Self-assembly of multi-component mitochondrial nucleoids via phase separation.

Mitochondria contain an autonomous and spatially segregated genome. The organizational unit of their genome is the nucleoid, which consists of mitochondrial DNA (mtDNA) and associated architectural proteins. Here, we show that phase separation is the primary physical mechanism for assembly and size control of the mitochondrial nucleoid (mt-nucleoid). The major mtDNA-binding protein TFAM spontaneously phase separates in vitro via weak, multivalent interactions into droplets with slow internal dynamics. TFAM and mtDNA form heterogenous, viscoelastic structures in vitro, which recapitulate the dynamics and behavior of mt-nucleoids in vivo. Mt-nucleoids coalesce into larger droplets in response to various forms of cellular stress, as evidenced by the enlarged and transcriptionally active nucleoids in mitochondria from patients with the premature aging disorder Hutchinson-Gilford Progeria Syndrome (HGPS). Our results point to phase separation as an evolutionarily conserved mechanism of genome organization.

Noncanonical autophagy is required for type I interferon secretion in response to DNA-immune complexes.

Toll-like receptor-9 (TLR9) is largely responsible for discriminating self from pathogenic DNA. However, association of host DNA with autoantibodies activates TLR9, inducing the pathogenic secretion of type I interferons (IFNs) from plasmacytoid dendritic cells (pDCs). Here, we found that in response to DNA-containing immune complexes (DNA-IC), but not to soluble ligands, IFN-α production depended upon the convergence of the phagocytic and autophagic pathways, a process called microtubule-associated protein 1A/1B-light chain 3 (LC3)-associated phagocytosis (LAP). LAP was required for TLR9 trafficking into a specialized interferon signaling compartment by a mechanism that involved autophagy-related proteins, but not the conventional autophagic preinitiation complex, or adaptor protein-3 (AP-3). Our findings unveil a new role for nonconventional autophagy in inflammation and provide one mechanism by which anti-DNA autoantibodies, such as those found in several autoimmune disorders, bypass the controls that normally restrict the apportionment of pathogenic DNA and TLR9 to the interferon signaling compartment.

DNA polymerase ß deficiency leads to neurodegeneration and exacerbates Alzheimer disease phenotypes.

We explore the role of DNA damage processing in the progression of cognitive decline by creating a new mouse model. The new model is a cross of a common Alzheimer's disease (AD) mouse (3xTgAD), with a mouse that is heterozygous for the critical DNA base excision repair enzyme, DNA polymerase ß. A reduction of this enzyme causes neurodegeneration and aggravates the AD features of the 3xTgAD mouse, inducing neuronal dysfunction, cell death and impairing memory and synaptic plasticity. Transcriptional profiling revealed remarkable similarities in gene expression alterations in brain tissue of human AD patients and 3xTg/Polß(+/-) mice including abnormalities suggestive of impaired cellular bioenergetics. Our findings demonstrate that a modest decrement in base excision repair capacity can render the brain more vulnerable to AD-related molecular and cellular alterations.

Cecal bascule presenting as internal hernia.

Cecal bascule, a rare subtype of cecal volvulus, presents diagnostic and management challenges. We report a case of cecal bascule presenting as an internal hernia in a 68-year-old male with no surgical history. Computed tomography revealed two areas of mesenteric swirling and a displaced cecum. Prompt surgical intervention included laparoscopic exploration, resection of a necrotic adhesive band, and cecopexy. This case is noteworthy because of the absence of predisposing factors like prior surgeries or inflammatory conditions. Management options for cecal bascule include resection and cecopexy, tailored to individual patient factors. Awareness among healthcare providers is crucial for the timely recognition and appropriate management of such cases. Further research is needed to refine management strategies and improve outcomes for these rare but potentially life-threatening conditions.

Omental infarction following robotic-assisted laparoscopic inguinal hernia repair.

Omental infarction (OI) is a rare condition with an overall incidence of less than 0.3%. It can occur spontaneously or can be secondary to trauma, surgery, and inflammation. While previously a diagnosis of exclusion, due to development in imaging technology, OI can now be identified based on CT findings. OI symptoms can mimic an acute abdomen, prompting potentially unnecessary surgical exploration. Treatment options range from conservative management to interventional radiology or surgical resection of the infarcted omentum. We are presenting the first case of OI following robotic-assisted inguinal hernia repair. This case highlights the importance of considering OI in differential diagnoses for patients presenting with acute abdominal pain, the utility of imaging workup in identifying OI, and guidance for conservative treatment approaches to reduce unnecessary surgical intervention.

