If you have occasion to study neurodegeneration, you will be struck by how many terrible high-profile diseases in this area seem to share a common theme. Alzheimer’s, ALS, progressive supranuclear palsy, Parkinson’s, Lewy body dementia, some types of frontotemporal dementia, Huntington’s, prion diseases such as BSE and more all feature abnormal protein aggregates that appear in neural tissues. There are plenty of variations, naturally. These aggregates happen in different types of cells, involve different key proteins that seem to have a variety of structural features that lead them into this process, and the resulting diseases affect different regions of the brain. But the overlap of such aggregation with disease is impossible to ignore, and believe me, no one has been ignoring it.
Ever since Alois Alzheimer noticed what we now call amyloid plaques in the brains of deceased dementia patients over a hundred years ago, every advance in neuroscience, cell biology, and instrumentation has been brought to bear on this problem. And it’s a measure of how complex such diseases are that we still don’t truly understand what’s going on and we still have no disease-modifying treatments for any of them. We’re still not sure how many of these aggregates are direct causes of the associated diseases, and how many might be side effects of some other disease process that’s more proximal. Even the ones where we have the most detailed knowledge have escaped us – Huntington’s for example. In that case, the protein that aggregates (Huntingtin) does so because it has a long “tail” of glutamine residues. This genetic basis for the disease was made clear in 1993. We know that if there are fewer than 28 of these, a patient will be completely normal. 28 to 35 of them takes you into a range where some signs might be picked up by post-mortem histopathlogy, but the affected patient is still asymptomatic. 36 to 40 glutamine repeats, that’s a danger zone. Patients in this range show disease, but its severity, age of onset, and progression are variable. And greater than 40 repeats means full-blown, progressive, and fatal Huntington’s.
Isn’t that enough clues? If this were a movie, the screenwriters would have us running into the labs with a cure in the third act, for sure. But we’re not even sure if the biggest problem in the disease is the amino acid repeats in the protein or the trinucleotide repeats in the precursor RNA. Even if we were absolutely sure that all we needed to do was to keep the Huntingtin protein from sticking to itself, we still can’t manage that. Drug discovery organizations have been screening for aggregation inhibitors for decades now, and I have seen more papers than I can possibly remember on compounds that (theoretically) keep amyloid, tau, Huntingtin, alpha-synuclein, and other such proteins from aggregating. To the best of my knowledge, most of these have not even made it into clinical trials, and the ones that do have a flat zero per cent success rate. On the other end of the process, Alzheimer’s especially has seen a whole list of attempts to clear out such aggregates, especially before they become pathological, but as my recent hand-wringing about aducanumab should make clear, I don’t see any successes so far in that approach, either.
This short review paper (open access) urges everyone to realize that these aggregates are even more complicated than they look. From one perspective that’s not such cheerful news, but we’re always better off facing reality in these situations. It’s easier to think of protein aggregation as happening with single proteins that have something wrong with them that form relatively pure clumps that are then cytotoxic. In vitro screening efforts often assume a picture something like this – there are a lot of systems where aggregation of protein constructs (usually through formation of fibril structures) is used as the basis for such a screen, and one recommendation I take from this new paper is to just stop doing that sort of thing entirely.
That’s because, as numerous references show, these aggregates in vivo are far from homogeneous lumps of toxic protein. Amyloid plaques, neurofibrillary tangles, and Lewy bodies have hundreds of different proteins in them. They have varying amounts of lipids, RNA species, and carbohydrates as well, and we’re not very far along in characterizing these. Lewy bodies have entire membranous organelles tangled up in them! The lack of detailed knowledge is partly because of the sheer complexity of the problem, and partly (as with the carbohydrates) because of deficiencies in our techniques for analyzing such things in general.
We also have deficiencies with some of the tools used in cell biology studies. Watching protein fibrils form from purified starting materials can tell you a lot from a structural biology perspective, but it’s not the same as what’s happening inside a neuron. Indeed, the structures of the fibrils themselves are different. One of the things I took from this paper is that my picture of the formation of the in vivo protein aggregates is way off – it’s easy, especially for a chemist, to imagine something like a messy precipitation, with random clumps of stuff coming out of solution. But in reality, these things are probably forming under rather specific and controlled conditions, and it may be really hard to recapitulate these outside of the cell. The details of the various post-translational modification of the aggregated proteins also argue for specific conditions rather than random fallout.
Even studying them inside the cell is tricky. Close study by advanced microscopy in wild-type cells and tissues shows that the structure and composition of even a single type of aggregate can vary according to what compartment of the cell it appears in, although these may appear superficially similar. As the paper points out, using protein overexpression systems leads to unnatural artifacts, as can tagging the relevant proteins with fluorescent groups. You’re always worried about such effects, of course, but when the very process you’re highlighting seem to depend on self-association of a key protein, then such assays are at their most vulnerable. At any rate, we have to rework our approaches here, because continuing to use (over)simplified assays mainly because they’re doable does not seem to be getting us very far.
Nobody likes to hear the “Gosh, it’s more complicated than we thought” news, but we get to hear it a lot in this business, and it’s generally an accurate view of the situation. Once in a great while it’s less complicated than we though, and those are memorable occasions, but mostly it’s like this when you study the causes of disease. Especially in neurology! The important thing is not to use this as an excuse to throw up your hands, but rather as a call to find something that can be improved.
The post Protein Aggregation Diseases first appeared on In the Pipeline.
