Polyglucosan Bodies Research Articles

A role for AGL ubiquitination in the glycogen storage disorders of Lafora and Cori’s disease

Cori's disease is a glycogen storage disorder characterized by a deficiency in the glycogen debranching enzyme, amylo-1,6-glucosidase,4-alpha-glucanotransferase (AGL). Here, we demonstrate that the G1448R genetic variant of AGL is unable to bind to glycogen and displays decreased stability that is rescued by proteasomal inhibition. AGL G1448R is more highly ubiquitinated than its wild-type counterpart and forms aggresomes upon proteasome impairment. Furthermore, the E3 ubiquitin ligase Malin interacts with and promotes the ubiquitination of AGL. Malin is known to be mutated in Lafora disease, an autosomal recessive disorder clinically characterized by the accumulation of polyglucosan bodies resembling poorly branched glycogen. Transfection studies in HepG2 cells demonstrate that AGL is cytoplasmic whereas Malin is predominately nuclear. However, after depletion of glycogen stores for 4 h, approximately 90% of transfected cells exhibit partial nuclear staining for AGL. Furthermore, stimulation of cells with agents that elevate cAMP increases Malin levels and Malin/AGL complex formation. Refeeding mice for 2 h after an overnight fast causes a reduction in hepatic AGL levels by 48%. Taken together, these results indicate that binding to glycogen crucially regulates the stability of AGL and, further, that its ubiquitination may play an important role in the pathophysiology of both Lafora and Cori's disease.

A Case of Adult Polyglucosan Body Disease

Adult polyglucosan body disease (APBD) is a rare neurological disease, characterized by adult onset (fifth to seventh decades), progressive sensorimotor or pure motor peripheral neuropathy, upper motor neuron symptoms, neurogenic bladder, and cognitive impairment. APBD is confirmed by a sural nerve biopsy that shows the widespread presence of polyglucosan bodies in the nerve. We report a 70 year old male patient who exhibited progressive weakness in all extremities and dementia. His electrodiagnostic studies showed sensorimotor polyneuropathy and muscle pathology that consisted of polyglucosan bodies located in small peripheral nerves. This is the first case of APBD reported in Korea.

Ground-Glass, Polyglucosan-Like Hepatocellular Inclusions: A “New” Diagnostic Entity

Ground-glass (GG) inclusions within hepatocytes are an important histopathologic marker of chronic hepatitis B virus (HBV) infection but may also be seen in Lafora's disease (myoclonus epilepsy), cyanamide alcohol aversion therapy, and type IV glycogenosis. We have noted a recent increased incidence of liver biopsy and postmortem specimens with GG inclusions associated with none of these etiologic factors. This study was undertaken to further delineate the clinical and liver pathologic features in such cases and their possible pathogenesis. Ten cases with GG inclusions (8 biopsy, 2 postmortem) were examined by light and electron microscopy, and the patients' clinical records were reviewed. Light microscopy demonstrated pale pink, oval to crescentic intracytoplasmic inclusions with a predilection for periportal hepatocytes but sometimes present throughout the lobules. The inclusions were intensely positive on periodic acid-Schiff stain and digested with diastase. Transmission electron microscopy of two cases showed non-membrane-bound cytoplasmic collections of granules with mild-to-moderate electron density, consistent with abnormal glycogen granules. The patients included 7 transplant recipients (liver, hematopoietic stem cell), 3 with type 2 diabetes and a child on chronic parenteral nutrition for short bowel syndrome. Medications included immunosuppressive agents, antibiotics, and insulin. GG hepatocellular inclusions may be seen in individuals without HBV infection or other recognized etiologies, appear to be composed of abnormal glycogen and closely resemble polyglucosan bodies described in humans, animals, and experimental models. The possible pathogenetic roles of disturbed glycogen metabolism and polypharmacotherapy are stressed.

