Werner syndrome (WS) is a premature aging disorder caused by WRN protein deficiency. Here, we report on the generation of a human WS model in human embryonic stem cells (ESCs). Differentiation of WRN-null ESCs to mesenchymal stem cells (MSCs) recapitulates features of premature cellular aging, a global loss of H3K9me3, and changes in heterochromatin architecture. We show that WRN associates with heterochromatin proteins SUV39H1 and HP1α and nuclear lamina-heterochromatin anchoring protein LAP2β. Targeted knock-in of catalytically inactive SUV39H1 in wild-type MSCs recapitulates accelerated cellular senescence, resembling WRN-deficient MSCs. Moreover, decrease in WRN and heterochromatin marks are detected in MSCs from older individuals. Our observations uncover a role for WRN in maintaining heterochromatin stability and highlight heterochromatin disorganization as a potential determinant of human aging.

Axon regeneration in the central nervous system normally fails, in part because of a developmental decline in the intrinsic ability of CNS projection neurons to extend axons. Members of the KLF family of transcription factors regulate regenerative potential in developing CNS neurons. Expression of one family member, KLF7, is down-regulated developmentally, and overexpression of KLF7 in cortical neurons in vitro promotes axonal growth. To circumvent difficulties in achieving high neuronal expression of exogenous KLF7, we created a chimera with the VP16 transactivation domain, which displayed enhanced neuronal expression compared with the native protein while maintaining transcriptional activation and growth promotion in vitro. Overexpression of VP16-KLF7 overcame the developmental loss of regenerative ability in cortical slice cultures. Adult corticospinal tract (CST) neurons failed to upregulate KLF7 in response to axon injury, and overexpression of VP16-KLF7 in vivo promoted both sprouting and regenerative axon growth in the CST of adult mice. These findings identify a unique means of promoting CST axon regeneration in vivo by reengineering a developmentally down-regulated, growth-promoting transcription factor. A xon regeneration generally fails after injury to the CNS, preventing substantial recovery. CNS regeneration is limited in part by the presence of extrinsic inhibitory molecules at the injury site, and also because adult CNS neurons possess an intrinsically low capacity for axon growth compared with embryonic or peripheral nervous system (PNS) neurons (1-3). Intrinsic regenerative capacity appears to be particularly low in the corticospinal tract (CST), an essential motor control pathway and important therapeutic target in humans (4). For instance, CST axons have shown mixed responses when inhibitory signals are neutralized (5, 6), and regenerate minimally or not at all into growth permissive tissue grafts that support some regeneration from propriospinal and brainstem neurons (7-10).The intrinsic molecular mechanisms that limit CNS axon growth remain unclear, but likely reflect a suboptimal pattern of regenerative gene expression (1, 11). In sensory neurons, overexpression of GAP43 and CAP23 or transcription factors, including ATF3, STAT3, or ID2, have produced modest gains in spinal regeneration (12-15). In the visual system, overexpression of p300 modestly increases growth (16), and knockdown of PTEN and SOCS3 results in substantial regeneration (17,18). In the CST, overexpression of the neurotrophin receptor TrkB enables regeneration into subcortical BDNF-secreting tissue grafts, and, notably, knockout of PTEN evokes regeneration in the spinal cord (19,20). The regenerative response evoked by these gene manipulations, including PTEN knockout, remains incomplete in terms of both the distance traveled by regenerating axons and the percent of neurons that respond to treatment.These findings illustrate the critical need to develop additional tools to enhance the intrinsic growth state of CNS neurons.We...

Specific point mutations in lamin A gene have been shown to accelerate aging in humans and mice. Particularly, a de novo mutation at G608G position impairs lamin A processing to produce the mutant protein progerin, which causes the Hutchinson Gilford progeria syndrome. The premature aging phenotype of Hutchinson Gilford progeria syndrome is largely recapitulated in mice deficient for the lamin A-processing enzyme, Zmpste24. We have previously reported that Zmpste24 deficiency results in genomic instability and early cellular senescence due to the delayed recruitment of repair proteins to sites of DNA damage. Here, we further investigate the molecular mechanism underlying delayed DNA damage response and identify a histone acetylation defect in Zmpste24 −/− mice. Specifically, histone H4 was hypoacetylated at a lysine 16 residue (H4K16), and this defect was attributed to the reduced association of a histone acetyltransferase, Mof, to the nuclear matrix. Given the reversible nature of epigenetic changes, rescue experiments performed either by Mof overexpression or by histone deacetylase inhibition promoted repair protein recruitment to DNA damage sites and substantially ameliorated agingassociated phenotypes, both in vitro and in vivo. The life span of Zmpste24 −/− mice was also extended with the supplementation of a histone deacetylase inhibitor, sodium butyrate, to drinking water. Consistent with recent data showing age-dependent buildup of unprocessable lamin A in physiological aging, aged wild-type mice also showed hypoacetylation of H4K16. The above results shed light on how chromatin modifications regulate the DNA damage response and suggest that the reversal of epigenetic marks could make an attractive therapeutic target against laminopathybased progeroid pathologies. E ukaryotic cells are equipped with a surveillance machinery to orchestrate the rapid detection and repair of DNA damage. When DNA damage occurs, chromatin surrounding the doublestrand breaks (DSBs) is altered and histones are modified to facilitate access for repair proteins (1). As a rapid response to DSB induction, the histone H2A variant, H2AX, is phosphorylated at Ser139 (γ-H2AX), which in turn interacts with MDC1, a DSB repair mediator, to facilitate the further recruitment of DNA repair proteins, such as 53BP1 and BRCA1 (2-4). Interestingly, γ-H2AX accumulation has been documented both in human senescent cells and in the fibroblasts of aged mice and primates (5-8). It has been proposed that these age-associated γ-H2AX foci contain nonrepairable DSBs and may have a role in initiating aging, especially because DSBs are very toxic and are one of the most lethal forms of DNA damage. Direct evidence for nonrepairable DNA damage as an inducer of premature aging has been obtained from mouse models that lack DNA repair proteins, such as ATM, Ku70, Ku80, DNA ligase IV, and Ercc1, as well as from humans with premature aging syndromes (9, 10). Together, these studies support the idea that the inability to recruit repair proteins to sites of DNA les...

