Periodic Reporting for period 4 - ALS-Networks (Defining functional networks of genetic causes for ALS and related neurodegenerative disorders)
Berichtszeitraum: 2021-10-01 bis 2022-09-30
There has been a steady rise in the prevalence and incidence of neurological diseases over the past decades. This is mainly due to the progressive aging of the general population.
Neurodegenerative diseases, including Alzheimer’s and related dementia, Parkinson and related movement disorders and Amyotrophic Lateral Sclerosis (ALS) play an extremely mportant impact for the society at the emotional, financial and social level. The majority of these neurodegenerative diseases are currently untreatable; thus representing a major economic burden for our society and in particular our health system.
Major genetic causes representing overall 5-10% of patients suffering from these diseases have been identified. Also, several specific genetic risk factors for each of these diseases have been mapped out. This genetic knowledge has also allowed to advance the understanding of pathophysiological features and clinical outcomes from these diseases. This genetic knowledge has allowed to define unexpected clinical spectrum connencting neurodegenerative diseases. A major recent advance has been the recognition of a spectrum connecting ALS to another major neurological disease, Frontotemporal Dementia (FTD). These disorders share major genetic and pathological markers thus providing evidence for important clinical overlap. Identification of this spectrum allows us to hypothesize that even though the initial site of onset and clinical progression could vary, several of these disorders could share molecular cascades that lead to neuronal demise.
The recognition of this genetic and pathological spectrum will certainly have consequences in treatment options for these neurodegenerative disorders. Unfortunately, a wide array of clnical trials have failed in the past years. Two major bottlenecks are evident to advance therapeutic insight and to identify avenues of treating ALS and FTD patients. The first major challenge is to be able to develop appropriate models to characterize pathophysiological mechanisms that feature certain clinical features and pathological markers of these neurodegenerative diseases. The second problematic that is critical to disease treatment is the ability to perform unbiased testing of pharmacological compounds in these models prior to the initiation of large-scale and very expensive clinical trials.
To achieve these major objectives and to overcome these bottlenecks, we proposed in the ERC project to develop animal models for the the major genetic causes of the ALS-FTD spectrum. These zebrafish models described in Aim 1 of this project will define how gain and loss of function of the major genetic factors can cause alterations of motor landmarks and appearance of neuropathological markers. Importantly, using these novel models, as part of the 2nd objective, I sought to understand and to delineate the shared molecular cascades that can lead to motor deficits and neuronal abnormalities. Finally, as part of the 3rd objective, I will target these molecular cascades using pharmacological compounds to test whether modifying the molecular cascades leading to neurodegeneration is directly associated with reduced or delayed motor deficits in vivo. Therefore, this project will increase our knowledge of major genetic factors in ALS and will have direct impact to the identification of novel therapeutic avenues for the clinical spectrum of ALS-FTD and related neurological diseases.
Neurodegenerative diseases, including Alzheimer’s and related dementia, Parkinson and related movement disorders and Amyotrophic Lateral Sclerosis (ALS) play an extremely mportant impact for the society at the emotional, financial and social level. The majority of these neurodegenerative diseases are currently untreatable; thus representing a major economic burden for our society and in particular our health system.
Major genetic causes representing overall 5-10% of patients suffering from these diseases have been identified. Also, several specific genetic risk factors for each of these diseases have been mapped out. This genetic knowledge has also allowed to advance the understanding of pathophysiological features and clinical outcomes from these diseases. This genetic knowledge has allowed to define unexpected clinical spectrum connencting neurodegenerative diseases. A major recent advance has been the recognition of a spectrum connecting ALS to another major neurological disease, Frontotemporal Dementia (FTD). These disorders share major genetic and pathological markers thus providing evidence for important clinical overlap. Identification of this spectrum allows us to hypothesize that even though the initial site of onset and clinical progression could vary, several of these disorders could share molecular cascades that lead to neuronal demise.
The recognition of this genetic and pathological spectrum will certainly have consequences in treatment options for these neurodegenerative disorders. Unfortunately, a wide array of clnical trials have failed in the past years. Two major bottlenecks are evident to advance therapeutic insight and to identify avenues of treating ALS and FTD patients. The first major challenge is to be able to develop appropriate models to characterize pathophysiological mechanisms that feature certain clinical features and pathological markers of these neurodegenerative diseases. The second problematic that is critical to disease treatment is the ability to perform unbiased testing of pharmacological compounds in these models prior to the initiation of large-scale and very expensive clinical trials.
