Assessment at the single-cell level identifies neuronal glutathione depletion as both a cause and effect of ischemia-reperfusion oxidative stress.

Oxidative stress contributes to neuronal death in brain ischemia-reperfusion. Tissue levels of the endogenous antioxidant glutathione (GSH) are depleted during ischemia-reperfusion, but it is unknown whether this depletion is a cause or an effect of oxidative stress, and whether it occurs in neurons or other cell types. We used immunohistochemical methods to evaluate glutathione, superoxide, and oxidative stress in mouse hippocampal neurons after transient forebrain ischemia. GSH levels in CA1 pyramidal neurons were normally high relative to surrounding neuropil, and exhibited a time-dependent decrease during the first few hours of reperfusion. Colabeling for superoxide in the neurons showed a concurrent increase in detectable superoxide over this interval. To identify cause-effect relationships between these changes, we independently manipulated superoxide production and GSH metabolism during reperfusion. Mice in which NADPH oxidase activity was blocked to prevent superoxide production showed preservation of neuronal GSH content, thus demonstrating that neuronal GSH depletion is result of oxidative stress. Conversely, mice in which neuronal GSH levels were maintained by N-acetyl cysteine treatment during reperfusion showed less neuronal superoxide signal, oxidative stress, and neuronal death. At 3 d following ischemia, GSH content in reactive astrocytes and microglia was increased in the hippocampal CA1 relative to surviving neurons. Results of these studies demonstrate that neuronal GSH depletion is both a result and a cause of neuronal oxidative stress after ischemia-reperfusion, and that postischemic restoration of neuronal GSH levels can be neuroprotective.

Robust neuroprosthetic control from the stroke perilesional cortex.

Intracortical brain-machine interfaces (BMIs) may eventually restore function in those with motor disability after stroke. However, current research into the development of intracortical BMIs has focused on subjects with largely intact cortical structures, such as those with spinal cord injury. Although the stroke perilesional cortex (PLC) has been hypothesized as a potential site for a BMI, it remains unclear whether the injured motor cortical network can support neuroprosthetic control directly. Using chronic electrophysiological recordings in a rat stroke model, we demonstrate here the PLC's capacity for neuroprosthetic control and physiological plasticity. We initially found that the perilesional network demonstrated abnormally increased slow oscillations that also modulated neural firing. Despite these striking abnormalities, neurons in the perilesional network could be modulated volitionally to learn neuroprosthetic control. The rate of learning was surprisingly similar regardless of the electrode distance from the stroke site and was not significantly different from intact animals. Moreover, neurons achieved similar task-related modulation and, as an ensemble, formed cell assemblies with learning. Such control was even achieved in animals with poor motor recovery, suggesting that neuroprosthetic control is possible even in the absence of motor recovery. Interestingly, achieving successful control also reduced locking to abnormal oscillations significantly. Our results thus suggest that, despite the disrupted connectivity in the PLC, it may serve as an effective target for neuroprosthetic control in those with poor motor recovery after stroke.

Microglial activation induced by the alarmin S100B is regulated by poly(ADP-ribose) polymerase-1.

Brain injury resulting from stroke or trauma can be exacerbated by the release of proinflammatory cytokines, proteases, and reactive oxygen species by activated microglia. The microglial activation resulting from brain injury is mediated in part by alarmins, which are signaling molecules released from damaged cells. The nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) has been shown to regulate microglial activation after brain injury, and here we show that signaling effects of the alarmin S100B are regulated by PARP-1. S100B is a protein localized predominantly to astrocytes. Exogenous S100B added to primary microglial cultures induced a rapid change in microglial morphology, upregulation of IL-1ß, TNFα, and iNOS gene expression, and release of matrix metalloproteinase 9 and nitric oxide. Most, though not all of these effects were attenuated in PARP-1(-/-) microglia and in wild-type microglia treated with the PARP inhibitor, veliparib. Microglial activation and gene expression changes induced by S100B injected directly into brain were likewise attenuated by PARP-1 inhibition. The anti-inflammatory effects of PARP-1 inhibitors in acutely injured brain may thus be mediated in part through effects on S100B signaling pathways. GLIA 2016;64:1869-1878.

