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1.
Tammy T. Nguyen Sang S. Oh David Weaver Agnieszka Lewandowska Dane Maxfield Max-Hinderk Schuler Nathan K. Smith Jane Macfarlane Gerald Saunders Cheryl A. Palmer Valentina Debattisti Takumi Koshiba Stefan Pulst Eva L. Feldman Gy?rgy Hajnóczky Janet M. Shaw 《Proceedings of the National Academy of Sciences of the United States of America》2014,111(35):E3631-E3640
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. 相似文献
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Nobuhiro Yamagata Toshiharu Ichinose Yoshinori Aso Pierre-Yves Pla?ais Anja B. Friedrich Richard J. Sima Thomas Preat Gerald M. Rubin Hiromu Tanimoto 《Proceedings of the National Academy of Sciences of the United States of America》2015,112(2):578-583
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. 相似文献
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Jeffrey P. Nguyen Frederick B. Shipley Ashley N. Linder George S. Plummer Mochi Liu Sagar U. Setru Joshua W. Shaevitz Andrew M. Leifer 《Proceedings of the National Academy of Sciences of the United States of America》2016,113(8):E1074-E1081
The ability to acquire large-scale recordings of neuronal activity in awake and unrestrained animals is needed to provide new insights into how populations of neurons generate animal behavior. We present an instrument capable of recording intracellular calcium transients from the majority of neurons in the head of a freely behaving Caenorhabditis elegans with cellular resolution while simultaneously recording the animal’s position, posture, and locomotion. This instrument provides whole-brain imaging with cellular resolution in an unrestrained and behaving animal. We use spinning-disk confocal microscopy to capture 3D volumetric fluorescent images of neurons expressing the calcium indicator GCaMP6s at 6 head-volumes/s. A suite of three cameras monitor neuronal fluorescence and the animal’s position and orientation. Custom software tracks the 3D position of the animal’s head in real time and two feedback loops adjust a motorized stage and objective to keep the animal’s head within the field of view as the animal roams freely. We observe calcium transients from up to 77 neurons for over 4 min and correlate this activity with the animal’s behavior. We characterize noise in the system due to animal motion and show that, across worms, multiple neurons show significant correlations with modes of behavior corresponding to forward, backward, and turning locomotion.How do patterns of neural activity generate an animal’s behavior? To answer this question, it is important to develop new methods for recording from large populations of neurons in animals as they move and behave freely. The collective activity of many individual neurons appears to be critical for generating behaviors including arm reach in primates (1), song production in zebrafinch (2), the choice between swimming or crawling in leech (3), and decision-making in mice during navigation (4). New methods for recording from larger populations of neurons in unrestrained animals are needed to better understand neural coding of these behaviors and neural control of behavior more generally.Calcium imaging has emerged as a promising technique for recording dynamics from populations of neurons. Calcium-sensitive proteins are used to visualize changes in intracellular calcium levels in neurons in vivo which serve as a proxy for neural activity (5). To resolve the often weak fluorescent signal of an individual neuron in a dense forest of other labeled cells requires a high magnification objective with a large numerical aperture that, consequently, can image only a small field of view. Calcium imaging has traditionally been performed on animals that are stationary from anesthetization or immobilization to avoid imaging artifacts induced by animal motion. As a result, calcium imaging studies have historically focused on small brain regions in immobile animals that exhibit little or no behavior (6).No previous neurophysiological study has attained whole-brain imaging with cellular resolution in a freely behaving unrestrained animal. Previous whole-brain cellular resolution calcium imaging studies of populations of neurons that involve behavior investigate either fictive locomotion (3, 7), or behaviors that can be performed in restrained animals, such as eye movements (8) or navigation of a virtual environment (9). One exception has been the development of fluorescence endoscopy, which allows recording from rodents during unrestrained behavior, although imaging is restricted to even smaller subbrain regions (10).Investigating neural activity in small transparent organisms allows one to move beyond studying subbrain regions to record dynamics from entire brains with cellular resolution. Whole-brain imaging was performed first in larval zebrafish using two-photon microscopy (7). More recently, whole-brain imaging was performed in Caenorhabditis elegans using two-photon (11) and light-field microscopy (12). Animals in these studies were immobilized, anesthetized, or both and thus exhibited no gross behavior.C. elegans’ compact nervous system of only 302 neurons and small size of only 1 mm make it ideally suited for the development of new whole-brain imaging techniques for studying behavior. There is a long and rich history of studying and quantifying the behavior of freely moving C. elegans dating back to the mid-1970s (13, 14). Many of these works involved quantifying animal body posture as the worm moved, for example as in ref. 15. To facilitate higher-throughput recordings of behavior, a number of tracking microscopes (16–18) or multiworm imagers were developed (19, 20) along with sophisticated behavioral analysis software and analytical tools (21, 22). Motivated in part to understand these behaviors, calcium imaging systems were also developed that could probe neural activity in at first partially immobilized (23) and then freely moving animals, beginning with ref. 24 and and then developing rapidly (17, 18, 25–29). One limitation of these freely moving calcium imaging systems is that they are limited to imaging only a very small subset of neurons and lack the ability to distinguish neurons that lie atop one another in the axial direction of the microscope. Despite this limitation, these studies, combined with laser-ablation experiments, have identified a number of neurons that correlate or affect changes in particular behaviors including the AVB neuron pair and VB-type motor neurons for forward locomotion; the AVA, AIB, and AVE neuron pairs and VA-type motor neurons for backward locomotion; and the RIV, RIB, and SMD neurons and the DD-type motor neurons for turning behaviors (17, 18, 25, 26, 28, 30, 31). To move beyond these largely single-cell studies, we sought to record simultaneously from the entire brain of C. elegans with cellular resolution and record its behavior as it moved about unrestrained. 相似文献
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Yichun Shuai Areekul Hirokawa Yulian Ai Min Zhang Wanhe Li Yi Zhong 《Proceedings of the National Academy of Sciences of the United States of America》2015,112(48):E6663-E6672
