Synaptic deposition of lysophospholipids at stresses - risk of the regional lipotoxicity for brain diseases

1 Introduction

Lipids are involved in many biological processes such as building cell membranes, keeping energy, and exerting cell signalings. As lipids are essential for the structural and functional integrity of cell membrane, the brain is enriched in lipids. The brain is especially abundant with two polyunsaturated fatty acids (PUFA) i.e., docosahexaenoic acid (DHA, 22:6) and arachidonic acid (AA, 20:4). Because of the presence of curvature (Figure 1A), the acyl chains associated with carbon double bonds supply more space between the membrane hydrophobic core, compared to saturated fatty acid chains. As DHA and arachidonic acids control fluidity and flexibility of membranes, the lipid control via biophysical properties influences neurotransmission (Bozek et al., 2015; Lauwers et al., 2016). For neurons to transmit impulses long distances over the presynaptic terminal, the presynaptic terminal is functionally dedicated to membrane remodeling. At dendrites, for example, the plasma membrane is extensively restructured to produce synaptic vesicles via endocytosis, which rapidly fuse with the presynaptic membrane for exocytosis upon opening of voltage-gated ion channels. For achieving this, the presynaptic terminal membrane contains numerous lipid-metabolizing phospholipases that cleave specific sites to alter lipid structures. Membraneous PUFA play vital roles in neurotransmission at physiological conditions, but are closely related to the occurrence of neuroinflammation at pathologic conditions (Bazinet and Layé, 2014).

Lipids account for up to 45% of the dry weight of the brain, and are categorized as glycerophospholipids (also known as phosphoglycerides), cholesterol, and sphingolipids. Glycerophospholipids consist of a glycerol backbone, two hydrophobic fatty acid chains attached to its structural nomenclature (sn) −1 and −2 carbons, while a polar head group attached to the sn-3 carbon via a phosphodiester linkage (Figure 1B). Glycerophospholipids at the synaptic membrane contain DHA or arachidonic acid at sn-1 and sn-2, and the presynaptic terminal contains lipid-metabolizing enzymes (Cremona et al., 1999; Di Paolo et al., 2004; Rohrbough et al., 2004). Structural details of ‘R’ group at the polar head (Figure 1B, red circles) yield different subtypes of glycerophospholipids with each composition (%) in the human brain i.e., phosphatidylethanolamine (PE) (35.6%), phosphatidylcholine (PC) (32.8%), phosphatidylserine (PS) (16.5%), and phosphatidylinositol (PI) (2.6%) (Javaid et al., 2021). The outer layer of neuronal cell membranes is rich in PC (Figure 1C) and sphingomyelin, while the inner layer is rich in PS, PI, and PE. PC is involved in maintaining the structural integrity of the neuronal/glial cell membranes (Bazinet and Layé, 2014).

Neuronal stresses produce reactive oxygen species (ROS) and cause excessive Ca²⁺ mobilization, overactivation of phospholipases, and upregulation of secondary cell responses (Yamashima, 2000; Yamashima et al., 2020; Yamashima et al., 2023a; Yamashima et al., 2023c; Yamashima et al., 2024). Stress-induced overactivation of phospholipase A₂ (PLA₂) causes breakdown of membrane PC, resulting in the increased generation of both lysophospholipids such as lysophosphatidylcholine (LysoPC, also called lysolecithins) and free fatty acids. The breakdown of LysoPC takes place by the enzyme autotaxin (ATX, also called lysophospholipase D), which yields lysophosphatidic acid (LysoPA) and free fatty acids (Jenkins and Frohman, 2005) (Figures 1C, 2A). Both LysoPC and LysoPA are known to disturb the fluidity and penetrability of neuronal membranes (Farooqui et al., 2006). Lipid peroxidation involves the insertion of a hydroperoxy group into the PUFA of phospholipids. Oxidative decomposition of PUFA initiates chain reactions that lead to the formation of a variety of carbonyl species (three to nine carbons in length), including α,β-unsaturated aldehydes (4-hydroxy-2-nonenal and acrolein), di-aldehydes (malondialdehyde and glyoxal), and keto-aldehydes (4-oxo-trans-2-nonenal) (Suzuki et al., 2010). In addition, free arachidonic acids act as a substrate for cyclooxygenases (COX) to produce eicosanoids including prostaglandins, thromboxanes, and leukotrienes, which further aggravate neurodegeneration via inflammatory cytokines (Yui et al., 2015).

