Choroid plexus mis-splicing and altered cerebrospinal fluid composition in myotonic dystrophy type 1.

Myotonic dystrophy type 1 is a dominantly inherited multisystemic disease caused by CTG tandem repeat expansions in the DMPK 3' untranslated region. These expanded repeats are transcribed and produce toxic CUG RNAs that sequester and inhibit activities of the MBNL family of developmental RNA processing factors. Although myotonic dystrophy is classified as a muscular dystrophy, the brain is also severely affected by an unusual cohort of symptoms, including hypersomnia, executive dysfunction, as well as early onsets of tau/MAPT pathology and cerebral atrophy. To address the molecular and cellular events that lead to these pathological outcomes, we recently generated a mouse Dmpk CTG expansion knock-in model and identified choroid plexus epithelial cells as particularly affected by the expression of toxic CUG expansion RNAs. To determine if toxic CUG RNAs perturb choroid plexus functions, alternative splicing analysis was performed on lateral and hindbrain choroid plexi from Dmpk CTG knock-in mice. Choroid plexus transcriptome-wide changes were evaluated in Mbnl2 knockout mice, a developmental-onset model of myotonic dystrophy brain dysfunction. To determine if transcriptome changes also occurred in the human disease, we obtained post-mortem choroid plexus for RNA-seq from neurologically unaffected (two females, three males; ages 50-70 years) and myotonic dystrophy type 1 (one female, three males; ages 50-70 years) donors. To test that choroid plexus transcriptome alterations resulted in altered CSF composition, we obtained CSF via lumbar puncture from patients with myotonic dystrophy type 1 (five females, five males; ages 35-55 years) and non-myotonic dystrophy patients (three females, four males; ages 26-51 years), and western blot and osmolarity analyses were used to test CSF alterations predicted by choroid plexus transcriptome analysis. We determined that CUG RNA induced toxicity was more robust in the lateral choroid plexus of Dmpk CTG knock-in mice due to comparatively higher Dmpk and lower Mbnl RNA levels. Impaired transitions to adult splicing patterns during choroid plexus development were identified in Mbnl2 knockout mice, including mis-splicing previously found in Dmpk CTG knock-in mice. Whole transcriptome analysis of myotonic dystrophy type 1 choroid plexus revealed disease-associated RNA expression and mis-splicing events. Based on these RNA changes, predicted alterations in ion homeostasis, secretory output and CSF composition were confirmed by analysis of myotonic dystrophy type 1 CSF. Our results implicate choroid plexus spliceopathy and concomitant alterations in CSF homeostasis as an unappreciated contributor to myotonic dystrophy type 1 CNS pathogenesis.

The complex role of inflammation and gliotransmitters in Parkinson's disease.

Our understanding of the role of innate and adaptive immune cell function in brain health and how it goes awry during aging and neurodegenerative diseases is still in its infancy. Inflammation and immunological dysfunction are common components of Parkinson's disease (PD), both in terms of motor and non-motor components of PD. In recent decades, the antiquated notion that the central nervous system (CNS) in disease states is an immune-privileged organ, has been debunked. The immune landscape in the CNS influences peripheral systems, and peripheral immunological changes can alter the CNS in health and disease. Identifying immune and inflammatory pathways that compromise neuronal health and survival is critical in designing innovative and effective strategies to limit their untoward effects on neuronal health.

CCR2 inhibition reduces tumor myeloid cells and unmasks a checkpoint inhibitor effect to slow progression of resistant murine gliomas.