Intracellular pH reduction prevents excitotoxic and ischemic neuronal death by inhibiting NADPH oxidase.

Sustained activation of N-methyl-d-aspartate (NMDA) -type glutamate receptors leads to excitotoxic neuronal death in stroke, brain trauma, and neurodegenerative disorders. Superoxide production by NADPH oxidase is a requisite event in the process leading from NMDA receptor activation to excitotoxic death. NADPH oxidase generates intracellular H(+) along with extracellular superoxide, and the intracellular H(+) must be released or neutralized to permit continued NADPH oxidase function. In cultured neurons, NMDA-induced superoxide production and neuronal death were prevented by intracellular acidification by as little as 0.2 pH units, induced by either lowered medium pH or by inhibiting Na(+)/H(+) exchange. In mouse brain, superoxide production induced by NMDA injections or ischemia-reperfusion was likewise prevented by inhibiting Na(+)/H(+) exchange and by reduced expression of the Na(+)/H(+) exchanger-1 (NHE1). Neuronal intracellular pH and neuronal Na(+)/H(+) exchange are thus potent regulators of excitotoxic superoxide production. These findings identify a mechanism by which cell metabolism can influence coupling between NMDA receptor activation and superoxide production.

Melatonin Reduces Hypoglycemia-Induced Neuronal Death in Rats.

Melatonin, N-aceyl-5-methoxytryptamine, is the main secretory product of the pineal gland and has neuroprotective effects on several brain injuries, including ischemic stroke. In the present study, we hypothesized that exogenous melatonin may decrease hypoglycemia-induced neuronal death through the prevention of superoxide generation. To test our hypothesis, hypoglycemia was induced by injecting human insulin (10 U/kg, i.p.) in rats. Melatonin injection was started immediately after hypoglycemia (10 mg/kg, i.p.). The first melatonin injection was performed at the end of a 30-min isoelectric EEG period. The second and third injections were administered at 1 and 3 h after the first injection. Reactive oxygen species generation, as detected by dihydroethidium staining, was significantly reduced by melatonin treatment. Neuronal injury was reduced by the treatment of melatonin in the hippocampal CA1 and dentate granule cells. Microglia activation was robust in the hippocampus after hypoglycemia, which was almost completely prevented by melatonin treatment. Hypoglycemia-induced cognitive impairment was also significantly prevented by melatonin treatment. The present study suggests that melatonin has therapeutic potential to prevent hypoglycemia-induced brain injury.

Stress hyperglycemia exacerbates inflammatory brain injury after stroke.

Post-stroke hyperglycemia occurs in 30% - 60% of ischemic stroke patients as part of the systemic stress response, but neither clinical evidence nor pre-clinical studies indicate whether post-stroke hyperglycemia affects stroke outcome. Here we investigated this issue using a mouse model of permanent ischemia. Mice were maintained either normoglycemic or hyperglycemic during the interval of 17 - 48 hours after ischemia onset. Post-stroke hyperglycemia was found to increase infarct volume, blood-brain barrier disruption, and hemorrhage formation, and to impair motor recovery. Post-stroke hyperglycemia also increased superoxide formation by peri-infarct microglia/macrophages. In contrast, post-stroke hyperglycemia did not increase superoxide formation or exacerbate motor impairment in p47 phox-/- mice, which cannot form an active superoxide-producing NADPH oxidase-2 complex. These results suggest that hyperglycemia occurring hours-to-days after ischemia can increase oxidative stress in peri-infarct tissues by fueling NADPH oxidase activity in reactive microglia/macrophages, and by this mechanism contribute to worsened functional outcome.

α-Synuclein triggers cofilin pathology and dendritic spine impairment via a PrPC-CCR5 dependent pathway.