ILT2 and ILT4 Drive Myeloid Suppression via Both Overlapping and Distinct Mechanisms.

Solid tumors are dense three-dimensional (3D) multicellular structures that enable efficient receptor-ligand trans interactions via close cell-cell contact. Immunoglobulin-like transcript (ILT)2 and ILT4 are related immune-suppressive receptors that play a role in the inhibition of myeloid cells within the tumor microenvironment. The relative contribution of ILT2 and ILT4 to immune inhibition in the context of solid tumor tissue has not been fully explored. We present evidence that both ILT2 and ILT4 contribute to myeloid inhibition. We found that although ILT2 inhibits myeloid cell activation in the context of trans-engagement by MHC-I, ILT4 efficiently inhibits myeloid cells in the presence of either cis- or trans-engagement. In a 3D spheroid tumor model, dual ILT2/ILT4 blockade was required for the optimal activation of myeloid cells, including the secretion of CXCL9 and CCL5, upregulation of CD86 on dendritic cells, and downregulation of CD163 on macrophages. Humanized mouse tumor models showed increased immune activation and cytolytic T-cell activity with combined ILT2 and ILT4 blockade, including evidence of the generation of immune niches, which have been shown to correlate with clinical response to immune-checkpoint blockade. In a human tumor explant histoculture system, dual ILT2/ILT4 blockade increased CXCL9 secretion, downregulated CD163 expression, and increased the expression of M1 macrophage, IFNγ, and cytolytic T-cell gene signatures. Thus, we have revealed distinct contributions of ILT2 and ILT4 to myeloid cell biology and provide proof-of-concept data supporting the combined blockade of ILT2 and ILT4 to therapeutically induce optimal myeloid cell reprogramming in the tumor microenvironment.

RAGE inhibits human respiratory syncytial virus syncytium formation by interfering with F-protein function.

Human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infection. Infection is critically dependent on the RSV fusion (F) protein, which mediates fusion between the viral envelope and airway epithelial cells. The F protein is also expressed on infected cells and is responsible for fusion of infected cells with adjacent cells, resulting in the formation of multinucleate syncytia. The receptor for advanced glycation end products (RAGE) is a pattern-recognition receptor that is constitutively highly expressed by type I alveolar epithelial cells. Here, we report that RAGE protected HEK cells from RSV-induced cell death and reduced viral titres in vitro. RAGE appeared to interact directly with the F protein, but, rather than inhibiting RSV entry into host cells, virus replication and budding, membrane-expressed RAGE or soluble RAGE blocked F-protein-mediated syncytium formation and sloughing. These data indicate that RAGE may contribute to protecting the lower airways from RSV by inhibiting the formation of syncytia, viral spread, epithelial damage and airway obstruction.

Kaposi sarcoma presenting as small bowel obstruction.

Kaposi sarcoma (KS) is a low-grade tumor of the vascular endothelium. The majority of individuals affected have advanced human immunodeficiency virus (HIV) or acquired immunodeficiency syndrome (AIDS). The disease typically manifests as cutaneous lesions but reports have shown that systemic disease is not uncommon. Because gastrointestinal KS is often asymptomatic, it is likely underdiagnosed. Those with symptoms can present with vague abdominal pain, nausea/vomiting or anemia. Rarely the tumors can cause bowel obstruction or perforation. We present a case of small bowel obstruction cause by KS tumors in a young transgender male to female patient with poorly controlled AIDS, supported by literature review of the clinical presentation, diagnosis and treatment recommendations.

AAV5-mediated manipulation of insulin expression in choroid plexus has long-term metabolic and behavioral consequences.

The choroid plexus (CP) is a source of trophic factors for the developing and mature brain. Insulin is produced in epithelial cells of the CP (EChPs), and its secretion is stimulated by Htr2c-mediated signaling. We modulated insulin expression in EChPs with intracerebroventricular injections of AAV5. Insulin overexpression in CP decelerates food intake, whereas its knockdown has the opposite effect. Insulin overexpression also results in reduced anxious behavior. Transcriptomic changes in the hypothalamus, especially in synapse-related processes, are also seen in mice overexpressing insulin in CP. Last, activation of Gq signaling in CP leads to acute Akt phosphorylation in neurons of the arcuate nucleus, indicating a direct action of CP-derived insulin on the hypothalamus. Taken together, our findings signify that CP is a relevant source of insulin in the central nervous system and that CP-derived insulin should be taken into consideration in future work pertaining to insulin actions in the brain.

