Mitochondria are highly dynamic organelles that provide essential metabolic functions and represent the major bioenergetic hub of eukaryotic cell. Therefore, maintenance of mitochondria activity is necessary for the proper cellular function and survival. To this end, several mechanisms that act at different levels and time points have been developed to ensure mitochondria quality control. An interconnected highly integrated system of mitochondrial and cytosolic chaperones and proteases along with the fission/fusion machinery represents the surveillance scaffold of mitostasis. Moreover, nonreversible mitochondrial damage targets the organelle to a specific autophagic removal, namely, mitophagy. Beyond the organelle dynamics, the constant interaction with the ubiquitin-proteasome-system (UPS) has become an emerging aspect of healthy mitochondria. Dysfunction of mitochondria and UPS increases with age and correlates with many age-related diseases including cancer and neurodegeneration. In this review, we discuss the functional cross talk of proteostasis and mitostasis in cellular homeodynamics and the impairment of mitochondrial quality control during ageing, cancer, and neurodegeneration. Cells express a pool of thousands of different proteins that need to be tightly controlled for proper cellular structure, organization, and function. The proteostasis network (PN) is an assembly of distinct dynamic molecular pathways that control the functionality of the proteome (proteome homeodynamics) during protein synthesis, folding, trafficking, and degradation. Failure of the PN is associated with broad range of diseases including cancer, neurodegeneration, and immunological and metabolic disorders . Ageing 2+ and metabolites required for cellular homeodynamics , the intramembrane space (Endonuclease G), and the inner membrane [Uncoupling Protein-2 and Uncoupling Protein-3 (UCP2 and UCP3)] –141. Nevmt was firstly described in mammalian cells as a mitochondrial stress response. Depletion of mtDNA or overexpression of a nuclear-encoded aggregation-prone protein in mitochondrial matrix induced increased gene expression of the mitochondrial molecular chaperone Hsp60 and of the protease ClpP ; Mfn homologs were later on also described in yeast (Fzo1) and in mammals (Mfn1 and Mfn2) [During fusion, mitochondria mix their genetic content in order to complement deficit of damaged mitochondria. In contrast to fission, mitochondrial fusion is operated by three dynamically related GTPases proteins, namely, Mfn1, Mfn2, and Optic Atrophy 1 (OPA1). Mfn1 and Mfn2 are implicated in the fusion of the mitochondrial outer membrane, whereas OPA1 is involved in the fusion of the inner membrane , 171. Mfnd Mfn2) , 173. Mend Mfn2) . Downregnd Mfn2) .OPA1 is a conserved large GTPase of the dynamin family, imported at the mitochondrial membrane by its N-terminal sequence. Opal is involved in cristea remodelling and inner membrane fusion , while m Drosophila resulted in reduction of mitochondria from dendrites and axons [Another important aspect of mitochondria dynamics is their motility and cellular distribution. The role and significance of this process are especially highlighted in neurons which need mitochondria energy at sites distant from the cell body . The trand axons .Studies on a knockout mouse model have demonstrated that attachment of the mitochondria to the microtubule can also be regulated by the protein syntaphilin (SNPH). Neuronal depletion of SNPH increased axonal mitochondrial motility, whereas overexpression of SNPH augmented the number of immobile mitochondria . Drosophila leads to a decrease of synaptic mitochondria [The fusion and fission processes are closely related to the mitochondria motility. Mitochondria fragmentation induced by loss of Mfn1 reduces the mitochondrial motility, while loss of Drp1 inchondria , 187. Onchondria .When a mitochondrial damage or unrepairable dysfunction occurs, selective removal of mitochondria by autophagy takes place; this process is known as mitophagy, a term proposed by Lemasters in 2005 .Autophagy is an evolutionarily conserved process that is responsible for the lysosome-mediated degradation of cytoplasmic components during a process where an isolated membrane named phagophore is generated upon autophagy signals . The fir Drosophila studies have shown that PINK1 and Parkin act in the same pathway since expression of Parkin in a background of mutated PINK1 in flies partially rescued the phenotype [One of the most described pathways of mitophagy is the PINK1/Parkin-mediated autophagy ; notablyhenotype –206.Mitochondrial depolarization stabilizes PINK1 on the outer mitochondrial membrane; this event directly phosphorylates Parkin and induces its recruitment in the mitochondria. Parkin then ubiquitinates the fusion proteins Mfn1 and Mfn2 and the proteins involved in mitochondrial trafficking, Miro1 and Miro2 , 203. MoSeveral studies link the PINK1/Parkin pathway to mitochondrial dynamics, namely, fission/fusion and motility. Specifically, PINK1 phosphorylates the fusion protein Mfn2 and this event, likely, induces recruitment of Parkin to mitochondria . MitofusLike mitofusins, Miro is also phosphorylated by PINK1 and ubiquitinated by Parkin. Parkin-dependent ubiquitination of Miro leads to proteasomal degradation and arrest of mitochondrial motility . MoreoveAdditional mechanisms that affect the PINK1/Parkin-mediated mitophagy include the activity of PI3K/AKT pathway in starvation conditions; this event attenuates mitophagy. On the other hand, mitophagy is enhanced by accumulation of unfolded proteins in the mitochondrial matrix or downregulation of the LONP1 peptidase (Human LON protease homolog) , 228. InDespite the growing knowledge about the PINK1/Parkin pathway involvement in mitophagy, most of the studies are performed in models with altered expression of Parkin. The majority of the cell systems are treated with CCCP, which totally depolarize the mitochondrial membrane resulting in Parkin overexpression ; it is tAlthough in the most studies mitophagy was induced artificially, in a recent work it was shown that constitutive mitophagy, which requires PINK1 and Parkin, occurs in mouse primary hippocampal neurons without mitochondrial membrane depolarization or drug treatment . Drosophila showed that the mitochondrial ubiquitin ligase 1 (MUL1) totally rescued the phenotype of PINK1/Parkin loss of function [In addition, loss of Drp1 leads to mitochondria ubiquitination, accumulation of damaged mitochondria, and p62 mitochondrial targeting, independently from Parkin , 234. Infunction . Other afunction . NIX nulfunction . Anotherfunction .Recently, two new Parkin-independent pathways have been described. Targeted overexpression of AMBRA1 at the mitochondrial outer membrane induces autophagy in both Parkin-dependent and Parkin-independent ways . SimilarAn additional mechanism for the removal of damaged mitochondria is the formation of mitochondria-derived vesicles (MDV) . MDV areOverall, mitophagy is an important mitochondrial quality control mechanism that effectively removes damaged mitochondria in order to prevent oxidative stress and cellular death. Considering the growing number of proteins involved in this process, the variation in mitophagic events, and its functional implication in ageing and age-related diseases, further detailed studies are needed to clarify and better understand this highly dynamic process.Ageing is a physiological process that occurs despite the presence of complex pathways of maintenance, defense, and repair, and it has been correlated with a number of diseases including cancer, neurodegenerative diseases, diabetes, and heart failure; notably, there are no evolutionary selected “gerontogenes” which function to cause ageing, while (among others) ageing correlates with increased proteome instability which leads to irreversible cellular damage and dysfunction , 242–246In relation to mitochondria, generation of a transgenic mouse model with mutated mtDNA provided the first genetic evidence that mutated mtDNA leads to premature ageing . Moreove Drosophila [Several studies have shown that caloric or dietary reduction increases lifespan –261. Insosophila . Thus, eThe “Warburg Effect” was proposed by Warburg and suggested that cancer cells have a metabolic shift toward aerobic glycolysis (rather than oxidative phosphorylation), reduced mitochondrial respiration, and functionally altered mitochondria in order to provide sufficient energy for their growth ; neverthAccumulation of mtDNA mutations along with increased levels of ROS  have been described as promoting factors of tumorigenesis –274; in κB), and the Hypoxia Inducible Factor 1α (HIF1α). The transcription factors NRF1 and NRF2 prompt the expression of the nuclear genes encoding subunits of the mitochondrial respiratory chain complexes and NRF2 activation increases synthesis of anabolic enzymes, NADPH production, and purine biosynthesis which all correlate with increased tumor growth [κB in tumorigenesis and mitochondria functionality has been adequately demonstrated in several studies [ Drosophila induces high levels of reactive oxygen species that originated from malfunctioning mitochondria, triggering an Nrf2-dependent upregulation of the proteasome subunits [Increased ROS levels, which largely originate from dysfunctional mitochondria, promote the activation of a number of transcription factors, including nuclear respiratory factor 2 (NRF2), the nuclear factor-kappa beta  and it also inhibits LON protease that (among others) degrades COX4-1 subunit [The high proliferative rate of tumor cells leads (among others) to insufficient blood supply with nutrient and oxygen. Therefore hypoxic conditions are a feature of tumor cellsn levels , 289. HI subunit , 291. In subunit , 293.Since increased ROS levels are a common feature of cancer cells therapeutic approaches that aim to decrease intracellular ROS levels have been considered as a possible method to inhibit cancer growth –296. Howα; a member of the PGC-1 family of coactivators) which is considered a key regulator of mitochondrial biogenesis and respiration. The PGC-1 family members potentiate the activity of other transcription factors and PGC-1α interacts with NRF1 and PPARα [α can also reduce the generation of mitochondrial ROS and it also regulates the mitochondrial fusion machinery by activating Mfn2 [α were found to be induced by the melanocyte-specific transcription factor (MITF) in melanoma cells, while growth and progression of these melanoma cells were strongly dependent on PGC-1α expression levels [α. Activation of Parkin promotes degradation of PARIS (a KRAB and zinc finger protein) which normally inhibits expression of PGC-1α by binding to insulin response sequences in the PGC-1α promoter [Another factor being activated during tumorigenesis is peroxisome proliferator-activated receptor gamma coactivator 1-alpha  tumors –308. RepFinally, alterations of the mitochondrial fusion/fission rate and machinery have been also observed in tumors. More specifically, several reports indicate that fission (linked to upregulation of Drp1 or downregulation of Mfn2) is increased in a variety of tumors, including lung cancer and invasive breast carcinoma , 314. Al Drosophila PINK1 or Parkin loss of function exhibits muscle and neuron degenerations which are highly reminiscent of Parkinson's Disease [Neuronal cells function and survival strongly depend on proper mitochondria functionality and activity, since axonal