Kanchan Garai

ApoE influences amyloid-β (Aβ) clearance despite minimal apoE/Aβ association in physiological conditions

Significance It has been proposed that differential physical interactions of apolipoprotein E (apoE) isoforms with soluble amyloid-β (Aβ) in brain fluids influence the metabolism of Aβ, providing a major mechanism to account for how APOE influences Alzheimer’s disease risk. The current study challenges this proposal and clearly shows that lipoproteins containing apoE isoforms are unlikely to play a significant role in Aβ metabolism by binding directly to Aβ in physiological fluids such as cerebrospinal fluid or interstitial fluid. Our in vitro and in vivo results suggest that apoE isoforms influence Aβ metabolism by competing for the same clearance pathways within the brain. 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.

Measuring size distribution in highly heterogeneous systems with fluorescence correlation spectroscopy.

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.

Unmasking the roles of N- and C-terminal flanking sequences from exon 1 of huntingtin as modulators of polyglutamine aggregation

Significance How do N- and C-terminal flanking sequences from exon 1 of the huntingtin protein modulate the mechanisms of polyglutamine aggregation? We answer this question using approaches that combine distinct probes of aggregation mechanisms with measurements of solubility and aggregate morphologies. The N- and C-terminal flanking sequence modules from exon 1 of huntingtin act as gatekeepers, whereby the N-terminal flanking sequence accelerates fibril formation while destabilizing nonfibrillar species, whereas the C-terminal flanking sequence reduces the overall driving force for aggregation. These results provide a mechanistic underpinning for observations regarding naturally occurring sequence contexts as modulators of polyglutamine toxicity. 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.

Quantitative analysis of the time course of Aβ oligomerization and subsequent growth steps using tetramethylrhodamine-labeled Aβ.

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 Aβ1–42 and Aβ1–40, with Aβ1–42 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 temperature-dependent 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.

Hydrogen/Deuterium Exchange and Electron-Transfer Dissociation Mass Spectrometry Determine the Interface and Dynamics of Apolipoprotein E Oligomerization

Apolipoprotein E, a 34 kDa protein, plays a key role in triglyceride and cholesterol metabolism. Of the three common isoforms (ApoE2, -3, and -4), only ApoE4 is a risk factor for Alzheimers disease. All three isoforms of wild-type ApoE self-associate to form oligomers, a process that may have functional consequences. Although the C-terminal domain, residues 216-299, of ApoE is believed to mediate self-association, the specific residues involved in this process are not known. Here we report the use of hydrogen/deuterium exchange (H/DX) coupled with enzymatic digestion to identify those regions in the sequence of full-length apoE involved in oligomerization. For this determination, we compared the results of H/DX of the wild-type proteins and those of monomeric forms obtained by modifying four residues in the C-terminal domain. The three wild-type and mutant isoforms show similar structures based on their similar H/DX kinetics and extents of exchange. Regions of the C-terminus (residues 230-270) of the ApoE isoforms show significant differences of deuterium uptake between oligomeric and monomeric forms, confirming that oligomerization occurs at these regions. To achieve single amino acid resolution, we examined the extents of H/DX by using electron transfer dissociation (ETD) fragmentation of peptides representing selected regions of both the monomeric and the oligomeric forms of ApoE4. From these experiments, we could identify the specific residues involved in ApoE oligomerization. In addition, our results verify that ApoE4 is composed of a compact structure at its N-terminal domain. Regions of C-terminal domain, however, appear to lack defined structure.

Structural differences between apoE3 and apoE4 may be useful in developing therapeutic agents for Alzheimer's disease.

Apolipoproteins E3 and E4, proteins with a molecular mass of 34.15 kDa, differ by a single amino acid change. ApoE4 contains an arginine residue at position 112, whereas apoE3 has a cysteine at this position. ApoE4 is the major risk factor for late-onset Alzheimer’s disease, whereas apoE3, the common isoform, is neutral with respect to this disease. Here, using literature data from both hydrogen-deuterium exchange and site-directed mutations, we suggest structural differences between these two isoforms that are distant from the site of the arginine-to-cysteine change. These structural differences involve sequences from both the N- and C-terminal domains, sequentially far apart but structurally close. In addition, these regions are close to regions that bind lipid and to a region involved in association of apoE monomers to higher molecular weight forms. We discuss the possibility that these regions could be targeted preferentially to affect the function of apoE4 relative to apoE3.

The binding of apolipoprotein E to oligomers and fibrils of amyloid-β alters the kinetics of amyloid aggregation.

