होम Molecular Genetics and Metabolism 32. Distinct features of disease phenotypes in two genetic models of NCL

32. Distinct features of disease phenotypes in two genetic models of NCL

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33. Assessment of neurological deterioration in subjects with LINCL

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31. TRPML1 downregulation is associated with changes in lysosomal enzyme levels

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Abstracts/Molecular Genetics and Metabolism 99 (2010) S8–S41

the effectiveness of intrathecal ERT as a treatment for another neurological symptom
of MPS I, cognitive decline.
This study is a 24-month open-label prospective randomized study in 16 MPS I
patients age 6 or older who have documented evidence of cognitive decline. The study
will test the safety and efficacy of intrathecal recombinant human a-L-iduronidase
(rhIDU) to reduce or stabilize cognitive decline by assessing the subjects at baseline
with neuropsychological, clinical, radiological, and biochemical evaluations and then
monitoring the change in these parameters during a regimen of first monthly, then
quarterly intrathecal treatments with rhIDU. The study will randomize subjects to a
treatment and a control group for 12 months, and then all subjects will receive treatment on a 12-month open-label continuation. If shown to be effective, intrathecal ERT
would be the only treatment for cognitive decline in patients who do not qualify for
and/or are unable to have hematopoietic stem cell transplantation.

28. Alpha-synuclein aggregation in Gaucher patients and carriers with
Jae Choia, Barbara Stubblefielda, Ozlem Goker-Alpana, Mark Cooksonb, Nahid Tayebia,
Ellen Sidranskya, aSectionon Molecular Neurogenetics, Medical Genetics Branch, NHGRI,
National Institutes of Health, Bethesda, MD, USA, bCell Biology and Gene Expression Unit,
Laboratory of Neurogenetics, NIA, National Institutes of Health, Bethesda, MD, USA
Recent findings demonstrate an increased frequency of mutations in glucocerebrosidase (GBA), the enzyme deficient in the lysosomal storage disorder, Gaucher disease (GD), among patients with synucleinopathies. Some patients have been observed
to develop both GD and parkinsonism. Neuropathology revealed Lewy bodies and
synuclein-positive inclusions in the vulnerable regions. In this study, samples from
cerebral cortex from subjects with different synucleinopathies and GBA mutations
were extr; acted. These include samples from patients with Lewy body dementia, Lewy
body variant Alzheimers disease and normal controls with and without GBA mutations. There were ten subjects with GBA mutations, four of them carrying homozygous mutations. The samples were homogenized and fractionated into total (PBSsoluble), soluble (SDS-soluble) and insoluble (Urea-soluble) fractions. Upon examination, most patients with GD with synucleinopathies were shown to exhibit aggregation of oligomeric forms of alpha-synuclein in the insoluble fraction, while others
showed monomeric form of alpha-synuclein in the same fraction. Those with extensive Lewy body pathologies tended to have more of the oligomeric forms of alphasynuclein. These studies indicate that patients with synucleinopathies carrying GBA
mutations show biochemical characteristics typical of Lewy body disorders.

29. Genetic and pharmacological chaperone modulation of brain GCase activity
affects synuclein accumulation in mice
Sean Clarka, Ying Sunb, You-Hai Xub, Lee Pellegrinoa, Yi Luna, Michelle Frascellaa,
Richie Khannaa, Gregory Grabowskib, Brandon Wustmana, Ken Valenzanoa, David
Lockharta, aAmicus Therapeutics, Cranbury, USA, bDivision of Human Genetics, Cincinnati
Childrens Hospital Medical Center, Cincinnati, USA
Pharmacological chaperones are orally available small molecules that bind and
stabilize a target protein, and increase cellular trafficking, levels and activity. Parkinsons disease (PD) and Dementia with Lewy Bodies (DLB) share both the accumulation
of – synuclein in the brain and a genetic risk factor: mutations in Gba which encodes
the lysosomal enzyme acid b-glucocerebrosidase (GCase). While Gba mutations occur
at a frequency of 0.4–6.2% in the general population, Gba carriers are found at a frequency of 2.3–31.3% in idiopathic PD, and up to 23% in DLB. In support of a biochemical basis for this genetic association, a Gba mutant mouse with reduced GCase
activity was found to accumulate both glucosylceramide (the substrate of GCase)
and endogenous synuclein in the brain. We tested the hypothesis that an increase
in GCase activity could prevent synuclein accumulation in mice that express normal
levels of endogenous, wild-type GCase but that overexpress wild-type human synuclein. We used the pharmacological chaperone AT 2101 to increase wild-type GCase
enzyme levels in the brain. Oral administration of AT 2101 for 12 weeks to mice overexpressing human synuclein prevented the accumulation of synuclein in the hippocampus that otherwise occurs in untreated animals. In addition, we have
investigated the effects of pharmacological chaperones in mice that express even
higher levels of human synuclein (the Thy-1-hSNCA model), and that have motor deficits as well as synuclein deposits even at an early age.

