α-Synuclein assembles into higher-order multimers upon membrane binding to promote SNARE complex formation

Physiologically, α-synuclein chaperones soluble NSF attachment protein receptor (SNARE) complex assembly and may also perform other functions; pathologically, in contrast, α-synuclein misfolds into neurotoxic aggregates that mediate neurodegeneration and propagate between neurons. In neurons, α-synuclein exists in an equilibrium between cytosolic and membrane-bound states. Cytosolic α-synuclein appears to be natively unfolded, whereas membrane-bound α-synuclein adopts an α-helical conformation. Although the majority of studies showed that cytosolic α-synuclein is monomeric, it is unknown whether membrane-bound α-synuclein is also monomeric, and whether chaperoning of SNARE complex assembly by α-synuclein involves its cytosolic or membrane-bound state. Here, we show using chemical cross-linking and fluorescence resonance energy transfer (FRET) that α-synuclein multimerizes into large homomeric complexes upon membrane binding. The FRET experiments indicated that the multimers of membrane-bound α-synuclein exhibit defined intermolecular contacts, suggesting an ordered array. Moreover, we demonstrate that α-synuclein promotes SNARE complex assembly at the presynaptic plasma membrane in its multimeric membrane-bound state, but not in its monomeric cytosolic state. Our data delineate a folding pathway for α-synuclein that ranges from a monomeric, natively unfolded form in cytosol to a physiologically functional, multimeric form upon membrane binding, and show that only the latter but not the former acts as a SNARE complex chaperone at the presynaptic terminal, and may protect against neurodegeneration.α-Synuclein is an abundant presynaptic protein that physiologically acts to promote soluble NSF attachment protein receptor (SNARE) complex assembly in vitro and in vivo (1–3). Point mutations in α-synuclein (A30P, E46K, H50Q, G51D, and A53T) as well as α-synuclein gene duplications and triplications produce early-onset Parkinson''s disease (PD) (4–10). Moreover, α-synuclein is a major component of intracellular protein aggregates called Lewy bodies, which are pathological hallmarks of neurodegenerative disorders such as PD, Lewy body dementia, and multiple system atrophy (11–14). Strikingly, neurotoxic α-synuclein aggregates propagate between neurons during neurodegeneration, suggesting that such α-synuclein aggregates are not only intrinsically neurotoxic but also nucleate additional fibrillization (15–18).α-Synuclein is highly concentrated in presynaptic terminals where α-synuclein exists in an equilibrium between a soluble and a membrane-bound state, and is associated with synaptic vesicles (19–22). The labile association of α-synuclein with membranes (23, 24) suggests that binding of α-synuclein to synaptic vesicles, and its dissociation from these vesicles, may regulate its physiological function. Membrane-bound α-synuclein assumes an α-helical conformation (25–32), whereas cytosolic α-synuclein is natively unfolded and monomeric (refs. 25, 26, 31, and 32; however, see refs. 33 and 34 and Discussion for a divergent view). Membrane binding by α-synuclein is likely physiologically important because in in vitro experiments, α-synuclein remodels membranes (35, 36), influences lipid packing (37, 38), and induces vesicle clustering (39). Moreover, membranes were found to be important for the neuropathological effects of α-synuclein (40–44).However, the relation of membrane binding to the in vivo function of α-synuclein remains unexplored, and it is unknown whether α-synuclein binds to membranes as a monomer or oligomer. Thus, in the present study we have investigated the nature of the membrane-bound state of α-synuclein and its relation to its physiological function in SNARE complex assembly. We found that soluble monomeric α-synuclein assembles into higher-order multimers upon membrane binding and that membrane binding of α-synuclein is required for its physiological activity in promoting SNARE complex assembly at the synapse.     

Molecular mechanism underlying β1 regulation in voltage- and calcium-activated potassium (BK) channels

Being activated by depolarizing voltages and increases in cytoplasmic Ca²⁺, voltage- and calcium-activated potassium (BK) channels and their modulatory β-subunits are able to dampen or stop excitatory stimuli in a wide range of cellular types, including both neuronal and nonneuronal tissues. Minimal alterations in BK channel function may contribute to the pathophysiology of several diseases, including hypertension, asthma, cancer, epilepsy, and diabetes. Several gating processes, allosterically coupled to each other, control BK channel activity and are potential targets for regulation by auxiliary β-subunits that are expressed together with the α (BK)-subunit in almost every tissue type where they are found. By measuring gating currents in BK channels coexpressed with chimeras between β1 and β3 or β2 auxiliary subunits, we were able to identify that the cytoplasmic regions of β1 are responsible for the modulation of the voltage sensors. In addition, we narrowed down the structural determinants to the N terminus of β1, which contains two lysine residues (i.e., K3 and K4), which upon substitution virtually abolished the effects of β1 on charge movement. The mechanism by which K3 and K4 stabilize the voltage sensor is not electrostatic but specific, and the α (BK)-residues involved remain to be identified. This is the first report, to our knowledge, where the regulatory effects of the β1-subunit have been clearly assigned to a particular segment, with two pivotal amino acids being responsible for this modulation.High-conductance voltage- and calcium-activated potassium (BK) channels are homotetrameric proteins of α-subunits encoded by the slo1 gene (1). These channels are expressed in virtually all mammalian tissues, where they detect and integrate membrane voltage and calcium concentration changes dampening the responsiveness of cells when confronted with excitatory stimuli. They are abundant in the CNS and nonneuronal tissues, such as smooth muscle or hair cells. This wide distribution is associated with an outstandingly large functional diversity, in which BK channel activity appears optimally adapted to the particular physiological demands of each cell type (2). On the other hand, small alterations in BK channel function may contribute to the pathophysiology of hypertension, asthma, cancer, epilepsy, diabetes, and other conditions in humans (3–8). Alternative splicing, posttranslational modifications, and regulation by auxiliary proteins have been proposed to contribute to this functional diversity (1, 2, 9–16).The BK channel α-subunit is formed by a single polypeptide of about 1,200 amino acids that contains all of the key structural elements for ion permeation, gating, and modulation by ions and other proteins. Tetramers of α-subunits form functional BK channels. Each subunit has seven hydrophobic transmembrane segments (S0–S6), where the voltage-sensor domain (VSD) and pore domain (PD) reside (2). The N terminus faces the extracellular side of the membrane, whereas the C terminus is intracellular. The latter contains four hydrophobic α-helices (S7–S10) and the main Ca²⁺ binding sites (2). VSDs formed by segments S1–S4 harbor a series of charged residues across the membrane that contributes to voltage sensing (2). Upon membrane depolarization, each VSD undergoes a rearrangement (17) that prompts the opening of a highly K⁺-selective pore formed by the four PDs that come together at the symmetry center of the tetramer.Although BK channel expression is ubiquitous, in most physiological scenarios their functioning is provided by their coassembly with auxiliary proteins, such as β-subunits. This coassembly brings channel activity into the proper cell/tissue context (11, 13). Four different β-subunits have been cloned (β1–β4) (18–24), all of which have been observed to modify BK channel function. Albeit to a different extent, all β-subunits modify the Ca²⁺ sensitivity, voltage dependence, and gating properties of BK channels, hence modifying plasma membrane excitability balance. Regarding auxiliary β-subunits, β1- and β2-subunits increase apparent Ca²⁺ sensitivity and decelerate macroscopic current kinetics (14, 20, 21, 25–30); β2 and β3 induce fast inactivation as well as an instantaneous outward rectification (20, 21, 24, 31, 32); and β4 slows down activation and deactivation kinetics (12, 23) and modifies Ca²⁺ sensitivity (12, 33, 34).It should be kept in mind that β-subunits are potential targets for different molecules that modulate channel function, such as alcohol (35), estrogens (15), hormones (36), and fatty acids (37, 38). Additionally, scorpion toxin affinity in BK channels would tend to increase when β1 is coexpressed with the α-subunit (22).To identify the molecular elements that give β1 the ability to modulate the voltage sensor of BK channels, we constructed chimeric proteins of β1/β2- and β1/β3-subunits by swapping their N and C termini, the transmembrane (TM) segments, and the extracellular loops and recorded their gating currents. Two lysine residues that are unique to the N terminus of β1 were identified to be sufficient for BK voltage-sensor modulation.     

