Optineurin is an autophagy receptor for damaged mitochondria in parkin-mediated mitophagy that is disrupted by an ALS-linked mutation

Mitophagy is a cellular quality control pathway in which the E3 ubiquitin ligase parkin targets damaged mitochondria for degradation by autophagosomes. We examined the role of optineurin in mitophagy, as mutations in optineurin are causative for amyotrophic lateral sclerosis (ALS) and glaucoma, diseases in which mitochondrial dysfunction has been implicated. Using live cell imaging, we demonstrate the parkin-dependent recruitment of optineurin to mitochondria damaged by depolarization or reactive oxygen species. Parkin’s E3 ubiquitin ligase activity is required to ubiquitinate outer mitochondrial membrane proteins, allowing optineurin to stably associate with ubiquitinated mitochondria via its ubiquitin binding domain; in the absence of parkin, optineurin transiently localizes to damaged mitochondrial tips. Following optineurin recruitment, the omegasome protein double FYVE-containing protein 1 (DFCP1) transiently localizes to damaged mitochondria to initialize autophagosome formation and the recruitment of microtubule-associated protein light chain 3 (LC3). Optineurin then induces autophagosome formation around damaged mitochondria via its LC3 interaction region (LIR) domain. Depletion of endogenous optineurin inhibits LC3 recruitment to mitochondria and inhibits mitochondrial degradation. These defects are rescued by expression of siRNA-resistant wild-type optineurin, but not by an ALS-associated mutant in the ubiquitin binding domain (E478G), or by optineurin with a mutation in the LIR domain. Optineurin and p62/SQSTM1 are independently recruited to separate domains on damaged mitochondria, and p62 is not required for the recruitment of either optineurin or LC3 to damaged mitochondria. Thus, our study establishes an important role for optineurin as an autophagy receptor in parkin-mediated mitophagy and demonstrates that defects in a single pathway can lead to neurodegenerative diseases with distinct pathologies.Damaged mitochondria are selectively turned over in eukaryotic cells via mitophagy, a process in which double-membraned autophagosomes sequester and ultimately degrade mitochondria via lysosomal fusion (1, 2). This process is regulated by phosphatase and tensin homolog-induced putative kinase protein 1 (PINK1) and parkin, two proteins linked to hereditary forms of Parkinson’s disease (3, 4). PINK1 is stabilized on the outer membrane of damaged mitochondria and recruits the E3 ubiquitin ligase parkin, which ubiquitinates proteins on the outer mitochondrial membrane (OMM) (5–13). Parkin-mediated ubiquitination of damaged mitochondria is followed by autophagosome formation and engulfment of mitochondria (1, 2). However, the proteins involved in dynamically recruiting autophagic machinery to ubiquitinated damaged mitochondria still remain elusive.Optineurin is an autophagy receptor, characterized by its ability to bind ubiquitin via its ubiquitin binding in ABIN (A20 binding and inhibitor of NF-κB) and NEMO (NF-κB essential modulator) (UBAN) domain (14) and the autophagosome-associated protein LC3 (microtubule-associated protein light chain 3) via its LC3 interacting region (LIR) domain (15). Optineurin regulates autophagosome maturation (16) and autophagic degradation of Salmonella and protein aggregates (15, 17). However, optineurin’s role in mitophagy has not been previously studied. Mutations in optineurin lead to primary open-angle glaucoma (18) and amyotrophic lateral sclerosis (ALS) (19), two neurodegenerative diseases in which mitochondrial defects have been observed (20, 21). Thus, optineurin may play a role in regulating the autophagic turnover of damaged mitochondria during mitophagy.Here, we use confocal live cell imaging to show that parkin is both necessary and sufficient to stabilize optineurin on the surface of damaged mitochondria. In the absence of parkin, optineurin puncta transiently localize to damaged mitochondria but do not remain stably associated. In cells expressing parkin, optineurin is recruited to mitochondria following parkin recruitment, and this recruitment is stabilized via the UBAN domain. Following optineurin recruitment, double FYVE-containing protein 1 (DFCP1) puncta transiently localize to parkin/optineurin-labeled damaged mitochondria to mark the initial site of autophagosome formation (22). This is followed by LC3 recruitment and subsequent autophagosome formation around optineurin-labeled damaged mitochondria. Importantly, we find that depletion of optineurin inhibits autophagosome recruitment to damaged mitochondria, leading to increased levels of the mitochondrial matrix protein Hsp60 and mtDNA content within cells. This defect in mitochondrial degradation is rescued by wild-type optineurin but not by the E478G UBAN mutant in optineurin causative for ALS (19) or by an optineurin LIR mutant unable to bind LC3 (15). Optineurin and p62, previously implicated in mitophagy (23–26), are independently recruited to distinct domains on damaged mitochondria. In contrast to our observations with optineurin, depletion of p62 did not inhibit LC3 recruitment or efficient degradation of damaged mitochondria. Thus, our study shows an important role for the autophagy receptor optineurin in parkin-mediated mitophagy and provides support for the hypothesis that defective mitochondrial quality control may contribute to ALS pathogenesis.     

STEP61 is a substrate of the E3 ligase parkin and is upregulated in Parkinson’s disease

Parkinson’s disease (PD) is characterized by the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc). The loss of SNc dopaminergic neurons affects the plasticity of striatal neurons and leads to significant motor and cognitive disabilities during the progression of the disease. PARK2 encodes for the E3 ubiquitin ligase parkin and is implicated in genetic and sporadic PD. Mutations in PARK2 are a major contributing factor in the early onset of autosomal-recessive juvenile parkinsonism (AR-JP), although the mechanisms by which a disruption in parkin function contributes to the pathophysiology of PD remain unclear. Here we demonstrate that parkin is an E3 ligase for STEP₆₁ (striatal-enriched protein tyrosine phosphatase), a protein tyrosine phosphatase implicated in several neuropsychiatric disorders. In cellular models, parkin ubiquitinates STEP₆₁ and thereby regulates its level through the proteasome system, whereas clinically relevant parkin mutants fail to do so. STEP₆₁ protein levels are elevated on acute down-regulation of parkin or in PARK2 KO rat striatum. Relevant to PD, STEP₆₁ accumulates in the striatum of human sporadic PD and in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned mice. The increase in STEP₆₁ is associated with a decrease in the phosphorylation of its substrate ERK1/2 and the downstream target of ERK1/2, pCREB [phospho-CREB (cAMP response element-binding protein)]. These results indicate that STEP₆₁ is a novel substrate of parkin, although further studies are necessary to determine whether elevated STEP₆₁ levels directly contribute to the pathophysiology of PD.Parkinson’s disease (PD) is a common motor disorder with clinical symptoms that include bradykinesia, resting tremor, rigidity, postural instability, and cognitive deficits (1–3). The pathophysiology of PD includes selective loss of dopaminergic neurons in the substantia nigra, with a progressive depletion of striatal dopamine and the presence of intraneuronal cytoplasmic inclusions known as Lewy bodies. Mutations of several genes are implicated in PD and are responsible for ∼10% of cases; the remaining cases are classified as sporadic PD. Although specific mutations in genes that include PARK2, PINK-1, LRRK2, and DJ-1 are known, the effects these mutations have on intracellular signaling and disease progression are not well understood and form an area of intense investigation (2, 4–6).STEP₆₁ (striatal-enriched protein tyrosine phosphatase) is a brain-specific phosphatase enriched in the striatum and in other regions, including cortex, hippocampus, and substantia nigra (7–9). STEP₆₁ levels are elevated in several disorders, including Alzheimer’s disease, schizophrenia, and fragile X syndrome (10–12). STEP₆₁ levels are normally regulated by the ubiquitin proteasome system, and disruption of this pathway leads to an accumulation of STEP₆₁ in both Alzheimer’s disease and schizophrenia (10, 11).Substrates of STEP₆₁ include ERK1/2, Pyk2, Fyn, the GluN2B subunit of the NMDA receptor, and the GluA2 subunit of the AMPA receptor. The current model of STEP₆₁ function is that it opposes the development of synaptic strengthening by dephosphorylating regulatory tyrosines on these substrates. In the case of the kinases, STEP₆₁-mediated dephosphorylation of the regulatory Tyr within the activation loop inactivates these enzymes (13–16). STEP-mediated dephosphorylation of Tyr residues in the glutamate receptor subunits results in internalization of GluN1/GluN2B and GluA1/GluA2 receptor complexes (17–20). As a result, STEP KO mice have an increase in the basal Tyr phosphorylation of its substrates, including ERK1/2 and its downstream target pCREB (21, 22).Overexpression of STEP disrupts synaptic function, and thereby contributes to cognitive and behavioral deficits (23). Consistent with this hypothesis, genetic or pharmacologic reduction of STEP activity in several disorders in which STEP levels are elevated reverses the biochemical and cognitive deficits that are present (19, 24), and STEP KO mice demonstrate enhanced hippocampal long-term potentiation and enhanced hippocampal- and amygdalar-dependent memory tasks (22, 25).Direct mutations of the E3 ligase parkin (PARK2) result in autosomal recessive juvenile parkinsonism (AR-JP), with early onset of PD symptoms (26, 27); disruption of parkin activity is also implicated in sporadic PD (28–30). Moreover, PD toxins such as MPTP, rotenone, paraquat, and 6-hydroxydopamine alter parkin levels or its ligase activity and result in the accumulation of parkin substrates (31–35). Identification of new parkin substrates and characterization of their role or roles in synaptic function should result in a better understanding the molecular basis of PD.Here we identify parkin as an E3 ligase that ubiquitinates STEP₆₁. STEP₆₁ levels are increased in human PD brains and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD models and are associated with a decrease in the phosphorylation of ERK1/2 and CREB. As an increase in STEP₆₁ expression disrupts synaptic function and contributes to the cognitive deficits in several disorders, these findings suggest that the increase in STEP₆₁ levels in PD may contribute to the pathophysiology of this disorder.     

