Natural Killer Cells Do Not Attenuate a Mouse-Adapted SARS-CoV-2-Induced Disease in Rag2-/- Mice.

This study investigates the roles of T, B, and Natural Killer (NK) cells in the pathogenesis of severe COVID-19, utilizing mouse-adapted SARS-CoV-2-MA30 (MA30). To evaluate this MA30 mouse model, we characterized MA30-infected C57BL/6 mice (B6) and compared them with SARS-CoV-2-WA1 (an original SARS-CoV-2 strain) infected K18-human ACE2 (K18-hACE2) mice. We found that the infected B6 mice developed severe peribronchial inflammation and rapid severe pulmonary edema, but less lung interstitial inflammation than the infected K18-hACE2 mice. These pathological findings recapitulate some pathological changes seen in severe COVID-19 patients. Using this MA30-infected mouse model, we further demonstrate that T and/or B cells are essential in mounting an effective immune response against SARS-CoV-2. This was evident as Rag2-/- showed heightened vulnerability to infection and inhibited viral clearance. Conversely, the depletion of NK cells did not significantly alter the disease course in Rag2-/- mice, underscoring the minimal role of NK cells in the acute phase of MA30-induced disease. Together, our results indicate that T and/or B cells, but not NK cells, mitigate MA30-induced disease in mice and the infected mouse model can be used for dissecting the pathogenesis and immunology of severe COVID-19.

Ercc1 DNA repair deficiency results in vascular aging characterized by VSMC phenotype switching, ECM remodeling, and an increased stress response.

Cardiovascular diseases are the number one cause of death globally. The most important determinant of cardiovascular health is a person's age. Aging results in structural changes and functional decline of the cardiovascular system. DNA damage is an important contributor to the aging process, and mice with a DNA repair defect caused by Ercc1 deficiency display hypertension, vascular stiffening, and loss of vasomotor control. To determine the underlying cause, we compared important hallmarks of vascular aging in aortas of both Ercc1Δ/- and age-matched wildtype mice. Additionally, we investigated vascular aging in 104 week old wildtype mice. Ercc1Δ/- aortas displayed arterial thickening, a loss of cells, and a discontinuous endothelial layer. Aortas of 24 week old Ercc1Δ/- mice showed phenotypical switching of vascular smooth muscle cells (VSMCs), characterized by a decrease in contractile markers and a decrease in synthetic markers at the RNA level. As well as an increase in osteogenic markers, microcalcification, and an increase in markers for damage induced stress response. This suggests that Ercc1Δ/- VSMCs undergo a stress-induced contractile-to-osteogenic phenotype switch. Ercc1Δ/- aortas showed increased MMP activity, elastin fragmentation, and proteoglycan deposition, characteristic of vascular aging and indicative of age-related extracellular matrix remodeling. The 104 week old WT mice showed loss of cells, VSMC dedifferentiation, and senescence. In conclusion, Ercc1Δ/- aortas rapidly display many characteristics of vascular aging, and thus the Ercc1Δ/- mouse is an excellent model to evaluate drugs that prevent vascular aging in a short time span at the functional, histological, and cellular level.

A model of human neural networks reveals NPTX2 pathology in ALS and FTLD.

Human cellular models of neurodegeneration require reproducibility and longevity, which is necessary for simulating age-dependent diseases. Such systems are particularly needed for TDP-43 proteinopathies1, which involve human-specific mechanisms2-5 that cannot be directly studied in animal models. Here, to explore the emergence and consequences of TDP-43 pathologies, we generated induced pluripotent stem cell-derived, colony morphology neural stem cells (iCoMoNSCs) via manual selection of neural precursors6. Single-cell transcriptomics and comparison to independent neural stem cells7 showed that iCoMoNSCs are uniquely homogenous and self-renewing. Differentiated iCoMoNSCs formed a self-organized multicellular system consisting of synaptically connected and electrophysiologically active neurons, which matured into long-lived functional networks (which we designate iNets). Neuronal and glial maturation in iNets was similar to that of cortical organoids8. Overexpression of wild-type TDP-43 in a minority of neurons within iNets led to progressive fragmentation and aggregation of the protein, resulting in a partial loss of function and neurotoxicity. Single-cell transcriptomics revealed a novel set of misregulated RNA targets in TDP-43-overexpressing neurons and in patients with TDP-43 proteinopathies exhibiting a loss of nuclear TDP-43. The strongest misregulated target encoded the synaptic protein NPTX2, the levels of which are controlled by TDP-43 binding on its 3' untranslated region. When NPTX2 was overexpressed in iNets, it exhibited neurotoxicity, whereas correcting NPTX2 misregulation partially rescued neurons from TDP-43-induced neurodegeneration. Notably, NPTX2 was consistently misaccumulated in neurons from patients with amyotrophic lateral sclerosis and frontotemporal lobar degeneration with TDP-43 pathology. Our work directly links TDP-43 misregulation and NPTX2 accumulation, thereby revealing a TDP-43-dependent pathway of neurotoxicity.

