- Open Access
Neuronal sphingosine kinase 2 subcellular localization is altered in Alzheimer’s disease brain
- Gaëlle Dominguez1,
- Marie-Lise Maddelein2,
- Mélanie Pucelle3,
- Yvan Nicaise4,
- Claude-Alain Maurage5, 6,
- Charles Duyckaerts7,
- Olivier Cuvillier†2 and
- Marie-Bernadette Delisle†1, 3, 4Email authorView ORCID ID profile
© The Author(s). 2018
Received: 30 January 2018
Accepted: 19 March 2018
Published: 3 April 2018
Alzheimer’s disease (AD) is characterized by the accumulation of β-amyloid (Aβ) peptides and hyperphosphorylated tau protein accompanied by neuronal loss. Aβ accumulation has been associated with an impaired sphingosine 1-phosphate (S1P) metabolism. S1P is generated by sphingosine kinases (SphKs), of which there are two isoenzymes SphK1 and SphK2, and degraded by the sphingosine 1-phosphate lyase (SPL). We previously reported, that both a decrease in SphK1 expression and an increase in SPL expression, correlated with amyloid deposits in the entorhinal cortex of AD brains, suggesting a global loss of pro-survival S1P in AD neurons. SphK2 contribution has also been examined in AD yielding to conflicting results that may reflect the complexity of SphK2 regulation. The subcellular localization of SphK2, hence the compartmentalization of generated S1P, is recognized to play a crucial role in dictating either its pro-survival or pro-apoptotic functions. We therefore aimed at studying the expression of SphK2 and notably its subcellular localization in brain tissues from patients with AD.
We report that a decrease in SphK2 protein cytosolic expression correlated with the density of amyloid deposits in a cohort of 25 post-mortem brains. Interestingly, we observed that the equilibrium between cytoplasmic and nuclear SphK2 is disrupted and showed that SphK2 is preferentially localized in the nucleus in AD brain extracts as compared to control extracts, with a marked increase of cleaved SphK2.
Our results suggest that a shift in the subcellular localization of the S1P generating SphK2 may compromise the well established pro-survival cytosolic S1P by favoring the production of nuclear S1P associated with adverse effects in AD pathogenesis.
Alzheimer’s disease (AD) currently affects over 46 million people in the world and this number is estimated to reach 73 million by 2030 . Gaining a better understanding of the disease is therefore critical to develop efficient treatments. AD is characterized by two paramount lesions: i) intracellular accumulation of hyperphosphorylated Tau protein forming neurofibrillary tangles (NFT) and neuropil threads; and ii) extracellular deposits of β-amyloid (Aβ) peptide, leading to both diffuse and neuritic plaques. These lesions are associated with neuronal loss, inflammatory process and synaptic dysfunction [16, 17]. The regions affected by both lesions are hierarchically involved. Initially confined to specific locations, frontal and entorhinal cortex, hippocampus, the lesions progressively involve most areas of the brain defining stages in the disease [6, 50].
Sphingolipids are ubiquitous lipid components of membranes that are metabolized to form signaling molecules associated with cellular activities important for health and disease. Evidences are accumulating to argue on their key role in neurodegeneration [9, 28]. One of the most important of these metabolites is sphingosine 1-phosphate (S1P), which is involved in the onset or the progression of pathological conditions including cancer, autoimmunity, cardiovascular conditions or diabetes . The S1P content in cells is low and is kept under control through a delicately regulated balance between its synthesis and its degradation. The balance between the intracellular levels of S1P and its metabolic precursors, ceramide and sphingosine, has been suggested to be a switch determining whether a cell proliferates or dies . S1P is generated by sphingosine kinases (SphKs), of which there are two isoenzymes (SphK1 and SphK2), and is degraded by the S1P lyase (SPL) to hexadecenal and ethanolamine phosphate . Once produced, S1P can work as an intracellular signaling molecule or be secreted to act as an autocrine or paracrine molecule by binding to five specific high-affinity G protein-coupled receptors (GPCR), named S1P 1–5 [13, 45, 46]. Subcellular localization of SphK1 and SphK2 isoenzymes and subsequent compartimentalization of generated S1P appear to be crucial in dictating the biological effect of S1P . It is well established that in order for SphK1 to mediate pro-proliferative and pro-survival signaling, it must relocalize from the cytoplasm to the plasma membrane. In contrast, the subcellular localization of SphK2 appears to be much more complex, like its biological effects . For instance, when present in the nuclei, SphK2 has been associated with DNA synthesis inhibition  or HDAC regulation . In the mitochondria, SphK2 cooperates with Bak and Bax to promote apoptosis . In response to serum deprivation or cell density, SphK2 can also localize in the endoplasmic reticulum (ER) to produce S1P that can fuel a sphingolipid ‘salvage’ pathway, resulting in generated pro-apoptotic ceramide . Two isoforms of SphK2 have been described [33, 42]. The best characterized short isoform (SphK2a or SphK2-S) is the one to which literature refers unless otherwise specified. The large isoform (SphK2b or SphK2-L) is not expressed in mice, and appears the predominant form in several human cell lines and tissues. SphK2b may be the more important in human physiologically .
