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Atherosclerosis, the major cause of myocardial infarction and stroke, results from converging inflammatory, metabolic, and biomechanical factors. Arterial lesions form at sites of low and disturbed blood flow but are suppressed by high laminar shear stress (LSS) mainly via transcriptional induction of the anti-inflammatory transcription factor, Kruppel-like factor 2 (Klf2). We therefore performed a whole genome CRISPR-Cas9 screen to identify genes required for LSS induction of Klf2. Subsequent mechanistic investigation revealed that LSS induces Klf2 via activation of both a MEKK2/3–MEK5–ERK5 kinase module and mitochondrial metabolism. Mitochondrial calcium and ROS signaling regulate assembly of a mitophagy- and p62-dependent scaffolding complex that amplifies MEKK–MEK5–ERK5 signaling. Blocking the mitochondrial pathway in vivo reduces expression of KLF2-dependent genes such as eNOS and inhibits vascular remodeling. Failure to activate the mitochondrial pathway limits Klf2 expression in regions of disturbed flow. This work thus defines a connection between metabolism and vascular inflammation that provides a new framework for understanding and developing treatments for vascular disease.

A subset of adult ventral tegmental area dopamine (DA) neurons expresses vesicular glutamate transporter 2 (VGluT2) and releases glutamate as a second neurotransmitter in the striatum, while only few adult substantia nigra DA neurons have this capacity. Recent work showed that cellular stress created by neurotoxins such as MPTP and 6-hydroxydopamine can upregulate VGluT2 in surviving DA neurons, suggesting the possibility of a role in cell survival, although a high level of overexpression could be toxic to DA neurons. Here we examined the level of VGluT2 upregulation in response to neurotoxins and its impact on postlesional plasticity. We first took advantage of anin vitroneurotoxin model of Parkinson's disease and found that this caused an average 2.5-fold enhancement ofVglut2mRNA in DA neurons. This could represent a reactivation of a developmental phenotype because using an intersectional genetic lineage-mapping approach, we find that >98% of DA neurons have a VGluT2+lineage. Expression of VGluT2 was detectable in most DA neurons at embryonic day 11.5 and was localized in developing axons. Finally, compatible with the possibility that enhanced VGluT2 expression in DA neurons promotes axonal outgrowth and reinnervation in the postlesional brain, we observed that DA neurons in female and male mice in which VGluT2 was conditionally removed established fewer striatal connections 7 weeks after a neurotoxin lesion. Thus, we propose here that the developmental expression of VGluT2 in DA neurons can be reactivated at postnatal stages, contributing to postlesional plasticity of dopaminergic axons.SIGNIFICANCE STATEMENTA small subset of dopamine neurons in the adult, healthy brain expresses vesicular glutamate transporter 2 (VGluT2) and thus releases glutamate as a second neurotransmitter in the striatum. This neurochemical phenotype appears to be plastic as exposure to neurotoxins, such as 6-OHDA or MPTP, that model certain aspects of Parkinson's disease pathophysiology, boosts VGluT2 expression in surviving dopamine neurons. Here we show that this enhanced VGluT2 expression in dopamine neurons drives axonal outgrowth and contributes to dopamine neuron axonal plasticity in the postlesional brain. A better understanding of the neurochemical changes that occur during the progression of Parkinson's disease pathology will aid the development of novel therapeutic strategies for this disease.

SummaryMidbrain dopamine (DA) neurons are key regulators of basal ganglia functions. The axonal domain of these neurons is highly complex, with a large subset of non-synaptic release sites and a smaller subset of synaptic terminals from which glutamate or GABA are released. The molecular mechanisms regulating the connectivity of DA neurons and their neurochemical identity are unknown. Here we tested the hypothesis that the trans-synaptic cell adhesion molecules neurexins (Nrxns) regulate DA neuron neurotransmission. Conditional deletion of all Nrxns in DA neurons (DAT::Nrxns KO) showed that loss of Nrxns does not impair the basic development and ultrastructural characteristics of DA neuron terminals. However, loss of Nrxns caused an impairment of DA transmission revealed as a reduced rate of DA reuptake following activity-dependent DA release, decreased DA transporter levels, increased vesicular monoamine transporter expression, and impaired amphetamine-induced locomotor activity. Strikingly, electrophysiological recording revealed an increase of GABA co-release from DA neuron axons in the striatum of the KO mice. These findings suggest that Nrxns act as key regulators of DA neuron connectivity and DA-mediated functions.HighlightsThe study provides the first direct evidence of the role of neurexins in dopaminergic neurons.The synaptic adhesion molecules, neurexins, are not required for maintaining the structure of dopamine neuron terminals.Neurexins regulate dopaminergic neurotransmission through regulation of dopamine reuptake, impacting amphetamine-induced locomotion.Deletion of Nrxns in DA neurons causes a region-specific increase of GABA release by DA neurons.

