Ganglioside microdomains on cellular and intracellular membranes regulate neuronal cell fate determination

Abstract

Gangliosides are sialylated glycosphingolipids (GSLs) with essential but enigmatic functions in brain activities and neural stem cell (NSC) maintenance. Our group has pioneered research on the importance of gangliosides for growth factor receptor signaling and epigenetic regulation of NSC activity and differentiation. The primary localization of gangliosides is on cell-surface microdomains and the drastic dose and composition changes during neural differentiation strongly suggest that they are not only important as biomarkers, but also are involved in modulating NSC fate determination. Ganglioside GD3 is the predominant species in NSCs and GD3-synthase knockout (GD3S-KO) revealed reduction of postnatal NSC pools with severe behavioral deficits. Exogenous administration of GD3 significantly restored the NSC pools and enhanced the stemness of NSCs with multipotency and self-renewal. Since morphological changes during neurogenesis require a huge amount of energy, mitochondrial functions are vital for neurogenesis. We discovered that a mitochondrial fission protein, the dynamin-related protein-1 (Drp1), as a novel GD3-binding protein, and GD3 regulates mitochondrial dynamics. Furthermore, we discovered that GM1 ganglioside promotes neuronal differentiation by an epigenetic regulatory mechanism. Nuclear GM1 binds with acetylated histones on the promoters of N-acetylgalactosaminyltransferase (GalNAcT; GM2 synthase) as well as on the NeuroD1 genes in differentiated neurons. In addition, epigenetic activation of the GalNAcT gene was detected as accompanied by an apparent induction of neuronal differentiation in NSCs responding to an exogenous supplement of GM1. GM1 is indeed localized in the nucleus where it can interact with transcriptionally active histones. Interestingly, GM1 could induce epigenetic activation of the tyrosine hydroxylase (TH) gene, with recruitment of nuclear receptor related 1 (Nurr1, also known as NR4A2), a dopaminergic neuron-associated transcription factor, to the TH promoter region. In this way, GM1 epigenetically regulates dopaminergic neuron specific gene expression. GM1 interacts with active chromatin via acetylated histones to recruit transcription factors at the nuclear periphery, resulting in changes in gene expression for neuronal differentiation. The significance is that multifunctional gangliosides modulate lipid microdomains to regulate functions of important molecules on multiple sites: the plasma membrane, mitochondrial membrane, and nuclear membrane. Versatile gangliosides could regulate functional neurons as well as sustain NSC functions via modulating protein and gene activities on ganglioside microdomains.

1. Introduction

Cells and most subcellular organelles are surrounded by biological lipid membranes that define the individual cellular shape and help maintain cellular organization. Biological membranes are highly heterogenous in structure and there exist diverse microdomains (also known as lipid rafts), which are enriched with sugar molecules, such as glycoshingolipids (GSLs). GSLs are unique amphipathic molecules that contain a hydrophilic carbohydrate portion and a hydrophobic lipid component (Fig. 1) (Yu and Itokazu, 2014; Yu et al., 2011). Ganglioside synthesis pathways (Fig. 1) were initially delineated by Yu and Ando (Yu and Ando, 1980). Glucosylceramide (GlcCer) synthase-deficient mice and lactosylceramide (LacCer) synthase-knockout (KO) mice show embryonic lethality (Kumagai et al., 2009; Nishie et al., 2010; Yamashita et al., 1999). These observations indicated that GSLs are essential for development. Gangliosides, sialic acid-containing GSLs, are found in virtually all vertebrate cells but they are particularly abundant in the nervous system (Yu and Itokazu, 2014; Yu et al., 2009; Yu et al., 2011). GM3 synthase (GM3S) is a critical enzyme for the synthesis of all gangliosides. Mutation of GM3S is associated with human autosomal recessive infantile-onset symptomatic epilepsy syndrome (Simpson et al., 2004) and Rett syndrome-like phenotype (Lee et al., 2016), and an alteration of the GM2 synthase (GM2S) gene was reported in patients with hereditary spastic paraplegias (Boukhris et al., 2013) and axonal Charcot-Marie-Tooth disease (Hong et al., 2021). These studies clearly demonstrated that deletions of gangliosides are associated with human diseases. GM2S- and GM3S-double deficient mice, which lack all gangliosides, die soon after weaning at 3 weeks of age (Yamashita et al., 2005), and exhibited sudden death from audiogenic seizures (Furukawa et al., 2014; Kawai et al., 2001). GD3S-KO mice show decreased postnatal neural stem cell (NSC) pools (Wang and Yu, 2013) and impaired postnatal neurogenesis (Wang et al., 2014). GM2S-KO mice exhibit impaired movement and have virtually all the neuropathological symptoms of Parkinson’s disease (PD) (Ledeen and Wu, 2015). A detailed discussion about functional impairment of ganglioside deficiencies can be found in Chapter 14 by Itokazu et al.

Fig. 1. Metabolic pathways and structure of glycosphingolipids (GSLs), including gangliosides.

The nomenclature for gangliosides and their components are based on that of Svennerholm and the IUPAC–IUBMB Joint Commission on Biochemical Nomenclature (1977; Svennerholm, 1963). Cer ceramide, CST cerebroside sulfotransferase (Gal3st1, sulfatide synthase), GalNAc-T N-acetylgalactosaminyltransferase I (B4galnt1, GA2/GM2/GD2/GT2 synthase), GalT-I galactosyltransferase I (B4galt6, lactosylceramide synthase), GalT-II galactosyltransferase II (B3galt4, GA1/GM1/GD1b/GT1c synthase), GalT-III galactosyltransferase III (Ugt8a, galactosylceramide synthase), GlcT glucosyltransferase (Ugcg, glucosylceramide synthase), ST-I sialyltransferase I (St3gal5, GM3 synthase), ST-II sialyltransferase II (St8Sia1, GD3 synthase), ST-III sialyltransferase III (St8Sia3, GT3 synthase), ST-IV sialyltransferase IV (St3gal2, GM1b/GD1a/GT1b/GQ1c synthase), ST-V sialyltransferase V (St8sia5, GD1c/GT1a/GQ1b/GP1c synthase), ST-VII sialyltransferase VII (St6galnac6, GD1aα/GT1aα/GQ1bα/GP1cα-synthase). Official symbols of genes are represented in italics in this figure legend.

