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We show that redox active iron can induce a regulated form of non-apoptotic cell death and tissue damage called ferroptosis that can contribute to secondary damage and functional loss in the acute and chronic periods after spinal cord injury (SCI) in young, adult, female mice. Phagocytosis of red blood cells at sites of hemorrhage is the main source of iron derived from hemoglobin after SCI. Expression of hemeoxygenase-1 that induces release of iron from heme, is increased in spinal cord macrophages 7 days after injury. While iron is stored safely in ferritin in the injured spinal cord, it can, however, be released by NCOA4-mediated shuttling of ferritin to autophagosomes for degradation (ferritinophagy). This leads to the release of redox active iron that can cause free radical damage. Expression of NCOA4 is increased after SCI, mainly in macrophages. Increase in the ratio of redox active ferrous (Fe 2+ ) to ferric iron (Fe 3+ ) is also detected after SCI by capillary electrophoresis inductively coupled mass spectrometry. These changes are accompanied by other hallmarks of ferroptosis, i.e., deficiency in various elements of the antioxidant glutathione (GSH) pathway. We also detect increases in enzymes that repair membrane lipids (ACSL4 and LPCAT3) and thus promote on-going ferroptosis. These changes are associated with increased levels of 4-hydroxynonenal (4-HNE), a toxic lipid peroxidation product. Mice with mild SCI (30 kdyne force) treated with the ferroptosis inhibitor (UAMC-3203-HCL) either early or delayed times after injury showed improvement in locomotor recovery and secondary damage. Cerebrospinal fluid and serum samples from human SCI cases show evidence of increased iron storage (ferritin), and other iron related molecules, and reduction in GSH. Collectively, these data suggest that ferroptosis contributes to secondary damage after SCI and highlights the possible use of ferroptosis inhibitors to treat SCI., (© 2024. The Author(s).)

This study aims to determine the effect of neurogenic, inflammatory, and infective fevers on acutely injured human spinal cord. In 86 patients with acute, severe traumatic spinal cord injuries (TSCIs; American Spinal Injury Association Impairment Scale (AIS), grades A-C) we monitored (starting within 72 h of injury, for up to 1 week) axillary temperature as well as injury site cord pressure, microdialysis (MD), and oxygen. High fever (temperature ≥38°C) was classified as neurogenic, infective, or inflammatory. The effect of these three fever types on injury-site physiology, metabolism, and inflammation was studied by analyzing 2864 h of intraspinal pressure (ISP), 1887 h of MD, and 840 h of tissue oxygen data. High fever occurred in 76.7% of the patients. The data show that temperature was higher in neurogenic than non-neurogenic fever. Neurogenic fever only occurred with injuries rostral to vertebral level T4. Compared with normothermia, fever was associated with reduced tissue glucose (all fevers), increased tissue lactate to pyruvate ratio (all fevers), reduced tissue oxygen (neurogenic + infective fevers), and elevated levels of pro-inflammatory cytokines/chemokines (infective fever). Spinal cord metabolic derangement preceded the onset of infective but not neurogenic or inflammatory fever. By considering five clinical characteristics (level of injury, axillary temperature, leukocyte count, C-reactive protein [CRP], and serum procalcitonin [PCT]), it was possible to confidently distinguish neurogenic from non-neurogenic high fever in 59.3% of cases. We conclude that neurogenic, infective, and inflammatory fevers occur commonly after acute, severe TSCI and are detrimental to the injured spinal cord with infective fever being the most injurious. Further studies are required to determine whether treating fever improves outcome. Accurately diagnosing neurogenic fever, as described, may reduce unnecessary septic screens and overuse of antibiotics in these patients.

