Gene Therapy Strategy for Alzheimer’s and Parkinson’s Diseases Aimed at Preventing the Formation of Neurotoxic Oligomers in SH-SY5Y Cells

We present here a gene therapy approach aimed at preventing the formation of Ca²⁺-permeable amyloid pore oligomers that are considered as the most neurotoxic structures in both Alzheimer’s and Parkinson’s diseases. Our study is based on the design of a small peptide inhibitor (AmyP53) that combines the ganglioside recognition properties of the β-amyloid peptide (Aβ, Alzheimer) and α-synuclein (α-syn, Parkinson). As gangliosides mediate the initial binding step of these amyloid proteins to lipid rafts of the brain cell membranes, AmyP53 blocks, at the earliest step, the Ca²⁺ cascade that leads to neurodegeneration. Using a lentivirus vector, we genetically modified brain cells to express the therapeutic coding sequence of AmyP53 in a secreted form, rendering these cells totally resistant to oligomer formation by either Aβ or α-syn. This protection was specific, as control mCherry-transfected cells remained fully sensitive to these oligomers. AmyP53 was secreted at therapeutic concentrations in the supernatant of cultured cells, so that the therapy was effective for both transfected cells and their neighbors. This study is the first to demonstrate that a unique gene therapy approach aimed at preventing the formation of neurotoxic oligomers by targeting brain gangliosides may be considered for the treatment of two major neurodegenerative disorders, Alzheimer’s and Parkinson’s diseases.     

A Review of the Common Neurodegenerative Disorders: Current Therapeutic Approaches and the Potential Role of Nanotherapeutics

Neurodegenerative disorders are primarily characterized by neuron loss. The most common neurodegenerative disorders include Alzheimer’s and Parkinson’s disease. Although there are several medicines currently approved for managing neurodegenerative disorders, a large majority of them only help with associated symptoms. This lack of pathogenesis-targeting therapies is primarily due to the restrictive effects of the blood–brain barrier (BBB), which keeps close to 99% of all “foreign substances” out of the brain. Since their discovery, nanoparticles have been successfully used for targeted delivery into many organs, including the brain. This review briefly describes the pathophysiology of Alzheimer’s, Parkinson’s disease, and amyotrophic lateral sclerosis, and their current management approaches. We then highlight the major challenges of brain-drug delivery, followed by the role of nanotherapeutics for the diagnosis and treatment of various neurological disorders.     

The Role of Extracellular Vesicles in Demyelination of the Central Nervous System

It is being increasingly demonstrated that extracellular vesicles (EVs) are deeply involved in the physiology of the central nervous system (CNS). Processes such as synaptic activity, neuron-glia communication, myelination and immune response are modulated by EVs. Likewise, these vesicles may participate in many pathological processes, both as triggers of disease or, on the contrary, as mechanisms of repair. EVs play relevant roles in neurodegenerative disorders such as Alzheimer’s or Parkinson’s diseases, in viral infections of the CNS and in demyelinating pathologies such as multiple sclerosis (MS). This review describes the involvement of these membrane vesicles in major demyelinating diseases, including MS, neuromyelitis optica, progressive multifocal leukoencephalopathy and demyelination associated to herpesviruses.     

GM3 Ganglioside Linked to Neurofibrillary Pathology in a Transgenic Rat Model for Tauopathy

Glycosphingolipids (GSLs) are amphipathic lipids composed of a sphingoid base and a fatty acyl attached to a saccharide moiety. GSLs play an important role in signal transduction, directing proteins within the membrane, cell recognition, and modulation of cell adhesion. Gangliosides and sulfatides belong to a group of acidic GSLs, and numerous studies report their involvement in neurodevelopment, aging, and neurodegeneration. In this study, we used an approach based on hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution tandem mass spectrometry (HRMS/MS) to characterize the glycosphingolipid profile in rat brain tissue. Then, we screened characterized lipids aiming to identify changes in glycosphingolipid profiles in the normal aging process and tau pathology. Thorough screening of acidic glycosphingolipids in rat brain tissue revealed 117 ganglioside and 36 sulfatide species. Moreover, we found two ganglioside subclasses that were not previously characterized—GT1b-Ac2 and GQ1b-Ac2. The semi-targeted screening revealed significant changes in the levels of sulfatides and GM1a gangliosides during the aging process. In the transgenic SHR24 rat model for tauopathies, we found elevated levels of GM3 gangliosides which may indicate a higher rate of apoptotic processes.     

