The management strategy for the treatment of non-small-cell lung cancer (NSCLC) has been transformed by our improved understanding of the cancer biology and concomitant development of novel systemic therapies. Complete surgical resection of NSCLC continues to offer the best chance for cure or local and regional disease control, and with improvements in minimally invasive techniques and enhanced recovery, the morbidity associated with surgical resection has been reduced. Patient-centered multi-disciplinary discussions that consider surgical therapy are associated with improved outcomes. Provided with promising novel therapeutic modalities including immune checkpoint inhibitors with or without chemotherapy, stereotactic radiotherapy, and targeted systemic therapies, indications for surgery continue to evolve and have expanded to include selected patients with advanced and metastatic disease.With recent strides made within the field of thoracic oncology, the management of NSCLC is evolving rapidly. Careful patient selection and timing of multi-modality therapy to permit the optimization of therapeutic benefit must be pursued. While chemotherapy and radiotherapy continue to have a role in the management of lung cancer, surgical therapy remains an essential component of lung cancer treatment in early, locally and regionally advanced, as well as in selected, cases of metastatic disease. Recent and most impactful advances in the treatment of lung cancer relate to the advent of immunotherapy and targeted therapy, molecular profiling, and predictive biomarker discovery. Many of these systemic therapies are a part of the standard of care in metastatic NSCLC, and their indications are expanding towards surgically operable lung cancer to improve survival outcomes. Numerous completed and ongoing clinical trials in the surgically operable NSCLC speak to the interest and importance of the multi-modality therapy even in earlier stages of NSCLC. In this review, we focus on the current standard of care indications for surgical therapy in stage I-IV NSCLC as well as on the anticipated future direction of multi-disciplinary lung cancer therapy. Lung cancer affects an estimated 2 million new patients each year and is associated with 1.76 million deaths per year making it the leading cause of cancer-related death in the world [The aims of surgical therapy for NSCLC are to perform a safe and effective operation in order to achieve complete resection with negative margins and adequate clearance of lymph node basins that are at risk or involved and to return a patient to a functional state to either undergo additional therapy or return to his or her pre-operative activities. This overarching principle incorporates a patient-centered approach, with critical and intricate patient selection, leading to therapeutic options that optimize oncologic benefit and minimize risks of complications while considering goals of care set by the patient. Pulmonary function tests and predicted post-operative values are used to identify the ability of a patient to undergo resection safely and are combined with cardiovascular status and additional deleterious comorbidities that may represent contraindications.The choice of procedure and approach comes with the decision to operate. There are trials investigating the outcomes from sublobar resections compared to lobectomies or pneumonectomies, providing data specifically regarding oncological outcomes relative to the size of the tumor and nodal status, setting the lobectomy as the standard and most common oncologic resection  while rePeri-operative mortality and morbidity continue to improve with the propagation of enhanced recovery after thoracic surgery (ERATS) pathways. The use of post-operative early ambulation, multimodal opioid-sparing analgesia , and redMediastinal lymph node sampling during index operation compared to complete dissection also remains a source of discussion. Comprehensive nodal assessment is integral to the principles of surgical therapy for lung cancer. Clearance of at-risk lymph nodes is a cornerstone of optimizing survival benefit and depends on mediastinal nodal disease status. While the evidence remains equivocal, a large randomized controlled trial (ACO-SOG Z0030) highlighted that in the event that systematic mediastinal and hilar lymph node sampling  is negatThe TNM  staging schema, originating in the 1970s , has conPatient-centered treatment is the cornerstone of lung cancer surgical management, and medically operable patients with stage 1 lung cancer should be considered for curative-intent pulmonary resection. Numerous studies have concluded that surgical management of this patient population is the standard of care and provides superior outcomes and locoregional control compared to other modalities, in part due to the benefits associated with mediastinal lymph node dissection both for further diagnostic and curative purposes ,19. FollMultiple investigations have shown that in the case of tumors less than 2 cm in size, a segmentectomy can lead to oncologically sufficient outcomes ,22,23,24For tumors 2.1–3 cm -in size, lobectomy remains the standard of care ,26 whileThe best alternative to surgical resection for stage I NSCLC is stereotactic ablative radiotherapy (SABR). Previous work subjected to ongoing discussion concluded that SABR showed non-inferiority to minimally invasive lobectomy with mediastinal lymph node dissection with similar 3-year overall survival between propensity-matched cohorts following multidisciplinary discussion and patient preference; however, these trials were slow to accrue and performed their analysis early ,34.Patients with stage 1b disease will have tumors larger than 3 cm but smaller than 4 cm . These pPatients with tumors between 3 and 4 cm will also benefit from a primary operation, the extent of which deserves deliberation. The options consist again of sublobar or lobar resection. A large retrospective study has shown that patients undergoing lobectomies for tumors between 2 and 5 cm were more likely to have >10 lymph nodes removed which was associated with improved survival and cancer-specific mortality . DespiteTherapy utilizing tyrosine kinase inhibitors, specifically osimertinib, has shown superiority in patients with EGFR mutations, with prolonged disease-free survival , a benefPatients suffering from stage 2 lung cancer will benefit from surgical resection as well, with aims of cure and locoregional control. However, patients with stage II also need systemic therapy, and this stage meets inclusion criteria for all ongoing neoadjuvant and adjuvant clinical trials as well as the standard of care adjuvant chemotherapy plus targeted or immunotherapy. Special attention must be placed on multidisciplinary discussions and multimodal protocols as the evidence for stage 2 disease is scarce due to a paucity of patients diagnosed at this stage. There are clinical trials investigating the optimal strategy to manage these patients using the currently available modalities, bolstering that the use of adjuvant chemotherapy has shown benefits in this cohort  even in Given the paucity of evidence surrounding the optimal extent of surgical resection, patient-centered decisions must be made. In a recent retrospective study that included over 60 patients with stage 2 lung cancer within a larger cohort of patients with stage 1 lung cancer, early results show that long-term outcomes were similar between sublobar resections and lobectomies ; howeverImmunotherapy has dramatically progressed in the last decade and has offered compelling neoadjuvant opportunities to synergistically enhance major pathologic response ,51. BlocDurvalumab was shown to lead to high major pathologic response rate and a 1-year event-free-survival rate of 73% when used in the perioperative setting with concurrent neoadjuvant chemotherapy in the phase 2 SAKK 16/14 study .EGFR or ALK alterations. While the oncologic benefits attained from immunotherapy combined with chemotherapy can be cultivated in order to optimize survival outcomes, the effect of immunotherapy on local tissue can potentially render operations more challenging. However, this should not change the trial enrollment or multimodality management as operative mortality and morbidity remained stable but should warrant attention during the peri-operative stage [The phase 3 trial Checkmate 816, further investigating the use of nivolumab plus chemotherapy versus chemotherapy alone, showed benefits associated with complete pathological response  when comve stage . This asve stage .Immunotherapy and targeted therapy influence advanced NSCLC management. Multiple trials are ongoing and will provide important evidence to add to the patient-centered treatment compendium. Efforts originating from lung cancer mutation consortiums are ongoing and leading the development and characterization of lung cancer allowing for optimization of therapeutic benefit from current agents. It is now becoming standard of care to offer up-front tumor molecular profiling for patients diagnosed with NSCLC with trials  investigThe incredible evolution of neoadjuvant therapy, with ongoing developments in detecting actionable oncogenic drivers, leads to continued patient-centered neoadjuvant treatment optimization, with surgical resection remaining a meaningful cornerstone of curative management.Stage 3 has been the most controversial lung cancer stage due to its heterogeneity and multiple treatment options yet historically overall poor outcomes. Controversies relate to resectability, single or multiple or “bulky” N2 nodal disease status, contralateral or N3 mediastinal nodal disease, types of neoadjuvant therapy, and the appropriate extent of surgical resection, if any, in this setting. Immunotherapy has been redefining treatment paradigms in this setting and after many years also improving survival . PatientDespite the results originating from a large phase III randomized clinical trial conducted by Albain et al., which showed an insignificant survival benefit associated with resection compared to primary chemo-radiation when patients required a larger resection such as a pneumonectomy , lobectoHistorically, for patients with stage III disease, with involvement of the ipsilateral mediastinal and/or the subcarinal lymph nodes (N2 disease), whether single-station or multi-station, oncologic benefit was obtained via induction chemotherapy or concurrent neoadjuvant chemoradiotherapy. While it remains the case that patients in this group, with N2 disease, are candidates for induction therapy , neoadjuPatients with stage III disease that is characterized as unresectable, comprising approximately 20% of all cases of lung cancer in the United States , will beHistorically, the standard of care for this group of patients has involved chemoradiotherapy  without Multiple large trials have investigated the use of immunotherapy or proton therapy in this cohort.The PACIFIC trial (phase 3), investigating the consolidative use of a PD-L1 inhibitor  for up to 12 months, in patients with stable unresectable stage III disease following chemoradiotherapy, irrespective of PD-L1 expression levels, showed that its use was associated with a prolonged progression-free survival, decreased rate of distant metastasis, and significantly increased time to distant metastasis (23.2 months vs. 14.6 months in placebo) ,78. ThesThe benefits of immunotherapy following primary chemoradiation are tenable and resulted in increased consultations for salvage surgical resection in this patient cohort following the development of local or regional recurrence sometimes months to years after the index therapy. While maintenance checkpoint inhibition provides improved outcomes, these sites of recurrence will often have developed significant therapy-related inflammation and fibrosis . Such saThe landscape of management of unresectable stage III disease is very quickly evolving, and close attention must be paid to guidelines that encompass multimodality and multidisciplinary management of this heterogeneous patient population. Additionally, there are several clinical trials testing or that have evaluated immunotherapy with radiation therapy for patients with unresectable stage III disease, which are beyond the scope of this review focused on resectable disease. For patients with stage IV disease, the presence or absence of select actionable genomic alterations and the PD-L1 tumor expression status guide the use of standard of care targeted therapies, immunotherapy and chemotherapy plus immunotherapy (with or without an antiangiogenic agent) . CurativSpecifically, local consolidative therapy with surgery or radiotherapy, in patients with stage IV oligometastatic disease that had not progressed following primary systemic therapy, has shown benefits in progression-free survival and overall survival compared to maintenance therapy or observation , which wUsing SABR has also shown oncologic benefit in this population, specifically, in patients with EGFR-negative metastatic NSCLC with up to 5 metastatic sites, with a PFS advantage of 9.7 months compared to 3.5 months in patients managed with maintenance chemotherapy alone . This thIn addition, while radiotherapy has a role in the treatment of this cohort, surgical management in patients with operable oligometastatic disease had led to long-term survival or progression-free survival, with similar rates of freedom from locoregional and systemic progression when compared to radiotherapy, accentuating that surgical management of oligometastatic stage IV lung cancer remains a reasonable option in selected patients .Currently there are ongoing clinical trials further exploring multimodal treatment strategies for patients with stage IV disease, such as the LONESTAR trial investigating the role of local consolidation therapy in patients receiving nivolumab and ipilimumab , or the Also known as superior sulcus tumors, Pancoast tumors arise in the lung apex and can invade surrounding soft tissue and will require adequate staging with MRI which will further establish extrapulmonary involvement ,94. As sPatients who do not respond to their primary non-operative therapy might require surgical salvage therapy for persistent or recurrent lung cancer. Patients with early-stage NSCLC who are offered primary SBRT can see a two-year local control rate of 96%  while inSimilarly, surgical salvage can benefit patients who fail definitive chemoradiotherapy, with a rate of local recurrence up to 35% . The cohSurgical salvage is feasible and may benefit patients who underwent primary targeted therapy  althoughRuminococcus and Akkermansia have been associated with enhanced major pathologic responses in patients with lung cancer managed with immunotherapy [Immunotherapy and targeted therapy are rapidly becoming the cornerstones in both operable and metastatic lung cancer. Multiple trials are ongoing and will provide important evidence to add to the patient-centered treatment compendium. Novel biomarker and therapeutic research, however, extends beyond known cancer characteristics. The gut microbiome is an immunological modulator affecting therapeutic responses and efficacy of certain immunotherapy agents such as PD-1, PD-L1, and CTLA-4 inhibitors and has been shown to be associated with positive or negative outcomes from therapy  and may otherapy . These eotherapy , an effeotherapy ,109, incotherapy .The role of radiotherapy in the neoadjuvant setting will also likely be evolving , with imThe field relating to lung cancer management is one of the most exciting there is in surgical oncology, with an incredibly motivated multidisciplinary team relentlessly working to pioneer individualized patient-centered care and tailor current therapies to maximize clinical benefit.


