The peripheral branch of sensory dorsal root ganglion (DRG) neurons regenerates readily after injury unlike their central branch in the spinal cord. However, extensive regeneration and reconnection of sensory axons in the spinal cord can be driven by the expression of α9 integrin and its activator kindlin-1 (α9k1), which enable axons to interact with tenascin-C. To elucidate the mechanisms and downstream pathways affected by activated integrin expression and central regeneration, we conducted transcriptomic analyses of adult male rat DRG sensory neurons transduced with α9k1, and controls, with and without axotomy of the central branch. Expression of α9k1 without the central axotomy led to upregulation of a known PNS regeneration program, including many genes associated with peripheral nerve regeneration. Coupling α9k1 treatment with dorsal root axotomy led to extensive central axonal regeneration. In addition to the program upregulated by α9k1 expression, regeneration in the spinal cord led to expression of a distinctive CNS regeneration program, including genes associated with ubiquitination, autophagy, endoplasmic reticulum (ER), trafficking, and signaling. Pharmacological inhibition of these processes blocked the regeneration of axons from DRGs and human iPSC-derived sensory neurons, validating their causal contributions to sensory regeneration. This CNS regeneration-associated program showed little correlation with either embryonic development or PNS regeneration programs. Potential transcriptional drivers of this CNS program coupled to regeneration include Mef2a, Runx3, E2f4, and Yy1. Signaling from integrins primes sensory neurons for regeneration, but their axon growth in the CNS is associated with an additional distinctive program that differs from that involved in PNS regeneration.SIGNIFICANCE STATEMENT Restoration of neurologic function after spinal cord injury has yet to be achieved in human patients. To accomplish this, severed nerve fibers must be made to regenerate. Reconstruction of nerve pathways has not been possible, but recently, a method for stimulating long-distance axon regeneration of sensory fibers in rodents has been developed. This research uses profiling of messenger RNAs in the regenerating sensory neurons to discover which mechanisms are activated. This study shows that the regenerating neurons initiate a novel CNS regeneration program which includes molecular transport, autophagy, ubiquitination, and modulation of the endoplasmic reticulum (ER). The study identifies mechanisms that neurons need to activate to regenerate their nerve fibers.

• MeSH
• axony * fyziologie   MeSH
• integriny metabolismus   MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• mícha metabolismus   MeSH
• nervové receptory fyziologie   MeSH
• poranění míchy * terapie   metabolismus   MeSH
• potkani Sprague-Dawley MeSH
• regenerace nervu fyziologie   MeSH
• spinální ganglia metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Recombinant adeno-associated viral vectors (AAVs) are an effective system for gene transfer. AAV serotype 2 (AAV2) is commonly used to deliver transgenes to retinal ganglion cells (RGCs) via intravitreal injection. The AAV serotype however is not the only factor contributing to the effectiveness of gene therapies. Promoters influence the strength and cell-selectivity of transgene expression. This study compares five promoters designed to maximise AAV2 cargo space for gene delivery: chicken β-actin (CBA), cytomegalovirus (CMV), short CMV early enhancer/chicken β-actin/short β-globulin intron (sCAG), mouse phosphoglycerate kinase (PGK), and human synapsin (SYN). The promoters driving enhanced green fluorescent protein (eGFP) were examined in adult C57BL/6J mice eyes and tissues of the visual system. eGFP expression was strongest in the retina, optic nerves and brain when driven by the sCAG and SYN promoters. CBA, CMV, and PGK had moderate expression by comparison. The SYN promoter had almost exclusive transgene expression in RGCs. The PGK promoter had predominant expression in both RGCs and AII amacrine cells. The ubiquitous CBA, CMV, and sCAG promoters expressed eGFP in a variety of cell types across multiple retinal layers including Müller glia and astrocytes. We also found that these promoters could transduce human retina ex vivo, although expression was predominantly in glial cells due to low RGC viability. Taken together, this promoter comparison study contributes to optimising AAV-mediated transduction in the retina, and could be valuable for research in ocular disorders, particularly those with large or complex genetic cargos.

