Principal Investigator:
Dr. Guy Rouleau, OC, OQ, MD, PhD, FRCPC, FRSC
Director, The Neuro
Chair, Department of Neurology and Neurosurgery, McGill University
PROJECT SUMMARY
Specific aims: Essential tremor (ET) is one of the most common neurological disorders affecting nearly 1% of the worldwide population. It causes uncontrollable shaking of multiple body parts including the hands, arms, head and legs. The disorder is quite debilitating, having an effect on the capacity of patients to perform daily activities such as eating and dressing.
Not much is known about why and how ET arises in certain individuals. We know that the main brain region responsible for ET is the cerebellum, the brain region that is required to control the coordination of movements. However, we have limited information on the role that the different cells in the cerebellum play in the disease. We previously used RNA sequencing to investigate gene expression changes in the cerebellum of ET patients. This method is unable to study gene expression changes in specific cell populations and lacks the required resolution to understand the role of each cell type in the disease.
We also know that certain drugs are better than others at treating essential tremor. Propranolol is the most prescribed drug in treating ET, but the mechanism behind its efficiency is unknown. We previously screened the effects of propranolol on gene expression in human neural stem cells as well as cerebellar cancer cells. Although we found that it affected the expression of genes related to ET, these cells do not accurately represent cells found in the cerebellum of ET patients.
We would like to better resolve these issues by using more relevant techniques and cellular models to understand 1) the role of different cerebellar cells in ET, 2) the effects that genetic risk factors for ET have on the expression of certain genes in the cerebellum and 3) the effects of propranolol on cerebellar cells derived from ET patients.
Aim 1: Assess the transcriptomic effects of propranolol on ET patient-derived cerebellar cells
Human induced pluripotent stem cells (hiPSCs) from ET patients that have positive responses to propranolol will be differentiated into cerebellar cell cultures containing mature PCs and other cerebellar neurons and treated with propranolol. Single-cell RNA sequencing will be used to compare the effects of propranolol on gene expression in the different cell populations of the cerebellum. Changes to cell morphology and electrophysiological properties will also be assessed. We aim to identify genes that explain positive responses to propranolol.
Aim 2: Characterize the cellular landscape of the essential tremor cerebellum
We will use single-cell RNA sequencing to study gene expression changes in all cell populations of the cerebellum. We aim to sequence close to 1 million cells from cerebelli of 16 ET patients and 80 healthy patients. We hope to identify gene expression changes in relevant cell types in ET such as PCs. We will also investigate the role of non-neuronal cells, such as oligodendrocytes and astrocytes, in the pathophysiology of ET. We hope to learn about the pathological features of different cell types in the cerebellum of ET patients.
Aim 3: Evaluating the effects of genetic risk factors on gene expression in cerebellar cells
Small changes to the genetic code can have a large effect on the expression of genes. These effects are mostly cell-type specific. Recent advances in single-cell RNA sequencing and population genetics have allowed us to link genetic events to expression events (called expression quantitative trait loci (eQTLs)) and infer the effects of genetic risk factors on gene expression in specific cells. We plan to leverage our large-scale single-cell cerebellar data set coupled with genotyping data to first map these single-cell eQTLs, and second assess how genetic risk factors for ET (identified in our last genome-wide association study) can affect the expression of certain genes in different cell types of the cerebellum. We hope to characterize the progression from genetic risk factor to functional effects on the cerebellum and thus better understand the pathophysiological mechanisms driving risk for ET.
Progress Report
January 2023
Essential tremor (ET) is one of the most common neurological disorders affecting nearly 1% of the worldwide population. It causes uncontrollable shaking of multiple body parts including the hands, arms, head and legs. The disorder is quite debilitating, having an effect on the capacity of patients to perform daily activities such as eating and dressing.
Based on previous studies we know that a brain region called the cerebellum is affected in the disease. This brain region controls the coordination of movements. The role of different cerebellar cells and neurons in ET is, however, unknown. Our aims focus on understanding how different cells in the cerebellum contribute to the disease.
[ Aim 1: hiPSC]
The closest way of studying how neurons behave in the human brain is to generate them from human stem cells. Human induced-pluripotent stem cells (hiPSCs) can be derived from blood cells. These hiPSCs can then be differentiated into different type of mature cells such as cerebellar neurons. Our goal is to study the effects of propranolol, the most common treatment for ET, on neurons from responsive and non-responsive ET patients. Starting from 4 hiPSCs lines (2 responsive and 2 non-responsive) we have generated hindbrain neural progenitor cells. These cells, under the right conditions, will be able to differentiate into different cerebellar neurons including Purkinje cells. These cells are thought to be responsible for tremor generation in ET. 4 more hiPSCs lines from 2 responsive and 2 non-responsive patients are currently being reprogrammed.
[Aim 2: scRNAseq tissues]
Single-cell RNA sequencing has enabled researchers to study the expression of RNA molecules in thousands of different cell types. We decided to use such technology to study how different cells of the cerebellum are being affected in ET. We performed single-cell RNA sequencing of 16 ET and 16 control samples from the cerebellar cortex. We found that the most dysregulated cell types in the ET cerebellum were Bergmann glia, oligodendrocytes, and oligodendrocyte progenitor cells. These cells are non-neuronal cell types and act as support cells for neurons. Dysfunctional processes in these cells might in turn affect the normal functioning of neurons such as Purkinje cells. We are in the process of sequencing close to 1M nuclei from ET and control individuals (16 cases and 80 controls) in order to elucidate how dysfunctional glial cells might alter the normal functioning of cerebellar neurons.
[Aim 3: eQTL]
Small changes to the genetic code (DNA) can have a large effect on the expression of genes. These effects are mostly cell-type specific. Recent advances in single-cell RNA sequencing and population genetics have allowed us to link genetic events to expression events (called expression quantitative trait loci (eQTLs)) and infer the effects of genetic risk factors on gene expression in specific cells. We will use our dataset generated in Aim 2 coupled with genotyping from these 96 individuals to build a single-cell eQTL atlas of the cerebellum. DNA has already been extracted from all frozen cerebellar tissues. Genotyping will be performed in the coming months. Our goal is to understand how genetic variation affect cell-type specific expression in the cerebellum. This will enable us to study the effects of genetic variation in ET on the expression of RNA molecules in cerebellar neurons.