A Review of the Evidence for Tryptophan and the Kynurenine Pathway as a Regulator of Stem Cell Niches in Health and Disease.

Stem cells are ubiquitously found in various tissues and organs in the body, and underpin the body's ability to repair itself following injury or disease initiation, though repair can sometimes be compromised. Understanding how stem cells are produced, and functional signaling systems between different niches is critical to understanding the potential use of stem cells in regenerative medicine. In this context, this review considers kynurenine pathway (KP) metabolism in multipotent adult progenitor cells, embryonic, haematopoietic, neural, cancer, cardiac and induced pluripotent stem cells, endothelial progenitor cells, and mesenchymal stromal cells. The KP is the major enzymatic pathway for sequentially catabolising the essential amino acid tryptophan (TRP), resulting in key metabolites including kynurenine, kynurenic acid, and quinolinic acid (QUIN). QUIN metabolism transitions into the adjoining de novo pathway for nicotinamide adenine dinucleotide (NAD) production, a critical cofactor in many fundamental cellular biochemical pathways. How stem cells uptake and utilise TRP varies between different species and stem cell types, because of their expression of transporters and responses to inflammatory cytokines. Several KP metabolites are physiologically active, with either beneficial or detrimental outcomes, and evidence of this is presented relating to several stem cell types, which is important as they may exert a significant impact on surrounding differentiated cells, particularly if they metabolise or secrete metabolites differently. Interferon-gamma (IFN-γ) in mesenchymal stromal cells, for instance, highly upregulates rate-limiting enzyme indoleamine-2,3-dioxygenase (IDO-1), initiating TRP depletion and production of metabolites including kynurenine/kynurenic acid, known agonists of the Aryl hydrocarbon receptor (AhR) transcription factor. AhR transcriptionally regulates an immunosuppressive phenotype, making them attractive for regenerative therapy. We also draw attention to important gaps in knowledge for future studies, which will underpin future application for stem cell-based cellular therapies or optimising drugs which can modulate the KP in innate stem cell populations, for disease treatment.

Altered Hippocampal and Striatal Expression of Endothelial Markers and VIP/PACAP Neuropeptides in a Mouse Model of Systemic Lupus Erythematosus.

Neuropsychiatric systemic lupus erythematosus (NPSLE) is one of the most common and severe manifestations of lupus; however, its pathogenesis is still poorly understood. While there is sparse evidence suggesting that the ongoing autoimmunity may trigger pathogenic changes to the central nervous system (CNS) microvasculature, culminating in inflammatory/ischemic damage, further evidence is still needed. In this study, we used the spontaneous mouse model of SLE (NZBWF1 mice) to investigate the expression of genes and proteins associated with endothelial (dys)function: tissue and urokinase plasminogen activators (tPA and uPA), intercellular and vascular adhesion molecules 1 (ICAM-1 and VCAM-1), brain derived neurotrophic factor (BDNF), endothelial nitric oxide synthase (eNOS) and Krüppel-like factor 4 (KLF4) and neuroprotection/immune modulation: pituitary adenylate cyclase-activating peptide (PACAP), vasoactive intestinal peptide (VIP), PACAP receptor (PAC1), VIP receptors 1 and 2 (VPAC1 and VPAC2). Analyses were carried out both in the hippocampus and striatum of SLE mice of two different age groups (2 and 7 months old), since age correlates with disease severity. In the hippocampus, we identified a gene/protein expression profile indicative of mild endothelial dysfunction, which increased in severity in aged SLE mice. These alterations were paralleled by moderate alterations in the expression of VIP, PACAP and related receptors. In contrast, we report a robust upregulation of endothelial activation markers in the striatum of both young and aged mice, concurrent with significant induction of the VIP/PACAP system. These data identify molecular signatures of endothelial alterations in the hippocampus and striatum of NZBWF1 mice, which are accompanied by a heightened expression of endogenous protective/immune-modulatory neuropeptides. Collectively, our results support the idea that NPSLE may cause alterations of the CNS micro-vascular compartment that cannot be effectively counteracted by the endogenous activity of the neuropeptides PACAP and VIP.

Systemic Rotenone Administration Causes Extra-Nigral Alterations in C57BL/6 Mice.

