The prominence of autophagic neuronal death in the ischemic penumbra and the neuroprotective efficacy of postischemic autophagy inhibition indicate that autophagy should be a primary target in the treatment of neonatal cerebral ischemia.

NMDA receptors (NMDARs) mediate ischemic brain damage, for which interactions between the C termini of NR2 subunits and PDZ domain proteins within the NMDAR signaling complex (NSC) are emerging therapeutic targets. However, expression of NMDARs in a non-neuronal context, lacking many NSC components, can still induce cell death. Moreover, it is unclear whether targeting the NSC will impair NMDAR-dependent prosurvival and plasticity signaling. We show that the NMDAR can promote death signaling independently of the NR2 PDZ ligand, when expressed in non-neuronal cells lacking PSD-95 and neuronal nitric oxide synthase (nNOS), key PDZ proteins that mediate neuronal NMDAR excitotoxicity. However, in a non-neuronal context, the NMDAR promotes cell death solely via c-Jun N-terminal protein kinase (JNK), whereas NMDAR-dependent cortical neuronal death is promoted by both JNK and p38. NMDARdependent pro-death signaling via p38 relies on neuronal context, although death signaling by JNK, triggered by mitochondrial reactive oxygen species production, does not. NMDAR-dependent p38 activation in neurons is triggered by submembranous Ca 2ϩ , and is disrupted by NOS inhibitors and also a peptide mimicking the NR2B PDZ ligand (TAT-NR2B9c). TAT-NR2B9c reduced excitotoxic neuronal death and p38-mediated ischemic damage, without impairing an NMDAR-dependent plasticity model or prosurvival signaling to CREB or Akt. TAT-NR2B9c did not inhibit JNK activation, and synergized with JNK inhibitors to ameliorate severe excitotoxic neuronal loss in vitro and ischemic cortical damage in vivo. Thus, NMDAR-activated signals comprise pro-death pathways with differing requirements for PDZ protein interactions. These signals are amenable to selective inhibition, while sparing synaptic plasticity and prosurvival signaling.

Activation of poly(ADP-ribose) polymerase (PARP) is an important factor in the pathogenesis of various cardiovascular and inflammatory diseases. Here, we report that the gender-specific inflammatory response is preferentially down-regulated by PARP in male animals. Female mice produce less tumor necrosis factor-␣ and macrophage inflammatory protein-1␣ in response to systemic inflammation induced by endotoxin than male mice and are resistant to endotoxin-induced mortality. Pharmacological inhibition of PARP is effective in reducing inflammatory mediator production and mortality in male, but not in female, mice. Ovariectomy partially reverses the protection seen in female mice. Endotoxin-induced PARP activation in circulating leukocytes is reduced in male, but not female, animals by pharmacological PARP inhibition, as shown by flow cytometry. Pretreatment of male mice with 17-␤-estradiol prevents endotoxin-induced hepatic injury and reduces poly(ADPribosyl)ation in vivo. In male, but not female, animals, endotoxin induces an impairment of the endothelium-dependent relaxant responses, which is prevented by PARP inhibition. In vitro oxidant-induced PARP activation is reduced in cultured cells placed in female rat serum compared with male serum. Estrogen does not directly inhibit the enzymatic activity of PARP in vitro. However, PARP and estrogen receptor ␣ form a complex, which binds to DNA in vitro, and the DNA binding of this complex is enhanced by estrogen. Thus, estrogen may anchor PARP to estrogen receptor ␣ and to the DNA and prevent its recognition of DNA strand breaks and hence its activation. In conclusion, the gender difference in the inflammatory response shows preferential modulation by PARP in male animals.

