Glycogen Accumulation In Neurons Research Articles

Autopsy indicates brainstem and spinal neuropathology in late‐onset Pompe Disease

Pompe disease (PD) is a neuromuscular disorder caused by a mutation in the gene encoding acid alpha-glucosidase (GAA), the enzyme responsible for lysosomal breakdown of glycogen. The rate of PD progression corresponds to the amount of residual GAA enzyme activity. Infantile-onset Pompe disease is caused a by a complete lack of GAA activity, with patients typically progressing to cardiorespiratory failure within the first year of life. Late-onset PD patients retain some GAA activity and present symptoms later in life, with fatality mainly associated with respiratory failure. This case study presents a histological analysis of the brainstem and spinal cord from a male PD patient who was diagnosed with late-onset PD at age 35. The patient was wheelchair dependent by age 38, displayed progressive respiratory insufficiency with requirement for nocturnal ventilation at age 40, required 24-hour non-invasive ventilation by age 43, and passed away from respiratory failure at age 54. Autopsy was performed approximately 8 hours after death with informed consent. The brain and spinal cord were preserved in a 20% formalin solution and embedded in paraffin for subsequent histological assessment. Tissue sections were cut at 4 µm and stained with periodic acid-Schiff (PAS) to assess glycogen content, cresyl violet to assess neuronal morphology, and Iba-1 to characterize microglia as a marker of neuroinflammation. Positive PAS confirmed glycogen accumulation in neurons throughout the medulla and spinal cord. However, striking differences in neuronal morphology were present across the neuraxis. Hypoglossal motoneurons in the medulla (n=46, soma size: 421 ± 159 µm2) and putative motoneurons in the ventral cervical horn (n=50, soma size: 487 ± 189 µm2) had an atrophied, elongated appearance. In contrast, lumbar (n=63, soma size: 1363 ± 677 µm2) and sacral motoneurons (n=31, soma size: 1411 ± 633 µm2) had the classic ballooned morphology seen in early-onset PD. The Iba-1 staining showed an accumulation of microglia in the white matter of the cervical cord. In contrast, Iba-1 staining was localized to the gray matter in the lumbo-sacral spinal cord. These histological data confirm glycogen accumulation and pathology in brainstem and spinal neurons in late-onset PD. Further, the markedly atrophied appearance of hypoglossal and mid-cervical motoneurons is consistent with neurodegeneration and the concept that effective PD therapy will need to target the central nervous system, in addition to skeletal and cardiac muscle.

Ultrastructural and diffusion tensor imaging studies reveal axon abnormalities in Pompe disease mice

Pompe disease (PD) is caused by lysosomal glycogen accumulation in tissues, including muscles and the central nervous system (CNS). The intravenous infusion of recombinant human acid alpha-glucosidase (rhGAA) rescues the muscle pathologies in PD but does not treat the CNS because rhGAA does not cross the blood–brain barrier (BBB). To understand the CNS pathologies in PD, control and PD mice were followed and analyzed at 9 and 18 months with brain structural and ultrastructural studies. T2-weighted brain magnetic resonance imaging studies revealed the progressive dilatation of the lateral ventricles and thinning of the corpus callosum in PD mice. Electron microscopy (EM) studies at the genu of the corpus callosum revealed glycogen accumulation, an increase in nerve fiber size variation, a decrease in the g-ratio (axon diameter/total fiber diameter), and myelin sheath decompaction. The morphology of oligodendrocytes was normal. Diffusion tensor imaging (DTI) studies at the corpus callosum revealed an increase in axial diffusivity (AD) and mean diffusivity (MD) more significantly in 9-month-old PD mice. The current study suggests that axon degeneration and axon loss occur in aged PD mice and are probably caused by glycogen accumulation in neurons. A drug crossing the BBB or a treatment for directly targeting the brain might be necessary in PD.

The Respiratory Phenotype of Pompe Disease Mouse Models.

