The translocator protein (18 kDa) and its role in neuropsychiatric disorders

Translocator protein (TSPO) is an 18 kDa translocator membrane protein expressed in the outer mitochondrial membrane of steroid-synthesizing cells in the central and peripheral nervous systems. TSPO is involved in cellular functions, including the regulation of cell proliferation, transport of cholesterol to the inner mitochondrial membranes of glial cells, regulation of mitochondrial quality control, and haem synthesis. In the brain, TSPO has been extensively used as a biomarker of injury and inflammation. Indeed, TSPO was up-regulated in several inflammatory and neurodegenerative diseases. In contrast, the expression of TSPO was decreased in peripheral blood from psychiatric patients. Since TSPO is involved in several mechanisms related to mitochondrial function and inflammatory alterations, therapeutic approaches focusing on the regulation of TSPO may provide a new avenue for the treatment of neuropsychiatric disorders. Based on the involvement of mitochondrial alterations in the neurobiology of neuropsychiatric disorders, this review will focus on the functions and physiological roles of TSPO and the potential of TSPO ligands as therapeutic strategies for the treatment of neuropsychiatric disorders.     

Translocator protein (18 kDa) TSPO: An emerging therapeutic target in neurotrauma

Traumatic brain injury (TBI) induces physical, cognitive, and psychosocial deficits that affect millions of patients. TBI activates numerous cellular mechanisms and molecular cascades that produce detrimental outcomes, including neuronal death and loss of function. The mitochondrion is one of the major targets of TBI, as seen by increased mitochondrial activity in activated and proliferating microglia (due to high energy requirements and/or calcium overload) as well as increased reactive oxygen species, changes in mitochondrial permeability transition, release of cytochrome c, caspase activation, reduced ATP levels, and cell death in neurons. Translocator protein (TSPO) is an 18-kDa outer mitochondrial membrane protein that interacts with the mitochondria permeability transition pore and binds with high affinity to cholesterol and various classes of drug ligands, including some benzodiazepines such as 4′-chlorodiazepam (Ro5-4864). Although TSPO levels in the brain are low, they are increased after brain injury and inflammation. This finding has led to the proposed use of TSPO expression as a marker of brain injury and repair. TSPO drug ligands have been shown to participate in the control of mitochondrial respiration and function, mitochondrial steroid and neurosteroid formation, as well as apoptosis. This review and commentary will outline our current knowledge of the benefits of targeting TSPO for TBI treatment and the mechanisms underlying the neuroprotective effects of TSPO drug ligands in neurotrauma.     

4′-Chlorodiazepam Protects Mitochondria in T98G Astrocyte Cell Line from Glucose Deprivation

The translocator protein (TSPO), formerly known as the peripheral-type benzodiazepine receptor (PBR), is considered an important regulator of steroidogenesis and a potential therapeutic target in neurological disorders. Previous evidence suggests that TSPO ligands can protect cells during injury and prevent apoptosis in central nervous system (CNS) cells. However, its actions on astrocytic cells under metabolic injury are not well understood. In this study, we explored whether 4′-chlorodiazepam (Ro5–4864), a TSPO ligand, might protect astrocyte mitochondria under glucose deprivation. Our results showed that 4′-chlorodiazepam preserved cell viability and reduced nuclear fragmentation in glucose-deprived cells. These effects were accompanied by a reduced production of free radicals and maintenance of mitochondrial functions in cells treated with 4′-chlorodiazepam. Finally, our findings suggest that TSPO might be involved in reducing oxidative stress by preserving mitochondrial functions in astrocytic cells exposed to glucose withdrawal.     

