Isolation of primary microglia from the human post-mortem brain: effects of ante- and post-mortem variables

Mizee, Mark R.; Miedema, Suzanne S M; Poel, Marlijn van der; Adelia,; Schuurman, Karianne; Strien, Miriam E. van; Melief, Jeroen; Smolders, Joost; Hendrickx, Debbie A.E.; Heutinck, Kirstin M.; Hamann, Jörg; Huitinga, Inge

doi:10.1186/s40478-017-0418-8

Cited by 88 publications

(84 citation statements)

References 41 publications

(61 reference statements)

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“…New insights may be obtained with microglia isolated from human post-mortem brain tissue (Mizee et al, 2017 ), or derived from induced pluripotent stem cells generated from AD patients (Abud et al, 2017 ). Moreover, because neuron-astrocyte-microglia communication plays a crucial role in AD pathogenesis, and microglia activation triggers astrocyte neurotoxicity (Liddelow et al, 2017 ), additional studies using 3D culture models that allow cell-to-cell interplay and best recapitulate AD (Kim et al, 2015 ; Choi et al, 2016 ; Lee et al, 2016 ) should be used to corroborate or complement our findings.…”

Section: Discussionmentioning

confidence: 99%

Key Aging-Associated Alterations in Primary Microglia Response to Beta-Amyloid Stimulation

Caldeira

Cunha

Vaz

et al. 2017

Front. Aging Neurosci.

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Alzheimer’s disease (AD) is characterized by a progressive cognitive decline and believed to be driven by the self-aggregation of amyloid-β (Aβ) peptide into oligomers and fibrils that accumulate as senile plaques. It is widely accepted that microglia-mediated inflammation is a significant contributor to disease pathogenesis; however, different microglia phenotypes were identified along AD progression and excessive Aβ production was shown to dysregulate cell function. As so, the contribution of microglia to AD pathogenesis remains to be elucidated. In this study, we wondered if isolated microglia cultured for 16 days in vitro (DIV) would react differentially from the 2 DIV cells upon treatment with 1000 nM Aβ1–42 for 24 h. No changes in cell viability were observed and morphometric alterations associated to microglia activation, such as volume increase and process shortening, were obvious in 2 DIV microglia, but less evident in 16 DIV cells. These cells showed lower phagocytic, migration and autophagic properties after Aβ treatment than the 2 DIV cultured microglia. Reduced phagocytosis may derive from increased CD33 expression, reduced triggering receptor expressed on myeloid cells 2 (TREM2) and milk fat globule-EGF factor 8 protein (MFG-E8) levels, which were mainly observed in 16 DIV cells. Activation of inflammatory mediators, such as high mobility group box 1 (HMGB1) and pro-inflammatory cytokines, as well as increased expression of Toll-like receptor 2 (TLR2), TLR4 and fractalkine/CX3C chemokine receptor 1 (CX3CR1) cell surface receptors were prominent in 2 DIV microglia, while elevation of matrix metalloproteinase 9 (MMP9) was marked in 16 DIV cells. Increased senescence-associated β-galactosidase (SA-β-gal) and upregulated miR-146a expression that were observed in 16 DIV cells showed to increase by Aβ in 2 DIV microglia. Additionally, Aβ downregulated miR-155 and miR-124, and reduced the CD11b+ subpopulation in 2 DIV microglia, while increased the number of CD86+ cells in 16 DIV microglia. Simultaneous M1 and M2 markers were found after Aβ treatment, but at lower expression in the in vitro aged microglia. Data show key-aging associated responses by microglia when incubated with Aβ, with a loss of reactivity from the 2 DIV to the 16 DIV cells, which course with a reduced phagocytosis, migration and lower expression of inflammatory miRNAs. These findings help to improve our understanding on the heterogeneous responses that microglia can have along the progression of AD disease and imply that therapeutic approaches may differ from early to late stages.

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Section: Discussionmentioning

confidence: 99%

Key Aging-Associated Alterations in Primary Microglia Response to Beta-Amyloid Stimulation

Caldeira

Cunha

Vaz

et al. 2017

Front. Aging Neurosci.

