Functional impairment of the nervous system with glycolipid deficiencies

Abstract

Patients with nervous system disorders suffer from impaired cognitive, sensory and motor functions that greatly inconvenience their daily life and usually burdens their family and society. It is difficult to achieve functional recovery for the damaged central nervous system (CNS) because of its limited ability to regenerate. Glycosphingolipids (GSLs) are abundant in the CNS and are known to play essential roles in cell-cell recognition, adhesion, signal transduction, and cellular migration, that are crucial in all phases of neurogenesis. Despite intense investigation of CNS regeneration, the roles of GSLs in neural regeneration remain unclear. Here we focus on the respective potentials of glycolipids to promote regeneration and repair of the CNS. Mice lacking glucosylceramide, lactosylceramide or gangliosides show lethal phenotypes. More importantly, patients with ganglioside deficiencies exhibit severe clinical phenotypes. Further, neurodegenerative diseases and mental health disorders are associated with altered GSL expression. Accumulating studies demonstrate that GSLs not only delimit physical regions but also play central roles in the maintenance of the biological functions of neurons and glia. We anticipate that the ability of GSLs to modulate behavior of a variety of molecules will enable them to ameliorate biochemical and neurobiological defects in patients. The use of GSLs to treat such defects in the human CNS will be a paradigm-shift in approach since GSL-replacement therapy has not yet been achieved in this manner clinically.

1. Introduction

In the nervous system of vertebrates, lipids are the most abundant organic compounds, and a variety of lipids control the biophysical nature of lipid membranes. Cells and most subcellular organelles are surrounded by biological lipid membranes that define the individual cellular shape and help maintain cellular organization. It was hypothesized that distinct lipid microdomains enriched in glycosphingolipids (GSLs) provide functional platforms and modulate protein activities associated with the biological membrane (Hakomori et al., 1998; Simons and Ikonen, 1997). GSL microdomains are considered to be multiple signaling centers, cellular protein sorting machines, and important in membrane trafficking pathways. Biological membranes are highly heterogenous in structure with diverse microdomains which are enriched with gangliosides, a group of GSLs containing sialic acid. Different microdomains regulate protein activities and cellular functions in stage-, cell-, and cellular site-specific manners. Those microdomains regulate growth factor signaling, immune signaling and immune checkpoints, and cell-cell interactions including cell adhesion and migration. A detailed discussion about ganglioside microdomains can be found in Chapter 10 by Itokazu and Yu. GSLs are unique amphipathic molecules containing a hydrophilic carbohydrate portion and a hydrophobic ceramide. Lipid portions are in the membrane and their carbohydrate heads are on surface of the membrane. Glycosylation of ceramide with glucose forms Glucosylceramide (GlcCer) (Fig. 1). Further glycosylations make more complex GSLs, including gangliosides. Ganglioside synthesis pathways (Fig. 1) were initially described by Yu and Ando (Yu and Ando, 1980) following their structural characterization of c-series trisialogangliosides. During neural development, changes in expression patterns of GSLs in the nervous system correlate with neurodevelopmental processes (Yu and Itokazu, 2014). For instance, the globo-series of GSLs are robustly expressed in fertilized eggs. As cell division proceeds, neolacto-series GSLs are expressed, followed by the ganglio-series of GSLs in the developing brain. In early embryonic rodent brains, GM3 and GD3 are the predominant GSLs. Then, b-series gangliosides, such as GD1b, GT1b, and GQ1b, start to increase, followed by increases in a-series gangliosides such as GM1, GD1a, and GT1a, while expression of GM3 and GD3 markedly decreases during development. In the adult brain, GM1, GD1a, GD1b, and GT1b became major GSL components. GD3 synthase (GD3S) activity (GM3 => GD3) decreased from late embryonic to adult brains (Yu et al., 1988). In contrast GM2 synthase (GM2S) activity (GM3 => GM2) increased at mid-embryonic development to adult. These changes indicate that ganglioside “pathway switch” from GD3 to the enzymatically catalyzed synthesis of complex gangliosides (including GM1) correlates with their functional roles in determining neural cell fate.

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 (B4galT5 & 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.

Patients with GM3-synthase (GM3S) mutations have symptomatic epilepsy syndrome with severe neuronal dysfunction (Fragaki et al., 2013; Simpson et al., 2004). Patients have marked increases of the immediate precursor of GM3, lactosylceramide (LacCer), and an increased accumulation of the globosides Gb3 and Gb4, in their plasma and fibroblasts. Mutation of the GM2S gene was found in patients with hereditary spastic paraplegias with additional neurological symptoms (Boukhris et al., 2013; Harlalka et al., 2013). Fibroblasts from affected patients have increases in the levels of GM3, LacCer, and Gb4 when compared with control subjects. Interestingly, the patients have sialylated Gb3 that is not expressed in unaffected individuals. Since patients with defects that affect their ganglioside biosynthetic enzymes exhibit devastating disorders, development of effective therapies is needed to restore ganglioside-dependent neuronal structure and functions.

Because, a majority of patients with neurodegenerative diseases have alterations in their GSL metabolism, those changes may contribute to their accompanying pathogenesis (Ariga, 2014). Concentrations of complex gangliosides such as GM1 are significantly lower in the brains of patients with Alzheimer's disease (AD) than healthy subjects (Ariga, 2017). Most patients with Parkinson’s disease (PD) show significant deficiency of complex gangliosides, including GM1, in the brain (Seyfried et al., 2018; Wu et al., 2012). In Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS), alterations in GSL expression are observed in the central nervous system (CNS), primarily in the form of decreased complex ganglioside levels (Ariga, 2014; Yu et al., 1982). The importance of complex gangliosides in the prevention of neurodegenerative diseases has been shown in animal models. In APP/PSEN1/GD3S-KO mice, increased GM1 (163.8%) may prevent AD development (Bernardo et al., 2009). Importantly, amyloid-beta peptides (Aβs) could not bind to the cells of GD3S-KO mice which have a significantly increased concentration of GM1, and no Aβ-associated neuropathology was found. In contrast,, APP^SL/GM2S-KO mouse brains, which are deficient in GM1, were found to have significantly increased Aβ levels and senile plaques (Oikawa et al., 2009). Furthermore, GM2S-KO mice exhibit impaired movement and have virtually all the neuropathological symptoms of PD (Wu et al., 2011; Wu et al., 2020). In this chapter, we will introduce these deficiency of GSLs in neurological disorders and describe possible therapeutic strategies. Phenotypes causing deficiencies of GSLs in the mouse nervous system are summarized in table 1.

Table 1.