Recent studies have identified molecular pathways driving forgetting and supported the notion that forgetting is a biologically active process. The circuit mechanisms of forgetting, however, remain largely unknown. Here we report two sets of Drosophila neurons that account for the rapid forgetting of early olfactory aversive memory. We show that inactivating these neurons inhibits memory decay without altering learning, whereas activating them promotes forgetting. These neurons, including a cluster of dopaminergic neurons (PAM-β′1) and a pair of glutamatergic neurons (MBON-γ4>γ1γ2), terminate in distinct subdomains in the mushroom body and represent parallel neural pathways for regulating forgetting. Interestingly, although activity of these neurons is required for memory decay over time, they are not required for acute forgetting during reversal learning. Our results thus not only establish the presence of multiple neural pathways for forgetting in Drosophila but also suggest the existence of diverse circuit mechanisms of forgetting in different contexts.Although forgetting commonly has a negative connotation, it is a functional process that shapes memory and cognition (1–4). Recent studies, including work in relatively simple invertebrate models, have started to reveal basic biological mechanisms underlying forgetting (5–15). In Drosophila, single-session Pavlovian conditioning by pairing an odor (conditioned stimulus, CS) with electric shock (unconditioned stimulus, US) induces aversive memories that are short-lasting (16). The memory performance of fruit flies is observed to drop to a negligible level within 24 h, decaying rapidly early after training and slowing down thereafter (17). Memory decay or forgetting requires the activation of the small G protein Rac, a signaling protein involved in actin remodeling, in the mushroom body (MB) intrinsic neurons (6). These so-called Kenyon cells (KCs) are the neurons that integrate CS–US information (18, 19) and support aversive memory formation and retrieval (20–22). In addition to Rac, forgetting also requires the DAMB dopamine receptor (7), which has highly enriched expression in the MB (23). Evidence suggests that the dopamine-mediated forgetting signal is conveyed to the MB by dopamine neurons (DANs) in the protocerebral posterior lateral 1 (PPL1) cluster (7, 24). Therefore, forgetting of olfactory aversive memory in Drosophila depends on a particular set of intracellular molecular pathways within KCs, involving Rac, DAMB, and possibly others (25), and also receives modulation from extrinsic neurons. Although important cellular evidence supporting the hypothesis that memory traces are erased under these circumstances is still lacking, these findings lend support to the notion that forgetting is an active, biologically regulated process (17, 26).Although existing studies point to the MB circuit as essential for forgetting, several questions remain to be answered. First, whereas the molecular pathways for learning and forgetting of olfactory aversive memory are distinct and separable (6, 7), the neural circuits seem to overlap. Rac-mediated forgetting has been localized to a large population of KCs (6), including the γ-subset, which is also critical for initial memory formation (21, 27). The site of action of DAMB for forgetting has yet to be established; however, the subgroups of PPL1-DANs implicated in forgetting are the same as those that signal aversive reinforcement and are required for learning (28–30). It leaves open the question of whether the brain circuitry underlying forgetting and learning is dissociable, or whether forgetting and learning share the same circuit but are driven by distinct activity patterns and molecular machinery (26). Second, shock reinforcement elicits multiple memory traces through at least three dopamine pathways to different subdomains in the MB lobes (28, 29). Functional imaging studies have also revealed Ca2+-based memory traces in different KC populations (31). It is poorly understood how forgetting of these memory traces differs, and it remains unknown whether there are multiple regulatory neural pathways. Notably, when PPL1-DANs are inactivated, forgetting still occurs, albeit at a lower rate (7). This incomplete block suggests the existence of an additional pathway(s) that conveys forgetting signals to the MB. Third, other than memory decay over time, forgetting is also observed through interference (32, 33), when new learning or reversal learning is introduced after training (6, 34, 35). Time-based and interference-based forgetting shares a similar dependence on Rac and DAMB (6, 7). However, it is not known whether distinct circuits underlie forgetting in these different contexts.In the current study, we focus on the diverse set of MB extrinsic neurons (MBENs) that interconnect the MB lobes with other brain regions, which include 34 MB output neurons (MBONs) of 21 types and ∼130 dopaminergic neurons of 20 types in the PPL1 and protocerebral anterior medial (PAM) clusters (36, 37). These neurons have been intensively studied in olfactory memory formation, consolidation, and retrieval in recent years (e.g., 24, 28–30, 38–48); however, their roles in forgetting have not been characterized except for the aforementioned PPL1-DANs. In a functional screen, we unexpectedly found that several Gal4 driver lines of MBENs showed significantly better 3-h memory retention when the Gal4-expressing cells were inactivated. The screen has thus led us to identify two types of MBENs that are not involved in initial learning but play important and additive roles in mediating memory decay. Furthermore, neither of these MBEN types is required for reversal learning, supporting the notion that there is a diversity of neural circuits that drive different forms of forgetting. 相似文献
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Qizong Yang Pavlo Kuzyk Igor Antonov Caleb J. Bostwick Andrea B. Kohn Leonid L. Moroz Robert D. Hawkins 《Proceedings of the National Academy of Sciences of the United States of America》2015,112(52):16030-16035
Hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channels are critical regulators of neuronal excitability, but less is known about their possible roles in synaptic plasticity and memory circuits. Here, we characterized the HCN gene organization, channel properties, distribution, and involvement in associative and nonassociative forms of learning in Aplysia californica. Aplysia has only one HCN gene, which codes for a channel that has many similarities to the mammalian HCN channel. The cloned acHCN gene was expressed in Xenopus oocytes, which displayed a hyperpolarization-induced inward current that was enhanced by cGMP as well as cAMP. Similarly to its homologs in other animals, acHCN is permeable to K+ and Na+ ions, and is selectively blocked by Cs+ and ZD7288. We found that acHCN is predominantly expressed in inter- and motor neurons, including LFS siphon motor neurons, and therefore tested whether HCN channels are involved in simple forms of learning of the siphon-withdrawal reflex in a semiintact preparation. ZD7288 (100 μM) significantly reduced an associative form of learning (classical conditioning) but had no effect on two nonassociative forms of learning (intermediate-term sensitization and unpaired