More than 90 percent of cattle in Tanzania belong to the indigenous Tanzania Short Horn Zebu (TSZ) population which has been classified into 12 strains based on historical evidence, morphological characteristics, and geographic distribution. However, specific genetic information of each TSZ population has been lacking and has caused difficulties in designing programs such as selection, crossbreeding, breed improvement or conservation. This study was designed to evaluate the genetic structure, assess genetic relationships, and to identify signatures of selection among cattle of Tanzania with the main goal of understanding genetic relationship, variation and uniqueness among them.Bos indicus SNP 80K BeadChip was used to genotype genome wide SNPs in 168 DNA samples obtained from three strains of TSZ cattle namely Maasai, Tarime and Sukuma as well as two comparative breeds; Boran and Friesian. Population structure and signatures of selection were examined using principal component analysis (PCA), admixture analysis, pairwise distances (FST), integrated haplotype score (iHS), identical by state (IBS) and runs of homozygosity (ROH). There was a low level of inbreeding (F~0.01) in the TSZ population compared to the Boran and Friesian breeds. The analyses of FST, IBS and admixture identified no considerable differentiation between TSZ trains. Importantly, common ancestry in Boran and TSZ were revealed based on admixture and IBD, implying gene flow between two populations. In addition, Friesian ancestry was found in Boran. A few common significant iHS were detected, which may reflect influence of recent selection in each breed or strain.The Illumina Population admixture and selection signatures could be applied to develop conservation plan of TSZ cattle as well as future breeding programs in East African cattle. The Tanzania Shorthorn Zebu (TSZ) is the major type of indigenous cattle in Tanzania and is comprised of a number of strains including Maasai, Sukuma, Tarime, Iringa Red, Mkalama Dun, Singida White, Mbulu, Gogo, Chagga, Pare, Fipa and Zanzibar. These strains of TSZ have considerable differences in terms of morphological features such as body size, coat color, horn size and orientation, adaptation to different ecological conditions  and they generally show some differences in the ability to withstand drought, heat stress as well as diseases and parasites , 2. The To improve productivity, the national development strategies for milk and beef production have since the 1960s, placed emphasis on the use of European, US or Asian breeds such as Friesian, Ayrshire, and Jersey (for milk production) and Simmental and Angus (for beef) among others for crossbreeding and upgrading the local cattle. Among all breeds imported in Tanzania, the Boran cattle which belongs to the Large East African Zebu (LEAZ) population, is the recommended animal for upgrading the TSZ for meat production whereas Friesian is the most preferred breed for milk production improvement. These animals are recommended because of their superior performance (meat and milk production) of their F1 or F2 crossbreds . These aSelection within a local population is a potential and sustainable strategy in developing countries such as Tanzania , 9. ThisWe designed this study to carry out the first comprehensive analysis of genetic variation within and among three strains of TSZ cattle and two comparative breeds; Boran and Friesian. To arrive at our conclusions, we first estimated the inbreeding levels using runs of homozygosity (ROH) and the genomic inbreeding coefficients (F-geno). The ROH represents genomic autozygosity occurring due to mating between selected and genomically related individuals. Both ROH and F-geno can be a good measure of breeding depression and reduced fitness or measures the probability that two genes at any locus in an individual are identical by descent (IBD) from the common ancestors . SecondlHe) and observed heterozygosity (Ho) in the Friesian breeds whereas the difference was smaller in TSZ strains and Boran breed “Ne) of the TSZ strains were greater than the values in Friesians. Nevertheless, Ne has decreased consistently during the last 1000 generations “Ne was greater than 1,000 in most indigenous breeds 300 generations ago and the number was reduced to 100 or less in the contemporary Tanzanian cattle populations “The ROH based inbreeding coefficient (F-ROH) and F-geno were calculated to estimate the level of inbreeding. The F-ROH ranged from 0.005 to 0.023 in TSZ strains, and was 0.012 and 0.018 in Boran and Friesian breeds respectively “ST and IBS “ST which ranged from 0.011 to 0.013 among the TSZ strains. We also saw differences between the TSZ strains and the Boran breed . When comparing TSZ strains with each other, FST was not higher than 0.4 at any loci and this result represented little difference between the strains “ST values of approximately 0.2 were obtained, suggesting a greater differentiation. Genetic relatedness between individuals (IBS) was shown to summarize similarity between breeds “The genetic variability among our animal populations and breeds was evaluated by estimation of FTo further illustrate the relationship among the animals analyzed, a principle component analysis (PCA) was carried out. Animals analyzed in this study were clearly distinguishable by three clusters. Using the first principal component (PC1), the first cluster was composed of the three strains of TSZ while the second and the third clusters were composed of the Boran and Friesian breeds, respectively “MDFIC), Stabilin 2 (STAB2), 5'-Nucleotidase Domain Containing 3 (NT5DC3), Heat shock protein 90kDa beta family member 1 (HSP90B1), Bovine dopamine receptor D5 (DRD5), Adipocyte determination and differentiation-dependent factor 1 (ADD1), Major facilitator superfamily domain-containing protein 10 (MFSD10), Small nuclear RNA activating complex polypeptide 1 (SNAPC1) and hypoxia inducible factor 1 alpha subunit (HIF1A) among others in the Maasai strain. In the Boran breed genes identified were Fibronectin type III domain containing 3B (FNDC3B), Solute carrier family 6 member 15 (SLC6A15) and Tetraspanin 19 (TSPAN19) genes. Whereas Gamma-aminobutyric acid type A receptor rho3 subunit (GABRR3), MYC induced nuclear antigen (MINA), cell division cycle 7 (CDC7), zinc finger protein 644 (ZFP644) and the Exostosin family 1 (EXT1) were among the 19 genes annotated in the Friesian breed. The signatures of selection detected among the animals in this study are presented in “Some genomic regions may be fixed in individuals within a population as a result of artificial or natural selection for reasons such as adaptability or productivity. In the present study, evidence for positive selection was determined by calculating the values of iHS which measured the relative decay of extended haplotype homozygosity (EHH) of the ancestral and derived core allele. Consequently eight regions (signatures of selection) have been under recent natural or artificial selection on chromosomes 4, 5, 6, 7, 10, 11 and 20 in the Maasai strain. Three signatures of selection were detected on chromosomes 1, 5 and 14 in the Boran breed. No genomic region appeared to be involved in recent selection in the Sukuma and Tarime strains. In the Friesian breed, nine genomic regions were identified on chromosomes 1, 3, 14, 16, 19, 20, 22 and 27. A considerable number of genes were identified from the signatures of selection observed in this study. This included 20 genes in the Maasai strain, three in Boran and 19 in Friesian. Among the genes identified were the MyoD family inhibitor domain—containing gene  could be the origins of both the African and the European cattle populations [Bos taurus) and input of Asian zebu genes were discovered in characteristically and morphologically distinct African breeds that were regarded as zebu [In addition, results of admixture, IBS, Fst, and AMOVA have shown traces of TSZ populations in the Boran and Friesian breeds and vice versa. This is probably because of the sharing of recent common ancestors due to migration or closer ancient lineages as previously observed . Both TSulations . Probablulations . The movulations , 20. TheMoreover the differentiation between TSZ and Friesian animals further demonstrates that most agro-pastoralists and pastoralists in rural areas do not crossbreed their indigenous breeds with dairy breeds such as Friesian. This is probably due to the fact that in agro-pastoral and pastoral communities the TSZ breed is preferred to exotic breeds or TSZ x Friesian crosses because of the adaptive characteristics of the TSZ breed to tolerate drought, feed shortages, poor quality forages and endemic diseases. Therefore, crossbreeding with exotic breeds is not a better option for achieving the long term existence of different strains of TSZ and increased productivity, but rather selection and interbreeding among the indigenous strains as different human ethnic groups intermingle.MDFIC gene which has been associated with modulatory roles in immune cells or immune system capabilities [HSP90B1 gene is involved in a function related to lactation [DRD5 gene has been implicated in the regulation of feeding behavior and energy homeostasis [HIF1A is one of the factors promoting vascular endothelial growth factor-induced angiogenesis during luteal development and contributes to establishing of luteal vascularization [FNDC3B and ADD1 genes were associated with economically important traits in beef cattle. The gene FNDC3B was associated with fat deposition [ADD1/SREBP-1c gene have been associated with fatty acid composition [TSPAN19 gene was identified as one of the candidate genes affecting mastitis in dairy cattle [EXT1 and other exostosin family genes including EXT2, EXTL1, EXTL2, and EXTL3 mediate the synthesis of the backbone of Heparan sulfate proteoglycans (the ubiquitous components of the extracellular matrix) which play important roles in tissue homeostasis [Finally, the signatures of selection are worth mentioning and create a desire for future evaluation in terms of animal adaptation to local environments and for implementing of population breeding improvement schemes. Based on the iHS, the function of candidate genes playing important roles in cattle and other livestock species were summarized. For example the bilities  as well bilities . The HSPactation  while theostasis , 27. In rization . The FNDposition , and insposition . The TSPy cattle . The glyeostasis . These gBos indicus) are widely spread in eastern, central and southern Africa [Bos indicus chip to analyze SNPs in cattle as it appears to solve the issues of possible biasness or errors in grouping the local animals (TSZ and Boran) into the respective groups [Bos taurus we presumed that both Bos indicus and Bos taurus have ancestry in Bos primigenius [It is also worth noting that the present classification system of Tanzania has grouped the TSZ animals into more than ten strains. Of these only three were available for this study. It is therefore our recommendation that more strains be sampled and studied using genome-wide association studies such as this. In addition, zebu cattle  of TSZ was larger than Tanzanian Friesians. Nonetheless Ne has considerably decreased not only in Friesians but in TSZ strains, representing a narrow genetic pool in contemporary EAZ. Besides, the mean length of ROH in the Maasai strain was 17 Mb which is greater than the values in Western dairy breeds [Bos indicus. In the analysis of clearly distinguished groups F-ROH appear to represent a reliable estimate of inbreeding, which is less dependent on the frequencies of allele and genotypes. Implementation of selection, breeding and population improvement schemes within the local population for adaptation or productivity enhancement under local environments as opposed to crossbreeding would prevent interbreeding which poses a risk of disappearance of the uniqueness of the indigenous breeds. Therefore, the genomic information identified in our study will provide an insight for the future breeding and conservation programs of cattle in Tanzania.To conclude, we have reported here the genetic relatedness or diversity, structure, admixture and overall relationships among cattle of Tanzania including three strains of the local TSZ population, Boran and Friesian breeds. Our results have shown that there