γ‐glutamyl transpeptidase 1 specifically suppresses green‐light avoidance via GABAA receptors in Drosophila

Drosophila larvae innately show light avoidance behavior. Compared with robust blue‐light avoidance, larvae exhibit relatively weaker green‐light responses. In our previous screening for genes involved in larval light avoidance, compared with control w¹¹¹⁸ larvae, larvae with γ‐glutamyl transpeptidase 1 (Ggt‐1) knockdown or Ggt‐1 mutation were found to exhibit higher percentage of green‐light avoidance which was mediated by Rhodopsin6 (Rh6) photoreceptors. However, their responses to blue light did not change significantly. By adjusting the expression level of Ggt‐1 in different tissues, we found that Ggt‐1 in malpighian tubules was both necessary and sufficient for green‐light avoidance. Our results showed that glutamate levels were lower in Ggt‐1 null mutants compared with controls. Feeding Ggt‐1 null mutants glutamate can normalize green‐light avoidance, indicating that high glutamate concentrations suppressed larval green‐light avoidance. However, rather than directly, glutamate affected green‐light avoidance indirectly through GABA, the level of which was also lower in Ggt‐1 mutants compared with controls. Mutants in glutamate decarboxylase 1, which encodes GABA synthase, and knockdown lines of the GABA_A receptor, both exhibit elevated levels of green‐light avoidance. Thus, our results elucidate the neurobiological mechanisms mediating green‐light avoidance, which was inhibited in wild‐type larvae.

     

The voltage‐gated calcium channel blocker lomerizine is neuroprotective in motor neurons expressing mutant SOD1, but not TDP‐43

Excitotoxicity and disruption of Ca²⁺ homeostasis have been implicated in amyotrophic lateral sclerosis (ALS) and limiting Ca²⁺ entry is protective in models of ALS caused by mutation of SOD1. Lomerizine, an antagonist of L‐ and T‐type voltage‐gated calcium channels and transient receptor potential channel 5 transient receptor potential channels, is well tolerated clinically, making it a potential therapeutic candidate. Lomerizine reduced glutamate excitotoxicity in cultured motor neurons by reducing the accumulation of cytoplasmic Ca²⁺ and protected motor neurons against multiple measures of mutant SOD1 toxicity: Ca²⁺ overload, impaired mitochondrial trafficking, mitochondrial fragmentation, formation of mutant SOD1 inclusions, and loss of viability. To assess the utility of lomerizine in other forms of ALS, calcium homeostasis was evaluated in culture models of disease because of mutations in the RNA‐binding proteins transactive response DNA‐binding protein 43 (TDP‐43) and Fused in Sarcoma (FUS). Calcium did not play the same role in the toxicity of these mutant proteins as with mutant SOD1 and lomerizine failed to prevent cytoplasmic accumulation of mutant TDP‐43, a hallmark of its pathology. These experiments point to differences in the pathogenic pathways between types of ALS and show the utility of primary culture models in comparing those mechanisms and effectiveness of therapeutic strategies.

     

Sulforaphane enhances temozolomide‐induced apoptosis because of down‐regulation of miR‐21 via Wnt/β‐catenin signaling in glioblastoma

Temozolomide (TMZ) has been widely used in the treatment of glioblastoma (GBM), although inherent or acquired resistance restricts the application. This study was aimed to evaluate the efficacy of sulforaphane (SFN) to TMZ‐induced apoptosis in GBM cells and the potential mechanism. Biochemical assays and subcutaneous tumor establishment were used to characterize the function of SFN in TMZ‐induced apoptosis. Our results revealed that β‐catenin and miR‐21 were concordantly expressed in GBM cell lines, and SFN significantly reduced miR‐21 expression through inhibiting the Wnt/β‐catenin/TCF4 pathway. Furthermore, down‐regulation of miR‐21 enhanced the pro‐apoptotic efficacy of TMZ in GBM cells. Finally, we observed that SFN strengthened TMZ‐mediated apoptosis in a miR‐21‐dependent manner. In conclusion, SFN effectively enhances TMZ‐induced apoptosis by inhibiting miR‐21 via Wnt/β‐catenin signaling in GBM cells. These findings support the use of SFN for potential therapeutic approach to overcome TMZ resistance in GBM treatment.

