Mitochondrial Ca2+ uptake pathways.

Calcium (Ca2+) plays diverse roles in all living organisms ranging from bacteria to humans. It is a structural element for bones, an essential mediator of excitation-contraction coupling, and a universal second messenger in the regulation of ion channel, enzyme and gene expression activities. In mitochondria, Ca2+ is crucial for the control of energy production and cellular responses to metabolic stress. Ca2+ uptake by the mitochondria occurs by the uniporter mechanism. The Mitochondrial Ca2+ Uniporter (MCU) protein has recently been identified as a core component responsible for mitochondrial Ca2+ uptake. MCU knockout (MCU KO) studies have identified a number of important roles played by this high capacity uptake pathway. Interestingly, this work has also shown that MCU-mediated Ca2+ uptake is not essential for vital cell functions such as muscle contraction, energy metabolism and neurotransmission. Although mitochondrial Ca2+ uptake was markedly reduced, MCU KO mitochondria still contained low but detectable levels of Ca2+. In view of the fundamental importance of Ca2+ for basic cell signalling, this finding suggests the existence of other currently unrecognized pathways for Ca2+ entry. We review the experimental evidence for the existence of alternative Ca2+ influx mechanisms and propose how these mechanisms may play an integral role in mitochondrial Ca2+ signalling.

The mitochondrial calcium uniporter inhibitor Ru265 increases neuronal excitability and reduces neurotransmission via off-target effects.

BACKGROUND AND PURPOSE: Excitotoxicity due to mitochondrial calcium (Ca2+) overloading can trigger neuronal cell death in a variety of pathologies. Inhibiting the mitochondrial calcium uniporter (MCU) has been proposed as a therapeutic avenue to prevent calcium overloading. Ru265 (ClRu(NH3)4(µ-N)Ru(NH3)4Cl]Cl3) is a cell-permeable inhibitor of the mitochondrial calcium uniporter (MCU) with nanomolar affinity. Ru265 reduces sensorimotor deficits and neuronal death in models of ischemic stroke. However, the therapeutic use of Ru265 is limited by the induction of seizure-like behaviours. EXPERIMENTAL APPROACH: We examined the effect of Ru265 on synaptic and neuronal function in acute brain slices and hippocampal neuron cultures derived from mice, in control and where MCU expression was genetically abrogated. KEY RESULTS: Ru265 decreased evoked responses from calyx terminals and induced spontaneous action potential firing of both the terminal and postsynaptic principal cell. Recordings of presynaptic Ca2+ currents suggested that Ru265 blocks the P/Q type channel, confirmed by the inhibition of currents in cells exogenously expressing the P/Q type channel. Measurements of presynaptic K+ currents further revealed that Ru265 blocked a KCNQ current, leading to increased membrane excitability, underlying spontaneous spiking. Ca2+ imaging of hippocampal neurons showed that Ru265 increased synchronized, high-amplitude events, recapitulating seizure-like activity seen in vivo. Importantly, MCU ablation did not suppress Ru265-induced increases in neuronal activity and seizures. CONCLUSIONS AND IMPLICATIONS: Our findings provide a mechanistic explanation for the pro-convulsant effects of Ru265 and suggest counter screening assays based on the measurement of P/Q and KCNQ channel currents to identify safe MCU inhibitors.

Differential effect of lithium on spermidine/spermine N1-acetyltransferase expression in suicidal behaviour.

An altered polyamine system has been suggested to play a key role in mood disorders and suicide, a hypothesis corroborated by the evidence that lithium inhibits the polyamine mediated stress response in the rat brain. Recent post-mortem studies have shown that spermidine/spermine N1-acetyltransferase (SAT1), the key regulator of cellular polyamine content, is under-expressed in brains from suicide victims compared to controls. In our study we tested the effect of in vitro lithium treatment on SAT1 gene and protein expression in B lymphoblastoid cell lines (BLCLs) from bipolar disorder (BD) patients who committed suicide (and for which BLCLs were collected prior to their death), BD patients with high and low risk of suicide and a sample of non-psychiatric controls. Baseline mRNA levels were similar in the four groups of subjects (p > 0.05). Lithium had no effect in suicide completers (p > 0.05) while it significantly increased SAT1 expression in the high risk (p < 0.001) and low risk (p < 0.01) groups as well as in controls (p < 0.001). Protein and mRNA levels were not correlated; lithium significantly reduced protein levels only in the control sample (p < 0.05). Our findings suggest that SAT1 transcription is influenced by lithium and that this effect is altered in BD patients who completed suicide, further supporting a role for polyamines in suicide.

