An electrostatic cluster guides Aß40 fibril formation in sporadic and Dutch-type cerebral amyloid angiopathy.

Cerebral amyloid angiopathy (CAA) is associated with the accumulation of fibrillar Aß peptides upon and within the cerebral vasculature, which leads to loss of vascular integrity and contributes to disease progression in Alzheimer's disease (AD). We investigate the structure of human-derived Aß40 fibrils obtained from patients diagnosed with sporadic or familial Dutch-type (E22Q) CAA. Using cryo-EM, two primary structures are identified containing elements that have not been observed in in vitro Aß40 fibril structures. One population has an ordered N-terminal fold comprised of two ß-strands stabilized by electrostatic interactions involving D1, E22, D23 and K28. This charged cluster is disrupted in the second population, which exhibits a disordered N-terminus and is favored in fibrils derived from the familial Dutch-type CAA patient. These results illustrate differences between human-derived CAA and AD fibrils, and how familial CAA mutations can guide fibril formation.

Constitutive activation and oncogenicity are mediated by loss of helical structure at the cytosolic boundary of thrombopoietin receptor mutant dimers.

Dimerization of the thrombopoietin receptor (TpoR) is necessary for receptor activation and downstream signaling through activated Janus kinase 2. We have shown previously that different orientations of the transmembrane (TM) helices within a receptor dimer can lead to different signaling outputs. Here we addressed the structural basis of activation for receptor mutations S505N and W515K that induce myeloproliferative neoplasms. We show using in vivo bone marrow reconstitution experiments that ligand-independent activation of TpoR by TM asparagine (Asn) substitutions is proportional to the proximity of the Asn mutation to the intracellular membrane surface. Solid-state NMR experiments on TM peptides indicate a progressive loss of helical structure in the juxtamembrane (JM) R/KWQFP motif with proximity of Asn substitutions to the cytosolic boundary. Mutational studies in the TpoR cytosolic JM region show that loss of the helical structure in the JM motif by itself can induce activation, but only when localized to a maximum of six amino acids downstream of W515, the helicity of the remaining region until Box 1 being required for receptor function. The constitutive activation of TpoR mutants S505N and W515K can be inhibited by rotation of TM helices within the TpoR dimer, which also restores helicity around W515. Together, these data allow us to develop a general model for activation of TpoR and explain the critical role of the JM W515 residue in the regulation of the activity of the receptor.

Mechanism of Activation of the Visual Receptor Rhodopsin.

Rhodopsin is the photoreceptor in human rod cells responsible for dim-light vision. The visual receptors are part of the large superfamily of G protein-coupled receptors (GPCRs) that mediate signal transduction in response to diverse diffusible ligands. The high level of sequence conservation within the transmembrane helices of the visual receptors and the family A GPCRs has long been considered evidence for a common pathway for signal transduction. I review recent studies that reveal a comprehensive mechanism for how light absorption by the retinylidene chromophore drives rhodopsin activation and highlight those features of the mechanism that are conserved across the ligand-activated GPCRs.

Impact of Aß40 and Aß42 Fibrils on the Transcriptome of Primary Astrocytes and Microglia.

Fibrillar amyloid ß-protein (Aß) deposits in the brain, which are primarily composed of Aß40 or Aß42 peptides, are key pathological features of Alzheimer's disease (AD) and related disorders. Although the underlying mechanisms are still not clear, the Aß fibrils can trigger a number of cellular responses, including activation of astrocytes and microglia. In addition, fibril structures of the Aß40 and Aß42 peptides are known to be polymorphic, which poses a challenge for attributing the contribution of different Aß sequences and structures to brain pathology. Here, we systematically treated primary astrocytes and microglia with single, well-characterized polymorphs of Aß40 or Aß42 fibrils, and performed bulk RNA sequencing to assess cell-specific changes in gene expression. A greater number of genes were up-regulated by Aß42 fibril-treated glial cells (251 and 2133 genes in astrocyte and microglia, respectively) compared with the Aß40 fibril-treated glial cells (191 and 251 genes in astrocytes and microglia, respectively). Immunolabeling studies in an AD rat model with parenchymal fibrillar Aß42 plaques confirmed the expression of PAI-1, MMP9, MMP12, CCL2, and C1r in plaque-associated microglia, and iNOS, GBP2, and C3D in plaque-associated astrocytes, validating markers from the RNA sequence data. In order to better understand these Aß fibril-induced gene changes, we analyzed gene expression patterns using the Ingenuity pathway analysis program. These analyses further highlighted that Aß42 fibril treatment up-regulated cellular activation pathways and immune response pathways in glial cells, including IL1ß and TNFα in astrocytes, and microglial activation and TGFß1 in microglia. Further analysis revealed that a number of disease-associated microglial (DAM) genes were surprisingly suppressed in Aß40 fibril treated microglia. Together, the present findings indicate that Aß42 fibrils generally show similar, but stronger, stimulating activity of glial cells compared with Aß40 fibril treatment.