were low levels of genetic differentiation between TSZ strains. The Boran breed was differentiated from TSZ while gene flow between them has occurred, which was supported by the analyses of IBS, admixture, and haplotype sharing. The levels of inbreeding were relatively low in TSZ compared with Western dairy cattle , 37 and y breeds , 37 and y breeds . In Friey breeds , but mayThree strains of TSZ, namely Maasai, Tarime and Sukuma as well as two other breeds (Boran and Friesian) of cattle were involved in this study. Animals from the Maasai, Tarime and Sukuma TSZ strains were sampled from pastoralist and agro-pastoralist herds in Manyara, Mara, and Simiyu regions respectively. From each strain 40 unrelated animals were randomly sampled from four distantly (approximately 15 to 20 km apart) local villages  in a region. In each village, we sampled a total of 10 animals from five herds . The owners were asked about the relationships of the animals in order to avoid sampling of related animals. For the Boran and Friesian breeds 40 unrelated animals per breed were sampled from the government farms at Sao Hill livestock multiplication unit (LMU) and Kitulo dairy farm, respectively. In these farms, breeding records were used to avoid sampling of related animals. From all animals . The PLINK software [All samples were genotyped using the GeneSeek Genomic Profiler Indicine HD Beadchip, an Illumina Infinium array consisting of 80K SNPs that were selected for the analysis of software  was emplsoftware , unmappehomozyg command. The mean length of ROH was calculated from the total length of ROH divided by the total number of ROH in each breed. In addition, inbreeding levels were inferred based on the difference between observed and expected genotype frequencies (F-geno). The F-geno was obtained from the sum of single marker F using Plink het [Ne) was calculated based on linkage disequilibrium (LD) in each breed/strain using the SNeP package [ST) was estimated [ST was estimated using the adegenet package [PLINK was used for the analyses of ROH, F-geno, expected (He) and observed heterozygosity (Ho) and IBS. Haplotype was phased using Beagle [Two approaches were applied to calculate the inbreeding coefficient. First, the inbreeding coefficient was calculated from the sum of ROH length divided by the total length of the autosomes (genomic size) in an individual  and was link het  within e package . To furtstimated . The FST package  in R. Thg Beagle . Haplotyadegenet package in R [admixture [adegenet [pegas package in R [To ascertain the patterns of genetic diversity among Tanzanian cattle populations principal component analysis (PCA) was performed. The PCA was developed after condensation of a large number of genotypes into a few synthetic variables or clusters using the age in R . The crodmixture  was useddmixture  using adadegenet . For theage in R , 49.rehh package [Analyses of signatures of selection can reveal genomic regions of interest from previous selection and can be one of the powerful tools for designing a breeding program in cattle or other livestock species. In this study, the integrated haplotype score (iHS) was estimated and used to compare the EHH based on the ancestral and derived core alleles of each of the populations , thus en package . This me package  or WikiP package  and finaet al., Report of the SUA/ILRI Cow Killer Project, unpublished). Participation in the study was on a voluntary basis upon acceptance through the verbal consent which can be evidenced with their willingness to fill out our questionnaire in another study [http://www.fao.org/fileadmin/user_upload/animalwelfare/tanzania.pdf). Although, there is no committee responsible for animal ethics in Tanzania, COSTECH research permits require researchers to adhere to the welfare of animals. Neither, the Institutional Animal Care and Use Committee (IACUC) was consulted as the main part of work was done in Tanzania. Laboratory analyses at GeneSeek were based on the protocols of the company (http://www.illumina.com).In Tanzania, research permits are provided by the Commission for Science and Technology (COSTECH). Permit No. SUA/ADM/R. 1/8 was issued by the SUA Vice Chancellor on behalf of COSTECH to undertake our survey and sampling in private farms in Manyara, Mara, and Simiyu regions as well as Sao Hill LMU and Kitulo dairy farms in Iringa region. In addition, permission was requested from all local authorities in the study areas whereas the verbal consent was obtained from each project participants after explaining the purpose and importance of the study prior to commencement of sampling. The decision of using the verbal consent was based on a previous experience of working with the Tanzanian farmers who understand better using either explained or visual consent , Tarime (red), Maasai (green), Boran (Purple) and Friesian (light blue) is plotted separately. X and Y axis represents generations and Ne respectively.(TIF)Click here for additional data file.S2 FigThe values of FST are plotted against genomic position across the genome. Comparisons between (A) Sukuma-Tarime, (B) Sukuma-Maasai, (C) Maasai-Tarime, (D) Boran-TSZ, (E) Friesian-TSZ are shown.(TIF)Click here for additional data file.S3 FigThe distance between strains or breeds was calculated based on Reynold's method.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table1-log10  of iHS>3, at least 5 significant values in interval are shown; 2Number of significant iHS in the region; 3Maximum -log10  of iHS in the region; 4Genes located within 300 kb from the maximum his.(DOCX)Click here for additional data file.


Tropical forest loss and fragmentation are due to increase in coming decades. Understanding how matrix dynamics, especially secondary forest regrowth, can lessen fragmentation impacts is key to understanding species persistence in modified landscapes. Here, we use a whole-ecosystem fragmentation experiment to investigate how bat assemblages are influenced by the regeneration of the secondary forest matrix. We surveyed bats in continuous forest, forest fragments and secondary forest matrix habitats, ~15 and ~30 years after forest clearance, to investigate temporal changes in the occupancy and abundance of old-growth specialist and habitat generalist species. The regeneration of the second growth matrix had overall positive effects on the occupancy and abundance of specialists across all sampled habitats. Conversely, effects on generalist species were negligible for forest fragments and negative for secondary forest. Our results show that the conservation potential of secondary forests for reverting faunal declines in fragmented tropical landscapes increases with secondary forest age and that old-growth specialists, which are often of most conservation concern, are the greatest beneficiaries of secondary forest maturation. Our findings emphasize that the transposition of patterns of biodiversity persistence in island ecosystems to fragmented terrestrial settings can be hampered by the dynamic nature of human-dominated landscapes. Habitat loss and fragmentation are pervasive and conspicuous features of this new historical context, which, in combination with other human-related threats, are compelling the planet into a “sixth wave of extinction”3.Humanity’s global footprint is so ubiquitous and far-reaching that many argue that we now live in a new geological epoch, the Anthropocene4. As large swaths of old-growth forest give way to expanding humanized landscapes, species persisting in forest remnants are left to endure the pervasive consequences of increased isolation and decreased area5. Landscape-wide assemblage dynamics in fragments created in the aftermath of deforestation are dependent, to a large extent, on the nature of the matrix within which forest patches are embedded6. Conservation science has traditionally conceived the modified matrix as a “sea” of hostile habitat, in which fragments act as “islands” and this analogy has guided much of the theory and practice of the field7. However, equating forest fragments with island ecosystems, while appropriate in some situations, fails to accommodate the heterogeneous and dynamic nature of most present-day modified landscapes9.The scars of the Anthropocene defaunation are being carved deep into the planet’s biodiversity strongholds, the tropical forests12. Mainland studies that also construed fragments as true islands, have arrived at similar pessimistic narratives14. However, direct comparisons between these two systems (true islands vs mainland) have revealed that assemblages persisting in forest patches embedded in terrestrial human-dominated landscapes defy the patterns exhibited by their water-embedded analogues15.Vertebrate assemblage dynamics in tropical land-bridge islands have painted a dire portrait of the consequences of forest fragmentation in true island systems16, providing myriad services and natural products to human populations worldwide, and key habitat for countless forest-dwelling species18. Although some fragmentation-related extinctions can be averted by forest regeneration19, the role of second growth in biodiversity conservation remains controversial22. Central to the debate is the capacity of secondary forest to preserve old-growth specialist species and to buffer the impacts of fragmentation on assemblages living in forest remnants23.Second growth nowadays constitutes the predominant type of forest cover across the tropics24, in forest fragments and secondary forest sites, ~15 and ~30 years after forest clearance in the early 1980s at the Biological Dynamics of Forest Fragments Project (BDFFP), the world’s largest and longest-running fragmentation experiment, located in the central Brazilian Amazon25  across the three main habitats of the BDFFP: continuous primary forest, primary forest fragments and secondary forest matrix. We predicted that the maturation between study periods of the secondary forest surrounding forest fragments would provide extra resources for old-growth specialists, leading to increases in occupancy and abundance in this group both within fragments and the secondary regrowth matrix. Conversely, we expected that the successional advance of the secondary vegetation would have diminished the availability of food resources for generalist bats , hence reducing their abundance in the same habitats. Additionally, since similarity in structure and floristic composition between secondary and primary forests increases with regeneration time28 we predicted bat assemblage similarity between continuous forest and secondary forest to be higher ~30 years after forest clearance (~2011) than half-way through the study period . Similarly, due to a reduction in fragment-matrix contrast, we predicted that assemblage similarity between forest fragments and continuous forest was going to increase over the same period.We surveyed bats, a taxon demonstrably sensitive to habitat modificationWe captured 4,028 bats in the first period  and 2,081 bats in the second period . Twenty-seven species were classified as specialists whereas 23 were classified as habitat generalists  or negative (secondary forest) Fig. . Model pBetween ~1996 and ~2011, only 3 and 4 of the 27 species classified as specialists decreased in occupancy respectively in fragments and secondary forest. Furthermore, statistical support for these declines was limited Fig. . During Assemblage similarity between continuous forest and fragments increased slightly with time for generalists when considering both occupancy and abundance but declined for specialists. For secondary forests, occupancy- and abundance-based assemblage similarities relative to continuous forest declined for both groups. However, statistical support for these trends was limited (Table 29). Consequently, in contrast to the evaluation of species responses to spatial features, which has received some attention in the bat conservation literature, temporal variation, and in particular how bat responses to fragmentation are shaped by changes in matrix condition, remains little explored24. Here, we show that most phyllostomid bat species and Pteronotus parnellii benefited from the increased permeability of the matrix associated with the maturation of the secondary regrowth surrounding the BDFFP fragments, and that specialist and generalist species exhibited contrasting