Increased production of reactive oxygen and nitrogen species has recently been implicated in the pathogenesis of cardiac and endothelial dysfunction associated with atherosclerosis, hypertension, and aging. Oxidant-induced cell injury triggers the activation of nuclear enzyme poly(ADP-ribose) polymerase (PARP), which in turn contributes to cardiac and vascular dysfunction in various pathophysiological conditions including diabetes, reperfusion injury, circulatory shock, and aging. Here, we investigated the effect of a new PARP inhibitor, INO-1001, on cardiac and endothelial dysfunction associated with advanced aging using Millar's new Aria pressure-volume conductance system and isolated aortic rings. Young adult (3 months old) and aging (24 months old) Fischer rats were treated for 2 months with vehicle, or the potent PARP inhibitor INO-1001. In the vehicle-treated aging animals, there was a marked reduction of both systolic and diastolic cardiac function and loss of endothelial relaxant responsiveness of aortic rings to acetylcholine. Treatment with INO-1001 improved cardiac performance in aging animals and also acetylcholine-induced, nitric oxide-mediated vascular relaxation. Thus, pharmacological inhibition of PARP may represent a novel approach to improve cardiac and vascular dysfunction associated with aging.Poly(ADP-ribose) polymerase (PARP) is the most abundant nuclear enzyme of eukaryotic cells. When activated by DNA single-strand breaks, PARP initiates an energy-consuming cycle by transferring ADP ribose units from NAD ϩ to nuclear proteins. This process results in rapid depletion of the intracellular NAD ϩ and ATP pools, slowing the rate of glycolysis and mitochondrial respiration, eventually leading to cellular dysfunction and death (reviewed in Virá g and Szabó 2002). PARP can also regulate the expression of various inflammatory mediators such as inducible nitric-oxide synthase, tumor necrosis factor-␣, and intracellular adhesion molecule-1. Suppression of expression of inducible nitric-oxide synthase, tumor necrosis factor-␣, and intracellular adhesion molecule-1 has been reported in PARP-deficient mice and in the presence of pharmacological inhibitors of the enzyme (reviewed in Virá g and Szabó 2002).Overactivation of PARP represents an important mechanism of tissue damage in pathological conditions associated with oxidative/nitrosative stress, including myocardial reperfusion injury, stroke, circulatory shock, autoimmune ␤-cell destruction, diabetic complications, and various forms of heart failure (Szabó et al., 1997;Thiemermann et al., 1997;Zingarelli et al., 1998; Soriano et al., 2001a,b; Pacher et al., 2002a-f;Virá g and Szabó 2002;Szabó 2004a

Recent studies showed that endocytosis is enhanced in neurons exposed to an excitototoxic stimulus. We here confirm and analyze this new phenomenon using dissociated cortical neuronal cultures. NMDA-induced uptake (FITC-dextran or FITC or horseradish peroxidase) occurs in these cultures and is due to endocytosis, not to cell entry through damaged membranes; it requires an excitotoxic dose of NMDA and is dependent on extracellular calcium, but occurs early, while the neuron is still intact and viable. It involves two components, NMDA-induced and constitutive, with different characteristics. Neither component involves specific binding of the endocytosed molecules to a saturable receptor. Strikingly, molecules internalized by the NMDA-induced component are targeted to neuronal nuclei. This component, but not the constitutive one, is blocked by a c-Jun N-terminal protein kinase inhibitor. In conclusion, an excitotoxic dose of NMDA triggers c-Jun N-terminal protein kinase-dependent endocytosis in cortical neuronal cultures, providing an in vitro model of the excitotoxicity-induced endocytosis reported in intact tissues.

Angiotensin II (AII) contributes to the pathogenesis of many cardiovascular disorders. Oxidant-mediated activation of poly(adenosine diphosphate-ribose) polymerase (PARP) plays a role in the development of endothelial dysfunction and the pathogenesis of various cardiovascular diseases. We have investigated whether activation of the nuclear enzyme PARP contributes to the development of AII-induced endothelial dysfunction. AII in cultured endothelial cells induced DNA single-strand breakage and dose-dependently activated PARP, which was inhibited by the AII subtype 1 receptor antagonist, losartan; the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, apocynin; and the nitric oxide synthase inhibitor, N-nitro-L-arginine methyl ester. Infusion of sub-pressor doses of AII to rats for 7 to 14 d induced the development of endothelial dysfunction ex vivo. The PARP inhibitors PJ34 or INO-1001 prevented the development of the endothelial dysfunction and restored normal endothelial function. Similarly, PARP-deficient mice infused with AII for 7 d were found resistant to the AII-induced development of endothelial dysfunction, as opposed to the wild-type controls. In spontaneously hypertensive rats there was marked PARP activation in the aorta, heart, and kidney. The endothelial dysfunction, the cardiovascular alterations and the activation of PARP were prevented by the angiotensin-converting enzyme inhibitor enalapril. We conclude that AII, via AII receptor subtype 1 activation and reactive oxygen and nitrogen species generation, triggers DNA breakage, which activates PARP in the vascular endothelium, leading to the development of endothelial dysfunction in hypertension.

