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Cited by 171Neuroinflammation and Neurodegeneration MechanismsAlzheimer's disease research and treatmentsDementia and Cognitive Impairment Research

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Glycoprotein NMB: a novel Alzheimer’s disease associated marker expressed in a subset of activated microglia

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Hüttenrauch, Melanie, Ogorek, Isabella, Klafki, Hans, Otto, Markus, Stadelmann, Christine, Weggen, Sascha, Wiltfang, Jens, Wirths, Oliver·2018·Acta Neuropathologica Communications
DOI 10.1186/s40478-018-0612-3PMC PMC6194687
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Abstract

Alzheimer's disease (AD) is an irreversible, devastating neurodegenerative brain disorder characterized by the loss of neurons and subsequent cognitive decline. Despite considerable progress in the understanding of the pathophysiology of AD, the precise molecular mechanisms that cause the disease remain elusive. By now, there is ample evidence that activated microglia have a critical role in the initiation and progression of AD. The present study describes the identification of Glycoprotein nonmetastatic melanoma protein B (GPNMB) as a novel AD-related factor in both transgenic mice and sporadic AD patients by expression profiling, immunohistochemistry and ELISA measurements. We show that GPNMB levels increase in an age-dependent manner in transgenic AD models showing profound cerebral neuron loss and demonstrate that GPNMB co-localizes with a distinct population of IBA1-positive microglia cells that cluster around amyloid plaques. Our data further indicate that GPNMB is part of a microglia activation state that is only present under neurodegenerative conditions and that is characterized by the up-regulation of a subset of genes including TREM2, APOE and CST7. In agreement, we provide in vitro evidence that soluble Aβ has a direct effect on GPNMB expression in an immortalized microglia cell line. Importantly, we show for the first time that GPNMB is elevated in brain samples and cerebrospinal fluid (CSF) of sporadic AD patients when compared to non-demented controls.The current findings indicate that GPNMB represents a novel disease-associated marker that appears to play a role in the neuroinflammatory response of AD.

