Polyglucosan Bodies Research Articles

Neuromuscular junction dysfunction in Lafora disease.

Lafora disease (LD), a fatal neurodegenerative disorder, is caused by mutations in the EPM2A gene encoding laforin phosphatase or NHLRC1 gene encoding malin ubiquitin ligase. LD symptoms include epileptic seizures, ataxia, dementia and cognitive decline. Studies on LD have primarily concentrated on the pathophysiology in the brain. A few studies have reported motor symptoms, muscle weakness and muscle atrophy. Intriguingly, skeletal muscles are known to accumulate Lafora polyglucosan bodies. Using laforin-deficient mice, an established model for LD, we demonstrate that LD pathology correlated with structural and functional impairments in the neuromuscular junction (NMJ). Specifically, we found impairment in NMJ transmission, which coincided with altered expression of NMJ-associated genes and reduced motor endplate area, fragmented junctions and loss of fully innervated junctions at the NMJ. We also observed a reduction in alpha-motor neurons in the lumbar spinal cord, with significant presynaptic morphological alterations. Disorganised myofibrillar patterns, slight z-line streaming and muscle atrophy were also evident in LD animals. In summary, our study offers insight into the neuropathic and myopathic alterations leading to motor deficits in LD.

518P Proteomic characterization of polyglucosan bodies in skeletal muscle in glycogenin-1 deficiency

518P Proteomic characterization of polyglucosan bodies in skeletal muscle in glycogenin-1 deficiency

Incidence and characterization of polyglucosan bodies in the cerebella of montserrat orioles (Icterus oberi).

Polyglucosan bodies are accumulations of insoluble glucose polymers and proteins that form intracytoplasmic inclusions in the brain, large numbers of which can be indicative of neurodegenerative diseases such as Lafora disease. Montserrat orioles (Icterus oberi) are an icterid passerine endemic to Montserrat with conservation populations maintained in captivity abroad. We demonstrate that polyglucosan bodies are unusually abundant in the cerebellar molecular and Purkinje cell layers and cerebellar peduncles of captive-bred and wild-caught Montserrat orioles. The bodies are periodic acid-Schiff positive and diastase resistant and label with concanavalin A and for ubiquitin, consistent with those seen in humans. We found no association of the polyglucosan bodies with concurrent neurological lesions or clinical signs, nor with EPM2A and EPM2B gene mutations associated with Lafora disease. We conclude that an abundance of cerebellar polyglucosan bodies may be a normal finding in aged Montserrat orioles and not a threat to the captive breeding population.

The Pgb1 locus controls glycogen aggregation in astrocytes of the aged hippocampus without impacting cognitive function.

In aged humans and mice, aggregates of hypobranched glycogen molecules called polyglucosan bodies (PGBs) accumulate in hippocampal astrocytes. PGBs are known to drive cognitive decline in neurological diseases but remain largely unstudied in the context of typical brain aging. Here, we show that PGBs arise in autophagy-dysregulated astrocytes of the aged C57BL/6J mouse hippocampus. To map the genetic cause of age-related PGB accumulation, we quantified PGB burden in 32 fully sequenced BXD-recombinant inbred mouse strains, which display a 400-fold variation in hippocampal PGB burden at 16-18 months of age. A major modifier locus was mapped to chromosome 1 at 72-75 Mb, which we defined as the Pgb1 locus. To evaluate candidate genes and downstream mechanisms by which Pgb1 controls the aggregation of glycogen, extensive hippocampal transcriptomic and proteomic datasets were produced for aged mice of the BXD family. We utilized these datasets to identify Smarcal1 and Usp37 as potential regulators of PGB accumulation. To assess the effect of PGB burden on age-related cognitive decline, we performed phenome-wide association scans, transcriptomic analyses as well as conditioned fear memory and Y-maze testing. Importantly, we did not find any evidence suggesting a negative impact of PGBs on cognition. Taken together, our study demonstrates that the Pgb1 locus controls glycogen aggregation in astrocytes of the aged hippocampus without affecting age-related cognitive decline.

