The Myoblast C2C12 Transfected with Mutant Valosin-Containing Protein Exhibits Delayed Stress Granule Resolution on Oxidative Stress.

Valosin-containing protein (VCP) mutations cause inclusion body myopathy with Paget disease and frontotemporal dementia. However, the mechanisms by which mutant VCP triggers degeneration remain unknown. Here, we investigated the role of VCP in cellular stress and found that the oxidative stressor arsenite and heat shock-activated stress responses evident by T-intracellular antigen-1-positive granules in C2C12 myoblasts. Granules also contained phosphorylated transactive response DNA-binding protein 43, ubiquitin, microtubule-associated protein 1A/1B light chains 3, and lysosome-associated membrane protein 2. Mutant VCP produced more T-intracellular antigen-1-positive granules than wild-type in the postarsenite exposure period. Similar results were observed for other granule components, indicating that mutant VCP delayed clearance of stress granules. Furthermore, stress granule resolution was impaired on differentiated C2C12 cells expressing mutant VCP. To address whether mutant VCP triggers dysregulation of the stress granule pathway in vivo, we analyzed skeletal muscle of aged VCPR155H-knockin mice. We found significant increments in oxidated proteins but observed the stress granule markers RasGAP SH3-binding protein and phosphorylated eukaryotic translation initiation factor 2α unchanged. The mixed results indicate that mutant VCP together with aging lead to higher oxidative stress in skeletal muscle but were insufficient to disrupt the stress granule pathway. Our findings support that deficiencies in recovery from stressors may result in attenuated tolerance to stress that could trigger muscle degeneration.

Tet2 regulates Sin3a recruitment at active enhancers in embryonic stem cells.

Tet2 is a member of the Ten-eleven translocation (Tet1/2/3) family of enzymes and is expressed in embryonic stem cells (ESCs). It demethylates DNA (catalytic functions) and partners with chromatin modifiers (noncatalytic functions) to regulate genes. However, the significance of these functions in ESCs is less defined. Using Tet2 catalytic mutant (Tet2m/m) and knockout (Tet2-/-) ESCs, we identified Tet2 target genes regulated by its catalytic dependent versus independent roles. Tet2 was enriched at their active enhancers and promoters to demethylate them. We also identified the histone deacetylase component Sin3a as a Tet2 partner, co-localizing at promoters and active enhancers. Tet2 deficiency diminished Sin3a at these regions. Tet2 and Sin3a co-occupancy overlapped with Tet1. Combined loss of Tet1/2, but not of their catalytic activities, reduced Sin3a at active enhancers. These findings establish Tet2 catalytic and noncatalytic functions as regulators of DNA demethylation and Sin3a recruitment at active enhancers in ESCs.

Comparative analysis of Tet2 catalytic-deficient and knockout bone marrow over time.

TET2 is a member of the Ten-eleven translocation (Tet) family of DNA dioxygenases that regulate gene expression by promoting DNA demethylation (enzymatic activity) and partnering with chromatin regulatory complexes (nonenzymatic functions). TET2 is highly expressed in the hematopoietic lineage, where its molecular functions are the subject of continuous investigations because of the prevalence of TET2 mutations in hematologic malignancies. Previously, we have implicated Tet2 catalytic and noncatalytic functions in the regulation of myeloid and lymphoid lineages, respectively. However, the impact of these functions of Tet2 on hematopoiesis as the bone marrow ages remains unclear. Here, we conducted comparative transplantations and transcriptomic analyses of 3-, 6-, 9-, and 12-month-old Tet2 catalytic mutant (Mut) and knockout (KO) bone marrow. Tet2 Mut bone marrow of all ages exclusively caused hematopoietic disorders of the myeloid lineage. In contrast, young Tet2 KO bone marrow developed both lymphoid and myeloid diseases, whereas older Tet2 KO bone marrow predominantly elicited myeloid disorders with shorter latency than age-matched Tet2 Mut bone marrow. We identified robust gene dysregulation in Tet2 KO Lin- cells at 6 months that involved lymphoma and myelodysplastic syndrome and/or leukemia-causing genes, many of which were hypermethylated early in life. There was a shift from lymphoid to myeloid gene deregulation in Tet2 KO Lin- cells with age, underpinning the higher incidence of myeloid diseases. These findings expand on the dynamic regulation of bone marrow by Tet2 and show that its catalytic-dependent and -independent roles have distinct impacts on myeloid and lymphoid lineages with age.

