Investigation of pathology,expression and proteomic profiles in human TREM2 variant postmortem brains with and without Alzheimer’s disease

Triggering receptor expressed on myeloid cells 2 TREM2 was identified as a risk factor for late onset Alzheimer’s disease (AD). Here we compared TREM2 cases with a variant (TREM2⁺) and cases without a TREM2 variant (TREM2⁻), considering pathological burden, inflammatory response and altered canonical pathways and biochemical functions between the cohorts. We hypothesised that TREM2⁺ cases would have a loss of function, indicating an altered inflammatory profile compared to TREM2⁻ cases. Immunohistochemistry was performed using antibodies against Aβ, tau and microglia markers in TREM2⁺ cases, with and without AD, which were compared to sporadic TREM2⁻ AD, familial AD and neurologically normal control cases. Aβ and tau load were measured along with the composition of Aβ plaques, in addition to microglial load and circularity. Expression and proteomic profiles were determined from the frontal cortex of selected cases. TREM2⁺ control cases had no Aβ or tau deposition. No differences in the amount of Aβ or tau, or the composition of Aβ plaques were observed between TREM2⁺ and TREM2⁻ SAD cases. There were no differences in microglial load observed between disease groups. However, the TREM2⁺ SAD cases showed more amoeboid microglia than the TREM2⁻ SAD cases, although no differences in the spatial relationship of microglia and Aβ plaques were identified. Visualisation of the canonical pathways and biological functions showed differences between the disease groups and the normal controls, clearly showing a number of pathways upregulated in TREM2⁺ SAD, TREM2⁻ SAD and FAD groups whilst, the TREM2⁺ controls cases showed a downregulation of the majority of the represented pathways. These findings suggest that the TREM2⁺ control group, although carrying the TREM2⁺ variant, have no pathological hallmarks of AD, have altered microglial and expression profiles compared to the TREM2⁺ SAD cases. This indicates that other unknown factors may initiate the onset of AD, with TREM2 influencing the microglial involvement in disease pathogenesis.     

TREM2 Protein Expression Changes Correlate with Alzheimer's Disease Neurodegenerative Pathologies in Post‐Mortem Temporal Cortices

Triggering receptor expressed by myeloid cells 2 (TREM2), a member of the immunoglobulin superfamily, has anti‐inflammatory phagocytic function in myeloid cells. Several studies have shown that TREM2 gene variant rs75932628‐T increased the risks for Alzheimer's disease (AD), Parkinson's disease, frontotemporal dementia and amyotrophic lateral sclerosis. It has been suggested that the risks could be resulted from the loss of TREM2 function caused by the mutation. Indeed, new evidence showed that several mutations in the immunoglobulin‐like V‐region led to low cell surface expression of TREM2 and reduced phagocytic function. Because of the emerging importance in understanding TREM2 expression and functions in human neurodegenerative diseases, we conducted biochemical and morphological studies of TREM2 expression in human post‐mortem temporal cortical samples from AD and normal cases. Increased expression of TREM2 protein was found to significantly correlate with increases of phosphorylated‐tau and active caspase 3, a marker of apoptosis, and also loss of the presynaptic protein SNAP25. Strong intensities of TREM2 immunoreactivity were observed in the microglia associated with amyloid plaques and in neuritic pathology‐enriched areas. Based on the findings that TREM2 expression correlated with neurodegenerative markers, further investigation on whether there is abnormality of TREM2 functions in AD brains with nonmutated TREM2 is needed.     

The microglial phagocytic role with specific plaque types in the Alzheimer disease brain

Alzheimer disease (AD) involves glial inflammation associated with amyloid plaques. The role of the microglial cells in the AD brain is controversial, as it remains unclear if the microglia form the amyloid fibrils of plaques or react to them in a macrophage-phagocytic role. Also, it is not known why microglia are preferentially associated with some amyloid plaque types. This review will provide substantial evidence to support the phagocytic role of microglia in the brain as well as explain why microglia are generally associated with specific plaque types that may be explained through their unique mechanisms of formation. In summary, the data presented suggests that plaque associated microglial activation is typically subsequent to specific amyloid plaque formations in the AD brain.     