Cell type-specific recognition of human metapneumoviruses (HMPVs) by retinoic acid-inducible gene I (RIG-I) and TLR7 and viral interference of RIG-I ligand recognition by HMPV-B1 phosphoprotein.

Human metapneumoviruses (HMPVs) are recently identified Paramyxoviridae that contribute to respiratory tract infections in children. No effective treatments or vaccines are available. Successful defense against virus infection relies on early detection by germ line-encoded pattern recognition receptors and activation of cytokine and type I IFN genes. Recently, the RNA helicase retinoic acid-inducible gene I (RIG-I) has been shown to sense HMPV. In this study, we investigated the abilities of two prototype strains of HMPV (A1 [NL\1\00] and B1 [NL\1\99]) to activate RIG-I and induce type I IFNs. Despite the abilities of both HMPV-A1 and HMPV-B1 to infect and replicate in cell lines and primary cells, only the HMPV-A1 strain triggered RIG-I to induce IFNA/B gene transcription. The failure of the HMPV-B1 strain to elicit type I IFN production was dependent on the B1 phosphoprotein, which specifically prevented RIG-I-mediated sensing of HMPV viral 5' triphosphate RNA. In contrast to most cell types, plasmacytoid dendritic cells displayed a unique ability to sense both HMPV-A1 and HMPV-B1 and in this case sensing was via TLR7 rather than RIG-I. Collectively, these data reveal differential mechanisms of sensing for two closely related viruses, which operate in cell type-specific manners.

A feeding jejunostomy causing an intussusception - case report and literature review.

Use of enteral nutrition has increased dramatically over the past two decades due to improved nutritional formulas, better quality feeding tubes and the ability to use less invasive endoscopic, fluoroscopic and laparoscopic techniques. Intussusception accounts for 1-5% of adult intestinal obstructions, with feeding tubes acting as a lead point in < 1% of cases. Since intussusception is rare, especially in adults, it is not always considered in the initial differential diagnosis of patients presenting with abdominal pain, nausea, vomiting or constipation. If left untreated, intussusception can eventually lead to bowel compromise, poor outcomes and even death. Therefore, prompt recognition and correction are necessary. We present a case of small bowel obstruction in an elderly male secondary to a tube-related intussusception. A review of the signs, symptoms and treatment recommendations is provided.

NAD+ augmentation with nicotinamide riboside improves lymphoid potential of Atm-/- and old mice HSCs.

NAD+ supplementation has significant benefits in compromised settings, acting largely through improved mitochondrial function and DNA repair. Elevating NAD+ to physiological levels has been shown to improve the function of some adult stem cells, with implications that these changes will lead to sustained improvement of the tissue or system. Here, we examined the effect of elevating NAD+ levels in models with reduced hematopoietic stem cell (HSC) potential, ATM-deficient and aged WT mice, and showed that supplementation of nicotinamide riboside (NR), a NAD+ precursor, improved lymphoid lineage potential during supplementation. In aged mice, this improved lymphoid potential was maintained in competitive transplants and was associated with transcriptional repression of myeloid gene signatures in stem and lineage-committed progenitor cells after NR treatment. However, the altered transcriptional priming of the stem cells toward lymphoid lineages was not sustained in the aged mice after NR removal. These data characterize significant alterations to the lineage potential of functionally compromised HSCs after short-term exposure to NR treatment.

Knockdown of FABP5 mRNA decreases cellular cholesterol levels and results in decreased apoB100 secretion and triglyceride accumulation in ARPE-19 cells.

To maintain normal retinal function, retinal pigment epithelial (RPE) cells engulf photoreceptor outer segments (ROS) enriched in free fatty acids (FFAs). We have previously demonstrated fatty acid-binding protein 5 (FABP5) downregulation in the RPE/choroidal complex in a mouse model of aging and early age-related macular degeneration. FABPs are involved in intracellular transport of FFAs and their targeting to specific metabolic pathways. To elucidate the role of FABP5 in lipid metabolism, the production of the FABP5 protein in a human RPE cell line was inhibited using RNA interference technology. As a result, the levels of cholesterol and cholesterol ester were decreased by about 40%, whereas FFAs and triglycerides were increased by 18 and 67% after siRNA treatment, respectively. Some species of phospholipids were decreased in siRNA-treated cells. Cellular lipid droplets were evident and apoB secretion was decreased by 76% in these cells. Additionally, we discovered that ARPE-19 cells could synthesize and secrete Apolipoprotein B100 (apoB100), which may serve as a backbone structure for the formation of lipoprotein particles in these cells. Our results indicate that FABP5 mRNA knockdown results in the accumulation of cellular triglycerides, decreased cholesterol levels, and reduced secretion of apoB100 protein and lipoprotein-like particles. These observations indicated that FABP5 plays a critical role in lipid metabolism in RPE cells, suggesting that FABP5 downregulation in the RPE/choroid complex in vivo might contribute to aging and early age-related macular degeneration.