From physiopathology to treatment of Alzheimer's disease

The natural and molecular history of familial or sporadic Alzheimer's disease (AD) shows that APP (amyloid protein precursor) dysfunction is a consensual central etiological factor in Alzheimer's disease (AD). This is demonstrated by 1) genetic defects involving APP gene or APP dysfunction (such as PS1 or PS2), leading to the formation of neocortical amyloid plaques in familial AD; 2) transgenic mice with these mutated genes that develop plaques; 3) both sporadic and familial AD develop plaques. But two alternatives to explain the physiopathology can be proposed: a gain of toxic function of AB peptide (reflected by the amyloid cascade hypothesis) or a loss of function of APP, a ubiquitous and well conserved protein with numerous possible neurotrophic activities. On the other hand, AD is also characterized by another inescapable degenerating process: tauopathy, an intraneuronal aggregation of tau proteins into neurofibrillary tangles. Remarkably enough, progression of tauopathy in neocortical areas fully explains the progressive clinical deficits of AD, from memory loss to aphasia, apraxia, agnosia. Also one has to bare in mind that most demented patients and most dementing neurodegenerative disorders have a tauopathy. From that, it is concluded that APP an Tau are solid therapeutic targets. But if we know that APP and Tau dysfunctions interact to boost neurodegeneration in AD, we still do no know what are the intraneuronal signaling pathways to activate or to inhibit to stop the degenerating process. There are many hypotheses and many possible approaches: the inhibition of toxicity of plaque, of AB protofibrils, or of AB oligomers inside or outside the neuron, using vaccination or ligands (Alzhemed). On the other hand, modulation of secretases that cleave APP by inhibiting those involved in the amyloidogenic pathway or by stimulating those of the non-amyloidogenic pathway, is a major route of research. Also modulation of kinases or phosphatases possibly involved in the aggregation of tau is also investigated. Because animal models are not perfectly relevant, at the end of the long and costly pathway of drug discovery, therapeutic trials are the only way to test these different hypotheses.     

Translocator protein 18 kDa is involved in the regulation of reactive gliosis

Translocator protein (18 kDa) (TSPO), previously known as peripheral-type benzodiazepine receptor, is a critical component of the mitochondrial permeability transition pore. Brain inflammation results in the induction of the expression of TSPO in glial cells and some TSPO ligands decrease reactive gliosis after brain injury. However, since some TSPO ligands are neuroprotective, their effects on reactive gliosis may be the consequence of a reduced neurodegeneration. To assess whether TSPO ligands can modulate reactive gliosis in absence of neuronal death, we have tested their effects on the inflammatory response induced in the hippocampus of male rats by the intracerebroventricular infusion of lipopolysaccharide (LPS). LPS treatment did not induce neuronal death, assessed by Fluoro jade-B staining, but increased the number of cells immunoreactive for vimentin and MHC-II, used as markers of reactive astrocytes and reactive microglia, respectively. Furthermore, LPS produced an increase in the number of proliferating microglia. The TSPO ligand PK11195 reduced the number of MHC-II immunoreactive cells and the proliferation of microglia in LPS treated rats. In contrast, another TSPO ligand, Ro5-4864, did not significantly affect the response of microglia to LPS. Neither PK11195 nor Ro5-4864 affected the LPS-mediated increase in the number of vimentin-immunoreactive astrocytes at the time point studied, although PK11195 reduced vimentin immunoreactivity. These findings identify TSPO as a potential target for controlling neural inflammation, showing that the TSPO ligand PK11195 may reduce microglia activation by a mechanism that is independent of the regulation of neuronal survival.     

Terminal hypothermic Tau.P301L mice have increased Tau phosphorylation independently of glycogen synthase kinase 3α/β

The microtubule‐associated protein Tau is responsible for a large group of neurodegenerative disorders, known as tauopathies, including Alzheimer's disease. Tauopathy result from augmented and/or aberrant phosphorylation of Tau. Besides aging and various genetic and epigenetic defects that remain largely unknown, an important non‐genetic agent that contributes is hypothermia, eventually caused by anesthesia. Remarkably, tauopathy in brains of hibernating mammals is not pathogenic, and, because it is fully reversible, is even considered to be neuroprotective. Here, we assessed the terminal phase of Tau.P301L mice and bigenic crosses with mice lacking glycogen synthase kinase 3 (GSK3)α completely, or GSK3β specifically in neurons. We also analysed biGT bigenic mice that co‐express Tau.P301L with GSK3β.S9A and develop severe forebrain tauopathy with age. We found that the precocious mortality of Tau.P301L mice was typified by hypothermia that aggravated Tau phosphorylation, but, surprisingly, independently of GSK3α/β. The important contribution of hypothermia at the time of death of mice with tauopathy suggests that body temperature should be included as a parameter in the analysis of pre‐clinical models, and, by extension, in patients suffering from tauopathy.     