A Phenotypic Change But Not Proliferation Underlies Glial Responses in Alzheimer Disease

Classical immunohistochemical studies in the Alzheimer disease (AD) brain reveal prominent glial reactions, but whether this pathological feature is due primarily to cell proliferation or to a phenotypic change of existing resting cells remains controversial. We performed double-fluorescence immunohistochemical studies of astrocytes and microglia, followed by unbiased stereology-based quantitation in temporal cortex of 40 AD patients and 32 age-matched nondemented subjects. Glial fibrillary acidic protein (GFAP) and major histocompatibility complex II (MHC2) were used as markers of astrocytic and microglial activation, respectively. Aldehyde dehydrogenase 1 L1 and glutamine synthetase were used as constitutive astrocytic markers, and ionized calcium-binding adaptor molecule 1 (IBA1) as a constitutive microglial marker. As expected, AD patients had higher numbers of GFAP⁺ astrocytes and MHC2⁺ microglia than the nondemented subjects. However, both groups had similar numbers of total astrocytes and microglia and, in the AD group, these total numbers remained essentially constant over the clinical course of the disease. The GFAP immunoreactivity of astrocytes, but not the MHC2 immunoreactivity of microglia, increased in parallel with the duration of the clinical illness in the AD group. Cortical atrophy contributed to the perception of increased glia density. We conclude that a phenotypic change of existing glial cells, rather than a marked proliferation of glial precursors, accounts for the majority of the glial responses observed in the AD brain.The role of glial cells in Alzheimer disease (AD), particularly the role of astrocytes and microglia, is a matter of growing interest. Pioneering immunohistochemical studies revealed an increased immunoreactivity for astrocytes and microglial cells in the cortex of AD patients, compared with that of nondemented elderly people, with the vast majority of these cells clustering around dense-core plaques.^1,2 Astrocytes are usually visualized with immunohistochemistry for glial fibrillary acidic protein (GFAP), but recent data suggest that only a subset of all astrocytes, labeled with aldehyde dehydrogenase 1 L1 (ALDH1L1), are also GFAP immunopositive.^3,4 Similarly, a number of microglial markers are available that do not completely overlap with one another and that also label blood and bone marrow mononuclear phagocytes,⁵ including major histocompatibility complex II [MHC2 (alias HLA-DP-DQ-DR)],⁶ CD11b (alias Mac-1, CR3),⁷ CD45 [alias leukocyte common antigen (LCA)],⁸ ionized calcium-binding adaptor molecule 1 [IBA1; alias allograft inflammatory factor 1 (AIF-1)],⁹ and CD68.¹⁰In AD, in various other neurodegenerative disorders, and in acute brain injuries such as stroke, trauma or infection, the terms reactive astrocytes and activated microglia are widely used to describe the characteristic morphology (ie, hypertrophy of soma and retraction and thickening of processes) of these glial cells. However, the astrocytic and microglial reactions observed in these conditions have also been often referred to as glial proliferation or glial hyperplasia.^11,12 To understand the role of glial cells in AD and their relationship to amyloid plaques and neurofibrillary tangles (NFTs), the pathological hallmarks of AD, it is important to know whether glial responses involve proliferation of glia or are due mainly to a phenotypic change in existing glia. With the present study, we sought to understand the relative contribution of cell proliferation versus phenotypic change to the increased number of astrocytes and microglial cells described in the AD brain by classical immunohistochemistry.To this end, we performed double-fluorescence immunohistochemical studies and unbiased stereology-based analyses on the temporal cortex of a large sample of AD patients and age-matched nondemented individuals. Double-labeling of astrocytes with GFAP and glutamine synthetase (GS) or ALDH1L1 enabled the visualization of many astrocytes that were not identifiable with GFAP immunohistochemistry alone, particularly in nondemented subjects. Stereology-based counts revealed that the total number of astrocytes does not differ between AD brain and normal aging brain, and that the number of astrocytes remains essentially constant through the clinical course of AD (although astrocytes increasingly become GFAP⁺ as the disease progresses). Double-labeling of microglia with IBA1 and MHC2 revealed that there are distinct subpopulations of microglial cells in the cortex, and that the number of MHC2⁺ activated microglia, but not the total number of microglial cells, is significantly increased in the AD brain. Thus, we conclude that glial responses in AD are largely due to a phenotypic change of resting glial cells, rather than to cell proliferation.     

