Final Report Summary - TOXICITY IN MND (Screening of candidate targets for astrocytic toxicity in motor neurone disease)
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by death of the neurons regulating muscle movement, called motor neurons. Failure of the breathing muscles is fatal and patients typically die within 2-5 years from diagnosis. ALS is a multifactorial disease where different cell types contribute to the pathologic mechanisms.
In 1993 researchers identified the first mutation associated with familial ALS, a mutation in superoxide dismutase 1 (SOD1), a gene involved in detoxification of radicals. This finding lead to the generation of transgenic animal models carrying the human mutant gene and developing neuronal degeneration similar to patients affected by ALS.
Experiments using the mouse models of this disease highlighted the role that different cell types play in determining onset or progression of disease. Recent studies have demonstrated that the cells normally involved in supporting the neurons in the brain and spinal cord, called glia, are implicated in disease development and progression.
With the discovery that adult human skin cells could be transformed into induced pluripotent stem
(iPS) cells and subsequently into nerve cells and glia, the field of ALS saw the opportunity to finally model not only the familial, but especially the sporadic disease in vitro.
In 2011 researchers managed to isolate cells from the brain and spinal cord that could be differentiated into glial cells, in particular astrocytes, one of the cells types supporting neurons. The studies conducted using these cells demonstrated that astrocytes from ALS patients are not able to support neurons; on the contrary, they induce their death.
Project objectives
In this study we aimed at identifying factors that determine or contribute to astrocyte toxicity against neurons. During the first year we planned to identify selected genes that contribute to astrocyte toxicity and neuronal death in ALS, with the final aim to use gene therapy approaches to silence these toxic genes and improve neuronal activity and survival.
In the second period of the Fellowship we aimed at screening about 4000 drugs to identify chemical compounds that, in a similar way, can decrease glia toxicity towards neurons.
Description of the work performed since the beginning of the project
We performed a gene expression study to look at what genes are more or less expressed in astrocytes from the ALS mouse model compared to control healthy mice. Using the bioinformatics expertise available in Sheffield, we identified several candidate genes and pathways potentially involved in astrocyte toxicity. In particular, in Columbus the Fellow focused on the role of the immune system, including genes belonging to or regulating the complement system, inflammation and the immune response adopted by glia.
Moreover, the Fellow joined the Kaspar lab when one graduate student had just started studying the effect of decreasing the expression of the superoxide dismutase 1 (SOD1) gene at different time points of the disease in the mouse model of ALS using a gene therapy approach. The study developed further into testing the safety of this approach for clinical trial.
Co-cultures of human astrocytes and motor neurons were set up to test the toxic properties of astrocytes from ALS patients.
The Fellow contributed to setting up a new method to develop glia cells from skin cells isolated from patients affected by familial or sporadic ALS patients and healthy individuals and then culture them with healthy neurons to test the effect of these supporting cells onto neuronal survival.
Decreasing the expression of selected genes involved in inflammation and immune system regulation in glial cells from ALS patients was extremely successful in rescuing neuronal survival in co-culture.
During the last year of this fellowship, the Fellow screened more than 1000 compounds on ALS astrocytes and healthy neurons to identify drugs that can rescue neuronal survival. We identified 17 compounds that are more effective in protecting neurons than Riluzole, the only FDA approved drug used on ALS patients.
In conclusion, this study has produced new models to study sporadic as well as familial ALS. Skin-derived neurons and glia are the only way to model the sporadic form of ALS, the variant that is not linked to any genetic mutation and, therefore, cannot be easily modelled. This study has brought to light new targets for gene therapy and drug screening to silence astrocyte toxicity, thus identifying new therapeutic treatments.
The new tools developed to model human glia in vitro have generated the unique opportunity to test a large number of chemical compounds on several different cell lines each representing a different form of ALS and a different patient.
Ultimately, this project has opened the door to personalised drug screenings and treatments (Fig1).
Fig1. Use of skin-derived astrocytes and neurons to model ALS and identify new drugs for disease treatment.
In 1993 researchers identified the first mutation associated with familial ALS, a mutation in superoxide dismutase 1 (SOD1), a gene involved in detoxification of radicals. This finding lead to the generation of transgenic animal models carrying the human mutant gene and developing neuronal degeneration similar to patients affected by ALS.
Experiments using the mouse models of this disease highlighted the role that different cell types play in determining onset or progression of disease. Recent studies have demonstrated that the cells normally involved in supporting the neurons in the brain and spinal cord, called glia, are implicated in disease development and progression.
With the discovery that adult human skin cells could be transformed into induced pluripotent stem
(iPS) cells and subsequently into nerve cells and glia, the field of ALS saw the opportunity to finally model not only the familial, but especially the sporadic disease in vitro.
In 2011 researchers managed to isolate cells from the brain and spinal cord that could be differentiated into glial cells, in particular astrocytes, one of the cells types supporting neurons. The studies conducted using these cells demonstrated that astrocytes from ALS patients are not able to support neurons; on the contrary, they induce their death.
Project objectives
In this study we aimed at identifying factors that determine or contribute to astrocyte toxicity against neurons. During the first year we planned to identify selected genes that contribute to astrocyte toxicity and neuronal death in ALS, with the final aim to use gene therapy approaches to silence these toxic genes and improve neuronal activity and survival.
In the second period of the Fellowship we aimed at screening about 4000 drugs to identify chemical compounds that, in a similar way, can decrease glia toxicity towards neurons.
Description of the work performed since the beginning of the project
We performed a gene expression study to look at what genes are more or less expressed in astrocytes from the ALS mouse model compared to control healthy mice. Using the bioinformatics expertise available in Sheffield, we identified several candidate genes and pathways potentially involved in astrocyte toxicity. In particular, in Columbus the Fellow focused on the role of the immune system, including genes belonging to or regulating the complement system, inflammation and the immune response adopted by glia.
Moreover, the Fellow joined the Kaspar lab when one graduate student had just started studying the effect of decreasing the expression of the superoxide dismutase 1 (SOD1) gene at different time points of the disease in the mouse model of ALS using a gene therapy approach. The study developed further into testing the safety of this approach for clinical trial.
Co-cultures of human astrocytes and motor neurons were set up to test the toxic properties of astrocytes from ALS patients.
The Fellow contributed to setting up a new method to develop glia cells from skin cells isolated from patients affected by familial or sporadic ALS patients and healthy individuals and then culture them with healthy neurons to test the effect of these supporting cells onto neuronal survival.
Decreasing the expression of selected genes involved in inflammation and immune system regulation in glial cells from ALS patients was extremely successful in rescuing neuronal survival in co-culture.
During the last year of this fellowship, the Fellow screened more than 1000 compounds on ALS astrocytes and healthy neurons to identify drugs that can rescue neuronal survival. We identified 17 compounds that are more effective in protecting neurons than Riluzole, the only FDA approved drug used on ALS patients.
In conclusion, this study has produced new models to study sporadic as well as familial ALS. Skin-derived neurons and glia are the only way to model the sporadic form of ALS, the variant that is not linked to any genetic mutation and, therefore, cannot be easily modelled. This study has brought to light new targets for gene therapy and drug screening to silence astrocyte toxicity, thus identifying new therapeutic treatments.
The new tools developed to model human glia in vitro have generated the unique opportunity to test a large number of chemical compounds on several different cell lines each representing a different form of ALS and a different patient.
Ultimately, this project has opened the door to personalised drug screenings and treatments (Fig1).
Fig1. Use of skin-derived astrocytes and neurons to model ALS and identify new drugs for disease treatment.