Protein phosphatase 5 protects neurons against amyloid-β toxicity

Amyloid-β (Aβ) is thought to promote neuronal cell loss in Alzheimer's disease, in part through the generation of reactive oxygen species (ROS) and subsequent activation of mitogen-activated protein kinase (MAPK) pathways. Protein phosphatase 5 (PP5) is a ubiquitously expressed serine/threonine phosphatase which has been implicated in several cell stress response pathways and shown to inactivate MAPK pathways through key dephosphorylation events. Therefore, we examined whether PP5 protects dissociated embryonic rat cortical neurons in vitro from cell death evoked by Aβ. As predicted, neurons in which PP5 expression was decreased by small-interfering RNA treatment were more susceptible to Aβ toxicity. In contrast, over-expression of PP5, but not the inactive mutant, PP5(H304Q), prevented MAPK phosphorylation and neurotoxicity induced by Aβ. PP5 also prevented cell death caused by direct treatment with H₂O₂, but did not prevent Aβ-induced production of ROS. Thus, the neuroprotective effect of PP5 requires its phosphatase activity and lies downstream of Aβ-induced generation of ROS. In summary, our data indicate that PP5 plays a pivotal neuroprotective role against cell death induced by Aβ and oxidative stress. Consequently, PP5 might be an effective therapeutic target in Alzheimer's disease and other neurodegenerative disorders in which oxidative stress is implicated.     

IFN-γ and IL-4 differentially shape metabolic responses and neuroprotective phenotype of astrocytes

Astrocytes can either exacerbate or ameliorate secondary degeneration at sites of injury in the CNS but the contextual basis for eliciting these opposing phenotypes is poorly understood. In this study, we demonstrate that the two major cytokines produced by Th1 and Th2 cells, interferon-γ (IFN-γ), and interleukin-4 (IL-4), respectively, contribute differentially to shaping a neuroprotective response in astrocytes. While IFN-γ protects the ability of oxidatively stressed murine astrocytes to clear extracellular glutamate in culture, IL-4 has no effect at any concentration that was tested (10–100 ng/mL). The enhanced release of neuroprotective thiols and lactate by astrocytes in response to T cell stimulation is mimicked by both IL-4 and IFN-γ. When co-administered, IL-4 abrogated the protective effect of low IFN-γ on the glutamate clearance function of oxidatively stressed astrocytes in a dose-dependent manner. Astrocyte-conditioned media obtained from cells cultured in the presence of IL-4 (10 or 100 ng/mL) or IFN-γ (10 ng/mL) decreased by ∼2-fold, neuronal apoptosis induced by oxidative stress in vitro . However, unlike IL-4, IFN-γ at high concentrations (100 ng/mL) was not neuroprotective. Our studies with IFN-γ and IL-4 suggest that a balanced Th1 and Th2 cytokine response might be needed for protecting two key astrocytic functions, glutamate clearance and thiol secretion and might be pertinent to neuroprotective approaches that are aimed at inhibition of an initial pro-inflammatory response to injury or its sustained boosting.     

Metabotropic glutamate receptor 3 activation prevents nitric oxide‐induced death in cultured rat astrocytes

Altered glial function may contribute to the initiation or progression of neuronal death in neurodegenerative diseases. Thus, modulation of astrocyte death may be essential for preventing pathological processes in the CNS. In recent years, metabotropic glutamate receptor (mGluR) activation has emerged as a key target for neuroprotection. We investigated the effect of subtype 3 mGluR (mGluR3) activation on nitric oxide (NO)‐induced astroglial death. A mGluR3 selective agonist, LY379268, reduced inducible NO synthase expression and NO release induced by bacterial lipopolysaccharide and interferon‐γ in cultured rat astrocytes. In turn, a NO donor (diethylenetriamine/NO) induced apoptotic‐like death in cultured astrocytes, which showed apoptotic morphology and DNA fragmentation, but no caspase 3 activation. LY379268 prevented astrocyte death induced by NO exposure, which correlates with a reduction in: phosphatidylserine externalization, p53 and Bax activation and mitochondrial permeability. The reported effects of LY379268 were prevented by the mGluR3 antagonist (s)‐α‐ethylglutamic acid. All together, these findings show the protective effect of mGluR3 activation on astroglial death and provide further evidence of a role of these receptors in preventing CNS injury triggered by several inflammatory processes associated with dysregulated NO production.     