In AD, a deregulation of the sphingolipid metabolism was originally shown in brain samples from AD patients and age-matched normal individuals, with reduced levels of S1P, together with elevated ceramide . Cellular studies showed an alteration of the ceramide/S1P balance where Aβ treatment of neuronal  or glial  cells was associated with apoptosis caused by enhanced ceramide production or decreased protective S1P production. We further showed that a decrease of SphK1, S1P1 receptor expression together with an increased SPL expression in neurons were correlated to Aβ deposits in entorhinal cortex from human AD brains . In line with these results, Couttas and coworkers showed that loss of activity of SphK1 and SphK2 was correlated with progression of AD lesions (Braak stages) . The role of SphK2 particularly in AD remains controversial. While some authors showed a decrease of activity of SphK2 in temporal cortex and hippocampus , others reported an increase of SphK2 activity in the frontal cortex  of human AD brains.
Therefore, the involvement of SphK2 in AD processes needs further explorations. It could depend on deregulation of its expression or subcellular localization. To answer the question about SphK2 implication in the deleterious effects of Aβ in AD, we used brain tissues from patients with AD. We herein report for the first time that SphK2 expression is inversely associated with the density of amyloid deposits in frontal and hippocampal area of brains from AD patients and demonstrated a shift of SphK2 from the cytosol to the nucleus in AD neurons, accompanied by a marked increase of cleaved SphK2 in the AD brain.
Materials and methods
Validation of anti-SphK2 antibody
E. coli BL21 strains were transformed by one of the pJ414 constructs - PET21 vectors expressing either SphK1 (403AA) or SphK2 (674AA) with an HIS tag. Forty ml of an overnight pre-culture of transformants grown at 37 °C in DYT medium supplemented with kanamycin (100 μg/ml) was used to inoculate 200 ml of DYT-KAN media. After 2 h of growth at 37 °C, SphK proteins expression was induced by 5 mM of Isopropyl β-d-1-thiogalactopyranoside (IPTG). Aliquots of the cultures were made 5 h after induction. Protein expression was confirmed by western blot analysis with mouse anti-histidine (Cusabio, Ref. CSB-MA000011M0m). The specificity of the anti-SphK2CT rabbit polyclonal antibody (Sigma, Ref. SAB4502433) was confirmed by western blot.
Human brain tissues
Paraffin embedded human brain tissues were provided by certified French biological resource centers from Lille (Neurobank Lille DC-2008-642) and Toulouse (Brain bank AC-2009-973) for immunohistochemistry and immunofluorescence studies. For western blot studies, human brain frozen tissues were provided by the national brain bank GIE Neuro-CEB (AC-2007-5) and the biological resource center of CHU Toulouse (Brain bank AC-2009-973). The utilization of postmortem material was approved by the corresponding biobank ethic committees. All cases were scored according to current criteria of NIA-Alzheimer’s Association . The assessment included Braak and Thal neuropathology stages [5, 50].
