We measured non-targeted lipidomics on 316 post-mortem brain samples and employed a multi-omics network approach to integrate proteomic data available for the same brain region of the same brains. Our key findings are: lower abundance of total LPE and LPC in symptomatic stages of AD compared to control or asymptomatic cases. Although brain lipid composition differs between brain regions and shows dynamic changes during development and aging, overall, the deficiency of brain PE and PE plasmalogens are associated with AD27–32, and a decreased ratio of PC/PE inhibits γ-secretase activity, resulting in less β-amyloid formation33. Consistent with this observation, the abundance of plasmalogens, PC_e (p = 0.087) and PE_e (p = 0.26), trended lower in SAD vs controls (Fig. S2). The PC/PE ratio was slightly decreased in controls relative to the SAD group, in line with decreased BACE1 and APP (Fig. S2 and S12). Lipid class variation may be due to membrane remodeling, consistent with phospholipid composition redistribution rather than a dramatic change in lipid metabolism, which is rarely seen in the brain34. Also, previous studies demonstrated that oleic acid-enriched TAG accumulates in ependymal cells in both postmortem AD brains and a transgenic AD mouse model is associated with the suppressed regeneration and homeostasis of neural stem cells35. In our LSEA results, we found that oleic acid enriched TAG was significantly elevated in SAD versus control case DLPFC, possibly suggesting the involvement of oleic acid enriched TAG in AD. Targeted lipidomics showed a decreased ratio of PC/PE was also observed in controls compared to SAD and AAD (Fig. S10d). In general, controls tended to have slightly higher levels than other groups of phospholipids incorporating omega-3 and omega-6 polyunsaturated fatty acids (PUFA), including DHA (omega-3), DPA (omega-3), AA (omega-6), and LA (omega-6) (Fig. S10b-c). A decrease of total PUFA in phospholipids has been observed in the prefrontal cortex in other AD studies30, possibly due to increased oxidative stress, which non-enzymatically degrades PUFA species34, potentially contributing to the reduced levels of PUFA in SAD patients. However, the mechanism underlying lower PUFA levels in AD patients remains unclear and the contribution of dietary PUFA to brain PUFA levels needs further study.
LPE 22:6 regiospecificity (sn-1 vs sn-2) of the glycerol backbone has not been specified in most prior studies and the physiological function of LPE might be regiospecific if not subject to regioselective enzyme-mediated transformation. Specifically, our study demonstrated that brain LPE 20:4 [sn-1] and LPE 22:6 [sn-1] showed significantly lower abundance in SAD than the control group. LPE is low in abundance in the brain and little is known regarding its relative changes in AD. In animal studies, brain LPE 20:4 and LPE 22:6 levels trend lower during aging36 or with impaired spatial cognitive abilities or memory37. Specifically, LPE 22:6 (omega-3) levels recover during the recovery of trauma brain injury38. In contrast, hippocampus LPE 18:1 and LPE 22:6 tend to be increased in vascular dementia39 and AD treatments for the inhibition of either PLA240 or BACE141 reduce LPE accumulation in brain. Even though the pathological significance of LPE is inconclusive in the brain, a few studies suggest the function of exogenous LPE species could stimulate neurite outgrowth in cell culture42–44. Hisano and others have reported the beneficial effect of LPE 18:1 [sn-1] on primary cortical neuron growth against glutamate-induced excitotoxicity via the activation of the GCPR-PLC-PKC-MAPK pathway, and compared to other phospholipids, LPE exhibited the most significant impact on neurite morphology as observed by an increase of microtubule associated protein 2-positive dendrites42,43. LPE isolated from Grifola frondosa mainly consists of LPE 18:2 [sn-1] exhibiting anti-apoptotic activity and enhancing neuronal differentiation through MAPK activation in PC-12 cells44. Moreover, in a mouse model, hepatic levels of LPE 22:6 [sn-1] are negatively associated with non-alcoholic steatohepatitis compared to control mice, and interestingly, highly unsaturated acyl chain in LPE shows a preference for the LPE [sn-1] form45. Taken together, this supports that the PUFA of LPE at its sn-1 position is potentially favorable for neuron function and lipid metabolism; however, more studies are needed to clarify roles of these species in brain pathology.