PP2A inhibitor SET promotes mTORC1 and Bmi1 signaling through Akt activation and maintains the colony-formation ability of cancer cells.

Protein phosphatase 2A (PP2A) is an essential tumor suppressor, with its activity often hindered in cancer cells by endogenous PP2A inhibitory proteins like SE translocation (SET). SET/PP2A axis plays a pivotal role in the colony-formation ability of cancer cells and the stabilization of c-Myc and E2F1 proteins implicated in this process. However, in osteosarcoma cell line HOS, SET knock-down (KD) suppresses the colony-formation ability without affecting c-Myc and E2F1. This study aimed to unravel the molecular mechanism through which SET enhances the colony-formation ability of HOS cells and determine if it is generalized to other cancer cells. Transcriptome analysis unveiled that SET KD suppressed mTORC1 signaling. SET KD inhibited Akt phosphorylation, an upstream kinase for mTORC1. PP2A inhibitor blocked SET KD-mediated decrease in phosphorylation of Akt and a mTORC1 substrate p70S6K. A constitutively active Akt restored decreased colony-formation ability by SET KD, indicating the SET/PP2A/Akt/mTORC1 axis. Additionally, enrichment analysis highlighted that Bmi-1, a polycomb group protein, is affected by SET KD. SET KD decreased Bmi-1 protein by Akt inhibition but not by mTORC1 inhibition, and exogenous Bmi-1 expression rescued the reduced colony formation by SET KD. Four out of eight cancer cell lines exhibited decreased Bmi-1 by SET KD. Further analysis of these cell lines revealed that Myc activity plays a role in SET KD-mediated Bmi-1 degradation. These findings provide new insights into the molecular mechanism of SET-regulated colony-formation ability, which involved Akt-mediated activation of mTORC1/p70S6K and Bmi-1 signaling.

Integrated transcriptome landscape of ALS identifies genome instability linked to TDP-43 pathology.

Amyotrophic Lateral Sclerosis (ALS) causes motor neuron degeneration, with 97% of cases exhibiting TDP-43 proteinopathy. Elucidating pathomechanisms has been hampered by disease heterogeneity and difficulties accessing motor neurons. Human induced pluripotent stem cell-derived motor neurons (iPSMNs) offer a solution; however, studies have typically been limited to underpowered cohorts. Here, we present a comprehensive compendium of 429 iPSMNs from 15 datasets, and 271 post-mortem spinal cord samples. Using reproducible bioinformatic workflows, we identify robust upregulation of p53 signalling in ALS in both iPSMNs and post-mortem spinal cord. p53 activation is greatest with C9orf72 repeat expansions but is weakest with SOD1 and FUS mutations. TDP-43 depletion potentiates p53 activation in both post-mortem neuronal nuclei and cell culture, thereby functionally linking p53 activation with TDP-43 depletion. ALS iPSMNs and post-mortem tissue display enrichment of splicing alterations, somatic mutations, and gene fusions, possibly contributing to the DNA damage response.

Partial RAG deficiency in humans induces dysregulated peripheral lymphocyte development and humoral tolerance defect with accumulation of T-bet+ B cells.

