Spivey, M. L. | bioRxiv Science

bioRxiv2026

TRPML1 positions lysosomes and regulates actin-membrane linkers in astrocyte processes

Lysosomes are critical for neuronal physiology and synaptic function, but their organization and roles within astrocytes, an integral component of the tripartite synapse, remain unknown. Here, we use a neuron-astrocyte coculture system that promotes stellate astrocyte morphology to investigate the trafficking of late endosomes and lysosomes (LELs) in astrocytes. As astrocyte branches mature, degradative activity becomes concentrated in the soma and LELs in branches undergo bidirectional motility that becomes progressively attenuated. We establish that the lysosomal cation channel TRPML1 drives LEL immobilization. TRPML1-mediated arrest involves myosin-Va tethering to the actin cytoskeleton, which may position LELs near peripheral astrocyte processes (PAPs), fine actin-enriched protrusions that can contact synapses. Strikingly, TRPML1 activity modulates the phosphorylation of ezrin, radixin, and moesin, actin-membrane linkers enriched in PAPs. These effects are rapid and transient, suggesting a role for lysosomal TRPML1 in regulating PAP membrane dynamics. Thus, TRPML1 positions LELs proximal to PAPs which may influence PAP structural plasticity and astrocyte-synapse contacts.

neuroscience↗

bioRxiv2025

Differential regulation of p62-ubiquitin conjugates in neurons versus astrocytes during cellular stress

Sequestosome 1/p62 (hereafter referred to as p62) is a multifunctional protein that orchestrates various cellular stress response pathways including autophagy, proteasome-mediated degradation, antioxidant defense, nutrient sensing, and inflammatory signaling. Mutations in distinct functional domains of p62 are linked with the neurodegenerative disease amyotrophic lateral sclerosis (ALS), underscoring its importance in neural cells. Neurons and astrocytes perform distinct roles in brain physiology and thus encounter a unique landscape of cellular stress. However, how p62 is regulated in these cell types in response to various stress modalities remains largely unexplored. Several functions for p62 depend on engagement with ubiquitinated substrates. Thus, we investigated how the regulation of p62-ubiquitin conjugates differs between neurons and astrocytes exposed to two stress modalities: lysosomal membrane damage and metabolic stress. Lysosomal damage triggered ubiquitin-dependent assembly of p62 puncta in both neurons and astrocytes. In contrast, nutrient deprivation elicited different responses between neurons and astrocytes. Neurons formed p62-ubiquitin structures more prominently and displayed a greater dependence on ubiquitin for p62 clustering. Together, these findings reveal cell-type-specific and stress-specific regulation of p62-ubiquitin conjugates, indicating that neurons and astrocytes can deploy distinct quality control strategies.

neuroscience↗