ZCCHC17) was found when directly comparing nerve-crushed muscles from old and adult mice. This analysis revealed key differences in muscle responses which may underlie the diminished ability of old mice to repair following nerve injury.Motor unit remodelling involving repeated denervation and re-innervation occurs throughout life. The efficiency of this process declines with age contributing to neuromuscular deficits. This study investigated differentially expressed genes (DEG) in muscle following peroneal nerve crush to model motor unit remodelling in C57BL/6 J mice. Muscle RNA was isolated at 3 days post-crush, RNA libraries were generated using poly-A selection, sequenced and analysed using gene ontology and pathway tools. Three hundred thirty-four DEG were found in quiescent muscle from (26mnth) old compared with (4-6mnth) adult mice and these same DEG were present in muscle from adult mice following nerve crush. Peroneal crush induced 7133 DEG in muscles of adult and 699 DEG in muscles from old mice, although only one DEG (The online version contains supplementary material available at 10.1186/s13395-021-00283-4. Loss of skeletal muscle mass and function occurs over a substantial portion of later life and plays a crucial role in the development of frailty, leading to increased risk of falls, immobility, loss of independence and declining quality of life , 93, 112The mechanisms responsible for the loss of muscle mass and function have not been fully elucidated, but it has been estimated from electromyography studies that there are 40% fewer motor units by age 70 , 84. In Caenorhabditis elegans and Zebrafish have identified key processes and signalling pathways involved in regrowth following damage [The process of axonal regeneration and regrowth is complex, but elegant studies in tractable models such g damage , 42, 97.g damage  and thesg damage . These fg damage , but theg damage , 98. Preg damage ; studiesThe present study examined the transcriptomic profile of anterior tibialis (AT) muscles from adult and old mice, as well as from a subset of adult and old mice that had undergone a crush injury to the peroneal nerve. We aimed to identify pathways underlying age-related decline in skeletal muscle and differences between the responses of muscle from adult and older mice to nerve crush.Adult (6–8 months) and old (26 months) C57BL/6 J male mice were obtained from Charles River . All animals were housed in a temperature-controlled room (22–25 °C) with food and water provided ad libitum on a 12-h light/dark cycle in the University of Liverpool animal facilities. All experimental procedures were performed under a UK Home Office licence  and complied with the UK Animals (Scientific Procedures) Act 1986 and received ethical approval from the University of Liverpool AWERB .n = 5/adult control, n = 5/ adult crush, n = 5/old control, n = 5/old crush.Mice were anaesthetised using isoflurane, the hind limb was shaved, antiseptic skin cleanser (Videne) applied and buprenorphine given . All animals were maintained under gaseous anaesthesia throughout and all surgery carried out as per Staunton et al. . A smallMice were killed by a Schedule 1 procedure  and the AT muscle dissected and frozen in liquid nitrogen. RNA extraction was performed using the RNeasy Fibrous Tissues kit  as per the manufacturer’s instructions. Approx. 30 mg of tissue was lysed and homogenised using an ultra turrax  in buffer RLT before proteinase K digestion steps, total RNA bound followed by several washing steps and DNase digestion and total RNA eluted. Total RNA integrity was confirmed using the Bioanalyzer . Ribosomal RNA (rRNA) was depleted from RNA samples by using the Ribo-Zero™ rRNA Removal Kit  in accordance with the instructions of the manufacturer.https://www.liverpool.ac.uk/genomic-research/).DNA free total RNA was selected for poly A using NEB Next® Poly(A) mRNA Magnetic isolation Module. RNA–Seq libraries were prepared from the enriched material using the NEBNext®Ultra™Directional RNA Library Prep Kit for Illumina®#E7420. Libraries were purified using AMPure XP beads. Each library was quantified using Qubit and the size distribution assessed using the Agilent 2100 Bioanalyser. The quantity was assessed using a Qubit® dsDNA HS Assay Kit, while the quality and average size was assessed using the High Sensitivity DNA Kit. Subsequently, qPCR assay, designed to specifically detect adapter sequences flanking the Illumina libraries, was performed using an Illumina® KAPA Library Quantification Kit . The RNA libraries were sequenced on an Illumina® HiSeq 4000 platform with version 1 chemistry using sequencing by synthesis (SBS) technology to generate 2 × 150 bp paired-end reads. Sequencing was performed at the Centre for Genomic Research, University of Liverpool  to produce FASTQ format. The raw FASTQ files were trimmed to remove Illumina adapter sequences using Cutadapt version 1.2.1 . The reaRaw counts were subsequently used as the input into R (version 1.2.5042) to utilise the DESeq2 package  for idenp values < 0.05, generated using the Benjamini-Hochberg approach and in some cases a 1.4 log2 fold change (Log2FC) from the DEG, were used to perform functional analysis, specifically enrichment analysis with Gene Ontology (GO) Biological Process using the Panther Classification System [Fold changes and the threshold of false discovery rate (FDR) adjusted n System , Kyoto En System  from Bion System  with moup value calculated for that network as described by Calvano et al. [http://string-db.org). For the adult control vs old control cohort, an additional analysis was performed using the SarcoAtlas database to compare results here to the mRNA profiles of the TA skeletal muscles of male C57BL/6JRj mice from adult (10 month) and old (30 months) (https://sarcoatlas.scicore.unibas.ch/) [Further bioinformatics to determine the interactions of age and/or crush-related DEG, canonical pathway, upstream regulator, causal/master regulators and network analyses from these were performed using Ingenuity Pathway Analysis package (IPA)  “Core Analysis” settings. The core analysis was carried out with the settings of indirect and direct relationships between molecules based on experimentally observed data, and data sources were considered in mouse databases in the Ingenuity Knowledge Base. All network scores were calculated by IPA as negative exponent of the o et al. . In addibas.ch/) , 41. ThiWe measured cluster separation as Euclidean distance between treatment groups and tested for statistical significance with the R ClusterSignificance package . EquivalFull data are available on the EBI array express database with accession number: E-MTAB-10601.RNAseq analysis was conducted on AT muscle from control adult and old male mice as well as mice that had undergone a peroneal nerve crush model of denervation and regeneration. The AT muscle was used for all studies, 2 × 150 bp paired-end reads were generated, then data processed, and a total of 29,842 different gene transcripts were detected across 20 samples. The Venn diagram in Fig. n = 5/cohort)  Table . The datGstp2), as well as