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An integrative approach to elucidate the mechanisms and dynamics of Huntingtin aggregation and inclusion formation in neuronal models of Huntington's Disease

Nathan Alain Denis Riguet

Despite the fact that the gene responsible for Huntington's disease (HD) is known, we still do not understand the underlying mechanisms leading to neurodegeneration and death. Identifying and understanding the mechanisms controlling mutant huntingtin (mHtt) aggregation and inclusion formation using different cellular and animal models is crucial to elucidate the molecular mechanisms underpinning the disease and to develop effective treatments to prevent or slow the progression of HD. At the mechanistic level, our work shows that mHtt aggregation and inclusion formation in the cytosol and nucleus occur via different mechanisms and lead to the formation of inclusions with distinct biochemical and ultrastructural properties. We show that mHtt cytoplasmic inclusion formation occurs via two phases: 1) a first phase involves the rapid formation of a dense fibrillar core and is driven predominantly by intermolecular interactions involving the polyQ domain via phase separation-like mechanisms; 2) a second phase is associated with the recruitment of soluble mHtt, fibril growth, and the active recruitment and sequestration of lipids, proteins, and membranous organelles. Our work points to this second phase as an important contributor to mHtt toxicity and suggests that targeting inclusion growth and maturation represent a promising therapeutic strategy. These observations suggest that the two types of inclusions may exert their toxicity via different mechanisms and may require different strategies to interfere with their formation, maturation and toxicity. Using primary neurons, we demonstrated that neuronal intranuclear inclusions evolve over time from small aggregates to large granulo-filamentous inclusions associated with cellular toxicity. In addition, our work underscores the failure of the cellular degradation machineries to clear mHtt inclusions, as well as the sequestration of other proteins, lipids and organelles. Finally, we would like to emphasize that our comparative analysis of tag-free and GFP-tagged mutant mHtt aggregation and inclusions formation will inform future efforts to develop models that reproduce HD pathology more faithfully and underscore the need for developing label-free techniques to investigate disease-relevant mechanisms that underpin inclusion formation.

EPFL2022

N-terminal mutant huntingtin deposition correlates with CAG repeat length and symptom onset, but not neuronal loss in Huntington's disease

Lorène Aeschbach, Hilal Lashuel, Nathan Alain Denis Riguet

Huntington's disease (HD) is caused by a CAG repeat expansion mutation in the gene encoding the huntingtin (Htt) protein, with mutant Htt protein subsequently forming aggregates within the brain. Mutant Htt is a current target for novel therapeutic strategies for HD, however, the lack of translation from preclinical research to disease-modifying treatments highlights the need to improve our understanding of the role of Htt protein in the human brain. This study aims to undertake an immunohistochemical screen of 12 candidate antibodies against various sequences along the Htt protein to characterize Htt distribution and expression in post-mortem human brain tissue microarrays (TMAs).Immunohistochemistry was performed on middle temporal gyrus TMAs comprising of up to 28 HD and 27 age -matched control cases, using 12 antibodies specific to various sequences along the Htt protein. From this study, six antibodies directed to the Htt N-terminus successfully immunolabeled human brain tissue. Htt aggregates and Htt protein expression levels for the six successful antibodies were subsequently quantified with a customized automated image analysis pipeline on the TMAs. A 2.5-12 fold increase in the number of Htt aggregates were detected in HD cases using antibodies MAB5374, MW1, and EPR5526, despite no change in overall Htt protein expression compared to control cases, suggesting a redistribution of Htt into aggregates in HD. MAB5374, MW1, and EPR5526 Htt aggregate numbers were positively correlated with CAG repeat length, and negatively corre-lated with the age of symptom onset in HD. However, the number of Htt aggregates did not correlate with the degree of striatal degeneration or the degree of cortical neuron loss. Together, these results suggest that longer CAG repeat lengths correlate with Htt aggregation in the HD human brain, and greater Htt cortical aggregate deposition is associated with an earlier age of symptom onset in HD. This study also reinforces that antibodies MAB5492, MW8, and 2B7 which have been utilized to characterize Htt in animal models of HD do not specif-ically immunolabel Htt aggregates in HD human brain tissue exclusively, thereby highlighting the need for validated means of Htt detection to support drug development for HD.

ACADEMIC PRESS INC ELSEVIER SCIENCE2022

The Nt17 Domain and its Helical Conformation Regulate the Aggregation, Cellular Properties and Neurotoxicity of Mutant Huntingtin Exon 1

Urszula Beata Cendrowska, Anass Chiki, Sean Michael Deguire, Giovanni Dietler, Hilal Lashuel, Anne-Laure Mahul Mellier, Nathan Alain Denis Riguet, Francesco Simone Ruggeri, Sophie Vieweg

Converging evidence points to the N-terminal domain comprising the first 17 amino acids of the Huntingtin protein (Nt17) as a key regulator of its aggregation, cellular properties and toxicity. In this study, we further investigated the interplay between Nt17 and the polyQ domain repeat length in regulating the aggregation and inclusion formation of exon 1 of the Huntingtin protein (Httex1). In addition, we investigated the effect of removing Nt17 or modulating its local structure on the membrane interactions, neuronal uptake, and toxicity of monomeric or fibrillar Httex1. Our results show that the polyQ and Nt17 domains synergistically modulate the aggregation propensity of Httex1 and that the Nt17 domain plays important roles in shaping the surface properties of mutant Httex1 fibrils and regulating their poly-Q-dependent growth, lateral association and neuronal uptake. Removal of Nt17 or disruption of its transient helical conformations slowed the aggregation of monomeric Httex1 in vitro, reduced inclusion formation in cells, enhanced the neuronal uptake and nuclear accumulation of monomeric Httex1 proteins, and was sufficient to prevent cell death induced by Httex1 72Q overexpression. Finally, we demonstrate that the uptake of Httex1 fibrils into primary neurons and the resulting toxicity are strongly influenced by mutations and phosphorylation events that influence the local helical propensity of Nt17. Altogether, our results demonstrate that the Nt17 domain serves as one of the key master regulators of Htt aggregation, internalization, and toxicity and represents an attractive target for inhibiting Htt aggregate formation, inclusion formation, and neuronal toxicity.

2021