University of California San Diego – Sanford Consortium of Regenerative Medicine
/by Chelsea's HopeSanford Consortium of Regenerative Medicine, University of California San Diego on June 12-13, 2014
The workshop was directed by Dr. Jack Dixon, organized by Dr. Carolyn Worby and Kim Rice, and was sponsored by Chelsea’s Hope Lafora Research Fund. It included a welcome address by Linda Gerber, a presentation and video titled “Kristen’s Story” by Kim and Jim Rice, a keynote address by Dr. Berge Minassian, eleven scientific presentations, and a round table discussion. More than 25 researchers, postdoctoral fellows, and students directly involved in Lafora research, and several Lafora parents attended the workshop. Here are the highlights from the presentations without scientific jargon as much as possible. I stressed the significance of the research to potential clinical treatments. As such, it might downplay the significance of a lot of exquisite fundamental research that was presented.
It is safe to assume that everybody knows (of) Dr. Minassian so he doesn’t need a long introduction. I would just say that he is a professor in the Department of Pediatrics at University of Toronto and a neurologist at The Hospital for Sick Children. His lab has discovered the genes responsible for Lafora disease, EPM2A encoding laforin and EPM2B, encoding malin, and hypothesized that they have a crucial role in glycogen metabolism. He is a strong proponent of the theory that the formation of Lafora bodies (LB) from poorly branched glycogen (polyglucosan chains) is the main cause of the Lafora disease. His lab showed in two seminal papers that mice with either Lafora disease mutations and mutations that reduce the glycogen production indeed did not develop the disease. They followed these mice well into old age, over two years, and they remain absolutely healthy! Moreover, this path of attacking the disease has been confirmed by two other labs, lead by Dr. DePaoli-‐Roach and Dr. Guinovart (more on that later). This independent confirmation will be extremely important when we will have to get approval for human tests. The crucial insight from these studies is that reducing glycogen production by 30-‐50% prevents the formation of LB and most importantly prevents the disease from developing. To reduce the production of glycogen three targets have been identified: the primer for the glycogen molecule, glycogenin (GYG), the protein targeting to glycogen (PTG) that activates glycogen synthase, and glycogen synthase (GS) itself, the enzyme that adds glucose molecules to glycogen.
Without getting into technical details, there are three stages in the production of an enzyme or protein: Thus, there are three levels where one can interfere, and all 3 are active research areas in Dr. Minassian’s lab.
Gene-RNA-Protein
Gene level: CRISPR is the newest technique in gene manipulation inspired by bacterial defense against viruses. It was developed at MIT by Dr. Feng Zhang and is a hot commodity. As of now it cannot be used to repair genes, just to silence them. You can imagine CRISPR as a very precise pair of scissors that can be targeted to a specific gene in a cell, making it inactive. Dr. Minassian roached Dr. Zhang last fall and initiated a research project for Lafora targeted CRISPR. Dr. Zhang delivered recently the CRISPRs against GS, PTG, and GYG with more than 50% efficiency! The next step is to test them in cell cultures and in mice, by employing an effective delivery method.
Through another collaborator, Dr. Brian Kaspar from Ohio State University, Dr. M. has access to a new adeno-‐associated virus called AAV9 that has very good attributes for gene therapy: it can cross the blood brain barrier (BBB); can populate large percentage of the brain without generating a negative reaction; does not integrate in the genome (no risk of complications due to altered DNA); stays active for long periods of times; is large enough to host CRISPRs. Dr.Kaspar has shown good results using AAV9 in mice with Amyotrophic Lateral Sclerosis (ALS). Dr. Minassian has experiments running using AAV9 and CRISPR in cell cultures and soon in mice. He is very excited and optimistic about these experiments.
Also at the gene level, AAV9 is employed to transport normal EPM2A and EPM2B genes in mice with LD. The virus will activate the genes inside the cell producing the missing laforin and malin protein and restoring their functionality in the glycogen metabolism. If successful this can be an effective cure for LD!
