CB1 receptor expression and signaling are required for dexamethasone-induced aversive memory consolidation.

The molecular processes that underlie long-term memory formation involve signaling pathway activation by neurotransmitter release, which induces the expression of immediate early genes, such as Zif268, having a key role in memory formation. In this work, we show that the cannabinoid CB1 receptor signaling is necessary for the effects of dexamethasone on the behavioral response in an inhibitory avoidance task, on dexamethasone-induced ERK phosphorylation, and on dexamethasone-dependent Zif268 expression. Furthermore, we provide primary evidence for the mechanism responsible for this crosstalk between cannabinoid and glucocorticoid-mediated signaling pathways, showing that dexamethasone regulates endocannabinoid metabolism by inhibiting the activity of the Fatty acid amide hydrolase (FAAH), an integral membrane enzyme that hydrolyzes endocannabinoids and related amidated signaling lipids. Our results provide novel evidence regarding the role of the endocannabinoid system, and in particular of the CB1 receptor, as a mediator of the effects of glucocorticoids on the consolidation of aversive memories.

Diazepam-Induced Down-Regulation of the GABAA receptor α1 Subunit, as mediated by the activation of L-Type Voltage-Gated calcium Channel/Ca2+/Protein kinase a signaling cascade.

Benzodiazepines are among the most prescribed drug class worldwide to treat disorders such as anxiety, insomnia, muscle spasticity, and convulsive disorders, and to induce presurgical sedation. Although benzodiazepines exhibit a high therapeutic index and low toxicity in short-term treatments, prolonged administration induces tolerance to most of their therapeutic actions. The mechanism of this tolerance remains unclear. The central actions of benzodiazepines are mediated by binding to GABAA receptors, which mediate most fast inhibitory transmission in the brain. The majority of GABAA receptors are composed of two α-(1-6), two ß-(1-3) and one γ-subunits (1-3). In a previous report, we demonstrated that the prolonged exposure of cerebrocortical neurons to diazepam produces a transcriptional repression of the GABAA receptor α1 subunit gene via a mechanism dependent on the activation of L-type voltage-gated calcium channels (L-VGCCs). The results reported here confirm that the diazepam-induced downregulation of the α1 subunit is contingent upon calcium influx from extracellular space. In addition, this regulatory mechanism involves the activation of protein kinase A (PKA) and is accompanied by the activation of two transcription factors, the cAMP-response element-binding protein (CREB) and the inducible cAMP early repressor (ICER). Together, our results suggest that diazepam s activation of an L-VGCC/Ca2+/PKA/CREB-ICER signaling pathway is responsible for the regulation of GABAA receptors. This elucidation of the intracellular signaling cascade activated by a prolonged benzodiazepine exposure, itself potentially involved in the development of tolerance, may contribute to locating molecular targets for future therapeutic interventions.

Fyn knockdown prevents levodopa-induced dyskinesia in a mouse model of Parkinson's disease.

Dopamine replacement by levodopa is the most widely used therapy for Parkinson's disease (PD), however patients often develop side effects, known as levodopa-induced dyskinesia (LID), that usually need therapeutic intervention. There are no suitable therapeutic options for LID, except for the use of the NMDA receptor antagonist amantadine, which has limited efficacy. The NMDA receptor is indeed the most plausible target to manage LID in PD and recently the kinase Fyn- one of its key regulators- became a new putative molecular target involved in LID. The aim of this work was to reduce Fyn expression to alleviate LID in a mouse model of PD. We performed intra-striatal delivery of a designed micro-RNA against Fyn (miRNA-Fyn) in 6-OHDA-lesioned mice treated with levodopa. The miRNA-Fyn was delivered either before or after levodopa exposure to assess its ability to prevent or revert dyskinesia. Pre-administration of miRNA-Fyn reduced LID with a concomitant reduction of FosB-ΔFosB protein levels -a marker of LID- as well as decreased phosphorylation of the NR2B-NMDA subunit, which is a main target of Fyn. On the other hand, post L-DOPA delivery of miRNA-Fyn was less effective to revert already established dyskinesia, suggesting that early blocking of Fyn activity might be a more efficient therapeutic approach. Together, our results provide proof of concept about Fyn as a plausible therapeutic target to manage LID, and validate RNA silencing as a potential approach to locally reduce striatal Fyn, rising new perspectives for RNA therapy interventions in PD.Significance StatementLevodopa induced dyskinesia (LID) is an incapacitant side effect of treatment in Parkinson's disease (PD). LID is a therapeutic challenge, lacking an effective pharmacological treatment, except for the use of inhibitors of the NMDA receptor, which have limited efficacy and may trigger untoward side effects. The kinase Fyn is a key regulator of NMDA function and a potential therapeutic target to control LID. Here, we show that RNA interference therapy to reduce the amount of Fyn mRNA in the adult brain is effective to prevent LID in a mouse model of PD, setting the grounds for future biomedical interventions to manage LID in PD.