N.P.3 08 Contribution of the nerve biopsy in hereditary peripheral neuropathies

Most cases of hereditary peripheral neuropathies (HPN) are now diagnosed by molecular biology, but the peripheral nerve biopsy (PNB) is still contributory in a few atypical cases. On the basis of a 30-year-experience of PNB, we wish to illustrate histopathological features which may inform the molecular biologist toward a specific genetic abnormality. A whole nerve biopsy is performed on the superficial peroneal or the sural nerve. Each case is prepared for light and electron microscopy (EM). On paraffin sections, small amyloid deposits scattered in the endoneurium, or intra-axonal polyglucosan bodies, can occasionally be disclosed. Semi-thin sections sometimes exhibit features of hypermyelination, i.e. tomaculae, or onion-bulb formations (OBF) indicating previous episodes of primary demyelination. EM is more informative, as it reveals nerve fiber modifications characteristic of certain types of HPN. Tomaculae correspond to hereditary neuropathy to pressure palsy, which presents in certain cases as acute symmetrical polyneuropathy, but tomaculae can also be the prominent feature in certain cases of Charcot-Marie-Tooth (CMT) disease type 1b. Other CMT1b families exhibit mainly features of uncompacted myelin lamellae. Myelin outfoldings are rarely observed and point to a recessive form, CMT4. Prominent OBF are present in every case of CMT1a and also in rare cases of chronic inflammatory demyelinating polyneuropathy (CIDP). Atypical OBF, mainly made of thin remnants of basal lamina are only observed in CMT infantile cases, so-called Dejerine-Sottas disease. Intra-axonal filament accumulation, with sometimes giant axons are seen in recessive cases, CMT4. Macrophage-associated demyelination is mainly observed in CIDP cases, but can reveal genuine cases of CMT1a presenting with a concurrent auto-immune inflammatory process. Occasionally, EM can disclose storage material in ceroid lipofuscinosis, adrenoleukodystrophy or Tangier’s disease. PNB can reveal familial pathology in pseudo-sporadic cases of any age and orientate molecular genetics investigation in rare cases.

The presence of polyglucosan bodies in temporal lobe epilepsy: its role and significance

Mesial temporal sclerosis (MTS) is an important pathology in temporal lobe epilepsy (TLE) and often associated with good surgical outcome, however prognostic factors for surgical outcome are conflicting. The authors examine seizure outcome after surgery for TLE due to MTS, with focus on the presence of polyglucosan bodies (PGBs), and its relation to the epileptogenic process. Records of 44 consecutive patients with intractable TLE who underwent anterior temporal lobectomy (ATL) at JFK medical center between 1994-2001 were reviewed. Details of clinical, electrophysiological evaluation, and outcome were collected. All slides were reviewed for the presence of MTS. In twenty-one patients with MTS, detailed histology of surgical specimens was reviewed and relative concentration of PGBs was assessed. The postoperative follow-up duration ranges between 28-74 months. Surgical outcome was evaluated according to Engel's classification. Data of fourteen females (mean age 32.8 years) and 7 males (mean age 34.2) were analyzed. PBGs were identified in 10 patients (8 females and 2 males). All 10 patients had good surgical outcome, with 6 patients (60%) being seizure free (class 1) and 4 patients as class 2. While 11 patients (6 males and 5 females) with absent PGB, 72.8% had good surgical outcome. Of these, 7 (63.8%) had class 1, 1 patient had class 2, and 3 patients had class 3&4. Assessing several risk factors, the only significant difference between the two groups was epilepsy duration, which was significantly longer in patients with PGBs (P = 0.011). The histological presence of PGB is associated with long duration of epilepsy and could be the consequence of epilepsy. Despite prolonged duration of epilepsy, the surgical outcome in PGBs +ve is similar to PGBs -ve patients.

