Recent advances in cell imaging have provided novel insight into microglial physiology in the brain. Microglia plays important roles in the healthy brain such as it is involved in synaptic stripping during the development or experience-dependent sensory modulation, on the other hand, it is the first to respond to acute brain injury or chronic neurodegenerative diseases so it can be neuroprotective and neurotoxic depending on the context, thus, microglia represents a double-edged sword. Interestingly, an increasing amount of data suggests that neuroinflammation and microglia may represent very important participants in the development of Alzheimer's disease. Therefore, this review will describe microglial- neuronal complicated relationships under physiological and pathological conditions; furthermore, participation of microglia directly in synaptic stripping under pathological conditions, neuronal loss and synaptic stripping in the animal model of AD and finally it will be discussed that microglial derived immune cytokine such asTgf — β1 and it main signaling cascade SMAD2/3 being not only novel players in AD pathology but also they may represent future therapeutic target for AD treatment.
Introduction. Alzheimer's disease is an age-associated neurodegenerative disease that is defined by extracellular amyloid deposits composed of αβ peptide and intracellular tangles composed of hyper-phosphorylated tau protein. Recent studies show that inflammation plays important role in the AD development. Microglia is myeloid-derived resident in the central nervous system that comprises 10-20 % of the non-neuronal cell population. By morphological heterogeneity,microglia is classified into quiescent/surveying, reactive/activated and amoeboid/phagocytic . Microglia servethe first to respond to acute brain injury or slowly progressing neurodegenerative diseases such as AD . Under pathologic conditions microglia undergoes transformation from resting to phagocytic and becomes activated in a process known as microglial priming . The priming is strictly regulated and triggered by the presence of stimuli such as accumulation ofcellular debris or/and of aberrant proteins. Activated microglia produces various soluble pro- and anti-inflammatory cytokines, free radicals, proteases which has effects such as preventing infection/inflammation spread, rapid clearance of apoptotic debris at the lesion site or aberrant protein accumulations and creation of permissive environment for repair but on the other hand this may promotes neurotoxicity .
Microglia may represent a part of “quad”-partite synapse by modulating LTP  as well as GABA-ergic synaptic transmission and participating of restructuring of neuronal circuits and synaptic tuning, process known as synaptic stripping, in a sensory experience dependent manner . Many in-vitro studies show that microglia express a variety of variety of receptors for neurotransmitters, neuropeptides,chemokines, cytokines which in turn allows it easily acquire the priming state. Many studies show that constantly primed pro-inflammatory microglia is neurotoxic and have detrimental effects on the nervous system homeostasis and proper functioning, thus, microglia represents a double-edged sword whose activity is much context-dependent.
Recent studies show that resting microglia monitors the CNS homeostasis by making brief physical contacts and it can rapidly change its phenotype from ramified/activated/phagocytic . Furthermore, two-photon in-vivo imaging and immuno-EM of double transgenic mice IBA1-EGFP-THY-1 GFP where both microglia and neurons were visualized simultaneously showed that resting microglia contacts neurons by direct physical contact in a neuronal activity dependent manner not only in healthy brain but also under ischemic conditions, so that prolonged microglia/neuronal contactswere shown to be associated with a reductions of number of spines by synaptic phagocytosis  (Wake et al., 2009), however, it should be noted that there was no direct evidence that the prolonged contacts triggered phagocytosis despite that the authors used relatively non- invasive laser-induced ischemia it cannot be excluded that microglia, being very reactive cells, was not already primed by anesthesia, cranial thinning as well as laser-induced phototoxicity. The other limitation is that it was the double - transgenic animal which in turn must affect others cells, so that the imaging might involve other cells such as astrocytes regardless that specific promoters to the microglia and thalamic/cortical neurons were used. Nevertheless, it was shown in-vivo under appropriate resolution and that microglia promotes synaptic stripping very rapidly under pathological conditions thereby providing promising directions in the study of microglia-neuron relationships so that understanding what cellular mechanism triggers/promotes such fast stripping may help to develop pharmacological drugs that would alleviate synaptic stripping under pathological conditions thereby partially solving cognitive problems resulting from neuronal disconnection.
Inflammation and Alzheimer's disease.
