Leishmania mexicana promotes pain-reducing metabolomic reprogramming in cutaneous lesions.

Cutaneous leishmaniasis (CL) is characterized by extensive skin lesions, which are usually painless despite being associated with extensive inflammation. The molecular mechanisms responsible for this analgesia have not been identified. Through untargeted metabolomics, we found enriched anti-nociceptive metabolic pathways in L. mexicana-infected mice. Purines were elevated in infected macrophages and at the lesion site during chronic infection. These purines have anti-inflammatory and analgesic properties by acting through adenosine receptors, inhibiting TRPV1 channels, and promoting IL-10 production. We also found arachidonic acid (AA) metabolism enriched in the ear lesions compared to the non-infected controls. AA is a metabolite of anandamide (AEA) and 2-arachidonoylglycerol (2-AG). These endocannabinoids act on cannabinoid receptors 1 and 2 and TRPV1 channels to exert anti-inflammatory and analgesic effects. Our study provides evidence of metabolic pathways upregulated during L. mexicana infection that may mediate anti-nociceptive effects experienced by CL patients and identifies macrophages as a source of these metabolites.

Short-term high-fat diet consumption impairs synaptic plasticity in the aged hippocampus via IL-1 signaling.

More Americans are consuming diets higher in saturated fats and refined sugars than ever before. These trends could have serious consequences for the older population because high-fat diet (HFD) consumption, known to induce neuroinflammation, has been shown to accelerate and aggravate memory declines. We have previously demonstrated that short-term HFD consumption, which does not evoke obesity-related comorbidities, produced profound impairments to hippocampal-dependent memory in aged rats. These impairments were precipitated by increases in proinflammatory cytokines, primarily interleukin-1 beta (IL-1ß). Here, we explored the extent to which short-term HFD consumption disrupts hippocampal synaptic plasticity, as measured by long-term potentiation (LTP), in young adult and aged rats. We demonstrated that (1) HFD disrupted late-phase LTP in the hippocampus of aged, but not young adult rats, (2) HFD did not disrupt early-phase LTP, and (3) blockade of the IL-1 receptor rescued L-LTP in aged HFD-fed rats. These findings suggest that hippocampal memory impairments in aged rats following HFD consumption occur through the deterioration of synaptic plasticity and that IL-1ß is a critical driver of that deterioration.

Neural stem and progenitor cells support and protect adult hippocampal function via vascular endothelial growth factor secretion.

Adult neural stem and progenitor cells (NSPCs) reside in the dentate gyrus (DG) of the hippocampus throughout the lifespan of most mammalian species. In addition to generating new neurons, NSPCs may alter their niche via secretion of growth factors and cytokines. We recently showed that adult DG NSPCs secrete vascular endothelial growth factor (VEGF), which is critical for maintaining adult neurogenesis. Here, we asked whether NSPC-derived VEGF alters hippocampal function independent of adult neurogenesis. We found that loss of NSPC-derived VEGF acutely impaired hippocampal memory, caused neuronal hyperexcitability and exacerbated excitotoxic injury. We also found that NSPCs generate substantial proportions of total DG VEGF and VEGF disperses broadly throughout the DG, both of which help explain how this anatomically-restricted cell population could modulate function broadly. These findings suggest that NSPCs actively support and protect DG function via secreted VEGF, thereby providing a non-neurogenic functional dimension to endogenous NSPCs.

In vivo Mouse Intervertebral Disc Degeneration Models and Their Utility as Translational Models of Clinical Discogenic Back Pain: A Comparative Review.

