Hematopoietic prostaglandin D synthase is expressed in microglia in the developing postnatal mouse brain

Hematopoietic prostaglandin D synthase (HPGDS) is a PGD(2)-synthesizing enzyme and is expressed in antigen-presenting cells, mast cells, and other immunocompetent cells. We here report the HPGDS expression in microglia and the migration pathway of microglia in the developing mouse brain as detected by HPGDS immunohistochemistry. Expression of HPGDS mRNA peaked at postnatal day (PND) 10, decreased gradually thereafter, and reached a plateau at PND 20. The mRNAs for target molecules of PGD(2), i.e., DP receptor (DPR) and CRTH2 receptor, showed developmental profiles overlapped to that of HPGDS. Most of the HPGDS(+) cells at PND 10 had morphological characteristics of ameboid microglia and gave positive immunostaining with microglia-specific markers such as RCA-1, F4/80, or ER-MP12. These specific markers became less detectable later on, but HPGDS was still expressed even in resting microglia. Thus, HPGDS is a useful marker for investigation of microglial development. Spaciotemporal evaluation of microglial development and migration with HPGDS immunostaining revealed the following three major possible migration pathways of microglia in the postnatal brain: from the lateral ventricle via subventricular zones to brain parenchyma; from the leptomeninges around the cerebellopontine angle to the cerebellar white matter; and from the overlying leptomeninges to the hippocampus, basal forebrain, and brainstem.     

DNA sensors are expressed in astrocytes and microglia in vitro and are upregulated during gliosis in neurodegenerative disease

The detection of nucleic acids by the innate immune system is an essential host response during viral infection. In recent years, a number of immune sensors capable of recognizing cytosolic DNA have been identified and include the PYHIN family members AIM2, IFI16, and p204 as well as the enzyme, cGAS. Activation of these receptors leads to the induction of antiviral genes including Type‐1 interferons and chemokines such as CCL5. We have carried out extensive expression profiling of these DNA sensors and other members of the PYHIN family in highly purified primary astrocytes and microglia and have demonstrated that both cell types express the majority of these proteins at the mRNA level. In microglia, several family members are highly upregulated in response to IFN‐β treatment while both cell types induce robust proinflammatory and antiviral cytokine production (e.g., IL‐6, CCL5, IFN‐β) in the presence of immune stimulatory DNA and RNA. The production of IL‐6 is partially dependent on the interferon receptor as is IFN‐β itself. Furthermore, we have found that p204 and AIM2 are upregulated in a Type I IFN dependent fashion in vivo, in a murine model of chronic neurodegeneration. Given the propensity of inflammatory responses to cause neuronal damage, increased expression and activation of these receptors, not only during viral infection but also during sterile inflammatory responses, has the potential to exacerbate existing neuroinflammation leading to further damage and impaired neurogenesis. GLIA 2015;63:812–825     

Identification of monocyte chemoattractant protein-1 in senile plaques and reactive microglia of Alzheimer's disease

Abstract It has been shown that human monocytes express monocyte chemoattractant protein-1 (MCP-1), an inflammatry factor, in response to non-fibrillar β-amyloid protein. Reactive microglia and inflammatory factors were reported to be present in β-amyloid deposits (senile plaques) in Alzheimer's disease, suggesting the presence of MCP-1 in senile plaques. To address this issue, we examined MCP-1-immunoreactivity in senile plaques using a mouse monoclonal anti-MCP-1 antibody. Monocyte chemoattractant protein-1 was found immunohistochemically in mature senile plaques and reactive microglia but not in immature senile plaques of brain tissues from five patients with Alzheimer's disease. These findings suggest that MCP-1-related inflammatory events induced by reactive microglia contribute to the maturation of senile plaques.     

Lipocalin-type prostaglandin D synthase (beta-trace) is upregulated in the alphaB-crystallin-positive oligodendrocytes and astrocytes in the chronic multiple sclerosis

Lipocalin-type prostaglandin D synthase (L-PGDS), which is mainly synthesized in leptomeningeal cells and oligodendrocytes (OLs) in rodents and humans, is secreted into the human cerebrospinal fluid (CSF) as beta-trace. L-PGDS protects OLs and neurones against apoptosis in twitcher mice, a murine model of Krabbe's disease, and is the second only to a stress protein, alphaB-crystallin, as the most abundant gene product upregulated in the demyelinating focus of multiple sclerosis (MS). Here we report that although the CSF level of L-PGDS is not increased in MS patients, L-PGDS is increased in the white matter of MS patients, especially in the shadow plaque as compared with the normal white matter. L-PGDS immunoreactivity was intensely expressed in OLs within the shadow plaques and in hypertrophied astrocytes within the chronic plaques of MS patients. Both L-PGDS-positive OLs and astrocytes expressed a stress protein, alphaB-crystallin. These results suggest that the upregulation of L-PGDS occurs in OLs and astrocytes as a stress reaction.     

