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Axonal growth is limited or absent following peripheral or central nervous system injury respectively, inhibiting repair. The identification of novel growth-promoting molecular mechanisms is therefore a priority. In the search for dietary-dependent mechanisms that control neuronal regenerative ability, we discovered that growth-promoting intermittent fasting (IF) induced leptin expression in sensory neurons of the dorsal root ganglia (DRG). Surprisingly, leptin signalling, whose canonical function is to control energy homeostasis, was found to be central to the IF-dependent regenerative phenotype. In fact, neuronal conditional deletion of the leptin receptor significantly impaired the regenerative response elicited by IF. Overexpression of leptin in vivo in DRG neurons enhanced axonal regeneration following peripheral sciatic nerve crush (SNC) and central spinal cord injury (SCI). Lastly, RNA sequencing following leptin overexpression in DRG neurons showed a significant increase in regenerative gene expression and transcription after SCI, indicating a role for leptin in inducing a euchromatic, transcriptionally active environment that facilitates nervous system repair after injury.

To explore the clinical features and genetic basis of a patient with glycogen storage disease type VI (GSD-VI).Clinical data of the patient was collected. Genomic DNA was extracted from peripheral blood samples of the proband and his parents. Genetic variants were detected by using whole exome sequencing. Candidate variants were verified by Sanger sequencing followed by bioinformatics analysis.The proband presented fasting hypoglycemia, hepatomegaly, growth retardation, transaminitis, metabolic acidosis and hyperlactatemia. Liver biopsy indicated GSD. Novel compound heterozygous PYGL gene variants (c.2089AG/c.158_160delACT) were detected in the proband. Compound heterozygosity was confirmed by Sanger sequencing of the patient's genomic DNA. Provean and MutationTaster predicted the two variants as deleterious and the variant sites are highly conserved.The compound heterozygous variants (c.2089AG/c.158_160delACT) of PYGL gene probably underlay the GSD in the patient. The two novel variants have expanded the spectrum of PYGL gene variants and provided the basis for genetic counseling of the family.

The interruption of spinal circuitry following spinal cord injury disrupts neural activity and is followed by a failure to mount an effective regenerative gene expression response resulting in permanent neurological disability. Functional recovery requires the enhancement of axonal and synaptic plasticity of spared as well as injured fibres, which need to sprout and/or regenerate to form new connections. Here we will investigate whether the combined epigenetic stimulation of the regenerative gene expression program with neuronal activity-dependent enhancement of neuroplasticity and guidance can overcome the current inability to promote neurological recovery in severe and chronic spinal cord injury. This will be carried out by delivering the small molecule CBP/p300 activator CSP-TTK21 in combination with environmental enrichment. We will deliver the CBP/p300 activator CSP-TTK21 weekly between week twelve and twenty following a severe transection model of spinal cord injury in the mouse. Data analysis will assess modifications in regenerative signalling, in motor and sensory axon sprouting and regeneration, in synaptic plasticity as well as in neurological sensorimotor recovery. This approach will advance our mechanistic understanding of regenerative failure and provide insight of whether combining growth promoting strategies with enhancement of neural activity is an effective therapeutic strategy in chronic spinal cord injury.

The interruption of spinal circuitry following spinal cord injury (SCI) disrupts neural activity and is followed by a failure to mount an effective regenerative response resulting in permanent neurological disability. Functional recovery requires the enhancement of axonal and synaptic plasticity of spared as well as injured fibres, which need to sprout and/or regenerate to form new connections. Here, we have investigated whether the epigenetic stimulation of the regenerative gene expression program can overcome the current inability to promote neurological recovery in chronic SCI with severe disability. We delivered the CBP/p300 activator CSP-TTK21 or vehicle CSP weekly between week 12 and 22 following a transection model of SCI in mice housed in an enriched environment. Data analysis showed that CSP-TTK21 enhanced classical regenerative signalling in dorsal root ganglia sensory but not cortical motor neurons, stimulated motor and sensory axon growth, sprouting, and synaptic plasticity, but failed to promote neurological sensorimotor recovery. This work provides direct evidence that clinically suitable pharmacological CBP/p300 activation can promote the expression of regeneration-associated genes and axonal growth in a chronic SCI with severe neurological disability.

