Mutation of the diamond-blackfan anemia gene Rps7 in mouse results in morphological and neuroanatomical phenotypes.

The ribosome is an evolutionarily conserved organelle essential for cellular function. Ribosome construction requires assembly of approximately 80 different ribosomal proteins (RPs) and four different species of rRNA. As RPs co-assemble into one multi-subunit complex, mutation of the genes that encode RPs might be expected to give rise to phenocopies, in which the same phenotype is associated with loss-of-function of each individual gene. However, a more complex picture is emerging in which, in addition to a group of shared phenotypes, diverse RP gene-specific phenotypes are observed. Here we report the first two mouse mutations (Rps7(Mtu) and Rps7(Zma)) of ribosomal protein S7 (Rps7), a gene that has been implicated in Diamond-Blackfan anemia. Rps7 disruption results in decreased body size, abnormal skeletal morphology, mid-ventral white spotting, and eye malformations. These phenotypes are reported in other murine RP mutants and, as demonstrated for some other RP mutations, are ameliorated by Trp53 deficiency. Interestingly, Rps7 mutants have additional overt malformations of the developing central nervous system and deficits in working memory, phenotypes that are not reported in murine or human RP gene mutants. Conversely, Rps7 mouse mutants show no anemia or hyperpigmentation, phenotypes associated with mutation of human RPS7 and other murine RPs, respectively. We provide two novel RP mouse models and expand the repertoire of potential phenotypes that should be examined in RP mutants to further explore the concept of RP gene-specific phenotypes.

Neuropeptide TLQP-21, a VGF internal fragment, modulates hormonal gene expression and secretion in GH3 cell line.

In the present study we demonstrated that TLQP-21, a biologically active peptide derived from the processing of the larger pro-VGF granin, plays a role in mammotrophic cell differentiation. We used an established in vitro model, the GH3 cell line, which upon treatment with epidermal growth factor develops a mammotrophic phenotype consisting of induction of prolactin expression and secretion, and inhibition of growth hormone. Here we determined for the first time that during mammotrophic differentiation, epidermal growth factor also induces Vgf gene expression and increases VGF protein precursor processing and peptide secretion. After this initial observation we set out to determine the specific role of the VGF encoded TLQP-21 peptide on this model. TLQP-21 induced a trophic effect on GH3 cells and increased prolactin expression and its own gene transcription without affecting growth hormone expression. TLQP-21 was also able to induce a significant rise of cytoplasmic calcium, as measured by Fura2AM, due to the release from a thapsigargin-sensitive store. TLQP-21-dependent rise in cytoplasmic calcium was, at least in part, dependent on the activation of phospholipase followed by phosphorylation of PKC and ERK. Taken together, the present results demonstrate that TLQP-21 contributes to differentiation of the GH3 cell line toward a mammotrophic phenotype and suggest that it may exert a neuroendocrine role in vivo on lactotroph cells in the pituitary gland.

Diamond-Blackfan anemia: genotype-phenotype correlations in Italian patients with RPL5 and RPL11 mutations.

BACKGROUND: Diamond-Blackfan anemia is a rare, pure red blood cell aplasia of childhood due to an intrinsic defect in erythropoietic progenitors. About 40% of patients display various malformations. Anemia is corrected by steroid treatment in more than 50% of cases; non-responders need chronic transfusions or stem cell transplantation. Defects in the RPS19 gene, encoding the ribosomal protein S19, are the main known cause of Diamond-Blackfan anemia and account for more than 25% of cases. Mutations in RPS24, RPS17, and RPL35A described in a minority of patients show that Diamond-Blackfan anemia is a disorder of ribosome biogenesis. Two new genes (RPL5, RPL11), encoding for ribosomal proteins of the large subunit, have been reported to be involved in a considerable percentage of patients. DESIGN AND METHODS: In this genotype-phenotype analysis we screened the coding sequence and intron-exon boundaries of RPS14, RPS16, RPS24, RPL5, RPL11, and RPL35A in 92 Italian patients with Diamond-Blackfan anemia who were negative for RPS19 mutations. RESULTS: About 20% of the patients screened had mutations in RPL5 or RPL11, and only 1.6% in RPS24. All but three mutations that we report here are new mutations. No mutations were found in RPS14, RPS16, or RPL35A. Remarkably, we observed a higher percentage of somatic malformations in patients with RPL5 and RPL11 mutations. A close association was evident between RPL5 mutations and craniofacial malformations, and between hand malformations and RPL11 mutations. CONCLUSIONS: Mutations in four ribosomal proteins account for around 50% of all cases of Diamond-Blackfan anemia in Italian patients. Genotype-phenotype data suggest that mutation screening should begin with RPL5 and RPL11 in patients with Diamond-Blackfan anemia with malformations.

TLQP-21, a neuroendocrine VGF-derived peptide, prevents cerebellar granule cells death induced by serum and potassium deprivation.

Different VGF peptides derived from Vgf, originally identified as a nerve growth factor responsive gene, have been detected in neurons within the central and peripheral nervous system and in various endocrine cells. In the current study, we have evaluated the ability of TLQP-21, a VGF-derived peptide, to protect, in a dose- and time-dependent manner, primary cultures of rat cerebellar granule cells (CGCs) from serum and potassium deprivation-induced cell death. We demonstrated that TLQP-21 increased survival of CGCs by decreasing the degree of apoptosis as assessed by cell viability and DNA fragmentation. Moreover, TLQP-21 significantly activated extracellular signal-regulated kinase 1/2, serine/threonine protein kinase, and c-jun N-terminal kinase phosphorylation, while decreased the extent of protein kinase C phosphorylation, as demonstrated by western blot analysis. In addition, TLQP-21 induced significant increase in intracellular calcium (as measured by fura-2AM) in about 60% of the recorded neurons. Taken together, the present results demonstrate that TLQP-21 promotes the survival of CGCs via pathways involving, within few minutes, modulation of kinases associated with CGCs survival, and by increasing intracellular calcium which can contribute to the neuroprotective effect of the peptide.