Low back pain is a leading cause of disability worldwide and studies have demonstrated intervertebral disc (IVD) degeneration as a major risk factor. While many in vitro models have been developed and used to study IVD pathophysiology and therapeutic strategies, the etiology of IVD degeneration is a complex multifactorial process involving crosstalk of nearby tissues and systemic effects. Thus, the use of appropriate in vivo models is necessary to fully understand the associated molecular, structural, and functional changes and how they relate to pain. Mouse models have been widely adopted due to accessibility and ease of genetic manipulation compared to other animal models. Despite their small size, mice lumbar discs demonstrate significant similarities to the human IVD in terms of geometry, structure, and mechanical properties. While several different mouse models of IVD degeneration exist, greater standardization of the methods for inducing degeneration and the development of a consistent set of output measurements could allow mouse models to become a stronger tool for clinical translation. This article reviews current mouse models of IVD degeneration in the context of clinical translation and highlights a critical set of output measurements for studying disease pathology or screening regenerative therapies with an emphasis on pain phenotyping. First, we summarized and categorized these models into genetic, age-related, and mechanically induced. Then, the outcome parameters assessed in these models are compared including, molecular, cellular, functional/structural, and pain assessments for both evoked and spontaneous pain. These comparisons highlight a set of potential key parameters that can be used to validate the model and inform its utility to screen potential therapies for IVD degeneration and their translation to the human condition. As treatment of symptomatic pain is important, this review provides an emphasis on critical pain-like behavior assessments in mice and explores current behavioral assessments relevant to discogenic back pain. Overall, the specific research question was determined to be essential to identify the relevant model with histological staining, imaging, extracellular matrix composition, mechanics, and pain as critical parameters for assessing degeneration and regenerative strategies.

Disruption of dopamine receptor 1 localization to primary cilia impairs signaling in striatal neurons.

A rod-shaped appendage called a primary cilium projects from the soma of most central neurons in the mammalian brain. The importance of cilia within the nervous system is highlighted by the fact that human syndromes linked to primary cilia dysfunction, collectively termed ciliopathies, are associated with numerous neuropathologies, including hyperphagia-induced obesity, neuropsychiatric disorders, and learning and memory deficits. Neuronal cilia are enriched with signaling molecules, including specific G protein-coupled receptors (GPCRs) and their downstream effectors, suggesting they act as sensory organelles that respond to neuromodulators in the extracellular space. We previously showed that GPCR ciliary localization is disrupted in neurons from mouse models of the ciliopathy Bardet-Biedl syndrome (BBS). Based on this finding we hypothesized that mislocalization of ciliary GPCRs may impact receptor signaling and contribute to the BBS phenotypes. Here, we show that disrupting localization of the ciliary GPCR dopamine receptor 1 (D1) in male and female mice, either by loss of a BBS protein or loss of the cilium itself, specifically in D1-expressing neurons, results in obesity. Interestingly, the weight gain is associated with reduced locomotor activity, rather than increased food intake. Moreover, loss of a BBS protein or cilia on D1-expressing neurons leads to a reduction in D1-mediated signaling. Together, these results indicate that cilia impact D1 activity in the nervous system and underscore the importance of neuronal cilia for proper GPCR signaling.SIGNIFICANCE STATEMENT:Most mammalian neurons possess solitary appendages called primary cilia. These rod-shaped structures are enriched with signaling proteins, such as G protein-coupled receptors (GPCRs), suggesting they respond to neuromodulators. This study examines the consequences of disrupting ciliary localization of the GPCR dopamine receptor 1 (D1) in D1-expressing neurons. Remarkably, mice that have either abnormal accumulation of D1 in cilia or loss of D1 ciliary localization become obese. In both cases the obesity is associated with lower locomotor activity rather than overeating. As D1 activation increases locomotor activity, these results are consistent with a reduction in D1 signaling. Indeed, we found that D1-mediated signaling is reduced in brain slices from both mouse models. Thus, cilia impact D1 signaling in the brain.

Chronic Cortical Inflammation, Cognitive Impairment, and Immune Reactivity Associated with Diffuse Brain Injury Are Ameliorated by Forced Turnover of Microglia.

