BRIEF COMMUNICATIONPTG Protein Depletion
Rescues Malin-Deficient
Lafora Disease in Mouse
Julie Turnbull, PhD,1
Jonathan R. Epp, PhD,2Danielle Goldsmith, BSc,1
Xiaochu Zhao, BSc,1 Nela Pencea, BSc,1,3
Peixiang Wang, PhD,1Paul W. Frankland, PhD,2
Cameron A. Ackerley, PhD,3and Berge A. Minassian, MD1,4,5
Ubiquitin ligases regulate quantities and activities oftarget proteins, often pleiotropically. The malin ubiquitin E3 ligase is reported to regulate autophagy, themisfolded protein response, microRNA silencing, Wnt
signaling, neuronatin-mediated endoplasmic reticulum
stress, and the laforin glycogen phosphatase. Malin
deficiency causes Lafora disease, pathologically characterized by neurodegeneration and accumulations ofmalformed glycogen (Lafora bodies). We show that
reducing glycogen production in malin-deficient miceby genetically removing PTG, a glycogen synthesis
activator protein, nearly completely eliminates Lafora
bodies and rescues the neurodegeneration, myoclonus,
seizure susceptibility, and behavioral abnormality. Glycogen synthesis downregulation is a potential therapy
for the fatal adolescence onset epilepsy Lafora
disease.
ANN NEUROL 2014;75:442–446

L

afora disease (LD) is a catastrophic cause of teenage
onset progressive myoclonus epilepsy. The initial
symptoms are headaches, decline in school performance,
myoclonus, visual hallucinations, and generalized seizures. By 5 years, myoclonus is frequent, thoughts andspeech are constantly interrupted by atypical and myoclonic absences, seizures are intractable, and a disinhibited dementia has set in. By 10 years, the patient is in avegetative state, myoclonus is constant, and convulsive
status epilepticus is frequent and soon fatal. Pathology
reveals widespread intraneuronal Lafora bodies (LB),
which consist of malformed glycogen molecules and asmall amount (<10%) of protein.1
In 50% of cases, LD is caused by mutations in
the EPM2A gene,1 which encodes a glycogen phosphatase, laforin. Glycogen possesses a minute but criticalamount of phosphate, which is regulated by laforin.
C 2014 Child Neurology Society/American Neurological Association
442 V

Absence of laforin results in glycogen hyperphosphorylation, which disturbs the construction of glycogen, preventing its elaborate spherical architecture essential to solubility.2–4 The malformed glycogen (called polyglucosan)
precipitates, aggregates, and accumulates into LB. Excess
neuronal glycogen, normal or malstructured, is neurotoxic, and is the likely basis of the neurodegenerationand epilepsy in LD.5,6 Although the role of phosphate
and the mechanisms of generation of glycogen spheresare poorly understood, it is clear that glycogen synthase(GS), the enzyme that forms the vast majority of glycogen’s interglucosidic (a1–4) bonds, is essential to glycogen synthesis, whether the final structure is normal or
polyglucosan.3 To treat LD by reducing polyglucosans,brain GS was recently genetically eliminated fromlaforin-lacking LD mice, which resulted in correction of
the LD phenotype, including elimination of LB, neurodegeneration, and seizure predisposition.7 Importantly,
the same result was obtained through partial reduction of
glycogen synthesis by genetically removing a protein,
PTG, that activates GS.8 These results indicated that LD
is potentially treatable by glycogen synthesis
downregulation.
In the remaining cases LD is caused by mutationsin the EPM2B gene encoding a ubiquitin E3 ligase,malin,1 which was shown to regulate laforin.9–11 Malin
was also suggested to regulate autophagy,11 the misfolded
protein response,12 microRNA gene silencing,13 Wnt signal transduction,14 and neuronatin-mediated endoplasmic reticulum stress,15 implying a possible complex causality in the EPM2B form of LD and therefore a lower
likelihood of response to therapies targeting glycogensynthesis regulation.
We here removed PTG from the malin-deficientmouse model of LD and show normalization of the LD