α-Synuclein Oligomers Induce Glutamate Release from Astrocytes and Excessive Extrasynaptic NMDAR Activity in Neurons, Thus Contributing to Synapse Loss.

Synaptic and neuronal loss are major neuropathological characteristics of Parkinson's disease. Misfolded protein aggregates in the form of Lewy bodies, comprised mainly of α-synuclein (αSyn), are associated with disease progression, and have also been linked to other neurodegenerative diseases, including Lewy body dementia, Alzheimer's disease, and frontotemporal dementia. However, the effects of αSyn and its mechanism of synaptic damage remain incompletely understood. Here, we show that αSyn oligomers induce Ca2+-dependent release of glutamate from astrocytes obtained from male and female mice, and that mice overexpressing αSyn manifest increased tonic release of glutamate in vivo In turn, this extracellular glutamate activates glutamate receptors, including extrasynaptic NMDARs (eNMDARs), on neurons both in culture and in hippocampal slices of αSyn-overexpressing mice. Additionally, in patch-clamp recording from outside-out patches, we found that oligomerized αSyn can directly activate eNMDARs. In organotypic slices, oligomeric αSyn induces eNMDAR-mediated synaptic loss, which can be reversed by the drug NitroSynapsin. When we expose human induced pluripotent stem cell-derived cerebrocortical neurons to αSyn, we find similar effects. Importantly, the improved NMDAR antagonist NitroSynapsin, which selectively inhibits extrasynaptic over physiological synaptic NMDAR activity, protects synapses from oligomeric αSyn-induced damage in our model systems, thus meriting further study for its therapeutic potential.SIGNIFICANCE STATEMENT Loss of synaptic function and ensuing neuronal loss are associated with disease progression in Parkinson's disease (PD), Lewy body dementia (LBD), and other neurodegenerative diseases. However, the mechanism of synaptic damage remains incompletely understood. α-Synuclein (αSyn) misfolds in PD/LBD, forming Lewy bodies and contributing to disease pathogenesis. Here, we found that misfolded/oligomeric αSyn releases excessive astrocytic glutamate, in turn activating neuronal extrasynaptic NMDA receptors (eNMDARs), thereby contributing to synaptic damage. Additionally, αSyn oligomers directly activate eNMDARs, further contributing to damage. While the FDA-approved drug memantine has been reported to offer some benefit in PD/LBD (Hershey and Coleman-Jackson, 2019), we find that the improved eNMDAR antagonist NitroSynapsin ameliorates αSyn-induced synaptic spine loss, providing potential disease-modifying intervention in PD/LBD.

Differential effects of HDAC inhibitors on PPN oscillatory activity in vivo.

The pedunculopontine nucleus (PPN) has long been known to be part of the reticular activating system (RAS) in charge of arousal and REM sleep. We previously showed that in vitro exposure to a HDAC Class I and II mixed inhibitor (TSA), or a specific HDAC class IIa inhibitor (MC 1568), decreased PPN gamma oscillations. Given the lack of information on systemic in vivo treatments on neuronal synaptic properties, the present study was designed to investigate the systemic effect of HDAC inhibitors (HDACi) on PPN rhythmicity. Rat pups were injected (acute, single dose) with TSA (4 or 20 mg/kg), MC1568 (4 or 20 mg/kg), or MS275 (20 or 100 mg/kg). Our results show that MC1568 (20 mg/kg) reduced mean frequency of PPN oscillations at gamma band, while increasing mean input resistance (Rm) of PPN neurons. For TSA (4 and 20 mg/kg), we observed reduced mean frequency of oscillations at gamma band and increased mean Rm of PPN neurons. Systemic administration of 20 mg/kg MC1568 and TSA effects on Rm were washed out after 60 min of in vitro incubation of PPN slices, suggesting an underlying functional recovery of PPN calcium-mediated gamma band oscillations over time. In addition, at a lower dose, 4 mg/kg, MC1568 and TSA induced higher mean amplitude gamma oscillations. Blocking HDAC class I might not have deleterious effects on gamma activity, but, more importantly, the inhibition of HDAC class I (at 100 mg/kg) increased gamma band oscillations. In conclusion, the present results in vivo validate our previous findings in vitro and further expand information on the effects of HDAC inhibition on PPN rhythmicity. PPN neurons require normal activity of HDAC class IIa in order to oscillate at gamma band.

