The Stephen N. Jasperson Study: Small Molecule Activators of Phospholipase D and Tau Clearance via Autophagic Flux

Principal Investigator: Dr. Haung (Ho) Yu
Columbia University, New York, NY

 

Stimulating autophagy, the brain’s “garbage disposal,” to accelerate the elimination of toxic tau protein buildup.

The build-up of the tau protein as a toxic aggregate is a hallmark of progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) and is likely a primary event in the onset and development of these devastating diseases.

While it is not known why these proteins accumulate, it is possible to reverse the buildup by stimulating a cellular pathway called autophagy. Autophagy is a mechanism within every cell to get rid of unwanted protein or other material, whereby an intracellular compartment called an autophagic vacuole is generated to envelop toxic proteins.

This recycling process is critical for cellular health and we understand that it may become dysfunctional with aging and disease. In an effort to combat the accumulation of proteins like tau in brain cells, we have developed a series of drugs that can promote the clearance of tau through autophagy. This has been proposed and examined by several groups, including our own, but what makes this drug development program unique is that it specifically targets the pathway for clearance.

While other research endeavors have focused on inducing the pathway, we have developed a novel series of drugs that facilitate the degradation of the autophagic vacuole by fusion with the lysosome, the ultimate degradation compartment. While the principle action of isolating tau in an autophagic vacuole is important, these vacuoles must also be degraded, as their presence itself may disrupt a cell’s normal function, as is the case in tauopathies, where the accumulation of autophagic vacuoles is observed in the brains of patients with PSP and CBD.

In this study, we will test these drugs in a brain slice model of PSP/CBD to identify if they facilitate the removal of abnormal tau and would have utility in slowing or ultimately reversing disease progression.

This study is generously funded by Newell Jasperson in honor of his son, Dr. Stephen N. Jasperson.

Results

The build-up of the tau protein as a toxic aggregate is a hallmark of progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) and is likely a primary event in the onset and development of these devastating diseases. While it is not known why these proteins accumulate, it is possible to reverse the buildup by stimulating a cellular pathway called autophagy. Autophagy is a mechanism within every cell to get rid of unwanted protein or other material, whereby an intracellular compartment called an autophagic vacuole is generated to envelop toxic proteins. This recycling process is critical for cellular health and we understand that it may become dysfunctional with aging and disease. In an effort to combat the accumulation of proteins like tau in brain cells, we have developed a series of drugs that can promote the clearance of tau through autophagy. This has been proposed and examined by several groups, including our own, but what makes this drug development program unique is that it specifically targets the pathway for clearance. While other research endeavors have focused on inducing the pathway, we have developed a novel series of drugs that facilitate the degradation of the autophagic vacuole by fusion with the lysosome, the ultimate degradation compartment. While the principle action of isolating tau in an autophagic vacuole is important, these vacuoles must also be degraded, as their presence itself may disrupt a cell’s normal function, as is the case in tauopathies, where the accumulation of autophagic vacuoles is observed in the brains of patients with PSP and CBD.

In this grant submission, we proposed to characterize a series of small molecules codenamed WHYKD and developed at Columbia University that we observed were able to reduce tau, the primary protein that aggregates in neurological disorders like progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), frontotemporal dementia (FTD) and Alzheimer’s disease. The primary goal of this application was to identify the specific mechanism of action and to query that target for drug development. Further, the aim of this grant was to use that target to take forward into drug development licensed through Columbia University. To fulfill this goal, we needed to identify the specific molecular target which we had narrowed down to an enzyme family involved in promoting the fusion of autophagic vacuoles which envelop aggregated proteins and other cytoplasmic cargo and the lysosome, the compartment where toxic or redundant proteins are degraded.

To being, we have used specific inhibitors that target the enzyme isoforms (1 and 2) and successfully blocked the action of the drug in our tau expressing cells. We have also demonstrated that we block the tau lowering effect. We are currently testing this in brain slices from tau mice to further validate this. Finally, we hope to complete the experiments in genetically modified mice that lack one of the 2 isoforms of the enzyme.  In triaging the three assays and what they represent in terms of stage of autophagic flux relative to PLD1 activity, measuring LC3-II has been a hallmark of autophagic vacuoles, but it tends to be a static marker indicating either induction of autophagy is taking place, or that clearance is blocked.  The criteria to measure that autophagic flux is occurring is by challenging the system with a lysosomal blocker to see if you have even greater increases in LC3-II (which suggests the system is clearing autophagic vacuoles) or by identifying decreased cargo (such as tau or aggregates).  In this case, PLD1 promotes the fusion of late vesicles/autophagic vacuoles with lysosomes by cleaving phosphatidyl choline into phospho-alcohols like phosphatidylethanol.  PLD1 also plays a minor role in autophagic induction, but largely is resident in late compartments like lysosomes.

Based on our work, we had 4 that exhibited submicromolar efficacy in the phenotypic screens. Further, future compound identification may benefit from using only the IC50 of mKAte2 and Proteostat given the high fidelity between the three assays and the mKate2+Proteostat. In the biochemical measurements of target, a similar profile (though not completely overlapping) was seen with tool compounds that may be beneficial, including WHYKD12, 15 and 19. 

Future:

While intended in this study, the use of PLD KO neurons was not possible due to low fecundity, hence use of pharmacological inhibitors as a contingency. A small breeder population has been maintained and their breeding is being monitored with the hope that the litters will be more frequent and with higher pup numbers to allow for use in biochemical testing. Alternates include using shRNA knockdown of PLD1 or PLD2 as opposed to pharmacological agents.

In addition to the phenotypic assay, the use of PLD1 truncated or full-length protein will aid in a mechanistic screen of new compounds or from a re-purposed library.  This assay will likely yield positive hits within the scope of identifying drug/compound targets that can be explored for therapeutic use.