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Priming at the site of natural infection typically elicits a protective T cell response against subsequent pathogen encounter. Here, we report the identification of a novel fungal antigen that we harnessed for mucosal vaccination and tetramer generation to test whether we can elicit protective, antigen-specific tissue resident memory (Trm) CD4+ T cells in the lung parenchyma. In contrast to expectations, CD69+, CXCR3+, CD103− Trm cells failed to protect against a lethal pulmonary fungal infection. Surprisingly, systemic vaccination induced a population of tetramer+ CD4+ T cells enriched within the pulmonary vasculature, and expressing CXCR3 and CX3CR1, that migrated to the lung tissue upon challenge and efficiently protected mice against infection. Mucosal vaccine priming of Trm may not reliably protect against mucosal pathogens.

The development of safe subunit vaccines requires adjuvants that augment immunogenicity of non-replicating protein-based antigens. Current vaccines against infectious diseases preferentially induce protective antibodies driven by adjuvants such as alum. However, the contribution of antibody to host defense is limited for certain classes of infectious diseases such as fungi, whereas animal studies and clinical observations implicate cellular immunity as an essential component of the resolution of fungal pathogens. Here, we decipher the structural bases of a newly identified glycoprotein ligand of Dectin-2 with potent adjuvancy, Blastomyces endoglucanase-2 (Bl-Eng2). We also pinpoint the developmental steps of antigen-specific CD4+ and CD8+ T responses augmented by Bl-Eng2 including expansion, differentiation and tissue residency. Dectin-2 ligation led to successful systemic and mucosal vaccination against invasive fungal infection and Influenza A infection, respectively. O-linked glycans on Bl-Eng2 applied at the skin and respiratory mucosa greatly augment vaccine subunit- induced protective immunity against lethal influenza and fungal pulmonary challenge., Author summary Fungal disease remains a challenging clinical and public health problem in part because there is no commercial vaccine available. The lack of suitable adjuvants is a critical barrier to developing safe and effective vaccines against fungal pathogens. Current adjuvants such as alum preferentially induce antibody responses which may be limited in mediating protection against fungi. Clinical observations and animal studies implicate cellular immunity as the essential component for the resolution of fungal infections. We have recently discovered an adjuvant that augments cell mediated immune responses and vaccine induced protection against fungi. Here, we identified the structural and mechanistic requirements by which this newly discovered adjuvant induces cell mediated immunity against fungi. As a proof of principle we also demonstrate that the adjuvant drives cellular immune responses against viruses such as influenza. We anticipate that our adjuvant can be used for vaccination with safe subunit vaccines against many microbial pathogens including viruses, intracellular bacteria, fungi and parasites that require cell mediated immune responses.

Dimorphic fungi in the genera Blastomyces, Histoplasma, Coccidioides, and Paracoccidioides are important human pathogens that affect human health in many countries throughout the world. Understanding the biology of these fungi is important for the development of effective treatments and vaccines. Gene editing is a critically important tool for research into these organisms. In recent years, gene targeting approaches employing RNA-guided DNA nucleases, such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9), have exploded in popularity. Here, we provide a detailed description of the steps involved in applying CRISPR/Cas9 technology to dimorphic fungi, with Blastomyces dermatitidis in particular as our model fungal pathogen. We discuss the design and construction of single guide RNA and Cas9-expressing targeting vectors (including multiplexed vectors) as well as introduction of these plasmids into Blastomyces using Agrobacterium-mediated transformation. Finally, we cover the outcomes that may be expected in terms of gene-editing efficiency and types of gene alterations produced. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Construction of CRISPR/Cas9 targeting vectors Support Protocol 1: Choosing protospacers in the target gene Basic Protocol 2: Agrobacterium-mediated transformation of Blastomyces Support Protocol 2: Preparation of electrocompetent Agrobacterium Support Protocol 3: Preparation and recovery of Blastomyces frozen stocks.

Airway epithelium is the first body surface to contact inhaled irritants and report danger. Here, we report how epithelial cells recognize and respond to aeroallergen alkaline protease 1 (Alp1) of Aspergillus sp., because proteases are critical components of many allergens that provoke asthma. In a murine model, Alp1 elicits helper T (Th) cell-dependent lung eosinophilia that is initiated by the rapid response of bronchiolar club cells to Alp1. Alp1 damages bronchiolar cell junctions, which triggers a calcium flux signaled through calcineurin within club cells of the bronchioles, inciting inflammation. In two human cohorts, we link fungal sensitization and/or asthma with SNP/protein expression of the mechanosensitive calcium channel, TRPV4. TRPV4 is also necessary and sufficient for club cells to sensitize mice to Alp1. Thus, club cells detect junction damage as mechanical stress, which signals danger via TRPV4, calcium, and calcineurin to initiate allergic sensitization.