downregulation of the complex 1-related gene NADH:ubiquinone oxidoreductase (Ndufa13), and upregulation of heat shock proteins (e.g. hsp8). The 5 significantly DEG that were most upregulated (in terms of Log2FC) were Leucine-rich repeat kinase 2/PARK8 (Lrrk2), Complement Factor H (Cfh), ATPase Na+/K+ Transporting Subunit Alpha 4 (Atp1a4), Kelch Repeat and BTB Domain Containing 7 (Kbtbd7) and Vitamin K-Dependent Protein S (Pros1). The 5 significantly DEG that were most downregulated (in terms of Log2FC) were Syntaxin 11 (Stx11), WD repeat and HMG-box DNA binding protein 1/CTF4 (Wdhd1), Angiopoietin-like protein 3 (Angptl3), TOX High Mobility Group Box Family Member 2 (Tox2) and Galactose-3-O-Sulfotransferase 3 .A total of 334 DEG were found from a comparison of muscle from adult and old control mice; 199 of which were upregulated and 135 downregulated in muscles of old compared with adult mice . The TRAF2-GSTP1 complex (GO:0097057) was the most significantly overrepresented GO term for cellular components, followed by endoplasmic reticulum exit site (GO:0070971), I band (GO:0031674) and Z disc (GO:0030018).Gene ontology (GO) analysis was conducted using PANTHER and when examining the DEG obtained from adult vs old controls, a total of 15 shared GO biological processes were reported, with approximately 25% of the DEG classified into the cellular process category (GO:0009987), which includes cell growth and/or cell maintenance processes Fig. A. StatisKEGG pathway enrichment analysis was performed using enrichKEGG; a total of 5 significantly enriched KEGG categories were uncovered, as shown in Fig. p value > 1.3 as well as the representative DEG in each canonical pathway are listed in Table Using the well-established Ingenuity Pathway Analysis (IPA) , the DEG from a comparison of muscle from adult vs old were further analysed based on an FDR of < 0.05 with a 1.4 log2 fold change cut off/threshold (Log2FC). A total of 16 significantly enriched canonical pathways were identified (when scored using Fisher’s Exact Test) as well as 17 upstream regulators and 7 causal networks (summarised outputs in Table SLC30A7), lipase E (LIPE), androgen receptor (AR) and collagen type VI alpha 1 chain (COL6A1). Four master regulators were identified for the Adult vs Old DEG expression data: TAR DNA binding protein (TARDBP), growth factor receptor bound protein 14 (GRB14), growth factor receptor bound protein 10 (GRB10) and CCAAT enhancer binding protein beta (CEBPB).The advanced analytics of IPA allow identification of upstream regulators that may be causal regulators of observed gene changes (“master regulators”). Using this method, the 5 most significant upstream regulators were dystrophin (DMD), solute carrier family 30 member 7 , Growth/differentiation factor 5/ BMP 14 (Gdf5), Neuronal acetylcholine receptor subunit alpha-9 (Chrna9), FAT Atypical Cadherin 2 (Fat2) and Cholinergic Receptor Nicotinic Gamma (Chrng) and the 5 most significantly downregulated genes were Synaptotagmin 8 (Syt8), Aquaporin 4 (Aqp4), Kelch Repeat And BTB Domain Containing 13 (Kbtbd13), Methyltransferase Like 21C (Mettl21c) and Monoacylglycerol O-Acyltransferase 2 (Mogat2).In order to understand why motor unit turnover differs with age and how different ages respond to injury, a peroneal nerve crush model was undertaken and the AT muscle from both control and crushed tissue was examined 3 days post nerve crush. RNAseq analysis detected a total of 29,842 genes, with 23% of those genes differentially expressed between adult control and crushed tissue . The three most significantly overrepresented GO molecular functions were structural constituent of ribosome (GO:0003735), rRNA binding (GO:0019843) and electron transfer activity (GO:0009055) .GO analysis with PANTHER on the up- and down-regulated DEG from adult control and adult crush samples revealed 21 enriched biological pathways. The top three enriched processes and the percentage of genes classified to these categories being cellular process , metabolic process  and biological regulation  . Figure p < 0.05, Fisher’s exact test, Table Ingenuity pathway analysis was conducted to further investigate the mechanisms behind the changes seen with nerve crush and reveal key canonical pathways, upstream regulators and causal networks. A total of 41 out of 516 detected canonical pathways were significantly enriched . A total 17 upstream regulators were significantly associated with the DEG from adult control compared to adult crush and included transcriptional regulators together with receptors and cytokines, the 5 most significant upstream regulators were: lipase E (LIPE), Oestrogen-Related Receptor Gamma (ESRRG), PPARG coactivator 1 alpha (PPARGC1A), Uncoupling protein 1  and Smoothelin Like 1 (SMTNL1). Master regulators identified included a variety of molecular types such as transcriptional regulators, transports and enzymes and the top 5 master regulators were Oestrogen-Related Receptor Gamma (ESRRG), Histone deacetylase 4 (HDAC4), Uncoupling protein 1/ Thermogenin (UCP1), Smoothelin Like 1 (SMTNL1) and Alpha-1-syntrophin (SNTA1).Several canonical pathways including those linked to protein homeostasis, integrin signalling, interleukin signalling, mitochondrial dysfunction and senescence were identified and the 10 most enriched pathways and the molecules associated to the specific pathways and respective log fold change values are presented in Table SOD2), the pan-macrophage marker CD68, Malic Enzyme 1 ) and Mitochondrial creatine kinase (MtCK), and the most enriched biological functions this network was associated with were energy production, molecular transport and nucleic acid metabolism.IPA analysis identified 25 significant networks in total, 3 of which were all associated with 35 focus molecules and had a score of 13. The top network is shown in Fig. n = 5 per group, FDR < 0.05, Table In order to understand how muscle from old mice responds to motor nerve crush injury, the AT muscle from 26-month-old mice was compared to 26-month-old muscle that had been subjected to peroneal nerve crush 3 days prior. The transcriptomic analysis revealed a total of 699 genes were differentially expressed out of the 29,842 that were detected (Chrng), Cation Channel Sperm Associated 1 (Catsper), Tumour Protein P53 Pathway Corepressor 1 (Trp53cor1), Lectin Mannose Binding 1 (Lman1l /ERGIC-53) and Chitinase 3-like-1 (Chil1). The 5 most downregulated genes (in terms of LogFC) were Fructosamine 3 Kinase (Fn3k), EMAP Like 6 (Eml6), Outer dense fibre protein 3-like protein 2 (Odf3l2), Potassium Voltage-Gated Channel Modifier Subfamily G Member 4 (Kcng4) and Leucine Rich Repeat Containing 38 (Lrrc38).The 5 most significantly upregulated DEG (in terms of LogFC) were Cholinergic Receptor Nicotinic Gamma Subunit . The statistically overrepresented cellular components were mitochondrial proton-transporting ATP synthase complex, coupling factor F(o) (GO:0000276), proton-transporting ATP synthase complex, coupling factor F(o) (GO:0045263) and mitochondrial proton-transporting ATP synthase complex (GO:0005753) . In terms of GO molecular functions, the 3 