Sandy Y. Chang

Prelamin A and lamin A appear to be dispensable in the nuclear lamina

Lamin A and lamin C, both products of Lmna, are key components of the nuclear lamina. In the mouse, a deficiency in both lamin A and lamin C leads to slow growth, muscle weakness, and death by 6 weeks of age. Fibroblasts deficient in lamins A and C contain misshapen and structurally weakened nuclei, and emerin is mislocalized away from the nuclear envelope. The physiologic rationale for the existence of the 2 different Lmna products lamin A and lamin C is unclear, although several reports have suggested that lamin A may have particularly important functions, for example in the targeting of emerin and lamin C to the nuclear envelope. Here we report the development of lamin C-only mice (Lmna(LCO/LCO)), which produce lamin C but no lamin A or prelamin A (the precursor to lamin A). Lmna(LCO/LCO) mice were entirely healthy, and Lmna(LCO/LCO) cells displayed normal emerin targeting and exhibited only very minimal alterations in nuclear shape and nuclear deformability. Thus, at least in the mouse, prelamin A and lamin A appear to be dispensable. Nevertheless, an accumulation of farnesyl-prelamin A (as occurs with a deficiency in the prelamin A processing enzyme Zmpste24) caused dramatically misshapen nuclei and progeria-like disease phenotypes. The apparent dispensability of prelamin A suggested that lamin A-related progeroid syndromes might be treated with impunity by reducing prelamin A synthesis. Remarkably, the presence of a single Lmna(LCO) allele eliminated the nuclear shape abnormalities and progeria-like disease phenotypes in Zmpste24-/- mice. Moreover, treating Zmpste24-/- cells with a prelamin A-specific antisense oligonucleotide reduced prelamin A levels and significantly reduced the frequency of misshapen nuclei. These studies suggest a new therapeutic strategy for treating progeria and other lamin A diseases.

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. This neuronal layering abnormality is due to defective neuronal migration, a process that is dependent on the organized movement of the nucleus within the cell. These studies establish an essential function for lamin B2 in neuronal migration and brain development.

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

Lamin B1 is essential for neuronal migration and progenitor proliferation during the development of the cerebral cortex. The observation of distinct phenotypes of Lmnb1- and Lmnb2-knockout mice and the differences in the nuclear morphology of cortical neurons in vivo suggest that lamin B1 and lamin B2 play distinct functions in the developing brain.

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. The prelamin A in HIV-PI-treated fibroblasts migrated more rapidly than nonfarnesylated prelamin A, comigrating with the farnesylated form of prelamin A that accumulates in ZMPSTE24-deficient fibroblasts. The accumulation of farnesyl-prelamin A in response to HIV-PI treatment was exaggerated in fibroblasts heterozygous for Zmpste24 deficiency. HIV-PIs inhibited the endoproteolytic processing of a GFP-prelamin A fusion protein. The HIV-PIs did not affect the farnesylation of HDJ-2, nor did they inhibit protein farnesyltransferase in vitro. HIV-PIs also did not inhibit the activities of the isoprenyl-cysteine carboxyl methyltransferase ICMT or the prenylprotein endoprotease RCE1 in vitro, but they did inhibit ZMPSTE24 (IC50: lopinavir, 18.4 ± 4.6 μM; tipranavir, 1.2 ± 0.4 μM). We conclude that the HIV-PIs inhibit ZMPSTE24, leading to an accumulation of farnesyl-prelamin A. The inhibition of ZMPSTE24 by HIV-PIs could play a role in the side effects of these drugs.

An absence of both lamin B1 and lamin B2 in keratinocytes has no effect on cell proliferation or the development of skin and hair