Mitochondria are rich in neurons to produce abundant ATP via the electron transport chain for preserving the brain function. Cardiolipin is an important component of the mitochondrial inner membranes that accounts for ∼20% of the total lipid content in mitochondria. It serves as a stabilizer of mitochondrial cristae and is vital for the integrity of the respiratory chain to produce ATP. However, cardiolipin is susceptible to lipid peroxidation, because it contains four molecules of linoleic acids. ROS oxidize both membrane-derived free arachidonic acids and mitochondrial linoleic acids to generate an endogenous lipid-peroxidation product such as 4-hydroxy-2-nonenal (4-HNE). It causes carbonylation of lysine, arginine, proline, and threonine residues of the substrate protein, leading to its loss-of-function modification. Most importantly, 4-HNE carbonylates a lysosomal stabilizer, heat-shock protein 70.1 (Hsp70.1), facilitates calpain-mediated cleavage of the carbonylated Hsp70.1, and causes lysosomal membrane rupture and neuronal death (Yamashima et al., 2020; Yamashima et al., 2024).

Although cell toxicity of 4-HNE has been elucidated in detail by the author’s group (Yamashima et al., 2020; Yamashima, 2023; Yamashima et al., 2023a; Yamashima et al., 2023b; Yamashima et al., 2023c; Yamashima et al., 2024), the molecular mechanism of toxicity of other lipid-breakdown products, especially lysophospholipids, remains incompletely elucidated. The gross spatiotemporal changes of lipidome in the brain are visualized by the novel methodology like mass spectrometry imaging in the living animals. However, to elucidate the mechanism of regional and/or subcellular lipotoxicity, detecting glycerophospholipid-breakdown products which were deposited within the specific area of the brain tissue i.e., synapses are indispensable. Herein, the author highlighted the possible toxicity of lysophospholipids which accumulate within the synaptic area at stresses. This review focuses on the ultrastructural visualization of lysophospholipids, generating at the synaptic area at stresses and triggering the regional lipotoxicity.

2 Membrane lipid peroxidation and generation of lysophospholipids

Synaptic membrane remodeling necessary for the neurotransmission can be achieved, as membranes are enriched in PUFA-containing lipids which make them flexible (Marszalek and Lodish, 2005; Takamori et al., 2006). Both arachidonic acids and DHA have higher conformational flexibility to interpose membrane proteins like receptors and channels between each phospholipids, which are indispensable for the synaptic transmission (Antonny et al., 2015). Consequently, the synaptic membrane lipids are flexible to transmit impulses, and can actively participate in the neurotransmission. At the same time, however, they are vulnerable to the oxidative stress, because unsaturated fatty acids within the membrane lipids are prone to the oxidative injury via the phospholipase overactivation at the excessive stresses. Although flexible changes of the unique lipidomes are indispensable for maintaining the synaptic function, their vulnerability to stresses leads to the occurrence of synaptic disorders (Lauwers, et al., 2016). Details of the spatial reorganization of lipids across the synapse membranes remain poorly elucidated until now, because of the insufficient bulk tissue homogenization losing regional information. There were technical limitations of the regional lipid assay which can accurately detect the molecular alterations occurring at the synapse membranes.

As ROS have been identified as a key causative factor in most of the brain diseases, the lipid peroxidation is considered one of the major causes of neuronal injury. Lipid peroxidation begins with the interaction of ROS with PUFA involved within the cell membrane (Yamashima et al., 2020). Activated PLA₂ catalyzes release of free arachidonic acids from the sn-2 position of glycerophospholipids (Figure 1C, lower violet arrow and circle) (Sun et al., 2004; 2014; Farooqui and Horrocks, 2006; Chen et al., 2008; Dennis et al., 2011; Hermann et al., 2014). Consequently, 4-HNE is generated from these arachidonic acids to carbonylate Hsp70.1, which triggers programmed neuronal necrosis (Yamashima et al., 2020; Yamashima, 2023; Yamashima et al., 2023a; Yamashima et al., 2023b; Yamashima et al., 2023c; Yamashima et al., 2024; Yamashima et al., 2024).

Cylindrical PC with two fatty acyl chains is appropriate for constituting cell membrane, while inverted cone LysoPC with one chain is prone to make vesicles via buddings (Figures 2B,C). According to the regional amount of LysoPC, the synapses show distinct response each other. The physiological synaptic stimuli are appropriate for making synaptic vesicles, whereas exaggerated stimuli at stresses are prone to make extramembranous, multilamellar bodies composing of LysoPC from synaptic vesicles (Figures 2C,D circle). Lysophospholipids are glycerophospholipids with only one fatty acyl chain, and constitute about 0.5%–6% of the total lipid weight of biological membranes at physiological conditions (Wilton, 2008). At neuronal stresses, however, generation of ROS causes an excessive generation of lysophospholipids. Appropriate amount of the endogenous lysophospholipids is physiologically important in the membrane-shaping process for the membrane phospholipid remodeling (Figure 1C) (Shindou et al., 2013). They are secreted into the blood, saliva, and tear, and activate a variety of G protein-coupled receptor (GPCR) for facilitating cell growth, differentiation, migration, cell-cell attachment, inflammation, and immunity (Ailte et al., 2016). However, generation of excessive lysophospholipids by the ROS-induced hydrolysis of membrane phospholipids in the brain may cause synaptic and neurological disorders.