most significantly overrepresented functions were pyrimidine nucleotide binding (GO:0019103), proton-transporting ATP synthase activity, rotational mechanism (GO:0046933) and NADH dehydrogenase activity (GO:0003954) .Gene ontology analysis of old control versus old crush DEG detected 19 associated biological processes Fig. A. The hiKEGG enrichment analysis revealed that 12 out of a total of 259 detected KEGG pathways were significantly enriched in the old and old crush DEG dataset (FDR < 0.05), the 10 most enriched pathways are shown in Fig. p value < 0.05. These include oxidative phosphorylation, mitochondrial dysfunction and sirtuin signalling which were the top 3 enriched pathways. The top 10 canonical pathways are shown in Table Twenty-two significantly canonical pathways enriched pathways were identified in the DEG obtained comparing old control vs old crushed tissue were those with a P < 0.05). The 5 most significantly enriched upstream regulators were peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) (PPARGC1A), smoothelin-like protein 1 (SMTNL1 often termed CHASM), mammalian target of rapamycin (MTOR), adiponectin receptor 1 (ADIPOR1) and prospero homeobox protein 1 (PROX1) , peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) (PPARGC1A), prospero homeobox protein 1 (PROX1), collagen alpha-1(VI) (COL6A1) and histone deacetylase 4 (HDAC4).Ten master regulators were identified in our dataset, that is, regulators that may not be listed directly in our dataset but are predicted to be key controllers that may activate or inhibit other pathways. The top 5 master regulators detected were smoothelin-like protein 1 , was differentially expressed . This suggests a substantial harmonisation of gene expression in both cohorts at this time point following crush. This can be visualised in the volcano plot where by ZCCHC17 is the single gene located at the top right of the plot in red .To directly explore how ageing might affect the muscle response to nerve injury, the DEG following crush from both adult and old cohorts were compared. 29,842 gene transcripts were analysed, but only one gene, Zinc Finger CCHC-Type Containing 17  and SET Nuclear Proto-Oncogene (SET), and binds to Syndecan Binding Protein 2 (SDCBP2), Zinc Finger And BTB Domain Containing 38 (ZBTB38), Jumonji Domain Containing 6 (JMJD6), amyloid precursor protein (APP), Pinin (PNN), Dynein Light Chain Tctex-Type 1 (DYNLT1), Nuclear Receptor Subfamily 2 Group C Member 2 (NR2C2), Cyclic GMP-AMP Synthase (CGAS), Autophagy Related 16 Like 1 (ATG16L1), HECT And RLD Domain Containing E3 Ubiquitin Protein Ligase 2 (HERC2), HECT Domain E3 Ubiquitin Protein Ligase 1 (HECTD1), Ribosomal RNA-processing protein 8 (RRP8), Ferritin Light Chain (FTL), Joining Chain Of Multimeric IgA And IgM (JCHAIN) and RNA-binding motif (RBM).IPA gene view categorises ZCCHC17 as a nucleolar protein involved in RNA and protein binding. The GO molecular functions that ZCCHC17 is associated with are metal ion binding, nucleic acid binding, protein binding and RNA binding. IPA proposes ZCCHC17 to be regulated by Kruppel Like Factor 3 ZCCHC17 has a low tissue specificity, but the RNAseq databases suggest it is predominantly expressed in neuronal tissues. Phenotypes, diseases and traits associated with this gene include abnormal cell differentiation, early cellular replicative senescence and oxidative stress [Expression data from the Human Protein Atlas show that e stress .ZCCHC17 were identified with STRING and the interacting partners are shown in Fig. Protein-protein interactions of The extensive motor neuron loss and NMJ degeneration seen in old mice associated with muscle loss prompted a comparison of the DEG found in AT muscles from control old mice with those seen post-nerve crush in adult mice. In order to investigate how similar the DEG changes that occur in muscle with ageing due to the presence of denervated muscle fibres in old age to those seen in muscle from adult mice following nerve crush, we compared these data and used statistical equivalence testing to obtain further information on the commonality between the two data sets.p = 0.78).Principal component analysis (PCA) across all 4 datasets . The PCHierarchical clustering by genes (rows) and population (columns) shows genes acting in a similar manner Fig. A. The meEquivalence testing was conducted to further explore the potential similarity between the two populations; this examined all the genes detected to assess what number of genes (and what genes) are statistically similar in expression between the adult crush vs old control populations. This non-parametric test used normalised gene counts and identified 2672/21,269 genes that were statistically similar in expression between the adult crush and the old control samples.The aim of this study was to use RNAseq analysis to examine differences in gene expression underlying age-related decline in skeletal muscle in mice, to identify whether the differences seen in old mice were related to the presence of actively degenerating or regenerating nerves, and to identify differences between the acute transcriptional responses of muscle between old and adult mice subject to nerve crush that might indicate why muscle from old mice shows poor re-innervation following nerve damage. The post-crush samples were obtained at 3 days following peroneal nerve crush, a time point previously shown by us as associated with significant loss of the distal axonal structure and an increase in generation of peroxides by the AT muscle mitochondria, although AT mass is preserved at that stage .The data obtained have identified DEG that occur in muscle with ageing, and show that all of the DEG found in muscle from old mice can be induced in adult mice by nerve crush. Only one DEG was found in adult mice following nerve crush compared with those seen in muscle of old mice following nerve crush indicating that differences between muscles from old and adult mice were minimised in the responses to crush injury.This study revealed the DEG for each contrast, few of which have previously been explored with respect to skeletal muscle ageing and denervation but provided some interesting predicted pathway enrichments and potential upstream regulators and network analyses. Whilst a range of DEG were detected which could be controlling how adult and aged skeletal muscles respond to injury/denervation, it is important to appreciate the limitations of this approach. It cannot be presumed that all the DEG are from the myofibres since the muscle tissue analysed contain a number of other cells including neuronal tissue satellite cells, fibroblasts, terminal Schwann cells (tSCs), adipocytes and immune cells which, although present in low amounts, might contribute to the data obtained and to overcome this one could conduct single cell RNAseq, or possibly single nucleus RNAseq would be appropriate. It is important to realise that aged motor neurons have altered properties which could participate in the observed phenotype. It is known that during ageing, the number of satellite cells decline in mice , 22 in aThe response of skeletal