Catherine Bartoli

Splicing-Directed Therapy in a New Mouse Model of Human Accelerated Aging

Antisense oligonucleotides reverse premature aging and extend life span in mutant mice that mimic aberrant splicing in progeria patients. Countering Careless Cutting Carpenters warn that one should “measure twice, cut once” to avoid unfixable assaults on building materials. Indeed, careless cutting lies at the heart of Hutchinson-Gilford progeria syndrome (HGPS). This premature aging disease is caused by a point mutation in the LMNA gene that activates a cryptic donor splice site in LMNA RNA; aberrant cutting and splicing results in the production of an mRNA that encodes progerin, a truncated form of the lamin A protein that is also produced in small amounts during normal aging. Until now, no model system has recapitulated the pathogenic LMNA splicing that occurs in HGPS patients. Here, Osorio et al. characterize such HGPS mutant mice mimics—called LmnaG609G/G609G mice—and show that antisense oligonucleotide–based therapy reverses various premature aging phenotypes and extends life span. Encoded by the LMNA gene, lamin A is a nuclear envelope protein that is important for nuclear stability, chromatin structure, and regulation of gene expression. Osorio et al. showed that the LmnaG609G/G609G mice produced reduced amounts of intact lamin A, accumulated progerin, displayed the nuclear abnormalities and transcriptional alterations seen in other progeroid models, and sported the key clinical features of human HGPS, such as a shortened life span, reduced size, disrupted metabolism, and enhanced bone and cardiovascular maladies relative to wild-type animals. The authors then used their newly characterized HGPS animal model to test the effects of antisense morpholino oligonucleotides that bound to and blocked the aberrant splice donor site in Lmna RNA. These reagents reduced progerin accumulation and corrected the nuclear abnormalities in both cultured mutant mouse and human HGPS fibroblasts. Furthermore, LmnaG609G/G609G mice that were treated with a combination of two antisense oligonucleotides that blocked aberrant splicing displayed reduced amounts of accumulated progerin, enhanced life expectancy, and a reversal of the phenotypical and molecular alterations associated with HGPS, including the righting of gene expression aberrations and normalization of blood glucose levels. Together, these findings provide preclinical proof of concept for the use of antisense oligonucleotide–based therapies in the treatment of HGPS. Furthermore, because progerin also accumulates during normal aging, the LmnaG609G/G609G mutant mice may be useful for preclinical testing of therapies designed to slow the human aging process and prevent age-related diseases. As the poet Ralph Waldo Emerson noted, “All diseases run into one—old age.” Hutchinson-Gilford progeria syndrome (HGPS) is caused by a point mutation in the LMNA gene that activates a cryptic donor splice site and yields a truncated form of prelamin A called progerin. Small amounts of progerin are also produced during normal aging. Studies with mouse models of HGPS have allowed the recent development of the first therapeutic approaches for this disease. However, none of these earlier works have addressed the aberrant and pathogenic LMNA splicing observed in HGPS patients because of the lack of an appropriate mouse model. Here, we report a genetically modified mouse strain that carries the HGPS mutation. These mice accumulate progerin, present histological and transcriptional alterations characteristic of progeroid models, and phenocopy the main clinical manifestations of human HGPS, including shortened life span and bone and cardiovascular aberrations. Using this animal model, we have developed an antisense morpholino–based therapy that prevents the pathogenic Lmna splicing, markedly reducing the accumulation of progerin and its associated nuclear defects. Treatment of mutant mice with these morpholinos led to a marked amelioration of their progeroid phenotype and substantially extended their life span, supporting the effectiveness of antisense oligonucleotide–based therapies for treating human diseases of accelerated aging.

Cytokines, chemokines, and cell adhesion molecules in inflammatory myopathies

The inflammatory myopathies include dermatomyositis (DM), polymyositis (PM), and sporadic inclusion‐body myositis (s‐IBM). In DM, the main immune effector response appears to be humoral and directed against the microvasculature, whereas in both PM and s‐IBM, cytotoxic CD8+ T cells and macrophages invade and eventually destroy nonnecrotic muscle fibers expressing major histocompatibility complex class I. The need for more specific and safer therapies in inflammatory myopathies has prompted researchers to better decipher the molecular events associated with inflammation and muscle fiber loss in these diseases. The complex specific migration of leukocyte subsets to target tissues requires a coordinated series of events, namely activation of leukocytes, adhesion to the vascular endothelium, and migration. Cell adhesion molecules (CAM) and chemokines play a major role in this multistep process. In addition, cytokines by stimulating CAM expression and orchestrating T‐cell differentiation also influence the immune response. This review focuses on recent advances in defining the molecular events involved in leukocyte trafficking in inflammatory myopathies. Specific topics include a concise summary of clinical features, pathological findings and immunopathology observed in inflammatory myopathies, background information about cytokines, chemokines and cell adhesion molecules, and the expression of these molecules in inflammatory myopathies. Muscle Nerve 28: 659–682, 2003

