Neuronal CC chemokines:the distinct roles of CCL21 and CCL2 in neuropathic pain

The development of neuropathic pain in response to peripheral nerve lesion for a large part depends on microglia located at the dorsal horn of the spinal cord. Thus the injured nerve initiates a response of microglia, which represents the start of a cascade of events that leads to neuropathic pain development. For long it remained obscure how a nerve injury in the periphery would initiate a microglia response in the dorsal horn of the spinal cord. Recently, two chemokines have been suggested as potential factors that mediate the communication between injured neurons and microglia namely CCL2 and CCL21. This assumption is based on the following findings. Both chemokines are not found in healthy neurons, but are expressed in response to neuronal injury. In injured dorsal root ganglion cells CCL2 and CCL21 are expressed in vesicles in the soma and transported through the axons of the dorsal root into the dorsal horn of the spinal cord. Finally, microglia in vitro are known to respond to CCL2 and CCL21. Whereas the microglial chemokine receptor involved in CCL21-induced neuropathic pain is not yet defined the situation concerning the receptors for CCL2 in microglia in vivo is even less clear. Recent results obtained in transgenic animals clearly show that microglia in vivo do not express CCR2 but that peripheral myeloid cells and neurons do. This suggests that CCL2 expressed by injured dorsal root neurons does not act as neuron-microglia signal in contrast to CCL21. Instead, CCL2 in the injured dorsal root ganglia (DRG) may act as autocrine or paracrine signal and may stimulate first or second order neurons in the pain cascade and/or attract CCR2-expressing peripheral monocytes/macrophages to the spinal cord

Microglia priming in the aging brain:Implications for neurodegeneration

The primary aim of the thesis “Microglia priming in the aging brain: Implications for neurodegeneration” was to understand microglia phenotypes associated with brain aging and the potential mechanisms for this age-associated change. Microglia in the aging brain assume a hypersensitive proinflammatory phenotype termed as “priming”. Brain aging is the degenerative result of multiple cellular insults. DNA damage accumulation and critical shortening of the ends of chromosomes in cells called telomeres are two well-studied mechanisms that cause organismal aging. In addition to aging mice, we utilized transgenic mouse models in which the above mentioned aging mechanisms are triggered artificially to accelerate aging. For this purpose, we utilized the ERCC1∆/- mice, a mouse model of DNA damage accumulation previously shown to suffer cognitive impairment and known to model a human progeroid disorder to understand microglia phenotype and functionality. In addition to aging mice, ERCC1∆/- proved to be a good mouse model to study age associated phenotypic changes in microglia. In the ERCC1∆/- mice, age associated microglia priming was shown to be a response to neuronal dysfunction as a result of genotoxic stress. The prominence of microglia priming in the white matter tracts of the brain suggests that the axons might be primarily affected by the accumulation of DNA damage. The exact mechanism by which DNA damage accumulation affects axonal function is yet to be worked out. However, a possible mechanism could be transcriptional blockage as a consequence of DNA damage accumulation inducing metabolic stress and axonal dystrophy in neurons. On the other hand, in a mouse model of telomere shortening, the most prominent change observed in microglia was the increased cytokine response to peripheral inflammation due to alterations in the blood brain barrier. The results show that telomere shortening occurs in microglia with brain aging. However, critical telomere shortening is not the reason for microglia priming in the aging brain. Brain endothelium particularly in the white matter are more susceptible to telomere attrition than microglia as a cell type. These studies together highlight two possible mechanisms that make the white matter of the aging brain a particularly vulnerable target in the aging brain. The thesis also highlights regional differences between gray and white matter in microglia priming in the aging mouse and human brain. Surprisingly, microglia priming in the white matter already begins at middle age in humans. The increased and persistent white matter microglia priming draws attention to the role of white matter changes particularly in axons in brain aging and explores the possible use of white matter priming as a predictive factor to envisage the progression of cognitive aging and onset of neurodegeneration. It is well known that other clinical parameters such as Aβ plaque pathology, tau accumulation do not linearly correlate to cognitive impairments in the elderly. Microglia-mediated neuroinflammation in the white matter together with functional assessment of white matter function using non-invasive approaches such as Diffusion Tensor Imaging (DTI) and pathological markers of axonal dystrophy could yield powerful predictive tools for the assessment of cognitive impairment and pathological progression of neurodegeneration in the elderly

