We are interested in determining how cell death occurs in age-related neurodegenerative disorders characterized by protein aggregation, such as Parkinson’s and Alzheimer’s disease. Our lab is focused on two related pathways that are critical for understanding the neurodegenerative process:
- Mechanisms that initiate and govern the conformational conversion of soluble proteins into insoluble amyloid fibrils
- The downstream neurotoxic effects of aggregated proteins with emphasis on the autophagic / lysosomal degradation system. We use analytic biochemical techniques and neurons generated from patient-derived induced pluripotent stem cells (iPSC) to study disease mechanisms of Parkinson’s disease and related amyloidoses
Culturing Patient-Derived human Neurons to Study Chronic Neurodegenerative Diseases
A) We generate induced pluripotent stem cells (iPSCs) from skin-punch fibroblast biopsies of patients with rare lysosomal and neurodegenerative disorders. iPSCs are generated by viral transduction of reprogramming factors Klf4, cMyc, Oct4, and Sox2. iPSCs are then differentiated into human neurons in vitro, providing a model to study mechanisms of amyloid formation and neurodegeneration. Neuronal marker biiiTubulin (green), midbrain maker tyrosine hydroxylase (red), and nuclei (blue) are shown in the merged image. B) Through careful culturing techniques, we have been able to age patient-derived neurons for hundreds of days. We use this model to study age-dependent pathological changes that occur in disease neurons, such as accumulation of a-synuclein (red) in non-synaptic locations of the neuron. A synaptic marker, synapsin, is shown in green.
Amyloid Formation in Neurons
The pathways that mediate amyloid formation in the brain are not well understood. Previous studies have shown that accumulating metabolic substrates of certain lysosomal storage diseases (LSDs), such as glucosylceramide found in Gaucher disease, influence the amyloid-forming pathway of a-synuclein (Mazzulli et al, Cell, 2011). We study neurons derived from patients with LSDs to learn how lysosomal dysfunction contributes to formation of toxic protein assemblies, including amyloid fibrils (species 3) and oligomeric intermediates (species 2). We also directly generate different pathogenic forms of a-synuclein made in vitro, with purified recombinant protein (electron micrograph of synthetic a-synuclein fibrils is shown on the right). Using this simple in vitro system allows for direct study of factors that influence the amyloid-forming pathway, as well as determining the relative neurotoxicity of different forms of a-synuclein.
Amyloid Toxicity and Pathways to the Lysosome
We are broadly interested in determining how amyloid causes cellular dysfunction, with a specific focus the lysosomal degradation system. The lysosome is a critical cellular component required for recycling and degrading damaged proteins, and mediates neuronal ‘self renewal.’ We are interested in determining how macromolecular degrading enzymes mature through the secretory pathway to reach the lysosomal compartment. Our data from Parkinson patient neurons indicates that pathogenic conformations of a-synuclein disrupt the trafficking process from the Endoplasmic Reticulum (A)-> Golgi (B)-> Lysosome (C), resulting in lysosomal enzyme depletion and dysfunction. We use Parkinson’s patient neurons that form amyloidogenic a-synuclein as a model to study how cells traffic hydrolases to the lysosome under stressful metabolic conditions.