responses to matrix regeneration across the study landscape.There is a lack of studies that directly investigate temporal trends in wildlife responses to fragmentation and even fewer that evaluate how matrix use changes through time 32 are not effectively sampled with mist-nets33. However, we anticipate that old-growth specialist aerial insectivores are likely to have benefited from the maturation of second growth surrounding the BDFFP fragments in a similar way than their phyllostomid counterparts and to the aerial insectivore P. parnellii.The sole reliance on mist-netting data precludes a complete overview of the effects of second forest regeneration on the BDFFP chiropteran fauna as a whole since, with the exception of et al.11 in the forest islands of the Chiew Larn reservoir in Thailand. Whereas most species of the mega-diverse bat assemblage at the BDFFP increased in occupancy and abundance across the second growth-dominated landscape, in the land-bridge island system in Thailand and, during a similar time window , most species became extinct in forest fragments surrounded by a static matrix (water). The recovery observed at the BDFFP was mostly due to the recolonization of previously deforested areas and forest fragments by specialist species, which increased in all sampled habitats during the second period. This recolonization is likely attributable to an increased diversity of food resources in the matrix, allowing to fulfil the energetic requirements of a larger set of species other than generalists. Additionally, many specialist bats at the BDFFP are highly edge-sensitive36 and consequently the maturation of the secondary vegetation adjoining fragment edges might have increased habitat suitability by reducing the magnitude of edge effects across the landscape. Notwithstanding major morphological and ecological differences between rodents and bats, the widely different trajectories exhibited by assemblages inhabiting true island systems11 and fragments embedded within a regenerating matrix (this study) highlight the potential of second growth forests to mitigate fragmentation-related extinctions.Our results contrast with the catastrophic faunal declines observed in rodent communities by Gibson 37, suggests that the second growth matrix at the BDFFP still acts as an environmental filter. This filtering shapes bat assemblages in a trait-mediated manner, selectively benefiting bat species with a phytophagous diet and reduced body mass39. Similar pervasive consequences of forest clearance can still be detected in birds42 and primates43 in the BDFFP landscape, highlighting that, although second growth can be of conservation significance, primary forest is of irreplaceable value44.In spite of the signs of recovery exhibited by specialist species across our study landscape ~30 years after forest clearance, our results do not support an increase in assemblage similarity between continuous forest and secondary forest over time. This, together with evidence that bat assemblages in smaller fragments (≤10 ha) and secondary forest sites still differ considerably from continuous forest in terms of species richness, evenness, composition and abundance45. Here, we show that the species richness of generalists and both the species richness and abundance of specialists have increased in our reference sites in continuous forest, indicating considerable temporal heterogeneity in undisturbed forest assemblages over a period of ~15 years. This suggests that space-for-time results may be undermined not only by confounding effects arising from spatial heterogeneity but also by constraints associated with the temporal heterogeneity of the assemblages inhabiting sites used as spatial surrogates. This shifting baseline somewhat limits our capacity to attribute the observed changes in fragment and secondary forest bat assemblages entirely to the effect of matrix maturation. However, the contrasting temporal trends in the species richness of generalists in continuous forest and secondary forest, i.e. increase in continuous forest vs. decrease in secondary forest, indicate that secondary forest regeneration plays an important role in the assemblage dynamics across the landscape. Yet, our limited knowledge of the extent of spatial and temporal dynamics of generalist and specialist species in continuous forest and how these fluctuations may relate to patterns in fragmented landscapes still precludes a full understanding of these systems and therefore should be a high priority for future research.Our results have important implications for the interpretation of land-use change studies using space-for-time approaches. Researchers rarely have the opportunity to collect data prior to the main disturbance events that mould humanized landscapes. Consequently, studies often have to rely on nearby sites where the target impact has not yet taken place and assume that these accurately mimic pre-disturbance conditions15) emphasizing that the transposition of patterns of biodiversity persistence in island ecosystems to fragmented terrestrial settings can be hampered by the dynamic nature of human-dominated landscapes, and consequently predictions under the island biogeographic framework can distort our understanding and misguide conservation strategies. Accordingly, and in light of the contrasting temporal trajectories of specialist and generalist species at the BDFFP, alternative theoretical frameworks, importantly, countryside biogeography46, in which species’ differential habitat affinities can be accommodated, emerge as better suited for forecasting biological changes in human-modified landscapes9.Despite the controlled, experimental conditions of the BDFFP, our findings add to an increasing body of evidence , tropical forest loss has increased by more than 2,000 km52. Although legal protection per se does not ensure long-term safeguarding of the services provided by second-growth forests, it represents a critical step towards their management. We therefore urge researchers, practitioners and policy makers to adopt similar protective measures, especially in areas where primary forest is scarce or highly fragmented.To a large extent, the conservation potential of the world’s tropical secondary rainforests depends on the legal framework underpinning their governance. In the Brazilian Amazon, the state of Pará has recently introduced legislation recommending protection of >20-year-old secondary forest (as identified through inspection of satellite images) as well as younger stands depending on the total stand basal area of native trees and palms53. The ability of species to persist in such dynamic landscapes will ultimately depend on the interaction between their intrinsic traits (e.g. mobility and life span), interspecific interactions and the availability of habitat capable of meeting their specific resource needs. Although hotly debated55, the “rescue” potential of secondary forests in these dynamic landscapes is far from negligible16. While adding to mounting evidence that secondary forests are of conservation value, our, and many other long-term studies at the BDFFP 25 and elsewhere in the tropics (e.g.)56 reveal that continuous primary forest and large (>100 ha) forest fragments are of overwhelming importance for the conservation of tropical biodiversity.Human-modified tropical landscapes are in continuous flux, with areas of secondary forest being converted to agricultural land and vice-versa. Vegetation disturbances, both anthropogenic and natural (e.g. fire), are irregular in space and time, moulding mosaic landscapes in which the classic split between fragments and matrix is blurredOur results show that specialist bats, which occurred at low abundances in secondary regrowth and in forest fragments ~15 years after the experimental clearing, have benefited from the increased permeability of the matrix associated with the maturation of the secondary forest in the matrix during the last 15 years. This suggests that matrix management, and specifically the management of regenerating (secondary) forest can majorly dictate the future of biodiversity in human-modified landscapes, including that remaining in fragments of natural vegetation.2 study area is non-flooded (terra firme) rainforest with a canopy height of ca. 23 m and emergent trees reaching 55 m57. The forest at the BDFFP is among the most biodiverse in the world (tree species richness often exceeding 280 species/ha)58 and, with the exception of the experimental fragmentation, has been sheltered from anthropogenic disturbances such as logging and fires. The climate is characterized by a dry season between June and October and annual rainfall varies from 1,900 to 3,500 mm. Eleven fragments were isolated from continuous forest by distances of 80–650 m in the early 1980s and are categorized into size classes of 1, 10 and 100 ha. Fragments were originally located within cattle ranches  but poor soils and low productivity dictated the abandonment of livestock activities and fragments became gradually surrounded by secondary forest dominated mainly by Vismia spp. and Cecropia spp.25. Following secondary forest proliferation, fragment isolation was maintained by clearing a 100 m-wide strip of regrowth at intervals of ~10 years around most experimental forest fragments35. During this study fragment re-isolation occurred between 1999 and 2001. For a description of the study landscape experimental manipulation and ecosystem-wide responses see Laurance et al.25.Bat surveys took place at the Biological Dynamics of Forest Fragments Project (BDFFP), approximately 80 km north of Manaus , state of Amazonas, Brazil  we sampled bats in forest fragments , secondary forest (seven sites) and continuous forest (six sites)  and 18 to 24 (fragments and continuous forest sites) ground-level mist nets (12 m × 2.5 m) placed along existing trails. Trails used for sampling forest fragments were located as close as possible to the centre of the fragment. We surveyed fragment and continuous forest sites on seven to 12 nights and secondary forest sites between three to seven nights. Total mist net effort was 8,757, 9,429 and 860 mist-net hours  for continuous forest, fragments and secondary forests, respectively. Captured bats were identified to species-level and had standard morphometric and demographic data collected. For this first study period, detailed site descriptions, methods and results for fragments and continuous forest can be found in Sampaio61 and Sampaio et al.59 and for secondary forest in Bobrowiec & Gribel60. Our analyses are restricted to ground-level captures in fragment and continuous forest interiors59 and to captures in Vismia- and Cecropia-dominated secondary forest60. Distance between sampling sites ranged from 148 m to 41 km and consequently some level of non-independence between bat assemblages of sites located closer together is plausible.During the first sampling period bats were surveyed from January 1996 to June 1999 in forest fragments and continuous forest sitesDuring the second period we re-surveyed all 19 sites between August 2011 and June 2013. The mist-netting protocol consisted of seven (secondary forest sites) and 14 (fragments and continuous forest sites) ground-level mist nets (12 × 2.5 m) placed at existing trails. Total mist net effort was 4,009, 3,963 and 1,941 mnh for continuous forest, fragments and secondary forests, respectively. Similarly to the first period, captured bats were identified and had standard morphometric and demographic data collected.62 and in accordance with Brazilian conservation and animal welfare laws. Sampling guidelines were approved by the ICMBio (Instituto Chico Mendes de Conservação da Biodiversidade) and research was conducted under permit number 26877–2.Bat capture and handling was conducted following guidelines approved by the American Society of MammalogistsPteronotus parnellii since all other captured species are inadequately sampled with ground-level mist-nets33. Taxonomy follows Gardner63.We restricted our analyses to phyllostomid bats and et al.64 to classify species into one of four groups: primary forest specialists, secondary forest specialists, generalists or too rare to classify. Classification was based on the whole dataset of 10,311 captures of 50 species sampled at the BDFFP between 1996 and 2014. Only a