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ɣ-aminobutyric acid (GABA) is a four‑carbon amino acid acting as the main inhibitory transmitter in the invertebrate and vertebrate nervous systems. The metabolism of GABA is well compartmentalized in the cell and the uptake of cytosolic GABA into the mitochondrial matrix is required for its degradation. A previous study carried out in the fruit fly Drosophila melanogaster indicated that the mitochondrial aspartate/glutamate carrier (AGC) is responsible for mitochondrial GABA accumulation. Here, we investigated the transport of GABA catalysed by the human and D. melanogaster AGC proteins through a well-established method for the study of the substrate specificity and the kinetic parameters of the mitochondrial carriers. In this experimental system, the D. melanogaster spliced AGC isoforms (Aralar1-PA and Aralar1-PE) and the human AGC isoforms (AGC1/aralar1 and AGC2/citrin) are unable to transport GABA both in homo- and in hetero-exchange with either glutamate or aspartate, i.e. the canonical substrates of AGC. Moreover, GABA has no inhibitory effect on the exchange activities catalysed by the investigated AGCs. Our data demonstrate that AGC does not transport GABA and the molecular identity of the GABA transporter in human and D. melanogaster mitochondria remains unknown., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The malate aspartate shuttle (MAS) plays a pivotal role in transporting cytosolic reducing equivalents - electrons - into the mitochondria for energy conversion at the electron transport chain (ETC) and in the process of oxidative phosphorylation. The MAS consists of two pairs of cytosolic and mitochondrial isoenzymes (malate dehydrogenases 1 and 2; and glutamate oxaloacetate transaminases 1 and 2) and two transporters (malate-2-oxoglutarate carrier and aspartate glutamate carrier (AGC), the latter of which has two tissue-dependent isoforms AGC1 and AGC2). While the inner mitochondrial membrane is impermeable to NADH, the MAS forms one of the main routes for mitochondrial electron uptake by promoting uptake of malate. Inherited bi-allelic pathogenic variants in five of the seven components of the MAS have been described hitherto and cause a wide spectrum of symptoms including early-onset epileptic encephalopathy. This review provides an overview of reported patients suffering from MAS deficiencies. In addition, we give an overview of diagnostic procedures and research performed on patient-derived cellular models and tissues. Current cellular models are briefly discussed and novel ways to achieve a better understanding of MAS deficiencies are highlighted., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Changes in the amount of daylight (photoperiod) alter physiology and behaviour 1,2 . Adaptive responses to seasonal photoperiods are vital to all organisms-dysregulation associates with disease, including affective disorders 3 and metabolic syndromes 4 . The circadian rhythm circuitry is implicated in such responses 5,6 , yet little is known about the precise cellular substrates that underlie phase synchronization to photoperiod change. Here we identify a brain circuit and system of axon branch-specific and reversible neurotransmitter deployment that are critical for behavioural and sleep adaptation to photoperiod. A type of neuron called mrEn1-Pet1 7 in the mouse brainstem median raphe nucleus segregates serotonin from VGLUT3 (also known as SLC17A8, a proxy for glutamate) to different axonal branches that innervate specific brain regions involved in circadian rhythm and sleep-wake timing 8,9 . This branch-specific neurotransmitter deployment did not distinguish between daylight and dark phase; however, it reorganized with change in photoperiod. Axonal boutons, but not cell soma, changed neurochemical phenotype upon a shift away from equinox light/dark conditions, and these changes were reversed upon return to equinox conditions. When we genetically disabled Vglut3 in mrEn1-Pet1 neurons, sleep-wake periods, voluntary activity and clock gene expression did not synchronize to the new photoperiod or were delayed. Combining intersectional rabies virus tracing and projection-specific neuronal silencing, we delineated a preoptic area-to-mrEn1Pet1 connection that was responsible for decoding the photoperiodic inputs, driving the neurotransmitter reorganization and promoting behavioural synchronization. Our results reveal a brain circuit and periodic, branch-specific neurotransmitter deployment that regulates organismal adaptation to photoperiod change., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Members of the SLC25 mitochondrial carrier family link cytosolic and mitochondrial metabolism and support cellular maintenance and growth by transporting compounds across the mitochondrial inner membrane. Their monomeric or dimeric state and kinetic mechanism have been a matter of long-standing debate. It is believed by some that they exist as homodimers and transport substrates with a sequential kinetic mechanism, forming a ternary complex where both exchanged substrates are bound simultaneously. Some studies, in contrast, have provided evidence indicating that the mitochondrial ADP/ATP carrier (SLC25A4) functions as a monomer, has a single substrate binding site, and operates with a ping-pong kinetic mechanism, whereby ADP is imported before ATP is exported. Here we reanalyze the oligomeric state and kinetic properties of the human mitochondrial citrate carrier (SLC25A1), dicarboxylate carrier (SLC25A10), oxoglutarate carrier (SLC25A11), and aspartate/glutamate carrier (SLC25A13), all previously reported to be dimers with a sequential kinetic mechanism. We demonstrate that they are monomers, except for dimeric SLC25A13, and operate with a ping-pong kinetic mechanism in which the substrate import and export steps occur consecutively. These observations are consistent with a common transport mechanism, based on a functional monomer, in which a single central substrate-binding site is alternately accessible., (© 2024. The Author(s).)

Exposure to stressors has profound effects on sleep that have been linked to serotonin (5-HT) neurons of the dorsal raphe nucleus (DR). However, the DR also comprises glutamatergic neurons expressing vesicular glutamate transporter type 3 (DR VGLUT3 ), leading us to examine their role. Cell-type-specific tracing revealed that DR VGLUT3 neurons project to brain areas regulating arousal and stress. We found that chemogenetic activation of DR VGLUT3 neurons mimics stress-induced sleep perturbations. Furthermore, deleting VGLUT3 in the DR attenuated stress-induced sleep perturbations, especially after social defeat stress. In the DR, VGLUT3 is found in subsets of 5-HT and non-5-HT neurons. We observed that both populations are activated by acute stress, including those projecting to the ventral tegmental area. However, deleting VGLUT3 in 5-HT neurons minimally affected sleep regulation. These findings suggest that VGLUT3 expression in the DR drives stress-induced sleep perturbations, possibly involving non-5-HT DR VGLUT3 neurons., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cholinergic striatal interneurons (ChIs) express the vesicular glutamate transporter 3 (VGLUT3) which allows them to regulate the striatal network with glutamate and acetylcholine (ACh). In addition, VGLUT3-dependent glutamate increases ACh vesicular stores through vesicular synergy. A missense polymorphism, VGLUT3-p.T8I, was identified in patients with substance use disorders (SUDs) and eating disorders (EDs). A mouse line was generated to understand the neurochemical and behavioral impact of the p.T8I variant. In VGLUT3 T8I/T8I male mice, glutamate signaling was unchanged but vesicular synergy and ACh release were blunted. Mutant male mice exhibited a reduced DA release in the dorsomedial striatum but not in the dorsolateral striatum, facilitating habit formation and exacerbating maladaptive use of drug or food. Increasing ACh tone with donepezil reversed the self-starvation phenotype observed in VGLUT3 T8I/T8I male mice. Our study suggests that unbalanced dopaminergic transmission in the dorsal striatum could be a common mechanism between SUDs and EDs., (© 2024. The Author(s).)

Social behavior is important for our well-being, and its dysfunctions impact several pathological conditions. Although the involvement of glutamate is undeniable, the relevance of vesicular glutamate transporter type 3 (VGluT3), a specific vesicular transporter, in the control of social behavior is not sufficiently explored. Since midbrain median raphe region (MRR) is implicated in social behavior and the nucleus contains high amount of VGluT3+ neurons, we compared the behavior of male VGluT3 knock-out (KO) and VGluT3-Cre mice, the latter after chemogenetic MRR-VGluT3 manipulation. Appropriate control groups were included. Behavioral test battery was used for social behavior (sociability, social discrimination, social interaction, resident intruder test) and possible confounding factors (open field, elevated plus maze, Y-maze tests). Neuronal activation was studied by c-Fos immunohistochemistry. Human relevance was confirmed by VGluT3 gene expression in relevant human brainstem areas. VGluT3 KO mice exhibited increased anxiety, social interest, but also aggressive behavior in anxiogenic environment and impaired social memory. For KO animals, social interaction induced lower cell activation in the anterior cingulate, infralimbic cortex, and medial septum. In turn, excitation of MRR-VGluT3+ neurons was anxiolytic. Inhibition increased social interest 24 h later but decreased mobility and social behavior in aggressive context. Chemogenetic activation increased the number of c-Fos+ neurons only in the MRR. We confirmed the increased anxiety-like behavior and impaired memory of VGluT3 KO strain and revealed increased, but inadequate, social behavior. MRR-VGluT3 neurons regulated mobility and social and anxiety-like behavior in a context-dependent manner. The presence of VGluT3 mRNA on corresponding human brain areas suggests clinical relevance., Competing Interests: The authors declare no competing financial interests. The agencies had no further role in study design and in the collection, analysis, or interpretation of the data., (Copyright © 2024 Fazekas et al.)

