α-Synuclein strain propagation is independent of cellular prion protein expression in a transgenic synucleinopathy mouse model.

The cellular prion protein, PrPC, has been postulated to function as a receptor for α-synuclein, potentially facilitating cell-to-cell spreading and/or toxicity of α-synuclein aggregates in neurodegenerative disorders such as Parkinson's disease. Previously, we generated the "Salt (S)" and "No Salt (NS)" strains of α-synuclein aggregates that cause distinct pathological phenotypes in M83 transgenic mice overexpressing A53T-mutant human α-synuclein. To test the hypothesis that PrPC facilitates the propagation of α-synuclein aggregates, we produced M83 mice that either express or do not express PrPC. Following intracerebral inoculation with the S or NS strain, the absence of PrPC in M83 mice did not prevent disease development and had minimal influence on α-synuclein strain-specified attributes such as the extent of cerebral α-synuclein deposition, selective targeting of specific brain regions and cell types, the morphology of induced α-synuclein deposits, and the structural fingerprints of protease-resistant α-synuclein aggregates. Likewise, there were no appreciable differences in disease manifestation between PrPC-expressing and PrPC-lacking M83 mice following intraperitoneal inoculation of the S strain. Interestingly, intraperitoneal inoculation with the NS strain resulted in two distinct disease phenotypes, indicative of α-synuclein strain evolution, but this was also independent of PrPC expression. Overall, these results suggest that PrPC plays at most a minor role in the propagation, neuroinvasion, and evolution of α-synuclein strains in mice that express A53T-mutant human α-synuclein. Thus, other putative receptors or cell-to-cell propagation mechanisms may have a larger effect on the spread of α-synuclein aggregates during disease.

The molecular determinants of a universal prion acceptor.

In prion diseases, the species barrier limits the transmission of prions from one species to another. However, cross-species prion transmission is remarkably efficient in bank voles, and this phenomenon is mediated by the bank vole prion protein (BVPrP). The molecular determinants of BVPrP's ability to function as a universal prion acceptor remain incompletely defined. Building on our finding that cultured cells expressing BVPrP can replicate both mouse and hamster prion strains, we systematically identified key residues in BVPrP that permit cross-species prion replication. We found that residues N155 and N170 of BVPrP, which are absent in mouse PrP but present in hamster PrP, are critical for cross-species prion replication. Additionally, BVPrP residues V112, I139, and M205, which are absent in hamster PrP but present in mouse PrP, are also required to enable replication of both mouse and hamster prions. Unexpectedly, we found that residues E227 and S230 near the C-terminus of BVPrP severely restrict prion accumulation following cross-species prion challenge, suggesting that they may have evolved to counteract the inherent propensity of BVPrP to misfold. PrP variants with an enhanced ability to replicate both mouse and hamster prions displayed accelerated spontaneous aggregation kinetics in vitro. These findings suggest that BVPrP's unusual properties are governed by a key set of amino acids and that the enhanced misfolding propensity of BVPrP may enable cross-species prion replication.

A single protective polymorphism in the prion protein blocks cross-species prion replication in cultured cells.

The bank vole (BV) prion protein (PrP) can function as a universal acceptor of prions. However, the molecular details of BVPrP's promiscuity for replicating a diverse range of prion strains remain obscure. To develop a cultured cell paradigm capable of interrogating the unique properties of BVPrP, we generated monoclonal lines of CAD5 cells lacking endogenous PrP but stably expressing either hamster (Ha), mouse (Mo), or BVPrP (M109 or I109 polymorphic variants) and then challenged them with various strains of mouse or hamster prions. Cells expressing BVPrP were susceptible to both mouse and hamster prions, whereas cells expressing MoPrP or HaPrP could only be infected with species-matched prions. Propagation of mouse and hamster prions in cells expressing BVPrP resulted in strain adaptation in several instances, as evidenced by alterations in conformational stability, glycosylation, susceptibility to anti-prion small molecules, and the inability of BVPrP-adapted mouse prion strains to infect cells expressing MoPrP. Interestingly, cells expressing BVPrP containing the G127V prion gene variant, identified in individuals resistant to kuru, were unable to become infected with prions. Moreover, the G127V polymorphic variant impeded the spontaneous aggregation of recombinant BVPrP. These results demonstrate that BVPrP can facilitate cross-species prion replication in cultured cells and that a single amino acid change can override the prion-permissive nature of BVPrP. This cellular paradigm will be useful for dissecting the molecular features of BVPrP that allow it to function as a universal prion acceptor.

