Creative destruction of the microtubule array

Comment on: Mukherjee S, et al. Cell Cycle 2012; 11:2359-66.Typical cells contain a dense array of microtubules that serves as a structural backbone and also provides a substrate against which molecular motor proteins generate force. Cells transitioning through the cell cycle or undergoing significant morphological changes must be able to tear apart the microtubule array and reconstruct it into new configurations, either partially or completely. The microtubule field was revolutionized in the 1980s with the introduction of the dynamic instability model,¹ now broadly recognized as a fundamental mechanism by which microtubule populations are reconfigured.² Dynamic instability involves the catastrophic disassembly of microtubules, generally from their plus ends, as well as the rapid reassembly of microtubules and selective stabilization of particular ones. Microtubules can be stabilized along their length by binding to various proteins and can be attached at their minus ends to structures such as the centrosome and “captured” at their plus ends by proteins in the cell’s cortex.² Given the contribution of these stabilizing and anchoring factors, additional mechanisms beyond dynamic instability are required to tear down previous microtubule structures so that new ones can be constructed. Borrowing from the field of economics, we refer to this as creative destruction.Various proteins such as stathmin³ and kinesin-13⁴ contribute to creative destruction by promoting loss of tubulin subunits from the ends of the microtubules. We find especially interesting a category of AAA enzymes called microtubule-severing proteins that use the energy of ATP hydrolysis to yank at tubulin subunits within the microtubule, thereby causing the lattice to break.⁵ If this occurs along the length of the microtubule, the microtubule will be severed into pieces. If this occurs at either of the two ends of the microtubule, the microtubule will lose subunits from that end. The first discovered and best-studied microtubule-severing proteins are katanin and spastin.Thanks to David Sharp and his colleagues at Albert Einstein College of Medicine, as well as other workers in the field, we now know that cells express at least five other AAA proteins with potential microtubule-severing properties, on the basis of sequence similarity to katanin and spastin in the AAA region.⁵ Two of these, called katanin-like-1 and katanin-like-2, are very similar to katanin. The three others are similar to one another, collectively termed fidgetins (fidgetin, fidgetin-like-1 and fidgetin-like-2). One possibility is that all seven of the microtubule-severing proteins are regulated similarly and are functionally redundant with one another. A more compelling possibility is that, while there is some functional redundancy, there is also a division of labor, with each severing protein displaying distinct properties and carrying out its own duties. Thus far, Sharp’s studies on mitosis support the latter scenario, with katanin, fidgetin and spastin having characteristic distributions within the spindle, resulting in unique phenotypes when depleted.⁶In a new article, Sharp’s group has confirmed that fidgetin has microtubule-severing properties. Interestingly, fidgetin depolymerizes microtubules preferentially from the minus end.⁷ In addition, the new work shows that in human U2OS cells, fidgetin targets to the centrosome, where most minus ends of microtubules are clustered, suggesting a scenario by which fidgetin suppresses microtubule growth from the centrosome as well as attachment to it. Consistent with this scenario, the authors show that experimental depletion of fidgetin reduces that speed of poleward tubulin flux as well as the speed of anaphase A chromatid-to-pole motion and also results in an increase in both the number and length of astral microtubules. Notably, this contrasts with katanin, which favors the plus ends of microtubules, for example, at the chromosome during cell division⁶ and at the leading edge of motile cells.⁸The authors close their article by pointing out that microtubule-severing is important beyond mitosis, for example, in the restructuring of the microtubule array in neurons and migrating cells, and we would point to plants as well.⁹ We previously described a mechanism called “cut and run,” wherein the severing of microtubules is important for motility within the microtubule array, as short microtubules are more mobile than long ones.⁹ Now, inspired by the work of Sharp and colleagues, we envision “creative destruction” as another way of understanding the crucial roles played by a diversity of microtubule-severing proteins in cells.     

Familial FTDP-17 Missense Mutations Inhibit Microtubule Assembly-promoting Activity of Tau by Increasing Phosphorylation at Ser202 in Vitro

In Alzheimer disease (AD), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) and other tauopathies, tau accumulates and forms paired helical filaments (PHFs) in the brain. Tau isolated from PHFs is phosphorylated at a number of sites, migrates as ∼60-, 64-, and 68-kDa bands on SDS-gel, and does not promote microtubule assembly. Upon dephosphorylation, the PHF-tau migrates as ∼50–60-kDa bands on SDS-gels in a manner similar to tau that is isolated from normal brain and promotes microtubule assembly. The site(s) that inhibits microtubule assembly-promoting activity when phosphorylated in the diseased brain is not known. In this study, when tau was phosphorylated by Cdk5 in vitro, its mobility shifted from ∼60-kDa bands to ∼64- and 68-kDa bands in a time-dependent manner. This mobility shift correlated with phosphorylation at Ser²⁰², and Ser²⁰² phosphorylation inhibited tau microtubule-assembly promoting activity. When several tau point mutants were analyzed, G272V, P301L, V337M, and R406W mutations associated with FTDP-17, but not nonspecific mutations S214A and S262A, promoted Ser²⁰² phosphorylation and mobility shift to a ∼68-kDa band. Furthermore, Ser²⁰² phosphorylation inhibited the microtubule assembly-promoting activity of FTDP-17 mutants more than of WT. Our data indicate that FTDP-17 missense mutations, by promoting phosphorylation at Ser²⁰², inhibit the microtubule assembly-promoting activity of tau in vitro, suggesting that Ser²⁰² phosphorylation plays a major role in the development of NFT pathology in AD and related tauopathies.Neurofibrillary tangles (NFTs)⁴ and senile plaques are the two characteristic neuropathological lesions found in the brains of patients suffering from Alzheimer disease (AD). The major fibrous component of NFTs are paired helical filaments (PHFs) (for reviews see Refs. 1–3). Initially, PHFs were found to be composed of a protein component referred to as “A68” (4). Biochemical analysis reveled that A68 is identical to the microtubule-associated protein, tau (4, 5). Some characteristic features of tau isolated from PHFs (PHF-tau) are that it is abnormally hyperphosphorylated (phosphorylated on more sites than the normal brain tau), does not bind to microtubules, and does not promote microtubule assembly in vitro. Upon dephosphorylation, PHF-tau regains its ability to bind to and promote microtubule assembly (6, 7). Tau hyperphosphorylation is suggested to cause microtubule instability and PHF formation, leading to NFT pathology in the brain (1–3).PHF-tau is phosphorylated on at least 21 proline-directed and non-proline-directed sites (8, 9). The individual contribution of these sites in converting tau to PHFs is not entirely clear. However, some sites are only partially phosphorylated in PHFs (8), whereas phosphorylation on specific sites inhibits the microtubule assembly-promoting activity of tau (6, 10). These observations suggest that phosphorylation on a few sites may be responsible and sufficient for causing tau dysfunction in AD.Tau purified from the human brain migrates as ∼50–60-kDa bands on SDS-gel due to the presence of six isoforms that are phosphorylated to different extents (2). PHF-tau isolated from AD brain, on the other hand, displays ∼60-, 64-, and 68 kDa-bands on an SDS-gel (4, 5, 11). Studies have shown that ∼64- and 68-kDa tau bands (the authors have described the ∼68-kDa tau band as an ∼69-kDa band in these studies) are present only in brain areas affected by NFT degeneration (12, 13). Their amount is correlated with the NFT densities at the affected brain regions. Moreover, the increase in the amount of ∼64- and 68-kDa band tau in the brain correlated with a decline in the intellectual status of the patient. The ∼64- and 68-kDa tau bands were suggested to be the pathological marker of AD (12, 13). Biochemical analyses determined that ∼64- and 68-kDa bands are hyperphosphorylated tau, which upon dephosphorylation, migrated as normal tau on SDS-gel (4, 5, 11). Tau sites involved in the tau mobility shift to ∼64- and 68-kDa bands were suggested to have a role in AD pathology (12, 13). It is not known whether phosphorylation at all 21 PHF-sites is required for the tau mobility shift in AD. However, in vitro the tau mobility shift on SDS-gel is sensitive to phosphorylation only on some sites (6, 14). It is therefore possible that in the AD brain, phosphorylation on some sites also causes a tau mobility shift. Identification of such sites will significantly enhance our knowledge of how NFT pathology develops in the brain.PHFs are also the major component of NFTs found in the brains of patients suffering from a group of neurodegenerative disorders collectively called tauopathies (2, 11). These disorders include frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17), corticobasal degeneration, progressive supranuclear palsy, and Pick disease. Each PHF-tau isolated from autopsied brains of patients suffering from various tauopathies is hyperphosphorylated, displays ∼60-, 64-, and 68-kDa bands on SDS-gel, and is incapable of binding to microtubules. Upon dephosphorylation, the above referenced PHF-tau migrates as a normal tau on SDS-gel, binds to microtubules, and promotes microtubule assembly (2, 11). These observations suggest that the mechanisms of NFT pathology in various tauopathies may be similar and the phosphorylation-dependent mobility shift of tau on SDS-gel may be an indicator of the disease. The tau gene is mutated in familial FTDP-17, and these mutations accelerate NFT pathology in the brain (15–18). Understanding how FTDP-17 mutations promote tau phosphorylation can provide a better understanding of how NFT pathology develops in AD and various tauopathies. However, when expressed in CHO cells, G272V, R406W, V337M, and P301L tau mutations reduce tau phosphorylation (19, 20). In COS cells, although G272V, P301L, and V337M mutations do not show any significant affect, the R406W mutation caused a reduction in tau phosphorylation (21, 22). When expressed in SH-SY5Y cells subsequently differentiated into neurons, the R406W, P301L, and V337M mutations reduce tau phosphorylation (23). In contrast, in hippocampal neurons, R406W increases tau phosphorylation (24). When phosphorylated by recombinant GSK3β in vitro, the P301L and V337M mutations do not have any effect, and the R406W mutation inhibits phosphorylation (25). However, when incubated with rat brain extract, all of the G272V, P301L, V337M, and R406W mutations stimulate tau phosphorylation (26). The mechanism by which FTDP-17 mutations promote tau phosphorylation leading to development of NFT pathology has remained unclear.Cyclin-dependent protein kinase 5 (Cdk5) is one of the major kinases that phosphorylates tau in the brain (27, 28). In this study, to determine how FTDP-17 missense mutations affect tau phosphorylation, we phosphorylated four FTDP-17 tau mutants (G272V, P301L, V337M, and R406W) by Cdk5. We have found that phosphorylation of tau by Cdk5 causes a tau mobility shift to ∼64- and 68 kDa-bands. Although the mobility shift to a ∼64-kDa band is achieved by phosphorylation at Ser^396/404 or Ser²⁰², the mobility shift to a 68-kDa band occurs only in response to phosphorylation at Ser²⁰². We show that in vitro, FTDP-17 missense mutations, by promoting phosphorylation at Ser²⁰², enhance the mobility shift to ∼64- and 68-kDa bands and inhibit the microtubule assembly-promoting activity of tau. Our data suggest that Ser²⁰² phosphorylation is the major event leading to NFT pathology in AD and related tauopathies.     