The recombination-activating genes (RAG) 1 and 2 are indispensable for diversifying the primary B cell receptor repertoire and pruning self-reactive clones via receptor editing in the bone marrow; however, the impact of RAG1/RAG2 on peripheral tolerance is unknown. Partial RAG deficiency (pRD) manifesting with late-onset immune dysregulation represents an 'experiment of nature' to explore this conundrum. By studying B cell development and subset-specific repertoires in pRD, we demonstrate that reduced RAG activity impinges on peripheral tolerance through the generation of a restricted primary B cell repertoire, persistent antigenic stimulation and an inflammatory milieu with elevated B cell-activating factor. This unique environment gradually provokes profound B cell dysregulation with widespread activation, remarkable extrafollicular maturation and persistence, expansion and somatic diversification of self-reactive clones. Through the model of pRD, we reveal a RAG-dependent 'domino effect' that impacts stringency of tolerance and B cell fate in the periphery.

Cooperation between KDM6B overexpression and TET2 deficiency in the pathogenesis of chronic myelomonocytic leukemia.

Loss-of-function TET2 mutations are recurrent somatic lesions in chronic myelomonocytic leukemia (CMML). KDM6B encodes a histone demethylase involved in innate immune regulation that is overexpressed in CMML. We conducted genomic and transcriptomic analyses in treatment naïve CMML patients and observed that the patients carrying both TET2 mutations and KDM6B overexpression constituted 18% of the cohort and 42% of patients with TET2 mutations. We therefore hypothesized that KDM6B overexpression cooperated with TET2 deficiency in CMML pathogenesis. We developed a double-lesion mouse model with both aberrations, and discovered that the mice exhibited a more prominent CMML-like phenotype than mice with either Tet2 deficiency or KDM6B overexpression alone. The phenotype includes monocytosis, anemia, splenomegaly, and increased frequencies and repopulating activity of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs). Significant transcriptional alterations were identified in double-lesion mice, which were associated with activation of proinflammatory signals and repression of signals maintaining genome stability. Finally, KDM6B inhibitor reduced BM repopulating activity of double-lesion mice and tumor burden in mice transplanted with BM-HSPCs from CMML patients with TET2 mutations. These data indicate that TET2 deficiency and KDM6B overexpression cooperate in CMML pathogenesis of and that KDM6B could serve as a potential therapeutic target in this disease.

PRDM paralogs antagonistically balance Wnt/ß-catenin activity during craniofacial chondrocyte differentiation.

Cranial neural crest cell (NCC)-derived chondrocyte precursors undergo a dynamic differentiation and maturation process to establish a scaffold for subsequent bone formation, alterations in which contribute to congenital birth defects. Here, we demonstrate that transcription factor and histone methyltransferase proteins Prdm3 and Prdm16 control the differentiation switch of cranial NCCs to craniofacial cartilage. Loss of either paralog results in hypoplastic and disorganized chondrocytes due to impaired cellular orientation and polarity. We show that these proteins regulate cartilage differentiation by controlling the timing of Wnt/ß-catenin activity in strikingly different ways: Prdm3 represses whereas Prdm16 activates global gene expression, although both act by regulating Wnt enhanceosome activity and chromatin accessibility. Finally, we show that manipulating Wnt/ß-catenin signaling pharmacologically or generating prdm3-/-;prdm16-/- double mutants rescues craniofacial cartilage defects. Our findings reveal upstream regulatory roles for Prdm3 and Prdm16 in cranial NCCs to control Wnt/ß-catenin transcriptional activity during chondrocyte differentiation to ensure proper development of the craniofacial skeleton.

A de novo paradigm for male infertility.

De novo mutations are known to play a prominent role in sporadic disorders with reduced fitness. We hypothesize that de novo mutations play an important role in severe male infertility and explain a portion of the genetic causes of this understudied disorder. To test this hypothesis, we utilize trio-based exome sequencing in a cohort of 185 infertile males and their unaffected parents. Following a systematic analysis, 29 of 145 rare (MAF < 0.1%) protein-altering de novo mutations are classified as possibly causative of the male infertility phenotype. We observed a significant enrichment of loss-of-function de novo mutations in loss-of-function-intolerant genes (p-value = 1.00 × 10-5) in infertile men compared to controls. Additionally, we detected a significant increase in predicted pathogenic de novo missense mutations affecting missense-intolerant genes (p-value = 5.01 × 10-4) in contrast to predicted benign de novo mutations. One gene we identify, RBM5, is an essential regulator of male germ cell pre-mRNA splicing and has been previously implicated in male infertility in mice. In a follow-up study, 6 rare pathogenic missense mutations affecting this gene are observed in a cohort of 2,506 infertile patients, whilst we find no such mutations in a cohort of 5,784 fertile men (p-value = 0.03). Our results provide evidence for the role of de novo mutations in severe male infertility and point to new candidate genes affecting fertility.

