Colin L. Stewart

Lamin A/C deficiency causes defective nuclear mechanics and mechanotransduction

Mutations in the lamin A/C gene (LMNA) cause a variety of human diseases including Emery-Dreifuss muscular dystrophy, dilated cardiomyopathy, and Hutchinson-Gilford progeria syndrome. The tissue-specific effects of lamin mutations are unclear, in part because the function of lamin A/C is incompletely defined, but the many muscle-specific phenotypes suggest that defective lamin A/C could increase cellular mechanical sensitivity. To investigate the role of lamin A/C in mechanotransduction, we subjected lamin A/C-deficient mouse embryo fibroblasts to mechanical strain and measured nuclear mechanical properties and strain-induced signaling. We found that Lmna-/- cells have increased nuclear deformation, defective mechanotransduction, and impaired viability under mechanical strain. NF-kappaB-regulated transcription in response to mechanical or cytokine stimulation was attenuated in Lmna-/- cells despite increased transcription factor binding. Lamin A/C deficiency is thus associated with both defective nuclear mechanics and impaired mechanically activated gene transcription. These findings suggest that the tissue-specific effects of lamin A/C mutations observed in the laminopathies may arise from varying degrees of impaired nuclear mechanics and transcriptional activation.

Homozygous Defects in LMNA, Encoding Lamin A/C Nuclear-Envelope Proteins, Cause Autosomal Recessive Axonal Neuropathy in Human (Charcot-Marie-Tooth Disorder Type 2) and Mouse

The Charcot-Marie-Tooth (CMT) disorders comprise a group of clinically and genetically heterogeneous hereditary motor and sensory neuropathies, which are mainly characterized by muscle weakness and wasting, foot deformities, and electrophysiological, as well as histological, changes. A subtype, CMT2, is defined by a slight or absent reduction of nerve-conduction velocities together with the loss of large myelinated fibers and axonal degeneration. CMT2 phenotypes are also characterized by a large genetic heterogeneity, although only two genes---NF-L and KIF1Bbeta---have been identified to date. Homozygosity mapping in inbred Algerian families with autosomal recessive CMT2 (AR-CMT2) provided evidence of linkage to chromosome 1q21.2-q21.3 in two families (Zmax=4.14). All patients shared a common homozygous ancestral haplotype that was suggestive of a founder mutation as the cause of the phenotype. A unique homozygous mutation in LMNA (which encodes lamin A/C, a component of the nuclear envelope) was identified in all affected members and in additional patients with CMT2 from a third, unrelated family. Ultrastructural exploration of sciatic nerves of LMNA null (i.e., -/-) mice was performed and revealed a strong reduction of axon density, axonal enlargement, and the presence of nonmyelinated axons, all of which were highly similar to the phenotypes of human peripheral axonopathies. The finding of site-specific amino acid substitutions in limb-girdle muscular dystrophy type 1B, autosomal dominant Emery-Dreifuss muscular dystrophy, dilated cardiomyopathy type 1A, autosomal dominant partial lipodystrophy, and, now, AR-CMT2 suggests the existence of distinct functional domains in lamin A/C that are essential for the maintenance and integrity of different cell lineages. To our knowledge, this report constitutes the first evidence of the recessive inheritance of a mutation that causes CMT2; additionally, we suggest that mutations in LMNA may also be the cause of the genetically overlapping disorder CMT2B1.

Astrocytes Promote Myelination in Response to Electrical Impulses

Myelin, the insulating layers of membrane wrapped around axons by oligodendrocytes, is essential for normal impulse conduction. It forms during late stages of fetal development but continues into early adult life. Myelination correlates with cognitive development and can be regulated by impulse activity through unknown molecular mechanisms. Astrocytes do not form myelin, but these nonneuronal cells can promote myelination in ways that are not understood. Here, we identify a link between myelination, astrocytes, and electrical impulse activity in axons that is mediated by the cytokine leukemia inhibitory factor (LIF). These findings show that LIF is released by astrocytes in response to ATP liberated from axons firing action potentials, and LIF promotes myelination by mature oligodendrocytes. This activity-dependent mechanism promoting myelination could regulate myelination according to functional activity or environmental experience and may offer new approaches to treating demyelinating diseases.