Increase in wasteosomes (corpora amylacea) in frontotemporal lobar degeneration with specific detection of tau, TDP-43 and FUS pathology

Wasteosomes (or corpora amylacea) are polyglucosan bodies that appear in the human brain with aging and in some neurodegenerative diseases, and have been suggested to have a potential role in a nervous system cleaning mechanism. Despite previous studies in several neurodegenerative disorders, their status in frontotemporal lobar degeneration (FTLD) remains unexplored. Our study aims to characterize wasteosomes in the three primary FTLD proteinopathies, assessing frequency, distribution, protein detection, and association with aging or disease duration. Wasteosome scores were obtained in various brain regions from 124 post-mortem diagnosed sporadic FTLD patients, including 75 participants with tau (FTLD-tau), 42 with TAR DNA-binding protein 43 (FTLD-TDP), and 7 with Fused in Sarcoma (FTLD-FUS) proteinopathies, along with 29 control subjects. The wasteosome amount in each brain region for the different FLTD patients was assessed with a permutation test with age at death and sex as covariables, and multiple regressions explored associations with age at death and disease duration. Double immunofluorescence studies examined altered proteins linked to FTLD in wasteosomes. FTLD patients showed a higher accumulation of wasteosomes than control subjects, especially those with FTLD-FUS. Unlike FTLD-TDP and control subjects, wasteosome accumulation did not increase with age in FTLD-tau and FTLD-FUS. Cases with shorter disease duration in FTLD-tau and FTLD-FUS seemed to exhibit higher wasteosome quantities, whereas FTLD-TDP appeared to show an increase with disease progression. Immunofluorescence studies revealed the presence of tau and phosphorylated-TDP-43 in the periphery of isolated wasteosomes in some patients with FTLD-tau and FTLD-TDP, respectively. Central inclusions of FUS were observed in a higher number of wasteosomes in FTLD-FUS patients. These findings suggest a role of wasteosomes in FTLD, especially in the more aggressive forms of FLTD-FUS. Detecting these proteins, particularly FUS, in wasteosomes from cerebrospinal fluid could be a potential biomarker for FTLD.

1H and 31P magnetic resonance spectroscopy reveals potential pathogenic and biomarker metabolite alterations in Lafora disease.

Lafora disease is a fatal teenage-onset progressive myoclonus epilepsy and neurodegenerative disease associated with polyglucosan bodies. Polyglucosans are long-branched and as a result precipitation- and aggregation-prone glycogen. In mouse models, downregulation of glycogen synthase, the enzyme that elongates glycogen branches, prevents polyglucosan formation and rescues Lafora disease. Mouse work, however, has not yet revealed the mechanisms of polyglucosan generation, and few in vivo human studies have been performed. Here, non-invasive in vivo magnetic resonance spectroscopy (1H and 31P) was applied to test scan feasibility and assess neurotransmitter balance and energy metabolism in Lafora disease towards a better understanding of pathogenesis. Macromolecule-suppressed gamma-aminobutyric acid (GABA)-edited 1H magnetic resonance spectroscopy and 31P magnetic resonance spectroscopy at 3 and 7 tesla, respectively, were performed in 4 Lafora disease patients and a total of 21 healthy controls (12 for the 1H magnetic resonance spectroscopy and 9 for the 31PMRS). Spectra were processed using in-house software and fit to extract metabolite concentrations. From the 1H spectra, we found 33% lower GABA concentrations (P = 0.013), 34% higher glutamate + glutamine concentrations (P = 0.011) and 24% lower N-acetylaspartate concentrations (P = 0.0043) in Lafora disease patients compared with controls. From the 31P spectra, we found 34% higher phosphoethanolamine concentrations (P = 0.016), 23% lower nicotinamide adenine dinucleotide concentrations (P = 0.003), 50% higher uridine diphosphate glucose concentrations (P = 0.004) and 225% higher glucose 6-phosphate concentrations in Lafora disease patients versus controls (P = 0.004). Uridine diphosphate glucose is the substrate of glycogen synthase, and glucose 6-phosphate is its extremely potent allosteric activator. The observed elevated uridine diphosphate glucose and glucose 6-phosphate levels are expected to hyperactivate glycogen synthase and may underlie the generation of polyglucosans in Lafora disease. The increased glutamate + glutamine and reduced GABA indicate altered neurotransmission and energy metabolism, which may contribute to the disease's intractable epilepsy. These results suggest a possible basis of polyglucosan formation and potential contributions to the epilepsy of Lafora disease. If confirmed in larger human and animal model studies, measurements of the dysregulated metabolites by magnetic resonance spectroscopy could be developed into non-invasive biomarkers for clinical trials.

