Back pain is a leading cause of global disability and is strongly associated with intervertebral disc (IVD) degeneration (IDD). Hallmarks of IDD include progressive cell loss and matrix degradation. The Akt signaling pathway regulates cellularity and matrix production in IVDs and its inactivation is known to contribute to a catabolic shift and increased cell loss via apoptosis. The PH domain leucine-rich repeat protein phosphatase (Phlpp1) directly regulates Akt signaling and therefore may play a role in regulating IDD, yet this has not been investigated. The aim of this study was to investigate if Phlpp1 has a role in Akt dysregulation during IDD. In human IVDs, Phlpp1 expression was positively correlated with IDD and the apoptosis marker cleaved Caspase-3, suggesting a key role of Phlpp1 in the progression of IDD. In mice, 3 days after IVD needle puncture injury, Phlpp1 knockout (KO) promoted Akt phosphorylation and cell proliferation, with less apoptosis. At 2 and 8 months after injury, Phlpp1 deficiency also had protective effects on IVD cellularity, matrix production, and collagen structure as measured with histological and immunohistochemical analyses. Specifically, Phlpp1-deletion resulted in enhanced nucleus pulposus matrix production and more chondrocytic cells at 2 months, and increased IVD height, nucleus pulposus cellularity, and extracellular matrix deposition 8 months after injury. In conclusion, Phlpp1 has a role in limiting cell survival and matrix degradation in IDD and research targeting its suppression could identify a potential therapeutic target for IDD. Today’s operative and non-operative treatments for IDD have low efficacy and the current focus on painful conditions has resulted in over-reliance on opioids and is a contributor to the current opioid crisis2, making the development of treatments to prevent IDD a priority.Back pain is a leading cause of global disability and is strongly associated with intervertebral disc (IVD) degeneration (IDD)6. IDD is biochemically characterized by apoptosis, IVD structural deterioration, and extracellular matrix (ECM) degradation including glycosaminoglycan (GAG) loss and increased catabolic enzymes11. It has been demonstrated that Akt signaling regulates apoptosis and ECM degradation in IVD cells14 and inactivation of Akt likely contributes to cell loss and poor healing capacity, which are known to accelerate IDD. Regulators of Akt signaling, such as phosphatase and tensin homolog (PTEN), protein phosphatase 2 A, and calcineurin15, can inhibit Akt activity by controlling its upstream signaling or by dephosphorylating Akt at Thr308 and/or Ser473.The causes for painful IDD are multifactorial and include risk factors such as heritability, lack of sport activities, complex loading, and trauma18. Phlpp1 is expressed in musculoskeletal tissues15 where it is involved in several conditions that are implicated with IDD, such as insulin resistance, obesity, and osteoarthritis22.The pleckstrin homology leucine-rich repeat protein phosphatase  more directly targets Akt1-3 and dephosphorylate the Ser473 activation site, thereby reducing its activity23, central nervous system24, and cartilage25, reduced Phlpp1 activation can promote healing. For example, Phlpp1 depletion in brain and heart tissues enhanced Akt activation and provided neuro-26 and cardioprotection27 after injury. In murine tibia, Phlpp1 depletion limited osteoarthritis progression by increasing the cellular content of articular cartilage after destabilization of the medial meniscal tibial ligament22. In contrast, inhibition of Phlpp1 decreased Akt phosphorylation in adult rat hippocampus17 and promoted neuronal apoptosis after spinal cord injury in mice28. However, Phlpp1 also acts as an important tumor suppressor by terminating Akt signaling, and thus induces apoptosis and suppresses tumorigenesis in a variety of cancers30. Taken together, although Phlpp1 might act via similar mechanisms in different tissues, the outcomes of Phlpp1 interactions are highly context-specific and its depletion can be both physiologic and pathophysiologic.In tissues with limited healing abilities, such as the heartThe role of Phlpp1 in IDD has not been established. Its tight regulation of Akt signaling suggests that it plays an essential role in Akt-regulated IVD cellularity and matrix homeostasis. The aim of this study was to determine whether Phlpp1 plays a key role in IDD progression in humans and mice. We hypothesized that Phlpp1 regulates cell proliferation, apoptosis, and matrix degradation in IDD by modulation of Akt phosphorylation. Utilizing human IVD tissues and in vivo Phlpp1 KO mouse IVD injury models, we (1) investigated the correlation of Phlpp1 expression with human IDD, and (2) mechanistically evaluated the role of Phlpp1 on IVD cellularity and matrix degradation in the progression of IDD by utilizing a needle puncture mouse model.R2 = 0.849; p < 0.001) and AF . In contrast, Phlpp1 expression was weakly correlated with aging in both NP  and AF  tissues  and AF  regions  and AF  tissues sp3 Fig. . A stronons Fig. . The proues Fig. , suggestTo investigate the role of Phlpp1 on Akt signaling during IVD injury, we performed needle punctures in tail IVDs of 4–5 month-old C57BL/6 J wild type (WT) and Phlpp1 KO mice and assessed Akt phosphorylation 3 days after injury Fig. . The sevThree days after needle puncture, AF cell proliferation was significantly increased in injured Phlpp1 KO IVDs compared to all other groups while only minor cell proliferation was observed in control Phlpp1 KO and WT mice, or in injured WT mice Fig. . Phlpp1-As expected, needle puncture injury significantly increased AF cell apoptosis in injured WT IVDs Fig. . In contTo assess the long-term effects of Phlpp1 in needle puncture induced injury, we examined the changes in disc height, a sensitive marker of IVD injury, 2 and 8 months after injury Fig. . We calcPicrosirius Red-Alcian Blue staining was performed to examine structural alterations in IVDs 2 and 8 months after needle puncture Fig. . Both CoIn injured IVDs, despite the severe loss of NP tissue 3 days after needle puncture, the NP region was filled with a collagen-rich ECM often containing chondrocyte-like cells at 2 months, suggesting an attempted healing response in both WT and Phlpp1 KO mice. However, the intense Picrosirius Red staining together with diminished Alcian Blue intensity suggested a loss of the GAG-rich matrix within the NP. Structurally, in both injured WT and Phlpp1 KO IVDs, the AF lamellae were bulged inwards and disorganized with a blurred AF-NP boundary Fig. .Eight months after needle puncture, structural defects accumulated further in injured WT IVDs, as demonstrated by pronounced cell loss in the NP and large fissures with fibrotic scar formation in both NP and AF tissues. In Phlpp1 KO mice, the collagenous matrix in the NP was maintained and the AF structure was not restored Fig. .33. The total histological score was significantly increased both in injured WT and Phlpp1 KO mice, and was not restored over time  imaging. In control IVDs of both WT and Phlpp1 KO mice, strong SHG signals were observed within AF tissues, whereas NP tissues had no or little SHG signal Fig. . Two monEight months after needle puncture injury, SHG intensities in AF tissues of WT and Phlpp1 KO mice were generally reduced when compared with the 2 month timepoint. Although control and injured AF tissues exhibited similar level of the SHG intensity in both genotypes, the SHG intensity in AF tissues was higher in Phlpp1 KO than WT mice at baseline. In the NP, enhanced SHG intensity was detected in Phlpp1 KO mice but not in WT mice. These findings demonstrated that Phlpp1 deficiency protected against the progression of degenerative changes in the IVDs after injury.To better understand the NP matrix composition of injured IVDs 2 and 8 months after injury, aggrecan content was assessed immunohistochemically. In control NP tissues of WT and Phlpp1 KO IVDs, aggrecan was deposited uniformly throughout the NP matrix except for the notochordal band. Needle puncture injury resulted in significantly decreased aggrecan content in NP tissues of both genotypes at 2 months Fig. . However35. In an acute response to injury, Phlpp1 depletion elevated Akt phosphorylation and cell proliferation while suppressing apoptosis. Over time, Phlpp1 KO promoted IVD healing from the puncture injury as indicated by improved DHI, increased NP cellularity, and matrix production 8 months after injury. Together, our data suggested that Phlpp1 is involved with the progression of IDD, and that it plays an important role in regulating cellularity and matrix accumulation in the IVD during healing of injuries via the Akt pathway.This study revealed an association between Phlpp1 expression and human IVD degeneration, and demonstrated that Phlpp1 depletion could prevent the progression of IDD after needle puncture injury in mice. In human IVDs, our data showed a strong and positive correlation of Phlpp1 with IDD and apoptosis, a known contributor of cell loss during IDD16. Aberrant expression of Phlpp1 has been reported in several diseases that depend on intact Akt signaling36, such as osteoarthritis22, diabetes37, or myocardial ischemia38. Dysregulated Akt signaling affects multiple downstream effectors involved in cell survival and proliferation39. Our data demonstrated that Phlpp1 depletion led to enhanced basal Akt phosphorylation in mice IVDs, which was further elevated in response to needle puncture injury where it increased cell proliferation and suppressed apoptosis. In vitro, enhanced Akt activity can attenuate NP cell apoptosis following mechanical overloading14, pro- inflammatory cytokine exposure40, or hyperosmotic stress41. In adipose and skeletal muscles and the heart of obese patients, impaired Akt activity induced by high levels of Phlpp1 limited glycogen synthesis20 and facilitated cardiomyocyte death27. Phlpp1 depletion in brain and heart tissues increased basal levels of Akt activation and cell survival facilitating neuroprotection and cardioprotection after injury, which were attenuated by Akt inhibition39. This broad literature highlighting increased cell survival with Phlpp1 depletion supports our data and together suggests a role of Phlpp1 in regulating IVD cell survival potentially via Akt signaling during IDD.Phlpp1 selectively inhibits cell survival throughout the suppression Akt signaling by dephosphorylating Akt2 and Akt3 at Ser47344 and resulted in the formation of large fissures and fibrotic tissue accumulation within the NP and AF regions 2 month after injury42. Although the acute response 3 days after injury did not resemble natural IDD in humans, the injury induced degenerative changes with further diminished tissue integrity, severe matrix breakdown, and cleft formation 8 months after injury, which simulated the natural progression of IDD.Consistent with previous studies, severe needle puncture injuries caused immediate NP herniation45. In line with our findings, in a post-traumatic osteoarthritis mouse model, Phlpp1 depletion protected against the progression of osteoarthritis by increasing cellular content and attenuating cartilage degradation22. In human IDD, unregulated proliferative cells