     

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.

     

Motor neuropathy‐associated mutation impairs Seipin functions in neurotransmission

Gain‐of‐toxic‐function mutations in Seipin (Asparagine 88 to Serine (N88S) and Serine 90 to Leucine (S90L) mutations, both of which disrupt the N‐glycosylation) cause autosomal dominant motor neuron diseases. However, the mechanism of how these missense mutations lead to motor neuropathy is unclear. Here, we analyze the impact of disruption of N‐glycosylation of Seipin on synaptic transmission by over‐expressing mutant Seipin in cultured cortical neurons via lentiviral infection. Immunostaining shows that over‐expressed Seipin is partly colocalized with synaptic vesicle marker synaptophysin. Electrophysiological recordings reveal that the Seipin mutation significantly decreases the frequency, but not the amplitudes of miniature excitatory post‐synaptic currents and miniature inhibitory post‐synaptic currents. The amplitude of both evoked excitatory post‐synaptic currents and inhibitory post‐synaptic current is also compromised by mutant Seipin over‐expression. The readily releasable pool and vesicular release probability of synaptic vesicles are both altered in neurons over‐expressing Seipin‐N88S, whereas neither γ‐amino butyric acid (GABA) nor α‐Amino‐3‐hydroxy‐5‐methyl‐4‐ isoxazolepropionic acid (AMPA) induced whole cell currents are affected. Moreover, electron microscopy analysis reveals decreased number of morphologically docked synaptic vesicles in Seipin‐N88S‐expressing neurons. These data demonstrate that Seipin‐N88S mutation impairs synaptic neurotransmission, possibly by regulating the priming and docking of synaptic vesicles at the synapse.

     

A dual role for AMP‐activated protein kinase (AMPK) during neonatal hypoxic–ischaemic brain injury in mice

Perinatal hypoxic–ischaemic encephalopathy (HIE) occurs in 1–2 in every 1000 term infants and the devastating consequences range from cerebral palsy, epilepsy and neurological deficit to death. Cellular damage post insult occurs after a delay and is mediated by a secondary neural energy failure. AMP‐activated protein kinase (AMPK) is a sensor of cellular stress resulting from ATP depletion and/or calcium dysregulation, hallmarks of the neuronal cell death observed after HIE. AMPK activation has been implicated in the models of adult ischaemic injury but, as yet, there have been no studies defining its role in neonatal asphyxia. Here, we find that in an in vivo model of neonatal hypoxia–ischaemic and in oxygen/glucose deprivation in neurons, there is pathological activation of the calcium/calmodulin‐dependent protein kinase kinase β (CaMKKβ)‐AMPKα1 signalling pathway. Pharmacological inhibition of AMPK during the insult promotes neuronal survival but, conversely, inhibiting AMPK activity prior to the insult sensitizes neurons, exacerbating cell death. Our data have pathological relevance for neonatal HIE as prior sensitization such as exposure to bacterial infection (reported to reduce AMPK activity) produces a significant increase in injury.

     

Differential regulation of CaMKIIα interactions with mGluR5 and NMDA receptors by Ca2+ in neurons

