Neuroinflammation is considered a key pathological process in neurodegenerative diseases of aging, including Alzheimer’s disease (AD). Many studies have defined phenotypes of reactive microglia, the brain-resident macrophages, with different antigenic markers to identify those potentially causing inflammatory damage. We took an alternative approach with the goal of characterizing the distribution of purinergic receptor P2RY12-positive microglia, a marker previously defined as identifying homeostatic or non-activated microglia. We examined the expression of P2RY12 by dual-color light and fluorescence immunohistochemistry using sections of middle temporal gyrus from AD, high plaque and low plaque non-demented cases in relation to amyloid beta (Aβ) plaques and phosphorylated tau, markers of pathology, and HLA-DR, IBA-1, CD68, and progranulin, microglial phenotype markers. In low plaque cases, P2RY12-positive microglia mostly had non-activated morphologies, while the morphologies of P2RY12-positive microglia in AD brains were highly variable, suggesting its expression could encompass a wider range of phenotypes than originally hypothesized. P2RY12 expression by microglia differed depending on the types of plaques or tangles they were associated with. Areas of inflammation characterized by lack of P2RY12-positive microglia around mature plaques could be observed, but many diffuse plaques showed colocalization with P2RY12-positive microglia. Based on these results, P2RY12 expression by microglia should not be considered solely a marker of resting microglia as P2RY12 immunoreactivity was identifying microglia positive for CD68, progranulin and to a limited extent HLA-DR, markers of activation.

Progranulin (PGRN) is a protein encoded by the GRN gene with multiple identified functions including as a neurotrophic factor, tumorigenic growth factor, anti-inflammatory cytokine and regulator of lysosomal function. A single mutation in the human GRN gene resulting in reduced PGRN expression causes types of frontotemporal lobar degeneration resulting in frontotemporal dementia. Prosaposin (PSAP) is also a multifunctional neuroprotective secreted protein and regulator of lysosomal function. Interactions of PGRN and PSAP affect their functional properties. Their roles in Alzheimer’s disease (AD), the leading cause of dementia, have not been defined. In this report, we examined in detail the cellular expression of PGRN in middle temporal gyrus samples of a series of human brain cases (n = 45) staged for increasing plaque pathology. Immunohistochemistry showed PGRN expression in cortical neurons, microglia, cerebral vessels and amyloid beta (Aβ) plaques, while PSAP expression was mainly detected in neurons and Aβ plaques, and to a limited extent in astrocytes. We showed that there were increased levels of PGRN protein in AD cases and corresponding increased levels of PSAP. Levels of PGRN and PSAP protein positively correlated with amyloid beta (Aβ), with PGRN levels correlating with phosphorylated tau (serine 205) levels in these samples. Although PGRN colocalized with lysosomal-associated membrane protein-1 in neurons, most PGRN associated with Aβ plaques did not. Aβ plaques with PGRN and PSAP deposits were identified in the low plaque non-demented cases suggesting this was an early event in plaque formation. We did not observe PGRN-positive neurofibrillary tangles. Co-immunoprecipitation studies of PGRN from brain samples identified only PSAP associated with PGRN, not sortilin or other known PGRN-binding proteins, under conditions used. Most PGRN associated with Aβ plaques were immunoreactive for PSAP showing a high degree of colocalization of these proteins that did not change between disease groups. As PGRN supplementation has been considered as a therapeutic approach for AD, the possible involvement of PGRN and PSAP interactions in AD pathology needs to be further considered.

New super-sensitive biomarker assay platforms for measuring Alzheimer’s disease (AD) core pathological markers in plasma have recently been developed and tested. Research findings from these technologies offer promising evidence for identifying the earliest stages of AD and correlating them with brain pathological progression. Here, we review findings using immunomagnetic reduction, one of these ultrasensitive technologies. The principles, technology and assays developed, along with selected published findings will be discussed. The major findings from this technology were significant increases of amyloid beta (Aβ) 42 and total tau (t-tau) levels in subjects clinically diagnosed with early AD when compared with cognitively normal control (NC) subjects. The composite marker of the product of Aβ42 and t-tau discriminated subjects with early AD from NC subjects with high accuracy. The potential of this technology for the purpose of early or preclinical disease stage detection has yet to be explored in subjects who have also been assessed with brain imaging and cerebrospinal fluid AD core biomarker measurements.