αIIbβ3 variants defined by next-generation sequencing: Predicting variants likely to cause Glanzmann thrombasthenia

Next-generation sequencing is transforming our understanding of human genetic variation but assessing the functional impact of novel variants presents challenges. We analyzed missense variants in the integrin αIIbβ3 receptor subunit genes ITGA2B and ITGB3 identified by whole-exome or -genome sequencing in the ThromboGenomics project, comprising ∼32,000 alleles from 16,108 individuals. We analyzed the results in comparison with 111 missense variants in these genes previously reported as being associated with Glanzmann thrombasthenia (GT), 20 associated with alloimmune thrombocytopenia, and 5 associated with aniso/macrothrombocytopenia. We identified 114 novel missense variants in ITGA2B (affecting ∼11% of the amino acids) and 68 novel missense variants in ITGB3 (affecting ∼9% of the amino acids). Of the variants, 96% had minor allele frequencies (MAF) < 0.1%, indicating their rarity. Based on sequence conservation, MAF, and location on a complete model of αIIbβ3, we selected three novel variants that affect amino acids previously associated with GT for expression in HEK293 cells. αIIb P176H and β3 C547G severely reduced αIIbβ3 expression, whereas αIIb P943A partially reduced αIIbβ3 expression and had no effect on fibrinogen binding. We used receiver operating characteristic curves of combined annotation-dependent depletion, Polyphen 2-HDIV, and sorting intolerant from tolerant to estimate the percentage of novel variants likely to be deleterious. At optimal cut-off values, which had 69–98% sensitivity in detecting GT mutations, between 27% and 71% of the novel αIIb or β3 missense variants were predicted to be deleterious. Our data have implications for understanding the evolutionary pressure on αIIbβ3 and highlight the challenges in predicting the clinical significance of novel missense variants.Next-generation sequencing is transforming our understanding of human genetic variation (1) and providing profound insights into the impact of both inherited and de novo variants on human health (2, 3). At the same time, the data from these studies present serious challenges in providing information to individuals who are found to have variant forms of different proteins. To highlight these challenges, in this report we describe our experience in analyzing missense variants of the platelet αIIbβ3 integrin receptor from The Human Genome Mutation Database (HGMD), the 1000 Genomes project (1000G), the United Kingdom 10K Whole Exome Sequencing project (U.K.10KWES), the United Kingdom 10K Whole Genome Sequencing project (U.K.10KWGS), and The National Heart, Lung and Blood Institute Exome Sequencing Project (ESP); the latter four sources encompass ∼32,000 alleles derived from 16,108 individuals.The αIIbβ3 receptor has a number of virtues as a model system. First, it is required for hemostasis because platelet aggregation requires cross-linking of the activated form of αIIbβ3 by macromolecular ligands (4). Thus, defects in its biogenesis, activation, or ligand binding lead to the rare bleeding diathesis Glanzmann thrombasthenia (GT), an autosomal recessive disorder (5, 6). Patients with GT come to medical attention because of their hemorrhagic symptoms, and thus have been carefully analyzed clinically and with tests of platelet function for nearly 50 y (5, 7). The biochemical and molecular abnormalities in GT have been studied for nearly 40 y (4, 6, 8–10). In the past 10 y, high-resolution crystallography, electron microscopy, and computational studies of the αIIbβ3 receptor have provided atomic-level information on the correlation between receptor structure and function (11–21). In addition, ethnic groups with relatively high prevalence of GT have been defined that share the same genetic abnormality based on founder mutations, and the dates that some of the mutations entered the population have been estimated (22–28). An on-line registry of GT abnormalities, including patient phenotypes was developed in 1997 (29) and currently contains 51 αIIb and 43 β3 missense variants linked to the disorder (sinaicentral.mssm.edu/intranet/research/glanzmann). The frequency of GT in the general population has not been established but it has a world-wide distribution, and based on data from hematologic practices, it is rare except in areas with a high rate of consanguineous mating (30).Second, alloimmune disorders, including neonatal thrombocytopenia and posttransfusion purpura, due to amino acid substitutions in either αIIb or β3, have been characterized at the molecular biological level and correlated with mechanisms of immunologic recognition (31).Third, inherited macrothrombocytopenia and anisothrombocytopenia have been associated with heterozygous missense variants or deletions in αIIb or β3. All of these appear to induce constitutive activation of the receptor and impair proplatelet formation (32–38).Fourth, αIIbβ3 contributes to pathological platelet thrombus formation in human ischemic cardiovascular disease and αIIbβ3 is a validated target for antithrombotic therapy (39–41).Fifth, αIIbβ3 is a member of the large integrin family of receptors, which includes 24 receptors derived from 18 α- and 8 β-subunits (41, 42). These receptors are involved in important biologic processes, including development, cell migration, homing, cell survival, and adaptive immunity (41–43). More is known about the structure–function relationships of αIIbβ3 than the other members of the group, and so it serves as the paradigmatic integrin receptor (44, 45).Sixth, 3D molecular models have been built based on crystallographic and NMR data to analyze the effects of novel amino acid substitutions on receptor structure and function and the generation of alloantigens (15, 46–53). The data from these models and assessments of the severity of the amino acid change in the variants have the potential to aid in predicting whether a novel variant is likely to affect receptor function and immunogenicity (54–59).     

Differential expression of estrogen receptor α, β1, and β2 in lobular and ductal breast cancer