Infections are prevalent after spinal cord injury (SCI), constitute the main cause of death and are a rehabilitation confounder associated with impaired recovery. We hypothesize that SCI causes an acquired lesion-dependent (neurogenic) immune suppression as an underlying mechanism to facilitate infections. The international prospective multicentre cohort study (SCIentinel; protocol registration DRKS00000122; n = 111 patients) was designed to distinguish neurogenic from general trauma-related effects on the immune system. Therefore, SCI patient groups differing by neurological level, i.e. high SCI [thoracic (Th)4 or higher]; low SCI (Th5 or lower) and severity (complete SCI; incomplete SCI), were compared with a reference group of vertebral fracture (VF) patients without SCI. The primary outcome was quantitative monocytic Human Leukocyte Antigen-DR expression (mHLA-DR, synonym MHC II), a validated marker for immune suppression in critically ill patients associated with infection susceptibility. mHLA-DR was assessed from Day 1 to 10 weeks after injury by applying standardized flow cytometry procedures. Secondary outcomes were leucocyte subpopulation counts, serum immunoglobulin levels and clinically defined infections. Linear mixed models with multiple imputation were applied to evaluate group differences of logarithmic-transformed parameters. Mean quantitative mHLA-DR [ln (antibodies/cell)] levels at the primary end point 84 h after injury indicated an immune suppressive state below the normative values of 9.62 in all groups, which further differed in its dimension by neurological level: high SCI [8.95 (98.3% confidence interval, CI: 8.63; 9.26), n = 41], low SCI [9.05 (98.3% CI: 8.73; 9.36), n = 29], and VF without SCI [9.25 (98.3% CI: 8.97; 9.53), n = 41, P = 0.003]. Post hoc analysis accounting for SCI severity revealed the strongest mHLA-DR decrease [8.79 (95% CI: 8.50; 9.08)] in the complete, high SCI group, further demonstrating delayed mHLA-DR recovery [9.08 (95% CI: 8.82; 9.38)] and showing a difference from the VF controls of -0.43 (95% CI: -0.66; -0.20) at 14 days. Complete, high SCI patients also revealed constantly lower serum immunoglobulin G [-0.27 (95% CI: -0.45; -0.10)] and immunoglobulin A [-0.25 (95% CI: -0.49; -0.01)] levels [ln (g/l × 1000)] up to 10 weeks after injury. Low mHLA-DR levels in the range of borderline immunoparalysis (below 9.21) were positively associated with the occurrence and earlier onset of infections, which is consistent with results from studies on stroke or major surgery. Spinal cord injured patients can acquire a secondary, neurogenic immune deficiency syndrome characterized by reduced mHLA-DR expression and relative hypogammaglobulinaemia (combined cellular and humoral immune deficiency). mHLA-DR expression provides a basis to stratify infection-risk in patients with SCI., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Background and Objectives: Spinal cord injury (SCI) disrupts the fine-balanced interaction between the CNS and immune system and can cause maladaptive aberrant immune responses. The study examines emerging autoantibody synthesis after SCI with binding to conformational spinal cord epitopes and surface peptides located on the intact neuronal membrane., Methods: This is a prospective longitudinal cohort study conducted in acute care and inpatient rehabilitation centers in conjunction with a neuropathologic case-control study in archival tissue samples ranging from acute injury (baseline) to several months thereafter (follow-up). In the cohort study, serum autoantibody binding was examined in a blinded manner using tissue-based assays (TBAs) and dorsal root ganglia (DRG) neuronal cultures. Groups with traumatic motor complete SCI vs motor incomplete SCI vs isolated vertebral fracture without SCI (controls) were compared. In the neuropathologic study, B cell infiltration and antibody synthesis at the spinal lesion site were examined by comparing SCI with neuropathologically unaltered cord tissue. In addition, the CSF in an individual patient was explored., Results: Emerging autoantibody binding in both TBA and DRG assessments was restricted to an SCI patient subpopulation only (16%, 9/55 sera) while being absent in vertebral fracture controls (0%, 0/19 sera). Autoantibody binding to the spinal cord characteristically detected the substantia gelatinosa, a less-myelinated region of high synaptic density involved in sensory-motor integration and pain processing. Autoantibody binding was most frequent after motor complete SCI (grade American Spinal Injury Association impairment scale A/B, 22%, 8/37 sera) and was associated with neuropathic pain medication. In conjunction, the neuropathologic study demonstrated lesional spinal infiltration of B cells (CD20, CD79a) in 27% (6/22) of patients with SCI, the presence of plasma cells (CD138) in 9% (2/22). IgG and IgM antibody syntheses colocalized to areas of activated complement (C9neo) deposition. Longitudinal CSF analysis of an additional single patient demonstrated de novo (IgM) intrathecal antibody synthesis emerging with late reopening of the blood-spinal cord barrier., Discussion: This study provides immunologic, neurobiological, and neuropathologic proof-of-principle for an antibody-mediated autoimmunity response emerging approximately 3 weeks after SCI in a patient subpopulation with a high demand of neuropathic pain medication. Emerging autoimmunity directed against specific spinal cord and neuronal epitopes suggests the existence of paratraumatic CNS autoimmune syndromes., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Introduction: This study on pyogenic spinal infections with intraspinal epidural involvement (PSI +) compared the outcome of patients with spinal cord injury (SCI) to those without (noSCI) taking diagnostic algorithm, therapy, and complications into account., Methods: Patients were enrolled in an ambispective study (2012-2017). Diagnostic and therapeutic algorithms, complications, and neurological outcome were analyzed descriptively. Survival was analyzed applying Kaplan-Meier method and Cox regression., Results: In total, 134 patients with a median (IQR) age of 72 (61-79) years were analyzed. Baseline characteristics were similar between the SCI (n = 55) and noSCI (n = 79). A higher percentage of endocarditis (9% vs. 0%; p = 0.03) was detected in the noSCI group. The majority (81%) received combinatorial therapy including spinal surgery and antibiotic treatment. The surgery complication rate was 16%. At discharge, improvement in neurologic function was present in 27% of the SCI patients. Length of stay, duration of ventilation and the burden of disease-associated complications were significantly higher in the SCI group (e.g., urinary tract infection, pressure ulcers). Lethality risk factors were age (HR 1.09, 95% CI 1.02-1.16, p = 0.014), and empyema/abscess extension (≥ 3 infected spinal segments, HR 4.72, 95% CI 1.57-14.20, p = 0.006), dominating over additional effects of Charlson comorbidity index, SCI, and type of treatment. The overall lethality rate was 11%., Conclusion: PSI + are associated with higher in-hospital mortality, particularly when multiple spinal segments are involved. However, survival is similar with (SCI) or without myelopathy (noSCI). If SCI develops, the rate of disease complications is higher and early specialized SCI care might be substantial to reduce complication rates., (© 2022. The Author(s).)