Cortical Microtubule Arrays Are Initiated from a Nonrandom Prepattern Driven by Atypical Microtubule Initiation

The ordered arrangement of cortical microtubules in growing plant cells is essential for anisotropic cell expansion and, hence, for plant morphogenesis. These arrays are dismantled when the microtubule cytoskeleton is rearranged during mitosis and reassembled following completion of cytokinesis. The reassembly of the cortical array has often been considered as initiating from a state of randomness, from which order arises at least partly through self-organizing mechanisms. However, some studies have shown evidence for ordering at early stages of array assembly. To investigate how cortical arrays are initiated in higher plant cells, we performed live-cell imaging studies of cortical array assembly in tobacco (Nicotiana tabacum) Bright Yellow-2 cells after cytokinesis and drug-induced disassembly. We found that cortical arrays in both cases did not initiate randomly but with a significant overrepresentation of microtubules at diagonal angles with respect to the cell axis, which coincides with the predominant orientation of the microtubules before their disappearance from the cell cortex in preprophase. In Arabidopsis (Arabidopsis thaliana) root cells, recovery from drug-induced disassembly was also nonrandom and correlated with the organization of the previous array, although no diagonal bias was observed in these cells. Surprisingly, during initiation, only about one-half of the new microtubules were nucleated from locations marked by green fluorescent protein-γ-tubulin complex protein2-tagged γ-nucleation complexes (γ-tubulin ring complex), therefore indicating that a large proportion of early polymers was initiated by a noncanonical mechanism not involving γ-tubulin ring complex. Simulation studies indicate that the high rate of noncanonical initiation of new microtubules has the potential to accelerate the rate of array repopulation.Higher plant cells feature ordered arrays of microtubules at the cell cortex (Ledbetter and Porter, 1963) that are essential for cell and tissue morphogenesis, as revealed by disruption of cortical arrays by drugs that cause microtubule depolymerization (Green, 1962) or stabilization (Weerdenburg and Seagull, 1988) and by loss-of-function mutations in a wide variety of microtubule-associated proteins (Baskin, 2001; Whittington et al., 2001; Buschmann and Lloyd, 2008; Lucas et al., 2011). The structure of these arrays is thought to control the pattern of cell growth primarily by its role in the deposition of cellulose microfibrils, the load-bearing component of the cell wall (Somerville, 2006). Functional relations between cortical microtubules and cellulose microfibrils have been proposed since the early sixties, even before cortical microtubules had been visualized (Green, 1962). Recent live-cell imaging studies have confirmed that cortical microtubules indeed guide the movement of cellulose synthase complexes that produce cellulose microfibrils (Paredez et al., 2006) and have shown further that microtubules position the insertion of most cellulose synthase complexes into the plasma membrane (Gutierrez et al., 2009). These activities of ordered cortical microtubules are proposed to facilitate the organization of cell wall structure, creating material properties that underlie cell growth anisotropy.While organization of the interphase cortical array appears to be essential for cell morphogenesis, this organization is disrupted during the cell cycle as microtubules are rearranged to create the preprophase band, spindle, and phragmoplast during mitosis and cytokinesis (for review, see Wasteneys, 2002). Upon completion of cytokinesis, an organized interphase cortical array is regenerated, but the pathway for this reassembly is not well understood.The plant interphase microtubule array is organized and maintained without centrosomes as organizing centers (for review, see Wasteneys, 2002; Bartolini and Gundersen, 2006; Ehrhardt and Shaw, 2006), and microtubule self-organization is proposed to play an important role in cortical microtubule array ordering (Dixit and Cyr, 2004). In electron micrographs, microtubules have been observed to be closely associated with the plasma membrane (Hardham and Gunning, 1978), and live-cell imaging provides evidence for attachment of microtubules to the cell cortex (Shaw et al., 2003; Vos et al., 2004). The close association to the plasma membrane restricts the cortical microtubules to a quasi two-dimensional plane where they interact through polymerization-driven collisions (Shaw et al., 2003; Dixit and Cyr, 2004). Microtubule encounters at shallow angles (<40°) have a high probability of leading to bundling, while microtubule encounters at steeper angles most likely result in induced catastrophes or microtubule crossovers (Dixit and Cyr, 2004). Several computational modeling studies have since shown that these types of interactions between surface-bound dynamical microtubules can indeed explain spontaneous coalignment of microtubules (Allard et al., 2010; Eren et al., 2010; Hawkins et al., 2010; Tindemans et al., 2010).The question of how the orientation of the cortical array is established with respect to the cell axis is less well understood. One possibility is that microtubules are selectively destabilized with respect to cellular coordinates (Ehrhardt and Shaw, 2006). Indeed, recent results from biological observations and modeling suggest that catastrophic collisions induced at the edges between cell faces or heighted catastrophe rates in cell caps could be sufficient to selectively favor microtubules in certain orientation and hence determine the final orientation of the array (Allard et al., 2010; Eren et al., 2010; Ambrose et al., 2011; Dhonukshe et al., 2012).To date, all models of cortical array assembly assume random initial conditions. However, experimental work by Wasteneys and Williamson (1989a, 1989b) in Nitella tasmanica showed that, during array reassembly after drug-induced disruption, microtubules were initially transverse. This was followed by a less ordered phase and later by the acquisition of the final transverse order. A nonrandom initial ordering was also observed in tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells by Kumagai et al. (2001), who concluded that the process of transverse array establishment starts with longitudinal order but did not provide quantitative data for the process of array assembly. The initial conditions for the cortical microtubule array formation are important to consider, as they may strongly influence the speed at which order is established and could even prevent it from being established over a biologically relevant time scale.In this study, we used live-cell imaging to follow and record the whole transition from the cortical microtubule-free state to the final transverse array and used digital tracking algorithms to quantify the microtubule order. Nucleation stands out as a central process to characterize during array initiation. Lacking a central body to organize microtubule nucleations, the higher plant cell has dispersed nucleation complexes (Wasteneys and Williamson, 1989a, 1989b; Chan et al., 2003; Shaw et al., 2003; Murata et al., 2005; Pastuglia et al., 2006; Nakamura et al., 2010). Therefore, we performed high time resolution observations to quantify nucleation complex recruitment, nucleation rates, and microtubule nucleation angles. We found evidence for a highly nonrandom initial ordering state that features diagonal microtubule orientation and an atypical microtubule initiation mechanism. Simulation analysis indicates that these atypical nucleations have the potential to accelerate the recovery of cortical array density.     