To achieve these major objectives and to overcome these bottlenecks, we proposed in the ERC project to develop animal models for the the major genetic causes of the ALS-FTD spectrum. These zebrafish models described in Aim 1 of this project will define how gain and loss of function of the major genetic factors can cause alterations of motor landmarks and appearance of neuropathological markers. Importantly, using these novel models, as part of the 2nd objective, I sought to understand and to delineate the shared molecular cascades that can lead to motor deficits and neuronal abnormalities. Finally, as part of the 3rd objective, I will target these molecular cascades using pharmacological compounds to test whether modifying the molecular cascades leading to neurodegeneration is directly associated with reduced or delayed motor deficits in vivo. Therefore, this project will increase our knowledge of major genetic factors in ALS and will have direct impact to the identification of novel therapeutic avenues for the clinical spectrum of ALS-FTD and related neurological diseases.
During the initial period of the project, I have focused on the experiments described in Aim 1 and 2 and am in the process of initiating the experiments of Aim 3. Below I describe in detail work provided during this initial period in order to achieve the following deliverables.
Aim 1 : The initial results from my team demonstrate that there is a synergy between gain and loss of function of C9orf72. We have lowered the expression of C9orf72 through the usage of antisense oligonucleotides and co-expressed one of the most prevalent pathological dipeptide repeats (DPRs), GP100. Lowered expression of C9orf72 is accompanied by accumulation and aggregation of GP100 with autophagy activation by rapamycin capable of reducing DPR aggregation and motor deficits. Furthermore, we observed selective motor neuron degeneration at the level of the spinal cord upon co-expression of GP100 alongside C9orf72 reduced levels. We demonstrate by genetic experiments and proteomics results that this motor neuron death is due to mitochondrial-dependent activation of apoptosis specifically in motor neurons. This is the first vertebrate model to replicate pathological features observed in ALS patients carrying the C9orf72 repeats where both lowered C9orf72 expression and dipeptide repeat pathological features are observed. We have also developed a number of deletion mutants for C9orf72 and have identified a zebrafish homozygous mutant with adult-onset degeneration and reduced viability. Overexpression of DPRs in this mutant line leads to exacerbated toxicity and motor deficits confirming the synergy of gain and loss of function for the C9orf72 mutation. We are currently generating lines where the expanded hexanucleotide or dipeptide repeats are being inserted in the C9orf72 locus. Alternatively, we will cross the deletion C9orf72 mutants with the transgenic lines overexpressing DPRs or hexanucleotide repeats.
We have developed a FUS deletion mutant line where we observe motor features associated with lowered evoked and spontaneous swimming and reduced viability at the larval stages of FUS deletion mutants where the expression of FUS has been inactivated as measured by Western blotting and proteomic analysis. We have also defined deficits at the level of the neuromuscular junction with these deficits restored by overexpression of WT human FUS. Importantly, in concordance with results observed in mouse model, we observed muscle defects in the FUS knockout model associated with alterations at the mitochondrial transcription with lowered rates of mitochondrial respiration measured in zebrafish homozygous mutants. Indeed a proteomics analysis reveals metabolic deficits that we are currently validating in this model as well as in pathological samples and biopsies from patients carrying FUS mutations. Similarly, we have developed zebrafish deletion mutants for the two TDP-43 orthologues in zebrafish. Unlike C9orf72 and FUS these deletion mutants do not display any major motor deficits. We are currently crossing these deletion mutants with an ALS-related mutant TDP-43 transgenic line developed by my team. Moreover, for the TDP-43 and FUS, we are currently developing kockin models that target to delete the Nuclear Localization Signal of the zebrafish FUS. Finally, we are also in the process of developing deletion line for the SQSTM1 gene, with particular interest in the functional domain (LIR and UBA domains) and analyzing the phenotypic features of these mutant zebrafish.
Aim 2 : To define common pathogenic mechanisms we have assessed the autophagy response in the models described in Aim 1. Zebrafish with combined gain and loss of function of C9orf72 display altered rated of autophagic flux as measured by the LC3 GFP/RFP probe. Similarly, there is an altered upregulation of the autophagy regulator, SQSTM1/p62 with these deficits rescued upon treatment with an activator of autophagy, the mTOR inhibitor, rapamycin. Similarly, upon TDP-43 depletion we observed altered rate of autophagy with the autophagy receptor, SQSTM1 and the kinase that is important for mTOR activation, TBK1 capable of rescuing deficits due to TDP-43 loss of function.