SummaryMidbrain dopamine (DA) neurons, a population of cells that are critical for motor control, motivated behaviors and cognition, release DA via an exocytotic mechanism from both their axonal terminals and their somatodendritic (STD) compartment. In Parkinson’s disease (PD), it is striking that motor dysfunctions only become apparent after extensive loss of DA innervation. Although it has been hypothesized that this resilience is due to the ability of many motor behaviors to be sustained through a basal tone of DA and diffuse transmission, experimental evidence for this is limited. Here we conditionally deleted the calcium sensor synaptotagmin-1 (Syt1) in DA neurons (cKODA mice) to abrogate most activity-dependent axonal DA release in the striatum and mesencephalon, leaving STD DA release intact. Strikingly, Syt1 cKODA mice showed intact performance in multiple unconditioned DA-dependent motor tasks, suggesting that activity-dependent DA release is dispensable for such basic motor functions. Basal extracellular levels of DA in the striatum were unchanged, suggesting that a basal tone of extracellular DA is sufficient to sustain basic movement. We also found multiple adaptations in the DA system of cKODA mice, similar to those happening at early stages of PD. Taken together, our findings reveal the striking resilience of DA-dependent motor functions in the context of a near-abolition of phasic DA release, shedding new light on why extensive loss of DA innervation is required to reveal motor dysfunctions in PD.

Chemical neurotransmission in the brain typically occurs through synapses, which are structurally and functionally defined as sites of close apposition between an axon terminal and a postsynaptic domain. Ultrastructural examinations of axon terminals established by monoamine neurons in the brain often failed to identify a similar tight pre- and postsynaptic coupling, giving rise to the concept of “diffuse” or “volume” transmission. Whether this results from intrinsic properties of such modulatory neurons remains undefined. Using an efficient co-culture model, we find that dopaminergic neurons establish an axonal arbor that is distinctive compared to glutamatergic or GABAergic neurons in both size and propensity of terminals to avoid direct contact with target neurons. Furthermore, while most dopaminergic varicosities express key proteins involved in exocytosis such as synaptotagmin 1, only ~20% of these are synaptic. The active zone protein bassoon was found to be enriched in a subset of dopaminergic terminals that are in proximity to a target cell. Irrespective of their structure, a majority of dopaminergic terminals were found to be active. Finally, we found that the presynaptic protein Nrxn-1αSS4- and the postsynaptic protein NL-1AB, two major components involved in excitatory synapse formation, play a critical role in the formation of synapses by dopamine neurons. Taken together, our findings support the idea that dopamine neurons in the brain are endowed with a distinctive developmental program that leads them to adopt a fundamentally different mode of connectivity, compared to glutamatergic and GABAergic neurons involved in fast point-to-point signaling.SIGNIFICANCE STATEMENTMidbrain dopamine (DA) neurons regulate circuits controlling movement, motivation, and learning. The axonal connectivity of DA neurons is intriguing due to its hyperdense nature, with a particularly large number of release sites, most of which not adopting a classical synaptic structure. In this study, we provide new evidence highlighting the unique ability of DA neurons to establish a large and heterogeneous axonal arbor with terminals that, in striking contrast with glutamate and GABA neurons, actively avoid contact with the target cells. The majority of synaptic and non-synaptic terminals express proteins for exocytosis and are active. Finally, our finding suggests that, NL-1A+B and Nrxn-1αSS4-, play a critical role in the formation of synapses by DA neurons.

While current effective therapies are available for the symptomatic control of PD, treatments to halt the progressive neurodegeneration still do not exist. Loss of dopamine neurons in the SNc and dopamine terminals in the striatum drive the motor features of PD. Multiple lines of research point to several pathways which may contribute to dopaminergic neurodegeneration. These pathways include extensive axonal arborization, mitochondrial dysfunction, dopamine's biochemical properties, abnormal protein accumulation of α-synuclein, defective autophagy and lysosomal degradation, and synaptic impairment. Thus, understanding the essential features and mechanisms of dopaminergic neuronal vulnerability is a major scientific challenge and highlights an outstanding need for fostering effective therapies against neurodegeneration in PD. This article, which arose from the Movement Disorders 2018 Conference, discusses and reviews the possible mechanisms underlying neuronal vulnerability and potential therapeutic approaches in PD. © 2019 International Parkinson and Movement Disorder Society.

