Clinical/ScientificNotes

Jean-Marie Girard, PhD
Scellig S.D. Stone, MD,PhD
Hannes Lohi, PhD
Christophe Blaszykowski,PhD
Catia Teixeira, PhD
Julie Turnbull, PhD
Afra Wang, MSc
Arman Draginov, BSc
Peixiang Wang, PhD
Xiao Chu Zhao, BSc
Cameron A. Ackerley,
PhD
Paul W. Frankland, PhD
Berge A. Minassian, MD

Editorial, page 21

Supplemental data at
www.neurology.org
Supplemental Data

100

PHOSPHORYLATION PREVENTS POLYGLUCOSAN
TRANSPORT IN LAFORA DISEASE

Neuronal distal axons have limited access to glucose,
their myelin sheaths preventing direct interface withblood, and their axoplasms being at great distancesfrom their cell bodies. Glycogen, the mammalian
glucose store, is characterized by extreme branching,which allows packing 55,000 glucoses/glycogen, andmaintaining solubility. In neurons, glycogen phosphorylase, the glycogen-digesting enzyme, is locatedin distal axons where it can generate large amounts of
glucose from glycogen.1
Two neurologic diseases, adult polyglucosan bodydisease (APBD) and Lafora disease (LD), are associated with neuronal formation of poorly branchedglycogen, termed polyglucosan, which precipitate
and accumulate into large masses called polyglucosan
or Lafora bodies. APBD is caused by mutations in
the GBE1 gene encoding the glycogen branching enzyme. APBD polyglucosans appear to be subject totransport from cell body to axons, accumulating exclusively in axons and axon hillocks, with no accumulation in the somatodendritic compartment.
Subcortical and spinal cord fiber tracts and peripheral nerves are replete with polyglucosans and often
obstructed. Expectedly, the disease is an axonopathy(onset ⬃age 50) with progressive upper and lower
motor, sensory, and bladder control deficits. MRI
shows diffuse subcortical signal abnormality, andnerve conduction studies and EMG axonal sensorimotor peripheral neuropathy.2 There is no epilepsy.
LD is a fatal progressive myoclonus epilepsy (onset ⬃age 15),3 with no axonopathy, caused by mutations of genes EPM2A (laforin) or EPM2B (malin).3,4
Laforin is a phosphatase that prevents accumulationof phosphate on glycogen.5 Malin is an E3 ubiquitin
ligase which regulates laforin.4 Phosphate accumulation on glycogen leads glycogen to unfold and precipitate.5 Glycogen synthase (GS), the enzyme thatelongates glycogen, remains bound to the precipitating glycogen, while branching enzyme does not.5
Elongation by GS without branching may explain
subsequent conversion of precipitated glycogen topolyglucosan. LD polyglucosans are identical to
APBD polyglucosans, except that they are phosphorylated.3–5,e5 They also differ in the neuronal compartNeurology 79

July 3, 2012

ment in which they accumulate, namely cell body anddendrites, gradually replacing the cytoplasms of countless dendrites. Axons are rarely affected. Subcortical
MRI signal and nerve conduction studies are normal.3
Likely, progressive overtaking of dendritic cytoplasms
underlies the progressive epilepsy of LD as does the accumulation in axons the axonopathy of APBD.3
The standard neuronal tracer dextran is a poorly
branched nonphosphorylated polyglucosan produced by fermenting bacteria. It is structurally similar to APBD and LD polyglucosans, differing inhaving ␣1– 6 instead of ␣1– 4 interglucosidic linkages. When injected in the brain, it is taken up byneuronal cell bodies and transported via active transport (not in vesicles) to axons and then distal axons.6Results. In this study we use dextran to explore why
LD polyglucosans accumulate in the somatodendritic compartment. First, we asked whether LD
neurons have a defect in their ability to transport
polyglucosans to axons. We injected fluorescently labeled dextran into M1/M2 primary motor cortex of
6 3-month-old epm2a⫺/⫺ and 6 wild-type miceand studied brain sections after 45 minutes and 24hours. At 45 minutes, dextran was present at the injection site (figure, A) within neurons (figure, C). At
24 hours, it had been transported to the distal axonsof M1/M2 neurons in the corpus callosum and striatum, equally in wild-type and epm2a⫺/⫺ mice (figure, A), indicating that polyglucosan transportmechanisms are intact. Next, we asked whether
phosphorylation, which characterizes LD polyglucosans, inhibits transport. We phosphorylated the dextran (20% of glucoses phosphorylated) (e-Methods
in appendix e-1 on the Neurology® Web site atwww.neurology.org) and injected 6 wild-type mice,
3 with phosphorylated and 3 with nonphosphorylated dextran (figure, B). Nonphosphorylated dextran traveled normally as above. Phosphorylated
dextran entered neurons normally (figure, D), but
was not transported (figure, B), indicating that polyglucosan phosphorylation prevents transport.
Discussion. The origin of the phosphorylation
which initiates LD pathogenesis was recently discovered: GS, while attaching glucoses to glycogen, epi-