Traumatic brain injury (TBI) is associated with an increased risk of cognitive, psychiatric, and neurodegenerative complications that may develop after injury. Increased microglial reactivity following TBI may underlie chronic neuroinflammation, neuropathology, and exaggerated responses to immune challenges. Therefore, the goal of this study was to force turnover of trauma-associated microglia that develop after diffuse TBI and determine whether this alleviated chronic inflammation, improved functional recovery and attenuated reduced immune reactivity to lipopolysaccharide (LPS) challenge. Male mice received a midline fluid percussion injury (mFPI) and 7 d later were subjected to a forced microglia turnover paradigm using CSF1R antagonism (PLX5622). At 30 d postinjury (dpi), cortical gene expression, dendritic complexity, myelin content, neuronal connectivity, cognition, and immune reactivity were assessed. Myriad neuropathology-related genes were increased 30 dpi in the cortex, and 90% of these gene changes were reversed by microglial turnover. Reduced neuronal connectivity was evident 30 dpi and these deficits were attenuated by microglial turnover. TBI-associated dendritic remodeling and myelin alterations, however, remained 30 dpi independent of microglial turnover. In assessments of functional recovery, increased depressive-like behavior, and cognitive impairment 30 dpi were ameliorated by microglia turnover. To investigate microglial priming and reactivity 30 dpi, mice were injected intraperitoneally with LPS. This immune challenge caused prolonged lethargy, sickness behavior, and microglial reactivity in the TBI mice. These extended complications with LPS in TBI mice were prevented by microglia turnover. Collectively, microglial turnover 7 dpi alleviated behavioral and cognitive impairments associated with microglial priming and immune reactivity 30 dpi.SIGNIFICANCE STATEMENT A striking feature of traumatic brain injury (TBI), even mild injuries, is that over 70% of individuals have long-term neuropsychiatric complications. Chronic inflammatory processes are implicated in the pathology of these complications and these issues can be exaggerated by immune challenge. Therefore, our goal was to force the turnover of microglia 7 d after TBI. This subacute 7 d postinjury (dpi) time point is a critical transitional period in the shift toward chronic inflammatory processes and microglia priming. This forced microglia turnover intervention in mice attenuated the deficits in behavior and cognition 30 dpi. Moreover, microglia priming and immune reactivity after TBI were also reduced with microglia turnover. Therefore, microglia represent therapeutic targets after TBI to reduce persistent neuroinflammation and improve recovery.

Sleep fragmentation engages stress-responsive circuitry, enhances inflammation and compromises hippocampal function following traumatic brain injury.

Traumatic brain injury (TBI) impairs the ability to restore homeostasis in response to stress, indicating hypothalamic-pituitary-adrenal (HPA)-axis dysfunction. Many stressors result in sleep disturbances, thus mechanical sleep fragmentation (SF) provides a physiologically relevant approach to study the effects of stress after injury. We hypothesize SF stress engages the dysregulated HPA-axis after TBI to exacerbate post-injury neuroinflammation and compromise recovery. To test this, male and female mice were given moderate lateral fluid percussion TBI or sham-injury and left undisturbed or exposed to daily, transient SF for 7- or 30-days post-injury (DPI). Post-TBI SF increases cortical expression of interferon- and stress-associated genes characterized by inhibition of the upstream regulator NR3C1 that encodes glucocorticoid receptor (GR). Moreover, post-TBI SF increases neuronal activity in the hippocampus, a key intersection of the stress-immune axes. By 30 DPI, TBI SF enhances cortical microgliosis and increases expression of pro-inflammatory glial signaling genes characterized by persistent inhibition of the NR3C1 upstream regulator. Within the hippocampus, post-TBI SF exaggerates microgliosis and decreases CA1 neuronal activity. Downstream of the hippocampus, post-injury SF suppresses neuronal activity in the hypothalamic paraventricular nucleus indicating decreased HPA-axis reactivity. Direct application of GR agonist, dexamethasone, to the CA1 at 30 DPI increases GR activity in TBI animals, but not sham animals, indicating differential GR-mediated hippocampal action. Electrophysiological assessment revealed TBI and SF induces deficits in Schaffer collateral long-term potentiation associated with impaired acquisition of trace fear conditioning, reflecting dorsal hippocampal-dependent cognitive deficits. Together these data demonstrate that post-injury SF engages the dysfunctional post-injury HPA-axis, enhances inflammation, and compromises hippocampal function. Therefore, external stressors that disrupt sleep have an integral role in mediating outcome after brain injury.