Increased glycogen storage in yeast results in less branched glycogen

Glycogen is a branched polymer of glucose, synthesized as a reserve of both energy and carbon. The branched nature of glycogen is important for its function and polyglucosan bodies, particles that contain a glycogen-like polymer with reduced branching, are a feature of several disease states. The degree of glycogen branching is thought to be governed by the balance between glycogen synthesis and branching activities. However, there have been reports that the intrinsic properties of individual branching enzymes govern the degree of branching. To address the relationship between synthesis and branching more fully, we made use of the yeast Saccharomyces cerevisiae. The glycogen content of yeast cells was manipulated by using different growth conditions or by the introduction of specific mutations. Whenever glycogen storage was elevated, the polysaccharide formed was found to be less branched but normal branching could be restored by overexpression of branching enzyme.

Age-Related Histological Changes in the Canine Substantia Nigra.

Age-related changes in the canine substantia nigra, were examined using immunostaining for tyrosine hydroxylase (TH), glial fibrillary acidic protein (GFAP), neurofilament (NF), ubiquitin, single stranded DNA (ssDNA), and alpha-synuclein (alphaSN). Brain sections from 34 necropsied dogs, ranging from 2 months to 18 years old, were used for this study. On general histological examinations, several age-related changes, including lipofuscin deposition, polyglucosan bodies, amorphous basophilic inclusions and eosinophilic crystal inclusions, were found in the aged dogs. Immunohistochemically, TH-positive neurons were located only in the substantia nigra. The number of TH-positive neurons was well preserved in all dogs examined, however, the ratio of TH-positive neurons to GFAP-positive glial cells tended to show slight decrease in aged dogs. By ssDNA immunostaining for apoptotic cells, there were no significant results in the number of ssDNA-positive neurons. The number of ubiquitin- and NF-positive swollen neurites was increased markedly in aged dogs. Ubiquitin immunostaining revealed a small number of basophilic and eosinophilic inclusions, although both types of inclusions were negative for NF. By alphaSN immunostaining, no neurons were immunoreactive and no basophilic or eosinophilic intracytoplasmic inclusions were revealed. These results indicate that in the substantia nigra of aged dogs the dopaminergic neurons are well preserved, but intracytoplasmic inclusions and ubiquitin-positive degenerative neurites are commonly found.

Polyglucosan body disease in a mixed-breed dog

AIM: To describe the histopathology of a previously unrecorded canine disease and deduce the cause of the lesions. METHODS: Formalin-fixed tissues were processed into paraffin wax and epoxy resin for light and electron microscopy of variously stained sections of liver, brain, heart muscle and kidney. RESULTS: Periodic acid Schiff (PAS) -positive bodies in liver and myocardium were typical of a polyglucosan body disease. Neurons contained coarse granular material that stained similarly to the polyglucosan bodies. CONCLUSION: The nature, distribution and histochemistry of lesions observed are consistent with a putative diagnosis of Glycogen storage disease type IV, an inherited metabolic defect associated with a deficiency of glycogen-branching enzyme not previously reported in dogs.

Enfermedad de Lafora. Confirmación diagnóstica por genética molecular de un nuevo caso.

Lafora s disease is a type of progressive myoclonic epilepsy with bad prognosis. Until now diagnosis was based on finding characteristic intracytoplasmatic polyglucosan bodies in biopsies of sweat secreting cells in the skin. Recently the gene responsible has been discovered. This permits firm diagnosis and screening of carriers. We present the case of a child diagnosed on molecular genetic studies.A 12 year old boy with a clinical history of three febrile seizures at the age of one year but no other abnormalities, presented a seizure of visual disorder with secondary generalization. There was no family history of seizures. Following a period of normality he had further seizures (clonic, visual and generalized myoclonic). The EEG showed generalized spike and wave activity, which was more marked after stimulation by light and became progressively worse. Neuroimaging studies were normal. In spite of treatment there was a progressive increase in visual and generalized myoclonic seizures together with deterioration of cognitive function and ataxia. Histological studies of the sweat glands showed homogeneous nodular deposits of intracytoplasmatic PAS+. Molecular studies of the EPM2A gene linked to chromosome 6q24 showed the presence of two mutations on the 1 and 4 exons.We describe a 12 year old patient with all the clinical features of Lafora type progressive myoclonic epilepsy in whom characteristic cytoplasmic bodies were found in the sweat gland biopsy. Molecular genetic studies of the EPM2A gene confirmed diagnosis of the disorder.