Numerous clinical and research investigations conducted during the last two decades have implicated excessive oxidative stress caused by high levels of reactive oxygen species (ROS) in the development of the severe and frequently progressive fibrotic process in Systemic Sclerosis (SSc). The role of excessive oxidative stress in SSc pathogenesis has been supported by the demonstration of increased levels of numerous biomarkers, indicative of cellular and molecular oxidative damage in serum, plasma, and other biological fluids from SSc patients, and by the demonstration of elevated production of ROS by various cell types involved in the SSc fibrotic process. However, the precise mechanisms mediating oxidative stress development in SSc and its pathogenetic effects have not been fully elucidated. The participation of the NADPH oxidase NOX4, has been suggested and experimentally supported by the demonstration that SSc dermal fibroblasts display constitutively increased NOX4 expression and that reduction or abrogation of NOX4 effects decreased ROS production and the expression of genes encoding fibrotic proteins. Furthermore, NOX4-stimulated ROS production may be involved in the development of certain endothelial and vascular abnormalities and may even participate in the generation of SSc-specific autoantibodies. Collectively, these observations suggest NOX4 as a novel therapeutic target for SSc. The most severe clinical and pathologic manifestations of Systemic Sclerosis (SSc) are the result of a fibrotic process characterized by the excessive and often progressive deposition of collagen and other extracellular matrix (ECM) macromolecules in skin and numerous internal organs ,2,3,4. VThere is extensive clinical and experimental evidence demonstrating increased oxidative stress in SSc including elevated serum and plasma levels of malondialdehyde (MDA), asymmetric dimethylarginine (ADMA), 8-isoprostane, and F2 isoprostane; and elevated 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG), a specific marker of DNA oxidation, in the urine of these patients ,25,26,27Extensive experimental evidence supports the role of increased oxidative stress in the development of SSc tissue fibrosis, including the demonstration that fibroblasts isolated from affected SSc skin produce elevated ROS levels compared to normal dermal fibroblasts and that elevated ROS levels induce molecular alterations characteristic of a fibrotic phenotype in these cells in vitro ,13,32,33Although there are numerous cellular sources of ROS production, several cell types intimately involved in SSc pathogenesis besides fibroblasts and endothelial cells have been shown to contain elevated ROS levels. For example, it has been described that monocytes isolated from SSc patients spontaneously release increased amounts of certain ROS , and thaUnder physiological conditions, ROS participate in multiple cellular functions and signaling pathways and exert these effects by regulating the expression of numerous genes ,46,47,48Following Murrell’s provocative hypothesis , numerouThe molecular mechanisms of ROS stimulation of the fibrotic process are highly complex and involve multiple molecular pathways, including the ROS-mediated activation of latent TGF-β1 and the phenotypic conversion of quiescent fibroblasts and endothelial cells into profibrotic myofibroblasts. The newly generated myofibroblasts are highly active mesenchymal cells capable of producing large amounts of interstitial fibrillar collagens and other fibrotic proteins. The elevated production of these proteins by the activated myofibroblasts and their exaggerated accumulation in the interstitial space of the affected organs results in severe and often progressive fibrotic alterations characteristic of SSc, as illustrated in Owing to the potential therapeutic relevance of ROS-mediated pro-fibrotic effects there have been numerous studies focused on the elucidation of the molecular pathways involved. Among these, a recent study showed that ROS induced a potent transcriptional inhibition of the anti-Wnt protein, Wnt inhibitory factor 1 (WIF-1), leading to activation of Wnt pathway-induced tissue fibrosis . Other iBesides ROS stimulation of the fibrotic process in SSc, there are some novel studies supporting the possibility that elevated ROS may also play a role in the development of the autoimmune component in SSc. In one of these studies, it has been shown that ROS may induce abnormal oxidation of DNA-topoisomerase-1 and that this molecular/structural modification may result in an increase in its antigenicity and, therefore, may be responsible for the production of anti-DNA-topoisomerase (Scl-70) autoantibodies, the prototypic autoantibody marker in diffuse SSc . The potThere are multiple biochemical pathways and enzymatic sources that are capable of intracellular ROS production. The most important include ROS production inside mitochondria, and ROS generation as a result of the activity of numerous intracellular enzymes, including lipoxygenases, cyclooxygenases, and xanthine oxidases ,46,47,48Numerous recent studies have indicated that NOX4 may play a crucial role in the initiation, establishment and progression of tissue fibrosis ,77,78,79The mechanisms involved in the regulation of NOX activity are quite complex and vary depending on the specific cellular and functional context. However, in contrast with other NOX enzymes, NOX4 does not require other protein subunits for its activity and, therefore, the extent and intensity of its effects are entirely dependent on the levels of expression of its corresponding gene . Given t2DCFDA, a compound that permeates live cells and that in the presence of ROS becomes oxidized and emits a bright fluorescence that can be quantitatively assessed. In order to analyze NOX4-specific effects, normal dermal fibroblasts were treated with TGF-β1 to stimulate NOX4 expression, followed by incubation with GKT137831, a highly specific dual inhibitor of NOX4/NOX1. The results confirmed the expected increase in ROS production induced by TGF-β1 and demonstrated a marked reduction in TGF-β-stimulated ROS production by the specific NOX4/NOX1 inhibitor. To directly examine the role of NOX4 on the expression of genes encoding ECM molecules and on the levels of production of type I collagen in SSc, cultured SSc dermal fibroblasts were transfected with specific siRNA against NOX4 resulting in a 60% reduction of NOX4 mRNA. The NOX4 RNA decrease resulted in a significant reduction in ROS production and, more importantly, caused a 20%–30% decrease in type I collagen protein levels secreted into the culture media of the SSc dermal fibroblasts. The results of this extensive study confirmed increased NOX4 expression and ROS production by SSc dermal fibroblasts and showed that stimulation of ROS production by NOX4 mediated potent pro-fibrotic effects, since a significant reduction of type I collagen production was observed as a result of NOX4 siRNA inhibition in these cells [Numerous studies have examined NOX4 expression levels in cultured normal and SSc human dermal fibroblasts and characterized its effects on molecular pathways involved in the exaggerated production and accumulation of fibrotic extracellular matrix (ECM) proteins in affected SSc tissues ,58,77,98se cells .Numerous studies have demonstrated that endothelial cells are capable of a remarkable change in their specific cellular phenotype and that under certain conditions they undergo a conversion into myofibroblastic mesenchymal cells through the complex process of EndoMT. During EndoMT, endothelial cells adopt a myofibroblast cell morphology and initiate the expression and production of mesenchymal cell-specific proteins, including α-smooth muscle actin, extra domain A fibronectin, and fibrillar type I and type III collagens ,100,101.As discussed extensively in previous sections, increased oxidative stress caused by excessive and unbalanced generation of ROS in affected tissues contributes to the development and progression of the fibrotic process in SSc. Furthermore, the elevated ROS levels in SSc are mediated by high levels of NOX4 activity. Based on this concept, it has been postulated that assessment of ROS production and of the main enzymatic pathways involved in ROS generation and production may allow to predict the clinical evolution of SSc and may also indicate a favorable therapeutic response of the disease process . Accordi2 of body surface). One group of six patients received cyclophosphamide plus the antioxidants, and the comparison group of seven patients received only cyclophosphamide. After six months of therapy, patients treated with cyclophosphamide plus antioxidants had a significantly lower rate of progression of skin involvement and their DLCO improved slightly, although the improvement was not statistically significant compared to patients treated with cyclophosphamide without antioxidants, in whom the pulmonary function studies showed a small deterioration [Regarding SSc, there are few clinical trials that have actually tested the therapeutic effectiveness of antioxidant therapies for the disease ,123,124.ioration . Anotherioration .Several studies examined the effects of NAC on cutaneous and pulmonary fibrotic involvement in SSc ,123,124.The few studies that examined oxidative stress inhibitors in SSc patients are summarized in Overall, the results of the clinical studies discussed above regarding the therapeutic effects of antioxidants for SSc have been negative or non-conclusive. Therefore, it will be necessary to perform further studies including well controlled placebo matched clinical trials to document any beneficial effects of antioxidant/NOX4 inhibitory therapeutic interventions on the development or progression of the fibrotic process in SSc. Furthermore, the participation of ROS/NOX occurs most likely during the earliest stages of development of the fibrotic process in SSc, therefore, antioxidant drugs should be administrated very early in the development of tissue fibrosis to increase their possible beneficial therapeutic effects. In addition, it should be emphasized that the combined use of antioxidant drugs that exert their effects through different molecular mechanisms may further result in improved disease-modifying effects for this disabling and frequently fatal disease.This review examines the abundant scientific literature and numerous recent experimental studies that provide strong evidence to indicate that exaggerated oxidative stress caused by increased levels of ROS is involved in the development of the SSc-associated fibrotic process and that NOX4 is the most important source of increased ROS in SSc ,33,34. F