Juxtaparanodal clustering of Shaker-like K+ channels in myelinated axons depends on Caspr2 and TAG-1

In myelinated axons, K+ channels are concealed under the myelin sheath in the juxtaparanodal region, where they are associated with Caspr2, a member of the neurexin superfamily. Deletion of Caspr2 in mice by gene targeting revealed that it is required to maintain K+ channels at this location. Furthermore, we show that the localization of Caspr2 and clustering of K+ channels at the juxtaparanodal region depends on the presence of TAG-1, an immunoglobulin-like cell adhesion molecule that binds Caspr2. These results demonstrate that Caspr2 and TAG-1 form a scaffold that is necessary to maintain K+ channels at the juxtaparanodal region, suggesting that axon–glia interactions mediated by these proteins allow myelinating glial cells to organize ion channels in the underlying axonal membrane.

Accelerated ageing in mice deficient in Zmpste24 protease is linked to p53 signalling activation.

Zmpste24 (also called FACE-1) is a metalloproteinase involved in the maturation of lamin A (Lmna), an essential component of the nuclear envelope. Both Zmpste24- and Lmna-deficient mice exhibit profound nuclear architecture abnormalities and multiple histopathological defects that phenocopy an accelerated ageing process. Similarly, diverse human progeroid syndromes are caused by mutations in ZMPSTE24 or LMNA genes. To elucidate the molecular mechanisms underlying these devastating diseases, we have analysed the transcriptional alterations occurring in tissues from Zmpste24-deficient mice. We demonstrate that Zmpste24 deficiency elicits a stress signalling pathway that is evidenced by a marked upregulation of p53 target genes, and accompanied by a senescence phenotype at the cellular level and accelerated ageing at the organismal level. These phenotypes are largely rescued in Zmpste24-/-Lmna+/- mice and partially reversed in Zmpste24-/-p53-/- mice. These findings provide evidence for the existence of a checkpoint response activated by the nuclear abnormalities caused by prelamin A accumulation, and support the concept that hyperactivation of the tumour suppressor p53 may cause accelerated ageing.

Lamins A and C but Not Lamin B1 Regulate Nuclear Mechanics

Mutations in the nuclear envelope proteins lamins A and C cause a broad variety of human diseases, including Emery-Dreifuss muscular dystrophy, dilated cardiomyopathy, and Hutchinson-Gilford progeria syndrome. Cells lacking lamins A and C have reduced nuclear stiffness and increased nuclear fragility, leading to increased cell death under mechanical strain and suggesting a potential mechanism for disease. Here, we investigated the contribution of major lamin subtypes (lamins A, C, and B1) to nuclear mechanics by analyzing nuclear shape, nuclear dynamics over time, nuclear deformations under strain, and cell viability under prolonged mechanical stimulation in cells lacking both lamins A and C, cells lacking only lamin A (i.e. “lamin C-only” cells), cells lacking wild-type lamin B1, and wild-type cells. Lamin A/C-deficient cells exhibited increased numbers of misshapen nuclei and had severely reduced nuclear stiffness and decreased cell viability under strain. Lamin C-only cells had slightly abnormal nuclear shape and mildly reduced nuclear stiffness but no decrease in cell viability under strain. Interestingly, lamin B1-deficient cells exhibited normal nuclear mechanics despite having a significantly increased frequency of nuclear blebs. Our study indicates that lamins A and C are important contributors to the mechanical stiffness of nuclei, whereas lamin B1 contributes to nuclear integrity but not stiffness.

A human iPSC model of Hutchinson Gilford Progeria reveals vascular smooth muscle and mesenchymal stem cell defects.

