Cell Culture and Stem Cell Biology (Officer In-charge: Dr. T.R. Raju)
This laboratory is well equipped with all the infrastructure and successfully functioning as a Cell Culture and Stem Cell Biology lab. Currently primary cultures of motor neurons, astrocytes, microglia, olfactory bulb and cell lines of motor neurons (NSC-34), Dopaminergic neurons (N27), Neuroblastoma (SHSY5Y) and glioblastoma (U251MG, U87MG) are grown in the cell culture laboratory. These cell lines are used as cellular model system to delineate the pathogenesis of ALS, Parkinson’s disease and Glioma. Several funded projects are being executed in this laboratory.
Completed research activities include the following:
1. To investigate the possibility that aggregation of phosphorylated neurofilaments might have occurred due to the deranged expression of the neurofilament subunits, we examined the expression of sub-units NF-H and NF-L in NSC-34 cells upon exposure to ALS-CSF by immunocytochemistry and Western blotting. We found stable expression of NF-H in NSC-34 cells upon exposure to ALS-CSF; however the expression of NF-L was up-regulated when compared to the control groups viz. NC and NALS.
2. Quantitative proteomic analysis by iTRAQ labelling was carried out to identify toxic factors present in CSF of Amyotrophic Lateral Sclerosis patients (ALS-CSF). The mass spectrometry results of 10 ALS-CSF samples compared with 10 normal CSF samples from patients undergoing orthopaedic surgery (N-CSF) showed up-regulation of approximately 31 proteins (showing more than 1.5 fold increase) and 17 proteins were down regulated (showing less than 0.5 fold decrease) in ALS-CSF samples. The 4 proteins (chitotriosidase (CHIT), osteopontin, chitinase-3-like protein 2 and chitinase-3-like protein 1), which were up-regulated in ALS-CSF were validated using ELISA. CHIT activity also showed a tenfold increase in ALS-CSF. Immunostaining revealed that microglial cells exposed to ALS-CSF expressed CHIT, but not the astrocytes. All these results indicate that CHIT may play an important role in pathogenesis of ALS.
Ongoing research activities include the following;
1. We have investigated mitochondrial dysfunction in the sporadic model of ALS. Proteomics of the mitochondrial proteins from the spinal cords of ALS-CSF injected animals revealed up-regulation and down-regulation of several key proteins, thereby identifying the pathways which lead to mitochondrial dysfunction.
2. Proteomics analysis of ALS-CSF revealed up-regulation of Chitotriosidase (CHIT-1) compared to normal CSF. The level of CHIT-1 was further evaluated by ELISA in a larger cohort of patients’ CSF, which showed a 17 fold increase in the CSF of ALS patients. To determine the biological significance of the up-regulated CHIT-1, its effect on NSC-34 motor neuronal cell line was investigated. CHIT-1 did not have a direct effect on the viability of NSC-34 cells as shown by MTT assay. The effect of CHIT-1 on the non-neuronal cells viz. astrocytes and microglia is currently being investigated.
3. Primary astrocytes were exposed to CSF from ALS patients (ALS-CSF) and compared with the normal controls (NC) and Non-ALS (NALS-CSF). The studies revealed a down-regulation of trophic factors VEGF and GDNF in ALS, while inflammatory markers like IL6, and TNF-a were found to be significantly upregulated, suggesting the role of neuroinflammation in the exacerbation of the disease. Cultures rich in microglia were derived from embryonic rat spinal cords. Up-regulation of chitotriosidase (CHIT) was also observed in the microglial cultures exposed to ALS-CSF. Reduced trophic support and enhanced activation of microglia with up-regulated CHIT, an indicator of neuroinflammation hints at crucial role of astroglia and microglia in eliciting inflammatory cascades in the neurodegeneration associated with ALS.
The laboratory currently has 2 ongoing funded projects (including one COE Grant) and 3 non funded projects. More than 25 researchers including PhD scholars, PDF, DM students and faculty from NIMHANS and other institutes are currently using the lab facility. The researchers have received awards for the research carried out in the laboratory and in the year 2013-2014, there were 2 publications from this lab in peer reviewed journals.
Workshops & Symposia conducted by the lab –
a. Cell to system (20 participants), Pre-conference workshop of 59th annual national conference of Association of Physoiologists & Pharmacologists of India, 26th November, 2013.
b. Hands on training of quantification techniques using Leica ‘Q- Win’ Software and Demonstration of Culture Techniques (25 participants) at Parkinson’s disease Education Programme, 5-7 March, 2013