Comorbidity scores are important predictors of in-hospital mortality after traumatic spinal cord injury (tSCI), but the impact of specific pre-existing diseases is unknown. This retrospective cohort study aims at identifying relevant comorbidities and explores the influence of end-of-life decisions. In-hospital mortality of all patients admitted to the study center after acute tSCI from 2011 to 2017 was assessed. A conditional inference tree analysis including baseline data, injury characteristics, and Charlson Comorbidity Index items was used to identify crucial predictors. End-of-life decisions were recorded. Three-hundred-twenty-one patients were consecutively enrolled. The median length of stay was 95.7 days (IQR 56.8-156.0). During inpatient care, 20 patients (6.2%) died. These patients were older (median: 79.0 (IQR 74.7-83.2) vs. 55.5 (IQR 41.4-72.3) years) and had a higher Charlson Comorbidity Index score (median: 4.0 (IQR 1.75-5.50) vs. 0.0 (IQR 0.00-1.00)) compared to survivors. Pre-existing kidney or liver disease were identified as relevant predictors of in-hospital mortality. End-of-life decisions were observed in 14 (70.0%) cases. The identified impairment of kidney and liver, important for drug metabolism and elimination, points to the need of careful decisions on pharmaceutical treatment regimens after tSCI. Appropriate reporting of end-of-life decisions is required for upcoming studies., (© 2022. The Author(s).)

