The psychiatric risk gene Cacna1c regulates mitochondrial function in cellular stress responses

Affective disorders such as major depression and bipolar disorder are among the most prevalent forms of mental illness, and their pathophysiology involves complex interactions between genetic and environmental risk factors. However, the underlying mechanisms explaining how genetic and environmental alterations affect the risk for psychiatric disorders are still largely unknown. Confirmed by several genome-wide association studies over the past ten years, CACNA1C represents one of the strongest and most replicable psychiatric risk genes. Besides genetic predispositions, environmental influences such as childhood maltreatment or chronic stress also contribute to disease vulnerability. In addition, increasing evidence suggests a crucial role for mitochondrial dysfunction, oxidative stress, excitotoxicity, and neuroinflammation in the development of major neuropsychiatric disorders. Furthermore, mitochondrial dysfunction in peripheral blood mononuclear cells (PBMCs) is currently being discussed as a potential biomarker for affective disorders supporting early diagnosis, control of disease progression, and evaluation of treatment response. In a translational setting, the present project focused on the effects of defined gene-environment interactions on brain mitochondrial integrity and function in order to provide new insights into pathophysiological mechanisms of affective disorders and to identify novel therapeutic targets with potential relevance for future treatment strategies. Using immortalized mouse hippocampal HT22 cells, a well-established model system to investigate glutamate-mediated oxidative stress, it was demonstrated that both siRNA-mediated Cacna1c gene silencing and L-type calcium channel (LTCC) blockade with nimodipine significantly prevented the glutamate-mediated rise in lipid peroxidation, excessive ROS formation, collapse of mitochondrial membrane potential, loss of ATP, reduction in mitochondrial respiration, and ultimately neuronal cell death. Moreover, both Cacna1c knockdown and pharmacological LTCC inhibition altered CaV1.2-dependent gene transcription, thereby suppressing the glutamate-induced expression of the inner mitochondrial membrane calcium uptake protein MCU. Accordingly, downregulation of Cacna1c substantially diminished the elevation in mitochondrial calcium levels after glutamate treatment. In the employed paradigm of oxidative glutamate toxicity, Cacna1c depletion also protected against detrimental mitochondrial fission and stimulated mitochondrial biogenesis without affecting mitophagy, thus promoting the turnover of mitochondria and preventing the accumulation of dysfunctional mitochondria in neuronal HT22 cells. These data imply that upstream genetic modifications, e.g. reduced CACNA1C expression, converge to control mitochondrial function, resulting in cellular resilience against oxidative stress. In primary cortical rat neurons, heterozygous Cacna1c knockout partially reduced Cacna1c expression but had no impact on either initial increase in [Ca2+]i or delayed perturbations in mitochondrial bioenergetics, ATP levels, and cell viability in response to glutamate-mediated excitotoxicity. Furthermore, Cacna1c mRNA and protein expression levels were subject to strong regulation and degradation in this model of neuronal excitotoxicity. Partial neuroprotection against long-term glutamate toxicity by pharmacological LTCC blockade highlighted a potential dose-effect-dependency and the involvement of LTCCs in this cell death pathway. In primary rat microglia cultures, both Cacna1c haploinsufficiency and nimodipine treatment were associated with reduced morphological changes and glycolytic metabolism upon lipopolysaccharide (LPS) stimulation. The LPS-induced shift from oxidative phosphorylation towards glycolysis seems essential for the inflammatory response, since the downstream release of NO, IL-1α, IL-1β, IL-6, IL-10, and TNF-α was also decreased in heterozygous Cacna1c as well as nimodipine-treated microglial cells. These results indicate a major functional role for CaV1.2-dependent signaling in the pro-inflammatory activation of microglia, the innate immune cells of the central nervous system. By simulating the interaction of psychiatric disease-relevant genetic and environmental factors in vivo, the present study additionally evaluated their potential effect on brain mitochondrial function using a constitutive heterozygous Cacna1c rat model in combination with a four-week exposure to either post-weaning social isolation, standard housing, or social and physical environmental enrichment during the juvenile developmental period. In this specific gene-environment setting, isolated mitochondria from prefrontal cortex and hippocampus, both representing particularly susceptible brain regions in neuropsychiatric disorders, did not reveal considerable differences in mitochondrial bioenergetics, respiratory chain complex protein levels, superoxide formation, and membrane potential between the investigated conditions. Finally, mitochondrial function was investigated in human PBMCs from probands recruited in the Marburg/Münster Affective Disorders Cohort Study (MACS). However, neither a family history of psychiatric disorders nor an experience of maltreatment during childhood had a significant effect on mitochondrial superoxide levels and respiratory parameters in PBMCs from healthy female subjects. Consequently, further research is required in order to shed more light on the early pathological mechanisms underlying neuropsychiatric disorders. Overall, the present findings suggest that the GWAS-confirmed psychiatric risk gene CACNA1C plays a significant role in oxidative stress as well as neuroinflammatory pathways with particular impact on mitochondrial integrity and function, thereby adding to a better understanding of the intracellular processes likely involved in the pathophysiology of CACNA1C-associated disorders. Thus, modulating L-type calcium signaling may offer an effective therapeutic strategy in psychiatric disorders, where neuronal atrophy and inflammation contribute to disease pathophysiology