Over-expression of X-linked inhibitor of apoptosis protein modulates multiple aspects of neuronal Ca2+ signaling.

X-linked inhibitor of apoptosis (XIAP) protects and preserves the function of neurons in both in vitro and in vivo models of excitotoxicity. Since calcium (Ca(2+)) overload is a pivotal event in excitotoxic neuronal cell death, we have determined whether XIAP over-expression influences Ca(2+)-signaling in primary cultures of mouse cortical neurons. Using cortical neuron cultures derived from wild-type (Wt) mice transiently transfected with XIAP or from transgenic mice that over-express XIAP, we show that XIAP opposes the rise in intracellular Ca(2+) concentration by a variety of triggers. Relative to control neurons, XIAP over-expression produced a slight, but significant, elevation of resting Ca(2+) concentrations. By contrast, the rise in intracellular Ca(2+) concentrations produced by N-methyl-D-aspartate receptor stimulation and voltage gated Ca(2+) channel activation were markedly attenuated by XIAP over-expression. The release of Ca(2+) from intracellular stores induced by the sarco/endoplasmic reticulum Ca(2+) ATPase inhibitor thapsigargin was also inhibited in neurons transiently transfected with XIAP. The pan-caspase inhibitor zVAD did not, however, diminish the rise in intracellular Ca(2+) concentrations elicited by L-glutamate suggesting that XIAP influences Ca(2+) signaling in a caspase-independent manner. Taken together, these findings demonstrate that the ability of XIAP to block excessive rises in intracellular Ca(2+) by a variety of triggers may contribute to the neuroprotective effects of this anti-apoptotic protein.

Dinuclear nitrido-bridged osmium complexes inhibit the mitochondrial calcium uniporter and protect cortical neurons against lethal oxygen-glucose deprivation.

Dysregulation of mitochondrial calcium uptake mediated by the mitochondrial calcium uniporter (MCU) is implicated in several pathophysiological conditions. Dinuclear ruthenium complexes are effective inhibitors of the MCU and have been leveraged as both tools to study mitochondrial calcium dynamics and potential therapeutic agents. In this study, we report the synthesis and characterization of Os245 ([Os2(µ-N)(NH3)8Cl2]3+) which is the osmium-containing analogue of our previously reported ruthenium-based inhibitor Ru265. This complex and its aqua-capped analogue Os245' ([Os2(µ-N)(NH3)8(OH2)2]5+) are both effective inhibitors of the MCU in permeabilized and intact cells. In comparison to the ruthenium-based inhibitor Ru265 (k obs = 4.92 × 10-3 s-1), the axial ligand exchange kinetics of Os245 are two orders of magnitude slower (k obs = 1.63 × 10-5 s-1) at 37 °C. The MCU-inhibitory properties of Os245 and Os245' are different (Os245 IC50 for MCU inhibition = 103 nM; Os245' IC50 for MCU inhibition = 2.3 nM), indicating that the axial ligands play an important role in their interactions with this channel. We further show that inhibition of the MCU by these complexes protects primary cortical neurons against lethal oxygen-glucose deprivation. When administered in vivo to mice (10 mg kg-1), Os245 and Os245' induce seizure-like behaviors in a manner similar to the ruthenium-based inhibitors. However, the onset of these seizures is delayed, a possible consequence of the slower ligand substitution kinetics for these osmium complexes. These findings support previous studies that demonstrate inhibition of the MCU is a promising therapeutic strategy for the treatment of ischemic stroke, but also highlight the need for improved drug delivery strategies to mitigate the pro-convulsant effects of this class of complexes before they can be implemented as therapeutic agents. Furthermore, the slower ligand substitution kinetics of the osmium analogues may afford new strategies for the development and modification of this class of MCU inhibitors.

Preserving and enhancing mitochondrial function after stroke to protect and repair the neurovascular unit: novel opportunities for nanoparticle-based drug delivery.