transport, neurotransmitter releasing, and ionic gradient can be severely impaired by dysfunctional mitochondria , 318. In Disease . Moreove Disease . Neverth Disease . Alpha-s Disease , 321; moβ (Aβ) plaques and neurofibrillary tangles (as result of the association of mainly fibrillar forms of Aβ and tau protein with microtubules), impaired mitochondrial trafficking, and increased ROS levels [β-peptide can accumulate at mitochondria and probably interacts with Drp1, while AD cellular models present decreased levels of Drp1 protein and increased expression of the Fis1 counterpart [β plaques formation impairs normal proteasomal function; this effect further fuels the formation of neurofibrillary tangles [AD is characterized by the formation of characteristic amyloid-S levels . Amyloidnterpart , 324. UP tangles , 326.Several other neurodegeneration diseases are associated with mitochondrial proteins dysfunction. Impaired fusion of the inner membrane due to Opal mutations leads to dominant optic atrophy, whether mutation of the outer membrane fusion protein Mfn2 is linked to peripheral neuropathy 30 Charcot-Marie-Tooth type 2A , 327. FuThe association of mitochondria function and dynamics with these neurological disorders highlights the central role of this organelle in proper functionality of neuronal cells. Besides mitochondria studies, even more data describe the UPS dysfunction in neurodegeneration disorders –335. NevThe vital role of mitochondria in cellular homeodynamics is clearly reflected in the severe effects of mitochondrial dysfunction on cellular functionality and human health, ageing, and age-related diseases .Despite the growing knowledge about the molecular mechanisms that impose on mitochondrial function and structural preservation several controversial questions remain to be answered. For example, although mitochondria dysfunction  seems to be a common feature in both neurodegeneration and cancer, the disease-specific alterations that determine the fate of the disorder need further detailed investigation efforts. In this line of research, the identification of the mitochondrial maintenance and/or signaling pathways that are specifically implicated in malignancy or neurodegeneration will, likely, reveal new disease-specific therapeutic approaches; similar efforts should aim at identifying how loss of mitostasis impacts on the progression of ageing. vice versa.An additional topic of exciting future research should of course relate to the identification of the molecular pathways that regulate the intense cross talk between the proteostatic and mitostatic modules in the young and aged somatic and reproductive tissues and how deterioration of one pathway affects the functionality of the other; these efforts will be particularly relevant given the UPS involvement in mitochondrial quality control and functionality andMoreover, the triggering event(s) that modulate the activation of the UPS systemic responses or mitophagy following mitochondrial damage clearly need further investigations. Most likely, the disrupted balance of ATP production  along with membrane depolarization and ROS accumulation influences the equilibrium between the selective removal of mitochondria by mitophagy or UPS-mediated degradation of damaged mitochondrial proteins.Finally, another aspect of significant importance relates to the question whether UPS is also involved in the degradation of proteins of the internal mitochondrial compartments.A better understanding of the mechanisms that regulate mitochondria quality control and their interconnection with the proteostasis modules  is relevant for human health since, besides the basic knowledge, mitochondria and proteasomes apart from impacting organismal healthspan are, likely, key therapeutic targets in main age-related diseases including cancer and neurodegeneration.


Remarkably, in the mid-late fetal and early postnatal neocortex of human FXS patients, NOS1 expression is severely diminished. Given the role of nitric oxide in diverse neural processes, including synaptic development and plasticity, the loss of NOS1 in FXS may contribute to the etiology of the disorder. Here, we outline the genetic and neurobiological data that implicate neocortical dysfunction in FXS, review the evidence supporting dysregulated nitric oxide signaling in the developing FXS neocortex and its contribution to the disorder, and discuss the implications for targeting nitric oxide signaling in the treatment of FXS and other psychiatric illnesses.A mechanistic understanding of the pathophysiology underpinning psychiatric disorders is essential for the development of targeted molecular therapies. For fragile X syndrome (FXS), recent mechanistic studies have been focused on the metabotropic glutamate receptor (mGluR) signaling pathway. This line of research has led to the discovery of promising candidate drugs currently undergoing various phases of clinical trial, and represents a model of how biological insights can inform therapeutic strategies in neurodevelopmental disorders. Although mGluR signaling is a key mechanism at which targeted treatments can be directed, it is likely to be one of many mechanisms contributing to FXS. A more complete understanding of the molecular and neural underpinnings of the disorder is expected to inform additional therapeutic strategies. Alterations in the assembly of neural circuits in the neocortex have been recently implicated in genetic studies of autism and schizophrenia, and may also contribute to FXS. In this review, we explore dysregulated nitric oxide signaling in the developing neocortex as a novel candidate mechanism of FXS. This possibility stems from our previous work demonstrating that neuronal  FMR1 as the X-linked gene affected in FXS , the leading inherited cause of intellectual disability (ID) and monogenic syndromic form of autism spectrum disorder (ASD), remains incompletely understood at the mechanistic level. Since the identification of d in FXS , signifiXS  theory of FXS, was proposed following observations linking mGluR signaling, long-term depression (LTD), protein synthesis, and FMRP function , 2002 ande novo proteins necessary for LTD have remained largely mysterious, and they are likely to represent only a subset of FMRP targets. Therefore, it is possible that additional mechanisms or brain regions are involved in the intellectual and behavioral manifestations of FXS. Elucidating these mechanisms is expected to uncover the neurobiology pertinent to FXS and provide novel targets toward molecular therapy.Since its original proposal, several lines of converging evidence have corroborated the mGluR theory. The majority of the data supporting excessive LTD in FXS is derived from studies of the hippocampus , a critinitric oxide synthase 1 (NOS1 or nNOS) to be translationally regulated by FMRP in the fetal human neocortex and severely reduced in the fetal and post-natal developing neocortex of FXS patients  signaling as a new candidate mechanism of FXS centered in the developing neocortex. In our recent work, we showed neuronal patients . As NO ide novo mutations in ASD simplex families preferentially affect genes encoding FMRP targets , Kcnd2 (Kv4.2), and DLG4 (PSD95)) ) , and mayNOS1 is species-dependent, being present in primate species, which exhibit conserved G-quartet sequences in NOS1, but absent in rodent species, which lack the NOS1 G-quartets. Consistent with this species difference, NOS1 has not been previously identified in the mouse brain to be an FMRP target mRNA . Although a large number of FMRP target mRNAs have been identified in the mouse brain  neocortex, however, we found NOS1 to be present not only in the interneurons, but also in the pyramidal neurons of two specific neocortical areas underlying higher cognitive functions: the fetal Broca’s area and orofacial motor cortex, and the ACC  corticofugal projection neurons, the output neurons of the neocortex , a transcription factor that is specifically expressed in and required for the development of corticofugal projection neurons . This coFigure 3; In the mid-fetal Broca’s area, NOS1 neurons coexpress FOXP2 , a gene In addition to the Broca’s area, a second neocortical region with important cognitive functions also exhibit pyramidal NOS1 expression during mid-fetal development. Starting from approximately 16 weeks post-conception, NOS1 is expressed in a large number of upper-layer (L2–L3) intracortical projection neurons of the ACC at significantly higher levels compared to the L5 neurons of the Broca’s area . NOS1-exCentrally positioned in the neocortex, the ACC is a key point of integration for cognitive and emotional processes, playing a crucial role in executive, social, and affective functions . The ACCNOS1 hypomorphic allele revealed homozygosity to be associated with attention deficit hyperactivity disorder (ADHD), impulsivity, and aggression .Consistent with a putative contribution of dysregulated NO signaling to FXS pathophysiology, NOS1 is likely to have a synaptic function as well. Subcellularly, NOS1 is significantly localized to the dendritic spines . This loeceptors . Interesine loss . TherefoThe development of molecular therapeutic interventions for neurodevelopmental disorders represents an immense challenge. In the past decades, these efforts have been impeded by the tremendous phenotypic and genotypic heterogeneity in the patient populations, the lack of specific knowledge on the molecular pathways, brain regions, or developmental timepoints most relevant to the pathophysiology of the disorders, and the identification of the appropriate primary endpoints . Recent FMR1 promoter and in an animal model of FXS , a second messenger elevated downstream of NO-mediated activation of soluble guanylate cyclase sGC; . Phosphoull mice  and deliull mice . Additioull mice . A drug ull mice . Furtherde novo variants uncovered shared genetic underpinnings in ASD and SCZ (CACNA1C and CACNB2, surpassed statistical threshold. This and previous studies associating CACNA1C with the individual disorders [Emerging genetic studies of multiple psychiatric disorders previously thought to be mechanistically distinct have implicated the same set of genes to carry disease risk for each disorder. A recent analysis of  and SCZ . In a re and SCZ . Remarkaisorders  stronglyNos1 null mice (The genetic and mechanistic evidence presented in this review suggest that NOS1 may similarly play a role in multiple psychiatric disorders. NOS1 activity is dependent on calcium signaling mediated via calmodulin. This calcium signaling may partially originate from L-type VGCCs. One study found depolarization-stimulated and spontaneous cGMP formation downstream of NOS1 activity to be blocked by the L-type calcium channel blocker nifedipine . Furtherull mice . TogetheThe pursuit of convergent mechanisms in multiple psychiatric disorders may help identify points at which disease pathways intersect and may lead to a more mechanistic nosology in psychiatry beyond descriptive classification. These studies are expected to not only lead to conceptual advances in the neurobiology of psychiatric disorders, but also to targets of molecular therapies with widespread utility. In particular, because NO signaling has been targeted pharmacologically for other indications, understanding NOS1 dysfunction in psychiatric disorders has tremendous potential to lead to the repurposing of efficacious, tested agents to treat these disorders.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.