RNA level: Dr. Minassian mentioned his collaboration with ISIS Pharmaceuticals, which specializes in anti-‐sense drugs that bind to the mRNA produced by that, effectively turning that gene “off”. He was excited to report that they delivered Anti-‐Sense Oligonucleotides (ASOs) that bring GS, PTG, and GYG down by 50%, the target he requested. ISIS has tested these in cell cultures and in normal mice. Dr. M. was not allowed to show the actual results due to a non-‐ disclosure agreement (NDA), but he has already injected ASOs in the brains of Lafora mice. The drawback of ASOs is that they cannot cross the BBB. Thus, they need to be injected in the brain ventricles or by a spinal tap t 2-‐3 months interval. A note on ISIS Pharmaceuticals: Currently has one approved drug (KYNAMRO for hypercholesterolemia), and various compounds in clinical trials for a variety of diseases such as Crohn’s disease, psoriasis, asthma, and cancer. They are collaborating with Biogen ($100 millions project) to develop antisense drugs for neurological disorders, which will advance the brain delivery methods for ASOs.
Protein Level: From Dr. Minassian updates we all know that his lab and collaborators are involved in a massive automatic screening of small molecules that can cross the BBB (more than 190,000) find GS inhibitors. In addition, Dr. M. has entered in an agreement with an unnamed pharmaceutical company to share 5,000 chemical compounds that, in their tests, were showing GS reduction. Unofficially, because he is restricted by NDA to show the actual results, he shared with us that 40 of those compounds indeed reduced the GS in the test assays! While he called this “fantastic progress” he was quick to caution that it does not mean automatically that those compounds are safe to be use in patients. Indeed he does not even know what those compounds are since they are identified just by a code. The company has the option to pursue the treatment with Dr. M. or they might just disclose to him what their chemical composition is. Right now Dr. M. is already embarked in the next step, determining the dosage required to produce 50% suppression of GS in cell cultures. After that of course they will be tested on mice for safety and effectiveness. Dr. Minassian is optimistic about this treatment avenue as the first line of attack on the disease. In his opinion this would allow the fastest translation to human tests.
Dissolving Lafora Bodies: Most of the researchers now agree that poorly branched glycogen accumulates into insoluble polyglucosan chains that form the LB that in turn produce neuronal degeneration. The research projects enumerated above all aim to reduce the production of glycogen in the brain and stop LB formation, halting the disease. Yet, to effectively reverse the disease, the Lafora bodies have to be removed. It was hypothesized at the Workshop that the cells might have cleaning mechanisms that are simply overwhelmed in LD. If the glycogen production is reduced they might slowly clean up the LB. Nevertheless, Dr. Minassian’s lab works on research to dissolve LB using amylase. The challenge is to deliver functional amylase to the brain. Two vehicles are now under investigation. AAV9 loaded with amylase was already injected in mice and the results are pending. The second involves CRM197 a mutated, non-‐toxic diphtheria toxin (DT) that is supplemented with amylase. The diphtheria toxin crosses the BBB and infects the neurons. It is hoped that this mutated version will safely transport amylase into the brain.
Lafora disease (LD) is caused by mutations in two genes (EPM2A and EPM2B) that in yet incompletely understood ways regulate glycogen metabolism. The AAV9 virus is a natural dweller of the human brain, has no difficulty crossing the blood-brain carrier, is non-immunogenic, and has a plasmid which neither integrates the human genome, nor is silenced. We are presently testing the replacement of Epm2a and Epm2b in LD mice to rescue their phenotypes. At the same time, it is now clear that Lafora bodies are the pathogenic insult to the brain, that preventing Lafora body formation prevents the disease, and that Lafora body formation can be prevented by reducing glycogen synthesis. We are using AAV9 to introduce CRISPR/Cas9 nucleases targeted against glycogen synthase, PTG, and glycogenin, to stop brain glycogen and thus Lafora body formation. Towards the same goal, we are testing antisense oligonucleotides and triple helix forming oligonucleotides against the same three targets. Again towards the same end, we are screening focused libraries of potential glycogen synthesis inhibiting small molecules, as well as large unselected libraries, in order to identify glycogen synthesis inhibitor compounds. Finally, we are working towards introducing amylase, the only known enzyme that can digest Lafora bodies, into the murine brain through inactivated diphtheria toxin in one set of experiments, and carried by AAV9 in another. We hope that one, likely several, of the above approaches will progress towards a therapy for LD.
University of California San Diego
Presented work on a neurodegenerative disease that has similarities with Lafora. His work in producing neurons carrying the mutations of the disease from epithelial cells by making stem cells has good potential in creating Lafora affected neurons for cell culture studies.