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.     

Dopamine release from transplanted neural stem cells in Parkinsonian rat striatum in vivo

Embryonic stem cell-based therapies exhibit great potential for the treatment of Parkinson’s disease (PD) because they can significantly rescue PD-like behaviors. However, whether the transplanted cells themselves release dopamine in vivo remains elusive. We and others have recently induced human embryonic stem cells into primitive neural stem cells (pNSCs) that are self-renewable for massive/transplantable production and can efficiently differentiate into dopamine-like neurons (pNSC–DAn) in culture. Here, we showed that after the striatal transplantation of pNSC–DAn, (i) pNSC–DAn retained tyrosine hydroxylase expression and reduced PD-like asymmetric rotation; (ii) depolarization-evoked dopamine release and reuptake were significantly rescued in the striatum both in vitro (brain slices) and in vivo, as determined jointly by microdialysis-based HPLC and electrochemical carbon fiber electrodes; and (iii) the rescued dopamine was released directly from the grafted pNSC–DAn (and not from injured original cells). Thus, pNSC–DAn grafts release and reuptake dopamine in the striatum in vivo and alleviate PD symptoms in rats, providing proof-of-concept for human clinical translation.Parkinson’s disease (PD) is a chronic progressive neurodegenerative disorder characterized by the specific loss of dopaminergic neurons in the substantia nigra pars compacta and their projecting axons, resulting in loss of dopamine (DA) release in the striatum (1). During the last two decades, cell-replacement therapy has proven, at least experimentally, to be a potential treatment for PD patients (2–7) and in animal models (8–15). The basic principle of cell therapy is to restore the DA release by transplanting new DA-like cells. Until recently, obtaining enough transplantable cells was a major bottleneck in the practicability of cell therapy for PD. One possible source is embryonic stem cells (ESCs), which can develop infinitely into self-renewable pluripotent cells with the potential to generate any type of cell, including DA neurons (DAns) (16, 17).Recently, several groups including us have introduced rapid and efficient ways to generate primitive neural stem cells (pNSCs) from human ESCs using small-molecule inhibitors under chemically defined conditions (12, 18, 19). These cells are nonpolarized neuroepithelia and retain plasticity upon treatment with neuronal developmental morphogens. Importantly, pNSCs differentiate into DAns (pNSC–DAn) with high efficiency (∼65%) after patterning by sonic hedgehog (SHH) and fibroblast growth factor 8 (FGF8) in vitro, providing an immediate and renewable source of DAns for PD treatment. Importantly, the striatal transplantation of human ESC-derived DA-like neurons, including pNSC–DAn, are able to relieve the motor defects in a PD rat model (11–13, 15, 19–23). Before attempting clinical translation of pNSC–DAn, however, there are two fundamental open questions. (i) Can pNSC–DAn functionally restore the striatal DA levels in vivo? (ii) What cells release the restored DA, pNSC–DAn themselves or resident neurons/cells repaired by the transplants?Regarding question 1, a recent study using nafion-coated carbon fiber electrodes (CFEs) reported that the amperometric current is rescued in vivo by ESC (pNSC–DAn-like) therapy (19). Both norepinephrine (NE) and serotonin are present in the striatum (24, 25). However, CFE amperometry/chronoamperometry alone cannot distinguish DA from other monoamines in vivo, such as NE and serotonin (Fig. S1) (see also refs. 26–28). Considering that the compounds released from grafted ESC-derived cells are unknown, the work of Kirkeby et al. was unable to determine whether DA or other monoamines are responsible for the restored amperometric signal. Thus, the key question of whether pNSC–DAn can rescue DA release needs to be reexamined for the identity of the restored amperometric signal in vivo.Regarding question 2, many studies have proposed that DA is probably released from the grafted cells (8, 12, 13, 20), whereas others have proposed that the grafted stem cells might restore striatal DA levels by rescuing injured original cells (29, 30). Thus, whether the grafted cells are actually capable of synthesizing and releasing DA in vivo must be investigated to determine the future cellular targets (residual cells versus pNSC–DAn) of treatment.To address these two mechanistic questions, advanced in vivo methods of DA identification and DA recording at high spatiotemporal resolution are required. Currently, microdialysis-based HPLC (HPLC) (31–33) and CFE amperometric recordings (34, 35) have been used independently by different laboratories to assess evoked DA release from the striatum in vivo. The major advantage of microdialysis-based HPLC is to identify the substances secreted in the cell-grafted striatum (33), but its spatiotemporal resolution is too low to distinguish the DA release site (residual cells or pNSC–DAn). In contrast, the major advantage of CFE-based amperometry is its very high temporal (ms) and spatial (μm) resolution, making it possible to distinguish the DA release site (residual cells or pNSC–DAn) in cultured cells, brain slices, and in vivo (34–39), but it is unable to distinguish between low-level endogenous oxidizable substances (DA versus serotonin and NE) in vivo.In the present study, we developed a challenging experimental paradigm of combining the two in vivo methods, microdialysis-based HPLC and CFE amperometry, to identify the evoked substance as DA and its release site as pNSC–DAn in the striatum of PD rats.     

Effects of lymphocyte profile on development of EBV-induced lymphoma subtypes in humanized mice