Experimental studies of neuroinflammation in Alzheimer’s disease (AD) have mostly investigated microglia, the brain-resident macrophages. This review focused on human microglia obtained at rapid autopsies. Studies employing methods to isolate and culture human brain microglia in high purity for experimental studies were discussed. These methods were employed to isolate human microglia for investigation of a number of features of neuroinflammation, including activation phenotypes, neurotoxicity, responses to abnormal aggregated proteins such as amyloid beta, phagocytosis, and the effects of aging and disease on microglia cellular properties. In recent years, interest in human microglia and neuroinflammation has been renewed due to the identification of inflammation-related AD genetic risk factors, in particular the triggering receptor expressed on myeloid cells (TREM)-2. Because of the difficulties in developing effective treatments for AD, there has been a general need for greater understanding of the functions of microglia in normal and AD brains. While most experimental studies on neuroinflammation have employed rodent microglia, this review considered the role of human microglia in experimental studies. This review focused on the development of in vitro methodology for the culture of postmortem human microglia and the key findings obtained from experimental studies with these cells.

Inflammation is considered a key pathological process in neurodegenerative diseases, including Alzheimer’s disease (AD) and Parkinson’s disease (PD), but there are still mechanisms not understood. In the brain, most microglia are performing essential homeostatic functions, but can also respond to pathogenic stimuli by producing harmful pro-inflammatory cytokines or free radicals. Distinguishing between damaging and homeostatic microglia in human diseased brain tissues is a challenge. This report describes findings using a monoclonal antibody to CD105/Endoglin (R&D Systems MAB1097) that identifies subtypes of activated microglia. CD105/Endoglin is a co-receptor for transforming growth factor beta (TGFβ) receptor that antagonizes TGFβ signaling. CD105/Endoglin is a marker for vascular endothelial cells, but was originally identified as a marker for activated macrophages. This antibody did not identify endothelial cells in brain sections, only microglia-like cells. In this study, we examined with this antibody tissue section from middle temporal gyrus derived from human brains from normal control subjects with low-plaque pathology, high-plaque pathology, and AD cases, and also substantia nigra samples from control and PD cases, in conjunction with antibodies to markers of pathology and microglia. In low-plaque pathology cases, CD105-positive microglia were mostly absent, but noticeably increased with increasing pathology. CD105-positive cells strongly colocalized with amyloid-beta plaques, but not phosphorylated tau positive tangles. In substantia nigra, strong microglial CD105 staining was observed in microglia associated with degenerating dopaminergic neurons and neuromelanin. In PD cases with few surviving dopaminergic neurons, this staining had decreased. By Western blot, this antibody identified polypeptide bands of 70 kDa in brain samples, and samples from microglia, macrophages, and brain endothelial cells. In comparison with other tested CD105 antibodies, this antibody did not recognize the glycosylated forms of CD105 on Western blots. Overall, the data indicate that this antibody and this marker could have utility for subtyping of microglia in pathologically-involved tissue.

The Arizona Study of Aging and Neurodegenerative Disorders/Brain and Body Donation Program at Banner Sun Health Research Institute (BSHRI) is a longitudinal clinicopathological study with a current enrollment of more than 900 living subjects for aging and neurodegenerative disease research. Annual clinical assessments are done by cognitive and movement neurologists and neuropsychologists. Brain and body tissues are collected at a median postmortem interval of 3.0 h for neuropathological diagnosis and banking. Since 2018, the program has undertaken banking of scalp fibroblasts derived from neuropathologically characterized donors with Alzheimer’s disease, Parkinson’s disease, and other neurodegenerative diseases. Here, we describe the procedure development and cell characteristics from 14 male and 15 female donors (mean ± SD of age: 83.6 ± 12.2). Fibroblasts from explant cultures were banked at passage 3. The results of mRNA analysis showed positive expression of fibroblast activation protein, vimentin, fibronectin, and THY1 cell surface antigen. We also demonstrated that the banked fibroblasts from a postmortem elderly donor were successfully reprogramed to human-induced pluripotent stem cells (hiPSCs). Taken together, we have demonstrated the successful establishment of a human autopsy-derived fibroblast banking program. The cryogenically preserved cells are available for request at the program website of the BSHRI.