Myofiber-type-dependent 'boulder' or 'multitudinous pebble' formations across distinct amylopectinoses.

At least five enzymes including three E3 ubiquitin ligases are dedicated to glycogen's spherical structure. Absence of any reverts glycogen to a structure resembling amylopectin of the plant kingdom. This amylopectinosis (polyglucosan body formation) causes fatal neurological diseases including adult polyglucosan body disease (APBD) due to glycogen branching enzyme deficiency, Lafora disease (LD) due to deficiencies of the laforin glycogen phosphatase or the malin E3 ubiquitin ligase and type 1 polyglucosan body myopathy (PGBM1) due to RBCK1 E3 ubiquitin ligase deficiency. Little is known about these enzymes' functions in glycogen structuring. Toward understanding these functions, we undertake a comparative murine study of the amylopectinoses of APBD, LD and PGBM1. We discover that in skeletal muscle, polyglucosan bodies form as two main types, small and multitudinous ('pebbles') or giant and single ('boulders'), and that this is primarily determined by the myofiber types in which they form, 'pebbles' in glycolytic and 'boulders' in oxidative fibers. This pattern recapitulates what is known in the brain in LD, innumerable dust-like in astrocytes and single giant sized in neurons. We also show that oxidative myofibers are relatively protected against amylopectinosis, in part through highly increased glycogen branching enzyme expression. We present evidence of polyglucosan body size-dependent cell necrosis. We show that sex influences amylopectinosis in genotype, brain region and myofiber-type-specific fashion. RBCK1 is a component of the linear ubiquitin chain assembly complex (LUBAC), the only known cellular machinery for head-to-tail linear ubiquitination critical to numerous cellular pathways. We show that the amylopectinosis of RBCK1 deficiency is not due to loss of linear ubiquitination, and that another function of RBCK1 or LUBAC must exist and operate in the shaping of glycogen. This work opens multiple new avenues toward understanding the structural determinants of the mammalian carbohydrate reservoir critical to neurologic and neuromuscular function and disease.

Proteomic investigations of adult polyglucosan body disease: insights into the pathobiology of a neurodegenerative disorder.

Inadequate glycogen branching enzyme 1 (GBE1) activity results in different forms of glycogen storage disease type IV, including adult polyglucosan body disorder (APBD). APBD is clinically characterized by adult-onset development of progressive spasticity, neuropathy, and neurogenic bladder and is histologically characterized by the accumulation of structurally abnormal glycogen (polyglucosan bodies) in multiple cell types. How insufficient GBE1 activity causes the disease phenotype of APBD is poorly understood. We hypothesized that proteomic analysis of tissue from GBE1-deficient individuals would provide insights into GBE1-mediated pathobiology. In this discovery study, we utilized label-free LC-MS/MS to quantify the proteomes of lymphoblasts from 3 persons with APBD and 15 age- and gender-matched controls, with validation of the findings by targeted MS. There were 531 differentially expressed proteins out of 3,427 detected between APBD subjects vs. controls, including pronounced deficiency of GBE1. Bioinformatic analyses indicated multiple canonical pathways and protein-protein interaction networks to be statistically markedly enriched in APBD subjects, including: RNA processing/transport/translation, cell cycle control/replication, mTOR signaling, protein ubiquitination, unfolded protein and endoplasmic reticulum stress responses, glycolysis and cell death/apoptosis. Dysregulation of these processes, therefore, are primary or secondary factors in APBD pathobiology in this model system. Our findings further suggest that proteomic analysis of GBE1 mutant lymphoblasts can be leveraged as part of the screening for pharmaceutical agents for the treatment of APBD.

