Protein Misfolding and Neurodegenerative Diseases

Autophagy-inducing compounds to reduce neuron loss in ALS and FTD

Autophagy is a critical, evolutionarily conserved pathway mediating the breakdown and recycling of cellular proteins and organelles. Emphasizing its pivotal nature, genetic autophagy dysfunction contributes to neurodegenerative conditions, cancer, infectious diseases and cardiac disorders. Substantial genetic, anatomic and clinical evidence indicates that the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are not only intimately connected with one another, but that autophagy is broadly neuroprotective in ALS and FTD. The deposition of protein rich inclusions within neurons is a signature pathology of ALS, FTD and related disorders, highlighting a fundamental deficiency in protein homeostasis underlying these and other neurodegenerative diseases. Likewise, stimulating autophagy extends neuronal survival and ameliorates symptoms of disease in cellular and animal models of ALS and FTD, making this pathway an important and potentially rich therapeutic target.

The central goal is the identification of novel, potent autophagy-inducing compounds with the potential to reduce neuron loss in ALS and FTD. Development of effective autophagy modulating drugs to treat these diseases has been hampered by fundamental deficiencies in available methods for measuring autophagic activity, or flux. To overcome these limitations, we created a unique human reporter cell line and a non-invasive imaging assay to measure autophagic flux in living cells, without the need for potentially confounding drug treatments or protein overexpression. The photoconvertible protein Dendra2 was introduced into the endogenous MAP1LC3B locus of human cell lines via CRISPR/Cas9 genome editing, enabling accurate and sensitive determinations of autophagy activity via optical pulse labeling. High-content screening of 6,000 tool compounds and FDA-approved drugs using this assay showed that many reported autophagy stimulators either fail to enhance autophagy, or conversely inhibit autophagy flux, reaffirming the importance of measuring pathway dynamics rather than the steady state abundance of intermediates. Within an expanded library of 24,000 compounds sampling a wide diversity of chemical space, we identified novel active compounds with profound effects on autophagy, validated by comparison with alternative methods. Further, we demonstrate that NVP-BEZ235, one of the most prominent autophagy activators identified herein, exhibited significant neuroprotective properties in a neuronal model of frontotemporal dementia and amyotrophic lateral sclerosis. These studies thus confirm the utility of the high-content autophagy assay we developed, while simultaneously highlighting new autophagy-modulating compounds that display promising therapeutic effects. Because protein misfolding and accumulation are conserved characteristics found in nearly all neurodegenerative disorders as well as age-related conditions such as heart disease and liver disease, the compounds identified herein may have extensive and wide-ranging therapeutic potential.

Novel BK channel activators

Spinocerebellar Ataxias (SCAs) are dominantly inherited debilitating neurodegenerative disorders causing progressive decline in balance, speech, and gait, often resulting in wheelchair confinement. There are no effective treatments for SCAs and a substantial need exists for treatments improving motor function and slowing neurodegeneration. In a series of recent publications supported by new data, we have identified loss of function of large-conductance calcium -activated potassium (BK) channels as a key feature disease in several SCAs. The current project proposes to use a high-throughput screening strategy on a novel automated patch-clamp electrophysiology platform to identify chemical agents to activate BK channels. The long-term goal of this work is to develop therapy for SCAs. The objective of this specific project is to identify agents that augment the opening of BK channels in the presence of low intracellular calcium that may serve as both molecular probes and lead molecules for future drug development.

The current work makes use of a high-throughput patch-clamp electrophysiology platform to identify novel chemical agents for activating BK channels. This represents a novel approach as it allows direct measurement of BK currents in order to identify agents that augment the opening of BK channels in the presence of low intracellular calcium. The work also leverages a novel scientific premise based on mouse genetic models, unbiased transcriptomics, and ion-channel expression studies that have identified BK channels as an appropriate therapeutic target for a variety of degenerative cerebellar ataxias.