Vanya Stefovska

Synaptic NMDA receptor activity boosts intrinsic antioxidant defenses.

Intrinsic antioxidant defenses are important for neuronal longevity. We found that in rat neurons, synaptic activity, acting via NMDA receptor (NMDAR) signaling, boosted antioxidant defenses by making changes to the thioredoxin-peroxiredoxin (Prx) system. Synaptic activity enhanced thioredoxin activity, facilitated the reduction of overoxidized Prxs and promoted resistance to oxidative stress. Resistance was mediated by coordinated transcriptional changes; synaptic NMDAR activity inactivated a previously unknown Forkhead box O target gene, the thioredoxin inhibitor Txnip. Conversely, NMDAR blockade upregulated Txnip in vivo and in vitro, where it bound thioredoxin and promoted vulnerability to oxidative damage. Synaptic activity also upregulated the Prx reactivating genes Sesn2 (sestrin 2) and Srxn1 (sulfiredoxin), via C/EBPβ and AP-1, respectively. Mimicking these expression changes was sufficient to strengthen antioxidant defenses. Trans-synaptic stimulation of synaptic NMDARs was crucial for boosting antioxidant defenses; chronic bath activation of all (synaptic and extrasynaptic) NMDARs induced no antioxidative effects. Thus, synaptic NMDAR activity may influence the progression of pathological processes associated with oxidative damage.

Sedative and anticonvulsant drugs suppress postnatal neurogenesis

Sedative and anticonvulsant drugs, which inhibit N‐methyl‐D‐aspartate receptor–mediated excitation or enhance GABA‐mediated action, may cause apoptotic neurodegeneration in the developing mammalian brain. Here we explored whether such agents influence early postnatal neurogenesis.

Anticancer agents are potent neurotoxins in vitro and in vivo

Neurotoxicity of anticancer agents complicates treatment of children with cancer. We investigated neurotoxic effects of common cytotoxic drugs in neuronal cultures and in the developing rat brain. When neurons were exposed to cisplatin (5–100μM), cyclophosphamide (5–100μM), methotrexate (5–100μM), vinblastin (0.1–1μM), or thiotepa (5–100μM), a concentration‐dependent neurotoxic effect was observed. Neurotoxicity was potentiated by nontoxic glutamate concentrations. The N‐methyl‐D‐aspartate receptor antagonist MK 801 (10μM), the AMPA receptor antagonists GYKI 52466 (10μM) and NBQX (10μM), and the pancaspase inhibitor Ac‐DEVD‐CHO (1nM) ameliorated neurotoxicity of cytotoxic drugs. To investigate neurotoxicity in vivo, we administered to 7‐day‐old rats the following: cisplatin (5–15mg/kg IP), cyclophosphamide (200–600mg/kg IP), thiotepa (15–45mg/kg), or ifosfamide (100–500mg/kg) and their brains were analyzed at 4 to 24 hours. Cytotoxic drugs produced widespread lesions within cortex, thalamus, hippocampal dentate gyrus, and caudate nucleus in a dose‐dependent fashion. Early histological analysis demonstrated dendritic swelling and relative preservation of axonal terminals, which are morphological features indicating excitotoxicity. After longer survival periods, degenerating neurons displayed morphological features consistent with active cell death. These results demonstrate that anticancer drugs are potent neurotoxins in vitro and in vivo; they activate excitotoxic mechanisms but also trigger active neuronal death. Ann Neurol 2004

Sulthiame but not levetiracetam exerts neurotoxic effect in the developing rat brain

Antiepileptic drugs (AEDs) used to treat seizures in pregnant women, infants, and young children can cause cognitive impairment. One mechanism implicated in the development of neurocognitive deficits is a pathologic enhancement of physiologically occurring apoptotic neuronal death in the developing brain. We investigated whether the newer antiepileptic drug levetiracetam (LEV) and the older antiepileptic drug sulthiame (SUL) have neurotoxic properties in the developing rat brain. SUL significantly enhanced neuronal death in the brains of rat pups ages 0 to 7 days at doses of 100 mg/kg and above, whereas LEV did not show this neurotoxic effect. Dosages of both drugs used in the context of this study comply with an effective anticonvulsant dose range applied in rodent seizure models. Thus, LEV is an AED which lacks neurotoxicity in the developing rat brain and should be considered in the treatment of epilepsy in pregnant women, infants, and toddlers once general safety issues have been properly addressed.