Mitochondrial dysfunction by TFAM depletion disrupts self-renewal and lineage differentiation of human PSCs by affecting cell proliferation and YAP response.

Genetic mitochondrial dysfunction is frequently associated with various embryonic developmental defects. However, how mitochondria contribute to early development and cell fate determination is poorly studied, especially in humans. Using human pluripotent stem cells (hPSCs), we established a Dox-induced knockout model with mitochondrial dysfunction and evaluated the effect of mitochondrial dysfunction on human pluripotency maintenance and lineage differentiation. The nucleus-encoded gene TFAM (transcription factor A, mitochondrial), essential for mitochondrial gene transcription and mitochondrial DNA replication, is targeted to construct the mitochondrial dysfunction model. The hPSCs with TFAM depletion exhibit the decrease of mtDNA level and oxidative respiration efficiency, representing a typical mitochondrial dysfunction phenotype. Mitochondrial dysfunction leads to impaired self-renewal in hPSCs due to proliferation arrest. Although the mitochondrial dysfunction does not affect pluripotent gene expression, it results in a severe defect in lineage differentiation. Further study in mesoderm differentiation reveals that mitochondrial dysfunction causes proliferation disability and YAP nuclear translocalization and thus together blocks mesoderm lineage differentiation. These findings provide new insights into understanding the mitochondrial function in human pluripotency maintenance and mesoderm differentiation.

Role of Mel1/Prdm16 in bone differentiation and morphology.

MEL1 (MDS1/EVI1-like gene 1/PRDM16), a zinc finger protein, is located near the chromosomal breakpoint at 1p36 in human acute myeloid leukemia (AML) cells with the t (1; 3) (p36; q21) translocation. Mel1/Prdm16 is not only a causative gene of leukemia, but also has multiple regulatory functions, such as the regulation of fat metabolism. To investigate the function of Mel1/Prdm16, we generated Mel1/Prdm16-deficient mice, but homozygous deficiency (Mel1/Prdm16-/-) was embryonic lethal at E 11.5. Heterozygous mice showed abnormal cartilage and bone formation in the postnatal skull and long bones, suggesting that Mel1/Prdm16 expression plays an important role in bone development. In osteoblast and chondrocyte cell lines, Mel1/Prdm16 promotes the differentiation of chondrocytes and regulates the differentiation of osteoblasts. Transient repression of the master regulator Runx2 is required for chondrocyte differentiation at an early stage of differentiation. However, in Mel1/Prdm16-suppressed ATDC5 cells, the initial suppression of Runx2 was lacking and its expression was upregulated at the beginning of differentiation, suggesting that chondrogenic differentiation is suppressed in Mel1/Prdm16+/- mesenchymal progenitor cells because Runx2 expression is upregulated during the early stage of differentiation. Thus, the Mel1/Prdm16 gene may be involved in the early repression of Runx2 expression during osteochondral differentiation and promote chondrogenic differentiation.

TET deficiency perturbs mature B cell homeostasis and promotes oncogenesis associated with accumulation of G-quadruplex and R-loop structures.

Enzymes of the TET family are methylcytosine dioxygenases that undergo frequent mutational or functional inactivation in human cancers. Recurrent loss-of-function mutations in TET proteins are frequent in human diffuse large B cell lymphoma (DLBCL). Here, we investigate the role of TET proteins in B cell homeostasis and development of B cell lymphomas with features of DLBCL. We show that deletion of Tet2 and Tet3 genes in mature B cells in mice perturbs B cell homeostasis and results in spontaneous development of germinal center (GC)-derived B cell lymphomas with increased G-quadruplexes and R-loops. At a genome-wide level, G-quadruplexes and R-loops were associated with increased DNA double-strand breaks (DSBs) at immunoglobulin switch regions. Deletion of the DNA methyltransferase DNMT1 in TET-deficient B cells prevented expansion of GC B cells, diminished the accumulation of G-quadruplexes and R-loops and delayed B lymphoma development, consistent with the opposing functions of DNMT and TET enzymes in DNA methylation and demethylation. Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated depletion of nucleases and helicases that regulate G-quadruplexes and R-loops decreased the viability of TET-deficient B cells. Our studies suggest a molecular mechanism by which TET loss of function might predispose to the development of B cell malignancies.