sub-set of these captures  was subsequently used in the joint species distribution models (see below). The method uses a multinominal model based on species relative abundance in both habitats (here defined as continuous primary forest vs forest fragments and secondary forest) and simultaneously minimizes bias due to different sampling effort between habitats and due to insufficient captures of rare species. Classification was conducted in R v.3.0.265 using function clamtest of the vegan package and was based on the super-majority specialization threshold (K = 2/3) and setting a significance level of P = 0.01. We conservatively grouped primary forest specialists and species too rare to classify into a single group and since only two species were assigned to the secondary forest category, they were lumped together with generalists. We therefore considered two functional groups in our analysis: primary forest species and species too rare to classify  and generalists and secondary forest specialists .We used the statistical approach developed by Chazdon 27 to relate the bat occurrence data to environmental covariates. As a sampling unit, we considered one mist-netting session in one site (n = 301 mist-netting sessions)  software for MatLab (HMSC-MatLab) to fit the model to the data, assuming the default priors described in the Supporting Information of Ovaskainen et al.27. We ran the model for 50,000 iterations out of which 15,000 were discarded as transient.To account for repeated measurements at the same sites, we assumed a site-level random effect, implemented at the community level using the latent factor approach of Ovaskainen We used the parameterized model to predict the expected species richness and number of captured individuals  in each habitat class and study period per survey visit. Species richness was computed as the sum (over the species) of the occurrence probabilities predicted by model 1. Number of individuals was computed as the sum (over the species) of species-specific abundances, computed as the product of occurrence probability (from model 1) and abundance conditional on presence (prediction of model 2 plus one). In these predictions, we standardized the mist netting effort to the mean value of a given habitat category across both study periods, and the percentage of secondary forest to the mean value of a particular habitat type during a given survey period. Capture effort was standardized within each habitat category and thus the results are comparable between periods but not across habitat types. Species-level responses were assessed by computing the difference between the occurrence probability and mean number of individuals expected to be captured per survey visit between the first and the second period.71. To characterize assemblage turnover, we computed assemblage similarity between the different habitat categories as well as between the two study periods. Assemblage similarity was defined as the correlation between model-predicted occurrence probabilities or abundances (both log-transformed)72 We performed these calculations for all species, and separately for generalists and specialists only.Turnover metrics are considered better suited to quantify biodiversity change in local assemblages through time than simple temporal trends of within-sample diversity The data used in this study are archived at 10.6084/m9.figshare.5907322.v2.Supplementary Information


Athina Markou and her colleagues discovered persistent changes in adult behavior following adolescent exposure to ethanol or nicotine consistent with increased risk for developing addiction. Building on Dr. Markou’s important work and that of others in the field, researchers at the Bowles Center for Alcohol Studies have found that persistent changes in behavior following adolescent stress or alcohol exposure may be linked to induction of immune signaling in brain.This study aims to illuminate the critical interrelationship of the innate immune system  in the neurobiology of addiction.This study reviews the relevant research regarding the relationship between the innate immune system and addiction.Emerging evidence indicates that TLRs in brain, particularly those on microglia, respond to endogenous innate immune agonists such as HMGB1 and microRNAs (miRNAs). Multiple TLRs, HMGB1, and miRNAs are induced in the brain by stress, alcohol, and other drugs of abuse and are increased in the postmortem human alcoholic brain. Enhanced TLR-innate immune signaling in brain leads to epigenetic modifications, alterations in synaptic plasticity, and loss of neuronal cell populations, which contribute to cognitive and emotive dysfunctions. Addiction involves progressive stages of drug binges and intoxication, withdrawal-negative affect, and ultimately compulsive drug use and abuse. Toll-like receptor signaling within cortical-limbic circuits is modified by alcohol and stress in a manner consistent with promoting progression through the stages of addiction. Several studies have found lasting changes in brain that contribute to the progression through the stages of addiction  , more recent “sterile inflammation” studies in brain led to the identification of these receptors as pattern recognition receptors (PRRs). Pattern recognition receptors have revolutionized our understanding of innate immune system signaling in brain. These receptors recognize and respond not only to specific molecular patterns present on foreign (exogenous) pathogens  but also to endogenous signaling molecules associated with cell damage, degeneration, or stress  Bianchi . To dateIn order for these innate immune signaling molecules to remain elevated for extended periods of abstinence, signaling amplification of these signaling molecules likely occurs. Ethanol causes activation of the transcription factor NF-κB, leading to increased PRR expression, cytokine induction, and DAMP release. This signaling system leads to amplification of the innate immune response in brain through an autocrine and paracrine process such that secreted cytokines, DAMPs, and microRNAs activate receptors on the cells of origin as well as surrounding cells. In addition, ethanol causes generation of reactive oxygen species that further activates NF-κB transcription of cytokines and DAMPs in an autocrine and paracrine fashion  and TNF receptor-associated factor 6 (TRAF6), which cause IκB and MAPK activation. Activation of IκB and MAPK in turn leads to downstream activation of the transcription factor NF-κB and subsequent cytokine induction , which are small, non-coding RNAs (∼22 nucleotides) that regulate innate immunity  vector into the central nucleus of the amygdala of alcohol-preferring rats diminished binge drinking, which was associated with reduced expression of TLR4  binge/intoxication, (2) withdrawal/negative affect, and (3) preoccupation/craving (Koob and Volkow There is also evidence implicating innate immune activation in the withdrawal/negative affect stage of addiction. Substance abuse is associated with the development of negative affective states, such as anxiety and dysphoria (Koob and Le Moal Stress causes induction of innate immune signaling in brain and mounting evidence indicates that immune activation can lead to alterations in affective states. As mentioned in the previous section, stress sensitizes microglia to inflammation in an HMGB1-dependent manner (Weber et al. Evidence supports an involvement of the innate immune system in affective disorders, but the mechanism remains to be elucidated fully. The literature provides evidence that a potential mechanism for innate immune involvement in the development of affective disorders involves alterations in the activity of neural circuitry that modulates affect. Administration of a typhoid vaccine was found to increase circulating levels of the proinflammatory cytokine IL-6 and significantly reduce mood (Harrison et al. Evidence also implicates the TLR-innate immune system in the third stage of addiction involving preoccupation with drug taking and craving, which is associated with a heightened likelihood of relapse. Plasma levels of the proinflammatory cytokines IL-8 and IL-1β were positively correlated with indices of alcohol craving in human alcohol-dependent subjects (Leclercq et al. The discovery that innate immune mechanisms contribute to the neurobiology of addiction provides a novel approach for the treatment of alcohol use disorders. While innate immune activation contributes to alcohol consumptive behaviors, it also contributes to alcohol-induced neurodegeneration (Crews et al.


This review details the manner in which the central nervous system regulates metabolic homeostasis in normal weight and obese rodents and humans. It includes a review of the homeostatic contributions of neurons located in the hypothalamus, the midbrain and limbic structures, the pons and the medullary area postrema, nucleus tractus solitarius, and vagus nucleus, and details how these brain regions respond to circulating levels of orexigenic hormones, such as ghrelin, and anorexigenic hormones, such as glucagon-like peptide 1 and leptin. It provides an insight as to how high intensity exercise may improve homeostatic control in overweight and obese subjects. Finally, it provides suggestions as to how further progress can be made in controlling the current pandemic of obesity and diabetes. According to the World Health Organization, non-communicable diseases are the leading cause of death worldwide, causing 39.5 million deaths  in 2015; ischemic heart disease was the leading cause of mortality, accounting for 17.7 million or 45% of deaths. These statistics have been linked to a concomitant worldwide increase in the prevalence of obesity-associated disorders , most particularly type 2 diabetes mellitus -mediated tonic inhibition of POMC/CART neurons including the suppression of anorexigenic signals from neurons located in the parabrachial nucleus of the pons. The anorexigenic effects of the parabrachial nucleus come from glutaminergic signals originating in the nucleus tractus solitarius in the medulla which services the vagus nerve  and melanocortin-4 (MC4R) receptors promotes energy expenditure and suppresses food intake. MC3R are expressed at high levels in the ventromedial and arcuate nuclei, whereas MC4R are widely spread throughout the central nervous system, most notably in the ventromedial nucleus  expressing neurons in the medulla send anorexigenic signals to the paraventricular nucleus (PVN) of the hypothalamus and that postnatal depletion of GLP-1 receptors in the PVN causes hyperphagic obesity  and peptide YY (PYY), and orexigenic signals from ghrelin Wren, . This arThe area postrema and NTS also contribute to non-homeostatic body weight regulation by expressing receptors for GDF15 (GDF15AL). GDF15LA ligation transmits anorexigenic signals to neurons located in the parabrachial and central amygdala nuclei. This pathway causes anorexia and weight loss in disorders associated with tissue stress or injury  and then to other areas in the limbic system, ultimately reaching the prefrontal cortex where conscious decisions regarding eating are made , liver , and gastrointestinal tract  Blasi, .