Alzheimer Disease (AD) is an age-associated neurodegenerative disease characterized by progressive loss of neurons leading to cognitive disturbances in patients. Pathophysiologically, the disease is defined by cerebral amyloid deposits composed of 4042 amino-acid peptide (aβ) and intracellular neurofibrillary tangles (NFT) comprised of hyperphosphorylated tau protein. It is believed that aberrant Amyloid Precursor Protein (APP) cleavage may lead to toxic aβ accumulation thereby leading to aβ oligomerization and plaques generation. A few retrospective epidemiological studies indicated than non-steroidal antiinflammatory drugs application reduced the risk of AD suggesting that inflammation plays a role in AD . Furthermore, starting from 1990's it has been proposed that it is the immune response to aberrant protein accumulation leads to AD development . For example, it was shown that detectable microglial activation in tau overexpressing mice takes place long before NFT manifestation and synapse loss; moreover, immune suppression not only increased lifespan but also attenuated tau pathology . Many in-vitro and in-vivo studies show that C- terminal of APP/ tau overexpression induce inflammation and immune response , but it should be noted that most of the studies explore primed microglial activity under pro- inflammatory systemic conditions induced by pathogens such as LPS injections, so whether aβ or tau can prime resting microglia directly warrants further investigation. On the other hand, Wallerian degeneration, may prime microglia and provoke a classical pro-inflammatory profile  thus it may be said aβ and tau trigger microglia activation indirectly participating in neuronal degeneration and indeed increase in inflammation- associated cytokines proteins is an invariant feature of AD compared with normal brain. This essay will particularly focus Ontransforming growth factor-ß(Tдƒ — β1 ) a multifunctional and a very contradictory cytokine strikingly increased in AD patients, for instance, immunofluorescent, in-vitro and MRI studies showed that TGF — β1 levels are increased in the frontal cortex, cerebrospinal fluid as well as along blood vessels, plaques, astrocytes, neurons and microglia of AD patients compared to age-matched healthy controls  so the exact role of TGF — β1 warrants further investigation. Interestingly, in contradiction to Tgf — β1 anti-inflammatory roles, some studies reported that TGF — β1 overexpression promote cerebral angiopathy very similar to AD at the very early stage of life . Moreover, enzyme-like immunosorbent assay showed that incubation with TGF-B1 promoted expression of pro- inflammatory cytokines by brain endothelial cells [1, 32] as well as pharmacological Tgf — ß1/SMAD2-3 blocking mitigated aβ depositions and improved aβ phagocytosis in APP overexpressing mouse [18,29,34] though TGF — β1 promotes APP upregulation via SMAD-3 binding to APP promoter (rev. in Ueberham et al., 2006), moreover, it seems that Tgf — β1 and aβ interact with each other and may even have synergistic effects in AD pathology. So, for instance, Tgf — β1 overexpressing mice developed almost double amount of aβ deposits compared to control animals [15, 25]. Moreover, as Tgf — β1 upregulates extracellular matrix proteins, it may impair aβ proteolytic degradation thereby promoting aβ accumulation . Although recent data suggest that AD hallmarks such as aβ and NFT interfere with Tgf — β1 signaling, so for example, immunofluorescent studies showed that aberrant pSMAD2-3 cellular localization such as cytoplasmic pSMAD2-3 bound to NFT  or nuclear overaccumulation of pSMAD2-3 . Chronic overexpression of Tgf — β1 promotes NFT accumulation and leads to microvascular degeneration in mice through ectopic pSMAD2-3 accumulations . On the other hand, in-vitro transwell assays, pharmacological, RT-PCR and biochemical studies show that Tgf — ß1/SMADs not only promote immediate aβ uptake by microglia (Huang et al., 2010) but also promote upregulation of scavenger receptors on its surface as well as reduce production of microglial cytotoxic NO ∣28∣; furthermore, Tgf — ß1/SMAD2-3 being critical for aβ induced microglial transformation from quiescent to phagocytic state  where blocking Tgf — β1 signaling in rat hippocampal slices amplifies significantly aβ induced neurotoxicity [6,34] whereas pre-treatment by Tgf — β1 increases neuronal resistance to αrβ-mediated cytotoxicity .
AD, microglia and microglial-derived Tgf — βT.
Recent paper by Fuhrmann and colleagues provided direct evidence that microglia is involved in neuronal loss and synaptic stripping in mice overexpressing both APP and tau , by means of two photon in-vivo imaging microscopy individual microglial cells and neurons were visualized simultaneously and were monitored for 28 days in the living animal. It was shown that microglia directly contacts neurons; moreover, based on microglial density and velocity of movement around neurons it was suggested that microglia participates directly in synaptic stripping and neuronal death at least in the animal modelof AD . However, it should be noted that it is not clear whether microglia induced neuronal death or was clearing cellular debris of dying/dead neurons due to aberrant overexpression of tau/APP, although KO of microglial receptor CX3CR1 (key receptor for microglial chemotaxis towards injured neurons) rescued neurons suggesting activated and cytotoxic microglia drives neuronal death directly. One of the limitations is that it should be noted that it is a 5xTg mice which in turn must have alternated the cellular phenotype so neurons might already be dying and microglia overactivated due to expression of proteins not only far from physiological levels but also in their aberrant form; laser photoxicityand cranial thinning. Furthermore, it should be noted that as the GFP/YFP could also be expressed in other cells such as astrocytes though the authors used cells specific promoter. Furthermore, altho ugh CX3CR1 KO rescued neurons there were no direct evidence that microglia induce neuronal death it might be that dying neurons due to many pathologic process promote immune cytotoxic response. Nevertheless, this paper provided in-vivo evidence under good resolution that microglia is involved in neuronal and synaptic loss as well as it provided evidence that CX3CR1 signaling cascade plays an important in the animal model of AD.