Epstein-Barr virus (EBV) infection causes both Hodgkin’s lymphoma (HL) and non-Hodgkin’s lymphoma (NHL). The present study reveals that EBV-induced HL and NHL are intriguingly associated with a repopulated immune cell profile in humanized mice. Newborn immunodeficient NSG mice were engrafted with human cord blood CD34⁺ hematopoietic stem cells (HSCs) for a 8- or 15-wk reconstitution period (denoted ^8whN and ^15whN, respectively), resulting in human B-cell and T-cell predominance in peripheral blood cells, respectively. Further, novel humanized mice were established via engraftment of hCD34⁺ HSCs together with nonautologous fetal liver-derived mesenchymal stem cells (MSCs) or MSCs expressing an active notch ligand DLK1, resulting in mice skewed with human B or T cells, respectively. After EBV infection, whereas NHL developed more frequently in B-cell–predominant humanized mice, HL was seen in T-cell–predominant mice (P = 0.0013). Whereas human splenocytes from NHL-bearing mice were positive for EBV-associated NHL markers (hBCL2⁺, hCD20⁺, hKi67⁺, hCD20⁺/EBNA1⁺, and EBER⁺) but negative for HL markers (LMP1⁻, EBNA2⁻, and hCD30⁻), most HL-like tumors were characterized by the presence of malignant Hodgkin’s Reed–Sternberg (HRS)-like cells, lacunar RS (hCD30⁺, hCD15⁺, IgJ⁻, EBER⁺/hCD30⁺, EBNA1⁺/hCD30⁺, LMP⁺/EBNA2⁻, hCD68⁺, hBCL2⁻, hCD20^-/weak, Phospho STAT6⁺), and mummified RS cells. This study reveals that immune cell composition plays an important role in the development of EBV-induced B-cell lymphoma.Epstein Barr virus (EBV) infects human B lymphocytes and epithelial cells in >90% of the human population (1, 2). EBV infection is widely associated with the development of diverse human disorders that include Hodgkin’s lymphoma (HL) and non-Hodgkin’s lymphomas (NHL), including diffused large B-cell lymphoma (DLBCL), follicular B-cell lymphoma (FBCL), endemic Burkitt’s lymphoma (BL), and hemophagocytic lymphohistiocytosis (HLH) (3).HL is a malignant lymphoid neoplasm most prevalent in adolescents and young adults (4–6). Hodgkin/Reed–Sternberg (HRS) cells are the sole malignant cells of HL. HRS cells are characterized by CD30⁺/CD15⁺/BCL6⁻/CD20^+/− markers and appear large and multinucleated owing to multiple nuclear divisions without cytokinesis. Although HRS cells are malignant in the body, surrounding inflammatory cells greatly outnumber them. These reactive nonmalignant inflammatory cells, including lymphocytes, histiocytes, eosinophils, fibroblasts, neutrophils, and plasma cells, compose the vast majority of the tumor mass. The presence of HRS cells in the context of this inflammatory cellular background is a critical hallmark of the HL diagnosis (4). Approximately 50% of HL cases are EBV-associated (EBVaHL) (7–11). EBV-positive HRS cells express EBV latent membrane protein (LMP) 1 (LMP1), LMP2A, LMP2B, and EBV nuclear antigen (EBNA) 1 (EBNA1), but lack EBNA2 (latency II marker) (12). LMP1 is consistently expressed in all EBV-associated cases of classical HL (13, 14). LMP1 mimics activated CD40 receptors, induces NF-κB, and allows cells to become malignant while escaping apoptosis (15).The etiologic role of EBV in numerous disorders has been studied in humanized mouse models in diverse experimental conditions. Humanized mouse models recapitulate key characteristics of EBV infection-associated disease pathogenesis (16–24). Different settings have given rise to quite distinct phenotypes, including B-cell type NHL (DLBCL, FBCL, and unspecified B-cell lymphomas), natural killer/T cell lymphoma (NKTCL), nonmalignant lymphoproliferative disorder (LPD), extremely rare HL, HLH, and arthritis (16–24). Despite considerable efforts (16–24), EBVaHL has not been properly produced in the humanized mouse setting model, owing to inappropriate animal models and a lack of in-depth analyses. After an initial report of infected humanized mice, HRS-like cells appeared to be extremely rare in the spleens of infected humanized mice; however, the findings were inconclusive (18). Here we report direct evidence of EBVaHL or HL-like neoplasms in multiple humanized mice in which T cells were predominant over B cells. Our study demonstrates that EBV-infected humanized mice display additional EBV-associated pathogenesis, including DLBCL and hemophagocytic lymphohistiocytosis (16, 17).     

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.     

Collapse of cooperation in evolving games

Game theory provides a quantitative framework for analyzing the behavior of rational agents. The Iterated Prisoner’s Dilemma in particular has become a standard model for studying cooperation and cheating, with cooperation often emerging as a robust outcome in evolving populations. Here we extend evolutionary game theory by allowing players’ payoffs as well as their strategies to evolve in response to selection on heritable mutations. In nature, many organisms engage in mutually beneficial interactions and individuals may seek to change the ratio of risk to reward for cooperation by altering the resources they commit to cooperative interactions. To study this, we construct a general framework for the coevolution of strategies and payoffs in arbitrary iterated games. We show that, when there is a tradeoff between the benefits and costs of cooperation, coevolution often leads to a dramatic loss of cooperation in the Iterated Prisoner’s Dilemma. The collapse of cooperation is so extreme that the average payoff in a population can decline even as the potential reward for mutual cooperation increases. Depending upon the form of tradeoffs, evolution may even move away from the Iterated Prisoner’s Dilemma game altogether. Our work offers a new perspective on the Prisoner’s Dilemma and its predictions for cooperation in natural populations; and it provides a general framework to understand the coevolution of strategies and payoffs in iterated interactions.Iterated games provide a framework for studying social interactions (1–6) that allows researchers to address pervasive biological problems such as the evolution of cooperation and cheating (2, 7–12). Simple examples such as the Iterated Prisoner’s Dilemma, Snowdrift, and Stag Hunt games (13–18) showcase a startling array of counterintuitive social behaviors, especially when studied in a population replicating under natural selection (16, 19–25). Despite the subject’s long history, a systematic treatment of all evolutionary robust cooperative outcomes for even the simple Iterated Prisoner’s Dilemma has only recently emerged (21, 26–29).Understanding the evolution of strategies in a population under fixed payoffs already poses a steep challenge. To complicate matters further, in many biological settings the payoffs themselves may also depend on the genotypes of the players. Changes to the payoff matrix have been studied in a number of contexts, including one-shot two-player games (13), payoff evolution without strategy evolution (30, 31), under environmental “shocks” to the payoff matrix (32–34), and using continuous games (22, 23, 35). Here we adopt a different approach, and we explicitly study the coevolutionary dynamics between strategies and payoffs in iterated two-player games. We decouple strategy mutations from payoff mutations, and we leverage results on the evolutionary robustness of memory-1 strategies with arbitrary payoff matrices to explore the relationship between payoff evolution and the prevalence of cooperation in a population. We identify a feedback between the costs and benefits of cooperation and the evolutionary robustness of cooperative strategies. Depending on the functional form (35) of the relationship between costs and benefits, this feedback may either reinforce the evolutionary success of cooperation or else precipitate its collapse. In particular, we show that cooperation will always collapse when there are diminishing returns for mutual cooperation.     

Loss of Miro1-directed mitochondrial movement results in a novel murine model for neuron disease

Defective mitochondrial distribution in neurons is proposed to cause ATP depletion and calcium-buffering deficiencies that compromise cell function. However, it is unclear whether aberrant mitochondrial motility and distribution alone are sufficient to cause neurological disease. Calcium-binding mitochondrial Rho (Miro) GTPases attach mitochondria to motor proteins for anterograde and retrograde transport in neurons. Using two new KO mouse models, we demonstrate that Miro1 is essential for development of cranial motor nuclei required for respiratory control and maintenance of upper motor neurons required for ambulation. Neuron-specific loss of Miro1 causes depletion of mitochondria from corticospinal tract axons and progressive neurological deficits mirroring human upper motor neuron disease. Although Miro1-deficient neurons exhibit defects in retrograde axonal mitochondrial transport, mitochondrial respiratory function continues. Moreover, Miro1 is not essential for calcium-mediated inhibition of mitochondrial movement or mitochondrial calcium buffering. Our findings indicate that defects in mitochondrial motility and distribution are sufficient to cause neurological disease.Motor neuron diseases (MNDs), including ALS and spastic paraplegia (SP), are characterized by the progressive, length-dependent degeneration of motor neurons, leading to muscle atrophy, paralysis, and, in some cases, premature death. There are both inherited and sporadic forms of MNDs, which can affect upper motor neurons, lower motor neurons, or both. Although the molecular and cellular causes of most MNDs are unknown, many are associated with defects in axonal transport of cellular components required for neuron function and maintenance (1–6).A subset of MNDs is associated with impaired mitochondrial respiration and mitochondrial distribution. This observation has led to the hypothesis that neurodegeneration results from defects in mitochondrial motility and distribution, which, in turn, cause subcellular ATP depletion and interfere with mitochondrial calcium ([Ca²⁺]_m) buffering at sites of high synaptic activity (reviewed in ref. 7). It is not known, however, whether mitochondrial motility defects are a primary cause or a secondary consequence of MND progression. In addition, it has been difficult to isolate the primary effect of mitochondrial motility defects in MNDs because most mutations that impair mitochondrial motility in neurons also affect transport of other organelles and vesicles (1, 8–11).In mammals, the movement of neuronal mitochondria between the cell body and the synapse is controlled by adaptors called trafficking kinesin proteins (Trak1 and Trak2) and molecular motors (kinesin heavy chain and dynein), which transport the organelle in the anterograde or retrograde direction along axonal microtubule tracks (7, 12–24). Mitochondrial Rho (Miro) GTPase proteins are critical for transport because they are the only known surface receptors that attach mitochondria to these adaptors and motors (12–15, 18, 25, 26). Miro proteins are tail-anchored in the outer mitochondrial membrane with two GTPase domains and two predicted calcium-binding embryonic fibroblast (EF) hand motifs facing the cytoplasm (12, 13, 25, 27, 28). A recent Miro structure revealed two additional EF hands that were not predicted from the primary sequence (29). Studies in cultured cells suggest that Miro proteins also function as calcium sensors (via their EF hands) to regulate kinesin-mediated mitochondrial “stopping” in axons (15, 16, 26). Miro-mediated movement appears to be inhibited when cytoplasmic calcium is elevated in active synapses, effectively recruiting mitochondria to regions where calcium buffering and energy are needed. Despite this progress, the physiological relevance of these findings has not yet been tested in a mammalian animal model. In addition, mammals ubiquitously express two Miro orthologs, Miro1 and Miro2, which are 60% identical (12, 13). However, the individual roles of Miro1 and Miro2 in neuronal development, maintenance, and survival have no been evaluated.We describe two new mouse models that establish the importance of Miro1-mediated mitochondrial motility and distribution in mammalian neuronal function and maintenance. We show that Miro1 is essential for development/maintenance of specific cranial neurons, function of postmitotic motor neurons, and retrograde mitochondrial motility in axons. Loss of Miro1-directed retrograde mitochondrial transport is sufficient to cause MND phenotypes in mice without abrogating mitochondrial respiratory function. Furthermore, Miro1 is not essential for calcium-mediated inhibition of mitochondrial movement or [Ca²⁺]_m buffering. These findings have an impact on current models for Miro1 function and introduce a specific and rapidly progressing mouse model for MND.     