The segmental premature aging disease Hutchinson-Gilford Progeria syndrome (HGPS) is caused by a truncated and farnesylated form of Lamin A called progerin. HGPS affects mesenchymal lineages, including the skeletal system, dermis, and vascular smooth muscle (VSMC). To understand the underlying molecular pathology of HGPS, we derived induced pluripotent stem cells (iPSCs) from HGPS dermal fibroblasts. The iPSCs were differentiated into neural progenitors, endothelial cells, fibroblasts, VSMCs, and mesenchymal stem cells (MSCs). Progerin levels were highest in MSCs, VSMCs, and fibroblasts, in that order, with these lineages displaying increased DNA damage, nuclear abnormalities, and HGPS-VSMC accumulating numerous calponin-staining inclusion bodies. Both HGPS-MSC and -VSMC viability was compromised by stress and hypoxia in vitro and in vivo (MSC). Because MSCs reside in low oxygen niches in vivo, we propose that, in HGPS, this causes additional depletion of the MSC pool responsible for replacing differentiated cells lost to progerin toxicity.

A perinuclear actin cap regulates nuclear shape

Defects in nuclear morphology often correlate with the onset of disease, including cancer, progeria, cardiomyopathy, and muscular dystrophy. However, the mechanism by which a cell controls its nuclear shape is unknown. Here, we use adhesive micropatterned surfaces to control the overall shape of fibroblasts and find that the shape of the nucleus is tightly regulated by the underlying cell adhesion geometry. We found that this regulation occurs through a dome-like actin cap that covers the top of the nucleus. This cap is composed of contractile actin filament bundles containing phosphorylated myosin, which form a highly organized, dynamic, and oriented structure in a wide variety of cells. The perinuclear actin cap is specifically disorganized or eliminated by inhibition of actomyosin contractility and rupture of the LINC complexes, which connect the nucleus to the actin cap. The organization of this actin cap and its nuclear shape-determining function are disrupted in cells from mouse models of accelerated aging (progeria) and muscular dystrophy with distorted nuclei caused by alterations of A-type lamins. These results highlight the interplay between cell shape, nuclear shape, and cell adhesion mediated by the perinuclear actin cap.

LBR and Lamin A/C Sequentially Tether Peripheral Heterochromatin and Inversely Regulate Differentiation

Eukaryotic cells have a layer of heterochromatin at the nuclear periphery. To investigate mechanisms regulating chromatin distribution, we analyzed heterochromatin organization in different tissues and species, including mice with mutations in the lamin B receptor (Lbr) and lamin A (Lmna) genes that encode nuclear envelope (NE) proteins. We identified LBR- and lamin-A/C-dependent mechanisms tethering heterochromatin to the NE. The two tethers are sequentially used during cellular differentiation and development: first the LBR- and then the lamin-A/C-dependent tether. The absence of both LBR and lamin A/C leads to loss of peripheral heterochromatin and an inverted architecture with heterochromatin localizing to the nuclear interior. Myoblast transcriptome analyses indicated that selective disruption of the LBR- or lamin-A-dependent heterochromatin tethers have opposite effects on muscle gene expression, either increasing or decreasing, respectively. These results show how changes in NE composition contribute to regulating heterochromatin positioning, gene expression, and cellular differentiation during development.

Teratocarcinoma stem cells and early mouse embryos contain only a single major lamin polypeptide closely resembling lamin B

The nuclear lamina in adult mammalian somatic cells is composed of three major proteins, lamins A, B, and C. The expression of these proteins during the differentiation of teratocarcinomas and mouse embryogenesis is described. Embryos up to day 8 of gestation and embryonal carcinoma (EC) cells express only a single lamin species closely resembling, if not identical to, lamin B. Lamins A and/or C were detected in fertilized eggs, but disappear during the first 2-4 cleavage divisions, only reappearing in 8 day post-implantation embryos. These two lamins are absent from EC cells, but are strongly expressed in some of their derivatives. These results show that cells of the early mouse embryo do not have a functional requirement for lamins A and C and imply that the structural organization of the nucleus may change fundamentally during embryogenesis.