Tet1 Suppresses p21 to Ensure Proper Cell Cycle Progression in Embryonic Stem Cells.

Ten eleven translocation 1 (Tet1) is a DNA dioxygenase that promotes DNA demethylation by oxidizing 5-methylcytosine. It can also partner with chromatin-activating and repressive complexes to regulate gene expressions independent of its enzymatic activity. Tet1 is highly expressed in embryonic stem cells (ESCs) and regulates pluripotency and differentiation. However, its roles in ESC cell cycle progression and proliferation have not been investigated. Using a series of Tet1 catalytic mutant (Tet1m/m), knockout (Tet1-/-) and wild type (Tet1+/+) mouse ESCs (mESCs), we identified a non-catalytic role of Tet1 in the proper cell cycle progression and proliferation of mESCs. Tet1-/-, but not Tet1m/m, mESCs exhibited a significant reduction in proliferation and delayed progression through G1. We found that the cyclin-dependent kinase inhibitor p21/Cdkn1a was uniquely upregulated in Tet1-/- mESCs and its knockdown corrected the slow proliferation and delayed G1 progression. Mechanistically, we found that p21 was a direct target of Tet1. Tet1 occupancy at the p21 promoter overlapped with the repressive histone mark H3K27me3 as well as with the H3K27 trimethyl transferase PRC2 component Ezh2. A loss of Tet1, but not loss of its catalytic activity, significantly reduced the enrichment of Ezh2 and H3K27 trimethylation at the p21 promoter without affecting the DNA methylation levels. We also found that the proliferation defects of Tet1-/- mESCs were independent of their differentiation defects. Together, these findings established a non-catalytic role for Tet1 in suppressing p21 in mESCs to ensure a rapid G1-to-S progression, which is a key hallmark of ESC proliferation. It also established Tet1 as an epigenetic regulator of ESC proliferation in addition to its previously defined roles in ESC pluripotency and differentiation.

Idax and Rinf facilitate expression of Tet enzymes to promote neural and suppress trophectodermal programs during differentiation of embryonic stem cells.

The Inhibitor of disheveled and axin (Idax) and its ortholog the Retinoid inducible nuclear factor (Rinf) are DNA binding proteins with nuclear and cytoplasmic functions. Rinf is expressed in embryonic stem cells (ESCs) where it regulates transcription of the Ten-eleven translocation (Tet) enzymes, promoting neural and suppressing mesendoderm/trophectoderm differentiation. Here, we find that Idax, which is not expressed in ESCs, is induced upon differentiation. Like Rinf, Idax facilitates neural and silences trophectodermal programs. Individual or combined loss of Idax and Rinf led to downregulation of neural and upregulation of trophectoderm markers during differentiation of ESCs to embryoid bodies as well as during directed differentiation of ESCs to neural progenitor cells (NPCs) and trophoblast-like cells. These defects resemble those of Tet-deficient ESCs. Consistently, Tet genes are direct targets of Idax and Rinf, and loss of Idax and Rinf led to downregulation of Tet enzymes during ESC differentiation to NPCs and trophoblast-like cells. While Idax and Rinf single and double knockout (DKO) mice were viable and overtly normal, DKO embryos had reduced expression of several NPC markers in embryonic forebrains and deregulated expression of selected trophoblast markers in placentas. NPCs derived from DKO forebrains had reduced self-renewal while DKO placentas had increased junctional zone and reduced labyrinth layers. Together, our findings establish Idax and Rinf as regulators of Tet enzymes for proper differentiation of ESCs.

Definitive hematopoietic stem cells minimally contribute to embryonic hematopoiesis.

Hematopoietic stem cells (HSCs) are rare cells that arise in the embryo and sustain adult hematopoiesis. Although the functional potential of nascent HSCs is detectable by transplantation, their native contribution during development is unknown, in part due to the overlapping genesis and marker gene expression with other embryonic blood progenitors. Using single-cell transcriptomics, we define gene signatures that distinguish nascent HSCs from embryonic blood progenitors. Applying a lineage-tracing approach to selectively track HSC output in situ, we find significantly delayed lymphomyeloid contribution. An inducible HSC injury model demonstrates a negligible impact on larval lymphomyelopoiesis following HSC depletion. HSCs are not merely dormant at this developmental stage, as they showed robust regeneration after injury. Combined, our findings illuminate that nascent HSCs self-renew but display differentiation latency, while HSC-independent embryonic progenitors sustain developmental hematopoiesis. Understanding these differences could improve de novo generation and expansion of functional HSCs.

