Microglial NMDA receptors drive pro-inflammatory responses via PARP-1/TRMP2 signaling.

Chronic neuroinflammation driven by microglia is a characteristic feature associated with numerous neurodegenerative diseases. While acute inflammation can assist with recovery and repair, prolonged microglial pro-inflammatory responses are known to exacerbate neurodegenerative processes. Yet, detrimental outcomes of extended microglial activation are counterbalanced by beneficial outcomes including phagocytosis and release of trophic factors promoting neuronal viability. Our past work has shown that the nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) is a key signaling hub driving pro-inflammatory microglia responses, but the signaling pathway maintaining PARP-1 activation remains elusive. While best understood for its role in promoting DNA repair, our group has shown that PARP-1 activity can be stimulated via Ca2+ influx-dependent ERK1/2-mediated phosphorylation. However, to date, the route of Ca2+ entry responsible for stimulating PARP-1 has not been identified. A likely candidate is via Ca2+ -permeable transient receptor potential melastatin 2 (TRPM2) channels activated downstream of PARP-1 in a cascade that involves ADP-ribose (ADPR) production by poly(ADP-ribose) glycohydrolase (PARG). Here we demonstrate that NMDA receptor (NMDAR) stimulation in primary cultured microglia induces their proliferation, morphological activation and release of pro-inflammatory mediators. These responses were contingent on the recruitment of PARP-1, PARG and Ca2+ permeable TRPM2 channels. Furthermore, we show that Ca2+ influx is necessary to activate PARP-1/TRPM2 signaling, in an ERK1/2-dependent, but DNA damage independent, manner. Our findings, showing that PARP-1/TRPM2 mediate the pro-inflammatory effects of NMDAR stimulation, provides a unifying mechanism linking elevated glutamate levels to chronic neuroinflammation.

Exposure to gestational diabetes mellitus induces neuroinflammation, derangement of hippocampal neurons, and cognitive changes in rat offspring.

BACKGROUND: Birth cohort studies link gestational diabetes mellitus (GDM) with impaired cognitive performance in the offspring. However, the mechanisms involved are unknown. We tested the hypothesis that obesity-associated GDM induces chronic neuroinflammation and disturbs the development of neuronal circuitry resulting in impaired cognitive abilities in the offspring. METHODS: In rats, GDM was induced by feeding dams a diet high in sucrose and fatty acids. Brains of neonatal (E20) and young adult (15-week-old) offspring of GDM and lean dams were analyzed by immunohistochemistry, cytokine assay, and western blotting. Young adult offspring of GDM and lean dams went also through cognitive assessment. Cultured microglial responses to elevated glucose and/or fatty acids levels were analyzed. RESULTS: In rats, impaired recognition memory was observed in the offspring of GDM dams. GDM exposure combined with a postnatal high-fat and sucrose diet resulted in atypical inattentive behavior in the offspring. These cognitive changes correlated with reduced density and derangement of Cornu Ammonis 1 pyramidal neuronal layer, decreased hippocampal synaptic integrity, increased neuroinflammatory status, and reduced expression of CX3CR1, the microglial fractalkine receptor regulating microglial pro-inflammatory responses and synaptic pruning. Primary microglial cultures that were exposed to high concentrations of glucose and/or palmitate were transformed into an activated, amoeboid morphology with increased nitric oxide and superoxide production, and altered their cytokine release profile. CONCLUSIONS: These findings demonstrate that GDM stimulates microglial activation and chronic inflammatory responses in the brain of the offspring that persist into young adulthood. Reactive gliosis correlates positively with hippocampal synaptic decline and cognitive impairments. The elevated pro-inflammatory cytokine expression at the critical period of hippocampal synaptic maturation suggests that neuroinflammation might drive the synaptic and cognitive decline in the offspring of GDM dams. The importance of microglia in this process is supported by the reduced Cx3CR1 expression as an indication of the loss of microglial control of inflammatory responses and phagocytosis and synaptic pruning in GDM offspring.

NF-κB transcriptional activation by TNFα requires phospholipase C, extracellular signal-regulated kinase 2 and poly(ADP-ribose) polymerase-1.