The neurovascular unit (NVU) is composed of vascular cells, glia, and neurons that form the basic component of the blood brain barrier. This intricate structure rapidly adjusts cerebral blood flow to match the metabolic needs of brain activity. However, the NVU is exquisitely sensitive to damage and displays limited repair after a stroke. To effectively treat stroke, it is therefore considered crucial to both protect and repair the NVU. Mitochondrial calcium (Ca2+) uptake supports NVU function by buffering Ca2+ and stimulating energy production. However, excessive mitochondrial Ca2+ uptake causes toxic mitochondrial Ca2+ overloading that triggers numerous cell death pathways which destroy the NVU. Mitochondrial damage is one of the earliest pathological events in stroke. Drugs that preserve mitochondrial integrity and function should therefore confer profound NVU protection by blocking the initiation of numerous injury events. We have shown that mitochondrial Ca2+ uptake and efflux in the brain are mediated by the mitochondrial Ca2+ uniporter complex (MCUcx) and sodium/Ca2+/lithium exchanger (NCLX), respectively. Moreover, our recent pharmacological studies have demonstrated that MCUcx inhibition and NCLX activation suppress ischemic and excitotoxic neuronal cell death by blocking mitochondrial Ca2+ overloading. These findings suggest that combining MCUcx inhibition with NCLX activation should markedly protect the NVU. In terms of promoting NVU repair, nuclear hormone receptor activation is a promising approach. Retinoid X receptor (RXR) and thyroid hormone receptor (TR) agonists activate complementary transcriptional programs that stimulate mitochondrial biogenesis, suppress inflammation, and enhance the production of new vascular cells, glia, and neurons. RXR and TR agonism should thus further improve the clinical benefits of MCUcx inhibition and NCLX activation by increasing NVU repair. However, drugs that either inhibit the MCUcx, or stimulate the NCLX, or activate the RXR or TR, suffer from adverse effects caused by undesired actions on healthy tissues. To overcome this problem, we describe the use of nanoparticle drug formulations that preferentially target metabolically compromised and damaged NVUs after an ischemic or hemorrhagic stroke. These nanoparticle-based approaches have the potential to improve clinical safety and efficacy by maximizing drug delivery to diseased NVUs and minimizing drug exposure in healthy brain and peripheral tissues.

Fingolimod attenuates gait deficits in mice subjected to experimental autoimmune encephalomyelitis.

Fingolimod, used to treat relapsing-remitting multiple sclerosis (RRMS), reduces motor deficits in mice with established experimental autoimmune encephalomyelitis (EAE). To better characterize the therapeutic effects of fingolimod, kinematic gait analysis was employed to precisely measure movements of a hindleg while EAE mice walked on a treadmill. Relative to the vehicle group, oral dosing with fingolimod, beginning after disease onset (1 mg/kg/day), increased hip heights and knee joint movements, and reduced spinal cord demyelination. These findings suggest that fingolimod preserves gait in RRMS patients by protecting motor circuits in the spinal cord.

Mechanism of action and therapeutic route for a muscular dystrophy caused by a genetic defect in lipid metabolism.

CHKB encodes one of two mammalian choline kinase enzymes that catalyze the first step in the synthesis of the membrane phospholipid phosphatidylcholine. In humans and mice, inactivation of the CHKB gene (Chkb in mice) causes a recessive rostral-to-caudal muscular dystrophy. Using Chkb knockout mice, we reveal that at no stage of the disease is phosphatidylcholine level significantly altered. We observe that in affected muscle a temporal change in lipid metabolism occurs with an initial inability to utilize fatty acids for energy via mitochondrial ß-oxidation resulting in shunting of fatty acids into triacyglycerol as the disease progresses. There is a decrease in peroxisome proliferator-activated receptors and target gene expression specific to Chkb-/- affected muscle. Treatment of Chkb-/- myocytes with peroxisome proliferator-activated receptor agonists enables fatty acids to be used for ß-oxidation and prevents triacyglyerol accumulation, while simultaneously increasing expression of the compensatory choline kinase alpha (Chka) isoform, preventing muscle cell injury.

Altered circadian activity and sleep/wake rhythms in the stable tubule only polypeptide (STOP) null mouse model of schizophrenia.