Rapamycin reversal of VEGF-C-driven lymphatic anomalies in the respiratory tract.

Lymphatic malformations are serious but poorly understood conditions that present therapeutic challenges. The goal of this study was to compare strategies for inducing regression of abnormal lymphatics and explore underlying mechanisms. CCSP-rtTA/tetO-VEGF-C mice, in which doxycycline regulates VEGF-C expression in the airway epithelium, were used as a model of pulmonary lymphangiectasia. After doxycycline was stopped, VEGF-C expression returned to normal, but lymphangiectasia persisted for at least 9 months. Inhibition of VEGFR-2/VEGFR-3 signaling, Notch, ß-adrenergic receptors, or autophagy and antiinflammatory steroids had no noticeable effect on the amount or severity of lymphangiectasia. However, rapamycin inhibition of mTOR reduced lymphangiectasia by 76% within 7 days without affecting normal lymphatics. Efficacy of rapamycin was not increased by coadministration with the other agents. In prevention trials, rapamycin suppressed VEGF-C-driven mTOR phosphorylation and lymphatic endothelial cell sprouting and proliferation. However, in reversal trials, no lymphatic endothelial cell proliferation was present to block in established lymphangiectasia, and rapamycin did not increase caspase-dependent apoptosis. However, rapamycin potently suppressed Prox1 and VEGFR-3. These experiments revealed that lymphangiectasia is remarkably resistant to regression but is responsive to rapamycin, which rapidly reduces and normalizes the abnormal lymphatics without affecting normal lymphatics.

Reconstrucción artroscópica de inestabilidad anterior traumática de hombro: resultados funcionales y recidiva a 2 años / Arthroscopic reconstruction of traumatic anterior shoulder instability: functional results and recurrence at 2 years

Introducción: la inestabilidad es una patología frecuente del hombro, cuyo tratamiento se puede realizar por vía artroscópica o mediante cirugía abierta. Las series iniciales de reconstrucción artroscópica reportaron una mayor tasa de recidiva. Sin embargo, en la actualidad los resultados de ambas técnicas son similares. Objetivo: Evaluar los resultados funcionales e índice de recurrencia de la reconstrucción artroscópica de la inestabilidad antero-inferior de hombro a 2 años de seguimiento. Material y métodos: se analizaron 65 hombros (64 pacientes) con diagnóstico de inestabilidad traumática anterior de hombro. El promedio de edad fue de 27 años (18-45). El seguimiento promedio fue de 23,2 meses (12-50). Tomamos como recidiva aquellos pacientes que habían sufrido luxación de hombro, y también a los pacientes que en la evaluación final de control presentaban test de aprehensión y recolocación positivos. Se evalúo rango de movilidad pre operatorio y al final del seguimiento. Los pacientes fueron evaluados funcionalmente con el Constant score, ASES score y Simple Shoulder Test. Se utilizó el score de Rowe para la evaluación al final del seguimiento. Resultados: Tuvimos 1 caso (1,5 por ciento) que presento luxación de hombro. Pero si considerando recidiva los pacientes que presentaron test de aprehensión y recolocación positivo tuvimos 5 casos más; lo que representa un total del 7,7 por ciento de recidiva pero sin luxación. Se observó una mejoría significativa de los scores funcionales. Conclusión: La reconstrucción artroscópica permite restaurar la estabilidad articular, obteniendo resultados funcionales aceptables, con alto índice de satisfacción de los pacientes y una tasa de recidiva aceptable menor al 10 por ciento en el seguimiento a los 2 años de operado. Tipo de estudio: Serie de Casos. Nivel de evidencia: IV