Coherent forms of physiologically relevant circuit activity manifest themselves as rhythmic oscillations in the LFPs. Frequencies of rhythmic oscillations that are most closely associated with animal behavior are in the range of 4–80 Hz, which is subdivided into theta (4–14 Hz), beta (15–29 Hz) and gamma (30–80 Hz) bands. Activation of mAChRs triggers rhythmic oscillations in these bands in the hippocampus and neocortex. Inhibitory responses mediated by GABAergic interneurons constitute a prominent feature of these oscillations, and indeed, appear to be their major underlying factor in many cases. An important issue is which interneurons are involved in rhythm generation. Besides affecting cellular and network properties directly, mAChRs can cause the mobilization of endogenous cannabinoids  that, by acting on the principal cannabinoid receptor of the brain, CB1R, regulate the release of certain neurotransmitters, including GABA. CB1Rs are heavily expressed on only a subset of interneurons and, at lower density, on glutamatergic neurons. Exogenous cannabinoids typically disrupt oscillations in the theta (θ) and gamma (γ) ranges, which probably contributes to the behavioral effects of these drugs. It is important to understand how neuronal circuit activity is affected by mAChR-driven eCBs, as this information will provide deeper insight into the actions of ACh itself, as well as into the effects of eCBs and exogenous cannabinoids in animal behavior. After covering some basic aspects of the mAChR system, this review will focus on recent findings concerning the mechanisms and circuitry that generate θ and γ rhythms in hippocampus and neocortex. The ability of optogenetic methods to probe the many roles of ACh in rhythm generation is highlighted.Activation of muscarinic acetylcholine (ACh) receptors (mAChRs) powerfully affects many neuronal properties as well as numerous cognitive behaviors. Small neuronal circuits constitute an intermediate level of organization between neurons and behaviors, and mAChRs affect interactions among cells that compose these circuits. Circuit activity is often assessed by extracellular recordings of the local field potentials (LFPs), which are analogous to  The numerous effects that acetylcholine (ACh) has in the nervous system are mediated by both muscarinic (mAChR) and nicotinic (nAChR) receptors. Initially, attention focused on the mAChRs, following the classical experiments of Otto Loewi that showed that chemical transmission at synapses in the heart was mediated by ACh acting at mAChRs. With the recognition that nAChRs are also present in the brain and are directly relevant to the understanding of, e.g., the addictive potency of nicotine and its importance in schizophrenia, an enormous effort has gone into investigating the nAChRs in the central nervous system, although work continued on the molecular structure and pharmacology of the mAChRs. In addition, electrophysiological studies have provided a wealth of data on their cellular actions, the ion channels that they control, and their downstream biochemical mechanisms. Yet, despite these efforts, there remain important gaps in our knowledge of how the mAChRs affect neuronal circuits. Neuronal oscillations are among the most prominent and readily detected signs of neuronal circuit behavior. Certain oscillations, particularly those in the theta  and gamma  frequency ranges, are widely believed to be essential for the performance of various behavioral and cognitive functions. A general cholinergic agonist, carbachol (CCh), is often used to induce these rhythms which are mediated by mAChRs in hippocampus and neocortex in model experimental systems. However, a full understanding of the cellular and molecular mechanisms of rhythm generation has not been achieved. GABAergic inhibitory interneurons are key elements in rhythm generation, and mAChRs affect their behavior in many ways.This review will highlight some new results on the generation of oscillations in hippocampus and neocortex; useful reviews of earlier work  cascade leading to intracellular Ca2+ mobilization, and activation of protein kinase C and mitogen-activated protein kinase (MAPK) pathways. M2 and M4 receptors (M2-class) couple to the pertussis-toxin sensitive Gi/o proteins and inhibit adenylyl cyclase activity.Muscarinic acetylcholine receptors (mAChRs) are G-protein coupled receptors of the Class A, rhodopsin-like family, with ACh being the main endogenous agonist , Ca2+ -dependent, non-selective cation current  , which in turn hyperpolarizes the neuron or inhibits transmitter release . M4 is very high almost everywhere in the brain, while M2 is found at much lower densities. M5 mRNA is relatively sparse except in hippocampal CA1 PCs and some scattered subcortical nuclei. M1-class receptors are often located on somato-dendritic regions of neurons, and their activation leads to membrane depolarization and increases in cellular excitability by enhancing the mixed-cation NaIn the hippocampal CA1 region, PCs provide excitatory output to other cortical and subcortical areas, and carry information about spatial location and episodic memories : the parv- albumin-expressing (PV+), fast-spiking interneurons, and cholecystokinin-expressing (CCK+) regular-spiking interneurons - and mGluR1a-expressing interneuron. Hippocampal interneurons in this class have their somata in the stratum oriens and the great bulk of their axonal arbor in the stratum lacunosum-moleculare, and are referred to as oriens-lacunosum moleculare (O-LM) cells. O-LM cells target the dendrites rather than perisomatic regions of PCs and have different phase preferences for firing within θ rhythm oscillations than do the BCs (or axo-axonic cells). Therefore O-LM cells play distinctive functional roles in regulating both PC excitability and temporal patterning of PC activity Buzsáki, .These classes of interneurons are not unique to the CA1 region; most other regions of the hippocampus and the neocortex have similar inhibitory configurations  cells also express M2 and M4 receptors to some extent and show biphasic responses when a cholinergic agonist is applied . These effects have been reported most frequently in hippocampal slices, but also occur in neocortical slices. Thus, the brain slice preparation has been an invaluable tool for studying the mechanisms by which ACh can generate oscillations at multiple levels, especially when considering the “inverse problem” of the LFP  θ might additionally require rhythmic inhibition onto interneurons from septal GABAergic afferents  is profound, reversible disinhibition of principal cells that was initially described in the hippocampus as a transient suppression of sIPSPs or sIPSCs that followed a brief, 1 or 2 s, depolarizing current injection into a CA1 PC  by co-activation of a GPCR pathway  actions and those inhibited by M2 mAChRs are of different classes. Undoubtedly, the former represented the CCK+/CB1R+ interneurons and the latter the CCK−/CB1R− interneurons, probably the PV+ cells, although the cells were not immunologically identified. The results from slices  isoform-specific, knock-out mice, β is an essential element in the G-protein signaling pathway between mAChRs, or mGluRs, and eCBs in hippocampus and cerebellum, with the different isoforms being predominant in different brain structures  and the absence of this enzyme in −/− mice  vector, which has a high tropism for neural tissue and can result in expression levels exceeding 90% in target cells , or ChR2 variant. In general the AAVs appear to have equal tropism for all cells, although selective transformation of inhibitory cells in the cortex was reported with low titer levels  and the PV+  cells are sharply segregated by their divergent properties  resistance of the target cells, and is frequency dependent, decreasing as the frequency of the voltage deflections through the junctional channels increases. When the leak resistance is low the coupling among cells is also low, because the currents, instead of passing via the gap junction into the coupled cells and depolarizing them, are shunted through the leak resistance to the extracellular space. When the leak resistance is high, the strength of electrical coupling increases. Interestingly the PV+ and CCK+ interneurons that are electrically coupled to each other are frequently also chemically coupled; that is, the target postsynaptic cells receive both electrical and chemical synaptic transmission from their upstream presynaptic partners  mAChRs potently stimulates the release of eCBs, an M1-specific PAM should enhance the ability of cholinergic agonists to mobilize eCBs. This hypothesis has not been tested, but could easily be investigated in 2+]i  somatic action potential activity is an accurate guide to the activity reaching the terminals; and (2) axons do not act independently of somata.However, under some circumstances axonal action potentials can be initiated independently of somatic depolarizations. These were initially described in the context of disease or other aberrant conditions, but new challenges to the simple picture have arisen in physiological contexts. In hippocampal slices, Dugladze et al.  report t−/–Fmr1 mice, there is enhanced coupling of mGluRs to eCB release at inhibitory synapses in both hippocampus . In the hippocampus of a mouse model of FXS, Studies of the mAChR system in the brain continue to yield exciting new insights and information on a wide variety of neurophysiological problems. Undoubtedly the future holds enormous promise for novel and valuable advances both in the basic understanding of this powerful and ubiquitous regulatory system, and in eventual clinical applications.This review was written jointly by all of the authors. The recent research referred to from the Alger laboratory was supervised by Bradley E. Alger and conducted by Daniel A. Nagode and Ai-Hui Tang, who also had major roles in the experimental design, data analysis, and interpretation of results.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.


