Characterizing the Transcriptomic Landscapes of Essential Tremor

IETF Funded Research

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.

 

January 2024

Decoding Essential Tremor: A single-cell transcriptomic journey to new answers

Essential Tremor (ET) touches the lives of almost 1% of people globally, bringing with it involuntary shaking in various parts of the body – hands, arms, head, and legs. These uncontrollable tremors can significantly impact daily activities. In our quest to unravel the sources behind the onset of ET, our team turned its attention to a crucial brain area known as the cerebellum. This region is like the conductor of an orchestra, in the sense that it orchestrates the coordination of movements throughout the body. However, despite our existing knowledge of the cerebellum’s involvement in ET, the specific roles played by the different cells and neurons present in the cerebellum remain a mystery. This is where the journey of our IETF proposal began, delving into the intricate world of cerebellar cells to understand their unique contributions to the challenges posed by ET.

To uncover the hidden dynamics within the cerebellum and how these influence ET, we are using different cutting-edge approaches: in vivo cerebral organoid modelling [aim 1], global analysis of gene expression at the single-cell level [aim 2], and genomic profiling [aim 3].

This will lead to a better understanding of the distinctive roles of various cells, concurrently this will shed light on the intricate interaction between cerebellar cells and how disruptions in this delicate choreography lead to the manifestations of ET.

¾Progress regarding the aims of our initial proposal has been providing novel and relevant information

[Aim 1] Mini-brains (cerebral organoids) for better insights.

In our recent progress, we are excited to share that we have been growing tiny brain-like structures called cerebellar organoids, using cells from people with ET. These 3D mini-brains will give us a closer look at what happens in the brain compared to traditional methods. The maturation of these mini-brains requires lengthy periods of time (120 days) for various cell types to differentiate, and our observations have already revealed that these mini-brains exhibit markers specific to cells that are relevant to ET (e.g. Purkinje cells, glia, and astrocytes).

Given that we want this model to offer insights into how a treatment commonly offered to ET patients works its benefits, we have used it to probe the impact of a commonly used medication. Therefore, we exposed mini-brains to propranolol. Our goal is to see how different cells respond over short (6 hours) and longer (7 days) periods. RNA (material carrying information from the genome to be expressed as regulatory molecules and proteins) was prepared for single-cell RNA sequencing (scRNA-seq), which will reveal the transcriptomic profile of each cell type found in the mini-brain. scRNA-seq reveals positive/negative alterations in the expression profile of the different cell types present in the mini-brain. Later this data will also complement the protein profiling (LC-MS/MS) that will be generated from lysates prepared from the mini-brains at the same time. This dual approach will allow us to cross-verify our findings, giving us a comprehensive picture of how genes and proteins are influenced.

Looking ahead, we are on the verge of having scRNA-seq data of 200,000 cells from the mini-brains of 8 different ET patients (4 propanolol responsive and 4 unresponsive) exposed to propranolol. This will identify specific genes that behave differently in response to treatment, providing crucial insights into the benefits (and lack thereof) of propranolol personalized to the genetic makeup of ET patients. Moreover, this could also identify cellular avenues that other treatments (e.g., FDA approved for other conditions) might target.

[Aim 2 & 3] Unlocking the genetic code of ET

Last year, our scRNA-seq investigation of the cerebellar tissue of 109 unaffected human donors and 16 ET patients identified a total of 14 cerebellar cell types, including rare neuronal populations (e.g. Purkinje layer interneurons, unipolar brush cells, and Golgi cells). In parallel, we generated whole-genome genotyping data from the same individuals to conduct an eQTL study. eQTL stands for expression Quantitative Trait Loci. In simpler terms, eQTL analysis helps to connect the dots between our genetic makeup and how our genes are used or expressed, providing insights into the genetic factors that influence traits or conditions. Our eQTL analysis zoomed in on thousands of genes that are regulated differently across various cell types of the cerebellum. Amongst those cell-type-specific genes, we found that the BACE2 gene was significantly dysregulated in the cerebellar oligodendrocytes of ET patients. Oligodendrocytes are a type of brain cell crucial for myelin production in cerebellar white matter.

The eQTL significance of BACE2 is an observation in line with our 2022 JAMA Neurology publication. Therefore, we are now delving into how specific genetic variations might contribute to ET, focusing on oligodendrocytes.

IMPACT: In essence, this journey is a collaborative effort – bringing together the expertise of scientists, the unique experiences of individuals with ET, and the collective hope for a future where the mysteries of this neurological disorder are unraveled and meaningful solutions emerge. Our project is on its way to characterizing the cellular landscape of ET cerebellar cortex and providing a better understanding of the causal relationship between genetic risk factors and affected cell types. In the same process, we are also constructing what may be the largest single-cell mapping of the human cerebellum. Finally, we believe that our work will contribute to an emerging paradigm shift in ET research, focusing on the role of oligodendrocytes in disease pathophysiology.