Cyr61 activates retinal cells and prolongs photoreceptor survival in rd1 mouse model of retinitis pigmentosa

Subretinal injections with glial cell line‐derived neurotrophic factor (GDNF) rescue morphology as well as function of rod cells in mouse and rat animal models of retinitis pigmentosa. At the same time, it is postulated that this effect is indirect, mediated by activation of retinal Müller glial (RMG) cells. Here, we show that Cyr61/CCN1, one of the secreted proteins up‐regulated in primary RMG after glial cell line‐derived neurotrophic factor stimulation, provides neuroprotective and pro‐survival capacities: Recombinant Cyr61 significantly reduced photoreceptor (PR) cells death in organotypic cultures of Pde6b^rd1 retinas. To identify stimulated pathways in the retina, we treated Pde6b^rd1 retinal explants with Cyr61 and observed an overall increase in activated Erk1/2 and Stat3 signalling molecules characterized by activation‐site‐specific phosphorylation. To identify Cyr61 retinal target cells, we isolated primary porcine PR, RMG and retinal pigment epithelium (RPE) cells and exposed them separately to Cyr61. Here, RMG as well as RPE cells responded with induced phosphorylation of Erk1/2, Stat3 and Akt. In PR, no increase in phosphorylation in any of the studied proteins was detected, suggesting an indirect neuroprotective effect of Cyr61. Cyr61 may thus act as an endogenous pro‐survival factor for PR, contributing to the complex repertoire of neuroprotective activities generated by RMG and RPE cells.

     

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.

     

Alterations in STriatal‐Enriched protein tyrosine Phosphatase expression,activation, and downstream signaling in early and late stages of the YAC128 Huntington's disease mouse model

Striatal neurodegeneration and synaptic dysfunction in Huntington's disease are mediated by the mutant huntingtin (mHtt) protein. MHtt disrupts calcium homeostasis and facilitates excitotoxicity, in part by altering NMDA receptor (NMDAR) trafficking and function. Pre‐symptomatic (excitotoxin‐sensitive) transgenic mice expressing full‐length human mHtt with 128 polyglutamine repeats (YAC128 Huntington's disease mice) show increased calpain activity and extrasynaptic NMDAR (Ex‐NMDAR) localization and signaling. Furthermore, Ex‐NMDAR stimulation facilitates excitotoxicity in wild‐type cortical neurons via calpain‐mediated cleavage of STriatal‐Enriched protein tyrosine Phosphatase 61 (STEP61). The cleavage product, STEP33, cannot dephosphorylate p38 mitogen‐activated protein kinase (MAPK), thereby augmenting apoptotic signaling. Here, we show elevated extrasynaptic calpain‐mediated cleavage of STEP61 and p38 phosphorylation, as well as STEP61 inactivation and reduced extracellular signal‐regulated protein kinase 1/2 phosphorylation (ERK1/2) in the striatum of 6‐week‐old, excitotoxin‐sensitive YAC128 mice. Calpain inhibition reduced basal and NMDA‐induced STEP61 cleavage. However, basal p38 phosphorylation was normalized by a peptide disrupting NMDAR‐post‐synaptic density protein‐95 (PSD‐95) binding but not by calpain inhibition. In 1‐year‐old excitotoxin‐resistant YAC128 mice, STEP33 levels were not elevated, but STEP61 inactivation and p38 and ERK 1/2 phosphorylation levels were increased. These results show that in YAC128 striatal tissue, enhanced NMDAR–PSD‐95 interactions contributes to elevated p38 signaling in early, excitotoxin‐sensitive stages, and suggest that STEP61 inactivation enhances MAPK signaling at late, excitotoxin‐resistant stages.

     

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.