In vivo imaging of microglial activation by positron emission tomography with [11C]PBR28 in the 5XFAD model of Alzheimer's disease

Microglial activation has been linked with deficits in neuronal function and synaptic plasticity in Alzheimer's disease (AD). The mitochondrial translocator protein (TSPO) is known to be upregulated in reactive microglia. Accurate visualization and quantification of microglial density by PET imaging using the TSPO tracer [¹¹C]‐R‐PK11195 has been challenging due to the limitations of the ligand. In this study, it was aimed to evaluate the new TSPO tracer [¹¹C]PBR28 as a marker for microglial activation in the 5XFAD transgenic mouse model of AD. Dynamic PET scans were acquired following intravenous administration of [¹¹C]PBR28 in 6‐month‐old 5XFAD mice and in wild‐type controls. Autoradiography with [³H]PBR28 was carried out in the same brains to further confirm the distribution of the radioligand. In addition, immunohistochemistry was performed on adjacent brain sections of the same mice to evaluate the co‐localization of TSPO with microglia. PET imaging revealed that brain uptake of [¹¹C]PBR28 in 5XFAD mice was increased compared with control mice. Moreover, binding of [³H]PBR28, measured by autoradiography, was enriched in cortical and hippocampal brain regions, coinciding with the positive staining of the microglial marker Iba‐1 and amyloid deposits in the same areas. Furthermore, double‐staining using antibodies against TSPO demonstrated co‐localization of TSPO with microglia and not with astrocytes in 5XFAD mice and human post‐mortem AD brains. The data provided support of the suitability of [¹¹C]PBR28 as a tool for in vivo monitoring of microglial activation and assessment of treatment response in future studies using animal models of AD. GLIA 2016;64:993–1006     

Axonal regeneration and neuroinflammation: roles for the translocator protein 18 kDa

After a traumatic injury of the nervous system or in the course of a neurodegenerative disease, the speed of axonal regeneration and the control of the inflammatory response are fundamental parameters of functional recovery. Spontaneous regeneration takes place in the peripheral nervous system, although the process is slow and often incomplete. There is currently no efficient treatment for enhancing axonal regeneration, including elongation speed and functional reinnervation. Ligands of the translocator protein 18 kDa (TSPO) are currently under investigation as therapeutic means for promoting neuroprotection, accelerating axonal regeneration and modulating inflammation. The mechanisms of action of TSPO ligands involve the regulation of mitochondrial activity and the stimulation of steroid biosynthesis. In the peripheral nervous system, TSPO expression is strongly up-regulated after injury, primarily in Schwann cells and macrophages, but also in neurones. Its levels return to low control values when nerve regeneration is completed, strongly supporting an important role in regenerative processes. We have demonstrated a role for the benzoxazine etifoxine in promoting axonal regeneration in the lesioned rat sciatic nerve, either after freeze-injury or complete transection. Etifoxine is already clinically approved for the treatment of anxiety disorders (Stresam(?) , Biocodex, Gentilly, France). Daily treatment with etifoxine resulted in a two-fold acceleration in axonal regeneration, as well as in a marked improvement of both the speed and quality of functional recovery. The neuroregenerative effects of etifoxine are likely to be mediated by TSPO, and they may involve an increased synthesis of pregnenolone and its metabolites, such as progesterone. After freeze-injury of the sciatic nerve, administration of etifoxine also strongly reduced the number of activated macrophages and decreased the production of the inflammatory cytokines tumour necrosis factor-α and interleukin-1β. Thus, this drug offers promise for the treatment of peripheral nerve injuries and axonal neuropathies. It may also be used as a lead compound in the development of new TSPO-based neuroprotective approaches.     

