Citations

The study establishes the presence of a robust mitophagic response to acute mitochondrial depolarization in hASM cells mediated through the canonical PINK1-Parkin pathway. By integrating quantitative mitochondrial labeling and biochemical analyses, we demonstrate increased mitochondrial damage and remodeling, accompanied by mitochondrial PINK1 accumulation and Parkin activation, resulting in ubiquitin-dependent signaling and lysosomal engulfment. These findings identify mitophagy as a critical adaptive mechanism for maintaining mitochondrial quality control in airway smooth muscle cells during metabolic stress.

Abstract Background Although novel treatments targeting amyloid‐beta (Aβ) have been recently approved by the FDA, there remains a critical need for alternative therapeutic strategies to delay the onset and progression of Alzheimer's disease (AD). Mitochondrial dysfunction and disrupted brain energy homeostasis are key hallmarks of early‐stage AD. Developing treatments that address these deficits could offer substantial benefits. We identified mitochondrial complex I (mtCI) as a druggable target and demonstrated that its mild inhibition using the small molecule CP2 improves brain energy homeostasis, restores synaptic and cognitive function, and activates neuroprotective signaling in AD mouse models. However, the mechanistic link between mtCI inhibition, glucose metabolism, and energy homeostasis remains unclear. Methods Cellular energy metabolism was assessed in neuroblastoma SH‐SY5Y cells expressing mutant human APP protein (APPswe) and in control cells. Glycolytic function, mitochondrial energetics, and fatty acid β‐oxidation (FAO) were measured using a Seahorse Analyzer. Glucose transporter (GLUT) translocation to the cell surface was evaluated using flow cytometry and immunofluorescence staining. Protein expression changes were analyzed via Western blot, and glucose uptake was quantified with the Glucose Uptake‐Glo™ assay. Additionally, metabolic flux analysis using 13C‐labeled glucose and targeted metabolomics by mass spectrometry were performed to measure specific metabolites. Results APPswe cells displayed significant reductions in glycolysis and spare respiratory capacity, indicative of impaired mitochondrial energy production under stress. These deficits were accompanied by reduced glucose uptake, which was partially compensated by an increase in FAO. At concentrations relevant to in vivo treatments, CP2 acutely enhanced GLUT translocation to the cell surface, increasing glucose uptake within one hour. Enhanced glucose utilization in OXPHOS machinery was confirmed by inhibition of PDH phosphorylation and increased conversion of pyruvate to acetyl‐CoA, fueling the TCA cycle and generating critical signaling metabolites essential for gene regulation. These mechanisms were mediated by AMPK. Conclusion Our findings suggest that mild inhibition of mtCI activates multiple neuroprotective mechanisms, including improved glucose metabolism and energy homeostasis. These consistent results in human cells and AD mouse models highlight the translational potential of this therapeutic approach for AD.

ABSTRACT Metastatic cancer to the pancreas is uncommon, accounting for only 2%–4% of pancreatic malignancies. Prostate cancer spreading to the pancreas is exceptionally rare. We present 2 cases of metastatic castration-resistant prostate cancer with histologic transformation from adenocarcinoma to neuroendocrine prostate cancer. Both patients developed new pancreatic lesions, in which endoscopic ultrasound-guided fine-needle biopsy confirmed the diagnosis. These cases highlight therapy-emergent neuroendocrine prostate cancer with loss of prostate lineage markers (prostate-specific antigen, NKX3.1), positive neuroendocrine markers (synaptophysin, INSM1, chromogranin), very high proliferative indices, and serum marker discordance that can mimic primary pancreatic cancer. These scenarios present a diagnostic challenge with an aggressive and complex nature, requiring multidisciplinary management. With newer therapies and prolonged survival of patients with prostate cancer, clinicians will likely encounter more cases in the future. Gastroenterologists can play a key role in diagnosis and palliative treatment.