Dynein at the nuclear envelope

Most cellular organelles are positioned through active transport by motor proteins. The authors discuss the evidence that dynein has important cell cycle-regulated functions in this context at the nuclear envelope.Most cellular organelles are positioned through active transport by motor proteins. This is especially important during cell division, a time when the organelles and genetic content need to be divided equally between the two daughter cells. Although individual proteins can attain their correct location by diffusion, larger structures are usually positioned through active transport by motor proteins. The main motor that transports cargoes to the minus ends of the microtubules is dynein. In nondividing cells, dynein probably transports or positions the nucleus inside the cells by binding to the nuclear envelope (NE; Burke & Roux, 2009). However, it appears that dynein also has important cell-cycle-regulated functions at the NE, as it is recruited to the NE every cell cycle just before cells enter mitosis (Salina et al, 2002; Splinter et al, 2010). Here, we discuss why dynein might be recruited to the NE for a brief period before mitosis.During late G2 or prophase the centrosomes separate to opposite sides of the nucleus, but remain closely associated with the NE during separation. This close association is probably mediated through NE-bound dynein, which ‘walks'' towards the minus ends of centrosomal microtubules, thereby pulling centrosomes towards the NE (Splinter et al, 2010; Gonczy et al, 1999; Robinson et al, 1999). We speculate that close association of centrosomes to the NE might have several functions. First, if centrosomes are not mechanically coupled to the NE, centrosome movement during separation will occur in random directions and chromosomes will not end up between the two separated centrosomes. In this scenario, individual kinetochores might attach more frequently to microtubules coming from both centrosomes (merotelic attachments), a defect that can result in aneuploidy, a characteristic of cancer. Second, centrosome-nuclear attachment also keeps centrosomes in close proximity to chromosomes, which might facilitate rapid capture of chromosomes by microtubules nucleated by the centrosomes after NE breakdown. This might not be absolutely essential, as chromosome alignment can occur in the absence of centrosomes. However, the spatial proximity of centrosomes and chromosomes at NE breakdown might improve the fidelity of kinetochore capture and chromosome alignment.In addition, dynein has also been suggested to promote centrosome separation in prophase in some systems (Gonczy et al, 1999; Robinson et al, 1999; Vaisberg et al, 1993), although not in others (Tanenbaum et al, 2008). Perhaps dynein, anchored at the NE just before mitosis, could exert force on microtubules emanating from both centrosomes, thereby pulling centrosomes apart. However, this force could also be produced by cortical dynein and specific inhibition of NE-associated or cortical dynein will be required to test which pool is responsible.Dynein has also been implicated in the process of NE breakdown itself, by promoting mechanical shearing of the NE. Two elegant studies showed that microtubules can tear the NE as cells enter mitosis (Salina et al, 2002; Beaudouin et al, 2002). One possibility is that microtubules growing into the NE mechanically disrupt it. Alternatively, NE-associated dynein might ‘walk'' along centrosomal microtubules and thereby pull on the NE, tearing it apart. However, testing the exact role of dynein in NE breakdown is complicated by the fact that centrosomes detach from the NE on inactivation of dynein and centrosomal microtubules stop growing efficiently into the NE. Thus, selective inhibition of dynein function will also be required to test this idea.Specific recruitment of dynein to the NE just before mitosis clearly suggests a role for dynein at the NE in preparing cells for mitosis. A major role of NE-associated dynein is to maintain close association of centrosomes with the NE during centrosome separation, which might be needed for efficient capture and alignment of chromosomes after NE breakdown, but additionally, NE-associated dynein could facilitate breakdown and contribute to centrosome separation in some systems.     

The Human Tau Interactome: Binding to the Ribonucleoproteome,and Impaired Binding of the Proline-to-Leucine Mutant at Position 301 (P301L) to Chaperones and the Proteasome

The tau protein is central to the etiology of several neurodegenerative diseases, including Alzheimer''s disease, a subset of frontotemporal dementias, progressive supranuclear palsy and dementia following traumatic brain injury, yet the proteins it interacts with have not been studied using a systematic discovery approach. Here we employed mild in vivo crosslinking, isobaric labeling, and tandem mass spectrometry to characterize molecular interactions of human tau in a neuroblastoma cell model. The study revealed a robust association of tau with the ribonucleoproteome, including major protein complexes involved in RNA processing and translation, and documented binding of tau to several heat shock proteins, the proteasome and microtubule-associated proteins. Follow-up experiments determined the relative contribution of cellular RNA to the tau interactome and mapped interactions to N- or C-terminal tau domains. We further document that expression of P301L mutant tau disrupts interactions of the C-terminal half of tau with heat shock proteins and the proteasome. The data are consistent with a model whereby a higher propensity of P301L mutant tau to aggregate may reflect a perturbation of its chaperone-assisted stabilization and proteasome-dependent degradation. Finally, using a global proteomics approach, we show that heterologous expression of a tau construct that lacks the C-terminal domain, including the microtubule binding domain, does not cause a discernible shift of the proteome except for a significant direct correlation of steady-state levels of tau and cystatin B.The tau protein is a member of the family of microtubule-associated proteins (MAPs)¹ that in humans is coded by the MAPT gene on chromosome 17q21.31 (1). Initially, described as a factor that binds to and stabilizes microtubules (MTs) (2), interest in the tau protein grew when it was shown to represent the main constituent of intracellular protein aggregates, termed neurofibrillary tangles (NFTs), observed in Alzheimer''s disease (3, 4). Similar tau aggregates have since been described in other, less common dementias, including progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick''s disease and dementia pugilistica, a form of dementia observed in athletes who had been exposed to repeated traumatic brain injury (5).Despite its early recognition as a MT binding molecule, the physiological function of the tau protein is still being debated (6). At least, in part, this uncertainty is born from the observation that tau knockout mice are rather nonconspicuous in their phenotype (7, 8). Ongoing attempts to define additional roles for this protein have, over the years, generated several hypotheses, including that the tau protein modulates neurite outgrowth and axonogenesis (8, 9), bridges the microtubule and actin cytoskeletons (10), and acts as a scaffold for tethering the Src family tyrosine kinase Fyn to PSD-95/NMDA receptor complexes (11). The predominant expression of tau in neuronal axons suggests a role in brain function. Significantly, in all tauopathies, a group term used to describe dementias with pathological tau protein involvement, the tau protein is observed to detach from microtubules and to form aggregates. There also is compelling evidence from a body of work with transgenic models that the cellular toxicity observed in the aforementioned dementias relies on the presence of the tau protein (12). Consequently, it seems plausible that the cellular toxicity observed in AD and other dementias does not relate to a loss of function of the tau protein but represents a gain of toxic function the protein exhibits in its microtubule-detached form.The tau molecule can be crudely subdivided into an amino-terminal projection domain (PD), a microtubule-binding domain (MTB), and a carboxy-terminal domain (C'') (13). The protein has long been known to exhibit some remarkable biochemical characteristics, including an ability to withstand harsh acid and heat treatments that would cause a majority of other proteins to precipitate (2, 14). These characteristics have been attributed to tau being natively unfolded and possessing a highly dynamic character (15). The tau protein is also known to be a substrate for several post-translational modifications (PTMs), and the list of tau modifying enzymes that have been described is long. In particular, tau phosphorylation has been recognized to occur in vivo and in disease, and tau hyperphosphorylation at sites within the MTB domain and at nearby sites flanking the MTB has been shown to promote detachment of tau from microtubules (16). There further is broad agreement in the field that levels of several other tau PTMs are raised in tauopathies, including nitration (17), ubiquitination (18), sumoylation (19), and truncation (20, 21). Less agreement exists on the degree to which specific PTMs contribute to disease manifestation in individual tauopathies (22). Lacking also are insights into other physiological protein interactions the tau protein engages in and, surprisingly, to our knowledge, no systematic screen for tau binders has been reported. Thus, except for its well-established binding to microtubules (2), members of the Src family of protein kinases (23, 24), Hsp70 (25)/Hsp90 (26, 27), and reports on its interaction with F-actin (28), ApoE3 (29), a subset of peptidyl-prolyl cis-trans isomerases (30, 31), α-synuclein (32), PACSIN1 (33), and negatively charged polymers, including nucleic acids (34, 35), relatively little is known about other nonenzymatic interactions the protein engages in.In an attempt to address this unmet need, we set out to define molecular interactors of the tau protein in the human neuroblastoma SH-SY5Y cell model. The study made use of advanced instrumentation and workflows that included comparative mass spectrometry based on isobaric tags. We observed that the tau interactome is dramatically enriched in cellular components involved in the regulation and execution of RNA-processing and translation. We document that the previously known ability of the tau protein to bind to nucleic acids is partially responsible but not sufficient by itself to explain this binding propensity of the tau protein. We narrowed down the binding preference of individual binders to N- and C-terminal domains within tau and document that several interactors, including 14-3-3 proteins, heat shock proteins, and the proteasome, exhibit a strong binding preference for the C terminus of tau. When comparing the interactomes of wild-type and mutant tau (P301L) linked to frontotemporal dementia, we observed that interactions with the aforementioned C-terminal tau binding partners are diminished for mutant tau. Despite the strong binding of tau to the ribonucleoproteome, its overexpression does not seem to affect the global steady-state levels of cellular proteins, and only the levels of cystatin B, a natural inhibitor of cysteine proteases, were modified and correlated directly with the levels of heterologously expressed tau.     