Behavior of sugar derivatives in procedure for ganglioside isolation

A common method of studying ganglioside metabolism is to measure the amounts of radioactivity incorporated into ganglioside from a radiolabeled precursor. This requires that radioactive nonganglioside material be completely removed from the ganglioside fraction. Nucleotide sugars and aminosugars comprise an important source of such contaminants. Therefore, we have studied their behaviors in several procedures currently employed to isolate gangliosides. Over 50% of the radioactivity associated with several nucleotide sugars added to a brain homogenate is extracted with chloroform/methanol (2∶1, v/v), and most of this is recovered in the upper phase of a Folch partition. Dialysis against water removes almost all of the free aminosugar but only 70% of nucleotide sugar. Treatment with alkaline phosphatase, phosphodiesterase and alkaline methanol followed by dialysis removes almost all of the nucleotide diphosphate sugars but only 88% of cytidine 5′-monophosphate sialic acid (CMP-NeuAc). Nucleotide sugars cannot be separated from gangliosides by Unisil or latrobead chromatography, but nucleotide diphosphate sugars and gangliosides are resolved with Sephadex LH-20 chromatography following treatment with phosphodiesterase and alkaline phosphatase. CMP-NeuAc was not satisfactorily separated from gangliosides using any of the procedures.     

Small GTPases of the Rab and Arf Families: Key Regulators of Intracellular Trafficking in Neurodegeneration

Small guanosine triphosphatases (GTPases) of the Rab and Arf families are key regulators of vesicle formation and membrane trafficking. Membrane transport plays an important role in the central nervous system. In this regard, neurons require a constant flow of membranes for the correct distribution of receptors, for the precise composition of proteins and organelles in dendrites and axons, for the continuous exocytosis/endocytosis of synaptic vesicles and for the elimination of dysfunctional proteins. Thus, it is not surprising that Rab and Arf GTPases have been associated with neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Both pathologies share characteristics such as the presence of protein aggregates and/or the fragmentation of the Golgi apparatus, hallmarks that have been related to both Rab and Arf GTPases functions. Despite their relationship with neurodegenerative disorders, very few studies have focused on the role of these GTPases in the pathogenesis of neurodegeneration. In this review, we summarize their importance in the onset and progression of Alzheimer’s and Parkinson’s diseases, as well as their emergence as potential therapeutical targets for neurodegeneration.     

Mechanism of Ganglioside Receptor Recognition by Botulinum Neurotoxin Serotype E

The botulinum neurotoxins are potent molecules that are not only responsible for the lethal paralytic disease botulism, but have also been harnessed for therapeutic uses in the treatment of an increasing number of chronic neurological and neuromuscular disorders, in addition to cosmetic applications. The toxins act at the cholinergic nerve terminals thanks to an efficient and specific mechanism of cell recognition which is based on a dual receptor system that involves gangliosides and protein receptors. Binding to surface-anchored gangliosides is the first essential step in this process. Here, we determined the X-ray crystal structure of the binding domain of BoNT/E, a toxin of clinical interest, in complex with its GD1a oligosaccharide receptor. Beyond confirmation of the conserved ganglioside binding site, we identified key interacting residues that are unique to BoNT/E and a significant rearrangement of loop 1228–1237 upon carbohydrate binding. These observations were also supported by thermodynamic measurements of the binding reaction and assessment of ganglioside selectivity by immobilised-receptor binding assays. These results provide a structural basis to understand the specificity of BoNT/E for complex gangliosides.     