SOD1) are a leading cause of ALS, responsible for up to 20% of familial cases. Although the exact mechanism by which mutant SOD1 causes disease remains unknown, multiple studies have shown that reduction of the mutant species leads to delayed disease onset and extension of lifespan of animal models. This makes SOD1 an ideal target for gene therapy coupling adeno associated virus vector (AAV) gene delivery with RNAi molecules. In this review we summarize the studies done thus far attempting to decrease SOD1 gene expression, using AAV vectors as delivery tools, and RNAi as therapeutic molecules. Current hurdles to be overcome, such as the need for widespread gene delivery through the entire central nervous system (CNS), are discussed. Continued efforts to improve current AAV delivery methods and capsids will accelerate the application of these therapeutics to the clinic.Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease caused by progressive loss of upper and lower motor neurons. Mutations in superoxide dismutase 1 ( SOD1) are responsible for over 20% of familial, or inherited, ALS cases  is a progressive neuromuscular disorder, resulting in loss of upper and lower motor neurons, leading to paralysis, and death within 3–5 years after diagnosis , the levels of an already existing protein. Nevertheless, since the Sod1+∕− mouse does have a mild phenotype, ideal therapies would be allelic specific, reducing only the mutant gene product. Meanwhile, future clinical trials could consider supplementation with SOD1 enzyme replacement therapy.A common therapeutic approach for gain of function diseases is reduction of the toxic gene product. Loss of Gene therapy for familial ALS has made substantial headway in recent years, especially as the number of preclinical studies and clinical trials using RNAi therapies and AAV vectors have increased rapidly  EIAV lentivirus vector pseudotyped with the Rabies G envelope glycoprotein, and specifically target the innervating neurons. Thus, it is not surprising that while disease onset was delayed, the length of disease duration was not increased further supporting the notion that motor neurons are mediators of disease onset, but progression is also dependent on expression of mutant SOD1 in glia  under cell specific promoters were infused into the lateral ventricles of neonate SOD1 mice  target mostly glia after an postnatal day 1 intravenous infusion . In the first study, adult SOD1 mice received an intravenous infusion of AAV6-shRNA vector resulting in 50% reduction in SOD1 protein in skeletal muscle. However, < 5% of motor neurons were transduced, and there was no apparent reduction in SOD1 levels in the spinal cord , or differences in shRNA efficacy. In a separate experiment, an shRNA against SOD1 was delivered by intramuscular injection of a lentivirus vector pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G) not capable of retrograde transport, or an AAV2 vector capable of retrograde transport. Both vectors reduced SOD1 protein levels in skeletal muscle, but the improvement in motor function was seen solely in the AAV2 treated SOD1 mouse  motor neuron degeneration in the disease phenotype. Ozdinler et al.  showed tSOD1, delivered into the brain lateral ventricles of SOD1G93A mice at post natal day 1  infusion into the lumbar spinal cord. This injection route leads to effective transduction of spinal cord motor neurons in adult non-human primates, but the percentage of transduced neurons in the brain remains limited. Studies using intrathecal infusions of AAV vectors in mice reported variable reproducibility, and limited vector spread to distal spinal cord regions and to the brain (Snyder et al., Multiple studies have used AAVs to deliver therapeutic RNAi molecules Table , but nonLS drafted the initial manuscript, and edited it into the final version. MSE edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The genetic and phenotypic heterogeneity of autism spectrum disorders (ASD) presents a substantial challenge for diagnosis, classification, research, and treatment. Investigations into the underlying molecular etiology of ASD have often yielded mixed and at times opposing findings. Defining the molecular and biochemical underpinnings of heterogeneity in ASD is crucial to our understanding of the pathophysiological development of the disorder, and has the potential to assist in diagnosis and the rational design of clinical trials. In this review, we propose that genetically diverse forms of ASD may be usefully parsed into entities resulting from converse patterns of growth regulation at the molecular level, which lead to the correlates of general synaptic and neural overgrowth or undergrowth. Abnormal brain growth during development is a characteristic feature that has been observed both in children with autism and in mouse models of autism. We review evidence from syndromic and non-syndromic ASD to suggest that entities currently classified as autism may fundamentally differ by underlying pro- or anti-growth abnormalities in key biochemical pathways, giving rise to either excessive or reduced synaptic connectivity in affected brain regions. We posit that this classification strategy has the potential not only to aid research efforts, but also to ultimately facilitate early diagnosis and direct appropriate therapeutic interventions. Autism spectrum disorders (ASD) are a group of neurodevelopmental disorders frequently characterized by impairments in social interactions, difficulties with language and communication, and the presence of repetitive, perseverative behaviors -phosphorylation-regulated kinase 1 A (DYRK1A) gene are associated with a type of syndromic ASD and intellectual disability that presents with microcephaly , and extracellular signal-regulated kinase (ERK1/2) signaling. Characterizing how the regulators and downstream correlates of these growth pathways are dysregulated in ASD will allow us to better classify forms of autism into broad, but more homogeneous subtypes. This knowledge will be valuable for (i) diagnosis of under- or over-growth phenotypic subtypes of ASD, (ii) selection of homogeneous patient populations for more rigorously controlled studies, and (iii) a potential biomarker that can be used to potentially assess efficacy of clinical trials. We propose that broadly stratifying patient populations in terms of under- or over-growth phenotypes based on the following signaling pathways would facilitate mechanistic and therapeutic insights into ASD, without necessitating an impractical fine categorization by precise genetic etiology.Neurotrophins, such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) were first identified as target-derived survival factors. However, growing body of evidence has indicated that neurotrophins regulate many aspects of neuronal structure and function, including neurodevelopment, differentiation, morphogenesis, and synaptic plasticity Poo, . SubsequOther trophic factors, such as glial-derived neurotrophic factor (GDNF), vascular endothelial growth factor (VEGF), and ciliary neurotrophic factor (CNTF), as well as insulin-like growth factor (IGF) also induce pro-growth signaling pathways. Like BDNF, NGF, NT-3, and NT-4, these additional trophic factors also signal through receptor tyrosine kinases to elicit downstream signaling cascades that promote growth in a variety of tissues, including neurons   and the eukaryotic translation initiation factor 4E-binding proteins (4E-BPs), are emerging as key players in autism pathogenesis  and analysis of copy number variations (CNV) have identified an enrichment of MAPK/ERK signaling components in patients with autism  and NF1 all ultimately leading to activation of ERK1/2. NF1 is a GTPase-activating protein (GAP) for Ras, an upstream activator of ERK1/2. Mutations in NF1 cause neurofibromatosis type 1, an autosomal inherited disorder with a high frequency of hyper-proliferative schwannoma cancers. More than 50% of individuals with mutations in NF1 also have cognitive impairments and recent studies report a significant increase in the incidence of ASD in NF1 patients  is the most common inherited cause of ASD and intellectual disability, affecting approximately 1 in 4000 males and 1 in 8000 females worldwide Peprah, . AccountFXS patients display intellectual disability and cognitive impairments, developmental delay and physical features consistent with overgrowth, such as macrocephaly, elongated facial morphology, and enlarged ears  levels in brain tissues from Fmr1 KO mice and FXS patients  levels in brains of FXS patients and Fmr1 KO mice  is a neurodevelopmental disorder that is caused by autosomal dominant mutations in the TSC1 or TSC2 tumor suppressor genes, which function as negative regulators of the mTOR signaling cascade. Loss of TSC1/2 function which enhances cell growth and promotes dysregulated metabolism leads to non-malignant tumor formation in the skin, brain, and other organs  and Tsc2 (CamKII-CRE Tsc2fl/fl) mutant mice as compared to wild-type controls. However, this experiment was performed in adult animals where compensatory changes could have occurred, and therefore, more thorough analysis of phospho-ERK1/2 levels throughout development may be required.Recent studies have also demonstrated that TSC1/2 may interact with the MAPK/ERK signaling cascade. This relationship was first appreciated in clinical studies which revealed that components of the ERK1/2 pathway are constitutively activated in TSC-associated brain lesions and tumor cell lines  is a regulator of gene expression which is present in cells throughout the body, but is particularly abundant in the brain. MeCP2 provides an interesting case in which the available evidence indicates that loss of MeCP2 (Rett Syndrome) can produce an “undergrowth” entity, while excess MeCP2 (MeCP2 duplication syndrome) conversely results in a presentation consistent with underlying “overgrowth.” Rett Syndrome (RTT), in 95% of cases, is caused by loss-of-function mutations in the X-linked MeCP2 and is predominately seen in females  as these mutations in males lead to perinatal lethality  was initially suspected to be associated with ASD found in a pair of monozygotic twins which had a Danio rerio while the recent development of a mouse loss-of-function model has begun to elucidate the function of the AUTS2 gene product. The function of AUTS2 has been recently addressed in animal models of perturbation in AUTS2. In zebrafish, reductions of AUTS2 by morpholinos knockdown results in microcephaly, reduced mobility and decreases in the number of neurons . Parallel to the effects observed with head size, deletion of this region often results in early-onset obesity, while duplication is associated with significantly reduced postnatal body weight and BMI  and/or duplication (dp/+) of the conserved syntenic region on chromosome 7  growth abnormalities emerge early in the pathogenesis of the disease, and (ii) manipulations and interventions that target growth-related pathways not only ameliorate growth defects but also lead to improvements in behavior.For therapies to yield maximal success, the initial identification and design should consider whether the form of ASD being treated could be generally classified as overgrowth or undergrowth. This sub-classification of growth phenotypes in ASD is critical as the same drug is unlikely to be effective under both circumstances. Aside from obvious growth defects such as macrocephaly and microcephaly, more subtle growth phenotypes might prove to be difficult to diagnose early in development. Ultimately, development of a biomarker that serves as a readout for growth status in ASD would be necessary in order to facilitate early detection and proper intervention. Possible development of biomarkers could include components of growth pathways highlighted in this review, such as ERK1/2, mTOR, and neurotrophic factors.Proper growth of functional neural circuits requires highly orchestrated control of intracellular signaling cascades and gene expression. These changes in neuronal growth depend upon the specific composition of gene regulatory networks present not only within each cell type, but within subcellular regions of a given neuron that give rise to the various neuronal processes, such as axons, dendrites, and spines, structures that are often dysregulated in forms of autism characterized by over- or undergrowth. Although CRE-driver lines have been highly informative for our understanding of cell type specific changes in behavior, gene expression, patterns of neuronal growth, and connectivity across various brain regions between wild-type mice and disease models, development of CRE-drivers requires a priori knowledge of the specific gene expression patterns unique to each cell type. Even within cells identified by such a CRE-driver system, there exists considerable heterogeneity in terms of gene expression. Each of the human brain's billions of neurons is unique not only in terms of its activity signature but also within the complex three-dimensional spatial environment of the brain that permits unique connectivity for each neuron. For these reasons, we predict that future single cell analyses of gene expression across ensembles of cells from wild-type controls and disease models will transform our understanding of these highly complex cells and gain better mechanistic insights into the signaling pathways that underlie over- or under-growth phenotypes in ASD.Many large-scale ASD studies have now been conducted, which have included mixed patient populations, with variations in multiple critical factors including age, diagnosis, and symptom severity. However, it has remained difficult to extract meaningful conclusions from these studies, in part, since molecular changes are likely to be different among forms of ASD with differing underlying etiologies. Identifying common molecular pathways that are dysregulated among various genetic causes of ASD is an important step in effectively stratifying ASD into endophenotypes from which therapeutic responses can be more readily anticipated. Shrinking the heterogeneity of the disorder would also make the study of ASD more tractable, allowing investigations in monogenic mouse models to be more appropriately applied to other genetic forms of ASD. Utilizing overgrowth and undergrowth phenotypes Figure  as a basThe 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.