30. Open-label Phase I/II clinical trial of pyrimethamine for the treatment of
chronic GM2 gangliosidosis
Joe Clarkea, Edwin Kolodnyb, Don Mahurana, Maria Fullerc, Michael Tropaka, Jack
Keimeld, Swati Satheb, Sophia Pesotchinskye, Brigette Rigata, aResearch Institute,
Hospital for Sick Children, Toronto, Canada, bNew York University Medical Center, New
York, USA, cWomens and Childrens Hospital, Adelaide, Australia, dUniversity of Minnesota,
Minneapolis, MN, USA, eLSVP International, Inc., Los Altos, USA
Background: Chronic GM2 gangliosidosis (CGM2G) is a lysosomal storage disease
caused by deficiency of hexosaminidase A (Hex A) due to mutations in either the
HEXA or HEXB genes. The disease is often the result of misfolding and premature degradation of the mutant polypeptide. In cellulo studies showed that mutant enzyme
activity was enhanced by exposure to pyrimethamine (PYR), a competitive inhibitor
of Hex A.
Aim: This is a Phase I/II clinical trial to evaluate the safety and efficacy of PYR
administered to adults with CGM2G.
Methodology: It is an open-label, two-center, dose escalation study, with plans to
enroll a total of 20 patients. In addition to safety data, information on efficacy is collected by measurement of plasma PYR levels, leukocyte and plasma Hex A activities
using MUGS as substrate, and leukocyte and plasma GM2 ganglioside levels. Patients
start on 25 mg PYR increasing every 4 weeks to a final dosage of 100 mg per day. All
patients receive folinic acid 5 mg daily throughout the study.
Results: A total of 10 patients have been enrolled (7 male, 3 female), ages 22–
50 yrs. The drug is well-tolerated. Preliminary analyses show significant enhancement of Hex A activities in leukocytes. Some patients exhibited subjective improvement in speech and muscle power.
Conclusion: The clinical trial is progressing without significant adverse events and
with some preliminary evidence that PYR treatment results in enhanced Hex A activity in leukocytes of patients with CGM2G.

31. TRPML1 downregulation is associated with changes in lysosomal enzyme
Grace Colletti, Mark Miedel, Yi Wang, Ora A. Weisz, Kirill Kiselyov, University of
Pittsburgh, Pittsburgh, USA
Mucolipidosis Type IV (MLIV) is an autosomal recessive, neurodegenerative lysosomal storage disorder that results from inactivating mutations in the gene MCOLN1
that encodes the lysosomal ion channel Mucolipin-1 (TRPML1). TRPML1s function in
the lysosome is undefined; however there is evidence supporting two models for
TRPML1 function. The ‘‘trafficking’’ model suggests that TRPML1 plays a role in the
trafficking or fusion of vesicles along the endocytic pathway. The ‘‘metabolic’’ model
argues that TRPML1 plays a role in regulating the ionic environment of the lysosome.
Recent data have shown that TRPML1 may regulate lysosomal pH as well as iron
uptake, supporting the latter model [Miedel, 2008. Journal of Experimental Medicine;
Dong, 2008. Nature]. TRPML1-mediated changes in lysosomal homeostasis could lead
to either reversible or chronic inactivation of lysosomal enzymes. To test this possibility, we have used proteomic approaches to identify changes in lysosomal enzyme
levels or processing upon RNAi mediated TRPML1 loss.
Lysosome-enriched subcellular fractions and whole cell extracts were subjected
to 2-D DIGE and western blot analyses. These studies revealed changes in the proteins
Annexin 2A, Cathepsin B, and Lysosomal acid lipase (LAL). Annexin 2A and Cathepsin
B have dramatically increased levels upon TRPML1 loss, while LAL levels are
decreased. These data provide insight into the early pathology of MLIV and suggest
a novel role for TRPML1 in regulating lysosomal function.

32. Distinct features of disease phenotypes in two genetic models of NCL
Susan Cotmana, Yi Caoa, Sunita Biswasa, Pavlina Wolfa, Ashish Masseyb, Ana Maria
Cuervob, Marcy MacDonalda, Jong-Min Leea, aMassachusetts General Hospital, Boston,
MA, USA, bAlbert Einstein College of Medicine, Bronx, NY, USA
CLN3 and CLN6 are novel genes mutated in juvenile and late-infantile Neuronal
Ceroid Lipofuscinosis (NCL), respectively. Research suggests that CLN3 and CLN6 proteins reside in late endosomes and lysosomes and endoplasmic reticulum, respectively, and that their loss of function leads to NCL disease with strongly
overlapping features including lysosomal accumulation of subunit c of the mitochondrial F0 ATP synthase. Here, we sought to establish precise genetic models of these
two forms of NCL for genotype–phenotype studies aimed at testing the hypothesis

