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Scholarly Works (76 results)

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UCLA Electronic Theses and Dissertations (2024)

Children with Hutchinson-Gilford progeria syndrome (HGPS) display symptoms resembling accelerated aging, with most succumbing in their mid-teens to complications from arteriosclerotic lesions in major arteries. HGPS arises from a de novo mutation in the LMNA gene, such as the Gly608Gly mutation, which creates an aberrant splice donor site, resulting in a deletion of 50 amino acids in prelamin A and the production of mutant protein called progerin. Progerin, unlike mature lamin A, retains a farnesyl lipid anchor, which contributes to cellular toxicity. In HGPS, a prominent vascular anomaly is reduced numbers of smooth muscle cells (SMCs) in the medial layer of large arteries, promoting arteriosclerotic disease. In order to study the underlying mechanisms of vascular disease in HGPS, we created a mouse model of HGPS (LmnaG609G) which exhibits disease phenotypes reminiscent of human HGPS, including the hallmark vascular disease. We also created an in vitro doxycycline-inducible cell culture system to investigate properties of progerin by studying the effects of progerin on the nuclear lamina meshwork, nuclear mechanics, integrity of the nuclear envelope, their relationship to nuclear morphology, DNA damage, and cell viability. In this dissertation, my goal was to understand the mechanisms underlying vascular pathology in HGPS, focusing on the interactions between progerin, lamin B1, and the integrity of the nuclear lamina meshwork in vascular SMCs. In Chapter 2, we investigated the role of mechanical stress on the vascular disease and showed that disrupting the LINC complex in smooth muscle cells reduces SMC loss in LmnaG609G mice. In Chapter 3, we hypothesized that progerin weakens the structural integrity of the nuclear envelope. Indeed, we showed that progerin resulted in nuclear membrane ruptures and found that it was responsible for vascular pathology in vivo. In Chapter 4, we investigated a more fundamental question—how does progerin induce abnormal biological effects in cells? We discovered that progerin forms an abnormal meshwork, characterized by clusters of large meshwork gaps. Unexpectedly, progerin had a dominant-negative effect on the nuclear lamina, thus further disrupting nuclear integrity. In Chapter 5, I summarize our key findings and discuss our current view on the mechanisms of vascular smooth muscle cell loss in HGPS.

    Cover page: Underlying Mechanisms of Vascular Disease in Hutchinson-Gilford Progeria Syndrome
    • Article
    • Peer Reviewed
    UCLA Previously Published Works (2017)
    Over the past few decades, several approaches have been used to image lipids in cells and tissues, but most have limited spatial resolution and sensitivity. Here, we discuss a relatively new approach, nanoscale secondary ion mass spectrometry imaging, that makes it possible to visualize lipids in cells and tissues in a quantitative fashion and with high spatial resolution and high sensitivity.
      Cover page: NanoSIMS imaging: an approach for visualizing and quantifying lipids in cells and tissues
      • Article
      • Peer Reviewed
      UCLA Previously Published Works (2014)
      Lamins B1 and B2 have a high degree of sequence similarity and are widely expressed from the earliest stages of development. Studies of Lmnb1 and Lmnb2 knockout mice revealed that both of the B-type lamins are crucial for neuronal migration in the developing brain. These observations naturally posed the question of whether the two B-type lamins might play redundant functions in the development of the brain. To explore that issue, Lee and coworkers generated "reciprocal knock-in mice" (knock-in mice that produce lamin B1 from the Lmnb2 locus and knock-in mice that produce lamin B2 from the Lmnb1 locus). Both lines of knock-in mice manifested neurodevelopmental abnormalities similar to those in conventional knockout mice, indicating that lamins B1 and B2 have unique functions and that increased production of one B-type lamin cannot compensate for the loss of the other.
        Cover page: Do lamin B1 and lamin B2 have redundant functions?
        • Article
        • Peer Reviewed
        UCLA Previously Published Works (2016)
        A variety of missense mutations in LMNA (the gene for lamin C and prelamin A) cause familial partial lipodystrophy (FPLD), a disease associated with reduced adipose tissue, particularly in the limbs. Several studies have reported that fibroblasts from FPLD subjects have an accumulation of prelamin A. Those findings were intriguing but also perplexing because many of the LMNA missense mutations associated with lipodystrophy are located in sequences distant from the sequences required for the farnesylation of prelamin A and ZMPSTE24-mediated conversion of prelamin A to mature lamin A. Here, we revisited the issue of prelamin A accumulation in the setting of FPLD mutations. We used western blots with lamin A/C antibodies and prelamin A-specific monoclonal antibodies to assess prelamin A levels in wild-type fibroblasts and fibroblasts carrying LMNA mutations associated with lipodystrophy (R482W, I299V, C591F, T528M). None of the mutant fibroblasts exhibited an accumulation of prelamin A. Also, the amount of prelamin A accumulation in response to lopinavir (an inhibitor of ZMPSTE24) was similar in wild-type and mutant fibroblasts. Thus, the LMNA lipodystrophy mutations that we examined did not lead to prelamin A accumulation, nor did they render those cells more susceptible to prelamin A accumulation when ZMPSTE24 was inhibited by lopinavir.