Systemic administration of rotenone replicates several pathogenic and behavioural features of Parkinson's disease (PD), some of which cannot be explained by deficits of the nigrostriatal pathway. In this study, we provide a comprehensive analysis of several neurochemical alterations triggered by systemic rotenone administration in the CNS of C57BL/6 mice. Mice injected with either 1, 3 or 10 mg/kg rotenone daily via intraperitoneal route for 21 days were assessed weekly for changes in locomotor and exploratory behaviour. Rotenone treatment caused significant locomotor and exploratory impairment at dosages of 3 or 10 mg/kg. Molecular analyses showed reductions of both TH and DAT expression in the midbrain, striatum and spinal cord, accompanied by altered expression of dopamine receptors and brain-derived neurotrophic factor (BDNF). Rotenone also triggered midbrain-restricted inflammatory responses with heightened expression of glial markers, which was not seen in extra-nigral regions. However, widespread alterations of mitochondrial function and increased signatures of oxidative stress were identified in both nigral and extra-nigral regions, along with disruptions of neuroprotective peptides, such as pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal peptide (VIP) and activity-dependent neuroprotective protein (ADNP). Altogether, this study shows that systemic rotenone intoxication, similarly to PD, causes a series of neurochemical alterations that extend at multiple CNS levels, reinforcing the suitability of this pre-clinical model for the study extra-nigral defects of PD.

Pro-Inflammatory and Pro-Apoptotic Effects of the Non-Protein Amino Acid L-Azetidine-2-Carboxylic Acid in BV2 Microglial Cells.

L-Azetidine-2-carboxylic acid (AZE) is a toxic non-protein coding amino acid (npAA) that is highly abundant in sugar and table beets. Due to its structural similarity with the amino acid L-proline, AZE can evade the editing process during protein assembly in eukaryotic cells and be misincorporated into L-proline-rich proteins, potentially causing protein misfolding and other detrimental effects to cells. In this study, we sought to determine if AZE treatment triggered pro-inflammatory and pro-apoptotic responses in BV2 microglial cells. BV2 microglial cells exposed to AZE at increasing concentrations (0−2000 µM) at 0, 3, 6, 12 and 24 h were assayed for cell viability (MTT) and nitric oxide release (Griess assay). Annexin V-FITC/propidium iodide (PI) staining was used to assess apoptosis. Real-time qPCR, Western blot and immunocytochemistry were used to interrogate relevant pro- and anti-inflammatory and other molecular targets of cell survival response. AZE (at concentrations > 1000 µM) significantly reduced cell viability, increased BAX/Bcl2 ratio and caused cell death. Results were mirrored by a robust increase in nitric oxide release, percentage of activated/polarised cells and expression of pro-inflammatory markers (IL-1ß, IL-6, NOS2, CD68 and MHC-2a). Additionally, we found that AZE induced the expression of the extracellular matrix degrading enzyme matrix metalloproteinase 9 (MMP-9) and brain derived neurotrophic factor (BDNF), two critical regulators of microglial motility and structural plasticity. Collectively, these data indicate that AZE-induced toxicity is associated with increased pro-inflammatory activity and reduced survival in BV2 microglia. This evidence may prompt for an increased monitoring of AZE consumption by humans.

Exploring the Pro-Phagocytic and Anti-Inflammatory Functions of PACAP and VIP in Microglia: Implications for Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic neuroinflammatory and demyelinating disease of the central nervous system (CNS), characterised by the infiltration of peripheral immune cells, multifocal white-matter lesions, and neurodegeneration. In recent years, microglia have emerged as key contributors to MS pathology, acting as scavengers of toxic myelin/cell debris and modulating the inflammatory microenvironment to promote myelin repair. In this review, we explore the role of two neuropeptides, pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP), as important regulators of microglial functioning during demyelination, myelin phagocytosis, and remyelination, emphasising the potential of these neuropeptides as therapeutic targets for the treatment of MS.

The Anxiolytic Drug Buspirone Prevents Rotenone-Induced Toxicity in a Mouse Model of Parkinson's Disease.

A pharmacological and genetic blockade of the dopamine D3 receptor (D3R) has shown to be neuroprotective in models of Parkinson's disease (PD). The anxiolytic drug buspirone, a serotonin receptor 1A agonist, also functions as a potent D3R antagonist. To test if buspirone elicited neuroprotective activities, C57BL/6 mice were subjected to rotenone treatment (10mg/kg i.p for 21 days) to induce PD-like pathology and were co-treated with increasing dosages of buspirone (1, 3, or 10 mg/kg i.p.) to determine if the drug could prevent rotenone-induced damage to the central nervous system (CNS). We found that high dosages of buspirone prevented the behavioural deficits caused by rotenone in the open field test. Molecular and histological analyses confirmed that 10 mg/kg of buspirone prevented the degeneration of TH-positive neurons. Buspirone attenuated the induction of interleukin-1ß and interleukin-6 expression by rotenone, and this was paralleled by the upregulation of arginase-1, brain-derived neurotrophic factor (BDNF), and activity-dependent neuroprotective protein (ADNP) in the midbrain, striatum, prefrontal cortex, amygdala, and hippocampus. Buspirone treatment also improved mitochondrial function and antioxidant activities. Lastly, the drug prevented the disruptions in the expression of two neuroprotective peptides, pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP). These results pinpoint the neuroprotective efficacy of buspirone against rotenone toxicity, suggesting its potential use as a therapeutic agent in neurodegenerative and neuroinflammatory diseases, such as PD.

