Crowded organelles, lipid accumulation, and abnormal membrane tubulation in cellular models of enhanced α-synuclein membrane interaction.

Previous work from our group showed that certain engineered missense mutations to the α-synuclein (αS) KTKEGV repeat motifs abrogate the protein's ability to form native multimers. The resultant excess monomers accumulate in lipid-membrane-rich inclusions associated with neurotoxicity exceeding that of natural familial Parkinson's disease mutants such as E46K. We presented an initial characterization of the lipid-rich inclusions and found similarities to the αS- and vesicle-rich inclusions that form in baker's yeast when αS is expressed. We also discussed, with some caution, a possible role of membrane-rich inclusions as precursors to filamentous Lewy bodies, the widely accepted hallmark pathology of Parkinson's disease and other synucleinopathies. In the meantime, advances in the microscopic characterization of Lewy bodies have highlighted the presence of crowded organelles and lipid membranes in addition to αS accumulation. This prompted us to revisit the αS inclusions caused by our repeat motif variants in neuroblastoma cells. In addition to our previous characterization, we found that these inclusions can often be seen by brightfield microscopy, overlap with endogenous vesicle markers in immunofluorescence experiments, stain positive for lipid dyes, and can be found to be closely associated with mitochondria. We also observed abnormal tubulation of membranes, which was subtle in inducible lines and pronounced in cells that transiently expressed high amounts of the highly disruptive KTKEGV motif mutant "KLKEGV". Membrane tubulation had been reported before as an αS activity in reductionist systems. Our in-cellulo demonstration now suggests that this mechanism could possibly be a relevant aspect of aberrant αS behavior in cells.

Focused ultrasound with anti-pGlu3 Aß enhances efficacy in Alzheimer's disease-like mice via recruitment of peripheral immune cells.

Pyroglutamate-3 amyloid-ß (pGlu3 Aß) is an N-terminally modified, pathogenic form of amyloid-ß that is present in cerebral amyloid plaques and vascular deposits. Here, we used focused ultrasound (FUS) with microbubbles to enhance the intravenous delivery of an Fc-competent anti-pGlu3 Aß monoclonal antibody, 07/2a mAb, across the blood brain barrier (BBB) in an attempt to improve Aß removal and memory in aged APP/PS1dE9 mice, an Alzheimer's disease (AD)-like model of amyloidogenesis. First, we demonstrated that bilateral hippocampal FUS-BBB disruption (FUS-BBBD) led to a 5.5-fold increase of 07/2a mAb delivery to the brains compared to non-sonicated mice 72 h following a single treatment. Then, we determined that three weekly treatments with 07/2a mAb alone improved spatial learning and memory in aged, plaque-rich APP/PS1dE9 mice, and that this improvement occurred faster and in a higher percentage of animals when combined with FUS-BBBD. Mice given the combination treatment had reduced hippocampal plaque burden compared to PBS-treated controls. Furthermore, synaptic protein levels were higher in hippocampal synaptosomes from mice given the combination treatment compared to sham controls, and there were more CA3 synaptic puncta labeled in the APP/PS1dE9 mice given the combination treatment compared to those given mAb alone. Plaque-associated microglia were present in the hippocampi of APP/PS1dE9 mice treated with 07/2a mAb with and without FUS-BBBD. However, we discovered that plaque-associated Ly6G+ monocytes were only present in the hippocampi of APP/PS1dE9 mice that were given FUS-BBBD alone or even more so, the combination treatment. Lastly, FUS-BBBD did not increase the incidence of microhemorrhage in mice with or without 07/2a mAb treatment. Our findings suggest that FUS is a useful tool to enhance delivery and efficacy of an anti-pGlu3 Aß mAb for immunotherapy either via an additive effect or an independent mechanism. We revealed a potential novel mechanism wherein the combination of 07/2a mAb with FUS-BBBD led to greater monocyte infiltration and recruitment to plaques in this AD-like model. Overall, these effects resulted in greater plaque removal, sparing of synapses and improved cognitive function without causing overt damage, suggesting the possibility of FUS-BBBD as a noninvasive method to increase the therapeutic efficacy of drugs or biologics in AD patients.