Sleep and circadian rhythm disruptions commonly occur in individuals with schizophrenia. Stable tubule only polypeptide (STOP) knockout (KO) mice show behavioral impairments resembling symptoms of schizophrenia. We previously reported that STOP KO mice slept less and had more fragmented sleep and waking than wild-type littermates under a light/dark (LD) cycle. Here, we assessed the circadian phenotype of male STOP KO mice by examining wheel-running activity rhythms and EEG/EMG-defined sleep/wake states under both LD and constant darkness (DD) conditions. Wheel-running activity rhythms in KO and wild-type mice were similarly entrained in LD, and had similar free-running periods in DD. The phase delay shift in response to a light pulse given early in the active phase under DD was preserved in KO mice. KO mice had markedly lower activity levels, lower amplitude activity rhythms, less stable activity onsets, and more fragmented activity than wild-type mice in both lighting conditions. KO mice also spent more time awake and less time in rapid eye movement sleep (REMS) and non-REMS (NREMS) in both LD and DD conditions, with the decrease in NREMS concentrated in the active phase. KO mice also showed altered EEG features and higher amplitude rhythms in wake and NREMS (but not REMS) amounts in both lighting conditions, with a longer free-running period in DD, compared to wild-type mice. These results indicate that the STOP null mutation in mice altered the regulation of sleep/wake physiology and activity rhythm expression, but did not grossly disrupt circadian mechanisms.

Nitric-oxide synthase mediates the ability of darbepoetin alfa to attenuate pre-existing spatial working memory deficits in rats subjected to transient global ischemia.

Erythropoietin has been reported to improve the behavioral performance of healthy mice in tests thought to depend on synaptic plasticity in the CA1 region of the hippocampus. We show here for the first time that a single injection of the erythropoietin analog darbepoetin alfa reverses pre-existing cognitive deficits in adult rats that had been subjected to transient global ischemia produced by four-vessel occlusion (4-VO). Quantification of neuronal density demonstrated that 12 min of 4-VO selectively killed more than 90% of CA1 neurons in the dorsal hippocampus. Rats that had sustained a bilateral loss of hippocampal CA1 neurons in this range (4-VO rats) displayed more errors and longer escape latencies in the Barnes maze compared with sham-operated controls. A single injection of darbepoetin alfa (5000 U/kg i.p.) 4 h before behavioral testing decreased deficits in escape latency for 4-VO rats but not sham-operated controls. This improvement in spatial working memory performance was correlated with increased levels of nitric-oxide metabolites in the ventral hippocampus. Systemic administration of the nitric-oxide synthase inhibitor N(G)-nitro-L-nitro-arginine methyl ester reversed the increase in nitric-oxide metabolites and improvements in spatial working memory produced by darbepoetin alfa (5000 U/kg, i.p.) at a dose (10 mg/kg, i.p.) that did not impair the spatial working memory performance of intact rats. Taken together, these findings suggest that darbepoetin alfa reverses pre-existing spatial working memory deficits resulting from transient global ischemia by increasing the activity of nitric-oxide synthase, an enzyme implicated in synaptic plasticity.

Homeostatic state of microglia in a rat model of chronic sleep restriction.

Chronic sleep restriction (CSR) negatively impacts brain functions. Whether microglia, the brain's resident immune cells, play any role is unknown. We studied microglia responses to CSR using a rat model featuring slowly rotating wheels (3 h on/1 h off), which was previously shown to induce both homeostatic and adaptive responses in sleep and attention. Adult male rats were sleep restricted for 27 or 99 h. Control rats were housed in locked wheels. After 27 and/or 99 h of CSR, the number of cells immunoreactive for the microglia marker ionized calcium-binding adaptor molecule-1 (Iba1) and the density of Iba1 immunoreactivity were increased in 4/10 brain regions involved in sleep/wake regulation and cognition, including the prelimbic cortex, central amygdala, perifornical lateral hypothalamic area, and dorsal raphe nucleus. CSR neither induced mitosis in microglia (assessed with bromodeoxyuridine) nor impaired blood-brain barrier permeability (assessed with Evans Blue). Microglia appeared ramified in all treatment groups and, when examined quantitatively in the prelimbic cortex, their morphology was not affected by CSR. After 27 h, but not 99 h, of CSR, mRNA levels of the anti-inflammatory cytokine interleukin-10 were increased in the frontal cortex. Pro-inflammatory cytokine mRNA levels (tumor necrosis factor-α, interleukin-1ß, and interleukin-6) were unchanged. Furthermore, cortical microglia were not immunoreactive for several pro- and anti-inflammatory markers tested, but were immunoreactive for the purinergic P2Y12 receptor. These results suggest that microglia respond to CSR while remaining in a physiological state and may contribute to the previously reported homeostatic and adaptive responses to CSR.