Mitochondrial mutations and dysfunction have been demonstrated in several age-related disorders including osteoarthritis, yet its relative contribution to pathogenesis remains unknown. Here we evaluated whether premature aging caused by accumulation of mitochondrial DNA mutations in PolgD275A mice predisposes to the development of knee osteoarthritis. Compared with wild type animals, homozygous PolgD275A mice displayed a specific bone phenotype characterized by osteopenia of epiphyseal trabecular bone and subchondral cortical plate. Trabecular thickness was significantly associated with osteocyte apoptosis rates and osteoclasts numbers were increased in subchondral bone tissues. While chondrocyte apoptosis rates in articular and growth plate cartilage were similar between groups, homozygous mitochondrial DNA mutator mice displayed elevated numbers of hypertrophic chondrocytes in articular calcified cartilage. Low grade cartilage degeneration, predominantly loss of proteoglycans, was present in all genotypes and the development of osteoarthritis features was not found accelerated in premature aging. Somatically acquired mitochondrial DNA mutations predispose to elevated subchondral bone turnover and hypertrophy in calcified cartilage, yet additional mechanical or metabolic stimuli would seem required for induction and accelerated progression of aging-associated osteoarthritis.

The retrotransposon long interspersed nuclear element-1 (LINE-1) can autonomously increase its copy number within a host genome through the retrotransposition process. LINE-1 is active in the germline and in neural progenitor cells, and its somatic retrotransposition activity has a broad impact on neural development and susceptibility to neuropsychiatric disorders. The method to quantify the genomic copy number of LINE-1 would be important in unraveling the role of retrotransposition, especially in the brain. However, because of the species-specific evolution of LINE-1 sequences, methods for quantifying the copy number should be independently developed. Here, we developed a quantitative PCR (qPCR) assay to measure the copy number of active LINE-1 subfamilies in mice. Using the assay, we investigated aging-associated alterations of LINE-1 copy number in several brain regions in wild-type mice and Polg+/D257A mice as a model for accelerated aging. We found that aged Polg+/D257A mice showed higher levels of the type GfII LINE-1 in the basal ganglia than the wild-type mice did, highlighting the importance of assays that focus on an individual active LINE-1 subfamily.

Background Mitochondrial dysfunction has been demonstrated in ageing and osteoarthritic tissues. However it remains unclear whether dysfunctional mitochondria are directly implied in the pathogenesis of osteoarthritis. Objectives We investigated knee joints of prematurely ageing mitochondrial DNA mutator mice ( Polg D275A ) to evaluate a causal relationship between mitochondrial dysfunction and different features of osteoarthritis. Methods Bone structural parameters and chondropathy were evaluated in knee joints of mice displaying increased mtDNA mutations rates and accelerated ageing, due to expression of a proofreading-deficient mtDNA polymerase, using micro-computed tomography and histopathological analysis. Results Homozygous mutants displayed osteopenia of the epiphyseal trabecular bone and subchondral cortical plate in comparison to wild type controls and heterozygous mutants. Osteopenia was associated with a strong increase of osteoclast numbers (0.88±0.30/mm bone perimeter) compared to heterozygous (0.25±0.03/mm) and wild type mice (0.12±0.04/mm). New bone formation was not observed. Wild type mice displayed only low grade cartilage degeneration (OARSI grade ≤1) due to loss of cartilage proteoglycans. Increased tibio-femoral chondropathy was not apparent in hetero- and homozygous mitochondrial DNA mutator mice. Conclusions Mitochondrial dysfunction and premature ageing in mice with somatically acquired mtDNA mutations predisposes to enhanced subchondral bone resorption as potential early step of osteoarthritis, but not to cartilage damage or new bone formation. This phenotype potentially corresponds to an osteoporotic osteoarthritis phenotype in humans. Disclosure of Interest None declared

Objective Mutations in nuclear-encoded mitochondrial DNA (mtDNA) polymerase (POLG) are known to cause autosomal dominant chronic progressive external ophthalmoplegia (adCPEO) with accumulation of multiple mtDNA deletions in muscles. However, no animal model with a heterozygous Polg mutation representing mtDNA impairment and symptoms of CPEO has been established. To understand the pathogenic mechanism of CPEO, it is important to determine the age dependency and tissue specificity of mtDNA impairment resulting from a heterozygous mutation in the Polg gene in an animal model. Methods We assessed behavioral phenotypes, tissue-specific accumulation of mtDNA deletions, and its age dependency in heterozygous PolgD257A knock-in mice carrying a proofreading-deficient mutation in the Polg. Results Heterozygous PolgD257A knock-in mice exhibited motor dysfunction in a rotarod test. Polg+/D257A mice had significant accumulation of multiple mtDNA deletions, but did not show significant accumulation of point mutations or mtDNA depletion in the brain. While mtDNA deletions increased in an age-dependent manner regardless of the tissue even in Polg+/+ mice, the age-dependent accumulation of mtDNA deletions was enhanced in muscles and in the brain of Polg+/D257A mice. Interpretation Heterozygous PolgD257A knock-in mice showed tissue-specific, age-dependent accumulation of multiple mtDNA deletions in muscles and the brain which was likely to result in neuromuscular symptoms. Polg+/D257A mice may be used as an animal model of adCPEO associated with impaired mtDNA maintenance.