Tau stabilizes microtubules by binding at the interface between tubulin heterodimers

The structure, dynamic behavior, and spatial organization of microtubules are regulated by microtubule-associated proteins. An important microtubule-associated protein is the protein Tau, because its microtubule interaction is impaired in the course of Alzheimer’s disease and several other neurodegenerative diseases. Here, we show that Tau binds to microtubules by using small groups of evolutionary conserved residues. The binding sites are formed by residues that are essential for the pathological aggregation of Tau, suggesting competition between physiological interaction and pathogenic misfolding. Tau residues in between the microtubule-binding sites remain flexible when Tau is bound to microtubules in agreement with a highly dynamic nature of the Tau–microtubule interaction. By binding at the interface between tubulin heterodimers, Tau uses a conserved mechanism of microtubule polymerization and, thus, regulation of axonal stability and cell morphology.Microtubules regulate cell division, cell morphology, intracellular transport, and axonal stability and, therefore, play crucial roles in cell function (1). Microtubules are built from tubulin heterodimers that polymerize into protofilaments and associate laterally into microtubules (2). Microtubule dynamics in neurons is modulated by several accessory proteins termed microtubule-associated proteins (3). However, little is known about the mechanism of assembly and stabilization of microtubules by microtubule-associated proteins.An important microtubule-associated protein is the protein Tau, which promotes formation of axonal microtubules, stabilizes them, and drives neurite outgrowth (4, 5). The adult human brain contains six isoforms of Tau, which are generated from a single gene by alternative splicing. The six isoforms are composed of either three or four repeats, with up to two N-terminal inserts, and range from 37 to 45 kDa (6). The 31- to 32-residue-long imperfect repeats are located in the carboxyl-terminal half of Tau and are highly conserved in several microtubule-associated proteins (7). The repeat domain is flanked by a proline-rich region that enhances binding to microtubules and microtubule assembly (8). Tau isoforms are developmentally regulated and have similar levels in the adult human brain (9).Impaired interaction of Tau with microtubules plays an important role in the pathology of several neurodegenerative diseases (10, 11). Dysregulation by genetic mutation or hyperphosphorylation affects the Tau–microtubule complex, leads to Tau detachment, causes instability and disassembly of microtubules, and, thus, perturbs axonal transport (12, 13). Microtubule-stabilizing drugs might therefore improve neuronal degeneration (14). When detached from microtubules, Tau can self-aggregate into insoluble aggregates through its hexapeptide motifs in the repeat domain (15). The deposition of aggregated Tau into neurofibrillary tangles and neuritic Tau pathology is one of the hallmarks in Alzheimer’s disease (16).Biochemical studies have shown that the repeat domain and the neighboring basic proline-rich region contribute strongly to microtubule binding (8, 17). In addition, a variety of binding sites and models of the Tau–microtubule complex were proposed (18–22). The models include binding of Tau to the outer surface of microtubules connecting tubulin subunits either across or along protofilaments (18). Tau might also reach into the interior of the microtubule wall near the binding site of the anticancer drug paclitaxel (19). Often these studies are complicated by the flexibility of the Tau protein, which belongs to the class of intrinsically disordered proteins (23, 24).Here, we studied the molecular mechanism of the interaction of Tau with microtubules by using a combination of NMR spectroscopy and mass spectrometry. We show that small groups of evolutionary conserved Tau residues bind dynamically at the interface between tubulin heterodimers, thereby promoting microtubule assembly and stabilization.     

High-speed atomic force microscopy reveals structural dynamics of amyloid β1–42 aggregates

Aggregation of amyloidogenic proteins into insoluble amyloid fibrils is implicated in various neurodegenerative diseases. This process involves protein assembly into oligomeric intermediates and fibrils with highly polymorphic molecular structures. These structural differences may be responsible for different disease presentations. For this reason, elucidation of the structural features and assembly kinetics of amyloidogenic proteins has been an area of intense study. We report here the results of high-speed atomic force microscopy (HS-AFM) studies of fibril formation and elongation by the 42-residue form of the amyloid β-protein (Aβ_1–42), a key pathogenetic agent of Alzheimer''s disease. Our data demonstrate two different growth modes of Aβ_1–42, one producing straight fibrils and the other producing spiral fibrils. Each mode depends on initial fibril nucleus structure, but switching from one growth mode to another was occasionally observed, suggesting that fibril end structure fluctuated between the two growth modes. This switching phenomenon was affected by buffer salt composition. Our findings indicate that polymorphism in fibril structure can occur after fibril nucleation and is affected by relatively modest changes in environmental conditions.Amyloid fibril accumulation is associated with numerous neurodegenerative diseases, including Alzheimer’s (AD) (1–3), prionoses (4–7), Parkinson’s (8–11), and Huntington''s (12). Nonhomologous genes encode the proteins involved in each disease, namely the amyloid β-protein (Aβ), prions (e.g., PrP, Sup35, Het-s), α-synuclein, and huntingtin, respectively. Each of these proteins assembles from a monomer state through a variety of intermediates to form insoluble amyloid fibrils that accumulate in brain tissues. The suggestion that brain Aβ accumulation and neurodegeneration are correlated remains an area of contention. Studies of human brain extracts and transgenic mice suggest such a correlation does not exist (13, 14), whereas other studies support this relationship (15). Amyloid deposition in the brain does correlate with progress from mild cognitive impairment to AD (16). The amount of brain amyloid in asymptomatic elderly people generally is less than in AD patients (17). Historically, amyloid fibrils have been regarded as the key pathologic agents in AD. This idea has been supplanted by theories in which oligomers are central (18, 19). A variety of studies support this view (20–23). In addition, oligomers appear to be more toxic to cultured cells than are fibrils (24, 25). Nevertheless, Aβ40 fibrils are neurotoxic (24, 26–28) and fibrillar Aβ appears to be associated with inflammation (29, 30) and oxidative damage (31, 32) in the brain. We believe that an unbiased assessment of working theories of disease causation does not allow one to conclude that the two theories are mutually exclusive. It is more likely that both types of assemblies are involved in AD pathogenesis. In fact, Lu et al. have provided support for this more inclusive theory by arguing that oligomers and fibrils exist in equilibrium (33). Such an equilibrium is thought to include, in addition, monomers and protofibrils (34).Although specific intermediates and fibrils have been studied in isolation at particular stages of Aβ assembly, it has been more difficult to observe structural transitions that may occur among assembly types. Techniques such as thioflavin-T (ThT) fluorescence, circular dichroism spectroscopy, and Fourier transform infrared spectroscopy are widely used to monitor development of β-sheet structure, but these methods do not provide information on aggregate tertiary or quaternary structure. Structural studies using X-ray crystallography or solid-state NMR have provided useful information on protein structure at the atomic level, but this information is static in nature and does not reveal aspects of the gross structural transitions among assembly states. Electron microscopy also is a static method.The process of fibril formation itself has been shown to be more complicated than originally thought. One reason is the huge conformational space of the intrinsically disordered Aβ monomer, which gives rise to many different oligomer structures, some of which are on-pathway for fibril formation and some of which are not. This diversity of prefibrillar structures is reflected in the structures of the fibrils that then form. Fibrils are polymorphic—Aβ forms fibrils with distinct structures depending on experimental conditions (35, 36). Additionally, different fibril types have different impacts on neurodegeneration (26, 33, 37–39). Thus, characterization of the structural dynamics of the fibril formation process is an important endeavor. Real-time visualization of monomer aggregation and fibril formation offers the possibility of understanding the dynamics of the system, developing hypotheses about assembly mechanisms, and elucidating aggregation mechanisms.In the present study, we used high-speed atomic force microscopy (HS-AFM) (40, 41) to study the dynamics of Aβ_1–42 assembly. We were able to visualize initial fibril nucleation and subsequent fibril elongation. We observed two distinct growth modes for Aβ_1–42 fibrils—one producing straight fibrils and one producing spiral fibrils—and, unexpectedly, morphological switching between these two modes.     