Adoptive Transfer of Immune Cells Into RAG2IL-2Rγ-Deficient Mice During Litomosoides sigmodontis Infection: A Novel Approach to Investigate Filarial-Specific Immune Responses.

Despite long-term mass drug administration programmes, approximately 220 million people are still infected with filariae in endemic regions. Several research studies have characterized host immune responses but a major obstacle for research on human filariae has been the inability to obtain adult worms which in turn has hindered analysis on infection kinetics and immune signalling. Although the Litomosoides sigmodontis filarial mouse model is well-established, the complex immunological mechanisms associated with filarial control and disease progression remain unclear and translation to human infections is difficult, especially since human filarial infections in rodents are limited. To overcome these obstacles, we performed adoptive immune cell transfer experiments into RAG2IL-2Rγ-deficient C57BL/6 mice. These mice lack T, B and natural killer cells and are susceptible to infection with the human filaria Loa loa. In this study, we revealed a long-term release of L. sigmodontis offspring (microfilariae) in RAG2IL-2Rγ-deficient C57BL/6 mice, which contrasts to C57BL/6 mice which normally eliminate the parasites before patency. We further showed that CD4+ T cells isolated from acute L. sigmodontis-infected C57BL/6 donor mice or mice that already cleared the infection were able to eliminate the parasite and prevent inflammation at the site of infection. In addition, the clearance of the parasites was associated with Th17 polarization of the CD4+ T cells. Consequently, adoptive transfer of immune cell subsets into RAG2IL-2Rγ-deficient C57BL/6 mice will provide an optimal platform to decipher characteristics of distinct immune cells that are crucial for the immunity against rodent and human filarial infections and moreover, might be useful for preclinical research, especially about the efficacy of macrofilaricidal drugs.

AIM2 Suppresses Inflammation and Epithelial Cell Proliferation during Glomerulonephritis.

Absent in melanoma-2 (AIM2) is an inflammasome-forming innate immune sensor for dsDNA but also exhibits inflammasome-independent functions such as restricting cellular proliferation. AIM2 is expressed in the kidney, but its localization and function are not fully characterized. In normal human glomeruli, AIM2 localized to podocytes. In patients with glomerulonephritis, AIM2 expression increased in CD44+-activated parietal epithelial cells within glomerular crescents. To explore AIM2 effects in glomerular disease, studies in Aim2 -/- mice were performed. Aim2-/- glomeruli showed reduced expression of Wilm tumor gene-1 (WT1), WT1-driven podocyte genes, and increased proliferation in outgrowth assays. In a nephrotoxic serum (NTS)-induced glomerulonephritis model, Aim2-/- (B6) mice exhibited more severe glomerular crescent formation, tubular injury, inflammation, and proteinuria compared with wild-type controls. Inflammasome activation markers were absent in both Aim2 -/- and wild-type kidneys, despite an increased inflammatory transcriptomic signature in Aim2 -/- mice. Aim2 -/- mice also demonstrated dysregulated cellular proliferation and an increase in CD44+ parietal epithelial cells during glomerulonephritis. The augmented inflammation and epithelial cell proliferation in Aim2 -/- (B6) mice was not due to genetic background, as Aim2 -/- (B6.129) mice demonstrated a similar phenotype during NTS glomerulonephritis. The AIM2-like receptor (ALR) locus was necessary for the inflammatory glomerulonephritis phenotype observed in Aim2 -/- mice, as NTS-treated ALR -/- mice displayed equal levels of injury as wild-type controls. Podocyte outgrowth from ALR -/- glomeruli was still increased, however, confirming that the ALR locus is dispensable for AIM2 effects on epithelial cell proliferation. These results identify a noncanonical role for AIM2 in suppressing inflammation and epithelial cell proliferation during glomerulonephritis.

Self-guarding of MORC3 enables virulence factor-triggered immunity.