training) or baseline responses. The HCN current is enhanced by nitric oxide (NO), which may explain the postsynaptic role of NO during conditioning. HCN current in turn enhances the NMDA-like current in the motor neurons, suggesting that HCN channels contribute to conditioning through this pathway.Hyperpolarization-activated, cyclic nucleotide-gated (HCN), cation nonselective ion channels generate hyperpolarization-activated inward currents (Ih) and thus tend to stabilize membrane potential (1–3). In addition, binding of cyclic nucleotides (cAMP and cGMP) to the C-terminal cyclic nucleotide binding domain (CNBD) enhances Ih and thus couples membrane excitability with intracellular signaling pathways (2, 4). HCN channels are widely important for numerous systemic functions such as hormonal regulation, heart contractility, epilepsy, pain, central pattern generation, sensory perception (4–15), and learning and memory (16–24).However, in previous studies it has been difficult to relate the cellular effects of HCN channels directly to their behavioral effects, because of the immense complexity of the mammalian brain. We have therefore investigated the role of HCN channels in Aplysia, which has a numerically simpler nervous system (25). We first identified and characterized an HCN gene in Aplysia, and showed that it codes for a channel that has many similarities to the mammalian HCN channel. We found that the Aplysia HCN channel is predominantly expressed in motor neurons including LFS neurons in the siphon withdrawal reflex circuit (26, 27). We therefore investigated simple forms of learning of that reflex in a semiintact preparation (28–30) and found that HCN current is involved in classical conditioning and enhances the NMDA-like current in the motor neurons. These results provide a direct connection between HCN channels and behavioral learning and suggest a postsynaptic mechanism of that effect. HCN current in turn is enhanced by nitric oxide (NO), a transmitter of facilitatory interneurons, and thus may contribute to the postsynaptic role of NO during conditioning. 相似文献
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Xiaofan Li Hansi Liu Jose Chu Luo Sarah A. Rhodes Liana M. Trigg Damian B. van Rossum Andriy Anishkin Fortunay H. Diatta Jessica K. Sassic David K. Simmons Bishoy Kamel Monica Medina Mark Q. Martindale Timothy Jegla 《Proceedings of the National Academy of Sciences of the United States of America》2015,112(9):E1010-E1019
We examined the origins and functional evolution of the Shaker and KCNQ families of voltage-gated K+ channels to better understand how neuronal excitability evolved. In bilaterians, the Shaker family consists of four functionally distinct gene families (Shaker, Shab, Shal, and Shaw) that share a subunit structure consisting of a voltage-gated K+ channel motif coupled to a cytoplasmic domain that mediates subfamily-exclusive assembly (T1). We traced the origin of this unique Shaker subunit structure to a common ancestor of ctenophores and parahoxozoans (cnidarians, bilaterians, and placozoans). Thus, the Shaker family is metazoan specific but is likely to have evolved in a basal metazoan. Phylogenetic analysis suggested that the Shaker subfamily could predate the divergence of ctenophores and parahoxozoans, but that the Shab, Shal, and Shaw subfamilies are parahoxozoan specific. In support of this, putative ctenophore Shaker subfamily channel subunits coassembled with cnidarian and mouse Shaker subunits, but not with cnidarian Shab, Shal, or Shaw subunits. The KCNQ family, which has a distinct subunit structure, also appears solely within the parahoxozoan lineage. Functional analysis indicated that the characteristic properties of Shaker, Shab, Shal, Shaw, and KCNQ currents evolved before the divergence of cnidarians and bilaterians. These results show that a major diversification of voltage-gated K+ channels occurred in ancestral parahoxozoans and imply that many fundamental mechanisms for the regulation of action potential propagation evolved at this time. Our results further suggest that there are likely to be substantial differences in the regulation of neuronal excitability between ctenophores and parahoxozoans.Voltage-gated K+ channels are highly conserved among bilaterian metazoans and play a central role in the regulation of excitation in neurons and muscle. Understanding the functional evolution of these channels may therefore provide important insights into how neuromuscular excitation evolved within the Metazoa. Three major gene families, Shaker, KCNQ, and Ether-a-go-go (EAG) encode all voltage-gated K+ channels in bilaterians (1, 2). In this study, we examine the functional evolution and origins of the Shaker and KCNQ gene families. Shaker family channels can be definitively identified by a unique subunit structure that includes both a voltage-gated K+ channel core and a family-specific cytoplasmic domain within the N terminus known as the T1 domain. T1 mediates assembly of Shaker family subunits into functional tetrameric channels (3, 4). KCNQ channels are also tetrameric but lack a T1 domain and use a distinct coiled-coil assembly domain in the C terminus (5, 6). KCNQ channels can be identified by the presence of this family-specific assembly motif and high amino acid conservation within the K+ channel core. Both channel families are found in cnidarians (1, 7) and thus predate the divergence of cnidarians and bilaterians, but their ultimate evolutionary origins have not yet been defined.Shaker family K+ channels serve diverse roles in the regulation of neuronal firing and can be divided into four gene subfamilies based on function and sequence homology: Shaker, Shab, Shal, and Shaw (8, 9). The T1 assembly domain is only compatible between subunits from the same gene subfamily (4, 10) and thus serves to keep the subfamilies functionally segregated. Shaker subfamily channels activate rapidly near action potential threshold and range from rapidly inactivating to noninactivating. Multiple roles for Shaker channels in neurons and muscles have been described, but their most unique and fundamental role may be that of axonal action potential repolarization. Shaker channels are clustered to the axon initial segment and nodes of Ranvier in vertebrate neurons (11–13) and underlie the delayed rectifier in squid giant axons (14). The Shaker subfamily is diverse in cnidarians (15, 16), and the starlet sea anemone Nematostella vectensis has functional orthologs of most identified Shaker current types observed in bilaterians (16).The Shab and Shal gene subfamilies encode somatodendritic delayed rectifiers and A currents, respectively (17–20). Shab channels are important for maintaining sustained firing (21, 22), whereas the Kv4-based A current modulates spike threshold and frequency (17). Shab and Shal channels are present in cnidarians, but cnidarian Shab channels have not been functionally characterized, and the only cnidarian Shal channels expressed to date display atypical voltage dependence and kinetics compared with bilaterian channels (23). Shaw channels are rapid, high-threshold channels specialized for sustaining fast firing in vertebrates (24, 25) but have a low activation threshold and may contribute to