Born in the cold: contrasted thermal exchanges and maintenance costs in juvenile and adult snow buntings on their breeding and wintering grounds.

Several species of passerines leave their nest with unfinished feather growth, resulting in lower feather insulation and increased thermoregulatory demands compared to adults. However, feather insulation is essential for avian species breeding at northern latitudes, where cold conditions or even snowstorms can occur during the breeding season. In altricial arctic species, increased heat loss caused by poor feather insulation during growth could be counter-adaptative as it creates additional energy demands for thermoregulation. Using flow-through respirometry, we compared resting metabolic rate at thermoneutrality (RMRt), summit metabolic rate (Msum) and heat loss (conductance) in adult and juvenile snow buntings on their summer and winter grounds. In summer, when buntings are in the Arctic, juveniles had a 12% higher RMRt, likely due to unfinished growth, and lost 14% more heat to the environment than adults. This pattern may result from juveniles fledging early to avoid predation at the cost of lower feather insulation. Surprisingly, an opposite pattern was observed at lower latitudes on their wintering grounds. Although they showed no difference in RMRt and Msum, adults were losing 12% more heat than juveniles. We suggest that this difference is due to poorer insulative property of plumage in adults stemming from energetic and time constraints encountered during their post-breeding molt. High plumage insulation in first-winter juvenile buntings could be adaptive to reduce thermoregulatory demands and maximize survival in the first winter of life, while adults could use behavioral strategies to compensate for their greater rate of heat loss.

VGF is required for recovery after focal stroke.

The high incidence of ischemic stroke worldwide and poor efficacy of neuroprotective drugs has increased the need for novel therapies in stroke recovery. Transcription of the neurosecretory protein VGF (non-acronym) is enhanced following ischemic stroke and proposed to be important for stroke recovery. To determine the requirement for VGF in recovery, we created Vgffl/fl:Nestin-Cre conditional knockout (Vgf cKO) mice and induced a photothrombotic focal ischemic stroke. Naïve Vgf cKO mice had significant less body weight in the absence of gross defects in brain size, cortical lamination, or deficits in locomotor activity compared to wildtype controls. Following a focal stroke, the Vgf cKO mice had greater deficits including impaired recovery of forepaw motor deficits at 2- and 4-weeks post stroke. The increase in deficits occurred in the absence of any difference in lesion size and was accompanied by a striking loss of stroke-induced migration of SVZ-derived immature neurons to the peri-infarct region. Importantly, exogenous adenoviral delivery of VGF (AdVGF) significantly improved recovery in the Vgf cKO mice and was able to rescue the immature neuron migration defect observed. Taken together, our results define a requirement for VGF in post stroke recovery and identify VGF peptides as a potential future therapeutic.

Warming in the land of the midnight sun: breeding birds may suffer greater heat stress at high- versus low-Arctic sites.

Rising global temperatures are expected to increase reproductive costs for wildlife as greater thermoregulatory demands interfere with reproductive activities. However, predicting the temperatures at which reproductive performance is negatively impacted remains a significant hurdle. Using a thermoregulatory polygon approach, we derived a reproductive threshold temperature for an Arctic songbird-the snow bunting (Plectrophenax nivalis). We defined this threshold as the temperature at which individuals must reduce activity to suboptimal levels (i.e. less than four-time basal metabolic rate) to sustain nestling provisioning and avoid overheating. We then compared this threshold to operative temperatures recorded at high (82° N) and low (64° N) Arctic sites to estimate how heat constraints translate into site-specific impacts on sustained activity level. We predict buntings would become behaviourally constrained at operative temperatures above 11.7°C, whereupon they must reduce provisioning rates to avoid overheating. Low-Arctic sites had larger fluctuations in solar radiation, consistently producing daily periods when operative temperatures exceeded 11.7°C. However, high-latitude birds faced entire, consecutive days when parents would be unable to sustain required provisioning rates. These data indicate that Arctic warming is probably already disrupting the breeding performance of cold-specialist birds and suggests counterintuitive and severe negative impacts of warming at higher latitude breeding locations.

