WO2025059648A2 - Non-caloric carbohydrate combinations to improve cognition during aging - Google Patents

Abstract

Non-caloric carbohydrates, i.e., carbohydrates that do not contribute calories to the diet, have emerged as a new avenue in the modulation of brain metabolism and the extracellular matrix in the context of cognitive aging. In this disclosure, non-caloric monosaccharides such as fucose and galactose were tested for their impact on protein glycosylation and brain metabolism as well as social behavior. The invention generally provides monosaccharide supplements and kits thereof as well as use methods to improve brain function in learning, memory, social interactions, group dynamics, and mood.

Description

NON-CALORIC CARBOHYDRATE COMBINATIONS TO IMPROVE COGNITION DURING AGING

GOVERNMENT SUPPORT CLAUSE

This invention was made with government support under R35 NS 116824 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

1. Technical Field

The invention relates to dietary supplements and kits thereof for enhancing brain function. More particularly, the invention relates to monosaccharide dietary supplements for improving brain function for enhancing learning, memory, social interactions, group dynamics, and mood.

2. Description of the Related Art

Glycogen is one of the fundamental biological macromolecules, which is critical for primary carbohydrate storage and energy metabolism. Glycogen is found in high levels in the liver and muscle, where it serves as systemic and local carbohydrate reserves. However, the machinery for glycogen synthesis and degradation is widely expressed throughout the body, and other tissues and organs have been increasingly recognized to critically depend on cellular glycogen (Brewer & Gentry, 2019; Dienel & Carlson, 2019; Obel et al., 2012). Through the lenses of newly developed technologies, spatially unique glycogen distribution in the brain, lung, testis, kidney, and bone marrow has been increasingly studied (Oe et al., 2016; Sun et al., 2019, 2021; Young et al., 2020, 2022).

Interestingly, glycogen displays heterogeneous patterns of distribution within the brain (Oe et al., 2019) and is observed in diverse cell types within the central nervous system (CNS), including neurons, astrocytes, and microglia (Brown & Ransom, 2007; Hertz et al., 2007). This has significance for key aspects of brain biology as well as implications for diseases that affect the brain. Current understanding of brain glycogen includes its pivotal roles in learning and memory, signaling events, neurotransmitter metabolism, and protein glycosylation, suggesting the role of glycogen in neurological disorders.

Glycogen is mainly composed of glucose molecules linked via glycoside bonds with a- 1,4 chains and a- 1,6 branches. This design allows the storage of up to -50000 glucose units. However, brain glycogen has been shown to be a rich source of some monosaccharides used for protein glycosylation (Sun et al., 2021). Protein glycosylation is a highly regulated posttranslational modification that is critical for multiple cellular functions including protein folding, cell-cell interactions, cell adhesion, proliferation, and inflammation (Ng & Freeze, 2018; Schwarz &Aebi, 2011; Sun et al., 2021).

Almost all the secreted and membrane-associated proteins of eukaryotic cells, including neuronal cells, are glycosylated with oligosaccharides. There are two key types of glycosylation: N-linked glycosylation at Asn residues and O-linked glycosylation at Ser or Thr residues.

More recently, a key pathway in brain carbohydrate metabolism that directly connects brain glycogen and N-linked protein glycosylation has been reported (Sun et al., 2021). It is well- established that both glycogen metabolism and N-linked protein glycosylation are crucial for synaptic modeling and memory formation. Perturbations in either pathway delay neuronal outgrowth and initiate neuro-inflammation that leads to cognitive delay, memory loss, and epilepsy (Dienel, 2019; Gibbs et al., 2006; Scott & Panin, 2014). In fact, multiple disorders are either directly caused by or have perturbed glycosylation as a hallmark of the disease, including congenital disorders of glycosylation (CDG), some cancers, Alzheimer's disease, Pompe disease (PD), and Lafora disease (LD) (Conroy et al., 2021; de Souza-Ferreira et al., 2023; Gaunitz et al., 2020; Hawkinson et al., 2021; Ng & Freeze, 2018; Sun et al., 2021; Wens et al., 2014). Furthermore, multiple GSD and CDG patients share many neurological symptoms, including epilepsy and cognitive impairment (Korlimarla et al., 2019; Nitschke et al., 2018; Piedade et al., 2022). While the novel route of hexosamine metabolism in the brain provides a direct metabolic connection between glycogen and N-glycans, key insights remain to be determined. Excitingly, these novel avenues of research have the potential to be therapeutically actionable for key glycogen-driven diseases of the CNS. BRIEF SUMMARY

The brain is an energy-demanding organ relying on a continuous supply of glucose. Brain glycogen promptly responds to changing cerebral energy demands and is degraded for glucose supply (glycogenolysis) in response to low-glucose levels in both astrocytes and neurons (Bastian et al., 2019; Dienel & Rothman, 2019; Guo et al., 2021). In addition, it has been increasingly appreciated in recent years that glycogen has pleiotropic roles that support normal brain function, and that dysregulation of glycogen metabolism contributes to multiple diseases (Bak & Walls, 2018; Dienel, 2019; DiNuzzo & Schousboe, 2019; Obel et al., 2012; Waitt et al., 2017).

Glucose released from glycogen can also be utilized as a substrate for protein glycosylation or provide fructose-6-phosphate for the synthesis of N-acetylglucosamine (GlcNAc), another building block monosaccharide for protein glycosylation. For protein glycosylation, fucose, galactose, mannose as well as sialic acid can be utilized in addition to glucose and GlcNAc. When these monosaccharides are present in extracellular environment, they enter cells by facilitated diffusion through the glucose transporter (GLUT) family.

Historically, the chemical heterogeneity of glycosylation has proven to be a barrier to study. However, emerging methods utilizing mass spectrometry and in situ enzymatic degradation of glycans known as MALDI-MSI provides improved sensitivity and spatial resolution.

In this disclosure, using this technology, protein glycosylation and behavioral changes were studied in mice, and based on the results, dietary supplements comprising monosaccharides are suggested to enhance learning, memory , social interactions, group dynamics, and mood in a subject who wishes to improve its brain function.

In various embodiments, dietary supplements are provided, which comprise or consist of one or more kinds of monosaccharides selected from the group of L-fucose, D-galactose, D- mannose, and N-acetyl D-glucosamine (GlcNAc)

In a specific embodiment, a dietary supplement comprises L-fucose, and the supplement may optionally comprise pharmaceutical excipients for oral administration. In certain embodiments, a dietary supplement comprises L-fucose and another monosaccharide selected from D-galactosc, D-mannosc, and GlcNAc, and the supplement comprises at least 50% L-fucose by weight.

In certain embodiments, a dietary supplement comprises L-fucose and other two monosaccharides selected from D-galactosc, D-mannosc, and GlcNAc, and the supplement comprises at least 50% L-fucose by weight.

In certain embodiments, a dietary supplement comprises L-fucose and D-galactose, D- mannose, and GlcNAc, and the supplement comprises at least 50% L-fucose by weight.

In other embodiments, a dietary supplement comprises one selected from D-galactose, D- mannose, or GlcNAc, and the supplement may optionally comprise pharmaceutical excipients for oral administration.

In some embodiments, the dietary supplement is formulated into solid, semi-solid, or liquid dosage form for oral administration, and the supplement is optionally formulated into multiple units of a dosage form. A unit may comprise a total weight of about 0.5-200 g of a monosaccharide or monosaccharides and optionally pharmaceutical excipients.

In addition, in certain embodiments, the dietary supplements aforementioned are packaged into a kit, and the kit comprises L-fucose supplement and at least one of the L-galactose-, L- mannose-, and/or GlcNAc supplements separately. In a kit, the supplements are all in the same dosage form or at least one of the supplements is in a different dosage form.

In a certain embodiment, a kit comprises a dietary supplement comprising L-fucose and D- galactose, and separately packed D-mannose supplement and/or GlcNAc supplement. In another embodiment, a kit comprises a dietary supplement comprising L-fucose and D-mannose, and separately packed D-galactose supplement and/or GlcNAc supplement. In another embodiment, a kit comprises a dietary supplement comprising L-fucose and GlcNAc, and separately packed D- galactose supplement and/or D-mannose supplement.

In a certain embodiment, a kit comprises a dietary supplement comprising L-fucose, D- galactose and D-mannose, and separately packed GlcNAc supplement. In another embodiment, a kit comprises a dietary supplement comprising L-fucose, D-mannose and GlcNAc, and separately packed D-galactose supplement. In another embodiment, a kit comprises a dietary supplement comprising L-fucosc, L-galactosc, and GlcNAc and separately packed D-mannosc supplement.

In a certain embodiment, a kit comprises a dietary supplement comprising L-fucose, D- galactose, D-mannose, and GlcNAc.

In some embodiments, each supplement is collectively packaged in a container or the individual units of each supplement are wrapped separately and packaged together in a container for each supplement or all together.

Further, a use method of aforementioned supplements and kits is provided for improving brain function to enhance learning, memory, social interactions, group dynamics, and mood in a subject; The method comprises orally administering a subject an effective amount of the monosaccharide supplement(s), and a daily dose is less than 5 g/kg, and optionally less than 2 g/kg. The supplement may be administered once a day, twice a day, or thrice a day every day, every other day, every three days, every four days, every five days, every six days, or once a week, for at least one month or more. When more than one supplement is co-administered, they can be administered at the same time or different times.

