Apolipoprotein E gene (APOE) alleles may shift the onset of Alzheimer's disease (AD) through apoE protein isoforms changing the probability of amyloid-β (Aβ) accumulation. It has been proposed that differential physical interactions of apoE isoforms with soluble Aβ (sAβ) in brain fluids influence the metabolism of Aβ, providing a mechanism to account for how APOE influences AD risk. In contrast, we provide clear evidence that apoE and sAβ interactions occur minimally in solution and in the cerebrospinal fluid of human subjects, producing apoE3 and apoE4 isoforms as assessed by multiple biochemical and analytical techniques. Despite minimal extracellular interactions with sAβ in fluid, we find that apoE isoforms regulate the metabolism of sAβ by astrocytes and in the interstitial fluid of mice that received apoE infusions during brain Aβ microdialysis. We find that a significant portion of apoE and sAβ compete for the low-density lipoprotein receptor-related protein 1 (LRP1)-dependent cellular uptake pathway in astrocytes, providing a mechanism to account for apoE's regulation of sAβ metabolism despite minimal evidence of direct interactions in extracellular fluids. We propose that apoE influences sAβ metabolism not through direct binding to sAβ in solution but through its actions with other interacting receptors/transporters and cell surfaces. These results provide an alternative frame work for the mechanistic explanations on how apoE isoforms influence the risk of AD pathogenesis.neurodegeneration | cholesterol efflux

Huntington disease is caused by mutational expansion of the CAG trinucleotide within exon 1 of the huntingtin (Htt) gene. Exon 1 spanning N-terminal fragments (NTFs) of the Htt protein result from aberrant splicing of transcripts of mutant Htt. NTFs typically encompass a polyglutamine tract flanked by an N-terminal 17-residue amphipathic stretch (N17) and a C-terminal 38-residue proline-rich stretch (C38). We present results from in vitro biophysical studies that quantify the driving forces for and mechanisms of polyglutamine aggregation as modulated by N17 and C38. Although N17 is highly soluble by itself, it lowers the saturation concentration of soluble NTFs and increases the driving force, vis-à-vis homopolymeric polyglutamine, for forming insoluble aggregates. Kinetically, N17 accelerates fibril formation and destabilizes nonfibrillar intermediates. C38 is also highly soluble by itself, and it lends its high intrinsic solubility to lower the driving force for forming insoluble aggregates by increasing the saturation concentration of soluble NTFs. In NTFs with both modules, N17 and C38 act synergistically to destabilize nonfibrillar intermediates (N17 effect) and lower the driving force for forming insoluble aggregates (C38 effect). Morphological studies show that N17 and C38 promote the formation of ordered fibrils by NTFs. Homopolymeric polyglutamine forms a mixture of amorphous aggregates and fibrils, and its aggregation mechanisms involve early formation of heterogeneous distributions of nonfibrillar species. We propose that N17 and C38 act as gatekeepers that control the intrinsic heterogeneities of polyglutamine aggregation. This provides a biophysical explanation for the modulation of in vivo NTF toxicities by N17 and C38. (2). The Htt gene with expanded CAG tracts can undergo erroneous splicing, and the resultant aberrant messenger RNA is translated into a mutant exon 1 version of Htt that is similar to toxic NTFs found in neuronal intranuclear inclusions (3). Exon 1 spanning NTFs typically include a polyglutamine tract that is flanked on its N terminus by an amphipathic 17-residue stretch (MATLEKLMKAFESLKSF) denoted as N17 and by a 38-residue proline-rich stretch on its C terminus (P 11 -QLPQPPPQAQPLLPQPQ-P 10 ) denoted as C38. The N17 sequence is conserved among higher mammals (SI Appendix, Fig. S1), and mutations within N17 impact the properties of NTFs (4, 5). N17 enhances the overall rate of aggregation, as measured by the rate of forming large insoluble species both in vitro (6) and in yeast (7). The C-terminal proline-rich region of exon 1 modulates polyglutamine aggregation and reduces the cellular toxicity of Htt exon 1 even when the polyglutamine tract is significantly expanded (8, 9).A molecular-level understanding of the synergy between the length of polyglutamine tracts and its flanking sequences is essential for inferring the roles of N17 and C38 in vivo. This requires a quantitative understanding of the driving forces, mechanisms, and morphologies for homopolymeric polyglutamine and t...

