Maternal Herpesviridae infection during pregnancy alters midbrain dopaminergic signatures in adult offspring.

BACKGROUND: Motor symptoms of Parkinson's disease (PD) are apparent after a high proportion of dopamine neurons in the substantia nigra have degenerated. The vast majority of PD cases are sporadic, and the underlying pathobiological causes are poorly understood. Adults exhibit great variability in the numbers of nigral dopamine neurons, suggesting that factors during embryonic or early life regulate the development and physiology of dopaminergic neurons. Furthermore, exposure to infections and inflammation in utero has been shown to affect fetal brain development in models of schizophrenia and autism. Here, we utilize a mouse maternal infection model to examine how maternal herpesvirus infection impacts dopaminergic neuron-related gene and protein expression in the adult offspring. METHODS: Pregnant mice were injected with murine cytomegalovirus (MCMV), murine gamma herpes virus-68 (MHV68) or phosphate buffered saline (PBS) at embryonic day 8.5. Offspring were sacrificed at eight weeks of age and midbrains were processed for whole genome RNA sequencing, DNA methylation analysis, targeted protein expression and high-performance liquid chromatography for quantification of dopamine and its metabolites. RESULTS: The midbrain of adult offspring from MHV68 infected dams had significantly decreased expression of genes linked to dopamine neurons (Th, Lmx1b, and Foxa1) and increased Lrrk2, a gene involved in familial PD and PD risk that associates with neuroinflammation. Deconvolution analysis revealed that the proportion of dopamine neuron genes in the midbrain was reduced. There was an overall increase in DNA methylation in the midbrain of animals from MHV68-infected dams and pathway analyses indicated mitochondrial dysfunction, with reductions in genes associated with ATP synthesis, mitochondrial respiratory chain, and mitochondrial translation in the offspring of dams infected with MHV68. TIGAR (a negative regulator of mitophagy) and SDHA (mitochondrial complex II subunit) protein levels were increased, and the levels of 3,4-dihydroxyphenylacetic acid (DOPAC) in the striatum were increased in these offspring compared to offspring from uninfected control dams. No such changes were observed in the offspring of dams infected with MCMV. CONCLUSION: Our data suggest that maternal infection with Herpesviridae, specifically MHV68, can trigger changes in the development of the midbrain that impact dopamine neuron physiology in adulthood. Our work is of importance for the understanding of neuronal susceptibility underlying neurodegenerative disease, with particular relevance for PD.

Upregulation of α-synuclein following immune activation: Possible trigger of Parkinson's disease.

Alpha-synuclein (α-syn) has been suggested to have many functions including, vesicle transport in neurons, transcriptional regulator, modulator of immune cell maturation and response, and a role as an antimicrobial peptide. This protein forms insoluble aggregates, called Lewy bodies, in several neurodegenerative diseases, termed synucleinopathies, including Parkinson's disease (PD), Multiple System Atrophy, and Lewy Body Dementia, and aggregates are also commonly found in Alzheimer's disease. Moreover, multiplications and point mutations in the gene cause rare autosomal dominant forms of parkinsonism, which resemble sporadic PD. It has been suggested that the accumulation of α-syn in the monomeric state followed by aggregation of the protein and seeding of further pathogenic α-syn aggregates are key steps in the pathogenesis of synucleinopathies. The triggers of α-syn aggregation in neurodegeneration are unknown, but inflammation caused by bacterial and viral pathogens or exposure to environmental toxins have been implicated. The purpose of this review is to present emerging evidence that α-syn may play a role in the immune response to pathogens. We present recent findings suggesting that upregulation of α-syn levels is a normal response to infections. We propose that under certain conditions (e.g., dysregulated inflammatory responses due to genetic predisposition and aging), monomeric α-syn will form oligomers that are taken up by nerve endings and undergo axonal transport to the central nervous system, where they can aggregate into pathogenic fibrils. Under unfavorable conditions, we suggest that this process can trigger neurodegenerative disease. Therefore, a deeper understanding of the roles of α-syn in the immune system could provide crucial insights into the origins of synucleinopathies.