express a high level of catabolic enzymes MMPs and ADAMTs, leading to accumulation of degradative matrix, which attenuates protein renewal and resistance to compression47. Cleaved aggrecan fragments in degenerated IVDs promote further degeneration by lowering the osmotic pressure, ultimately leading to a loss of IVD height48. In our model, Phlpp1 KO prevented the progression of DHI loss and IVD matrix breakdown, likely by accumulation of aggrecan 8 months after injury compared with WT mice. However, the SHG intensity within the AF was generally decreased in 12 months old WT and Phlpp1 KO mice. A recent study by Xiao et al.49 demonstrated a strong correlation between AF collagen damage and age by using a collagen-hybridizing peptide (which integrates into damaged collagen) and observed a significant increase in damaged collagen between 6–8 months and 10–12 months old mice. Given that the damage to the secondary and tertiary protein structure of collagen is also detectable by decreased SHG intensity50, their findings supported the lower SHG intensity detected in the AF tissues of 12 months old mice relative to 6 months group. In addition, SHG imaging demonstrated increased collagenous matrix in the NP of injured Phlpp1 KO mice, indicating that Phlpp1 depletion mitigated the catabolic shift.Annular needle puncture injuries can cause an imbalance between catabolic and anabolic activity in IVDs by upregulating MMP3, and downregulating BMP-2 and -751. Using a conditional knockout model would allow targeting only a single marker, and to our knowledge no specific markers exist that are uniquely expressed in both NP, AF, and endplate cells, without being present in surrounding tissues. One limitation of using mouse IVDs to study IVD degeneration is that mice maintain a subpopulation of notochordal cells throughout life while human IVDs lose their notochordal cells during adolescence. Yet, the needle puncture resulted in complete herniation of the notochordal cell rich NP 3 days after injury. Although we are not able to study the direct effect of Phlpp1 depletion on NP cells, the positive correlation of Phlpp1 with IVD degeneration and apoptosis strongly suggests a role of Phlpp1 in human NP and AF during IDD, thus future research is warranted to verify the protective role of Phlpp1 deficiency in human NP cell culture studies.For this study we used a global Phlpp1 KO mouse model. We cannot exclude that the systemic depletion of Phlpp1 affected the IVD healing response in Phlpp1 KO mice by altering surrounding tissues such as tendons, ligaments, or muscles. However, we did not observe any morphological differences when comparing mature WT and Phlpp1 KO vertebral structures, suggesting that global Phlpp1-deletion had no or only minimal effects on spinal structures. The use of a global knockout allowed us to assess the effect of Phlpp1 deficiency on the entire IVD, which is composed of three distinct tissues with NP cells originating from the notochord, whereas AF and endplate cells originate from the sclerotome53 and the pro-apoptotic kinase Mst155, which likely explains the protective effect of Phlpp1 depletion on cell proliferation but less pronounced effects on Akt phosphorylation. Phlpp1 deficiency also activates the negative feedback regulation of the Akt pathway, where PTEN, a lipid phosphatase, participates in the feedback loop by dephosphorylating PI3K to balance the level of phosphorylated Akt49. PTEN is an another phosphatase that has several off-target effects. One of its important functions is participating in the repair and response of DNA damage56. PTEN-deficient neurons exhibit seizures, autism-related disorders, and are highly prone to cancer, which do not emerge in Phlpp1-deficient mice29.Besides Akt, Phlpp1 also directly controls other cell survival signaling pathways, including PKC and p70 S6 kinase58. As Phlpp1 expression was increased with degeneration grades in human IVDs, local administration of Phlpp inhibitors via suitable biomaterials in herniated or mildly and moderately degenerated IVDs would alleviate the continued deterioration of IVD matrix and enhance cell survival via AKT signaling. Contrary to its pathological effects, deregulated activation of PI3K/AKT signaling is also known to play a role in tumorigenesis19 and care has to be taken when developing drugs for enhancing cell proliferation and IVD metabolism. The specificity of Phlpp1 on regulating anabolic and apoptotic signaling pathways, which are essential for IVD healing, makes it a promising target for preventing the progression of IDD and its deficiency has a potential to improve cell survival and matrix production in IVDs.In summary, Phlpp1 was associated with increased human IVD degeneration and increased cleavage of caspase-3. To mechanistically identify the role of Phlpp1, a global Phlpp1 KO mouse model was used. Depletion of Phlpp1 activated Akt signaling, enhanced cell proliferation, and suppressed apoptosis, which likely promoted increased cellularity and cartilaginous matrix deposition in the NP region, preventing further progression of IDD. Small molecule Phlpp inhibitors have been synthesized and confirmed to improve cell survival and promote matrix production36 prior to fixation in zinc formalin  and embedding in methyl-methacrylate .Human lumbar IVDs (Thompson grade 1–5) from autopsy were isolated from spinal segments . All animal protocols were approved by the Mount Sinai Institutional Animal Care and Use Committee (protocol# 2017-0018). Phlpp1 KO mice were obtained from Alexandra Newton’s lab at the University of California, San Diego.n = 24) and C57BL/6 J  mice. Needle punctures are an accepted model to simulate the progression of IDD in vivo44. IVD injuries were performed under general anesthesia (2% isoflurane in oxygen) and sterile conditions. Caudal IVDs  were identified by palpation and exposed via a 2–4 mm dorsolateral incision. After confirming the location of IVD using a M60 microscope , full AF needle punctures were created using 26 G syringe needles to a depth of 50% of dorsal-ventral width. The needle diameter  was chosen based on a previous study44. Incisions were sutured  and mice were closely monitored to ensure the absence of intraoperative complications and allowed free activity in their cages with ad libitum access to food and water. Mice were killed via carbon dioxide inhalation at 3 days, 2 months, and 8 months after surgery.To investigate the effect of Phlpp1 KO on IVD healing, caudal needle puncture injuries were induced in 4–5 months old Phlpp1 KO  and protein-blocking . IVD sections were incubated with anti Phlpp1 antibody  for 1 h at room temperature, followed by incubation with an alkaline phosphatase-conjugated secondary antibody . Samples were then incubated with a red chromogenic staining solution , counterstained in toluidine blue, and mounted . To detect clCasp3, specimens were incubated with anti clCasp3 antibody  followed by incubation with a horseradish peroxidase-conjugated secondary antibody . Samples were then incubated with a diaminobenzidine-based horseradish peroxidase substrate  to visualize clCasp3 expression. For double staining with Phlpp1, samples were incubated with blocking solution, stained with anti Phlpp1 antibody, and followed the same steps as described above. Normal rabbit serum  served as a negative control. Samples stained with anti Phlpp1/negative control or anti clCasp3/negative control served as controls for nonspecific double staining. Samples were imaged under bright-field (Phlpp1) or differential interference contrast (Phlpp1/clCasp3 co-expression) microscopy with standardized exposure times .Decalcified and methyl-methacrylate embedded human lumbar IVDs were assessed for expression of Phlpp1 and clCasp3. Five-μm thick mid- sagittal sections sections were de-plasticized in a series of xylene, and 2-ethoxyethanol60. Immunohistochemistry was performed for pAkt1  and aggrecan . For aggrecan, antigen retrieval was performed by applying 0.8% hyaluronidase for 1 h at 37 °C. Following 30 min incubation in blocking solution, samples were incubated for 1 h at room temperature with an anti-aggrecan antibody . Next, samples were incubated with the secondary, alkaline phosphatase-conjugated secondary antibody  and visualized using alkaline phosphatase substrate , counterstained with Mayer’s hematoxylin, and mounted. For pAkt1 staining, samples were incubated for 30 min in blocking solution and incubated with anti pAkt1 antibody  for 1 h at room temperature, followed by incubation with the secondary horseradish peroxidase-conjugated antibody . pAkt staining was visualized using diaminobenzidine-based horseradish peroxidase substrate , counterstained with toluidine blue, and mounted. Normal rabbit serum  served as negative control for pAkt and aggrecan staining. All Samples were imaged using bright-field microscopy  with standardized exposure times. PAkt immunopositive cells were identified in the AF only (owing to complete NP herniation at day 3) and aggrecan-immunopositive areas were identified in the NP region by calculating the percentage of stained NP tissues, which accumulated in the NP region 2 and 8 months after injury. The immunopositive area and total NP regions were identified by two independent observers who were blinded to experimental groups.Decalcified IVD-vertebrae segments were embedded in paraffin and 5-μm-thick mid-sagittal sections were used for histology and immunohistochemistry. IVD morphology was visualized by Picrosirius Red-Alcian Blue staining (PRAB)n = 7, injured WT n = 6, control Phlpp1 KO n = 10, injured Phlpp1 KO n = 8; for 8 months: n = 5 per group) were semi-quantitatively graded within eight parameters for signs of degeneration, based on a scoring system published by Tam et al.33. One section per IVD was used for analysis. Scores within five categories were added together for a final score out of 14. All sections were examined by two researchers blinded to the experimental groups, and then averaged for analysis.IVD sections of PRAB staining were used to score the degree of degeneration based on histological appearances of the IVD. Sections  were used and the NP and AF areas were used for cell counting using ImageJ. Cell numbers from two hematoxylin counterstained sections were averaged. All sections were examined by two researchers blinded to the experimental groups.Hematoxylin stained mid-sagittal 5-μm thick sections of PRAB staining and immunohistochemistry for aggrecan were used to quantify the cell number in the NP and AF of injured IVDs. Two hematoxylin counterstained sections (one from PRAB staining and one from aggrecan immunostaining) per IVD (2 months: n = 7 per group) by EdU assay according to manufacturer’s instructions . In brief: EdU  was injected subcutaneously 24 h before sacrifice. Histological IVD-vertebrae sections were stained using an EdU assay kit . One section per IVD was used for analysis. Proliferation was assessed 3 days after injury because this early timepoint should approximately correspond with the end of the initial acute inflammatory response to needle puncture, and the start of the proliferative phase of wound healing.To determine the acute response to needle puncture injury, cell proliferation was assessed day 3 