Uveal melanoma (UM) is the most common primary cancer of the eye and has a strong propensity for metastasis. Although there have been many recent improvements in the diagnosis and treatment of UM, and only 2-4% of patients present with detectable metastasis, up to half of patients are at risk for dying of metastatic disease. Clinicopathologic factors are not accurate enough for individualized patient care. Chromosomal alterations have been used for prognostic purposes, but the routine clinical use of these methods is limited by their susceptibility to sampling error resulting from tumor heterogeneity, limited clinical validation, lack of standardized testing platforms, and high technical failure rates. In contrast, the DecisionDx-UM gene expression profile test is a stand-alone platform which requires no other information for maximal prognostic accuracy and which circumvents many of the drawbacks of chromosomal methods through the use of a highly sensitive microfluidics, PCR-based platform that simultaneously measures the expression of 15 carefully selected genes from primary uveal melanoma samples obtained by fine needle biopsy. Low metastatic risk is reported as Class 1, and high metastatic risk as Class 2. The test allows patients to be stratified into risk categories such that high-risk patients can be offered intensive metastatic surveillance and adjuvant therapy while low-risk patients can be spared these interventions. This test is now used as part of the standard of care in many ocular oncology centers.  Brachytherapy, proton beam radiotherapy, and enucleation are highly effective treatments for primary uveal melanomaTM gene expression profile (GEP) test was designed to address these challenges by providing a highly accurate, stand-alone prognostic test that is straightforward to interpret, requiring no additional information or analysis. The DecisionDx-UM test simultaneously measures the expression of 15 genes from primary uveal melanoma samples obtained by fine needle biopsy in order to determine the likelihood of metastasis. Low metastatic risk is reported as Class 1 whereas high metastatic risk is reported as Class 2. The DecisionDx-UM test allows for implementation of a risk-appropriate metastatic surveillance strategy and accurate identification of high-risk patients who may be eligible for targeted adjuvant therapy prior to the detection of metastasisThe DecisionDx-UMThe DecisionDx-UM test uses RNA extracted from a fine needle aspiration biopsy (FNAB) sample, formalin-fixed paraffin embedded (FFPE) post-enucleation specimens, or resected tumorThe assay includes 12 genes that are differentially expressed in Class 1 versus Class 2 tumors (Table 1), and 3 control genes  that are expressed at similar levels in Class 1 and Class 2 tumorsThe DecisionDx-UM test is currently performed in a single College of American Pathologists (CAP) accredited, Clinical Laborary Improvement Amendments (CLIA) certified laboratory. The test has been performed for over 1500 patients with uveal melanoma in more than 100 ocular oncology centers in North America. Similar to the results recently published for the Collaborative Ocular Oncology Group (COOG) studySignificance of Class 1 Versus Class 2 DesignationThe DecisionDx-UM test assesses the molecular signature of a patient’s tumor and designates it as Class 1 or Class 2Biologically, the GEP of Class 1 tumors closely resembles that of normal uveal melanocytes and low-grade uveal melanocytic tumors. Conversely, the GEP of Class 2 tumors resembles that of primitive neural/ectodermal stem cellsUveal melanoma is the most common primary cancer in the eye, with an incidence of 1200-1500 new cases each yearReviews: The technique of GEP has been compared to other methods of prognostication for uveal melanoma, including clinicopathologic staging and chromosomal testing, and it has been found to be superior to these other optionsProfessional Organization Reviews: Gene expression profiling for prognostication is recommended by the American Joint Committee on Cancer (AJCC) in their classification guidelines for uveal melanomaHealth Plan/Payer Reviews: Four external, independent review organization appeals have been completed for “experimental and investigational” and “not medically necessary” payment denials, and all four external appeals concluded that the DecisionDx-UM test was medically necessary and reasonable . Specifics include:Independent review request submitted by Blue Cross Blue Shield of North Carolina to the North Carolina Department of Insurance (NCDOI). NCDOI contracted the review to the Medwork Independent Review organization. The appeal concluded that the DecisionDx-UM test was medically necessary.Independent review request submitted by Medical Mutual of Ohio. Medical Mutual contracted with National Medical Reviews, Inc. The appeal outcome concluded that the DecisionDx-UM test was medically necessary.Independent review request submitted by United Healthcare to Medical Care Ombudsman Program (MCMC). The appeal outcome concluded that the DecisionDx-UM test was not experimental or investigational and that the test did meet covered health service requirements .Independent review request submitted by Cigna of Colorado. The appeal outcome concluded that the DecisionDx-UM test was not experimental or investigational.Analytic Validity:Test accuracy and reliability in identifying gene expression profileA pubmed search with key words uveal melanoma and gene expression, chromosomal, monosomy 3, staging, or prognosis was conducted in the English literature. The pertinent orginal articles were acquired and reviewed for data and additional references.The DecisionDx-UM test is a GEP assay that analyzes RNA expression of 15 selected genes, including 3 endogenous controls and 12 class discriminating genes (Table 1). Initially, there was concern that an RNA-based test may not be clinically feasible due to RNA instability. However, a standard operating procedure was developed to rapidly stabilize the RNA immediately after biopsy, which eliminated RNA degradation as a major source of technical failure.In the DecisionDx-UM test, a gene is considered undetectable if its amplification product registers no Ct value after 40 cycles of quantitative PCR, and a sample is considered a technical failure if 1 or more endogenous control genes or at least 3 discriminating genes are undetectable. A large, prospective multicenter study reported high analytic sensitivity for the test, demonstrating interpretable GEP results in more than 97% of FNAB cases, a higher success rate than that of competing DNA-based testsTechnical concordance studies are performed for the DecisionDx-UM test at regular intervals, comparing the classification results obtained in the clinical laboratory to those from the research laboratory. With about 150 samples tested to date, there has been 100% concordance between the laboratories . Further, the DecisionDx-UM test is regularly evaluated for algorithmic proficiency, which is the ability of the computer algorithm that generates class assignments to render the same result when performed by independent operators. With about 400 samples analyzed to date, there has been 100% concordance between operators .Intratumoral heterogeneity for monosomy 3 is estimated to occur in 14-18% of uveal melanomasClinical Validity:Test accuracy and reliability in assigning accurate prognosis in uveal melanoma and comparison to other existing methodsRetrospective studies from multiple independent institutions have shown that the general technique of GEP is superior in predicting metastasis in patients with uveal melanoma than clinical, pathologic or chromosomal methodsWhile cytogenetic markers have been widely studied as prognostic factors of uveal melanoma metastasis, the accuracy of methods used to identify cytogenetic abnormalities has been less than sufficient for clinical applicability. For example, the use of fluorescence in situ hybridization for the identification of monosomy 3 in tumors has resulted in as much as a 50% technical failure rateThe NCCN task force on evaluating the clinical utility of tumor markers in oncology stated that the “clinical utility of a marker should be determined in a prospective clinical trial, as is required for new drugs”Clinical Utility:net benefit of test in improving health outcomesThe potential benefits of the DecisionDx-UM test are three-fold. First, the test provides patients with information that most of them desire for personal planning. In two recent studies, 97% of patients reported that they desired to have prognostic informationSecond, the test identifies patients at high risk of metastasis who may benefit from more intensive metastatic surveillance, while sparing low-risk patients of these measures. High intensity surveillance with more frequent and sensitive testing than standard Collaborative Ocular Melanoma Study (COMS) guidelines allows detection and treatment of metastatic disease at an earlier stage, when locoregional treatments such as hepatic chemoembolization and targeted systemic therapies may be more effectiveThird, the DecisionDx-UM test allows accurate stratification to allow entry of high-risk patients into clinical trials to assess the efficacy of adjuvant therapies. Several such trials are already underway, and many more are expected to begin in the coming months as more rationally designed targeted therapies are identifiedClinical use of the DecisionDx-UM test has been recently documented in two independent studies:(1) Study 1. A blinded survey of ocular oncologists that was presented at the Retina 2012 subspecialty section of the annual meeting of the American Academy of Ophthalmology demonstrated that results of the DecisionDx-UM test impact the follow-up surveillance regimen of patients with uveal melanoma(2) Study 2. This study involved the systematic review of Medicare cases for 100% of Medicare patients (n=237) who had a reported DecisionDx-UM test result between August, 2010 and January, 2012The utility of the DecisionDx-UM test has not been assessed for uveal melanomas that have undergone radiation therapy previously. Therefore, collection of the tissue sample must be performed prior to radiation therapy. Also, the test is not recommended for highly necrotic samples.The DecisionDx-UM test is the only prognostic tool in uveal melanoma that has been prospectively validated in a multi-center study, and it is used by the majority of ocular oncology centers in North America for routine clinical prognostic testingDiscriminating genes comprising the DecisionDx- UMTM gene expression profile prognostic test for uveal melanomaDr. Harbour is the inventor of intellectual property used in the study and receives royalties from its commercialization. He is a paid consultant for Castle Biosciences, licensee of intellectual property presented in this article.