Niemann Pick type C1 (NPC1) is a glysosomal storage disease that may share important characteristics with Lafora disease. I’ll discuss our work, which has led to several surprising conclusions about NPC1 that might be relevant to the mechanism of disease action and potential therapy. We studied purified human neurons derived from induced pluripotent stem cells carrying NPC1 mutations. We found that loss of NPC1 protein in human neurons leads to trapped cholesterol in a late endosomal compartment and persistent activation of autophagy. This persistent activation of autophagy may be a response to the trapped cholesterol or to a cholesterol starvation response and appears to also result in large numbers of fragmented, depolarized mitochondria that generate elevated levels of reactive oxygen. Our hypothesis is that this mitochondrial fragmentation may be more toxic to neurons then trapping of cholesterol, especially in light of evidence that cholesterol can be trafficked by alternative pathways in NPCI neurons. We suggest that autophagy activation in neurodegenerative diseases characterized by accumulations of different materials may all activate autophagy persistently, which could lead to a common phenotype of toxic
fragmented depolarized mitochondria.
Her work independently confirmed that reducing glycogen presence in neurons rescues Lafora disease, and answer affirmatively that Lafora bodies are causative of the Lafora disease. An experiment treating Lafora mice rapamycin+verapamil (increasing autophagy, the cell cleaning mechanism), showed no effect on the progression of the disease. Her lab works independently of Dr. Minassian to identify GS inhibitors by screening small molecules from the ChemBridge libraries (193,000 compounds). She announced finding 29 potentially active molecular compounds, as well as 5 active compounds from the list of 1,200 FDA approved drugs. More tests are required before on cell cultures and mice to confirm the results.
Lafora disease (LD) is an autosomal recessive, teenage-onset fatal form of progressive myoclonus epilepsy characterized by accumulation of poorly branched, insoluble glycogen into structures termed Lafora bodies (LB). The disease results from mutations in the EPM2A gene, which encodes laforin, a dual specificity phosphatase, or in the EPM2B gene, which codes for malin, a putative E3 ubiquitin ligase. Studies from human patients and from mouse models of the disease support the involvement of a glycogen-associated phosphate in the formation of LB. A number of mechanisms, mostly based on in vitro or on overexpressing cultured cell lines, have been proposed. Some studies have implicated protein targeting to glycogen (PTG), a protein phosphatase 1 glycogen targeting subunit that dephosphorylates and activates glycogen synthase (GS) in the disease. It has been suggested that malin, recruited to glycogen by binding to laforin, ubiquitylates PTG, GS and glycogen debranching enzyme (AGL) so that these glycogen metabolizing enzymes are ubiquitylated and consequently degraded. Malin has also been suggested to degrade laforin. Other mechanisms proposed to be involved or even causative of the disease include autophagy, proteosomal activity and ER stress. An important question is whether Lafora bodies are causative of the Lafora disease. Different proposed mechanisms for the pathogenesis of the disease will be discussed as well as potential approaches for treatment. (Supported by NIH grants DK27221 and NS056454)
2 CIBERER. Centro de Investigación Biomédica en Red de Enfermedades Raras. Valencia. Spain.
3 FIHCUV-INCLIVA. Valencia. Spain
4 Dept. Physiology. Medicine School. University of Valencia. Valencia. Spain
5 Centro de Investigación Príncipe Felipe. Valencia. Spain.
6 Sistemas Genómicos, Paterna. Spain.
His group seems to support the theory that LBs are not the actual cause of the disease. Using fibroblasts from LD patients and samples from LD mouse brains, they found mitochondrial alterations, signs of oxidative stress and a deficiency in antioxidant enzymes. He is working on finding molecular compounds to reduce the oxidative stress. Personal note: NAC (N-Acetyl-Cysteine) has antioxidant effect. In the discussion that followed his presentation most scientists agreed that oxidative stress is
present, but it is caused by glycogen accumulation.
Lafora Disease (LD, OMIM 254780, ORPHA501) is a fatal neurodegenerative disorder characterized by the presence of glycogen-like intracellular inclusions called Lafora bodies and caused, in the vast majority of cases, by mutations in either EPM2A or EPM2B genes, encoding respectively laforin and malin. In the last years, several reports have revealed molecular details of these two proteins and have identified several processes affected in LD, but the pathophysiology of the disease still remains largely unknown. Since autophagy impairment has been reported as a characteristic treat in both Lafora disease cell and animal models, and since there is a link between autophagy and mitochondrial performance, we sought to determine if mitochondrial function could be also altered in those models. Using fibroblasts from LD patients, deficient in laforin or malin, we found mitochondrial alterations, oxidative stress and a deficiency in antioxidant enzymes involved in the detoxification of reactive oxygen species (ROS). Similar results were obtained in brain tissue samples from transgenic mice deficient in either the EPM2A or EPM2B genes. Furthermore, in a proteomic analysis of brain tissue obtained from Epm2b-/- mice, we observed an increase in a modified form of peroxirredoxin-6, an antioxidant enzyme involved in other neurological pathologies, thus corroborating an alteration of the redox condition. These data support that oxidative stress produced by an increase in ROS production and an impairment of the antioxidant enzyme response to this stress play an important role in the development of LD.
