This sequence of events will include initial defense against excessive reactive oxygen species production, compensation mechanisms by the unfolded protein response (UPRmt), mitochondrial dynamics and elimination by mitophagy.Mitochondria undergo continuous challenges in the course of their life, from their generation to their degradation. These challenges include the management of reactive oxygen species, the proper assembly of mitochondrial respiratory complexes and the need to balance potential mutations in the mitochondrial DNA. The detection of damage and the ability to keep it under control is critical to fine-tune mitochondrial function to the organismal energy needs. In this review, we will analyze the multiple mechanisms that safeguard mitochondrial function in light of  The eukaryote cell is an exceptionally complex organization of macromolecules. While initial crucial steps in the origin of eukaryote life might include the development of flexible cell surface, complex cytoskeletal organizations and heavily specialized cell compartmentalization, the ability of the cell to communicate with the external milieu has been vital for the efflorescence of specialized tissues and multicellular organisms.The origins of mitochondria probably find their place in the engulfment and symbiotic establishment of a proteobacteria into the protoeukaryotic cell. Mitochondria act as the cellular powerhouses and play a central role in bioenergetics and metabolism of amino acids and lipids, since they host fatty acid β-oxidation, the Kreb’s cycle  cycle) and oxidative phosphorylation (OXPHOS) 2 and ROS. An imbalance between ROS generation and the organismal/cellular system's ability for clearance, promotes oxidative damage to lipids, nucleic acids and proteins Nevertheless, mitochondrial physiology engages into a number of significant challenges. One of them, intrinsic to its respiratory function, is the management of OMitochondria have historically been viewed as relatively passive generators of the ATP that is necessary to thermodynamically drive many cellular biochemical reactions. However, mitochondria have also developed mechanisms to communicate with the rest of the cell, probably to ensure that cells do not commit to a biological event that mitochondria cannot metabolically sustain . The biochemical nature of these molecules is very broad and includes mitochondrial DNA (mtDNA) fragments, mitochondrial lipids (e.g.: cardiolipin), metabolites and small peptides  with similar function to humanin and whose levels decreased with age Figure 1). This allows coupling chromatin-dependent gene regulation with the metabolic state of the cell Metabolites released from the mitochondria to the nucleus also act as retrograde signaling systems, and many of them constitute substrates for chromatin-modifying enzymes . ROS react with the redox-sensitive cysteine residues of tyrosine phosphatases leading to their transient inactivation, which favors unopposed kinase activity 26C. elegans30Mitochondria are also an important source for ROS. ROS molecules have long been known as being damaging and pernicious agents to the cell, resulting in oxidative stress . However, ROS play a critical role as signaling molecules to maintain physiological functions. In this regard, superoxide anions and hydrogen peroxide were probed to activate signaling pathways controlled by tyrosine phosphorylation such as NF-κB, PKC, MAPK or JNK 242) levels. This proteobactium helped in the removal of the O2 that was being diffused inside the host cell from the environment 322 consumption to ATP synthesis allowed the sustainability of eukaryotic cellular bioenergetics as we know them today. In addition, aerobic metabolism spurred the generation of new metabolites such as steroids, alkaloids and isoflavonoids 34The integration of an α-proteobacterium within an ancient host cell around 1.45 billion years ago was key for the survival and replication of the host in an environment with increasing oxygen , but also to the remodeling of protein structure and function, for example, via post-translational modifications 2−), hydrogen peroxide (H2O2) and the hydroxyl free radical (OH-). O2− causes the formation of other reactive species: it can directly produce OH- or indirectly through the dismutation of H2O2. H2O2 has low reactivity, but high penetrability in cell membranes and when it accumulates, it is highly toxic to cells 372O2 can be converted to OH- in the presence of Fe2+38OExcessive ROS production or an ineffective antioxidant response results in oxidative stress 374546472− to H2O2 and O2, and they are considered as one of the most powerful antioxidant agents in cells  2O2, SOD1 can translocate to the nucleus, where it binds to DNA promoters and favors the expression of oxidative resistance and repair genes 5558Eukaryotic cells harbor complex antioxidant strategies to protect against an uncontrolled increase in free radicals. This includes both enzymatic and non-enzymatic mechanisms (i.e. vitamins). The enzymatic response is primarily mediated by superoxide dismutases (SOD), catalases, thioredoxin reductases or glutathione peroxidases 2O2 into water and molecular oxygen by utilizing iron or manganese as a cofactor, thereby completing the detoxification initiated by SOD 612O2 to H2O and O2 is carried out by another type of enzymes known as glutathione peroxidases (GPx), which use a selenium intermediate and glutathione as substrates 64662O2, it has been proposed that the glutathione redox cycle is a source of protection against mild oxidative stress as compared to CAT, which protect against severe oxidative stress Catalases (CAT) can break down H+2O2 into water 7172Thioredoxin reductases (TrxRs) are enzymes characterized by the redox activity of its flavin adenine nucleotide (FAD) group, which helps reducing thioredoxins (Trxs) by the oxidation of NADPH to NADP2−, also contributes to the antioxidant response by inducing the NRF2/KEAP pathway mediated by fumarate accumulation and ERK5 activation The expression of ROS metabolizing enzymes can be directly controlled by oxidative stress. Primary responses to ROS are modulated by the cooperation of the NF-κB, AP1 and MAPK pathways. When these responses are not sufficient to counteract the increase in ROS, the NRF2/KEAP cascade is activated to induce antioxidant defenses and minimize oxidative damage Overexpression or knockout of the ROS metabolizing enzymes has helped in the understanding of the function and activity of the different groups of antioxidant enzymes, as well as how their dysregulation can lead to disease. For instance, SOD3 ablation in adult mice caused increased lung superoxide content, inflammation, respiratory acidosis and reached 85% mortality within six days mt), the endoplasmic reticulum UPR (UPRER) and the cytosolic heat-shock response (HSR), exist as quality control mechanisms against proteostatic stress cues that put at risk cellular homeostasis, and that can lead, ultimately, to cell death and apoptosis.To guarantee the appropriate folding, assembly and turnover of proteins under normal and stress conditions, cells modulate the levels of proteases and chaperones involved in protein quality control. In eukaryotes, the cytosol, ER and mitochondria are all exposed to nascent polypeptides, thus each compartment requires specific protein-folding machinery and responds differently upon unfolded polypeptides, signaling to the nucleus to induce the expression of organelle-specific chaperones. The unfolded protein response (UPR) encompasses, thus, a collection of signaling pathways that evolved to restore an efficient protein-folding environment. The mitochondrial UPR (UPRmt), characterized by the upregulation of nuclear genes encoding mitochondrial molecular chaperones and proteases, in order to ensure the functional integrity of the mitochondrial proteome mtper se is enough to reduce the abundance or aggregation of misfolded proteins. Similarly, it is not clear whether the removal of these aggregates is required for the potential recovery of mitochondrial function after UPRmt.One of the first hints to the existence of a mitochondrial UPR was provided by the Hoogenraad laboratory in a simple yet pioneering experiment overexpressing OTC-Δ - a mutant form of the mitochondrial matrix protein Ornithine transcarbamylase - as a way to promote protein misfolding and aggregation. This artificial paradigm to induce variations in the stoichiometry of the mitochondrial- and nuclear-encoded proteins in the mitochondrial matrix led to the increased expression of HSP60 and CLPP (ATP-dependent caseinolytic protease proteolytic subunit) in cultured mammalian cells 8686mt include mtDNA depletion, impaired mitochondrial protein quality control or OXPHOS dysregulations. These defects influence one another, as OXPHOS or mitochondrial proteostasis perturbations reduce the rate of protein import inside mitochondria by increasing the inner mitochondrial membrane (IMM) proton permeability, which consequently dissipates the proton gradient and causes precursor proteins to remain outside the mitochondria 88Some of the factors triggering the UPR2 in the process The mitochondrial genome encodes 13 proteins that are constituents of the OXPHOS respiratory complexes, but the rest of the mitochondrial proteome is encoded in nuclear genes, synthetized in the cytosol and imported to the different mitochondrial compartments via the TOM (translocase of the outer membrane) and TIM (translocase of the inner membrane) channels 90mt molecular mechanism has been mostly characterized in C. elegans due to the relative ease of using this organism to perform genetic screenings. In worms, the response is characterized by the action of regulators including the mitochondrial matrix protease CLPP, the ATP-binding cassette (ABC) transporter HAF-1 and the basic leucine zipper (bZIP) transcription factor ATFS-1. Briefly, upon mitochondrial proteostatic stress, CLPP proteolytic activity degrades misfolded proteins in the mitochondrial matrix, and the small peptides are then transferred across the IMM via HAF-1. Afterwards, these peptides are moved through the outer mitochondrial membrane (OMM) by passive diffusion to the cytosol . This activates ATFS-1, which translocates to the nucleus thanks to a nuclear localization sequence (NLS) and activates the ubiquitin-like protein UBL-5 to form a complex with the transcription factor DVE-1 94. Recent studies also demonstrate that chromatin is specifically remodelled during mitochondrial dysfunction to activate the UPRmt - responsive genes. The histone methyltransferase MET2 in concert with LIN65 promote global chromatin condensation, while the histone demethylases JMJD-3.1 and JMJD-1.2 maintain the promoters of UPRmt-induced genes in a transcriptionally competent state, and this structure is further stabilized by the DVE-1/UBL-5 complex to facilitate ATFS-1 access to chaperone promoters Although initially discovered in mammals mt response gene sets encode as well for mitochondrial proteases, ROS detoxification enzymes, and mitochondrial protein import components mt in C. elegans is different to that of mammalian cells. This emphasizes that the mechanisms of proteostatic regulation differ between organisms and that equivalences between models should be taken with some caution. For instance, although some of the components of the pathway, such as the mitochondrial chaperones and the quality control protease CLPP were shown to be conserved from C. elegans to mammals, the closest mammalian homologs of DVE-1, SATB1 and SATB2, are unable to stimulate the UPRmtOther UPRmt is mediated by the CHOP- C/EBPβ system 87Figure 3b). Once expressed, the CHOP- C/EBPβ system binds to specific gene promoters, characterized by the presence of the CHOP binding site and two mitochondrial unfolded protein response elements (MURE1 and MURE2), promoting the transcription of UPRmt -responsive genes. The