Fetal alcohol spectrum disorder (FASD) encompasses the range of deleterious outcomes of prenatal alcohol exposure (PAE) in the affected offspring, including developmental delay, intellectual disability, attention deficits, and conduct disorders. Several factors contribute to the risk for and severity of FASD, including the timing, dose, and duration of PAE and maternal factors such as age and nutrition. Although poorly understood, genetic factors also contribute to the expression of FASD, with studies in both humans and animal models revealing genetic influences on susceptibility. In this article, we review the literature related to the genetics of FASD in humans, including twin studies, candidate gene studies in different populations, and genetic testing identifying copy number variants. Overall, these studies suggest different genetic factors, both in the mother and in the offspring, influence the phenotypic outcomes of PAE. While further work is needed, understanding how genetic factors influence FASD will provide insight into the mechanisms contributing to alcohol teratogenicity and FASD risk and ultimately may lead to means for early detection and intervention. Prenatal alcohol exposure (PAE) is linked to miscarriage, stillbirth, preterm birth, and sudden infant death syndrome  and is cAlthough public health guidelines continue to discourage alcohol use during pregnancy, PAE is the leading cause of preventable developmental disability worldwide. The estimated incidence of FASD is high both in the United States (10 per 1000 children ) and worDespite the high prevalence of FASD, which is likely underestimated, most children exposed to alcohol prenatally do not develop FASD . StudiesWhile FASD essentially is contingent on exposure, genotypes of both mother and offspring appear to also influence FASD and the severity of alcohol teratogenicity, shifting reaction range to exposure. The role of genetic influences in FASD is known from preclinical studies, with different strains of mice, rats, chickens, and fish having been shown to have different susceptibilities to PAE effects . For exaSome of the most compelling evidence for genetic influences on FASD susceptibility and severity is derived from twin studies, small and few that these studies have been. Early case studies in dizygotic (DZ) twins born to mothers with moderate to high alcohol use during pregnancy led to the knowledge that one twin may often be more affected than the other ,22,23,24A more recent, although similarly small, study from the Fetal Alcohol Syndrome Diagnostic and Prevention Network also supports high heritability for FASD . In addiTwin studies and studies of other pairs of individuals at differing degrees of relationship can be confounded by ascertainment bias, with pairs of children with FASD perhaps being more likely to be recognized. However, as far as is known, this is not the case for the heritability of FASD, leaving the conclusion that FASD is highly heritable, although more influenced by environmental factors in different aspects that lie within the spectrum of FASD. Discordant diagnosis in DZ twins also provides evidence for the significance of offspring genotype in PAE vulnerability rather than the maternal genotype; however, as discussed in the next sections, maternal genotype can also be a significant factor in FASD vulnerability. While these studies have provided evidence for a genetic contribution to FASD, only two studies thus far have included both MZ and DZ twins, with only 5 and 9 MZ twin pairs and only 11 and 39 DZ twin pairs. Thus, further studies and larger cohorts are needed to validate these findings, particularly including more MZ twins.ADH1B gene are Arg48His  and Arg370Cys  [ADH1B His48 and ADH1B Cys370 metabolizing ethanol to acetaldehyde more than 70–80 times faster [ADH1B His48 has an allele frequency greater than 60% in Chinese, Japanese, and Korean populations [ADH1B Cys370 has an allele frequency of approximately 22–25% in African Americans [ADH1B Arg48 and Arg370 (ADH1B*1) are predominant in European white individuals. In addition to these common ADH variants, the Lys504 allele of the Glu504Lys ALDH2 polymorphism encodes a low-activity variant of the enzyme and is present in up to half of people in certain East Asian populations [ADH1B His48, ALDH2 Lys504, or both are less likely to develop AUD because of the flushing reaction caused by the acetaldehyde-induced release of histamine. Paradoxically, these individuals are also more vulnerable to the toxic effects of alcohol, including DNA damage and cancer, because many individuals with the Asian flush do consume at least moderate amounts of alcohol [As has long been recognized and more recently confirmed in genome-wide association studies (GWASs) , genetics faster ,31. Thesulations , and ADHmericans . On the ulations . People  alcohol . This vuADH genotype in FASD vulnerability is dependent on the developmental timing of gene expression, with ADH1A being expressed in the early fetal liver, ADH1B being detectable after the first trimester, and ADH1C being expressed in the liver after birth [It is perhaps unsurprising that alcohol metabolic gene variants have also been associated with differential teratogenicity of PAE . Partly,er birth . Furtherer birth ; therefoADH variants contribute to FASD susceptibility has been accumulated from several populations. A study on African American women at the University Health Center in Detroit, Michigan stratified subjects by maternal alcohol intake levels and ADH1B genotype [ADH1B Cys370 (ADH1B*3) allele was associated with faster development in the offspring of drinking mothers but not non-drinking mothers, with the offspring of Cys370 carriers being developmentally more like offspring of non-drinking women. The protective effects of the Cys370 allele are thought to be related to its higher Vmax for alcohol metabolism, thus decreasing peak alcohol levels reached in these carriers and also suggesting that alcohol, rather than acetaldehyde, acts as the prominent teratogen. However, it could also be due to the tendency of Cys370 carriers to flush, thus reducing or altering the pattern of alcohol consumption. Because the study enrolled participants with minimal differences in periconceptional alcohol intake within each genotype, differences in alcohol intake by genotype should not account for the differences in offspring outcomes. Interestingly, offspring with the ADH1B Cys370 allele similarly showed improved development, an unexpected finding given the low activity of ADH during gestation. The same group later investigated whether the ADH1B genotype influenced facial morphology in the same cohort of patients. By analyzing photographs of 247 offspring of mothers with a range of alcohol use during pregnancy, researchers found the Cys370 allele, whether in mother, offspring, or both, had a protective effect against alcohol-induced facial dysmorphology [Evidence that the common genotype . In the rphology .ADH genotype on FASD were later studied in the Detroit Longitudinal Prenatal Alcohol Exposure Cohort, where offspring were assessed at infancy and 7.5 years. This study of a cohort of 263 mother–child pairs of African ancestry found that mothers carrying at least one copy of the Cys370 allele had protective effects on birth size as well as behavioral and cognitive outcomes at both time points. In contrast with the previously mentioned study, there was no consistent effect on the infant genotype [The potential long-term effects of the genotype . Alternagenotype .ADH genotypes altering risk for FASD. In a study of 56 mothers and their FAS children in a population of Khoisan–Caucasian mixed-ancestry women in the Western Cape Province of South Africa, the allele frequency of ADH1B His48 (ADH1B*2) was lower in FAS children and mothers [ADH1B His48 allele is associated with a lower frequency of drinking. Although ADH1B Cys370 was shown to be protective against FASD in studies of African American women in the Detroit studies [ADH/ALDH genotypes in a mixed-race population was performed on 404 high-risk pregnant women and 139 infants in the Boston, Massachusetts, area. In this study, mothers who were ADH1B Cys370 carriers drank more and had an increased risk for having children with growth restriction and/or FAS facial features [ADH1B Cys370 conferred protection against FASD. The authors attributed this discrepancy to differences in the phenotypes measured between studies, but it may instead be due to differences in timing and quantity of drinking by the mothers in these two studies conducted at different times and in different populations.Studies from other populations have also shown trends for  mothers . These f studies ,40,41, tfeatures , and 60%ADH1B rs1229984, a rare variant associated with accelerated ethanol clearance, was associated with lower alcohol consumption during pregnancy and a higher likelihood to abstain from alcohol use altogether [ADH genotype in the effects of moderate alcohol drinking on cognitive and behavioral outcomes in offspring was also investigated in the ALSPAC cohort. In an analysis of ten single nucleotide polymorphisms (SNPs) in four ADH genes, four variants were related to differences in IQ present in children of mothers who drank moderately during pregnancy [Further studies have been conducted from the Avon Longitudinal Study of Parents and Children (ALSPAC), a population-based study in England investigating the effect of environmental factors on child health and development. In a Mendelian randomization study of 7410 white women, polymorphisms in four ADH genes predicted patterns of maternal alcohol intake. One non-synonymous variant, together . The rolregnancy . The preregnancy . The ADHADH variants which accelerate alcohol metabolism appear to have a protective effect against FASD. This could be due to differences in levels and developmental alcohol consumption, although some of the studies at least controlled for level of exposure. The protective effect of ADH alleles could also be due to a decrease in peak alcohol levels, implicating ethanol rather than acetaldehyde as the primary teratogenic agent. Despite the well-known toxicity and carcinogenicity of acetaldehyde, studies in mice support the idea that ethanol is the primary teratogen in FASD, showing that inhibiting ADH activity, but not ALDH, increases alcohol teratogenicity [ADH genotype has been shown to confer differential vulnerability in some but not all of the studies. The contribution of fetal metabolism of ethanol is estimated to be only 3–4% of the maternal rate [ADH variants lead to protection or vulnerability is unknown, but, as was observed earlier, the ADH1B gene is not expressed early in gestation, and thereby fetal ADH1B genotype might well be serving as a surrogate for maternal genotype and alcohol metabolic capacity, at least at early developmental timepoints. While initially low, the fetal capacity to metabolize alcohol does reach around 84% in the newborn [ADH genotype may be dependent on the timing of exposure, with fetal genotype expected to be a greater factor at later stages in development. The relationship of ADH polymorphisms to FASD risk requires further investigation of fetal and maternal genotypes in the context of the timing of alcohol exposure and may be more feasible in humanized genetic animal models.Further studies are warranted to elucidate the role of ADH genes in FASD vulnerability. Thus far, studies are limited to relatively small sample sizes in specific individuals of particular demographics. Although conflicting evidence exists, genicity ,54. Diregenicity . Thus, anal rate ; therefo newborn , and difBeyond polymorphisms altering alcohol metabolism, and still in advance of genome-wide studies, some other genetic variants involved in general developmental processes or alcohol use have been implicated in FASD . The eviCYP17, which encodes cytochrome P450c17α which acts in estrogen biosynthesis. Mutations in CYP17 are associated with maternal hormone imbalance and intrauterine growth restriction (IUGR), the latter being a commonly reported feature of FASD. In a hospital-based case-control study in Liverpool comparing low to normal birthweight pregnancies (N = 180 controls and 90 cases), a polymorphism in the untranslated region of CYP17 was more frequent in mothers of babies with IUGR if they had consumed alcohol during pregnancy [IGF2/H19 is a parentally imprinted gene critical for normal placental and embryonic growth and has been previously implicated in altered DNA methylation after PAE in both mice and humans [CTCF6 (rs10732516), a zinc finger protein that regulates IGF2, was previously implicated in newborn and adult height [CTCF6 rs10732516 was associated with both