Cellular and Molecular Characterization of Microglia:A Unique Immune Cell Population

Microglia are essential for the development and function of the adult brain. Microglia arise from erythro-myeloid precursors in the yolk sac and populate the brain rudiment early during development. Unlike monocytes that are constantly renewed from bone marrow hematopoietic stem cells throughout life, resident microglia in the healthy brain persist during adulthood via constant self-renewal. Their ontogeny, together with the absence of turnover from the periphery and the singular environment of the central nervous system, make microglia a unique cell population. Supporting this notion, recent genome-wide transcriptional studies revealed specific gene expression profiles clearly distinct from other brain and peripheral immune cells. Here, we highlight the breakthrough studies that, over the last decades, helped elucidate microglial cell identity, ontogeny, and function. We describe the main techniques that have been used for this task and outline the crucial milestones that have been achieved to reach our actual knowledge of microglia. Furthermore, we give an overview of the "microgliome" that is currently emerging thanks to the constant progress in the modern profiling techniques

The neuroprotective role of microglial cells against amyloid beta-mediated toxicity in organotypic hippocampal slice cultures

During Alzheimer’s disease (AD) progression, microglial cells play complex roles and have potentially detrimental as well as beneficial effects. The use of appropriate model systems is essential for characterizing and understanding the roles of microglia in AD pathology. Here, we used organotypic hippocampal slice cultures (OHSCs) to investigate the impact of microglia on amyloid beta (Aβ)-mediated toxicity. Neurons in OHSCs containing microglia were not vulnerable to cell death after 7 days of repeated treatment with Aβ1-42 oligomer-enriched preparations. However, when clodronate was used to remove microglia, treatment with Aβ1-42 resulted in significant neuronal death. Further investigations indicated signs of endoplasmic reticulum stress and caspase activation after Aβ1-42 challenge only when microglia were absent. Interestingly, microglia provided protection without displaying any classic signs of activation, such as an amoeboid morphology or the release of pro-inflammatory mediators (e.g., IL-6, TNF-α, NO). Furthermore, depleting microglia or inhibiting microglial uptake mechanisms resulted in significant more Aβ deposition compared to that observed in OHSCs containing functional microglia, suggesting that microglia efficiently cleared Aβ. Because inhibiting microglial uptake increased neuronal cell death, the ability of microglia to engulf Aβ is thought to contribute to its protective properties. Our study argues for a beneficial role of functional ramified microglia whereby they act against the accumulation of neurotoxic forms of Aβ and support neuronal resilience in an in situ model of AD pathology

Antidepressant treatment is associated with epigenetic alterations of Homer1 promoter in a mouse model of chronic depression

Background: Understanding the neurobiology of depression and the mechanism of action of therapeutic measures is currently a research priority. We have shown that the expression of the synaptic protein Homer1a correlates with depression-like behavior and its induction is a common mechanism of action of different antidepressant treatments. However, the mechanism of Homer1a regulation is still unknown. Methods: We combined the chronic despair mouse model (CDM) of chronic depression with different antidepressant treatments. Depression-like behavior was characterized by forced swim and tail suspension tests, and via automatic measurement of sucrose preference in IntelliCage. The Homer1 mRNA expression and promoter DNA methylation were analyzed in cortex and peripheral blood by qRT-PCR and pyrosequencing. Results: CDM mice show decreased Homer1a and Homer1b/c mRNA expression in cortex and blood samples, while chronic treatment with imipramine and fluoxetine or acute ketamine application increases their level only in the cortex. The quantitative analyses of the methylation of 7 CpG sites, located on the Homer1 promoter region containing several CRE binding sites, show a significant increase in DNA methylation in the cortex of CDM mice. In contrast, antidepressant treatments reduce the methylation level. Limitations: Homer1 expression and promotor methylation were not analyzed in different blood cell types. Other CpG sites of Homer1 promoter should be investigated in future studies. Our experimental approach does not distinguish between methylation and hydroxymethylation. Conclusions: We demonstrate that stress-induced depression-like behavior and antidepressant treatments are associated with epigenetic alterations of Homer1 promoter, providing new insights into the mechanism of antidepressant treatment