Phenotypes/symptoms of GSL synthase-KO mice

Disrupted		Lost GSLs	Increased GSL	GSL expression	Phenotypes of the nervous system	Reference
Synthase	Gene
GleCerS	Ugcg	major GSLs except GalCer-based GSLs		GalCer, GM4 Sulfatide	Embryonic lethal cKO (nestin-Cre) died at P11-24	Yamashita et al., 1999 Jennemann et al., 2005
LacCerS	B4gallt5 B4galt6	LacCer, gangliosides	GlcCer	GlcCer, GalCer Sulfatide, GM4	Embryonic lethal cKO (nestin-Cre) died by P30	Kumagai et al., 2009 Yoshihara et al., 2018
GalCer	Ugt8	GalCer, GM4 sulfatide,	GlcCer	GlcCer-based GSLs such as gangliosides	Some animals dyed (P8–30), progressive loss of function in the hindlimbs (P60) Died before myelinogenesis (P30), smaller size, whole body tremor, loss of locomotor activity	Coetzee et al., 1996 Bosio et al., 1996
SulfatideS	Gal3st1	sulfatide		except for sulfatide	Hindlimb weakness, tremor, progressive ataxia	Honke et al., 2002
GM3S	St3gal5	a-, b,- c-series gangliosides, GM4	GM1b GD1α Sulfatide	0-seriese GlcCer, LacCer GalCer Sulfatide	Enhanced insulin sensitivity, reduced development of insulin resistance Attention-deficit hyperactivity disorder (ADHD) Enhancement of osteoarthritis with aging Hearing loss, abnormal auditory structure and function Hyperactive, susceptible to seizures, seizure-induced aberrant neurogenesis Autism spectrum disorder, dysregulated inflammation	Yamashita et al., 2003 Niimi et al., 2011 Sasazawa et al., 2014 Yoshikawa et al., 2015 Tang et al., 2020 Strekalova et al., 2021
GD3S	St8si1	b,- c-series gangliosides,	GM1 GD1a GM2 GM3	0-, a-seriese GlcCer, LacCer GalCer Sulfatide GM4	Impaired regenerative capacity of damaged hypoglossal nerves Thermal hyperalgesia and mechanical allodynia Functional impairment of EGF induced neural stem cell (NSC) self-renewal Reduced NSC pools at SVZ & DG, impaired neurogenesis at OB & DG Morphological abnormalities, impaired regenerative capacity of sciatic nerve Lesser bone loss with aging (10 month old) Reduced visual acuity and contrast sensitivity	Okada et al., 2002 Handa et al., 2005 Wang & Yn 2013 Wang et al., 2014 Ribeiro-Reseude et al., 2014 Yo et al., 2019 Abreu et al., 2021
GM2S	B4galnt1	complex gangliosides including GM1	GD3 GM3	GM3, GD3, GT3 GlcCer, LacCer GalCer Sulfatide GM4	Reduction in the neural conduction velocity Sterility & aspermatogenic of male Demyelinatioii, axonal degeneration, and reduced expression of myelin-associated glycoprotein (MAG) Motor deficits, gradual abnormalities with aging Dysregulated Ca(2+) homeostasis in cultured granule neurons Parkinsonian pathology: loss of dopaminergic neurons in the substantia nigra parts compacta Declined GDNF signaling Urinary and bladder dysfunction Disrupted membrane microdoinains and dispersed prion proteins in the brain	Takamiya et al., 1996 Takamiya et al., 1998 Sheikh et al., 1999 Chiavegatto et al., 2000 Wu et al., 2001,2004 Wu et al., 2011,2012 Hadaczek et al., 2015 Gil-Tommee et al., 2019 Kobayashi et al., 2019
GD3S/GM2S(DKO)		all gangliosides other than GM3	GM3	GM3, GlcCer, LacCer GalCer, sulfatide, GM4	50% of KO mice died by 13 weeks of age, a sudden death, lethal seizures by sound, male sterility Peripheral nerve degeneration, skin lesion primarily on the face Smaller brain, memory & learning deterioration, progressively motor & sensory dysfunctions with aging Upregulated inflammatory cytokine genes similar to AD, disrupted microdomain, increased glial numbers	Kawai et al., 2001 Inoue et al., 2002 Tajima et al., 2009 Ohmi et al., 2009, 2011
GM3S/GM2S(DKO)		all gangliosides	LacCer, SM3	LacCer, GalCer-deriva.	Severe neurodegenerative disease leads to death soon after weaning	Yamashita et al., 2005

2. GlcCer synthase-KO mouse

GlcCer synthase (GlcCerS, uridine diphosphate (UDP)-glucose:ceramide glucosyltransferase; Ugcg, GlcT-1, EC2.4.1.80), catalyzes the first glycosylation step in glycosphingolipid synthesis, and was originally cloned by Hirabayashi's group (Ichikawa et al., 1996). GlcCerS-knockout (KO) mice die between embryonic day 6.5 and 9.5 (E6.5-E9.5) (Yamashita et al., 1999). Conditional KO mice having a floxed GlcCerS gene in combination with the Nestin gene promoter-driven Cre transgene was developed (Jennemann et al., 2005). In these mice, gangliosides were deleted at stage E15.5 by Nestin-promoted GlcCerS gene deletion in neuronal cells. Central nervous system specific KO mice of GlcCerS died from postnatal day 11 to 24 (P11-P24). Soon after birth, the conditional GlcCerS-KO mice displayed dysfunction of cerebellum and peripheral nerves. Significant loss of axon branching and diminished dendritic complexity were observed. It was found that myelin sheaths were broadened and focally severely disorganized. Deficiency of GSL in conditional GlcCerS-KO brains caused alterations in expression of numerous mRNAs needed for brain development and neuronal homeostasis. These results demonstrated that importance of GSLs not only on membrane properties but also on regulating gene expression for brain development and neuronal differentiation. Cerebellar Purkinje cell-specific GlcCerS-KO mice were bred by crossing L7 gene promoter-driven Cre transgenic mice with GlcCerS-flanked mice (Watanabe et al., 2010).

This conditional GlcCerS-KO mouse line showed numerous axonal swellings, dystrophic morphology of axons, and subsequent neuronal degeneration. In the swollen axons, accumulations of mitochondria, synaptic vesicle proteins, and microtubule-associated motor proteins, were seen. Thus, axonal transport deficits are caused by GlcCer deficiency. Further, loss of GlcCerS disrupted myelin sheaths by causing formation of doubly myelinated axons, axons enveloped by an additional concentric myelin sheath. The excessive paranodal loops and doubly-myelinated sheaths were shaped preceding Purkinje cell loss. On the other hand, mice having a specific absence of GlcCerS in oligodendrocytes, made by crossing mice carrying a gene encoding the myelin-associated enzyme 2’, 3’ cyclic nucleotide 3’ phosphodiesterase (CNP) in combination with a gene promoter-driven Cre and GlcCerS-flanked mice, revealed no phenotype in the CNS (Saadat et al., 2010). Based on these observations, neuronal GlcCer and derivative GSLs are suggested to be necessary for regulating axonal maintenance and stabilizing myelin sheath formation.

