Terminally differentiated effector memory T cells associate with cognitive and AD-related biomarkers in an aging-based community cohort.

BACKGROUND AND PURPOSE: The immune response changes during aging and the progression of Alzheimer's disease (AD) and related dementia (ADRD). Terminally differentiated effector memory T cells (called TEMRA) are important during aging and AD due to their cytotoxic phenotype and association with cognitive decline. However, it is not clear if the changes seen in TEMRAs are specific to AD-related cognitive decline specifically or are more generally correlated with cognitive decline. This study aimed to examine whether TEMRAs are associated with cognition and plasma biomarkers of AD, neurodegeneration, and neuroinflammation in a community-based cohort of older adults. METHODS: Study participants from a University of Kentucky Alzheimer's Disease Research Center (UK-ADRC) community-based cohort of aging and dementia were used to test our hypothesis. There were 84 participants, 44 women and 40 men. Participants underwent physical examination, neurological examination, medical history, cognitive testing, and blood collection to determine plasma biomarker levels (Aß42/Aß40 ratio, total tau, Neurofilament Light chain (Nf-L), Glial Fibrillary Acidic Protein (GFAP)) and to isolate peripheral blood mononuclear cells (PBMCs). Flow cytometry was used to analyze PBMCs from study participants for effector and memory T cell populations, including CD4+ and CD8+ central memory T cells (TCM), Naïve T cells, effector memory T cells (TEM), and effector memory CD45RA+ T cells (TEMRA) immune cell markers. RESULTS: CD8+ TEMRAs were positively correlated with Nf-L and GFAP. We found no significant difference in CD8+ TEMRAs based on cognitive scores and no associations between CD8+ TEMRAs and AD-related biomarkers. CD4+ TEMRAs were associated with cognitive impairment on the MMSE. Gender was not associated with TEMRAs, but it did show an association with other T cell populations. CONCLUSION: These findings suggest that the accumulation of CD8+ TEMRAs may be a response to neuronal injury (Nf-L) and neuroinflammation (GFAP) during aging or the progression of AD and ADRD. As our findings in a community-based cohort were not clinically-defined AD participants but included all ADRDs, this suggests that TEMRAs may be associated with changes in systemic immune T cell subsets associated with the onset of pathology.

IL-1R1 signaling in TBI: assessing chronic impacts and neuroinflammatory dynamics in a mouse model of mild closed-head injury.

Neuroinflammation contributes to secondary injury cascades following traumatic brain injury (TBI), with alternating waves of inflammation and resolution. Interleukin-1 (IL-1), a critical neuroinflammatory mediator originating from brain endothelial cells, microglia, astrocytes, and peripheral immune cells, is acutely overexpressed after TBI, propagating secondary injury and tissue damage. IL-1 affects blood-brain barrier permeability, immune cell activation, and neural plasticity. Despite the complexity of cytokine signaling post-TBI, we hypothesize that IL-1 signaling specifically regulates neuroinflammatory response components. Using a closed-head injury (CHI) TBI model, we investigated IL-1's role in the neuroinflammatory cascade with a new global knock-out (gKO) mouse model of the IL-1 receptor (IL-1R1), which efficiently eliminates all IL-1 signaling. We found that IL-1R1 gKO attenuated behavioral impairments 14 weeks post-injury and reduced reactive microglia and astrocyte staining in the neocortex, corpus callosum, and hippocampus. We then examined whether IL-1R1 loss altered acute neuroinflammatory dynamics, measuring gene expression changes in the neocortex at 3, 9, 24, and 72 h post-CHI using the NanoString Neuroinflammatory panel. Of 757 analyzed genes, IL-1R1 signaling showed temporal specificity in neuroinflammatory gene regulation, with major effects at 9 h post-CHI. IL-1R1 signaling specifically affected astrocyte-related genes, selectively upregulating chemokines like Ccl2, Ccl3, and Ccl4, while having limited impact on cytokine regulation, such as Tnfα. This study provides further insight into IL-1R1 function in amplifying the neuroinflammatory cascade following CHI in mice and demonstrates that suppression of IL-1R1 signaling offers long-term protective effects on brain health.

Effects of advanced age upon astrocyte-specific responses to acute traumatic brain injury in mice.