Cognitive dysfunction and dementia are critical symptoms of Lewy Body dementias (LBD). Specifically, alpha-synuclein (αSyn) accumulation in the hippocampus leading to synaptic dysfunction is linked to cognitive deficits in LBD. Here, we investigated the pathological impact of αSyn on hippocampal neurons. We report that either αSyn overexpression or αSyn pre-formed fibrils (PFFs) treatment triggers the formation of cofilin-actin rods, synapse disruptors, in cultured hippocampal neurons and in the hippocampus of synucleinopathy mouse models and of LBD patients. In vivo, cofilin pathology is present concomitantly with synaptic impairment and cognitive dysfunction. Rods generation prompted by αSyn involves the co-action of the cellular prion protein (PrPC) and the chemokine receptor 5 (CCR5). Importantly, we show that CCR5 inhibition, with a clinically relevant peptide antagonist, reverts dendritic spine impairment promoted by αSyn. Collectively, we detail the cellular and molecular mechanism through which αSyn disrupts hippocampal synaptic structure and we identify CCR5 as a novel therapeutic target to prevent synaptic impairment and cognitive dysfunction in LBD.

Nuclear factor erythroid 2-related factor 2 facilitates neuronal glutathione synthesis by upregulating neuronal excitatory amino acid transporter 3 expression.

Astrocytes support neuronal antioxidant capacity by releasing glutathione, which is cleaved to cysteine in brain extracellular space. Free cysteine is then taken up by neurons through excitatory amino acid transporter 3 [EAAT3; also termed Slc1a1 (solute carrier family 1 member 1)] to support de novo glutathione synthesis. Activation of the nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant responsive element (ARE) pathway by oxidative stress promotes astrocyte release of glutathione, but it remains unknown how this release is coupled to neuronal glutathione synthesis. Here we evaluated transcriptional regulation of the neuronal cysteine transporter EAAT3 by the Nrf2-ARE pathway. Nrf2 activators and Nrf2 overexpression both produced EAAT3 transcriptional activation in C6 cells. A conserved ARE-related sequence was found in the EAAT3 promoter of several mammalian species. This ARE-related sequence was bound by Nrf2 in mouse neurons in vivo as observed by chromatin immunoprecipitation. Chemical activation of the Nrf2-ARE pathway in mouse brain increased both neuronal EAAT3 levels and neuronal glutathione content, and these effects were abrogated in mice genetically deficient in either Nrf2 or EAAT3. Selective overexpression of Nrf2 in brain neurons by lentiviral gene transfer was sufficient to upregulate both neuronal EAAT3 protein and glutathione content. These findings identify a mechanism whereby Nrf2 activation can coordinate astrocyte glutathione release with neuronal glutathione synthesis through transcriptional upregulation of neuronal EAAT3 expression.

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.

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.

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.

Hyperglycemia promotes tissue plasminogen activator-induced hemorrhage by Increasing superoxide production.

OBJECTIVE: Risk of intracerebral hemorrhage is the primary factor limiting use of tissue plasminogen activator (tPA) for stroke. Clinical studies have established an association between admission hyperglycemia and the risk of hemorrhage with tPA use, independent of prior diabetes. Here we used an animal model of tPA-induced reperfusion hemorrhage to determine if this clinical association reflects a true causal relationship. METHODS: Rats underwent 90 minutes of focal ischemia, and tPA infusion was begun 10 minutes prior to vessel reperfusion. Glucose was administered during ischemia to generate blood levels ranging from 5.9 ± 1.8mM (normoglycemia) to 21 ± 2.3mM. In some studies, apocynin was administered to block superoxide production by nicotinamide adenine dinucleotide phosphate (NADPH). Brains were harvested 1 hour or 3 days after reperfusion to evaluate the effects of hyperglycemia and apocynin on oxidative stress, blood-brain barrier breakdown, infarct volume, and hemorrhage volume. RESULTS: Rats that were hyperglycemic during tPA infusion had diffusely increased blood-brain barrier permeability in the postischemic territory, and a 3- to 5-fold increase in intracerebral hemorrhage volumes. The hyperglycemic rats also showed increased superoxide formation in the brain parenchyma and vasculature during reperfusion. The effects of hyperglycemia on superoxide production, blood-brain barrier disruption, infarct size, and hemorrhage were all attenuated by apocynin. INTERPRETATION: These findings demonstrate a causal relationship between hyperglycemia and hemorrhage in an animal model of tPA stroke treatment, and suggest that this effect of hyperglycemia is mediated through an increase in superoxide production by NADPH oxidase.