T Cells Limit Accumulation of Aggregate Pathology Following Intrastriatal Injection of α-Synuclein Fibrils.

BACKGROUND: α-Synuclein (α-syn) is the predominant protein in Lewy-body inclusions, which are pathological hallmarks of α-synucleinopathies, such as Parkinson's disease (PD) and multiple system atrophy (MSA). Other hallmarks include activation of microglia, elevation of pro-inflammatory cytokines, as well as the activation of T and B cells. These immune changes point towards a dysregulation of both the innate and the adaptive immune system. T cells have been shown to recognize epitopes derived from α-syn and altered populations of T cells have been found in PD and MSA patients, providing evidence that these cells can be key to the pathogenesis of the disease.ObjectiveTo study the role of the adaptive immune system with respect to α-syn pathology. METHODS: We injected human α-syn preformed fibrils (PFFs) into the striatum of immunocompromised mice (NSG) and assessed accumulation of phosphorylated α-syn pathology, proteinase K-resistant α-syn pathology and microgliosis in the striatum, substantia nigra and frontal cortex. We also assessed the impact of adoptive transfer of naïve T and B cells into PFF-injected immunocompromised mice. RESULTS: Compared to wildtype mice, NSG mice had an 8-fold increase in phosphorylated α-syn pathology in the substantia nigra. Reconstituting the T cell population decreased the accumulation of phosphorylated α-syn pathology and resulted in persistent microgliosis in the striatum when compared to non-transplanted mice. CONCLUSION: Our work provides evidence that T cells play a role in the pathogenesis of experimental α-synucleinopathy.

Tryptophan Metabolites Are Associated With Symptoms and Nigral Pathology in Parkinson's Disease.

BACKGROUND: The objective of this study was to determine whether neurotoxic kynurenine metabolites, induced by inflammation, in plasma and cerebrospinal fluid (CSF) are associated with symptom severity and nigral pathology in Parkinson's disease (PD). METHODS: Clinical and MRI data were obtained from 97 PD and 89 controls. We used ultra-performance liquid chromatography to quantify kynurenine metabolites and high-sensitivity multiplex assays to quantify inflammation in plasma and CSF. We evaluated group-wise differences as well as associations between the biomarkers, motor and nonmotor symptoms, and nigral R2* (MRI metric reflecting iron content). RESULTS: PD subjects had >100% higher 3-hydroxykynurenine and 14% lower 3-hydroxyanthranilic acid in plasma. The 3-HK in plasma was closely associated with both symptom severity and disease duration. PD subjects also had 23% lower kynurenic acid in the CSF. Higher CSF levels of the excitotoxin quinolinic acid were associated with more severe symptoms, whereas lower levels of the neuroprotective kynurenic acid were linked to olfactory deficits. An elevated quinolinic acid/picolinic acid ratio in the CSF correlated with higher R2* values in the substantia nigra in the entire cohort. Plasma C-reactive protein and serum amyloid alpha were associated with signs of increased kynurenine pathway activity in the CSF of PD patients, but not in controls. CONCLUSIONS: In PD, the kynurenine pathway metabolite levels are altered in both the periphery and the central nervous system, and these changes are associated with symptom severity. Replication studies are warranted in other cohorts, and these can also explore if kynurenine metabolites might be PD biomarkers and/or are involved in PD pathogenesis. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Retinoic acid inducible gene-I mediated detection of bacterial nucleic acids in human microglial cells.