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Figures

Fig. 1 GPNMB mRNA expression increases in an age- and dose-dependent manner in some Alzheimer’s disease mouse models. (a) Cerebral GPNMB mRNA levels increased in an age-dependent manner in APP/PS1KI compared to PS1KI control mice. (b) In 5XFAD mice, levels of GPNMB mRNA started to be significantly increased at 7 months of age when compared to wild-type (WT) control mice. 12-month-old 5XFAD mice showed even higher GPNMB levels. (c) In 12-month-old APP23 mice, GPNMB gene expression levels were not increased compared to WT animals. All data are given as mean ± SD. ***P < 0.001; **P < 0.01; *P < 0.05
Fig. 1 GPNMB mRNA expression increases in an age- and dose-dependent manner in some Alzheimer’s disease mouse models. (a) Cerebral GPNMB mRNA levels increased in an age-dependent manner in APP/PS1KI compared to PS1KI control mice. (b) In 5XFAD mice, levels of GPNMB mRNA started to be significantly increased at 7 months of age when compared to wild-type (WT) control mice. 12-month-old 5XFAD mice showed even higher GPNMB levels. (c) In 12-month-old APP23 mice, GPNMB gene expression levels were not increased compared to WT animals. All data are given as mean ± SD. ***P < 0.001; **P < 0.01; *P < 0.05CC BY · PubMed Central
Fig. 2 GPNMB co-localizes with IBA1-positive microglia cells in 5XFAD brains. (a-c) Double immunostaining with antibodies against GPNMB and IBA1 revealed a cellular co-localization in 12-month-old 5XFAD brains, while no co-localization was seen with the astrocytic marker GFAP or the neuronal marker NeuN. Scale bar: A-C = 33 μm
Fig. 2 GPNMB co-localizes with IBA1-positive microglia cells in 5XFAD brains. (a-c) Double immunostaining with antibodies against GPNMB and IBA1 revealed a cellular co-localization in 12-month-old 5XFAD brains, while no co-localization was seen with the astrocytic marker GFAP or the neuronal marker NeuN. Scale bar: A-C = 33 μmCC BY · PubMed Central
Fig. 3 Increased GPNMB protein levels and cerebral expression pattern in 5XFAD mice. (a) Abundant GPNMB staining was detected throughout the whole brain in 12-month-old 5XFAD mice, e.g. in the subiculum. (b) High-power view of GPNMB-positive cells surrounding an amyloid plaque core. (c) Quantitative analysis of GPNMB protein levels in TBS- and SDS-soluble brain fractions of 12-month-old WT, 5XFAD and APP23 mice revealed a highly significant increase in GPNMB levels in 5XFAD mice compared to WT animals, while no elevated levels could be observed in APP23 mice. (d) GPNMB immunoreactivity was also demonstrated in the spinal cord (SC) of 12-month-old 5XFAD mice. GPNMB was abundantly expressed throughout the whole SC tissue and showed co-localization with IBA1-positive microglia cells (parallel sections). (e) As demonstrated by RT-PCR analyses, 5XFAD mice showed a highly significant increase in GPNMB mRNA levels in the SC when compared to WT controls. However, no such differences were observed in peripheral liver samples serving as a non-neuronal control tissue. Enzyme-linked immunosorbent assay revealed highly increased GPNMB protein level in 5XFAD SC tissue compared to WT controls (f). All data are given as mean ± SD. ***P < 0.001. Scale bar: A = 133 μm, B = 25 μm; D = 200 μm (upper panel), 20 μm (lower panel)
Fig. 3 Increased GPNMB protein levels and cerebral expression pattern in 5XFAD mice. (a) Abundant GPNMB staining was detected throughout the whole brain in 12-month-old 5XFAD mice, e.g. in the subiculum. (b) High-power view of GPNMB-positive cells surrounding an amyloid plaque core. (c) Quantitative analysis of GPNMB protein levels in TBS- and SDS-soluble brain fractions of 12-month-old WT, 5XFAD and APP23 mice revealed a highly significant increase in GPNMB levels in 5XFAD mice compared to WT animals, while no elevated levels could be observed in APP23 mice. (d) GPNMB immunoreactivity was also demonstrated in the spinal cord (SC) of 12-month-old 5XFAD mice. GPNMB was abundantly expressed throughout the whole SC tissue and showed co-localization with IBA1-positive microglia cells (parallel sections). (e) As demonstrated by RT-PCR analyses, 5XFAD mice showed a highly significant increase in GPNMB mRNA levels in the SC when compared to WT controls. However, no such differences were observed in peripheral liver samples serving as a non-neuronal control tissue. Enzyme-linked immunosorbent assay revealed highly increased GPNMB protein level in 5XFAD SC tissue compared to WT controls (f). All data are given as mean ± SD. ***P < 0.001. Scale bar: A = 133 μm, B = 25 μm; D = 200 μm (upper panel), 20 μm (lower panel)CC BY · PubMed Central
Fig. 4 GPNMB/IBA-1-positive microglia cells cluster around individual plaque cores in 5XFAD brains. Triple immunofluorescence staining using antibodies against GPNMB (a), Aβ (b) and IBA1 (c) demonstrated the spatial co-localization of GPNMB-positive microglia cells around amyloid plaque cores in 12-month-old 5XFAD brains (d). Even though APP23 mice showed numerous activated microglia cells (g) clustered around amyloid plaques (f), no GPNMB signal could be detected (e,h). (i) RT-PCR analyses revealed significantly increased mRNA levels of CST7, TREM2, APOE, CLEC7A and CCL2 in 5XFAD brains when compared to WT and APP23 mice. However, levels of AIF1 and TMEM119 were comparable in all groups tested. All data are given as mean ± SD. ***P < 0.001; **P < 0.01. Scale bar: A-H = 33 μm
Fig. 4 GPNMB/IBA-1-positive microglia cells cluster around individual plaque cores in 5XFAD brains. Triple immunofluorescence staining using antibodies against GPNMB (a), Aβ (b) and IBA1 (c) demonstrated the spatial co-localization of GPNMB-positive microglia cells around amyloid plaque cores in 12-month-old 5XFAD brains (d). Even though APP23 mice showed numerous activated microglia cells (g) clustered around amyloid plaques (f), no GPNMB signal could be detected (e,h). (i) RT-PCR analyses revealed significantly increased mRNA levels of CST7, TREM2, APOE, CLEC7A and CCL2 in 5XFAD brains when compared to WT and APP23 mice. However, levels of AIF1 and TMEM119 were comparable in all groups tested. All data are given as mean ± SD. ***P < 0.001; **P < 0.01. Scale bar: A-H = 33 μmCC BY · PubMed Central