BACKGROUND: The nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) is required for pro-inflammatory effects of TNFα. Our previous studies demonstrated that PARP-1 mediates TNFα-induced NF-κB activation in glia. Here, we evaluated the mechanisms by which TNFα activates PARP-1 and PARP-1 mediates NF-κB activation. METHODS: Primary cultures of mouse cortical astrocytes and microglia were treated with TNFα and suitable signaling pathway modulators (pharmacological and molecular). Outcome measures included calcium imaging, PARP-1 activation status, NF-κB transcriptional activity, DNA damage assesment and cytokine relesease profiling. RESULTS: TNFα induces PARP-1 activation in the absence of detectable DNA strand breaks, as measured by the PANT assay. TNFα-induced transcriptional activation of NF-κB requires PARP-1 enzymatic activity. Enzymatic activation of PARP-1 by TNFα was blocked in Ca(2+)-free medium, by Ca(2+) chelation with BAPTA-AM, and by D609, an inhibitor of phoshatidyl choline-specific phospholipase C (PC-PLC), but not by thapsigargin or by U73112, an inhibitor of phosphatidyl inisitol-specific PLC (PI -PLC). A TNFR1 blocking antibody reduced Ca(2+) influx and PARP-1 activation. TNFα-induced PARP-1 activation was also blocked by siRNA downregulation of ERK2 and by PD98059, an inhibitor of the MEK / ERK protein kinase cascade. Moreover, TNFα-induced NF-κB (p65) transcriptional activation was absent in cells expressing PARP-1 that lacked ERK2 phosphorylation sites, while basal NF-κB transcriptional activation increased in cells expressing PARP-1 with a phosphomimetic substitution at an ERK2 phophorylation site. CONCLUSIONS: These results suggest that TNFα induces PARP-1 activation through a signaling pathway involving TNFR1, Ca(2+) influx, activation of PC-PLC, and activation of the MEK1 / ERK2 protein kinase cascade. TNFα-induced PARP-1 activation is not associated with DNA damage, but ERK2 mediated phosphorylation of PARP-1.

Inhibition of NADPH oxidase activation reduces EAE-induced white matter damage in mice.

BACKGROUND: To evaluate the role of NADPH oxidase-mediated reactive oxygen species (ROS) production in multiple sclerosis pathogenesis, we examined the effects of apocynin, an NADPH oxidase assembly inhibitor, on experimental autoimmune encephalomyelitis (EAE). METHODS: EAE was induced by immunization with myelin oligodendrocyte glycoprotein (MOG (35-55)) in C57BL/6 female mice. Three weeks after initial immunization, the mice were analyzed for demyelination, immune cell infiltration, and ROS production. Apocynin (30 mg/kg) was given orally once daily for the entire experimental course or after the typical onset of clinical symptom (15 days after first MOG injection). RESULTS: Clinical signs of EAE first appeared on day 11 and reached a peak level on day 19 after the initial immunization. The daily clinical symptoms of EAE mice were profoundly reduced by apocynin. The apocynin-mediated inhibition of the clinical course of EAE was accompanied by suppression of demyelination, reduced infiltration by encephalitogenic immune cells including CD4, CD8, CD20, and F4/80-positive cells. Apocynin reduced MOG-induced pro-inflammatory cytokines in cultured microglia. Apocynin also remarkably inhibited EAE-associated ROS production and blood-brain barrier (BBB) disruption. Furthermore, the present study found that post-treatment with apocynin also reduced the clinical course of EAE and spinal cord demyelination. CONCLUSIONS: These results demonstrate that apocynin inhibits the clinical features and neuropathological changes associated with EAE. Therefore, the present study suggests that inhibition of NADPH oxidase activation by apocynin may have a high therapeutic potential for treatment of multiple sclerosis pathogenesis.

Poly(ADP-ribose) polymerase-1-induced NAD(+) depletion promotes nuclear factor-κB transcriptional activity by preventing p65 de-acetylation.

