Fluid flow-induced activation of subcellular AMPK and its interaction with FAK and Src.

AMP-activated protein kinase (AMPK) is a metabolic energy sensor that plays a critical role in cancer cell survival and growth. While the physical microenvironment is believed to influence tumor growth and progression, its role in AMPK regulation remains largely unknown. In the present study, we evaluated AMPK response to mechanical forces and its interaction with other mechano-responsive signaling proteins, FAK and Src. Using genetically encoded biosensors that can detect AMPK activities at different subcellular locations (cytosol, plasma membrane, nucleus, mitochondria, and Golgi apparatus), we observed that AMPK responds to shear stress in a subcellular location-dependent manner in breast cancer cells (MDA-MB-231). While normal epithelial cells (MCF-10A) also similarly responded to shear stress, they are less sensitive to shear stress compared to MDA-MB-231 cells. Inhibition of FAK and Src significantly decreased the basal activity level of AMPK at all five subcellular locations in MDA-MB-231 cells and selectively blocked shear stress-induced AMPK activation. Moreover, testing with cytoskeletal drugs revealed that myosin II might be the critical mediator of shear stress-induced AMPK activation in MDA-MB-231 cells. These findings suggest that breast cancer cells and normal epithelial cells may have different mechanosensitivity in AMPK signaling and that FAK and Src as well as the myosin II-dependent signaling pathway are involved in subcellular AMPK mechanotransduction in breast cancer cells.

Safety and efficacy of deferiprone for pantothenate kinase-associated neurodegeneration: a randomised, double-blind, controlled trial and an open-label extension study.

BACKGROUND: Pantothenate kinase-associated neurodegeneration (PKAN) is a rare genetic disorder characterised by progressive generalised dystonia and brain iron accumulation. We assessed whether the iron chelator deferiprone can reduce brain iron and slow disease progression. METHODS: We did an 18-month, randomised, double-blind, placebo-controlled trial (TIRCON2012V1), followed by a pre-planned 18-month, open-label extension study, in patients with PKAN in four hospitals in Germany, Italy, England, and the USA. Patients aged 4 years or older with a genetically confirmed diagnosis of PKAN, a total score of at least 3 points on the Barry-Albright Dystonia (BAD) scale, and no evidence of iron deficiency, neutropenia, or abnormal hepatic or renal function, were randomly allocated (2:1) to receive an oral solution of either deferiprone (30 mg/kg per day divided into two equal doses) or placebo for 18 months. Randomisation was done with a centralised computer random number generator and with stratification based on age group at onset of symptoms. Patients were allocated to groups by a randomisation team not masked for study intervention that was independent of the study. Patients, caregivers, and investigators were masked to treatment allocation. Co-primary endpoints were the change from baseline to month 18 in the total score on the BAD scale (which measures severity of dystonia in eight body regions) and the score at month 18 on the Patient Global Impression of Improvement (PGI-I) scale, which is a patient-reported interpretation of symptom improvement. Efficacy analyses were done on all patients who received at least one dose of the study drug and who provided a baseline and at least one post-baseline efficacy assessment. Safety analyses were done for all patients who received at least one dose of the study drug. Patients who completed the randomised trial were eligible to enrol in a single-arm, open-label extension study of another 18 months, in which all participants received deferiprone with the same regimen as the main study. The trial was registered on ClinicalTrials.gov, number NCT01741532, and EudraCT, number 2012-000845-11. FINDINGS: Following a screening of 100 prospective patients, 88 were randomly assigned to the deferiprone group (n=58) or placebo group (n=30) between Dec 13, 2012, and April 21, 2015. Of these, 76 patients completed the study (49 in the deferiprone group and 27 in the placebo group). After 18 months, the BAD score worsened by a mean of 2·48 points (SE 0·63) in patients in the deferiprone group versus 3·99 points (0·82) for patients in the control group (difference -1·51 points, 95% CI -3·19 to 0·16, p=0·076). No subjective change was detected as assessed by the PGI-I scale: mean scores at month 18 were 4·6 points (SE 0·3) for patients in the deferiprone group versus 4·7 points (0·4) for those in the placebo group (p=0·728). In the extension study, patients continuing deferiprone retained a similar rate of disease progression as assessed by the BAD scale (1·9 points [0·5] in the first 18 months vs 1·4 points [0·4] in the second 18 months, p=0·268), whereas progression in patients switching from placebo to deferiprone seemed to slow (4·4 points [1·1] vs 1·4 points [0·9], p=0·021). Patients did not detect a change in their condition after the additional 18 months of treatment as assessed by the PGI-I scale, with mean scores of 4·1 points [0·2] in the deferiprone-deferiprone group and of 4·7 points [0·3] in the placebo-deferiprone group. Deferiprone was well tolerated and adverse events were similar between the treatment groups, except for anaemia, which was seen in 12 (21%) of 58 patients in the deferiprone group, but was not seen in any patients in the placebo group. No patient discontinued therapy because of anaemia, and three discontinued because of moderate neutropenia. There was one death in each group of the extension study and both were secondary to aspiration. Neither of these events was considered related to deferiprone use. INTERPRETATION: Deferiprone was well tolerated, achieved target engagement (lowering of iron in the basal ganglia), and seemed to somewhat slow disease progression at 18 months, although not significantly, as assessed by the BAD scale. These findings were corroborated by the results of an additional 18 months of treatment in the extension study. The subjective PGI-I scale was largely unchanged during both study periods, indicating that might not be an adequate tool for assessment of disease progression in patients with PKAN. Our trial provides the first indication of a decrease in disease progression in patients with neurodegeneration with brain iron accumulation. The extensive information collected and long follow-up of patients in the trial will improve the definition of appropriate endpoints, increase the understanding of the natural history, and thus help to shape the design of future trials in this ultra-orphan disease. FUNDING: European Commission, US Food and Drug Administration, and ApoPharma Inc.