Alcohol dependence results in long-lasting neuroadaptive changes in meso-corticolimbic system, especially in the nucleus accumbens (NAc), which drives relapse-like ethanol drinking upon abstinence or withdrawal. Within NAc, altered glutamate homeostasis is one of the neuroadaptive changes caused by alcohol dependence. Accumbal glutamate homeostasis is tightly maintained through glutamate transporter 1 (GLT-1) and cystine-glutamate antiporter (xCT). But the role of GLT-1 and xCT in relapse-like ethanol drinking is poorly understood. Here, we used alcohol-preferring (P) rats in relapse-like ethanol drinking paradigm to (a) determine the effect of relapse-like ethanol drinking on gene and protein expression of GLT-1 and xCT in NAc, measured by quantitative polymerase chain reaction (qPCR) and Western blot, respectively; (b) examine if glutamate uptake is affected by relapse-like ethanol drinking in NAc, measured by radioactive glutamate uptake assay; (c) elucidate if upregulation of either/both GLT-1 or/and xCT through ceftriaxone is/are required to attenuate relapse-like ethanol drinking. The GLT-1 or xCT protein expression was suppressed during ceftriaxone treatments through microinjection of GLT-1/xCT anti-sense vivo-morpholinos. We found that relapse-like ethanol drinking did not affect the gene and protein expression of GLT-1 and xCT in NAc. The glutamate uptake was also unaltered. Ceftriaxone (200 mg/kg body weight, i.p.) treatments during the last 5 days of abstinence attenuated relapse-like ethanol drinking. The suppression of GLT-1 or xCT expression prevented the ceftriaxone-induced attenuation of relapse-like ethanol drinking. These findings confirm that upregulation of both GLT-1 and xCT within NAc is crucial for ceftriaxone-mediated attenuation of relapse-like ethanol drinking., (© 2022 Society for the Study of Addiction.)

Background: Myelin-associated glycoprotein (MAG) is a key molecule involved in the nurturing effect of myelin on ensheathed axons. MAG also inhibits axon outgrowth after injury. In preclinical stroke models, administration of a function-blocking anti-MAG monoclonal antibody (mAb) aimed to improve axon regeneration demonstrated reduced lesion volumes and a rapid clinical improvement, suggesting a mechanism of immediate neuroprotection rather than enhanced axon regeneration. In addition, it has been reported that antibody-mediated crosslinking of MAG can protect oligodendrocytes (OLs) against glutamate (Glu) overload by unknown mechanisms., Purpose: To unravel the molecular mechanisms underlying the protective effect of anti-MAG therapy with a focus on neuroprotection against Glu toxicity., Results: MAG activation (via antibody crosslinking) triggered the clearance of extracellular Glu by its uptake into OLs via high affinity excitatory amino acid transporters. This resulted not only in protection of OLs but also nearby neurons. MAG activation led to a PKC-dependent activation of factor Nrf2 (nuclear-erythroid related factor-2) leading to antioxidant responses including increased mRNA expression of metabolic enzymes from the glutathione biosynthetic pathway and the regulatory chain of cystine/Glu antiporter system xc - increasing reduced glutathione (GSH), the main antioxidant in cells. The efficacy of early anti-MAG mAb administration was demonstrated in a preclinical model of excitotoxicity induced by intrastriatal Glu administration and extended to a model of Experimental Autoimmune Encephalitis showing axonal damage secondary to demyelination., Conclusions: MAG activation triggers Glu uptake into OLs under conditions of Glu overload and induces a robust protective antioxidant response., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Mutations in TUBB4A are associated with a spectrum of neurologic disorders categorized as TUBB4A -related leukoencephalopathy. Affected children can present with global developmental delay or normal early development, followed by a variable loss of skills over time. Further research is needed to characterize the factors associated with the divergent developmental trajectories in this rare monogenic disorder because this phenotypic spectrum is not fully explained by genotype alone.To characterize early psychomotor features, developmental milestones and age of disease onset were collected from medical records (n=54 individuals). Three subcohorts were identified: individuals with the common p.Asp249Asn variant vs all other genotypes with either early (<12 months of age) or late onset of presentation. Individuals with the p.Asp249Asn variant or those with non-p.Asp249Asn genotypes with later disease onset attained key milestones, including head control, sitting, and independent walking. Subjects with early-onset, non-p.Asp249Asn-associated disease were less likely to achieve developmental milestones. Next, we defined the developmental severity as the percentage of milestones attained by age 2 years. The mild form was defined as attaining at least 75% of key developmental milestones. Among cohort categorized as mild, individuals with p.Asp249Asn variant were more likely to lose acquired abilities when compared with non-p.Asp249Asn individuals.Our results suggest multiple influences on developmental trajectory, including a strong contribution from genotype and age of onset. Further studies are needed to identify additional factors that influence overall outcomes to better counsel families and to design clinical trials with appropriate clinical endpoints.

Background: In man two mitochondrial aspartate/glutamate carrier (AGC) isoforms, known as aralar and citrin, are required to accomplish several metabolic pathways. In order to fill the existing gap of knowledge in Drosophila melanogaster, we have studied aralar1 gene, orthologue of human AGC-encoding genes in this organism., Methods: The blastp algorithm and the "reciprocal best hit" approach have been used to identify the human orthologue of AGCs in Drosophilidae and non-Drosophilidae. Aralar1 proteins have been overexpressed in Escherichia coli and functionally reconstituted into liposomes for transport assays., Results: The transcriptional organization of aralar1 comprises six isoforms, three constitutively expressed (aralar1-RA, RD and RF), and the remaining three distributed during the development or in different tissues (aralar1-RB, RC and RE). Aralar1-PA and Aralar1-PE, representative of all isoforms, have been biochemically characterized. Recombinant Aralar1-PA and Aralar1-PE proteins share similar efficiency to exchange glutamate against aspartate, and same substrate affinities than the human isoforms. Interestingly, although Aralar1-PA and Aralar1-PE diverge only in their EF-hand 8, they greatly differ in their specific activities and substrate specificity., Conclusions: The tight regulation of aralar1 transcripts expression and the high request of aspartate and glutamate during early embryogenesis suggest a crucial role of Aralar1 in this Drosophila developmental stage. Furthermore, biochemical characterization and calcium sensitivity have identified Aralar1-PA and Aralar1-PE as the human aralar and citrin counterparts, respectively., General Significance: The functional characterization of the fruit fly mitochondrial AGC transporter represents a crucial step toward a complete understanding of the metabolic events acting during early embryogenesis., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The mammalian cochlea cannot regenerate functional hair cells (HCs) spontaneously. Atoh1 overexpression as well as other strategies are unable to generate functional HCs. Here, we simultaneously upregulated the expression of Gfi1, Pou4f3, and Atoh1 in postnatal cochlear supporting cells (SCs) in vivo, which efficiently converted SCs into HCs. The newly regenerated HCs expressed HC markers Myo7a, Calbindin, Parvalbumin, and Ctbp2 and were innervated by neurites. Importantly, many new HCs expressed the mature and terminal marker Prestin or vesicular glutamate transporter 3 (vGlut3), depending on the subtypes of the source SCs. Finally, our patch-clamp analysis showed that the new HCs in the medial region acquired a large K + current, fired spikes transiently, and exhibited signature refinement of ribbon synapse functions, in close resemblance to native wild-type inner HCs. We demonstrated that co-upregulating Gfi1, Pou4f3, and Atoh1 enhances the efficiency of HC generation and promotes the functional maturation of new HCs., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cochlear outer hair cells (OHCs) are known to uniquely participate in auditory processing through their electromotility, and like inner hair cells, are also capable of releasing vesicular glutamate onto spiral ganglion (SG) neurons: in this case, onto the sparse Type II SG neurons. However, unlike glutamate signaling at the inner hair cell-Type I SG neuron synapse, which is robust across a wide spectrum of sound intensities, glutamate signaling at the OHC-Type II SG neuron synapse is weaker and has been hypothesized to occur only at intense, possibly damaging sound levels. Here, we tested the ability of the OHC-Type II SG pathway to signal to the brain in response to moderate, nondamaging sound (80 dB SPL) as well as to intense sound (115 dB SPL). First, we determined the VGluTs associated with OHC signaling and then confirmed the loss of glutamatergic synaptic transmission from OHCs to Type II SG neurons in KO mice using dendritic patch-clamp recordings. Next, we generated genetic mouse lines in which vesicular glutamate release occurs selectively from OHCs, and then assessed c-Fos expression in the cochlear nucleus in response to sound. From these analyses, we show, for the first time, that glutamatergic signaling at the OHC-Type II SG neuron synapse is capable of activating cochlear nucleus neurons, even at moderate sound levels. SIGNIFICANCE STATEMENT Evidence suggests that cochlear outer hair cells (OHCs) release glutamate onto Type II spiral ganglion neurons only when exposed to loud sound, and that Type II neurons are activated by tissue damage. Knowing whether moderate level sound, without tissue damage, activates this pathway has functional implications for this fundamental auditory pathway. We first determined that OHCs rely largely on VGluT3 for synaptic glutamate release. We then used a genetically modified mouse line in which OHCs, but not inner hair cells, release vesicular glutamate to demonstrate that moderate sound exposure activates cochlear nucleus neurons via the OHC-Type II spiral ganglion pathway. Together, these data indicate that glutamate signaling at the OHC-Type II afferent synapse participates in auditory function at moderate sound levels., (Copyright © 2021 the authors.)