          Cover page: LMNA missense mutations causing familial partial lipodystrophy do not lead to an accumulation of prelamin A
          • Article
          • Peer Reviewed
          UCLA Previously Published Works (2015)
          The nuclear lamina, along with associated nuclear membrane proteins, is a nexus for regulating signaling in the nucleus. Numerous human diseases arise from mutations in lamina proteins, and experimental models for these disorders have revealed aberrant regulation of various signaling pathways. Previously, we reported that the inner nuclear membrane protein Lem2, which is expressed at high levels in muscle, promotes the differentiation of cultured myoblasts by attenuating ERK signaling. Here, we have analyzed mice harboring a disrupted allele for the Lem2 gene (Lemd2). No gross phenotypic defects were seen in heterozygotes, although muscle regeneration induced by cardiotoxin was delayed. By contrast, homozygous Lemd2 knockout mice died by E11.5. Although many normal morphogenetic hallmarks were observed in E10.5 knockout embryos, most tissues were substantially reduced in size. This was accompanied by activation of multiple MAP kinases (ERK1/2, JNK, p38) and AKT. Knockdown of Lem2 expression in C2C12 myoblasts also led to activation of MAP kinases and AKT. These findings indicate that Lemd2 plays an essential role in mouse embryonic development and that it is involved in regulating several signaling pathways. Since increased MAP kinase and AKT/mTORC signaling is found in other animal models for diseases linked to nuclear lamina proteins, LEMD2 should be considered to be another candidate gene for human disease.
            Cover page: Nuclear envelope protein Lem2 is required for mouse development and regulates MAP and AKT kinases.
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            • Peer Reviewed
            UCLA Previously Published Works (2009)
            A common strategy for conditional knockout alleles is to “flox” (flank with loxP sites) a 5′ exon within the target gene. Typically, the floxed exon does not contain a unit number of codons so that the Cre-mediated recombination event yields a frameshift and a null allele. Documenting recombination within the genomic DNA is often regarded as sufficient proof of a frameshift, and the analysis of transcripts is neglected. We evaluated a previously reported conditional knockout allele for the β-subunit of protein farnesyltransferase. The recombination event in that allele—the excision of exon 3—was predicted to yield a frameshift. However, following the excision of exon 3, exon 4 was skipped by the mRNA splicing machinery, and the predominant transcript from the mutant allele lacked exon 3 and exon 4 sequences. The “∆exon 3–4 transcript” does not contain a frameshift but rather is predicted to encode a protein with a short in-frame deletion. This represents a significant concern when studying an enzyme, since an enzyme with partial function could lead to erroneous conclusions. With thousands of new conditional knockout alleles under construction within mouse mutagenesis consortiums, the protein farnesyltransferase allele holds an important lesson—to characterize knockout alleles at both the DNA and RNA levels.
              Cover page: Caution! Analyze transcripts from conditional knockout alleles
              • Article
              • Peer Reviewed
              UCLA Previously Published Works (2014)
              Much of the work on nuclear lamins during the past 15 years has focused on mutations in LMNA (the gene for prelamin A and lamin C) that cause particular muscular dystrophy, cardiomyopathy, partial lipodystrophy, and progeroid syndromes. These disorders, often called "laminopathies," mainly affect mesenchymal tissues (e.g., striated muscle, bone, and fibrous tissue). Recently, however, a series of papers have identified important roles for nuclear lamins in the central nervous system. Studies of knockout mice uncovered a key role for B-type lamins (lamins B1 and B2) in neuronal migration in the developing brain. Also, duplications of LMNB1 (the gene for lamin B1) have been shown to cause autosome-dominant leukodystrophy. Finally, recent studies have uncovered a peculiar pattern of nuclear lamin expression in the brain. Lamin C transcripts are present at high levels in the brain, but prelamin A expression levels are very low-due to regulation of prelamin A transcripts by microRNA 9. This form of prelamin A regulation likely explains why "prelamin A diseases" such as Hutchinson-Gilford progeria syndrome spare the central nervous system. In this review, we summarize recent progress in elucidating links between nuclear lamins and neurobiology.
