Pink1/Parkin deficiency alters circulating lymphocyte populations and increases platelet-T cell aggregates in rats.

Parkinson's disease (PD) is the most common progressive neurodegenerative movement disorder and results from the selective loss of dopaminergic neurons in the substantia nigra pars compacta. Pink1 and Parkin are proteins that function together in mitochondrial quality control, and when they carry loss-of-function mutations lead to familial forms of PD. While much research has focused on central nervous system alterations in PD, peripheral contributions to PD pathogenesis are increasingly appreciated. We report Pink1/Parkin regulate glycolytic and mitochondrial oxidative metabolism in peripheral blood mononuclear cells (PBMCs) from rats. Pink1/Parkin deficiency induces changes in the circulating lymphocyte populations, namely increased CD4 + T cells and decreased CD8 + T cells and B cells. Loss of Pink1/Parkin leads to elevated platelet counts in the blood and increased platelet-T cell aggregation. Platelet-lymphocyte aggregates are associated with increased thrombosis risk, and venous thrombosis is a cause of sudden death in PD, suggesting targeting the Pink1/Parkin pathway in the periphery has therapeutic potential.

Parkinson's disease relevant pathological features are manifested in male Pink1/Parkin deficient rats.

Animal disease models are important for neuroscience experimentation and in the study of neurodegenerative disorders. The major neurodegenerative disorder leading to motor impairments is Parkinson's disease (PD). The identification of hereditary forms of PD uncovered gene mutations and variants, such as loss-of-function mutations in PTEN-induced putative kinase 1 (Pink1) and the E3 ubiquitin ligase Parkin, two proteins involved in mitochondrial quality control, that could be harnessed to create animal models. However, to date, such models have not reproducibly recapitulated major aspects of the disease. Here, we describe the generation and phenotypic characterization of a combined Pink1/Parkin double knockout (dKO) rat, which reproducibly exhibits PD-relevant abnormalities, particularly in male animals. Motor dysfunction in Pink1/Parkin dKO rats was characterized by gait abnormalities and decreased rearing frequency, the latter of which was responsive to levodopa treatment. Pink1/Parkin dKO rats exhibited elevated plasma levels of neurofilament light chain and significant loss of tyrosine hydroxylase expression in the substantia nigra pars compacta (SNpc). Glial cell activation was also observed in the SNpc. Pink1/Parkin dKO rats showed elevated plasma and reduced cerebrospinal levels of alpha-synuclein as well as the presence of alpha-synuclein aggregates in the striatum. Further, the profile of circulating lymphocytes was altered, as elevated CD3+CD4+ T cells and reduced CD3+CD8+ T cells in Pink1/Parkin dKO rats were found. This coincided with mitochondrial dysfunction and infiltration of CD3+ T cells in the striatum. Altogether, the Pink1/Parkin dKO rats exhibited phenotypes similar to what is seen with PD patients, thus highlighting the suitability of this model for mechanistic studies of the role of Pink1 and Parkin in PD pathogenesis and as therapeutic targets.

Robotic Arm-Assisted versus Manual Total Knee Arthroplasty: A Propensity Score-Matched Analysis.

The purpose of this study was to compare (1) operative time, (2) in-hospital pain scores, (3) opioid medication use, (4) length of stay (LOS), (5) discharge disposition at 90-day postoperative, (6) range of motion (ROM), (7) number of physical therapy (PT) visits, (8) emergency department (ED) visits, (9) readmissions, (10) reoperations, (11) complications, and (12) 1-year patient-reported outcome measures (PROMs) in propensity matched patient cohorts who underwent robotic arm-assisted (RA) versus manual total knee arthroplasty (TKA). Using a prospectively collected institutional database, patients who underwent RA- and manual TKA were the nearest neighbor propensity score matched 3:1 (255 manual TKA:85 RA-TKA), accounting for various preoperative characteristics. Data were compared using analysis of variance (ANOVA), Kruskal-Wallis, Pearson's Chi-squared, and Fisher's exact tests, when appropriate. Postoperative pain scores, opioid use, ED visits, readmissions, and 1-year PROMs were similar between the cohorts. Manual TKA patients achieved higher maximum flexion ROM (120.3 ± 9.9 versus 117.8 ± 10.2, p = 0.043) with no statistical differences in other ROM parameters. Manual TKA had shorter operative time (105 vs.113 minutes, p < 0.001), and fewer PT visits (median [interquartile range] = 10.0 [8.0-13.0] vs. 11.5 [9.5-15.5] visits, p = 0.014). RA-TKA had shorter LOS (0.48 ± 0.59 vs.1.2 ± 0.59 days, p < 0.001) and higher proportion of home discharges (p < 0.001). RA-TKA and manual TKA had similar postoperative complications and 1-year PROMs. Although RA-TKA patients had longer operative times, they had shorter LOS and higher propensity for home discharge. In an era of value-based care models and the steady shift to outpatient TKA, these trends need to be explored further. Long-term and randomized controlled studies may help determine potential added value of RA-TKA versus manual TKA. This study reflects level of evidence III.