The cell surface glycoprotein CD44 has various types of splicing variants, which contribute to its multiple distinct cellular functions. Recently, it was reported that the CD44v8-10 isoform interacts with the system Xc(-) transporter-related protein (xCT), and inhibits the accumulation of reactive oxygen species by promoting the synthesis of the antioxidant glutathione in human tumour cells. In this study, we investigated the expression and function of CD44 variants and xCT in canine tumours. From semi-quantitative reverse transcription polymerase chain reaction analysis, the mRNA expression of the CD44v8-10 isoform was observed in canine tumour tissues as well as human cases. The overexpression of CD44v8-10 may promote the synthesis of glutathione and enhance the resistance to radiation of canine breast tumour cells. Furthermore, canine xCT mRNA expression was significantly upregulated in the canine breast tumour tissues as compared to the normal tissues surrounding the tumours. To investigate the function of canine xCT, we treated canine tumour cells with the xCT inhibitor sulfasalazine. Consequently, the sulfasalazine-treated cells were more sensitive to oxidative stress than the non-treated cells. Taken together, these results suggested that CD44v8-10 and xCT play important roles in the therapy resistance of canine tumours as well as human tumours., (© 2020 The Authors Veterinary Medicine and Science Published by John Wiley & Sons Ltd.)

Amino acids are delivered into developing wheat grains to support the accumulation of storage proteins in the starchy endosperm, and transporters play important roles in regulating this process. RNA-seq, RT-qPCR, and promoter-GUS assays showed that three amino acid transporters are differentially expressed in the endosperm transfer cells (TaAAP2), starchy endosperm cells (TaAAP13), and aleurone cells and embryo of the developing grain (TaAAP21), respectively. Yeast complementation revealed that all three transporters can transport a broad spectrum of amino acids. RNAi-mediated suppression of TaAAP13 expression in the starchy endosperm did not reduce the total nitrogen content of the whole grain, but significantly altered the composition and distribution of metabolites in the starchy endosperm, with increasing concentrations of some amino acids (notably glutamine and glycine) from the outer to inner starchy endosperm cells compared with wild type. Overexpression of TaAAP13 under the endosperm-specific HMW-GS (high molecular weight glutenin subunit) promoter significantly increased grain size, grain nitrogen concentration, and thousand grain weight, indicating that the sink strength for nitrogen transport was increased by manipulation of amino acid transporters. However, the total grain number was reduced, suggesting that source nitrogen remobilized from leaves is a limiting factor for productivity. Therefore, simultaneously increasing loading of amino acids into the phloem and delivery to the spike would be required to increase protein content while maintaining grain yield., Competing Interests: The authors have declared that no competing interests exist.

Amacrine cells of the retina are conspicuously variable in their morphologies, their population demographics, and their ensuing functions. Vesicular glutamate transporter 3 (VGluT3) amacrine cells are a recently characterized type of amacrine cell exhibiting local dendritic autonomy. The present analysis has examined three features of this VGluT3 population, including their density, local distribution, and dendritic spread, to discern the extent to which these are interrelated, using male and female mice. We first demonstrate that Bax -mediated cell death transforms the mosaic of VGluT3 cells from a random distribution into a regular mosaic. We subsequently examine the relationship between cell density and mosaic regularity across recombinant inbred strains of mice, finding that, although both traits vary across the strains, they exhibit minimal covariation. Other genetic determinants must therefore contribute independently to final cell number and to mosaic order. Using a conditional KO approach, we further demonstrate that Bax acts via the bipolar cell population, rather than cell-intrinsically, to control VGluT3 cell number. Finally, we consider the relationship between the dendritic arbors of single VGluT3 cells and the distribution of their homotypic neighbors. Dendritic field area was found to be independent of Voronoi domain area, while dendritic coverage of single cells was not conserved, simply increasing with the size of the dendritic field. Bax -KO retinas exhibited a threefold increase in dendritic coverage. Each cell, however, contributed less dendrites at each depth within the plexus, intermingling their processes with those of neighboring cells to approximate a constant volumetric density, yielding a uniformity in process coverage across the population. SIGNIFICANCE STATEMENT Different types of retinal neuron spread their processes across the surface of the retina to achieve a degree of dendritic coverage that is characteristic of each type. Many of these types achieve a constant coverage by varying their dendritic field area inversely with the local density of like-type neighbors. Here we report a population of retinal amacrine cells that do not develop dendritic arbors in relation to the spatial positioning of such homotypic neighbors; rather, this cell type modulates the extent of its dendritic branching when faced with a variable number of overlapping dendritic fields to approximate a uniformity in dendritic density across the retina., (Copyright © 2021 the authors.)

Neurological diseases were often characterized by progressive neuronal death, and emerging evidences suggested that ferroptosis may be an active driver of multiple neurodegenerative diseases. However, the mechanisms underlying ferroptosis in neuron cells are unclear. Here, we demonstrated that ferroptotic stimuli caused injury in neuron-like PC12 cells by modulating the expression of proteins involved in iron metabolism and lipid peroxidation at multiple levels, such as altering iron import/export, activating ferritinophagy, and decreasing glutathione (GSH) level. Nuclear factor erythroid 2-related factor 2 (Nrf2) regulates multiple genes involved in ferroptosis, however, its exact role remain elusive. Our mechanistic inquiry revealed that Nrf2 expression enhanced iron storage capacity by increasing ferritin heavy chain 1 (FTH1) expression in PC12 cells. Moreover, Nrf2 alleviated the decrease in GSH level by promoting the expression of genes related to GSH synthesis, including solute carrier family 7 member 11 (SLC7A11) and cysteine ligase (GCL). The contribution of Nrf2 on ferroptosis resistance was further verified by increasing cell tolerance to oxidative stress. Furthermore, Nfe2l2 (Nrf2) knockdown sensitized cells to ferroptotic cell death. Taken together, our findings suggested that iron accumulation caused by altering iron metabolism and the decrease of GSH content are key factors in determining ferroptosis in PC12 cells, and Nrf2 inhibits ferroptosis by combating iron-induced oxidative stress. Our present study provided new clues for the intervention and prevention against ferroptosis-associated neurological diseases., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Innocuous mechanical stimuli, such as rubbing or stroking the skin, relieve itch through the activation of low-threshold mechanoreceptors. However, the mechanisms behind this inhibition remain unknown. We presently investigated whether stroking the skin reduces the responses of superficial dorsal horn neurons to pruritogens in male C57BL/6J mice. Single-unit recordings revealed that neuronal responses to chloroquine were enhanced during skin stroking, and this was followed by suppression of firing below baseline levels after the termination of stroking. Most of these neurons additionally responded to capsaicin. Stroking did not suppress neuronal responses to capsaicin, indicating state-dependent inhibition. Vesicular glutamate transporter 3 (VGLUT3)-lineage sensory nerves compose a subset of low-threshold mechanoreceptors. Stroking-related inhibition of neuronal responses to chloroquine was diminished by optogenetic inhibition of VGLUT3-lineage sensory nerves in male and female Vglut3-cre / NpHR-EYFP mice. Conversely, in male and female Vglut3-cre/ChR2-EYFP mice, optogenetic stimulation of VGLUT3-lineage sensory nerves inhibited firing responses of spinal neurons to pruritogens after the termination of stimulation. This inhibition was nearly abolished by spinal delivery of the κ-opioid receptor antagonist nor-binaltorphimine dihydrochloride, but not the neuropeptide Y receptor Y1 antagonist BMS193885. Optogenetic stimulation of VGLUT3-lineage sensory nerves inhibited pruritogen-evoked scratching without affecting mechanical and thermal pain behaviors. Therefore, VGLUT3-lineage sensory nerves appear to mediate inhibition of itch by tactile stimuli. SIGNIFICANCE STATEMENT Rubbing or stroking the skin is known to relieve itch. We investigated the mechanisms behind touch-evoked inhibition of itch in mice. Stroking the skin reduced the activity of itch-responsive spinal neurons. Optogenetic inhibition of VGLUT3-lineage sensory nerves diminished stroking-evoked inhibition, and optogenetic stimulation of VGLUT3-lineage nerves inhibited pruritogen-evoked firing. Together, our results provide a mechanistic understanding of touch-evoked inhibition of itch., (Copyright © 2020 the authors.)

