KAUR, SUPINDER et al. (2015) International Worm Meeting "Statins in management of Neurodegenerative Parkinson's disease: Studies employing Transgenic C.elegans Model.."

Kaur, Supinder, Nazir, Aamir

[International Worm Meeting, 2015]

Parkinson's disease (PD) is the second most common form of neurodegenerative disease associated with alpha synuclein aggregation, dopaminergic neurodegeneration, reduced dopamine levels leading to impaired motor functions. Presently management of PD is restricted to providing symptomatic relief only. In the light of beneficial effects observed upon chronic use of statins in PD, statins are being looked upon as possible candidates for its management. However, various studies have resulted in mixed conclusions pointing towards both positive and negative implications of statin use. Modulation of PD through chronic statin use is often attributed to the cholesterol independent i.e. pleiotropic effects of statins. In order to investigate the current scientific notion, we took up this study in a cholesterol auxotroph organism, C. elegans. In the present study using the C.elegans model of PD, we have studied the effect of statins on alpha synuclein accumulation. To begin with we studied the three common types of statins viz. Atorvastatin, Simvastatin and Rosuvastatin for their effect on alpha synuclein levels. Rosuvastatin being the most potent candidate was carried forward for further studies including estimation of dopamine levels through repellent assay and HPLC, lifespan assay, motor functions, autophagy and mRNA expression of genes linked to autophagy and protein sorting/packaging machinery. Rosuvastatin was found to be most potent of all statins and exhibited significant lowering of alpha-synuclein levels as checked through fluorescence microscopy and Western Blotting. Rosuvastatin treatment showed increased levels of autophagy in terms of punctate LGG-1::GFP expression, increased motility in terms of increased number of thrashes and elevated dopamine levels. The dopamine levels were found elevated in both wild-type worms and worms expressing alpha-synuclein levels. A moderate extension in mean life span was also observed which was specific to the disease condition. Our studies establish the beneficial effects of Rosuvastatin through its pleiotropic effects and we hypothesize that Rosuvastatin being a safe lipid drug should be explored further to accomplish therapeutic or supportive roles in PD management. .

Jadiya, Pooja et al. (2013) International Worm Meeting "Sirtuin Mediated Neuroprotection and its Association with Autophagy and Apoptosis: Studies Employing Transgenic C. elegans Model."

Nazir, Aamir, Jadiya, Pooja

[International Worm Meeting, 2013]

Sirtuins, NAD+-dependent protein deacetylases, are well known for their role in longevity. Aging is the major known risk factor for the onset of Parkinson's disease (PD). In our previous studies, SIRT1 induction by calorie restriction (CR) diet regimen has been shown to protect against dopaminergic neurodegeneration via sir-2.1 mediated pathway. Taking the studies forward, we endeavoured to understand the role of nicotinamide adenine dinucleotide activated protein deacetylase Sir2p/Sirt1 in calorie restriction mediated prevention of Parkinsonism employing transgenic Caenorhabditis elegans expressing human alpha synuclein. Our findings provide evidences towards the role of calorie restriction in reducing a-synuclein aggregation, mitochondrial, lipid content and ROS in human a-synuclein expressing strain of C. elegans. RNAi of sir-2.1 enhanced aggregation of alpha synuclein but sir-2.1 silenced worms raised on reduced calorie diet didn't show protective effect in reducing protein aggregation which concluded that protective effects of calorie restriction was mediated by NAD-dependent histone deacetylase activity of sir-2.1. We next focused on the mechanism of the sir-2.1 mediated neuroprotective effect via autophagy. Assessment of autophagy in C. elegans was performed using a transgenic strain DA2123 strain, expressing a GFP-tagged LGG-1 protein. Worms with RNAi induced gene silencing of sir-2.1 showed decreased expression of GFP-tagged LGG-1 protein and decreased mRNA level of different autophagy genes including lgg-1,bec-1,atg-5,atg-7 and atg-13 in qPCR studies. To further examine the signaling pathways which could link SIRT1 to the regulation of autophagic degradation, we analyzed the expression of different apoptosis genes including ced-4, cep-1, lin-35, jkk-1 and jnk-1. In our studies, silencing of sir-2.1 showed significantly up-regulation of ced-4 (Apaf-1) & cep-1 (p53 ortholog- DNA damage pathway) apoptosis genes. Our study emphasizes the protective role of sir-2.1 on autophagosome formation, which is associated with the p53 and apaf-1 dependent signaling pathways which are well-known stress resistance mediators.

Kaur, Supinder et al. (2011) International Worm Meeting "Studies on Toxicity of Nano Iron Employing Model System Caenorhabditis elegans."

