Estimating Neuroprotective Effects of Nigral AAV-NRTN Using Selective Reaction Monitoring to Quantitatively Measure p-erk1/2 Signaling Strength in Nigral DA Neurons
MJFF Research Grant, 2011
Pre-clinical studies indicate that neurotrophic factor gene therapy has the potential to attenuate, delay and perhaps prevent the progressive loss of nigral DA neurons in aging and neurotoxic models of PD. The outcome of a recent Phase II clinical trial suggests that optimization of delivery and/or targeting strategies will be required to realize the true potential of neurotrophic factor gene therapy in the clinic. Recent findings from pre-clinical studies demonstrate that properly scaled and targeted AAV-Neurturin (NRTN) to the substantia nigra (SN) is safe, effective, and causes no weight loss in pre-clinical models. Additional, unpublished findings suggest that SN targeting of AAV-NRTN to degenerating nigrostriatal neurons results in more robust NRTN immunohistochemical signal, along with greater dopaminergic cell preservation against 6-OHDA compared to that achieved with striatal (STR) targeting alone. Importantly, immunoreactivity for a cellular marker of neurotrophic factor-mediated bioactivity, phosphorylated extracellular regulated kinase 1/2 (p-ERK1/2), appeared more robust following SN delivery of AAV-NRTN or combined SN+STR delivery, providing qualitative evidence for more robust neurotrophic intracellular signaling following SN and SN+STR targeting versus STR only targeting. However, to achieve important topographic resolution regarding the precise location of the changes in intracellular signaling pathways (such as p-ERK 1/2 activation), new techniques, such as targeted mass spectrometry, are needed to enable accurate and sensitive quantification of specific protein modifications in an antigen/antibody-independent manner from histological sections.
The overall goals of this grant application are (1) to demonstrate ‘proof of concept’ that a targeted mass spectrometry (MS) approach may be a powerful tool to quantitatively and sensitively measure strength of signaling pathways activated in DA neurons (and presumably other neuronal populations, as well) as a result of neurotrophic gene transfer, thus enabling rigorous and robust assessment of outcomes after treatment strategies in ways that cannot otherwise be done by conventional immunohistochemical approaches; and (2) to generate data demonstrating that targeting degenerating SN neuronal cell bodies with AAV-NRTN offers advantages over striatal (i.e., nerve terminal) targeting alone for inducing a robust neurtrophic response in degenerating neurons- a possibility that has not yet been fully explored, and indeed runs counter to current dogma.
Relevance to Diagnosis/Treatment of Parkinson’s Disease:
Successful completion of these studies will provide new and valuable information on how this targeted MS approach might be useful to quantify other proteins and their post-translational modifications in healthy or diseased SN in an antibody-independent manner and/or interrogate other signaling pathways in brain or CSF to help guide PD diagnosis or assess treatments.
Completion of the proposed studies will achieve the following:
1) Proof of concept on use of a targeted mass spectrometry-based approach to quantify proteins in DA neurons as a measure of protective neurotrophic activity induced by AAV-NRTN injections into both the pre-clinical SNpc+STR (substantia nigra pars compacta plus striatum) or the STR only.
2) Additional proof of principle that nigral targeting of AAV-NRTN offers significant advantages over STR-only targeting by providing quantitative evidence for greater neurotrophic factor signaling, thus supporting the basis for an ongoing clinical trial of AAV-NRTN in PD subjects, and potentially future trials using any neurotrophic factor approach.
INTERIM PROGRESS REPORT
Quantification of the phosphoproteome induced by an intrastriatal 6-OHDA lesion and the “rescuing” effects of AAV-NRTN gene therapy has enabled rigorous and robust assessment of signaling outcomes in ways that cannot otherwise be done by conventional immunohistochemical approaches. Particular peptides of interest rescued by AAV-NRTN with consensus 6-OHDA sensitive sites identified in both phases of the project included multiple phosphosites on MBP, CAMK2B, MAP1A, CRMP2/DPYSL2, and SIRT2. The sites on SIRT2 and CRMP2 have been published previously to regulate protein lysine deacetylase activity and growth cone formation, respectively. Completion of the proposed studies represents ‘proof of concept’ that a tandem mass spectrometry (MS) approach is a powerful tool to quantitatively and sensitively measure strength of signaling pathways activated in the nigrostriatal pathway as a result of NRTN neurotrophic gene transfer. Because this tandem MS approach allows quantification of the phosphoproteome in an antibody-independent manner and interrogation of multiple signaling pathways in an unbiased (hypothesis-indpendent) manner, it may help identify novel biomarkers in CSF for PD diagnosis or treatment evaluation.
Associate Professor of Physiology at Emory University School of Medicine
Location: Atlanta, Georgia, United States
Assistant Professor of Biochemistry and Neurology at Emory University School of Medicine
Location: Atlanta, Georgia
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