Periodic Report Summary 1 - MIRNAS/22Q11DS (NEUROGENESIS IN 22Q11.2 DELETION SYNDROME: ROLE OF microRNAs)
22q11.2 deletion syndrome (22q11DS or DiGeorge syndrome) is an autosomal disease affecting 1:4000 live births, and refers to a group of related syndromes associated by hemizygous deletion of a 1.5 to 3 Mb region within Chromosome 22. Individuals affected by 22q11.2DS have impaired cognitive functions and a high incidence of psychiatric disorders, including bipolar disorder, autism spectrum disorders, and depression; moreover, microdeletion of the 22q11 locus is the highest known genetic risk factor associated with schizophrenia (Karayiorgou M and Gogos JA, 2004). Haploinsufficiency of DiGeorge critical region 8 (Dgcr8), a gene frequently deleted in 22q11DS patients that encodes for DGCR8, an essential protein for microRNAs biogenesis, has been directly associated to some of the phenotypic deficits of the syndrome (Stark KL, et al., 2008). Thus, impaired miRNA biogenesis observed in an adult mouse model of 22q11DS contributes to the neuroanatomical and psychiatric defects of 22q11DS affected individuals (Stark KL, et al., 2008).
microRNAs (miRNAs) are 20−25 nucleotides long non-coding RNAs that have been found in a wide variety of organisms, and have been shown to regulate gene expression post-transcriptionally by targeting mRNAs for destabilization and translational repression (Filipowicz W, et al., 2008). miRNAs biogenesis is initiated in the nucleus by the Microprocessor, a protein complex comprising the type III RNase DROSHA that provides the catalytic activity and the double-strand RNA binding protein DGCR8 indispensable for substrate recognition, and upon export to the cytoplasm, the biogenesis process is completed by the cytosolic RNase DICER that cleaves the double-stranded miRNA into its mature form. Finally, one strand of the miRNA is loaded into a complex of proteins that mediates its inhibitory action toward the target mRNA (Filipowicz W, et al., 2008).
Despite a possible neurodevelopmental origin for the neuroanatomical defects observed in 22q11DS has been hypothesized, little is known about the early brain development of mouse models of 22q11DS or the possible causative role of miRNAs dysfunction at early stages of brain development of 22q11DS affected individuals. Our hypothesis is that impaired miRNA biogenesis during brain development might contribute to the physiopathology in 22q11DS.
Using LgDel mice (having a hemizygous deletion that includes most the genes encoded in the human 22q11.2 region. Merscher et al., 2001), which recapitulate some of the neuroanatomical and psychiatric defects observed in 22q11DS patients, we sought to investigate the role of altered miRNAs biogenesis in early processes of brain development. Through gene expression profiling of RNAs extracted from LgDel and WT embryonic cortices, we identified a pool of mature miRNAs significantly downregulated and several deregulated mRNAs in embryonic cortices of LgDel mouse embryos if compared to cortices of WT embryos. Moreover, by bionformatic analyses we identified among the deregulated mRNAs in LgDel cortices, several putative targets of downregulated miRNAs. These results are therefore compatible with our hypothesis that impaired miRNA biogenesis during brain development is responsible for some of the defects observed in brain 22q11DS. Moreover, to investigate the effects of hemizygous deletion of Dgcr8 for cortical development, we used in utero electroporation to achieve selective labeling of newborn glutamatergic projection neurons. By this means, we found a migratory/maturation defect of glutamatergic projection neurons, upon hemizygous deletion of Dgcr8 gene. Finally, in vitro experiments with primary embryonic cortical neurons from LgDel mice mirrored the defects observed in vivo upon DGCR8 reduction. Taken together, our results provide a possible mechanism to explain the neuroanatomical defects observed in brain of 22q11DS.
In parallel we designed and tested in cell lines a luciferase reporter plasmid to monitor the activity of the Microprocessor complex, thus paving the way for future screenings of compounds able to restore DGCR8 function in 22q11DS patients.
References:
Filipowicz W, Bhattacharyya SN, Sonenberg N. Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat Rev Genet. 2008 Feb;9(2):102-14. doi: 10.1038/nrg2290.
Karayiorgou M, Gogos JA. The molecular genetics of the 22q11-associated schizophrenia. Brain Res Mol Brain Res. 2004 Dec 20;132(2):95-104.