Mitochondrial DNA (mtDNA) mutations are thought to have a causative role in age-related pathologies. We have shown previously that mitochondrial mutator mice (PolgD257A/D257A), harboring a proofreading-deficient version of the mtDNA polymerase gamma (POLG), accumulate mtDNA mutations in multiple tissues and display several features of accelerated aging. Calorie restriction (CR) is known to delay the onset of age-related diseases and to extend the lifespan of a variety of species, including rodents. In the current study we investigated the effects of CR on the lifespan and healthspan of mitochondrial mutator mice. Long-term CR did not increase the median or maximum lifespan of PolgD257A/D257A mice. Furthermore, CR did not reduce mtDNA deletions in the heart and muscle, accelerated sarcopenia, testicular atrophy, nor improve the alterations in cardiac parameters that are present in aged mitochondrial mutator mice. Therefore, our findings suggest that accumulation of mtDNA mutations may interfere with the beneficial action of CR in aging retardation.

A causal role for mitochondrial DNA (mtDNA) mutagenesis in mammalian aging is supported by recent studies demonstrating that the mtDNA mutator mouse, harboring a defect in the proofreading-exonuclease activity of mitochondrial polymerase gamma, exhibits accelerated aging phenotypes characteristic of human aging, systemic mitochondrial dysfunction, multisystem pathology, and reduced lifespan. Epidemiologic studies in humans have demonstrated that endurance training reduces the risk of chronic diseases and extends life expectancy. Whether endurance exercise can attenuate the cumulative systemic decline observed in aging remains elusive. Here we show that 5 mo of endurance exercise induced systemic mitochondrial biogenesis, prevented mtDNA depletion and mutations, increased mitochondrial oxidative capacity and respiratory chain assembly, restored mitochondrial morphology, and blunted pathological levels of apoptosis in multiple tissues of mtDNA mutator mice. These adaptations conferred complete phenotypic protection, reduced multisystem pathology, and prevented premature mortality in these mice. The systemic mitochondrial rejuvenation through endurance exercise promises to be an effective therapeutic approach to mitigating mitochondrial dysfunction in aging and related comorbidities.

Recent reports describe hematopoietic abnormalities in mice with targeted instability of the mitochondrial genome. However, these abnormalities have not been fully described. We demonstrate that mutant animals develop an age-dependent, macrocytic anemia with abnormal erythroid maturation and megaloblastic changes, as well as profound defects in lymphopoiesis. Mice die of severe fatal anemia at 15 months of age. Bone-marrow transplantation studies demonstrate that these abnormalities are intrinsic to the hematopoietic compartment and dependent upon the age of donor hematopoietic stem cells. These abnormalities are phenotypically similar to those found in patients with refractory anemia, suggesting that, in some cases, the myelodysplastic syndromes are caused by abnormalities of mitochondrial function.

Mitochondrial DNA (mtDNA) mutations may contribute to aging and age-related diseases. Previously, we reported that accumulation of mtDNA mutations is associated with age-related hearing loss in mice carrying a mutator allele of the mitochondrial Polg DNA polymerase. To elucidate the role of mtDNA mutations in the pathogenesis of age-related hearing loss or presbycusis, we performed large scale gene expression analysis to identify mtDNA mutation-responsive genes and biological process categories associated with mtDNA mutations by comparing the gene expression patterns of cochlear tissues from 9-month-old mitochondrial mutator and control mice. mtDNA mutations were associated with transcriptional alterations consistent with impairment of energy metabolism, induction of apoptosis, cytoskeletal dysfunction, and hearing dysfunction in the cochlea of aged mitochondrial mutator mice. TUNEL staining and caspase-3 immunostaining analysis demonstrated that the levels of apoptotic markers were significantly increased in the cochleae of mitochondrial mutator mice compared to age-matched controls. These observations support a new model of how mtDNA mutations impact cochlear function whereby accumulation of mtDNA mutations lead to mitochondrial dysfunction, an associated impairment of energy metabolism, and the induction of an apoptotic program. The data presented here provide the first global assessment at the molecular level of the pathogenesis of age-related disease in mitochondrial mutator mice and reveal previously unrecognized biological pathways associated with mtDNA mutations.

AIM We have applied diffusion tensor imaging (DTI) to interrogate microstructural changes in white matter integrity in a widely used middle cerebral artery occlusion (MCAO) model of cerebral ischemia. MATERIAL AND METHODS We performed ex vivo DTI 35 days after 60 minutes transient focal ischemia in male spontaneously hypertensive rats and generated fractional anisotropy (FA), mean, axial and radial diffusivity maps. Regions of interest corresponding to external capsule (EC), corpus callosum (CC) and internal capsule (IC) were compared among sham and stroked rats. We compared tractographic projections of white matter fiber patterns and examined white matter integrity by Luxol fast blue histological analysis. We also determined infarct lesion volumes at 24 hours post-ischemia by T2-weighted magnetic resonance imaging (MRI) or at 35 days by histological staining with cresyl violet. RESULTS We found alterations in EC and IC, but not CC, as represented by decreased FA and increased mean, axial and radial diffusivities. The size of the ischemic lesion detected subacutely by T2-weighted MRI or at 35 days by histological staining correlated with the decline in FA in the affected structures. Tractography revealed disruption of fiber trajectories through the EC and reorientation of fibers within the caudate/putamen of rats subjected to MCAO. Similarly, loss of white matter integrity in the EC and increased white matter density in the caudate/putamen along the infarct border zone was evidenced by Luxol fast blue staining. CONCLUSION Diffusion tensor imaging therefore allows for monitoring of white matter injury and reorganization in hypertensive rats.

