An apparent core/shell architecture of polyQ aggregates in the aging Caenorhabditis elegans neuron

Abstract

Huntington's disease is caused by a polyglutamine (polyQ) expansion in the huntingtin protein which results in its abnormal aggregation in the nervous system. Huntingtin aggregates are linked to toxicity and neuronal dysfunction, but a comprehensive understanding of the aggregation mechanism in vivo remains elusive. Here, we examine the morphology of polyQ aggregates in Caenorhabditis elegans mechanosensory neurons as a function of age using confocal and fluorescence lifetime imaging microscopy. We find that aggregates in young worms are mostly spherical with homogenous intensity, but as the worm ages aggregates become substantially more heterogeneous. Most prominently, in older worms we observe an apparent core/shell morphology of polyQ assemblies with decreased intensity in the center. The fluorescence lifetime of polyQ is uniform across the aggregate indicating that the dimmed intensity in the assembly center is most likely not due to quenching or changes in local environment, but rather to displacement of fluorescent polyQ from the central region. This apparent core/shell architecture of polyQ aggregates in aging C. elegans neurons contributes to the diverse landscape of polyQ aggregation states implicated in Huntington's disease.

Intermediate Filaments Associate with Aggresome-like Structures in ProteostressedC. elegansNeurons and Influence Large Vesicle Extrusions as Exophers

“…Interestingly, we noted that association of IFDs and HttQ128::CFP can adopt multiple relative configurations, being mostly coincident for smaller presumptively newly formed aggresomes (Figure 4) but more frequently exhibiting less complete “covering” with advancing time or when relative expression of HttQ128::CFP and IFDs is unbalanced (Figure 4, Supplementary Figure 14). Dynamic patterns of polyQ concentrations and maturation have been reported in vivo in C. elegans [49]) and in HEK cells [50]; our data further support a dynamic interaction of IFDs and aggregates, and challenge the concept that aggresomes are static structures in which IFs fully “cage” sequestered materials (see Discussion).…”

“…In this case, we note that the IFD compartment often does not appear to fully surround the HttQ128 signal. Biophysical analyses support heterogeneity in aggregate properties in vivo in polyQ touch neurons [49]. Top row : Adult touch neuron soma displaying the three main patterns of interaction shown when expressing mScarlet::IFD-1 and HttQ128::CFP.…”

The polyglutamine amyloid nucleus in living cells is a monomer with competing dimensions of order

“…A cryoEM tomography study revealed a slab-like architecture of 51:Q amyloids in cells, with occasional restrictions to an apparent protofilament width of approximately 2 nm (Galaz-Montoya et al, 2021). Fluorescence microscopy studies of aging C. elegans neurons expressing Q:128, fused to the oligomerizing N-terminus of huntingtin and C-terminal CFP, revealed spherical aggregates with an apparently soft core and hard shell (Fisher et al, 2021).…”

“…Second, the protein may partition into "off-pathway" oligomers or condensates at high concentration, within which the nucleating conformation is disfavored. PolyQ has been observed to form non-amyloid oligomers and condensates, which has led to the prevailing paradigm that phase separation precedes and facilitates the nucleation of conformational order (Borcherds et al, 2021;Camino et al, 2021;Crick et al, 2013Crick et al, , 2006Dignon et al, 2020;Fisher et al, 2021;Halfmann et al, 2011;Peskett et al, 2018;Posey et al, 2018;Vitalis and Pappu, 2011;Yang and Yang, 2020). The second explanation above would seem to contradict this paradigm.…”

“…The physiological concentration of huntingtin in the human brain, estimated to be low nanomolar (Macdonald et al, 2014), is likely too low for liquid-liquid phase separation in the presence of even one binding partner at its physiological concentration (Posey et al, 2018). Notwithstanding limited reports to the contrary (Fisher et al, 2021;Peskett et al, 2018;Wan et al, 2021) --which we respectfully attribute to known effects of flanking domain interactions and partitioning into pre-existing protein compartments (Duennwald et al, 2006a(Duennwald et al, , 2006bJiang et al, 2017) --our findings corroborate prior demonstrations that polyQ does not phase separate prior to amyloid formation, whether expressed in human neuronal cells (Colby et al, 2006;Kakkar et al, 2016), C. elegans body wall muscle cells (Sinnige et al, 2021), or [pin -] yeast cells (Duennwald et al, 2006a(Duennwald et al, , 2006bJiang et al, 2017).…”