bioinformatics approach by Aldridge et al. (2007) identified seven genes UPRmt -responsive, including YMEL1L1, mitochondrial thioredoxin 2 (Trx2), NDUFB2 (subunit of complex I) or CLPP mt is questionable, as its promoter contains both an UPRER and an UPRmt response element mt87mt that senses excess of unfolded proteins in the IMS and that is independent of CHOP 105In mammals, the UPRmt in mammals, in a mechanism dependent on the phosphorylation of the eukaryotic translation initiation factor 2 (eIF2α) 108Induction of the integrated stress response (ISR) has also been reported to coordinate the UPRet al. (2010) developed a mouse model carrying a mutation in TWINKLE - a mtDNA helicase - that lead to the accumulation of mtDNA deletion, resulting in respiratory chain deficiency and the induction of the MSR et al. (2017) showed the overlapping between the MSR and the ISR via the specific activation of ATF4, which resulted in the upregulation of pathways related to biosynthesis of amino acids, in particular serine Moreover, several mitochondrial stress responses (MSRs) are induced by other factors different to protein misfolding and that can trigger the ISR. For instance, Tyynismaa Mitochondrial function is also altered by its communication with other cellular organelles, besides the nucleus. Mitochondria physical interaction with the ER has many functional implications, including calcium exchange or lipid transfer, but also targets the ER as one of the first sites when mitochondrial function is disrupted. The association between defective mitochondria and the ER has been demonstrated to increase ER stress, resulting in incorrect protein folding ER). The UPRER was the first stress response identified 117121+2 and induces Caspase12 cleavage and consequently apoptosis +2 released from the ER by inositol-1,4,5-triphosphate (InsP3) and also sustains lipid biosynthesis, which occurs at sites of ER membranes attached to mitochondria that contain phospholipid and glycosphingolipid biosynthetic enzymes 126The accumulation of toxic, unfolded proteins in the ER leads to ER stress and the activation of the ER unfolded protein response  demonstrated that suppression of protein synthesis due to ER stress had an effect on the assembly of the COX complex by disruption of the synthesis of COX subunits and ATP-dependent enzymes Multiple models of mitochondrial dysfunction are characterized by ER stress. Elevated levels of free Caet al. (2017) reported that, in the absence of HSF1, the levels of NAD+ and ATP are not sustained in hepatic cells, and this increased protein acetylation and impaired mitochondrial integrity. Furthermore, the activity of HSF1 upon low levels of mitochondrial stress regulates cytoplasmic proteostasis and healthspan in worms ER - the intricate regulation between the cytosolic UPR and mitochondrial function.The cytosolic UPR is mediated mainly by the heat-shock factor (HSF) family of proteins, maintaining protein-folding homeostasis in the cytosol. In eukaryotes, ribosome-associated chaperones such as NAC (nascent chain associated complex) and RAC (ribosome associated complex) interact with the ribosome and bind to hydrophobic elements of newly synthesized polypeptides, facilitating folding or polypeptide transfer to downstream chaperones 133140Damaged mitochondria that are not salvageable by the UPR can be identified and specifically degraded through a process called mitophagy . This process helps eliminate the impaired mitochondria that could harm the rest of the functional network, and will be discussed in the next section.Mitochondrial dynamics are the repetitive cycles of fission and fusion of the mitochondrial network. These are highly orchestrated events influenced by a variety of physiological and environmental cues. For instance, nutrient supply: nutrient overload is linked to a fragmentation of the mitochondrial network, while mitochondria elongate under starvation 145148149150152154Fission and fusion events act as a quality control mechanism by removing defective mitochondria or by selecting the organelles with the optimal matrix metabolites, intact mtDNA copies and mitochondrial membrane components. This interplay between mitochondrial dynamics and mitophagy - the selective removal of mitochondria by the autophagic machinery - assures the homeostasis of the cell. When one of these elements fails, dysfunctional mitochondria are not properly removed from the cellular pool, generally leading to higher amounts of ROS production and increased susceptibility to release cytochrome c and apoptosis-inducing factor (AIF) 156et al. (2008) showed, by measuring the bioenergetic profile during fusion/fission events in COS7 and INS1 cells, that mitochondria tend to maintain a stable membrane potential ΔΨm (within ( 2.7 mV) m and, in the majority of the cases, one daughter mitochondria will be depolarized while the other will hyperpolarize (ΔΨm difference > 5 mV), resulting in asymmetric daughter mitochondria. Depolarization below a certain ΔΨm is associated with impaired mitochondrial function and acts as the trigger for mitophagy 163Generally, mitochondrial fragmentation precedes mitophagy as mitochondria that are smaller are easier to be engulfed by the autophagosomes (APs) 158159160161The perspective of mitochondrial fission acting upstream of mitophagy was validated by genetic manipulation of the pro-fission proteins Fis1 (Fission 1 protein) and Drp1 (Dynamin-related protein 1). The mammalian Drp1 is a GTPase that localizes mostly in the cytosol, but that can cycle on and off the OMM, where it can be docked by a set of different proteins, such as Fis1, the mitochondrial fission factor (Mff) or MiD49/51 163Following mitochondrial fission and depolarization, Parkin and PINK1 (PTEN-induced putative kinase 1) are recruited to the OMM to tag the organelle for mitophagy. PINK1 is a serine/threonine kinase that identifies and targets specific mitochondria for degradation. Healthy mitochondria maintain a stable membrane potential that facilitates the import of PINK1 into the mitochondrial matrix, where it is cleaved and degraded m depolarization can also act as regulators of mitophagy by promoting Opa1 cleavage. Opa1 regulates mitochondrial fusion and cristae structure in the IMM 175178Mitochondrial ATP status and ΔΨFigure 4). Nevertheless, it should also be noted that mitophagy can be modulated in a PINK1/Parkin - independent manner, upon hypoxia or targeting by other OMM proteins. For instance, Nix is an OMM receptor that activates the apoptotic machinery and favors the elimination of mitochondria during red blood cell differentiation and elimination in erythrocytes 182in vivoOverall, sustained membrane depolarization results in cleavage of Opa1 in the IMM, accumulation of PINK1/Parkin and ubiquitination of Mfn1/2 in the OMM, and this promotes the damaged and targeted mitochondria to be engulfed by the AP  allowed the visualization of mtDNA nucleoid dynamics after complete fusion of the mitochondria. Rho0 cells recovered their ΔΨm after fusion with wild type cells, and this was prevented by the deletion of Mfn1/2 and Opa1. Interestingly, motility of the newly fused mitochondria was increased 192+2 oscillations also contributes to increased fusion rate et al. (2017), reporting that unsaturated fatty acids impede outer membrane fusion, establishing a mechanistic crosstalk between mitofusins and fatty acid desaturation Mitochondrial fusion contributes to the maintenance of a stable ΔΨet al. (2008) that, after fission, fusion occurs preferentially between mitochondria with higher ΔΨm, as compared to the subpopulation of non-fusing mitochondria that presented depolarized ΔΨm. The depolarization of an individual mitochondrion is also accompanied by reduced levels of Opa1, and this decreases its probability of undergoing fusion m and loss of Opa1 activity, would promote targeting of mitochondria for autophagy. The selectivity of mitochondrial fusion not only prevents the migration of damaged components into active mitochondria, but is also an isolation step that creates a segregated population that is available for autophagy .Then, what determines whether mitochondria should be rescued by fusion from autophagy? Redistribution has an impact upon mitochondria function. Non-selective fusion would contribute to damaged mitochondria impairing the activity and efficiency of the healthy population. It was demonstrated by Twig Nevertheless, fusion is not only a compensatory, but also a protective mechanism of mitochondria against metabolic and stress challenges, carried out by modification of the key players in the fusion machinery. Upon starvation, the increase in cAMP levels activates PKA, which phosphorylates Drp1 at Ser637 and keeps it in the cytosol, leading to unopposed mitochondrial fusion. Elongated mitochondria are spared from autophagic degradation, present higher oligomerization of ATPase and increased efficiency of ATP production. Therefore, mitochondrial elongation during starvation protects cells from death Mitochondrial fusion has been suggested to provide a new treatment for mitochondria-related diseases such as diabetes, muscular dystrophies or neurodegenerative disorders, hence the increasing interest in developing activators of mitochondrial fusion Therefore, the elimination of damaged mitochondria by mitophagy and the elongation of the functional mitochondrial network by fusion could act as complementary processes to guarantee cell homeostasis and disease prevention. The choice of one or another to preserve mitochondrial health might depend on multiple parameters, including the degree of mitochondrial damage, the basal mitochondrial turnover rate or the intrinsic ability of the tissue/cell type to mobilize mitochondria for degradation.Mitochondrial biogenesis is the process of increasing cellular mitochondrial mass. In most cases, mitochondrial biogenesis occurs in response to energy deficit, triggered either by increased cellular energy demand or by impaired ATP synthesis. Hence, it is not surprising that both situations share, at least in part, common mechanisms to increase mitochondrial copy number.One possible starting point for mitochondrial biogenesis in situations of energy stress might be found in the activation of the AMP-activated protein kinase (AMPK). AMPK is a heterotrimeric enzyme composed by an α, β and γ subunit, all of which can be present as different isoforms per se, but might alter the ability of PGC-1α to interact with other proteins AMPK influences mitochondrial biogenesis, in part but not exclusively, through the activation of the transcriptional coactivator PGC-1α (peroxisome proliferator activator receptor gamma coactivator 1a). PGC-1α was originally described as a cold-induced transcriptional coactivator in brown adipose tissue 206208+-dependent protein deacylase SIRT1 210+ is high enough to make physiological intracellular levels of NAD+ rate-limiting for SIRT1 activity + sensor in the cell, hence responding to metabolic and oxidative stress challenges. AMPK has been shown to increase intracellular NAD+ levels through metabolic rewiring upon fatty acid availability 2 delivery or oxidative stress defenses, including PPARα, ERRγ and Forkhead-O-box transcription factors Interestingly, AMPK influences the activity of another key regulator of cellular metabolism and mitochondrial function, the NADPGC-1α, however, is a nuclear coactivator, and mitochondrial biogenesis requires the replication and transcription of the mitochondrial genome. Bringing light into these issues, it was shown that the nuclear expression of the mitochondrial transcription factor A (TFAM), the key controller of mitochondrial DNA replication, is controlled via the nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2) mtAMPK exemplifies how a mitochondrial-related cue  can be transformed into a metabolic adaptation. Nevertheless, mitochondrial biogenesis and PGC-1α can be controlled