a decrease in newborn head circumference (HC) and decreased methylation at the H19 imprinting coding region (ICR) of IGF2 in alcohol-exposed placentas [IGF2 and CTCF6 in PAE-induced reductions in HC. Finally, variants affecting N-glycosylation have also been associated with an increased risk of FAS. FAS has several overlapping clinical presentations with congenital disorders of glycosylation, and in a study of 25 FAS patients and 20 controls, rare variants in genes associated with N-glycosylation presented at a higher frequency in patients, suggesting these variants might increase the risk for FAS [Associations of both maternal and offspring genes related to development have been made in a few studies, albeit with small sample sizes. These genes include regnancy , suggestd humans . An SNP t height . In placlacentas . Only 146 rs107326, a zincSLC44A1 out of 243 tested SNPs associated with choline were significantly associated with increased cognitive performance [Whether genetic variants might impact the effectiveness of FASD interventions has also been investigated. Dietary choline intake during pregnancy has been shown to improve memory, cognition, and attention in young children, and choline supplementation has been shown to reduce some deficits consequent to PAE . Fifty-tformance . These SPDYN), ADH1C, and the glutamate metabotropic receptor (GRM8) and drinking behaviors at different stages of pregnancy [ADH1B and ALDH2 as previously mentioned), and polygenic scores (PGSs) derived from them presently account for up to 5% of the variation in AUD [In addition to genes that influence development, studies have also investigated genes associated with alcohol use in general. The Danish National Birth Cohort (DNCB) examined the extent to which candidate genes for smoking, alcohol use, and obesity predicted the use of these substances as well as weight gain during pregnancy. A relatively small GWAS of 1937 mothers revealed associations at 39 genes including prodynorphin , a microdeletion at 18q12.3-q21.1 was detected in the affected twin . DeletioIn this review, we summarized findings investigating potential genetic influences on FASD. Genetic variation both within the mother and the infant may alter FASD, maternal genetic variation perturbing alcohol metabolism and alcohol intake, both of which largely determine fetal alcohol exposure, as well as variation related to the uterine environment. Fetal genotype can also alter alcohol metabolism and, critically, a variety of fetal genetic variations can either directly cause malformations resembling FASD or, as is likely, interact with alcohol exposure to mediate differences in teratogenicity of alcohol. Early twin case studies first established the potential link between genetic factors and PAE outcomes, with several cases of DZ twins presenting discordant FASD diagnosis, despite virtually equivalent PAE and uterine environments. Later twin studies, which included MZ twins, further support a genetic component of FASD, with MZ twins showing 100% concordance, reflecting a remarkably high heritability that still needs to be confirmed via larger studies of twins and other pairs of relatives, or by identification of genes responsible. The effect of common variants for the alcohol-metabolizing enzyme ADH has been explored in populations of different ancestries, with different variants showing some protective effects in different populations. Candidate gene studies in smaller cohorts have linked other variants related to growth and nutrient metabolism to FASD outcome, and polymorphisms linked to AUD showed some associations with drinking behaviors surrounding pregnancy in a GWAS study. Genetic testing for FASD has revealed CNVs that were both associated with the condition, including a microdeletion found in one affected twin in a discordance DZ pair, and also those that potentially explain phenotypes in children incorrectly diagnosed with FASD. Overall, these studies provide some gene-based evidence that heritability influences the variability in FASD and may partially explain why not all children with PAE manifest FASD phenotypes.ADH variants, a wide range of drinking patterns during pregnancy, and a variety of maternal factors which influence FASD, large, deeply phenotype longitudinal studies will be ultimately needed to isolate the effects of individual genes and to identify the cumulative, polygenic contributions of many genes that may be combined into polygenic risk scores and used both in research and, one day, clinically. Importantly, this would also facilitate the identification of novel genes and pathways to FASD which are largely unknown at this point.As mentioned earlier in this review, a significant limitation in the interpretation of existing studies is the small sample sizes. Twin studies included only 5 and 9 MZ twins and 11 and 39 DZ twins, and the candidate genes ranged from 45 to 404 mother–offspring dyads. Studies performed in the Avon Longitudinal Study of Parents and Children (ALSPAC) and The Danish National Birth Cohort (DNCB) had sample sizes of 1900 to 7400, which while an improvement is still relatively small in the era of large-cohort genomic studies. For example, GWA studies of height and body mass, far easier phenotypes to assess, are conducted on greater than one million individuals. Given the known impact of ancestry, particularly for the Both determining diagnosis and elucidating the etiology of FASD are complicated by the number of associated comorbid conditions. A recent study found 428 comorbid conditions with FASD, with the most commonly occurring conditions being related to congenital malformations, deformities, and chromosomal abnormalities . This inGiven the wide range of phenotypic outcomes of PAE as well as the number of factors that contribute to severity, understanding the potential genetic contributions to FASD will likely need to focus on specific phenotypes  within specific populations  and account for the different factors that influence developmental outcomes . GWASs and other genomic studies may validate but will hopefully also expand our knowledge of molecular pathways to FASD, for which there are now many theories. In addition to further genetic and genomic studies in larger populations for identifying risk or resilience variants for FASD, findings across different teratogenic syndromes and neurodevelopmental disorders as well as complementary non-human models will continue to be fundamental in understanding the genetic contributions to PAE phenotypes and FASD risk factors. FASD represents a complex interplay of environments, including the environment of the mother, the environment within the mother, and the environmental exposure to alcohol. Understanding the interaction of both maternal and offspring genes within these environments across the trajectory of development poses a challenge for scientists and clinicians in identifying genetic factors that might confer different vulnerabilities. As there is no cure for FASD, identifying these genetic vulnerabilities could aid in early detection and intervention preventing FASD or mitigating the consequences of PAE.


Aberrant cell death can cause inflammation and inflammation-related diseases. While the link between cell death and inflammation has been widely established in mouse models, evidence supporting a role for cell death in the onset of inflammatory and autoimmune diseases in patients is still missing. In this review, we discuss how the lessons learnt from mouse models can help shed new light on the initiating or contributing events leading to immune-mediated disorders. In addition, we discuss how multiomic approaches can provide new insight on the soluble factors released by dying cells that might contribute to the development of such diseases. The field of cell death has undergone quite a substantial evolution over the past three decades. During the 1990s and the first years of the new century, scientists were mainly focused on how the knowledge of cell death pathways could have helped improve cancer therapy, to eliminate as many cancer cells as possible, with little interest for the pathophysiological roles of cell death . In the Cytokines of the tumour necrosis factor (TNF) family are crucial regulators of cell death, inflammation and autoimmunity . TNF recUpon binding to its cognate ligand, TNF, TNFR1 trimerizes and initiates the formation of a receptor-bound complex called complex-I or TNFR1-associated signalling complex (TNFR1-RSC) . The firThe TNF-signalling pathway is tightly controlled by a number of checkpoints that rely on ubiquitination-, phosphorylation-, gene-expression-dependent and proteolytic events ,29. In cBoth the conjugation and hydrolysis of ubiquitin moieties represent one of the most studied checkpoint mechanisms that control complex-II formation and restrain TNF cytotoxicity . Amongstcpdm (chronic proliferative dermatitis mice)  or reduction in linear chains (SHARPIN) in TNFR1-induced complex-I . The pheis mice) , which cammation . This coammation ; and  and one boy from a consanguineous marriage, with homozygous deletion of two nucleotides (c.121_122delCT) in HOIL-1 . Few weeThe first HOIP homozygous missense mutation (L72P) patient was reported in 2015 . This paVery recently, a non-synonymous variant of SHARPIN was identified as genetic risk factor for LOAD (late-onset Alzheimer’s disease) in a cohort of 202 Japanese individuals . This vaThe different LUBAC mutant mouse models described above have been instrumental in unveiling the physiological role of linear ubiquitination and how linear ubiquitin chains orchestrate inflammatory programs. Equally important was the fact that they allowed to unravel the link between cell death and inflammation and the potent inflammatory potential of cell death activation. Mouse work revealed that the individual LUBAC components contribute to optimal gene activation following stimulation of immune receptors, including TNFR1, but, most importantly, they limit the killing activity of TNF. Therefore, the predominant phenotypic effect triggered in mice by their absence is cell death and cell death-induced inflammation. However, the phenotypic similarities between LUBAC mutant mice and patients are limited to some features of glycogen deposition and heart defects. In the human setting, loss of function mutations of HOIP, HOIL-1 or SHARPIN do not only result in autoinflammation, but also in immunodeficiency, glycogen storage disorders (HOIP and HOIL-1) and neurodegeneration (SHARPIN). This might indicate that (i) in humans, the gene activatory functions of LUBAC are as or more predominant than its cell death inhibitory functions, although the occurrence of cell death has not been fully analysed in patients; and (ii) those individuals carrying LUBAC mutations that escape lethality in utero, might have backup systems in place to regulate cell death and inflammation that are not completely dependent on LUBAC. An extensive cell death analysis in LUBAC mutant patients, using classical cell death markers might help elucidate the role of LUBAC in controlling organism homeostasis, and find therapeutic strategies to improve the care of LUBAC mutant patients.OTULIN is the only known linear chain-specific deubiquitinase . The amiThis view was subsequently challenged by another study that, using a catalytically inactive mutant of OTULIN in mice, showed that the primary effect of linear chains accumulation is not NF-κB hyperactivation but rather complex-II-mediated cell death, in the form of apoptosis and necroptosis  Table 1Table 1, Gumby mutation, which is a spontaneous mutation in OTULIN (W96R), results in embryonic lethality resembling the report on OTULIN catalytically inactive mice [Importantly, OTULIN has also been implicated in signalling events that are different from cell death and inflammation per se. For example, the ive mice . Gumby mive mice . Whetherive mice .The OTULIN-mutant mouse models were extremely useful to unveil the link between OTULIN mutations and excessive linear chains in ORAS/Otulipenia. In addition, they allowed to understand why infliximab has such therapeutic benefits in patients. At present, and similar to LUBAC-mutant patients, evidence that OTULIN absence can unleash inflammatory cell death is still missing. One could speculate that absence of OTULIN activity can induce cell death only in some cell types, while in others sustained NF-κB activation would be the main outcome. Future analysis employing cell death-specific stainings will be required to understand the mechanisms of hyperinflammation and the respective contributions of cell death and NF-κB in OTULIN mutant patients.