Contribution of reactive oxygen species to cerebral amyloid angiopathy,vasomotor dysfunction,and microhemorrhage in aged Tg2576 mice

Cerebral amyloid angiopathy (CAA) is characterized by deposition of amyloid β peptide (Aβ) within walls of cerebral arteries and is an important cause of intracerebral hemorrhage, ischemic stroke, and cognitive dysfunction in elderly patients with and without Alzheimer’s Disease (AD). NADPH oxidase-derived oxidative stress plays a key role in soluble Aβ-induced vessel dysfunction, but the mechanisms by which insoluble Aβ in the form of CAA causes cerebrovascular (CV) dysfunction are not clear. Here, we demonstrate evidence that reactive oxygen species (ROS) and, in particular, NADPH oxidase-derived ROS are a key mediator of CAA-induced CV deficits. First, the NADPH oxidase inhibitor, apocynin, and the nonspecific ROS scavenger, tempol, are shown to reduce oxidative stress and improve CV reactivity in aged Tg2576 mice. Second, the observed improvement in CV function is attributed both to a reduction in CAA formation and a decrease in CAA-induced vasomotor impairment. Third, anti-ROS therapy attenuates CAA-related microhemorrhage. A potential mechanism by which ROS contribute to CAA pathogenesis is also identified because apocynin substantially reduces expression levels of ApoE—a factor known to promote CAA formation. In total, these data indicate that ROS are a key contributor to CAA formation, CAA-induced vessel dysfunction, and CAA-related microhemorrhage. Thus, ROS and, in particular, NADPH oxidase-derived ROS are a promising therapeutic target for patients with CAA and AD.Cerebral amyloid angiopathy (CAA) is characterized by amyloid deposition within walls of leptomeningeal and cortical arterioles. Among the several types of amyloid proteins causing CAA, fibrillar amyloid β (Aβ) is by far the most common (1). This pathological form of Aβ is also the major constituent of neuritic plaques in patients with Alzheimer’s disease (AD) (2). Aβ is a 39- to 43-amino acid peptide that is produced from the amyloid precursor protein (APP) via sequential proteolytic cleavage processed by β- and γ-secretases (3, 4). Aβ₄₀ is the predominant Aβ species present in CAA whereas Aβ₄₂ is the major Aβ species present in neuritic plaques. CAA is a very common disorder, pathologically affecting about one-third of all elderly patients (>60 y of age) and about 90% of patients with AD (5, 6). CAA is a well-recognized cause of intracerebral hemorrhage (7, 8). It is also a major contributor to ischemic stroke and dementia (2, 9–12)—two conditions in which CAA-induced impairment in cerebral arteriole function is likely to play a fundamental role (13).Multiple lines of evidence indicate that soluble Aβ monomers and insoluble Aβ fibrils in the form of CAA cause significant cerebrovascular (CV) impairment. Ex vivo studies with isolated cerebral arterioles show that synthetic Aβ₄₀ (and to a lesser degree Aβ₄₂) induces direct vessel constriction, enhanced response to vasoconstrictors, and reduced response to vasodilators (14–22). Similar results have been demonstrated with synthetic Aβ₄₀ topically applied to the cerebral cortex (23, 24), results that are generally supported by in vivo studies (20, 23, 25). For example, Iadecola and coworkers have shown that young APP transgenic mice (Tg2576) exposed to elevated levels of Aβ₄₀ and Aβ₄₂ (but no CAA) have reduced baseline cerebral blood flow (CBF) and decreased CBF responses to topical vasodilators (23, 24, 26). We have shown similar CV deficits in young Tg2576 mice (13). Moreover, we provided the most direct evidence to date that endogenous soluble Aβ plays a causal role in these CV deficits when we found that depletion of soluble Aβ via γ-secretase inhibition restores CV function in young Tg2576 mice (13).Fibrillar Aβ in the form of CAA produces even greater degrees of CV impairment. Evidence for this notion comes from several experimental studies from different laboratories that show reduced pial arteriole responses (27) and diminished CBF responses (27, 28) to a variety of vasodilatory stimuli in aged APP mice with CAA vs. young APP mice without CAA. Our past work examining pial arteriole function in young vs. aged Tg2576 mice shows similar age-dependent CV deficits (13). Moreover, multiple additional observations from our study show that CAA (and not prolonged exposure to soluble Aβ and/or mutant APP) is the principle cause of the severe CV dysfunction noted in aged Tg2576 mice: (i) The severity of the vasomotor deficits noted in these mice is dependent on the presence and extent of CAA; (ii) even small amounts of CAA are associated with profound vasomotor impairment; and (iii) the CV dysfunction noted in CAA-ladened arteries is poorly responsive to depletion of soluble Aβ via γ-secretase inhibition (13).Regarding the mechanism of soluble Aβ-induced CV deficits, increased reactive oxygen species (ROS) are strongly implicated. Cerebral arterioles exposed to exogenous Aβ₄₀ develop significant oxidative stress (29), and various anti-ROS strategies have been shown to improve Aβ₄₀-induced vessel dysfunction (16, 23). Similarly, cerebral arterioles of young APP mice producing elevated levels of endogenous Aβ₄₀ and Aβ₄₂ (but no CAA) display oxidative stress (19), and the CV deficits found in these mice can be attenuated by both genetic and pharmacologic anti-ROS interventions (15, 19, 20, 30). In particular, ROS derived from NADPH oxidase—one of two major sources of ROS in the cerebrovasculature (31–33)—have been implicated (28–30, 34, 35).Regarding the mechanism of CAA-induced CV deficits, far less is known; however, three recent findings suggest that ROS may play a role. First, CAA-affected vessels were shown to have significantly greater oxidative stress than CAA-free vessels of aged Tg2576 mice (36). Second, genetic knockdown of mitochondrial superoxide dismutase 2 (SOD2)—which increases mitochondria-derived ROS—was shown to exacerbate CAA pathology in aged APP mice (37). Third, genetic depletion of the catalytic subunit Nox2 of NADPH oxidase was shown to reduce oxidative stress and improve CV function in aged Tg2576 mice (28, 35). Importantly, the latter studies did not examine for the presence of CAA, nor did they assess for the effect of CAA on cerebral arteriole function (28, 35). To address this critical knowledge gap, we examined the effect of the NADPH oxidase inhibitor, apocynin, and the nonspecific ROS scavenger, tempol, on CAA-induced CV dysfunction in aged Tg2576 mice. The effect of these agents on CAA formation and CAA-related microhemorrhage was also examined.     

