Molecular mechanisms underlying williams syndrome.

A. OVERAL AIM AND KEY OBJECTIVES: Williams Syndrome (WS) is a rare neurodevelopmental disorder, caused by a hemizygous deletion of a 2 cM region on chromosome band 7q 11.23. Characteristic features of WS include supravalvular aortic stenosis (SVAS), connective tissue abnormalities, transient hypercalcemia, growth retardation, hyperacusis, anxiety disorder, attention-deficit/hyperactivity disorder and mental retardation. Cognitive dysfunction in WS is characterized by a profound difficulty in visuo-spatial information processing, whereas linguistic abilities are relatively preserved. The WS critical region spans approximately 16 genes and identification of patients with small deletions has demonstrated that haploinsufficiency for the elastin (ELN) gene is responsible for cardiovascular anomalies. Similar studies with other patients have linked hemizygous deletion of the LIM-kinase 1 (LIMK1) gene to visuo-spatial cognition deficits in WS, but these resuits have been challenged. We have shown that the CYLN2 gene, which encodes the microtubule associated protein CLIP-115, is localized within the WS critical region, telomeric to the LIMK1 gene. Since CLIP-115 is mainly detected in the dendritic compartment of a variety of neurons in the brain, we have proposed that hemizygous deletion of the CYLN2 gene may underlie part of the neurologic phenotype in WS. CLIP-115 is a member of the small family of "cytoplasmic linker proteins", or CLIPs. This name was originally coined to describe the main features of another protein, CLIP-170, which was proposed to link endocytotic vesicles to microtubules. More recently CLIP-170 and -115 have been shown to associate specifically with the growing (plus) ends of microtubules and a CLIP-170 homologue in fission yeast was shown to function as anti-catastrophe factor. We have discovered that "CLIP-associating proteins" (CLASPs), which colocalize with the CLIPs at microtubule distal ends, are involved in the regulation of microtubule dynamics in motile fibroblasts. We have proposed that CLIP-CLASP interactions are necessary for proper regulation of microtubule stability in polarized cells. In WS microtubule plus ends may have altered dynamics, because of diminished CLIP-115 levels. To test whether hemizygous deletion of the CYLN2 gene underlies (part of) the neurologic abnormalities in WS, we have generated an inducible knock out of the gene in mice, by placing lox-sites up- and downstream of the gene. After the 3' end lox-site a lacZ reporter gene was added. We generated two types of mice: 1) a strain in which the 5'- and 3' end lox-sites, including the reporter gene, are present and the CYLN2 gene is still intact (CLIP-T mice); 2) a strain in which the CYLN2 gene is deleted and instead of CLIP-115 lacZ is expressed (CLIP-L mice, see figure 1, PGN 9, part 2 of this proposal). CLIP-T mice serve as an excellent control for the CLIP-L mice in behavioural experiments, since both lines have the same genetic background, except for the presence/absence of the CYLN2 gene. We have shown that CLIP-115 is expressed in the CLIP-T mice, but not in the CLIP-L mice. Conversely, in bram sections of the CLIP-L mice lacZ is expressed, whereas in the CLIP-T mice blue staining is absent. These data revealed that CLIP-115 is very abundantly expressed in pyramidal neurons of the hippocampus and less so in the amygdala and in Purkinje cells of the cerebellum. Based upon these observations we performed a number of simple behavioural assays, which demonstrate that deletion of the CYLN2 gene causes specific behavioural deficits. Lack of CLIP-115 also causes growth retardation in mice. These problems already occur in heterozygous animals, linking haploinsufficiency for the CYLN2 gene to neurodevelopmental features in WS. The central aims of the present research proposal are: 1) to explore the cell biology and physiology of a deficiency of CLIP-115; 2) to link the dysfunctioning in CYLN2 knock out mice to a deficiency of CLIP-115 in (neuronal) develonment, in adult tissue/cells, or both. We will address these issues along 4 lines of research (key objectives): 1) investigation of the cell biology of the CYLN2 gene inactivation; 2) investigation of the spatio-temporal effect of the inactivation