The role of estrogen receptor (ER) α as a target in treatment of breast cancer is clear, but those of ERβ1 and ERβ2 in the breast remain unclear. We have examined expression of all three receptors in surgically excised breast samples from two archives: (i): 187 invasive ductal breast cancer from a Japanese study; and (ii) 20 lobular and 24 ductal cancers from the Imperial College. Samples contained normal areas, areas of hyperplasia, and in situ and invasive cancer. In the normal areas, ERα was expressed in not more than 10% of epithelium, whereas approximately 80% of epithelial cells expressed ERβ. We found that whereas ductal cancer is a highly proliferative, ERα-positive, ERβ-negative disease, lobular cancer expresses both ERα and ERβ but with very few Ki67-positive cells. ERβ2 was expressed in 32% of the ductal cancers, of which 83% were postmenopausal. In all ERβ2-positive cancers the interductal space was filled with dense collagen, and cell nuclei expressed hypoxia-inducible factor 1α. ERβ2 expression was not confined to malignant cells but was strong in stromal, immune, and endothelial cells. In most of the high-grade invasive ductal cancers neither ERα nor ERβ was expressed, but in the high-grade lobular cancer ERβ was lost and ERα and Ki67 expression were abundant. The data show a clear difference in ER expression between lobular and ductal breast cancer and suggest (i) that tamoxifen may be more effective in late than in early lobular cancer and (ii) a potential role for ERβ agonists in preventing in situ ductal cancers from becoming invasive.Despite decades of research, the etiology of breast cancer remains unclear. It is currently thought that most breast cancers occur in the normal terminal duct lobular unit and progress in a stepwise fashion over time (1). Ductal carcinoma in situ (DCIS) means the cancer has not spread beyond the duct into any normal surrounding breast tissue and is thought by some to be the direct precursor of invasive ductal carcinoma (IDC).Estrogens play an important role in normal breast development as well as breast cancer progression (2). Most of the effects of estrogen are mediated through its two receptors: estrogen receptor α (ERα) and β (ERβ) (3). ERα is expressed in 50–80% of breast tumors, and its presence is the main indicator for antihormonal therapy (4). ERβ was first discovered in 1996, and its role in breast cancer is still being explored (5–7).The first step in understanding the role of ERβ in breast cancer was to define the expression pattern of ERβ in the normal human breast and in various stages of cancer. Since its discovery, several laboratories have reported ERβ expression in clinical samples (8–28). Most of these studies investigated the expression of ERβ in invasive breast cancer samples (12–15, 17, 19, 21–23). Some studies have reported ERβ expression in invasive breast cancer and normal breast tissue (11, 18, 26–28), but few have compared the expression of ERβ in the normal tissue, DCIS, and IDC within the same sample. Usually tumor samples are taken from one patient and normal tissue from another patient (8–10). Samples taken from different patients have intrinsic limitation (i.e., they cannot account for variations between different patients). In addition, because tumors are heterogeneous, core biopsies do not fully reflect the histological and biological diversity of breast tumors (29).The roles of ERβ1 and its splice variant ERβ2 in breast cancer are still unclear. As reviewed by Murphy and Leygue (30), some studies show a loss of ERβ1 as ductal cancer progresses, but others do not. Some studies show ERβ2 as a marker of bad prognosis (31), and others not (19). Some of these differences may be due to differences in antibody use and differences in tissue fixation and handling.When ERα and ERβ are coexpressed in breast cancer it is unclear whether tamoxifen treatment will be successful. This is because tamoxifen acts as an agonist of ERβ at activator protein 1 (AP-1) sites (32) and thus should oppose the antiproliferative effects of the tamoxifen–ERα complex. Yan et al. (33) have found that expression of ERβ predicts tamoxifen benefit in patients with ERα-negative early breast cancer, whereas Esslimani-Sahla et al (23) have found that low ERβ level is an independent marker, better than ERα level, to predict tamoxifen resistance. Although apparently saying different things, these two results actually agree with each other: in ERα-negative breast cancer, estrogen is not driving proliferation, so tamoxifen via ERβ may interfere with another growth signaling pathway. In ERα-positive cancers whose proliferation is driven by E2, tamoxifen with ERβ would oppose the antiproliferative effects of the ERα–tamoxifen complex.Investigation of the expression pattern of ERβ in normal tissue, DCIS, and IDC is important to understand the function of this receptor in the progression of breast cancer. We have a set of samples obtained from surgical excision of breast tumors from women before pharmacological intervention. The cohorts include lobular cancer, which has not yet been thoroughly studied for ERβ expression. Lobular cancer is an ERα-positive form of breast cancer characterized by loss of E-cadherin and relatively low proliferation rate. It is accompanied by a resistance to anoikis (34). It accounts for 10–15% of diagnosed breast cancer, and there are still many questions about the optimal therapeutic approach to this cancer. We have explored the changes in expression of the two ERs using identical protocols and reagents in different developmental stages of breast cancer within each patient.     

Self-assembly of alkynylplatinum(II) terpyridine amphiphiles into nanostructures via steric control and metal–metal interactions

A series of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing the hydrophilic oligo(para-phenylene ethynylene) with two 3,6,9-trioxadec-1-yloxy chains was designed and synthesized. The mononuclear alkynylplatinum(II) terpyridine complex was found to display a very strong tendency toward the formation of supramolecular structures. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would lead to the formation of nanotubes or helical ribbons. These desirable nanostructures were found to be governed by the steric bulk on the platinum(II) terpyridine moieties, which modulates the directional metal−metal interactions and controls the formation of nanotubes or helical ribbons. Detailed analysis of temperature-dependent UV-visible absorption spectra of the nanostructured tubular aggregates also provided insights into the assembly mechanism and showed the role of metal−metal interactions in the cooperative supramolecular polymerization of the amphiphilic platinum(II) complexes.Square-planar d⁸ platinum(II) polypyridine complexes have long been known to exhibit intriguing spectroscopic and luminescence properties (1–54) as well as interesting solid-state polymorphism associated with metal−metal and π−π stacking interactions (1–14, 25). Earlier work by our group showed the first example, to our knowledge, of an alkynylplatinum(II) terpyridine system [Pt(tpy)(C ≡ CR)]⁺ that incorporates σ-donating and solubilizing alkynyl ligands together with the formation of Pt···Pt interactions to exhibit notable color changes and luminescence enhancements on solvent composition change (25) and polyelectrolyte addition (26). This approach has provided access to the alkynylplatinum(II) terpyridine and other related cyclometalated platinum(II) complexes, with functionalities that can self-assemble into metallogels (27–31), liquid crystals (32, 33), and other different molecular architectures, such as hairpin conformation (34), helices (35–38), nanostructures (39–45), and molecular tweezers (46, 47), as well as having a wide range of applications in molecular recognition (48–52), biomolecular labeling (48–52), and materials science (53, 54). Recently, metal-containing amphiphiles have also emerged as a building block for supramolecular architectures (42–44, 55–59). Their self-assembly has always been found to yield different molecular architectures with unprecedented complexity through the multiple noncovalent interactions on the introduction of external stimuli (42–44, 55–59).Helical architecture is one of the most exciting self-assembled morphologies because of the uniqueness for the functional and topological properties (60–69). Helical ribbons composed of amphiphiles, such as diacetylenic lipids, glutamates, and peptide-based amphiphiles, are often precursors for the growth of tubular structures on an increase in the width or the merging of the edges of ribbons (64, 65). Recently, the optimization of nanotube formation vs. helical nanostructures has aroused considerable interests and can be achieved through a fine interplay of the influence on the amphiphilic property of molecules (66), choice of counteranions (67, 68), or pH values of the media (69), which would govern the self-assembly of molecules into desirable aggregates of helical ribbons or nanotube scaffolds. However, a precise control of supramolecular morphology between helical ribbons and nanotubes remains challenging, particularly for the polycyclic aromatics in the field of molecular assembly (64–69). Oligo(para-phenylene ethynylene)s (OPEs) with solely π−π stacking interactions are well-recognized to self-assemble into supramolecular system of various nanostructures but rarely result in the formation of tubular scaffolds (70–73). In view of the rich photophysical properties of square-planar d⁸ platinum(II) systems and their propensity toward formation of directional Pt···Pt interactions in distinctive morphologies (27–31, 39–45), it is anticipated that such directional and noncovalent metal−metal interactions might be capable of directing or dictating molecular ordering and alignment to give desirable nanostructures of helical ribbons or nanotubes in a precise and controllable manner.Herein, we report the design and synthesis of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing hydrophilic OPEs with two 3,6,9-trioxadec-1-yloxy chains. The mononuclear alkynylplatinum(II) terpyridine complex with amphiphilic property is found to show a strong tendency toward the formation of supramolecular structures on diffusion of diethyl ether in dichloromethane or dimethyl sulfoxide (DMSO) solution. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would result in nanotubes or helical ribbons in the self-assembly process. To the best of our knowledge, this finding represents the first example of the utilization of the steric bulk of the moieties, which modulates the formation of directional metal−metal interactions to precisely control the formation of nanotubes or helical ribbons in the self-assembly process. Application of the nucleation–elongation model into this assembly process by UV-visible (UV-vis) absorption spectroscopic studies has elucidated the nature of the molecular self-assembly, and more importantly, it has revealed the role of metal−metal interactions in the formation of these two types of nanostructures.     