Immunotherapy directed at the PD-L1/PD-1 axis has produced treatment advances in various human cancers. Unfortunately, progress has not extended to glioblastoma (GBM), with phase III clinical trials assessing anti-PD-1 monotherapy failing to show efficacy in newly diagnosed and recurrent tumors. Myeloid-derived suppressor cells (MDSCs), a subset of immunosuppressive myeloid derived cells, are known to infiltrate the tumor microenvironment of GBM. Growing evidence suggests the CCL2-CCR2 axis is important for this process. This study evaluated the combination of PD-1 blockade and CCR2 inhibition in anti-PD-1-resistant gliomas. CCR2 deficiency unmasked an anti-PD-1 survival benefit in KR158 glioma-bearing mice. CD11b+/Ly6Chi/PD-L1+ MDSCs within established gliomas decreased with a concomitant increase in overall CCR2+ cells and MDSCs within bone marrow of CCR2-deficient mice. The CCR2 antagonist CCX872 increased median survival as a monotherapy in KR158 glioma-bearing animals and further increased median and overall survival when combined with anti-PD-1. Additionally, combination of CCX872 and anti-PD-1 prolonged median survival time in 005 GSC GBM-bearing mice. In both models, CCX872 decreased tumor associated MDSCs and increased these cells within the bone marrow. Examination of tumor-infiltrating lymphocytes revealed an elevated population, increased IFNγ expression, indicating enhanced cytolytic activity, as well as decreased expression of exhaustion markers in CD4+ and CD8+ T cells following combination treatment. These data establish that combining CCR2 and PD-1 blockade extends survival in clinically relevant murine glioma models and provides the basis on which to advance this combinatorial treatment toward early-phase human trials.

Methamphetamine Dysregulation of the Central Nervous System and Peripheral Immunity.

Methamphetamine (METH) is a potent psychostimulant that increases extracellular monoamines, such as dopamine and norepinephrine, and affects multiple tissue and cell types in the central nervous system (CNS) and peripheral immune cells. The reinforcing properties of METH underlie its significant abuse potential and dysregulation of peripheral immunity and central nervous system functions. Together, the constellation of METH's effects on cellular targets and regulatory processes has led to immune suppression and neurodegeneration in METH addicts and animal models of METH exposure. Here we extensively review many of the cell types and mechanisms of METH-induced dysregulation of the central nervous and peripheral immune systems. SIGNIFICANCE STATEMENT: Emerging research has begun to show that methamphetamine regulates dopaminergic neuronal activity. In addition, METH affects non-neuronal brain cells, such as microglia and astrocytes, and immunological cells of the periphery. Concurrent disruption of bidirectional communication between dopaminergic neurons and glia in the CNS and peripheral immune cell dysregulation gives rise to a constellation of dysfunctional neuronal, cell, and tissue types. Therefore, understanding the pathophysiology of METH requires consideration of the multiple targets at the interface between basic and clinical neuroscience.

Microglia and Other Myeloid Cells in Central Nervous System Health and Disease.

Mononuclear macrophages derived from the bone marrow (myeloid cells) are key cellular components of the innate immune system in different organs. In this minireview, we are focused on both brain and blood macrophages, known as microglia and monocytes, respectively. We provide a succinct summary of the cells' functions under both normal and pathologic conditions, with particular reference to common neurodegenerative disorders, such as Alzheimer and Parkinson disease. SIGNIFICANCE STATEMENT: In this minireview, we aim to summarize available literature on microglial and myeloid involvement in CNS disease, directing the reader toward relevant and translatable interpretations of myeloid cell function in CNS health and neurodegeneration.

Development of a Fluorescently Labeled Ligand for Rapid Detection of DAT in Human and Mouse Peripheral Blood Monocytes.

The dopamine transporter (DAT) is one of the key regulators of dopamine (DA) signaling in the central nervous system (CNS) and in the periphery. Recent reports in a model of Parkinson's disease (PD) have shown that dopamine neuronal loss in the CNS impacts the expression of DAT in peripheral immune cells. The mechanism underlying this connection is still unclear but could be illuminated with sensitive and high-throughput detection of DAT-expressing immune cells in the circulation. Herein, we have developed fluorescently labeled ligands (FLL) that bind to surface-expressing DAT with high affinity and selectivity. The diSulfoCy5-FLL (GC04-38) was utilized to label DAT in human and mouse peripheral blood mononuclear cells (PBMCs) that were analyzed via flow cytometry. Selective labeling was validated using DAT KO mouse PBMCs. Our studies provide an efficient and highly sensitive method using this novel DAT-selective FLL to advance our fundamental understanding of DAT expression and activity in PBMCs in health and disease and as a potential peripheral biomarker.

