Through direct interactions with gene promoters, enhancers give rise to spatially and temporally precise gene expression profiles in distinct cell or tissue types. In the brain, the accurate regulation of these intricate expression programs across different neuronal classes gives rise to an incredible cellular and functional diversity. Newly developed technologies have recently allowed more accurate enhancer mapping and more sophisticated enhancer manipulation, producing rapid progress in our understanding of enhancer biology. Furthermore, identification of disease-linked genetic variation in enhancer regions has highlighted the potential influence of enhancers in brain health and disease. This review outlines the key role of enhancers as transcriptional regulators, reviews the current understanding of enhancer regulation in neuronal development, function and dysfunction and provides our thoughts on how enhancers can be targeted for technological and therapeutic goals.Enhancers are non-coding DNA elements that function in  Xenopus oocytes discovered a sequence upstream of the H2A gene that could modulate transcription levels in an orientation-independent fashion. Additionally, deletion of these elements resulted in lower expression levels [SV40 gene. This cis-acting DNA sequence was found to modulate and increase transcription of a distal gene more than 200-fold, independent of its orientation and location relative to the target gene. Several studies of the viral SV40 enhancer identified many additional characteristic enhancer features, such as DNase hypersensitivity (indicative of open chromatin) and transcription factor (TF) binding [Complex and precise spatiotemporal gene expression patterns orchestrate the wide diversity of cell fate and function. Non-coding DNA elements, such as enhancers, regulate these intricate expression programs at any given time to ensure proper development and function of specific tissues and cell types . Enhancen levels , suggest binding ,4,5,6. T binding .Enhancers are probably best understood in murine embryonic stem cells and the context of cellular development, followed by cancer as a large portion of research has focused on these areas. Nevertheless, the brain provides a unique window to explore how enhancers contribute to cell fate and function and also to understand how enhancers can be dysregulated in disease. In the brain, enhancers help to ensure cell- and tissue-specific gene expression profiles, defining which genes are active during neuronal specification and which genes remain accessible in adult neurons. In adult neurons, the storage of information requires the dynamic regulation of gene expression patterns to allow for neuronal plasticity, learning and memory formation and long-term adaptations of behavior to an environment. Furthermore, a subset of enhancers shows activity-dependent induction in response to neuronal activation and behavioral experience, suggesting a regulatory role in transcription and brain function.A significant fraction of non-coding genomic space is dedicated to enhancers, with current estimates suggesting that vertebrate genomes may encode tens of thousands or even millions of active enhancers ,9,10,11.(1)an open chromatin structure, indicated by DNase hypersensitivity,(2)specific epigenetic modifications,(3)defined molecular interactions, and(4)bi-directional transcription starting in the center of the enhancer leading outward on opposite stands ,27,28,29Enhancers and enhancer states are characterized bycis regulatory elements and discuss potential functions of enhancer clustering and phase separation in gene regulation.This section will explore the prominent features of enhancers, contrast enhancers to other cis regulatory elements share architectural similarities and enhancers provide a key example of this. Distal enhancers and gene-proximal promoters are defined by many structural and functional features [FAF2 promoter interacts with three other promoters as an enhancer and that deletion of this promoter decreased expression of all three target genes [Many features ,30. Tradfeatures . The eukfeatures . Likewisfeatures ,33. Builfeatures ,34,35,36et genes . More reOne common way in which enhancers regulate gene expression occurs via direct interaction with promoters through physical enhancer-promoter loops. This enhancer looping mechanism allows distal enhancers to act on genes that are thousands  of bases upstream or downstream of the gene locus and provides another layer of control over dynamic transcriptional responses to internal and external stimuli.Several factors regulate and stabilize this looping process, including TFs like YY1, the CCCTC-binding factor CTCF and the Mediator/Cohesin complex. CTCF, a TF often found at transactivation domain (TAD) boundaries, is thought to stabilize loop anchors . CTCF fuEnhancer regions are typically more conserved across species than surrounding regions in the genome. In fact, initial genome-wide enhancer screens used sequence conservation as an indicator for enhancers . While dAs enhancers are characterized by open chromatin, techniques based on chromatin accessibility such as mapping of DNase hypersensitive sites or Assay for Transposase Accessible Chromatin Sequencing (ATAC-seq) are often used to identify enhancers across the genome ,44. ThisInterestingly, histones in enhancer regions show an enrichment of H2A.Z and H3.3 histone variants. These variants are thought to be less stable (and easier to displace) than traditional histones, allowing for more dynamic mobility and easier access for TFs to bind ,47. In aEnhancer regions are subject to dynamic epigenetic modifications and binding of chromatin remodelers such as histone acetyltransferases (HATs). CBP and p300, two closely related HATs, target histone tails as well as transcription machinery including RNAP2 in TAD domains. While histone acetylation creates a more accessible and permissive chromatin structure at enhancers and improves TF recruitment, RNAP2 acetylation promotes its release into the gene body ,49,50. NThe histone methyltransferases Kmt7  has been found to bind enhancers in muscle tissue in addition to HATs. Set7 binding increases H3K4me1 at enhancers and is also associated with increased target gene expression . Other gThe binding of epigenetic modifiers is also crucial for TF function and the ability of enhancers to drive gene expression. Enhancer activation is thought to require the binding of several TFs, often including lineage-specific and motif-dependent factors that respond to signaling cascades to integrate extrinsic and intrinsic signals. These early TFs then prime the enhancer region and reinforce binding of additional TFs and coactivators. The combinatorial binding of different TFs and coactivators then gives rise to discrete expression patterns across different tissues and cell types.Extensive investigation of developmentally regulated enhancers in stem cells has shown how master TFs like Oct4, Sox2 and Nanog can identify or predict enhancer activity and that TF function often depends on the binding of coactivators that harbor chromatin remodeler activity or mediate long-range interactions ,55. In pRecent reports have suggested that a subset of TFs is biased towards different regulatory elements like enhancers or promoters. AP1, a TF composed of FOS/JUN heterodimers, is biased towards enhancer elements, whereas EGR1 and SP1 are biased towards promoters and promoter activity. In contrast, a subset of TFs, including RFX, is capable of generating both enhancer and promoter activities .Fos gene in response to neuronal depolarization [The key role of TF binding in enhancer activation is not limited to developmental processes, as a striking subset of enhancers show activity-dependent TF binding. For example, CBP and Npas4 were found to bind enhancers surrounding the rization  and manyrization . Surprisrization .The term super enhancers (SEs) refers to clusters of enhancers that are characterized by increased size, abundant TF and Mediator complex occupation and increased capability to induce gene expression compared to “typical” individual enhancer loci. SEs dictate expression patterns of entire groups of functionally related genes. Whyte et al. identified SEs that regulate cell identity genes in mouse myotubes, T helper cells and macrophages . On a geAnother hallmark of active enhancers is bi-directional transcription, which originates in the center of the enhancer and produces RNA transcripts from both DNA strands in opposing directions. The act of transcription itself has been suggested to keep the enhancer locus in a more permissive and active state, accessible for TFs, epigenetic modifiers and transcription machinery. Nevertheless, these transcripts arising from enhancers , were initially thought to result from their close proximity to RNAP2 and actively transcribed genes. Thus, eRNAs were not initially included in models of enhancer function and their specific roles have remained controversial. However, over the last decade striking evidence for the functional relevance of eRNAs has emerged. Several studies have demonstrated that eRNAs interact with several key molecules in enhancer function including the Mediator complex, transcriptional repressors and activators, as well as epigenetic machinery ,61,62,63(1)the number of participating components, (2)all possible chemical modifications of their residues,(3)and the rate at which these modifications are facilitated or inhibited.Phase separation describes molecular condensates within eukaryotic cells that create compartments of high protein and nucleic acid density, such as the nucleolus, nuclear speckles, stress granules and many other compartments. The formation and specific composition of phase-separated compartments is tightly regulated to provide the opportunity for controlled environments optimal for specific reactions. The investigation of phase separation has recently gained a lot of momentum, as striking discoveries have demonstrated that the components of these condensates can change over time depending on the state of cell cycle or in response to certain stimuli (reviewed in Reference ). IntrigSpecifically, SEs are suggested to rely on high levels of such cooperativity between numerous interaction partners, as they typically accumulate higher numbers of interacting molecules than traditional enhancers. Overall, this model addresses how phase separation could play a role in transcriptional control and explains how it might contribute to previously observed characteristics of SEs.Phase separation as a mechanism for enhancer function is particularly interesting as it would enable concentration of a variety of components necessary for regulated gene transcription. Sabari et al. were the first to demonstrate the participation of SEs in phase separation by showing that coactivators Mediator and BRD4 (a member of the BET family), associate with SEs in nuclear condensates. Disruption of the condensates by 1,6-Hexanediol resulted in loss of Mediator and BRD4 binding at SEs and loss of RNAP2 occupancy at SEs and their target genes. This work suggests a role of coactivators in the compartmentalization and local concentration of SEs and transcription machinery , perhapsEnhancers control complex spatiotemporal expression programs throughout development and into adulthood and so initial studies on neuronal enhancers focused on identification of enhancer loci in the developing CNS. In a pioneering study, Nord et al. measured H3K27 acetylation with Chromatin immunoprecipitation sequencing (ChIP-Seq) as a readout of active enhancer loci across seven developmental stages ranging from embryonic day 11.5 to postnatal day 56 to identify enhancer activity profiles in three different tissues including the mouse forebrain ,67. ThisBuilding on these findings, subsequent studies have investigated developmental changes in chromatin accessibility and enhancer activity in specific cell types in the cerebellum ,61,62,63While initial studies of enhancers in the 1980s established a cell type specific role for enhancers , later gcis-regulatory elements, including promoters and enhancers and created a map of chromatin accessibility in neuronal and non-neuronal nuclei across 14 brain regions of human postmortem tissue. This study revealed