In addition, the supplement(s) can be added to food products, including cereal, oatmeal, gruel, jelly pudding, fruit jam, yogurt, ice cream, milk, soy milk, chocolate milk shake, cocoa, coffee, tea, fruit juice, energy drink, and/or to meals, including soup, salad, cooked vegetables, meat, starchy food, and dairy.

It can also be contemplated to use the individual or combination of the monosaccharides disclosed here to enhance or reduce neurotransmitter release from the presynaptic neurons and/or the activity of neurotransmitter receptors on the postsynaptic neurons as well as ion channel conductance in the CNS by parenterally administering an effective amount of the monosaccharide(s) to a subject in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Illustration of the basic concept of the invention.

Figure 2. Experimental design for L-fucose treatment. Figure 3. The effect of L- fucose treatment on mouse physiology. (A) Body weight change. (B) Glucose tolerance test.

Figure 4. The effect of L-fucose treatment on protein glycosylation. (A) Strategy for N- glycosylation and O-glycosylation analysis. (B) O-GlcNAc Western blot. (C) Jacalin Lectin blot.

Figure 5. The effect of L-fucose treatment on metabolism. (A) GC-MS workflow for the analysis of metabolic changes. (B) Heatmap of the top 50 most changed polar metabolites. (C) Principal Component Analysis (PCA) of polar metabolites. (D) Comparison of the levels of fucose, stearic acid, and cholesterol in fucose-treated mice and water-control mice.

Figure 6. The effect of L-fucose treatment on glycogen in the brain. (A) GC-MS of glycogen in the whole brain. (B) Glycogen in the cortex, cerebellum, and hippocampus detected by immunohistochemical (IHC) images.

Figure ?. The effect of L-fucose treatment on behavior. (A) Social memory. (B) social dominance.

Figure 8. Experimental design for galactose treatment.

Figure 9. The effect of galactose treatment on protein glycolysis. (A) Strategy for N-glycosylation and O-glycosylation analysis. (B) Fluorescence intensity analysis of O-GlcNAc Western blot and Jacalin Lectin blot. (C) MALDLMSI analysis of N-glycan in the brains from galactose treated mice and water control mice. (D) Clustering hcatmap analysis of top 10 N-linkcd glycan features of brain slices from water (Wat) and galactose (Gal) treated mice separated by brain region (cerebellum [CB], frontal cortex [FC], and hippocampus [HC]).

Figure 10. The effect of galactose treatment on metabolism. (A) GC-MS workflow for the analysis of metabolic changes. (B) Principal Component Analysis (PCA) of polar metabolites. (C) Heatmap of the top 10 most changed polar metabolites. (D) Comparison of glycogen level in the brain from galactose-treated mice and water-control mice. (E) Leloir pathway for galactose.

Figure 11. The effect of galactose treatment on behavior. (A) Anxiety test. (B) Social dominance test.

Figure 12. Summary of the effects of galactose treatment.

Figure 13. Chemical structures of L-fucose, D-galactose, D-mannose, and N-acetylglucosamine. Figure 14. Oral supplement glucosamine as a major driver of human dementia

DETAILED DESCRIPTION

A. Overview

Protein glycosylation is necessary for proper central nervous system (CNS) function, as it is required for protein folding and cell-to-cell communication. For example, congenital disorders of glycosylation (CDGs) are diseases caused by defects in glycosylation (i.e., addition of carbohydrate motifs on proteins). They are often characterized by neurological symptoms like cognitive decline, epilepsy, and ataxia. In some CDGs, supplementation with glycan-composing monosaccharides has emerged as an intriguing therapeutic strategy, alleviating disease symptoms with minimal adverse effects. However, the impact of such supplementation on brain metabolism and glycosylation in healthy individuals remains unknown.

In this disclosure, the effect of dietary supplementation comprising monosaccharides, in particular glycan-composing monosaccharides, and in more particular L-fucose as well as D- galactose, on the brain function was shown in mice compared with water-treated controls. To this end, using matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) and lectin blots, mouse brain N-glycosylation and O-glycosylation were quantified, respectively. Notably, L-fucose treated mice exhibited increased levels of O-glycosylation and decreased levels of brain glycogen. In addition, analysis of metabolism by gas chromatography-mass spectrometry (GC-MS) revealed significant changes in stearic acid and cholesterol in the brain of L-fucose treated mice. Interestingly, these metabolic alterations were associated with behavioral changes.

Such non-caloric carbohydrates may interact with certain metabolic pathways, thereby facilitating a homeostatic environment in the brain. They might enhance neuronal connectivity and plasticity by influencing neurotransmitter synthesis and release, as well as modulating the extracellular matrix (ECM) components. The alteration of ECM can result in improved cellular communication, reduced inflammation, and restoration of the integrity of the blood-brain barrier. Collectively, these effects might lead to the preservation of cognitive function in aging, presenting non-caloric carbohydrates as potential therapeutic agents in slowing or possibly reversing cognitive decline associated with aging. In summary, the disclosure highlights that dietary supplementation with monosaccharides in healthy mice influence brain metabolism, glycosylation and behavior to different extent, suggesting non-caloric monosaccharides can be leveraged to improve the cognitive aging process.

The practice of the invention will employ, unless indicated specifically to the contrary, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA techniques, genetics, immunology, and cell biology that are within the skill of the art, many of which are described below for the purpose of illustration.

B. Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred embodiments of supplements, methods and materials are described herein. For the purposes of the present invention, the following terms are defined below.

The articles “a,” “an,” and “the” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives.

As used herein, the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In particular embodiments, the terms “about” or “approximately” when preceding a numerical value indicates the value plus or minus a range of 15%, 10%, 5%, or 1%.

As used herein, the term “consists essentially of’ (and grammatical variants thereof), as applied to the compositions and methods of the present invention, means that the compositions/methods may contain additional components so long as the additional components do not materially alter the composition/method. The term “materially alter,” as applied to a composition/mcthod, refers to an increase or decrease in the effectiveness of the composition/method of at least about 20% or more.

As used herein, the term “supplement” or “dietary supplement” refers to an edible product intended to be added to or supplement the diet, which is different from conventional food. Generally, a dietary supplement is intended to prevent, treat, or cure diseases, and it functions like a drug. Dietary supplements are orally taken and manufactured in many forms, including tablets, capsules, soft gels, gummies, bars, powder, granules, syrups, and solutions. Examples of supplements include vitamins (such as multivitamins or individual vitamins like biotin, folic acid, and vitamin D), minerals (such as calcium, magnesium, and iron), botanicals or herbs (such as echinacea and ginger), botanical compounds (such as caffeine and curcumin), amino acids (such as tryptophan and glutamine), and live microbials (commonly referred to as “probiotics”).

As used herein, the term “subject” or “subject in need,” which can be used interchangeably with “patient,” refers to a mammal, in particular a primate, and in more particular a human individual, which is to be treated with a supplement or supplements comprising at least one monosaccharide as taught herein for the improvement of brain functions. In various embodiments, the subject can be a healthy individual (e.g., adult male, adult female, adolescent male, adolescent female) who wishes to improve his/her brain function without the prescription of a physician or other health worker or a patient (e.g., adult male, adult female, adolescent male, adolescent female, male child, female child) under the care of a physician or other health worker in a hospital or other care facilities as an inpatient or outpatient.

As used herein, the term "treatment" or “treat” in the context of pharmacological or medical meaning refers to intervention of disease, disorder, condition or one or more symptoms thereof to obtain a desired physiological and/or clinical effect. "Treatment" or “treat” includes, but is not limited to, administering one or more drugs or agents (e.g., dietary supplements) comprising at least one active ingredient to a subject using any known method for purposes such as: inhibiting the disease, disorder, condition, or one or more symptoms thereof; slowing or delaying the progress of the disease, disorder, condition, or one or more symptoms thereof; stabilizing (i.e., not worsening) a state of the disease, disorder, condition, or one or more symptoms thereof; and relieving, palliating, alleviating, or ameliorating the severity of the disease, disorder, condition, or one or more symptoms thereof; or preventing remission, whether partial or total and whether detectable or undetectable. “Treatment,” or “treat” docs not necessarily indicate complete eradication or cure of a non-degenerative neurological condition, or associated symptoms thereof. In some embodiment, treatment comprises improvement of at least one brain function such as learning, memory, social interactions, group dynamics, and mood. The improvement may be partial or complete. Improvement in brain function may be measured using any method accepted in the art.

As used herein, the term “pharmaceutical excipient” or “pharmaceutically acceptable excipient” refers to a substance other than the active ingredient(s) included in pharmaceutical dosage forms. They are considered as inert substances, i.e., they do not have any active role in therapeutics, but they are added to improve stability, bioavailability, and manufacturability. Pharmaceutical excipients can comprise: (1) fillers or extenders including, but not limited to, as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders including, but not limited to, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3) humectants including, but not limited to, glycerol; (4) disintegrating agents including, but not limited to, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators including, but not limited to, quaternary ammonium compounds; (7) wetting agents including, but not limited to, cetyl alcohol and glycerol monostearate; (8) absorbents including, but not limited to, kaolin and bentonite clay; (9) lubricants including, but not limited to, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin or vegetable capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like. By way of example, a pharmaceutically acceptable carrier, additive, or excipient can include sodium citrate or calcium carbonate. For example, excipients are preservatives to prevent contamination (e.g., thimerosal), adjuvants to help stimulate a stronger immune response (e.g., aluminum salts), stabilizers to keep the 3D structure of proteins during transportation and storage (starch, gelatin, cellulose, cellulose derivatives, polyvinylpyrrolidone, and polyethylene glycol), surfactants to prevent protein aggregation, amino acids, antioxidants, buffer (acetate, citrate, histidine, succinate, phosphate, and hydroxymethylaminomethane (Tris).), cytoprotectant (e.g., disaccharidcs such as sucrose and trehalose), and other additives including mannitol, BSA, scrum, and skim milk. Examples of suitable pharmaceutical excipients are described in “Remington's Pharmaceutical Sciences.”

