In vivo base editing rescues Hutchinson-Gilford progeria syndrome in mice.

Hutchinson-Gilford progeria syndrome (HGPS or progeria) is typically caused by a dominant-negative C•G-to-T•A mutation (c.1824 C>T; p.G608G) in LMNA, the gene that encodes nuclear lamin A. This mutation causes RNA mis-splicing that produces progerin, a toxic protein that induces rapid ageing and shortens the lifespan of children with progeria to approximately 14 years1-4. Adenine base editors (ABEs) convert targeted A•T base pairs to G•C base pairs with minimal by-products and without requiring double-strand DNA breaks or donor DNA templates5,6. Here we describe the use of an ABE to directly correct the pathogenic HGPS mutation in cultured fibroblasts derived from children with progeria and in a mouse model of HGPS. Lentiviral delivery of the ABE to fibroblasts from children with HGPS resulted in 87-91% correction of the pathogenic allele, mitigation of RNA mis-splicing, reduced levels of progerin and correction of nuclear abnormalities. Unbiased off-target DNA and RNA editing analysis did not detect off-target editing in treated patient-derived fibroblasts. In transgenic mice that are homozygous for the human LMNA c.1824 C>T allele, a single retro-orbital injection of adeno-associated virus 9 (AAV9) encoding the ABE resulted in substantial, durable correction of the pathogenic mutation (around 20-60% across various organs six months after injection), restoration of normal RNA splicing and reduction of progerin protein levels. In vivo base editing rescued the vascular pathology of the mice, preserving vascular smooth muscle cell counts and preventing adventitial fibrosis. A single injection of ABE-expressing AAV9 at postnatal day 14 improved vitality and greatly extended the median lifespan of the mice from 215 to 510 days. These findings demonstrate the potential of in vivo base editing as a possible treatment for HGPS and other genetic diseases by directly correcting their root cause.

Transient induction of telomerase expression mediates senescence and reduces tumorigenesis in primary fibroblasts.

Telomerase is an enzymatic ribonucleoprotein complex that acts as a reverse transcriptase in the elongation of telomeres. Telomerase activity is well documented in embryonic stem cells and the vast majority of tumor cells, but its role in somatic cells remains to be understood. Here, we report an unexpected function of telomerase during cellular senescence and tumorigenesis. We crossed Tert heterozygous knockout mice (mTert+/- ) for 26 generations, during which time there was progressive shortening of telomeres, and obtained primary skin fibroblasts from mTert+/+ and mTert-/- progeny of the 26th cross. As a consequence of insufficient telomerase activities in prior generations, both mTert+/+ and mTert-/- fibroblasts showed comparable and extremely short telomere length. However, mTert-/- cells approached cellular senescence faster and exhibited a significantly higher rate of malignant transformation than mTert+/+ cells. Furthermore, an evident up-regulation of telomerase reverse-transcriptase (TERT) expression was detected in mTert+/+ cells at the presenescence stage. Moreover, removal or down-regulation of TERT expression in mTert+/+ and human primary fibroblast cells via CRISPR/Cas9 or shRNA recapitulated mTert-/- phenotypes of accelerated senescence and transformation, and overexpression of TERT in mTert-/- cells rescued these phenotypes. Taking these data together, this study suggests that TERT has a previously underappreciated, protective role in buffering senescence stresses due to short, dysfunctional telomeres, and preventing malignant transformation.