post injury (n = 4 per group) were washed with PBS then permeabilized using 0.5% Triton-X/PBS (PBST) for 15 min to detect cell death in situ  according to the manufacturer’s instructions. A positive control section was treated with DNase 1  for 30 min at room temperature and a negative control section was processed by depriving of terminal transferase enzyme. One section per IVD was used for analysis. Images were captured using a Zeiss  microscope at × 20 magnification and number of TUNEL-positive cells were counted using the ImageJ interface for selected IVD regions. All sections were examined by two researchers blinded to the experimental groups, and then averaged for analysis.Deparaffinized IVD sections . One section per IVD was used for analysis. Tissue sections were excited with a two photon tunable Coherent Chameleon Vision II laser at 910 nm. The SHG emission was collected in the backward direction using the dedicated Olympus WLPLN water-immersive × 25 objective with a numerical aperture of 1.05. This emission was recorded by a photomultiplier tube (PMT) at 440 ± 10 nm to capture the SHG signal at 455 nm. All parameters  were held constant between 2 and 8 months timepoints to allow comparison of image intensity within each timepoint. To allow for comparison between 2 and 8 months timpoints, the 8 months SHG images were pre-processed to the intensity levels of the 2 months timepoint using a conversion factor determined experimentally by scanning a standard tissue section with the two sets of imaging parameters. All image processing and quantification was performed in Fiji . Maximum intensity z-projection of 10–12 optical sections was performed on mosaic images. The background was subtracted using a 50-pixel rolling ball radius. The posterior AF was manually contoured and the mean grey value was measured to quantify the SHG intensity of the posterior AF and the central NP region.Unstained paraffin-embedded slides were deparaffinized and sealed to a coverslip with acrylic mount media before imaging and imaged with an Olympus FV1000 MPE laser-scanning microscope  at the Icahn School of Medicine at Mount Sinai Microscopy Core  and 8 months  post surgery. Immediately after sacrifice, mice tails were imaged using a high-resolution radiographic system . IVD heights and lengths of the adjacent vertebrae were measured using ImageJ. DHI was determined by normalizing the IVD height to the lengths of the adjacent vertebrae using the formula: DHI = 2 × (D + E + F)/(A + B + C + G + H + I) .DHI was determined 2 months  were used to assess the effect of injury and genotype. Significant differences between groups were assessed by Bonferroni post hoc testing. Post hoc testing was done to compare injury versus control within each genotype. Differences in healing responses for WT versus Phlpp1 KO were identified from significant interactions in the two-way ANOVA. Power analysis was performed using G*POWER to determine the sample size . Statistical analyses were performed using Graphpad Prism7 . A


Acute myeloid leukemia (AML) is a genetically heterogeneous malignancy for which treatment options have been largely limited to cytotoxic chemotherapy for the past four decades. Next-generation sequencing and other approaches have identified a spectrum of genomic and epigenomic alterations that contribute to AML initiation and maintenance. The key role of epigenetic modifiers and the reversibility of epigenetic changes have paved the way for evaluation of a new set of drug targets, and facilitated the design of novel candidate treatment strategies. More recently, seven new targeted therapies have been FDA-approved demonstrating successful implementation of the past decades' research. In this review, we will summarize the most recent advances in targeted therapeutics designed for a focused group of key epigenetic regulators in AML, outline their mechanism of action and their current status in clinical development. Furthermore, we will discuss promising new approaches for epigenetic targeted treatment in AML which are currently being tested in pre-clinical trials. Epigenetic regulation of the state of a cell involves various dynamic and reversible post-translational modifications of DNA and histone proteins. These modifications in their totality regulate the accessibility of DNA for the transcription machinery, thereby determining which specific genomic loci are transcriptionally active or repressed . The besFLT3 and KIT), transcription factors (such as CEBPA and RUNX1), chromatin modifiers (such as MLL and ASXL1) or direct and indirect regulators of DNA methylation  , The Cancer Genome Atlas TCGA, 2006), the International Cancer Genome Consortium ICGC, 2008), and the European Community initiative BLUEPRINT (2011) have been critical in defining the regulatory networks in different normal hematopoietic cell types as well as how they are deregulated in myeloid malignancies 003, The . The key, the Intnd TET2) , 8. Alth, and the+ AML alone or in combination with conventional chemotherapy; the multi-kinase inhibitor midostaurin for newly diagnosed FLT3-mutated AML in combination with conventional chemotherapy; and the FLT3, AXL, and ALK inhibitor gilteritinib for relapsed or refractory FLT3-mutated AML  and arsenic trioxide (ATO) for treatment of acute promyelocytic leukemia  and the ated AML –19. IDH1ated AML . As a geated AML  and so rWhile these new developments in FDA approval are welcome, there remains much to do to improve the outcome of patients with myeloid malignancies. In this perspective, we will discuss a discrete set of candidate epigenetic therapeutic targets currently under evaluation in AML: the lysine demethylase LSD1, the protein methyltransferases EZH2, DOT1L, and PRMT5, and the BET bromodomain proteins. We will describe the importance of these transcriptional activators and repressors in different AML subtypes as well as their targeting potential, possible limitations and potential toxicities. We will summarize their current status in clinical development. For detailed review of other equally important targets, such as DNMTs and HDACs the reader is referred to recent comprehensive reviews –22. FinaHistone methylation and demethylation are tightly regulated, dynamic processes that regulate transcriptional activation or repression depending on the location of the modification. Methylation is generated by specific histone methyltransferases (HMTs), such as MLL, DOT1L, and EZH2. As for other histone modifications, methylation can be reversed by two classes of demethylases (KDM): the larger Jumonji domain family and the smaller lysine-specific demethylase (LSD) family.LSD1/KDM1A is a flavin-adenine dinucleotide (FAD)-dependent histone demethylase , 24 withMLL gene translocations  and iadademstat , have been evaluated in phase I trials in patients with relapsed or refractory AML . While tocations , 38. PreEZH2 is the catalytic subunit of Polycomb Repressive Complex 2 (PRC2) which is responsible for maintaining transcriptional repression of its target genes through tri-methylation of H3 K27 , 40 Fig. This hiMLL-rearranged AML, the dual EZH1–EZH2 inhibitor DS3201b has entered phase 1 as monotherapy in patients with refractory acute leukemia (NCT03110354)  where S-adenosyl methionine-competitive inhibitors, such as GSK2816126, CPI1205, and the most promising, tazemetostat (EPZ-6438), have been evaluated. Another is MAK683 which is an EED-binding complex disrupter under investigation in refractory lymphoma and solid malignancies (NCT02900651). Phase I data for tazemetostat demonstrated an acceptable safety profile and some objective responses (NCT01897571) resulting in initiation of a number of follow on single agent and combination studies . On the basis of pre-clinical studies demonstrating the functional importance of PRC2 in 3110354) . HoweverMLL (KMT2A) occurs in around 5% of AML cases, predominantly resulting in an MLL-AF9 fusion gene, although other partner genes occur less frequently .The S-adenosyl methionine-competitive DOT1L inhibitor pinometostat (EPZ-5676) displays great specificity for DOT1L over other histone methyltransferases –63. Prec in vivo . Phase Iompleted   family of proteins, and was identified as a potential cancer therapeutic based on results of a genome-wide shRNA screening in MLL-dependent AML cells , 89. BRDpression . Geneticenhancer , but alsex vivo patient samples or mouse models, in particular in specific subtypes with MLL rearrangement, or those with mutations in NPM1, FLT3 or IDH2, or EVI1 overexpression  and histone deacetylases (HDACs) catalyze the dynamic and reversible acetylation of histone and non-histone proteins, and are involved in major epigenetic regulatory mechanism of gene transcription  in normaMOZ or MLL in leukemia  has been demonstrated to have potent anti-proliferative and pro-differentiation activity in AML cell lines and primary patient samples  . MEN1 seMEIS1  (Figure MEIS1 . InteresMEIS1 , genetic(in vivo , highlig(in vivo , 116, whHOXA and MEIS gene expression, thereby releasing the differentiation block associated with MLL-rearranged leukemia (MLL translocations (in vivo is still an important hurdle to overcome.Several small molecules and tool compounds targeting the interactions of components of the MLL complex have been recently developed. Inhibitors targeting MEN1-MLL have been shown to reverse leukemia –121. Simocations  or CEBPAocations . HoweverEpigenetic regulators are central players in the initiation and maintenance of hematopoietic malignancies, an observation which has resulted in myriad opportunities for development of targeted therapies. In particular, where epigenetic mechanisms are specifically disordered in malignant but not normal blood cells, there exists the potential for a significant therapeutic window. Along these lines, there has been significant progress in understanding the role of epigenetic modifications and their modifiers in cancer in general and in AML in particular. The discovery and development of small-molecule inhibitors targeting certain epigenetic regulators has already led to opportunities for clinical trial evaluation and potential patient benefit; impending trial results will inform on efficacy and safety. While the evaluation of many of these compounds for their single agent activity is an essential first step, it will be critical to test efficacy in combination with either standard-of-care chemotherapies or novel therapeutics, to determine their optimal role in the treatment of leukemia. In time, personalization of therapeutic regimens according to patient cytogenetics and molecular mutations may become of essential importance. A final point is that the interdependence of cancer epigenetics and immunological responses has to be taken in consideration. In the setting of leukemia, this includes the therapeutic modality of allogeneic stem cell transplantation. It is clear that epigenetic therapies can induce cellular responses in tumor cells that interact with the immune system and which may contribute to their efficacy; for example, effects of adoptive immunotherapies and immune checkpoint inhibitors can be potentiated by epigenetic therapies . As suchBW and TS conceived and wrote 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.