VP343 Glycogen storage disease type IV without polyglucosan bodies: report of three cases and literature review

VP343 Glycogen storage disease type IV without polyglucosan bodies: report of three cases and literature review

Muscle Pathology in a Case of Glycogen Storage Disease III (P6-8.014)

<h3>Objective:</h3> To describe histopathological features on muscle biopsy in glycogen storage disease III (GSDIII). <h3>Background:</h3> GSDIII is a rare disease caused by autosomal recessive mutations in <i>AGL</i> which encodes the glycogen debranching enzyme. Enzyme deficiency leads to glycogen accumulation primarily in liver, heart and skeletal muscle. Clinical presentation varies, and given the rarity, there is a paucity of literature on the skeletal muscle histopathology. <h3>Design/Methods:</h3> We describe a patient suspected to have GSD based on muscle biopsy and subsequently confirmed on exome sequencing. <h3>Results:</h3> A 53-year-old man with history of congenital hepatomegaly and hypertrophic cardiomyopathy presented with 3 years of proximal muscle weakness and atrophy. Family history was notable for elevated liver enzymes in his mother. Examination showed symmetric proximal weakness with reduced reflexes. Electrodiagnostic testing revealed a myopathic pattern with muscle membrane irritability. CK was elevated at 7207 U/L, but testing was otherwise unremarkable including GAA enzyme activity and myositis and LGMD panels. Muscle biopsy showed a severe vacuolar myopathy with large vacuoles filled with lightly basophilic PAS-positive and diastase sensitive material, indicative of normal glycogen and arguing against polyglucosan bodies. Additional myopathic features included significant myofiber size variation, ring fibers, and necrotic and regenerating myofibers. Atrophic nonspecific esterase-positive myofibers were also present. Acid phosphatase was negative and electron microscopy demonstrated abundant non-membrane bound sarcoplasmic glycogen, consistent with a non-lysosomal glycogen storage myopathy. Subsequent exome sequencing revealed two clinically significant variants in the <i>AGL</i> gene, c.3965delT (p.V1322AfsX27) and c.256 C&gt;T (p.Q86X). <h3>Conclusions:</h3> In GSDIII, muscle biopsy shows prominent vacuolar pathology with abundant non-membrane bound sarcoplasmic glycogen deposits. In contrast to Pompe disease, acid phosphatase is negative, demonstrating the absence of lysosomal activation in GSDIII. <b>Disclosure:</b> Dr. Alrasheed has nothing to disclose. Dr. Mozaffar has received personal compensation in the range of $10,000-$49,999 for serving as a Consultant for Alexion Pharmaceuticals. Dr. Mozaffar has received personal compensation in the range of $10,000-$49,999 for serving as a Consultant for Argenx. Dr. Mozaffar has received personal compensation in the range of $10,000-$49,999 for serving as a Consultant for Sanofi-Genzyme. Dr. Mozaffar has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Amicus. Dr. Mozaffar has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Modis Therapeutics. Dr. Mozaffar has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for UCB/ Ra Pharmaceuticals. Dr. Mozaffar has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Maze Therapeutics. Dr. Mozaffar has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Spark Therapeutics. Dr. Mozaffar has received personal compensation in the range of $5,000-$9,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Sarepta. Dr. Mozaffar has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for National Institutes of Health. Dr. Mozaffar has received personal compensation in the range of $5,000-$9,999 for serving on a Speakers Bureau for Argenx. Dr. Mozaffar has received personal compensation in the range of $500-$4,999 for serving as an officer or member of the Board of Directors for Neuromuscular Disease Foundation. The institution of Dr. Mozaffar has received research support from NIH. The institution of Dr. Mozaffar has received research support from Muscular Dystrophy Association. The institution of Dr. Mozaffar has received research support from Sanofi-Genzyme. The institution of Dr. Mozaffar has received research support from Argenx. The institution of Dr. Mozaffar has received research support from Amicus Therapeutics. The institution of Dr. Mozaffar has received research support from Spark Therapeutics. The institution of Dr. Mozaffar has received research support from Audentes Therapeutics. The institution of Dr. Mozaffar has received research support from Cartesian. Dr. Mozaffar has received personal compensation in the range of $500-$4,999 for serving as a Study Section Member with NIH. Mari Perez-Rosendahl has nothing to disclose.