Microglial response to amyloid plaques in APPsw transgenic mice.

Microglial activation is central to the inflammatory response in Alzheimer's Disease (AD). A recently described mouse line, Tg(HuAPP695.K670N/M671L)2576, expressing human amyloid precursor protein with a familial AD gene mutation, age-related amyloid deposits, and memory deficits, was found to develop a significant microglial response using Griffonia simplicifolia lectin or phosphotyrosine probe to identify microglia Both Griffonia simplicifolia lectin and phosphotyrosine staining showed increased numbers of intensely labeled, often enlarged microglia clustered in and around plaques, consistent with microglial activation related to beta-amyloid formation. Using quantitative image analysis of coronal phosphotyrosine-immunostained sections, transgene-positive 10- to 16-month-old, hemizygous, hybrid Tg2576 (APPsw) animals showed significantly increased microglial density and size in plaque-forming areas of hippocampus and frontal, entorhinal, and occipital cortex. Quantitative analysis of microglia as a function of distance from the center of plaques (double labeled for A beta peptide and microglia) revealed highly significant, two- to fivefold elevations in microglial number and area within plaques compared with neighboring regions. Tg2576 beta-amyloid-plaque-forming mice should be a useful system for assessing the consequences of the microglial-mediated inflammatory response to beta-amyloid and developing anti-inflammatory therapeutic strategies for Alzheimer's disease. These results provide the first quantitative link between beta-amyloid plaque formation and microglial activation in an animal model with neuritic plaques and memory deficits.     

Amyloid-beta peptides induce several chemokine mRNA expressions in the primary microglia and Ra2 cell line via the PI3K/Akt and/or ERK pathway

Alzheimer's disease (AD) is characterized by the presence of senile plaques composed primarily of amyloid-beta peptide (Abeta) in the brain. Microglia have been reported to surround these Abeta plaques, which have opposite roles, provoking a microglia-mediated inflammatory response that contributes to neuronal cell loss or the removal of Abeta and damaged neurons. To perform these tasks microglia migrate to the sites of Abeta secretion. We herein analyzed the process of chemokine expression induced by Abeta stimulation in primary murine microglia and Ra2 microglial cell line. We found that Abeta1-42 induced the expressions of CCL7, CCL2, CCL3, CCL4 and CXCL2 in the microglia. The signal transduction pathway for the expression of CCL2 and CCL7 mRNA induced by Abeta1-42 was found to depend on phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK), whereas the pathway for CCL4 depended only on PI3K/Akt. These inflammatory chemokine expressions by Abeta stimulation emphasize the contribution of neuroinflammatory mechanisms to the pathogenesis of AD.     

TGF-β1 blockade of microglial chemotaxis toward Aβ aggregates involves SMAD signaling and down-regulation of CCL5

Background  

Overactivated microglia that cluster at neuritic plaques constantly release neurotoxins, which actively contribute to progressive neurodegeneration in Alzheimer's disease (AD). Therefore, attenuating microglial clustering can reduce focal neuroinflammation at neuritic plaques. Previously, we identified CCL5 and CCL2 as prominent chemokines that mediate the chemotaxis of microglia toward beta-amyloid (Aβ)aggregates. Although transforming growth factor-β1 (TGF-β1) has been shown to down-regulate the expression of chemokines in activated microglia, whether TGF-β1 can reduce the chemotaxis of microglia toward neuritic plaques in AD remains unclear.     

