Reciprocal knock-in mice to investigate the functional redundancy of lamin B1 and lamin B2.

Lamins B1 and B2 (B-type lamins) have very similar sequences and are expressed ubiquitously. In addition, both Lmnb1- and Lmnb2-deficient mice die soon after birth with neuronal layering abnormalities in the cerebral cortex, a consequence of defective neuronal migration. The similarities in amino acid sequences, expression patterns, and knockout phenotypes raise the question of whether the two proteins have redundant functions.

Farnesylation of lamin B1 is important for retention of nuclear chromatin during neuronal migration.

The role of protein farnesylation in lamin A biogenesis and the pathogenesis of progeria has been studied in considerable detail, but the importance of farnesylation for the B-type lamins, lamin B1 and lamin B2, has received little attention. Lamins B1 and B2 are expressed in nearly every cell type from the earliest stages of development, and they have been implicated in a variety of functions within the cell nucleus. To assess the importance of protein farnesylation for B-type lamins, we created knock-in mice expressing nonfarnesylated versions of lamin B1 and lamin B2.

Understanding the roles of nuclear A- and B-type lamins in brain development.

The nuclear lamina is composed mainly of lamins A and C (A-type lamins) and lamins B1 and B2 (B-type lamins). Dogma has held that lamins B1 and B2 play unique and essential roles in the nucleus of every eukaryotic cell. Recent studies have raised doubts about that view but have uncovered crucial roles for lamins B1 and B2 in neuronal migration during the development of the brain.

Regulation of prelamin A but not lamin C by miR-9, a brain-specific microRNA.

Lamins A and C, alternatively spliced products of the LMNA gene, are key components of the nuclear lamina. The two isoforms are found in similar amounts in most tissues, but we observed an unexpected pattern of expression in the brain. Western blot and immunohistochemistry studies showed that lamin C is abundant in the mouse brain, whereas lamin A and its precursor prelamin A are restricted to endothelial cells and meningeal cells and are absent in neurons and glia.

Are B-type lamins essential in all mammalian cells?

The B-type lamins are widely assumed to be essential for mammalian cells. In part, this assumption is based on a highly cited study that found that RNAi-mediated knockdown of lamin B1 or lamin B2 in HeLa cells arrested cell growth and led to apoptosis. Studies indicating that B-type lamins play roles in DNA replication, the formation of the mitotic spindle, chromatin organization and regulation of gene expression have fueled the notion that B-type lamins must be essential.

Deficiencies in lamin B1 and lamin B2 cause neurodevelopmental defects and distinct nuclear shape abnormalities in neurons.

Neuronal migration is essential for the development of the mammalian brain. Here, we document severe defects in neuronal migration and reduced numbers of neurons in lamin B1-deficient mice. Lamin B1 deficiency resulted in striking abnormalities in the nuclear shape of cortical neurons; many neurons contained a solitary nuclear bleb and exhibited an asymmetric distribution of lamin B2.

Investigating the purpose of prelamin A processing.

Lmna yields two major protein products in somatic cells, lamin C and prelamin A. Mature lamin A is produced from prelamin A by four posttranslational processing steps-farnesylation of a carboxyl-terminal cysteine, release of the last three amino acids of the protein, methylation of the farnesylcysteine, and the endoproteolytic release of the carboxyl-terminal 15 amino acids of the protein (including the farnesylcysteine methyl ester). Although the posttranslational processing of prelamin A has been conserved in vertebrate evolution, its physiologic significance remains unclear.

LINCing lamin B2 to neuronal migration: growing evidence for cell-specific roles of B-type lamins.

Nuclear lamins are major components of the nuclear lamina, and play essential roles in supporting the nucleus and organizing nuclear structures. While a large number of clinically important mutations have been mapped to the LMNA gene in humans, very few mutations have been associated with the B-type lamins. We have shown that lamin B2-deficiency in mice results in severe brain abnormalities.

Direct synthesis of lamin A, bypassing prelamin a processing, causes misshapen nuclei in fibroblasts but no detectable pathology in mice.

Lamin A, a key component of the nuclear lamina, is generated from prelamin A by four post-translational processing steps: farnesylation, endoproteolytic release of the last three amino acids of the protein, methylation of the C-terminal farnesylcysteine, and finally, endoproteolytic release of the last 15 amino acids of the protein (including the farnesylcysteine methyl ester). The last cleavage step, mediated by ZMPSTE24, releases mature lamin A. This processing scheme has been conserved through vertebrate evolution and is widely assumed to be crucial for targeting lamin A to the nuclear envelope.

Abnormal development of the cerebral cortex and cerebellum in the setting of lamin B2 deficiency.