Background The role of mitochondrial dysfunction has long been implicated in age-related brain pathology, including Alzheimer’s disease (AD). However, the mechanism by which mitochondrial dysfunction may cause neurodegeneration in AD is unclear. To model mitochondrial dysfunction in vivo, we utilized mice that harbor a knockin mutation that inactivates the proofreading function of mitochondrial DNA polymerase γ (PolgA D257A), so that these mice accumulate mitochondrial DNA mutations with age. PolgA D257A mice develop a myriad of mitochondrial bioenergetic defects and physical phenotypes that mimic premature ageing, with subsequent death around one year of age. Results We crossed the D257A mice with a well-established transgenic AD mouse model (APP/Ld) that develops amyloid plaques. We hypothesized that mitochondrial dysfunction would affect Aβ synthesis and/or clearance, thus contributing to amyloidogenesis and triggering neurodegeneration. Initially, we discovered that Aβ42 levels along with Aβ42 plaque density were increased in D257A; APP/Ld bigenic mice compared to APP/Ld monogenic mice. Elevated Aβ production was not responsible for increased amyloid pathology, as levels of BACE1, PS1, C99, and C83 were unchanged in D257A; APP/Ld compared to APP/Ld mice. However, the levels of a major Aβ clearance enzyme, insulin degrading enzyme (IDE), were reduced in mice with the D257A mutation, suggesting this as mechanism for increased amyloid load. In the presence of the APP transgene, D257A mice also exhibited significant brain atrophy with apparent cortical thinning but no frank neuron loss. D257A; APP/Ld mice had increased levels of 17 kDa cleaved caspase-3 and p25, both indicative of neurodegeneration. Moreover, D257A; APP/Ld neurons appeared morphologically disrupted, with swollen and vacuolated nuclei. Conclusions Overall, our results implicate synergism between the effects of the PolgA D257A mutation and Aβ in causing neurodegeneration. These findings provide insight into mechanisms of mitochondrial dysfunction that may contribute to the pathogenesis of AD via decreased clearance of Aβ.

Mitochondrial DNA (mtDNA) mutations are hypothesized to play a pathogenic role in aging and age-related neurodegenerative diseases such as Parkinson's disease (PD). In support of this, high levels of somatic mtDNA mutations in “POLG mutator” mice carrying a proofreading-deficient form of mtDNA polymerase a (Polg(D257A)) lead to a premature aging phenotype. However, the relevance of this finding to the normal aging process has been questioned as the number of mutations is greater even in young POLG mutator mice, which shows no overt phenotype, than levels achieved during normal aging in mice. Vulnerability of dopaminergic neurons to 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) increases with age, and we hypothesized that this may result in part from the accumulation with age of somatic mtDNA mutations. If correct, then levels of mutations in young (2–3 month old) POLG mutator mice should be sufficient to increase vulnerability to MPTP. In contrast, we find that susceptibility to MPTP in both heterozygous and homozygous POLG mutator mice at this young age is not different from that of wild type littermate controls as measured by levels of tyrosine hydroxylase positive (TH+) striatal terminals, striatal dopamine and its metabolites, a marker of oxidative damage, or stereological counts of TH+ and total substantia nigra neurons. These unexpected results do not support the hypothesis that somatic mtDNA mutations contribute to the age-related vulnerability of dopaminergic neurons to MPTP. It remains possible that somatic mtDNA mutations influence vulnerability to other stressors, or require additional time for the deleterious consequences to manifest. Furthermore, the impact of the higher levels of mutations present at older ages in these mice was not assessed in our study, although a prior study also failed to detect an increase in vulnerability to MPTP in older mice. With these caveats, the current data do not provide evidence for a role of somatic mtDNA mutations in determining the vulnerability to MPTP.

Mitochondrial DNA (mtDNA) mutations are thought to have a causal role in many age-related pathologies. Here we identify mtDNA deletions as a driving force behind the premature aging phenotype of mitochondrial mutator mice, and provide evidence for a homology-directed DNA repair mechanism in mitochondria that is directly linked to the formation of mtDNA deletions. In addition, our results demonstrate that the rate at which mtDNA mutations reach phenotypic expression differs markedly among tissues, which may be an important factor in determining the tolerance of a tissue to random mitochondrial mutagenesis.

