Calcium sensor STIM1 regulates gene expression and synaptic connectivity of Purkinje neurons

Sreeja Kumari Dhanya’s interview with Bio Patrika hosting “Vigyan Patrika”, a series of author interviews. Dhanya is a Ph.D. student working under the guidance of Prof. Gaiti Hasan in National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore. She completed her master’s in Medical Biotechnology from Manipal School of Life Sciences (MSLS) at Manipal Academy of Higher Education (MAHE), Manipal. Her doctoral research focuses on understanding the role of STIM1 (Stromal Interaction Molecule), a calcium sensor molecule present in the endoplasmic reticulum (ER) and investigating its role in regulating gene expression, excitability and synaptic connectivity in mouse Purkinje neurons (PNs). Her research has significantly contributed to uncovering novel aspects of intracellular calcium signaling in regulating neuronal functions and physiology in mammalian system. Dhanya, as her long-term goal, envisages exploring signaling mechanisms involved in various neurodegenerative disorders and identifying novel therapeutic insights. Here, Dhanya talks about her work on ‘Purkinje Neurons with Loss of STIM1 Exhibit Age-Dependent Changes in Gene Expression and Synaptic Components’ published in Journal of Neuroscience.

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How would you explain your paper’s key results to the non-scientific community?

Calcium in neurons plays an important role in regulating various cellular processes such as neurotransmission, gene expression, etc. Proper control of calcium flux between the cytosol and intracellular calcium stores (endoplasmic reticulum and mitochondria) is required for maintaining normal neuronal functions. Cytosolic calcium levels depend on both the calcium entering from the extracellular space through stimulation of the receptors and those released from the intracellular stores through endoplasmic reticulum (ER) resident calcium channel, the inositol 1,4,5-trisphosphate receptor 1 (IP3R1). Previous studies have reported mutations in IP3R1 are associated with spinocerebellar ataxias 15 and 16 (SCA 15/16) where Purkinje neurons undergo neurodegeneration leading to impaired coordination of vertebrate movements.

Refilling of calcium into the ER is initiated by an ER resident calcium sensor protein, STIM1 through interaction with pore channel Orai and transient receptor potential channel TRPC3 in Purkinje neurons. Previous studies have reported a correlation between loss of STIM1 and metabotropic Glutamate receptor 1 (mGluR1) controlling synaptic transmission and neuronal calcium homeostasis in Purkinje neurons. However, molecular mechanisms involved in affecting synaptic plasticity with loss of STIM1 is not known. To understand how altered intracellular calcium signaling leads to age-dependent deficits in Purkinje neuron function, as observed in neurodegenerative disorders like SCAs, we investigated cellular and molecular changes in the Purkinje neurons of mice with Purkinje neuron- specific knockout of STIM1. We observed that loss of STIM1 in the Purkinje neurons caused motor learning and coordination deficits in mice suggesting the importance of STIM1 in regulating membrane excitability and calcium transients following mGluR1 activation. Loss of STIM1 would reduce calcium levels in ER thereby attenuating intracellular calcium release upon mGluR1 stimulation and loss of STIM1-induced store-operated calcium entry.

Figure 1: Schematic representation of intracellular calcium signaling in Purkinje neurons. Activation of mGluR1 stimulates IP3 mediated calcium release from ER through IP3R and dearth of calcium in ER is sensed by STIM1. Store operated calcium entry is initiated through interaction of STIM1 with Orai and transient receptor potential channel TRPC3. Calcium entered into the cytosol moves to ER through sarcoendoplasmic reticulum calcium transport ATPase (SERCA) [Image created using Biorender (BioRender.com)].

It has been well known that gene expression changes upon reduced store-operated calcium entry in non-excitable cells. We further investigated the transcriptional profile of mature STIM1 knockout Purkinje neurons. Interestingly, we observed that loss of STIM1 in Purkinje neurons significantly altered the expression of genes belonging to key biological pathways such as calcium signaling, synaptic signaling, endocytic recycling and neurite development amongst others. The differential gene expression correlated with altered dendritic patterns and greater climbing fiber-Purkinje neuron synapses in ageing STIM1PKO mice suggesting long-term changes in synapse formation with loss of STIM1 in Purkinje neurons.

Overall, our data provide a novel role of STIM1 in regulating the gene expression profile and synaptic connectivity of Purkinje neurons. These findings are relevant in the context of uncovering the mechanisms by which dysregulated calcium signals impact molecular and cellular pathways involved in multiple neurodegenerative disorders.

