4.1 Subtype-specific proteome alterations in sCJD
The sCJD has an unusual degree of phenotypic heterogeneity. Based on observations that a certain disease phenotype is associated with prion type and codon 129 genotype, six different sCJD subtypes (MM1, MM2, MV1, MV2, VV1, and VV2) were distinguished. In this study, the two most frequent ones (MM1 and VV2) were analyzed for proteome changes using a differential proteomic approach.
The MM1 subtype is a myoclonic type with cognitive impairment accompanied by mental and visual signs at the onset of disease. In turn, the VV2 subtype is an ataxic type, where dementia usually develops later in the progression of the disease. Differences between subtypes are also clearly found in the histopathology of the brain. For instance, the topography of the MM1-associated lesions shows that the rostral are more severely affected than the caudal brain regions, while in the VV2, it is other way around . Such a high degree of heterogeneity might suggest the involvement of different molecular pathways in the sCJD pathogenesis, depending on which infectious prion strain is involved . The proteomic data we obtained support this hypothesis.
Out of the 34 differentially expressed proteins identified, only seven showed a common alteration in both analyzed sCJD subtypes. The remaining proteins displayed a subtype-specific difference in the expression level. Interestingly, some of them, such as aconitate hydratase or macrophage migration inhibitory factor, showed a lower expression level in the MM1, while they showed a higher one in the VV2 subtype.
Since both codon 129 genotype and PrPSc type have an influence on the expression level of the brain protein, one could ask if codon 129 genotype alone may also influence the state of the brain proteome. We screened expression levels of sCJD differentially expressed proteins in the control group, which consists of three different codon 129 genotypes (MM, MV, and VV), and we did not find significant differences between genotypes for almost all of the proteins. Three proteins are the exception: septin-11, and both 14–3-3 isoforms. It seems that the presence of methionine in the codon 129 is associated with an increased level of septin-11, and the presence of valine in the codon 129 is associated with an increased level of 14–3-3. This fact might explain why these proteins did not fulfill the significance requirement in the statistical evaluation.
The sCJD-related proteins identified in this study can be classified in a few different functional blocks. The most striking difference between subtypes was found within proteins involved in the cell cycle and death, as well as in ones related to the cell structure and motility.
Supporting Information File 2 provides a detailed description of the biological function of sCJD-related proteins, as well some already known links to prion pathophysiology or other neurodegenerative processes.
Two independent studies showed that four major human prion strains largely correlate with widely accepted sCJD classification into six subtypes: MM1 + MV1, MV2 + VV2, MM1, and VV2 [10, 11]. It is likely that these prion strains are characterized by different conformation of PrPSc, which may affect interactions of cellular prion protein with PrPSc and/or other molecular partners in a way that different molecular pathways are involved in the pathogenesis of prion diseases. For instance, MM1-sCJD-associated cofilin-1, which regulates actin cytoskeleton dynamics, is a known interactor of both cellular prion protein and PrPSc [12, 13]. The disrupted interaction between cofilin-1 and PrP might cause the formation of actin rods, which leads to cellular transport deficits and synaptic dysfunction, as it is observed in other neurodegenerative diseases (reviewed in ).
4.2 Rab3a recycling in sCJD
Synaptic vesicle exocytosis serves as the nervous system's main form of cell-to-cell communication, and it is a multi-step, tightly regulated mechanism. Modifications to any of these steps may change the strength of synaptic connections or their integrity. Growing evidence indicates that synaptic dysfunction is a key process in the development of many neurodegenerative disorders, including sCJD. Furthermore, it is very likely that synaptic failure and loss already occur before the onset of the disease. For instance, scrapie-infected mice show changes in motivational behavior long before the appearance of motor signs, and this correlates with the initial loss of presynaptic terminals in the dorsal hippocampus [15, 16].
Our proteomic data indicated that some of the sCJD-related proteins are involved in signal transduction and neuronal activity. Interestingly, for almost all of them, a decreased level of expression was found in both investigated subtypes. The exception is αGDI in the VV2 subtype, where one out of three differentially expressed isoforms detected on 2D maps showed 3-fold upregulation. Due to some posttranslational modification, most probably phosphorylation [17, 18], these isoforms vary in their isoelectric point, while molecular weight remains the same. Therefore, 1D analysis via Western blot revealed only one band confirming a slight but significant increase in the αGDI level when compared to the control group.
αGDI regulates the function of several Rab proteins, including Rab3a [19, 20], which is crucial for the Ca2+-dependent exocytosis of synaptic vesicles [21, 22]. Rabs are functionally active when located on the membrane, but they are inactive when associated with GDI in the cytoplasm. Thus, Rab distribution between membranes and cytoplasm indicates its functional state. Therefore, we investigated Rab3a levels in both the membrane and the cytosol fraction, and we found that the pool of membrane-bound Rab3a is significantly increased in both sCJD subtypes. This suggests that either Rab3a is accumulating on the membrane due to its altered cycling or that there is some compensation mechanism that increases levels of the active Rab3a form. Since αGDI was also identified as a sCJD-related protein, we tended to assume that disrupted αGDI-mediated Rab3a recycling may cause Rab3a accumulation on the membrane.
It has been already shown that the major pool of the cytosolic αGDI form is mainly in a complex with Rab3a, while the minor pool of membrane-associated αGDI forms a complex with chaperons, including Hsp90. Moreover, αGDI interaction with the Hsp90-containing chaperone complex is required for efficient Rab3a recovery from the membrane and Ca2+-dependent neurotransmitter release [23, 24]. In our studies, we observed subtype-specific differential expression of the Hsp90. In both, cytosol and membrane of the VV2 subtype, a highly elevated Hsp90 level positively correlates with an elevated αGDI level. In the case of the MM1 subtype, a decreased level was found in both fractions. Moreover, αGDI and Hsp90 cytosol versus membrane ratios were significantly lower in the MM1 when compared to the control group and the other sCJD subtypes. This suggests an altered cellular distribution, which might also affect their biological activity.
It should be mentioned that these subtype-specific alterations of Rab3a recycling could be influenced by other factors, which were not analyzed in these studies. Moreover, using our approach we were not able to show whether only a particular isoform of αGDI or Hsp90, or rather all of them are involved in Rab3a recycling.