muscle to ageing was examined by comparison of DEG between muscles of adult and old mice. This comparison revealed 334 DEG, from a total of 29,842 genes identified. Only a small proportion of these genes have previously been studied in relation to skeletal muscle biology. Pathway enrichment analysis was undertaken to identify how such differential gene expression could contribute to age-related loss of murine skeletal muscle mass and function.Lrrk2) a gene associated to the neurological disorder Parkinson’s disease was found to be differentially expressed [Several of the processes identified are linked to neurological degeneration or regeneration. Thus, the 5 most upregulated DEG found from the comparison between data from control muscle of adult and old mice were associated with different biological processes. Leucine-rich repeat kinase 2/PARK8 ; this gene encodes an integral membrane protein responsible for establishing the electrochemical gradient of Na+ and K+ ions [2+) transients involved in muscle contraction [2+ release, impairments that have been suggested to explain deficits in physical performance in muscle from older animals [Another strongly upregulated DEG was ATPase Na+/K+ Transporting Subunit Alpha 4 Atpa4; this traction , and thi animals .STX11) was downregulated with age. Changes in syntaxins have been linked to impaired neurotransmission [Syntaxins, a family of SNARE proteins, are membrane proteins primarily localised to the presynaptic active zone and involved exocytosis of vesicles. Syntaxin 11 , but this has not previously been associated with muscle physiology [Pros1 gene (Protein S) was found to be significantly downregulated in muscle of old mice. Little previous work has been reported on Pros1, but a study by Murton et al. [Other DEG identified were associated with processes such as DNA damage regulation  signalling as one of the top canonical pathways associated with ageing skeletal muscle. IL-9 and its receptor (IL-9R) are mainly expressed in neuronal and immune cells [IPA upstream analysis identified interleukin-9 (ne cells  and are ne cells  and thisne cells  found thne cells .DMD). It appears surprising that this large protein was indicated in this type of analysis but UR analytics showed it to be associated with 4 target genes in the dataset . Dystrophin appears to have regulatory roles in addition to the well-known structural role. This has been shown by studies in dystrophin-deficient (mdx) mice and many studies have now identified compensatory proteins to DMD which are thought to be involved in the maintenance of the neuromuscular junction architecture and in muscle homeostasis  were also revealed using the advanced analytics in IPA. The UR identification methods examine links to DEG through coordinated expressions to potential UR that can be transcription factors or any gene or small molecule that has been observed experimentally to affect gene expression. The top UR detected between samples from old and adult mice was the key structural protein dystrophin  (as mentioned above) and Thbs4 were deemed neuromuscular junction related. Thromonospondin (Thbs4), a protein that regulates skeletal muscle integrity and its susceptibility to muscular dystrophy [A study by Pannérec et al.  assessedystrophy , was sigystrophy  dataset.ystrophy , the comTARDBP; this gene encodes TDP-43, a highly conserved and ubiquitously expressed protein involved in transcription and RNA splicing regulation. Hyper-phosphorylated and ubiquitinated TDP-43 deposits are associated with several neurodegenerative diseases where the deposits produced act as inclusion bodies in the brain and spinal cord [CEBPB) was also found to be a master regulator and this particular gene highlights the potential of satellite cell involvement with skeletal muscle ageing. Recent studies show CEBPB as a novel regulator of satellite cell self-renewal during muscle cell regeneration [CEBPB protein has also recently been associated with ATF4 whereby it is thought to mediate muscle atrophy [One of the top master regulators identified was nal cord . Olivé enal cord  have indneration , 69. The atrophy .GPX3) and Glutathione S-transferases genes (including GTSP1) as well as stress response genes (ATP6V) and several chaperones specifically heat shock proteins (including HSPA8) in a similar manner to the datasets of Murgia et al. [The pathways and specific DEG outlined above were obtained from examining muscle tissue and so a limitation of this study was that we were unable to confirm the cellular source and data on any changes in skeletal muscle fibre type proportions were not available in our current study. Murgia et al.  conductea et al. , support2O2 generation following denervation and this increased mitochondrial H2O2 release is already apparent within 3 days of nerve transection. The effect of this activation of specific degradatory pathways is unclear although we speculate that initially this may reflect an attempt to restore innervation, since products such as cytokines are released from the muscle fibre and some cytokines have been proposed to stimulate axonal sprouting [CHRNA9), cholinergic receptor nicotinic gamma (CHRNG) and growth differentiation factor 5 (GDF5) and the canonical pathways identified include mTOR signalling which is key to regulation of muscle protein homeostasis. The KEGG analysis identified pathways induced in the muscle of adult mice following nerve crush that are associated with neurodegenerative disorders, such as Huntington’s, Parkinson’s and Alzheimer’s diseases  [UCP1) [7133 skeletal muscle genes showed differential changes in expression following crush injury to the peroneal nerve in adult mice. Experimental data has shown that sectioning of motor nerves or nerve crush leads to the rapid activation of degenerative pathways in the denervated muscle, including an increased mitochondrial generation of reactive oxygen species , increasprouting . The larses Fig. B. While PARGC1A) , 51, 73 ) [UCP1) , 44, 53.Most surprisingly, a comparison of the DEG in skeletal muscle resulting from nerve crush in adult mice with the DEG in skeletal muscle of control old compared with control adult mice revealed that all of the 344 DEG associated with ageing in control muscle were induced by nerve crush in muscles of adult mice although the full extent of DEGs seen in adult mice with crush is considerably greater. Thus, the DEG seen in control muscles of old mice do not reflect the full extent of gene changes following crush in adult mice. These data indicate that, in terms of DEG, all of the changes seen in muscle during ageing are reproduced as part of the pattern of DEG seen in adult mice by crush of the peroneal nerve.These results emphasise the critical importance of motor nerve loss and regeneration (i.e. motor unit turnover) in the transcriptional changes seen with ageing in skeletal muscle and fundamentally influence the way in which ageing induced changes in skeletal muscle gene expression are interpreted. This conclusion about the importance of motor neuron and motor unit loss in the causes of ageing related muscle loss has been implied