Different microdomains regulate protein activities and cellular functions in biological membranes. GSLs help organize microdomains, and regulate growth factor signaling, immune signaling and immune checkpoints, cell-cell interactions including cell adhesion and migration. Growth factor receptors (GFRs) and their signaling are involved in many cellular functions. In carcinoma cells, for example, GM3 (but neither GD3 nor GM1) inhibited epidermal growth factor (EGF)-induced autophosphorylation of EGFR (Bremer et al., 1986). Inhibition was caused by the interaction of the N-linked GlcNAc termini of EGFR with the oligosaccharide portion of GM3 (Hanai et al., 1988). Ganglioside-containing microdomains provide a platform for the initiation of growth factor signaling in neuronal cells. It has been reported that GluR2-containing AMPARs bind selectively to GM1, while AMPAR-trafficking complexes containing Thorase, Nicalin, N-ethylmaleimide-sensitive factor, and its attachment protein γ-SNAP, bind selectively to ganglioside GT1b (Prendergast et al., 2014). Specific gangliosides clearly control structures and functions of recently described distinct microdomains for receptor trafficking and for other protein activities. Intriguingly, it has been reported that GM1 ganglioside is bound by the angiotensin-converting enzyme-2 recognized by the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for COVID-19 infection (Nguyen et al., 2021). Membrane lipids have been revealed to be important not only for separating intracellular from extracellular environments, but also for providing functional platforms for numerous molecular interactions. More specifically, GSLs are essential for maintenance of the biological functions of neuronal cells.

2. Ganglioside multifunction in cell membranes and intracellular organelles.

It has been revealed that functional gangliosides regulate the activity of a variety of membrane proteins. Early studies reported that gangliosides have growth factor like activities for neuronal functions (Ferrari et al., 1983; Schwartz and Spirman, 1982; Tsuji et al., 1983). Specific gangliosides control protein functions of biological membranes, including plasma membrane, mitochondrial membrane, nuclear membrane, etc. Signaling of neurotrophic factors is regulated by gangliosides (e.g., GD3 for EGF and GM1 for GDNF) (Hadaczek et al., 2015; Itokazu et al., 2018; Wang and Yu, 2013). It has also been reported that GM1 in plasma membranes modulates the integrin-FAK signaling pathway to promote neurite outgrowth and cell migration (Itokazu et al., 2014; Palazzo et al., 2004; Wu et al., 2007). We found that GD3 regulates mitochondrial dynamics by mediating dynamin related protein-1 (Drp1) (Tang et al., 2020b). Furthermore, we found that GM1 binds to other mitochondrial proteins to regulate mitochondrial functions (Itokazu et al., unpublished data). With regard to nuclear gangliosides, we demonstrated that nuclear GM1 binds to the neurogenic promoter region to enhance neuronal differentiation (Itokazu et al., 2016b; Tsai et al., 2016). Nuclear receptor related 1 (Nurr1, also known as NR4A2) is an essential transcription factor for differentiation, maturation, and maintenance of midbrain dopaminergic neurons, including activation of the tyrosine hydroxylase (TH) promoter. We discovered that GM1 can recruit Nurr1 on TH promoter to enhance expression (Itokazu et al., 2021) and that GD3 increased expression of the NSC-associated transcription factor, SRY (sex determining region Y)-box 2 (SOX2) (Itokazu et al., 2019). These are just some examples of how gangliosides can modulate lipid microdomains on membranes, to regulate functions of different molecules on them. In sum, the versatile nature of gangliosides allows them to modulate several aspects, such as, growth factor signaling, mitochondrial function, gene regulation, and altered NSC activities.

3. Ganglioside microdomains for neuronal cell fate determination

NSCs are fundamental cells that are capable of differentiating into various cell types in the nervous system. Gangliosides undergo dramatic qualitative and quantitative developmental changes that are correlated with cellular proliferation, differentiation, and biological functions of nerve cells (Fig. 2). For most mammals, neurogenesis commences during early embryonic stages and is almost complete shortly after birth; it continues to occur at a much slower pace and in a limited manner throughout the entire adult life. In the adult brain of mammals, neurogenesis persists primarily in two germinal zones, the subventricular zone (SVZ) of the lateral ventricles (Doetsch et al., 1999; Doetsch et al., 1997) and the subgranular zone (SGZ) in the dentate gyrus of the hippocampus (Seri et al., 2001; Suhonen et al., 1996). In the adult SVZ, different cell types that have distinct functional roles are present in a NSC niche (Fig. 3). Type B cells are radial glial cell (RGC)-like cells and are considered as NSCs. Type B NSCs are slow dividing (duration of cell cycle >15 days) and express glial fibrillary acidic protein (GFAP). The quiescent type B1 radial glial cells are morphologically and phenotypically distinguished from the activated cells (no contacting cilium, EGFR+). Quiescent NSCs express platelet-derived growth factor receptor beta (PDGFRβ), and loss of PDGFRβ was reported to release the NSCs from quiescence (Delgado et al., 2021). Type C cells are transient amplifying cells that are rapidly proliferating (duration of cell cycle about 13 hr) and express the transcription factor Mash1. Type A cells are neuronal precursors that have already committed to differentiate into neurons, and these cells express polysialic acid-neural cell adhesion molecules (PSA-NCAM) and a cell cycle of about 13 hr (Morshead, 2004). Ependymal cells are lined on the wall of the ventricle and have multi-motile cilia, which are important for controlling the flow of cerebrospinal fluid (CSF). Multipotency of the ependymal cells has been reported (Johansson et al., 1999), although this is still not settled (Chiasson et al., 1999; Doetsch et al., 1999; Laywell et al., 2000). Further research reported that ependymal cells have been shown to be the most quiescent type of NSCs whose cell cycle is strictly regulated and reinitiated under specific circumstances. In certain restricted situations, a subpopulation of ependymal cells may develop into neurons, and these cells are considered to be NSCs (Carlen et al., 2009; Coskun et al., 2008). In the SGZ, five types of cells have been described (Filippov et al., 2003). Type 1 cells are considered quiescent neural progenitors that are RGC-like cells and largely equivalent to Type B NSCs in the SVZ. Type 2 cells express Nestin, and this cell type has been classified into two cell populations. Type 2a cells are amplifying neural progenitors that are similar to Type C transient amplifying cells in the SVZ. Type 2b and 3 cells are neuroblasts that express PSA-NCAM and correspond with Type A neuronal precursors (Encinas et al., 2006; Steiner et al., 2006). The other type of cells are mature granule neurons. Recently, it has been reported that Mash1⁺ cells do not amplify and are therefore not Type 2a amplifying neural progenitors that can directly differentiate into early neuroblasts without mitosis (Lugert et al., 2012).