Purpose: To investigate the association of age with delay in spine surgery and the effects on neurological outcome after traumatic spinal cord injury (SCI)., Methods: Ambispective cohort study (2011-2017) in n = 213 patients consecutively enrolled in a Level I trauma center with SCI care in a metropolitan region in Germany. Age-related differences in the injury to surgery interval and conditions associated with its delay (> 12 h after SCI) were explored using age categories or continuous variables and natural cubic splines. Effects of delayed surgery or age with outcome were analyzed using multiple logistic regression., Results: The median age of the study population was 58.8 years (42.0-74.6 IQR). Older age (≥ 75y) was associated with a prolonged injury to surgery interval of 22.8 h (7.2-121.3) compared to 6.6 h (4.4-47.9) in younger patients (≤ 44y). Main reasons for delayed surgery in older individuals were secondary referrals and multimorbidity. Shorter time span to surgery (≤ 12 h) was associated with higher rates of ASIA impairment scale (AIS) conversion (OR 4.22, 95%CI 1.85-9.65), as mirrored by adjusted spline curves (< 20 h 20-25%, 20-60 h 10-20%, > 60 h < 10% probability of AIS conversion). In incomplete SCI, the probability of AIS conversion was lower in older patients [e.g., OR 0.09 (0.02-0.44) for'45-59y' vs.' ≤ 44y'], as confirmed by spline curves (< 40y 20-80%, ≥ 40y 5-20% probability)., Conclusion: Older patient age complexifies surgical SCI care and research. Tackling secondary referral to Level I trauma centers and delayed spine surgery imposes as tangible opportunity to improve the outcome of older SCI patients., (© 2021. The Author(s).)

In five patients with acute, severe thoracic traumatic spinal cord injuries (TSCIs), American spinal injuries association Impairment Scale (AIS) grades A-C, we induced cord hypothermia (33 °C) then rewarming (37 °C). A pressure probe and a microdialysis catheter were placed intradurally at the injury site to monitor intraspinal pressure (ISP), spinal cord perfusion pressure (SCPP), tissue metabolism and inflammation. Cord hypothermia-rewarming, applied to awake patients, did not cause discomfort or neurological deterioration. Cooling did not affect cord physiology (ISP, SCPP), but markedly altered cord metabolism (increased glucose, lactate, lactate/pyruvate ratio (LPR), glutamate; decreased glycerol) and markedly reduced cord inflammation (reduced IL1β, IL8, MCP, MIP1α, MIP1β). Compared with pre-cooling baseline, rewarming was associated with significantly worse cord physiology (increased ICP, decreased SCPP), cord metabolism (increased lactate, LPR; decreased glucose, glycerol) and cord inflammation (increased IL1β, IL8, IL4, IL10, MCP, MIP1α). The study was terminated because three patients developed delayed wound infections. At 18-months, two patients improved and three stayed the same. We conclude that, after TSCI, hypothermia is potentially beneficial by reducing cord inflammation, though after rewarming these benefits are lost due to increases in cord swelling, ischemia and inflammation. We thus urge caution when using hypothermia-rewarming therapeutically in TSCI.

Acute spinal cord injury (SCI) causes systemic immunosuppression and life-threatening infections, thought to result from noradrenergic overactivation and excess glucocorticoid release via hypothalamus-pituitary-adrenal axis stimulation. Instead of consecutive hypothalamus-pituitary-adrenal axis activation, we report that acute SCI in mice induced suppression of serum norepinephrine and concomitant increase in cortisol, despite suppressed adrenocorticotropic hormone, indicating primary (adrenal) hypercortisolism. This neurogenic effect was more pronounced after high-thoracic level (Th1) SCI disconnecting adrenal gland innervation, compared with low-thoracic level (Th9) SCI. Prophylactic adrenalectomy completely prevented SCI-induced glucocorticoid excess and lymphocyte depletion but did not prevent pneumonia. When adrenalectomized mice were transplanted with denervated adrenal glands to restore physiologic glucocorticoid levels, the animals were completely protected from pneumonia. These findings identify a maladaptive sympathetic-neuroendocrine adrenal reflex mediating immunosuppression after SCI, implying that therapeutic normalization of the glucocorticoid and catecholamine imbalance in SCI patients could be a strategy to prevent detrimental infections.

