Polarity [v0.4.3] ((INSTALL))
One goal of regenerative medicine is to rejuvenate tissues and extend lifespan by restoring the function of endogenous aged stem cells. However, evidence that somatic stem cells can be targeted in vivo to extend lifespan is still lacking. Here, we demonstrate that after a short systemic treatment with a specific inhibitor of the small RhoGTPase Cdc42 (CASIN), transplanting aged hematopoietic stem cells (HSCs) from treated mice is sufficient to extend the healthspan and lifespan of aged immunocompromised mice without additional treatment. In detail, we show that systemic CASIN treatment improves strength and endurance of aged mice by increasing the myogenic regenerative potential of aged skeletal muscle stem cells. Further, we show that CASIN modifies niche localization and H4K16ac polarity of HSCs in vivo. Single-cell profiling reveals changes in HSC transcriptome, which underlie enhanced lymphoid and regenerative capacity in serial transplantation assays. Overall, we provide proof-of-concept evidence that a short systemic treatment to decrease Cdc42 activity improves the regenerative capacity of different endogenous aged stem cells in vivo, and that rejuvenated HSCs exert a broad systemic effect sufficient to extend murine health- and lifespan.
Cell polarity is critical for asymmetric cell division, which underlies the self-renewal and differentiation capacity of somatic stem cells over time4,5. Cdc42 is a small RhoGTPase that cycles between an active (GTP-bound) and an inactive (GDP-bound) state and plays a central role in cell polarity establishment in most organisms ranging from yeast to mammals6,7. With physiological aging, Cdc42-GTP levels increase in several tissues in mice and humans. The constitutive gain of function of Cdc42 in mice is achieved by genetic deletion of Cdc42GAP (or Arhgap1 also known as p50RhoGAP), which is an ubiquitously expressed negative regulator of Cdc42, that catalyzes GTP hydrolysis by Cdc42 leading to Cdc42 inactivation. In Cdc42GAP knock-out mice, Cdc42-GTP is not hydrolyzed and levels of active Cdc42 persist elevated in the cells, without affecting activity levels of other small RhoGTPases. Importantly, Cdc42GAP knock-out results in a premature aging-like phenotype in mice that affects several organs and overall mouse fitness8. In detail, Cdc42GAP mice present with 2- to 3-fold higher Cdc42-GTP levels in different tissues and compared to wild-type littermates their lifespan is significantly shorter. They also show a reduction in body mass, loss of subdermal adipose tissue, severe lordokyphosis, muscle atrophy, osteoporosis, impaired wound-healing and hair regeneration, anemia, and other hematopoietic phenotypes. Consistently, the increased activity of Cdc42 with aging has also been shown to impair the function of several somatic stem cells in different tissues, and as a consequence, to negatively affect at least blood, skin, and intestine tissue homeostasis and regeneration9,10,11,12,13,14,15,16.
Recently, we reported that the systemic treatment of aged mice with a Cdc42 activity-specific inhibitor (CASIN)17 for only 4 consecutive days significantly extends their average and maximum lifespan18. Considering the importance of Cdc42 activity in cell polarity and for stem cell function9,10,11,12,13,14,15,16, here we investigated whether this brief systemic treatment with CASIN improves the capacity of endogenous aged stem cells to regenerate tissues and the extent to which this affects murine health- and lifespan.
The ability to restore or rejuvenate aged tissues by targeting endogenous stem cells is a central goal of regenerative medicine. However, systemic rejuvenation of aged stem cells remains a challenge and it is still unclear to what degree do stem cells contribute to overall organism health- and lifespan. Here, we show that a brief systemic treatment of aged mice with the Cdc42-activity inhibitor CASIN17 improves the regenerative potential of endogenous aged MuSCs and HSCs in vivo. We report that after CASIN treatment aged MuSCs divisional kinetic and myogenic capacity in vitro are enhanced and, after injuring the muscle in vivo with Ntx, tissue regeneration is improved. Supporting that the MuSC improvement after CASIN might contribute to extend mouse healthspan, CASIN mice performed better than aged control mice in endurance and strength tests in steady-state and also after Ntx damage. Moreover, we report on systemic CASIN affecting Cdc42 and tubulin polarity as well as H4K16ac epigenetic polarity in aged HSCs. The data on H4K16ac obtained by histological analyses of whole mount BM sections aligns to those previously reported on the DNA-methylation-based epigenetic clock18 and strongly support at least some traits of epigenetic rejuvenation in HSCs after systemic CASIN treatment. Furthermore, the histological analysis shows an intriguing effect of CASIN also on aged HSC localization, which after the treatment is closer to arteries and endosteum, like young blood stem cells. At the transcriptional level, stress response and inflammation constitute the major signaling pathways targeted by CASIN in vivo. Consistently, we have previously reported a significant reduction in the levels of inflammatory cytokines (IL1α, IL1β, and INFγ) in peripheral blood serum of aged mice after in vivo CASIN treatment18. These same cytokines were also shown by others to play critical roles in aging of the blood and other tissues54,55.
