3.2. Specific Identification of Various Lipid Isoforms after Aβ(1–40) and Aβ(1–42) Treatment from Hippocampal Neurones and Glial Cells Using Tandem Mass Spectrometry Analysis
To corroborate whether Aβ(1–40) and Aβ(1–42) preferentially influence specific sphingolipid or glycerophospholipids isoforms, researchers treated hippocampal neurones at DIV 12 and glial cells for 3 h and 12 h with Aβ(1–40) and Aβ(1–42) and subsequently analysed them by tandem MS. Here, the characterisation of altered lipid isoforms for hippocampal neurones and glial cells after 3 h and 12 h Aβ(1–40) and Aβ(1–42) treatment are shown, focussing on sphingolipids such as ceramides (Cer), dihydroceramides (DHCer), lactosylceramides (LacCer), monohexosylceramides (HexCer), sphingosine (So), sphinganine (Sa), sphingosine-1-phosphate (Sa1P), sphinganine-1-phosphate (So1P), sphingosylphosphorylcholine (SPC), sphingomylines (SM), dihydrosphingomylines (DHSM), and glycerophospholipids such as phosphatidylcholine (PC), lyso-phosphatidylcholine (LPC), lyso-phosphatidylethanolamine (LPE), lyso-phosphatidylglycerol (LPG), lyso-phosphatidylserine (LPS), Lyso-platelet-activating factor (Lyso-PAF) (Figure 2, Figure 3 and Figure 4). All results are presented as a log10 transformation and were performed in triplicate. Furthermore, ratios between the Aβ-treated groups and their negative control (DMSO) were calculated, setting the negative control (DMSO) to 0. After taking the ratios, the Z-score was taken for the total data set. Changes in lipid isoforms are shown as tendencies. This means that the red colours refer to an increase of lipid quantities and the green colour indicates a reduction compared to their negative control (DMSO) which was set to 0 as baseline parameter (Figure 2, Figure 3 and Figure 4).
Figure 2. Heat-map-based mass spectrometry analysis of sphingolipid classes from hippocampal neurones and glial cells after 3 h and 12 h Aβ treatment. Here, researchers examined different sphingolipid isoforms: ceramides (Cer), dihydroceramides (DHCer), lactosylceramides (LacCer), and monohexosylceramides (HexCer) after 3 h and 12 h treatment with (1 µM) Aβ(1–40) and (1 µM) Aβ(1–42) of hippocampal neurons at DIV 12 and glial cells (all groups, from three independent experiments (n = 3)). Changes of these lipid classes are shown as logarithmic (log10) relative intensity (arbitrary unit); the green colour refers to a reduction, and the red colour refers to an increase of lipid levels compared to their negative control (DMSO), which was set to 0 as baseline. Both hippocampal neurones and glial cells showed an increased and reduced intensity of Cer, DHCer, LacCer, and HexCer isoforms after both 3 h and 12 h Aβ(1–40) and Aβ(1–42) treatment. Different changes in the lipid classes are shown between treated hippocampal neurones and glial cells. Aβ-treated hippocampal neurones showed prominent increases and decreases in Cer isoforms and LacCer and HexCer isoforms after 3 h and 12 h of Aβ(1–40) and Aβ(1–42) treatment. There was both a prominent intensity increase and a decrease of DHCer isoforms after 3 h and 12 h of Aβ(1–40) and Aβ(1–42) treatment. Glial cells showed prominent lipid changes of Cer, DHCer, LacCer, and HexCer isoforms after 3 h and 12 h of Aβ(1–40) or Aβ(1–42) treatment.
Figure 3. Heat-map-based mass spectrometry analysis of sphingophospholipid classes from hippocampal neurones and glial cells after 3 h and 12 h Aβ treatment. Here, researchers examined different sphingophospholipid isoforms; sphingosine (d18:1 So), sphinganine (d18:0 Sa), sphingosine-1-phosphate (d18:1 So1P), sphinganine-1-phosphate (d18:1 Sa1P), sphingomyelines (SM) and dihydrosphingomyelines (DHSM) after 3 h and 12 h treatment with (1 µM) Aβ(1–40) and (1 µM) Aβ(1–42) hippocampal neurones at DIV 12 and glial cells (all groups, from three independent experiments (n = 3)). Changes of these lipid classes are shown as logarithmic (log10) relative intensity (arbitrary unit); the green colour refers to a reduction and the red colour refers to an increase in lipid levels compared to their negative control (DMSO), which was set to 0 as baseline. Major intensity changes in lipid classes of d18:1 So, d18:0 Sa, d18:1 So1P, d18:1 Sa1P after 3 h and 12 h Aβ(1–40) and Aβ(1–42) treatment of hippocampal neurones and glial cells were observed. Specific lipid intensity changes after Aβ(1–40) and Aβ(1–42) treatment of hippocampal neurones of very long fatty acid SM compared to Aβ-treated glial cells were detected.