Characteristics of patients included in the immunohistochemistry study
AD (n = 25)
Age at death (years)
Female gender, n (%)
Post mosterm interval
“A” Thal - n of patients (%)
3 (4 and 5)
1 (1 and 2)
“B” Braak Stage - n of patients (%)
3 (V and VI)
2 (III and IV)
1 (I and II)
Preparation of human brain total homogenates
Characteristics of patients included in the western blot study
Controls (n = 6)
AD (n = 10)
Age at death (years)
Female gender, n (%)
Post mosterm interval
“A” Thal - n of patients (%)
3 (4 and 5)
1 (1 and 2)
“B” Braak Stage - n of patients (%)
3 (V and VI)
2 (III and IV)
1 (I and II)
Preparation of nuclei-enriched fractions and cytoplasm-enriched fraction
The same cases except one control (AD = 10, Control = 5) were included in this experiment. Frozen tissue samples were lysed as aforementioned in HEPES buffer (pH 7.4) (20 mM HEPES, 10 mM KCl, 2 mM MgCl2, 1 mM EDTA, 0.25 M saccharose, and EDTA-free protease inhibitor cocktail). Nuclei-enriched fractions and cytoplasmic-enriched fractions were prepared by sequential centrifugation. Nuclei were pelleted by centrifugation (500 g for 10 min at 4 °C) then sonicated at 4 °C and centrifuged at 9000 g for 30 min, in Tris buffer (pH 7.4) (50 mM Tris-HCl, 250 mM NaCl, 1 mM EDTA, 0.1% Triton and EDTA-free protease inhibitor cocktail). Supernatants were removed, and the membrane and cytoplasmic fractions were separated by centrifugation at 16,000 g (30 min at 4 °C). Isolation of nuclear and cytosolic fractions was confirmed by western blot with primary antibodies directed against rabbit polyclonal PARP (GeneTex, Ref. GTX100573, 1:1000) and rabbit polyclonal LDH (GeneTex, Ref. GTX114525, 1:10,000).
Total protein concentration was assessed on the supernatant with the BCA Protein Assay (Interchim). Samples were heated at 95 °C for 5 min with 5% β-mercapto-ethanol, 0.05% bromophenol blue. 150 μg of total proteins were loaded by lane in precast gels with Stain-Free technology (4–15% Mini-PROTEAN® TGX Stain-Free™, Ref. 456–8083). The proteins were separated by electrophoresis at 90 V in a MiniProtean Tetra System (Bio-Rad Laboratories, Irvine, CA). After migration and semi-dry transfer (Transblot Turbo, Bio-Rad), nitrocellulose membranes were blocked with 5% skimmed milk, and washed 3 times with Tris-buffered saline buffer containing 0.05% Tween-20 (TBST). Blots were probed with a primary antibody directed against rabbit polyclonal SphK2 (Sigma, Ref. SAB4502433, 1:500) and β-amyloid (Sigma, Ref. A8354, 1:1000). After an overnight incubation at 4 °C, the membranes were washed with TBST, labeled with a peroxidase-conjugated anti-rabbit (dilution 1:3000) secondary antibody (Bio-Rad) and revealed by chemiluminescence (Clarity kit, Bio-Rad). Bands were quantified by densitometry with ImageLab (Bio-Rad) software. The density of total protein, detected by UV light, was used to normalize the signals [20, 23].
Characteristics of patients included in the immunofluorescence study
Controls (n = 6)
AD (n = 9)
Age at death (years)
Female gender, n (%)
Post mosterm interval
“A” Thal - n of patients (%)
3 (4 and 5)
1 (1 and 2)
“B” Braak Stage - n of patients (%)
3 (V and VI)
2 (III and IV)
1 (I and II)
Statistical analysis was carried out with a multilevel linear mixed model to take into account non independent data. For cortical area, due to the poor representativeness of fields 1 (non tissular zone and pial surface) and 10 (proximal white matter), these fields were not included in statistical analysis. To avoid experimental bias related to the number of neurons per field, the immunohistochemistry data were expressed as percentage of variation relative to the total number neurons. For correlation analyses, the Spearman’s correlation coefficient, R, was determined. Correlations were estimated as significant at p < 0.05. Comparisons between groups were analyzed using one-way analyses of variance (ANOVAs). Statistical analyses were performed using the Statview 5.0 software.
SphK2 antibody is specific for SphK2 and does not cross-react with the SphK1 isoenzyme
Negative correlation between amyloid deposits and the percentage of SphK2 positive neurons in AD brain
The percentage of SphK2 positive neurons (cytoplasm) was inversely correlated with the amount of amyloid deposits (Aβ surface percentage) in the frontal cortex (r = 0.037, p = 0.007; Fig. 2b1), in the entorhinal cortex (r = 0.062, p < 0.001; Fig. 2b2) and in CA1 (r = 0.29, p < 0.001; Fig. 2b3). This negative correlation was also found for the focal plaques (frontal cortex: r = 0.044, p = 0.003; entorhinal cortex: r = 0.064, p < 0.001; and CA1: r = 0.38, p < 0.001; Fig. 2c). From the coefficient values, we noted that the relationship between the amount of amyloid and the percentage of SphK2 positive neurons was stronger in the CA1 area than in the cortical regions (frontal and entorhinal).