resting potential in Drosophila (19, 26, 27). A Caenorhabditis elegans Shaw has slow kinetics but a high activation threshold (28), and a single expressed cnidarian Shaw channel has the opposite: a low activation threshold but relatively fast kinetics (29). Thus, the ancestral properties and function of Shaw channels is not yet understood. Further functional characterization of cnidarian Shab, Shal, and Shaw channels would provide a better understanding of the evolutionary status of the Shaker family in early parahoxozoans.KCNQ family channels underlie the M current in vertebrate neurons (30) that regulates subthreshold excitability (31). The M current provides a fundamental mechanism for regulation of firing threshold through the Gq G-protein pathway because KCNQ channels require phosphatidylinositol 4,5-bisphosphate (PIP2) for activation (32, 33). PIP2 hydrolysis and subsequent KCNQ channel closure initiated by Gq-coupled receptors produces slow excitatory postsynaptic potentials, during which the probability of firing is greatly increased (32, 33). The key functional adaptations of KCNQ channels for this physiological role that can be observed in vitro are (i) a requirement for PIP2 to couple voltage-sensor activation to pore opening (34, 35), and (ii) a hyperpolarized voltage–activation curve that allows channels to open below typical action potential thresholds. Both key features are found in vertebrate (30, 34, 36–38), Drosophila (39), and C. elegans (40) KCNQ channels, suggesting they may have been present in KCNQ channels in a bilaterian ancestor. Evolution of the M current likely represented a major advance in the ability to modulate the activity of neuronal circuits, but it is not yet clear when PIP2-dependent KCNQ channels first evolved.Here, we examine the origins and functional evolution of the Shaker and KCNQ gene families. If we assume the evolution of neuronal signaling provided a major selective pressure for the functional diversification of voltage-gated K+ channels, then we can hypothesize that the appearance of these gene families might accompany the emergence of the first nervous systems or a major event in nervous system evolution. Recent phylogenies that place the divergence of ctenophores near the root of the metazoan tree suggest that the first nervous systems, or at least the capacity to make neurons, may have been present in a basal metazoan ancestor (41–43) (Fig. S1). One hypothesis then is that much of the diversity of metazoan voltage-gated channels should be shared between ctenophores and parahoxozoans [cnidarians, bilaterians, and placozoans (44)]. However, genome analysis indicates that many “typical” neuronal genes are missing in ctenophores and the sponges lack a nervous system, leading to the suggestion that extant nervous systems may have evolved independently in ctenophores and parahoxozoans (42, 45). Thus, a second hypothesis is that important steps in voltage-gated K+ channel evolution might have occurred separately in ctenophores and parahoxozoans. We tested these hypotheses by carefully examining the phylogenetic distribution and functional evolution of Shaker and KCNQ family K+ channels. Our results support a model in which major innovations in neuromuscular excitability occurred specifically within the parahoxozoan lineage. 相似文献
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Jean-Marc Devaud Thomas Papouin Julie Carcaud Jean-Christophe Sandoz Bernd Grünewald Martin Giurfa 《Proceedings of the National Academy of Sciences of the United States of America》2015,112(43):E5854-E5862
Learning theories distinguish elemental from configural learning based on their different complexity. Although the former relies on simple and unambiguous links between the learned events, the latter deals with ambiguous discriminations in which conjunctive representations of events are learned as being different from their elements. In mammals, configural learning is mediated by brain areas that are either dispensable or partially involved in elemental learning. We studied whether the insect brain follows the same principles and addressed this question in the honey bee, the only insect in which configural learning has been demonstrated. We used a combination of conditioning protocols, disruption of neural activity, and optophysiological recording of olfactory circuits in the bee brain to determine whether mushroom bodies (MBs), brain structures that are essential for memory storage and retrieval, are equally necessary for configural and elemental olfactory learning. We show that bees with anesthetized MBs distinguish odors and learn elemental olfactory discriminations but not configural ones, such as positive and negative patterning. Inhibition of GABAergic signaling in the MB calyces, but not in the lobes, impairs patterning discrimination, thus suggesting a requirement of GABAergic feedback neurons from the lobes to the calyces for nonelemental learning. These results uncover a previously unidentified role for MBs besides memory storage and retrieval: namely, their implication in the acquisition of ambiguous discrimination problems. Thus, in insects as in mammals, specific brain regions are recruited when the ambiguity of learning tasks increases, a fact that reveals similarities in the neural processes underlying the elucidation of ambiguous tasks across species.Learning can be categorized into two levels of complexity termed elemental and configural (nonelemental) (1–3). Simple and unambiguous links between events characterize elemental learning (4). By contrast, ambiguity and nonlinearity characterize configural learning, where associations involve conjunctions of elemental stimuli, which may have different, contradictory outcomes. As a consequence, solving configural tasks typically requires treating stimulus conjunctions as being different from the simple sum of their elemental components (5–8). For example, in a negative patterning task (9–11), subjects have to discriminate a nonreinforced conjunction of two elements A and B from its reinforced elements (i.e., AB– vs. A+ and B+), which requires treating AB as being different from the simple sum of A and B (12, 13). The ambiguity of the task lies in the fact that each element (A and B) is as often reinforced (when presented alone) as nonreinforced (when presented as a compound). In mammals, different brain structures have been associated with these two learning forms: Whereas the hippocampus seems to be dispensable for learning elemental associations (6, 8), it is required for fast formation of conjunctive representations during learning tasks, such as spatial learning or contextual fear conditioning (6, 8, 10, 14–19). Moreover, the cortical system is necessary to form configural representations over extended training, thus supporting the learning of nonlinear discriminations,Here, we ask whether the specialization of different brain centers for learning tasks of different complexity is a property that can be extended to an insect brain. Insects offer the possibility of studying sophisticated behaviors and simultaneously accessing the neural bases of these behaviors (20). Several studies have shown that insects, in particular the honey bee Apis mellifera, possess