Catherine Marchand-Verrecchia

NMDA Receptor Activation Inhibits α-Secretase and Promotes Neuronal Amyloid-β Production

Acute brain injuries have been identified as a risk factor for developing Alzheimers disease (AD). Because glutamate plays a pivotal role in these pathologies, we studied the influence of glutamate receptor activation on amyloid-β (Aβ) production in primary cultures of cortical neurons. We found that sublethal NMDA receptor activation increased the production and secretion of Aβ. This effect was preceded by an increased expression of neuronal Kunitz protease inhibitory domain (KPI) containing amyloid-β precursor protein (KPI-APP) followed by a shift from α-secretase to β-secretase-mediated APP processing. This shift is a result of the inhibition of the α-secretase candidate tumor necrosis factor-α converting enzyme (TACE) when associated with neuronal KPI-APPs. This KPI-APP/TACE interaction was also present in AD brains. Thus, our findings reveal a cellular mechanism linking NMDA receptor activation to neuronal Aβ secretion. These results suggest that even mild deregulation of the glutamatergic neurotransmission may increase Aβ production and represent a causal risk factor for developing AD.

Deleterious poly(ADP-ribose)polymerase-1 pathway activation in traumatic brain injury in rat

Traumatic brain injury produces nitric oxide and reactive oxygen species. Peroxynitrite, resulting from the combination of nitric oxide and superoxide anions, triggers DNA strand breaks, leading to the activation of poly(ADP-ribose)polymerase-1. As excessive activation of this enzyme induces cell death, we examined the production of nitrosative stress, the activation of poly(ADP-ribose)polymerase-1, and the role of this enzyme in the outcomes of traumatic brain injury produced by fluid percussion in rats. Immunohistochemistry showed that 3-nitrotyrosine, an indicator of nitrosative stress, and poly(ADP-ribose), a marker of poly(ADP-ribose)polymerase-1 activation, were present as early as 30 min post-injury, and that persisted for 72 h. The poly(ADP-ribose)polymerase inhibitor, 3-aminobenzamide, at 10 and 30 mg/kg, significantly improved the neurological deficit, with a 60% reduction in the brain lesion volume and inhibition of poly(ADP-ribose)polymerase-1 activation. Thus, poly(ADP-ribose)polymerase-1 is involved in the neurological consequences of traumatic brain injury and may be a promising therapeutic target in clinical treatment of acute brain trauma.

l-NAME reduces infarction, neurological deficit and blood–brain barrier disruption following cerebral ischemia in mice

The role of nitric oxide (NO) in the development of post-ischemic cerebral infarction has been extensively examined, but fewer studies have investigated its role in other outcomes. In the present study, we first determined the temporal evolution of infarct volume, NO production, neurological deficit and blood-brain barrier disruption in a model of transient focal cerebral ischemia in mice. We then examined the effect of the nonselective NO-synthase inhibitor N(omega)-nitro-L-arginine-methylester (L-NAME). L-NAME given at 3 mg/kg 3 h after ischemia reduced by 20% the infarct volume and abolished the increase in brain NO production evaluated by its metabolites (nitrites/nitrates) 48 h after ischemia. L-NAME with this protocol also reduced the neurological deficit evaluated by the grip test and decreased by 65% the extravasation of Evans blue, an index of blood-brain barrier breakdown. These protective activities of L-NAME suggest that NO has multiple deleterious effects in cerebral ischemia.

Detrimental Role of Bradykinin B2 Receptor in a Murine Model of Diffuse Brain Injury

Inhibition of the bradykinin B2 receptor type (B2R) has been shown to improve neurological outcome in models of focal traumatic brain injury. However, the involvement of B2R in trauma-induced diffuse injury has not yet been explored. This is an important point, since in humans a pattern of diffuse injury is commonly found in severely injured patients and has been associated with a poor neurological outcome and prognosis. Using the non-peptide B2R antagonist LF 16-0687 Ms and B2R null (B2R-/-) mice, we investigated the role of B2R in a model of closed head trauma (CHT). LF 16-0687 Ms given 30 min after injury reduced the neurological deficit by 26% and the cerebral edema by 22% when evaluated 4 h after CHT. Neurological function after CHT was improved in B2R-/- mice compared to B2R+/+ mice, although there was no difference in the development of brain edema. Treatment with LF 16-0687 Ms and B(2)R gene deletion decreased the accumulation of neutrophils at 24 h after CHT (50% and 36%, respectively). In addition, the inducible NO synthase (iNOS) mRNA level increased markedly, and this was reduced by LF 16-0687 Ms. Taken together, these data support a detrimental role of B2R in the development of the neurological deficit and of the inflammatory secondary damage resulting from diffuse traumatic brain injury. Therefore, blockade of bradykinin B2 receptors might represent an attractive therapeutic approach in the pharmacological treatment of traumatic brain injury.

LF 16-0687 Ms, a bradykinin B2 receptor antagonist, reduces ischemic brain injury in a murine model of transient focal cerebral ischemia.