TULP3 is required for localization of membrane-associated proteins ARL13B and INPP5E to primary cilia.

The primary cilia are known as biosensors that transduce signals through the ciliary membrane proteins in vertebrate cells. The ciliary membrane contains transmembrane proteins and membrane-associated proteins. Tubby-like protein 3 (TULP3), a member of the tubby family, has been shown to interact with the intraflagellar transport-A complex (IFT-A) and to be involved in the ciliary localization of transmembrane proteins, although its role in the ciliary entry of membrane-associated proteins has remained unclear. Here, to determine whether TULP3 is required for the localization of ciliary membrane-associated proteins, we generated and analyzed TULP3-knockout (KO) hTERT RPE-1 (RPE1) cells. Immunofluorescence analysis demonstrated that ciliary formation was downregulated in TULP3-KO cells and that membrane-associated proteins, ADP-ribosylation factor-like 13B (ARL13B) and inositol polyphosphate-5-phosphatase E (INPP5E), failed to localize to primary cilia in TULP3-KO cells. These defects in the localization of ARL13B and INPP5E in TULP3-KO cells were rescued by the exogenous expression of wild-type TULP3, but not that of mutant TULP3 lacking the ability to bind IFT-A. In addition, the expression of TUB protein, another member of the tubby family whose endogenous expression is absent in RPE1 cells, also rescued the defective ciliary localization of ARL13B and INPP5E in TULP3-KO cells, suggesting that there is functional redundancy between TULP3 and TUB. Our findings indicate that TULP3 participates in ciliogenesis, and targets membrane-associated proteins to primary cilia via binding to IFT-A.

Endothelial cell-derived endothelin-1 is involved in abnormal scar formation by dermal fibroblasts through RhoA/Rho-kinase pathway.

Hypertrophic scars and keloids are characterized by excessive dermal deposition of extracellular matrix due to fibroblast-to-myofibroblast differentiation. Endothelin-1 (ET-1) is primarily produced by vascular endothelial cells and plays multiple roles in the wound-healing response and organ fibrogenesis. In this study, we investigated the pathophysiological significance of ET-1 and involvement of RhoA, a member of the Rho GTPases, in hypertrophic scar/keloid formation. We found that ET-1 expression on dermal microvascular endothelial cells (ECs) in hypertrophic scars and keloids was higher than that in normal skin and mature scars. We also confirmed that ET-1 induced myofibroblast differentiation and collagen synthesis in cultured human dermal fibroblasts through the RhoA/Rho-kinase pathway. Finally, since hypertrophic scar/keloid formation was most prominent in areas exposed to mechanical stretch, we examined how mechanical stretch affected ET-1 secretion in human dermal microvascular ECs, and found that mechanical stretch increased ET-1 gene expression and secretion from ECs. Taken together, these results suggest that dermal microvascular ECs release ET-1 in response to mechanical stretch, and thereby contribute to the formation of hypertrophic scars and keloids through the RhoA/Rho-kinase pathway.

SCYL1 arginine methylation by PRMT1 is essential for neurite outgrowth via Golgi morphogenesis.

Arginine methylation is a common posttranslational modification that modulates protein function. SCY1-like pseudokinase 1 (SCYL1) is crucial for neuronal functions and interacts with γ2-COP to form coat protein complex I (COPI) vesicles that regulate Golgi morphology. However, the molecular mechanism by which SCYL1 is regulated remains unclear. Here, we report that the γ2-COP-binding site of SCYL1 is arginine-methylated by protein arginine methyltransferase 1 (PRMT1) and that SCYL1 arginine methylation is important for the interaction of SCYL1 with γ2-COP. PRMT1 was colocalized with SCYL1 in the Golgi fraction. Inhibition of PRMT1 suppressed axon outgrowth and dendrite complexity via abnormal Golgi morphology. Knockdown of SCYL1 by small interfering RNA (siRNA) inhibited axon outgrowth, and the inhibitory effect was rescued by siRNA-resistant SCYL1, but not SCYL1 mutant, in which the arginine methylation site was replaced. Thus, PRMT1 regulates Golgi morphogenesis via SCYL1 arginine methylation. We propose that SCYL1 arginine methylation by PRMT1 contributes to axon and dendrite morphogenesis in neurons.