Fig. 2.

Neuronal differentiation and concurrent changes in ganglioside expression. The synthesis of GD3 is switched into the synthesis of complex gangliosides (GM1, GD1a, GD1b, and GT1b). During neuronal differentiation, the expression of complex a-series gangliosides, especially GM1, is augmented by a GM1-modulated epigenetic gene regulation mechanism of GalNAcT. Nuclear GM1 modulates transcriptional activity for neuronal differentiation. NSC, neural stem cell; NPC, neuronal progenitor cell.

Fig. 3.

Neural stem cell niche at the subventricular zone (SVZ) on the surface of the lateral ventricle in the adult mouse brain. (A) Type B cells are radial glial cell (RGC)-like cells and have been considered as neural stem cells (NSCs). Gangliosides seem to regulate transition between quiescent type B1 radial glial cells and activated type B2 NSCs. Type C cells are transient amplifying cells that are rapidly proliferating. Type A cells are neuronal precursors that have already committed to becoming neurons expressing PSA-NCAM on the cell surface. (B) Nestin+/GFAP+ radial glia-like NSCs are significantly reduced in the SVZ of GD3S-KO mice (1-month-old). In the 6-month-old SVZ, Nestin+/GFAP+ cells are nearly absent from the GD3S-KO brains (data not shown). Consistent with this observation, the number of Nestin+/GFAP+ radial glia-like cells at the dentate gyrus (DG) of GD3S-KO mice is also significantly reduced.

With regard to gangliosides, we have shown that GD3 is the predominant ganglioside in NSCs, and it can serve as a convenient cell surface marker of these cells (Nakatani et al., 2010). The interaction of GD3 with EGFR plays a crucial role in maintaining the self-renewal capacity of NSCs by directing the EGFR through the recycling pathway rather than through the degradative pathway after endocytosis (Wang and Yu, 2013). Deletion of GD3 in NSCs reduced stem cell pools at the SVZ and dentate gyrus (DG) in the adult mouse brain (Fig. 3B) (Wang et al., 2014). Thus, GD3 plays a crucial role in the long-term maintenance of NSC populations in the DG of both the hippocampus and the SVZ of postnatal mouse brain. We have reported that efficient histone acetylation of glycosyltransferase (GT) genes contributes to the developmental alteration of ganglioside expression in mouse brain (Suzuki et al., 2011). Further, we have demonstrated that acetylation of histones H3 and H4 on the GM2/GD2S gene promoter leads to recruitment of trans-activation factors specificity protein 1 (Sp1) and activating protein 2 (AP-2) during neuronal differentiation (Tsai and Yu, 2014). We also found that nuclear GM1 binds with acetylated histones on the promoters of the GM2/GD2S gene as well as on the neurogenic transcription factor, NeuroD1 gene, in differentiated neurons from neuronal progenitor cells (NPCs) (Tsai et al., 2016). More recently, we showed that administration of GD3 augments the expression of the multipotent marker, SOX2, in cells at the SVZ and DG in the adult mouse brain (Itokazu et al., 2019). Our most recent work demonstrated that GM1 enhances neuronal differentiation and up-regulates dopaminergic neuron-specific genes via recruitment of the Nurr1 transcription factor (Itokazu et al., 2021). These examples illustrate the importance of nuclear membrane gangliosides in regulating neural development and neuronal differentiation of NSCs.

4. GD3-EGFR

We fiirst discovered that GD3 is the predominant ganglioside species in NSCs (>80%) (Nakatani et al., 2010). The expression of GD3 and Nestin was found in identical cells at the SVZ of postnatal mice. Surprisingly, no significant difference in the proliferation rate and expression of lineage-associated markers was detected between wild-type (WT) and GD3S-KO NSCs that were cultured in the presence of FGF-2 but in the absence of EGF (Yu and Yanagisawa, 2007). To clarify whether GD3 modulates the self-renewal capacity or cell fate determination of NSCs, NSCs from GD3-synthase (GD3S)-knockout (KO) mice were cultured with EGF. Intriguingly, GD3S-KO NSCs that were cultured with EGF showed dramatically inhibited cell proliferation (Wang and Yu, 2013). The expression of Nestin and EGFR was also strongly down-regulated and the MAPK/ERK pathway signaling was impaired in GD3S-KO NSCs. Additionally, EGFR degradation and the reduction of p-EGFR and p-ERK1/2 in the GD3S-KO NSCs correlated with EGF stimulation. Subsequently a decrease of the MAPK/ERK proliferation pathway was identified in GD3S-KO NSCs. Furthermore, surface expression of membrane EGFRs is significantly reduced in GD3S-KO NSCs. Correspondingly, GD3 and EGFR were observed to be co-localized in NSCs, and they interacted in the microdomains of the cell surface as well as in intracellular vesicles. Interestingly, EGFR turned out to exist in non-lipid microdomain fractions in GD3S-KO NSCs. One of the most fascinating discovery was that EGFR is localized in GD3 ganglioside-enriched microdomains, and GD3 is essential for the maintenance of the self-renewal capacity in NSCs by recruiting EGFR to the microdomains to sustain the EGF-induced downstream signaling (Wang and Yu, 2013).