Translational regulation in the brain by TDP-43 phase separation.

The in vivo physiological function of liquid-liquid phase separation (LLPS) that governs non-membrane-bound structures remains elusive. Among LLPS-prone proteins, TAR DNA-binding protein of 43 kD (TDP-43) is under intense investigation because of its close association with neurological disorders. Here, we generated mice expressing endogenous LLPS-deficient murine TDP-43. LLPS-deficient TDP-43 mice demonstrate impaired neuronal function and behavioral abnormalities specifically related to brain function. Brain neurons of these mice, however, did not show TDP-43 proteinopathy or neurodegeneration. Instead, the global rate of protein synthesis was found to be greatly enhanced by TDP-43 LLPS loss. Mechanistically, TDP-43 LLPS ablation increased its association with PABPC4, RPS6, RPL7, and other translational factors. The physical interactions between TDP-43 and translational factors relies on a motif, the deletion of which abolished the impact of LLPS-deficient TDP-43 on translation. Our findings show a specific physiological role for TDP-43 LLPS in the regulation of brain function and uncover an intriguing novel molecular mechanism of translational control by LLPS.

Traumatic Brain Injury Causes Chronic Cortical Inflammation and Neuronal Dysfunction Mediated by Microglia.

Traumatic brain injury (TBI) can lead to significant neuropsychiatric problems and neurodegenerative pathologies, which develop and persist years after injury. Neuroinflammatory processes evolve over this same period. Therefore, we aimed to determine the contribution of microglia to neuropathology at acute [1 d postinjury (dpi)], subacute (7 dpi), and chronic (30 dpi) time points. Microglia were depleted with PLX5622, a CSF1R antagonist, before midline fluid percussion injury (FPI) in male mice and cortical neuropathology/inflammation was assessed using a neuropathology mRNA panel. Gene expression associated with inflammation and neuropathology were robustly increased acutely after injury (1 dpi) and the majority of this expression was microglia independent. At 7 and 30 dpi, however, microglial depletion reversed TBI-related expression of genes associated with inflammation, interferon signaling, and neuropathology. Myriad suppressed genes at subacute and chronic endpoints were attributed to neurons. To understand the relationship between microglia, neurons, and other glia, single-cell RNA sequencing was completed 7 dpi, a critical time point in the evolution from acute to chronic pathogenesis. Cortical microglia exhibited distinct TBI-associated clustering with increased type-1 interferon and neurodegenerative/damage-related genes. In cortical neurons, genes associated with dopamine signaling, long-term potentiation, calcium signaling, and synaptogenesis were suppressed. Microglial depletion reversed the majority of these neuronal alterations. Furthermore, there was reduced cortical dendritic complexity 7 dpi, reduced neuronal connectively 30 dpi, and cognitive impairment 30 dpi. All of these TBI-associated functional and behavioral impairments were prevented by microglial depletion. Collectively, these studies indicate that microglia promote persistent neuropathology and long-term functional impairments in neuronal homeostasis after TBI.SIGNIFICANCE STATEMENT Millions of traumatic brain injuries (TBIs) occur in the United States alone each year. Survivors face elevated rates of cognitive and psychiatric complications long after the inciting injury. Recent studies of human brain injury link chronic neuroinflammation to adverse neurologic outcomes, suggesting that evolving inflammatory processes may be an opportunity for intervention. Here, we eliminate microglia to compare the effects of diffuse TBI on neurons in the presence and absence of microglia and microglia-mediated inflammation. In the absence of microglia, neurons do not undergo TBI-induced changes in gene transcription or structure. Microglial elimination prevented TBI-induced cognitive changes 30 d postinjury (dpi). Therefore, microglia have a critical role in disrupting neuronal homeostasis after TBI, particularly at subacute and chronic timepoints.