NAADP-induced EL Ca2+ signals may be amplified by the endoplasmic reticulum (ER) through the Ca2+-induced Ca2+ release mechanism (CICR). Herein, we aimed at assessing for the first time the role of EL Ca2+ signaling in primary cultures of human metastatic colorectal carcinoma (mCRC) by exploiting Ca2+ imaging and molecular biology techniques. The lysosomotropic agent, Gly-Phe β-naphthylamide (GPN), and nigericin, which dissipates the ΔpH which drives Ca2+ refilling of acidic organelles, caused massive Ca2+ release in the presence of a functional inositol-1,4,5-trisphosphate (InsP3)-sensitive ER Ca2+ store. Liposomal delivery of NAADP induced a transient Ca2+ release that was reduced by GPN and NED-19, a selective TPC antagonist. Pharmacological and genetic manipulations revealed that the Ca2+ response to NAADP was triggered by TPC1, the most expressed TPC isoform in mCRC cells, and required ER-embedded InsP3 receptors. Finally, NED-19 and genetic silencing of TPC1 reduced fetal calf serum-induced Ca2+ signals, proliferation, and extracellular signal-regulated kinase and Akt phoshorylation in mCRC cells. These data demonstrate that NAADP-gated TPC1 could be regarded as a novel target for alternative therapies to treat mCRC.Nicotinic acid adenine dinucleotide phosphate (NAADP) gates two-pore channels 1 and 2 (TPC1 and TPC2) to elicit endo-lysosomal (EL) Ca Acidic EL vesicles, including endosomes and lysosomes, provide an additional Ca2+ reservoir that is recruited by extracellular ligands through the generation of the novel Ca2+-releasing second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP) i in the absence of extracellular Ca2+ (0Ca2+)  A, which al store .2+ refilling [2+]i under 0Ca2+ conditions  activity, which prevents Ca2+ sequestration into ER lumen and results in depletion of the ER Ca2+ pool. We have recently shown that pretreating mCRC cells with 30 μM CPA for 30 min under 0Ca2+ conditions is sufficient to deplete the InsP3-sensitive ER Ca2+ store in mCRC cells i. NAADP is therefore unlikely to evoke extracellular Ca2+ entry in mCRC cells. Control experiments revealed that liposomal formulations devoid of NAADP did not cause any Ca2+ signal (2+ response to NAADP (1:20) was impaired by disruption of acidic stores with GPN  (p < 0.05) reduced both the percentage of responding cells  B, which ng cells C and theresponse D. n = 4) higher than TPC2  reduced by silencing TPC1 with a selective small interfering RNA (siRNA) (p < 0.05) reduced both the percentage of mCRC cells responding to NAADP (1:20)  B, which P (1:20) C and the upon depletion of the ER Ca2+ store with CPA  under 0Ca2+ conditions i. We finally illustrated that NAADP-dependent TPC1-mediated Ca2+ signals underlie FBS-induced endogenous Ca2+ release and proliferation in mCRC cells. These data endorse the view that TPCs may provide alternative targets to design novel anticancer therapies and that the therapeutic outcome of their inhibition should be investigated in vivo. Herein, we demonstrated for the first time that a functional lysosomal Ca2+]i? Second, is the lysosomal Ca2+ store functionally coupled to the main ER Ca2+ store, as observed in normal cell types? Third, is NAADP per se able to mobilize lysosomal Ca2+ through TPCs? Herein, we first demonstrated that a robust endogenous Ca2+ release could be evoked by challenging mCRC cells with two structurally and mechanistically unrelated agents which target the lysosomal Ca2+ content. Accordingly, both the lysosomotropic compound GPN and the ionophore nigericin induced a transient increase in [Ca2+]i under 0Ca2+ conditions that was associated to the loss of Lysotracker Red fluorescence. These findings strongly suggested that an acidic Ca2+ store was present in mCRC cells and could be depleted to elevate intracellular Ca2+ levels. Acidic vesicles may establish quasi-synaptic MCSs with the ER, which confers them with the unique role of triggering physiologically relevant Ca2+ signals by inducing ER-dependent Ca2+ release via CICR i evoked by lysosomal Ca2+ release also in mCRC cells. Notably, a recent mathematical model confirmed that local lysosomal Ca2+ leak may be amplified into a regenerative Ca2+ wave by juxtaposed ER-resident InsP3Rs i. NAADP induced a transient Ca2+ signal that was associated to endogenous Ca2+ release, but not to extracellular Ca2+ entry. To the best of our knowledge, this was the first demonstration that NAADP per se was able to increase the [Ca2+]i in a cancer cell line other than cervical cancer HeLa cells i.Subsequently, we used a liposomal preparation to load mCRC cells with the membrane-impermeant NAADP and examine the following increase in i was monitored by measuring, for each ROI, the ratio of the mean fluorescence emitted at 510 nm when exciting alternatively at 340 and 380 nm (shortly termed “ratio”). An increase in [Ca2+]i causes an increase in the ratio [mCRC cells were loaded with 4 µM fura-2 acetoxymethyl ester  in PSS for 30 min at 37 °C and 5% COshown in . After whe ratio . Ratio mTPCN1 and TPCN2, and for RyR1, RyR2 and RyR3 .Total RNA was extracted from mCRC cells using the QIAzol Lysis Reagent . Single cDNA was synthesized from RNA (1 μg) using random hexamers and M-MLV Reverse Transcriptase . Reverse transcription was always performed in the presence or absence (negative control) of the reverse transcriptase enzyme. qRT-PCR was performed in triplicate using 1 μg cDNA and specific primers (intron-spanning primers) for and RyR3 . Brieflyg for 10 min. The cell pellets were resuspended with RIPA buffer  containing 0.1 mg/mL PMSF, Protease Inhibitor Cocktails  and Phosphatase Inhibitor (1 mM Sodium Orthovanadate). The homogenates were solubilized in Laemmli buffer [Cells were diluted in PBS and centrifuged 1000× i buffer  and 30 µProtein contents of all the samples were determined by the Bradford’s  method usiRNA targeting TPC1 was purchased by Sigma-Aldrich Inc. MISSION esiRNA . Scrambled siRNA was used as negative control. Briefly, once the monolayer cells had reached 50% confluency, the medium was removed and the cells were added with Opti-MEM I reduced serum medium without antibiotics . siRNAs  were diluted in Opti-MEM I reduced serum medium and mixed with Lipofectamine™ RNAiMAX transfection reagent  pre-diluted in Opti-MEM), according to the manufacturer’s instructions. After 20 min incubation at room temperature, the mixes were added to the cells and incubated at 37 °C for 5 h. Transfection mixes were then completely removed and fresh culture media was added. The effectiveness of silencing was determined by immunoblotting see  and sileLysosomal compartment was visualized in vital cells by incubating the cultures in the presence of LysoTraker Red, as described in . Briefly2+ release in response to each agonist (NAADP and FBS) or drug (GPN and nigericin) was measured as the difference between the ratio at the peak of intracellular Ca2+ mobilization and the mean ratio of 1 min baseline before the peak. Pooled data are given as mean ± SE and statistical significance (p < 0.05) was evaluated by the Student’s t-test for unpaired observations. Data relative to Ca2+ signals are presented as mean ± SE, while the number of cells analyzed is indicated in the corresponding bar histograms.All the data have been collected from mCRC cells deriving from at least three coverslips from three independent experiments. The amplitude of intracellular CaFura-2/AM and Lysotracker Red were obtained from Molecular Probes . All the chemicals were of analytical grade and obtained from Sigma Chemical Co. .2+ store is present and functionally coupled to ER-dependent Ca2+ release through InsP3Rs in mCRC cells. NAADP stimulates TPC1 to mobilize lysosomal Ca2+ and mediate 20% FBS-induced mCRC proliferation. TPC1 stimulates proliferation by recruiting the Ca2+-dependent ERK and PI3K/Akt phoshorylation cascades. These data extend to cancer cells previous findings on normal cells and lend further support to the notion that NAADP-induced TPC activation could be targeted to eradicate secondary metastasis in cancer patients.In conclusion, this study demonstrated for the first time that a functional lysosomal Ca


T-type Ca2+ channels are particularly overexpressed in various cancers. Because of their activation profile at membrane potentials close to rest and the generation of a window current, T-type Ca2+ channels may regulate a variety of Ca2+-dependent cellular processes, including cell proliferation, survival, and differentiation. The expression of T-type Ca2+ channels is of special interest as a target for therapeutic interventions.Although voltage-activated Ca T-. T-2+ is tissues . Voltageynthesis . OscillaG1 phase . Ca2+/call cycle . InhibitG2 phase ,42,48. Iblastoma ,88.2+ channels can also regulate cell migration, which may have important implications in the development of metastases. In the fibrosarcoma cell line HT1080, mibefradil causes a concentration-dependent inhibition of cell motility and invasion [2+ channels in glioblastoma cells is altered by endostatin, a C-terminal proteolytic fragment of collagen. Endostatin inhibits T-type Ca2+ channel function, resulting in a significant reduction in cell migration [Expression of T-type Cainvasion . Similarinvasion . The proigration .2+ channels support malignant growth through cellular survival. Autophagy is a process that helps to maintain cellular homeostasis. Through catabolism, proteins and damaged organelles can be degraded and recycled [v3.1 and/or Cav3.2 T-type Ca2+ channels cause autophagy impairment in melanoma cells (2+ channel function is inhibited in M16 or JG melanoma cancer cells [2+ channels can alter cell proliferation by promoting apoptosis. Many different cell lines undergo apoptosis when T-type Ca2+ channels are inhibited [v3.1 induced apoptosis in MCF-7 cells [T-type Carecycled . In cancrecycled . Pharmacma cells . The exper cells . This leer cells . As discnhibited ,42,43,45-7 cells .2+ channels have been implicated in the progression of various cancers. Ca2+ influx via T-type Ca2+ channels can activate various signaling pathways. Ca2+ activates calmodulin, which in turn controls the expression of many different CDKs that can affect cell cycle regulation and proliferation. Identifying T-type Ca2+ channel expression in tumors and characterizing their function may introduce additional treatment alternatives, particularly for patients who are unresponsive to standard therapies. Pharmacological inhibitors of T-type Ca2+ channels can induce apoptosis and inhibit proliferation in certain cancer cell lines and also increase sensitivity to certain traditional chemotherapy agents. Thus, T-type Ca2+ channels have the potential to become a promising therapeutic target in certain cancers.T-type Ca