−/−Tnfaip3, the myel-KOTnfaip3  and ZnF7mutTnfaip3 mutant mice were extremely useful to strengthen the link between cell death and inflammation, and to gain a better understanding of the cause of the inflammatory diseases displayed by A20 mutant patients. A20-deficient mice suffered from severe multiorgan inflammation, including, but not limited to, liver, kidneys and joints. Similarly, although milder, mice lacking A20 in myeloid cells and those bearing a ZnF7 mutant version of A20 developed arthritis [A20 is a deubiquitinase enzyme that exhibits 88.1% amino acid sequence conservation between human and mouse. It was identified in 1990 as a NF-κB target gene , which hrthritis  , rheumatoid arthritis, psoriasis and diabetes ,73,74,75−/−Tnfaip3 and ZnF7mutTnfaip3 mice. These new potential mouse models would broaden the possibility to study HA20 and find novel therapeutic approaches.A20 is another example of how mouse models can be extremely valuable to accelerate the understanding of (i) the genetic cause of a human pathology, or group of pathologies, and (ii) the molecular mechanisms driving the pathology. At the same time, the genetic studies in humans can indicate how to refine the existing mouse models to develop better preclinical disease models. For example, although the A20 mutant mice develop a set of diseases that quite closely recapitulate the patient’s clinical features, it is surprising that almost all the human mutations are found in the OTU catalytic domain , and no mutation has ever been found in the ZnF7 domain . One expFinally, while the mouse models clearly indicated that the aetiology of the systemic inflammation observed in A20 mutant mice is cell death, in patients there is still lack of evidence supporting this possibility. Similar to what was highlighted for LUBAC and OTULIN mutant patients, cell death marker stainings, (phospho)proteomic and ubiquitinome analysis on patients’ samples might help determine the contribution of cell death vs. NF-κB hyperactivation to the disease.XIAP is an E3 ligase enzyme that belongs to the IAP (inhibitor of apoptosis) family . Human a−/−Xiap mice as a model [−/−Xiap mice are very sensitive to intestinal inflammation triggers, such as DSS or the pathogenic bacterial strain Helicobacter hepaticus. Importantly, delivery of antimicrobial peptide to the intestine, by means of adenoviruses, allowed XIAP-deficient mice to clear the H. hepaticus [Mouse models were again of great help to gain insights into the role of XIAP in the regulation of immune system responses . Indeed, a model ,78. In oGenomic sequencing approaches have decisively contributed to identify the cause of many inflammation-related genetic diseases. The combination of genetic studies with biochemical studies has then helped dissect the molecular mechanisms underlying these genetic diseases and provided sound scientific basis for therapeutic intervention. The question that can be raised now is: How can different omic approaches further help understand the aetiology of human inflammatory diseases? It is established that TNF often plays an eminent role in inflammatory syndromes and that TNF-induced cell death, rather than TNF-induced gene activation, might be the decisive factor for the development of these syndromes. Therefore, the next question to be addressed now relates to the nature of factors released by dying cells that can instigate inflammatory processes.Different cell death forms have different inflammatory potential. It is widely accepted that, while necroptosis and pyroptosis are inflammatory cell death forms, because of membrane rupture and intracellular content spillage, apoptosis is immunologically silent . InteresThe last decade saw an impressive body of work that enabled us to understand how cell death modulates inflammatory diseases and how this was associated with human genetics. It is not always expected that mouse models completely recapitulate the human settings. However, model organisms spanning from 2D cells/organoids to invertebrates and mice bring us closer to the identification of aetiological factors of chronic inflammation and autoimmune disorders in humans. Excitingly, the gap between mice and men is becoming smaller with the advancement in technologies and preclinical animal models. Currently, mouse work has become the springboard for human studies with the ultimate purpose to design novel therapies that improve the care of patients affected by inflammatory/autoimmune diseases.


Infertility is a highly prevalent condition, affecting 9–20% of couples worldwide. Among the identifiable causes, the male factor stands out in about half of infertile couples, representing a growing problem. Accordingly, there has been a decline in both global fertility rates and sperm counts in recent years. Remarkably, nearly 80% of cases of male infertility (MI) have no clinically identifiable aetiology. Among the mechanisms likely plausible to account for idiopathic cases, oxidative stress (OS) has currently been increasingly recognized as a key factor in MI, through phenomena such as mitochondrial dysfunction, lipid peroxidation, DNA damage and fragmentation and finally, sperm apoptosis. In addition, elevated reactive oxygen species (ROS) levels in semen are associated with worse reproductive outcomes. However, despite an increasing understanding on the role of OS in the pathophysiology of MI, therapeutic interventions based on antioxidants have not yet provided a consistent benefit for MI, and there is currently no clear consensus on the optimal antioxidant constituents or regimen. Therefore, there is currently no applicable antioxidant treatment against this problem. This review presents an approach aimed at designing an antioxidant strategy based on the particular biological properties of sperm and their relationships with OS. Infertility is defined as the failure to achieve a spontaneous pregnancy despite 1 year of practicing regular sexual intercourse without a contraceptive  and a maIdentifiable causes of male infertility comprise as few as 10% of the total cases, with around 5% being sperm transport disorders  and the The redox state of a biological system can be defined as the balance between its oxidative elements and the capacity of its antioxidant elements. The main oxidant elements are reactive oxygen species (ROS), a group of highly reactive oxidant agents, including radical anion superoxide, hydrogen peroxide, hydroxyl radical and singlet oxygen. At low concentrations, ROS are actually essential for normal cell homeostasis and are involved in various cell signalling pathways . ConversDespite its epidemiological relevance and its widely accepted association to OS, no antioxidant strategy has shown consistent benefit on male infertility. This review focuses on the role of OS in idiopathic dysspermatogenesis and unexplained male infertility, and new approaches on the development of therapeutic strategies against male infertility.In 1994, Aitken proposed the loss of sperm function subsequent to the induction of peroxidative damage by ROS originating from the spermatozoa as a theory for male infertility . Since tBefore further exploring the pathophysiological mechanisms behind the relationship between OS and male infertility, it is imperative to properly characterise the oxidant and antioxidant elements playing in the particular setting of human spermatozoa. The cellular oxidative homeostasis in spermatozoa relies on two groups of components that interact in maintaining this balance: ROS and antioxidants.2−), primarily originating in mitochondria via the slippage of an electron from the electron transport chain (ETC) to molecular oxygen during oxidative phosphorylation, (2) hydrogen peroxide (H2O2) and the (3) hydroxyl radical (·OH) . Th. Th75]. Excessive mitochondrial ROS production is a milestone in the creation of the OS state that underlies male infertility. A crucial trait of this phenomenon is that it establishes a self-perpetuating, vicious cycle in which OS further promotes the generation of excessive ROS generation at a mitochondrial level. This self-perpetuating feature is enabled by the production of aldehydes such as acrolein, malondialdehyde and 4-hydroxynonenal generated by lipid peroxidation, which, in the first instance, carry out an oxidative attack on histidine, lysine and valine residues in mitochondrial proteins, particularly succinate dehydrogenase, an enzyme located in the inner mitochondrial membrane that participates in the ETC . This leLipid peroxidation is a result of the oxidative attack of ROS over PUFAs, which are abundant molecules in spermatozoa membranes, particularly docosahexaenoic and arachidonic acids , since t2O2 [As previously discussed, the self-perpetuating cycle of ROS generation at a mitochondrial level results in high levels of peroxidation, loss of mitochondrial membrane potential, membrane damage in the midpiece of the spermatozoa and decreased sperm motility ,79. Subs2O2 . This is2O2 . Further2O2 , since mMoreover, nuclear DNA sperm is found in a quasicrystalline state, making penetration by endonucleases highly unlikely , possiblThe apoptotic process of sperm is critical in male infertility because of its close association to DNA damage, under the premise that apoptosis is initiated during spermatogenesis but the process is arrested due to the profound conformational changes of male germ cells following the meiotic process, which removes the apoptotic intracellular machinery. Consequently, DNA damage could also be attributed to an abortive apoptotic process ,84.Spermatozoa are proposed to be particularly susceptible to DNA damage, given both a lesser degree of condensation for mitochondrial DNA and the absence of DNA repair systems for nuclear DNA . AlthougTwo main mechanisms for DNA damage in the context of male infertility have been described: endonuclease-mediated cleavage and ROS . Interes2− generation in sperm mitochondria [Consistent with Aitkens’ hypothesis, an early stage in this process and probable triggering event seems to be the induction of Ochondria ,89.Types of DNA damage identified on spermatozoa include base mismatch, abasic sites, base modifications, DNA adducts and crosslink, pyrimidine dimers, single strand breaks and double strand breaks. All of the above can lead to SDF, and except for base mismatch and pyrimidine dimers, all are induced by ROS overproduction .2O2, which is able to penetrate the nucleus due to its low molecular weight and lack of electric charge [Indeed, ROS overproduction induces SDF among other deleterious effects . Severalc charge ,91. Furtc charge ,93,94. TSingle strand breaks (SSB) and double strand breaks (DSB) are both main elements of SDF . SSB areThere are several methods available for the evaluation of sperm DNA fragmentation/integrity including: TUNEL assay , sperm cRegarding the effects of DNA damage and sperm DNA fragmentation on human reproduction, SDF has been inversely associated with probability of natural pregnancy in a Danish cohort . FurtherThe current management of SDF and OS in unexplained infertility focuses on the use of several interventions, with a variable degree of supporting evidence: antioxidant supplementation ,110, lifTaking into account the specific intracellular phenomena and sensitive sperm-development timeframes, we hypothesise that positive achievements will result from interventions based on the relationship between the time course of stages of spermatogenesis and OS. Thus, these processes could be favoured throughout the spermatogenesis cycle by the pertinent effect of appropriate time-related pharmacological agents in order to improve the clinical outcomes of male infertility.Aiming to properly contextualise the reasoning underlying our proposal, key aspects of sperm development are described below:(1)dark- and Apale-spermatogonia undergo continuous mitosis for renewal of the germ cell line, while Apale-spermatogonia commit to differentiation and divide to form B-spermatogonia, subsequently dividing to form preleptotene spermatocytes [Mitosis: Progenitor Aatocytes .