Sox/SOX genes, remarkable progress has been made in illuminating their roles as key players in the regulation of multiple developmental and physiological processes. SOX TFs govern diverse cellular processes during development, such as maintaining the pluripotency of stem cells, cell proliferation, cell fate decisions/germ layer formation as well as terminal cell differentiation into tissues and organs. However, their roles are not limited to development since SOX proteins influence survival, regeneration, cell death and control homeostasis in adult tissues. This review summarized current knowledge of the roles of SOX proteins in control of central nervous system development. Some SOX TFs suspend neural progenitors in proliferative, stem-like state and prevent their differentiation. SOX proteins function as pioneer factors that occupy silenced target genes and keep them in a poised state for activation at subsequent stages of differentiation. At appropriate stage of development, SOX members that maintain stemness are down-regulated in cells that are competent to differentiate, while other SOX members take over their functions and govern the process of differentiation. Distinct SOX members determine down-stream processes of neuronal and glial differentiation. Thus, sequentially acting SOX TFs orchestrate neural lineage development defining neuronal and glial phenotypes. In line with their crucial roles in the nervous system development, deregulation of specific SOX proteins activities is associated with neurodevelopmental disorders (NDDs). The overview of the current knowledge about the link between SOX gene variants and NDDs is presented. We outline the roles of SOX TFs in adult neurogenesis and brain homeostasis and discuss whether impaired adult neurogenesis, detected in neurodegenerative diseases, could be associated with deregulation of SOX proteins activities. We present the current data regarding the interaction between SOX proteins and signaling pathways and microRNAs that play roles in nervous system development. Finally, future research directions that will improve the knowledge about distinct and various roles of SOX TFs in health and diseases are presented and discussed.The SOX proteins belong to the superfamily of transcription factors (TFs) that display properties of both classical TFs and architectural components of chromatin. Since the cloning of the  SOX gene family.The development of multicellular organisms and the maintenance of homeostasis in adulthood are achieved by complex control of basic cellular processes such as the maintenance of pluripotent stem cells, cell fate decision, differentiation, proliferation, and cell death. One of the key mechanisms involved in the control of developmental processes is based on the transcriptional regulation of gene expression. Through the activation and repression of the target genes, transcription factors (TFs) determine the fate of cells within tissues, organs and organisms, controlling the development. Most TFs act within complex regulatory networks, enabling combinatorial regulation of gene expression within the cells. Numerous families of genes encoding TFs involved in the control of embryonic development have been discovered, including the Sry (Sex-determining Region Y), a founder member of the Sox gene family, was discovered in 1990 as a sex-determining gene necessary and sufficient to specify the male phenotype  standing for Sry-related HMG box genes  and B2 (SOX14 and SOX21)  . SOX prod SOX21) . Apart fe genome . MajoritThe SOX proteins display properties of both classical TFs and architectural components of chromatin  and to maintain their pluripotent and proliferative state. The numerous evidences reveal the roles of SOX proteins in preservation of stem cell characteristics.As mentioned above, SOX proteins have the ability to pair off with various types of TFs and regulatory functions of SOX proteins usually require the cooperation with interacting partner factors . SOX2, tThis pluripotency gene regulatory network relies on direct physical interaction between SOX2, NANOG, and OCT4 . ThroughOCT4 gene expression in pluripotent embryonal carcinoma stem cells NT2/D1 with constitutive SOX2 overexpression . Reprogramming is achieved by overexpression of stem cell-associated genes in differentiated cells, such as adult fibroblasts . SOX2 isSox2, other members of the Sox gene family may also be involved in the reprogramming process. Sox1 yields iPSCs with a similar efficiency as Sox2, while Sox3, Sox15, and Sox18 genes are also capable to generate iPSCs, although with decreased efficiency  through prebinding by SOX3 . FurtherTogether these interesting data demonstrated that sequentially acting SOX TFs orchestrate neural lineage development including neuronal-, astrocyte- and oligodendrocyte-specific gene expression.SoxB1 genes are expressed in the neural precursor cells, while Sox21, Sox4, and Sox11 are mostly expressed in neural cells committed to neuronal differentiation  is essential for neuronal differentiation  positive neurons upon RA induced neural differentiation of NT2/D1 cells  and gain- and loss-of-function studies indicated that Sox9 was required for multipotentiality and maintenance of NSCs during development , characterized by impairment of neuronal function during brain development. SOX gene variants associated with NDDs are listed in Recent literature data has revealed that OX genes to NDDs is still not clear. Both deletions and duplications of SOX3 gene were detected in patients with intellectual disability  of lateral ventricle and in the subgranular zone (SGZ) of the dentate gyrus in the hippocampus   and subset of neuronal progenitor cells (Type 2a) in SGZ . Functioactor 5) .In the adult mammalian brain, the expression of SOX4 and SOX11 proteins is detected prominently in the both main neurogenic niches, SGZ  and SVZ miR-124, which targets Sox9, increase SOX9 expression and decrease neurogenesis  in the hippocampus of 2 months old 5xFAD mice, which represent a transgenic model of Alzheimer’s disease. Immunohistochemical analysis revealed a significant decrease in the number of cells expressing SOX1, SOX2, and SOX21 TFs within the SGZ of 5xFAD mice in comparison to their non-transgenic counterparts. Our comparative study also revealed, for the first time, significant difference in the number of SOX1 positive cells between genders in both, transgenic and non-transgenic animals . ConsideSox9 in NSCs caused defects in the specification of oligodendrocytes and astrocytes, the two main types of glial cells in CNS, and induced a transient increase in the number of motoneurons  in its promoter  enhancer , by binding to low-affinity SOX binding sites in Ccnd1 promoter, by interactions with TCF/LEF proteins and by recruitment of GRO/TLE (Groucho/Transducin-like Enhancers) co-repressors. Upon differentiation, pro-neural proteins reduce SOXB1 level, thus only the high affinity SOX binding sites stay occupied, while loss of binding to low-affinity sites de-represses Ccnd1 and promotes proliferation of progenitor cells  (Retinoic acid exerts its pleiotropic effects through binding to retinoic acid receptors (RARs), members of the nuclear receptor superfamily (reviewed by  (RAREs) , atypica (RAREs)  and comp (RAREs) . Ligand  (RAREs) .Our group conducted comprehensive analyses of the SOXB1 protein expression during RA induced neural differentiation of NT2/D1 cells . ObtaineSox2 expression in mouse P19 and F9 embryonal carcinoma cell lines  in neural-specific target genes (Dbx1 (Developing brain homeobox 1)  . In anotple RARE  confirmsMany evolutionary conserved microRNAs (miRNAs) present key factors in fine regulation of self-renewal and proliferation of NSCs and NPCs . By inteSox2, thus regulating the transition from NSCs to postmitotic and differentiated cells  stage until terminal maturation of neurons and macroglia  resembling the embryonic human brain regarding the cell types, cells’ architectures and maturation . The 3D elopment . CombineIn the past decade, the “omics” technologies, such as genomics, transcriptomics, miRNomics, and proteomics have become integrated parts of the research in biology and medicine, enabling progress in collecting, processing and integrating huge amounts of health-related information . While gAdvance in proteomic technologies enables mapping of specific gene interactome providing the insights into the network of interacting partners. Thus, the study of Nanog interactome identified SOX2 as interacting factor . MappingAlthough many important roles during neural development have been assigned to SOX TFs, we strongly believe that many novel functions are yet to be discovered.All authors wrote the manuscript and contributed to literature collection. MiS designed the concept of the manuscript and supervised and edited the manuscript. MiS, DD, and DSN contributed to the preparation of the tables. MaS, AL, MM, and DSN designed the figures. 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.