through other means, for examples in situations of cancer or cellular proliferation Mitochondria are a key source for fuel and intermediate metabolites that are critical for cellular proliferation, differentiation, growth and function. The clear link between mitophagy and human diseases suggests a potential applicability of this process as a therapeutic target. However, some aspects need to be taken into consideration for the development of therapeutic strategies, including the tissue to be targeted or the severity of the disease. In physiological conditions, mitochondrial turnover takes place at very different rates depending on the tissue examined, and may even vary in the mitochondrial population of tissues of the same type mt are still too vaguely defined. It will be important to define a clear standardization in the evaluation of UPRmt as, in fact, most works do not characterize mitochondrial protein aggregation.The different mechanisms ensuring mitochondrial quality control may gradually enter into scene, starting by basic defense systems such as the antioxidant responses against oxidative damage resulting from increased production of ROS. When these defenses are not sufficient and affect protein function and folding, mitochondria communicates to the nucleus to activate the unfolded protein response mediated by chaperones and proteases to restore protein homeostasis. Mitochondrial dynamics and mitophagy will also act reciprocally to maintain the quality, not just of individual organelles, but of the entire mitochondrial network. When this process is compromised, different pathologies arise, out of the accumulation of dysfunctional mitochondria or defective fusion/fission mediators. It must be clarified, however, that many of these mechanisms might occur simultaneously in a mitochondrial population, or even in the same mitochondrion. The boundaries and independence of these responses are therefore unclear. Similarly, some mitochondrial stress responses, such as the mammalian UPRStudying organelles in isolation has been indispensable for constructing our main body of knowledge on mitochondrial physiology. However, mitochondria do not behave as isolated entities in the cytosol, but as signaling organelles that communicate continuously with other cellular membranes and within themselves. Mitochondria impact nuclear gene expression by retrograde signaling, but also influence metabolite transfer through the physical contact sites with other organelles, such as the ER, lipid droplets, peroxisomes and lysosomes


Genetics has led to a new focus regarding approaches to the most prevalent diseases today. Ascertaining the molecular secrets of neurodegenerative diseases will lead to developing drugs that will change natural history, thereby affecting the quality of life and mortality of patients. The sequencing of candidate genes in patients suffering neurodegenerative pathologies is faster, more accurate, and has a lower cost, thereby enabling algorithms to be proposed regarding the risk of neurodegeneration onset in healthy persons including the year of onset and neurodegeneration severity. Next generation sequencing has resulted in an explosion of articles regarding the diagnosis of neurodegenerative diseases involving exome sequencing or sequencing a whole gene for correlating phenotypical expression with genetic mutations in proteins having key functions. Many of them occur in neuronal glia, which can trigger a proinflammatory effect leading to defective proteins causing sporadic or familial mutations. This article reviews the genetic diagnosis techniques and the importance of bioinformatics in interpreting results from neurodegenerative diseases. Risk scores must be established in the near future regarding diseases with a high incidence in healthy people for defining prevention strategies or an early start for giving drugs in the absence of symptoms. Advances have been made regarding chronic disease therapy due to an understanding of altered molecular mechanisms in cells from different bodily organs. One of the most fascinating advances during the last decade has occurred in the field of genetics concerning new sequencing techniques; this concerns identifying genotypic aberrations leading to the determination dysfunctional phenotypic expressions using large bioinformatic databases. Sequencing a genome or exome for clinical applications has now entered medical practice. Several thousand tests have already been ordered for patients to establish a diagnosis regarding rare diseases that are clinically unrecognizable, or baffling, but have a suspected genetic origin.Neurological diseases are disorders of the brain, spinal cord, and the nerves. There are more than 600 neurological diseases , the majThe neuroinflammatory reaction caused by neurons and non-neuronal cells in neurodegenerative diseases (NDs) is persistent due to many triggering factors leading to the mutation of genes altering proteins implicated in the development of neurodegeneration such as the beta amyloid protein in AD, the alpha-synuclein protein in PD, and the superoxide dismutase (SOD)-1 mutation in amyotrophic lateral sclerosis (ALS), as discussed later on . The selThe field of neurology is not the exception in the explosion of articles/material proclaiming the usefulness of genetics in identifying genetic risk factors and regarding diagnosis specificity. NDs have been the target for intensive research in the field of genetics due to their great impact on morbimortality of adult patients. This article has thus been aimed at reviewing recent advances in the genetic diagnosis of the following ND: AD, PD, and ALS.In 1977, Sanger et al. established the most commonly used method, until recently, for sequencing a determined fragment of deoxyribonucleic acid (DNA) . Figure NGS incorporates technologies that produce millions of short DNA sequences at low cost and in a short time, read mostly in the 25 to 700 bp length range for a gene suspected of producing a disease . It reprA person’s DNA consists of more than 3000 million nucleotides in the genome, whilst the exome . An exome is little more than 1% of the genome; up to 85% of all mutations causing disease in Mendelian disorders are found within the encoding exons, thereby still being the most requested method . WES is DNA must be isolated and fragmented to enable sequencing the exome; the resulting DNA fragments are a mixture of introns and exons. The following step consists of separating the exons from the rest of the genome and sequencing them; the exome must be amplified, and during amplification, fragmented DNA becomes exposed to the surface containing the whole exome sequence. Then, the complementary regions (just exons) bind (making hybrids). The remaining DNA (introns) becomes eliminated in the process. Finally, the exons are sequenced and compared to a reference sequence; thousands of variants appear  from such a comparison. All the variants are filtered until the variants causing a particular disease are detected. Just exon variants can be selected from the 60,000 variants, thereby reducing the number of variants to be considered to 13,000. This process is repeated until just a few variants are obtained (about 10). It is then possible to observe which variants are shared with other members of a (target) family ,15.The disadvantages of WES can be divided into two groups: enrichment mechanisms and coverage difficulties. Regarding enrichment mechanisms, this is the most relevant inconvenience, especially because when “enriching” the exome by hybridization or amplification, not only are artifacts introduced into the sequencing and amplify the DNA by PCR, but elements also become introduced as not all of the regions can be amplified in the same pattern. Regarding coverage, difficulties arise due to enrichment measures and amplification addressing regions with weak coverage or regions lacking coverage, and various mutations not being found in the exome  and not being able to be detected ,17. WGS will surely replace WES because of its reduced cost; from a methodological point of view, it is better to have fewer extras, have homogeneous coverage, and a better analysis strategy. Knowledge has been gained every day when evaluating elements related to parts of the genome which are unrelated to the exon. Other cases where using WES would be indicated would be an unclear phenotype, atypical clinical presentations, challenging cases regarding their interpretation, and evaluating results. Other cases would involve a negative result where the cost can be assumed and when a genetic diagnosis is required which involves cutting-edge technology ,14,16,17The Human Phenotype Ontology (HPO) project provides standardized terminology  regarding the phenotypical abnormalities found in human genetic syndromes. Phenotypical characteristics are formally represented as terms on a directed acyclic graph. Multiple paternity allows the representation of different aspects regarding phenotypical abnormalities . The HPOThe strategy of filtering possible variants is designed to highlight rare or de novo mutations as well as high penetrance mutations modifying proteins. The filtering strategy substantially reduces the list of candidate variants found in expression concerning the confirmation of their functionality in an individual. Studies by Van Deerlin et al. discussed the importance of putting advances in genetic knowledge regarding NDs into practice, highlighting the correct use of nomenclature, changes in the approach to identified variants, ethical aspects arising from managing the information, the resources available for accessing information, and the genetic counselling that patients and their families-care-givers should receive regarding the diagnosis of a disease having a defined inheritance pattern .Identifying mutations in ND-related genes is important regarding different areas of knowledge such as basic research, clinical research, clinical characteristics, and identifying biomarkers and images. The scope of this article focused on identifying the mutations related to each disease.The main cause of dementia affects around 35 million patients around the world ; it is pThe APP gene’s official name is the amyloid beta 4 (A4) precursor protein; APP is mainly found in the CNS. Kang et al. cloned the gene from chromosome 21 ,36. RymaMost variants with an identified risk in more than 20 genes, determined by genome-wide association studies (GWAS), have not been recognized as affecting protein structure or function, only conferring 10% to 20% of risk of disease . Other vAPOE, a component of senile plaques , has beeIdentifying single-nucleotide polymorphisms (SNP) in the human genome and the development of SNP genotyping technologies have led to the genetic understanding of commonly occurring complex diseases ,47. MoreGWAS use large platforms consisting of different SNP markers; it is worth mentioning late onset AD association studies . GWAS haThe clinical implications of genetic discoveries have been mentioned in different studies ,64,65 wiAmyotrophic lateral sclerosis (or Lou Gehrig’s disease) is one of the main NDs of the motor neurons, leading to death within 3 to 5 years following the onset of symptoms. Its prevalence is 1 in 300 people and produces large-scale disability in patients suffering from it. Superoxide dismutase Cu/Zn or SOD1 was the first ALS-associated gene to be identified in 1993 . Recent Even though GWAS has broadened the panorama of genes in different diseases like ALS, other factors associated with the onset of NDs have been reported in studies on twins and families having established hereditary components. Further in-depth studies are needed into the genetics as well as into polygenetics and epigenetics to ensure personalized medicine by finding the factors triggering diseases ,69,70.Evidence regarding genetic traits in ALS has emerged from studies on twins, demonstrating