(2)Meiosis: Diploid spermatocytes (2n2c) undergo 2 meiotic divisions to form haploid spermatids (nc). Primary spermatocytes are the first cells to undergo meiosis . During (3)Spermiogenesis: Consists of the transformation of round spermatids into elongated, mature spermatozoa. It includes the loss of cytoplasm, migration of cytoplasmic organelles, formation of the acrosome from the Golgi apparatus, formation of the flagellum from the centriole, nuclear compaction to about 10% of former size, and reorganisation of mitochondria around the sperm midpiece . Moreove(4)Spermiation: The mature spermatozoon is freed of its anchorage to the Sertoli cell, being released into the tubule lumen.Spermatogenesis, the process by which spermatogonial cells become spermatozoa, is a complex process occurring within seminiferous tubules of the testis whose exhaustive description is beyond the aim of this work but can be broadly divided into four phases: (1) mitosis; (2) meiosis; (3) spermiogenesis and (4) spermiation . In humaOur proposal includes 3 antioxidants see , whose rOmega-3 (ω-3) polyunsaturated fatty acids. PUFAs comprise five substances: eicosapentaenoic acid (EPA), alpha-linolenic-acid, stearidonic acid, docosahexaenoic acid (DHA), and docosapentaenoic acid. Of particular relevance are long-chain ω-3 PUFAs such as EPA and DHA, which are abundant in the body lipids of fatty fish, the liver of white lean fish, and the blubber of marine mammals .EPA and DHA are highly susceptible to free-radical oxidation , yet theThe consumption of long chain ω-3 PUFAs has shown improvements in sperm SOD and CAT activities , sperm cResveratrol. Resveratrol is a phenolic compound derived from stilbene naturally found in foods such as grapes, nuts, cranberries and red wine . It poss2− and metal-induced ROS, thereby protecting cell membranes from lipid peroxidation and DNA from ROS-mediated damage [The antioxidant properties that resveratrol exhibits are partly due to being an excellent scavenger of ·OH, Od damage .Moreover, this compound has been shown to act through the transcription factor named nuclear factor erythroid 2–related factor 2 (Nrf2) pathway, as it downregulates Keap1 , a proteFurthermore, the upregulation of the Nrf2 pathway by resveratrol could be important in the maintenance of mitochondrial homeostasis and structural integrity . The aboInterestingly, resveratrol was also found to have a significant inhibitory effect on the NF-κB signalling pathway after cellular exposure to metal-induced radicals .In 2020, Illiano et al. treated 20 patients with idiopathic infertility with a resveratrol-based multivitamin supplement containing a dose of 150 mg of resveratrol daily, for 6 months, after which they showed improved sperm motility and concentration compared to baseline measurements . LimitatMelatonin. Melatonin is an ancient molecule with presence throughout Eukarya and Bacteria domains, and first appeared as a way to mitigate ROS production secondary to aerobic metabolism . Since iIt exhibits antioxidant activity through various direct and indirect mechanisms: ROS scavenging ,143,144,Melatonin is an amphiphilic molecule, with the capacity to cross biological membranes and reach any cellular and subcellular compartment, owing to this property its high rate of distribution and the ability to exert its antioxidant activity ,152. TheMoreover, studies in mice showed melatonin’s ability to reverse the effects of cyanide, an ETC Complex IV inhibitor . FurtherFurthermore, in recent work Malmir and colleagues studied the in vitro effect of melatonin over spermatozoa collected from men with idiopathic asthenoteratozoospermia. DNA fragmentation measured by TUNEL, sperm chromatin dispersion and malondialdehyde levels were significantly reduced while mitochondrial membrane potential and viability were significantly increased in the melatonin group compared to control .Given its stability during digestion and fast absorption, melatonin possesses a high bioavailability . Oral adThese compounds share a few characteristics that might be relevant to the effectiveness of the regimen: they exert their antioxidant capacities through various mechanisms, many of which overlap. In addition, the three of them have been shown to promote the activity of intrinsic, enzymatic antioxidant systems through the Nrf2 pathway. In the case of PUFAs, the aim of their use during the mitotic phase of spermatogenesis is to generate non-hypoxic preconditioning of male germ cells in order to strengthen their enzymatic antioxidant defences. Moreover, we propose the use of resveratrol during meiosis because of its simultaneous role as direct and indirect antioxidant, aiming to neutralise the possible exogenous ROS before the most vulnerable moment of spermatogenesis: spermiogenesis. Furthermore, melatonin is the antioxidant proposed for the spermiogenesis phase due to the following features: (i) multiple antioxidant capabilities and mitochondrial stabilising properties; (ii) ample distribution throughout the cell given its amphiphilic nature and the proposed active transport at a mitochondrial level; and (iii) prolonged effect due to the analogous actions of its active metabolites. This proposal (graphically presented in As we have established, several reports support the association of high seminal ROS levels both with impaired sperm parameters ,167 and Therefore, the use of antioxidants as a treatment for male infertility presents a reasonable and biologically plausible intervention. Despite how promising it seems, few studies have been able to prove a consistent benefit of antioxidant-based therapy by themselves and evidence is largely controversial see . FurtherIt is worth taking into account that for most RCT and other trials using antioxidant treatment for male infertility, there is considerable variability regarding sample selection and general inclusion criteria. Despite the amount of evidence supporting OS as a core element in male infertility pathophysiology, idiopathic male infertility cohorts are highly heterogeneous in terms of the specific abnormalities in spermiogram and other clinical evidence that leads to their inclusion in this group. In other words, the concept of idiopathic male infertility becomes an umbrella term for a myriad of clinical conditions that lead to dysfunction of male reproductive function.A necessary approach to improve the outcomes in future therapeutic interventions could be to effectively establish OS is in fact a main factor in the pathophysiology of their particular reproductive dysfunction by adding high OS biomarker levels as an inclusion criteria for the use of an antioxidant regimen on idiopathic male infertility. Regarding measurements and outcomes, the determination of said biomarkers during and after the intervention becomes mandatory along with sperm quality and in vivo fertility follow-ups in order to establish consistent correlations.Concerning the components and structure of the antioxidant regimen itself, there is still need for further evidence on the role that commonly cited antioxidants have under in vivo conditions in human sperm. Nevertheless, given the fact that male fertility is a continuous, highly complex process with an equally nuanced pathophysiology, future antioxidant regimens should entail a dynamic, longitudinal approach to antioxidant compound selection and timing of interventions.


Peripheral nerve injury (PNI) is one of the most common concerns in trauma patients. Despite significant advances in repair surgeries, the outcome can still be unsatisfactory, resulting in morbidities such as loss of sensory or motor function and reduced quality of life. This highlights the need for more supportive strategies for nerve regrowth and adequate recovery. Multifunctional cytokine transforming growth factor-β (TGF-β) is essential for the development of the nervous system and is known for its neuroprotective functions. Accumulating evidence indicates its involvement in multiple cellular and molecular responses that are critical to peripheral nerve repair. Following PNI, TGF-β is released at the site of injury where it can initiate a series of phenotypic changes in Schwann cells (SCs), modulate immune cells, activate neuronal intrinsic growth capacity, and regulate blood nerve barrier (BNB) permeability, thus enhancing the regeneration of the nerves. Notably, TGF-β has already been applied experimentally in the treatment of PNI. These treatments with encouraging outcomes further demonstrate its regeneration-promoting capacity. Herein, we review the possible roles of TGF-β in peripheral nerve regeneration and discuss the underlying mechanisms, thus providing new cues for better treatment of PNI. Unlike the nerves in the central nervous system, peripheral nerves have significant potential to regenerate and repair after injuries . DespiteChallenges associated with meaningful functional recovery after PNI primarily include the rate of regeneration, specificity of regeneration, segmental nerve defects and degeneration of the target-end organs . The seaTransforming growth factor-β (TGF-β) proteins are multifunctional cytokines. TGF-β is a well-studied factor involved in various physiological processes like cell proliferation, differentiation, wound healing and immune response. It is also implicated in the pathogenesis of several diseases such as connective tissue diseases, skeletal diseases, fibrosis, and cancer progression . They reBased on the extent of damage to the connective tissues and the axons of the nerve, PNI was first classified by Seddon into three categories, namely neurapraxia, axonotmesis, neurotmesis, and by Sunderland into five degrees (I–V) . The milWallerian degeneration is the first process after nerve injury and occurs in the distal end of the damage 24–48 h after the injury. During this process, the distal axons, the adjacent myelin and other axonal components start to degenerate to clear the undesired debris . RemovalWhile Wallerian degeneration takes place in the distal stump and regrowing axons extend from the proximal stump, SCs experience a series of injury-induced changes that involve both the loss and gain of cellular phenotypes, namely reprogramming or transdifferentiation . ReprogrBesides clearance of debris and formation of bands of Büngner, SCs also help create a microenvironment that favors axonal regeneration and recruitment of macrophages. They are vital sources of endogenous neurotrophic factors, promoting neuronal survival and axonal regeneration after acute or chronic nerve injuries . PreviouFollowing PNI, adult SCs are reprogrammed into a repair-competent state . As mentDedifferentiation is an important part and an indicator of the beginning of SC reprogramming. The capacity of TGF-β to promote SC dedifferentiation is supported by current findings. In purified SCs, treatment with TGF-β1 induces inhibition of myelin P0 expression, indicating the blockage of the myelination process . Using ain vitro and in vivo  . These f in vivo . TGF-β c in vivo . Notably in vivo . At the  in vivo .Repair SCs exhibit both bipolar and unipolar morphologies, which enhances their migratory capacity and thus contributes to the formation of bands of Büngner . StudiesBecause of the inhibitory effects of myelin on neural regeneration, rapid clearance of myelin is of vital importance . SCs inivia the production of chemokines, including macrophage inflammatory protein and monocyte chemoattractant protein-1 , a milieu molecule for TGF-β expressed on myeloid cells  . RAGs arAs numerous basic studies have reported on the pro-regenerative effects of TGF-β in various cellular responses in peripheral nerve regeneration, the translation of this knowledge into clinical practice for treating PNI is attractive. As mentioned above, TGF-β enhances neural regeneration by local addition. After chronic nerve injury, TGF-β plus forskolin improves axonal recovery by upregulating RAGs and activation of SCs and macrophages . TGF-β cNerve tissue engineering has emerged as a constructive strategy for PNI treatment. Nerve guidance conduits (NGCs), the core of nerve tissue engineering, have gained attention recently. Research indicates that NGCs combined with cells  and neurotrophic factors are highly effective in promoting axonal growth. This combination  serves both as a scaffold and a carrier of molecular and mechanical signals in the process of regeneration, thus offering a prospective method of peripheral nerve repair . SCs arevia both canonical and non-canonical signaling pathways. This can offer another possible reason for the negative impacts of TGF-β on regeneration . In this review, we conclude that TGF-β can guide these critical processes toward a regeneration-promoting direction by regulating the behavior of major cells involved (summarized in In recent years, with the development of tissue engineering, NGCs have become a hot topic in the field of peripheral nerve regeneration. Combined with cells and neurotrophic factors, NGCs can provide physical support and mimic the regenerative microenvironment of PNS, thereby greatly promoting the regenerative effects. TGF-β is abundantly present in the natural nerve bridge and participates in multiple regeneration processes. It can help optimize the interaction between cells in the scaffold and enhance its regenerative capacity. However, the role of TGF-β in NGCs is rather neglected. It has seldom been included in the currently available design of NGCs, unlike other factors such