FigurePhosphorylation inhibits dextran transport in brain(A) Transport of nonphosphorylated dextran (10 kDa fluoro-ruby dextran) is identical in wt and epm2a⫺/⫺ mice. Red fluorescence, dextran at the injection
site; cc ⫽ corpus callosum; str ⫽ striatum; bars, 100 ␮m. (B) Phosphorylated dextran is not transported in wt mice. (⫺), nonphosphorylated dextran; (⫹),
phosphorylated dextran. (C, D) Lack of phosphorylated dextran transport is not due to lack of neuronal entry. Confocal imaging at 45 minutes shows that
phosphorylated dextran (D) is within neuronal cytoplasms, similar to nonphosphorylated dextran (C). Arrowheads, examples of neurons containing dendrites; blue, nuclei; red, dextran; green, Nissl; bars, 50 ␮m. Methods for brain injections and dextran phosphorylation are detailed in appendix e-1.

sodically introduces phosphates by enzymatic error,normally corrected by laforin.5 The present study indicates that the phosphorylation may also underlie
the somatodendritic localization of polyglucosans, by
preventing their removal into axons. Two caveats ofour study are that dextran is not exactly identical toLD and APBD polyglucosans, and its phosphorylation in our experiments (⬃20%) exceeds that of LD
polyglucosans (⬃1.26%).e5
LD includes defects in autophagy and protein
ubiquitination/clearance in addition to polyglucosan
formation. In a recent study, preventing polygluco-

san formation by downregulating GS in LD mice
prevented myoclonus and neurodegeneration, andcured the disease, highlighting the role of polyglucosans.7 The present study suggests that the epileptogenesis initiated by accumulating polyglucosans
occurs in the somatodendritic domain of affected
neurons. Downregulating GS would prevent this accumulation, with important therapeutic significance.
From the Program in Genetics and Genome Biology (J.-M.G., H.L.,
J.T., A.D., P.W., X.C.Z., B.A.M.), Program in Neuroscience(S.S.D.S., C.T., A.W., P.W.F.), Department of Pathology andLaboratory Medicine (C.A.A.), and Department of Paediatrics
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101