ST6GALNAC5 Expression Decreases the Interactions between Breast Cancer Cells and the Human Blood-Brain Barrier

The ST6GALNAC5 gene that encodes an α2,6-sialyltransferase involved in the biosynthesis of α-series gangliosides, was previously identified as one of the genes that mediate breast cancer metastasis to the brain. We have shown that the expression of ST6GALNAC5 in MDA-MB-231 breast cancer cells resulted in the expression of G_D1α ganglioside at the cell surface. By using a human blood-brain barrier in vitro model recently developed, consisting in CD34⁺ derived endothelial cells co-cultivated with pericytes, we show that ST6GALNAC5 expression decreased the interactions between the breast cancer cells and the human blood-brain barrier.     

The Role of Gut Microbiota and Gut–Brain Interplay in Selected Diseases of the Central Nervous System

The gut microbiome has attracted increasing attention from researchers in recent years. The microbiota can have a specific and complex cross-talk with the host, particularly with the central nervous system (CNS), creating the so-called “gut–brain axis”. Communication between the gut, intestinal microbiota, and the brain involves the secretion of various metabolites such as short-chain fatty acids (SCFAs), structural components of bacteria, and signaling molecules. Moreover, an imbalance in the gut microbiota composition modulates the immune system and function of tissue barriers such as the blood–brain barrier (BBB). Therefore, the aim of this literature review is to describe how the gut–brain interplay may contribute to the development of various neurological disorders, combining the fields of gastroenterology and neuroscience. We present recent findings concerning the effect of the altered microbiota on neurodegeneration and neuroinflammation, including Alzheimer’s and Parkinson’s diseases, as well as multiple sclerosis. Moreover, the impact of the pathological shift in the microbiome on selected neuropsychological disorders, i.e., major depressive disorders (MDD) and autism spectrum disorder (ASD), is also discussed. Future research on the effect of balanced gut microbiota composition on the gut–brain axis would help to identify new potential opportunities for therapeutic interventions in the presented diseases.     

Ganglioside Composition Distinguishes Anaplastic Ganglioglioma Tumor Tissue from Peritumoral Brain Tissue: Complementary Mass Spectrometry and Thin-Layer Chromatography Evidence

Gangliosides serve as antitumor therapy targets and aberrations in their composition strongly correlate with tumor growth and invasiveness. Anaplastic ganglioglioma is a rare, poorly characterized, malignant neuronal–glial tumor type. We present the first comparative characterization of ganglioside composition in anaplastic ganglioglioma vs. peritumoral and healthy brain tissues by combining mass spectrometry and thin-layer chromatography. Anaplastic ganglioglioma ganglioside composition was highly distinguishable from both peritumoral and healthy tissue despite having five to six times lower total content. Ten out of twelve MS-identified ganglioside classes, defined by unique glycan residues, were represented by a large number and considerable abundance of individual species with different fatty acid residues (C16–C24) in ceramide portions. The major structurally identified class was tumor-associated GD3 (>50%) with 11 species; GD3 (d18:1/24:0) being the most abundant. The dominant sphingoid base residue in ganglioside ceramides was sphingosine (d18:1), followed by eicosasphingosine (d20:1). The peritumoral tissue ganglioside composition was estimated as normal. Specific ganglioside composition and large variability of ganglioside ceramide structures determined in anaplastic ganglioglioma demonstrate realistic ganglioside expression patterns and correspond to the profile of high-grade malignancy brain tumors.     