It is significant to note that GD3 interacts with EGFR, an important mitogen receptor for the self-renewal of NSCs in the microdomain. Endocytosis is a basic cellular process that is used by cells to internalize a variety of molecules. Cells are estimated to internalize, via endocytosis, about half their plasma membrane per hour (Steinman et al., 1983). GSLs are recognized to undergo endocytosis. Once internalized, GSLs can be (1) recycled to the plasma membrane; (2) sorted to the Golgi complex; or (3) degraded in the lysosome. Since EGFR expression is reduced in GD3S-KO NSCs, internalization of its ligand EGF was investigated. It was found that the number of NSCs with internalized biotinylated-fluorescent-EGF was significantly reduced in the absence of GD3. Since it is known that EGFR can be recruited to the endosomes for recycling or sorted to lysosomes for degradation, it was found that in the GD3S-KO NSCs, EGFR exhibited increased co-localization with the lysosome-associated membrane protein 1 (LAMP1) in lysosomes as well as diminished co-localization with a marker of recycling endosomes, Ras-related protein Rab11 (Rab11) (Fig. 4). The co-localization with Early Endosome Antigen 1 (EEA1) also showed a mild decrease in the GD3S-KO NSCs. Pursuing the endocytosed EGFR indicated that a large amount of EGFR in GD3S-KO NSCs underwent the endosomal–lysosomal degradative pathway, while a greater proportion of the EGFR was subject to the recycling pathway in the WT NSCs than in GD3S-KO NSCs. Thus, the interaction of GD3 and EGFR in NSCs is responsible for sustaining EGFR surface expression and downstream signaling to maintain the self-renewal of NSCs (Wang and Yu, 2013). Our research demonstrated that this interaction functions as: 1) a “platform” to initiate EGFR downstream signaling to induce NSC self-renewal; and 2) a “director” for the recycling of EGFR after endocytosis. In postnatal brain, GD3 is required for the long-term maintenance of NSCs. Deficiency in GD3 leads to developmental and behavioral deficits, such as depression (Wang et al., 2014).

Fig. 4.

Interaction of GD3 with EGFR modulates self-renewal of NSCs. The decrease in EGFR expression is accompanied by an accelerated loss of the self-renewal property in the GD3S-KO NSCs. EGFR (red) is expressed in GD3 (blue)-rich microdomains on neural stem cell (NSC) surface. GD3 regulates the endocytosis of EGFR. With GD3, EGFR is co-localized with a marker of recycling endosomes, Rab11. On the other hand, EGFR showed significantly increased co-localization with the lysosomal marker LAMP1 in GD3S-KO NSCs. The interaction of GD3 and EGFR in NSCs is responsible for sustaining EGFR surface expression and downstream signaling to maintain self-renewal of the cells.

5. GD3 regulates mitochondrial dynamics by interacting with Drp1

Mitochondrial lipid membranes influence mitochondrial functions, including the electron transport chain activities, nucleotide transport, mitochondrial protein import, membrane properties, ATP synthesis, cell death signaling, and mitochondrial dynamics. The morphological changes that occur as NSCs mature to neurons during neurogenesis requires a huge amount of energy (Son and Han, 2018). The mitochondrion, the main intracellular organelle for producing adenosine triphosphate (ATP), plays a crucial role in adult neurogenesis (Beckervordersandforth et al., 2017; Son and Han, 2018). To understand how GD3 regulates adult neurogenesis, we performed a mass spectrometric (MS) analysis-based proteomic screen for GD3-protein interactions. We first performed affinity chromatography using the anti-GD3 antibody R24 followed by MS analysis. We identified Dynamin-1-like protein (Drp1), a mitochondrial fission protein, as a GD3-interacting protein (Tang et al., 2020b). Drp1 is a GTPase that regulates mitochondrial fission and plays important roles in the regulation of mitochondrial dynamics. To investigate whether GD3 affects mitochondrial dynamics, we examined mitochondrial morphology in the nascent granule neurons. At all stages, mitochondria had a tubular morphology. GD3S-KO neurons had shorter mitochondria with a smaller aspect ratio (major axis/minor axis) than their WT mates, suggesting that mitochondria were fragmented. The neurite mitochondrial index, an index for mitochondrial density, was also significantly reduced in GD3S-KO neurons. Further Western blot analysis demonstrated that Drp1 levels were increased and the activated-Drp1 (phosphorylation of Drp1 at serine 616) was higher in GD3S-KO mitochondria. In contrast, there were no significant changes on the expression of the fusion proteins mitofusion 2 (Mfn2). Image analysis showed that the density and average size of the mitochondrial Drp1 punctate distribution was significantly increased in GD3S-KO neurons. To understand how GD3 regulates the Drp1 protein level, we measured the half-life of Drp1 in control and GD3S-KO neurons and found that the half-life of Drp1 in control cells was about 15 hours. However, it was dramatically increased in GD3S-KO neurons, with no detectable reduction in 20 hours. These findings suggest that GD3 may participate in regulating the clearance of mitochondrial Drp1 complexes. These findings suggest that the loss of GD3 resulted in mitochondrial fragmentation by increased Drp1-induced mitochondrial fissions in newborn neurons. (Fig. 5) (Tang et al., 2020b).

Fig. 5.

GD3 regulates mitochondrial Drp1 complex turnover. GD3 was found to be associated with mitochondrial fission protein-Drp1. Without GD3, Drp1 levels are increased and aberrant mitochondrial fragmentation is augmented in newborn neurons (lower cartoon). GD3 seems to regulate the clearance of mitochondrial Drp1 complexes to maintain healthy mitochondria.