Parkinson’s disease is a neurodegenerative disorder with motor symptoms linked to the loss of dopaminergic neurons in the substantia nigra compacta. Although the mechanisms that trigger the loss of dopaminergic neurons are unclear, mitochondrial dysfunction and inflammation are thought to have key roles1,2. An early-onset form of Parkinson’s disease is associated with mutations in the PINK1 kinase and PRKN ubiquitin ligase genes3. PINK1 and Parkin (encoded by PRKN) are involved in the clearance of damaged mitochondria in cultured cells4, but recent evidence obtained using knockout and knockin mouse models have led to contradictory results regarding the contributions of PINK1 and Parkin to mitophagy in vivo5–8. It has previously been shown that PINK1 and Parkin have a key role in adaptive immunity by repressing presentation of mitochondrial antigens9, which suggests that autoimmune mechanisms participate in the aetiology of Parkinson’s disease. Here we show that intestinal infection with Gram-negative bacteria in Pink1−/− mice engages mitochondrial antigen presentation and autoimmune mechanisms that elicit the establishment of cytotoxic mitochondria-specific CD8+ T cells in the periphery and in the brain. Notably, these mice show a sharp decrease in the density of dopaminergic axonal varicosities in the striatum and are affected by motor impairment that is reversed after treatment with l-DOPA. These data support the idea that PINK1 is a repressor of the immune system, and provide a pathophysiological model in which intestinal infection acts as a triggering event in Parkinson’s disease, which highlights the relevance of the gut–brain axis in the disease10. In mice lacking PINK1, bacterial infection in the intestine results in mitochondrial antigen presentation and generation of CD8+ T cells, and infected mice develop motor impairments, suggesting that PINK1 suppresses autoimmunity.

Dopamine (DA) neurons of the substantia nigra pars compacta (SNc) are uniquely vulnerable to neurodegeneration in Parkinson’s disease (PD). We hypothesize that their large axonal arbor is a key factor underlying their vulnerability, due to increased bioenergetic, proteostatic and oxidative stress. In keeping with this model, other DAergic populations with smaller axonal arbors are mostly spared during the course of PD and are more resistant to experimental lesions in animal models. Aiming to improve mouse PD models, we examined if neonatal partial SNc lesions could lead to adult mice with fewer SNc DA neurons that are endowed with larger axonal arbors because of compensatory mechanisms. We injected 6-hydroxydopamine (6-OHDA) unilaterally in the SNc at an early postnatal stage at a dose selected to induce loss of approximately 50% of SNc DA neurons. We find that at 10- and 90-days after the lesion, the axons of SNc DA neurons show massive compensatory sprouting, as revealed by the proportionally smaller decrease in tyrosine hydroxylase (TH) in the striatum compared to the loss of SNc DA neuron cell bodies. The extent and origin of this axonal sprouting was further investigated by AAV-mediated expression of eYFP in SNc or ventral tegmental area (VTA) DA neurons of adult mice. Our results reveal that SNc DA neurons have the capacity to substantially increase their axonal arbor size and suggest that mice designed to have reduced numbers of SNc DA neurons could potentially be used to develop better mouse models of PD, with elevated neuronal vulnerability.Graphical abstract textWe describe a technique to induce the loss of approximately 50% of SNc DA neurons in neonate mice using unilateral intranigral 6-OHDA (left panel).Compensatory axonal sprouting was observed in the striatum as early as 10 days following the lesion (at P15), with effects lasting until adulthood (P90).Conditional AAV-mediated expression of eYFP (green) reveals SNc DA neurons, projecting to the dorsal striatum (middle panel), and not VTA DA neurons, projecting to the ventral striatum (right panel), as the main source of compensatory axonal sprouting.Graphical abstract

Dopamine (DA) neurons can release DA not just from axon terminals, but also from their somatodendritic (STD) compartment thought a mechanism that is still incompletely understood. Using voltammetry in mouse mesencephalic brain slices, we find that STD DA release has low capacity, is stable in response to electrical but not optogenetic train pulses and shows a calcium sensitivity that is comparable to that of axonal release. It is also strikingly more resilient compared to axonal release in a 6‐ hydroxydopamine model of Parkinson’s disease plasticity. We find that the molecular mechanism of STD DA release differs from axonal release with regards to the implication of synaptotagmin (Syt) calcium sensors. While individual constitutive knock-out Syt4 and Syt7 is not sufficient to reduce STD DA release, removal of both isoforms reduces this release by ~50%, leaving axonal release unimpaired. Our works unveils clear differences in the mechanisms of STD and axonal DA release.