Frontal cortex and hippocampal AD brain extracts show high amyloid content
In addition to 150–250 kDa bands corresponding to higher MW Aβ oligomers, bands of 27 kDa were detected. This molecular masses apparently correspond to multiples of trimeric Aβ and could thus represent hexameric (Aβ*27) Aβ assemblies. High levels of total Aβ (high MW oligomers and Aβ*27) were observed in AD group in comparison with the control group (F(1,38) = 10.7; p = 0.002). When each structure was considered separately (Fig. 3b), statistical analysis revealed a significant increase of total Aβ in frontal cortex (p = 0.009) and an increase at the margin of statistical significance in the hippocampal area (p = 0.06). No difference was found in the cerebellum (p > 0.5).
With regard to the Aβ*27 oligomers, there was a significant difference between groups (F(1,38) = 7.9; p = 0.008), with a significant increase in hippocampal area (p = 0.028; Fig. 3c) and an increase in the frontal to the margin of the significance (p = 0.08). No difference was found in cerebellum (p = 0.99).
Higher MW Aβ oligomers (F(1,38) = 8.6; p = 0.006) content were markedly increased in frontal cortex (p = 0.02) but not significantly in hippocampal area (p = 0.095) and in the cerebellum (p = 0.4), in AD group (data not shown).
These data are in accordance with the evolution of Aβ deposits and the distinct sequences in which the regions of the brain are hierarchically involved according to the Thal stages (Phase 1: neocortical Aβ deposits, Phase 2: hippocampal area deposits, Phase 5: cerebellum is the latest structure affected by Aβ deposits) .
Specific increased expression of a cleaved SphK2 form in AD brains
The SphK2 immunoblots showed two major bands in brain lysates, a 75 kDa band corresponding to the large isoform of SphK2 (SphK2b) and a protein of lower molecular weight which was considered as a cleaved fragment of SphK2 (Fig. 3a, low row). A cleaved fragment of SphK2 has been previously described in extraneural cell models (Jurkat T cells, murine fibroblasts, spleen cells) in relation to apoptotic process .
The quantification of total SphK2 (full length and cleaved forms) revealed an increase of its expression in AD samples, in frontal cortex (F(1,14) = 5.4; p = 0.04) but not in the hippocampal area and cerebellum (p > 0.5 for both comparisons). However, full length SphK2 was not different between AD and control groups (data not shown) (p > 0.05). In contrast, the cleaved SphK2 fragments (Fig. 3d) was higher in AD extracts as compared to control (F(1,38) = 7.6; p = 0.009). More specifically, an increase was observed in the frontal cortex (F(1,14) = 5.8; p = 0.03) and in the hippocampal area, to the margin of the significance (F(1,14) = 4.1; p = 0.06). No difference was found in cerebellum (p > 0.08).
Correlation analyses were made to confirm relationships between individual expression of a cleaved SphK2 form and Aβ levels (Fig. 3e). The individual expression of cleaved SphK2 form correlated positively with Aβ*27 levels in frontal cortex (r = 0.35, p = 0.016; Fig. 3e1) and in hippocampal area (r = 0.48, p = 0.003; Fig. 3e2), but not in cerebellum (p > 0.5; Fig. 3e3).
A positive correlation was also noted between higher MW Aβ oligomers and cleaved SphK2 in frontal cortex (r = 0.47, p = 0.003) and in hippocampal area (r = 0.73, p = 0.001) (data not shown). In the cerebellum, higher MW Aβ oligomers were expressed at a low level in one AD case (Fig. 3a3). Cleaved SphK2 was also found in this case.
The subcellular localization of SphK2 is altered in AD brains
The quantification of total SphK2 (full length and cleaved forms) revealed a differential expression between the two subcellular fractions in control group (F(1,18) = 12.9; p = 0.002). SphK2 was less expressed in the nuclei-enriched fraction (1.22E8 ± 0.23E8 densitometry units) than in the cytoplasmic-enriched fraction (2.47E8 ± 0.25E8 densitometry units). This difference was found in the frontal cortex (p = 0.04) and in the hippocampal area (p = 0.01). In AD group (F(1,38) = 0.35; p = 0.5), no difference was found between the nuclear (2.75E8 ± 0.63E8 densitometry units) and the cytoplasmic (3.22E8 ± 0.48E8 densitometry units) (data not shown).