     

HIV‐1 Tat C modulates NOX2 and NOX4 expressions through miR‐17 in a human microglial cell line

HIV‐1 invades CNS in the early course of infection, which can lead to the cascade of neuroinflammation. NADPH oxidases (NOXs) are the major producers of reactive oxygen species (ROS), which play important roles during pathogenic insults. The molecular mechanism of ROS generation via microRNA‐mediated pathway in human microglial cells in response to HIV‐1 Tat protein has been demonstrated in this study. Over‐expression and knockdown of microRNAs, luciferase reporter assay, and site‐directed mutagenesis are main molecular techniques used in this study. A significant reduction in miR‐17 levels and increased NOX2, NOX4 expression levels along with ROS production were observed in human microglial cells upon HIV‐1 Tat C exposure. The validation of NOX2 and NOX4 as direct targets of miR‐17 was done by luciferase reporter assay. The over‐expression and knockdown of miR‐17 in human microglial cells showed the direct role of miR‐17 in regulation of NOX2, NOX4 expression and intracellular ROS generation. We demonstrated the regulatory role of cellular miR‐17 in ROS generation through over‐expression and knockdown of miR‐17 in human microglial cells exposed to HIV‐1 Tat C protein.

     

Inhibition of Rho‐kinase protects cerebral barrier from ischaemia‐evoked injury through modulations of endothelial cell oxidative stress and tight junctions

Ischaemic strokes evoke blood–brain barrier (BBB) disruption and oedema formation through a series of mechanisms involving Rho‐kinase activation. Using an animal model of human focal cerebral ischaemia, this study assessed and confirmed the therapeutic potential of Rho‐kinase inhibition during the acute phase of stroke by displaying significantly improved functional outcome and reduced cerebral lesion and oedema volumes in fasudil‐ versus vehicle‐treated animals. Analyses of ipsilateral and contralateral brain samples obtained from mice treated with vehicle or fasudil at the onset of reperfusion plus 4 h post‐ischaemia or 4 h post‐ischaemia alone revealed these benefits to be independent of changes in the activity and expressions of oxidative stress‐ and tight junction‐related parameters. However, closer scrutiny of the same parameters in brain microvascular endothelial cells subjected to oxygen–glucose deprivation ± reperfusion revealed marked increases in prooxidant NADPH oxidase enzyme activity, superoxide anion release and in expressions of antioxidant enzyme catalase and tight junction protein claudin‐5. Cotreatment of cells with Y‐27632 prevented all of these changes and protected in vitro barrier integrity and function. These findings suggest that inhibition of Rho‐kinase after acute ischaemic attacks improves cerebral integrity and function through regulation of endothelial cell oxidative stress and reorganization of intercellular junctions.

     

Valproic acid induces neuronal cell death through a novel calpain‐dependent necroptosis pathway

A growing body of evidence indicates that valproic acid (VPA), a histone deacetylase inhibitor used to treat epilepsy and mood disorders, has histone deacetylase‐related and ‐unrelated neurotoxic activity, the mechanism of which is still poorly understood. We report that VPA induces neuronal cell death through an atypical calpain‐dependent necroptosis pathway that initiates with downstream activation of c‐Jun N‐terminal kinase 1 (JNK1) and increased expression of receptor‐interacting protein 1 (RIP‐1) and is accompanied by cleavage and mitochondrial release/nuclear translocation of apoptosis‐inducing factor, mitochondrial release of Smac/DIABLO, and inhibition of the anti‐apoptotic protein X‐linked inhibitor of apoptosis (XIAP). Coinciding with apoptosis‐inducing factor nuclear translocation, VPA induces phosphorylation of the necroptosis‐associated histone H2A family member H2AX, which is known to contribute to lethal DNA degradation. These signals are inhibited in neuronal cells that express constitutively activated MEK/ERK and/or PI3‐K/Akt survival pathways, allowing them to resist VPA‐induced cell death. The data indicate that VPA has neurotoxic activity and identify a novel calpain‐dependent necroptosis pathway that includes JNK1 activation and RIP‐1 expression.