Impaired LEF1 Activation Accelerates iPSC-Derived Keratinocytes Differentiation in Hutchinson-Gilford Progeria Syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is a detrimental premature aging disease caused by a point mutation in the human LMNA gene. This mutation results in the abnormal accumulation of a truncated pre-lamin A protein called progerin. Among the drastically accelerated signs of aging in HGPS patients, severe skin phenotypes such as alopecia and sclerotic skins always develop with the disease progression. Here, we studied the HGPS molecular mechanisms focusing on early skin development by differentiating patient-derived induced pluripotent stem cells (iPSCs) to a keratinocyte lineage. Interestingly, HGPS iPSCs showed an accelerated commitment to the keratinocyte lineage than the normal control. To study potential signaling pathways that accelerated skin development in HGPS, we investigated the WNT pathway components during HGPS iPSCs-keratinocytes induction. Surprisingly, despite the unaffected ß-catenin activity, the expression of a critical WNT transcription factor LEF1 was diminished from an early stage in HGPS iPSCs-keratinocytes differentiation. A chromatin immunoprecipitation (ChIP) experiment further revealed strong bindings of LEF1 to the early-stage epithelial developmental markers K8 and K18 and that the LEF1 silencing by siRNA down-regulates the K8/K18 transcription. During the iPSCs-keratinocytes differentiation, correction of HGPS mutation by Adenine base editing (ABE), while in a partial level, rescued the phenotypes for accelerated keratinocyte lineage-commitment. ABE also reduced the cell death in HGPS iPSCs-derived keratinocytes. These findings brought new insight into the molecular basis and therapeutic application for the skin abnormalities in HGPS.

Mechanisms controlling the smooth muscle cell death in progeria via down-regulation of poly(ADP-ribose) polymerase 1.

Hutchinson-Gilford progeria syndrome (HGPS) is a severe human premature aging disorder caused by a lamin A mutant named progerin. Death occurs at a mean age of 13 y from cardiovascular problems. Previous studies revealed loss of vascular smooth muscle cells (SMCs) in the media of large arteries in a patient with HGPS and two mouse models, suggesting a causal connection between the SMC loss and cardiovascular malfunction. However, the mechanisms of how progerin leads to massive SMC loss are unknown. In this study, using SMCs differentiated from HGPS induced pluripotent stem cells, we show that HGPS SMCs exhibit a profound proliferative defect, which is primarily caused by caspase-independent cell death. Importantly, progerin accumulation stimulates a powerful suppression of PARP1 and consequently triggers an activation of the error-prone nonhomologous end joining response. As a result, most HGPS SMCs exhibit prolonged mitosis and die of mitotic catastrophe. This study demonstrates a critical role of PARP1 in mediating SMC loss in patients with HGPS and elucidates a molecular pathway underlying the progressive SMC loss in progeria.

A synaptogenic amide N-docosahexaenoylethanolamide promotes hippocampal development.

Docosahexaenoic acid (DHA), the n-3 essential fatty acid that is highly enriched in the brain, increases neurite growth and synaptogenesis in cultured mouse fetal hippocampal neurons. These cellular effects may underlie the DHA-induced enhancement of hippocampus-dependent learning and memory functions. We found that N-docsahexaenoylethanolamide (DEA), an ethanolamide derivative of DHA, is a potent mediator for these actions. This is supported by the observation that DHA is converted to DEA by fetal mouse hippocampal neuron cultures and a hippocampal homogenate, and DEA is present endogenously in the mouse hippocampus. Furthermore, DEA stimulates neurite growth and synaptogenesis at substantially lower concentrations than DHA, and it enhances glutamatergic synaptic activities with concomitant increases in synapsin and glutamate receptor subunit expression in the hippocampal neurons. These findings suggest that DEA, an ethanolamide derivative of DHA, is a synaptogenic factor, and therefore we suggest utilizing the term 'synaptamide'. This brief review summarizes the neuronal production and actions of synaptamide and describes other N-docosahexaenoyl amides that are present in the brain.

Ultraviolet radiation protection potentials of Methylene Blue for human skin and coral reef health.