The cell-permeable mitochondrial calcium uniporter inhibitor Ru265 preserves cortical neuron respiration after lethal oxygen glucose deprivation and reduces hypoxic/ischemic brain injury.

The mitochondrial calcium (Ca2+) uniporter (MCU) mediates high-capacity mitochondrial Ca2+ uptake implicated in ischemic/reperfusion cell death. We have recently shown that inducible MCU ablation in Thy1-expressing neurons renders mice resistant to sensorimotor deficits and forebrain neuron loss in a model of hypoxic/ischemic (HI) brain injury. These findings encouraged us to compare the neuroprotective effects of Ru360 and the recently identified cell permeable MCU inhibitor Ru265. Unlike Ru360, Ru265 (2-10 µM) reached intracellular concentrations in cultured cortical neurons that preserved cell viability, blocked the protease activity of Ca2+-dependent calpains and maintained mitochondrial respiration and glycolysis after a lethal period of oxygen-glucose deprivation (OGD). Intraperitoneal (i.p.) injection of adult male C57Bl/6 mice with Ru265 (3 mg/kg) also suppressed HI-induced sensorimotor deficits and brain injury. However, higher doses of Ru265 (10 and 30 mg/kg, i.p.) produced dose-dependent increases in the frequency and duration of seizure-like behaviours. Ru265 is proposed to promote convulsions by reducing Ca2+ buffering and energy production in highly energetic interneurons that suppress brain seizure activity. These findings support the therapeutic potential of MCU inhibition in the treatment of ischemic stroke but also indicate that such clinical translation will require drug delivery strategies which mitigate the pro-convulsant effects of Ru265.

Neuronal mitochondrial calcium uniporter deficiency exacerbates axonal injury and suppresses remyelination in mice subjected to experimental autoimmune encephalomyelitis.

High-capacity mitochondrial calcium (Ca2+) uptake by the mitochondrial Ca2+ uniporter (MCU) is strategically positioned to support the survival and remyelination of axons in multiple sclerosis (MS) by undocking mitochondria, buffering Ca2+ and elevating adenosine triphosphate (ATP) synthesis at metabolically stressed sites. Respiratory chain deficits in MS are proposed to metabolically compromise axon survival and remyelination by suppressing MCU activity. In support of this hypothesis, clinical scores, mitochondrial dysfunction, myelin loss, axon damage and inflammation were elevated while remyelination was blocked in neuronal MCU deficient (Thy1-MCU Def) mice relative to Thy1 controls subjected to experimental autoimmune encephalomyelitis (EAE). At the first sign of walking deficits, mitochondria in EAE/Thy1 axons showed signs of activation. By contrast, cytoskeletal damage, fragmented mitochondria and large autophagosomes were seen in EAE/Thy1-MCU Def axons. As EAE severity increased, EAE/Thy1 axons were filled with massively swollen mitochondria with damaged cristae while EAE/Thy1-MCU Def axons were riddled with late autophagosomes. ATP concentrations and mitochondrial gene expression were suppressed while calpain activity, autophagy-related gene mRNA levels and autophagosome marker (LC3) co-localization in Thy1-expressing neurons were elevated in the spinal cords of EAE/Thy1-MCU Def compared to EAE/Thy1 mice. These findings suggest that MCU inhibition contributes to axonal damage that drives MS progression.

Experimental autoimmune encephalomyelitis accelerates remyelination after lysophosphatidylcholine-induced demyelination in the corpus callosum.

Experimental autoimmune encephalomyelitis (EAE) and lysophosphatidylcholine (LPC)-induced demyelination were combined to study remyelination in a pro-inflammatory context. Two groups of female C57BL/6 mice were subjected either to EAE (EAE mice) or injected with just complete Freund's adjuvant (CFA) and pertussis toxin (PTX) followed by bilateral LPC and phosphate buffered saline injections in the corpus callosum on day 7 (CFA controls). Relative to CFA controls, EAE accelerated remyelination and increased innate immune cell activation, lymphocyte infiltration and cytokine gene expression in the LPC lesions. However, compared to CFA mice, remyelination was reduced (day 14) suggesting this aggressive immune response also compromised myelin repair in EAE mice.