differences between neuronal and non-neuronal cell types, as well as differences among neurons from different brain regions [More recently, examination of enhancer activity states has been extended to postmortem brain samples to begin defining cell class and cell type dependent enhancer programs in the human brain. Fullard et al. investigated  regions . Similar regions . This woHow is enhancer activity regulated in a spatiotemporal precise manner? One route of regulation is enhancer accessibility to interaction partners, which is determined by epigenetic modifications at enhancers. For example, a recent study found that the epigenetic state of enhancers affects gene expression and neuronal migration patterns. The histone methyltransferase PRDM16 is a crucial regulator of the epigenetic state of enhancers in the embryonic cortex and is thought to deposit H3K9me1 and H3K4me1 . This stOnce enhancers are accessible, cell identity is mediated by coordinated binding of specific TFs to specific enhancers. Rhee et al. characterized how initial TF binding dictates differentiation and cell identity and how the interplay between different TFs ensures stable expression patterns in mature motor neurons . More spIn the context of brain function, one important question emerges: how do enhancers regulate genes in response to neuronal stimulation? Activity-dependent gene regulation is a hallmark of key neuronal phenomena, including long-term potentiation, homeostatic plasticity, long-term memory formation and adaptive behavior. Therefore, a central focus of ongoing attempts to understand enhancer function in the central nervous system has utilized activity-responsive enhancers found near genes that are rapidly controlled by neuronal depolarization and other stimuli.Fos, Rgs2 and Nr4a2). Activity-dependent recruitment of Creb, Mef2, Npas4 and Fos to enhancer regions further demonstrated the importance of this activity-dependent mechanism for enhancer regulation [Addressing this question, a pioneering study by Kim et al. discovered ~12,000 activity-regulated enhancers in murine neurons. Enhancer activation was accompanied by stimulus-dependent CBP recruitment to enhancers near activity-regulated genes  result in different combinations of enhancer activation. Together, these findings highlight how combinatorial enhancer activation can distinguish between different stimuli and potentially fine tune and adjust the transcriptional response to a certain stimulus [The best characterized activity responsive enhancers are located around the activity ,57,58 anstimulus . ImportaArc gene. This immediate early gene encodes a cytoskeleton protein crucial for synaptic plasticity and has been shown to underlie enhancer mediated regulation [Another well studied activity dependent enhancer is located upstream of the murine gulation ,75. Neurgulation . Similargulation ,77, indugulation . Reelin-Together with their cell type specific nature, enhancers’ ability to respond to neuronal activity is extremely important to understand how different cells in circuit or brain region function on a cellular as well as on a network level . This unNumerous studies have linked neurological diseases to mutations and variants in DNA. While in some cases a specific gene mutation can be identified as the cause for a disease, for many diseases of the brain this is not the case. However, a large number of sequence variants have been linked to such diseases via genome-wide association studies. Interestingly, the majority of disease linked SNPs fall into non-coding regions and are increasingly linked to enhancer function. As normal enhancer activity requires a complex sequence of interaction partner binding and chromatin remodeling to induce transcription a, the loGenetic mutations could interfere with enhancer function and disrupt transcriptional regulation in various ways . First, CDH10 and CDH9 genes and a prominent locus for GWAS hits for ASD linked variants. This ASD-associated locus exhibits enhancer activity that regulates expression in cortical layer II/III, striatal and cerebellar neurons in human BAC2 transgenic mice [Neurodevelopmental disorders—Prevalent neurodevelopmental disorders have been linked directly to polymorphisms in enhancer regions. Autism Spectrum Disorder (ASD) presents with a wide range of clinical symptoms and numerous mutations and genetic variants have been linked to increased ASD susceptibility. The 5p14.1 locus is a region between the cadherin nic mice .FMR1 gene, resulting in impaired CTCF binding. Aberrant CTCF binding could disturb enhancer looping to target promoters and result in altered gene expression levels. Neurological diseases—Even though enhancers are typically associated with development, enhancer dysregulation does not only lead to neurodevelopmental problems but has also been implicated in neurodegenerative and psychiatric diseases, as well as in mental health. Alzheimer’s disease (AD) is a devastating progressive neurodegenerative disease characterized by dementia and loss of cognitive function. Various studies have identified and linked mutations and sequence variants to AD, including APP, APOE4 and BIN1 [BIN1 expression [PM20D1 locus with AD risk [PM20D1 locus interacts via CTCF-mediated chromatin loops with an AD-associated haplotype that shows enhancer-like characteristics. This region displayed an enrichment for epigenetic enhancer marks and increased expression in a luciferase assay and the interaction with PM20DI was haplotype dependent. Interestingly, PM20DI expression increased in response to neurotoxicity and overexpression alleviated cell death, decreased Aβ levels and improved cognitive function. Therefore, risk-haplotype carriers might be left with an impaired cellular defense in AD as a result of decreased PDM20DI expression. Together these studies highlight the potential impact of enhancer function on AD predisposition and manifestation.A number of diseases have been associated with short tandem repeats  and thesand BIN1 ,82,83. Hpression ,83. Anot AD risk . The PM2SNCA gene was identified as one of the strongest risk loci associated with sporadic PD and mutations of the gene have been shown to cause familiar PD [SNCA in patient derived pluripotent stem cells [SNCA expression. This study demonstrates how sequence variants in enhancers can impair enhancer functions such as proper TF binding and thereby promote disease-related expression imbalances.Parkinson’s disease (PD) is one of the most common neurodegenerative disorders and is characterized by late disease onset and complex interactions between genetic and environmental risk factors. The iliar PD ,86. Intrem cells . Alpha-sPsychiatric disorders—Schizophrenia (SZ) is one of the most common psychiatric disorders characterized by positive, negative and cognitive symptoms and a complex interplay between genetic and environmental risk factors. There is increasing evidence for an enrichment of enhancers and promoters in SZ-linked gene variants and several SZ associated SNPs have been shown to fall into non-coding regions with enhancer activity ,70,88,89GAL enhancer. As proper GAL expression is required for food and alcohol intake as well as the regulation of mood, these SNPs could also have implications in alcoholism and obesity. Interestingly, the more conserved sequence shows strong enhancer activity while the less conserved and disease-linked variant was associated with decreased gene induction capacity [Alcohol dependence and depression are psychiatric disorders that often occur together. A number of genetic variants likely influence the development of both disorders . Two SNPcapacity . Thus, eAs we continue to study disease associated SNPs, the importance of enhancer function becomes abundantly clear. Future work will not only identify other disease-linked regions with enhancer activity but also help understand the underlying mechanisms of enhancer function and their contribution to disease manifestation.In addition to mutations in enhancer elements directly, mutations in interaction partners can also obstruct enhancer function and result in disease-related expression patterns. For example, mutations of the Mediator complex, which is crucial for enhancer looping, have been associated with a number of neurological and neurodevelopmental disorders ,94,95,96With growing understanding of the importance of enhancers in brain health and disease, future research will need to elucidate the mechanisms and dynamics of enhancer activity in response to neuronal stimulation. While the ability of enhancers to respond to stimuli such as neuronal depolarization has been appreciated for nearly a decade, the role of different enhancers in regulating the response of the same gene to different stimuli is much less clear. This activity could potentially provide a key mechanism by which a specific context or experience can be translated and integrated into specific transcriptional response patterns. Thus, a critical area of focus should be to unravel how enhancer activation and inactivation is mediated in different brain regions and in response to different intrinsic and extrinsic stimuli. This knowledge will help explain how enhancers regulate and fine tune transcriptional responses in a specific context and how the disruption of enhancer activity contributes to disease formation.Likewise, even though we know several key events that regulate enhancer activation , recent discoveries in the field of phase separation open up intriguing possibilities for how groups of enhancers are co-regulated. While there is initial evidence linking enhancers to phase-separated condensates, it is unclear how this is regulated and whether this is a general mechanism for enhancer function. Nevertheless, the concept of phase separation offers exciting possibilities for dynamic enhancer regulation and allows for quick responses to various signals. This mechanism could explain how enhancers can rapidly accumulate and induce complex gene expression programs in response to a stimulus. Simultaneously, phase separation could also create condensates that promote degradation of other proteins and transcripts to further balance the cellular transcriptome and achieve a balanced cellular composition.Technological advances have provided a number of important avenues to utilize enhancers to understand the basic biology of the nervous system. For example, recent efforts have capitalized on the cell-type specific nature of many enhancers to drive gene expression in a highly cell-type or brain region specific manners. While previous technologies  enable Technological innovations have also generated useful ways to dissect enhancer function, identify mechanistic interactions and distinguish biochemical roles of specific proteins, nucleic acids and molecules see . For exa(1)use of cell-penetrant antisense oligonucleotide strategies to modulate functional enhancer RNAs, (2)targeting enhancer-linked proteins  using small molecule drugs,(3)or use of enhancer sequences to drive expression in gene therapy approaches.Finally, it will also be important to investigate how enhancer dynamics contribute to brain health and disease, as this provides another layer in which enhancer mediated gene regulation is vulnerable to disruption. Thus, in addition to transcriptional control as a research tool, advances in our understanding of enhancer biology could also provide promising new therapeutic avenues. Enhancer activity could potentially be targeted to correct aberrant gene expression caused by dysregulation of the enhancer  but might also be used to alleviate dysregulated gene expression in conditions with other underlying enhancer-independent mechanisms. As enhancers and particularly SEs can regulate multiple genes, targeting SEs could alleviate aberrant gene expression of entire gene programs or pathways simultaneously. Furthermore, by identifying genes that are regulated by disease-linked SEs, we could discover previously unknown elements in disease mechanisms as well novel therapeutic targets. These targets could be harnessed in the near future in several ways, includingAdditionally, future strategies could leverage human GWAS datasets with gene editing to enable pursuit of even more targeted therapeutic approaches.Box 1Box 1: Technical challenges to studying