NF-κB is a transcription factor that integrates pro-inflammatory and pro-survival responses in diverse cell types. The activity of NF-κB is regulated in part by acetylation of its p65 subunit at lysine 310, which is required for transcription complex formation. De-acetylation at this site is performed by sirtuin 1(SIRT1) and possibly other sirtuins in an NAD(+) dependent manner, such that SIRT1 inhibition promotes NF-κB transcriptional activity. It is unknown, however, whether changes in NAD(+) levels can influence p65 acetylation and cellular inflammatory responses. Poly(ADP-ribose)-1 (PARP-1) is an abundant nuclear enzyme that consumes NAD(+) in the process of forming (ADP-ribose)polymers on target proteins, and extensive PARP-1 activation can reduce intracellular NAD(+) concentrations. Here we tested the idea that PARP-1 activation can regulate NF-κB transcriptional activity by reducing NAD(+) concentrations and thereby inhibiting de-acetylation of p65. Primary astrocyte cultures were treated with the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) to induce PARP-1 activation. This resulted in sustained acetylation of p65 and increased NF-κB transcriptional activity as monitored by a κB-driven eGFP reporter gene. These effects of MNNG were negated by a PARP-1 inhibitor, in PARP-1(-/-) cells, and in PARP-1(-/-) cells transfected with a catalytically inactive PARP-1 construct, thus confirming that these effects are mediated by PARP-1 catalytic activity. The effects of PARP-1 activation were replicated by a SIRT1 inhibitor, EX-527, and were reversed by exogenous NAD(+). These findings demonstrate that PARP-1-induced changes in NAD(+) levels can modulate NF-κB transcriptional activity through effects on p65 acetylation.

Cortical astrocyte N-methyl-D-aspartate receptors influence whisker barrel activity and sensory discrimination in mice.

Astrocytes express ionotropic receptors, including N-methyl-D-aspartate receptors (NMDARs). However, the contribution of NMDARs to astrocyte-neuron interactions, particularly in vivo, has not been elucidated. Here we show that a knockdown approach to selectively reduce NMDARs in mouse cortical astrocytes decreases astrocyte Ca2+ transients evoked by sensory stimulation. Astrocyte NMDAR knockdown also impairs nearby neuronal circuits by elevating spontaneous neuron activity and limiting neuronal recruitment, synchronization, and adaptation during sensory stimulation. Furthermore, this compromises the optimal processing of sensory information since the sensory acuity of the mice is reduced during a whisker-dependent tactile discrimination task. Lastly, we rescue the effects of astrocyte NMDAR knockdown on neurons and improve the tactile acuity of the animal by supplying exogenous ATP. Overall, our findings show that astrocytes can respond to nearby neuronal activity via their NMDAR, and that these receptors are an important component for purinergic signaling that regulate astrocyte-neuron interactions and cortical sensory discrimination in vivo.

Poly(ADP-ribose) polymerase 2 contributes to neuroinflammation and neurological dysfunction in mouse experimental autoimmune encephalomyelitis.

BACKGROUND: Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis characterized by entry of activated T cells and antigen presenting cells into the central nervous system and subsequent autoimmune destruction of nerve myelin. Previous studies revealed that non-selective inhibition of poly(ADP-ribose) polymerases (PARPs) 1 and 2 protect against neuroinflammation and motor dysfunction associated with EAE, but the role of the PARP-2 isoform has not yet been investigated selectively. RESULTS: EAE was induced in mice lacking PARP-2, and neurological EAE signs, blood-spine barrier (BSB) permeability, demyelination and inflammatory infiltration were monitored for 35 days after immunization. Mice lacking PARP-2 exhibited significantly reduced overall disease burden and peak neurological dysfunction. PARP-2 deletion also significantly delayed EAE onset and reduced BSB permeability, demyelination and central nervous system (CNS) markers of proinflammatory Th1 and Th17 T helper lymphocytes. CONCLUSIONS: This study represents the first description of a significant role for PARP-2 in neuroinflammation and neurological dysfunction in EAE.

CX3CR1 protein signaling modulates microglial activation and protects against plaque-independent cognitive deficits in a mouse model of Alzheimer disease.