Despite demonstrating only partial efficacy in preventing new infections, available HIV prevention interventions could offer a powerful strategy when combined. In anticipation of combination HIV prevention programs and research studies we estimated the population-level impact of combining effective scalable interventions at high population coverage, determined the factors that influence this impact, and estimated the synergy between the components.We used a mathematical model to investigate the effect on HIV incidence of a combination HIV prevention intervention comprised of high coverage of HIV testing and counselling, risk reduction following HIV diagnosis, male circumcision for HIV-uninfected men, and antiretroviral therapy (ART) for HIV-infected persons. The model was calibrated to data for KwaZulu-Natal, South Africa, where adult HIV prevalence is approximately 23%.Compared to current levels of HIV testing, circumcision, and ART, the combined intervention with ART initiation according to current guidelines could reduce HIV incidence by 47%, from 2.3 new infections per 100 person-years (pyar) to 1.2 per 100 pyar within 4 years and by almost 60%, to 1 per 100 pyar, after 25 years. Short-term impact is driven primarily by uptake of testing and reductions in risk behaviour following testing while long-term effects are driven by periodic HIV testing and retention in ART programs. If the combination prevention program incorporated HIV treatment upon diagnosis, incidence could be reduced by 63% after 4 years and by 76% (to about 0.5 per 100 pyar) after 15 years. The full impact of the combination interventions accrues over 10–15 years. Synergy is demonstrated between the intervention components.High coverage combination of evidence-based strategies could generate substantial reductions in population HIV incidence in an African generalized HIV epidemic setting. The full impact could be underestimated by the short assessment duration of typical evaluations. Three decades into the fight to prevent new HIV infections, no single intervention has been found to have sufficiently high efficacy, acceptability, feasibility, and affordability to individually control the generalized HIV epidemic in sub-Saharan Africa. However, a growing number of interventions have been shown to partially reduce HIV risk and have been demonstrated to be deliverable at scale in Africa. These include reducing sexual risk behaviour by increasing condom use and decreasing partner acquisition rate, reducing the likelihood of HIV acquisition by male circumcision for HIV uninfected men HIV testing is the crucial entry point to effective HIV prevention. For infected persons, knowledge of HIV status enables referral to care and assessment for ART. Testing can also motivate male circumcision for HIV uninfected men and reductions in risk behaviour, particularly among newly diagnosed persons with HIV Implementation and field evaluation of wide-scale HIV testing coupled with combination HIV prevention interventions are urgently needed. Program evaluations and clinical trials are underway Some previous model projections have suggested that ART alone could achieve high impact on HIV incidence In this article, we use a mathematical model of the HIV epidemic in South Africa to study HIV incidence under rapid and high testing coverage that is repeated every four years and the following interventions: risk reduction following HIV diagnosis (the first HIV-positive test), medical male circumcision, and ART initiation following current WHO guidelines (CD4 count ≤350 cells/ml). First, the interventions are considered individually to disentangle and characterize their impacts. Secondly, we estimate the population-level impact of a feasible implementation of a combination intervention and examine how trials to measure these effects might be designed. Thirdly, we studied the implications of expanding ART initiation to be upon HIV diagnosis. Finally, we determine the factors that influence this impact and quantify the interaction between the components in the combined intervention at the short and the long terms. The HIV settings of South Africa are ideal for our study because of very high HIV prevalence (17% among 15–49 year-old men and women) and because trials of a combination of deliverable prevention interventions are being planned in similar settings.et al. based on analyses of cohort data from rural Uganda We constructed a compartmental mathematical model to represent transmission of HIV in the heterosexual adult population in KwaZulu-Natal (KZN), South Africa. A full technical specification of the model and its parameterization is given in current standard scenario in 3 but most treatment initiation occurs at CD4 count <100 cells/mm3 due to the low rate of testing The intervention scenarios investigated focused on three partially-effective HIV interventions and were based on achieving 90% HIV testing of adults over one year (for example through community home based counselling and testing (HBCT)) and repeated every four years (in the text below we refer to this as ‘community testing’). The three interventions are –1) Risk behaviour reduction for individuals newly diagnosed with HIV, 2) male circumcision for HIV uninfected men, and 3) ART initiation for HIV infected persons. A combination prevention intervention consists of the effects of these three interventions acting together in KZN population and we studied two possible combination interventions . EstimatAssumed coverage levels of the community testing and the interventions were chosen to be optimistic but potentially achievable. Community testing increases the proportion of adults that ‘know’ their HIV status to 90%; new rounds of community testing are implemented every 4 years, allowing persons infected in the interim or not tested in previous rounds to learn their status. No testing is assumed to take place between the rounds of the four-yearly community testing and sensitivity analyses incorporated lower testing levels in each HIV testing. The model allows retesting individuals and tracks whether they have been infected since their last HIV test or remain uninfected . HIV-infCurrent KZN circumcision prevalence 27%  is assum3 and current South Africa and WHO guidelines, to initiate ART at CD4 ≤ 350 cells/mm33. Individuals receiving ART stop doing so (‘drop out’) at a rate of 14.5% per year HIV infected individuals who are aware of their HIV status are implicitly assumed to have regular CD4 count monitoring and thus access ART promptly after their CD4 cell count falls below a chosen threshold  we introduce a measure of synergy through calculating extra reduction in incidence. Let the incidence under current-standard at time t be 0(t)I and the incidence under the individual interventions 1, 2, 3, …, n be 1(t), I2(t), I3(t),…, In(t),I respectively. If the incidence when the interventions are acting together is c,I then:Measuring the interaction between co-existing interventions: to tease out any interaction between the interventions Synergy is non-zero, have synergy if Synergy is positive, and otherwise they are redundant: that is one or more of the components are preventing what the rest of the components are already preventing. To be noted here is that the above synergy measure is a time varying quantity because incidence varies with time.The combined interventions have interaction if First, we disentangle the time variation of the impact of the three interventions mentioned above by having them acting alone in the population of KZN. Secondly, we estimate the joint impact when all of these interventions are in action ; . Risk be3 on HIV incidence is initially small but grows quickly as a substantial proportion of HIV-diagnosed adults initiate ART and then reach later stages of infection while being less infectious than they otherwise would be To disentangle the effect of ART from risk behaviour reduction among newly-identified HIV-infected adults in , we assu3 (3 slightly improves the impact compared to ART at ≤200 cells/mm3 (33% (32%–34%) versus 24% (23%–25%) reduction in HIV incidence at 4 years). In contrast, with immediate ART initiation upon diagnosis the impact on HIV incidence would be more substantial; our model predicts a 53% reduction in incidence at 4 years compared to the current standard; growing to 66% at 10 years and 67% at 15 years  in a combination intervention that is based on community testing . Under this scenario, the reduction in incidence would be greater than under Combination I at the short and long terms. At 4 years the reduction in incidence is 63% (60%–65%) versus 47% (43%–50%) for Combination I, and at 15 years the reduction in incidence is 76% (71%–81%) versus 52% (45%–60%) for Combination I . A compaThe continuation of HIV community testing, immediate initiation of ART, and circumcision in Combination II intervention would achieve two major goals of HIV prevention interventions. First, the rate of new HIV infections would eventually be reduced to a low level: 0.3 new infections per 100 pyar after 25 years. Second, it would reduce the rate of HIV deaths and simultaneously bring the rate of new HIV infections below the rate of AIDS death - a point defined as the ‘AIDS Transition’ At high levels of coverage for the interventions, the interaction among them could lead to unneeded excessive protection; this is the case for example when large numbers of individuals are exposed to two interventions that reduce the same risk. Newly diagnosed individuals with HIV can be exposed to ART and risk reduction. 3, and male circumcision for HIV-uninfected men. The model is parameterized to the epidemiological context of KZN and includes important pragmatic assumptions about initiating treatment based on knowledge of HIV serostatus, repeating community testing every four years to identify newly-infected adults and those not previously tested, and assuming realistic drop-out rates from ART, suboptimal viral suppression due to imperfect adherence on ART, incomplete uptake of circumcision with coverage consistent with targets in the South Africa’s national plan, and modest and temporary changes in sexual risk behaviours following HIV testing We mathematically modelled a combination prevention package based on the platform of testing 90% of adults in KwaZulu-Natal, South Africa through community HIV testing campaigns every four years coupled with risk behaviour reduction following HIV diagnosis, ART initiation for HIV-infected adults according to current WHO and South African guidelines at CD4 ≤ 350 cells/mmThe combination of intervention components that act over different time scales with different impact on HIV incidence. The combined effects of these interventions reduce both the risk of acquisition and transmission of HIV, and could generate rapid, sustained, and substantial reductions in HIV incidence. Further, our results suggest that high coverage HIV testing every four years coupled with high uptake of circumcision and ART initiation upon HIV diagnosis could reduce HIV incidence and death rates to as low as 0.3% per year, significantly reducing the scale of the epidemic. By utilizing a measure for synergy we showed that although the effects of circumcision, ART, and risk behaviour reduction coexist in the population as a result of the combination intervention, the effects substantially strengthen each other particularly if the assumed levels of coverage for these interventions are maintained.These findings have important implications for HIV prevention prioritization and program planning. Large reductions in HIV incidence in high prevalence settings in sub-Saharan Africa can be attained with ambitious, but feasible, assumptions about scale-up coverage and impact of HIV testing, male circumcision, and ART initiation and retention. Our assumed ambitious levels of coverage are premised on high coverage in pilot work and preliminary studies; HIV testing rates in large scale HBCT programs in Uganda and South Africa have exceeded 80% We showed that maintaining high coverage levels of testing and risk reduction, circumcision, and HIV treatment would not compromise the synergy between these interventions. Optimizing the prevention benefits of each and every intervention of these to combat HIV is important due to the imperfections in the current HIV population-level intervention programs such as the suboptimal coverage and adherence levels, the drop out from ART, and the temporary nature of the reduction in sexual risk behaviour after HIV diagnoses.We have not incorporated the impact of circumcision provision to HIV-infected men, because there is little evidence of direct effect of circumcision on HIV transmission to women Although there have been other model projections suggesting single HIV interventions could achieve similar levels of impact, these have required more demanding assumptions such as universal testing every year and perfect adherence on ART that may not be consistently attainable The rapid increase in the numbers of individuals in need of HIV care and circumcision services, in Combination I and II, demands parallel increases in the capacity and the accessibility of HIV care and circumcision clinics. A parallel increase in active follow up would also be required to achieve ART timely initiation, high circumcision uptake, and the sustainability of reduced risk behaviours. The waiting period between the four-yearly testing waves might facilitate task shifting to active follow up of persons newly diagnosed with HIV. We have not investigated the financial obligations and implications of any of our scenarios. Recent studies have illustrated that among non-traditional counselling and testing methods, door-to-door HIV counselling and testing in Ugandan population has the lowest cost Our findings have significant implications for studies that aim to measure the impact of combination HIV prevention. First, the full impact of combination prevention interventions will be achieved over 10–15 years. Because most forms of impact evaluations of combination HIV prevention interventions, including community-randomized trials and programmatic assessments, are anticipated to examine impact over a much shorter period (e.g. 2–3 years), those studies could underestimate the full benefit of combination packages. Second, our model found that a determinant of short-term impact of a combination package is risk behaviour reduction following HIV diagnosis as a product of community testing. If the degree of risk reduction is less than we assumed, the ability to measure the impact on HIV incidence in a short-term evaluation is reduced. The time to see different effect magnitudes for individual components is an important consideration in the duration and interpretation of community-randomized trials of combination prevention, and argues for longer periods of observation, and monitoring intermediate biologic markers such as community viral load and key process outcomes such as number of men circumcised. The growing difference between the impacts of Combination I and Combination II over time indicates the importance of longer periods in comparing the effectiveness of different combination prevention packages. Lastly, the small relative differences in the impact of single interventions at the short-term , could mAlthough the efficacy of topical or oral pre-exposure prophylaxis (PrEP) in reducing HIV risk has been demonstrated The goal of this model was to evaluate the population-level impact on HIV incidence if high coverage levels of available strategies are achieved in KZN, South Africa. The numerical estimates presented are specific to KZN, and the impact of interventions in different contexts might vary. In settings where a lower proportion of men are already circumcised, the impact of the combination intervention could be greater and more sensitive to the uptake of circumcision. We have incorporated the uncertainty in identifying the epidemiological context of KZN using a rejection-sampling approach. Prioritizing interventions for specific age, gender, or demographic group was not the focus of this modelling exercise. Therefore, the model is not age-structured and has no details about how sexual partnerships are formed between members of different demographic groups In summary, combination HIV prevention represents the best hope for a significant impact in reducing population HIV incidence in hyperendemic countries. These theoretical model results indicate that achieving high coverage of evidence-based combination HIV prevention interventions will have a greater population impact than any of the individual interventions, and could lead to large reductions in population HIV incidence in four years with increased impact thereafter. It is now time to implement this combination prevention approach in a high prevalence sub-Saharan setting, with a rigorous evaluation conducted with sufficient coverage levels and duration in order to assess the impact on population HIV incidence.Supplementary Material S1(DOC)Click here for additional data file.


