The role of S‐nitrosylation of kainate‐type of ionotropic glutamate receptor 2 in epilepsy induced by kainic acid

Epilepsy is a chronic brain disease affecting millions of individuals. Kainate receptors, especially kainate‐type of ionotropic glutamate receptor 2 (GluK2), play an important role in epileptogenesis. Recent data showed that GluK2 could undergo post‐translational modifications in terms of S‐nitrosylation (SNO ), and affect the signaling pathway of cell death in cerebral ischemia‐reperfusion. However, it is unclear whether S‐nitrosylation of GluK2 (SNO ‐GluK2) contributes to cell death induced by epilepsy. Here, we report that kainic acid‐induced SNO ‐GluK2 is mediated by GluK2 itself, regulated by neuronal nitric oxide synthase (nNOS ) and the level of cytoplasmic calcium in vivo and in vitro hippocampus neurons. The whole‐cell patch clamp recordings showed the influence of SNO ‐GluK2 on ion channel characterization of GluK2‐Kainate receptors. Moreover, immunohistochemistry staining results showed that inhibition of SNO ‐GluK2 by blocking nNOS or GluK2 or by reducing the level of cytoplasmic calcium‐protected hippocampal neurons from kainic acid‐induced injury. Finally, immunoprecipitation and western blotting data revealed the involvement of assembly of a GluK2‐PSD 95‐nNOS signaling complex in epilepsy. Taken together, our results showed that the SNO ‐GluK2 plays an important role in neuronal injury of epileptic rats by forming GluK2‐PSD 95‐nNOS signaling module in a cytoplasmic calcium‐dependent way, suggesting a potential therapeutic target site for epilepsy.

     

G5G2.5 core‐shell tecto‐dendrimer specifically targets reactive glia in brain ischemia

Secondary neuronal death is a serious stroke complication. This process is facilitated by the conversion of glial cells to the reactive pro‐inflammatory phenotype that induces neurodegeneration. Therefore, regulation of glial activation is a compelling strategy to reduce brain damage after stroke. However, drugs have difficulties to access the CNS , and to specifically target glial cells. In the present work, we explored the use core‐shell polyamidoamine tecto‐dendrimer (G5G2.5 PAMAM ) and studied its ability to target distinct populations of stroke‐activated glial cells. We found that G5G2.5 tecto‐dendrimer is actively engulfed by primary glial cells in a time‐ and dose‐dependent manner showing high cellular selectivity and lysosomal localization. In addition, oxygen‐glucose deprivation or lipopolysaccharides exposure in vitro and brain ischemia in vivo increase glial G5G2.5 uptake; not being incorporated by neurons or other cell types. We conclude that G5G2.5 tecto‐dendrimer is a highly suitable carrier for targeted drug delivery to reactive glial cells in vitro and in vivo after brain ischemia.

     

Expression of the purine biosynthetic enzyme phosphoribosyl formylglycinamidine synthase in neurons

Purines are metabolic building blocks essential for all living organisms on earth. De novo purine biosynthesis occurs in the brain and appears to play important roles in neural development. Phosphoribosyl formylglycinamidine synthase (FGAMS , also known as PFAS or FGARAT ), a core enzyme involved in the de novo synthesis of purines, may play alternative roles in viral pathogenesis. To date, no thorough investigation of the endogenous expression and localization of de novo purine biosynthetic enzymes has been conducted in human neurons or in virally infected cells. In this study, we characterized expression of FGAMS using multiple neuronal models. In differentiated human SH ‐SY 5Y neuroblastoma cells, primary rat hippocampal neurons, and in whole‐mouse brain sections, FGAMS immunoreactivity was distributed within the neuronal cytoplasm. FGAMS immunolabeling in vitro demonstrated extensive distribution throughout neuronal processes. To investigate potential changes in FGAMS expression and localization following viral infection, we infected cells with the human pathogen herpes simplex virus 1. In infected fibroblasts, FGAMS immunolabeling shifted from a diffuse cytoplasmic location to a mainly perinuclear localization by 12 h post‐infection. In contrast, in infected neurons, FGAMS localization showed no discernable changes in the localization of FGAMS immunoreactivity. There were no changes in total FGAMS protein levels in either cell type. Together, these data provide insight into potential purine biosynthetic mechanisms utilized within neurons during homeostasis as well as viral infection.