The regenerative potential of mammalian peripheral nervous system (PNS) neurons after injury is critically limited by their slow axonal regenerative rate1. Since a delayed target re-innervation leads to irreversible loss of function of target organs2, accelerated axonal regeneration is required to enhance functional outcomes following injury. Regenerative ability is influenced by both injury-dependent and injury-independent mechanisms3. Among the latter, environmental factors such as exercise and environmental enrichment have been shown to affect signalling pathways that promote axonal regeneration4. Several of these pathways, including modifications in gene transcription and protein synthesis, mitochondrial metabolism and release of neurotrophins, can be activated by intermittent fasting (IF)5,6. IF has in turn been shown to increase synaptic plasticity7,8 and neurogenesis9, partially sharing molecular mechanisms with axonal regeneration. However, whether IF influences the axonal regenerative ability remains to be investigated. Here we show that IF promotes axonal regeneration after sciatic nerve crush in the mouse via an unexpected mechanism that relies upon the gram + gut microbiome and an increase of the gut bacteria-derived metabolite indole-3-propionic acid (IPA) in the serum. IPA production by Clostridium sporogenes is required for efficient axonal regeneration, and delivery of IPA after sciatic injury significantly enhances axonal regeneration, accelerating recovery of sensory function. Mechanistically, RNA sequencing analysis from sciatic dorsal root ganglia suggested a role for neutrophil chemotaxis in the IPA-dependent regenerative phenotype that was confirmed by the inhibition of neutrophil chemotaxis. Our results demonstrate for the first time the ability of a microbiome derived metabolite, such as IPA, in facilitating regeneration and functional recovery of sensory axons via an immune-mediated mechanism. Since IPA is a naturally occurring metabolite with a very favourable toxicity profile, it represents a realistic translational possibility for human axonal injuries.

The regenerative potential of mammalian peripheral nervous system neurons after injury is critically limited by their slow axonal regenerative rate

Regeneration after injury occurs in axons that lie in the peripheral nervous system but fails in the central nervous system, thereby limiting functional recovery. Differences in axonal signalling in response to injury that might underpin this differential regenerative ability are poorly characterized. Combining axoplasmic proteomics from peripheral sciatic or central projecting dorsal root ganglion (DRG) axons with cell body RNA-seq, we uncover injury-dependent signalling pathways that are uniquely represented in peripheral versus central projecting sciatic DRG axons. We identify AMPK as a crucial regulator of axonal regenerative signalling that is specifically downregulated in injured peripheral, but not central, axons. We find that AMPK in DRG interacts with the 26S proteasome and its CaMKIIα-dependent regulatory subunit PSMC5 to promote AMPKα proteasomal degradation following sciatic axotomy. Conditional deletion of AMPKα1 promotes multiple regenerative signalling pathways after central axonal injury and stimulates robust axonal growth across the spinal cord injury site, suggesting inhibition of AMPK as a therapeutic strategy to enhance regeneration following spinal cord injury.

Cyclin-dependent-like kinase 5 (CDKL5) gene mutations lead to an X-linked disorder that is characterized by infantile epileptic encephalopathy, developmental delay, and hypotonia. However, we found that a substantial percentage of these patients also report a previously unrecognized anamnestic deficiency in pain perception. Consistent with a role in nociception, we found that CDKL5 is expressed selectively in nociceptive dorsal root ganglia (DRG) neurons in mice and in induced pluripotent stem cell (iPS)-derived human nociceptors. CDKL5-deficient mice display defective epidermal innervation, and conditional deletion of CDKL5 in DRG sensory neurons impairs nociception, phenocopying CDKL5 deficiency disorder in patients. Mechanistically, CDKL5 interacts with calcium/calmodulin-dependent protein kinase II α (CaMKIIα) to control outgrowth and transient receptor potential cation channel subfamily V member 1 (TRPV1)-dependent signaling, which are disrupted in both CDKL5 mutant murine DRG and human iPS-derived nociceptors. Together, these findings unveil a previously unrecognized role for CDKL5 in nociception, proposing an original regulatory mechanism for pain perception with implications for future therapeutics in CDKL5 deficiency disorder.

Reactive oxygen species (ROS) contribute to tissue damage and remodelling mediated by the inflammatory response after injury. Here we show that ROS, which promote axonal dieback and degeneration after injury, are also required for axonal regeneration and functional recovery after spinal injury. We find that ROS production in the injured sciatic nerve and dorsal root ganglia requires CX3CR1-dependent recruitment of inflammatory cells. Next, exosomes containing functional NADPH oxidase 2 complexes are released from macrophages and incorporated into injured axons via endocytosis. Once in axonal endosomes, active NOX2 is retrogradely transported to the cell body through an importin-β1-dynein-dependent mechanism. Endosomal NOX2 oxidizes PTEN, which leads to its inactivation, thus stimulating PI3K-phosporylated (p-)Akt signalling and regenerative outgrowth. Challenging the view that ROS are exclusively involved in nerve degeneration, we propose a previously unrecognized role of ROS in mammalian axonal regeneration through a NOX2-PI3K-p-Akt signalling pathway.