From the 1Program in Genetics and Genome Biology, 2Program in Neurosciences and Mental Health, 3Division of Pathology, Department of
Pathology and Laboratory Medicine, the Hospital for Sick Children,
4
Division of Neurology, Department of Paediatrics, the Hospital for Sick
Children and University of Toronto, and 5Institute of Medical Sciences,
University of Toronto, Toronto, Ontario, Canada.
Address correspondence to Dr Minassian, Program in Genetics andGenome Biology, the Hospital for Sick Children, Toronto, Ontario,
Canada. E-mail: berge.minassian@sickkids.ca
Received Aug 7, 2013, and in revised form Jan 6, 2014. Accepted for
publication Jan 6, 2014.
View this article online at wileyonlinelibrary.com. DOI: 10.1002/ana.
24104

Turnbull et al: Malin-Deficient Lafora Diseasephenotype, including resolution of LB formation, neurodegeneration, myoclonus, seizure predisposition, andcontextual fear response. LD due to EPM2B mutations is
as responsive to glycogen synthesis downregulation as LD
due to EPM2A mutations.

Materials and Methods
Mice
All animal procedures were approved by the Toronto Centre
for Phenogenomics and the Hospital for Sick Children animal care committees. Both malin-deficient (Epm2b2/2; malinknockout [MKO])16 and PTG-deficient (Ptg2/2; PTG
knockout [PKO])8 mouse lines have been previously
described. To generate MKO mice lacking PTG, MKO andPKO mice were bred, and the resultant double heterozygous
mice (Epm2b1/2/Ptg1/2) crossed together to generate thedouble-knockout (DKO) animals used in this work. All mice
were studied at 7 to 10 months of age, unless otherwise
indicated.

Pathology and Biochemistry
Mice were sacrificed by cervical dislocation, and tissues were
fixed in 10% formalin. Periodic acid-Schiff staining with diastase predigestion (PASD) to stain LB and glial fibrillary
acidic protein staining to assess gliosis were as described previously.8 For LB counts, images of PASD-stained brain sections at 320 magnification were opened in Photoshop(Adobe, San Jose, CA) for color adjustments. Magenta was
set to 2200 and blue to 1300, and images were inverted toshow LB as white. Adjusted images were then opened in
Volocity (PerkinElmer, Waltham, MA), and objects were
found using intensity. Objects <200lm and >20lm werecounted as LB.
For electron microscopy, mice were perfused with 2.5%
glutaraldehyde in 0.1M phosphate buffer (pH 7.4), and tissues
were processed as described.8
GS activity and glycogen measurements were performed
as previously described.10

Myoclonus and Seizure Susceptibility
Myoclonus measurements were performed as previously
described, with slight modifications.8 Briefly, mice were videotaped for 4-hour periods. Recordings were then analyzed
and myoclonus was counted during 5-minute periods where
the mouse was visible from head to tail. Data are reported asmeans 6 standard error. Significance was evaluated using an
unpaired Student t test.
Seizure susceptibility was assessed as the response tokainic acid, injected intraperitoneally (15mg/kg). Mice wereclassified susceptible if they developed convulsive seizures within
1 hour postinjection.

Contextual Fear Conditioning
Prior to testing, mice were handled for 2 minutes per day for
3 days. On testing day, they were transported to the testing
March 2014

FIGURE 1: Lafora bodies do not form and there is no gliosis
in malin-deficient mice that lack PTG. (A, C, E, G) Periodic
acid-Schiff staining with diastase predigestion stain witharrows pointing to Lafora bodies. (B, D, F, H) Glial fibrillary
acidic protein immunostaining. (I, J) Lafora body counts inhippocampus. DKO 5 malin and PTG double knockout;
LB 5 Lafora bodies; MKO 5 malin knockout; PTGKO 5 PTG
knockout; WT 5 wild type. Bars 5 100lm in A, C, E, and G;
200lm B, D, F, and H.

room and placed inside Plexiglas chambers with stainless steel
grid floors (Colbourn Instruments, Whitehall, PA). Two
minutes later a foot shock was delivered (1mA, 2-second
duration). Mice remained in the chambers for an additional
minute before being returned to their home cages. Twentyfour hours later, the mice were returned to the same conditioning chambers for 5 minutes, during which time no shock
was delivered. The time spent freezing, a measure of fear
memory, was calculated automatically using the FreezeFramesoftware package (Actimetrics, Wilmette, IL).