Prostate adenocarcinoma (CaP) is the most frequent cancer and second leading cause of cancer death among men in the Western world . Hh signOur work has shown that Hh signaling can be regulated in part by a protein normally expressed in the prostate, protease nexin 1  . PN1 is in vivo. Indeed, inducing expression of PN1 in several different prostate cancer cells was as effective in the inhibition of proliferation as treatment with the Hh-pathway inhibitor cyclopamine.Our new study features several components. Firstly, we show that PN1 inhibits Hh signaling by reduction in the hedgehog ligand, Sonic (SHH). This regulation is dependent upon uptake of PN1 through the LRP receptor and on the inhibitory activity of PN1. The immediate functional impact of PN1 is to reduce proliferation of prostate metastatic cells both in culture and Hh signaling inhibition also substantially reduced neo-angiogenesis. Xenografts from PC3 prostate cancer cells expressed high levels of SHH, producing small, disorganized vessels typical of tumour vasculature. These results are in line with other reports linking the Hedgehog pathway with the formation of new blood vessels and increases in tumor growth and metastasis . By contThe capacity to reduce tumor growth and vessel formation suggests a new physiological significance for PN1 and perhaps other serpins with similar functions. Interestingly, PN1 features some similarities to maspin, a tumor suppressor and angiogenesis blocker. Maspin has also been reported to reduce uPA-mediated CaP . HoweverAs a regulatory toggle, we have shown that PN1 is also a target of MMP9-mediated degradation. The localisation of PN1 in the ECM allows for its regulation by MMP9, an important factor in metastasis that can cleave collagen and basement membrane to drive this process. We extended these findings to show that higher MMP9 levels are inverse to PN1 levels and that this ratio is important in controlling hedgehog signaling. We demonstrate competing regulation of hedgehog signaling by PN1 and MMP9 in tissue culture, normal prostate tissues, xenografts, and orthotopic prostate cancers.The presence of PN1 in normal and BPH (benign prostate hypertrophy) tissues and decline in higher Gleason score samples may indicate that its loss is a marker of malignant transition in the prostate epithelium, amplifying Hh signaling. Indeed, one aim was to utilize Hh signalling as a stratifier of intermediate grade prostate cancer as the transitional status of these patients often makes treatment uncertain. Implications are that pharmacological inhibition of hedgehog signaling might be useful in this subset of patients. Thus, PN1 itself might be a potential therapeutic agent and, in combination with other SHH inhibitors, may be useful in the encouragement of CaP growth lag or inhibition. This work further indicates the potential important of hedgehog signaling in prostate cancer (Figure


Down syndrome (DS) is caused by the overexpression of genes on triplicated regions of human chromosome 21 (Hsa21). While the resulting physiological and behavioral phenotypes vary in their penetrance and severity, all individuals with DS have variable but significant levels of cognitive disability. At the core of cognitive processes is the phenomenon of synaptic plasticity, a functional change in the strength at points of communication between neurons. A wide variety of evidence from studies on DS individuals and mouse models of DS indicates that synaptic plasticity is adversely affected in human trisomy 21 and mouse segmental trisomy 16, respectively, an outcome that almost certainly extensively contributes to the cognitive impairments associated with DS. In this review, we will highlight some of the neurophysiological changes that we believe reduce the ability of trisomic neurons to undergo neuroplasticity-related adaptations. We will focus primarily on hippocampal networks which appear to be particularly impacted in DS and where consequently the majority of cellular and neuronal network research has been performed using DS animal models, in particular the Ts65Dn mouse. Finally, we will postulate on how altered plasticity may contribute to the DS cognitive disability. Down syndrome (DS) results from the triplication of genes on human chromosome 21 (Hsa21) and is associated with a range of phenotypes including craniofacial changes , susceptThe phenomenon of synaptic plasticity has been strongly linked to cognitive processes, such as learning and memory , 9. SynaThe link between synaptic plasticity and cognitive processes such as learning and memory is frequently studied within the hippocampus, a structure involved in diverse cognitive processes such as those related to acquisition, coding, storing, and recalling information in physical or perceived spatial environments –16. Multin utero and persisting throughout life. The neocortical development of DS fetuses appears normal up to at least gestational week 22 [The basis for altered synaptic plasticity in DS can be found in changes in the physical structure of the dendrites. Alterations in the shape and densities of dendrites would be expected to adversely affect the information storage capacity of neural networks by reducing the number of potential sites for plasticity to occur. Consistent with this idea and the observed deficits in cognition associated with DS, examination of postmortem brain tissue from DS individuals reveals profound alterations in dendritic and neuronal densities and morphology across many regions of the brain beginning  week 22 –21. By 4 week 22 , 20. In  week 22 . These c week 22  since, d week 22 .  During the early postnatal period, significant deficits in brain weight and gross morphology as well as myelination and neuronal densities and morphology appear . InitialThese morphological and cognitive deficits are consistent with aberrant synaptic plasticity, and, indeed, while difficult to measure directly in human subjects, evidence suggests that plasticity is reduced at least in the motor cortex of DS individuals . AdditioOver the past few decades, several mouse models of Down syndrome have been developed to further our understanding of the link between enhanced gene dosage and DS phenotypes such as altered plasticity and cognition. The Tc1 mouse model carries an almost complete, freely segregating copy of Hsa21, but the chromosome is present in only approximately 50% of cells making this a mosaic model of DS . InteresSimilar to the Ts65Dn mouse but with smaller triplicated Mmu16 segments are the Ts1Cje and Ts1Rhr mouse models. These mice display phenotypes similar to Ts65Dn mice including hippocampal dysfunction; however, the severity of the deficits is reduced . The redOlig1/2 [Mouse models of DS, including the Ts65Dn strain, show similar detrimental changes in neuronal and dendritic morphologies observed in humans. The neocortex of Ts65Dn mice contains fewer excitatory neurons but an increased number of a subset of inhibitory neurons relative to euploid controls, a phenotype that was reversed by normalizing the expression levels of Olig1/2 . AdditioOlig1/2 . In the Olig1/2 . Such a Olig1/2 , again sOlig1/2 . Beyond Synaptic plasticity in the hippocampus is often investigated in the context of long-term potentiation (LTP) in which high-frequency activation of specific inputs in the hippocampus results in a long-lasting potentiation of synaptic responses along the excited afferent pathway. First described in the anesthetized rabbit , this phA antagonists picrotoxin [A receptors in hippocampal slices from Ts1Cje and Ts1Rhr mice rescues LTP deficits in the dentate gyrus in these DS mouse models as well [As outlined above, structural changes suggest that inhibition is exaggerated in the trisomic hippocampus. Consistent with this idea is the observation that LTP in the dentate gyrus and CA1 regions of Ts65Dn hippocampal slices, induced by high-frequency stimulation and theta burst protocols, respectively, can be rescued by the GABAcrotoxin , 76. Blo as well , 68. A s as well .In addition to suppressed LTP, hippocampi from Ts65Dn mice show enhanced long-term depression (LTD) in response to sustained activation of excitatory synapses , 78. ThiChanges in intracellular calcium concentrations are important triggers for many intracellular signaling cascades including those underlying LTP and LTD . For exaActivation of postsynaptic NMDA receptors (NMDARs) concomitant with the depolarization of the postsynaptic membrane is sufficient to relieve the magnesium block of NMDA channels leading to an influx of calcium into the intracellular postsynaptic space. In the case of LTP, the rise of intracellular calcium leads to the activation of calcium calmodulin-dependent protein kinase II (CaMKII), a necessary step for initiating NMDAR-dependent LTP . BlockinOne of the substrates targeted by CaMKII during the initial expression of LTP is the serine 831 residue on GluR1 subunits of AMPA receptors , 95. ThiProtein kinase A (PKA) also plays a critical role in establishing LTP. In particular, evidence suggests that it is involved in initiating the protein synthesis required for the late phase of LTP , 98. BloPKA plays a role in LTD where its substrates, such as GluR1, show increased dephosphorylation following induction , 103. DeIn addition to GluR1 subunits, both CaMKII and PKA converge on another common effector, the mitogen-activated protein kinase (MAPK/ERK), that is associated with a host of synaptic-plasticity-related cellular processes . In the α, GluR1, and NMDA receptor subunit 1 [Brain-derived neurotrophic factor (BDNF) contributes to LTP by stimulating protein synthesis. In activating postsynaptic TrkB receptors, BDNF stimulates the PI3K pathway  which caubunit 1 . In Ts65ubunit 1 . Consistubunit 1 . As BDNFubunit 1 , these lExamination of BDNF levels in DS mouse models presents a complex picture. In Ts65Dn mice, levels of BDNF in the frontal cortex are diminished . In the The discrepancies between observations in Ts65Dn and Ts1Cje BDNF levels may reflect how BDNF expression is distributed in these structures, elevated in some subregions or subcellular compartments while diminished in others, resulting in an increased functional effect despite reduced global