Autophagy and inflammation are 2 fundamental biological processes involved in both physiological and pathological conditions. Through its crucial role in maintaining cellular homeostasis, autophagy is involved in modulation of cell metabolism, cell survival, and host defense. Defective autophagy is associated with pathological conditions such as cancer, autoimmune disease, neurodegenerative disease, and senescence. Inflammation represents a crucial line of defense against microorganisms and other pathogens, and there is increasing evidence that autophagy has important effects on the induction and modulation of the inflammatory reaction; understanding the balance between these 2 processes may point to important possibilities for therapeutic targeting. This review focuses on the crosstalk between autophagy and inflammation as an emerging field with major implications for understanding the host defense on the one hand, and for the pathogenesis and treatment of immune-mediated diseases on the other hand. Autophagy is the physiological cellular process through which intracellular components undergo lysosome-mediated self-digestion and recycling.ATG) genes and proteins are involved in the induction of autophagy ,Mycobacterium tuberculosis,IRGM polymorphisms with tuberculosis in West Africans, and increased Bacillus Calmette-Guérin (BCG) vaccination efficacy through enhanced antigen presentation.In addition to PRRs, several proinflammatory cytokines including TNF (tumor necrosis factor)de facto mechanism of evasion from the host defense. In line with this, human immunodeficiency virus (HIV) infection of CD4 lymphocytes reduces their content of BECN1 and MAP1LC3B-II, 2 crucial components for the induction of autophagy.M. tuberculosis is also able to inhibit autophagy and phagolysosome biogenesis, a process that greatly contributes to the escape from the bactericidal mechanisms of the host.In addition, it must be underlined that some infectious agents are able to manipulate and inhibit autophagy, a In this review we will further focus on the modulatory effects of autophagy on inflammation, a broad concept with important consequences for the pathogenesis not only of infections, but of autoinflammatory diseases and malignancies as well.Innate immunity represents the first line of defense against pathogens. It requires adequate pathogen recognition, which triggers activation of inflammation resulting in recruitment of immune cells, such as phagocytes, secretion of cytokines and chemokines, which in turn help recruit other immune cells and prime dendritic cells (DCs) for activation of adaptive immune responses. An increasing number of studies have recently shown that autophagy has important effects on the induction of the inflammatory reaction. However, when the inflammatory process is not properly controlled, autophagy can be detrimental for the host. In atherosclerosis, excessively stimulated autophagy can lead to endothelial cell death that can contribute to plaque destabilization, maintaining the inflammatory status of the plaque.Atg16l1 knockout mice respond with increased production of IL1B after stimulation with LPS. This was due to an exaggerated activation of CASP1 in the Atg16l1-deficient mice, mediated by the adapter molecule TICAM.Production of cytokines from the IL1 family, and especially IL1B, is one of the main mechanisms through which innate and adaptive immune responses are induced by an infection. The regulation of the production of this cytokine is thus crucial for understanding inflammation and host defense. Probably the best studied aspect of the interaction between autophagy and inflammation is represented by the effects of autophagy on inflammasome activation and IL1B release. An overview of the interplay between autophagy and IL1B-inflammasome activation is depicted in Several mechanisms have been proposed to mediate these anti-inflammatory effects of autophagy. During homeostatic conditions autophagy is responsible for the clearing of the cytoplasm of nonfunctional mitochondria and other organelles. If autophagy is defective this leads to an accumulation of depolarized mitochondria, that release inflammasome activators such as reactive oxygen species (ROS) or mitochondrial DNA (mtDNA).IL1B mRNA transcription is elevated when autophagy is inhibited, whereas no effects are observed on CASP1 activation.The important role of autophagy for IL1B secretion was also confirmed in human primary cells, in which inhibition of autophagy leads to increased production of IL1B.Autophagy does not only have inhibitory effects on inflammasome activation, but also on inflammatory mediators that are independent of CASP1 activation. In line with this, autophagy suppresses CAPN/calpain-dependent IL1B activation, and this effect is linked to the release of ROS as well.In addition to these effects on proinflammatory cytokines, autophagy also amplifies TLR signaling to improve the delivery of microbial ligands to cytoplasmic receptors such as TLR7 that promote the synthesis of type I IFN.These data suggest therefore that inflammation and autophagy are intertwined processes during host defense, and derangements in the crosstalk between these 2 processes can have important consequences for the pathogenesis and treatment of several diseases.Due to the mechanisms described above, autophagy has emerged as a central player in the pathogenesis of immune-mediated diseases, including autoinflammatory and autoimmune diseases, infections, and cancer.ATG16L1, IRGM, and ULK1, have been reproducibly associated with susceptibility and clinical outcome of the disease, demonstrating the important role of autophagy in CD.ATG16L1 variant in particular, as has been demonstrated for defective Paneth cell function,IRGM promoter polymorphism risk allele influences IRGM expression, at least in part through posttranscriptional regulation by miRNAs.CALCOCO2/NDP52 mutations in CD patients, a gene that codes for an important factor in phagophore targeting of intracellular bacteria (The first major autoinflammatory disease in which autophagy has been suggested to play a significant role is Crohn disease (CD). CD is a chronic inflammatory disorder of the intestine caused by a combination of multiple factors including defective host response, altered mucosal barrier function, and exaggerated cytokine production. Genetic variants of important autophagy genes such as bacteria .66,67FiIn addition to CD, important insights have also been provided by the assessment of autophagy induction and inflammasome activation in patients with chronic granulomatous diseases (CGD). CGD is a primary immunodeficiency characterized by defective ROS production due to mutations in the proteins forming the NOX (NADPH oxidase) complex.ATG5 associated with systemic lupus erythematosus (SLE)ATG5 genetic variants are functionally linked to a differential ATG5 expression and exhibit gene-gene interactions with ATG7 and IRGM,Epidemiological studies assessing genetic variants in autophagy genes demonstrated their association with susceptibility to autoimmune disease as well, such as variants in SLE is characterized by deregulation of autoantibody-producing B cells, infiltration of target organs by inflammatory T cells and aberrant immune cell activation. The latter is caused by abnormal activation of myeloid cells triggered by the presence of immune complexes composed of self-DNA from apoptotic cells and antibodies generated by autoreactive B cells.In rheumatoid arthritis (RA), a chronic inflammatory disorder affecting the joints, autophagy has a critical role in TNF-induced joint destruction ; TNF activates autophagy by induction of BECN1 and ATG7 and the conversion from MAP1LC3B-I to MAP1LC3B-II in osteoclasts, providing a mechanism for TNF-associated synovial inflammation and bone resorption.In the fight against pathogenic microorganisms, autophagy  contributes to both innate immune defense and to development of adaptive immune memory, and excellent recent reviews have described these mechanisms in great detail.Pseudomonas aeruginosa, and cleavage of TICAM1 by CASP1 inhibits autophagy.S. pneumonia.Within the context of innate host defense, autophagy provides the machinery for intracellular killing of microbes by means of phagophore engulfment and lysosomal fusion. Proper designation of microorganisms for autophagy processing is facilitated by protein adaptors such as SQSTM1/p62, NBR1 and CALCOCO2, that are activated downstream of microbial recognition by pattern-recognition receptors , providing anchors for phagophore assembly around the selected cargo.+ T-effector function, but is crucial for long-term cell survival and memory.+ memory was reported during influenza and MCMV infections.Atg5 knockout mice display a defective lymphocyte survival and proliferation.Autophagy is also important for the induction of T cell responses, and recent studies have underlined its crucial role for lymphocyte survival and memory. During infection with lymphocytic choriomeningitis virus autophagy is not required for proliferation and CD8M. tuberculosis induces ubiquitin-mediated targeting in macrophages, resulting in delivery of the pathogen to autolysosomes. This response requires the autophagy receptors SQSTM1, CALCOCO2 and the DNA-responsive kinase TBK1 (TANK-binding kinase 1),At the level of autophagy-inflammation interaction, host defense mechanisms are directly influenced by the modulation of proinflammatory cytokines, as described above in detail. However, the host defense against various classes of microorganisms is not influenced to the same extent. One of the most important infections for which autophagy makes a major impact is tuberculosis. Several studies have demonstrated the crucial role of autophagy for elimination of mycobacteria.Toxoplasma gondii,Salmonella spp.Listeria monocytogenes.118 and Rift valley virus,+ lymphocytes.Cryptococcus neoformans, through mechanisms involving the nonlytic exocytosis of the fungus, but also interfering with production of the inflammatory mediators IL6 and CXCL10/IP-10 (chemokine [C-X-C motif] ligand 10).Candida albicans has been demonstrated,Autophagy is also important in host defense against other intracellular pathogens such as M. tuberculosis latently infects human immune cells by concealed residence in the autophagosome and by preventing lysosomal fusion.Salmonella in turn attempts to inhibit autophagy and increase its survival by activating PTK2/focal adhesion kinase and MTOR.Listeria monocytogenes and Shigella, as well as viral pathogens such as HIV, have evolved different strategies to evade the autophagy machinery by inhibition of autophagosome formation or by modulation of positive  and negative upstream regulators  of autophagy.Notably, pathogens have also evolved strategies to evade the immune response by taking advantage of the autophagy machinery. For instance, ATG16L1 polymorphism impairs defense mechanisms against Salmonella typhimurium and increases susceptibility