Our multi-omics analyzes revealed that three lipid co-expression modules consisting of either LPE only or a combination of LPE and LPC correlate more strongly to AD clinical traits. Particularly, the M4 lipid module, mainly driven by LPE 22:6 [sn-1] and LPE 20:4, was associated with the AD protein network modules MAPK/metabolism (black), post-synaptic density (green), and Cell-ECM interaction (greenyellow); these were highly associated with AD neuropathology and cognitive function. Johnson et al. (2022) 8 demonstrated that the MAPK/metabolism module enriched in proteins co-localized to Aβ plaques and Tau neurofibrillary tangles, was strongly associated with cognitive decline, and trended towards enrichment for AD genetic risk. The green post-synaptic density module was associated with cognitive preservation and enriched in proteins positively correlated to cognitive resilience, while negatively correlated to tau microtubule-binding domain (MTBR) peptide levels. The cell-ECM interaction module was positively associated with MTBR and correlated to cognition decline prior to the adjustment of neuropathology. Both post-synaptic density and Cell-ECM interaction modules correlating to MTBR tended to be altered in AD. Although the physiological significance of LPE in brain function is rarely studied, Lee and others have demonstrated that LPE increases intracellular calcium influx in PC-12 neuron cells and SH-SY5Y neuroblastoma cells46,47 while LPE induces Calcium flux independent of LPAR48. Age-related alterations in neuronal calcium (Ca2+) largely contribute to AD pathology and studies of brain tissues have shown significant changes in levels of proteins or genes directly involved in neuronal Ca2+ signaling49. Briefly, Ca2+ dysregulation is associated with familial AD mutations such as in presenilin-1 (PSEN-1) and APP, and the disruption of Ca2+ handling is linked to Tau or Aβ accumulation50. Aβ interacts with Ca2+-related receptors (NMDAR, AMPAR) and channels (VGCC), and further induces Ca2+ excess influx to cytoplasm. Other unbalanced Ca2+ conditions in AD also include Ca2+ from endoplasmic reticulum (ER) leakage (RyR, IP3R, and SERCA), mitochondrial Ca2+ overload, and dysfunction of Ca2+ buffering proteins49–51. Calcium signaling plays an important role in synaptic plasticity triggering several kinase cascades such as calcium/ calmodulin-regulated protein kinases (CaMK), the cAMP-dependent protein kinase A (PKA), PKC, and MAPK/ERKs51. Although in our proteomic dataset, most of these calcium signaling-related proteins were not significantly different among these three AD phenotypes, proteins related to activation of NMDAR and post synaptic signaling transmission were significantly up-regulated in controls vs SAD including glutamate ionotropic receptor AMPA type subunit 1–3 ,GRIA1-3 (AMPAR), voltage-dependent calcium channel gamma-2-4, CACNG2-4 (VGCC), PKC alpha binding protein (PICK1), and Calcium/calmodulin-dependent protein kinase type IV (CAMK4) (Fig. S12c). APP and PSEN-1 double knockin mouse models demonstrate that a decrease of AMPAR efficacy is relevant to synaptic downscaling, an early onset phenomenon in AD, and AMPAR is crucial for long-term potentiation (LTP), critical for memory encoding as well as memory flexibility52. Moreover, treatment using electromagnetic fields (EMF) has shown that human plasma levels of LPE 20:4 and LPE 22:6 are increased after the exposure to high-voltage electric potential and an increase of LPE is possibly due to elevated hydrolysis by phospholipase A2 (PLA2) 53,54. Mostly, EMF therapy has been reported to be associated with VGCC stimulation in various cell types55. Based on our results and literature, LPE species exhibit potential as therapeutic targets for AD via calcium homeostasis in brain.