The retina possesses few types of neurons so; it is considered an excellent model for understanding the neural mechanisms underlying basic neural information processing in the brain. Glutamate is the major excitatory neurotransmitter in the vertebrate central nervous system and retina. The present study was carried out to characterize the expression pattern of vesicular glutamate transporter 2 (Vglut2) and 3 (Vglut3) and glutamate receptor 1 (GluR1) and 2 (GluR2) mRNAs in the retina of adult laughing dove (Streptopelia senegalensis) by RT-PCR and in situ hybridization histochemistry. The cerebellum of adult laughing dove was used as a positive control in this study. Vglut2 mRNA was highly expressed only in the granular layer of the cerebellum while Vglut3 mRNA was weakly expressed only in the Purkinje cells layer. In the retina, Vglut2 mRNA was highly expressed in the ganglion cell layer and moderately expressed in the inner nuclear layer while Vglut3 mRNA was moderately expressed only in the inner nuclear layer. GluR1 mRNA was intensely expressed in the Purkinje cells layer while GluR2 mRNA signals were highly detectable in both granular and Purkinje cells layers of the cerebellum. In the retina, moderate expression of GluR1 and intense expression of GluR2 was found in both ganglion cell layer and the internal half of inner nuclear layer mostly amacrine cells. These results suggest that some retinal neuronal cells in the adult laughing dove are glutamatergic. Therefore, GluR1 and 2 are suggested as useful markers for glutamatergic retinal neuronal cells in the adult laughing doves., Competing Interests: Declaration of Competing Interest Authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Sphingolipids are abundant and essential molecules in eukaryotes that have crucial functions as signaling molecules and as membrane components. Sphingolipid biosynthesis starts in the endoplasmic reticulum with the condensation of serine and palmitoyl-CoA. Sphingolipid biosynthesis is highly regulated to maintain sphingolipid homeostasis. Even though, serine is an essential component of the sphingolipid biosynthesis pathway, its role in maintaining sphingolipid homeostasis has not been precisely studied. Here we show that serine uptake is an important factor for the regulation of sphingolipid biosynthesis in Saccharomyces cerevisiae. Using genetic experiments, we find the broad-specificity amino acid permease Gnp1 to be important for serine uptake. We confirm these results with serine uptake assays in gnp1Δ cells. We further show that uptake of exogenous serine by Gnp1 is important to maintain cellular serine levels and observe a specific connection between serine uptake and the first step of sphingolipid biosynthesis. Using mass spectrometry-based flux analysis, we further observed imported serine as the main source for de novo sphingolipid biosynthesis. Our results demonstrate that yeast cells preferentially use the uptake of exogenous serine to regulate sphingolipid biosynthesis. Our study can also be a starting point to analyze the role of serine uptake in mammalian sphingolipid metabolism., Competing Interests: The authors have declared that no competing interests exist

Case: We are reporting the third unrelated case of cerebral aspartate-glutamate carrier isoform 1 (AGC1) deficiency. Patient is a 21-month-old Yemeni male who presented with refractory seizure disorder and developmental arrest. Neuroimaging showed cerebral volume loss and diminished N-acetylaspartate (NAA) peak. Whole exome sequencing revealed a homozygous novel missense variant in the SLC25A12 gene. Patient's seizure frequency abated drastically following initiation of ketogenic diet., Discussion and Conclusion: Cerebral AGC1 deficiency results in dysfunction of mitochondrial malate aspartate shuttle, thereby prohibiting myelin synthesis. There are significant phenotypic commonalities between our patient and previously reported cases including intractable epilepsy, psychomotor delay, cerebral atrophy, and diminished NAA peak. Our report also provides evidence regarding beneficial effect of ketogenic diet in this rare neurometabolic epilepsy., Competing Interests: Disclosure The authors report no conflicts of interest in this work., (Georg Thieme Verlag KG Stuttgart · New York.)

In the yeast Saccharomyces cerevisiae, the transcriptional factor Msn2 plays an essential role in response to a variety of environmental stresses by activating the transcription of many genes that contain the stress-responsive elements in the promoters. We previously reported that overexpression of the MSN2 gene confers tolerance to various stresses in industrial yeast strains. Recently, the overexpression of MSN2 was shown to increase the amount of the amino acid permease Gnp1 on the plasma membrane, leading to the increased uptake of proline into the cell, suggesting a novel link between the Msn2-mediated stress response and amino acid homeostasis in yeast. Here, we found that overexpression of MSN2 increased ubiquitinated protein levels with reduced free ubiquitin. Among deubiquitinating enzymes (DUBs), it was revealed that the loss of Ubp6 depleted the free ubiquitin level and decreased tolerance to the toxic amino acid analogues. The overexpression of UBP6 in MSN2-overexpressing cells clearly complemented the impaired tolerance towards the toxic amino acid analogues. Both the protein level and the plasma-membrane localization of Gnp1 were increased in ubp6-deleted cells, as shown in MSN2-overexpressing cells. These results suggest that an excess level of Msn2 impairs endocytic degradation of Gnp1 through dysfunction of Ubp6 and other DUBs., (Copyright © 2019 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

PEX34, encoding a peroxisomal protein implicated in regulating peroxisome numbers, was identified as a high copy suppressor, capable of bypassing impaired acetate utilization of agc1∆ yeast. However, improved growth of agc1∆ yeast on acetate is not mediated through peroxisome proliferation. Instead, stress to the endoplasmic reticulum and mitochondria from PEX34 overexpression appears to contribute to enhanced acetate utilization of agc1∆ yeast. The citrate/2-oxoglutarate carrier Yhm2p is required for PEX34 stimulated growth of agc1∆ yeast on acetate medium, suggesting that the suppressor effect is mediated through increased activity of a redox shuttle involving mitochondrial citrate export. Metabolomic analysis also revealed redirection of acetyl-coenzyme A (CoA) from synthetic reactions for amino acids in PEX34 overexpressing yeast. We propose a model in which increased formation of products from the glyoxylate shunt, together with enhanced utilization of acetyl-CoA, promotes the activity of an alternative mitochondrial redox shuttle, partially substituting for loss of yeast AGC1., (© FEMS 2019.)

The aim of the present study was to test the hypothesis that vesicular glutamate transporter 3 (VGluT3) deficiency is associated with cognitive impairments. Male VGluT3 knockout (KO) and wild type (WT) mice were exposed to a behavioral test battery covering paradigms based on spontaneous exploratory behavior and reinforcement-based learning tests. Reversal learning was examined to test the cognitive flexibility. The VGluT3 KO mice clearly exhibited the ability to learn. The social recognition memory of KO mice was intact. The y-maze test revealed weaker working memory of VGluT3 KO mice. No significant learning impairments were noticed in operant conditioning or holeboard discrimination paradigm. In avoidance-based learning tests (Morris water maze and active avoidance), KO mice exhibited slightly slower learning process compared to WT mice, but not a complete learning impairment. In tests based on simple associations (operant conditioning, avoidance learning) an attenuation of cognitive flexibility was observed in KO mice. In conclusion, knocking out VGluT3 results in mild disturbances in working memory and learning flexibility. Apparently, this glutamate transporter is not a major player in learning and memory formation in general. Based on previous characteristics of VGluT3 KO mice we would have expected a stronger deficit. The observed hypolocomotion did not contribute to the mild cognitive disturbances herein reported, either., (Copyright © 2019 Elsevier Inc. All rights reserved.)

