Shiga toxin is a potent cytotoxin. Infection with Shiga toxin-producing Escherichia coli (STEC) causes not only bloody diarrhea and hemolytic uremic syndrome (HUS) but also neurological disorders. Disorders in central nervous system (CNS) function are the main cause of sudden death in patients with STEC-HUS. CNS complications occur in approximately 30% of STEC-HUS patients. Shiga toxin has been demonstrated to affect the CNS through globotriaosylceramide (Gb3) localized in neurons. Shiga toxin binds to Galα(1,4)Galβ (galabiose), which is located terminally in the oligosaccharide chain of Gb3 and galabiosylceramide (Gb2). Gb2 and its structural isomer, lactosylceramide (LacCer), are ceramide dihexosides (CDH). Gb2 is synthesized by α-1,4-galactosyltransferase (A4GALT), which transfers the galactose residue from UDP-galactose to galactosylceramide (GalCer). While Gb2 has been reported to be in the liver, kidney, and urine, its presence in cerebrospinal fluid (CSF) has not been reported. In this study, we isolated CDH fractions from CSF of patients with idiopathic normal pressure hydrocephalus. Purified CDH fractions showed positive reaction with Shiga toxin. The isolated CDH fractions were analyzed by hydrophilic interaction chromatography/mass spectrometry (HILIC/MS). HILIC columns, which are widely used for separating isomeric glycolipids, were effective for separating LacCer and Gb2. HILIC/MS revealed the presence of Gb2 and LacCer in the fractions. Gb2 was also detected in CSF from neurologically normal control subjects. HILIC/MS also revealed the presence of Gb2 having hydroxy fatty acids (HFA) as their N-acyl chains in the isolated CDH fractions, suggesting that Gb2 is synthesized from HFA-containing GalCer. In the CNS, GalCer is synthesized in oligodendrocytes, and more than half of them contains HFA. GalCer and A4GALT have been suggested to localize in pia mater. Pia mater forms choroid plexus, which secretes CSF. Although Gb2 expression in pia mater or choroid plexus has not been reported, Gb2 might be synthesized by A4GALT from GalCer in choroid plexus and secreted into CSF. This is the first report of the existence of Gb2 in human CSF. Although it is not yet clear whether Gb2 is localized in the CNS, it will be important to determine whether CNS complications in patients with STEC-HUS is mediated also by Gb2, not only by Gb3.


Hisako AKIYAMA obtained her Ph.D. in Cell Biology in March 2011 from Ochanomizu University in Japan. From April 2011 to March 2013, she worked as a research fellow in Ochanomizu University and she discovered a new function of acid β-glucosidase (GBA1) as a transglucosidase to form glucosylated cholesterol (GlcChol) in vitro. Impaired GBA1 function is associated with Gaucher disease and Parkinson’s disease (PD). In 2013, she received a research fellowship from RIKEN and moved to RIKEN, Brain Science Institute as the Special Postdoctoral Researcher under the supervision of Dr. Yoshio Hirabayashi. During her postdoc, she discovered the existence of GlcChol in the brain. Although GlcChol was thought to be the sole sterylglucoside in vertebrate, GlcChol was found to form a group of glucosylated sterols including a plant type glucosylated sitosterol and a bacterial type galactosylated cholesterol (GalChol) in vertebrate brain. In 2016, she was appointed as a research scientist in RIKEN, Center for Brain Science and started a project to identify enzyme responsible for metabolizing the newly found-brain glycosylated sterols in vivo. She established a method to quantify the brain glycosylated sterols by liquid chromatography/mass spectrometry (LC/MS) and found that GBA1 and neutral β-glucosidase GBA2 regulate formation and degradation of the brain glycosylated sterols. Impaired GBA2 function is associated with cerebellar ataxia and spastic paraplegia. In 2018, she received a research grant “Fostering Joint International Research” from Japan Society for the Promotion of Science and she visited at Leiden Institute of Chemistry in Leiden University as a visiting researcher from August 2018 to February 2019. During that period, she discovered that GBA2 is responsible for GalChol formation. After returning from Leiden University, she started a new project aiming to develop an analytical platform using LC/MS to understand biological functions of brain glycolipids including the glycosylated sterols. During the development, she discovered that galabiosylceramide is present in human cerebrospinal fluid and that brain glycosylated sterols levels are changed in PD model animals. Her project on brain glycolipids offers a potential for predictive medicine as a source of novel diagnostic biomarkers as well as drug targets for therapies of brain diseases such as PD.