The Kinase Fyn As a Novel Intermediate in L-DOPA-Induced Dyskinesia in Parkinson's Disease.

Dopamine replacement therapy with L-DOPA is the treatment of choice for Parkinson's disease; however, its long-term use is frequently associated with L-DOPA-induced dyskinesia (LID). Many molecules have been implicated in the development of LID, and several of these have been proposed as potential therapeutic targets. However, to date, none of these molecules have demonstrated full clinical efficacy, either because they lie downstream of dopaminergic signaling, or due to adverse side effects. Therefore, discovering new strategies to reduce LID in Parkinson's disease remains a major challenge. Here, we have explored the tyrosine kinase Fyn, as a novel intermediate molecule in the development of LID. Fyn, a member of the Src kinase family, is located in the postsynaptic density, where it regulates phosphorylation of the NR2B subunit of the N-methyl-D-aspartate (NMDA) receptor in response to dopamine D1 receptor stimulation. We have used Fyn knockout and wild-type mice, lesioned with 6-hydroxydopamine and chronically treated with L-DOPA, to investigate the role of Fyn in the induction of LID. We found that mice lacking Fyn displayed reduced LID, ΔFosB accumulation and NR2B phosphorylation compared to wild-type control mice. Pre-administration of saracatinib (AZD0530), an inhibitor of Fyn activity, also significantly reduced LID in dyskinetic wild-type mice. These results support that Fyn has a critical role in the molecular pathways affected during the development of LID and identify Fyn as a novel potential therapeutic target for the management of dyskinesia in Parkinson's disease.

Is it All Said for NSAIDs in Alzheimer's Disease? Role of Mitochondrial Calcium Uptake.

OBJECTIVES: Epidemiological data suggest that non-steroidal anti-inflammatory drugs (NSAIDs) may protect against Alzheimer's disease (AD). Unfortunately, recent trials have failed in providing compelling evidence of neuroprotection. Discussion as to why NSAIDs effectivity is uncertain is ongoing. Possible explanations include the view that NSAIDs and other possible disease-modifying drugs should be provided before the patients develop symptoms of AD or cognitive decline. In addition, NSAID targets for neuroprotection are unclear. Both COX-dependent and independent mechanisms have been proposed, including γ-secretase that cleaves the amyloid precursor protein (APP) and yields amyloid ß peptide (Aß). METHODS: We have proposed a neuroprotection mechanism for NSAIDs based on inhibition of mitochondrial Ca2+ overload. Aß oligomers promote Ca2+ influx and mitochondrial Ca2+ overload leading to neuron cell death. Several non-specific NSAIDs including ibuprofen, sulindac, indomethacin and Rflurbiprofen depolarize mitochondria in the low µM range and prevent mitochondrial Ca2+ overload induced by Aß oligomers and/or N-methyl-D-aspartate (NMDA). However, at larger concentrations, NSAIDs may collapse mitochondrial potential (ΔΨ) leading to cell death. RESULTS: Accordingly, this mechanism may explain neuroprotection at low concentrations and damage at larger doses, thus providing clues on the failure of promising trials. Perhaps lower NSAID concentrations and/or alternative compounds with larger dynamic ranges should be considered for future trials to provide definitive evidence of neuroprotection against AD.

Levetiracetam inhibits oligomeric Aß-induced glutamate release from human astrocytes.