As used herein, the term "administering" refers to introducing an agent (e.g., monosaccharide supplement) into a subject, and can be performed using any of the various methods for drug delivery known to those skilled in the art. Agent administration routes include, but are not limited to oral administration, parenteral administration (subcutaneous (SC/SQ: <1 mL), intravenous (IV: 1-20 mL), intradermal (ID: <0.2 mL), intramuscular (IM: < 4 mL), intraperitoneal (IP), intraarterial, intracardiac, intraarticular, intrathecal (IT), intracisternal magna (ICM), intracerebroventricular (ICV), and intraspinal bolus injection or drip infusion), rectal administration by way of suppositories or enema, local/topical administration directly into or onto a target tissue, nasal administration (nebulizer, nasal spray, inhalation), or administration by any route or method that can deliver a therapeutically effective amount of the drug or agent to the cells or tissue to which it is targeted.

The terms “co-administration” or “co-administering” as used herein refers to the administration of an agent (e.g., monosaccharide supplement) before, concurrently, or after the administration of another substance such that the biological effects of either substance synergistically overlap.

As used herein, a "therapeutically effective amount” or an “effective amount” refers to a quantity of an agent (e.g., monosaccharide supplement) that is capable of achieving a desired effect, e.g., an improvement in learning, memory, social interactions, group dynamics, and mood. It can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the supplement to generate a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of a supplement are outweighed by the therapeutically beneficial effects. The “effective amount” needs to be determined considering the drug or agent pharmacodynamics and pharmacokinetics to achieve the intended result.

As used herein, the term “dosage form” refers to a pharmaceutical preparation in which a specific mixture of active ingredients (e.g., monosaccharide supplement) and inactive components (excipients) are formulated in a particular shape or form to facilitated administration and accurate delivery of active ingredients, and/or to be presented in the market. Solid dosage forms include powder, granules, capsules, tablets/pills, cachets, troches, lozenges, gummies, suppositories. Optionally, a tablet dosage form can be fast dissolving, extended release (XR) or long-acting (LA), sustained release (SR), controlled release (CR), delayed release (DR), or enteric coating formulation. Semi-solid dosage forms include ointment, creams, paste, gels, poultices. Liquid dosage forms include collodions, droughts, elixirs, emulsions, suspension, enemas, gargles, linctuses, lotion, liniments, mouth washes, nasal drop, paints, solutions, syrups. Gaseous dosage forms include aerosols, inhalations, and sprays. The dosage form for oral administration can include standard excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical excipients for each dosage form are described in “Remington's Pharmaceutical Sciences.”

As used herein, the term “brain function” refers to neuronal activity related to abilities to think, plan, and make decisions, memories, emotions, speech and language functions, movements (motor function), balance and coordination, perception of various sensations including pain, automatic behavior such as breathing, heart rate, sleep and temperature control, and regulation of organ function, and fight or flight response (stress response). In the context of the invention disclosed herein, brain function refers to cognitive function, i.e., the mental processes involved in learning, thinking, understanding and remembering things. It includes many complex brain activities, such as attention (i.e., the ability to choose and concentrate on relevant stimuli), memory (short-term memory, intermediate-term memory (ITM or medium-term memory), long-term memory, working memory, procedural memory, and prospective memory), processing speed, executive functions, decision making, language abilities, perception, reasoning, and problem solving. Memory is a key cognitive function to acquire, store, and retrieve information, and some aspects of memory include: consolidation (the process of stabilizing and integrating memories into long-term storage) and retrieval (the process of accessing, selecting, and reactivating stored memories). Cognitive function can be affected by age-related changes, such as brain shrinkage, decreased number of synapses, and decreased number of receptors for neurotransmitters, and may affect emotion and/or behavior. As used herein, the term, “improving” or “enhancing” generally refers to the ability of a supplement to produce or cause a greater physiological response (i.c., measurable downstream effect), as compared with normal, untreated, or control-treated individuals or a previous response of the individual receiving the supplement. For example, the physiological response may be increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, or greater. Such measurable physiological responses include brain functions described above.

The term “glycosylation” refers to the process by which a carbohydrate is covalently attached to a target macromolecule. The target macromolecules are typically proteins and lipids, which serve various functions. The building block monosaccharides for glycosylation include glucose, N-acetylglucosamine (GlcNAc), fucose, mannose, galactose, sialic acid, N- acetylgalactosamine (GalNAc), glucuronic acid (GlcA), iduronic acid (IdoA), 5-N- acetylneuraminic acid (Neu5Ac), fructose, and xylose (Xyl). These monosaccharides enter cells by facilitated diffusion through the glucose transporter (GLUT) family of membrane transporters encoded by the SLC2 genes. There are two major types of glycosylation, N-glycosylation and O- glycosylation.

N-glycosylation occurs while the polypeptide is being synthesized. The initial N-glycan structure (2 GlcNAc residues, 9 mannose residues and 3 glucose residues) is synthesized in the endoplasmic reticulum (ER) as a branched structure on a lipid anchor (dolichol pyrophosphate), and then co-translationally, "en bloc" transferred to an asparagine residue within a specific N- glycosylation acceptor sequence (Asn-X-Ser or Asn-X-Thr, where X is any amino acid except Pro or Asp) of a nascent recipient polypeptide. The initial N-glycan is linked via N-acetylglucosamine of the glycan to an Asp residue of the target polypeptide. In the ER and then in the Golgi apparatus, some of the sugar residues are removed from the N-linked glycan structure ("trimming") by glucosidases and mannosidases, and additional monosaccharide residues such as GlcNAc, galactose, mannose, fucose, or sialic acid are added ("processing") in the Golgi apparatus. The common core of N-glycan is

Man a (1^6)

Man P (l- 4) - GlcNAc 0 (1~ ) - GlcNAc - protein

Man a (1~ 3) There is enormous diversity among the oligosaccharides of N-glycan.

O-glycosylation is a post translational process in the Golgi apparatus. It starts by adding a monosaccharide GalNAc (from UDP-GalNAc) to the OH-group of a serine or threonine residue on a polypeptide, which is catalyzed by GalNAc transferase (GalNAcT). After the initiation, serial addition of monosaccharides takes place by corresponding glycosyltransfcrasc. Many O-glycans are extended into long chains with variable size, but O-glycans are less branched than most N- glycans and are commonly biantennary structures. Unlike N-glycosylation, a consensus amino acid sequence comprising Ser or Thr residue for GalNAc addition has not been found. O-glycan biosynthesis is simpler than N-glycan in that there is no need of transferring a lipid-linked precursor oligosaccharide glycan to a protein.

Recently, another type of glycosylation has been discovered, which is known as O-GlcNAc glycosylation. O-GlcN Acylation, termed O-P-GlcNAc or simply O-GlcNAc, is a unique type of glycosylation that adds a monosaccharide, P-N-acetylglucosamine, to serine or threonine hydroxyl moieties on a poplypeptide. O-GlcNAc, which is catalyzed by O-GlcNAc transferase (OGT), is different from N- or O-linked glycosylation in that (i) it occurs mostly in intracellular compartments such as the nucleus, mitochondria, and cytoplasm; (ii) generally, only a single GlcNAc moiety is added without elongation or more complex structures; and (iii) O-GlcNAc attachment and removal is a dynamic process that occurs multiple times in the life of a protein. However, there is another type of O-GlcNAc, EGF O-GlcNAc, which is different from the aforementioned intracellular, single sugar unit O-GlcNAcylation in that (i) it occurs outside the cell membrane and specifically on extracellular proteins containing epidermal growth factor (EGF) repeats; (ii) it is catalyzed by a different enzyme called EOGT in the endoplasmic reticulum (ER), (iii) it can be further elongated to form more complex glycan structures by the addition of galactose; and (iv) unlike dynamic intracellular, single sugar unit O-GlcNAcylation, modification of EGF repeats by O-GlcNAc is static. Intracellular O-GlcNAcylation seems to be involved in the regulation of epigenetics, gene expression, translation, protein degradation, signal transduction, mitochondrial bioenergetics, the cell cycle, and protein localization. (Essentials of Glycobiology, Cold Spring Harbor Laboratory Press; 2022).

C. Protein Glycosylation and Brain function Almost all the secreted proteins and membrane-associated proteins of eukaryotic cells are glycosylated, and there arc many membrane-associated proteins involved in brain function, c.g., neurotransmitter transporters and membrane proteins in the vesicles for neurotransmitter storage and release in a presynaptic nerve terminal, and neurotransmitter receptors on a postsynaptic nerve dendrite or soma (i.e., cell body) region as well as various ion channel proteins in the whole CNS system, including astrocytes. For example, in the presynaptic terminal of cholinergic neurons, Na⁺-dependent choline transporter, vesicular acetylcholine transporters (VAChT), voltage gated calcium channel, vesicle-associated membrane proteins (VAMPs, e.g., v-SNARE, synaptoprevin), synaptosomal nerve-associated proteins (SNAPs), and acetylcholine autoreceptor (both nicotinic and muscarinic) are glycosylated membrane-associated proteins. In the presynaptic terminal of adrenergic neurons, Na⁺-dependent earner for tyrosine transport into the cell, vesicular monoamine transporters (VMATs), voltage gated calcium channel, VAMPs, SNAPs, and norepinephrine transporter to reabsorb norepinephrine from the synaptic cleft are glycosylated membrane-associated proteins.

