ALCAM on human oligodendrocytes mediates CD4 T cell adhesion.

Multiple sclerosis is a chronic neuroinflammatory disorder characterized by demyelination, oligodendrocyte damage/loss and neuroaxonal injury in the context of immune cell infiltration in the CNS. No neuroprotective therapy is available to promote the survival of oligodendrocytes and protect their myelin processes in immune-mediated demyelinating diseases. Pro-inflammatory CD4 Th17 cells can interact with oligodendrocytes in multiple sclerosis and its animal model, causing injury to myelinating processes and cell death through direct contact. However, the molecular mechanisms underlying the close contact and subsequent detrimental interaction of Th17 cells with oligodendrocytes remain unclear. In this study we used single cell RNA sequencing, flow cytometry and immunofluorescence studies on CNS tissue from multiple sclerosis subjects, its animal model and controls to characterize the expression of cell adhesion molecules by mature oligodendrocytes. We found that a significant proportion of human and murine mature oligodendrocytes express melanoma cell adhesion molecule (MCAM) and activated leukocyte cell adhesion molecule (ALCAM) in multiple sclerosis, in experimental autoimmune encephalomyelitis and in controls, although their regulation differs between human and mouse. We observed that exposure to pro-inflammatory cytokines or to human activated T cells are associated with a marked downregulation of the expression of MCAM but not of ALCAM at the surface of human primary oligodendrocytes. Furthermore, we used in vitro live imaging, immunofluorescence and flow cytometry to determine the contribution of these molecules to Th17-polarized cell adhesion and cytotoxicity towards human oligodendrocytes. Silencing and blocking ALCAM but not MCAM limited prolonged interactions between human primary oligodendrocytes and Th17-polarized cells, resulting in decreased adhesion of Th17-polarized cells to oligodendrocytes and conferring significant protection of oligodendrocytic processes. In conclusion, we showed that human oligodendrocytes express MCAM and ALCAM, which are differently modulated by inflammation and T cell contact. We found that ALCAM is a ligand for Th17-polarized cells, contributing to their capacity to adhere and induce damage to human oligodendrocytes, and therefore could represent a relevant target for neuroprotection in multiple sclerosis.

Pro-inflammatory T helper 17 directly harms oligodendrocytes in neuroinflammation.

T helper (Th)17 cells are considered to contribute to inflammatory mechanisms in diseases such as multiple sclerosis (MS). However, the discussion persists regarding their true role in patients. Here, we visualized central nervous system (CNS) inflammatory processes in models of MS live in vivo and in MS brains and discovered that CNS-infiltrating Th17 cells form prolonged stable contact with oligodendrocytes. Strikingly, compared to Th2 cells, direct contact with Th17 worsened experimental demyelination, caused damage to human oligodendrocyte processes, and increased cell death. Importantly, we found that in comparison to Th2 cells, both human and murine Th17 cells express higher levels of the integrin CD29, which is linked to glutamate release pathways. Of note, contact of human Th17 cells with oligodendrocytes triggered release of glutamate, which induced cell stress and changes in biosynthesis of cholesterol and lipids, as revealed by single-cell RNA-sequencing analysis. Finally, exposure to glutamate decreased myelination, whereas blockade of CD29 preserved oligodendrocyte processes from Th17-mediated injury. Our data provide evidence for the direct and deleterious attack of Th17 cells on the myelin compartment and show the potential for therapeutic opportunities in MS.

Analysis of the microglia transcriptome across the human lifespan using single cell RNA sequencing.

BACKGROUND: Microglia are tissue resident macrophages with a wide range of critically important functions in central nervous system development and homeostasis. METHOD: In this study, we aimed to characterize the transcriptional landscape of ex vivo human microglia across different developmental ages using cells derived from pre-natal, pediatric, adolescent, and adult brain samples. We further confirmed our transcriptional observations using ELISA and RNAscope. RESULTS: We showed that pre-natal microglia have a distinct transcriptional and regulatory signature relative to their post-natal counterparts that includes an upregulation of phagocytic pathways. We confirmed upregulation of CD36, a positive regulator of phagocytosis, in pre-natal samples compared to adult samples in situ. Moreover, we showed adult microglia have more pro-inflammatory signature compared to microglia from other developmental ages. We indicated that adult microglia are more immune responsive by secreting increased levels of pro-inflammatory cytokines in response to LPS treatment compared to the pre-natal microglia. We further validated in situ up-regulation of IL18 and CXCR4 in human adult brain section compared to the pre-natal brain section. Finally, trajectory analysis indicated that the transcriptional signatures adopted by microglia throughout development are in response to a changing brain microenvironment and do not reflect predetermined developmental states. CONCLUSION: In all, this study provides unique insight into the development of human microglia and a useful reference for understanding microglial contribution to developmental and age-related human disease.