BACKGROUND: Older-age individuals are at the highest risk for disability from a traumatic brain injury (TBI). Astrocytes are the most numerous glia in the brain, necessary for brain function, yet there is little known about unique responses of astrocytes in the aged-brain following TBI. METHODS: Our approach examined astrocytes in young adult, 4-month-old, versus aged, 18-month-old mice, at 1, 3, and 7 days post-TBI. We selected these time points to span the critical period in the transition from acute injury to presumably irreversible tissue damage and disability. Two approaches were used to define the astrocyte contribution to TBI by age interaction: (1) tissue histology and morphological phenotyping, and (2) transcriptomics on enriched astrocytes from the injured brain. RESULTS: Aging was found to have a profound effect on the TBI-induced loss of astrocyte function needed for maintaining water transport and edema-namely, aquaporin-4. The aged brain also demonstrated a progressive exacerbation of astrogliosis as a function of time after injury. Moreover, clasmatodendrosis, an underrecognized astrogliopathy, was found to be significantly increased in the aged brain, but not in the young brain. As a function of TBI, we observed a transitory refraction in the number of these astrocytes, which rebounded by 7 days post-injury in the aged brain. Transcriptomic data demonstrated disproportionate changes in genes attributed to reactive astrocytes, inflammatory response, complement pathway, and synaptic support in aged mice following TBI compared to young mice. Additionally, our data highlight that TBI did not evoke a clear alignment with the previously defined "A1/A2" dichotomy of reactive astrogliosis. CONCLUSIONS: Overall, our findings point toward a progressive phenotype of aged astrocytes following TBI that we hypothesize to be maladaptive, shedding new insights into potentially modifiable astrocyte-specific mechanisms that may underlie increased fragility of the aged brain to trauma.

Simulation of P2X-mediated calcium signalling in microglia.

KEY POINTS: A computational model of P2X channel activation in microglia was developed that includes downfield Ca2+ -dependent signalling pathways. This model provides quantitative insights into how diverse signalling pathways in microglia converge to control microglial function. ABSTRACT: Microglia function is orchestrated through highly coupled signalling pathways that depend on calcium (Ca2+ ). In response to extracellular ATP, transient increases in intracellular Ca2+ driven through the activation of purinergic receptors, P2X and P2Y, are sufficient to promote cytokine synthesis. Although the steps comprising the pathways bridging purinergic receptor activation with transcriptional responses have been probed in great detail, a quantitative model for how these steps collectively control cytokine production has not been established. Here we developed a minimal computational model that quantitatively links extracellular stimulation of two prominent ionotropic purinergic receptors, P2X4 and P2X7, with the graded production of a gene product, namely the tumour necrosis factor α (TNFα) cytokine. In addition to Ca2+ handling mechanisms common to eukaryotic cells, our model includes microglia-specific processes including ATP-dependent P2X4 and P2X7 activation, activation of nuclear factor of activated T-cells (NFAT) transcription factors, and TNFα production. Parameters for this model were optimized to reproduce published data for these processes, where available. With this model, we determined the propensity for TNFα production in microglia, subject to a wide range of ATP exposure amplitudes, frequencies and durations that the cells could encounter in vivo. Furthermore, we have investigated the extent to which modulation of the signal transduction pathways influence TNFα production. Our results suggest that pulsatile stimulation of P2X4 via micromolar ATP may be sufficient to promote TNFα production, whereas high-amplitude ATP exposure is necessary for production via P2X7. Furthermore, under conditions that increase P2X4 expression, for instance, following activation by pathogen-associated molecular factors, P2X4-associated TNFα production is greatly enhanced. Given that Ca2+ homeostasis in microglia is profoundly important to its function, this computational model provides a quantitative framework to explore hypotheses pertaining to microglial physiology.