higher-order cognitive abilities (5, 21), which raises the question of which neural mechanisms support these capacities in a brain whose size is only 1 mm3 (22).The mushroom bodies (MBs) are paired structures in the insect brain that have been historically associated with olfactory learning and memory. Their function has been extensively studied in a variety of elemental learning protocols, mainly in the honey bee and the fruit fly Drosophila melanogaster (23–29). In both species, MBs play a fundamental role for the encoding, storing, and retrieval of appetitive and aversive elemental memories, but no study has clearly established their role for nonelemental learning and memory (30). In fruit flies, this missing information may be due to the incapacity of these insects to solve nonelemental problems, such as negative patterning (31). By contrast, honey bees exhibit elaborated nonelemental learning abilities (32–36), which have been suggested to require intact MB function (5).Here, we used a combination of nonelemental conditioning protocols, disruption of MB function, and optophysiological recordings of neural activity to determine whether MBs are necessary for nonelemental forms of learning. Our results show that acquisition of olfactory patterning discriminations is impaired in bees in which neural activity in the MBs was blocked by procaine injection (37, 38), but not in control animals injected with saline solution. By contrast, MB blockade by procaine affected neither olfactory processing upstream of the MBs nor elemental olfactory discriminations. To uncover the neural mechanisms underlying the necessity of MBs for patterning discriminations, we focused on GABAergic feedback neurons (39), which provide inhibitory feedback to the MBs of the bee (40–43). We blocked GABAergic signaling by locally injecting picrotoxin (PTX), a GABA antagonist, into the MB calyces or into the MB lobes. We show that GABAergic feedback to the calyces—but not to the lobes—is required for patterning discriminations. These results uncover a previously unidentified role for MBs: namely, the disambiguation between elemental and conjunctive odor representations, thus supporting the learning of nonlinear discriminations. 相似文献
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Cedric Bardy Mark van den Hurk Tameji Eames Cynthia Marchand Ruben V. Hernandez Mariko Kellogg Mark Gorris Ben Galet Vanessa Palomares Joshua Brown Anne G. Bang Jerome Mertens Lena B?hnke Leah Boyer Suzanne Simon Fred H. Gage 《Proceedings of the National Academy of Sciences of the United States of America》2015,112(20):E2725-E2734
Human cell reprogramming technologies offer access to live human neurons from patients and provide a new alternative for modeling neurological disorders in vitro. Neural electrical activity is the essence of nervous system function in vivo. Therefore, we examined neuronal activity in media widely used to culture neurons. We found that classic basal media, as well as serum, impair action potential generation and synaptic communication. To overcome this problem, we designed a new neuronal medium (BrainPhys basal + serum-free supplements) in which we adjusted the concentrations of inorganic salts, neuroactive amino acids, and energetic substrates. We then tested that this medium adequately supports neuronal activity and survival of human neurons in culture. Long-term exposure to this physiological medium also improved the proportion of neurons that were synaptically active. The medium was designed to culture human neurons but also proved adequate for rodent neurons. The improvement in BrainPhys basal medium to support neurophysiological activity is an important step toward reducing the gap between brain physiological conditions in vivo and neuronal models in vitro.Induced pluripotent stem cell (iPSC) technology is currently being used to model human diseases in vitro and may contribute to the discovery and validation of new pharmacological treatments (1–3). In particular, neuroscientists have seized the opportunity to culture neurons from patients with neurological and psychiatric disorders and have demonstrated that phenotypes associated with particular disorders can be recapitulated in the dish (4–7). However, the basic culture conditions for growing neurons in vitro have not been updated to reflect fundamental principles of brain physiology. Currently, most human neuronal cultures are grown in media based on DMEM/F12 (4, 5, 7–24), Neurobasal (25–30), or a mixture of DMEM and Neurobasal (DN) (31–34). To promote neuronal differentiation and survival, a variety of supplements, such as serum, growth factors, hormones, proteins, and antioxidants, are typically added to these basal media. Although these media were designed and optimized to promote neuronal survival in vitro, they were not tested for their ability to support fundamental neuronal functions. Using several electrophysiological techniques such as patch clamping, calcium imaging, and multielectrode arrays, we found that widely used tissue culture media (e.g., DMEM basal, Neurobasal, serum) actually impaired neurophysiological functions.We identified several neuroactive components in these media that acutely interfered with neuronal function. To solve these issues, we designed a chemically defined basal medium: BrainPhys basal. We used human neurons in vitro to demonstrate in a series of experiments that this new medium, combined with the appropriate supplements, better supports important neuronal functions while sustaining cell survival in vitro. Notably, BrainPhys-based medium better mimics the environment present in healthy living brains, unlike previous media based on DMEM, Neurobasal or serum. Although BrainPhys basal was specifically designed for the culturing of mature human neurons, our studies also showed that BrainPhys provided a functional environment for ex vivo brain slices and for culturing rodent primary neurons. 相似文献
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Sarit Avrani Debbie Lindell 《Proceedings of the National Academy of Sciences of the United States of America》2015,112(17):E2191-E2200
Prochlorococcus is an abundant marine cyanobacterium that grows rapidly in the environment and contributes significantly to global primary production. This cyanobacterium coexists with many cyanophages in the oceans, likely aided by resistance to numerous co-occurring phages. Spontaneous resistance occurs frequently in Prochlorococcus and is often accompanied by a pleiotropic fitness cost manifested as either a reduced growth rate or enhanced infection by other phages. Here, we assessed the fate of a number of phage-resistant Prochlorococcus strains, focusing on those with a high fitness cost. We found that phage-resistant strains continued evolving toward an improved growth rate and a narrower resistance range, resulting in lineages with phenotypes intermediate between those of ancestral susceptible wild-type and initial resistant substrains. Changes in growth rate and resistance range often occurred in independent events, leading to a decoupling of the selection pressures acting on these phenotypes. These changes were largely the result of additional, compensatory mutations in noncore genes located in genomic