Acetylcholine and norepinephrine are the most important examples of CNS neurotransmitters, but there are many other CNS neurotransmitters released from the presynaptic nerve terminal, and there are receptors corresponding to those neurotransmitters on the postsynaptic nerve dendrite or soma region, many of which are glycosylated membrane associate proteins. Examples of neurotransmitters include acetylcholine, norepinephrine, epinephrine, dopamine, serotonin, melatonin, histamine, gamma-aminobutyric acid, glycine, glutamate, aspartate, purines (ATP, UTP, and UDP), arachidonic acid, nitric oxide, carbon monoxide. In addition, there are neuropeptides released in the CNS system, including somatostatin, betaendorphin, Leu/Met enkephalin and related opioid peptides, substance P, bradykinin, galanin, orexin, neuropeptide Y (NPY), neurotensin, and some enteric nervous system peptides such as vasoactive intestinal peptide, gastrin-releasing peptide (GRP), calcitonin gene related peptides (CGRP), cholecystokinin, gastrin, etc. Many of the receptors for those neuropeptides are also glycosylated membrane associate proteins.

Furthermore, there are various ion channels in addition to the aforementioned voltage gated calcium channels, including voltage gated ion channels for Na⁺, K⁺ and Cl" and ligand gated ion channels for Ca²⁺, Na⁺, K⁺ and Cl" in neurons and astrocytes. Their conductance change is directly related to neuronal excitation (e.g., increased conductance of Na⁺ channel or decreased conductance of Cl- or K+ channel) or inhibition (e.g., increased conductance of Cl- or K+ channel), which is critical for various brain activity. Since ion channels are transmembrane proteins, most of them are also glycosylated on the extracellular side.

Considering these aspects of protein glycosylation, it may be possible to improve brain function such as learning, memory, social interactions, group dynamics, and mood by increasing monosaccharide pools in the brain for protein glycosylation. For example, memory sensitization is caused by synaptic pathway facilitation and memory habituation caused by inhibition of the synaptic pathways in the cerebral cortex, which is also important for learning. Neuronal excitation and inhibition in the limbic system including hypothalamus, hippocampus and amygdala are known to be important for behavioral and motivational mechanisms of the brain and leaning. Therefore, glycosylation of the proteins in the CNS system is very important for brain function and may affect learning, memory, social interactions, group dynamics, and mood.

Based on the description above, it can be contemplated to use monosaccharides of glycosylation building block to enhance or reduce neurotransmitter release from the presynaptic neurons and/or the activity of neurotransmitter receptors on the postsynaptic neurons as well as ion channel conductance in the CNS in a subject in need thereof.

D. Monosaccharide Supplements

Supplements herein include, but are not limited to, one or more monosaccharides, especially building block monosaccharides for glycosylation. Generally, a monosaccharide is the basic unit to form carbohydrates or saccharides, which contain three elements - carbon, hydrogen, and oxygen. Some of these monosaccharides also contain nitrogen. Sometimes, sugar derivatives are also considered monosaccharides. One example is amino sugars, in which one or more OH groups of the monosaccharide are replaced by an amino group, which is often acetylated. For example, one OH group at C2 of glucose and galactose are replaced with an amino group to form glucosamine and galactosamine, respectively. N-acetylglucosamine (GlcNAc) is an acetylate form of glucosamine.

There are numerous kinds of monosaccharides, and they are classified according to the chemical nature of their carbonyl group (aldose and ketose) and the number of their C atoms (triose, tetrose, pentose, hexose, heptose, etc.). For example, glucose, galactose, and mannose are all aldo-hcxoscs having the formula CeH 12O6. In aldo-hcxoscs, there arc four C chiral centers, and consequently 2⁴=16 stereoisomers (i.e., isomers that differ in spatial arrangement of atoms, rather than order of atomic connectivity) are possible. Sugars that differ only by the configuration around one C atom are known as epimers (e.g., D-glucose and D-mannose). The assignment of D or L chirality is determined according to the Fisher convention, i.e., rotation of polarized light to the right (D) or to the left (L). D sugars and L sugars are enantiomers to each other, which are a special case of stereoisomers, which are molecules non-superimposable on their mirror images. In all D aldo-hexose sugars, the -OH at C5 (the asymmetric center farthest from their carbonyl group) is on the right in a Fisher projection. Most natural sugars are D-chirality.

Fucose (chemical formula C6H12O5) is a monosaccharide, and it has two structural features distinguishing it from other six-carbon monosaccharides: the lack of a hydroxyl group on the carbon at the 6-position (C-6) (thereby making it a deoxy sugar) and the L-configuration. L-fucose is equivalent to 6-deoxy-l-galactose, and it is commonly found in many N- and O-linked glycans and glycolipids produced by mammalian cells. Fucosylated glycans are constructed by fucosyltransferases, which require the substrate GDP-fucose. In mammalian cells, GDP-fucose is synthesized by two pathways; the GDP-mannose-dependent de novo pathway and the free fucosedependent salvage pathway. (Daniel I. Becker DI and Lowe JB, Fucose: biosynthesis and biological function in mammals, Glycobiology, 13(7):41R-53R.)

E. Monosaccharide supplements

A supplement will comprise an effective amount of a monosaccharide or monosaccharides selected from the group consisting of L-fucose, D-galactose, D-mannose, and N-acetyl D- glucosamine (GlcNAc) to improve brain function for enhancing learning, memory, social interactions, group dynamics, and mood in a subject. In certain embodiments, the supplement does not include glucose, acetylated mannose, N-acetylneuraminic acid, N-acetylagalactosamine, arabinose, arabinogalactan, glucuronic acid, galacturonic acid, iduronic acid and/or glucosamine.

Based on the animal study results presented in this disclosure using about 2 g/kg/d monosaccharides, a daily use amount of the monosaccharides enumerated herein (L-fucose, D- galactose, D-mannose, and N-acetyl D-glucosamine (GlcNAc)) contained in the supplements for human use may be within a range of 0.01-5 g/kg/d, and for 50-100 kg adult body weight, daily dose may be about 0.5-500 g, or any intervening range therein. These amounts may relate to individual monosaccharides or to a combination of monosaccharides at certain ratios. In an embodiment, a supplement comprising a combination of monosaccharides will include one monosaccharide as the predominant monosaccharide in terms of relative amounts.

In an embodiment, each monosaccharide is formulated as individual dosage forms. In another embodiment, 2 different monosaccharides arc formulated together as a single dosage form. In another embodiment, 3 different monosaccharides are formulated together as a single dosage form. In another embodiment, 4 different monosaccharides are formulated together as a single dosage form.

In some embodiments, a supplement comprises about 0.5 gram, about 1 gram, about 2 gram, about 5 gram, about 10 gram, about 20 gram, about 50 gram, or any intervening amount between 0.5 - 200 gram as a single unit of a single monosaccharide constituent or as a single unit of combination of multiple monosaccharides.

In a certain embodiment, a supplement is provided, which comprises or consists of L- fucose. In another embodiment, a supplement is provided, which comprises or consists of L-fucose and pharmaceutical excipients. In other embodiments, a supplement is provided, which comprises L-fucose as the main ingredient of at least 50% by weight of the supplement and at least one of other monosaccharides selected from D-galactose, D-mannose, and GlcNAc with or without pharmaceutical excipients. However, it can also be contemplated that a supplement comprises L- fucose and one, two or three other monosaccharides at a similar ratio, e.g., 1: 1:1:1.

In some embodiments, a supplement is provided that comprises L-fucose and another monosaccharide selected from D-galactosc, D-mannosc, and GlcNAc, and the ratio between L- fucose and the other monosaccharide is about 1: 0.1-1. In related embodiments, supplements are provided that include L-fucose at least 50% by weight of the supplement.

In some embodiments, a supplement is provided that comprises L-fucosc and two other monosaccharides selected from D-galactose, D-mannose, and GlcNAc, and the ratio between L- fucose and the other two monosaccharides is about 1: 0.1-0.5: 0.1-0.5. In related embodiments, supplements are provided that include L-fucose at least 50% by weight of the supplement. In some embodiments, a supplement is provided that comprises L-fucose, D-galactose, D- mannosc, and GlcNAc, and the ratio between L-fucosc and the other three monosaccharides is about 1 : 0.1-0.3: 0.1-0.3: 0.1-0.3. In a related embodiment, a supplement is provided that includes L-fucose at least 50% by weight of the supplement.

In an embodiment, a supplement is provided that comprises or consists of D-galactosc and optionally pharmaceutical excipients. In another embodiment, a supplement is provided that includes D-galactose and at least one of other monosaccharides selected from L-fucose, D- mannose, and GlcNAc, wherein the supplement includes D-galactose at least 50% by weight of the supplement.

In an embodiment, a supplement is provided that comprises or consists of D-mannose and optionally pharmaceutical excipients. In another embodiment, a supplement is provided that includes D-mannose and at least one of other monosaccharides selected from L-fucose, D- galactose, and GlcNAc, wherein the supplement includes D-mannose at least 50% by weight of the supplement.