Aberrant microglial activation has been proposed to contribute to the cognitive decline in Alzheimer disease (AD), but the underlying molecular mechanisms remain enigmatic. Fractalkine signaling, a pathway mediating the communication between microglia and neurons, is deficient in AD brains and down-regulated by amyloid-ß. Although fractalkine receptor (CX3CR1) on microglia was found to regulate plaque load, no functional effects have been reported. Our study demonstrates that CX3CR1 deficiency worsens the AD-related neuronal and behavioral deficits. The effects were associated with cytokine production but not with plaque deposition. Ablation of CX3CR1 in mice overexpressing human amyloid precursor protein enhanced Tau pathology and exacerbated the depletion of calbindin in the dentate gyrus. The levels of calbindin in the dentate gyrus correlated negatively with those of tumor necrosis factor α and interleukin 6, suggesting neurotoxic effects of inflammatory factors. Functionally, removing CX3CR1 in human amyloid precursor protein mice worsened the memory retention in passive avoidance and novel object recognition tests, and their memory loss in the novel object recognition test is associated with high levels of interleukin 6. Our findings identify CX3CR1 as a key microglial pathway in protecting against AD-related cognitive deficits that are associated with aberrant microglial activation and elevated inflammatory cytokines.

Recurrent/moderate hypoglycemia induces hippocampal dendritic injury, microglial activation, and cognitive impairment in diabetic rats.

BACKGROUND: Recurrent/moderate (R/M) hypoglycemia is common in type 1 diabetes. Although mild or moderate hypoglycemia is not life-threatening, if recurrent, it may cause cognitive impairment. In the present study, we sought to determine whether R/M hypoglycemia leads to neuronal death, dendritic injury, or cognitive impairment. METHODS: The experiments were conducted in normal and in diabetic rats. Rats were subjected to moderate hypoglycemia by insulin without anesthesia. Oxidative stress was evaluated by 4-Hydroxy-2-nonenal immunostaining and neuronal death was determined by Fluoro-Jade B staining 7 days after R/M hypoglycemia. To test whether oxidative injury caused by NADPH oxidase activation, an NADPH oxidase inhibitor, apocynin, was used. Cognitive function was assessed by Barnes maze and open field tests at 6 weeks after R/M hypoglycemia. RESULTS: The present study found that oxidative injury was detected in the dendritic area of the hippocampus after R/M hypoglycemia. Sparse neuronal death was found in the cortex, but no neuronal death was detected in the hippocampus. Significant cognitive impairment and thinning of the CA1 dendritic region was detected 6 weeks after hypoglycemia. Oxidative injury, cognitive impairment, and hippocampal thinning after R/M hypoglycemia were more severe in diabetic rats than in non-diabetic rats. Oxidative damage in the hippocampal CA1 dendritic area and microglial activation were reduced by the NADPH oxidase inhibitor, apocynin. CONCLUSION: The present study suggests that oxidative injury of the hippocampal CA1 dendritic region by R/M hypoglycemia is associated with chronic cognitive impairment in diabetic patients. The present study further suggests that NADPH oxidase inhibition may prevent R/M hypoglycemia-induced hippocampal dendritic injury.

Microglial activation induced by brain trauma is suppressed by post-injury treatment with a PARP inhibitor.

BACKGROUND: Traumatic brain injury (TBI) induces activation of microglia. Activated microglia can in turn increase secondary injury and impair recovery. This innate immune response requires hours to days to become fully manifest, thus providing a clinically relevant window of opportunity for therapeutic intervention. Microglial activation is regulated in part by poly(ADP-ribose) polymerase-1 (PARP-1). Inhibition of PARP-1 activity suppresses NF-kB-dependent gene transcription and thereby blocks several aspects of microglial activation. Here we evaluated the efficacy of a PARP inhibitor, INO-1001, in suppressing microglial activation after cortical impact in the rat. METHODS: Rats were subjected to controlled cortical impact and subsequently treated with 10 mg/kg of INO-1001 (or vehicle alone) beginning 20 - 24 hours after the TBI. Brains were harvested at several time points for histological evaluation of inflammation and neuronal survival, using markers for microglial activation (morphology and CD11b expression), astrocyte activation (GFAP), and neuronal survival (NeuN). Rats were also evaluated at 8 weeks after TBI using measures of forelimb dexterity: the sticky tape test, cylinder test, and vermicelli test. RESULTS: Peak microglial and astrocyte activation was observed 5 to 7 days after this injury. INO-1001 significantly reduced microglial activation in the peri-lesion cortex and ipsilateral hippocampus. No rebound inflammation was observed in rats that were treated with INO-1001 or vehicle for 12 days followed by 4 days without drug. The reduced inflammation was associated with increased neuronal survival in the peri-lesion cortex and improved performance on tests of forelimb dexterity conducted 8 weeks after TBI. CONCLUSIONS: Treatment with a PARP inhibitor for 12 days after TBI, with the first dose given as long as 20 hours after injury, can reduce inflammation and improve histological and functional outcomes.