Fluorescence correlation spectroscopy (FCS) is a sensitive and widely used technique for measuring diffusion. FCS data are conventionally modeled with a finite number of diffusing components and fit with a least-square fitting algorithm. This approach is inadequate for analyzing data obtained from highly heterogeneous systems. We introduce a Maximum Entropy Method based fitting routine (MEMFCS) that analyzes FCS data in terms of a quasicontinuous distribution of diffusing components, and also guarantees a maximally wide distribution that is consistent with the data. We verify that for a homogeneous specimen (green fluorescent protein in dilute aqueous solution), both MEMFCS and conventional fitting yield similar results. Further, we incorporate an appropriate goodness of fit criterion in MEMFCS. We show that for errors estimated from a large number of repeated measurements, the reduced chi(2) value in MEMFCS analysis does approach unity. We find that the theoretical prediction for errors in FCS experiments overestimates the actual error, but can be empirically modified to serve as a guide for estimating the goodness of the fit where reliable error estimates are unavailable. Finally, we compare the performance of MEMFCS with that of a conventional fitting routine for analyzing simulated data describing a highly heterogeneous distribution containing 41 diffusing species. Both methods fit the data well. However, the conventional fit fails to reproduce the essential features of the input distribution, whereas MEMFCS yields a distribution close to the actual input.

Although amyloid β (Aβ) is a critical player in the pathology of Alzheimer's disease, there is currently little Information on the rate and extent of formation of oligomers that lead to the presence of Aβ fibrils observed in amyloid plaques. Here we describe a unique method to monitor the full time course of Aβ aggregation. In this method, Aβ is labeled with tetramethylrhodamine at a lysine residue on the N-terminal end. During aggregation, the fluorescence is quenched in a time-dependent manner in three distinct phases: an early oligomerization phase, an intermediate phase, and a growth phase. The oligomerization phase can be characterized as a monomer-dimer-trimer process for which we have determined the rate and equilibrium constants. The rate constants differ markedly between Aβ 1-42 and Aβ 1-40 , with Aβ 1-42 showing a greater oligomerization propensity. The intermediate phase reflects slow clustering and reorganization of the oligomers, whereas the growth phase ultimately results in the formation of fibrillar material. The data are consistent with a conformational change being an important rate-limiting step in the overall aggregation process. The rates of all phases are highly sensitive to temperature and pH, with the pH-dependent data indicating important roles for lysine and histidine residues. From the temperaturedependent data, activation energies of oligomerization and fibrillization are estimated to be 5.5 and 12.1 kCal/mol, respectively. The methodologies presented here are simple and can be applied to other amyloidogenic peptides or proteins.oligomer formation | fluorescence quenching | kinetics of aggregation | nucleation A lzheimer's disease (AD), the most prevalent form of neurodegenerative diseases, is characterized by deposition of senile plaques in the brain. These plaques contain aggregates of the amyloid β (Aβ) peptides Aβ 1-42 and Aβ 1-40 . In vitro, both peptides self-assemble into soluble oligomers and insoluble fibrils in a time-dependent manner. There has been extensive structure-function characterization of the in vitro aggregation process using a large number of available methods (1). The most widely used method follows changes in the fluorescence of Thioflavin T (ThT). This dye is particularly useful because of the large fluorescence increase observed during the final phase of aggregation coincident with the formation of β-strand structure. This assay, however, does not report on intermediates in the aggregation process and therefore cannot detect soluble oligomers that lack well-defined β-strand structure. It is critical to understand the nature of these small oligomers, because recent experiments suggest that they may be the major cytotoxic species for AD (2-5).Data in the literature present a highly complex picture of the oligomer formation, starting from dimers to spherical oligomers and linear protofibrils comprised of a large number of monomers. Biophysical characterization of the low-molecular-weight oligomers, however, has been difficult because of their small size and meta...