These data, together with the recent data supporting an improved activity of aged intestinal and hair follicle stem cells after CASIN treatment13,15 support that the increased activity of Cdc42 with aging impairs the function of several somatic stem cells in different tissues59. Therefore, systemic treatment with CASIN can elicit distinct positive biological effects in vivo, which might depend on the doses and way of administration. Besides, recently Cdc42 activity has been shown to limit the lifespan of the budding yeast, hinting at a phylogenetically conserved mechanism of the Cdc42-polarity axis in affecting organism aging6.
Genomic comparison and characteristic analysis of the two opposite-polarity monospore strains of Morchella importuna. Genomic comparison and characteristic analysis of the top N50 sequences between the two monospore strains, M04M24 and M04M26. The outermost loop is the scaffold fragment of the two strains, with different scaffolds indicated in different colors; M04M24 is on the right, M04M26 is on the left. The scaffolds that were larger than the respective N50 values are numbered 1 to 17 for M04M26, and from 1 to 24 for M04M24. The innermost circle (f) is the gene collinearity between the two strain scaffolds of the outermost ring (a), and the inner color corresponds to the outer color of the scaffold. (b) shows the distribution of the four different types of repeat sequences in the genome, and from outside to inside are the three RNA repeat sequences: long terminal repeat (LTR) green lines, long interspersed nuclear element (LINE) purple lines, short interspersed nuclear element (SINE) blue lines, and DNA repeat sequences: red lines, with black lines in the innermost cycle displayed as the synthesis of four repeat sequences. (c) (the histogram) represents the percentage of GC in the genome. (d) shows the single-nucleotide polymorphism (SNP) frequency of the genome in the form of a histogram, and the ordinate is the SNP number per 20 kb of genome. (e) shows the gene expression of RNA sequencing (RNA-seq) samples in the form of heat map. Red indicates that FPKM (Fragments Per Kilobase Million) is more than 100, orange is 100 > FPKM > 10, green is 10 > FPKM > 0, and black indicates FPKM = 0.
Configuration options include the voltage range, polarity, and output current. The voltage range can also be limited by two user-accessible potentiometers. The PD200 is suited to a wide range of applications including electro-optics, ultrasound, vibration control, nanopositioning systems, and piezoelectric motors.
Migratory cells use distinct motility modes to navigate different microenvironments, but it is unclear whether these modes rely on the same core set of polarity components. To investigate this, we disrupted actin-related protein 2/3 (Arp2/3) and the WASP-family verprolin homologous protein (WAVE) complex, which assemble branched actin networks that are essential for neutrophil polarity and motility in standard adherent conditions. Surprisingly, confinement rescues polarity and movement of neutrophils lacking these components, revealing a processive bleb-based protrusion program that is mechanistically distinct from the branched actin-based protrusion program but shares some of the same core components and underlying molecular logic. We further find that the restriction of protrusion growth to one site does not always respond to membrane tension directly, as previously thought, but may rely on closely linked properties such as local membrane curvature. Our work reveals a hidden circuit for neutrophil polarity and indicates that cells have distinct molecular mechanisms for polarization that dominate in different microenvironments.
While actin polymerization serves as a key ingredient in generating the positive and negative feedback loops that give rise to polarity, we lack an understanding of how specific types of actin networks provide each kind of feedback. Immune cells assemble multiple actin networks at different subcellular locations that carry out distinct functions to support migration: Arp2/3-dependent assembly of branched actin networks at the leading edge contributes to cell guidance/steering and protrusion extension, while actomyosin bundles near the trailing edge provide contractile force to lift the cell rear and squeeze the cell body forward [20,21]. Along with these functional differences, the types of actin networks that immune cells and other migratory cells employ for migration vary with microenvironment [21,22]. The role of actin dynamics in migration is complex and likely depends on the type of actin network, its subcellular location, and the extracellular environment. Existing tools to probe the role of actin networks in both the positive and negative feedback loops needed for polarity are fairly crude and have largely been based on pharmacological perturbations that target all actin polymer [4,6,7,10,18,23,24]. More surgical experiments are needed to clarify how different subcellular actin networks contribute to polarity generation under different environmental conditions. 041b061a72