The mitochondrial DNA (mtDNA) polymerase γ (POLG) mutator mice provide the first experimental evidence that high levels of somatic mtDNA mutations can be functionally significant. Here we report that older homozygous, but not heterozygous, POLG mice show significant reductions in striatal dopaminergic terminals as well as deficits in motor function. However, resting oxygen consumption, heat production, mtDNA content and mitochondrial electron transport chain activities are significantly decreased at older ages in both homozygous and heterozygous mice. These results indicate that high levels of somatic mtDNA mutations can contribute to dopaminergic dysfunction and to behavioral and metabolic deficits.

Changes in intestinal absorption of nutrients are important aspects of the aging process. To address this issue, we investigated the impact of accelerated mitochondrial DNA mutations on the stem/progenitor cells in the crypts of Lieberkühn in mice homozygous for a mitochondrial DNA polymerase gamma mutation, Polg D257A, that exhibit accelerated aging phenotype. As early as 3–7 mo of age, the small intestine was significantly enlarged in the PolgD257A mice. The crypts of the PolgD257A mice contained 20% more cells than those of their wild-type littermates and exhibited a 10-fold increase in cellular apoptosis primarily in the stem/progenitor cell zones. Actively dividing cells were proportionally increased, yet a significantly smaller proportion of cells was in the S phase of the cell cycle. Stem cell-derived organoids from PolgD257A mice failed to develop fully in culture and exhibited fewer crypt units, indicating an impact of the mutation on the intestinal epithelial stem/progenitor cell maintenance. In addition, epithelial cell migration along the crypt-villus axis was slowed and less organized, and the ATP content in the villi was significantly reduced. On a high-fat, high-carbohydrate diet, PolgD257A mice showed significantly restricted absorption of excess lipids accompanied by an increase in fecal steatocrits. We conclude that the PolgD257A mutation causes cell cycle dysregulation in the crypts leading to the age-associated changes in the morphology of the small intestine and contributes to the restricted absorption of dietary lipids.

Diabetes and the development of its complications have been associated with mitochondrial DNA (mtDNA) dysfunction, but causal relationships remain undetermined. With the objective of testing whether increased mtDNA mutations exacerbate the diabetic phenotype, we have compared mice heterozygous for the Akita diabetogenic mutation (Akita) with mice homozygous for the D257A mutation in mitochondrial DNA polymerase gamma (Polg) or with mice having both mutations (Polg-Akita). The Polg-D257A protein is defective in proofreading and increases mtDNA mutations. At 3 mo of age, the Polg-Akita and Akita male mice were equally hyperglycemic. Unexpectedly, as the Polg-Akita males aged to 9 mo, their diabetic symptoms decreased. Thus, their hyperglycemia, hyperphagia and urine output declined significantly. The decrease in their food intake was accompanied by increased plasma leptin and decreased plasma ghrelin, while hypothalamic expression of the orexic gene, neuropeptide Y, was lower and expression of the anorexic gene, proopiomelanocortin, was higher. Testis function progressively worsened with age in the double mutants, and plasma testosterone levels in 9-mo-old Polg-Akita males were significantly reduced compared with Akita males. The hyperglycemia and hyperphagia returned in aged Polg-Akita males after testosterone administration. Hyperglycemia-associated distal tubular damage in the kidney also returned, and Polg-D257A-associated proximal tubular damage was enhanced. The mild diabetes of female Akita mice was not affected by the Polg-D257A mutation. We conclude that reduced diabetic symptoms of aging Polg-Akita males results from appetite suppression triggered by decreased testosterone associated with damage to the Leydig cells of the testis.

Stroke is a major public health concern, with ~795,000 strokes occurring in the United States each year, resulting in over 130,000 deaths annually, and 4.8 million stroke survivors (CDC Risk Survey 2008). The estimated direct and indirect cost of stroke for 2010 is $73.7 billion (CDC Risk Survey 2008). Clinical treatment options for ischemic stroke (which accounts for ~87% of stroke cases) are limited to chemical or mechanical clot-busting interventions during a short time-window following stroke. Therefore, the development of post-ischemic therapies to reduce the mortality and disability associated with stroke would have clear public health benefits. Multipotent mesenchymal stromal cell (MSC; also sometimes called mesenchymal stem cell) transplantation has shown protection against stroke in animal models, reducing infarct volumes and improving behavioral function (Chen et al. 2001; Li et al. 2001); transplanted MSCs, however, often do not show show long-term survival and integration into the brain. This has led many investigators to believe that paracrine mechanisms underlie the benefits of MSC transplantation. Indeed, neural stem cell derivatives, such as neuroepithelial cells, may be better suited than MSCs to promote neuronal replacement due to their neuroectodermal developmental origin (Kelly et al. 2004; Jiang et al. 2006; Darsalia et al. 2007; Fong et al. 2007; Daadi et al. 2008). Transplanted cells face a hostile, inflammatory environment in the near term post-ischemic cerebrum, which may contribute to the limited survival of the engrafted stem cells. MSC transplantation induces gene expression changes that suggest an altered inflammatory response following ischemia (Ohtaki et al. 2008). In addition to MSC promotion of endogenous repair and replacement processes, this inflammatory modulation (Aggarwal & Pittenger 2005) may provide for an ameliorated prosurvival and/or pro-differentiation milieu for subsequent transplantation of other stem cell types, such as neuroepithelial cells. Addressing the issues of potential allograft rejection and limited long-term cell survival/replacement will be important to the clinical application of stem cell therapies, regardless of which type of stem cell is used. The immunosuppressive attributes of MSCs put them under consideration for use in co-transplantation approaches to mitigate graft rejection concerns.