Ultrastructural localization of butyrylcholinesterase in senile plaques in the brains of aged and Alzheimer disease patients

Histochemical localization of butyrylcholinesterase has been carried out in primitive, perivascular, and classic plaques in the brains of both nondemented and Alzheimer disease (AD) patients. Butyrylcholinesterase histochemistry has been compared to amyloid β-protein (AβP) immunocytochemistry in adjacent sections. In small primitive plaques, most of the butyrylcholinesterase reaction product appears ultrastructurally located over plasma membranes of healthy-looking cell processes. In more extensive primitive plaques, butyrylcholinesterase reaction product also decorates amyloid filaments, which become identifiable as delicate wisps. In classic plaques, large aggregates of butyrylcholinesterase reaction product colocalize with bundles of amyloid filaments, as well as with the compact amyloid core. Thus, deposition of butyrylcholinesterase in senile plaques follows a close parellelism with the progressive aggregation of amyloid β-protein, supporting the possibility that cholinesterases may play some role in the maturation of these structures.     

Clustering of plaques contributes to plaque growth in a mouse model of Alzheimer’s disease

Amyloid-β (Aβ) plaque deposition plays a central role in the pathogenesis of Alzheimer’s disease (AD). Post-mortem analysis of plaque development in mouse models of AD revealed that plaques are initially small, but then increase in size and become more numerous with age. There is evidence that plaques can grow uniformly over time; however, a complementary hypothesis of plaque development is that small plaques cluster and grow together thereby forming larger plaques. To investigate the latter hypothesis, we studied plaque formation in APPPS1 mice using in vivo two-photon microscopy and immunohistochemical analysis. We used sequential pre- and post-mortem staining techniques to label plaques at different stages of development and to detect newly emerged plaques. Post-mortem analysis revealed that a subset (22 %) of newly formed plaques appeared very close (<40 μm) to pre-existing plaques and that many close plaques (25 %) that were initially separate merged over time to form one single large plaque. Our results suggest that small plaques can cluster together, thus forming larger plaques as a complementary mechanism to simple uniform plaque growth from a single initial plaque. This study deepens our understanding of Aβ deposition and demonstrates that there are multiple mechanisms at play in plaque development.     

Cholesterol accumulates in senile plaques of Alzheimer disease patients and in transgenic APP(SW) mice

Mounting evidence suggests that cholesterol may contribute to the pathogenesis of Alzheimer disease (AD). We examined whether cholesterol might be present in senile plaques, a hallmark neuropathological feature of AD. We employed 2 different fluorometric-staining techniques (filipin staining and an enzymatic technique) for the determination of cholesterol in brains of postmortem confirmed AD patients and in nondemented, age-matched histopathologically normal controls. AD patient brains showed abnormal accumulation of cholesterol in congophilic/birefringent dense cores of senile plaques that was essentially absent in histopathologically normal controls. To determine whether increased senile plaque-associated cholesterol occurred generally in all plaques or was restricted to a specific subset, quantitative analysis was performed. Data indicate abnormal accumulation of cholesterol in cores of mature plaques but not in diffuse or immature plaques. Additionally, transgenic mice that overexpress the "Swedish" amyloid precursor protein (Tg APP(SW), line 2576) exhibited a similar pattern of abnormal cholesterol accumulation in mature, congophilic amyloid plaques at 24 months of age that was absent in their control littermates or in 8-month-old Tg APP(SW) mice (an age prior to amyloid deposition). Taken together, our results imply a link between cholesterol and AD pathogenesis and suggest that cholesterol plays an important role in the formation and/or progression of senile plaques.     