For each subcellular fraction, we analyzed separately full length and cleaved SphK2 fragments in AD group as compared to control. The results are expressed as percentage variation in AD versus control group (Fig. 4b and c).
Full length SphK2 expression was increased in the nuclei enriched fraction (F(1,28) = 3.9; p = 0.050) but was decreased in cytoplasmic enriched fraction (F(1,28) = 5.4; p = 0.027) in AD group relative to the control (Fig. 4b). Post-hoc analyses showed a reduction of cytoplasmic full length SphK2 mainly in the hippocampal area (p < 0.001).
A rise of cleaved SphK2 (Fig. 4c) was observed in nuclear fractions (F(1,28) = 10.4; p = 0.003) and in cytoplasmic fractions (F(1,28) = 3.9; p = 0.050) in AD group relative to control group especially in frontal cortex (nuclear p = 0.0015 et cytoplasmic p = 0.08; Fig. 4c1). To a lesser extent, yet not significantly, this change of expression was seen in hippocampal area (nuclear p = 0.17 and cytoplasmic p = 0.13; Fig. 4c2).
These data suggest a shift of full length SphK2 from cytosol to the nucleus examined in all brain structures, accompanied by an accumulation of the cleaved SphK2 in the nucleus that would be more significant in the frontal cortex, in AD.
Immunofluorescence study shows that SphK2 is preferentially localized in the nucleus
The density of neurons and of amyloid deposits were inversely correlated in the frontal (r = 0.14, p = 0.0004) and entorhinal cortex (r = 0.15, p = 0.002), but not in the CA1 (r = 0.02, p = 0.25). This negative correlation was only related to the presence of focal deposits in all structure (frontal cortex: r = 0.13, p = 0.0006, entorhinal cortex: r = 0.13, p = 0.0045, CA1: r = 0.11, p = 0.005) (data not shown).
In agreement with data shown in Fig. 2, immunofluoresence microscopy revealed that the percentage of SphK2 stained surface in neuron (nuclear and cytoplasmic compartments) was decreased (F(1,837) = 49.0; p < 0.0001; Fig. 5b) in the AD group as compared to control for all structures (frontal cortex: p = 0.033; entorhinal cortex and CA1: p < 0.0001). The nuclear/cytoplasmic ratio of SphK2 stained surface was increased in AD group (F(1,837) = 113.5; p < 0.0001). This change of expression was seen in the frontal cortex, the entorhinal cortex and the CA1 (p < 0.001 for all comparisons) as compared to the control subjects, demonstrating a disturbance of the SphK2 subcellular localization (Fig. 5c). This observation was reinforced by the increase of SphK2 nuclear/cytoplasmic ratio of staining intensity in AD group, for the three structures (p ≤ 0.001 for the three structures) (Fig. 5d).
A deregulation of sphingolipid metabolism and more particularly S1P signaling has been recently stressed in AD (reviewed in [7, 9, 28, 38, 52]). Initial work in neuronal cell models showed that apoptosis induced by Aβ peptide was dependent on increased production of pro-apoptotic ceramide  or decreased activity of SphK1 leading to decreased content of pro-survival S1P . This deregulation of the ceramide/S1P balance with increase in ceramide and decrease in S1P was seen in patients to correlate with the level of Aβ peptide and phosphorylated tau . More recently, two independent studies established an alteration of S1P metabolism with a decrease in SphK1 or SphK2 expression and enzymatic activity and increase in S1P lyase, the enzyme that degrades S1P [8, 12]. Again, enzymatic deregulation leading to decreased S1P correlated with Aβ deposits and progression of AD lesions [8, 12].
Besides the likely role for SphK1 isoform, SphK2 deregulation may contribute more significantly to AD lesions and progression. Indeed, SphK2 was found as the predominant isoenzyme in the rodent brain [3, 31, 33], and SphK2 activity and mRNA levels are considerably higher than that of SphK1 [3, 12]. Using mice with germline knockout of SphK2, it was recently shown that SphK2 is responsible to the largest part for S1P synthesis in the brain . As aforementioned, the role of SphK2 has been examined in AD patients yet the results remain so far controversial. If a decrease in S1P level was linked to the reduction of SphK2 activity in the hippocampus and the temporal cortex , an increase of SphK2 activity has been reported in the frontal cortex . These ambiguous results may simply reflect the complexity of SphK2 regulation and function. Depending on its subcellular compartment, SphK2 can generate S1P that will favor cell death or proliferation and survival .
