Mitochondrial DNA depletion and multiple deletions syndromes (MDDS) constitute a group of mitochondrial diseases defined by dysfunctional mitochondrial DNA (mtDNA) replication and maintenance. As is the case for many other mitochondrial diseases, the options for the treatment of these disorders are rather limited today. Some aggressive treatments such as liver transplantation or allogeneic stem cell transplantation are among the few available options for patients with some forms of MDDS. However, in recent years, significant advances in our knowledge of the biochemical pathomechanisms accounting for dysfunctional mtDNA replication have been achieved, which has opened new prospects for the treatment of these often fatal diseases. Current strategies under investigation to treat MDDS range from small molecule substrate enhancement approaches to more complex treatments, such as lentiviral or adenoassociated vector-mediated gene therapy. Some of these experimental therapies have already reached the clinical phase with very promising results, however, they are hampered by the fact that these are all rare disorders and so the patient recruitment potential for clinical trials is very limited. Mitochondria are organelles located in the cytoplasm of most eukaryotic cells and are involved in many important cellular metabolic processes, among which the synthesis of ATP has been classically considered their main function . Mitocho2 into electrochemical energy which is finally used to phosphorylate ADP to ATP, the main energy currency in the cell. Electrons from these reduced compounds flow through different substrates by the catalysis of four complexes (I–IV) of the electron transport chain (ETC). This flow is coupled to the pumping of protons from the mitochondrial matrix to the intermembrane space, creating a proton electrochemical gradient across the mitochondrial inner membrane that finally drives the phosphorylation of ADP to ATP through the catalysis of the ATP synthase, the fifth complex of the OXPHOS system [The mitochondrial pathway leading to ATP synthesis is known as oxidative phosphorylation (OXPHOS), and it is mediated by the transformation of the energy contained in the reduced substrates NADH and FADHS system .The endosymbiont hypothesis postulates that mitochondria and plastids originated from prokaryotic progenitors associated with ancestor hosts to generate the first eukaryotic cells , and thiAs indicated above, 13 subunits of the mitochondrial OXPHOS system  are encoded by mtDNA. All other subunits of the five OXPHOS complexes, as well as all other proteins located within mitochondria and needed for their function (more than 1100 proteins) are encoded by the nuclear DNA (nDNA), synthesized in the cytosol and then imported to mitochondria . TherefoAlthough mitochondria have several functions, the term “mitochondrial disease” classically refers to those diseases caused by dysfunctional OXPHOS which results in insufficient ATP synthesis in affected tissues . This enAlternatively, OXPHOS dysfunction can be caused by mutations in nuclear genes encoding subunits of OXPHOS complexes, as well as other factors involved in several processes needed for correct mitochondrial function: factors needed for the ETC complexes assembly, factors regulating mitochondrial protein import, enzymes of the coenzyme Q synthesis pathway, factors involved in mitochondrial translation, maintenance of the mitochondrial lipid milieu or in mitochondrial dynamics, and all factors needed for the correct mtDNA replication and maintenance. Ultimately, mutations in these genes also lead to OXPHOS impairment and therefore to an insufficiency of energy production .Mitochondria contain multiple copies of mtDNA whose integrity and maintenance are critical for the faithful expression of all mtDNA-encoded OXPHOS subunits and correct mitochondrial function. As indicated above, some mitochondrial diseases caused by mutations in nuclear genes affect proteins needed for mtDNA replication and maintenance. When the proteins encoded by these genes do not work properly, dysfunctional mtDNA replication results in quantitative alterations  and/or qualitative alterations . For this reason, this specific group of disorders is known as mitochondrial DNA depletion and multiple deletions syndromes (MDDS). These diseases are somehow peculiar because they respond to Mendelian inheritance, but the somatic aberrations (not inherited from the mother) in the mtDNA molecules confer them some molecular features typical of primary mtDNA disorders, such as different heteroplasmy degrees in different tissues.https://pubmed.ncbi.nlm.nih.gov/29517884/, accessed on 22 May 2021). However, the knowledge acquired in recent years, as the genetic causes of these disorders are gradually being elucidated, indicates that both mtDNA depletion and multiple deletions may be the result of the same genetic defect and therefore primarily triggered by the same dysfunctional process . Most genes associated with depletion in some patients have been found to cause multiple deletions in other patients, even when sharing the same mutation. In some cases, both molecular features may coexist in the same patient [Traditionally, mtDNA maintenance defects have been regarded as two different groups of disorders: mtDNA depletion syndromes, characterized by the reduction in mtDNA copy number in affected tissues, and commonly found in paediatric patients with variable and often severe phenotypes ,13 and m patient . AlthougThe clinical presentation of the different MDDS forms is heterogeneous, even for groups of patients with mutations in the same gene. MDDS are associated with a wide phenotypic spectrum that affects different tissues, ranging from mild forms with presentation in adulthood to severe forms with infantile onset. Moreover, the mutations and phenotypes overlap, which means that alterations in one gene can lead to different phenotypes and similar phenotypes can be caused by mutations in different genes. All these factors make it difficult to associate a clear phenotype to these syndromes in some cases, and therefore to establish their diagnosis and classification.POLG [TWNK [LIG3 causing a mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) like phenotype [mtDNA replication is entirely dependent on nuclear encoded proteins that are synthesized in the cytosol and imported into mitochondria. Interestingly, mutations in genes encoding most of the factors constituting the mtDNA replisome ,49 have POLG  and TWNKLG [TWNK  as causahenotype .POLG that has the catalytic activities , and a homodimeric subunit formed by two small peptides of 55 kDa, each encoded by POLG2, which acts as a DNA binding factor providing processivity to the complex [POLG are the most frequent genetic cause of MDDS  [POLG , affecting both polymerase and proofreading activities, and resulting in mtDNA depletion, multiple deletions and point mutations [The first protein in this list is polymerase gamma, the mitochondrial replicase . It is a complex . Mutatioiseases) . More thutations ,52.POLG cause many different syndromes with variable severities . These include Alpers–Huttenlocher Syndrome (AHS), sensory ataxic neuropathy, disarthrya and ophthalmoparesis (SANDO), neurogaostrointestinal encephalomyopatic syndrome (MNGIE type), and dominant or recessive progressive external ophthalmoplegia (PEO). A recent study compiling 155 patients with mutations in POLG found that neurological, ophthalmological and gastrointestinal are the most frequently reported symptoms. Moreover, the symptoms are associated with the age of onset, with feeding difficulties and seizures being the most common symptoms in people under 12 years, and ataxia, peripheral neuropathy and seizures in patients between 12 and 40 years of age and ptosis, PEO and ataxia in patients with onset above 40 years [POLG2, encoding the ancillary subunit of polymerase gamma, are much rarer than those found in POLG, but since the number of patients with new pathogenic mutations in POLG2 is increasing, it has become evident that the range of phenotypes is also variable [Mutations in 40 years . Mutatiovariable ,54, inclvariable .TWNK  were associated with cases of autosomal dominant progressive external ophthalmoplegia (adPEO) with multiple mtDNA deletions [TWNK found in adPEO patients with multiple deletions affect its helicase activity, stall the replication fork and cause mtDNA depletion in vitro, and also in some in vivo conditions (transgenic mice expressing adPEO variants) [TWNK mutations also cause mtDNA depletion, associated with more severe phenotypes, such as infantile onset spinocerebellar ataxia (IOSCA) [POLG and TWNK [TYMP [Additionally, in 2001, mutations in eletions . This geeletions . Mutatioariants) . These o (IOSCA) , or othe (IOSCA) ,61. Inteand TWNK , but to NK [TYMP   and other long-ago known proteins participating in mtDNA replication such as mtSSB and TFAM. The range of different phenotypes associated with these disorders is also wide, from mild or moderate myopathy to severe phenotypes with multisystem involvement.As shown in The second group of genes associated with MDDS is constituted by genes encoding enzymes that catalyse reactions of dNTP metabolism. Since mtDNA needs a balanced supply of dNTPs for replication and repair, it is not surprising that mutations in these genes cause mtDNA alterations. Some of the MDDS that are currently treated with specific therapies belong to this group and their therapeutic approaches are targeted to modify/correct dNTP homeostasis see  below. TThere are two anabolic pathways to obtain dNTPs in cells: the de novo pathway that is fully active in dividing cells only, and the salvage pathway that recycles deoxyribonucleosides from endogenous turnover and from the diet and takes place in both dividing and non-dividing cells. In addition, catabolism (consecutive dNTP dephosphorylation and further degradation of the resulting deoxyribonucleosides) also contributes to dNTP homeostasis. Alterations in both anabolic and catabolic pathways are involved in MDDS .2 are used as substrate precursors to obtain nucleoside diphosphates (NDPs), which are then reduced to deoxyribonucleoside diphosphates (dNDPs) by the ribonucleotide reductase (RNR). RNR is a key enzyme in the de novo pathway that works as a heterotetramer composed of two copies of a large catalytic subunit (R1 encoded by RRM1) and two copies of a small subunit that can be presented in two isoforms . During S phase, the complex RNR is composed by 2R1/2R2 while in cells out of the S phase, the transcription of subunit R2 is inhibited, R2 subunits are degraded, and R1 thus solely interacts with p53R2, which is expressed throughout the cell cycle. The activity of the complex 2R1/2R2 is higher than 2R1/2p53R2 because in the S phase, the needs of dNTPs are much higher than in the rest of the cell cycle [The main source of nucleotides (which includes dNTPs) is the de novo pathway, which takes place in the cytosol and is fully active in S phase. Small molecules such as amino acids and