In an embodiment, a supplement is provided that comprises or consists of GlcNAc, and optionally pharmaceutical excipients. In another embodiment, a supplement is provided that includes GlcNAc and at least one of other monosaccharides selected from L-fucose, D-galactose, and D-mannose, wherein the supplement includes GlcNAc at least 50% by weight of the supplement.

E Formulations and dosage forms

The dietary supplements disclosed herein are formulated into solid, semi-solid, or liquid dosage form for oral administration. The supplement can be formulated as powder, granules, premix, gelcaps, capsules, tablet (solid, chewable, lozenges, time-release), pills, caplets, dragees, bars, gummies, gels, pastes, solutions, syrups, elixir, emulsions, suspensions and the like, for oral administration. The solid, semisolid or liquid dosage form may be packed in a capsule (gelatin or vegetable capsule). The monosaccharide supplements may be formulated by combining the active ingredient(s) with pharmaceutically acceptable excipients suitable for oral delivery well known in the art. The term “pharmaceutically acceptable” refers to being suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio within the scope of sound medical judgment. There is virtually no limit to other reagents that may also be included in the supplements, provided that the additional reagents do not adversely affect the desired cognitive improvement.

As used herein “pharmaceutically acceptable carrier, or excipient” includes without limitation any adjuvant, diluent, glidant, sweetening agent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surfactant, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals. Exemplary pharmaceutically acceptable carriers include, but are not limited to, to sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffins, silicones, bentonites, silicic acid, zinc oxide; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and any other compatible substances employed in pharmaceutical formulations. Techniques for formulation of drugs may be found in Remington: The Science and Practice of Pharmacy. 22nd Edition. Pharmaceutical Press. 2012 or later version, which is incorporated herein by reference in its entirety. The nature of the formulation will depend on the intended routc(s) of administration.

For oral solid formulations, pharmaceutically acceptable excipients can include, cellulose preparations (e.g., maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmcthyl-ccllulosc, sodium carboxymcthylccllulosc), synthetic polymers (e.g., polyvinylpyrrolidone (PVP)), granulating agents; and binding agents. If desired, disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. The supplement may be coated with dragee cores using materials known in the art optionally containing gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dye stuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Capsule formulations for oral administration can be made of gelatin, vegetable cellulose, and a plasticizer, such as glycerol or sorbitol, and contain the active ingredients in admixture with excipients such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers; and it can contain suitable liquid components, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.

Liquid dosage formulations for oral administration may include pharmaceutically acceptable solutions, beverage, suspensions, syrups, elixirs, and emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil. As suitable dispersing or suspending agents for aqueous suspensions, synthetic natural gums, such as tragacanth, acacia, alginate, dextran, sodium carboxymethyl cellulose, methylcellulose, polyvinylpyrrolidone or gelatin can be added. A beverage adapted for oral administration of monosaccharide supplement can be water or other liquids, such as juices, iced tea, hot tea, and soda.

Liquid preparations for oral administration may also be prepared as a dry formulation for reconstitution with water or other suitable liquids with additives such as suspending agents (e.g., , methyl cellulose or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); nonaqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); preservatives (e.g., methyl or propyl p-hydroxybenzoates or sorbic acid); and artificial or natural colors and/or sweeteners.

In certain embodiments, the monosaccharide supplements can be provided as food additives or food products. As used herein, “food additives” refer to any liquid, semi-solid, or solid material that is intended to be added to a food product, for example, an agent having a distinct taste and/or flavor or a physiological effect. As used herein, “food product” refers to an edible product comprising protein, carbohydrate and/or fat, which can be used for energy supply, generation of building block molecules for growth, repair and other vital processes of the body. Food products may also be other dietary supplements such as minerals, vitamins, amino acids, etc. For example, the supplement(s) can be added to food products, including cereal, oatmeal, gruel, jelly pudding, fruit jam, yogurt, ice cream, milk, soy milk, chocolate milk shake, cocoa, coffee, tea, fruit juice, and energy drink, and/or to meals, including soup, salad, cooked vegetables and meat, starchy food, and dairy.

The foregoing exemplary formulations and dosages are merely illustrative and not necessarily limiting. In various embodiments, other combinations and dosages of monosaccharide supplements can be formulated.

G. Kits

Optionally, the monosaccharide supplements may be formulated into one or more “unit dosage” forms, which can be individually or collectively packaged. Herein, the “unit dosage” refers to an amount of a monosaccharide to be taken each time according to a dosing schedule. For example, a supplement comprising L-fucose can be formulated as bars packed individually for one time use or as powder or granules collectively packed in a container for multi-time use.

When multiple supplements are packaged as multiple units of dosage forms, the dosage forms of each supplement can be the same dosage form or different dosage forms. In some embodiments, all supplements can be in a dosage form of a concentrated solution, or one supplement as capsules and the other supplement(s) gummies in a kit.

It can also be contemplated that in a kit, one supplement comprising L-fucose and D- galactose is formulated as a pudding-like- or yogurt-like product, each unit of which are individually packed in a plastic container; another supplement comprising D-mannose is formulated as syrup, each unit of which are individually packed in a plastic container; and the other supplement comprising GlcNAc is formulated as powder, which is collectively packed in a container or individually packed with one time use amount in paper pouches.

In some embodiments, the supplements are formulated as multiple tablets and/or capsules comprising less than 5 g of the supplement. In the case where a supplement is formulated as multiple capsules, the capsule (gelatin or vegetable) may comprise a small amount (less than 5 g) liquid, semi-solid, or solid dosage form of the supplement. For example, a kit may comprise L- fucose and L-galactose supplements as tablets, L-mannose supplement as jelly, and GlcNAc as granules packed in capsules. The foregoing combinations are merely illustrative and not necessarily limiting. In various embodiments, other combinations of the monosaccharide supplements arc contemplated herein.

Further, with multiple dosage forms, a subject may select individual supplements and the quantities thereof to suit its particular needs. The individual supplements are provided in an integrated package containing some or all of the monosaccharides, which may be bundled together in various packaging systems e.g., a pack or dispenser device, such as an FDA approved kit, that can contain one or more unit dosage forms that collectively comprise the complete supplement.

It will be appreciated that these kits/packaging systems are intended to be illustrative and not limiting. Using the teachings provided herein, numerous alternative packaging will be available to provide the supplements contemplated herein.

In addition, the packaging systems/kits optionally include labeling and/or instructional materials providing directions (i.e., protocols) for the practice of the methods or use of the supplements of this invention.

H. Administration and dosing schedule

In various embodiments, contemplated methods comprise administering supplements disclosed herein to a subject who wishes to improve brain function by enhancing learning capability, memory, social interactions, group dynamics, and mood.

A suitable route of administration for the monosaccharide supplements disclosed herein is preferably oral administration route. However, it can be contemplated that each monosaccharide or a combination of monosaccharides can be administered through parenteral administration route such as intrathecal (IT), intracisternal magna (ICM), intracerebroventricular (ICV) and intravenous (IV) injection if need be in accordance with standard methods well known to those of skill in the art. For injection, the monosaccharides described herein can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks solution, Ringer's solution, or physiological saline buffer and/or in certain emulsion formulations. The solution(s) can contain suitable excipients such as suspending, stabilizing and/or dispersing agents. In certain embodiments, the monosaccharide supplements can be provided in powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water or saline, before use. The supplement is orally or parenterally administered once a day, twice a day, or thrice a day every day, every other day, every three days, every four days, every five days, every six days, or once a week, for at least one month or more. Each supplement monosaccharide can be administered at the same time or different times.

Although the supplements arc intended to be self-administered, i.c., taken by the patient without medical or parental supervision, in some embodiments, administration of the supplement may be under the direction of a physician or adult if the individual taking the supplement is a minor or requires supervision.

Subjects/individuals that may benefit from the methods described herein include individuals that are adults with normal brain functions, and adults otherwise healthy but also possess reduced brain function due to various neurological disorders.

In further embodiments, the method contemplated herein comprises measuring brain function of the individual before supplementation and throughout the period of supplementation at either regular or irregular’ intervals. The initial brain function assessment may serve as a baseline to measure the improvement in brain function provided by the supplement provided herein. In addition, the individual receiving the supplement may be compared with a group of subjects who receive placebo. Methods for measuring brain function may be given by a psychologist or qualified professional either in person or remotely. In addition, brain function can be assessed using computerized assessment programs, or any art-accepted method.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results.

EXAMPLES Example 1 , General Methods

1.1. Mouse model

Female C57BL/6J mice were purchased from Jackson Laboratories (JAX:000664) and housed in a climate-controlled environment with a 12 (light)/12 (dark) hours cycle (light period starting at 7:00) with temperature and humidity maintained at 72°F (22°C) and at least 30%, respectively. Water and solid diet (Standard Envigo 2919) provided ad libitum throughout the study by Automatic Watering and on wire bar lid, respectively. Animals were housed in edge cage systems with micro barrier tops (NexGen Mouse 500, Allentown; 193 mm W x 178 mm H x 397 mm D) on corn cob bedding and were provided with tissue material for nesting and a transfer tunnel for enrichment. Males and females were housed in the same room.