enhancers.Identifying enhancer regionsBased on our knowledge about the chromatin structures and epigenetic signatures of enhancer regions, ChIP-seq for these characteristic histone marks has evolved as common screening method for enhancers. Often these studies focus on H3K27 acetylation when searching for active enhancers. RNA-seq data can complement these searches and help identify active enhancers as well. One of the challenges in studying enhancers is that they typically only function in specific cell types. As a result, there are limitations when studying neuronal enhancers in many common and easy to use dividing cell lines. This also highlights the importance of single-cell techniques to study enhancers. More recently developed high-throughput ATAC-seq techniques to study chromatin accessibility in single cells have great potential to drive enhancer research forward with increased cellular resolution ,108. HowAssessing enhancer functionHigh throughput enhancer reporter assays such as massively parallel reporter assays (MPRAs)  or STARRIdentifying enhancer targetsWhile enhancers often regulate nearby genes, they can act over long distances or even regulate multiple genes . It therTechnological advances that allow mapping of RNA-genome interactions (MARGI) are particularly promising in revealing eRNA interactions on a genome-wide scale. An atlas of non-coding RNA interactions could help identify previously unknown enhancer and eRNA targets and provide a deeper understanding for how non-coding RNAs affect chromatin conformation and transcriptional regulation genome-wide ,113,114.Studying enhancers vs eRNAsEnhancer transcription is commonly used as a marker of active enhancer, which leads to the next question: How do we dissociate genomic enhancers from their enhancer RNAs when studying their function? Traditional mutations of the enhancer region do not allow us to tease apart which observed effects are based on the genomic enhancer and which are based on its eRNA. Even new genome and epigenome editing tools such as the TALEN or CRISPR-dCas9 systems, in which chromatin remodelers, and transcriptional activators or repressors can be targeted to the enhancer, cannot fully dissociate the two functional units. In addition to direct RNA knockdown approaches that don’t affect the underlying DNA, recent studies have employed CRIPSR-Display, a tool that allows for RNA tethering to the enhancer to study the effects of non-coding RNAs in a more direct fashion ,103,115.Enhancers are key genomic features that not only define the development of the central nervous system but also contribute to numerous neurodevelopmental, neuropsychiatric and neurodegenerative diseases. Our understanding of enhancer function in the brain is rapidly accelerating, in part due to innovative technologies that overcome many previous limitations . In addi


Human brain organoids, generated from pluripotent stem cells, have emerged as a promising technique for modeling early stages of human neurodevelopment in controlled laboratory conditions. Although the applications for disease modeling in a dish have become routine, the use of these brain organoids as evolutionary tools is only now getting momentum. Here, we will review the current state of the art on the use of brain organoids from different species and the molecular and cellular insights generated from these studies. Besides, we will discuss how this model might be beneficial for human health and the limitations and future perspectives of this technology. Gaining a clearer understanding of human brain evolution is crucial to interpreting how human genetic variants lead to disease. Therefore, understanding the evolutionary path of the modern human brain will likely illuminate the origins of conditions such as autism, dementia, or schizophrenia, which are considerable burdens to all present and future human societies.What is it that makes us uniquely human? Among several features of our species, perhaps the most impactful characteristic consists of our sophisticated brains and all the abilities and advanced lifestyles we all enjoy. However, a complex brain came with a cost. The vulnerability for human-specific neurological disorders might well be an undesired evolutionary trade-off. Although many genetic mutations might also cause diseases in other animals, the genomic landscape of humans makes our species more susceptible to certain neurological disordersIn utero experimental access to ape brains is not always possible. Instead, scientists have relied on less invasive passive alternatives, such as ultrasound and functional imaging. Most of our understanding of normal and pathological brain conditions comes from postmortem tissues that represent only a blurry snapshot of a highly dynamic tissue. Also, the neuroanatomical and functional heterogeneity that individuals have because of their genetic and environmental backgrounds adds another obstacle to fully understanding the typical and unhealthy progression of neurodevelopment3.Our knowledge of human brain development is deficient and there are gaps in the critical steps leading to the cellular organization and the formation of functional networks that are the basis of cognition. The limited accessibility of the human  brain has blocked our understanding of neurodevelopment, especially at very early stages. During embryogenesis, a limited number of pluripotent embryonic stem cells will give rise to a multi-cellular and complex nervous system in the brain, which orchestrates many of the autonomous and non-autonomous functions of the body.5 and molecular profile of the developing human embryonic and fetal brain9. These structures can grow free floating in media or embedded in matrigel and develop different brain regions using endogenous patterning cues11. Alternatively, cells could be patterned early on by exposure to specific cocktails of factors, coaxing the identity of the cells toward a specific brain region13. Differently patterned organoids independently created could be fused, exchanging signals among the different brain regions, stimulating cell exchange16 or reciprocal projections17. The “lego-organoid” approach can recreate specific circuit formations in a more controlled and reproducible fashion than non-patterned cerebral organoids.Brain organoids, generated from pluripotent stem cells, are multi-cellular, three-dimensional self-assemble miniaturized structures that mimic the dynamic organization21 and autism22. Interestingly, the fact that brain organoids can be generated from induced pluripotent stem cells (iPSCs)24, reprogrammed from somatic cells of any species, offered an unprecedented opportunity to compare early stages of human neurodevelopment with those of other primates, including our closest NHP relatives: the chimpanzees (Pan troglodytes) and bonobos (Pan paniscus)27. These three species have very similar genomes, including nearly 98% of alignable genomic sequence28. However, cellular and molecular phenotypes, especially at similar stages of development, are difficult to establish, mainly owing to limited access to live embryonic material from humans and NHPs. Thus, the study and manipulation of brain organoids in a dish are novel and promising evolutionary tools25.Human brain organoid has recently contributed to the understanding of several neurological conditions  and PIWIL2—were found upregulated in human cells. The experimental manipulation of A3B and PIWIL2 levels in the pluripotent stem cells supported a causal inverse relationship with L1 retrotransposition. Increased levels of L1 retrotransposition suggested that NHP-derived iPSCs would be more susceptible or permissive to new insertions. An increased copy number of species-specific L1 elements in the genome of chimpanzees was observed when compared with humans. The data support the idea that increased L1 mobility in NHPs might not be limited to the pluripotent stage and may also occur in the germline during primate evolution. Thus, it is possible to speculate that the activity of L1 elements has differentially shaped the human genome and still has adaptive significance.The first comparative study between human and NHP iPSCs revealed unexpected differential regulation of long interspersed element-1  endogenous retrotransposons36. AGS can be caused by mutations in the three-prime repair exonuclease I (TREX1) gene37. Curiously, rodent models of AGS do not mimic the severe neurological aspect of the human condition38. However, AGS-derived brain organoids do mimic the neurodegeneration and striking microcephaly seen during patient neurodevelopmentin utero39. Neuronal death in the brain organoids was caused, at least partially, by a substantial exposure of type I interferon, a pro-inflammatory cytokine secreted by astrocytes. The innate immune reaction in the astrocytes was triggered by the accumulation of L1 retrotransposons in the cell because of TREX1 absence. L1 elements are different between humans and other animals40. Evidence that L1 activity could contribute to other neurological disorders, such as Huntington’s disease41, Down syndrome, and Alzheimer’s disease42, and aging43 has recently been provided. It is an attractive hypothesis that the sequence-specific L1 differences might contribute to several human conditions, creating the potential for unconventional treatments using reverse transcriptase inhibitors, such as anti-HIV drugs.The RNA from L1 elements might also have an impact on human health. Aicardi–Goutières syndrome (AGS) type I is characterized by a dramatic neuronal loss, leading to lifelong disability46. Moreover, brain organoids from NHPs revealed differences in viral replication rates of the Brazilian strain compared with the African Zika virus at the same developmental stage. This observation might suggest that the Zika virus can adapt to different primates. Evidence for specific mutations in the circulating Brazilian Zika virus has emerged47 but it is unclear whether these variants were responsible for the dramatic phenotypes seen in the affected human babies. The abundance of human brain organoids was also a positive feature to rapidly screen for drugs that could prevent infection49 or block viral replication and eventual vertical transmission50.Endogenous viruses are not the only ones affecting human evolution. Human brain organoids played an essential role in the causal link demonstration between the Brazilian Zika virus and the microcephaly outbreak in Brazil. The relatively slow human neurodevelopment, compared with that of other experimental animal models mimicked by the human brain organoids in a dish, allowed investigators to dissect how the virus could infect neural progenitor cells leading to defects in the cortical plate51. The ability to create cortical brain organoids from human and other NHP iPSCs provides a unique opportunity to study the expansion of the neocortex in a dish52. By contrasting brain organoids of humans with those of chimpanzees, it was possible to determine a subtle differentiation between timing and lengthening of prometaphase-metaphase in human apical mitosis that is specific to proliferating neural progenitor cells54. It is possible that subtle differences at very early stages have a more dramatic impact later in development and thus set humans apart from other primates.In the past, testing hypotheses about human brain evolution was restricted to manipulations in animals or non-relevant cell types. Owing to cellular reprogramming, it is now possible to compare differences and similarities between human neurodevelopment and that of other primates without the use of embryonic materials that are ethically and technically difficult to access55. Several long non-coding RNAs (lncRNAs) were detected in specific cell types and stages of differentiation in all of the species. LncRNAs are implicated in several molecular mechanisms, including the regulation of neurodevelopment57. The conservation of pattern expression on these tissue-specific lncRNAs in all of these primates indicates a possible role in transition stages during neurodevelopment. Another recent work that contrasted brain organoids from human and NHP iPSCs also described gene network conservation among primates while identifying a set of 261 genes that are human-specific58. Some of these genes overlap