Is microglial dystrophy a form of cellular senescence? An analysis of senescence markers in the aged human brain.

Aging can cause morphological transformation in human microglia indicative of cell senescence, termed microglial dystrophy. However, cellular senescence is characterized by additional changes, such as an irregular cell cycle arrest, and a variety of metabolic and molecular changes including a senescence-associated secretory phenotype, dysfunction of degradation mechanisms, and altered DNA damage response. Here, we tested whether dystrophic microglia display customary markers of cell senescence by performing double and triple staining in sections of the temporal lobe and brain stem from 14 humans. We found that markers related to oxidative damage, such as upregulation of 8-hydroxy-2'-deoxyguanosine (8-OHdG), hemeoxygenase-1 (HO-1), and y-H2AX, as well as inclusion of lipofuscin, do not or only exceptionally colocalize with dystrophic microglia. Further, we did not observe a decline in lamin B1 around nuclear laminae in either dystrophic or ramified microglia within the same microscopic field. Only ferritin expression, which is known to increase with aging in CNS microglia, was frequently observed in dystrophic, but rarely in ramified microglial cells. We conclude that neither dystrophic nor ramified microglia in human brain exhibit significant expression of conventional senescence markers associated with oxidative stress, and that ferritin is the dominant immunophenotypic change related to microglial aging. We suggest that multiple pathogenic mechanisms other than those driving cellular senescence contribute to dystrophic transformation of microglia.

Dystrophic microglia in late-onset Alzheimer's disease.

Here, we summarize current understanding of functional involvement of microglial cells in the most common neurodegenerative disease to affect humans, which is sporadic or late-onset Alzheimer's disease (LOAD). Our review narrowly focuses on insights obtained from post-mortem neuropathological examinations of human brains paying particular attention to microglia as these cells have long been implicated as pivotal players in the cellular processes that lead to AD-type neurodegeneration. Although complete understanding of the roles played by microglia in AD neurodegeneration remains elusive, our studies thus far have illuminated microglial involvement in LOAD, showing that microglial dystrophy, the morphological manifestation of senescence, can be integrated with other hallmark pathological features of AD, such as intraneuronal neurofibrillary degeneration (NFD) and extracellular deposits of amyloid-beta (Aß) protein. We have demonstrated an in situ correlation between microglial dystrophy and presence of NFD suggesting that neurodegeneration is secondary to aging-related microglial deterioration, a concept founded on the notion that proper neuronal function is dependent on presence of healthy microglia. Diseased or weakened glia are detrimental for neuronal well-being because their ability to provide neuronal support may be impaired. Our most recent work also links microglial dystrophy with Aß deposits by showing that there is a chronic, yet futile microglial reaction to insoluble amyloid deposits. This inability of microglia to remove aggregated amyloid (a foreign body) causes microglial exhaustion and thereby exacerbates already ongoing aging-dependent microglial deterioration. An eventual total loss of functional microglia in advanced LOAD promotes widespread NFD, dementia, and brain failure.

Microglia senescence occurs in both substantia nigra and ventral tegmental area.

During aging humans lose midbrain dopamine neurons, but not all dopamine regions exhibit vulnerability to neurodegeneration. Microglia maintain tissue homeostasis and neuronal support, but microglia become senescent and likely lose some of their functional abilities. Since aging is the greatest risk factor for Parkinson's disease, we hypothesized that aging-related changes in microglia and neurons occur in the vulnerable substantia nigra pars compacta (SNc) but not the ventral tegmental area (VTA). We conducted stereological analyses to enumerate microglia and dopaminergic neurons in the SNc and VTA of 1-, 6-, 9-, 18-, and 24-month-old C57BL/J6 mice using sections double-stained with tyrosine hydroxylase (TH) and Iba1. Both brain regions show an increase in microglia with aging, whereas numbers of TH+ cells show no significant change after 9 months of age in SNc and 6 months in VTA. Morphometric analyses reveal reduced microglial complexity and projection area while cell body size increases with aging. Contact sites between microglia and dopaminergic neurons in both regions increase with aging, suggesting increased microglial support/surveillance of dopamine neurons. To assess neurotrophin expression in dopaminergic neurons, BDNF and TH mRNA were quantified. Results show that the ratio of BDNF to TH decreases in the SNc, but not the VTA. Gait analysis indicates subtle, aging-dependent changes in gait indices. In conclusion, increases in microglial cell number, ratio of microglia to dopamine neurons, and contact sites suggest that innate biological mechanisms compensate for the aging-dependent decline in microglia morphological complexity (senescence) to ensure continued neuronal support in the SNc and VTA.