Kinetics of nucleotide-dependent structural transitions in the kinesin-1 hydrolysis cycle

To dissect the kinetics of structural transitions underlying the stepping cycle of kinesin-1 at physiological ATP, we used interferometric scattering microscopy to track the position of gold nanoparticles attached to individual motor domains in processively stepping dimers. Labeled heads resided stably at positions 16.4 nm apart, corresponding to a microtubule-bound state, and at a previously unseen intermediate position, corresponding to a tethered state. The chemical transitions underlying these structural transitions were identified by varying nucleotide conditions and carrying out parallel stopped-flow kinetics assays. At saturating ATP, kinesin-1 spends half of each stepping cycle with one head bound, specifying a structural state for each of two rate-limiting transitions. Analysis of stepping kinetics in varying nucleotides shows that ATP binding is required to properly enter the one-head–bound state, and hydrolysis is necessary to exit it at a physiological rate. These transitions differ from the standard model in which ATP binding drives full docking of the flexible neck linker domain of the motor. Thus, this work defines a consensus sequence of mechanochemical transitions that can be used to understand functional diversity across the kinesin superfamily.Kinesin-1 is a motor protein that steps processively toward microtubule plus-ends, tracking single protofilaments and hydrolyzing one ATP molecule per step (1–6). Step sizes corresponding to the tubulin dimer spacing of 8.2 nm are observed when the molecule is labeled by its C-terminal tail (7–10) and to a two-dimer spacing of 16.4 nm when a single motor domain is labeled (4, 11, 12), consistent with the motor walking in a hand-over-hand fashion. Kinesin has served as an important model system for advancing single-molecule techniques (7–10) and is clinically relevant for its role in neurodegenerative diseases (13), making dissection of its step a popular ongoing target of study.Despite decades of work, many essential components of the mechanochemical cycle remain disputed, including (i) how much time kinesin-1 spends in a one-head–bound (1HB) state when stepping at physiological ATP concentrations, (ii) whether the motor waits for ATP in a 1HB or two-heads–bound (2HB) state, and (iii) whether ATP hydrolysis occurs before or after tethered head attachment (4, 11, 14–20). These questions are important because they are fundamental to the mechanism by which kinesins harness nucleotide-dependent structural changes to generate mechanical force in a manner optimized for their specific cellular tasks. Addressing these questions requires characterizing a transient 1HB state in the stepping cycle in which the unattached head is located between successive binding sites on the microtubule. This 1HB intermediate is associated with the force-generating powerstroke of the motor and underlies the detachment pathway that limits motor processivity. Optical trapping (7, 19, 21, 22) and single-molecule tracking studies (4, 8–11) have failed to detect this 1HB state during stepping. Single-molecule fluorescence approaches have detected a 1HB intermediate at limiting ATP concentrations (11, 12, 14, 15), but apart from one study that used autocorrelation analysis to detect a 3-ms intermediate (17), the 1HB state has been undetectable at physiological ATP concentrations.Single-molecule microscopy is a powerful tool for studying the kinetics of structural changes in macromolecules (23). Tracking steps and potential substeps for kinesin-1 at saturating ATP has until now been hampered by the high stepping rates of the motor (up to 100 s⁻¹), which necessitates high frame rates, and the small step size (8.2 nm), which necessitates high spatial precision (7). Here, we apply interferometric scattering microscopy (iSCAT), a recently established single-molecule tool with high spatiotemporal resolution (24–27) to directly visualize the structural changes underlying kinesin stepping. By labeling one motor domain in a dimeric motor, we detect a 1HB intermediate state in which the tethered head resides over the bound head for half the duration of the stepping cycle at saturating ATP. We further show that at physiological stepping rates, ATP binding is required to enter this 1HB state and that ATP hydrolysis is required to exit it. This work leads to a significant revision of the sequence and kinetics of mechanochemical transitions that make up the kinesin-1 stepping cycle and provides a framework for understanding functional diversity across the kinesin superfamily.     

Distinct dopamine neurons mediate reward signals for short- and long-term memories

Drosophila melanogaster can acquire a stable appetitive olfactory memory when the presentation of a sugar reward and an odor are paired. However, the neuronal mechanisms by which a single training induces long-term memory are poorly understood. Here we show that two distinct subsets of dopamine neurons in the fly brain signal reward for short-term (STM) and long-term memories (LTM). One subset induces memory that decays within several hours, whereas the other induces memory that gradually develops after training. They convey reward signals to spatially segregated synaptic domains of the mushroom body (MB), a potential site for convergence. Furthermore, we identified a single type of dopamine neuron that conveys the reward signal to restricted subdomains of the mushroom body lobes and induces long-term memory. Constant appetitive memory retention after a single training session thus comprises two memory components triggered by distinct dopamine neurons.Memory of a momentous event persists for a long time. Whereas some forms of long-term memory (LTM) require repetitive training (1–3), a highly relevant stimulus such as food or poison is sufficient to induce LTM in a single training session (4–7). Recent studies have revealed aspects of the molecular and cellular mechanisms of LTM formation induced by repetitive training (8–11), but how a single training induces a stable LTM is poorly understood (12).Appetitive olfactory learning in fruit flies is suited to address the question, as a presentation of a sugar reward paired with odor induces robust short-term memory (STM) and LTM (6, 7). Odor is represented by a sparse ensemble of the 2,000 intrinsic neurons, the Kenyon cells (13). A current working model suggests that concomitant reward signals from sugar ingestion cause associative plasticity in Kenyon cells that might underlie memory formation (14–20). A single activation session of a specific cluster of dopamine neurons (PAM neurons) by sugar ingestion can induce appetitive memory that is stable over 24 h (19), underscoring the importance of sugar reward to the fly.The mushroom body (MB) is composed of the three different cell types, α/β, α′/β′, and γ, which have distinct roles in different phases of appetitive memories (11, 21–25). Similar to midbrain dopamine neurons in mammals (26, 27), the structure and function of PAM cluster neurons are heterogeneous, and distinct dopamine neurons intersect unique segments of the MB lobes (19, 28–34). Further circuit dissection is thus crucial to identify candidate synapses that undergo associative modulation.By activating distinct subsets of PAM neurons for reward signaling, we found that short- and long-term memories are independently formed by two complementary subsets of PAM cluster dopamine neurons. Conditioning flies with nutritious and nonnutritious sugars revealed that the two subsets could represent different reinforcing properties: sweet taste and nutritional value of sugar. Constant appetitive memory retention after a single training session thus comprises two memory components triggered by distinct reward signals.     

PP2A methylation controls sensitivity and resistance to β-amyloid–induced cognitive and electrophysiological impairments

Elevated levels of the β-amyloid peptide (Aβ) are thought to contribute to cognitive and behavioral impairments observed in Alzheimer’s disease (AD). Protein phosphatase 2A (PP2A) participates in multiple molecular pathways implicated in AD, and its expression and activity are reduced in postmortem brains of AD patients. PP2A is regulated by protein methylation, and impaired PP2A methylation is thought to contribute to increased AD risk in hyperhomocysteinemic individuals. To examine further the link between PP2A and AD, we generated transgenic mice that overexpress the PP2A methylesterase, protein phosphatase methylesterase-1 (PME-1), or the PP2A methyltransferase, leucine carboxyl methyltransferase-1 (LCMT-1), and examined the sensitivity of these animals to behavioral and electrophysiological impairments caused by exogenous Aβ exposure. We found that PME-1 overexpression enhanced these impairments, whereas LCMT-1 overexpression protected against Aβ-induced impairments. Neither transgene affected Aβ production or the electrophysiological response to low concentrations of Aβ, suggesting that these manipulations selectively affect the pathological response to elevated Aβ levels. Together these data identify a molecular mechanism linking PP2A to the development of AD-related cognitive impairments that might be therapeutically exploited to target selectively the pathological effects caused by elevated Aβ levels in AD patients.Multiple observations suggest a role for the serine/threonine protein phosphatase 2A (PP2A) in the molecular pathways that underlie Alzheimer’s disease (AD). Analyses conducted on postmortem AD brains have found reduced PP2A expression and activity, and studies conducted in animal models have found that inhibiting PP2A produces AD-like tau pathology and cognitive impairment (1–3). One of the ways in which PP2A may affect AD is through its role as the principal tau phosphatase (4–7). PP2A also interacts with a number of kinases implicated in AD including glycogen synthase kinase 3β (GSK3β), cyclin-dependent kinase 5 (CDK5), and ERK and JNK as well as amyloid precursor protein and the NMDA and metabotropic glutamate receptors (reviewed in ref. 2).PP2A is a heterotrimeric protein composed of a catalytic, scaffolding, and regulatory subunit. Each subunit is encoded by multiple genes and splice isoforms, and the subunit composition of a particular PP2A molecule determines its subcellular distribution and substrate specificity (reviewed in ref. 2). One of the ways in which PP2A activity is regulated is through C-terminal methylation of the catalytic subunit (reviewed in refs. 8 and 9). Impaired methyl-donor metabolism is a risk factor for AD (10, 11), and PP2A dysregulation caused by impaired methylation is thought to be one of the molecular mechanisms contributing to this increased risk (12–14). Methylation promotes the formation of PP2A holoenzymes that contain Bα regulatory subunits (7, 13, 15–19), and these forms of PP2A exhibit the greatest tau phosphatase activity (6, 7).PP2A methylation is catalyzed in vivo by the methyl transferase, leucine carboxyl methyltransferase 1 (LCMT-1) (20–22), and its demethylation is catalyzed by the methylesterase, protein phosphatase methylesterase 1 (PME-1) (23–25). To explore the role of PP2A in AD further, we generated lines of transgenic mice that overexpress these enzymes and tested their effect on the sensitivity of animals to electrophysiogical and behavioral impairments caused by β-amyloid (Aβ). We found that LCMT-1 overexpression protected animals from Aβ-induced impairments, whereas overexpression of PME-1 worsened Aβ neurotoxicity. Neither transgene affected endogenous Aβ levels, suggesting that they acted by altering the response to Aβ rather than Aβ production. We also found that PME-1 and LCMT-1 overexpression were without effect on the electrophysiological response to picomolar Aβ application, suggesting that they selectively affected the response to pathological Aβ concentrations. Together these data indicate that this pathway has potential as a therapeutic avenue for AD that acts not by targeting Aβ production but by selectively altering the response to pathological levels of Aβ.     

