Faculty Bibliography

Parkinson's disease and other synucleinopathies are characterized by the presence of intra-neuronal protein aggregates enriched in the presynaptic protein alpha-synuclein. alpha-synuclein is considered an intrinsically disordered 14 kDa monomer, and although poorly understood, its transition to higher-order multimeric species may play central roles in healthy neurons and during Parkinson's disease pathogenesis. In this study, we demonstrate that alpha-synuclein exists as defined, subcellular-specific species that change characteristics in response to oxidative stress in neuroblastoma cells and in response to Parkinson's disease pathogenesis in human cerebellum and frontal cortex. We further show that the phosphorylation patterns of different alpha-synuclein species are subcellular specific and dependent on the oxidative environment. Using high-performance liquid chromatography and mass spectrometry, we identify a Parkinson's disease enriched, cytosolic ~36-kDa alpha-synuclein species which can be recapitulated in Parkinson's disease model neuroblastoma cells. The characterization of subcellular-specific alpha-synuclein features in neurodegeneration will allow for the identification of neurotoxic alpha-synuclein species, which represent prime targets to reduce alpha-synuclein pathogenicity.

Background/UNASSIGNED:Multiple studies demonstrate the benefit of a vegan diet on cardiovascular risk factors when compared to no intervention or usual dietary patterns. The aim of this study is to evaluate the effect of a vegan diet versus the American Heart Association (AHA)-recommended diet on inflammatory and glucometabolic profiles in patients with angiographically defined coronary artery disease (CAD). Study Design/UNASSIGNED:This study is a randomized, open label, blinded end-point trial of 100 patients with CAD as defined by ≥50% diameter stenosis in a coronary artery ≥2 mm in diameter on invasive angiography. Participants are randomized to 8 weeks of either a vegan or AHA-recommended diet (March 2014 and February 2017). Participants are provided weekly groceries that adhere to the guidelines of their diet. The primary endpoint is high sensitivity C-reactive concentrations. Secondary endpoints include anthropometric data, other markers of inflammation, lipid parameters, glycemic markers, endothelial function, quality of life data, and assessment of physical activity. Endpoints are measured at each visit (baseline, 4 weeks, and 8 weeks). Dietary adherence is measured by two weekly 24-hour dietary recalls, a 4-day food record during the week prior to each visit, and both plasma and urine levels of trimethylamine-N-oxide at each visit. Conclusion/UNASSIGNED:This study is the first to comprehensively assess multiple indices of inflammation and glucometabolic profile in a rigorously conducted randomized trial of patients with CAD on a vegan versus AHA-recommended diet.

Mammalian polymerase theta (Poltheta) is a multifunctional enzyme that promotes error-prone DNA repair by alternative nonhomologous end joining (alt-NHEJ). Here we present structure-function analyses that reveal that, in addition to the polymerase domain, Poltheta-helicase activity plays a central role during double-strand break (DSB) repair. Our results show that the helicase domain promotes chromosomal translocations by alt-NHEJ in mouse embryonic stem cells and also suppresses CRISPR-Cas9- mediated gene targeting by homologous recombination (HR). In vitro assays demonstrate that Poltheta-helicase activity facilitates the removal of RPA from resected DSBs to allow their annealing and subsequent joining by alt-NHEJ. Consistent with an antagonistic role for RPA during alt-NHEJ, inhibition of RPA1 enhances end joining and suppresses recombination. Taken together, our results reveal that the balance between HR and alt-NHEJ is controlled by opposing activities of Poltheta and RPA, providing further insight into the regulation of repair-pathway choice in mammalian cells.

Calvarial defects pose a continued clinical dilemma for reconstruction. Advancements within the fields of stem cell biology and tissue engineering have enabled researchers to develop reconstructive strategies using animal models. We review the utility of various animal models and focus on the mouse, which has aided investigators in understanding cranial development and calvarial bone healing. The murine model has also been used to study regenerative approaches to critical-sized calvarial defects, and we discuss the application of stem cells such as bone marrow-derived mesenchymal stromal cells, adipose-derived stromal cells, muscle-derived stem cells, and pluripotent stem cells to address deficient bone in this animal. Finally, we highlight strategies to manipulate stem cells using various growth factors and inhibitors and ultimately how these factors may prove crucial in future advancements within calvarial reconstruction using native skeletal stem cells.