Extraordinarily rapid proliferation of cultured muscle satellite cells from migratory birds.

Migratory birds experience bouts of muscle growth and depletion as they prepare for, and undertake prolonged flight. Our studies of migratory bird muscle physiology in vitro led to the discovery that sanderling (Calidris alba) muscle satellite cells proliferate more rapidly than other normal cell lines. Here we determined the proliferation rate of muscle satellite cells isolated from five migratory species (sanderling; ruff, Calidris pugnax; western sandpiper, Calidris mauri; yellow-rumped warbler, Setophaga coronata; Swainson's thrush, Catharus ustulatus) from two families (shorebirds and songbirds) and with different migratory strategies. Ruff and sanderling satellite cells exhibited rapid proliferation, with population doubling times of 9.3 ± 1.3 and 11.4 ± 2 h, whereas the remaining species' cell doubling times were greater than or equal to 24 h. The results indicate that the rapid proliferation of satellite cells is not associated with total migration distance but may be related to flight bout duration and interact with lifespan.

Limited heat tolerance in an Arctic passerine: Thermoregulatory implications for cold-specialized birds in a rapidly warming world.

Arctic animals inhabit some of the coldest environments on the planet and have evolved physiological mechanisms for minimizing heat loss under extreme cold. However, the Arctic is warming faster than the global average and how well Arctic animals tolerate even moderately high air temperatures (T a) is unknown.Using flow-through respirometry, we investigated the heat tolerance and evaporative cooling capacity of snow buntings (Plectrophenax nivalis; ≈31 g, N = 42), a cold specialist, Arctic songbird. We exposed buntings to increasing T a and measured body temperature (T b), resting metabolic rate (RMR), rates of evaporative water loss (EWL), and evaporative cooling efficiency (the ratio of evaporative heat loss to metabolic heat production).Buntings had an average (±SD) T b of 41.3 ± 0.2°C at thermoneutral T a and increased T b to a maximum of 43.5 ± 0.3°C. Buntings started panting at T a of 33.2 ± 1.7°C, with rapid increases in EWL starting at T a = 34.6°C, meaning they experienced heat stress when air temperatures were well below their body temperature. Maximum rates of EWL were only 2.9× baseline rates at thermoneutral T a, a markedly lower increase than seen in more heat-tolerant arid-zone species (e.g., ≥4.7× baseline rates). Heat-stressed buntings also had low evaporative cooling efficiencies, with 95% of individuals unable to evaporatively dissipate an amount of heat equivalent to their own metabolic heat production.Our results suggest that buntings' well-developed cold tolerance may come at the cost of reduced heat tolerance. As the Arctic warms, and this and other species experience increased periods of heat stress, a limited capacity for evaporative cooling may force birds to increasingly rely on behavioral thermoregulation, such as minimizing activity, at the expense of diminished performance or reproductive investment.

Species-specific metabolic responses of songbird, shorebird, and murine cultured myotubes to n-3 polyunsaturated fatty acids.

Migratory birds may benefit from diets rich in polyunsaturated fatty acids (PUFAs) that could improve exercise performance. Previous investigations suggest that different types of birds may respond differently to PUFA. We established muscle myocyte cell culture models from muscle satellite cells of a migratory passerine songbird (yellow-rumped warbler, Setophaga coronata coronata) and a nonpasserine shorebird (sanderling, Calidris alba). We differentiated and treated avian myotubes and immortalized murine C2C12 myotubes with n-3 PUFA docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), and with monounsaturated oleic acid (OA) to compare effects on aerobic performance, metabolic enzyme activities, key fatty acid (FA) transporters, and expression of peroxisome proliferator-activated receptors (PPARs). Sanderling and C2C12 myotubes increased expression of PPARs with n-3 PUFA treatments, whereas expression was unchanged in yellow-rumped warblers. Both sanderlings and yellow-rumped warblers increased expression of fatty acid transporters, whereas C2C12 cells decreased expression following n-3 PUFA treatments. Only yellow-rumped warbler myotubes increased expression of some metabolic enzymes, whereas the sanderling and C2C12 cells were unchanged. PUFA supplementation in C2C12 myotubes increased mitochondrial respiratory chain efficiency, whereas sanderlings increased proton leak-associated respiration and maximal respiration (measurements were not made in warblers). This research indicates that songbirds and shorebirds respond differently to n-3 PUFA and provides support for the hypothesis that n-3 PUFA increase the aerobic capacity of migrant shorebird muscle, which may improve overall endurance flight performance.