the inheritance in the onset of sporadic ALS, ranging from 0.38 to 0.78  ,72. HoweIn 2014, Keller et al. estimated important genetic factors by meta-analysis using GWES databases, and found a 21% probability of inheritance, identifying 17 regions of the genome having high significance for the disease . InteresThe genes related to familial inheritance in ALS (in order of importance) are SOD1, encoding a copper/zinc superoxide dismutase ,76 whoseTAF15 [C9orf72 [C21orf2, myelin-associated oligodendrocyte basic protein (MOBP), and SCDF1 [The unc-13 homolog A (UNC13A), elongator acetyltransferase complex subunit 3 (ELP3), homeostatic iron regulator (HFE), angiogenin (ANG), neurofilament heavy (NEFH), and EWS RNA binding protein 1 (EWSR1) genes have been reported to be associated with sporadic ALS ,100,101,[C9orf72 , C21orf2nd SCDF1 . Recent nd SCDF1 . Different studies have been carried out on chimeric mice, leading to the inclusion of the SOD1 gene: they have shown cell complex pathways and molecular injury at neuromuscular junctions and in the cell body of the motor neurons ,109,110.The immune and glial cells of transgenic mice with the SOD1 gene affect motor neuron development, highlighting a multifactorial disease with different mechanisms leading to neuron injury, involving non-neuron cells such as astrocytes, oligodendrocytes, and microglia for its rapid progression . NeuroinNicolas et al. identified a new gene associated with ALS such as the kinesin family member 5A (KIF5A) using GWAS. In this study, 20,876 cases of ALS were compared with 59,804 controls . MutatioThe UNC13A gene in the variant rs12608932 is associated with a lower survival in patients with ALS, according to the study by Diekstra et al. . A totalThis is the second most common cause of progressive ND; it affects 30 to 190 patients in 100,000 inhabitants with an average age regarding symptom onset of 60-years old . PD is cNGS technology has been used by several researchers for finding mutations in candidate genes in familial PD, both autosomal dominant and recessive ones having incomplete penetrance. Chartier-Harlin et al.  studied Genes identified with those to whom the risk of developing PD can be attributed, can be classified according to the disease’s clinical presentations in cases of autosomal dominant and autosomal recessive PD . The firOn the other hand, it is important to bear in mind that the genome that does not encode a protein can generate progression to a disease by affecting the normal expression of a gene. The therapeutic possibility of constructing oligonucleotic antisequencing of the exons is known. However, there are microRNAs, unions introns/exons, repetitive RNA, and a large number of non-conforming RNA . Matsu eGenetic tests for diagnosis or detection are necessary for detecting a genetic alteration/aberration in an affected person or one at risk. Test capacity for/ability to detect a genetic alteration depends on many factors including gene location, the nature of the mutation, test sensitivity , test specificity , and reproducibility  . There a


NADPH oxidases (NOX) are enzyme complexes that have received much attention as key molecules in the development of vascular dysfunction. NOX have the primary function of generating reactive oxygen species (ROS), and are considered the main source of ROS production in endothelial cells. The endothelium is a thin monolayer that lines the inner surface of blood vessels, acting as a secretory organ to maintain homeostasis of blood flow. The enzymatic production of nitric oxide (NO) by endothelial NO synthase (eNOS) is critical in mediating endothelial function, and oxidative stress can cause dysregulation of eNOS and endothelial dysfunction. Insulin is a stimulus for increases in blood flow and endothelium-dependent vasodilation. However, cardiovascular disease and type 2 diabetes are characterized by poor control of the endothelial cell redox environment, with a shift toward overproduction of ROS by NOX. Studies in models of type 2 diabetes demonstrate that aberrant NOX activation contributes to uncoupling of eNOS and endothelial dysfunction. It is well-established that endothelial dysfunction precedes the onset of cardiovascular disease, therefore NOX are important molecular links between type 2 diabetes and vascular complications. The aim of the current review is to describe the normal, healthy physiological mechanisms involved in endothelial function, and highlight the central role of NOX in mediating endothelial dysfunction when glucose homeostasis is impaired. Type 2 diabetes (T2D) is a metabolic disease, characterized by hyperglycemia, which often coincides with macro- and micro-vascular complications. Early onset of T2D increases the risk of cardiovascular disease (CVD), and the age at which patients are diagnosed with T2D is declining . The proThe pathogenesis of T2D is not completely understood, however, there are two key precursors that lead to development of this disease: (1) peripheral insulin resistance and (2) β-cell dysfunction . In a noThe vascular endothelium is an active endocrine organ involved in the regulation of vascular tone and maintenance of vascular homeostasis. This monolayer of tissue lines the inner wall of blood vessels, hence positioning endothelial cells in direct contact with the flowing blood and resultant shear stress. Endothelial cell exposure to shear stress leads to transmission of mechanical signals to tightly control lumen size . In addiphox, p22phox, p47phox, and p67phox in skeletal muscle.NADPH oxidases (NOX) are membrane-bound enzyme complexes that are considered potent stimulators of ROS in endothelial cells, particularly under conditions of hyperglycemia ,22. In pThis review will present the normal healthy mechanisms involved in generation of nitric oxide (NO) and highlight the recent progress in understanding the contributions of hyperglycemia-induced NOX activation in endothelial dysfunction, with a focus on the interplay between NOX and eNOS regulation. In addition, we will describe the interactions between endothelial NOX and maintenance of glucose homeostasis.A healthy endothelium modulates homeostasis of the vasculature in response to physical and chemical stimuli by secreting vasoactive molecules that act in autocrine, paracrine, or endocrine fashions. These endothelial-derived factors control a number of physiological outcomes including vessel tone, diameter, vascular smooth muscle cell (VSMC) proliferation, platelet activation, and leukocyte adhesion. In turn, the endothelium is considered a strong indicator of cardiovascular health, and several non-invasive techniques have been developed to assess endothelial function . The imp4) core . W. W2O2 pr2O2 in obesity and diabetes promotes excess mitochondrial ROS production, a phenomenon termed ROS-induced ROS release [Thus, these studies in adipose tissue provide evidence of dynamic roles for NOX4 in mediating insulin sensitivity. NOX4 activation during the onset of insulin resistance is likely increased to enhance vascular hyperpolarizing effects and insulin signaling effects; whereas advanced diabetes progression hampers the beneficial effects of NOX4, with oxidative stress instead being induced. The distinct subcellular localization of NOX4 compared with other NOX isoforms may explain the differing functions of NOX4 throughout insulin resistance progression. NOX4 is the only NOX isoform identified with mitochondrial localization , and it  release . Overpro release ,202. It  release .2O2 in the vasculature. However, the interactions between NOX4, NOS/NO and other mediators of endothelial function in T2D remain unknown.In conclusion, it is clear that hyperglycemia and T2D are strong stimulators of NOX1- and NOX2- dependent endothelial dysfunction. Excess superoxide production derived from NOX1 and NOX2 in response to high glucose or a diabetic setting results in impaired endothelial function and insulin resistance. Conversely, NOX4 has emerged as a major intermediary of glucose homeostasis, which is displayed by the array of metabolic derangements that occur when NOX4 is absent. Collectively, the in vitro and in vivo studies have progressed the understanding of mechanisms that activate the different NOX isoforms and the subsequent molecules that are targeted downstream. Uncoupling of eNOS appears to be a shared mechanism between NOX1 and NOX2 that mediates impairments in endothelium-dependent vasodilation and exacerbated oxidative stress. Endothelial NOX2 activation in response to high glucose and insulin alters the balance between Raf/MAPK-dependent vasoconstriction and PI3K/Akt-dependent vasodilation in favor of constriction. Whereas, NOX4 enhances insulin signaling via inhibition of PTP1B to counteract the onset of insulin resistance. The protective effects of NOX4 are further described by the less injurious nature of HDespite advances in defining the molecular interlinks between diabetic-induced NOX activation and endothelial dysfunction, there remain significant gaps in the literature. The pre-clinical studies have established NOX as central molecules in the regulation of endothelial function; however, it is unclear whether these mechanisms can be translated to humans. Recent developments in methodology to detect NOX-derived ROS in humans by our group provides an opportunity to investigate therapeutic approaches that may reduce endothelial dysfunction in humans . In addiThe available evidence indicates the various endothelial NOX isoforms are promising targets to establish the molecular links between CVD and T2D. Physiological NOX activation is a predominant source of ROS production in the endothelium, and helps to maintain insulin signaling and vascular function. However, dysregulated glucose homeostasis has emerged as a major stimulus to provoke vascular pathology. Hyperglycemia and hyperinsulinemia are significant stimuli of aberrant NOX activation and contribute to endothelial dysfunction. The overstimulation of specific isoforms of NOX in settings of T2D creates a cytotoxic environment, where eNOS uncoupling diminishes the ability of the endothelium to promote vasorelaxation and deliver nutrients . Animal