3 Role of lysophosphatidylcholine (LysoPC) in neurodegenerative diseases

PC is the second abundant component of biological membranes of mammalian cells (Watson, 2015), while LysoPC is endogenously produced by the cleavage of the membranous PC via PLA₂ (Figures 1C, 2A). Since the surface area of the polar head group is larger than that of the fatty acyl chain, the inverted cone of LysoPC (Figure 2B) is appropriate for making synaptic vesicles via buddings, but at the same time, it is prone to make lamellar structures via filopodia (Figures 2C,D) (Saadeldin et al., 2023). In the physiological conditions, LysoPC is present at very low concentrations in the body fluids, because it is catabolized through opposite pathways via separate enzymes such as PLA₂ or LysoPC acyltransferase (LPCAT). The breakdown of PC to LysoPC occurs by PLA₂, whereas LPCAT converts LysoPC back to PC via the phospholipid remodeling called Lands’ cycle (Figure 1C) (Lands, 1958).

Excess LysoPC in the brain is generated not only in acute stress conditions such as ischemia, trauma, irradiation etc., but also in chronic neurodegenerative diseases and brain aging. The number of carbon atoms and double bonds in the fatty acyl chain makes many subtypes of LysoPC which show diverse responses. For example, in the mouse model with the transient occlusion of the right middle cerebral artery (MCA), Zheng et al. (2020) demonstrated that the level of PC (16:0/16:0) in the affected hemisphere (RC) decreased on days 1, 3 and 7 after the occlusion, whereas LysoPC (16:0) level increased significantly, compared to the control hemisphere (LC) (Figure 3A) (Zheng et al., 2020). The rat model of permanent MCA occlusion also showed similar data on the mass spectrometry imaging. LysoPC (16:0) accumulation was increased in the brain at 2 h and 24 after the occlusion, although it remained until 5 and 20 days (Figure 3B) (Nielsen, 2016). In the brain, LysoPC disrupts barrier disruption of the microvasculature, mediates pericyte loss, and induces inflammation and oligodendrocyte demyelination (Law et al., 2019). Using the brain explant culture of mice, Zheng et al. (2020) showed that LysoPC (16:0), but not PC (16:0/16:0), is detrimental to the integrity of neuronal growth cones and neuronal viability. LysoPA induce collapses of the growth cone more drastically than LysoPC (Figure 3C, red arrows) (Zheng et al., 2020). LysoPC enhances the oligomer formation of amyloid β₄₂ and neurotoxic protein aggregation, which facilitate occurrence of neurodegenerative diseases (Sheikh et al., 2015; Kaya et al., 2017).

Previous data suggest that oxidative stress-induced activation of PLA₂ generates not only LysoPC but also LysoPS and LysoPE in the acute phase of brain injuries. LysoPS is a signaling lipid that regulates immunological and neurological processes via membrane receptors, including stimulation of phagocytosis by macrophages (Frasch and Bratton, 2012). LysoPS was observed at day 5 in the infarcted area, but not at an earlier time point (Nielsen et al., 2016). LysoPE is also a class of phospholipid derived from PE which is the most abundant structural component of cell membranes. LysoPE was shown to mobilize intracellular Ca²⁺ through G protein-coupled receptors in the human SHSY5Y neuroblastoma cells (Lee et al., 2017). The lipidomics assay has been used as a metabolomic approach for investigating the quantitative and qualitative profiles of lipid metabolites in the serum, tissue, and cells (Dennis, 2009). Presumably, LysoPS and LysoPE similarly damage the growth cone like LysoPA.

Recently, research has begun to focus on the accurate analysis of diverse LysoPC species, the toxicity of which differs according to carbon chain length and number of double bonds (Ren et al., 2022). However, it remains unelucidated where LysoPC accumulates at the subcellular level in the brain. In the patients with Alzheimer’s disease and mild cognitive impairment, the plasma levels of five types of PC are lower, whereas those of LysoPC (C18:1 or C18:2) are higher, as compared to the healthy subjects (Klavins et al., 2015). Both the decrease of certain types of PC and the increase of LysoPC were thought to be due to the aberrant activity of PLA₂, because amyloid β₄₂ peptides increase its activity in the brain of Alzheimer’s disease (Hicks et al., 2008; Lee et al., 2011). Since several LysoPC species were increased within the frontal cortex of the postmortem Alzheimer brains (Grimm et al., 2011), it is likely that PC involved in its neuronal and glial membranes was pathologically cleaved by PLA₂. However, it is difficult to detect LysoPC in the living brain, which has accumulated in the restricted region via ROS generating at pathological conditions. The plasma level of LysoPC ranges from 125 to 143 nM/mL in healthy conditions, but its level increases not only in Alzheimer’s disease but also in chronic diseases such as diabetes, ovarian cancer, and renal failure (Rabini et al., 1994; Okita et al., 1997; Sasagawa et al., 1998). Excessive LysoPC activates multiple signaling pathways via GPCR G2A and Toll-like receptors, which are involved in the consecutive oxidative stress and inflammatory responses, and occurrence of these diseases (Rabini et al., 1994; Okita et al., 1997; Sasagawa et al., 1998; Law et al., 2019).