levels. Conversely, the differences in observed BDNF levels could be related to the different numbers of genes overexpressed in these two mouse lines , 65, 122GABA B receptors, the type 2 G-protein-activated inward rectifying potassium (GIRK2) channel may act to dampen the expression of LTP in Ts65Dn hippocampus through a shunting mechanism. As mentioned above, postsynaptic calcium influx is critical for LTP and LTD in the hippocampus. This initiating step relies heavily upon depolarization of the postsynaptic membrane to relieve the voltage-dependent magnesium block of NMDA channels. Any phenomenon that reduces the ability of the postsynaptic membrane to depolarize would thus be expected to adversely affect plasticity. Through its coupling to Kcnj6 which is located on the chromosomal segment triplicated in DS and Ts65Dn mice, and, consequently, elevated expression levels have been found in the Ts65Dn hippocampus [in vitro [+/−) resulted in a gene dosage-dependent change in the resting membrane potential and facilitation of LTP in GIRK2 knockout mice [GIRK2 is encoded by the gene pocampus , 123. Atpocampus  and CA1 in vitro . Selectiout mice . Selectiout mice . B receptors which, in pyramidal neurons, are in closest proximity to GIRK2-contaning potassium channels near glutamatergic synapses on dendritic spines [B receptors are functionally linked to GIRK channels, and, indeed, whole-cell GABAB-mediated potassium currents are exaggerated in Ts65Dn hippocampal neurons [B-mediated currents is also found in the dentate gyrus where the presynaptic release probability of GABA is increased [B and other G-protein coupled receptors, appear to act as a break on synaptic plasticity in the Ts65Dn hippocampus.These effects on LTP and LTD could be mediated through GABAc spines . GABAB r neurons , 128. Inncreased . Thus, GThe hippocampus receives major inputs from the entorhinal cortex (EC) which converge on CA1 pyramidal through two main pathways: the perforant pathway (PP) and the temporoammonic (TA) pathway . The PP Electroencephalogram (EEG) recordings from DS individuals suggest that such a preferential suppression of high-frequency information flow may result from overexpression of Hsa21 genes. Compared to controls, DS individuals have increased power at low EEG frequencies and a corresponding reduction in power at higher frequencies . SimilarA number of studies using the Ts65Dn mouse model of DS have examined the possibility of pharmacologically reversing cognitive deficits (reviewed in ). Of parA receptor antagonist, picrotoxin to hippocampal slices from Ts65Dn mice rescues LTP in the dentate gyrus [A receptor antagonists  improves cognition in Ts65Dn mice suggesting that the efficacy of this class of pharmacological agents could be tested for reversing impaired cognition in DS [A receptors can induce seizures, translating these findings to humans requires great caution. Careful screening of similar drugs or design of pharmacological compounds with similar blocking capabilities but reduced propensities for inducing seizures may prove to be effective treatments. Currently, a small molecule targeting GABAA receptors developed by F. Hoffmann-La Roche Ltd  is in clinical trials with the goal of safely improving cognition in DS individuals. Application of the GABAte gyrus  and CA1 te gyrus . Chronicon in DS . HoweverA receptors.Braudeau et al. , 135 areAnother pharmacological avenue targets aberrant NMDA receptor-mediated signaling apparently present in Ts65Dn mice. The uncompetitive NMDA receptor antagonist memantine improves the cognitive performance of Ts65Dn mice  and normIn addition to pharmacological approaches, behavioral therapies have been shown to improve cognition in Ts65Dn mice. When housed in enriched environments (larger cage with novel objects such as toys and running wheels), trisomic mice performed as well as euploid littermates in the Morris water maze and had normalized hippocampal LTP , 137. InThe benefits of environmental enrichment appear to be linked in part to regulation of excess inhibition in the neocortex and hippocampus. Release of GABA from synaptosomes isolated from the hippocampus and neocortex is elevated in Ts65Dn mice, an effect that is reversed by environmental enrichment . In adulDeficits in neurogenesis in the dentate gyrus and forebrain subventricular zone in Ts65Dn mice are also reversed following environmental enrichment  and may Early behavioral intervention techniques designed to improve development in DS children show great promise , 142 sugSynaptic plasticity is believed to be the process central to learning and memory. This belief is bolstered by experiments where drugs that normalize aberrant plasticity in hippocampal slices isolated from mouse models of DS also confer improvements in cognition in in intact adult mice. The initiation and maintenance of plastic changes involve structural and compositional modifications of synapses that depend on intracellular signaling cascades. The reduced excitatory neuronal densities and deficits in dendritic morphologies present in individuals with DS diminish the capacity of their neural networks in general to undergo neuroplastic adaptations. Combined with the deficits in signaling pathways reported in Ts65Dn mice, evidence strongly suggests that synaptic plasticity is severely impaired in DS neural networks. By understanding how plasticity is perturbed, we can design therapies to reverse these phenotypes and ultimately improve cognition in DS individuals.


In humans converging evidence indicates that affective aspects of touch are signaled by low threshold mechanoreceptive C tactile (CT) afferents. Analyses of electrophysiological recordings, psychophysical studies in denervated subjects, and functional brain imaging, all indicate that CT primary afferents contribute to pleasant touch and provide an important sensory underpinning of social behavior. Considering both these pleasant and social aspects of gentle skin-to-skin contact, we have put forward a framework within which to consider CT afferent coding properties and pathways—the CT affective touch hypothesis. Recent evidence from studies in mice suggests that CTs, when activated, may have analgesic or anxiolytic effects. However, in neuropathic pain conditions, light touch can elicit unpleasant sensations, so called tactile allodynia. In humans, tactile allodynia is associated with reduced CT mediated hedonic touch processing suggesting loss of the normally analgesic effect of CT signaling. We thus propose that the contribution of CT afferents to tactile allodynia is mainly through a loss of their normally pain inhibiting role. In contrast, unmyelinated low-threshold mechanoreceptive afferents (C-LTMRs) have been known to exist in the hairy skin of mammals since 1939  afferents conducting impulses at high speed   was weak, vague, and inconsistent. In some trials the subject reported no sensations at all. In others, they reported a sensation of light touch which was barely detectable and difficult to describe. One of the subjects (GL) reported that she began to feel more touch sensations in her daily life once she had had the experience of touch perception from the affected skin areas during the experiments and had become aware of this type of perceptual experience. Although the two neuronopathy subjects were not able to give a concise or detailed description of the sensation elicited by CT stimulation, they both reported, independent of each other, that it was a pleasant touch experience with no hint of pain, tickle, or itch. None of the two neuronopathy subjects feel tickle in the affected skin areas which contradicts the old hypothesis that CTs may signal a tickling sensation , as less pleasant than do matched controls and also differ in their rating patterns across stimulation velocities. Hence, these observations further support the notion that CT afferents make a critical contribution to the perception of affective touch. We have also examined patients with a hereditary disorder associated with a nerve growth factor beta (NGFB) gene mutation causing a denervation pattern opposite to that of the neuronopathy subjects GL and IW. Carriers of the NGFB mutation show a reduction in density of thinly myelinated and unmyelinated nerve fibers, thus likely including CT afferents, whereas their Aβ afferents are intact. Their condition has been classified as hereditary sensory and autonomic neuropathy type V (HSAN-V). We have addressed the relationship between C fiber function and pleasant touch perception in 10 HSAN-V individuals from a unique population of carriers  is used to study brain responses to touch stimuli in neurologically intact subjects and in neuronopathy subjects lacking Aβ afferents, different sensory areas are activated by Aβ and CT afferents. In healthy subjects soft brush stroking activates the classical somatosensory areas S1 and S2 as well as insular cortex, notably the posterior part of the contralateral insular cortex  for brush stroking on the hairy skin of the forearm  knock-out mouse, which functionally disconnects signaling from C-LTMRs by preventing glutamate release  promoting conditioned place preference in mice, indicating that such activation is positively reinforcing and/or anxiolytic (Vrontou et al., The topic of C-LTMRs in pain inhibition also ties back to the finding of pharmacogenetic activation of MRGPRB4Based on this animal literature we set out to examine the contribution of CT afferents to the allodynic condition in humans using the heat capsaicin model of dynamic tactile allodynia (Liljencrantz et al., Considering a possible analgesic effect of C-LTMR signaling (Lu and Perl, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background/objective: Polybrominated diphenyl ethers (PBDEs) and their hydroxylated (OH-) or methoxylated forms have been detected in humans. Because this raises concern about adverse effects on the developing brain, we reviewed the scientific literature on these mechanisms.2+) homeostasis, and intracellular signaling pathways.Data synthesis: Many rodent studies reported behavioral changes after developmental, neonatal, or adult exposure to PBDEs, and other studies documented subtle structural and