We have developed a transgenic zebrafish line with the GFP inserted in the G3BP frame. This line is being used to purify for the first time stress granules and to define their dynamics using the in vivo models described in Aim 1. We have assessed also physiological (heat-shock) and chemical (arsenite treatment) protocols to induce formation of stress granules in vivo.
Finally, we have developed protocols to purify motor neurons by fluorescent cell sorting and are performing transcriptomic analysis in all our mutant lines. The altered transcripts will be validated in motor neurons using ViewRNA ISH cell assay to identify shared transcriptional signature for the major genetic causes of ALS.
Aim 1 : The initial results from my team demonstrate that there is a synergy between gain and loss of function of C9orf72. We have lowered the expression of C9orf72 through the usage of antisense oligonucleotides and co-expressed one of the most prevalent pathological dipeptide repeats (DPRs), GP100. Lowered expression of C9orf72 is accompanied by accumulation and aggregation of GP100 with autophagy activation by rapamycin capable of reducing DPR aggregation and motor deficits. Furthermore, we observed selective motor neuron degeneration at the level of the spinal cord upon co-expression of GP100 alongside C9orf72 reduced levels. We demonstrate by genetic experiments and proteomics results that this motor neuron death is due to mitochondrial-dependent activation of apoptosis specifically in motor neurons. This is the first vertebrate model to replicate pathological features observed in ALS patients carrying the C9orf72 repeats where both lowered C9orf72 expression and dipeptide repeat pathological features are observed. We have also developed a number of deletion mutants for C9orf72 and have identified a zebrafish homozygous mutant with adult-onset degeneration and reduced viability. Overexpression of DPRs in this mutant line leads to exacerbated toxicity and motor deficits confirming the synergy of gain and loss of function for the C9orf72 mutation. We are currently generating lines where the expanded hexanucleotide or dipeptide repeats are being inserted in the C9orf72 locus. Alternatively, we will cross the deletion C9orf72 mutants with the transgenic lines overexpressing DPRs or hexanucleotide repeats.
We have developed a FUS deletion mutant line where we observe motor features associated with lowered evoked and spontaneous swimming and reduced viability at the larval stages of FUS deletion mutants where the expression of FUS has been inactivated as measured by Western blotting and proteomic analysis. We have also defined deficits at the level of the neuromuscular junction with these deficits restored by overexpression of WT human FUS. Importantly, in concordance with results observed in mouse model, we observed muscle defects in the FUS knockout model associated with alterations at the mitochondrial transcription with lowered rates of mitochondrial respiration measured in zebrafish homozygous mutants. Indeed a proteomics analysis reveals metabolic deficits that we are currently validating in this model as well as in pathological samples and biopsies from patients carrying FUS mutations. Similarly, we have developed zebrafish deletion mutants for the two TDP-43 orthologues in zebrafish. Unlike C9orf72 and FUS these deletion mutants do not display any major motor deficits. We are currently crossing these deletion mutants with an ALS-related mutant TDP-43 transgenic line developed by my team. Moreover, for the TDP-43 and FUS, we are currently developing kockin models that target to delete the Nuclear Localization Signal of the zebrafish FUS. Finally, we are also in the process of developing deletion line for the SQSTM1 gene, with particular interest in the functional domain (LIR and UBA domains) and analyzing the phenotypic features of these mutant zebrafish.
Aim 2 : To define common pathogenic mechanisms we have assessed the autophagy response in the models described in Aim 1. Zebrafish with combined gain and loss of function of C9orf72 display altered rated of autophagic flux as measured by the LC3 GFP/RFP probe. Similarly, there is an altered upregulation of the autophagy regulator, SQSTM1/p62 with these deficits rescued upon treatment with an activator of autophagy, the mTOR inhibitor, rapamycin. Similarly, upon TDP-43 depletion we observed altered rate of autophagy with the autophagy receptor, SQSTM1 and the kinase that is important for mTOR activation, TBK1 capable of rescuing deficits due to TDP-43 loss of function.
We have developed a transgenic zebrafish line with the GFP inserted in the G3BP frame. This line is being used to purify for the first time stress granules and to define their dynamics using the in vivo models described in Aim 1. We have assessed also physiological (heat-shock) and chemical (arsenite treatment) protocols to induce formation of stress granules in vivo.