Neural mechanisms of transient neocortical beta rhythms: Converging evidence from humans,computational modeling,monkeys, and mice

Human neocortical 15–29-Hz beta oscillations are strong predictors of perceptual and motor performance. However, the mechanistic origin of beta in vivo is unknown, hindering understanding of its functional role. Combining human magnetoencephalography (MEG), computational modeling, and laminar recordings in animals, we present a new theory that accounts for the origin of spontaneous neocortical beta. In our MEG data, spontaneous beta activity from somatosensory and frontal cortex emerged as noncontinuous beta events typically lasting <150 ms with a stereotypical waveform. Computational modeling uniquely designed to infer the electrical currents underlying these signals showed that beta events could emerge from the integration of nearly synchronous bursts of excitatory synaptic drive targeting proximal and distal dendrites of pyramidal neurons, where the defining feature of a beta event was a strong distal drive that lasted one beta period (∼50 ms). This beta mechanism rigorously accounted for the beta event profiles; several other mechanisms did not. The spatial location of synaptic drive in the model to supragranular and infragranular layers was critical to the emergence of beta events and led to the prediction that beta events should be associated with a specific laminar current profile. Laminar recordings in somatosensory neocortex from anesthetized mice and awake monkeys supported these predictions, suggesting this beta mechanism is conserved across species and recording modalities. These findings make several predictions about optimal states for perceptual and motor performance and guide causal interventions to modulate beta for optimal function.Beta band rhythms (15–29 Hz) are a commonly observed activity pattern in the brain. They are found with magnetoencephalography (MEG) (1–4), EEG (5, 6), and local field potential (LFP) recordings from neocortex (7–9) and are preserved across species (10). Local beta oscillations and their coordination between regions are implicated in numerous functions, including sensory perception, selective attention, and motor planning and initiation (2, 3, 6, 7, 9, 11–15). Neocortical beta oscillations are disrupted in various neuropathologies, most notably Parkinson’s disease (PD), in which treatments that alleviate motor symptoms also reverse the neocortical beta disruption (16, 17). Although associations between beta and performance suggest a crucial role in brain function, beta rhythmicity might not be important per se but instead may be an epiphenomenal consequence of other important processes. Discovering how beta emerges at the cellular and network levels is crucial to understanding why beta is such a clear predictor of performance in many domains.A major, unresolved point of debate concerns the locus of beta generation. One prominent view is that beta is generated in basal ganglia and thalamic structures and that neocortical beta is an entrained reflection of these inputs. Alternatively, beta may emerge within the neocortex as a consequence of internal dynamics. An intermediate view, supported by the model presented here, is that beta emerges in the neocortex but is dependent on extrinsic synaptic drive that could originate from basal ganglia/thalamus. Consistent with the first view, beta has been robustly observed in LFP signals from basal ganglia nuclei including the subthalamic nucleus, striatum, and globus pallidus (18, 19), and computational models have proposed mechanisms by which beta rhythms can emerge via interactions within and between these circuits (20, 21). Other studies have suggested that the neocortex itself has unique properties that generate beta rhythms through spike-mediated synaptic and electrical interactions within local circuits (22–25) or that beta in early-sensory neocortical areas could be driven in a top-down manner from frontal cortex during attentive states (26).Understanding the temporal and spectral nature of a specific beta signal is critical to uncovering its mechanism of generation and its role in the precise local circuit and context in which it is observed. A common view of beta “rhythms” is that they are sustained in time for many cycles, up to seconds in duration. The view of beta as a sustained rhythm is consistent with several papers that have reported what appears to be a continuous, high-power increase in beta activity, for example during a planning or “hold” period in a motor task or during the allocation of attention in sensory neocortices (2, 27–29). Data showing such effects are almost always averaged spectrograms or averaged power spectral density measurements taken from many individual trials aligned to functionally relevant events. However, burst-like or intermittent periods of high beta power occurring stochastically within the time-averaged period could appear as continuous rhythms in averaged spectrograms, despite not ever actually being sustained. Several recent studies have shown that in nonaveraged data beta oscillations often emerge transiently, typically lasting <150 ms (2, 4, 30–32).Here, we combined human MEG, computational neural modeling, and laminar recordings in animals to propose a new theory to explain the origin of spontaneous beta activity that emerges transiently and intermittently in the awake mammalian neocortex. Building on our prior work (1, 2, 15, 33), we studied MEG-measured spontaneous beta activity in two brain areas: (i) primary somatosensory neocortex (SI), where beta emerges as part of the so-called “mu rhythm” and typically contains a complex of alpha and beta events, and (ii) frontal cortex, specifically the right inferior frontal cortex (IFC), where beta can be expressed without a strong alpha signal. We previously have shown that beta activity in each of these areas is coordinated, with synchrony in the beta band increasing during inattention (15) and increased beta power in SI predicting failed detection (2).To uncover the neural mechanisms specific to beta generation, we first quantified beta’s manifestation on individual trials. We verified that beta events in each area were independent of alpha events and were transient in time. Further, we found beta events had a consistent temporal profile. To study the cellular and network level mechanisms creating this beta activity, we used a biophysically principled model of a laminar cortical circuit designed specifically to simulate human MEG/EEG-measured primary current source signals (1, 34–37). In keeping the output of our model in close agreement with our MEG data, we arrived at the prediction that these beta events are not inherited linearly from subcortical structures or generated by the spiking interactions in local neocortical circuits. Rather, our data suggest that transient beta events emerge locally in neocortex from the integration of synchronous bursts of subthreshold excitatory synaptic drive that simultaneously target both proximal and distal dendrites of pyramidal neurons (PNs), such that the distal input is sufficiently strong and lasts a beta period.Our beta theory predicted a specific neocortical laminar current profile during beta events that was supported by laminar recordings in mice and monkeys. In sum, our model accurately reproduces beta events found in SI and in higher-order frontal area IFC and accurately reflects the data generated in distinct species, in distinct recording modalities (MEG and invasive laminar electrophysiological recordings), and in distinct brain states (anesthetized versus awake-behaving).     