with recent chromosomal segmental duplications59. Interestingly, increased activation of the PI3K/AKT/mTOR pathway was validated in the radial glia cells of the outer subventricular zone of human fetal brain tissues, possibly contributing to the neocortex expansion during evolution. The creation of a “brain organoid zoo”, which has representatives of different primates and other species, might help us to resolve critical molecular and cellular steps to comprehend brain evolution better.Cortical tissues generated from human, chimpanzee, orangutan, and rhesus iPSC-derived brain organoids were also used for a dynamic transcriptional analysis60, malaria vulnerability 61, Alzheimer’s disease (absence of neurofibrillary tangles)62, and susceptibility to certain cancers63 as well as other differences65. The identification and characterization of the cellular and molecular mechanism that distinguishes humans from our closest relatives at early stages of embryogenesis and in specific types of cells are likely to become a new resource for evolutionary studies66. In this context, recent advances in iPSC-derived neural progenitor cells and brain organoids are an attractive tool to dissect cellular and molecular events that contribute to the uniqueness of the human brain. However, this artificialin vitro approach is not without serious inherent limitations. Most comparative studies are standardizing the growth of the brain organoids using human culture conditions. This “humanized” situation is likely masking important differences among species. The use of “neutral backgrounds”, such as transplanting different primate cells in the mouse brain54, could mitigate this concern. Other limitations are intrinsic to the brain organoid model: lack of specific cell types, cellular stress, organized brain regions, and endogenous vascularization. The use of patterned brain organoids might help to reduce experimental variability and increase confidence in the data, especially when differences are subtle. Thus, owing to these limitations, validation or confirmation of findings in primary tissues67 or even intact functional brain68 is the gold standard in this field.Our natural interest in understanding the processes that make us human goes beyond mere anthropocentrism and philosophical debates about the human condition. Novel knowledge gained from interspecies comparisons can potentially contribute to biomedical advances. For example, humans and other primates can be distinguished by AIDS progression68. Finally, the perspective to extrapolate the comparative approach of modern humans to other extinct hominins, such as Neanderthals and Denisovans, by using human brain organoids carrying ancestral genetic variants will lead to an entire new field70.Nonetheless, it is expected that some of these technical shortcomings might be resolved in the next few years (reviewed inAGS, Aicardi–Goutières syndrome; iPSC, induced pluripotent stem cell; L1 or LINE-1, long interspersed element-1; lncRNA, long non-coding RNA; NHP, non-human primate; TREX1, three-prime repair exonuclease I


Platelet activating factor is a lipid mediator of inflammation, and in recent decades, it has emerged as an important factor in tumor outcomes. Platelet activating factor acts by specific binding to its receptor, which is present in both tumor cells and cells that infiltrate tumors. Pro-tumorigenic effects of platelet activating factor receptor in tumors includes promotion of tumor cell proliferation, production of survival signals, migration of vascular cells and formation of new vessels and stimulation of dendritic cells and macrophages suppressor phenotype. In experimental models, blocking of platelet activating factor receptor reduced tumor growth and increased animal survival. During chemotherapy and radiotherapy, tumor cells that survive treatment undergo accelerated proliferation, a phenomenon known as tumor cell repopulation. Work from our group and others showed that these treatments induce overproduction of platelet activating factor-like molecules and increase expression of its receptor in tumor cells. In this scenario, antagonists of platelet activating factor markedly reduced tumor repopulation. Here, we note that combining chemo- and radiotherapy with platelet activating factor antagonists could be a promising strategy for cancer treatment. Platelet-activating factor (PAF) was first described in the early 1970s by Benveniste et al. de novo synthesis, which is responsible for the functional regulation of plasma membranes PAF production relies on inflammatory stimuli or stress, such as that induced by radiotherapy and chemotherapy 5The receptor that binds PAF is a GPCR (G-protein coupled receptor), cloned by Sugimoto et al. 2In this review, we will first discuss the effects of PAF in tumor cells and then the PAF effects on cells from the tumor microenvironment, such as macrophages and endothelial cells. Finally, the effect of PAFR antagonists on cancer treatment and in tumor cell repopulation after radio- and chemotherapy will be addressed.The expression of PAFR is elevated in several human tumor lineages [e.g., Kaposi's sarcoma cells PAF also contributes to the malignant development of esophageal squamous cell carcinoma by stimulating PI3K/AKT activation +/CD8+ lymphocyte infiltration in the tumors implanted in PAFRKO, suggesting that PAFR may be involved in the recruitment of these cells into the tumor stroma +; high iNOS; low arginase; low IL10) than WT mice, whereas those from WT mice presented the M2 phenotype marker (CD206+). Thus, in the absence of PAFR, the tumor infiltrating macrophages acquire an activated phenotype, which is associated with the reduced tumor growth observed in PAF KO animals In solid tumors, neoplastic cells grow in the stroma, which is primarily composed of neoformed vessels, connective tissue  and several cell types recruited from the bloodstream, constituting the tumor inflammatory infiltrate (mainly macrophages and lymphocytes) The PAF/PAFR axis can also suppress the immune response by inducing tolerogenic dendritic cells 44PAFR signaling can also promote modifications in the tumor microenvironment by inducing angiogenesis through activation of matrix remodeling metalloproteases (MMPs) and stimulation of vascular endothelial growth factor (VEGF) production via NFkB activation 47These results indicate that PAFR signaling may have direct and indirect effects in promoting cancer progression and growth and that interfering with PAFR pathways may control tumor growth.Based on the prosurvival and proliferative effects of PAF in tumor cells described here, it is likely that the signaling pathways evoked by PAFR might protect tumor cells during chemo- and radiotherapy. Although PAF was shown to enhance cell death induced by chemotherapy in an epidermal carcinoma model transduced with PAFR Several years later, studies from Onuchic et al. In an ovarian cancer model, the same chemotherapeutic agent, cisplatin, led to an increase in PAFR mRNA and protein levels, which was mediated by NFkB and HIF-1α. Silencing of PAFR with siRNA or with a PAFR antagonist increased cisplatin-induced cell death in human ovarian carcinoma cells, indicating that PAF/PAFR might be involved in tumor cell survival after genotoxic stress. Using a SKOV-3-luciferase xenograft model, the authors also showed that the combined treatment with cisplatin and a PAFR antagonist (ginkgolide B) inhibited tumor progression However, it has been demonstrated in melanoma that chemotherapy can generate PAFR agonists, which might reduce chemotherapeutic efficacy in this tumor type. The chemotherapeutic agents etoposide, dacarbazine, and cisplatin generate PAF-like molecules in a time- and dose-dependent manner in B16F10 cells. Furthermore, in a model of dual injection of B16F10 cells in WT mice, intratumoral treatment with melphalan or etoposide in one tumor augmented the growth of untreated tumors in a PAFR-dependent manner Following chemo- and radiotherapy, stressed and dying cancer cells release a variety of substances, including reactive oxygen as well as nitrogen species along with cytokines, such as IL-6, IL-8, and TNF-α, which can activate PAFR agonist production in the tumor microenvironment Under these conditions, surviving tumor cells undergo cell proliferation, a phenomenon known as tumor cell repopulation. In a recent Opinion article, Ichim and Tait (2016) Several groups have demonstrated that the so-called cancer stem cells (CSCs) are responsible for tumor repopulation. Evidence from preclinical studies demonstrated that during chemotherapy, there is an increase in the frequency of a subpopulation of quiescent or CSCs that re-enter the cell cycle, contributing to tumor repopulation 632 secretion by irradiated carcinoma cells and PAFR antagonists prevented PGE2 production and COX2 expression 2 production was dependent on PAF in our studies. This finding can be explained by the fact that the PAFR pathway is often associated with arachidonic acid release Interestingly, in 2012, we demonstrated that PAF is at least partially responsible for this mitogenic effect of dying cells. In this study, we observed that the injection of a subtumorigenic dose of murine melanoma cells admixed with apoptotic cells promoted tumor engraftment and growth, and this effect was dependent on PAFR activation In fact, the COX2/PGE2 axis is involved in resistance and the stemness phenotype in many malignancies In addition, stressed and dying cancer cells can also release pro-oxidative stressors that can act directly on GPC to produce oxidized GPC (ox-GPC), which is a potent PAFR ligand 70feeder effect that significantly increased tumor cell proliferation, and this effect was markedly reduced by PAFR antagonists added to the co-cultures. In addition, when PAFR antagonists were added before irradiation, we found a significant decrease in tumor cell viability, suggesting that PAFR ligands protect tumor cells from death induced by radiotherapy + and PAFR– tumor cells admixed with irradiated tumor cells were injected subcutaneously in RAGKO mice, and tumor growth was monitored for 30 days. PAFR+ tumors became significantly larger and more vascularized than PAFR- tumors. Repopulation in PAFR+ tumors was accompanied by an increase in protumoral macrophage (CD206+) infiltration Recently, our group reported data that support the concept that the presence of PAFR ligands in the tumor microenvironment is a possible mechanism underlying tumor repopulation following chemo- and radiotherapy. Working with human and mouse cancer-derived cell lines, we first showed that tumor cells produce PAFR ligands after undergoing gamma irradiation. The irradiated cells co-cultured with live tumor cells exerted a In prostate cancer, PAFR was overexpressed in response to irradiation, and the PAFR antagonist ginkgolide B reduced cell viability. The radiosensitizing effect of this antagonist was observed in xenograft tumors, and this effect was shown to be mediated by the interaction between PAFR and beclin-1, leading to inactivation of autophagy Based on these studies, we propose that the PAF/PAFR axis may constitute an important route for acquired resistance after chemo- and radiotherapy in a direct or indirect manner, as highlighted previously Treatments based on cell death trigger homeostatic mechanisms within tissues that favor the survival of surrounding cells. Tumor cell repopulation following chemo- and radiotherapy is the major drawback of these treatments. We have discussed here the mechanisms of treatment failure focusing on the PAF/PAFR axis. During these therapies, cell stress and oxidative processes generate a wide range of oxidized phospholipids that bind to PAFR. Our group has shown that blocking PAFR with selective antagonists was very effective in reducing tumor cell repopulation following chemo- and radiotherapy. Thus, PAFR antagonists could improve the efficacy of radiotherapy through inhibition of tumor repopulation. The association of radiotherapy with PAFR antagonists could be a novel and efficient therapeutic alternative in cancer treatment.Silva Junior IA and Andrade LN wrote the manuscript. Jancar S wrote the manuscript and participated in the conception and review of the study. Chammas R. participated in the conception and review of the study.