Apolipoprotein B100 secretion by cultured ARPE-19 cells is modulated by alteration of cholesterol levels.

Cholesteryl ester rich apolipoprotein B100 (apoB100) lipoproteins accumulate in Bruch's membrane before the development of age-related macular degeneration. It is not known if these lipoproteins come from the circulation or local ocular tissue. Emerging, but incomplete evidence suggests that the retinal pigmented epithelium (RPE) can secrete lipoproteins. The purpose of this investigation was to determine (i) whether human RPE cells synthesize and secrete apoB100, and (ii) whether this secretion is driven by cellular cholesterol, and if so, (iii) whether statins inhibit this response. The established, human derived ARPE-19 cells challenged with 0-0.8 mM oleic acid accumulated cellular cholesterol, but not triglycerides. Oleic acid increased the amount of apoB100 protein recovered from the medium by both western blot analysis and (35) S-radiolabeled immunoprecipitation while negative stain electron microscopy showed lipoprotein-like particles. Of nine statins evaluated, lipophilic statins induced HMG-CoA reductase mRNA expression the most. The lipophilic Cerivastatin (5 µM) reduced cellular cholesterol by 39% and abrogated apoB100 secretion by 3-fold. In contrast, the hydrophilic statin Pravastatin had minimal effect on apoB100 secretion. These data suggest that ARPE-19 cells synthesize and secrete apoB100 lipoproteins, that this secretion is driven by cellular cholesterol, and that statins can inhibit apoB100 secretion by reducing cellular cholesterol.

Short-term NAD+ supplementation prevents hearing loss in mouse models of Cockayne syndrome.

Age-related hearing loss (ARHL) is one of the most common disorders affecting elderly individuals. There is an urgent need for effective preventive measures for ARHL because none are currently available. Cockayne syndrome (CS) is a premature aging disease that presents with progressive hearing loss at a young age, but is otherwise similar to ARHL. There are two human genetic complementation groups of CS, A and B. While the clinical phenotypes in patients are similar, the proteins have very diverse functions, and insight into their convergence is of great interest. Here, we use mouse models for CS (CSA -/- and CSB m/m ) that recapitulate the hearing loss in human CS patients. We previously showed that NAD+, a key metabolite with various essential functions, is reduced in CS and associated with multiple CS phenotypes. In this study, we report that NAD+ levels are reduced in the cochlea of CSB m/m mice and that short-term treatment (10 days) with the NAD+ precursor nicotinamide riboside (NR), prevents hearing loss, restores outer hair cell loss, and improves cochlear health in CSB m/m mice. Similar, but more modest effects were observed in CSA -/- mice. Remarkably, we observed a reduction in synaptic ribbon counts in the presynaptic zones of inner hair cells in both CSA -/- and CSB m/m mice, pointing to a converging mechanism for cochlear defects in CS. Ribbon synapses facilitate rapid and sustained synaptic transmission over long periods of time. Ribeye, a core protein of synaptic ribbons, possesses an NAD(H) binding pocket which regulates its activity. Intriguingly, NAD+ supplementation rescues reduced synaptic ribbon formation in both CSA -/- and CSB m/m mutant cochleae. These findings provide valuable insight into the mechanism of CS- and ARHL-associated hearing loss, and suggest a possible intervention.

Oxidized low density lipoproteins induce a pathologic response by retinal pigmented epithelial cells.