Results
MKO Mice Lacking PTG Have Drastic Reduction
in LB and No Neurodegeneration
MKO mice have profuse LB in multiple tissues including skeletal muscle and brain.16 MKO mice lacking
PTG (DKO mice) are similar to wild-type mice
(Fig 1).
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DKO Mice Have No Abnormal Myoclonus andDramatically Improved Seizure Susceptibility
Although both laforin-deficient and MKO mice havespontaneous convulsive seizures, these are very infrequent and do not serve as a useful outcome measure.
Spontaneous myoclonus and kainic acid seizure susceptibility, however, are readily quantifiable in these mice.
Regarding myoclonus, wild-type mice have a certain
amount of myoclonus, which is drastically increased in
LD mice.8,17
Measurement of spontaneous myoclonus showedthat DKO mice lack the abnormal increased myoclonusof their MKO littermates and are indistinguishable fromwild-type mice (see Fig 3D). After kainic acid injection,
no wild-type (0 of 4), all MKO (7 of 7), 1 of 6 PKO,
and 1 of 6 DKO mice developed convulsive seizures.

FIGURE 2: Malin-deficient mice lacking PTG do not exhibit
the neurodegeneration of Lafora disease. Cerebellar Purkinje cells are shown. (A, C, E, G) Low-power electron micrographs of representative cerebellar Purkinje cells; wild-type(WT), PTG knockout (PTGKO), and malin and PTG double
knockout (DKO) neurons have normal full cytoplasms withsmooth taut plasma membrane, whereas the malin knockout(MKO) cell has a dark, condensed cytoplasm and retracted
plasma membrane; arrows indicate plasma membranes. (B,D, F, H) Electron micrographs of synaptic contacts (arrows)
with Purkinje cell membranes; multiple normal synapses
with smooth Purkinje cell plasma membrane are seen in WT,PTGKO, and DKO, and poor contacts with a distorted
plasma membrane in MKO; note the Lafora bodies (LB) in
the MKO cell. Bars 5 2lm in A, C, and G; 500nm in B, D, F,
and H; 10lm in E.

Neurodegeneration in LD mouse models, laforindeficient or MKO, is characterized by shrinkage anddarkening of neurons, and retraction of neuronal nuclear
and plasma membranes, the latter associated with loss ofsynaptic contacts. Gliosis, most prominently in the hippocampus, is also present.7,8,17 DKO mice have no gliosis (see Fig 1) and have normal neuronal morphology(Fig 2).
The reduction in GS activity in the DKO micecompared to MKO was 50% (Fig 3A). The reduction
in glycogen was 30 to 50% (see Fig 3B, C).
444

FIGURE 3: Malin-deficient mice lacking PTG have decreased
glycogen synthase (GS) activity (A) and glycogen (B, C), anddo not have abnormal myoclonus (D) or contextual fear conditioning (E). Results in A are ratios: GS activity with no glucose 6-phosphate (G6P) added divided by GS activity in thepresence of 8mM G6P. G6P is an allosteric activator and at
8mM leads to maximal GS activation; the ratios therefore
reflect the extent of the enzyme’s activation in each genotype. In A, t test statistics are: malin knockout (MKO) tomalin and PTG double knockout (DKO), p 5 0.04, n 5 8–10
mice per genotype; in B and C t test statistics are: MKO towild type (WT) and MKO to DKO, p < 0.005, n 5 5–7 mice
per genotype. In D, t test statistics are: MKO to WT,
p 5 0.008; MKO to DKO, p 5 0.003; n 5 6–8 mice per genotype. In E, analysis of variance statistics are: MKO to WT,
p 5 0.008; MKO to DKO, p 5 0.019; n 5 14–18 mice per
genotype. *Statistically significant. PTGKO 5 PTG knockout.