Restoring tripartite glutamatergic synapses: A potential therapy for mood and cognitive deficits in Gulf War illness.

Gulf War illness is associated with a combination of exposure to war-related chemical agents and traumatic stress. Currently, there are no effective treatments, and the pathophysiology remains elusive. Neurological problems are among the most commonly reported symptoms. In this study, we investigated the glutamatergic system in the hippocampi of mice exposed to war-related chemical agents and stress. Mice developed Gulf War illness-like symptoms, including mood deficits, cognitive impairments, and fatigue. They exhibited the following pathological changes in hippocampi: elevated extracellular glutamate levels, impaired glutamatergic synapses, astrocyte atrophy, loss of interneurons, and decreased neurogenesis. LDN/OSU-215111 is a small-molecule that can strengthen the structure and function of both the astrocytic processes and the glutamatergic synapses that together form the tripartite synapses. We found that LDN/OSU-215111 effectively prevented the development of mood and cognitive deficits in mice when treatment was implemented immediately following the exposure. Moreover, when symptoms were already present, LDN/OSU-215111 still significantly ameliorated these deficits; impressively, benefits were sustained one month after treatment cessation, indicating disease modification. LDN/OSU-215111 effectively normalized hippocampal pathological changes. Overall, this study provides strong evidence that restoration of tripartite glutamatergic synapses by LDN/OSU-215111 is a potential therapy for Gulf War illness.

Enhancement of tripartite synapses as a potential therapeutic strategy for Alzheimer's disease: a preclinical study in rTg4510 mice.

BACKGROUND: The lack of effective treatment options for Alzheimer's disease (AD) is of momentous societal concern. Synaptic loss is the hallmark of AD that correlates best with impaired memory and occurs early in the disease process, before the onset of clinical symptoms. We have developed a small-molecule, pyridazine-based series that enhances the structure and function of both the glial processes and the synaptic boutons that form the tripartite synapse. Previously, we have shown that these pyridazine derivatives exhibit profound efficacy in an amyloid precursor protein AD model. Here, we evaluated the efficacy of an advanced compound, LDN/OSU-0215111, in rTg4510 mice-an aggressive tauopathy model. METHODS: rTg4510 mice were treated orally with vehicle or LDN/OSU-0215111 (10 mg/kg) daily from the early symptomatic stage (2 months old) to moderate (4 months old) and severe (8 months old) disease stages. At each time point, mice were subjected to a battery of behavioral tests to assess the activity levels and cognition. Also, tissue collections were performed on a subset of mice to analyze the tripartite synaptic changes, neurodegeneration, gliosis, and tau phosphorylation as assessed by immunohistochemistry and Western blotting. At 8 months of age, a subset of rTg4510 mice treated with compound was switched to vehicle treatment and analyzed behaviorally and biochemically 30 days after treatment cessation. RESULTS: At both the moderate and severe disease stages, compound treatment normalized cognition and behavior as well as reduced synaptic loss, neurodegeneration, tau hyperphosporylation, and neuroinflammation. Importantly, after 30 days of treatment cessation, the benefits of compound treatment were sustained, indicating disease modification. We also found that compound treatment rapidly and robustly reduced tau hyperphosphorylation/deposition possibly via the inhibition of GSK3ß. CONCLUSIONS: The results show that LDN/OSU-0215111 provides benefits for multiple aspects of tauopathy-dependent pathology found in Alzheimer's disease including tripartite synapse normalization and reduction of toxic tau burden, which, in turn, likely accounted for normalized cognition and activity levels in compound-treated rTg4510 mice. This study, in combination with our previous work regarding the benefit of pyridazine derivatives against amyloid-dependent pathology, strongly supports pyridazine derivatives as a viable, clinically relevant, and disease-modifying treatment for many of the facets of Alzheimer's disease.

Insulin Modulates Excitatory Synaptic Transmission and Synaptic Plasticity in the Mouse Hippocampus.

