1st year Research Reviews – 2012


Gustavo H.B. Maegawa, MD, PhD
Johns Hopkins School of Medicine, Department of Pediatrics
Baltimore, MD
“Induced-neuronal (iN) cells as tools to study the pathogenesis of neurological manifestations in MPS-II.”

2012 1st yr fig 1

Fig. 1. Induced-neuronal cell from a MPS-II patient. Skin cells from a patient with neuronopathic MPS-II carrying mutation (IDS-D334G) were initially established. Through viral transfection, these cells then received three different transcription factors Pou3f2, Ascl1, Myt1l. The induced- neuronal (iN) cell showed to express two neuronal markers are shown, MAP2 (green) and b-tubulin (bTub – red).

A.  Background and Objectives
Mucopolysaccharidosis type II (MPS-II) is a genetic disease caused by the inability to breakdown large molecules called glycosaminoglycans. MPS-II is caused by the deficiency of an enzyme located in the lysosome, essential recycling units present in each cell. In MPS-II, the accumulation of undegraded material results in dysfunction of the lysosomes, compromising the entire cells and ultimately multiple organs/systems. The mechanisms how the storage of glycosaminoglycans can severely affect brain causing severe mental disabilities have not been fully elucidated. Using a new technology, we are now able to convert skin cells into brain cells, called “induced-neuronal cells” also known as iN cells. Our premise is that the induced- neuronal (iN) cells from patients can be key research tools to study mechanisms causing the neurological problems in MPSII. In this project, we aim to convert skin cells from MPS-II patients into the iN cells and determine how they can be used to study brain disease in MPS-II. The results of this project will provide cell-model to study the brain disease in the MPS-II, which can subsequently result in the discovery of novel treatments for the neurological problems commonly seen in affected patients.

B.  Current Findings and Results
We have established some iN cells from cultured skin cells (fibroblasts) from our patients with MPS-II. We were able to generate iN cells from the MPS-II skin cells, we called fibroblasts (Fig.1).
In tissue culture conditions, in order to make skin cells to convert into neurons, we insert specific transcription factors, which are key genes that function regulating the expression of other genes. Three transcription factors, Pou3f2 (known as Brn2)Ascl1 and Myt1l can “re-activate” and/or “inactivate” other genes that are only expressed during the development of the anterior part of the brain, called “forebrain” [1, 2]. To insert the genes into the skin cells, we used virus, more specifically lentivirus, which are used as a vehicle to carry and insert the specific transcription factors.

Beyond the conversion from skin to brain cells, which we had demonstrated some preliminary results in the proposal of this project, our objective is to improve the conversion rate of skin cells from MPS-II patients producing increased number of iN cells [1, 2]. This will allow us to perform further studies to investigate what specific pathways are disturbed.
We have been working with specific synthetic molecules that were previously shown to facilitate the process to mature brain cells. These molecules may assist the conversion cultured mature fibroblasts into iN cells. Previous studies have shown that blocking enzymes located in specific steps of the metabolism (e.g. SMAD signaling and glycogen synthase kinase-3β, GSK-3β), can promote a highly efficient neural differentiation of human embryonic stem cells and induced pluripotent stem cells (iPSCs) [3]. In addition, based on a recent report [4], we used these molecules to inhibit GSK-3β and  SMAD pathway to augment the rate of iN cell conversion from MPS-II fibroblasts (fig.2).

To optimize the conversion from MPS-II patients fibroblasts, we used both SB-431542 (SB), potent inhibitor of activin-like kinase 5 (ALK5) at 0.5 µM (in medium), and noggin, an inhibitor of bone morphogenic protein (BMP) at 100 ng/mL (in medium). These two small molecules promote inhibition of SMAD pathway. In addition, we also used the 1-azakenpaullone (CHIR99021) at 0.5 µM, which is a potent and selective GSK3β inhibitor [4]. We are currently doing experiments to investigate the degree of sphingolipid accumulation and presence of neurodegenerative markers in the iN cells generated. We are also working on methods to increase the number of iN cells to perform further assays to study the calcium metabolism.

C.  Significance
The results from the studies here described indicate that generation of neuronal cells (iN cells) directly from skin cells in culture. The iN cells will allow us to study the neurodegenerative process that occurs in neuronapathic MPS-II. Studying these iN cells from MPS-II patients, we will be able to tease out what molecular disturnaces can explain the progressive developmental and cognitive impairment in the neuronopathic form of MPS-II. These will ultimately results in targets that can be explored to develop therapeutic agents to treat the neurological problems in MPS-II.

D.  Plans
Beyond the aims proposed, we also plan to develop an enrichment method to make a larger number of iN cells available to perform several assays. We are currently working on evaluating cell membrane expression markers including the NCAM which can then be used to enrich IN cells by two methods: fluorescence-activated cell sorting (FACS) or using magnetic beads followed by magnetic chromatography.