A recently identified mechanism for oligomeric Aß-induced glutamate release from astrocytes involves intracellular Ca elevation, potentially by Ca-dependent vesicular release. Evidence suggests that levetiracetam (LEV; Keppra), an antiepileptic drug, can improve cognitive performance in both humans with mild cognitive impairment and animal models of Alzheimer disease. Because LEV acts by modulating neurotransmitter release from neurons by interaction with synaptic vesicles, we tested the effect of LEV on Aß-induced astrocytic release of glutamate. We used a fluorescence resonance energy transfer-based glutamate sensor (termed SuperGluSnFR), whose structure is based on the ligand-binding site of glutamate receptors, to monitor glutamate release from primary cultures of human astrocytes exposed to oligomeric amyloid-ß peptide 1-42 (Aß42). We found that LEV (10 µM) inhibited oligomeric Aß-induced astrocytic glutamate release. In addition, we show that this Aß-induced glutamate release from astrocytes is sensitive to tetanus neurotoxin, an inhibitor of the vesicle release machinery. Taken together, our evidence suggests that LEV inhibits Aß-induced vesicular glutamate release from astrocytes and thus may underlie, at least in part, the ability of LEV to reduce hyperexcitability in Alzheimer disease.

Elevated glucose and oligomeric ß-amyloid disrupt synapses via a common pathway of aberrant protein S-nitrosylation.

Metabolic syndrome (MetS) and Type 2 diabetes mellitus (T2DM) increase risk for Alzheimer's disease (AD). The molecular mechanism for this association remains poorly defined. Here we report in human and rodent tissues that elevated glucose, as found in MetS/T2DM, and oligomeric ß-amyloid (Aß) peptide, thought to be a key mediator of AD, coordinately increase neuronal Ca(2+) and nitric oxide (NO) in an NMDA receptor-dependent manner. The increase in NO results in S-nitrosylation of insulin-degrading enzyme (IDE) and dynamin-related protein 1 (Drp1), thus inhibiting insulin and Aß catabolism as well as hyperactivating mitochondrial fission machinery. Consequent elevation in Aß levels and compromise in mitochondrial bioenergetics result in dysfunctional synaptic plasticity and synapse loss in cortical and hippocampal neurons. The NMDA receptor antagonist memantine attenuates these effects. Our studies show that redox-mediated posttranslational modification of brain proteins link Aß and hyperglycaemia to cognitive dysfunction in MetS/T2DM and AD.

Protection from cyanide-induced brain injury by the Nrf2 transcriptional activator carnosic acid.

Cyanide is a life-threatening, bioterrorist agent, preventing cellular respiration by inhibiting cytochrome c oxidase, resulting in cardiopulmonary failure, hypoxic brain injury, and death within minutes. However, even after treatment with various antidotes to protect cytochrome oxidase, cyanide intoxication in humans can induce a delayed-onset neurological syndrome that includes symptoms of Parkinsonism. Additional mechanisms are thought to underlie cyanide-induced neuronal damage, including generation of reactive oxygen species. This may account for the fact that antioxidants prevent some aspects of cyanide-induced neuronal damage. Here, as a potential preemptive countermeasure against a bioterrorist attack with cyanide, we tested the CNS protective effect of carnosic acid (CA), a pro-electrophilic compound found in the herb rosemary. CA crosses the blood-brain barrier to up-regulate endogenous antioxidant enzymes via activation of the Nrf2 transcriptional pathway. We demonstrate that CA exerts neuroprotective effects on cyanide-induced brain damage in cultured rodent and human-induced pluripotent stem cell-derived neurons in vitro, and in vivo in various brain areas of a non-Swiss albino mouse model of cyanide poisoning that simulates damage observed in the human brain. Cyanide, a potential bioterrorist agent, can produce a chronic delayed-onset neurological syndrome that includes symptoms of Parkinsonism. Here, cyanide poisoning treated with the proelectrophillic compound carnosic acid, results in reduced neuronal cell death in both in vitro and in vivo models through activation of the Nrf2/ARE transcriptional pathway. Carnosic acid is therefore a potential treatment for the toxic central nervous system (CNS) effects of cyanide poisoning. ARE, antioxidant responsive element; Nrf2 (NFE2L2, Nuclear factor (erythroid-derived 2)-like 2).

Susceptibility to excitotoxicity in aged hippocampal cultures and neuroprotection by non-steroidal anti-inflammatory drugs: role of mitochondrial calcium.