The relationship between the morphological subtypes of microglia and Alzheimer’s disease neuropathology

Microglial associations with both the major Alzheimer’s disease (AD) pathognomonic entities, β‐amyloid‐positive plaques and tau‐positive neurofibrillary tangles, have been noted in previous investigations of both human tissue and mouse models. However, the precise nature of their role in the pathogenesis of AD is debated; the major working hypothesis is that pro‐inflammatory activities of activated microglia contribute to disease progression. In contrast, others have proposed that microglial dystrophy with a loss of physiological and neuroprotective activities promotes neurodegeneration. This immunohistochemical study sought to gain clarity in this area by quantifying the morphological subtypes of microglia in the mildly‐affected primary visual cortex (PVC), the moderately affected superior frontal cortex (SFC) and the severely affected inferior temporal cortex (ITC) of 8 AD cases and 15 age and gender‐matched, non‐demented controls with ranging AD‐type pathology. AD cases had increased β‐amyloid and tau levels compared to controls in all regions. Neuronal loss was observed in the SFC and ITC, and was associated with atrophy in the latter. A major feature of the ITC in AD was a decrease in ramified (healthy) microglia with image analysis confirming reductions in arborized area and skeletal complexity. Activated microglia were not associated with AD but were increased in non‐demented controls with greater AD‐type pathology. Microglial clusters were occasionally associated with β‐amyloid‐ and tau‐positive plaques but represented less than 2% of the total microglial population. Dystrophic microglia were not associated with AD, but were inversely correlated with brain pH suggesting that agonal events were responsible for this morphological subtype. Overall these novel findings suggest that there is an early microglial reaction to AD‐type pathology but a loss of healthy microglia is the prominent feature in severely affected regions of the AD brain.     

Peripherally administered human umbilical cord blood cells reduce parenchymal and vascular beta-amyloid deposits in Alzheimer mice

Modulation of immune/inflammatory responses by diverse strategies including amyloid-beta (Abeta) immunization, nonsteroidal anti-inflammatory drugs, and manipulation of microglial activation states has been shown to reduce Alzheimer's disease (AD)-like pathology and cognitive deficits in AD transgenic mouse models. Human umbilical cord blood cells (HUCBCs) have unique immunomodulatory potential. We wished to test whether these cells might alter AD-like pathology after infusion into the PSAPP mouse model of AD. Here, we report a marked reduction in Abeta levels/beta-amyloid plaques and associated astrocytosis following multiple low-dose infusions of HUCBCs. HUCBC infusions also reduced cerebral vascular Abeta deposits in the Tg2576 AD mouse model. Interestingly, these effects were associated with suppression of the CD40-CD40L interaction, as evidenced by decreased circulating and brain soluble CD40L (sCD40L), elevated systemic immunoglobulin M (IgM) levels, attenuated CD40L-induced inflammatory responses, and reduced surface expression of CD40 on microglia. Importantly, deficiency in CD40 abolishes the effect of HUCBCs on elevated plasma Abeta levels. Moreover, microglia isolated from HUCBC-infused PSAPP mice demonstrated increased phagocytosis of Abeta. Furthermore, sera from HUCBC-infused PSAPP mice significantly increased microglial phagocytosis of the Abeta1-42 peptide while inhibiting interferon-gammainduced microglial CD40 expression. Increased microglial phagocytic activity in this scenario was inhibited by addition of recombinant CD40L protein. These data suggest that HUCBC infusion mitigates AD-like pathology by disrupting CD40L activity.     

3D-Reconstruction of microglia and amyloid in APP23 transgenic mice: no evidence of intracellular amyloid

Microglia cells are closely associated with compact amyloid plaques in Alzheimer's disease (AD) brains. Although activated microglia seem to play a central role in the pathogenesis of AD, mechanisms of microglial activation by beta-amyloid as well as the nature of interaction between amyloid and microglia remain poorly understood. We previously reported a close morphological association between activated microglia and congophilic amyloid plaques in the brains of APP23 transgenic mice at both the light and electron microscopic levels [25]. In the present study, we have further examined the structural relationship between microglia and amyloid deposits by using postembedding immunogold labeling, serial ultrathin sectioning, and 3-dimensional reconstruction. Although bundles of immunogold-labeled amyloid fibrils were completely engulfed by microglial cytoplasm on single sections, serial ultrathin sectioning and three-dimensional reconstruction revealed that these amyloid fibrils are connected to extracellular amyloid deposits. These data demonstrate that extracellular amyloid fibrils form a myriad of finger-like channels with the widely branched microglial cytoplasm. We conclude that in APP23 mice a role of microglia in amyloid phagocytosis and intracellular production of amyloid is unlikely.     