Correction to: C3aR signaling and gliosis in response to neurodevelopmental damage in the cerebellum.

Following publication of the original article, the authors noticed missing labels in Fig. 1a.

C3aR signaling and gliosis in response to neurodevelopmental damage in the cerebellum.

BACKGROUND: Conditional ablation of the Smarca5 gene in mice severely impairs the postnatal growth of the cerebellum and causes an ataxic phenotype. Comparative gene expression studies indicated that complement-related proteins were upregulated in the cerebellum of Smarca5 mutant mice. Complement proteins play critical roles within innate immune signaling pathways and, in the brain, are produced by glial cells under both normal and pathological conditions. The C3 complement protein-derived signaling peptide, C3a, has been implicated in contributing to both tissue damage and repair in conditions such as multiple sclerosis and stroke. Here, we investigated whether C3a receptor (C3aR) signaling promoted damage or repair in the developing cerebellum of Smarca5 mutant mice. METHODS: Brain and cerebellum lysates from single Smarca5 conditional knockout (Smarca5 cKO) mice, C3aR1 KO mice, or double mutant mice were used for qRT-PCR and immunoblotting to assess the contribution of C3aR to the Smarca5 cKO brain pathology. Immunohistochemistry was used to characterize alterations to astroglia and phagocyte cells in the developing cerebellum of each of the genotypes. RESULTS: C3aR signaling was observed to limit gliosis and promote granule neuron survival during postnatal cerebellar development. In Smarca5 cKO mice, disorganized astroglia with increased GFAP expression develops concurrently with cerebellar granule neuron loss and phagocyte invasion over the first 10 days following birth. Potential ligand precursors of C3aR-VGF and C3-were found to have upregulated expression and/or altered processing during this time. Phagocytes (microglia and macrophages) in both the control and Smarca5 mutant mice were the only cells observed to express C3aR. Loss of C3aR in the Smarca5 cKO cerebellum resulted in increased numbers of apoptotic cells and early phagocyte invasion into the external granule cell layer, as well as an exacerbated disorganization of the Bergmann glia. The loss of C3aR expression also attenuated an increase in the expression of the efferocytosis-related protein, MerTK, whose transcript was upregulated ~ 2.5-fold in the Smarca5 mutant cerebellum at P10. CONCLUSIONS: This data indicates that C3aR can play an important role in limiting astrogliosis and regulating phagocyte phenotypes following developmental cell loss in the brain.

Recovery from impaired muscle growth arises from prolonged postnatal accretion of myonuclei in Atrx mutant mice.

Reduced muscle mass due to pathological development can occur through several mechanisms, including the loss or reduced proliferation of muscle stem cells. Muscle-specific ablation of the α-thalassemia mental retardation syndrome mutant protein, Atrx, in transgenic mice results in animals with a severely reduced muscle mass at three weeks of age; yet this muscle mass reduction resolves by adult age. Here, we explore the cellular mechanism underlying this effect. Analysis of Atrx mutant mice included testing for grip strength and rotorod performance. Muscle fiber length, fiber volume and numbers of myofiber-associated nuclei were determined from individual EDL or soleus myofibers isolated at three, five, or eight weeks. Myofibers from three week old Atrx mutant mice are smaller with fewer myofiber-associated nuclei and reduced volume compared to control animals, despite similar fiber numbers. Nonetheless, the grip strength of Atrx mutant mice was comparable to control mice when adjusted for body weight. Myofiber volume remained smaller at five weeks, becoming comparable to controls by 8 weeks of age. Concomitantly, increased numbers of myofiber-associated nuclei and Ki67+ myoblasts indicated that the recovery of muscle mass likely arises from the prolonged accretion of new myonuclei. This suggests that under disease conditions the muscle satellite stem cell niche can remain in a prolonged active state, allowing for the addition of a minimum number of myonuclei required to achieve a normal muscle size.

