LEGISLACION DE PERÚ

Dyx1c1 was the first gene associated with dyslexia susceptibility (Taipale et al

2003). While the results of RNAi experiments suggest a role in neuronal

migration (Wang et al 2006), and protein interaction studies suggest possible roles in chaperonin function (Chen et al 2009) and estrogen receptor trafficking

(Massinen et al 2009), a genetic test for the required functions of Dyx1c1 has

been lacking. In order to elucidate the required functions of Dyx1c1, we have analyzed the phenotype of a Dyx1c1 knockout mouse in which exons 2,3 and 4 were deleted. Western blot analysis confirmed that the mutation results in a complete loss of Dyx1c1 protein expression. In breeding experiments between heterozygous mice the ratio of homozygous KO, heterozygous, and WT mice was 1:4:2, indicating possible embryonic lethality. The surviving Dyx1c1 knockout mice display severe developmental abnormalities that include Situs Inversus, a reversal in the typical left-right asymmetry of thoracic and abdominal organs and early onset hydrocephaly. These two phenotypes are often associated with Primary Ciliary Dyskinesia in mice. We therefore examined cilia structure and motility in the ventricles of knockout mice. Immunostaining for acetylated tubulin, and gamma tubulin indicated that the cilia of ependymal cells lining the lateral, third, and fourth ventricles were intact and not appreciably decreased in number. In contrast, live cell imaging and an Indian ink flow assay indicated a lack of ciliary motility. In WT mice, a cilia beat frequency of 7.5 beats/second was

observed in lateral, third and fourth ventricles using DIC video microscopy of brain slices. However, in Dyx1c1 KOs cilia were completely immotile and displayed no apparent beating. In an additional assay for the motility of cilia in the lateral ventricles a small volume of Indian ink was applied to the surface of the ventricle and the dispersion and flow of ink was imaged. In wt mice an anteriorly directed flow was apparent with an approximate velocity of 96 mm/sec, while in the KO mice the Indian ink showed only a passive diffusion in all directions at a rate of 6 mm/sec. When the ultrastructure of the WT and KO cilia was examined, we found that the inner and outer dynein arms that are required for the ciliary motility were missing in the KO. In sum, the analysis of the Dyx1c1 KO thus indicates that Dyx1c1 is required for motility of cilia.

2.2 Introduction

Cilia, hair-like organelles projecting from the surface of nearly all polarized cell types, serve essential roles in cellular signaling and motility. The basic structure of motile cilia and the related organelle flagella is evolutionarily conserved. In most motile cilia, a ring of nine peripheral microtubule doublets surrounds a central pair apparatus of single microtubules that connect to the nine peripheral doublets by radial spokes (9+2 structure). Motile monocilia present at the mouse node during early embryogenesis are an exception, lacking the central pair apparatus (9+0 structure)72,134. Distinct multi-protein dynein complexes attached at regular intervals to the peripheral microtubule doublets contain molecular

motors that drive and regulate ciliary motility. Specifically, ODAs are responsible for beat generation, whereas both the IDAs and the nexin link–dynein regulatory complexes (N-DRCs) regulate ciliary and flagellar beating pattern and frequency. Identifying the proteins responsible for the correct assembly of this molecular machinery is critical to understanding the causes of motile cilia–related diseases134.

PCD (MIM 244400), a rare genetic disorder affecting approximately 1 in 20,000 individuals, is caused by immotile or dyskinetic cilia. Loss of ciliary function in upper and lower airways causes defective mucociliary airway clearance and, subsequently, chronic inflammation that regularly progresses to destructive airway disease (bronchiectasis). Organ laterality defects are also observed, with approximately half of individuals with PCD exhibiting situs inversus and, more rarely, situs ambiguus, which can associate with complex congenital heart disease. Dysfunctional sperm tails (flagella) frequently cause male infertility in individuals with PCD. Another consequence of ciliary dysfunction, particularly evident in mouse models, is hydrocephalus caused by disrupted flow of cerebrospinal fluid through the cerebral aqueduct connecting the third and fourth brain ventricles. Although ciliary dysmotility is not sufficient for hydrocephalus formation in humans owing to morphological differences between the mouse and human brains, the incidence of hydrocephalus, secondary to aqueduct closure, is higher in individuals with PCD85,90,135.

Genetic analyses of individuals with PCD have identified several autosomal recessive mutations in genes encoding axonemal subunits of the ODA

complexes and related components. In addition, recessive mutations in CCDC3996 (MIM 613798) and CCDC4097 (MIM 613799) have been linked to PCD with severe tubular disorganization and defective nexin links. Mutations in the radial spoke head genes RSPH4A and RSPH9 as well as in HYDIN can cause intermittent or complete loss of the central-apparatus microtubules135. Two X-linked PCD variants associated with syndromic cognitive dysfunction and retinal degeneration are caused by mutations in OFD1 (MIM 311200) and RPGR (MIM 312610), respectively. Another functional class of proteins emerging from the identification of PCD-causing mutations are proteins involved in the cytoplasmic preassembly of both ODAs and IDAs, including those encoded by DNAAF260 (also known as KTU; MIM 612517), DNAAF1104 (also known as LRRC50; MIM 613190), DNAAF3102 (also known as C19orf51; MIM 614566) and the recently identified LRRC6103 _{(MIM 614930).}

DYX1C1 (MIM 608706) was initially identified as a candidate dyslexia gene, owing to both a single balanced translocation t(2;15)(q11;q21) coincidentally segregating with dyslexia in a family and subsequent SNP association studies112. Follow-up gene association studies have provided both positive and negative support for association with dyslexia. Taipale et al. (2003) showed that DYX1C1 is expressed nearly ubiquitously in adult tissues, including brain, and that the protein can be detected by immunocytochemistry in the nuclei and cytoplasm of neurons and glia in human neocortex. The protein domains of DYX1C1 include an N-terminal p23 and three C-terminal TPR domains. DYX1C1, when over- expressed in cell lines, can interact with Hsp70, Hsp90 and an E-3 ubiquitin

ligase, CHIP, suggesting that the protein may also be involved in the degradation of unfolded proteins127,128. Wang and colleagues performed the characterization of the developmental function of DYX1C1 by employing in utero RNAi in embryonic rat neocortex. The results from this study indicate that DYX1C1 plays a role in the migration of neocortical neurons and more specifically is required for the transition out of the multipolar stage of migration. Dyx1c1 RNAi decreased the migration of neurons causing them to accumulate in a multipolar stage of migration. The impairment was rescued by overexpression of Dyx1c1, and the C- terminal TPR domains were sufficient to rescue migration. Using truncation mutants, they found that the C-terminus of DYX1C1 biases the cellular localization of DYX1C1 to the cytoplasm, while the N-terminus alone can localize to the nucleus and cytoplasm in cell lines and in developing neurons124,125. Molecular and cellular analyses of DYX1C1 have indicated potential functional roles in estrogen receptor trafficking, and recent proteomic and gene expression studies have suggested a possible role in cilia121,129,133.