DNA double strand breaks (DSB) play a pivotal role for cellular damage, which is a hazard encountered in toxicology and radiation protection, but also exploited e.g. in eradicating tumors in radiation therapy. It is still debated whether and in how far clustering of such DNA lesions leads to an enhanced severity of induced damage. Here we investigate - using focused spots of ionizing radiation as damaging agent - the spatial extension of DNA lesion patterns causing cell inactivation. We find that clustering of DNA damage on both the nm and µm scale leads to enhanced inactivation compared to more homogeneous lesion distributions. A biophysical model interprets these observations in terms of enhanced DSB production and DSB interaction, respectively. We decompose the overall effects quantitatively into contributions from these lesion formation processes, concluding that both processes coexist and need to be considered for determining the resulting damage on the cellular level. DSB are frequently induced by reactive oxygen species or other oxidative stress, by interaction with chemicals5 or radiation6, by replication stress or are even stimulated by cellular processes within certain phases and types of eukaryotic cell division like meiosis7. DSB stimulated by these processes are usually repaired with high fidelity by various repair pathways. This is also valid for DSB induced by ionizing radiation2, but there a relatively larger fraction remains unrepaired or gets misrepaired leading to e.g. chromosomal aberrations or gene mutations4. This suggested the relevance of complex damage, where in general DSB interaction with further, proximate damage is meant to be a substantial contribution for cell inactivation. The interaction partner could be in some basepairs distance, but also possible DSB-DSB interaction within a µm range is discussed8.Among DNA lesions, double strand breaks (DSB) formed by two single strand breaks (SSB) in close proximity on the scale of some nanometers are the key elementary lesions for cell inactivation10 which is usually attributed to the highly localized energy deposition along their path: There, secondary electrons are produced at a high rate, carry the energy outwards and give rise to the formation of a corridor of high ionization density - the so-called track structure - whose lateral dose profile provides very high local doses of up to about 107 Gy. Such high ionization densities cause a larger number of induced DSB per Gy organized along tracks11, cause ‘dirty’ DNA ends at the DSB, and facilitate the interaction of neighbored DSB as they are induced spatially correlated. Therefore the energy loss of radiation per path length, also expressed as linear energy transfer (LET), is an important factor parameterizing the resulting biologic effect. In contrast to such high-LET radiation, for low LET ion radiation, as e.g. for high energetic protons, local doses are moderate. Thus DNA lesions are distributed much more homogeneous, similar to DNA lesions induced by X-ray irradiation covering more homogeneously the exposed volumes. It is thus commonly accepted that the lesions induced by high LET irradiation are much harder to repair than clean ends or individual, well separated DSB, resulting in a larger number of e.g. chromosomal aberrations6.Considering radiation as a damaging agent, it is well known and supported by a plethora of dedicated experiments that the radiation effect to cells and tissues is not uniquely determined by the average energy dose to which the cells are exposed. For instance, in comparison with photon radiation, heavy ions are more effective in stopping cell proliferation and inducing mutations12 or a characteristic scale for mobility for the induced damage in random walk processes. So far there is no general answer on how the overall observed effect modifications are affected by processes on different levels of damage localization.From a formal perspective, a coexistence of any two elementary lesions in sufficient proximity can act synergistically and result in a more complex lesion whose effect is larger than the sum of the effects of both isolated lesions. Such interaction mechanisms go along with a spatial scale of proximity and an associated biologic target, i.e. a conformation level of the DNA14. In contrast, also the µm scale is known to have relevance since studies on the interaction range for chromosome aberrations15, and is also given different interpretation in terms of DNA conformation16. Also other scales of lesion interaction and the possible coexistence of scales were proposed17. Independent of this a number of studies investigated the phenomenon of sublethal damage, where lethality was committed only after a second dose of the damaging agent19. However, a rigorous investigation of which scales are of importance and what their relative contribution is in different experimental settings is lacking. This work presents an experimental and modelling approach to disentangle the relevance of the different scales of DNA damage.In the literature the relevance of the nanometer scale has been investigated, stimulated by the formation processes of DSB. This has led to the notion of complex DSB, where the complexity refers to additional lesions within some nm distanceIn our experiments, we analyzed the impact of DNA damage induction on CHO cells using cellular survival level measured by colony formation as endpoint, which is regarded as a ‘gold standard’ in radiation biology. Clonogenic cell survival is a clean and representative endpoint which reflects the final level of damage after all cellular repair or misrepair processes have been completed.As DNA damaging agent we have used ionizing radiation, allowing to vary the spatial distribution of damages by the appropriate choice of radiation type and irradiation conditions. As reference case we have chosen conditions where DNA damages are distributed throughout the target in an approximately random manner, thus maximizing the mean distance between individual DSB and minimizing the probability of mutual interaction. This condition is fulfilled for low-LET radiation like e.g. photon radiation. Identical damage distribution patterns can be achieved also with high energetic protons using conventional broad beam irradiation , if the mean number of DSB induced per proton traversal through the cell is significantly below 1.0. According to the minimized DSB interaction probability under these conditions, allowing for optimal repair of radiation damage, photon and high energy proton radiation are expected to exhibit the lowest effectiveness.The concentration of particle traversals in small, subcellular regions, leaving the space in between these irradiation spots unaffected; in this case interaction can occur between DSB induced by different ion tracks (inter-track interaction) within the spots, while the total number of DSB is kept constant.An increase of the energy deposition per particle traversal and with that the number of DSB induced per traversal e.g. by choosing heavier particles like Li or C ions. In this case, damage interaction can occur between DSBs of a single track, which are produced preferentially along the trajectory of the ion.A combination of the two above mentioned options.We have compared these reference conditions with situations where DSB were induced in a clustered, spatially correlated manner, using the following options:21, where a defined number of ion traversals can be applied either in a homogenous, random distribution or in sub-micrometer sized focused beam spots22. For the latter, electrostatic deflection of beam paths is employed to direct individual particles to pre-specified locations (see details in Methods).In our experiments we have realized all these options by using the capabilities of the ion beam microprobe SNAKEIn a first step, we have compared the effects of randomly distributed low LET proton traversals  with spatially bunched proton spots, where the mean dose was 1.7 Gy in both conditions. The corresponding local energy deposition patterns and DSB distributions are depicted schematically in Fig. 24. For protons this is not the case as the number of DSB induced per track is well below 1.0.In a second step, we have used bunched high LET particle irradiation  with its pronounced local dose distribution on the nm scale Fig. , leading2 grid. While for the smallest mesh width used in this study (3.82 × 3.82 µm2) on average about 4.6 spots hit the cell nuclei based on the mean nuclear area of 67.6 µm2 (standard deviation 24.8 µm²), for the largest grid (10.8 × 10.8 µm2) only 0.58 spots are applied on average to a cell nucleus and 44% of cell nuclei remain unhit.In the experiments the Gaussian beams were elliptical with approximately 0.6 µm and 1.2 µm FWHM; the same spot geometry was used for all experiments described. In order to vary the number of ions per spot and to correspondingly vary the local DNA damage density, different mesh widths were applied, where an increase in the mesh width was compensated by a corresponding increase in the number of ions per spot to keep a mean dose of approximately 1.7 Gy fixed. The different beam delivery modes were verified with a DNA damage marker . For instance, 117 protons were focused to each spot of a 5.4 × 5.4 µmTable Figure Impressively, simply by focusing protons to spots of 117 particles each 27, microdosimetry28 and target theory29 on the three scales in a simple way, using only SSB and DSB as elementary DNA lesions. The model constants of a yield of 1250 SSB and 30 DSB per Gy and cell nucleus for photon radiation, a maximum distance of 25 bp for SSB to result in a DSB, and cubic domains according to 2 Mbp as size of chromatin loops for DSB interaction  are taken as a fixed part of the model and were originally obtained by experimental data31. The lesion statistics predicted by this approach are shown in Table S1 along with further model details in the supplementary information.To quantify the radiation effect, three spatial scales of DNA damage formation are implemented in LEM and the corresponding processes are hypothesized to be relevant for the cellular survival after irradiation : (i) On the nm level the interaction of SSB to DSB gives rise to an enhanced production of DSB per Gy for high local energy concentrations; (ii) on the µm scale the interaction of DSB within chromatin loops of the DNA 30 nm fiber leads to even more complex lesions which are harder to repair for the cell; (iii) on the 10 µm scale the hit statistics of a cell population may decrease the overall effect to correct for unhit cells. Notably, neither higher complexity levels of individual DSB on the nm scale nor the formation of chromosome aberrations are explicitly considered in the model, although the exploited effects of reference radiation imlicitly include the latter process. After all, LEM conceptually combines approaches developed in the framework of nanodosimetryThe LEM has been adapted to the experimental conditions of this work by an implementation of the 2D Gaussian beam spots of a specified number of ions delivered to cells. The model results are displayed in Fig. Dx-ray needed for the induction of an effect by the high energy photon irradiation to the dose D where the same effect is obtained by the radiation type under investigation,Encouraged by the model agreement to the data we also analyzed systematically the relative impact of the considered mechanisms on the overall radiation damage with the model. The efficiency of radiation induced effects is usually expressed by the relative biological effectiveness (RBE), which is defined as the ratio of the dose RBEx is an effect modifying factor on the spatial scale x. In this approach RBEnm is just the enhancement of the DSB yield, often referred as RBEDSB. The RBEµm reflects the enhancement in the effect by the interaction of DSB on the µm scale. The factor RBE10 µm is a hit statistic correction factor. It should be noted that the three RBEx factors correspond to a hierarchy of processes. For example, a strong SSB interaction will result in more DSB and thus also amplify the DSB interaction. Hence RBEµm depends implicitly on the value of RBEnm, and the processes are not independent, Nevertheless the factorization approach gives useful information as the terms are directly related to the individual processes. The different RBEx therefore can be regarded as the relative contributions to the overall RBE, corresponding to number and proximity of DSB and to hit statistics. This implies that each of the underlying mechanisms has the capability to modify the RBE, and the factorization is a possible way of quantifying their relative importance.Considering that any of the three spatial scales pointed out potentially modifies the radiation effectiveness, the observed effects can be thought of being amplified by