islands, although genetic reversions were also observed. Additionally, a mutator strain was identified. The similarity of the evolutionary pathway followed by multiple independent resistant cultures and clones suggests they undergo a predictable evolutionary pathway. This process serves to increase both genetic diversity and infection permutations in Prochlorococcus populations, further augmenting the complexity of the interaction network between Prochlorococcus and its phages in nature. Last, our findings provide an explanation for the apparent paradox of a multitude of resistant Prochlorococcus cells in nature that are growing close to their maximal intrinsic growth rates.Large bacterial populations are present in the oceans, playing important roles in primary production and the biogeochemical cycling of matter. These bacterial communities are highly diverse (1–4) yet form stable and reproducible bacterial assemblages under similar environmental conditions (5–7).These bacteria are present together with high abundances of viruses (phages) that have the potential to infect and kill them (8–11). Although studied only rarely in marine organisms (12–16), this coexistence is likely to be the result of millions of years of coevolution between these antagonistic interacting partners, as has been well documented for other systems (17–20). From the perspective of the bacteria, survival entails the selection of cells that are resistant to infection, preventing viral production and enabling the continuation of the cell lineage. Resistance mechanisms include passively acquired spontaneous mutations in cell surface molecules that prevent phage entry into the cell and other mechanisms that actively terminate phage infection intracellularly, such as restriction–modification systems and acquired resistance by CRISPR-Cas systems (21, 22). Mutations in the phage can also occur that circumvent these host defenses and enable the phage to infect the recently emerged resistant bacterium (23).Acquisition of resistance by bacteria is often associated with a fitness cost. This cost is frequently, but not always, manifested as a reduction in growth rate (24–27). Recently, an additional type of cost of resistance was identified, that of enhanced infection whereby resistance to one phage leads to greater susceptibility to other phages (14, 15, 28).Over the years, a number of models have been developed to explain coexistence in terms of the above coevolutionary processes and their costs (16, 29–32). In the arms race model, repeated cycles of host mutation and virus countermutation occur, leading to increasing breadths of host resistance and viral infectivity. However, experimental evidence generally indicates that such directional arms race dynamics do not continue indefinitely (25, 33, 34). Therefore, models of negative density-dependent fluctuations due to selective trade-offs, such as kill-the-winner, are often invoked (20, 33, 35, 36). In these models, fluctuations are generally considered to occur between rapidly growing competition specialists that are susceptible to infection and more slowly growing resistant strains that are considered defense specialists. Such negative density-dependent fluctuations are also likely to occur between strains that have differences in viral susceptibility ranges, such as those that would result from enhanced infection (30).The above coevolutionary processes are considered to be among the major mechanisms that have led to and maintain diversity within bacterial communities (32, 35, 37–39). These processes also influence genetic microdiversity within populations of closely related bacteria. This is especially the case for cell surface-related genes that are often localized to genomic islands (14, 40, 41), regions of high gene content, and gene sequence variability among members of a population. As such, populations in nature display an enormous degree of microdiversity in phage susceptibility regions, potentially leading to an assortment of subpopulations with different ranges of susceptibility to coexisting phages (4, 14, 30, 40).Prochlorococcus is a unicellular cyanobacterium that is the numerically dominant photosynthetic organism in vast oligotrophic expanses of the open oceans, where it contributes significantly to primary production (42, 43). Prochlorococcus consists of a number of distinct ecotypes (44–46) that form stable and reproducible population structures (7). These populations coexist in the oceans with tailed double-stranded DNA phage populations that infect them (47–49).Previously, we found that resistance to phage infection occurs frequently in two high-light–adapted Prochlorococcus ecotypes through spontaneous mutations in cell surface-related genes (14). These genes are primarily localized to genomic island 4 (ISL4) that displays a high degree of genetic diversity in environmental populations (14, 40). Although about a third of Prochlorococcus-resistant strains had no detectable associated cost, the others came with a cost manifested as either a slower growth rate or enhanced infection by other phages (14). In nature, Prochlorococcus seems to be growing close to its intrinsic maximal growth rate (50–52). This raises the question as to the fate of emergent resistant Prochlorococcus lineages in the environment, especially when resistance is accompanied with a high growth rate fitness cost.To begin addressing this question, we investigated the phenotype of Prochlorococcus strains with time after the acquisition of resistance. We found that resistant strains evolved toward an improved growth rate and a reduced resistance range. Whole-genome sequencing and PCR screening of many of these strains revealed that these phenotypic changes were largely due to additional, compensatory mutations, leading to increased genetic diversity. These findings suggest that the oceans are populated with rapidly growing Prochlorococcus cells with varying degrees of resistance and provide an explanation for how a multitude of presumably resistant Prochlorococcus cells are growing close to their maximal known growth rate in nature. 相似文献
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Arnulf B. A. Graf Richard A. Andersen 《Proceedings of the National Academy of Sciences of the United States of America》2014,111(49):17630-17635