+ neurons in the nucleus accumbens -6; when injected into the third ventricle of obese rats, IL-6 reduced food intake and restored the anorexigenic effects of insulin and leptin by promoting IL-10-mediated inhibition of IOther studies have shown the beneficial effects of exercise on CNS responses to leptin or insulin. Krawczewski and co-authors found that voluntary exercise in a mouse strain susceptible to diet-induced obesity decreased fat mass & increased energy expenditure via activation of leptin receptor-positive neurons in the ventromedial nucleus; in this study, intracerebral leptin did not decrease body weight or food intake in sedentary mice fed high fat diets but did reduce body weight in exercising mice. They concluded that exercise leads to the maintenance of a lower body weight and leaner composition by improving CNS leptin action in obese animals  was reduced  but did not produce compensatory changes in appetite or energy intake  or 36 min at 75% VO2peak and another nine men ran for 45 or 90 min at 70% VO2peak; in both groups, plasma levels of acylated ghrelin and hunger ratings fell and remained suppressed for up to 1.5 h  is reported to decrease ghrelin levels and energy intake in obese subjects  of areas involved in autonomic nervous system and somatomotor control, including the parabrachial nucleus, the medial portion of the NTS, and medullary areas containing the area postrema  or reduce  food intake. High intensity exercise can also cause transient increases in plasma levels of the gut hormones GLP-1, PP, and PYY  of high energy density foods, although it had no effect on food preferences. It is not known whether ghrelin, which binds to GHSRs in the amygdala and dopaminergic neurons, played a role in this effect  exert their anorexigenic effects in the area postrema, NTS, and dorsal motor nucleus of the vagus. Blood levels of PP and PYY may increase transiently following high intensity exercise.Following bariatric surgery the vagal NTS and vaso-vagal pathways play a major role in restoring normal function to the pancreas , liver , and gastrointestinal tract .Please see Table 2), nearly five times as high in persons with class 1 obesity (30.0–34.9 kg/m2), and nearly fifteen times as high in persons with class 2/3 obesity (BMI > 35.0 kg/m2) when compared to normal weight controls. Severely obese individuals were particularly prone to develop diabetes mellitus followed by a vascular event  was twice as high in overweight individuals  has become a survival risk for many of us.2peak of at least 60% for a protracted period of time  in four healthy men undergoing a two-phase exercise regimen: cycling for 6 min at 77% VOFurther research is needed to determine the safety and efficacy of anorexigenic peptides such as GLP-1 and GLP-1 analogs, GDF15, and BDNF in inducing weight loss in obese individuals. Trials of exercise training done in low temperature environments, where energy demands are inherently higher, should be assessed. Studies should be done to determine whether exercise training done early in life causes epigenetic changes that have long term beneficial effects on CNS regulation of metabolic homeostasis. Efforts should be made to find medications that can safely downregulate hedonic dietary habits. And, in accordance with World Health Organization recommendations, all school systems should include programs that require children and adolescents to participate in activities that allow them to accumulate at least 60 min of moderate to intense physical activity daily.JS is the author of this manuscript and takes sole responsibility for the accuracy of its content.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This year, more than 100 participants, among which senior and young scientists from Europe, USA, North Africa and Japan, had an intense and open exchange of achievements and ideas. This open and informal communication among researchers has been a constant hallmark of all the IMYA meetings. The global yeast death community was embraced and inspired by the lively and warm atmosphere of Bari, the capital city of Apulia, and its beautiful surroundings, with colorful landscapes, historical and artistic heritage, tastes and scents that reflect the interface between Eastern and Western culture.Over the last 14 years, the field of yeast regulated cell death (RCD) has been expanding to more and more biomedical research themes, including aging, human diseases, cell stress response, metabolism and systems biology. The 12Frank Madeo (Austria), a pioneer of yeast apoptosis studies. He presented data on the anti-aging effects of spermidine in different model organisms, including yeast, nematodes and flies. He demonstrated that this ubiquitous polycation induces autophagy and significantly reduces age-related oxidative protein damage in mice. Spermidine supplementation was also shown to have a strong cardio protective effect on diastolic parameters. These findings support the evidence that autophagy mediates cytoprotection against a variety of noxious agents, thereby conferring longevity when induced at the whole-organism level. Outstanding scholars gave their plenary lectures in several thematic sessions as follows.The meeting started with the keynote lecture of Marie Hardwick (USA), the first speaker of the opening session on fungal cell death pathways reported the identification of certain death-resistant deletion strains through a genome wide screen analysis in Saccharomyces cerevisiae. These strains were knockouts for all subunits of a vesicle trafficking complex involved in responding to amino acid levels, thus supporting the evidence that yeast cell death and nutrient-sensing pathways merge and can affect survival following stress conditions.The perception of mammalian "cell death" has evolved from being a passive incident to an active process as supported by the characterization of several active death pathways, such as apoptosis, necroptosis, or pyroptosis. In contrast, although the evidence of apoptosis in dying fungal cells is clear, less is known about its molecular mechanisms and the impact of the environmental cues on cell fate control. Campbell Gourlay (UK) presented results on the conserved link between environmental signals, cytoskeletal integrity and cell fate control. Particularly, he showed that defects in actin regulation lead to the inability to control the fidelity of MAPK signaling pathways. He observed that the yeast filamentous growth and mating MAPK pathways become inappropriately activated in response to a reduction in actin dynamics and that actin stabilization leads to the constitutive activation of the MAPK controlled cell wall integrity pathway. He also observed that the de-regulation of MAPK signaling leads to vacuole dysfunction, ROS production and cell death that can be reversed by porin deletion.Katrina Cooper (USA), who is working on cyclin C-mediated cell death, proposed a model in two steps for cyclin C release from the nucleus to the cytoplasm, representing a key cell-fate checkpoint control. She found that first cyclin C is phosphorylated by the MAP kinase Slt2 permitting its disassociation from the Mediator component Med13 and after that Med13 destruction allows full cyclin C release and prevents re-accumulation of the cyclin in the nucleus.Manuela Côrte-Real  analyzed the selective anticancer activity of lactoferrin showing that this glycoprotein activates a mitochondria- and caspase-dependent apoptotic process in yeast, which led to the hypothesis that lactoferrin could act as a V-H+-ATPase inhibitor in highly metastatic cancer line cells. In addition, she showed that bovine lactoferrin causes intracellular acidification in metastatic breast cancer cells. Her data demonstrate how yeast can provide relevant clues in the identification of the molecular target/mechanisms of action of lactoferrin.Benedikt Westermann (Germany) added information to this understanding by presenting data on the unexpected link between mitochondrial transport and fusion. Genetic analysis, live cell microscopy and simulations in silico showed that this link is critical for a correct mitochondrial partitioning and that mitochondrial quantity causes a severe decline of replicative lifespan of daughter cells. This suggests that coordination of mitochondrial transport, fusion, and fission is critical for asymmetric division and rejuvenation of daughter cells.During cell division, the partitioning of cell organelles involves both stochastic and ordered mechanisms and budding yeast is a useful model to study organelle inheritance during asymmetric cell division. Michal Eisenberg-Bord (Israel) discussed lipid droplets and their key functions in bioenergetics homeostasis and membrane biosynthesis. In particular, she focused on a specialized lipid droplet subpopulation equipped with a unique set of surface proteins that is localized in proximity of the nucleus vacuole junction. By using high-throughput screening, she identified two Lipid Droplet Organization (Ldo) factors, which are required for proper localization of Pdr16 residing in this lipid droplet subpopulation.Jens Nielsen (Sweden) addressed how different parts of cellular metabolism are connected, how metabolism can be modelled at the genome-scale and how incorporation of protein crowding and proteome homeostasis may be key determinants for cellular function. He also showed how regulation of metabolism can be studied using different omics analysis.As metabolism represents the core of cellular functions and since most cellular processes interact with metabolism, a highly complex network is established. Hiroshi Takagi (Japan) presented data on the signaling molecule nitric oxide (NO) and the flavoprotein Tah18, which is involved in nitric oxide synthase-like activity. He reported that cell death induced by Tah18-dependent NOS activity can be prevented by enhancing the interaction between Tah18 and its molecular partner Dre2. Appropriate nitric oxide levels were found to confer cellular tolerance either to high temperature, via Mac1-mediated Sod1 activation, or to low levels of hydrogen peroxide. In contrast, under severe stress conditions, such as high levels of hydrogen peroxide, excess NO may induce cell death. Tah18-dependent NO production may thus exhibit two opposite effects in yeast, similar as in mammalian cells.Francesco Cecconi  discussed the role of AMBRA1, the activating molecule in beclin 1-regulated autophagy, and he placed this in the context of evolution and development. Beyond AMBRA1’s recognized role in embryonic development, neurological disorders and carcinogenesis, he showed that AMBRA1 can coordinate a cell response to starvation or other stresses by translocation of the autophagosome core complex to ER, by regulative ubiquitination and stabilization of the kinase ULK1, and by selective mitochondria removal and cell cycle down-regulation. AMBRA1 appears also to be targeted by different regulators, such as Culling-dependent degradation, caspase cleavage and several modifications, from phosphorylation to ubiquitination.Autophagy is a conserved process that functions during cellular stress and nutrient starvation as part of an adaptive response to maintain homeostasis and quality control. Ted Powers (USA) discussed the role of the rapamycin-insensitive TORC2 as a positive regulator of autophagy, specifically under amino acid starvation. He described the architecture and the activity of this regulatory pathway, showing that TORC2, through its downstream target kinase Ypk1, inhibits calcineurin. On the other hand, calcineurin activation requires Mid1, a calcium channel regulatory protein and when TORC2-Ypk1 signaling is compromised, the normal mitochondrial respiration is perturbed resulting in the accumulation of reactive oxygen species and the inhibition of the general amino acid control and autophagy.Zdena Palková (Czech Republic) focused on the regulatory role of mitochondria and certain mitochondrial pathways during cell subpopulation longevity as well as on the regulation of metabolic differentiation and the production of specific metabolic proteins and transporters in specific cell subpopulations.Similar as multicellular organisms, growing yeast cells are able to differentiate in organized communities, such as colonies. In such colonies, cells initiate various metabolic reprogramming leading to the formation of cell subpopulations that mutually interact in order to fulfill specific tasks. Tiago Outeiro opened this part of IMYA12 with an inspiring talk in memory of his mentor Susan Lindquist, who pioneered on the use of budding yeast as model system for studying human disease, evolution, and biomaterials. The following sessions then ensued.It is well known that yeast offers an excellent tool to study the molecular basis of human diseases as it: allows mimicking a variety of physio-pathological aspects under different environmental conditions. By avoiding complex ethical issues associated with mammalian models or human subjects, the use of yeast models enable the discovery of novel targets via high-throughput screens, thereby accelerating therapeutic research provided that such targets are further validated in more complex models.  