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“…Tissue was obtained in accordance with the Declaration of Helsinki and the Amsterdam UMC Research Code provided by the Medical Ethics Committee. Human primary peripheral blood mononuclear cells (PBMCs) and T cells were isolated from blood using the Macs Pan T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and human primary microglia was isolated as previously described (35).…”

Section: Human Primary Cellsmentioning

confidence: 99%

Increased expression of miR142 and miR155 in glial and immune cells after traumatic brain injury may contribute to neuroinflammation via astrocyte activation

et al. 2020

Self Cite

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Traumatic brain injury (TBI) is associated with the pathological activation of immune‐competent cells in the brain, such as astrocytes, microglia and infiltrating immune blood cells, resulting in chronic inflammation and gliosis. This may contribute to the secondary injury after TBI, thus understanding of these processes is crucial for the development of effective treatments of post‐traumatic pathologies. MicroRNAs (miRNAs, miRs) are small noncoding RNAs, functioning as posttranscriptional regulators of gene expression. The increased expression of inflammation‐associated microRNAs miR155 and miR142 has been reported after TBI in rats. However, expression of these miRNAs in the human brain post‐TBI is not studied and their functions are not well understood. Moreover, circulating miR155 and miR142 are candidate biomarkers. Therefore, we characterized miR142 and miR155 expression in the perilesional cortex and plasma of rats that underwent lateral fluid‐percussion injury, a model for TBI and in the human perilesional cortex post‐TBI. We demonstrated higher miR155 and miR142 expression in the perilesional cortex of rats 2 weeks post‐TBI. In plasma, miR155 was associated with proteins and miR142 with extracellular vesicles, however their expression did not change. In the human perilesional cortex miR155 was most prominently expressed by activated astrocytes, whereas miR142 was expressed predominantly by microglia, macrophages and lymphocytes. Pro‐inflammatory medium from macrophage‐like cells stimulated miR155 expression in astrocytes and overexpression of miR142 in these cells further potentiated a pro‐inflammatory state of activated astrocytes. We conclude that miR155 and miR142 promote brain inflammation via astrocyte activation and may be involved in the secondary brain injury after TBI.

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“…The interphase containing the microglia was transferred to a new tube (the myelin and erythrocyte layers were discarded) and washed twice with DMEM supplemented with 10% FCS, 1% Pen/Strep, 1% gentamycin, and 25 mM HEPES (Invitrogen). Negative selection of granulocytes (previous method only) and positive selection of microglia with anti-CD15-and anti-CD11b-conjugated magnetic microbeads (Miltenyi Biotec), respectively, were performed by magnetic activated cell sorting (MACS) according to the manufacturer's protocol [36]. Brie y, cells were incubated with 10 μl CD15 microbeads for 15 min at 4°C, washed, suspended in bead buffer (0.5% BSA, 2 mM EDTA in PBS, pH 7.2), and transferred to an MS column placed in a magnetic holder.…”

Section: Phagocytosis Assaymentioning

confidence: 99%

Defective phagocytic function of induced microglia-like cells is correlated with rapid progression of sporadic ALS

Noh¹,

Kwon²,

Oh³

et al. 2020

Preprint

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Background: Microglia play a key role in determining the progression of amyotrophic lateral sclerosis (ALS), yet their precise role in ALS has not been identified in humans. The objective of this study was to identify the functional characteristics of microglia and related factors in patients with sporadic ALS that is rapidly progressing.Methods: After confirming that microglia-like cells (iMGs) induced by human monocytes could recapitulate the main signatures of brain microglia, serial comparative studies were conducted to delineate functional differences in iMGs from patients with slowly progressive ALS [ALS(S), n = 14] versus rapidly progressive ALS [ALS(R), n = 15].Results: Despite an absence of significant differences in the expression of microglial homeostatic genes, ALS(R)-iMGs preferentially showed defective phagocytosis and an exaggerated pro-inflammatory response to LPS stimuli compared to ALS(S)-iMGs. Transcriptome analysis revealed that the perturbed phagocytosis seen in ALS(R)-iMGs was closely associated with decreased NCKAP1 (NCK-associated protein 1)-mediated abnormal actin polymerization. NCKAP1 overexpression was sufficient to rescue impaired phagocytosis in ALS(R)-iMGs. To leverage our findings and identify biological markers of rapidly progressing ALS, we measured plasma miRNA-214-3p (negative regulator targeting NCKAP1) levels. Post-hoc analysis indicated that decreased NCKAP1 expression in iMGs and a concomitant increase in plasma miRNA-214-3p levels was correlated with rapid progression of ALS.Interpretation: Our data suggest that microglial NCKAP1 may be an alternative therapeutic target in rapidly progressive sporadic ALS. In addition, miRNA-214-3p levels could be a serological biomarker for predicting the speed of disease progression.

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Isolation of primary microglia from the human post-mortem brain: effects of ante- and post-mortem variables

Cited by 88 publications

References 41 publications

Key Aging-Associated Alterations in Primary Microglia Response to Beta-Amyloid Stimulation

Key Aging-Associated Alterations in Primary Microglia Response to Beta-Amyloid Stimulation

Increased expression of miR142 and miR155 in glial and immune cells after traumatic brain injury may contribute to neuroinflammation via astrocyte activation

Defective phagocytic function of induced microglia-like cells is correlated with rapid progression of sporadic ALS

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