There are a few reports on the lipidomic profiling of the serum of the patients with mental disorders such as migraine, anxiety, depression, etc. For example, comparing migraine patients with the healthy subjects, Ren et al. (2022) found a significant upregulation of LysoPC (20:0), and downregulation of both LysoPC (16:0, 24:0,14:0, 15:0, 22:0, 18:2, 22:5) and LysoPE (18:1, 18:2, 22:5, 18:3, 22:6, 22:4, 20:4, 20:3) in the serum of migraine patients (Ren et al., 2022). Although implication of the oxidative stress was suggested, the mechanism of such LysoPC and LysoPE alterations has been unelucidated. It is probable that different alterations of the enzyme activity of phospholipases might be observed between the brain tissue and the serum at neuronal stresses/injuries. When oxidative stress occurs, for example, generation of free radicals can activate enzyme activity of PLA₂ and upregulate hydrolysis of PC or PE in the brain tissue, whereas antioxidants in the serum may disrupt PLA₂ and downregulate generation of LysoPC and LysoPE (Ren et al., 2022). One must keep it in mind that the serum concentration of lysophospholipids is not always in parallel with that involved in the synaptic area.

4 Role of lysophosphatidic acids (LysoPA) in brain disorders

For the past three decades, the involvement of LysoPC-derived LysoPA (1-acyl-2-sn-glycerol-3-phosphoate) in the pathologies of various central nervous system (CNS) disorders has come to light (Tigyi et al., 1996; Chuprun et al., 1997; Sayas et al., 1999; Sayas et al., 2002; Sayas et al., 2006; Moolenaar et al., 2004; Frisca et al., 2012; Castilla-Ortega et al., 2014; Crack et al., 2014; Gotoh et al., 2019; Herr et al., 2020; Geraldo et al., 2021; Dedoni et al., 2024). As described below, the regional accumulation of LysoPC, followed by the accumulation of LysoPA, is related to the occurrence of the excessive Ca²⁺ mobilization. LysoPA is one of the most basic natural phospholipids, having only one fatty acyl chain which consists of a glycerol backbone connected to a phosphate group (Figures 1, 2). It is a proinflammatory, bioactive phospholipid being generated from LysoPC mainly by the enzymatic action of ATX (Aoki et al., 2008; Perrakis and Moolenaar, 2014). As LysoPA is water-soluble, it is ubiquitously found in the cerebrospinal fluid (CSF), serum, and biological fluids such as saliva, urine, and seminal fluid (Baker et al., 2000; Choi and Chun, 2013). LysoPA acts through binding to the specific G protein-coupled LysoPA receptors (LPAR) to induce pleiotropic effects (Frisca et al., 2012). LPAR activate different downstream signaling mediators, including phospholipase C, Akt, mitogen-activated protein kinase and Rho-associated protein kinase (Kranenburg and Moolenaar, 2001; Kim and Adelstein, 2011). The cellular responses elicited by LysoPA are involved in the proliferation, survival, migration, cytoskeleton reorganization, differentiation, and the DNA synthesis in physiologic conditions. On the other hand, an excessive amount of LysoPA interferes with the structure of the cytoskeleton and causes neurite retraction by an increase in the phosphorylation of tau proteins in pathologic conditions (Tigyi et al., 1996; Chuprun et al., 1997; Sayas et al., 1999; 2002; 2006; Moolenaar et al., 2004). The abnormal functioning of the ATX-LysoPA axis and excess generation of LysoPA may be closely related to the onset and progression of synaptic disorders, which leads to the progression of neurological symptoms, mental disorders, or neurodegenerative diseases.

Castilla-Ortega et al. analyzed effects of the LysoPA (18:1) injection into the rat midbrain upon behaviour changes (Castilla-Ortega et al., 2014). They showed that LysoPA (18:1) increased immunoreactivities of c-Fos and LPAR in the dorsal periaqueductal gray matter (Figure 4A, DLPAG). In the forced swimming test, LysoPA (18:1) dose-dependently increased depression-like behaviour, as evaluated by the immobility time (Figure 4B). As the LPAR receptor was associated with emotional learning and memory (Pedraza et al., 2013), the excess LysoPA signaling obviously contributes to neuropsychiatric disorders. In addition, LysoPA has a potential role also in the brain trauma. Crack et al. (2014) reported that LysoPA levels are increased significantly in the CSF taken from the mice subjected to the cortical impact injury. They showed that blocking LysoPA with the specific anti-LysoPA antibody significantly reduced lesion volume on both the gross inspection and MRI (Figure 4C), and improved neurological outcomes after the traumatic brain injury (Crack et al., 2014). These data altogether suggest implication of LysoPA for the occurrence of neuronal injury and symptoms in rodents.