Abstracts/Molecular Genetics and Metabolism 99 (2010) S8–S41
that CLN3 and CLN6 proteins function at distinct steps in a common pathway. Cell
biological screening of the mutant neuronal precursor cells (CbCln6 and CbCln3 cells)
revealed altered ER marker stain in homozygous CbCln6 mutant cells, and both
genetic models exhibited reduced endocytosis and altered Lysotracker stain but with
distinct subcellular rearrangements of these organelles. Pathways-based global gene
expression analyses further identified overlapping yet distinct alterations, including
commonly altered pathways related to energy metabolism, ion transport, proteolysis/degradation, and trafficking, and unique ER-related changes in CbCln6 mutant
cells. Investigation of abnormal ATP synthase subunit c accumulation revealed that
autophagy is disrupted by CLN3 and CLN6 NCL disease mutations, though likely via
distinct mechanisms. Therefore, these data support the hypothesis that the common
disease features of variant late-infantile NCL and juvenile NCL result from defects at
different steps in common or overlapping pathways that lead to abnormal accumulation of ATP synthase subunit c protein and neuronal cell dysfunction.

33. Assessment of neurological deterioration in subjects with LINCL
Ronald Crystal, Joan & Stanford Weill Medical College of Cornell University, New York, NY,
Late infantile ceroid lipofuscinosis (LINCL) is a rare, rapidly progressing lysosomal
storage disease. The rareness of the patients as well as the possibility of non-uniform
progression depending on genotype mean that limited data is available that delineates
the natural history of disease progression. The primary focus of this study is to use clinical rating scales and magnetic resonance imaging methods to define the natural history
of LINCL and to provide objective and sensitive surrogates for neurological status and
for the assessment of the impact of experimental treatments in children with LINCL.
To achieve this goal we have three aims: (1) recruit children with LINCL and perform
serial neurological assessments and MRI studies; (2) using this data, expand the spectrum of existing quantitative MRI parameters and derive normal ranges and correlate
with neurological status and specific mutations; and (3) extract additional parameters
from MRI data including volumes of brain substructures, local metabolite levels by
magnetic resonance spectroscopy and local diffusion weighted imaging. Together,
these parameters will be applicable to future clinical studies of novel therapies for
LINCL, and should be transferable to other neurological lysosomal storage diseases.

34. Macroautophagy is defective in mucolipin 1-deficient mouse neurons
Cyntia Curcio-Morellia, Florie A. Charlesa, Matthew C. Micsenyib, Yi Caoa,
Bhuvarahamurthy Venugopala, Marsha F. Browninga, Kostantin Dobrenisb, Susan L.
Cotmana, Steven U. Walkleyb, Susan A. Slaugenhaupta, aCenter for Human Genetic
Research, Massachusetts General Hospital/Harvard Medical School, Boston, USA,
Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine,
Bronx, USA
Mucolipidosis Type IV is a neurodegenerative lysosomal disease clinically characterized by psychomotor retardation, visual impairment and achlorhydria. Prior to this
study, functional characterization in MLIV cells has been limited to fibroblast cultures
gleaned from patients. Here we report the generation and characterization of neuronal
cultures from the cerebrum of Mcoln1 / embryos. The Mcoln1 / neuronal cultures
show an increase in the number and size of LysoTracker positive-vesicles. Electron
microscopy studies reveal significant membranous intracytoplasmic storage bodies,
which correlate with the storage morphology observed in cerebral cortex of Mcoln1 /
pups. Using this neuronal model system, we show that macroautophagy is defective
in mucolipin-1 deficient neurons and that LC3-II levels are significantly elevated. This
increase appeared to be mediated by Beclin-1 and not by the mammalian target of rapamycin. P62/SQSTM1 levels were increased in Mcoln1 / neuronal cultures. Treatment
with rapamycin plus protease inhibitors failed to increase levels of LC3-II in Mcoln1 /
neuronal cultures, suggesting that the lack of mucolipin-1 affects macroautophagy flux.
This study describes, for the first time, a defect in autophagy in mucolipin-1 deficient
neurons, which provides new insight into the neuronal pathogenesis of this disease.