Cover Image for this Issue: doi: 10.1111/jnc.14169 .

     

Vitamin C modulates glutamate transport and NMDA receptor function in the retina

Vitamin C (in the reduced form ascorbate or in the oxidized form dehydroascorbate) is implicated in signaling events throughout the central nervous system (CNS ). In the retina, a high‐affinity transport system for ascorbate has been described and glutamatergic signaling has been reported to control ascorbate release. Here, we investigated the modulatory role played by vitamin C upon glutamate uptake and N ‐methyl‐d ‐aspartate (NMDA ) receptor activation in cultured retinal cells or in intact retinal tissue using biochemical and imaging techniques. We show that both forms of vitamin C, ascorbate or dehydroascorbate, promote an accumulation of extracellular glutamate by a mechanism involving the inhibition of glutamate uptake. This inhibition correlates with the finding that ascorbate promotes a decrease in cell surface levels of the neuronal glutamate transporter excitatory amino acid transporter 3 in retinal neuronal cultures. Interestingly, vitamin C is prone to increase the activity of NMDA receptors but also promotes a decrease in glutamate‐stimulated [³H] MK 801 binding and decreases cell membrane content of NMDA receptor glutamate ionotropic receptor subunit 1 (GluN1) subunits. Both compounds were also able to increase cAMP response element‐binding protein phosphorylation in neuronal nuclei in a glutamate receptor and calcium/calmodulin kinase‐dependent manner. Moreover, the effect of ascorbate is not blocked by sulfinpyrazone and then does not depend on its uptake by retinal cells. Overall, these data indicate a novel molecular and functional target for vitamin C impacting on glutamate signaling in retinal neurons.

     

Single cocaine exposure does not alter striatal pre‐synaptic dopamine function in mice: an [18F]‐FDOPA PET study

Cocaine is a recreational drug of abuse that binds to the dopamine transporter, preventing reuptake of dopamine into pre‐synaptic terminals. The increased presence of synaptic dopamine results in stimulation of both pre‐ and post‐synaptic dopamine receptors, considered an important mechanism by which cocaine elicits its reinforcing properties. However, the effects of acute cocaine administration on pre‐synaptic dopamine function remain unclear. Non‐invasive imaging techniques such as positron emission tomography have revealed impaired pre‐synaptic dopamine function in chronic cocaine users. Similar impairments have been seen in animal studies, with microdialysis experiments indicating decreased basal dopamine release. Here we use micro positron emission tomography imaging techniques in mice to measure dopamine synthesis capacity and determine the effect of acute cocaine administration of pre‐synaptic dopamine function. We show that a dose of 20 mg/kg cocaine is sufficient to elicit hyperlocomotor activity, peaking 15–20 min post treatment (p < 0.001). However, dopamine synthesis capacity in the striatum was not significantly altered by acute cocaine treatment (: 0.0097 per min vs. 0.0112 per min in vehicle controls, p > 0.05). Furthermore, expression levels of two key enzymes related to dopamine synthesis, tyrosine hydroxylase and aromatic l ‐amino acid decarboxylase, within the striatum of scanned mice were not significantly affected by acute cocaine pre‐treatment (p > 0.05). Our findings suggest that while the regulation of dopamine synthesis and release in the striatum have been shown to change with chronic cocaine use, leading to a reduced basal tone, these adaptations to pre‐synaptic dopaminergic neurons are not initiated following a single exposure to the drug.

