in-vivo-calcium-imaging-neuronal-ensembles-networks-primary-sensory

<em>In Vivo</em> Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Trigeminal Ganglia

Calcium imaging is an important tool for monitoring signaling mechanisms that either influx Ca2+ into the cytosol or release Ca2+ from intracellular Ca2+ ...

University of Alabama at Birmingham

Incheon National University

The human FAM190A gene undergoes frequent alteration in human cancer, most commonly involving in-frame deletions in exon 9 or exons 9 & 10. These deletions form novel peptide sequences, serving as presumptive cancer-specific neo antigens. However, it remains elusive whether these in-frame deletions of FAM190A could induce oncogenic properties in vivo. In this study, we aimed to explore the functional significance of in-frame deletions in FAM190A genes. We generated two deletion mutant forms, FAM190A and FAM190A, and examined their gain-of-function effects in vitro and in vivo. Global transcript profiling in NIH3T3 cells revealed that the transcripts displaying altered expression following introduction of FAM190A and FAM190A were significantly enriched for genes assigned to cellular movement and cell-to-cell signaling, respectively. Furthermore, ectopic expression of FAM190A and FAM190A induced in vivo tumor formation in nu/nu mice. Taken together, our results are the first to demonstrate the in vivo oncogenic properties of in-frame deletions in the FAM190A gene and indicate that these transcript variants might be clinically applicable as therapeutic targets in patients with cancer.

Functional evaluation of alternative splicing in the FAM190A gene.

Senescence is defined as irreversible cell cycle arrest and constitutes a major driving force in diseases related to aging or premature aging. Recent studies indicate that activation of the serine/threonine protein kinase B-raf (BRAF) plays important roles in oncogene-induced senescence. However, it remains elusive whether BRAF inhibition might be effective for abrogating senescence. In this study, we assessed several BRAF inhibitors to identify compounds that ameliorate senescence and revealed SB590885 as an effective agent. Senescence-ameliorating effect upon BRAF inhibition was evident from the observation that SB590885 treatment increased cellular proliferation but diminished senescent phenotypes. Moreover, BRAF inhibition induced the mitochondrial functional recovery along with the metabolic reprogramming, which comprises two salient features that are altered in senescent cells. Furthermore, mitochondrial metabolic reprogramming via BRAF inhibition was a prerequisite for senescence amelioration. Taken together, our data revealed a novel mechanism in which senescence amelioration is mediated by mitochondrial metabolic reprogramming upon BRAF inhibition.

Mitochondrial metabolic reprograming via BRAF inhibition ameliorates senescence.

Previously, we uncovered a novel mechanism in which senescence is controlled by mitochondrial functional recovery upon Ataxia-telangiectasia mutated (ATM) inhibition. However, it remains elusive how ATM controls signaling pathways to achieve restorative effect. In this study, we performed microarray and found that p53 pathway was differentially expressed upon ATM inhibition. We found that ATM inhibition yields senescence amelioration through p53-dependent manner. The restorative effect was also afforded by direct p53 inhibition. Furthermore, mitochondrial metabolic reprogramming via p53 inhibition was a prerequisite for senescence amelioration. Taken together, our data indicated that p53 pathway functions as potential target for ATM-mediated senescence amelioration.

ATM mediated-p53 signaling pathway forms a novel axis for senescence control.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for  autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).

Senescence is a phenomenon defined by alterations in cellular organelles and is the primary cause of aging and aging-related diseases. Recent studies have shown that oncogene-induced senescence is driven by activation of serine/threonine protein kinases (AKT1, AKT2 and AKT3). In this study, we evaluated twelve AKT inhibitors and revealed GDC0068 as a potential agent to ameliorate senescence. Senescence-ameliorating effect was evident from the finding that GDC0068 yielded lysosomal functional recovery as observed by reduction in lysosomal mass and induction in autophagic flux. Furthermore, GDC0068-mediated restoration of lysosomal function activated the removal of dysfunctional mitochondria, resulting in restoration of mitochondrial function. Together, our findings revealed a unique mechanism by which senescence is recovered by functional restoration of lysosomes and mitochondria through modulation of AKT activity.