He studied the Lafora bodies in mouse brains. He identified LBs of type I, that appear first, are irregular in shape and in large numbers, as well as LBs of type II, that appear later, are spherical in appearance with complex architecture, and their number is much less. His hypothesis is that the LB type II are in fact the results of the neurons processing the LB type I. Also, he sowed large differences in the number of LBs in different areas of the brain.
Lafora disease (LD) is an autosomal recessive, always fatal progressive myoclonus epilepsy with rapid cognitive and neurologic deterioration. One of the pathological hallmarks of LD is the presence of cytoplasmic PAS+ polyglucosan inclusions called Lafora bodies (LBs). Current clinical and neuropathological views consider LBs to be the cause of neurological derangement of patients. A systematic study of the ontogeny and structural features of the LBs has not been done in the past. Therefore, we undertook a detailed microscopic analysis of the neuropile of a Laforin-deficient (epm2a-/-) mice model. Wild type and epm2a-/- mice were sacrificed at different ages and their encephalon processed for light microscopy. Luxol-fast-blue, PAS, Bielschowski techniques, as well as immunocytochemistry (TUNEL, Caspase-3, Apaf-1, Cytochrome-C and Neurofilament L antibodies) were used. Young null mice (11 days old) showed necrotic neuronal death in the absence of LBs. Both cell death and LBs showed a progressive increment in size and number with age. LBs type I emerged at two weeks of age and were distributed in somata and neurites. LBs type II appeared around the second month of age and always showed a complex architecture and always restricted to neuronal somata. Their number was considerable less than LBs type I. Bielschowski method showed neurofibrillary degeneration and senile-like plaques. These changes were more prominent in the hippocampus and ventral pons. Neurofibrillary tangles were already present in 11 days-old experimental animals, whereas senile-like plaques appeared around the third to fourth month of life. The null mice encephalons of null mice were not uniformly affected: Diencephalic structures were spared whereas cerebral cortex, basal ganglia, pons, hippocampus and cerebellum were notoriously affected. This uneven distribution was present even within the same structure, i.e.,: hippocampal sectors. Of special relevance was the observation of the presence of immunoreactivity to neurofilament L on the external rim of LBs type II. Perhaps, LBs type II is not the cul-de-sac of a metabolic abnormality. Instead, we suggest that LB type II is a highly specialized structural and functional entity that emerges as a neuronal response to major carbohydrate metabolism impairment. Early necrotic cell death, neurocytoskeleton derangement, different structural and probably functional profiles for both forms of LBs, a potential relationship between the external rim of the LB type II and the cytoskeleton and an uneven distribution of these abnormalities indicate that LD is both a complex neurodegenerative, and glycogen metabolism disorder. Acknowledgements: We are deeply grateful to Drs. J. Dixon, Carolyn Worby and Matthew Gentry for their comments and suggestions, as well as for supporting us with their light and electron microscopic facilities. We also thank Timo Meerlo for providing his valuable support at the EM lab.
He studied the sensitivity of Lafora mice to the administration of convulsant drugs of different doses. Not surprisingly the Lafora mice were more sensitive (had seizures faster and at lower doses) that healthy mice. Since this is a standard test by which the anti-epileptic treatments are evaluated, his work paved the way to show the effectiveness of LD. It will be very useful in persuading the NIH and FDA to agree on human tests.