Near-infrared fluorescence molecular imaging of amyloid beta species and monitoring therapy in animal models of Alzheimer’s disease

Near-infrared fluorescence (NIRF) molecular imaging has been widely applied to monitoring therapy of cancer and other diseases in preclinical studies; however, this technology has not been applied successfully to monitoring therapy for Alzheimer’s disease (AD). Although several NIRF probes for detecting amyloid beta (Aβ) species of AD have been reported, none of these probes has been used to monitor changes of Aβs during therapy. In this article, we demonstrated that CRANAD-3, a curcumin analog, is capable of detecting both soluble and insoluble Aβ species. In vivo imaging showed that the NIRF signal of CRANAD-3 from 4-mo-old transgenic AD (APP/PS1) mice was 2.29-fold higher than that from age-matched wild-type mice, indicating that CRANAD-3 is capable of detecting early molecular pathology. To verify the feasibility of CRANAD-3 for monitoring therapy, we first used the fast Aβ-lowering drug LY2811376, a well-characterized beta-amyloid cleaving enzyme-1 inhibitor, to treat APP/PS1 mice. Imaging data suggested that CRANAD-3 could monitor the decrease in Aβs after drug treatment. To validate the imaging capacity of CRANAD-3 further, we used it to monitor the therapeutic effect of CRANAD-17, a curcumin analog for inhibition of Aβ cross-linking. The imaging data indicated that the fluorescence signal in the CRANAD-17–treated group was significantly lower than that in the control group, and the result correlated with ELISA analysis of brain extraction and Aβ plaque counting. It was the first time, to our knowledge, that NIRF was used to monitor AD therapy, and we believe that our imaging technology has the potential to have a high impact on AD drug development.Alzheimer’s disease (AD) has been considered incurable, because none of the clinically tested drugs have shown significant effectiveness (1–4). Therefore, seeking effective therapeutics and imaging probes capable of assisting drug development is highly desirable. The amyloid hypothesis, in which various Aβ species are believed to be neurotoxic and one of the leading causes of AD, has been considered controversial in recent years because of the failures of amyloid beta (Aβ)-based drug development (1, 3, 5–8). However, no compelling data can prove that this hypothesis is wrong (2, 5), and no other theories indicate a clear path for AD drug development (4). Thus the amyloid hypothesis is still an important framework for AD drug development (1–5, 9–14). Additionally, Kim and colleagues (15) recently reported that a 3D cell-culture model of human neural cells could recapture AD pathology. In this study, their finding that the accumulation of Aβs could drive tau pathology provided strong support for the amyloid hypothesis (15).It is well known that Aβ species, including soluble monomers, dimers, oligomers, and insoluble fibrils/aggregates and plaques, play a central role in the neuropathology of AD (2, 5). Initially, it was thought that insoluble deposits/plaques formed by the Aβ peptides in an AD brain cause neurodegeneration. However, studies have shown that soluble dimeric and oligomeric Aβ species are more neurotoxic than insoluble deposits (16–21). Furthermore, it has been shown that soluble and insoluble species coexist during disease progression. The initial stage of pathology is represented by an excessive accumulation of Aβ monomers resulting from imbalanced Aβ clearance (22, 23). The early predominance of soluble species gradually shifts with the progression of AD to a majority of insoluble species (24, 25). Therefore imaging probes capable of detecting both soluble and insoluble Aβs are needed to monitor the full spectrum of amyloidosis pathology in AD.Thus far, three Aβ PET tracers have been approved by the Food and Drug Administration (FDA) for clinical applications. However, they are not approved for positive diagnosis of AD; rather, they are recommended for excluding the likelihood of AD. The fundamental limitation of these three tracers and others under development is that they bind primarily to insoluble Aβs, not the more toxic soluble Aβs (26–32). Clearly, work remains to be done in developing imaging probes based on Aβs, and imaging probes capable of diagnosing AD positively are undeniably needed.Numerous agents reportedly are capable of inhibiting the generation and aggregation of Aβs in vitro; however, only few have been tested in vivo. Partially, this lack of testing arises from the lack of reliable imaging methods that can monitor the agents’ therapeutic effectiveness in vivo. PET tracers, such as ¹¹C-Pittsburgh compound B (¹¹C-PiB) and ¹⁸F-AV-45, recently have been adapted to evaluate the efficacy of experimental AD drugs in clinical trials (33). However, they are rarely used to monitor drug treatment in small animals (30–32), likely because of the insensitivity of the tracers for Aβ species [particularly for soluble species (34–36)], complicated experimental procedures and data analysis in small animals, the high cost of PET probe synthesis and scanning, and the use of radioactive material. Therefore, a great demand for imaging agents that could be used in preclinical drug development to monitor therapeutic effectiveness in small animals remains unmet.Because of its low cost, simple operation, and easy data analysis, near infrared fluorescence (NIRF) imaging is generally more suitable than PET imaging for animal studies. Several NIRF probes for insoluble Aβs have been reported (37–45). It has been almost 10 y since the first report of NIRF imaging of Aβs by Hintersteiner et al. in 2005 (41). However, to the best of our knowledge, successful application of NIRF probes for monitoring therapeutic efficacy has not yet been reported. Our group recently has designed asymmetrical CRANAD-58 to match the hydrophobic (LVFF) and hydrophilic (HHQK) segments of Aβ peptides and demonstrated its applicability for the detection of both insoluble and soluble Aβs in vitro and in vivo (46). In this report, CRANAD-3 was designed to enhance the interaction with Aβs by replacing the phenyl rings of curcumin with pyridyls to introduce potential hydrogen bonds. Additionally, we demonstrated, for the first time to our knowledge, that the curcumin analog CRANAD-3 could be used as an NIRF imaging probe to monitor the Aβ-lowering effectiveness of therapeutics.     

