A hallmark of major neurodegenerative diseases is that cell death is selective. In Huntington's disease, for example, the offending protein is expressed ubiquitously, but a subpopulation of neurons from the striatum is among the first to succumb1
. A key unsolved mystery in the field is whether the production of toxic structures, or the capacity of the protein homeostasis system itself, varies sufficiently among subpopulations of neurons to account for this cell-type specificity. Frustratingly, the best-suited method to investigate these processes, metabolic pulse labeling, yields results that are confounded by protein aggregation and toxicity and lacks resolution to uncover cell-to-cell variation in protein homeostasis.
Neurodegenerative diseases are also characterized by abnormal accumulation of misfolded proteins2
. We therefore wondered whether the accumulation leads to neurodegeneration. At least two perspectives, which are not mutually exclusive, are possible. The first focuses on the misfolded proteins themselves, emphasizing their ability to adopt structures as monomers or aggregates that confer potentially toxic functions and lead to neurodegeneration3,4
. The second focuses on the limited capacity of the cell to handle misfolded proteins5
, whereby doses of misfolded protein exceeding a certain capacity trigger widespread misfolding of other metastable proteins, resulting in complex loss-of-function phenotypes that mediate neurodegeneration.
To understand how a protein's propensity to misfold and how individual cellular responses to protein misfolding relate to neurodegeneration, we examined the effect of disease-associated polyQ expansions on the mean lifetime of a fragment of huntingtin, the protein that causes Huntington's disease, at the single-cell level in living neurons. Using an optical pulse-chase method, we found that polypeptides with polyQ expansions that are not bound to an inclusion body have much shorter mean lifetimes than otherwise identical versions with shorter polyQ stretches, indicating that neurons recognize disease-associated polyQ peptides specifically and effectively target them for degradation. Clearing the polyQ-expanded polypeptide was particularly dependent on autophagy, and expression of Nrf2, a transcription factor that mediates stress responses, reduced the mean lifetime of polyQ-expanded polypeptides and increased neuronal survival. To our surprise, single-cell analysis showed that the mean lifetimes of identical polypeptides varied widely from neuron to neuron, indicating that the cellular environment in which a protein is expressed has a major role in its stability. The shorter the mean lifetime of a polyQ-expanded polypeptide in cortical and striatal neurons, the longer those neurons tended to live, suggesting that the proteostasis system is a major determinant of the vulnerability of susceptible neurons to aggregation-prone proteins. Proteostasis was also an important predictor of longevity in cerebellar neurons, which are relatively spared in Huntington's disease, though additional factors substantially contributed to their ability to resist toxicity from a mutant form of a fragment of the protein that causes Huntington's disease, huntingtin (mHttex1). Therefore, we conclude that differences in turnover capacity contribute to cell susceptibility to toxic proteins, and this might help explain how the same aggregation-prone protein causes neurodegeneration in some neuronal subpopulations and spares others.
We developed a new technology that enables measurement of the mean lifetime of aggregation-prone proteins in live cells and ensures that the measurements are not confounded by cell death. We applied the technology to a model of Huntington's disease and found that neurons selectively destabilize polypeptides with disease-causing polyQ expansions. We showed that neurons selectively clear diffuse mHttex1
, distinguishing it from wild-type forms and targeting the mutant form for accelerated clearance. By contrast, mHttex1
molecules that aggregate into inclusion bodies are cleared much more slowly6,7
, offering an explanation for how mHtt accumulates in Huntington's disease.