Objective: Genetically engineered mice lacking expression of either laforin (Epm2a-/-) or malin (Epm2b-/-) display a number of neurological and behavioral abnormalities similar to those found in patients suffering from Lafora disease. Both Epm2a-/- and Epm2b-/- mice show altered motor activity, impaired motor coordination, and episodic memory deficits. They also present different degrees of spontaneous epileptic activity. Epm2a-/- mice present tonic-clonic seizures, and both Epm2a-/- and Epm2b-/- mice show spontaneous single spikes, spike-wave, polyspikes, and polyspike-wave complexes with correlated myoclonic jerks. Intracellular inclusions immunostained for ubiquitin were abundant in the same regions as PAS-positive inclusions. Number and size of PAS-positive and ubiquitin immunostained Lafora aggregates increased with age in both mutants. The objective of this study is to analyzed the sensitivity of Epm2a-/- and Epm2b-/- mice to the administration of the convulsant drug pentylenetetrazol (PTZ), an antagonist of the γ-aminobutiric acid type A (GABAA) receptor, commonly used to induce epileptic tonic-clonic seizures in laboratory animals. Methods: PTZ-induced epileptic activity, including myoclonic jerks and tonic-clonic seizures, was analyzed in 2 age groups of mice comprising representative samples of young adult and aged mice, after administration of PTZ at sub-convulsive and convulsive doses. Results: Epm2a-/- and Epm2b-/- mice showed a lower convulsive threshold after PTZ injections at sub-convulsive doses. A lower convulsive threshold and shorter latencies to develop epileptic seizures were observed after PTZ injections at convulsive doses. Different patterns of generalized seizures and of discharges were observed in Epm2a-/-and Epm2b-/- mice. Significance: Epm2a-/- and Epm2b-/- mice present an increased sensitivity to the convulsant agent PTZ, reflecting different degrees of increased GABAA receptormediated hyperexcitability. Grant/Other Support: SAF2010-18586 and ACCI 13-742/112.08
His research is at the fundamental scientific level. He is trying to understand the exact molecular structure of laforin and the role of laforin in glycogen metabolism. He presented exquisite detailed models of laforin and showed the possible impact of several of the Lafora mutations in its functionality. His work is very important in understanding Lafora disease and could lead to different treatment avenues. In his own words: ”Our hope is that we can [] identify some small molecules that will be beneficial for some mutations. I see this type of work as the second wave of translational work. My hope is that the first wave is successful and we can devise a treatment that slows disease progress while we continue to work on this second wave.“
Research into Lafora disease (LD) and starch metabolism are surprisingly linked by a family of enzymes that we recently discovered called glucan phosphatases. Glucans are the most abundant polymer in plants, with cellulose serving as the structural component and starch as the energy reserve. Instead of starch, humans utilize glycogen as their primary carbohydrate energy storage molecule. Recent discoveries show that the metabolism of both starch and glycogen is dependent on the action of glucan phosphatases. Plants release energy from starch via a recently identified three-step process: dikinases phosphorylate starch, amylases degrade it until they reach a phosphate moiety, and glucan phosphatases dephosphorylate starch to reset the cycle. In the absence of glucan phosphatases, starch becomes hyperphosphorylated and starch granules grow in size while plants are unable to access the energy stored in the starch and plant growth is stunted. The EPM2A gene encodes laforin and recessive mutations in EPM2A result in LD. We demonstrated that laforin is a human glucan phosphatase that removes phosphate from phosphorylated glucans, e.g. glycogen. In the absence of laforin activity, glycogen transforms into a hyper-phosphorylated, water-insoluble, starch-like Lafora body (LB). LBs are the suspected cause of neuronal apoptosis, neurodegeneration, and eventual death of LD patients. We have determined the first structures of glucan phosphatases. Using these structures, we gained new insights into the molecular basis of this medically relevant enzyme family. Additionally, we have defined their unique mechanisms of catalysis, substrate specificity, and interaction with glucans. We identified glucan-interacting platforms necessary for substrate engagement and dephosphorylation. Some LD patient mutations are within these newly identified regions. Cumulatively, we define the role of laforin in glycogen metabolism, establish how laforin mutants contribute to the fundamental biology of LB formation, and determine laforin’s molecular role in LD. This structure/function approach allows us to obtain atomic level insights that can be translated into diagnoses, bioassays, and putative treatments.
He studied the plant starch metabolism that is the equivalent of glycogen metabolism in animals. He recently switched to studying Lafora bodies. In his work he purified LBs and identified what proteins and enzymes are present in LBs. The goal is to identify all proteins involved in LBs formation. Most importantly he developed a method to purify LBs and this is very valuable service for other researchers. I know that Dr. Minassian plans to use purified LBs from Oliver for his amylase research.
