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Defining functional networks of genetic causes for ALS and related neurodegenerative disorders

Periodic Reporting for period 1 - ALS-Networks (Defining functional networks of genetic causes for ALS and related neurodegenerative disorders)

Reporting period: 2017-04-01 to 2018-09-30

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.

Due to technological advances in our ability to perform genetic sequencing, over the last two decades, we understand much better the genetic component of these diseases. 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. More importantly, therapeutic approaches that are efficient for one of these major disorders, such as ALS could be successful for FTD and related dementia.

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 that will allow us advance our understanding on the ALS-FTD spectrum, we proposed in the ERC project to develop animal models for the the major genetic causes of these disorders.
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 to advance and to complete these objectives of the project. Importantly, I am in the process of initiating the experiments described in Aim 3 by preparing the tools and expertise to undertake this objective.
Below we 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 caspase 3 and 9 that result in apoptotic cell death. Indeed, blocking the mitochondrial permeability pore using DXXX will reduce the motor neuron degeneration and lower the motor deficits in this novel zebrafish model combining gain and loss of function features of C9orf72. 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.

During this period, we have also developed a number of deletion mutants for C9orf72 and have identified a zebrafish homozygous mutant with a frameshift mutation in the initial translation site of the C9orf72 orthologue leads to 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 that we are developing in the lab or a transgenic line that overexpresses hexanucleotide repeats.

SImilarly, we have developed zebrafish deletion mutants for the two TDP-43 orthologues in zebrafish. Unlike C9orf72 and FUS these deleion 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. Similarly to C9orf72 lines, we are also developing kockin lines where the last exon is inserted in the TDP-43 locus and developing mutants using CRISPR/Cas9s that target to delete the Nuclear Localization Signal of the zebrafish TDP-43.

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. These results describing this novel model are currently under review.
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 at the level of amino-acids that we are currently validating in this model as well as in pathological samples and biopsies from patien
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 targeting the orthologous genes in zebrafish and to overexpress in the same time the ALS-related mutations. During this initial period, we have analyzed most of these lines and have zebrafish models with specific motor deficits for C9orf72, TDP-43 and FUS. 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.
We are currently defining how the epistatic interactions amongst these major genetic factors in ALS for the first time in vertebrate disease models.

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 motor neuron as well as other neuronal populations 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 models that we have generated and to understand what is common amongst major genetic markers in 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 advance but also to propose novel and innovative therapeutic avenues, we are in the process to develop a platform to screen pharmacological compounds. This platform will optimize the detection of mobile and immobile zebrafish larvae and embryos using light and fluorescent microscopy. This platform will enable to perform long-duration pharmacological treatments 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.
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