BACKGROUND: Bacterial meningitis and meningoencephalitis are associated with devastating neuroinflammation. We and others have demonstrated the importance of glial cells in the initiation of immune responses to pathogens invading the central nervous system (CNS). These cells use a variety of pattern recognition receptors (PRRs) to identify common pathogen motifs and the cytosolic sensor retinoic acid inducible gene-1 (RIG-I) is known to serve as a viral PRR and initiator of interferon (IFN) responses. Intriguingly, recent evidence indicates that RIG-I also has an important role in the detection of bacterial nucleic acids, but such a role has not been investigated in glia. METHODS: In this study, we have assessed whether primary or immortalized human and murine glia express RIG-I either constitutively or following stimulation with bacteria or their products by immunoblot analysis. We have used capture ELISAs and immunoblot analysis to assess human microglial interferon regulatory factor 3 (IRF3) activation and IFN production elicited by bacterial nucleic acids and novel engineered nucleic acid nanoparticles. Furthermore, we have utilized a pharmacological inhibitor of RIG-I signaling and siRNA-mediated knockdown approaches to assess the relative importance of RIG-I in such responses. RESULTS: We demonstrate that RIG-I is constitutively expressed by human and murine microglia and astrocytes, and is elevated following bacterial infection in a pathogen and cell type-specific manner. Additionally, surface and cytosolic PRR ligands are also sufficient to enhance RIG-I expression. Importantly, our data demonstrate that bacterial RNA and DNA both trigger RIG-I-dependent IRF3 phosphorylation and subsequent type I IFN production in human microglia. This ability has been confirmed using our nucleic acid nanoparticles where we demonstrate that both RNA- and DNA-based nanoparticles can stimulate RIG-I-dependent IFN responses in these cells. CONCLUSIONS: The constitutive and bacteria-induced expression of RIG-I by human glia and its ability to mediate IFN responses to bacterial RNA and DNA and nucleic acid nanoparticles raises the intriguing possibility that RIG-I may be a potential target for therapeutic intervention during bacterial infections of the CNS, and that the use of engineered nucleic acid nanoparticles that engage this sensor might be a method to achieve this goal.

Inflammation and kynurenine pathway dysregulation in post-partum women with severe and suicidal depression.

Depression during pregnancy and the post-partum is common, with severe cases resulting in suicidal behavior. Despite the urgent and unmet medical need, the biological underpinnings of peri-partum depression remain unclear. It has been suggested that it is triggered by dynamic changes of the immune system during pregnancy and at delivery. Therefore, we investigated whether a pro-inflammatory status in plasma, together with changes in the kynurenine pathway activity, is associated with the development of severe depression and suicidal behavior in the post-partum. Our cross-sectional study targets a unique, understudied population in which the pronounced severity of symptoms required hospitalization. We analyzed plasma IL-1ß, IL-2, IL-6, IL-8, TNF-α, tryptophan, serotonin, kynurenine, nicotinamide, quinolinic- and kynurenic acids in post-partum women diagnosed with peripartum onset depression (PPD) and healthy controls (n = 165). We assessed depression severity using the Edinburgh Postnatal Depression Scale and suicidality using the Columbia-Suicide Severity Rating Scale. We found that increased plasma IL-6 and IL-8 and reductions of serotonin, IL-2 and quinolinic acid were associated with the severity of depressive symptoms and increased the risk for PPD. Moreover, women with lower serotonin levels were at an increased risk for suicidal behavior, even when adjusting for depression severity, psychosocial factors, age BMI, and medication. Our results indicate that severe depression in the post-partum involves dysregulation of the immune response and the kynurenine pathway, with a concomitant reduction in serotonin levels. We propose that inflammatory cytokines and the kynurenine pathway are potential treatment targets in PPD, opening up the possibility of novel therapeutic strategies targeting the peripartum.

Microglia affect α-synuclein cell-to-cell transfer in a mouse model of Parkinson's disease.