Blast-induced axonal degeneration in the rat cerebellum in the absence of head movement.

Blast exposure can injure brain by multiple mechanisms, and injury attributable to direct effects of the blast wave itself have been difficult to distinguish from that caused by rapid head displacement and other secondary processes. To resolve this issue, we used a rat model of blast exposure in which head movement was either strictly prevented or permitted in the lateral plane. Blast was found to produce axonal injury even with strict prevention of head movement. This axonal injury was restricted to the cerebellum, with the exception of injury in visual tracts secondary to ocular trauma. The cerebellar axonal injury was increased in rats in which blast-induced head movement was permitted, but the pattern of injury was unchanged. These findings support the contentions that blast per se, independent of head movement, is sufficient to induce axonal injury, and that axons in cerebellar white matter are particularly vulnerable to direct blast-induced injury.

Recovery of consolidation after sleep following stroke-interaction of slow waves, spindles, and GABA.

Sleep is known to promote recovery after stroke. Yet it remains unclear how stroke affects neural processing during sleep. Using an experimental stroke model in rats along with electrophysiological monitoring of neural firing and sleep microarchitecture, here we show that sleep processing is altered by stroke. We find that the precise coupling of spindles to global slow oscillations (SOs), a phenomenon that is known to be important for memory consolidation, is disrupted by a pathological increase in "isolated" local delta waves. The transition from this pathological to a physiological state-with increased spindle coupling to SO-is associated with sustained performance gains during recovery. Interestingly, post-injury sleep could be pushed toward a physiological state via a pharmacological reduction of tonic γ-aminobutyric acid (GABA). Together, our results suggest that sleep processing after stroke is impaired due to an increase in delta waves and that its restoration can be important for recovery.

Neuronal Oxidative Stress Promotes α-Synuclein Aggregation In Vivo.

Both genetic and environmental factors increase risk for Parkinson's disease. Many of the known genetic factors influence α-synuclein aggregation or degradation, whereas most of the identified environmental factors produce oxidative stress. Studies using in vitro approaches have identified mechanisms by which oxidative stress can accelerate the formation of α-synuclein aggregates, but there is a paucity of evidence supporting the importance of these processes over extended time periods in brain. To assess this issue, we evaluated α-synuclein aggregates in brains of three transgenic mouse strains: hSyn mice, which overexpress human α-synuclein in neurons and spontaneously develop α-synuclein aggregates; EAAT3-/- mice, which exhibit a neuron-specific impairment in cysteine uptake and resultant neuron-selective chronic oxidative stress; and double-transgenic hSyn/EAAT3-/- mice. Aggregate formation was evaluated by quantitative immunohistochemistry for phosphoserine 129 α-synuclein and by an α-synuclein proximity ligation assay. Both methods showed that the double transgenic hSyn/EAAT3-/- mice exhibited a significantly higher α-synuclein aggregate density than littermate hSyn mice in each brain region examined. Negligible aggregate formation was observed in the EAAT3-/- mouse strain, suggesting a synergistic rather than additive interaction between the two genotypes. A similar pattern of results was observed in assessments of motor function: the pole test and rotarod test. Together, these observations indicate that chronic, low-grade neuronal oxidative stress promotes α-synuclein aggregate formation in vivo. This process may contribute to the mechanism by which environmentally induced oxidative stress contributes to α-synuclein pathology in idiopathic Parkinson's disease.

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.

Facilitated neurogenesis in the developing hippocampus after intake of theanine, an amino acid in tea leaves, and object recognition memory.

