Richardson, C. E. | Search bioRxiv

bioRxiv2024

TFEB/HLH-30-mediated expansion of neuronal lysosomal capacity in early adulthood protects dendrite maintenance during aging in Caenorhabditis elegans

Lysosomes are essential for neuronal homeostasis, providing degradation and recycling functions necessary to support neurons complex operations and long lifespans. However, the regulation of lysosomal degradative capacity in healthy neurons is poorly understood. Here, we investigate the role of HLH-30, the sole Caenorhabditis elegans homolog of Transcription Factor EB (TFEB), a master regulator of lysosome biogenesis and autophagy that it is thought to predominantly function in the context of starvation or stress. We demonstrate that HLH-30 is dispensable for neuronal development but acts cell-intrinsically to expand lysosomal degradative capacity during early adulthood. Loss of HLH-30 leads to lysosomal dysfunction and delayed turnover of synaptic vesicle proteins from the synapse. Notably, we show that basal HLH-30 activity is sufficient to expand neuronal lysosomal capacity without nuclear enrichment, in contrast to the nuclear translocation associated with starvation- and stress-induced activation of TFEB and HLH-30. Furthermore, we show that neuronal lysosomal function declines with age in wild-type animals, and this corresponds to a decrease in basal HLH-30-mediated transcription. We further demonstrate that basal HLH-30 activity is crucial for neuron maintenance: lysosomal dysfunction due to inadequate HLH-30 activity leads to dendrite degeneration and aberrant outgrowths. In summary, our study establishes a critical role for HLH-30/TFEB in promoting lysosomal capacity to preserve neuronal homeostasis and structural integrity of mature neurons in vivo.

cell biology↗

bioRxiv2024

Visualizing turnover of synaptic vesicle protein Synaptogyrin/SNG-1 in vivo using a new method, ARGO (Analysis of Red Green Offset)

In long-lived cells such as neurons, proteostasis involves the regulated degradation and replacement of proteins to ensure their quality and appropriate abundance. Synaptic vesicle (SV) protein turnover in neurons is important for controlling the SV pool size to maintain appropriate levels of neurotransmission; yet, it is incompletely understood, partly due to limited tools for quantifying protein turnover in vivo. We present ARGO (Analysis of Red-Green Offset), a fully genetically encoded, ratiometric fluorescence imaging method that visualizes and quantifies protein turnover with subcellular resolution in vivo. ARGO is inexpensive, modular, and scalable for use in genetically tractable experimental organisms. Using ARGO, we examine the turnover of Synaptogyrin/SNG-1, an evolutionarily conserved, integral SV protein, in C. elegans neurons. We show that the SNG-1 turnover rate is consistent across presynapses within a single neuron but varies between neuron classes. Notably, we find SNG-1 and can exist in two distinct, non-intermixing populations within each presynapse. Further, we present an initial mutant analysis of uba-1, the sole E1 ubiquitin ligase in C. elegans, showing that we can detect slowed SNG-1 turnover even though steady-state SNG-1 abundance is not increased compared to wild-type. These results provide new hints for the regulation of SV pool size. Significance Statement- In long-lived cells, a proteins rates of synthesis and degradation together determine its abundance, yet regulation of protein turnover is largely unknown due to the lack of simple methods for in vivo quantification. - Using C. elegans, the authors develop ARGO, a genetically encoded, microscopy approach that quantifies a proteins turnover. Results suggest that synaptic vesicle protein Synaptogyrin/SNG-1 can partition into two presynaptic pools with distinct half-lives. - This provides a powerful tool to study protein turnover, reveals an unexpected complexity in SNG-1 turnover, and lays the groundwork for future investigations of synaptic vesicle protein compartmentalization, sorting, and degradation.

cell biology↗