Antibody-directed extracellular proximity biotinylation reveals that Contactin-1 regulates axo-axonic innervation of axon initial segments.

Axon initial segment (AIS) cell surface proteins mediate key biological processes in neurons including action potential initiation and axo-axonic synapse formation. However, few AIS cell surface proteins have been identified. Here, we use antibody-directed proximity biotinylation to define the cell surface proteins in close proximity to the AIS cell adhesion molecule Neurofascin. To determine the distributions of the identified proteins, we use CRISPR-mediated genome editing for insertion of epitope tags in the endogenous proteins. We identify Contactin-1 (Cntn1) as an AIS cell surface protein. Cntn1 is enriched at the AIS through interactions with Neurofascin and NrCAM. We further show that Cntn1 contributes to assembly of the AIS extracellular matrix, and regulates AIS axo-axonic innervation by inhibitory basket cells in the cerebellum and inhibitory chandelier cells in the cortex.

Antibody-directed extracellular proximity biotinylation reveals Contactin-1 regulates axo-axonic innervation of axon initial segments.

Axon initial segment (AIS) cell surface proteins mediate key biological processes in neurons including action potential initiation and axo-axonic synapse formation. However, few AIS cell surface proteins have been identified. Here, we used antibody-directed proximity biotinylation to define the cell surface proteins in close proximity to the AIS cell adhesion molecule Neurofascin. To determine the distributions of the identified proteins, we used CRISPR-mediated genome editing for insertion of epitope tags in the endogenous proteins. We found Contactin-1 (Cntn1) among the previously unknown AIS proteins we identified. Cntn1 is enriched at the AIS through interactions with Neurofascin and NrCAM. We further show that Cntn1 contributes to assembly of the AIS-extracellular matrix, and is required for AIS axo-axonic innervation by inhibitory basket cells in the cerebellum and inhibitory chandelier cells in the cortex.

Low-density lipoprotein receptor-related protein 1 (LRP1) is a novel receptor for apolipoprotein A4 (APOA4) in adipose tissue.

Apolipoprotein A4 (APOA4) is one of the most abundant and versatile apolipoproteins facilitating lipid transport and metabolism. APOA4 is synthesized in the small intestine, packaged onto chylomicrons, secreted into intestinal lymph and transported via circulation to several tissues, including adipose. Since its discovery nearly 4 decades ago, to date, only platelet integrin αIIbß3 has been identified as APOA4 receptor in the plasma. Using co-immunoprecipitation coupled with mass spectrometry, we probed the APOA4 interactome in mouse gonadal fat tissue, where ApoA4 gene is not transcribed but APOA4 protein is abundant. We demonstrate that lipoprotein receptor-related protein 1 (LRP1) is the cognate receptor for APOA4 in adipose tissue. LRP1 colocalized with APOA4 in adipocytes; it interacted with APOA4 under fasting condition and their interaction was enhanced during lipid feeding concomitant with increased APOA4 levels in plasma. In 3T3-L1 mature adipocytes, APOA4 promoted glucose uptake both in absence and presence of insulin in a dose-dependent manner. Knockdown of LRP1 abrogated APOA4-induced glucose uptake as well as activation of phosphatidylinositol 3 kinase (PI3K)-mediated protein kinase B (AKT). Taken together, we identified LRP1 as a novel receptor for APOA4 in promoting glucose uptake. Considering both APOA4 and LRP1 are multifunctional players in lipid and glucose metabolism, our finding opens up a door to better understand the molecular mechanisms along APOA4-LRP1 axis, whose dysregulation leads to obesity, cardiovascular disease, and diabetes.

Ankyrin-R regulates fast-spiking interneuron excitability through perineuronal nets and Kv3.1b K+ channels.

Neuronal ankyrins cluster and link membrane proteins to the actin and spectrin-based cytoskeleton. Among the three vertebrate ankyrins, little is known about neuronal Ankyrin-R (AnkR). We report AnkR is highly enriched in Pv+ fast-spiking interneurons in mouse and human. We identify AnkR-associated protein complexes including cytoskeletal proteins, cell adhesion molecules (CAMs), and perineuronal nets (PNNs). We show that loss of AnkR from forebrain interneurons reduces and disrupts PNNs, decreases anxiety-like behaviors, and changes the intrinsic excitability and firing properties of Pv+ fast-spiking interneurons. These changes are accompanied by a dramatic reduction in Kv3.1b K+ channels. We identify a novel AnkR-binding motif in Kv3.1b, and show that AnkR is both necessary and sufficient for Kv3.1b membrane localization in interneurons and at nodes of Ranvier. Thus, AnkR regulates Pv+ fast-spiking interneuron function by organizing ion channels, CAMs, and PNNs, and linking these to the underlying ß1 spectrin-based cytoskeleton.

The major cap-binding protein eIF4E regulates lipid homeostasis and diet-induced obesity.

Obesity is a global epidemic leading to increased mortality and susceptibility to comorbidities, with few viable therapeutic interventions. A hallmark of disease progression is the ectopic deposition of lipids in the form of lipid droplets in vital organs such as the liver. However, the mechanisms underlying the dynamic storage and processing of lipids in peripheral organs remain an outstanding question. Here, we show an unexpected function for the major cap-binding protein, eIF4E, in high-fat-diet-induced obesity. In response to lipid overload, select networks of proteins involved in fat deposition are altered in eIF4E-deficient mice. Specifically, distinct messenger RNAs involved in lipid metabolic processing and storage pathways are enhanced at the translation level by eIF4E. Failure to translationally upregulate these mRNAs results in increased fatty acid oxidation, which enhances energy expenditure. We further show that inhibition of eIF4E phosphorylation genetically-and by a potent clinical compound-restrains weight gain following intake of a high-fat diet. Together, our study uncovers translational control of lipid processing as a driver of high-fat-diet-induced weight gain and provides a pharmacological target to treat obesity.