3. LacCer synthase-KO mouse

LacCer is a common precursor and is required for the synthesis of all GSLs of GlcCer derivatives. LacCer is a key intermediate for a majority of GSLs including the ganglioside-, globo-, isoglobo-, lacto-, and neolacto-GSLs. GlcCer is synthesized by GlcCerS by transfer of glucose from UDP-glucose to ceramide, and then LacCer is synthesized by LacCer synthase (LacCerS) catalyzing the transfer of galactose from UDP-galactose to GlcCer. LacCerS is mainly synthesized by beta-1,4-galactosyltransferase 5 (B4galt5, EC 2.4.1.274) during early mouse embryogenesis (Kumagai et al., 2009). The B4galt5-KO mice die by E10.5 and their growth is delayed by one to 1.5 days from E7.5 (Kumagai et al., 2009; Nishie et al., 2010). The embryonic lethality seen in the β4GalT5-KO mice is similar to that seen in the GlcCerS-KO mice. Although LacCerS activities are 38% and 52% in B4Galt5 conditional KO (Nestin-Cre) and in B4galt6 KO mice respectively, LacCerS activity were completely absent in brain of the conditional B4Galt5 and 6 double KO (D-cKO) (Yoshihara et al., 2018). The D-cKO mice revealed growth retardation and motor deficits at 2 weeks and the mice died by the time they were 1-month-old. Axon-oligodendroglial communication was severely impaired in the CNS of the D-cKO mice.

GSLs, including gangliosides, are known to modulate cell proliferation, cell adhesion, cell migration, and cell signal transduction (Todeschini et al., 2007; Yu and Itokazu, 2014), and GSLs clustered at cell surfaces interact with transmembrane proteins such as integrins to facilitate cell-cell and cell-extracellular matrix adhesions. For example, focal adhesion kinase (FAK) is a key cytoplasmic tyrosine kinase and important mediator of intracellular signaling by integrins. Intriguingly, the consequence of deletion of FAK signaling is similar to that seen with GSL-KO mice as FAK-KO mice die at about E8.5-10 (Ilic et al., 1995). GlcCerS-KO and LacCerS-KO mice also die at this stage during embryogenesis. GSLs provide specific microdomains needed for essential functions during both embryonic development as well as postnatal life.

4. GalCer synthase-KO mouse and sulfatide synthase-KO mouse

Galactosylceramide (GalCer) is synthesized by addition of galactose to ceramide with UDP-galactose as the donor in a reaction catalyzed by the enzyme UDP-galactose:ceramide galactosyltransferase (CGT, GalCer synthase; GalCerS, Ugt8, EC2.4.2.62). GalCerS-KO mice did not have GalCer, or either its sialylated derivative, GM4, or sulfated derivative, sulfatide, but did have GlcCer in their myelin (Coetzee et al., 1996). These KO mice showed severe generalized tremor, mild ataxia, and conduction deficits induced by reduced insulative capability of the myelin sheath. GalCerS-KOmice developed age-related progressive hind limb paralysis and extensive myelin vacuolation of the ventral region of the spinal cord. The growth of GalCerS-KO mice is retarded and most of them died before the end of myelinogenesis (P25-P30) (Bosio et al., 1996). After increasing loss of locomotor activity and development of a very conspicuous gait pattern, KO mice develop a whole body tremor. In an open field test GalCerS-KO mice showed minimal activity. Those reports suggest that lack of membrane-stabilizing GalCer and its derivatives altered membrane structure on the molecular level in both the CNS and the peripheral nervous system (PNS). GalCer is the major component of the myelin membrane that is a unique plasma membrane of oligodendrocytes found in the CNS and Schwann cells in PNS and is essential to facilitate saltatory nerve conduction. Oligodendrocyte-specific overexpression of GalCerS induced by proteolipid protein (PLP) promoter, completely restored the multiple phenotypes seen in the GalCerS-KO mice (Zoller et al., 2005). These mice had no change in the total amount of GalCer in their brains supporting the conclusion that specific loss of GalCerS in oligodendrocytes is responsible for the myelin structural and functional deficits seen in mice with a GalCer deficiency.

Sulfatide (sulfogalactosylceramide), a major sulfoglycolipid in the brain, is a major lipid component of the myelin sheath and is synthesized in both oligodendrocytes in the CNS as well as Schwann cells in the PNS. Sulfatide synthase (SulfatideS, 3'-phosphoadenosine-5'-phosphosulfate-cerebroside sulfotransferase, cerebroside sulfotransferase, CST, Gal3st1, EC 2.8.2.11) adds sulfate to GalCer. SulfatideS-KO mice that lack sulfatides, display hindlimb weakness before 6 weeks of age and subsequently developed an apparent tremor and progressive ataxia (Honke et al., 2002). SulfatideS-KO mice had milder phenotypes than those of GalCerS-KO mice, though SulfatideS-KO mice displayed abnormalities in paranodal junctions and males were infertile. During oligodendrocyte development, O4 (sulfatide; HSO3-3Galβ1-1′Cer) and O1 antigens (galactosylceramide; GalCer; Galβ1-1′Cer), have been utilized as specific markers to define immature and mature oligodendrocytes, respectively. The O1 and O4 antigens play important roles as modulators of oligodendrocyte development and function as well as major components of the myelin sheath to facilitate nerve conduction. Interestingly, the number of oligodendrocytes is increased in sulfatideS-KO mice, indicating that the O4 antigen, sulfatide, is a critical molecule for the negative regulation of terminal differentiation of oligodendrocytes (Hirahara et al. 2004).

5. GM3 synthase-KO mouse

In early embryonic CNS, the pattern of ganglioside expression is characterized by the expression of a large amount of simple gangliosides, such as GM3 and GD3. In the later developmental stages, more complex gangliosides prevail, particularly the gangliotetraose gangliosides GM1, GD1a, GD1b, and GT1b, which account for more than 90% of total gangliosides (Yu et al., 2009). GM3S (sialyltransferase I, ST-I, CMP-sialic acid:lactosylceramide alpha2,3-sialyltransferase; St3gal5, EC 2.4.99.9) is a critical enzyme for the synthesis of all complex gangliosides. GM3 is a precursor molecule in the synthesis of a-, b-, and c- series gangliosides. ST-1 also catalyzes the synthesis of GM4 from GalCer. GM3S-KO mice are unable to synthesize GM3 ganglioside, other GM3 derived gangliosides (Yamashita et al., 2003), and GM4 (Chisada et al., 2009). The KO-mice have GM1b and GD1α as major gangliosides in the brain. Hearing ability were absent in GM3S-KO mice and completely lost by P17 of age, showing a deformity in hair cells in the organ of Corti (Yoshikawa et al., 2009). Abnormal distribution and trafficking of stereocilia and kinocilia were observed in cochlear hair cells in GM3S KO mice, suggesting the essential role of GM3 for the proper function of auditory hair cells (Yoshikawa et al., 2015). In the visual system, thinner retina in GM3-KO mouse is observed, although neither histopathological abnormalities in the retina nor abnormal optic nerve functions were found in the KO mice (Hiraoka et al., 2019).

GM3S-KO mice display attention-deficit hyperactivity disorder (ADHD), which includes hyperactivity, reduced anxiety, lower attention, and increased impulsive behaviors (Niimi et al., 2011). Although both male and female GM3S-KO mice showed increased activity and enhanced methylphenidate hydrochloride sensitivity, GM3S-KO male mice had more severe inattention and impulsivity behaviors. GM3S-KO mice have substantial abnormalities in dominant behavior and neutral sociability. GM3S-KO mice also displayed impaired inhibitory learning, anxiety-like and stereotyped behaviors, abnormal social interaction, moderate motor deficits, systemic inflammation, and signs of hypomyelination (Strekalova et al., 2021). These phenotypes in GM3S-KO mice reflect an autism spectrum disorder (ASD)-like syndrome. Molecular analysis showed decreased expression of myelin protein proteolipid protein-1 (Plp1) and abnormal expression of proinflammatory cytokines in the KO-mice. This study suggested that a deficiency in a-, b- and c-series gangliosides in the brain correlates with abnormalities seen in ASD.