Dendritic cells (DCs) excel at cross-presenting antigens, but their effectiveness as cancer vaccine is limited. Here, we describe an innovative vaccine approach using mesenchymal stromal cells (MSCs) engineered to express the immunoproteasome (IPr) complex (MSC-IPr). Such modification instilled efficient antigen cross-presenting abilities associated with enhanced major histocompatibility complex (MHC)I and CD80 expression, de novo expression of interleukin-12 along with higher chemokine secretion. In addition, the cross-presentation capacity of MSC-IPr is highly dependent on AMPK signaling, oxidative phosphorylation and production of reactive oxygen species. Compared to DCs, MSC-IPr hold the ability to cross-present a vastly different epitope repertoire, which translates into potent re-activation of T-cell immunity against EL4 lymphoma and B16 melanoma tumors. Moreover, therapeutic vaccination of animals with pre-established tumors efficiently controls cancer growth, an effect further enhanced when combined with antibodies targeting PD1, CTLA4, LAG3 or 4-1BB under both autologous and allogeneic settings. MSC-IPr constitute therefore a novel subset of non-hematopoietic antigen-presenting cells suitable for the future design of universal cell-based cancer vaccines.

D-amphetamine maintenance therapy shows promise as a treatment for people with cocaine addiction. Preclinical studies using Long Access (LgA) cocaine self-administration procedures suggest D-amphetamine may act by preventing tolerance to cocaine’s effects at the dopamine transporter (DAT). However, Intermittent Access (IntA) cocaine self-administration better reflects human patterns of use, is especially effective in promoting addiction-relevant behaviors, and instead of tolerance, produces psychomotor, incentive, and neural sensitization. We asked, therefore, how D-amphetamine maintenance during IntA influences cocaine use and cocaine’s potency at the DAT. Male rats self-administered cocaine intermittently (5 min ON, 25 min OFF x10; 5-h/session) for 14 sessions, with or without concomitant D-amphetamine maintenance therapy during these 14 sessions (5 mg/kg/day via s.c. osmotic minipump). We then assessed responding for cocaine under a progressive ratio schedule, responding under extinction and cocaine-primed reinstatement of drug seeking. We also assessed the ability of cocaine to inhibit dopamine uptake in the nucleus accumbens core using fast scan cyclic voltammetry ex vivo. IntA cocaine self-administration produced psychomotor (locomotor) sensitization, strong motivation to take and seek cocaine, and it increased cocaine’s potency at the DAT. D-amphetamine co-administration suppressed the psychomotor sensitization produced by IntA cocaine experience. After cessation of D-amphetamine treatment, the motivation to take and seek cocaine was also reduced, and sensitization of cocaine’s actions at the DAT was reversed. Thus, treatment with D-amphetamine might reduce cocaine use by preventing sensitization-related changes in cocaine potency at the DAT, consistent with an incentive-sensitization view of addiction.

BackgroundD-amphetamine maintenance therapy shows promise as a treatment for people with cocaine addiction. Preclinical studies using Long Access (LgA) cocaine self-administration procedures suggest D-amphetamine may act by preventing tolerance to cocaine’s effects at the dopamine transporter (DAT). However, Intermittent Access (IntA) cocaine self-administration better reflects human patterns of use, is especially effective in promoting addiction-relevant behaviors, and instead of tolerance, produces psychomotor, incentive, and neural sensitization. We asked, therefore, how D-amphetamine maintenance during IntA influences cocaine use and cocaine’s potency at the DAT.MethodsMale rats self-administered cocaine intermittently (5 minutes ON, 25 minutes OFF x 10) for 14 sessions, with or without concomitant D-amphetamine (5 mg/kg/day via s.c. osmotic minipump). In Experiment 1, psychomotor sensitization, responding for cocaine under a progressive ratio schedule, responding under extinction and cocaine-primed relapse were assessed. In Experiment 2, rats self-administered cocaine or saline intermittently, with or without D-amphetamine, and the ability of cocaine to inhibit dopamine uptake in the nucleus accumbens core was assessed using fast scan cyclic voltammetry ex vivo.ResultsIntA cocaine self-administration produced psychomotor sensitization, strong motivation to take and seek cocaine, and it increased cocaine’s potency at the DAT. The co-administration of D-amphetamine suppressed both the psychomotor sensitization and high motivation for cocaine produced by IntA experience, and also reversed sensitization of cocaine’s actions at the DAT, leaving baseline DAT function unchanged.ConclusionsTreatment with D-amphetamine might reduce cocaine use by preventing sensitization-related changes in cocaine potency at the DAT, consistent with an incentive-sensitization view of addiction.