Hyperglutamylation of Tubulin Can either Stabilize or Destabilize Microtubules in the Same Cell

In most eukaryotic cells, tubulin is subjected to posttranslational glutamylation, a conserved modification of unclear function. The glutamyl side chains form as branches of the primary sequence glutamic acids in two biochemically distinct steps: initiation and elongation. The length of the glutamyl side chain is spatially controlled and microtubule type specific. Here, we probe the significance of the glutamyl side chain length regulation in vivo by overexpressing a potent side chain elongase enzyme, Ttll6Ap, in Tetrahymena. Overexpression of Ttll6Ap caused hyperelongation of glutamyl side chains on the tubulin of axonemal, cortical, and cytoplasmic microtubules. Strikingly, in the same cell, hyperelongation of glutamyl side chains stabilized cytoplasmic microtubules and destabilized axonemal microtubules. Our observations suggest that the cellular outcomes of glutamylation are mediated by spatially restricted tubulin interactors of diverse nature.Microtubules are dynamic elements of the cytoskeleton that are assembled from heterodimers of α- and β-tubulin. Once assembled, tubulin subunits undergo several conserved posttranslational modifications (PTMs) that diversify the external and luminal surfaces of microtubules (51). Two tubulin PTMs, glycylation and glutamylation, collectively known as polymodifications, form peptide side chains that are attached to the γ-carboxyl groups of glutamic acids in the primary sequence of the C-terminal tails (CTTs) of α- and β-tubulin (14, 36). Glutamylated microtubules are abundant in projections of neurons (14), axonemes (8, 15, 17), and centrioles/basal bodies (5, 31) and are detectable in the mitotic spindle and on a subset of cytoplasmic network microtubules (1, 5). The modifying enzymes, tubulin glutamic acid ligases (tubulin E-ligases), belong to the family of proteins related to the tubulin tyrosine ligase (TTL), known as TTL-like (TTLL) proteins (22, 50, 53). Tubulin glutamylation appears to be important in vivo. A knockdown of the TTLL7 E-ligase mRNA in cultured neurons inhibits the outgrowth of neurites (20). A loss of PGs1, a protein associated with TTLL1 E-ligase (22, 37), disorganizes sperm axonemes in the mouse (11), and a morpholino knockdown of TTLL6 E-ligase expression in zebrafish inhibits the assembly of olfactory cilia (33). The biochemical consequences of tubulin glutamylation in vivo are poorly understood, but the emerging model is that this PTM regulates interactions between microtubules and microtubule-associated proteins (MAPs) (6, 7, 19, 27).The ciliate Tetrahymena thermophila has 18 types of diverse microtubules that are all assembled in a single cell. Although most, if not all, of these microtubules are glutamylated, the length of glutamyl side chains is spatially regulated (8, 53). Minimal side chains composed of a single glutamic acid (monoglutamylation) are present on the cytoplasmic and nuclear microtubules, whereas elongated side chains are present on the basal bodies and axonemes (53). In Tetrahymena, Ttll6Ap is a β-tubulin-preferring E-ligase (22), with a strong if not exclusive, side chain elongating activity (50). Here, by overproducing Ttll6Ap in vivo, we explore the consequences of glutamyl side chain hyper-elongation. Unexpectedly, we show that in the same cells, hyperelongation of glutamyl side chains stabilizes cell body and destabilizes axonemal microtubules. The simplest explanation of these data is that, in vivo, the cellular outcomes of tubulin glutamylation are mediated by diverse microtubule type-specific MAPs. To our knowledge, we are first to report that excessive tubulin glutamylation can either stabilize or destabilize microtubules in the same cell.     

Regulation of Microtubule Dynamic Instability in Vitro by Differentially Phosphorylated Stathmin

Stathmin is an important regulator of microtubule polymerization and dynamics. When unphosphorylated it destabilizes microtubules in two ways, by reducing the microtubule polymer mass through sequestration of soluble tubulin into an assembly-incompetent T2S complex (two α:β tubulin dimers per molecule of stathmin), and by increasing the switching frequency (catastrophe frequency) from growth to shortening at plus and minus ends by binding directly to the microtubules. Phosphorylation of stathmin on one or more of its four serine residues (Ser¹⁶, Ser²⁵, Ser³⁸, and Ser⁶³) reduces its microtubule-destabilizing activity. However, the effects of phosphorylation of the individual serine residues of stathmin on microtubule dynamic instability have not been investigated systematically. Here we analyzed the effects of stathmin singly phosphorylated at Ser¹⁶ or Ser⁶³, and doubly phosphorylated at Ser²⁵ and Ser³⁸, on its ability to modulate microtubule dynamic instability at steady-state in vitro. Phosphorylation at either Ser¹⁶ or Ser⁶³ strongly reduced or abolished the ability of stathmin to bind to and sequester soluble tubulin and its ability to act as a catastrophe factor by directly binding to the microtubules. In contrast, double phosphorylation of Ser²⁵ and Ser³⁸ did not affect the binding of stathmin to tubulin or microtubules or its catastrophe-promoting activity. Our results indicate that the effects of stathmin on dynamic instability are strongly but differently attenuated by phosphorylation at Ser¹⁶ and Ser⁶³ and support the hypothesis that selective targeting by Ser¹⁶-specific or Ser⁶³-specific kinases provides complimentary mechanisms for regulating microtubule function.Stathmin is an 18-kDa ubiquitously expressed microtubule-destabilizing phosphoprotein whose activity is modulated by phosphorylation of its four serine residues, Ser¹⁶, Ser²⁵, Ser³⁸, and Ser⁶³ (1–7). Several classes of kinases have been identified that phosphorylate stathmin, including kinases associated with cell growth and differentiation such as members of the mitogen-activated protein kinase (MAPK)2 family, cAMP-dependent protein kinase (1–5, 8–11), and kinases associated with cell cycle regulation such as cyclin-dependent kinase 1 (3, 12–14). Phosphorylation of stathmin is required for cell cycle progression through mitosis and for proper assembly/function of the mitotic spindle (3, 13–16). Inhibition of stathmin phosphorylation produces strong mitotic phenotypes characterized by disassembly and disorganization of mitotic spindles and abnormal chromosome distributions (3, 13–14).Stathmin is known to destabilize microtubules in two ways. One is by binding to soluble tubulin and forming a stable complex that cannot polymerize into microtubules, consisting of one molecule of stathmin and two molecules of tubulin (T2S complex) (17–24). Addition of stathmin to microtubules in equilibrium with soluble tubulin results in sequestration of the tubulin and a reduction in the level of microtubule polymer (17–18, 22, 25–28). In addition to reducing the amount of assembled polymer, tubulin sequestration by stathmin has been shown to increase the switching frequency at microtubule plus ends from growth to shortening (called the catastrophe frequency) as the microtubules relax to a new steady state (17, 29). The second way is by binding directly to microtubules (27–30). The direct binding of stathmin to microtubules increases the catastrophe frequency at both ends of the microtubules and considerably more strongly at minus ends than at plus ends (27). Consistent with its strong catastrophe-promoting activity at minus ends, stathmin increases the treadmilling rate of steady-state microtubules in vitro (27). These results have led to the suggestion that stathmin might be an important cellular regulator of minus-end microtubule dynamics (27).Phosphorylation of stathmin diminishes its ability to regulate microtubule polymerization (3, 14, 25–26). Phosphorylation of Ser¹⁶ or Ser⁶³ appears to be more critical than phosphorylation of Ser²⁵ and Ser³⁸ for the ability of stathmin to bind to soluble tubulin and to inhibit microtubule assembly in vitro (3, 25). Inhibition of stathmin phosphorylation induces defects in spindle assembly and organization (3, 14) suggesting that not only soluble tubulin-microtubule levels are regulated by phosphorylation of stathmin, but the dynamics of microtubules could also be regulated in a phosphorylation-dependent manner.It is not known how phosphorylation at any of the four serine residues of stathmin affects its ability to regulate microtubule dynamics and, specifically, its ability to increase the catastrophe frequency at plus and minus ends due to its direct interaction with microtubules. Thus, we determined the effects of stathmin individually phosphorylated at either Ser¹⁶ or Ser⁶³ and doubly phosphorylated at both Ser²⁵ and Ser³⁸ on dynamic instability at plus and minus ends in vitro at microtubule polymer steady state and physiological pH (pH 7.2). We find that phosphorylation of Ser¹⁶ strongly reduces the direct catastrophe-promoting activity of stathmin at plus ends and abolishes it at minus ends, whereas phosphorylation of Ser⁶³ abolishes the activity at both ends. The effects of phosphorylation of individual serines correlated well with stathmin''s reduced abilities to form stable T2S complexes, to inhibit microtubule polymerization, and to bind to microtubules. In contrast, double phosphorylation of Ser²⁵ and Ser³⁸ did not alter the ability of stathmin to modulate dynamic instability at the microtubule ends, its ability to form a stable T2S complex, or its ability to bind to microtubules. The data further support the hypotheses that phosphorylation of stathmin on either Ser¹⁶ or Ser⁶³ plays a critical role in regulating microtubule polymerization and dynamics in cells.     