     

Phosphoinositide and Erk signaling pathways mediate activity‐driven rodent olfactory sensory neuronal survival and stress mitigation

Olfactory sensory neurons (OSNs) are the initial site for olfactory signal transduction. Therefore, their survival is essential to olfactory function. In the current study, we demonstrated that while odorant stimulation promoted rodent OSN survival, it induced generation of reactive oxygen species in a dose‐ and time‐dependent manner as well as loss of membrane potential and fragmentation of mitochondria. The MEK‐Erk pathway played a critical role in mediating these events, as its inhibition decreased odorant stimulation‐dependent OSN survival and exacerbated intracellular stress measured by reactive oxygen species generation and heat‐shock protein 70 expression. The phosphoinositide pathway, rather than the cyclic AMP pathway, mediated the odorant‐induced activation of the MEK‐Erk pathway. These findings provide important insights into the mechanisms of activity‐driven OSN survival, the role of the phosphoinositide pathway in odorant signaling, and demonstrate that odorant detection and odorant stimulation‐mediated survival proceed via independent signaling pathways. This mechanism, which permits independent regulation of odorant detection from survival signaling, may be advantageous if not diminished by repeated or prolonged odor exposure.

     

Ephrin‐A3 reverse signaling regulates hippocampal neuronal damage and astrocytic glutamate transport after transient global ischemia

Increasing evidence indicates that the Eph receptors and their ephrin ligands are involved in the regulation of interactions between neurons and astrocytes. Moreover, astrocytic ephrin‐A3 reverse signaling mediated by EphA4 receptors is necessary for controlling the abundance of glial glutamate transporters. However, the role of ephrin‐A3 reverse signaling in astrocytic function and neuronal death under ischemic conditions remains unclear. In the present study, we found that the EphA4 receptor and its ephrin‐A3 ligand, which were distributed in neurons and astrocytes, respectively, in the hippocampus showed a coincident up‐regulation of protein expression in the early stage of ischemia. Application of clustered EphA4 decreased the expressions of astrocytic glutamate transporters together with astrocytic glutamate uptake capacity through activating ephrin‐A3 reverse signaling. In consequence, neuronal loss was aggravated in the CA1 region of the hippocampus accompanied by impaired hippocampus‐dependent spatial memory when clustered EphA4 treatment was administered prior to transient global ischemia. These findings indicate that EphA4‐mediated ephrin‐A3 reverse signaling is a crucial mechanism for astrocytes to control glial glutamate transporters and prevent glutamate excitotoxicity under pathological conditions.

     

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.

     

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.

     

A conserved sequence in calmodulin regulated spectrin‐associated protein 1 links its interaction with spectrin and calmodulin to neurite outgrowth

Calmodulin regulated spectrin‐associated protein 1 (CAMSAP1) is a vertebrate microtubule‐binding protein, and a representative of a family of cytoskeletal proteins that arose with animals. We reported previously that the central region of the protein, which contains no recognized functional domain, inhibited neurite outgrowth when over‐expressed in PC12 cells [Baines et al., Mol. Biol. Evol. 26 (2009), p. 2005]. The CKK domain (DUF1781) binds microtubules and defines the CAMSAP/ssp4 family of animal proteins (Baines et al. 2009). In the central region, three short well‐conserved regions are characteristic of CAMSAP‐family members. One of these, CAMSAP‐conserved region 1 (CC1), bound to both βIIΣ1‐spectrin and Ca²⁺/calmodulin in vitro. The binding of Ca²⁺/calmodulin inhibited spectrin binding. Transient expression of CC1 in PC12 cells inhibited neurite outgrowth. siRNA knockdown of CAMSAP1 inhibited neurite outgrowth in PC12 cells or primary cerebellar granule cells: this could be rescued in PC12 cells by wild‐type CAMSAP1‐enhanced green fluorescent protein, but not by a CC1 mutant. We conclude that CC1 represents a functional region of CAMSAP1, which links spectrin‐binding to neurite outgrowth.