Uncovering tau in wasteosomes (corpora amylacea) of Alzheimer's disease patients.

Brain corpora amylacea, recently renamed as wasteosomes, are polyglucosan bodies that appear during aging and some neurodegenerative conditions. They collect waste substances and are part of a brain cleaning mechanism. For decades, studies on their composition have produced inconsistent results and the presence of tau protein in them has been controversial. In this work, we reanalyzed the presence of this protein in wasteosomes and we pointed out a methodological problem when immunolabeling. It is well known that to detect tau it is necessary to perform an antigen retrieval. However, in the case of wasteosomes, an excessive antigen retrieval with boiling dissolves their polyglucosan structure, releases the entrapped proteins and, thus, prevents their detection. After performing an adequate pre-treatment, with an intermediate time of boiling, we observed that some brain wasteosomes from patients with Alzheimer's disease (AD) contained tau, while we did not detect tau protein in those from non-AD patients. These observations pointed the different composition of wasteosomes depending on the neuropathological condition and reinforce the role of wasteosomes as waste containers.

Deciphering the Polyglucosan Accumulation Present in Lafora Disease Using an Astrocytic Cellular Model.

Lafora disease (LD) is a neurological disorder characterized by progressive myoclonus epilepsy. The hallmark of the disease is the presence of insoluble forms of glycogen (polyglucosan bodies, or PGBs) in the brain. The accumulation of PGBs is causative of the pathophysiological features of LD. However, despite the efforts made by different groups, the question of why PGBs accumulate in the brain is still unanswered. We have recently demonstrated that, in vivo, astrocytes accumulate most of the PGBs present in the brain, and this could lead to astrocyte dysfunction. To develop a deeper understanding of the defects present in LD astrocytes that lead to LD pathophysiology, we obtained pure primary cultures of astrocytes from LD mice from the postnatal stage under conditions that accumulate PGBs, the hallmark of LD. These cells serve as novel in vitro models for studying PGBs accumulation and related LD dysfunctions. In this sense, the metabolomics of LD astrocytes indicate that they accumulate metabolic intermediates of the upper part of the glycolytic pathway, probably as a consequence of enhanced glucose uptake. In addition, we also demonstrate the feasibility of using the model in the identification of different compounds that may reduce the accumulation of polyglucosan inclusions.

Histological, immunohistochemical and ultrastructural features of polyglucosan bodies in uterine smooth muscle of pet rabbits (Oryctolaguscuniculus)

We document the frequency and morphological and immunohistochemical features of inclusion bodies in uterine smooth muscle cells in 56 (76%) of 74 investigated pet rabbits (Oryctolagus cuniculus). Inclusion bodies began to appear at the age of 2 years and their frequency increased with age (P = 0.047, r = 0.33). They ranged from 5 to 20 μm in diameter, were slightly basophilic to amphophilic with well-delimited oval bodies in haematoxylin and eosin-stained tissue sections and formed in the cytoplasm of the uterine smooth muscle cells with displacement of the cell nuclei. The inclusion bodies were positive with periodic acid–Schiff, Best's carmine, Lugol's iodine and Grocott's methenamine silver methods. They were immunoreactive to a monoclonal antibody raised against human polyglucosan and negative with monoclonal antibodies for several intermediate filament proteins. Electron microscopy revealed that they were non-membranous structures composed of electron-dense amorphous material. The morphological, histochemical, immunohistochemical and ultrastructural features of the inclusion bodies in the rabbi uteri were similar to those of human polyglucosan bodies (PGBs). PGBs appear to occur at a high frequency in the uterus of rabbits, which are known to be susceptible to uterine diseases.