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Turnbull et al: Malin-Deficient Lafora Disease

MKO Mice Have Abnormal Contextual Fear
Conditioning, Which Is Corrected in DKO Mice
Behavioral tests in LD mouse models are highly sensitive tounknown modifying effects in genetic background. Three
separate MKO mouse lines, including the present mice,
have been studied behaviorally. Ataxia, motor, and memory
deficits were present in 1 (a C57/BL6 and 129Sv mix)11 but
not in another (pure C57/BL6),17 and are not present in
our MKO mice (a C57/BL6 and 129S6/SvEvTac mix; data
not shown). Our mice do, however, have a clearly abnormal
freezing fear response in contextual fear conditioning testing. This defect is fully corrected in their DKO littermates(see Fig 3E).

glycogen synthesis by removing PTG prevents LD inmice with laforin deficiency suggested that interventions
aimed at partially downregulating GS activity would betherapeutic in LD patients with EPM2A mutations. The
present work shows that LD patients with EPM2B mutations, half of all LD patients, are equally likely torespond. Known GS inhibitors (eg, rapamycin used in
tuberous sclerosis),20 new small molecule inhibitors that
could be identified through high-throughput screens, andgenetic approaches such as antisense oligonucleotides
against GS can all now be parts of the potential armamentarium that can be developed to treat LD, which isamong the most severe of adolescence onset neurologicaldiseases.

Discussion
In silico analyses that identified laforin’s carbohydrate
binding domain were soon followed by experiments that
unequivocally established that at least 1 function of
laforin involves binding and acting on glycogen.3,6 In
laforin-deficient mice, preventing brain glycogen synthesis completely (by genetically removing GS) or partially(by removing the GS activator PTG) eliminated polyglucosan formation and LB. These results indicated that in
LD caused by laforin deficiency, LB are due to a disturbance in glycogen metabolism, and that loss of laforin’s
glycogen-related function underlies this disturbance.
Elimination of LB led to elimination of neurodegeneration, myoclonus, and seizure susceptibility, suggestingthat LB underlie these neurological features of LD.7,8
Malin lacks a carbohydrate binding domain andhas numerous attributed functions.9,11–14,18 Here weshow that interfering with glycogen production byremoving PTG from MKO mice results in eliminationof LB. This indicates that in LD caused by malin deficiency, LB are due to a disturbance in glycogen metabolism, and that of malin’s many possible functions thosethat are glycogen-related underlie LB formation. As in
laforin-deficient mice, removal of LB from MKO micecorrects neurodegeneration, myoclonus, and seizure susceptibility, confirming that LB are pathogenic in LD,
and not mere epiphenomena. For the first time, ourresults show that removal of LB also corrects a behavioral
phenotype, suggesting that the globality of the neurological disorder in LD, neurodegeneration, epilepsy, andbehavioral disturbance, is secondary to the LD
glycogenosis.
Fifty percent reduction of GS activity is harmless,as indicated by the finding that parents of patients who
have no glycogen synthesis in the brain (glycogen storage
disease type 0b) are healthy.19 Absence of PTG reduces
glycogen by approximately 30%8 (30–50% in the present
study). Our previous work showing that reducing
March 2014

Acknowledgment
This work was supported by the Canadian Institutes of
Health Research, the Ontario Brain Institute (EpLink
project), the Ontario Ministry of Research and Innovation (J.R.E.), and families and friends of patients with
LD worldwide. B.A.M. holds the University of Toronto
Michael Bahen Chair in Epilepsy Research. J.T. was supported by a fellowship from Epilepsy Canada.

Authorship
J.T. and J.R.E. contributed equally to this work.

Potential Conflicts of Interest
Nothing to report.

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