Proteomic Analysis of Dorsal Root Ganglia in a Mouse Model of Paclitaxel-Induced Neuropathic Pain.

Paclitaxel is a chemotherapy drug widely used for the treatment of various cancers based on its ability to potently stabilize cellular microtubules and block division in cancer cells. Paclitaxel-based treatment, however, accumulates in peripheral system sensory neurons and leads to a high incidence rate (over 60%) of chemotherapy induced peripheral neuropathy. Using an established preclinical model of paclitaxel-induced peripheral neuropathy (PIPN), we examined proteomic changes in dorsal root ganglia (DRG) of adult male mice that were treated with paclitaxel (8 mg/kg, at 4 injections every other day) relative to vehicle-treated mice. High throughput proteomics based on liquid chromatography electrospray ionization mass spectrometry identified 165 significantly altered proteins in lumbar DRG. Gene ontology enrichment and bioinformatic analysis revealed an effect of paclitaxel on pathways for mitochondrial regulation, axonal function, and inflammatory purinergic signaling as well as microtubule activity. These findings provide insight into molecular mechanisms that can contribute to PIPN in patients.

Protein Painting Mass Spectrometry in the Discovery of Interaction Sites within the Acetylcholine Binding Protein.

Nicotinic acetylcholine receptors (nAChRs) are a family of ligand-gated ion channel receptors that contribute to cognition, memory, and motor control in many organisms. The pharmacological targeting of these receptors, using small molecules or peptides, presents an important strategy for the development of drugs that can treat important human diseases, including neurodegenerative disorders. The Aplysia californica acetylcholine binding protein (Ac-AChBP) is a structural surrogate of the nAChR with high homology to the extracellular ligand binding domain of homopentameric nAChRs. In this study, we optimized protein-painting-based mass spectrometry to identify regions of interaction between the Ac-AChBP and several nAChR ligands. Using molecular dyes that adhere to the surface of a solubilized Ac-AChBP complex, we identified amino acid residues that constitute a contact site within the Ac-AChBP for α-bungarotoxin, choline, nicotine, and amyloid-ß 1-42. By integrating innovation in protein painting mass spectrometry with computational structural modeling, we present a new experimental tool for analyzing protein interactions of the nAChR.

The human acetylcholinesterase C-terminal T30 peptide activates neuronal growth through alpha 7 nicotinic acetylcholine receptors and the mTOR pathway.

Acetylcholinesterase (AChE) is a highly conserved enzyme responsible for the regulation of acetylcholine signaling within the brain and periphery. AChE has also been shown to participate in non-enzymatic activity and contribute to cellular development and aging. In particular, enzymatic cleavage of the synaptic AChE isoform, AChE-T, is shown to generate a bioactive T30 peptide that binds to the ⍺7 nicotinic acetylcholine receptor (nAChR) at synapses. Here, we explore intracellular mechanisms of T30 signaling within the human cholinergic neural cell line SH-SY5Y using high performance liquid chromatography (HPLC) coupled to electrospray ionization mass spectrometry (ESI-MS/MS). Proteomic analysis of cells exposed to (100 nM) T30 for 3-days reveals significant changes within proteins important for cell growth. Specifically, bioinformatic analysis identifies proteins that converge onto the mammalian target of rapamycin (mTOR) pathway signaling. Functional experiments confirm that T30 regulates neural cell growth via mTOR signaling and ⍺7 nAChR activation. T30 was found promote mTORC1 pro-growth signaling through an increase in phosphorylated elF4E and S6K1, and a decrease in the autophagy LC3B-II protein. These findings are corroborated in hippocampal neurons and show that T30 promotes dendritic arborization. Taken together, our findings define mTOR as a novel pathway activated by T30 interaction with the nAChR and suggest a role for this process in human disease.

A Novel Bioactive Peptide, T14, Selectively Activates mTORC1 Signalling: Therapeutic Implications for Neurodegeneration and Other Rapamycin-Sensitive Applications.

T14 modulates calcium influx via the α-7 nicotinic acetylcholine receptor to regulate cell growth. Inappropriate triggering of this process has been implicated in Alzheimer's disease (AD) and cancer, whereas T14 blockade has proven therapeutic potential in in vitro, ex vivo and in vivo models of these pathologies. Mammalian target of rapamycin complex 1 (mTORC1) is critical for growth, however its hyperactivation is implicated in AD and cancer. T14 is a product of the longer 30mer-T30. Recent work shows that T30 drives neurite growth in the human SH-SY5Y cell line via the mTOR pathway. Here, we demonstrate that T30 induces an increase in mTORC1 in PC12 cells, and ex vivo rat brain slices containing substantia nigra, but not mTORC2. The increase in mTORC1 by T30 in PC12 cells is attenuated by its blocker, NBP14. Moreover, in post-mortem human midbrain, T14 levels correlate significantly with mTORC1. Silencing mTORC1 reverses the effects of T30 on PC12 cells measured via AChE release in undifferentiated PC12 cells, whilst silencing mTORC2 does not. This suggests that T14 acts selectively via mTORC1. T14 blockade offers a preferable alternative to currently available blockers of mTOR as it would enable selective blockade of mTORC1, thereby reducing side effects associated with generalised mTOR blockade.