Eva Klinman, M.D., Ph.D. (Year 1)

ABSTRACT

Neurodegenerative conditions including Alzheimer’s disease (AD) are challenging to study due to a lack of tractable models that reproduce the onset, progression, and sporadic nature of the diseases. Most human studies rely on neurons generated from induced pluripotent stem cells (iPSCs) through a process that strips them of their
age-associated epigenetic and cellular characteristics. Consequently, neurons differentiated from iPSCs show characteristics of fetal neurons, limiting their ability to capture cellular properties intrinsic to aged neurons. We have developed a novel and highly efficient method of generating human neurons via microRNA-induced direct
conversion of skin fibroblasts. This approach retains the age-associated epigenetic signatures of the original donor cells and natively exhibits disease-associated cellular changes in multiple neurodegenerative conditions including AD. The microRNA direct conversion system allows neurons generated from donors of different ages to be compared with those from patients with various stages of AD. Ultimately, we aim to understand aging as distinct from disease, to provide personalized medical predictions to determine an individual’s risk of developing AD, as well as create a platform for drug discovery using age-appropriate human cells.

This proposal focuses on the behavior of the microtubule-binding protein tau and the impact of microtubule changes on autophagosomes and mitochondrial resilience as a function of advancing age and sporadic AD. Tau dysregulation and accumulation of tau “tangles” are required for the development of sporadic AD, and both aging and AD have shown perturbations to the health and function of autophagosomes and mitochondria. We hypothesize that advanced age increases axonal accumulation of tau, resulting in an age-related decline in cellular transport. We posit that these cytoskeletal changes become pathologic in at-risk individuals when combined with age-related mitochondrial fragility and decreased autophagosome maturation. These concepts will be tested by evaluating the motility, function, and distribution of mitochondria and autophagosomes in directly converted neurons derived from healthy young and old individuals using live-cell imaging and confocal microscopy. Physiologic age-related changes will be contrasted with those observed in neurons from donors with sporadic late-onset AD. Neurons will additionally be treated with siRNA targeting tau to establish the role of tau during aging and disease. Our findings will provide fundamental insights into cytoskeletal dynamics and the resulting behavior of neuronal organelles in healthy aging and AD.

Ultimately, this work will guide future pharmaceutical advances by providing a neuronal model for drug testing to assist with both anti-aging medications and targeted AD therapeutics. Our next steps include testing small molecule activators of mitochondrial fusion as well as autophagy enhancers to determine if these chemicals can improve the survival of AD patient neurons. In the future, we hope that individualized assessment of patient-derived microRNA-induced neurons may be used to predict neurodegenerative disease risk in patients.

Lay Summary
Aging poses the biggest risk for developing Alzheimer’s disease and other neurodegenerative diseases. It is difficult to study how aging affects the human brain because there are not good models of Alzheimer’s disease. The brains of animals do not age the same way as human brains, and animals do not develop Alzheimer’s disease. Human brain cells (neurons) can be grown in a dish, but traditional culture methods create cells that act like infant neurons and not like adult neurons.

This project uses a new technique that converts human skin cells into neurons that keep the age-related markers of the person who provided the skin sample. This allows us to compare living neurons from many types of people including young people, old people, and people with Alzheimer’s disease. The focus of this project is on structures inside the cell that support its internal organization and shape, as well as mitochondria which supply the cell with energy, and autophagosomes which act as cellular trash collectors. We predict that structures inside the cell become more fragile with age and that this results in the breakdown of normal movement and debris collection. When these processes go wrong, certain at-risk cells may sicken and develop signs of Alzheimer’s disease. By examining living cells under a microscope from people of different ages and disease severity, we hope to clarify the role that these changes play in the risk of developing Alzheimer’s disease as people age.