Harnessing the mitochondrial vulnerability of neurons from Huntington´s disease patients for uncovering novel therapeutic targets

• Applicant: Professor Alessandro Prigione, Ph.D.
• Subject Area: Molecular and Cellular Neurology and Neuropathology
• Term: from 2020 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 423776278

Huntington’s disease (HD) is an incurable neurodegenerative disorder caused by an abnormal CAG expansion in the gene Huntingtin (HTT). HD is characterized by specific neuronal loss, suggesting that neurons may be particularly susceptible to the cellular toxicity of mutant HTT (mHTT). This susceptibility may be a consequence of the neuronal dependence on mitochondrial function, which is impaired in HD. mHTT disrupts mitochondrial dynamics, mitochondrial membrane potential (MMP), mitochondrial metabolism, and mitochondrial calcium homeostasis. It can also trigger apoptotic cell death. These mitochondrial defects may occur early in the disease process and may contribute to the decompensation of neurons and their ultimate loss. In this project, we aim to address the vulnerability to mitochondrial impairment of neurons carrying mHTT and exploit it for the discovery of potential novel therapeutic targets for HD. We will take advantage of induced pluripotent stem cells (iPSCs) that allow the generation of early neurons that do not contain the numerous secondary cellular defects that may accumulate over time in the patient brain. As preliminary work, we generated iPSCs from four HD patients and four age and gender-matched controls. We successfully established a neuronal differentiation protocol that generates a pure neuronal population in a rapid and robust manner. We used CRISPR/Cas9 genome-editing technology to obtain isogenic iPSCs carrying different CAG repeats within the same patient-specific genomic background. Genome-edited cells will allow us to address the consequences of CAG repeat length and of physiological and pathological huntingtin on the functionality of human neurons. We propose to investigate in details the mitochondrial function of neurons from controls, HD patients, and genome-edited lines. We will first analyze mitochondrial properties and integrity at the basal level and upon metabolic and mitochondrial stresses. We will perform RNA-seq to identify the pathways that are most affected by mHTT in early neurons. We will assess whether modulation of these pathways can affect the mitochondrial and cellular health of HD neurons differently from that of control neurons. This will be done using a multiplexed high-content analysis (HCA)-based assay that we have developed, which assesses in live-cells the function of mitochondria and the ability of the neurons to develop complex branches. Overall, we wish to uncover the mechanisms underlying the susceptibility to mitochondrial toxicity of neurons carrying mHTT in order to identify potential targets of intervention. By understanding which stressors and which biological pathways affect neuronal homeostasis in HD, it may be possible to devise future strategies to preserve neuronal mitochondrial function and homeostasis and to interfere with the early toxic consequences of mutant HTT in the patients.

• DFG Programme: Research Grants