COll cycle . The dNDll cycle ,68, and ll cycle ,70,71. Tll cycle .RRM2B, have to date been associated with MDDS. As mentioned above, RRM2B encodes p53R2, the p53-regulated small subunit of RNR. Initially, mutations in this gene were associated with a fatal encephalomyopathic form of MDDS with renal tubulopathy and profound mtDNA depletion [Mutations in one gene belonging to the de novo pathway, epletion ; howeverepletion , or domiepletion ,75,76,77As indicated above, the dNTP salvage pathway recycles deoxyribonucleosides (dNs) derived from diet or from cellular metabolism  to dNTPs. It takes place in both cytosol and mitochondria and it is not cell-cycle regulated. It relies on two parallel sets of enzymes in cytosol and mitochondria that sequentially phosphorylate dNs to dNTPs. The first phosphorylation (from dN to dNMP) is the rate-limiting step; in the cytosol, it is catalysed by thymidine kinase 1 (TK1), which phosphorylates thymidine, dThd , and deoxycytidine kinase (dCK) that phosphorylates deoxycytidine (dCtd), deoxyadenosine (dAdo) and deoxyguanosine (dGuo). The equivalent enzymes in mitochondria are thymidine kinase 2 (TK2), which phosphorylates the pyrimidine dNs , and deoxyguanosine kinase (dGK), which phosphorylates the purine dNs (dAdo and dGuo). dNMPs produced in these first steps are further phosphorylated by nucleoside monophosphate kinases (NMPKs) and nucleoside diphosphate kinases (NDPKs), before finally obtaining dNTPs.TK2 and DGUOK (encoding TK2 and dGK) are associated with severe MDDS [TK2 cause a myopathic form with a considerable clinical variability, ranging from severe presentations that result as fatal in infancy or childhood, associated with marked mtDNA depletion in muscle, to less severe presentations that are usually associated with mtDNA multiple deletions, with slower clinical progression or even onset in adulthood [DGUOK mutations cause severe hepatopathy or hepatocerebral syndrome with mtDNA depletion in infants, although myopathic cases with multiple deletions have also been reported [Mutations in ere MDDS ,28. Mutadulthood . DGUOK mreported ,80.NT5M [dNTPs are catabolized through consecutive dephosphorylating reactions  and further degradation of the resulting dNs. Several specific enzymes catalyse some catabolic reactions biochemically different than those mediated by the anabolic enzymes, thus constituting independent regulatory catabolic steps. 5′-nucleotidases, with several isoforms including a mitochondrial isoform encoded by NT5M , catalysNT5M . SAMHD1,NT5M , and dCTNT5M ,84. dN dTYMP, and it is the only nucleotide/nucleoside catabolic enzyme associated with MDDS. Mutations in TYMP cause mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), a recessive disorder characterized by gastrointestinal dismotility, cachexia, ptosis, progressive external ophtalmoplegia, peripheral neuropathy and leukoencephalopathy [The latter enzyme, TP, is encoded by alopathy ,26,85. Aalopathy ,87,88,89alopathy . As a coalopathy ,63,91. AThe de novo pathway is active in replicating cells, providing elevated dNTP cellular levels needed for nuclear DNA replication. Post-mitotic cells downregulate the de novo pathway, have reduced dNTP levels, and thus become much more dependent on the cytosolic and mitochondrial salvage pathways. In the particular case of the dThd, the cytosolic salvage pathway is also inhibited, because TK1 is downregulated. Therefore, the salvage of dThd mainly depends on the mitochondrial TK2 in quiescent cells .As mentioned above, biochemical evidence indicates that deoxyribonucleotides can be exchanged between cytosol and mitochondria through the inner mitochondrial membrane ,68, and SLC28 genes) encoding the concentrative nucleoside transporters CNT (CNT1-3), and the solute carrier family 29 (SLC29 genes) encoding the equilibrative nucleoside transporters ENT (ENT1-4). Concentrative and equilibrative transporters are located in the plasma membranes of cells, with different distribution in different cells and even different regions of the plasma membrane to promote vectorial nucleoside transport through the cell [On the other hand, plasma membranes are impermeable to nucleotides because they do not have transporters enabling the internalization of these charged compounds . Insteadthe cell ,94. Thusthe cell .Although mutations in some nucleoside transporters may be pathogenic ,97, to dA functional mitochondrial dynamics is essential for mtDNA replication because the fission and fusion of the organelle allow mitochondria to exchange components of the matrix, thus facilitating a balanced composition of the mitochondrial dNTP pool and mtDNA replication enzymes, as well as an even distribution of mtDNA molecules throughout the cellular mitochondrial network ,99.OPA1 encodes an alternatively spliced isoform, OPA1-exon4b, that is associated with the mitochondrial inner membrane and directly interacts with the nucleoids, allowing their distribution within the mitochondrial network. OPA1-exon4b also promotes mtDNA replication by interacting with and regulating the replisome [Mitochondria mix through the coordinated fusion of the outer and inner membranes by the action of three GTPases: mitofusin 1 (MFN1) and 2 (MFN2) mediate the fusion of the outer mitochondrial membrane while the dynamin-related protein OPA1 is essential for inner membrane fusion . In addieplisome . Thus, iMFN2 and OPA1 cause MDDS, presenting as optic atrophy, myopathy, axonal neuropathy and Charcot–Marie–Tooth disease in the case of MFN2 [OPA1 [MSTO1, is associated with the mitochondrial outer membrane and participates in mitochondrial distribution and network formation [MSTO1 cause myopathy and ataxia [MICOS13 encodes MIC13, a subunit of the mitochondrial contact site and cristae junction organizing system (MICOS). Mutations in this gene cause liver mtDNA depletion, OXPHOS deficiency and fewer cristae structures [SPG7, and AFG3L2, are components of the m-AAA protease complex, located in the inner mitochondrial membrane. This proteolytic complex degrades misfolded proteins and regulates ribosome assembly [AFG3L2 destabilize OPA1 leading to mitochondrial fragmentation [SPG7 and AFG3L2 have been associated with PEO accompanied by movement disorders with mtDNA multiple deletions in muscle [Mutations in  of MFN2  and optiN2 [OPA1 . Proteinormation . Mutatiod ataxia . MICOS13ructures ,102. Parassembly ,103. Recentation . Mutation muscle ,33,105.SLC25A4, AGK, SUCLA2, SUCLG1, ABAT, MPV17) as related with nucleotide metabolism [SLC25A4 encodes ANT1, the muscle isoform of the ADP/ATP mitochondrial translocator . ATP and ADP nucleotides are not mtDNA building blocks, therefore, the reason why mutations in this gene produce mtDNA multiple deletions seems not to be directly related with dNTP imbalance. Mutations in this gene were associated with myopathy with mtDNA multiple deletions as early as 2000 [Approximately one third of all genes associated with MDDS to date encode proteins that do not have a clear functional link with mtDNA replication. This group constitutes the last category of genes listed in tabolism , however as 2000 .AGK have been associated with mtDNA depletion in Sengers syndrome [SUCLA2 and SUCLG1 have been associated with encephalopathy with mtDNA depletion [SUCLA2 and SUCLG1 disrupt dNTP homeostasis because of this molecular association has not been supported by experimental data. ABAT encodes the mitochondrial enzyme gamma-aminobutyrate aminotransferase, which catalyses the catabolic deamination of the neurotransmitter gamma-aminobutyric acid (GABA). The main reason that this enzyme has been claimed to have a role in dNTP homeostasis relies on the observation that dNTP addition rescues mtDNA depletion, as observed in ABAT-deficient cultured cells [MPV17 mutants in vitro and in vivo [SLC25A4, AGK, SUCLA2, SUCLG1, ABAT and MPV17 remain to be clarified.Similarly, mutations in syndrome ,107. Thisyndrome , it has epletion ,41. Thesepletion , the assed cells . In facted cells , and theed cells ,111. In ed cells . Similar in vivo ) has a rSLC25A21, SLC25A10) encoding two mitochondrial dicarboxylate transporters have been found in patients with mtDNA depletion [GFER encodes a growth factor whose mutations cause myopathy, congenital cataract and developmental delay. In vitro, mutations in GFER are associated with mtDNA multiple deletions in patients’ myoblasts but not in fibroblasts [FBXL4 encodes a protein located in the mitochondrial intermembrane space that belongs to the F-box family, whose exact function is not yet known. Although proteins of this family participate in Skp1-cullin1-F-box (SCF)-dependent proteasomal ubiquitin-dependent protein catabolic process, FBXL4 has also been associated with mitochondrial dynamics [Fbxl4 knockout mice have revealed that the molecular phenotype caused by FBXL4 mutations  may be patients . C1QBP . It is expected that new cases of MDDS patients with mutations in these genes will confirm their association with mtDNA replication disorders.Three of the genes quoted at the OMIM database were associated with mtDNA depletion  have been reported in isolated single cases to date , and foMDDS are mitochondrial disorders. The OXPHOS dysfunction derived from the somatic mtDNA alteration is the factor causing the clinical presentation in MDDS patients. As with other mitochondrial diseases, treatments conceived to ameliorate mitochondrial dysfunction are often applied to MDDS patients even when acting through non-targeted or unspecific mechanisms, and some of the experimental approaches under preclinical investigation have also been explored in MDDS models.MDDS are diseases with poor prognosis in the majority of patients and there is currently no curative therapy for any of them . TherefoPOLG, TWNK and others [Seizures is one of the serious manifestations that may occur in MDDS patients (observed in some forms caused by mutations in d others ). They cd others . Nasogasd others .Special diets are also a possibility used in some mitochondrial diseases ,120. OptMPV17 patients [Supplementation with key compounds of the ETC is a widely used treatment for MDDS. For instance, succinate and ubiquinone have been used to slow down liver dysfunction in patients , and trepatients , levocarpatients . Neverthpatients  and therPhysical therapy can help maintain muscle tone and function . MoreoveSeveral non-targeted experimental strategies have been proposed to fight mitochondrial dysfunction, such as the activation of mitochondrial biogenesis to compensate ATP depletion , the inhTreatments addressing primary mutations in mtDNA, such as mitochondrial replacement therapy  or mtDNAThe first patients with mtDNA depletion and multiple deletions were reported long ago ,137, andAlthough there are other mitochondrial diseases caused by the toxic effect of accumulated metabolites , mitochoSince dThd and dUrd are filterable water-soluble molecules, the first therapeutic approach for MNGIE was haemodialysis. However, this method was ineffective because the reduction in circulating dThd levels was suboptimal  and