Theanine, γ-glutamylethylamide, is one of the major amino acid components in green tea. In this study, cognitive function and the related mechanism were examined in theanine-administered young rats. Newborn rats were fed theanine through dams, which were fed water containing 0.3% theanine, and then fed water containing 0.3% theanine after weaning. Theanine level in the brain was under the detectable limit 6 weeks after the start of theanine administration. Theanine administration did not influence locomotor activity in the open-field test. However, rearing behavior was significantly increased in theanine-administered rats, suggesting that exploratory activity is increased by theanine intake. Furthermore, object recognition memory was enhanced in theanine-administered rats. The increase in exploratory activity in the open-field test seems to be associated with the enhanced object recognition memory after theanine administration. On the other hand, long-term potentiation (LTP) induction at the perforant path-granule cell synapse was not changed by theanine administration. To check hippocampal neurogenesis, BrdU was injected into rats 3 weeks after the start of theanine administration, and brain-derived neurotropic factor (BDNF) level was significantly increased at this time. Theanine intake significantly increased the number of BrdU-, Ki67-, and DCX-labeled cells in the granule cell layer 6 weeks after the start of theanine administration. This study indicates that 0.3% theanine administration facilitates neurogenesis in the developing hippocampus followed by enhanced recognition memory. Theanine intake may be of benefit to the postnatal development of hippocampal function.

Coordinated increase of reliable cortical and striatal ensemble activations during recovery after stroke.

Skilled movements rely on a coordinated cortical and subcortical network, but how this network supports motor recovery after stroke is unknown. Previous studies focused on the perilesional cortex (PLC), but precisely how connected subcortical areas reorganize and coordinate with PLC is unclear. The dorsolateral striatum (DLS) is of interest because it receives monosynaptic inputs from motor cortex and is important for learning and generation of fast reliable actions. Using a rat focal stroke model, we perform chronic electrophysiological recordings in motor PLC and DLS during long-term recovery of a dexterous skill. We find that recovery is associated with the simultaneous emergence of reliable movement-related single-trial ensemble spiking in both structures along with increased cross-area alignment of spiking. Our study highlights the importance of consistent neural activity patterns across brain structures during recovery and suggests that modulation of cross-area coordination can be a therapeutic target for enhancing motor function post-stroke.

α-synuclein aggregates induce c-Abl activation and dopaminergic neuronal loss by a feed-forward redox stress mechanism.

Oxidative stress and α-synuclein aggregation both drive neurodegeneration in Parkinson's disease, and the protein kinase c-Abl provides a potential amplifying link between these pathogenic factors. Suppressing interactions between these factors may thus be a viable therapeutic approach for this disorder. To evaluate this possibility, pre-formed α-synuclein fibrils (PFFs) were used to induce α-synuclein aggregation in neuronal cultures. Exposure to PFFs induced oxidative stress and c-Abl activation in wild-type neurons. By contrast, α-synuclein - deficient neurons, which cannot form α-synuclein aggregates, failed to exhibit either oxidative stress or c-Abl activation. N-acetyl cysteine, a thiol repletion agent that supports neuronal glutathione metabolism, suppressed the PFF - induced redox stress and c-Abl activation in the wild-type neurons, and likewise suppressed α-synuclein aggregation. Parallel findings were observed in mouse brain: PFF-induced α-synuclein aggregation in the substantia nigra was associated with redox stress, c-Abl activation, and dopaminergic neuronal loss, along with microglial activation and motor impairment, all of which were attenuated with oral N-acetyl cysteine. Similar results were obtained using AAV-mediated α-synuclein overexpression as an alternative means of driving α-synuclein aggregation in vivo. These findings show that α-synuclein aggregates induce c-Abl activation by a redox stress mechanism. c-Abl activation in turn promotes α-synuclein aggregation, in a feed-forward interaction. The capacity of N-acetyl cysteine to interrupt this interaction adds mechanistic support its consideration as a therapeutic in Parkinson's disease.