Neuroprotective strategies, including free radical scavengers, ion channel modulators, and anti-inflammatory agents, have been extensively explored in the last 2 decades for the treatment of neurological diseases. Unfortunately, none of the neuroprotectants has been proved effective in clinical trails. In the current study, we demonstrated that methylene blue (MB) functions as an alternative electron carrier, which accepts electrons from NADH and transfers them to cytochrome c and bypasses complex I/III blockage. A de novo synthesized MB derivative, with the redox center disabled by N-acetylation, had no effect on mitochondrial complex activities. MB increases cellular oxygen consumption rates and reduces anaerobic glycolysis in cultured neuronal cells. MB is protective against various insults in vitro at low nanomolar concentrations. Our data indicate that MB has a unique mechanism and is fundamentally different from traditional antioxidants. We examined the effects of MB in two animal models of neurological diseases. MB dramatically attenuates behavioral, neurochemical, and neuropathological impairment in a Parkinson disease model. Rotenone caused severe dopamine depletion in the striatum, which was almost completely rescued by MB. MB rescued the effects of rotenone on mitochondrial complex I-III inhibition and free radical overproduction. Rotenone induced a severe loss of nigral dopaminergic neurons, which was dramatically attenuated by MB. In addition, MB significantly reduced cerebral ischemia reperfusion damage in a transient focal cerebral ischemia model. The present study indicates that rerouting mitochondrial electron transfer by MB or similar molecules provides a novel strategy for neuroprotection against both chronic and acute neurological diseases involving mitochondrial dysfunction.

Although neurofibrillary tangle (NFT) formation is a central event in both familial and sporadic Alzheimer's disease (AD), neither cellular origin nor functional consequence of the NFTs are fully understood. This largely is due to the lack of available in vivo models for neurofibrillary degeneration (NFD). NFTs have only been identified in transgenic mice, bearing a transgene for a rare hereditary neurodegenerative disease, frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP17). Epidemiological evidence suggests a much higher occurrence of dementia in stroke patients. This may represent the underlying cause of the pathogenesis of sporadic AD, which accounts for the majority of AD cases. We examined pathological markers of AD in a rodent stroke model. Here we show that after transient cerebral ischemia, hyperphosphorylated tau accumulates in neurons of the cerebral cortex in the ischemic area, forms filaments similar to those present in human neurodegenerative tauopathies and colocalizes with markers of apoptosis. As a potential underlying mechanism, we were able to determine that transient ischemia induced tau hyperphosphorylation and NFT-like conformations are associated with aberrant activation of cyclin dependent kinase 5 (Cdk5) and can be rescued by delivery of a potent, but non-specific cyclin dependent kinase inhibitor, roscovitine to the brain. Our study further indicates that accumulation of p35 and its calpain-mediated cleavage product, p25 may account for the deregulation of Cdk5 induced by transient ischemia. We conclude that Cdk5 may be the principal protein kinase responsible for tau hyperphosphorylation and may be a hallmark of the tauopathies in this stroke model.