Unbiased chemoproteomic profiling of small-molecule interactions with endogenous proteins is important for drug discovery. For meaningful results, all protein classes have to be tractable, including G protein-coupled receptors (GPCRs). These receptors are hardly tractable by affinity pulldown from lysates. We report a capture compound (CC)-based strategy to target and identify GPCRs directly from living cells. We synthesized CCs with sertindole attached to the CC scaffold in different orientations to target the dopamine D2 receptor (DRD2) heterologously expressed in HEK 293 cells. The structure-activity relationship of sertindole for DRD2 binding was reflected in the activities of the sertindole CCs in radioligand displacement, cell-based assays, and capture compound mass spectrometry (CCMS). The activity pattern was rationalized by molecular modelling. The most-active CC showed activities very similar to that of unmodified sertindole. A concentration of DRD2 in living cells well below 100 fmol used as an experimental input was sufficient for unambiguous identification of captured DRD2 by mass spectrometry. Our new CCMS workflow broadens the arsenal of chemoproteomic technologies to close a critical gap for the comprehensive characterization of drug-protein interactions., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Histone deacetylase (HDAC) inhibitors such as suberoylanilide hydroxamic acid (SAHA) are not commonly used in clinical practice for treatment of B-cell lymphomas, although a subset of patients with refractory or relapsed B-cell lymphoma achieved partial or complete remissions. Therefore, the purpose of this study was to identify molecular features that predict the response of B-cell lymphomas to SAHA treatment. We designed an integrative approach combining drug efficacy testing with exome and captured target analysis (DETECT). In this study, we tested SAHA sensitivity in 26 B-cell lymphoma cell lines and determined SAHA-interacting proteins in SAHA resistant and sensitive cell lines employing a SAHA capture compound (CC) and mass spectrometry (CCMS). In addition, we performed exome mutation analysis. Candidate validation was done by expression analysis and knock-out experiments. An integrated network analysis revealed that the Src tyrosine kinase Gardner-Rasheed feline sarcoma viral (v-fgr) oncogene homolog (FGR) is associated with SAHA resistance. FGR was specifically captured by the SAHA-CC in resistant cells. In line with this observation, we found that FGR expression was significantly higher in SAHA resistant cell lines. As functional proof, CRISPR/Cas9 mediated FGR knock-out in resistant cells increased SAHA sensitivity. In silico analysis of B-cell lymphoma samples (n = 1200) showed a wide range of FGR expression indicating that FGR expression might help to stratify patients, which clinically benefit from SAHA therapy. In conclusion, our comprehensive analysis of SAHA-interacting proteins highlights FGR as a factor involved in SAHA resistance in B-cell lymphoma., (Copyright © 2016 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Introduction: The approved analgesic and anti-inflammatory drugs ibuprofen and indometacin block the small GTPase RhoA, a key enzyme that impedes axonal sprouting after axonal damage. Inhibition of the Rho pathway in a central nervous system-effective manner requires higher dosages compared with orthodox cyclooxygenase-blocking effects. Preclinical studies on spinal cord injury (SCI) imply improved motor recovery after ibuprofen/indometacin-mediated Rho inhibition. This has been reassessed by a meta-analysis of the underlying experimental evidence, which indicates an overall effect size of 20.2% regarding motor outcome achieved after ibuprofen/indometacin treatment compared with vehicle controls. In addition, ibuprofen/indometacin may also limit sickness behaviour, non-neurogenic systemic inflammatory response syndrome (SIRS), neuropathic pain and heterotopic ossifications after SCI. Consequently, 'small molecule'-mediated Rho inhibition after acute SCI warrants clinical investigation., Methods and Analysis: Protocol of an investigator-initiated clinical open-label pilot trial on high-dose ibuprofen treatment after acute traumatic, motor-complete SCI. A sample of n=12 patients will be enrolled in two cohorts treated with 2400 mg/day ibuprofen for 4 or 12 weeks, respectively. The primary safety end point is an occurrence of serious adverse events, primarily gastroduodenal bleedings. Secondary end points are pharmacokinetics, feasibility and preliminary effects on neurological recovery, neuropathic pain and heterotopic ossifications. The primary safety analysis is based on the incidence of severe gastrointestinal bleedings. Additional analyses will be mainly descriptive and casuistic., Ethics and Dissemination: The clinical trial protocol was approved by the responsible German state Ethics Board, and the Federal Institute for Drugs and Medical Devices. The study complies with the Declaration of Helsinki, the principles of Good Clinical Practice and all further applicable regulations. This safety and pharmacokinetics trial informs the planning of a subsequent randomised controlled trial. Regardless of the result of the primary and secondary outcome assessments, the clinical trial will be reported as a publication in a peer-reviewed journal., Trial Registration Number: NCT02096913; Pre-results., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