toward Helicobacter pylori infection.ATG16L1 and IRGM are associated with sepsis, potentially through modulation of immunoparalysis.M. tuberculosis infections, although autophagy profoundly influences the host immune response against this pathogen.Candida spp, indicating the redundant role of autophagy in anticandidal host defense.Genetic variants of autophagy genes have been investigated for their effects on antimicrobial immune responses, which revealed that the CD-associated Both autophagy and inflammation are involved in several steps of carcinogenesis and cancer progression. Autophagy has been reported to have both antitumoral effects and tumor-promoting effects in cancer. The generally accepted explanation for this apparent ambiguous role of autophagy in cancer is that its role might differ in the different stages of carcinogenesis and in different tumor types and is related to changes in the tumor microenvironment and treatment. In addition, the dual role of autophagy in cancer might reflect the interplay between autophagy and other fundamental biological processes such as apoptosis, senescence, cell metabolism, and the immune system.Becn1, results in autophagy deficiency, leads to spontaneous development of malignant tumors, and stimulates development of hepatitis-B-induced premalignant liver lesions.ATG genes have been described in different malignant tumors.AKT1 and class I phosphoinositide 3-kinase (PI3K) and somatic or germ-line mutations leading to inactivation of the tumor suppressor gene PTEN are often found in malignant tumors.In the early stages of cancer development, defects in autophagy machinery or inhibition of autophagy in premalignant cells might result in impaired degradation and removal of damaged organelles, unfolded proteins and ROS. These cytosolic components, which might accumulate in the cells, produced as a result of activation of oncogenic pathways result in DNA damage and maintain the carcinogenic phenotype.Conversely, in the more advanced stages of tumor development and progression, once the carcinogenic phenotype has been established, the cancer cells rely on autophagy to support the requirements for energy and provide substrates necessary for proliferation. This is especially important for surviving in an unfavorable microenvironment characterized by severe hypoxia and nutrient-deficient conditions. Therefore activation of autophagy in this context could be regarded as a survival factor, promoting cell proliferation and tumor progression. This is particularly relevant in the context of therapeutic response to cytotoxic agents and ionizing radiation. The strong induction of autophagy observed as a result of treatment with ionizing radiation or chemotherapy may contribute to cell survival and the acquired resistance to therapy in many cancer cells including breast cancer, pancreatic cancer, and malignant glioma.The relationship between inflammation and cancer has long been recognized. Many chronic inflammatory conditions, such as inflammatory bowel disease, chronic hepatitis, or pancreatitis are clearly recognized as precursors of malignancies in the respective organs.It is well known that tumor cells rely on aerobic glycolysis to support their energy requirements and to enable increased protein production .Although not an inflammatory mechanism itself, it is also important to underline that antigen recognition and presentation is the cornerstone of the specific immune responses against malignant cells. Hence, proper functioning of these processes heavily depends on the integrity of the autophagy machinery. It has for instance been shown that autophagy facilitates antigen presentation by tumor cells, such as human embryonic kidney cells (HEK293T) and melanoma cells, thereby facilitating the recognition of these cells by immune cells and their subsequent removal.In addition, autophagy induces cellular senescence, a physiological state of sustained cell cycle arrest characterized by a senescence-associated secretory phenotype. This consists of increased expression and secretion of several cytokines, chemokines, growth factors, and proteases in the senescent cells,ATG7 siRNA sensitizes the cells to TNFSF10-induced apoptosis, indicating that modulation of autophagy holds important therapeutic potential for anticancer treatment.The interplay between autophagy and inflammation is particularly interesting in the context of therapeutic implications, and the concerted targeting of these 2 processes can be explored for novel strategies. TNFSF10/TRAIL  induces cell death in different cancer cells and therefore contributes to immune cell-mediated cytotoxicity.atg5fl/fl mice have an accelerated growth compared to tumors developing in autophagy-competent KRAS; Atg5fl/+ animals. In this model, the autophagy deficient tumors also show increased infiltration with FOXP3+ regulatory T cells and increased expression of ENTPD1/CD39 ecto-ATPase. These effects can be reversed by pharmacological inhibition of ENTPD1, thus supporting the concept that autophagy deficiency contributes to carcinogenesis through inhibition of anticancer immunosurveillance.Moreover, autophagy plays an important role in the response to chemotherapy of some tumors, in particular through influencing the immune response in the tumor microenvironment. Treatment with chemotherapy can induce immunogenic cell death.Therefore, these data indicate that the immunogenic responses and immunosurveillance against cancer are dependent on the interplay between autophagy and inflammation. In addition the autophagy status contributes significantly to tumorigenesis and to the efficacy of chemo- and radiotherapy.Autophagy influences several key components of the immune response, and thus plays a critical role in regulating inflammatory responses. It has even been suggested that autophagy evolved as a primordial host defense mechanism of eukaryotes.Despite the increasing knowledge about the role of autophagy in modulation of inflammation, this field is still only in its beginnings. Little is known about the involvement of the autophagy-related SQSTM1-like receptors for the modulation of inflammation, although some initial data have suggested important effects.Finally and most importantly, modulation of autophagy activity may represent a promising therapeutic approach for a wide range of inflammatory conditions. In this respect, a recent study reported the development of an autophagy-inducing peptide, Tat-BECN1, which decreased the replication of several human pathogens in vitro, and reduced mortality in mice infected with chikungunya or West Nile virus.


Xenopus laevis, to address molecular strategies that may mediate RBP diplomacy and conflict resolution for coordinated control of mRNA translational output. Continued study of regulated ribonucleoprotein complex dynamics promises valuable new insights into mRNA translational control and may suggest novel therapeutic strategies for the treatment of disease.Regulated mRNA translation plays a key role in control of cell cycle progression in a variety of physiological and pathological processes, including in the self-renewal and survival of stem cells and cancer stem cells. While targeting mRNA translation presents an attractive strategy for control of aberrant cell cycle progression, mRNA translation is an underdeveloped therapeutic target. Regulated mRNAs are typically controlled through interaction with multiple RNA binding proteins (RBPs) but the mechanisms by which the functions of distinct RBPs bound to a common target mRNA are coordinated are poorly understood. The challenge now is to gain insight into these mechanisms of coordination and to identify the molecular mediators that integrate multiple, often conflicting, inputs. A first step includes the identification of altered mRNA ribonucleoprotein complex components that assemble on mRNAs bound by multiple, distinct RBPs compared to those recruited by individual RBPs. This review builds upon our knowledge of combinatorial control of mRNA translation during the maturation of oocytes from  Control of mRNA translation is critical for regulation of cell cycle progression during key physiological and pathological processes ,5,6,7,8.Recent technical advances have provided unprecedented insights into the ribonucleoprotein complex components that are involved in mRNA translation ,30,31,32cis is an emergent issue as there is growing experimental evidence that mRNAs with common RBP interactions are differentially regulated within the same cell context ,5251,52]oncogene ,38,53. Doncogene ,54,55. Tradation ,57,58. Mradation . As willXenopus mRNAs encoding key cell cycle control proteins hitherto characterized, reveals that their 3' UTRs contain multiple, distinct cis element RBP binding sites (reviewed in  binding protein , the mosTo assess possible Musashi and CPEB interactions more directly, we performed co-association analyses. Musashi1 was found to associate with both CPEB1 and CPEB4 A. These To determine if the Musashi interaction with CPEB1 or with CPEB4 was a consequence of direct binding, we employed the yeast two hybrid protein:protein association assay. This assay indicated that while Musashi1:Musashi1 homodimerization was the result of direct association, Musashi1 did not associate directly with either CPEB1 or CPEB4 . We concThe coordinated dynamic assembly and disassembly of shared protein complexes would allow communication between RBPs for reinforcement of similar functions and resolution of opposing functions. Such communication may be mediated through prevention of co-factor association within a shared mRNP in one cell context and facilitation of association in response to extracellular cues. There is evidence for such communication between Musashi1 and CPEB1. Musashi-directed mRNA translational activation is reduced in the absence of activating phosphorylation of CPEB1, which would preclude dissociation of the CPEB1:PARN complex . An inteRegulated mRNA translation is required to control the transitions between phases of the vertebrate cell cycle, including cell cycle entry and cell cycle exit. An understanding of the complex coordination of the components that make up mRNA-specific regulatory complexes presents both a challenge and an opportunity in control of mRNA translation. The challenge is in identifying, among the many distinct proteins that converge upon mRNA translation, those that may be amenable to manipulation . The opportunity lies with the fact that if the key regulator of translation of a specific mRNA is not a good target for manipulation, knowledge of the mRNP complex that interacts with the regulator may enable a work-around by manipulation of co-associated proteins. A better understanding of the mechanisms by which mRNA translation is modulated and coordinated may lead to development of novel approaches for control of cell cycle progression and cell fate, including anti-cancer therapy and stem cell lineage manipulation for regenerative medicine.