Brain damage after insult and cognitive decline are related to excitotoxicity and strongly influenced by aging, yet mechanisms of aging-dependent susceptibility to excitotoxicity are poorly known. Several non-steroidal anti-inflammatory drugs (NSAIDs) may prevent excitotoxicity and cognitive decline in the elderly by an unknown mechanism. Interestingly, after several weeks in vitro, hippocampal neurons display important hallmarks of neuronal aging in vivo. Accordingly, rat hippocampal neurons cultured for several weeks were used to investigate mechanisms of aging-related susceptibility to excitotoxicity and neuroprotection by NSAIDs. We found that NMDA increased cytosolic Ca(2+) concentration in young, mature and aged neurons but only promoted apoptosis in aged neurons. Resting Ca(2+) levels and responses to NMDA increased with time in culture which correlated with changes in expression of NMDA receptor subunits. In addition, NMDA promoted mitochondrial Ca(2+) uptake only in aged cultures. Consistently, specific inhibition of mitochondrial Ca(2+) uptake decreased apoptosis. Finally, we found that a series of NSAIDs depolarized mitochondria and inhibited mitochondrial Ca(2+) overload, thus preventing NMDA-induced apoptosis in aged cultures. We conclude that mitochondrial Ca(2+) uptake is critical for age-related susceptibility to excitotoxicity and neuroprotection by NSAIDs. Rat hippocampal neurons aged in culture were used to investigate mechanisms of age-related susceptibility to excitotoxicity and neuroprotection by non-steroidal anti-inflammatory drugs (NSAIDs). Old neurons display enhanced resting calcium and responses to NMDA along with increased expression of NMDA receptor subunits NR1 and NR2A altogether favoring mitochondrial calcium overload. NSAIDs protect neurons against excitotoxicity acting on mitochondrial calcium uptake. NMDA, N methyl d-aspartate.

S-nitrosylation-mediated redox transcriptional switch modulates neurogenesis and neuronal cell death.

Redox-mediated posttranslational modifications represent a molecular switch that controls major mechanisms of cell function. Nitric oxide (NO) can mediate redox reactions via S-nitrosylation, representing transfer of an NO group to a critical protein thiol. NO is known to modulate neurogenesis and neuronal survival in various brain regions in disparate neurodegenerative conditions. However, a unifying molecular mechanism linking these phenomena remains unknown. Here, we report that S-nitrosylation of myocyte enhancer factor 2 (MEF2) transcription factors acts as a redox switch to inhibit both neurogenesis and neuronal survival. Structure-based analysis reveals that MEF2 dimerization creates a pocket, facilitating S-nitrosylation at an evolutionally conserved cysteine residue in the DNA binding domain. S-Nitrosylation disrupts MEF2-DNA binding and transcriptional activity, leading to impaired neurogenesis and survival in vitro and in vivo. Our data define a molecular switch whereby redox-mediated posttranslational modification controls both neurogenesis and neurodegeneration via a single transcriptional signaling cascade.

Differential effects of synaptic and extrasynaptic NMDA receptors on Aß-induced nitric oxide production in cerebrocortical neurons.

Oligomerized amyloid-ß (Aß) peptide is thought to contribute to synaptic damage, resulting in dysfunctional neuronal networks in patients with Alzheimer's disease. It has been previously suggested that Aß may be detrimental to neuronal health, at least in part, by triggering oxidative/nitrosative stress. However, the mechanisms underlying this process remain to be elucidated. Here, using rat primary cerebrocortical cultures, we demonstrate that Aß1-42 oligomers trigger a dramatic increase in intracellular nitric oxide (NO) concentration via a process mediated by activation of NMDA-type glutamate receptors (NMDARs). Considering that synaptic NMDARs and extrasynaptic NMDARs (eNMDARs) can have opposite effects on neuronal viability, we explored their respective roles in Aß-induced increases in NO levels. Surprisingly, after pharmacological isolation of eNMDARs, we discovered that eNMDARs are primarily responsible for the increase in neuronal NO triggered by Aß oligomers. Moreover, we found that the eNMDAR-mediated increase in NO can produce S-nitrosylation of Drp1 (dynamin-related protein 1) and Cdk5 (cyclin-dependent kinase 5), targets known to contribute to Aß-induced synaptic damage. These results suggest that pharmacological intervention specifically aimed at eNMDARs may decrease Aß-induced nitrosative stress and thus ameliorate neurotoxic damage to synapses.

Aß induces astrocytic glutamate release, extrasynaptic NMDA receptor activation, and synaptic loss.