S. cerevisiae have significantly contributed to our understanding of the cellular mechanisms underlying neurodegenerative diseases, such as Parkinson’s (PD) and Alzheimer’s diseases (AD), for which the pathobiology remains largely unclear. Tiago Outeiro (Germany) has investigated the role of alpha-synuclein in PD in a yeast model. He presented data about the effect of post-translational modifications, such as phosphorylation and glycation, on alpha-synuclein aggregation and toxicity. His findings on phosphorylation of alpha-synuclein at a specific serine residue have been validated in mammalian cell models and may open novel perspectives for therapeutic intervention in synucleopathies.Studies inDina Petranovic (Sweden) presented results on the expression in yeast of two forms of the amyloid β-peptide (Aβ), which typically deposits in the amyloid plaques found in AD brain, but whose mechanisms of toxicity are not fully understood. By comparative analysis of the physiology, molecular markers and genome-wide transcriptional responses in strains constitutively expressing either Aβ1-42 or Aβ1-40, she identified some mechanisms that seem to be implicated in the toxicity of Aβ peptides. She also described recent findings on a mutant protein, named ubiquitin B protein+1 (UBB+1), which accumulates in an age-dependent manner and appears to correlate with AD. Differently from other data, low level of UBB+1 can induce a protective response assisting yeast cells to cope better with misfolded proteins during chronological aging, including the reduction of the Aβ toxicity associated to lower levels of Aβ aggregates.Joris Winderickx (Belgium) has been using yeast as a model to decipher fundamentals of age-associated human disorders. Particularly, he focused on the interplay of PKA-, TORC1- and Sch9- controlled nutrient-sensitive pathways that have a profound impact on growth, stress resistance and longevity. He gave an overview on recent findings about the connections of intracellular pH and calcium homeostasis with these pathways and he discussed their effect on the overall cell physiology.Richard Y. Zhao (USA) presented evidence that fission yeast can be used as a surrogate system for the rapid identification and genome-wide functional analysis of Zika virus (ZIKV) proteins. ZIKV causes various congenital neurologic disorders including microcephaly in newborns and Guillain-Barré Syndrome in adults. Through the yeast studies, seven ZIKV proteins were identified that cause different levels of cytopathic effects including cell growth restriction, cell cycle dysregulation and cell death. Those initial findings generated from yeast provide a primary reference for future mammalian studies on their potential links to ZIKV-induced neurologic disorders. Ongoing mammalian translation studies were also presented and discussed.Ali Gargouri (Tunisia) described the effects of p53 ectopic expression in S. cerevisiae by illustrating different aspects: the occurrence and analysis of apoptosis, the structural and functional connection with mitochondria, the impact on gene expression and screening of intragenic or over-expressing p53-inactivating mutations. He also showed the selection of biomolecules suppressing the p53-mediated cell death, including a very potent macromolecule, extracted from Nigella sativa that is able to rescue apoptosis without affecting p53 expression.Carlo Bruschi (Austria) underlined the complex apoptotic dynamics seen following chromosome translocation events that depend on the subsequent genomic rearrangements and epigenetic alteration of the primary homeostatic structure of the chromatin. In particular, he reported death rates and expression profiles of apoptosis-related genes in yeast cells in which the primary event of chromosome translocation was induced ad hoc by Bridge-Induced Translocation (BIT) technology. He also showed that the BIT system was successfully applied to reproduce in yeast precisely the peculiar chromosome translocation associated with acute myeloid leukemia and that this yeast model can be used to test the constitutive expression of human P53.S. cerevisiae has been successfully used as a model organism in phenotypic screens aiming to identifying molecules of pharmacological interest or with anti-aging properties, to understand mechanism of action of drug or to reveal novel drug targets and target pathways. Ralf Braun (Germany) presented results on the expression and clearance of the nuclear RNA-binding neurotoxic protein, TDP-43, which accumulates and aggregates in the cytoplasm of patients affected by motor neuron disorder amyotrophic lateral sclerosis. He demonstrated that TDP-43 interferes with its own degradation via the lysosomal clearance pathway, potentially by entrapping its pivotal components. Modulating the fine balance between cell survival and protein degradation systems might delay the loss of neuronal activities during disease progression.Paula Ludovico  showed how metabolite supplementation, such as certain amino acids, can have anti-aging effects when cells suffer proteotoxic stress elicited by the heterologous expression of human α-synuclein (α-syn). These effects are associated with a decreased accumulation of reactive oxygen species, Sch9-mediated increased superoxide dismutase activity and alterations in the mitochondrial network. These findings might be an attractive mechanism to rescue cells from α-syn mediated toxicity and for future pharmacological research designed to improve the prognosis of age-associated diseases. Charles Boone (Canada) presented the quantitative genetic interaction data produced by genome-scale Synthetic Genetic Array (SGA) experiments with S. cerevisiae. He outlined the functional interactions and the relationships genotype-phenotype, revealing the potential of genetic interactions for assembly of a hierarchical model of cell function, including modules corresponding to protein complexes and pathways, biological processes, and cellular compartments. He also introduced the in fieri work on the extent and composition of the global yeast trigenic interaction network.Systems biology studies in yeast are supporting the development and understanding of the complexity and interplay of biological networks with their dynamics, basic principles, rules and balanced orchestrated functions in direct interaction with the environment.Duccio Cavalieri  discussed the physiological significance of PCD in the framework of ecological interactions of "natural" yeast strains with different life span and different levels of toxins production, such as ethanol and acetic acid, in sugar rich environment. He presented evidences for the role of released Hsp12p and Pau5p in modulating cell growth under the effect of quorum sensing molecules and determining yeast community level fitness in different ecological settings. Maurizio Bettiga (Sweden) presented results on the relationships between plasma membrane properties and acetic acid stress tolerance. He demonstrated that an increase of the sphingolipids content in the plasma membrane is associated with acetic acid stress response in Zygosaccharomyces bailii. These studies point to the possibility to engineer the membrane composition of an industrial yeast strain towards reduced permeability as to obtain higher acetic acid tolerance.Several industrial yeast-based processes, such as the production of ethanol from lignocelluloses or wine making, are inhibited by the accumulation of organic acids, including acetic acid, which is known to inhibit metabolism and affect cell viability. Thus, one of the central issues for the improvement of their robustness is to gain insight into the molecular mechanisms of acetic acid toxicity.International Meeting on Yeast Aging and Apoptosis, though maintaining the winning-horse acronym IMYA. The next meeting, i.e. IMYA13, will be organized in Leuven, Belgium. Thus, "tot binnenkort in België"!With 37 selected oral presentations and 26 posters, IMYA12 has been a showcase for the latest advances in the knowledge on the molecular basis of many fundamental cellular processes governing cell homeostasis. IMYA12 also presented cutting-edge achievements in the exploitation of yeast as outstanding model for deciphering at the system level different aspects related to biomedical and bio-industrial science. With the aim of witnessing this new fascinating era of yeast research, the IMYA Scientific Advisory Board has decided to update the title of the conference series into


Control of the bandgap is achieved using heteroepitaxial growth of new structural phases on SrTiO3 (001) substrates. Compared to bulk crystals, anomalous bandgap narrowing is obtained in the FE state of KNO and NNO films. This effect opposes polarization-induced bandgap widening, which is typically found for FE materials. Transmission electron microscopy and spectroscopic ellipsometry measurements indicate that the formation of higher-symmetry structural phases of KNO and NNO produces the desirable red shift of the absorption spectrum towards visible light, while simultaneously stabilizing robust FE order. Tuning of optical properties in FE films is of interest for nanoscale photonic and optoelectronic devices.Perovskite-type ferroelectric (FE) crystals are wide bandgap materials with technologically valuable optical and photoelectric properties. Here, versatile engineering of electronic transitions is demonstrated in FE nanofilms of KTaO Such fields are comparable to the intra-atomic electric field of FEs, which is anticipated to generate nonlinear effects and additional functionalities.Optical and photonic devices have long been employing strong optoelectronic effects , 2, nonlP) is stabilized at the paraelectric–ferroelectric (PE–FE) phase transition. Compared to the PE phase, the bandgap of the FE phase widens by gΔE  and in-plane [110] directions in NNO (s = 0%). Thus, compared to bulk crystals, the structural symmetry is lowered in nanofilms of KTO [At room temperature, the bulk crystal structure of KTO is cubic 1/2 ∝ (E − Ei) and (αE)2 ∝ (E − Ed) for indirect and direct transitions  and the presence of FE polarization ) as shown in figure n(T) of the KTO nanofilm increases on cooling at high temperatures, which is typical for the PE state [n below 650 K, however, evidences the presence of FE polarization [n(T) curve. The non-monotonic positive slope of n(T) for the KNO nanofilm indicates a stable FE state up to at least 780 K (the maximum temperature in this measurement). This upward shift of the FE phase transition is in qualitative agreement with the strain–temperature phase diagram of epitaxial perovskite FE films [n(T) around 485 K in the KNO crystal, does not exist in the tetragonal KNO nanofilm. An absence of bulk-like phase transitions has also been found for FE BaTiO3 films [Our results suggest that new structural phases of epitaxial nanofilms are primarily responsible for complex changes in the electronic band structure. Most significantly, these include bandgap narrowing in the presence of robust FE polarization for structural phases of KNO and NNO with higher symmetry than their bulk crystals. Enhancements of the FE phases in the KTO, KNO, and NNO nanofilms are confirmed by the temperature evolution of the index of refraction  for KTO on the other, suggest that new structural phases affect the electronic transitions more than FE polarization. A more detailed theoretical analysis of this anomalous bandgap behavior using FP calculations is difficult. While approaches based on density functional theory (DFT) are widely employed in the search for new materials with reduced bandgaps –57, theinal ones . This re4.In summary, heteroepitaxial growth allows for the creation of perovskite FE nanofilms with new crystal phases and controllable lattice strain. The formation of new structural phases with a different electronic band structure compared to bulk crystal prototypes significantly modifies the optical properties. Bandgap alterations as large as 0.3–0.8 eV are inferred from our optical measurements and, depending on nanofilm material and structure, either a widening or narrowing of the bandgap is realized. Most significantly, the formation of higher-symmetry structural phases of NNO and KNO allows for considerable bandgap narrowing in the presence of enhanced FE polarization. We note that these promising results, which could raise the efficiency of photovoltaic, photostrictive, and photocatalytic systems operating under solar light, are still early in our understanding of bandgap behavior in FEs and that details related to structural symmetry need to be carefully examined in future experimental and theoretical studies.


