widespread-transduction-mouse-neocortical-neurons-subarachnoid

Widespread Transduction of Mouse Neocortical Neurons by Subarachnoid Injection of AAV2

Recombinant adeno-associated viruses are a flexible and powerful tool for the delivery and expression of various genes of interest in many areas of ...

Russian Academy of Sciences

Several pharmacological, dietary, and genetic interventions that increase mammalian lifespan are known, but general principles of lifespan extension remain unclear. Here, we performed RNA sequencing (RNA-seq) analyses of mice subjected to 8 longevity interventions. We discovered a feminizing effect associated with growth hormone regulation and diminution of sex-related differences. Expanding this analysis to 17 interventions with public data, we observed that many interventions induced similar gene expression changes. We identified hepatic gene signatures associated with lifespan extension across interventions, including upregulation of oxidative phosphorylation and drug metabolism, and showed that perturbed pathways may be shared across tissues. We further applied the discovered longevity signatures to identify new lifespan-extending candidates, such as chronic hypoxia, KU-0063794, and ascorbyl-palmitate. Finally, we developed GENtervention, an app that visualizes associations between gene expression changes and longevity. Overall, this study describes general and specific transcriptomic programs of lifespan extension in mice and provides tools to discover new interventions.

Identification and Application of Gene Expression Signatures Associated with Lifespan Extension.

In the current review, we aim to discuss the principles and the perspectives of using the genetic constructs based on AAV vectors to regulate astrocytes' activity. Practical applications of optogenetic approaches utilizing different genetically encoded opsins to control astroglia activity were evaluated. The diversity of astrocytic cell-types complicates the rational design of an ideal viral vector for particular experimental goals. Therefore, efficient and sufficient targeting of astrocytes is a multiparametric process that requires a combination of specific AAV serotypes naturally predisposed to transduce astroglia with astrocyte-specific promoters in the AAV cassette. Inadequate combinations may result in off-target neuronal transduction to different degrees. Potentially, these constraints may be bypassed with the latest strategies of generating novel synthetic AAV serotypes with specified properties by rational engineering of AAV capsids or using directed evolution approach by searching within a more specific promoter or its replacement with the unique enhancer sequences characterized using modern molecular techniques (ChIP-seq, scATAC-seq, snATAC-seq) to drive the selective transgene expression in the target population of cells or desired brain regions. Realizing these strategies to restrict expression and to efficiently target astrocytic populations in specific brain regions or across the brain has great potential to enable future studies.

Genetic Constructs for the Control of Astrocytes' Activity.

Ischemic cerebral stroke is one of the leading causes of death and disability in humans. However, molecular processes underlying the development of this pathology remain poorly understood. There are major gaps in our understanding of metabolic changes that occur in the brain tissue during the early stages of ischemia and reperfusion. In particular, it is generally accepted that both ischemia (I) and reperfusion (R) generate reactive oxygen species (ROS) that cause oxidative stress which is one of the main drivers of the pathology, although ROS generation during I/R was never demonstrated in vivo due to the lack of suitable methods. In the present study, we record for the first time the dynamics of intracellular pH and HO during I/R in cultured neurons and during experimental stroke in rats using the latest generation of genetically encoded biosensors SypHer3s and HyPer7. We detect a buildup of powerful acidosis in the brain tissue that overlaps with the ischemic core from the first seconds of pathogenesis. At the same time, no significant HO generation was found in the acute phase of ischemia/reperfusion. HyPer7 oxidation in the brain was detected only 24 h later. Comparison of in vivo experiments with studies on cultured neurons under I/R demonstrates that the dynamics of metabolic processes in these models significantly differ, suggesting that a cell culture is a poor predictor of metabolic events in vivo.

In vivo dynamics of acidosis and oxidative stress in the acute phase of an ischemic stroke in a rodent model.

The engineered expression of K channels has been proposed as a potential treatment for epilepsy due to their exceptional ability to hyperpolarize neurons. A number of rodent models of gene therapy have yielded promising outcomes. However, the prevailing viral delivery methods for transgenes lack external control over expression, which may lead to the overproduction of K channel subunits and subsequent adverse effects. AAV-based expression of the KCNN4 gene in excitatory neurons has recently been demonstrated to suppress seizures by decreasing neuronal spiking activity. In this study, we examine the effects of overexpression of KCNN4, a gene encoding a pore-forming subunit of KCa3.1 channels, in neurons and HEK293 cells at the cellular and subcellular levels. We employ patch-clamp electrophysiology, immunocytochemistry, and imaging of tagged channel subunits to gain insights into the consequences of KCNN4 overexpression. Our results show that at higher expression levels, the number of channels at the cell membrane decreases, while the engineered expression of the KCa3.1 channel shows a peak in efficiency. Furthermore, our experiments demonstrate that KCNN4 overexpression results in decreased availability of other channels on the membrane and compromised functionality of other channels of the cells. These findings raise an important issue regarding the potential side effects of channel-based gene therapy for neurological disorders. It is critical to consider these side effects in order to successfully translate animal models into clinical trials.

Article	Year
Identification and Application of Gene Expression Signatures Associated with Lifespan Extension. Cell metabolism\| PubMed ID: 31353263	2019
Genetic Constructs for the Control of Astrocytes' Activity. Cells\| PubMed ID: 34202359	2021
In vivo dynamics of acidosis and oxidative stress in the acute phase of an ischemic stroke in a rodent model. Redox biology\| PubMed ID: 34773835	2021
Potassium KCa3.1 channel overexpression deteriorates functionality and availability of channels at the outer cellular membrane. Scientific reports\| PubMed ID: 39929947	2025

Anastasia A. Borodinova

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