Molecular Basis of the Disease State

The infectious agent of CJD is a prion, a normal protein that has misfolded and is capable of inducing misfolds in normal versions of itself. In the case of CJD and other prion diseases, the protein in question is PRNP, a membrane glycoprotein containing five unstable octapeptide repeats (Zahn et al., 2000; Knaus et al., 2001). The protein is composed of three alpha helices and two antiparallel beta sheets. When the protein is converted to the disease state, beta-sheet character is greatly increased and much of the protein takes on a beta-sheet secondary structure. At the current, there are only a few studies that have been able to make progress in the elucidation of the transition between healthy and disease-state PRNP.

Figure 1. (a)Prion protein in its wild-type conformation (b)the disease-state prion protein. Note the increased beta-sheet character in the disease-state prion. The two versions are well documented, but the mechanism for the transition between these two states and how it is caused by the latter state is still a gray area for research [Image from Sussex University Website via Google images]
            Initial studies examining the mechanism of how these prions eventually result in the diseases state indicated that insoluble fibrils of prions accumulated in neurons, killing them (Prusiner, 1982). There is certainly still evidence of this being the case. In fact, it is likely these aggregates of prions that cause the vacuolization so characteristic of CJD brains, lending them their spongiform appearance. However, studies coming after Prusiner’s initial discoveries have indicated that, on top of the plaques of protein forming on the cells (remember PRNP is on the membrane), it also has something to do with the normal PRNP already present in the cells (Tobler et al., 1996).

Tobler’s clever experiment in PRNP knockout mice was able to strengthen the evidence supporting PRNP as the major causative factor in CJD, as well as tell us something about the nature of its pathology. The authors took wild-type mice and PRNP knockout mice and infected them with misfolded PRNP. Although aggregates formed in the knockout mice, they did not show a severe pathology when compared to the infected wild-type mice, who suffered from CJD-like symptoms and died. This showed that endogenous PRNP was necessary for full disease-state CJD to develop in mouse models. Following this, researchers began to stipulate that, without a supply of PRNP in the cell, the prions could do little to actually reproduce themselves. There needed to be active transcription of PRNP to provide the wild-type protein serving as the precursor to the disease-state protein.

Building on top of this knowledge, another study in 2011 indicated that upregulation of PRNP expression might be present in CJD models. Kaski et al. show that specific haplotypes (combinations of alleles at adjacent loci which are often inherited together) indicate higher risk for CJD. Indeed, all patients screened were homozygous for a methionine at the polymorphic codon 129. In other words, the residue at this locus varies between individuals, but all CJD patients screened were homozygous for a methionine here. Oddly, these patients did not have a family history of dementia or other neurodegenerative symptoms. This indicated that merely having two copies of this allele was insufficient to cause the disease outright, merely increasing the patient’s susceptibility. Additionally, there were other genetic factors screened, and patients were also found to all be homozygous at two other SNP loci. In healthy individuals with this haplotype containing these SNPs, blood concentrations of PRNP was higher than in the rest of the population, implying that these individuals may produce more PRNP than others, perhaps as a result of these alleles. These results, taken together support the idea that a constant supply of normal PRNP is necessary for the CJD disease state by showing that individuals with higher production (or at least, higher blood concentrations) of PRNP are at more risk for CJD.

In the past year, a study that went nigh unnoticed (in the Journal of Chromatography) indicated that PRNP was capable of binding to the H3 histone protein (Cai et al., 2013). Although the authors do not connect this to Kaski’s study, it is possible that some sort of expression regulation is occurring in this relationship. Histones are often modified in order to change chromatin organization and increase or decrease the expression of genes or gene clusters, so PRNP’s interactions with the H3 histone may yet be revealed to have a physiological relevance to the disease state.

In terms of structural analyses, there have been numerous performed on PRNP. Perhaps the most relevant and important of these was Kong et al.’s 2013 paper. In their study the authors use in silico modeling to identify portions of the protein contributing to thermodynamic instability. They identify numerous cavities in the protein that hinder hydrophobic packing and make the protein unstable. They also identify a residue, Val209, which can be mutated to increase stability without changing the overall fold of the protein. The V209M version of the protein was much more difficult to convert into the disease state version in vitro.

Figure 2. Illustrated here is the V209M mutation. On top is the human wild-type version of the gene, and on the bottom is the more-stabilized, better packed mutant. [Adapted from Kong et al. 2013]
In addition, when mice were grown up with the V209M mutation, they were shown to be largely impervious to infection with prions, indicating that the mutation stabilized the protein enough that it could not be transformed by the diseased version. This represents a landmark step toward elucidating the mechanism of conversion, which is key to understanding and treating CJD and other prion diseases.