WW domain-containing oxidoreductase (Wwox) is a putative tumor suppressor. Several germline mutations of Wwox have been associated with infant neurological disorders characterized by epilepsy, growth retardation, and early death. Less is known, however, about the pathological link between Wwox mutations and these disorders or the physiological role of Wwox in brain development. In this study, we examined age-related expression and histological localization of Wwox in forebrains as well as the effects of loss of function mutations in the Wwox gene in the immature cortex of a rat model of lethal dwarfism with epilepsy ( lde/lde ). Immunostaining revealed that Wwox is expressed in neurons, astrocytes, and oligodendrocytes. lde/lde cortices were characterized by a reduction in neurite growth without a reduced number of neurons, severe reduction in myelination with a reduced number of mature oligodendrocytes, and a reduction in cell populations of astrocytes and microglia. These results indicate that Wwox is essential for normal development of neurons and glial cells in the cerebral cortex.

Noise-induced excitotoxicity is thought to depend on glutamate. However, the excitotoxic mechanisms are unknown, and the necessity of glutamate for synapse loss or regeneration is unclear. Despite absence of glutamatergic transmission from cochlear inner hair cells in mice lacking the vesicular glutamate transporter-3 ( Vglut3 KO ), at 9-11 weeks, approximately half the number of synapses found in Vglut3 WT were maintained as postsynaptic AMPA receptors juxtaposed with presynaptic ribbons and voltage-gated calcium channels (Ca V 1.3). Synapses were larger in Vglut3 KO than Vglut3 WT In Vglut3 WT and Vglut3 +/- mice, 8-16 kHz octave-band noise exposure at 100 dB sound pressure level caused a threshold shift (∼40 dB) and a loss of synapses (>50%) at 24 h after exposure. Hearing threshold and synapse number partially recovered by 2 weeks after exposure as ribbons became larger, whereas recovery was significantly better in Vglut3 WT Noise exposure at 94 dB sound pressure level caused auditory threshold shifts that fully recovered in 2 weeks, whereas suprathreshold hearing recovered faster in Vglut3 WT than Vglut3 +/- These results, from mice of both sexes, suggest that spontaneous repair of synapses after noise depends on the level of Vglut3 protein or the level of glutamate release during the recovery period. Noise-induced loss of presynaptic ribbons or postsynaptic AMPA receptors was not observed in Vglut3 KO , demonstrating its dependence on vesicular glutamate release. In Vglut3 WT and Vglut3 +/- , noise exposure caused unpairing of presynaptic ribbons and presynaptic Ca V 1.3, but not in Vglut3 KO where Ca V 1.3 remained clustered with ribbons at presynaptic active zones. These results suggest that, without glutamate release, noise-induced presynaptic Ca 2+ influx was insufficient to disassemble the active zone. However, synapse volume increased by 2 weeks after exposure in Vglut3 KO , suggesting glutamate-independent mechanisms. SIGNIFICANCE STATEMENT Hearing depends on glutamatergic transmission mediated by Vglut3, but the role of glutamate in synapse loss and repair is unclear. Here, using mice of both sexes, we show that one copy of the Vglut3 gene is sufficient for noise-induced threshold shift and loss of ribbon synapses, but both copies are required for normal recovery of hearing function and ribbon synapse number. Impairment of the recovery process in mice having only one functional copy suggests that glutamate release may promote synapse regeneration. At least one copy of the Vglut3 gene is necessary for noise-induced synapse loss. Although the excitotoxic mechanism remains unknown, these findings are consistent with the presumption that glutamate is the key mediator of noise-induced synaptopathy., (Copyright © 2019 the authors.)

The soil bacterium Bacillus subtilis can get into contact with growth-inhibiting substances, which may be of anthropogenic origin. Glyphosate is such a substance serving as a nonselective herbicide. Glyphosate specifically inhibits the 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase, which generates an essential precursor for de novo synthesis of aromatic amino acids in plants, fungi, bacteria and archaea. Inhibition of the EPSP synthase by glyphosate results in depletion of the cellular levels of aromatic amino acids unless the environment provides them. Here, we have assessed the potential of B. subtilis to adapt to glyphosate at the genome level. In contrast to Escherichia coli, which evolves glyphosate resistance by elevating the production and decreasing the glyphosate sensitivity of the EPSP synthase, B. subtilis primarily inactivates the gltT gene encoding the high-affinity glutamate/aspartate symporter GltT. Further adaptation of the gltT mutants to glyphosate led to the inactivation of the gltP gene encoding the glutamate transporter GltP. Metabolome analyses confirmed that GltT is the major entryway of glyphosate into B. subtilis. GltP, the GltT homologue of E. coli also transports glyphosate into B. subtilis. Finally, we found that GltT is involved in uptake of the herbicide glufosinate, which inhibits the glutamine synthetase., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The atypical vesicular glutamate transporter VGLUT3 is present in subpopulations of GABAergic interneurons in the cortex and the hippocampus, in subgroups of serotoninergic neurons in raphe nuclei, and in cholinergic interneurons in the striatum. C56BL/6N mice that no longer express VGLUT3 (VGLUT3 -/- ) display anxiety-associated phenotype, increased spontaneous and cocaine-induced locomotor activity and decreased haloperidol-induced catalepsy. Inbred mouse strains differ markedly in their sensitivity to anxiety and behavioral responses elicited by drugs. The purpose of this study was to investigate strain differences in VGLUT3 expression levels and its potential correlates with anxiety and reward-guided behaviors. Five inbred mouse lines were chosen according to their contrasted anxiety and drugs sensitivity: C57BL/6N, C3H/HeN, DBA/2J, 129/Sv, and BALB/c. VGLUT3 protein expression was measured in different brain areas involved in reward or mood regulation (such as the striatum, the hippocampus, and raphe nuclei) and genetic variations in Slc17a8, the gene encoding for VGLUT3, have been explored. These five inbred mouse strains express very different levels of VGLUT3, which cannot be attributed to the genetic variation of the Slc17a8 locus. Furthermore, mice behavior in the open field, elevated plus maze, spontaneous- and cocaine-induced locomotor was highly heterogeneous and only partially correlated to VGLUT3 levels. These data highlight the fact that one single gene polymorphism could not account for VGLUT3 expression variations, and that region specific VGLUT3 expression level variations might play a key role in the modulation of discrete behaviors., (© 2018 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.)

Background: Inflammatory myopathies are characterized by infiltration of inflammatory cells into muscle. Typically, immune-mediated disorders such as polymyositis, dermatomyositis and inclusion body myositis are diagnosed., Objective: A small family of dogs with early onset muscle weakness and inflammatory muscle biopsies were investigated for an underlying genetic cause., Methods: Following the histopathological diagnosis of inflammatory myopathy, mutational analysis including whole genome sequencing, functional transport studies of the mutated and wild-type proteins, and metabolomic analysis were performed., Results: Whole genome resequencing identified a pathological variant in the SLC25A12 gene, resulting in a leucine to proline substitution at amino acid 349 in the mitochondrial aspartate-glutamate transporter known as the neuron and muscle specific aspartate glutamate carrier 1 (AGC1). Functionally reconstituting recombinant wild-type and mutant AGC1 into liposomes demonstrated a dramatic decrease in AGC1 transport activity and inability to transfer reducing equivalents from the cytosol into mitochondria. Targeted, broad-spectrum metabolomic analysis from affected and control muscles demonstrated a proinflammatory milieu and strong support for oxidative stress., Conclusions: This study provides the first description of a metabolic mechanism in which ablated mitochondrial glutamate transport markedly reduced the import of reducing equivalents into mitochondria and produced a highly oxidizing and proinflammatory muscle environment and an inflammatory myopathy.

The mitochondrial ATP-Mg/P i carrier is responsible for the calcium-dependent regulation of adenosine nucleotide concentrations in the mitochondrial matrix, which allows mitochondria to respond to changing energy requirements of the cell. The carrier is expressed in mitochondria of fungi, plants and animals and belongs to the family of mitochondrial carriers. The carrier is unusual as it consists of three separate domains: (i) an N-terminal regulatory domain with four calcium-binding EF-hands similar to calmodulin, (ii) a loop domain containing an amphipathic α-helix and (iii) a mitochondrial carrier domain related to the mitochondrial ADP/ATP carrier. This striking example of three domains coming together from different origins to provide new functions represents an interesting quirk of evolution. In this review, we outline how the carrier was identified and how its physiological role was established with a focus on human isoforms. We exploit the sequence and structural information of the domains to explore the similarities and differences to their closest counterparts; mitochondrial ADP/ATP carriers and proteins with four EF-hands. We discuss how their combined function has led to a mechanism for calcium-regulated transport of adenosine nucleotides. Finally, we compare the ATP-Mg/P i carrier with the mitochondrial aspartate/glutamate carrier, the only other mitochondrial carrier regulated by calcium, and we will argue that they have arisen by convergent rather than divergent evolution. © 2018 The Authors. IUBMB Life published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology, 70(12):1222-1232, 2018., (© 2018 The Authors. IUBMB Life published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology.)

