Osteoarthritis as an Umbrella Term for Different Subsets of Humans Undergoing Joint Degeneration: The Need to Address the Differences to Develop Effective Conservative Treatments and Prevention Strategies

Osteoarthritis (OA) of joints such as the knee and hip are very prevalent, and the number of individuals affected is expected to continue to rise. Currently, conservative treatments after OA diagnosis consist of a series of increasingly invasive interventions as the degeneration and pain increase, leading very often to joint replacement surgery. Most interventions are focused on alleviating pain, and there are no interventions currently available that stop and reverse OA-associated joint damage. For many decades OA was considered a disease of cartilage, but it is now considered a disease of the whole multi-tissue joint. As pain is the usual presenting symptom, for most patients, it is not known when the disease process was initiated and what the basis was for the initiation. The exception is post-traumatic OA which results from an overt injury to the joint that elevates the risk for OA development. This scenario leads to very long wait lists for joint replacement surgery in many jurisdictions. One aspect of why progress has been so slow in addressing the needs of patients is that OA has been used as an umbrella term that does not recognize that joint degeneration may arise from a variety of mechanistic causes that likely need separate analysis to identify interventions unique to each subtype (post-traumatic, metabolic, post-menopausal, growth and maturation associated). A second aspect of the slow pace of progress is that the bulk of research in the area is focused on post-traumatic OA (PTOA) in preclinical models that likely are not clearly relevant to human OA. That is, only ~12% of human OA is due to PTOA, but the bulk of studies investigate PTOA in rodents. Thus, much of the research community is failing the patient population affected by OA. A third aspect is that conservative treatment platforms are not specific to each OA subset, nor are they integrated into a coherent fashion for most patients. This review will discuss the literature relevant to the issues mentioned above and propose some of the directions that will be required going forward to enhance the impact of the research enterprise to affect patient outcomes.     

Overexpression of TcNTPDase-1 Gene Increases Infectivity in Mice Infected with Trypanosoma cruzi

Ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) are enzymes located on the surface of the T. cruzi plasma membrane, which hydrolyze a wide range of tri-/-diphosphate nucleosides. In this work, we used previously developed genetically modified strains of Trypanosoma cruzi (T. cruzi), hemi-knockout (KO +/−) and overexpressing (OE) the TcNTPDase-1 gene to evaluate the parasite infectivity profile in a mouse model of acute infection (n = 6 mice per group). Our results showed significantly higher parasitemia and mortality, and lower weight in animals infected with parasites OE TcNTPDase-1, as compared to the infection with the wild type (WT) parasites. On the other hand, animals infected with (KO +/−) parasites showed no mortality during the 30-day trial and mouse weight was more similar to the non-infected (NI) animals. In addition, they had low parasitemia (45.7 times lower) when compared with parasites overexpressing TcNTPDase-1 from the hemi-knockout (OE KO +/−) group. The hearts of animals infected with the OE KO +/− and OE parasites showed significantly larger regions of cardiac inflammation than those infected with the WT parasites (p < 0.001). Only animals infected with KO +/− did not show individual electrocardiographic changes during the period of experimentation. Together, our results expand the knowledge on the role of NTPDases in T. cruzi infectivity, reenforcing the potential of this enzyme as a chemotherapy target to treat Chagas disease (CD).     

Identification of the Role of TGR5 in the Regulation of Leydig Cell Homeostasis

Understanding the regulation of the testicular endocrine function leading to testosterone production is a major objective as the alteration of endocrine function is associated with the development of many diseases such as infertility. In the last decades, it has been demonstrated that several endogenous molecules regulate the steroidogenic pathway. Among them, bile acids have recently emerged as local regulators of testicular physiology and particularly endocrine function. Bile acids act through the nuclear receptor FXRα (Farnesoid-X-receptor alpha; NR1H4) and the G-protein-coupled bile acid receptor (GPBAR-1; TGR5). While FXRα has been demonstrated to regulate testosterone synthesis within Leydig cells, no data are available regarding TGR5. Here, we investigated the potential role of TGR5 within Leydig cells using cell culture approaches combined with pharmacological exposure to the TGR5 agonist INT-777. The data show that activation of TGR5 results in a decrease in testosterone levels. TGR5 acts through the PKA pathway to regulate steroidogenesis. In addition, our data show that TGR5 activation leads to an increase in cholesterol ester levels. This suggests that altered lipid homeostasis may be a mechanism explaining the TGR5-induced decrease in testosterone levels. In conclusion, the present work highlights the impact of the TGR5 signaling pathway on testosterone production and reinforces the links between bile acid signaling pathways and the testicular endocrine function. The testicular bile acid pathways need to be further explored to increase our knowledge of pathologies associated with impaired testicular endocrine function, such as fertility disorders.     

