Neurodegeneration: new clues on inclusions

AbstractThe rare neurological disorders frontotemporal dementia and British dementia have been linked to two mutant genes whose products constitute the fibrils that define the two disease pathologies. Two recent studies add to the mounting circumstantial case that protein fibrillization, inside (neurofibrillary tangles) or outside (amyloid plaques) of the neuron, may be pathogenic and suggest that either or both of these mechanisms could initiate Alzheimer’s disease

A century-old debate on protein aggregation and neurodegeneration enters the clinic

The correlation between neurodegenerative disease and protein aggregation in the brain has long been recognized, but a causal relationship has not been unequivocally established, in part because a discrete pathogenic aggregate has not been identified. The complexity of these diseases and the dynamic nature of protein aggregation mean that, despite progress towards understanding aggregation, its relationship to disease is difficult to determine in the laboratory. Nevertheless, drug candidates that inhibit aggregation are now being tested in the clinic. These have the potential to slow the progression of Alzheimer's disease, Parkinson's disease and related disorders and could, if administered presymptomatically, drastically reduce the incidence of these diseases. The clinical trials could also settle the century-old debate about causality

Observation of metastable Aβ amyloid protofibrils by atomic force microscopy

AbstractBackground: Brain amyloid plaque, a diagnostic feature of Alzheimer's disease (AD), contains an insoluble fibrillar core that is composed primarily of variants of the β-amyloid protein (Aβ). As Aβ amyloid fibrils may initiate neurodegeneration, the inhibition of fibril formation is a possible therapeutic strategy. Very little is known about the early steps of the process, however.Results: Atomic force microscopy was used to follow amyloid fibril formation in vitro by the Aβ variants Aβ1-40 and Aβ1-42. Both variants first form small ordered aggregates that grow slowly and then rapidly disappear, while prototypical amyloid fibrils of two discrete morphologies appear. Aβ1-42 aggregates much more rapidly than Aβ1-40, which is consistent with its connection to early-onset AD. We propose that the metastable intermediate species be called Aβ amyloid protofibrils.Conclusions: Aβ protofibrils are likely to be intermediates in the in vitro assembly of Aβ amyloid fibrils, but their in vivo role has yet to be determined. Numerous reports of a nonfibrillar form of Aβ aggregate in the brains of individuals who are predisposed to AD suggest the existence of a precursor form, possibly the protofibril. Thus, stabilization of Aβ protofibrils may be a useful therapeutic strategy

Improving Binding Specificity of Pharmacological Chaperones That Target Mutant Superoxide Dismutase-1 Linked to Familial Amyotrophic Lateral Sclerosis Using Computational Methods

We recently described a set of drug-like molecules obtained from an in silico screen that stabilize mutant superoxide dismutase-1 (SOD-1) linked to familial amyotrophic lateral sclerosis (ALS) against unfolding and aggregation but exhibited poor binding specificity toward SOD-1 in presence of blood plasma. A reasonable but not a conclusive model for the binding of these molecules was proposed on the basis of restricted docking calculations and site-directed mutagenesis of key residues at the dimer interface. A set of hydrogen bonding constraints obtained from these experiments were used to guide docking calculations with compound library around the dimer interface. A series of chemically unrelated hits were predicted, which were experimentally tested for their ability to block aggregation. At least six of the new molecules exhibited high specificity of binding toward SOD-1 in the presence of blood plasma. These molecules represent a new class of molecules for further development into clinical candidates

The UCH-L1 gene encodes two opposing enzymatic activities that affect alpha-synuclein degradation and Parkinson's disease susceptibility

The assumption that each enzyme expresses a single enzymatic activity in vivo is challenged by the linkage of the neuronal enzyme ubiquitin C-terminal hydrolase-L1 (UCH-L1) to Parkinson's disease (PD). UCH-L1, especially those variants linked to higher susceptibility to PD, causes the accumulation of alpha-synuclein in cultured cells, an effect that cannot be explained by its recognized hydrolase activity. UCH-L1 is shown here to exhibit a second, dimerization-dependent, ubiquityl ligase activity. A polymorphic variant of UCH-L1 that is associated with decreased PD risk (S18Y) has reduced ligase activity but comparable hydrolase activity as the wild-type enzyme. Thus, the ligase activity as well as the hydrolase activity of UCH-L1 may play a role in proteasomal protein degradation, a critical process for neuronal health