KATP Channel Opener Diazoxide Prevents Neurodegeneration: A New Mechanism of Action via Antioxidative Pathway Activation

Pharmacological modulation of ATP-sensitive potassium channels has become a promising new therapeutic approach for the treatment of neurodegenerative diseases due to their role in mitochondrial and cellular protection. For instance, diazoxide, a well-known ATP-sensitive potassium channel activator with high affinity for mitochondrial component of the channel has been proved to be effective in animal models for different diseases such as Alzheimer's disease, stroke or multiple sclerosis. Here, we analyzed the ability of diazoxide for protecting neurons front different neurotoxic insults in vitro and ex vivo. Results showed that diazoxide effectively protects NSC-34 motoneurons from glutamatergic, oxidative and inflammatory damage. Moreover, diazoxide decreased neuronal death in organotypic hippocampal slice cultures after exicitotoxicity and preserved myelin sheath in organotypic cerebellar cultures exposed to pro-inflammatory demyelinating damage. In addition, we demonstrated that one of the mechanisms of actions implied in the neuroprotective role of diazoxide is mediated by the activation of Nrf2 expression and nuclear translocation. Nrf2 expression was increased in NSC-34 neurons in vitro as well as in the spinal cord of experimental autoimmune encephalomyelitis animals orally administered with diazoxide. Thus, diazoxide is a neuroprotective agent against oxidative stress-induced damage and cellular dysfunction that can be beneficial for diseases such as multiple sclerosis

Forebrain microglia from wild-type but not adult 5xFAD mice prevent amyloid-beta plaque formation in organotypic hippocampal slice cultures

The role of microglia in amyloid-beta (A beta) deposition is controversial. In the present study, an organotypic hippocampal slice culture (OHSC) system with an in vivo-like microglial-neuronal environment was used to investigate the potential contribution of microglia to A beta plaque formation. We found that microglia ingested A beta, thereby preventing plaque formation in OHSCs. Conversely, A beta deposits formed rapidly in microglia-free wild-type slices. The capacity to prevent A beta plaque formation was absent in forebrain microglia from young adult but not juvenile 5xFamilial Alzheimer's disease (FAD) mice. Since no loss of A beta clearance capacity was observed in both wild-type and cerebellar microglia from 5xFAD animals, the high A beta(1-42) burden in the forebrain of 5xFAD animals likely underlies the exhaustion of microglial A beta clearance capacity. These data may therefore explain why A beta plaque formation has never been described in wild-type mice, and point to a beneficial role of microglia in AD pathology. We also describe a new method to study A beta plaque formation in a cell culture setting

Enhanced adenosine A(1) receptor and Homer1a expression in hippocampus modulates the resilience to stress-induced depression-like behavior

Resilience to stress is critical for the development of depression. Enhanced adenosine A1 receptor (A1R) signaling mediates the antidepressant effects of acute sleep deprivation (SD). However, chronic SD causes long-lasting upregulation of brain A1R and increases the risk of depression. To investigate the effects of A1R on mood, we utilized two transgenic mouse lines with inducible A1R overexpression in forebrain neurons. These two lines have identical levels of A1R increase in the cortex, but differ in the transgenic A1R expression in the hippocampus. Switching on the transgene promotes robust antidepressant and anxiolytic effects in both lines. The mice of the line without transgenic A1R overexpression in the hippocampus (A1Hipp-) show very strong resistance towards development of stress-induced chronic depression-like behavior. In contrast, the mice of the line in which A1R upregulation extends to the hippocampus (A1Hipp+), exhibit decreased resilience to depression as compared to A1Hipp-. Similarly, automatic analysis of reward behavior of the two lines reveals that depression resistant A1Hipp-transgenic mice exhibit high sucrose preference, while mice of the vulnerable A1Hipp + line developed stress-induced anhedonic phenotype. The A1Hipp + mice have increased Homer1a expression in hippocampus, correlating with impaired long-term potentiation in the CA1 region, mimicking the stressed mice. Furthermore, virus-mediated overexpression of Homer1a in the hippocampus decreases stress resilience. Taken together our data indicate for first time that increased expression of A1R and Homer1a in the hippocampus modulates the resilience to stress-induced depression and thus might potentially mediate the detrimental effects of chronic sleep restriction on mood