Polypharmacological Perturbation of the 14-3-3 Adaptor Protein Interactome Stimulates Neurite Outgrowth

Targeting protein-protein interactions (PPIs) is a promising approach in the development of drugs for many indications. 14-3-3 proteins are a family of phosphoprotein-binding molecules with critical functions in dozens of cell signaling networks. 14-3-3s are abundant in the central nervous system, and the small molecule fusicoccin-A (FC-A), a tool compound that can be used to manipulate 14-3-3 PPIs, enhances neurite outgrowth in cultured neurons. New semisynthetic FC-A derivatives with improved binding affinity for 14-3-3 complexes have recently been developed. Here, we use a series of screens that identify these compounds as potent inducers of neurite outgrowth through a polypharmacological mechanism. Using proteomics and X-ray crystallography, we discover that these compounds extensively regulate the 14-3-3 interactome by stabilizing specific PPIs, while disrupting others. These results provide new insights into the development of drugs to target 14-3-3 PPIs, a potential therapeutic strategy for CNS diseases. Kaplan et al. identify a derivative of the small molecule fusicoccin-A (FC-A) that stimulates neurite outgrowth by regulating dozens of protein-protein interactions (PPIs) in the 14-3-3 adaptor protein interactome. This compound, FC-NCPC, bidirectionally stabilizes and inhibits 14-3-3 PPIs enriched within the Rap1 signaling network, suggesting new targets for CNS indications

3-Dimensional Immunostaining and Automated Deep-Learning Based Analysis of Nerve Degeneration

Multiple sclerosis (MS) is an autoimmune and neurodegenerative disease driven by inflammation and demyelination in the brain, spinal cord, and optic nerve. Optic neuritis, characterized by inflammation and demyelination of the optic nerve, is a symptom in many patients with MS. The optic nerve is the highway for visual information transmitted from the retina to the brain. It contains axons from the retinal ganglion cells (RGCs) that reside in the retina, myelin forming oligodendrocytes and resident microglia and astrocytes. Inflammation, demyelination, and axonal degeneration are also present in the optic nerve of mice subjected to experimental autoimmune encephalomyelitis (EAE), a preclinical mouse model of MS. Monitoring the optic nerve in EAE is a useful strategy to study the presentation and progression of pathology in the visual system; however, current approaches have relied on sectioning, staining and manual quantification. Further, information regarding the spatial load of lesions and inflammation is dependent on the area of sectioning. To better characterize cellular pathology in the EAE model, we employed a tissue clearing and 3D immunolabelling and imaging protocol to observe patterns of immune cell infiltration and activation throughout the optic nerve. Increased density of TOPRO staining for nuclei captured immune cell infiltration and Iba1 immunostaining was employed to monitor microglia and macrophages. Axonal degeneration was monitored by neurofilament immunolabelling to reveal axonal swellings throughout the optic nerve. In parallel, we developed a convolutional neural network with a UNet architecture (CNN-UNet) called BlebNet for automated identification and quantification of axonal swellings in whole mount optic nerves. Together this constitutes a toolkit for 3-dimensional immunostaining to monitor general optic nerve pathology and fast automated quantification of axonal defects that could also be adapted to monitor axonal degeneration and inflammation in other neurodegenerative disease models

Collapsin Response Mediator Protein 4 (CRMP4) Facilitates Wallerian Degeneration and Axon Regeneration following Sciatic Nerve Injury

In contrast to neurons in the CNS, damaged neurons from the peripheral nervous system (PNS) regenerate, but this process can be slow and imperfect. Successful regeneration is orchestrated by cytoskeletal reorganization at the tip of the proximal axon segment and cytoskeletal disassembly of the distal segment. Collapsin response mediator protein 4 (CRMP4) is a cytosolic phospho-protein that regulates the actin and microtubule cytoskeleton. During development, CRMP4 promotes growth cone formation and dendrite development. Paradoxically, in the adult CNS, CRMP4 impedes axon regeneration. Here, we investigated the involvement of CRMP4 in peripheral nerve injury in male and female Crmp4/ mice following sciatic nerve injury. We find that sensory axon regeneration and Wallerian degeneration are impaired in Crmp4/ mice following sciatic nerve injury. In vitro analysis of dissociated dorsal root ganglion (DRG) neurons from Crmp4/ mice revealed that CRMP4 functions in the proximal axon segment to promote the regrowth of severed DRG neurons and in the distal axon segment where it facilitates Wallerian degeneration through calpain-dependent formation of harmful CRMP4 fragments. These findings reveal an interesting dual role for CRMP4 in proximal and distal axon segments of injured sensory neurons that coordinately facilitate PNS axon regeneration.Fil: Girouard, Marie Pier. McGill University. Montreal Neurological Institute and Hospital; CanadáFil: Simas, Tristan. McGill University. Montreal Neurological Institute and Hospital; CanadáFil: Hua, Luyang. McGill University. Montreal Neurological Institute and Hospital; CanadáFil: Morquette, Barbara. McGill University. Montreal Neurological Institute and Hospital; CanadáFil: Khazaei, Mohamad R.. McGill University. Montreal Neurological Institute and Hospital; CanadáFil: Unsain, Nicolas. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Johnstone, Aaron D.. McGill University. Montreal Neurological Institute and Hospital; CanadáFil: Rambaldi, Isabel. McGill University. Montreal Neurological Institute and Hospital; CanadáFil: Sanz, Ricardo L.. McGill University. Montreal Neurological Institute and Hospital; CanadáFil: Di Raddo, Marie Eve. McGill University; CanadáFil: Gamage, Kanchana. University of Virginia; Estados UnidosFil: Yong, Yu. University of Virginia; Estados UnidosFil: Willis, Dianna. Weill Cornell Medicine; Estados Unidos. Burke Neurological Institute; Estados UnidosFil: Verge, Valerie M. K.. University of Saskatchewan; CanadáFil: Barker, Philip A.. McGill University. Montreal Neurological Institute and Hospital; Canadá. University of British Columbia; CanadáFil: Deppmann, Christopher. University of Virginia; Estados UnidosFil: Fournier, Alyson. McGill University. Montreal Neurological Institute and Hospital; Canad