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.

Background: Aging results in a progressive loss of skeletal muscle, a condition known as sarcopenia. Mitochondrial DNA (mtDNA) mutations accumulate with aging in skeletal muscle and correlate with muscle loss, although no causal relationship has been established. Methodology/Principal Findings: We investigated the relationship between mtDNA mutations and sarcopenia at the gene expression and biochemical levels using a mouse model that expresses a proofreading-deficient version (D257A) of the mitochondrial DNA Polymerase c, resulting in increased spontaneous mtDNA mutation rates. Gene expression profiling of D257A mice followed by Parametric Analysis of Gene Set Enrichment (PAGE) indicates that the D257A mutation is associated with a profound downregulation of gene sets associated with mitochondrial function. At the biochemical level, sarcopenia in D257A mice is associated with a marked reduction (35–50%) in the content of electron transport chain (ETC) complexes I, III and IV, all of which are partly encoded by mtDNA. D257A mice display impaired mitochondrial bioenergetics associated with compromised state-3 respiration, lower ATP content and a resulting decrease in mitochondrial membrane potential (Dym). Surprisingly, mitochondrial dysfunction was not accompanied by an increase in mitochondrial reactive oxygen species (ROS) production or oxidative damage. Conclusions/Significance: These findings demonstrate that mutations in mtDNA can be causal in sarcopenia by affecting the assembly of functional ETC complexes, the lack of which provokes a decrease in oxidative phosphorylation, without an increase in oxidative stress, and ultimately, skeletal muscle apoptosis and sarcopenia.

The operating microscope is a fixture of modern surgical facilities, and it is a critically important factor in the success of many of the most complex and difficult surgical interventions used in medicine today. The rise of this key surgical tool reflects advances in understanding the principles of optics and vision that have occurred over centuries. The development of reading spectacles in the late 13th century led to the construction of early compound microscopes in the 16th and 17th centuries by Lippershey, Janssen, Galileo, Hooke, and others. Perhaps surprisingly, Leeuwenhoek's simple microscopes of this era offered improved performance over his contemporaries' designs. The intervening years saw improvements that reduced the spherical and chromatic aberrations present in compound microscopes. By the late 19th century, Carl Zeiss and Ernst Abbe ushered the compound microscope into the beginnings of the modern era of commercial design and production. The introduction of the microscope into the operating room by Nylén in 1921 initiated a revolution in surgical practice that gained momentum throughout the 1950s with multiple refinements, the introduction of the Zeiss OPMI series, and Kurze's application of the microscope to neurosurgery in 1957. Many of the refinements of the last 50 years have greatly improved the handling and practical operation of the surgical microscope, considerations which are equally important to its optical performance. Today's sophisticated operating microscopes allow for advanced real-time angiographic and tumor imaging. In this paper the authors discuss what might be found in the operating rooms of tomorrow.

Mitochondria have occupied a central place in theories on the underlying cellular mechanisms of eukaryotic aging for several decades and much debate has ensued regarding the role of oxidative stress and mitochondrial genomic damage in these processes. Mouse models with greatly enhanced mitochondrial mutagenesis have produced dramatic aging-like phenotypes but recent results have led some to reassess whether such models are relevant to naturally occurring aging mechanisms. Here, we discuss the evolving insight that may be gained from these models regarding the contribution of mitochondrial DNA mutations to aging.

Mitochondrial DNA (mtDNA) accumulates both base-substitution mutations and deletions with aging in several tissues in mammals. Here, we examine the evidence supporting a causative role for mtDNA mutations in mammalian aging. We describe and compare human diseases and mouse models associated with mitochondrial genome instability. We also discuss potential mechanisms for the generation of these mutations and the means by which they may mediate their pathological consequences. Strategies for slowing the accumulation and attenuating the effects of mtDNA mutations are discussed.

Mitochondrial DNA (mtDNA) accumulates both base-substitution mutations and deletions with aging in several tissues in mammals. Here, we examine the evidence supporting a causative role for mtDNA mutations in mammalian aging. We describe and compare human diseases and mouse models associated with mitochondrial genome instability. We also discuss potential mechanisms for the generation of these mutations and the means by which they may mediate their pathological consequences. Strategies for slowing the accumulation and attenuating the effects of mtDNA mutations are discussed.