Structural basis for the sheddase function of human meprin β metalloproteinase at the plasma membrane

Ectodomain shedding at the cell surface is a major mechanism to regulate the extracellular and circulatory concentration or the activities of signaling proteins at the plasma membrane. Human meprin β is a 145-kDa disulfide-linked homodimeric multidomain type-I membrane metallopeptidase that sheds membrane-bound cytokines and growth factors, thereby contributing to inflammatory diseases, angiogenesis, and tumor progression. In addition, it cleaves amyloid precursor protein (APP) at the β-secretase site, giving rise to amyloidogenic peptides. We have solved the X-ray crystal structure of a major fragment of the meprin β ectoprotein, the first of a multidomain oligomeric transmembrane sheddase, and of its zymogen. The meprin β dimer displays a compact shape, whose catalytic domain undergoes major rearrangement upon activation, and reveals an exosite and a sugar-rich channel, both of which possibly engage in substrate binding. A plausible structure-derived working mechanism suggests that substrates such as APP are shed close to the plasma membrane surface following an “N-like” chain trace.Physiological processes in the extracellular milieu and the circulation require finely tuned concentrations of signal molecules such as cytokines, growth factors, receptors, adhesion molecules, and peptidases. Many of these proteins are synthesized as type-I membrane protein variants or precursors consisting of a glycosylated N-terminal ectoprotein, a transmembrane helix, and a C-terminal cytosolic tail. Their localization at the cell surface restricts their field of action to autocrine or juxtacrine processes. However, to act at a distance in paracrine, synaptic, or endocrine events, they have to be released from the plasma membrane into the extracellular space as soluble factors through “protein ectodomain shedding” (1, 2). This entails limited proteolysis and is a major posttranslational regulation mechanism that affects 2–4% of the proteins on the cell surface, occurs at or near the plasma membrane (3), and apparently follows a common release mechanism (2). It may also proteolytically inactivate proteins to terminate their function on the cell surface (4). Peptidases engaged in such processing are “sheddases” and the most studied transmembrane sheddases are members of the adamalysin/ADAMs (4, 5) and matrix-metalloproteinase (MMP) (6) families within the metzincin clan of metallopeptidases (MPs) (7–9). These include ADAM-8, -9, -10, -12, -15, -17, -19, -28, and -33 (1, 4) and membrane-type 1 (MT1)-MMP, MT3-MMP, and MT5-MMP (2, 6, 10). Other confirmed transmembrane sheddases are the aspartic proteinases BACE-1 and -2 (ref. 11 and references therein) and the malarian parasite serine proteinases, PfSUB2, PfROM1, and PfROM4 (12). Distinct sheddases may participate in intercalating processes, with disparate physiological consequences: ADAM-9, -10 (α-secretases), and -17 contribute to the nonamyloidogenic pathway of human amyloid precursor protein (APP) processing, whereas BACE-1 (β-secretase) participates in the amyloidogenic pathway. Whereas the former generates innocuous peptides, the latter gives rise to the toxic β-amyloid peptides believed to be responsible for Alzheimer’s disease (11). In several instances, shedding at the membrane surface is followed by a “regulated intramembrane proteolysis” step within the membrane (1). This is the case for the processing of Notch ligand Delta1 and of APP, both carried out by γ-secretase after action of an α/β-secretase (11), and for signal-peptide peptidase, which removes remnants of the secretory protein translocation from the endoplasmic membrane (13).Recently, human meprin β (Mβ) was found to specifically process APP in vivo, which may contribute to Alzheimer’s disease (14, 15). It was also reported to activate cell-anchored α-secretase ADAM-10 and to be widely expressed in brain, intestine, kidney, and skin (14, 16–18). Disruption of Mβ in mice affects embryonic viability, birth weight, and renal gene expression profiles (19). The enzyme was further identified as a sheddase or proteolytic regulator at the plasma membrane of interleukin-1β (20), interleukin-18 (21), tumor growth factor α (22), procollagen III (23), epithelial sodium channel (24), E-cadherin (25), tenascin-C (26), and vascular endothelial growth factor A (27). Further substrates include fibroblast growth factor 19 and connective tissue growth factor. Altered expression and activity of the enzyme are associated with pathological conditions such as inflammatory bowel disease (28), tumor progression (29), nephritis (30), and fibrosis (23).Mβ is a 679-residue secreted multidomain type-I membrane MP that belongs to the astacin family within the metzincins (7, 9, 16, 31, 32). The enzyme is glycosylated and assembles into either disulfide-linked homodimers or heterodimers with the closely related meprin α-subunit (33). Mβ homodimers are essentially membrane bound but may also be shed from the surface by ADAM-10 and -17 (34, 35). To assess function, working mechanism, and activation of Mβ, we analyzed the structure of the major ectoprotein of mature Mβ (MβΔC) and of its zymogen, promeprin β (pMβΔC). With regard to transmembrane sheddases, to date only the structures of the isolated monomeric catalytic domains of ADAM-17 [Protein Data Bank (PDB) access code 1BKC], ADAM-33 (PDB 1R55), MT1-MMP (PDB 1BUV), MT3-MMP (PDB 1RM8), BACE-1 (PDB 1FKN), and BACE-2 (PDB 2EWY) have been described. Accordingly, this is a unique structural report of a multidomain oligomeric transmembrane sheddase. This report has allowed us a better understanding of the structural basis for latency and activation of this MP and to derive a plausible working mechanism for shedding of glycosylated type-I membrane substrates such as APP at the extracellular membrane surface.     

TNF-α–stimulated fibroblasts secrete lumican to promote fibrocyte differentiation

In healing wounds and fibrotic lesions, fibroblasts and monocyte-derived fibroblast-like cells called fibrocytes help to form scar tissue. Although fibrocytes promote collagen production by fibroblasts, little is known about signaling from fibroblasts to fibrocytes. In this report, we show that fibroblasts stimulated with the fibrocyte-secreted inflammatory signal tumor necrosis factor-α secrete the small leucine-rich proteoglycan lumican, and that lumican, but not the related proteoglycan decorin, promotes human fibrocyte differentiation. Lumican competes with the serum fibrocyte differentiation inhibitor serum amyloid P, but dominates over the fibroblast-secreted fibrocyte inhibitor Slit2. Lumican acts directly on monocytes, and unlike other factors that affect fibrocyte differentiation, lumican has no detectable effect on macrophage differentiation or polarization. α2β1, αMβ2, and αXβ2 integrins are needed for lumican-induced fibrocyte differentiation. In lung tissue from pulmonary fibrosis patients with relatively normal lung function, lumican is present at low levels throughout the tissue, whereas patients with advanced disease have pronounced lumican expression in the fibrotic lesions. These data may explain why fibrocytes are increased in fibrotic tissues, suggest that the levels of lumican in tissues may have a significant effect on the decision of monocytes to differentiate into fibrocytes, and indicate that modulating lumican signaling may be useful as a therapeutic for fibrosis.During wound healing, monocytes leave the circulation, enter the tissue, and differentiate into fibroblast-like cells called fibrocytes (1–6). Fibrocytes are also found in the scar tissue-like lesions associated with fibrotic diseases such as pulmonary fibrosis, congestive heart failure, cirrhosis of the liver, and nephrogenic systemic fibrosis (3, 7–11). Fibrocytes express markers of both hematopoietic cells (CD34, CD45, FcγR, LSP-1, MHC class II) and stromal cells (collagens, fibronectin, and matrix metalloproteases) (2, 3, 12–14). Fibrocytes also promote angiogenesis by secreting VEGF, bFGF, IL-8, and PDGF (15). A key question about fibrocyte differentiation and fibrosis is why fibrocytes are readily observed in fibrotic lesions, but are rarely observed in healthy tissues (3, 10, 16–19).Fibrosis is a dynamic process involving many cells besides fibrocytes (20, 21). In fibrotic lesions, tissue-resident fibroblasts proliferate and produce excessive amounts of extracellular matrix (ECM) that distorts tissue architecture, leading to tissue destruction (21, 22). Fibrocytes secrete a variety of cytokines including IL-13, TGF-β, CTGF, and TNF-α that promote the proliferation, migration, and extracellular matrix production by the local fibroblasts (15, 23–26). Conversely, fibroblasts secrete a variety of factors that promote leukocyte entry, survival, and retention during inflammation (27–30). An intriguing possibility is that a runaway positive feedback loop involving unknown signals from fibrocyte-activated fibroblasts back to fibrocytes may lead to the persistence of fibrotic lesions.In this report, we show that fibroblasts stimulated with TNF-α secrete the small leucine-rich proteoglycan lumican, and that lumican promotes fibrocyte differentiation. In addition, we show that in a mouse pulmonary fibrosis model as well as human patients with pulmonary fibrosis, there appears to be an increase in lumican levels in the lungs, suggesting that pulmonary fibrosis may be in part due to elevated lumican levels. These data suggest that lumican may be one of the unknown signals from fibroblasts to fibrocytes that mediates part of a fibroblast-fibrocyte feedback loop that potentiates fibrosis.     