As signaling mediators, only a small amount of LysoPA and its precursor LysoPC are sufficient at the extracellular and intracellular compartment of the normal brain (Dedoni et al., 2024). Therefore, increments of the LysoPA signaling caused by the excessive generation in the synaptic area at stresses, are thought to be related to the occurrence of various neurological diseases, including major depression, schizophrenia, Alzheimer’s disease, and Parkinson’s disease, because the patients of these diseases show higher levels of LysoPA or ATX, as compared to the control individuals (Roberts et al., 2005; Yang et al., 2015; Ahmad et al., 2020; Riya, et al., 2020; Dedoni et al., 2024). In Alzheimer’s disease, an abnormal LysoPA metabolism leading initially to the activation of LPAR, subsequently to GSK-3 activation, facilitates tau phosphorylation, formation of neurofibrillary tangles, extracellular aggregation of amyloid β, and microglial activation (Herr et al., 2020; Geraldo et al., 2021; Dedoni et al., 2024). Neurofibrillary tangles are pathological intercellular accumulations of phosphorylated tau protein, and LysoPA-induced neurite retraction is associated with an increase in the phosphorylated tau protein. Not only the CSF but also the serum may reflect molecular dynamics in the brain, but LysoPA levels in both showed no differences between the patients of major depression and healthy subjects (Gotoh et al., 2019). However, it is probable that LysoPA levels involved in the brain tissue were not precisely reflected to the CSF or serum, especially when LysoPA levels are predominantly higher at the restricted area of the brain, for example, within the synaptic area.

Excess LysoPA triggers occurrence of neurological symptoms and brain diseases, because the synaptic overload of LysoPA would cause disorders of the synaptic transmission. The regional concentrations of LysoPA in the brain tissue are measured quantitatively by the enzyme-linked immunosorbent assay (ELISA) or the matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). However, the subcellular identification of water-soluble LysoPA is extremely difficult, because of the very small amount involved. On the contrary, the subcellular distribution of its precursor, water-insoluble LysoPC can be visualized in the brain tissue by electron microscopy, if an appropriate methodology of the ultrastructural fixation is utilized.

5 Electron microscopic observation of LysoPC using tannic acid

Due to the absence of primary amines, PC involved in the biological membranes incompletely reacts with the conventional fixatives such as glutaraldehyde and OsO₄. Since PC is grossly extracted during dehydration with ethanol or acetone during the tissue preparation, the double membrane structures had not been observed in detail by the conventional electron microscopic analysis. In 1972, however, Mizuhira and Futaesaku introduced a novel ultrastructural fixation methodology by adding a small amount of tannic acid (galloylglucose, C₇₆H₅₂O₄₆) to glutaraldehyde, in order to preserve PC in the cell membrane and observe distinct double membrane structures (Mizuhira and Futaesaku, 1972). Subsequently, focusing the synthetic PC, and type II pneumocytes which contain a high percentage of saturated PC dipalmitoyl, Kalina and Pease (1977) could observe highly-ordered, myelin-like multilamellar structures, using this unique fixation. Of the repeating periodicity of ∼45 Å lamellae, the hydrophobic (electron-lucent) layer was 25–30 Å, while the hydrophilic (electron-dense) zone was 15–20 Å (Kalina and Peace, 1977). Using tannic acid prior to osmication, the author also observed pulmonary surfactant-like multilamellar structures within human meningioma cells (Yamashima and Yamashita, 1990).

Tannic acid fixation is necessary for both visualizing PC in the membrane clearly and detecting LysoPC at the synaptic area in the very natural state, compared with conventional ultrastructural methods. The mordanting effect of tannic acid was thought to be due to the direct interaction of its carboxyl or hydroxyl groups with the quaternary ammonium radical of choline. Therefore, PE, PI or PS do not form a complex with tannic acid. As LysoPA is water-soluble, and devoid of choline base, the ultrastructural observation is impossible. On the contrary, even after the utilization of organic solvents, the synthetic PC-tannic acid complex is well preserved via forming aggregates (Figure 5A), and can be observed by electron microscopy as highly-ordered, multilamellar structures after stabilization with OsO₄ (Figure 5B) and by a polarizing microscope as a growing structure (Figure 5C). On thin-layer chromatography, the interaction of choline base (CH) with tannic acid (TA) was not observed without osmication, but it appeared as a distinctive precipitation spot after exposing the plate to OsO₄ vapor (Figure 5A, CH-TA) (Kalina and Peace, 1977). By electron microscopy also, omission of osmication resulted in an almost complete loss of multilamellar structures with leaving distorted structures. Among the synthetic phospholipids tested including PC, PE, PI, PS, sphingomyelin, and phosphatidic acid, only PC and sphingomyelin which contain choline base and can react with tannic acid, were observed as multilamellar structures.