Until now, the lack of commercial SphK2 antibodies validated for western blotting and immunohistochemistry has remained a drawback for a better understanding of the specific functions of SphK2. In this study, we used a SphK2 antibody raised against the C-terminal sequence of SphK2, the specificity of which for the SphK2 isoenzyme was confirmed using human SphK1 and SphK2 recombinant proteins.
We herein report that a decrease in cytoplasmic SphK2 protein expression correlates with the density of amyloid deposits in AD brains. Interestingly, we observed that the equilibrium between cytoplasmic and nuclear SphK2 was disrupted and showed that SphK2 is preferentially localized in the nucleus in AD brain extracts as compared to control extracts, with a marked increase of cleaved SphK2.
More precisely, we showed that cytoplasmic expression of SphK2 in neurons was inversely correlated to Aβ deposits, whatever their type (diffuse or focal) in the CA1 and to a lesser extent in the frontal and entorhinal cortex. This is in keeping with our previous results on SphK1 cytoplasmic expression, which was also negatively correlated to the density of Aβ deposit , suggesting the involvement of both isoenzymes in the deregulation of this signaling pathway. Western blot analyses corroborated the absence of variation of SphK2 expression in whole brain lysates of AD patients when compared to control brains as previously reported [8, 49]. Cieslik et al., using PC12 cells, similarly showed that exposure to Aβ1–42 had no effect on SphK2 . These results showing an absence of variation of total SphK2 at both cellular and tissular levels suggest there could be a change in subcellular localization of SphK2 without any alteration of its total expression. As a matter of fact, both nuclei-enriched and the cytoplasmic-enriched fractions showed an alteration of SphK2 expression with a loss of expression in cytoplasm accompanied with an increased expression in nucleus in hippocampal area and to a lesser extent in frontal cortex. Immunofluorescence studies confirmed that nuclear and cytoplasmic expressions of SphK2 were modified in favor of a cytoplasmic decrease and a nuclear increase expression in AD brains.
Taking advantage of an antibody directed against the C-terminal portion of SphK2, the presence of a low molecular weight protein (~ 50 kDa) could be detected on immunoblots. This form matches with the truncated fragment of SphK2 cleaved by caspase-1 at the level of the D138 according to Weigert et al., and online software ExPASy (PeptideCutter) . In this truncated fragment, all the domains of SphK2 are preserved including the catalytic domain and the ligand binding domain, suggesting that cleaved SphK2 could preserve its enzymatic activity. Its distribution in both compartments is in agreement with theoretical predictions based on online software WoLF PSORT 20, which establishes that cleaved SphK2 could be localized in both cytoplasmic and nuclear compartments. Caspase-1-mediated cleavage of SphK2, by removing the nuclear localization signal (NLS), would prevent the entry in the nucleus of a cytosolic cleaved SphK2. This is in favor of a cleavage of SphK2 in the nucleus. Actually, it has been reported that inactivated caspase-1 can be translocated to the nucleus and processed into an active form in parallel with apoptosis . Our data show that an increase of cleaved SphK2 in AD group correlated positively with the amount of amyloid oligomers, particularly in hippocampus and in entorhinal cortex. Yet, the significance and function of such cleaved SphK2 requires further interrogation.
Similar to SphK1, SphK2 has a pro-survival effect when localized in the cytoplasm from where it can be targeted to the plasma membrane to produce S1P, which can be then secreted to exert autocrine and paracrine effect through its specific receptors . Thus, our results suggest that, during AD, there is a decrease in the pro-survival cytoplasmic SphK2 similar to previous findings showing a decrease in pro-survival cytoplasmic SphK1 [8, 12]. We also previously reported that S1P receptor subtype 1 was decreased in AD  and recent data suggest that pharmacological agonists of S1P1 (FTY720, SEW2871) can improve cognitive function in AD rat model [1, 2]. Collectively, these data indicate that cytosolic SphK1/SphK2 signaling and potential subsequent S1P receptor signaling could be linked to a pro-survival role of S1P in AD pathogenesis, and that strategies aimed at rescuing impaired cytosolic SphK1/SphK2 signaling could be beneficial for patients.
