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.

Poly(ADP-ribose) polymerase-1 regulates microglia mediated decrease of endothelial tight junction integrity.

Alzheimer's disease pathology includes, beside neuronal damage, reactive gliosis and reduced blood-brain barrier (BBB) integrity. Microglia are intimately associated with the BBB and upon AD pathology, pro-inflammatory responses of microglia could contribute to BBB damage. To study whether microglia can directly affect BBB integrity, the effects of amyloid beta (Aß) -stimulated primary murine microglia on co-cultured mouse brain endothelial cells (bEnd3) and murine astrocyte cultures were assessed. We also assessed whether microglial phenotype modulation via poly(ADP-ribose) polymerase-1 (PARP-1) inhibition/ablation can reverse microglial impact on these BBB forming cells. Unstimulated microglia promoted expression of tight junction proteins (TJPs), zonula ocluden-1 (ZO-1) and occludin in co-cultured endothelia cells, whereas Aß-stimulated microglia reduced endothelial expression of ZO-1 and occludin. Astrocytes co-cultured with microglia showed elevated glial fibrillary acidic protein (GFAP) expression, which was further increased if microglia had been stimulated with Aß. Aß induced microglial release of nitric oxide (NO) and tumour necrosis factor alpha (TNFα), which resulted in reduced endothelial expression of TJPs and increased paracellular permeability. Microglial PARP-1 inhibition attenuated these Aß-induced events. These findings demonstrate that PARP-1 mediated microglial responses (NO and TNFα) can directly reduce BBB integrity by promoting TJP degradation, increasing endothelial cell permeability and inducing astrogliosis. PARP-1 as a modulator of microglial phenotype can prevent microglial BBB damaging events, and thus is a potential therapeutic target.

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.

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.

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.

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.

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.

Poly(ADP-ribose) polymerase-1 promotes microglial activation, proliferation, and matrix metalloproteinase-9-mediated neuron death.

Activated microglia contribute to cell death in ischemic and neurodegenerative disorders of the CNS. Microglial activation is regulated in part by NF-kappaB, and the nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) enhances NF-kappaB binding to DNA. In this study, the role of PARP-1 in microglia-mediated neurotoxicity was assessed using microglia from wild-type (wt) and PARP-1-/- mice. Cultured microglia were incubated with TNF-alpha, a cytokine that is up-regulated in many neurological disorders. When stimulated with TNF-alpha, wt microglia proliferated, underwent morphological changes characteristic of activation, and killed neurons placed in coculture. The effects of TNF-alpha were markedly attenuated both in PARP-1-/- microglia and in wt microglia treated with the PARP enzymatic inhibitor 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2h)-isoquinolinone. These effects were also blocked by (E)-3-(4-methylphenylsulfonyl)-2-propenenenitrile, which inhibits translocation of NF-kappaB to the nucleus. TNF-alpha also up-regulated microglial release of matrix metalloproteinase-9 (MMP-9), an enzyme with potential neurotoxic properties that is transcriptionally regulated by NF-kappaB. This up-regulation was blocked in PARP-1-/- microglia and in wt microglia by the PARP inhibitor 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2h)-isoquinolinone. Microglia from MMP-9-/- mice were used to evaluate the contribution of MMP-9 to microglial neurotoxicity. MMP-9-/- microglia treated with TNF-alpha showed substantially reduced neurotoxicity relative to the wt microglia. TNF-alpha-stimulated wt microglia treated with the MMP inhibitor ilomastat also showed reduced neurotoxicity. These findings suggest that PARP-1 activation is required for both TNF-alpha-induced microglial activation and the neurotoxicity resulting from TNF-alpha-induced MMP-9 release.