transient. Three hours after the dialysis, plasma dThd levels returned to their initial values, even after several consecutive repeated rounds of dialysis . More reThe results obtained with haemodialysis in MNGIE patients revealed that urine dThd is reabsorbed in the kidney, so it was proposed to inhibit this reabsorption to contribute to eliminate this metabolite from patients, but finding a drug that selectively inhibits the reabsorption of pyrimidine dNs without affecting the reabsorption of other compounds is difficult .Regarding toxic metabolite accumulation in MDDS other than MNGIE, the existence of an oxidative environment favours the oxidation of cysteine to cystine, which accumulates in several tissues mainly affecting kidneys and eyes. The use of cysteamine bitartrate that catalyses the conversion of cystine to cysteine, was proposed to prevent the formation of cystine crystals. Two clinical trials assessed its efficacy in MDDS . This approach is based on the ex vivo encapsulation of the recombinant TP enzyme within patient’s erythrocytes to be reinfused into the patient . This stAs mentioned above, platelets and white blood cells are among the richest reservoirs of TP in humans ,143. ThiTo gain a first proof of concept for this approach, the effect of platelet infusions from healthy donors was studied in vitro and in MNGIE patients. Patients treated with this strategy reduced dThd and dUrd levels in blood, but this reduction was transient and suboptimal due to the short half-life of platelets (8–9 days) . In factThe first report of two patients treated with AHSCT confirmed that, after successful engraftment, circulating levels of dThd and dUrd were lowered to undetectable or barely detectable levels . HoweverDGUOK, MPV17 and others. Liver transplantation is an option for these patients, although limited to cases of isolated liver involvement, because the multi-organ affectation would not be corrected with this intervention [MPV17 have been treated with this strategy, but its efficacy is controversial because more than half of the transplanted children died in the post-transplantation period [https://pubmed.ncbi.nlm.nih.gov/22593919/ accessed on 22 May 2021). A recent work has reported that liver transplant is only effective in patients with mutations in MPV17 with late onset and mild phenotype [DGUOK patients since most of them show severe hepatic dysfunction. The results have been highly uneven, with some transplanted patients surviving longer than 5 years, while in other cases non-transplanted patients had a better outcome [POLG can benefit from this treatment, but only if they do not manifest Alpers–Huttenlocher syndrome, because transplantation does not revert the brain alterations [POLG, but only one of them survived the procedure [The liver is a severely affected organ in some forms of MDDS such as those caused by mutations in rvention . Some pan period   or catabolic (TYMP) reactions , and the results of the follow-up of this cohort showed that the treatment dramatically changed the course of the disease . The subDGUOK cells [RRM2B, encoding the p53R2 subunit of the de novo enzyme ribonucleotide reductase [TYMP, encoding the catabolic enzyme TP, it has been suggested that the treatment with dCtd could also be considered, because mtDNA depletion seems to be the consequence of secondary dCTP depletion as a consequence of dThd excess [There is preclinical evidence obtained in vitro that this strategy enhances mtDNA replication in other diseases caused by anabolic defects in dNTP metabolism. Several studies have confirmed this result in OK cells ,170,181 eductase . In the d excess .POLG and MPV17 [POLG, as this is the most prevalent gene among all those causing MDDS. In this particular case, it was verified that dN supplementation (which changes dNTP composition) does not induce somatic mutations in de novo synthesized mtDNA [Interestingly, similar positive effects of dN supplementation on mtDNA replication have been observed in vitro and in vivo for mutations in genes not directly related with dNTP metabolism, such as nd MPV17 ,111,181.ed mtDNA . The bioPolg knock in mouse has a dysfunctional proof-reading exonuclease activity and does not present mtDNA depletion [A449T/A449TPolg mouse [POLG mutation in patients) could be a more adequate model to test this therapy, although its phenotype is also limited .In any case, further in vivo preclinical testing is needed to confirm the efficacy of dNs as a treatment for MDDS other than TK2 deficiency. Some existing animal models are not adequate for this purpose. For example, the mutator epletion , and it 9T mouse   vectors. AAV are preferably used over other viral vectors because of the low risk of random insertion into nuclear genome as they are episomic, and because of their long-term persistence in cells . HoweverSLC24A4, encoding the heart/muscle isoform of the mitochondrial adenine nucleotide translocator (ANT1). AVV vectors carrying the wild-type version of Slc25A4 were generated. After some in vitro tests showing the effects of AAV transduction on mutant myoblasts obtained from the knockout mice, the vectors were injected in the skeletal muscle of neonatal mice, which increased Ant1 expression and partially reverted the OXPHOS dysfunction, as assessed by histochemical and immunohistochemical methods [The first preclinical study of gene therapy for a MDDS was conducted in 2005, using a knockout murine model of the myopathy caused by mutations in  methods .MPV17. This study faced some difficulties derived from the frequently observed fact that genetically modified mice do not fully recapitulate the disease they are intended to model. Although the Mpv17 knockout mouse shows many molecular features that recapitulate those observed in the human disease (the most relevant one being mtDNA depletion in the liver), the mice fail to develop any functional phenotype other than late-onset kidney dysfunction [Mpv17 knockout mice developed severe liver cirrhosis and liver failure after a few weeks. The authors treated Mpv17 knockout mice with AAV2/8 carrying the correct human coding sequence of MPV17 by intravenous injection, and the treatment rescued mtDNA levels and OXPHOS function, and prevented the liver phenotype induced by the ketogenic diet [AAV-mediated gene therapy has been also tested in a model of the hepatocerebral form of MDDS caused by mutations in function . Howevernic diet .Mpv17 knockout model, the one available model for MNGIE (the double knockout for Tymp and Upp1) only recapitulates the biochemical imbalances of the disease [Gene therapy has been studied for MNGIE. For this disease, targeting a specific tissue is not the main objective because, as previously mentioned, the pursued effect is to clear toxic nucleosides on a systemic level. Whatever organ or cells accessible to the blood stream may be a good target to transduce the clearing enzyme TP. As indicated for the murine  disease .Two different strategies have been tested. The first one targeted the hematopoietic tissue, which is one of the richest sources of TP in humans (but not in mice) using lentiviral vectors. This approach demonstrated a reduction in dThd and dUrd levels observed in the animal model to barely detectable levels . The effHowever, this strategy was associated with increased mortality of the animals, most likely owing to the aggressive conditioning of recipient mice ,191. TheAll gene therapy approaches discussed above have shown very promising results. Unfortunately, there are several big steps to take before they can be translated to patients. One of the main limitations is the amount of vector needed for transduction in humans. In the case of the AAV vectors targeting the liver, we also need to consider the fact that the transduction efficiency is lower in humans as compared to that observed in mice ,197,198.OPA1 is found to regulate the organization of respiratory complexes in mice with mutations in genes not related to MDDS [As some MDDS are caused by mutations in factors regulating mitochondrial dynamics, strategies aimed at restoring these processes and improving mitochondrial shape have been considered. Moderate overexpression of  to MDDS . Additio to MDDS . AlthougMFN2 can be potentially treated with a strategy that uses a small mitofusin agonist that activates MFN2, thus promoting mitochondrial fusion [MFN2 p.Thr105Met mutant mice.MDDS caused by mutations in l fusion . This woAnother strategy targeting mitochondrial shape consists of the use of Szeto–Schiller (SS) peptides. SS peptides enter mitochondria and bind cardiolipin, a lipid of the inner mitochondrial membrane that regulates respiratory chain complexes and cristae structures. Elamipretide  is an SS peptide that has been found to be able to correct mitochondrial ultrastructure ,203. AgaPOLG mutants [POLG mutations, demonstrating its ability to rescue mtDNA levels [POLG deficiency. Although it is known that CLO is an antiarrhythmic acting as a potassium channel blocker, its mechanism of action in rescuing mtDNA levels is not known [A yeast-based screening assay of 1500 chemical compounds has identified clofilium tyosilate (CLO) to be useful to correct mtDNA instability in  mutants . This coA levels . Furtherot known .Many therapeutic approaches have been suggested or applied to treat mitochondrial diseases in general, and more specifically for MDDS. The complexity and variability of these disorders makes it difficult in many cases to definitely conclude whether they have demonstrated substantial efficacy. Those in clinical trial phases are expected to reach this milestone at the specific expected time points defined in the trial.DGUOK, POLG, MPV17 and TYMP, or AHSCT for patients with mutations in TYMP, have shown variable success and to date are the only available choices for patients that, without treatment, could face a fatal outcome. One of the potential alternatives for some of these forms of MDDS is the implementation of gene therapy approaches. As indicated at the end of the corresponding Currently, MDDS have very limited options for therapy, although significant progresses have been achieved in recent years and the situation is significantly different now than it was ten years ago. Some aggressive treatments, such as liver transplantation for mutations in POLG, MPV17, DGUOK or others [The potential improvement that may constitute the dN-treatment if it is finally approved for TK2 deficiency should be also underlined. Robust clinical results show that this therapy changes the clinical course of the disease, at least for paediatric patients . Importar others ,182,206.The special importance of the availability of in vivo models to test these therapies should be also noted. Experimental evidence supporting the efficacy of some of the experimental approaches discussed above has only been obtained in vitro, and we should be very cautious before using these approaches in patients, unless their efficacy is additionally confirmed in vivo using animal models. There are several genetically modified mouse strains that aim