The Goα splice variants Go1α and Go2α are subunits of the most abundant G-proteins in brain, Go1 and Go2. Only a few interacting partners binding to Go1α have been described so far and splice variant-specific differences are not known. Using a yeast two-hybrid screen with constitutively active Go2α as bait, we identified Rap1GTPase activating protein (Rap1GAP) and Girdin as interacting partners of Go2α, which was confirmed by co-immunoprecipitation. Comparison of subcellular fractions from brains of wild type and Go2α-/- mice revealed no differences in the overall expression level of Girdin or Rap1GAP. However, we found higher amounts of active Rap1-GTP in brains of Go2α deficient mutants, indicating that Go2α may increase Rap1GAP activity, thereby effecting the Rap1 activation/deactivation cycle. Rap1 has been shown to be involved in neurite outgrowth and given a Rap1GAP-Go2α interaction, we found that the loss of Go2α affected axonal outgrowth. Axons of cultured cortical and hippocampal neurons prepared from embryonic Go2α-/- mice grew longer and developed more branches than those from wild-type mice. Taken together, we provide evidence that Go2α regulates axonal outgrowth and branching., (© 2012 International Society for Neurochemistry.)

The α-subunit of Go2 is a regulator of dopamine (DA) homeostasis. Deletion of the protein results in an imbalance of the direct and indirect DA pathway by reducing D1 and increasing D2 receptors. As a result, cocaine-induced behavioral sensitization is abolished. Here we show that repeated amphetamine injections in Go2α-/- mice induced a similar D1/D2 receptor ratio shift as cocaine but surprisingly the knockouts developed normal behavioral sensitization. DA receptor signaling following either cocaine or amphetamine treatment was also similar in Go2α-/- mice suggesting another mechanism involved in the differential behavioral response. Evidence is increasing that DA-glutamate interactions in the striatum determine psychostimulant action. In this line, repeated amphetamine injections led to a twofold increase in the amount of the NMDA receptor subunit NR2B in Go2α-/- mice resulting in an enhanced inhibition of the indirect DA pathway. This effect is not seen after cocaine treatment. Furthermore, amphetamine but not cocaine treatment maintained the ratio between the glutamate receptor mGluR1/5 interacting proteins Homer and Homer1a in the knockouts thereby sustaining the direct pathway. We conclude that amphetamine provokes behavioral sensitization in Go2α-/- mice by an enhanced inhibition of the indirect pathway without disturbing the direct pathway thereby overcoming the imbalance in the DArgic system., (© 2010 The Authors. Journal Compilation © 2010 International Society for Neurochemistry.)