Stark KL, Xu B, Bagchi A, Lai WS, Liu H, Hsu R, Wan X, Pavlidis P, Mills AA, et al. Altered brain microRNA biogenesis contributes to phenotypic deficits in a 22q11-deletion mouse model. Nat Genet. 2008 Jun;40(6):751-60. Epub 2008 May 11.
Merscher S, Funke B, Epstein JA, Heyer J, Puech A, et al. TBX1 is responsible for cardiovascular defects in velo-cardio-facial/DiGeorge syndrome. Cell. 2001 Feb 23;104(4):619-29.
microRNAs (miRNAs) are 20−25 nucleotides long non-coding RNAs that have been found in a wide variety of organisms, and have been shown to regulate gene expression post-transcriptionally by targeting mRNAs for destabilization and translational repression (Filipowicz W, et al., 2008). miRNAs biogenesis is initiated in the nucleus by the Microprocessor, a protein complex comprising the type III RNase DROSHA that provides the catalytic activity and the double-strand RNA binding protein DGCR8 indispensable for substrate recognition, and upon export to the cytoplasm, the biogenesis process is completed by the cytosolic RNase DICER that cleaves the double-stranded miRNA into its mature form. Finally, one strand of the miRNA is loaded into a complex of proteins that mediates its inhibitory action toward the target mRNA (Filipowicz W, et al., 2008).
Despite a possible neurodevelopmental origin for the neuroanatomical defects observed in 22q11DS has been hypothesized, little is known about the early brain development of mouse models of 22q11DS or the possible causative role of miRNAs dysfunction at early stages of brain development of 22q11DS affected individuals. Our hypothesis is that impaired miRNA biogenesis during brain development might contribute to the physiopathology in 22q11DS.
Using LgDel mice (having a hemizygous deletion that includes most the genes encoded in the human 22q11.2 region. Merscher et al., 2001), which recapitulate some of the neuroanatomical and psychiatric defects observed in 22q11DS patients, we sought to investigate the role of altered miRNAs biogenesis in early processes of brain development. Through gene expression profiling of RNAs extracted from LgDel and WT embryonic cortices, we identified a pool of mature miRNAs significantly downregulated and several deregulated mRNAs in embryonic cortices of LgDel mouse embryos if compared to cortices of WT embryos. Moreover, by bionformatic analyses we identified among the deregulated mRNAs in LgDel cortices, several putative targets of downregulated miRNAs. These results are therefore compatible with our hypothesis that impaired miRNA biogenesis during brain development is responsible for some of the defects observed in brain 22q11DS. Moreover, to investigate the effects of hemizygous deletion of Dgcr8 for cortical development, we used in utero electroporation to achieve selective labeling of newborn glutamatergic projection neurons. By this means, we found a migratory/maturation defect of glutamatergic projection neurons, upon hemizygous deletion of Dgcr8 gene. Finally, in vitro experiments with primary embryonic cortical neurons from LgDel mice mirrored the defects observed in vivo upon DGCR8 reduction. Taken together, our results provide a possible mechanism to explain the neuroanatomical defects observed in brain of 22q11DS.
In parallel we designed and tested in cell lines a luciferase reporter plasmid to monitor the activity of the Microprocessor complex, thus paving the way for future screenings of compounds able to restore DGCR8 function in 22q11DS patients.
References:
Filipowicz W, Bhattacharyya SN, Sonenberg N. Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat Rev Genet. 2008 Feb;9(2):102-14. doi: 10.1038/nrg2290.
Karayiorgou M, Gogos JA. The molecular genetics of the 22q11-associated schizophrenia. Brain Res Mol Brain Res. 2004 Dec 20;132(2):95-104.
Stark KL, Xu B, Bagchi A, Lai WS, Liu H, Hsu R, Wan X, Pavlidis P, Mills AA, et al. Altered brain microRNA biogenesis contributes to phenotypic deficits in a 22q11-deletion mouse model. Nat Genet. 2008 Jun;40(6):751-60. Epub 2008 May 11.
Merscher S, Funke B, Epstein JA, Heyer J, Puech A, et al. TBX1 is responsible for cardiovascular defects in velo-cardio-facial/DiGeorge syndrome. Cell. 2001 Feb 23;104(4):619-29.