to model these diseases . This constitutes a good record if we consider that MDDS are very rare disorders. Moreover, there is growing optimism in the possibility of expanding the potential therapy options for these disorders due to the large number of preclinical data that researchers in the field have gathered in recent years. This is a growing group of diseases, with mutations in new genes causing MDDS found almost every year. However, they are well delimited by the presence of one or more of the hallmarks that define this group of disorders . Many of the genes associated with MDDS encode proteins belonging to the mtDNA replication machinery, dNTP metabolism or mitochondrial dynamics, and these biological processes are the subject of in-depth investigation by the scientific community working in the mitochondrial field. Without a doubt, the combination of relatively well-known pathomechanisms of these disorders and an increasing knowledge of the basic biochemical and molecular processes involved in mtDNA replication has contributed to the implementation of many novel therapy approaches for MDDS. Our efforts should now be dedicated to maintaining and further improving our knowledge in this field, thus generating new approaches and refining those already under preclinical and clinical investigation, and to overcome the aforementioned barriers hindering their final implementation into medical practice.The potential therapy choices for patients with mtDNA maintenance disorders have significantly increased in the last ten years. The data collected in this review indicate that there are currently nine active interventional clinical trials for seven different treatments, recruiting patients with mutations in five different genes (


This study examined socioeconomic inequalities in compliance with precautions and health behavior changes during the coronavirus disease 2019 (COVID-19) outbreak using a representative Korean sample.This exploratory study utilized around 210,000 participants aged ≥25 years in the Korean Community Health Survey 2020. Socioeconomic status was measured with educational attainment and household income. Outcomes included non-compliance with 8 precaution measures and deterioration in 6 health behaviors. The relative inequality index (RII) was calculated to quantify the degree of inequality by education and income level. RII values >1.0 indicate that deprived people have a higher frequency of health problems, and RII values <1.0 conversely indicate a higher frequency of health problems in more advantaged groups.People with lower education or income levels tended to have higher rates of non-compliance with COVID-19 safety precautions . Lower education and income levels were associated with an increased smoking amount  and sleep duration changes . On the contrary, higher education and income levels were associated with decreased physical activity  and increased delivery food consumption . However, increased alcohol drinking was associated with lower education and income levels in younger men , but with higher levels in younger women .Our findings suggest the need to develop customized strategies, considering the characteristics of the target population, to decrease the burden and impact of the COVID-19 outbreak. The coronavirus disease 2019 (COVID-19) outbreak was first reported in December 2019 and continued to spread worldwide . AdditioWe analyzed an anonymized research dataset obtained from the Korean Community Health Survey (KCHS) for 2020. The KCHS is a nationwide survey of health interviews conducted by the Korea Disease Control and Prevention Agency (KCDA), aiming to establish a standardized community survey with which to develop health projects for all local districts. Detailed information on the study design and aims of the KCHS has been previously reported . BrieflyEducational attainment was categorized into 4 groups: elementary school or less, middle school, high school, and college or more (reference group). Equivalized household income was defined as the total monthly household income divided by the square root of the number of household members. Participants were divided into 4 groups according to the quartiles of equivalized household income, from Q1 (lowest) to Q4 . The quartiles of monthly household income were separately calculated by gender and age (< 65 or ≥ 65) considering that those aged ≥ 65 were mostly economically inactive. Self-reported responses for compliance with COVID-19 safety precautions and deterioration of health behaviors during the COVID-19 outbreak were utilized as the main outcomes. First, compliance with COVID-19 safety precautions was evaluated through questions asking whether the participants, in the last week, (1) covered their mouth while coughing; (2) practiced regular disinfection; (3) practiced regular ventilation; (4) wore a mask in indoor facilities such as public transportation, restaurants, and department stores; (5) wore a mask when it was hard to keep a distance; (6) adhered to recommendations on the minimal physical distance (2 meters); (7) refrained from visiting patients in hospitals; and (8) refrained from going out. These items had responses of “completely complied,” “complied,” “failed to comply,” or “not applicable.” Participants were dichotomized according to responses of “failed to comply.” Health behavior changes were evaluated using questions asking whether participants experienced changes in (1) physical activity including walking or exercise; (2) sleep duration; (3) amount of instant meal and soda consumption; (4) amount of delivery food consumption; (5) alcohol drinking; and (6) amount of smoking. These questions had responses of “increased,” “did not change,” “decreased,” or “not applicable.” Health behavior deterioration was captured as increases in the smoking amount, alcohol drinking, consumption of instant food and soda, or consumption of delivery food; changes in sleep duration; and decreases in physical activity.The demographic characteristics of the participants were presented as weighted mean± standard deviation or frequency and weighted proportion. Logistic regression models were used to estimate the associations between SES  and outcomes of interest . Age-adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using the PROC SURVEYLOGISTIC procedure to apply stratification, primary sampling units, and population weights. Additionally, the analysis of compliance with precautions was adjusted for COVID-19-related characteristics including quarantine/isolation experience due to COVID-19 infection and recent experience of fever/coughing. For health behavior changes, smoking status (past/current), alcohol drinking frequency, sleep duration, and moderate physical activity (yes/no) were additionally adjusted in the models for increased smoking amount, increased alcohol drinking, changed sleep duration, and decreased physical activity. Furthermore, the age-adjusted frequencies of non-compliance with precautions and health behavior deterioration were estimated using the direct standardization method based on the 2015 Korean census.The relative index of inequality (RII) was developed to quantify the relative inequality gap between those who are positioned in the lowest and the highest SES categories . The RIIThe data in this study were obtained from the KCHS, KDCA, Ministry for Health and Welfare in Korea. Our data from the KCHS are freely available if researchers submit appropriate institutional review board clearance to the KDCA. This study was approved by the Institutional Review Board of Severance Hospital at Yonsei University College of Medicine (IRB No: 4-2021-1256).Overall, in the nationwide health survey conducted in 2020, significant socioeconomic discrepancies were observed in compliance with COVID-19 precautions and in health behavior changes. People with lower education or lower income levels were more likely to fail to comply with safety precautions, but the relative degree of inequality differed by types of precaution measure and by respondents’ gender and age. Socioeconomic discrepancies in health behavior deterioration showed variation not only in their magnitude, but also in the direction of inequality indicators.Our findings of SES inequalities in safety precautions align with those of a previous study analyzing the data of 6,000 individuals from the United States, the United Kingdom, Italy, China, Japan, and Korea, in that high-income people adopted more self-protective behaviors such as mask-wearing during the COVID-19 pandemic . In the Socioeconomic inequalities were also observed in deteriorating health behavior after the COVID-19 outbreak, but the direction and magnitude were different depending on the type of health behavior. Our study findings align with those of a previous study with 1,809 United States participants recruited through convenience sampling, which reported that people with higher educational attainment tended to decrease tobacco use or increase alcohol consumption during the COVID-19 outbreak . In the In the current study, socioeconomic inequalities in increases in smoking amounts and changes in sleep duration were more prominent in the younger group. Among young people, who are likely to be economically active, those with lower education or income levels showed greater fear of financial difficulties during COVID-19 ,23. StudAn advantage of our study is that it used a representative sample that covered all administrative districts of Korea and used data collected according to a standardized questionnaire and quality control program. However, several limitations should be considered. First, there is a possibility of measurement error as both compliance with COVD-19 precautions and changes in health behaviors were measured based on self-report surveys. We cannot completely rule out the possibility that these measurement errors may have varied depending on the socioeconomic level. Second, our study could be considered as an exploratory analysis in the association of SES with precaution compliance and health behavior changes. Confirmatory studies are required to further evaluate socioeconomic inequalities. Third, the RII is a relative indicator of inequality and is useful for comparing the degree of inequality between different health outcomes or subgroups, but these findings need to be supplemented with data on absolute inequality levels.In conclusion, this study of a representative Korean sample found that socioeconomic inequalities existed in compliance with COVID-19 precautions and in health behavior deterioration. People with low SES were more likely to fail to comply with safety precautions, but the magnitude of inequality varied by gender, age, and type of precaution measure. The socioeconomic inequality in health behaviors differed not only in its magnitude, but also in the direction of inequality indicators. Our findings suggest that it is necessary to develop a customized strategy that considers the characteristics of the target population to reduce the burden and impact of the COVID-19 outbreak.


