The activity of neural circuits in the brain relies on the generation and propagation of neuronal action potentials. The axon initial segment (AIS) is the proximal region of the axon, typically 20 to 60 µm long, responsible for both action potential initiation and the maintenance of neuronal polarity. Changes in the molecular composition, length, or position of the AIS can significantly alter intrinsic neuronal excitability. Disruption of the AIS has been observed to cause axons to acquire dendritic characteristics. Previous studies indicate that AIS disruption is a common occurrence in various neurological diseases, including autism, Alzheimer's disease, stroke, bipolar disorder, schizophrenia, and amyotrophic lateral sclerosis. Notably, rescuing AIS integrity and function has shown promise in ameliorating neurological symptoms in Alzheimer's disease and Angelman Syndrome mouse models. However, our incomplete understanding of AIS components hinders a comprehensive grasp of the structural and functional regulation of the AIS in both health and disease.
Traditional methods for identifying AIS components, such as antibody staining and pharmacological screening, face limitations in specificity, availability, cost, and throughput. Although enzyme-mediated proximity labeling methods provide proteome-wide mapping with submicron- to nanometer-scale spatial resolution, they depend on introducing exogenous fusion proteins which may lead to mislocalization. For example, BioID technology, while used to identify the AIS proteome in cultured hippocampal neurons, suffers from spatial specificity issues due to bait protein mislocalization. The current challenges in AIS component research demand a technique for endogenous bait protein proximity labeling.
Recently, a research group led by Prof. Peng Zou at Peking University, in collaboration with Prof. Matthew N. Rasband’s group at Baylor College of Medicine, published a paper in Nature Communications titled "Immunoproximity Biotinylation Reveals the Axon Initial Segment Proteome" This study introduced a new technology based on immunoproximity chemical labeling, providing high spatial specificity data on the neuronal AIS proteome and illustrating dynamic changes in AIS components during neuronal development. The research identified novel AIS components, such as SCRIB, and characterized their localization and protein-protein interactions using super-resolution fluorescence imaging and biochemical methods. These findings contribute valuable data resources for understanding the regulation of AIS structure and function.
The authors first established an Immunoproximity Labeling technology (IPL) for the AIS. By utilizing antibodies against the AIS protein NF186, they directed the peroxidase enzyme HRP to the AIS, enabling targeted labeling of endogenous proteins without genetic manipulation. Furthermore, through HRP catalysis with hydrogen peroxide, they generated biotin phenol radicals with a diffusion radius of 100 nm, covalently labeling proteins adjacent to NF186 and effectively covering AIS components. Immunofluorescence imaging and immunoblot analysis demonstrated the high spatial resolution capabilities of IPL.
Fig. 1 Profiling the axon initial segment proteome with immunoproximity labeling.
Subsequently, the authors utilized multiple neuronal subcellular structures, encompassing the cell body, dendrites, and axons, as spatial references. They achieved high-quality AIS proteomic data by integrating 10-plex TMT peptide labeling and ratiometric quantitative proteomics, effectively subtracting background and reference signals. Data analysis revealed that the captured 1403 proteins nearly encompassed all reported AIS molecules (see Fig. 1), spanning extracellular matrix proteins, AIS membrane proteins, various voltage-gated ion channels, and cytoskeletal-related proteins. Notably, well-known high-expression AIS molecules such as AnkG, TRIM46, NFASC, Nav1.2, and βIV-spectrin ranked among the top 5 in the data list. Consequently, the authors compiled a protein data list with extensive coverage of the mature neuronal AIS.
To investigate dynamic changes in AIS components during development, the authors selected three time points: early development at DIV7, mature stage at DIV14, and fully mature stage at DIV21. Employing the IPL strategy, they provided AIS datasets for each period. Analysis of potential core components during development revealed dynamic changes in AIS components: the expression levels of most proteins increased with AIS development, exemplified by AnkG and Nav1.2, while a few proteins, including TRIM46 involved in early neuronal polarity establishment, exhibited significant decreases. Consequently, the authors constructed a dynamic map of the AIS proteome aligned with the physiological function during neuronal development.
Finally, guided by DIV14 proteomic data, the authors employed a CRISPR/Cas9-dependent HiUGE gene knock-in strategy to characterize the top three proteins lacking previous studies in AIS. This involved adding endogenous tags to the proteins of interest. Imaging results showcased varied expression levels of these three proteins in AIS, notably the scaffold protein SCRIB displaying a highly polarized distribution at the AIS (Fig. 2b and c). Subsequent experiments, employing both in vitro and in vivo gene tagging strategies alongside antibody staining, further confirmed the polarized distribution of SCRIB in the AIS. Through a CRISPR/Cas9-dependent gene knockout strategy and immunoprecipitation experiments, the authors uncovered the enrichment of SCRIB in the AIS, recruited by the AIS core component AnkG. Notably, the N-terminal domain of SCRIB was identified as involved in its interaction with AnkG.
Fig. 2 Validating AIS candidates via fluorescence microscopy.
In summary, this study introduced an immunoproximity labeling technology to profile the components of the action potential initiation domain, the AIS, unveiling its dynamic changes during neuronal development. The proteomic data obtained will guide future research on AIS components, establishing a foundation for a deeper understanding of the mechanisms that regulate neuronal excitability and contribute to the onset of pathological defects.
Dr. Peng Zou is affiliated with the College of Chemistry and Molecular Engineering, Peking University, the Peking-Tsinghua Center for Life Sciences, and the Peking University IDG/McGovern Institute for Brain Research. Dr. Wei Zhang, a former postdoctoral researcher at the College of Chemistry and Molecular Engineering, Peking University, and the Peking-Tsinghua Center for Life Sciences, is the first author of the paper. This work was supported by the National Natural Science Foundation of China, the Ministry of Science and Technology, the Beijing National Laboratory for Molecular Sciences, the Key Laboratory of Bioorganic and Molecular Engineering of the Ministry of Education, the Beijing Institute for Brain Research, the National Institutes of Neurological Disorders and Stroke, and the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation.
Original link for the paper:: https://www.nature.com/articles/s41467-023-44015-2