Pathogens use virulence factors to inhibit the immune system1. The guard hypothesis2,3 postulates that hosts monitor (or 'guard') critical innate immune pathways such that their disruption by virulence factors provokes a secondary immune response1. Here we describe a 'self-guarded' immune pathway in human monocytes, in which guarding and guarded functions are combined in one protein. We find that this pathway is triggered by ICP0, a key virulence factor of herpes simplex virus type 1, resulting in robust induction of anti-viral type I interferon (IFN). Notably, induction of IFN by ICP0 is independent of canonical immune pathways and the IRF3 and IRF7 transcription factors. A CRISPR screen identified the ICP0 target MORC34 as an essential negative regulator of IFN. Loss of MORC3 recapitulates the IRF3- and IRF7-independent IFN response induced by ICP0. Mechanistically, ICP0 degrades MORC3, which leads to de-repression of a MORC3-regulated DNA element (MRE) adjacent to the IFNB1 locus. The MRE is required in cis for IFNB1 induction by the MORC3 pathway, but is not required for canonical IFN-inducing pathways. As well as repressing the MRE to regulate IFNB1, MORC3 is also a direct restriction factor of HSV-15. Our results thus suggest a model in which the primary anti-viral function of MORC3 is self-guarded by its secondary IFN-repressing function-thus, a virus that degrades MORC3 to avoid its primary anti-viral function will unleash the secondary anti-viral IFN response.

Deficiency of autism risk factor ASH1L in prefrontal cortex induces epigenetic aberrations and seizures.

ASH1L, a histone methyltransferase, is identified as a top-ranking risk factor for autism spectrum disorder (ASD), however, little is known about the biological mechanisms underlying the link of ASH1L haploinsufficiency to ASD. Here we show that ASH1L expression and H3K4me3 level are significantly decreased in the prefrontal cortex (PFC) of postmortem tissues from ASD patients. Knockdown of Ash1L in PFC of juvenile mice induces the downregulation of risk genes associated with ASD, intellectual disability (ID) and epilepsy. These downregulated genes are enriched in excitatory and inhibitory synaptic function and have decreased H3K4me3 occupancy at their promoters. Furthermore, Ash1L deficiency in PFC causes the diminished GABAergic inhibition, enhanced glutamatergic transmission, and elevated PFC pyramidal neuronal excitability, which is associated with severe seizures and early mortality. Chemogenetic inhibition of PFC pyramidal neuronal activity, combined with the administration of GABA enhancer diazepam, rescues PFC synaptic imbalance and seizures, but not autistic social deficits or anxiety-like behaviors. These results have revealed the critical role of ASH1L in regulating synaptic gene expression and seizures, which provides insights into treatment strategies for ASH1L-associated brain diseases.

Acute depletion of the ARID1A subunit of SWI/SNF complexes reveals distinct pathways for activation and repression of transcription.

The ARID1A subunit of SWI/SNF chromatin remodeling complexes is a potent tumor suppressor. Here, a degron is applied to detect rapid loss of chromatin accessibility at thousands of loci where ARID1A acts to generate accessible minidomains of nucleosomes. Loss of ARID1A also results in the redistribution of the coactivator EP300. Co-incident EP300 dissociation and lost chromatin accessibility at enhancer elements are highly enriched adjacent to rapidly downregulated genes. In contrast, sites of gained EP300 occupancy are linked to genes that are transcriptionally upregulated. These chromatin changes are associated with a small number of genes that are differentially expressed in the first hours following loss of ARID1A. Indirect or adaptive changes dominate the transcriptome following growth for days after loss of ARID1A and result in strong engagement with cancer pathways. The identification of this hierarchy suggests sites for intervention in ARID1A-driven diseases.

Gfi1 Loss Protects against Two Models of Induced Diabetes.