Deposition of amyloid-β (Aβ) in Alzheimer’s disease (AD) is strongly correlated with the APOE genotype. However, the role of apolipoprotein E (apoE) in Aβ aggregation has remained unclear. Here we have used different apoE preparations, such as recombinant protein or protein isolated from cultured astrocytes, to examine the effect of apoE on the aggregation of both Aβ1–40 and Aβ1–42. The kinetics of aggregation, measured by the loss of fluorescence of tetramethylrhodamine-labeled Aβ, is shown to be dramatically slowed by the presence of substoichiometric concentrations of apoE. Using these concentrations, we conclude that apoE binds primarily to and affects the growth of oligomers that lead to the nuclei required for fibril growth. At higher apoE concentrations, the protein also binds to Aβ fibrils, resulting in fibril stabilization and a slower rate of fibril growth. The aggregation of Aβ1–40 is dependent on the apoE isoform, being the most dramatic for apoE4 and less so for apoE3 and apoE2. Our results indicate that the detrimental role of apoE4 in AD could be related to apoE-induced stabilization of the soluble but cytotoxic oligomeric forms and intermediates of Aβ, as well as fibril stabilization.

The association−dissociation behavior of the ApoE proteins: kinetic and equilibrium studies.

The apolipoprotein E family consists of three major protein isoforms: apolipoprotein E4 (ApoE4), ApoE3, and ApoE2. The isoforms, which contain 299 residues, differ only by single-amino acid changes, but of the three, only ApoE4 is a risk factor for Alzheimer’s disease. At micromolar concentrations, lipid-free ApoE exists predominantly as tetramers. In more dilute solutions, lower-molecular mass species predominate. Using fluorescence correlation spectroscopy (FCS), intermolecular fluorescence resonance energy transfer (FRET), and sedimentation methods, we found that the association−dissociation reaction of ApoE can be modeled with a monomer−dimer−tetramer process. Equilibrium constants have been determined from the sedimentation data, while the individual rate constants for association and dissociation were determined by measurement of the kinetics of dissociation of ApoE and are in agreement with the equilibrium constants. Dissociation kinetics as measured by intermolecular FRET show two phases reflecting the dissociation of tetramer to dimer and of dimer to monomer, with dissociation from tetramer to dimer being more rapid than the dissociation from dimer to monomer. The rate constants differ for the different ApoE isoforms, showing that the association−dissociation process is isoform specific. Strikingly, the association rate constants are almost 2 orders of magnitude slower than expected for a diffusion-controlled process. Dissociation kinetics were also monitored by tryptophan fluorescence in the presence of acrylamide and the data found to be consistent with the monomer−dimer−tetramer model. The approach combining multiple methods establishes the reaction scheme of ApoE self-association.

Mass Spectrometry-based Protein Footprinting Characterizes the Structures of Oligomeric Apolipoprotein E2, E3, and E4

The three common isoforms of apolipoprotein E (ApoE) differ at two sites in their 299 amino acid sequence; these differences modulate the structure of ApoE to affect profoundly the isoform associations with disease. The ε4 allele in particular is strongly associated with Alzheimers disease. The study of the structural effects of these mutation sites in aqueous media is hampered by the aggregation proclivity of each ApoE isoform. Hence, understanding the differences between isoforms has thus far relied on lower resolution biophysical measurements, mutagenesis, homology studies, and the use of truncated ApoE variants. In this study, we report two comparative studies of the ApoE family by using the mass spectrometry-based protein footprinting methods of FPOP and glycine ethyl ester (GEE) labeling. The first experiment examines the three full-length WT isoforms in their tetrameric state and finds that the overall structures are similar, with the exception of M108 in ApoE4 which is more solvent-accessible in this isoform than in ApoE2 and ApoE3. The second experiment provides clear evidence, from a comparison of the footprinting results of the wild-type proteins and a monomeric mutant, that several residues in regions 183-205 and 232-251 are involved in self-association.

Quasihomogeneous nucleation of amyloid beta yields numerical bounds for the critical radius, the surface tension, and the free energy barrier for nucleus formation

Amyloid aggregates are believed to grow through a nucleation mediated pathway, but important aggregation parameters, such as the nucleation radius, the surface tension of the aggregate, and the free energy barrier toward aggregation, have remained difficult to measure. Homogeneous nucleation theory, if applicable, can directly relate these parameters to measurable quantities. We employ fluorescence correlation spectroscopy to measure the particle size distribution in an aggregating solution of Alzheimers amyloid beta molecule (Abeta(1-40)) and analyze the data from a homogeneous nucleation theory perspective. We observe a reproducible saturation concentration and a critical dependence of various aspects of the aggregation process on this saturation concentration, which supports the applicability of the nucleation theory to Abeta aggregation. The measured size distributions show a valley between two peaks ranging from 5 to 50 nm, which defines a boundary for the value of the nucleation radius. By carefully controlling the conditions to inhibit heterogeneous nucleation, we can hold off nucleation in a 25 times supersaturated solution for at least up to 3 h at room temperature. This quasi-homogeneous kinetics implies that at room temperature, the surface energy of the Abeta/water interface is > or =4.8 mJ/m(2), the free energy barrier to nucleation (at 25 times supersaturation) is > or =1.93x10(-19) J, and the number of monomers in the nucleus is > or =29.