Columba livia) is a goodmodel for evaluating the vestibular system involved with posturalmaintenance during movement.To evaluate whether the pigeon injection in horizontal semicircular canal (HSCC), immunohistochemistry forGluR2/3 and GluR4 AMPA and computerized histomorphology reconstruction.Our results show the same distribution pattern with ipsilateral projectionsto vestibular nuclear complex (VNC) from the HSCC, with the majority oflabeled fibers being, long, thin, with few varicosities and manyramifications. Horizontal semicircular canal projections achieve neuronsbelonging to all nuclei of the VNC with exception of dorsal portion oflateral vestibular nucleus and this area express GluR2/3 and GluR4 AMPAreceptors reinforcing the idea of glutamate participation in theseconnections.Pigeon is an appropriated experimental model to study of projections of HSCCand reinforcing the information that the vestibular system has strongrelation with the fast responses necessary for postural control. Moreover,its phylogenetic organization apparently conservation, also seems to be afundamental characteristic for vertebrates. Distinctanimal classes utilize diverse locomotor strategies, but all of these properties aredependent of peripheral information offered by the vestibular organs and theexistence of brainstem nuclei that receive and process such data, in order toprovide descendent outputs to somatic motor neurons,,,,,-Briefly speaking, the vestibular inputs of vertebrates come from the ampullae’s andmacula’s hair cells present in the cupula of the semicircular canals (ampullarycrista) and in the interior of the sacculus and the utricle (saccular macula andutricular macula), respectively,,,--,,-The VNC is composed of the superior vestibular nucleus (SVN), lateral vestibularnucleus, medial vestibular nucleus and the descendent vestibular nucleus; althoughsome additional small neuronal populations also participate,Some species, such as primates, sustain complex motor actions with the horizontalplane movement prevailing. An avian is also able to keep their head in parallelstance to the surface during their walking and flying, suggesting that thehorizontal semicircular canal (HSCC) is the preponderant source of information,similar for mammalian speciesAll experiments were conducted according to an approved institutional animalexperimentation protocol ,according to the Guide for the Care and Use of Laboratory Animals in Research of theNational Institutes of Health, USA.Columba livia) weighing 360to 420 g with no sex distinction were maintained in an animal facility room at 21°Cwith a 12 h-12 h dark-light cycle with food and water adlibitum.Fifteen adult (12 to 24 months) pigeons (–1 of phosphate buffer (PB), pH 7.4All the animals were anesthetized by intramuscular injections of xylazine (1 mg/100 gof body weight) and ketamine (5 mg/100 g). The bony labyrinth was exposed and thehorizontal canal was reached by an upper aperture to avoid damage to the auricularspace that was used to fix the pigeon at the stereotaxic. A final perforation wascompleted with an insertion of a glass borosilicate micropipette tip (15 to 25 µm)used to inject the tracer solution, constituted by about 0.2 to 0.3 µL of a 10%solution of BDA of 10,000 molecular weight (BDA 10 kDa) dissolved in 0.1 molLAfter 7–10 days the pigeons were deeply anesthetized as above, perfused and thebrains were then removed, post-fixed and immersed in a cryoprotective solutionThe histochemical detection with BDA label was performed with eleven animalsdistributed in five compartments, visualized using 0.05% diamino benzidine  and 0.03% hydrogen peroxide in PB with the additionof 1% nickel sulfateWith the last four pigeons, a double labeling was done between the BDA and glutamatereceptors of the AMPA type: GluR1 , GluR4  or a single antibody to identify the presence of GluR2 and/orGluR3 . Theantibodies were diluted 1:500 in PB, incubated in biotin secondary antiserum,processed with the ABC-Elite kit (Vector Labs) and finally revealed by DABThe images were captured by selecting brain regions with a 10× ocular of a NikonEclipse E 800 , a microscopeequipped with a CCD camera  connected to aMacintosh desktop computer, and figures were prepared using the Adobe Photoshopsoftware. No attempt was made to quantify the labeled structures or to evaluate theintensity of the markers, but when needed, we estimated the cellular size bymeasuring along the larger neuronal axis as priory described-Some general controls for the specificity of the AMPA immuno-staining were previouslyreported,The obtained results were very similar for all animals assayed (the case number 2 wasselected as a model) in which BDA was injected into the HSCC of pigeons. As shown inThe vestibular ganglion (VG) of the injected side offered an unequivocal presenceof neurons filled with BDA, almost dorsal, with virtually none positionedventrally . Also viNone BDA-labeled fibers were found contralateral to the injection side . In addiConcerning typology, the majority of the BDA-labeling was found to be in long andthin fibers containing few varicose and many ramifications, preferentially inthe rostral SVN . These fNo label was found in the dorsal LVN , but manThe rostral MVN hold limits with the SVN and brachium conjunctive, presentingvery few thin-like with and without varicose .Moving to the caudal to initiate the intermediate portion, just into theappearance of the LVN, the number dramatically increases and many varicose canbe observed in the tiny fibers as well many terminal-like arborizations can nowbe observed  and 5. IMany BDA-containing fibers were detected all over the DVN . The rosThe immunoblotting assay shows a good reactivity for all three immune-markersstudied further, indicating the constitutive expression of these AMPA receptorsin the hindbrain region of the adult pigeons .The occurrence of AMPA-type containing neurons coincides with the massivelocation of the terminal fields of the BDA-filled fibers assuming to be HSCCprojecting terminals  and 8.,,,,-Several aspects of the VNC, like cellular morphometric and input-outputconnection, have been extensively described in numerous speciesAnalyzing the VG neurons, we also observed that the presence of bipolar neuronsheld distinct sizes, all of them fulfilled BDA, occupying the superior dorsalarea of the ganglion as predicted by Dickman and FangAfter passing by the restiform body and the rostral part of the tangentialnucleus, the BDA filled fibers comprise two paths: 1) run lateral to medial,adjoining the central and caudal portions of the VNC; 2) ascend dorsal torostral to arrive at the most lateral and superior portion of the VNC.,,Even presenting minor differences in innervations pattern and terminal fields,the HSCC projections achieve possible synaptic contacts with neurons belongingto all nuclei of the VNC, except those present in the dorsal LVN. This featureof the double vestibular input appears to be a very conservative archetypealready described in primates,,As long as the fibers reach their target, they grow tiny varicosed branchesacquiring a constitution of terminal fields. In the SVN these rich varicosefibers were found preferentially in the central-medial portion while in the LVNthey were, in majority, found in the central-dorsal region in the ventralportion since the dorsal LVN seems to be a recipient free zone from peripheralterminals. In the MVN these structures were found in the dorsomedial portionjust below the fourth ventricle while the entire DVN, especially in the centralpart, seems to be tagged, although, decreasing in intensity along theirrostral-caudal axis. Again, these broad descriptions come into a conservativeelement being similar to some species of monkeysA few discrepancies were observed; for instance, HSCC in chinchillas, in whichthere is a supposed preference of rostral innervation in the LVN form,-,,,Since the peripheral vestibular inputs are carried out by the neurotransmitterglutamateet al.GluR2/3 and GluR4-expressing neurons are uniformly distributed over all lengthsin SVN, and the central part of these nuclei is the place in which we found apreponderance of BDA-filled terminal fibers . In addiet al.etal.In the LVN, MVN and DVN  and 11 wThe efficiency of the information carried out by the HSCC inputs in controllinghead movements with ocular adjustments and their influence in postural tonusdepends not just in the final destination of these fibers but in theneurochemical nature of these connections as well.,,,GluR2/3 and GluR4 subunits of AMPA receptors were found in all targeting regionsof the HSCC in all pigeon vestibular nuclei suggesting its involvement inexcitatory postsynaptic transmission in vestibular neurons, similar to findingsin other specieset al.The SVN is considered the most convey center for the integration of ocularreflexes mediated by semicircular canals, thus, being the cranial motor nucleione of the SVN efferent targets,,,,,,The LVN participates in modulating postural adjustments of the neurons of thespinal cord through vestibulospinal lateral tract, consisting of projections oftheir dorsal and ventral portions,,,By the medial vestibulospinal tract, the MVN influences the postural control and the horizontal vestibulocochlear reflex bysending projections to the cervical and thoracic regions of the spinal cord,,++ in the vestibular immature neurons, coinciding withthe absence of GluR2 for first day postnatal,++channels AMPA is due to increase expression of the subunit GluR2 in the neuronsvestibular nuclei,Different subunits of the vestibular nuclei glutamatergic receptors are involvedin synaptic formation during the development and in synaptic plasticity both forin normal and pathological conditionsOur data show that three different types of BDA filled fibers, from HSCC, spread forall CNV nuclei, except for the dorsal portion of lateral vestibular nucleus. Thepresence of terminals fields in CNV neurons was evidenced after GluR2/3, GluR4 andNeuN immunoreactivity.Projections of HSCC for CNV are the main output signal to the spinal cord,reinforcing the information that the vestibular system has strong relation with thefast responses necessary for postural control. Moreover, its phylogeneticorganization apparently conservation, also seems to be a fundamental characteristicfor vertebrates.