A Comprehensive Assessment of Qualitative and Quantitative Prodromal Parkinsonian Features in Carriers of Gaucher Disease—Identifying Those at the Greatest Risk

Carriers of GBA1 gene variants have a significant risk of developing Parkinson’s disease (PD). A cohort study of GBA carriers between 40–75 years of age was initiated to study the presence of prodromal PD features. Participants underwent non-invasive tests to assess different domains of PD. Ninety-eight unrelated GBA carriers were enrolled (43 males) at a median age (range) of 51 (40–74) years; 71 carried the N370S variant (c.1226A > G) and 25 had a positive family history of PD. The Montreal Cognitive Assessment (MoCA) was the most frequently abnormal (23.7%, 95% CI 15.7–33.4%), followed by the ultrasound hyperechogenicity (22%, 95% CI 14–32%), Unified Parkinson’s Disease Rating Scale part III (UPDRS-III) (17.2%, 95% CI 10.2–26.4%), smell assessment (12.4%, 95% CI 6.6–20.6%) and abnormalities in sleep questionnaires (11%, 95% CI 5.7–19.4%). Significant correlations were found between tests from different domains. To define the risk for PD, we assessed the bottom 10th percentile of each prodromal test, defining this level as “abnormal”. Then we calculated the percentage of “abnormal” tests for each subject; the median (range) was 4.55 (0–43.5%). Twenty-two subjects had more than 15% “abnormal” tests. The limitations of the study included ascertainment bias of individuals with GBA-related PD in relatives, some incomplete data due to technical issues, and a lack of well-characterized normal value ranges in some tests. We plan to enroll additional participants and conduct longitudinal follow-up assessments to build a model for identifying individuals at risk for PD and investigate interventions aiming to delay the onset or perhaps to prevent full-blown PD.     

Dynamic Regulation of Mitochondrial [Ca2+] in Hippocampal Neurons

While neuronal mitochondria have been studied extensively in their role in health and disease, the rules that govern calcium regulation in mitochondria remain somewhat vague. In the present study using cultured rat hippocampal neurons transfected with the mtRCaMP mitochondrial calcium sensor, we investigated the effects of cytosolic calcium surges on the dynamics of mitochondrial calcium ([Ca²⁺]m). Cytosolic calcium ([Ca²⁺]c) was measured using the high affinity sensor Fluo-2. We recorded two types of calcium events: local and global ones. Local events were limited to a small, 2–5 µm section of the dendrite, presumably caused by local synaptic activity, while global events were associated with network bursts and extended throughout the imaged dendrite. In both cases, cytosolic surges were followed by a delayed rise in [Ca²⁺]m. In global events, the rise lasted longer and was observed in all mitochondrial clusters. At the end of the descending part of the global event, [Ca²⁺]m was still high. Global events were accompanied by short and rather high [Ca²⁺]m surges which we called spikelets, and were present until the complete decay of the cytosolic event. In the case of local events, selective short-term responses were limited to the part of the mitochondrial cluster that was located directly in the center of [Ca²⁺]c activity, and faded quickly, while responses in the neighboring regions were rarely observed. Caffeine (which recruits ryanodine receptors to supply calcium to the mitochondria), and carbonyl cyanide m-chlorophenyl hydrazine (CCCP, a mitochondrial uncoupler) could affect [Ca²⁺]m in both global and local events. We constructed a computational model to simulate the fundamental role of mitochondria in restricting calcium signals within a narrow range under synapses, preventing diffusion into adjacent regions of the dendrite. Our results indicate that local cytoplasmic and mitochondrial calcium concentrations are highly correlated. This reflects a key role of signaling pathways that connect the postsynaptic membrane to local mitochondrial clusters.     

Postconditioning by Delayed Administration of Ciclosporin A: Implication for Donation after Circulatory Death (DCD)

Heart transplantation is facing a shortage of grafts. Donation after Circulatory Death (DCD) would constitute a new potential of available organs. In the present work, we aimed to evaluate whether Postconditioning (ischemic or with ciclosporin-A (CsA)) could reduce ischemia-reperfusion injury in a cardiac arrest model when applied at the start of reperfusion or after a delay. An isolated rat heart model was used as a model of DCD. Hearts were submitted to a cardiac arrest of 40 min of global warm ischemia (37 °C) followed by 3 h of 4 °C-cold preservation, then 60 min reperfusion. Hearts were randomly allocated into the following groups: control, ischemic postconditioning (POST, consisting of two episodes each of 30 s ischemia and 30 s reperfusion at the onset of reperfusion), and CsA group (CsA was perfused at 250 nM for 10 min at reperfusion). In respective subgroups, POST and CsA were applied after a delay of 3, 10, and 20 min. Necrosis was lower in CsA and POST versus controls (p < 0.01) whereas heart functions were improved (p < 0.01). However, while the POST lost its efficacy if delayed beyond 3 min of reperfusion, CsA treatment surprisingly showed a reduction of necrosis even if applied after a delay of 3 and 10 min of reperfusion (p < 0.01). This cardioprotection by delayed CsA application correlated with better functional recovery and higher mitochondrial respiratory index. Furthermore, calcium overload necessary to induce mitochondrial permeability transition pore (MPTP) opening was similar in all cardioprotection groups, suggesting a crucial role of MPTP in this delayed protection of DCD hearts.     