Functional restoration of lysosomes and mitochondria through modulation of AKT activity ameliorates senescence.

The establishment of cell lines with a high protein production is the most crucial objective in the field of biopharmaceuticals. To this end, efforts have been made to increase transgene expression through promoter improvement, but the efficiency or stability of protein production was insufficient for use in commercial production. Here, we developed a novel strategy to increase the efficiency and stability of protein production by hybridizing a promoter that exhibits higher expression levels at the transient level with a promoter that exhibits higher stability at the stable level. Expression levels of transgenes by each promoter were measured at transient and stable levels for five single promoters: Rous sarcoma virus (RSV), cytomegalovirus (CMV), human phosphoglycerate kinase (hPGK), simian virus 40 (SV40), and zebrafish ubiquitin B (Ubb). The hPGK promoter enabled high-yield transgene expression at transient levels and the SV40 promoter enabled sustained expression at stable levels. Therefore, hPGK and SV40 promoters were selected as candidates for establishing hybrid promoters and two hybrid promoters were constructed; one hybrid promoter in which the SV40 promoter is added before the hPGK promoter (a.k.a. SKYI) and the other hybrid promoter in which the SV40 promoter is added after the hPGK promoter (a.k.a. SKYII). Of the two hybrid promoters, the hybrid promoter SKYII promoted high-yield transgene expression at both transient and stable levels compared to single hPGK and SV40. Together, our findings open new doors in the field of biopharmaceuticals by presenting a novel promoter platform that can be used for high-yield and sustained protein production.

A novel hybrid promoter capable of continuously producing proteins in high yield.

One of the major factors inducing senescence is reactive oxygen species (ROS) produced from dysfunctional mitochondria. Therapeutic strategies that reduce mitochondrial ROS generation are considered essential for rejuvenating senescence, but effective methods have not yet been established. Here, we screened phenylpropanoids (PPs), secondary metabolites produced in response to oxidative stress in plants, and identified dehydroacteoside as a potential candidate. Dehydroacteoside restored mitochondrial function, thereby reducing mitochondrial ROS generated by inefficient electron transport. Furthermore, senescence-associated phenotypes were restored by dehydroacteoside-mediated ROS reduction. Using RNA sequencing, we identified TVP23C-CDRT4 as a gene that plays a critical role in dehydroacteoside-mediated senescence rejuvenation. Knockdown of TVP23C-CDRT4 showed similar effects to dehydroacteoside, reducing ROS and subsequently restoring senescence-associated phenotypes. Taken together, our study uncovered a novel mechanism by which dehydroacteoside reduces mitochondrial ROS generation, thereby restoring senescence. Our findings open the way to a new field of anti-aging therapy aimed at controlling senescence by modulating ROS production in mitochondria.

Article	Year
Functional evaluation of alternative splicing in the FAM190A gene. Genes & genomics\| PubMed ID: 30298359	2019
Mitochondrial metabolic reprograming via BRAF inhibition ameliorates senescence. Experimental gerontology\| PubMed ID: 31421186	2019
ATM mediated-p53 signaling pathway forms a novel axis for senescence control. Mitochondrion\| PubMed ID: 32949791	2020
Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition). Autophagy\| PubMed ID: 33634751	2021
Functional restoration of lysosomes and mitochondria through modulation of AKT activity ameliorates senescence. Experimental gerontology\| PubMed ID: 36657533	2023
A novel hybrid promoter capable of continuously producing proteins in high yield. Biochemical and biophysical research communications\| PubMed ID: 36774687	2023
Dehydroacteoside rejuvenates senescence via TVP23C-CDRT4 regulation. Experimental gerontology\| PubMed ID: 40472904	2025

Joon Tae Park

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