Structural interactions of a voltage sensor toxin with lipid membranes

Protein toxins from tarantula venom alter the activity of diverse ion channel proteins, including voltage, stretch, and ligand-activated cation channels. Although tarantula toxins have been shown to partition into membranes, and the membrane is thought to play an important role in their activity, the structural interactions between these toxins and lipid membranes are poorly understood. Here, we use solid-state NMR and neutron diffraction to investigate the interactions between a voltage sensor toxin (VSTx1) and lipid membranes, with the goal of localizing the toxin in the membrane and determining its influence on membrane structure. Our results demonstrate that VSTx1 localizes to the headgroup region of lipid membranes and produces a thinning of the bilayer. The toxin orients such that many basic residues are in the aqueous phase, all three Trp residues adopt interfacial positions, and several hydrophobic residues are within the membrane interior. One remarkable feature of this preferred orientation is that the surface of the toxin that mediates binding to voltage sensors is ideally positioned within the lipid bilayer to favor complex formation between the toxin and the voltage sensor.Protein toxins from venomous organisms have been invaluable tools for studying the ion channel proteins they target. For example, in the case of voltage-activated potassium (Kv) channels, pore-blocking scorpion toxins were used to identify the pore-forming region of the channel (1, 2), and gating modifier tarantula toxins that bind to S1–S4 voltage-sensing domains have helped to identify structural motifs that move at the protein–lipid interface (3–5). In many instances, these toxin–channel interactions are highly specific, allowing them to be used in target validation and drug development (6–8).Tarantula toxins are a particularly interesting class of protein toxins that have been found to target all three families of voltage-activated cation channels (3, 9–12), stretch-activated cation channels (13–15), as well as ligand-gated ion channels as diverse as acid-sensing ion channels (ASIC) (16–21) and transient receptor potential (TRP) channels (22, 23). The tarantula toxins targeting these ion channels belong to the inhibitor cystine knot (ICK) family of venom toxins that are stabilized by three disulfide bonds at the core of the molecule (16, 17, 24–31). Although conventional tarantula toxins vary in length from 30 to 40 aa and contain one ICK motif, the recently discovered double-knot toxin (DkTx) that specifically targets TRPV1 channels contains two separable lobes, each containing its own ICK motif (22, 23).One unifying feature of all tarantula toxins studied thus far is that they act on ion channels by modifying the gating properties of the channel. The best studied of these are the tarantula toxins targeting voltage-activated cation channels, where the toxins bind to the S3b–S4 voltage sensor paddle motif (5, 32–36), a helix-turn-helix motif within S1–S4 voltage-sensing domains that moves in response to changes in membrane voltage (37–41). Toxins binding to S3b–S4 motifs can influence voltage sensor activation, opening and closing of the pore, or the process of inactivation (4, 5, 36, 42–46). The tarantula toxin PcTx1 can promote opening of ASIC channels at neutral pH (16, 18), and DkTx opens TRPV1 in the absence of other stimuli (22, 23), suggesting that these toxin stabilize open states of their target channels.For many of these tarantula toxins, the lipid membrane plays a key role in the mechanism of inhibition. Strong membrane partitioning has been demonstrated for a range of toxins targeting S1–S4 domains in voltage-activated channels (27, 44, 47–50), and for GsMTx4 (14, 50), a tarantula toxin that inhibits opening of stretch-activated cation channels in astrocytes, as well as the cloned stretch-activated Piezo1 channel (13, 15). In experiments on stretch-activated channels, both the d- and l-enantiomers of GsMTx4 are active (14, 50), implying that the toxin may not bind directly to the channel. In addition, both forms of the toxin alter the conductance and lifetimes of gramicidin channels (14), suggesting that the toxin inhibits stretch-activated channels by perturbing the interface between the membrane and the channel. In the case of Kv channels, the S1–S4 domains are embedded in the lipid bilayer and interact intimately with lipids (48, 51, 52) and modification in the lipid composition can dramatically alter gating of the channel (48, 53–56). In one study on the gating of the Kv2.1/Kv1.2 paddle chimera (53), the tarantula toxin VSTx1 was proposed to inhibit Kv channels by modifying the forces acting between the channel and the membrane. Although these studies implicate a key role for the membrane in the activity of Kv and stretch-activated channels, and for the action of tarantula toxins, the influence of the toxin on membrane structure and dynamics have not been directly examined. The goal of the present study was to localize a tarantula toxin in membranes using structural approaches and to investigate the influence of the toxin on the structure of the lipid bilayer.     

Helical arrays of U-shaped ATP synthase dimers form tubular cristae in ciliate mitochondria

F₁F_o-ATP synthases are universal energy-converting membrane protein complexes that synthesize ATP from ADP and inorganic phosphate. In mitochondria of yeast and mammals, the ATP synthase forms V-shaped dimers, which assemble into rows along the highly curved ridges of lamellar cristae. Using electron cryotomography and subtomogram averaging, we have determined the in situ structure and organization of the mitochondrial ATP synthase dimer of the ciliate Paramecium tetraurelia. The ATP synthase forms U-shaped dimers with parallel monomers. Each complex has a prominent intracrista domain, which links the c-ring of one monomer to the peripheral stalk of the other. Close interaction of intracrista domains in adjacent dimers results in the formation of helical ATP synthase dimer arrays, which differ from the loose dimer rows in all other organisms observed so far. The parameters of the helical arrays match those of the cristae tubes, suggesting the unique features of the P. tetraurelia ATP synthase are directly responsible for generating the helical tubular cristae. We conclude that despite major structural differences between ATP synthase dimers of ciliates and other eukaryotes, the formation of ATP synthase dimer rows is a universal feature of mitochondria and a fundamental determinant of cristae morphology.F₁F_o-ATP synthases are ubiquitous, highly conserved energy-converting membrane protein complexes. ATP synthases produce ATP from ADP and inorganic phosphate (P_i) by rotary catalysis (1, 2) using the energy stored in a transmembrane electrochemical gradient. The ∼600-kDa monomer of the mitochondrial ATP synthase is composed of a soluble F₁ subcomplex and a membrane-bound F_o subcomplex (3). The main components of the F₁ subcomplex are the (αβ)₃ hexamer and the central stalk (4). The F_o subcomplex includes a rotor ring of 8–15 hydrophobic c subunits (5), the peripheral stalk, and several small hydrophobic stator subunits. Protons flowing through the membrane part of the F_o subcomplex drive the rotation of the c-ring (6–9). The central stalk transmits the torque generated by c-ring rotation to the catalytic head of the F₁ subcomplex, where it induces conformational changes of the α and β subunits that result in phosphate bond formation and the generation of ATP. The catalytic (αβ)₃ hexamer is held stationary relative to the membrane region by the peripheral stalk (10, 11). Several high-resolution structures of the F₁/rotor ring complexes have been solved by X-ray crystallography (12–16), and the structure of the complete assembly has been determined by cryoelectron microscopy (cryo-EM) (10, 17–20).In mitochondria, the ATP synthase forms dimers in the inner membrane. In fungi, plants, and metazoans, the dimers are V-shaped and associate into rows along the highly curved ridges of lamellar cristae (19–22). F_o subcomplexes of the two monomers in the dimer interact in the lipid bilayer via a number of hydrophobic stator subunits (20, 23–25). Coarse-grained molecular dynamics simulations have suggested that the V-shape of the ATP synthase dimers induces local membrane curvature, which in turn drives the association of ATP synthase dimers into rows (20). The exact role of the dimer rows is unclear, however rows of ATP synthase dimers have been proposed to promote the formation of lamellar cristae in yeast (20, 26).So far, all rows of ATP synthase dimers observed by electron cryotomography have been more or less straight (19–22, 27). However, an earlier deep-etch freeze-fracture study of mitochondria from the ciliate Paramecium multimicronucleatum revealed double rows of interdigitating 10-nm particles on helical tubular cristae (28). These particles were interpreted as ATP synthases, which, if correct, would suggest that the mitochondrial ATP synthase can assemble into rows that differ significantly from the standard geometry found in lamellar cristae (19, 21, 22).To investigate the helical rows in more detail, we performed electron cryotomography of isolated mitochondrial membranes from Paramecium tetraurelia. Using subtomogram averaging, we show that these helical rows do indeed consist of ATP synthase molecules, as suggested by Allen et al. (28). However, unlike the V-shaped dimers of metazoans, the ATP synthase of this species forms U-shaped dimers, which have new and unusual structural features. When assembled into the helical rows, the ATP synthase monomers interdigitate, whereas the U-shaped dimers align side by side. Thus, rows of ATP synthase dimers seem to be a universal feature of all mitochondria. We propose that the particular shape of the P. tetraurelia ATP synthase dimer induces its assembly into helical rows, which in turn cause the formation of the helical tubular cristae of ciliates.