Novel TNF receptor-1 inhibitors identified as potential therapeutic candidates for traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) begins with the application of mechanical force to the head or brain, which initiates systemic and cellular processes that are hallmarks of the disease. The pathological cascade of secondary injury processes, including inflammation, can exacerbate brain injury-induced morbidities and thus represents a plausible target for pharmaceutical therapies. We have pioneered research on post-traumatic sleep, identifying that injury-induced sleep lasting for 6 h in brain-injured mice coincides with increased cortical levels of inflammatory cytokines, including tumor necrosis factor (TNF). Here, we apply post-traumatic sleep as a physiological bio-indicator of inflammation. We hypothesized the efficacy of novel TNF receptor (TNF-R) inhibitors could be screened using post-traumatic sleep and that these novel compounds would improve functional recovery following diffuse TBI in the mouse. METHODS: Three inhibitors of TNF-R activation were synthesized based on the structure of previously reported TNF CIAM inhibitor F002, which lodges into a defined TNFR1 cavity at the TNF-binding interface, and screened for in vitro efficacy of TNF pathway inhibition (IκB phosphorylation). Compounds were screened for in vivo efficacy in modulating post-traumatic sleep. Compounds were then tested for efficacy in improving functional recovery and verification of cellular mechanism. RESULTS: Brain-injured mice treated with Compound 7 (C7) or SGT11 slept significantly less than those treated with vehicle, suggesting a therapeutic potential to target neuroinflammation. SGT11 restored cognitive, sensorimotor, and neurological function. C7 and SGT11 significantly decreased cortical inflammatory cytokines 3 h post-TBI. CONCLUSIONS: Using sleep as a bio-indicator of TNF-R-dependent neuroinflammation, we identified C7 and SGT11 as potential therapeutic candidates for TBI.

Retention of normal glia function by an isoform-selective protein kinase inhibitor drug candidate that modulates cytokine production and cognitive outcomes.

BACKGROUND: Brain p38α mitogen-activated protein kinase (MAPK), a potential therapeutic target for cognitive dysfunction based on the neuroinflammation-synaptic dysfunction cycle of pathophysiology progression, offers an innovative pharmacological strategy via inhibiting the same activated target in both glia and neurons, thereby enhancing the possibility for efficacy. The highly selective, brain-penetrant p38αMAPK inhibitor MW150 attenuates cognitive dysfunction in two distinct Alzheimer's disease (AD)-relevant models and avoids the problems encountered with previous mixed-kinase inhibitor drug candidates. Therefore, it is essential that the glial effects of this CNS-active kinase inhibitor be addressed in order to anticipate future use in clinical investigations. METHODS: We explored the effects of MW150 on glial biology in the AD-relevant APP/PS1 knock-in (KI) mouse model where we previously showed efficacy in suppression of hippocampal-dependent associative and spatial memory deficits. MW150 (2.5 mg/kg/day) was administered daily to 11-12-month-old KI mice for 14 days, and levels of proinflammatory cytokines IL-1ß, TNFα, and IL-6 measured in homogenates of mouse cortex using ELISA. Glial markers IBA1, CD45, CD68, and GFAP were assessed by immunohistochemistry. Microglia and amyloid plaques were quantified by immunofluorescence staining followed by confocal imaging. Levels of soluble and insoluble of Aß40 and Aß42 were measured by ELISA. The studies of in vivo pharmacodynamic effects on markers of neuroinflammation were complemented by mechanistic studies in the murine microglia BV2 cell line, using live cell imaging techniques to monitor proliferation, migration, and phagocytosis activities. RESULTS: Intervention with MW150 in KI mice during the established therapeutic time window attenuated the increased levels of IL-1ß and TNFα but not IL-6. MW150 treatment also increased the IBA1+ microglia within a 15 µm radius of the amyloid plaques, without significantly affecting overall microglia or plaque volume. Levels of IBA1, CD45, CD68, GFAP, and Aß40 and Aß42 were not affected by MW150 treatment. MW150 did not significantly alter microglial migration, proliferation, or phagocytosis in BV2 cells. CONCLUSIONS: Our results demonstrate that MW150 at an efficacious dose can selectively modulate neuroinflammatory responses associated with pathology progression without pan-suppression of normal physiological functions of microglia.

Closed head injury in an age-related Alzheimer mouse model leads to an altered neuroinflammatory response and persistent cognitive impairment.

Epidemiological studies have associated increased risk of Alzheimer's disease (AD)-related clinical symptoms with a medical history of head injury. Currently, little is known about pathophysiology mechanisms linked to this association. Persistent neuroinflammation is one outcome observed in patients after a single head injury. Neuroinflammation is also present early in relevant brain regions during AD pathology progression. In addition, previous mechanistic studies in animal models link neuroinflammation as a contributor to neuropathology and cognitive impairment in traumatic brain injury (TBI) or AD-related models. Therefore, we explored the potential interplay of neuroinflammatory responses in TBI and AD by analysis of the temporal neuroinflammatory changes after TBI in an AD model, the APP/PS1 knock-in (KI) mouse. Discrete temporal aspects of astrocyte, cytokine, and chemokine responses in the injured KI mice were delayed compared with the injured wild-type mice, with a peak neuroinflammatory response in the injured KI mice occurring at 7 d after injury. The neuroinflammatory responses were more persistent in the injured KI mice, leading to a chronic neuroinflammation. At late time points after injury, KI mice exhibited a significant impairment in radial arm water maze performance compared with sham KI mice or injured wild-type mice. Intervention with a small-molecule experimental therapeutic (MW151) that selectively attenuates proinflammatory cytokine production yielded improved cognitive behavior outcomes, consistent with a link between neuroinflammatory responses and altered risk for AD-associated pathology changes with head injury.