While the loss of GRN is primarily associated with neurodegeneration, the biological functions of the secreted growth factor-like protein are more diverse, ranging from wound healing, inflammation, vasculogenesis, and metabolic regulation to tumor cell growth and metastasis. To date, no disease-modifying treatments exist for FTD, but different therapeutic approaches to boost GRN levels in the central nervous system are currently being developed (including AAV-mediated GRN gene delivery as well as anti-SORT1 antibody therapy). In this review, we provide an overview of the multifaceted regulation of GRN levels and the corresponding therapeutic avenues. We discuss the opportunities, advantages, and potential drawbacks of the diverse approaches. Additionally, we highlight the therapeutic potential of elevating GRN levels beyond patients with loss-of-function mutations in GRN.Frontotemporal dementia (FTD) is a neurodegenerative disease, leading to behavioral changes and language difficulties. Heterozygous loss-of-function mutations in  GRN, MAPT, and C9orf72) explaining the majority of the genetic cases  is a neurodegenerative disease, leading to behavioral changes and language difficulties. Previously considered a rare disease entity, FTD is in fact the third leading cause of all dementia cases  positive inclusions, and FTLD-TDP/FTD-U for ubiquitin and transactive response DNA-binding protein 43 (TDP-43) positive inclusions  refers to the underlying neuropathology. FTLD neuropathological subtypes were established based on the proteins found in the neuropathological inclusions. The two main subtypes are FTLD-tau for microtubule-associated protein tau  were tau-negative. In 2006, mutations in the neighboring GRN gene were discovered, explaining the genetic linkage to the same chromosomal locus , Valosin-Containing Protein (VCP), ubiquilin 2 (UBQLN2), TANK-Binding Kinase 1 (TBK1), sequestosome 1 (SQSTM1), and T cell–restricted intracellular antigen 1 (TIA1) explain residual cases of genetic FTD  is the most common clinical manifestation of frontotemporal lobar degeneration, occurring in 60 percent of cases. When language is primarily impaired the term primary progressive aphasia is used (PPA)  gene is located on chromosome 17q21, 1.7 Mb centromeric of MAPT, and consists of 12 protein-coding exons and a 5′ non-coding exon. The mammalian GRN gene codes for a repetition of seven and a half granulin domains. Each granulin domain is encoded by two neighboring exons, contributing either the N- or C-terminal half of the domain. The unique structure of the evolutionary conserved granulin motif is reviewed in Palfree et al.  , GRN cerebrospinal fluid (CSF) levels rise with disease progression and correlate with cognitive impairment and with the expression of the microglia derived soluble TREM2 (triggering receptor expressed on myeloid cells 2) , as the majority of GRN mutations are nonsense and frameshift mutations that introduce premature termination codons (PTC). One therapeutic option could therefore be to block NMD in order to salvage truncated transcripts or to promote read-through of nonsense codons. The efficacy of these approaches hinges on whether the proteins resulting from nonsense suppression retain substantial function. A study in rodents, focusing on the most prevalent nonsense mutation (GRNR493X), provided proof of principle for the inhibition of NMD by use of antisense oligonucleotides (ASO) as well as by genetic and pharmacological intervention  as an enhancer of GRN expression and validated their results in cells from patients with frontotemporal dementia . The compound induced GRN in rodent brain and in FTD-GRN patient-derived lymphoblasts but failed to increase GRN levels in patients at the doses tested . In July 2020, Prevail Therapeutics started the first GRN-AAV Phase 1/2 clinical trial in 15 people with FTD due to a GRN mutation. The study evaluates a different GRN-AAV9 vector, administered in combination with immunosuppressive drugs .Gene therapy, the As an alternative to gene therapy, direct intracerebroventricular delivery of recombinant GRN could allow a more controlled and dosed delivery of GRN. Whereas this delivery method has been safely tested before for other recombinant proteins  penetrance. The protein named PTV:PGRN (Protein Transport Vehicle) was engineered to bind to the human transferrin receptor, facilitating transport across the BBB. Preliminary reports state that intravenously administered PTV:PGRN can correct inflammatory and lysosomal lipid alterations in GRN expression. The group identified different GRN transcripts with short and long 5′ untranslated regions (UTR). Expression of the long 5′UTR, containing an upstream open reading frame, stalled the ribosomal translation of GRN and affected mRNA stability. Similar repression mechanisms by 5′UTRs have been demonstrated for the mRNAs of BACE1 and ADAM10. Selective repression of GRN translation could contribute to the differential expression of for example neurons and microglia, although to our knowledge, this has not been investigated.Research conducted by Capell et al.  providedGRN mRNA stability and translation. The 3′UTR of progranulin contains a predicted miR-29b binding site and the knockdown of miR-29b was shown to increase the secretion of GRN in NIH3T3 cells , rs5848, in the 3’UTR of the GRN gene was overrepresented in FTLD cases and was predicted in silico to increase the binding of miRNA-659, resulting in decreased GRN expression  . The completed Phase 1 study (NCT03636204) demonstrated that AL001 treatment could normalize GRN CSF levels in symptomatic and asymptomatic GRN mutation carriers  as a regulator of GRN  kinase and phosphoinositide 3 (PI3) kinase pathways, it does not bind a classical growth factor receptor nor does the mode of growth factor signaling resemble that of other growth factors like PDGF or EGF  receptors  as well as The interaction of GRN with a multitude of extracellular, transmembrane, intracellular, and nuclear proteins is remarkable  and by mode of administration . The resulting pattern of (ectopic) GRN expression will therefore influence the biological effect and possible side-effects. Close monitoring of GRN protein levels will be needed to ensure expression within a physiological range, as elevated GRN levels have been associated with cancer, metabolic disease, and chronic inflammatory states.In recent years, significant progress has been made towards the development of therapies modulating GRN levels. While raising GRN has always seemed straightforward, the multitude of mechanisms at play in regulating GRN levels pose extra challenges for the development of therapeutic agents. With a common goal to raise GRN levels, every therapeutic intervention differs in the extent of action depending on the biological strategy used (e.g., affected by viral vector tropism or depending on GRN mutations provides a unique starting point for further research. The recent extensive documentation of fluid, neuroimaging and cognitive biomarkers for FTD will undoubtedly prove to be crucial in monitoring treatment response and guiding therapeutic development (Benussi et al., The current development and utilization of GRN therapy in FTD patients with JT, CV, and PVD wrote the manuscript. All authors made substantial contributions to the discussion of the content, reviewed and edited the article. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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How does the information in the genome program the functions of the wide variety of cells in the body? While the development of biological organisms appears to follow an explicit set of genomic instructions to generate the same outcome each time, many biological mechanisms harness molecular noise to produce variable outcomes. Non-deterministic variation is frequently observed in the diversification of cell surface molecules that give cells their functional properties, and is observed across eukaryotic clades, from single-celled protozoans to mammals. This is particularly evident in immune systems, where random recombination produces millions of antibodies from only a few genes; in nervous systems, where stochastic mechanisms vary the sensory receptors and synaptic matching molecules produced by different neurons; and in microbial antigenic variation. These systems employ overlapping molecular strategies including allelic exclusion, gene silencing by constitutive heterochromatin, targeted double-strand breaks, and competition for limiting enhancers. Here, we describe and compare five stochastic molecular mechanisms that produce variety in pathogen coat proteins and in the cell surface receptors of animal immune and neuronal cells, with an emphasis on the utility of non-deterministic variation. Despite the shocking complexity of eukaryotic life, eukaryotic genomes often contain less than 20,000 protein-coding genes. While most genes are expressed in a deterministic manner, a variety of molecular mechanisms have been discovered that expand the coding capacity of the genome by expressing cell surface molecules in a quasi-random manner. Expression systems that accomplish cell surface molecule diversification make use of genomic rearrangement, RNA splicing, and epigenetic restriction to create a vast array of molecular variants from a limited amount of DNA. In this way, the static information within genomes can generate a wider diversity of cells throughout the body or across unicellular populations.This diversity is crucial for the proper functioning of many different biological systems. The immune system, for example, relies on diverse antigen receptors to bind to and recognize an incredible range of potential pathogens and harmful molecules. Without the stochastic mechanisms driving this variation in expression, the proper functioning of the immune system would be severely compromised. The nervous system is similar in that it also relies on diversity in gene expression for proper functioning. Like the immune system, neurons in chemosensory systems express diverse receptors to bind a wide array of environmental molecules. In addition, neurons, even of the same class, must be sufficiently different from one another in order to properly identify self vs. non-self. Finally, pathogens also diversify their surface molecules in an arms race with the adaptive immune system. Thus, understanding the mechanisms that produce non-deterministic cellular heterogeneity is an important area of study.Trypanosoma brucei, pathogen identification by B cell and T cell receptors, neuronal self-avoidance through expression of Dscams and protocadherins, and the perception of stimuli through the olfactory system. While other reviews have compared subsets of these systems, here we broaden the scope of the comparison by considering both single-celled organisms and animals and by considering both neuronal and barrier functions  by the parasite unctions . In addiVSGs. Lastly, in three of these systems, there are feedback mechanisms downstream of selection that can act to help correct any flaws that were made during selection. In antigenic (VSG) variation, this feedback is whether or not the cell survives the host immune system. In V(D)J recombination, feedback takes place within the germinal center when higher affinity B cells win the competition for antigen. In olfactory receptor choice, the feedback mechanism allows the cell to choose a different olfactory receptor gene if it initially chose a flawed one—or stops the cell from choosing another gene if the one it already chose is functional. Although similar feedback processes may take place in Dscam and protocadherin expression, they have not yet been discovered.There are many similarities