Zhuhao Wu

Single-cell transcriptomic studies have revealed remarkable molecular heterogeneity across neuronal and glial populations during development, aging, and disease. However, dissociation-based approaches disrupt tissue architecture, and RNA measures alone incompletely capture cellular function. These limitations are particularly relevant in neurodegenerative diseases, where pathology develops within cellular microenvironments and diseased cells progressively lose canonical cell-state identity markers. Spatial proteomic approaches are beginning to address this critical gap by enabling high-dimensional molecular profiling within the intact tissue, preserving the spatial relationships between vulnerable cells and their surrounding cellular environments across scales ranging from subcellular single-cell imaging to large-volume three-dimensional mapping. Here, we discuss emerging spatial proteomic technologies and their applications in aging and neurodegeneration, emphasizing how integration with complementary modalities, including transcriptomics, glycomics, three-dimensional genome architecture, and quantitative neuropathology, is providing new insight into disease biology. We further highlight how these approaches are beginning to accelerate therapeutic target validation, biomarker discovery, and treatment response monitoring. Realizing the full potential of spatial proteomics will require continued advances in cross-platform harmonization, analytical standardization, and computational methods capable of integrating increasingly complex, multimodal datasets.

The X-linked RNA helicase DDX3X is a high-risk gene for autism spectrum disorder and intellectual disability primarily in females. Individuals with pathogenic variants in DDX3X have pervasive and often severe motor manifestations. Using mouse models, we studied the circuits underlying the motor phenotypes associated with Ddx3x deficiency and examined the feasibility of an early intervention to ameliorate motor trajectories. We show that Ddx3x controls both the development and later plasticity of motor circuits connecting the motor cortex with the pontine nuclei. Ddx3x haploinsufficiency during development affects the dendritic arborization of corticopontine neurons. Juvenile-onset Ddx3x loss in corticopontine neurons is sufficient to induce adult motor impairments overlapping with those caused by chemogenetic disruption of corticopontine circuits. Further, Ddx3x mutant mice engage corticopontine circuits in skilled motor tasks in a sex-dependent manner. Last, Ddx3x haploinsufficient female mice undergoing a juvenile motor intervention show long-lasting specific benefits in their adult motor function. Together, these findings establish Ddx3x as an essential regulator of corticopontine neuron development and function and provide an early motor intervention to ameliorate motor outcomes in a mouse model of DDX3X haploinsufficiency.

AlphaFold3 has shown promise as a tool for predicting antibody-antigen binding, yet its performance across large datasets has not been fully characterized. In this study, 3401 experimentally validated antibody-antigen complexes were sourced from the Structural Antibody Database and screened alongside 23798 negative controls to benchmark AlphaFold3’s binding prediction capabilities. Confidence metrics including Predicted Aligned Error and Interface Predicted Template Modeling score were used to achieving a maximum recall of 53% at 100 inference seeds. Several factors were found to influence prediction accuracy: a notable bias was observed toward antibodies derived from X-ray crystallography structures versus those from electron microscopy, and positive prediction rates were found to decrease with increasing target protein size and surface area. In contrast, neither the amino acid composition or lengths of the complementarity determining regions, nor training data leakage were found to introduce significant bias. An innate false positive rate of approximately 3% was identified, with AF3 shown to hallucinate plausible binding interfaces across the surface of decoy targets while avoiding disordered regions. Epitope mapping using DockQ, epitope shift, and antibody displacement revealed that approximately 34% of false negatives retained the correct epitope location despite poor structural alignment, suggesting that conformation refinement tools could recover additional true binding predictions. These findings provide a comprehensive characterization of AlphaFold3’s strengths and limitations for antibody screening in computational drug discovery. Key Messages: AlphaFold3 has a recall of 50% and an innate false positive prediction rate of 3%. False negative predictions can still feature the correct epitope despite poor RMSD. Factors such as disorder and target size impact accuracy.

Cancer radiotherapy inevitably damages normal tissues, yet therapeutic activation of pro-survival or regenerative pathways frequently increases the risk of cancer recurrence. Strategies that selectively restore normal tissue without fueling malignant regrowth remain largely undefined. Using the slow dividing yet radio-responsive salivary gland and adjacent oral squamous cell carcinoma (OSCC) as paired in vivo models, we established a high-throughput genetic screening platform to quantify post-irradiation clonal expansion in both normal and malignant epithelium. Comparative screening revealed depletion of the salivary gland dominant SWI/SNF core ATPase Smarca2 as the top-ranking selective driver of salivary epithelial regeneration, in contrast to most candidates that promoted expansion in both tissues. Smarca2 depletion specifically enhanced regeneration of acinar cells, the lineage most refractory to recovery after irradiation, by suppressing terminal differentiation programs and enabling transcriptional activation of key regenerative genes. In OSCC cells, however, this regenerative switch was uncoupled from Smarca2 depletion due to tumor-specific chromatin state remodeling. Notably, this mechanism is conserved in human salivary gland tissue and can be induced using SMARCA2-targeting PROTAC degrader, a therapeutic class under active development for cancer treatment. Together, our findings suggest that Smarca2 depletion is a potent lineage-specific mechanism to replenish radiation damaged salivary epithelium without promoting OSCC expansion, a paradigm that is underexplored.