Total gangliosides,ganglioside species and the activity of neuraminidase in different brain regions and spinal cord of normal and undernourished rats

Total gangliosides, their concentrations and the distribution of individual ganglioside species were determined in the spinal cord, pons-medulla, midbrain, cerebellum, hypothalamus, cerebral cortex, olfactory lobes and the rest of the forebrain, of 18- and 33-day-old normal and food-restricted and rehabilitated rats. The activity of neuraminidase in the different regions of the brain and spinal cord was determined. Differences in total gangliosides as well as individual species in different regions of the brain were observed. Among brain regions, while total ganglioside content was significantly reduced in the case of the cerebellum, the hypothalamus was the most affected in the distribution of ganglioside species and the activity of neuraminidase was decreased. The significance of the distribution of ganglioside species in different brain regions in relation to the activity of neuraminidase is discussed.     

Lactational changes in concentration and distribution of ganglioside molecular species in human breast milk from Chinese mothers

Gangliosides play a critical role in human brain development and function. Human breast milk (HBM) is an important dietary source of gangliosides for the growing infant. In this study, ganglioside concentrations were measured in the breast milk from a cross‐sectional sample of Chinese mothers over an 8‐month lactation period. The average total ganglioside concentration increased from 13.1 mg/l during the first month to 20.9 mg/l by 8 months of lactation. The average concentration during the typically solely breast‐feeding period of 1?6 months was 18.9 mg/l. This is the first study to report the relative distribution of the individual ganglioside molecular species through lactation for any population group. The ganglioside molecular species are made up of different fatty acid moieties that influence the physical properties of these gangliosides, and hence affect their function. The GM₃ molecular species containing long‐chain acyl fatty acids had the most prominent changes, increasing in both concentration and relative distribution. The equivalent long‐chain acyl fatty acid GD₃ molecular species typically decreased in concentration and relative distribution. The lactational trends for both concentration and relative distribution for the very long‐chain acyl fatty acid molecular species were more varied. The major GM₃ and GD₃ molecular species during lactation were d40:1 and d42:1, respectively. An understanding of ganglioside molecular species distribution in HBM is essential for accurate application of mass spectrometry methods for ganglioside quantification.     

Lipids of retina: I. Analysis of gangliosides in beef retina by thin layer chromatography

Lipids were extracted from beef retina by chloroform-methanol (2∶1); the gangliosides were removed from the total lipid extract by partitioning into water and chromatographing on thin layer plates coated with silica gel. The analytical methods are described for estimating ganglioside components, i.e., N-acetyl neuraminic acid, hexoses, hexosamine, sphingosine and fatty acids, in the presence of silica gel. Major gangliosides present in beef retina have been tentatively identified as follows: a ganglioside containing two N-acetyl neuraminyl groups but no hexosamine; two gangliosides containing two N-acetyl neuraminyl groups and one hexosamine; and a ganglioside with three N-acetyl neuraminyl moieties and one hexosamine.     

A comparison of the ganglioside distributions of fat tissues in various animals by two-dimensional thin layer chromatography

The ganglioside distributions of various fat tissues from human, rabbit, rat, mouse, chicken and frog were compared with pig adipose gangliosides by two-dimensional thin layers chromatography. It was found that there is a remarkable species variation in ganglioside distribution, especially in the composition and relative concentration of complex gangliosides. Differing from pig adipose tissues, those of human, rabbit, rat, mouse, chicken, but not frog, contained GM3 as a most abundant ganglioside. The data for human, rabbit and chicken indicated a simple distribution of only NeuActype gangliosides, while those for rat and mouse indicated a rather complicated pattern containing both NeuAc- and NeuGc-type gangliosides. The ganglioside pattern of the frog fat body differed markedly from those of mammalian fat tissues because of the presence of three different, unsual monosialosylgangliosides as major components. In other respects, a substantial amount of disialosylgangliosides was commonly found in all animal fat tissues.     