Quiescent and self-renewing hematopoietic stem cells (HSCs) rely on glycolysis rather than on mitochondrial oxidative phosphorylation (OxPHOS) for energy production. HSC reliance on glycolysis is considered an adaptation to the hypoxic environment of the bone marrow (BM) and reflects the low energetic demands of HSCs. Metabolic rewiring from glycolysis to mitochondrial-based energy generation accompanies HSC differentiation and lineage commitment. Recent evidence, however, highlights that alterations in mitochondrial metabolism and activity are not simply passive consequences but active drivers of HSC fate decisions. Modulation of mitochondrial activity and metabolism is therefore critical for maintaining the self-renewal potential of primitive HSCs and might be beneficial for ex vivo expansion of transplantable HSCs. In this review, we emphasize recent advances in the emerging role of mitochondria in hematopoiesis, cellular reprograming, and HSC fate decisions. Hematopoiesis is a complex process that allows sustained production of each of the blood cell lineages throughout the lifespan of an individual. Vast numbers of adult mature blood cells are constantly generated from hematopoietic stem cells (HSCs) through a series of lineage-committed progenitor cells . HSCs reMitochondria are very complex and highly dynamic organelles. They are the major source of adenosine-5′-triphosphate (ATP) production through oxidative phosphorylation and sustained electron transport chain (ETC) activity. Mitochondrial OxPHOS is fueled by the tricarboxylic acid (TCA) cycle that converts pyruvate to acetyl-CoA. In addition, mitochondria serve as biosynthetic and signaling organelles . The intQuiescent HSCs predominantly reside in regions of the bone marrow (BM) cavity termed niches, which provide a unique landscape with a low oxygen tension , 17. As α (HIF1α). In turn, HIF1α drives and regulates a metabolic program that limits the engagement of the TCA cycle and sustains glycolysis as a main source of energy [HSCs display unique properties and functions that distinguish them from more committed progenitors and mature blood cells. HSCs are predominantly quiescent, and their metabolic wiring and reliance on glycolysis are distinct from those of committed progenitors and the other cells in the BM that encompass primarily lineage-differentiated cells , 5, 27. f energy .Enhanced mitochondrial activity is detrimental to the functional identity of HSCs and maintenance of their numbers. Loss of mitochondrial carrier homolog 2 (MTCH2) enhances OxPHOS activity and intracellular ROS levels, triggering the entry of HSCs into the cell cycle and loss of their self-renewal potential . By contSdhD gene, which encodes for one of the subunits of the mitochondrial complex II, is characterized by impaired survival of both HSCs and progenitors belonging to different lineages [In spite of the high preference for glycolysis, mitochondria in HSCs are not completely inactive. In fact, HSCs residing in the BM depend on mitochondrial activity and metabolism for their differentiation and survival. Suppression of OxPHOS activity in HSCs upon the loss of Ptpmt1, a PTEN-like mitochondrial phosphatase, impairs the early differentiation of HSCs and results in defective hematopoiesis . Moreovelineages . Of intelineages . Whereaslineages . CollectWhile the metabolic switch from glycolysis to mitochondrial OxPHOS activity and metabolism is required to meet the robust energy and metabolic demands imposed by differentiation, the precise mechanism underlying this switch remains elusive. It is likely that this metabolic rewiring is more complex than a simple switch from one form of energy production to another one. Instead, it might be the result of a series of events that occur before the onset of the well-known “metabolic switch.” Importantly, these events might engage mechanisms that not only impact but also act in concert with this “metabolic switch” to coordinate and control the balance between HSC self-renewal and differentiation.The role of mitochondria in HSC fate decisions and function is not merely limited to the metabolic switch but involves concomitant alterations of the mitochondrial features and properties. Distinctive mitochondrial membrane potential and mass between HSCs and cells at different stages of hematopoiesis have been reported. Indeed, both low mitochondrial activity and membrane potential mark the self-renewing murine and human HSCs , 33, 34 +cells (UCB-CD34+) [+ cells enriched in cells with long-term repopulating potential exhibits low levels of both mitochondrial mass and membrane potential as opposed to more differentiated cells [+ cells into functional HSCs and acquisition of the CD90 phenotype triggered by treatment with a histone deacetylase inhibitor, valproic acid (VPA), are accompanied by a significant decline in mitochondrial mass and DNA content [The role and extent of mitochondrial membrane potential and mass on murine HSC self-renewal and commitment are similar to those observed during ex vivo studies performed with human umbilical cord blood-derived CD34B-CD34+) . The subed cells . Interesed cells . Unlike  content , 38. Imp content  , which is currently emerging as one of the main mitochondrial quality control mechanisms for HSC self-renewal. Interestingly, one of the key elements of the UPRmt, SIRT7, protects the HSC pool challenged by stress by suppressing mitochondrial biogenesis [It appears that the activation of autophagy is not balanced by enhanced mitochondrial biogenesis. In support of this notion is the evidence that mTOR pathway inhibition contributes to HSC quiescence not only by promoting autophagy but also by repressing mitochondrial biogenesis , 43–47. ogenesis . This evHSC fate decision with regard to self-renewal or commitment is monitored and regulated by ROS, a by-product of the bioenergetic metabolism. Although critical for physiological processes including activation of signal transduction pathways and fighting pathogens, excessive ROS can impair cellular functions by causing oxidative damage to lipids, proteins, RNA, and DNA. While the mitochondria are the major sources of ROS generation, they are also the main targets of ROS leading to vicious cycles of mitochondrial damage and energetic catastrophe. As discussed above, such damage and a complete failure in mitochondrial activity eventually lead to HSC exhaustion and impaired differentiation. Excessive ROS contribute to HSC aging and senescence, and at even higher levels, ROS induce HSC cell death .Although evidence points towards the need for a tight control of ROS levels to prevent tissue damage and cell death, it is becoming clear that ROS might function as a rheostat that regulates cell fate decisions. At low levels, ROS maintain quiescence and the long-term repopulating capacity of HSCs . At modeα and HIF2α [α and HIF2α in HSC function [κB), mTOR, and epigenetic regulators such as histone deacetylases (HDACs), are tightly interconnected. Together, they control, in a coordinated manner, the equilibrium between quiescence, active cycling, and differentiation of HSCs [Regulation of ROS levels in HSCs is highly complex and involves regulation of both HSC metabolic activity and their antioxidant defense mechanisms , 58, 59.nd HIF2α , 58, 59,nd HIF2α , 46, 60.function , 62 has function , 64. In  of HSCs , 65, 66. of HSCs . SIRT1 c of HSCs –74. It i of HSCs , 76. In  of HSCs . Notably of HSCs , 79.2+-mediated mitochondrial functions contributes to the maintenance of self-renewing murine HSCs during cell divisions through upregulation of p53-related genes [+cells with VPA treatment [The orchestration of the redox status of HSCs is monitored by the antioxidant defense mechanism, which relies on the activity of the scavenger antioxidant enzymes such as MnSOD. Sirtuin family proteins, primarily SIRT3, are required to retain the regenerative capacity of aged HSCs and to limit ROS production by enhancing MnSOD activity . The loned genes . At the reatment . To prevreatment . Consistreatment . TogetheMitochondrial bioenergetics and structure are tightly linked. Mitochondrial dynamics, including the modulation in mitochondrial ultrastructure, has been suggested to play a fundamental role in mitochondrial metabolism and, therefore, in determining stem cell fate , 86. Eme+, is also required for the maintenance of the lymphoid potential of HSCs, suggesting that mitochondrial dynamics influences HSC fate through numerous mechanisms [Mitochondrial dynamics and morphology are orchestrated by the mitochondrial fission/fusion machinery that relies on the activity of shaping proteins such as optic atrophy (OPA1), mitofusin-1 and mitofusin-2 (Mfn1 and Mfn2), and dynamin-related protein 1 (Drp1). Indeed, the shape of mitochondria is continuously defined by antagonistic and balanced activities of fusion and fission proteins . Aberratchanisms .