Biology subjects

Oliveri, F.

Publications and source records attributed to Oliveri, F..

4 recordsLinked to original sources

bioRxiv2024

Single-nuclei sequencing of skeletal muscle reveals subsynaptic-specific transcripts involved in neuromuscular junction maintenance

The neuromuscular junction (NMJ) is the synapse formed between motor neurons and skeletal muscle fibers. Its stability relies on the continued expression of genes in a subset of myonuclei, called NMJ myonuclei. Here, we use single-nuclei RNA-sequencing (snRNA-seq) to identify numerous undescribed NMJ-specific transcripts. To elucidate how the NMJ transcriptome is regulated, we also performed snRNA-seq on sciatic nerve transected, botulinum toxin injected and Musk knockout muscles. These data show that NMJ gene expression is not only driven by agrin-Lrp4/MuSK signaling, but is also affected by electrical activity and trophic factors other than agrin. By selecting three previously undescribed NMJ genes Etv4, Lrtm1 and Pdzrn4, we further characterize novel contributors to NMJ stability and function. AAV-mediated overexpression and AAV-CRISPR/Cas9-mediated knockout show that Etv4 is sufficient to upregulate expression of [~]50% of the NMJ genes in non-synaptic myonuclei, while muscle-specific knockout of Pdzrn4 induces NMJ fragmentation. Further investigation of Pdzrn4 revealed that it localizes to the Golgi apparatus and interacts with MuSK protein. Collectively, our data provide a rich resource of NMJ transcripts, highlight the importance of ETS transcription factors at the NMJ and suggest a novel pathway for NMJ post-translational modifications.

molecular biology↗

bioRxiv2024

Muscle fiber Myc is dispensable for muscle growth and forced expression severely perturbs homeostasis

The oncogenic transcription factor Myc stimulates many growth processes including cell cycle progression and ribosome biogenesis. Myc expression is low in adult skeletal muscle, but is upregulated upon growth stimuli. Furthermore, muscle fiber Myc overexpression recapitulates many aspects of growth-related gene expression, suggesting Myc may mediate pro-growth responses to anabolic stimuli, such as exercise. Here, we tested this hypothesis by examining mouse models in which Myc was specifically eliminated or overexpressed in skeletal muscle fibers or muscle stem cells (MuSC). While muscle fiber Myc expression increased during muscle growth and Myc expression in MuSCs was required for successful muscle regeneration, muscle fiber Myc expression was dispensable for post-natal, mechanical overload or PKB/Akt-induced muscle growth in mice. Similarly, constitutive Myc expression did not promote skeletal muscle hypertrophy, but instead impaired muscle fiber structure and function within days. These data question the role of Myc in skeletal muscle growth.

physiology↗

bioRxiv2023

Fast, multiplexable and highly efficient somatic gene deletions in adult mouse skeletal muscle fibers using AAV-CRISPR/Cas9

Molecular screens comparing different disease states to identify candidate genes rely on the availability of fast, reliable and multiplexable systems to interrogate genes of interest. CRISPR/Cas9- based reverse genetics is a promising method to eventually achieve this. However, such methods are sorely lacking for multi-nucleated muscle fibers, since highly efficient nuclei editing is a requisite to robustly inactive candidate genes. Here, we couple Cre-mediated skeletal muscle fiber-specific Cas9 expression with myotropic adeno-associated virus-mediated sgRNA delivery to establish a system for highly effective somatic gene mutation in mice. Using well-characterized genes, we show that local or systemic inactivation of these genes copy the phenotype of traditional gene-knockout mouse models. Thus, this proof-of-principle study establishes a method to unravel the function of individual genes or entire signaling pathways in adult skeletal muscle fibers without the cumbersome requirement of generating knockout mice.

molecular biology↗

bioRxiv2021

Dual roles of mTORC1-dependent activation of the ubiquitin-proteasome system in muscle proteostasis

Muscle size is controlled by the PI3K-PKB/Akt-mTORC1-FoxO pathway, which integrates signals from growth factors, energy and amino acids to activate protein synthesis and inhibit protein breakdown. While mTORC1 activity is necessary for PKB/Akt-induced muscle hypertrophy, its constant activation alone induces muscle atrophy. Here we show that this paradox is based on mTORC1 activity promoting protein breakdown through the ubiquitin-proteasome system (UPS) by simultaneously inducing ubiquitin E3 ligase expression via feedback inhibition of PKB/Akt and proteasome biogenesis via Nuclear Factor Erythroid 2-Like 1 (Nrf1). Muscle growth was restored by reactivation of PKB/Akt, but not by Nrf1 knockdown, implicating ubiquitination as the limiting step. However, both PKB/Akt activation and proteasome depletion by Nrf1 knockdown led to an immediate disruption of proteome integrity with rapid accumulation of damaged material. These data highlight the physiological importance of mTORC1-mediated PKB/Akt inhibition and point to juxtaposed roles of the UPS in atrophy and proteome integrity.

physiology↗