TGF — β1 is a pleiotropic cytokine, primarily produced by microglial at the very early stages of inflammation, that is involved in cell proliferation/ differentiation and apoptosis . TGF — β KO mice die at 3-4 weeks postnatally from severe neuroinflammation; moreover, in-vitro cultured primary neurons deprived of TGF — β signaling had reduced survival; some studies show that TGF — β overexpression protects from neurons fromacute and chronic cytotoxicity. For instance,in-vitro microarray, pharmacological and immunocytochemical studies showed Tgf — β1 inhibited primed microglial reactivity by inhibiting microglial proliferation [3,19] bydownregulating of pro-inflammatorycytokines , free radicals and upregulating of not only anti-inflammatory molecules [14, 22, 23]regulating microglial quiescence [4, 27] but also those involved in chemotaxis and even LTP . In contrast, some studies report contradictory data, so for example, TGF — βoverexpression was reported to be pro-inflammatory and exacerbatingexcitotoxicity in newborn mice ; moreover, mice overexpressing TGF — β were often hydrocephalic ; in-vitro studies show that TGF — β treatment promotes neuronal cell death and induce and microgliosis.
TGF — βl- SMAD2-3 signaling cascade.
The biological effects of TGF-Bs are exerted through TGF — β type I and type II receptors that belong to the family of serinethreonine kinases. Upon ligand binding, type II phosphorylates type I followedby phosphorylation of SMAD proteins . Phosphorylated SMAD (pSMAD) form oligomers composed of SMAD2 and SMAD3/SMAD4 and translocate to the nucleus where they directly bind to promoter sequence via specific SMAD binding element with following gene expression  such as cell migratory genes , for example, CX3-CR1 [1,23].Recent in-vitro RT-PCR, immunocytochemical, genetic interference a ndstudy of brain microglial and primary microglial cell cultures by DeSimone and colleagues showed that Tgf — β1 promotes microglial chemotaxis through purinergic receptors such as P2Y1 and P2y12 as well as upregulates proteins such as Arg-1 that promote microglial quiescent and thus neuroprotective state. This study showed that Tgf — β1 is a negative regulator of microglial activation but it promotes chemotaxis and increase migratory response of resting microglia.However, it should be noted that ADP concentrations used in-vitro were far from physiological range but several two-photon in-vivo imaging studies proved that ADP/PY12 represent microglial chemotactic signaling pathway under resting conditions [8,13]. Furthermore, primary cultures should be considered in caution as there might be another cell types such as astrocytes which may also control microglial physiology that were also affected on by ADP as well as microglia present there might possess different morphological states; moreover, under experimental conditions microglia may have altered phenotype. Nevertheless, this study showed that 7⅛ƒ — β1 directly involved in microglial chemotaxis towards injured neurons, promotion of its own expression and microglial motility genes upregulation.
Therefore, as the data above suggest loss of Tgf — β1-SMAD signaling cascade might represent important stage in ADdevelopment by overactivation of resting microglia. Nevertheless, it is not clear whether aberrant SMAD localization emanates from AD-associated abnormal protein aggregation orTgf — β1 -SMAD promoting "permissive environment” for AD development by promoting microglial chemotaxis and toxicity in the inflamed brain. It is clear that activated microglia represent immune response to overcome aberrant protein accumulations however it is not clear whether microglial response drives cellular death or there are agents/factors, such as age, pathological conditions that oversensitize microgliathereby leading to neurodegeneration. To summarize, this review described microglial function in adult healthy and AD brains; moreover, it described one of the central chemokines such asTgf — β1 that clearly plays a role in AD by modulating microglial state, furthermore, this essay discussed papers by Wake and colleague that showed that microglia directly involved in synaptic stripping in healthy and ischemic conditions in-vivo ; by Furhmann and colleagues that showed that microglia involved in neuronal and synaptic loss in the animal model of AD and KO of microglial receptors resulted in neuronal rescue in vivo  and paper by De-Simone and colleagues that showed that Tgf -β1 promotes microglial chemotaxis towards injured in neurons . Altogether, they provide some consistent evidence that microglia and Tgf — β1 /SMAD signaling may represent a potential target in AD treatment, so for example, modulation of immune response in AD might help alleviate the problem of cognitive disturbance by modulating the process of neuronal death and synaptic stripping and thus preserving neuronal connectivity.
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