6. GD3 amplifies SOX2 expression and GM1 promotes DCX expression

Significantly reduced cellularity is observed in the postnatal SVZ and DG in GD3S-KO mice (Wang et al., 2014). The absolute numbers of bromodeoxyuridine (BrdU)+ and SOX2+ cells were reduced in both the SVZ and DG of GD3S-KO mice compared with age-matched WT animals. There was no significant difference in the percentage of BrdU+ versus DAPI+ cells and SOX2+ cells versus DAPI+ cells between different groups. This observation suggested that the reduction of the number of BrdU+ and SOX2+ cells was due to the reduction of the whole NSC pool. To investigate the functional roles of GD3 in postnatal brains, GD3 was intracerebroventricularly (icv) infused into adult (6-month-old) GD3S-KO mice employing a mini-pump for 7 days. In the SVZ of GD3S-KO mice, BrdU positive newly generated and SOX2 positive self-renewal or multipotent cells were less than that in the WT control (Fig. 6). On the other hand, GD3 treatment increased SOX2 positive self-renewal or multipotent cells in the SVZ of GD3S-KO mice. In both neurogenic regions, SVZ and DG of GD3S-KO mice, the number of BrdU+/SOX2+ newly generated multipotent cells were significantly increased following GD3 infusion. These data indicate that infusion of GD3 could restore NSCs in both the SVZ and DG to maintain their properties at early NSC stages. Interestingly, GD3S-KO mice had more doublecortin (DCX)+ and BrdU+ cells than WT controls and GD3 injection restored normal levels in the SVZ and DG. This data suggests that GD3 maintains the multipotent state of NSCs (SOX2+/BrdU+) and controls their differentiation. In this way, GD3 plays a crucial role in the long-term maintenance of NSC populations in the DG of hippocampus and SVZ of postnatal mouse brains (Itokazu et al., 2019; Itokazu et al., 2018; Wang et al., 2014). Moreover, the impaired neurogenesis in the adult GD3S-KO mice led to depression-like behaviors. Our results provide direct evidence linking ganglioside deficiency to behavioral deficits, and support a crucial role of gangliosides in the long-term maintenance of adult neurogenesis (Itokazu et al., 2018; Wang et al., 2014).

Fig. 6.

GD3 promotes SOX2 expression to maintain NSC stages and suppress further differentiation. GD3S-KO mice showed thinner SVZ and DG with reduced cellularity. GD3S-KO mice showed significantly decreased percentage of SOX2 and BrdU double labeled cells versus DAPI+ cells and an increased percentage of DCX and BrdU double labeled cells. On the contrary, intracerebroventricularly (icv) infused GD3 significantly increased the percentage of SOX2 and BrdU double labeled cells versus DAPI+ cells and decreased percentage of DCX and BrdU double labeled cells versus DAPI+ cells compared to GD3S-KO mice. The data in (A) were quantified cell staining photomicrographs of (B). Green, BrdU; blue, nuclear DAPI; and red, SOX2 or DCX. SOX2 maintains stemness such as multipotency and self-renewal. DCX is expressed in cells committed immature neuron stages, including neuronal precursor cells. GD3 infusion can sustain the NSC pool, and prevent them from differentiating in postnatal brains.

The loss of NSCs is known to occur during normal aging, and it has been hypothesized that an accelerated loss of the NSC pool is one of the mechanisms for transition from normal aging to neurodegenerative diseases, such as Alzheimer’s disease (AD). Therefore, sustaining endogenous neurogenesis has been suggested as an important target for treatment and prevention of AD. The 5XFAD transgenic mice with two point mutations in presenilin1 (M146L & L286V) and the Florida (I716V), London (V717I), and Swedish (KM670/671NL) mutations in the amyloid precursor protein have severe pathological phenotypes. The 5XFAD mice are utilized as a preclinical AD mouse model extensively and this model showed a significantly decreased percentage of BrdU and SOX2 double labeled cells versus DAPI+ cells compared to WT mice. To examine the physiological roles of GD3 on postnatal neurogenesis in the DG of AD model mice, GD3 was administered into the 5XFAD mouse brains. GD3 infusion augmented self-renewal and NSCs expressing the multipotent marker, SOX2, in the DG (Itokazu et al., 2019). These data suggest that icv infusion of GD3 could be an effective means to maintain neurogenesis in brains of the 5XFAD mouse AD model.

During neuronal differentiation, the concentration of GD3, which is the predominant ganglioside in NSCs, is rapidly decreased. Concomitantly, the levels of “brain-type” gangliosides such as GM1, GD1a, GD1b and GT1b continuously increase in young animals, reaching a plateau during adulthood. These pattern changes follow closely with the up-regulation of GM2/GD2S expression (Ngamukote et al., 2007). The dramatic changes in the expression profile of gangliosides clearly reflect the biological need for GalNAc-containing ganglio-series gangliosides at particular stages of brain development and neuronal differentiation. Throughout neuronal development, GM1-expressing cells are considered as neuronal progenitor cells and neurons. To understand the functional roles of GM1 on postnatal neurogenesis in the DG of the AD model mouse, GM1 was administered into the 5XFAD mouse brains. GM1 increases BrdU+/DCX+ newly generated immature neuronal cells in 5XFAD mouse brains (Itokazu et al., 2019). As expected, the combinatorial infusion (GD3 and GM1) had a synergistic effect. These results demonstrated that icv infusion of gangliosides GD3 and GM1 simultaneously could enhance neurogenesis in adult mouse brain. For promoting adult endogenous neurogenesis, GD3 restored NSC pools while GM1 enhanced neuronal differentiation by cells in the DG of brains from AD model mice. The combinatorial use of gangliosides (GD3 and GM1) to promote endogenous neurogenesis for the treatment of neurodegenerative diseases could be a new intervention.