Lateral unicompartmental knee arthroplasty through a lateral parapatellar approach has high early survivorship.

BACKGROUND: The literature suggests lateral unicompartmental knee arthroplasties are associated with low revision rates. However, there are fewer reports describing techniques for lateral unicompartmental arthroplasty and whether technique influences ROM and function compared to reports for medial unicompartmental arthroplasty. QUESTIONS/PURPOSES: We report our indications for lateral unicompartmental arthroplasty, how we perform this procedure, and the subsequent Knee Society scores, ROM, and revision and reoperation rates. PATIENTS AND METHODS: From a retrospective review of electronic records from 2004 through 2008, we identified 93 patients who had 100 lateral unicompartmental arthroplasties. Indications were complete lateral bone-on-bone arthrosis with a correctible deformity and maintenance of the medial joint space on varus stress radiographs or isolated lateral disease by diagnostic arthroscopy. Average age was 68 years. Seventy percent of patients were women. At followup, we obtained Knee Society scores and ROM. Minimum followup was 24 months (average, 39 months; range, 24-81 months). RESULTS: At followup, Knee Society scores averaged 46 for pain, 94 for clinical, and 89 for function, and ROM averaged 124°. Three patients had reoperations: one an open reduction and internal fixation for fracture at 2 years postoperatively, one an arthroscopy for a medial meniscal tear, and one a revision for pain. CONCLUSIONS: Based on our observations, we believe complete cartilage loss laterally and correctible deformity with maintenance of the medial joint on varus stress radiographs are reasonable indications for lateral unicompartmental arthroplasty. We recommend a lateral parapatellar approach can be utilized. The early reoperation and revision rates were low.

Hyperphosphorylated Human Tau Accumulates at the Synapse, Localizing on Synaptic Mitochondrial Outer Membranes and Disrupting Respiration in a Mouse Model of Tauopathy.

Neurogenerative disorders, such as Alzheimer's disease (AD), represent a growing public health challenge in aging societies. Tauopathies, a subset of neurodegenerative disorders that includes AD, are characterized by accumulation of fibrillar and hyperphosphorylated forms of microtubule-associated protein tau with coincident mitochondrial abnormalities and neuronal dysfunction. Although, in vitro, tau impairs axonal transport altering mitochondrial distribution, clear in vivo mechanisms associating tau and mitochondrial dysfunction remain obscure. Herein, we investigated the effects of human tau on brain mitochondria in vivo using transgenic htau mice at ages preceding and coinciding with onset of tauopathy. Subcellular proteomics combined with bioenergetic assessment revealed pathologic forms of tau preferentially associate with synaptic over non-synaptic mitochondria coinciding with changes in bioenergetics, reminiscent of an aged synaptic mitochondrial phenotype in wild-type mice. While mitochondrial content was unaltered, mitochondrial maximal respiration was impaired in synaptosomes from htau mice. Further, mitochondria-associated tau was determined to be outer membrane-associated using the trypsin protection assay and carbonate extraction. These findings reveal non-mutant human tau accumulation at the synapse has deleterious effects on mitochondria, which likely contributes to synaptic dysfunction observed in the context of tauopathy.

Physiologically Relevant Concentrations of Dolutegravir, Emtricitabine, and Efavirenz Induce Distinct Metabolic Alterations in HeLa Epithelial and BV2 Microglial Cells.