means of both SSB and DSB interaction and damped by hit statistics. This suggests an approach to disentangle the contribution of these mechanisms asRBEx, and Fig. α, and restrict to this limiting case, which reflects the modification of the initial slope of dose response curves. The overall RBE reveals the typical RBE-LET characteristics with an effect increase up to the pronounced maximum at 150 keV/µm. Beyond, the RBE decreases again due to the overkill effect. At the high LET end the hook occurs as for high LET values two values of kinetic energy are associated with the same LET, due to the distinct properties of ions decelerating in a surrounding medium32.The LEM allows an explicit evaluation of the RBEnm factor increases monotonically in the entire LET range and goes up to approximately 5–10 at very high LET33. For low LET instead merely DSB interaction on the µm scale causes most of the RBE increase. For increasing LET this process gets less important again due to saturation: If there are more than two DSB interacting within the sensitive volume the damage is not enhanced compared to when only two DSB interact within that domain, as in both cases a complex lesion on the µm scale is formed. Thus the relative effect per particle decreases and RBEµm eventually drops below 1. The third modification by means of the RBE10µm is a pure damping factor which can only take values smaller than 1. It gets relevant for high LET where individual tracks have a high cell inactivation capability so that cells are killed if they are hit at least once, or survive if they are not hit. Hence, the nuclear area distribution which determines the number of hits per nucleus becomes important. To gain further insight in the meaning of the effect decomposition for applications, we also considered the case when the RBE is plotted against the remaining range of the carbon ions in water up to where particles stop in the so-called Bragg peak as used in clinical applications such as heavy ion cancer therapy or in material sciences in the supplementary information . As turns out, in the Bragg peak region, all scales are of importance and may not be neglected for a proper RBE prediction.The DSB yield given by the Concerning our experimental data, ignoring a µm lesion interaction mechanism precludes understanding the enormous effect enhancement we observed simply due to µm focusing of low LET protons  as compared to a more homogeneous distribution . As long as isolated DSB are formed by any kind of stress factor the relevance of such isolated DSB for affecting proliferation is small. Instead, the interaction of DSB and therefore their local concentration seems to be a prerequisite that DSB may lead to more severe consequences on the cell level.In combination with previous findings, these observations suggest that different types of complex damage may exist, associated with different spatial extensions and biological targets, whose relevance depends on the distribution of more elementary DNA lesions.43. In addition, complementary to the spatial relevant scales addressed in the present work, various temporal scales are known to be of importance in DNA repair, and numerous approaches attempt to gain insight in their mechanistic underpinning. For example, it is well established that the time to repair DSB and the fraction of unrepaired DSB after one day of observation increases for high LET irradiation44, but can be modified by accumulating factors involved in DSB repair pathways46. Finally, at the level of connected tissues or organs, further spatial scales of radiation damage may occur, as is e.g. evident from recent experiments with so-called minibeams48, where very high doses of either low or high LET radiation are delivered in grids of tiny mm sized beams, while in between no irradiation is delivered. Such irradiation scenarios are usually well tolerated by various tissues, indicating intercellular repair processes on the mm scale and opening new perspectives for radiation therapy of cancer or other diseases.Many projects in DNA repair research focus on revealing the nature of complex damage: Modern microscopy techniques exhibited clustering of damage markers such as γH2AX or their substructure, which might be associated to LET dependent damage complexitySummarizing, all these approaches promise further insight in the role of complexity and repair of radiation damage. The present work formulates expectations for some of the properties of complex damage. The exploited microbeam techniques and theoretical effect modelling may play an important role in future studies.22. A short description of the adaption is given in the supplementary information.The cell survival experiments were carried out using Chinese hamster ovary cells, CHO-K1. Standard cell culture conditions were used as described in the supplementary information. For cell survival experiments it is essential that all seeded cells get irradiated. For this purpose the cell cultivation setup was adapted and modified as described in20. The proton, lithium and carbon ion beams left the beam transport vacuum through 7.5 µm Kapton foil. After an air gap of ~30 µm and the cell carrier foil (5 µm) the ions hit the cells. The energy loss in the materials was calculated with TRIM, the LET values with SRIM49. Resulting energies and LET values at the target are given in Table Ion irradiation was performed at the Scanning Ion Microprobe SNAKE installed at the Munich 14 MV tandem accelerator22. Spots of roughly 0.6 × 1.2 µm² were prepared by magnetic quadrupole focusing. The measured spot sizes for different beam times and the method of measurement are shown within Table The main parts of the irradiation setup and processes for spot irradiation have already been described inTo suppress systematic errors all broad beam irradiations were performed in the same setup. There the focused microbeam was scanned very fast many times over the (500 × 500) µm² field and particles were counted until the necessary number for the total area was reached before moving the cell sample to irradiate the next field. This approach allows switching immediately from spot to random irradiation under the otherwise same conditions. For all cell samples, the irradiation container was mounted in irradiation position, visualized by phase contrast microscopy and the size and position of the entire irradiation field were adjusted to cover the whole cultivation area. The total irradiation procedure takes 15–20 minutes.The size of the irradiated spots was characterized for each experiment using fluorescent nuclear track detectors (FNTD) for protons and track etch detectors (polycarbonate or CR 39) for lithium and carbon ions, see Table Immediately after irradiation the cells were removed from the mylar foil by trypsinization and cell concentration was determined using the Fuchs-Rosenthal cell counting chamber (NanoEntek). This chamber differs from other cell counting chambers by its large surface area and greater chamber depth. This design allowed accurate determination of small cell counts. Cell counting has been performed four times for each cell sample. The cell suspension was diluted in a dose-specific manner and reseeded into 12 well plates (Corning), where either 100 or 200 cells were seeded per well in typically 24 or 36 wells. The plates were incubated under standard culture conditions. On the fifth day after irradiation medium was removed from the wells and cells were rinsed once with PBS. The colonies were fixed with methanol and stained with 0.1% crystal violet. Plates were washed with water and then left for drying at room temperature. The counting of stained colonies has been performed by an automatic counting bioreader (Biosys). Only colonies consisting of 50 or more cells were counted as surviving cells. Colony numbers were mostly exceeding 10, assuring in combination with the large number of wells a sufficient statistical precision. The Plating Efficiency (PE) is described as the percentage of cells that are able to divide and form colonies. It was found to be 0.52 ± 0.08 from sham irradiated cells, where the uncertainty is the standard deviation among different beam times according to Table For all used ion species data from multiple irradiated samples obtained in multiple beam times are available (c.f. Table −1 (15 mA) and a distance of 42 cm from the source to cells. The irradiation dose ranged from 0.5 to 8 Gy. Survival data of two independent experiments with at least triplicates are shown in Fig. S2. A least square fit reveals α = (0.156 ± 0.045) Gy−1 and β = (0.0235 ± 0.0055) Gy−2 and a covariance Cov = 0.00025 Gy−3 for the common dose response curves obtained from both experiments. The uncertainties arise from the propagation of the fit parameter standard errors of the two independent experiments and can be interpreted as 68% confidence intervals.Reference survival curves with X-rays have been determined using the same cell containers under identical culture conditions as for spot irradiations in order to have a proper characterization of the radiation sensitivity of the cells . The cells were exposed by a 200 kV X-ray irradiator irradiation facility  with a dose rate of 1.21 Gy mint, marking the transition to a linear dose response regime. According to an empiric relation50 the latter was chosen as Dt = 4 Gy + 1.1 α/β. Further details of the model steps are described in the supplementary information and in26.The LEM predicts the effects of ion beam radiation based on (i) amorphous track structure, i.e. the local dose as a continuous function reflecting the local energy concentration averaged over a large ensemble of tracks., and (ii) the photon dose response curve for the endpoint under consideration. The latter is parameterized by the measured linear-quadratic coefficients α and β and the threshold dose DFor the present work the adapted LEM code for Gaussian beam spots allows a prediction of the effect of single spots to the cell nuclei. For this the geometric properties as determined from the nuclear size distribution were considered in the LEM calculations. The distribution of the number of spots to the nuclei has been obtained by a simple Monte Carlo algorithm as explained above. Using this information and the effect per spot the weighted overall survival was calculated.LEM simulations have been performed individually for each beam time, taking into account the determined spot sizes. Uncertainties in the predicted effects were quantified from the uncertainties of the input photon parameters. In a next step the modelling results corresponding to individual beam times have been pooled in the same way as the experimental data, and uncertainties have been propagated. To substantiate our modelling results we verified via a sensitivity analysis by manually detuning model input parameters that within the uncertainty levels of the photon dose response parameters the general conclusions of our work did not change.Supplementary Information


