Within the “Prion Respiration” project we used N2a cells as model to investigate the proteomic consequences following the infection of two different prion strains, denotes as 22L and RML.
The relative apparent abundance of the proteins between sample groups has been examined with the goal of comparing the global effects of two prion strains on the proteome profiles of isolated mitochondria from uninfected and prion infected N2a cells. A total of 72 and 87 differentially expressed proteins (DEP) was identified in RML and 22L prion infected cells, respectively. Interestingly, these DEP have a strong association with each prion strain suggesting a strain dependent effect on the mitochondrial proteome.
Conserved DEP among the two cell lines include upregulated proteins involved, for instance, in the regulation of reactive oxygen species, while down-regulated DEP mainly include functions such as mitochondrion organization and calcium homeostasis.
More specifically, the RML prion infection resulted in the down-regulation of proteins involved in the oxidative phosphorylation system, OXPHOS, including CI, CIV and CV subunits, assembly factors and electron transfer components. Mitochondria from N2a cells infected with the 22L prion strain display different DEP signatures with mainly upregulated proteins involved in the oxidative stress response and protein unfolding response.
Proteomic differences in the OXPHOS observed in RML and 22L prion-infected cells may have an impact on the expression levels of key scaffolding proteins responsible for respiratory complexes formation as the AF. To test this hypothesis we run WB experiments using antibodies targeting three key AF such as ACAD9, ECSIT and NDUFAF1 and the CI subunit NDUFV1 and the outer membrane mitochondrial channel VDAC1.
We examined whether the chemical induction of mitochondrial dysfunctions cause severe changes in the expression levels of these proteins compared to untreated cells. The antibiotic chloramphenicol (Chl) was used as mitochondrial inhibitor able to cause OXPHOS dysfunctions.
Analysis on total protein content in the untreated cells showed that ACAD9 expression levels remain constant in the three cell lines, while ECSIT and NDUFAF1 are upregulated in both 22L and RML prion-infected cells. NDUFV1 levels appear down-regulated in 22L and RML N2a cells suggesting a decrease in the content of CI subunits as documented in our differential proteomic analysis.
We propose that the increased expression of these AF components represents an adaptative change of the cell to reduced levels of CI, as inferred by our proteomic analysis. During prion replication, AF may be required to stabilize partially assembled and intermediate modules of CI in cases where CI subunits are damaged, poorly expressed or assembly is compromised.
This preliminary study lays the foundation for further investigation on proteomic and cell biology experiments on the role of AF during different neurodegenerative diseases using different cellular and in vivo models for neurodegeneration.
The rationale of the “Prion Respiration” project and these preliminary results have been disseminated through international congresses and seminars. These events include: one poster presentation (at the “ESRF User Meeting 2020”, February 3-5 2020,Grenoble, France), one oral presentation in a congress (1st Italian Crystallographic Association - Biological Macromolecules Group Meeting, February 20-21 2020, Fiesole, Italy), one seminar at the University of Verona – Italy (on November 8 2019) and two seminars at the University of Trento – Italy (on November 11 2019 and on April 28 2020).
