Liquid-liquid phase separation has emerged as a unique form of intracellular organization through membraneless gel-like structures known as biomolecular condensates (BMCs). The formation of BMCs represents a novel form of protein and nucleic acid compartmentalization that plays pivotal roles in human health and disease. Among other processes, BMCs are involved in neuronal mRNA trafficking, local protein synthesis during memory consolidation, and the stabilization of both pre- and post-synaptic components essential for neuronal communication. TDP-43, α-synuclein and Tau undergo phase separation, forming toxic BMCs that have been found in the brains of patients with amyotrophic lateral sclerosis, Parkinson’s disease, and Alzheimer’s disease (AD), respectively. Tau is a microtubule-associated protein expressed in the nervous system that works as a scaffold component with crucial roles in neuronal plasticity and migration. Hyperphosphorylation of Tau represents a pathological switch that promotes the formation of cytotoxic Tau aggregates commonly observed in AD. Tau also undergoes phase separation, but the precise physiological and pathological functions of Tau-BMCs still need to be fully understood. Electron microscopy (EM) and fluorescence recovery after photobleaching (FRAP) are among the most used imaging techniques to investigate the structure, composition and dynamics of BMCs. However, EM only provides static information, and FRAP does not achieve sufficient spatial resolution. The development of super-resolution imaging (SRI) has revolutionized neuroscience research, offering unprecedented insights into how the brain works and providing exceptional levels of temporal and spatial resolution, necessary for the study of elusive structures such as BMCs.
Combining large-scale phosphoproteomic analysis with single-molecule SRI in live neurons, we revealed that Tau molecules undergo liquid-liquid phase separation, generating presynaptic nanoclusters whose density and number are regulated by synaptic activity. Tau translocates from the axon into the presynapse in an activity-dependent manner, controlling the mobility of the recycling pool of SVs and forming sub-diffraction synaptic nano-BMCs. At the post-synapse, we also found that a mutant version of Tau associated with frontotemporal dementia (P301L-Tau) forms aberrant BMCs that sequester the Src kinase Fyn, leading to enhanced clustering and accelerated synaptotoxicity. Moreover, our results showed that targeting the formation of these Tau gel-like aggregates prevents Fyn aberrant clustering.
Our results show how SRI can be used to uncover novel mechanisms that control neuronal communication based on the formation of Tau nano-BMCs. Dysregulation of these transient Tau condensates may produce “seeds” that sequester neuronal proteins, amplifying their toxic activity and leading to the development of neurological disorders.