Ezrin and alpha-smooth muscle actin are immunohistochemical prognostic markers in conventional osteosarcomas

Ezrin is a cytoskeleton linker protein that is actively involved in the metastatic process of cancer cells. We have searched for a prognostic value of ezrin and some of its partners: α-smooth muscle actin and CD44H in 37 patients with an osteosarcoma. Automate immunohistochemistry (IHC) with anti-ezrin, α-smooth muscle actin and CD44H antibodies was performed in 66 specimens: 37 biopsies before chemotherapy, 16 resected tumours of “poor” responders and 13 metastases. The messenger RNA (mRNA) levels of ezrin of 13 frozen biopsies and 4 metastases were evaluated by real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR). All results were correlated to the following clinical data. Ezrin expression by IHC was found in 62% of 37 biopsies in the different histological subtypes. A good correlation was found between positive or negative samples by IHC and mRNA levels. Ezrin expression was recorded in 84.5% of metastastic samples. The mean expression of ezrin was higher in metastases than biopsies (p = 0.024). In multivariate analysis, ezrin was an independent prognostic marker for event-free survival and overall survival (OS) with p < 0.001 and p = 0.003, respectively, and α-smooth muscle actin for OS only (p = 0.024). Our findings suggest that ezrin and α-smooth muscle actin are predictive IHC prognostic markers for patients with an osteosarcoma.

Expression of PECAM-1/CD31 isoforms in human brain gliomas

Microvascular proliferation is a histopathological hallmark of glioblastomas and anaplastic oligodendrogliomas. Platelet endothelial cell adhesion molecule 1 (PECAM-1/CD31) is involved in angiogenesis. PECAM-1 mediates homophilic and heterophilic interactions (with glycosaminoglycans and αVβ3), but deletion of exon 14 results in a loss of heterophilic adhesion. Expression of various PECAM-1 isoforms was searched for in brain gliomas, showing microvascular proliferation (glioblastomas and anaplastic oligodendrogliomas) or not (oligodendrogliomas). In addition, expression of αVβ3 in some tumors was studied by immunohistochemistry. Various tissues and the HUVEC primary cell line were used as controls.Immunohistochemistry showed that PECAM-1 was expressed by all endothelial cells in all tissues and by some tumor cells in glioblastomas and anaplastic oligodendrogliomas. Microvascular proliferation always expressed αVβ3. In addition, some tumor cells in anaplastic oligodendroglioma and glioblastomas expressed it. In all samples examined, PECAM-1 exists under at least two transcriptional isoforms: the whole length molecule and an isoform made by the splicing of exon 14. Western blot analysis revealed in all cases 130 and 110 kDa bands corresponding to the mature form and its precursor respectively.These results suggest that splicing of exon 14 occurs in vivo in various normal and tumoral tissues and may modulate PECAM-1 adhesion according to the presence or not of other PECAM-1 ligands such as αVβ3.Expression of PECAM-1 by tumor cells in glioblastomas and anaplastic oligodendrogliomas may favour angiogenesis by specific PECAM-1 interactions between glial and endothelial cells.

Molecular characterization of the response to chemotherapy in conventional osteosarcomas: Predictive value of HSD17B10 and IFITM2

The therapy regimen of high‐grade osteosarcoma includes chemotherapy followed by surgical resection and postoperative chemotherapy. The degree of necrosis following definitive surgery remains the only reliable prognostic factor and is used to guide the choice of postoperative chemotherapy. The aim of this study was to find molecular markers able to classify patients with an osteosarcoma as good or poor responders to chemotherapy before beginning treatment. Gene expression screening of 20 nonmetastatic high‐grade osteosarcoma patients was performed using cDNA microarray. Expression of selected relevant genes was validated using QRT‐PCR. Immunohistochemistry on tissue microarrays sections of 73 biopsies was performed to investigate protein expression. Fluorescent in situ hybridization was performed for RPL8 gene. We have found that HSD17B10 gene expression was up‐regulated in poor responders and that immunohistochemistry expression of HSD17B10 on biopsy before treatment was correlatedto response to chemotherapy. Other results include correlationof IFITM2, IFITM3, and RPL8 gene expression to chemotherapy response. A statistical correlation was found between polysomy 8 or gain of RPL8 and good response to chemotherapy. These data suggest that HSD17B10, RPL8, IFITM2, and IFITM3 genes are involved in the response to the chemotherapy and that HSD17B10 may be a therapeutic target. RPL8 and IFITM2 may be useful in the assessment at diagnosis and for stratifying patients taking part in randomized trials.