functional alterations in brains of PBDE-exposed animals. Functional effects have been observed on synaptic plasticity and the glutamate–nitric oxide–cyclic guanosine monophosphate pathway. In the brain, changes have been observed in the expression of genes and proteins involved in synapse and axon formation, neuronal morphology, cell migration, synaptic plasticity, ion channels, and vesicular neurotransmitter release. Cellular and molecular mechanisms include effects on neuronal viability (via apoptosis and oxidative stress), neuronal differentiation and migration, neurotransmitter release/uptake, neurotransmitter receptors and ion channels, calcium  the role of oxidative metabolism in producing active metabolites of PBDEs and their impact on brain development; b) concentrations of parent and OH-PBDEs in the brain; and c) interactions between different environmental contaminants during exposure to mixtures, which may increase neurotoxicity.Conclusions: The increased hazard for developmental neurotoxicity by hydroxylated (OH-)PBDEs compared with their parent congeners via direct neurotoxicity and thyroid disruption clearly warrants further investigation into  Fire safety standards, including the application of flame-retardant chemicals, have been established in modern societies to reduce deaths and injuries as well as economic impact of fires. Bromine, like other halogens, quenches free radicals generated in a fire with high efficiency, thereby preventing the propagation of a flame. In Europe and North America, brominated flame retardants (BFRs) are therefore commonly used in household applications. BFRs were established as the major chemical flame retardant in the 1970s after the discovery of the adverse effects of polychlorinated biphenyls (PCBs) on human and environmental health and in experimental studies  and hydroxylated (OH-)PBDEs in vitro . Interesin vitro . Human ein vitro .Since the start of this century, exposure to BFRs has been associated with  neurotoxicity . RecentlIn this review we extend knowledge from previous reviews of underlying mechanisms of adverse effects on the nervous system by exposure to PBDEs  and alsoin vivo toxicity studies have revealed only moderate effects of PBDEs at high exposure concentrations, mainly in the liver and thyroid gland  and have been found to bind to transthyretin (TTR), a thyroid hormone transport protein, and thyroid receptors α and β (4 (hypothyroidism), were confirmed in vivo in rodents exposed prenatally and/or neonatally (3) in serum of rats exposed to PBDEs . Decreases in synaptic plasticity were related to hippocampal BDE-209 concentrations, which were highest after intragastric exposure during the lactational period compared with after exposure via the mother  or directly to the pups after weaning  2–9] to BDE-99 resulted in increased activity of the glutamate–nitric oxide–cyclic guanosine monophosphate pathway in rat cerebellum . This paSeveral studies have revealed effects of PBDEs on neurotransmitter receptor function and brain protein levels . EffectsCell viability. In several in vitro studies, tetra- and pentaBDEs were found to induce apoptotic cell death in primary neurons or neuronal cell lines, which appeared to be a consequence of oxidative stress , 10 μM]. Instead, a larger proportion of the neural stem cells differentiated into glial cells  have been shown to affect several aspects of inter- and intraneuronal signaling, including inhibition of neurotransmitter uptake in synaptosomes and neurotransmitter vesicles and alterations in calcium (Ca2+) homeostasis (2+]i). At 2 μM, BDE-47 induced only subtle changes in Ca2+ homeostasis that manifested as increased frequency and duration of fluctuations in [Ca2+]i, whereas no effects were observed on [Ca2+]i when PC12 cells were exposed to BDE-49 , BDE-99, BDE-100 , or BDE-153  showed no effect]; although the potency to disturb calcium homeostasis depended on the presence of large atomic groups (bromine atom or aromatic ring) adjacent to the OH, the hydroxylation position was of lesser importance  receptor and antagonistic effects on the α4β2 nicotinic acetylcholine (nACh) receptor (expressed in Xenopus oocytes) were demonstrated for 6-OH-BDE-47, whereas these effects were not observed for BDE-47  affect all levels of neurotransmission, from presynaptic neurotransmitter homeostasis, intracellular signaling, and neurotransmitter release to postsynaptic neurotransmitter receptors. For some neurotoxic end points, it is evident that OH-PBDEs have a higher potency than their parent congeners (2+ release (or inhibition of Ca2+ uptake) suggests that mitochondria are a primary target for PBDE toxicity. This is supported by the observation of accumulation of PBDEs in mitochondria in mouse primary neurons (2+) signaling pathways. Influence of transient alterations in [Ca2+]i on brain development is not unlikely, because Ca2+ signals are essential in (early) brain development chronic exposure  has especially been proven to be very sensitive to neurotoxic effects (reviewed by 4-binding globulin (TBG), which is the main transporter protein for thyroid hormones in humans across the blood–brain barrier  brain is detected in wildlife  and toxi barrier . Becausein vitro effects related to neuronal signaling, there is a scientific rationale to use concentration addition as the default precautionary approach for risk assessment of mixtures of PBDEs, PCBs, and their metabolites. Because of the possible additivity of these environmental pollutants at levels below effective concentrations of the individual congeners and because of the higher sensitivity of the developing brain, mixture effects must be taken into account for human risk assessment for developmental neurotoxicity and investigated further.For most classes of chemicals, risk assessment is based on exposure and hazard assessment of single compounds, with the exception of the toxic equivalency approach for dioxins and dioxin-like compounds . In realAlthough the use of PBDEs has been greatly reduced in most countries because of voluntary and legislative measures, humans and wildlife will still be exposed to PBDEs and their metabolites for many years to come because of their environmental persistence and the fact that consumer products with these flame retardants are still present in many homes. This also underscores the necessity of implementing better hazard and risk assessment strategies for identifying human health risks of new chemicals, such as novel halogenated flame retardants. More knowledge on oxidative metabolism and the possible bioactivation of PBDEs, the sensitivity to neurotoxicity during specific processes of brain development, the concentrations of (OH-)PBDEs in the brain, and the interactions between different environmental contaminants during simultaneous exposure is currently required to further improve the assessment of neurotoxic risk of PBDEs, their metabolites, and related environmental contaminants.(148 KB) PDFClick here for additional data file.


Carpodacus mexicanus), we have found that retinal carotenoid accumulation varies significantly among individuals and is related to dietary carotenoid intake. If diet-induced changes in retinal carotenoid accumulation alter spectral sensitivity, then they have the potential to affect visually mediated foraging performance.For many bird species, vision is the primary sensory modality used to locate and assess food items. The health and spectral sensitivities of the avian visual system are influenced by diet-derived carotenoid pigments that accumulate in the retina. Among wild House Finches  and dimmer low-contrast (red-filtered) lighting conditions. In experiment one, zeaxanthin-supplemented birds had significantly increased retinal carotenoid levels, but declined in foraging performance in the high-contrast condition relative to astaxanthin-supplemented birds that showed no change in retinal carotenoid accumulation. In experiments one and two combined, we found that retinal carotenoid concentrations predicted relative foraging performance in the low- vs. high-contrast light conditions in a curvilinear pattern. Performance was positively correlated with retinal carotenoid accumulation among birds with low to medium levels of accumulation (∼0.5–1.5 µg/retina), but declined among birds with very high levels (>2.0 µg/retina).Our results suggest that carotenoid-mediated spectral filtering enhances color discrimination, but that this improvement is traded off against a reduction in sensitivity that can compromise visual discrimination. Thus, retinal carotenoid levels may be optimized to meet the visual demands of specific behavioral tasks and light environments. Food detection is a major selective pressure shaping the visual systems of animals, and a primary goal of visual ecologists is to understand the links between the environment, foraging behavior, and the physiology and function of the visual system Foraging may also have a much more direct influence on the performance of the visual system because it determines the availability of nutrients necessary for the development, maintenance, and function of the eye. For example, retinal  is an essential component of the photopigments of all animals and must be acquired from food, and diet-derived carotenoid pigments act as intraocular filters to protect the eye and tune spectral sensitivities of photoreceptors in many species Among vertebrates, birds have some of the most complex and capable visual systems and are a model for the study of visual ecology de novo, but must acquire them through their diet, and carotenoid accumulation in the retina is sensitive to recent dietary pigment intake Taeniopygia guttata) and crimson rosellas (Platycercus elegans) and observed subtle shifts in the absorbance of specific types of oil droplets in specific regions of the retina. They concluded that these small changes were unlikely to affect spectral sensitivity; however this was not tested directly.Carotenoids are particularly interesting components of the visual system because their accumulation 1) is dependent upon environmental availability and acquisition, and 2) may be traded off among multiple functions in the body, including antioxidant