as IL-4, brain-derived neurotrophic factor and GDNF (reviewed by ZY and JW contributed to design, drafted, and critically revised the manuscript. CZ and JH contributed to conception, design and critically revised the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Danio rerio)—a well-established model for studying osteogenesis and mineralogenesis—has recently gained momentum as a model to study ectopic calcification disorders. In this review, we outline the mechanisms of ectopic mineralization in zebrafish, provide insights into zebrafish mutants that share phenotypic similarities with human pathological mineralization disorders, list the compounds capable of rescuing mutant phenotypes, and describe current methods to induce and characterize ectopic calcification in zebrafish.Ectopic calcification refers to the pathological accumulation of calcium ions in soft tissues and is often the result of a dysregulated action or disrupted function of proteins involved in extracellular matrix mineralization. While the mouse has traditionally been the go-to model organism for the study of pathologies associated with abnormal calcium deposition, many mouse mutants often have exacerbated phenotypes and die prematurely, limiting the understanding of the disease and the development of effective therapies. Since the mechanisms underlying ectopic calcification share some analogy with those of bone formation, the zebrafish ( Calcium is central to human physiology, being a major player in the homeostasis of mineralized tissues , but also of soft tissues, where it is involved in blood clotting, muscle contraction, nerve function, and regulation of heart rhythm, among other processes ,2. UnderEctopic calcification was long thought to be an aging-dependent disorder, but significant data has recently come forward indicating that it may also result from dysfunctional anti-mineralizing proteins ,8. In thTwo distinct forms of ectopic calcifications have been described in humans. Metastatic calcification is characterized by an accumulation of calcium in soft tissues following an increase in serum levels, while dystrophic calcification results from an accumulation of calcium at pathologically altered sites . DespiteMgp null mice die within the first two months of age due to extensive mineralization of arteries and subsequent rupture [Animal models of ectopic calcification are critical to better understand the disease pathophysiology and allow the screening of novel therapeutics . While t rupture .Danio rerio) has emerged as a valuable model to gain insights into the mechanisms underlying the development of human diseases as it brings intrinsic advantages over mammalian models. Among those, it is worth mentioning that (i) a single breeding event can produce hundreds of embryos for testing, allowing a robust statistical analysis; (ii) embryos and larvae are optically clear and develop externally, allowing easy and direct visualization of the developmental process; (iii) amenability to genetic manipulation and availability of tools for genetic screening and editing; and (iv) a fast development throughout adulthood, allowing genetic experiments to be performed within a short period [pubmed.ncbi.nlm.nih.gov (accessed on 7 December 2022)”, we also outline the current tools, techniques, and mutants available to gain insights into mechanisms of ectopic calcification and calcium-associated pathologies, and list compounds with the capacity to rescue mutant phenotypes and contribute to the next-generation therapeutics to prevent or treat ectopic calcification disorders.The zebrafish  the mechanisms underlying calcium metabolism, (ii) the sites of calcium-phosphate crystal (predominantly in the form of hydroxyapatite) deposition, and (iii) a phenotypic response that mimics human mineralization disorders.Although calcium intake occurs in the kidney and intestine in mammals and the gills and yolk skin (during development) in fish, the major principles of calcium transport and hormonal control were found to be conserved from zebrafish to mammals ,23. For Several zebrafish models of Mendelian genetic disorders share phenotypic similarities with the acquired forms of metastatic and dystrophic calcifications and may serve as genetically controlled systems to study human calcification disorders. Recent studies on small teleost fishes have allowed the identification of various genetic factors that contribute to ectopic calcification; they are further discussed below and summarized in Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1)—and pseudoxanthoma elasticum (PXE)—caused by mutations in the gene ATP binding cassette subfamily C member 6 (ABCC6)—are rare autosomal recessive genetic disorders associated with arterial and cartilage calcification, and ectopic calcifications of elastic fibers, respectively [enpp1 mutants were shown to develop ectopic calcification of the skin, cartilage, heart, intracranial space, and notochord sheet, independently of osteoblast activity. They also displayed higher osteoclast activity at sites of ectopic calcification, and bisphosphonates could efficiently rescue associated phenotypes [abcc6a mutants showed defects in vertebral calcification and displayed ectopic calcification in soft tissues [abcc6a mutants [Generalized arterial calcification of infancy (GACI)—caused by mutations in the gene ectively ,42. Zebrenotypes . Zebrafi tissues . Since A mutants . Abcc6 knockout mice [abcc6a mutants [abcc6a zebrafish mutants [abcc6a mutants treated with minocycline, a DNA damage response inhibitor, had reduced occurrences of pathological mineralization [Vitamin K was proposed to be central to the pathophysiology of GACI and PXE as patients usually show a reduction in circulating vitamin K, which may be a countermeasure against pathological mineralization . While tout mice , it rest mutants ,43. Intelization .αKlotho and Fgf23 null mice are difficult to maintain as they die by three months of age; this represents a major bottleneck in the use of these murine models [Chronic kidney disease (CKD) patients are likely to suffer from ectopic calcifications due to a dysregulated mineral metabolism and pathological alterations in KLOTHO and Fibroblast Growth Factor (FGF) 23 . Mice dee models ,48.αklotho expression is detected in the adult kidney and fgf23 is continuously expressed in the corpuscles of Stannius, an endocrine gland close to the nephron that contributes to calcium homeostasis, where Fgf23 is responsible for adjusting and regulating calcium metabolism [αklotho and fgf23 have a short lifespan [αklotho, fgf23, and ennp1 zebrafish mutants have consistently shown an increase in osteoclast activity around mineralized soft tissues, hinting at the existence of osteoclasts that develop as a response to ectopic calcifications [In zebrafish, tabolism ,54. As flifespan . Howeverlifespan . Indeed,ications ,34,55,56Myogenesis regulating glycosidase (MYORG) have been linked to the development of autosomal recessive PFBC [Myorg knockout mice at nine months of age [myorg expression was knocked-down using morpholinos  is a rare progressive neurodegenerative disorder characterized by bilateral brain calcifications and associated with symptoms of dementia, Parkinsonism, and dystonia ,58. In 2ive PFBC ,60. The pholinos  exhibitegolgin b1 (golgb1) and activin A receptor, type 1 like (acvr1l) have been associated with the development of hyperphosphatemic familial tumoral calcinosis (HFTC) [golgin b1 and acvr1l develop ectopic calcification, it is unknown whether these fish simply failed to do so, or it was not observed or reported as it was not the focus of these studies. So far, mutations in the zebrafish orthologs of many single-gene human diseases—associated with ectopic mineralization disorders—faithfully mimicked disease phenotype and show pathological calcium deposition. Therefore, it is likely that many zebrafish mutants for genes associated with calcium mineralization disorders—whose phenotype has yet to be published—may develop ectopic mineralization phenotypes. Zebrafish mutants that may develop ectopic calcifications, and are therefore promising targets for the development of new models, are summarized in Mutations in the genes s (HFTC)  and fibrs (HFTC) —rare diss (HFTC) ,62. Whilsa18644 studies—targeting exon 3—have been reported and the phenotype closely matches the loss-of-αKlotho function previously reported in zebrafish [Most zebrafish models initially used to study ectopic mineralization have been developed through forward genetic approaches. In a screening to identify novel regulators of osteogenesis and bone mineralization, a mutant named dragonfish (dgf) showed ectopic mineralization in the craniofacial and axial skeleton . This muebrafish .abcc6a-specific MOs in zebrafish eggs induced cardiac malformations and developmental defects in 8 dpf morphant larvae similar to those observed in GACI patients [Reverse genetics has also been successfully applied to zebrafish. Morpholino antisense oligomers (MOs) are the preferred method of gene knockdown in zebrafish ,66 and tpatients . Similarpatients . CRISPR/patients ,69 makinpatients . As manyThe detection and quantification of calcium deposition in zebrafish can be achieved through a variety of techniques that are based on the optical detection of changes in luminescence, fluorescence, or absorbance of an organic indicator that specifically binds to calcium ions . SeveralVon Kossa’s staining is also a very popular method to detect the presence of abnormal deposits of calcium-phosphate crystals in the body. This histological method is based on the transformation of calcium phosphate salts into silver phosphate salts, which undergo photochemical degradation when illuminated with a UV light, leading to the formation of dark silver deposits . While iHistological techniques used to detect ectopic calcification have limitations associated with the physical sectioning of hard tissues that often result in tissue loss or incomplete tissue sectioning. In addition, visualization of calcium deposition in elongated structures  and in a single section is extremely difficult. Micro-computed tomography (micro-CT) is commonly used for the 3D imaging and analysis of skeletal structures and other calcified tissues ,81; it uThe identification of compounds that induce phenotypical changes similar to those observed in pathological conditions is of the utmost interest as these compounds allow quick, easy, and cost-effective access to in vitro or in vivo disease models. Although the precise mechanisms that lead to ectopic calcification remain to be better understood, molecules that disrupt the balance between pro- and anti-mineralizing pathways have often been associated with pathological mineralization ,91.Vitamin K vitamers are central to bone mineralization through their role in the carboxylation of several vitamin K-dependent bone-related proteins, such as osteocalcin, matrix Gla protein, and Gla-rich protein ,93,94. WTherapeutic strategies to efficiently counteract the accumulation of calcium ions in soft tissues responsible for ectopic mineralization disorders have yet to be developed. Antiresorptive drugs have been tried, but they were only able to delay and not prevent disease progression ,106. TheEctopic calcification disorders are medical conditions that affect the well-being and life expectancy of many people worldwide and for which therapeutics remain to be developed. To improve the current scenario, the complex mechanisms underlying these disorders must be better understood. Until recently, most of the information regarding pathological calcification was collected from human patients and mouse models, two biological systems with inherent limitations. Zebrafish models have recently emerged with the potential to provide new and fast insights into pathological mechanisms, as they can overcome some of these limitations  and accelerate the collection of data with phenotypic alterations occurring at early stages of development. As for mammalian systems, zebrafish ectopic calcification models develop calcification in most soft tissues, and the detection of sites of ectopic calcification could be detected early during development . Thanks to the Zebrafish Mutation Project “zmp.buschlab.org (accessed on 7 December 2022)”, there are currently dozens of available mutants with genes associated with calcium mineralization disorders that remain to be analyzed and studied  and eggs