of CLIP-115; 2) investigation of the electrophysiology of the different types of CYLN2 knock out mice; 3) investigation of the effect of deletion of other genes in the WS critical region (in particular the LIMK1 gene), in conjunction with the CYLN2 gene, in mice. B. APPROACH 1,2) CLIP-115 is expressed in fibroblasts, derived from CLIP-T mice, but not in celis from CLIP-L mice. These fibroblasts will be used to investigate the effect of a CLIP-115 deficiency on the distribution of other plus end binding protens and on microtubule dynamics, in a relatively simple cell culture system. In the mean time, CLIP-T mice (which have an intact CYLN2 gene surrounded by loxsites) will be crossed with mouse lines expressing a fusion protein of the Cre recombinase, linked to the tamoxifen-responsive ligand binding domain (LBD). Since the Cre-LBD protein will be placed under control of different promoters, we can control deletion of the CYLN2 gene in a highly specific spatio-temporal fashion. Ceil lines will be derived from these mice and both cell lines and mice will be investigated at the cel! biological level (described above), at the behavioural level (which includes rotarod assays and fear conditioning experiments, in which the CLIP-L mice were shown to behave poorly) and at the electrophysiological level (see point 3). In addition we will determine growth curves of the different mice. These results together will show in which ceil types CLIP-115 is required, potentially linking cell-specific CLIP-115 deficits to specific cell biological and/or behavioural deficiencies. 3) All different types of knock out mice will be tested both in a cell physiological as well as in a systems physiological manner, to determine whether lack of CLIP-115 influences the electrophysiological properties of specific neuronal circuits. It will be particularly interesting to compare hippocampal deficits with those of the cerebellum, since behavioural experiments indicate that (parts of) these brain areas are affected in CLIP-L mice. In addition, we have shown that in the CLIP-L mice hippocampal LTP is affected specifically. These mice will therefore be examined morphologically for dendritic abnomralities. 4) One of the outstanding questions in WS research is whether deletion of other genes, apart from CYLN2 and ELN, contributes to the development of the disorder, in particular to neurodevelopmental aspects of the disease. It has been shown that a region on mouse chromosome 5 is syntenic to the WS critical region on human chromosome 7q11.23 and that the murine genes are arranged in the same order as on the human chromosome. We have applied Cre-lox technology to remove the region including LIMK1 and CYLN2 genes from the mouse genome, using the available lox-site at the 3'end of the CYLN2 gene as a basis. ES cells which contained the 3'end CYLN2 lox-site were targeted with the 5'end LIMK1 construct and homologously targeted ES cell clones were treated with Cre recombinase. The phenotype of mice, in which both CYLN2 and LIMK1 genes have been affected, will be compared to that of CLIP-L mice, in which only the CYLN2 gene is absent. C, D. ELEMENTS OF INNOVATION AND RELEVANCE FOR HEALTH CARE. The generation of Cre-LBD/CLIP-T mice provides us with an innovative system to analyze the spatio-temporal effects of deletion of the CYLN2 gene in the mouse and to compare this to features of WS. Results from these studies may prove valuable for potential future therapeutic strategies in WS. Application of the Cre-LBD system to specific cells/tissues in the mouse is still a relatively unexplored area of research. We have shown that in our CLIP-T construct, deletion of the CYLN2 gene can be exactly monitored (by the apparition of LacZ). Thus, we will be able to determine which cells are receptive to Cre-LBD. This is of fundamental importance for future research for the whole scientific community. CLIP-115 is part of a growing number of proteins that regulate microtubule dynamics from the plus ends. More knowledge as to how microtubules behave in vivo is of great relevance to health care. That this does not only apply to WS is clear when one looks at the number of drugs that are targeted to microtubules (e.g. anti-cancer drugs) and that are being designed to affect microtubule dynamics.