Synaptic loss is the cardinal feature linking neuropathology to cognitive decline in Alzheimer's disease (AD). However, the mechanism of synaptic damage remains incompletely understood. Here, using FRET-based glutamate sensor imaging, we show that amyloid-ß peptide (Aß) engages α7 nicotinic acetylcholine receptors to induce release of astrocytic glutamate, which in turn activates extrasynaptic NMDA receptors (eNMDARs) on neurons. In hippocampal autapses, this eNMDAR activity is followed by reduction in evoked and miniature excitatory postsynaptic currents (mEPSCs). Decreased mEPSC frequency may reflect early synaptic injury because of concurrent eNMDAR-mediated NO production, tau phosphorylation, and caspase-3 activation, each of which is implicated in spine loss. In hippocampal slices, oligomeric Aß induces eNMDAR-mediated synaptic depression. In AD-transgenic mice compared with wild type, whole-cell recordings revealed excessive tonic eNMDAR activity accompanied by eNMDAR-sensitive loss of mEPSCs. Importantly, the improved NMDAR antagonist NitroMemantine, which selectively inhibits extrasynaptic over physiological synaptic NMDAR activity, protects synapses from Aß-induced damage both in vitro and in vivo.

Study of neurotoxic intracellular calcium signalling triggered by amyloids.

Neurotoxicity in Alzheimer's disease (AD) is associated to dishomeostasis of intracellular Ca(2+) induced by amyloid ß peptide (Aß) species. Understanding of the effects of Aß on intracellular Ca(2+) homeostasis requires preparation of the different Aß assemblies including oligomers and fibrils and the testing of their effects on cytosolic and mitochondrial Ca(2+) in neurons. Procedures for cerebellar granule cell culture, preparation of Aß species as well as fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca(2+) in neurons are described.

17beta-estradiol promotes cementoblast proliferation and cementum formation in experimental periodontitis.

BACKGROUND: Periodontitis is a chronic inflammatory condition that leads to the destruction of the tooth-supporting tissues. Its treatment includes the arrest of the inflammatory process and, in some circumstances, the restoration of the lost anatomy and function, including the formation of new cementum, periodontal ligament (PDL), and bone. With this goal, we investigated the effects of low concentrations of 17beta-estradiol on human cementoblast proliferation and its possible regenerative potential in vivo. METHODS: Human cementum-derived cells obtained from a healthy human premolar were isolated and characterized by immunocytochemistry. Cell proliferation assays were performed to test the effects of 100 nM 17beta-estradiol and enamel matrix derivative (EMD). Three-wall intrabony periodontal defects were created in beagle dogs. After 1 month of plaque accumulation, 0.225 mg 17beta-estradiol impregnated in a collagen sponge was applied to randomly selected defects (test group), whereas a collagen sponge impregnated in a culture medium was applied to the control group. After 3 months, specimens were obtained, and tissue regeneration was assessed by histometric analysis. RESULTS: Cells spreading out from human tooth-layer explants were able to form cell colonies, produce a mineral matrix, and express osteocalcin, indicating they were cementoblast-like cells. In contrast, PDL fibroblasts did not express osteocalcin. 17beta-estradiol, but not EMD, increased the rate of human cementoblast cell proliferation in vitro by 2.5-fold. Histometric results from the treated periodontal defects revealed that 17beta-estradiol promoted the formation of 2.94 mm of new cementum, (45% of the defects) compared to 1.54 mm of new cementum in the control group (28% of the defects). Furthermore, the test group showed an inhibition of epithelial downgrowth and a gain of new connective tissue attachment. CONCLUSION: 17beta-estradiol promoted human cementoblast cell proliferation in vitro and periodontal regeneration in an experimental periodontitis model.

Mitochondria and calcium flux as targets of neuroprotection caused by minocycline in cerebellar granule cells.

