K. P. Mohanakumar, Ph.D.
Chief Scientist, Cell Biology & Physiology Division, Indian Institute of Chemical Biology, Kolkata
Neuroprotective and neurodestructive effects of Ayurvedic drug constituents: Parkinson’s disease
The present study reports the good and the bad entities in an Indian traditional medicine used for treating Parkinson’s disease (PD). A prospective clinical trial on the effectiveness of Ayurvedic medication in a population of PD patients revealed significant benefits, which has been attributed to L-DOPA present in the herbs . Later studies revealed better benefits with one of the herbs alone, compared to pure L-DOPA in a clinical trial conducted in UK , and in several studies conducted on animal models of PD in independent laboratories world over [3-5]. We have adapted strategies to segregate molecules from the herb, and then carefully removed L-DOPA contained therein, and tested each of these sub-fractions for anti-PD activity in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, rotenone and 6-hydroxydopamine -induced parkinsonian animal models, and transgenic mitochondrial cybrids. We report here two classes of molecules contained in the herb, one of which possessed severe pro-parkinsonian (phenolic amine derivatives) and the other having excellent anti-parkinsonian potential (substituted tetrahydroisoquinoline derivatives). The former has been shown to cause severe dopamine depletion in the striatum of rodents, when administered acutely or chronically. It also caused significant behavioral aberrations, leading to anxiety and depression . The latter class of molecules administered in PD animal model , caused reversal of behavioral dysfunctions and significant attenuation of striatal dopamine loss. These effects were comparable or better than the effects of the anti-PD drugs, selegiline or L-DOPA. The mechanism of action of the molecule has been found to be novel, at the postsynaptic receptor signaling level, as well as cellular α-synuclein oligomerization and specifically at mitochondria. The molecule helped in maintaining mitochondrial ETC complex activity and stabilized cellular respiration, and mitochondrial fusion-fission machinery with specific effect on the dynamin related protein 1. Although there existed significant medical benefits that could be derived to patients due to the synergistic actions of several molecules present in a traditional preparation, accumulated data in our hands suggest complicated mechanisms of actions of Ayurvedic medication. Our results also provide great hope for extracting, synthesizing and optimizing the activity of anti-parkinsonian molecules present in traditional Ayurvedic herbs, and for designing novel drugs with novel mechanisms of action.
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R. Manjunath, Ph.D.
Associate Professor, Dept of Biochemistry, Indian Institute of Science, Bengaluru, India
REGULATION OF THE MHC COMPLEX AND HLA SOLUBILISATION BY THE FLAVIVIRUS, JAPANESE ENCEPHALITIS VIRUS
Viral encephalitis caused by Japanese encephalitis virus (JEV) and West Nile Virus (WNV) is a mosquito-borne disease that is prevalent in different parts of India and other parts of South East Asia. JEV is a positive single stranded RNA virus that belongs to the Flavivirus genus of the family Flaviviridae. The genome of JEV is about 11 kb long and codes for a polyprotein which is cleaved by both host and viral encoded proteases to form 3 structural and 7 non-structural proteins. It is a neurotropic virus which infects the central nervous system (CNS) and causes death predominantly in newborn children and young adults. JEV follows a zoonotic life-cycle involving mosquitoes and vertebrate, chiefly pigs and ardeid birds, as amplifying hosts. Humans are infected when bitten by an infected mosquito and are dead end hosts. Its structural, pathological, immunological and epidemiological aspects have been well studied. After entry into the host following a mosquito bite, JEV infection leads to acute peripheral neutrophil leucocytosis in the brain and leads to elevated levels of type I interferon, macrophage-derived chemotactic factor, RANTES,TNF-α and IL-8 in the serum and cerebrospinal fluid.
Major Histocompatibility Complex (MHC) molecules play a very important role in adaptive immune responses. Along with various classical MHC class I molecules, other non-classical MHC class I molecules play an important role in modulating innate immune responses. Our lab has shown the activation of cytotoxic T-cells (CTLs) during JEV infection and CTLs recognize non-self peptides presented on MHC molecules and provide protection by eliminating infected cells. However, along with proinflammatory cytokines such as TNFα, they may also cause immunopathology within the JEV infected brain. Both JEV and WNV, another related flavivirus have been shown to increase MHC class I expression. Infection of human foreskin fibroblast cells (HFF) by WNV results in upregulation of HLA expression. Data from our lab has also shown that JEV infection upregulates classical as well as nonclassical (class Ib) MHC antigen expression on the surface of primary mouse brain astrocytes and mouse embryonic fibroblasts.
