Justin Legleiter

Accelerating Amyloid-β Fibrillization Reduces Oligomer Levels and Functional Deficits in Alzheimer Disease Mouse Models

Many proteins suspected of causing neurodegenerative diseases exist in diverse assembly states. For most, it is unclear whether shifts from one state to another would be helpful or harmful. We used mutagenesis to change the assembly state of Alzheimer disease (AD)-associated amyloid-β (Aβ) peptides. In vitro, the “Arctic” mutation (AβE22G) accelerated Aβ fibrillization but decreased the abundance of nonfibrillar Aβ assemblies, compared with wild-type Aβ. In human amyloid precursor protein (hAPP) transgenic mice carrying mutations adjacent to Aβ that increase Aβ production, addition of the Arctic mutation markedly enhanced the formation of neuritic amyloid plaques but reduced the relative abundance of a specific nonfibrillar Aβ assembly (Aβ*56). Mice overexpressing Arctic mutant or wild-type Aβ had similar behavioral and neuronal deficits when they were matched for Aβ*56 levels but had vastly different plaque loads. Thus, Aβ*56 is a likelier determinant of functional deficits in hAPP mice than fibrillar Aβ deposits. Therapeutic interventions that reduce Aβ fibrils at the cost of augmenting nonfibrillar Aβ assemblies could be harmful.

Mutant Huntingtin Fragments Form Oligomers in a Polyglutamine Length-dependent Manner in Vitro and in Vivo

Huntington disease (HD) is caused by an expansion of more than 35–40 polyglutamine (polyQ) repeats in the huntingtin (htt) protein, resulting in accumulation of inclusion bodies containing fibrillar deposits of mutant htt fragments. Intriguingly, polyQ length is directly proportional to the propensity for htt to form fibrils and the severity of HD and is inversely correlated with age of onset. Although the structural basis for htt toxicity is unclear, the formation, abundance, and/or persistence of toxic conformers mediating neuronal dysfunction and degeneration in HD must also depend on polyQ length. Here we used atomic force microscopy to demonstrate mutant htt fragments and synthetic polyQ peptides form oligomers in a polyQ length-dependent manner. By time-lapse atomic force microscopy, oligomers form before fibrils, are transient in nature, and are occasionally direct precursors to fibrils. However, the vast majority of fibrils appear to form by monomer addition coinciding with the disappearance of oligomers. Thus, oligomers must undergo a major structural transition preceding fibril formation. In an immortalized striatal cell line and in brain homogenates from a mouse model of HD, a mutant htt fragment formed oligomers in a polyQ length-dependent manner that were similar in size to those formed in vitro, although these structures accumulated over time in vivo. Finally, using immunoelectron microscopy, we detected oligomeric-like structures in human HD brains. These results demonstrate that oligomer formation by a mutant htt fragment is strongly polyQ length-dependent in vitro and in vivo, consistent with a causative role for these structures, or subsets of these structures, in HD pathogenesis.

Identifying polyglutamine protein species in situ that best predict neurodegeneration

SUMMARY Polyglutamine (polyQ) stretches exceeding a threshold length confer a toxic function on proteins that contain them and cause at least nine neurological disorders. The basis for this toxicity threshold is unclear. Although polyQ expansions render proteins prone to aggregate into inclusion bodies (IBs), IB formation may be a neuronal coping response to more toxic forms of polyQ. The exact structure of these more toxic forms is unknown. Here we show that monoclonal antibody (mAb) 3B5H10 recognizes a species of polyQ protein in situ that strongly predicts neuronal death. The epitope selectively appears among some of the many low-molecular weight conformational states expanded polyQ assumes and disappears in higher molecular-weight aggregated forms, such as IBs. These results suggest that protein monomers and possibly small oligomers containing expanded polyQ stretches can adopt a conformation that is recognized by 3B5H10 and is toxic or closely related to a toxic species.