Pancreatic beta cells play a central role in regulating glucose homeostasis by secreting the hormone insulin. Failure of beta cells due to reduced function and mass and the resulting insulin insufficiency can drive the dysregulation of glycemic control, causing diabetes. Epigenetic regulation by DNA methylation is central to shaping the gene expression patterns that define the fully functional beta cell phenotype and regulate beta cell growth. Establishment of stage-specific DNA methylation guides beta cell differentiation during fetal development, while faithful restoration of these signatures during DNA replication ensures the maintenance of beta cell identity and function in postnatal life. Lineage-specific transcription factor networks interact with methylated DNA at specific genomic regions to enhance the regulatory specificity and ensure the stability of gene expression patterns. Recent genome-wide DNA methylation profiling studies comparing islets from diabetic and non-diabetic human subjects demonstrate the perturbation of beta cell DNA methylation patterns, corresponding to the dysregulation of gene expression associated with mature beta cell state in diabetes. This article will discuss the molecular underpinnings of shaping the islet DNA methylation landscape, its mechanistic role in the specification and maintenance of the functional beta cell phenotype, and its dysregulation in diabetes. We will also review recent advances in utilizing beta cell specific DNA methylation patterns for the development of biomarkers for diabetes, and targeting DNA methylation to develop translational approaches for supplementing the functional beta cell mass deficit in diabetes. DNA metpatterns –22, and patterns   after implantation, and maintenance of these patterns by Dnmt1 ensures continuity of the epigenetic state through cell division  during gametogenesis [reviewed in –40. Suchdivision –40. Linedivision ]. OveralThe pancreas is a multifunctional organ, performing both exocrine and endocrine functions, namely the secretion of digestive enzymes and hormones . The exoDe novo methylation patterning plays a critical role in this stepwise differentiation process; with Dnmt3a being the primary de novo DNA methyltransferase in the endocrine lineage, while Dnmt3b is restricted to the acinar lineage (in vitro differentiation of human pluripotent stem cells (hPSCs) into pancreatic islets revealed that pluripotency genes undergo DNA hypermethylation to enable chromatin silencing, and that hypomethylation of lineage specific genes drives stage-specific chromatin activation during endocrine specification  locus, which encodes a master regulator of alpha cell identity  and lactate dehydrogenase A (Ldha), leads to their repression and allows the coupling of insulin secretion to glucose levels  and beta cell immaturity genes (Dlk1 and MafB) led to impaired glucose stimulated insulin secretion (GSIS) and reduced beta cell mass due to beta cell-death (via DNA methylation patterning. The link between metabolic changes and DNA methylation is underscored by the dependence of DNA methyltransferase and DNA demethylases on metabolites such as S-adenosyl methionine (SAM) and a-ketoglutarate (a-KG), respectively  compared to early neonatal life (1-2 weeks) . This isnization , and polnization –98. It inization . Collectnization . This maNKAP, APLN (both X-linked), and SPESP1 , that showed higher methylation and reduced mRNA in female islets, were found to regulate insulin secretion in functional assays. In addition, this study identified two miRNA loci that were differentially methylated and expressed between male and female islets, correlating with the observed sex differences in islet expression of multiple target genes for these miRNAs  presents a unique challenge for the cell to faithfully transmit the epigenetic patterns that define the differentiated phenotype, from the parent- to daughter-cells. The maintenance methyltransferase Dnmt1 serves to maintain beta cell identity during cell division by reestablishing the DNA methylation patterns required for the continued repression of alpha cell lineage, such as the methylation of Arx . Consequell mass . The DNAer cells . Notably and Arx , suggest and Arx . A recenidentity , suggestidentity Figure 3In light of the above discussion, it is conceivable that changes in nutrition and metabolism at any point in postnatal life can alter the DNA methylation patterns underlying beta cell identity and maturity, which can in turn impact the functional response of beta cells. Such plasticity of the beta cell epigenome is essential for adaptation to changing insulin demands. However, altered nutrition during the sensitive growth phase in early life, or sustained high demand for insulin in adult life, can disrupt the epigenetic program and lead to an insufficiency of functional beta cell mass, due to poor growth or poor adaptation, respectively.A large number of studies across multiple species point to a strong link between developmental perturbations such as poor intrauterine nutrition and fetal growth impairment, and diabetes development in later life, and suggest that early epigenetic events can contribute to disease risk and pathogenesis , 107–110As discussed earlier, DNA methylation homeostasis directly coupled with cellular metabolism and nutrient quality. The availability of the Dnmt co-factor S-adenosyl methionine (SAM). Cellular levels of SAM are determined by biochemical reactions involving methyl-donor vitamins such as folate and vitamin B12, amino acids such as methionine, choline, taurine, as well as flux through the TCA cycle . Thus, aIn line with the above discussion, intrauterine growth restriction (IUGR) leads to impaired pancreatic development, reduced functional beta cell mass, and higher risk for diabetes , 132. A KCNQ1, which encodes a potassium channel essential for beta cell function, have been shown to impart diabetes susceptibility when inherited maternally, due to altered DNA methylation in early development , resulting in unchecked beta cell expansion and excessive beta cell mass, which causes hyperinsulinemia, and hypoglycemia  2. Severc donors , 158, rec donors . Similarfunction .vice versa. Functional analysis of some of these candidates in human islets using RNAi based knockdown or exposure to stress stimuli underscored the relevance of these genes to dysregulation of beta cell homeostasis in T2D. These analyses, along with functional annotation, revealed epigenetic dysregulation of genes related to pathways such as transcriptional control , ER-stress, function, and survival , in diabetic islets  were differentially expressed  gene have been widely used to detect beta cell death in the context of T1D  cells derived from subjects with T1D, which are otherwise known to differentiate poorly  and 5-aza-cytidine  are approved for use as anti-tumor agents . In a ree poorly . DNA deme poorly . Thus, wCDKN1C was recently used to stimulate the expansion of adult human beta cells that typically do not replicate. Using a TALEN dependent targeting of TET1, Kaestner and colleagues simulated the DNA methylation patterns associated with beta cell hyper-proliferation in Beckwith Wiedemann Syndrome (BWS), where hypomethylation of the CDKN1C locus leads to reduced p57Kip2 and beta-cell hyper-proliferation. Using this approach, they were able to achieve the demethylation of CDKN1C locus and consequent reduction of p57Kip2 levels in human islets, and successfully induce replication of adult human beta cells  that simulate an in vivo scenario with epigenetic targeting and/or small molecule epigenetic modifiers, future work could dramatically improve the quality of stem cell derived islet products. Data generated from genome-wide DNA methylation profiling studies along with mechanistic work on the dysregulation of DNA methylation of key regulatory pathways (such as the imprinted DLK1-MEG3 locus in T2D), is likely to inspire the development of novel therapeutic approaches for augmenting beta cell function and mass. Given the specificity of epigenetic engineering, such approaches may only have minimal off-target effects, and are thus likely to emerge as powerful therapeutic tools.Recent advances in genome editing using molecular tools such as CRISPR/Cas9 and TALEN now enable us to modify the DNA methylation patterns at specific regions in the genome, to either remedy disease specific epigenetic changes or introduce epigenetic patterns with potential therapeutic benefits. Locus specific epigenetic engineering has been used to target Dnmt or Tet enzymes to drive methylation or demethylation of specific genes . In a reta cells . In anot lineage . Targeterotocols . In thisrotocols . This prThe epigenome serves as a mechanism to adapt the behavior of a cell in response to changes in its environment, by interpreting the genome in a context specific manner and facilitating a transcriptional response accordingly. Methylation of the cytosine within the CpG residues in DNA is one of the most thoroughly studied epigenetic modifications, and one that can be faithfully inherited from parent to daughter cell during cell-division. DNA methylation is indispensable for shaping stage- and cell-type specific transcriptional programs, starting from early embryonic differentiation events to the maintenance of fully differentiated cellular phenotypes . While iin vitro data suggests that acute changes in DNA methylation can occur in the islets , the Wanek Family Project to Cure Type 1 Diabetes at City of Hope, and Human Islet Research Network (NIH) UC4 DK104162 (to SD).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.


Lipids modulate the function of many ion channels, possibly through direct lipid-protein interactions. The recent outpouring of ion channel structures by cryo-EM has revealed many lipid binding sites. Whether these sites mediate lipid modulation of ion channel function is not firmly established in most cases. However, it is intriguing that many of these lipid binding sites are also known sites for other allosteric modulators or drugs, supporting the notion that lipids act as endogenous allosteric modulators through these sites. Here, we review such lipid-drug binding sites, focusing on pentameric ligand-gated ion channels and transient receptor potential channels. Notable examples include sites for phospholipids and sterols that are shared by anesthetics and vanilloids. We discuss some implications of lipid binding at these sites including the possibility that lipids can alter drug potency or that understanding protein-lipid interactions can guide drug design. Structures are only the first step toward understanding the mechanism of lipid modulation at these sites. Looking forward, we identify knowledge gaps in the field and approaches to address them. These include defining the effects of lipids on channel function in reconstituted systems using asymmetric membranes and measuring lipid binding affinities at specific sites using native mass spectrometry, fluorescence binding assays, and computational approaches. Lipids regulate the structure and function of ion channels, which are embedded in complex and dynamic lipid membrane environments. Classic examples include cholesterol activation of the nicotinic acetylcholine receptor nAchR;  and phosLipids may regulate ion channels by direct binding to specific sites or indirect effects on membrane physical properties . The recAR), mediate synaptic neurotransmission and determine neuronal excitability. Over four decades ago, the nAchR from Torpedo muscle was the first ion channel found to be sensitive to its lipid environment. The nAchR requires cholesterol, phosphatidylethanolamine (PE), and anionic phospholipids, such as phosphatidylserine (PS), for maximal channel responses to agonist  and GABA(A) receptor , which lines the channel pore, the intermediate ring consists of M1 and M3, and the outer ring consists of M4, which has the greatest exposure to lipids . BetweenARs ; a POPC density was identified in this site , which produced a significant shift of the TMD helices relative to a structure without POPC . CompareAR in complex with the general anesthetics, propofol, and diazepam, show putative lipid densities in this site at each interface of this heteromeric channel  or diazepam (β2/α1 and γ2/β2). Interestingly, a phospholipid density is also observed at the α1/γ2 interface, which has been called an orphan site as no anesthetic is known to bind to this site  simulations. Coarse-grained MD simulations of the GlyR in a neuronal-like membrane show increased binding of cholesterol at this site in the agonist-bound conformation compared to apo . The stehis site . The simhis site . Similarhis site . Consisthis site .In summary, the outer leaflet intersubunit site in pLGICs is a key binding site for allosteric modulators. Structural and computational studies indicate that phospholipids and steroids bind to this site, with certain lipids possibly favoring agonist-bound, activated conformations.AR by binding to this site at the α1/β3 interface  shows a density at this site consistent with PS  and all TRPM channels . These helices comprise the voltage-sensing like domain  and pore domain (S5–S6), connected by the critical S4–S5 linker. Also, lining the inner membrane interface are the N-terminal pre-S1 domain and the C-terminal TRP domain . Amidst r lipids .The vanilloid binding site, which binds capsaicin (the active component of chili peppers) in TRPV1, is an inner leaflet pocket bound by S3/S4 of the VSLD, the S4-S5 linker, and the pore-forming S6 of the adjacent subunit  phospholipid that was resolved in the apo, closed structure of TRPV1 in asolectin nanodiscs . Unlike A lipid occupies the equivalent vanilloid binding site in many other TRP channel structures, some of which show evidence of state-dependent lipid binding. Recent structures of TRPV3 in NW11 asolectin nanodiscs at different temperatures illuminate the mechanism of heat activation of TRPV3. The closed structure shows a phospholipid in this site that is excluded from this pocket by a conformational change in the heat-activated open structure , similarIn many TRP channels, the VSLD, which comprised the S1–S4 helix bundle, forms an inner leaflet ligand binding pocket . Ligands2, a major phosphoinositide in the plasma membrane inner leaflet. Both positive and negative regulatory effects of PIP2 have been reported in TRPV1 and other TRP channels, and there is evidence to suggest that multiple distinct binding sites account for these varied effects  in the S4–S5 linker and the N-linker before the pre-S1 helix  understanding the exact effects of lipids on ion channel function and (2) determining lipid binding affinities at specific sites in different conformational states. Such understanding taken together will be necessary to answer the essential question of whether certain lipids regulate ion channel function through a specific site(s) or through indirect effects on lipid bilayer properties. We highlight several approaches and suggest new developments to these techniques to address the above knowledge gaps.Lipid regulation of ion channel function is best determined in model membranes where lipid composition can be precisely controlled. The necessity of reconstituting biochemical quantities of pure, functional protein has made such experiments challenging and mostly limited to prokaryotic ion channels. However, advances in large-scale expression of mammalian membrane proteins  and soluFunctional studies of ligand-gated ion channels also require the capability to examine gating kinetics, including processes such as rapid activation in response to agonist and slow desensitization in the continued presence of agonist. This can be achieved with a stopped-flow fluorometric ion flux assay , which mAR. While crystal structures only show neurosteroid binding to an inner leaflet intersubunit site  range from 2 to 10 nm  simulations, with unparalleled spatiotemporal resolution, have also been leveraged to characterize binding of lipids to a wide range of membrane proteins including ion channels . A majorEnhanced sampling techniques may also be useful to improve modeling of the equilibrium ensemble of membrane protein systems . These tHigh-resolution structures of ion channels, especially those embedded in lipid nanodiscs, show multiple lipid-drug binding sites in pLGIC and TRP channel families. While these sites are generally conserved among multiple members of each family, they are also the sites of action of some highly specific and potent ligands such as ivermectin in GluCl, capsaicin in TRPV1, and GNE551 in TRPA1. Thus, drug discovery efforts targeting these lipid binding sites in different channel subtypes may yield specific new modulators/inhibitors as was the case for TRPV5 . CurrentWWLC drafted the manuscript. MJA and JTP wrote and edited the manuscript. All authors contributed to the article and approved the submitted version.This work was funded by the National Institutes of Health  and the International Anesthesia Research Society (Frontiers in Anesthesia Research Award).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.