Clathrin-dependent endocytosis is mediated by a tightly regulated network of molecular interactions that provides essential protein-protein and protein-lipid binding activities. Here we report the hydrolysis of the ␣-and ␤2-subunits of the tetrameric adaptor protein complex 2 by calpain. Calcium-dependent ␣-and ␤2-adaptin hydrolysis was observed in several rat tissues, including brain and primary neuronal cultures. Neuronal ␣-and ␤2-adaptin cleavage was inducible by glutamate stimulation and was accompanied by the decreased endocytosis of transferrin. Heterologous expression of truncated forms of the ␤2-adaptin subunit significantly decreased the membrane recruitment of clathrin and inhibited clathrin-mediated receptor endocytosis. Moreover, the presence of truncated ␤2-adaptin sensitized neurons to glutamate receptor-mediated excitotoxicity. Proteolysis of ␣-and ␤2-adaptins, as well as the accessory clathrin adaptors epsin 1, adaptor protein 180, and the clathrin assembly lymphoid myeloid leukemia protein, was detected in brain tissues after experimentally induced ischemia and in cases of human Alzheimer disease. The present study further clarifies the central role of calpain in regulating clathrin-dependent endocytosis and provides evidence for a novel mechanism through which calpain activation may promote neurodegeneration: the sensitization of cells to glutamate-mediated excitotoxicity via the decreased internalization of surface receptors.

Proton spectroscopy allows the simultaneous quantification of a high number of metabolite concentrations termed the neurochemical profile. The spin echo full intensity acquired localization (SPECIAL) scheme with an echo time of 2.7 ms was used at 9.4T for excitation of a slab parallel to a home-built quadrature surface coil in conjunction with phase encoding in the two remaining spatial dimensions to yield an effective spatial resolution of 1.7 L. The absolute concentrations of at least 10 metabolites were calculated from the spectra of individual voxels using LCModel analysis. The calculated concentrations were used for constructing quantitative metabolic maps of the neurochemical profile in normal and pathological rat brain. Summation of individual spectra was used to assess the neurochemical profile of unique brain regions, such as corpus callosum, in rat for the first time. Following focal ischemia in rat pups, imaging the neurochemical profile indicated increased choline groups in the ischemic core and increased glutamine in the penumbra, which is proposed to reflect glutamate excitotoxicity. We conclude that it is feasible to achieve a sensitivity that is sufficient for quantitative mapping of the neurochemical profile at microliter spatial resolution. In the past decade we and others have established that proton MR localized spectroscopy with echo times on the order of 2 ms allows measuring metabolite concentrations in the brain (1-4). Approximately 18 metabolite concentrations that can be measured at 9.4T results in a neurochemical profile (2) consisting of putative markers implicated in myelination (phosphoethanolamine [PE] [Gln]). Short echo time proton spectroscopy enables us to quantify compounds with spin systems including strongly and/or weakly Jcoupled multiplets as well as singlet resonances without the need for correcting effects of T 2 relaxation times. Most studies to date have used single-voxel localized spectroscopy to obtain such neurochemical profiles only from one selected volume at a time.On the other hand, spectroscopic imaging can be used to obtain this information simultaneously from many voxels, amounting to a regional mapping of the neurochemical profile. It is also likely to yield important insight in diseases with complex regional distribution of metabolites such as focal ischemia or multiple sclerosis. Most studies based on spectroscopic imaging techniques have used long echo times, thereby limiting the obtained concentrations to prominent singlets of NAA, total creatine (tCr), and choline-containing compounds (Cho), and in some cases to a few J-coupled resonances, such as Lac and glutamate ϩ glutamine (Glx) (5,6) due to the additional signal loss incurred by J-modulation of strongly coupled spin systems.A number of practical implementations of short-echotime spectroscopic imaging in brain have been reported. In human studies short-echo-time measurements were used either for obtaining localized spectra and metabolite concentrations from specific regions of the brain (7-14) or fo...