Radhika Puttagunta

Mutations in a novel gene encoding a CRAL-TRIO domain cause human Cayman ataxia and ataxia/dystonia in the jittery mouse.

Cayman ataxia is a recessive congenital ataxia restricted to one area of Grand Cayman Island. Comparative mapping suggested that the locus on 19p13.3 associated with Cayman ataxia might be homologous to the locus on mouse chromosome 10 associated with the recessive ataxic mouse mutant jittery. Screening genes in the region of overlap identified mutations in a novel predicted gene in three mouse jittery alleles, including the first mouse mutation caused by an Alu-related (B1 element) insertion. We found two mutations exclusively in all individuals with Cayman ataxia. The gene ATCAY or Atcay encodes a neuron-restricted protein called caytaxin. Caytaxin contains a CRAL-TRIO motif common to proteins that bind small lipophilic molecules. Mutations in another protein containing a CRAL-TRIO domain, alpha-tocopherol transfer protein (TTPA), cause a vitamin E–responsive ataxia. Three-dimensional protein structural modeling predicts that the caytaxin ligand is more polar than vitamin E. Identification of the caytaxin ligand may help develop a therapy for Cayman ataxia.

HDAC inhibition promotes neuronal outgrowth and counteracts growth cone collapse through CBP/p300 and P/CAF-dependent p53 acetylation

Neuronal outgrowth is guided by both extrinsic and intrinsic factors, involving transcriptional regulation. The acetylation of histones and transcription factors, which facilitates promoter accessibility, ultimately promotes transcription, and depends on the balance between histone deacetylases (HDACs) and histone acetyltransferases (HATs) activities. However, a critical function for specific acetylation modifying enzymes in neuronal outgrowth has yet to be investigated. To address this issue, we have used an epigenetic approach to facilitate gene expression in neurons, by using specific HDAC inhibitors. Neurons treated with a combination of HDAC and transcription inhibitors display an acetylation and transcription-dependent increase in outgrowth and a reduction in growth cone collapse on both ‘permissive’ (poly-D-lysine, PDL) and ‘non-permissive’ substrates (myelin and chondroitin sulphate proteoglycans (CSPGs)). Next, we specifically show that the expression of the histone acetyltransferases CBP/p300 and P/CAF is repressed in neurons by inhibitory substrates, whereas it is triggered by HDAC inhibition on both permissive and inhibitory conditions. Gene silencing and gain of function experiments show that CBP/p300 and P/CAF are key players in neuronal outgrowth, acetylate histone H3 at K9-14 and the transcription factor p53, thereby initiating a pro-neuronal outgrowth transcriptional program. These findings contribute to the growing understanding of transcriptional regulation in neuronal outgrowth and may lay the molecular groundwork for the promotion of axonal regeneration after injury.

A p53-CBP/p300 transcription module is required for GAP-43 expression, axon outgrowth, and regeneration

Transcription regulates axon outgrowth and regeneration. However, to date, no transcription complexes have been shown to control axon outgrowth and regeneration by regulating axon growth genes. Here, we report that the tumor suppressor p53 and its acetyltransferases CBP/p300 form a transcriptional complex that regulates the axonal growth-associated protein 43, a well-characterized pro-axon outgrowth and regeneration protein. Acetylated p53 at K372-3-82 drives axon outgrowth, GAP-43 expression, and binds specific elements on the neuronal GAP-43 promoter in a chromatin environment through CBP/p300 signaling. Importantly, in an axon regeneration model, both CBP and p53 K372-3-82 are induced following axotomy in facial motor neurons, where p53 K372-3-82 occupancy of GAP-43 promoter is enhanced as shown by in vivo chromatin immunoprecipitation. Finally, by comparing wild-type and p53 null mice, we demonstrate that the p53/GAP-43 transcriptional module is specifically switched on during axon regeneration in vivo. These data contribute to the understanding of gene regulation in axon outgrowth and may suggest new molecular targets for axon regeneration.

PCAF-dependent epigenetic changes promote axonal regeneration in the central nervous system.

Axonal regenerative failure is a major cause of neurological impairment following central nervous system (CNS) but not peripheral nervous system (PNS) injury. Notably, PNS injury triggers a coordinated regenerative gene expression programme. However, the molecular link between retrograde signalling and the regulation of this gene expression programme that leads to the differential regenerative capacity remains elusive. Here we show through systematic epigenetic studies that the histone acetyltransferase p300/CBP-associated factor (PCAF) promotes acetylation of histone 3 Lys 9 at the promoters of established key regeneration-associated genes following a peripheral but not a central axonal injury. Furthermore, we find that extracellular signal-regulated kinase (ERK)-mediated retrograde signalling is required for PCAF-dependent regenerative gene reprogramming. Finally, PCAF is necessary for conditioning-dependent axonal regeneration and also singularly promotes regeneration after spinal cord injury. Thus, we find a specific epigenetic mechanism that regulates axonal regeneration of CNS axons, suggesting novel targets for clinical application.

Epigenetic Regulation of Axon Outgrowth and Regeneration in CNS Injury: The First Steps Forward

Inadequate axonal sprouting and lack of regeneration limit functional recovery following neurologic injury, such as stroke, brain, and traumatic spinal cord injury. Recently, the enhancement of the neuronal regenerative program has led to promising improvements in axonal sprouting and regeneration in animal models of axonal injury. However, precise knowledge of the essential molecular determinants of this regenerative program remains elusive, thus limiting the choice of fully effective therapeutic strategies. Given that molecular regulation of axonal outgrowth and regeneration requires carefully orchestrated waves of gene expression, both temporally and spatially, epigenetic changes may be an ideal regulatory mechanism to address this unique need. While recent evidence suggests that epigenetic modifications could contribute to the regulation of axonal outgrowth and regeneration following axonal injury in models of stroke, and spinal cord and optic nerve injury, a number of unanswered questions remain. Such questions require systematic investigation of the epigenetic landscape between regenerative and non-regenerative conditions for the potential translation of this knowledge into regenerative strategies in human spinal and brain injury, as well as stroke.