35. Disease brain endothelia provide unique molecular signatures for CNSdirected enzyme therapy
Beverly Davidson, Yong-Hong Chen, Michael Chang, University of Iowa, Iowa City, IA,


Vascular endothelial cells vary among organs and as a consequence of disease status. Moreover, transduced endothelial cells demonstrate basolateral secretion of gene
product, illustrating the potential for correction of the vascularized tissue. Therefore,
the brain vascular endothelium represents a promising target for gene therapies of
neurodegenerative disorders caused by lysosomal storage disease (LSD), in which
the recombinant enzyme expressed in, and secreted from, the vascular endothelia will
be endocytosed by underlying neurons and glia, decreasing neuropathology. Here we
tested the hypothesis that endothelia lining these vessels can be harnessed to create a
cellular reservoir of enzyme replacement therapy to diseased brain. In our approach
to develop brain vascular endothelium targeted AAV, we used animals with central
nervous system (CNS) deficits due to LSD. We screened a phage library invivo to identify peptides that mediate selective and efficient binding to brain endothelial cells in
diseased and wildtype mice. Surprisingly, epitopes binding diseased brain were distinct from those panned from normal brain. Moreover, different epitopes were
panned out of different disease models, implying a unique vascular signature
imparted by the disease state. Importantly, presentation of these epitopes on the capsid of adeno-associated virus (AAV) expanded the biodistribution of IV-injected AAV
from predominantly liver to include the CNS. Peripheral injection of the epitope-modified AAVs expressing the enzymes lacking in LSD mice reconstituted enzyme activity
throughout the brain and improved disease phenotypes in two distinct models.

36. Intrathecal enzyme replacement therapy treats meningeal storage and spinal
cord compression in MPS I dogs
Patricia Dicksona, N.M. Ellinwoodb, A. Dierenfeldb, K. Klineb, J. Parkesb, S. Hansonc, C.
Vited, A. Mlikotica, A. Chena, W. Grossb, M. Haskinsd, K. Pondere, S. Lea, aLA Biomed at
Harbor-UCLA, Torrance, USA, bAnimal Science, Iowa State University, Ames, USA,
Veterinary Neurology Center, Tustin, USA, dSchool of Veterinary Medicine, University of
Pennsylvania, Philadelphia, USA, eWashington University School of Medicine, St. Louis, USA
Intrathecal enzyme replacement therapy (IT ERT) was studied long-term and
from birth in MPS I dogs for treatment of spinal cord compression. Four MPS I dogs
age 15–16 mo. received weekly IV ERT + IT ERT (duration 1 year, group Adult IV + IT).
Four MPS I dogs received only weekly IV ERT (group Adult IV). Four MPS I dogs 7–
23 days old received weekly IV ERT + IT ERT for 65–81 weeks (group Early IV + IT).
Eight MPS I dogs 3–9 days old received IV ERT weekly at 0.58 mg/kg/wk (n = 4, Early
Low IV) or 1.57 mg/kg/wk (n = 4, Early High IV). At study start, all adult dogs had compression of their spinal cord, three of four had a large third ventricle and all had minor
neurologic signs. Two Adult IV + IT dogs showed improvement in neurologic examination; two showed no change. All four Adult IV dogs had worsened neurologic examination. No Early treated dog developed myelopathy, though four in Early Low IV or
IV + IT group developed tenderness over areas of intervertebral narrowing. Meninges
GAG was lower than age-matched untreated MPS I dogs in Early Low IV and Early
High IV groups, but was lowest in IV + IT groups, reaching normal levels in two Early
IV + IT dogs (mean of cervical, thoracic, and lumbar areas). Spinal MRI are being evaluated. IT ERT may stabilize or reverse neurologic signs of spinal cord compression and
prevent meningeal GAG storage in MPS I dogs.

37. Cervical cord compression in mucopolysaccharidosis diseases
Brenda Diethelm-Okita, Chester B. Whitley, University of Minnesota, Minneapolis, USA
Background: Because of its debilitative effect, cervical cord compression is an
important complication of mucopolysaccharidosis (MPS) conditions. Via pachymeningitis cervicalis, cervical cord compression leads to loss of sensation and function in the
extremities causing, impaired gait, loss of manual dexterity, and decreased bowel and
bladder control. The standard treatment has been decompressive laminectomy, a surgical procedure with uncertain outcomes, which may be syndrome dependant. Of the
new treatment modalities, hematopoietic stem cell transplantation (HSCT) reduces
the GAG level in the CNS but, because of the blood–brain barrier, enzyme replacement
therapy (ERT) does not.
Hypothesis: We assess the outcomes associated with surgical de-compression for
a cohort of patients with MPS at the University of Minnesota, and from the literature
review. Outcomes were assessed through a ratings scale ranging from 0 to 4, where 0
equals death, 1 equals significant loss of function, 2 equals minor loss of function, 3
equals no change, and 4 is equivalent to improvement over pre-operative condition.
Scores were assigned by physician assessment.
Results: Literature review suggests that surgical correction for cord compression is
successful over 90% of the time (19 of 22 patients, average score = 3.7). In contrast
75% of the cohort of patients at the University of Minnesota had less than favorable
outcomes (6 of 8, x = 1.3). Our results indicate that surgical correction of cervical cord
compression in MPS disorders may have poorer outcomes than previously reported,