Immunophenotyping Tracks Motor Progression in Parkinson's Disease Associated with a TH Mutation.

Background: Parkinson's disease (PD) is the second most common neurodegenerative disorder, with genetic factors accounting for about 15% of cases. There is a significant challenge in tracking disease progression and treatment response, crucial for developing new therapies. Traditional methods like imaging, clinical monitoring, and biomarker analysis have not conclusively tracked disease progression or treatment response in PD. Our previous research indicated that PD patients with increased dopamine transporter (DAT) and tyrosine hydroxylase (TH) in peripheral blood mononuclear cells (PBMCs) might show disease progression and respond to levodopa treatment. Objective: This study evaluates whether DAT- and TH-expressing PBMCs can monitor motor progression in a PD patient with a heterozygous TH mutation. Methods: We conducted a longitudinal follow-up of a 46-year-old female PD patient with a TH mutation, assessing her clinical features over 18 months through DaT scans and PBMC immunophenotyping. This was compared with idiopathic PD patients (130 subjects) and healthy controls (80 age/sex-matched individuals). Results: We found an increase in DAT+ immune cells concurrent with worsening motor scores (UPDRS-III). Following levodopa therapy, unlike idiopathic PD patients, TH+ immune cell levels in this patient remained high even as her motor scores improved. Conclusions: Longitudinal immunophenotyping in this PD patient suggests DAT+ and TH+ PBMCs as potential biomarkers for tracking PD progression and treatment efficacy, supporting further exploration of this approach in PD research.

Methamphetamine induces a low dopamine transporter expressing state without altering the total number of peripheral immune cells.

Methamphetamine is a widely abused psychostimulant and one of the main targets of dopamine transporter (DAT). Methamphetamine reduces DAT-mediated dopamine uptake and stimulates dopamine efflux leading to increased synaptic dopamine levels many folds above baseline. Methamphetamine also targets DAT-expressing peripheral immune cells, reduces wound healing and increases infection susceptibility. Peripheral immune cells such as myeloid cells, B cells and T cells express DAT. DAT activity on monocytes and macrophages exhibits immune suppressive properties via an autocrine paracrine mechanism, where deletion or inhibition of DAT activity increases inflammatory responses. In this study, utilizing a mouse model of daily single dose of methamphetamine administration, we investigated the impact of the drug on DAT expression in peripheral immune cells. We found in methamphetamine-treated mice that DAT expression was down-regulated in most of the innate and adaptive immune cells. Methamphetamine did not increase or decrease the total number of innate and adaptive immune cells but changed their immunophenotype to low-DAT-expressing phenotype. Moreover, serum cytokine distributions were altered in methamphetamine-treated mice. Therefore, resembling its effect in the CNS, in the periphery, methamphetamine regulates DAT expression on peripheral immune cell subsets, potentially describing methamphetamine regulation of peripheral immunity.

Who Knew? Dopamine Transporter Activity Is Critical in Innate and Adaptive Immune Responses.

The dopamine transporter (DAT) regulates the dimension and duration of dopamine transmission. DAT expression, its trafficking, protein-protein interactions, and its activity are conventionally studied in the CNS and within the context of neurological diseases such as Parkinson's Diseases and neuropsychiatric diseases such as drug addiction, attention deficit hyperactivity and autism. However, DAT is also expressed at the plasma membrane of peripheral immune cells such as monocytes, macrophages, T-cells, and B-cells. DAT activity via an autocrine/paracrine signaling loop regulates macrophage responses to immune stimulation. In a recent study, we identified an immunosuppressive function for DAT, where blockade of DAT activity enhanced LPS-mediated production of IL-6, TNF-α, and mitochondrial superoxide levels, demonstrating that DAT activity regulates macrophage immune responses. In the current study, we tested the hypothesis that in the DAT knockout mice, innate and adaptive immunity are perturbed. We found that genetic deletion of DAT (DAT-/-) results in an exaggerated baseline inflammatory phenotype in peripheral circulating myeloid cells. In peritoneal macrophages obtained from DAT-/- mice, we identified increased MHC-II expression and exaggerated phagocytic response to LPS-induced immune stimulation, suppressed T-cell populations at baseline and following systemic endotoxemia and exaggerated memory B cell expansion. In DAT-/- mice, norepinephrine and dopamine levels are increased in spleen and thymus, but not in circulating serum. These findings in conjunction with spleen hypoplasia, increased splenic myeloid cells, and elevated MHC-II expression, in DAT-/- mice further support a critical role for DAT activity in peripheral immunity. While the current study is only focused on identifying the role of DAT in peripheral immunity, our data point to a much broader implication of DAT activity than previously thought. This study is dedicated to the memory of Dr. Marc Caron who has left an indelible mark in the dopamine transporter field.