7. GM1 binds to neuronal gene promoter regions

In an initial study, we investigated the epigenetic regulation of two key GTs, GM2S and GD3S, in embryonic, postnatal, and adult mouse brains. Interestingly, the temporal expression patterns of GM2S and GD3S mRNAs are correlated with histone H3 and H4 acetylation (AcH3/AcH4) of the gene’s 5’ flanking region in chromatin (Suzuki et al., 2011). These observations suggest that the 5’ region of the GM2S and GD3S genes could be targets for epigenetic regulation by histone modifications. Further, we have demonstrated that acetylation of histones H3 and H4 on the GM2S gene promoter leads to recruitment of trans-activation factors Sp1 and AP-2. When the cellular histone deacetylase activity was globally inhibited by valproic acid (VPA), more GM2S or GD3S mRNA was detected, which could be triggered due to a loading boost of the transcription factors AP-2 and Sp1 on the promoter region. Individually knocking down Histone deacetylases 1 (HDAC1) and HDAC2 gene expression increased the levels of AcH3 and AcH4 on the GM2S gene (Tsai and Yu, 2014). Intriguingly, when both HDAC1 and HDAC2 were knocked down, the expression of GM2S mRNA was up-regulated and AP-2- or Sp1-loading was significantly increased, reflecting the elevated level of histone acetylation on the GM2S gene. However, this was not the case for the GD3S gene. Our results indicated that transcription of GM2S and GD3S could be regulated by different HDAC isoforms, since double-knockdown by si-HDAC1 and si-HDAC2 led to GM2S gene trans-activation, but not GD3S (Tsai and Yu, 2014). In addition, NPCs cultured with GM1 supplementation exhibited a significantly enhanced neurogenic effect (Tsai and Yu, 2014). The GM1, but not GD3, enhanced GM2S expression of mRNA, while the mRNA level of GD3S remained unchanged. The presence of ectopic GM1 resulted in enrichment of the acetylated histones on the gene loci of GM2S, but not GD3S, which was accompanied by the recruitment of the transcription factors AP-2 and Sp1 within the gene promoter region. This observation suggests the possibility that GM1 generates a positive feedback loop to promote neuronal differentiation and to produce more brain-type gangliosides, such as GM1, GD1a, GD1b and GT1b (Tsai and Yu, 2014).

To further study the significance of nuclear GM1, Neuro 2a cells were treated with photoactivatable and clickable (pac) GM1 (pacGM1). PacGM1 in isolated nuclei was visualized using click chemistry-mediated tagging with fluorophores. Figure 7A shows that exogenous pacGM1 is indeed delivered into the nucleus. GM1 is co-localized with Lamin B1 (a protein of the nuclear lamina) or Nucleoporin (a protein of the nuclear pore complex) (Tsai et al., 2016) on the nuclear periphery of neurons induced from NPCs. Chromatin immuno-precipitation (ChIP) assay showed that the promoter regions of the GM2S and NeuroD1 genes are associated with GM1 (Fig. 7B). In situ hybridization assays revealed that GM1 and the promoter region of the GM2S gene are in close proximity in the nucleus of a neuron. Recently, Proximity Ligation Assays (PLAs) have been developed to detect the formation of lipid-protein interactions by immunohistochemistry (Itokazu et al., 2021; Kong et al., 2018). Using this technology, we isolated nuclei from WT mice and performed PLAs to detect GM1 and acetylated histone complex. Each PLA probe contains a unique short DNA strand attached to it. If the PLA probes are in close proximity (<40 nm), the DNA strands interact and generate circle-forming DNA used for enzymatic ligation. The ligated DNA is amplified via rolling circle amplification using a polymerase. Several-hundredfold replication of the DNA circle labels complementary oligonucleotide probes that yield a high intensity of fluorescence (red). The PLA signals in Figure 7C demonstrate that GM1 is indeed co-localized with acetylated histone H3, i.e., GM1 is localized on active chromatin in the nucleus (1.94 ± 0.297 PLA signals in nucleus). The result clearly indicates that GM1 is localized in the nucleus where it can interact with transcriptionally active histones (Itokazu et al., 2021; Tsai et al., 2016).

Fig. 7.

Nuclear GM1 localizes the nuclear periphery and accumulates on the activated GM2S and NeuroD1 genes in neuronal cells. (A) A photoactivatable diazirine ring for the UV-light-induced covalent linkage of photoactivatable GM1 to proteins in close proximity. Photoactivatable and clickable (pac) GM1 (pacGM1) was tagged with TAMRA (Carboxytetramethylrhodamine) on isolated nuclei of Neuro 2a cells. pacGM1 proves that exogenous GM1 (red) can be delivered to the inside of the nucleus. Nuclei were co-stained with Lamin B1 (LB1; green). (B) Chromatin immuno-precipitation (ChIP) assay shows that GM1 interacts with GM2S gene and NeuroD1 promoter regions, but not the GD3S gene in the differentiated neurons from NSCs. The amount of specific DNA fragments (GM2S +0, GD3S +0 or NeuroD1 +0) co-precipitated with GM1 was analyzed by quantitative real-time PCR. The data indicated the relative GM1 binding ability in neurons. (C) Proximity Ligation Assay (PLA) using anti-GM1 (mouse antibody, MINUS) and acetylated histone H3 anti (rabbit antibody, PLUS) in isolated nuclei from adult mouse brain.