Nazir, Aamir, Kaur, Supinder, Sammi, Shreesh Raj, Jadiya, Pooja

[International Worm Meeting, 2011]

The rapidly evolving field of scientific and medical research is making numerous advances in terms of newer technologies aimed at bettering human health and quality of life. Amongst such advances, use of nanomaterials in various forms and facets has gained a lot of momentum in recent years. Nano particles find use in diverse processes such as drug delivery, bio- sensing, bio-imaging, blood vessel repair, magnetic cell therapy, hyperthermic cancer treatment and water purification. Having a widespread utility, nanomaterials reach humans via various means, thus making it relevant to assess their safety using efficient, sensitive and reliable methods. Amongst various models employed for studying toxicity and safety of chemicals, C. elegans is a fairly new addition and is proving to be a model of immense utility considering its powerful genetics, well dissected out developmental biology, homology of gene sequences with that of humans and ease of laboratory use. Of late, C. elegans is also well suited to large scale toxicity screening campaigns. We conducted the present study with a three-fold aim: 1) to use model system C. elegans for toxicity studies on nanoparticles; 2) to assess the safety of nano particle iron that finds widespread use in processes like water purification, disease treatment; and 3) to study the effect of particle size on the toxicity of iron (nano vs bulk) in C. elegans model. We first studied the absorption of nanoparticles inside the worm by feeding the worms with fluorescent nanoparticles. In subsequent studies nematodes were exposed to three different concentrations (20mM, 200mM and 2mM) of nanoparticle and bulk Iron (in the form of Fe2O3). Worms were subsequently studied for effects on locomotion, longevity, fertility, oxidative stress, apoptosis, mitochondrial content, ATP levels and for effects on expression of sod-1, sod-2, sod-3, mtl-1, mtl-2 and hsp-60. Our data reveals that nano iron induces significant effect on longevity of the nematodes as the life span was reduced by 17% in case of worms treated with 2mM nanoparticle Fe2O3; worms treated with equal concetntration of bulk Fe2O3 did not exhibit a reduced longevity as compared to control subjects. Similarly, nano iron exhibited a significant effect on generation of reactive oxygen species thus increasing the amount of oxidative stress within the worms. We conclude that: 1) C. elegans could successfully be employed in studying toxicity of nanoparticles; 2) nano iron did exhibit whole-organismal toxic effects at 2mM concentration in nematode C. elegans; and 3) nanoparticulate form of iron, at tested concentrations, induces comparatively more whole-organismal toxicity than that of the bulk form.

Jadiya, Pooja et al. (2011) International Worm Meeting "Dietary Epigenetics and Neurodegenerative Diseases: Effect of Calorie Restriction on Dopaminergic Neurodegeneration in Caenorhabditis elegans."

Nazir, Aamir, Sammi, Shreesh Raj, Kaur, Supinder, Jadiya, Pooja

[International Worm Meeting, 2011]

The fact, that causal factors of most common diseases often involve both-susceptibility genes and their interactions with exogenous/endogenous environment, is well established. The conceptual approaches to role of such epigenetics in human disease conditions have been well conceived. Amongst various diseases that are modulated by such factors, age associated neurodegenerative diseases are a class of syndromes that have intrigued researchers for decades. C. elegans models of age associated neurodegenerative diseases have provided several meaningful insights into the understanding of these disease conditions. We employ transgenic nematode models towards understanding various mechanistic aspects of such diseases. In the present study we employed the transgenic C. elegans model (Pdat-1::GFP) expressing green fluorescence protein (GFP) specifically in eight dopaminergic (DA) neurons. Selective degeneration of dopaminergic neurons was induced by treatment of worms with 6-hydroxy dopamine (6-OHDA) as previously described. In subsequent studies, we explored the role of calorie restriction in modulating the 6-OHDA induced neuronal damage. Worms were treated with 25mM 6-OHDA to induce degeneration of DA neurons and were raised either on control diet (normal concentration of bacteria OP50) or on 10,000 fold diluted OP50. It was observed that worms raised on the reduced calorie diet exhibited a protective effect on dopaminergic neurons with ADE and PDE neurons exhibiting an enhanced and complete expression of GFP; CEP neurons exhibited a marginal increase in GFP expression. When quantified using Image J analysis, the mean fluorescence (GFP) intensity corresponding to a healthy dopaminergic neurophysiology, was 6.32 plus or minus 0.56 arbitrary units in control worms, 1.92 plus or minus 0.15 arbitrary units in 6-OHDA treated subjects (a 3.3 fold reduction vs. control) and 5.28 plus or minus 0.61 arbitrary units in case of worms fed with a 10,000 fold diluted OP50 (a 2.75 fold increase as compared to 6-OHDA treated subjects). We further studied the effect of 6-OHDA induced dopaminergic neurodegeneration and calorie restriction on mitochondrial content, ATP levels and oxidative stress in the worms. Our studies provide evidence that calorie restriction affords significant protection against the dopaminergic neurodegeneration, that might have implications for neurodegenerative Parkinson's disease.