across these five examples  and is able to switch which VSG is expressed in order to evade host immune systems . The field has primarily focused on these first two mechanisms, with telomere exchange still relatively underexplored, so we will focus more on gene conversion and in situ switching in this review.Multiple overlapping mechanisms accomplish this dynamic VSG switching . The preion site . VSG expsilenced . A thirdVSG expression sites plays a part in their repression   that promote transcription of  (ESAGs) . They conscribed  , active BES are localized to an extranucleolar region termed the expression site body  complex  and VSG exclusion 2 (VEX2). VEX1 has been shown to positively regulate the active VSG site in cis while also negatively regulating all other VSGs in trans . VEX1 binds to VEX2 independently of transcription, and together, they are responsible for VSG exclusion . The BESs, along with many of the silent VSG arrays, are located in sub-telomeric regions of the genome . These processes allow for a truly outstanding level of diversity to emerge from just a few germline-level genes, preparing the immune system for any antigen it might face without taking up very much genomic space. Much as learning mechanisms in the nervous system allow animals to relate arbitrary sensory stimuli to the contexts in which they are experienced, selective processes during B and T cell development in the context of an immune response shape cellular immune responses according to the “meaning” of self, benign, or pernicious antigens.The coexistence of host and pathogen has largely driven the diversification of both the host’s immune surveillance and the pathogen’s antigenic determinants . MammaliAntibodies, which are immunoglobulin proteins, possess variable binding surfaces that can recognize diverse antigens. These antibodies can be secreted in the serum or bound to the surface of B lymphocytes to form B cell receptors (BCRs). T lymphocytes also have surface receptors (TCRs) that recognize antigens in combination with antigen presenting major histocompatibility complex (MHC) proteins. Like TCRs and BCRs, MHC proteins are present in the population as diverse alleles. While TCR and BCR diversity is generated via somatic mechanisms, population-level MHC diversity is maintained at the germline level via balancing selection.Immunoglobulins are composed of covalently-linked heavy and light chains, both of which possess a variable N-terminus that recognizes antigens and a constant C-terminus that can recruit effectors . Here, w6 potential combinatorial products that can arise from these V(D)J and heavy-light chain pairings, and final protein products are additionally varied by junctional diversification that occurs during recombination. Recombination is induced by RAG1/2 (recombination-activating gene) which target discrete locations within the immunoglobulin loci through conserved and repeated DNA sequence elements (In V(D)J recombination, the N termini of the heavy and light chains are rearranged to bring distinct V (variable) segments, followed by the D (diversity) segments, and then the J (joining) segments into proximity with the constant region  Schroed. The twoelements   proteins are diversified during lymphocyte maturation via insertion of LRR modules from flanking regions of the locus (reviewed in While V(D)J recombination removes V regions proximal to D or J segments, distal V options remain intact, and each V has its own upstream promoter. Nevertheless, transcription always begins at the V region most proximal to D/J and therefore ensures that only a single V—the most proximal—is included in the transcript . This seIg alleles are transcribed biallelically early in B cell development, indicating that transcriptional activation alone does not govern the allelic exclusion of these loci (Ig genes that have not undergone proper V(D)J recombination possess premature stop codons that prevent production of functional protein J recombination of one allele induces the suppression of the second non-recombined allele, preventing subsequent recombination and productive transcription . This isEarly models suggested that the low rate of recombination allowed for a probabilistic first-come, first-serve mechanism where allelic selection was purely based on which allele happened to recombine first , but conThe vast pool of antigen receptors allows for modest binding and subsequent detection of most antigens, but once lymphocytes are activated they undergo numerous rounds of selection to increase their affinity for the antigen. We will focus on the positive selection of antigen-selective B lymphocytes in the germinal center of lymph nodes. Of course, stochastic production of TCRs and BCRs can also lead to dangerous autoimmune reactions; these are minimized due to distinct processes of negative selection that occur during lymphocyte development (reviewed in BCR locus undergoes a significant increase in the rate of point mutations compared to the rest of the genome (Ig genes (Ig strands at a high rate. DNA damage response processes then generate base substitutions at and around the lesion created by the deoxy-uracil (Somatic hypermutation (SHM), a key process in affinity maturation, functions to diversify BCRs and promote the adaptive immune response. During SHM, the e genome . These mIg genes . AID funy-uracil .Lymph node germinal centers (GCs) are the site of B lymphocyte clonal selection that drives affinity maturation to produce memory B cells and antibody-secreting plasma cells . The GC interclonal competition, competition eventually progresses to intraclonal competition between variants generated by SHM  family of immunoglobulins. Via alternative splicing, the Drosophila Dscam1 locus encodes up to 38,016 distinct Dscam isoforms, all of which contain the same basic structure: an ectodomain comprised of 10 immunoglobulin (Ig) domains and six fibronectin type III repeats, a transmembrane domain, and a C-terminal cytoplasmic tail (Ig2 (12 variants), Ig3 (48 variants), Ig7 (33 variants), and the transmembrane domains (2 variants). This means that for 38,016 distinct isoforms there are potentially  distinct ectodomains; at least 18,048 of these ectodomains are confirmed to support “homophilic” binding, or binding between identical isoforms  neurons  and can be cleaved by proteolysis in vivo . In mammin vivo  , the exon six variants of the Dscam1 locus are maintained in a repressed state until a selection event specifically activates a variant for expression  and promote binding of serine-arginine rich (SR) proteins  as the hnRNP responsible for global repression of the exon 6 cluster . Hrp36 wDscam1 exon 6 cluster appears to be determined by the binding ability of different selector sequences, which may be modulated by splicing factors and RNA-binding proteins (RBPs) such as SR proteins and hnRNPs . It is nt times . These pnt times . Future Experimental evidence and comparative genomic analyses indicate that selection of exons 4, 9, and 17 also relies on competing RNA secondary structures, which may be recognized by distinct but overlapping sets of RBPs. This is discussed further elsewhere . There iStudies that reduced the number of possible Dscams underline the importance of great isoform variety. Regarding self-avoidance, studies found that reducing the Dscam repertoire to just one isoform produced marked neural circuit defects in MB and olfactory receptor (OR) neurons . In anotWhile a large variety of isoforms is clearly required, it is unclear whether any one isoform is necessary for normal patterning. In particular, studies that serially deleted different exon 4 variants (thereby eliminating particular Ig2 domains) did not produce observable phenotypes in MB or da neurons, indicating that self-avoidance does not require any specific isoform . On the Vertebrate protocadherins function analogously to invertebrate Dscams in that both systems mediate neuronal self-avoidance . As withDSCAM gene is conserved in mammals, it does not encode diverse isoforms. In some tissues, including the vertebrate eye, DSCAM has been shown to play a deterministic role in synaptic matching , beta (PCDHB), and gamma (PCDHG) clusters, arranged in tandem along chromosome 5 in humans. The PCDHA genes are encoded by a set of 15 large variable (V) exons that precede 3 constant (C) exons  exons  (Wu and C) exons . Each V  C exons . The PCDct genes .PCDHA cluster, PCDHAC1 and PCDHAC2, and the PCDHGC3, PCDHGC4, and PCDHGC5 from the PCDHG cluster are very similar to each other but not to other V exons  , has also been identified downstream of the PCDHG cluster (PCDHG cluster expression. This suggests the existence of distinct PCDHG enhancer element(s) (Xγ elements) whose exact genomic location is unknown (The promoters preceding nt (CSE) , which hnt (CSE) . With thA V exon  (Chen anA V exon . Similar cluster : The PCDSE sites . A long- cluster . Deletio unknown .PCDHA only), must escape repressive methylation established by DNMT3B during embryogenesis , and exon sequence , which is located within the intron between the second and third C exons, lacks a CSE site but is still able to mediate DNA-looping through interaction with cohesin alone , but expression patterns are not well classified , which recognize odorant molecules to detect smells. Buck and Axel correctly estimated that there are roughly 1,000 OR genes in the mammalian genome  genes are organized in large clusters in the genome and, like n genome . StochasORs of mice and humans reveals that they share many subfamilies of OR genes. However, homology relationships are difficult to discern at the gene level, and mouse subfamilies typically include more OR genes than human subfamilies , hylation .OR genes per OSN precedes the eventual translation of just a single OR per neuron J recombination, clustered VSG expression instead follows a temporal bias for which the probability of certain VSGs appears to change throughout the course of infection, with mosaic VSGs emerging in the later stages of infection. Within the confines of these spatial, cell type, and temporal restrictions the available genes and gene segments still seem to be stochastically distributed. The mechanisms that restrict stochasticity are largely unknown.These systems also exhibit context-driven restrictions and biases regarding which genes or gene segments are chosen. Within the olfactory epithelium, OSNs are spatially segregated into different zones, and each OR is expressed only within a single zone. Dscams and PCDHs show a similar restriction, but based on cell type rather than spatial location, as the pattern of variable domains expressed appears to vary between different populations of neurons. VSG expression site is active relies heavily on nuclear localization and chromatin remodeling, a feature shared by the other four systems. During VSG, OR, and antigen receptor selection the inactive alleles are repressed in heterochromatic regions, with VSG and antigen receptor loci localized to the nuclear periphery and OR gene arrays compacted into repressive foci. The selected allele in these systems is found in accessible euchromatic regions, which for antigen receptors is in the nuclear center, for ORs entails the active gene escaping the repressive foci, and for VSG genes requires the single active expression site to localize to the expression body. These nuclear localization events are intimately tied to chromatin modifications and DNA methylation. VSG expression sites localized to the expression body show significant nucleosome depletion. Interestingly, the active VSG expression site is enriched with H3K10 acetylation as well as H3K4 trimethylation. H3K4me3 is an activating signature that is also found in the