Insights into Cerebral Amyloid Angiopathy Type 1 and Type 2 from Comparisons of the Fibrillar Assembly and Stability of the Aß40-Iowa and Aß40-Dutch Peptides.

Two distinct diseases are associated with the deposition of fibrillar amyloid-ß (Aß) peptides in the human brain in an age-dependent fashion. Alzheimer's disease is primarily associated with parenchymal plaque deposition of Aß42, while cerebral amyloid angiopathy (CAA) is associated with amyloid formation of predominantly Aß40 in the cerebral vasculature. In addition, familial mutations at positions 22 and 23 of the Aß sequence can enhance vascular deposition in the two major subtypes of CAA. The E22Q (Dutch) mutation is associated with CAA type 2, while the D23N (Iowa) mutation is associated with CAA type 1. Here we investigate differences in the formation and structure of fibrils of these mutant Aß peptides in vitro to gain insights into their biochemical and physiological differences in the brain. Using Fourier transform infrared and nuclear magnetic resonance spectroscopy, we measure the relative propensities of Aß40-Dutch and Aß40-Iowa to form antiparallel structure and compare these propensities to those of the wild-type Aß40 and Aß42 isoforms. We find that both Aß40-Dutch and Aß40-Iowa have strong propensities to form antiparallel ß-hairpins in the first step of the fibrillization process. However, there is a marked difference in the ability of these peptides to form elongated antiparallel structures. Importantly, we find marked differences in the stability of the protofibril or fibril states formed by the four Aß peptides. We discuss these differences with respect to the mechanisms of Aß fibril formation in CAA.

Magic angle spinning NMR of G protein-coupled receptors.

G protein-coupled receptors (GPCRs) have a simple seven transmembrane helix architecture which has evolved to recognize a diverse number of chemical signals. The more than 800 GPCRs encoded in the human genome function as receptors for vision, smell and taste, and mediate key physiological processes. Consequently, these receptors are a major target for pharmaceuticals. Protein crystallography and electron cryo-microscopy have provided high resolution structures of many GPCRs in both active and inactive conformations. However, these structures have not sparked a surge in rational drug design, in part because GPCRs are inherently dynamic and the structural changes induced by ligand or drug binding to stabilize inactive or active conformations are often subtle rearrangements in packing or hydrogen-bonding interactions. NMR spectroscopy provides a sensitive probe of local structure and dynamics at specific sites within these receptors as well as global changes in receptor structure and dynamics. These methods can also capture intermediate states and conformations with low populations that provide insights into the activation pathways. We review the use of solid-state magic angle spinning NMR to address the structure and activation mechanisms of GPCRs. The focus is on the large and diverse class A family of receptors. We highlight three specific class A GPCRs in order to illustrate how solid-state, as well as solution-state, NMR spectroscopy can answer questions in the field involving how different GPCR classes and subfamilies are activated by their associated ligands, and how small molecule drugs can modulate GPCR activation.

Human cerebral vascular amyloid contains both antiparallel and parallel in-register Aß40 fibrils.

The accumulation of fibrillar amyloid-ß (Aß) peptides alongside or within the cerebral vasculature is the hallmark of cerebral amyloid angiopathy (CAA). This condition commonly co-occurs with Alzheimer's disease (AD) and leads to cerebral microbleeds, intracranial hemorrhages, and stroke. CAA also occurs sporadically in an age-dependent fashion and can be accelerated by the presence of familial Aß mutant peptides. Recent studies using Fourier transform infrared (FTIR) spectroscopy of vascular Aß fibrils derived from rodents containing the double E22Q/D23N mutations indicated the presence of a novel antiparallel ß-sheet structure. To address whether this structure is associated solely with the familial mutations or is a common feature of CAA, we propagated Aß fibrils from human brain vascular tissue of patients diagnosed with nonfamilial CAA. Aß fibrils were isolated from cerebral blood vessels using laser capture microdissection in which specific amyloid deposits were removed from thin slices of the brain tissue. Transmission electron microscopy revealed that these deposits were organized into a tight meshwork of fibrils, which FTIR measurements showed could serve as seeds to propagate the growth of Aß40 fibrils for structural studies. Solid-state NMR measurements of the fibrils propagated from vascular amyloid showed they contained a mixture of parallel, in-register, and antiparallel ß-sheet structures. The presence of fibrils with antiparallel structure derived from vascular amyloid is distinct from the typical parallel, in-register ß-sheet structure that appears in fibrils derived from parenchymal amyloid in AD. These observations reveal that different microenvironments influence the structures of Aß fibrils in the human brain.