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Aberrant mitosis occurs in many tauopathy-related neurodegenerative diseases and is believed to precede the formation of neurofibrillary tangles. In this study, we report for the first time that transient cerebral ischemia induces aberrant mitotic proteins and hyperphosphorylation of tau protein with neurofibrillary tanglelike conformational epitopes in adult female rat cortex. Following transient cerebral ischemia in rats, initiation of apoptosis precedes and is potentially integrated with subsequent aberrant mitosis and tau hyperphosphorylation. Furthermore, inhibition of mitosis-related cyclindependent kinases (Cdks) by roscovitine significantly reduced the hyperphosphorylation of tau. Administration of the female sex steroid and potent neuroprotective agent, 17␤-estradiol, reduced ischemia-reperfusioninduced cerebral damage and the subsequent aberrant mitosis and tauopathies. These results provide a neuropathological basis for the higher prevalence of dementia in stroke patients and support the hypothesis that apoptosis and aberrant mitosis are integrated pathological events in neurons that may play a critical role in the development of Alzheimer's disease and other tauopathy-related neuropathology.The prevalence of dementia in ischemic stroke patients is nine times higher than controls at 3 months (1) and 4 -12 times higher than in controls at 4 years after a lacunar infarct (2). Many of these dementias developed progressively, and cerebral damage is believed to be the direct cause of cognitive decline in only half of these cases (3). Alzheimer's disease (AD) 1 is the most prevalent dementia (4) and shares common neuropathology features with stroke. Amyloid angiopathy can lead to cerebral hemorrhage (5), Alz-50-immunoreactive granules are found around cerebral infarction after a stroke (6), amyloid precursor protein accumulates following transient focal ischemia (7), and ApoE4 is a genetic risk factor for both AD and stroke (8).Neurofibrillary tangles (NFTs), whose major component is hyperphosphorylated tau (9), are observed in many neurodegenerative diseases (10, 11). Recent reports indicate that aberrant mitotic activation, as well as DNA replication, in terminally differentiated neurons appears to be involved in the pathogenesis of AD (12, 13). Many cyclin-dependent kinases, including Cdc2 and cyclin B1, as well as many other mitotic markers, are elevated in nearly all tauopathy-related neurodegenerative diseases (14). These protein kinases regulate cell cycle progression into mitosis in developing and proliferating cells. If cell cycle re-entrance is forced by ectopically driving an oncogene with a neuronal-specific promotor, the targeted neurons will die rather than divide (15-18). In AD and other tauopathy diseases, induction of Cdc2 and cyclin B1, as well as aberrant DNA replication, is observed in neurons containing NFTs (13). However, completion of successful nuclear divisions in differentiated neurons has never been reported. These inappropriately induced mitotic protein kinases can phosphorylat...

Oxidative stress is implicated in neurodegenerative diseases including stroke, Alzheimer's disease and Parkinson's disease, and has been extensively studied as a potential target for therapeutic intervention. Pyruvate, a natural metabolic intermediate and energy substrate, exerts antioxidant effects in brain and other tissues susceptible to oxidative stress. We tested the protective effects of pyruvate on hydrogen peroxide (H 2 O 2 ) toxicity in human neuroblastoma SK-N-SH cells and the mechanisms underlying its protection. Hydrogen peroxide insult resulted in 85% cell death, but cotreatment with pyruvate dose-dependently attenuated cell death. At concentrations of ≥ 1 mM, pyruvate totally blocked the cytotoxic effects of H 2 O 2 . Pyruvate exerted its protective effects even when its administration was delayed up to 2 hr after H 2 O 2 insult. As a scavenger of reactive oxygen species (ROS), pyruvate dose-dependently attenuated H 2 O 2 -induced ROS formation, assessed from 2,7-dichlorofluorescein diacetate fluorescence. Furthermore, pyruvate suppressed superoxide production by submitochondrial particles, and attenuated oxidative stress-induced collapse of the mitochondrial membrane potential. Collectively, these results suggest pyruvate protects neuronal cells through its antioxidant actions on mitochondria.

Methylene blue (MB), the first lead chemical structure of phenothiazine and other derivatives, is commonly used in diagnostic procedures and as a treatment for methemoglobinemia. We have previously demonstrated that MB could function as an alternative mitochondrial electron transfer carrier, enhance cellular oxygen consumption, and provide protection in vitro and in rodent models of Parkinson’s disease and stroke. In the present study, we investigated the structure-activity relationships of MB in vitro using MB and six structurally related compounds. MB reduces mitochondrial superoxide production via alternative electron transfer that bypasses mitochondrial complexes I-III. MB mitigates reactive free radical production and provides neuroprotection in HT-22 cells against glutamate, IAA and rotenone toxicity. Distinctly, MB provides no protection against direct oxidative stress induced by glucose oxidase. Substitution of a side chain at MB’s 10-nitrogen rendered a 1000-fold reduction of the protective potency against glutamate neurototoxicity. Compounds without side chains at positions 3 and 7, chlorophenothiazine and phenothiazine, have distinct redox potentials compared to MB and are incapable of enhancing mitochondrial electron transfer, while obtaining direct antioxidant actions against glutamate, IAA, and rotenone insults. Chlorophenothiazine exhibited direct antioxidant actions in mitochondria lysate assay compared to MB, which required reduction by NADH and mitochondria. MB increased complex IV expression and activity, while 2-chlorphenothiazine had no effect. Our study indicated that MB could attenuate superoxide production by functioning as an alternative mitochondrial electron transfer carrier and as a regenerable anti-oxidant in mitochondria.