Figure 4. Heat map-based mass spectrometry analysis of glycerophospholipid classes from hippocampal neurones and glial cells after 3 h and 12 h Aβ treatment. Here, researchers examined different glycerophospholipids isoforms: phosphatidylcholine (PC), lyso-phosphatidylcholine (LPC), lyso-phosphatidylethanolamine (LPE), lyso-phosphatidylglycerol (LPG), lyso-phosphatidylserine (LPS), and lyso-platelet-activating factor (Lyso-PAF) after 3 h and 12 h treatment with (1 µM) Aβ(1–40) and (1 µM) Aβ(1–42) of hippocampal neurones at DIV 12 and glial cells (all groups, from three independent experiments (n = 3)). Changes in these lipid classes are shown as logarithmic (log10) relative intensity (arbitrary unit); the green colour refers to a reduction and the red colour refers to an increase in lipid levels compared to their negative control (DMSO), which was set to 0 as baseline. Hippocampal neurones specifically showed PC (38:0) intensity changes after 3 h and 12 h Aβ(1–40) and Aβ(1–42) treatment compared to an overall reduction in PC for Aβ(1–40) and Aβ(1–42)-treated glial cells. LPC, LPG, LPS, and Lyso-PAF showed specific changes after 3 h and 12 h Aβ(1–40) and Aβ(1–42) treatment in both hippocampal neurones and glial cells.
Ceramides (Cer)
Both hippocampal neurones and glial cells showed alterations in Cer isoforms after 3 h and 12 h Aβ(1–40) and Aβ(1–42) treatment (Figure 2). Neurones showed a reduction in C14 Cer after 3 h Aβ(1–40) treatment. An increase of C14 Cer was observed after 3 h and 12 h of Aβ(1–40) and Aβ(1–42) treatment. Other ceramide isoforms (C16 Cer, C18:0 Cer, C20 Cer, C22 Cer, C24:1 Cer) showed an increase in 3 h Aβ(1–40)-and 12 h Aβ(1–42)-treated neurons. Aβ-treated glial cells showed a strong increase after 3 h Aβ(1–40) and Aβ(1–42) treatment for C14 Cer. After 12 h, Aβ(1–42)-treated glial cells showed a strong reduction compared to Aβ(1–40) for C14 Cer. A weak reduction in C18:1 Cer for 3 h Aβ(1–42) and 12 h Aβ(1–40) (Figure 2) was also observed. Other ceramide isoforms (C16 Cer, C18:0 Cer, C20 Cer, C22 Cer, C24:1 Cer) showed a weak increase after 12 h Aβ(1–42)-treatment on glial cells. An increase was observed in C24 Cer after 3 h Aβ(1–40) as well as after 3 h and 12 h Aβ(1–42) in treated neurones. No changes were observed in C24 Cer of Aβ(1–40)- and Aβ(1–42)-treated glial cells (Figure 2).