The profiling of subproteomes from complex mixtures on the basis of small molecule interactions shared by members of protein families or small molecule interaction domains present in a subset of proteins is an increasingly important approach in functional proteomics. Capture Compound Mass Spectrometry (CCMS) is a novel technology to address this issue. CCs are trifunctional molecules that accomplish the reversible binding of target protein families to a selectivity group (small molecule), covalent capturing of the bound proteins by photoactivated cross-linking through a reactivity group, and pullout of the small molecule-protein complexes through a sorting function, e.g. biotin. Here we present the design, synthesis, and application of a new Capture Compound to target and identify cAMP-binding proteins in complex protein mixtures. Starting with modest amounts of total protein mixture (65-500 microg), we demonstrate that the cAMP-CCs can be used to isolate bona fide cAMP-binding proteins from lysates of Escherichia coli, mammalian HepG2 cells, and subcellular fractions of mammalian brain, respectively. The identified proteins captured by the cAMP-CCs range from soluble cAMP-binding proteins, such as the catabolite gene activator protein from E. coli and regulatory subunits of protein kinase A from mammalian systems, to cAMP-activated potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channels from neuronal membranes and specifically synaptosomal fractions from rat brain. The latter group of proteins has never been identified before in any small molecule protein interaction and mass spectrometry-based proteomics study. Given the modest amount of protein input required, we expect that CCMS using the cAMP-CCs provides a unique tool for profiling cAMP-binding proteins from proteome samples of limited abundance, such as tissue biopsies.

Ca(2+)-dependent activator proteins of secretion (CAPS) 1 and 2 are essential regulators of synaptic vesicle and large dense core vesicle priming in mammalian neurons and neuroendocrine cells. CAPS1 appears to have an additional and as yet unexplained function in vesicular catecholamine uptake or storage as CAPS1-deficient chromaffin cells exhibit strongly reduced vesicular catecholamine levels. Here we describe a role of CAPS proteins in vesicular monoamine uptake. Both CAPS1 and CAPS2 promote monoamine uptake and storage mediated by the vesicular monoamine transporters VMAT1 and VMAT2. Monoamine uptake of vesicular preparations from embryonic brains of CAPS1 deletion mutants is decreased as compared with corresponding preparations from wild type littermates, and anti-CAPS1 or anti-CAPS2 antibodies inhibit monoamine sequestration by synaptic vesicles from adult mouse brain. In addition, overexpression of CAPS1 or CAPS2 enhances vesicular monoamine uptake in Chinese hamster ovary cells that stably express VMAT1 or VMAT2. CAPS function has been linked to the heterotrimeric GTPase G(o), which modulates vesicular monoamine uptake. We found that the expression of CAPS1 is decreased in brain membrane preparations from mice lacking G(o2)alpha, which may explain the reduced monoamine uptake by G(o2)alpha-deficient synaptic vesicles. Accordingly, anti-CAPS1 antibodies do not further reduce monoamine uptake by G(o2)alpha-deficient synaptic vesicles, whereas antibodies directed against CAPS2, whose expression is not altered in G(o2)alpha-deficient brain, still reduce monoamine uptake into G(o2)alpha-deficient vesicles. We conclude that CAPS proteins are involved in optimizing vesicular monoamine uptake and storage mediated by VMAT1 and VMAT2.

The alpha-subunits of the trimeric Go class of GTPases, comprising the splice variants Go1alpha and Go2alpha, are abundantly expressed in brain and reside on both plasma membrane and synaptic vesicles. Go2alpha is involved in the vesicular storage of monoamines but its physiological relevance is still obscure. We now show that genetic depletion of Go2alpha reduces motor activity induced by dopamine-enhancing drugs like cocaine, as repeated injections of cocaine fail to provoke behavioral sensitization in Go2alpha(-/-) mice. In Go2alpha(-/-) mice, D1 receptor signaling in the striatum is attenuated due to a reduced expression of Golf alpha and Gs alpha. Following cocaine treatment, Go2alpha(-/-) mice have lower D1 and higher D2 receptor amounts compared to wild-type mice. The lack of behavioral sensitization correlates with reduced dopamine levels in the striatum and decreased expression of tyrosine hydroxylase. One reason for the neurochemical changes may be a reduced uptake of monoamines by synaptic vesicles from Go2alpha(-/-) mice as a consequence of a lowered set point for filling. We conclude that Go2alpha optimizes vesicular filling which is instrumental for normal dopamine functioning and for the development of drug-induced behavioral sensitization.