Mitochondria, Microglia, and the Immune System—How Are They Linked in Affective Disorders?

Major depressive disorder (MDD) is a severe mood disorder and frequently associated with alterations of the immune system characterized by enhanced levels of circulating pro-inflammatory cytokines and microglia activation in the brain. Increasing evidence suggests that dysfunction of mitochondria may play a key role in the pathogenesis of MDD. Mitochondria are regulators of numerous cellular functions including energy metabolism, maintenance of redox and calcium homeostasis, and cell death and therefore modulate many facets of the innate immune response. In depression-like behavior of rodents, mitochondrial perturbation and release of mitochondrial components have been shown to boost cytokine production and neuroinflammation. On the other hand, pro-inflammatory cytokines may influence mitochondrial functions such as oxidative phosphorylation, production of adenosine triphosphate, and reactive oxygen species, thereby aggravating inflammation. There is strong interest in a better understanding of immunometabolic pathways in MDD that may serve as diagnostic markers and therapeutic targets. Here, we review the interaction between mitochondrial metabolism and innate immunity in the pathophysiology of MDD. We specifically focus on immunometabolic processes that govern microglial and peripheral myeloid cell functions, both cellular components involved in neuroinflammation in depression-like behavior. We finally discuss microglial polarization and associated metabolic states in depression-associated behavior and in MDD

Cytochrome c Oxidase Inhibition by ATP Decreases Mitochondrial ROS Production

This study addresses the eventual consequence of cytochrome c oxidase (CytOx) inhibition by ATP at high ATP/ADP ratio in isolated rat heart mitochondria. Earlier, it has been demonstrated that the mechanism of allosteric ATP inhibition of CytOx is one of the key regulations of mitochondrial functions. It is relevant that aiming to maintain a high ATP/ADP ratio for the measurement of CytOx activity effectuating the enzymatic inhibition as well as mitochondrial respiration, optimal concentration of mitochondria is critically important. Likewise, only at this concentration, were the differences in ΔΨ(m) and ROS concentrations measured under various conditions significant. Moreover, when CytOx activity was inhibited in the presence of ATP, mitochondrial respiration and ΔΨ(m) both remained static, while the ROS production was markedly decreased. Consubstantial results were found when the electron transport chain was inhibited by antimycin A, letting only CytOx remain functional to support the energy production. This seems to corroborate that the decrease in mitochondrial ROS production is solely the effect of ATP binding to CytOx which results in static respiration as well as membrane potential

Interaction of the Psychiatric Risk Gene Cacna1c With Post-weaning Social Isolation or Environmental Enrichment Does Not Affect Brain Mitochondrial Bioenergetics in Rats