Methylene blue (MB) is a century-old medicine, a laboratory dye, and recently shown as a premier antioxidant that combats ROS-induced cellular aging in human skins. Given MB's molecular structure and light absorption properties, we hypothesize that MB has the potential to be considered as a sunscreen active for UV radiation protection. In this study, we tested the effects of MB on UVB ray-induced DNA double-strand breaks in primary human keratinocytes. We found that MB treatment reduced DNA damages caused by UVB irradiation and subsequent cell death. Next, we compared MB with Oxybenzone, which is the most commonly used chemical active ingredient in sunscreens but recently proven to be hazardous to aquatic ecosystems, in particular to coral reefs. At the same concentrations, MB showed more effective UVB absorption ability than Oxybenzone and significantly outperformed Oxybenzone in the prevention of UVB-induced DNA damage and the clearance of UVA-induced cellular ROS. Furthermore, unlike Oxybenzone, MB-containing seawater did not affect the growth of the coral species Xenia umbellata. Altogether, our study suggests that MB has the potential to be a coral reef-friendly sunscreen active ingredient that can provide broad-spectrum protection against UVA and UVB.

Acetyl-Ile-Gly-Leu protects neurons from Abeta(1-42) induced toxicity in vitro and in V337M human tau-expressing mice.

AIMS: We previously reported that the neurotoxicity of amyloid beta protein (Abeta(1-42), 10 nM) was blocked by an Abeta-derived tripeptide, Abeta(32-34) (Ile-Gly-Leu, IGL), suggesting that IGL may be a lead compound in the design of Abeta antagonists. In the present study, three stable forms of IGL peptide with acetylation of its N-terminal and/or amidation of its C-terminal (acetyl-IGL, IGL-NH(2) and acetyl-IGL-NH(2)) were synthesized and examined for their effects on Abeta-induced neurotoxicity. MAIN METHODS: Phosphatidylinositol 4-kinase type II (PI4KII) activity was measured using recombinant human PI4KIIalpha kinase and cell viability was assessed in primary cultured hippocampal neurons. To test effects in vivo, 1.5 microl of 100 nM Abeta and/or 100 nM acetyl-IGL was injected into the hippocampal CA1 region of right hemisphere in transgenic mice expressing V337M human tau protein. Four weeks later, behavior performance in the Morris water maze was tested and after another 2 weeks, sections of brain were prepared for immunohistochemistry. KEY FINDINGS: Among the three modified tripeptides, acetyl-IGL attenuated the Abeta-induced inhibition of PI4KII activity as well as enhancement of glutamate neurotoxicity in primary cultured rat hippocampal neurons. Injection of Abeta into the hippocampus of mice impaired spatial memory and increased the number of degenerating neurons in bilateral hippocampal regions. Co-injection of acetyl-IGL prevented the learning impairment as well as the neuronal degeneration induced by Abeta. SIGNIFICANCE: These results suggest that a modified tripeptide, acetyl-IGL, may be effective in the treatment of Alzheimer's disease.

Diminished Canonical ß-Catenin Signaling During Osteoblast Differentiation Contributes to Osteopenia in Progeria.

Patients with Hutchinson-Gilford progeria syndrome (HGPS) have low bone mass and an atypical skeletal geometry that manifests in a high risk of fractures. Using both in vitro and in vivo models of HGPS, we demonstrate that defects in the canonical WNT/ß-catenin pathway, seemingly at the level of the efficiency of nuclear import of ß-catenin, impair osteoblast differentiation and that restoring ß-catenin activity rescues osteoblast differentiation and significantly improves bone mass. Specifically, we show that HGPS patient-derived iPSCs display defects in osteoblast differentiation, characterized by a decreased alkaline phosphatase activity and mineralizing capacity. We demonstrate that the canonical WNT/ß-catenin pathway, a major signaling cascade involved in skeletal homeostasis, is impaired by progerin, causing a reduction in the active ß-catenin in the nucleus and thus decreased transcriptional activity, and its reciprocal cytoplasmic accumulation. Blocking farnesylation of progerin restores active ß-catenin accumulation in the nucleus, increasing signaling, and ameliorates the defective osteogenesis. Moreover, in vivo analysis of the Zmpste24-/- HGPS mouse model demonstrates that treatment with a sclerostin-neutralizing antibody (SclAb), which targets an antagonist of canonical WNT/ß-catenin signaling pathway, fully rescues the low bone mass phenotype to wild-type levels. Together, this study reveals that the ß-catenin signaling cascade is a therapeutic target for restoring defective skeletal microarchitecture in HGPS. © 2018 American Society for Bone and Mineral Research.