Nrf2 (nuclear factor erythroid 2-related factor 2), a transcription factor that controls expression of several proteins that are related to cellular antioxidant capacity, such as the subunit xCT of the system x c - , is dysregulated in diabetes. Recently, it was described that system x c - is decreased in the retina after 3 weeks of diabetes. So, in the present work, the temporal relationship between xCT and Nrf2 in the retina of diabetic animals was investigated. Diabetes was induced in male Wistar rats (200 g) by a single injection of streptozotocin, and retinas were collected after 1, 2, and 6 months of diabetes induction. Expression of xCT, Nrf2 activity, and binding to antioxidant-responsive element (ARE) sequence were evaluated. Glutathione and reactive oxygen species (ROS) levels were also assessed. After 1 month of diabetes, Nrf2 activity, xCT expression, and glutathione levels were reduced whereas ROS were increased. Although glutathione and ROS levels remain unchanged until later stages, Nrf2 activity and xCT expression returned to normal levels after 2 months. However, they were decreased again at 6 months of diabetes. Accordingly, Nrf2 binding to xCT ARE sequence followed the same pattern of Nrf2 activity and xCT expression. These data showed that retinal xCT expression is regulated by Nrf2 in diabetic condition. The results also demonstrated a temporal relationship between Nrf2 and system x c - which could be implicated in the initiation of oxidative stress in retina in diabetes.

Folate metabolism in the brain is critically important and serves a number of vital roles in nucleotide synthesis, single carbon metabolism/methylation, amino acid metabolism, and mitochondrial translation. Genetic defects in almost every enzyme of folate metabolism have been reported to date, and most have neurological sequelae. We report 2 patients presenting with a neurometabolic disorder associated with biallelic variants in the MTHFS gene, encoding 5,10-methenyltetrahydrofolate synthetase. Both patients presented with microcephaly, short stature, severe global developmental delay, progressive spasticity, epilepsy, and cerebral hypomyelination. Baseline CSF 5-methyltetrahydrolate (5-MTHF) levels were in the low-normal range. The first patient was treated with folinic acid, which resulted in worsening cerebral folate deficiency. Treatment in this patient with a combination of oral L-5-methyltetrahydrofolate and intramuscular methylcobalamin was able to increase CSF 5-MTHF levels, was well tolerated over a 4 month period, and resulted in subjective mild improvements in functioning. Measurement of MTHFS enzyme activity in fibroblasts confirmed reduced activity. The direct substrate of the MTHFS reaction, 5-formyl-THF, was elevated 30-fold in patient fibroblasts compared to control, supporting the hypothesis that the pathophysiology of this disorder is a manifestation of toxicity from this metabolite., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The coordinated distribution of nitrogen to source leaves and sinks is essential for supporting leaf metabolism while also supplying sufficient nitrogen to seeds for development. This study aimed to understand how regulated amino acid allocation to leaves affects photosynthesis and overall plant nitrogen use efficiency in Arabidopsis ( Arabidopsis thaliana ) and how soil nitrogen availability influences these processes. Arabidopsis plants with a knockout of AAP2 , encoding an amino acid permease involved in xylem-to-phloem transfer of root-derived amino acids, were grown in low-, moderate-, and high-nitrogen environments. We analyzed nitrogen allocation to shoot tissues, photosynthesis, and photosynthetic and plant nitrogen use efficiency in these knockout plants. Our results demonstrate that, independent of nitrogen conditions, aap2 plants allocate more nitrogen to leaves than wild-type plants. Increased leaf nitrogen supply positively affected chlorophyll and Rubisco levels, photosynthetic nitrogen use efficiency, and carbon assimilation and transport to sinks. The aap2 plants outperformed wild-type plants with respect to growth, seed yield and carbon storage pools, and nitrogen use efficiency in both high and deficient nitrogen environments. Overall, this study demonstrates that increasing nitrogen allocation to leaves represents an effective strategy for improving carbon fixation and photosynthetic nitrogen use efficiency. The results indicate that an optimized coordination of nitrogen and carbon partitioning processes is critical for high oilseed production in Arabidopsis, including in plants exposed to limiting nitrogen conditions., (© 2018 American Society of Plant Biologists. All rights reserved.)

In the auditory system, type I spiral ganglion neurons (SGNs) convey complex acoustic information from inner hair cells (IHCs) to the brainstem. Although SGNs exhibit variation in physiological and anatomical properties, it is unclear which features are endogenous and which reflect input from synaptic partners. Using single-cell RNA sequencing, we derived a molecular classification of mouse type I SGNs comprising three subtypes that express unique combinations of Ca 2+ binding proteins, ion channel regulators, guidance molecules, and transcription factors. Based on connectivity and susceptibility to age-related loss, these subtypes correspond to those defined physiologically. Additional intrinsic differences among subtypes and across the tonotopic axis highlight an unexpectedly active role for SGNs in auditory processing. SGN identities emerge postnatally and are disrupted in a mouse model of deafness that lacks IHC-driven activity. These results elucidate the range, nature, and origins of SGN diversity, with implications for treatment of congenital deafness., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Impairment in glutamate neurotransmission mediates the development of dependence upon nicotine (NIC) and ethanol (EtOH). Previous work indicates that continuous access to EtOH or phasic exposure to NIC reduces expression of the glutamate transporter-1 (GLT-1) and cystine/glutamate antiporter (xCT) but not the glutamate/aspartate transporter (GLAST). Additionally, metabotropic glutamate receptors (mGluRs) expression was affected following exposure to EtOH or NIC. However, little is known about the effects of EtOH and NIC co-consumption on GLT-1, xCT, GLAST, and mGluR1 expression. In this study, peri-adolescent female alcohol preferring (P) rats were given binge-like access to water, sucrose (SUC), SUC-NIC, EtOH, or EtOH-NIC for four weeks. The present study determined the effects of these reinforcers on GLT-1, xCT, GLAST, and mGluR1 expression in the nucleus accumbens (NAc), hippocampus (HIP) and prefrontal cortex (PFC). GLT-1 and xCT expression were decreased in the NAc following both SUC-NIC and EtOH-NIC. In addition, only xCT expression was downregulated in the HIP in both of these latter groups. Also, glutathione peroxidase (GPx) activity in the HIP was reduced following SUC, SUC-NIC, EtOH, and EtOH-NIC consumption. Similar to previous work, GLAST expression was not altered in any brain region by any of the reinforcers. However, mGluR1 expression was increased in the NAc in the SUC-NIC, EtOH, and EtOH-NIC groups. These results indicate that peri-adolescent binge-like drinking of EtOH or SUC with or without NIC may exert differential effects on astroglial glutamate transporters and receptors. Our data further parallel some of the previous findings observed in adult rats., (Copyright © 2018. Published by Elsevier Inc.)

Precise mouse genetic studies rely on specific tools that can label specific cell types. In mouse cochlea, previous studies suggest that vesicular glutamate transporter 3 (vGlut3), also known as Slc17a8, is specifically expressed in inner hair cells (IHCs) and loss of vGlut3 causes deafness. To take advantage of its unique expression pattern, here we generate a novel vGlut3-P2A-iCreER knockin mouse strain. The P2A-iCreER cassette is precisely inserted before stop codon of vGlut3, by which the endogenous vGlut3 is intact and paired with iCreER as well. Approximately, 10.7%, 85.6% and 41.8% of IHCs are tdtomato + when tamoxifen is given to vGlut3-P2A-iCreER/+; Rosa26-LSL-tdtomato/+ reporter strain at P2/P3, P10/P11 and P30/P31, respectively. Tdtomato + OHCs are never observed. Interestingly, besides IHCs, glia cells, but not spiral ganglion neurons (SGNs), are tdtomato+, which is further evidenced by the presence of Sox10+/tdtomato+ and tdtomato+/Prox1(Gata3 or Tuj1)-negative cells in SGN region. We further independently validate vGlut3 expression in SGN region by vGlut3 in situ hybridization and antibody staining. Moreover, total number of tdtomato + glia cells decreased gradually when tamoxifen is given from P2/P3 to P30/P31. Taken together, vGlut3-P2A-iCreER is an efficient genetic tool to specifically target IHCs for gene manipulation, which is complimentary to Prestin-CreER strain exclusively labelling cochlear outer hair cells (OHCs)., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Early-onset epileptic encephalopathies (EOEEs) are a genetically heterogeneous collection of severe epilepsies often associated with psychomotor regression. Mutations in SZT2, a known seizure threshold regulator gene, are a newly identified cause of EOEE. We present an individual with EOEE, macrocephaly, and developmental regression with compound heterozygous mutations in SZT2 as identified by whole exome sequencing. Serial imaging characterized the novel finding of progressive loss of central myelination. This case expands our clinical understanding of the SZT2-phenotype and emphasizes the role of this gene in the diagnostic investigation for EOEE and leukoencephalopathies., (© 2018 Wiley Periodicals, Inc.)

