Tissue resident memory CD8+ T cells are present but not critical for demyelination and neurodegeneration in a mouse model of multiple system atrophy.

Multiple system atrophy (MSA) is rare, fast progressing, and fatal synucleinopathy with alpha-synuclein (α-syn) inclusions located within oligodendroglia called glial cytoplasmic inclusions (GCI). Along with GCI pathology there is severe demyelination, neurodegeneration, and neuroinflammation. In post-mortem tissue, there is significant infiltration of CD8+ T cells into the brain parenchyma, however their role in disease progression is unknown. To determine the role of CD8+ T cells, a modified AAV, Olig001-SYN, was used to selectively overexpress α-syn in oligodendrocytes modeling MSA in mice. Four weeks post transduction, we observed significant CD8+ T cell infiltration into the striatum of Olig001-SYN transduced mice recapitulating the CD8+ T cell infiltration observed in post-mortem tissue. To understand the role of CD8+ T cells, a CD8 knockout mice were transduced with Olig001-SYN. Six months post transduction into a mouse lacking CD8+ T cells, demyelination and neurodegeneration were unchanged. Four weeks post transduction, neuroinflammation and demyelination were enhanced in CD8 knockout mice compared to wild type controls. Applying unbiased spectral flow cytometry, CD103+, CD69+, CD44+, CXCR6+, CD8+ T cells were identified when α-syn was present in oligodendrocytes, suggesting the presence of tissue resident memory CD8+ T (Trm) cells during MSA disease progression. This study indicates that CD8+ T cells are not critical in driving MSA pathology but are needed to modulate the neuroinflammation and demyelination response.

IFNγ drives neuroinflammation, demyelination, and neurodegeneration in a mouse model of multiple system atrophy.

Multiple system atrophy (MSA) is a rare and fatal synucleinopathy characterized by insoluble alpha-synuclein (α-syn) cytoplasmic inclusions located within oligodendroglia. Neuroinflammation, demyelination, and neurodegeneration are correlated with areas of glia cytoplasmic inclusions (GCI) pathology, however it is not known what specifically drives disease pathogenesis. Recent studies have shown that disease pathologies found in post-mortem tissue from MSA patients can be modeled in rodents via a modified AAV overexpressing α-syn, Olig001-SYN, which has a 95% tropism for oligodendrocytes. In the Olig001-SYN mouse model, CD4+ T cells have been shown to drive neuroinflammation and demyelination, however the mechanism by which this occurs remains unclear. In this study we use genetic and pharmacological approaches in the Olig001-SYN model of MSA to show that the pro-inflammatory cytokine interferon gamma (IFNγ) drives neuroinflammation, demyelination, and neurodegeneration. Furthermore, using an IFNγ reporter mouse, we found that infiltrating CD4+ T cells were the primary producers of IFNγ in response to α-syn overexpression in oligodendrocytes. Results from these studies indicate that IFNγ expression from CD4+ T cells drives α-syn-mediated neuroinflammation, demyelination, and neurodegeneration. These results indicate that targeting IFNγ expression may be a potential disease modifying therapeutic strategy for MSA.

Disease mechanisms as subtypes: Inflammation in Parkinson disease and related disorders.

Neuroinflammation is a core feature of Parkinson disease (PD) and related disorders. Inflammation is detectable early in PD and persists throughout the disease state. Both the innate and the adaptive arms of the immune system are engaged in both human PD as well as in animal models of the disease. The upstream causes of PD are likely multiple and complex, which makes targeting of disease-modifying therapies based on etiological factors difficult. Inflammation is a broadly shared common mechanism and likely makes an important contribution to progression in most patients with manifest symptoms. Development of treatments targeting neuroinflammation in PD will require an understanding of the specific immune mechanisms which are active, their relative effects on both injury and neurorestoration, as well as the role of key variables likely to modulate the immune response: age, sex, the nature of the proteinopathies present, and the presence of copathologies. Studies characterizing the specific state of immune response in individuals and groups of people affected by PD will be essential to the development of targeted disease-modifying immunotherapies.

Assessing the Association of Mitochondrial Function and Inflammasome Activation in Murine Macrophages Exposed to Select Mitotoxic Tri-Organotin Compounds.

BACKGROUND: Mitochondrial function is implicated as a target of environmental toxicants and found in disease or injury models, contributing to acute and chronic inflammation. One mechanism by which mitochondrial damage can propagate inflammation is via activation of the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family, pyrin domain-containing receptor (NLRP)3 inflammasome, a protein complex that processes mature interleukin (IL)-1ß. IL-1ß plays an important role in the innate immune response and dysregulation is associated with autoinflammatory disorders. OBJECTIVE: The objective was to evaluate whether mitochondrial toxicants recruit inflammasome activation and IL-1ß processing. METHOD: Murine macrophages (RAW 264.7) exposed to tri-organotins (triethyltin bromide (TETBr), trimethyltin hydroxide (TMTOH), triphenyltin hydroxide (TPTOH), bis(tributyltin)oxide) [Bis(TBT)Ox] were examined for pro-inflammatory cytokine induction. TMTOH and TETBr were examined in RAW 264.7 and bone marrow-derived macrophages for mitochondrial bioenergetics, reactive oxygen species (ROS) production, and inflammasome activation via visualization of aggregate formation, caspase-1 flow cytometry, IL-1ß enzyme-linked immunosorbent assay and Western blots, and microRNA (miRNA) and mRNA arrays. RESULTS: TETBr and TMTOH induced inflammasome aggregate formation and IL-1ß release in lipopolysaccharide (LPS)-primed macrophages. Mitochondrial bioenergetics and mitochondrial ROS were suppressed. Il1a and Il1b induction with LPS or LPS+ATP challenge was diminished. Differential miRNA and mRNA profiles were observed. Lower miR-151-3p targeted cyclic adenosine monophosphate (cAMP)-mediated and AMP-activated protein kinase signaling pathways; higher miR-6909-5p, miR-7044-5p, and miR-7686-5p targeted Wnt beta-catenin signaling, retinoic acid receptor activation, apoptosis, signal transducer and activator of transcription 3, IL-22, IL-12, and IL-10 signaling. Functional enrichment analysis identified apoptosis and cell survival canonical pathways. CONCLUSION: Select mitotoxic tri-organotins disrupted murine macrophage transcriptional response to LPS, yet triggered inflammasome activation. The differential response pattern suggested unique functional changes in the inflammatory response that may translate to suppressed host defense or prolong inflammation. We posit a framework to examine immune cell effects of environmental mitotoxic compounds for adverse health outcomes. https://doi.org/10.1289/EHP8314.

