Loss of Mitochondrial Tusc2/Fus1 Triggers a Brain Pro-Inflammatory Microenvironment and Early Spatial Memory Impairment.

Brain pathological changes impair cognition early in disease etiology. There is an urgent need to understand aging-linked mechanisms of early memory loss to develop therapeutic strategies and prevent the development of cognitive impairment. Tusc2 is a mitochondrial-resident protein regulating Ca2+ fluxes to and from mitochondria impacting overall health. We previously reported that Tusc2-/- female mice develop chronic inflammation and age prematurely, causing age- and sex-dependent spatial memory deficits at 5 months old. Therefore, we investigated Tusc2-dependent mechanisms of memory impairment in 4-month-old mice, comparing changes in resident and brain-infiltrating immune cells. Interestingly, Tusc2-/- female mice demonstrated a pro-inflammatory increase in astrocytes, expression of IFN-γ in CD4+ T cells and Granzyme-B in CD8+T cells. We also found fewer FOXP3+ T-regulatory cells and Ly49G+ NK and Ly49G+ NKT cells in female Tusc2-/- brains, suggesting a dampened anti-inflammatory response. Moreover, Tusc2-/- hippocampi exhibited Tusc2- and sex-specific protein changes associated with brain plasticity, including mTOR activation, and Calbindin and CamKII dysregulation affecting intracellular Ca2+ dynamics. Overall, the data suggest that dysregulation of Ca2+-dependent processes and a heightened pro-inflammatory brain microenvironment in Tusc2-/- mice could underlie cognitive impairment. Thus, strategies to modulate the mitochondrial Tusc2- and Ca2+- signaling pathways in the brain should be explored to improve cognitive health.

Tubular CPT1A deletion minimally affects aging and chronic kidney injury.

Kidney tubules use fatty acid oxidation (FAO) to support their high energetic requirements. Carnitine palmitoyltransferase 1A (CPT1A) is the rate-limiting enzyme for FAO, and it is necessary to transport long-chain fatty acids into mitochondria. To define the role of tubular CPT1A in aging and injury, we generated mice with tubule-specific deletion of Cpt1a (Cpt1aCKO mice), and the mice were either aged for 2 years or injured by aristolochic acid or unilateral ureteral obstruction. Surprisingly, Cpt1aCKO mice had no significant differences in kidney function or fibrosis compared with wild-type mice after aging or chronic injury. Primary tubule cells from aged Cpt1aCKO mice had a modest decrease in palmitate oxidation but retained the ability to metabolize long-chain fatty acids. Very-long-chain fatty acids, exclusively oxidized by peroxisomes, were reduced in kidneys lacking tubular CPT1A, consistent with increased peroxisomal activity. Single-nuclear RNA-Seq showed significantly increased expression of peroxisomal FAO enzymes in proximal tubules of mice lacking tubular CPT1A. These data suggest that peroxisomal FAO may compensate in the absence of CPT1A, and future genetic studies are needed to confirm the role of peroxisomal ß-oxidation when mitochondrial FAO is impaired.

Pharmacological Modulation of Energy and Metabolic Pathways Protects Hearing in the Fus1/Tusc2 Knockout Model of Mitochondrial Dysfunction and Oxidative Stress.

Tightly regulated and robust mitochondrial activities are critical for normal hearing. Previously, we demonstrated that Fus1/Tusc2 KO mice with mitochondrial dysfunction exhibit premature hearing loss. Molecular analysis of the cochlea revealed hyperactivation of the mTOR pathway, oxidative stress, and altered mitochondrial morphology and quantity, suggesting compromised energy sensing and production. Here, we investigated whether the pharmacological modulation of metabolic pathways using rapamycin (RAPA) or 2-deoxy-D-glucose (2-DG) supplementation can protect against hearing loss in female Fus1 KO mice. Additionally, we aimed to identify mitochondria- and Fus1/Tusc2-dependent molecular pathways and processes critical for hearing. We found that inhibiting mTOR or activating alternative mitochondrial energetic pathways to glycolysis protected hearing in the mice. Comparative gene expression analysis revealed the dysregulation of critical biological processes in the KO cochlea, including mitochondrial metabolism, neural and immune responses, and the cochlear hypothalamic-pituitary-adrenal axis signaling system. RAPA and 2-DG mostly normalized these processes, although some genes showed a drug-specific response or no response at all. Interestingly, both drugs resulted in a pronounced upregulation of critical hearing-related genes not altered in the non-treated KO cochlea, including cytoskeletal and motor proteins and calcium-linked transporters and voltage-gated channels. These findings suggest that the pharmacological modulation of mitochondrial metabolism and bioenergetics may restore and activate processes critical for hearing, thereby protecting against hearing loss.

Tubular ß-catenin and FoxO3 interactions protect in chronic kidney disease.