Abundant copathologies of polyglucosan bodies, frontotemporal lobar degeneration with TDP-43 inclusions and ageing-related tau astrogliopathy in a family with a GBE1 mutation.

Adult polyglucosan body disease (APBD) is a progressive neurogenetic disorder caused by 1,4-alpha-glucan branching enzyme 1 (GBE1) mutation with an accumulation of polyglucosan bodies (PBs) in the central and peripheral nervous systems as a pathological hallmark. Here, we report two siblings in a family with a GBE1 mutation with prominent frontotemporal lobar degeneration with TAR DNA-binding protein 43 (FTLD-TDP) and ageing-related tau astrogliopathy (ARTAG) copathologies with PBs in the central nervous system. Whole-genome sequencing (WGS) followed by Sanger sequencing (SS) was performed on three affected and two unaffected siblings in a pedigree diagnosed with familial frontotemporal dementia. Out of the affected siblings, autopsies were conducted on two cases, and brain samples were used for biochemical and histological analyses. Brain sections were stained with haematoxylin and eosin and immunostained with antibodies against ubiquitin, tau, amyloid β, α-synuclein, TDP-43 and fused in sarcoma (FUS). A novel single nucleotide deletion in GBE1, c.1280delG, was identified, which is predicted to result in a reading frameshift, p.Gly427Glufs*9. This variant segregated with disease in the family, is absent from population databases and is predicted to cause loss of function, a known genetic mechanism for APBD. The affected siblings showed a greater than 50% decrease in GBE protein levels. Immunohistochemical analysis revealed widespread FTLD-TDP (type A) and ARTAG pathologies as well as PBs in the brains of two affected siblings for whom an autopsy was performed. This is the first report of a family with several individuals with a FTD clinical phenotype and underlying copathologies of APBD, FTLD-TDP and ARTAG with a segregating GBE1 loss-of-function mutation in affected siblings. The finding of copathologies of APBD and FTLD-TDP suggests these processes may share a disease mechanism resulting from this GBE1 mutation.

Lafora disease in a Chihuahua dog: a case report

The aging process is still not fully understood, although it has been studied for centuries. One of the processes in the brain during aging is the accumulation of extracellular and intracellular deposits of amyloid and lipofuscin. Deposits of various polyglucosan bodies (PGBs) are also found in brain tissue. The accumulation of the Lafora bodies (LB), a type of PGBs, can cause the Lafora disease (LD). Initial signs of the disease in humans are tonic-clonic seizures with blindness and myoclonus seizures. Normally, all haematological and biochemical indices are within the reference range in dogs with this disease. In this case, a 7-year-old Chihuahua dog with tonic-clonic seizures was presented. According to history, neurological examination, and blood test, toxic, metabolic, and infectious causes for the seizures were ruled out. The dog was started on phenobarbital 2.5 mg/kg twice daily per os. Two years later, the dog died due to complications caused by a duodenal foreign body. Postmortem examination revealed hyperaemic meninges and an enlarged, oedematous brain with flattened gyri and narrowed sulci. Histopathological examination revealed multifocal to diffuse, randomly distributed PGBs that were positive on periodic acid-Schiff (PAS) staining. The diagnosis of LD in dogs is of great importance because they are a good experimental model for neurological studies of neurodegenerative diseases in humans.

Glial Contributions to Lafora Disease: A Systematic Review.

Lafora disease (LD) is a neurodegenerative condition characterized by the accumulation of polyglucosan bodies (PBs) throughout the brain. Alongside metabolic and molecular alterations, neuroinflammation has emerged as another key histopathological feature of LD. To investigate the role of astrocytes and microglia in LD, we performed a systematic review according to the PRISMA statement. PubMed, Scopus, and Web-of-Science database searches were performed independently by two reviewers. Thirty-five studies analyzing the relationship of astrocytes and microglia with LD and/or the effects of anti-inflammatory treatments in LD animal models were identified and included in the review. Although LD has long been dominated by a neuronocentric view, a growing body of evidence suggests a role of glial cells in the disease, starting with the finding that these cells accumulate PBs. We discuss the potential meaning of glial PB accumulations, the likely factors activating glial cells, and the possible contribution of glial cells to LD neurodegeneration and epilepsy. Given the evidence for the role of neuroinflammation in LD, future studies should consider glial cells as a potential therapeutic target for modifying/delaying LD progression; however, it should be kept in mind that these cells can potentially assume multiple reactive phenotypes, which could influence the therapeutic response.