Human Oligodendrocyte Myelination Potential; Relation to Age and Differentiation.

OBJECTIVE: Myelin regeneration in the human central nervous system relies on progenitor cells within the tissue parenchyma, with possible contribution from previously myelinating oligodendrocytes (OLs). In multiple sclerosis, a demyelinating disorder, variables affecting remyelination efficiency include age, severity of initial injury, and progenitor cell properties. Our aim was to investigate the effects of age and differentiation on the myelination potential of human OL lineage cells. METHODS: We derived viable primary OL lineage cells from surgical resections of pediatric and adult brain tissue. Ensheathment capacity using nanofiber assays and transcriptomic profiles from RNA sequencing were compared between A2B5+ antibody-selected progenitors and mature OLs (non-selected cells). RESULTS: We demonstrate that pediatric progenitor and mature cells ensheathed nanofibers more robustly than did adult progenitor and mature cells, respectively. Within both age groups, the percentage of fibers ensheathed and ensheathment length per fiber were greater for A2B5+ progenitors. Gene expression of OL progenitor markers PDGFRA and PTPRZ1 were higher in A2B5+ versus A2B5- cells and in pediatric A2B5+ versus adult A2B5+ cells. The p38 MAP kinases and actin cytoskeleton-associated pathways were upregulated in pediatric cells; both have been shown to regulate OL process outgrowth. Significant upregulation of "cell senescence" genes was detected in pediatric samples; this could reflect their role in development and the increased susceptibility of pediatric OLs to activating cell death responses to stress. INTERPRETATION: Our findings identify specific biological pathways relevant to myelination that are differentially enriched in human pediatric and adult OL lineage cells and suggest potential targets for remyelination enhancing therapies. ANN NEUROL 2022;91:178-191.

Diverse injury responses of human oligodendrocyte to mediators implicated in multiple sclerosis.

Early multiple sclerosis lesions feature relative preservation of oligodendrocyte cell bodies with dying back retraction of their myelinating processes. Cell loss occurs with disease progression. Putative injury mediators include metabolic stress (low glucose/nutrient), pro-inflammatory mediators (interferon γ and tumour necrosis factor α), and excitotoxins (glutamate). Our objective was to compare the impact of these disease relevant mediators on the injury responses of human mature oligodendrocytes. In the current study, we determined the effects of these mediators on process extension and survival of human brain derived mature oligodendrocytes in vitro and used bulk RNA sequencing to identify distinct effector mechanisms that underlie the responses. All mediators induced significant process retraction of the oligodendrocytes in dissociated cell culture. Only metabolic stress (low glucose/nutrient) conditions resulted in delayed (4-6 days) non-apoptotic cell death. Metabolic effects were associated with induction of the integrated stress response, which can be protective or contribute to cell injury dependent on its level and duration of activation. Addition of Sephin1, an agonist of the integrated stress response induced process retraction under control conditions and further enhanced retraction under metabolic stress conditions. The antagonist ISRIB restored process outgrowth under stress conditions, and if added to already stressed cells, reduced delayed cell death and prolonged the period in which recovery could occur. Inflammatory cytokine functional effects were associated with activation of multiple signalling pathways (including Jak/Stat-1) that regulate process outgrowth, without integrated stress response induction. Glutamate application produced limited transcriptional changes suggesting a contribution of effects directly on cell processes. Our comparative studies indicate the need to consider both the specific injury mediators and the distinct cellular mechanisms of responses to them by human oligodendrocytes to identify effective neuroprotective therapies for multiple sclerosis.

Striatal Direct Pathway Targets Npas1+ Pallidal Neurons.