醒脑静对Aβ25-35诱导的SH-SY5Y细胞线粒体损伤的保护作用

目的 观察醒脑静注射液对Aβ_25-35诱导的SH-SY5Y细胞线粒体损伤的保护作用。方法 将培养的SH-SY5Y细胞随机分为5组,即(1)正常对照组:细胞正常培养27 h;(2)AD细胞模型组:细胞正常培养3 h,随后用老化的Aβ_25-35(25μmol/L)处理24 h;(3)醒脑静低浓度组:醒脑静注射液(5 μl/mL)预处理细胞3 h,随后用老化的Aβ_25-35(25 μmol/L)处理24 h;(4)醒脑静中浓度组:醒脑静注射液(10 μl/mL)预处理细胞3 h,随后用老化的Aβ_25-35(25μmol/L)处理24 h;(5)醒脑静高浓度组:醒脑静注射液(20 μl/mL)预处理细胞3 h,随后用老化的Aβ_25-35(25 μmol/L)处理24 h; 处理后各组细胞利用MTT法检测细胞存活率,紫外分光光度法检测细胞内ATP含量,流式细胞仪检测线粒体膜电位水平。结果 AD细胞模型组细胞存活率较正常对照组降低(P<0.05),醒脑静不同浓度组细胞存活率较AD细胞模型组均明显升高(P<0.05); AD细胞模型组细胞内ATP含量、线粒体膜电位水平较正常对照组下降(P<0.05),醒脑静不同浓度组细胞内ATP含量、线粒体膜电位水平较AD细胞模型组显著升高(P<0.05)。结论 醒脑静预处理可抑制Aβ_25-35诱导的SH-SY5Y细胞凋亡,提高细胞存活率,其机制可能与醒脑静提高细胞内ATP含量及线粒体膜电位水平而发挥线粒体保护作用有关。     

Translocator protein (18 kDa)/peripheral benzodiazepine receptor specific ligands induce microglia functions consistent with an activated state

In the brain, translocator protein (18 kDa) (TSPO), previously called peripheral benzodiazepine receptor (PBR), is a glial protein that has been extensively used as a biomarker of brain injury and inflammation. However, the functional role of TSPO in glial cells is not well characterized. In this study, we show that the TSPO-specific ligands R-PK11195 (PK) and Ro5-4864 (Ro) increased microglia proliferation and phagocytosis with no effect on migration. Both ligands increased reactive oxygen species (ROS) production, and this effect may be mediated by NADPH-oxidase. PK and Ro also produced a small but detectable increase in IL-1β release. We also examined the effect of PK and Ro on the expression of proinflammatory genes and cytokine release in lipopolysaccharide (LPS) and adenosine triphosphate (ATP) activated microglia. PK or Ro had no effect on LPS-induced increase of pro-inflammatory genes, but they both decreased the ATP-induced increase of COX-2 gene expression. Ro, but not PK, enhanced the LPS-induced release of IL-1β. However, Ro decreased the ATP-induced release of IL-1β and TNF-α, and PK decreased the ATP-induced release of TNF-α. Exposure to Ro in the presence of LPS increased the number of apoptotic microglia, an effect that could be blocked by PK. These findings show that TSPO ligands modulate cellular functions consistent with microglia activation. Further, when microglia are activated, these ligands may have therapeutic potential by reducing the expression of pro-inflammatory genes and cytokine release. Finally, Ro-like ligands may be involved in the elimination of activated microglia via apoptosis.     

Effects of Translocator Protein (18 kDa) Ligands on Microglial Activation and Neuronal Death in the Quinolinic-Acid-Injected Rat Striatum

There is evidence that excitotoxicity and prolonged microglial activation are involved in neuronal death in neurodegenerative disorders. Activated microglia express various molecules, including the translocator protein 18 kDa (TSPO; formerly known as the peripheral benzodiazepine receptor) on the outer mitochondrial membrane. The TSPO is a novel target for neuroprotective treatments which aim to reduce microglial activation. The effect of PK 11195 and three other TSPO ligands on the level of microglial activation and neuronal survival was evaluated in a quinolinic acid (QUIN) rat model of excitotoxic neurodegeneration. All three ligands were neuroprotective at a level comparable to PK 11195. All of the ligands decreased microglial activation following the injection of QUIN but had no effect on astrogliosis. Interestingly, we also observed neuroprotective effects from the vehicle, dimethyl sulfoxide (DMSO).     