BACKGROUND: Cell-to-cell propagation of α-synuclein (α-syn) aggregates is thought to contribute to the pathogenesis of Parkinson's disease (PD) and underlie the spread of α-syn neuropathology. Increased pro-inflammatory cytokine levels and activated microglia are present in PD and activated microglia can promote α-syn aggregation. However, it is unclear how microglia influence α-syn cell-to-cell transfer. METHODS: We developed a clinically relevant mouse model to monitor α-syn prion-like propagation between cells; we transplanted wild-type mouse embryonic midbrain neurons into a mouse striatum overexpressing human α-syn (huα-syn) following adeno-associated viral injection into the substantia nigra. In this system, we depleted or activated microglial cells and determined the effects on the transfer of huα-syn from host nigrostriatal neurons into the implanted dopaminergic neurons, using the presence of huα-syn within the grafted cells as a readout. RESULTS: First, we compared α-syn cell-to-cell transfer between host mice with a normal number of microglia to mice in which we had pharmacologically ablated 80% of the microglia from the grafted striatum. With fewer host microglia, we observed increased accumulation of huα-syn in grafted dopaminergic neurons. Second, we assessed the transfer of α-syn into grafted neurons in the context of microglia activated by one of two stimuli, lipopolysaccharide (LPS) or interleukin-4 (IL-4). LPS exposure led to a strong activation of microglial cells (as determined by microglia morphology, cytokine production and an upregulation in genes involved in the inflammatory response in the LPS-injected mice by RNA sequencing analysis). LPS-injected mice had significantly higher amounts of huα-syn in grafted neurons. In contrast, injection of IL-4 did not change the proportion of grafted dopamine neurons that contained huα-syn relative to controls. As expected, RNA sequencing analysis on striatal tissue revealed differential gene expression between LPS and IL-4-injected mice; with the genes upregulated in tissue from mice injected with LPS including several of those involved in an inflammatory response. CONCLUSIONS: The absence or the hyperstimulation of microglia affected α-syn transfer in the brain. Our results suggest that under resting, non-inflammatory conditions, microglia modulate the transfer of α-syn. Pharmacological regulation of neuroinflammation could represent a future avenue for limiting the spread of PD neuropathology.

Murine astrocytes are responsive to the pro-inflammatory effects of IL-20.

Glia are key regulators of inflammatory responses within the central nervous system (CNS) following infection or trauma. We have previously demonstrated the ability of activated astrocytes to rapidly produce pro-inflammatory mediators followed by a transition to an anti-inflammatory cytokine production profile that includes the immunosuppressive cytokine interleukin (IL)-10 and the closely related cytokines IL-19 and IL-24. IL-20, another member of the IL-10 family, is known to modulate immune cell activity in the periphery and we have previously demonstrated that astrocytes constitutively express the cognate receptors for this cytokine. However, the ability of glia to produce IL-20 remains unclear and the effects of this pleiotropic cytokine on glial immune functions have not been investigated. In this study, we report that primary murine and human astrocytes are not an appreciable source of IL-20 following challenge with disparate bacterial species or their components. Importantly, we have determined that astrocyte are responsive to the immunomodulatory actions of this cytokine by showing that recombinant IL-20 administration upregulates microbial pattern recognition receptor expression and induces release of the inflammatory mediator IL-6 by these cells. Taken together, these data suggest that IL-20 acts in a dissimilar manner to other IL-10 family members to augment the inflammatory responses of astrocytes.

Murine astrocytes produce IL-24 and are susceptible to the immunosuppressive effects of this cytokine.