Similar concentric, multilamellar bodies were observed by electron microscopy in the human neurodegenerative diseases such as Tay-Sachs disease and Niemann-Pick disease (Becker and Yates, 1991; Otterbach and Stoffel, 1995). These multilamellar bodies were observed within the neuronal cell soma which contain abundant lysosomes, because both diseases were essentially lysosome-related disorders. For example, Niemann-Pick disease is defined as a sphingolipid storage disease, being based on the intracellular accumulation of cholesterol, glycosphingolipids, and lysobisphosphatidic acid especially within the lysosomal lumen (Zervas et al., 2001; Vance, 2006). Both immunohistochemical and immunoelectron microscopic analyses of lysosomal cathepsin enzymes in the control and postischemic CA1 neurons, showed negligible immunoreactivity at the synaptic area in dendritic spines, with most of the immunoreactivity gathered at the perinuclear soma (Yamashima et al., 1998). Although lysosome-related organelles and autophagosomes are known to occur locally at synapses, the lysosome-like structures in the CA1 neurons had a distance from the multilamellar bodies, as shown in Figure 8. The multilamellar bodies described in this review were distinct from those described in Tay-Sachs disease and Niemann-Pick disease, because the former was observed at the synaptic area in the dendritic spines rather than the perinuclear soma, having negligible relations to lysosomes which gathered in the perinuclear cytoplasm. Taken these together, the multilamellar bodies described here were thought to be breakdown products of phospholipids involved within the synaptic vesicular membranes, rather than aggregates of sphingolipid or cholesterol which were stocked within lysosomes.

Synaptic damages at stresses were accurately observed by light microscopy as a decrease of the synaptic spines (Figures 6A,B) (Duman et al., 2016), but the conventional electron microscopic analysis could not detect the relevant alterations at the ultrastructural level (Figures 6C,D) (Martin et al., 1994). The precise ultrastructural observation especially of the injured neurons was difficult for the previous investigators, because the preservation of PC and/or LysoPC was insufficient by the conventional ultrastructural fixation methodology using glutaraldehyde and OsO₄. Especially, ultrastructure of degenerating neurons associated with breakdown of membraneous lipids was less preserved with the conventional fixatives. In the cortex of aged macaque monkeys, for example, Martin et al. (1994) could not observe distinct multilamellar bodies but distorted lamella-like inclusions in the dystrophic dendrites by the conventional ultrastructural fixation in both the degenerating dendrites (Figure 6C-d, red arrow) and the presynaptic terminals (Figure 6D-t, red arrow) (Martin et al., 1994). The similar lamella-like inclusions were frequently observed at the high-magnification of the normal synapse (Figure 6E, red circle) (cited from the home page of Okinawa Institute of Science and Technology Graduate University, https://www.oist.jp/news-center/photos/high-magnification-image-synapse-obtained-electron-microscopy). These data indicate that formation of the distorted lamella is often seen at the synapses by the improper ultrastructural fixation or at those after stresses. However, the previous researchers did not mention the significance of such distorted lamellar structures. They were interpreted as unavoidable artifacts occurring during the conventional electron microscopic fixation.

After the long-term exposure to microwaves in rats, impairments of the spatial learning and memory occurred (Figure 7A), and light microscopic analysis showed a relevant increase of degenerated granular neurons of the dentate gyrus (Figure 7B, blue arrows). Conventional electron microscopic analysis, however, could reveal only a partial decrease of synaptic vesicles, and thickening of the synaptic plate (Figures 7C,D, red arrows) in the presynaptic terminals (Wang et al., 2023). Interestingly, distorted lamellae were seen in the close vicinity of synaptic vesicles (Figures 7C,D, red circles), which indicated their origin from the synaptic vesicle membranes. Similarly, decrease of the synapse number and dysfunction with the reduced volume was observed by the light microscopic observation in the hippocampus and the prefrontal cortex of the individuals and experimental animals which were exposed to long-term stresses (Duman et al., 2016). However, the conventional electron microscopic observation of the injured/aged brain could not show relevant ultrastructural changes as observed by the light microscopy (Figures 6A,B, 7B), although distorted lamella-like structures were a common finding at the synapses (Figures 6C,D, red arrows; E, red circle, Figures 7C,D, red circles). It is suggested from these data that formation of distorted lamellae at synapses is a common response to brain stresses/aging which occurs from rodents to humans (Martin et al., 1994; Wang et al., 2023), but was unfortunately paid little attentions until now.