Mitochondrial volume and quality are tightly regulated by two key mechanisms: removal of dysfunctional/damaged mitochondria by selective autophagy (mitophagy) and mitochondrial biogenesis. Disruption of either one or both processes results in the accumulation of dysfunctional mitochondria and increased oxidative stress. Emerging evidence suggest that mitophagy and mitochondrial biogenesis are tightly coordinated, working in concert to maintain mitochondrial homeostasis and quality control. Previously, we reported that tumor necrosis factor α (TNFα), a proinflammatory cytokine produced during inflammatory airway disease, promotes mitochondrial biogenesis by enhancing peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) signaling, the nodal regulator of mitochondrial biogenesis, thereby increasing mitochondrial volume density in human airway smooth muscle (hASM) cells. Additional evidence indicates that TNFα induces mitochondrial depolarization, which is known to trigger a mitophagy pathway involving PTEN-induced putative kinase 1 (PINK1) and Parkin, an E3 ubiquitin ligase. Mitochondrial depolarization facilitates the mitochondrial accumulation of PINK1 and its activation by autophosphorylation at ser228 (pPINK1 s228 ). pPINK1 s228 promotes the recruitment and phosphorylation of Parkin at ser65 (pParkin s65 ), which ubiquitylates several mitochondrial associated proteins promoting the removal of damaged mitochondria. The decrease in the number of functional mitochondria by mitophagy can be offset by increasing mitochondrial biogenesis through the upregulation of PGC-1α signaling. Therefore, we hypothesize that PARIS (ZNF746), a transcriptional repressor of PGC-1α, is a target of PINK1/Parkin-mediated ubiquitination and degradation in hASM cells. In the present study, primary hASM cells were isolated from bronchiolar tissue of 6 subjects with no history of chronic pulmonary disease or smoking and separated into the following treatment groups: 1) FCCP (1 µM, 6 h; positive control), a proton ionophore that induces mitochondrial depolarization; 2) TNFα (20 ng/mL, 6 h); and 3) untreated control. We observed that FCCP and TNFα-induced mitochondrial depolarization triggered mitochondrial translocation of PINK1 and increased phosphorylation of pPINK1 s228 . Additionally, phosphorylation of pParkin s65 and ubiquitination of several mitochondrial associated proteins was increased. Immunoprecipitation of ubiquitinated proteins revealed increased ubiquitination of PARIS, while total expression of PARIS was decreased. Mitochondria and lysosomes were labelled with MitoTracker green (200 nM) and LysoTracker red (100 nM), respectively, and imaged in 3D (Z optical slice of 0.5 μm) to demonstrate the coefficient for colocalization of mitochondria with lysosomes. We observed increased formation of mitophagosomes, characteristic of mitophagy. Our findings demonstrate that TNFα-induces both mitophagy and mitochondrial biogenesis via the activation of PINK1/Parkin-mediated mitophagy pathway. Such coordination of mitophagy and mitochondrial biogenesis has significant ramifications for the maintenance of mitochondrial homeostasis, quality control and cellular energy metabolism. Supported by NIH grant HL157984 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

INTRODUCTION: Patients with inflammatory bowel disease (IBD) are at an increased risk of infections. Before the COVID-19 pandemic, respiratory syncytial virus (RSV) followed predictable seasonal patterns, which have been recently disrupted. This study aimed to investigate whether severe acute respiratory syndrome (SARS) coronavirus 2 (CoV-2) infection is associated with an increased risk of RSV infection in patients with IBD compared with those without a history of SARS-CoV-2 infection. METHODS: This retrospective cohort study used the TriNetX database to identify patients aged 18 years and older with IBD and SARS-CoV-2 infection (IBD-SARS-CoV-2 cohort) during the 2022 and 2023 RSV seasons. A 1:1 propensity score matching was used to compare patients with IBD but no history of SARS-CoV-2 infection (IBD non-SARS-CoV-2 cohort). RESULTS: In the 2022 IBD-SARS-CoV-2 cohort (mean age: 53.7 ± 17.6 years; 59% female; 77% White), the RSV infection risk was 0.47%, higher than 0.19% in the matched IBD non-SARS-CoV-2 cohort (adjusted odds ratio [aOR]: 2.4; 95% CI: 1.5–3.6). The risk was highest 30–60 days after SARS-CoV-2 infection (aOR: 2.9; 95% CI: 1.7–4.9), particularly in those aged 60 years and older (aOR: 2.5; 95% CI: 1.3–4.5). The use of systemic corticosteroids (aOR: 2.3; 95% CI: 1.1–4.6) or immune-modifying therapies (aOR: 3.9; 95% CI: 2–7.1) was associated with higher RSV infection risk. Similar trends were observed during the 2023 RSV season, with no significant differences in RSV-related hospitalizations. DISCUSSION: Adults with IBD have a higher risk of RSV infection after SARS-CoV-2 infection, particularly those receiving steroids or immune therapies. SARS-CoV-2 infection may have contributed to the recent RSV infection surge in this population, warranting further research.

INTRODUCTION: Studies suggest that the generation of durable T-cell immunity following coronavirus disease 2019 (COVID-19) vaccination protects against severe disease. The aim of this study was to measure cell-mediated immune response (CMIR) 1–2 months and 6 months after a third dose of a COVID-19 mRNA vaccine. METHODS: This prospective study (HumoRal and CellULar initial and Sustained immunogenicity in patients with inflammatory bowel disease [IBD]) evaluated CMIR at 28–65 days (t 1 ) after dose 2, 28–65 days (t 2 ) (n = 183) and 6 months (±45 days) (t 3 ) (n = 167) after a third dose of an mRNA COVID-19 vaccine. A small cohort had blood sample available 28–65 days (t 4 ) (n = 55) after a fourth dose. Primary outcomes were CMIR at (t 2 ) and (t 3 ). Secondary outcomes included the effect of immunosuppressing IBD medications on CMIR and response at (t 4 ). RESULTS: All patients had measurable CMIR at all time points. CMIR increased at t 2 compared with that at t 1 (median 1,467 responding cells per million (interquartile range [IQR] 410–5,971) vs 313 (94–960) P < 0.001). There was no significant waning in t 2 vs t 3 or significant boosting at t 4 . Those on anti–tumor necrosis factor monotherapy had a higher CMIR compared with those not on this therapy at t 2 (4,132 [IQR 1,136–8,795] vs 869 [IQR 343–3,221] P < 0.001) and t 3 (2,843 [IQR 596–6,459] vs 654 [IQR 143–2,067] P < 0.001). In univariable analysis, anti–tumor necrosis factor monotherapy was associated with a higher CMIR at t 2 ( P < 0.001) and t 3 ( P < 0.001) and confirmed in a multivariable model ( P < 0.001). DISCUSSION: A third dose of a COVID-19 vaccine boosts CMIR, and the response is sustained in patients with IBD.