Targeting innate immunity for neurodegenerative disorders of the central nervous system.

Neuroinflammation is critically involved in numerous neurodegenerative diseases, and key signaling steps of innate immune activation hence represent promising therapeutic targets. This mini review series originated from the 4th Venusberg Meeting on Neuroinflammation held in Bonn, Germany, 7-9th May 2015, presenting updates on innate immunity in acute brain injury and chronic neurodegenerative disorders, such as traumatic brain injury and Alzheimer disease, on the role of astrocytes and microglia, as well as technical developments that may help elucidate neuroinflammatory mechanisms and establish clinical relevance. In this meeting report, a brief overview of physiological and pathological microglia morphology is followed by a synopsis on PGE2 receptors, insights into the role of arginine metabolism and further relevant aspects of neuroinflammation in various clinical settings, and concluded by a presentation of technical challenges and solutions when working with microglia and astrocyte cultures. Microglial ontogeny and induced pluripotent stem cell-derived microglia, advances of TREM2 signaling, and the cytokine paradox in Alzheimer's disease are further contributions to this article. Neuroinflammation is critically involved in numerous neurodegenerative diseases, and key signaling steps of innate immune activation hence represent promising therapeutic targets. This mini review series originated from the 4th Venusberg Meeting on Neuroinflammation held in Bonn, Germany, 7-9th May 2015, presenting updates on innate immunity in acute brain injury and chronic neurodegenerative disorders, such as traumatic brain injury and Alzheimer's disease, on the role of astrocytes and microglia, as well as technical developments that may help elucidate neuroinflammatory mechanisms and establish clinical relevance. In this meeting report, a brief overview on physiological and pathological microglia morphology is followed by a synopsis on PGE2 receptors, insights into the role of arginine metabolism and further relevant aspects of neuroinflammation in various clinical settings, and concluded by a presentation of technical challenges and solutions when working with microglia cultures. Microglial ontogeny and induced pluripotent stem cell-derived microglia, advances of TREM2 signaling, and the cytokine paradox in Alzheimer's disease are further contributions to this article.

Diffuse traumatic brain injury induces prolonged immune dysregulation and potentiates hyperalgesia following a peripheral immune challenge.

BACKGROUND: Nociceptive and neuropathic pain occurs as part of the disease process after traumatic brain injury (TBI) in humans. Central and peripheral inflammation, a major secondary injury process initiated by the traumatic brain injury event, has been implicated in the potentiation of peripheral nociceptive pain. We hypothesized that the inflammatory response to diffuse traumatic brain injury potentiates persistent pain through prolonged immune dysregulation. RESULTS: To test this, adult, male C57BL/6 mice were subjected to midline fluid percussion brain injury or to sham procedure. One cohort of mice was analyzed for inflammation-related cytokine levels in cortical biopsies and serum along an acute time course. In a second cohort, peripheral inflammation was induced seven days after surgery/injury with an intraplantar injection of carrageenan. This was followed by measurement of mechanical hyperalgesia, glial fibrillary acidic protein and Iba1 immunohistochemical analysis of neuroinflammation in the brain, and flow cytometric analysis of T-cell differentiation in mucosal lymph. Traumatic brain injury increased interleukin-6 and chemokine ligand 1 levels in the cortex and serum that peaked within 1-9 h and then resolved. Intraplantar carrageenan produced mechanical hyperalgesia that was potentiated by traumatic brain injury. Further, mucosal T cells from brain-injured mice showed a distinct deficiency in the ability to differentiate into inflammation-suppressing regulatory T cells (Tregs). CONCLUSIONS: We conclude that traumatic brain injury increased the inflammatory pain associated with cutaneous inflammation by contributing to systemic immune dysregulation. Regulatory T cells are immune suppressors and failure of T cells to differentiate into regulatory T cells leads to unregulated cytokine production which may contribute to the potentiation of peripheral pain through the excitation of peripheral sensory neurons. In addition, regulatory T cells are identified as a potential target for therapeutic rebalancing of peripheral immune homeostasis to improve functional outcome and decrease the incidence of peripheral inflammatory pain following traumatic brain injury.