active regions of the OR, PCDH, and antigen receptor loci. Histone modifications are prominent features of these systems. The antigen receptor loci are further activated by H3K9 acetylation and H3K36 trimethylation, whereas the inactive locus is enriched for repressive H3K9 di- and trimethylation. H3K9 methylation is also utilized to repress OR loci alongside H4K20 di- and trimethylation.The process determining which cis-acting elements are also a common feature activating the selected sequences throughout many of these systems. Antigen receptors and PCDH loci share mechanistically similar downstream enhancer elements that loop back to the loci and activate the selected promoter, in both cases scanning for the first available promoter sequence either at the most proximal V segment or hypomethylated PCDH segment. Enhancers play a role not only in inducing expression of the selected OR gene, but also in carrying out the selection process, as enhancers coalesce first in cis, and then in trans onto a given OR gene for selective expession. Even though Dscam selection occurs at the RNA level, an enhancer-like element (the docking site-selector sequence stem-loop) occurs in the secondary structure of the transcript. Reminiscent of the previous systems, the stochastic binding of variant selector sequences to the docking site (which forms the stem-loop) dictates the pattern of exon splicing. While VSG expression has not been shown to require enhancer interactions, the selected VSG still must be located within the active expression site. Further research into whether a limiting enhancer selects a VSG for selection at a particular time will be of great interest.Singular Trypanosoma brucei, immune system pathogen identification by B cell and T cell receptors, neuronal self-avoidance through diverse expression and self-binding processes of Dscams and protocadherins, and the perception of stimuli through the olfactory system, we have shown that the role of diversity is crucial to the survival and function of organisms through both internal and environmental interactions. Common processes such as monoallelic expression, epigenetic regulation, and specificity of binding of variable domains were discussed. It is important to recognize that all five systems studied are examples of surface molecules that utilize the stochastic selection of genetic material to create cellular diversity. Like aleatoric music and the world-generating algorithms of sandbox games, these systems maximize limited genetic information to construct complex and unpredictable outcomes.In this review, we have emphasized the importance of stochastic processes in achieving variability in barrier and nervous systems. By analyzing immune system evasion by the parasite


PADI3 are linked to human alopecia. Furthermore, we propose mechanisms of PADI4 dependent fine-tuning of the hair follicle lineage progression. Finally, we discuss citrullination in the context of inflammatory alopecia. We present how infiltrating neutrophils establish a citrullination-driven self-perpetuating proinflammatory circuitry resulting in T-cell recruitment and activation contributing to hair follicle degeneration. In summary, we aim to provide a comprehensive perspective on how citrullination modulates hair follicle regeneration and contributes to inflammatory alopecia.In this Review article, we focus on delineating the expression and function of Peptidyl Arginine Delminases (PADIs) in the hair follicle stem cell lineage and in inflammatory alopecia. We outline our current understanding of cellular processes influenced by protein citrullination, the PADI mediated posttranslational enzymatic conversion of arginine to citrulline, by exploring citrullinomes from normal and inflamed tissues. Drawing from other stem cell lineages, we detail the potential function of PADIs and specific citrullinated protein residues in hair follicle stem cell activation, lineage specification and differentiation. We highlight PADI3 as a mediator of hair shaft differentiation and display why mutations in  Hair follicles (HFs) undergo phases of destruction and regeneration throughout the lifespan of an organism. Regeneration and hair formation depend on balanced stem cell renewal and differentiation, integrating transcriptional and epigenetic regulation with microenvironmental niche-derived cues. Failure to coordinate signaling, or respond to inflammatory signals, deregulates hair follicle lineage progression, abrogates regeneration, and commonly leads to alopecia. Posttranslational modifications are enzymatically catalyzed amino acid alterations, which constitute a non-genetic mechanism for modulating protein function. Whereas our understanding of how posttranslational modifications affects stem cell lineage progression and disease progression is far from complete, extensive work identifying histone modification “codes” acting to maintain cell states as well as to ensure proper lineage progression, suggest that the impact of protein modifications requires further investigation.Here, we focus on how citrullination contributes to hair follicle lineage progression during regeneration, and how citrullination-mediated inflammation acts to perpetuate disease progression in inflammatory alopecia. We hope that this work will inspire scientists to explore new citrullination paved avenues towards the understanding of skin and skin disease.via enzymatic alteration of an existing peptide.Protein citrullination, or deimination, is the conversion of the positively charged amino acid arginine to neutral citrulline by replacement of the arginine side chain imine with an ureido group . Peptidyin vitro  result in chromatin de-condensation and transcriptional activation , whereasSkin immune cell infiltration is a hallmark of inflammatory alopecia. Activated neutrophils and T-cells express enzymatically active PADI2 and PADI4 which contributes to several aspects of the inflammatory process. For example, H3R8 citrullination at pro-inflammatory TNF-α and IL-8 promoters drive cytokine expression  in activEven though we do not fully understand PADI enzyme substrate specificity, it is clear that individual enzymes display distinct substrate preferences. Although tissue and disease-associated alterations in the citrullinome correlate to enzyme activity and substrate abundance, aspects of substrate recognition may be context-dependent, relying on, for example, a tissue-specific set of co-factors.Here we aim to delineate the function of PADIs from a hair follicle-centric perspective, using the wealth of existing hair follicle stem cell (HFSC) expression profiling as the starting point for understanding the impact of citrullination on HFSC lineage progression. A functional role for PADIs in epidermal differentiation is well established and comprehensively summarized elsewhere .Padi expression, mRNAs of both Padi3 and Padi4 are found in activated hair germ and bulge stem cells  initiate regeneration, primed hair germ stem cells are the first to divide, followed by proliferation of bulge HFSCs . Whereasem cells  and Padiair germ , suggestignature , and Padferation  indicatiferation . It is pde novo DNA methylases DNMT3A and DNMT3B  and medulla lineages, Padi4 is preferentially expressed in the cortical hair shaft and medulla lineages   , LEF1 Chget gene . Furtherromoters , indicatAnalogously, PADI2 skews effector T-cell specification towards a Th17 fate at the expense of Th2 , mechaniIn addition to citrullination of transcription factors and consequently modulating their binding affinity towards DNA, other co-factors or their subcellular localization , PADI4 cPadi1/3 are co-expressed in the differentiated inner root sheath and medulla lineages, Padi1 expression is significantly lower than that of Padi3, identifying PADI3 as the likely main contributor of protein citrullination in the IRS and medulla. PADI1/3 protein localization correlates well with mRNA expression, as well as global citrullination pattern in both human and mouse hair follicles  , a strucPADI3 mutations are linked to uncombable hair syndrome, manifesting as frizzy and fair hair resistant to combing flat , a marker normally associated with the epidermal lineage. Interestingly, HDAC1 binds to promoters of epidermal differentiation genes in epidermal progenitor cells  and uprePadi4 and Hdac1 by RNAi resulted in reduced H3 citrullination, increased H3 acetylation, and acquisition of H3R2 and H4R3 dimethylation marks. Interestingly, downregulation of Hdac1 alone resulted in less pronounced epigenetic alterations, demonstrating that PADI4 and HDAC1 work cooperatively to favor citrullination and deacetylation of the pS2 promoter  2 . Both nor states . Since tr states , PRC2-caConsidering the complexities of hair follicle formation, it is perhaps surprising that hair follicles are at all able to faithfully regenerate. In addition, hair follicle regeneration is continuously challenged by injurious and infectious insults. Despite this, an infection rarely elicits systemic responses but is contained at the site of transgression, a feature likely stemming from several intricate defence mechanisms employed by the hair follicle epithelium . FailureUpon microbial invasion, epithelial cells in the hair follicle release antimicrobial peptides  togetherCollapse of the immune privilege is a likely contributory mechanism leading to alopecia. Lesioned human scalp shows upregulation of MHC class I and II  and downPTPN22 (Protein Tyrosine Phosphatase Non-Receptor Type 22) is associated with severe forms of alopecia areata , a scarring alopecia developing in scalp regions exposed to repetitive mechanical stress and as a consequence, inflammation  , are ablThe expression of pro-inflammatory cytokines is known to be regulated by NF-κB signaling, a transcriptional effector whose activity is influenced by direct citrullination in immune cells, as well as indirectly by the presence of citrullinated H3 in NETs. Interestingly, LPS-induced neutrophil activation leads to endogenous NF-κB dependent expression of pro-inflammatory TNF-α and IL-1β  Sun et . In thisAdditionally, The Cathelicidin antimicrobial peptide LL-37 is found in NETs where it binds DNA, forming an immunogenic complex insensitive to DNAse I , therebyPADI2 and PADI4 localized to NETs readily citrullinates LL-37  Figure . CitrullActivated T-cells are important mediators of hair follicle destruction . In addiPadi2 or Padi4, or treatment with PADI-inhibitor Cl-amidine, resulted in decreased Th1 and Th17 cell numbers and production of associated cytokine IFN- γ, while Th2 cell numbers were increased . However the potential synergies between histone citrullination at the Despite an increasing number of reports delineating the protein expression of PADI enzymes, the tools used to identify PADIs and their enzymatic activity are still underdeveloped, adding up to a discordant and sometimes even contradictory picture. Here we have chosen to rely predominantly on mRNA expression for detailing cell populations that express PADI enzymes in the hair follicle and inflammation. In cases where protein characterization is available, mRNA and protein expression profiles largely overlap. However challenging to visualize, there is reason to believe that the function of these elusive proteins is far more complex than first perceived. Our current understanding of citrullination suggests that the functional outcome of citrullination is context dependent and dynamically regulated. The deiminase activity of PADIs is thus employed by most cells, in one way or the other, to achieve an array of functions.In view of citrullination as a stable, and possibly irreversible, modification it is intuitive to localize PADIs to differentiated IRS and medulla