Pathomorphogenesis of Glycogen-Ground Glass Hepatocytic Inclusions (Polyglucosan Bodies) in Children after Liver Transplantation.

Seventeen out of 764 liver biopsies from transplanted (Tx) livers in children showed glycogen-ground glass (GGG) hepatocytic inclusions. The inclusions were not present in pre-Tx or in the explanted or donor’s liver. Under the electron microscope (EM), the stored material within the cytosol appeared as non-membrane-bound aggregates of electron-lucent globoid or fibrillar granules, previously described as abnormally structured glycogen and identified as Polyglucosan bodies (PB). The appearance of GGG in our children was analogous to that of PB-GGG occurring in a number of congenital diseases due to gene mutations such as Lafora’s d., Andersen’s d., Adult Polyglucosan Body Disease and glycogenin deficiency. The same type of GGG was previously reported in the liver of patients undergoing transplants, immunosuppressive or antiblastic treatment. To explore the potential mechanism of GGG formation, we examined whether the drugs after whose treatment this phenomenon was observed could have a role. By carrying out molecular docking, we found that such drugs somehow present a high binding affinity for the active region of glycogenin, implicating that they can inactivate the protein, thus preventing its interaction with glycogen synthase (GS), as well as the maturation of the nascent glycogen towards gamma, beta or alfa glycogen granules. We could also demonstrate that PG inclusions consist of a complex of PAS positive material (glycogen) and glycogen-associated proteins, i.e., glicogenin-1 and -2 and ubiquitin. These features appear to be analogous to congenital GGG, suggesting that, in both cases, they result from the simultaneous dysregulation of glycogen synthesis and degradation. Drug-induced GGG appear to be toxic to the cell, despite their reversibility.

Lafora Disease and Alpha-Synucleinopathy in Two Adult Free-Ranging Moose (Alces alces) Presenting with Signs of Blindness and Circling.

Simple SummaryReports of behavioral signs, such as blindness and circling in free-ranging moose from different parts of the world, have spurred comprehensive pathological investigation to find the causes of the disease that have clinical relevance. In this case study, brains collected from two adult free-ranging moose (Alces alces) cows that were seemingly blind and found walking in circles were examined by light and electron microscopy with further ancillary testing. Here, we report for the first time Lafora disease and alpha-synucleinopathy in two wild free-ranging moose cows who presented with abnormal behavior and blindness, with similar neuronal polyglucosan body (PGB) accumulations identified in humans and other animals. Microscopic analysis of the hippocampus of brain revealed inclusion bodies resembling PGBs (Lafora disease) in the neurons with ultrastructural findings of aggregates of branching filaments, consistent with polyglucosan bodies. Furthermore, α-synuclein immunopositivity was noted in the hippocampus, with accumulations of small granules ultrastructurally distinct from PGBs and morphologically compatible with alpha-synucleinopathy (Lewy body). The apparent blindness found in these moose could be related to an injury associated with secondary bacterial invasion; however, an accumulation of neurotoxicants (PGBs and α-synucleins) in retinal ganglion cells could also be the cause. Lafora disease and alpha-synucleinopathy were considered in the differential diagnosis of the young adult moose who presented with signs of blindness and behavioral signs such as circling.Lafora disease is an autosomal recessive glycogen-storage disorder resulting from an accumulation of toxic polyglucosan bodies (PGBs) in the central nervous system, which causes behavioral and neurologic symptoms in humans and other animals. In this case study, brains collected from two young adult free-ranging moose (Alces alces) cows that were seemingly blind and found walking in circles were examined by light and electron microscopy. Microscopic analysis of the hippocampus of the brain revealed inclusion bodies resembling PGBs in the neuronal perikaryon, neuronal processes, and neuropil. These round inclusions measuring up to 30 microns in diameter were predominantly confined to the hippocampus region of the brain in both animals. The inclusions tested α-synuclein-negative by immunohistochemistry, α-synuclein-positive with PAS, GMS, and Bielschowsky’s staining; and diastase-resistant with central basophilic cores and faintly radiating peripheral lines. Ultrastructural examination of the affected areas of the hippocampus showed non-membrane-bound aggregates of asymmetrically branching filaments that bifurcated regularly, consistent with PGBs in both animals. Additionally, α-synuclein immunopositivity was noted in the different regions of the hippocampus with accumulations of small granules ultrastructurally distinct from PGBs and morphologically compatible with alpha-synucleinopathy (Lewy body). The apparent blindness found in these moose could be related to an injury associated with secondary bacterial invasion; however, an accumulation of neurotoxicants (PGBs and α-synuclein) in retinal ganglions cells could also be the cause. This is the first report demonstrating Lafora disease with concurrent alpha-synucleinopathy (Lewy body neuropathy) in a non-domesticated animal.