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

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

Microglial activation occurs late during preclinical Alzheimer's disease.

Sporadic Alzheimer's disease (AD) is marked by a lengthy preclinical phase during which patients are nonsymptomatic but show pathology in variable manifestations. Whether or not neuroinflammation occurs in such nondemented individuals is unknown. We evaluated the medial temporal lobe of 66 nondemented subjects, aged 42-93, in terms of tau pathology, Aß deposition, and microglial activation. We show that 100% of subjects had neurofibrillary degeneration (NFD), 35% had Aß deposits, and 8% revealed microglial activation in individuals where early amyloid formation was apparent by Congo Red staining. Amyloid-induced neuroinflammatory clusters of Iba1, CD68, and ferritin-positive microglia were evident in the immediate vicinity of aggregated Aß. Microglia in the adjacent neuropil were nonactivated. Thus, neuroinflammation in AD represents a highly localized phagocyte reaction, essentially a foreign body response, geared toward removal of insoluble Aß. Because clustered microglia in some amyloid plaques were dystrophic and ferritin-positive, we hypothesize that these cells were exhausted by their attempts to remove the aggregated, insoluble Aß. Our findings show that the sequence of pathologic events in AD begins with tau pathology, followed by Aß deposition, and then by microglial activation. Because only 8% of our subjects revealed all three hallmark pathologic features, we propose that these nondemented individuals were near the threshold of transitioning from nonsymptomatic to symptomatic disease. The onset of neuroinflammation in AD may thus represent a tipping point in AD pathogenesis. Our study suggests that the role of microglia in AD pathogenesis entails primarily the attempted removal of potentially toxic, extracellular material.

HIV-1 Tat regulation of dopamine transmission and microglial reactivity is brain region specific.

The transactivator of transcription protein, HIV-1 Tat, is linked to neuroAIDS, where degeneration of dopamine neurons occurs. Using a mouse model expressing GFAP-driven Tat protein under doxycycline (Dox) regulation, we investigated microglial-neuronal interactions in the rostral substantia nigra pars compacta (SNc). Immunohistochemistry for microglia and tyrosine hydroxylase (TH) showed that the ratio of microglia to dopamine neurons is smaller in the SNc than in the ventral tegmental area (VTA) and that this difference is maintained following 7-day Dox exposure in wild type animals. Administration of Dox to wild types had no effect on microglial densities. In addressing the sensitivity of neurons to potentially adverse effects of HIV-1 Tat, we found that HIV-1 Tat exposure in vivo selectively decreased TH immunoreactivity in the SNc but not in the VTA, while levels of TH mRNA in the SNc remained unchanged. HIV-1 Tat induction in vivo did not alter the total number of neurons in these brain regions. Application of Tat (5 ng) into dopamine neurons with whole-cell patch pipette decreased spontaneous firing activity. Tat induction also produced a decline in microglial cell numbers, but no microglial activation. Thus, disappearance of dopaminergic phenotype is due to a loss of TH immunoreactivity rather than to neuronal death, which would have triggered microglial activation. We conclude that adverse effects of HIV-1 Tat produce a hypodopamine state by decreasing TH immunoreactivity and firing activity of dopamine neurons. Reduced microglial numbers after Tat exposure in vivo suggest impaired microglial functions and altered bidirectional interactions between dopamine neurons and microglia.

Methamphetamine neurotoxicity, microglia, and neuroinflammation.