The accumulation of apolipoprotein B100 lipoproteins in Bruch membrane is an early event thought to promote age-related macular degeneration (AMD). Immunohistochemistry using an anti-oxidized low density lipoprotein antibody on 10 AMD specimens showed staining in Bruch membrane including basal deposits, a marker of AMD. To determine whether retinal pigmented epithelial cells develop a pathologic phenotype after interaction with lipoproteins, ARPE-19 cells were exposed to low density lipoproteins (LDL) or oxidized LDLs (oxLDL). Analysis using the Affymetrix U133 Plus 2.0 (Affymetrix, Inc., Santa Clara, CA, USA) gene chip showed physiological and pathological transcriptional responses after LDL and oxLDL treatment, respectively. LDL induced a down-regulation of cholesterol biosynthesis genes while oxLDL induced transcriptional alterations in genes related to lipid metabolism, oxidative stress, inflammation and apoptosis. Electrospray mass spectrometry showed that oxLDL, but not LDL induced large cellular increases of sphingomyelin, ceramides, and cholesteryl esters. With TUNEL labeling, oxLDL caused 14.6% apoptosis compared to <1% after LDL. Addition of an inhibitor of sphingomyelin synthase inhibited this apoptosis by 41%. These data support the hypothesis that oxidized lipoproteins are one trigger for initiating early events in the pathogenesis of AMD.

Cell cycle-dependent phosphorylation regulates RECQL4 pathway choice and ubiquitination in DNA double-strand break repair.

Pathway choice within DNA double-strand break (DSB) repair is a tightly regulated process to maintain genome integrity. RECQL4, deficient in Rothmund-Thomson Syndrome, promotes the two major DSB repair pathways, non-homologous end joining (NHEJ) and homologous recombination (HR). Here we report that RECQL4 promotes and coordinates NHEJ and HR in different cell cycle phases. RECQL4 interacts with Ku70 to promote NHEJ in G1 when overall cyclin-dependent kinase (CDK) activity is low. During S/G2 phases, CDK1 and CDK2 (CDK1/2) phosphorylate RECQL4 on serines 89 and 251, enhancing MRE11/RECQL4 interaction and RECQL4 recruitment to DSBs. After phosphorylation, RECQL4 is ubiquitinated by the DDB1-CUL4A E3 ubiquitin ligase, which facilitates its accumulation at DSBs. Phosphorylation of RECQL4 stimulates its helicase activity, promotes DNA end resection, increases HR and cell survival after ionizing radiation, and prevents cellular senescence. Collectively, we propose that RECQL4 modulates the pathway choice of NHEJ and HR in a cell cycle-dependent manner.

WRN regulates pathway choice between classical and alternative non-homologous end joining.

Werner syndrome (WS) is an accelerated ageing disorder with genomic instability caused by WRN protein deficiency. Many features seen in WS can be explained by the diverse functions of WRN in DNA metabolism. However, the origin of the large genomic deletions and telomere fusions are not yet understood. Here, we report that WRN regulates the pathway choice between classical (c)- and alternative (alt)-nonhomologous end joining (NHEJ) during DNA double-strand break (DSB) repair. It promotes c-NHEJ via helicase and exonuclease activities and inhibits alt-NHEJ using non-enzymatic functions. When WRN is recruited to the DSBs it suppresses the recruitment of MRE11 and CtIP, and protects the DSBs from 5' end resection. Moreover, knockdown of Wrn, alone or in combination with Trf2 in mouse embryonic fibroblasts results in increased telomere fusions, which were ablated by Ctip knockdown. We show that WRN regulates alt-NHEJ and shields DSBs from MRE11/CtIP-mediated resection to prevent large deletions and telomere fusions.

NAD+ Replenishment Improves Lifespan and Healthspan in Ataxia Telangiectasia Models via Mitophagy and DNA Repair.

Ataxia telangiectasia (A-T) is a rare autosomal recessive disease characterized by progressive neurodegeneration and cerebellar ataxia. A-T is causally linked to defects in ATM, a master regulator of the response to and repair of DNA double-strand breaks. The molecular basis of cerebellar atrophy and neurodegeneration in A-T patients is unclear. Here we report and examine the significance of increased PARylation, low NAD+, and mitochondrial dysfunction in ATM-deficient neurons, mice, and worms. Treatments that replenish intracellular NAD+ reduce the severity of A-T neuropathology, normalize neuromuscular function, delay memory loss, and extend lifespan in both animal models. Mechanistically, treatments that increase intracellular NAD+ also stimulate neuronal DNA repair and improve mitochondrial quality via mitophagy. This work links two major theories on aging, DNA damage accumulation, and mitochondrial dysfunction through nuclear DNA damage-induced nuclear-mitochondrial signaling, and demonstrates that they are important pathophysiological determinants in premature aging of A-T, pointing to therapeutic interventions.