The aetiologies of obesity and type 2 diabetes are incredibly complex, but the potential role of paternal influences remains relatively understudied. A better understanding of paternal influences on offspring risk of obesity and type 2 diabetes could have profound implications for public health, clinical practice and society. In this review, we outline potential biological and social mechanisms through which fathers might exert an impact on the health of their offspring. We also present a systematically compiled overview of the current evidence linking paternal factors to offspring development of obesity and type 2 diabetes throughout the life course. Although evidence is accumulating to support paternal associations with offspring outcomes, more high-quality research is needed to overcome specific methodological challenges and provide stronger causal evidence.The online version of this article (10.1007/s00125-019-4919-9) contains peer-reviewed but unedited supplementary material including a slideset of the figures for download, which is available to authorised users. Research on the early-life origins of health and disease has traditionally focused on the maternal (mostly intrauterine) impact on offspring health , 2. AlthIn this review, we discuss the implications of paternal impacts on offspring risk of obesity and type 2 diabetes. We then outline potential biological and social mechanisms through which fathers might exert an impact on the health of their offspring, before providing an overview of the current evidence linking paternal factors to offspring development of obesity and type 2 diabetes throughout the life course. Finally, we discuss future research challenges and suggest potential strategies to overcome them.Appreciation of the idea that fathers might influence risk of obesity and type 2 diabetes in their offspring would challenge assumptions about the causal primacy of maternal effects in pregnancy ) found a link between paternal adiposity (including BMI and high-fat-diet-induced obesity) and obesity- or type 2 diabetes-related offspring outcomes . Of the 12 null studies, two investigated paternal BMI and found no evidence of association with offspring obesity-related traits . In addition to paternal adiposity, the other top five most studied paternal factors were genetic factors , glycaemic exposures, such as diabetes and hyperglycaemia , demographic factors such as socioeconomic position and age , and health behaviours such as smoking and drinking alcohol .Of 128 studies, 78 (61%) were conducted in humans and 50 (39%) were conducted in animals (mainly rodents [n = 79 [62%]) did not explicitly explore potential mechanisms linking paternal factors to offspring obesity or type 2 diabetes. Of those that did, the most commonly explored mechanisms were postnatal , genetic imprinting , sperm epigenetics , paternal genetics  and perinatal offspring epigenetics .The majority of studies  as human studies (30/78 [38%]), although this can be explained by 20 human studies where the exposure, and therefore the mechanism, was postnatal. Mechanisms involving genetics or prenatal factors were far more likely to be explored in animal studies  than human studies (10/78 [13%]).In line with what we see in the developmental origins of health and disease field more widely , 2, compHowever, we also consider that a high proportion of non-null studies could indicate publication bias and/or be an artefact of our search strategy . Our review aimed to provide an overview of the current literature on paternal impacts on offspring obesity and type 2 diabetes. Although beyond the scope of this paper, a more detailed review and meta-analysis to assess average effect estimates and evidence of heterogeneity and small-study (publication) bias is warranted. A previous systematic review and meta-analysis of human studies found only modest average effects of various paternal exposures on cardiometabolic outcomes and identified some issues with the quality of evidence .Most of the studies we identified were human observational studies and causal evidence for paternal impacts (in humans at least) is lacking. Most studies did not explore the biological or social mechanisms through which fathers might influence obesity and type 2 diabetes risk in offspring, but identification and clarification of such mechanisms would be one way to strengthen causal inference.There are some specific challenges to the study of paternal effects that must be tackled by future research. In human observational studies, two major challenges are confounding and bias. When studying paternal direct effects, a major source of confounding is likely to arise from high correlation with maternal exposures due to shared environments and assortative mating . CompareA better understanding of paternal influences on offspring risk of obesity and type 2 diabetes could have profound implications for public health, clinical practice and society. There are multiple possible mechanisms through which paternal exposures might influence offspring health and development. Evidence is accumulating to support paternal associations with offspring outcomes; however, more high quality research is needed to overcome specific methodological challenges and provide stronger causal evidence.Slideset of figures(PPTX 377 kb)ESM Table(XLSX 52 kb)


A receptor function, the antidepressant activity of allopregnanolone is attributed to an increase in GABAergic inhibition. In addition, allopregnanolone may stabilize normal mood by decreasing the activity of stress-responsive dentate granule cells and thereby sustain resilience behavior. Therefore, allopregnanolone may augment and extend its antidepressant activity by fostering resilience. The recent structural resolution of the neurosteroid binding domain of GABAA receptors will expedite the development of more selective ligands as a potential new class of central nervous system drugs.The GABAergic deficit hypothesis of depression states that a deficit of GABAergic transmission in defined neural circuits is causal for depression. Conversely, an enhancement of GABA transmission, including that triggered by selective serotonin reuptake inhibitors or ketamine, has antidepressant effects. Brexanolone, an intravenous formulation of the endogenous neurosteroid allopregnanolone, showed clinically significant antidepressant activity in postpartum depression. By allosterically enhancing GABA MDD is difficult to treat, in part because it is a phenotypically and etiologically heterogeneous syndrome4. Accordingly, it is challenging to conceive of a single mechanism that could account for most forms of this disease and of a treatment that might alleviate symptoms in the majority of patients. Indeed, current first-line antidepressants that are designed to modulate monoaminergic transmitter systems separate from placebo in only about 50% of clinical trials5, are effective in fewer than two thirds of patients subjected to one or two regimens of treatment6, and act with a delay of several weeks to months7. Even among patients who respond to these treatments, only a small fraction show remission. These features often lead to endless and futile pursuits of an effective treatment and illustrate the enormous unmet need for better antidepressant therapies. Here, we summarize the GABAergic deficit hypothesis of depression and its clinical support by the neurosteroid brexanolone, which largely acts by enhancing GABAergic inhibition.Major depressive disorder (MDD) represents the most common cause of total psychophysiological disability with a worldwide lifetime prevalence of 12 to 20% and estimated annual costs to the US economy of more than $100 billion9. Consistent with this hypothesis, clinical studies over the past 15 years have provided compelling evidence that MDD is associated with diverse defects in GABAergic neurotransmission. This includes well-replicated findings of reduced brain levels of GABA12, reduced expression of glutamic acid decarboxylase (GAD) as the principal enzyme responsible for GABA synthesis by GABAergic interneurons14, reduced density or function of GABAergic interneurons17, and reduced expression and function of the principal receptors for GABA known as GABAA receptors20. Together, these changes explain the markedfunctional defects in cortical GABAergic inhibition observed in patients with MDD21.The GABAergic deficit hypothesis of MDD posits that diverse defects in GABAergic neural inhibition can causally contribute to common phenotypes of MDD and conversely that the efficacy of current and future antidepressant therapies depends on their ability to restore GABAergic neurotransmission22 but may have distinct developmental origins23. Compared with other neuropsychiatric disorders, MDD shows low heritability of about 38%24. Even this low heritability remains unexplained as attempts to replicate the identification of candidate genes of MDD have been failing26. Therefore, rather than relying on genetic models to explore disease mechanism, pre-clinical models of MDD are often based on the notion that chronic stress represents a major environmental vulnerability and precipitating factor of MDD. Consistent with a causative role of stress for MDD, chronic exposure of rodents to stress results in diverse behavioral alterations in a direction opposite to those induced by antidepressant drug treatment, and antidepressant drug treatments prevent or ameliorate the detrimental effects of stress in these models28. Chronic stress also results in reduced production and survival of adult-born hippocampal granule cell neurons and these cells are essential for at least some of the behavioral actions of antidepressants29. Importantly, stress-induced behavioral alterations of rodents are associated with impairment of GABAergic interneurons, reduced expression of GAD and of the vesicular and plasma membrane transporters for GABA, and reduced density and function of GABAergic synapses34. In addition, chronic stress leads to marked deficits in the synthesis of endogenous GABA-potentiating neurosteroids, as detailed below. Lastly, chronic stress also leads to a shift in the chloride reversal potential to more depolarized membrane potentials, which renders GABAergic inhibition ineffective36. In corticotropin-releasing hormone (CRH) neurons of the hypothalamus, corresponding stress-induced loss of inhibitory drive leads to chronic hypothalamic–pituitary–adrenal (HPA) axis activation35. Thus, stress-induced defects in GABAergic inhibition are self-perpetuating because they exacerbate stress-induced glutamate release and lead to chronically dysregulated stress axis function. Conversely, mechanisms that enhance GABAergic inhibition are predicted to confer stress resilience, a process that has been described by the American Psychological Association as “adapting well in the face of adversity, trauma, tragedy threats or significant sources of stress” .Beyond MDD, GABAergic deficits are also broadly implicated in anxiety disorders, which are highly comorbid with MDDcausally contribute to stress-induced anxiety- and depressive-like symptoms is available from analyses of GABAA receptor mutant mice. Knockout mice that were rendered heterozygous for the γ2 subunit  exhibit anxiety- and depression-related behavior, defects in hippocampal neurogenesis, cognitive deficits in emotional pattern separation, and chronic HPA axis activation that are expected of an animal model of MDD40. Some of these same behavioral defects have been described in mice lacking the α2 subunit of GABAA receptors41 or the neurosteroid binding site of α2 GABAA receptors42 and in mice with genetically reduced GABA synthesis43.Some of the most compelling evidence that defects in GABA transmission can+/− mice further have in common that they result in similar homeostatic-like downregulation of ionotropic glutamate receptors (AMPA and NMDA receptors) and glutamatergic synaptic transmission . Such defects in functional neural connectivity and their rescue by antidepressant therapies represent functional hallmarks of MDD48. Importantly, chronic treatment of γ2+/− mice with the norepinephrine (NE) reuptake inhibitor desipramine is able to similarly normalize the behavior of γ2+/− mice along with normalization of HPA axis function in these mice40. Chronic stress–induced or optogenetic activation of NE neurons of the locus coerulus (LC) that project to dopaminergic (DA) neurons in the ventral tegmental area (VTA) mediates resilience to chronic social defeat stress of mice49. In the VTA, LC-derived NE acts through α1- and β3-adrenergic receptors to induce homeostatic alterations of ion channel expression in DA neurons that contribute to stress resilience. Accordingly, we speculate that hyperexcitability of LC NE neurons in γ2+/− mice facilitates the antidepressant action of NE reuptake inhibitors to induce slow homeostatic changes in DA neurons that underlie stress resilience. Thus, both conventional antidepressants and ketamine can act to overcome genetic  defects in GABAergic synaptic transmission, albeit by entirely different mechanisms. Moreover, directly and deliberately increasing the excitability of certain subsets of GABAergic interneurons in mice has robust anxiolytic and antidepressant-like behavioral and biochemical consequences50. Collectively, these findings lend strong support to the GABAergic deficit hypothesis of MDD and suggest that certain  agents that enhance GABAergic inhibition may have antidepressant properties9. For example, benzodiazepines, which act as positive allosteric modulators of GABAA receptors and are first-line treatments for anxiety disorders, have only limited efficacy as antidepressants51, even though they are often used to augment conventional antidepressants and to treat comorbidities of MDD, such as anxiety and insomnia55. Limited antidepressant efficacy of benzodiazepines may be due to tolerance, which is thought to involve chronic drug-induced degradation of major subsets of GABAA receptors and corresponding loss of inhibitory synapses57. However, two negative allosteric modulators of α5-GABAA receptors—L-655,708 and MRK-016—have been shown to exhibit rapid antidepressant-like activity in a chronic stress model of rodents58. Notably, in contrast to benzodiazepines and comparable to ketamine (discussed in the following), these agents act by transient disinhibition of neural circuits, which results in antidepressant-like activity in the “drug-off” situation.Chronic stress and defects in GABAergic transmission of γ2mission 44–46. 