Methamphetamine (METH) is an illicit psychostimulant that is subject to abuse worldwide. While the modulatory effects of METH on dopamine neurotransmission and its neurotoxicity in the central nervous system are well studied, METH's effects on modulating microglial neuroimmune functions and on eliciting neuroinflammation to affect dopaminergic neurotoxicity has attracted considerable attention in recent years. The current review illuminates METH-induced neurotoxicity from a neuropathological perspective by summarizing studies reporting microglial activation after METH administration in rodents. Assessing microglial reactivity in terms of the cells' morphology and immunophenotype offers an opportunity for comprehensive and objective assessment of the severity and nature of METH-induced neuronal perturbations in the CNS and can thus contribute to a better understanding of the nature of METH toxicity. We reach the conclusion here that the intensity of microglial activation reported in the majority of animal models after METH administration is quite modest, indicating that the extent of dopaminergic neuron damage directly caused by this neurotoxicant is relatively minor. Our conclusion stands in contrast to claims of excessive and detrimental neuroinflammation believed to contribute and exacerbate METH neurotoxicity. Thus, our analysis of published studies does not support the idea that suppression of microglial activity with anti-inflammatory agents could yield beneficial effects in terms of treating addiction disorders.

Astrocytic expression of the CXCL12 receptor, CXCR7/ACKR3 is a hallmark of the diseased, but not developing CNS.

Based on our previous demonstration of CXCR7 as the major mediator of CXCL12 signaling in cultured astrocytes, we have now compared astrocytic expression of the CXCL12 receptors, CXCR7 and CXCR4, during CNS development and disease. In addition, we asked whether disease-associated conditions/factors affect expression of CXCL12 receptors in astrocytes. In the late embryonic rat brain, CXCR7+/GFAP+ cells were restricted to the ventricular/subventricular zone while CXCR4 was widely absent from GFAP-positive cells. In the early postnatal and adult brain, CXCR7 and CXCR4 were almost exclusively expressed by GFAP-immunoreactive astrocytes forming the superficial glia limitans. Contrasting the situation in the intact CNS, a striking increase in astrocytic CXCR7 expression was detectable in the cortex of rats with experimental brain infarcts, in the spinal cord of rats with experimental autoimmune encephalomyelitis (EAE) and after mechanical compression, as well as in the in infarcted human cerebral cortex and in the hippocampus of Alzheimer's disease patients. None of these pathologies was associated with substantial increases in astrocytic CXCR4 expression. Screening of various disease-associated factors/conditions further revealed that CXCR7 expression of cultured cortical astrocytes increases with IFNγ as well as under hypoxic conditions whereas CXCR7 expression is attenuated following treatment with IFNß. Again, none of the treatments affected CXCR4 expression in cultured astrocytes. Together, these findings support the hypothesis of a crucial role of astrocytic CXCR7 in the progression of various CNS pathologies.

Inhomogeneous distribution of Iba-1 characterizes microglial pathology in Alzheimer's disease.

Microglial dystrophy has recently been described as a morphological phenotype of microglia that differs from resting and activated states by spheroid formation and cytorrhexis. In thick sections immunolabeled for HLA-DR or Iba-1 dystrophic microglial processes lose their typical, homogeneous staining pattern and appear to be fragmented or clustered. In this study, we performed double immunofluorescence and electron microscopy to determine if this labeling pattern indeed reflects complete separation of microglial processes from the soma. Using Iba-1/CD68 and Iba-1/MHC class II, as microglial markers, we observed that isolated Iba-1 fragments were still connected to each other by segments of the microglial process immune positive for CD68 or MHC class II. Ultrathin serial sections of two Iba-1 fragments which appeared to be disconnected from each other at the light microscopical level revealed a still existing "bridge" with a diameter of around 0.182 µm. Therefore, microglial dystrophy may reflect alterations of the cytoskeleton ultimately leading to slow cytorrhexis. GLIA 2016;64:1562-1572.

Microglia in dementia with Lewy bodies.