Background Age is the most prominent risk factor for osteoarthritis and mitochondrial DNA dysfunction has been repeatedly described in human osteoarthritic chondrocytes. Mice expressing a proofreading-deficient mitochondrial DNA polymerase (POLG) mutant accumulate an excess of reactive oxygen species and apoptosis and develop a premature aging phenotype. While these mice display a reduction in bone density, it is unclear whether they are more prone to developing osteoarthritis. Objectives In this study, we assessed the histomorphometric properties of subchondral bone and cartilage tissues in premature aging mice. Methods Mice carrying heterozygous (wt/mut n= 5) or homozygous (mut/mut n= 4) D257A mutations in POLG were compared with wildtype littermates (wt n =7). Animals were aged between eleven and fifteen months. Sagittal histological sections from knee joints were stained with Safranin- O /Fast Green and cartilage degeneration was assessed using OARSI scores. Subchondral bone area fraction (B.Ar/T.Ar) and osteoclast numbers (Oc.N/mm perimeter) between epiphyses and articular cartilage was determined on tissues stained for tartrate-resistant acid phosphatase (TRAP) and methylene blue using bone histomorphometric analyses. Results Wild type mice revealed only low grade cartilage degeneration (OARSI score p Conclusions Mice with premature aging due to accumulation of mtDNA mutations display increased bone remodelling favouring subchondral bone resorption. Additional biomechanical factors might be required in this premature aging phenotype for development of osteoarthritis. Disclosure of Interest None declared

Whether mitochondrial mutations cause mammalian aging, or are merely correlated with it, is an area of intense debate 1 . Here, we use a new, highly sensitive assay 2 to redefine the relationship between mitochondrial mutations and age. We measured the in vivo rate of change of the mitochondrial genome at a single‐base pair level in mice, and we demonstrate that the mutation frequency in mouse mitochondria is more than ten times lower than previously reported. Although we observed an 11-fold increase in mitochondrial point mutations with age, we report that a mitochondrial mutator mouse 3 was able to sustain a 500-fold higher mutation burden than normal mice, without any obvious features of rapidly accelerated aging. Thus, our results strongly indicate that mitochondrial mutations do not limit the lifespan of wild-type mice. Mitochondria are functionally diverse organelles with a central role in both oxidative phosphorylation 4 and apoptosis 5 . Accordingly, postmitotic tissues with high rates of oxygen consumption, such as brain and heart, are exceptionally sensitive to mitochondrial dysfunction 6 . The mitochondrial theory of aging postulates that a lifelong accumulation of mitochondrial DNA (mtDNA) mutations in multiple tissues eventually results in mitochondrial failure, which, in synergy with downstream processes such as apoptosis, results in loss of cellularity and the progressive decline of tissue functioning known as aging 3,7 . These mutations may compromise the integrity of the electron transport chain and increase the formation of reactive oxygen species (ROS), creating a vicious cycle of mutagenesis that continuously amplifies the production of cytotoxic oxygen radicals 8,9 . Although several studies have quantified either tissue-specific mutation frequencies 10 or mutation accumulation as a function of age 11 , most methods rely heavily on PCR and cloning-based strategies 12,13 , techniques that are limited in throughput and that may be confounded by polymerase infidelity on damaged templates and by cloning artifacts. Therefore, we explored the relationship between age and mutation accumulation in mtDNA with an adaptation of the random mutation capture (RMC) assay 2,14 , a quantitative

Mutations in mitochondrial DNA (mtDNA) accumulate during aging, but their significance to longevity and age-associated disease has been uncertain. Recently, in support of the hypothesis that mtDNA integrity is important, we have shown that age-associated diseases arise more rapidly in mice where mtDNA mutations and increased levels of apoptosis occur at higher rates than normal due to expression of an error-prone mtDNA polymerase. Further studies in this model may provide deeper insights into the relationship between mitochondria, aging, and susceptibility to age-associated diseases, such as cancer. (Cancer Res 2006; 66(15): 7386-9)

It has been postulated that the differences in longevity observed between organisms of different sexes within a species can be attributed to differences in oxidative stress. It is generally accepted that differences are due to the higher female estrogen levels. However, in some species males live the same or longer despite their lower estrogen values. Therefore, in the present study, we analyze key parameters of mitochondrial bioenergetics, oxidative stress and apoptosis in the B6 (C57Bl/6J) mouse strain. There are no differences in longevity between males and females in this mouse strain, although estrogen levels are higher in females. We did not find any differences in heart, skeletal muscle and liver mitochondrial oxygen consumption (State 3 and State 4) and ATP content between male and female mice. Moreover, mitochondrial H(2)O(2) generation and oxidative stress levels determined by cytosolic protein carbonyls and concentration of 8-hydroxy-2'-deoxyguanosine in mitochondrial DNA were similar in both sexes. In addition, markers of apoptosis (caspase-3, caspase-9 and mono- and oligonucleosomes: the apoptosis index) were not different between male and female mice. These data show that there are no differences in mitochondrial bioenergetics, oxidative stress and apoptosis due to gender in this mouse strain according with the lack of differences in longevity. These results support the Mitochondrial Free Radical Theory of Aging, and indicate that oxidative stress generation independent of estrogen levels determines aging rate.

Mutations in mitochondrial DNA (mtDNA) accumulate in tissues of mammalian species and have been hypothesized to contribute to aging. We show that mice expressing a proofreading-deficient version of the mitochondrial DNA polymerase g (POLG) accumulate mtDNA mutations and display features of accelerated aging. Accumulation of mtDNA mutations was not associated with increased markers of oxidative stress or a defect in cellular proliferation, but was correlated with the induction of apoptotic markers, particularly in tissues characterized by rapid cellular turnover. The levels of apoptotic markers were also found to increase during aging in normal mice. Thus, accumulation of mtDNA mutations that promote apoptosis may be a central mechanism driving mammalian aging.