The presence of Lafora bodies (LBs) in various tissues of Lafora disease (LD) patients has first been reported over 100 years ago. LBs are PAS-positive deposits consisting in large part of polyglucosan – a carbohydrate that is usually referred to as abnormally structured, highly phosphorylated, insoluble glycogen. It is generally assumed that LB accumulation in neurons triggers LD symptoms, but the mechanisms leading to LB formation are still uncertain. Since LBs also contain a significant amount of protein, we reasoned that among these we could possibly find enzymes and/or other protein factors involved in LB metabolism. Therefore, we developed a workflow for the isolation of native LBs from different tissues of 9-months-old laforin-deficient (Epm2a-/-) mice and subsequently identified associated proteins using high-sensitivity mass spectrometry. In total, we identified and semi-quantitatively analyzed 143 proteins. 51 of these were found in LBs from at least two of the three tissues analyzed (brain, heart, and skeletal muscle). Many of these generally highly abundant candidates represent proteins known to relate to glycogen metabolism underlining the feasibility and significance of our study. In addition, we identified a number of proteins potentially or de facto involved in disease-related processes, including autophagy, apoptosis, inflammatory response, ubiquitin proteasome system and multidrug resistance. Verification of expression levels of these candidates in different tissues of Epm2a-/- and wild-type mice is still ongoing, but we are positive that our data confirm the involvement in LD establishment of different pathways making LD a complex neurodegenerative disease.
Working with cultured neurons, genetically modified mice and flies, his lab found that neurons contain low amounts of glycogen and the mechanism to metabolize it. He showed that mice without glycogen were less able to learn, and that glycogen has positive effects in neuronal survival in stress induced by hypoxia, and in situations that require intense brain activity. His work with Lafora mice showed that reducing glycogen synthase by 50% prevents the disease, independently confirming Dr. Minassian’s results by a different method. Moreover, mice without Lafora mutations but with increased PTG expression (increased glycogen production) showed formation of accumulations similar to LB. Their work also revealed that glycogen accumulation is the cause for the neurodegeneration, as opposed to the hypothesis that blames autophagy impairment.
The metabolic activity of the brain is largely supported by externally provided glucose. However, some glucose is stored locally in the form of glycogen, mainly in astrocytes, which can degrade this polymer to provide neurons with energy-rich substrates. Strikingly, neurons accumulate large glycogen-like deposits under a variety of disease conditions, i.e. Lafora disease (LD) and Adult polyglucosan body disease (APBD). Working with primary cultured neurons, as well as with genetically modified mice and flies, we have found that—against general belief—neurons contain low but measurable amounts of glycogen and that, in addition to glycogen synthase, these cells express the brain isoform of glycogen phosphorylase, thus allowing glycogen to be fully metabolized. Most importantly, neuronal glycogen metabolism protects cultured neurons from hypoxia-induced death and flies from hypoxia-induced stupor. To study whether glycogen is primarily responsible for the neurodegeneration in Lafora disease, we generated malin knockout mice with impaired (totally or partially) glycogen synthesis. These animals did not show the increase in markers of neurodegeneration, the impairments in electrophysiological properties of hippocampal synapses, or the susceptibility to kainate-induced epilepsy seen in the malin knockout model. Interestingly, the autophagy impairment that has been described in malin knockout animals was also rescued in this double knockout model. Conversely, two other mouse models in which glycogen is over-accumulated in the brain, independently of the lack of malin, showed altered autophagy. Our findings change the current view of the role of glycogen in the brain and reveal that neuronal glycogen metabolism participates in tolerance to hypoxia. They also reveal that glycogen accumulation accounts for the neurodegeneration, as well as the impaired autophagy, observed in the malin knockout model.
He is another convert from plant metabolism research, working now with Dr. Minassian in Toronto. He studied in detail the functionality of laforin on glycogen and provided insight from comparisons with plant starch metabolism. His work has a significant contribution to understanding exactly how LD develops.