Transgenic expression of neuronal dystonin isoform 2 partially rescues the disease phenotype of the dystonia musculorum mouse model of hereditary sensory autonomic neuropathy VI.

A newly identified lethal form of hereditary sensory and autonomic neuropathy (HSAN), designated HSAN-VI, is caused by a homozygous mutation in the bullous pemphigoid antigen 1 (BPAG1)/dystonin gene (DST). The HSAN-VI mutation impacts all major neuronal BPAG1/dystonin protein isoforms: dystonin-a1, -a2 and -a3. Homozygous mutations in the murine Dst gene cause a severe sensory neuropathy termed dystonia musculorum (dt). Phenotypically, dt mice are similar to HSAN-VI patients, manifesting progressive limb contractures, dystonia, dysautonomia and early postnatal death. To obtain a better molecular understanding of disease pathogenesis in HSAN-VI patients and the dt disorder, we generated transgenic mice expressing a myc-tagged dystonin-a2 protein under the regulation of the neuronal prion protein promoter on the dt(Tg4/Tg4) background, which is devoid of endogenous dystonin-a1 and -a2, but does express dystonin-a3. Restoring dystonin-a2 expression in the nervous system, particularly within sensory neurons, prevented the disorganization of organelle membranes and microtubule networks, attenuated the degeneration of sensory neuron subtypes and ameliorated the phenotype and increased life span in these mice. Despite these improvements, complete rescue was not observed likely because of inadequate expression of the transgene. Taken together, this study provides needed insight into the molecular basis of the dt disorder and other peripheral neuropathies including HSAN-VI.

Modulation of antigenic location converts chronic into acute infection by forcing CD8+ T cell recognition.

Pathogens that reside in the phagosomes of infected cells persist despite the presence of potent T cell responses. We addressed the mechanism of immune evasion by using a mouse model of Salmonella typhimurium (ST). Recombinants of ST were generated that translocated antigen to the cytosol or phagosomes of infected cells. We find that the kinetics of antigen presentation and CD8(+) T cell priming is accelerated by cytosolic antigen delivery, although the magnitude of CD8(+) T cell response is not influenced by antigenic location. More importantly, only those targets that readily display antigen on the cell surface, owing to antigenic translocation to the cytosol, are recognized and killed by CD8(+) T cells. Thus, vaccination approaches developed to control phagosomal pathogens should incorporate methods for modulating antigen presentation such that infected target cells can be readily recognized by CD8(+) T cells.

MAP1B and clathrin are novel interacting partners of the giant cyto-linker dystonin.

Dystonin is a large multidomain cytoskeletal-associated protein that plays an essential role in the nervous system. Loss of dystonin results in neuromuscular dysfunction and early death in a mouse mutant called dystonia musculorum. Conserved among related proteins, the plakin domain is a defining feature of all major dystonin isoforms, yet its interactions have not been explored in detail. The purpose of the present study was to identify novel interacting partners of the plakin domain of the neuronal isoform of dystonin (dystonin-a). Newly identified interacting proteins discovered through a pull-down assay were validated using coimmunoprecipitation, coimmunofluorescence, and proximity ligation assays. Microtubule associated protein 1B (MAP1B), a microtubule stabilizing protein, and clathrin heavy chain, the major component of the clathrin triskelion, were identified as interaction partners for dystonin-a. Increased levels of phosphorylated MAP1B suggest a misregulation of MAP1B and a potentially novel component of the dt pathology. This work will further facilitate our understanding of how cytoskeletal proteins can affect and regulate neurodegenerative disorders.

CD8+ T cells primed in the periphery provide time-bound immune-surveillance to the central nervous system.

