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ABSTRACT OF THE THESISPACSIN1, an intracellular neuronal protein, activates LRP1 dependent cell signaling in Schwann CellsbyMark Carter as listed on UC San Diego official Academic RecordsMaster of Science in BiologyUniversity of California San Diego, 2019Professor Wendy Campana, ChairPeripheral nerves have the unique ability to regenerate following injury, as opposed to nerves in the central nervous system (CNS) which do not spontaneously regenerate. The ability of peripheral nerves to regenerate is due to the remarkable and versatile principle glia cell of the peripheral nervous system (PNS), the Schwann cell (SC). SCs have the ability to detect injury in peripheral nerves, such as the dorsal root ganglion (DRG), and transform phenotypically to facilitate nerve repair. Downstream cell-signaling pathways and changes in gene expression that drive SC phenotypic transformation in injury have been previously described as well as SC receptors that detect injury. LDL receptor-related protein (LRP1) is a receptor for numerous ligands, including protein components of degenerated myelin. After injury, LRP1 is dramatically upregulated in SCs and functions with the N-methyl-D-aspartate receptor (NMDA-R) to trigger cell signaling in response to ligands. Using a capture tech¬nology and LC-MS/MS to identify novel LRP1 ligands in the injured nerve, we identified Protein kinase C and casein kinase substrate in neurons protein 1, PACSIN1, as a LRP1ligand. Not much is known of PACSIN1 as it pertains to the PNS injury. Herein, I demonstrate, for the first time, that PACSIN 1 is specifically localized in the cell bodies and axons of both large and small sensory neurons in the dorsal root ganglia (DRGs), confirming neuronal specificity. I hypothesized that after injury PACSIN1 is released by dorsal root ganglia (DRG) neurons and binds to Schwann cell LRP1 receptor. Indeed, when rhPACSIN was added to primary SC cultures, LRP1-dependent cell signaling was activated. PACSIN1 robustly activated c-Jun, the SC repair protein, and ERK1/2 in SCs within 10 minutes. This response was blocked when LRP1 was genetically silenced or when the NMDA-Receptor was pharmacologically antagonized with MK801. These data further confirm that the NMDAR is an active co-receptor for LRP1 in SCs. Finally, I demonstrate, for the first time, that expression of PACSIN1 in DRGs is significantly decreased by 5 days after sciatic nerve injury, suggesting that PACSIN1 may play a role in SC signaling in early stages after injury. We conclude that the Schwann cell LRP1 receptor is indeed responsive to intracellular neuronal proteins released at injury. Our results support a model in which SC LRP1 serves as a major injury-induced receptor in the peripheral nervous system.

Neural stem cell (NSC) damage has been reported in patients with Alzheimer’s disease. Intracellular Aβ plays a vital role in NSC damage. Heparan sulfate proteoglycans are potent mediators of Aβ enrichment in the brain. We hypothesized the heparan sulfate proteoglycan glypican 4 (Gpc4) regulates Aβ internalization by NSCs. We evaluated Gpc4 expression in NSCs from P0–P2 generations using immunofluorescence. Adenovirus and lentivirus were used to regulate Gpc4 expression in NSCs and APP/PS1 mice, respectively. Co-immunoprecipitation was used to determine the relationship between Gpc4, Aβ, and low-density lipoprotein receptor-related protein 1 (LRP1). Intracellular Aβ concentrations were detected using enzyme-linked immunosorbent assay and immunofluorescence. The role of Gpc4/LRP1 on toxic/physical Aβ-induced effects was evaluated using the JC-1 kit, terminal deoxynucleotidyl transferase dUPT nick end labeling, and western blotting. Gpc4 was stably expressed in NSCs, neurons, and astrocytes. Gpc4 was upregulated by Aβ in NSCs and regulated Aβ internalization. Gpc4 attenuation reduced Aβ uptake; Gpc4 overexpression increased Aβ uptake. Gpc4 regulated Aβ internalization through LRP1 and contributed to Aβ internalization and toxic/physical concentrations of Aβ-induced mitochondrial membrane potential and cell apoptosis, partly via LRP1. Therefore, Gpc4 is a key regulator of Aβ enrichment in NSCs. Inhibiting Gpc4 rescued the Aβ-induced toxic effect and attenuated the nontoxic Aβ enrichment into intracellular toxic concentrations. Gpc4 contributed to Aβ internalization and toxic/physical concentrations of Aβ-induced mitochondrial membrane potential damage and cell apoptosis, partly via LRP1. These findings suggest a potential role of Gpc4 in treating Alzheimer’s disease at an early stage, by targeting NSCs.

Low density lipoprotein receptor-related protein 1 (LRP1) is involved in the catabolism of many ligands, including factor VIII (FVIII) and alpha-2-macroglobulin (α2M). Transfer of FVIII to LRP1 is currently believed to be preceded by pre-concentration on the cell surface, by interacting with a so far unidentified component. In the present study, we used confocal microscopy and flow cytometry to compare endocytosis of FVIII and α2M using U87MG cells. The results show that α2M is rapidly internalized and does not compete for LRP1 mediated internalization of FVIII. FVIII endocytosis did not occur in the presence of receptor-associated-protein (RAP), but FVIII remained visible as a striated fluorescent pattern at the cell borders. In the presence of Von Willebrand Factor (VWF), no FVIII was observed on or within the cells, suggesting that VWF blocks interaction with both cell surface and LRP1. The same dual inhibition has previously been observed for FVIII C1 domain directed monoclonal antibody KM33. Elimination of the KM33 epitope by replacing FVIII C1 residues 2091-2095 and 2155-2160 for the homologues from factor V (FV), however, did not impair FVIII endocytosis. These membrane spikes alone were insufficient for cellular uptake, because FV was neither internalized by U87MG cells nor capable of effectively competing for FVIII endocytosis. These results show that FVIII endocytosis is driven by interaction with LRP1, but at the same time involves the spikes in the C1 domain that have been implicated in lipid binding.