                Cover page: Nuclear Lamins and Neurobiology
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                • Peer Reviewed
                UCLA Previously Published Works (2018)
                The nuclear lamina, an intermediate filament meshwork lining the inner nuclear membrane, is formed by the nuclear lamins (lamins A, C, B1, and B2). Defects or deficiencies in individual nuclear lamin proteins have been reported to elicit nuclear blebs (protrusions or outpouchings of the nuclear envelope) and increase susceptibility for nuclear membrane ruptures. It is unclear, however, how a complete absence of nuclear lamins would affect nuclear envelope morphology and nuclear membrane integrity (i.e., whether nuclear membrane blebs or protrusions would occur and, if not, whether cells would be susceptible to nuclear membrane ruptures). To address these issues, we generated mouse embryonic fibroblasts (MEFs) lacking all nuclear lamins. The nuclear lamin-deficient MEFs had irregular nuclear shapes but no nuclear blebs or protrusions. Despite a virtual absence of nuclear blebs, MEFs lacking nuclear lamins had frequent, prolonged, and occasionally nonhealing nuclear membrane ruptures. By transmission electron microscopy, the inner nuclear membrane in nuclear lamin-deficient MEFs have a "wavy" appearance, and there were discrete discontinuities in the inner and outer nuclear membranes. Nuclear membrane ruptures were accompanied by a large increase in DNA damage, as judged by γ-H2AX foci. Mechanical stress increased both nuclear membrane ruptures and DNA damage, whereas minimizing transmission of cytoskeletal forces to the nucleus had the opposite effects.
                  Cover page: Fibroblasts lacking nuclear lamins do not have nuclear blebs or protrusions but nevertheless have frequent nuclear membrane ruptures
                  • Article
                  • Peer Reviewed
                  UCLA Previously Published Works (2014)
                  Lamins A and C (products of the LMNA gene) are found in roughly equal amounts in peripheral tissues, but the brain produces mainly lamin C and little lamin A. In HeLa cells and fibroblasts, the expression of prelamin A (the precursor to lamin A) can be reduced by miR-9, but the relevance of those cell culture studies to lamin A regulation in the brain was unclear. To address this issue, we created two new Lmna knock-in alleles, one (Lmna(PLAO-5NT)) with a 5-bp mutation in a predicted miR-9 binding site in prelamin A's 3' UTR, and a second (Lmna(PLAO-UTR)) in which prelamin A's 3' UTR was replaced with lamin C's 3' UTR. Neither allele had significant effects on lamin A levels in peripheral tissues; however, both substantially increased prelamin A transcript levels and lamin A protein levels in the cerebral cortex and the cerebellum. The increase in lamin A expression in the brain was more pronounced with the Lmna(PLAO-UTR) allele than with the Lmna(PLAO-5NT) allele. With both alleles, the increased expression of prelamin A transcripts and lamin A protein was greater in the cerebral cortex than in the cerebellum. Our studies demonstrate the in vivo importance of prelamin A's 3' UTR and its miR-9 binding site in regulating lamin A expression in the brain. The reduced expression of prelamin A in the brain likely explains why children with Hutchinson-Gilford progeria syndrome (a progeroid syndrome caused by a mutant form of prelamin A) are spared from neurodegenerative disease.
                    Cover page: New Lmna knock-in mice provide a molecular mechanism for the ‘segmental aging’ in Hutchinson–Gilford progeria syndrome
                    • Article
                    • Peer Reviewed
                    UCLA Previously Published Works (2016)
                    GPIHBP1, a GPI-anchored protein in capillary endothelial cells, is crucial for the lipolytic processing of triglyceride-rich lipoproteins (TRLs). GPIHBP1 shuttles lipoprotein lipase (LPL) to its site of action in the capillary lumen and is essential for the margination of TRLs along capillaries - such that lipolytic processing can proceed. GPIHBP1 also reduces the unfolding of the LPL catalytic domain, thereby stabilizing LPL catalytic activity. Many different GPIHBP1 mutations have been identified in patients with severe hypertriglyceridemia (chylomicronemia), the majority of which interfere with folding of the protein and abolish its capacity to bind and transport LPL. The discovery of GPIHBP1 has substantially revised our understanding of intravascular triglyceride metabolism but has also raised many new questions for future research.
                      Cover page: GPIHBP1 and Plasma Triglyceride Metabolism
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