1.2. Method details

1.2.1. Sugar supplementation by oral gavage

4-Month-old female C57BL/6 mice were individually housed 3 days prior to the beginning of gavage period. At 6 h of light period, 250 pL of water or IM solutions of D-galactose (2.00 g/kg/d; Sigma #G5388, Lot BCBZ5583), L-fucose (1.82 g/kg/d; Chem-Impex #00736, Lot #002995-190226-01), D-mannose (2.00 g/kg/d; Sigma, #M8574), D-glucosamine hydrochloride (2.39 g/kg/d; GlcN-HCl; Sigma #1514, Lot #BCBX5411), D-trehalose dihydrate (4.20 g/kg/d; Chem-Impex #00766, Lot #001112- 10440006) or N-acetyLD-glucosamine (2.46 g/kg/d; GlcNAc; Sigma #A4106, Lot #SLCF8395) were administered orally using plastic feeding tube (Instech #FTP-20-38; 20 ga X 38 mm). Solutions were prepared fresh daily. Mice were treated 5 days per week for 3.5 weeks. Body weight was monitored daily prior to each oral administration.

1.2.2. Glucose Tolerance Test

Mice were fasted for 6 hours (starting at the beginning of light period) prior to performing the glucose tolerance test. Mice were administered a bolus injection of glucose (2g/kg body weight) via oral gavage and blood was collected by tail puncture. Blood glucose was measured using the Dario Smail Glucometer before (0 minute) and 15, 30, 60, and 120 minutes after glucose administration.

1.2.3. Tissue collection Mice were sacrificed by cervical dislocation and blood, brain, lungs, liver, heart and quadriceps collected within 3 minutes. Resected tissues were washed once with PBS, twice with diH2O, and blotted dry. Tissues were fixed either by snap-freezing in liquid nitrogen, by slow- freezing on an isopentane bath chilled over dry ice or by overnight incubation in 10% NBF then storage in 70% ethanol. The formalin-fixed tissue was then embedded in paraffin (FFPE) and cut for use in downstream analyses.

1.2.4. Tissue preparation for GC-MS

Snap-frozen tissues were pulverized using a Freezer/Mill Cryogenic Grinder (model 6875D, SPEX SamplePrep). Polar metabolites were then extracted from 20 mg of pulverized tissue using an ice-cold 1 : 1 HPLC-grade methanol/water mixture with 20 pM L-norvaline (Sigma- Aldrich). Centrifugation (21,000 g at 4°C for 10 minutes) was used to separate the pellet — containing lipids, proteins, and glycogen — and polar fractions. The polar fraction was then removed and snap-frozen in liquid nitrogen and stored at -80°C. The pellet was subsequently washed with methanol and dried using a CentriVap Benchtop Vacuum Concentrator connected to a CentriVap -105°C Cold Trap (Labconco) at 10-3 mBar for 30 min. The pellet was then hydrolyzed by incubating in 3M HO at 95°C for 2 hours with regular mixing. Hydrolysis was quenched using an equal volume of 100% methanol with 40 pM L-norvaline and the supernatant was collected after centrifugation (15,000 rpm at 4°C for 10 minutes). The supernatant was then dried by vacuum centrifugation at 10-3 mBar for 4 hours. Before derivatization, polar samples were moved to v-shaped amber glass chromatography vials before being dried for 2 hours with a vacuum centrifuge at 10-3 mBar.

1.2.5. Sample derivatization

Dry polar and protein fractions were derivatized using methoxyamine HO (20 mg/mL) in pyridine and N-methyl-trimethylsilyl-trifluoroacetamide (MSTFA; Thermo Fisher Scientific). To the polar fraction, 50 pL of methoxyamine HC1 was added and the samples incubated at 30°C for 1.5 hours. For the protein fractions, 70 pL of methoxyamine HC1 were added and, after the incubation period, centrifuged at 15,000 rpm for 10 minutes. Then, 50 pL of the supernatant was transferred to a v-shaped amber glass chromatography vial. After, 80 pL of MSTFA were added to each sample and they were incubated at 37 °C for 30 minutes. 1 .2.6. GC-MS quantification

An Agilent 8890 gas-chromatography (GC) system coupled to a 5977C single quadrupole mass spectrometry detector equipped with an InertPlus Electron Ionization (El) source was used during this study. Protocols were similar to previously described (Young et al., 2020). Briefly, 1 pL of dcrivatizcd sample was injected in a J&W HP-5ms Ultra Inert GC column (Agilent Technologies, #19091S-433UI) in a 1:10 split mode. Ultra high purity helium (Airgas #UN1046) flow was set-up at 0.687 mL/min with a pressure of 4.3 psi. Initial temperature was held at 60°C for 1 min, then increased to 325°C with a 10°C/min and held at 325°C for 10 min. El energy was set up at 70 eV and the temperature of the transfer line at 290°C. Source temperature was adjusted to 250°C for polar and 280°C for non-polar samples. Data was acquired in scan mode (50-550 m/z) and metabolite. El fragmentation pattern and retention time was matched to metabolites from the Fiehn 2013 Metabolomics RTL Database (available from Agilent) in the Automated Mass Spectral Deconvolution and Identification System (AMDIS) software for identification and further confirmed with ultrapure standard purchased from Sigma. L-norvalinc served as internal control to assess proper sample process and derivatization. Relative abundance was calculated using the Data Extraction for Stable Isotope-labeled Metabolites (DEXSI) software package and normalized on total protein abundance as determined by adding amino acid signals from the non-polar fraction.

1.2.7. Slide preparation for MALDI-MSI

FFPE tissue was cut into 4 um slices onto positively charged slides. Slides were then dewaxed by incubating at 60°C in a humidity chamber for 1 hour followed by two xylene washes (3 minutes each) and a rehydration series (one 1 -minute wash in 100% ethanol, 95% ethanol, 70% ethanol, and two 3-minute washes in water). Antigen retrieval was then performed by submerging slides in a citraconic anhydride buffer in a mailer and placed into a vegetable steamer for 25 minutes. The citraconic anhydride buffer was prepared by diluting 25 pL of citraconic anhydride (Thermo) in 50 mL of water and adjusted to pH 3.0 with HC1. After incubation in the vegetable steamer, the buffer was allowed to cool and was replaced with water. Slides were then dried in a desiccator prior to enzyme application.

1.2.8. N-glycan and glycogen MALDI-MSI

T1 A 200 pL mixture of peptide N-glycosidase F (PNGase F; 20mg/slide) and isoamylase (10 units/slidc) were sprayed onto each slide using an automated HTX spraying station (HTX). The spray nozzle was heated to 45 °C with a spray velocity of 900 m/min. Slides were subsequently incubated in a humidified chamber at 37 °C for 2 hours. The slides were then desiccated and the ionization matrix (a-cyano-4-hydroxycinnamic acid matrix [0.021 g CHCA in 3 mL 50% acetonitrile/50% water and 12 mL TFA]) was applied using the HTX sprayer. MALDI-MSI was performed using a Bruker timsTOF fleX using a 46 pm X 46 pm laser raster to produce 50 pm x 50 pm pixels. Imaging was performed using positive ion mode and with the settings of MS 1 scan, 50 X 50 pm pixels from a 46 X 46 pm raster scan range, 37% laser power, 1 burst of 300 shots, 10,000 Hz frequency, and a range of m/z 700-4000. SCiLs Lab 2023a pro (Bruker Daltronic) was used to process raw data by matching accurate mass with previously published peaklists (Conroy et al., 2023; Conroy et al., 2021; Wang et al., 2022; Wang et al., 2022; Wang et al., 2015). Export to a pixel-by-pixel tabular format was performed using SCiLS lab R package and corresponding API (available from Bruker) using peak area normalized to total ion count (TIC).

1.2.9. Immunohistochemistry staining

FFPE tissue was sliced at 4 pm and stained at the UF Molecular Pathology Core (RRID: SCR_016601). Immunohistochemistry stains were performed for glycogen (IV58B6 [1:50], and polyglucosan bodies (PGBs; KM279 [1 :50]). Slides were incubated in high pH Target Retrieval Solution (Dako Omnis) for 20 min at 95°C for antigen retrieval and primary antibodies were added to each slide and incubated for 1 hour. M.O.M. ImmPRESS HRP Polymer kit (Vector, #MP-2400) was used according to manufacturer’s instructions for blocking and biotinylated secondary application. DAB was then added to the tissue for 5 minutes and subsequently quenched with water. Digital images for stained slides were obtained using a PhenoImager HT slide scanner (Akoya Biosciences) at 20x resolution. Data analysis was performed using QuPath (Bankhead et al., 2017).

1.2.10. Protein preparation for blotting

Proteins were extracted in 500 pl RIPA buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris- HC1, 0.5% sodium deoxycholate, 0.2% SDS, 1% Igepal) supplemented with 0.5 pM PUGNAc and cOmplete EDTA-free Protease Inhibitor Cocktail from 20 mg of pulverized tissues. Tissue was resuspended with a syringe and incubated on ice for Ih with intermittent vortexing, then centrifuged 20 min at 15,000 g. Protein concentration from supernatant was estimated using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). For further O-GlcNAc western blotting and lectin blotting, 150 pg proteins were incubated for Ih at 37°C in presence of 6 pg PNGase F Prime (N-Zymes) and then stored at -80°C.