Attenuation of traumatic brain injury-induced cognitive impairment in mice by targeting increased cytokine levels with a small molecule experimental therapeutic.

BACKGROUND: Evidence from clinical studies and preclinical animal models suggests that proinflammatory cytokine overproduction is a potential driving force for pathology progression in traumatic brain injury (TBI). This raises the possibility that selective targeting of the overactive cytokine response, a component of the neuroinflammation that contributes to neuronal dysfunction, may be a useful therapeutic approach. MW151 is a CNS-penetrant, small molecule experimental therapeutic that selectively restores injury- or disease-induced overproduction of proinflammatory cytokines towards homeostasis. We previously reported that MW151 administered post-injury (p.i.) is efficacious in a closed head injury (CHI) model of diffuse TBI in mice. Here we test dose dependence of MW151 to suppress the target mechanism (proinflammatory cytokine up-regulation), and explore the therapeutic window for MW151 efficacy. METHODS: We examined suppression of the acute cytokine surge when MW151 was administered at different times post-injury and the dose-dependence of cytokine suppression. We also tested a more prolonged treatment with MW151 over the first 7 days post-injury and measured the effects on cognitive impairment and glial activation. RESULTS: MW151 administered up to 6 h post-injury suppressed the acute cytokine surge, in a dose-dependent manner. Administration of MW151 over the first 7 days post-injury rescues the CHI-induced cognitive impairment and reduces glial activation in the focus area of the CHI. CONCLUSIONS: Our results identify a clinically relevant time window post-CHI during which MW151 effectively restores cytokine production back towards normal, with a resultant attenuation of downstream cognitive impairment.

Time-dependent effects of CX3CR1 in a mouse model of mild traumatic brain injury.

BACKGROUND: Neuroinflammation is an important secondary mechanism that is a key mediator of the long-term consequences of neuronal injury that occur in traumatic brain injury (TBI). Microglia are highly plastic cells with dual roles in neuronal injury and recovery. Recent studies suggest that the chemokine fractalkine (CX3CL1, FKN) mediates neural/microglial interactions via its sole receptor CX3CR1. CX3CL1/CX3CR1 signaling modulates microglia activation, and depending upon the type and time of injury, either protects or exacerbates neurological diseases. METHODS: In this study, mice deficient in CX3CR1 were subjected to mild controlled cortical impact injury (CCI), a model of TBI. We evaluated the effects of genetic deletion of CX3CR1 on histopathology, cell death/survival, microglia activation, and cognitive function for 30 days post-injury. RESULTS: During the acute post-injury period (24 h-15 days), motor deficits, cell death, and neuronal cell loss were more profound in injured wild-type than in CX3CR1(-/-) mice. In contrast, during the chronic period of 30 days post-TBI, injured CX3CR1(-/-) mice exhibited greater cognitive dysfunction and increased neuronal death than wild-type mice. The protective and deleterious effects of CX3CR1 were associated with changes in microglia phenotypes; during the acute phase CX3CR1(-/-) mice showed a predominant anti-inflammatory M2 microglial response, with increased expression of Ym1, CD206, and TGFß. In contrast, increased M1 phenotypic microglia markers, Marco, and CD68 were predominant at 30 days post-TBI. CONCLUSION: Collectively, these novel data demonstrate a time-dependent role for CX3CL1/CX3CR1 signaling after TBI and suggest that the acute and chronic responses to mild TBI are modulated in part by distinct microglia phenotypes.

The p38α MAPK regulates microglial responsiveness to diffuse traumatic brain injury.