Despite the large number of studies that have examined the acute effects of different warm up modalities (WU) on physical performance, none of them have documented the time course of potential performance recovery in tennis players. The aim of this study was twofold: (a) to analyze and compare the acute effects of two different WU modalities  on physical performance  in elite junior players, as well as (b) to monitor the time course of any WU-induced changes after 30 and 60 min of simulated match-play. Twelve junior elite players completed both WUs modalities (TWU and DWU) in a counterbalanced order on separate days. In each experimental session, counter movement jump (CMJ), 20-m sprint, tennis serve speed and accuracy tests were performed before (immediately after TWU or DWU) during (30 min) and after 60 min of a simulated match play. Measures were compared via four factorial (WU intervention and time) repeated measures ANOVAs. There were main effects of WU (TWU and DWU) throughout the time for all the variables analysed. The results indicate that DWU routine led to significantly faster 20 m sprint times and higher CMJs as well as faster and more accurate tennis serves at both post warm-up and 30 min match-play testing moments in comparison with the scores reported by the TWU routine . No significant intergroup differences were found at 60-min match-play testing moment in any variable (except for the 20 m sprint). Therefore, the findings of this study recommend for optimal performance in these elite tennis players, DWU routines should be performed prior to formal training and competition rather than TWU routines. Tennis has experienced a significant increase in popularity in recent years, with more than 75 million people participating both, at recreational or professional levels . Like alLow to moderate intensity aerobic activity is an important element of the WU, increasing muscle temperature which is directly responsible for a number of mechanisms important for short term performance  . In contParallel to the study of the effects of SS on force and power production, researchers have studied the use of so-called dynamic stretching exercises (DS) in WUs. The DS are basically active aerobic activities combined with sport-specific whole body movements that have been designed to be an extension of the general WU, under the term of “dynamic warm-up” (DWU). In contrast to TWU, evidence exists indicating that DWU induce improvements in strength and power performance . AlthougThese different effects of the TWU and DWU on maximal muscular performance, along with the fact that both WU modalities have demonstrated similar effectiveness to improve joint range of motion , 28, havHowever, analysing the body of literature regarding the acute effects of the TWU and DWU on sports performance, some limitations are noted, which should be clarified before recommendations to athletes can be made. For instance, a recent meta-analysis regarding the acute effects of pre-participation SS on muscle performance has pointed out that some of the studies have used overall stretch durations on a single muscle group , ranging from 90s to over 8 min . These SIn addition, most of the studies that examined the time course of the dose response effects of the TWU on muscle performance –39, althTherefore, there is a clear need for applied research studies that analyse and compare the acute and retentive effects of realistic TWU and DWU protocols on sports-specific performance measures. The knowledge about which pre-participation WU modality (TWU or DWU) can optimize better the maximal muscle performance seems to be essential for tennis players, as they must perform high-intensity short-term motor activities  since the beginning until the end of a match .Therefore, the aim of the present study was twofold: (a) to analyse and compare the acute effects of two different WU modalities (TWU and DWU) on physical performance  in elite junior tennis players; and (b) to monitor the time course of any WU-induced changes after 30 and 60 min of simulated match-play.Twelve internationally ranked (top 100 ITF Juniors rankings) male junior tennis players  completed this study. All the players participated on average in 14–16 h of training  per week, plus 3.2 ± 0.4 matches per week, including singles and doubles. None of the participants reported any current or ongoing neuromuscular diseases or musculoskeletal injuries and none of them were taking any dietary or performance supplements that might be expected to affect performance during the study. Before any participation, experimental procedures and potential risks were explained fully to the participants, and a written informed consent was obtained from them and their parents/guardians. The Institutional Research Ethics committee (University Miguel Hernandez of Elche [Spain]), conformed to the recommendations of the Declaration of Helsinki, specifically approved this study.The study took place at the beginning of the summer competition season (April–May). A counter-balanced crossover-study design, in which participants performed all interventions, was used. Participants visited our laboratory on three occasions, with 2-weeks rest between sessions. The first visit was a familiarisation session, including the different testing procedures and WU exercises, and the following two visits were the experimental sessions.During each experimental session, participants began by completing one of the two interventions: TWU or DWU, which were counterbalanced per person to avoid carryover effects. The assessments of the (a) jumping height, (b) 20 m sprint time, (c) serve speed and (d) accuracy were carried out before (post-warm-up), during (30´match-play) and after (60´match-play) a 60 min simulated tennis match-play. The order of the tests was consistent through the experimental sessions and is displayed in To reduce the interference of uncontrolled variables, all the participants were instructed to maintain their habitual lifestyle and normal dietary intake before and during the study. The participants were told not to exercise on the day before a test and to consume their last (caffeine-free) meal at least 3 hours before the scheduled test time.Each WU intervention lasted about 15–20 min. Aside from the stretching component, each WU followed the exact procedure in both experimental sessions (TWU and DWU), consisting of the following given below. Participants started performing at least a 4–5 min of self-paced general WU consisting of low- to moderate-intensity (self-perceived) running including forward/backwards movements, sidestepping and general mobilization , followed by 10 minutes of a designated stretching routine (SS for TWU or DS for DWU) before completing 6–8 min of tennis-specific intensity activities . Following these stretching routines, participants performed 3 sets of ballistic exercises with a 15 s rest period between each set. Exercises included single hop jumps (5 repetitions), alternate leg bounds , service motion throws without a tennis ball (5 repetitions each arm) and short (2–3 m) accelerations and decelerations in different directions (3 repetitions forwards and 5 repetitions side to side).The rationale of using this WU structure  was to replicate the WU structure that is recommended by the ITF and usually performed by tennis players .Participants performed 6–8 min of DS  performing 3 sets, from low to high intensity, with a 15 s rest period between each set. Each exercise was performed in a controlled manner through a range of motion required in many sports .The SS routine consisted of exercises designed to stretch the plantar flexors , hip flexors (hamstrings), hip extensors , hip adductors, quadriceps, posterior shoulder, triceps, shoulder external, pectoralis, deltoid, biceps brachii, and forearm extensors and flexors. Exercises were selected based on previous literature , 7, 18 aA CMJ without arm swing was performed on a contact platform  according to Bosco et al. . During Each player performed 2 maximal CMJs interspersed with 45 s of passive recovery, and the best height for each was recorded.Owing to its good reproducibility, linear sprint tests ranging from 10 to 30 m are used as general measures of linear acceleration and speed . Time duA radar gun  was used to measure serve speed, following the methods previously described . Before For serve accuracy, players performed another 5 maximum effort serves with approximate the same spin (flat or slice) to the "advantage" service box. The accuracy scores were determined by counting the number of times the ball landed within a designated target perimeter. Target dimensions were designed considering similar methodologies, available resources, through discussion with coaches and athletes and preliminary trials . The tar2k) and coefficients of variation  were calculated for each measure using the method previously described by Hopkins [Before data collection, the intra and inter-session reliability of the jumping height, 20 m sprint time, serve speed and accuracy measures was determined using a test-retest design . Thus, t Hopkins .Means ± standard deviations (SD) were used to describe variables. Data normality and homoscedasticity were confirmed before inferential analysis through Kolmogorov-Smirnov and Levene’s test, respectively.Four factorial (WU intervention and time) repeated measures ANOVAs were used to analyse and compare TWU and DWU responses for each of the three testing moments . Each participant’s change score was expressed as a percentage of baseline score (post-warm-up) via analysis of log-transformed values, to reduce bias arising from non-uniformity of error. Errors of measurement and individual responses expressed as coefficients of variation were also estimated. In addition, the analysis determines the chances that the true effects were medium, substantial or trivial when a value for the smallest worthwhile change is entered. Thus, the intra and inter-session CV determined during the pilot study was considered the smallest substantial/worthwhile change for each of the variables for intra and inter-groups comparisons respectively. The experiment-wise type I error rate was set at p < 0.05 and protected by adjusting the critical p values for each RMANOVA using a Holms correction.The qualitative descriptors proposed by Batterham & Hopkins  were useExcellent intra- and inter-session reliability scores were demonstrated for all the performance measures analysed (ICC > 0.80 and CV < 5–10%) . The intEffects of TWU and DWU routines on CMJ, 20 m sprint time, serve speed and accuracy throughout the three different testing moments are presented in The primary findings of the present study showed that performing a DWU routine including active aerobic activities, DS and tennis-specific skills led to higher jumping heights, faster sprint times as well as superior serve performance compared with a TWU routine with SS followed by tennis-specific skills. In addition, the results also showed that the differences between interventions  appeared to be more evident immediately after the WU  and were gradually reducing their magnitude throughout the simulated match-play until disappearing at 60 min of the game. Although from a statistical point of view the magnitude of these differences could be considered small in almost all performance measures , these changes could be considered as relevant for tennis performance. Since margins between winning and losing in tennis are very small, fractions of a second in a short sprint prior to the ball hitting or loss of centimetres in jump height during a service may be essential components of tennis tasks and contribute to competent performances .Our findings are not consistent with the few previous studies that have investigated the acute effects of different WU modalities (i.e. TWU and DWU) on specific explosive qualities (sprint and jumping), showing that practice attempts of the required tasks or sports specific dynamic movements may offset some of the potential negative effects of SS when included prior to dynamic movements –9, 53. TAnother important finding of the current study is that the physical performance measures obtained by the DWU intervention were stable throughout the 60 min of simulated match-play (linear tendency), suggesting a transitory negative effect of the TWU routine on performance. As the current study design did not contemplate a control group, this last-mentioned suggestion should be considered as a hypothesis based on the previously reported literature results.Transitory negative effects of the SS on muscular performance have also been reported in previously studies. For example, Mizuno et al.  found thServe performance measures (speed and accuracy) showed the largest percentage reductions after the TWU routine  and at 30 min of match-play (3 and 4% respectively), compared to the DWU. These findings have very important implications regarding the design of effective WU strategies in tennis, as the serve has been considered the most stable and predictable measure of on-court tennis performance and therefore, essential to achieve high levels of performance . PresentTo the best of our knowledge this is the first study the time-interval of the negative effects of a TWU intervention on the physical performance of elite junior tennis players in a simulated match-play situation, and unfortunately, comparisons are not possible.Although this is the first study analysing the acute effects of two different WU modalities (TWU and DWU) on physical performance in elite junior players before, during and after 60 min of simulated match-play, some limitations should be noted. The study did not analyse the effects of the pre-participation WU designed for this study without the stretching component, we are not able to know if the DS component by itself could have had any positive effect on the physical performance. However, previous studies have reported significant improvements in jumping height, sprinting times and serve speed when a WU with a DS component was compared to a WU without any stretch component , 30, 31.In conclusion, the findings of the present study showed that the use of DS as a part of tennis-specific WU results in superior performance levels  before and during a 60 min simulated match-play, compared to the same WU routine with SS instead of DS in these elite junior male tennis players. In addition, the results found that these negative effects of using SS routines after a tennis-specific WU can last for almost 30 min of a simulated competitive situation.In young elite tennis players, a 15 min WU routine, combining a cardiovascular activation followed by DS and tennis-specific skills exercises, should be recommended in order to achieve high specific performance levels . It seems important to suggest that, especially at young ages, coaches and strength and conditioning specialists should educate the players in order to be able to distinguishing between the routine used for improving flexibility  and the one used as part of the WU process, targeting to activate the muscle groups involved in a specific performance task . Moreove


