Distinguishing post-translational modifications in dominantly inherited frontotemporal dementias: FTLD-TDP Type A (GRN) vs Type B (C9orf72).

AIMS: Frontotemporal dementias are neuropathologically characterised by frontotemporal lobar degeneration (FTLD). Intraneuronal inclusions of transactive response DNA-binding protein 43 kDa (TDP-43) are the defining pathological hallmark of approximately half of the FTLD cases, being referred to as FTLD-TDP. The classification of FTLD-TDP into five subtypes (Type A to Type E) is based on pathologic phenotypes; however, the molecular determinants underpinning the phenotypic heterogeneity of FTLD-TDP are not well known. It is currently undetermined whether TDP-43 post-translational modifications (PTMs) may be related to the phenotypic diversity of the FTLDs. Thus, the investigation of FTLD-TDP Type A and Type B, associated with GRN and C9orf72 mutations, becomes essential. METHODS: Immunohistochemistry was used to identify and map the intraneuronal inclusions. Sarkosyl-insoluble TDP-43 was extracted from brains of GRN and C9orf72 mutation carriers post-mortem and studied by Western blot analysis, immuno-electron microscopy and mass spectrometry. RESULTS: Filaments of TDP-43 were present in all FTLD-TDP preparations. PTM profiling identified multiple phosphorylated, N-terminal acetylated or otherwise modified residues, several of which have been identified for the first time as related to sarkosyl-insoluble TDP-43. Several PTMs were specific for either Type A or Type B, while others were identified in both types. CONCLUSIONS: The current results provide evidence that the intraneuronal inclusions in the two genetic diseases contain TDP-43 filaments. The discovery of novel, potentially type-specific TDP-43 PTMs emphasises the need to determine the mechanisms leading to filament formation and PTMs, and the necessity of exploring the validity and occupancy of PTMs in a prognostic/diagnostic setting.

Folate-targeted intraoperative fluorescence, OTL38, in robotic-assisted laparoscopic partial nephrectomy.

OBJECTIVE: To investigate the safety and efficacy of OTL38, a folate-targeted, intraoperative fluorescence agent, in patients undergoing robotic-assisted laparoscopic partial nephrectomy. METHODS: Patients with proven or suspected localized renal cell carcinoma at a single academic institution were selected from 2016 to 2018. Patients received one dose of OTL38 at 0.025 mg/kg prior to robotic-assisted laparoscopic partial nephrectomy. The da Vinci Fluorescence Imaging Vision System was used to identify the tumor and inspect for residual disease after resection. Immunohistochemistry was performed to quantify folate receptor alpha in both the tumor and surrounding normal parenchyma. Patient follow-up was 1 month. Outcome data included descriptive statistics of the patient cohort and surgeon and pathologist surveys. RESULTS: Ten cases were performed. Mean patient age was 62.9 years (range = 50-70). Mean tumor size was 2.45 cm. Pathologic tumor stages ranged from T1a-T3a. Histologic tumor types included clear cell, chromophobe, type 1 papillary renal cell carcinoma and oncocytoma. The tumors did not fluoresce, while the surrounding normal parenchyma did show fluorescence. No adverse reactions were seen. Staining for folate receptor alpha was localized to the proximal renal tubules. Average staining in normal surrounding renal parenchyma was significantly greater than staining observed in tumor tissue (0.2086 vs 0.0467; p = 0.002). The mean difference in staining between tumor tissue and surrounding normal renal parenchyma was 0.1619 (95% CI = 0.0796-0.2442). CONCLUSIONS: Based on our initial experience, OTL38 shows potential as a safe, effective and easy to use tool to improve visualization and resection of renal tumors.

Aging-associated skeletal muscle defects in HER2/Neu transgenic mammary tumor model.