Neuregulin-1 and erbB4 immunoreactivity is associated with neuritic plaques in Alzheimer disease brain and in a transgenic model of Alzheimer disease

Neuregulin-1 (NRG-1) regulates developmental neuronal survival and synaptogenesis, astrocytic differentiation, and microglial activation. Given these NRG-1 actions, we hypothesized that the synaptic loss, gliosis, inflammation, and neuronal death occurring in Alzheimer disease (AD) is associated with altered expression of NRG-1 and its receptors (the erbB2, erbB3, and erbB4 membrane tyrosine kinases). We examined the expression and distribution of NRG-1 and the erbB kinases in the hippocampus of AD patients and cognitively normal controls and in transgenic mice that coexpress AD-associated mutations of the beta amyloid precursor protein (APP(K670N,M671L)) and presenilin-1 (PS1(M146L)). In the hippocampi of both control humans and wild type mice, NRG-1 and the 3 erbB receptors are expressed in distinct cellular compartments of hippocampal neurons. All 4 molecules are associated with neuronal cell bodies, but only NRG-1, erbB2, and erbB4 are present in synapse-rich regions. In AD and in the doubly transgenic mouse, erbB4 is expressed by reactive astrocytes and microglia surrounding neuritic plaques. In AD brains, microglia and, to a lesser extent, dystrophic neurites, also upregulate NRG-1 in neuritic plaques, suggesting that autocrine and/or paracrine interactions regulate NRG-1 action within these lesions. NRG-1 and erbB4, as well as erbB2, are similarly associated with neuritic plaques in the doubly transgenic mice. Thus, in AD the hippocampal distribution of NRG-1 and erbB4 is altered. The similarities between the alterations in the expression of NRG-1 and its receptors in human AD and in APP(K670N;M671L)/PS1(M146L) mutant mice suggests that this animal model may be very informative in deciphering the potential role of these molecules in AD.     

COX‐1‐dependent prostaglandin D2 in microglia contributes to neuropathic pain via DP2 receptor in spinal neurons

Cyclooxygenase (COX) enzyme synthesizes prostaglandins (PGs) from arachidonic acid and exists as two major isozymes, COX‐1 and COX‐2. The crucial role of prostaglandins in the pathogenesis of inflammatory pain in peripheral tissue and the spinal cord has been established; however its expression dynamics after peripheral nerve injury and its role in neuropathic pain are not clear. In this study, we examined the detailed expression patterns of genes for COX, PGD2 and thromboxane A2 synthases and their receptors in the spinal cord. Furthermore, we explored the altered gene expression of these molecules using the spared nerve injury (SNI) model. We also examined whether these molecules have a role in the development or maintenance of neuropathic pain. We found a number of interesting results in this study, the first was that COX‐1 was constitutively expressed in the spinal cord and up‐regulated in microglia located in laminae I‐II after nerve injury. Second, COX‐2 mRNA expression was induced in blood vessels after nerve injury. Third, TXA2 synthase and hematopoietic PGD synthase mRNAs were dramatically increased in the microglia after nerve injury. Finally, we found that intrathecal injection of a COX‐1 inhibitor and DP2 receptor antagonist significantly attenuated the mechanical allodynia. Our findings indicate that PGD2 produced by microglia is COX‐1 dependent, and that neurons in the spinal cord can receive PGD2 from microglia following peripheral nerve injury. We believe that PGD2 signaling via DP2 signaling pathway from microglia to neurons is one of the triggering factors for mechanical allodynia in this neuropathic pain model.     

S100A6 overexpression within astrocytes associated with impaired axons from both ALS mouse model and human patients

Astrogliosis is one of the earliest pathological changes observed in neurodegenerative diseases in general and in amyotrophic lateral sclerosis (ALS) in particular. ALS is characterized by selective degeneration of motoneurons. There are 2 forms of the disease: sporadic ALS (SALS), comprising 90%-95% of cases, and familial ALS (FALS), comprising 5%-10% of cases. FALS is an age-dependent autosomal dominant disorder in which mutations in the homodimeric enzyme Cu/ Zn superoxide dismutase 1 (SOD1) is linked to the disease. The animal model for this disease is a transgenic mouse expressing the mutated human SOD1(G93A) gene. Here we show by immunohistochemistry and double immunofluorescence that astrocytes located near impaired axons of motoneurons that were selectively programmed to die overexpressed S100A6, a Ca2+/Zn2+ binding protein able to translocate into the nucleus. Transgenic mice overexpressing the mutated human SOD1 gene and patients suffering from SALS showed this selective astrocytic S100A6 expression. For instance, the pyramidal tract could be macroscopically detected on S100A6-labeled spinal cord and brainstem sections from SALS patients. Transgenic mice overexpressing the non-mutated SOD1 gene did not overexpress S100A6, although glial fibrillary associated protein astrogliosis was seen. Although these results do not give any clue about the beneficial or detrimental role played by S100A6, its induction may be assumed to appropriately serve some function(s).     