Cells and tissues are continuously exposed to both chemical and physical stimuli and dynamically adapt and respond to this variety of external cues to ensure cellular homeostasis, regulated development and tissue-specific differentiation. Alterations of these pathways promote disease progression—a prominent example being cancer. Rho GTPases are key regulators of the remodeling of cytoskeleton and cell membranes and their coordination and integration with different biological processes, including cell polarization and motility, as well as other signaling networks such as growth signaling and proliferation. Apart from the control of GTP–GDP cycling, Rho GTPase activity is spatially and temporally regulated by post-translation modifications (PTMs) and their assembly onto specific protein complexes, which determine their controlled activity at distinct cellular compartments. Although Rho GTPases were traditionally conceived as targeted from the cytosol to the plasma membrane to exert their activity, recent research demonstrates that active pools of different Rho GTPases also localize to endomembranes and the nucleus. In this review, we discuss how PTM-driven modulation of Rho GTPases provides a versatile mechanism for their compartmentalization and functional regulation. Understanding how the subcellular sorting of active small GTPase pools occurs and what its functional significance is could reveal novel therapeutic opportunities. Cell motility is essential for tissue development and homeostasis in multicellular organisms, but its dysregulation plays a crucial role in pathological processes such as tumor invasion and metastasis. The actin cytoskeleton is the engine that drives polarized cell behavior through the spatial and temporal control of cell protrusiveness, adhesion, contractility and rear retraction. Changes in the actin cytoskeleton modulate not only focal adhesion dynamics and cell contraction but also the physical properties  of the cell, including its nucleus. Characterizing how these cellular systems function in an integrated manner is essential to understand cell migration. The ability of tumor cells (TCs) to actively migrate through tissue structures and colonize both adjacent and distant sites is intensively studied because metastatic disease is by far the prime death cause in cancer patients ,2. AdditThe dynamic architecture of the actin cytoskeleton is a key parameter in these complex processes . The basThe Rho family of proteins make up a major branch of the Ras superfamily of small GTPases. To date, 22 human genes encoding at least 25 proteins were described, the best characterized ‘classical’ members being RhoA, Rac1 and Cdc42. They share a high degree of amino acid sequence homology, indicating high biological relevance and selective pressure, especially regarding residues directly involved in the binding and hydrolysis of guanine nucleotides ,9.As molecular switches, Rho small GTPases cycle between an active GTP-bound form and an inactive GDP-bound state, and mutations in positions 12 or 61  result in catalytically deficient Rho proteins, thus considered to drive constitutive activation of downstream signaling. The control of the balance between the GTP-bound active state and the GDP-bound inactive state constitutes a first essential regulatory layer onto which several mechanisms converge ,11. GuanAdditional studies based on the characterization of substrate specificities, interactomes and localization revealed at systems-level how GEFs and GAPs contextualize and spatiotemporally control Rho signaling to provide novel aspects about the emergent organization principles of Rho signaling and establishing a model by which GEFs and GAPs provide positional information based on the placement of the enzymes on dedicated cellular structures and the assembly of additional signaling network components .Detailed studies based on structural and mutational analysis identified a series of regions and amino acid residues within amino-terminal regions—termed switch I and II—in RhoGTPases as essential for the exchange between the GDP- and GTP-bound states and also for efficient interaction with multiple effectors, GEFS and GAPs. However, several observations indicate that sequences outside the effector domain might also be participating in effector-mediated functions. The importance of sequences beyond the core effector domain in effector activation is exemplified by mutagenesis analysis of activated Rac1. While some residues are conserved in Rho, Rac and Cdc42 (but not Ras) proteins , other Rac1 residues are neither conserved in Ras nor in other Rho GTPases . These residues are surface-exposed residues and, consequently, logical candidates for involvement in effector interactions. The differential impairment in Rac1 signaling, actin reorganization and transformation exhibited by these mutants support the contribution of these residues to interaction(s) with different effectors [A second mechanism regulating Rho GTPase activation state is driven by Guanine nucleotide Dissociation Inhibitors (GDIs). This family of regulatory factors controls Rho signaling through different mechanisms. Many GDIs bind the inactive, GDP-bound form of Rho-family signaling, impairing GTP exchange by GEFs and maintaining GTPases in an inactive state . HoweverRhoGDIs not only maintain small GTPases in their inactive GDP-bound form but also act as chaperones for small GTPases protecting them from degradation. A recent systematic study shows that the three different RhoGDIs  interact with all possible Rho family small GTPases raising the possibility of a differential functional modulation depending on the RhoGDI binding isoform . MechaniThe third and perhaps most complex mechanism of modulation of GTPases is through subcellular compartmentalization. A large body of literature considers most signaling initiated by Rho GTPases occurs at or around the PM; however, it is becoming clear that functionally active Rho GTPases, their GEFs and GAPs, and their effectors can be localized to different intracellular compartments beyond the PM. Despite the potential relevance of these novel intracellular pools, little is known about the mechanisms underlying its modulation.Post-translational modifications of small GTPases are an additional regulatory layer that could integrate functional modulation and subcellular localization for the tight spatiotemporal control of GTPase activity. A wide range of PTMs was reported for small GPTases, including phosphorylation, ubiquitylation, SUMOylation and lipid modifications ,25 Tabl, which m2), superoxide (O−2), hydrogen peroxide (H2O2) and peroxynitrite (ONOO−) can alter protein activity [Different cell metabolic activities producing oxidizing molecular species can be sources of oxidative stress upon dysregulation. Elevated levels of oxidizing Reactive Oxygen and Nitrogen Species (ROS and RNS) damage different cell structures such as DNA and membrane lipids and contribute to several pathological processes, including cancer ,27. Thioactivity . In contactivity .in vitro [Several Ras superfamily small GTPases are redox-sensitive, and consensus acceptor motifs C and CGNKXD) were identified. The impact of redox agents on these redox-sensitive GTPases is similar to that of GEFs, in that they perturb GTPase nucleotide binding interactions resulting in the enhancement of guanine nucleotide exchange ,31. Somein vitro , and thein vitro .Importantly, small GTPases can also modulate the production of ROS, which can be considered part of their downstream effectors. For example, NADPH oxidases are prominent sources of ROS and affect redox signaling ,35. Rac1in vitro and in vivo. This phosphorylation does not modify its GTPase activity, nor its interaction with GEFs or GAPs, but significantly increases its interaction with RhoGDI [in vitro. Overexpression of a Rac1-Tyr64Glu mutant (a phosphomimetic construct) suggests that phosphorylation at Tyr64 on Rac1 negatively regulates cell spreading, focal adhesion localization and its binding to GTP, RhoGDI, GEFs and its effector protein p21-activated kinase (PAK) [The substantial evolutionary conservation of several phosphorylation acceptor residues in Rho GTPases supports their potential general role as modulation switches. RhoA was the first Rho GTPase shown to be phosphorylated. Cyclic AMP (cAMP)-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) phosphorylate RhoA on serine 188 both h RhoGDI ,39,40. Ph RhoGDI  and Cdc4h RhoGDI . Epidermse (PAK) .Clostridium difficile toxin A (TcdA), a potent inactivator of Ras superfamily GTPases, through irreversible glycosylation [Rho GTPases are also substrates for serine/threonine phosphorylation. RhoA is phosphorylated on Ser26 by Mst3 , which leads to an inactive RhoA status . Rac1 issylation .Rac1 Thr108 phosphorylation by an extracellular signal-regulated kinase (ERK) in response to EGF was also reported, leading to decreased Rac1 activity, partially through intervening Rac1 interaction with phospholipase C-γ1 (PLC-γ1). Of note, this is the only phosphorylation site reported to be directly involved in Rac1 nuclear localization .The impact on the subcellular compartmentalization of small GTPases was characterized for several of these modifications. Phosphorylation of cargoes shuttling between nucleus and cytoplasm is a prominent regulatory mechanism controlling gene expression, cell growth and proliferation. Phosphorylation and dephosphorylation modulate trafficking in a cargo-specific manner, and at present, it can be difficult to predict how a phosphorylation event affects the nucleocytoplasmic trafficking of a given protein ,51.Ubiquitylation, the covalent attachment of ubiquitin to Lys residues in a target protein, can lead to degradation of the substrate or the modulation of its subcellular compartmentalization and/or activity . SeveralSmall Ubiquitin-like Modifiers (SUMO) are small proteins remarkably similar in their structure to ubiquitin, although divergent in their amino acid sequence that is covalently attached to lysine residues in the target protein through an analogous but distinct process and machinery. Four distinct paralogs exist in humans. While recent reports show that various members of the Ras small GTPase family are targeted for SUMO conjugation ,59, the With the exception of RhoBTB1 and RhoBTB2, post-translational modification by lipids was described for all Rho GTPases , modulatPrenylation, the modification of proteins by isoprenoids, is known to control the localization and activity of several proteins, with a broad impact on cell regulation ,66,67. MPrenylation confers hydrophobicity to the C-terminus of GTPases, dramatically increasing their capacity to interact with cellular membranes. The prenylated motif can determine both the specific subcellular compartmentalization of proteins to specific domains of the PM or endomembranes and the activity of the modified protein ,72,73. WInterestingly, there is a level of specificity in the attachment of prenyl moieties to small GTPases: whereas the Rho GTPases are mainly geranylgeranylated , Ras subThe type of prenylation in GTPases seems to be critical to define their subcellular sorting. This is highlighted by the example of RhoB, which can be modified by both geranyl and farnesyl groups: geranylgeranylated RhoB localizes to late endosomes, while the farnesylated form is detected predominantly at the PM . Rac1, RRho GTPase prenylation also regulates their binding to GDI proteins ,79. GDIsSmall GTPase prenylation also has an impact on their guanosine nucleotide binding state, and non-prenylated Rho GTPase