previously on the basis of physiological  and struSix hundred ninety-nine DEG were detected in muscle from old mice following nerve crush. This was much less than the 7133 DEG found in muscle of adult mice following nerve crush despite the similar levels of total genes detected . The implication of this is that muscle from old mice had a diminished response to nerve crush. Muscle from aged mice has been claimed to have multiple interacting dysfunctional systems , such asp value 0.01). This Zinc Finger CCHC-Type Containing 17 (ZCCHC17) protein  is localised to the nucleolus and interacts with multiple metal ions, nucleic acids, proteins and RNA binding proteins [ZCCHC17 has recently been proposed as a master regulator in Alzheimer’s disease and is expressed in neuronal cells where levels are seen to drastically decrease early in AD, before significant neuronal cell loss or gliosis. ZCCHC17 is also believed to support the expression of a network of synaptic genes and linked to calcium signalling and protein mannosylation [ZCCHC17 may be a potential driver for both of these disorders.In contrast, when the muscles from adult and old mice post nerve crush were directly compared there was only one DEG (ZCCHC17) which was upregulated in the muscle from old mice —a cell adhesion protein that is involved in synapse formation, axonal guidance and neuronal-glial cell interactions [NPTN—a key neuronal cell adhesion molecule associated with neurite outgrowth and neuronal and synaptic plasticity [NGB) which is believed to be a stress sensor and promotes neuronal survival [An original aim of the study was to identify differential responses between skeletal muscle of old and adult mice post nerve crush that might explain the age-related differences in nerve repair capacity. It was hypothesised that a contributor to the defective re-innervation that occurs in muscle from old mice is a failure to release appropriate “sprouting factors” from muscle. Identification of specific axonal “sprouting factors” released from transiently denervated muscle fibres (and which are not appropriately released from muscle of old mice) is thought to be potential therapeutic targets to help retain appropriate innervation of muscle fibres during ageing. It was anticipated that neurotrophins such as NGF, BDNF, NT3 and NT5 would be identified. Whilst several typical neurotrophins were detected within the muscle bulk of adult and old mice and could be acting as potential regulators of the maintenance, function and regeneration of skeletal muscle, they were not differentially expressed. Other genes that were detected that could also be playing a role in NMJ maintenance and repair included Neurofascin  by direct comparison of DEGs in muscles from old mice following nerve crush to muscles of adult mice following nerve crush, there were 7133 DEG induced by nerve crush in muscles of adult mice compared with 699 DEG induced by nerve crush in muscles from old mice. The identification of only a single DEG in the direct comparison of nerve crushed old vs nerve crushed adult muscle is therefore surprising and presumably reflects the substantial increased variability in baseline gene expression that occurs with ageing.A comparison of the top 10 canonical pathways identified by IPA in muscles from old nerve-crushed vs control old mice Table  with thoThe findings of this study should be considered in light of some limitations. The availability of mice only allowed the examination of these effects in male mice. The examination of these effects in female mice is warranted although there is little evidence of any effect of sex on the rate of denervation seen in old age or differential effects following acute denervation. A lack of tissue availability excluded analysis of the effect of a change in muscle fibre type composition on our findings although the AT is predominantly type II and so we anticipate that fibre type will have played little role in our findings. The study focussed on the responses to nerve crush at a relatively early time point and so we were not able to exclude any additional effects at later time points. Data from the analysis of whole muscle tissue should always be examined with caution since muscle tissue contains a range of cell types which contribute to the local environment. An added complexity of analysis of RNASeq data is that in some instances this does not reflect the protein content of the tissue. However, the data are important in identifying differential control of critical pathways and provide a valid outcome despite the caution needed in interpretation.In conclusion, this study of differentially expressed genes in AT muscles from adult and old mice and adult and old mice that had undergone a crush injury to the peroneal nerve has characterised the transcriptional changes underlying age-related decline in skeletal muscle and differences between the responses of muscle from adult and older mice to nerve crush. Data have identified 344 DEG that occur in muscle with ageing and indicate that all of these DEG can be induced in muscle from adult mice by nerve crush suggesting a major component of nerve degeneration/remodelling in the changes in muscles of old mice. Although only one DEG was found by direct comparison of muscles from old mice following nerve crush and muscles of adult mice following nerve crush, there were 7133 DEG induced by nerve crush in muscles of adult mice compared with 699 DEG induced by nerve crush in muscles from old mice and further analysis of these genes may indicate key differences in the DEG response of skeletal muscle from old mice which underlie the diminished ability of muscle from old mice to repair following nerve injury.Additional file 1. Supplementary Table S1


Cervical cancer is the fourth most common cancer in women . Current18F]fluoro-2-deoxy-D-glucose ([18F]FDG) positron emission tomography combined with computed tomography (PET/CT) imaging plays an important role in treatment stratification in oncology. In cervical cancer, parameters such as the standard uptake value (SUV), metabolic tumour volume (MTV) or total lesion glycolysis (TLG) have been proposed as prognostic factors, although none has been integrated in the clinical decision algorithms FDG PET images of cervical cancer and evaluated the performance of different classifiers combined with different feature selection (FS) methods to predict DFS. We hypothesised that a PET-based radiomics signature would have a significant prognostic value, higher or complementary to standard clinical parameters. We then evaluated the predictive value of tumour to liver ratios (TLR) of radiomic features FDG PET images using the semi-automatic Fuzzy Local Adaptive Bayesian (FLAB) algorithm with 2 classes FDG uptake in cervical cancer when using conventional metrics such as the SUVmax, MTV or TLG [18F]FDG PET/CT alone , 5018F]FCT alone –22. FurtCT alone , 29, altCT alone . This is3.We also looked at various parameters known to affect the reproducibility and robustness of such process. Feature selection in particular is an important step of the analysis process as it improves the