Manganese Health Research Program: Phase 2, Core 11

Project Objectives:

• To determine the role of octapeptide repeat sequences on manganese-induced prion protein accumulation and stability.
• To determine whether deletion or repeat insertions of the octapeptide sequence in prion protein modify manganese-induced proteinase K-resistant prion protein (PrPsc), protein aggregation and neurotoxicity in scrapie–infected neuronal cells.

Project Description:

Prion disease is a devastating neurodegenerative disorder that causes fatalities in animals and humans. Unlike conventional infectious diseases, prion diseases are caused by an abnormally folded host-encoded prion protein that accumulates in the central nervous system. The cellular function of this protein remains to be elucidated but studies have suggested it is a metalloprotein with a binding affinity for divalent cations.  Emerging studies have shown that prion proteins contain octapeptide-repeat regions that bind to several divalent metals, including manganese (Mn) and copper (Cu), and that the metal binding may influence the confirmation and metabolism of prion proteins. Therefore, the long term objective of our project is to determine whether divalent metal Mn plays any role in the pathogenesis of prion diseases. During the previous funding period, we reported that normal prion protein impairs manganese transport and protects the cells from manganese-induced oxidative stress, mitochondrial dysfunction, cellular antioxidant depletion, and apoptosis.  We also reported that Mn treatment results in increased prion protein levels in mouse neuronal cells. During the current funding period, we continued to study the interaction of manganese with prion protein and made several interesting observations. Mn treatment in mouse brain slice cultures upregulated prion protein by stabilizing the protein in a time-dependent manner. Since manganese has been suggested to compete with copper for binding to the octapeptide repeat region of prion protein, we examined whether Cu treatment upregulates and stabilizes the prion protein as it does with Mn. We found copper treatment not only upregulated prion protein but also caused the shedding of prion proteins into the extracellular milieu.  Additional studies with another divalent metal cadium (Cd) revealed that the metal can potently inhibit proteasomal activity, which results in greatly increased formation of high molecular weight ubiquitinated proteins.  Immunohistochemical analysis also revealed a dramatic increase in the formation of oligomers after Cd treatment, which leads to ubiquitinated PrP, but did not lead to formation of PK-resistant PrP.  Further, we examined Mn-neurotoxicity in scrapie-infected neuronal cells and found that infected cells are more resistant to both Mn-induced cytotoxicity and Mn-induced apoptosis.   We partially attribute this response to the inability of manganese to access its binding site in the scrapie protein.  The Mn-binding site may be masked due to the presence of the scrapie prion protein in the oligomeric form and/or aggregates. Current studies are underway to determine the role of octapeptide repeats in manganese-induced stabilization of prion protein and its relevance to the pathogenesis of prion diseases.

Research Progress/Key Key research accomplishments:

We were funded last year to study the effect of manganese on prion protein function, turnover and proteinase K resistance and protein aggregation in cell culture models of prion diseases. We have made significant progress in the proposed studies, and several interesting discoveries are summarized below:

• Manganese upregulated prion protein by increasing the stability of the protein,
• Manganese neither altered the prion mRNA levels nor inhibited proteasomal function,
• Manganese reduced the proteinase-K dependent proteolysis of prion protein.
• Similar to manganese, copper upregulated prion protein independent of transcriptional activation and proteasomal degradation.
• Low dose copper enhances the antioxidant capacity of prion protein.
• Copper also reduced the proteinase-K dependent proteolytic rate of PrPc, suggesting a common mechanism of protein stability by manganese and copper.
• Copper induces the shedding of prion protein into the extracellular milieu.
• Manganese induced toxicity in the scrapie infected SN56sc cell model of prion diseases but the toxicity was less than the toxicity induced in uninfected cells.
• Manganese did not alter the proteasome activity in uninfected and scrapie infected cells.
• Currently, we are studying the role of octapeptide sequences in manganese-induced prion protein upregulation. 

Publications resulting from the project:

• Choi C. J., Anantharam, V., Nicholson, EM., Richt, J.A, Kanthasamy, A. and Kanthasamy, A.G (2009). Manganese stabilizes cellular prion protein and alters the Rate of Proteinase-K-Dependent Limited Proteolysis in Neuronal cells.  Prepared for submission to Tox. Sci
• Choi C. J., Anantharam, V., Nicholson, E.M., Richt, J.A, Kanthasamy, A. and Kanthasamy, A.G (2006).  Copper upregulates cellular Prion proteins and inhibits the rate of Proteinase-K dependent limited proteolysis in neuronal cells. Under preparation.
• Choi C. J., Anantharam, V., Nicholson, EM., Richt, J.A, Kanthasamy, A. and Kanthasamy, A.G (2009). Manganese stabilizes cellular prion protein and alters the Rate of Proteinase-K-Dependent Limited Proteolysis.  Presented at the 2008 Neuroprion meeting at Madrid, Oct 8-10, 2008.
• Choi, C.J., Anantharam, V., Saetveit, N.J., Houk, R.S., Kanthasamy, A., Kanthasamy AG. (2007), Normal cellular prion protein protects against manganese-induced oxidative stress and apoptotic cell death. Toxicol. Sci., 98: 495-509.
• Anantharam V, Kanthasamy A, Choi CJ, Martin DP, Latchoumycandane C, Richt JA, Kanthasamy AG. (2008) Opposing roles of prion protein in oxidative stress- and ER stress-induced apoptotic signaling. Free Radical Biol. Med. 45(11):1530-41.

Last updated: May12, 2009