Functional metabolism of storage carbohydrates is vital to plants and animals. Besides chemical similarities of animal glycogen and plant amylopectin both types of polyglucans contain low amounts of phosphate esters whose abundance varies. In the model plant Arabidopsis insufficiency in starch phosphorylation or dephosphorylation results in largely impaired starch turnover and starch accumulation. In humans deficiency of the glycogen phosphatase laforin leads to the progressive neurodegenerative epilepsy, Lafora disease. Patients lacking laforin progressively accumulate unphysiologically structured insoluble glycogen-derived particles (Lafora bodies) in many tissues including brain. We could show that glucosyl C6 phosphate, which was believed absent in glycogen but has a crucial role in plant starch metabolism, is present in many glycogen preparations examined. Several NMR techniques independently proved the existence of 6-phosphoglucosyl residues in glycogen and confirmed the recently described phosphorylation sites C2 and C3. Additionally, carbon C6 is severely hyperphosphorylated in glycogen of Lafora disease mice, and laforin is capable of removing C6 phosphate from glycogen. Gradual glycogen degradation experiments revealed that C6 phosphate is more abundant in central parts of the glycogen molecules and in regions possessing longer glucan chains. Likewise, glycogen of Lafora disease mice consistently contains a higher proportion of longer chains while most short chains were reduced as compared to wild type.The results imply that, as in starch metabolism, 6-phosphoglucosyl residues in glycogen metabolism have an important function that is related to branching and therefore to glycogen structure which is impaired in Lafora disease. Better understanding of the enzymology underlying glycogen phosphorylation will lead to new possibilities of Lafora disease treatment.
He discovered that LBs are formed from hyper-phosphorylated glycogen, and introduced the hypothesis that laforin is involved in glycogen metabolism. His current work is on the actual mechanism by which the GS is building up the glycogen molecule and the effect of lack of laforin on this process. Very important fundamental research, that could open additional treatment avenues.
Lafora bodies, which contain abnormally branched, hyper-phosphorylated glycogen, are a consistent feature of Lafora disease and suppression of their formation by genetically limiting glycogen accumulation alleviates symptoms in mouse models of the disease. Mammalian glycogen normally contains small amounts of covalent phosphate (perhaps one phosphate per 500-1500 glucoses). Current evidence suggests that the phosphate exists as monoesters at C2, C3 and C6 of constituent glucose residues. Laforin, the phosphatase encoded by EPM2A, one of the two most common Lafora disease genes, catalyzes the hydrolysis of all three linkages in vitro. Furthermore, in mice lacking laforin, the phosphorylation state of glycogen is elevated suggesting that laforin functions as a glycogen phosphatase in vivo. The origin of the phosphate in glycogen is still under investigation. Whelan had originally postulated that phosphate, as a bridging C1-C6 diester, was introduced by a glucose-1-P transferase that utilized UDP-glucose as donor, subsequent hydrolysis possibly generating the C6 monester. This activity has not been further characterized and transfer of the beta-phosphate of UDP-glucose to glycogen by muscle extracts was eliminated by disruption of the glycogen synthase gene. This result led to the observation that purified glycogen synthase was able to introduce the beta-phosphate of UDP-glucose into glycogen at a low rate, consistent with a catalytic side reaction or error. We postulated that such a reaction could account for C2, and perhaps C3, phosphorylation via the formation of cyclic glucose phosphodiester intermediates during catalysis. This hypothesis is supported by a crystal structure of glycogen synthase obtained with glucose-1,2-cyclic phosphate (GCP) bound at the catalytic site of glycogen synthase and consistent with the proposed mechanism. Furthermore, incubation of glycogen synthase with UDPglucose in the absence of acceptor resulted in the formation of GCP. These results provide a plausible explanation for the occurrence of C2 phosphomonoesters in glycogen. [Supported by NIH grants DK27221 and NS056454]
He did not present any scientific work; his presentation was about funding a potential
opportunity for epilepsy through NIH, to create a “Center without walls”.
