Spinocerebellar ataxia-1 (SCA1) is a late onset neurodegenerative disease caused by the expansion of a polyglutamine repeat within ataxin-1 protein. The toxic effects triggered by mutant ataxin-1 result in degeneration of the neurons in cerebellum, brain stem and spinocerebellar tracts. The targeted overexpression of mutant ataxin-1 in cerebellar Purkinje cells (PCs) of the SCA1 transgenic mice results in the formation of cytoplasmic vacuoles in PCs. These vacuoles appear early on before the onset of behavioral abnormalities. Interestingly, we found that vacoules contain S100B and vimentin proteins, which normally localize to neighboring Bergmann glia (BG). Further, immunohistochemical and specialized silver stain analysis revealed that vacuolar formation is associated with alterations in the morphology of dendritic spines of PCs. To gain insights into the mechanisms of vacuolar formation, we investigated if vacuoles in SCA1 PCs have an autophagic origin or are a consequence of some other event. We examined the expression levels (by Western blotting) of microtubule-associated protein light chain 3 (LC3)-I and LC3-II, and the degradation levels of p62 (a LC3 partner) in the cerebellar fractions prepared from pre-symptomatic SCA1 and age-matched wild-type mice. No p62 degradation was observed; however, LC3-II/(LC3-I + LC3-II) ratios were significantly altered in SCA1 mice indicating changes in the autophagic flux. In addition, LC3 localized to PC vacuoles. Further, we observed a co-localization of myo-inositol monophosphatase 1 (IMPA1) with S100B in PC vacuoles. IMPA1 is present in PC spines and has been implicated in autophagy. In vitro studies using purified IMPA1 and S100B demonstrated that S100B interacted with and activated IMPA1. Both apo and Ca2+-bound S100B were found to activate IMPA1, depending on substrate concentration. IMPA1 is regulated by another calcium-binding protein calbindin-D28k (CaB), since we reported earlier that the CaB levels are reduced in SCA1 PCs, the activation of IMPA1 by S100B may modulate CaB-dependent inositol signaling. This may cause BG–PC interface to degenerate resulting in vacuolar formation. In sum, these data indicate that vacuoles appearing early in SCA1 PCs could be developing through some unknown autophagic mechanism.
Improving drug delivery to the kidney using renal-targeted therapeutics is a promising but underdeveloped area. We aimed to develop a kidney-targeting construct for renal-specific drug delivery. Elastin-like polypeptides (ELPs) are nonimmunogenic protein-based carriers that can stabilize attached small-molecule and peptide therapeutics. We modified ELP at its NH-terminus with a cyclic, seven-amino acid kidney-targeting peptide (KTP) and at its COOH-terminus with a cysteine residue for tracer conjugation. Comparative in vivo pharmacokinetics and biodistribution in rat and swine models and in vitro cell binding studies using human renal cells were performed. KTP-ELP had a longer plasma half-life than ELP in both animal models and was similarly accumulated in kidneys at levels fivefold higher than untargeted ELP, showing renal levels 15- to over 150-fold higher than in other major organs. Renal fluorescence histology demonstrated high accumulation of KTP-ELP in proximal tubules and vascular endothelium. Furthermore, a 14-day infusion of a high dose of ELP or KTP-ELP did not affect body weight, glomerular filtration rate, or albuminuria, or induce renal tissue damage compared with saline-treated controls. In vitro experiments showed higher binding of KTP-ELP to human podocytes, proximal tubule epithelial, and glomerular microvascular endothelial cells than untargeted ELP. These results show the high renal selectivity of KTP-ELP, support the notion that the construct is not species specific, and demonstrate that it does not induce acute renal toxicity. The plasticity of ELP for attachment of any class of therapeutics unlocks the possibility of applying ELP technology for targeted treatment of renal disease in future studies.
S100B, a glial secreted protein is believed to play a major role in neurodegeneration in Alzheimer's disease, Down syndrome, traumatic brain injury and spinocerebellar ataxia type 1 (SCA1). SCA1 is a trinucleotide repeat disorder in which the expanded polyglutamine mutation in the protein ataxin-1 primarily targets Purkinje cells (PCs) of the cerebellum. Currently, the exact mechanism of S100B mediated PC damage in SCA1 is not clear. However, here we show that S100B may act via the activation of the RAGE signaling pathway resulting in oxidative stress mediated injury to mutant ataxin-1 expressing neurons. To combat S100B mediated neurodegeneration, we have designed a selective thermally responsive S100B inhibitory peptide, Synb1-ELP-TRTK. Our therapeutic polypeptide was developed using three key elements: (1) the elastin-like polypeptide (ELP), a thermally responsive polypeptide, (2) the TRTK12 peptide, a known S100B inhibitory peptide and (3) a cell penetrating peptide, Synb1, to enhance intracellular delivery. Binding studies revealed that our peptide, Synb1-ELP-TRTK, interacts with its molecular target S100B and maintains a high S100B binding affinity as comparable with the TRTK12 peptide alone. In addition, in vitro studies revealed that Synb1-ELP-TRTK treatment reduces S100B uptake in SHSY5Y cells. Furthermore, the Synb1-ELP-TRTK peptide decreased S100B induced oxidative damage to mutant ataxin-1 expressing neurons. To test the delivery capabilities of ELP based therapeutic peptides to the cerebellum; we treated mice with fluorescently labeled Synb1-ELP and observed that thermal targeting enhanced peptide delivery to the cerebellum. Here, we have laid the framework for thermal based therapeutic targeting to regions of the brain, particularly the cerebellum. Overall, our data suggests that thermal targeting of ELP based therapeutic peptides to the cerebellum is a novel treatment strategy for cerebellar neurodegenerative disorders.
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