Down-regulation of Cl- pump ClP55 subunit induced enhancement of glutamate neurotoxicity in cultured rat hippocampal neurons.

To test whether the increased intracellular Cl- concentration ([Cl-]i) is responsible for the enhanced glutamate toxicity, antisense oligonucleotide of ClP55, a Cl- -ATPase/pump associated protein, was transfected in cultured rat hippocampal neurons. Neuronal [Cl-]i in the antisense oligonucleotide-transfected culture increased to a level 3- to 4-fold higher than that in control. Glutamate exposure (10 microM, 10 min) increased neuronal apoptosis and decreased Akt-pS473 level in the antisense oligonucleotide-transfected neurons, but not in control or sense oligonucleotide-transfected ones, suggesting the responsibility of elevated [Cl-]i in the enhancement of glutamate neurotoxicity.

Protective effects of Abeta-derived tripeptide, Abeta(32-34), on Abeta(1-42)-induced phosphatidylinositol 4-kinase inhibition and neurotoxicity.

We previously reported that the neurotoxicity of pathophysiological concentrations of amyloid beta proteins (Abetas, 0.1-10nM) as assessed by the inhibition of type II phosphatidylinositol 4-kinase (PI4KII) activity and the enhancement of glutamate toxicity was blocked by a short fragment of Abeta, Abeta(31-35). Such protective effects of shorter fragments derived from Abeta(31-35) were examined in this study to reach the shortest effective peptide, using recombinant human PI4KII and primary cultured rat hippocampal neurons. Among the peptides tested (Abeta(31-34), Abeta(31-33), Abeta(31-32), Abeta(32-35), Abeta(33-35), Abeta(34-35), Abeta(32-34), Abeta(33-34) and Abeta(32-33)), Abeta(31-34), Abeta(32-35) and Abeta(32-34) blocked both the Abeta(1-42)-induced inhibition of PI4KII activity and enhancement of glutamate toxicity on cell viability. The shortest peptide among them, Abeta(32-34), showed a dose-dependent protective effect with 50% effective concentration near 1nM, while Abeta(34-32), with a reverse amino acid sequence for Abeta(32-34), showed no protective effects. Thus, a tripeptide, Abeta(32-34) i.e. Ile-Gly-Leu, may be available as a lead compound for designing effective Abeta antagonists.

Soybean-derived phosphatidylinositol inhibits in vivo low concentrations of amyloid beta protein-induced degeneration of hippocampal neurons in V337M human tau-expressing mice.

In our previous reports using primary cultured rat hippocampal neurons, pathophysiological concentrations (< or =10 nM) of amyloid beta proteins (Abetas) showed neurotoxicity via a phosphatidylinositol metabolism disorder, and soybean-derived phosphatidylinositol protected the neurons against the Abeta's neurotoxicity. In the present study, such a neurotoxic effect of Abeta and a neuroprotective effect of phosphatidylinositol were examined in vivo using transgenic mice expressing V337 M human tau. Intrahippocampal CA1 injection of 1.5 mul of 100 nM or 1 microM Abeta25-35 increased the number of degenerating neurons with an apoptotic feature in bilateral hippocampal CA1, CA2, CA3 and dentate gyrus regions in 1 month, demonstrating an in vivo neurotoxic effect of Abeta at lower concentrations after diffusion. Intrahippocampal co-injection or intracerebroventricular administration of 1.5 microl of 500 nM phosphatidylinositol prevented the Abeta25-35-induced neuronal degeneration in all the hippocampal regions, while co-injection of another acidic phospholipid, phosphatidylserine (1.5 microl, 500 nM) with Abeta25-35 showed no protective effects. Thus, exogenously applied phosphatidylinositol appeared to minimize the toxic effects of Abeta in vivo. These results suggest that soybean-derived phosphatidylinositol may be effective in the treatment of Alzheimer's disease.