Nuclear lamins are components of the nuclear lamina, a structural scaffolding for the cell nucleus. Defects in lamins A and C cause an array of human diseases, including muscular dystrophy, lipodystrophy, and progeria, but no diseases have been linked to the loss of lamins B1 or B2. To explore the functional relevance of lamin B2, we generated lamin B2-deficient mice and found that they have severe brain abnormalities resembling lissencephaly, with abnormal layering of neurons in the cerebral cortex and cerebellum.

The posttranslational processing of prelamin A and disease.

Human geneticists have shown that some progeroid syndromes are caused by mutations that interfere with the conversion of farnesyl-prelamin A to mature lamin A. For example, Hutchinson-Gilford progeria syndrome is caused by LMNA mutations that lead to the accumulation of a farnesylated version of prelamin A. In this review, we discuss the posttranslational modifications of prelamin A and their relevance to the pathogenesis and treatment of progeroid syndromes.

Activating the synthesis of progerin, the mutant prelamin A in Hutchinson-Gilford progeria syndrome, with antisense oligonucleotides.

Hutchinson-Gilford progeria syndrome (HGPS) is caused by point mutations that increase utilization of an alternate splice donor site in exon 11 of LMNA (the gene encoding lamin C and prelamin A). The alternate splicing reduces transcripts for wild-type prelamin A and increases transcripts for a truncated prelamin A (progerin). Here, we show that antisense oligonucleotides (ASOs) against exon 11 sequences downstream from the exon 11 splice donor site promote alternate splicing in both wild-type and HGPS fibroblasts, increasing the synthesis of progerin.

Development of the vertebral morphogenetic field in the mouse: interactions between Crossveinless-2 and Twisted Gastrulation.

Crossveinless-2 (Cv2), Twisted Gastrulation (Tsg) and Chordin (Chd) are components of an extracellular biochemical pathway that regulates Bone Morphogenetic Protein (BMP) activity during dorso-ventral patterning of Drosophila and Xenopus embryos, the formation of the fly wing, and mouse skeletogenesis. Because the nature of their genetic interactions remained untested in the mouse, we generated a null allele for Cv2 which was crossed to Tsg and Chd mutants to obtain Cv2; Tsg and Cv2; Chd compound mutants. We found that Cv2 is essential for skeletogenesis as its mutation caused the loss of multiple bone structures and posterior homeotic transformation of the last thoracic vertebra.

Crossveinless-2 Is a BMP feedback inhibitor that binds Chordin/BMP to regulate Xenopus embryonic patterning.

Vertebrate Crossveinless-2 (CV2) is a secreted protein that can potentiate or antagonize BMP signaling. Through embryological and biochemical experiments we find that: (1) CV2 functions as a BMP4 feedback inhibitor in ventral regions of the Xenopus embryo; (2) CV2 complexes with Twisted gastrulation and BMP4; (3) CV2 is not a substrate for tolloid proteinases; (4) CV2 binds to purified Chordin protein with high affinity (K(D) in the 1 nM range); (5) CV2 binds even more strongly to Chordin proteolytic fragments resulting from Tolloid digestion or to full-length Chordin/BMP complexes; (6) CV2 depletion causes the Xenopus embryo to become hypersensitive to the anti-BMP effects of Chordin overexpression or tolloid inhibition. We propose that the CV2/Chordin interaction may help coordinate BMP diffusion to the ventral side of the embryo, ensuring that BMPs liberated from Chordin inhibition by tolloid proteolysis cause peak signaling levels.

HIV-protease inhibitors block the enzymatic activity of purified Ste24p.

We reported that several HIV protease inhibitors (HIV-PIs) interfere with the endoproteolytic processing of two farnesylated proteins, yeast a-factor and mammalian prelamin A. We proposed that these drugs interfere with prelamin A processing by blocking ZMPSTE24, an integral membrane zinc metalloproteinase known to play a critical role in its processing. However, because all of the drug inhibition studies were performed with cultured fibroblasts or crude membrane fractions rather than on purified enzyme preparations, no definitive conclusions could be drawn.

A potent HIV protease inhibitor, darunavir, does not inhibit ZMPSTE24 or lead to an accumulation of farnesyl-prelamin A in cells.

HIV protease inhibitors (HIV-PIs) are key components of highly active antiretroviral therapy, but they have been associated with adverse side effects, including partial lipodystrophy and metabolic syndrome. We recently demonstrated that a commonly used HIV-PI, lopinavir, inhibits ZMPSTE24, thereby blocking lamin A biogenesis and leading to an accumulation of prelamin A. ZMPSTE24 deficiency in humans causes an accumulation of prelamin A and leads to lipodystrophy and other disease phenotypes.