Loss of Hsp110 Leads to Age-Dependent Tau Hyperphosphorylation and Early Accumulation of Insoluble Amyloid β

Accumulation of tau into neurofibrillary tangles is a pathological consequence of Alzheimer''s disease and other tauopathies. Failures of the quality control mechanisms by the heat shock proteins (Hsps) positively correlate with the appearance of such neurodegenerative diseases. However, in vivo genetic evidence for the roles of Hsps in neurodegeneration remains elusive. Hsp110 is a nucleotide exchange factor for Hsp70, and direct substrate binding to Hsp110 may facilitate substrate folding. Hsp70 complexes have been implicated in tau phosphorylation state and amyloid precursor protein (APP) processing. To provide evidence for a role for Hsp110 in central nervous system homeostasis, we have generated hsp110^−/− mice. Our results show that hsp110^−/− mice exhibit accumulation of hyperphosphorylated-tau (p-tau) and neurodegeneration. We also demonstrate that Hsp110 is in complexes with tau, other molecular chaperones, and protein phosphatase 2A (PP2A). Surprisingly, high levels of PP2A remain bound to tau but with significantly reduced activity in brain extracts from aged hsp110^−/− mice compared to brain extracts from wild-type mice. Mice deficient in the Hsp110 partner (Hsp70) also exhibit a phenotype comparable to that of hsp110^−/− mice, confirming a critical role for Hsp110-Hsp70 in maintaining tau in its unphosphorylated form during aging. In addition, crossing hsp110^−/− mice with mice overexpressing mutant APP (APPβsw) leads to selective appearance of insoluble amyloid β42 (Aβ42), suggesting an essential role for Hsp110 in APP processing and Aβ generation. Thus, our findings provide in vivo evidence that Hsp110 plays a critical function in tau phosphorylation state through maintenance of efficient PP2A activity, confirming its role in pathogenesis of Alzheimer''s disease and other tauopathies.Diseases like Alzheimer''s disease (AD) and other tauopathies are defined by the expression of neurofibrillary tangles (NFTs) deposited mainly in neurons. The NFTs are aggregates of the hyperphosphorylated tau (p-tau) (3, 74). Normal tau increases microtubule stability, but tau can be hyperphosphorylated under disease conditions and released from microtubules (3, 5, 6). The molecular mechanisms involved in the formation of NFTs are not completely understood. However, accumulation of abnormal p-tau and NFTs causes neurodegeneration (3). A number of protein kinases, including glycogen synthase kinase 3 (GSK3) and cyclin-dependent protein kinase 5 (CDK5), have been shown to phosphorylate tau at Thr231 and Ser262 as well as several other sites that flank the microtubule binding repeat, leading to tangles of paired helical filaments (PHFs) similar to those observed in the brains of patients with AD (54, 72). Evidence shows that GSK3 physically interacts with tau and is thought to be the main contributor to the formation of NFTs and amyloid β (Aβ) plaques in AD patients (18, 53, 54). Phosphorylation of GSK3a/b at S9/S21 which is inhibitory to its activity during insulin signaling, leads to phosphorylation of tau in neurons (80). GSK3a/b phospho-S9/S21, p-tau, and 14-3-3zeta have been isolated in a 500-kDa complex, and the interaction has been shown to result in tau phosphorylation by GSK3 (1, 80). Although not well characterized, p-tau has been shown to be dephosphorylated by the B family regulatory subunit of the heterotrimeric PP2A holoenzyme (76). There are two protein phosphatase 2A (PP2A) binding sites on microtubule tau binding repeats, perhaps allowing tau to be more efficiently dephosphorylated by PP2A catalytic subunit (76).Both GSK3 and CDK5 are also known to be involved in the phosphorylation of amyloid precursor protein (APP) at Thr668 and APP processing and Aβ production (53, 58). Studies suggest that amyloid peptide can activate GSK3 signaling, and the increase in GSK3 activity can then contribute to abnormal APP processing. Indeed, reduction in GSK3 activity reduces amyloid peptide production in murine AD models (18, 53, 57, 71). Reduction in PP2A activity leads to altered APP regulation as well (26, 43). Additional molecules that affect tau hyperphosphorylation and APP processing are the peptidyl prolyl isomerases (9, 36, 51). Deletion of Pin1 isomerase in vivo leads to p-tau and neurodegeneration (42). Crossing Pin1-deficient mice with transgenic mice expressing mutant APP (APPβsw) leads to abnormal APP processing and accumulation of toxic amyloid β42 (Aβ42) species. Pin1, therefore, is implicated in isomerization of tau, perhaps facilitating its dephosphorylation (42). The presence of Pin1 has been implicated in promoting nonamyloidogenic processing of APP and reduction in toxic Aβ42 production (51).Hsp70/Hsc70 has been shown to preferentially bind to a hyperphosphorylated form of tau in the diseased human brain (49). Cross talk between the ubiquitin proteasome system (UPS) and molecular chaperones might also be critical in regulating the deposition and toxicity of tau (8, 16). These results suggest that the activity of Hsp70 and Hsp90 preserve the native structure and function of tau protein. Hsp70 and the C-terminal Hsp70-interacting protein (Chip) have been shown to regulate tau ubiquitination and degradation (11, 12, 21, 52, 65). Interestingly, Chip and βAPP interact, and Chip and Hsp70/90 expression have been shown to lower the cellular levels of Aβ and reduce Aβ toxicity in vitro (39). Misfolded proteins are either degraded through the UPS or are folded, at least in part, by the Hsps (4, 7).Eukaryotic cells possess a class of heat shock proteins (Hsps) related to the Hsp70 family. This Hsp100 family of proteins contains Hspa41 (Apg1 or OSP94), Hsp94 (Apg2), and Hsp110 (2, 17, 28, 61, 70, 77, 78). They were initially considered to be “holdases” that keep denatured proteins in solution, and no client proteins have been described for them (14, 15, 56, 62). Hsp110 interacts with Hsp70 and increases its ATPase activity (15, 56, 62). The main function of Hsp110 appears to be a nucleotide exchange factor (NEF) for Hsp70 (14, 64). In general, Hsp110 is known to induce suppression of aggregation and protein refolding, and it protects proteins from the damaging effects of various stresses; however, its physiological function in mammalian cells remains unknown (15, 60). In these studies, we examined the role of Hsp110 in central nervous system (CNS) homeostasis in vivo. We have found that hsp110^−/− mice exhibit an age-dependent accumulation of p-tau that is associated with pathological features, such as the appearance of NFTs and neurodegeneration. We also show that lack of Hsp110 leads to accelerated pathology as evidenced by the early appearance of senile plaques containing Aβ42 (a major toxic species [46]) in an AD transgenic mouse model. At the biochemical level, we show that Hsp110 interacts with tau, a number of Hsps, GSK3, Pin1, and PP2A. Furthermore, tau immunocomplexes pulled down from hsp110^−/− brain extracts possess elevated levels of PP2A, but the pulled-down PP2A has significantly lower activity than the PP2A from wild-type mice. Our studies therefore suggest a critical role for Hsp110 in maintaining the proper folding environment that is required for phosphorylation and dephosphorylation of tau and APP processing in vivo.     