Dihydroceramides (DHCer)
In Aβ(1–42)-treated glial cells, C14 DHCer, C16 DHCer and C18:0 DHCer decreased after 3 h and increased after 12 h treatment. However, in glial cells C14 DHCer and C18:0 DHCer was increased after 3 h of Aβ(1–40) treatment. After 3 h Aβ(1–40)- and Aβ(1–42)-treatment on glial cells, a reduction in C20 DHCer and an increase in C22 DHCer was observed. After 12 h there was a decrease in Aβ(1–40)- and an slight increase in Aβ(1–42)-treated glial cells. C24:1 DHCer showed a weak reduction after 3 h Aβ(1–40) and Aβ(1–42) treatment as well as an increase after 12 h Aβ(1–42) treatment in glial cells. After 3 h and 12 h Aβ treatment, glial cells showed a decrease in C24 DHCer (Figure 2). Aβ(1–40)- and Aβ(1–42)-treated hippocampal neurones showed a specific decrease in C20 DHCer after 3 h, and no changes after 12 h Aβ(1–40) and Aβ(1–42) treatment. C22 DHCer showed a decrease in 3 h Aβ(1–40)- and 12 h Aβ(1–42)-treated hippocampal neurones. An increase in C22 DHCer was seen in 3 h Aβ(1–42)- and 12 h Aβ(1–40)-treated hippocampal neurones (Figure 2). Other Dihydroceramides isoforms (C14 DHCer, C16 DHCer, C24:1 DHCer, C24 DHCer) showed a weak increase in 3 h Aβ(1–40)-treated neurons. However, 3 h Aβ(1–42) treatment showed a reduction in C14 DHCer, C16 DHCer, C24:1 DHCer, and increase in C24 DHCer (Figure 2).
Lactosylceramides (LacCer)
Glial cells treated with Aβ(1–40) and Aβ(1–42) showed a reduction in all LacCer isoforms after 12 h. In 3 h Aβ(1–42)-and 12 h Aβ(1–40)-treated glial cells, decreases in C18:0 LacCer and C22 LacCer and an increase after 12 h Aβ(1–42) treatment were seen. In C16 LacCer and C24:1 LacCer an increase after 12 h Aβ(1–40) and Aβ(1–42) treatment on glial cells was seen. A reduction was observed in C16 LacCer after 3 h Aβ(1–40) and a decrease after 3 h Aβ(1–42) treatment on glial cells. After 3 h, Aβ(1–40)-treated hippocampal neurones showed an increase in C18:0 LacCer. A decrease in C16 LacCer and C18 LacCer was seen in 3 h Aβ(1–42)-treated hippocampal neurons (Figure 2). A slight decrease was observed in C18:0 LacCer after 12 h Aβ(1–40) and Aβ(1–42)-treatment on neurones as well as in C22 LacCer after 3 h Aβ(1–42) treatment.
Monohexosylceramides (HexCer)
After Aβ(1–40) treatment, C16 HexCer showed a strong reduction in 12 h-treated hippocampal neurones and in 3 h-treated glial cells, whereas 3 h Aβ(1–42)-treated glial cells and 3 h Aβ(1–40)- and Aβ(1–42)-treated hippocampal neurones showed an increase (Figure 2). C22 HexCer and C24 HexCer showed strong increases after 12 h Aβ(1–40) and decreases in 12 h Aβ(1–42) for glial cells and a slight reduction in 12 h Aβ(1–40)- and a strong increase in 12 h Aβ(1–42)-treated hippocampal neurones. After 3 h of Aβ(1–40)- and Aβ(1–42)-treated hippocampal neurons and glial cells, a strong increase was seen in C22 HexCer. C24 HexCer showed an increase in 3 h Aβ(1–40)- and Aβ(1–42)-treated glial cells as well as in 3 h Aβ(1–42)-treated hippocampal neurons. Slight increases were observed in C14 HexCer, C18:0 HexCer, and C20 HexCer after 3 h Aβ(1–40) treatment on hippocampal neurones. However, C14 HexCer showed an weak increase after 3 h Aβ(1–40) and 12 h Aβ(1–42) treatment on hippocampal neurones. A slight reduction was seen in C14 DHCer after 3 h Aβ(1–42) and 12 h Aβ(1–40) treatment on glial cells. C18:0 HexCer showed an increase after 3 h Aβ(1–40) and 12 h Aβ(1–42) on hippocampal neurones. A slight decrease in C18:0 HexCer was observed after 3 h Aβ(1–42) treatment (Figure 2).
Sphingosine (So) and Sphingosine-1-Phosphate (So1P)
Hippocampal neurones and glial cells showed weak changes of d18:1 So after both 3 h and 12 h Aβ(1–40) and Aβ(1–42) treatments (Figure 3). There was a decrease in d18:1 So1P after 3 h Aβ(1–42) treatment in hippocampal neurones and an increase after 12 h Aβ(1–42) treatment in glial cells. A reduction in d18:1 So1P was also observed after 3 h and 12 h Aβ(1–40) treatment of hippocampal neurons (Figure 3). An increase in d18:1 So1P for both 3 h Aβ(1–40)-treated glial cells and 12 h Aβ(1–42)-treated hippocampal neurones was observed (Figure 3).