Two glutamate receptors, metabotropic glutamate receptor 5 (mGluR5), and ionotropic NMDA receptors (NMDAR), functionally interact with each other to regulate excitatory synaptic transmission in the mammalian brain. In exploring molecular mechanisms underlying their interactions, we found that Ca²⁺/calmodulin‐dependent protein kinase IIα (CaMKIIα) may play a central role. The synapse‐enriched CaMKIIα directly binds to the proximal region of intracellular C terminal tails of mGluR5 in vitro. This binding is state‐dependent: inactive CaMKIIα binds to mGluR5 at a high level whereas the active form of the kinase (following Ca²⁺/calmodulin binding and activation) loses its affinity for the receptor. Ca²⁺ also promotes calmodulin to bind to mGluR5 at a region overlapping with the CaMKIIα‐binding site, resulting in a competitive inhibition of CaMKIIα binding to mGluR5. In rat striatal neurons, inactive CaMKIIα constitutively binds to mGluR5. Activation of mGluR5 Ca²⁺‐dependently dissociates CaMKIIα from the receptor and simultaneously promotes CaMKIIα to bind to the adjacent NMDAR GluN2B subunit, which enables CaMKIIα to phosphorylate GluN2B at a CaMKIIα‐sensitive site. Together, the long intracellular C‐terminal tail of mGluR5 seems to serve as a scaffolding domain to recruit and store CaMKIIα within synapses. The mGluR5‐dependent Ca²⁺ transients differentially regulate CaMKIIα interactions with mGluR5 and GluN2B in striatal neurons, which may contribute to cross‐talk between the two receptors.

     

The mitochondrial uncoupler DNP triggers brain cell mTOR signaling network reprogramming and CREB pathway up‐regulation

Mitochondrial metabolism is highly responsive to nutrient availability and ongoing activity in neuronal circuits. The molecular mechanisms by which brain cells respond to an increase in cellular energy expenditure are largely unknown. Mild mitochondrial uncoupling enhances cellular energy expenditure in mitochondria and can be induced with 2,4‐dinitrophenol (DNP), a proton ionophore previously used for weight loss. We found that DNP treatment reduces mitochondrial membrane potential, increases intracellular Ca²⁺ levels and reduces oxidative stress in cerebral cortical neurons. Gene expression profiling of the cerebral cortex of DNP‐treated mice revealed reprogramming of signaling cascades that included suppression of the mammalian target of rapamycin (mTOR) and insulin – PI3K – MAPK pathways, and up‐regulation of tuberous sclerosis complex 2, a negative regulator of mTOR. Genes encoding proteins involved in autophagy processes were up‐regulated in response to DNP. CREB (cAMP‐response element‐binding protein) signaling, Arc and brain‐derived neurotrophic factor, which play important roles in synaptic plasticity and adaptive cellular stress responses, were up‐regulated in response to DNP, and DNP‐treated mice exhibited improved performance in a test of learning and memory. Immunoblot analysis verified that key DNP‐induced changes in gene expression resulted in corresponding changes at the protein level. Our findings suggest that mild mitochondrial uncoupling triggers an integrated signaling response in brain cells characterized by reprogramming of mTOR and insulin signaling, and up‐regulation of pathways involved in adaptive stress responses, molecular waste disposal, and synaptic plasticity.

     

Ionizing radiation causes increased tau phosphorylation in primary neurons

Radiotherapy is the major treatment modality for primary and metastatic brain tumors which involves the exposure of brain to ionizing radiation. Ionizing radiation can induce various detrimental pathophysiological effects in the adult brain, and Alzheimer's disease and related neurodegenerative disorders are considered to be late effects of radiation. In this study, we investigated whether ionizing radiation causes changes in tau phosphorylation in cultured primary neurons similar to that in Alzheimer's disease. We demonstrated that exposure to 0.5 or 2 Gy γ rays causes increased phosphorylation of tau protein at several phosphorylation sites in a time‐ and dose‐dependent manner. Consistently, we also found ionizing radiation causes increased activation of GSK3β, c‐Jun N‐terminal kinase and extracellular signal‐regulated kinase before radiation‐induced increase in tau phosphorylation. Specific inhibitors of these kinases almost fully blocked radiation‐induced tau phosphorylation. Our studies further revealed that oxidative stress plays an important role in ionizing radiation‐induced tau phosphorylation, likely through the activation of c‐Jun N‐terminal kinase and extracellular signal‐regulated kinase, but not GSK3β. Overall, our studies suggest that ionizing radiation may cause increased risk for development of Alzheimer's disease by promoting abnormal tau phosphorylation.