Acute and long-term proteome changes induced by oxidative stress in the developing brain.

The developing mammalian brain experiences a period of rapid growth during which various otherwise innocuous environmental factors cause widespread apoptotic neuronal death. To gain insight into developmental events influenced by a premature exposure to high oxygen levels and identify proteins engaged in neurodegenerative and reparative processes, we analyzed mouse brain proteome changes at P7, P14 and P35 caused by an exposure to hyperoxia at P6. Changes detected in the brain proteome suggested that hyperoxia leads to oxidative stress and apoptotic neuronal death. These changes were consistent with results of histological and biochemical evaluation of the brains, which revealed widespread apoptotic neuronal death and increased levels of protein carbonyls. Furthermore, we detected changes in proteins involved in synaptic function, cell proliferation and formation of neuronal connections, suggesting interference of oxidative stress with these developmental events. These effects are age-dependent, as they did not occur in mice subjected to hyperoxia in adolescence.

Cannabinoids enhance susceptibility of immature brain to ethanol neurotoxicity

Marijuana and alcohol are most widely abused drugs among women of reproductive age. Neurocognitive deficits have been reported in children whose mothers used marijuana during pregnancy. Maternal consumption of ethanol is known to cause serious developmental deficits

Neurotrophin potentiation of iron-induced spinal cord injury

Previous studies have shown that pretreatment with neurotrophins can potentiate the vulnerability of cultured neurons to excitotoxic and free radical-induced necrosis, in contrast to their well known neuroprotective effects against apoptosis. Here we tested the hypothesis that this unexpected injury-potentiating effect of neurotrophins would also take place in the adult rat spinal cord. Fe(3+)-citrate was injected stereotaxically into spinal cord gray matter in adult rats in amounts sufficient to produce minimal tissue injury 24 h later. Twenty-four-hour pretreatment with brain-derived neurotrophic factor, neurotrophin-3, or neurotrophin-4/5, but not nerve growth factor, markedly enhanced tissue injury in the gray matter as evidenced by an increase in the damaged area, as well as the loss of neurons and oligodendrocytes. Consistent with maintained free radical mediation, the neurotrophin-potentiated iron-induced spinal cord damage was blocked by co-application of the antioxidant N-tert-butyl-(2-sulfophenyl)-nitrone. These data support the hypothesis that the overall neuroprotective properties of neurotrophins in models of acute injury to the spinal cord may be limited by an underlying potentiation of free radical-mediated necrosis.

Activation of caspase-1 dependent interleukins in developmental brain trauma

Focal mechanical cortical trauma triggers diffuse apoptotic neurodegeneration in the developing rat brain which is associated with invasion of brain tissue with inflammatory mediators. We hypothesized that caspase-1 and the two caspase-1-processed cytokines, interleukin (IL)-1beta and IL-18, are involved in trauma-induced neuronal cell death in the developing brain. 7-day-old Wistar rats or C57/BL6 mice were subjected to head trauma using a weight drop device. Animals were sacrificed at defined time points following trauma and brains were processed for histology and molecular analyses. Neuronal cell death in the immature brain peaked at 12-24 h and was accompanied by a marked increase of mRNA and protein levels for caspase-1, IL-1beta and IL-18 within 2 to 12 h following the injury. Caspase-1 levels were elevated for 72 h, whereas IL-1beta decreased earlier at 48 h. IL-18 remained high over a period of 3 days and decreased to normal levels by day 7 after the injury. Intraperitoneal injection of recombinant human IL-18-binding protein (IL-18BP), a specific inhibitor of IL-18, attenuated traumatic brain injury. Mice deficient in IL-18 (IL-18-/-) were protected against trauma-induced brain damage. These findings indicate that IL-18 is involved in trauma-induced neuronal cell death in the immature rodent brain and might serve as a potential therapeutic target.