1.2.11. Western blotting

Lysates were mixed in 2X Laemmli sample buffer (Bio-Rad, #1610737) with 5% P- mercaptoethanol and incubated at 37°C for Ih. Then, 10 pg of proteins were loaded on Mini- Protean or Criterion TGX Stain-Free gel with a 4-15% gradient of polyacrylamide (Bio-Rad #4568086 and #5678085), run in a running buffer containing 25 mM Tris, 192 mM glycine and 1% SDS and transferred on 0.45 pm LF-PVDF membrane (Bio-Rad #1704275) using a TransBlot Turbo system (Bio-Rad). Stain-free total proteins were imaged with a ChemiDoc MP (Bio-Rad) for normalization. Membrane was blocked in Carbo-Free Blocking Solution (Vector Laboratories #SP-5040-125) and incubated overnight with the RL2 monoclonal anti-O-GlcNAc antibody conjugated with AlexaFluor 647 (1: 1,000; Abeam, #ab201994) before imaging. Same membrane was then probed for O-GlcNAcase (OGA) using the anti-MGEA5 polyclonal antibody (1:2,000, overnight; Proteintech #14711-1-AP) and a secondary Goat Anti-Mouse IgG (H+L)-HRP Conjugate (1 :2,500, Ih; Bio-Rad #1706516). Clarity Max Western ECL Substrate (Bio-Rad #1705062) applied and chemiluminescence captured. Images were analyzed using the volume tool of ImageLab software (Bio-Rad).

1.2.12. Lectin blotting

Protein migration and transfer were performed as described above for Western blotting. After transfer, Stain-free signal was captured and LF-PVDF membrane was blocked in Carbo-Free Blocking Solution (Vector Laboratories #SP-5040-125). Membrane was incubated overnight with 10 pg/mL of biotinylated lectins, including Jacalin, PNA, VVL and SNA (Vector Laboratories, #B- 1155-5, #B-1075-5, #B-1235-2, #B-1305-2), washed and further incubated with Streptavidin conjugated with AlexaFluor 568 (1 pg/mL; Thermo Fisher #S 11226) for Ih. Fluorescence was captured on ChemiDoc MP, and images analyzed using the volume tool of ImageLab software (Bio-Rad).

1.2.13. Tube Dominance Test At 3 weeks of treatment, mice were trained for the tube dominance test by walking through the tube five times through each side across three sessions. After training, randomized, pairwise matches between the treated and control mice were carried out across four sessions. Matches started at the first physical interaction and ended when one mouse had all limbs outside of the tube. A loss was considered a retreat if the mice left the tube without force. Every mouse participated in the same number of matches and started equally on both extremities of the tube. Mice were handled for 30 seconds before each match to reduce stress, and tube, surfaces and gloves were cleaned with 70% ethanol between every match.

1.2.14. Open Field Test

Mice were transferred to a corner of the open field maze (54 cm X 54 cm) and monitored for 5 minutes. Open field surface was delimited in 9 boxes of equal surface and time in locomotion, number of boxes entered and time spent in the central and outer boxes was calculated. Video of each session was analyzed by three independent researchers. Between each mouse, 70% ethanol was used to clean the maze and gloves and allowed to dry prior to introducing the next mouse.

1.2.15. 5-Trial Social Memory Test

During the 3rd week of gavage, and for 5 consecutive days, subject mice were individually placed into a new cage for the 5 -trial social memory test and were allowed to habituate for 10 minutes. Then, a novel mouse was introduced to the cage and the time that the subject mice spent investigating the novel mice was measured over a period of 5 minutes before removing the novel mice and transferring subject mice back to their original cage. Pairings between subject and novel mice were conserved across all sessions. Videos were analyzed by three independent researchers.

1.2.16. Quantification and Statistical Analysis

Statistical analyses were carried out using GraphPad Prism 10.0. All numerical data are presented as mean ± SD. XY analysis was performed using multiple t-tests, grouped analysis using two-way ANOVA and column analysis using one-way ANOVA. A p-value less than 0.05 was considered statistically significant. The statistical parameters for each experiment can be found in the figures and figure legends.

1.2.17. Behavioral testing information Behavioral tests were selectively performed to determine changes in different aspects of cognition (emotion, social interaction, and memory). The tube dominance test was used to quantify dominance, a trait primarily associated with the prefrontal cortex, exemplified by mice across treatment conditions. In particular, this test has previously been used to characterize a mouse model of schizophrenia, a pathology known to involve prefrontal cortex in humans, that correlated with significantly higher tube dominance victory rate in mice.

The open field test was used to detect anxiety-like symptoms in mice, associated with the amygdala. For instance, diazepam, a drug used to treat anxiety in humans, induced a decrease in anxiety-like behavior in mice as determined using the open field test. 5-trial social memory test tested social memory associated with hippocampus and amygdala.

Example 2, Fucose treatment

2.1. Experimental design

The experimental design is illustrated in Figure 2. Four-month-old C57BL/6 mice underwent daily supplementation of 2g/kg of L- fucose for 3.5 weeks by oral gavage, and conservation of normal glucose metabolism was investigated with a glucose tolerance test.

At the end of the gavage period, mice were euthanized by cervical dislocation. The left hemispheres of the brains were formalin-fixed and paraffin-embedded (FFPE) for analysis through matrix-assisted laser desorption ionization-mass spectrometry imagery (MALDI-MSI), and the right hemispheres were flash-frozen in liquid nitrogen for metabolomics using gas chromatography-mass spectrometry (GC-MS). Distribution of N-glycans and glycosylation is determined by MALDI-MSI from a single FFPE slide. Further, the impact of L-fucose on the behavior was investigated after 2 weeks of L- fucose dietary supplementation by a 5-trial social memory test (hippocampus and amygdala) and a tube dominance test (frontal cortex and hypothalamus).

2.2. L-fucosc and Physiology

Figure 3 (A) shows body weight changes of mice on water control or L-fucose supplemented diet over the gavage period. There was no difference of body weight changes between L-fucose-treated and water-control mice. Figure 3 (B) shows blood glucose levels during an oral glucose tolerance test. After 6 hours of fasting, mice were given an oral bolus of glucose (2g/kg). Blood glucose was measured for 2 hours using a Dario glucometer. No significant difference was observed between the water controls and the L-fucosc treated mice. In summary, dietary supplementation with L-fucose had no impact on mice’s body weight and glucose tolerance.

2.3. L-fucosc and Protein Glycosylation

For protein N-glycosylation measurement, mice brain slides were sprayed with PNGase F to cleave N-glycans that were analyzed by MALDI-MSI. After PNGase F treatment, protein lysates were separated on SDS-PAGE, transferred on PVDF membrane, and O-GlcNAc and O- glycans were detected by Western blot and lectin blot, respectively. The lectin used, jacalin, recognized several O-glycans including Tn antigen, sialyl-T antigen, core 1 and core 3 glycans. The brains of L-fucose treated mice exhibited significantly higher levels of O-GlcNAcylation and O-glycosylation than the brains of water control mice, demonstrating that dietary supplementation with L-fucose slightly increased protein O-glycosylation (Figure 4).

2.4. L-fucose and Metabolism

Dietary supplementation with L-fucose had limited impact on the whole brain metabolism. Figure 5 (B) is a heatmap of the top 50 most changed polar metabolites and (C) is the result of Principal Monosaccharide Analysis (PCA) of polar metabolites. Either heatmap or PCA was not able to distinguish L-fucose-treated mice from water controls, suggesting no major impact of L- fucose on general brain metabolism. However, Figure 5 (D) shows that the levels of three metabolites, stearic acid, cholesterol and fucose were significantly changed. The level of fucose increased in samples from L-fucose-treated mice, suggesting that L-fucose was able to access the brain, and the levels of stearic acid and cholesterol decreased upon L-fucosc treatment. This result demonstrates the role of L-fucose to suppress lipid accumulation.

2.5. L-fucose and Glycogen

Dietary supplementation with L-fucose decreases brain glycogen content. Whole brain glycogen, as measured by GC-MS, looks less abundant in the brain of fucose treated mice compared with water controls. Despite a huge variability of glycogen accumulation in the water group, the accumulation of glycogen in L-fucose treated mice significantly decreased on immunohistochemical (IHC) images of 3 different hrain regions (cortex, cerebellum and hippocampus) using the anti-glycogen antibody IV58B6.

2.6. L-fucose and Behavior

Dietary supplementation with L-fucose induces behavior changes in WT mice.

On the 3rd week on gavage, each mice underwent a 5-trial social memory test. This consists of measuring the time mice spend investigating the same novel mouse for 5 consecutive days. No significative change was observed.

On the 4th week of gavage, social dominance and aggressiveness were assessed with a tube dominance test. Treated and control mice are placed at both extremities of a tube they cannot cross in and need to push each other to exit. The mouse staying inside is declared winner. L-fucose treated mice won all their matches against water control, suggesting that fucose increased dominance and/or aggressiveness.

In summary, it was demonstrated that L-fucose slightly increases brain O-glycosylation while having no impact on basal metabolism, except decreasing lipid accumulation. Furthermore, the main effects induced by L-fucose supplementation appear to be a decrease in glycogen accumulation and cognitive changes.

Example 3. Galactose treatment

3.1. Experimental Design

The experimental design is illustrated in Figure 8. C57/BL6 mice were treated daily with either galactose (2g/kg) or an equivalent volume of water for 3.5 weeks. Then, brain tissue was harvested, and each hemisphere was divided and fixed by either formalin fixation (left hemisphere) for MALDLMSI analysis or freezing (right hemisphere) for GC-MS analysis. MALDLMSI was performed to see regional changes in protein N-glycosylation, and GC-MS was performed to see whole-brain metabolomics. In addition, behavior experiments started at 2.5 weeks with open field test and tube dominance test.