Nuclear lamins are usually classified as A-type (lamins A and C) or B-type (lamins B1 and B2). A-type lamins have been implicated in multiple genetic diseases but are not required for cell growth or development. In contrast, B-type lamins have been considered essential in eukaryotic cells, with crucial roles in DNA replication and in the formation of the mitotic spindle. Knocking down the genes for B-type lamins (LMNB1, LMNB2) in HeLa cells has been reported to cause apoptosis. In the current study, we created conditional knockout alleles for mouse Lmnb1 and Lmnb2, with the goal of testing the hypothesis that B-type lamins are crucial for the growth and viability of mammalian cells in vivo. Using the keratin 14-Cre transgene, we bred mice lacking the expression of both Lmnb1 and Lmnb2 in skin keratinocytes (Lmnb1(Δ/Δ)Lmnb2(Δ/Δ)). Lmnb1 and Lmnb2 transcripts were absent in keratinocytes of Lmnb1(Δ/Δ)Lmnb2(Δ/Δ) mice, and lamin B1 and lamin B2 proteins were undetectable. But despite an absence of B-type lamins in keratinocytes, the skin and hair of Lmnb1(Δ/Δ)Lmnb2(Δ/Δ) mice developed normally and were free of histological abnormalities, even in 2-year-old mice. After an intraperitoneal injection of bromodeoxyuridine (BrdU), similar numbers of BrdU-positive keratinocytes were observed in the skin of wild-type and Lmnb1(Δ/Δ)Lmnb2(Δ/Δ) mice. Lmnb1(Δ/Δ)Lmnb2(Δ/Δ) keratinocytes did not exhibit aneuploidy, and their growth rate was normal in culture. These studies challenge the concept that B-type lamins are essential for proliferation and vitality of eukaryotic cells.

Eliminating the Synthesis of Mature Lamin A Reduces Disease Phenotypes in Mice Carrying a Hutchinson-Gilford Progeria Syndrome Allele

Hutchinson-Gilford progeria syndrome is caused by the synthesis of a mutant form of prelamin A, which is generally called progerin. Progerin is targeted to the nuclear rim, where it interferes with the integrity of the nuclear lamina, causes misshapen cell nuclei, and leads to multiple aging-like disease phenotypes. We created a gene-targeted allele yielding exclusively progerin (LmnaHG) and found that heterozygous mice (LmnaHG/+) exhibit many phenotypes of progeria. In this study, we tested the hypothesis that the phenotypes elicited by the LmnaHG allele might be modulated by compositional changes in the nuclear lamina. To explore this hypothesis, we bred mice harboring one LmnaHG allele and one LmnaLCO allele (a mutant allele that produces lamin C but no lamin A). We then compared the phenotypes of LmnaHG/LCO mice (which produce progerin and lamin C) with littermate LmnaHG/+ mice (which produce lamin A, lamin C, and progerin). LmnaHG/LCO mice exhibited improved body weight curves (p < 0.0001), reduced numbers of spontaneous rib fractures (p < 0.0001), and improved survival (p < 0.0001). In addition, LmnaHG/LCO fibroblasts had fewer misshapen nuclei than LmnaHG/+ fibroblasts (p < 0.0001). A likely explanation for these differences was uncovered; the amount of progerin in LmnaHG/LCO fibroblasts and tissues was lower than in LmnaHG/+ fibroblasts and tissues. These studies suggest that compositional changes in the nuclear lamina can influence both the steady-state levels of progerin and the severity of progeria-like disease phenotypes.

Assessing the efficacy of protein farnesyltransferase inhibitors in mouse models of progeria

Hutchinson-Gilford progeria syndrome (HGPS) is caused by the accumulation of a farnesylated form of prelamin A (progerin). Previously, we showed that blocking protein farnesylation with a farnesyltransferase inhibitor (FTI) ameliorates the disease phenotypes in mouse model of HGPS (LmnaHG/+). However, the interpretation of the FTI treatment studies is open to question in light of recent studies showing that mice expressing a nonfarnesylated version of progerin (LmnanHG/+) develop progeria-like disease phenotypes. The fact that LmnanHG/+ mice manifest disease raised the possibility that the beneficial effects of an FTI in LmnaHG/+ mice were not due to the effects of the drug on the farnesylation of progerin, but may have been due to unanticipated secondary effects of the drug on other farnesylated proteins. To address this issue, we compared the ability of an FTI to improve progeria-like disease phenotypes in both LmnaHG/+ and LmnanHG/+ mice. In LmnaHG/+ mice, the FTI reduced disease phenotypes in a highly significant manner, but the drug had no effect in LmnanHG/+ mice. The failure of the FTI to ameliorate disease in LmnanHG/+ mice supports the idea that the beneficial effects of an FTI in LmnaHG/+ mice are due to the effect of drug on the farnesylation of progerin.