Finally, we have developed protocols to purify motor neurons by fluorescent cell sorting and are performing transcriptomic analysis in all our mutant lines. The altered transcripts will be validated in motor neurons using ViewRNA ISH cell assay to identify shared transcriptional signature for the major genetic causes of ALS.
This project seeks to better understand the functional and genetic network unravelled by identification of major genetic and pathological markers for the ALS-FTD spectrum. Importantly, the aim of this project was to connect these major genetic causes but to also be able to identify common and shared cellular disease markers and to validate these in other animal models of disease and in pathological tissue from ALS patients. Finally, I wanted to define whether modulating these crucial pathogenic cascades had important consequences and were able to reverse phenotypic features in the animal models developed during the course of the ERC project. Therefore, the goal of this project was to extend the understanding of the ALS-FTD clinical spectrum and to propose therapies for patients affected by these major neurodegenerative disorders.
As described in the work performed during the initial period, we have developed a range of zebrafish models for ALS genetic causes. During this initial period, we have analyzed most of these lines. For this we have optimized the usage of CRISP/Cas9 to derive deletion mutants and a range of transgenic lines with inducible or constitutive expression to achieve appropriate loss of function and gain of function features similar to pathological hallmarks that have been described in patients.
Moreover, we have optimized methods to be able to rapidly and efficiently purify specific cellular populations of neurons using fluorescent cell soring. Using this method, we can obtain pure neuronal populations (including motor neurons) that can be analyzed for precise immunolocalization of a range of pathogenic markers of disease. Furthermore, we have also performed and optimized protocols to perform transcriptomics, proteomics and metabolomics analysis in these neuronal populations. These analyses will allow us to precisely understand the cellular processes that are shared amongst the different genetic causes of ALS. Moreover, we aim to define the common pathways that are initiated upon motor neuron degeneration and lead to motor deficits in vertebrate models of disease.
Significantly, we aim to validate these markers and we are already establishing whether certain key deregulated pathways (mitophagy and autophagy) in pathological tissue obtained from sporadic and familial ALS patients as well as complementary disease models. To propose novel and innovative therapeutic avenues, we are in the process to develop a platform to screen pharmacological compounds. This platform will enable to perform long-duration pharmacological treatments in zebrafish models and optimize the analysis of these results as described in the 3rd aim of this project.
Therefore, this project provides a blue-print of translational research going from the understanding of the genetic mutation in a model organism to pharmacological treatments aimed to halt the pathogenic processes associated leading to neurodegeneration. We are confident that this approach will allow to a better understanding of the genetic interactions amongst major genetic factors in ALS, identification of novel pathogenic processes and therapeutic avenues that can be fast-tracked to clinical trials for ALS and related neurodegenerative disorders.
As described in the work performed during the initial period, we have developed a range of zebrafish models for ALS genetic causes. During this initial period, we have analyzed most of these lines. For this we have optimized the usage of CRISP/Cas9 to derive deletion mutants and a range of transgenic lines with inducible or constitutive expression to achieve appropriate loss of function and gain of function features similar to pathological hallmarks that have been described in patients.
Moreover, we have optimized methods to be able to rapidly and efficiently purify specific cellular populations of neurons using fluorescent cell soring. Using this method, we can obtain pure neuronal populations (including motor neurons) that can be analyzed for precise immunolocalization of a range of pathogenic markers of disease. Furthermore, we have also performed and optimized protocols to perform transcriptomics, proteomics and metabolomics analysis in these neuronal populations. These analyses will allow us to precisely understand the cellular processes that are shared amongst the different genetic causes of ALS. Moreover, we aim to define the common pathways that are initiated upon motor neuron degeneration and lead to motor deficits in vertebrate models of disease.
Significantly, we aim to validate these markers and we are already establishing whether certain key deregulated pathways (mitophagy and autophagy) in pathological tissue obtained from sporadic and familial ALS patients as well as complementary disease models. To propose novel and innovative therapeutic avenues, we are in the process to develop a platform to screen pharmacological compounds. This platform will enable to perform long-duration pharmacological treatments in zebrafish models and optimize the analysis of these results as described in the 3rd aim of this project.
Therefore, this project provides a blue-print of translational research going from the understanding of the genetic mutation in a model organism to pharmacological treatments aimed to halt the pathogenic processes associated leading to neurodegeneration. We are confident that this approach will allow to a better understanding of the genetic interactions amongst major genetic factors in ALS, identification of novel pathogenic processes and therapeutic avenues that can be fast-tracked to clinical trials for ALS and related neurodegenerative disorders.