Project leader: Franco COTELLI (franco.cotelli@unimi.it)

Project leader: Franco COTELLI (franco.cotelli@unimi.it)

Location: Department of Biosciences, University of Milan, Italy

RESEARCH PROJECT SUMMARY

Cerebral cavernous malformations (CCMs) are neurovascular abnormalities composed of enlarged thin-walled capillary clusters, which may cause several neural deficits. In human, the onset of this disorder has been related to sporadic or heritable mutations in, at least, one of these three genes, KRIT1 (CCM1), OSM (CCM2) and PDCD10 (CCM3) [1]. A specific mutation in the gene KRIT1 is responsible for the majority of malformation and in vitro experiments revealed that its silencing inactivate the NOTCH pathway [2,3].

In the last years zebrafish has been well established as a great model organism for studying embryonic development, human illness and heritable disorders. This is due to light transparency, very small size and genetic tractability of the embryos. Zebrafish has also proven to be a good model for comparative studies, which demonstrated a striking degree of anatomical and functional conservation between zebrafish and mammals [4,5].

Thanks to all these features it turned out to be a powerful model system to study CCMs. Previous works characterized in zebrafish loss-of-function mutations of ccm1 and functional knockdown of ccm3, which result in dilation of thin-walled vessels and over-branching of cranial vessels [6-8]. All these defects may be related to alterations of the Notch pathway.

1. 
   Labauge P, Denier C, Bergametti F, Tournier-Lasserve E (2007). Genetics of cavernous angiomas. Lancet Neurol; 6:237-244.
   
2. 
   Eerola I, Plate KH, Spiegel R, Boon LM, Mulliken JB, Vikkula M (2000). KRIT1 is mutated in hyperkeratotic cutaneous capillary-venous malformation associated with cerebral capillary malformation. Hum Mol Gen; 9:1351–1355.
   
3. 
   Wüstehube J, Bartol A, Liebler SS, Brütsch R, Zhu Y, Felbor U, Sure U, Augustin HG, Fischer A (2010). Cerebral cavernous malformation protein CCM1 inhibits sprouting angiogenesis by activating DELTA-NOTCH signaling. Proc Natl Acad Sci U S A; 107:12640-14645.
   
4. 
   Zon LI (1999). Zebrafish: a new model for human disease. Genome Res; 9:99-100.
   
5. 
   Vogel AM, Weinstein BM (2000). Studying vascular development in the zebrafish. Trends Cardiovasc Med; 10:352-360.
   
6. 
   Kleaveland B, Zheng X, Liu JJ, Blum Y, Tung JJ, Zou Z, Sweeney SM, Chen M, Guo L, Lu MM, Zhou D, Kitajewski J, Affolter M, Ginsberg MH, Kahn ML (2009). Regulation of cardiovascular development and integrity by the heart of glass-cerebral cavernous malformation protein pathway. Nat Med; 15:169-176. Erratum in: Nat Med;15:584. Sweeney, Shawn M [added].
   
7. 
   Hogan BM, Bussmann J, Wolburg H, Schulte-Merker S (2008). ccm1 cell autonomously regulates endothelial cellular morphogenesis and vascular tubulogenesis in zebrafish. Hum Mol Genet; 17:2424-2432.
   
8. 
   Yoruk B, Gillers BS, Chi NC, Scott IC (2012). Ccm3 functions in a manner distinct from Ccm1 and Ccm2 in a zebrafish model of CCM vascular disease. Dev Biol; 362:121-131
   
9. 
   Gulino A, Di Marcotullio L, Screpanti I (2010). The multiple functions of Numb. Exp Cell Res; 316:900-906.
   
10. 
    Liu L, Lanner F, Lendahl U, Das D (2011). Numblike and Numb differentially affect p53 and Sonic Hedgehog signaling. Biochem Biophys Res Commun; 413:426-431.
    
11. 
    Bassi A, Fieramonti L, D'Andrea C, Mione M, Valentini G (2011). In vivo label-free three-dimensional imaging of zebrafish vasculature with optical projection tomography. J Biomed Opt; 16: 100502.
    

1. Project leader: STEFANO DUGA (stefano.duga@unimi.it)

3. RESEARCH PROJECT SUMMARY

Parkinson disease (PD) is a degenerative disorder of the central nervous system, characterized by the progressive death of dopaminergic neurons in the substantia nigra, associated with resting tremor, bradykinesia, postural instability, and rigidity. PD is a complex disorder caused by the combination of so-far largely unidentified environmental factors and of predisposing susceptibility genetic components. Despite extensive studies, only a minority of such genetic factors are known.

Even though most forms of PD are sporadic, some rare monogenic forms, with a clear “Mendelian” inheritance, have been reported, leading to the identification of several loci for familial PD. However, for some of them, the specific gene remains elusive. Intriguingly, the clinical presentations and neuropathological findings of hereditary forms of PD are often indistinguishable from the sporadic ones, raising the possibility that common pathophysiologic mechanisms underlie both hereditary and sporadic PD.

In the past few years, the study of the genetic variants predisposing to sporadic PD took advantage of genome wide association studies (GWAS). However, the 5 published GWAS did not add much to what was previously known, explaining only a very small portion of the total heritability of the disease. This suggests that the missing heritability could be, at least in part, due to rare higher-impact variants. In this frame, the rapid advancement of sequencing technologies has made it possible to capture, by targeted hybridization and deep-sequencing, all genetic variations within exonic regions of the genome, including splice junctions and miRNAs: the so called “whole-exome” approach.

This project aims to discover new genes involved in recessive Mendelian form of PD by a next-generation sequencing approach. The specific aims are: 1) to capture and sequence the exome of PD probands of recessive families (only affected siblings with consanguineous parents) and to select the variants potentially causative; 2) to verify if selected variants are specific for PD analyzing a large panel of cases and controls; 3) to search the candidate genes in a group of potentially recessive PD cases; 4) to evaluate the functional role of identified mutations

To accomplish theses goals, we will focus on 10 PD patients derived from 7 families with consanguineous parents and 2 or more affected siblings selected from the DNA Bank of the Parkinson Institute of Milan, which is based in our laboratory. Such cases are highly suggestive of a recessive form of PD and represent a valuable resource for the identification of PD causative genes as their inbred nature overcomes several of the limitations that exist in the use of exome sequencing. The exome-sequencing experiments will be performed in collaboration with Prof. John Landers of the University of Massachusetts Medical School, Worcester.