Abnormal splicing of LMNA gene or aberrant processing of prelamin A results in progeroid syndrome. Here we show that lamin A interacts with and activates SIRT1. SIRT1 exhibits reduced association with nuclear matrix (NM) and decreased deacetylase activity in the presence of progerin or prelamin A, leading to rapid depletion of adult stem cells (ASCs) in Zmpste24(-/-) mice. Resveratrol enhances the binding between SIRT1 and A-type lamins to increases its deacetylase activity. Resveratrol treatment rescues ASC decline, slows down body weight loss, improves trabecular bone structure and mineral density, and significantly extends the life span in Zmpste24(-/-) mice. Our data demonstrate lamin A as an activator of SIRT1 and provide a mechanistic explanation for the activation of SIRT1 by resveratrol. The link between conserved SIRT1 longevity pathway and progeria suggests a stem cell-based and SIRT1 pathway-dependent therapeutic strategy for progeria.

Embryonic neurons, peripheral neurons, and CNS neurons in zebrafish respond to axon injury by initiating pro-regenerative transcriptional programs that enable axons to extend, locate appropriate targets, and ultimately contribute to behavioral recovery. In contrast, many long-distance projection neurons in the adult mammalian CNS, notably corticospinal tract (CST) neurons, display a much lower regenerative capacity. To promote CNS repair, a long-standing goal has been to activate pro-regenerative mechanisms that are normally missing from injured CNS neurons. Sox11 is a transcription factor whose expression is common to a many types of regenerating neurons, but it is unknown whether suboptimal Sox11 expression contributes to low regenerative capacity in the adult mammalian CNS. Here we show in adult mice that dorsal root ganglion neurons (DRGs) and CST neurons fail to upregulate Sox11 after spinal axon injury. Furthermore, forced viral expression of Sox11 reduces axonal dieback of DRG axons, and promotes CST sprouting and regenerative axon growth in both acute and chronic injury paradigms. In tests of forelimb dexterity, however, Sox11 overexpression in the cortex caused a modest but consistent behavioral impairment. These data identify Sox11 as a key transcription factor that can confer an elevated innate regenerative capacity to CNS neurons. The results also demonstrate an unexpected dissociation between axon growth and behavioral outcome, highlighting the need for additional strategies to optimize the functional output of stimulated neurons.

A de novo G608G mutation in LMNA gene leads to Hutchinson–Gilford progeria syndrome. Mice lacking the prelamin A-processing metalloprotease, Zmpste24, recapitulate many of the progeroid features of Hutchinson–Gilford progeria syndrome. Here we show that A-type lamins interact with SUV39H1, and prelamin A/progerin exhibits enhanced binding capacity to SUV39H1, protecting it from proteasomal degradation and, consequently, increasing H3K9me3 levels. Depletion of Suv39h1 reduces H3K9me3 levels, restores DNA repair capacity and delays senescence in progeroid cells. Remarkably, loss of Suv39h1 in Zmpste24−/− mice delays body weight loss, increases bone mineral density and extends lifespan by ∼60%. Thus, increased H3K9me3 levels, possibly mediated by enhanced Suv39h1 stability in the presence of prelamin A/progerin, compromise genome maintenance, which in turn contributes to accelerated senescence in laminopathy-based premature aging. Our study provides an explanation for epigenetic alterations in Hutchinson–Gilford progeria syndrome and a potential strategy for intervention by targeting SUV39H1-mediated heterochromatin remodelling.

The brain communicates with the spinal cord through numerous axon tracts that arise from discrete nuclei, transmit distinct functions, and often collateralize to facilitate the coordination of descending commands. This complexity presents a major challenge to interpreting functional outcomes from therapies that target supraspinal connectivity after injury or disease, while the wide distribution of supraspinal nuclei complicates the delivery of therapeutics. Here we harness retrograde viral vectors to overcome these challenges. We demonstrate that injection of AAV2-Retro to the cervical spinal cord of adult female mice results in highly efficient transduction of supraspinal populations throughout the brainstem, midbrain, and cortex. Some supraspinal populations, including corticospinal and rubrospinal neurons, were transduced with Ͼ90% efficiency, with robust transgene expression within 3 d of injection. In contrast, propriospinal and raphe spinal neurons showed much lower rates of retrograde transduction. Using tissue clearing and light-sheet microscopy we present detailed visualizations of descending axons tracts and create a mesoscopic projectome for the spinal cord. Moreover, chemogenetic silencing of supraspinal neurons with retrograde vectors resulted in complete and reversible forelimb paralysis, illustrating effective modulation of supraspinal function. Retrograde vectors were also highly efficient when injected after spinal injury, highlighting therapeutic potential. These data provide a global view of supraspinal connectivity and illustrate the potential of retrograde vectors to parse the functional contributions of supraspinal inputs.The complexity of descending inputs to the spinal cord presents a major challenge in efforts deliver therapeutics to widespread supraspinal systems, and to interpret their functional effects. Here we demonstrate highly effective gene delivery to diverse supraspinal nuclei using a retrograde viral approach and combine it with tissue clearing and 3D microscopy to map the descending projectome from brain to spinal cord. These data highlight newly developed retrograde viruses as therapeutic and research tools, while offering new insights into supraspinal connectivity.

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