Exoskeleton technology has made significant advances during the last decade, resulting in a considerable variety of solutions for gait assistance and rehabilitation. The mechanical design of these devices is a crucial aspect that affects the efficiency and effectiveness of their interaction with the user. Recent developments have pointed towards compliant mechanisms and structures, due to their promising potential in terms of adaptability, safety, efficiency, and comfort. However, there still remain challenges to be solved before compliant lower limb exoskeletons can be deployed in real scenarios. In this review, we analysed 52 lower limb wearable exoskeletons, focusing on three main aspects of compliance: actuation, structure, and interface attachment components. We highlighted the drawbacks and advantages of the different solutions, and suggested a number of promising research lines. We also created and made available a set of data sheets that contain the technical characteristics of the reviewed devices, with the aim of providing researchers and end-users with an updated overview on the existing solutions.The online version of this article (10.1186/s12984-019-0517-9) contains supplementary material, which is available to authorized users. Robotic wearable exoskeletonsCurrently, there is a large variety of designs of lower limb compliant exoskeletons aimed at gait rehabilitation or assistance. However, there is a lack of detailed information about the mechanical components of these devices, which has been largely overlooked by previous reviews e.g. –9). Thes. Thes9])In this work, we refer to ‘compliant exoskeleton’ as a system that includes compliant properties derived from non-rigid actuation system and/or structure. Our review focused on three particular aspects: the actuation technology, the structure of the exoskeleton and the interface attachment componentsWe have gathered the mechanical and actuation characteristics of 52 devices into standardized data sheets  AND (“exoskeleton*” OR “rehabilitat*” OR “orthotic*” OR “orthos*” OR (“wearable” AND “robot*”)) OR “exosuit” OR “exo-suit”), which returned 1131 studies. We excluded: publications focusing on upper limb robots; non-actuated compliant exoskeletons; solutions where compliance was achieved through control; studies that did not report any mechanical information on the robot; and studies not related to either assistance or rehabilitation. The above process resulted in a total of 105 publications, which covered 52 different lower limb exoskeletons.To simplify and structure the information, we classified the compliant exoskeletons according to the mechanical component that results in their intrinsic compliant performance: (i) exoskeletons with compliant actuators  and rigid structure; (ii) exoskeletons with soft structure , Variable Stiffness Actuators (VSAs), and pneumatic actuators. As shown in Fig. Variable Stiffness Actuators (VSAs) are implemented in eight exoskeletons. These actuators are a variation of SEAs, in which the degree of compliance can be mechanically modulated to change the actuator’s output characteristics (e.g. output stiffness) . These aFigure Figure The selection of the spring stiffness is critical when designing a SEA or a VSA. Multiple selection criteria have been used . The mosFigure Compliant or spring-like behaviour can also be achieved by pneumatic actuators, which depend on input air flow rate to contract and/or expand . Eight oExoskeletons with compliant actuators normally have rigid structures, composed of mechanical links and transmission mechanisms placed in parallel with the user’s limbs. This rigid configuration hinders a full kinematic compatibility with human joints , 49. In To adapt to the user-specific morphology, the foot length, pelvis width and inter-joint distance are the primary design parameters. Moltedo et al.  proposedFigure The braces, cuffs, straps and orthopaedic components used in the reviewed exoskeletons are based on a broad variety of materials and configurations. The majority 30) of exoskeletons with compliant actuators have only one brace per leg segment (i.e. thigh or shank). Five exoskeletons have two braces per segment. Five other exoskeletons present a combined solution, i.e. two braces for thigh and one for shank. Some solutions are based on orthopaedic commercial braces in order to reduce costs  of exosk, 40, butExoskeletons structures composed by non-rigid components such as textiles take advantage of compliance for providing compatibility with human kinematics and dynamics, with potential to improve comfort, safety, efficiency and functionality , 71–73. The actuation mechanism of soft exoskeletons is usually composed of motors with different gearbox mechanisms , the type of support depends on the level of the lesion and its severity . When thGait disorders and lower limb impairments are also related to ageing . In thisSolutions based on SEAs and VSAs are highly heterogeneous, resulting in a strong non-linear relationship between maximum torque and weight. This, together with the scarce of technical data reported, make it difficult to identify one best design option. Most of the actuators are the result of a trial-and-error design process, based on past experience and the specific needs of the application. The selection of the elastic component type and spring stiffness remains a major open challenge for SEA or VSA designs. From the control point of view, higher stiffness is preferred in order to increase the bandwidth of the system . HoweverVSAs have been proposed as a solution to make robots more adaptable to user’s residual abilities and biomechanical restrictions . NeverthWe found several compliant exoskeletons with pneumatic actuators in the literature. Their dependence on off-board air supply restricts the ambulatory applications of these exoskeletons. Most of the publications did not include sufficient technical details with respect to, for instance, air flow rate, pressure level for contraction and expansion and other technical characteristics of the artificial muscle . This information, in our opinion, would have been beneficial to compare the different solutions and therefore help the convergence on successful design strategies.Modularity, defined as the application of the same actuator to different active DoF, is a common practice in exoskeleton design as a strategy to reduce costs as well as effort in manufacturing, development and tuning . While sThe structure’s total weight and weight distribution have considerable impact on the functional performance and metabolic consumption . SimulatInappropriate physical contact between the user and the robot is an issue potentially affecting pain and discomfort , 94, andSoft exoskeletons present three main advantages with respect to compliant exoskeletons with rigid structures. First, the cable transmission allows optimizing the number and location of the actuators, with direct effects on the weight and inertia of the device. Second, the soft structure strongly reduce the misalignments and kinematic incompatibility between user and device . Third, Only two out of the 52 reviewed exoskeletons included a combination of soft structure and compliant actuation. This choice is promising, and likely to lead to lighter devices with higher torque actuation performance with respect to current solutions Fig. -B 44, 7, 744, 73Choosing the right actuation type, the structure, and the physical interfaces and number of DoFs is a hard problem, which strongly depends on the application. Compliant exoskeletons with SEAs or VSAs are the most popular choice for ambulatory solutions for activities of daily living. This justifies the huge number of exoskeletons with this actuation type and the broad range of different existing designs. Pneumatic and soft exoskeletons with off-board actuators are preferred for non-ambulatory gait rehabilitation and assistance applications in the clinical setting. Soft structures are usually preferred in exoskeletons for gait restoration for individuals who still retain some walking ability Fig.  71]. Co. Co71]. A challenging issue is the ergonomic adaptation of exoskeletons to a wide range of patients within a clinical environment. In this case, designers have to use up-to-date anthropometric databases when defining the dimensions of the structures and interfaces. Scanning patient’s limbs and obtaining the brace with additive manufacturing techniques is a promising, and low cost, solution . A critiA particularly relevant problem related to the use of exoskeletons in rehabilitation or assistance is their effect on balance. Users’ balance may be compromised when using ambulatory unilateral or bilateral exoskeletons, due to the weight of the device and its behaviour. In addition, the loss of walking functions in most patients is frequently associated with balance disorders . As a reThe studies carried out to date have seldom included the user’s subjective perception of the exoskeleton in their evaluations. Including the users’ opinion can shed light to the design process, in particular the design of the structure and the interface . SatisfaIn this review, we described and compared the mechanical design choices of 52 lower limb compliant exoskeletons. We have limited our analysis to solutions that included mechanical compliant properties in the actuation and/or structure, excluding all those devices whose compliant behaviour is obtained by control strategies, namely virtual or active compliance. We focused on three main technical aspects, i.e. the actuators, the structure, and the interface attachment components. Compliant actuators are still heterogeneous and there are not clear established criteria for their design. The robotics community would highly benefit from specific guidelines that can speed up the development and the uptake of these technologies. Promising directions go in favour of more compact and personalized actuators, dimensioned on joint- and patient-specific torque requirements. These solutions will require extra efforts in terms of mechanical design with respect to traditional modular approaches, but are likely to produce more lightweight and efficient solutions. The choice of the structure material and morphology is also a crucial factor, with important effects on comfort and actuation principles.The physical interface between human and exoskeleton is a factor that has been particularly overlooked by current research. It should be seriously considered in the future to account for comfort, adaptation and efficiency problems. The soft exoskeleton technology, despite having several potential advantages over classic rigid approaches, needs to be further analysed in order to become a useful tool for rehabilitation and assistive applications.Under the DoF perspective, we found a great heterogeneous picture, with unclear correlation between technical solutions and pathologies addressed. Future efforts should be devoted in clarifying whether and how the number, type (active/passive) and distribution of DoF affects the performance of the different targeted populations.Finally, we observed a clear lack of technical information, metrics and benchmarks of performance across the reviewed literature. This input would be of great help to evaluate and compare the different devices on a standardized basis , 110. InAdditional file 1:Compliant lower limb exoskeletons: A comprehensive review on mechanical design principles. Technical characteristics of 52 compliant exoskeletons reviewed in the article including information about their actuation system, structure, interface attachment components, applicability, control strategies and related publications (PDF 18064 kb)


The best characterised signalling pathway activated by both CGRP and adrenomedullin is stimulation of adenylate cyclase via Gs. However, it is clear that in some circumstances the peptides can activate other signal transduction pathways, e.g., increases in intracellular calcium. Many of these signalling pathways can be observed in cultured cells but it is important also to examine isolated tissues to discover the full repertoire of transduction events. In the rat cerebellum there are receptors that respond to both CGRP and adrenomedullin. These seem to be located postsynaptically on Parallel Fibre nerve terminals and modulate transmission to Purkinje cells. Adrenomedullin acts via cAMP, apparently to augment neurotransmitter release. By contrast, CGRP decreases transmitter release, via a non-cAMP mediated pathway. We are currently examining the role of NO and tyrosine kinases in the responses to these peptides.