From the Cover: PNAS Plus: Nicastrin functions to sterically hinder γ-secretase–substrate interactions driven by substrate transmembrane domain

γ-Secretase is an intramembrane-cleaving protease that processes many type-I integral membrane proteins within the lipid bilayer, an event preceded by shedding of most of the substrate’s ectodomain by α- or β-secretases. The mechanism by which γ-secretase selectively recognizes and recruits ectodomain-shed substrates for catalysis remains unclear. In contrast to previous reports that substrate is actively recruited for catalysis when its remaining short ectodomain interacts with the nicastrin component of γ-secretase, we find that substrate ectodomain is entirely dispensable for cleavage. Instead, γ-secretase–substrate binding is driven by an apparent tight-binding interaction derived from substrate transmembrane domain, a mechanism in stark contrast to rhomboid—another family of intramembrane-cleaving proteases. Disruption of the nicastrin fold allows for more efficient cleavage of substrates retaining longer ectodomains, indicating that nicastrin actively excludes larger substrates through steric hindrance, thus serving as a molecular gatekeeper for substrate binding and catalysis.Regulated intramembrane proteolysis (RIP) involves the cleavage of a wide variety of integral membrane proteins within their transmembrane domains (TMDs) by a highly diverse family of intramembrane-cleaving proteases (I-CLiPs) (1). I-CLiPs are found in all forms of life and govern many important biological functions, including but not limited to organism development (2), lipid homeostasis (3), the unfolded protein response (4), and bacterial quorum sensing (5). As the name implies, RIP must be tightly regulated to ensure that the resultant signaling events occur only when prompted by the cell and to prevent cleavage of the many nonsubstrate “bystander” proteins present within cellular membranes. Despite this, very little is known about the molecular mechanisms by which I-CLiPs achieve their exquisite specificity. Although traditional soluble proteases maintain substrate specificity by recognizing distinct amino acid sequences flanking the scissile bond, substrates for intramembrane proteases have little to no sequence similarity.Recent work on rhomboid proteases has demonstrated that this family of I-CLiPs achieves substrate specificity via a mechanism that is dependent on the transmembrane dynamics of the substrate rather than its sequence of amino acids (6, 7). Here, rhomboid possesses a very weak binding affinity for substrate and, in a rate-driven reaction, only cleaves those substrates that have unstable TMD helices that have had time to unfold into the catalytic active site, where they are cleaved before they can dissociate from the enzyme–substrate complex. Although it may be tempting to speculate that this is a conserved mechanism for all I-CLiPs, rhomboid is the only family of I-CLiPs that does not require prior activation of substrate through an initial cleavage by another protease (8). Specifically, site-2 protease substrates must be first cleaved by site-1 protease (9), signal peptide peptidase substrates are first cleaved by signal peptidase (10), and ectodomain shedding by α- or β-secretase is required before γ-secretase cleavage of its substrates (11, 12). These facts suggest that the diverse families of I-CLiPs likely have evolved fundamentally different mechanisms by which they recognize and cleave their substrates.Presenilin/γ-secretase is the founding member of the aspartyl family of I-CLiPs. The importance of γ-secretase function in biology and medicine is highlighted by its cleavage of the notch family of receptors, which is required for cell fate determination in all metazoans (2, 13–16), and of the amyloid precursor protein (APP), which is centrally implicated in Alzheimer’s disease (AD) (14, 17). In addition to APP and notch, γ-secretase has over 90 other reported substrates, many of which are involved in important signaling events (12, 18). Despite this, little is known about the mechanism by which γ-secretase binds and cleaves its substrates. Currently, the only known prerequisite for a substrate to be bound and hydrolyzed by γ-secretase is that it be a type-I integral membrane protein that first has most of its ectodomain removed by a sheddase, either α- or β-secretases (11, 12, 19). How γ-secretase selectively recognizes ectodomain-shed substrates and recruits them for catalysis while at the same time preventing cleavage of nonsubstrates remains unsettled.γ-Secretase is a multimeric complex composed of four integral membrane proteins both necessary and sufficient for full activity: presenilin, nicastrin, Aph-1, and Pen-2 (20–24). Presenilin is the proteolytic component, housing catalytic aspartates on TMDs 6 and 7 of its nine TMDs (17, 25, 26). After initial complex formation, the mature proteolytically active complex is formed when presenilin undergoes auto-proteolysis, resulting in N- and C-terminal fragments (NTF and CTF, respectively) (17, 27, 28), a process thought to be stimulated by the three-TMD component Pen-2 (29). The seven-TMD protein Aph-1 is believed to play a scaffolding role in complex formation (30, 31). Nicastrin is a type-I integral membrane protein with a large, heavily glycosylated ectodomain (32–34) that contains multiple stabilizing disulfide bridges (24, 34).The ectodomain of nicastrin is structurally homologous to a bacterial amino peptidase (34). Although nicastrin lacks the specific amino acids required for peptidase activity, it has been proposed to bind the N terminus of ectodomain-shed substrate, thereby directing substrate TMD to the γ-secretase active site for cleavage (35, 36). This mechanism has been suggested to depend on a key binding interaction between the free amine at the N terminus of the shortened substrate ectodomain and E333 of the vestigial amino peptidase domain of nicastrin (35, 36). However, the importance of nicastrin in substrate recognition has been questioned (37, 38), and although an initial high-resolution structure of γ-secretase suggested a role for nicastrin in substrate recognition (24), the most recent structures of the γ-secretase complex and the nicastrin ectodomain reveal that E333 is actually buried within the interior of nicastrin and resides on the opposite side of the complex relative to the active site (39, 40). Although this makes it unlikely that nicastrin is involved in direct substrate binding barring a large, energy-intensive conformational change, the basic mechanism of substrate recognition by γ-secretase remains controversial and requires resolution.Here, we demonstrate that nicastrin functions to sterically exclude substrates based on ectodomain size rather than actively recruit them for catalysis. This blocking mechanism allows γ-secretase to distinguish substrate from nonsubstrate and explains why substrate ectodomain shedding by α- or β-secretases is a prerequisite for γ-secretase catalysis. In contrast to rhomboid, γ-secretase apparently binds substrate TMD tightly, making the nicastrin steric hindrance mechanism necessary to prevent cleavage of nonectodomain-shed substrates and nonsubstrates alike.     