Although both neuropsychiatric and neurodegenerative diseases are a serious global health concern, the exact pathogenesis of these diseases still remains incompletely elucidated. Now, we had better pay attentions to the implication of stress-induced multilamellar lysophospholipids which accumulated in the synaptic area for the occurrence of cognitive and mental disorders as well as neurodegenerative diseases. Therefore, electron microscopic observation after the tannic acid fixation is uniquely necessary for detecting LysoPC compared with conventional ultrastructural methods.

6 Possible role of multilamellar LysoPC for neurodegeneration

During the ischemia/reperfusion injury of the brain, the ischemic phase creates anaerobic conditions in which intracellular Ca²⁺ mobilization occurs, whereas the reperfusion phase initiates oxidative stress that generates ROS (Yamashima, 2000). Both synergically cause activation of membranous enzymes and harmful changes in the membrane lipids. The critical event in such a series of harmful events in the neuron is unwanted accumulation of lipid-breakdown products that may exert the regional lipotoxicity. To elucidate the exact mechanism of stress-induced lipotoxicity at the synapses, it's indispensable to detect the breakdown products at the subcellular level. As mentioned above, it is difficult to detect them by the conventional ultrastructural fixation. However, when tannic acid is added to the conventional fixatives, the synthetic PC (Figure 5B) can be observed as distinct multilamellar structures. So, the author focused on the effect of transient brain ischemia upon the synaptic ultrastructure of hippocampal neurons of the Japanese macaque monkeys, using tannic acid. The distorted lamella-like structures as observed by the conventional fixatives, were observed more clearly as multilamellar bodies in the monkey experimental paradigm by the addition of tannic acid to the conventional fixatives (Figures 8–10).

Although multilamellar structures were negligible at the synaptic area of the control (non-ischemic) monkey neurons (Figure 8A), they were visualized clearly in the presynaptic areas of the degenerating hippocampal CA1 neurons from immediately after ischemia until day 8 by the electron microscopic observation using 0.1% tannic acid (Figures 8B, 9) (Mizuhira and Futaesaku, 1972; Schrijvers et al., 1989; Yamashima and Yamashita, 1990). The degenerating CA1 neurons after ischemia showed disintegrity of synaptic membranes (Figure 9, yellow rectangle) and decrease of synaptic vesicles (Figure 9, circles), being associated with membrane buddings (Figure 8B, circles) and generation of multilamellar bodies in its vicinity (Fig, 9, arrows), as seen in vitro (Figures 5B,C). Further, the ischemia-resistant granular neurons of the hippocampal dentate gyrus also showed deposition of multilamellar phospholipids after transient brain ischemia (Figure 10B, red arrows), being compared to the non-ischemic controls (Figure 10A). Since polar heads of PE and PS contain primary amines, they should have been fixed as unilamellar vesicular structures (Figure 10B, yellow circles) (Yamashima and Yamashita, 1990) within the enlarged dendritic spines (Figure 10B, asterisks).

The presynaptic membrane is extensively restructured to produce synaptic vesicles which are trafficked to rapidly fuse with the synapses upon opening of voltage-gated ion channels at physiological conditions (Lauwers et al., 2016). To achieve such synaptic function, the presynaptic membrane contains numerous lipid-metabolizing enzymes that locally alter lipidome (Cremona et al., 1999; Rohrbough et al., 2004; Di Paolo and De Camilli, 2006). At the brain stresses/injuries, however, this may lead to rapid changes in the lipid concentration, organization, and metabolism with the local accumulation of lysophospholipids. Since multilamellar bodies in the postischemic CA1 neurons were in continuity with the disintegrated presynaptic membranes (Figure 9, red arrows), they were thought to have enlarged via engulfing degenerating membranes of the synaptic vesicles (Figure 9, red circles). These well-preserved, ordered, multilamellar bodies were scarcely observed without the tannic acid treatment, but could be clearly observed by the 0.1% tannic acid treatment (Figures 8–10). Therefore, these extramembraneous multilamellar bodies were considered LysoPC which contains a choline base. LysoPC was produced by the breakdown of PC which was involved within the synaptic vesicular membranes via the increased PLA₂ activity at the ischemia/reperfusion injury (Figures 1, 2). These multilamellar bodies were seen only at the synaptic area, showing a remarkable contrast with alveolar type II pneumocytes and human meningioma cells in which multilamellar structures were observed mainly within and around lysosomes in the perinuclear soma (Yamashima and Yamashita, 1990; Becker and Yates, 1991; Otterbach and Stoffel, 1995; Zervas et al., 2001; Vance, 2006).