generalisation of the results. It is known that each FS method and each classifier has its own limitations, and indeed, like other researchers in various settings, we did observe a large performance variability when using different methods for discretisation, FS and classification , 19–22. Multi-center radiomics studies imply features variability mainly due to distinct imaging devices and protocols. This variability is among the major obstacles preventing generalisation of radiomics signatures in the clinical practice. Different methods have been developed to decrease radiomic features variability across clinical centers. Harmonising acquisition and reconstruction parameters, for instance through the EANM EARL initiative might contribute reaching such goal. A key finding of the present study is that the TLR perform better across scanners than the native radiomics features. As each patient acts as his own control, it should not come as a surprise finding the ratios as more stable than the native features. ComBat is another method that was previously successfully used for that purpose. It was shown to align features distributions across different clinical centers as well as to improve predictive ability of radiomic features namely in cervical cancer DFS prediction . In our There is currently no satisfying answer to the only clinically relevant question when it comes to introducing ML and radiomics in the clinical field of nuclear medicine: Is there a “universal” feature set or ML strategy that could predict the outcome, in this case DFS in cervical cancer patients, independently of the PET/CT device and imaging methodology?. Indeed, in lung cancer for instance, Parmar et al.  and Sun Our study presents some limitations. The tumour segmentations were done by a single observer, using the semi-automatic FLAB algorithm. Using fully automatic and thus possibly more reproducible segmentation methods  55] mig mig55] mDespite these limitations, to the best of our knowledge, this is the first study to investigate TLR radiomic features in predicting DFS in cervical cancer. All the features calculations were performed using a software which is documented and fully in line with IBSI standardisation, which should help other research groups reproduce our results. Our study follows the TRIPOD guidelines  Supplem and scorFuture research should include combining different image modalities  23] and and23] aIn this multicentre series, a ML algorithm with 10 TLR radiomic features combined with the clinical information resulted in the best predictor of cancer recurrence. Despite these encouraging results, one cannot ignore the persistence of significant dependency of the features on the local acquisition settings as well as the poor F-score performance. Hence, further works need to be done prior to large-scale clinical testing.ESM 118F] FDG PET protocol description according to Scanner. (PDF 363 kb)A) Patients [ESM 2B) Oncoradiomics features description (PDF 585 kb)ESM 3F1-score and F2-score, precision, recall and AUCpr values of the best models using ComBat harmonization (PDF 125 kb)C) Bootstrap mean AUC, ESM 4D) TRIPOD adherence data extraction checklist (PDF 871 kb)


The highly secure blood–brain barrier (BBB) restricts drug access to the brain, limiting the molecular toolkit for treating central nervous system (CNS) diseases to small, lipophilic drugs. Development of a safe and effective BBB modulator would revolutionise the treatment of CNS diseases and future drug development in the area. Naturally, the field has garnered a great deal of attention, leading to a vast and diverse range of BBB modulators. In this review, we summarise and compare the various classes of BBB modulators developed over the last five decades—their recent advancements, advantages and disadvantages, while providing some insight into their future as BBB modulators. Passive diffusion is a general route of access across the BBB, but certain restrictions prevent most molecules from successfully utilising this pathway. In the past 20 years, a range of lipid diffusion models have shown correlations with various physicochemical properties for predicting a drug’s BBB permeability. While certain models have been debated [The blood–brain barrier (BBB) is the term used to describe the fortified microvascular network of the CNS, noted for its remarkably heightened molecular specificity—facilitating nutrient supply, whilst impeding the access of harmful substances to the brain. BBB passage of endogenous biomolecules is generally achieved via transcellular and paracellular pathways. Transcellular processes facilitate the transport of molecules  debated , a commoConsidering the physicochemical properties governing BBB permeability presented in between the gaps of neighbouring cells. This process is restricted and generally limited to diffusion of small ions and water molecules. The remarkably restrictive properties of the BBB’s paracellular flux is due to the high concentration of tight junctions (TJs) found within the intercellular cleft between neighbouring brain microvascular endothelial cells (BMECs). Tight junctions are composed of a branching network of ‘sealing strands’, with each strand formed between integral membrane proteins embedded in the membranes of neighbouring endothelial cells. The extracellular domains of each transcellular protein associate with one another, forming stable barriers that can also adapt to the environment and exhibit significant plasticity. Tight junction proteins include occludin, claudins and junctional adhesion molecules. The strength and rigidity of these proteins is due to their interactions on the inner side of the cell with a membrane-associated cytoplasmic protein; the most prominent of which are zonula occludens (ZOs). Zonula occludens act as scaffolding proteins by binding to both TJ proteins and the cytoskeleton via actin filaments. The resultant structural organisation creates a network of strongly embedded TJ proteins, giving rise to tightly sealed paracellular gaps  deconstruct cell–cell contacts, widening their tight junctions and facilitating paracellular transport of chemical species larger than small monoatomic ions. Both approaches have advantages and limitations. While transcellular modulators facilitate a more selective solute passage, drug uptake is far less efficient. Transcellular-mediated systems are dependent in both energy and active binding; thus, their kinetics are restricted. Conversely, paracellular modulation enhances passive broadscale passage of solutes, whereby it effectively ‘opens the floodgates’—the opening of which however can be attenuated. Here, we provide a comprehensive summary of the various BBB modulator classes, comparing their advantages and limitations while analysing their current status towards implementation in the clinic.BBB modulators have been under investigation for five decades  with mulFocused ultrasound (FUS) is an approach that utilises sound waves to induce a mechanical or thermal effect on targeted tissue without the need for radiation or surgical intervention . SimilarThe disruption of