In plants, the vacuole builds up the cellular turgor and represents an important component in cellular responses to diverse stress stimuli. Rapid volume changes of cells, particularly of motor cells, like guard cells, are caused by variation of osmolytes and consequently of the water contents in the vacuole. Moreover, directed solute uptake into or release out of the large central vacuole allows adaptation of cytosolic metabolite levels according to the current physiological requirements and specific cellular demands. Therefore, solute passage across the vacuolar membrane, the tonoplast, has to be tightly regulated. Important principles in vacuolar transport regulation are changes of tonoplast transport protein abundances by differential expression of genes or changes of their activities, e.g., due to post-translational modification or by interacting proteins. Because vacuolar transport is in most cases driven by an electro-chemical gradient altered activities of tonoplast proton pumps significantly influence vacuolar transport capacities. Intense studies on individual tonoplast proteins but also unbiased system biological approaches have provided important insights into the regulation of vacuolar transport. This short review refers to selected examples of tonoplast proteins and their regulation, with special focus on protein phosphorylation. The vacuole represents a characteristic organelle of plant cells and formerly two types of vacuoles were distinguished: protein storage vacuoles and lytic vacuoles. However, this classification is rather artificial and several facts and recent data suggest that it is not as clear as previously assumed. E.g., in one cell, storage, and lytic vacuoles can exist in parallel and also their total number is quite variable and ranges from many small organelles to only one large central vacuole that can occupy up to 80% of the cell volume. Moreover, differentiation, and hence classification of vacuoles  becomes further complicated by the fact that vacuolar protein compositions and stored contents show broad variation not only during development or in different tissues but also in short-term response to different environmental conditions, stressors etc. Due to the broad spectrum of variations vacuoles can be considered as multifunctional organelles involved in diverse cellular processes and adapted to all stages of a plants life.Vacuoles represent cellular storages for primary metabolites, proteins, pigments, metal, and nonmetal ions, they allow sequestration of xenobiotics and other toxic compounds, and generally are an essential component for the constitution of the cellular turgor pressure. The tonoplast harbors various membrane proteins: proton pumps, channel proteins, ABC transporters, tonoplast intrinsic proteins (TIPs) and diverse solute carriers. The relative abundance of these proteins and their respective activities/regulation determine the specific function of plant vacuoles either in adjustment of cellular – mainly cytosolic – metabolite levels (homeostasis), in detoxification, in plant growth, and plant movement  or in plant adaptation to abiotic stress situations . During the past decades, the biochemical properties and physiological functions of several tonoplast proteins have been elucidated . FurtherThis review gives special attention to the latest knowledge about the regulation of tonoplast protein activities, particularly by phosphorylation, because these factors represent important control mechanisms in fast adaptation of specific functional properties.+/H+ antiporters and sugar transporters.To maintain vacuolar flexibility transport processes across the tonoplast have to be constantly controlled and altered. This can be achieved by modified expression of genes encoding tonoplast proteins. Corresponding studies have been carried out for example with genes encoding V-ATPase subunits . HoweverXenopus oocytes and phosphorylation of the heterologously expressed protein was demonstrated with the help of bovine protein kinase A  HvSTP1/2 of the Arabidopsis TMTs exhibit(s) 12 phosphorylated loop residues  from the TMTs . A charathe TMTs . For a lresidues . Five oforylated . After pArabidopsis leaf extract . Attached proteins were analyzed by mass spectrometry (in vivo and that it mediates TMT1 loop phosphorylation at least in vitro. Transport measurements with isolated Arabidopsis vacuoles either in presence or absence of recombinant VIK1 helped to clarify the impact of phosphorylation on TMT activity. Vacuolar glucose uptake was significantly stimulated by addition of ATP as phosphate donor and heterologously (Escherichia coli) expressed, purified VIK1. Furthermore, VIK1 t-DNA insertion lines phenocopy TMT loss-of-function mutants in important aspects: seedlings exhibit reduced growth and fresh weight in liquid media with high glucose concentrations  can be applied. However, it is sometimes more advisable to conduct a more goal-oriented approach. To identify proteins that might interact with the large hydrophilic TMT loop trometry . In totatrometry . Subsequtrations . The obsArabidopsis mutants lacking TMTs show lower glucose and fructose uptake into the vacuole and significantly reduced (vacuolar) glucose and fructose contents compared to wild type plants. The differences in sugar transport and contents of wild type and mutant plants become more pronounced when plants are exposed to cold temperatures, an abiotic stress situation inducing cellular sugar accumulation and vacuolar sequestration . Of course these findings are only a first step in understanding the regulation of TMT-type proteins and continuing efforts are required to explain how TMTs are incorporated in the complex network of sugar sensing and sugar partitioning of plant cells. The TMT interacting kinase VIK1 belongs to the RAF related group C of MAPKKKs and within this group VIK1 is a member of the subgroup C1. All six members of the C1 subgroup (VIK1 and VIK1-like 1–5) exhibit an ankyrin repeat motif that is missing in all other MAPKKKs. Generally, ankyrin repeats are known to be involved in protein–protein interactions , the protein kinase (SOS2) that regulates SOS1 activity, and the calcium binding protein (SOS3) that on the other hand regulates SOS2  pathway and comprises at least three proteins: the Nates SOS2 . Apart f AtNHX1; . Howeverscovered .In vivo interaction of SOS2 and V-ATPase subunits B1 and B2 were confirmed by yeast two-hybrid analyses (+/H+ antiporter (AtNHX1) into the vacuole. The data underline the central role of SOS2 as a hub to activate transport proteins that remove Na2+ from the cytosol (either to the apoplast or the vacuole) and to activate proteins that provide energy for these transport activities.Interestingly, it has also been reported that SOS2 interacts with the vacuolar ATPase  the vacuolar Cater CAX1 . This in2+ is one central element in signal transduction and most abiotic, biotic but also hormonal stimuli cause changes in free cytosolic concentrations of this ion  calmodulin, (2) calcineurin, and (3) calcium dependent protein kinases , use chemical energy of ATP or pyrophosphate to pump protons into the vacuolar lumen. Proton pumping activity causes considerable acidification of the vacuolar lumen and leads to establishment of a significant electrochemical gradient  that faces the cytosol and catalyzes ATP hydrolysis and a membrane spanning entity  that performs proton transport. Each multimeric entity is composed by a subset of proteins that can vary in its composition. In vérasion”) berries soften and start to accumulate massive amounts of glucose and fructose in the vacuole. Activity measurements and protein quantification revealed that both vacuolar proton pumps, V-PPase and V-ATPase, showed increased activity  in the vacuole. Cold acclimation was shown to increase abundance and hence activity of the V-ATPase resulting in higher vacuolar glucose and fructose uptake capacity , sensitivity to salt stress remains rather unaffected whereas nutrient storage is markedly impaired. Moreover, reduced transcript levels of vha-a1 resulted in a SOS-like phenotype. These results suggest that the TGN/EE is important for sodium removal from the cytosol and that the energy of the corresponding transport process is provided by the TGN/EE located V-ATPase  cytosolic sodium can be imported via the tonoplast Na AtNHX1; , (ii) tr AtNHX1; . These oV-ATPase .Because of its role in balancing cytosolic levels of major metabolites and ions, and because of its function in different organelles the activity and subcellular distribution of the V-ATPase has to be tightly regulated. This can happen on transcriptional, translational, and post-translational level. There is rising evidence that this important proton pump is not only regulated by gene expression, also post-translational modification seems to be a relevant mechanism. However, up to now our knowledge on this type of V-ATPase modification is still incomplete.AtWNK8; with no K (lysine)) was shown to bind and phosphorylate subunit C at multiple sites (2+-dependent protein kinase (CDPK) has been identified that activates V-ATPase and thereby stimulates vacuolar acidification. CDPK was shown to be involved in vacuolation in the endosperm but does not influence cytoplasmic Ca2+ increase or gene expression that is typically induced by gibberellic acid during endosperm reserve mobilization. Accordingly, CDPK is supposed to fulfill a specific function in tonoplast transport regulation in aleuron cells  and with the B1 subunit of the V-ATPase (VHA-B1). The corresponding proteins  form a complex that exclusively occurs in the nucleus. Moreover, no interaction with other V-ATPase subunits, not even with the relative homologous forms of VHA-B1 (VHA-B2 or VHA-B3) could be identified. This observation suggests that nuclear located interaction of HXK1, VHA-B1, and RPTB5 is highly specific (glucose insensitive (gin) phenotype comparable to that of HXK1 mutants . Accordingly, the nuclear complex of the three proteins might be physiologically relevant for glucose sensing and related signaling pathways. Furthermore, the function of VHA-B1 in glucose signaling seems to be independent of its role in V-ATPase function because other VHA mutants (vha-A9 and vha-E1) are lethal (Hexokinase1 (HXK1) fulfills diverse signaling functions particularly in regulation of gene expression and hence might be regarded as the most important glucose sensor of plants. In specific . Therefoe lethal  or absene lethal .Taken together these analyses provide evidence that the V-ATPase is regulated by diverse mechanisms and that at least one subunit of the complex has an additional, independent regulatory function.In vitro studies revealed that V-ATPases of plants as well as those from mammals and fungi can be inactivated by oxidizing agents  and that the activity can be restored by reducing agents. The subunit A contains three cysteine residues  that have been proposed to be the target for this redox modulation. Indirect evidence suggests that redox modulation of the V-ATPase also occurs in vivo because yeast mutants with impaired glutathione biosynthesis shows reduced proton pumping activity. In Arabidopsis in detail. Mutation of the three conserved cysteine residues of VHA-A to serine and complementation of a VHA-A null mutant with the modified VHA-A genes revealed that oxidative inhibition does not represent a primary regulatory mechanism in plant V-ATPases. Mutations of cysteine residues 256 and 535 to serine in VHA-A resulted in a fully complemented vha-a phenotype comparable to that of wild type plants. Solely, mutation of cysteine 279 to serine did not fully compensate the impaired growth phenotype. Even under conditions of a fully oxidized glutathione pool in root cells, corresponding vacuolar pH was totally unaffected in all complementation lines  whereas expression of other subunit genes seems to be rather tissue-independent (vha-a). There is also increasing evidence for V-ATPase regulation by post-translational modification (phosphorylation), by interaction with regulatory proteins  or enzymes generally known from primary metabolism . Other regulatory principles that have been described for V-ATPases from yeast, insects and mammals have not been observed yet or are of minor importance in plants (redox modulation). Different targeting of subunits can also be seen as a mechanism of regulation (vha-a1 versus vha-a2 and3). The detailed complexity of subunit targeting and assembly in different cellular membranes remains to be addressed in future studies. Finally, subunit B1 in complex with HXK1 and RPTB5 exhibits a regulatory function in nuclear gene expression that is apparently independent of the proton pumping activity of the holoenzyme.Tonoplast proteins and accordingly vacuolar transport processes are regulated by diverse mechanisms and by this become adapted to the specifically required function of the vacuole. Transcriptional regulation, increase or decrease in protein abundance and also post-translational modifications were discovered for diverse tonoplast proteins.in vitro activity measurements, mutant proteins, mutant plants lacking the respective kinase, etc.). Although progress has been made concerning phosphorylation of tonoplast proteins specific important details are missing. For example, interaction of the kinase VIK1 with the TMT protein, VIK1 mediated phosphorylation of the TMT loop and stimulation of TMT activity due to presence of VIK1 have been demonstrated, however, it is still unclear which specific TMT amino acid residue(s) represent the actual targets of VIK1 and which alterations of the biochemical properties are induced by specific modifications the TMTs. Interestingly, there is also evidence that TMT proteins are capable of sucrose transport, identified by patch clamping of isolated Arabidopsis vacuoles . Subsequently, the impact of the identified modification on the protein activity has to be clarified (e.g., by vacuoles , and it 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.