DNA polymerases δ and λ cooperate in repairing double-strand breaks by microhomology-mediated end-joining in Saccharomyces cerevisiae

Maintenance of genome stability is carried out by a suite of DNA repair pathways that ensure the repair of damaged DNA and faithful replication of the genome. Of particular importance are the repair pathways, which respond to DNA double-strand breaks (DSBs), and how the efficiency of repair is influenced by sequence homology. In this study, we developed a genetic assay in diploid Saccharomyces cerevisiae cells to analyze DSBs requiring microhomologies for repair, known as microhomology-mediated end-joining (MMEJ). MMEJ repair efficiency increased concomitant with microhomology length and decreased upon introduction of mismatches. The central proteins in homologous recombination (HR), Rad52 and Rad51, suppressed MMEJ in this system, suggesting a competition between HR and MMEJ for the repair of a DSB. Importantly, we found that DNA polymerase delta (Pol δ) is critical for MMEJ, independent of microhomology length and base-pairing continuity. MMEJ recombinants showed evidence that Pol δ proofreading function is active during MMEJ-mediated DSB repair. Furthermore, mutations in Pol δ and DNA polymerase 4 (Pol λ), the DNA polymerase previously implicated in MMEJ, cause a synergistic decrease in MMEJ repair. Pol λ showed faster kinetics associating with MMEJ substrates following DSB induction than Pol δ. The association of Pol δ depended on RAD1, which encodes the flap endonuclease needed to cleave MMEJ intermediates before DNA synthesis. Moreover, Pol δ recruitment was diminished in cells lacking Pol λ. These data suggest cooperative involvement of both polymerases in MMEJ.DNA double-strand breaks (DSBs) are toxic lesions that can be repaired by two major pathways in eukaryotes: nonhomologous end-joining (NHEJ) and homologous recombination (HR) (1). Although HR repairs DSBs in a template-dependent, high-fidelity manner, NHEJ functions to ligate DSB ends together using no or very short (1–4 bp) homology. Recently, a new pathway was identified in eukaryotes, which uses microhomologies (MHs) to repair a DSB and does not require the central proteins used in HR (Rad51, Rad52) or NHEJ (Ku70–Ku80) (2–5). In mammalian cells, this pathway of repair is known as alternative end-joining (Alt-EJ) and is often but not always associated with MHs, whereas in budding yeast, the commensurate pathway, MH-mediated end-joining (MMEJ), will typically use 5–25 bp of MH (6, 7). These pathways are associated with genomic rearrangements, and cancer genomes show evidence of MH-mediated rearrangements (8–12). In addition, eukaryotic genomes contain many dispersed repetitive elements that can lead to genome rearrangements when recombination occurs between them (13–16). Therefore, controlling DSB repair in the human genome, which features a variety of repeats, is especially important given the fact that recombination between repetitive elements has been implicated in genomic instability associated with disease (17–20).The original characterization of Alt-EJ in mammalian cells suggested it did not represent a significant DNA repair pathway and only operated in the absence of functional HR and NHEJ pathways. More recent analyses demonstrate a physiological role of Alt-EJ during DNA repair in the presence of active HR and NHEJ pathways (2, 12, 21, 22). Furthermore, examination of I-SceI–induced translocation junctions in mammalian cells revealed the frequent presence of MHs (23, 24). NHEJ-deficient and p53-null mice develop pro–B-cell lymphomas, and nonreciprocal translocations characterized by small MHs are found at their break point junctions (25–28). Similarly, in human cancers, many translocation break point junctions contain MHs, suggesting a role for Alt-EJ in cancer development (29–31) and resistance to chemotherapy and genetic disease (32–36). Hence, the presence of many short repetitive sequences in the human genome is likely to increase rearrangements mediated by MHs following the creation of a DSB.MMEJ is a distinct DSB repair pathway that operates in the presence of functional NHEJ and HR pathways (10, 37). The genetic requirements of MMEJ are being studied in the model eukaryote Saccharomyces cerevisiae and involve components traditionally considered specific to the NHEJ (Pol λ) and HR (Rad1–Rad10, Rad59, and Mre11–Rad50–Xrs2) pathways (4, 5, 10, 38). Although being clearly independent of the central NHEJ factor Ku70–Ku80 heterodimer (10, 37), the involvement of the key HR factor Rad52 in MMEJ remains uncertain. It has been reported that Rad52 is required for MMEJ repair (4, 10, 38), whereas in another assay system Rad52 suppresses MMEJ repair (37). More recently, it has been proposed that the replication protein A (RPA) regulates pathway choice between HR and MMEJ (37). In addition, several models have been proposed that identify specific pathways that may use MHs for the repair of DNA damage (39–41). Despite current advancements in our understanding of MMEJ, the precise involvement of DNA polymerases in supporting the repair of DSBs using MHs remains poorly understood. DNA polymerase λ (also called Pol4 in budding yeast) and its human homolog Pol λ are considered to be the primary candidates for the DNA polymerases working in NHEJ and MMEJ (4, 5, 42–46). Both genetic and biochemical evidence shows that Pol δ is recruited during HR to extend Rad51-dependent recombination intermediates (47–50). Recent analysis using pol32 mutants (5, 10) implicated the Pol32 subunit of Pol δ in MMEJ. Pol32 and Pol31 were also identified as subunits of the DNA polymerase zeta complex (Pol ζ) (51, 52), but previous analysis showed no effect of rev3 mutants in MMEJ (10). REV3 encodes the catalytic subunit of Pol ζ. However, an involvement of Pol δ had not been demonstrated directly before, and it is possible that Pol32 could act in conjunction with yet another DNA polymerase.Here, we report the development of a series of interchromosomal MMEJ assays in diploid S. cerevisiae to assess the mechanisms underlying the repair of DSBs using varying MHs. We focus on diploid cells, as they represent the natural state of budding yeast, which is a diplontic organism (53). The yeast mating-type switching system represents a mechanism to return haploid yeast as efficiently as possible to diploidy (54). Using a combination of genetic, molecular, and in vivo chromatin immunoprecipitation (ChIP) experiments, we provide compelling evidence for a direct involvement of Pol δ in coordinating with Pol λ in MMEJ in budding yeast.     

DNMT3a epigenetic program regulates the HIF-2α oxygen-sensing pathway and the cellular response to hypoxia