In our study, a decreased ratio of LPE to PE was observed in SAD compared to controls, indicating possibly inactive hydrolysis of PE to LPE in SAD. PLA2 is responsible for catalyzing the hydrolysis of phospholipids to lyso-phospholipids and free fatty acids, and the reduction of the hippocampus group IVA isoform of PLA2 (PLA2G4A) has been shown to ameliorate Aβ-dependent deficits in a hAPP mouse model56. However, this PLA2 isoform was not detected in the DLPFC proteomic dataset reported by Johnson et al8 possibly due to differential expression in different brain regions. A slightly higher expression of PLA2G15 was observed in SAD (P = 0.064), while the protein levels of other PLA2 isoforms tended to be similar in controls versus SAD, but did not reach statistical significance. PLA2G15 acts as a lysosomal phospholipase preferring PC as substrate. In a PLA2G15 knockout mouse model, a nearly 2-fold increase in both PC and LPC levels in alveolar lavage fluid is observed57. Unlike the strong effects observed in knock out studies, the subtle change in brain PLA2G15 abundance in our proteomic data cannot fully reflect LPC/PC or LPE/PE ratio changes in our lipidomic analysis; however, the mechanism by which a decreased level of total LPE or specifically, LPE 22:6 [sn-1] occurs in SAD brain needs more study. In contrast, myelin disruption is associated with accumulated amyloid plaques58. Myelin lipid disruption is specific to the edges of the white matter in the corpus callosum, while no disruption of the myelin sheath is observed in gray matter. Together with myelin loss in white matter, depletion of PE and increased LPE are observed in a 5xFAD mouse model58. In addition to LPE release from PE through PLA2 hydrolysis, the uptake of plasma LPE can be regulated by the Sodium-dependent LPC symporter (Mfsd2a) and transferred to the brain through the blood brain barrier59. However, this is difficult to validate because Mfsd2a protein was not detected and we lack information about circulating LPE. Despite the complexity introduced by regional differences, cell types shift, and neuropathological conditions, changes in LPE levels associated with AD brain alterations has been highlighted in several AD studies37,41,58,60, indicating the significance of LPE in AD. Moreover, LPE metabolism has been reported being altered by obesity61 and by brown fat activity62, and notably, mid-life obesity links to a higher risk of dementia63. However, direct evidence regarding the role of LPE, specifically LPE 22:6 [sn-1], on AD pathology and the mechanism underlying LPE loss in AD requires further investigation.
In network analysis, the M4 module is strongly associated with the M3 module. Among the hub lipids in the M3 module, LPC 22:6 shares greater similarity with LPE 22:6 in the M4 module, both in terms of pathway and higher abundance in controls than the SAD group. At the lipid class level, lower LPC concentrations have been reported in the prefrontal cortex64, and frontal cortex65 of patients with AD compared to controls, aligning with our findings in the DLPFC in this study. DHA (C22:6) constitutes nearly 50% of PUFA content in the brain66, and depending on the brain regions, a characteristic of AD is the presence of lower DHA levels in brain phospholipids, ranging from about 15–60%67. However, dietary DHA, in the form of free DHA, is either resynthesized into TG or bound to albumin in the blood. The former is delivered to peripheral tissues through lipoprotein rather than the brain68,69, and the latter is released from albumin and transported along the outer membrane in BBB via passive diffusion70. In contrast, dietary LPC 22:6 is the brain’s preferred source of DHA through Mfsd2a59, which improves cognitive function compared free DHA supplements in a mouse study68,69.
Utilizing a proteomic dataset from iPSC-derived neurons71 from matched ROSMAP individuals, we found that LPE 22:6 is highly associated with Echinoderm microtubule-associated protein-like 4 (EML4), which is essential for the stabilization of microtubules (Fig. S13a). In line with our results, the lipid M4 module containing LPE 22:6 and LPE 20:4 correlates to a post-synaptic density module that is negatively associated with MTBR levels. According to Lagomarsono’s finding71, the protein levels of PPP1CA, a core catalytic component of protein phosphatase 1 (PP1), are significantly lower in the AD brain while PPP1R1A, a negative regulator of PP1, shows higher steady state levels. Aβ42/37 treatment in neuronal cells reduces PPP1CA protein levels and subsequently, influences tau phosphorylation and aggregation, indicating the contribution of Aβ-reduced PP1 activity to disturbed tau proteostasis. Interestingly, our correlation analysis (Fig. S13b-c) showed that LPE 22:6 has a negative correlation to PPP1R1A (cor=-0.58, p = 0.0015) and a positive correlation to PPP1CA (cor = 0.28, P = 0.16), suggesting a possible role of LPE 22:6 in AD development.
Our integrative multi-omics results revealed that LPE, including LPE 22:6 [sn-1], is strongly related to AD disease-associated protein modules such as the modules representing MAPK/metabolism, post-synaptic density, and cell-ECM interaction, implicating that LPE species may serve as promising therapeutic targets and even possibly as dietary supplementation for treatment of AD. These primary results provide future directions for studying the underlying mechanisms of LPE in AD brain and underscore the utility of future studies in cellular systems to uncover LPE-relevant neuronal biology in processes of different cell types contributing to AD pathologies.