Genetic studies on the functional relevance of the protein prenyltransferases in skin keratinocytes

The modification of proteins with farnesyl or geranylgeranyl lipids, a process called protein prenylation, facilitates interactions of proteins with membrane surfaces. Protein prenylation is carried out by a pair of cytosolic enzymes, protein farnesyltransferase (FTase) and protein geranylgeranyltransferase type I (GGTase-I). FTase and GGTase-I have attracted interest as therapeutic targets for both cancer and progeria, but very little information exists on the importance of these enzymes for homeostasis of normal tissues. One study actually suggested that FTase is entirely dispensable. To explore the importance of the protein prenyltransferases for normal tissues, we used conditional knockout alleles for Fntb and Pggt1b (which encode the beta-subunits of FTase and GGTase-I, respectively) and a keratin 14-Cre transgene to create mice lacking FTase or GGTase-I in skin keratinocytes. Keratinocyte-specific Fntb knockout mice were viable but developed severe alopecia. Although hair follicles appeared normal during development, they were morphologically abnormal after birth, and ultrastructural and immunohistochemical studies revealed many apoptotic cells. The interfollicular epidermis of Fntb-deficient mice appeared normal; however, keratinocytes from these mice could not proliferate in culture. As expected, non-farnesylated prelamin A and non-farnesylated DNAJA1 accumulated in Fntb-deficient keratinocytes. Keratinocyte-specific Pggt1b knockout mice survived development but died shortly after birth. Like Fntb-deficient keratinocytes, Pggt1b-deficient keratinocytes did not proliferate in culture. Thus, both FTase and GGTase-I are required for the homeostasis of skin keratinocytes.

Inhibitors of protein geranylgeranyltransferase-I lead to prelamin A accumulation in cells by inhibiting ZMPSTE24.

Protein farnesyltransferase (FTase) inhibitors, generally called “FTIs,” block the farnesylation of prelamin A, inhibiting the biogenesis of mature lamin A and leading to an accumulation of prelamin A within cells. A recent report found that a GGTI, an inhibitor of protein geranylgeranyltransferase-I (GGTase-I), caused an exaggerated accumulation of prelamin A in the presence of low amounts of an FTI. This finding was interpreted as indicating that prelamin A can be alternately prenylated by GGTase-I and that inhibiting both protein prenyltransferases leads to more prelamin A accumulation than blocking FTase alone. Here, we tested an alternative hypothesis—GGTIs are not specific for GGTase-I, and they lead to prelamin A accumulation by inhibiting ZMPSTE24 (a zinc metalloprotease that converts farnesyl–prelamin A to mature lamin A). In our studies, commonly used GGTIs caused prelamin A accumulation in human fibroblasts, but the prelamin A in GGTI-treated cells exhibited a more rapid electrophoretic mobility than prelamin A from FTI-treated cells. The latter finding suggested that the prelamin A in GGTI-treated cells might be farnesylated (which would be consistent with the notion that GGTIs inhibit ZMPSTE24). Indeed, metabolic labeling studies revealed that the prelamin A in GGTI-treated fibroblasts is farnesylated. Moreover, biochemical assays of ZMPSTE24 activity showed that ZMPSTE24 is potently inhibited by a GGTI. Our studies show that GGTIs inhibit ZMPSTE24, leading to an accumulation of farnesyl–prelamin A. Thus, caution is required when interpreting the effects of GGTIs on prelamin A processing.

Severe hepatocellular disease in mice lacking one or both CaaX prenyltransferases

Protein farnesyltransferase (FTase) and protein geranylgeranyltransferase-I (GGTase-I) add 15- or 20-carbon lipids, respectively, to proteins that terminate with a CaaX motif. These posttranslational modifications of proteins with lipids promote protein interactions with membrane surfaces in cells, but the in vivo importance of the CaaX prenyltransferases and the protein lipidation reactions they catalyze remain incompletely defined. One study concluded that a deficiency of FTase was inconsequential in adult mice and led to little or no tissue pathology. To assess the physiologic importance of the CaaX prenyltransferases, we used conditional knockout alleles and an albumin–Cre transgene to produce mice lacking FTase, GGTase-I, or both enzymes in hepatocytes. The hepatocyte-specific FTase knockout mice survived but exhibited hepatocellular disease and elevated transaminases. Mice lacking GGTase-I not only had elevated transaminases but also had dilated bile cannaliculi, hyperbilirubinemia, hepatosplenomegaly, and reduced survival. Of note, GGTase-I–deficient hepatocytes had a rounded shape and markedly reduced numbers of actin stress fibers. Hepatocyte-specific FTase/GGTase-I double-knockout mice closely resembled mice lacking GGTase-I alone, but the disease was slightly more severe. Our studies refute the notion that FTase is dispensable and demonstrate that GGTase-I is crucial for the vitality of hepatocytes.