species exhibit increased GTP binding through mechanisms as yet poorly understood ,83. A poBecause of the pervasiveness as a mechanism to regulate GTPases and the extensive information from the mutational analysis of the CAAX motif across several proteins, prenylation was intensively investigated as a pharmacological target. However, available combinations of inhibitors of farnesyltransferase (for targeting Ras GTPases) and GGTase I (for Rho GTPases) exhibit significant toxicity and thus have limited clinical potential ,91, althStatins have emerged as a means to pharmacologically modulate GTPase signaling. Statins inhibit hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase (HMGCR), the rate-limiting enzyme for mevalonate/cholesterol biosynthesis that catalyzes the conversion of HMG-CoA to mevalonic acid. This step is also required for the synthesis of isoprenoid intermediates, such as farnesylpyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), the precursors needed for GTPase prenylation ,93. WhilWhile not as common as prenylation of the CAAX box, some Rho family members additionally require the covalent addition of a palmitate acyl chain to the adjacent C-terminal hypervariable domain for appropriate membrane association and regulation .S-palmitoylation is the covalent attachment of a palmitate molecule  to the side chain of Cys residues. This PTM regulates the subcellular localization and trafficking of substrates. It may also directly modulate protein–protein interactions by physically masking binding sites or by forcing a binding site into close membrane proximity, hence reducing availability for protein interaction. Palmitoylation can regulate either retention or anterograde trafficking of proteins at the ER-Golgi interface and towards the endosomal system . N-termiAlthough some proteins spontaneously autopalmitoylate, most palmitoylation events are catalyzed by the zinc finger DHHC-type-containing (ZDHHC) family of palmitoyl S-transferases (PATs), comprised of 23 distinct proteins in mammals. While PATs can exhibit multiple localizations, ER and Golgi are the prime sites of protein palmitoylation activity in mammalian cells, and three specific ZDHHC enzymes  are preferentially targeted to the PM ,101,102.Multiple cancer-associated proteins are palmitoylated. A classic example is provided by the Ras family of small GTPases. In addition to being irreversibly farnesylated at a conserved C-terminal CAAX sequence, H-Ras, N-Ras and the K-Ras4A isoform (but not the K-Ras-4B isoform) are reversibly palmitoylated at one (N-Ras and K-Ras-4A) or two (H-Ras) C- terminal residues. Only the dually lipidated  forms of H-Ras, N-Ras and K-Ras 4A are properly localized to the PM and capable of transforming cells ,106,107.Regarding the two other main members of the Rho family, RhoA is not susceptible to palmitoylation, while the ubiquitously expressed major Cdc42 protein isoform is not palmitoylated, a novel brain-specific alternative splicing variant enriched in dendritic spines was reported as a palmitoylation substrate ,112,113.Although palmitoylation confers a relatively labile anchorage by itself, its combination with other lipid modifications or polybasic domains can significantly enhance protein attachment to the PM and segregation into specific membrane microdomains enriched in cholesterol, known as ‘lipid rafts’ or cholesterol-enriched plasma membrane microdomains (CEMMs); this is exemplified by Rac1 .Interestingly, the weakness of the association conferred by palmitoylation is an important feature allowing for the dynamic trafficking across different intracellular compartments, such as endosomes. RhoB was the first Rho GTPase described as associating with the endosomal compartment ; subsequBy reinforcing the key role of appropriate palmitoylation for RhoGTPase intracellular regulation, the ‘creation’ of a novel palmitoylation site in Cdc42─by substitution of the Arg 186 residue in position 186 to Cys—sequesters this protein at the Golgi, impairing its shuttling to the PM. This overpalmitoylated form of Cdc42 fails to sustain actin filament polymerization and induces exacerbated pro-inflammatory cytokine production due to increased NF-κB activation  Table 1Table 1.3) over phosphatidylserine, PIP2 and other abundant anionic phospholipids, and cholesterol. The biological relevance of this selectivity for PA and PIP3 is evidenced when depleting the PM of PA or PIP3, which decreases Rac1 PM binding and nanoclustering [3; thus, reduced palmitoylation also reduces Rac1 affinity for PA and PIP3 [Together with PTMs and in coordination with their lipidation, GTPases are critically regulated by their non-covalent interaction with specific lipid species across different cellular membranes. Rac1 is able to form spatially segregated nanoclusters at PM domains of distinct lipid composition by selectively associating with phosphatidic acid (PA) and phosphoinositol -trisphosphate  is essential for maintaining the unique biochemical identity of the nucleus. The nucleus is a special membrane-bound compartment: two concentric lipid bilayers, continuous with the ER system―the ONM (outer-nuclear-membrane) and the INM (inner-nuclear-membrane), separated by the perinuclear space (PNS)―and with each other at nuclear pores, act as a strong physical barrier. Despite their similarities in lipid composition, the INM and ONM are biochemically distinct: while the ONM has a certain continuity with the rough endoplasmic reticulum (RER), the INM exhibits a distinct set of integral membrane proteins, which provide docking sites for lamins, chromatin and via interaction with ONM proteins, the cytoskeleton ,131.Apart from separating the genome and its associated functions from the cell, the nucleus determines the interaction of cells with their physical environment and their ability to migrate through three-dimensional tissue fiber meshworks. With a distinct rigidity, conferred in large part by the nuclear lamina, and significantly larger dimensions as compared with the surrounding cytoplasm, the nucleus of interphase cells most often defines the limit diameter through which migrating cells can squeeze. The nucleus is also a receptor to both mechanotransduction networks signaling from peripheral structures , as well as direct forces through its physical connection with the actin cytoskeleton. As such, the nucleus critically contributes to cell mechanoadaptation. Finally, physical forces can have a variable impact on the integrity of the nucleus and the genome, depending on nuclear deformability, a phenomenon that contributes to tumor cell behavior and evolution.PTMs regulating small GTPases can regulate the nuclear import and function of many nuclear proteins. Prominent nuclear proteins subjected to prenylation are prelamin A and B type lamins, intermediate filament proteins that polymerize to form the nuclear lamina lining the inner side of the INM. Both are farnesylated and carboxymethylated, but prelamin A is further processed by endoproteolysis to mature lamin A, which lacks the final 18 amino acids, including the modified Cys residue. In cells from patients with Hutchinson–Gilford progeria syndrome, a mutant prelamin A protein, progerin, cannot release its prenylated carboxyl-terminal moiety and therefore remains permanently associated with the nuclear envelope (NE), causing aberrant nuclear morphology. Thus, prenylation is important in the nuclear localization of lamins, but it requires to be properly controlled and removed to ensure cell homeostasis. Palmitoylation can also regulate protein shuttling from the PM to the nucleus. Although palmitoylation was first studied for its impact on protein trafficking through the secretory pathway en route to the PM, several nuclear proteins were identified as targets of palmitoylation. Palmitoylation can modulate the nuclear import of transcription factors ,133, incDifferent species of Rho GTPases localize to this compartment in a regulated manner ,87,137. A fraction of total RhoA localizes to the nucleus at steady-state, and its activity is controlled by the GEF Net1. DNA damage signals such as ionizing radiation (IR) modulate nuclear RhoA, but the specific mechanisms underlying its nucleocytoplasmic shuttling and functional relevance are still being investigated . Recent Apart from the C-terminal polybasic region, which favors its nuclear translocation ,142, RacWhile the functional relevance of Rac1 signaling at the NE or inside the nucleus is still incompletely understood, nuclear actin polymerization control appears to be a key downstream output. The formation of distinct nuclear actin structures can be observed upon induction of different forms of cell stress ,146, butNuclear actin was associated with most nuclear functions, ranging from RNA biogenesis to DNA damage responses and repair. Actin pools are found as a constitutive component of all three types of RNA polymerase  ,151,153 Recent studies demonstrate that pre-mRNA splicing can be modulated by nuclear Rac1 . As an ein vitro [The additional, prominent potential functions for nuclear actin, closely related to the dynamical control of the cytoskeleton by Rho GTPases, are physically scaffolding and conferring plasticity to the cell nucleus. In fact, classic experiments have shown that tugging on integrin adhesion receptors results in distortion of the nucleus, reflecting a physical linkage between the cell surface and the nucleus through the cytoskeleton . Local Rin vitro .These questions are particularly relevant in the context of genomic integrity. Genotoxic agents activate Rac1 and promote actin polymerization in the nucleus ,162,163.Further, the nucleus is subjected to direct mechanical cues and constitutes an emerging mechanotransducer structure in the cell. Different mechanisms determine nuclear mechanics: (i) regulation of lamins, and lamin B receptor expression levels, modulate nuclear envelope integrity and deformability ; (ii) thRecent studies reveal that mechanical stress can induce conformational changes in (and modulate the PTM of) nuclear envelope proteins . For exaRecent findings reveal how mechanical stimulation by stretching induces an increased expression of RhoA and Rac1, with deAn intriguing additional potential layer of coupled regulation arises when considering that nuclear membranes are continuous with the ER. Mechanical stretching of the NE is expected to increase membrane tension at the adjacent RER . Of noteWhile Rac1 nuclear expression levels clearly correlate with malignancy, nuclear Cdc42 localization correlates with ER-positive, low-grade tumors . Recent The last aspect worth mentioning regarding nuclear membrane dynamics and the regulation of nuclear pools of Rho GTPases pertains to the control of its lipid composition. Despite the presence of a plethora of biologically active lipids at nuclear membranes and nucleoplasmic structures, such as glycosphingolipids, the main phospholipid component is phosphatidylcholine ,183, togAn additional virtually unexplored field is the relevance of the nuclear localization of different disease-associated GTPase mutants. While activating mutations in KRas, NRas and HRas genes have long been recognized and occur in many types of cancer, genetic dysregulation of Rho family GTPases, such as Rac1 and RhoA, have only recently been characterized as the result of extensive cancer genome-sequencing efforts and are associated with rather specific types of cancer.Regarding Rac1, the paradigm example we have focused on above, its overexpression has long been reported in many cancers, most frequently correlating with