Study of Neurological Pathological Mechanisms of and Neurodegenerative Diseases in Neurons and Glia Derived From Induced Stem Cells (iPS) in Patients and Healthy Family Controls.

1. INTRODUCTION Neurological and neurodegenerative diseases have a major impact in families and in the national health service due to the lack in many cases of effective and long-lasting therapies. The lack of these therapeutic strategies is due in large part to the difficulty of modeling these pathologies in vitro. In fact, the impossibility of being able to cultivate human neurons in vitro has forced the use of animal cell models that do not adequately recapitulate the complexity of these human pathologies. For this reason it is necessary to proceed with the development of in vitro models of human origin that reproduce the molecular and biochemical characteristics of these diseases.

The discovery of cellular reprogramming allowed the generation of pluripotent stem cells from the conversion of somatic cells taken from adult individuals. This technology is based on the expression of the 4 genes OCT4, SOX2, KLF4 and c-MYC that synergistically are sufficient to convert somatic cells into induced stem cells called iPS (1,2). For this technology Prof. S. Yamanaka from the University of Kyoto was awarded the Nobel Prize for Medicine in 2012 (3). Human iPS-induced stem cells can be maintained in vitro in a stable manner over time and then be differentiated into any specialized cell including neurons and glial cells. In this way, therefore, it is possible to generate human neurons from adult individuals suffering from neurological pathologies, allowing the study of pathophysiological mechanisms in cells affected by these diseases. Research in recent years has established many reliable protocols to differentiate human iPS cells into different subtypes of neurons (glutamatergics, GABAergics, dopaminergics), astrocytes, oligodendrocytes and microglia (4-6). Thanks to these procedures it was possible to generate neuronal and glial models of many neurological diseases by creating very useful systems for studying pathological processes and identifying new therapeutic targets for some genetic forms of Alzheimer's, Parkinson's and autism (7-9).

The proposing group already has great experience in generating iPS cells by reprogramming and in differentiating them into neurons and glias useful for neurological disease cellular studies. As an example, Dr. Broccoli's group has generated iPS cells from patients with Parkinson's disease and mutations in the OPA1 gene (8). The study of neurons differentiated by these iPS cells allowed to identify mitochondrial defects at the base of neuronal dysfunctions and to identify for the first time how the degeneration of dopaminergic neurons also depends on a moving mode of cell death called necroptosis (8).

The investigators therefore propose to establish lines of iPS cells from patients with genetic mutations responsible for neurological and neurodegenerative diseases to generate neuronal and glial models in vitro for the study of pathological mechanisms and the validation of new future experimental therapies. 2. PURPOSE AND DRAWING OF THE STUDY The aim of this study is to generate iPS-induced stem cell lines from patients with neurological and neurodegenerative diseases to differentiate into neurons and glial cells to study the pathological cellular and molecular processes of these diseases. These in vitro cultures will also be used to validate molecules or experimental therapeutic approaches. IPS cells will be generated by the reprogramming of isolated 10-mL cells of peripheral venous blood.

The study therefore involves taking 10 ml of peripheral blood from