Background: Although several approaches have revealed much about individual factors that regulate pancreatic development, we have yet to fully understand their complicated interplay during pancreas morphogenesis. Gfi1 is transcription factor specifically expressed in pancreatic acinar cells, whose role in pancreas cells fate identity and specification is still elusive. Methods: In order to gain further insight into the function of this factor in the pancreas, we generated animals deficient for Gfi1 specifically in the pancreas. Gfi1 conditional knockout animals were phenotypically characterized by immunohistochemistry, RT-qPCR, and RNA scope. To assess the role of Gfi1 in the pathogenesis of diabetes, we challenged Gfi1-deficient mice with two models of induced hyperglycemia: long-term high-fat/high-sugar feeding and streptozotocin injections. Results: Interestingly, mutant mice did not show any obvious deleterious phenotype. However, in depth analyses demonstrated a significant decrease in pancreatic amylase expression, leading to a diminution in intestinal carbohydrates processing and thus glucose absorption. In fact, Gfi1-deficient mice were found resistant to diet-induced hyperglycemia, appearing normoglycemic even after long-term high-fat/high-sugar diet. Another feature observed in mutant acinar cells was the misexpression of ghrelin, a hormone previously suggested to exhibit anti-apoptotic effects on ß-cells in vitro. Impressively, Gfi1 mutant mice were found to be resistant to the cytotoxic and diabetogenic effects of high-dose streptozotocin administrations, displaying a negligible loss of ß-cells and an imperturbable normoglycemia. Conclusions: Together, these results demonstrate that Gfi1 could turn to be extremely valuable for the development of new therapies and could thus open new research avenues in the context of diabetes research.

In Vivo Renin Activity Imaging in the Kidney of Progeroid Ercc1 Mutant Mice.

Changes in the renin-angiotensin system, known for its critical role in the regulation of blood pressure and sodium homeostasis, may contribute to aging and age-related diseases. While the renin-angiotensin system is suppressed during aging, little is known about its regulation and activity within tissues. However, this knowledge is required to successively treat or prevent renal disease in the elderly. Ercc1 is involved in important DNA repair pathways, and when mutated causes accelerated aging phenotypes in humans and mice. In this study, we hypothesized that unrepaired DNA damage contributes to accelerated kidney failure. We tested the use of the renin-activatable near-infrared fluorescent probe ReninSense680™ in progeroid Ercc1d/- mice and compared renin activity levels in vivo to wild-type mice. First, we validated the specificity of the probe by detecting increased intrarenal activity after losartan treatment and the virtual absence of fluorescence in renin knock-out mice. Second, age-related kidney pathology, tubular anisokaryosis, glomerulosclerosis and increased apoptosis were confirmed in the kidneys of 24-week-old Ercc1d/- mice, while initial renal development was normal. Next, we examined the in vivo renin activity in these Ercc1d/- mice. Interestingly, increased intrarenal renin activity was detected by ReninSense in Ercc1d/- compared to WT mice, while their plasma renin concentrations were lower. Hence, this study demonstrates that intrarenal RAS activity does not necessarily run in parallel with circulating renin in the aging mouse. In addition, our study supports the use of this probe for longitudinal imaging of altered RAS signaling in aging.

Characterization of subcellular localization of eukaryotic clamp loader/unloader and its regulatory mechanism.

Proliferating cell nuclear antigen (PCNA) plays a critical role as a processivity clamp for eukaryotic DNA polymerases and a binding platform for many DNA replication and repair proteins. The enzymatic activities of PCNA loading and unloading have been studied extensively in vitro. However, the subcellular locations of PCNA loaders, replication complex C (RFC) and CTF18-RFC-like-complex (RLC), and PCNA unloader ATAD5-RLC remain elusive, and the role of their subunits RFC2-5 is unknown. Here we used protein fractionation to determine the subcellular localization of RFC and RLCs and affinity purification to find molecular requirements for the newly defined location. All RFC/RLC proteins were detected in the nuclease-resistant pellet fraction. RFC1 and ATAD5 were not detected in the non-ionic detergent-soluble and nuclease-susceptible chromatin fractions, independent of cell cycle or exogenous DNA damage. We found that small RFC proteins contribute to maintaining protein levels of the RFC/RLCs. RFC1, ATAD5, and RFC4 co-immunoprecipitated with lamina-associated polypeptide 2 (LAP2) α which regulates intranuclear lamin A/C. LAP2α knockout consistently reduced detection of RFC/RLCs in the pellet fraction, while marginally affecting total protein levels. Our findings strongly suggest that PCNA-mediated DNA transaction occurs through regulatory machinery associated with nuclear structures, such as the nuclear matrix.