Identical oligomeric and fibrillar structures captured from the brains of R6/2 and knock-in mouse models of Huntington's disease

Huntingtons disease (HD) is a late-onset neurodegenerative disorder that is characterized neuropathologically by the presence of neuropil aggregates and nuclear inclusions. However, the profile of aggregate structures that are present in the brains of HD patients or of HD mouse models and the relative contribution of specific aggregate structures to disease pathogenesis is unknown. We have used the Seprion ligand to develop a highly sensitive enzyme-linked immunosorbent assay (ELISA)-based method for quantifying aggregated polyglutamine in tissues from HD mouse models. We used a combination of electron microscopy, atomic force microscopy (AFM) and sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) to investigate the aggregate structures isolated by the ligand. We found that the oligomeric, proto-fibrillar and fibrillar aggregates extracted from the brains of R6/2 and HdhQ150 knock-in mice were remarkably similar. Using AFM, we determined that the nanometre globular oligomers isolated from the brains of both mouse models have dimensions identical to those generated from recombinant huntingtin exon 1 proteins. Finally, antibodies that detect exon 1 Htt epitopes differentially recognize the ligand-captured material on SDS–PAGE gels. The Seprion-ligand ELISA provides an assay with good statistical power for use in preclinical pharmacodynamic therapeutic trials or to assess the effects of the genetic manipulation of potential therapeutic targets on aggregate load. This, together with the ability to identify a spectrum of aggregate species in HD mouse tissues, will contribute to our understanding of how these structures relate to the pathogenesis of HD and whether their formation can be manipulated for therapeutic benefit.

Collagen VI protects neurons against Abeta toxicity.

Amyloid-β (Aβ) peptides, widely presumed to cause Alzheimers disease, increased mouse neuronal expression of collagen VI through a mechanism involving transforming growth factor signaling. Reduction of collagen VI augmented Aβ neurotoxicity, whereas treatment of neurons with soluble collagen VI blocked the association of Aβ oligomers with neurons, enhanced Aβ aggregation and prevented neurotoxicity. These results identify collagen VI as an important component of the neuronal injury response and demonstrate its neuroprotective potential.

Identification of Novel Potentially Toxic Oligomers Formed in Vitro from Mammalian-derived Expanded huntingtin Exon-1 Protein

Huntington disease is a genetic neurodegenerative disorder that arises from an expanded polyglutamine region in the N terminus of the HD gene product, huntingtin. Protein inclusions comprised of N-terminal fragments of mutant huntingtin are a characteristic feature of disease, though are likely to play a protective role rather than a causative one in neurodegeneration. Soluble oligomeric assemblies of huntingtin formed early in the aggregation process are candidate toxic species in HD. In the present study, we established an in vitro system to generate recombinant huntingtin in mammalian cells. Using both denaturing and native gel analysis, we have identified novel oligomeric forms of mammalian-derived expanded huntingtin exon-1 N-terminal fragment. These species are transient and were not previously detected using bacterially expressed exon-1 protein. Importantly, these species are recognized by 3B5H10, an antibody that recognizes a two-stranded hairpin conformation of expanded polyglutamine believed to be associated with a toxic form of huntingtin. Interestingly, comparable oligomeric species were not observed for expanded huntingtin shortstop, a 117-amino acid fragment of huntingtin shown previously in mammalian cell lines and transgenic mice, and here in primary cortical neurons, to be non-toxic. Further, we demonstrate that expanded huntingtin shortstop has a reduced ability to form amyloid-like fibrils characteristic of the aggregation pathway for toxic expanded polyglutamine proteins. Taken together, these data provide a possible candidate toxic species in HD. In addition, these studies demonstrate the fundamental differences in early aggregation events between mutant huntingtin exon-1 and shortstop proteins that may underlie the differences in toxicity.

Hsp70 and Hsp40 functionally interact with soluble mutant huntingtin oligomers in a classic ATP-dependent reaction cycle.

Inclusion bodies of aggregated mutant huntingtin (htt) fragments are a neuropathological hallmark of Huntington disease (HD). The molecular chaperones Hsp70 and Hsp40 colocalize to inclusion bodies and are neuroprotective in HD animal models. How these chaperones suppress mutant htt toxicity is unclear but might involve direct effects on mutant htt misfolding and aggregation. Using size exclusion chromatography and atomic force microscopy, we found that mutant htt fragments assemble into soluble oligomeric species with a broad size distribution, some of which reacted with the conformation-specific antibody A11. Hsp70 associated with A11-reactive oligomers in an Hsp40- and ATP-dependent manner and inhibited their formation coincident with suppression of caspase 3 activity in PC12 cells. Thus, Hsp70 and Hsp40 (DNAJB1) dynamically target specific subsets of soluble oligomers in a classic ATP-dependent reaction cycle, supporting a pathogenic role for these structures in HD.

Biophysical insights into how surfaces, including lipid membranes, modulate protein aggregation related to neurodegeneration.