Prevention of hypoglycemia-induced neuronal death by minocycline.

Diabetic patients who attempt strict management of blood glucose levels frequently experience hypoglycemia. Severe and prolonged hypoglycemia causes neuronal death and cognitive impairment. There is no effective tool for prevention of these unwanted clinical sequelae. Minocycline, a second-generation tetracycline derivative, has been recognized as an anti-inflammatory and neuroprotective agent in several animal models such as stroke and traumatic brain injury. In the present study, we tested whether minocycline also has protective effects on hypoglycemia-induced neuronal death and cognitive impairment. To test our hypothesis we used an animal model of insulin-induced acute hypoglycemia. Minocycline was injected intraperitoneally at 6 hours after hypoglycemia/glucose reperfusion and injected once per day for the following 1 week. Histological evaluation for neuronal death and microglial activation was performed from 1 day to 1 week after hypoglycemia. Cognitive evaluation was conducted 6 weeks after hypoglycemia. Microglial activation began to be evident in the hippocampal area at 1 day after hypoglycemia and persisted for 1 week. Minocycline injection significantly reduced hypoglycemia-induced microglial activation and myeloperoxidase (MPO) immunoreactivity. Neuronal death was significantly reduced by minocycline treatment when evaluated at 1 week after hypoglycemia. Hypoglycemia-induced cognitive impairment is also significantly prevented by the same minocycline regimen when subjects were evaluated at 6 weeks after hypoglycemia. Therefore, these results suggest that delayed treatment (6 hours post-insult) with minocycline protects against microglial activation, neuronal death and cognitive impairment caused by severe hypoglycemia. The present study suggests that minocycline has therapeutic potential to prevent hypoglycemia-induced brain injury in diabetic patients.

N-acetylcysteine prevents loss of dopaminergic neurons in the EAAC1-/- mouse.

OBJECTIVE: Dopaminergic neuronal death in Parkinson's disease (PD) is accompanied by oxidative stress and preceded by glutathione depletion. The development of disease-modifying therapies for PD has been hindered by a paucity of animal models that mimic these features and demonstrate an age-related progression. The EAAC1(-/-) mouse may be useful in this regard, because EAAC1(-/-) mouse neurons have impaired neuronal cysteine uptake, resulting in reduced neuronal glutathione content and chronic oxidative stress. Here we aimed to (1) characterize the age-related changes in nigral dopaminergic neurons in the EAAC1(-/-) mouse, and (2) use the EAAC1(-/-) mouse to evaluate N-acetylcysteine, a membrane-permeable cysteine pro-drug, as a potential disease-modifying intervention for PD. METHODS: Wild-type mice, EAAC1(-/-) mice, and EAAC1(-/-) mice chronically treated with N-acetylcysteine were evaluated at serial time points for evidence of oxidative stress, dopaminergic cell death, and motor abnormalities. RESULTS: EAAC1(-/-) mice showed age-dependent loss of dopaminergic neurons in the substantia nigra pars compacta, with more than 40% of these neurons lost by age 12 months. This neuronal loss was accompanied by increased nitrotyrosine formation, nitrosylated α-synuclein, and microglial activation. These changes were substantially reduced in mice that received N-acetylcysteine. INTERPRETATION: These findings suggest that the EAAC1(-/-) mouse may be a useful model of the chronic neuronal oxidative stress that occurs in PD. The salutary effects of N-acetylcysteine in this mouse model provide an impetus for clinical evaluation of glutathione repletion in PD.

