Adeno-associated viruses (AAVs) are preferred gene therapy vectors because of their versatility, durability and safety profile. Here, we demonstrate extensive chimerism, manifesting as pervasive barcode swapping, among complex recombinant AAV (rAAV) libraries that are packaged as a pool. The observed chimerism is length and homology dependent but capsid independent, in some cases affecting the majority of packaged rAAV genomes. These results have implications for the design and deployment of functional rAAV libraries. Pooled production of recombinant AAV vectors leads to frequent genetic recombination.
Aggregation of the protein tau defines tauopathies, the most common age-related neurodegenerative diseases, which include Alzheimer’s disease and frontotemporal dementia. Specific neuronal subtypes are selectively vulnerable to tau aggregation, dysfunction, and death. However, molecular mechanisms underlying cell-type-selective vulnerability are unknown. To systematically uncover the cellular factors controlling the accumulation of tau aggregates in human neurons, we conducted a genome-wide CRISPRi screen in induced pluripotent stem cell (iPSC)-derived neurons. The screen uncovered both known and unexpected pathways, including UFMylation and GPI anchor biosynthesis, which control tau oligomer levels. We discovered that the E3 ubiquitin ligase CRL5SOCS4 controls tau levels in human neurons, ubiquitinates tau, and is correlated with resilience to tauopathies in human disease. Disruption of mitochondrial function promotes proteasomal misprocessing of tau, generating disease-relevant tau proteolytic fragments and changing tau aggregation in vitro. These results systematically reveal principles of tau proteostasis in human neurons and suggest potential therapeutic targets for tauopathies.
Animal locomotion relies on rhythmic body movements driven by central pattern generators (CPGs): neural circuits that produce oscillating output without oscillating input. However, the circuit structure of a CPG for walking is not known in any animal. To identify the cells and synapses that underlie rhythmic leg movement in walking flies, we developed dynamic simulations of the Drosophila ventral nerve cord (VNC) connectomes. A computational activation screen of descending neurons from the central brain identified DNg100-a known command neuron for walking-as the top driver of rhythmic leg motor activity. Simulated network pruning isolated a minimal rhythm-generating circuit consisting of one inhibitory and two excitatory interneurons; this three-neuron circuit was necessary and sufficient for motor rhythms across all six legs and in four connectome datasets. Simulations also predicted that a separate descending pathway (DNb08) drives rhythmic leg movements, which we confirmed experimentally using optogenetics in behaving flies. Our results reveal the cellular identity and synaptic structure of a putative CPG circuit for walking and other rhythmic leg movements in flies.
Cis-acting regulatory enhancer elements are powerful tools for achieving cell type-specific genetic access in adeno-associated virus (AAV) delivery platforms. However, a significant bottleneck in enhancer discovery remains the accurate characterization of their in vivo expression patterns, which currently relies on labor-intensive, gold-standard one-by-one validation. Here, we evaluate multiple barcoded, multiplexed strategies for accelerated profiling of enhancer-driven expression at cell type resolution. As a proof-of-concept, we test small pools of well-validated enhancer AAVs with known activity across diverse cell types in the mouse brain. Despite extensive optimization and testing, we encounter substantial technical and biological noise, including chimeric AAV packaging products, that obscure true enhancer expression patterns. These effects are particularly pronounced for weaker enhancers and enhancers active in less abundant cell subpopulations. These findings highlight the challenges inherent to multiplexed enhancer AAV screening, the importance of careful enhancer pool design, and the complexity of enhancer AAV biology in vivo.
Patterns of brain activity moving in waves occur across brain regions and species, yet their spatial organization, anatomical basis, and brain-wide distribution remain unclear. Using cortex-wide imaging and electrophysiology in awake mice, we revealed a prominent wave motif across spatial scales. Waves frequently formed rotational patterns centered on somatosensory cortex and sweeping across somatotopic maps. Axonal architecture within sensory cortex exhibited a matching circular arrangement. Rotating waves were mirrored between hemispheres and between sensory and motor cortex and were coordinated with subcortical spiking. Bilaterally cutting the circular circuitry diminished rotating waves. Rotating waves were modulated across behavioral states, evoked by sensory inputs, and recruited during correct visuomotor performance. These results establish that rotating waves are sculpted by axonal architecture across diverse brain systems and behavioral contexts.