8. Nuclear GM1 promotes neuronal gene expression

GM1 has been reported to have an important role in both neuronal differentiation and neuronal function. Importantly, aged GM2S-KO mice (lacking GM1) present with movement dysfunction and have virtually all the neuropathologies of PD (Ledeen and Wu, 2018; Wu et al., 2012; Wu et al., 2020). We determined whether GM1 regulates TH gene expression and found that qPCR analyses of the mRNA level for TH expression were substantially decreased in the substantia nigra pars compacta of GM2S-KO mice. Interestingly, intranasal administration of GM1 for 28 days restored normal TH expression (Itokazu et al., 2021). These data suggested that GM1 is an important regulatory factor in modulating TH gene expression. Next, we analyzed dopaminergic neuron-specific gene expression utilizing Neuro 2a cells after treatment with GM1 or GD3. While TH expression was not detected in untreated cells, it increased dramatically in cells treated with GM1. ChIP assay showed that ectopic GM1 significantly induced epigenetic activation of the TH gene, including augmentation of acetylated histone H3. Moreover, GM1 recruited the dopaminergic neuron-associated transcription factor, Nurr1, to the TH promoter region. This result demonstrates that GM1 promotes the interaction of Nurr1 with the TH gene promoter for activating its gene expression. In addition, GM1 also recruited paired-like homeodomain transcription factor 3 (Pitx3), a critical transcription factor for the survival of midbrain dopaminergic neurons (Itokazu et al., 2021). Since transcriptional activity of Nurr1 could be stimulated by GM1, it is possible that nuclear GM1 could modulate nuclear membrane and chromatin structure to enhance gene expression for augmenting dopaminergic neurons. Fundamental cellular processes are governed by changes in chromatin architecture that regulate neuronal gene expression during differentiation and development. Interestingly, our investigations suggest that nuclear gangliosides can modulate epigenetic gene expression for neuronal differentiation and neuronal functions. Ganglioside expression profiles are known to be closely associated with pathogenic mechanisms of neurodegenerative diseases of the central nervous system. Regulating gene expression by nuclear gangliosides is a novel mechanism to control cellular activity to rescue or protect neurons in neurodegenerative diseases.

9. Other gangliosides for NSC specification

GD3 regulates NSC activities, and GM1s control neuronal differentiation and neuronal functions. Other gangliosides have also been reported to be involved in NSC fate determination. A2B5 antigens, including the c-series gangliosides (Fig. 1), are well known markers for progenitor cells in the nervous system and are the first GSL antigens expressed in glial lineage cells (Eisenbarth et al., 1979; Kasai and Yu, 1983; Raff et al., 1983; Saito et al., 2001). A2B5 is a monoclonal antibody originally developed using chicken embryonic retina cells as the immunogen (Eisenbarth et al., 1979), and those antigens have been established as the c-series gangliosides, including GQ1c, GT1c, and GT3 (Kasai and Yu, 1983; Saito et al., 2001). A2B5 antibody recognizes the Neu5Acα2-8-Neu5Acα2-8Neu5Acα-or α2,8-trisialosyl (triSia) structure (Inoko et al., 2010). c-Series gangliosides are phylogenetically conserved and developmentally regulated. The significant expression of c-series gangliosides is in the brain of lower vertebrates, such as fish, and in the brain of mammalian embryos, but not adult brains (Ando and Yu, 1979; Freischutz et al., 1994, 1995; Rosner et al., 1988; Yu and Ando, 1980). c-Series gangliosides comprise up to 70% of the total gangliosides in adult zebrafish brain (Viljetic et al., 2012). Therefore, the damaged zebrafish brains that largely contain A2B5⁺ cells, have high regeneration capacity (Kishimoto et al., 2012). GT3-synthase (GT3S or ST-III), an enzyme for the synthesis for c-series gangliosides (A2B5 antigens), is highly expressed in proliferating NSCs of mammals (Itokazu and Yu, 2014; Koon et al., 2015). A population of GFAP-expressing cells is considered to be NSCs as well as astrocytes. A2B5+/GFAP+ cells have a lipid composition distinct from mature astrocytes as it is more analogous to stem or progenitor cells (Itokazu et al., 2016a). During maturation of mammalian brain, the concentration of c-series gangliosides decreases drastically, and this decrease in the synthesis of c-series gangliosides is compensated for by a pathway shift in favor of the accretion of a- and b-series gangliosides (Itokazu et al., 2018; Ngamukote et al., 2007). c-Series gangliosides may modulate the immature stage of stem or progenitor cells, albeit further research is needed to address functional interactions of c-series gangliosides and stem cell or progenitor activities.

Chol-1α gangliosides (GT1aα and GQ1bα, Fig. 1) are minor species in the brain and serve as unique markers of cholinergic neurons (Ando et al., 1992; Hirabayashi et al., 1992). Chol-1α ganglioside expression is developmentally regulated and their concentrations increase with aging in the rat brain (Derrington and Borroni, 1990). It has been reported that the treatment with anti-Chol-1α monoclonal antibody inhibits the release of acetylcholine from synaptosomes, and remarkably suppressed memory and learning abilities (Ando et al., 2004). Conversely, addition of Chol-1α gangliosides isolated from synaptosomes induced high affinity choline uptake into synaptosomes and enhanced synthesis of acetylcholine. Accordingly, Chol-1α gangliosides may contribute to maintaining cognitive functions such as memory and learning. Furthermore, Chol-1α gangliosides appeared to alleviate the decreased synaptic functions of aged brains (Ando, 2014; Ando et al., 1998). Chol-1α gangliosides were found to be expressed in NSC, in vitro (Ngamukote et al., 2007). In total, Chol-1α antigens may play an important role in cholinergic synaptic transmission and participate in cognitive function, although the detailed mechanisms need to be addressed.

GM3S is a key enzyme involved in the biosynthesis of all majour gangliosides (Fig. 1). GM3S deficiency causes an absence of GM3 and all downstream gangliosides. We found that GM3S-KO mice were significantly more susceptible (270 % higher in seizure score) to seizures than WT mice. In the hippocampal DG, loss of GM3 aggravates seizure-induced aberrant neurogenesis. An increased number of BrdU+/DCX+ immature neurons migrated from the DG to the hilus, and newborn cells were mislocalized in GD3S-KO brains. (Tang et al., 2020a). This result indicates that GM3 and its downstream gangliosides are important regulators of epilepsy and play an important role in placing adult newborn neurons at the right position.