BACKGROUND: Glia are key regulators of inflammatory responses within the central nervous system (CNS) following infection or trauma. We have previously demonstrated the ability of activated glia to rapidly produce pro-inflammatory mediators followed by a transition to an anti-inflammatory cytokine production profile that includes the immunosuppressive cytokine interleukin (IL)-10 and the closely related cytokine IL-19. IL-24, another member of the IL-10 family, has been studied in a number of inflammatory conditions in the periphery and is known to modulate immune cell activity. However, the ability of glia to produce IL-24 remains unclear and the effects of this pleiotropic cytokine on glial immune functions have not been investigated. METHODS: In this study, we have assessed whether primary murine glia produce IL-24 following stimulation and evaluated the effect of this cytokine on the immune responses of such cells. We have utilized RT-PCR and immunoblot analyses to assess the expression of IL-24 and its cognate receptors by astrocytes following challenge with bacteria or their components. Furthermore, we have determined the effect of recombinant IL-24 on astrocyte immune signaling and responses to clinically relevant bacteria using RT-PCR and specific capture ELISAs. RESULTS: We demonstrate that astrocytes express IL-24 mRNA and release detectable amounts of this cytokine protein in a delayed manner following bacterial challenge. In addition, we have determined that glia constitutively express the cognate receptors for IL-24 and show that such expression can be increased in astrocytes following activation. Importantly, our results indicate that IL-24 exerts an immunosuppressive effect on astrocytes by elevating suppressor of cytokine signaling 3 expression and limiting IL-6 production following challenge. Furthermore, we have demonstrated that IL-24 can also augment the release of IL-10 by bacterially challenged astrocytes and can induce the expression of the potentially neuroprotective mediators, glutamate transporter 1, and cyclooxygenase 2. CONCLUSIONS: The expression of IL-24 and its cognate receptors by astrocytes following bacterial challenge, and the ability of this cytokine to limit inflammatory responses while promoting the expression of immunosuppressive and/or neuroprotective mediators, raises the intriguing possibility that IL-24 functions to regulate or resolve CNS inflammation following bacterial infection in order to limit neuronal damage.

The Interleukin-10 Family of Cytokines and Their Role in the CNS.

Resident cells of the central nervous system (CNS) play an important role in detecting insults and initiating protective or sometimes detrimental host immunity. At peripheral sites, immune responses follow a biphasic course with the rapid, but transient, production of inflammatory mediators giving way to the delayed release of factors that promote resolution and repair. Within the CNS, it is well known that glial cells contribute to the onset and progression of neuroinflammation, but it is only now becoming apparent that microglia and astrocytes also play an important role in producing and responding to immunosuppressive factors that serve to limit the detrimental effects of such responses. Interleukin-10 (IL-10) is generally considered to be the quintessential immunosuppressive cytokine, and its ability to resolve inflammation and promote wound repair at peripheral sites is well documented. In the present review article, we discuss the evidence for the production of IL-10 by glia, and describe the ability of CNS cells, including microglia and astrocytes, to respond to this suppressive factor. Furthermore, we review the literature for the expression of other members of the IL-10 cytokine family, IL-19, IL-20, IL-22 and IL-24, within the brain, and discuss the evidence of a role for these poorly understood cytokines in the regulation of infectious and sterile neuroinflammation. In concert, the available data indicate that glia can produce IL-10 and the related cytokines IL-19 and IL-24 in a delayed manner, and these cytokines can limit glial inflammatory responses and/or provide protection against CNS insult. However, the roles of other IL-10 family members within the CNS remain unclear, with IL-20 appearing to act as a pro-inflammatory factor, while IL-22 may play a protective role in some instances and a detrimental role in others, perhaps reflecting the pleiotropic nature of this cytokine family. What is clear is that our current understanding of the role of IL-10 and related cytokines within the CNS is limited at best, and further research is required to define the actions of this understudied family in inflammatory brain disorders.

Human microglia and astrocytes constitutively express the neurokinin-1 receptor and functionally respond to substance P.