NAD+ depletion is necessary and sufficient for poly(ADP-ribose) polymerase-1-mediated neuronal death.

Poly(ADP-ribose)-1 (PARP-1) is a key mediator of cell death in excitotoxicity, ischemia, and oxidative stress. PARP-1 activation leads to cytosolic NAD(+) depletion and mitochondrial release of apoptosis-inducing factor (AIF), but the causal relationships between these two events have been difficult to resolve. Here, we examined this issue by using extracellular NAD(+) to restore neuronal NAD(+) levels after PARP-1 activation. Exogenous NAD(+) was found to enter neurons through P2X(7)-gated channels. Restoration of cytosolic NAD(+) by this means prevented the glycolytic inhibition, mitochondrial failure, AIF translocation, and neuron death that otherwise results from extensive PARP-1 activation. Bypassing the glycolytic inhibition with the metabolic substrates pyruvate, acetoacetate, or hydroxybutyrate also prevented mitochondrial failure and neuron death. Conversely, depletion of cytosolic NAD(+) with NAD(+) glycohydrolase produced a block in glycolysis inhibition, mitochondrial depolarization, AIF translocation, and neuron death, independent of PARP-1 activation. These results establish NAD(+) depletion as a causal event in PARP-1-mediated cell death and place NAD(+) depletion and glycolytic failure upstream of mitochondrial AIF release.

EAAC1 gene deletion alters zinc homeostasis and exacerbates neuronal injury after transient cerebral ischemia.

EAAC1 is a neuronal glutamate and cysteine transporter. EAAC1 uptake of cysteine provides substrate for neuronal glutathione synthesis, which plays a key role in both antioxidant defenses and intracellular zinc binding. Here we evaluated the role of EAAC1 in neuronal resistance to ischemia. EAAC1(-/-) mice subjected to transient cerebral ischemia exhibited twice as much hippocampal neuronal death as wild-type mice and a corresponding increase in microglial activation. EAAC1(-/-) mice also had elevated vesicular and cytosolic zinc concentrations in hippocampal CA1 neurons and an increased zinc translocation to postsynaptic neurons after ischemia. Treatment of the EAAC1(-/-) mice with N-acetyl cysteine restored neuronal glutathione concentrations and normalized basal zinc levels in the EAAC1(-/-) mice. Treatment of the EAAC1(-/-) mice with either N-acetyl cysteine or with zinc chelators reduced ischemia-induced zinc translocation, superoxide production, and neuron death. These findings suggest that cysteine uptake by EAAC1 is important for zinc homeostasis and neuronal antioxidant function under ischemic conditions.

Poly(ADP-ribose)polymerase-1 modulates microglial responses to amyloid ß.

BACKGROUND: Amyloid ß (Aß) accumulates in Alzheimer's disease (AD) brain. Microglial activation also occurs in AD, and this inflammatory response may contribute to disease progression. Microglial activation can be induced by Aß, but the mechanisms by which this occurs have not been defined. The nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) regulates microglial activation in response to several stimuli through its interactions with the transcription factor, NF-κB. The purpose of this study was to evaluate whether PARP-1 activation is involved in Aß-induced microglial activation, and whether PARP-1 inhibition can modify microglial responses to Aß. METHODS: hAPP(J20) mice, which accumulate Aß with ageing, were crossed with PARP-1(-/-) mice to assess the effects of PARP-1 depletion on microglial activation, hippocampal synaptic integrity, and cognitive function. Aß peptide was also injected into brain of wt and PARP-1(-/-) mice to directly determine the effects of PARP-1 on Aß-induced microglial activation. The effect of PARP-1 on Aß-induced microglial cytokine production and neurotoxicity was evaluated in primary microglia cultures and in microglia-neuron co-cultures, utilizing PARP-1(-/-) cells and a PARP-1 inhibitor. NF-κB activation was evaluated in microglia infected with a lentivirus reporter gene. RESULTS: The hAPP(J20) mice developed microglial activation, reduced hippocampal CA1 calbindin expression, and impaired novel object recognition by age 6 months. All of these features were attenuated in hAPP(J20)/PARP-1(-/-) mice. Similarly, Aß(1-42) injected into mouse brain produced a robust microglial response in wild-type mice, and this was blocked in mice lacking PARP-1 expression or activity. Studies using microglial cultures showed that PARP-1 activity was required for Aß-induced NF-κB activation, morphological transformation, NO release, TNFα release, and neurotoxicity. Conversely, PARP-1 inhibition increased release of the neurotrophic factors TGFß and VEGF, and did not impair microglial phagocytosis of Aß peptide. CONCLUSIONS: These results identify PARP-1 as a requisite and previously unrecognized factor in Aß-induced microglial activation, and suggest that the effects of PARP-1 are mediated, at least in part, by its interactions with NF-κB. The suppression of Aß-induced microglial activation and neurotoxicity by PARP-1 inhibition suggests this approach could be useful in AD and other disorders in which microglial neurotoxicity may contribute.