Erythropoietin Receptor (EpoR) Agonism Is Used to Treat a Wide Range of Disease

The erythropoietin receptor (EpoR) was discovered and described in red blood cells (RBCs), stimulating its proliferation and survival. The target in humans for EpoR agonists drugs appears clear—to treat anemia. However, there is evidence of the pleitropic actions of erythropoietin (Epo). For that reason, rhEpo therapy was suggested as a reliable approach for treating a broad range of pathologies, including heart and cardiovascular diseases, neurodegenerative disorders (Parkinson’s and Alzheimer’s disease), spinal cord injury, stroke, diabetic retinopathy and rare diseases (Friedreich ataxia). Unfortunately, the side effects of rhEpo are also evident. A new generation of nonhematopoietic EpoR agonists drugs (asialoEpo, Cepo and ARA 290) have been investigated and further developed. These EpoR agonists, without the erythropoietic activity of Epo, while preserving its tissue-protective properties, will provide better outcomes in ongoing clinical trials. Nonhematopoietic EpoR agonists represent safer and more effective surrogates for the treatment of several diseases such as brain and peripheral nerve injury, diabetic complications, renal ischemia, rare diseases, myocardial infarction, chronic heart disease and others.In principle, the erythropoietin receptor (EpoR) was discovered and described in red blood cell (RBC) progenitors, stimulating its proliferation and survival. Erythropoietin (Epo) is mainly synthesized in fetal liver and adult kidneys (1–3). Therefore, it was hypothesized that Epo act exclusively on erythroid progenitor cells. Accordingly, the target in humans for EpoR agonists drugs (such as recombinant erythropoietin [rhEpo], in general, called erythropoiesis-simulating agents) appears clear (that is, to treat anemia). However, evidence of a kaleidoscope of pleitropic actions of Epo has been provided (4,5). The Epo/EpoR axis research involved an initial journey from laboratory basic research to clinical therapeutics. However, as a consequence of clinical observations, basic research on Epo/EpoR comes back to expand its clinical therapeutic applicability.Although kidney and liver have long been considered the major sources of synthesis, Epo mRNA expression has also been detected in the brain (neurons and glial cells), lung, heart, bone marrow, spleen, hair follicles, reproductive tract and osteoblasts (6–17). Accordingly, EpoR was detected in other cells, such as neurons, astrocytes, microglia, immune cells, cancer cell lines, endothelial cells, bone marrow stromal cells and cells of heart, reproductive system, gastrointestinal tract, kidney, pancreas and skeletal muscle (18–27). Conversely, Sinclair et al.(28) reported data questioning the presence or function of EpoR on nonhematopoietic cells (endothelial, neuronal and cardiac cells), suggesting that further studies are needed to confirm the diversity of EpoR. Elliott et al.(29) also showed that EpoR is virtually undetectable in human renal cells and other tissues with no detectable EpoR on cell surfaces. These results have raised doubts about the preclinical basis for studies exploring pleiotropic actions of rhEpo (30).For the above-mentioned data, a return to basic research studies has become necessary, and many studies in animal models have been initiated or have already been performed. The effect of rhEpo administration on angiogenesis, myogenesis, shift in muscle fiber types and oxidative enzyme activities in skeletal muscle (4,31), cardiac muscle mitochondrial biogenesis (32), cognitive effects (31), antiapoptotic and antiinflammatory actions (33–37) and plasma glucose concentrations (38) has been extensively studied. Neuro- and cardioprotection properties have been mainly described. Accordingly, rhEpo therapy was suggested as a reliable approach for treating a broad range of pathologies, including heart and cardiovascular diseases, neurodegenerative disorders (Parkinson’s and Alzheimer’s disease), spinal cord injury, stroke, diabetic retinopathy and rare diseases (Friedreich ataxia).Unfortunately, the side effects of rhEpo are also evident. Epo is involved in regulating tumor angiogenesis (39) and probably in the survival and growth of tumor cells (25,40,41). rhEpo administration also induces serious side effects such as hypertension, polycythemia, myocardial infarction, stroke and seizures, platelet activation and increased thromboembolic risk, and immunogenicity (42–46), with the most common being hypertension (47,48). A new generation of nonhematopoietic EpoR agonists drugs have hence been investigated and further developed in animals models. These compounds, namely asialoerythropoietin (asialoEpo) and carbamylated Epo (Cepo), were developed for preserving tissue-protective properties but reducing the erythropoietic activity of native Epo (49,50). These drugs will provide better outcome in ongoing clinical trials. The advantage of using nonhematopoietic Epo analogs is to avoid the stimulation of hematopoiesis and thereby the prevention of an increased hematocrit with a subsequent procoagulant status or increased blood pressure. In this regard, a new study by van Rijt et al. has shed new light on this topic (51). A new nonhematopoietic EpoR agonist analog named ARA 290 has been developed, promising cytoprotective capacities to prevent renal ischemia/reperfusion injury (51). ARA 290 is a short peptide that has shown no safety concerns in preclinical and human studies. In addition, ARA 290 has proven efficacious in cardiac disorders (52,53), neuropathic pain (54) and sarcoidosis-induced chronic neuropathic pain (55). Thus, ARA 290 is a novel nonhematopoietic EpoR agonist with promising therapeutic options in treating a wide range of pathologies and without increased risks of cardiovascular events.Overall, this new generation of EpoR agonists without the erythropoietic activity of Epo while preserving tissue-protective properties of Epo will provide better outcomes in ongoing clinical trials (49,50). Nonhematopoietic EpoR agonists represent safer and more effective surrogates for the treatment of several diseases, such as brain and peripheral nerve injury, diabetic complications, renal ischemia, rare diseases, myocardial infarction, chronic heart disease and others.     

Risk of vascular events in patients with polymyalgia rheumatica

Background:

Polymyalgia rheumatica is one of the most common inflammatory rheumatologic conditions in older adults. Other inflammatory rheumatologic disorders are associated with an excess risk of vascular disease. We investigated whether polymyalgia rheumatica is associated with an increased risk of vascular events.

Methods:

We used the General Practice Research Database to identify patients with a diagnosis of incident polymyalgia rheumatica between Jan. 1, 1987, and Dec. 31, 1999. Patients were matched by age, sex and practice with up to 5 patients without polymyalgia rheumatica. Patients were followed until their first vascular event (cardiovascular, cerebrovascular, peripheral vascular) or the end of available records (May 2011). All participants were free of vascular disease before the diagnosis of polymyalgia rheumatica (or matched date). We used Cox regression models to compare time to first vascular event in patients with and without polymyalgia rheumatica.

Results:

A total of 3249 patients with polymyalgia rheumatica and 12 735 patients without were included in the final sample. Over a median follow-up period of 7.8 (interquartile range 3.3–12.4) years, the rate of vascular events was higher among patients with polymyalgia rheumatica than among those without (36.1 v. 12.2 per 1000 person-years; adjusted hazard ratio 2.6, 95% confidence interval 2.4–2.9). The increased risk of a vascular event was similar for each vascular disease end point. The magnitude of risk was higher in early disease and in patients younger than 60 years at diagnosis.