3.2. Protein Glycosylation MALDI-MSI was used to measure regional differences in N-glycosylation and glycogen architecture between water-treated mice and galactosc-trcatcd mice. PNGascF is an enzyme that cleaves GlcNAc-Asn bond to release glycan. Analysis of protein N-glycan abundance revealed no significant changes in glycosylation profiles following galactose treatment (Figure 9), and unbiased clustering analysis of regional N-glycan profiles could not differentiate between treatment conditions. After PNGase F treatment, O-glycans were probed using the Jacalin lectin and an antibody specific for O-GlcNAc. There was no significant change in O- GlcNAc or O-glycosylation between water-treated mice and galactose-treated mice.

3.3. Metabolism

GC-MS was used to measure metabolic profiles of the brain from water-treated mice and galactose-treated mice. Analysis of polar metabolites revealed significant changes in cerebral metabolism following galactose treatment, and principal monosaccharide analysis (PCA) of polar metabolites revealed treatment-dependent grouping. In addition, unbiased clustering analysis distinguished the two treatment groups, and showed a significant increase of central carbon metabolism in galactose treated mice, demonstrating that glycogen levels tend to increase after gavage of galactose (Figure 10).

3.4. Behavior

Next, changes in behavior were monitored starting after 2.5 weeks after diet supplementation of galactose until time of sacrifice (Figure 11). The open field test demonstrated that mice treated with galactose showed significantly more anxiety-like behavior. In addition, the tube dominance test demonstrated that galactose-treated mice were more aggressive than the control mice.

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Claims

CLAIMS What is claimed is:

1. A dietary supplement, wherein the supplement comprises, consists essentially of, or consists of one or more monosaccharides selected from the group of L-fucose, D-galactose, D- mannose, and N-acetyl D-glucosamine (GlcNAc), wherein the dietary supplement optionally lacks glucose, acetylated mannose, N- acetylneuraminic acid, N-acetylagalactosamine, arabinose, arabinogalactan, glucuronic acid, galacturonic acid, iduronic acid and/or glucosamine.

2. The dietary supplement of claim I , wherein the supplement comprises L-fucose, and wherein the supplement may optionally comprise pharmaceutical excipients for oral administration.

3. The dietary supplement of claim 2, wherein the supplement further comprises D- galactose, and wherein the supplement comprises at least 50% L-fucose by weight.

4. The dietary supplement of claim 2, wherein the supplement further comprises D- mannose, and wherein the supplement comprises at least 50% L-fucose by weight.

5. The dietary supplement of claim 2, wherein the supplement further comprises GlcNAc, and wherein the supplement comprises at least 50% L-fucose by weight.

6. The dietary supplement of claim 2, wherein the supplement further comprises D- galactose and D-mannose, and wherein the supplement comprises at least 50% L-fucose by weight.

7. The dietary supplement of claim 2, wherein the supplement further comprises D- galactose and GlcNAc, and wherein the supplement comprises at least 50% L-fucose by weight.

8. The dietary supplement of claim 2, wherein the supplement further comprises D-mannose and GlcNAc, and wherein the supplement comprises at least 50% L-fucose by weight.

9. The dietary supplement of claim 2, wherein the supplement further comprises D- galactose, D-mannose, and GlcNAc, and wherein the supplement comprises at least 50% L- fucose by weight.

10. The dietary supplement of claim 1 , wherein the supplement comprises D-galactose, and wherein the supplement may optionally comprise pharmaceutical excipients for oral administration.

11. The dietary supplement of claim 1, wherein the supplement comprises D-mannose, and wherein the supplement may optionally comprise pharmaceutical excipients for oral administration.

12. The dietary supplement of claim 1, wherein the supplement comprises GlcNAc, and wherein the supplement may optionally comprise pharmaceutical excipients for oral administration.

13. The dietary supplement of any one of the preceding claims, wherein the supplement is formulated into solid, semi-solid, or liquid dosage form for oral administration.

14. The dietary supplement of claim 13, wherein the supplement is optionally formulated into multiple units of a dosage form, and wherein a unit comprises a total weight of about 0.5-200 g of a monosaccharide or monosaccharides and optionally pharmaceutical excipients.

15. The dietary supplement of any of claims 1-14, wherein the dietary supplement lacks glucosamine.

16. The dietary supplement of any of claims 1-14, wherein the dietary supplement lacks glucose.

17. The dietary supplement of any of claims 1-14, wherein the dietary supplement lacks acetylated mannose.

18. The dietary supplement of any of claims 1-14, wherein the dietary supplement lacks N- acetylneuraminic acid.

19. The dietary supplement of any of claims 1-14, wherein the dietary supplement lacks N- acety lagalacto s amine .

20. The dietary supplement of any of claims 1-14, wherein the dietary supplement lacks arabinose.

21 . The dietary supplement of any of claims 1-14, wherein the dietary supplement lacks arabinogalactan.

22. The dietary supplement of any of claims 1-14, wherein the dietary supplement lacks glucuronic acid.

23. The dietary supplement of any of claims 1-14, wherein the dietary supplement lacks galacturonic acid.

24. The dietary supplement of any of claims 1-14, wherein the dietary supplement lacks iduronic acid.

25. A kit comprising the dietary supplement of claim 2, wherein the kit further comprises at least one of the supplements of claims 10 to 12, and wherein the supplements are all in the same dosage form or at least one of the supplements is in a different dosage form.

26. A kit comprising the dietary supplement of claim 3, wherein the kit further comprises the dietary supplements of claims 11 and/or 12, and wherein the supplements are in the same dosage form or in different dosage forms.

27. A kit comprising the dietary supplement of claim 4, wherein the kit further comprises the dietary supplements of claims 10 and/or 12, wherein the supplements are in the same dosage form or in different dosage forms.

28. A kit comprising the dietary supplement of claim 5, wherein the kit further comprises the dietary supplements of claims 10 and/or 11 , wherein the supplements are in the same dosage form or in different dosage forms.

29. A kit comprising the dietary supplement of claim 6, wherein the kit further comprises the dietary supplement of claim 12, wherein the supplements are in the same dosage form or in different dosage forms.

30. A kit comprising the dietary supplement of claim 7, wherein the kit further comprises the dietary supplement of claim 11, wherein the supplements are in the same dosage form or in different dosage forms.

31 . A kit comprising the dietary supplement of claim 8, wherein the kit further comprises the dietary supplement of claim 10, wherein the supplements arc in the same dosage form or in different dosage forms.

32. A kit comprising the dietary supplement of claim 9, and optionally any dietary supplements of claim 2 and claims 10-12.

33. The kit of any one of claims 25-32, wherein each supplement is collectively packaged in a container or the individual units of each supplement are wrapped separately and packaged together in a container for each supplement or all together.

34. A method for improving brain function to enhance learning, memory, social interactions, group dynamics, and mood in a subject, the method comprising orally administering the subject an effective amount of the monosaccharide supplement(s) of any one of claims 2-24 and 39-44, wherein daily dose is less than 5 g/kg, and optionally less than 2 g/kg.

35. The method of claim 34, wherein the supplement is administered once a day, twice a day, or thrice a day every day, every other day, every three days, every four days, every five days, every six days, or once a week, for at least one month or more.

36. The method of claim 34 or 35, wherein when more than one supplement is coadministered, they can be administered at the same time or different times.

37. The methods of any one of claims 34-36, wherein the supplement(s) is/are added to food products, including cereal, oatmeal, gruel, jelly pudding, fruit jam, yogurt, ice cream, milk, soy milk, chocolate milk shake, cocoa, coffee, tea, fruit juice, and energy drink, and/or to meals, including soup, salad, cooked vegetables and meat, starchy food, and dairy.

38. Use of at least one monosaccharide selected from L-fucose, D-galactose, D-mannose, and/or GlcNAc to enhance or reduce neurotransmitter release from the presynaptic neurons, the activity of neurotransmitter receptors on the postsynaptic neurons, and/or ion channel conductance in the central nervous system by parenterally administering an effective amount of the monosaccharide(s) to a subject in need thereof.

39. The dietary supplement of claim 1 , wherein the supplement comprises, consists essentially of, or consists of a monosaccharide of D-galactose, and, optionally, at least one of L- fucose, D-mannose, GlcNAc, and/or an excipient.

40. The dietary supplement of claim 1, wherein the supplement comprises, consists essentially of, or consists of a monosaccharide of D-mannosc, and, optionally, at least one of D- galactose, L-fucose, GlcNAc, and/or an excipient.

41. The dietary supplement of claim 1, wherein the supplement comprises, consists essentially of, or consists of a monosaccharide of GlcNAc, and, optionally, at least one of D- galactose, D-mannose, L-fucose, and/or an excipient.

42. The dietary supplement of claim 1, wherein the supplement comprises, consists essentially of, or consists of two different monosaccharides, in particular L-fucose and D- galactose; L-fucose and D-mannose; or L-fucose and GlcNAc, and optionally an excipient.

43. The dietary supplement of claim 1, wherein the supplement comprises three different monosaccharides, in particular L-fucose, D-galactose and D-mannose; L-fucose, D-galactose and GLcNAc; or L-fucose, D-mannose and GlcNAc.

44. The dietary supplement of claim 1, wherein the supplement comprises four different monosaccharides, in particular L-fucose, D-galactose, D-mannose, and GlcNAc.