Little is known about the organizational and functional connectivity of the corticospinal (CS) circuits that are essential for voluntary movement. Here, we map the connectivity between CS neurons in the forelimb motor and sensory cortices and various spinal interneurons, demonstrating that distinct CS-interneuron circuits control specific aspects of skilled movements. CS fibers originating in the mouse motor cortex directly synapse onto premotor interneurons, including those expressing Chx10. Lesions of the motor cortex or silencing of spinal Chx10 + interneurons produces deficits in skilled reaching. In contrast, CS neurons in the sensory cortex do not synapse directly onto premotor interneurons, and they preferentially connect to Vglut3 + spinal interneurons. Lesions to the sensory cortex or inhibition of Vglut3 + interneurons cause deficits in food pellet release movements in goal-oriented tasks. These findings reveal that CS neurons in the motor and sensory cortices differentially control skilled movements through distinct CS-spinal interneuron circuits., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Glutamatergic neurons, characterized by vesicular glutamate transporters (VGluT1-3) provide the main excitation in the brain. Their disturbances have been linked to various brain disorders, which could be also modeled by the contextual fear test in rodents. We aimed to characterize the participation of VGluT3 in the development of contextual fear through its contribution to hypothalamic-pituitary-adrenocortical axis (HPA) regulation using knockout (KO) mice. Contextual fear conditioning was induced by foot shock and mice were examined 1 and 7 d later in the same environment comparing wild type with KO. Foot shock increased the immobility time without context specificity. Additionally, foot shock reduced open arm time in the elevated plus maze (EPM) test, and distance traveled in the open field (OF) test, representing the generalization of fear. Moreover, KO mice spent more time with freezing during the contextual fear test, less time in the open arm of the EPM, and traveled a smaller distance in the OF, with less entries into the central area. However, there was no foot shock and genotype interaction suggesting that VGluT3 does not influence the fear conditioning, rather determines anxiety-like characteristic of the mice. The resting hypothalamic CRH mRNA was higher in KO mice with reduced stressor-induced corticosterone elevations. Immunohistochemistry revealed the presence of VGluT3 positive fibers in the paraventricular nucleus of hypothalamus, but not on the hypophysis. As a summary, we confirmed the involvement of VGluT3 in innate fear, but not in the development of fear memory and generalization, with a significant contribution to HPA alterations. Highlights VGluT3 KO mice show innate fear without significant influence on fear memory and generalization. A putative background is the higher resting CRH mRNA level in their PVN and reduced stress-reactivity.

To determine whether responses during infrared neural stimulation (INS) result from the direct interaction with spiral ganglion neurons (SGNs), we tested three genetically modified deaf mouse models: Atoh1-cre; Atoh1 f/f (Atoh1 conditional knockout, CKO), Atoh1-cre; Atoh1 f/kiNeurog1 (Neurog1 knockin, KI), and the Vglut3 knockout (Vglut3 -/- ) mice. All animals were exposed to tone bursts and clicks up to 107 dB (re 20 µPa) and to INS, delivered with a 200 µm optical fiber. The wavelength (λ) was 1860 nm, the radiant energy (Q) 0-800 µJ/pulse, and the pulse width (PW) 100-500 µs. No auditory responses to acoustic stimuli could be evoked in any of these animals. INS could not evoke auditory brainstem responses in Atoh1 CKO mice but could in Neurog1 KI and Vglut3 -/- mice. X-ray micro-computed tomography of the cochleae showed that responses correlated with the presence of SGNs and hair cells. Results in Neurog1 KI mice do not support a mechanical stimulation through the vibration of the basilar membrane, but cannot rule out the direct activation of the inner hair cells. Results in Vglut3 -/- mice, which have no synaptic transmission between inner hair cells and SGNs, suggested that hair cells are not required.

Maintenance of the homeostasis in a constantly changing environment is a fundamental process of life. Disturbances of the homeostatic balance is defined as stress response and is induced by wide variety of challenges called stressors. Being the main excitatory neurotransmitter of the central nervous system glutamate is important in the adaptation process of stress regulating both the catecholaminergic system and the hypothalamic-pituitary-adrenocortical axis. Data are accumulating about the role of different glutamatergic receptors at all levels of these axes, but little is known about the contribution of different vesicular glutamate transporters (VGluT1-3) characterizing the glutamatergic neurons. Here we summarize basic knowledge about VGluTs, their role in physiological regulation of stress adaptation, as well as their contribution to stress-related psychopathology. Most of our knowledge comes from the VGluT3 knockout mice, as VGluT1 and 2 knockouts are not viable. VGluT3 was discovered later than, and is not as widespread as the VGluT1 and 2. It may co-localize with other transmitters, and participate in retrograde signaling; as such its role might be unique. Previous reports using VGluT3 knockout mice showed enhanced anxiety and innate fear compared to wild type. Moreover, these knockout animals had enhanced resting corticotropin-releasing hormone mRNA levels in the hypothalamus and disturbed glucocorticoid stress responses. In conclusion, VGluT3 participates in stress adaptation regulation. The neuroendocrine changes observed in VGluT3 knockout mice may contribute to their anxious, fearful phenotype.

Objective: To quantify the abundance of dural afferent neurons expressing transient receptor potential channel melastatin 8 (TRPM8), vesicular glutamate transporter 3 (VGLUT3), and neurofilament 200 (NF200) in adult mice., Background: With the increasing use of mice as a model system to study headache mechanisms, it is important to understand the composition of dural afferent neurons in mice. In a previous study, we have measured the abundance of mouse dural afferent neurons that express neuropeptide calcitonin gene-related peptide as well as two TRP channels TRPV1 and TRPA1, respectively. Here, we conducted quantitative analysis of three other dural afferent subpopulations in adult mice., Methods: We used the fluorescent tracer Fluoro-Gold to retrogradely label dural afferent neurons in adult mice expressing enhanced green fluorescent protein in discrete subpopulations of trigeminal ganglion (TG) neurons. Mechanoreceptors with myelinated fibers were identified by NF200 immunoreactivity. We also conducted Ca 2+ -imaging experiments to test the overlap between TRPM8 and VGLUT3 expression in mouse primary afferent neurons (PANs)., Results: The abundance of TRPM8-expressing neurons in dural afferent neurons was significantly lower than that in total TG neurons. The percentages of dural afferent neurons expressing VGLUT3 and NF200 were comparable to those of total TG neurons, respectively. TRPM8 agonist menthol evoked Ca 2+ influx in less than 7% VGLUT3-expressing PANs in adult mice., Conclusions: TG neurons expressing TRPM8, VGLUT3, and NF200 all innervate adult mouse dura. TRPM8 and VGLUT3 are expressed in distinct subpopulations of PANs in adult mice. These results provide an anatomical basis to investigate headache mechanisms in mouse models., (© 2017 American Headache Society.)

Introduction: Hyporesponsiveness of human lamina propria immune cells to microbial and nutritional antigens represents one important feature of intestinal homeostasis. It is at least partially mediated by low expression of the innate response receptors CD11b, CD14, CD16 as well as the cystine-glutamate transporter xCT on these cells. Milieu-specific mechanisms leading to the down-regulation of these receptors on circulating monocytes, the precursor cells of resident macrophages, are mostly unknown., Methods: Here, we addressed the question whether the short chain fatty acid n-butyrate, a fermentation product of the mammalian gut microbiota exhibiting histone deacetylase inhibitory activity, is able to modulate expression of these receptors in human circulating monocytes., Results: Exposure to n-butyrate resulted in the downregulation of CD11b, CD14, as well as CD16 surface expression on circulating monocytes. XCT transcript levels in circulating monocytes were also reduced following exposure to n-butyrate. Importantly, treatment resulted in the downregulation of protein and gene expression of the transcription factor PU.1, which was shown to be at least partially required for the expression of CD16 in circulating monocytes. PU.1 expression in resident macrophages in situ was observed to be substantially lower in healthy when compared to inflamed colonic mucosa., Conclusions: In summary, the intestinal microbiota may support symbiosis with the human host organism by n-butyrate mediated downregulation of protein and gene expression of innate response receptors as well as xCT on circulating monocytes following recruitment to the lamina propria. Downregulation of CD16 gene expression may at least partially be caused at the transcriptional level by the n-butyrate mediated decrease in expression of the transcription factor PU.1 in circulating monocytes., (© 2017 The Authors. Immunity, Inflammation and Disease Published by John Wiley & Sons Ltd.)