The classic basal ganglia circuit model asserts a complete segregation of the two striatal output pathways. Empirical data argue that, in addition to indirect-pathway striatal projection neurons (iSPNs), direct-pathway striatal projection neurons (dSPNs) innervate the external globus pallidus (GPe). However, the functions of the latter were not known. In this study, we interrogated the organization principles of striatopallidal projections and their roles in full-body movement in mice (both males and females). In contrast to the canonical motor-promoting response of dSPNs in the dorsomedial striatum (DMSdSPNs), optogenetic stimulation of dSPNs in the dorsolateral striatum (DLSdSPNs) suppressed locomotion. Circuit analyses revealed that dSPNs selectively target Npas1+ neurons in the GPe. In a chronic 6-hydroxydopamine lesion model of Parkinson's disease, the dSPN-Npas1+ projection was dramatically strengthened. As DLSdSPN-Npas1+ projection suppresses movement, the enhancement of this projection represents a circuit mechanism for the hypokinetic symptoms of Parkinson's disease that has not been previously considered. In sum, our results suggest that dSPN input to the GPe is a critical circuit component that is involved in the regulation of movement in both healthy and parkinsonian states.SIGNIFICANCE STATEMENT In the classic basal ganglia model, the striatum is described as a divergent structure: it controls motor and adaptive functions through two segregated, opposing output streams. However, the experimental results that show the projection from direct-pathway neurons to the external pallidum have been largely ignored. Here, we showed that this striatopallidal subpathway targets a select subset of neurons in the external pallidum and is motor-suppressing. We found that this subpathway undergoes changes in a Parkinson's disease model. In particular, our results suggest that the increase in strength of this subpathway contributes to the slowness or reduced movements observed in Parkinson's disease.

Dissociable Roles of Pallidal Neuron Subtypes in Regulating Motor Patterns.

We have previously established that PV+ neurons and Npas1+ neurons are distinct neuron classes in the external globus pallidus (GPe): they have different topographical, electrophysiological, circuit, and functional properties. Aside from Foxp2+ neurons, which are a unique subclass within the Npas1+ class, we lack driver lines that effectively capture other GPe neuron subclasses. In this study, we examined the utility of Kcng4-Cre, Npr3-Cre, and Npy2r-Cre mouse lines (both males and females) for the delineation of GPe neuron subtypes. By using these novel driver lines, we have provided the most exhaustive investigation of electrophysiological studies of GPe neuron subtypes to date. Corroborating our prior studies, GPe neurons can be divided into two statistically distinct clusters that map onto PV+ and Npas1+ classes. By combining optogenetics and machine learning-based tracking, we showed that optogenetic perturbation of GPe neuron subtypes generated unique behavioral structures. Our findings further highlighted the dissociable roles of GPe neurons in regulating movement and anxiety-like behavior. We concluded that Npr3+ neurons and Kcng4+ neurons are distinct subclasses of Npas1+ neurons and PV+ neurons, respectively. Finally, by examining local collateral connectivity, we inferred the circuit mechanisms involved in the motor patterns observed with optogenetic perturbations. In summary, by identifying mouse lines that allow for manipulations of GPe neuron subtypes, we created new opportunities for interrogations of cellular and circuit substrates that can be important for motor function and dysfunction.SIGNIFICANCE STATEMENT Within the basal ganglia, the external globus pallidus (GPe) has long been recognized for its involvement in motor control. However, we lacked an understanding of precisely how movement is controlled at the GPe level as a result of its cellular complexity. In this study, by using transgenic and cell-specific approaches, we showed that genetically-defined GPe neuron subtypes have distinct roles in regulating motor patterns. In addition, the in vivo contributions of these neuron subtypes are in part shaped by the local, inhibitory connections within the GPe. In sum, we have established the foundation for future investigations of motor function and disease pathophysiology.

Regional and age-related diversity of human mature oligodendrocytes.