A number of studies in tetraplegic humans and healthy nonhuman primates (NHPs) have shown that neuronal activity from reach-related cortical areas can be used to predict reach intentions using brain–machine interfaces (BMIs) and therefore assist tetraplegic patients by controlling external devices (e.g., robotic limbs and computer cursors). However, to our knowledge, there have been no studies that have applied BMIs to eye movement areas to decode intended eye movements. In this study, we recorded the activity from populations of neurons from the lateral intraparietal area (LIP), a cortical node in the NHP saccade system. Eye movement plans were predicted in real time using Bayesian inference from small ensembles of LIP neurons without the animal making an eye movement. Learning, defined as an increase in the prediction accuracy, occurred at the level of neuronal ensembles, particularly for difficult predictions. Population learning had two components: an update of the parameters of the BMI based on its history and a change in the responses of individual neurons. These results provide strong evidence that the responses of neuronal ensembles can be shaped with respect to a cost function, here the prediction accuracy of the BMI. Furthermore, eye movement plans could be decoded without the animals emitting any actual eye movements and could be used to control the position of a cursor on a computer screen. These findings show that BMIs for eye movements are promising aids for assisting paralyzed patients.Brain–machine interfaces (BMIs) have been successfully used to predict reaches and arm movements (1–7). However, little effort has been concentrated on building a BMI based on eye movements. This gap is surprising because the motor and neuronal mechanisms of eye movements are very well understood and arguably simpler than those of arm movements. Specifically, eye movements are rapid and ballistic. The lateral intraparietal cortex (LIP) is ideally suited to be the site for a BMI based on eye movements (8). LIP neurons are known to encode eye movement plans, among other signals such as eye position (9–16). We recently showed that eye movement plans can be accurately predicted from the responses of populations of LIP neurons using Bayesian inference (16). The aim of the present study was twofold. First, a BMI was used with small neuronal ensembles of LIP neurons to predict, in real time, eye movement plans without the animals actually making eye movements. Second, the BMI application induced learning-related changes in the saccade system. Learning can produce changes in reach areas, but how learning-related changes occur at the level of LIP neuronal ensembles is still unclear (17, 18).Here, we show that the intended eye movement activity can be used to accurately position a cursor on a computer screen. These results suggest that an eye movement BMI can be used as a prosthetic to assist locked-in patients who cannot produce eye movements. Moreover, such an eye movement BMI can also be used to assist tetraplegic persons to decode intended limb movements by providing an extra channel of target position information (19). Learning, defined as an increase in the prediction accuracy, occurred at the level of neuronal ensembles, particularly for difficult predictions. The population learning had two components: an update of the parameters of the BMI based on its history and a change in the responses of individual neurons. These results provide strong evidence that the responses of neuronal ensembles can be shaped with respect to a cost function, which here is the prediction accuracy of the BMI. Such learning adds additional support for the utility of an eye movement BMI based on LIP activity. 相似文献
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Robert E. Ricklefs Diana C. Outlaw Maria Svensson-Coelho Matthew C. I. Medeiros Vincenzo A. Ellis Steven Latta 《Proceedings of the National Academy of Sciences of the United States of America》2014,111(41):14816-14821
The malaria parasites (Apicomplexa: Haemosporida) of birds are believed to have diversified across the avian host phylogeny well after the origin of most major host lineages. Although many symbionts with direct transmission codiversify with their hosts, mechanisms of species formation in vector-borne parasites, including the role of host shifting, are poorly understood. Here, we examine the hosts of sister lineages in a phylogeny of 181 putative species of malaria parasites of New World terrestrial birds to determine the role of shifts between host taxa in the formation of new parasite species. We find that host shifting, often across host genera and families, is the rule. Sympatric speciation by host shifting would require local reproductive isolation as a prerequisite to divergent selection, but this mechanism is not supported by the generalized host-biting behavior of most vectors of avian malaria parasites. Instead, the geographic distribution of individual parasite lineages in diverse hosts suggests that species formation is predominantly allopatric and involves host expansion followed by local host–pathogen coevolution and secondary sympatry, resulting in local shifting of parasite lineages across hosts.Cospeciation of symbionts with their hosts has been recognized in parasites with strong vertical transmission (1, 2), viruses that spread by direct contact (3), and bacterial and viral symbionts passed from mother to offspring through the egg (4). Species formation in parasites that are transmitted between hosts by vectors is less well-understood (5, 6). Poor matching between the phylogenetic trees of vector-borne hemosporidian (malaria) parasites and their North American avian hosts suggests a predominance of host shifting compared with cospeciation (7) (reviewed in a broader context in ref. 8). Whether host shifting occurs primarily between closely related hosts and in geographic sympatry, and whether rates of host shifting followed by species formation are sufficient to explain the contemporary diversity of hemosporidian parasites, have not been determined.Many species of hemosporidian parasites have been described and named based primarily on the microscopic morphology of meronts and gametocytes in blood smears (9). The more recent discovery of hundreds of lineages based on DNA sequence variation in the mitochondrial cytochrome b gene (cyt b) (5, 10, 11) requires, however, a different species concept based on analysis of independent components of the genome (12–16). Recent estimates of the rate of molecular evolution in hemosporidian mitochondrial genes imply that the contemporary malaria parasites of vertebrates might have descended from a common ancestor within the past 20 (17) or 40 Ma (18) or, perhaps, a longer time period (19). Although an appropriate calibration for the rate of hemosporidian evolution remains unsettled (20, 21), host shifting almost certainly has been frequent, likely across great host distances at times, over the recent history of the group.Speciation in sympatry (i.e., in the absence of geographic barriers to gene flow through local host specialization) might follow host shifting if mating between parasites was assortative with respect to vertebrate host or if different hosts imposed strong disruptive selection on parasites (22). However, despite some documented feeding preferences (23–25), dipteran vectors of avian malaria parasites do not seem to be sufficiently specialized to isolate populations of parasites on different hosts (26–29). In addition, many parasite species and many parasite lineages distinguished by DNA sequence variation occur locally across broad ranges of hosts without apparent differentiation, at least in the mitochondrial cytochrome b gene (30–32) and several nuclear markers (12, 14). Alternatively, host shifting in one allopatric population of a parasite species could be followed, after sufficient host–pathogen coevolution and evolutionary differentiation to produce reproductive incompatibility, by secondary sympatry, thereby increasing local parasite diversity.Here, we examine recent nodes in an mtDNA-based phylogeny of New World hemosporidian parasites to