RA–RAR-β counteracts myelin-dependent inhibition of neurite outgrowth via Lingo-1 repression

Central nervous system injury results in the release of molecules that inhibit neuronal regeneration, but retinoic acid counteracts this effect by inhibiting Lingo-1.

Retinoic acid signaling in axonal regeneration.

Following an acute central nervous system (CNS) injury, axonal regeneration and functional recovery are extremely limited. This is due to an extrinsic inhibitory growth environment and the lack of intrinsic growth competence. Retinoic acid (RA) signaling, essential in developmental dorsoventral patterning and specification of spinal motor neurons, has been shown through its receptor, the transcription factor RA receptor β2 (RARβ2), to induce axonal regeneration following spinal cord injury (SCI). Recently, it has been shown that in dorsal root ganglion neurons (DRGs), cAMP levels were greatly increased by lentiviral RARβ2 expression and contributed to neurite outgrowth. Moreover, RARβagonists, in cerebellar granule neurons (CGN) and in the brain in vivo, induced phosphoinositide 3-kinase dependent phosphorylation of AKT that was involved in RARβ-dependent neurite outgrowth. More recently, RA-RARβpathways were shown to directly transcriptionally repress a member of the inhibitory Nogo receptor (NgR) complex, Lingo-1, under an axonal growth inhibitory environment in vitro as well as following spinal injury in vivo. This perspective focuses on these newly discovered molecular mechanisms and future directions in the field.

The MDM4/MDM2-p53-IGF1 axis controls axonal regeneration, sprouting and functional recovery after CNS injury.

Regeneration of injured central nervous system axons is highly restricted, causing neurological impairment. To date, although the lack of intrinsic regenerative potential is well described, a key regulatory molecular mechanism for the enhancement of both axonal regrowth and functional recovery after central nervous system injury remains elusive. While ubiquitin ligases coordinate neuronal morphogenesis and connectivity during development as well as after axonal injury, their role specifically in axonal regeneration is unknown. Following a bioinformatics network analysis combining ubiquitin ligases with previously defined axonal regenerative proteins, we found a triad composed of the ubiquitin ligases MDM4, MDM2 and the transcription factor p53 (encoded by TP53) as a putative central signalling complex restricting the regeneration program. Indeed, conditional deletion of MDM4 or pharmacological inhibition of MDM2/p53 interaction in the eye and spinal cord promote axonal regeneration and sprouting of the optic nerve after crush and of supraspinal tracts after spinal cord injury. The double conditional deletion of MDM4-p53 as well as MDM2 inhibition in p53-deficient mice blocks this regenerative phenotype, showing its dependence upon p53. Genome-wide gene expression analysis from ex vivo fluorescence-activated cell sorting in MDM4-deficient retinal ganglion cells identifies the downstream target IGF1R, whose activity and expression was found to be required for the regeneration elicited by MDM4 deletion. Importantly, we demonstrate that pharmacological enhancement of the MDM2/p53-IGF1R axis enhances axonal sprouting as well as functional recovery after spinal cord injury. Thus, our results show MDM4-MDM2/p53-IGF1R as an original regulatory mechanism for CNS regeneration and offer novel targets to enhance neurological recovery.media-1vid110.1093/brain/awv125_video_abstractawv125_video_abstract.

DNA methylation temporal profiling following peripheral versus central nervous system axotomy.

The regulatory mechanisms responsible for the gene expression pattern associated with axotomy-dependent signaling affecting the neuronal phenotype, including the axonal regenerative program, remain unclear. To further this understanding, we recently performed DNA methylation temporal profiling in lumbar dorsal root ganglia (DRG) after axotomy of the central spinal (non-regenerating) and of the peripheral sciatic nerve (regenerating) axonal branches. DNA methylation microarrays for mouse gene promoters and CpG islands (Roche/NimbleGen) were employed after immunoprecipitation of 5-methylcytosine-DNA. Here we provide a detailed data descriptor of this DNA methylation dataset, which allows in depth evaluation of the experimental design, assessment of data reproducibility and a full interactive operator-based systematic data analysis. In fact, we offer a methylation ‘hit’ scoring map of the whole microarray data in a workable spreadsheet that allows data sorting by genes, conditions or hits of interests that is ready for functional gene annotation and classification. This dataset allows investigators bioinformatic comparison to other epigenetic and gene expression datasets and further experimental characterization of the role of DNA methylation in axotomy-dependent pathways.

PP4-dependent HDAC3 dephosphorylation discriminates between axonal regeneration and regenerative failure

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. Graphical Abstract Following central nervous system (CNS) spinal injury, protein phosphatase 4/2 activity is not induced since calcium levels remain unchanged compared to uninjured conditions. HDAC3 remains phosphorylated and occupies deacetylated chromatin contributing to its compaction inhibiting gene expression. Following peripheral nervous system (PNS) sciatic injury, protein phosphatase 4/2 activity is induced by calcium. HDAC3 is dephosphorylated leading to its inhibition and release from chromatin sites contributing to increase in histone acetylation and in the expression of regeneration associated genes (RAGs).