HIV protease inhibitors block the zinc metalloproteinase ZMPSTE24 and lead to an accumulation of prelamin A in cells.

HIV protease inhibitors (HIV-PIs) target the HIV aspartyl protease, which cleaves the HIV gag-pol polyprotein into shorter proteins required for the production of new virions. HIV-PIs are a cornerstone of treatment for HIV but have been associated with lipodystrophy and other side effects. In both human and mouse fibroblasts, we show that HIV-PIs caused an accumulation of prelamin A.

A farnesyltransferase inhibitor improves disease phenotypes in mice with a Hutchinson-Gilford progeria syndrome mutation.

Hutchinson-Gilford progeria syndrome (HGPS) is caused by the production of a truncated prelamin A, called progerin, which is farnesylated at its carboxyl terminus. Progerin is targeted to the nuclear envelope and causes misshapen nuclei. Protein farnesyltransferase inhibitors (FTI) mislocalize progerin away from the nuclear envelope and reduce the frequency of misshapen nuclei.

A protein farnesyltransferase inhibitor ameliorates disease in a mouse model of progeria.

Progerias are rare genetic diseases characterized by premature aging. Several progeroid disorders are caused by mutations that lead to the accumulation of a lipid-modified (farnesylated) form of prelamin A, a protein that contributes to the structural scaffolding for the cell nucleus. In progeria, the accumulation of farnesyl-prelamin A disrupts this scaffolding, leading to misshapen nuclei.

Selective deletion of the Hnf1beta (MODY5) gene in beta-cells leads to altered gene expression and defective insulin release.

Hepatocyte nuclear factor 1alpha (HNF1alpha) and HNF1beta (or vHNF1) are closely related transcription factors expressed in liver, kidney, gut, and pancreatic beta-cells. Many HNF1 target genes are involved in carbohydrate metabolism. Human mutations in HNF1alpha or HNF1beta lead to maturity-onset diabetes of the young (MODY3 and MODY5, respectively), and patients present with impaired glucose-stimulated insulin secretion.

Mouse Crossveinless-2 is the vertebrate homolog of a Drosophila extracellular regulator of BMP signaling.

The Dpp/BMP signaling pathway is highly conserved between vertebrates and invertebrates. The recent molecular characterization of the Drosophila crossveinless-2 (cv-2) mutation by Conley and colleagues introduced a novel regulatory step in the Dpp/BMP pathway (Development 127 (2000) 3945). The CV-2 protein is secreted and contains five cysteine-rich (CR) domains similar to those observed in the BMP antagonist Short gastrulation (Sog) of Drosophila and Chordin (Chd) of vertebrates.

Mouse Crossveinless-2 is the vertebrate homolog of a Drosophila extracellular regulator of BMP signaling.

The Dpp/BMP signaling pathway is highly conserved between vertebrates and invertebrates. The recent molecular characterization of the Drosophila crossveinless-2 (cv-2) mutation by Conley and colleagues introduced a novel regulatory step in the Dpp/BMP pathway (Development 127 (2000) 3945). The CV-2 protein is secreted and contains five cysteine-rich (CR) domains similar to those observed in the BMP antagonist Short gastrulation (Sog) of Drosophila and Chordin (Chd) of vertebrates.

Chordin-like CR domains and the regulation of evolutionarily conserved extracellular signaling systems.

In fruit flies as well as in humans the Short gastrulation (Sog)/Chordin protein functions as an antagonist of the signaling of decapentaplegic (Dpp)/bone morphogenetic protein (BMP) in the extracellular space. Such antagonism inhibits Dpp/BMP signaling by blocking its binding to the receptor. Modulation of Dpp/BMP signaling is phylogenetically conserved and is a key step for the establishment of the dorso-ventral axis in vertebrates and invertebrates.

Bile system morphogenesis defects and liver dysfunction upon targeted deletion of HNF1beta.

The inactivation of the Hnf1beta gene identified an essential role in epithelial differentiation of the visceral endoderm and resulted in early embryonic death. In the present study, we have specifically inactivated this gene in hepatocytes and bile duct cells using the Cre/loxP system. Mutant animals exhibited severe jaundice caused by abnormalities of the gallbladder and intrahepatic bile ducts (IHBD).

Neuralin-1 is a novel Chordin-related molecule expressed in the mouse neural plate.

Cysteine-rich repeats (CRs) of the type described in Chordin constitute conserved domains present in an expanding family of secreted molecules. These motifs were shown to mediate directly the antagonism of BMP signaling by Chordin and play a major role during development. Here we report the cloning and expression pattern of neuralin-1, a new member of the chordin family.