BACKGROUND: The tachykinin substance P (SP) is recognized to exacerbate inflammation at peripheral sites via its target receptor, neurokinin 1 receptor (NK-1R), expressed by leukocytes. More recently, SP/NK-1R interactions have been associated with severe neuroinflammation and neuronal damage. We have previously demonstrated that NK-1R antagonists can limit neuroinflammatory damage in a mouse model of bacterial meningitis. Furthermore, we have since shown that these agents can attenuate bacteria-induced neuronal and glial inflammatory mediator production in nonhuman primate (NHP) brain explants and isolated neuronal cells, and following in vivo infection. METHODS: In the present study, we have assessed the ability of NHP brain explants, primary human microglia and astrocytes, and immortalized human glial cell lines to express NK-1R isoforms. We have utilized RT-PCR, immunoblot analysis, immunofluorescent microscopy, and/or flow cytometric analysis, to quantify NK-1R expression in each, at rest, or following bacterial challenge. Furthermore, we have assessed the ability of human microglia to respond to SP by immunoblot analysis of NF-kB nuclear translocation and determined the ability of this neuropeptide to augment inflammatory cytokine release and neurotoxic mediator production by human astrocytes using an ELISA and a neuronal cell toxicity assay, respectively. RESULTS: We demonstrate that human microglial and astrocytic cells as well as NHP brain tissue constitutively express robust levels of the full-length NK-1R isoform. In addition, we demonstrate that the expression of NK-1R by human astrocytes can be further elevated following exposure to disparate bacterial pathogens or their components. Importantly, we have demonstrated that NK-1R is functional in both human microglia and astrocytes and show that SP can augment the inflammatory and/or neurotoxic immune responses of glial cells to disparate and clinically relevant bacterial pathogens. CONCLUSIONS: The robust constitutive and functional expression of the full-length NK-1R isoform by human microglia and astrocytes, and the ability of SP to augment inflammatory signaling pathways and mediator production by these cells, support the contention that SP/NK-1R interactions play a significant role in the damaging neuroinflammation associated with conditions such as bacterial meningitis.

Aprepitant limits in vivo neuroinflammatory responses in a rhesus model of Lyme neuroborreliosis.

BACKGROUND: Substance P (SP) is produced at high levels in the central nervous system (CNS), and its target receptor, neurokinin 1 receptor (NK-1R), is expressed by glia and leukocytes. This tachykinin functions to exacerbate inflammatory responses at peripheral sites. Moreover, SP/NK-1R interactions have recently been associated with severe neuroinflammation and neuronal damage. We have previously demonstrated that NK-1R antagonists can limit neuroinflammatory damage in a mouse model of bacterial meningitis. Furthermore, we have since shown that these agents can attenuate Borrelia burgdorferi-induced neuronal and glial inflammatory mediator production in non-human primate brain explants and isolated neuronal cells. METHODS: In the present study, we have assessed the role played by endogenous SP/NK-1R interactions in damaging CNS inflammation in an established rhesus macaque model that faithfully reproduces the key clinical features of Lyme neuroborreliosis, using the specific NK-1R antagonist, aprepitant. We have utilized multiplex ELISA to quantify immune mediator levels in cerebrospinal fluid, and RT-PCR and immunoblot analyses to quantify cytokine and NK-1R expression, respectively, in brain cortex, dorsal root ganglia, and spinal cord tissues. In addition, we have assessed astrocyte number/activation status in brain cortical tissue by immunofluorescence staining and confocal microscopy. RESULTS: We demonstrate that aprepitant treatment attenuates B. burgdorferi-induced elevations in CCL2, CXCL13, IL-17A, and IL-6 gene expression in dorsal root ganglia, spinal cord, and/or cerebrospinal fluid of rhesus macaques at 2 to 4 weeks following intrathecal infection. In addition, we demonstrate that this selective NK-1R antagonist also prevents increases in total cortical brain NK-1R expression and decreases in the expression of the astrocyte marker, glial fibrillary acidic protein, associated with B. burgdorferi infection. CONCLUSIONS: The ability of a centrally acting NK-1R inhibitor to attenuate B. burgdorferi-associated neuroinflammatory responses and sequelae raises the intriguing possibility that such FDA-approved agents could be repurposed for use as an adjunctive therapy for the treatment of bacterial CNS infections.