Mitochondrial glutathione protects against cell death induced by oxidative and nitrative stress in astrocytes

The major cellular antioxidant, glutathione, is mostly localized in the cytosol but a small portion is found in mitochondria. We have recently shown that highly selective depletion of mitochondrial glutathione in astrocytes in culture markedly increased cell death induced by the peroxynitrite donor, 3-morpholino-syndnonimine. The present study was aimed at characterizing the increase in susceptibility arising from mitochondrial glutathione loss and testing the possibility that elevating this metabolite pool above normal values could be protective. The increased vulnerability of astrocytes with depleted mitochondrial glutathione to Sin-1 was confirmed. Furthermore, these cells showed marked increases in sensitivity to hydrogen peroxide and also to high concentrations of the nitric oxide donor, S-nitroso-N-acetyl-penicillamine. The increase in cell death was mostly due to necrosis as indicated by substantially increased release of lactate dehydrogenase and staining of nuclei with propidium iodide but little change in annexin V staining and caspase 3 activation. The enhanced cell loss was blocked by prior restoration of the mitochondrial glutathione content. It was also essentially fully inhibited by treatment with cyclosporin A, consistent with a role for the mitochondrial permeability transition in the development of cell death. Susceptibility to the classical apoptosis inducer, staurosporine, was only affected to a small extent in contrast to the response to the other substances tested. Incubation of normal astrocytes with glutathione monoethylester produced large and long-lasting increases in mitochondrial glutathione content with much smaller effects on the cytosolic glutathione pool. This treatment reduced cell death on exposure to 3-morpholino-syndnonimine or hydrogen peroxide but not S-nitroso-N-acetyl-pencillamine or staurosporine. These findings provide evidence for an important role for mitochondrial glutathione in preserving cell viability during periods of oxidative or nitrative stress and indicate that increases in this glutathione pool can confer protection against some of these stressors.     

Energy metabolism in glutamatergic neurons,GABAergic neurons and astrocytes in primary cultures

Several aspects of energy metabolism (glucose utilization, lactate production,¹⁴CO₂ production from labeled glucose, glutamate or pyruvate, oxygen consumption and contents of ATP and phosphocreatine) were measured in cerebellar granule cells (glutamatergic) in primary cultures and compared with corresponding data for cerebral cortical neurons (mainly GABA-ergic) and astrocytes. Cerebellar granule cells and astrocytes were metabolically more active than cerebral cortical neurons. Glutamate which is utilized as a major metabolic fuel as astrocytes and, to a lesser extent, in cerebral cortical neurons, was virtually not oxidized in cerebellar granule cells.Special Issue dedicated to Prof. Holger Hydén.     

Dopamine protects neurons against glutamate-induced excitotoxicity

Glutamate excitotoxicity is responsible for neuronal death in acute neurological disorders including stroke, trauma and neurodegenerative disease. Loss of calcium homeostasis is a key mediator of glutamate-induced cell death. The neurotransmitter dopamine (DA) is known to modulate calcium signalling, and here we show that it can do so in response to physiological concentrations of glutamate. Furthermore, DA is able to protect neurons from glutamate-induced cell death at pathological concentrations of glutamate. We demonstrate that DA has a novel role in preventing delayed calcium deregulation in cortical, hippocampal and midbrain neurons. The effect of DA in abolishing glutamate excitotoxicity can be induced by DA receptor agonists, and is abolished by DA receptor antagonists. Our data indicate that the modulation of glutamate excitotoxicity by DA is receptor-mediated. We postulate that DA has a major physiological function as a safety catch to restrict the glutamate-induced calcium signal, and thereby prevent glutamate-induced cell death in the brain.     