Ultrastructural changes in paravertebral muscles associated with degenerative spondylolisthesis.

The paravertebral muscle of 30 patients with spondylolisthesis and 30 control patients were investigated histologically. To propose myopathologic paravertebral muscle changes in cases of degenerative lumbar spondylolisthesis. The stability of the vertebral column is based on both active and passive systems. The passive system is composed of the vertebrae, the intervertebral discs, and the ligaments. Surrounding muscles and tendons constitute the active system. The autochthonous back muscles take over support functions if the passive system is ineffective. In some cases, muscles are overstrained for a long period, ultimately leading to muscular changes. This study was performed to determine the histopathologic correlates of this permanent strain. Between July 1998 and July 1999, paravertebral muscle biopsies were performed for 30 patients with monosegmental degenerative spondylolisthesis undergoing posterior lumbar interbody fusion. The tissue samples were submitted to histologic analysis including immune and enzyme histochemistry and electron microscopy. In addition, the muscle fibers were submitted to morphometry. Severe pathologic alterations were found. The findings showed that 22 patients (73.3%) had ragged red fibers with evident ultrastructural mitochondrial anomalies. The cristae appeared irregular in 12 patients (40%) Type 1 paracrystalline inclusions were detected in five samples (16.6%) and dense bodies in eight (26.6%). Fibers with ubiquitin-positive inclusions were detected by immunohistochemistry in 13 patients (43.3%). As shown by the electron microscope, these corresponded to granulofilamentous inclusions and polyglucosan bodies. The samples were submitted to genetical analysis because biochemical studies showed reduced activity of the respiratory chain enzymes. Normal mitochondrial deoxyribonucleic acids of unchanged length were detected. Apart from nonspecific myopathic changes such as those observed in rimmed vacuoles and rods, increased numbers of polyglucosan bodies were detected. This increase in polyglucosan bodies currently has not been described in patients with otherwise normal muscles.

Surprises of genetic engineering: a possible model of polyglucosan body disease.

The authors previously reported the generation of a knockout mouse model of Pompe disease caused by the inherited deficiency of lysosomal acid alpha-glucosidase (GAA). The disorder in the knockout mice (GAA-/-) resembles the human disease closely, except that the clinical symptoms develop late relative to the lifespan of the animals. In an attempt to accelerate the course of the disease in the knockouts, the authors increased the level of cytoplasmic glycogen by overexpressing glycogen synthase (GSase) or GlutI glucose transporter. GAA-/- mice were crossed to transgenic mice overexpressing GSase or GlutI in skeletal muscle. Both transgenics on a GAA knockout background (GS/GAA-/- and GlutI/GAA-/-) developed a severe muscle wasting disorder with an early age at onset. This finding, however, is not the major focus of the study. Unexpectedly, the mice bearing the GSase transgene, but not those bearing the GlutI transgene, accumulated structurally abnormal polysaccharide (polyglucosan) similar to that observed in patients with Lafora disease, glycogenosis type IV, and glycogenosis type VII. Ultrastructurally, the periodic acid-Schiff (PAS)-positive polysaccharide inclusions were composed of short, amorphous, irregular branching filaments indistinguishable from classic polyglucosan bodies. The authors show here that increased level of GSase in the presence of normal glycogen branching enzyme (GBE) activity leads to polyglucosan accumulation. The authors have further shown that inactivation of lysosomal acid alpha-glucosidase in the knockout mice does not contribute to the process of polyglucosan formation. An imbalance between GSase and GBE activities is proposed as the mechanism involved in the production of polyglucosan bodies. The authors may have inadvertently created a "muscle polyglucosan disease" by simulating the mechanism for polyglucosan formation.

