Title: The role of Synaptic Genes NRXN1 and SHANK3 in Autism: an induced pluripotent stem cell study
Research group: Autism Research Centre, Department of Psychiatry
Supervisor: Professor Simon Baron-Cohen
Advisor: Dr Mark Kotter, Clinical Neurosciences; Dr Deepak Srivastava, King’s College London
- Can you give us a short background into what your PhD was about?
Autism is a complex neurodevelopmental condition with hundreds of genes associated with its pathophysiology. Several autism related genes have been reported to be strongly associated with the structural or functional aspects of neuronal connections or synapses. Accumulation of mutations or deletions in such risk genes correlate with the clinical diagnosis of autism. However, it is poorly understood how the loss of function of these synaptic genes contribute to the altered neuronal network physiology and atypical communication between brain cells.
Synapses are highly specialized structures in the mammalian brain and functional sites of neuronal connections for facilitating information processing across brain regions. Autism is often theorised as a condition caused by aberrant synaptogenesis due to various mutations of synaptic genes. In my PhD, I studied two such autism related synaptic genes, NRXN1 and SHANK3.
The advent of human induced pluripotent stem cell (iPSC) technology has paved new avenues towards establishment of human cellular models to study neuropsychiatric conditions. iPSCs could be non-invasively generated by reprogramming human somatic cells, such as keratinocytes or hair samples, with a combination of defined transcription factors in the laboratory. During my PhD, I synthesised and characterised cortical neurons from iPSCs, derived from individuals with autism carrying deletions in NRXN1, SHANK3 and typically developing controls; to elucidate the morphological and electrophysiological cellular phenotypes associated with autism.
- How would you sum up your main findings?
The research in my PhD thesis aims to first test the efficacy and suitability of an iPSC derived neuronal model for studying autism related synaptic genes. I utilised an inducible transcriptional overexpression system or a “molecular switch” that can be turned on by biochemical intervention to trigger gene expression that results in generation of cortical neurons from iPSCs. My findings show that these induced neurons (iNeurons), derived by NGN2 transcription factor overexpression by a method called “forward programming”, express autism related genes, post synaptic density molecules and synaptic receptors. iNeurons also demonstrate mature electrophysiological activity relevant for studying autism related functional phenotypes, within only four weeks of neuronal induction.
Secondly, my work aims to study the effect of pre-synaptic NRXN1 and post-synaptic SHANK3 deletions on neuronal morphology and synaptic network activity. The neurons derived from both NRXN1 and SHANK3 mutant iPSCs showed aberrant morphological phenotypes such as reduced neurite length and branching points. The electrophysiological study using multi-electrode arrays elucidated that only NRXN1 mutant neurons fire at much higher levels like being in a hyper-excited state or epileptic seizures compared to controls.
This study proposes a novel in vitro model for generating more homogenous and functional neurons compared to the existing methods adopted in the field of autism research. It has been exemplified that the effect deletions in synaptic genes such as NRXN1 and SHANK3 on mature cellular phenotypes can be effectively studied using iNeurons as a model system.
- What made you want to do a PhD?
I was born in a Bengali family to academic parents and while growing up in my hometown Calcutta, surprisingly I was most passionate about music, poetry, painting and theatre. Thanks to my schoolteachers, that I discovered Chemistry as my favourite subject of creativity and design and eventually studied Biochemistry in bachelors and masters. While I was very happy directing stage theatre and forming my amateur band, I got excited about Neuroscience while reading articles on Neuro-aesthetics, a philosophical discourse at the intersection of brain sciences and aesthetic principles, which led me to pursue scientific research projects at the National Brain Research Centre, India.
I was introduced to “autism” while volunteering at an art exhibition of paintings by autistic children in New Delhi which made me curious as a researcher to pursue cognitive and behavioural research with autistic children in a clinical setting. In my PhD research proposal, I formulated a hypothesis based on my training in Biochemistry/Neuroscience and tiny little understanding of autism. Fortunately, I could pursue my dream journey from Calcutta to Cambridge when St John’s college kindly accepted me, and I was nominated from India in Life Sciences category and generously awarded the Commonwealth Scholarship. Reminiscing the roller coaster ride of mixed emotions of past couple of decades, I honestly feel that I have never consciously planned my life or career. However, I have always tried to follow a trajectory led by my passion and heart (or the brain).
- What was your best day during your PhD?
In the second year of my PhD, I was conducting gene editing experiments on iPSC lines from autistic individuals and repeatedly failing for several months. It was extremely frustrating for a while until I managed to optimise and establish an alternative method using virus particles. Finally, after six months of struggle when I could successfully visualize green fluorescent protein labelled autistic neurons under the microscope, it was a moment euphoria!
- What was your worst day during your PhD?
At the very final stage of my PhD, the laboratory was shut down due to the pandemic situation. The samples that I had collected at different timepoints from several iPSC-line derived neurons for the past ten months could not proceed for RNA sequencing. It was my worst day as I realized that I have just lost a big dataset and a couple chapters of my thesis.
- Do you have any words of advice to future PhD students in Psychiatry?
I would strongly recommend any future graduate student to explore learning and networking opportunities beyond academia by participating in societies and activities of one’s choice and benefit from the intellectually stimulating ecosystem that Cambridge offers. The most enlightening discussions I remember during my PhD happened over formal dinners or informal coffee meetings with some brightest minds from very diverse fields. Also, this long journey requires a lot of commitment and perseverance; and one is most likely to face difficult phases either professionally or personally. Therefore, it is important to look after oneself, reach out for help if needed and take time off from long hours at lab, especially when experimental failures happen. It is often much easier to solve problems and succeed with a fresh mind than a sleep-deprived one! Last but not the least, I do believe that the mistakes that we make inadvertently are the best learning experiences in the long run; and a PhD as a degree in Philosophy is quite a life changing experience to treasure.
- What do you hope to do next?
Since completion of my PhD, I have been working at the Clinical Neurosciences department as a postdoctoral research associate. Besides, I am currently preparing myself to embark on a new journey beyond academia to explore opportunities within the industry. I am deeply passionate about bench-to-bedside translational and clinical research and aspire to grow as a scientist in an innovation-led collaborative environment, acquire leadership training and most importantly pursue cutting-edge science that can potentially impact the quality of human life.