Heat stress severely affects plant growth and crop production. It is therefore urgent to uncover the mechanisms underlying heat stress responses of plants and establish the strategies to enhance heat tolerance of crops. The chloroplasts and mitochondria are known to be highly sensitive to heat stress. Heat stress negatively impacts on the electron transport chains, leading to increased production of reactive oxygen species (ROS) that can cause damages on the chloroplasts and mitochondria. Disruptions of photosynthetic and respiratory metabolisms under heat stress also trigger increase in ROS and alterations in redox status in the chloroplasts and mitochondria. However, ROS and altered redox status in these organelles also activate important mechanisms that maintain functions of these organelles under heat stress, which include HSP-dependent pathways, ROS scavenging systems and retrograde signaling. To discuss heat responses associated with energy regulating organelles, we should not neglect the energy regulatory hub involving TARGET OF RAPAMYCIN (TOR) and SNF-RELATED PROTEIN KINASE 1 (SnRK1). Although roles of TOR and SnRK1 in the regulation of heat responses are still unknown, contributions of these proteins to the regulation of the functions of energy producing organelles implicate the possible involvement of this energy regulatory hub in heat acclimation of plants. Heat stress caused by unusual temperature increase severely affects plant growth and crop production. It has been also estimated that global temperature will increase 2.0–4.9 °C by the end of 21st century . I. I145]. rt chain . These rrt chain ,151. It rt chain ; thus, s2O2 is depending on the functions of ASCORBATE PEROXIDASE (APX) that catalyzes oxidation of ascorbate to monodehydroascorbate. Reduction of monodehydroascorbate to ascorbate is carried out by the functions of MONODEHYDROASCORBATE REDUCTASES and GSH-DEPENDENT DEHYDROASCORBATE REDUCTASES [2O2, which can affect functions of the electron transport chain and oxidative phosphorylation [2O2 [Glutathione (GSH) is known to play important roles in the regulation of ROS and redox homeostasis as well as energy demand in the whole plant cell level ,153,154.DUCTASES . Mitochorylation ,161,162.ion [2O2 ,123,163.2− and H2O2. Of these, H2O2 with longer half-life compared to •O2− moves into the cytosol and functions as a signaling molecule. It was demonstrated that in Arabidopsis, H2O2 interacts with the endoplasmic reticulum and induces release of transcription factors such as ANAC013 and ANAC017, key regulators of mitochondrial retrograde signaling [Aox transcript [Links between the chloroplasts and mitochondria can be also supported by the findings associated with retrograde signaling. The chloroplasts and mitochondria share the same components of retrograde signaling, such as ROS, ANAC017, ABI4 and PAP. Both chloroplasts and mitochondria produce •Oignaling ,142. Of ignaling . ABI4 anignaling . ABI4 acignaling . In contanscript . These Wanscript , suggestanscript . In contanscript ,105. It anscript . To elucThe functions of TOR and SnRK1 to modulate growth and stress response signals depending on energy status have been extensively studied ,31,32. HArabidopsis, AKIN10, a kinase subunit of SnRK1 was shown to be activated in response to the inhibition of electron transport at PSII [Arabidopsis mutant deficient in ethylene response (etr1 mutants) showed impaired PSII efficiency, leading to energy depletion that can activate AKIN10. Taken together, these results indicate that TOR and SnRK1 might control energy usage via modulating activity of photosynthesis depending on the energy status in cells.Glucose and sucrose derived from photosynthesis as well as light signals activate TOR signaling via promoting auxin signals, which enhance cell division in the shoot and root meristems ,168,169. at PSII . Integra at PSII . An ArabSnRK1 can be activated by hypoxia accompanied by suppression of mitochondrial aerobic respiration and ATP synthesis ,173. UndAox expression [Aox expression is integrated with the primary energy/stress signaling pathways [Involvement of SnRK1 in the regulation of AOX was demonstrated in previous studies. SnRK1 reprograms stress acclimatory mechanisms by modulating expression of the stress response genes and phosphorylation of the key metabolic proteins involved in mitochondrial retrograde signaling and the TCA cycle . CYCLIN-pression ,177,178.pression . SnRK1-dpathways . In contcis element in the promoter of stress response genes functions under starvation [Based on the links of TOR and SnRK1 with functions of the chloroplasts and mitochondria, we can speculate that TOR- and SnRK1-dependent mechanisms might be also associated with ROS regulatory systems. It has been well known that ROS producing enzymes, RBOHs are involved in a broad range of processes underlying growth, development and stress responses in plants . Thus, tarvation ,181. In arvation , processarvation ,168,169.arvation ,184. Decarvation .Previous studies indicated the possible links between TOR-dependent mechanisms with heat response pathways. Glucose-dependent TOR signaling was shown to induce expression of HSPs through the modulation of TOR-E2Fa signaling accompanied by enhanced heat tolerance ,186. GluThe findings above indicate that the TOR- and SnRK1-dependent mechanisms might play important roles in the regulation of heat responses in plants, because the processes in the chloroplasts and mitochondria which are linked with TOR- and SnRK1 are known to be heat-sensitive. In future studies, it is necessary to investigate how photosynthetic and respiratory processes damaged by heat stress can be modulated via functions of TOR and SnRK1.Effects of heat stress on functions of the chloroplasts and mitochondria are mainly associated with excess production of ROS and altered redox status. ROS produced via dysfunctions of the chloroplasts and mitochondria can accelerate the damage on these organelles. However, increase in ROS production and alteration in redox status in these organelles can also trigger the acclimatory mechanisms of plants to heat stress. Indeed, many researchers have already known that ROS production needs to be strictly modulated under stresses including heat stress. The heat response mechanism activated by ROS include HSFs- and HSPs-dependent pathways. The findings described above suggest that plants possess various ROS-dependent pathways associated with the chloroplastic and mitochondrial signals to activate HSFs and HSPs in response to heat stress. Activation of heat responses via multiple pathways might be essential to flexibly tailor heat responses depending on intensity and duration of heat stress as well as growth stages of plants. We can expect that effects of heat stress on the chloroplasts and mitochondria might be different depending on such diverse situations with different energy demand.Aox transcript. Even only for mitochondrial retrograde signaling, several key components responsible for the regulation of AOX expression have been found. In contrast to photosynthesis, to some extent, respiration can be activated by heat stress to provide energy required for protection of cells, indicating the significance of the maintenance of mitochondrial functions for the heat acclimation of plants. Thus, the modulation of ROS level via AOX might be a key process to protect cells against heat stress.Another key player regulating the heat acclimation of cells might be AOX in the mitochondria. Previous studies revealed various mechanisms that activate expression of ROS and redox regulation might be essential to integrate functions of the chloroplasts and mitochondria via the exchange of metabolites and retrograde signaling, leading to the proper balance between ATP production and carbon level in cells. Significance of ROS in the regulation of carbon level and energy production could be also supported by the integrations of ROS regulatory systems with sugar metabolisms proposed in a previous review . However


Arabidopsis thaliana and briefly discuss future prospects for this field.Temperature is a key factor in determining plant growth and development, geographical distribution, and seasonal behavior. Plants accurately sense subtle changes in ambient temperature and alter their growth and development accordingly to improve their chances of survival and successful propagation. Thermomorphogenesis encompasses a variety of morphological changes that help plants acclimate to warm environmental temperatures. Revealing the molecular mechanism of thermomorphogenesis is important for breeding thermo-tolerant crops and ensuring food security under global climate change. Plant adaptation to elevated ambient temperature is regulated by multiple signaling pathways and epigenetic mechanisms such as histone modifications, histone variants, and non-coding RNAs. In this review, we summarize recent advances in the mechanism of epigenetic regulation during thermomorphogenesis with a focus on the model plant  Temperature plays an important role in plant growth and development and ambient temperature affects the entire plant life cycle , plants undergo adaptive growth, which leads to a variety of morphological changes termed thermomorphogenesis  to promote flowering and restricts H3K4me2 on the auxin biosynthesis-related gene YUCCA8 (YUC8) promoter  to promote hypocotyl elongation in warmer temperature  regulates H3K4me2 demethylation in Arabidopsis  is an important marker for transcriptional elongation and positively regulates transcription rate   , indicating a diminished response to warm temperatures , and it appears that HDA19 binding is related to the expression of these genes. HDA19 inhibits photomorphogenesis in Arabidopsis , red light (RL) and blue light (BL) conditions  to synergistically inhibit temperature responses  are reported to be involved in thermomorphogenesis. Interestingly, different HDACs have different or even opposite functions in thermomorphogenesis  and in the gene body, causing nucleosomes to wrap DNA more tightly, which influences the ability of RNA polymerase (Pol) II to transcribe genes in plants  and arp6 both have elevated HEAT SHOCK PROTEIN 70 (HSP70) expression along with early flowering, long hypocotyls, petiole elongation, and other warm temperature-related phenotypes (Kumar and Wigge FLOWERING LOCUS T (FT) for transcriptional activation and control plants timing of flowering in response to temperature , which are the pivotal transcriptional activators of the heat shock response in eukaryotes. As the temperature rises, HSFA1 binds to temperature-responsive genes and initiates transcription by dynamically facilitating H2A.Z loss, thus antagonizing the function of H2A.Z in response to warm temperature , the dominant red-light photoreceptor, exists in two photo-interconvertible forms, Pr  and Pfr  , also known as HISTONE‐ASSOCIATED PROTEIN5 (HAP5) or CCAAT BINDING FACTOR C (CBF‐C) has been reported to regulate light‐induced H2A.Z deposition associated with hypocotyl elongation‐related genes, repressing related gene expression in a light‐dependent manner  regulate the post-transcriptional expression of genes and include microRNAs (miRNAs) and small interfering RNAs (siRNAs). Small RNAs play key roles in regulating plant growth and development, signal transduction, stress responses, and genome stability  was down-regulated at warm temperatures and the flinc mutants flowered earlier than wild type, confirming the regulatory role of FLINC in thermomorphogenesis  occur widely in eukaryotes (Statello et al. PIF7 adopts a loose conformation, leading to enhanced translation efficiency (Chung et al. The metabolism and processing of mRNAs is particularly important in environmental temperature responses. For example, alternative splicing modulates plant responses to high temperature (Ling et al. Plant cell types have distinct functions and the synergies among different cell types determine the plasticity of plant development. Revealing the functions of cell types, even at the single-cell level, is crucial to understanding the environmental adaptation of plants (Rich-Griffin et al. The role of epigenetic factors in defining individual cell types and the extent to which they vary during plant development or under environmental stress remains unknown. Epigenome studies based on single-cell behavior have tremendous potential for exploring the regulatory mechanisms of plant perception and responses to changes in environmental temperature. In addition, the explosion of single-cell genomics in recent years has created an opportunity to jointly reveal the variation and differential use of genomic information in different cell types of plant species during development, differentiation, and response to environmental stresses at multiple levels Perkel . The comThermomorphogenesis of plants is closely related to their adaptation to global climate change, as global warming has a profound impact on plant morphology, development, and geographical distribution (Lee et al.