Bradykinin promotes neuronal damage and brain edema through the activation of the B2 receptor. The neuroprotective effect of LF 16‐0687 Ms, a B2 receptor antagonist, has been described when given prior to induction of transient focal cerebral ischemia in rat, but there are no data regarding the consequence of a treatment when given after injury. Therefore, in a murine model of transient middle cerebral artery occlusion (MCAO), we evaluated the effect of LF 16‐0687 Ms given prior to and/or after the onset of ischemia on neurological deficit, infarct volume and inflammatory responses including cerebral edema, blood–brain barrier (BBB) disruption and neutrophil accumulation. LF 16‐0687 Ms (1, 2 and 4 mg kg−1) administered 0.5 h before and, 1.25 and 6 h after MCAO, decreased the infarct volume by a maximum of 33% and significantly improved the neurological recovery. When given at 0.25 and 6.25 h after MCAO, LF 16‐0687 Ms (1.5, 3 and 6 mg kg−1) decreased the infarct volume by a maximum of 25% and improved the neurological score. Post‐treatment with LF 16‐0687 Ms (1.5 mg kg−1) significantly decreased brain edema (−28%), BBB disruption (−60%) and neutrophil accumulation (−65%) induced by ischemia. Physiological parameters were not modified by LF 16‐0687 Ms. These data emphasize the role of bradykinin B2 receptor in the development of infarct lesion, neurological deficit and inflammatory responses resulting from transient focal cerebral ischemia. Therefore, B2 receptor antagonist might represent a new therapeutic approach in the pharmacological treatment of stroke.

Beneficial effects of PJ34 and INO-1001, two novel water-soluble poly(ADP-ribose) polymerase inhibitors, on the consequences of traumatic brain injury in rat

Traumatic brain injury produces peroxynitrite, a powerful oxidant which triggers DNA strand breaks, leading to the activation of poly(ADP-ribose)polymerase-1 (PARP-1). We previously demonstrated that 3-aminobenzamide, a PARP inhibitor, is neuroprotective in a model of traumatic brain injury induced by fluid percussion in rat, suggesting that PARP-1 could be a therapeutic target. In order to confirm this hypothesis, we investigated the effects of PJ34 and INO-1001, two PARP inhibitors from structural classes other than benzamide, on the post-traumatic consequences. Pre- and post-treatments with PJ34 (30 mg/kg/day) and INO-1001 (10 mg/kg/day) decrease the neurological deficit at 3 days post-injury and this deficit is still reduced at 7 days. These neurological recovery-promoting effects are associated with the inhibition of PARP-1 activation caused by trauma, as demonstrated by abolishment of immunostaining of poly(ADP-ribose). Thus, the present work strengthens strongly the concept that PARP-1 inhibition may be a suitable approach for the treatment of brain trauma.

Cortical calcium increase following traumatic brain injury represents a pitfall in the evaluation of Ca2+-independent NOS activity

In this report, our findings highlighted the presence of a high level of calcium in the cortex following traumatic brain injury (TBI) in a rat model of fluid percussion-induced brain injury. This calcium increase represents a pitfall in the assessment of Ca2+-independent nitric oxide synthase (NOS) activity supposed to play a role in the secondary brain lesion following TBI. The so-called Ca2+-independent NOS activity measured in the injured cortex 72 h after TBI had the pharmacological profile of a Ca2+-dependent NOS and was therefore inhibited with a supplement of calcium chelator. The remaining activity was very low and iNOS protein was hardly immunodetected on the same sample used for NOS activity assay. The concentration of calcium chelator used in the assay should be revised and adjusted consequently to make sure that the calcium-free condition is achieved for the assay. Otherwise, the findings tend towards an overestimation of Ca2+-independent and underestimation of Ca2+-dependent NOS activities. The revised Ca2+-independent NOS activity assay was then tested, in relation with the amount of iNOS protein, in a model of LPS-induced neuroinflammation. Taken together, precautions should be taken when assessing the Ca2+-independent enzymatic activity in cerebral tissue after a brain insult.

Deleterious activation of poly(ADP-ribose)polymerase-1 in brain after in vivo oxidative stress.

Oxidative stress has been shown to be implicated in the pathogenesis of central nervous system injuries such as cerebral ischemia and trauma, and chronic neurodegenerative diseases. In vitro studies show that oxidative stress, particularly peroxynitrite, could trigger DNA strand breaks, which lead to the activation of repairing enzymes including Poly(ADP-ribose) Polymerase-1 (PARP-1). As excessive activation of this enzyme induces cell death, we examined whether such a cascade also occurs in vivo in a model of oxidative stress in rat brain. For this purpose, the mitochondrial toxin malonate, which promotes free radical production, was infused into the left striatum of rats. Immunohistochemistry showed that 3-nitrotyrosine, an indicator of nitrosative stress, and poly(ADP-ribose), a marker of poly(ADP-ribose)polymerase-1 activation, were present as early as 1 h after malonate, and that they persisted for 24 h. The PARP inhibitor, 3-aminobenzamide, significantly reduced the lesion and inhibited PARP-1 activation induced by malonate. These results demonstrate that oxidative stress induced in vivo in the central nervous system leads to the activation of poly(ADP-ribose)polymerase-1, which contributes to neuronal cell death.