Matrix metalloproteinase 9 regulates cell death following pilocarpine-induced seizures in the developing brain.

Matrix metalloproteinases (MMPs) are involved in tissue repair, cell death and morphogenesis. We investigated the role of the gelatinases MMP-2 and MMP-9 in the pathogenesis of neuronal death induced by prolonged seizures in the developing brain. Seven-day-old rats, MMP-9 knockout mice and transgenic rats overexpressing MMP-9 received intraperitoneal injections of pilocarpine, 250 mg/kg, to induce seizures. After 6-72 h pups were sacrificed, tissue from different brain regions was isolated and expression of MMP-9 mRNA and protein was analyzed by real-time PCR or Western blot. Additionally, brains were fixed and processed for TUNEL-staining, immunohistochemistry and in situ zymography. We found increased numbers of TUNEL-positive cells 24 h after pilocarpine-induced seizures, most pronounced in cortical areas and the dentate gyrus, and less pronounced in thalamus. At 6-24 h, MMP-9 mRNA levels showed significant elevation compared to sham-treated controls; this effect resolved by 48 h, whereas MMP-2 mRNA levels remained stable. Cortical gelatinolytic activity, monitored by in situ zymography, was enhanced following pilocarpine-induced seizures. The MMP inhibitor GM 6001 ameliorated cell death following pilocarpine-induced seizures in infant rats. MMP-9 knockout mice were less susceptible to seizure-induced brain injury. Transgenic rats overexpressing MMP-9 were equally susceptible to seizure-induced brain injury as wild type rats. Our results suggest a significant contribution of MMP-9 to cell death after pilocarpine-induced seizures in the developing brain. As indicated by Western blot analysis, MMP-9 activation may be linked to activation of the Erk/CREB-pathway. The findings implicate involvement of MMP-9 in the pathophysiology of brain injury following seizures in the developing brain.

Mechanisms of disease: motoneuron disease aggravated by transgenic expression of a functionally modified AMPA receptor subunit.

Abstract: To reveal whether increased Ca2+ permeability of glutamate AMPA channels triggered by the transgene for GluR‐B(N) induces decline in motor functions and neurodegeneration in the spinal cord, we evaluated growth, motor coordination, and spinal reflexes in transgenic GluR‐B(N) and wild‐type (wt) mice. To reveal whether the transgenic GluR‐B(N) expression aggravates the course of motoneuron disease in SOD1 mice, we mated heterozygous GluR‐B(N) and SOD1 [C57BL6Ico‐TgN(hSOD1‐G93A)1Gur] mice to generate double‐transgenic progeny. The phenotypic sequelae in mice carrying mutations were evaluated by monitoring growth, motor coordination, and survival. Neuronal degeneration was assessed by morphological and stereological analysis of spinal cord and brain. We found that transgenic expression in mice of GluR‐B(N)‐containing glutamate AMPA receptors with increased Ca2+ permeability leads to a late‐onset degeneration of neurons in the spinal cord and decline of motor functions. Neuronal death progressed over the entire life span, but manifested clinically in late adulthood, resembling the course of a slow neurodegenerative disorder. Additional transgenic expression of mutated human SOD1 accelerated disease progression, aggravated severity of motor decline, and decreased survival. These observations reveal that moderate, but persistently elevated Ca2+ influx via glutamate AMPA channels causes degeneration of spinal motoneurons and motor decline over the span of life. These features resemble the course of sporadic amyotrophic lateral sclerosis (ALS) in humans and suggest that modified function of glutamate AMPA channels may be causally linked to pathogenesis of ALS.