PARP-DNA trapping ability of PARP inhibitors jeopardizes astrocyte viability: Implications for CNS disease therapeutics.

There is emerging interest in the role of poly(ADP-ribose) polymerase-1 (PARP-1) in neurodegeneration and potential of its therapeutic targeting in neurodegenerative disorders. New generations of PARP inhibitors exhibit polypharmacological properties; they do not only block enzymatic activity with lower doses, but also alter how PARP-1 interacts with DNA. While these new inhibitors have proven useful in cancer therapy due to their ability to kill cancer cell, their use in neurodegenerative disorders has an opposite goal: cell protection. We hypothesize that newer generation PARP-1 inhibitors jeopardize the viability of dividing CNS cells by promoting DNA damage upon the PARP-DNA interaction. Using enriched murine astrocyte cultures, our study evaluates the effects of a variety of drugs known to inhibit PARP; talazoparib, olaparib, PJ34 and minocycline. Despite similar PARP enzymatic inhibiting activities, we show here that these drugs result in varied cell viability. Talazoparib and olaparib reduce astrocyte growth in a dose-dependent manner, while astrocytes remain unaffected by PJ34 and minocycline. Similarly, PJ34 and minocycline do not jeopardize DNA integrity, while treatment with talazoparib and olaparib promote DNA damage. These two drugs impact astrocytes similarly in basal conditions and upon nitrosative stress, a pathological condition typical for neurodegeneration. Mechanistic assessment revealed that talazoparib and olaparib promote PARP trapping onto DNA in a dose-dependent manner, while PJ34 and minocycline do not induce PARP-DNA trapping. This study provides unique insight into the selective use of PARP inhibitors to treat neurodegenerative disorders whereby inhibition of PARP enzymatic activity must occur without deleteriously trapping PARP onto DNA.

Zinc triggers microglial activation.

Microglia are resident immune cells of the CNS. When stimulated by infection, tissue injury, or other signals, microglia assume an activated, "ameboid" morphology and release matrix metalloproteinases, reactive oxygen species, and other proinflammatory factors. This innate immune response augments host defenses, but it can also contribute to neuronal death. Zinc is released by neurons under several conditions in which microglial activation occurs, and zinc chelators can reduce neuronal death in animal models of cerebral ischemia and neurodegenerative disorders. Here, we show that zinc directly triggers microglial activation. Microglia transfected with a nuclear factor-kappaB (NF-kappaB) reporter gene showed a severalfold increase in NF-kappaB activity in response to 30 microm zinc. Cultured mouse microglia exposed to 15-30 microm zinc increased nitric oxide production, increased F4/80 expression, altered cytokine expression, and assumed the activated morphology. Zinc-induced microglial activation was blocked by inhibiting NADPH oxidase, poly(ADP-ribose) polymerase-1 (PARP-1), or NF-kappaB activation. Zinc injected directly into mouse brain induced microglial activation in wild-type mice, but not in mice genetically lacking PARP-1 or NADPH oxidase activity. Endogenous zinc release, induced by cerebral ischemia-reperfusion, likewise induced a robust microglial reaction, and this reaction was suppressed by the zinc chelator CaEDTA. Together, these results suggest that extracellular zinc triggers microglial activation through the sequential activation of NADPH oxidase, PARP-1, and NF-kappaB. These findings identify a novel trigger for microglial activation and a previously unrecognized mechanism by which zinc may contribute to neurological disorders.