     

A dual role for Integrin α6β4 in modulating hereditary neuropathy with liability to pressure palsies

Peripheral myelin protein 22 (PMP 22) is a component of compact myelin in the peripheral nervous system. The amount of PMP 22 in myelin is tightly regulated, and PMP 22 over or under‐expression cause Charcot‐Marie‐Tooth 1A (CMT 1A) and Hereditary Neuropathy with Pressure Palsies (HNPP ). Despite the importance of PMP 22 , its function remains largely unknown. It was reported that PMP 22 interacts with the β4 subunit of the laminin receptor α6β4 integrin, suggesting that α6β4 integrin and laminins may contribute to the pathogenesis of CMT 1A or HNPP . Here we asked if the lack of α6β4 integrin in Schwann cells influences myelin stability in the HNPP mouse model. Our data indicate that PMP 22 and β4 integrin may not interact directly in myelinating Schwann cells, however, ablating β4 integrin delays the formation of tomacula, a characteristic feature of HNPP . In contrast, ablation of integrin β4 worsens nerve conduction velocities and non‐compact myelin organization in HNPP animals. This study demonstrates that indirect interactions between an extracellular matrix receptor and a myelin protein influence the stability and function of myelinated fibers.

     

Alteration of sphingolipid metabolism as a putative mechanism underlying LPS‐induced BBB disruption

Septic encephalopathy with confusion and agitation occurs early during sepsis and contributes to the severity of the disease. A decrease in the sphingosine‐1‐phosphate (S1P) blood levels has been shown in patients and in animal models of sepsis. The lipid mediator S1P is known to be involved in endothelial barrier function in a context‐dependent manner. We utilized lipopolysaccharide (LPS )‐injected mice as a model for septic encephalopathy and first performed tracer permeability assays to assess the blood–brain barrier (BBB ) breakdown in vivo. At time points corresponding to the BBB breakdown post LPS injection, we aimed to characterize the regulation of the sphingolipid signaling pathway at the BBB during sepsis. We measured sphingolipid concentrations in blood, in mouse brain microvessels (MBMV s), and brain tissue. We also analyzed the expression of S1P receptors, transporters, and metabolizing enzymes in MBMV s and brain tissue. Primary mouse brain microvascular endothelial cells (MBMEC s) were isolated to evaluate the effects of LPS on transendothelial electrical resistance (TEER ) as a measure of permeability in vitro . We observed a relevant decrease in S1P levels after LPS injection in all three compartments (blood, MBMV s, brain tissue) that was accompanied by an increased expression of the S1P receptor type 1 and of sphingosine kinase 1 on one hand and of the S1P degrading enzymes lipid phosphate phosphatase 1 (LPP 1) and S1P phosphatase 1 on the other hand, as well as a down‐regulation of sphingosine kinase 2. Application of LPS to a monolayer of primary MBMEC s did not alter TEER , but serum from LPS ‐treated mice lead to a breakdown of the barrier compared to serum from vehicle‐treated mice. We observed profound alterations of the sphingolipid metabolism at the BBB after LPS injection that point toward a therapeutic potential of drugs interfering with this pathway as novel approach for the detrimental overwhelming immune response in sepsis.

Read the Editorial Highlight for this article on page 115 . Cover Image for this Issue: doi. 10.1111/jnc.14161 .

     

Mitochondrial dysfunction in human skeletal muscle biopsies of lipid storage disorder

