Funded Studies
Objective/Rationale:
We demonstrated that Ceruloplasmin (Cp), an enzyme present in cerebrospinal fluid (CSF), is oxidized in the CSF of PD patients. Among oxidative modifications, the asparagine deamidation is of interest because Cp’s aminoacid-sequence contains specific motifs that upon deamidation may acquire binding properties to membrane receptors. If Cp deamidation occurs in the oxidative milieu of the neurodegenerative disease, it is conceivable that out of to lose its physiological function, Cp might gains new detrimental pro-adhesive function.
Project Description:
To validate the hypothesis that the oxidized Cp present in the CSF of PD patients might contributes to pathological mechanisms we first assess by immunocapture and mass spectrometry analysis whether the specific motifs of the endogenous Cp are deamidated in vivo in the CSF of PD patients, like we known it occurs in vitro, using CSF from healthy subjects as control; then we will define whether the deamidated Cp from PD patients became able to bind membrane receptors and favours cellular adhesion. After that we will investigate by using protein microarrays whether pro-adhesive oxidized/deamidated Cp is able to transduce an intracellular signal throughout the receptors engagement. Finally we will look whether this binding affects cellular functions like, proliferation and viability, morphological tissue organization.
Relevance to Diagnosis/Treatment of Parkinson’s Disease:
The effects that oxidative stress can cause in CNS of PD patients, through Cp oxidation might have important clinical implications both as biomarker and for therapeutic intervention. If validated, the hypothesis of the Cp gain of pro-adhesive function would highlight a completely new scenario of possibly detrimental pathological mechanisms like dysfunction of the choroid plexus epithelia barrier, or modification of the subventricular neural stem cell niche altering the balance between cells injury and repair.
Anticipated Outcome:
This research will allow us to define whether the spontaneous modifications that occur in protein aging are accelerated in the CSF of PD patients as consequence of changes in environmental redox status of CSF. Results will indicate whether the gain of pro-adhesive function of Cp will disclose new pathological mechanisms mediated by anomalous signal transduction of cells expressing membrane receptors, that may be the potential targets of innovative therapeutic strategies.
Final Outcome
Our results indicated that the ceruloplasmin present in the cerebrospinal fluid (CSF) of Parkinson’s disease (PD) patients undergoes to peculiar modification (namely deamidation) in a specific NGR-motif, as consequence of structural changes induced by pro-oxidant pathological environment. Upon oxidation and deamidation, ceruloplasmin loss its physiological ferroxidase activity with detrimental consequences on brain iron homeostasis. In addition to loss of function, the pathological CSF from both PD and Alzheimer’s disease fosters ceruloplasmin deamidation that results in a NGR-mediated gain of new binding function to integrins, a family of cell surface receptor molecules. Via integrin binding, the deamidated-ceruloplasmin promotes epithelial and astrocytes cells adhesion and spreading, suggesting that modified ceruloplasmin may affect cellular functions. Indeed, the oxidized/deamidated-ceruloplasmin is able to transduce an intracellular signal via integrin-binding that results in cell cycle arrest and inhibition of cell proliferation. These effects are evident on epithelial and neural stem cells but are not effective on neuronal and astrocytes cell lines, indicating a cells specificity.
We found that in PD brain the ceruloplasmin seems to be included in plaques as possible consequence of protein aggregate formation. Indeed, we demonstrated that protein aging under oxidative conditions induced ceruloplasmin-aggregate formation possibly mediated by an highly reactive intermediate generated during NGR-deamidation.
In conclusion, we identified a specific protein modification occurring in the CSF of PD patients that fosters loss of physiological function and gain of new function, both events may have important role in Parkinson’s disease pathological mechanisms.
Presentations & Publications
Articles:
Barbariga M, Curnis F, Spitaleri A, Andolfo A, Zucchelli C, Lazzaro M, Magnani G, Musco G, Corti A, Alessio M. Oxidation-induced structural changes of ceruloplasmin foster NGR-motifs deamidation that promote integrin binding and signalling. J Biol Chem 298: 3736-48, 2014.
Barbariga M, Curnis F, Andolfo A, Zanardi A, Lazzaro M, Conti A, Magnani G, Volontè MA. Coppi E, Ferrari L, Corti A, Alessio M. Ceruloplasmin functional changes in Parkinson's disease-cerebrospinal fluid. (2014, submitted)
Abstracts:
Barbariga M, Olivieri S, Conti A, Curnis F, Andolfo A, Iannaccone S, Campanella A, Codazzi F, Magnani G, Corti A, Alessio M. Ceruloplasmin oxidation, a feature of Parkinson’s and Alzheimer’s diseases, inhibits ferroxidase activity and promotes cellular iron retention. IBIS, International Bioiron Society, 5th Congress, Biennal World Meeting. April 14-18, 2013 university College Lonson, London UK.
June 2014