60 and is only briefly recapitulated here. Key aspects of the antidepressant mechanism of ketamine are that it involves brief inhibition of GABAergic interneurons61 followed by a transient surge in glutamate release62 (lasting at most 1 hour) that then triggers the release of brain-derived neurotrophic factor (BDNF)63 and a wave of synaptogenesis64. The ensuing increase in synapse density, the corresponding restoration of neural connectivity, and normalized emotional behavior are all observed in the drug-off state and last for several days. Notably, ketamine-induced synaptogenesis and antidepressant behavioral response are drastically enhanced by GABAergic deficits, as observed in γ2+/− mice46 and also evident in animals exposed to chronic stress66, perhaps because neural hyperexcitability in these models facilitates the ketamine-induced glutamate surge and BDNF release. Importantly, restoration of glutamatergic synapses in γ2+/− mice is associated with even more dramatic formation and pre- and post-synaptic potentiation of GABAergic synapses46, which appears to ensure that inhibitory and excitatory synaptic transmission remain balanced. Indeed, we found no evidence in the literature that ketamine treatment triggers seizures, despite the glutamate surge and evidence of a reduced seizure threshold in patients with MDD67. Similar to restoration of glutamatergic synapses, ketamine-induced strengthening of GABAergic synapses is long-lasting and observed in the drug-off situation46 and temporally separated from the initial direct action of ketamine at GABAergic interneurons mentioned above. Here, we propose thatdirect and potent pharmacological enhancement of GABAergic transmission  will act accordingly to transiently dampen glutamate release and allow for lasting recovery of glutamatergic and GABAergic synaptic transmission beyond the end of treatment , 3α,5β-tetrahydro-progesterone , and 3α,5α tetrahydrodeoxycorticosterone .Neurosteroids are metabolites of cholesterol-derived steroid hormones synthesized in the brain by neurons and astrocytes. They act as potent, endogenous, positive allosteric modulators of GABA68, which results in an increase of the mean channel open time of the GABA-activated chloride channel and a prolonged inhibitory post-synaptic current. However, when acting on extra-synaptic receptors that are kept tonically active by ambient concentrations of GABA, the allopregnanolone-induced prolonged decay kinetic results in an increased amplitude of the tonic current. In addition to potentiation of GABAA receptor channel function70, allopregnanolone and its synthetic derivatives may potentiate GABA transmission by promoting the cell surface expression of GABAA receptors71. The impact on phasic or tonic inhibition can be strikingly different because of the type of GABAA receptor subtype involved. The prototypic synaptic receptor contains α1, β2/3, and γ2 subunits and is sensitive to physiological concentrations of neurosteroids68. However, in certain cells, such as dentate gyrus (DG) granule cells or cerebellar granule cells, allopregnanolone at low concentrations (10–100 nM) selectively enhances tonic inhibition with little or no effect on phasic conductance. This appears to be due largely to the preponderance of highly neurosteroid-sensitive extra-synaptic δ subunit–containing receptors with the subunit combinations α4,β3,δ, and α6,β2,3,δ74. In addition, phosphorylation of the β3 subunit by protein kinase C appears to promote neurosteroid sensitivity of extra-synaptic receptors while limiting that of synaptic receptors75. In some neurons, the strict division of GABAA receptors into synaptic and extra-synaptic receptors mediating phasic and tonic inhibition, respectively, has become an oversimplification76. The therapeutic action of brexanolone  is likely to comprise an enhancement of both phasic and tonic inhibition.Neurosteroids such as allopregnanolone have little effect on the rise time or the amplitude of GABA-induced synaptic currents but primarily prolong the decay kinetics of the GABA-gated ion channel77 and in plasma79. Plasma allopregnanolone was similarly decreased in postpartum “blues”80 (but see below), post-traumatic stress disorder82, schizophrenia negative symptomatology83, pain84, and pharmacologically induced panic attacks85 but did not reach significance in general anxiety disorder86. Conversely, the 3β isomer of allopregnanolone antagonizes GABAA receptor function87 and is increased in panic attacks88. Based on studies of postmortem brain, changes in the neurosteroid synthesis pathways were also proposed to contribute to the pathologies of neurodegenerative and inflammatory diseases (Alzheimer’s and Parkinson’s disease and multiple sclerosis)89.The downregulation of neurosteroid biosynthesis has been implicated as a possible contributor to various psychiatric conditions, as shown in a number of clinical trials. In patients with MDD, allopregnanolone and pregnanolone were decreased in cerebrospinal fluid (CSF)90, chronic stress and pharmacological induction of panic attacks84 result in reduced levels of neurosteroids. In animal models of chronic stress, the concentration of allopregnanolone was decreased in serum93 and in selected corticolimbic brain areas94. This decrease was attributed to stress-induced downregulation of the 5α-hydroxysteroid-dehydrogenase, the rate-limiting enzyme in the synthesis of allopregnanolone96. Moreover, the stress-induced reduction of allopregnanolone was associated with heightened depressive/anxiety-like behavioral phenotypes, increased fear and aggression behavior, dysregulation of the HPA axis97, and impaired adult hippocampal neurogenesis99.Because chronic stress is a major risk factor for depression, the influence of chronic stress on neurosteroids has become a major focus. In striking contrast to acute stress, which increases allopregnanolone levels101. Furthermore, allopregnanolone and pregnanolone produced anxiolytic-like effects in various animal models of anxiety102. Micro-infusion of allopregnanolone identified the amygdala as being relevant for anxiolysis103 and both the hippocampus and amygdala for overcoming learned helplessness104. These results support a role of allopregnanolone in ameliorating symptoms of depression and anxiety and thereby support the view that a pathological deficit of GABAergic transmission contributes to these disorders105.Administration of allopregnanolone either before or after a period of chronic stress was able to alleviate the symptoms of depressive/anxiety behavior, prevent or normalize HPA axis dysfunction, and restore neurogenesis and cognitive deficits in transgenic mouse models of Alzheimer’s disease107. This effect, as shown for selective serotonin reuptake inhibitors (SSRIs), was independent of their ability to inhibit serotonin reuptake110 and in the case of fluoxetine is thought to involve inhibition of a microsomal dehydrogenase that oxidizes allopregnanolone to 5α-dihydroprogesterone111.With allopregnanolone being able to overcome depressive-like behavior, the question arose whether classic antidepressant drugs would act via an enhancement of neurosteroid levels. In animal models of depression, multiple antidepressants  normalized corticolimbic levels of allopregnanolone concomitant with reduced anxiety-like, fear, and aggression behavior77. This finding was extended to a range of antidepressants (SSRIs and tricyclic antidepressants), which normalized plasma allopregnanolone levels concomitant with an improvement of depression109. A normalization of plasma allopregnanolone and pregnenolone was also seen following 3 weeks of mirtazapine treatment in patients with major depression79. Notably, the mirtazapine-induced maximal increases in these neurosteroids preceded their maximal clinical effects by about 2 weeks, suggesting that they are part of the pharmacological response mechanism rather than a subsequent measure of clinical improvement. These pre-clinical and clinical findings support the view that the neurosteroidogenic action of SSRIs may constitute a major part of their therapeutic effectiveness in patients with depressive disorders.These findings gave reason to test whether the clinical effectiveness of SSRIs was linked to normalizing the allopregnanolone level. Indeed, in patients with unipolar depression, the decreased allopregnanolone level, measured in CSF, was normalized after 8 to 10 weeks of treatment with fluoxetine or fluvoxamine and correlated with an improved symptomatology112. The risk of depression in women becomes significantly increased during the postpartum period, and nearly 20% of mothers have PPD, which is frequently preceded by antenatal anxiety- and depression-related symptoms or chronic stress as the strongest predictors115. PPD is frequently attributed to a maladaptation to peripartum fluctuations in reproductive hormone levels during pregnancy and the postpartum period117. Plasma allopregnanolone concentrations rise in parallel with progesterone throughout pregnancy, reaching the highest level in the third trimester and decreasing abruptly after childbirth80.Postpartum depression (PPD) is an important public health issue as it affects women at a highly vulnerable time and can affect the cognitive and emotional development of the child118. The resting-state functional connectivity within corticolimbic regions implicated in depression was attenuated compared with healthy postpartum women114. Similarly, emotionally normal (euthymic) women with a history of PPD showed stronger signs of depression than controls in tests of withdrawal from supra-physiological gonadal steroid levels119. Additional factors that have been implicated in the pathophysiology of PPD include the lactogenic hormones oxytocin and prolactin, thyroid function, and a hyperactivity of the HPA axis117. As outlined below, the potential importance of GABAA receptor plasticity in PPD has been derived largely from animal studies120.Nevertheless, peripartum changes in gonadal hormones affect the emotional brain in vulnerable women. PPD was characterized by abnormal activation of the same brain regions implicated in non-puerperal major depressionA receptor expression as shown for the GABAA receptor γ2 and δ subunit in mouse and rat hippocampus121. Within days after parturition, GABAergic transmission and the level of GABAA receptor expression rebound to control levels122. This fluctuation in receptor expression is considered to be a homeostatic response to the elevated levels of pregnenolone and allopregnanolone in plasma and brain during rodent pregnancy and their rapid return to control levels postpartum123.Rodent models suggest that both phasic and tonic GABAergic inhibition in the brain are decreased during pregnancy in parallel with a decrease in GABAA receptor plasticity. Mice that lacked the GABAA receptor δ subunit partly or fully (Gabrd+/− andGabrd−/−) exhibited PPD-like behavior (reduced latency to immobility in the Porsolt forced swim test and reduced sucrose preference) and abnormal maternal behavior (reduced