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Loss of the Neuroprotective Factor Sphingosine 1-phosphate Early in Alzheimer's Disease Pathogenesis

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Loss of the Neuroprotective Factor Sphingosine 1-phosphate Early in Alzheimer's Disease Pathogenesis

Timothy A Couttas et al. Acta Neuropathol Commun.

Abstract

Background: The greatest genetic risk factor for late-onset Alzheimer's disease (AD) is the ϵ4 allele of Apolipoprotein E (ApoE). ApoE regulates secretion of the potent neuroprotective signaling lipid Sphingosine 1-phosphate (S1P). S1P is derived by phosphorylation of sphingosine, catalysed by sphingosine kinases 1 and 2 (SphK1 and 2), and SphK1 positively regulates glutamate secretion and synaptic strength in hippocampal neurons. S1P and its receptor family have been subject to intense pharmacological interest in recent years, following approval of the immunomodulatory drug Fingolimod, an S1P mimetic, for relapsing multiple sclerosis.

Results: We quantified S1P levels in six brain regions that are differentially affected by AD pathology, in a cohort of 34 post-mortem brains, divided into four groups based on Braak neurofibrillary tangle staging. S1P declined with increasing Braak stage, and this was most pronounced in brain regions most heavily affected by AD pathology. The S1P/sphingosine ratio was 66% and 64% lower in Braak stage III/IV hippocampus (p = 0.010) and inferior temporal cortex (p = 0.014), respectively, compared to controls. In accordance with this change, both SphK1 and SphK2 activity declined with increasing Braak pathology in the hippocampus (p = 0.032 and 0.047, respectively). S1P/sphingosine ratio was 2.5-fold higher in hippocampus of ApoE2 carriers compared to ApoE4 carriers, and multivariate regression showed a significant association between APOE genotype and hippocampal S1P/sphingosine (p = 0.0495), suggesting a new link between APOE genotype and pre-disposition to AD.

Conclusions: This study demonstrates loss of S1P and sphingosine kinase activity early in AD pathogenesis, and prior to AD diagnosis. Our findings establish a rationale for further exploring S1P receptor pharmacology in the context of AD therapy.

Figures

Figure 1
Figure 1
S1P levels decline during AD pathogenesis. (A – F) S1P levels, expressed as a ratio to total sphingosine, in human hippocampus (A), inferior temporal GM (B), inferior temporal WM (C), superior frontal GM (D), superior frontal WM (E), and cerebellum (F) tissue samples. Samples were divided into four groups based on Braak NFT pathology, as detailed in Results text. Horizontal bars indicate the mean. Statistical significance was determined by a one-way ANOVA, followed by Dunnett’s post test, as described in Methods.
Figure 2
Figure 2
Ceramide levels in hippocampus and temporal GM remain relatively constant. Levels of the four most abundant ceramide species in (A) hippocampus and (B) temporal GM were determined by LC-MS/MS. Ceramide content is expressed relative to tissue mass. Mean and standard deviation for each of the Braak stage groups are shown. Statistical significance was determined by one-way ANOVA, followed by Dunnett’s post test to compare different Braak groupings to the control group.
Figure 3
Figure 3
Reduced SphK1 activity in the hippocampus of Braak III/IV subjects. SphK1 activity in hippocampus (A) and temporal GM (B) tissue extracts of control (n = 9), Braak stage I/II (n = 8), Braak III/IV (n = 7), and Braak V/VI (n = 10) brains. Results shown are the mean and standard error for combined results derived from two (B) or three (A) independent assays. (C) SphK1 protein levels in the hippocampus were determined by western blotting and normalised to housekeeping gene β-actin. (D) Example western blot showing SphK1 protein in hippocampus tissue samples. Braak stage is indicated for each sample. Statistical significance was determined using a one-way ANOVA and Dunnett’s post test.
Figure 4
Figure 4
SphK2 activity declines during AD pathogenesis. SphK2 activity in hippocampus (A) and temporal GM (B) tissue extracts of control (n = 9), Braak stage I/II (n = 8), Braak III/IV (n = 7), and Braak V/VI (n = 10) brains. SphK2 activity was assayed as described in methods. Results shown are mean and standard error for combined results from two independent enzyme activity assays. Statistical significance was determined using a one-way ANOVA and Dunnett’s post test.
Figure 5
Figure 5
S1P phosphatase activity increases in temporal GM of AD brains. Total S1P phosphatase activity in hippocampus (A) and temporal GM (B) extracts of control (n = 9), Braak stage I/II (n = 8), Braak III/IV (n = 7), and Braak V/VI (n = 10) brains. Overall association between S1P phosphatase activity activity and Braak stage was significant (P < 0.0001 by one-way ANOVA) for temporal GM but not hippocampus (P = 0.063). Sgpp1 (C) and Sgpp2 (D) protein levels in the temporal GM tissue extracts were determined by western blotting and normalised to housekeeping gene β-actin. (E) Example western blots for Sgpp1 and Sgpp2 protein. Braak stage is indicated for each sample.
Figure 6
Figure 6
Aβ overproduction in mice does not cause a reduction in normalised S1P. Soluble and insoluble Aβ40 and Aβ42 (A), S1P (B), and sphingosine (C), were quantified in cortical tissue from APPswe/PS1ΔE9 (n = 4) or control C57BL/6 (n = 4) mice. S1P/sphingosine ratio is shown in (D). Mean and standard deviation are shown. Statistical significance of differences between the two sample groups was tested with t-tests, adjusted for multiple comparisons: *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 7
Figure 7
Hippocampal S1P level is related to APOE genotype. (A) S1P/sphingosine ratio in the hippocampus of subjects grouped according to APOE genotype: ϵ2/ϵ3 (n = 5), ϵ3/ϵ3 (n = 16), ϵ3/ϵ4 (n = 11). (B) S1P/sphingosine data after removing subjects with Braak III-VI pathology: ϵ2/ϵ3 (n = 5), ϵ3/ϵ3 (n = 7), ϵ3/ϵ4 (n = 5). Horizontal bars indicate the mean.

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