Mutations in the LRRK2 gene are present in 5-13% of familial PD and 1-5% of sporadic PD cases. Most of the research on PD-related LRRK2 mutations has focused on mutations in the kinase domain, viz. the G2019S mutation which elevates the kinase activity of the enzyme. This mutation is predominant in Caucasian PD patients, but its prevalence in the Indian population is less than 0.1% . On the other hand, the I1371V mutation has much higher prevalence in East Indian PD patients, but has hardly been studied. It is crucial that PD treatment strategies take these ethnic differences into consideration. Current disease-modeling and drug-development programs mostly use animal models that cannot replicate these human ethnicity variations. It is also well-established that humans and mice have significant developmental, genetic and physiological differences. Induced pluripotent stem-cells (iPSCs) derived from patients, however, can help overcome many of these limitations. Experimental modeling of human disorders using iPSCs helps us define the cellular and molecular mechanisms underlying diseases and has revolutionized the way we study monogenic, complex and epigenetic disorders. We have generated the first iPSC line from an Indian patient carrying the I1371V mutation in the LRRK2 enzyme. We are now investigating the functional aspects of the cellular pathogenesis of the I1371V mutation.
Role of phosphorylated α-synuclein in dopaminergic neuron dysfunction
Among the several post-translational modifications of α-synuclein that are known to occur in Parkinson’s disease, phosphorylation at the serine 129 region is the most common, leading to the accumulation of Lewy Bodies that is a pathological hallmark of PD. Approximately 90% of the α-synuclein deposited in Lewy Bodies is extensively phosphorylated at the serine 129 region, compared to only 4% or less in a normal healthy brain. The key kinases involved in the phosphorylation of α-synuclein are POLO-Like kinase (PLK), Casein Kinase2 (CK2), Leucine Rich Repeat Kinase 2 (LRRK2) and G-Protein Coupled Receptor Kinase 5 (GRK5). Inhibition of these individual kinases through siRNA has been shown to decrease phosphorylation of α-synuclein in WT and A53T α-synuclein transfected SH-SY5Y cells. While there are studies indicating an association between α-synuclein aggregation and reduction in dopamine release, the importance of α-synuclein phosphorylation to the process of vesicular dopamine release, their exocytosis and recycling, along with changes in intracellular calcium upon physiological stimulation and Store-Operated Ca2+ Entry (SOCE) are unreported. This is now under investigation.
Parkinson’s disease pathogenesis by astrocytes: differential response to different forms of extracellular α-synuclein
Our work focuses on the pathology caused by astrocytes due to their association with wild type or mutated forms of α-synuclein. Many in vivo and post-mortem studies have indicated the presence of monomeric and/or aggregated species of α-synuclein in dystrophic astrocytes, suggesting the deterioration of the cellular niche (astrocytes) that paves the way for neurodegeneration. However, the cellular and molecular mechanisms behind this is yet to be elucidated. Dysfunction of astrocytes in terms of defence against oxidative stress and other stress-indicative markers, glutamate clearance, calcium dynamics in presence of physiological stimulation, ER and lysosomal impairments, accumulation of other modified species of α-synuclein and the crosstalk between affected astrocytes and dopaminergic neurons – these are a few of the topics addressed in our research. We hope this understanding will help us design novel therapeutic strategies targeting the niche cells.
Astrocytes derived from Indian Ethnicity LRRK2 I1371V mutation Patient-Specific iPSCs and their non-cell autonomous role on the neurodegeneration of Parkinson’s disease.
Astrocytes, the structural support cells of the brain, also play a crucial role in the survival and functionality of neuronal cells through metabolic delivery of nutrients and/or anti-oxidants under physiological conditions. They are further capable of responding to pathological stimuli and consequently bring about changes in neuronal cells through neuron-glia crosstalk. These glial cells are now emerging as critical players in the pathogenesis of Parkinson’s disease. Among the many genes associated with PD is LRRK2, which represents the most common cause of inherited or familial PD. Several pathogenic missense LRRK2 mutations have been identified, the locations of which lie in different functional domains of LRRK2. While the G2019S mutation has been studied the most, the dysfunction or the morphological changes of the astrocytes caused due to the LRRK2 I1371V mutation (prevalent in India) is our subject of focus. We are evaluating the effect of the LRRK2 I1371V mutation on astrocytic fate commitment, cell vulnerability, cellular deregulation in reference to intracellular calcium response, lysosomal degradation, and the astrocytes’ neuroprotective functions on dopaminergic neurons.
Human Dental Pulp Stem Cells in the treatment of Diabetic Neuropathy: Does exogenous stem cell transplantation correct endogenous stem cell dysfunction?
Indications of endogenous Bone Marrow Mesenchymal Stem Cell (BM-MSC) dysfunction in diabetes have surfaced of late. Impaired cellular and molecular interactions between vascular and neurological components form the basis of disease progression in Diabetic Neuropathy. Our lab focuses on investigating BM-MSC dysfunction in pre-DN and post-DN rats, particularly with regard to their migratory/homing properties toward chemokines and cytokines, and the underlying intracellular calcium response. These experiments aid in answering some critical questions – does BM dysfunction precede the onset of neuropathy? Are endogenous DN-BMMSCs unable to protect DN-Schwann cells? And if BM-MSC dysfunction is indeed an early event, is that why chronic diabetic complications develop?
Biodistribution of transplanted human stem cells: Where do the cells go?
One of the biggest challenges in treatment strategies involving mesenchymal stem cell (MSC) therapy for any disease is determining the optimal route of transplantation and dosage. Key questions of how long MSCs dwell in the body and what tissues they migrate to need to be answered clearly so as to design the dose and frequency of MSC transplantations appropriately.
In our lab, we have tried to find answers to these questions by plotting the biodistribution of transplanted MSCs or its exosomes. Using minimally-invasive Near Infra-Red (NIR) whole animal and tissue imaging, we determine MSC/exosome retention time, course of movement, homing to niche and localization in organs.
The NIR range provides high sensitivity owing to least autofluorescence of animal tissue in this spectral region. In addition, it provides a higher depth of tissue penetration and therefore a higher signal-to-noise ratio. The technique requires minimal manipulation of the cells, and relies on simply tagging the plasma membrane with lipophilic NIR dyes that are quick, more stable, and do not require special handling.
Exosomes as a Drug Delivery tool
Exosomes are naturally occurring nano-sized vesicles produced by all cells, and range in size from 30-150nm. They carry the characteristics of their parent cell, have low immunogenicity, high bio-compatibility and minimum toxicity. DPSC-exosomes display similar neuro-modulatory and neuroprotective mechanism as DPSC themselves. Thus, they are an ideal delivery vesicle that crosses the BBB easily, unlike the cells they originate from. Being a neurodegenerative disorder, Parkinson’s Disease is difficult to treat as most of the drugs are administered as prodrugs that fail to provide the required concentration in the brain, owing to the BBB.
Exosomes are HLA-DR negative and bypass the mononuclear-phagocyte system to deliver their cargo without getting destroyed (Hall et al 2016).
Antioxidant-loaded exosomes delivered directly through the intranasal route maximize their availability in the midbain, avoiding the first pass metabolism and systemic distribution as observed in other routes of administration.