Characterization of microcarrier cultures of neurons and astrocytes from cerebral cortex and cerebellum

In the present investigation a method is described for culturing cerebellar granule cells (glutamatergic neurons), cerebral cortical neurons (GABAergic neurons) and cortical astrocytes on Cytodex 3 microcarriers. It was possible to obtain a high yield of attached neurons and astrocytes on the microcarriers and the cell specific characteristics such as the ability to release neurotransmitter (neurons) and a high activity of glutamine synthetase (astrocytes) were preserved. This system, allowing mixtures of neurons and astrocytes at any given ratio to be produced, may constitute an attractive model system by which the interaction between neurons and astrocytes with regard to exchange of neurotransmitter precursors as well as other compounds may be studied.     

Neuropeptide Y receptors activation protects rat retinal neural cells against necrotic and apoptotic cell death induced by glutamate

It has been claimed that glutamate excitotoxicity might have a role in the pathogenesis of several retinal degenerative diseases, including glaucoma and diabetic retinopathy. Neuropeptide Y (NPY) has neuroprotective properties against excitotoxicity in the hippocampus, through the activation of Y₁, Y₂ and/or Y₅ receptors. The principal objective of this study is to investigate the potential protective role of NPY against glutamate-induced toxicity in rat retinal cells (in vitro and in an animal model), unraveling the NPY receptors and intracellular mechanisms involved. Rat retinal neural cell cultures were prepared from newborn Wistar rats (P3-P5) and exposed to glutamate (500 μM) for 24 h. Necrotic cell death was evaluated by propidium iodide (PI) assay and apoptotic cell death using TUNEL and caspase-3 assays. The cell types present in culture were identified by immunocytochemistry. The involvement of NPY receptors was assessed using selective agonists and antagonists. Pre-treatment of cells with NPY (100 nM) inhibited both necrotic cell death (PI-positive cells) and apoptotic cell death (TUNEL-positive cells and caspase 3-positive cells) triggered by glutamate, with the neurons being the cells most strongly affected. The activation of NPY Y₂, Y₄ and Y₅ receptors inhibited necrotic cell death, while apoptotic cell death was only prevented by the activation of NPY Y₅ receptor. Moreover, NPY neuroprotective effect was mediated by the activation of PKA and p38K. In the animal model, NPY (2.35 nmol) was intravitreally injected 2 h before glutamate (500 nmol) injection into the vitreous. The protective role of NPY was assessed 24 h after glutamate (or saline) injection by TUNEL assay and Brn3a (marker of ganglion cells) immunohistochemistry. NPY inhibited the increase in the number of TUNEL-positive cells and the decrease in the number of Brn3a-positive cells induced by glutamate. In conclusion, NPY and NPY receptors can be considered potential targets to treat retinal degenerative diseases, such as glaucoma and diabetic retinopathy.     

Aging‐associated formaldehyde‐induced norepinephrine deficiency contributes to age‐related memory decline