Advanced brain-wide mapping is critical for addressing complex questions in neuroscience. However, current imaging methods are limited by throughput, resolution and signal-to-noise ratio, constraining their broader applicability. Here, we present confocal Airy beam integrated with single-photon oblique light-sheet tomography (CAB-OLST): a system that integrates single-photon excitation with a scanned Airy beam light sheet, virtual slit detection and automated mechanical sectioning. CAB-OLST enables high-throughput, high-resolution and high-signal-to-noise ratio volumetric imaging, achieving an optical resolution of 0.77 μm × 0.49 μm × 2.61 μm. This allows for mouse brain-wide cell type distribution mapping at a voxel size of 0.37 μm × 0.37 μm × 1.77 μm in 10 h and single-neuron projectome imaging with a voxel size of 0.26 μm × 0.26 μm × 1.06 μm over 58 h. Compared to existing light-sheet and point-scanning systems, CAB-OLST provides a scalable and robust platform for comprehensive neuronal morphology reconstruction and high-precision cell atlas generation.

DDX3X is an X-linked RNA helicase that escapes X chromosome inactivation and is expressed at higher levels in female brains. Mutations in DDX3X are associated with intellectual disability (ID) and autism spectrum disorder (ASD) and are predominantly identified in females (DDX3X syndrome). Using cellular and mouse models, we show that Ddx3x mediates sexual dimorphisms in brain development at a molecular, cellular, and behavioral level. During cortical neuronal development, Ddx3x sustains a female-biased signature of enhanced ribosomal biogenesis and mRNA metabolism. Compared to male neurons, female neurons display larger nucleoli, higher expression of a set of ribosomal proteins, and a higher cytoplasm-to-nucleus ratio of ribosomal RNA. All these sex dimorphisms are obliterated by Ddx3x loss. Ddx3x regulates dendritic arborization complexity in a sex- and dose-dependent manner in both female and male neurons. Ddx3x modulates the development of dendritic spines but only in female neurons. Further, ablating Ddx3x conditionally in forebrain neurons is sufficient to yield sex-specific changes in developmental outcomes and motor function. Together, these findings pose Ddx3x as a mediator of sexual differentiation during neurodevelopment and open new avenues to understand sex differences in health and disease.

Glioblastoma (GBM) infiltrates the brain and can be synaptically innervated by neurons, which drives tumor progression^1,2. Synaptic inputs onto GBM cells identified so far are largely short-range and glutamatergic^3,4. The extent of GBM integration into the brain-wide neuronal circuitry remains unclear. Here we applied rabies virus- and herpes simplex virus-mediated trans-monosynaptic tracing^5,6 to systematically investigate circuit integration of human GBM organoids transplanted into adult mice. We found that GBM cells from multiple patients rapidly integrate into diverse local and long-range neural circuits across the brain. Beyond glutamatergic inputs, we identified various neuromodulatory inputs, including synapses between basal forebrain cholinergic neurons and GBM cells. Acute acetylcholine stimulation induces long-lasting elevation of calcium oscillations and transcriptional reprogramming of GBM cells into a more motile state via the metabotropic CHRM3 receptor. CHRM3 activation promotes GBM cell motility, whereas its downregulation suppresses GBM cell motility and prolongs mouse survival. Together, these results reveal the striking capacity for human GBM cells to rapidly and robustly integrate into anatomically diverse neuronal networks of different neurotransmitter systems. Our findings further support a model wherein rapid connectivity and transient activation of upstream neurons may lead to a long-lasting increase in tumor fitness.

We conducted a large-scale whole-brain morphometry study by analyzing 3.7 peta-voxels of mouse brain images at the single-cell resolution, producing one of the largest multi-morphometry databases of mammalian brains to date. We registered 204 mouse brains of three major imaging modalities to the Allen Common Coordinate Framework (CCF) atlas, annotated 182,497 neuronal cell bodies, modeled 15,441 dendritic microenvironments, characterized the full morphology of 1876 neurons along with their axonal motifs, and detected 2.63 million axonal varicosities that indicate potential synaptic sites. Our analyzed six levels of information related to neuronal populations, dendritic microenvironments, single-cell full morphology, dendritic and axonal arborization, axonal varicosities, and sub-neuronal structural motifs, along with a quantification of the diversity and stereotypy of patterns at each level. This integrative study provides key anatomical descriptions of neurons and their types across a multiple scales and features, contributing a substantial resource for understanding neuronal diversity in mammalian brains.