+ cells is accompanied by a profound remodeling of the mitochondrial network comprised of morphologically small and globular mitochondria [The current understanding of mitochondrial architecture in HSCs and the role of fission/fusion in retaining of HSC self-renewal potential is very limited. A better understanding has been achieved by studies performed during reprograming of somatic cells into induced pluripotent stem cells (iPSs). During reprograming to iPCs, mitochondria undergo significant remodeling accompanied by an early wave of mitochondrial fragmentation due to increased fission and Drp1 activity . In factchondria . This ceHSC reliance on glycolysis has been perceived as an adaptation to the hypoxic niche of the BM until now. Current evidence emphasized in this review, however, suggests that hypoxia might not be the only cause of limited mitochondrial metabolism in HSCs. In fact, numerous studies highlight the plasticity of mitochondria and their profound role in controlling the self-renewal and maintenance of HSCs. Important cues regarding the mechanisms and roles of mitochondria as drivers of HSC fate might be applied to improve efforts aimed at manipulation and ex vivo expansion of HSCs from UCBs. Ex vivo-expanded HSCs have potential therapeutic benefits in regenerative medicine to be used as allogeneic grafts for transplantation and/or gene therapy for monogenetic inherited blood disorders.The use of UCBs for bone marrow transplantation is restricted due to the limited number of HSCs within a single unit. Several ex vivo strategies including the aryl hydrocarbon receptor antagonist (SR-1), pyrimidoindole derivative (UM-171), and VPA are currently utilized to overcome this limitation and expand to a great degree the numbers of transplantable HSCs , 99, 100Ex vivo HSC cultures have been reported to induce stress. Such stress can impact mitochondrial function and activity, compromising therefore the characteristics of primary and clinically relevant HSCs. Overcoming this stress by limiting mitochondrial potential and activity presents an exciting target by which to expand the numbers of cycling HSCs while they retain their self-renewal and primitive characteristics. Remarkably, VPA treatment in ex vivo cultures has been reported to trigger both the acquisition and retention of a transcriptome and primitive mitochondrial profile with low activity, characteristic of primary functional HSCs , 38. Bot


Intensive research on sperm ion channels has identified members of several ion channel families in both mouse and human sperm. Gene knock-out studies have unequivocally demonstrated the importance of the calcium and potassium conductances in sperm for fertility. In both species, the calcium current is carried by the highly complex cation channel of sperm (CatSper). In mouse sperm, the potassium current has been conclusively shown to be carried by a channel consisting of the pore forming subunit SLO3 and auxiliary subunit leucine-rich repeat-containing 52 (LRRC52). However, in human sperm it is controversial whether the pore forming subunit of the channel is composed of SLO3 and/or SLO1. Deciphering the role of the proton-specific Hv1 channel is more challenging as it is only expressed in human sperm. However, definitive evidence for a role in, and importance for, human fertility can only be determined through studies using clinical samples.This review aims to provide insight into the role of sperm ion channels in human fertilization as evidenced from recent studies of sperm from infertile men. We also summarize the key discoveries from mouse ion channel knock-out models and contrast the properties of mouse and human CatSper and potassium currents. We detail the evidence for, and consequences of, defective ion channels in human sperm and discuss hypotheses to explain how defects arise and why affected sperm have impaired fertilization potential.Relevant studies were identified using PubMed and were limited to ion channels that have been characterized in mouse and human sperm. Additional notable examples from other species are included as appropriate.in vivo and CatSper-null sperm also fail to fertilize at IVF. Sperm-lacking potassium currents are capable of fertilizing at IVF, albeit at a much lower rate. However, additional complex and heterogeneous ion channel dysfunction has been reported in sperm from infertile men, the causes of which are unknown. Similarly, the nature of the functional impairment of affected patient sperm remains elusive. There are no reports of studies of Hv1 in human sperm from infertile men.There are now well-documented fundamental differences between the properties of CatSper and potassium channel currents in mouse and human sperm. However, in both species, sperm lacking either channel cannot fertilize Recent studies using sperm from infertile men have given new insight and critical evidence supporting the supposition that calcium and potassium conductances are essential for human fertility. However, it should be highlighted that many fundamental questions remain regarding the nature of molecular and functional defects in sperm with dysfunctional ion channels. The development and application of advanced technologies remains a necessity to progress basic and clinical research in this area, with the aim of providing effective screening methodologies to identify and develop treatments for affected men in order to help prevent failed ART cycles. Conversely, development of drugs that block calcium and/or potassium conductances in sperm is a plausible strategy for producing sperm-specific contraceptives. A fundamental reason for this is a lack of understanding of the cellular and molecular control of the physiological functions of spermatozoa  nor its regulation by P4 in human sperm could be further characterized due to technical challenges of applying patch-clamp electrophysiology to whole sperm. Initial attempts at characterization of channels in boar, sea urchin and mouse sperm relied on fusion of the cell membrane to planar bilayers  impossible. A significant reason for this is that CatSper is a highly complex multi-subunit ion channel made up of at least 10 subunits  suggests the potential for significantly divergent function and/or regulation. Indeed, the weight of evidence now suggests that mouse CatSper is not a reliable surrogate for studying channel function in human sperm , also localized to the flagellum. However, although the current characteristics resembled those of mouse ICatSper, human ICatSper is strongly potentiated by P4 and PGE1. Comparison of the characteristics of P4-induced Ca2+ influx and potentiation of ICatSper confirmed that the action of P4 on human sperm is mediated by activation of CatSper  and even then only ~40% of sperm respond  present in the principal piece  [Ca2+]i influx  that is proposed to contribute to generation of Cai influx , though xcursion . Howeverxcursion  is much xcursion . A possiCatSper is blocked by a bewildering array of chemically unrelated compounds . Semen parameters were again consistent with asthenoteratozoospermia (2+]i response to screen sperm from patients undergoing ART (either on the day of treatment or following failed/low fertilization) followed by patch clamp analysis. Using this approach, two cases were observed where sperm failed completely to respond to P4, and IVF was simultaneously affected by failed fertilization  P4 elicited a normal increase in [Ca2+]i and fertilization was achieved. In stark contrast to mouse CatSper-null sperm, there were no gross abnormalities in sperm motility in samples that were refractory to P4 although in these cases P4 did not stimulate penetration into viscous media, thus demonstrating physiological impairment. Hyperactivation induced by pharmacological mobilization of Ca2+ from intracellular stores was normal in sperm cell samples from both patients  an episodic loss of the P4-induced i to a level sufficient to potentiate IKSper. Reciprocally, activation of IKSper hyperpolarizes Vm that may reduce CatSper opening, suggesting a connection between activity of CatSper and regulation of membrane potential , which was caused either by negligible IKSper or enhanced inward conductance , samples where sperm exhibited these abnormalities were significantly more likely to achieve ‘poor’ levels of fertilization (<25% fertilization in cycles with four or more metaphase II oocytes) than those with a more negative membrane potential. Exome analysis of one patient (patient D) whose cells had a stable lesion in outward conductance, did not reveal any causative mutations. This study reveals that abnormalities of K+ conductance and/or Vm regulation are both common and complex, and that fertilization potential of affected cells is severely diminished  are elevated in capacitated cells and are potentiated by the endocannabinoid anandamide but blocked by Zn2+. The robust currents seen in human sperm contrast sharply with the extremely limited ability of mouse sperm to conduct protons. In human sperm, activation of Hv1 is proposed to facilitate capacitation through H+ efflux, which elevates pHi and activates CatSper conductance. Therefore, functionally, Hv1 activity is permissive for development of hyperactivated motility, enhanced flagellar rotation and the acrosome reaction  has been recorded using patch clamp electrophysiology in human sperm. This current has properties that closely resemble heterologously expressed TMEM16A CaCC. Inhibition of CaCC effectively reduces the acrosome reaction induced by solubilized recombinant zona pellucida 3 (ZP3) protein  is a protein kinase A (PKA)-activated anion channel that is localized to the equatorial segment of the sperm head in human and mouse  induces the acrosome reaction in human sperm, although does not affect motility  (see 2+ stores). Several studies provide evidence for CCE in human sperm  channels form a large family of Ca CatSper . CCE invtivation . Howeverv 1.2, 1.4-1.9) are also proposed to have a functional role in human sperm. They are immunolocalized to different regions of human sperm, and pharmacological activation has a positive effect on motility  of these other channels in sperm function and fertilization and the incidence and consequences of their malfunction.To date, reports of definitive abnormalities of patient sperm ion channels are limited to those affecting IIon channel expression requires the successful co-ordination of a complex series of events involving transcriptional regulation, post-translational quality control, assembly, trafficking and insertion into the plasma membrane. Sequestration is governed by internalization, recycling and degradation