Sphinganine (Sa) and Sphinganine-1-Phosphate (Sa1P)
A prominent reduction was seen in d18:0 Sa for glial cells after 3 h and 12 h Aβ(1–40) and Aβ(1–42) treatment (Figure 3). In hippocampal neurones d18:0 Sa was slightly increased after 3 h Aβ(1–40) treatment and slightly reduced after 12 h Aβ(1–42) treatment. A reduction was also observed in d18:0 Sa1 P after 3 h Aβ(1–40)- and Aβ(1–42)-treated hippocampal neurones, as well as 3 h and 12 h Aβ(1–40)-treated glial cells. No strong reduction was observed in d18:0 Sa1P after 12 h Aβ(1–40) and Aβ(1–42) treatment of hippocampal neurones as well as in 3 h Aβ(1–42)-treated glial cells (Figure 3).
Sphingosylphosphorylcholine (SPC)
Both, hippocampal neurones and glial cells showed alterations in SPC 16:0 after 3 h Aβ(1–40) and Aβ(1–42) treatment. The most prominent decrease in SPC 16:0 was shown after 3 h of Aβ(1–40) and Aβ(1–42) treatment for both hippocampal neurones and glial cells. A decrease was also observed after 12 h of Aβ(1–42) treatment for both hippocampal neurones and glial cells (Figure 3).
Sphingomyelines (SM)
All SM isoforms (C14 SM, C16 SM, C18:1 SM, C18:0 SM, C20 SM, C22 SM, C24:1 SM, C24 SM, C26:1 SM, and C26 SM) showed a slight increase after 3 h Aβ(1–40) and Aβ(1–42) treatment on hippocampal neurones, and glial cells after 3 h and 12 h Aβ(1–40) and Aβ(1–42) treatment (Figure 3). The most prominent reductions were observed in C26 SM for glial cells after 3 h Aβ(1–40) and 12 h Aβ(1–42) treatment and in C26:1 SM for hippocampal neurones after 3 h and 12 h Aβ(1–40) and Aβ(1–42) treatment. 3 h and 12 h Aβ(1–40)-treated hippocampal neurones showed a strong increase in C26 SM (Figure 3).
Dihydrosphingomyelines (DHSM)
Almost all DHSM isoforms (C14 DHSM, C16 DHSM, C18:1 DHSM, C18:0 DHSM, C20 DHSM, C22 DHSM, C24:1 DHSM, C24 DHSM, C26:1 DHSM and C26 DHSM) showed an increase after 3 h Aβ(1–40) and Aβ(1–42) and 12 h Aβ(1–40) treatment, as well as a decrease after 12 h Aβ(1–42) treatment of hippocampal neurones. The most prominent reduction in DHSM for hippocampal neurones was observed after 3 h in C14 DHSM after Aβ(1–40) treatment and C26 DHSM after Aβ(1–42) treatment, and for all DHSM isoforms after 12 h Aβ(1–42) treatment (Figure 3). However, the most prominent increase in DHSM for hippocampal neurones was observed after 3 h in C14 DHSM after Aβ(1–42) treatment and C26 DHSM after Aβ(1–40) treatment, and for all DHSM isoforms after 12 h Aβ(1–40) treatment (Figure 3). Almost all DHSM isoforms (C14 DHSM, C16 DHSM, C18:1 DHSM, C18:0 DHSM, C20 DHSM, C22 DHSM, C24:1 DHSM, C24 DHSM, C26:1 DHSM, and C26 DHSM) from glial cells treated with Aβ were increased, whereas in glial cells, the most prominent increase are observed after 3 h Aβ(1–40) treatment for C14 DHSM and after 12 h Aβ(1–42) treatment for C18:0 DHSM, C20 DHSM, and C22 DHSM. Additionally, glial cells showed a prominent decrease in C14 DHSM after 12 h Aβ(1–42) (Figure 3).