Neuropathology after traumatic brain injury (TBI) is the result of both the immediate impact injury and secondary injury mechanisms. Unresolved post-traumatic glial activation is a secondary injury mechanism that contributes to a chronic state of neuroinflammation in both animal models of TBI and human head injury patients. We recently demonstrated, using in vitro models, that p38α MAPK signaling in microglia is a key event in promoting cytokine production in response to diverse disease-relevant stressors and subsequent inflammatory neuronal dysfunction. From these findings, we hypothesized that the p38α signaling pathway in microglia could be contributing to the secondary neuropathologic sequelae after a diffuse TBI. Mice where microglia were p38α-deficient (p38α KO) were protected against TBI-induced motor deficits and synaptic protein loss. In wild-type (WT) mice, diffuse TBI produced microglia morphological activation that lasted for at least 7 d; however, p38α KO mice failed to activate this response. Unexpectedly, we found that the peak of the early, acute phase cytokine and chemokine levels was increased in injured p38α KO mice compared with injured WT mice. The increased cytokine levels in the p38α KO mice could not be accounted for by more infiltration of macrophages or neutrophils, or increased astrogliosis. By 7 d after injury, the cytokine and chemokine levels remained elevated in injured WT mice but not in p38α KO mice. Together, these data suggest that p38α balances the inflammatory response by acutely attenuating the early proinflammatory cytokine surge while perpetuating the chronic microglia activation after TBI.

The p38alpha mitogen-activated protein kinase limits the CNS proinflammatory cytokine response to systemic lipopolysaccharide, potentially through an IL-10 dependent mechanism.

BACKGROUND: The p38α mitogen-activated protein kinase (MAPK) is a well-characterized intracellular kinase involved in the overproduction of proinflammatory cytokines from glia. As such, p38α appears to be a promising therapeutic target for neurodegenerative diseases associated with neuroinflammation. However, the in vivo role of p38α in cytokine production in the CNS is poorly defined, and prior work suggests that p38α may be affecting a yet to be identified negative feedback mechanism that limits the acute, injury-induced proinflammatory cytokine surge in the CNS. METHODS: To attempt to define this negative feedback mechanism, we used two in vitro and two in vivo models of neuroinflammation in a mouse where p38α is deficient in cells of the myeloid lineage. RESULTS: We found that p38α in myeloid cells has an important role in limiting amplitude of the acute proinflammatory cytokine response to a systemic inflammatory challenge. Moreover, we identified IL-10 as a potential negative feedback mechanism regulated by p38α. CONCLUSIONS: Our data suggest that p38α regulates a proper balance between the pro- and anti-inflammatory cytokine responses to systemic inflammation, and that if circulating IL-10 levels are not elevated to counter-balance the increased systemic proinflammatory responses, the spread of the inflammatory response from the periphery to the CNS is exaggerated.

Targeting astrocytes ameliorates neurologic changes in a mouse model of Alzheimer's disease.

Astrocytes are the most abundant cell type in the brain and play a critical role in maintaining healthy nervous tissue. In Alzheimer's disease (AD) and most other neurodegenerative disorders, many astrocytes convert to a chronically "activated" phenotype characterized by morphologic and biochemical changes that appear to compromise protective properties and/or promote harmful neuroinflammatory processes. Activated astrocytes emerge early in the course of AD and become increasingly prominent as clinical and pathological symptoms progress, but few studies have tested the potential of astrocyte-targeted therapeutics in an intact animal model of AD. Here, we used adeno-associated virus (AAV) vectors containing the astrocyte-specific Gfa2 promoter to target hippocampal astrocytes in APP/PS1 mice. AAV-Gfa2 vectors drove the expression of VIVIT, a peptide that interferes with the immune/inflammatory calcineurin/NFAT (nuclear factor of activated T-cells) signaling pathway, shown by our laboratory and others to orchestrate biochemical cascades leading to astrocyte activation. After several months of treatment with Gfa2-VIVIT, APP/PS1 mice exhibited improved cognitive and synaptic function, reduced glial activation, and lower amyloid levels. The results confirm a deleterious role for activated astrocytes in AD and lay the groundwork for exploration of other novel astrocyte-based therapies.

Early stage drug treatment that normalizes proinflammatory cytokine production attenuates synaptic dysfunction in a mouse model that exhibits age-dependent progression of Alzheimer's disease-related pathology.

