Regional N-Glycan and Lipid Analysis from Tissues Using MALDI-Mass Spectrometry Imaging

N-glycans and lipids are structural metabolites that play important roles in cellular processes. Both show unique regional distribution in tissues; therefore, spatial analyses of these metabolites are crucial to our understanding of cellular physiology. Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is an innovative technique that enables in situ detection of analytes with spatial distribution. This workflow details a MALDI-MSI protocol for the spatial profiling of N-glycans and lipids from tissues following application of enzyme and MALDI matrix. For complete details on the use and execution of this protocol, please refer to Drake et al. (2018) and Andres et al. (2020)

Astrocytic glycogen accumulation drives the pathophysiology of neurodegeneration in Lafora disease

The hallmark of Lafora disease, a fatal neurodegenerative disorder, is the accumulation of intracellular glycogen aggregates, called Lafora bodies. Until recently, it was widely believed that brain Lafora bodies were present exclusively in neurons and thus that Lafora disease pathology derived from their accumulation in this cell population. However, recent evidence indicates that Lafora bodies are also present in astrocytes. To define the role of astrocytic Lafora bodies in Lafora disease pathology, we deleted glycogen synthase specifically from astrocytes in a mouse model of the disease (malinKO). Strikingly, blocking glycogen synthesis in astrocytes-thus impeding Lafora bodies accumulation in this cell type-prevented the increase in neurodegeneration markers, autophagy impairment, and metabolic changes characteristic of the malinKO model. Conversely, mice that overaccumulate glycogen in astrocytes showed an increase in these markers. These results unveil the deleterious consequences of the deregulation of glycogen metabolism in astrocytes and change the perspective that Lafora disease is caused solely by alterations in neuron

BRAIN GLYCOGEN – BEYOND ENERGY STORAGE IN GLYCOGEN STORAGE DISEASES

Glycogen is a carbohydrate molecule that is traditionally viewed as a convenient and easily accessible energy storage form of glucose. However,emerging evidence supports the role of glycogen as more than a glucose storage form. During the last 20 years, glycogen has been shown to play pivotal roles in learning and memory, signaling events, viscosity, protein glycosylation, and be acritical hallmark in devastating diseases. Not only, does glycogen play a role as an energy substrate and critical metabolite during energy deprivation, glycogen is central for neurotransmitter homeostasis, tumor initiation, and ontributes to proper protein glycosylation in the brain. In this work, the crucial role of glycogen homeostasis in the healthy and diseased brain is elucidated with a focus on our emerging understanding of glycogen as a critical metabolite. These aspects will be discussed concerning our understanding of diseases with a focus on Glycogen Storage Diseases (GSDs), utilizing Lafora disease (LD) and Glucose transporter 1 deficiency syndrome (G1D) as model systems. Although these diseases have different genetic causes, they share several clinical and biochemical characteristics, including perturbed glycogen metabolism. Utilizing LD as a primary disease, we elucidated glycogen’sinfluence on neurotransmitter metabolism, the metabolic profile as a biomarker, and brain glycosylation. LD is classified as a glycogen storage disease and progressive myoclonus epilepsy caused by mutations in the genes EPM2A and EMP2B, encoding the glycogen phosphatase, laforin, and the E3 ubiquitin ligase, malin. Although laforin and malin are structurally and functionally different proteins, they are both involved in glycogen metabolism. Importantly, mutations in either gene lead to this devasting and fatal disease clinically presenting with increasing and worsening treatment-resistant epileptic seizure, loss of muscular control, dementia, and severe cognitive decline. The first symptoms generally start in the second decade of individuals who appears to develop normally, and the disease eventually leads to a vegetative state followed by death typically around 11 years after disease onset. A key hallmark of LD is the formation of insoluble glycogen-like aggregates known as Lafora bodies (LBs). LBs form in multiple tissues and several laboratories, using various mouse models, have demonstrated that LBs drive disease progression. However, the mechanism of disease is poorly understood, and there is a lack of robust biomarkers. To that end, we aimed to determine the metabolic phenotype of LD mouse and human samples by mass spectrometry analysis. The results revealed complex disturbances of central carbon metabolism in LD brain with perturbed oxidative glucose metabolism in both brain slices and primary cultures of neurons and astrocytes. Results from LD patient CSF samples indicate perturbations linked to neurotransmitter and glycogen metabolism as well as the hexosamine pathway.The distinct metabolic profile and perturbations in the hexosamine pathway suggest a connection to other carbohydrate pathways, including glycosylation.Indeed, we recently demonstrated that brain glycogen plays a key role in brain glycosylation, a posttranslational modification, and LBs disrupt this homeostasis. To further elucidate this aspect of glycogen metabolism, we here characterized glycogen and glycosylation in an LD mouse model, utilizing cutting-edge techniques such as Matrix-Assisted Laser Desorption Ionization Mass Spectrometry Imaging and Gas Chromatography-Mass Spectrometry.Furthermore, we determined how sustained oral administration of glucosamine, a building block for glycosylation and brain glycogen, impacts glycogen metabolism, glycosylation, and behavior in mice. Overall, oral supplementation of glucosamine positively affects the biochemical characteristics of the disease and impacts mouse behavior. Thus, we provide evidence of a crucial connection between perturbed glycogen metabolism and glycosylation in LD.Collectively, this work demonstrates divergent roles of glycogen as more than a glucose cache. We show that glycogen perturbations affect protein glycosylation and the potential value of a metabolic profile as a biomarker for GSDs. Future work will continue to elucidate our understanding of glycogen in health and disease

Hippocampal disruptions of synaptic and astrocyte metabolism are primary events of early amyloid pathology in the 5xFAD mouse model of Alzheimer's disease

Abstract Alzheimer’s disease (AD) is an unremitting neurodegenerative disorder characterized by cerebral amyloid-β (Aβ) accumulation and gradual decline in cognitive function. Changes in brain energy metabolism arise in the preclinical phase of AD, suggesting an important metabolic component of early AD pathology. Neurons and astrocytes function in close metabolic collaboration, which is essential for the recycling of neurotransmitters in the synapse. However, this crucial metabolic interplay during the early stages of AD development has not been sufficiently investigated. Here, we provide an integrative analysis of cellular metabolism during the early stages of Aβ accumulation in the cerebral cortex and hippocampus of the 5xFAD mouse model of AD. Our electrophysiological examination revealed an increase in spontaneous excitatory signaling in the 5xFAD hippocampus. This hyperactive neuronal phenotype coincided with decreased hippocampal tricarboxylic acid (TCA) cycle metabolism mapped by stable 13C isotope tracing. Particularly, reduced astrocyte TCA cycle activity and decreased glutamine synthesis led to hampered neuronal GABA synthesis in the 5xFAD hippocampus. In contrast, the cerebral cortex of 5xFAD mice displayed an elevated capacity for oxidative glucose metabolism, which may suggest a metabolic compensation in this brain region. We found limited changes when we explored the brain proteome and metabolome of the 5xFAD mice, supporting that the functional metabolic disturbances between neurons and astrocytes are early primary events in AD pathology. In addition, synaptic mitochondrial and glycolytic function was selectively impaired in the 5xFAD hippocampus, whereas non-synaptic mitochondrial function was maintained. These findings were supported by ultrastructural analyses demonstrating disruptions in mitochondrial morphology, particularly in the 5xFAD hippocampus. Collectively, our study reveals complex regional and cell-specific metabolic adaptations in the early stages of amyloid pathology, which may be fundamental for the progressing synaptic dysfunctions in AD