Gueven, N. | Search bioRxiv

bioRxiv2024

Single-cell fluorescence imaging reveals heterogeneity in senescence biomarkers and identifies rapamycin-responsive sub-populations

Cellular senescence is a state of irreversible cell cycle arrest accompanied by a distinctive inflammatory secretory profile known as the senescence-associated secretory phenotype (SASP). While various biomarkers, such as senescence-associated beta-galactosidase (SA-{beta}gal), EdU incorporation, P21, and P16, are used to identify senescent cells, no single biomarker universally defines cellular senescence, and current methods often fail to address heterogeneity in biomarker expression levels. This study leverages single-cell fluorescence imaging to assess multiple senescence markers including SA-{beta}gal enzymatic activity, P21 and IL-6 expression, and nuclear and cell area, in chemotherapy-induced (mitomycin C) and oxidative stress-induced (D-galactose) senescence models in human fibroblasts. Our findings reveal significant heterogeneity in SA-{beta}gal activity and distinct sub-populations within senescent cells. Nuclear and cell area measurements emerged as robust indicators of cellular senescence, displaying similar variability across individual cells. Importantly, we identified specific nuclear area sub-populations that strongly correlate with IL-6 expression levels, demonstrating a relationship between the heterogeneous expression of senescence biomarkers and the SASP. To address this heterogeneity, we introduced an induction threshold method to more accurately quantify the percentage of cells expressing senescence biomarkers. Furthermore, in both senescence models, we observed that rapamycin, a well-known senomorphic agent, selectively targets specific biomarker-expressing sub-populations. This study underscores the value of assessing cellular heterogeneity in senescence research and provides an improved approach for analysing senescence markers in diverse cellular contexts.

cell biology↗

bioRxiv2023

HDAC6 inhibition as a mechanism to prevent axon degeneration in the mSOD1G93A mouse model of ALS.

The loss of upper and lower motor neurons, and their axons is central to the loss of motor function and death in amyotrophic lateral sclerosis (ALS). Due to the diverse range of genetic and environmental factors that contribute to the pathogenesis of ALS, there have been difficulties in developing effective therapies for ALS. One dichotomy emerging in the field is that protection of the neuronal cell soma itself does not prevent axonal vulnerability and degeneration, suggesting the need for targeted therapeutics to prevent axon degeneration. Post-translational modifications of protein acetylation can alter the function, stability and half-life of individual proteins, and can be enzymatically modified by histone acetyltransferases (HATs) and histone deacetyltransferases (HDACs), which add, or remove acetyl groups, respectively. Maintenance of post-translational microtubule acetylation has been suggested as a potential mechanism to stabilise axons and prevent axonal loss and neurodegeneration in ALS. This study has utilized an orally dosed HDAC6 specific inhibitor, ACY-738, prevent deacetylation and stabilize microtubules in the mSOD1G93A mouse model of ALS. Furthermore, co-treatment with riluzole was performed to determine any effects or drug interactions and potentially enhance preclinical research translation. This study shows ACY-738 treatment increased acetylation of microtubules in the spinal cord of mSOD1G93A mice, reduced lower motor neuron degeneration in the lumbar spinal cord of female mice, ameliorated reduction in peripheral nerve axon puncta size, but did not prevent overt motor function decline. The current study also shows peripheral nerve axon puncta size to be partially restored after treatment with riluzole and highlights the importance of co-treatment to measure the potential effects of therapeutics in ALS. HighlightsO_LIACY-738 inhibits HDAC6 and leads to increased microtubule acetylation in spinal cord of mSOD1G93A mice. C_LIO_LIACY-738 treatment reduces lower motor neuron degeneration in the lumbar spinal cord of mSOD1G93A mice. C_LIO_LIACY-738 treatment restores peripheral nerve axon puncta size of mSOD1G93A mice. C_LIO_LIACY-738 treatment does not prevent overt motor function decline mSOD1G93A mice. C_LIO_LIRiluzole treatment partially restores peripheral nerve axon puncta size in mSOD1G93A mice. C_LI

neuroscience↗