Laforin is a cell membrane and endoplasmic reticulum–associated protein tyrosine phosphatase

Lafora disease (LD) is the only progressive myoclonus epilepsy with polyglucosan bodies. Among conditions with polyglucosan bodies, LD is unique for the subcellular location of its polyglucosans in neuronal perikarya and dendrites and not in axons. Here we report that the protein encoded by the EPM2A gene, which is mutated in LD, localizes at the plasma membrane and the endoplasmic reticulum and that it is a functional protein tyrosine phosphatase. The significance of these findings in the epilepsy of LD and in the origin and characteristic subcellular location of Lafora bodies is discussed. Ann Neurol 2001;49:271–275

Laforin is a cell membrane and endoplasmic reticulum-associated protein tyrosine phosphatase

Lafora disease (LD) is the only progressive myoclonus epilepsy with polyglucosan bodies. Among conditions with polyglucosan bodies, LD is unique for the subcellular location of its polyglucosans in neuronal perikarya and dendrites and not in axons. Here we report that the protein encoded by the EPM2A gene, which is mutated in LD, localizes at the plasma membrane and the endoplasmic reticulum and that it is a functional protein tyrosine phosphatase. The significance of these findings in the epilepsy of LD and in the origin and characteristic subcellular location of Lafora bodies is discussed.

Probable Adult Polyglucosan Body Disease

Adult polyglucosan body disease is a clinicopathologic entity characterized by progressive upper and lower motor neuron dysfunction, sensory loss in the lower extremities, sphincter dysfunction, and occasionally dementia. Pathologically, numerous large polyglucosan bodies are noted in peripheral nerves, cerebral hemispheres, and the spinal cord, as well as in other systemic tissues. We present a case of probable adult polyglucosan body disease based on clinical history and examination, magnetic resonance images, and sural nerve biopsy findings.

Structural brain aging in inbred mice: potential for genetic linkage.

To identify genetic factors involved in brain aging, we have initiated studies assessing behavioral and structural changes with aging among inbred mouse strains. Cognitive performance of C57BL/6J mice is largely maintained with aging, and stereological analysis revealed no significant age-related change in neuron number, synaptic bouton number or glial number in the hippocampus. Moreover, no change in cortical neuron number and cholinergic basal forebrain neuron number has been found in this strain. 129Sv/J mice have more pronounced age-related cognitive deficits, although hippocampal and basal cholinergic forebrain neuron number also appear unchanged with aging. Differences in neurogenesis and neuron vulnerability in the adult CNS of C57BL/6, 129/Sv and other inbred strains have been reported, which in turn may have important consequences for brain aging. Age-related lesions, such as thalamic eosinophilic inclusions and hippocampal clusters of polyglucosan bodies also vary greatly among inbred strains although the functional significance of these lesions is not clear. The continued assessment of such age-related structural and behavioral changes among inbred mouse strains offers the potential to identify genes that control age-related changes in brain structure and function.

Mutation spectrum and predicted function of laforin in Lafora's progressive myoclonus epilepsy.

Lafora's disease is a progressive myoclonus epilepsy with pathognomonic inclusions (polyglucosan bodies) caused by mutations in the EPM2A gene. EPM2A codes for laforin, a protein with unknown function. Mutations have been reported in the last three of the gene's exons. To date, the first exon has not been determined conclusively. It has been predicted based on genomic DNA sequence analysis including comparison with the mouse homologue. 1) To detect new mutations in exon 1 and establish the role of this exon in Lafora's disease. 2) To generate hypotheses about the biological function of laforin based on bioinformatic analyses. 1) PCR conditions and components were refined to allow amplification and sequencing of the first exon of EPM2A. 2) Extensive bioinformatic analyses of the primary structure of laforin were completed. 1) Seven new mutations were identified in the putative exon 1. 2) Laforin is predicted not to localize to the cell membrane or any of the organelles. It contains all components of the catalytic active site of the family of dual-specificity phosphatases. It contains a sequence predicted to encode a carbohydrate binding domain (coded by exon 1) and two putative glucohydrolase catalytic sites. The identification of mutations in exon 1 of EPM2A establishes its role in the pathogenesis of Lafora's disease. The presence of potential carbohydrate binding and cleaving domains suggest a role for laforin in the prevention of accumulation of polyglucosans in healthy neurons.