In Parkinson’s disease, the most vulnerable neurons are found in the ventral tier of the substantia nigra (SN), while the adjacent dopamine (DA) neurons of the ventral tegmental area (VTA) are mostly spared. Although a significant subset of adult VTA DA neurons expressesVglut2, a vesicular glutamate transporter, and release glutamate as a second neurotransmitter in the striatum, only very few adult SN DA neurons have this capacity. Previous work has demonstrated that lesions created by neurotoxins such as MPTP and 6-hydroxydopamine (6-OHDA) can upregulate the expression of Vglut2 in surviving DA neurons. Currently, the molecular mechanisms explaining the plasticity of Vglut2 expression in DA neurons are unknown, as are the physiological consequences for DA neuron function and survival. Here we aimed to characterize the developmental expression pattern of Vglut2 in DA neurons and the role of this transporter in post-lesional plasticity in these neurons. Using an intersectional genetic lineage-mapping approach, based on Vglut2-Cre and TH-Flpo drivers, we first found that more than 98% of DA neurons expressedVglut2at some point in their embryonic development. Expression of this transporter was detectable in most DA neurons until E11.5 and was found to be localized in developing axons. Moderate enhancement of VGLUT2 expression in primary DA neurons caused an increase in axonal arborization length. Compatible with a developmental role, constitutive deletion of Vglut2 caused a regional defect in TH-innervation of the dorsal striatum in E18.5 embryos. Moreover, using anin vitroneurotoxin model, we demonstrate thatVglut2expression can be upregulated in post-lesional DA neurons by 2.5-fold, arguing that the developmental expression ofVglut2in DA neurons can be reactivated at postnatal stages and contribute to post-lesional plasticity of dopaminergic axons. In support of this hypothesis, we find fewer mesostriatial dopaminergic projections in the striatum of conditional Vglut2 KO mice 7 weeks after a neurotoxic lesion, compared to control animals. Thus, we propose here that one of the functions of Vglut2 in adult DA neurons is to promote post-lesional recovery of meso-striatal axons.

Dopamine (DA) is a key regulator of circuits controlling movement and motivation. A subset of midbrain DA neurons has been shown to express the vesicular glutamate transporter (VGLUT)2, underlying their capacity for glutamate release. Glutamate release is found mainly by DA neurons of the ventral tegmental area (VTA) and can be detected at terminals contacting ventral, but not dorsal, striatal neurons, suggesting the possibility that target-derived signals regulate the neurotransmitter phenotype of DA neurons. Whether glutamate can be released from the same terminals that release DA or from a special subset of axon terminals is unclear. Here, we provide in vitro and in vivo data supporting the hypothesis that DA and glutamate-releasing terminals in mice are mostly segregated and that striatal neurons regulate the cophenotype of midbrain DA neurons and the segregation of release sites. Our work unveils a fundamental feature of dual neurotransmission and plasticity of the DA system.-Fortin, G. M., Ducrot, C., Giguère, N., Kouwenhoven, W. M., Bourque, M.-J., Pacelli, C., Varaschin, R. K., Brill, M., Singh, S., Wiseman, P. W., Trudeau, L.-E. Segregation of dopamine and glutamate release sites in dopamine neuron axons: regulation by striatal target cells.

Many mutations in genes encoding proteins such as Parkin, PTEN-induced putative kinase 1 (PINK1), protein deglycase DJ-1 (DJ-1 or PARK7), leucine-rich repeat kinase 2 (LRRK2), and α-synuclein have been linked to familial forms of Parkinson's disease (PD). The consequences of these mutations, such as altered mitochondrial function and pathological protein aggregation, are starting to be better understood. However, little is known about the mechanisms explaining why alterations in such diverse cellular processes lead to the selective loss of dopamine (DA) neurons in the substantia nigra (SNc) in the brain of individuals with PD. Recent work has shown that one of the reasons for the high vulnerability of SNc DA neurons is their high basal rate of mitochondrial oxidative phosphorylation (OXPHOS), resulting from their highly complex axonal arborization. Here, we examined whether axonal growth and basal mitochondrial function are altered in SNc DA neurons from Parkin-, Pink1-, or DJ-1–KO mice. We provide evidence for increased basal OXPHOS in Parkin-KO DA neurons and for reduced survival of DA neurons that have a complex axonal arbor. The surviving smaller neurons exhibited reduced vulnerability to the DA neurotoxin and mitochondrial complex I inhibitor MPP+, and this reduction was associated with reduced expression of the DA transporter. Finally, we found that glial cells play a role in the reduced resilience of DA neurons in these mice and that WT Parkin overexpression rescues this phenotype. Our results provide critical insights into the complex relationship between mitochondrial function, axonal growth, and genetic risk factors for PD.

