Role of TNFa and IGF-1 in neuronal apoptosis

Project Associates/Assistants
Ajay Yadav
Anjana Kalita

Collaborators
E Hussain, Jamia Millia Islamia, New Delhi

The project is aimed at understanding the signaling pathways, mechanisms of actions and cross talk between TNFa and IGF-1. The objectives of the study are to (i) study the effect of TNF in the system under study and identify the signal transduction pathways associated with action of TNF, (ii) establish the effect of IGF-1 in the system under study and identify the signal transduction pathways associated with action of IGF-1, and (iii) identify potential cross talk points between the various signaling cascades initiated by TNFa and IGF-1 leading to inhibition of neurodegeneration by IGF-1.

In order to further our knowledge of the role of cytokines and growth factor signaling during neuro-degenerative processes we looked at a pathway which has recently been implicated to play an important role. The fundamental components of this molecular circuitry consist of the receptor associated Janus tyrosine kinase (JAK) and latent cytoplasmic transcription factors termed ‘signal transducers and activators of transcription (STAT)’. Activated dimerized STAT translocates to the nucleus and increase transcription of number of genes including those of suppressors of cytokine signaling (SOCS). SOCS in turns suppresses this JAK-STAT pathway. Current evidence suggests that SOCS may be important regulators of cytokine actions in the CNS in diverse physiologic and pathophysiologic states. We also have included another pro-inflammatory cytokine IL-6 in our study, which has been demonstrated to be upregulated during neuro-degenerative processes. Our studies with human cell lines demonstrate that IL-6 induces apoptosis in a dose dependant manner.

In order to understand the role of SOCS in cytokine signaling in the brain, we have cloned human SOCS1, SOCS2, SOCS3, SOCS4, SOCS5 and SOCS7 in both cloning and expressions vectors by RTPCR followed by direct cloning of the PCR products into cloning vector pGEMT followed by sub-cloning of some of them into expression vectors like EGFP and pCDNA. The constructs were then sequenced completely. Efforts are now on to generate stable transfectants expressing these constructs. We have observed that the SOCS1 cellular localization is different from other SOCS genes. Efforts are on to understand the effect of the cytokines on these SOCS transfected cells. Preliminary experiments using RTPCR, have revealed that SOCS3 gene gets upregulated upon IGF-1 stimulation but not by TNFa or IL-6. We also have looked at upstream level, where we observe that IGF-1 activates STAT3 which dissociates from the JAK to dimerize. SOCS2 gene expression on the other hand is not altered upon stimulation with any of the above cytokines.

Primary neuronal cell culture

In the reporting year we have also standardized the procedure for primary neuronal cell culture, which involves development of timed pregnancies in rats, dissection of the cortex from embryonic day 18 to postnatal day 1 rats, subsequent dissociation of the disuse into individual cells, culturing of these cells in appropriate media conditions and finally making them differentiate into the neuronal phenotype. Presently we can keep the cultures alive for 15-20 days which is sufficient for all our experiments.

Subsequently we have then gone on to establish the effect of TNFa, IL-6 and IGF-1 on these primary cortical cells, where we observed that increasing doses of TNF is inhibitory to neuronal cell survival. IL-6 on the other hand inhibits survival to a lesser extent than TNFa. IGF-1 in both instances is not only able to inhibit the pro-apoptotic abilities of both TNFa and IL-6, but also leads to better differentiation and neuritic networking thus proving to a good survival factor for the dying neurons. We have cloned the rat SOCS1, SOCS2, SOCS3 and SOCS7 by RTPCR followed by direct cloning into pGEMT and EGFP expression vectors. Using these primary neuronal cultures we have observed, that both SOCS1 and SOCS2 gene expression is upregulated by TNFa but inhibited by IGF-1. SOCS7 on the other hand is inhibited by both TNF and IGF-1. IL-6 does not appear to have any effect on SOCS gene expression. This is an interesting observation because so far SOCS gene expression has been shown to be altered by IL-6, but not by TNFa in the brain.

Publications

Original peer-reviewed articles

1.     Kenchappa P, Yadav A, Singh G, Nandana S and Banerjee K (2004) Rescue of TNFa inhibited neuronal cells by IGF-1 involves akt and c-jun N-terminal kinases. J Neurosci Res (in press).