Development of a Novel ELISA for Sensitive Quantitation of Glycogen and Polyglucosan Bodies

Many glycogen storage diseases, e.g., Lafora disease and Pompe disease, are characterized by aberrant glycogen‐like aggregates known as polyglucosan bodies (PGBs). Despite the clinical relevance of PGB accumulation, there are limited options to rapidly assess PGB levels in tissues and biofluids. Additionally, there are limitations to current pre‐clinical methods that rely on enzymatic degradation assays and mass spectrometry in quantitating PGBs. We recently developed novel sandwich ELISAs for the quantitation of both glycogen and PGBs. By utilizing the specificities of glycogen and PGB antibodies, we can specifically capture glycogen or PGBs and detect them utilizing a unique carbohydrate binding module (CBM). We establish the specificity of the assays and the sensitivity of the sandwich ELISAs compared to other techniques used for quantitation. We also demonstrate the applicability of the ELISA using multiple mouse models of Lafora disease and Pompe disease to quantify PGB loads in the brain. Moving forward, these ELISAs could be used in both research and clinical settings to accurately determine PGB levels.

AAV-Mediated Artificial miRNA Reduces Pathogenic Polyglucosan Bodies and Neuroinflammation in Adult Polyglucosan Body and Lafora Disease Mouse Models

Adult polyglucosan body disease (APBD) and Lafora disease (LD) are autosomal recessive glycogen storage neurological disorders. APBD is caused by mutations in the glycogen branching enzyme (GBE1) gene and is characterized by progressive upper and lower motor neuron dysfunction and premature death. LD is a fatal progressive myoclonus epilepsy caused by loss of function mutations in the EPM2A or EPM2B gene. These clinically distinct neurogenetic diseases share a common pathology. This consists of time-dependent formation, precipitation, and accumulation of an abnormal form of glycogen (polyglucosan) into gradually enlarging inclusions, polyglucosan bodies (PBs) in ever-increasing numbers of neurons and astrocytes. The growth and spread of PBs are followed by astrogliosis, microgliosis, and neurodegeneration. The key defect in polyglucosans is that their glucan branches are longer than those of normal glycogen, which prevents them from remaining in solution. Since the lengths of glycogen branches are determined by the enzyme glycogen synthase, we hypothesized that downregulating this enzyme could prevent or hinder the generation of the pathogenic PBs. Here, we pursued an adeno-associated virus vector (AAV) mediated RNA-interference (RNAi) strategy. This approach resulted in approximately 15% reduction of glycogen synthase mRNA and an approximately 40% reduction of PBs across the brain in the APBD and both LD mouse models. This was accompanied by improvements in early neuroinflammatory markers of disease. This work represents proof of principle toward developing a single lifetime dose therapy for two fatal neurological diseases: APBD and LD. The approach is likely applicable to other severe and common diseases of glycogen storage.