The circadian clock has been implicated in addiction and several forms of depression [1, 2], indicating interactions between the circadian and the reward systems in the brain [3-5]. Rewards such as food, sex, and drugs influence this system in part by modulating dopamine neurotransmission in the mesolimbic dopamine reward circuit, including the ventral tegmental area (VTA) and the ventral striatum (NAc). Hence, changes in dopamine levels in these brain areas are proposed to influence mood in humans and mice [6-10]. To establish a molecular link between the circadian-clock mechanism and dopamine metabolism, we analyzed the murine promoters of genes encoding key enzymes important in dopamine metabolism. We find that transcription of the monoamine oxidase A (Maoa) promoter is regulated by the clock components BMAL1, NPAS2, and PER2. A mutation in the clock gene Per2 in mice leads to reduced expression and activity of MAOA in the mesolimbic dopaminergic system. Furthermore, we observe increased levels of dopamine and altered neuronal activity in the striatum, and these results probably lead to behavioral alterations observed in Per2 mutant mice in despair-based tests. These findings suggest a role of circadian-clock components in dopamine metabolism highlighting a role of the clock in regulating mood-related behaviors.

Dephosphorylation of NFAT by the Ca(2+)-calmodulin-dependent Ser/Thr protein phosphatase calcineurin is a bottleneck of T cell receptor-dependent activation of T cells. In dimeric complexes with immunophilins, the immunosuppressants cyclosporine A (CsA) and tacrolimus (FK506) block this process by inhibition of the enzymatic activity of calcineurin. We have identified the pyrazolopyrimidine compound NCI3 as a novel inhibitor of calcineurin-NFAT signaling. Similar to CsA and FK506, NCI3 inhibits dephosphorylation and nuclear translocation of NFAT, IL-2 production and proliferation of stimulated human primary T cells with IC(50) values from 2 to 4.5 microM. However, contrary to CsA and FK506, NCI3 neither blocks calcineurin;s phosphatase activity nor requires immunophilins for inhibiting NFAT activation. Our data suggest that NCI3 binds to calcineurin and causes an allosteric change interfering with NFAT dephosphorylation in vivo but not in vitro. NCI3 acts not only on the endogenous calcineurin but also on a C-terminally truncated, constitutively active version of calcineurin. The novel inhibitor described herein will be useful in better defining the cellular regulation of calcineurin activation and may serve as a lead for the development of a new type of immunosuppressants acting not by direct inhibition of the calcineurin phosphatase activity.

The activity of vesicular monoamine transporters (VMATs) is down-regulated by the G-protein alpha-subunits of G(o2) and G(q), but the signaling pathways are not known. We show here that no such regulation is observed when VMAT1 or VMAT2 are expressed in Chinese hamster ovary (CHO) cells. However, when the intracellular compartments of VMAT-expressing CHO cells are preloaded with different monoamines, transport becomes susceptible to G-protein-dependent regulation, with differences between the two transporter isoforms. Epinephrine induces G-protein-mediated inhibition of transmitter uptake in CHOVMAT1 cells but prevents inhibition induced by dopamine in CHOVMAT2 cells. Epinephrine also antagonizes G-protein-mediated inhibition of monoamine uptake by VMAT2 expressing platelets or synaptic vesicles. In CHOVMAT2 cells G-protein-mediated inhibition of monoamine uptake can be induced by 5-hydroxytryptamine (serotonin) 1B receptor agonists, whereas alpha1 receptor agonists modulate uptake into CHOVMAT1 cells. Accordingly, 5-hydroxytryptamine 1B receptor antagonists prevent G-protein-mediated inhibition of uptake in partially filled platelets and synaptic vesicles expressing VMAT2. CHO cells expressing VMAT mutants with a shortened first vesicular loop transport monoamines. However, no or a reduced G-protein regulation of uptake can be initiated. In conclusion, vesicular content is involved in the activation of vesicle associated G-proteins via a structure sensing the luminal monoamine content. The first luminal loop of VMATs may represent a G-protein-coupled receptor that adapts vesicular filling.