A norepinephrine (NE) deficiency has been observed in aged rats and in patients with Alzheimer's disease and is thought to cause cognitive disorder. Which endogenous factor induces NE depletion, however, is largely unknown. In this study, we investigated the effects of aging‐associated formaldehyde (FA) on the inactivation of NE in vitro and in vivo, and on memory behaviors in rodents. The results showed that age‐related DNA demethylation led to hippocampal FA accumulation, and when this occurred, the hippocampal NE content was reduced in healthy male rats of different ages. Furthermore, biochemical analysis revealed that FA rapidly inactivated NE in vitro and that an intrahippocampal injection of FA markedly reduced hippocampal NE levels in healthy adult rats. Unexpectedly, an injection of FA (at a pathological level) or 6‐hydroxydopamine (6‐OHDA, a NE depletor) can mimic age‐related NE deficiency, long‐term potentiation (LTP) impairments, and spatial memory deficits in healthy adult rats. Conversely, an injection of NE reversed age‐related deficits in both LTP and memory in aged rats. In agreement with the above results, the senescence‐accelerated prone 8 (SAMP8) mice also exhibited a severe deficit in LTP and memory associated with a more severe NE deficiency and FA accumulation, when compared with the age‐matched, senescence‐resistant 1 (SAMR1) mice. Injection of resveratrol (a natural FA scavenger) or NE into SAMP8 mice reversed FA accumulation and NE deficiency and restored the magnitude of LTP and memory. Collectively, these findings suggest that accumulated FA is a critical endogenous factor for aging‐associated NE depletion and cognitive decline.     

Triheptanoin protects against status epilepticus‐induced hippocampal mitochondrial dysfunctions,oxidative stress and neuronal degeneration

Triheptanoin, the triglyceride of heptanoate, is anaplerotic (refills deficient tricarboxylic acid cycle intermediates) via the propionyl‐CoA carboxylase pathway. It has been shown to be neuroprotective and anticonvulsant in several models of neurological disorders. Here, we investigated the effects of triheptanoin against changes of hippocampal mitochondrial functions, oxidative stress and cell death induced by pilocarpine‐induced status epilepticus (SE ) in mice. Ten days of triheptanoin pre‐treatment did not protect against SE , but it preserved hippocampal mitochondrial functions including state 2, state 3 ADP , state 3 uncoupled respiration, respiration linked to ATP synthesis along with the activities of pyruvate dehydrogenase complex and oxoglutarate dehydrogenase complex 24 h post‐SE . Triheptanoin prevented the SE ‐induced reductions of hippocampal mitochondrial superoxide dismutase activity and plasma antioxidant status as well as lipid peroxidation. It also reduced neuronal degeneration in hippocampal CA 1 and CA 3 regions 3 days after SE . In addition, heptanoate significantly reduced hydrogen peroxide‐induced cell death in cultured neurons. In situ hybridization localized the enzymes of the propionyl‐CoA carboxylase pathway, specifically Pcc α, Pcc β and methylmalonyl‐CoA mutase to adult mouse hippocampal pyramidal neurons and dentate granule cells, indicating that anaplerosis may occur in neurons. In conclusion, triheptanoin appears to have anaplerotic and antioxidant effects which contribute to its neuroprotective properties.

     

Targeted deletion of GD3 synthase protects against MPTP‐induced neurodegeneration

Parkinson's disease is a debilitating neurodegenerative condition for which there is no cure. Converging evidence implicates gangliosides in the pathogenesis of several neurodegenerative diseases, suggesting a potential new class of therapeutic targets. We have shown that interventions that simultaneously increase the neuroprotective GM1 ganglioside and decrease the pro‐apoptotic GD3 ganglioside – such as inhibition of GD3 synthase (GD3S) or administration of sialidase – are neuroprotective in vitro and in a number of preclinical models. In this study, we investigated the effects of GD3S deletion on parkinsonism induced by 1‐methyl‐4phenyl‐1,2,3,6‐tetrahydropyridine (MPTP). MPTP was administered to GD3S?/? mice or controls using a subchronic regimen consisting of three series of low‐dose injections (11 mg/kg/day × 5 days each, 3 weeks apart), and motor function was assessed after each. The typical battery of tests used to assess parkinsonism failed to detect deficits in MPTP‐treated mice. More sensitive measures – such as the force‐plate actimeter and treadmill gait parameters – detected subtle effects of MPTP, some of which were absent in mice lacking GD3S. In wild‐type mice, MPTP destroyed 53% of the tyrosine‐hydroxylase (TH)‐positive neurons in the substantia nigra pars compacta (SNc) and reduced striatal dopamine 60.7%. In contrast, lesion size was only 22.5% in GD3S?/? mice and striatal dopamine was reduced by 37.2%. Stereological counts of Nissl‐positive SNc neurons that did not express TH suggest that neuroprotection was complete but TH expression was suppressed in some cells. These results show that inhibition of GD3S has neuroprotective properties in the MPTP model and may warrant further investigation as a therapeutic target.     