Introduction: Shoulder Instability is a frequent pathology and its treatment can be done by arthroscopic or open surgery. Initial series of arthroscopic reconstruction reports high failure rates. However both technique have similar results. Purpose: To evaluate outcomes and recurrence of the arthroscopic reconstruction of the shoulder instability at 2 year follow up. Method: We evaluate 65 shoulder (64 patients) with traumatic anterior-inferior shoulder dislocation. Mean age was 27 years (18-45). Mean follow-up was 23,2 months (12-50). We defined has recurrence patients with shoulder dislocation o patients with positive apprehension or relocation test at the last control test. We evaluate range of motion prior surgery and at the end of follow up. Patients were evaluated functionally with Constant Score, ASES and Simple Shoulder Test. We use the Rowe score to evaluate the last follow up. Results: Recurrence rate was 1,5% (1 cases) with shoulder dislocation and 7,7% (5 cases) with positive apprehension or relocation test. A significant improvement of functional outcomes scores was observed. Conclusion: Arthroscopic reconstruction restores joint stability, with very good clinical outcomes and high rate of patient satisfaction with a recurrence rate below 10% at 2 years follow-up. Study Design: Cases Series. Level of Evidence: IV

Reconstrucción artroscópica de inestabilidad anterior traumática de hombro: resultados funcionales y recidiva a 2 años / Arthroscopic reconstruction of traumatic anterior shoulder instability: functional results and recurrence at 2 years

Introducción: la inestabilidad es una patología frecuente del hombro, cuyo tratamiento se puede realizar por vía artroscópica o mediante cirugía abierta. Las series iniciales de reconstrucción artroscópica reportaron una mayor tasa de recidiva. Sin embargo, en la actualidad los resultados de ambas técnicas son similares. Objetivo: Evaluar los resultados funcionales e índice de recurrencia de la reconstrucción artroscópica de la inestabilidad antero-inferior de hombro a 2 años de seguimiento. Material y métodos: se analizaron 65 hombros (64 pacientes) con diagnóstico de inestabilidad traumática anterior de hombro. El promedio de edad fue de 27 años (18-45). El seguimiento promedio fue de 23,2 meses (12-50). Tomamos como recidiva aquellos pacientes que habían sufrido luxación de hombro, y también a los pacientes que en la evaluación final de control presentaban test de aprehensión y recolocación positivos. Se evalúo rango de movilidad pre operatorio y al final del seguimiento. Los pacientes fueron evaluados funcionalmente con el Constant score, ASES score y Simple Shoulder Test. Se utilizó el score de Rowe para la evaluación al final del seguimiento. Resultados: Tuvimos 1 caso (1,5 por ciento) que presento luxación de hombro. Pero si considerando recidiva los pacientes que presentaron test de aprehensión y recolocación positivo tuvimos 5 casos más; lo que representa un total del 7,7 por ciento de recidiva pero sin luxación. Se observó una mejoría significativa de los scores funcionales. Conclusión: La reconstrucción artroscópica permite restaurar la estabilidad articular, obteniendo resultados funcionales aceptables, con alto índice de satisfacción de los pacientes y una tasa de recidiva aceptable menor al 10 por ciento en el seguimiento a los 2 años de operado. Tipo de estudio: Serie de Casos. Nivel de evidencia: IV (AU)

Introduction: Shoulder Instability is a frequent pathology and its treatment can be done by arthroscopic or open surgery. Initial series of arthroscopic reconstruction reports high failure rates. However both technique have similar results. Purpose: To evaluate outcomes and recurrence of the arthroscopic reconstruction of the shoulder instability at 2 year follow up. Method: We evaluate 65 shoulder (64 patients) with traumatic anterior-inferior shoulder dislocation. Mean age was 27 years (18-45). Mean follow-up was 23,2 months (12-50). We defined has recurrence patients with shoulder dislocation o patients with positive apprehension or relocation test at the last control test. We evaluate range of motion prior surgery and at the end of follow up. Patients were evaluated functionally with Constant Score, ASES and Simple Shoulder Test. We use the Rowe score to evaluate the last follow up. Results: Recurrence rate was 1,5% (1 cases) with shoulder dislocation and 7,7% (5 cases) with positive apprehension or relocation test. A significant improvement of functional outcomes scores was observed. Conclusion: Arthroscopic reconstruction restores joint stability, with very good clinical outcomes and high rate of patient satisfaction with a recurrence rate below 10% at 2 years follow-up. Study Design: Cases Series. Level of Evidence: IV (AU)