     

Nidogen‐1 is a common target of microRNAs MiR‐192/215 in the pathogenesis of Hirschsprung's disease

Recent studies have emphasized the important role of microRNA (miRNA) clusters and common target genes in disease progression. Despite the known involvement of the miR‐192/215 family in many human diseases, its biological role in Hirschsprung disease (HSCR) remains undefined. In this study, we explored the role of the miR‐192/215 family in the pathogenesis of HSCR. Quantitative real‐time PCR and western blotting measured relative expression levels of miRNAs, mRNAs, and proteins in 80 HSCR patients and 77 normal colon tissues. Targets were evaluated by dual‐luciferase reporter assays, and the functional effects of miR‐192/215 on human 293T and SH‐SY5Y cells were detected by the Transwell assay, CCK8 assay and flow cytometry. MiR‐192/215 was significantly down‐regulated in HSCR tissue samples, and their knockdown inhibited cell migration and proliferation in the human 293T and SH‐SY5Y cell lines. Nidogen 1 (NID1) was confirmed as a common target gene of miR‐192/215 by dual‐luciferase reporter gene assay and its expression was inversely correlated with that of miR‐192/215 in tissue samples and cell lines. Silencing of NID1 could rescue the extent of the suppressing effects by miR‐192/215 inhibitor. The down‐regulation of miR‐192/215 may contribute to HSCR development by targeting NID1.

     

Interferon‐γ induces leucine‐rich repeat kinase LRRK2 via extracellular signal‐regulated kinase ERK5 in macrophages

The gene encoding leucine‐rich repeat kinase 2 (LRRK2) comprises a major risk factor for Parkinson's disease. Recently, it has emerged that LRRK2 plays important roles in the immune system. LRRK2 is induced by interferon‐γ (IFN‐γ) in monocytes, but the signaling pathway is not known. Here, we show that IFN‐γ‐mediated induction of LRRK2 was suppressed by pharmacological inhibition and RNA interference of the extracellular signal‐regulated kinase 5 (ERK5). This was confirmed by LRRK2 immunostaining, which also revealed that the morphological responses to IFN‐γ were suppressed by ERK5 inhibitor treatment. Both human acute monocytic leukemia THP‐1 cells and human peripheral blood monocytes stimulated the ERK5‐LRRK2 pathway after differentiation into macrophages. Thus, LRRK2 is induced via a novel, ERK5‐dependent IFN‐γ signal transduction pathway, pointing to new functions of ERK5 and LRRK2 in human macrophages.

     

Coordinated activation of AMP‐activated protein kinase,extracellular signal‐regulated kinase,and autophagy regulates phorbol myristate acetate‐induced differentiation of SH‐SY5Y neuroblastoma cells

We explored the interplay between the intracellular energy sensor AMP‐activated protein kinase (AMPK), extracellular signal‐regulated kinase (ERK), and autophagy in phorbol myristate acetate (PMA)‐induced neuronal differentiation of SH‐SY5Y human neuroblastoma cells. PMA‐triggered expression of neuronal markers (dopamine transporter, microtubule‐associated protein 2, β‐tubulin) was associated with an autophagic response, measured by the conversion of microtubule‐associated protein light chain 3 (LC3)‐I to autophagosome‐bound LC3‐II, increase in autophagic flux, and expression of autophagy‐related (Atg) proteins Atg7 and beclin‐1. This coincided with the transient activation of AMPK and sustained activation of ERK. Pharmacological inhibition or RNA interference‐mediated silencing of AMPK suppressed PMA‐induced expression of neuronal markers, as well as ERK activation and autophagy. A selective pharmacological blockade of ERK prevented PMA‐induced neuronal differentiation and autophagy induction without affecting AMPK phosphorylation. Conversely, the inhibition of autophagy downstream of AMPK/ERK, either by pharmacological agents or LC3 knockdown, promoted the expression of neuronal markers, thus indicating a role of autophagy in the suppression of PMA‐induced differentiation of SH‐SY5Y cells. Therefore, PMA‐induced neuronal differentiation of SH‐SY5Y cells depends on a complex interplay between AMPK, ERK, and autophagy, in which the stimulatory effects of AMPK/ERK signaling are counteracted by the coinciding autophagic response.