The administration of exogenous insulin into the hippocampus has the potential to enhance cognitive function and exert other beneficial effects. Elucidating the neurobiological substrates of insulin action and its underlying physiological mechanisms may further improve treatment efficacy. Previous work has shown that insulin affects synaptic plasticity, however there are discrepancies and contradictory conclusions between studies. Here, we used extracellular field recordings in mouse hippocampal slices to investigate how insulin acutely modulates synaptic transmission and synaptic plasticity, both of which are correlated with learning and memory processes. Our data demonstrate that insulin application inhibited basal excitatory synaptic transmission and promoted long-term potentiation (LTP) induction at hippocampal Schaffer collateral-CA1 synapses. Under similar conditions, insulin strongly activated the PI3K/AKT pathway, but had only a weak effect on the MAPK/ERK pathway. Although insulin-induced inhibition of field excitatory post-synaptic potentials (fEPSPs) was previously termed insulin-long-term depression (insulin-LTD), insulin application potentiated recovery from classically induced LTD. Further analysis suggests suppression of presynaptic neurotransmitter release contributed to the insulin-LTD. At low concentrations, insulin primarily inhibited fEPSPs; however, at high concentration, its effects were of mixed inhibition and enhancement in different recordings. Moreover, a broad spectrum protein kinase C blocker, cannabinoid receptor type 1 activator, or a high glucose concentration inhibited fEPSPs per se, and disturbed insulin's effect on fEPSP. Insulin also caused depotentiation during LTP expression and triggered depression during LTD recovery. Given the essential roles of dynamic synaptic transmission and plasticity in learning and memory, our data provide more evidence that insulin application may actively modulate hippocampal-dependent cognitive events.

Pyridazine-derivatives Enhance Structural and Functional Plasticity of Tripartite Synapse Via Activation of Local Translation in Astrocytic Processes.

Excitatory amino acid transporter 2 (EAAT2) is primarily located in perisynaptic astrocytic processes (PAP) where it plays a critical role in synaptic glutamate homeostasis. Dysregulation of EAAT2 at the translational level has been implicated in a myriad of neurological diseases. We previously discovered that pyridazine analogs can activate EAAT2 translation. Here, we sought to further refine the site and mechanism of compound action. We found that in vivo, compound treatment increased EAAT2 expression only in the PAP of astrocytes where EAAT2 mRNA also was identified. Direct application of compound to isolated PAP induced de novo EAAT2 protein synthesis, indicating that compound activates translation locally in the PAP. Using a screening process, we identified a set of PAP proteins that are rapidly up-regulated following compound treatment and a subset of these PAP proteins may be locally synthesized in the PAP. Importantly, these identified proteins are associated with the structural and functional capacity of the PAP, indicating compound enhanced plasticity of the PAP. Concomitantly, we found that pyridazine analogs increase synaptic protein expression in the synapse and enhance hippocampal long-term potentiation. This was not dependent upon compound-mediated local translation in neurons. This suggests that compound enhances the structural and functional capacity of the PAP which in turn facilitates enhanced plasticity of the tripartite synapse. Overall, this provides insight into the mechanism action site of pyridazine derivatives as well as the growing appreciation of the dynamic regulation and functional aspects of the PAP at the tripartite synapse.

Dissipation of transmembrane potassium gradient is the main cause of cerebral ischemia-induced depolarization in astrocytes and neurons.