poor prognosis and therapeutic resistance ,182,183.Exosome-sequencing efforts have revealed Rac1 activating mutations in malignant melanoma patients. Most of these mutations affect codon 29, changing Pro to either Ser or, less commonly, Leu, which promotes the quick exchange of GDP to GTP and thus altering signaling output. The 3D structure of Rac1 Pro29Ser in the presence of non-hydrolyzable GTP analogs has shown significant differences in the folding of the switch-I motif, which resembles more closely that of Ras. These mutations represent a typical UV signature and occur in up to 9% of sun-exposed, cutaneous transformations. The downstream effects of these mutations are not fully characterized and likely complex and contextual, beyond the relative accumulation of Rac1-GTP. Expression of PD-L1 is specifically affected by Rac1 Pro29Ser but no other Rac1 mutants . Rac1 PrVirtually no information exists on what the lipid modification status of each of these mutants is and how they are affected regarding their subcellular partitioning. This information could hold the key to new therapeutic approaches against the disorders these mutations are associated with.Rho GTPases govern many essential processes, including cell adhesion, survival, cell cycle and migration, which underlie their contribution to disease and justifies their exploration as important therapeutic targets in cancer and other disorders—particularly in relation to their regulatory activity on cytoskeletal dynamics and PM organization. This perspective was recently expanded by the ability of Rho GTPases to signal from compartments different from the PM, such as endosomes and the nucleus. A deeper understanding of the mechanisms underlying the regulated sorting of RhoGTPases across these compartments is thus warranted because currently available small compounds for the modulation of GTPase activity are limited in their efficacy and safety and do not discriminate between subcellular pools that are profoundly distinct in their function. PTMs of RhoGTPases are critical regulators of their subcellular distribution and functional regulation, but we need to fully characterize their interplay and integration with other cellular processes. The role of RhoGTPases as determinants of nuclear function and mechanoadaptation is one such emerging field, increasingly important to understand tumor cell migration and its link with gene expression reprogramming and genome integrity. In this regard, two main non-exclusive mechanisms were identified: (i) nuclear actin dynamics and its control on nuclear membrane deformability and nuclear function; and (ii) modulation of nuclear architecture through integrin signaling and cytoskeletal components such as perinuclear actin fibers, by virtue of specialized linking structures. Improved insights into the interplay between mechanics and post-translational modifications of RhoGTPases could help us to better understand how their spatiotemporally regulated signaling at distinct compartments occurs.Furthermore, considering that specialized membranous compartments can handle specific metabolic processes, it is possible that RhoGTPases activity inside these compartments holds the key to the coupling between mechanoadaptation and metabolism. It is plausible that regulation of small GTPase trafficking into the nucleus can be part of a safeguard mechanism that degrades and thereby maintains the appropriate levels of the active form of GTPases with the nucleocytoplasmic shuttling of RhoGTPases as a potential mechanism to regulate cell homeostasis.An emerging concept is the potential role of GTPases in the control of cellular homeostasis through its implication in autophagic processes at the cytosolic level. The possibility exists that the nuclear pool of small GTPases can also contribute to modulate nuclear homeostasis through the control of a novel and unexplored mechanism such as the nucleophagy, by selectively removing damaged or non-essential nuclear material from the cell. Thus, an altered nucleocytoplasmic shuttling could explain the potential contribution of small nuclear GTPases to tumor progression and invasion. Current and future challenges reside in fully understanding the molecular principles governing these functions and their integration with cell behavior  at the systems level.


Brain plasticity is widely accepted as the core neurophysiological basis of memory and is generally defined by activity-dependent changes in synaptic efficacy, such as long-term potentiation (LTP) and long-term depression (LTD). By using diverse induction protocols like high-frequency stimulation (HFS) or spike-timing dependent plasticity (STDP), such crucial cognition-relevant plastic processes are shown to be impaired in Alzheimer’s disease (AD). In AD, the severity of the cognitive impairment also correlates with the level of disruption of neuronal network dynamics. Currently under debate, the named amyloid hypothesis points to amyloid-beta peptide 1–42 (Aβ42) as the trigger of the functional deviations underlying cognitive impairment in AD. However, there are missing functional mechanistic data that comprehensively dissect the early subtle changes that lead to synaptic dysfunction and subsequent neuronal network collapse in AD. The convergence of the study of both, mechanisms underlying brain plasticity, and neuronal network dynamics, may represent the most efficient approach to address the early triggering and aberrant mechanisms underlying the progressive clinical cognitive impairment in AD. Here we comment on the emerging integrative roles of brain plasticity and network oscillations in AD research and on the future perspectives of research in this field. Brain is constantly changing throughout life with modifications at synaptic levels namely plastic changes that involve morphologic and physiological modifications . Brain pUnderlying rhythmic activity within the network also conditions plastic processes. For instance, spike timing-dependent plasticity (STDP), a ubiquitous Hebbian learning rule  in whichPlasticity processes also participate in how the brain reacts to lesions and injuries  and its From the past 20 years, there is growing evidence indicating that subtle synaptic changes precede neuronal and synaptic loss typical of AD . Particuin vitro, ex vivo, and in vivo AD models as well as different electrophysiological approaches. This offers several advantages on the field toward the understanding of particular features of the pathology while brings a complex picture of the disease. Some of the diverse studies on plasticity are outlined in In recent years, a wealth of data has been accumulated on the study of plasticity in AD by using a plethora of available and newly generated ex vivo AD-model) or hippocampal Aβ42 injections in vivo likely serve as a feasible prototype for cognition-relevant neuronal network dynamics in AD, since several synaptic and neuronal AD features has been replicated , and normal LTP at 7 days old in the hippocampal CA1 area in response to HFS of Schaffer collaterals. By contrast, at 14–16 months old, the same model exhibits hyper-excitable synaptic transmission, enhanced PPF, and unstable LTP . Interes5xFAD mice start to develop visible Aβ deposits as early as 2 months of age, consistent with their dramatically accelerated Aβ1-42 production. This Aβ deposition first emerges in the subiculum area of the hippocampus and in the layer 5 of somatosensory cortex, and then rapidly increases with age, spreading to fill much of the hippocampus and cortex by 9 months of age . In anotex vivo, the authors proposed that soluble Aβ might trigger the decrease of synaptic plasticity in neocortical pyramidal cell networks during early stages of AD pathogenesis by preferentially targeting postsynaptic AMPA receptors   and NMDAin vivo . Interesin vivo . In addiin vivo . A permain vivo . During in vivo . In previn vivo . Howeverin vivo stimulation of fast-spiking interneurons at γ frequency altered microglia morphology inducing a shift from pro-inflammatory to phagocytic phenotype that resulted in a significant reduction of the amyloid load in 5xFAD mouse model and improved network performance  contribution to the overall burden of AD cases could be considered negligible, its discovery has boosted the generation of a diverse transgenic mouse models carrying a combination of human mutations that triggers pathogenic events sharing commonalities with human sporadic AD, such as Aβ deposition and progressive cognitive decline.Accordingly, some features of the mentioned models give rise to differences on timely physiological deviations between models, particularly if the focus is turned onto earlier time points of the disease progression. The contribution of Despite either the controversy or the ample commonalities found among the current studies in diverse AD models, such studies suggest but do not dissect out the earliest changes in the neuronal networks dynamics at the root of the progressive deterioration of timing precision activity. Although APP-overexpressing transgenic mice have been important tools in AD research, concerns exist regarding the interference of the non-physiologically high levels of APP and its proteolytic fragments with normal brain function. Such APP processing products are not increased in human AD , and thein vivo phenomena showing that theta-nested gamma oscillations induced LTP is impaired in the hippocampal CA1 area of slices treated with acute Aβ42. In the study, optogenetic activation of SST has successfully restored theta-nested gamma oscillations-induced LTP from Aβ42-induced impairment. Finally, the emergence and termination of some forms of plasticity during critical periods has been generally addressed in quiescent states out of considering a more physiological scenario where external inputs such as ongoing neuronal networks oscillations command the sign of plasticity as it may occurs in vivo. Furthermore, despite the clear contribution of Aβ42 to the pathology, putative source of other mediators of the functional collapse remains elusive or mainly consider involvement of astrocytes while microglia involvement remains poorly studied.A widely number of different protocols are used to induce LTP such as theta burst stimulation (TBS), which is based on the hippocampal rhythm within theta band frequency (4–8 Hz) . Comparein vitro and ex vivo) may find a plausible target for plasticity studies in humans within the transcranial magnetic stimulation (TMS) technique. TMS could cross-validate diverse features of such experimental advances regardless possible differences between the mechanisms underlying TMS-induced LTP-like or LTD-like and mechanisms supporting experimental plasticity  to the emergence, loss, sign and forms of synaptic plasticity across brain development in healthy condition, thus helping to set the basis for the comprehensive study of plastic processes during the progression of cognitive-compromised disorders, majorly AD. Finally, regardless controversies, from this complex picture a promising horizon is rising targeting and based on basic, clinical and therapeutic AD research. Probably the commonalities among the diverse methods and models have set the basis for the current approaches. The success in restoring neuronal dynamics by optogenetic stimulation of key neuronal populations in AD models and reliability of TMS in human neurological disorders offers a suitable scenario where putative therapeutic tools could emerge.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.Both authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.