The Wnt/ß-catenin signaling pathway plays an important role in renal development and is reexpressed in the injured kidney and other organs. ß-Catenin signaling is protective in acute kidney injury (AKI) through actions on the proximal tubule, but the current dogma is that Wnt/ß-catenin signaling promotes fibrosis and development of chronic kidney disease (CKD). As the role of proximal tubular ß-catenin signaling in CKD remains unclear, we genetically stabilized (i.e., activated) ß-catenin specifically in murine proximal tubules. Mice with increased tubular ß-catenin signaling were protected in 2 murine models of AKI to CKD progression. Oxidative stress, a common feature of CKD, reduced the conventional T cell factor/lymphoid enhancer factor-dependent ß-catenin signaling and augmented FoxO3-dependent activity in proximal tubule cells in vitro and in vivo. The protective effect of proximal tubular ß-catenin in renal injury required the presence of FoxO3 in vivo. Furthermore, we identified cystathionine γ-lyase as a potentially novel transcriptional target of ß-catenin/FoxO3 interactions in the proximal tubule. Thus, our studies overturned the conventional dogma about ß-catenin signaling and CKD by showing a protective effect of proximal tubule ß-catenin in CKD and identified a potentially new transcriptional target of ß-catenin/FoxO3 signaling that has therapeutic potential for CKD.

Characterization of genetically engineered mouse hepatoma cells with inducible liver functions by overexpression of liver-enriched transcription factors.

New cell sources for the research and therapy of organ failure could significantly alleviate the shortage of donor livers that are available to patients who suffer from liver disease. Liver carcinoma derived cells, or hepatoma cells, are the ideal cells for developing bioartificial liver systems. Such cancerous liver cells are easy to prepare in large quantities and can be maintained over long periods under standard culture conditions, unlike primary hepatocytes. However, hepatoma cells possess only a fraction of the functions of primary hepatocytes. In a previous study, by transducing cells with liver-enriched transcription factors that could be inducibly overexpressed-hepatocyte nuclear factor (HNF)1α, HNF1ß, HNF3ß [FOXA2], HNF4α, HNF6, CCAAT/enhancer binding protein (C/EBP)α, C/EBPß and C/EBPγ-we created mouse hepatoma cells with high liver-specific gene expression called the Hepa/8F5 cell line. In the present study, we performed functional and genetic analyses to characterize the Hepa/8F5 cell line. Further, in three-dimensional cultures, the function of these cells improved significantly compared to parental cells. Ultimately, these cells might become a new resource that can be used in basic and applied hepatic research.

Enhanced Hepatic Functions of Genetically Modified Mouse Hepatoma Cells by Spheroid Culture for Drug Toxicity Screening.

While hepatic cell lines are mainly used for in vitro drug induced toxicity studies, they exhibit limited functionalities. To overcome this, the authors have employed genetically engineered mouse hepatoma cells, Hepa/8F5, wherein expression of liver enriched transcription factors is induced by doxycycline leading to increased functionality. Further enhancement in functionality is achieved by spheroid culture in a previously developed 3D cell culture platform. Cells are seeded in presence of temperature-responsive poly(N-isopropylacrylamide) on poly(N-isopropylacrylamide--co-gelatin) cryogel scaffold based high throughput platform. Cells seeded in presence of poly(N-isopropylacrylamide) and induced with doxycycline exhibited highest functionalities. There is an increase of ≈26, 36, and 39% in albumin secretion, ammonia removal, and CYP3A4 activity, respectively. Morphological analysis showed arrest in cell proliferation and enlarged nucleus in presence of doxycyline and spheroid formation in presence of poly(N-isopropylacrylamide). Drug induced liver toxicity studies revealed that cells induced with doxycycline are resistive to tamoxifen but sensitive to acetaminophen whereas, cultures initiated in presence of poly(N-isopropylacrylamide) are resistive to both the drugs which is indicative of diffusional barrier of the spheroids. The authors conclude that Hepa/8F5 cells show enhanced functionality in cryogel based spheroid culture platform which can be successfully used for high throughput screening of hepatic toxicity in vitro.

Three-dimensional culture of a genetically modified hepatoma cell line using macroporous gelatin beads.

Hepatoma cells are a candidate cell source for bio-artificial livers. However, they exhibit reduced liver functions compared with primary hepatocytes. In our previous study, genetically engineered mouse hepatoma cells were created by transduction with vectors mediating inducible overexpression of eight liver-enriched transcription factors. Upon the induction of the liver-enriched transcription factors transduced, the cells expressed both phenotypic and genotypic liver functions at high levels. In the present study, we performed three-dimensional culture of these cells using macroporous gelatin beads. When immobilized on the macroporous gelatin beads, these cells exhibited further enhancement in liver functionality, including increased albumin secretion, ammonia removal and cytochrome P450 activity. The levels of these functions were significantly enhanced compared to monolayer culture. The method is simple and scalable, and provides highly functional cells that can be used in basic and applied fields of hepatic research.