Summary Dendritic cells (DCs) excel at cross-presenting antigens, but their effectiveness as cancer vaccine is limited. Here, we describe a vaccination approach using mesenchymal stromal cells (MSCs) engineered to express the immunoproteasome complex (MSC-IPr). Such modification instills efficient antigen cross-presentation abilities associated with enhanced major histocompatibility complex class I and CD80 expression, de novo production of interleukin-12, and higher chemokine secretion. This cross-presentation capacity of MSC-IPr is highly dependent on their metabolic activity. Compared with DCs, MSC-IPr hold the ability to cross-present a vastly different epitope repertoire, which translates into potent re-activation of T cell immunity against EL4 and A20 lymphomas and B16 melanoma tumors. Moreover, therapeutic vaccination of mice with pre-established tumors efficiently controls cancer growth, an effect further enhanced when combined with antibodies targeting PD-1, CTLA4, LAG3, or 4-1BB under both autologous and allogeneic settings. Therefore, MSC-IPr constitute a promising subset of non-hematopoietic antigen-presenting cells suitable for designing universal cell-based cancer vaccines., Graphical abstract, Highlights • Stable expression of the immunoproteasome in MSCs instills a pro-inflammatory phenotype • Induced metabolic alterations drive antigen cross-presentation by MSC-IPr • Engineered MSC-IPr elicit potent anti-tumoral immunity • The immunopeptidome of MSC-IPr is distinct from dendritic cells, Abusarah et al. demonstrate that mesenchymal stromal cells engineered to express the immunoproteasome (MSC-IPr) exhibit distinct antigen presentation properties, display altered metabolic signatures, and present a unique set of antigens compared with standard dendritic cells. These attributes make them ideal candidates for the future design of cell-based cancer vaccines.

Mitochondria play a crucial role in neuronal survival through efficient energy metabolism. In pathological conditions, mitochondrial stress leads to neuronal death, which is regulated by the anti-apoptotic BCL-2 family of proteins. MCL-1 is an anti-apoptotic BCL-2 protein localized to mitochondria either in the outer membrane (OM) or inner membrane (Matrix), which have distinct roles in inhibiting apoptosis and promoting bioenergetics, respectively. While the anti-apoptotic role for Mcl1 is well characterized, the protective function of MCL-1 Matrix remains poorly understood. Here, we show MCL-1OM and MCL-1Matrix prevent neuronal death through distinct mechanisms. We report that MCL-1Matrix functions to preserve mitochondrial energy transduction and improves respiratory chain capacity by modulating mitochondrial oxygen consumption in response to mitochondrial stress. We show that MCL-1Matrix protects neurons from stress by enhancing respiratory function, and by inhibiting mitochondrial permeability transition pore opening. Taken together, our results provide novel insight into how MCL-1Matrix may confer neuroprotection under stress conditions involving loss of mitochondrial function.

This work was supported by Byggmastare Olle Engkvist foundation and Major Gosta Linds foundation (ES), and from National Institute of Neurological Disease and Stroke Grant NS029436 (MN), Canadian Institutes of Health Research and the Brain Canada (Multi-Investigator Research Initiative) and Krembil Foundations (L-ET), Consejo Nacional de Ciencia y Tecnologia (CONACYT) Mexico (Grant #128332) (MM).

A nanothin block copolymer (BCP) brush-layer film adsorbed on glass nanofibers is shown to address the long-standing challenge of forming a template for the deposition of dense and well dispersed nanoparticles on highly curved surfaces, allowing the development of an improved nanosensor for neurotransmitters. We employed a polystyrene-block-poly(4-vinylpyridine) BCP and plasmonic gold nanoparticles (AuNPs) of 52 nm in diameter for the fabrication of the nanosensor on pulled fibers with diameters down to 200 nm. The method is simple, using only solution processes and a plasma cleaning step. The templating of the AuNPs on the nanofiber surprisingly gave rise to more than one order of magnitude improvement in the surface-enhanced Raman scattering (SERS) performance for 4-mercaptobenzoic acid compared to the same AuNPs aggregated on identical fibers without the use of a template. We hypothesize that a wavelength-scale lens formed by the nanofiber contributes to enhancing the SERS performance, to the extent that it can melt the glass nanofiber under moderate laser power. We then show the capability of this nanosensor to detect the co-release of the neurotransmitters dopamine and glutamate from living mouse brain dopaminergic neurons with a sensitivity one order of magnitude greater than with aggregated AuNPs. The simplicity of fabrication and the far superior performance of the BCP-templated nanofiber demonstrates the potential of this method to efficiently pattern nanoparticles on highly curved surfaces and its application as molecular nanosensors for cell physiology.