10. Future Studies

The simple ganglioside, GM3, is synthesized by attachment of sialic acid to LacCer (Fig. 1). This reaction, catalyzed by GM3S, is followed by enzymatic reactions that split ganglioside biosynthesis into two distinct pathways: to GD3 or to GM2/GM1 (Fig. 1). Addition of a second sialic acid residue to GM3, a reaction catalyzed by GD3S, results in synthesis of GD3. However, if GalNAc is the next sugar residue added to GM3, a reaction catalyzed by GM2S, ganglioside biosynthesis will exclusively switch to GM1 subsequent to formation of GM2. The switch to GD3 or GM2 (then GM1) is critical for NSCs or neuronal differentiation, respectively. GD3 is a robust ganglioside in NSCs, and loss of GD3 impairs stemness of NSCs. During neural differentiation, however, GD3 is decreased and complex GalNAc containing “postnatal brain type” gangliosides are increased, implying a switch from a decrease in glial cells and an increase in neuronal cells. We refer to this as the ganglioside “pathway switch” (Fig. 9).

Fig. 9.

Ganglioside “pathway switch” from GD3 to complex gangliosides (including GM1) by glycosyltransferases (GTs), has functional roles in determining neural cell fate.

We are currently analyzing several regulatory mechanisms of GT activities on NSC fate determination. Results from the PLA experiment, shown in Fig. 10, demonstrated that GM3S and GM2S are co-localized in differentiating neuronal cells, but not in undifferentiated NSCs. This exciting observation supports the hypothesis that GM3S and GM2S are able to form a protein complex that drives expression of the GTs needed to convert GM3 to more complex gangliosides, such as GM1.

Fig. 10.

Enzyme complex formation of GTs regulates ganglioside expression and neuronal differentiation and functions. Co-expression of GM3S and GM2S in differentiating cells. NSCs were analyzed by Proximity Ligation Assay (PLA) using antibodies against endogenous GM3S and GM2S. After induction of neural differentiation (by retinoic acid), the PLA signal of GM3S/GM2S dramatically increased. It is now co-localized, in differentiating cells allowing for enzyme complex formation and “pathway switch” of the gangliosides from GD3 synthesis to generate more GM1.

Despite accumulating evidence obtained using GT-KO mice, the specific roles of gangliosides in determining the stage and cell-lineage determination of NSCs remain unclear. Furthermore, global ganglioside-KO mice, such as the GD3S-KO mice, cannot investigate if gangliosides regulate adult/postnatal neurogenesis, since their gangliosides are eliminated before birth. To resolve this issue, detailed research on ganglioside conditional KO mice is urgently required. Studies with GD3S/GM2S conditional KO mice are significant and will reveal more about the roles of gangliosides, including those in cell-, stage-, and disease-specific biological functions. We are investigating the molecular mechanisms of ganglioside-modulating receptor activities, including direct interaction of GD3 and growth factor receptors. We are studying the functional links between postnatal neurogenesis regulated by gangliosides, such as GD3, and the behavioral abnormalities of GD3S-KO mice. Further, specific complex formations of GT and gangliosides are predicted to induce GM2S activation leading to synthesis of more GalNAc-containing gangliosides such as GM2 and GM1, and reduced GD3. Ganglioside compositions are partially epigenetically regulated during development and differentiation. The novel epigenetic regulatory mechanisms for GTs will contribute to a better understanding of the “pathway switch” observed during differentiation. Our published data support our hypothesis that up-regulation of complex ganglioside GM1 induces epigenetic maturation of neurons.

Although recent studies of gangliosides have shed light on their roles in modulating signaling pathways during cellular differentiation and reprograming, mice deficient in some of these molecules show only subtle phenotypic abnormalities compared with the WT animals in early development. Clearly, the biological functions of one glycoconjugate can be substituted by another, albeit with less efficiency. Nonetheless, aberrant ganglioside expression becomes progressively more serious in the adult stage and pathogenic conditions. The “biological redundancy” can be considered for more pivotal roles of these molecules. Ganglioside expression profiles are connected not only with NSC fate determination but also with respective pathogenic mechanisms of neurodegenerative diseases. Epigenetic and post-translational regulation of cell surface and intracellular ganglioside microdomains will provide additional clues underlying the pathogenic mechanisms, which may be useful in developing novel strategies for disease treatment and neuronal regeneration. Future studies on ganglioside microdomains will prove fruitful in this regard.

Fig. 8.

GM1 induces epigenetic activation of the TH or GM2S gene via recruitment of transcription factors on promoter regions. GM1 facilitates binding of acetylated histones (AcH) and transcription factors on promoters to increase their expression via opening the chromatin. The nuclear GM1-lipid domains may serve as a docking site at the nuclear periphery for specific active chromatins for neuronal differentiation and for maintaining neuronal functions.

Abbreviations

AP-2

Activating protein 2

BrdU

Bromodeoxyuridine

Cer

Ceramide

CNS

Central nervous system

CSF

Cerebrospinal fluid

DCX

Doublecortin

Drp1

Dynamin related protein-1

EGFR

Epidermal growth factor receptor

GalCer

Galactosylceramide

GalNAcT

N-acetylgalactosaminyltransferase

GD3

Disialoganglioside 3

GD3S

GD3 synthase

GFAP

Glial fibrillary acidic protein

GlcCer

Glucosylceramide

GM1

Monosialoganglioside 1

GM2S

GM2 synthase

GM3S

GM3 synthase

GSL

Glycosphingolipid

GT

Glycosyltransferase

LacCer

Lactosylceramide

MS

Mass spectrometry

NEC

Neuroepithelial cell

Nurr1

Nuclear receptor related 1, also known as NR4A2

NPC

Neural progenitor cell

NSC

Neural stem cell

PDGF

Platelet-derived growth factor

PLA

Proximity Ligation Assay

PSA-NCAM

Polysialic acid-neural cell adhesion molecule

RGC

Radial glial cell

SGZ

Subgranular zone

SOX2

SRY (sex determining region Y)-box 2

Sp1

Specificity protein 1

ST

Sialyltransferase

SVZ

Subventricular zone

TH

Tyrosine hydroxylase