Fig. 5 Soluble Aβ induces GPNMB mRNA expression in immortalized microglia cells. (a) Treatment of BV2 cells with Aβ-containing medium or synthetic Aβ1–42 peptides resulted in a highly significant increase in GPNMB mRNA expression, while LPS treatment had no effect on GPNMB levels. In contrast, LPS treatment caused microglia activation as shown by upregulation of the pro-inflammatory cytokines IL1-β and TNFα (b, c). While LPS treatment led to decreased expression of APOE and CLEC7A, conditioned Aβ-containing media induced these DAM phenotype-associated genes (d, e). MITF levels did not change following treatment with Aβ-conditioned medium or synthetic Aβ although GPNMB levels were clearly elevated (f). All data are given as mean ± SD. * P < 0.05; **P < 0.01; ***P < 0.001
Fig. 5 Soluble Aβ induces GPNMB mRNA expression in immortalized microglia cells. (a) Treatment of BV2 cells with Aβ-containing medium or synthetic Aβ1–42 peptides resulted in a highly significant increase in GPNMB mRNA expression, while LPS treatment had no effect on GPNMB levels. In contrast, LPS treatment caused microglia activation as shown by upregulation of the pro-inflammatory cytokines IL1-β and TNFα (b, c). While LPS treatment led to decreased expression of APOE and CLEC7A, conditioned Aβ-containing media induced these DAM phenotype-associated genes (d, e). MITF levels did not change following treatment with Aβ-conditioned medium or synthetic Aβ although GPNMB levels were clearly elevated (f). All data are given as mean ± SD. * P < 0.05; **P < 0.01; ***P < 0.001CC BY · PubMed Central
Fig. 6 Increased GPNMB protein levels in brain tissue and cerebrospinal fluid (CSF) of sporadic Alzheimer‘s disease cases. GPNMB immunoreactivity was detected in microglial cells surrounding amyloid plaque cores and in the vicinity of blood vessel walls (a, e). (b) High-power view of the plaque core in (a). In addition, GPNMB-positive amoeboid microglia were detected in plaque-free areas (c, f), while GPNMB-positive microglia were only occasionally observed in samples from non-demented control patients (d). (g) In the TBS-soluble brain fractions, higher levels of GPNMB were detected in AD cases when compared to non-demented controls (NDC), however, without reaching statistical significance (p = 0.06). (h) No differences were detected between the two groups in SDS-soluble brain fractions. (i) The amount of GPNMB in the CSF of AD patients was significantly higher than in control patients. (j) No significant difference in GPNMB serum levels could be detected between controls and AD. All data are given as mean ± SD. *P < 0.05. Scale bar: A,D,E = 50 μm; B,C,F = 20 μm
Fig. 6 Increased GPNMB protein levels in brain tissue and cerebrospinal fluid (CSF) of sporadic Alzheimer‘s disease cases. GPNMB immunoreactivity was detected in microglial cells surrounding amyloid plaque cores and in the vicinity of blood vessel walls (a, e). (b) High-power view of the plaque core in (a). In addition, GPNMB-positive amoeboid microglia were detected in plaque-free areas (c, f), while GPNMB-positive microglia were only occasionally observed in samples from non-demented control patients (d). (g) In the TBS-soluble brain fractions, higher levels of GPNMB were detected in AD cases when compared to non-demented controls (NDC), however, without reaching statistical significance (p = 0.06). (h) No differences were detected between the two groups in SDS-soluble brain fractions. (i) The amount of GPNMB in the CSF of AD patients was significantly higher than in control patients. (j) No significant difference in GPNMB serum levels could be detected between controls and AD. All data are given as mean ± SD. *P < 0.05. Scale bar: A,D,E = 50 μm; B,C,F = 20 μmCC BY · PubMed Central
Fig. 7 Schematic summary of results and hypothesis: In Alzheimer’s disease (AD) mouse models with profound neuron loss (e.g. 5XFAD), GPNMB-positive activated microglia cells cluster around individual amyloid plaque cores. In contrast, microglia cells are persistently GPNMB-negative in healthy wild-type mice or AD models showing abundant plaque pathology without neuron loss. Under neurodegenerative conditions, microglia switch from a homeostatic to a disease-associated phenotype, which has been called “microglial neurodegenerative phenotype” (MGnD) or “disease-associated microglia” (DAMs), and which is characterized by the upregulation of a subset of genes including GPNMB. The precise role of GPNMB in the central nervous system is not yet known, however, evidence indicates that it has anti-inflammatory and regenerative functions. Individual components of the scheme have been taken from: http://smart.servier.com
Fig. 7 Schematic summary of results and hypothesis: In Alzheimer’s disease (AD) mouse models with profound neuron loss (e.g. 5XFAD), GPNMB-positive activated microglia cells cluster around individual amyloid plaque cores. In contrast, microglia cells are persistently GPNMB-negative in healthy wild-type mice or AD models showing abundant plaque pathology without neuron loss. Under neurodegenerative conditions, microglia switch from a homeostatic to a disease-associated phenotype, which has been called “microglial neurodegenerative phenotype” (MGnD) or “disease-associated microglia” (DAMs), and which is characterized by the upregulation of a subset of genes including GPNMB. The precise role of GPNMB in the central nervous system is not yet known, however, evidence indicates that it has anti-inflammatory and regenerative functions. Individual components of the scheme have been taken from: http://smart.servier.comCC BY · PubMed Central