determine the degree to which lineage formation is associated with host shifting. Although our phylogenetic reconstruction is based on a single mitochondrial gene (cyt b), phylogenies based on genes from the mitochondrial, nuclear, and apicoplast genomes are broadly consistent for the relatively recent nodes considered in this analysis (6, 12–14, 33–35). In addition, analyses of avian hemosporidian parasites based on multiple independent markers have distinguished mtDNA-defined lineages on the basis of significant linkage disequilibrium (13).We distinguish as species the lineages that differ in their mtDNA cytochrome b gene sequence (by as few as 2 nt) and, for the most part, occur in either different hosts in the same local area or the same or different hosts in different geographic areas (32, 36). In some cases, closely related lineages occur in the same host locally. Sister lineages in this analysis differ by an average of about 1% sequence divergence, although some sequences separated by as little as a single nucleotide can exhibit consistent host or geographic differences. Inference concerning the mode of species formation is based primarily on host and geographic distributions of these hemosporidian mtDNA lineages. However, the correspondence between lineages and reproductively isolated species is poorly resolved (13, 37, 38). Each node was designated as either sympatric or allopatric depending on whether the descendant lineages occurred on the same West Indian islands or in the same regions within larger continental areas. The status of closely related parasite lineages occurring locally in the same host species is ambiguous, but these lineages might reflect genetic variation within a parasite species.Previous analyses have suggested that host shifting, rather than codivergence, predominates species formation in the hemosporidian parasites of birds (5, 7). We find this most frequently to be the case in this analysis, and we discuss whether species formation by host shifting occurs primarily in sympatry or allopatry. 相似文献
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Hea-Jin Jung Chika Nobumori Chris N. Goulbourne Yiping Tu John M. Lee Angelica Tatar Daniel Wu Yuko Yoshinaga Pieter J. de Jong Catherine Coffinier Loren G. Fong Stephen G. Young 《Proceedings of the National Academy of Sciences of the United States of America》2013,110(21):E1923-E1932
The role of protein farnesylation in lamin A biogenesis and the pathogenesis of progeria has been studied in considerable detail, but the importance of farnesylation for the B-type lamins, lamin B1 and lamin B2, has received little attention. Lamins B1 and B2 are expressed in nearly every cell type from the earliest stages of development, and they have been implicated in a variety of functions within the cell nucleus. To assess the importance of protein farnesylation for B-type lamins, we created knock-in mice expressing nonfarnesylated versions of lamin B1 and lamin B2. Mice expressing nonfarnesylated lamin B2 developed normally and were free of disease. In contrast, mice expressing nonfarnesylated lamin B1 died soon after birth, with severe neurodevelopmental defects and striking nuclear abnormalities in neurons. The nuclear lamina in migrating neurons was pulled away from the chromatin so that the chromatin was left “naked” (free from the nuclear lamina). Thus, farnesylation of lamin B1—but not lamin B2—is crucial for brain development and for retaining chromatin within the bounds of the nuclear lamina during neuronal migration.The nuclear lamina is an intermediate filament meshwork that lies beneath the inner nuclear membrane. This lamina provides structural support for the nucleus and also interacts with nuclear proteins and chromatin, thereby affecting many functions within the cell nucleus (1, 2). In mammals, the main protein components of the nuclear lamina are lamins A and C (A-type lamins) and lamins B1 and B2 (B-type lamins).Both B-type lamins and prelamin A (the precursor of lamin A) terminate with a CaaX motif, which triggers three posttranslational modifications (3–5): farnesylation of the carboxyl-terminal cysteine (the “C” in the CaaX motif) (6), endoproteolytic cleavage of the last three amino acids (the -aaX) (7, 8), and carboxyl methylation of the newly exposed farnesylcysteine (9, 10). Prelamin A undergoes a second endoproteolytic cleavage event, mediated by zinc metalloproteinase STE24 (ZMPSTE24), which removes 15 additional amino acids from the carboxyl terminus, including the farnesylcysteine methyl ester (11–14). Lamins B1 and B2 do not undergo the second cleavage step and therefore retain their farnesyl lipid anchor.The discovery that Hutchinson-Gilford progeria syndrome (HGPS) is caused by a LMNA mutation yielding an internally truncated farnesyl–prelamin A (15) has focused interest in the farnesylation of nuclear lamins. This interest has been fueled by the finding that disease phenotypes in mouse models of HGPS could be ameliorated by blocking protein farnesylation with a protein farnesyltransferase inhibitor (FTI) (16–19). Most recently, children with HGPS seemed to show a positive response to FTI treatment (20).The prospect of using an FTI to treat children with HGPS naturally raises the issue of the importance of protein farnesylation for lamin B1 and lamin B2. The B-type lamins are expressed in all mammalian cells and have been highly conserved during vertebrate evolution. The B-type lamins have been reported to participate in many functions within the cell nucleus, including DNA replication (21) and the formation of the mitotic spindle (22). Recently, both lamin B1 and lamin B2 have been shown to be important for neuronal migration within the developing brain (23–26). A deficiency of either protein causes abnormal layering of cortical neurons (23, 24). Coffinier et al. (24) proposed that the neuronal migration defect might be the consequence of impaired integrity of the nuclear lamina. Whether the farnesylation of lamin B1 or lamin B2 is important for neuronal migration is not known.Over the past few years, it has become increasingly clear that mouse models are important for elucidating the in vivo relevance of lamin posttranslational processing. In the case of prelamin A, cell culture studies suggested that protein farnesylation plays a vital role in the targeting of prelamin A to the nuclear rim (27–29), but recent studies with gene-targeted mice have raised questions about the in vivo relevance of those findings. For example, knock-in mice that produce mature lamin A directly (bypassing prelamin A synthesis and protein farnesylation) are free of disease, and the nuclear rim positioning of lamin A in the tissues of mice is quite normal (30).To assess the importance of protein farnesylation for B-type lamins, we reasoned that mouse models would be even more important because these lamins are important for neuronal migration in the developing brain, a complex process that requires the use of animal models. In the present study, we investigated the in vivo functional relevance of protein farnesylation in B-type lamins by creating knock-in mice expressing nonfarnesylated versions of lamin B1 and lamin B2. 相似文献
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