DAT and TH expression marks human Parkinson's disease in peripheral immune cells.

Parkinson's disease (PD) is marked by a loss of dopamine neurons, decreased dopamine transporter (DAT) and tyrosine hydroxylase (TH) expression. However, this validation approach cannot be used for diagnostic, drug effectiveness or investigational purposes in human patients because midbrain tissue is accessible postmortem. PD pathology affects both the central nervous and peripheral immune systems. Therefore, we immunophenotyped blood samples of PD patients for the presence of myeloid derived suppressor cells (MDSCs) and discovered that DAT+/TH+ monocytic MDSCs, but not granulocytic MDSCs are increased, suggesting a targeted immune response to PD. Because in peripheral immune cells DAT activity underlies an immune suppressive mechanism, we investigated whether expression levels of DAT and TH in the peripheral immune cells marks PD. We found drug naïve PD patients exhibit differential DAT+/TH+ expression in peripheral blood mononuclear cells (PBMCs) compared to aged/sex matched healthy subjects. While total PBMCs are not different between the groups, the percentage of DAT+/TH+ PBMCs was significantly higher in drug naïve PD patients compared to healthy controls irrespective of age, gender, disease duration, disease severity or treatment type. Importantly, treatment for PD negatively modulates DAT+/TH+ expressing PBMCs. Neither total nor the percentage of DAT+/TH+ PBMCs were altered in the Alzheimer's disease cohort. The mechanistic underpinning of this discovery in human PD was revealed when these findings were recapitulated in animal models of PD. The reverse translational experimental strategy revealed that alterations in dopaminergic markers in peripheral immune cells are due to the disease associated changes in the CNS. Our study demonstrates that the dopaminergic machinery on peripheral immune cells displays an association with human PD, with exciting implications in facilitating diagnosis and investigation of human PD pathophysiology.

Functional characterization of the biogenic amine transporters on human macrophages.

Monocyte-derived macrophages (MDMs) are key players in tissue homeostasis and diseases regulated by a variety of signaling molecules. Recent literature has highlighted the ability for biogenic amines to regulate macrophage functions, but the mechanisms governing biogenic amine signaling in and around immune cells remain nebulous. In the CNS, biogenic amine transporters are regarded as the master regulators of neurotransmitter signaling. While we and others have shown that macrophages express these transporters, relatively little is known of their function in these cells. To address these knowledge gaps, we investigated the function of norepinephrine transporter (NET) and dopamine transporter (DAT) on human MDMs. We found that both NET and DAT are present and can uptake substrate from the extracellular space at baseline. Not only was DAT expressed in cultured MDMs, but it was also detected in a subset of intestinal macrophages in situ. Surprisingly, we discovered a NET-independent, DAT-mediated immunomodulatory mechanism in response to LPS. LPS induced reverse transport of dopamine through DAT, engaging an autocrine/paracrine signaling loop that regulated the macrophage response. Removing this signaling loop enhanced the proinflammatory response to LPS. Our data introduce a potential role for DAT in the regulation of innate immunity.

TNFα increases tyrosine hydroxylase expression in human monocytes.