After vaccination, memory CD8(+) T cells migrate to different organs to mediate immune surveillance. In most nonlymphoid organs, following an infection, CD8(+) T cells differentiate to become long-lived effector-memory cells, thereby providing long-term protection against a secondary infection. In this study, we demonstrated that Ag-specific CD8(+) T cells that migrate to the mouse brain following a systemic Listeria infection do not display markers reminiscent of long-term memory cells. In contrast to spleen and other nonlymphoid organs, none of the CD8(+) T cells in the brain reverted to a memory phenotype, and all of the cells were gradually eliminated. These nonmemory phenotype CD8(+) T cells were found primarily within the choroid plexus, as well as in the cerebrospinal fluid-filled spaces. Entry of these CD8(+) T cells into the brain was governed primarily by CD49d/VCAM-1, with the majority of entry occurring in the first week postinfection. When CD8(+) T cells were injected directly into the brain parenchyma, cells that remained in the brain retained a highly activated (CD69(hi)) phenotype and were gradually lost, whereas those that migrated out to the spleen were CD69(low) and persisted long-term. These results revealed a mechanism of time-bound immune surveillance to the brain by CD8(+) T cells that do not reside in the parenchyma.

Formins in cell signaling.

The founding formin homology protein family members were implicated early on as being involved in regulating cytoskeletal remodeling pathways, as formin protein mutations in Drosophila and yeast lead to obvious actin cytoskeleton defects. The discovery that these proteins associated directly with small Rho family GTPases confirmed these results and greatly enhanced our understanding of their function. The mammalian diaphanous-related formins (DRFs) were subsequently recognized as being involved in activation of serum response factor (SRF), tying formins to transcriptional regulation. In the past few years, much progress has been made in demonstrating how DRFs act as both downstream effectors and upstream modulators of Rho GTPase signaling. These functions are important for regulation of both actin and microtubule cytoskeletal structures, and affect cellular processes such as the establishment of polarity, vesicle movement, and focal adhesion remodeling. The connection of DRFs to the SH3 domain-containing protein, Src, has also been described as being important to several basic cellular functions. While still unresolved, extensive work has been carried out on how DRFs mediate SRF activation, and the importance of this to the regulation of cytoskeletal structure. This review will focus on the role of formins in cytoplasmic signal transduction pathways and the downstream effects on the regulation of gene expression.

INF1 is a novel microtubule-associated formin.

Formin proteins, characterized by the presence of conserved formin homology (FH) domains, play important roles in cytoskeletal regulation via their abilities to nucleate actin filament formation and to interact with multiple other proteins involved in cytoskeletal regulation. The C-terminal FH2 domain of formins is key for actin filament interactions and has been implicated in playing a role in interactions with microtubules. Inverted formin 1 (INF1) is unusual among the formin family in having the conserved FH1 and FH2 domains in its N-terminal half, with its C-terminal half being composed of a unique polypeptide sequence. In this study, we have examined a potential role for INF1 in regulating microtubule structure. INF1 associates discretely with microtubules, and this association is dependent on a novel C-terminal microtubule-binding domain. INF1 expressed in fibroblast cells induced actin stress fiber formation, coalignment of microtubules with actin filaments, and the formation of bundled, acetylated microtubules. Endogenous INF1 showed an association with acetylated microtubules, and knockdown of INF1 resulted in decreased levels of acetylated microtubules. Our data suggests a role for INF1 in microtubule modification and potentially in coordinating microtubule and F-actin structure.

Dystonin/Bpag1 is a necessary endoplasmic reticulum/nuclear envelope protein in sensory neurons.

Dystonin/Bpag1 proteins are cytoskeletal linkers whose loss of function in mice results in a hereditary sensory neuropathy with a progressive loss of limb coordination starting in the second week of life. These mice, named dystonia musculorum (dt), succumb to the disease and die of unknown causes prior to sexual maturity. Previous evidence indicated that cytoskeletal defects in the axon are a primary cause of dt neurodegeneration. However, more recent data suggests that other factors may be equally important contributors to the disease process. In the present study, we demonstrate perikaryal defects in dorsal root ganglion (DRG) neurons at stages preceding the onset of loss of limb coordination in dt mice. Abnormalities include alterations in endoplasmic reticulum (ER) chaperone protein expression, indicative of an ER stress response. Dystonin in sensory neurons localized in association with the ER and nuclear envelope (NE). A fusion protein ofthe dystonin-a2 isoform, which harbors an N-terminal transmembrane domain, associated with and reorganized the ER in cell culture. This isoform also interacts with the NE protein nesprin-3alpha, but not nesprin-3beta. Defects in dt mice, as demonstrated here, may ultimately result in pathogenesis involving ER dysfunction and contribute significantly to the dt phenotype.