Deconstructing the transmembrane core of class A G protein-coupled receptors.

Class A G protein-coupled receptors have evolved to recognize ligands ranging from small-molecule odorants to proteins. Although they are among the most diverse membrane receptors in eukaryotic organisms, they possess a highly conserved core within their seven-transmembrane helix framework. The conservation of the transmembrane core has led to the idea of a common mechanism by which ligand binding is coupled to the outward rotation of helix H6, the hallmark of an active receptor. Nevertheless, there is still no consensus on the mechanism of coupling or on the roles of specific residues within the core. Recent insights from crystallography and NMR spectroscopy provide a way to decompose the core into its essential structural and functional elements that shed new light on this important region.

Erratum: Dimeric Transmembrane Orientations of APP/C99 Regulate γ-Secretase Processing Line Impacting Signaling and Oligomerization.

[This corrects the article DOI: 10.1016/j.isci.2020.101887.].

Anti-Parallel ß-Hairpin Structure in Soluble Aß Oligomers of Aß40-Dutch and Aß40-Iowa.

The amyloid-ß (Aß) peptides are associated with two prominent diseases in the brain, Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA). Aß42 is the dominant component of cored parenchymal plaques associated with AD, while Aß40 is the predominant component of vascular amyloid associated with CAA. There are familial CAA mutations at positions Glu22 and Asp23 that lead to aggressive Aß aggregation, drive vascular amyloid deposition and result in degradation of vascular membranes. In this study, we compared the transition of the monomeric Aß40-WT peptide into soluble oligomers and fibrils with the corresponding transitions of the Aß40-Dutch (E22Q), Aß40-Iowa (D23N) and Aß40-Dutch, Iowa (E22Q, D23N) mutants. FTIR measurements show that in a fashion similar to Aß40-WT, the familial CAA mutants form transient intermediates with anti-parallel ß-structure. This structure appears before the formation of cross-ß-sheet fibrils as determined by thioflavin T fluorescence and circular dichroism spectroscopy and occurs when AFM images reveal the presence of soluble oligomers and protofibrils. Although the anti-parallel ß-hairpin is a common intermediate on the pathway to Aß fibrils for the four peptides studied, the rate of conversion to cross-ß-sheet fibril structure differs for each.

Dimeric Transmembrane Orientations of APP/C99 Regulate γ-Secretase Processing Line Impacting Signaling and Oligomerization.

Amyloid precursor protein (APP) cleavage by the ß-secretase produces the C99 transmembrane (TM) protein, which contains three dimerization-inducing Gly-x-x-x-Gly motifs. We demonstrate that dimeric C99 TM orientations regulate the precise cleavage lines by γ-secretase. Of all possible dimeric orientations imposed by a coiled-coil to the C99 TM domain, the dimer containing the 33Gly-x-x-x-Gly37 motif in the interface promoted the Aß42 processing line and APP intracellular domain-dependent gene transcription, including the induction of BACE1 mRNA, enhancing amyloidogenic processing and signaling. Another orientation exhibiting the 25Gly-x-x-x-Gly29 motif in the interface favored processing to Aß43/40. It induced significantly less gene transcription, while promoting formation of SDS-resistant "Aß-like" oligomers, reminiscent of Aß peptide oligomers. These required both Val24 of a pro-ß motif and the 25Gly-x-x-x-Gly29 interface. Thus, crossing angles imposed by precise dimeric orientations control γ-secretase initial cleavage at Aß48 or Aß49, linking the former to enhanced signaling and Aß42 production.

Copper stabilizes antiparallel ß-sheet fibrils of the amyloid ß40 (Aß40)-Iowa variant.