Morphological and emerging molecular studies have provided evidence for heterogeneity within the oligodendrocyte population. To address the regional and age-related heterogeneity of human mature oligodendrocytes (MOLs) we applied single-cell RNA sequencing to cells isolated from cortical/subcortical, subventricular zone brain tissue samples, and thoracolumbar spinal cord samples. Unsupervised clustering of cells identified transcriptionally distinct MOL subpopulations across regions. Spinal cord MOLs, but not microglia, exhibited cell-type-specific upregulation of immune-related markers compared to the other adult regions. SVZ MOLs showed an upregulation of select number of development-linked transcription factors compared to other regions; however, pseudotime trajectory analyses did not identify a global developmental difference. Age-related analysis of cortical/subcortical samples indicated that pediatric MOLs, especially from under age 5, retain higher expression of genes linked to development and to immune activity with pseudotime analysis favoring a distinct developmental stage. Our regional and age-related studies indicate heterogeneity of MOL populations in the human CNS that may reflect developmental and environmental influences.

Parvalbumin+ and Npas1+ Pallidal Neurons Have Distinct Circuit Topology and Function.

The external globus pallidus (GPe) is a critical node within the basal ganglia circuit. Phasic changes in the activity of GPe neurons during movement and their alterations in Parkinson's disease (PD) argue that the GPe is important in motor control. Parvalbumin-positive (PV+) neurons and Npas1+ neurons are the two principal neuron classes in the GPe. The distinct electrophysiological properties and axonal projection patterns argue that these two neuron classes serve different roles in regulating motor output. However, the causal relationship between GPe neuron classes and movement remains to be established. Here, by using optogenetic approaches in mice (both males and females), we showed that PV+ neurons and Npas1+ neurons promoted and suppressed locomotion, respectively. Moreover, PV+ neurons and Npas1+ neurons are under different synaptic influences from the subthalamic nucleus (STN). Additionally, we found a selective weakening of STN inputs to PV+ neurons in the chronic 6-hydroxydopamine lesion model of PD. This finding reinforces the idea that the reciprocally connected GPe-STN network plays a key role in disease symptomatology and thus provides the basis for future circuit-based therapies.SIGNIFICANCE STATEMENT The external pallidum is a key, yet an understudied component of the basal ganglia. Neural activity in the pallidum goes awry in neurologic diseases, such as Parkinson's disease. While this strongly argues that the pallidum plays a critical role in motor control, it has been difficult to establish the causal relationship between pallidal activity and motor function/dysfunction. This was in part because of the cellular complexity of the pallidum. Here, we showed that the two principal neuron types in the pallidum have opposing roles in motor control. In addition, we described the differences in their synaptic influence. Importantly, our research provides new insights into the cellular and circuit mechanisms that explain the hypokinetic features of Parkinson's disease.

Npas1+-Nkx2.1+ Neurons Are an Integral Part of the Cortico-pallido-cortical Loop.

Within the basal ganglia circuit, the external globus pallidus (GPe) is critically involved in motor control. Aside from Foxp2+ neurons and ChAT+ neurons that have been established as unique neuron types, there is little consensus on the classification of GPe neurons. Properties of the remaining neuron types are poorly defined. In this study, we leverage new mouse lines, viral tools, and molecular markers to better define GPe neuron subtypes. We found that Sox6 represents a novel, defining marker for GPe neuron subtypes. Lhx6+ neurons that lack the expression of Sox6 were devoid of both parvalbumin and Npas1. This result confirms previous assertions of the existence of a unique Lhx6+ population. Neurons that arise from the Dbx1+ lineage were similarly abundant in the GPe and displayed a heterogeneous makeup. Importantly, tracing experiments revealed that Npas1+-Nkx2.1+ neurons represent the principal noncholinergic, cortically-projecting neurons. In other words, they form the pallido-cortical arm of the cortico-pallido-cortical loop. Our data further show that pyramidal-tract neurons in the cortex collateralized within the GPe, forming a closed-loop system between the two brain structures. Overall, our findings reconcile some of the discrepancies that arose from differences in techniques or the reliance on preexisting tools. Although spatial distribution and electrophysiological properties of GPe neurons reaffirm the diversification of GPe subtypes, statistical analyses strongly support the notion that these neuron subtypes can be categorized under the two principal neuron classes: PV+ neurons and Npas1+ neurons.SIGNIFICANCE STATEMENT The poor understanding of the neuronal composition in the external globus pallidus (GPe) undermines our ability to interrogate its precise behavioral and disease involvements. In this study, 12 different genetic crosses were used, hundreds of neurons were electrophysiologically characterized, and >100,000 neurons were histologically- and/or anatomically-profiled. Our current study further establishes the segregation of GPe neuron classes and illustrates the complexity of GPe neurons in adult mice. Our results support the idea that Npas1+-Nkx2.1+ neurons are a distinct GPe neuron subclass. By providing a detailed analysis of the organization of the cortico-pallidal-cortical projection, our findings establish the cellular and circuit substrates that can be important for motor function and dysfunction.