Anti-Aging Potentials of Methylene Blue for Human Skin Longevity.

Oxidative stress is the major cause of skin aging that includes wrinkles, pigmentation, and weakened wound healing ability. Application of antioxidants in skin care is well accepted as an effective approach to delay the skin aging process. Methylene blue (MB), a traditional mitochondrial-targeting antioxidant, showed a potent ROS scavenging efficacy in cultured human skin fibroblasts derived from healthy donors and from patients with progeria, a genetic premature aging disease. In comparison with other widely used general and mitochondrial-targeting antioxidants, we found that MB was more effective in stimulating skin fibroblast proliferation and delaying cellular senescence. The skin irritation test, performed on an in vitro reconstructed 3D human skin model, indicated that MB was safe for long-term use, and did not cause irritation even at high concentrations. Application of MB to this 3D skin model further demonstrated that MB improved skin viability, promoted wound healing and increased skin hydration and dermis thickness. Gene expression analysis showed that MB treatment altered the expression of a subset of extracellular matrix proteins in the skin, including upregulation of elastin and collagen 2A1, two essential components for healthy skin. Altogether, our study suggests that MB has a great potential for skin care.

Lamin A and microtubules collaborate to maintain nuclear morphology.

Lamin A (LA) is a critical structural component of the nuclear lamina. Mutations within the LA gene (LMNA) lead to several human disorders, most striking of which is Hutchinson-Gilford Progeria Syndrome (HGPS), a premature aging disorder. HGPS cells are best characterized by an abnormal nuclear morphology known as nuclear blebbing, which arises due to the accumulation of progerin, a dominant mutant form of LA. The microtubule (MT) network is known to mediate changes in nuclear morphology in the context of specific events such as mitosis, cell polarization, nucleus positioning and cellular migration. What is less understood is the role of the microtubule network in determining nuclear morphology during interphase. In this study, we elucidate the role of the cytoskeleton in regulation and misregulation of nuclear morphology through perturbations of both the lamina and the microtubule network. We found that LA knockout cells exhibit a crescent shape morphology associated with the microtubule-organizing center. Furthermore, this crescent shape ameliorates upon treatment with MT drugs, Nocodazole or Taxol. Expression of progerin, in LA knockout cells also rescues the crescent shape, although the response to Nocodazole or Taxol treatment is altered in comparison to cells expressing LA. Together these results describe a collaborative effort between LA and the MT network to maintain nuclear morphology.

Attenuation of amyloid beta (Abeta)-induced inhibition of phosphatidylinositol 4-kinase activity by Abeta fragments, Abeta20-29 and Abeta31-35.

We previously reported that pathophysiological concentrations of amyloid beta protein (Abeta25-35, 0.1-10 nM) directly inhibited type II phosphatidylinositol 4-kinase (PI4KII) activity in neuronal plasma membranes, which resulted in the enhanced glutamate neurotoxicity. In the present study, we examined the effects of Abeta fragments, Abeta20-29 and Abeta31-35, on the 10 nM Abeta25-35- or Abeta1-42-induced inhibition of PI4KII activity. Both of the peptide fragments recovered the inhibition of rat brain plasma membrane PI4KII activity over the concentration range of 0.1-5 nM. Such protection by the Abeta fragments was observed in the 10 nM Abeta25-35-induced inhibition of recombinant human PI4KII, suggesting that these Abeta fragments blocked the inhibition on PI4KII molecule. The Abeta25-35-induced enhancement of glutamate neurotoxicity was also completely inhibited in the presence of these fragments. Thus, Abeta20-29 and Abeta31-35 ameliorated the Abeta-enhanced glutamate neurotoxicity probably through attenuation of Abeta-induced inhibition of PI4KII activity.

Chloride-dependency of amyloid beta protein-induced enhancement of glutamate neurotoxicity in cultured rat hippocampal neurons.