Correlation between ERK1 and STAT3 expression and chemoresistance in patients with conventional osteosarcoma

BackgroundThe standard therapy regimen of conventional osteosarcoma includes neoadjuvant chemotherapy followed by surgical resection and postoperative chemotherapy. The percentage of necrotic tissue following induction chemotherapy is assessed by using the Huvos grading system, which classifies patients as “poor responders” (PR) and “good responders” (GR). The aim of this study was to identify molecular markers expressed differentially between good and poor responders to neoadjuvant chemotherapy in order to predict the response to chemotherapy in conventional osteosarcomas before beginning treatment.MethodsSuppression Substractive Hybridization (SSH) was performed by using cDNA from frozen biopsy specimens. Expression of selected relevant genes identified by SSH was validated by using QRT-PCR. Immunohistochemistry (IHC) on tissue microarray (TMA) sections of 52 biopsies was performed to investigate protein expression in an independent cohort.ResultsERK1 and STAT3 mRNA level were significantly different between PR and GR in an independent cohort. Phosphorylated STAT3 and ERK1 expressions by IHC on TMA were correlated with poor response to chemotherapy.ConclusionsOur results suggest that ERK1 and STAT3 expression are good predictive markers for chemotherapy response and that inhibitors might be used in combination with common chemotherapeutic drugs in conventional osteosarcomas.

Antisense-Based Progerin Downregulation in HGPS-Like Patients’ Cells

Progeroid laminopathies, including Hutchinson-Gilford Progeria Syndrome (HGPS, OMIM #176670), are premature and accelerated aging diseases caused by defects in nuclear A-type Lamins. Most HGPS patients carry a de novo point mutation within exon 11 of the LMNA gene encoding A-type Lamins. This mutation activates a cryptic splice site leading to the deletion of 50 amino acids at its carboxy-terminal domain, resulting in a truncated and permanently farnesylated Prelamin A called Prelamin A Δ50 or Progerin. Some patients carry other LMNA mutations affecting exon 11 splicing and are named “HGPS-like” patients. They also produce Progerin and/or other truncated Prelamin A isoforms (Δ35 and Δ90) at the transcriptional and/or protein level. The results we present show that morpholino antisense oligonucleotides (AON) prevent pathogenic LMNA splicing, markedly reducing the accumulation of Progerin and/or other truncated Prelamin A isoforms (Prelamin A Δ35, Prelamin A Δ90) in HGPS-like patients’ cells. Finally, a patient affected with Mandibuloacral Dysplasia type B (MAD-B, carrying a homozygous mutation in ZMPSTE24, encoding an enzyme involved in Prelamin A maturation, leading to accumulation of wild type farnesylated Prelamin A), was also included in this study. These results provide preclinical proof of principle for the use of a personalized antisense approach in HGPS-like and MAD-B patients, who may therefore be eligible for inclusion in a therapeutic trial based on this approach, together with classical HGPS patients.

Erratum to: Differential Brain, Cognitive and Motor Profiles Associated with Partial Trisomy. Modeling Down Syndrome in Mice

We hypothesize that the trisomy 21 (Down syndrome) is the additive and interactive outcome of the triple copy of different regions of HSA21. Because of the small number of patients with partial trisomy 21, we addressed the question in the Mouse in which three chromosomal regions located on MMU10, MMU17 and MMU16 carries almost all the HSA21 homologs. Male mice from four segmental trisomic strains covering the D21S17-ETS2 (syntenic to MMU16) were examined with an exhaustive battery of cognitive tests, motor tasks and MRI and compared with TS65Dn that encompasses D21S17-ETS2. None of the four strains gather all the impairments (measured by the effect size) of TS65Dn strain. The 152F7 strain was close to TS65Dn for motor behavior and reference memory and the three other strains 230E8, 141G6 and 285E6 for working memory. Episodic memory was impaired only in strain 285E6. The hippocampus and cerebellum reduced sizes that were seen in all the strains indicate that trisomy 21 is not only a hippocampus syndrome but that it results from abnormal interactions between the two structures.

An overview of treatment strategies for Hutchinson-Gilford Progeria syndrome

ABSTRACT Hutchinson-Gilford progeria syndrome (HGPS) is a sporadic, autosomal dominant disorder characterized by premature and accelerated aging symptoms leading to death at the mean age of 14.6 years usually due to cardiovascular complications. HGPS is caused by a de novo point mutation in the LMNA gene encoding the intermediate filament proteins lamins A and C which are structural components of the nuclear lamina. This mutation leads to the production of a truncated toxic form of lamin A, issued from aberrant splicing and called progerin. Progerin accumulates in HGPS cells’ nuclei and is a hallmark of the disease. Small amounts of progerin are also produced during normal aging. HGPS cells and animal preclinical models have provided insights into the molecular and cellular pathways that underlie the disease and have also highlighted possible mechanisms involved in normal aging. This review reports recent medical advances and treatment approaches for patients affected with HGPS.

Selecting the Right Species: Practical Information on Organism Models

Most of the attempt to model autism spectrum disorders (ASD) resorts to the mouse. Other species are available for modeling the brain and psychiatric disorders. The present chapter focuses on species of interest offering prospects for modeling behavior and nervous system: Caenorhabditis elegans , Drosophila , zebra fi sh, mouse, dog, and nonhuman primates. Yeast will be briefl y considered for its contribution to neurochemistry. The chapter provides practical information for the maintenance of Caenorhabditis elegans , Drosophila, and zebra fi sh. Mouse will be the target of another chapter.