To investigate how a deficiency of GM3 is involved in seizure-susceptibility, we induced seizures with chemoconvulsants (including kainate and pilocarpine) in GM3S-KO mice. We found enhanced susceptibility to seizure induced by chemoconvulsants in GM3S-KO mice compared to wild-type (WT) mice. In the hippocampal dentate gyrus (DG), loss of GM3 aggravates seizure-induced aberrant neurogenesis. An increased number of bromodeoxyuridine (BrdU)+/ doublecortin+ (DCX+) immature neurons abnormally migrated from the DG to the hilus, and were mislocalized in GM3S-KO brains (Tang et al., 2020a). Although GM3S-KO mice displayed reduced body weight and hyperactivity, naive animals showed no difference in adult neurogenesis in the dentate gyrus or morphological abnormality in the brain. These data indicate that GM3 and its downstream gangliosides are important regulators of epilepsy and play an important role to place adult newborn neurons at the right position.

GM3S-KO mice display higher insulin sensitivity and low grade inflammatory states (Nagafuku et al., 2015; Yamashita et al., 2003), and were protected from high-fat diet-induced insulin resistance. GM3S gene-deficiency in obese diabetic mice (KK-Ay; GM3S-KO) significantly ameliorated their obese phenotype compared to that seen with KK-Ay mice, although the KO mice did develop obesity when given high fat diets (Inamori et al., 2018). Since the GM3S-KO mice were protected from high-fat diet-induced insulin resistance, GM3 ganglioside might be a negative regulator of insulin signaling and a potential therapeutic target in type 2 diabetes.

GM3S-KO mice have exacerbated inflammatory arthritis in the mouse model of rheumatoid arthritis (RA), a chronic systemic inflammatory disorder (Tsukuda et al., 2012). GM3S-KO mice showed severe pathologic osteoarthritis after interleukin-induced inflammation (Sasazawa et al., 2014). GM3-deficiency leads to the expression of matrix metalloprotease (MMP)-13, disintegrin, metalloprotease with thrombospondin type I motifs (ADAMTS)-5 secretion, and induced chondrocyte apoptosis. The expression of GM3S by chondrocytes suppressed the expression of MMP13 and ADAMTS-5, which resulted in reduced cartilage degradation even after interleukin-stimulation. These studies indicate that gangliosides have a suppressive capacity in the development of inflammation. The discrepancy of inflammatory states in GM3S-KO mice suggests that a variety of microdomains with different gangliosides and GSLs in individual T cells at local environments may contribute to the pathogenesis or resilience of allergic diseases. Since there are no treatments that block the progression of these diseases, functional gangliosides could be an effective therapeutic for the treatment of inflammatory disorders.

6. GD3 synthase-KO mouse

GD3 ganglioside is a major b-series ganglioside expressed in immature vertebrate neuroepithelial cells (Goldman et al., 1984). GD3 was detected in neural tubes early in development using the GD3-specific monoclonal antibody R24 (Rosner et al., 1992) and GD3 is the predominant ganglioside species (>80%) in embryonic and postnatal neural stem cells (NSCs) (Nakatani et al., 2010). Its postnatal expression levels in the CNS rapidly declines, and becomes undetectable during neural development. The expression of GD3 is limited to NSCs that express Nestin and stage-specific embryonic antigen-1 (SSEA-1), with expression levels drastically decreased during differentiation. GD3 expressing cells do not have neuronal linage markers such as microtubule-associated protein 2 (MAP2) and neuron-specific beta-III tubulin (Tuj-1). GD3S (sialyltransferase II, ST-II, alpha-N-acetylneuraminate alpha-2,8-sialyltransferase; St8sia1, EC 2.4.99.8)-KO mice do not express b- and c- series gangliosides (Okada et al., 2002). GD3S-KO mice had impaired regenerative capacity of damaged hypoglossal nerves and their surviving neuron number was significantly reduced to 50% after damage. Apparently, b- and/or c- series gangliosides are critical in the protection and repair of damaged nerves.

Our studies discovered that GD3 regulates the self-renewal of NSCs via epidermal growth factor (EGF) signaling by interacting with EGF receptors (EGFR) in the microdomains of the plasma membrane preventing their degradation (Wang and Yu, 2013). Thus GD3 on the cell-surface of NSCs can facilitate signal transduction in response to extrinsic stimuli. The self-renewal capability is greatly reduced in higher passage numbers of NSCs taken from both embryonic and postnatal GD3S-KO mice. The results support the hypothesis that GD3 has a role in the maintenance of NSCs. Interestingly, the NSC populations and neurogenesis sites of GD3S-KO mice differ little from those of WT mice in two germinal zones, the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) in the DG of the hippocampus during the embryonic and early postnatal stages (Wang et al., 2014).

Both 1- and 6-month-old GD3S-KO mice exhibit a progressive reduction in cell number in the thinner SVZ. The cellularity of the DG showed no significant difference in 1-month-old GD3S-KO mice, however, the cell number in the DG of 6-month-old GD3S-KO mice was significantly reduced compared with their WT littermates. This progressive reduction of cellular pool might be caused by impaired maintenance of the NSC population in the GD3S-KO mouse brain. BrdU-labeled cells in the SVZ were reduced in GD3S-KO mice compared with their WT littermates at 1- and 6-months of age. Moreover, SRY (sex determining region Y)-box 2 (SOX2)-expressing cells were also reduced in the SVZ of GD3S-KO mice compared with WT mice at both 1- and 6-months. These observations indicate that a GD3-deficiency leads to a decline of SOX2-positive NSC cells in mice as young as 1 month. The Nestin and GFAP double-positive radial glia-like cells at both the SVZ and SGZ were also significantly reduced in GD3S-KO mice compared with their WT littermates. These results indicate that GD3 is required to maintain the postnatal NSC pool. Most importantly, the impaired neurogenesis in the adult GD3S-KO mice led to depression-like behaviors. Our research provides direct evidence linking ganglioside deficiency to behavioral deficits, and support a crucial role for gangliosides in the long-term maintenance of adult neurogenesis and NSC activities (Itokazu et al., 2018; Wang et al., 2014).