Cerebral amyloid angiopathy (CAA) is a vascular disorder that primarily involves deposition of the 40-residue-long ß-amyloid peptide (Aß40) in and along small blood vessels of the brain. CAA is often associated with Alzheimer's disease (AD), which is characterized by amyloid plaques in the brain parenchyma enriched in the Aß42 peptide. Several recent studies have suggested a structural origin that underlies the differences between the vascular amyloid deposits in CAA and the parenchymal plaques in AD. We previously have found that amyloid fibrils in vascular amyloid contain antiparallel ß-sheet, whereas previous studies by other researchers have reported parallel ß-sheet in fibrils from parenchymal amyloid. Using X-ray fluorescence microscopy, here we found that copper strongly co-localizes with vascular amyloid in human sporadic CAA and familial Iowa-type CAA brains compared with control brain blood vessels lacking amyloid deposits. We show that binding of Cu(II) ions to antiparallel fibrils can block the conversion of these fibrils to the more stable parallel, in-register conformation and enhances their ability to serve as templates for seeded growth. These results provide an explanation for how thermodynamically less stable antiparallel fibrils may form amyloid in or on cerebral vessels by using Cu(II) as a structural cofactor.

A Conserved Proline Hinge Mediates Helix Dynamics and Activation of Rhodopsin.

Despite high-resolution crystal structures of both inactive and active G protein-coupled receptors (GPCRs), it is still not known how ligands trigger the large structural change on the intracellular side of the receptor since the conformational changes that occur within the extracellular ligand-binding region upon activation are subtle. Here, we use solid-state NMR and Fourier transform infrared spectroscopy on rhodopsin to show that Trp2656.48 within the CWxP motif on transmembrane helix H6 constrains a proline hinge in the inactive state, suggesting that activation results in unraveling of the H6 backbone within this motif, a local change in dynamics that allows helix H6 to swing outward. Notably, Tyr3017.48 within activation switch 2 appears to mimic the negative allosteric sodium ion found in other family A GPCRs, a finding that is broadly relevant to the mechanism of receptor activation.

MPL mutations in essential thrombocythemia uncover a common path of activation with eltrombopag dependent on W491.

Mutations in the MPL gene encoding the human thrombopoietin receptor (TpoR) drive sporadic and familial essential thrombocythemias (ETs). We identified 2 ET patients harboring double mutations in cis in MPL, namely, L498W-H499C and H499Y-S505N. Using biochemical and signaling assays along with partial saturation mutagenesis, we showed that L498W is an activating mutation potentiated by H499C and that H499C and H499Y enhance the activity of the canonical S505N mutation. L498W and H499C can activate a truncated TpoR mutant, which lacks the extracellular domain, indicating these mutations act on the transmembrane (TM) cytosolic domain. Using a protein complementation assay, we showed that L498W and H499C strongly drive dimerization of TpoR. Activation by tryptophan substitution is exquisitely specific for position 498. Using structure-guided mutagenesis, we identified upstream amino acid W491 as a key residue required for activation by L498W or canonical activating mutations such as S505N and W515K, as well as by eltrombopag. Structural data point to a common dimerization and activation path for TpoR via its TM domain that is shared between the small-molecule agonist eltrombopag and canonical and novel activating TpoR mutations that all depend on W491, a potentially accessible extracellular residue that could become a target for therapeutic intervention.

Influence of the familial Alzheimer's disease-associated T43I mutation on the transmembrane structure and γ-secretase processing of the C99 peptide.

Extracellular deposition of ß-amyloid (Aß) peptides in the brain is a hallmark of Alzheimer's disease (AD). Upon ß-secretase-mediated cleavage of the ß C-terminal fragment (ß-CTF) from the Aß precursor protein, the γ-secretase complex produces the Aß peptides associated with AD. The familial T43I mutation within the transmembrane domain of the ß-CTF (also referred to as C99) increases the ratio between the Aß42 and Aß40 peptides largely due to a decrease in Aß40 formation. Aß42 is the principal component of amyloid deposits within the brain parenchyma, and an increase in the Aß42/Aß40 ratio is correlated with early-onset AD. Using NMR and FTIR spectroscopy, here we addressed how the T43I substitution influences the structure of C55, the minimal sequence containing the entire extracellular and transmembrane (TM) domains of C99 needed for γ-secretase processing. 13C NMR chemical shifts indicated that the T43I substitution increases helical structure within the TM domain of C55. These structural changes were associated with a shift of the C55 dimer to the monomer and an increase in the tilt of the TM helix relative to the membrane normal in the T43I mutant compared with that of WT C55. The A21G (Flemish) mutation was previously found to increase secreted Aß40 levels; here, we combined this mutation in the extracellular domain of C99 with T43I and observed that the T43I/A21G double mutant decreases Aß40 formation. We discuss how the observed structural changes in the T43I mutant may decrease Aß40 formation and increase the Aß42/Aß40 ratio.