In 2013-2014 (25 years after CFTR was discovered) the Cystic Fibrosis Foundation together with Vertex Pharmaceuticals looked to complete the cure for Cystic Fibrosis (CF) and started phase III trials on the corrector drug VX-809 (moves the CFTR or cystic fibrosis transmembrane regulator gene to the cell surface) in combination with the doorman, Kalydeco (opens channels to let chloride flow in and out) to correct the Delta F 508 mutation. In April 2014, at the AAN annual meeting, Massimo Pandolfo revealed results of phase I trials of antioxidant therapy in Friedreich’s Ataxia designed to correct the disrupted iron-sulfur biosynthesis produced by reduced expression of the mitochondrial protein FRATAXIN and resulting oxidative stress. Here, we in the research community for Lafora PME, marvel at, admire and envy these advances. For many years, many of us have talked about finding a cure. Eain Cornford and his team, part of our epilepsy program at UCLA/VA in West LA, worked for over 10 years delivering laforin contained in immunoliposomes as the “Trojan horse” and obtained best results when delivered in utero in pregnant mice with Lafora disease. Translating this technique to humans would be an enormous challenge and Dr. Cornford has since retired and so our work on the blood-brain-barrier and immunoliposome delivery of laforin is at a standstill. Today, most of the research community in Lafora PME know what has to be done — in vitro curative drug trials in Lafora PME using induced pluripotent stem cells that contain EPM2A or EPM2B mutations and in vitro curative drug trials using neuronal cells in culture that contain the EPM2A or EPM2B mutations, introduced by CRISPR technology. From here, the path would be drug trials in transgenic mice and then drug trials in humans with LD. The community knows what has to be done, what enterprise it should be. How do you get the funds to support such an enterprise? On May 12, 2014, NINDS released a Notice of Intent to publicly announce a Funding
Opportunity for the NINDS Epilepsy Centers without Walls Program on Disease Modification or Prevention (U54). The estimated publication date is June 2014 with first estimated application date being November 2014. See more at: http://grants.nih.gov/grants/guide/notice-files/NOT-NS-14-028.html. So, can we unite the Lafora disease research community in a common goal and find a cure for LD? Here is our “once in a generation” opportunity. NINDS has budgeted 20 million for worthy projects. To get you thinking about a center without walls for Lafora disease, here are some questions I pose to you today:
1) How do you want to organize? The initial spark and PI and leadership should come from the basic science labs. I envision a central core clinical database with data on all LD patients, worldwide, that would be open to all collaborating site PIs. There would also be a central core of skin fibroblasts and even IPSCs that would be open to collaborating site PIs and to several collaborating research units, worldwide, who are working on correcting disease mechanisms. To help the hunt for curative drugs, this core should help and acquire such drugs for testing from pharma, NIH, private university labs depending on disease mechanisms to be tested and assays developed (122,000 chemicals were tested in CF).
2) Which disease mechanism(s) should be corrected? I list several possibilities below and invite you to suggest other disease mechanisms that you view as being the crucial defects that should be corrected.
3) Should disease mechanisms be tested first in Knockin transgenic models of more common mutations? Examples: p.R241X in exon4 of EPM2A; p.G2795S
in DSPD of EPM2A; missense mutation p.P69A in RING domain of NHLRC1.
4) In addition to in vitro drug trials, accelerated approval and compassionate use of a drug that corrects the crucial disease mechanism would need to be approved by IRBs and FDA. So, for the minutes allotted for my talk let’s instead discuss the disease mechanisms you consider crucial for correction if we are to cure Lafora PME. Can we come to agreements and chart a course – realizing that not one academic team can take on this challenge, and no pharma company would embark on such an expensive drug search in a rare disease and hope to recoup investments. This is why such an opportunity to create a center without walls for Lafora disease is timely, ripe and we can unite as a group and compete for the NIH funds. Clearly the world community wants to see CURES in the epilepsies. This is the second $20 million investment by NIH for the epilepsies. Which epilepsy should take a higher priority than Lafora disease—the most rapidly progressive fatal epilepsy? Suggested disease mechanisms that should be corrected Which would you target to develop a CURE in Lafora Disease?
I. Decrease glycogen synthesis
Why? Because of 2 hypothesis in LD —
1) increased glycogen phosphorylation
2) increased glycogen synthase
A. Decrease Phosphate Incorporation from UDP-glucose during glycogen synthesis
OR
B. Decrease Hyperphosphorylation of glucose/C6 carbons in glycogen
OR
C. Increase GSK3 ser9 phosphatase to inactivate glycogen synthase
II. Resist Endoplasmic Reticulum Stress and Apoptosis
III. Reverse proteasomal dysfunctions and ER-stress
Stress increases glucose 6 phosphate allosterically hyperactivating glycogen synthase 1 whose dephosphorylation is increased by Laforin
IV. Decrease mRNA decapping enzyme Dcp1a and facilitate micro RNA gene silencing
V. Enhance Autophagosome Biogenesis inhibited by Laforin Mutations
VI. Serum glucocorticoid-induced Kinase1 (SGK1) with Laforin mutations which impair glucose transporter and decrease glucose update and lead to glycogen accumulations….therefore, SGK1 should be inhibited
Dr. Dixon led the round table discussion and started by asking for opinions regarding the usefulness of the Symposium. All scientists agreed that it was very beneficial for them and voted to organize it every two years. The next one in 2016 is going to take place again in San Diego. The funding opportunity proposed by Dr. Escueta was discussed, but no consensus was reached. Dr. Gentry was charged with reporting the Workshop proceedings to the NIH Rare Disease program director.
Chelsea’s Hope, Post Office Box 348626, Sacramento, CA 95834
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