We also found that depletion of GD3 decreased dendritic arborization and spines of nascent granule neurons in the DG, and compromised integration of newborn DG neurons in hippocampal circuitry in the adult brain (Tang et al., 2020b). The novel object recognition task was performed to test hippocampus-dependent memory of GD3S-KO mice. WT mice spent more time exploring the novel object than the familiar one that was used in the training session, while the GD3S-KO mice spent significantly less time exploring the unfamiliar object, implying inferior recognition memory performance. Barnes maze task, a hippocampal-dependent spatial memory test, was also performed. GD3S-KO mice displayed a significant increase in escape latency to find the hidden chamber, showing a spatial memory defect. We identified dynamin-1-like protein (Drp1) that is recruited to the outer mitochondrial membrane and critical for GTP-dependent mitochondrial fission, essential for maintenance of the mitochondrial network, as a GD3-interacting protein. Observation of dsRed-labeled mitochondria demonstrated that mitochondria were highly fragmented in the DG of GD3S-KO mice compared with WT. Consistent with this observation, we found an increased level of Drp1 and significantly increased levels of activated-Drp1 (phosphorylation of Drp1 at serine 616), as well as excessive mitochondrial fragmentation in the GD3S-KO mice. The reduced number of mitochondria in GD3S-KO neurons might cause the fewer arborization of dendrites and spine formation. These findings demonstrated that GD3 has an important role in the cytoarchitecture of the mature neuron through the regulation of mitochondrial fusion-fission dynamics, which affect hippocampal memory formation.

We reported that GD3 is the major ganglioside in developing and postnatal retina of vertebrates (Seyfried and Yu, 1985; Seyfried et al., 1982). Abreu et al. found that a significant reduction of retinal ganglion cells and optic nerve in GD3S-KO mice (Abreu et al., 2021). Cell number of the outer nuclear layer, where photoreceptors are expressed, was also decreased in GD3S-KO mice. Consistent with these results, electrophysiological function and contrast sensitivity was reduced in GD3S-KO mice. These findings suggested that GD3 plays a key role in the optomotor system by maintaining retinal ganglion cells and photoreceptors.

Under particular conditions, GD3S-KO mice showed both motor and sensory dysfunctions. Axonal thickness and myelination of the sciatic nerve was reduced in GD3S-KO mice which leads to a reduced pain threshold with a hyperalgesic response (Handa et al., 2005; Ribeiro-Resende et al., 2014). Notable motor deficits were also observed in GD3S-KO mice at high speeds of the rotarod motor test. Regenerative ability of GD3-KO sciatic nerve was found to be lower with a decreased axonal growth rate after crush injury. Neurite growth of dorsal root ganglia after lesioning was reduced in GD3S-KO whereas exogenous GD3 administration dramatically restored regeneration by increasing and recruiting β1-integrin to the growing neurites. These results indicate that GD3 and its derivative b- and c-series gangliosides have functional roles during PNS maintenance and regeneration. Meanwhile, in the CNS, GD3S-KO mice show decreased postnatal NSC pools (Wang and Yu, 2013) and impaired postnatal neurogenesis leads to depressive symptoms (Wang et al., 2014). Consequently, GD3 has strong potential to restore both CNS and PNS dysfunctions.

7. GM2 synthase-KO mouse

For synthesis of further complex gangliosides, UDP-N-acetylgalactosamine (GalNAc) is added to GM3 (Fig. 1) by GM2S (UDP-GaINAc:GM3 N-acetylgalactosaminyl-transferase; GalNAcT, beta 1,4-N-acetylgalactosaminyltransferase; B4galnt1, EC 2.4.1.92 ). GM2S is one of the key enzymes needed for synthesis of the major “brain-type” gangliosides, including GM1, GD1a, GD1b, and GT1b. GM2S-KO mice do not express GalNAc-containing gangliosides, and only express GM3 and GD3 as major gangliosides but total amount of brain gangliosides is not different between WT and KO mice (Sheikh et al., 1999; Takamiya et al., 1996). GM2S-KO mice present morphological abnormalities, including axonal degeneration, decreased myelination, and demyelination in the CNS and PNS at 12–16 weeks of age and motor deficits at 8-12 months (Chiavegatto et al., 2000). GM1 is involved in Ca²⁺ homeostasis and nuclear GM1 was proposed to function as a modulator of nuclear Ca²+ during axonogenesis. Cultured cerebellar granule neurons from GM2S-KO mice showed persistent elevation of intracellular Ca²⁺ leading to apoptosis, which was averted by exogenous GM1 or a GM1 membrane permeant analog, LIGA20 (Wu et al., 2004; Wu et al., 2001). GM2S-KO mice exhibit the moderate phenotype of PD including motor disability on aging, gastrointestinal and sympathetic cardiac disorders, and memory dysfunction (Wu et al., 2011; Wu et al., 2012; Wu et al., 2020). The primary and rate-limiting step in the successive biosynthesis of catecholamines including dopamine, is conversion of L-tyrosine into L-3, 4-dihydroxyphenylalanine (L-DOPA) catalyzed by tyrosine hydroxylase (TH). Consistently, expression levels of the rate-determining enzyme, TH in the dopaminergic neurons of the substantia nigra pars compacta are reduced in GM2S-KO mice along with an increase in alpha-synuclein (aSyn) levels.

Expression of TH is promoted by glial cell derived neurotrophic factor (GDNF) signaling. GDNF recruits and activates GDNF receptors, such as GDNF family receptor alpha-1 (GFRα-1) and rearranged during transfection (RET). GM2S-KO mice (i.e. GM1-deficient mice) showed decreased complex formation of GDNF receptors, suggesting that GM1 is necessary for the GFRα-1-RET complex formation on the cell surface that facilitates intracellular functions induced by GDNF signaling. A GM1 analog, LIGA20, restores the activation of GDNF signaling and the expression of TH in GM1-KO mice despite its possible cytotoxicity (Hadaczek et al., 2015). GM2S-KO mice also exhibit PD-like non-motor symptoms such as detrusor muscle underactivity, which results in decreased voiding compared to that of WT littermates. Enlarged bladders with elevated pro nerve growth factor (NGF) which causes an adverse effect on bladder function, and abnormal bladder nerve myelination were also observed in GM2S KO mice (Gil-Tommee et al., 2019). We also found that mRNA expression of TH is significantly down-regulated in brains of GM2S-KO mice, and intranasally administrated GM1 reached the brain and restored TH expression through the recruitment of Nurr1 transcription factor on active chromatin (Itokazu et al., 2021). Those results strongly suggest that PD symptoms due to reduced biosynthesis of dopamine might be restored by administration of GM1.

8. Both GD3S/GM2S-KO mice (GM3 only mice)

GD3S/GM2S double KO mice expressed GM3 as the major ganglioside. In contrast to the single mutant mice, GD3S/GM2S double KO mice displayed a sudden death phenotype and were extremely susceptible to induction of lethal seizures by sound stimulus (Kawai et al., 2001). After weaning, GD3S/GM2S double KO mice had an extremely high mortality rate and 50% of KO mice died by 13 weeks of age. Like GM2S-KO mice, male GD3S/GM2S double KO mice displayed sterility due to a defect in sperm maturation. GD3S/GM2S double KO mice showed enhanced susceptibility to lethal seizure. Induced tonic-clonic seizure caused the death of around 90% of GD3S/GM2S double KO mice, although neither GD3S-KO nor GM2S-KO mice died from induced seizures. Whole brains of GD3S/GM2S double KO mice had about a 20% reduction in weight after about 30 weeks of age and progressively poorer motor function after 6-8 months (Tajima et al., 2009).