endothelial cells by ultrasound was first reported in 1953 by Lehmann et Herrick  who repoAs previously mentioned, doxorubicin cannot cross the BBB and is an ineffective chemotherapeutic agent for glioblastoma, unless highly invasive procedures are employed . The incCisplatin is a common anticancer drug used to treat extracranial cancers. Its high plasma protein binding affinity leads to a low bioavailability of 5–10%, which combined with its high toxicity and poor BBB penetration, makes it an ineffective brain cancer treatment without reaching systemically toxic levels in patients ,46. A goAnother Cisplatin-ferrying nanoparticle conjugate is a PEGylated polyaspartic acid organic nanoparticle that displayed enhanced BBB and Blood–Tumour Barrier (BTB) permeability when used in conjunction with MRgFUS. After three MRgFUS sessions, a significant 6-fold increase in nanoparticle BBB/BTB traversal was measured in one in vivo glioma model (9 L), and a staggering 26-fold in another model F98; . In addiMRgFUS gene delivery has also produced noteworthy results when utilising viral vectors as vehicles for transporting genes ,52,53,54Recently, a phase I clinical trial was carried out to determine if MRgFUS could induce amyloid-β clearance from the brain parenchyma of five Alzheimer’s patients . No signAnother phase I clinical study utilising MRgFUS in the delivery of anticancer drugs across the BBB/BTB was evaluated . MainpriMRgFUS BBB modulation was evaluated as a method for enabling treatment of amyotrophic lateral sclerosis (ALS) in a phase I clinical study involving four patients . Gadolinv/v aq solution), injected within a 30 to 40 s timeframe is required [Hyperosmolar agents are well-established TJ modulators that have been utilised as BBB modulators for five decades . Dehydrarequired . BBB disrequired ,64,65. Rrequired . Despiterequired .The toxicity of mannitol has been contested, with some studies reporting seizures among patients in addition to adverse cardiac effects . AmeliorAs mannitol is indiscriminate in its BBB disruption, widescale neural distribution of therapeutics can lead to systemic neurotoxicity in medications of considerate toxicity. Chemotherapeutics such as cisplatin, bleomycin, 5-fluorouracil, mitomycin, and vincristine are acutely neurotoxic and are incompatible with mannitol-based BBBD, while less neurotoxic agents such as cyclophosphamide, methotrexate, and carboplatin may be utilised in conjunction ,72.Noteworthy is the generally inconsistent duration of barrier disruption reported by researchers. For example, Joshi et al. observed that barrier opening duration was inconsistent even within one subject; rabbit brain vasculature opened up for various periods of time at different sites, as observed by optical measurements . On averAlthough mannitol’s use as a BBB modulator was initially reported five decades ago, its presence in this area remains relevant and continues to be implemented with cutting edge therapies such as gene silencing  and nanoTo augment the specificity of mannitol BBBD, a method that utilises microcatheters to deliver therapeutics to a specific vascular territory—Superelective Intra-Arterial Infusion (SIACI)—has been developed, enabling the delivery of a high drug concentration at the desired endovascular site . This taInterestingly, arabinose has also been reported to disrupt the BBB  in a sim2+ levels [Endogenous inflammatory mediators have a marked effect on vascular permeabilisation. Histamine and leukotrienes are particularly well understood in this regard ,93. All + levels . Doses o+ levels ,101 , Cisplatin 125-fold), Vancomycin (15-fold) and Gentamicin (12-fold) in the ipsilateral hemisphere compared to injection in the absence of alkylglycerols -fold, Va,127. In Investigating alkylglycerol’s mechanism of action, Hülper et al. found cytoplasmic redistribution and internalisation of both claudin-5 and β-catenin upon exposure to both 1-O-pentylglycerol and 2-O-hexyldiglycerol, as revealed by immunohistochemistry . Since iSodium Caprate (C10), is a medium-chain fatty acid  that incThe BBBD of sodium caprate was confirmed by Preston et al., whereby 15–25 mM modulated the BBB reversibly for 1 h, increasing mannitol (tracer) flux 10-fold—levels similar to hyperosmolar and alkylglycerols agents . HoweverCoadministration of an anti-hypertensive agent alleviated the high blood pressure, removing mannitol flux inconsistencies among the subject population . Sodium Mechanistic insight was provided by Del Vecchio et al., whereby claudin-5 trans interactions were decreased, coupled with a 61% claudin-5 reduction at cell contacts and F-actin internalisation in MDCK-II cells . Immunos2A adenosine receptor agonist used as a vasodilator during pharmacologic stress tests, has been reported to increase BBB permeability by inhibiting the predominantly expressed [2A receptors [Regadenoson, an Axpressed  efflux txpressed ,144, as xpressed   in endothelial cells as a target for BBB modulation. S1P receptors are G protein-coupled receptors (GPCRs), that regulate cell migration, adhesion, survival and proliferation . Endotherenchyma . Interesrenchyma .The size-selective BBB opening observed by Yanagida et al. is similar to that previously reported by Keaney et al., where both claudin-5 and occludin were knocked out ; howeverNS1619, a BK channel activator, has been shown to induce a 4-fold increase in paracellular flux of a 44 kDa tracer across the blood–tumour barrier (BTB) by downregulating expression of both claudin-5 and occludin  Table 3Table 3. Wang et al. have recently described the intra-arterial injection of NEO100—a highly purified monoterpene (S)-perillyl alcohol, which has been shown to reversibly and dose-dependently open the BBB without toxicity in vivo . Full reIn a recent publication, Breitkreuz-Korff et al. reported M01; a derivative of Nalidixic acid, having a potent effect on BBB modulation . Intrave1 and B2. B1 expression is activated in response to chronic pain and inflammation, whilst B2 is constitutively expressed and mediates vasodilation and tissue permeability [2 is a GPCR that, upon activation, results in a reduction in cAMP levels, upregulation of myosin light-chain kinase phosphorylation with subsequent opening of TJs by actin–myosin contraction and the formation of stress fibers [2, maintains an enhanced plasma half-life relative to bradykinin and exhibits efficacy as a BBB modulator [A common effect observed by inflammatory mediators is their ability to increase BBB permeability temporarily  [Zonula toxin (Zot) is a 45 kDa protein produced by the pathogenic bacterium cholerae . Zot inccholerae . Zot is cholerae , which icholerae . The 12 cholerae  and endoae [app)  , was isolated , and wasThe refinement of Zot to a hexapeptide simplifies ease of synthesis; unfortunately however, potency was reduced by five orders of magnitude  Table 3Table 3. 