Mechanotransduction of mitochondrial AMPK and its distinct role in flow-induced breast cancer cell migration.

The biophysical microenvironment of the tumor site has significant impact on breast cancer progression and metastasis. The importance of altered mechanotransduction in cancerous tissue has been documented, yet its role in the regulation of cellular metabolism and the potential link between cellular energy and cell migration remain poorly understood. In this study, we investigated the role of mechanotransduction in AMP-activated protein kinase (AMPK) activation in breast cancer cells in response to interstitial fluid flow (IFF). Additionally, we explored the involvement of AMPK in breast cancer cell migration. IFF was applied to the 3D cell-matrix construct. The subcellular signaling activity of Src, FAK, and AMPK was visualized in real-time using fluorescent resonance energy transfer (FRET). We observed that breast cancer cells (MDA-MB-231) are more sensitive to IFF than normal epithelial cells (MCF-10A). AMPK was activated at the mitochondria of MDA-MB-231 cells by IFF, but not in other subcellular compartments (i.e., cytosol, plasma membrane, and nucleus). The inhibition of FAK or Src abolished flow-induced AMPK activation in the mitochondria of MDA-MB-231 cells. We also observed that global AMPK activation reduced MDA-MB-231 cell migration. Interestingly, specific AMPK inhibition in the mitochondria reduced cell migration and blocked flow-induced cell migration. Our results suggest the linkage of FAK/Src and mitochondria-specific AMPK in mechanotransduction and the differential role of AMPK in breast cancer cell migration depending on its subcellular compartment-specific activation.

Monitoring clinical progression with mitochondrial disease biomarkers.

Mitochondrial disorders are genetically determined metabolic diseases due to a biochemical deficiency of the respiratory chain. Given that multi-system involvement and disease progression are common features of mitochondrial disorders they carry substantial morbidity and mortality. Despite this, no disease-modifying treatments exist with clear clinical benefits, and the current best management of mitochondrial disease is supportive. Several therapeutic strategies for mitochondrial disorders are now at a mature preclinical stage. Some are making the transition into early-phase patient trials, but the lack of validated biomarkers of disease progression presents a challenge when developing new therapies for patients. This update discusses current biomarkers of mitochondrial disease progression including metabolomics, circulating serum markers, exercise physiology, and both structural and functional imaging. We discuss the advantages and disadvantages of each approach, and consider emerging techniques with a potential role in trials of new therapies.

Subcellular domain-dependent molecular hierarchy of SFK and FAK in mechanotransduction and cytokine signaling.

Focal adhesion kinase (FAK) and Src family kinases (SFK) are known to play critical roles in mechanotransduction and other crucial cell functions. Recent reports indicate that they reside in different microdomains of the plasma membrane. However, little is known about their subcellular domain-dependent roles and responses to extracellular stimuli. Here, we employed fluorescence resonance energy transfer (FRET)-based biosensors in conjunction with collagen-coupled agarose gels to detect subcellular activities of SFK and FAK in three-dimensional (3D) settings. We observed that SFK and FAK in the lipid rafts and nonrafts are differently regulated by fluid flow and pro-inflammatory cytokines. Inhibition of FAK in the lipid rafts blocked SFK response to fluid flow, while inhibition of SFK in the non-rafts blocked FAK activation by the cytokines. Ex-vivo FRET imaging of mouse cartilage explants showed that intermediate level of interstitial fluid flow selectively decreased cytokine-induced SFK/FAK activation. These findings suggest that SFK and FAK exert distinctive molecular hierarchy depending on their subcellular location and extracellular stimuli.

Cardiac involvement in hereditary myopathy with early respiratory failure: A cohort study.

OBJECTIVE: To assess whether hereditary myopathy with early respiratory failure (HMERF) due to the c.951434T>C; (p.Cys31712Arg) TTN missense mutation also includes a cardiac phenotype. METHOD: Clinical cohort study of our HMERF cohort using ECG, 2D echocardiogram, and cross-sectional cardiac imaging with MRI or CT. RESULTS: We studied 22 participants with the c.951434T>C; (p.Cys31712Arg) TTN missense mutation. Three were deceased. Cardiac conduction abnormalities were identified in 7/22 (32%): sustained atrioventricular tachycardia (n = 2), atrial fibrillation (n = 2), nonsustained atrial tachycardia (n = 1), premature supraventricular complexes (n = 1), and unexplained sinus bradycardia (n = 1). In addition, 4/22 (18%) had imaging evidence of otherwise unexplained cardiomyopathy. These findings are supported by histopathologic correlation suggestive of myocardial cytoskeletal remodeling. CONCLUSIONS: Coexisting cardiac and skeletal muscle involvement is not uncommon in patients with HMERF arising due to the c.951434T>C; (p.Cys31712Arg) TTN mutation. All patients with pathogenic or putative pathogenic TTN mutations should be offered periodic cardiac surveillance.

Mitochondrial Causes of Epilepsy: Evaluation, Diagnosis, and Treatment.

Mitochondrial disorders are frequently associated with seizures. In this review, the authors discuss the seizure patterns and distinguishing features of mitochondrial epilepsy, alongside the indications for investigating, and how to investigate epilepsy from a mitochondrial perspective. Finally, they discuss management strategies for this complex group of patients.