Liver kinase B1 depletion from astrocytes worsens disease in a mouse model of multiple sclerosis

Liver kinase B1 (LKB1) is a ubiquitously expressed kinase involved in the regulation of cell metabolism, growth, and inflammatory activation. We previously reported that a single nucleotide polymorphism in the gene encoding LKB1 is a risk factor for multiple sclerosis (MS). Since astrocyte activation and metabolic function have important roles in regulating neuroinflammation and neuropathology, we examined the serine/threonine kinase LKB1 in astrocytes in a chronic experimental autoimmune encephalomyelitis mouse model of MS. To reduce LKB1, a heterozygous astrocyte-selective conditional knockout (het-cKO) model was used. While disease incidence was similar, disease severity was worsened in het-cKO mice. RNAseq analysis identified Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriched in het-cKO mice relating to mitochondrial function, confirmed by alterations in mitochondrial complex proteins and reductions in mRNAs related to astrocyte metabolism. Enriched pathways included major histocompatibility class II genes, confirmed by increases in MHCII protein in spinal cord and cerebellum of het-cKO mice. We observed increased numbers of CD4+ Th17 cells and increased neuronal damage in spinal cords of het-cKO mice, associated with reduced expression of choline acetyltransferase, accumulation of immunoglobulin-γ, and reduced expression of factors involved in motor neuron survival. In vitro, LKB1-deficient astrocytes showed reduced metabolic function and increased inflammatory activation. These data suggest that metabolic dysfunction in astrocytes, in this case due to LKB1 deficiency, can exacerbate demyelinating disease by loss of metabolic support and increase in the inflammatory environment.     

Inducible nitric oxide synthase immunoreactivity in the Alzheimer disease hippocampus: association with Hirano bodies, neurofibrillary tangles, and senile plaques

Inducible nitric oxide synthase (iNOS) is involved in the generation of nitric oxide, a molecule with multiple biological activities. Although iNOS expression may be part of antimicrobial armamentarium, inappropriate expression of iNOS can potentially lead to damage to the host. In this report, we determined the expression of iNOS by immunocytochemistry in the hippocampus of the Alzheimer brains (AD) as well as in young and old normal brains. The results showed localization of iNOS immunoreactivity to Hirano bodies of the AD hippocampus. In addition, small granular iNOS immunoreactive profiles were detected associated with senile plaques and extracellular neurofibrillary tangles. In the hippocampus of control brains, morphologically similar profiles were immunoreactive for iNOS, but in far fewer numbers than in AD hippocampus. The results suggest that iNOS is expressed in a subset of pyramidal neurons in the AD hippocampus, and that iNOS may be involved in the pathogenesis of neuronal degeneration in AD.     

Fluoro-Jade B staining as useful tool to identify activated microglia and astrocytes in a mouse transgenic model of Alzheimer's disease

Fluoro-Jade B is known as a high affinity fluorescent marker for the localization of neuronal degeneration during acute neuronal distress. However, one study suggested that fluoro-Jade B stains reactive astroglia in the primate cerebral cortex. In this study, we analyzed the staining of fluoro-Jade B alone or combined with specific markers for detection of glial fibrillary acidic protein (GFAP) or activated CD68 microglia in the double APP(SL)/PS1 KI transgenic mice of Alzheimer's disease (AD), which display a massive neuronal loss in the CA1 region of the hippocampus. Our results showed that fluoro-Jade B did not stain normal and degenerating neurons in this double mouse transgenic model. Fluoro-Jade B was co-localized with Abeta in the core of amyloid deposits and in glia-like cells expressing Abeta. Furthermore, fluoro-Jade B was co-localized with CD68/macrosialin, a specific marker of activated microglia, and with GFAP for astrocytes in APP(SL)/PS1 KI transgenic mice of AD. Taken together, these findings showed that fluoro-Jade B can be used to label activated microglia and astrocytes which are abundant in the brain of these AD transgenic mice. It could stain degenerating neurons as a result of acute insult while it could label activated microglia and astrocytes during a chronic neuronal degenerative process such as AD for example.     