Cancer is a complex and heterogeneous disease thought to be caused by multiple genetic lesions. The recent finding that enzymes of the tricarboxylic acid (TCA) cycle are mutated in cancer rekindled the hypothesis that altered metabolism might also have a role in cellular transformation. Attempts to link mitochondrial dysfunction to cancer uncovered the unexpected role of small molecule metabolites, now known as oncometabolites, in tumorigenesis. In this review, we describe how oncometabolites can contribute to tumorigenesis. We propose that lesions of oncogenes and tumour suppressors are only one of the possible routes to tumorigenesis, which include accumulation of oncometabolites triggered by environmental cues. •Altered metabolism is an emerging aspect of tumorigenesis.•Damaged mitochondria accumulate metabolites that can activate oncogenic cascades.•Environmental and nutritional cues trigger the accumulation of these metabolites. These discoveries led Knudson and colleagues to hypothesise a “multiple-hit” model of tumorigenesis, where multiple genetic alterations are required to achieve full blown transformation MYC-induced anchorage-independent growth in both human and mouse cellular models Cancer is a complex and multifactorial disease. Although its malignant features have been known for centuries, it was not until the advent of modern biology that the molecular determinants of cancer transformation have been elucidated. In 1911, pioneering work from Peyton Rous showed that avian sarcomas were transmissible to other healthy fowls through a filtrate of the tumours devoid of cells (FH)(SDH)(IDH)oncometabolites. Recent data suggest that reprogramming of cellular metabolism occurs both as direct and indirect consequence of oncogenic mutations and that environmental cues, such as hypoxia, could affect the metabolic phenotype of cancer cells and the abundance of oncometabolites, amplifying oncogenic cascades. In this review we describe the main oncogenic functions of oncometabolites and how their abundance can be affected by genetic mutations and environmental cues.The discovery that mutations in the metabolic genes Fumarate hydratase 2SDH was the first mitochondrial enzyme found mutated in cancer 2.1SDH are found in familial paraganglioma and pheochromocytoma Sdhb-/- cells SDH is an enzyme of the TCA cycle involved in the conversion of succinate to fumarate and a key component of the mitochondrial respiratory chain. This enzyme is composed of four subunits, SDHA, SDHB, SDHC, SDHD; and two assembly factors, SDHF1 and SDHF2 One of the most striking features of SDH-deficient cells is the accumulation of succinate 2.2FH mutations followed by the loss of heterozygosity of the second allele cause Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC) FH is also mutated in paraganglioma, pheochromocytoma FH is an enzyme of the TCA cycle that converts fumarate to malate. Whilst homozygous FH mutations cause fumaric aciduria, a condition associated with infantile encephalopathy and brain malformations ABL-1) as potential driver in fumarate-dependent tumorigenesis in vitro and in vivo. Mechanistically, through ABL-1 activation, fumarate stimulates an antioxidant response mediated by transcription factor NFE2-related factor 2 (NRF2), and a metabolic rewiring through activation of mammalian target of rapamycin (mTOR)-HIF axis succinationp21, a major player in senescence induction, induces transformation of benign cysts in FH-deficient mice indicating the tumour-suppressive role of senescence in FH-mediated tumorigenesis Fumarate has been implicated in tumorigenesis of HLRCC and, for this reason, included in the list of oncometabolites 2.3+-dependent mitochondrial enzyme, core component of the TCA cycle. The other two IDH isoforms (IDH1 and IDH2) are NADP+-dependent proteins expressed respectively in cytosol and mitochondria IDH1 and IDH2 were found in gliomas SDH and FH, IDH mutations are gain-of-function mutations and confer to the enzyme the ability to produce the oncometabolite D-2-hydroxyglutarate (D-2HG) d-2-hydroxyglutarate dehydrogenases (L2HGDH and D2HGDH) IDH1-mutant tumours exhibit DNA hypermethylation in gliomas TET2 and IDH1/2 mutations are mutually exclusive in AML tumours PDGFRA) Isocitrate dehydrogenases (IDHs) are homodimeric enzymes responsible for the reversible oxidative decarboxylation of isocitrate to aKG. Three different isoforms of IDH have been described, which have distinct subcellular compartmentalisation. IDH3 is a NAD350 for TETs and KDMs The brief description above suggests that signalling cascades elicited by fumarate, succinate, and 2HG, share some common targets but also exhibit distinct features, which can explain the different types of tumours associated with their accumulation . The con4The observation that 2HG is sufficient to promote tumorigenesis 2-HG is not the only oncometabolite whose levels are altered by hypoxia. During ischemia, succinate significantly accumulates in multiple organs and its oxidation is responsible for ROS generation during reperfusion 5Oncometabolites are emerging as key components of the communication between mitochondria and the nucleus. Chronic accumulation of these small molecules triggered by genetic or environmental cues may alter the epigenetic landscape of the cell eliciting oncogenic signalling cascades. This new paradigm of tumorigenesis challenges the role of gene mutations as the exclusive driving mechanism of tumorigenesis and suggests that the latter may be only one of the possible mechanisms that lead to transformation.The authors declare no competing interests.MS and CF jointly wrote the manuscript.MRC Core Funding to the MRC Cancer Unit.MS is a Research Associate in the laboratory of CF. CF is a group leader at the MRC Cancer Unit, University of Cambridge, Cambridge, UK. MS and CF are funded by an


The Neuronal Ceroid Lipofuscinoses  are a group of inherited neurodegenerative disorders with variable age of onset, characterized by the lysosomal accumulation of autofluorescent ceroid lipopigments. The endoplasmic reticulum (ER) is a critical organelle for normal cell function. Alteration of ER homeostasis leads to accumulation of misfolded protein in the ER and to activation of the unfolded protein response. ER stress and the UPR have recently been linked to the NCLs. In this review, we will discuss the evidence for UPR activation in the NCLs, and address its connection to disease pathogenesis. Further understanding of ER-stress response involvement in the NCLs may encourage development of novel therapeutical agents targeting these pathogenic pathways. •ER-stress activation has been linked to various neurodegenerative diseases.•ER-stress is a common patho-mechanism in four forms of NCL.•Pharmacological modulation of UPR could provide new treatment for NCL. Thirteen genetically distinct genes have been identified that, when mutated, result in abnormal lysosomal function and an excessive accumulation of ceroid lipofuscin in neurons and other cell types. The NCL family of proteins is comprised of four lysosomal enzymes , a soluble lysosomal protein (CLN5), secreted proteins (GRN/CLN11), and several proteins that display different subcellular localisations . Unfortunately, the precise function and localisation of many of the NCL proteins are still unclear, which has made targeted therapy development challenging. In this review we present and discuss the pathogenic role of ER stress in the listed NCL subtypes.1.1CLN4 (DNAJC5) and CLN11 (GRN) genes encode, respectively, a cytosolic and extracellular proteins The NCLs are the most common autosomal recessive neurodegenerative storage disorders, and have no effective treatment. The hallmark characteristic is the presence of autofluorescent storage material in lysosomes resembling ceroid and lipofuscin lipopigments that also accumulate in disease or during the normal ageing process. This storage material is observed in many tissues and cell types. However, the pathological effect of the disease is most prominent in the central nervous system (CNS) and the eyes. There are several types of NCLs, each of which is caused by mutation in a distinct gene and differ for age of onset, disease progression and severity, and the morphological appearance of the storage material 1.2Proteins destined for secretion, membranes or organelles begin their maturation in the ER where they fold, oligomerise, and are frequently subjected to covalent modifications in the process of becoming biologically active. The secretory pathway comprises the endoplasmic reticulum (ER), ER exit sites (ERES), the ER-to-Golgi intermediate compartment (ERGIC), the Golgi complex and post-Golgi carriers en route to their final destination. To exit the ER, newly synthesised proteins must pass a tightly controlled quality check COPII-coated vesicles transport cargo proteins from the ER to the Golgi; COPI-coated vesicles transport cargo in the retrograde direction (from the cis-Golgi back to the ER) and between Golgi cisternae. Clathrin-coated vesicles form at the plasma membrane and the trans-Golgi network (TGN) and subsequently fuse with endosomes or lysosomes. By coupling cargo selection to vesicle formation, cells can achieve efficient protein sorting as an in-built outcome of the transport pathway itself. In addition, some cargoes exit the ER without concentration into vesicles or interaction with the vesicle coat Understanding how protein-folding influences ER–Golgi traffic is crucial, since premature exit from the ER results in a lack of folding and of access to the degradation machinery. Cargo adaptors and cargo receptors in both the ER and Golgi could play direct roles in determining the fidelity of transport. ER receptors discriminate between fully folded proteins and those that need further chaperone action; Golgi receptors bind misfolded cargo for retrieval back to the ER. However, whilst this general role may suffice for some misfolded proteins it is unlikely to apply to all proteins. Many incompletely folded proteins are actually transport-competent beyond the ER 1.32+ storage, lipid and carbohydrate metabolism. Cellular stress, induced by internal or external cues, activates several orchestrated processes aimed at either restoring cellular homeostasis (adaptive UPR) or committing to cell death . Those processes named ‘ER stress response’ comprise various aspects such as unfolded protein response activation, autophagy, and mitochondrial response. When one of the elements of the ER stress response is impaired, as often occurs under pathological conditions, overall cellular homeostasis may be perturbed The ER is a complex, dynamic organelle and is the first compartment in the secretory pathway responsible for the synthesis, modification and export of newly synthesised proteins. The ER is also involved in Ca1.3.1The overall protein folding status is relayed to the cytosol, nucleus and other organelles such as lysosomes and mitochondria. During times of ER stress, protein load and translation are decreased and the folding capacity of the ER increased in an attempt to restore the ER equilibrium. To reduce the protein load, transcription of genes encoding secretory proteins is downregulated, misfolded proteins are cleared through ERAD, and a general reduction of protein translation is imposed at the ER. More chaperones are synthesised and the volume of the ER is increased in order to dilute the unfolded protein load and to host more chaperone machineries in an attempt to increase the protein folding capacity. If the aforementioned mechanisms fail to rescue and restore cell homeostasis then apoptosis could be triggered through the intrinsic pathway in an attempt to prevent further damage at the level of tissue or organism The protein folding machinery of the ER consists of three classes of proteins, foldases, molecular chaperones and lectins. Foldases directly promote protein folding and molecular chaperones prevent