Figure 2. Schematic representation showing a novel role of STIM1 mediated calcium signaling in mouse Purkinje neurons [Image created using Biorender (BioRender.com)].

The findings provide evidence for the novel role of STIM1 dependent calcium homeostasis and signaling in regulating the expression of multiple key components of neurite development and synaptic organization in ageing animals.

What are the possible consequences of these findings for your research area?

Dysregulation of intracellular calcium signaling in Purkinje neurons has been proposed as a possible mechanism in the pathogenesis of many neurodegenerative disorders. More specifically, individuals with mutations of the IP3R are associated with SCA 15/16. However, the precise molecular mechanism underlying neuronal dysfunction and motor coordination deficits is poorly understood. Our study elucidates a potential mechanism by which altered intracellular calcium signaling affects neuronal morphology and synaptic connectivity of Purkinje neurons thereby affecting motor behaviour. The findings provide evidence for the novel role of STIM1 dependent calcium homeostasis and signaling in regulating the expression of multiple key components of neurite development and synaptic organization in ageing animals. The outcome opens a path to discovering new therapeutic interventions alleviating the degenerative changes associated with neurodegenerative disorders.

What was the exciting moment (eureka moment) during your research?

The most exciting moments were when we found that the loss of STIM1 affects multiple gene expression across various biological pathways and the transcriptional alterations are age-dependent, evident in older mice. This suggested a possible molecular mechanism acting downstream of the STIM1 and reflected on the various possible biological networks affected on dysregulated intracellular calcium signaling. To our surprise, we also found that the gene expression changes correlated with altered dendritic morphology and greater climbing fiber-Purkinje neuron synapses further indicating the impairment in error encoding during motor learning tasks.

What do you hope to do next?

Our study identifies the functional significance of STIM1 mediated calcium signaling on Purkinje function and synaptic connectivity. My immediate goal is to investigate whether we could rescue the motor deficits and other neuronal dysfunction observed in the STIM1 knockout mice by altering Septin 7 levels exclusively in the Purkinje neurons. The rationale behind this study was influenced by the novel discovery from our lab where Septin 7 which are GTP-binding proteins was found to function as a ‘molecular brake’ on the activation of Orai channels (pore channel present on plasma membrane) in Drosophila neurons and human neural progenitor cells. Partial genetic depletion of dSeptin7 was found to rescue the flight defects in Drosophila with IP3R mutations or STIM knockdown. This indicates store independent opening of dOrai channels and enhancement in the intracellular calcium entry on lowering dSEPT7 levels, thereby compensating for the reduced function of IP3R or STIM in Drosophila neurons. We are interested to further investigate if this negative regulation of Orai by Septin7 is conserved among mammals and to explore the molecular and behaviour modulations in Purkinje neuron-specific STIM1-SEPT7 knockout mouse model.

Where do you seek scientific inspiration?

My scientific inspiration comes from my school days where I was curious to seek answers to scientific problems. I was always fascinated by the discoveries and the innovative way by which each question was addressed. I was curious to understand the complex system of life and rediscover the intricate machinery that works in a programmed manner inside a living organism. The amazing training and guidance that I received at MSLS, Manipal during my Master’s motivated me to explore more into life sciences and gave me immense confidence to pursue research as my career. I am very fortunate to take up my Ph.D. journey under the mentorship of Prof. Gaiti Hasan as her tremendous support and guidance always instigate a drive to develop my scientific thinking and explore more on discovering novel findings.

How do you intend to help Indian science improve?

I believe that young minds at their early stages of education should be motivated to develop the desire for science and develop an interest in scientific reasoning. According to my perspective, students should be made aware of the various opportunities available in India and provide proper guidance to pursue research careers. There should be more efforts to help the young researchers establish their own labs and provide adequate support through various funding agencies. I would certainly go for my post-doctoral research in the field of neuroscience to expand my knowledge, skills and expertise so that I can contribute to the identification of potential targets and collaborate with pharmaceutical domains to develop therapeutic drugs against neurodegeneration.

Reference

Sreeja Kumari Dhanya and Gaiti Hasan. Purkinje Neurons with Loss of STIM1 Exhibit Age-Dependent Changes in Gene Expression and Synaptic Components. Journal of Neuroscience 28 April 2021, 41 (17) 3777-3798; DOI: https://doi.org/10.1523/JNEUROSCI.2401-20.2021

Email: dhanyask@ncbs.res.in

Prof. Gaiti Hasan Lab: https://www.ncbs.res.in/faculty/gaiti

Edited by: Pragya Gupta

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