9. Both GM3S/GM2S-KO mice (ganglioside null mice)

Both GM2S and GM3S double KO mice are unable to synthesize gangliosides, which are the major GSL class in the nervous system (Yamashita et al., 2005). Those mice have increased amounts of LacCer and sulfated LacCer (SM3) in their brains. At 2 weeks of age, GM3S/GM2S double KO mice developed motor dysfunctions including hind limb weakness, ataxia, and tremors. The double KO mice died soon after weaning at 3 weeks of age, exhibited sudden death from audiogenic seizures, and revealed striking vacuolar pathology with axonal degeneration and perturbed axon–glia connections. In animals that survived to 1 month of age, the brains of GM3S/GM2S double KO mice were smaller than those from WT mice (Furukawa et al., 2014). These studies indicated that deletions of gangliosides are associated with human diseases.

10. Diseases caused by ganglioside alterations

10.1. Diseases caused by congenital mutations of ganglioside metabolism

Autosomal recessive lysosomal storage disorders are caused by dysfunctions of lysosomal hydrolases or small lipid-binding proteins. In gangliosidosis, degradation pathways of ganglioside are blocked, and certain gangliosides accumulate. Both GM1 gangliosidosis and Morquio type B disease (Mucopolysaccharidosis type IVB) are caused by mutations in the β-galactosidase gene (GBL1) encoding lysosomal acid β-galactosidase (E.C.3.2.1.23). Lack of β-galactosidase activity leads to accumulation of β-linked galactose-containing glycoconjugates. GM1 gangliosidosis is a severe neuronopathic disorder with progressive neurodegeneration. Accumulation of GM1 in the brain contributes to neurologic symptoms of GM1 gangliosidosis. Morquio type B disease is caused by different mutations in the GBL1 gene from those of GM1 gangliosidosis and major accumulation of galactose containing keratan sulfate and oligosaccharides has been shown. In addition, accumulation of GM2 (Tay–Sachs Disease, Sandhoff Disease, GM2 Activator Deficiency), GlcCer (Gaucher disease), and other sphingolipids leads to lysosomal storage diseases with central nervous system involvement (Sandhoff et al., 2018).

Gangliosides 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). 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), refractory early onset seizures with failure to thrive and psychomotor delay by a respiratory chain dysfunction and mitochondrial membrane potential perturbation (Fragaki et al., 2013), Salt & Pepper syndrome characterized with severe intellectual disability, epilepsy, scoliosis, choreoathetosis, dysmorphic facial features and altered dermal pigmentation (Boccuto et al., 2014), Rett syndrome-like phenotype (Lee et al., 2016), hearing loss (Yoshikawa et al., 2015), progressive microcephaly, psychomotor stagnation, dyskinetic movements, somatic growth failure (Bowser et al., 2019), mild to severe dystonia with poor visual tracking and quadriparesis, (Wang and Kilbane, 2021), skin dyspigmentation of freckle-like hyperpigmented macules on the extremities (Wang et al., 2013). GM3S dysfunction causes refractory epilepsy and mitochondrial dysfunction (Fragaki et al., 2013). The patients have developmental stagnation, blindness, required feeding tubes (poor feeding, vomiting and failure to thrive), seizure activity (started within the first year of life), generalized tonic-clonic seizures, tonic spasms, episodes of eye deviation, startle from sleep, startle myoclonus. Those seizure controls are difficult, requiring multiple medications and, for some cases vagus nerve stimulators. They are unable to sit unsupported, reach or walk and nonverbal. Eye contact and visual function deteriorate, caused by optic atrophy and cortical impairment. Electroencephalograms (EEG) ultimately showed multifocal epileptiform discharges. Furthermore, severe auditory impairment observed in GM3S-deficient patients suggests ganglioside microdomains maintain the structural and functional integrity of cochlear hair cells (Inokuchi et al., 2017). Deficiency of GM3S in patients have much severe phenotypes as compared to GM3S-KO mice. GM3S/GM2S double KO mice more closely reflect human GM3S deficiency. Gangliosides in nervous system have much more important functions and biological significant in human life than murine models.

An alteration of the GM2S gene was reported in patients of hereditary spastic paraplegias that is characterized as a heterogeneous disorder with progressive spasticity, weakness of the lower limbs, hyperextended knees and gait abnormalities, additional neurological manifestations (Boukhris et al., 2013; Harlalka et al., 2013; Wakil et al., 2014) and axonal Charcot-Marie-Tooth disease, a hereditary motor and sensory neuropathy (Hong et al., 2021). These clinical features of patients are reasonably similar to the abnormal phenotypes detected in GM2S-KO mice. While a GM3S mutation may affect neuronal development, a deficiency in GM2S results in milder phenotypes with slower disease progression.

10.2. Ganglioside deficiency in neurodegenerative diseases

Aging is the principal risk factor for the development and progression of neurodegenerative diseases. Gangliosides undergo dramatic qualitative and quantitative developmental changes that correlate with cellular events such as proliferation, differentiation, and neuronal function. In early embryonic brains, the pattern of gangliosides is characterized by the expression of several simple gangliosides, such as GM3 and GD3. GD3 is the predominant ganglioside species in NSCs (Nakatani et al., 2010), while in later developmental stages, more complex gangliosides prevail, particularly GM1, GD1a, GD1b, and GT1b through increases in GM2S levels. Ganglioside metabolism is closely associated with the pathology of neurodegenerative diseases. For instance, alterations of GD3, GM1 and other ganglioside levels were reported in AD patients and mice (Ariga, 2017; Ariga et al., 2008; Barrier et al., 2007; Svennerholm, 1994). The potential involvement of gangliosides in AD pathogenesis and disease treatment has been reviewed by us (Ariga et al., 1998; Ariga et al., 2008; Ariga et al., 2010; Ariga et al., 2011; Yu et al., 2012). Progressive imbalance of cell membrane lipid composition is a physicochemical property that changes during normal aging, and further disruptions in it are observed in neurodegenerative diseases. Ganglioside synthesis which occurs in sequential steps is controled mainly by glycosyltransferases (GTs; ganglioside synthases) found predominantly in the Golgi apparatus. Golgi fragmentation is observed in Aβ-treated primary neurons and in the hippocampus in a transgenic AD [B6C3Tg(APPswe,Psen1dE9)85Dbo/J] (Joshi et al., 2014) mouse model. It is expected that GT (ganglioside synthase) activities in different regions of the Golgi apparatus are impaired by the disrupted Golgi structure. Ganglioside composition is altered in the brains of AD patients; major brain-type gangliosides, including GM1, are significantly decreased in the hippocampus in addition to several other areas important for cognition, such as frontal and temporal cortices, parietal cortex, basal telencephalon, and frontal white matter (Ariga, 2017; Ariga et al., 2008; Svennerholm, 1994). A progressive alteration in brain gangliosides, e.g., reduced cortical GM1, is exhibited by patients and in multiple transgenic mouse models of AD.