Membrane potential (VM) depolarization occurs immediately following cerebral ischemia and is devastating for the astrocyte homeostasis and neuronal signaling. Previously, an excessive release of extracellular K+ and glutamate has been shown to underlie an ischemia-induced VM depolarization. Ischemic insults should impair membrane ion channels and disrupt the physiological ion gradients. However, their respective contribution to ischemia-induced neuronal and glial depolarization and loss of neuronal excitability are unanswered questions. A short-term oxygen-glucose deprivation (OGD) was used for the purpose of examining the acute effect of ischemic conditions on ion channel activity and physiological K+ gradient in neurons and glial cells. We show that a 30 min OGD treatment exerted no measurable damage to the function of membrane ion channels in neurons, astrocytes, and NG2 glia. As a result of the resilience of membrane ion channels, neuronal spikes last twice as long as our previously reported 15 min time window. In the electrophysiological analysis, a 30 min OGD-induced dissipation of transmembrane K+ gradient contributed differently in brain cell depolarization: severe in astrocytes and neurons, and undetectable in NG2 glia. The discrete cellular responses to OGD corresponded to a total loss of 69% of the intracellular K+ contents in hippocampal slices as measured by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). A major brain cell depolarization mechanism identified here is important for our understanding of cerebral ischemia pathology. Additionally, further understanding of the resilient response of NG2 glia to ischemia-induced intracellular K+ loss and depolarization should facilitate the development of future stroke therapy.

Molecular determinants of acidic pH-dependent transport of human equilibrative nucleoside transporter 3.

Equilibrative nucleoside transporters (ENTs) translocate hydrophilic nucleosides across cellular membranes and are essential for salvage nucleotide synthesis and purinergic signaling. Unlike the prototypic human ENT members hENT1 and hENT2, which mediate plasma membrane nucleoside transport at pH 7.4, hENT3 is an acidic pH-activated lysosomal transporter partially localized to mitochondria. Recent studies demonstrate that hENT3 is indispensable for lysosomal homeostasis, and that mutations in hENT3 can result in a spectrum of lysosomal storage-like disorders. However, despite hENT3's prominent role in lysosome pathophysiology, the molecular basis of hENT3-mediated transport is unknown. Therefore, we sought to examine the mechanistic basis of acidic pH-driven hENT3 nucleoside transport with site-directed mutagenesis, homology modeling, and [3H]adenosine flux measurements in mutant RNA-injected Xenopus oocytes. Scanning mutagenesis of putative residues responsible for pH-dependent transport via hENT3 revealed that the ionization states of Asp-219 and Glu-447, and not His, strongly determined the pH-dependent transport permissible-impermissible states of the transporter. Except for substitution with certain isosteric and polar residues, substitution of either Asp-219 or Glu-447 with any other residues resulted in robust activity that was pH-independent. Dual substitution of Asp-219 and Glu-447 to Ala sustained pH-independent activity over a broad range of physiological pH (pH 5.5-7.4), which also maintained stringent substrate selectivity toward endogenous nucleosides and clinically used nucleoside drugs. Our results suggest a putative pH-sensing role for Asp-219 and Glu-447 in hENT3 and that the size, ionization state, or electronegative polarity at these positions is crucial for obligate acidic pH-dependent activity.

G-Protein-Coupled Receptor Signaling in Cilia.

G-protein-coupled receptors (GPCRs) are the largest and most versatile family of signaling receptors in humans. They respond to diverse external signals, such as photons, proteins, peptides, chemicals, hormones, lipids, and sugars, and mediate a myriad of functions in the human body. Signaling through GPCRs can be optimized by enriching receptors and downstream effectors in discrete cellular domains. Many GPCRs have been found to be selectively targeted to cilia on numerous mammalian cell types. Moreover, investigations into the pathophysiology of human ciliopathies have implicated GPCR ciliary signaling in a number of developmental and cellular pathways. Thus, cilia are now appreciated as an increasingly important nexus for GPCR signaling. Yet, we are just beginning to understand the precise signaling pathways mediated by most ciliary GPCRs and how they impact cellular function and mammalian physiology.

Oligodendrocytes contribute to motor neuron death in ALS via SOD1-dependent mechanism.