Pompe disease is a glycogen storage disease caused by a deficiency in acid α-glucosidase (GAA), a hydrolase necessary for the degradation of lysosomal glycogen. This deficiency in GAA results in muscle and neuronal glycogen accumulation, which causes respiratory insufficiency. Pompe disease mouse models provide a means of assessing respiratory pathology and are important for pre-clinical studies of novel therapies that aim to treat respiratory dysfunction and improve quality of life. This review aims to compile and summarize existing manuscripts that characterize the respiratory phenotype of Pompe mouse models. Manuscripts included in this review were selected utilizing specific search terms and exclusion criteria. Analysis of these findings demonstrate that Pompe disease mouse models have respiratory physiological defects as well as pathologies in the diaphragm, tongue, higher-order respiratory control centers, phrenic and hypoglossal motor nuclei, phrenic and hypoglossal nerves, neuromuscular junctions, and airway smooth muscle. Overall, the culmination of these pathologies contributes to severe respiratory dysfunction, underscoring the importance of characterizing the respiratory phenotype while developing effective therapies for patients.

Pompe disease gene therapy: neural manifestations require consideration of CNS directed therapy.

Pompe disease is a neuromuscular disease caused by a deficiency of the lysosomal enzyme acid alpha-glucosidase leading to lysosomal and cytoplasmic glycogen accumulation in neurons and striated muscle. In the decade since availability of first-generation enzyme replacement therapy (ERT) a better understanding of the clinical spectrum of disease has emerged. The most severe form of early onset disease is typically identified with symptoms in the first year of life, known as infantile-onset Pompe disease (IOPD). Infants are described at floppy babies with cardiac hypertrophy in the first few months of life. A milder form with late onset (LOPD) of symptoms is mostly free of cardiac involvement with slower rate of progression. Glycogen accumulation in the CNS and skeletal muscle is observed in both IOPD and LOPD. In both circumstances, multi-system disease (principally motoneuron and myopathy) leads to progressive weakness with associated respiratory and feeding difficulty. In IOPD the untreated natural history leads to cardiorespiratory failure and death in the first year of life. In the current era of ERT clinical outcomes are improved, yet, many patients have an incomplete response and a substantial unmet need remains. Since the neurological manifestations of the disease are not amenable to peripheral enzyme replacement, we set out to better understand the pathophysiology and potential for treatment of disease manifestations using adeno-associated virus (AAV)-mediated gene transfer, with the first clinical gene therapy studies initiated by our group in 2006. This review focuses on the preclinical studies and clinical study findings which are pertinent to the development of a comprehensive gene therapy strategy for both IOPD and LOPD. Given the advent of newborn screening, a significant focus of our recent work has been to establish the basis for repeat administration of AAV vectors to enhance neuromuscular therapeutic efficacy over the life span.

Neuropathology in respiratory-related motoneurons in young Pompe (Gaa−/−) mice

Respiratory and/or lingual dysfunction are among the first motor symptoms in Pompe disease, a disorder resulting from absence or dysfunction of the lysosomal enzyme acid α-glucosidase (GAA). Here, we histologically evaluated the medulla, cervical and thoracic spinal cords in 6 weeks old asymptomatic Pompe (Gaa−/−) mice to determine if neuropathology in respiratory motor regions has an early onset. Periodic acid-Schiff (PAS) staining indicated glycogen accumulation was exclusively occurring in Gaa−/− hypoglossal, mid-cervical and upper thoracic motoneurons. Markers of DNA damage (Tunel) and ongoing apoptosis (Cleaved Caspase 3) did not co-localize with PAS staining, but were prominent in a medullary region which included the nucleus tractus solitarius, and also in the thoracic spinal dorsal horn. We conclude that respiratory-related motoneurons are particularly susceptible to GAA deficiency and that neuronal glycogen accumulation and neurodegeneration may occur independently in early stage disease. The data support early therapeutic intervention in Pompe disease.

Changing shapes of glycogen-autophagy nexus in neurons: perspective from a rare epilepsy.