Overproduction of proinflammatory cytokines in the CNS has been implicated as a key contributor to pathophysiology progression in Alzheimer's disease (AD), and extensive studies with animal models have shown that selective suppression of excessive glial proinflammatory cytokines can improve neurologic outcomes. The prior art, therefore, raises the logical postulation that intervention with drugs targeting dysregulated glial proinflammatory cytokine production might be effective disease-modifying therapeutics if used in the appropriate biological time window. To test the hypothesis that early stage intervention with such drugs might be therapeutically beneficial, we examined the impact of intervention with MW01-2-151SRM (MW-151), an experimental therapeutic that selectively attenuates proinflammatory cytokine production at low doses. MW-151 was tested in an APP/PS1 knock-in mouse model that exhibits increases in AD-relevant pathology progression with age, including increases in proinflammatory cytokine levels. Drug was administered during two distinct but overlapping therapeutic time windows of early stage pathology development. MW-151 treatment attenuated the increase in microglial and astrocyte activation and proinflammatory cytokine production in the cortex and yielded improvement in neurologic outcomes, such as protection against synaptic protein loss and synaptic plasticity impairment. The results also demonstrate that the therapeutic time window is an important consideration in efficacy studies of drugs that modulate glia biological responses involved in pathology progression and suggest that such paradigms should be considered in the development of new therapeutic regimens that seek to delay the onset or slow the progression of AD.

Clinically relevant intronic splicing enhancer mutation in myelin proteolipid protein leads to progressive microglia and astrocyte activation in white and gray matter regions of the brain.

INTRODUCTION: Mutations in proteolipid protein (PLP), the most abundant myelin protein in the CNS, cause the X-linked dysmyelinating leukodystrophies, Pelizaeus-Merzbacher disease (PMD) and spastic paraplegia type 2 (SPG2). Point mutations, deletion, and duplication of the PLP1 gene cause PMD/SPG2 with varying clinical presentation. Deletion of an intronic splicing enhancer (ISEdel) within intron 3 of the PLP1 gene is associated with a mild form of PMD. Clinical and preclinical studies have indicated that mutations in myelin proteins, including PLP, can induce neuroinflammation, but the temporal and spatial onset of the reactive glia response in a clinically relevant mild form of PMD has not been defined. METHODS: A PLP-ISEdel knockin mouse was used to examine the behavioral and neuroinflammatory consequences of a deletion within intron 3 of the PLP gene, at two time points (two and four months old) early in the pathological progression. Mice were characterized functionally using the open field task, elevated plus maze, and nesting behavior. Quantitative neuropathological analysis was for markers of astrocytes (GFAP), microglia (IBA1, CD68, MHCII) and axons (APP). The Aperio ScanScope was used to generate a digital, high magnification photomicrograph of entire brain sections. These digital slides were used to quantify the immunohistochemical staining in ten different brain regions to assess the regional heterogeneity in the reactive astrocyte and microglial response. RESULTS: The PLP-ISEdel mice exhibited behavioral deficits in the open field and nesting behavior at two months, which did not worsen by four months of age. A marker of axonal injury (APP) increased from two months to four months of age. Striking was the robust reactive astrocyte and microglia response which was also progressive. In the two-month-old mice, the astrocyte and microglia reactivity was most apparent in white matter rich regions of the brain. By four months of age the gliosis had become widespread and included both white as well as gray matter regions of the brain. CONCLUSIONS: Our results indicate, along with other preclinical models of PMD, that an early reactive glia response occurs following mutations in the PLP gene, which may represent a potentially clinically relevant, oligodendrocyte-independent therapeutic target for PMD.

Proinflammatory and Immunomodulatory Gene and Protein Expression Patterns in Spinal Cord and Spleen Following Acute and Chronic High Thoracic Injury.

Introduction: In addition to paralysis and loss of sensation, high-level spinal cord injury (SCI) causes sympathetic dysfunction that can lead to autonomic dysreflexia (AD) and chronic immune suppression involving splenic leukopenia. Evidence has shown that treatment with either gabapentin or blockade of TNFα mitigates maladaptive plasticity and the underlying hemodynamic dysfunction, spleen atrophy, and immune dysfunction associated with AD. Because significant improvements long term was noted following treatments only during acute stages of recovery, we sought to systematically examine changes in proinflammatory and immunomodulatory cytokines to ascertain the reason. Methods: Adult female Wistar rats underwent complete T4 spinal transection before euthanasia at systematic intervals from 3 days to 8 weeks after injury. Using qRT-PCR and meso scale discovery (MSD) assays, the gene and protein expression of TNFα and IFNγ in the spleen, upper thoracic (T4-9) and lumbosacral (L5-S6) spinal cords were analyzed. Results: We found that spleen atrophy occurs in a biphasic manner compared to naïve controls, with significant decreases in the spleen mass noted at 3 days and 8 weeks after injury. Splenic TNFα mRNA and protein levels did not change significantly over time, while IFNγ gene expression dipped acutely with trends for increased protein levels at more chronic time points. TNFα protein increased significantly only in thoracic spinal cord segments from 3 to 14 days post-injury. IFNγ mRNA and protein levels remained unelevated in injured spinal cords over time, with trends for increased protein levels at 2 and 8 weeks in the lumbosacral segments. Discussion: Novel temporal-spatial cytokine expression profiles reveal that TNFα protein levels are increased solely in upper thoracic segments after high thoracic SCI, while IFNγ remains unaltered. Splenic leukopenia and latent systemic immunosuppression are not associated with altered TNFα or IFNγ expression in the spleen or spinal cord.