In our previous studies, pathophysiological concentrations of amyloid-beta (Abeta) proteins increased intracellular Cl(-) concentration ([Cl(-)]i) and enhanced glutamate neurotoxicity in primary cultured neurons, suggesting Cl(-)-dependent changes in glutamate signaling. To test this possibility, we examined the effects of isethionate-replaced low Cl(-) medium on the Abeta-induced enhancement of glutamate neurotoxicity in the primary cultured rat hippocampal neurons. In a normal Cl(-) (135 mM) medium, treatment with 10 nM Abeta25-35 for 2 days increased neuronal [Cl(-)]i to a level three times higher than that of control as assayed using a Cl(-)-sensitive fluorescent dye, while in a low Cl(-) (16 mM) medium such an Abeta25-35-induced increase in [Cl(-)]i was not observed. The Abeta treatment aggravated glutamate neurotoxicity in a normal Cl(-) medium as measured by mitochondrial reducing activity and lactate dehydrogenase (LDH) release, while in a low Cl(-) medium the Abeta treatment did not enhance glutamate toxicity. Upon such Abeta plus glutamate treatment under a normal Cl(-) condition, activated anti-apoptotic molecule Akt (Akt-pS473) level monitored by Western blot significantly decreased to 74% of control. Under a low Cl(-) condition, a resting Akt-pS473 level was higher than that under a normal Cl(-) condition and did not significantly change upon Abeta plus glutamate treatment. Tyrosine phosphorylation levels of 110 and 60 kDa proteins (pp110 and pp60) increased upon Abeta plus glutamate treatment under a normal Cl(-), but not low Cl(-), condition. These findings indicated that Abeta-induced enhancement of glutamate neurotoxicity is Cl(-)-dependent. Chloride-sensitive Akt pathway and tyrosine phosphorylation of proteins (pp110 and pp60) may be involved in this process.

Methylene blue alleviates nuclear and mitochondrial abnormalities in progeria.

Hutchinson-Gilford progeria syndrome (HGPS), a fatal premature aging disease, is caused by a single-nucleotide mutation in the LMNA gene. Previous reports have focused on nuclear phenotypes in HGPS cells, yet the potential contribution of the mitochondria, a key player in normal aging, remains unclear. Using high-resolution microscopy analysis, we demonstrated a significantly increased fraction of swollen and fragmented mitochondria and a marked reduction in mitochondrial mobility in HGPS fibroblast cells. Notably, the expression of PGC-1α, a central regulator of mitochondrial biogenesis, was inhibited by progerin. To rescue mitochondrial defects, we treated HGPS cells with a mitochondrial-targeting antioxidant methylene blue (MB). Our analysis indicated that MB treatment not only alleviated the mitochondrial defects but also rescued the hallmark nuclear abnormalities in HGPS cells. Additional analysis suggested that MB treatment released progerin from the nuclear membrane, rescued perinuclear heterochromatin loss and corrected misregulated gene expression in HGPS cells. Together, these results demonstrate a role of mitochondrial dysfunction in developing the premature aging phenotypes in HGPS cells and suggest MB as a promising therapeutic approach for HGPS.

Anxiolytic agent, dihydrohonokiol-B, recovers amyloid beta protein-induced neurotoxicity in cultured rat hippocampal neurons.

The effects of anxiolytic honokiol derivative, dihydrohonokiol-B (DHH-B), on amyloid beta protein (Abeta(25-35), 10 nM)-induced changes in Cl(-)-ATPase activity, intracellular Cl- concentration ([Cl-]i) and glutamate neurotoxicity were examined in cultured rat hippocampal neurons. DHH-B (10 ng/ml) recovered Abeta-induced decrease in neuronal Cl(-)-ATPase activity without any changes in the activities of Na+/K+-ATPase and anion-insensitive Mg2+-ATPase. A GABA(C) receptor antagonist (1,2,5,6,-tetrahydropyridin-4-yl) methyl-phosphinic acid (TPMPA, 15 microM), inhibited the protective effects of DHH-B on Cl(-)-ATPase activity. DHH-B reduced Abeta-induced elevation of [Cl-]i as assayed using a Cl(-)-sensitive fluorescent dye, and prevented Abeta-induced aggravation of glutamate neurotoxicity. These data suggest that DHH-B exerts the neuroprotective action against Abeta through GABA(C) receptor stimulation.