In brain, glycogen metabolism is predominantly restricted to astrocytes but it also indirectly supports neuronal functions. Increased accumulation of glycogen in neurons is mysteriously pathogenic triggering neurodegeneration as seen in “Lafora disease” (LD) and in other transgenic animal models of neuronal glycogen accumulation. LD is a fatal neurodegenerative disorder with excessive glycogen inclusions in neurons. Autophagy, a pathway for bulk degradation of obsolete cellular constituents also degrades metabolites like lipid and glycogen. Recently, defects in this pathway emerged as a plausible reason for glycogen accumulation in neurons in LD, although some contradictions prevail. Albeit surprising, a reciprocal regulation of autophagy by glycogen in neurons has also just been proposed. Notably, increasing evidences of interaction between proteins of autophagy and glycogen metabolism from diverse model systems indicate a conserved, dynamic, and regulatory cross-talk between these two pathways. Concerning these findings, we herein provide certain models for the molecular basis of this cross-talk and discuss its potential implication in the pathophysiology of LD.

Neuronal glycogen synthesis contributes to physiological aging.

Glycogen is a branched polymer of glucose and the carbohydrate energy store for animal cells. In the brain, it is essentially found in glial cells, although it is also present in minute amounts in neurons. In humans, loss-of-function mutations in laforin and malin, proteins involved in suppressing glycogen synthesis, induce the presence of high numbers of insoluble polyglucosan bodies in neuronal cells. Known as Lafora bodies (LBs), these deposits result in the aggressive neurodegeneration seen in Lafora’s disease. Polysaccharide-based aggregates, called corpora amylacea (CA), are also present in the neurons of aged human brains. Despite the similarity of CA to LBs, the mechanisms and functional consequences of CA formation are yet unknown. Here, we show that wild-type laboratory mice also accumulate glycogen-based aggregates in the brain as they age. These structures are immunopositive for an array of metabolic and stress-response proteins, some of which were previously shown to aggregate in correlation with age in the human brain and are also present in LBs. Remarkably, these structures and their associated protein aggregates are not present in the aged mouse brain upon genetic ablation of glycogen synthase. Similar genetic intervention in Drosophila prevents the accumulation of glycogen clusters in the neuronal processes of aged flies. Most interestingly, targeted reduction of Drosophila glycogen synthase in neurons improves neurological function with age and extends lifespan. These results demonstrate that neuronal glycogen accumulation contributes to physiological aging and may therefore constitute a key factor regulating age-related neurological decline in humans.

Deleterious effects of neuronal accumulation of glycogen in flies and mice

Under physiological conditions, most neurons keep glycogen synthase (GS) in an inactive form and do not show detectable levels of glycogen. Nevertheless, aberrant glycogen accumulation in neurons is a hallmark of patients suffering from Lafora disease or other polyglucosan disorders. Although these diseases are associated with mutations in genes involved in glycogen metabolism, the role of glycogen accumulation remains elusive. Here, we generated mouse and fly models expressing an active form of GS to force neuronal accumulation of glycogen. We present evidence that the progressive accumulation of glycogen in mouse and Drosophila neurons leads to neuronal loss, locomotion defects and reduced lifespan. Our results highlight glycogen accumulation in neurons as a direct cause of neurodegeneration.

Spinal Delivery of AAV Vector Restores Enzyme Activity and Increases Ventilation in Pompe Mice

Pompe disease is a form of muscular dystrophy due to lysosomal storage of glycogen caused by deficiency of acid α-glucosidase (GAA). Respiratory failure in Pompe disease has been attributed to respiratory muscle dysfunction. However, evaluation of spinal tissue from Pompe patients and animal models indicates glycogen accumulation and lower motoneuron pathology. We hypothesized that restoring GAA enzyme activity in the region of the phrenic motor nucleus could lead to improved breathing in a murine Pompe model (the Gaa(-/-) mouse). Adeno-associated virus serotype 5 (AAV5), encoding either GAA or green fluorescent protein (GFP), was delivered at the C(3)-C(4) spinal level of adult Gaa(-/-) mice and the spinal cords were harvested 4 weeks later. AAV5-GAA injection restored spinal GAA enzyme activity and GAA immunostaining was evident throughout the cervical ventral horn. The periodic acid Schiff (PAS) method was used to examine neuronal glycogen accumulation, and spinal PAS staining was attenuated after AAV5-GAA injection. Lastly, plethysmography revealed that minute ventilation was greater in unanesthetized AAV5-GAA versus AAV5-GFP treated Gaa(-/-) mice at 1-4 months postinjection. These results support the hypothesis that spinal cord pathology substantially contributes to ventilatory dysfunction in Gaa(-/-) mice and therefore requires further detailed evaluation in patients with Pompe disease.