Dopamine receptors mediate strategy abandoning via modulation of a specific prelimbic cortex-nucleus accumbens pathway in mice.

The ability to abandon old strategies and adopt new ones is essential for survival in a constantly changing environment. While previous studies suggest the importance of the prefrontal cortex and some subcortical areas in the generation of strategy-switching flexibility, the fine neural circuitry and receptor mechanisms involved are not fully understood. In this study, we showed that optogenetic excitation and inhibition of the prelimbic cortex-nucleus accumbens (NAc) pathway in the mouse respectively enhances and suppresses strategy-switching ability in a cross-modal spatial-egocentric task. This ability is dependent on an intact dopaminergic tone in the NAc, as local dopamine denervation impaired the performance of the animal in the switching of tasks. In addition, based on a brain-slice preparation obtained from Drd2-EGFP BAC transgenic mice, we demonstrated direct innervation of D2 receptor-expressing medium spiny neurons (D2-MSNs) in the NAc by prelimbic cortical neurons, which is under the regulation by presynaptic dopamine receptors. While presynaptic D1-type receptor activation enhances the glutamatergic transmission from the prelimbic cortex to D2-MSNs, D2-type receptor activation suppresses this synaptic connection. Furthermore, manipulation of this pathway by optogenetic activation or administration of a D1-type agonist or a D2-type antagonist could restore impaired task-switching flexibility in mice with local NAc dopamine depletion; this restoration is consistent with the effects of knocking down the expression of specific dopamine receptors in the pathway. Our results point to a critical role of a specific prelimbic cortex-NAc subpathway in mediating strategy abandoning, allowing the switching from one strategy to another in problem solving.

Developmental trajectory of oligodendrocyte progenitor cells in the human brain revealed by single cell RNA sequencing.

Characterizing the developmental trajectory of oligodendrocyte progenitor cells (OPC) is of great interest given the importance of these cells in the remyelination process. However, studies of human OPC development remain limited by the availability of whole cell samples and material that encompasses a wide age range, including time of peak myelination. In this study, we apply single cell RNA sequencing to viable whole cells across the age span and link transcriptomic signatures of oligodendrocyte-lineage cells with stage-specific functional properties. Cells were isolated from surgical tissue samples of second-trimester fetal, 2-year-old pediatric, 13-year-old adolescent, and adult donors by mechanical and enzymatic digestion, followed by percoll gradient centrifugation. Gene expression was analyzed using droplet-based RNA sequencing (10X Chromium). Louvain clustering analysis identified three distinct cellular subpopulations based on 5,613 genes, comprised of an early OPC (e-OPC) group, a late OPC group (l-OPC), and a mature OL (MOL) group. Gene ontology terms enriched for e-OPCs included cell cycle and development, for l-OPCs included extracellular matrix and cell adhesion, and for MOLs included myelination and cytoskeleton. The e-OPCs were mostly confined to the premyelinating fetal group, and the l-OPCs were most highly represented in the pediatric age group, corresponding to the peak age of myelination. Cells expressing a signature characteristic of l-OPCs were identified in the adult brain in situ using RNAScope. These findings highlight the transcriptomic variability in OL-lineage cells before, during, and after peak myelination and contribute to identifying novel pathways required to achieve remyelination.

Rapid and efficient generation of oligodendrocytes from human induced pluripotent stem cells using transcription factors.