protection, immune system performance, and body coloration Carpodacus mexicanus). The house finch is a common North American passerine and a model species for the study of sexual selection and the evolution of elaborate ornaments In this study, we examined the influence of dietary carotenoid supplementation and retinal carotenoid accumulation on the visually mediated foraging behavior of the house finch .ad libitum access to tap water and a very low carotenoid (0.078±0.031 µg/g) base diet of sunflower seeds. The greenhouse was illuminated with sunlight, and throughout the study the birds were maintained on a natural photoperiod. The birds were fed the base diet for eight weeks to minimize retinal carotenoid variation stemming from dietary differences in the wild. In weeks seven and eight of the initial depletion period, we tested foraging performance (see below) and in week nine we randomly assigned birds to one of three diet treatments: 1) control – four males and four females received the base diet and tap water with a non-carotenoid vitamin supplement ; 2) zeaxanthin – five males and five females received a supplement of zeaxanthin beadlets  suspended in their drinking water and the vitamin supplement; and 3) astaxanthin - five males and five females received a supplement of astaxanthin beadlets  suspended in their drinking water and the vitamin supplement. The birds were given the supplements ad libitum each weekday for eight weeks (weeks 9–16), with plain tap water provided on weekends. At the start of week 17 and continuing through week 18, all birds were returned to the base seed and tap-water diet and we again tested foraging performance (see below). Carotenoids deplete from the retina relatively slowly compared to other tissues, requiring ≥4 weeks of deprivation to cause significant declines In June 2009, we captured 14 adult male and 14 adult female house finches on the campus of Arizona State University in Tempe, Arizona, USA in baited basket traps , while the remaining 14 birds continued on the unsupplemented sunflower seed diet. Supplementation continued for eight to ten weeks and, following a depletion period as described in experiment one, we tested the foraging performance of all birds (see below). At the conclusion of the mate choice tests, we euthanized all birds and collected retinas to directly measure carotenoid accumulation (see below).In November 2009, we captured and housed 27 female house finches to study the influence of dietary carotenoid supplementation on female mate choice behavior (data not presented here). We trapped these finches as described in experiment one and maintained them on a sunflower seed diet. In January 2010, we randomly selected 13 females and supplemented their drinking water with carotenoids  dyed with red food coloring  full, unfiltered fluorescent light , or (2) red-filtered-light created by placing filters  over the fluorescent lights , Fig. S1In experiment one, prior to the second foraging performance test (week 16), we measured the food color preferences of all birds following the methods of Bascuñán et al. We quantified amounts of specific carotenoid types in the left retina of each bird using high performance liquid chromatography (HPLC). Extraction procedures, analytical methods, and the results of experiment 1 are reported in Toomey & McGraw Analyses were carried out in SPSS13 , and values are reported as mean ± SE throughout. To examine the influence of lighting conditions on the number of food pellets eaten, we used repeated-measures analyses of variance (rmANOVA), with the number of food pellets eaten in each lighting condition as the within-subjects factor and sex as a between-subjects factor. Because the number of pellets eaten differed significantly between lighting conditions (§3b), we tested the effects of dietary carotenoid supplementation on foraging performance in separate rmANOVAs for full and red-light, with the number of pellets eaten before and after supplementation as within-subjects factors and sex and supplementation treatment as between-subjects factors. Food color preferences were tested using rmANOVA, with seed color as the within-subjects factor and sex and supplementation treatment as the between-subjects factors. Non-significant interaction terms were removed from the models, Greenhouse-Geisser corrections were used when the models deviated from the assumptions of sphericity, and the significance level was set to α = 0.05.To test the relationship between our direct measures of retinal carotenoid accumulation and changes in foraging performance, we carried out separate repeated-measures analyses of covariance (rmANCOVA), with the number of food pellets eaten before and after supplementation as the repeated measure, sex as a between-subjects factor, and total retina carotenoid concentration as a covariate, under each lighting condition. Concentrations of all six retinal carotenoid types were significantly intercorrelated Because dietary supplementation had a relatively limited effect on the accumulation of retinal carotenoids The effects of dietary supplementation on retinal carotenoid accumulation are reported elsewhere . All retinal carotenoid types were significantly positively intercorrelated , with the exception of galloxanthin and an unidentified carotenoid .Female finches receiving the zeaxanthin-supplemented diet had significantly higher retinal carotenoid levels than females maintained on the low-carotenoid diet . The number of food pellets eaten in individual trials ranged from 0–24 under red light, and 3–27 under full light, and all individuals consumed pellets under each lighting condition in at least one of the three trials.Prior to carotenoid supplementation, birds ate significantly fewer food pellets in the low-contrast, red-filtered-lighting condition than in unfiltered full-light , but not control birds . Supplementation had no significant effect on foraging in the red-light condition ; females declined over time (pre: 15.00±0.93 vs. post: 12.4±0.95), while males remained relatively constant (pre: 11.3±0.93 vs. post: 12.0±0.95). There was a significant increase in the number of food items eaten in the red-filtered-light condition over time across all diet treatments . In the subset of birds for which we observed behavior during the trials, the amount of time spent actively foraging did not differ significantly between lighting conditions , the sexes , or among treatment groups . Over the course of these trials, we occasionally observed the birds making errors, picking up the distracters, manipulating them in their bills, and subsequently rejecting them.There was a significant effect of dietary carotenoid supplementation on number of food pellets eaten in the full-light condition . In separate analyses of the different retinal carotenoid types, concentrations of retinal galloxanthin and ε-carotene were significantly negatively correlated with the change in the number of food pellets eaten in full-light . Concentrations of other retinal carotenoid types were not significantly correlated with the decline in foraging performance . The temporal improvement in foraging performance in red-filtered-light was not significantly related to retinal carotenoid accumulation .Consistent with the treatment effects described above, retinal carotenoid levels, measured at the conclusion of experiment one, significantly predicted the change in the number of food pellets eaten in full-light before and after supplementation , with finches eating significantly more red dyed seeds than all other colors . There was no significant effect of dietary carotenoid supplementation on seed color preference  or on the total amount of food eaten .Seed consumption differed significantly by color type . However, across sexes and treatment groups, total retinal carotenoid concentration was a significant positive predictor of relative foraging performance in the low-contrast condition .Foraging performance, measured as the relative number of pellets eaten in the low- vs. high- contrast condition, in the post-supplementation period did not differ significantly among diet treatments or between the sexes . There was no significant linear relationship between retinal carotenoid accumulation and foraging performance . However, an inspection of the plotted data suggested that the relationship was not linear; we therefore fitted a quadratic relationship between retinal accumulation and foraging performance and found a significant fit , with the peak at 1.58 µg/retina, which falls just outside the 90th percentile of retinal carotenoid accumulation for wild birds , retinal carotenoid accumulation is developmentally plastic in response to light environment, such that chicks reared in dim environments develop less absorbent oil droplets with presumably lower carotenoid levels A carotenoid-mediated trade-off in avian visual function is supported by patterns of retinal carotenoid accumulation observed among species and individuals reared under varying light intensity. The retinas of nocturnal species (e.g. owls) have relatively pale oil droplets that presumably contain lower concentrations of carotenoids, which is hypothesized to improve their visual sensitivity under low light conditions Linking visual foraging performance to retinal carotenoid accumulation is particularly intriguing because carotenoid-based male plumage coloration plays an important role in house finch mate choice The visual pigment sensitivities of birds are considered to be highly conserved among species The trade-off between chromatic and luminance detection is an important force shaping the evolution of the visual system Figure S1Images of experimental food and lighting conditions. (A) A sample image of the red food pellets and inedible gray paper distracters presented to the birds; (B) Unfiltered full-lighting conditions in our study room (left panel) compared to the red-filtered-lighting conditions (right panel).(DOC)Click here for additional data file.Methods S1A detailed description of the methods used to measure the irradiance of the room lights and food and background spectral reflectance and the calculations of avian visual contrasts.(DOC)Click here for additional data file.