Minocycline, an antibiotic of the tetracycline family, has attracted considerable interest for its theoretical therapeutic applications in neurodegenerative diseases. However, the mechanism of action underlying its effect remains elusive. Here we have studied the effect of minocycline under excitotoxic conditions. Fluorescence and bioluminescence imaging studies in rat cerebellar granular neuron cultures using fura2/AM and mitochondria-targeted aequorin revealed that minocycline, at concentrations higher than those shown to block inflammation and inflammation-induced neuronal death, inhibited NMDA-induced cytosolic and mitochondrial rises in Ca(2+) concentrations in a reversible manner. Moreover, minocycline added in the course of NMDA stimulation decreased Ca(2+) intracellular levels, but not when induced by depolarization with a high K(+) medium. We also found that minocycline, at the same concentrations, partially depolarized mitochondria by about 5-30 mV, prevented mitochondrial Ca(2+) uptake under conditions of environmental stress, and abrogated NMDA-induced reactive oxygen species (ROS) formation. Consistently, minocycline also abrogates the rise in ROS induced by 75 microM Ca(2+) in isolated brain mitochondria. In search for the mechanism of mitochondrial depolarization, we found that minocycline markedly inhibited state 3 respiration of rat brain mitochondria, although distinctly increased oxygen uptake in state 4. Minocycline inhibited NADH-cytochrome c reductase and cytochrome c oxidase activities, whereas the activity of succinate-cytochrome c reductase was not modified, suggesting selective inhibition of complexes I and IV. Finally, minocycline affected activity of voltage-dependent anion channel (VDAC) as determined in the reconstituted system. Taken together, our results indicate that mitochondria are a critical factor in minocycline-mediated neuroprotection.

Mitochondrial Ca2+ overload underlies Abeta oligomers neurotoxicity providing an unexpected mechanism of neuroprotection by NSAIDs.

Dysregulation of intracellular Ca(2+) homeostasis may underlie amyloid beta peptide (Abeta) toxicity in Alzheimer's Disease (AD) but the mechanism is unknown. In search for this mechanism we found that Abeta(1-42) oligomers, the assembly state correlating best with cognitive decline in AD, but not Abeta fibrils, induce a massive entry of Ca(2+) in neurons and promote mitochondrial Ca(2+) overload as shown by bioluminescence imaging of targeted aequorin in individual neurons. Abeta oligomers induce also mitochondrial permeability transition, cytochrome c release, apoptosis and cell death. Mitochondrial depolarization prevents mitochondrial Ca(2+) overload, cytochrome c release and cell death. In addition, we found that a series of non-steroidal anti-inflammatory drugs (NSAIDs) including salicylate, sulindac sulfide, indomethacin, ibuprofen and R-flurbiprofen depolarize mitochondria and inhibit mitochondrial Ca(2+) overload, cytochrome c release and cell death induced by Abeta oligomers. Our results indicate that i) mitochondrial Ca(2+) overload underlies the neurotoxicity induced by Abeta oligomers and ii) inhibition of mitochondrial Ca(2+) overload provides a novel mechanism of neuroprotection by NSAIDs against Abeta oligomers and AD.

Bioluminescence imaging of mitochondrial Ca2+ dynamics in soma and neurites of individual adult mouse sympathetic neurons.

Changes in the cytosolic Ca(2+) concentration ([Ca(2+)](c)) are essential for triggering neurotransmitter release from presynaptic nerve terminals. Calcium-induced Ca(2+) release (CICR) from the endoplasmic reticulum (ER) may amplify the [Ca(2+)](c) signals and facilitate neurotransmitter release in sympathetic neurons. In adrenal chromaffin cells, functional triads are formed by voltage-operated Ca(2+) channels (VOCCs), CICR sites and mitochondria. In fact, mitochondria take up most of the Ca(2+) load entering the cells and are essential for shaping [Ca(2+)](c) signals and exocytosis. Here we have investigated the existence of such functional triads in sympathetic neurons. The mitochondrial Ca(2+) concentration ([Ca(2+)](m)) in soma and neurites of individual mouse superior cervical ganglion (SCG) neurons was monitored by bioluminescence imaging of targeted aequorins. In soma, Ca(2+) entry through VOCCs evoked rapid, near millimolar [Ca(2+)](m) increases in a subpopulation of mitochondria containing about 40% of the aequorin. Caffeine evoked a similar [Ca(2+)](m) increase in a mitochondrial pool containing about 30% of the aequorin and overlapping with the VOCC-sensitive pool. These observations suggest the existence of functional triads similar to the ones described in chromaffin cells. In neurites, mitochondria were able to buffer [Ca(2+)](c) increases resulting from activation of VOCCs but not those mediated by caffeine-induced Ca(2+) release from the ER. The weaker Ca(2+) buffering by mitochondria in neurites could contribute to facilitate Ca(2+)-induced exocytosis at the presynaptic sites.