A Novel Transgenic Rat Model of Robust Cerebral Microvascular Amyloid with Prominent Vasculopathy.

Accumulation of fibrillar amyloid ß protein in blood vessels of the brain, a condition known as cerebral amyloid angiopathy (CAA), is a common pathology of elderly individuals, a prominent comorbidity of Alzheimer disease, and a driver of vascular cognitive impairment and dementia. Although several transgenic mouse strains have been generated that develop varying levels of CAA, consistent models of associated cerebral microhemorrhage and vasculopathy observed clinically have been lacking. Reliable preclinical animal models of CAA and microhemorrhage are needed to investigate the molecular pathogenesis of this condition. Herein, we describe the generation and characterization of a novel transgenic rat (rTg-DI) that produces low levels of human familial CAA Dutch/Iowa E22Q/D23N mutant amyloid ß protein in brain and faithfully recapitulates many of the pathologic aspects of human small-vessel CAA. rTg-DI rats exhibit early-onset and progressive accumulation of cerebral microvascular fibrillar amyloid accompanied by early-onset and sustained behavioral deficits. Comparable to CAA in humans, the cerebral microvascular amyloid in rTg-DI rats causes capillary structural alterations, promotes prominent perivascular neuroinflammation, and produces consistent, robust microhemorrhages and small-vessel occlusions that are readily detected by magnetic resonance imaging. The rTg-DI rats provide a new model to investigate the pathogenesis of small-vessel CAA and microhemorrhages, to develop effective biomarkers for this condition and to test therapeutic interventions.

Disaggregation of Aß42 for Structural and Biochemical Studies.

The amyloid-ß (Aß) peptides that form the amyloid fibrils associated with Alzheimer's disease are generated by sequential proteolysis of the amyloid precursor protein by ß- and γ-secretase. The two predominant Aß peptides, Aß40 and Aß42, differ by two amino acids, are soluble as monomers at low concentration (and/or low temperature) and are normally cleared from the brain parenchyma. In order to study the structure and assembly of these peptides, they are often synthesized using solid-phase peptide synthesis and purified. Here, we outline the method we use to prepare monomeric Aß for structural and biochemical studies.

ß-Sheet Structure within the Extracellular Domain of C99 Regulates Amyloidogenic Processing.

Familial mutations in C99 can increase the total level of the soluble Aß peptides produced by proteolysis, as well as the Aß42/Aß40 ratio, both of which are linked to the progression of Alzheimer's disease. We show that the extracellular sequence of C99 forms ß-sheet structure upon interaction with membrane bilayers. Mutations that disrupt this structure result in a significant increase in Aß production and, in specific cases, result in an increase in the amount of Aß42 relative to Aß40. Fourier transform infrared and solid-state NMR spectroscopic studies reveal a central ß-hairpin within the extracellular sequence comprising Y10-E11-V12 and L17-V18-F19 connected by a loop involving H13-H14-Q15. These results suggest how familial mutations in the extracellular sequence influence C99 processing and provide a structural basis for the development of small molecule modulators that would reduce Aß production.

G Protein-Coupled Receptors Contain Two Conserved Packing Clusters.

G protein-coupled receptors (GPCRs) have evolved a seven-transmembrane helix framework that is responsive to a wide range of extracellular signals. An analysis of the interior packing of family A GPCR crystal structures reveals two clusters of highly packed residues that facilitate tight transmembrane helix association. These clusters are centered on amino acid positions 2.47 and 4.53, which are highly conserved as alanine and serine, respectively. Ala2.47 mediates the interaction between helices H1 and H2, while Ser4.53 mediates the interaction between helices H3 and H4. The helical interfaces outside of these clusters are lined with residues that are more loosely packed, a structural feature that facilitates motion of helices H5, H6, and H7, which is required for receptor activation. Mutation of the conserved small side chain at position 4.53 within packing cluster 2 is shown to disrupt the structure of the visual receptor rhodopsin, whereas sites in packing cluster 1 (e.g., positions 1.46 and 2.47) are more tolerant to mutation but affect the overall stability of the protein. These findings reveal a common structural scaffold of GPCRs that is important for receptor folding and activation.