Mitochondria regulate the balance between lipid metabolism and storage in the skeletal muscle. Altered lipid transport, metabolism and storage influence the bioenergetics, redox status and insulin signalling, contributing to cardiac and neurological diseases. Lipid storage disorders (LSD s) are neurological disorders which entail intramuscular lipid accumulation and impaired mitochondrial bioenergetics in the skeletal muscle causing progressive myopathy with muscle weakness. However, the mitochondrial changes including molecular events associated with impaired lipid storage have not been completely understood in the human skeletal muscle. We carried out morphological and biochemical analysis of mitochondrial function in muscle biopsies of human subjects with LSD s (n  = 7), compared to controls (n  = 10). Routine histology, enzyme histochemistry and ultrastructural analysis indicated altered muscle cell morphology and mitochondrial structure. Protein profiling of the muscle mitochondria from LSD samples (n  = 5) (vs. control, n  = 5) by high‐throughput mass spectrometric analysis revealed that impaired metabolic processes could contribute to mitochondrial dysfunction and ensuing myopathy in LSD s. We propose that impaired fatty acid and respiratory metabolism along with increased membrane permeability, elevated lipolysis and altered cristae entail mitochondrial dysfunction in LSD s. Some of these mechanisms were unique to LSD apart from others that were common to dystrophic and inflammatory muscle pathologies. Many differentially regulated mitochondrial proteins in LSD are linked with other human diseases, indicating that mitochondrial protection via targeted drugs could be a treatment modality in LSD and related metabolic diseases.

Cover Image for this Issue: doi: 10.1111/jnc.14177 .

     

Defective trafficking of Kv2.1 channels in MPTP‐induced nigrostriatal degeneration

Intracellular protein trafficking is tightly regulated, and improper trafficking might be the fundamental provocateur for human diseases including neurodegeneration. In neurons, protein trafficking to and from the plasma membrane affects synaptic plasticity. Voltage‐gated potassium channel 2.1 (Kv2.1) is a predominant delayed rectifier potassium (K⁺) current, and electrical activity patterns of dopamine (DA) neurons within the substantia nigra are generated and modulated by the orchestrated function of different ion channels. The pathological hallmark of Parkinson's disease (PD) is the progressive loss of these DA neurons, resulting in the degeneration of striatal dopaminergic terminals. However, whether trafficking of Kv2.1 channels contributes to PD remains unclear. In this study, we demonstrated that MPTP/MPP⁺ increases the surface expression of the Kv2.1 channel and causes nigrostriatal degeneration by using a subchronic MPTP mouse model. The inhibition of the Kv2.1 channel by using a specific blocker, guangxitoxin‐1E, protected nigrostriatal projections against MPTP/MPP⁺ insult and thus facilitated the recovery of motor coordination. These findings highlight the importance of trafficking of Kv2.1 channels in the pathogenesis of PD.

     

Validated multi‐step approach for in vivo recording and analysis of optogenetically evoked glutamate in the mouse globus pallidus

Precise quantification of extracellular glutamate concentrations upon neuronal activation is crucial for the understanding of brain function and neurological disorders. While optogenetics is an outstanding method for the correlation between distinct neurons and their role in circuitry and behavior, the electrochemically inactive nature of glutamate has proven challenging for recording upon optogenetic stimulations. This difficulty is due to the necessity for using enzyme‐coated microelectrodes and the risk for light‐induced artifacts. In this study, we establish a method for the combination of in vivo optogenetic stimulation with selective measurement of glutamate concentrations using enzyme‐coated multielectrode arrays and amperometry. The glutamatergic subthalamic nucleus (STN ), which is the main electrode target site in deep brain stimulation treatment of advanced Parkinson′s disease, has recently proven opotogenetically targetable in Pitx2‐Cre‐transgenic mice and was here used as model system. Upon stereotactic injection of viral Channelrhodopsin2‐eYFP constructs into the STN , amperometric recordings were performed at a range of optogenetic stimulation frequencies in the globus pallidus, the main STN target area, in anesthetized mice. Accurate quantification was enabled through a multi‐step analysis approach based on self‐referencing microelectrodes and repetition of the experimental protocol at two holding potentials, which allowed for the identification, isolation and removal of photoelectric and photoelectrochemical artifacts. This study advances the field of in vivo glutamate detection with combined optogenetics and amperometric recordings by providing a validated analysis framework for application in a wide variety of glutamate‐based approaches in neuroscience.