By exerting as a proinflammatory lipid, abnormal levels of LysoPC are closely related to diseases. Therefore, the precise analysis of the subcellular LysoPC is indispensable to elucidate the regional lipotoxicity in the brain. For example, lipidomics such as liquid chromatography coupled with mass spectrometry (LC-MS) or matrix-assisted laser desorption ionization mass-spectrometry (MALDI-MS), can detect LysoPC involved in the fluids or tissues. LC-MS has advantages of excellent resolution and sensitivity involved in the body fluid samples, while MALDI-MS are suitable for the solid, tissue samples (Mallah, et al., 2019). Lipidomics has an advantage in the quantitative analysis, but has a disadvantage in the spational and qualitative analyses. The latter can be achieved only by electron microscopy, using samples treated by the conventional fixatives mixed with tannic acid.

Because of enhancing the membrane excitability, exposure to the endogenous LysoPC has emerged as one of the key factors of neuronal and brain injuries in various mental disorders and Alzheimer’s disease (Qin et al., 2009; Chiarini et al., 2020). Lyso PC and other subtypes of lysophospholipids such as LysoPS and LysoPE can be altogether visualized as multilamellar or unilamellar structures (Figure 10B) in the synaptic area at neuronal stresses/aging by electron microscopy using tannic acid. As the total amount is very small, and catabolized by ATX and related enzymes like LPCAT, lysophospholipids including Lyso PC, LysoPS and LysoPE are hardly detected even by the novel lipidomic assays. In addition, as they are extracted during treatments with organic solvents, the chemical assay would be extremely difficult. If not only LysoPC but also the lipid-peroxidation product such as 4-HNE were continuously generated at the synaptic area via the circumferential stresses, the regional liotoxicity would be a serious cause of neuronal injuries and/or neurodegenerative diseases (Yamashima et al., 2020; Yamashima, 2023; Yamashima et al., 2023a; Yamashima et al., 2023b; Yamashima et al., 2023c; Yamashima et al., 2024).

The study of human diseases is complicated by the difficulty of obtaining fresh brain samples at the early stages of the disease. However, during the neurosurgical intervention for brain tumor, trauma, epilepsy, hydrocephalus, stereotactic neurosurgery etc., biopsy of the tiny (1–3 mm) piece of the human cortical tissue with the informed consent, would greatly help elucidate the pathogenesis of neurodegenerative diseases via the ultrastructural observation after the tannic acid treatment.

7 Conclusion

Oxidative stress/ischemia leading to PLA₂ activation, drive PC hydrolysis and LysoPC/LysoPA accumulation, which in turn increases membrane excitability and contributes to synaptic/neuronal dysfunction. Despite the recent lipidomics of lysophospholipids, detecting their subcellular localization within the restricted brain region is difficult, because most of LysoPC is extracted by the organic solvents while LysoPA is water-soluble being inappropriate for the morphologic observation. Multilamellar bodies i.e., LysoPC were ultrastructurally observed at the synaptic area of the monkey neurons after brain ischemia. Presumably, LysoPA would be generated nearby via the hydrolysis of LysoPC. PC breakdown products at stresses are the marker appropriate for the assessment of the neuronal damage. Using conventional lipidomic approaches, it is difficult to detect regional LysoPC accumulation in the restricted brain tissues, because of the bulk tissue homogenization losing regional information. A tannic acid-based electron microscopy technique to visualize PC and LysoPC, would provide an interesting perspective for elucidating the role of stress-induced phospholipid breakdown in the occurrence of neurodegenerative diseases.

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Footnotes

Edited by: Paola Maycotte, Instituto Mexicano del Seguro Social, Mexico

Reviewed by: Purva V. Gade, University of Massachusetts Medical School, United States

Santosh Kapil Kumar Gorti, IN8Bio, Inc., United States

Footnotes

AA, arachidonic acid; ATX, autotaxin; CA1, cornu Ammonis 1; CH-TA, choline-tannic acid; CNS, central nervous system; COX, cyclooxygenase; CSF, cerebrospinal fluid; DHA, docosahexaenoic acid; ELISA, enzyme-linked immunosorbent assay; GPCR, G protein-coupled receptor; Hsp70.1, heat-shock protein 70.1; 4-HNE, 4-hydroxy-2-nonenal; LC-MS, liquid chromatography-mass spectrometry; LPAR, lysophosphatidic acid A receptor; LPCAT, lysophosphatidylcholine acyltransferase; LysoPA, lysophosphatidic acid; LysoPC, lysophosphatidylcholine; MALDI-MS, matrix assisted laser desorption ionization-mass spectrometry; MCA, middle cerebral artery; MRS, magnetic resonance spectroscopy; OsO₄, osmium tetroxide; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; PLA₂, phospholipase A₂; PUFA, polyunsaturated fatty acids; ROS, reactive oxygen species; sn, structural nomenclature.