Microglia, the resident brain phagocytes, likely play a key role in human immunodeficiency virus (HIV) infection of the central nervous system (CNS) and subsequent neuropathogenesis; however, the nature of the infection-induced changes that yield damaging CNS effects and the stimuli that provoke microglial activation remains elusive, especially in the current era of using antiretroviral (ARV) drugs for ARV therapy (ART). Altered microglial metabolism can modulate cellular functionality and pathogenicity in neurological disease. While HIV infection itself alters brain energy metabolism, the effect of ARV drugs, particularly those currently used in treatment, on metabolism is understudied. Dolutegravir (DTG) and emtricitabine (FTC) combination, together with tenofovir (TAF or TDF), is one of the recommended first line treatments for HIV. Despite the relatively good tolerability and safety profile of FTC, a nucleoside reverse transcriptase inhibitor, and DTG, an integrase inhibitor, adverse side effects have been reported and highlight a need to understand off-target effects of these medications. We hypothesized that similar to previous ART regimen drugs, DTG and FTC side effects involve mitochondrial dysfunction. To increase detection of ARV-induced mitochondrial effects, highly glycolytic HeLa epithelial cells were forced to rely on oxidative phosphorylation by substituting galactose for glucose in the growth media. We assessed ATP levels, resazurin oxidation-reduction (REDOX), and mitochondrial membrane potential following 24-hour exposure (to approximate effects of one dose equivalent) to DTG, FTC, and efavirenz (EFV, a known mitotoxic ARV drug). Further, since microglia support productive HIV infection, act as latent HIV cellular reservoirs, and when dysfunctional likely contribute to HIV-associated neurocognitive disorders, the experiments were repeated using BV2 microglial cells. In HeLa cells, FTC decreased mitochondrial REDOX activity, while DTG, similar to EFV, impaired both mitochondrial ATP generation and REDOX activity. In contrast to HeLa cells, DTG increased cellular ATP generation and mitochondrial REDOX activity in BV2 cells. Bioenergetic analysis revealed that DTG, FTC, and EFV elevated BV2 cell mitochondrial respiration. DTG and FTC exposure induced distinct mitochondrial functional changes in HeLa and BV2 cells. These findings suggest cell type-specific metabolic changes may contribute to the toxic side effects of these ARV drugs.

Discovery of candidate HIV-1 latency biomarkers using an OMICs approach.

Infection with HIV-1 remains uncurable due to reservoirs of latently infected cells. Any potential cure for HIV will require a mechanism to identify and target these cells in vivo. We created a panel of Jurkat cell lines latently infected with the HIV DuoFlo virus to identify candidate biomarkers of latency. SWATH mass spectrometry was used to compare the membrane proteomes of one of the cell lines to parental Jurkat cells. Several candidate proteins with significantly altered expression were identified. The differential expression of several candidates was validated in multiple latently infected cell lines. Three factors (LAG-3, CD147,CD231) were altered across numerous cell lines, but the expression of most candidate biomarkers was variable. These results confirm that phenotypic differences in latently infected cells exists and identify additional novel biomarkers. The variable expression of biomarkers across different cell clones suggests universal antigen-based detection of latently infected cells may require a multiplex approach.

Inactivation of XPF Sensitizes Cancer Cells to Gemcitabine.

Gemcitabine (2', 2'-difluorodeoxycytidine; dFdC) is a deoxycytidine analog and is used primarily against pancreatic cancer. The cytotoxicity of gemcitabine is due to the inhibition of DNA replication. However, a mechanism of removal of the incorporated dFdC is largely unknown. In this report, we discovered that nucleotide excision repair protein XPF-ERCC1 participates in the repair of gemcitabine-induced DNA damage and inactivation of XPF sensitizes cells to gemcitabine. Further analysis identified that XPF-ERCC1 functions together with apurinic/apyrimidinic endonuclease (APE) in the repair of gemcitabine-induced DNA damage. Our results demonstrate the importance of the evaluation of DNA repair activities in gemcitabine treatment.

Proteomic profiling of HIV-infected T-cells by SWATH mass spectrometry.

Viral pathogenesis results from changes in host cells due to virus usurpation of the host cell and the innate cellular responses to thwart infection. We measured global changes in protein expression and localization in HIV-1 infected T-cells using subcellular fractionation and the Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS) proteomic platform. Eight biological replicates were performed in two independent experimental series. In silico merging of both experiments identified 287 proteins with altered expression (p < .05) between control and infected cells- 172 in the cytoplasm, 84 in the membrane, and 31 in nuclei. 170 of the proteins are components of the NIH HIV interaction database. Multiple Reaction Monitoring and traditional immunoblotting validated the altered expression of several factors during infection. Numerous factors were found to affect HIV infection in gain- and loss-of-expression infection assays, including the intermediate filament vimentin which was found to be required for efficient infection.