     

Machado–Joseph disease/spinocerebellar ataxia type 3: lessons from disease pathogenesis and clues into therapy

This editorial highlights a study by Rodriguez, Sanchez‐Moran et al. (2019) in the current issue of the Journal of Neurochemistry, in which the authors describe a microcephalic boy carrying the novel heterozygous de novo missense mutation c.560A> G; p.Asp187Gly in Cdh1/Fzr1 encoding the APC/C E3‐ubiquitin ligase cofactor CDH1. A functional characterization of mutant APC/C^CDH1 confirms an aberrant division of neural progenitor cells, a condition known to determine the mouse brain cortex size. These data suggest that APC/C^CDH1 may contribute to the regulation of the human brain size.

     

Protein components of post‐synaptic density lattice,a backbone structure for type I excitatory synapses

It is essential to study the molecular architecture of post‐synaptic density (PSD ) to understand the molecular mechanism underlying the dynamic nature of PSD , one of the bases of synaptic plasticity. A well‐known model for the architecture of PSD of type I excitatory synapses basically comprises of several scaffolding proteins (scaffold protein model). On the contrary, ‘PSD lattice’ observed through electron microscopy has been considered a basic backbone of type I PSD s. However, major constituents of the PSD lattice and the relationship between the PSD lattice and the scaffold protein model, remain unknown. We purified a PSD lattice fraction from the synaptic plasma membrane of rat forebrain. Protein components of the PSD lattice were examined through immuno‐gold negative staining electron microscopy. The results indicated that tubulin, actin, α‐internexin, and Ca²⁺/calmodulin‐dependent kinase II are major constituents of the PSD lattice, whereas scaffold proteins such as PSD ‐95, SAP 102, GKAP , Shank1, and Homer, were rather minor components. A similar structure was also purified from the synaptic plasma membrane of forebrains from 7‐day‐old rats. On the basis of this study, we propose a ‘PSD lattice‐based dynamic nanocolumn’ model for PSD molecular architecture, in which the scaffold protein model and the PSD lattice model are combined and an idea of dynamic nanocolumn PSD subdomain is also included. In the model, cytoskeletal proteins, in particular, tubulin, actin, and α‐internexin, may play major roles in the construction of the PSD backbone and provide linker sites for various PSD scaffold protein complexes/subdomains.

     

News & ISN April 2019

We are very sad that the ISN lost its President Kazuhiro Ikenaka, Professor and Chairman at National Institute for Physiological Sciences (NIPS), Director of Okazaki Institute of Integrative Biology. JNeurochem published an Obituary to value his outstanding achievements: Akio Wanaka et al. (2019) OBITUARY Kazuhiro Ikenaka (1952‐2018). https://doi.org/10.1111/jnc.14679

     

Connectivity between surface and interior in catalytic subunits of acetylcholinesterases inferred from their X-ray structures

Catalytic activity and function of acetylcholinesterase (AChE; EC 3.1.1.7) have been recognized and studied for over a century and its quaternary and primary structures for about half a century, and its tertiary structure has been known for about 33 years. Clear understanding of relationships between the structure and the function is still pending for this enzyme. Hundreds of crystallographic, static snapshots of AChEs from different sources reveal largely one general backbone conformation with narrow entry into the active center gorge, tightly fit to accept one acetylcholine (ACh) molecule, in contrast to its high catalytic turnover. This short review of available X-ray structures of AChEs from electric ray Torpedo californica, mouse and human, finds some limited, yet consistent deviations in conformations of selected secondary structure elements of AChE relevant for its function. The observed conformational diversity of the acyl pocket loop of AChE, unlike the large Ω-loop, appears consistent with structurally dynamic INS data and solution-based SAXS experiments to explain its dominant role in controlling the size of the active center gorge opening, as well as connectivity between the immediate surroundings of the buried active Ser, and catalytically relevant sites on the AChE surface.