Mitochondrial membrane potential decrease caused by loss of PINK1 is not due to proton leak,but to respiratory chain defects

Mutations in PTEN-induced putative kinase 1 (PINK1) cause a recessive form of Parkinson's disease (PD). PINK1 is associated with mitochondrial quality control and its partial knock-down induces mitochondrial dysfunction including decreased membrane potential and increased vulnerability against mitochondrial toxins, but the exact function of PINK1 in mitochondria has not been investigated using cells with null expression of PINK1. Here, we show that loss of PINK1 caused mitochondrial dysfunction. In PINK1-deficient (PINK1^?/?) mouse embryonic fibroblasts (MEFs), mitochondrial membrane potential and cellular ATP levels were decreased compared with those in littermate wild-type MEFs. However, mitochondrial proton leak, which reduces membrane potential in the absence of ATP synthesis, was not altered by loss of PINK1. Instead, activity of the respiratory chain, which produces the membrane potential by oxidizing substrates using oxygen, declined. H₂O₂ production rate by PINK1^?/? mitochondria was lower than PINK1^+/+ mitochondria as a consequence of decreased oxygen consumption rate, while the proportion (H₂O₂ production rate per oxygen consumption rate) was higher. These results suggest that mitochondrial dysfunctions in PD pathogenesis are caused not by proton leak, but by respiratory chain defects.     

Hyperphosphorylated Tau in an α-synuclein-overexpressing transgenic model of Parkinson's disease

Although clinically distinct diseases, tauopathies and synucleinopathies share a common genesis and mechanisms, leading to overlapping degenerative changes within neurons. In human postmortem striatum of Parkinson’s disease (PD) and PD with dementia, we have recently described elevated levels of tauopathy, indexed as increased hyperphosphorylated Tau (p‐Tau). Here we assessed tauopathy in striatum of a transgenic animal model of PD, overexpressing human α‐synuclein under the platelet‐derived growth factor promoter. At 11 months of age, large and progressive increases in p‐Tau in transgenic mice, hyperphosphorylated at sites reminiscent of Alzheimer’s disease, were noted, along with elevated levels of α‐synuclein and glycogen synthase kinase 3β phosphorylated at Tyr216 (p‐GSK‐3β), a major kinase involved in the hyperphosphorylation of Tau. Differential Triton X‐100 extraction of striata showed the presence of aggregated α‐synuclein in the transgenic mice, along with p‐Tau and p‐GSK‐3β, which was also confirmed through immunohistochemistry. After p‐Tau formation, both Tau and microtubule‐associated protein 1 (MAP1) dissociated from the cytoskeleton, consistent with the diminished ability of these cytoskeleton‐binding proteins to bind microtubules. Increases in free tubulin and actin were also noted, indicative of cytoskeleton remodeling and destabilization. In vivo magnetic resonance imaging of the transgenic animals showed a reduction in brain volume of transgenic mice, indicating substantial atrophy. From immunohistochemical studies, α‐synuclein, p‐Tau and p‐GSK‐3β were found to be overexpressed and co‐localized in large inclusion bodies, reminiscent of Lewy bodies. The elevated state of tauopathy seen in these platelet‐derived growth factor–α‐synuclein mice provides further confirmation that PD may be a tauopathic disease.     

Exploring the bi‐directional relationship between autophagy and Alzheimer’s disease

Alzheimer's disease (AD) is characterized by β‐amyloid (Aβ) deposition and Tau phosphorylation, in which its pathogenesis has not been cleared so far. The metabolism of Aβ and Tau is critically affected by the autophagy. Abnormal autophagy is thought to be involved in the pathogenesis of AD, regulating autophagy may become a new strategy for AD treatment. In the early stage of AD, the presence of Aβ and Tau can induce autophagy to promote their clearance by means of mTOR‐dependent and independent manners. As AD progress, the autophagy goes aberrant. As a result, Aβ and Tau generate continually, which aggravates both autophagy dysfunction and AD. Besides, several related genes and proteins of AD can also adapt autophagy to make an effect on the AD development. There seems to be a bi‐directional relationship between AD pathology and autophagy. At present, this article reviews this relationship from these aspects: (a) the signaling pathways of regulating autophagy; (b) the relationships between the autophagy and the processing of Aβ; (c) Aβ and Tau cause autophagy dysfunction; (d) normal autophagy promotes the clearance of Aβ and Tau; (e) the relationships between the autophagy and both genes and proteins related to AD: TFEB, miRNAs, Beclin‐1, Presenilin, and Nrf2; and (f) the small molecules regulating autophagy on AD therapy. All of the above may help to further elucidate the pathogenesis of AD and provide a theoretical basis for clinical treatment of AD.     