Fatty acid and long chain base composition of adrenal medulla gangliosides

Five ganglioside fractions from bovine adrenal medulla were analyzed with respect to their fatty acid and long chain base compositions. The five fractions included two hematosides and three hexasamine-containing species, the latter having chromatographic properties comparable to the major gangliosides of brain. The fatty acid compositions of all five were similar: 22∶0 was the most abundant, but significant amounts of 16∶0, 18∶0, 24∶0 and 24∶1 were also present. No hydroxy fatty acids were detected. The principal long chain base in each fraction was 4-sphingenine (sphingosine), with lesser amounts of the C₁₆ and C₁₇ homologues. Minor quantities of the corresponding saturated bases were also detected. These were identified by two gas liquid chromatography methods: (a) trimethylsilyl ether derivatives, (b) aldehydes formed by periodate oxidation of the long chain bases. No 4-eicosasphingenine (C₂₀-sphingosine), characteristic of brain gangliosides, was found in any of the fractions. The results demonstrate that gangliosides of the adrenal medulla show tissue specificity in fatty acid and long chain base composition which is independent of carbohydrate structure.     

Behavior of gangliosides on dialysis

Mixed brain gangliosides or individual ganglioside GM₁ were dissolved in one of the following solvents: (a) water, (b) 0.1 M aqueous KC1, or (c) methanol-aqueous 0.1 M KC1 at concentrations ranging from 10⁻⁸ to 10⁻³ M, and were submitted to dialysis against distilled water for up to 4 days. No significant loss of gangliosides on dialysis was observed when gangliosides were dissolved in water or 0.1 M aqueous KC1, but a loss occurred when the ganglioside solution contained methanol; the loss was greater at the lowest ganglioside concentration. However, losses ceased to occur after 1 day when methanol was removed from the dialysis sac.     

Analytical separation of nonlipid water soluble substances and gangliosides from other lipids by dextran gel column chromatography

A column chromatographic procedure is reported utilizing a dextran gel (Sephadex) for the complete separation of the major lipid classes from water-soluble nonlipids. Lipids other than gangliosides are eluted first with chloroform/methanol 19/1 saturated with water, gangliosides with chloroform/methanol/water containing acetic acid, and water-soluble nonlipids with methanol/water 1/1. Results for adult human whole brain, grey and white matter, and normal infant whole brain lipids are presented. With beef brain lipid as sample the ganglioside fraction is essentially pure, but with human brain lipid samples only about 70% of the second fraction is ganglioside. All ganglioside and water soluble nonlipid of a human spleen chloroform/methanol extract was separated from lipids with the procedure. Control studies with P³²O₄≡ and C₁₄ labelled glucose showed that all counts were present in fraction 3. Similar studies with C₁₄ labelled amino acids (glycine, serine, alanine, phenylalanine) showed that only phenylalanine counts were eluted in fraction 2 along with the gangliosides. The procedure was applied for removal of large amounts of ammonium acetate from DEAE cellulose column fractions and for complete removal of adsorbent and salts from lipids eluted from thin-layer chromatograms. After passage through the dextran gel columns, lipids eluted from thin-layer chromatograms were found to give infrared spectra identical to those of pure samples obtained by other procedures.     

Peptidomic Approaches and Observations in Neurodegenerative Diseases

Mass spectrometry (MS), with its immense technological developments over the last two decades, has emerged as an unavoidable technique in analyzing biomolecules such as proteins and peptides. Its multiplexing capability and explorative approach make it a valuable tool for analyzing complex clinical samples concerning biomarker research and investigating pathophysiological mechanisms. Peptides regulate various biological processes, and several of them play a critical role in many disease-related pathological conditions. One important example in neurodegenerative diseases is the accumulation of amyloid-beta peptides (Aβ) in the brain of Alzheimer’s disease (AD) patients. When investigating brain function and brain-related pathologies, such as neurodegenerative diseases, cerebrospinal fluid (CSF) represents the most suitable sample because of its direct contact with the brain. In this review, we evaluate publications applying peptidomics analysis to CSF samples, focusing on neurodegenerative diseases. We describe the methodology of peptidomics analysis and give an overview of the achievements of CSF peptidomics over the years. Finally, publications reporting peptides regulated in AD are discussed.