Aberrant cardiolipin metabolism is associated with cognitive deficiency and hippocampal alteration in tafazzin knockdown mice.

Cardiolipin (CL) is a key mitochondrial phospholipid essential for mitochondrial energy production. CL is remodeled from monolysocardiolipin (MLCL) by the enzyme tafazzin (TAZ). Loss-of-function mutations in the gene which encodes TAZ results in a rare X-linked disorder called Barth Syndrome (BTHS). The mutated TAZ is unable to maintain the physiological CL:MLCL ratio, thus reducing CL levels and affecting mitochondrial function. BTHS is best known as a cardiac disease, but has been acknowledged as a multi-syndrome disorder, including cognitive deficits. Since reduced CL levels has also been reported in numerous neurodegenerative disorders, we examined how TAZ-deficiency impacts cognitive abilities, brain mitochondrial respiration and the function of hippocampal neurons and glia in TAZ knockdown (TAZ kd) mice. We have identified for the first time the profile of changes that occur in brain phospholipid content and composition of TAZ kd mice. The brain of TAZ kd mice exhibited reduced TAZ protein expression, reduced total CL levels and a 19-fold accumulation of MLCL compared to wild-type littermate controls. TAZ kd brain exhibited a markedly distinct profile of CL and MLCL molecular species. In mitochondria, the activity of complex I was significantly elevated in the monomeric and supercomplex forms with TAZ-deficiency. This corresponded with elevated mitochondrial state I respiration and attenuated spare capacity. Furthermore, the production of reactive oxygen species was significantly elevated in TAZ kd brain mitochondria. While motor function remained normal in TAZ kd mice, they showed significant memory deficiency based on novel object recognition test. These results correlated with reduced synaptophysin protein levels and derangement of the neuronal CA1 layer in hippocampus. Finally, TAZ kd mice had elevated activation of brain immune cells, microglia compared to littermate controls. Collectively, our findings demonstrate that TAZ-mediated remodeling of CL contributes significantly to the expansive distribution of CL molecular species in the brain, plays a key role in mitochondria respiratory activity, maintains normal cognitive function, and identifies the hippocampus as a potential therapeutic target for BTHS.

Use of a poly(ADP-ribose) polymerase inhibitor to suppress inflammation and neuronal death after cerebral ischemia-reperfusion.

BACKGROUND AND PURPOSE: Most stroke patients do not present for medical treatment until several hours after onset of brain ischemia. Consequently, neuroprotective strategies are required with comparably long therapeutic windows. Poly(ADP-ribose) polymerase inhibitors such as PJ34 are known to suppress microglial activation, a postischemic event that may contribute to neuronal death. We evaluated the effects of PJ34 administered 8 hours after transient forebrain ischemia. METHODS: Rats were subjected to 10 minutes of forebrain ischemia and treated with PJ34 for 7 days beginning 8 hours after reperfusion. Activated microglia and infiltrating macrophages were evaluated at serial time points between zero and 14 days after ischemia by immunostaining for CD11b. CA1 neuronal survival was evaluated 7 days after ischemia. RESULTS: Rats treated with PJ34 showed a near-complete inhibition of microglia/macrophage activation (evaluated on day 5) and an 84% reduction in CA1 neuronal death. CONCLUSIONS: Administration of PJ34 as late as 8 hours after transient ischemia-reperfusion has a large protective effect on CA1 survival. This effect may be mediated by suppression of the postischemic brain inflammatory response.

Multiple roles for poly(ADP-ribose)polymerase-1 in neurological disease.

Poly(ADP-ribose)polymerase-1 (PARP-1) is a nuclear protein activated by DNA damage. PARP-1 activation is associated in DNA repair, cell death and inflammation. Since oxidative stress induced robust DNA damage and wide spread inflammatory responses are common pathologies of various CNS diseases, the interest toward PARP-1 as a therapeutic target has peaked. This review introduces mechanism of PARP-1 activation, the role of PARP-1 in cell physiology and pathology, and discusses the potential of PARP-1 inhibition as a therapy in acute and chronic CNS diseases.