The effect of oxygenation level on cerebral post-traumatic apoptotsis is modulated by the 18-kDa translocator protein (also known as peripheral-type benzodiazepine receptor) in a rat model of cortical contusion

Aims: Hyperbaric hyperoxia has been shown to reduce apoptosis in brain injury. As the 18-kDa translocator protein (TSPO), also known as peripheral-type benzodiazepine receptor, is closely associated with the mitochondrial transition pore and because of its role in mitochondrial respiration and apoptosis, we hypothesized that reduction of apoptosis by hyperoxia may involve the TSPO. Methods: TSPO and transferase-mediated dUTP nick end labelling (TUNEL) immunopositivity was first assessed in cortical contusion, created by dynamic cortical deformation, by immunohistochemistry in rats exposed to normoxia [(dynamic cortical deformation (DCD)], normobaric hyperoxia or hyperbaric hyperoxia [hyperbaric oxygen therapy (HBO)]. In a second step, transmembrane mitochondrial potential (Δψ_M) and caspase 9 activity were assessed in the injured area in comparison with the noninjured hemisphere. Measurements were performed in DCD and HBO groups. A third group receiving both HBO and the TSPO ligand PK11195 was investigated as well. Results: TSPO correlated quantitatively and regionally with TUNEL immunopositivity in the perilesional area. Hyperoxia reduced both the number of TSPO expressing and TUNEL positive cells in the perilesional area, and this effect proved to be pressure dependent. After contusion, we demonstrated a dissipation of Δψ_M in isolated mitochondria and an elevation of caspase 9 activity in tissue homogenates from the contused area, both of which could be substantially reversed by hyperbaric hyperoxia. This protective effect of hyperoxia was reversed by PK11195. Conclusions: The present findings suggest that the protective effect of hyperoxia may be due to a negative regulation of the proapoptotic function of mitochondrial TSPO, including conservation of the mitochondrial membrane potential.     

S-52, a novel nootropic compound, protects against β-amyloid induced neuronal injury by attenuating mitochondrial dysfunction

Accumulating evidence suggests that β‐amyloid (Aβ)‐induced oxidative DNA damage and mitochondrial dysfunction may initiate and contribute to the progression of Alzheimer's disease (AD). This study evaluated the neuroprotective effects of S‐52, a novel nootropic compound, on Aβ‐induced mitochondrial failure. In an established paradigm of moderate cellular injury induced by Aβ, S‐52 was observed to attenuate the toxicity of Aβ to energy metabolism, mitochondrial membrane structure, and key enzymes in the electron transport chain and tricarboxylic acid cycle. In addition, S‐52 also effectively inhibited reactive oxygen species accumulation dose dependently not only in Aβ‐harmed cells but also in unharmed, normal cells. The role of S‐52 as a scavenger of free radicals is involved in the antioxidative effect of this compound. The beneficial effects on mitochondria and oxidative stress extend the neuroprotective effects of S‐52. The present study provides crucial information for better understanding the beneficial profiles of this compound and discovering novel potential drug candidates for AD therapy. © 2012 Wiley Periodicals, Inc.     

Green tea epigallocatechin-3-gallate (EGCG) and other flavonoids reduce Alzheimer's amyloid-induced mitochondrial dysfunction

Amyloid-β (Aβ)-induced mitochondrial dysfunction may play a role in the onset and progression of Alzheimer's disease (AD). Therefore, therapeutics targeted to improve mitochondrial function could be beneficial. Plant-derived flavonoids have shown promise in improving certain AD phenotypes, but the overall mechanism of action(s) through which flavonoids protect from AD is still unknown. To identify flavonoids and other natural products that may correct amyloid-induced mitochondrial dysfunction, 25 natural products were screened for their ability to restore altered mitochondrial membrane potential (MMP), reactive oxygen species (ROS) production, or ATP levels in neuroblastoma cells expressing mutant amyloid-β protein precursor (AβPP). Epigallocatechin-3-gallate (EGCG) and luteolin were identified as the top two mitochondrial restorative compounds from the in vitro screen. EGCG was further tested in vivo to determine its effects on brain mitochondrial function in an AβPP/PS-1 (presenilin 1) double mutant transgenic mouse model of AD. EGCG treatment restored mitochondrial respiratory rates, MMP, ROS production, and ATP levels by 50 to 85% in mitochondria isolated from the hippocampus, cortex, and striatum. The results of this study lend further credence to the notion that EGCG and other flavonoids, such as luteolin, are 'multipotent therapeutic agents' that not only reduce toxic levels of brain Aβ, but also hold the potential to protect neuronal mitochondrial function in AD.     

