ß-barrel pore formed by OsVDAC4 on inserting into membranes. This study forms a platform to carry out studies of the interaction of OsVDAC4 with putative modulators.

Repression of the glucose-inducible outer-membrane protein OprB during utilization of aromatic compounds and organic acids in Pseudomonas putida CSV86.

Pseudomonas putida CSV86 shows preferential utilization of aromatic compounds over glucose. Protein analysis and [¹4C]glucose-binding studies of the outer membrane fraction of cells grown on different carbon sources revealed a 40 kDa protein that was transcriptionally induced by glucose and repressed by aromatics and succinate. Based on 2D gel electrophoresis and liquid chromatography-tandem mass spectrometry analysis, the 40 kDa protein closely resembled the porin B of P. putida KT2440 and carbohydrate-selective porin OprB of various Pseudomonas strains. The purified native protein (i) was estimated to be a homotrimer of 125 kDa with a subunit molecular mass of 40 kDa, (ii) displayed heat modifiability of electrophoretic mobility, (iii) showed channel conductance of 166 pS in 1 M KCl, (iv) permeated various sugars (mono-, di- and tri-saccharides), organic acids, amino acids and aromatic compounds, and (v) harboured a glucose-specific and saturable binding site with a dissociation constant of 1.3 µM. These results identify the glucose-inducible outer-membrane protein of P. putida CSV86 as a carbohydrate-selective protein OprB. Besides modulation of intracellular glucose-metabolizing enzymes and specific glucose-binding periplasmic space protein, the repression of OprB by aromatics and organic acids, even in the presence of glucose, also contributes significantly to the strain's ability to utilize aromatics and organic acids over glucose.