The pathophysiology of neuropsychiatric disorders involves complex interactions between genetic and environmental risk factors. Confirmed by several genome-wide association studies, Cacna1c represents one of the most robustly replicated psychiatric risk genes. Besides genetic predispositions, environmental stress such as childhood maltreatment also contributes to enhanced disease vulnerability. Both, Cacna1c gene variants and stressful life events are associated with morphological alterations in the prefrontal cortex and the hippocampus. Emerging evidence suggests impaired mitochondrial bioenergetics as a possible underlying mechanism of these regional brain abnormalities. In the present study, we simulated the interaction of psychiatric disease-relevant genetic and environmental factors in rodents to investigate their potential effect on brain mitochondrial function using a constitutive heterozygous Cacna1c rat model in combination with a four-week exposure to either post-weaning social isolation, standard housing, or social and physical environmental enrichment. Mitochondria were isolated from the prefrontal cortex and the hippocampus to evaluate their bioenergetics, membrane potential, reactive oxygen species production, and respiratory chain complex protein levels. None of these parameters were considerably affected in this particular gene-environment setting. These negative results were very robust in all tested conditions demonstrating that Cacna1c depletion did not significantly translate into altered bioenergetic characteristics. Thus, further investigations are required to determine the disease-related effects on brain mitochondria

Free Fatty Acids in Bone Pathophysiology of Rheumatic Diseases

Obesity—in which free fatty acid (FFA) levels are chronically elevated—is a known risk factor for different rheumatic diseases, and obese patients are more likely to develop osteoarthritis (OA) also in non-weight-bearing joints. These findings suggest that FFA may also play a role in inflammation-related joint damage and bone loss in rheumatoid arthritis (RA) and OA. Therefore, the objective of this study was to analyze if and how FFA influence cells of bone metabolism in rheumatic diseases. When stimulated with FFA, osteoblasts from RA and OA patients secreted higher amounts of the proinflammatory cytokine interleukin (IL)-6 and the chemokines IL-8, growth-related oncogene α, and monocyte chemotactic protein 1. Receptor activator of nuclear factor kappa B ligand (RANKL), osteoprotegerin, and osteoblast differentiation markers were not influenced by FFA. Mineralization activity of osteoblasts correlated inversely with the level of FFA-induced IL-6 secretion. Expression of the Wnt signaling molecules, axin-2 and β-catenin, was not changed by palmitic acid (PA) or linoleic acid (LA), suggesting no involvement of the Wnt signaling pathway in FFA signaling for osteoblasts. On the other hand, Toll-like receptor 4 blockade significantly reduced PA-induced IL-8 secretion by osteoblasts, while blocking Toll-like receptor 2 had no effect. In osteoclasts, IL-8 secretion was enhanced by PA and LA particularly at the earliest time point of differentiation. Differences were observed between the responses of RA and OA osteoclasts. FFA might therefore represent a new molecular factor by which adipose tissue contributes to subchondral bone damage in RA and OA. In this context, their mechanisms of action appear to be dependent on inflammation and innate immune system rather than Wnt-RANKL pathways

Blood Oxygenation Level-Dependent Cerebrovascular Reactivity-Derived Steal Phenomenon May Indicate Tissue Reperfusion Failure After Successful Endovascular Thrombectomy

In acute ischemic stroke due to large-vessel occlusion (LVO), the clinical outcome after endovascular thrombectomy (EVT) is influenced by the extent of autoregulatory hemodynamic impairment, which can be derived from blood oxygenation level-dependent cerebrovascular reactivity (BOLD-CVR). BOLD-CVR imaging identifies brain areas influenced by hemodynamic steal. We sought to investigate the presence of steal phenomenon and its relationship to DWI lesions and clinical deficit in the acute phase of ischemic stroke following successful vessel recanalization.From the prospective longitudinal IMPreST (Interplay of Microcirculation and Plasticity after ischemic Stroke) cohort study, patients with acute ischemic unilateral LVO stroke of the anterior circulation with successful endovascular thrombectomy (EVT; mTICI scale ≥ 2b) and subsequent BOLD-CVR examination were included for this analysis. We analyzed the spatial correlation between brain areas exhibiting BOLD-CVR-associated steal phenomenon and DWI infarct lesion as well as the relationship between steal phenomenon and NIHSS score at hospital discharge.Included patients (n = 21) exhibited steal phenomenon to different extents, whereas there was only a partial spatial overlap with the DWI lesion (median 19%; IQR, 8-59). The volume of steal phenomenon outside the DWI lesion showed a positive correlation with overall DWI lesion volume and was a significant predictor for the NIHSS score at hospital discharge.Patients with acute ischemic unilateral LVO stroke exhibited hemodynamic steal identified by BOLD-CVR after successful EVT. Steal volume was associated with DWI infarct lesion size and with poor clinical outcome at hospital discharge. BOLD-CVR may further aid in better understanding persisting hemodynamic impairment following reperfusion therapy