Microglial activation (neuroinflammation) is often cited as a pathogenic factor in the development of neurodegenerative diseases. However, there are significant caveats associated with the idea that inflammation directly causes either α-synuclein pathology or neurofibrillary degeneration (NFD). We have performed immunohistochemical studies on microglial cells in five cases of dementia with Lewy bodies (DLB), median age 87, and nine cases of non-demented (ND) controls, median age 74, using tissue samples from the temporal lobe and the superior frontal gyrus. Three different antibodies known to label microglia and macrophages were employed: iba1, anti-CD68, and anti-ferritin. All DLB cases showed both α-synuclein pathology (Lewy bodies and neurites) and NFD ranging from Braak stage II to IV. In contrast, all controls were devoid of α-synuclein pathology but did show NFD ranging from Braak stage I to III. Using iba1 labeling, our current results show a notable absence of activated microglia in all cases with the exception of two controls that showed small focal areas of microglial activation and macrophage formation. Both iba1 and ferritin antibodies revealed a mixture of ramified and dystrophic microglial cells throughout the regions examined, and there were no measurable differences in the prevalence of dystrophic microglial cells between DLB and controls. Double-labeling for α-synuclein and iba1-positive microglia showed that cortical Lewy bodies were surrounded by both ramified and dystrophic microglial cells. We found an increase in CD68 expression in DLB cases relative to controls. Since microglial dystrophy has been linked to NFD and since it did not appear to be worse in DLB cases over controls, our findings support the idea that the additional Lewy body pathology in DLB is not the result of intensified microglial dystrophy. CD68 is likely associated with lipofuscin deposits in microglial cells which may be increased in DLB cases because of impaired proteostasis. Overall, we conclude that neurodegenerative changes in DLB are unlikely to result directly from activated microglia but rather from dysfunctional ones.

Presence of severe neuroinflammation does not intensify neurofibrillary degeneration in human brain.

This study investigated the allegedly causal relationship between microglial activation and neurofibrillary degeneration (NFD) typical of Alzheimer's disease (AD) by determining if presence of extreme microglial activation coincides with intensified NFD. We performed comparative histopathological analyses of NFD and microglial reactivity in 18 primary subjects ranging from 4 to 51 years of age. Ten of these subjects (median age 34) died from infectious disease (HIV, sepsis) and CNS trauma, while eight subjects (median age 32.5) died from non-infectious conditions (controls). Second, we also examined two 52-year-old subjects with Down syndrome where one had comorbid sepsis and the other one did not. We found that all 10 subjects with infectious/traumatic diagnoses showed severe neuroinflammation, while the 8 control subjects completely lacked neuroinflammatory changes. However, all 18 primary subjects were found to show the same early-stage, pretangle neuropathology of Braak stage 1a and 1b, that is, they exhibited primarily subcortical NFD in the locus coeruleus and sporadic lesions in the transentorhinal cortex. Similarly, the two subjects with Down syndrome showed the same high levels of NFD (Braak stage VI) irrespective of the comorbid sepsis-related neuroinflammation present in one of these individuals. Collectively, our findings show that despite rampant microglial activation in all subjects with neuroinflammatory conditions the extent of NFD is at the same level as seen in non-inflamed controls. These findings demonstrate that microglial activation does not initiate or exacerbate NFD, and we conclude that CNS inflammation is unlikely to be causally involved in the development of NFD characteristic of AD dementia.

Droplet Degeneration of Hippocampal and Cortical Neurons Signifies the Beginning of Neuritic Plaque Formation.