processes. Therefore, disruption of the regulatory molecules involved in these events can culminate in defective channel expression  and undetectable in sperm. In contrast, LRRC52−/− animals express SLO3 in sperm  were imposed on the cells  current (+ conductance through CatSper and/or K+ channel (both of which are permeable to Na+), or even activity of TRPV4, newly identified as the depolarizing current DSper  of flagellar proteins that is necessary for development of hyperactivated motility. The P-Tyr levels in mutant sperm were unregulated, resulting in elevated phosphorylation of a wide range of proteins, including 11 structural proteins , potentially providing molecular insight into the impaired motility of CatSper null sperm. In wild-type cells the organization of the CatSper domain was intact in cells exhibiting hyperactivated motility. However, a remarkably heterogeneous pattern of channel subunit and regulatory molecule delocalization was observed in non-hyperactivated cells during capacitation, to the extent that these domains became degraded in some cells. Therefore, the role and regulation of endocytosis of ion channels and the sperm proteasome on channel expression merit close examination. In both Catsper zeta and Efcab9-null mouse sperm, which exhibit lower channel protein expression and consequently lower ICatSper, the overall quadrilateral banding is maintained but the linear domains become disrupted  intracellular pH, the complex limits channel activity. However, alkalinisation weakens their interaction to permit calcium ion influx that can bind to Ca2+-binding domains of EFCAB9. This further weakens its interaction with CATSPER ZETA to maximize CatSper conductance  had undergone a vasovasostomy. While this observation could be coincidental, particularly given the pregnancy rate achieved following vasectomy reversal  other ion channels. Similarly, effects on downstream protein phosphorylation could potentially affect a multitude of interconnected signalling pathways that determine sperm fertilizing capability. Defects may manifest as impairment(s) of motility, reduced or absent binding/interaction with the female tract and the cumulus-oocyte complex or failed acrosome reaction , a CatSper-dependent response in mouse sperm that is believed to direct swimming up the oviduct, against the flow of oviductal fluid  the concentration of P4 and consequent effects on ICatSper and IKSper null sperm was within the normal range (assessed by CASA). However, whereas IKSper null sperm were stimulated by P4 to penetrate viscous medium (≥100% increase compared to control), stimulation of ICatSper null sperm with P4 had no effect  in both ICatSper and IKSper can cause male subfertility. However, identification of causal defects (genetic and non-genetic) and the precise molecular and functional impairments that arise as a consequence, remains a significant but exciting challenge. Development of new technologies is critical, both to illuminate our understanding as well as facilitating progress towards the development of routine diagnostic tests and individualized medicine to advance patient treatment.Patch clamp electrophysiology has revolutionized our understanding of ion channel function in both the healthy and dysfunctional spermatozoon. Indeed, from our studies of sperm samples from patients, we hypothesize that loss of Iin vitro techniques to enable malfunctioning gametes to fertilize the oocyte. A primary aim of research in reproductive medicine should be, where possible, to restore normal gamete function so that these selection mechanisms can operate and fertilization can occur naturally . This is a major challenge, not least because of our dearth of current understanding (see, for example, Reproductive physiology is shaped by selective processes. During natural fertilization, the male and female tracts assist in preparing (CatSper and IKSper may make effective contraceptives. The sperm-specific nature of ICatSper and IKSper (see However, comprehension of human spermatogenesis, sperm behaviours and fertilization at a molecular level will advance both causes. Here we have argued that understanding the expression and functioning of human sperm ion channels is necessary for the development of improved therapies for male infertility but equally recent studies show that compounds that inhibit Iin vivo. Recent investigations have used transgenic mouse sperm that express fluorescent markers to permit live imaging in/ex vivo (in vivo fertilization in humans is currently beyond our imagination but argue that sperm plasma membrane ion channels will play a central role.It is clearly critical to observe and understand the mysteries of sperm /ex vivo  by concl


Giardia strains and how these contribute to the different clinical outcomes of infection are unknown. In addition, full characterization of their interaction with host cells and mechanisms of secretion from the parasite remain lacking.Infectious diseases are better understood when virulence factors associated with host interactions are identified and characterized. In giardiasis, several molecules associated to virulence have been identified –3. TheseGiardia trophozoites with intestinal epithelial cells in vitro. The first study, from the Svärd group, was published in PLOS Neglected Tropical Diseases [Giardia strains, GS (Assemblage B) and WB (Assemblage A), commonly used in studies of the parasite and prototypes for the two assemblages most often found in human infections. The number of identified proteins released by WB was greater than those released by GS, either when analyzing axenic cultures or in coculture with human intestinal CaCo2 cells.Two recent studies provide a valuable characterization of host–parasite interactions in giardiasis, delineating the parasite secretome and its interaction with intestinal cells. These studies both use proteomic analysis of proteins secreted by the parasite to assess the interactome of Diseases  and idenThe second study, from Dubourg and colleagues, also used proteomics to investigate the secretome of the same two strains, WB and GS . This grThe real value of these studies is the comprehensive analysis of the types of proteins secreted, their relative amounts, and their impacts on host cells. The two most abundant proteins secreted into the medium after interaction with intestinal cells, for both GS and WB, were arginine deiminase (ADI) and ornithine carbamoyltransferase (OCT). These enzymes cooperate in catabolizing arginine and producing ATP in the parasite cytosol. ADI has also been suggested to deplete arginine and limit nitric oxide production in the host as a mechanism of immune evasion , 12 as wGiardia. Several abundant molecules in the parasite secretome, e.g., ADI, EF-1α, and enolase, lack secretion signal peptides. Ma’ayeh and colleagues identified two types of vesicles ranging in size from 100 to 250 nm in the supernatant of parasite cultures, confirming a recent report of microvesicle release from the parasite [Ma’ayeh and colleagues also highlight the importance of the nonconventional secretory pathways present in parasite . HoweverGiardia protease from the WB strain (named by the authors as giardiapain-1) can induce apoptosis in rat intestinal epithelial cells (IEC-6) and Madin Darby Canine Kidney (MDCK) epithelial cells and degrade proteins at tight junctions. The same group has shown that WB trophozoites constitutively expressing a variant-specific surface protein that has cysteine protease activity (VSP9B10A) induced damage in IEC-6 or MDCK cell monolayers. Similarly, Buret’s group showed in recent studies that proteases from the Assemblage A parasite strain NF can induce cleavage of the cytoskeletal protein villin in CaCo2 cells [Giardia impacts intestinal tissue in vivo, whether only some or all the proteases secreted by Giardia contribute to the symptomatology of the disease and whether interstrain variation in any of these virulence factors contributes to the different clinical outcomes observed in patients.The work from Ma’ayeh, along with other recent studies, suggests that several candidate virulence factors whose distribution between symptomatic and subclinical infections might be highly informative. In addition to ADI and OCT, Ma’ayeh and colleagues report that both GS and WB release 13 different cathepsin B proteases, and Ortega-Pierres and colleagues  have reco2 cells  and degro2 cells . Proteaso2 cells , 8. FurtGiardia secretome studies mentioned above lay the groundwork for new investigations crucial to understanding pathogenesis in Giardia. Numerous studies have examined the association between parasite assemblage and virulence in humans. However, the genotyping approaches used to define assemblages rely on sequences of housekeeping genes, usually 18S rRNA, β-giardin, glutamate dehydrogenase, and/or triosephosphate isomerase. What is lacking in the literature is how virulence factors are distributed among parasite assemblages and individual isolates. Both GS and WB were isolated from patients with symptomatic infections, and GS has been shown to induce symptoms in human volunteers [Giardia has been possible for 20 years, although strategies for knocking down or knocking out genes in this parasite are much more difficult. Host responses play a pivotal role in pathogenesis in giardiasis as well, as hypermotility and sucrase deficiency are both reduced in immunodeficient animals, and studies of the intersection between parasite virulence factors and host immune response would also be of interest. Finally, the small intestine is home to a robust microbiota, and several studies suggest that interactions among Giardia, the host, and these additional microbes all contribute to the eventual outcome of Giardia infection [The lunteers . There infection , 20.