Association of Microglia with Amyloid Plaques in Brains of APP23 Transgenic Mice

Microglia are a key component of the inflammatory response in the brain and are associated with senile plaques in Alzheimer's disease (AD). Although there is evidence that microglial activation is important for the pathogenesis of AD, the role of microglia in cerebral amyloidosis remains obscure. The present study was undertaken to investigate the relationship between beta-amyloid deposition and microglia activation in APP23 transgenic mice which express human mutated amyloid-beta precursor protein (betaPP) under the control of a neuron-specific promoter element. Light microscopic analysis revealed that the majority of the amyloid plaques in neocortex and hippocampus of 14- to 18- month-old APP23 mice are congophilic and associated with clusters of hypertrophic microglia with intensely stained Mac-1- and phosphotyrosine-positive processes. No association of such activated microglia was observed with diffuse plaques. In young APP23 mice, early amyloid deposits were already of dense core nature and were associated with a strong microglial response. Ultrastructurally, bundles of amyloid fibrils, sometimes surrounded by an incomplete membrane, were observed within the microglial cytoplasm. However, microglia with the typical characteristics of phagocytosis were associated more frequently with dystrophic neurites than with amyloid fibrils. Although the present observations cannot unequivocally determine whether microglia are causal, contributory, or consequential to cerebral amyloidosis, our results suggest that microglia are involved in cerebral amyloidosis either by participating in the processing of neuron-derived betaPP into amyloid fibrils and/or by ingesting amyloid fibrils via an uncommon phagocytotic mechanism. In any case, our observations demonstrate that neuron-derived betaPP is sufficient to induce not only amyloid plaque formation but also amyloid-associated microglial activation similar to that reported in AD. Moreover, our results are consistent with the idea that microglia activation may be important for the amyloid-associated neuron loss previously reported in these mice.     

Insights into TREM2 biology by network analysis of human brain gene expression data

Rare variants in TREM2 cause susceptibility to late-onset Alzheimer's disease. Here we use microarray-based expression data generated from 101 neuropathologically normal individuals and covering 10 brain regions, including the hippocampus, to understand TREM2 biology in human brain. Using network analysis, we detect a highly preserved TREM2-containing module in human brain, show that it relates to microglia, and demonstrate that TREM2 is a hub gene in 5 brain regions, including the hippocampus, suggesting that it can drive module function. Using enrichment analysis we show significant overrepresentation of genes implicated in the adaptive and innate immune system. Inspection of genes with the highest connectivity to TREM2 suggests that it plays a key role in mediating changes in the microglial cytoskeleton necessary not only for phagocytosis, but also migration. Most importantly, we show that the TREM2-containing module is significantly enriched for genes genetically implicated in Alzheimer's disease, multiple sclerosis, and motor neuron disease, implying that these diseases share common pathways centered on microglia and that among the genes identified are possible new disease-relevant genes.     

Modeling microglial activation in Alzheimer’s disease with human postmortem microglial cultures

Alzheimer’s disease (AD) is a uniquely human disorder. Despite intense research, the lack of availability of model systems has hindered AD studies though in recent years transgenic mouse models have been produced, which develop AD-like amyloid beta peptide (Aβ) plaques. For the study of inflammatory changes in AD brains, these transgenic mice may have limitations due to differences in the innate immune system of humans and rodents. Many studies of inflammatory processes in AD have focused on the role of activated microglia. Over the last 8 years, our research has focused on the properties of human microglia cultured from brain tissues of AD and non-demented (ND) individuals. As these are the cells observed to be activated in AD tissues, they represent a useful system for modeling the inflammatory components of AD.