Hypoglossal motoneuron pathology in a mouse model of Pompe disease

Pompe disease is a lysosomal storage disorder associated with acid α‐glucosidase (GAA) deficiency. Both diaphragm and upper airway muscle dysfunction occur in Pompe disease and this may reflect central nervous system (CNS) pathology. We used a murine model of Pompe disease to test the hypothesis that systemic GAA deficiency is associated with hypoglossal (XII) motoneuron pathology and altered XII motor output. Brainstem tissue was harvested from adult Gaa−/− mice and the periodic acid Schiff (PAS) method was used to examine neuronal glycogen accumulation. Semi‐thin (2 μm) plastic sections showed extensive cytoplasmic glycogen accumulation in XII motoneuron soma. Phrenic and XII bursting was recorded in anesthetized and ventilated Gaa−/−and B6/129 mice before and after bilateral vagotomy. Vagotomy caused a robust increase in XII inspiratory burst amplitude in B6/129 mice (238 ± 44 %baseline; P < 0.01) but had little impact on burst amplitude in Gaa−/−mice (130 ± 23 %baseline; P > 0.05). Regression analysis revealed that phrenic and XII burst amplitudes were significantly correlated in B6/129 (r2 = 0.27, P < 0.05) but not Gaa−/−mice (r2 = 0.08, P = 0.27). This finding may indicate that the progression of neuropathology differs between XII and phrenic motoneurons in Gaa−/−mice. We conclude that CNS GAA deficiency is associated with glycogen accumulation in XII motoneuron cell bodies and altered XII motor output.

Hypoglossal Neuropathology and Respiratory Activity in Pompe Mice

Pompe disease is a lysosomal storage disorder associated with systemic deficiency of acid α-glucosidase (GAA). Respiratory-related problems in Pompe disease include hypoventilation and upper airway dysfunction. Although these problems have generally been attributed to muscular pathology, recent work has highlighted the potential role of central nervous system (CNS) neuropathology in Pompe motor deficiencies. We used a murine model of Pompe disease to test the hypothesis that systemic GAA deficiency is associated with hypoglossal (XII) motoneuron pathology and altered XII motor output during breathing. Brainstem tissue was harvested from adult Gaa−/− mice and the periodic acid Schiff method was used to examine neuronal glycogen accumulation. Semi-thin (2 μm) plastic sections showed widespread medullary neuropathology with extensive cytoplasmic glycogen accumulation in XII motoneuron soma. We next recorded efferent XII bursting in anesthetized and ventilated Gaa−/− and B6/129 mice both before and after bilateral vagotomy. The coefficient of variation of respiratory cycle duration was greater in Gaa−/− compared to B6/129 mice (p < 0.01). Vagotomy caused a robust increase in XII inspiratory burst amplitude in B6/129 mice (239 ± 44% baseline; p < 0.01) but had little impact on burst amplitude in Gaa−/− mice (130 ± 23% baseline; p > 0.05). We conclude that CNS GAA deficiency results in substantial glycogen accumulation in XII motoneuron cell bodies and altered XII motor output. Therapeutic strategies targeting the CNS may be required to fully correct respiratory-related deficits in Pompe disease.

Glycogen accumulation in spinal cord motor neurons due to partial ischemia.

Partial ischemia of the spinal cord in adult cats was induced by abdominal aortic ligation. The most striking abnormality was an accumulation of glycogen in large motor neurons and astroglia in the peripheral anterior horns. Little or no histological and ultrastructural abnormalities were present in these regions. The first glycogen deposits appeared after 1/2 h in glial cells, whereas glycogen accumulation in neurons was first noticeable 1 h after ligation reaching a maximum in 24 h. A gradual decrease occurred with disappearance of glycogen at 10 days. Increase in UDPG-transferase was found preceeding glycogen appearance, and increase in glycogen-phosphorylase activity occurred later concurrent with glycogen accumulation. This unique neuronal glycogen deposition may be due to the UDPG-transferase normally present in γ-motor neurons of the anterior horns. Other possible mechanisms are also considered.