Treatment of locally advanced squamous cell carcinomas of the head and neck (SCCHN) remains unsatisfactory. Although the addition of concurrent radiochemotherapy (RCT) or the combination of radiotherapy with blockade of the epidermal growth factor receptor (EGFR) have improved outcomes over radiotherapy alone, further optimization is urgently needed. The introduction of immune checkpoint inhibitors is currently revolutionizing cancer treatment. Clinical evidence has recently been provided in melanoma that immune checkpoint blockade may cooperate with radiation. Therefore, we searched in the literature for the evidence of combining immune checkpoint inhibitors with radiotherapy in primary treatment of SCCHN.A substantial amount of previous studies has dissected the molecular mechanisms of immune evasion in SCCHN. The biological effects of radio- and chemotherapy in tumor cells and the immune cell microenvironment were characterized in detail, revealing significant interference of both types of treatment with anti-tumor immunity. This extensive review of the literature revealed considerable amount of evidence that addition of immune checkpoint inhibitors might boost the immunomodulatory potential of radiotherapy and RCT regimens in SCCHN.Promising activity of immune checkpoint inhibitors has already been reported for metastatic/recurrent SCCHN. Given the immunogenic effect of radiotherapy and its enhancement by chemotherapy, combination of radiotherapy or RCT with this new type of immunotherapy might represent a valuable option for improvement of curative treatment modalities in SCCHN. Patients with SCCHN completing radiotherapy-based treatment remain at considerable risk for local relapse within the radiation field, regional recurrence in the neck and hematogenous spread of tumor cells with the potential to form distant metastases. Primary radiochemotherapy (RCT) applied concurrently with cisplatin still cures less than 40 % of patients , and in During the last decade, there has been increasing interest in combining RCT with molecularly targeted agents. Most targeted approaches for radiosensitization tested so far have been directed against molecular pathways within cancer cells in order to increase the magnitude of DNA damage or to inhibit cellular mechanisms which interfere with tumor cell DNA repair, thereby increasing the efficacy of RCT. Based on the overexpression of EGFR in the majority of SCCHN cases and its causative role in radioresistance , 5 the EIn principle, the defense by the immune system against pathogenic microbes and toxins from the environment is divided into two general types of processes: the innate immunity and the adaptive immunity. Innate immunity recognizes and fights microbial invaders at the site of infection. In contrast, adaptive immunity is serving to eliminate host cells infected with viruses by recognizing peptides from intracellular viral proteins loaded onto major histocompatibility complex (MHC) molecules and displayed on the host cell surface. The adaptive immune system is also able to recognize mutated proteins in tumor cells via the same mechanism.There are multiple mechanisms by which a tumor cell harboring immunogenic mutations can elicit adaptive immune responses, as schematically summarized in Fig. There are several ways for SCCHN to evade recognition by the adaptive immune system, as schematically illustrated in Fig. A number of both genetic and environmental mechanisms allow such immune escape and have been described in SCCHN (for a recent detailed review see ), includIonizing radiation has been used for more than a century to treat cancer , on the Mechanistically, radiotherapy has been shown to augment the afferent as well as efferent arms of cancer immunity. The induction of a specific T cell response to tumor cells (afferent immunity) has been observed in multiple studies. Almost 20 years ago molecular pathways were first described that were activated by treatment-induced cell stress (in particular after treatment with anthracyclins and ionizing radiation) and which induced a modality of cell death that was highly efficient in eliciting immune responses . The immRadiotherapy can also influence the efficacy of tumor cell destruction (efferent immunity) within the radiation field by altering tumor cell characteristics or the tissue microenvironment. Tumor cells in which the damage from radiation has not been sufficient to induce cell death show increased expression of MHC class-I antigen-presenting molecules  and adheIt has been known for some time that irradiated tissues often show very strong changes in the local cytokine milieu. As a result of their action, a cascade of pro-inflammatory processes are triggered. Secretion of interferon-γ enhances expression of MHC class-I by cancer cells, sustaining and extending the initial effects of radiation to allow efficient recognition and killing by T cells , 34. In Immune effects within the radiation field, however, are not sufficient for cure, as effective treatment of a high-risk primary cancer has to secure not only local but also systemic control of the disease. In principle, the nature of the adaptive immune system should be mechanistically well suited for systemic tumor surveillance. There is emerging evidence that radiotherapy can also be associated with immune destruction of distant metastases, pointing towards efferent immunity outside of the radiotherapy field. This phenomenon termed abscopal anti-tumor effect was already described in 1953 . ClinicaThe knowledge gained from mechanistic studies on the immunomodulatory effects of radiation mentioned above has changed the way the response to radiotherapy with/without chemotherapy in patients with cancer is now interpreted, by acknowledging the essential role of the host immune system for the success of radiotherapy. Importantly, these indirect effects of radiation – within and outside the field of treatment – also suggest new treatment possibilities, including combinations with established or novel forms of immunotherapy.de-novo immunity but release the effector phase of immunity  were the first three immune checkpoint inhibitors which received FDA approval for the treatment of metastatic melanoma. A three-arm phase III trial in melanoma  answeredxicities .There are at least eight anti-PD-1/PD-L1 antibodies currently in clinical development, covering phases I to III. In addition, the preclinical and early clinical development of inhibitors against other immune checkpoints, such as T cell immunoglobulin mucin receptor 3 (TIM3) and lymphocyte activation gene 3 protein (LAG3), and against co-stimulatory molecules, such as OX40 and CD137, are underway. Final results from several successful phase III trials with ipilimumab, nivolumab and pembrolizumab improving overall survival of metastatic cancer have been reported in melanoma and lung cancer, and it can be expected from the data available for a broad range of other histologies that this novel class of agents will be firmly established in modern treatment of many cancers.In recurrent/metastatic SCCHN, several PD-1/PD-L1 blocking agents are currently being investigated, with most mature information on nivolumab and pembrolizumab. The phase 1b multicohort trial Keynote-012 tested the efficacy of the anti-PD-1 antibody pembrolizumab for treatment of PD-L1+ in recurrent/metastatic SCCHN . A best Early evidence of clinical activity in SCCHN were also reported from multi-arm expansion studies of anti-PD-L1 antibodies  receiving cisplatin-based RCT . In addiIn locally advanced SCCHN the magnitude of immune suppression could also be linked to the efficacy of RCT, however, a direct role of immune checkpoint proteins remains to be established. Low numbers of tumor-infiltrating cytotoxic CD8+ T cells before treatment were significantly correlated with poor outcome after RCT in several studies –53 but tPrecise biomarkers to identify patients who benefit from immune checkpoint inhibition alone or in combination with RCT still have to be established. Current data in multiple cancers reveal that verification of PD-L1 overexpression by immunohistochemistry is associated with improved clinical outcome of anti-PD-1 therapy. However, the presence of robust responses in some patients with low or undetectable expression of PD-L1 complicates the issue of PD-L1 as an exclusive predictive biomarker . In the While there is much excitement around the phenomenon of a radiation-induced anticancer immune response and combining radio(chemo)therapy with immunotherapy, numerous questions remain to be addressed in clinical trials. A major challenge is to identify not only the optimal immune checkpoint inhibitor as partner for a given radiotherapy schedule but also the best chronological sequence for their combined application. Preclinical evidence can serve as guidance in treatment schedule and clinical trial development. As outlined above, danger signals induced by radiation lead to the recruitment of immune cells into the tumor. However, cells of the immune system are also vulnerable to radiation, as their exposure to ionizing radiation induces apoptosis in mature natural killer (NK) cells as well as T and B cells. Since radiotherapy is generally delivered in daily fractions, re-irradiation of the tumor might therefore damage infiltrating immune cells that display cytolytic activity themselves or might significantly reduce the capacity of DCs to activate effector T cells. In addition, DCs may find a hostile environment for T cell activation in draining lymph nodes, which represent their natural surrounding for interaction with T cells, as draining lymph nodes are systematically included into the radiation field in SCCHN. Conversely, however, the induction of immunogenic cell death (ICD) by each daily radiotherapy fraction might transiently generate a favorable milieu for immune activation within the tumor tissue, which may vanish again after termination of radiotherapy. In support of the latter, concurrent application of an anti-PD-L1 antibody together with fractionated radiotherapy significantly improved tumor control in a xenograft model . In contBesides the optimal time schedule also the optimal type and dosing of chemotherapy has to be established, if desired to be included into the treatment regimen. Significant differences in the ability of chemotherapeutic drugs to induce ICD have been reported previously . CisplatTaxanes including docetaxel and paclitaxel which are also common combination partners of radiotherapy in locally advanced SCCHN are known to modulate antitumor immune responses as well . SimilarIntriguingly, investigations on the immunogenic effects of chemotherapeutic drugs revealed also their direct interference with immune checkpoint expression. Treatment of DCs in vitro with platinum-based compounds strongly enhanced their potential to activate T cells which was caused by downregulation of PD-L2 in DCs . This efOverall, these preclinical observations provide a sound rationale for investigating immune checkpoint inhibitors with radiotherapy alone as well as in combination with standard cisplatin-based as well as taxane-based RCT. In addition, there is accumulating data that the efficacy of cetuximab-based regimens in treatment of recurrent/metastatic SCCHN is not only based on the inhibition of EGFR signaling pathways but also on the activation of Fcγ receptor-positive NK cells leading to DC maturation and activation of cytotoxic T cells . A combiThe toxicity of radiotherapy is mostly occurring directly at the irradiation site. Mucositis, xerostomia and swallowing dysfunctions are common side effects in radiotherapy of head and neck cancers. Clearly, the extent of early and late toxicity is dependent on the radiation technique and the applied dose: Intensity-modulated radiotherapy, which conforms closely to the tumor volume, avoids or minimizes exposure to unaffected tissue and thereby significantly reduces local side effects of irradiation . On the The introduction of immune checkpoint inhibitors into cancer treatment has been celebrated as the breakthrough of the year 2013. Impressive activity was proven in metastatic melanoma and lung cancer, and promising results were presented for recurrent/metastatic SCCHN. Given the immunogenic effect of radiotherapy and its enhancement by chemotherapy or cetuximab, it remains to be determined whether immune checkpoint inhibitors could further increase the activation of adaptive immunity and ultimately improve overall current cure rates of locally advanced SCCHN.CTLA-4, cytotoxic T-lymphocyte protein 4; DCs: dendritic cells; DNA, deoxyribonucleic acid; EGFR, epidermal growth factor receptor; HMGB1, high-mobility group box 1; ICD, immunogenic cell death; IDO, indoleamine 2:3-dioxygenase; LAG3, lymphocyte activation gene 3 protein; MHC, major histocompatibility complex; NK, natural killer; NSCLC, non-small cell lung cancer; PD-1, programmed cell death receptor 1; PD-L1, programmed cell death 1 ligand 1; RCT, radiochemotherapy; SCCHN, squamous cell carcinoma of the head and neck; TCR, T cell receptor; TIM3, T cell immunoglobulin mucin receptor 3; Treg, regulatory T cells