An association between mitochondria and microglia effector function. What do we think we know?

While resident innate immune cells of the central nervous system, the microglia, represent a cell population unique in origin, microenvironment, and longevity, they assume many properties displayed by peripheral macrophages. One prominent shared property is the ability to undergo a metabolic switch towards glycolysis and away from oxidative phosphorylation (OXPHOS) upon activation by the pro-inflammatory stimuli lipopolysaccharide. This shift serves to meet specific cellular demands and allows for cell survival, similar to the Warburg effect demonstrated in cancer cells. In contrast, normal survelliance phenotype or stimulation to a non-proinflammatory phenotype relies primarily on OXPHOS and fatty acid oxidation. Thus, mitochondria appear to function as a pivotal signaling platform linking energy metabolism and macrophage polarization upon activation. These unique shifts in cell bioenergetics in response to different stimuli are essential for proper effector responses at sites of infection, inflammation, or injury. Here we present a summary of recent developments as to how these dynamics characterized in peripheral macrophages are displayed in microglia. The new insights provided by an increased understanding of metabolic reprogramming in macrophages may allow for translation to the CNS and a better understanding of microglia heterogeneity, regulation, and function.

Mitochondrial-related effects of pentabromophenol, tetrabromobisphenol A, and triphenyl phosphate on murine BV-2 microglia cells.

The predominant reliance on bromated flame retardants (BFRs) is diminishing with expanded use of alternative organophosphate flame retardants. However, exposure related issues for susceptible populations, the developing, infirmed, or aged, remain given environmental persistence and home-environment detection. In this regard, reports of flame retardant (FR)-related effects on the innate immune system suggest process by which a spectrum of adverse health effects could manifest across the life-span. As representative of the nervous system innate immune system, the current study examined changes in microglia following exposure to representative FRs, pentabromophenol (PBP), tetrabromobisphenol A (2,2',6,6',-tetrabromo-4,4'-isopropylidine diphenol; TBBPA) and triphenyl phosphate (TPP). Following 18hr exposure of murine BV-2 cells, at dose levels resulting in ≥80% viability (10 and 40 µM), limited alterations in pro-inflammatory responses were observed however, changes were observed in mitochondrial respiration. Basal respiration was altered by PBP; ATP-linked respiration by PBP and TBBPA, and maximum respiration by all three FRs. Basal glycolytic rate was altered by PBP and TBBPA and compensatory glycolysis by all three. Phagocytosis was decreased for PBP and TBBPA. NLRP3 inflammasome activation was assessed using BV-2-ASC (apoptosis-associated speck-like protein containing a CARD) reporter cells to visualize aggregate formation. PBP, showed a direct stimulation of aggregate formation and properties as a NLRP3 inflammasome secondary trigger. TBBPA showed indications of possible secondary triggering activity while no changes were seen with TPP. Thus, the data suggests an effect of all three FRs on mitochondria metabolism yet, different functional outcomes including, phagocytic capability and NLRP3 inflammasome activation.

CHIP Regulates Aquaporin-2 Quality Control and Body Water Homeostasis.

The importance of the kidney distal convoluted tubule (DCT) and cortical collecting duct (CCD) is highlighted by various water and electrolyte disorders that arise when the unique transport properties of these segments are disturbed. Despite this critical role, little is known about which proteins have a regulatory role in these cells and how these cells can be regulated by individual physiologic stimuli. By combining proteomics, bioinformatics, and cell biology approaches, we found that the E3 ubiquitin ligase CHIP is highly expressed throughout the collecting duct; is modulated in abundance by vasopressin; interacts with aquaporin-2 (AQP2), Hsp70, and Hsc70; and can directly ubiquitylate the water channel AQP2 in vitro shRNA knockdown of CHIP in CCD cells increased AQP2 protein t1/2 and reduced AQP2 ubiquitylation, resulting in greater levels of AQP2 and phosphorylated AQP2. CHIP knockdown increased the plasma membrane abundance of AQP2 in these cells. Compared with wild-type controls, CHIP knockout mice or novel CRISPR/Cas9 mice without CHIP E3 ligase activity had greater AQP2 abundance and altered renal water handling, with decreased water intake and urine volume, alongside higher urine osmolality. We did not observe significant changes in other water- or sodium-transporting proteins in the gene-modified mice. In summary, these results suggest that CHIP regulates AQP2 and subsequently, renal water handling.