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, 16 (1), 48

Astrocytic Ceramide as Possible Indicator of Neuroinflammation

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Astrocytic Ceramide as Possible Indicator of Neuroinflammation

Nienke M de Wit et al. J Neuroinflammation.

Abstract

Background: Neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease dementia (PDD), and frontotemporal lobar dementia (FTLD) are characterized by progressive neuronal loss but differ in their underlying pathological mechanisms. However, neuroinflammation is commonly observed within these different forms of dementia. Recently, it has been suggested that an altered sphingolipid metabolism may contribute to the pathogenesis of a variety of neurodegenerative conditions. Especially ceramide, the precursor of all complex sphingolipids, is thought to be associated with pro-apoptotic cellular processes, thereby propagating neurodegeneration and neuroinflammation, although it remains unclear to what extent. The current pathological study therefore investigates whether increased levels of ceramide are associated with the degree of neuroinflammation in various neurodegenerative disorders.

Methods: Immunohistochemistry was performed on human post-mortem tissue of PDD and FTLD Pick's disease cases, which are well-characterized cases of dementia subtypes differing in their neuroinflammatory status, to assess the expression and localization of ceramide, acid sphingomyelinase, and ceramide synthase 2 and 5. In addition, we determined the concentration of sphingosine, sphingosine-1-phosphate (S1P), and ceramide species differing in their chain-length in brain homogenates of the post-mortem tissue using HPLC-MS/MS.

Results: Our immunohistochemical analysis reveals that neuroinflammation is associated with increased ceramide levels in astrocytes in FTLD Pick's disease. Moreover, the observed increase in ceramide in astrocytes correlates with the expression of ceramide synthase 5. In addition, HPLC-MS/MS analysis shows a shift in ceramide species under neuroinflammatory conditions, favoring pro-apoptotic ceramide.

Conclusions: Together, these findings suggest that detected increased levels of pro-apoptotic ceramide might be a common denominator of neuroinflammation in different neurodegenerative diseases.

Keywords: Acid sphingomyelinase; Ceramide; Ceramide synthase; Neurodegenerative diseases; Neuroinflammation; Sphingolipids.

Conflict of interest statement

Ethics approval and consent to participate

See materials and methods section ‘Post-mortem human brain tissue’.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Figures

Fig. 1
Fig. 1
Increased activation of microglia in FTD-Pi. Quantitative analysis of the immunoreactive area for microglia (HLA-DR) in control (n = 5), PDD (n = 5), and FTD-Pi (n = 5). The values represent the mean ± S.E.M. Statistical significance (Kruskal-Wallis test, with Dunn’s multiple correction) indicated with asterisks: **p < 0.01
Fig. 2
Fig. 2
Increased levels of ceramide in FTD-Pi. a Immunohistochemical staining for ceramide in the inferior frontal gyrus of non-demented controls, PDD, and FTD-Pi. DAB (brown) was used as chromogen and hematoxylin (blue) was used for counterstaining of the nucleus. White arrows indicate astrocytes. Bar: 50 μm. b Quantitative analysis of the immunoreactive area for ceramide in FTD-Pi (n = 5), PDD (n = 5), and controls (n = 5). The values represent the mean ± S.E.M. c Correlation analysis of the immunoreactivity levels of ceramide with astrocytes (GFAP). Statistical significance (Kruskal-Wallis test, with Dunn’s multiple correction) indicated with asterisks: **p < 0.01
Fig. 3
Fig. 3
Increased levels of ceramide in specifically astrocytes in FTD-Pi. Colocalization studies indicated that a ceramide containing cells (green) were immunopositive for GFAP (red), indicative of astrocytes. Nuclei were counterstained with Hoechst (blue). Bar: 20 μm. b Quantitative analysis of the fluorescent intensity of ceramide in astrocytes in control (n = 5), PDD (n = 5), and FTD-Pi (n = 5). The values represent the mean ± S.E.M. Statistical significance (Kruskal-Wallis test, with Dunn’s multiple correction) indicated with asterisks: **p < 0.01
Fig. 4
Fig. 4
Ceramide does not colocalize with microglia. Colocalization studies indicated that ceramide containing cells (red) were not immunopositive for HLA-DR (green), indicative for microglia. Nuclei were counterstained with Hoechst (blue). Bar: 20 μm
Fig. 5
Fig. 5
ASM expression is not specific for astrocytes. a Immunohistochemical staining for ASM in the inferior frontal gyrus of non-demented controls, PDD, and FTD-Pi. DAB (brown) was used as chromogen and hematoxylin (blue) was used for counterstaining of the nucleus. Black arrows indicate microglia. White arrows indicate astrocytes. Bar: 50 μm. b Quantitative analysis of the immunoreactive area for ASM in FTD-Pi (n = 4), PDD (n = 5), and controls (n = 5). The values represent the mean ± S.E.M
Fig. 6
Fig. 6
The expression of ceramide synthase 5, and not 2, in astrocytes correlates with the increase of ceramide in astrocytes. a The relative abundance normalized to GAPDH of the different CerSs in astrocytes where CerS2 and CerS5 relative mRNA expression are the highest. b Immunofluorescent double labeling of CerS2 (green) and ceramide (red) shows no colocalization, whereas c CerS5 (green) does colocalize with ceramide (red). Nuclei were counterstained with Hoechst (gray). Bar: 20 μm. d Correlation analysis of the immunoreactivity levels of ceramide with CerS5 expressed in astrocytes. e Quantitative analysis of CerS5 in control (n = 5), PDD (n = 5), and FTD-Pi (n = 5). The values represent the mean ± S.E.M, *p < 0.05
Fig. 7
Fig. 7
Altered ceramide levels in FTD-Pi compared to controls. a Levels of ceramides of different chain-lengths, b sphingosine, and c S1P were quantified by use of HPLC MS/MS in brain homogenates of FTD-Pi (n = 5), PDD (n = 5), and controls (n = 5). Data show the mean percentage ± S.E.M. Statistical significance between groups (Kruskal-Wallis test, with Dunn’s multiple correction) is indicated with asterisk: *p < 0.05

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