In this review, we summarize data by our group and others on the use of microglia for AD-related inflammatory research, with emphasis on results using human postmortem brain microglia. A range of products have been shown to be produced by human postmortem microglia, both constitutively and in response to treatment with Aβ, including proinflammatory cytokines such as interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF) , and macrophage colony stimulating factor (M-CSF), along with complement proteins, especially C1q, superoxide radicals and neurotoxic factors. In our studies, we have demonstrated that there was a significant difference between AD and ND microglia in terms of their secretion of M-CSF and C1q. We also discuss the role of putative Aβ microglial receptors, particular recent data showing a role for the receptor for advanced glycation endproducts (RAGE) in mediating the responses of human microglia to Aβ. Finally, our studies on the use of an Aβ spot paradigm to model microglia interactions with plaques demonstrated that many of the features of AD inflammation can be modeled with postmortem brain derived microglia.     

Differential pathways for interleukin-1β production activated by chromogranin A and amyloid β in microglia

Although chromogranin A (CGA) is frequently present in Alzheimer's disease (AD), senile plaques associated with microglial activation, little is known about basic difference between CGA and fibrillar amyloid-β (fAβ) as neuroinflammatory factors. Here we have compared the interleukin-1β (IL-1β) production pathways by CGA and fAβ in microglia. In cultured microglia, production of IL-1β was induced by CGA, but not by fAβ. CGA activated both nuclear factor–κB (NF-κB) and pro–caspase-1, whereas fAβ activated pro–caspase-1 only. For the activation of pro–caspase-1, both CGA and fAβ needed the enzymatic activity of cathepsin B (CatB), but only fAβ required cytosolic leakage of CatB and the NLRP3 inflammasome activation. In contrast, fAβ induced the IL-1β secretion from microglia isolated from the aged mouse brain. In AD brain, highly activated microglia, which showed intense immunoreactivity for CatB and IL-1β, surrounded CGA-positive plaques more frequently than Aβ-positive plaques. These observations indicate differential pathways for the microglial IL-1β production by CGA and fAβ, which may aid in better understanding of the pathological significance of neuroinflammation in AD.     

Donepezil suppresses intracellular Ca<Superscript>2+</Superscript> mobilization through the PI3K pathway in rodent microglia

Background

Microglia are resident innate immune cells which release many factors including proinflammatory cytokines or nitric oxide (NO) when they are activated in response to immunological stimuli. Pathophysiology of Alzheimer’s disease (AD) is related to the inflammatory responses mediated by microglia. Intracellular Ca²⁺ signaling is important for microglial functions such as release of NO and cytokines. In addition, alteration of intracellular Ca²⁺ signaling underlies the pathophysiology of AD, while it remains unclear how donepezil, an acetylcholinesterase inhibitor, affects intracellular Ca²⁺ mobilization in microglial cells.

Methods

We examined whether pretreatment with donepezil affects the intracellular Ca²⁺ mobilization using fura-2 imaging and tested the effects of donepezil on phagocytic activity by phagocytosis assay in rodent microglial cells.

Results

In this study, we observed that pretreatment with donepezil suppressed the TNFα-induced sustained intracellular Ca²⁺ elevation in both rat HAPI and mouse primary microglial cells. On the other hand, pretreatment with donepezil did not suppress the mRNA expression of both TNFR1 and TNFR2 in rodent microglia we used. Pretreatment with acetylcholine but not donepezil suppressed the TNFα-induced intracellular Ca²⁺ elevation through the nicotinic α7 receptors. In addition, sigma 1 receptors were not involved in the donepezil-induced suppression of the TNFα-mediated intracellular Ca²⁺ elevation. Pretreatment with donepezil suppressed the TNFα-induced intracellular Ca²⁺ elevation through the PI3K pathway in rodent microglial cells. Using DAF-2 imaging, we also found that pretreatment with donepezil suppressed the production of NO induced by TNFα treatment and the PI3K pathway could be important for the donepezil-induced suppression of NO production in rodent microglial cells. Finally, phagocytosis assay showed that pretreatment with donepezil promoted phagocytic activity of rodent microglial cells through the PI3K but not MAPK/ERK pathway.