Arabidopsis thaliana have established a basic molecular framework for the acquisition of cell fate and generation of cell polarity in these compartments, as well as describing some of the key signals and receptors required to produce stomata in organized patterns and in environmentally optimized numbers. Here we present parallel analyses of stomatal developmental pathways at morphological and molecular levels and describe the innovations made by particular clades of plants.Plant development has a significant postembryonic phase that is guided heavily by interactions between the plant and the outside environment. This interplay is particularly evident in the development, pattern and function of stomata, epidermal pores on the aerial surfaces of land plants. Stomata have been found in fossils dating from more than 400 million years ago. Strikingly, the morphology of the individual stomatal complex is largely unchanged, but the sizes, numbers and arrangements of stomata and their surrounding cells have diversified tremendously. In many plants, stomata arise from specialized and transient stem-cell like compartments on the leaf. Studies in the flowering plant  Plants conquered land more than 400 million years ago. In the fossil record, the appearance of these pioneer species is contemporaneous with the appearance of structures on their surfaces called stomata. Each stoma  consists of paired epidermal guard cells, a pore between them and an airspace in the photosynthetic mesophyll tissue subtending it. The function of stomata is to regulate gas exchange between the plant and its surroundings. On short timescales (minutes to hours), the opening and closing of the stomatal pore by turgor-driven changes in guard cell shape is a key regulatory step in maintaining water and carbon dioxide balance. Work from many laboratories has defined the intracellular signal transduction cascades that mediate changes in pore size in response to hormone and environmental signals .2 concentration [The current view is that stomata arose only once during evolution . In earlntration . The stoPaleobotanical analyses utilizing the fossils of the early land plants and their currently living descendants have been combined with phylogenetic analyses to address the origins of stomata Figures  and 2A. Arabidopsis[In the most recently derived plant group  there is a dedicated epidermal lineage that produces stomata. In dicot plants, such as the research model abidopsis, these labidopsis  transcription factors, SPEECHLESS (SPCH), MUTE and FAMA [SPCH leads to a complete loss of the stomatal lineage whereas overexpression of SPCH leads to ectopic asymmetric cell divisions [The regulation of stomatal development is best understood at a molecular level in and FAMA -24  and SCRM2, form heterodimers with SPCH, MUTE and FAMA and promote all three stomatal fate transitions [scrm-D mutant converts the epidermis into stomata, a phenotype identical to MUTE overexpression, whereas double mutants of ICE1/SCRM and SCRM2 resemble spch[. Interestingly, ICE1/SCRM has been shown to be involved in cold stress response [Proteins encoded by the paralogous bHLHs, nsitions . A semidmble spch. Interesresponse . Since sresponse  and deveresponse , it is pMore signal integration occurs via mitogen-activated protein kinases (MAPKs) which regulate stomatal development and stress responses through a three-step phosphorylation cascade. MAPK kinase kinase YODA, MAPK kinases (MKK4/5/7/9) and MAPKs (MPK3/6) are essential for normal stomatal spacing -32. SPCHUpstream of the intracellular signalling cascades, genetic studies have revealed that stomatal spacing is regulated by secreted peptides of the EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) family -41, by tEPF1 or EPF2 results in more stomata, but mutant and overexpression phenotypes indicate that EPF2 prevents entry into the stomatal lineage whereas EPF1 acts later. Their paralogue, STOMAGEN/EPFL9, by contrast, is expressed in the underlying cell layer (mesophyll) and travels to the epidermis to promote stomatal differentiation [tmm background [CHAL/EPFL4/EPFL5 are expressed in internal tissues and their loss leads to a compromised growth phenotype resembling loss of ER[in vitro[in planta, TMM can heterodimerize with ER and ERL1. Clarifying the physical interactions and in vivo activities of the four receptors with the 11 members of the EPF family looks to be an exciting future area of research in Arabidopsis. Homologues of ERf, TMM and EPFLs are found in diverse species, including monocots and mosses, indicating that the potential for conserved signaling systems exist. To date, however, no experimental information is available outside of Arabidopsis.EPF1 and EPF2 peptides are stomatal lineage-expressed and regulate the number and orientation of asymmetric divisions -41. Lossntiation ,47. EPF1ntiation -41,46,47ntiation . Althougoss of ER. Thus, toss of ER. Recentl[in vitro and thatArabidopsis are established by asymmetric cell divisions, and these unusual and unequal divisions involve several other novel, plant-specific, proteins: BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL) [BASL mutants display misoriented asymmetric cell division and overexpression of BASL leads to ectopic outgrowths in the positions where BASL is peripherally concentrated [POLAR, its localization is dependent on BASL suggesting that they act in the same pathway [Stomatal lineages in E (BASL)  and POLAE (BASL) . BASL diE (BASL) . BASL muentrated . Hence, entrated . Althoug pathway .Arabidopsis stomatal development. Loss of PAN1 leads to mislocalization of actin and the nucleus. This disrupts asymmetric cell divisions and results in abnormal subsidiary cells [One of the major differences between dicot and monocot  stomatal pathways is that, in the latter, subsidiary cells are recruited from cell files flanking the stomatal lineage. This process requires the generation of a highly polarized cell division that is specifically oriented toward the GMC. After formation of a GMC , neighboring subsidiary mother cells (SMCs) divide asymmetrically to produce small subsidiary cells next to the GMC Figures  and 4. Sry cells ,53. Recery cells . PAN1, Rry cells .Arabidopsis, five bHLH genes are major determinants of the identities and behaviors of different stomatal lineage precursors. SPCH, MUTE and FAMA are fairly restricted in their expression pattern to subsets of the stomatal lineage whereas ICE1/SCRM and SCRM2 are expressed throughout the lineage and in additional non-stomatal lineage cells. When considering the diversity of stomatal pattern in nature, it is interesting to think about how the expression, regulation and function (and existence) of this class of regulators may change. Moreover, one might ask whether the heterodimeric partnership between SPCH, MUTE and FAMA with ICE1/SCRM and SCRM2 could be ancient or whether this is a new innovation.As described above, in . SPCHMUTE and FAMA belong to the group IA bHLHs [Selaginella (a model lycophyte) and Physcomitrella (a model moss) [http://www.phytozome.net/). In the incomplete transcriptome and genome sequences from plant lineages predating the emergence of stomata, neither group IA nor group III bHLH genes are obvious [personal communication). Based on sequences currently available, in all cases where group 1A members can be distinguished, there is also a group III bHLH, suggesting that their partnership can be ancient. A group 1A homologue from Physcomitrella can partially complement Arabidopsis mute and fama, but not spch mutants [Physcomitrella; in this moss, no early asymmetric divisions are evident and instead a single GMC is specified and undergoes incomplete cytokinesis to form two connected guard cells  . Distinc obvious  , FAMA, is most highly conserved in terms of expression pattern and loss of function phenotypes in both rice and Arabidopsis[In grasses, the positions of stomata are determined and fixed at early stages of leaf development and amplifying divisions are not present. In fact, only the differentiation (GMC to guard cells) step is similar between grasses and s Figure . Nonethee plants . Perhapse plants . OsSPCH2of AtSPCH. Overexpabidopsis.Arabidopsis, POLAR and BASL, do not. BASL does not resemble any other Arabidopsis proteins and only in the congeneric A. lyrata is there a significantly similar sequence. In ArabidopsisPOLAR is moderately similar to another gene (POLAR-LIKE1) and homologues of POLAR and POLAR-LIKE1 can be found in closely related (dicot) species such as poplar (POPTR B9IL54). Already in rice, however, the sequence similarity becomes restricted to a very small domain of the proteins. It is interesting to consider whether this inability to find such homologues is because the function of BASL and POLAR is required only in the dicots, or because similar functions are carried out by different genes and the apparent uniqueness of these proteins represents either fast substitution rates or that their roles can be served by other proteins. For example, scaffold proteins that bind others together into complexes play important roles in polarity generation in yeast and animals, yet these scaffolds are often not well conserved at the sequence level and consist primarily of multiple interaction surfaces.Homologues of the cell fate regulators and many of the signaling components discussed above appear in many plant species ,58,59. I2 availability, the guard cells must be able to sense such environmental factors. Guard cells in angiosperms appear to sense many of these factors autonomously, and key kinases (OST1), phosphatases (PP2C) and receptors for the “drought stress” hormone, ABA (PYR1) have been identified in Arabidopsis[2 and ABA responsiveness in non-vascular plants have come to different conclusions about when the sensing of these different environmental cues arose. Monitoring stomatal pore closure in response to ABA, [Selaginella moellendorffii and Physcomitrella patens could partially restore the ability of Arabidopsis ost1 stomata to respond to ABA. Moreover, knockout of PpOST1-1 significantly attenuated ABA response in P. patens stomata [A developmental approach concerns itself with the correct specification and pattern of stomata. From a physiological point of view, however, the behavior of these final products is key. Modulation of the stomatal pore aperture depends on coordinated morphologies of the guard cell pair and, particularly in the case of the grasses, on the coordination of guard cells and the specialized subsidiary cells that are obligate parts of the stomatal complex . For stonases OST, phospha to ABA,  conclude to ABA,  and Chat to ABA,  showed t stomata . The dif stomata .Arabidopsis has been used as a model genetic system for the analysis of cell fate, cell polarity and cell to cell communication. The nature of the gene products identified in such analysis, coupled with the long tradition of evaluating the numbers and patterns of stomata in diverse plants for taxonomic purposes makes this system a useful natural laboratory to look at the parallel evolution of genes and developmental trajectories. As the number of completed plant genomes increases and tools for experimental manipulation of non-model species develop, we believe there will be an excellent opportunity to test the roles of candidate cell fate- and cell signaling factor-encoding genes in creating developmental diversity.Stomatal development in The authors declare that they have no competing interests.AV and DCB and designed the study, wrote the manuscript and prepared the figures. All authors read and approved the final manuscript.