Interpretation:

Patients with polymyalgia rheumatica have an increased risk of vascular events. This risk is greatest in the youngest age groups. As with other forms of inflammatory arthritis, patients with polymyalgia rheumatica should have their vascular risk factors identified and actively managed to reduce this excess risk.Inflammatory rheumatologic disorders such as rheumatoid arthritis,^1,2 systemic lupus erythematosus,^2,3 gout,⁴ psoriatic arthritis^2,5 and ankylosing spondylitis^2,6 are associated with an increased risk of vascular disease, especially cardiovascular disease, leading to substantial morbidity and premature death.^2–6 Recognition of this excess vascular risk has led to management guidelines advocating screening for and management of vascular risk factors.^7–9Polymyalgia rheumatica is one of the most common inflammatory rheumatologic conditions in older adults,¹⁰ with a lifetime risk of 2.4% for women and 1.7% for men.¹¹ To date, evidence regarding the risk of vascular disease in patients with polymyalgia rheumatica is unclear. There are a number of biologically plausible mechanisms between polymyalgia rheumatica and vascular disease. These include the inflammatory burden of the disease,^12,13 the association of the disease with giant cell arteritis (causing an inflammatory vasculopathy, which may lead to subclinical arteritis, stenosis or aneurysms),¹⁴ and the adverse effects of long-term corticosteroid treatment (e.g., diabetes, hypertension and dyslipidemia).^15,16 Paradoxically, however, use of corticosteroids in patients with polymyalgia rheumatica may actually decrease vascular risk by controlling inflammation.¹⁷ A recent systematic review concluded that although some evidence exists to support an association between vascular disease and polymyalgia rheumatica,¹⁸ the existing literature presents conflicting results, with some studies reporting an excess risk of vascular disease^19,20 and vascular death,^21,22 and others reporting no association.^23–26 Most current studies are limited by poor methodologic quality and small samples, and are based on secondary care cohorts, who may have more severe disease, yet most patients with polymyalgia rheumatica receive treatment exclusively in primary care.²⁷The General Practice Research Database (GPRD), based in the United Kingdom, is a large electronic system for primary care records. It has been used as a data source for previous studies,²⁸ including studies on the association of inflammatory conditions with vascular disease²⁹ and on the epidemiology of polymyalgia rheumatica in the UK.³⁰ The aim of the current study was to examine the association between polymyalgia rheumatica and vascular disease in a primary care population.     

Effect of ambient air pollution on the incidence of appendicitis

Background

The pathogenesis of appendicitis is unclear. We evaluated whether exposure to air pollution was associated with an increased incidence of appendicitis.

Methods

We identified 5191 adults who had been admitted to hospital with appendicitis between Apr. 1, 1999, and Dec. 31, 2006. The air pollutants studied were ozone, nitrogen dioxide, sulfur dioxide, carbon monoxide, and suspended particulate matter of less than 10 μ and less than 2.5 μ in diameter. We estimated the odds of appendicitis relative to short-term increases in concentrations of selected pollutants, alone and in combination, after controlling for temperature and relative humidity as well as the effects of age, sex and season.

Results

An increase in the interquartile range of the 5-day average of ozone was associated with appendicitis (odds ratio [OR] 1.14, 95% confidence interval [CI] 1.03–1.25). In summer (July–August), the effects were most pronounced for ozone (OR 1.32, 95% CI 1.10–1.57), sulfur dioxide (OR 1.30, 95% CI 1.03–1.63), nitrogen dioxide (OR 1.76, 95% CI 1.20–2.58), carbon monoxide (OR 1.35, 95% CI 1.01–1.80) and particulate matter less than 10 μ in diameter (OR 1.20, 95% CI 1.05–1.38). We observed a significant effect of the air pollutants in the summer months among men but not among women (e.g., OR for increase in the 5-day average of nitrogen dioxide 2.05, 95% CI 1.21–3.47, among men and 1.48, 95% CI 0.85–2.59, among women). The double-pollutant model of exposure to ozone and nitrogen dioxide in the summer months was associated with attenuation of the effects of ozone (OR 1.22, 95% CI 1.01–1.48) and nitrogen dioxide (OR 1.48, 95% CI 0.97–2.24).

Interpretation

Our findings suggest that some cases of appendicitis may be triggered by short-term exposure to air pollution. If these findings are confirmed, measures to improve air quality may help to decrease rates of appendicitis.Appendicitis was introduced into the medical vernacular in 1886.¹ Since then, the prevailing theory of its pathogenesis implicated an obstruction of the appendiceal orifice by a fecalith or lymphoid hyperplasia.² However, this notion does not completely account for variations in incidence observed by age,^3,4 sex,^3,4 ethnic background,^3,4 family history,⁵ temporal–spatial clustering⁶ and seasonality,^3,4 nor does it completely explain the trends in incidence of appendicitis in developed and developing nations.^3,7,8The incidence of appendicitis increased dramatically in industrialized nations in the 19th century and in the early part of the 20th century.¹ Without explanation, it decreased in the middle and latter part of the 20th century.³ The decrease coincided with legislation to improve air quality. For example, after the United States Clean Air Act was passed in 1970,⁹ the incidence of appendicitis decreased by 14.6% from 1970 to 1984.³ Likewise, a 36% drop in incidence was reported in the United Kingdom between 1975 and 1994¹⁰ after legislation was passed in 1956 and 1968 to improve air quality and in the 1970s to control industrial sources of air pollution. Furthermore, appendicitis is less common in developing nations; however, as these countries become more industrialized, the incidence of appendicitis has been increasing.⁷Air pollution is known to be a risk factor for multiple conditions, to exacerbate disease states and to increase all-cause mortality.¹¹ It has a direct effect on pulmonary diseases such as asthma¹¹ and on nonpulmonary diseases including myocardial infarction, stroke and cancer.^11–13 Inflammation induced by exposure to air pollution contributes to some adverse health effects.^14–17 Similar to the effects of air pollution, a proinflammatory response has been associated with appendicitis.^18–20We conducted a case–crossover study involving a population-based cohort of patients admitted to hospital with appendicitis to determine whether short-term increases in concentrations of selected air pollutants were associated with hospital admission because of appendicitis.     

Effectiveness of interventions designed to reduce the use of imaging for low-back pain: a systematic review

Background:Rates of imaging for low-back pain are high and are associated with increased health care costs and radiation exposure as well as potentially poorer patient outcomes. We conducted a systematic review to investigate the effectiveness of interventions aimed at reducing the use of imaging for low-back pain.Methods:We searched MEDLINE, Embase, CINAHL and the Cochrane Central Register of Controlled Trials from the earliest records to June 23, 2014. We included randomized controlled trials, controlled clinical trials and interrupted time series studies that assessed interventions designed to reduce the use of imaging in any clinical setting, including primary, emergency and specialist care. Two independent reviewers extracted data and assessed risk of bias. We used raw data on imaging rates to calculate summary statistics. Study heterogeneity prevented meta-analysis.Results:A total of 8500 records were identified through the literature search. Of the 54 potentially eligible studies reviewed in full, 7 were included in our review. Clinical decision support involving a modified referral form in a hospital setting reduced imaging by 36.8% (95% confidence interval [CI] 33.2% to 40.5%). Targeted reminders to primary care physicians of appropriate indications for imaging reduced referrals for imaging by 22.5% (95% CI 8.4% to 36.8%). Interventions that used practitioner audits and feedback, practitioner education or guideline dissemination did not significantly reduce imaging rates. Lack of power within some of the included studies resulted in lack of statistical significance despite potentially clinically important effects.Interpretation:Clinical decision support in a hospital setting and targeted reminders to primary care doctors were effective interventions in reducing the use of imaging for low-back pain. These are potentially low-cost interventions that would substantially decrease medical expenditures associated with the management of low-back pain.Current evidence-based clinical practice guidelines recommend against the routine use of imaging in patients presenting with low-back pain.^1–3 Despite this, imaging rates remain high,^4,5 which indicates poor concordance with these guidelines.^6,7Unnecessary imaging for low-back pain has been associated with poorer patient outcomes, increased radiation exposure and higher health care costs.⁸ No short- or long-term clinical benefits have been shown with routine imaging of the low back, and the diagnostic value of incidental imaging findings remains uncertain.^9–12 A 2008 systematic review found that imaging accounted for 7% of direct costs associated with low-back pain, which in 1998 translated to more than US$6 billion in the United States and £114 million in the United Kingdom.¹³ Current costs are likely to be substantially higher, with an estimated 65% increase in spine-related expenditures between 1997 and 2005.¹⁴Various interventions have been tried for reducing imaging rates among people with low-back pain. These include strategies targeted at the practitioner such as guideline dissemination,^15–17 education workshops,^18,19 audit and feedback of imaging use,^7,20,21 ongoing reminders⁷ and clinical decision support.^22–24 It is unclear which, if any, of these strategies are effective.²⁵ We conducted a systematic review to investigate the effectiveness of interventions designed to reduce imaging rates for the management of low-back pain.     