Rapid and efficient protocols to generate oligodendrocytes (OL) from human induced pluripotent stem cells (iPSC) are currently lacking, but may be a key technology to understand the biology of myelin diseases and to develop treatments for such disorders. Here, we demonstrate that the induction of three transcription factors (SOX10, OLIG2, NKX6.2) in iPSC-derived neural progenitor cells is sufficient to rapidly generate O4+ OL with an efficiency of up to 70% in 28 d and a global gene-expression profile comparable to primary human OL. We further demonstrate that iPSC-derived OL disperse and myelinate the CNS of Mbpshi/shiRag-/- mice during development and after demyelination, are suitable for in vitro myelination assays, disease modeling, and screening of pharmacological compounds potentially promoting oligodendroglial differentiation. Thus, the strategy presented here to generate OL from iPSC may facilitate the studying of human myelin diseases and the development of high-throughput screening platforms for drug discovery.

Helper CD4 T cells expressing granzyme B cause glial fibrillary acidic protein fragmentation in astrocytes in an MHCII-independent manner.

During inflammatory processes of the central nervous system, helper T cells have the capacity to cross the blood-brain barrier and injure or kill neural cells through cytotoxic mechanisms. Glial fibrillary acidic protein (GFAP) is an intermediate filament protein that is part of the astrocyte cytoskeleton that can become fragmented in neuroinflammatory conditions. The mechanism of action by which helper T cells with cytotoxic properties injure astrocytes is not completely understood. Primary human astrocytes were obtained from fetal brain tissue. Human helper (CD4+ ) T cells were isolated from peripheral blood mononuclear cells and activated with the superantigen staphylococcal enterotoxin E (SEE). Granzyme B was detected by enzyme linked immunosorbent assay and intracellular flow cytometry. GFAP fragmentation was monitored by western blotting. Cell death was monitored by lactic acid dehydrogenase release and terminal biotin-dUTP nick labeling (TUNEL). Astrocyte migration was monitored by scratch assay. Adult human oligodendrocytes were cultured with sublethally injured astrocytes to determine support function. Helper T cells activated with SEE expressed granzyme B but not perforin. Helper T cells released granzyme B upon contact with astrocytes and caused GFAP fragmentation in a caspase-dependent, MHCII-independent manner. Sublethally injured astrocytes were not apoptotic; however, their processes were thin and elongated, their migration was attenuated, and their ability to support oligodendrocytes was reduced in vitro. Helper T cells can release granzyme B causing sublethal injury to astrocytes, which compromises the supportive functions of astrocytes. Blocking these pathways may lead to improved resolution of neuroinflammatory lesions.

Sublethal oligodendrocyte injury: A reversible condition in multiple sclerosis?

OBJECTIVE: Degeneration of oligodendroglial distal processes has been identified as an early event in multiple sclerosis (MS) lesion development. Our objective was to further define the development of the "dying-back" oligodendrocyte lesion in situ and to model the development and potential reversibility of such responses using dissociated cultures of adult human brain-derived oligodendrocytes. METHODS: In situ analyses were performed on glutaraldehyde-fixed thin sections of clinically acute and pathologically active cases of MS. In vitro studies were conducted using adult human brain-derived oligodendrocytes challenged by metabolic stress conditions (low nutrient/glucose). RESULTS: In situ analyses indicated a spectrum of myelin changes in the presence of morphologically intact oligodendrocytes; these included degeneration of the inner cytoplasmic tongue with increasing sizes of intramyelinic bleb formation that could result in radial fractures of the myelin sheath. Macrophages with ingested myelin fragments were identified only once the fragmentation was established. In vitro studies indicated that oligodendrocyte process retraction, which was linked to reduced glycolytic respiratory activity, is reversible until a critical time point. Subsequent cell death was not linked to caspase-3-dependent programs. Gene expression studies conducted at the latest reversible time point revealed reduced expression of pathways associated with cell process outgrowth and myelination, as well as with metabolic activity. INTERPRETATION: Our findings reveal the potential to protect and possibly restore myelin elaborated by existent oligodendrocytes in early and evolving MS lesions, and suggest the necessity of ongoing studies of the mechanisms underlying subsequent adult human oligodendrocyte cell death. Ann Neurol 2017;81:811-824.

Oligodendrogliopathy in Multiple Sclerosis: Low Glycolytic Metabolic Rate Promotes Oligodendrocyte Survival.