     

Susceptibility to excitotoxicity in aged hippocampal cultures and neuroprotection by non‐steroidal anti‐inflammatory drugs: role of mitochondrial calcium

Brain damage after insult and cognitive decline are related to excitotoxicity and strongly influenced by aging, yet mechanisms of aging‐dependent susceptibility to excitotoxicity are poorly known. Several non‐steroidal anti‐inflammatory drugs (NSAIDs) may prevent excitotoxicity and cognitive decline in the elderly by an unknown mechanism. Interestingly, after several weeks in vitro, hippocampal neurons display important hallmarks of neuronal aging in vivo. Accordingly, rat hippocampal neurons cultured for several weeks were used to investigate mechanisms of aging‐related susceptibility to excitotoxicity and neuroprotection by NSAIDs. We found that NMDA increased cytosolic Ca²⁺ concentration in young, mature and aged neurons but only promoted apoptosis in aged neurons. Resting Ca²⁺ levels and responses to NMDA increased with time in culture which correlated with changes in expression of NMDA receptor subunits. In addition, NMDA promoted mitochondrial Ca²⁺ uptake only in aged cultures. Consistently, specific inhibition of mitochondrial Ca²⁺ uptake decreased apoptosis. Finally, we found that a series of NSAIDs depolarized mitochondria and inhibited mitochondrial Ca²⁺ overload, thus preventing NMDA‐induced apoptosis in aged cultures. We conclude that mitochondrial Ca²⁺ uptake is critical for age‐related susceptibility to excitotoxicity and neuroprotection by NSAIDs.

     

Lipid peroxidation is essential for α‐synuclein‐induced cell death

Parkinson's disease is the second most common neurodegenerative disease and its pathogenesis is closely associated with oxidative stress. Deposition of aggregated α‐synuclein (α‐Syn) occurs in familial and sporadic forms of Parkinson's disease. Here, we studied the effect of oligomeric α‐Syn on one of the major markers of oxidative stress, lipid peroxidation, in primary co‐cultures of neurons and astrocytes. We found that oligomeric but not monomeric α‐Syn significantly increases the rate of production of reactive oxygen species, subsequently inducing lipid peroxidation in both neurons and astrocytes. Pre‐incubation of cells with isotope‐reinforced polyunsaturated fatty acids (D‐PUFAs) completely prevented the effect of oligomeric α‐Syn on lipid peroxidation. Inhibition of lipid peroxidation with D‐PUFAs further protected cells from cell death induced by oligomeric α‐Syn. Thus, lipid peroxidation induced by misfolding of α‐Syn may play an important role in the cellular mechanism of neuronal cell loss in Parkinson's disease.

     

Pleiotrophin differentially regulates the rewarding and sedative effects of ethanol

Pleiotrophin (PTN) is a cytokine with important roles in dopaminergic neurons. We found that an acute ethanol (2.0 g/kg, i.p.) administration causes a significant up‐regulation of PTN mRNA and protein levels in the mouse prefrontal cortex, suggesting that endogenous PTN could modulate behavioural responses to ethanol. To test this hypothesis, we studied the behavioural effects of ethanol in PTN knockout (PTN^?/?) mice and in mice with cortex‐ and hippocampus‐specific transgenic PTN over‐expression (PTN‐Tg). Ethanol (1.0 and 2.0 g/kg) induced an enhanced conditioned place preference in PTN^?/? compared to wild type mice, suggesting that PTN prevents ethanol rewarding effects. Accordingly, the conditioning effects of ethanol were completely abolished in PTN‐Tg mice. The ataxic effects induced by ethanol (2.0 g/kg) were not affected by the genotype. However, the sedative effects of ethanol (3.6 g/kg) tested in a loss of righting reflex paradigm were significantly reduced in PTN‐Tg mice, suggesting that up‐regulation of PTN levels prevents the sedative effects of ethanol. These results indicate that PTN may be a novel genetic factor of importance in alcohol use disorders, and that potentiation of the PTN signalling pathway may be a promising therapeutic strategy in the treatment of these disorders.