Effect of nasal balloon autoinflation in children with otitis media with effusion in primary care: an open randomized controlled trial

Background:Otitis media with effusion is a common problem that lacks an evidence-based nonsurgical treatment option. We assessed the clinical effectiveness of treatment with a nasal balloon device in a primary care setting.Methods:We conducted an open, pragmatic randomized controlled trial set in 43 family practices in the United Kingdom. Children aged 4–11 years with a recent history of ear symptoms and otitis media with effusion in 1 or both ears, confirmed by tympanometry, were allocated to receive either autoinflation 3 times daily for 1–3 months plus usual care or usual care alone. Clearance of middle-ear fluid at 1 and 3 months was assessed by experts masked to allocation.Results:Of 320 children enrolled, those receiving autoinflation were more likely than controls to have normal tympanograms at 1 month (47.3% [62/131] v. 35.6% [47/132]; adjusted relative risk [RR] 1.36, 95% confidence interval [CI] 0.99 to 1.88) and at 3 months (49.6% [62/125] v. 38.3% [46/120]; adjusted RR 1.37, 95% CI 1.03 to 1.83; number needed to treat = 9). Autoinflation produced greater improvements in ear-related quality of life (adjusted between-group difference in change from baseline in OMQ-14 [an ear-related measure of quality of life] score −0.42, 95% CI −0.63 to −0.22). Compliance was 89% at 1 month and 80% at 3 months. Adverse events were mild, infrequent and comparable between groups.Interpretation:Autoinflation in children aged 4–11 years with otitis media with effusion is feasible in primary care and effective both in clearing effusions and improving symptoms and ear-related child and parent quality of life. Trial registration: ISRCTN, No. 55208702.Otitis media with effusion, also known as glue ear, is an accumulation of fluid in the middle ear, without symptoms or signs of an acute ear infection. It is often associated with viral infection.^1–3 The prevalence rises to 46% in children aged 4–5 years,⁴ when hearing difficulty, other ear-related symptoms and broader developmental concerns often bring the condition to medical attention.^3,5,6 Middle-ear fluid is associated with conductive hearing losses of about 15–45 dB HL.⁷ Resolution is clinically unpredictable,^8–10 with about a third of cases showing recurrence.¹¹ In the United Kingdom, about 200 000 children with the condition are seen annually in primary care.^12,13 Research suggests some children seen in primary care are as badly affected as those seen in hospital.^7,9,14,15 In the United States, there were 2.2 million diagnosed episodes in 2004, costing an estimated $4.0 billion.¹⁶ Rates of ventilation tube surgery show variability between countries,^17–19 with a declining trend in the UK.²⁰Initial clinical management consists of reasonable temporizing or delay before considering surgery.¹³ Unfortunately, all available medical treatments for otitis media with effusion such as antibiotics, antihistamines, decongestants and intranasal steroids are ineffective and have unwanted effects, and therefore cannot be recommended.^21–23 Not only are antibiotics ineffective, but resistance to them poses a major threat to public health.^24,25 Although surgery is effective for a carefully selected minority,^13,26,27 a simple low-cost, nonsurgical treatment option could benefit a much larger group of symptomatic children, with the purpose of addressing legitimate clinical concerns without incurring excessive delays.Autoinflation using a nasal balloon device is a low-cost intervention with the potential to be used more widely in primary care, but current evidence of its effectiveness is limited to several small hospital-based trials²⁸ that found a higher rate of tympanometric resolution of ear fluid at 1 month.^29–31 Evidence of feasibility and effectiveness of autoinflation to inform wider clinical use is lacking.^13,28 Thus we report here the findings of a large pragmatic trial of the clinical effectiveness of nasal balloon autoinflation in a spectrum of children with clinically confirmed otitis media with effusion identified from primary care.     

Gut microbiota of healthy Canadian infants: profiles by mode of delivery and infant diet at 4 months

Background:

The gut microbiota is essential to human health throughout life, yet the acquisition and development of this microbial community during infancy remains poorly understood. Meanwhile, there is increasing concern over rising rates of cesarean delivery and insufficient exclusive breastfeeding of infants in developed countries. In this article, we characterize the gut microbiota of healthy Canadian infants and describe the influence of cesarean delivery and formula feeding.

Methods:

We included a subset of 24 term infants from the Canadian Healthy Infant Longitudinal Development (CHILD) birth cohort. Mode of delivery was obtained from medical records, and mothers were asked to report on infant diet and medication use. Fecal samples were collected at 4 months of age, and we characterized the microbiota composition using high-throughput DNA sequencing.

Results:

We observed high variability in the profiles of fecal microbiota among the infants. The profiles were generally dominated by Actinobacteria (mainly the genus Bifidobacterium) and Firmicutes (with diverse representation from numerous genera). Compared with breastfed infants, formula-fed infants had increased richness of species, with overrepresentation of Clostridium difficile. Escherichia–Shigella and Bacteroides species were underrepresented in infants born by cesarean delivery. Infants born by elective cesarean delivery had particularly low bacterial richness and diversity.

Interpretation:

These findings advance our understanding of the gut microbiota in healthy infants. They also provide new evidence for the effects of delivery mode and infant diet as determinants of this essential microbial community in early life.The human body harbours trillions of microbes, known collectively as the “human microbiome.” By far the highest density of commensal bacteria is found in the digestive tract, where resident microbes outnumber host cells by at least 10 to 1. Gut bacteria play a fundamental role in human health by promoting intestinal homeostasis, stimulating development of the immune system, providing protection against pathogens, and contributing to the processing of nutrients and harvesting of energy.^1,2 The disruption of the gut microbiota has been linked to an increasing number of diseases, including inflammatory bowel disease, necrotizing enterocolitis, diabetes, obesity, cancer, allergies and asthma.¹ Despite this evidence and a growing appreciation for the integral role of the gut microbiota in lifelong health, relatively little is known about the acquisition and development of this complex microbial community during infancy.³Two of the best-studied determinants of the gut microbiota during infancy are mode of delivery and exposure to breast milk.^4,5 Cesarean delivery perturbs normal colonization of the infant gut by preventing exposure to maternal microbes, whereas breastfeeding promotes a “healthy” gut microbiota by providing selective metabolic substrates for beneficial bacteria.^3,5 Despite recommendations from the World Health Organization,⁶ the rate of cesarean delivery has continued to rise in developed countries and rates of breastfeeding decrease substantially within the first few months of life.^7,8 In Canada, more than 1 in 4 newborns are born by cesarean delivery, and less than 15% of infants are exclusively breastfed for the recommended duration of 6 months.^9,10 In some parts of the world, elective cesarean deliveries are performed by maternal request, often because of apprehension about pain during childbirth, and sometimes for patient–physician convenience.¹¹The potential long-term consequences of decisions regarding mode of delivery and infant diet are not to be underestimated. Infants born by cesarean delivery are at increased risk of asthma, obesity and type 1 diabetes,¹² whereas breastfeeding is variably protective against these and other disorders.¹³ These long-term health consequences may be partially attributable to disruption of the gut microbiota.^12,14Historically, the gut microbiota has been studied with the use of culture-based methodologies to examine individual organisms. However, up to 80% of intestinal microbes cannot be grown in culture.^3,15 New technology using culture-independent DNA sequencing enables comprehensive detection of intestinal microbes and permits simultaneous characterization of entire microbial communities. Multinational consortia have been established to characterize the “normal” adult microbiome using these exciting new methods;¹⁶ however, these methods have been underused in infant studies. Because early colonization may have long-lasting effects on health, infant studies are vital.^3,4 Among the few studies of infant gut microbiota using DNA sequencing, most were conducted in restricted populations, such as infants delivered vaginally,¹⁷ infants born by cesarean delivery who were formula-fed¹⁸ or preterm infants with necrotizing enterocolitis.¹⁹Thus, the gut microbiota is essential to human health, yet the acquisition and development of this microbial community during infancy remains poorly understood.³ In the current study, we address this gap in knowledge using new sequencing technology and detailed exposure assessments²⁰ of healthy Canadian infants selected from a national birth cohort to provide representative, comprehensive profiles of gut microbiota according to mode of delivery and infant diet.