Phosphatidylcholines (PC) and Lyso-Phosphatidylcholines (LPC)
A prominent decrease in PC (38:0) in hippocampal neurones after 3 h Aβ(1–40) as well as 12 h Aβ(1–40) and Aβ(1–42) was observed, as well as an strong increase after 3 h Aβ(1–42) treatment. All other PC isoforms (PC (28:0), PC (30:0), PC (30:1), PC (32:0), PC (32:1), PC (34:1), PC (34:2), PC (36:1), PC (36:2), PC (36:2), PC (36:3), PC (38:0), PC (38:1), PC (38:2), PC (38:3), PC (38:4)) showed a decrease in 3 h Aβ(1–40)- and 12 h Aβ(1–40)-treated glial cells. Beside PC (28:0), a weak increase was observed after 12 h Aβ(1–42) treatment in glial cells. The following PC isoforms (PC (28:0), PC (30:1), PC (32:0), PC (32:1), PC (34:1), PC (36:1), PC (38:0), PC (38:1), PC (38:2), PC (38:3)) were reduced after 3 h Aβ(1–40) treatment of glial cells. Glial cells also showed a prominent increase in PC (36:0) after 3 h Aβ(1–40) and Aβ(1–42) treatment. No changes were observed in PC (38:0) after 12 h Aβ(1–42) treatment of glial cells. LPC (20:0) showed a strong increase after 3 h Aβ(1–40) treatment of hippocampal neurones and in 3 h Aβ(1–42)- and 12 h Aβ(1–40)-treated glial cells (Figure 4). A prominent decrease was observed in LPC (20:0) in 3 h Aβ(1–40) and 12 h Aβ(1–42) treated glial cells and a weak decrease in 12 h Aβ(1–40)-treated hippocampal neurones (Figure 4).
Lyso-Phosphatidylethanolamine (LPE)
A reduction in all LPE isoforms (LPE (16:0), LPE (16:1), LPE (18:0), LPE (18:1), LPE (18:2), and LPE (20:4)) was shown in 3 h Aβ(1–40)- and Aβ(1–42)-treated hippocampal neurones. A slight decrease in all LPE isoforms (LPE (16:0), LPE (16:1), LPE (18:0), LPE (18:1), LPE (18:2), and LPE (20:4)) was shown in 3 h Aβ(1–42)-treated glial cells (Figure 4). An increase was observed in all LPE isoforms (LPE (16:0), LPE (16:1), LPE (18:0), LPE (18:1), LPE (18:2), and LPE (20:4)) in 12 h Aβ(1–42)-treated glial cells (Figure 4). 12 h Aβ(1–40)-treated hippocampal neurones showed an decrease in LPE (16:1), LPE (18:0), LPE (18:2), and LPE (20:4) as well as an increase in LPE (16:0), LPE (18:1) isoforms. An increase of LPE (16:0), LPE (18:1), and LPE (20:4)) was seen in 12 h Aβ(1–42)-treated hippocampal neurones.
Lyso-Phosphatidylglycerol (LPG)
LPG (14:1) and LPG (16:1) showed a reduction in 12 h Aβ(1–40)-treated hippocampal neurones. Glial cells showed a strong increase after 3 h Aβ(1–40) and Aβ(1–42) treatment in LPG (14:1) and after 12 h Aβ(1–40) treatment in LPG (16:1). In addition, LPG (16:1) showed a decrease after 3 h Aβ(1–40) and Aβ(1–42) treatment of glial cells (Figure 4).
Lyso-Phosphatidylserine (LPS)
LPS (18:2) showed an increase in 3 h Aβ(1–40)- and Aβ(1–42)-treated glial cells as well as in 3 h and 12 h Aβ(1–40)-treated hippocampal neurones. A reduction was observed in Aβ(1–42)-treated hippocampal neurons after 3 h and 12 h (Figure 4). A decrease was observed in LPS (18:0) after 3 h Aβ(1–40)- and Aβ(1–42)-treated glial cells. LPS (16:0) was reduced in 3 h Aβ(1–40)-treated hippocampal neurones, as well as in 12 h Aβ(1–40)-treated glial cells (Figure 4).
Lyso-Platelet-Activating Factor (Lyso-PAF)
Lyso-PAF showed a reduction in 12 h Aβ(1–40)-treated glial cells, as well as in hippocampal neurones after 3 h and 12 h, for both Aβ(1–40) and Aβ(1–42) treatment. A prominent increase of lyso-PAF was observed in 3 h Aβ(1–40)- and Aβ(1–42)-treated glial cells (Figure 4).