UNLABELLED: Multiple sclerosis (MS) lesions feature demyelination with limited remyelination. A distinct injury phenotype of MS lesions features dying back of oligodendrocyte (OL) terminal processes, a response that destabilizes myelin/axon interactions. This oligodendrogliopathy has been linked with local metabolic stress, similar to the penumbra of ischemic/hypoxic states. Here, we developed an in vitro oligodendrogliopathy model using human CNS-derived OLs and related this injury response to their distinct bioenergetic properties. We determined the energy utilization properties of adult human surgically derived OLs cultured under either optimal or metabolic stress conditions, deprivation of growth factors, and glucose and/or hypoxia using a Seahorse extracellular flux analyzer. Baseline studies were also performed on OL progenitor cells derived from the same tissue and postnatal rat-derived cells. Under basal conditions, adult human OLs were less metabolically active than their progenitors and both were less active than the rat cells. Human OLs and progenitors both used aerobic glycolysis for the majority of ATP production, a process that contributes to protein and lipid production necessary for myelin biosynthesis. Under stress conditions that induce significant process retraction with only marginal cell death, human OLs exhibited a significant reduction in overall energy utilization, particularly in glycolytic ATP production. The stress-induced reduction of glycolytic ATP production by the human OLs would exacerbate myelin process withdrawal while favoring cell survival, providing a potential basis for the oligodendrogliopathy observed in MS. The glycolytic pathway is a potential therapeutic target to promote myelin maintenance and enhance repair in MS. SIGNIFICANCE STATEMENT: The neurologic deficits that characterize multiple sclerosis (MS) reflect disruption of myelin (demyelination) within the CNS and failure of repair (remyelination). We define distinct energy utilization properties of human adult brain-derived oligodendrocytes and oligodendrocyte progenitor cells under conditions of metabolic stress that model the initial relapsing and subsequent progressive phases of MS. The observed changes in energy utilization affect both cell survival and myelination capacity. These processes may be amenable to therapeutic interventions to limit the extent of cumulative tissue injury and to promote repair in MS.

Npas1+ Pallidal Neurons Target Striatal Projection Neurons.

UNLABELLED: Compelling evidence demonstrates that the external globus pallidus (GPe) plays a key role in processing sensorimotor information. An anatomical projection from the GPe to the dorsal striatum has been described for decades. However, the cellular target and functional impact of this projection remain unknown. Using cell-specific transgenic mice, modern monosynaptic tracing techniques, and optogenetics-based mapping, we discovered that GPe neurons provide inhibitory inputs to direct and indirect pathway striatal projection neurons (SPNs). Our results indicate that the GPe input to SPNs arises primarily from Npas1-expressing neurons and is strengthened in a chronic Parkinson's disease (PD) model. Alterations of the GPe-SPN input in a PD model argue for the critical position of this connection in regulating basal ganglia motor output and PD symptomatology. Finally, chemogenetic activation of Npas1-expressing GPe neurons suppresses motor output, arguing that strengthening of the GPe-SPN connection is maladaptive and may underlie the hypokinetic symptoms in PD. SIGNIFICANCE STATEMENT: An anatomical projection from the pallidum to the striatum has been described for decades, but little is known about its connectivity pattern. The authors dissect the presynaptic and postsynaptic neurons involved in this projection, and show its cell-specific remodeling and strengthening in parkinsonian mice. Chemogenetic activation of Npas1(+) pallidal neurons that give rise to the principal pallidostriatal projection increases the time that the mice spend motionless. This argues that maladaptive strengthening of this connection underlies the paucity of volitional movements, which is a hallmark of Parkinson's disease.

Direct and indirect effects of immune and central nervous system-resident cells on human oligodendrocyte progenitor cell differentiation.