The retrosigmoid approach is a work-horse approach to the cerebellopontine angle (CPA), providing access from the foremen magnum to the tentorium. Indications for this approach are variable such as resection of meningiomas, acoustic neuromas and epidermoid tumors, treatment of vascular lesions of vertebrobasilar system, vascular decompression of cranial nerves (V, VII, IX, X), cranial nerve neurectomies, and intrinsic lesions of the cerebellum and brainstem. In this video, we demonstrate the use of retrosigmoid craniotomy for resection of a large CPA meningioma, delineating all steps including positioning, mapping.The video can be found here: http://youtu.be/kISkYS16Brk.

Mitochondrial DNA (mtDNA) mutations lead to decrements in mitochondrial function and accelerated rates of these mutations has been linked to skeletal muscle loss (sarcopenia). The purpose of this study was to investigate the effect of mtDNA mutations on mitochondrial quality control processes in skeletal muscle from animals (young; 3-6 months and older; 8-15 months) expressing a proofreading-deficient version of mtDNA polymerase gamma (PolG). This progeroid aging model exhibits elevated mtDNA mutation rates, mitochondrial dysfunction, and a premature aging phenotype that includes sarcopenia. We found increased expression of the mitochondrial biogenesis regulator peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) and its target proteins, nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (Tfam) in PolG animals compared to wild-type (WT) (P

The majority of human tumor cells, and particularly those of mesenchymal origin, have the ability to grow in semi-solid medium (i.e., anchorage-independent growth; refs. 1–5), a phenotype, which aside from some hematopoeitic cells, is rarely observed in nonneoplastic human cells. Treatment of rodent or human fibroblasts with mutagens has been shown to induce anchorage-independent growth at frequencies consistent with mutation at a single, dominant-acting gene (6,7), or perhaps pool of genes, any locus of which is permissive for the phenotype in its mutant form. Mutagen-induced acquisition of anchorage-independent growth in many aneuploid, immortal rodent cell lines has been shown to correlate well with acquisition of tumorigenic growth (8), whereas with diploid human fibroblasts, mutagen treatment has been shown to induce anchorage-independent cell populations that do not yield progressively growing tumors (9), and, consistent with this, the anchorage-independent cell clones senesce at passage levels similar to non-anchorage-independent cell clones (6). When neo-plastic transformation is viewed in the context of a multiple-step transition from normal to neoplastic cell (10), mutagen-induced anchorage-independence is likely to represent one of these discrete steps, where this acquired phenotype is related mechanistically to the final tumorigenic state.

Studies on the etiology of MDS have been hampered by the lack of relevant animal model systems. Numerous murine models of MDS/MPD overlap syndromes have been described, but MDS/MPD overlap syndromes are now felt to be clinically distinct from MDS. Likewise, a number of murine models of myeloid leukemia have been developed in which dysplastic features arise during conversion to frank leukemia. Here we describe the hematopoietic phenotype of the PolgD257A mitochondrial mutator mouse and demonstrate that this model system fulfills the myeloid dysplasia criteria outlined in the Bethesda Proposals (Blood, 100, 238, 2002). The PolgD257A allele eliminates the proofreading activity of Polg, the sole mitochondrial DNA (mtDNA) polymerase, and PolgA/A mice rapidly accumulate mtDNA mutations. Starting at 9 months of age, PolgA/A mice display multiple features of accelerated aging and develop a progressive and ultimately lethal anemia (Science, 309, 481, 2005). In data obtained at time of necropsy for other studies, the hemoglobin of young PolgA/A mice is identical to that of controls but by 10 months of age begins to fall below 10 g/dL. Overall, while Polg+/+ mice maintain a hemoglobin of greater than 10 g/dL through 30 months of age, the hemoglobin of PolgA/A drops below 5 g/dL as the mice succumb to their anemia at 13–16 months of age. As the hemoglobin of aging PolgA/A mice falls the MCV demonstrates a striking increase, from 53.6 fL to 69.8 fL. In order to confirm these intial findings, complete blood counts have been assessed prospectively in mice of all three genotypes (Polg+/+, Polg+/A, and PolgA/A; 5 mice per genotype) every other month up to 8 months of age and monthly thereafter. Full data up to 11 months of age, as well as 12 month data from all 5 PolgA/A mice, will be presented. As expected, up to eight months of age no significant difference in hemoglobin levels are apparent, but by eight months PolgA/A mice demonstrate a significant increase in MCV (51.5 fL) compared to either Polg+/A (44.9, p=0.0015) or Polg+/+ (44.5, p=0.0010) littermate controls. The disparity in MCV between PolgA/A mice and the other genotypes is progressive, with the MCV at 9 months increasing to 53.4 fL in PolgA/A mice (N=5), compared to 44.8 (p