Outcome Prediction in Patients with Severe COVID-19 Requiring Extracorporeal Membrane Oxygenation—A Retrospective International Multicenter Study

The role of veno-venous extracorporeal membrane oxygenation therapy (V-V ECMO) in severe COVID-19 acute respiratory distress syndrome (ARDS) is still under debate and conclusive data from large cohorts are scarce. Furthermore, criteria for the selection of patients that benefit most from this highly invasive and resource-demanding therapy are yet to be defined. In this study, we assess survival in an international multicenter cohort of COVID-19 patients treated with V-V ECMO and evaluate the performance of several clinical scores to predict 30-day survival. Methods: This is an investigator-initiated retrospective non-interventional international multicenter registry study (NCT04405973, first registered 28 May 2020). In 127 patients treated with V-V ECMO at 15 centers in Germany, Switzerland, Italy, Belgium, and the United States, we calculated the Sequential Organ Failure Assessment (SOFA) Score, Simplified Acute Physiology Score II (SAPS II), Acute Physiology And Chronic Health Evaluation II (APACHE II) Score, Respiratory Extracorporeal Membrane Oxygenation Survival Prediction (RESP) Score, Predicting Death for Severe ARDS on V-V ECMO (PRESERVE) Score, and 30-day survival. Results: In our study cohort which enrolled 127 patients, overall 30-day survival was 54%. Median SOFA, SAPS II, APACHE II, RESP, and PRESERVE were 9, 36, 17, 1, and 4, respectively. The prognostic accuracy for all these scores (area under the receiver operating characteristic—AUROC) ranged between 0.548 and 0.605. Conclusions: The use of scores for the prediction of mortality cannot be recommended for treatment decisions in severe COVID-19 ARDS undergoing V-V ECMO; nevertheless, scoring results below or above a specific cut-off value may be considered as an additional tool in the evaluation of prognosis. Survival rates in this cohort of COVID-19 patients treated with V-V ECMO were slightly lower than those reported in non-COVID-19 ARDS patients treated with V-V ECMO

Framework and baseline examination of the German National Cohort (NAKO)

The German National Cohort (NAKO) is a multidisciplinary, population-based prospective cohort study that aims to investigate the causes of widespread diseases, identify risk factors and improve early detection and prevention of disease. Specifically, NAKO is designed to identify novel and better characterize established risk and protection factors for the development of cardiovascular diseases, cancer, diabetes, neurodegenerative and psychiatric diseases, musculoskeletal diseases, respiratory and infectious diseases in a random sample of the general population. Between 2014 and 2019, a total of 205,415 men and women aged 19–74 years were recruited and examined in 18 study centres in Germany. The baseline assessment included a face-to-face interview, self-administered questionnaires and a wide range of biomedical examinations. Biomaterials were collected from all participants including serum, EDTA plasma, buffy coats, RNA and erythrocytes, urine, saliva, nasal swabs and stool. In 56,971 participants, an intensified examination programme was implemented. Whole-body 3T magnetic resonance imaging was performed in 30,861 participants on dedicated scanners. NAKO collects follow-up information on incident diseases through a combination of active follow-up using self-report via written questionnaires at 2–3 year intervals and passive follow-up via record linkages. All study participants are invited for re-examinations at the study centres in 4–5 year intervals. Thereby, longitudinal information on changes in risk factor profiles and in vascular, cardiac, metabolic, neurocognitive, pulmonary and sensory function is collected. NAKO is a major resource for population-based epidemiology to identify new and tailored strategies for early detection, prediction, prevention and treatment of major diseases for the next 30 years. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10654-022-00890-5