BACKGROUND: Neuritic plaques contain neural and microglial elements, and amyloid-ß protein (Aß), but their pathogenesis remains unknown. OBJECTIVE: Elucidate neuritic plaque pathogenesis. METHODS: Histochemical visualization of hyperphosphorylated-tau positive (p-tau+) structures, microglia, Aß, and iron. RESULTS: Disintegration of large projection neurons in human hippocampus and neocortex presents as droplet degeneration: pretangle neurons break up into spheres of numerous p-tau+ droplets of various sizes, which marks the beginning of neuritic plaques. These droplet spheres develop in the absence of colocalized Aß deposits but once formed become encased in diffuse Aß with great specificity. In contrast, neurofibrillary tangles often do not colocalize with Aß. Double-labelling for p-tau and microglia showed a lack of microglial activation or phagocytosis of p-tau+ degeneration droplets but revealed massive upregulation of ferritin in microglia suggesting presence of high levels of free iron. Perl's Prussian blue produced positive staining of microglia, droplet spheres, and Aß plaque cores supporting the suggestion that droplet degeneration of pretangle neurons in the hippocampus and cortex represents ferroptosis, which is accompanied by the release of neuronal iron extracellularly. CONCLUSION: Age-related iron accumulation and ferroptosis in the CNS likely trigger at least two endogenous mechanisms of neuroprotective iron sequestration and chelation, microglial ferritin expression and Aß deposition, respectively, both contributing to the formation of neuritic plaques. Since neurofibrillary tangles and Aß deposits colocalize infrequently, tangle formation likely does not involve release of neuronal iron extracellularly. In human brain, targeted deposition of Aß occurs specifically in response to ongoing ferroptotic droplet degeneration thereby producing neuritic plaques.

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

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

Differential Glial Chitotriosidase 1 and Chitinase 3-like Protein 1 Expression in the Human Primary Visual Cortex and Cerebellum after Global Hypoxia-Ischemia.

Here, we studied the neuroinflammation- and ischemia-related glial markers chitotriosidase 1 (CHIT1) and chitinase-3-like protein 1 (CHI3L1, alias YKL-40) in the human striate cortex and cerebellum at different time points after global hypoxic-ischemic brain injury (HIBI). Both regions differ considerably in their glial cell population but are supplied by the posterior circulation. CHIT1 and CHI3L1 expression was compared to changes in microglial (IBA1, CD68), astrocytic (GFAP, S100ß), and neuronal markers (H&E, neurofilament heavy chain, NfH; calretinin, CALR) using immunohistochemistry and multiple-label immunofluorescence. Initial striatal cortical and cerebellar Purkinje cell damage, detectable already 1/2 d after HIBI, led to delayed neuronal death, whereas loss of cerebellar NfH-positive stellate and CALR-positive granule cells was variable. During the first week post-HIBI, a transient reduction of IBA1-positive microglia was observed in both regions, and fragmented/clasmatodendritic cerebellar Bergmann glia appeared. In long-term survivors, both brain regions displayed high densities of activated IBA1-positive cells and CD68-positive macrophages, which showed CHIT1 co-localization in the striate cortex. Furthermore, enlarged GFAP- and S100ß-positive astroglia emerged in both regions around 9-10 d post-HIBI, i.e., along with clearance of dead neurons from the neuropil, although GFAP-/S100ß-positive gemistocytic astrocytes that co-expressed CHI3L1 were found only in the striate cortex. Thus, only GFAP-/S100ß-positive astrocytes in the striate cortex, but not cerebellar Bergmann glia, differentiated into CHI3L1-positive gemistocytes. CHIT1 was co-expressed almost entirely in macrophages in the striate cortex and not cerebellum of long-term survivors, thereby indicating that CHIT1 and CHI3L1 could be valuable biomarkers for monitoring the outcome of global HIBI.

Functional characterization of the biogenic amine transporters on human macrophages.

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

Microglial pathology in Down syndrome.

Subjects with Down syndrome (DS) inevitably develop histopathological features pathognomonic of Alzheimer's disease (AD), and DS can therefore be considered a human model of AD. Similar to AD, microglial activation has been reported in DS and the idea that detrimental neuroinflammation plays a key role in the pathogenesis of neurodegeneration is firmly embedded. However, recent work from this laboratory has offered evidence for an alternative view regarding the role of microglial cells in AD pathogenesis by showing presence of dystrophic (senescent) rather than activated microglia in both the AD and DS brain. In this report, we build on previously published observations in human brain and offer a detailed analysis of microglial senescent pathology in the temporal cortices of 6 DS cases in their 40s, a critical age bracket where virtually all DS subjects acquire neurofibrillary degeneration characteristic of AD. Our findings using both Iba1 and anti-ferritin immunostaining of microglial cells show that coincident with the appearance of tau pathology in DS subjects there is consistent presence of dystrophic microglial cells and conspicuous absence of activated microglia using both markers. The extent of microglial pathology varied among the individual DS cases, but they all revealed decreased numbers of normal microglia ranging from 19 to 85% of the controls. Nearly all of the ferritin-positive microglia, which constitute a subset of the total Iba1-reactive microglial population, exhibited dystrophic morphology. In its most severe form dystrophy was evident as total fragmentation of the cells' cytoplasm (cytorrhexis), which likely reflects terminal degeneration of microglia. Severely dystrophic, ferritin-positive cells were often found to be colocalized with tau-positive senile plaques. Our findings help to consolidate the idea that microglial degeneration and neurofibrillary degeneration are closely linked events in a human model of AD. They suggest that microglial degeneration follows a gradually progressive course that increases in its severity in parallel with the progression of AD neurodegenerative changes.