Interestingly, crosstalk between autophagy and apoptosis impacts several pathologies, including multiple rheumatic diseases. The purpose of this Special Issue is also to investigate the bioactive properties of drugs with antitumor activity, focusing particularly on the role of anthraquinone derivatives in the regulation of cell death and autophagy crosstalk. This Special Issue of Cells brings together the most recent advances in understanding the various aspects of crosstalk between autophagy and apoptosis and the interconnected signaling pathways, implying therapeutic perspectives for the utility of its modulation in an anti-cancer setting.Autophagy and apoptosis represent two fundamental pathophysiological mechanisms of cell fate regulation. However, the signaling pathways of these processes are significantly interconnected through various mechanisms of crosstalk. Indeed, autophagy/apoptosis crosstalk is still an emerging field, in which an increasing number of molecules are involved, including, for example, PINK1 and ERLINs. On the other hand, this crosstalk involves signal transduction pathways which are strongly dependent on Ca Autophagy and apoptosis represent two fundamental pathophysiological mechanisms of cell fate regulation. Novel evidence supports the view of a strict interconnection of the signaling pathways of these processes through various mechanisms of crosstalk that several studies contributed to elucidating. An example is provided by the study of Brunelli et al. who investigated the role of PINK1 in the crosstalk between autophagy and apoptosis [2+ [2+ functions as a second messenger are critical in coordinating fundamental physiological functions, including cell survival and growth, neuronal development and/or the maintenance of cell functions. The coordination among proteins/pumps/Ca2+ channels and Ca2+ storage in different organelles is critical in preserving cytosolic Ca2+ levels that keep the spatial resolution needed for cellular homeostasis. Ca2+ homeostasis is regulated by a store-operated Ca2+ entry (SOCE) mechanism that is activated by the depletion of Ca2+ from internal ER stores. Ca2+ has been shown to control opposing functions, such as autophagy, that promote cell survival; on the other hand, Ca2+ also regulates programmed cell death processes, including apoptosis. Recently, it has been evident that a complex network of lipid–lipid and lipid–protein interactions promotes the activation of a variety of signaling pathways regulating cell homeostasis [On the other hand, the crosstalk of autophagy and apoptosis involves signal transduction pathways which are strongly dependent on Caeostasis . In thiseostasis . Howevereostasis , but areeostasis . In parteostasis . The proIndeed, crosstalk between autophagy and apoptosis impacts several pathologies, including multiple rheumatic diseases. Since mitochondria are significant regulators in maintaining cartilage homeostasis, turnover of mitochondria through mitochondrial biogenesis and mitochondrial degradation may play an important role in the pathogenesis of osteoarthritis (OA). Kim et al. discuss the role of mitochondrial dysfunction in OA pathogenesis, identifying the peroxisome proliferator-activated receptor-gamma co-activator 1-alpha (PGC1α) as a potent regulator . They deThe purpose of this Special Issue is also to investigate the bioactive properties of drugs with antitumor activities, focusing on their role in the regulation of autophagy and cell death crosstalk that triggers the uncontrolled expansion of tumor cells. In this vein, an example is represented by anthraquinone derivatives, which may affect autophagy and/or apoptosis and promote oxidative changes in cancer cells ,11. In pAltogether, the articles in this Special Issue further improve the knowledge of the mechanisms regulating cell fate, analyzing either the signaling pathways regulating cell death and survival or the bioactive properties of drugs acting on the crosstalk between autophagy and apoptosis. This understanding may drive the development of a new therapeutic strategy for cancer and/or autoimmune diseases.


Cells is devoted to the recent advances in the identification of the molecular basis of NDDs, as well as the pathophysiological basis of these disorders. We invited original research articles and reviews on the “Pathophysiological Mechanisms of Neurodevelopmental Disorders”. Compiling articles that explore these mechanisms from molecular to cellular, tissue and animal model perspectives, as well as potential therapeutic strategies, this Special Issue successfully features various achievements in this rapidly expanding field by collating new knowledge and presents the state of the art for a wide range of NDDs.Technological advancements in next-generation DNA sequencing have enabled elucidation of many genetic causes of neurodevelopmental disorders (NDDs) over the last two decades. However, the investigation of the molecular mechanisms underlying the pathophysiology of NDDs has advanced far less quickly, because multiple interdisciplinary experiments, which are generally low throughput, are essential for this purpose. This Special Issue of The organized structure and spatial organization of the human brain is regulated by highly coordinated developmental events, such as neural cell proliferation, migration and differentiation. Sapir et al. employed a developmental perspective to describe and understand the etiology of common cortical malformations and their manifestation in the human brain .As for the organella function, the endosomal recycling is executed by the two protein complexes, Retromer and Retriever, whose defects cause dysregulation of many membrane proteins and result in several disorders, including NDDs. Saitoh reviewed the biological and developmental roles of the two complexes, and the consequences of defects in endosomal recycling . Rho family small GTPases regulate cellular signaling and cytoskeletal dynamics, playing a pivotal role in cell adhesion, migration, and cell-cycle progression. Among the Rac subfamily of Rho GTPases, the proper functions of Rac1 and Rac3 are crucial for neural development, and pathogenic variants affecting their biological processes are implicated in NDDs. Scala et al. reviewed the current pertinent literature on Rac-related disorders with a primary neurological involvement and provided an overview of the current knowledge on the pathophysiological mechanisms involved in the neuro-RACopathies [RAC1/3 genes, dysregulated RAC1/3 functions resulting from pathogenic variations of genes encoding guanine nucleotide-exchange factors (GEFs) and GTPase-activating proteins (GAPs) have been involved in the pathogenesis of NDDs. A RhoGAP, Oligophrenin-1 (OPHN1), is enriched at the synapse and plays a critical role in cytoskeleton remodeling and vesicle recycling. The OPHN1 gene mutations are associated with X-linked ID (XLID). Cresto et al. clarified that OPHN1 function in synapses is differentially affected during maturation of the brain, which provides some opportunities for early therapeutic intervention [In addition to a direct deleterious effect produced by genetic variants in the rvention .CNKSR2, which encodes a scaffolding molecule CNKSR2, is also responsible for XLID. CNKSR2 interacts with different molecules and regulates the MAP kinase (mitogen-activated protein kinase) cascade and Rho family small GTPase signaling. Ito and Nagata reviewed the possible pathophysiological mechanism of XLID by disrupting CNKSR2 functions, and summarized molecular features, neuronal function and NDD-related variations of CNKSR2 [f CNKSR2 . Protein phosphorylation plays critical roles in a variety of intracellular signaling pathways in the brain, of which dysregulation has been implicated in NDDs. Ahammad et al. reported a novel online database to provide information about the phosphorylation signals identified using the “KANPHOS”  database . The autGTF2I). By studying a novel murine model with selective deletion of Gtf2i in the excitatory neurons of the forebrain, Grad et al. demonstrated the substantial effect a single gene deletion can exert on miRNA regulation and brain structure and advanced our understanding of WS [Williams syndrome (WS) is a multisystem NDD caused by a de novo hemizygous deletion of ~26 genes from chromosome 7q11.23, including the transcription factor II-I  inherited from their healthy parents. While subsequent HPLC analysis clarified N-glycan-processing defects, MAN1B1 activity was compromised in patient-derived lymphocytes.Congenital disorders of glycosylation (CDG)—inherited metabolic diseases caused by defects in glycosylation—are associated with ID. Okamoto et al. examined two siblings with ID, dysmorphic features, early onset epileptic encephalopathy, cerebral infarction, etc. . Whole-eSeizure threshold 2 (SZT2) is a component of the KICSTOR complex which regulates the mechanistic target of Rapamycin complex 1 (mTORC1). Mutations in this gene cause NDDs. Cattelani et al. performed a systematic interactome analysis of SZT2 and identified clusters of proteins related to autophagy, ciliogenesis regulation, neurogenesis and neurodegenerative processes . They thNR2F1 gene, encoding a transcriptional regulator [Bertacchi et al. focused on the pathophysiology of Bosch–Boonstra–Schaaf Optic Atrophy Syndrome (BBSOAS), a recently described monogenic neurodevelopmental syndrome caused by the haploinsufficiency of egulator . The autAUTS2 has emerged as a crucial gene associated with a wide range of NDDs. Hori et al. summarize the knowledge regarding AUTS2, including its molecular and cellular functions in neurodevelopment, its genetics and its role in behaviors [AUTS2 is involved in transcriptional regulation, neuronal migration and neuritogenesis during brain development, while AUTS2 postnatally regulates the number of excitatory synapses to maintain the balance between excitation and inhibition in neural circuits. Notably, ehaviors .Deficits in the inhibitory circuits by interneurons that regulate the excitatory–inhibitory (E–I) balance toward excitation have been implicated in NDDs, including Fragile X syndrome (FXS). Indeed, manipulating the interneuron activity ameliorated the symptoms in the FXS mouse model, which makes it reasonable to conceptualize FXS as an interneuronopathy. While the ways in which the developmental profiles of the inhibitory circuit go awry in FXS are still poorly understood, recent works have uncovered several developmental alterations in the functional properties of interneurons. Nomura reviewed the recent evidence of the inhibitory alterations and interneuron dysfunction in FXS and discussed the therapeutic potential of correcting disrupted E–I balance in FXS . Chromosomal microarray testing reliably detects minute changes in genomic copy numbers. Haploinsufficiency is considered to be the pathogenic mechanism when the deletion of a gene is related to NDDs. While loss-of-function mutations may be evaluated using next-generation sequencing, increased copy numbers may exhibit different clinical symptoms; the additional copies of a gene may have a dominant negative effect. In addition, gene mutations with dominant negative effects are responsible for causing the clinical symptoms. Currently, the diagnostic yield of genomic alterations using comprehensive analysis is less than 50%, indicating the existence of more subtle alterations or genomic changes in the untranslated regions, while copy-neutral inversions and insertions may be related. Yamamoto outlined the genetic factors of NDDs and reviewed the current state of diagnosis by genomic medicine .The Axon initial segment (AIS), proximally located at the interface between the axon and cell body, has characteristic molecular and structural properties, and is a key domain for the control of neuronal excitability by homeostatic mechanisms. The AIS has high plasticity in normal developmental processes and pathological activities including NDDs. Fujitani et al. provided an overview of the molecular and structural characteristics of AIS, as well as AIS regulation through axo-axonic synapses, and axo−glial interactions . They th


