Proteomic Basis for Chronic Anatomical Plasticity of Synapses
Overview: We are interested in how the anatomical stability versus plasticity of brain wiring is accomplished, and how it is altered by disease. In addiction the rewiring of synaptic connections driven by biochemical changes underlies altered behavior. We study the mechanistic relationship of neuroproteomic patterns to forebrain synaptic turnover and rearrangement during the loss of synapses driven by trauma in spinal cord injury and traumatic brain injury, as well as by misfolded protein aggregation in Alzheimer's Disease and Fronto-Temporal Lobar Degeneration (R35NS097283; U01AG058608; R01AG034924; RF1AG053000; P50AG047270). Our goal is to define the molecular interactions driving synapse loss and the signal transduction mechanisms that link Aßo accumulation to synaptic degeneration. By understanding the basic protein chemistry of synapse stability control in the adult brain across this spectrum of conditions, the basis for disruption in addiction will be clarified.
Our previous investigation of the neurotoxic signaling of amyloid beta oligomers (Aßo) has uncovered critical roles for PrP-C (cellular prion protein), mGluR5 (metabotropic glutamate receptor 5), Fyn (tyrosine-protein kinase Fyn), and Pyk2 (protein-tyrosine kinase 2-beta). Both genetic and pharmacological inhibition of PrP-C, mGluR5, Fyn, and Pyk2 have been demonstrated to rescue synapse loss as well as memory and learning deficits in transgenic mouse models of Alzheimer’s disease. Further, the Fyn inhibitor AZD0530 has been demonstrated safe in Alzheimer’s disease patients and a Phase 2a study is underway (NCT02167256). Previously we have shown that there are early changes in synaptic gene expression in the APPswe/PS1dE9 and are linked to inflammatory changes. To extend our understanding of the neurotoxic signaling induced by Aßo, as well as identify signaling molecules downstream of Fyn and Pyk2, we conducted a proteomic label-free mass spectrometry study of 8 groups of mice with various genetic or pharmacological manipulations: in one (WT vs APPswe/PS1dE9)x(WT vs Pyk2) and in another (WT vs APPswe/PS1dE9)x(Veh vs AZD0530).
We are studying the entire proteome with a focus on synaptic proteins, as well as samples enriched for phospho-proteins. Recent data have revealed increased JNK3 levels and phospho-JNK3 in APPswe/PS1∆E9 mice. In WT mice, loss of Pyk2 does not alter these levels, but the elevation in AD mice is suppressed to WT levels by deletion of Pyk2 (Cox et al, 2019). These unbiased data fit with previous evidence for JNK activation in AD mouse models and with Pyk2 activation of JNK in non-neuronal cells. We have now utilized these data to guide histological and immunoblot studies in APPswe/PS1dE9 mice. We find that p-Jun levels are increased in the hippocampus and cerebral cortex of these mice. Because JNK activation has been linked to neuronal apoptosis, this expands the role of the Aß/PrP/mGluR5/Fyn/Pyk2 signaling pathway from synaptic dysfunction to cell loss.
Due to the essential but ill-defined role of Pyk2 in AD signal transduction we extracted Pyk2 interacting proteins from brain by affinity chromatography. Isolated proteins were analyzed by mass spectrometry, and Graf1 a rhoA regulating protein was identified. We verified that Graf1 mediates Aßo driven, Pyk2-mediated effects on dendritic spine anatomical loss (Lee et al, 2019).
Literature Cited
Cox, T. O. et al. (2019) Anti-PrP(C) antibody rescues cognition and synapses in transgenic alzheimer mice. Ann Clin Transl Neurol 6, 554-574.
Lee, S., Salazar, S. V., Cox, T. O. & Strittmatter, S. M. (2019) Pyk2 Signaling through Graf1 and RhoA GTPase Is Required for Amyloid-beta Oligomer-Triggered Synapse Loss. J Neurosci 39, 1910-1929.