Pramipexole protects against MPTP toxicity in non-human primates

The neurotoxin MPTP induces nigral dopaminergic cell death in primates and produces a partial model of Parkinson's disease (PD). Pramipexole is a D2/D3 dopamine receptor agonist used in the symptomatic treatment of PD, and which also protects neuronal cells against dopaminergic toxins in vitro. We now demonstrate that pramipexole partially prevents MPTP toxicity in vivo in a primate species. Common marmosets were repeatedly treated with pramipexole either before, coincidentally with, or after low-dose MPTP treatment designed to induce a partial lesion of the substantia nigra. Animals pretreated with pramipexole had a significantly greater number of surviving tyrosine hydroxylase (TH) positive neurones in the pars compacta of the substantia nigra. Pramipexole pretreatment also prevented degeneration of striatal dopamine terminals. Treatment with pramipexole concurrently with MPTP or following MPTP did not prevent TH-positive cell loss. Pramipexole pretreatment appears to induce adaptive changes that protect against dopaminergic cell loss in primates.     

A single intramuscular injection of rAAV‐mediated mutant erythropoietin protects against MPTP‐induced parkinsonism

Erythropoietin (Epo) is neuroprotective in a number of preparations, but can lead to unacceptably high and even lethal hematocrit levels. Recent reports show that modified Epo variants confer neuroprotection in models of glaucoma and retinal degeneration without raising hematocrit. In this study, neuroprotective effects of two Epo variants (EpoR76E and EpoS71E) were assessed in a model of Parkinson's disease. The constructs were packaged in recombinant adeno‐associated viral (rAAV) vectors and injected intramuscularly. After 3 weeks, mice received five daily injections of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) and were killed 5 weeks later. The MPTP‐lesioned mice pretreated with rAAV.eGFP (negative control) exhibited a 7‐ to 9‐Hz tremor and slower latencies to move on a grid test (akinesia). Both of these symptomatic features were absent in mice pretreated with either modified Epo construct. The rAAV.eGFP‐treated mice lesioned with MPTP exhibited a 41% reduction in tyrosine hydroxylase (TH)‐positive neurons in the substantia nigra. The rAAV.EpoS71E construct did not protect nigral neurons, but neuronal loss in mice pretreated with rAAV.EpoR76E was only half that of rAAV.eGFP controls. Although dopamine levels were normal in all groups, 3,4‐dihydroxyphenylacetic acid (DOPAC) was significantly reduced only in MPTP‐lesioned mice pretreated with rAAV.eGFP, indicating reduced dopamine turnover. Analysis of TH‐positive fibers in the striatum showed normalized density in MPTP‐lesioned mice pretreated with rAAV.EpoS71E, suggesting that enhanced sprouting induced by EpoS71E may have been responsible for normal behavior and dopaminergic tone in these mice. These results show that systemically administered rAAV‐generated non‐erythropoietic Epo may protect against MPTP‐induced parkinsonism by a combination of neuroprotection and enhanced axonal sprouting.     