There are a vast number of neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD), associated with the rearrangement of specific proteins to non-native conformations that promotes aggregation and deposition within tissues and/or cellular compartments. These diseases are commonly classified as protein-misfolding or amyloid diseases. The interaction of these proteins with liquid/surface interfaces is a fundamental phenomenon with potential implications for protein-misfolding diseases. Kinetic and thermodynamic studies indicate that significant conformational changes can be induced in proteins encountering surfaces, which can play a critical role in nucleating aggregate formation or stabilizing specific aggregation states. Surfaces of particular interest in neurodegenerative diseases are cellular and subcellular membranes that are predominately comprised of lipid components. The two-dimensional liquid environments provided by lipid bilayers can profoundly alter protein structure and dynamics by both specific and non-specific interactions. Importantly for misfolding diseases, these bilayer properties can not only modulate protein conformation, but also exert influence on aggregation state. A detailed understanding of the influence of (sub)cellular surfaces in driving protein aggregation and/or stabilizing specific aggregate forms could provide new insights into toxic mechanisms associated with these diseases. Here, we review the influence of surfaces in driving and stabilizing protein aggregation with a specific emphasis on lipid membranes.

The Interaction of Polyglutamine Peptides With Lipid Membranes is Regulated by Flanking Sequences Associated with Huntingtin

Background: Huntington disease (HD) is caused by an expanded polyglutamine (poly(Q)) domain in huntingtin (htt), leading to aggregation. Results: Specific flanking sequences adjacent to the poly(Q) domain modulate htt aggregation on lipid bilayers. Conclusion: Lipid-mediated htt aggregation may lead to membrane dysfunction in HD. Significance: Flanking sequences may play a role in membrane dysfunction associated with Huntington disease. Huntington disease (HD) is caused by an expanded polyglutamine (poly(Q)) repeat near the N terminus of the huntingtin (htt) protein. Expanded poly(Q) facilitates formation of htt aggregates, eventually leading to deposition of cytoplasmic and intranuclear inclusion bodies containing htt. Flanking sequences directly adjacent to the poly(Q) domain, such as the first 17 amino acids on the N terminus (Nt17) and the polyproline (poly(P)) domain on the C-terminal side of the poly(Q) domain, heavily influence aggregation. Additionally, htt interacts with a variety of membraneous structures within the cell, and Nt17 is implicated in lipid binding. To investigate the interaction between htt exon1 and lipid membranes, a combination of in situ atomic force microscopy, Langmuir trough techniques, and vesicle permeability assays were used to directly monitor the interaction of a variety of synthetic poly(Q) peptides with different combinations of flanking sequences (KK-Q35-KK, KK-Q35-P10-KK, Nt17-Q35-KK, and Nt17-Q35-P10-KK) on model membranes and surfaces. Each peptide aggregated on mica, predominately forming extended, fibrillar aggregates. In contrast, poly(Q) peptides that lacked the Nt17 domain did not appreciably aggregate on or insert into lipid membranes. Nt17 facilitated the interaction of peptides with lipid surfaces, whereas the poly(P) region enhanced this interaction. The aggregation of Nt17-Q35-P10-KK on the lipid bilayer closely resembled that of a htt exon1 construct containing 35 repeat glutamines. Collectively, this data suggests that the Nt17 domain plays a critical role in htt binding and aggregation on lipid membranes, and this lipid/htt interaction can be further modulated by the presence of the poly(P) domain.

Amyloid-forming proteins alter the local mechanical properties of lipid membranes.

A diverse number of diseases, including Alzheimers disease, Huntingtons disease, and type 2 diabetes, are characterized by the formation of fibrillar protein aggregates termed amyloids. The precise mechanism by which aggregates are toxic remains unclear; however, these proteins have been shown to interact strongly with lipid membranes. We investigated morphological and mechanical changes in model lipid bilayers exposed to amyloid-forming proteins by reconstructing the tapping forces associated with atomic force microscopy (AFM) imaging in solution. Tip/sample tapping forces contain information regarding mechanical properties of surfaces. Interpretation of the mechanical changes in the bilayers was aided by numerical simulations of the entire AFM experiment. Amyloid-forming proteins disrupted distinct regions of the bilayer morphology, and these regions were associated with decreased Youngs modulus and adhesive properties. These changes in bilayer mechanical properties upon exposure to amyloid-forming proteins may represent a common mechanism leading to membrane dysfunction in amyloid diseases.