PACAP and VIP Modulate LPS-Induced Microglial Activation and Trigger Distinct Phenotypic Changes in Murine BV2 Microglial Cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are two structurally related immunosuppressive peptides. However, the underlying mechanisms through which these peptides regulate microglial activity are not fully understood. Using lipopolysaccharide (LPS) to induce an inflammatory challenge, we tested whether PACAP or VIP differentially affected microglial activation, morphology and cell migration. We found that both peptides attenuated LPS-induced expression of the microglial activation markers Iba1 and iNOS (### p < 0.001), as well as the pro-inflammatory mediators IL-1ß, IL-6, Itgam and CD68 (### p < 0.001). In contrast, treatment with PACAP or VIP exerted distinct effects on microglial morphology and migration. PACAP reversed LPS-induced soma enlargement and increased the percentage of small-sized, rounded cells (54.09% vs. 12.05% in LPS-treated cells), whereas VIP promoted a phenotypic shift towards cell subpopulations with mid-sized, spindle-shaped somata (48.41% vs. 31.36% in LPS-treated cells). Additionally, PACAP was more efficient than VIP in restoring LPS-induced impairment of cell migration and the expression of urokinase plasminogen activator (uPA) in BV2 cells compared with VIP. These results suggest that whilst both PACAP and VIP exert similar immunosuppressive effects in activated BV2 microglia, each peptide triggers distinctive shifts towards phenotypes of differing morphologies and with differing migration capacities.

Assessing the Anti-Inflammatory Activity of the Anxiolytic Drug Buspirone Using CRISPR-Cas9 Gene Editing in LPS-Stimulated BV-2 Microglial Cells.

Buspirone is an anxiolytic drug with robust serotonin receptor 1A (Htr1a) agonist activities. However, evidence has demonstrated that this drug also targets the dopamine D3 receptor (Drd3), where it acts as a potent antagonist. In vivo, Drd3 blockade is neuroprotective and reduces inflammation in models of Parkinson's disease. To test if buspirone also elicited anti-inflammatory activities in vitro, we generated stable Drd3-/- and Htr1a-/- BV2 microglial cell lines using CRISPR-Cas9 technology and then tested the effects of buspirone after lipopolysaccharide (LPS) challenge. We found that LPS exposure had no effect on cell viability, except in Htr1a-/- cells, where viability was reduced (p < 0.001). Drug treatment reduced viability in Drd3-/- cells, but not in WT or Htr1a-/- cells. Buspirone counteracted LPS-induced NO release, NOS2, IL-1ß and TNF-α gene expression in WT cells, whereas it exerted limited effects in Drd3-/- or Htr1a-/- microglia. In summary, our findings indicate that buspirone attenuates microglial polarization after LPS challenge. These results also highlight some major effects of Drd3 or Htr1a genetic ablation on microglial biology, raising important questions on the complex role of neurotransmitters in regulating microglia functions.

Dopamine: an immune transmitter.

The dopaminergic system controls several vital central nervous system functions, including the control of movement, reward behaviors and cognition. Alterations of dopaminergic signaling are involved in the pathogenesis of neurodegenerative and psychiatric disorders, in particular Parkinson's disease, which are associated with a subtle and chronic inflammatory response. A substantial body of evidence has demonstrated the non-neuronal expression of dopamine, its receptors and of the machinery that governs synthesis, secretion and storage of dopamine across several immune cell types. This review aims to summarize current knowledge on the role and expression of dopamine in immune cells. One of the goals is to decipher the complex mechanisms through which these cell types respond to dopamine, in order to address the impact this has on neurodegenerative and psychiatric pathologies such as Parkinson's disease. A further aim is to illustrate the gaps in our understanding of the physiological roles of dopamine to encourage more targeted research focused on understanding the consequences of aberrant dopamine production on immune regulation. These highlights may prompt scientists in the field to consider alternative functions of this important neurotransmitter when targeting neuroinflammatory/neurodegenerative pathologies.