Most, if not all, peripheral immune cells in humans and animals express tyrosine hydroxylase (TH), the rate limiting enzyme in catecholamine synthesis. Since TH is typically studied in the context of brain catecholamine signaling, little is known about changes in TH production and function in peripheral immune cells. This knowledge gap is due, in part, to the lack of an adequately sensitive assay to measure TH in immune cells expressing lower TH levels compared to other TH expressing cells. Here, we report the development of a highly sensitive and reproducible Bio-ELISA to quantify picogram levels of TH in multiple model systems. We have applied this assay to monocytes isolated from blood of persons with Parkinson's disease (PD) and to age-matched, healthy controls. Our study unexpectedly revealed that PD patients' monocytes express significantly higher levels of TH protein in peripheral monocytes relative to healthy controls. Tumor necrosis factor (TNFα), a pro-inflammatory cytokine, has also been shown to be increased in the brains and peripheral circulation in human PD, as well as in animal models of PD. Therefore, we investigated a possible connection between higher levels of TH protein and the known increase in circulating TNFα in PD. Monocytes isolated from healthy donors were treated with TNFα or with TNFα in the presence of an inhibitor. Tissue plasminogen activator (TPA) was used as a positive control. We observed that TNFα stimulation increased both the number of TH+ monocytes and the quantity of TH per monocyte, without increasing the total numbers of monocytes. These results revealed that TNFα could potentially modify monocytic TH production and serve a regulatory role in peripheral immune function. The development and application of a highly sensitive assay to quantify TH in both human and animal cells will provide a novel tool for further investigating possible PD immune regulatory pathways between brain and periphery.

α-Synuclein-induced dysregulation of neuronal activity contributes to murine dopamine neuron vulnerability.

Pathophysiological damages and loss of function of dopamine neurons precede their demise and contribute to the early phases of Parkinson's disease. The presence of aberrant intracellular pathological inclusions of the protein α-synuclein within ventral midbrain dopaminergic neurons is one of the cardinal features of Parkinson's disease. We employed molecular biology, electrophysiology, and live-cell imaging to investigate how excessive α-synuclein expression alters multiple characteristics of dopaminergic neuronal dynamics and dopamine transmission in cultured dopamine neurons conditionally expressing GCaMP6f. We found that overexpression of α-synuclein in mouse (male and female) dopaminergic neurons altered neuronal firing properties, calcium dynamics, dopamine release, protein expression, and morphology. Moreover, prolonged exposure to the D2 receptor agonist, quinpirole, rescues many of the alterations induced by α-synuclein overexpression. These studies demonstrate that α-synuclein dysregulation of neuronal activity contributes to the vulnerability of dopaminergic neurons and that modulation of D2 receptor activity can ameliorate the pathophysiology. These findings provide mechanistic insights into the insidious changes in dopaminergic neuronal activity and neuronal loss that characterize Parkinson's disease progression with significant therapeutic implications.

A novel approach to study markers of dopamine signaling in peripheral immune cells.

Human monocytes express known markers of dopamine synthesis, storage and clearance, including dopamine transporter (DAT), tyrosine hydroxylase (TH), all subtypes of dopamine receptors and vesicular monoamine transporter 2 (VMAT2). Immunohistochemical and immunofluorescent methodologies have traditionally been employed to determine DAT and TH expression in the CNS, their detection in the blood and specifically in the peripheral monocytes has not been studied by flow cytometry. Flow cytometry assays are widely used in medicine and in basic, preclinical or clinical research to quantify physical and chemical characteristics of target cell populations. Here, we have established a highly sensitive and reproducible flow cytometry panel to detect and quantify DAT and TH expression in freshly isolated or cryopreserved human peripheral monocytes. In healthy humans (n = 41 biological replicates), we show baseline DAT and TH expressing monocytes constitute ~12% of the peripheral blood mononuclear cell (PBMC) fraction when examined in fresh isolation from whole blood. Using an identical flow cytometry panel, we found that cryopreservation of PBMCs using multiple techniques resulted in altered PBMC populations as compared to fresh isolation and relative to one another. Among these, we identified an optimum cryopreservation method for detecting TH and DAT in cryopreserved PBMCs. Our data provide a sensitive and reproducible approach to examine dopamine signaling in peripheral human immune cells. This approach can be applied to study peripheral dopamine signaling under healthy and potentially under disease conditions. The use of dopamine signaling could also be explored as a technique to monitor therapeutic interventions particularly those targeting DAT and TH in the periphery.