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Authors
Hüttenrauch, M., Ogorek, I., Klafki, H., Otto, M., Stadelmann, C., Weggen, S., Wiltfang, J., & Wirths, O.
Affiliations
  • Universitätsmedizin Göttingen (DE)
  • Heinrich Heine University Düsseldorf (DE)
  • Universität Ulm (DE)
  • German Center for Neurodegenerative Diseases (DE)
Corresponding author
Wirths, Oliver
ORCID
Venue
Acta Neuropathologica Communications
Publisher
Springer Science and Business Media LLC
Published
2018-10-19
Volume
6
Issue
1
Pages
108-108
ISSN
2051-5960 (ISSN-L)
DOI
10.1186/s40478-018-0612-3
PMC
PMC6194687
OpenAlex
W2898010909
Cited by
166
References
64
Citations by year
2026: 22 · 2025: 23 · 2024: 27 · 2023: 22 · 2022: 15 · 2021: 34 · 2020: 12 · 2019: 8 · 2018: 1
FWCI
6.18
Type
journal article
Language
en
Open access
Licence
Locations
  • Acta Neuropathologica Communications (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • GoeScholar The Publication Server of the Georg-August-Universität Göttingen (Georg-August-Universität Göttingen) (submittedVersion, cc-by)
  • DZNE Pub (submittedVersion, cc-by)
Funders
Alzheimer Forschung Initiative (16013)
Topics
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Alzheimer's disease research and treatments
  • Health Sciences › Medicine › Psychiatry and Mental health › Dementia and Cognitive Impairment Research
Keywords
GPNMB, Alzheimer's disease, microglial activation, disease biomarkers, amyloid plaques, TREM2, apolipoprotein E, cystatin F, amyloid-beta, neuroinflammation, cerebrospinal fluid, transgenic mouse model, sporadic Alzheimer's disease
MeSH terms
Age Factors, Aged, Aged, 80 and over, Alzheimer Disease, Animals, Brain, Cell Line, Transformed, Disease Models, Animal, Female, Glial Fibrillary Acidic Protein, Humans, Lipopolysaccharides, Male, Membrane Glycoproteins, Mice, Transgenic, Mutation, Peptide Fragments, Phosphopyruvate Hydratase, Up-Regulation, Amyloid beta-Peptides, Amyloid beta-Protein Precursor, Microglia, Mice, Presenilin-1

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