The localization of molecularly distinct microglia populations to Alzheimer's disease pathologies using QUIVER.

New histological techniques are needed to examine protein distribution in human tissues, which can reveal cell shape and disease pathology connections. Spatial proteomics has changed the study of tumor microenvironments by identifying spatial relationships of immunomodulatory cells and proteins and contributing to the discovery of new cancer immunotherapy biomarkers. However, the fast-expanding toolkit of spatial proteomic approaches has yet to be systematically applied to investigate pathological alterations in the aging human brain in health and disease states. Moreover, post-mortem human brain tissue presents distinct technical problems due to fixation procedures and autofluorescence, which limit fluorescence methodologies. This study sought to develop a multiplex immunohistochemistry approach (visualizing the immunostain with brightfield microscopy). Quantitative multiplex Immunohistochemistry with Visual colorimetric staining to Enhance Regional protein localization (QUIVER) was developed to address these technical challenges. Using QUIVER, a ten-channel pseudo-fluorescent image was generated using chromogen removal and digital microscopy to identify unique molecular microglia phenotypes. Next, the study asked if the tissue environment, specifically the amyloid plaques and neurofibrillary tangles characteristic of Alzheimer's disease, has any bearing on microglia's cellular and molecular phenotypes. QUIVER allowed the visualization of five molecular microglia/macrophage phenotypes using digital pathology tools. The recognizable reactive and homeostatic microglia/macrophage phenotypes demonstrated spatial polarization towards and away from amyloid plaques, respectively. Yet, microglia morphology appearance did not always correspond to molecular phenotype. This research not only sheds light on the biology of microglia but also offers QUIVER, a new tool for examining pathological alterations in the brains of the elderly.

Terminally differentiated effector memory T cells associate with cognitive and AD-related biomarkers in an aging-based community cohort.

Background and Purpose: The immune response changes during aging and the progression of Alzheimer's disease (AD) and related dementia (ADRD). Terminally differentiated effector memory T cells (called TEMRA) are important during aging and AD due to their cytotoxic phenotype and association with cognitive decline. However, it is not clear if the changes seen in TEMRAs are specific to AD-related cognitive decline specifically or are more generally correlated with cognitive decline. This study aimed to examine whether TEMRAs are associated with cognition and plasma biomarkers of AD, neurodegeneration, and neuroinflammation in a community-based cohort of older adults. Methods: Study participants from a University of Kentucky Alzheimer's Disease Research Center (UK-ADRC) community-based cohort of aging and dementia were used to test our hypothesis. There were 84 participants, 44 women and 40 men. Participants underwent physical examination, neurological examination, medical history, cognitive testing, and blood collection to determine plasma biomarker levels (Aß42/Aß40 ratio, total tau, Neurofilament Light chain (Nf-L), Glial Fibrillary Acidic Protein (GFAP)) and to isolate peripheral blood mononuclear cells (PBMCs). Flow cytometry was used to analyze PBMCs from study participants for effector and memory T cell populations, including CD4+ and CD8+ central memory T cells (TCM), Naïve T cells, effector memory T cells (TEM), and effector memory CD45RA+ T cells (TEMRA) immune cell markers. Results: CD8+ TEMRAs were positively correlated with Nf-L and GFAP. We found no significant difference in CD8+ TEMRAs based on cognitive scores and no associations between CD8+ TEMRAs and AD-related biomarkers. CD4+ TEMRAs were associated with cognitive impairment on the MMSE. Gender was not associated with TEMRAs, but it did show an association with other T cell populations. Conclusion: These findings suggest that the accumulation of CD8+ TEMRAs may be a response to neuronal injury (Nf-L) and neuroinflammation (GFAP) during aging or the progression of AD and ADRD. As our findings in a community-based cohort were not clinically-defined AD participants but included all ADRDs, this suggests that TEMRAs may be associated with changes in systemic immune T cell subsets associated with the onset of pathology.