Identification of FcγRI, II and III on normal human brain ramified microglia and on microglia in senile plaques in Alzheimer's disease

Using monoclonal antibodies to the three known human leukocyte IgG receptors, FcγR, we examined the expression of FcγR in normal brains and in Alzheimer's disease. We found FcγRI, II and III immunoreactivity in senile plaques and on ramified microglia throughout the cortex and white matter of normal and Alzheimer's disease brains. FcγRI expression was independently confirmed by a murine isotype binding study. These findings suggest that intrinsic FcγR may play an important role in normal and disordered immune-related processes in the brain. The support the idea that microglia are brain macrophages.     

铁和老年斑在阿尔茨海默病转基因小鼠脑内的沉积及其对磁共振T2弛豫时间的影响

目的 观察老年斑和铁在阿尔茨海默病(AD)转基因小鼠脑内皮质和海马处的沉积及其对磁共振活体扫描T2弛豫时间的影响.方法 2、4、6、8、10、12、14、16月龄的AD转基因小鼠各2只,分为幼龄组、成龄组和老龄组,8月龄野生型C57BL/6J小鼠2只作为对照,依次进行磁共振活体扫描,测量皮质、海马、丘脑、胼胝体、纹状体等结构的T2弛豫时间,扫描完成后,进行铁和老年斑的组织学染色,计算皮质和海马处老年斑的数目及含量、铁颗粒的数目及含量.结果 各组皮质及海马T2弛豫时间(ms)分别为:野生组:49.5±2.1、51.6±1.1;幼龄组(2、4月龄):49.7±0.5、50.7±0.7;成龄组(6、8、10月龄):47.2±0.8、47.7±0.9;老龄组(12、14、16月龄):44.6±0.8、45.3±0.4.各组皮质及海马T2弛豫时间差异有统计学意义(皮质F=18.620,海马F=67.925,均P＜0.01).组间比较示成龄组较幼龄组(皮质q=4.284,海马q=7.902;均P＜0.01)及野生组(皮质q=4.424,P＜0.05,海马q=11.450,P＜0.01)小鼠皮质及海马T2弛豫时间缩短;而老龄组较成龄组小鼠皮质及海马T2弛豫时间缩短(皮质q=4.812,海马q=7.034,均P＜0.01).组织学染色示4月龄转基因小鼠皮质和海马开始出现老年斑的沉积,6月龄开始出现铁的沉积,并且随着小鼠月龄的增加,皮质和海马处老年斑和铁的数目及含量均明显增加.老年斑和铁的沉积具有明显的正相关(r=0.931,P＜0.01),老年斑的含量、铁的含量与T2弛豫时间均具有明显的负相关(分别r=-0.884、-0.827,均P＜0.01).结论 老年斑和铁的沉积可能共同影响T2弛豫时间,提示T2弛豫时间可以作为早期诊断AD和抗痴呆药物筛选的一个敏感性指标.     

Increased hippocampal plaques and tangles in patients with Alzheimer disease with a lifetime history of major depression

CONTEXT: The hallmark pathological changes in Alzheimer disease (AD) are abundant plaque and tangle formation, especially in the temporal lobes and hippocampus. Although there is increasing evidence that major depression may interact with neuropathological processes in AD, there have been no studies of neuropathological changes in AD as a function of history of major depression. OBJECTIVE: To test the hypothesis that neuritic plaques and neurofibrillary tangles are more pronounced in the hippocampus of patients with AD with a lifetime history of major depressive disorder, as compared with patients with AD without depression history. DESIGN: Postmortem study. SETTING: Nursing home. PARTICIPANTS: The brains of 52 patients with AD without a lifetime history of major depression were compared with the brains of 50 patients with AD with a lifetime history of major depression. MAIN OUTCOME MEASURES: Neuropathological ratings from the Consortium to Establish a Registry in Alzheimer Disease battery. RESULTS: Brains of patients with AD with a lifetime history of depression showed higher levels of both plaque (P<.005) and tangle (P<.002) formation within the hippocampus than brains of patients with AD without a history of depression. Post hoc analyses showed that patients with AD who had a history of depression exhibited more rapid cognitive decline than patients without a history of depression (P<.004). Furthermore, within the group of patients with AD with a history of depression, patients who exhibited concurrent depression at the time of first diagnosis of AD exhibited more pronounced neuropathological changes in the hippocampus (P<.006). CONCLUSIONS: In AD, the presence of a lifetime history of depression corresponds to increases in AD-related neuropathological changes within the hippocampus. These changes go along with more rapid cognitive decline in patients with AD with a history of depression, and are more pronounced in patients with AD suffering from depression early on in the disease process, suggesting an interaction between major depression and AD neuropathology.