Sammi, Shreesh Raj et al. (2011) International Worm Meeting "A Three-pronged Method Employing Caenorhabditis elegans for Large Scale Screening of Potential Pharmacological Agents against Alzheimer's Disease."

Sammi, Shreesh Raj, Nazir, Aamir, Jadiya, Pooja, Kaur, Supinder

[International Worm Meeting, 2011]

Large scale screening of potential pharmacological compounds is usually a pilot task requiring larger efforts. Efficient and quick screenings rely on established models that can provide initial information on a high-throughput scale with a better sensitivity. Diseases like Alzheimer's disease (AD) have found no cure yet; though there has been an immense increase in understanding of the disease employing non-mammalian model systems like nematode C. elegans. The broad understanding that has been achieved, thus far, proves beyond doubt, the involvement of multiple factors that lead to devastating neurodegenerative AD. Amongst various factors involved, increased amyloid beta aggregation, increased reactive oxygen species and decreased acetylcholine levels are the hallmarks of AD. Thus any potential pharmacological agent or a possible combination therapy should be targeted at these multiple factors. Studies in this direction require robust model systems that could be used for high throughput screening of chemicals. In our research studies, we employ transgenic nematode strain CL2006 that expresses 'human' amyloid beta and serves as a model for Alzheimer's disease. The model has previously been shown to exhibit aggregation of amyloid beta and a proteomic expression pattern similar to human Alzheimer's patients. Herein we propose the use of a transgenic C. elegans based three pronged methodology that, we believe, could be immensely beneficial in carrying out large scale screening campaigns of potential pharmacological agents exhibiting anti-Alzheimer activity. Specifically we propose that studies on: a) amyloid beta expression (qualitative and quantitative), b) Acetylcholine activity and c) quantification of reactive oxygen species (ROS), in transgenic C. elegans model of Alzheimer's disease, could prove to be an effective regime of tests towards carrying out large scale screening campaigns. Since Alzheimer's disease is a multifactorial disease, a set of three tests targeting different factors could help in identifying potential pharmacologicals specifically suited to treat the multiple factors of the disease. Our studies demonstrate the feasibility of these methods aimed at establishing a sensitive test regime for screening drugs against Alzheimer's disease.

Haque, Rizwanul et al. (2015) International Worm Meeting "Insulin Modulates the Outcome of alpha Synuclein Aggregation via Daf-2/Daf-16 Signaling Pathway by Acting as Antagonist for DAF-2 Receptor in Transgenic C. elegans Model of Parkinson's Disease."

Nazir, Aamir, Jadiya, Pooja, Haque, Rizwanul, Kumar, Lalit, Shamsuzzama, Shamsuzzama, Fatima, Soobiya

[International Worm Meeting, 2015]

Insulin signaling is an important pathway in multiple cellular functions and organismal aging across the taxa. Recent findings have linked defective insulin signaling to neurodegenerative Parkinson's disease (PD). In genetic model system Caenorhabditis elegans, insulin signaling is regulated by DAF-2, a tyrosine kinase receptor orthologous to the mammalian insulin/IGF receptor, which phosphorylates downstream transcription factor DAF-16 via AKT protein kinase activation. Despite crucial evidences on association of insulin signaling with PD, the exact nature of molecular events and genetic associations are yet to be understood. We employed transgenic C. elegans strain harboring human alpha-synuclein::YFP transgene, towards studying the aggregation pattern of alpha-synuclein, a PD associated endpoint, under 10 and 15 U/ml biphasic isophane insulin (Huminsulin®) treatment and DAF-16/DAF-2 knockdown conditions, independently and in combination. We observed that the aggregation was increased when DAF-16 was knocked down independently or alongwith a co-treatment of insulin and decreased when under DAF-2 was knocked down independently or alongwith a co-treatment of insulin; whereas Insulin treatment per se, reduced the aggregation and associated effects in a concentration dependent manner. Our results depicted that Human insulin decreases alpha-synuclein aggregation via DAF-2/DAF-16 pathway by acting as an antagonist for DAF-2 receptor. Knockdown of reported DAF-2 agonist (INS-6) and antagonists (INS-17 and INS-18) also resulted in a similar effect on alpha-synuclein aggregation. Further by utilizing bioinformatic tools, we compared the structural differences among agonists and antagonists including human insulin. Our results suggest that any deviation from the normal insulin signaling may greatly affect the alpha-synuclein aggregation pattern; further human insulin treatment and DAF-16 expression play a protective role against alpha-synuclein aggregation and its associated effects, thus providing interesting avenues for further research and possible therapeutic interventions.