There are no reports that have discussed the expression of these molecules on other cells like endothelial and astrocyte that play an important role in viral invasion in humans. We have studied the expression of human classical class I molecules HLA-A, -B, -C and the non-classical HLA molecules, HLA-E as well as HLA-F in immortalized human brain microvascular endothelial cells (HBMEC), human endothelial cell line (ECV304), human glioblastoma cell line (U87MG) and human foreskin fibroblast cells (HFF). Nonclassical MHC molecules such as mouse Qa-1b and its human homologue, HLA-E have been shown to be the ligand for the inhibitory NK receptor, NKG2A/CD94 and may bridge innate and adaptive immune responses. We show that JEV infection of HBMEC and ECV 304 cells upregulates the expression of HLA-A, and –B antigens as well as HLA-E and HLA-F. Increased expression of total HLA-E upon JEV infection was also observed in other human cell lines as well like, human amniotic epithelial cells, AV-3, FL and WISH cells. Further, we show for the first time that soluble HLA-E (sHLA-E) was released from infected ECV and HBMECs. In contrast, HFF cells showed only upregulation of cell-surface HLA-E expression while U87MG, a human glioblastoma cell line neither showed any cell-surface induction nor its solubilization. This shedding of sHLA-E was found to be dependent on matrix metalloproteinase (MMP) and an important MMP, MMP-9 was upregulated during JEV infection. Treatment with IFNγ resulted in the shedding of sHLA-E from ECV as well as U87MG but not from HFF cells. Also, sHLA-E was shed upon treatment with IFNβ and both IFNβ and TNFα, when present together caused an additive increase in the shedding of sHLA-E. HLA-E is an inhibitory ligand for CD94/NKG2A receptor of Natural Killer cells. Thus, MMP mediated solubilization of HLA-E from infected endothelial cells may have important implications in JEV pathogenesis including its ability to compromise the blood brain barrier.
Andrey Panteleyev, Ph.D.
Vice Chair, Division of Molecular Biology, NBICS Centre-Kurchatov Institute, Moscow, Russia
The system of PAS proteins (HIF and AhR) as an interface between environment and skin homeostasis
Regulation of normal skin functions as well as etiology of many skin diseases are both tightly linked to the environmental impact. Nevertheless, molecular aspects of skin-environment communication and mechanisms coordinating skin response to a plurality of environmental stressors remain poorly understood.
Our studies along with the work of other groups have identified the family of PAS dimeric transcription factors as an essential sensory and regulatory component of communication between skin and the environment. This protein family comprises a number of hypoxia-induced factors (HIF-alpha proteins), aryl hydrocarbon receptor (AhR), AhR nuclear translocator (ARNT), and several proteins implicated in control of rhythmic processes (Clock, Period, and Bmal proteins). Together, various PAS proteins (and first of all ARNT – as the central dimerization partner in the family) control such pivotal aspects of cell physiology as drug/xenobiotic metabolism, hypoxic and UV light response, ROS activity, pathogen defense, overall energy balance and breathing pathways.
In his presentation Dr. Panteleyev will focus on the role of ARNT activity and local hypoxia in control of keratinocyte differentiation and cornification. His recent work revealed that ARNT negatively regulates expression of late differentiation genes through modulation of amphiregulin expression and downstream alterations in activity of EGFR pathway. All these effects are highly dependent on epigenetic mechanisms such as histone deacetylation. Characterisation of hypoxia as a key microenvironmental factor in the skin and the role of HIF pathway in control of dermal vasculature and epidermal functions is another major focus of Dr. Panteleyev’s presentation.
In general, the studies of Dr. Panteleyev’s laboratory provide an insight into the PAS-dependent maintenance of skin homeostasis and point to the potential role of these proteins in pathogenesis of environmentally-modulated skin diseases such as barrier defects, desquamation abnormalities, psoriasis, etc.
Tim Guilliams, Ph.D.
Junior Associate Fellow at the Centre for Science and Policy, University of Cambridge
From Camels to Worms: Novel Approaches for Drug Discovery in Parkinson’s Disease
The discovery of novel treatments for neurodegenerative diseases, such as Parkinson’s disease, represents one of the biggest scientific challenges of the 21st century. The development of new tools and models to study the mechanisms underlying neurotoxicity is therefore essential. During my talk, I will outline new strategies for drug design and innovation used during my PhD at the University of Cambridge, which include the combination of fluorescent nematode worms, camelid antibody fragment technology and chemical compounds. These novel approaches will help us to gain insights into the key pathogenic steps involved in Parkinson’s disease and potentially lead to new therapeutic strategies.