nesting behavior and pup care)121. Remarkably, the mice were behaviorally unremarkable until an animal was exposed to pregnancy and the postpartum state. Thus, reproductive events unmask the genetic susceptibility to affective dysregulation. The abnormal postpartum behavior inGabrd+/− mice was ameliorated by THIP, a GABA analogue with preferential affinity to GABAA receptors containing the δ subunit121 and the neuroactive steroid SGE-516115.A transgenic animal model of PPD supports the view that the pathophysiology of PPD may be related to a deficit of GABA115. CRH neurons in the paraventricular nucleus (PVN) of the hypothalamus govern the HPA axis and are critical for mounting the physiological response to stress. Chemogenetic activation of CRH neurons in the PVN was sufficient to induce abnormal postpartum behavior. Similarly, when GABAergic currents were reduced selectively in CRH neurons (KCC2/Crh mice), a depression-related phenotype and a deficit in maternal behavior were apparent in the postpartum period. In wild-type mice, the stress-induced activation of the HPA axis and the corresponding elevation of circulating corticosterone are normally blunted during pregnancy and postpartum124. The inability to blunt this stress-induced HPA axis activation in this model is thought to contribute to PPD. The neuroactive steroid SGE-516 ameliorated the behavioral deficits caused by the dysregulation of the HPA axis115.Another animal model suggests that the dysregulation of the HPA axis is sufficient to induce abnormal postpartum behavior125. In a subsequent phase III study with 246 patients, performed in two parts using the same design but two doses of drug, the HAM-D score was again significantly and clinically meaningfully decreased. The HAM-D total score mean reductions versus placebo were −5.5 and −3.7 points for the high and low dose, respectively (90 and 60 μg/kg per hour), and dizziness and somnolence were potential side effects126. Notably, brexanolone is very rapidly cleared from plasma, which explains the need for continuous drug infusion127. The higher dose explored above (90 μg/kg per hour) results in peak steady-state plasma concentrations of allopregnanolone (50 ng/mL) equivalent to those naturally reached in the third trimester of pregnancy128. The antidepressant drug effect was shown to become significant within 48 hours of drug infusion for both doses. Moreover, the mean reduction in HAM-D total scores observed for the high-dose treatment at the end of the study (day 30) was similar in magnitude to that observed at the end of the 60-hour infusion. Thus, in the context of PPD, brexanolone showed a rapid mode of action that is reminiscent of that of ketamine in MDD and appears to result in durable clinical improvement  received a 60-hour continuous infusion of brexanolone at a dose of up to 60 μg/kg per hour or placebo. Women who received treatment, in comparison with those who received placebo, had a significant and clinically meaningful reduction in mean total score on the 17-item HAMILTON Rating Scale for Depression (HAM-D) at the 60-hour time pointovement . Brexano80, there is no consistent evidence of abnormal basal circulating levels of allopregnanolone in PPD130. With allopregnanolone levels being normal, the antidepressant action of brexanolone is attributed to an enhancement of GABAA receptor function, thereby supporting the GABAergic deficit hypothesis of depression. In addition, emerging evidence suggests that allopregnanolone has anti-inflammatory effectsin vitro, a property that could contribute to its antidepressant activityin vivo78. The antidepressant properties of brexanolone in PPD may be applicable to other forms of depression that are less clearly linked to altered neurosteroid physiology and that may be associated with defects in phasic rather than tonic GABAergic inhibition118. Indeed, emerging evidence suggests that the anxiolytic effects of endogenous neurosteroids  are mediated in part by α2 subunit–containing synaptic GABAA receptors42. Moreover, a synthetic derivative of allopregnanolone  is currently in phase 3 clinical development for PPD and MDD131. Notably, potentiation of GABA transmission by brexanolone and potentially also zuranolone, unlike SSRIs that exhibit therapeutic delays of weeks or months, confers rapid and lasting antidepressant effects that are observed in the drug-off situation, reminiscent of mechanisms of ketamine. Interestingly, since allopregnanolone promotes proliferation of progenitor cells and restores neurogenesis in disease states132, it may also support neurogenesis-dependent resilience behavior, as outlined below.Although a decrease in serum allopregnanolone (but not progesterone) was reported in one study in women with postpartum “blues”Studies of stress resilience have opened up a fundamentally new way of understanding an individual’s response to adverse life events such as trauma, tragedy, and chronic stress and its ability to avoid deleterious behavioral changes such as anxiety disorders, post-traumatic stress disorder, or depression. Although resilience, as defined in humans, is difficult to relate to animal studies, animal models are indispensable in the search for biological determinants of resilience . This is all the more important as mechanisms that promote resilience to stress hold the promise of enabling the development of more efficacious antidepressant therapies.134. This is interpreted as resilience behavior and is associated with many distinct changes, particularly in the brain’s reward regions135. These changes include homeostatic adaptations of dopamine neurons in the VTA that prevent chronic stress–induced aberrant hyperexcitability of these cells137, the induction of immediate early gene products in the medial prefrontal cortex (mPFC)138, the sustainment of GABAergic inhibition and reduction of excitatory activity in the nucleus accumbens139, the prevention of spine density loss in the mPFC and hippocampus140, and epigenetic genomic changes that promote resilience in later life because of greater maternal care142.After chronic social defeat stress, about 40% of the stressed mice do not exhibit social avoidance or anhedonia in subsequent testing143. Chemogenetic inhibition of immature adult-born neurons in ventral DG (vDG)in vivo promoted susceptibility to social defeat stress. In contrast, increasing neurogenesis by inducible deletion of the proapoptotic geneBax (iBax mice) selectively from adult neural progenitor cells conferred resilience to chronic stress as measured by the time spent socially interacting with a novel mouse and the time spent exploring the center in an open field. On the cellular level, a subset of mature DG cells was identified as stress-responsive cells that were active preferentially during attack (17% of cells on defeat day 1 to 34% of cells on defeat day 10). The activity of the stress-responsive mature cells was decreased when neurogenesis was increased. Thus, immature adult-born granule cells inhibit mature stress-responsive granule cells in the vDG, which protects the animals from chronic stress–induced depressive and anxiety-like consequences. The inhibition of mature granule cells by immature adult-born cells is likely to involve activation of hilar GABAergic interneurons that are known to confer a strong inhibitory influence on mature granule cells144. Neurosteroids are predestined to inhibit DG granule cells because of their high level of expression of δ subunit–containing GABAA receptors, which are highly neurosteroid-sensitive74.More recently, adult neurogenesis in the DG of the hippocampus has been implicated in conferring resilience to the detrimental depressive-like consequences of chronic stress exposure of miceThe finding that GABAergic interneuron activity can support resilience may give rise to a potential GABAergic hypothesis of resilience, which conceivably is independent of the prerequisite of adult neurogenesis. Decreased GABAergic interneuron activity would be expected to reduce resilience behavior whereas enhancing GABAergic transmission would sustain it. The neurosteroid allopregnanolone appears to be a case in point. Its clinical effectiveness as an antidepressant in treating PPD might be supported, at least in part, by its ability to foster resilience.A receptors beyond that achieved by allopregnanolone is required. The recent molecular x-ray resolution of the neurosteroid binding domain is an essential step forward. In chimeric homopentameric GABAA receptor constructs, the neurosteroid THDOC was bound at the bottom of the transmembrane domain across each of the subunit interfaces147 and similar findings are expected for heteropentameric GABAA receptors148. These studies provide a structural framework for the development of more selective ligands acting at the neurosteroid site for the treatment of affective disorders, including PPD and MDD, but also pain and epilepsy.The demonstration of the clinical effectiveness of allopregnanolone in PPD lends new support to the GABAergic deficit hypothesis of MDD. This finding bodes well for further investigations of ligands for the neurosteroid site as a new class of drugs for affective disorders. To achieve this goal, a differentiation of GABA


DNA repair system has evolved to maintain the genomic integrity to defend against both endogenous and exogenous sources of DNA damage, such as endogenous factors include reactive oxygen species, replication errors or mistakes in meiosis and exogenous factors include ultraviolet (UV) radiation, ionizing radiation (IR), and some other chemicals or chemotherapeutic agents. Multiple repair pathways  can be aroused from the diverse forms of DNA lesions including mismatch paired bases, small deletions or insertions, and DNA single or double-strand breaks. These repair pathways also exert crosstalk with others to complete the whole DNA repair process.The deficient DNA repair causing prolonged existence of DNA damages can lead to genes mutations, chromosome rearrangements, genomic instability, and finally carcinogenesis. Indeed, defects in DNA repair pathways contribute to many heritable cancer predisposition syndromes; however, cancer-related DNA repair deficiency may also occur in sporadic cancer case. Defective DNA repair is common in carcinogenesis and plays a critical role in cancer progression. For example, genetic mutations in DNA mismatch repair genes are involved in reducing mismatch repair and increasing the risk to colon and uterine tumors; BRCA1, BRCA2, and PALB2 genes mutations result in defective homologous recombination repair and are associated with the carcinogenesis of breast and ovarian cancer. In these years, many cancer-related germline mutations in DNA repair have been reported; thus to detect these genetic variations gives us a chance to evaluate the cancer risk of the individual with these mutations.Furthermore, these defects in DNA repair pathways may have therapeutic implications for clinical practice. Most recently, a series of therapeutic strategy have been exploited, such as platinum chemotherapies and PARP inhibitors in homologous recombination defected breast and ovarian cancers and inhibitors of immune checkpoints CTLA-4, PD1/PD-L1 in the case of mismatch repair deficiency cancers. These observations give the direction for further research to investigate the defects in DNA repair pathways that may serve as very useful biomarkers for the choice of suitable oncotherapy.This special issue includes 4 high-quality peer-reviewed articles and 1 review that brings us new ideas and findings in DNA repair in different types of cancers focusing on the genetic changes in DNA repair genes, the influences from these changes to carcinogenesis, and also the therapeutic implications. We have reasons to believe that these articles will enlighten and motivate not only the new inspirations but also the scientific advances in the study of DNA repair in cancer.