Epigenetic regulation of gene expression by DNA methylation plays a central role in the maintenance of cellular homeostasis. Here we present evidence implicating the DNA methylation program in the regulation of hypoxia-inducible factor (HIF) oxygen-sensing machinery and hypoxic cell metabolism. We show that DNA methyltransferase 3a (DNMT3a) methylates and silences the HIF-2α gene (EPAS1) in differentiated cells. Epigenetic silencing of EPAS1 prevents activation of the HIF-2α gene program associated with hypoxic cell growth, thereby limiting the proliferative capacity of adult cells under low oxygen tension. Naturally occurring defects in DNMT3a, observed in primary tumors and malignant cells, cause the unscheduled activation of EPAS1 in early dysplastic foci. This enables incipient cancer cells to exploit the HIF-2α pathway in the hypoxic tumor microenvironment necessary for the formation of cellular masses larger than the oxygen diffusion limit. Reintroduction of DNMT3a in DNMT3a-defective cells restores EPAS1 epigenetic silencing, prevents hypoxic cell growth, and suppresses tumorigenesis. These data support a tumor-suppressive role for DNMT3a as an epigenetic regulator of the HIF-2α oxygen-sensing pathway and the cellular response to hypoxia.Metazoan life is dependent upon the use of molecular oxygen for an array of metabolic processes. Tissue hypoxia occurs during periods of imbalance between oxygen supply and consumption. One of the primary cellular responses to hypoxia is the activation of the hypoxia-inducible factor (HIF) program (1–4). HIF consists of oxygen-regulated α-subunits HIF-1α and HIF-2α and a constitutively expressed β-subunit (HIF-β). In the presence of oxygen, a series of nonheme Fe(II)- and 2-oxoglutarate–dependent dioxygenase oxygen sensors, referred to as HIF prolylhydroxylases (HIF PHDs), promote the hydroxylation of key proline residues on the HIF-α subunits (5, 6). This serves as a recognition site for the von Hippel-Lindau (VHL) tumor-suppressor protein, which mediates ubiquitination and proteasomal degradation of HIF-1α and HIF-2α (7–9). Hypoxia inhibits HIF PHDs, allowing HIF-1α and HIF-2α to evade VHL recognition and assemble with HIF-β to produce the active heterodimeric HIF factor. Once activated, HIF-1α and HIF-2α cooperate through common and distinct pathways to regulate hypoxic gene expression and cellular adaptation to hypoxia (10).A notable feature of the HIF response is the differential expression pattern of HIF-1α and HIF-2α in normal tissues. HIF-1α mRNA is ubiquitous and constitutively expressed in adult cells. In stark contrast, HIF-2α mRNA is detected in a few cell types of adult tissues and is typically not expressed by epithelia (11). This suggests a physiological necessity to fine-tune the HIF program depending upon the cellular settings by negatively regulating the HIF-2α gene (EPAS1) upstream of the HIF oxygen-sensing enzymes. The negative regulation of EPAS1 is often compromised in cancers, as HIF-2α mRNA is observed in the vast majority of overt tumors (11–13). This is particularly evident in renal cancer. Elegant studies by the Maxwell group (13) and others (14) revealed that HIF-2α mRNA is absent in human kidney tubule epithelia but present in dysplastic foci of the nephron. In these incipient renal tumor cells, HIF-2α may function as an oncoprotein (15), collaborating with, or activating, multiple growth-promoting pathways including cancer stewards c-myc (16), ras (17), and EGFR (18, 19). Silencing of HIF-2α suppresses tumorigenesis of various genetically diverse cancers, further highlighting its central role in malignancy (16, 17, 20, 21), although this depends on the experimental context (22). Therefore, EPAS1 is silent in adult epithelia but undergoes unscheduled activation in several malignancies, driving proliferation in the hypoxic tumor microenvironment (23).A clue to the mechanisms involved in the unscheduled activation of EPAS1 during early tumorigenesis may reside in its promoter, which harbors an enrichment of cytosine and guanine bases that often serve as sites of DNA methylation and epigenetic gene silencing (24–27). Cytosine methylation is catalyzed by a family of DNA methyltransferases (DNMTs) including DNMT1, DNMT3a, and DNMT3b. DNMT1 maintains the methylation pattern from the template strand to the newly synthesized strand during DNA replication (28). DNMT3a and DNMT3b are de novo methyltransferases that establish postreplicative methylation patterns (29). Alterations in DNA methylation patterns are common in tumors and likely play a central role in aberrant gene expression that characterizes the malignant phenotype (26, 30, 31). This is particularly evident for DNMT3a, as recent studies have identified mutations in DNMT3a in patients with acute myeloid leukemia (32, 33) or down-regulation of DNMT3a mRNA in a variety of solid tumors (34). It is suggested that DNMT3a is a tumor-suppressor gene and that its mutation, or mRNA down-regulation, contributes to reducing global DNMT3a methyltransferase activity (35, 36). Currently, a key challenge is to link aberrant methylation profiles commonly observed in malignant lesions, including alterations in the DNMT3a epigenetic program, to genes that directly promote the tumorigenic phenotype.Here we show that DNMT3a methylates and silences EPAS1 in normal cells. Loss of DNMT3a observed in primary tumors and malignant cells causes unscheduled EPAS1 activation. This allows emerging cancer cells to exploit the HIF-2α program that facilitates cancer cell traverse of the hypoxic barrier and formation of tumors larger than the diffusion limit of oxygen. We suggest that the DNMT3a epigenetic program is a gatekeeper of the hypoxic cancer cell phenotype.     

Intramuscular injection of α-synuclein induces CNS α-synuclein pathology and a rapid-onset motor phenotype in transgenic mice

It has been hypothesized that α-synuclein (αS) misfolding may begin in peripheral nerves and spread to the central nervous system (CNS), leading to Parkinson disease and related disorders. Although recent data suggest that αS pathology can spread within the mouse brain, there is no direct evidence for spread of disease from a peripheral site. In the present study, we show that hind limb intramuscular (IM) injection of αS can induce pathology in the CNS in the human Ala53Thr (M83) and wild-type (M20) αS transgenic (Tg) mouse models. Within 2–3 mo after IM injection in αS homozygous M83 Tg mice and 3–4 mo for hemizygous M83 Tg mice, these animals developed a rapid, synchronized, and predictable induction of widespread CNS αS inclusion pathology, accompanied by astrogliosis, microgliosis, and debilitating motor impairments. In M20 Tg mice, starting at 4 mo after IM injection, we observed αS inclusion pathology in the spinal cord, but motor function remained intact. Transection of the sciatic nerve in the M83 Tg mice significantly delayed the appearance of CNS pathology and motor symptoms, demonstrating the involvement of retrograde transport in inducing αS CNS inclusion pathology. Outside of scrapie-mediated prion disease, to our knowledge, this findiing is the first evidence that an entire neurodegenerative proteinopathy associated with a robust, lethal motor phenotype can be initiated by peripheral inoculation with a pathogenic protein. Furthermore, this facile, synchronized rapid-onset model of α-synucleinopathy will be highly valuable in testing disease-modifying therapies and dissecting the mechanism(s) that drive αS-induced neurodegeneration.Synucleinopathies are a group of diseases defined by the presence of amyloidogenic α-synuclein (αS) inclusions that can occur in neurons and glia of the central nervous system (CNS) (1–4). In Parkinson disease (PD), a causative role for αS has been established via the discovery of mutations in the αS gene SNCA resulting in autosomal-dominant PD (4–11). Although αS inclusions (e.g., Lewy bodies) are the hallmark pathology of PD, how they contribute to disease pathogenesis remains controversial (1, 3, 4, 12).Postmortem studies have suggested that αS pathology may spread following neuroanatomical tracts (13–15) and between cells (16–18). αS pathology has also been found in the peripheral nervous system (PNS): for example, in the enteric and pelvic plexus (19, 20). And it has been suggested that αS pathology might originate in the nerves of the PNS and spread to the CNS (14). Experimentally, it has been reported that intracerebral injections of preformed amyloidogenic αS fibrils in nontransgenic (nTg) and αS transgenic (Tg) mice induce the formation of intracellular αS inclusions that appear to progress from the site of injection (21–26). Collectively, these studies support the notion that αS inclusion pathology may propagate via a prion-like conformational self-templating mechanism (27, 28). A caveat of the direct intracerebral injection of αS is that this CNS invasive surgical procedure directly alters brain homeostasis that could influence or facilitate the formation of brain pathologies, especially because incidents such as traumatic brain injury can promote the formation of αS pathology (29). Here, we report that the intramuscular (IM) injection of fibrillar (fib) αS in M83 Tg mice expressing human Ala53Thr (A53T) αS can result in the rapid and synchronized development of hind limb motor weakness and robust widespread CNS αS pathology. Additionally, similar injection into M20 Tg mice expressing human wild-type αS, which do not intrinsically develop pathology, leads to the induction of CNS αS pathology as early as 4 mo postinjection.