Nearly 60 years ago Otto Warburg proposed, in a seminal publication, that an irreparable defect in the oxidative capacity of normal cells supported the switch to glycolysis for energy generation and the appearance of the malignant phenotype Warburg, . Curious It has been shown that leukemia cells generate significant amounts of lactate even in the presence of adequate amounts of oxygen  provides electrons for OXPHOS in a Krebs cycle- and glucose-independent manner thereby supporting mitochondrial bioenergetics and the proliferative capability of various cell types , and succinyl-CoA cataplerosis from the cycle provides carbon skeletons for the synthesis of heme groups , the canonical regulator of glycolysis that carries out the conversion of fructose-6-phosphate into fructose-1,6-bisphosphate. Recent evidence suggests that in lymphoma (Nb2-11) and leukemia (TF-1) cell lines, PFK-1 has been post-translationally modified via a proteinase K-type of mechanism resulting in a shorter polypeptide which maintains catalytic activity, but is missing the allosteric sites required for inhibition by citrate, ATP, and phosphoenolpyruvate  , in which pharmacologic or genetic inhibition of Bcl-2 rapidly diminishes oxygen consumption capacity and increases ROS generation prior to the onset of apoptosis  (Fiegl et al., A preponderance of evidence supports Warburg’s observation that cancer cells rely on glycolysis to meet their energy demands, although common, generalized defects in mitochondrial oxidative capacity have yet to be identified for the vast majority of cancers. In fact, more recent evidence has confirmed that cancer cells maintain the ability to oxidize pyruvate, albeit under conditions that were pharmacologically induced by DCA. Nevertheless, the concomitant ROS generation caused by this process is conceivably cytotoxic and, as such, could impinge on cell viability (Bonnet et al., Can the observations of Weinhouse and Warburg be reconciled? The evidence presented here suggests that, at least in leukemia cells, they can. On one hand, our recent data support Feodor Lynen’s hypothesis that increased lactate generation can be promoted under normal oxygen tension through mitochondrial uncoupling – induced by coculture with bone marrow mesenchymal stromal cells – in the absence of permanent, transmissible defects in oxygen consumption capacity. On the other hand, our observation that leukemia cells sustain molecular oxygen reduction rates utilizing electrons derived from FAO is in agreement with the work of Weinhouse utilizing transplantable hepatomas (Bloch-Frankenthal et al., While is important to point out that this review has focused predominantly in hematological malignancies, our data parallels recent observations in solid tumors where glutamine-dependent, glucose-independent Krebs cycle activity has been reported in glioblastoma and melanoma cells (Scott et al., Based on analysis of the literature and our own data, we propose a paradigm Figure  for mitoThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Retinal neurons are highly vulnerable to a diverse array of neurotoxic stimuli that leads to their degeneration, which is a major contributor to blindness. This review summarizes the role of epigenetic factors in mediating neuronal homeostasis and survival to protect against cell death and neurodegenerative conditions. Studies in human patients and mouse models implicate numerous chromatin modifications in neuroprotective processes including histone protein acetylation and methylation, DNA methylation, and ATP-dependent nucleosome remodeling. Recent research has begun to uncover specific epigenetic mechanisms invoked by neurotoxic stimuli. Continued investigation in this area will be the key to the generation of therapeutic strategies for the intervention of retinal neurodegenerative diseases. The development of the nervous system from primordial structures occurs through a highly organized and intricately regulated series of events involving cell proliferation, differentiation, migration and maturation, culminating in the establishment of numerous neuronal cell types that participate in complex neural circuits and networks. Once fully differentiated, neurons are required to carry out their functions for the lifespan of the organism as most are considered irreplaceable if lost. While adult neurogenesis does occur in select regions of the central nervous system (CNS), the vast majority of neuronal cell types cannot be regenerated naturally from existing cellular pools. This implies that neurons are required to remain intact and functional in order for proper neuronal communication to persist and to avoid deficits in neural activity that may occur through accumulated losses over time. In order to overcome the wide range of insults and stressors encountered over a lifetime, neurons must be able to rapidly react to changing conditions and appropriately modify gene expression to maintain cellular integrity and functionality.To quickly respond to extracellular cues and signals, the cell relies on epigenetic mechanisms of transcriptional regulation. Changes in gene expression occur as a result of alterations in the structure of chromatin that either allow or restrict access to the regulatory regions that control transcriptional activity. The basic subunit of chromatin is the nucleosome, consisting of 146 base pairs of DNA wrapped around an octamer of two copies of each of the core histone proteins H2A, H2B, H3, and H4 [The development of the retina follows defined genetic programs that have been extensively characterized , 6. An iHistone proteins can be post-translationally modified by a variety of enzyme activities, resulting in their acetylation, methylation, phosphorylation, sumoylation, ubiquitination, ADP-ribosylation, deimination, and β-N-acetylglucosamination . These cThe transfer of an acetyl group to the lysine side chains of histone proteins causes the neutralization of the positive charge on the lysine residues and can therefore disrupt the interactions between histones and DNA within nucleosomes. Acetylation has been described for all of the core histone proteins and is generally associated with chromatin relaxation and transcriptional activation, while deacetylation favors chromatin condensation and transcriptional repression. The extent of post-translational acetylation is determined by the enzymatic activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs). It is the net effect of both HAT and HDAC activities that determines the overall acetylation level of histones and impacts the transcriptional status of particular genes.Crx knockout mice, binding of HATs to target promoters and the levels of acetylated histone H3 on these promoters was reduced [The acetylation of histones has been the most extensively studied chromatin modification in the context of neurodegenerative processes. Despite the different pathophysiologies associated with various neurodegenerative diseases, in vivo models demonstrate that a common feature of the degenerative process in the affected neurons is a reduction in the global levels of histone acetylation –13. Cons reduced , and exp reduced .ataxin-7 results in inhibition of HAT function, reduced H3 acetylation and reduced Crx occupancy of target genes [ataxin-7 mutant mice also exhibit dramatic reorganization of chromatin correlated with reduced expression and abnormal distribution of the linker histone H1c in rod photoreceptor nuclei; however, global histone acetylation levels are unchanged [Similar to CBP and p300, the GCN5-HAT component of the multi-subunit SPT3-TAF9-ADA-GCN5 acetyltransferase (STAGA) transcription coactivator complex can interact with Crx and is associated with histone H3 acetylation on its target gene promoters . The intet genes , and cauet genes , 19. In nchanged . It is pnchanged . Neverthrd1 mouse model of retinitis pigmentosa, reduced histone acetylation levels were detected in photoreceptors but not other retinal cell types that do not undergo degeneration [rd1 photoreceptor cells undergoing apoptosis which could be protected from cell death by exposure to HDAC I/II inhibitors [This is further supported by the important role of HDACs that has been demonstrated in various mouse models of retinal neurodegeneration. In the neration . This wahibitors . HDAC inhibitors . Howeverhibitors , indicathibitors , 26 or ehibitors , 28 furtrd10 mice, where it co-localizes with apoptotic photoreceptors as well as pro-apoptotic proteins in the outer nuclear layer of the retina at the peak of cell death, and after which, its retinal expression is dramatically reduced [rd10 photoreceptors due to its cellular mislocalization and reduced level of expression, therefore resulting in the degeneration of these cells.Class III HDACs are also involved in mediating neuronal survival. A neuroprotective role for the Sirt1 histone deacetylase has been demonstrated in various neurodegenerative disease conditions –32. Cons reduced . It is hrd1 mice [rd1 mice, despite the ability of HDAC6 to rescue neurodegeneration in a Drosophila model of spinobulbar muscular atrophy [Individual HDACs also appear to function in distinct neuronal survival pathways. Specific inactivation of HDAC1 in post-mitotic primary neurons results in significant cell death, while increased HDAC1 activity is protective against neurotoxicity in vivo . HDAC4 hrd1 mice . Accordird1 mice . In cont atrophy . In opti atrophy . Only HD atrophy . This seHistone methylation is a non-charge neutralizing modification that occurs on specific lysine and arginine residues of histones H3 and H4. Methylation of particular lysine side chains can act as either permissive  or repressive  marks in the context of transcription. However, the extent of methylation of these residues can also affect their function in transcriptional activation or repression, with differential effects mediated by mono-, di- or trimethylation, thereby adding another layer of complexity , 39.pcd) mouse is characterized by postnatal loss of several neuronal cell types including retinal photoreceptors [Similar to histone acetylation, histone methylation appears to be involved in the maintenance of different retinal cell types. The Purkinje cell degeneration  protein-containing repressive complexes with gene promoters . Two DNADnmt1 in mouse CNS precursor cells in vivo leads to global DNA hypomethylation and postnatal death of neurons in multiple brain regions [Dnmt1 deletion targeted to the dorsal forebrain causes severe and progressive degeneration of cortical and hippocampal neurons due to hypomethylation-induced apoptotic cell death occurring both pre- and postnatally [Dnmt3a in the CNS, global DNA methylation was unperturbed; however, mutant mice exhibited loss of hypoglossal motor neurons located in the brainstem, abnormal neuromuscular junctions, motor defects, and premature death [DNMT3b mutations in human mental retardation [A number of studies involving CNS-specific knockouts of Dnmts implicate altered DNA methylation in neurodegenerative phenotypes. Conditional deletion of  regions . Dnmt1 dtnatally . This watnatally , 51. There death . This mare death –57, furtardation , 59 indiAPP) gene was shown to be hypomethylated in older individuals, suggesting a link between enhanced transcriptional potential of a pathogenic gene and increased susceptibility to neurodegeneration with age [APP and beta-site APP cleaving enzyme 1 (BACE1) in aged monkeys with AD-like neuropathology [These effects of impaired Dnmt activity and resulting reduction in DNA methylation levels have been extended to primate models of neurodegeneration. In postmortem analyses of human brains, the promoter region of the Alzheimer’s disease (AD)-causing amyloid precursor protein  and that signal DNA damage (phosphorylation of the histone variant H2AX) [DNA damage is recognized as a significant mediator of neuronal cell death , 86. Incnt H2AX) . DefectsMitochondrial dysfunction is associated with various forms of retinal and brain degeneration, and mutation of mitochondrial genes can lead to neurodegenerative diseases , 97. MitBDNF expression is controlled by a number of epigenetic factors [Atrx-knockout mouse retinas may result from misregulation of a common pathway involving all three factors that functions to promote retinal neuron survival.Neurotrophins are growth factors important for supporting neuronal function and survival and exert their neurotrophic effects through receptor-mediated intracellular signaling pathways. A number of these factors have demonstrated roles in neuroprotection of various retinal cell types in vitro and when administered to mouse models of retinal degeneration , 98. Bra factors  and can  factors . Neurotr factors , 107. Gi factors  and the  factors , it is tNR2B repression [NR2B expression and NMDA receptor activity [NR2B expression was also shown to be regulated by the histone methyltransferase Setdb1 [Excitotoxicity is caused by the hyperactivity of glutamate receptors on the cell surface of neurons due to excessive levels of the excitatory neurotransmitter glutamate, which in turn produces multiple adverse effects that lead to neuronal cell death. Excitotoxic neuronal death is a common feature of acute and chronic neurodegenerative conditions affecting various regions of the CNS including the inner retina , 109. Stpression , while Hactivity . NR2B exe Setdb1 , indicate Setdb1 .Complementing the established role for epigenetic regulation in neuronal development and function are studies detailing the importance of chromatin remodeling for long-term survival. We have summarized the current knowledge of epigenetic strategies employed by neurons to withstand a lifetime of genetic and environmental insults in order to evade death. As these mechanisms continue to emerge, it is becoming clear that chromatin regulation of neuroprotection is complex and is impacted by a wide range of neurotoxic stimuli. Failure of these mechanisms leads to cellular dysfunction and neuronal loss, the key hallmarks of human neurodegenerative diseases.The development of effective treatments for retinal degeneration and other neurodegenerative disorders requires a thorough understanding of the molecular events that control the survival of neurons and their demise in these diseases. The finding that changes in DNA and histone covalent modifications contribute to altered gene expression that leads to neuronal cell compromise can be exploited to generate therapeutic approaches targeted to chromatin modifying activities. For example, the association of reduced histone acetylation levels with neurodegenerative conditions has led to the extensive investigation of HDAC inhibitors as well as the proposal of HAT activators as therapeutics for neuronal degeneration , 119. Ho