2012 1st yr fig 2

Fig. 2. Small molecules increase the conversion rate of skin cells to induced-neuronal cells. Skin cells from a MPS-II patient with neuronopathic form carrying IDS Q277X mutation showed increased number of iN cells when skin cells were exposed to noggin (100 ng/mL) and SB-431542 (0.5 uM) and CHIR99021 (0.5 uM). The iN cells are shown here staining positively for the neuronal marker MAP2 (green) and showing the typical morphological characteristics of neurons.




Shunji Tomatsu, MD, PhD
Nemours Children’s Clinic – Delaware Valley of the Nemours Foundation
Wilmington, DE
“Development of Long Circulating Enzyme Replacement Therapy for MPS IVA.”


Progress report

Specific Aim: Assess the effectiveness of ERT using chemically modified GALNS (PerT-GALNS) in bone regions of a murine model of MPS IVA.

I.    MPS VII treated by long-circulating enzyme: To evaluate the effectiveness of long-circulating PerT-GUS in reducing the skeletal pathology, we treated MPS VII mice for 12 weeks beginning at 5 weeks of age with PerT-GUS or native GUS and used micro-CT, radiographs, and quantitative histopathological analysis for assessment of bones. Micro-CT findings showed PerT-GUS treatedmice had a significantly lower BMD. Histopathological analysis also showed reduced storage material and a more organized growth plate in PerT-GUS treated mice compared with native GUS treated mice. Long term treatment with PerT-GUS from birth up to 57 weeks also significantly improved bone lesions demonstrated by micro-CT, radiographs and quantitative histopathological assay. In conclusion, long-circulating PerT-GUS provides a significant impact to rescue of bone lesions and CNS involvement.

II.    Develop a new MPS IVA mouse model with skeletal dysplasia.  We have previously developed three types of MPS IVA mouse models that exhibit no detectable enzyme activity of N-acetylgalactosamine-6-sulfate sulfatase (GALNS) and accumulated glycosaminoglycans in multiple tissues including cartilage, ligament, and bone marrow. However, the synthesis of keratan sulfate (KS) in mice is limited compared to human, and these mouse models do not exhibit apparent skeletal symptom due to the fact that in mice there is an absence of KS in aggrecan. Currently, we are developing a new MPS IVA mouse model which contains a transgene (cDNA: hACAN) expressing human aggrecan enriched in KS, an inactive human GALNS (tolerant to the human GALNS protein), and mouse Galns with a knock-in mutation.

Cell line: PerT-GALNS
Purification of PerT-GALNS: The Chinese hamster ovarian cell line to produce human GALNS was established. The M6P and mannose recognition sites on GALNS are located in the carbohydrate portion of the enzyme. To inactivate exposed carbohydrates, the purified enzyme was treated with sodium metaperiodate followed by sodium borohydride and collected as PerT-GALNS.

This strategy can be applied to GALNS in MPS IVA. In a preliminary study, PerT GALNS was prolonged to a t1/2 of 3 h, while native GALNS was cleared with a t1/2 of 3 min. We have developed a comprehensive method to characterize the skeletal pathology in MPS mice including the use of micro-CT and quantitative histopathological assessment as shown above.

GALNS Uptake by Human Primary Chondrocytes: M6PR-mediated uptake was determined by adding 4,000 units of GALNS or PerT-GALNS and 2mM M6P in growth medium to confluent GALNS-deficient fibroblasts. After incubation at 37°C and 5% CO2 for 2 h, the cells were solubilized in 0.5 ml of 1% sodium desoxycholate. Extracts was assayed for GALNS activity and protein, and values was expressed as units of enzyme taken up per mg of cell protein per hour of uptake.

PerT-GALNS was not taken up by the GALNS-deficient fibroblasts, while GALNS was taken up as described previously.

Publication lists (2012-2013)

Yasudaet al. Pathogenesis of Morquio A syndrome: An autopsied case reveals systemic storage disorder, Mol. Genet. Metab. 2013109(3):301-11.

Tomatsu et al. Therapies of Mucopolysaccharidosis IVA (Morquio A Syndrome). Expert Opinion on Orphan Drugs (in press).

Tomatsu et al. Morquio A syndrome: Diagnosis and current and future therapies. Pediatric Endocrinology Reviews (submitted).


MPS III Grand Challenge Grant – with support from Team Sanfilippo

Dr. Brian Bigger
Stem Cell & Neurotherapies Group
Manchester, UK
“Evaluation of high dose genistein aglycone in the treatment of mucopolysaccharide disease
types IIIA, B and C

This clinical trial is beginning Fall 2013.