Activation of murine microglial cell lines by lipopolysaccharide and interferon-gamma causes NO-mediated decreases in mitochondrial and cellular function

Activation of murine microglial and macrophage cell lines with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) resulted in the induction of the inducible form of nitric oxide synthase (NOS) and the release of micromolar amounts of NO into the surrounding medium. The synthesis of NO was associated with increased cellular membrane damage as assessed by trypan blue dye exclusion and the leakage of lactate dehydrogenase into the cell culture medium. However, the synthesis and release of cytokines was largely unaffected. NO-mediated cell damage was also accompanied by a marked decrease in the intracellular levels of reduced glutathione and ATP. In addition, significant inhibition of mitochondrial respiratory chain enzyme activities was seen following cellular activation. However, citrate synthase activity (a mitochondrial matrix enzyme) was not detectable in the extracellular supernatants, suggesting preservation of the integrity of the mitochondrial inner membrane following activation. These effects were largely prevented by the addition of the NOS inhibitor, N-guanidino monomethyl L-arginine during the activation period. Our observations demonstrate that induction of NOS activity in microglia results in damage to the plasma membrane leading to a loss of glutathione, complex-specific inhibition of the mitochondrial electron transport chain and depletion of cellular ATP. Our data suggest that pharmacological modulation of NOS activity in activated microglia in vivo may prevent cellular damage to bystander cells such as neurons, astrocytes and oligodendrocytes, as well as to microglia themselves.     

Glucose deprivation produces a prolonged increase in sensitivity to glutamate in cultured rat cortical neurons

In this study we investigated whether the link between mitochondrial dysfunction and deregulation of Ca(2+) homeostasis preceding excitotoxic cell death is mediated by cellular deenergization. Glycolytic and/or mitochondrial ATP synthesis was inhibited with 2-deoxy-D-glucose (2DG) and oligomycin, respectively. Changes in cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) and mitochondrial membrane potential were simultaneously measured in response to low (10 microM) glutamate concentrations, using the fluorescence dyes fura-2FF and rhodamine 123. 2DG, which blocks glycolysis and also inhibits mitochondrial respiration due to depletion of pyruvate, greatly increased and accelerated glutamate-induced elevation of [Ca(2+)](c) and mitochondrial depolarization. The 2DG-induced hypersensitivity to glutamate was observed even after 150-min washout of 2DG with glucose-containing medium, suggesting a permanent deterioration of mitochondrial function. Prior blockade of only glycolytic (2DG with pyruvate) or only mitochondrial (oligomycin) ATP synthesis did not affect neuronal sensitivity to glutamate. Collectively, these studies show that to maintain the sensitivity of neurons to glutamate at control levels at least one of the cellular sources of ATP production must be intact. Either glycolysis or oxidative phosphorylation can effectively support Ca(2+) homeostasis in cultured forebrain neurons.     

Oxidative stress,mitochondria, and apoptosis

This article emphasizes the importance of mitochondria, the cellular ATP level, and the liberation of certain mitochondrial proteins for the execution phase of apoptosis. Destabilization of mitochondria results in release of these proteins. Oxidative stress and altered cellular Ca2+ homeostasis, considered to be mediators of apoptosis, synergistically decrease the mitochondrial membrane potential and lower the cellular ATP level. Conversely, stabilization of the mitochondrial membrane potential, e.g., by the protooncogene bcl-2, prevents cell death. An important process underlying mitochondrial destabilization is oxidant-induced mitochondrial Ca2+ release followed by re-uptake ("Ca2+ cycling"). Tumor necrosis factor-a induces oxygen radicals in mitochondria through ceramides, and the recently discovered mitochondrial nitric oxide synthase profoundly stimulates Ca2+ release from mitochondria through formation of nitrogen monoxide and peroxynitrite.