Phenotype-Dependent Coexpression Gene Clusters: Application to Normal and Premature Ageing.

Hutchinson Gilford progeria syndrome (HGPS) is a rare genetic disease with symptoms of aging at a very early age. Its molecular basis is not entirely clear, although profound gene expression changes have been reported, and there are some known and other presumed overlaps with normal aging process. Identification of genes with agingor HGPS-associated expression changes is thus an important problem. However, standard regression approaches are currently unsuitable for this task due to limited sample sizes, thus motivating development of alternative approaches. Here, we report a novel iterative multiple regression approach that leverages co-expressed gene clusters to identify gene clusters whose expression co-varies with age and/or HGPS. We have applied our approach to novel RNA-seq profiles in fibroblast cell cultures at three different cellular ages, both from HGPS patients and normal samples. After establishing the robustness of our approach, we perform a comparative investigation of biological processes underlying normal aging and HGPS. Our results recapitulate previously known processes underlying aging as well as suggest numerous unique processes underlying aging and HGPS. The approach could also be useful in detecting phenotype-dependent co-expression gene clusters in other contexts with limited sample sizes.

An inhibitory role of progerin in the gene induction network of adipocyte differentiation from iPS cells.

Lipodystrophies, characterized by partial or complete loss of adipose tissue, have been associated with mutations in the lamin A gene. It remains unclear how lamin A mutants interfere with adipose tissue formation. Hutchinson-Gilford progeria syndrome (HGPS) presents the most severe form of lamin A-associated diseases, whose patients show a complete loss of subcutaneous fat. Using iPSCs reprogrammed from HGPS fibroblasts, we induced adipocyte formation from iPSC derived embryoid bodies or from iPSC derived mesenchymal stem cells. Both approaches revealed a severe lipid storage defect in HGPS cells at late differentiation stage, faithfully recapitulating HGPS patient phenotype. Expression analysis further indicated that progerin inhibited the transcription activation of PPARγ2 and C/EBPα, but had little effects on the early adipogenic regulators. Our experiments demonstrate two comparable approaches of in vitro modeling lipodystrophies with patient-specific iPSCs, and support a regulatory role of lamin A in the terminal differentiation stage of adipogenesis.

Automated image analysis of nuclear shape: what can we learn from a prematurely aged cell?

The premature aging disorder, Hutchinson-Gilford progeria syndrome (HGPS), is caused by mutant lamin A, which affects the nuclear scaffolding. The phenotypic hallmark of HGPS is nuclear blebbing. Interestingly, similar nuclear blebbing has also been observed in aged cells from healthy individuals. Recent work has shown that treatment with rapamycin, an inhibitor of the mTOR pathway, reduced nuclear blebbing in HGPS fibroblasts. However, the extent of blebbing varies considerably within each cell population, which makes manual blind counting challenging and subjective. Here, we show a novel, automated and high throughput nuclear shape analysis that quantitatively measures curvature, area, perimeter, eccentricity and additional metrics of nuclear morphology for large populations of cells. We examined HGPS fibroblast cells treated with rapamycin and RAD001 (an analog to rapamycin). Our analysis shows that treatment with RAD001 and rapamycin reduces nuclear blebbing, consistent with blind counting controls. In addition, we find that rapamycin treatment reduces the area of the nucleus, but leaves the eccentricity unchanged. Our nuclear shape analysis provides an unbiased, multidimensional "fingerprint" for a population of cells, which can be used to quantify treatment efficacy and analyze cellular aging.