Senescence is a developmental mechanism that contributes to embryonic growth and patterning.

Senescence is a form of cell-cycle arrest linked to tumor suppression and aging. However, it remains controversial and has not been documented in nonpathologic states. Here we describe senescence as a normal developmental mechanism found throughout the embryo, including the apical ectodermal ridge (AER) and the neural roof plate, two signaling centers in embryonic patterning. Embryonic senescent cells are nonproliferative and share features with oncogene-induced senescence (OIS), including expression of p21, p15, and mediators of the senescence-associated secretory phenotype (SASP). Interestingly, mice deficient in p21 have defects in embryonic senescence, AER maintenance, and patterning. Surprisingly, the underlying mesenchyme was identified as a source for senescence instruction in the AER, whereas the ultimate fate of these senescent cells is apoptosis and macrophage-mediated clearance. We propose that senescence is a normal programmed mechanism that plays instructive roles in development, and that OIS is an evolutionarily adapted reactivation of a developmental process.

p21 maintains senescent cell viability under persistent DNA damage response by restraining JNK and caspase signaling.

Cellular senescence is a permanent state of cell cycle arrest that protects the organism from tumorigenesis and regulates tissue integrity upon damage and during tissue remodeling. However, accumulation of senescent cells in tissues during aging contributes to age-related pathologies. A deeper understanding of the mechanisms regulating the viability of senescent cells is therefore required. Here, we show that the CDK inhibitor p21 (CDKN1A) maintains the viability of DNA damage-induced senescent cells. Upon p21 knockdown, senescent cells acquired multiple DNA lesions that activated ataxia telangiectasia mutated (ATM) and nuclear factor (NF)-κB kinase, leading to decreased cell survival. NF-κB activation induced TNF-α secretion and JNK activation to mediate death of senescent cells in a caspase- and JNK-dependent manner. Notably, p21 knockout in mice eliminated liver senescent stellate cells and alleviated liver fibrosis and collagen production. These findings define a novel pathway that regulates senescent cell viability and fibrosis.

T Cells Expressing a Modified FcγRI Exert Antibody-Dependent Cytotoxicity and Overcome the Limitations of CAR T-cell Therapy against Solid Tumors.

The pioneering design of chimeric antigen receptor (CAR) T-cell therapy demonstrated the potential of reprogramming the immune system. Nonetheless, T-cell exhaustion, toxicity, and suppressive microenvironments limit their efficacy in solid tumors. We previously characterized a subset of tumor-infiltrating CD4+ T cells expressing the FcγRI receptor. Herein, we detail engineering of a receptor, based on the FcγRI structure, allowing T cells to target tumor cells using antibody intermediates. These T cells showed effective and specific cytotoxicity only when an appropriate antibody was added. Only target-bound antibodies activated these cells, while free antibodies were internalized without activation. Their cytotoxic activity was correlated to target protein density, therefore targeting tumor cells with high antigen density while sparing normal cells with low or no expression. This activation mechanism prevented premature exhaustion. Furthermore, during antibody-dependent cytotoxicity these cells secreted attenuated cytokine levels compared with CAR T cells, thereby enhancing their safety profile. These cells eradicated established melanomas, infiltrated the tumor microenvironment, and facilitated host immune cell recruitment in immunocompetent mice. In NOD/SCID gamma mice the cells infiltrate, persist, and eradicate tumors. As opposed to CAR T-cell therapies, which require changing the receptor across different types of cancer, our engineered T cells remain the same across tumor types, while only the injected antibody changes. Overall, we generated a highly flexible T-cell therapy capable of binding a wide range of tumor cells with high affinity, while preserving the cytotoxic specificity only to cells expressing high density of tumor-associated antigens and using a single manufacturing process.

Mating-increases trypsin in female Drosophila hemolymph.

Male-derived accessory gland proteins (Acps) are transferred to the female reproductive tract during mating and affect female reproductive maturation and behavior. Some Acps subsequently enter the female hemolymph. We hypothesized that humoral proteases are the primary effectors of Acp bioactivity by processing (activating) and/or degrading them. To test this hypothesis we examined the fate of one Acp, Drosophila melanogaster Sex Peptide (Acp70A, DrmSP), which possesses several putative serine-protease cleavage sites, in hemolymph of unmated and mated females. In D. melanogaster, DrmSP induces post-mating non-receptivity and enhances oogenesis. To determine if serine proteases regulate the duration of DrmSP activity in mated females, we performed kinetic analysis of cleavage of a synthetic N-terminal truncated DrmSP(8-36) (T-SP) with hemolymph of unmated versus mated females. We found that T-SP is cleaved more rapidly and completely in mated female hemolymph. Using LC-MS/MS analyses, we identified its primary cleavage sites, indicating that trypsin was the major endopeptidase regulating T-SP in hemolymph. This was verified in vitro by utilizing specific chromogenic serine-protease substrates and inhibitors. We propose that post-mating cleavage of DrmSP in the female hemolymph regulates the duration of the rapidly induced post-mating responses in D. melanogaster and that this is a specific example of Acp bioactivity regulated by hemolymph serine proteases.

Directed elimination of senescent cells by inhibition of BCL-W and BCL-XL.

Senescent cells, formed in response to physiological and oncogenic stresses, facilitate protection from tumourigenesis and aid in tissue repair. However, accumulation of such cells in tissues contributes to age-related pathologies. Resistance of senescent cells to apoptotic stimuli may contribute to their accumulation, yet the molecular mechanisms allowing their prolonged viability are poorly characterized. Here we show that senescent cells upregulate the anti-apoptotic proteins BCL-W and BCL-XL. Joint inhibition of BCL-W and BCL-XL by siRNAs or the small-molecule ABT-737 specifically induces apoptosis in senescent cells. Notably, treatment of mice with ABT-737 efficiently eliminates senescent cells induced by DNA damage in the lungs as well as senescent cells formed in the epidermis by activation of p53 through transgenic p14(ARF). Elimination of senescent cells from the epidermis leads to an increase in hair-follicle stem cell proliferation. The finding that senescent cells can be eliminated pharmacologically paves the way to new strategies for the treatment of age-related pathologies.

Rapid, reproducible transduction of select forebrain regions by targeted recombinant virus injection into the neonatal mouse brain.

Viral vectors can mediate long-term gene expression in different regions of the brain. Recombinant adeno-associated virus (rAAV) and Lenti virus (LV) have both gained prominence due to their ability to achieve specific transduction of various neuronal populations. Whilst widespread gene delivery has been obtained by targeted injection of rAAV in various brain structures, LV has also been utilized for infection of stem cell populations for cell lineage tracing. Both viral vector systems are most commonly used for gene delivery in mature brains, but the great potential of somatic gene delivery into the neonate brain has not been systematically exploited. Here we provide a systematic guideline for efficient stereotaxic virus delivery into different neuronal populations of the neonate brain. We demonstrate region specific recombination of a 'stop-floxed' Rosa26 reporter allele upon targeted injection of rAAV vectors expressing Cre-recombinase at postnatal day zero (P0). In addition, utilizing LV, we show efficient transduction of P0 subventricular zone stem cells with subsequent labeling of approximately 20% of migrating neuroblasts along the rostral migratory stream (RMS) into the olfactory bulb. In summary, we report on an optimized protocol for facile, reproducible, high-throughput virus-based gene transfer into neonatal brains of wild-type and genetically altered mice, which allows targeted transduction of different brain regions and distinct neuronal populations.