S100 proteins in Corpora Amylacea from normal human brain

Corpora amylacea (C.A.) also named polyglucosan bodies (P.B.) are one of the hallmarks of normal brain aging. Although their functions are not yet clear, C.A. increase in number in patients suffering from neurodegenerative diseases. C.A. contain 88% of hexoses and 4% of proteins. Most of the proteins in C.A. are aging or stress proteins such as heat shock proteins, ubiquitinated proteins and advanced glycation end products which are also proinflammatory products. Stimulated by the potential role played by some S100 proteins in the inflammatory process which may be triggered in C.A., we investigated, by immunohistochemistry, the presence of different S100 proteins (S100A1, S100A2, S100A3, S100A4, S100A5, S100A6, S100A8, S100A9, S100A12 and S100B) in C.A. from normal human brain. Among the ten S100 proteins analyzed, nine (S100A) were detected in C.A. Three S100 proteins (S100A8, S100A9, S100A12) which are highly expressed in activated macrophages and used as inflammatory markers were detected in C.A. S100A8 was, in addition, found in thick neuronal processes from the pons. One (S100B) could not be found in C.A. although it was highly expressed in astrocytes. In C.A., the staining intensity was estimated by computer-assisted microscopy and gave the following order: S100A1≅S100A8≅S100A9>S100A5≥S100A4>S100A12>S100A6>S100A2=S100A3. The potential inflammatory role played by S100 proteins in C.A. is discussed.

Glycogenosomes in the aging rat brain: their occurrence in the visual pathways.

The significance of glycogenosomes (glycogen bodies), frequently seen in peripheral neurites of aging rats, is unknown and their occurrence elsewhere in nervous tissue is poorly documented. During the course of another study these bodies were observed by light microscopy in the visual pathways of aging rats where they have not previously been noted, and this report documents their occurrence, localisation and changes in density with age. Using the periodic acid-Schiff stain, small brightly red-staining bodies, digested by diastase and containing beta-glycogen particles, were seen in increasing numbers in the neuropil of the superior colliculi in brain sections from animals of 5 months of age onwards. From 1 year until more than 2 years of age they steadily became more numerous in the outer one third of the superior colliculus, but remained small, rarely exceeding 4 microm. They were also found at later times in small numbers lying singly in the optic tract, the optic chiasm and optic nerves, although rarely in lateral geniculate nuclei. Similar bodies were also found to accumulate with age in the retinal photoreceptor cell layer. Changes in their densities and size with age in both regions have been documented and it is suggested that, while their occurrence in retinal photoreceptor cells may be due to sustained light damage leading to mitochondrial oxidative stress, it is difficult to implicate this mechanism for their occurrence in retino-tectal nerve fibres. The role of physical trauma, suggested for the presence of these bodies in aging peripheral axons, can be excluded and they appear not to be related to polyglucosan bodies.

Typical polyglucosan bodies are present in the sweat gland lumina in Lafora's disease

Typical polyglucosan bodies are present in the sweat gland lumina in Lafora's disease

Amylaceous (Polyglucosan) Bodies in Familial Cerebral Atrophy of Early Onset

An 8-year-old girl and her 4-year-old sister presented with psychomotor retardation during the 1st year of life. This was followed by spasticity, seizures, and in the older patient, progressive loss of faculties and death. Computed tomographic and magnetic resonance imaging scans demonstrated progressive cerebral atrophy in both patients. Postmortem examination in the older sibling showed diffuse atrophy of the supragranular layers of the cerebral cortex, atrophy and gliosis of the white matter, and accumulation of numerous amylaceous (polyglucosan) bodies within the cytoplasm of cell processes. The inclusions were closely associated with atrophy of the parenchyma. The findings suggest that this disorder is different from other childhood diseases in which amylaceous bodies accumulate within the brain.