The nuclear SphK2 signaling appears much more complex. It could be deleterious in AD pathogenesis. Interestingly, in cancer models, retinoic acid signaling was found to be deregulated by nuclear SphK2/S1P signaling, with S1P acting as an antagonist for the retinoic acid receptor (RAR) beta [47, 48]. Retinoic acid represents an antioxidant that can affect cognition [4, 43]. More specifically, retinoic acid can modulate the hippocampus-dependent memory influencing the neuronal plasticity (changes in synaptic strength, numbers of synapses and neurons) . The disruption of retinoid signaling has been implicated in the pathogenesis of AD . The expressions of genes that are engaged in the production of Aβ including β-secretase enzyme (BACE), presenilin 1 or presenilin 2 are regulated by retinoids [29, 41]. Finally, the protective role of retinoic acid in the pathogenesis of AD has been demonstrated in transgenic APP/PS1 mouse model. Indeed, its prolonged administration produced a reduction of amyloid deposits and lead to an improvement of memory . Thus, the disruption of acid retinoic signaling by the nuclear SphK2/S1P signaling may have an implication of neurodegenerative processes in AD and in the memory disorders. On the other hand, nuclear SphK2 may have a beneficial effect via its role in epigenetic regulation, on synaptic plasticity and memory processes. Indeed, the nuclear S1P produced by SphK2 inhibits histone deacetylases HDAC1/2 and regulated histone acetylation . FTY720, a synthetic analog of sphingosine which is phosphorylated by nuclear SphK2, induced an improvement of memory process [19, 25] and a better survival of neuronal progenitors in hippocampus . It could be hypothesized that the nuclear-localized SphK2 is involved in a compensatory attempt process to restore neuronal function.
The cellular functions of SphK2 are complex and the mechanism(s) that may drive the differences in its subcellular localization remain(s) largely unanswered question and will require further investigation. Similarly, future work is needed to understand the significance of the presence of cleaved SphK2 in the neuron nuclei. Nevertheless, from our findings it is reasonable to suggest that a change in the subcellular localization of the S1P generating SphK2 may compromise the well-established pro-survival cytosolic S1P by favoring the production of nuclear S1P associated with deleterious effects in AD pathogenesis.
This research was supported by a grant from the association France Alzheimer (grant no. SM2015#345). G. Dominguez was supported by this grant. The authors would like to thank Dr. Johnatan Ceccom, Dr. Christian Touriol, Pr Emmanuelle Uro-Coste and Pr Jean-Michel Senard as well as his team for their advices and assistance and Lorenne Robert, Najat Loukh, Celia Martron and Serge Estaque for technical assistance. They are grateful to Dr. Pascal Demange for generous gift of reagents: PERT21 vectors expressing either SphK1 or SphK2. The authors would like to thank Sabrina Leclere-Turbant and Marie-Claire Artaud-Botté from the GiE Neuro-CEB as well as Lille Neurobank. They are grateful to Maryline Calise for administrative help. They acknowledge the Cellular Imaging Facility Rangueil-I2MC/TRI Plateform (confocal microscopy) and the CMEAB-Rangueil Medicine Faculty/TRI Plateform (virtual slides).
All sources of funding for the research reported should be declared. The role of the funding body in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript should be declared.
GD and YN carried out double labeling immunoassays and immunoblot experiments and participated in drafting the manuscript. GD and MBD performed statistical analysis and data interpretation. MP and GD carried out immunofluorescence experiment and analyzed data. MLM carried out immunoblot experiment, analyzed data and participated in the draft of the manuscript. CAM and CD provided paraffin embedded material and associated data for staging, advices in the conception of used methodology, participated in drafting and reviewing the manuscript. GD, MLM, OC and MBD conceptualized the design of the study, analyzed and interpreted data, and wrote the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The study was approved by the corresponding Brain Banks' committees (Cérébrothèque/CRB CHU Toulouse N°AC-2009-973, Brain Bank NeuroCEB Paris N°AC-2007-5 and Neurobank Lille N° DC-2008-642). In all cases, in accordance with the French Bioethical Laws, the consents were signed by the patients themselves or their next of kin in their name to permit an autopsy and neuropathological investigation. The national computerized registry of objection to autopsy was systematically consulted. The above cited Brain Banks have been declared at the Ministry of Higher Education and Research and have received approval to distribute samples.
Consent for publication
Written informed consent was obtained from the patient for the publication of this report and any accompanying images as required in the concerned authorized biological resource centers.
The authors declare that they have no competing interests.
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