Oligodendrocytes have recently been implicated in the pathophysiology of amyotrophic lateral sclerosis (ALS). Here we show that, in vitro, mutant superoxide dismutase 1 (SOD1) mouse oligodendrocytes induce WT motor neuron (MN) hyperexcitability and death. Moreover, we efficiently derived human oligodendrocytes from a large number of controls and patients with sporadic and familial ALS, using two different reprogramming methods. All ALS oligodendrocyte lines induced MN death through conditioned medium (CM) and in coculture. CM-mediated MN death was associated with decreased lactate production and release, whereas toxicity in coculture was lactate-independent, demonstrating that MN survival is mediated not only by soluble factors. Remarkably, human SOD1 shRNA treatment resulted in MN rescue in both mouse and human cultures when knockdown was achieved in progenitor cells, whereas it was ineffective in differentiated oligodendrocytes. In fact, early SOD1 knockdown rescued lactate impairment and cell toxicity in all lines tested, with the exclusion of samples carrying chromosome 9 ORF 72 (C9orf72) repeat expansions. These did not respond to SOD1 knockdown nor did they show lactate release impairment. Our data indicate that SOD1 is directly or indirectly involved in ALS oligodendrocyte pathology and suggest that in this cell type, some damage might be irreversible. In addition, we demonstrate that patients with C9ORF72 represent an independent patient group that might not respond to the same treatment.

Deterministic transfection drives efficient nonviral reprogramming and uncovers reprogramming barriers.

Safety concerns and/or the stochastic nature of current transduction approaches have hampered nuclear reprogramming's clinical translation. We report a novel non-viral nanotechnology-based platform permitting deterministic large-scale transfection with single-cell resolution. The superior capabilities of our technology are demonstrated by modification of the well-established direct neuronal reprogramming paradigm using overexpression of the transcription factors Brn2, Ascl1, and Myt1l (BAM). Reprogramming efficiencies were comparable to viral methodologies (up to ~9-12%) without the constraints of capsid size and with the ability to control plasmid dosage, in addition to showing superior performance relative to existing non-viral methods. Furthermore, increased neuronal complexity could be tailored by varying BAM ratio and by including additional proneural genes to the BAM cocktail. Furthermore, high-throughput NEP allowed easy interrogation of the reprogramming process. We discovered that BAM-mediated reprogramming is regulated by AsclI dosage, the S-phase cyclin CCNA2, and that some induced neurons passed through a nestin-positive cell stage. FROM THE CLINICAL EDITOR: In the field of regenerative medicine, the ability to direct cell fate by nuclear reprogramming is an important facet in terms of clinical application. In this article, the authors described their novel technique of cell reprogramming through overexpression of the transcription factors Brn2, Ascl1, and Myt1l (BAM) by in situ electroporation through nanochannels. This new technique could provide a platform for further future designs.

ASICs and neuropeptides.

The acid sensing ion channels (ASICs) are proton-gated cation channels expressed throughout the nervous system. ASICs are activated during acidic pH fluctuations, and recent work suggests that they are involved in excitatory synaptic transmission. ASICs can also induce neuronal degeneration and death during pathological extracellular acidosis caused by ischemia, autoimmune inflammation, and traumatic injury. Many endogenous neuromodulators target ASICs to affect their biophysical characteristics and contributions to neuronal activity. One of the most unconventional types of modulation occurs with the interaction of ASICs and neuropeptides. Collectively, FMRFamide-related peptides and dynorphins potentiate ASIC activity by decreasing the proton-sensitivity of steady state desensitization independent of G protein-coupled receptor activation. By decreasing the proton-sensitivity of steady state desensitization, the FMRFamide-related peptides and dynorphins permit ASICs to remain active at more acidic basal pH. Unlike the dynorphins, some FMRFamide-related peptides also potentiate ASIC activity by slowing inactivation and increasing the sustained current. Through mechanistic studies, the modulation of ASICs by FMRFamide-related peptides and dynorphins appears to be through distinct interactions with the extracellular domain of ASICs. Dynorphins are expressed throughout the nervous system and can increase neuronal death during prolonged extracellular acidosis, suggesting that the interaction between dynorphins and ASICs may have important consequences for the prevention of neurological injury. The overlap in expression of FMRFamide-related peptides with ASICs in the dorsal horn of the spinal cord suggests that their interaction may have important consequences for the treatment of pain during injury and inflammation. This article is part of the Special Issue entitled 'Acid-Sensing Ion Channels in the Nervous System'.