2-activating motif. Subsequent PAR-2 activation would increase PKCα-dependent ZO-1 and myosin 1C serine/threonine phosphorylation, inducing selective disassociation of ZO-1 from its TJ protein-binding partners  and myosin 1C [Investigations into the mechanism of action of AT-1002 have revealed that the hexapeptide bears homology resembling a PARyosin 1C   mimicking the second half of the first ECL have shown barrier opening effects in epithelial and endothelial cells ,202. Pepα-carbon stereochemical centers from L to D-configurations (D-aa-C5C2) created a more efficacious and pharmacokinetically stable peptide [Dithmer et al. developed a claudin-5-targeting peptide mimicking the first ECL loop of claudin-5 which displayed BBB modulating activity . C5C2 de peptide . An in v peptide . Due to  peptide , a simil2 to ≈900 Ωcm2 was observed following OCC2 administration, while no significant TEER change was observed for OCC1—a synthetic peptide based on the first extracellular domain of occludin [A synthetic peptide based on the second extracellular loop of chicken occludin (OCC2) has been shown to reversibly disrupt the transepithelial permeability of the barrier in Xenopus kidney epithelial cells. A TEER reduction of ≈6000 ΩVcmoccludin . OCC2 reoccludin . Conversoccludin . Interesoccludin ,211. Desoccludin , the selClostridium perfringens iota-toxin (amino acids 421–664) [Deviating from targeting proteins found at bicellular TJs (bTJs), Angubindin-1, a section from the Ib domain of the 421–664)  was repo421–664) . Angubin421–664) . Since tGintonin, an extract from ginseng, was reported to rapidly modulate the BBB and grant high BBB traversal for macromolecules between 10–70 kDa . An in vMechanistic investigations into Gintonin’s effect at the BBB have revealed that the active component(s) bind to lysophosphatidic acid receptors 1 and 3 (LPA-1/3). Their activation, along with downstream ROCK pathway activity, induces morphological changes and subsequent redistribution of VE-cadherin, claudin-5, occludin and ZO-1 away from TJs; creating temporarily vulnerable paracellular gaps . DespiteSimilar to the approach of targeting claudin-5 via synthetic ECL peptides taken by Staat et al. , HashimoThe high specificity of antibodies has also been applied to transcellular shuttling of therapeutics across the BBB. Iduronate 2-sulfatase (IDS), a therapeutic enzyme used to treat lysosomal storage diseases, was incorporated by Ullman et al. into the FC domain of human Immunoglobulin G1 (IgG1) . ConjugaClaudin-5 silencing by siRNA has been shown to enhance BBB permeability towards biotin (600Da)  in additMore recently, siRNA conjugates were shown to penetrate brain microvascular endothelial cells in vitro. A block copolymeric nanoparticle coated with a transferrin receptor peptide substrate to facilitate receptor-mediated transcytosis encapsulated P-glycoprotein (P-gp) siRNA and reduced P-gp expression by up to 52%. The resultant curbing of P-gp efflux enhanced the BBB permeability of rhodamine 123 by up to 27% relative to standard P-gp activity, 92 h after cell treatment  Table 3Table 3. Antisense oligonucleotides represent a novel class of synthetic nucleic acid molecules that are capable of bringing about changes in gene expression, as detailed by Rinaldi et Wood . In thisCurrently, over half (25) of the active clinical trials in the area of BBB modulation are considering the application of focused ultrasound-based BBB disruption (FUS BBBD) in the treatment of patients with CNS diseases . AdditioClinical trial progression in the area has been hindered by a number of challenges identified at the Focused Ultrasound & Blood–Brain Barrier Workshop held in 2017 by the Focused Ultrasound Foundation (FUF) . For exaA potential downside to the technology is the reliance of MRgFUS on MRI guidance which places a heavy dependency on expensive infrastructure currently not found in most clinics worldwide. If this technique is deemed safe and effective for treating a wide range of CNS diseases, patient demand will be immense and aggressive expansion of MRI infrastructure will be required as the current state would render MRgFUS BBBD an unfeasible approach.® developed by CarThera [® is a low intensity ultrasound transducer that is precisely implanted into the cranium of patients over the target site and removes the need for real-time MRI guidance, requiring only an ultrasound source. A drawback of the device requiring implantation following either surgical resection of a tumour, via burr hole or via routine surgery under local anaesthesia is also a strength of the device, as barriers to successfully delivering ultrasound via FUS such as skull thickness and unevenness are removed.Treatments that adopt single or infrequent dosing regimens such as gene therapy might be particularly suited to overcoming potential clinical limitations . Recent CarThera ,232. SonAs mentioned prior, mannitol is indiscriminate with respect to BBB disruption; however, this may be advantageous in the context of therapy for CNS diseases affecting widespread brain tissue, such as neurological genetic disorders and metastasised cancers ,234,235.A recent preprint by Linville et al. describes the reversible opening of the BBB following administration of the 26-residue amphipathic peptide, melittin . Melittiα-tocopherol as a guide-ligand to their ASO construct  tumor vasculature in mice enabled increased levels of docetaxel to cross the brain tumor vasculature relative to normal BBB vasculature . HoweverAlthough completed phase I clinical trials  have deeImportantly, FUS is not the only approach to report this occurrence. Mannitol-based BBB modulation was reported to induce a sterile inflammation response (SIR) resemblant to FUS just minutes after intracarotid arterial administration of mannitol . Burks eWhile a large cohort of studies deem the reported SIR to be safe and manageable, inducing immune cell trafficking across the BBB may not be compatible in treating autoimmune CNS diseases such as multiple sclerosis; these modulative approaches may aggravate an already highly active disease. Hence, alternative approaches to BBB modulation will likely need to be developed alongside these approaches to increase the likelihood of delivering successful therapies for general and autoimmune CNS diseases alike.Gaining control over BBB regulation opens up the possibility of facilitating delivery of targeted therapeutics to treat brain tumours  and neurodegenerative disorders. This review has highlighted the key developments helping to drive the field forward and enable both the potent and selective modulation of the BBB. Due to the extensive surface area of the vascular system, a systemically distributed compound that avoids a device or surgical conjunctive therapy will likely need to be of exceptional potency to facilitate efficacious BBB opening and negate untoward side effects. In this regard, we strongly advocate investigation into additional targets and pathways that may offer gains in selectivity over other areas of the body. The latest clinical trial data points towards the potential future implementation of a drug–device combination using either FUS or SIACI to ensure highly localised drug delivery to the CNS, and stands to revolutionise current treatment options once the safety profiles of these approaches have been comprehensively investigated.