Although the biological significance of adult hippocampal neurogenesis in humans is still under debate, the detection of adult neurogenesis in certain brain regions supports the hypothesis that the adult brain exhibits more plasticity than previously thought, and this affects memory and the pathogenesis of neurodegenerative diseases (Gage, 2021). Emerging evidence from AD patients and mouse models supports an active level of neurogenesis in early/moderate onset of the disease, that then becomes sluggish in brains of patients with late/severe AD. Diminished neurogenesis occurs in normal aging, and it is hypothesized that an accelerated loss of the NSC pool is one mechanism for transition from a healthy to an AD brain. Impaired adult hippocampal neurogenesis occurs before signs of cognitive and memory deficits in AD (Berger et al., 2020; Moreno-Jimenez et al., 2019; Tobin et al., 2019). Therefore, promotion of endogenous neurogenesis has been suggested as an important target for treatment and prevention of AD (Choi et al., 2018; Mu and Gage, 2011). Essential cellular processes in NSC fate-determination and self-renewal are heavily influenced by cell surface glycoconjugates, including gangliosides. To examine the physiological roles of GD3 and GM1 on postnatal neurogenesis in the brain of AD model mice, gangliosides (GD3 or GM1 and combination) were intracerebroventricularly (icv) administrated into the 5XFAD mouse brain (Fig. 2). Our data clearly show that GD3 augments self-renewal and cells expressing the multipotent marker, SOX2, in DG (Itokazu et al., 2019). On the other hand, GM1 increases BrdU+/ DCX+ newly generated immature neurons in 5XFAD mouse brains. As expected, the combinational infusion of GD3 and GM1 had a synergistic effect. The data evidenced that exogenous gangliosides can restore the NSC pool in AD brains in the 5XFAD mouse model. Remarkably, Lars Svennerholm et al. described that icv administration of GM1 to AD patients could stop the continuous deterioration of nerve processes and increased the turnover of transmitter substances, although the molecular mechanisms underlying the positive effects of GM1 in AD brains were not determined (Svennerholm et al., 2002).

Fig. 2.

GD3 and GM1 restore the number of SOX2+ and DCX+ cells in the AD mouse brain. The 5XFAD transgenic mouse is an AD model with two point mutations in presenilin1 (M146L & L286V) and the Florida (I716V), London (V717I), and Swedish (KM670/671NL) mutations in the amyloid precursor protein. 5xFAD mice showed a significantly decreased percentage of both SOX2/BrdU and DCX/ BrdU double labeled cells compared to WT mice. GD3 and GM1 (5 mg/kg bodyweight/day GD3 or GM1) were introduced into the brains of (10-week-old) 5XFAD mice (via icv for 7 days by micro-osmotic pump at a flow rate of 0.5 μL/hr) and then injected with BrdU to assess its neurogenic potential with co-staining of lineage-associated markers. For the combinational experiment, GD3 was first infused for 7 days and then GM1 was infused for 7 days. (n = 3), *p < .05. WT = wild type. GD3 restores the number of SOX2+ cells and GM1 restores DCX+ neuronal cells in the DG of 5xFAD. The combinational infusion (GD3 and GM1) had a synergistic effect. The data demonstrated that GD3 amplifies NSCs and then GM1 promotes neuronal differentiation. WT: levels of wild-type; NSC, neural stem cell; NPC, neuronal progenitor cell.

Accumulations of aSyn is a major pathological hallmark in PD, and gangliosides bind to aSyn with high affinity to stabilize an alpha-helical state and inhibit fibrillation (Bartels et al., 2014; Martinez et al., 2007). Importantly, aSyn has a ganglioside binding domain, which has the most critical residues for binding GM1 (Di Scala et al., 2016). As expected, intranasally administrated GM1 dramatically reduced aSyn levels, not just monomeric but oligomeric aSyn in substantia nigra pars compacta of a PD mouse model, which is known as Hualpha-Syn(A53T) transgenic line G2-3 (referred to as A53T PD mouse) (Fig. 3) (Itokazu et al., 2021). Increasing disproportion of the lipid composition of cell is a physicochemical property altered with normal aging, and advanced disruptions to these processes are observed in neurodegenerative diseases, including in PD and AD brains. It is reported that the ganglioside composition is altered in the brains of PD patients, e.g., major “brain-type” gangliosides, such as GM1, are significantly decreased (Seyfried et al., 2018; Wu et al., 2012). In earlier studies, intraperitoneally injected GM1 was shown to restore TH activity and dopamine content in striatum of rodents after surgical and chemical (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MPTP) damage (Hadjiconstantinou et al., 1986; Toffano et al., 1983). A lot of rodent research on the effectiveness of GM1 therapy followed with positive results.

Fig. 3.

Intranasally administered GM1 reduced aSyn monomer, oligomers, and aggregates in A53T PD mouse brain. aSyn has a ganglioside binding domain. Intranasally infused GM1 (5mg/kg/day for 28 days) removed aSyn in A53T PD mouse brain (8-month-old). (A) aSyn and Aβ have functional ganglioside binding domain at K34-K45 (red), and R5-K16 (red), respectively. (B) Brain extracts from substantia nigra of WT and PD mouse brain were loaded for SDS PAGE. aSyn monomer is shown a band around 15-20k Da. Nasal GM1 infusion significantly reduced monomer, oligomer and total aSyn. Total (C), oligomer (D; 30-260 kDa), and monomer (E) aSyn levels were quantified by imageJ and normalized by actin (data not shown). n=3 mice/group, male.

To date, there is no definitive proven disease-modifying therapy for patients with PD. Bovine-derived GM1 has been extensively studied in many PD patients over many years in Jay Schneider et al.’s long-term NIH-funded research (Schneider, 2021). The safety profile of GM1 trials were impressive with no serious adverse events reported and no clinically significant abnormalities in blood chemistry, hematology measures, or urinalysis results. There were no antigenicity by ganglioside treatment. Although improvement of movement disorders and potential beneficial mechanisms of action of GM1 in PD were observed, use of GM1 from bovines was abandoned due to concern of possible contamination of GM1 supplies with abnormal prion protein from bovine spongiform encephalopathy (BSE)-affected brains. Another problem is the lack of efficient routes of administering GM1 in a manner that permits it to efficiently cross the blood–brain barrier (BBB). Intravenous or intramuscular administration of GM1to patients with stroke or spinal cord injury was found to be safe, no adverse effects, and most importantly the patients showed neurological improvement (Magistretti et al., 2019).

Results of our studies of mouse models demonstrate that intranasally administered GM1 was successfully delivered to various brain tissues including the olfactory bulb, cortex, midbrain, germinal zones, and cerebellum (Itokazu et al., 2021). PD is a neurodegenerative disease characterized by the loss of midbrain dopaminergic neurons with a subsequent decrease in the concentration of striatal dopamine. The expression level of TH was markedly reduced in the substantia nigra and striatum in the brains of A53T PD mice, and intranasally infused ganglioside GM1 dramatically restored it (Fig. 4). This suggests that the chronic dopamine depletion seen in PD-model mice and possibly PD patients might be restored by nasal administration of GM1. GDNF downstream transcriptional factors, Nurr1 and Pitx3, are involved in the expression of the TH needed for survival of substantia nigra dopaminergic neurons (Maxwell et al., 2005; Peng et al., 2011). Nurr1 is also essential for the expression of a GDNF receptor, Ret, suggesting a regulatory loop of GDNF signaling (Volakakis et al., 2015). However, GDNF treatment of PD-model cells, rodents, and patients with PD failed to protect midbrain dopaminergic neurons (Decressac et al., 2011; Kambey et al., 2021; Manfredsson et al., 2020; Mesa-Infante et al., 2022; Nutt et al., 2003). Since the expression of GDNF receptors is regulated by GM1 (Hadaczek et al., 2015), it is likely that GDNF alone (without GM1) does not activate further signaling. In our research, GM1 restored nuclear localization of Nurr1 in brains of A53T PD mice and recruited Nurr1 and PitX3 to the TH promoter to activate gene expression. Further, exogenous GM1 itself induced GM2S expression to augment endogenous GM1 by activating a chromatin-based epigenetic mechanism (Itokazu et al., 2016; Tsai and Yu, 2014). We propose that nuclear ganglioside microdomains modulate gene transcription during disease progression and regeneration of cells in diseased brains.