Synergistic Benefits of Combined Aerobic and Cognitive Training on Fluid Intelligence and the Role of IGF-1 in Chronic Stroke.

BACKGROUND: Paired exercise and cognitive training have the potential to enhance cognition by "priming" the brain and upregulating neurotrophins. METHODS: Two-site randomized controlled trial. Fifty-two patients >6 months poststroke with concerns about cognitive impairment trained 50 to 70 minutes, 3× week for 10 weeks with 12-week follow-up. Participants were randomized to 1 of 2 physical interventions: Aerobic (>60% VO2peak using <10% body weight-supported treadmill) or Activity (range of movement and functional tasks). Exercise was paired with 1 of 2 cognitive interventions (computerized dual working memory training [COG] or control computer games [Games]). The primary outcome for the 4 groups (Aerobic + COG, Aerobic + Games, Activity + COG, and Activity + Games) was fluid intelligence measured using Raven's Progressive Matrices Test administered at baseline, posttraining, and 3-month follow-up. Serum neurotrophins collected at one site (N = 30) included brain-derived neurotrophic factor (BDNF) at rest (BDNFresting) and after a graded exercise test (BDNFresponse) and insulin-like growth factor-1 at the same timepoints (IGF-1rest, IGF-1response). RESULTS: At follow-up, fluid intelligence scores significantly improved compared to baseline in the Aerobic + COG and Activity + COG groups; however, only the Aerobic + COG group was significantly different (+47.8%) from control (Activity + Games -8.5%). Greater IGF-1response at baseline predicted 40% of the variance in cognitive improvement. There was no effect of the interventions on BDNFresting or BDNFresponse; nor was BDNF predictive of the outcome. CONCLUSIONS: Aerobic exercise combined with cognitive training improved fluid intelligence by almost 50% in patients >6 months poststroke. Participants with more robust improvements in cognition were able to upregulate higher levels of serum IGF-1 suggesting that this neurotrophin may be involved in behaviorally induced plasticity.

Inhibitor of apoptosis protein (IAP) profiling in experimental autoimmune encephalomyelitis (EAE) implicates increased XIAP in T lymphocytes.

In multiple sclerosis (MS) and its widely accepted animal model, experimental autoimmune encephalomyelitis (EAE), the failure of autoreactive immune cells to undergo apoptosis is thought to contribute to CNS tissue damage and disease progression. Promoting apoptosis of myelin-reactive immune cells in diseases such as MS, may delay disease progression and decrease the frequency and severity of relapses. X-linked inhibitor of apoptosis (XIAP) is a potent anti-apoptotic protein that inhibits intrinsic, extrinsic, and c-Jun amino-terminal kinase mediated apoptosis and was the only member of the inhibitor of apoptosis (IAP) family upregulated in whole blood from EAE mice. Similar increases in XIAP were also observed in both peripheral and encephalitogenic T lymphocytes. Increased XIAP expression in T cells within areas of demyelination in the CNS suggests that XIAP may be enhancing cell survival and thereby contributing to disease pathology.

Increased X-linked inhibitor of apoptosis protein (XIAP) expression exacerbates experimental autoimmune encephalomyelitis (EAE).

Dysregulated apoptotic signaling has been implicated in most forms of cancer and many autoimmune diseases, such as multiple sclerosis (MS). We have previously shown that the anti-apoptotic protein X-linked inhibitor of apoptosis (XIAP) is elevated in T cells from mice with experimental autoimmune encephalomyelitis (EAE). In MS and EAE, the failure of autoimmune cells to undergo apoptosis is thought to exacerbate clinical symptoms and contribute to disease progression and CNS tissue damage. Antisense-mediated knockdown of XIAP, in vivo, increases the susceptibility of effector T cells to apoptosis, thus attenuating CNS inflammation and thereby alleviating the clinical signs of EAE. We report for the first time, generation of transgenic mice whereby the ubiquitin promoter drives expression of XIAP (ubXIAP), resulting in increased XIAP expression in a variety of tissues, including cells comprising the immune system. Transgenic ubXIAP mice and wild-type (WT) littermates were immunized with myelin oligodendrocyte glycoprotein (MOG35-55) in complete Freund's adjuvant and monitored daily for clinical symptoms of EAE over a 21-day period. The severity of EAE was increased in ubXIAP mice relative to WT-littermates, suggesting that XIAP overexpression enhanced the resistance of T cells to apoptosis. Consistent with this finding, T cells derived from MOG35-55-immunized ubXIAP mice and cultured in the presence of antigen were more resistant to etoposide-mediated apoptosis compared to WT-littermates. This work identifies XIAP is an important apoptotic regulator in EAE and a potential pharmacological target for treating autoimmune diseases such as MS.