In multiple sclerosis, successful remyelination within the injured CNS is largely dependent on the survival and differentiation of oligodendrocyte progenitor cells. During inflammatory injury, oligodendrocytes and oligodendrocyte progenitor cells within lesion sites are exposed to secreted products derived from both infiltrating immune cell subsets and CNS-resident cells. Such products may be considered either proinflammatory or anti-inflammatory and have the potential to contribute to both injury and repair processes. Within the CNS, astrocytes also contribute significantly to oligodendrocyte biology during development and following inflammatory injury. The overall objective of the current study was to determine how functionally distinct proinflammatory and anti-inflammatory human immune cell subsets, implicated in multiple sclerosis, can directly and/or indirectly (via astrocytes) impact human oligodendrocyte progenitor cell survival and differentiation. Proinflammatory T cell (Th1/Th17) and M1-polarized myeloid cell supernatants had a direct cytotoxic effect on human A2B5(+) neural progenitors, resulting in decreased O4(+) and GalC(+) oligodendrocyte lineage cells. Astrocyte-conditioned media collected from astrocytes pre-exposed to the same proinflammatory supernatants also resulted in decreased oligodendrocyte progenitor cell differentiation without an apparent increase in cell death and was mediated through astrocyte-derived CXCL10, yet this decrease in differentiation was not observed in the more differentiated oligodendrocytes. Th2 and M2 macrophage or microglia supernatants had neither a direct nor an indirect impact on oligodendrocyte progenitor cell differentiation. We conclude that proinflammatory immune cell responses can directly and indirectly (through astrocytes) impact the fate of immature oligodendrocyte-lineage cells, with oligodendrocyte progenitor cells more vulnerable to injury compared with mature oligodendrocytes.

Parvalbumin+ Neurons and Npas1+ Neurons Are Distinct Neuron Classes in the Mouse External Globus Pallidus.

Compelling evidence suggests that pathological activity of the external globus pallidus (GPe), a nucleus in the basal ganglia, contributes to the motor symptoms of a variety of movement disorders such as Parkinson's disease. Recent studies have challenged the idea that the GPe comprises a single, homogenous population of neurons that serves as a simple relay in the indirect pathway. However, we still lack a full understanding of the diversity of the neurons that make up the GPe. Specifically, a more precise classification scheme is needed to better describe the fundamental biology and function of different GPe neuron classes. To this end, we generated a novel multicistronic BAC (bacterial artificial chromosome) transgenic mouse line under the regulatory elements of the Npas1 gene. Using a combinatorial transgenic and immunohistochemical approach, we discovered that parvalbumin-expressing neurons and Npas1-expressing neurons in the GPe represent two nonoverlapping cell classes, amounting to 55% and 27% of the total GPe neuron population, respectively. These two genetically identified cell classes projected primarily to the subthalamic nucleus and to the striatum, respectively. Additionally, parvalbumin-expressing neurons and Npas1-expressing neurons were distinct in their autonomous and driven firing characteristics, their expression of intrinsic ion conductances, and their responsiveness to chronic 6-hydroxydopamine lesion. In summary, our data argue that parvalbumin-expressing neurons and Npas1-expressing neurons are two distinct functional classes of GPe neurons. This work revises our understanding of the GPe, and provides the foundation for future studies of its function and dysfunction. SIGNIFICANCE STATEMENT: Until recently, the heterogeneity of the constituent neurons within the external globus pallidus (GPe) was not fully appreciated. We addressed this knowledge gap by discovering two principal GPe neuron classes, which were identified by their nonoverlapping expression of the markers parvalbumin and Npas1. Our study provides evidence that parvalbumin and Npas1 neurons have different topologies within the basal ganglia.

Non-steroidal anti-inflammatory drug indometacin enhances endogenous remyelination.

Multiple sclerosis is the most frequent demyelinating disease in the CNS that is characterized by inflammatory demyelinating lesions and axonal loss, the morphological correlate of permanent clinical disability. Remyelination does occur, but is limited especially in chronic disease stages. Despite effective immunomodulatory therapies that reduce the number of relapses the progressive disease phase cannot be prevented. Therefore, promotion of neuroprotective and repair mechanisms, such as remyelination, represents an attractive additional treatment strategy. A number of pathways have been identified that may contribute to impaired remyelination in MS lesions, among them the Wnt/ß-catenin pathway. Here, we demonstrate that indometacin, a non-steroidal anti-inflammatory drug (NSAID) that has been also shown to modulate the Wnt/ß-catenin pathway in colorectal cancer cells promotes differentiation of primary human and murine oligodendrocytes, myelination of cerebellar slice cultures and remyelination in cuprizone-induced demyelination. Our in vitro experiments using GSK3ß inhibitors, luciferase reporter assays and oligodendrocytes expressing a mutant, dominant stable ß-catenin indicate that the mechanism of action of indometacin depends on GSK3ß activity and ß-catenin phosphorylation. Indometacin might represent a promising treatment option to enhance endogenous remyelination in MS patients.