Nano-carrier mediated delivery of small molecule activators of proteasome and autophagy enhancers

Amyotrophic lateral sclerosis (ALS, a common form of motor neuron disease) and frontotemporal dementia (FTD), which in most cases, are associated with abnormal cytoplasmic accumulations of a 43kDa TAR DNA-binding protein (TDP43) in diseased neurons. ALS/FTD pathogenesis implies deficiencies in protein degradation pathways importantly ubiquitin-proteasome system (UPS) and autophagy-lysosomal pathway (ALP). Given the fact that maintaining TDP43 homeostasis and removing TDP43 aggregations are promising therapeutical approaches resulting in functional recovery in cell and animal models of ALS/FTD, we designed experimental cell models based on tetracycline-inducible stable HEK293 cell lines expressing wild-type and cytoplasmic specific variant TDP43 (TDP43^HA-WT and TDP43^HA-dNLS, respectively) and investigated the regulation of UPS and ALP pathways engaged in TDP43 clearance process using D,L sulforaphane (SFN) and SFN loaded lipid nanocarriers (SFN.LN). SFN.LN was 54 nm in size with a narrow polydispersity index and semi-round shape morphology. Results from experimental cell models demonstrated that SFN significantly decreased the urea soluble fractions of TDP43 (without any changes in RIPA soluble fractions) through UPS and ALP is not effectively involved in this model. Accordingly, improved proteasome activity decreased levels of TDP43 in a transgenic zebrafish (Danio rerio) model with overexpressed human TDP43. Moreover, studying different subcellular fractions of TDP43 at the nucleus and cytoplasmic compartments showed that SFN.LN improved the clearance rate of both RIPA and urea soluble fractions of TDP43^HA-dNLS and urea soluble TDP43^HA-WT in the cytoplasm compared to incubation with free SFN. At the nucleus fraction, urea soluble TDP43^HA-dNLS was not detected and SFN.LN did not affect RIPA soluble TDP43^HA-WT. Notably, this work provides several experimental models for understanding the accumulation and fate of insoluble (urea soluble) TDP43 and demonstrates that SFN.LN might provide cytoplasmic-specific delivery of SFN and enhance clearance of cytoplasmic inclusions of TDP43 through stimulation of UPS, which in turn could provide a new therapeutical strategy for neurodegenerative diseases suffering from TDP43 proteinopathies specifically ALS/FTD.