Evaluation of Cisplatin-Induced Pathology in the Larval Zebrafish Lateral Line

Cisplatin is an effective anticancer agent, but also causes permanent hearing loss by damaging hair cells—the sensory receptors essential for hearing. There is an urgent clinical need to protect cochlear hair cells in patients undergoing cisplatin chemotherapy. The zebrafish lateral line organ contains hair cells and has been frequently used in studies to screen for otoprotective compounds. However, these studies have employed a wide range of cisplatin dosages and exposure times. We therefore performed a comprehensive evaluation of cisplatin ototoxicity in the zebrafish lateral line with the goal of producing a standardized, clinically relevant protocol for future studies. To define the dose- and time-response patterns of cisplatin-induced hair-cell death, we treated 6-day-old larvae for 2 h in 50 µM–1 mM cisplatin and allowed them to recover. We observed delayed hair cell death, which peaked at 4–8 h post-exposure. Cisplatin also activated a robust inflammatory response, as determined by macrophage recruitment and phagocytosis of hair cells. However, selective depletion of macrophages did not affect hair cell loss. We also examined the effect of cisplatin treatment on fish behavior and found that cisplatin-induced lateral line injury measurably impaired rheotaxis. Finally, we examined the function of remaining hair cells that appeared resistant to cisplatin treatment. We observed significantly reduced uptake of the cationic dye FM1-43 in these cells relative to untreated controls, indicating that surviving hair cells may be functionally impaired. Cumulatively, these results indicate that relatively brief exposures to cisplatin can produce hair cell damage and delayed hair cell death. Our observations provide guidance on standardizing methods for the use of the zebrafish model in studies of cisplatin ototoxicity.     

Chemical Element Profiling in the Sera and Brain of Bipolar Disorders Patients and Healthy Controls

Bipolar Disorder (BD) is a severe recurrent affective mood disorder characterized by a wide range of lifelong mood swings, varying between depressive and manic states. BD affects more than 1% of the world’s population irrespective of nationality, ethnic origin, or socioeconomic status and is one of the main causes of disability among young people, leading to cognitive and functional impairment and raised mortality, particularly death by suicide. Trace elements play a vital role in many biochemical and physiological processes. Compelling evidence shows that element toxicity might play a crucial role in the onset and progression of neurodegenerative disorders, but their involvement in mood disorders has been scarcely studied. In the present investigation, we determined the concentration of 26 elements in the serum of BD patients before and after treatment and in postmortem brain samples from BD patients and compared them with matched controls. The only element that was reduced significantly in the serum following treatment was vanadium (V). Furthermore, the concentration of Al, B, Cu, K, Mg and V were significantly lower in the pre-frontal cortex of BD patients compared with those of the controls. A comparison of Spearman’s rank correlation coefficients between the elements in the serum and brain of BD patients and control groups pointed to boron and aluminum as being involved in the disease. These results suggest that there is a disturbance in the elements’ homeostasis and the inter-elements’ relationship in the brain of BD patients and advocate a thorough examination of the possible involvement of chemical elements in different stages of the disease.     

Microbiota-Derived Extracellular Vesicles Detected in Human Blood from Healthy Donors

The microbiota constitutes an important part of the holobiont in which extracellular vesicles (EVs) are key players in health, especially regarding inter- and intra-kingdom communications. Analysis of EVs from the red blood cell concentrates of healthy donors revealed variable amounts of OmpA and LPS in 12 of the 14 analyzed samples, providing indirect experimental evidence of the presence of microbiota EVs in human circulating blood in the absence of barrier disruption. To investigate the role of these microbiota EVs, we tracked the fusion of fluorescent Escherichia coli EVs with blood mononuclear cells and showed that, in the circulating blood, these EVs interacted almost exclusively with monocytes. This study demonstrates that bacterial EVs constitute critical elements of the host–microbiota cellular communication. The analysis of bacterial EVs should thus be systematically included in any characterization of human EVs.     

FUS Microphase Separation: Regulation by Nucleic Acid Polymers and DNA Repair Proteins

Fused in sarcoma (FUS) is involved in the regulation of RNA and DNA metabolism. FUS participates in the formation of biomolecular condensates driven by phase transition. FUS is prone to self-aggregation and tends to undergo phase transition both with or without nucleic acid polymers. Using dynamic light scattering and fluorescence microscopy, we examined the formation of FUS high-order structures or FUS-rich microphases induced by the presence of RNA, poly(ADP-ribose), ssDNA, or dsDNA and evaluated effects of some nucleic-acid-binding proteins on the phase behavior of FUS–nucleic acid systems. Formation and stability of FUS-rich microphases only partially correlated with FUS’s affinity for a nucleic acid polymer. Some proteins—which directly interact with PAR, RNA, ssDNA, and dsDNA and are possible components of FUS-enriched cellular condensates—disrupted the nucleic-acid-induced assembly of FUS-rich microphases. We found that XRCC1, a DNA repair factor, underwent a microphase separation and formed own microdroplets and coassemblies with FUS in the presence of poly(ADP-ribose). These results probably indicated an important role of nucleic-acid-binding proteins in the regulation of FUS-dependent formation of condensates and imply the possibility of the formation of XRCC1-dependent phase-separated condensates in the cell.