BACKGROUND: Loss of skeletal muscle volume and resulting in functional limitations are poor prognostic markers in breast cancer patients. Several molecular defects in skeletal muscle including reduced MyoD levels and increased protein turn over due to enhanced proteosomal activity have been suggested as causes of skeletal muscle loss in cancer patients. However, it is unknown whether molecular defects in skeletal muscle are dependent on tumor etiology. METHODS: We characterized functional and molecular defects of skeletal muscle in MMTV-Neu (Neu+) mice (n= 6-12), an animal model that represents HER2+ human breast cancer, and compared the results with well-characterized luminal B breast cancer model MMTV-PyMT (PyMT+). Functional studies such as grip strength, rotarod performance, and ex vivo muscle contraction were performed to measure the effects of cancer on skeletal muscle. Expression of muscle-enriched genes and microRNAs as well as circulating cytokines/chemokines were measured. Since NF-κB pathway plays a significant role in skeletal muscle defects, the ability of NF-κB inhibitor dimethylaminoparthenolide (DMAPT) to reverse skeletal muscle defects was examined. RESULTS: Neu+ mice showed skeletal muscle defects similar to accelerated aging. Compared to age and sex-matched wild type mice, Neu+ tumor-bearing mice had lower grip strength (202±6.9 vs. 179±6.8 g grip force, p=0.0069) and impaired rotarod performance (108±12.1 vs. 30±3.9 seconds, P<0.0001), which was consistent with reduced muscle contractibility (p<0.0001). Skeletal muscle of Neu+ mice (n=6) contained lower levels of CD82+ (16.2±2.9 vs 9.0±1.6) and CD54+ (3.8±0.5 vs 2.4±0.4) muscle stem and progenitor cells (p<0.05), suggesting impaired capacity of muscle regeneration, which was accompanied by decreased MyoD, p53 and miR-486 expression in muscles (p<0.05). Unlike PyMT+ mice, which showed skeletal muscle mitochondrial defects including reduced mitochondria levels and Pgc1ß, Neu+ mice displayed accelerated aging-associated changes including muscle fiber shrinkage and increased extracellular matrix deposition. Circulating "aging factor" and cachexia and fibromyalgia-associated chemokine Ccl11 was elevated in Neu+ mice (1439.56±514 vs. 1950±345 pg/ml, p<0.05). Treatment of Neu+ mice with DMAPT significantly restored grip strength (205±6 g force), rotarod performance (74±8.5 seconds), reversed molecular alterations associated with skeletal muscle aging, reduced circulating Ccl11 (1083.26 ±478 pg/ml), and improved animal survival. CONCLUSIONS: These results suggest that breast cancer subtype has a specific impact on the type of molecular and structure changes in skeletal muscle, which needs to be taken into consideration while designing therapies to reduce breast cancer-induced skeletal muscle loss and functional limitations.

Complementary Embryonic and Adult Cell Populations Enhance Myocardial Repair in Rat Myocardial Injury Model.

We compared the functional outcome of Isl-1+ cardiac progenitors, CD90+ bone marrow-derived progenitor cells, and the combination of the two in a rat myocardial infarction (MI) model. Isl-1+ cells were isolated from embryonic day 12.5 (E12.5) rat hearts and expanded in vitro. Thy-1+/CD90+ cells were isolated from the bone marrow of adult Sprague-Dawley rats by immunomagnetic cell sorting. Six-week-old female Sprague-Dawley rats underwent permanent left anterior descending (LAD) coronary artery ligation and received intramyocardial injection of either saline, Isl-1+ cells, CD90+ cells, or a combination of Isl-1+ and CD90+ cells, at the time of infarction. Cells were delivered transepicardially to the peri-infarct zone. Left ventricular function was assessed by transthoracic echocardiography at 1- and 4-week post-MI and by Millar catheterization (-dP/dt and +dP/dt) at 4-week post-MI. Fluorescence in situ hybridization (Isl-1+cells) and monochrystalline iron oxide nanoparticles labeling (MION; CD90+ cells) were performed to assess biodistribution of transplanted cells. Only the combination of cells demonstrated a significant improvement of cardiac function as assessed by anterior wall contractility, dP/dt (max), and dP/dt (min), compared to Isl-1+ or CD90+ cell monotherapies. In the combination cell group, viable cells were detected at week 4 when anterior wall motion was completely restored. In conclusion, the combination of Isl-1+ cardiac progenitors and adult bone marrow-derived CD90+ cells shows prolonged and robust myocardial tissue repair and provides support for the use of complementary cell populations to enhance myocardial repair.

Blocking HIF signaling via novel inhibitors of CA9 and APE1/Ref-1 dramatically affects pancreatic cancer cell survival.

Pancreatic ductal adenocarcinoma (PDAC) has reactive stroma that promotes tumor signaling, fibrosis, inflammation, and hypoxia, which activates HIF-1α to increase tumor cell metastasis and therapeutic resistance. Carbonic anhydrase IX (CA9) stabilizes intracellular pH following induction by HIF-1α. Redox effector factor-1 (APE1/Ref-1) is a multifunctional protein with redox signaling activity that converts certain oxidized transcription factors to a reduced state, enabling them to upregulate tumor-promoting genes. Our studies evaluate PDAC hypoxia responses and APE1/Ref-1 redox signaling contributions to HIF-1α-mediated CA9 transcription. Our previous studies implicated this pathway in PDAC cell survival under hypoxia. We expand those studies, comparing drug responses using patient-derived PDAC cells displaying differential hypoxic responses in 3D spheroid tumor-stroma models to characterize second generation APE1/Ref-1 redox signaling and CA9 inhibitors. Our data demonstrates that HIF-1α-mediated CA9 induction differs between patient-derived PDAC cells and that APE1/Ref-1 redox inhibition attenuates this induction by decreasing hypoxia-induced HIF-1 DNA binding. Dual-targeting of APE1/Ref-1 and CA9 in 3D spheroids demonstrated that this combination effectively kills PDAC tumor cells displaying drastically different levels of CA9. New APE1/Ref-1 and CA9 inhibitors were significantly more potent alone and in combination, highlighting the potential of combination therapy targeting the APE1-Ref-1 signaling axis with significant clinical potential.