The molecular mechanisms discriminating between regenerative failure and success remain elusive. While a regeneration-competent peripheral nerve injury mounts a regenerative gene expression response in bipolar dorsal root ganglia (DRG) sensory neurons, a regeneration-incompetent central spinal cord injury does not. This dichotomic response offers a unique opportunity to investigate the fundamental biological mechanisms underpinning regenerative ability. Following a pharmacological screen with small molecule inhibitors targeting key epigenetic enzymes in DRG neurons we identified HDAC3 signalling as a novel candidate brake to axonal regenerative growth. In vivo, we determined that only a regenerative peripheral but not a central spinal injury induces an increase in calcium, which activates protein phosphatase 4 that in turn dephosphorylates HDAC3 thus impairing its activity and enhancing histone acetylation. Bioinformatics analysis of ex vivo H3K9ac ChIPseq and RNAseq from DRG followed by promoter acetylation and protein expression studies implicated HDAC3 in the regulation of multiple regenerative pathways. Finally, genetic or pharmacological HDAC3 inhibition overcame regenerative failure of sensory axons following spinal cord injury. Together, these data indicate that PP4-dependent HDAC3 dephosphorylation discriminates between axonal regeneration and regenerative failure.

In the version of this Article originally published, the affiliations for Roland A. Fleck and Jose Antonio Del Rio were incorrect due to a technical error that resulted in affiliations 8 and 9 being switched. The correct affiliations are: Roland A. Fleck: 8Centre for Ultrastructural Imaging, Kings College London, London, UK. Jose Antonio Del Rio: 2Cellular and Molecular Neurobiotechnology, Institute for Bioengineering of Catalonia, Barcelona, Spain; 9Department of Cell Biology, Physiology and Immunology, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain; 10Centro de Investigacion Biomedica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Barcelona, Spain. This has now been amended in all online versions of the Article.

Src homology-2 domain-containing protein tyrosine phosphatase (Shp2), a novel proto-oncogenic protein, is an important target in cancer therapy research. Approximately 2000 plant extracts were screened to find its natural specific inhibitors, with the ethyl acetate (EtOAc) active extract of the root of Angelica dahurica showing considerable inhibitory effects (IC(50)=21.6 mg/L). Bioguided isolation of EtOAc extract led to 13 compounds, including 10 fatty acids and derivatives. All these compounds were isolated from the plant for the first time. The inhibitory effects of these compounds on the enzyme activities of Shp2, VH1-related human protein (VHR), and hematopoietic protein tyrosine phosphatase (HePTP) were investigated. 8Z,11Z-Feptadecadienoic acid (4), 14Z,17Z-tricosadienoic acid (5), caffeic acid (9), and 2-hydroxy-3-[(1-oxododecyl) oxy]propyl-β-d-glucopyranoside (11) showed considerable selective inhibition of Shp2 activity. After treatment of HepG2 cells with the compounds, only compound 5, a polyunsaturated fatty acid, strongly induced poly (ADP-ribose) polymerase (PARP) cleavage in a dose- and time-dependent manner and increased the activities of caspase-3, caspase-8, and caspase-9 at 100 μM. Compound 5 also inhibited colony formation of HepG2 cells in a dose-dependent manner. Thus, this study reported fatty acids as specific Shp2 inhibitors and provided the molecular basis of one active compound as novel potential anticancer drug.

Shp2 is a member of the protein tyrosine phosphatase (PTP) family, which regulates a variety of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation. Using a recombinant Shp2-GST protein as the target and GST as a counter target, we have identified two classes of single-stranded DNA aptamers that selectively bind to Shp2 with a K(d) in the nanomolar range. Structural studies of the most abundant sequence in the enriched library, HJ24, revealed a parallel G-quadruplex as the core binding domain. Furthermore, this aptamer was found to be an effective inhibitor of Shp2 phosphatase, an effect which was readily reversed by using the cDNA of HJ24. In view of these characteristics, this aptamer has the potential to be used for further development of Shp2 assays and therapeutics for the treatment of Shp2-dependent cancers and other diseases.

Tyrosine phosphatase Src-homology phosphotyrosyl phosphatase 2 (Shp2) was identified as a potential molecular target for therapeutic treatment of diabetes and obesity. However, there is still no systematic research on the enhancers for the Shp2 enzyme. The present study established a novel powerful model for the high-throughput screening of Shp2 enhancers and successfully identified a new specific Shp2 enhancer, oleanolic acid, from Chinese herbs.