High resolution imaging techniques allowed examining somatic Ca2+ signals and, accordingly, the recruitment of hippocampal interneurons in awake behaving animals. However, little is still known about dendritic Ca2+ activity in interneurons during different behavioral states. Here, we used two-photon Ca2+ imaging in mouse hippocampal CA1 interneurons to reveal Ca2+ signal patterns in interneuron dendrites during animal locomotion and immobility. Despite overall variability in dendritic Ca2+ transients (CaTs) across different cells and dendritic branches, we report consistent behavior state-dependent organization of Ca2+ signaling in interneurons. As such, spreading regenerative CaTs dominated in dendrites during locomotion, whereas both spreading and localized Ca2+ signals were seen during immobility. Thus, these data indicate that while animal locomotion is associated with widespread Ca2+ elevations in interneuron dendrites that may reflect regenerative activity, local CaTs that may be related to synaptic activity become apparent during animal quiet state.Hippocampal inhibitory interneurons exhibit a large diversity of dendritic Ca It may have different spatial and temporal ranges of action, and can exert various functions from induction of synaptic plasticity and local tuning of neuronal firing to the regulation of the expression of genes involved in neurodegenerative processes  channels and NMDA receptors. These mechanisms are involved in generation of dendritic regenerative activity in form of back-propagating somatic action potentials (bAPs) and local Ca2+ and Na+ spikes generated in single or multiple dendritic branches  evoked by bAPs (bAP-CaTs) decline with distance from the soma due to a large K+ and low Na+ channel density  followed by Ca2+ release from internal stores is important for generating the supralinear Ca2+ signals, which control the direction of long-term plasticity at excitatory synapses located distally  cells, may have a relatively high density of Na+ channels in their dendrites and, subsequently, exhibit more wide-spread back-propagation of somatic APs  interneurons of awake head-fixed animals running on a treadmill. We found that dendritic CaTs (dCaTs) exhibit the behavior-state fluctuations, such as regenerative activity during locomotion and dendrite-autonomous local signals during immobility. This state-dependent Ca2+ signaling suggests that distinct forms of synaptic plasticity can be induced in interneurons of awake mice during different behavioral states.To begin examining the functional aspects of interneuron dendritic computations Experiments were performed on male C57BL/6 mice (P50–70) according to the procedures approved by the Animal Protection Committee of Université Laval (protocol #15-097-1). Mice were anesthetized deeply with ketamine/xylazine mixture (10/100 mg/kg) and fixed in a stereotaxic frame. A small (~0.5–1.0 mm) craniotomy was made over the hippocampus . For single cell dendritic imaging a low titer AAV1.Syn.GCaMP6f.WPRE.SV40 (Penn Vector Core) was diluted [1:4 in phosphate buffer saline ] and injected (one injection of 100 nL) at a depth of ~1,250 μm below the dura surface, resulting in the expression of GCaMP6f in a sparse population of CA1 neurons  followed by a hippocampal window and head-plate implantation surgery  began ~5 days after window implantation and continued until mice routinely ran back and forth and demonstrated stable running speed values as described previously  tuned to 900 nm and a 25× objective . The laser power was modulated using a Pockels cell and reached at the sample (after the objective) 10–75 mW. Green GCaMP6f fluorescence was routed to external photomultiplier tubes (PMTs) . The Leica LAS software was used for microscope control and image acquisition. Image series  in each plane were acquired at 47 Hz in single plane acquisitions. Imaging sessions lasted up to 30 ± 15 min and then the mouse was placed back in its home cage. Can = 10 interneurons; 11 cells were excluded from the analysis because of a high brain motion during morphological z-series preventing accurate dendrite tracing). The dendritic distance to soma was calculated along the dendrite using the maximal projections of morphological z-series.Analysis was performed using IgorPro  and custom scripts written in MatLab (The MathWorks). The time-series were motion corrected using whole frame cross-correlation, as described previously  or Mann-Whitney test .For CaT analysis, regions of interest (ROIs) were drawn manually on the mean soma or dendrite images following the outline of the structure of interest, and traces of ΔF/F vs. time were generated for each ROI as previously described  during immobility and locomotion. The imaging fields of view (207 × 207 μm) were selected to have in average 3–5 cells with clearly identifiable dendritic branches connected to the interneuron soma in the same focal plane  was similar in soma and dendrites when compared between different cells and behavioral states . Overall, like in soma, the average amplitude of dCaTs was significantly higher during locomotion , indicative of significant summation of dCaTs during spreading events. The remaining segments showed no change  or a slight decrease in dCaTs  when compared to the local events. Taken together, these data point to a dominant summation of dCaTs during spreading events, but also highlight the segment-specific variability in local dendritic signaling, likely due to specific spatio-temporal arrangements of excitatory and inhibitory inputs converging onto interneuron dendrites.Furthermore, during immobility state, dCaTs were more variable within a given cell. Overall, the variance in dCaTs was significantly higher in segments showing local events than in segments showing spreading events . As some2+ activity in hippocampal O/A interneurons of awake mice during locomotion and immobility. Our data showed that, despite a large variability in dCaTs across the cells as well as within different dendritic segments of the same interneuron, dendritic Ca2+ activity in interneurons reflects the animal behavior, as different types of Ca2+ signals and dendrite-soma interactions were observed in specific behavioral states. As a rule, Ca2+ signals had larger amplitude and could invade the entire dendritic tree in the focus of observation during locomotion. While this type of Ca2+ activity was also present during immobility, the signal amplitude was significantly lower in both soma and dendrites. Moreover, a significant fraction of dendrites showed spatially restricted CaTs, which were not seen by soma. Based on these data, we propose a scenario in which high soma-dendrite coupling, likely due to bAPs or regenerative activity and associated dCaTs, may facilitate the spike-timing-dependent or Hebbian forms of synaptic plasticity in O/A interneurons during animal locomotion. In contrast, during animal quiet state, this type of activity can be reduced or even replaced by local dendritic Ca2+ signaling, which, if occurs at the hyperpolarized level of membrane potential, may facilitate the anti-Hebbian plasticity mechanisms.Using high-resolution two-photon imaging, we explored dendritic Ca2+ signals in interneurons are more complex than in PCs as they arise from activation of different mechanisms, including the postsynaptic Ca2+-permeable receptors, such as NMDA, CP-AMPAR , which are restricted to individual dendritic microdomains or branches. Consistent with activation of local excitatory inputs within single dendritic segments, spatially restricted postsynaptic dCaTs were observed in our study in vivo during both locomotion and immobility states. In addition, bAP-CaTs can be reliably evoked in proximal dendrites of CA1 interneurons in vitrovia VGCCs and Ca2+ release  events of large amplitude can be generated in some interneurons at rest  with periodic sharp-wave-associated ripples  are recorded in the CA1 hippocampus, with CA1 PCs firing mostly during SWRs. The IS3 and VIP-LRP cells are active during LIA but do not participate to SWRs (Francavilla et al., 2+ activity in interneurons has been consistently associated with induction of Hebbian and anti-Hebbian long-term potentiation (LTP; Perez et al., 2+ sources involved (Lamsa et al., 2+ activity induce synaptic plasticity in vivo and which learning rules may be specific for interneurons in behaving animals performing cognitive tasks.Dendritic CaRF and VV performed the experiments. RF, OM and LT analyzed the data. RF and LT wrote 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.