Multiple discoveries made since the identification of vesicular glutamate transporters (VGLUTs) two decades ago revealed that many neuronal populations in the brain use glutamate in addition to their "primary" neurotransmitter. Such a mode of cotransmission has been detected in dopamine (DA), acetylcholine (ACh), serotonin (5-HT), norepinephrine (NE) and surprisingly even in GABA neurons. Interestingly, work performed by multiple groups during the past decade suggests that the use of glutamate as a cotransmitter takes different forms in these different populations of neurons. In the present review, we will provide an overview of glutamate cotransmission in these different classes of neurons, highlighting puzzling differences in: (1) the proportion of such neurons expressing a VGLUT in different brain regions and at different stages of development; (2) the sub-cellular localization of the VGLUT; (3) the localization of the VGLUT in relation to the neurons' other vesicular transporter; and (4) the functional role of glutamate cotransmission.

Summary Antigen presentation is essential for establishing immune tolerance and for immune responses against infectious disease and cancer. Although antigen presentation can be mediated by autophagy, here we demonstrate a pathway for mitochondrial antigen presentation (MitAP) that relies on the generation and trafficking of mitochondrial-derived vesicles (MDVs) rather than on autophagy/mitophagy. We find that PINK1 and Parkin, two mitochondrial proteins linked to Parkinson's disease (PD), actively inhibit MDV formation and MitAP. In absence of PINK1 or Parkin, inflammatory conditions trigger MitAP in immune cells, both in vitro and in vivo. MitAP and the formation of MDVs require Rab9 and Sorting nexin 9, whose recruitment to mitochondria is inhibited by Parkin. The identification of PINK1 and Parkin as suppressors of an immune-response-eliciting pathway provoked by inflammation suggests new insights into PD pathology.

Human mesenchymal stromal cells (MSC) have been shown to dampen immune response and promote tissue repair, but the underlying mechanisms are still under investigation. Herein, we demonstrate that umbilical cord-derived MSC (UC-MSC) alter the phenotype and function of monocyte-derived dendritic cells (DC) through lactate-mediated metabolic reprogramming. UC-MSC can secrete large quantities of lactate and, when present during monocyte-to-DC differentiation, induce instead the acquisition of M2-macrophage features in terms of morphology, surface markers, migratory properties and antigen presentation capacity. Microarray expression profiling indicates that UC-MSC modify the expression of metabolic-related genes and induce a M2-macrophage expression signature. Importantly, monocyte-derived DC obtained in presence of UC-MSC, polarize naive allogeneic CD4+ T-cells into Th2 cells. Treatment of UC-MSC with an inhibitor of lactate dehydrogenase strongly decreases lactate concentration in culture supernatant and abrogates the effect on monocyte-to-DC differentiation. Metabolic analysis further revealed that UC-MSC decrease oxidative phosphorylation in differentiating monocytes while strongly increasing the spare respiratory capacity proportional to the amount of secreted lactate. Because both MSC and monocytes are recruited in vivo at the site of tissue damage and inflammation, we propose the local increase of lactate concentration induced by UC-MSC and the consequent enrichment in M2-macrophage generation as a mechanism to achieve immunomodulation.

Calcium sensors detect intracellular calcium changes and interact with downstream targets to regulate many functions. Neuronal Calcium Sensor-1 (NCS-1) or Frequenin is widely expressed in the nervous system, and involved in neurotransmission, synaptic plasticity and learning. NCS-1 interacts with and regulates dopamine D2 receptor (D2R) internalization and is implicated in disorders like schizophrenia and substance abuse. However, the role of NCS-1 in behaviors dependent on dopamine signaling in the striatum, where D2R is most highly expressed, is unknown. We show that Ncs-1 deletion in the mouse decreases willingness to work for food. Moreover, Ncs-1 knockout mice have significantly lower activity-dependent dopamine release in the nucleus accumbens core in acute slice recordings. In contrast, food preference, responding for conditioned reinforcement, ability to represent changes in reward value, and locomotor response to amphetamine are not impaired. These studies identify novel roles for NCS-1 in regulating activity-dependent striatal dopamine release and aspects of motivated behavior.

Parkinson’s disease (PD) is a neurodegenerative disorder that has been shown to be influenced by the intestinal milieu. The gut microbiota is altered in PD patients, and murine studies have begun suggesting a causative role for the gut microbiota in progression of PD. We have previously shown that repeated infection with the intestinal murine pathogen Citrobacter rodentium resulted in the development of PD-like pathology in Pink1−/- mice compared to wild-type littermates. This addendum aims to expand this work by characterizing the gut microbiota during C. rodentium infection in our Pink1−/- PD model. We observed little disturbance to the fecal microbiota diversity both between infection timepoints and between Pink1−/- and wild-type control littermates. However, the level of short-chain fatty acids appeared to be altered over the course of infection with butyric acid significantly increasing in Pink1−/- mice and isobutyric acid increasing in wild-type mice.

