Progress Report Year 1 MPS/ISMRD Grant to Heather Flanagan-Steet – Investigating the Role of
Cathepsin Proteases in MLII Cardiac Pathology
In the last year we have made progress (detailed below) on several aspects of the aims outlined in thisgrant. In addition to progress on the proposed studies, we have also recently made two additional
advances that will have a major impact on our ongoing pursuit of molecular mediators of MLII cardiac
pathology. These include 1) the generation of a TALEN-mediated MLII mutant line and 2) the isolation ofa separate MLII mutant line from a sperm TILLING screen. These two unique zebrafish lines are essential
to confirm all of our morpholino-based findings, for analyses of later stage aspects of disease, and forsmall molecule screens. When published we will credit MPS/ISMRD for contributing to the establishmentof these vital tools.
Aim 1: Assess individual contribution of cathepsin K and L toward cardiac defects in ML zebrafish.
1) Cathepsin expression and generation of transgenic animals. Immunohistochemical analyses ofcathepsin K and L demonstrate that both proteases are expressed in multiple heart tissues, including themyocardium and epicardium. These analyses have guided the choice of tissue specific promoters fortransgenic constructs that will be used to generate zebrafish expressing fluorophor tagged WT andmutant versions of Cts K and L. Toward this goal, we have now successfully shown that following mRNA
injection, the CtsK-mCherry fusion protein is both expressed and active in developing embryos.
Establishing these parameters was critical before proceeding with transgenesis. Therefore the final DNA
constructs for transgenesis are currently in production at Gene Art. Initial injections for the generation oftransgenic lines should be completed by early next year.
2) Inhibition of cathespin K in MLII. To assess ctsK’s contribution toward ML cardiac pathology, its
expression or activity was inhibited in the MLII background. CtsK expression was genetically inhibitedusing one of two gene specific morpholinos and its activity reduced pharmacologically using the FDAapproved cathepsin K inhibitor Odanacatib. Preliminary analyses of multiple aspects of cardiac
morphology and function, including formation of the AV valve and unidirectional blood flow, suggest bothtreatments substantially improve MLII cardiac development. Additional experiments to confirm andextend these findings are currently underway.
Aim 2 – Determine whether increased cathepsin activity impacts TGFß signaling in MLII hearts.

1) Are there differences in either TGFß or BMP signaling in WT and MLII hearts? In combination with TGFßand BMP-reporter transgenic animals, immunohistochemical analyses of pSmad levels is being used toassess differences in signaling between WT and MLII hearts. In parallel studies pharmacologicalinhibition of TGFß/BMP receptor activation is being used to address the pathogenic significance of noted
differences in pathway activation. These studies are currently underway. Once these analyses arecompleted we will address whether genetic or pharmacological inhibition of cathepsin K also affects
pSmad levels and reporter transgene activation in MLII hearts.
Aim 3 – Determine which patient mutations are pathogenic for cardiac dysfunction.

To identify which patient mutations are associated with altered cardiac morphology and function, wehave introduced mRNA bearing specific lesions in the morphant background and compared their ability
to rescue heart phenotypes with ML animals co-injected with WT mRNA. We have analyzed threedifferent sets of mutations including several within the DMAP domain (including K732N), threemutations within Notch domain 1 (C442Y, C461G, and C468S), and one mutation in Notch domain 2. Thus
far analyses have been limited to gross morphology. Unlike WT gnptab mRNA, which reduces cardiac
edema, restores normal cardiac morphology, and increases blood flow in 85% of the animals analyzed,

mRNA bearing the K732N mutation did not significantly restore any of the tested parameters. Therefore,
as previously noted in the cartilage, the DMAP domain is essential for normal phosphotransferase
function – and mutations within it are highly likely to be pathogenic in both the cartilage/bone and theheart. In contrast, preliminary analyses of the three Notch domain 1 mutations suggest that the C442Y
lesion, which has been associated with MLIII, is less pathogenic in the heart than the ML-intermediate
lesion C468S. This is supported by the fact that mRNA“rescue” experiments show improved cardiacmorphology and reduced edema in 85% of the MLII animals injected with the C442Y-containing mRNA
versus only 40% recovery with the mRNA carrying the C468S lesion. mRNAs bearing either the C461G(Notch 1/MLIII) or C505Y (Notch2/MLIII) rescued gross cardiac pathology ~60% of the time, suggestingthat in addition to differences in pathogenic severity between MLII and MLIII, different mutations within
a single ML class may have different degrees of pathogenicity. More in depth experiments are currently
underway. Collectively these data support the idea that genotype-phenotype correlations can beestablished in this system, and as such may eventually combine with patient data to predict the severity
and course of mutation specific tissue pathologies.
Summary of progress to date:
Although all studies are still in progress and much work remains, we are highly encouraged by thefindings to date. First, the fact that both cathepsin K knockdown and treatment with Odanacatib
improved MLII heart development is very promising. These data not only support a central role forcathepsin(s) in primary pathogenesis but point to FDA-approved drugs like Odanacatib for future
consideration. Although these findings are preliminary and need to be independently confirmed, they arecertainly encouraging. Second, the fact that both cathepsin K and TGFß signaling may be involved in
cardiac pathology suggests common mechanisms may underlie ML skeletal and heart dysfunction. If true,
this would not only be an important advance for future scientific investigation, but also mean that anindividual modality may therapeutically improve both systems. Third, the tools generated thus far andthose underway will serve as novel platforms to both further investigate these and other emerging
mechanisms and to rapidly screen the efficacy of potential drugs.
Comment on year 1 budget residual funds:
As clear from the financial report of year 1’s expenditures there was a $2300 carry-over. I wanted toqualify that this money has essentially been spent, but because the “reagents” are in production they will
not post on this year’s financial report. $800 of the remaining balance will be payed to the Zebrafish
TILLING Project Consortium at The Fred Hutchinson Cancer Research Center. This money covers part ofthe cost for them to screen for the gnptab mutant, which is now identified and being raised for shipment
to us. The remaining $1500 is also already allocated to pay Gene Art for synthesizing one of the large
DNA constructs necessary to generate the cathespin transgenic animals.