Dystonin/Bpag1--a link to what?

The dystonin/Bpag1 cytoskeletal interacting proteins play important roles in maintaining cytoarchitecture integrity in skin and in the neuromuscular system. The most profound phenotype observed in the dystonin mutant dystonia musculorum (dt) mice is a severe movement disorder, attributed in large part to sensory neuron degeneration. The molecular basis for this phenotype is currently not clear, despite several studies indicating possible causes for the pathology in dt mice. Complicating the picture of what essential dystonin functions are lost in dt mice is the fact that our understanding of the very nature of what dystonin is has evolved greatly over the past decade. Elucidating the roles of dystonin most relevant to neuronal function and survival should help to shed light on some of the common mechanisms underlying neurodegeneration.

A Bpag1 isoform involved in cytoskeletal organization surrounding the nucleus.

Bpag1/dystonin proteins are giant cytoskeletal interacting proteins postulated to cross-link cytoskeletal filaments and thereby maintain cellular integrity. Loss of function of non-epithelial Bpag1 isoforms results in neuromuscular dysfunction and early postnatal death in mice. Multiple Bpag1 transcripts have been described, including those encoding protein isoforms that vary at the N-terminal end. Here, we have analyzed the subcellular localizations and cytoskeletal interactions of two isoforms, termed Bpag1a1 and Bpag1a2. We demonstrate that novel sequence at the 5' end of the Bpag1a2 transcript codes for an N-terminal transmembrane domain and targets the protein to the perinuclear region of the cell. Furthermore, we show that the endogenous Bpag1a2 protein is also present in the perinuclear region in myoblast cells. Differences in Bpag1a1 and Bpag1a2 with respect to the extent of their interactions with microtubules and microfilaments are also described, with Bpag1a2 fusion protein serving largely to associate with microfilaments surrounding the nucleus and Golgi apparatus. The overall structure and subcellular localizations of Bpag1a2 indicate possible functions in nuclear envelope structuring, nuclear tethering, and organization of membranous structures surrounding the nucleus.

Genetic alterations at the Bpag1 locus in dt mice and their impact on transcript expression.

The dystonin/Bpag1 gene encodes several tissue-specific alternatively spliced transcripts that encode cytoskeletal binding proteins. These various isoforms are necessary for maintaining the structural integrity of epithelial, neural, and muscle tissues. Mutations in the dystonin/Bpag1 gene cause dystonia musculorum (dt), a hereditary neuropathy of the mouse characterized by the progressive degeneration of sensory neurons. Several dt mutant alleles exist, most of which have arisen through spontaneous mutations. In this article we demonstrate that the dt locus encodes 107 exons spanning 400 kb. The high frequency of occurrence of spontaneous dt mutants may therefore be a result of the large size of the gene. Analysis of genomic DNA from several dt spontaneous mutant alleles, dt(24J), dt(27J), dt(Alb), and dt(Frk), shows a deletion of the central portion of the gene in dt(Alb) but no large rearrangements or deletions in the other alleles. These other alleles likely have small deletions or rearrangements, or point mutations. To determine the impact of the known and unknown mutations on transcript levels, RT-PCR was performed to detect various coding regions of the dystonin/Bpag1 transcripts in brain and muscle from multiple dt alleles: dt(Tg4), dt(Alb), dt(24J), dt(27J), and dt(Frk). With the exception of dt(Frk), reduced transcript levels were observed for all alleles tested. Such alterations likely result in reduced or absent dystonin/Bpag1 protein levels. Thus, distinct genetic defects lead to a common outcome of reduced transcript expression causing the same phenotype in multiple dt alleles.