Neurodegenerative disease: amyloid pores from pathogenic mutations

Alzheimer's and Parkinson's diseases are associated with the formation in the brain of amyloid fibrils from beta-amyloid and alpha-synuclein proteins, respectively. It is likely that oligomeric fibrillization intermediates (protofibrils), rather than the fibrils themselves, are pathogenic, but the mechanism by which they cause neuronal death remains a mystery. We show here that mutant amyloid proteins associated with familial Alzheimer's and Parkinson's diseases form morphologically indistinguishable annular protofibrils that resemble a class of pore-forming bacterial toxins, suggesting that inappropriate membrane permeabilization might be the cause of cell dysfunction and even cell death in amyloid diseases

Abeta protofibrils possess a stable core structure resistant to hydrogen exchange

Protofibrils are transient structures observed during in vitro formation of mature amyloid fibrils and have been implicated as the toxic species responsible for cell dysfunction and neuronal loss in Alzheimer's disease (AD) and other protein aggregation diseases. To better understand the roles of protofibrils in amyloid assembly and Alzheimer's disease, we characterized secondary structural features of these heterogeneous and metastable assembly intermediates. We chromatographically isolated different size populations of protofibrils from amyloid assembly reactions of Abeta(1-40), both wild type and the Arctic variant associated with early onset familial AD, and exposed them to hydrogen-deuterium exchange analysis monitored by mass spectrometry (HX-MS). We show that HX-MS can distinguish among unstructured monomer, protofibrils, and fibrils by their different protection patterns. We find that about 40% of the backbone amide hydrogens of Abeta protofibrils are highly resistant to exchange with deuterium even after 2 days of incubation in aqueous deuterated buffer, implying a very stable, presumably H-bonded, core structure. This is in contrast to mature amyloid fibrils, whose equally stable structure protects about 60% of the backbone amide hydrogens over the same time frame. We also find a surprising degree of specificity in amyloid assembly, in that wild type Abeta is preferentially excluded from both protofibrils and fibrils grown from an equimolar mixture of wild type and Arctic mutant peptides. These and other data are interpreted and discussed in terms of the role of protofibrils in fibril assembly and in disease

Alpha-synuclein, especially the Parkinson's disease-associated mutants, forms pore-like annular and tubular protofibrils

Two mutations in the alpha-synuclein gene (A30P and A53T) have been linked to autosomal dominant early-onset Parkinson's disease (PD). Both mutations promote the formation of transient protofibrils (prefibrillar oligomers), suggesting that protofibrils are linked to cytotoxicity. In this work, the effect of these mutations on the structure of alpha-synuclein oligomers was investigated using electron microscopy and digital image processing. The PD-linked mutations (A30P and A53T) were observed to affect both the morphology and the size distribution of alpha-synuclein protofibrils (measured by analytical ultracentrifugation and scanning transmission electron microscopy). The A30P variant was observed to promote the formation of annular, pore-like protofibrils, whereas A53T promotes formation of annular and tubular protofibrillar structures. Wild-type alpha-synuclein also formed annular protofibrils, but only after extended incubation. The formation of pore-like oligomeric structures may explain the membrane permeabilization activity of alpha-synuclein protofibrils. These structures may contribute to the pathogenesis of PD

The impact of the E46K mutation on the properties of alpha-synuclein in its monomeric and oligomeric states

The third and most recently identified Parkinson's disease-linked variant of the neuronal protein alpha-synuclein to be identified (E46K) results in widespread brain pathology and early onset Parkinson symptoms (Zarranz et al. (2004) Ann. Neurol. 55, 164-173). Herein, we present biochemical and biophysical characterization of E46K alpha-synuclein in various states of aggregation. Circular dichroism and nuclear magnetic resonance spectroscopy illustrate that the E46K mutation results in subtle changes in the conformation of the monomeric protein both free in solution and in the presence of SDS micelles. However, it does not alter the overall helical propensity of the protein in the presence of phospholipids. E46K alpha-synuclein formed insoluble fibrils in vitro more rapidly than the wild type protein, and electron microscopy revealed that E46K alpha-synuclein fibrils possess a typical amyloid ultrastructure. E46K alpha-synuclein protofibrils, soluble aggregates that form during the transition from the monomeric form to the fibrillar form of alpha-synuclein, were characterized by electron microscopy and gel filtration and were found to include annular species. The unique ability of a subfraction of E46K and wild type alpha-synuclein protofibrils containing porelike species to permeabilize lipid vesicles was demonstrated in vitro using a real-time chromatographic method. In contrast to simplistic expectations, the total amount of protofibrils and the amount of permeabilizing activity per mole protein in the protofibril fraction were reduced by the E46K mutation. These results suggest that if the porelike activity of alpha-synuclein is important for neurotoxicity, there must be factors in the neuronal cytoplasm that reverse the trends in the intrinsic properties of E46K versus WT alpha-synuclein that are observed in vitro