protein aggregation by shielding unfolded regions. The ER-resident chaperones comprise the HSP70 family, GRP78, LHS1/GRP170, their co-chaperones, which belong to the DnaJ and GrpE families, and an HSP90 paralogue, HSP90B1, which recognises partially folded proteins N-acetylglucosamine-1-phosphotransferase complex leading to abnormal sorting of newly synthesised lysosomal proteins and accumulation of storage material in the lysosomes, a similar phenotype to that observed in mucolipidosis type II (MLII) Three ER membrane sensors regulate the UPR: inositol-requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6) and double-stranded RNA-activated protein kinase-like ER kinase (PERK), all of which can be activated by perturbed ER homeostasis. Each activates specialised transcriptional programmes mediated by distinct transducers PERK, an ER transmembrane kinase, functions as an ER-stress sensor, influencing redox homeostasis and the ER-mitochondria interface 1.3.2Apoptosis is a highly sophisticated and regulated programme that requires activation of different molecular events. Studies indicate that there are two main apoptotic pathways: extrinsic (death receptor pathway) and intrinsic . The extrinsic pathway is triggered by transmembrane receptors belonging to the tumor necrosis factor (TNF) superfamily. These receptors contain a cytoplasmic “death domain” that transmits a signal from the cell surface into the cell, activating a signal cascade that culminates in activation of caspase 8. Activated caspase 8 cleaves downstream substrates including other caspases, thereby activating the execution phase of the apoptotic process 2+ ions, which destabilise mitochondrial membranes and activating caspase 9 with subsequent activation of caspase 3 and cleavage of PARP leading to cell death In addition, accumulation of a protein called growth-associated protein 43 (GAP43) in the ER is also able to trigger ER-dependent apoptosis In addition, IRE1 pathway activation can lead to Jun/JNK mediated apoptosis Apoptosis under ER stress is therefore the result of fine-tuning between different branches of the UPR pathway. Whether or not ER stress culminates in cell death is determined by multiple different factors, such as the intensity and duration of the stress. Evidence for a role of ER stress-mediated apoptosis in different neurodegenerative diseases make this process an attractive target for therapy. Thus, a better understanding of the mechanisms that orchestrate the ER stress mediated apoptosis may help to shape future therapeutic strategies.1.3.3Accumulating data indicate that ER stress is also a potent trigger of autophagy Whereas the induction of autophagy by ER stress is conserved from yeast to mammals, the signalling pathways responsible for autophagy induction and its cellular consequences appear to vary according to the cell type and the stimulus. Recently, it has been shown that the transcription factors TFE3 and TFEB A better understanding of the signalling pathways controlling autophagy and the cellular fate in response to ER stress will hopefully open new possibilities for the treatment of the numerous diseases related to ER stress.1.3.42+ homeostasis in response to a specific signalling pathway. PERK is enriched at the ER side of the MAMs where it plays an important role in integrity 2+ transfer Many regulatory members of UPR link with mitochondrial regulation and function Moreover, in skeletal muscle ATF6 is an important partner of PCG1 , which is associated with mitochondrial biogenesis The link between ER and mitochondrial stress is still under debate even though defects in this axis have been linked to various neurodegenerative disease 1.4A common feature of many neurodegenerative disorders is the accumulation and deposit of misfolded and unfolded proteins, which trigger ER stress affecting neuronal homeostasis and lead to cell death 2+ homeostasis 2+ caused by accumulation of toxic products are able to trigger ER stress eventually leading to motor neuron loss In Parkinson's disease, ER stress together with abnormal protein degradation plays an important role in the pathophysiology of PD, contributing to the selective death of dopaminergic neurons M1-gangliosidosis, Pelizaeus-Merzbacher disease (PMD) and the NCLs. Evidence suggests that ER stress and the UPR response accompany GM1-gangliosidosis M1-ganglioside ER stress has been shown to play a role also in the pathophysiology of paediatric diseases such as hereditary spastic paraplegia (HSP), an infantile inherited autosomal dominant neurological disorder characterized by spastic weakness of the lower extremities CLN4 gene cause the adult onset form of NCL known as Kufs disease. CLN4 or DNAJC5 encodes cysteine-string protein α (CSPα). CSPα is a chaperone that ensures protein folding CLN11/GRN (progranulin) has been suggested to play a role in lysosomes CLN13 (CTSF) CLN10 (CTSD) have been tightly related to protein degradation, apoptosis and autophagy In addition, various reports suggested a link between ER stress and four forms of NCL disease  . For theAs mentioned earlier, a strong correlation between ER stress and the disease gene has been described in four NCLs subtypes: CLN1, CLN3, CLN6 and CLN8 diseases. We now review the pathogenetic role of ER stress in these listed NCL subtypes .Fig. 2Mu22.1PPT1/CLN1Most cases of infantile NCL are caused by inactivating mutations in palmitoyl-protein thioestarase-1 (PPT1), encoded by 2+ dyshomeostasis with activation of caspase 9 and 3 N-oxide dihydride (TMAO) or tauroursodeoxycholic acid (TUDCA) It has been reported that some PPT1 variants containing disease-causing mutations are not correctly transported to the lysosomes, but are retained in the ER due to improper folding 2.2CLN3 gene. More than 40 disease-causing mutations have been identified in CLN3 (http://www.ucl.ac.uk/ncl) CLN3 results in multiple transcripts that include a frame-shift and premature termination or loss of several internal exons and an in-frame C terminus and retained protein function CLN3 encodes a 438 amino acid transmembrane protein and despite being identified more than two decades ago, the function of this highly conserved protein remain elusive. Levels of endogenous CLN3 protein are extremely low. A significant localisation of the CLN3 orthologue of fission and budding yeasts, Btn1p, has been shown at the Golgi complex 2+ homeostasis and autophagy CLN3 is an anti-apoptotic gene in neuronal precursor cells and in at least some cancers Drosophila as a CLN3 disease model, ER and mitochondrial functions were found to be impaired by oxidative stress The majority of juvenile cases of NCL, with an age of onset ranging between 5 and 7 years of age, are caused by mutations in the Recently, it has been shown that treatment of lymphoblast cells from patients with fibrates, drugs able to induce peroxisome proliferator-activated receptor-α (PPAR-α) activation, abrogates the depolarization of mitochondrial membrane potential, normalises defective autophagy and increases overall cell viability Together, the evidences so far therefore strongly suggest that ER stress activation may play a pivotal role in the pathogenesis of CLN3 disease, and certainly this requires further study.2.3CLN6 cause CLN6 disease nclf) models There is little current understanding of how mutations in nclf mouse disease model which carries a 1 bp insertion in the orthologous gene II(atsm) that is protective in amyotrophic lateral sclerosis-induced Zip7 upregulation, promotes Zn redistribution and restores Zn-dependent functions in primary mouse Cln6 deficient neurons and astrocytes Accumulation of biometals such as zinc, copper, manganese and cobalt has been described in Merino and South Hampshire sheep models of CLN6 disease 2.4CLN8 is mutated in two distinct clinical NCL phenotypes: late infantile and a mutation-specific juvenile-onset variant, progressive epilepsy with mental retardation (EPMR) also known as Northern Epilepsy Cln8mnd mouse model, which exhibits a disease phenotype similar to that of late-infantile CLN8 disease patients mnd mice defective lipids and protein transport and ER/mitochondria Ca2+ exchange are linked to alteration in MAMs Recently, molecular partners of CLN8 have been described to be involved in autophagy, apoptosis, as well as vesicular trafficking and lipid transport 3Experimental evidences suggest that several organelles are capable of sensing and relaying pro-apoptotic signals, culminating in the proteolytic activation of caspases and cell death. Each organelle is likely to be uniquely poised and equipped to sense specific stimuli related to their function and structure. For example, the ER is the major site for folding, modification and assembly of newly synthesised proteins and has evolved a stress response pathway to cope with the accumulation of unfolded or misfolded proteins. Activation of ER stress has been shown to induce the expression of chaperones, attenuate translation and degrade misfolded proteins in attempt to alleviate the stress. Prolonged ER stress results in irreparable damage leading to apoptosis. This is the case observed in some of the NCL types described in this review . Indeed,2+ dyshomeostasis contribute to ER stress activation. In CLN3 disease, ER-stress mediated apoptosis might play an important role. A global screen to identify genetic interactions with the fission yeast orthologue btn1 highlighted a conserved key signalling hub in which multiple components functionally relate to this conserved disease gene btn1 are unable to respond to the applied stresses, although the pathways themselves are still functional In CLN1 disease, both ROS and Ca2+ are important cofactors for protein post-translational modification, with defects in manganese homeostasis leading to misfolding of proteins and their accumulation in the ER and ER stress. In CLN8 disease, a mismanagement of the ER stress response is linked with lipid homeostasis and alteration of MAMs.In CLN6 disease in particular, accumulation of biometals could lead to ER-stress. Biometals such as MnTo date, the evidences reported are strongly suggestive that the dramatic neuronal loss observed in NCLs is caused by ER stress-mediated apoptosis, making this process an attractive target for therapy. The apoptotic pathway is not one single linear path, but is a complex and articulated pathway that can be activated via different stimuli and branches of the UPR. Given the various localizations and functions of the different CLN proteins, we cannot exclude that their mutation challenges the quality control and activates the ER stress/UPR in slightly different ways, but still resulting in a similar apoptotic phenotype. In addition, some mutant proteins are transport competent but not functional once reaching their final destination. Therefore, a better understanding of the interplay between the various pathways leading to the apoptotic pathway and arising from defects in the quality control process as well as how some proteins may escape quality control in the ER could greatly contribute to elucidating the pathogenesis of the various NCL diseases and provide specific therapeutic approaches. In addition, it is possible that alteration in one NCL gene influences the expression and localization of other NCL proteins, thereby generating a complex scenario in which modulation of the phenotype of various NCL diseases is the result of the combined alteration of expression of different NCL proteins.3.1The UPR pathway represents a possible common target to treat multiple forms of NCL disease. Therapeutically, it is therefore crucial to understand the role of the UPR in the pathogenesis of the NCLs. In addition, the pharmacological modulation of UPR, either by pro-apoptotic or pro-survival signalling, could provide new treatment strategies not only for the NCLs but many diseases, including other neurodegenerative disorders, heart disease and cancer.Transparency document.Image 1