Targeting apoptosis to treat multiple sclerosis.

Accumulating evidence implicates a failure of myelin-reactive immune cells to undergo apoptosis in the pathological events contributing to multiple sclerosis (MS). We have recently demonstrated that members of the inhibitor of apoptosis (IAP) family of anti-apoptotic genes are elevated in peripheral blood immune cells (monocytes, T cells) of patients with aggressive forms of MS (secondary progressive) or those with relapsing-remitting MS suffering a disease replase. These findings suggest that the IAPs may be novel diagnostic markers for distinguishing subtypes of MS. Moreover, antisense-mediated knockdown of the IAP family member known as X-linked IAP (XIAP) reverses paralysis in an animal model of MS suggesting that treatments targeting XIAP, and perhaps other IAPs, may have utility in the treatment of MS.

Tamoxifen-induced knockdown of the mitochondrial calcium uniporter in Thy1-expressing neurons protects mice from hypoxic/ischemic brain injury.

The mitochondrial calcium uniporter (MCU) mediates high-capacity mitochondrial calcium uptake that stimulates energy production. However, excessive MCU activity can cause ischemic heart injury. To examine if the MCU is also involved in hypoxic/ischemic (HI) brain injury, we have generated conditional MCU knockout mice by tamoxifen (TMX) administration to adult MCU-floxed (MCUfl/fl) mice expressing a construct encoding Thy1-cre/ERT2-eYFP. Relative to TMX/Thy1-cre/ERT2-eYFP controls, HI-induced sensorimotor deficits, forebrain neuron loss and mitochondrial damage were decreased for conditional MCU knockout mice. MCU knockdown by siRNA-induced silencing in cortical neuron cultures also reduced cell death and mitochondrial respiratory deficits following oxygen-glucose deprivation. Furthermore, MCU silencing did not produce metabolic abnormalities in cortical neurons observed previously for global MCU nulls that increased reliance on glycolysis for energy production. Based on these findings, we propose that brain-penetrant MCU inhibitors have strong potential to be well-tolerated and highly-efficacious neuroprotectants for the acute management of ischemic stroke.

Pioglitazone is superior to quetiapine, clozapine and tamoxifen at alleviating experimental autoimmune encephalomyelitis in mice.

Recent evidence suggests that clozapine and quetiapine (atypical antipsychotics), tamoxifen (selective-estrogen receptor modulator) and pioglitazone (PPARγ agonist) may improve functional recovery in multiple sclerosis (MS). We have compared the effectiveness of oral administration of these drugs, beginning at peak disease, at reducing ascending paralysis, motor deficits and demyelination in mice subjected to experimental autoimmune encephalomyelitis (EAE). Mice were immunized with an immunogenic peptide corresponding to amino acids 35-55 of the myelin oligodendrocyte glycoprotein (MOG35-55) in complete Freund's adjuvant and injected with pertussis toxin to induce EAE. Unlike clozapine, quetiapine and tamoxifen, administration of pioglitazone beginning at peak disease decreased both clinical scores and lumbar white matter loss in EAE mice. Using kinematic gait analysis, we found that pioglitazone also maintained normal movement of the hip, knee and ankle joints for at least 44 days after MOG35-55 immunization. This long-lasting preservation of hindleg joint movements was accompanied by reduced white matter loss, microglial and macrophage activation and the expression of pro-inflammatory genes in the lumbar spinal cords of EAE mice. These results support clinical findings that suggest pioglitazone may reduce the progressive loss of motor function in MS by decreasing inflammation and myelin damage.