To date, genetic, descriptive and mechanistic aspects of the ASDs have been investigated using mouse models and post-mortem brain tissue. More recently, the technology to generate stem cells from patients' samples has brought a new avenue for modeling ASD through 2D and 3D neuronal models that are derived from a patient's own cells, with the goal of building new therapeutic strategies for treating ASDs. This review analyses how studies performed on mouse models and human samples can complement each other, advancing our current knowledge into the pathophysiology of the ASDs. Regardless of the genetic and phenotypic heterogeneities of ASDs, convergent information regarding the molecular and cellular mechanisms involved in these disorders can be extracted from these models. Thus, considering the complexities of these disorders, patient-derived models have immense potential to elucidate molecular deregulations that contributed to the different autistic phenotypes. Through these direct investigations with the human  Autism is a complex, multigenic and multifactorial neurodevelopmental disorder that was originally defined based on three clinical criteria (i) abnormalities in social interactions, (ii) deficits in verbal and non-verbal communication and (iii) the presence of stereotypical and repetitive behaviors . As the Studies on syndromic forms of autism, as well as candidate gene approaches and genome-wide association studies, have led to the identification of more than 70 loci for ASDs in the human genome . Those lin vitro tool to decipher how perturbations in specific genes or pathways may be either involved or the causative mechanisms in the development of ASDs, with the ultimate goal of developing new therapeutics. Multiple genetic susceptibility loci identified in ASDs strongly implies that these disorders are linked to genetic variants and risk factors across multiple genes that are best studied in human models. This review aims to discuss the strengths and limitations of mouse models, human post-mortem brain tissue and iPSCs in studying ASDs, and to summarize how these studies combined have contributed to advance our understanding into the molecular and cellular mechanisms that potentially cause ASDs.Over the past decade, technology has developed to derive induced pluripotent stem cells (iPSCs) from the somatic cells of patients with many different types of diseases. In the context of ASDs, this approach enables us to differentiate and grow human neurons from patients, within a dish, presenting a new Animal models have been a useful tool in understanding how genes linked to ASDs may contribute to the pathogenesis of these disorders. In order to study ASDs, a combination of genetic, chemically induced, and environmental models have been generated. In the genetic models, ASDs-associated genes have been inactivated to observe distinct phenotypes within the mice that can include altered gene expression, cell morphology, and behavioral or social deficits.Fmr1 , present abnormal dendritic spine morphology, although one limitation of this model is that autistic traits often differ from one strain to another (Fmr1 knockout mice by S6K1 (a translation regulator) deletion can stabilize neurological function (Fmr1 knockout leads to a disruption of synaptic protein interactions that notably involve metabotropic glutamate receptor subunit 5 (mGluR5) and Homer scaffold protein. Homer1a acts as a dominant negative isoform that prevents the normal interaction with mGluR5, and its deletion rescues, in H1a/Fmr1 double knockouts mice, mGluR5 signaling , Rett Syndrome, and Tuberous Sclerosis  are stud another . Restorafunction . Fmr1 knignaling .Fmr1 knockout mice  as Viaat-Mecp2y−/ mice lack Mecp2 in GABAergic neurons, causing RTT-like features that include the development of stereotypes, self-injury, compulsive behavior and progressive motor dysfunction  is caused, in the majority of cases, by loss of function mutations in the CP2 gene . Mouse mfunction . Activatfunction . Interesckground . This stTSC1 and TSC2 are known to cause Tuberous Sclerosis (TSC)  . Actuallis (TSC) . The mTOis (TSC) , and itsreatment . A TSC mreatment . These mShank3 display impairments in NMDA receptor signaling function, synaptic trafficking  their function, (ii) their potential implication in the disease and (iii) on imaging studies. As such, several morphological studies have been performed on the cerebellum, which controls balance, posture, and regulation of fine movements  as it isMorphological studies of the cerebellum have shown it to be decreased in size accompanied by a decrease in the overall number of Purkinje cells in autistic patients relative to controls –53. SimiOther morphological studies were conducted on post-mortem brain tissue from patients diagnosed with syndromic ASDs, namely Fragile X and Rett Syndromes, two disorders that present with opposing morphological phenotypes. A decreased number of dendritic spines has been observed in the frontal cortex and in the CA1 regions of patient with Rett syndrome , 61 wherRELN gene, which had been associated with autism  an accumulation of mitochondria in the dendrites and (ii) a reduced mitochondrial potential membrane  a down-regulation in the expression of genes involved in neuronal development and cell signaling  and (ii)TRPC6 gene translocation. IGF1 treatment restored dendritic arborization that was decreased in TPRC6-disrupted neurons which also had decreased expression in synaptic proteins such as Homer and PSD95 —an HDAC inhibitor and an antiepileptic drug commonly used for bipolar disease treatment—and lithium, led to an increase in SHANK3 expression and its recruitment to post-synaptic sites (A screening of 202 compounds was performed on iPSC-derived cortical neurons from patients with ic sites  as well in vitro model for studying the impact of ASD-associated mutations and genetic vulnerability factors on the development and progression of the disease. Thus, many reasons can justify using human iPSC-derived models to increase our understanding of ASDs.Studies using iPSC-derived cells have shown that common processes are deregulated in syndromic and idiopathic forms of autism characterized by impairment in neuronal differentiation process, an imbalance between inhibitory and excitatory neurotransmission as well as mitochondrial deficits which corroborate genetic data as well as post-mortem brain studies. Several molecules such as rapamycin and IGF1, that have been also tested in mouse models seem to be effective in human iPSC-derived cells. The literature concerning the potential effect of VPA and lithium on mouse models is inconclusive. IPSC-derived cells from patients constitute a critical Interestingly, differential expression of human-specific genes was shown between control and brains of patients with ASDs . Along tThe current review aims to bring together findings in the field of ASDs provided by genetics, mouse models, post-mortem brain studies and iPSC-derived studies. All these approaches provide complementary information suggesting that ASDs are underpinned by dysregulation in the brain developmental program which include alterations of activity-dependent development, mitochondrial function as well as an imbalanced excitation-inhibition equilibrium . Those sTD, GM, and CR defined the scope of the review. GM and CR wrote the manuscript and prepared the tables. GB, TD, and LB corrected, edited, and formatted the 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.


Phospho-proteomic studies have confirmed that phosphorylation is a common mechanism to regulate protein function in the chloroplast, including the enzymes of starch metabolism. In addition to the photosynthetic machinery protein kinases (STN7 and STN8) and their cognate protein phosphatases PPH1 (TAP38) and PBCP, multiple other protein kinases and phosphatases have now been localized to the chloroplast. Here, we build a framework for understanding protein kinases and phosphatases, their regulation, and potential roles in starch metabolism. We also catalog mapped phosphorylation sites on proteins of chloroplast starch metabolism to illustrate the potential and mostly unknown roles of protein phosphorylation in the regulation of starch biology. Arabidopsis thaliana genome  with its ortholog in Arabidopsis being STN7 (Given the abundance of protein phosphorylation in prokaryotes it is no surprise that an organelle derived from prokaryotes (plastids) has many events controlled by this process. Consistent with this are the growing proteomic datasets that demonstrate widespread protein phosphorylation in the chloroplast. ing STN7 . Thylakoing STN7 , with STing STN7 . We refeing STN7 .1 but remind readers to be cautious about information collated from multiples studies. Many of these mass spectrometry studies do not report false discovery rates (FDRs) for peptide identification, there can be wrongly assigned phosphorylation sites, and most of these studies are not quantitative, and thus no information of phosphorylation stoichiometry is known. Due to the sensitivity of mass spectrometry, many of these sites will have very low stoichiometry, but this may also reflect the dynamic nature of covalent modifications and a vast array of conditions that experiments were performed under. We refer readers to excellent discussions of this data in  and metabolic conditions. The original publications leading to the PhosPhAT dataset are found on the website. Although glucan water-dikinase 2 or GWD2 (cytosolic), β-amylase 5 (cytosolic), β-amylase 7 (nuclear) and β-amylase 8 (nuclear) are phospho-proteins, they are excluded from this table because they are not plastid localized  of the sites from Supplementary Table The first notable feature of this table is that most starch machinery proteins are phospho-proteins, and most are phosphorylated at multiple sites, including chloroplast transit peptides (cTP), with phosphorylation predominantly on serine and threonine residues  were described . Evidenc to date , as doesFigure 2). CSK and PKUs are of prokaryotic origin and based on sequence do not have activation loops (CSK) or clearly defined activation loops (PKUs). The PKUs appear to have four of the twelve conserved eukaryotic protein kinase domains, including a ‘DFG’ motif .STN 7 and 8 were initially characterized to phosphorylate thylakoid associated proteins. Intuitively, it is hard to rationalize phosphorylation of ‘soluble’ stromal enzymes by thylakoid bound protein kinases, yet a recent phosphoproteomic study identified several non-thylakoid proteins as substrates of these thylakoid bound enzymes . That st2+/Mn2+-dependent enzymes (PP2C/PPM), the protein tyrosine phosphatases (PTP) and the aspartate-based enzymes , the Mg enzymes . The maj enzymes . With th enzymes , yet non enzymes .in vitro and achieve substrate specificity and regulation by association with additional proteins or regulatory subunits  (Table 1) have been localized to chloroplasts. Arabidopsis protein kinases and phosphatases. Using this information, they formally demonstrated 6 different PP2C cTPs could localize red fluorescent protein (RFP) to the chloroplast, strongly suggesting the endogenous enzymes reside there. To date, no biological functions for these 6 PP2Cs are known. We refer readers to Box 1 of The PP2C enzymes  are serine/threonine specific and have proliferated in plants with 80 annotated in  humans) . PPH1 an humans) . PPH1 is humans) . Other PProtein tyrosine phosphatase family biochemistry has determined that many proteins placed in the PTP group based on sequence are in fact not protein phosphatases, but dephosphorylate other molecules  . The besAlthough we have focused on the starch machinery of Arabidopsis photosynthetic tissue, several key works on the phosphorylation of these proteins comes from the endosperm (storage starch) of maize. Multiple phospho-proteomic and focused studies have demonstrated phosphorylation of many starch synthesis and degradative enzymes underscoring the key role of this protein covalent modification in starch biology . It is u32P-ATP, could readily label multiple proteins in the extract, consistent with phospho-proteomic studies discussed above. Second, they identified 3 phosphorylation sites on SBEIIb and perhaps most importantly, they uncovered two peaks of Ca2+-dependent protein kinase activity. The identify of these protein kinases and the function of SBEIIb phosphorylation has yet to be resolved.Starch metabolic enzymes can be soluble in the stroma or bound to the starch granule- either surface associated, or within the granule. Protein phosphorylation is prevalent in the plastid and clearly the starch machinery is controlled in this fashion-typically at multiple sites and with multiple protein kinases suggesting multiple factors/conditions feed into regulating these enzymes. How does phosphorylation affect these proteins? It could alter enzyme activity, control protein–protein interactions, localization in the cell, protein turnover, or even association with starch. All of these questions coupled with the observed degree of phosphorylation of the starch machinery really tell us we are still just at the tip of the iceberg in terms of understanding the role of protein kinases and phosphatases in starch synthesis and degradation.in vitro, protein kinases display substrate specificity based on short sequence motifs, and phosphatases, in general, do not. This meant discovering protein phosphatase substrates using a biochemical approach has been hampered and results from this approach often confusing (Protein kinases have been more studied than protein phosphatases for multiple factors, with one key reason being that onfusing . The reconfusing , 2017. Ionfusing  and PP6 onfusing  and for onfusing . HoweverCW-G, JJ, KM, AK, AV, and GM all participated in the writing of this review.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.