Conclusions

These suggest that donepezil could directly modulate the microglial function through the PI3K pathway in the rodent brain, which might be important to understand the effect of donepezil in the brain.

     

Amyloid cored plaques in Tg2576 transgenic mice are characterized by giant plaques,slightly activated microglia,and the lack of paired helical filament-typed,dystrophic neurites

We examined the brains of Tg2576 transgenic mice carrying human amyloid precursor protein with the Swedish mutation and Alzheimer's disease (AD) by means of immunohistochemistry and electron microscopy to clarify the characteristics of amyloid-associated pathology in the transgenic mice. In 12- to 29-month-old Tg2576 mice, congophilic cored plaques in the neocortex and hippocampus were labeled by all of the Abeta1-, Abeta40- and 42-specific antibodies, as seen in the classical plaques in AD. However, large-sized (>50 micro m in core diameter) plaques were seen more frequently in the older mice (18-29 months) than in those with AD (approximately 20% vs 2% in total cored plaques), and Tg2576 mice contained giant plaques (>75 micro m in core diameter), which were almost never seen in the brain of those with AD. Neither thread-like structures nor peripheral coronas were observed in the cored plaques of the transgenic mice in the silver impregnations. Immunohistochemically, plaque-accompanied microglia showed a slight enlargement of the cytoplasm with consistent labeling of Mac-1 and macrosialin (murine CD68), and with partial labeling of Ia antigen and macrophage-colony stimulating factor receptor. Ultrastructurally, the microglia surrounding the extracellular amyloid fibrils in the large, cored plaques showed some organella with phagocytic activity, such as secondary lysosomal, dense bodies, but intracellular amyloid fibrils were not evident. Dystrophic neurites in the plaques of the transgenic mice contained many dense multilaminar bodies, but no paired helical filaments. Our results suggest that giant cored plaques without coronas or paired helical filament-typed, dystrophic neurites are characteristic in Tg2576 mice, and that plaque-associated microglia in transgenic mice are activated to be in phagocytic function but not sufficient enough to digest extracellularly deposited amyloid fibrils.     

Alpha1-antichymotrypsin induces TNF-α production and NF-κB activation in the murine N9 microglial cell line

Microglia are known to accumulate in senile plaques of Alzheimer's disease (AD) together with a set of proteins including α₁-antichymotrypsin (ACT). To investigate the biological effects of the interaction between ACT and microglia, we examined cytokine production by the murine N9 microglial cell line after ACT treatment. Real-time PCR analysis and specific immunoassays demonstrate that ACT triggers mRNA expression and release of TNF-α by N9 microglial cells. Furthermore, we show that ACT induces a significant increase in NF-κB nuclear translocation. Taken together, these data demonstrate that ACT might contribute to the inflammatory mechanisms present in AD senile plaques.     

Microglial changes accompanying the promotion of retinal ganglion cell axonal regeneration into peripheral nerve grafts

Intravitreal injection of the microglia inhibitor tuftsin 1-3 leads to an increase in retinal ganglion cell axonal regeneration into peripheral nerve grafts and a decrease in phagocytic cells in the retina. However, the relation of phagocytic cells and particularly microglia towards axonal regeneration remains unclear. Initially, to assess this, tuftsin 1-3's effect on axonal regeneration was reexamined by doing a dose-response study. Optimal doses were found to be 2.5 g/ml and 250 g/ml in rats and hamsters respectively. We then studied retinal phagocytic cells in rats. Microglial cells were classified as resting or activated based on their morphology following OX42 immunolabelling. In controls, most microglial cells were in the resting state. Optic nerve cut led to an increase in the total number of microglia and a ten-fold elevation in the proportion of activated cells; changes were more pronounced at the optic nerve stump. Anastomosis of an autologous segment of sciatic nerve to the stump of the freshly cut optic nerve minimized the overall increase in microglia, and combined with 2.5 g/ml tuftsin 1-3, lead to a marked blunting of activation. Preservation within the retina of a higher proportion of resting over active form of microglia, and not the prevention of microglial proliferation per se, may be a crucial factor in allowing additional retinal ganglion cell axons to regenerate into peripheral nerve grafts.