Cellular mitochondrial function can be assessed using high-resolution respirometry that measures the O2 consumption rate (OCR) across a number of cells. However, a direct measurement of cellular mitochondrial function provides valuable information and physiological insight. In the present study, we used a quantitative histochemical technique to measure the activity of succinate dehydrogenase (SDH), a key enzyme located in the inner mitochondrial membrane, which participates in both the tricarboxylic acid (TCA) cycle and electron transport chain (ETC) as Complex II. In this study, we determine the maximum velocity of the SDH reaction (SDHmax) in individual human airway smooth muscle (hASM) cells. To measure SDHmax, hASM cells were exposed to a solution containing 80 mM succinate and 1.5 mM nitroblue tetrazolium (NBT, reaction indicator). As the reaction proceeded, the change in optical density (OD) due to the reduction of NBT to its diformazan (peak absorbance wavelength of 570 nm) was measured using a confocal microscope with the pathlength for light absorbance tightly controlled. SDHmax was determined during the linear period of the SDH reaction and expressed as mmol fumarate/liter of cell/min. We determine that this technique is rigorous and reproducible, and reliable for the measurement of mitochondrial function in individual cells.

Mitophagy is a regulated multistep process of selective removal of dysfunctional/damaged mitochondria by autophagy and is critical for the regulation of mitochondrial quality control in many physiological processes. The best characterized pathway of mitophagy involves PTEN-induced kinase 1 (PINK1) and E3-ubiquitin ligase Parkin. Upon mitochondrial depolarization, PINK1 stabilizes on the outer mitochondrial membrane (OMM) and recruits Parkin. After recruitment, PINK1 phosphorylates Parkin at Ser65, thus activating Parkin which promotes the ubiquitination of OMM proteins resulting in the subsequent lysosomal degradation of the mitochondria. Previously, we reported that tumor necrosis factor α (TNFα), a proinflammatory cytokine produced during inflammatory airway diseases, increases force generation and ATP hydrolysis, and results in increased mitochondrial fragmentation in human airway smooth muscle (hASM) cells. We also showed that TNFα induces PGC1α signaling, thus promoting mitochondrial biogenesis and increased mitochondrial volume density. In the present study, we hypothesize that TNFα induces mitophagy to promote selective clearance of dysfunctional mitochondria, thus maintaining a healthy mitochondrial pool to meet the increased energy demand in hASM cells. Experiments were performed in hASM cells dissociated from bronchiolar tissue samples collected during bronchiolar surgery from female and male donors with no current history of smoking or respiratory diseases. The hASM cells were serum deprived for 48 h and the phenotype was confirmed prior to experimentation. hASM cells were separated into three 6-h treatment groups: 1) TNFα (20 ng/mL), 2) FCCP (5 μM; positive control), and 3) untreated controls. The mitochondria and lysosomes in hASM cells were labelled with MitoTracker green (200 nM) and LysoTracker red (100 nM), respectively and imaged in 3D (Z optical slice of 0.5 μm) using an oil-immersion ×60/1.4 NA objective on a Nikon Eclipse A1 laser scanning confocal system. The 3D images were used to determine the coefficient for colocalization of mitochondria with lysosomes, lysosomal volume density, mitochondrial volume density, and mitochondrial complexity index (MCI). Protein expression and phosphorylation status of PINK1, Parkin, phospho-Parkin, VDAC (voltage-dependent anion channel), and LAMP1 (Lysosomal-associated membrane protein 1) were quantified by western blot. We observed that TNFα and FCCP treatment increased lysosomal volume density and mitochondrial colocalization with lysosomes in hASM cells, indicating the formation of mitophagosomes, characteristic of mitophagy. Consistent with previous findings, TNFα treatment increased mitochondrial volume density and decreased MCI, indicative of increased mitochondrial fragmentation. In addition, TNFα treatment increased the expression and mitochondrial translocation of PINK1 and Parkin. The results suggest that TNFα activates PINK1/Parkin-mediated mitophagy in hASM cells. Supported by NIH grants HL157984 (GCS) and HL105355 (OARR) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.