The Role of Microglia in Sporadic Alzheimer's Disease.

Microglia constitute the brain's immune system and their involvement in Alzheimer's disease has been discussed. Commonly, and in line with the amyloid/neuroinflammation cascade hypothesis, microglia have been portrayed as potentially dangerous immune effector cells thought to be overactivated by amyloid and producing neurotoxic inflammatory mediators that lead to neurofibrillary degeneration. We disagree with this theory and offer as an alternative the microglial dysfunction theory stating that microglia become impaired in their normally neuroprotective roles because of aging, i.e., they become senescent and aging neurons degenerate because they lack the needed microglial support for their survival. Thus, while the amyloid cascade theory relies primarily on genetic data, the dysfunction theory incorporates aging as a critical etiological factor. Aging is the greatest risk factor for the sporadic (late-onset) and most common form of Alzheimer's disease, where fully penetrant genetic mutations are absent. In this review, we lay out and discuss the human evidence that supports senescent microglial dysfunction and conflicts with the amyloid/neuroinflammation idea.

Beyond Activation: Characterizing Microglial Functional Phenotypes.

Classically, the following three morphological states of microglia have been defined: ramified, amoeboid and phagocytic. While ramified cells were long regarded as "resting", amoeboid and phagocytic microglia were viewed as "activated". In aged human brains, a fourth, morphologically novel state has been described, i.e., dystrophic microglia, which are thought to be senescent cells. Since microglia are not replenished by blood-borne mononuclear cells under physiological circumstances, they seem to have an "expiration date" limiting their capacity to phagocytose and support neurons. Identifying factors that drive microglial aging may thus be helpful to delay the onset of neurodegenerative diseases, such as Alzheimer's disease (AD). Recent progress in single-cell deep sequencing methods allowed for more refined differentiation and revealed regional-, age- and sex-dependent differences of the microglial population, and a growing number of studies demonstrate various expression profiles defining microglial subpopulations. Given the heterogeneity of pathologic states in the central nervous system, the need for accurately describing microglial morphology and expression patterns becomes increasingly important. Here, we review commonly used microglial markers and their fluctuations in expression in health and disease, with a focus on IBA1 low/negative microglia, which can be found in individuals with liver disease.

TNFα increases tyrosine hydroxylase expression in human monocytes.

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

Microglia: biology and pathology.

The past 20 years have seen a gain in knowledge on microglia biology and microglia functions in disease that exceeds the expectations formulated when the microglia "immune network" was introduced. More than 10,000 articles have been published during this time. Important new research avenues of clinical importance have opened up such as the role of microglia in pain and in brain tumors. New controversies have also emerged such as the question of whether microglia are active or reactive players in neurodegenerative disease conditions, or whether they may be victims themselves. Premature commercial interests may be responsible for some of the confusion that currently surrounds microglia in both the Alzheimer and Parkinson's disease research fields. A critical review of the literature shows that the concept of "(micro)glial inflammation" is still open to interpretation, despite a prevailing slant towards a negative meaning. Perhaps the most exciting foreseeable development concerns research on the role of microglia in synaptic plasticity, which is expected to yield an answer to the question whether microglia are the brain's electricians. This review provides an analysis of the latest developments in the microglia field.