Stine Knudsen, MD, PhD
Bo Biering-Sørensen, MD
Birgitte R. Kornum, PhD
Eva R. Petersen, MD
Jette D. Ibsen, MD
Steen Gammeltoft, MD,
Dr Med Sci
Emmanuel Mignot, MD,PhD
Poul J. Jennum, MD,
Dr Med Sci(B.A.M.), The Hospital for Sick Children, Toronto; Department ofSurgery (S.S.D.S.), University of Toronto, Toronto, Canada; Department of Veterinary Biosciences and Program in Molecular Medicine (H.L.), University of Helsinki and Folkhälsan ResearchCenter, Helsinki, Finland; and Department of Chemistry (C.B.,
B.A.M.), University of Toronto, Toronto, Canada.
Author contributions: Dr. Girard, Dr. Lohi, and Dr. Minassianconceived the study. Dr. Girard, Dr. Blaszykowski, and A.
Draginov prepared and analyzed the phosphorylated dextran.
Dr. Stone performed the mouse injections and fluorescence analyses with contributions by Dr. Teixeira and A. Wang and under
the supervision of Dr. Frankland. Dr. Turnbull, Dr. P. Wang,and Dr. Ackerley provided important conceptual insights. X.C.
Zhao took care of the animals and their genotyping. Dr. Girard
and Dr. Minassian wrote the paper. Dr. Minassian supervised
the overall work.
Acknowledgment: The authors thank Pr. Michael Thompson, Department of Chemistry, The University of Toronto, for his support
during the dextran phosphorylation.
Study funding: This work was funded by the Canadian Institutes ofHealth Research.
Dr. Girard reports no disclosures. Dr. Stone has received funding
from a CIHR fellowship and the Surgeon Scientist program at theUniversity of Toronto. Dr. Lohi, Dr. Blaszykowski, and Dr. Teixeira report no disclosures. Dr. Turnbull has received funding froman NSERC Canada Graduate Scholarship. A. Wang, A. Draginov,Dr. P. Wang, X. Zhao, Dr. Ackerley, and Dr. Frankland report nodisclosures. Dr. Minassian has received funding from CIHR. Go toNeurology.org for full disclosures.
Received June 17, 2011. Accepted in final form October 17, 2011.

Correspondence & reprint requests to Dr. Minassian: berge.
minassian@sickkids.ca

EARLY IVIg TREATMENT HAS NO EFFECT ON
POST-H1N1 NARCOLEPSY PHENOTYPE OR
HYPOCRETIN DEFICIENCYCSF parameters were normal. His score on the Epworth Sleepiness Scale (ESS) was 18/24. Polysomnography (PSG) revealed short sleep latency, sleep onset
REM periods (SOREMPs), and increased REM sleep
muscle activations. The multiple sleep latency test(MSLT) mean score was 2.1 minutes with SOREMPs
in 4/5 naps. CSF hypocretin-1 was very low (⬍10 pg/mL) and HLA-type was DQB1*06:01;03:01. Serumwas streptolysin O (ASO) positive (200 IU).
On February 14, we initiated IVIg 1 g/kg/day for
2 days after obtaining informed consent. The treatment was temporarily complicated by headache anda moderate rise in CSF leukocytes. Immediately afterIVIg treatment he reported a reduction of cataplexy(3– 8 attacks/day) and sleepiness (ESS 15–17). Objectively, however, there was no obvious change. For
example, he was still wheelchair-bound by cataplexy.
Moreover, hypnagogic hallucinations and sleep paralysis appeared on February 16 and 28, respectively.
On February 22, post-IVIg CSF hypocretin-1 remained ⬍10 pg/mL. Likewise, PSG parameters wereunchanged and a MSLT mean sleep latency of 36 swith SOREMPs in 5/5 naps. We refrained from administering further IVIg due to the adverse reaction
to the treatment and lack of apparent efficacy.

We previously suggested clinical effects of early IV immunoglobulin (IVIg) treatment in sporadic narcolepsy
with cataplexy (NC).1 Below we present the first postH1N1 vaccination NC case treated with IVIg, treated
19 days after the clear and abrupt onset of the disease.
Level of evidence. This is a single observational
study without controls (evidence level IV).
Case report. On October 14, 2010, a 21-year-oldDanish/Japanese man received H1N1vaccination (Influvac). He had previously received Pandemrix vaccination in autumn 2009. Apart from chronic asthma, hewas generally healthy. On January 22, 2011, he developed extreme sleepiness, characterized by 14 –16 hours
sleep/night and 5 naps/day. His sleep was disrupted byawakenings and dream-enacting behavior. On January
26, the first cataplectic attacks emerged, triggered bylaughter, jokes, witty replies, excitement, and exercise.
We examined him on February 8. He presented
with multiple sleep attacks and intermittent status cataplecticus (series of partial attacks of the face/knees/
hands mixed with falls/near-falls). Sleep paralysis andhypnagogic hallucinations were absent. Neurologic examination, brain MRI, EEG, and routine blood and102

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Copyright © 2012 by AAN Enterprises, Inc.
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Discussion. This first attempt to immunomodulate a post-H1N1 narcoleptic phenotype with