Microglia are resident myeloid cells of the central nervous system (CNS), activated in the brains of various neurological diseases. Microglia are ontogenetically and functionally distinct from monocyte-derived macrophages that infiltrate the CNS under pathological conditions. However, a lack of specific markers that distinguish resident microglia from circulating blood-derived macrophages in human brain tissues hampers accurate evaluation of microglial contributions to the human brain pathology. By comparative analysis of five comprehensive microglial transcriptome datasets, we identified an evolutionarily conserved protein TMEM119 as the most promising candidate for human microglial markers. TMEM119 was expressed on immortalized human microglia, in which the expression levels were not elevated by exposure to lipopolysaccharide, IFNγ, IL-4, IL-13 or TGFβ1. Notably, TMEM119 immunoreactivity was expressed exclusively on a subset of Iba1(+) CD68(+) microglia with ramified and amoeboid morphologies in the brains of neurodegenerative diseases, such as Alzheimer's disease (AD), whereas Iba1(+) CD68(+) infiltrating macrophages do not express TMEM119 in demyelinating lesions of multiple sclerosis and necrotic lesions of cerebral infarction. TMEM119 mRNA levels were elevated in AD brains, although the protein levels were not significantly different between AD and non-AD cases by western blot and morphometric analyses. TMEM119-positive microglia did not consistently express polarized markers for M1 (CD80) or M2 (CD163, CD209) in AD brains. These results suggest that TMEM119 serves as a reliable microglial marker that discriminates resident microglia from blood-derived macrophages in the human brain.

Ghrelin is a 28-amino-acid peptide that is the endogenous ligand for the pituitary growth hormone secretagogue receptor (GHS-R). Ghrelin is mainly produced from the stomach, but it is also expressed by various other tissues, including the CNS under normal conditions. Physiologically, ghrelin regulates appetite, gut motility, and GH release from the anterior pituitary, as well as cardiovascular and immune systems. Recent studies also indicate that ghrelin and GHS-R may play an important autocrine/paracrine role in neoplastic conditions. In order to clarify the role of ghrelin/GHS-R in gliomas, the present study assessed the expression of ghrelin and its functional receptor, GHS-R1a, in 39 glioblastomas (GBs), 13 anaplastic astrocytomas (AAs) and 11 diffuse astrocytomas (DAs) using immunohistochemical analyses. Immunohistochemical staining was evaluated as follows: no staining; 1+, 0-10% positive cells; 2+, 10-50% positive cells; 3+, >50% positive cells. Ghrelin expression was detected in 52 of 63 cases of which 38, 13 and one were scored as 3+, 2+ and 1+, respectively. GHS-R1a expression was detected in 45 of 63 cases of which 29, 15 and one were scored as 3+, 2+ and 1+, respectively. Ghrelin immunoreactivity was observed in 38 of 39 GBs, 12 of 13 AAs and two of 11 DAs. GHS-R1a immunoreactivity was observed in 39 of 39 GBs, five of 13 AAs, and one of 11 DAs. AAs and GBs showed moderate or strong immunostaining of ghrelin/GHS-R1a in the tumor cells and in proliferating microvessels. Patients were classified into lower to moderate-score, and high-score ghrelin/GHS-R categories according to the principal component and cluster analyses. Multivariate analysis of overall survival indicated that there was a significant difference (P = 0.0001) in the survival rate between these two groups. The combined results indicated that expression of the ghrelin/GHS-R1a axis increases the growth of AAs and GBs through an autocrine/paracrine mechanism.

It has been shown that high expression of certain immune checkpoint molecules, including those of the programmed death protein 1/programmed death ligand 1 (PD-1/PD-L1) axis, can be utilized to regulate immunosuppression in the microenvironment of malignant neoplasms. For the purpose of clarifying the immune-escape mechanism of primary central nervous system lymphomas (PCNSLs), particularly in Epstein-Barr virus (EBV)-positive cases, markers for PD-1, PD-L1, tumor-associated macrophages (TAMs), and tumor-infiltrating lymphocytes (TILs) in 39 surgical specimens of PCNSLs (17 EBV-positive, 22 EBV-negative) were investigated by immunohistochemistry. Staining for PD-L1 was scored as follows: (-), no staining; (1+), 0-30% positive cells; (2+), 30-60% positive cells; and (3+), >60% positive cells. In EBV-positive cases, PD-L1 was detected in both lymphoma cells and TAMs in 12/17 cases, and in TAMs only in 4/17 cases. The mean number of PD-1, TIA-1 (a marker for cytotoxic T-cells), and FOXP3 (a marker for regulatory T-cells)-positive TILs in EBV-positive cases was 36.4 ± 45.9, 390 ± 603, and 9.88 ± 15.1, respectively. In EBV-negative cases, PD-L1 was detected in both lymphoma cells and TAMs in 11/22 cases, and in TAMs only in 4/22 cases. The mean of PD-1, TIA-1 and FOXP3-positive lymphocytes in EBV-negative cases was 67.3 ± 82.0, 158 ± 206 and 9.32 ± 17.5, respectively. We found no significant difference in the number of FOXP3-positive, lymphocytes between EBV-positive and negative cases. However, there were significantly higher numbers of PD-1-positive lymphocytes in the former, and significantly higher numbers of TIA-1-positive lymphocytes in the latter (P < 0.05). The combined data indicate that expression of PD-L1 by lymphoma cells and TAMs mediate the trafficking of TILs, which may explain the immune-escape process of PCNSLs. In addition, EBV infection appears to affect the trafficking mechanism of TILs, and may thus play an important role in the microenvironment immunity of these tumors.

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BACKGROUNDExpanded GGGGCC hexanucleotide repeats located in the noncoding region of the chromosome 9 open reading frame 72 (C9orf72) gene represent the most common genetic abnormality for familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Formation of nuclear RNA foci, accumulation of repeat-associated non-ATG-translated dipeptide-repeat proteins, and haploinsufficiency of C9orf72 are proposed for pathological mechanisms of C9ALS/FTD. However, at present, the physiological function of C9orf72 remains largely unknown.METHODSBy searching on a bioinformatics database named COXPRESdb composed of the comprehensive gene coexpression data, we studied potential C9orf72 interactors.RESULTSWe identified the ATP/GTP binding protein 1 (AGTPBP1) gene alternatively named NNA1 encoding a cytosolic carboxypeptidase whose mutation is causative of the degeneration of Purkinje cells and motor neurons as the most significant gene coexpressed with C9orf72. We verified coexpression and interaction of AGTPBP1 and C9orf72 in transfected cells by immunoprecipitation and in neurons of the human brain by double-labeling immunohistochemistry. Furthermore, we found a positive correlation between AGTPBP1 and C9orf72 mRNA expression levels in the set of 21 human brains examined.CONCLUSIONSThese results suggest that AGTPBP1 serves as a C9orf72 interacting partner that plays a role in the regulation of neuronal function in a coordinated manner within the central nervous system.