Plasmonic nanostructures have found increasing utility due to the increased popularity that surface-enhanced Raman scattering (SERS) has achieved in recent years. SERS has been incorporated into an ever-growing list of applications, with bioanalytical and physiological analyses having emerged as two of the most popular. Thus far, the transition from SERS studies of cultured cells to SERS studies involving tissue has been gradual and limited. In most cases, SERS measurements in more intact tissue have involved nanoparticles distributed throughout the tissue or localized to specific regions via external functionalization. Performing highly localized measurements without the need for global nanoparticle uptake or specialized surface modifications would be advantageous to the expansion of SERS measurements in tissue. To this end, this work provides critical insight with supporting experimental evidence into performing SERS measurements with nanosensors inserted in tissues. We address two critical steps that are otherwise underappreciated when other approaches to performing SERS measurements in tissue are used. Specifically, we demonstrate two mechanical routes for controlled positioning and inserting the nanosensors into the tissue, and we discuss two means of focusing on the nanosensors both before and after they are inserted into the tissue. By examining the various combinations of these steps, we provide a blueprint for performing SERS measurements with nanosensors inserted in tissue. This blueprint could prove useful for the general development of SERS as a tool for bioanalytical and physiological studies and for more specialized techniques such as SERS-optophysiology.

Histamine H-3 receptors are widely distributed Gi-coupled receptors whose activation reduces neuronal activity and inhibits release of numerous neurotransmitters. Although these receptors are abundantly expressed in the striatum, their modulatory role on activity-dependent dopamine release is not well understood. Here, we observed that histamine H-3 receptor activation indirectly diminishes dopamine overflow in the ventral striatum by reducing cholinergic interneuron activity. Acute brain slices from C57BL/6 or channelrhodopsin-2-transfected DAT-cre mice were obtained, and dopamine transients evoked either electrically or optogenetically were measured by fast-scan cyclic voltammetry. The H-3 agonist alpha-methylhistamine significantly reduced electrically-evoked dopamine overflow, an effect blocked by the nicotinic acetylcholine receptor antagonist dihydro-beta-erythroidine, suggesting involvement of cholinergic interneurons. None of the drug treatments targeting H-3 receptors affected optogenetically evoked dopamine overflow, indicating that direct H-3-modulation of dopaminergic axons is unlikely. Next, we used qPCR and confirmed the expression of histamine H-3 receptor mRNA in cholinergic interneurons, both in ventral and dorsal striatum. Activation of H-3 receptors by alpha-methylhistamine reduced spontaneous firing of cholinergic interneurons in the ventral, but not in the dorsal striatum. Resting membrane potential and number of spontaneous action potentials in ventral-striatal cholinergic interneurons were significantly reduced by alpha-methylhistamine. Acetylcholine release from isolated striatal synaptosomes, however, was not altered by alpha-methylhistamine. Together, these results indicate that histamine H-3 receptors are important modulators of dopamine release, specifically in the ventral striatum, and that they do so by decreasing the firing rate of cholinergic neurons and, consequently, reducing cholinergic tone on dopaminergic axons. (C) 2018 IBRO. Published by Elsevier Ltd. All rights reserved.

Parkinson's disease is a neurodegenerative disorder with motor symptoms linked to the loss of dopaminergic neurons in the substantia nigra compacta. Although the mechanisms that trigger the loss of dopaminergic neurons are unclear, mitochondrial dysfunction and inflammation are thought to have key roles

Summary Although the mechanisms underlying the loss of neurons in Parkinson's disease are not well understood, impaired mitochondrial function and pathological protein aggregation are suspected as playing a major role. Why DA (dopamine) neurons and a select small subset of brain nuclei are particularly vulnerable to such ubiquitous cellular dysfunctions is presently one of the key unanswered questions in Parkinson's disease research. One intriguing hypothesis is that their heightened vulnerability is a consequence of their elevated bioenergetic requirements. Here, we show for the first time that vulnerable nigral DA neurons differ from less vulnerable DA neurons such as those of the VTA (ventral tegmental area) by having a higher basal rate of mitochondrial OXPHOS (oxidative phosphorylation), a smaller reserve capacity, a higher density of axonal mitochondria, an elevated level of basal oxidative stress, and a considerably more complex axonal arborization. Furthermore, we demonstrate that reducing axonal arborization by acting on axon guidance pathways with Semaphorin 7A reduces in parallel the basal rate of mitochondrial OXPHOS and the vulnerability of nigral DA neurons to the neurotoxic agents MPP + (1-methyl-4-phenylpyridinium) and rotenone. Blocking L-type calcium channels with isradipine was protective against MPP + but not rotenone. Our data provide the most direct demonstration to date in favor of the hypothesis that the heightened vulnerability of nigral DA neurons in Parkinson's disease is directly due to their particular bioenergetic and morphological characteristics.