Objective: To identify the gene defect in patients with hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) who are negative for TUBB4A mutations., Methods: We performed homozygosity mapping and whole exome sequencing (WES) to detect the disease-causing variant. We used a Taqman assay for population screening. We developed a luciferase reporter construct to investigate the effect of the promoter mutation on expression., Results: Sixteen patients from 14 families from different countries fulfilling the MRI criteria for H-ABC exhibited a similar, severe clinical phenotype, including lack of development and a severe epileptic encephalopathy. The majority of patients had a known Roma ethnic background. Single nucleotide polymorphism array analysis in 5 patients identified one large overlapping homozygous region on chromosome 13. WES in 2 patients revealed a homozygous deletion in the promoter region of UFM1 . Sanger sequencing confirmed homozygosity for this variant in all 16 patients. All patients shared a common haplotype, indicative of a founder effect. Screening of 1,000 controls from different European Roma panels demonstrated an overall carrier rate of the mutation of 3%-25%. Transfection assays showed that the deletion significantly reduced expression in specific CNS cell lines., Conclusions: UFM1 encodes ubiquitin-fold modifier 1 (UFM1), a member of the ubiquitin-like family involved in posttranslational modification of proteins. Its exact biological role is unclear. This study associates a UFM1 gene defect with a disease and sheds new light on possible UFM1 functional networks., (Copyright © 2017 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

One of the major players in the pathophysiology of cerebral ischemia is disrupted homeostasis of glutamatergic neurotransmission, resulting in elevated extracellular glutamate (Glu) concentrations and excitotoxicity-related cell death. In the brain, Glu concentrations are regulated by Glu transporters, including Glu transporter-1 (GLT-1) and cystine/Glu antiporter (system xc-). Modulation of these transporters by administration of ceftriaxone (CEF, 200 mg/kg, i.p.) or N-acetylcysteine (NAC, 150 mg/kg, i.p.) for 5 days before focal cerebral ischemia may induce brain tolerance to ischemia by significantly limiting stroke-related damage and normalizing Glu concentrations. In the present study, focal cerebral ischemia was induced by 90-minute middle cerebral artery occlusion (MCAO). We compared the effects of CEF and NAC pretreatment on Glu concentrations in extracellular fluid and cellular-specific expression of GLT-1 and xCT with the effects of two reference preconditioning methods, namely, ischemic preconditioning and chemical preconditioning in rats. Both CEF and NAC significantly reduced Glu levels in the frontal cortex and hippocampus during focal cerebral ischemia, and this decrease was comparable with the Glu level achieved with the reference preconditioning strategies. The results of immunofluorescence staining of GLT-1 and xCT on astrocytes, neurons and microglia accounted for the observed changes in extracellular Glu levels to a certain extent. Briefly, after MCAO, the expression of GLT-1 on astrocytes decreased, but pretreatment with CEF seemed to prevent this downregulation. In addition, every intervention used in this study seemed to reduce xCT expression on astrocytes and neurons. The results of this study indicate that modulation of Glu transporter expression may restore Glu homeostasis. Moreover, our results suggest that CEF and NAC may induce brain tolerance to ischemia by influencing GLT-1 and system xc- expression levels. These transporters are presumably good targets for the development of novel therapies for brain ischemia.

We hypothesized that both day of gestation and maternal nutrition would alter the relative mRNA expression of neutral and acid AA transporters , , , , and . Crossbred Angus heifers ( = 49) were synchronized, bred via AI, assigned to nutritional treatment (100% of NRC requirements for 0.45 kg/d gain [control heifers {CON}] and 60% of CON [restricted heifers {RES}]), and ovariohysterectomized on d 16, 34, or 50 of gestation ( = 6 to 9/d). Nonbred, nonpregnant (NB-NP) controls were ovariohysterectomized on d 16 of the estrous cycle ( = 6) after synchronization. The resulting arrangement was a 2 × 3 factorial + 1 (CON vs. RES × d 16, 34, or 50 + NB-NP controls). Tissues collected included caruncular endometrium (CAR), intercaruncular endometrium (ICAR), fetal membranes (FM; chorioallantois; d 16 and 34), cotyledonary placenta (COT; d 50 only), intercotyledonary placenta (ICOT; d 50 only), and amnion (AMN; d 50 only]). Relative expression of , , , , and was determined for each tissue using NB-NP CAR and NB-NP ICAR tissues for the baseline; for FM, endometrium from NB-NP controls served as the baseline. In CAR, no day × treatment interaction was observed ( > 0.05). However, day of gestation affected relative expression of , where expression on d 16 was greater ( < 0.01) than expression on d 34 and 50. Additionally, relative expression of and was greater ( ≤ 0.05) in pregnant heifers compared with NB-NP heifers. For ICAR, was influenced by a day × treatment interaction ( < 0.01), where expression in d 16 RES was greater ( ≤ 0.05) than that of any other day or nutritional treatment. Furthermore, expression in d 16 CON was greater ( ≤ 0.05) than that in d 50 RES, with those in d 34 CON and RES and d 50 CON being intermediate. In addition, was affected by day of gestation, where expression on d 16 was greater ( < 0.01) than that on d 34 and 50. A day × treatment interaction was not observed ( > 0.05) in FM; however, expression on d 34 was greater ( = 0.02) than on d 50, with that on d 16 being intermediate. Day of gestation also affected expression of , where expression on d 34 and 50 was greater ( < 0.01) than that on d 16. These data support our hypothesis in that both day of gestation and maternal nutrition affected the relative mRNA expression of AA transporter in ICAR, whereas day of gestation has a greater effect on the relative mRNA expression of other neutral and acidic AA transporters in the various tissues studied.

Cholinergic neurons in the basal forebrain project heavily to the main olfactory bulb, the first processing station in the olfactory pathway. The projections innervate multiple layers of the main olfactory bulb and strongly influence odor discrimination, detection, and learning. The precise underlying circuitry of this cholinergic input to the main olfactory bulb remains unclear, however. Here, we identify a specific basal forebrain cholinergic projection that innervates select neurons concentrated in the internal plexiform layer of the main olfactory bulb. Optogenetic activation of this projection elicits monosynaptic nicotinic and GABAergic currents in glomerular layer-projecting interneurons. Additionally, we show that the projection co-expresses markers for GABAergic neurotransmission. The data thus implicate neurotransmitter co-transmission in the basal forebrain regulation of this inhibitory olfactory microcircuit.Cholinergic neurons innervate multiple layers in the main olfactory bulb but the precise circuitry of this input is not known. Here the authors show that VGLUT3 + cholinergic neurons selectively innervate deep short axon cells in specific layers and elicit robust monosynaptic GABAergic and nicotinic postsynaptic currents.

ARALAR/AGC1 (aspartate-glutamate mitochondrial carrier 1) is an important component of the NADH malate-aspartate shuttle (MAS). AGC1-deficiency is a rare disease causing global cerebral hypomyelination, developmental arrest, hypotonia, and epilepsy (OMIM ID #612949); the aralar-KO mouse recapitulates the major findings in humans. This study was aimed at understanding the impact of ARALAR-deficiency in brain lactate levels as a biomarker. We report that lactate was equally abundant in wild-type and aralar-KO mouse brain in vivo at postnatal day 17. We find that lactate production upon mitochondrial blockade depends on up-regulation of lactate formation in astrocytes rather than in neurons. However, ARALAR-deficiency decreased cell respiration in neurons, not astrocytes, which maintained unchanged respiration and lactate production. As the primary site of ARALAR-deficiency is neuronal, this explains the lack of accumulation of brain lactate in ARALAR-deficiency in humans and mice. On the other hand, we find that the cytosolic and mitochondrial components of the glycerol phosphate shuttle are present in astrocytes with similar activities. This suggests that glycerol phosphate shuttle is the main NADH shuttle in astrocytes and explains the absence of effects of ARALAR-deficiency in these cells., (© 2017 International Society for Neurochemistry.)