Enhancement of glutamate uptake mediates the neuroprotection exerted by activating group II or III metabotropic glutamate receptors on astrocytes

We investigated whether the activation of astroglial group II and III metabotropic glutamate receptors (mGluRs) could exert neuroprotective effects and whether the neuroprotection was related to glutamate uptake. Our results showed that the activation of astroglial group II or III mGluRs exerted neuroprotection against 1-methyl-4-phenylpyridinium (MPP+) astroglial conditioned medium-induced neurotoxicity in midbrain neuron cultures. Furthermore, MPP+ decreased glutamate uptake of primary astrocytes and C6 glioma cells, which was recovered by activating group II or III mGluRs. Specific group II or III mGluRs antagonists completely abolished the neuroprotective effects and the enhancement of glutamate uptake of their respective agonists. Our results showed that the primary cultured rat astrocytes and C6 glioma cells expressed receptor proteins for group II mGluR2/3, group III mGluR4, mGluR6 and mGluR7. C6 glioma cells expressed mRNA for group II mGluR3, group III mGluR4, mGluR6, mGluR7 and mGluR8. In conclusion, we confirmed that the activation of astroglial mGluRs exerted neuroprotection, and demonstrated that the mechanism underlying this protective role was at least partially related to the enhancement of glutamate uptake.     

Ellagic acid protects dopamine neurons from rotenone‐induced neurotoxicity via activation of Nrf2 signalling

Parkinson's disease (PD) is the second most prevalent central nervous system (CNS) degenerative disease. Oxidative stress is one of key contributors to PD. Nuclear factor erythroid‐2‐related factor 2 (Nrf2) is considered to be a master regulator of many genes involved in anti‐oxidant stress to attenuate cell death. Therefore, activation of Nrf2 signalling provides an effective avenue to treat PD. Ellagic acid (EA), a natural polyphenolic contained in fruits and nuts, possesses amounts of pharmacological activities, such as anti‐oxidant stress and anti‐inflammation. Recent studies have confirmed EA could be used as a neuroprotective agent in neurodegenerative diseases. Here, mice subcutaneous injection of rotenone (ROT)‐induced DA neuronal damage was performed to investigate EA‐mediated neuroprotection. In addition, adult Nrf2 knockout mice and different cell cultures including MN9D‐enciched, MN9D‐BV‐2 and MN9D‐C6 cell co‐cultures were applied to explore the underlying mechanisms. Results demonstrated EA conferred neuroprotection against ROT‐induced DA neurotoxicity. Activation of Nrf2 signalling was involved in EA‐mediated DA neuroprotection, as evidenced by the following observations. First, EA activated Nrf2 signalling in ROT‐induced DA neuronal damage. Second, EA generated neuroprotection with the presence of astroglia and silence of Nrf2 in astroglia abolished EA‐mediated neuroprotection. Third, EA failed to produce DA neuroprotection in Nrf2 knockout mice. In conclusion, this study identified EA protected against DA neuronal loss via an Nrf2‐dependent manner.     

S‐allyl cysteine activates the Nrf2‐dependent antioxidant response and protects neurons against ischemic injury in vitro and in vivo

Stroke is a devastating clinical condition for which an effective neuroprotective treatment is currently unavailable. S‐allyl cysteine (SAC), the most abundant organosulfur compound in aged garlic extract, has been reported to possess neuroprotective effects against stroke. However, the mechanisms underlying its beneficial effects remain poorly defined. The present study tests the hypothesis that SAC attenuates ischemic neuronal injury by activating the nuclear factor erythroid‐2‐related factor 2 (Nrf2)‐dependent antioxidant response in both in vitro and in vivo models. Our findings demonstrate that SAC treatment resulted in an increase in Nrf2 protein levels and subsequent activation of antioxidant response element pathway genes in primary cultured neurons and mice. Exposure of primary neurons to SAC provided protection against oxygen and glucose deprivation‐induced oxidative insults. In wild‐type (Nrf2^+/+) mice, systemic administration of SAC attenuated middle cerebral artery occlusion‐induced ischemic damage, a protective effect not observed in Nrf2 knockout (Nrf2^?/?) mice. Taken together, these findings provide the first evidence that activation of the Nrf2 antioxidant response by SAC is strongly associated with its neuroprotective effects against experimental stroke and suggest that targeting the Nrf2 pathway may provide therapeutic benefit for the treatment of stroke.