Fig. 4.

Intranasally administered GM1 restored dopaminergic neurons in the PD mouse brain. Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of dopamine, and it is regularly used as a marker for dopaminergic neurons. Patients with PD exhibit neurodegeneration of dopaminergic neurons in the substantia nigra. We found a markedly reduced expression of TH in the A53T PD mouse brain (8-month-old). Intranasally infused GM1 (5mg/kg/day for 28 days) increased expression of TH in the substantia nigra in brains of A53T PD mice. (Lower images) The striatal projection of dopaminergic neurons (TH+) are vigorously decreased, on the other hand, intranasal infusion of GM1 recovered the TH+ axons in the striatum. The data shows that GM1 (green) and TH (purple) levels were dramatically restored in substantia nigra and striatum by nasal GM1. This suggests chronic dopamine depletion in the striatum of PD-model mice and possibly of PD patients might be restored by intranasally administration of GM1. Green: TH, Purple: GM1, Blue: nuclear DAPI.

Disruption of mitochondrial homeostasis and mitochondrial dysfunction play a vital role in the pathogenesis of several neurodegenerative diseases, and mitochondrial dynamics are critical for hippocampal dependent learning and memory (Simmons et al., 2020; Trinh et al., 2021). Figure 5 shows that a major component of the outer mitochondrial membrane, voltage-dependent anion channel 1 (VDAC1) known to regulate mitochondrial functions, is drastically downregulated in an A53T PD mouse brain. Intranasally administered GD3 or GM1 dramatically restored VDAC1 levels in an A53T PD mouse brain. We have shown that GD3 regulates mitochondrial dynamics by mediating Drp1 (Tang et al., 2020b). We also found that GM1 binds to other mitochondrial proteins to regulate mitochondrial functions (Itokazu et al., unpublished data). To cure neurodegenerative diseases, targeting mitochondrial dysfunction with functional gangliosides is another promising approach for the development of future therapies.

Fig. 5.

Intranasally administered GD3 and GM1 restored expression of VDAC1, a major component of the outer mitochondrial membrane known to regulate mitochondrial functions. Intranasally infused GD3 and/or GM1 (5mg/kg/day for 28 days) increased VDAC1 expression in dopaminergic neurons within the substantia nigra pars compacta (data not show) and cortex of an A53T PD mouse brain (8-month-old).

11. Future Studies

From the foregoing, significance of glycolipid deficiencies in the nervous system can be clearly seen. Mice deficient in some GSLs show only subtle phenotypic abnormalities compared with wild-type animals during early development. Clearly, the biological function of one glycoconjugate can be substituted by another (McGonigal et al., 2019), albeit with less efficiency. Notably, aberrant ganglioside expression becomes incrementally more serious in the adult stage and pathogenic conditions. The “biological redundancy” indicates that GSLs play more pivotal roles than general molecules that lack any biological function overlap in the nervous system. Evidently, patients with ganglioside deficiency have more severe clinical phenotypes than those of ganglioside KO mice, although mice completely deleted in gangliosides do have lethal phenotypes. Many patients with neurological disorders suffer from immense pain, immobility, disease progression, and problems with everyday needs. The lack of satisfying medications for these individuals reflects the lack of knowledge of the diseases which in turn underlies the paucity of effective therapies.

Biological membranes are highly heterogenous in structure and there exist diverse microdomains (also known as lipid rafts), which are enriched with GSLs. GSLs are unique amphipathic molecules that contain a hydrophilic carbohydrate portion and a hydrophobic lipid component. GSLs are a class of lipids highly enriched in the nervous system, with a wide variety and complexity. GSLs appear to be essential for neuronal development and neuronal functions. The significance is that GSLs modulate lipid microdomains to regulate functions of important molecules on plasma, mitochondrial, nuclear, and other biological membranes. Multifunctional gangliosides regulate specific cells in distinct stages via modulating protein and gene activities through the GSL microdomains. Despite cumulative evidence obtained by studying ganglioside-KO mice, the specific roles of gangliosides in the stage-, cell-, and disease-specific significance remain unclear. GD3S/GM2S-double KO mouse (GM3 only mouse) has severe phenotypes as described above. Neuronal expression of a-series gangliosides significantly abrogated the lethal phenotype and partially attenuated the neurodegenerative manifestations. Studies with ganglioside conditional KO mice with other disease models will help clarify cell-, stage-, and disease-specific biological functions of gangliosides.

Overall, ganglioside deficiency contributes to development of neurodegenerative diseases and mental health disorders. Adult neurogenesis is suggested to contribute to a healthy life. An adult human generates 700 new neurons daily in the hippocampus, with a gradual decline during aging (Spalding et al., 2013). Adult neurogenesis declines more in patients with neurodegenerative diseases and mental disorders (Berger et al., 2020; Moreno-Jimenez et al., 2019). Human subjects with higher numbers of DCX+ immature neurons tend to have higher cognitive performance (Moreno-Jimenez et al., 2019; Tobin et al., 2019). Reduced numbers of DCX+ cells were observed in patients with mild cognitive impairment and AD, and an accelerated reduction in later stages of pathology. In early to moderate stages of neurodegenerative diseases, GM1 would be sufficient to maintain neuronal functions. On the other hand, at more severe stages of diseases, first GD3 is needed to amplify NSCs and then GM1 to support neuronal differentiation of NSCs. Both GM1 and GD3 can reduce levels of neurotoxic proteins in the brains of people with neurodegenerative diseases. For depression treatment, GD3 may be needed first to increase the number of NSCs. The use of a combination of GD3 and GM1 represents a powerful means to slow down the progression of disease and restore dysfunctional neurons. Since the population of people with age-related neurodegenerative diseases and mental disorders is increasing, GSL research will benefit millions of patients, their families and society.

Abbreviations

AD

Alzheimer's Disease

BrdU

Bromodeoxyuridine

Cer

Ceramide

CNS

Central nervous system

DG

Dentate gyrus

GalCer

Galactosylceramide

GalNAc

N-acetylgalactosamine

GlcCer

Glucosylceramide

GSL

Glycosphingolipid

LacCer

Lactosylceramide

NSC

Neural stem cell

PD

Parkinson’s disease

PNS

Peripheral nervous system

SGZ

Subgranular zone

ST

Sialyltransferase

SVZ

Subventricular zone