Periodic Reporting for period 5 - ALS-Networks (Defining functional networks of genetic causes for ALS and related neurodegenerative disorders)
Periodo di rendicontazione: 2022-10-01 al 2023-05-31
Prevalence of neurological diseases is on the rise, including Amyotrophic Lateral Sclerosis (ALS). A major challenge to better treat these disorders is to be able to develop appropriate models and to perform unbiased testing of compounds prior to the initiation of large-scale and very expensive clinical trials. In this project we proposed to develop animal models for the major ALS genes. Importantly, using these novel models, shared molecular cascades that can lead to motor deficits and neuronal abnormalities will be identified and will target these molecular cascades using pharmacological compounds.
During the initial period of the project, I have focused on the experiments described in Aim 1 and 2 and have finalized 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 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. 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 TBK1 gene 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.
Aim 3: As mentioned above, apart rapamycin we have identified compounds that lower the neuromuscular deficits. These include the mitophagy activator, urolithin as well as the acetyl-carnitine that targets the metabolic deficits in ALS. In collaboration with clinicians, we have initiated a small clinical trial for salbutamol that targets the ER stress response.
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 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. 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 TBK1 gene 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.
Aim 3: As mentioned above, apart rapamycin we have identified compounds that lower the neuromuscular deficits. These include the mitophagy activator, urolithin as well as the acetyl-carnitine that targets the metabolic deficits in ALS. In collaboration with clinicians, we have initiated a small clinical trial for salbutamol that targets the ER stress response.
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 through the usage of CRISP/Cas9 to derive deletion mutants and a range of transgenic lines to mirror 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 sorting. 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 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.
These markers allow us to focus on 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.
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.
As described in the work performed during the initial period, we have developed a range of zebrafish models for ALS genetic causes through the usage of CRISP/Cas9 to derive deletion mutants and a range of transgenic lines to mirror 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 sorting. 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 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.
These markers allow us to focus on 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.
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.