Alterations in the sphingolipid metabolism of Parkinson’s Disease (PD) could be a potential diagnostic feature. Only around 10–15% of PD cases can be diagnosed through genetic alterations, while the remaining population, idiopathic PD (iPD), manifest without validated and specific biomarkers either before or after motor symptoms appear. Therefore, clinical diagnosis is reliant on the skills of the clinician, which can lead to misdiagnosis. IPD cases present with a spectrum of non-specific symptoms  that can occur up to 20 years before motor function loss  and formal clinical diagnosis. Prodromal alterations in metabolites and proteins from the pathways underlying these symptoms could act as biomarkers if they could be differentiated from the broad values seen in a healthy age-matched control population. Additionally, these shifts in metabolites could be integrated with other emerging biomarkers/diagnostic tests to give a PD-specific signature. Here we provide an up-to-date review of the diagnostic value of the alterations in sphingolipids pathway in PD by focusing on the changes in definitive PD (postmortem confirmed brain data) and their representation in “probable PD” cerebrospinal fluid (CSF) and blood. We conclude that the trend of holistic changes in the sphingolipid pathway in the PD brain seems partly consistent in CSF and blood, and could be one of the most promising pathways in differentiating PD cases from healthy controls, with the potential to improve early-stage iPD diagnosis and distinguish iPD from other Parkinsonism when combined with other pathological markers. Parkinson’s disease (PD) is the second most prevalent progressive neurodegenerative disorder with no definitive diagnosis available for idiopathic cases (iPD), which accounts for the majority of cases 85–90%) . Current–90% . CuAntemortem iPD diagnosis is urgently required as, currently, the definitive diagnosis of iPD is available only postmortem by confirmation of neuronal loss in the SN together with the presence of Lewy bodies . The proOther novel imaging techniques are emerging as potential tools for the prodromal diagnosis of iPD. For instance, neuromelanin imaging MRI and quantitative susceptibility mapping ,11,12 diNeuronal loss is preceded by long-lasting alterations in cellular mechanisms, the metabolites of which could be identified for prodromal diagnosis of iPD. Since the initial discovery that familial parkinsonism can be caused by multiplications and mutations in the SNCA gene, there has been a worldwide focus on the role of α-synuclein (αSyn) in the progression of PD . Indeed,Among pathways implicated in PD, the dysfunction of the protein clearance system is the closest cellular mechanism that could explain why misfolded protein accumulation in the brain appears as a core pathology in PD. The accumulation of intra-cellular αSyn implicates protein clearance systems; the ubiquitin-proteasome system  and the The autophagy-lysosomal pathway could be one of the promising pathways in differentiating PD from controls. This pathway orchestrates lipid metabolism, given the fact that lysosomes host multiple lipid hydrolysis enzymes and act as the hub for the degradation of various types of macromolecules . Lipids One of the most common risk factors for iPD is mutations in the glucosylceramidase beta (GBA) gene, which is associated with early disease onset, severe cognitive dysfunction and a higher mortality rate from diagnosis compared to noncarriers ,31,32. TSince PD and the GBA gene association were reported in 1996, many studies have explored the phenotypic impact of GCase activity in PD, yet data remain inconclusive ,36,37. TFor misfolded proteins to be cleared, ubiquitin ligases are required to tag them with ubiquitin molecules to signal proteolysis. These ubiquitylated proteins are then handed over to the 26S proteasome—the most used proteasome in mammals with a 20S catalytic core and 19S regulatory particles at both ends that break misfolded proteins into small peptides. Non-proteasomal endopeptidases and aminopeptidases then digest these small peptides into monomeric amino acids for reused in new protein synthesis .Failures in the ubiquitin-proteasome system (UPS) in PD are reported in both familial and iPD cases. In familial cases, an E3 ubiquitin ligase, parkin, is associated with early-stage PD (<30 years) . InteresAutophagy is a conserved lysosomal degradation process for selective removal of damaged subcellular organelles , midbody ring structures, ribosomes and aggresomes . This prSphingolipid pathway impairment is not restricted to only PD neurons or PD CSF but seems to be a systemic feature of PD evidenced by reduced GCase activity in blood and fibroblasts , which aThe GCase enzyme is a 536 amino acid length lysosomal-membrane-anchored protein that catalyzes glucosylceramide into glucose and ceramide , and gluIn definitive PD cases, which are diagnosed by postmortem confirmation of αSyn accumulation into Lewy bodies, reduced GCase activity is reported in both GBA mutation carriers and iPD ,36. In tGCase enzyme activity depends on several regulatory mechanisms including structure-functional changes, pH and the availability of its activator SapC; disruptions in any of these steps could potentially affect downstream GCase activity. The GBA gene located at 1q21 encodes the lysosome-membrane-anchored GCase enzyme and its mutations are implicated in iPD . After tThe prosaposin gene (PSAP) encodes a precursor protein that forms into sphingolipid activator proteins (SAPs)—saposin A-D—that facilitate lysosomal hydrolysis of sphingolipids . In lysoThe inconsistency of data on GCase activity is also represented at the substrate level. Here, the conflict starts from brain data, with some reports of no accumulation of GCase substrates (glucosylceramide and glucosylsphingosine) in the PD-GBA brain , yet theThe GCase enzyme interacts with sphingolipid pathways and changes in specific substrates could have variable indications in PD diagnosis; therefore, there is growing interest in a comprehensive analysis of sphingolipids pathway for biomarker discovery.Post-mitotic neuron survival is partly governed by sphingolipids through modulating the autophagy process (as reviewed by ). AutophCeramides are the precursor of complex sphingolipids, which are implicated in PD pathology (as reviewed in ) and cogThe data on other components of the sphingolipid pathway is also limited; however, reduced activity of the enzymes involved in this pathway, and in some cases, the accumulation of corresponding substrates, introduce a systemic disruption in sphingolipid metabolism in PD subjects . Here, GSphingolipids’ pathway dysregulation is becoming indicated in prodromal PD. The GBA gene mutations are listed as an intermediate-strength genetic factor for prodromal PD, where the total PD risk in mutation carriers at age 65 is 9 times higher (18% in PD versus 2% in age-matched controls) . InteresAltogether, data on using sphingolipids pathway as a diagnostic tool are promising but limited, and future studies with large patient populations and stratified controls could help confirm the specificity of different parts of the sphingolipid catabolism pathway and their respective diagnostic value for iPD.Integrating metabolites representing iPD pathophysiology with lower specificity and sensitivity could enhance the accuracy of iPD differentiation from other parkinsonism. To the best of our knowledge, there is no published evidence using this approach, yet preliminary data on biomarker integration support enhanced diagnostic accuracy. For instance, a combination of GCase, cathepsin D and β-hexosaminidase improves diagnostic accuracy—from an area under the curve value of 0.72; with a sensitivity of 0.67; and specificity of 0.77 for only GCase activity, to an area under the curve value of 0.77; sensitivity, 0.71; and specificity, 0.85 for the combination in differentiating PD from healthy controls . This imIntegrated biomarker set accuracy can also be further improved by incorporating markers from other generalist and specific pathways that represent the same trend of changes in CSF and/or serum as those seen in the PD brain such as 3-hydroxykynurenine ,95, apolA definitive antemortem diagnosis of iPD remains an urgent and unaddressed need, especially during the prodromal stage. Several lines of clinical evidence on the diagnostic potential of GCase activity and its substrate accumulation in iPD are inconclusive; however, a growing interest in a comprehensive analysis of GCase activity in the context of the sphingolipid catabolism pathway is promising for non-invasive differentiation of iPD from healthy age-matched controls. Since PD is a multisystem disease with shared pathology with other atypical parkinsonism, future investigations with the integration of potential biomarkers from the sphingolipid pathway, imaging data and clinical symptoms could help improve iPD diagnostic accuracy in the clinic in early stages. Earlier diagnosis may also allow improved therapeutic interventions, where a wider therapeutic window for neuroprotection could be exploited.