Rab GTPases regulate membrane trafficking at the stages of vesicle formation, movement and fusion with target compartments. Rab11 GTPase coordinates trafficking at biosynthetic and endocytic recycling routes acting at the trans-Golgi network (TGN), post-Golgi vesicles and recycling endosomes. Rab11 Binding Protein (Rab11BP) has long been described as a potential Rab11 effector but its function remains unknown. The structure of Rab11BP includes a Rab11 binding domain and several domains presumably involved in protein-protein interactions, which include an FFAT-like domain, a proline rich domain and seven WD40 repeats typical of scaffold proteins. Here we used shRNA silencing experiments to first evaluate whether Rab11BP is involved in the Rab11-dependent endocytic recycling of transferring receptor (TfR) and then assessed the protein traffic between the endoplasmic reticulum (ER) and Golgi. We silenced Rab11BP expression with shRNA using lentiviral transduction or microinjection. Rab11BP-silenced cells showed normal TfR endocytosis and recycling analyzed by FACS. However, the distribution of KDELR-GFP and the retrograde-impaired mutant KDELR(D193N)-GFP indicated that Rab11BP functions in Golgi-to-ER retrograde trafficking. Rab11BP silencing led the KDELRGFP to change its distribution from a predominant ER location to an accumulation at the cis-Golgi, colocalizing with Giantin, while the Golgi-retained mutant KDELR(D193N)-GFP remained unaffected. This indicates an impaired retrograde Golgi-to-ER transport without affecting the anterograde transport from ER to Golgi, which likely impact on the ER function of KDEL-bearing chaperones. Rab11BP silencing decreased TGN46, furin, M6PR and calnexin protein levels and induced the characteristic fragmentation of the TGN associated with an impaired Golgi-to-ER transport. These results indicate that Rab11BP is required for retrograde transport from the Golgi-to-ER contributing to the maintenance of the Golgi structure and homeostasis. As to our knowledge Rab11 has not been involved in this step of the biosynthetic trafficking, our results suggest that Rab11BP might have functions independently of Rab11

Netrins and semaphorins, members of the neuronal guidance cue family, exhibit a rich biology with significant roles that extend beyond chemotactic guidance of the axons to build the neuronal patterns of the body. Screening of adult tissues and specific cellular subsets have illuminated that these proteins are also abundantly expressed under both steady state and pathological scenarios. This observation suggests that, in addition to their role in the development of the axonal tree, these proteins possess additional novel functions in adult physiopathology. Notably, a series of striking evidence has emerged in the literature describing their roles as potent regulators of both innate and adaptive immunity, providing extra dimension to our knowledge of neuronal guidance cues. In this review, we summarize the key complex roles of netrins and semaphorins outside the central nervous system (CNS) with focus on their immunomodulatory functions that impact pathophysiological conditions.

BACKGROUND AIMS/OBJECTIVE:Regenerative medicine employs human mesenchymal stromal cells (MSCs) for their multi-lineage plasticity and their pro-regenerative cytokine secretome. Adipose-derived mesenchymal stromal cells (ASCs) are concentrated in fat tissue, and the ease of harvest via liposuction makes them a particularly interesting cell source. However, there are various liposuction methods, and few have been assessed regarding their impact on ASC functionality. Here we study the impact of the two most popular ultrasound-assisted liposuction (UAL) devices currently in clinical use, VASER (Solta Medical) and Lysonix 3000 (Mentor) on ASCs. METHODS:). ASC yield, viability, osteogenic and adipogenic differentiation capacity and in vivo regenerative performance were assessed. RESULTS:Both UAL samples demonstrated equivalent ASC yield and viability. VASER UAL ASCs showed higher osteogenic and adipogenic marker expression, but a comparable differentiation capacity was observed. Soft tissue healing and neovascularization were significantly enhanced via both UAL-derived ASCs in vivo, and there was no significant difference between the cell therapy groups. CONCLUSIONS:Taken together, our data suggest that UAL allows safe and efficient harvesting of the mesenchymal stromal cellular fraction of adipose tissue and that cells harvested via this approach are suitable for cell therapy and tissue engineering applications.

The extinct 'New World stilt-legged', or NWSL, equids constitute a perplexing group of Pleistocene horses endemic to North America. Their slender distal limb bones resemble those of Asiatic asses, such as the Persian onager. Previous palaeogenetic studies, however, have suggested a closer relationship to caballine horses than to Asiatic asses. Here, we report complete mitochondrial and partial nuclear genomes from NWSL equids from across their geographic range. Although multiple NWSL equid species have been named, our palaeogenomic and morphometric analyses support the idea that there was only a single species of middle to late Pleistocene NWSL equid, and demonstrate that it falls outside of crown group Equus. We therefore propose a new genus, Haringtonhippus, for the sole species H. francisci. Our combined genomic and phenomic approach to resolving the systematics of extinct megafauna will allow for an improved understanding of the full extent of the terminal Pleistocene extinction event.