lineages, where citrullination acts to mediate structural rigidity of the hair shaft. However, citrullinated proteins are also linked to progenitor proliferation and immune cell activation, cell types continuously responding to changing environmental cues, indicating that citrullination can be used by cells in a more multifaceted way than so far appreciated. Functionally, citrullination is seldom an end in itself, but rather acts in conjunction with other types of posttranslational modifications, be it on histones or otherwise. As such, citrullination seems to be a precision tool used to fine-tune the molecular functionalities a cell requires to maintain the complex interplay of protein and gene regulation, normally and in disease.Recent findings have unveiled the broad implications PADIs may have on stem cell maintenance and differentiation in diverse stem cell niches. In the HFSC lineage, PADI expression correlate with HFSC activation, progenitor specification and hair shaft lineage differentiation, positioning PADIs at key HF lineage transition points. Whereas PADI1 and PADI3 in the regenerating hair follicle are currently exclusively associated with citrullination of structural hair proteins, work detailing the PADI1 and PADI3 HF cell state specific citrullinomes would likely provide new insights into the HF specific role of PADI1/3, in differentiating as well as self-renewing cell populations. Taking into account the redundancy of PADI1 and PADI3 in the HF, it is likely that establishment of a mouse line genetically devoid of both PADI1/3 expression would be required to grasp the full functional magnitude of these enzymes in the regenerating hair follicle.In contrast to the relatively narrow functionality linked to PADI1 and PADI3, the implications of PADI4 expression in the hair follicle cast a wider web. PADI4 has a known role in regulating stem cell renewal and specification in stem cell lineages, and expression in activated HFSCs and progenitor cells suggests PADI4 could act analogously in HFs. The ability to citrullinate DNA methylases, modulate the function of key HF lineage determining transcription factors, synergizing with histone deacetylases and antagonizing arginine methylases indicates that the action of PADI4 in the regenerating hair follicle could be complex and modulate all stages of regeneration. Although aspects of epigenetic regulation of HFSC lineage progression is well established, how citrullination impinges on the HF epigenetic landscape is completely unknown. Delineating potential PADI4 dependent fine-tuning of the epigenetic landscape suggest an overall role in maintaining self-renewal and restricting differentiation.in vitro  would correlate to citrullination within the regulatory transcriptional landscape brought about by TCR signaling, either via citrullination of transcription factors or histones.Additionally, it is noteworthy that human activity . AlthougGiven the contribution of PADIs to autoinflammatory diseases such as rheumatoid arthritis, systemic lupus erythematosus and psoriasis, it is likely that the pathogenesis of alopecia follows similar disease trajectories. Both immune cell involvement and the cytokine profile driving the pro-inflammatory environment in alopecia match those observed in citrullination-associated autoimmune diseases. Although there are obvious gaps in our understanding of how PADIs directly and indirectly influence hair follicle regeneration normally and during inflammatory alopecia, drawing from other stem cell niches or inflammatory diseases enables identification of common mechanisms likely applicable to the hair follicle. Continuous technological and methodological development will undoubtedly further our understanding of citrullination in the skin, normally and during inflammatory disease.


The corticothalamic (CT) pathways emanate from either Layer 5 (L5) or 6 (L6) of the neocortex and largely outnumber the ascending, thalamocortical pathways. The CT pathways provide the anatomical foundations for an intricate, bidirectional communication between thalamus and cortex. They act as dynamic circuits of information transfer with the ability to modulate or even drive the response properties of target neurons at each synaptic node of the circuit. L6 CT feedback pathways enable the cortex to shape the nature of its driving inputs, by directly modulating the sensory message arriving at the thalamus. L5 CT pathways can drive the postsynaptic neurons and initiate a transthalamic corticocortical circuit by which cortical areas communicate with each other. For this reason, L5 CT pathways place the thalamus at the heart of information transfer through the cortical hierarchy. Recent evidence goes even further to suggest that the thalamus via CT pathways regulates functional connectivity within and across cortical regions, and might be engaged in cognition, behavior, and perceptual inference. As descending pathways that enable reciprocal and context-dependent communication between thalamus and cortex, we venture that CT projections are particularly interesting in the context of hierarchical perceptual inference formulations such as those contemplated in predictive processing schemes, which so far heavily rely on cortical implementations. We discuss recent proposals suggesting that the thalamus, and particularly higher order thalamus via transthalamic pathways, could coordinate and contextualize hierarchical inference in cortical hierarchies. We will explore these ideas with a focus on the auditory system. Then, we will discuss the participation of L5 CT projections in transthalamic pathways . Finally, we propose that the thalamus and the CT pathways participate in the coordination and contextualization of hierarchical inference in cortical hierarchies  Mumford, .drivers and modulators. Drivers are the main conduits of information and strongly activate the postsynaptic neuron, whereas modulators serve to modify the processing of information carried by driver inputs without changing the basic nature (such as the receptive field shape) of the message to be relayed. In this context, L5 CT projection provides driver input to thalamic neurons, similarly to the ascending, feedforward inputs whereas L6 cortical feedback modulates thalamic relay neurons, performing similar operations as the classical neuromodulators do  receive a reciprocal feedback input from L6 but no input from L5, whereas higher order nuclei  receive two distinct cortical inputs: one from L6 that is a reciprocal feedback, and another from L5 that is non-reciprocal  as the L6 fibers passing through it . When thvia TRN). This reduction was due to short-term synaptic depression of the TRN-thalamus synapse, with a minimal contribution from the intrinsically mediated reductions in TRN spiking  cortical interneurons  noted, the question remains as to whether the low-frequency phase reset mechanism unraveled by Guo et al.  noted, t. This enIn summary, the prominent feedback projections from cortical L6 to thalamus have intrigued auditory scientists since many decades, as displayed by the numerous studies dedicated to investigating their role in thalamic function. However, as we have reviewed above, L6 CT projections cannot be studied with classical techniques that are blind to the complexity of the circuits to which they belong. In recent years, the interest in meticulously studying these circuits and their roles in auditory processing and behavior has increased tremendously. This was largely propelled by the advent of new techniques that now enable the selective manipulation of L6a projections in the awake behaving animal. These studies have just started to unveil the powerful roles L6a feedback circuits have in auditory perception and behavior.Some CT neurons emanate from deep layer 6 (L6b) and have long been known to primarily innervate higher order thalamic nuclei . HoweverThe fact that using Ntsr1-Cre mice to manipulate Ntsr1 neurons engages neurons from both sublayers 6a and 6b makes it difficult to disambiguate their distinct roles and connectivity. Recently, it has become possible to selectively target a subpopulation of neurons in L6b using expression of the type 1a dopamine receptor  participate in frequency-dependent inter-areal interactions that modify the relative hierarchical positions of cortical areas  as higher order circuits that relay information between cortical regions is now recognized to be incomplete. Multiple lines of evidence suggest that the thalamus operates as a master regulator of functional cortical connectivity within and between cortical areas via transthalamic pathways is unknown , suppressing congruent incoming sensory signals by “explaining away” whatever differences, or prediction errors, they can by inferring likely causes for the discrepancies . Since pyramidal cells in deep cortical layers are thought to convey predictions by top-down connections  connections ; and (2) the ability to convey predictions of precision (second order predictions) that carry context information in the form of salience or precision ascribed to these prediction errors in order to change their gain . Whether L6 can perform these tasks is still unknown, but the remarkable ability of L6 to switch the excitatory-inhibitory balance of thalamic neurons , suppression of repeated sounds coexists with enhancement to rare, surprising sounds in single neurons, the so-called stimulus-specific adaptation SSA;  or neurovia CT pathways, seems to discriminate between neurons that encode prediction errors , and those that perform other type of computations , the pulvinar is uniquely positioned to provide sensory and contextual information to cortical computations  cortical areas and higher order thalamic nuclei (via L5 projections, posterior expectations about precision in the cells of the pulvinar (Kanai et al., via precision-weighted gain control of ascending prediction errors (Kanai et al., Kanai et al.  proposedc nuclei . This moThe diversity and abundance of CT circuits that tightly link the cortex to the thalamus underscores the functional importance of these circuits to brain function. In recent years, the use of genetic tools to selectively manipulate layer or cell-type specific neuronal populations combined with electrophysiology in the awake animal opened the possibility to directly test the role of CT pathways in behavior (e.g., L6a CT circuit; Guo et al., In this review we have paid particular attention to the transthalamic circuits mediated by L5 CT neurons. The disclosure of their roles, although still incipient, largely contributed to pull apart the old, corticocentric ideas that the thalamus is a passive relay center receiving instructions from the cortex. The existence of transthalamic corticocortical routes challenges the prevailing view that cortical areas communicate with each other exclusively by means of direct, hierarchical corticocortical connections (Felleman and van Essen, via transthalamic pathways is broadcasted to other subcortical centers (Williamson and Polley, A remarkable difference between transthalamic and direct corticocortical routes lies on their extrinsic connection targets. Direct projections involve axons without subcortically directed branches (Bourassa and Deschênes, As descending pathways that enable reciprocal and context-dependent communication between thalamus and cortex, we venture that CT projections are particularly interesting in the context of hierarchical perceptual inference formulations such as those contemplated in predictive processing schemes Friston, , which sFMA and MSM: conceptualization and planning. FMA: writing—original draft and review and editing. MSM: review and editing. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.