• MeSH
• aktiny genetika   metabolismus   MeSH
• cytomegalovirové infekce * genetika   metabolismus   MeSH
• Dependovirus genetika   metabolismus   MeSH
• genetické vektory genetika   MeSH
• lidé MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• Parvovirinae * genetika   MeSH
• retinální gangliové buňky metabolismus   MeSH
• transdukce genetická MeSH
• transgeny MeSH
• zelené fluorescenční proteiny genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Adeno-associated viral vectors are widely used as vehicles for gene transfer to the nervous system. The promoter and viral vector serotype are two key factors that determine the expression dynamics of the transgene. A previous comparative study has demonstrated that AAV1 displays efficient transduction of layer V corticospinal neurons, but the optimal promoter for transgene expression in corticospinal neurons has not been determined yet. In this paper, we report a side-by-side comparison between four commonly used promoters: the short CMV early enhancer/chicken β actin (sCAG), human cytomegalovirus (hCMV), mouse phosphoglycerate kinase (mPGK) and human synapsin (hSYN) promoter. Reporter constructs with each of these promoters were packaged in AAV1, and were injected in the sensorimotor cortex of rats and mice in order to transduce the corticospinal tract. Transgene expression levels and the cellular transduction profile were examined after 6 weeks. The AAV1 vectors harbouring the hCMV and sCAG promoters resulted in transgene expression in neurons, astrocytes and oligodendrocytes. The mPGK and hSYN promoters directed the strongest transgene expression. The mPGK promoter did drive expression in cortical neurons and oligodendrocytes, while transduction with AAV harbouring the hSYN promoter resulted in neuron-specific expression, including perineuronal net expressing interneurons and layer V corticospinal neurons. This promoter comparison study contributes to improve transgene delivery into the brain and spinal cord. The optimized transduction of the corticospinal tract will be beneficial for spinal cord injury research.

• MeSH
• Dependovirus * genetika   MeSH
• genetické vektory genetika   MeSH
• krysa rodu rattus MeSH
• myši MeSH
• promotorové oblasti (genetika) MeSH
• pyramidové dráhy * MeSH
• transdukce genetická MeSH
• transgeny MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Investigating the molecular mechanisms governing developmental axon growth has been a useful approach for identifying new strategies for boosting axon regeneration after injury, with the goal of treating debilitating conditions such as spinal cord injury and vision loss. The picture emerging is that various axonal organelles are important centers for organizing the molecular mechanisms and machinery required for growth cone development and axon extension, and these have recently been targeted to stimulate robust regeneration in the injured adult central nervous system (CNS). This review summarizes recent literature highlighting a central role for organelles such as recycling endosomes, the endoplasmic reticulum, mitochondria, lysosomes, autophagosomes and the proteasome in developmental axon growth, and describes how these organelles can be targeted to promote axon regeneration after injury to the adult CNS. This review also examines the connections between these organelles in developing and regenerating axons, and finally discusses the molecular mechanisms within the axon that are required for successful axon growth.

• MeSH
• lidé MeSH
• organely metabolismus   patologie   MeSH
• poranění míchy * metabolismus   patologie   terapie   MeSH
• regenerace nervu * MeSH
• růstové kužele metabolismus   patologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Regeneration after spinal cord injury is a goal of many studies. Although the most obvious target is to recover motor function, restoration of sensation can also improve the quality of life after spinal cord injury. For many patients, recovery of sensation in the perineal and genital area is a high priority. Currently there is no experimental test in rodents for measuring changes in sensation in the perineal and genital area after spinal cord injury. The aim of our study was to develop a behavioural test for measuring the sensitivity of the perineal and genital area in rats. We have modified the tape removal test used routinely to test sensorimotor deficits after stroke and spinal cord injury to test the perineal area with several variations. A small piece of tape (approximately 1 cm2) was attached to the perineal area. Time to first contact and to the removal of the tape was measured. Each rat was trained for 5 consecutive days and then tested weekly. We compared different rat strains (Wistar, Sprague-Dawley, Long-Evans and Lewis), both genders, shaving and non-shaving and different types of tape. We found that the test was suitable for all tested strains, however, Lewis rats achieved the lowest contact times, but this difference was significant only for the first few days of learning the task. There were no significant differences between gender and different types of tape or shaving. After training the animals underwent dorsal column lesion at T10 and were tested at day 3, 8, 14 and 21. The test detected a sensory deficit, the average time across all animals to sense the stimulus increased from 1'32 up to 3'20. There was a strong relationship between lesion size and tape detection time, and only lesions that extended laterally to the dorsal root entry zone produced significant sensory deficits. Other standard behavioural tests (BBB, von Frey, ladder and Plantar test) were performed in the same animals. There was a correlation between lesion size and deficit for the ladder and BBB tests, but not for the von Frey and Plantar tests. We conclude that the tape removal test is suitable for testing perineal sensation in rats, can be used in different strains and is appropriate for monitoring changes in sensation after spinal cord injury.

• MeSH
• adaptace psychologická * MeSH
• chování zvířat MeSH
• druhová specificita MeSH
• fyzikální stimulace MeSH
• krysa rodu rattus MeSH
• kůže zranění   MeSH
• perineum zranění   fyziologie   MeSH
• pohlavní orgány zranění   MeSH
• poranění míchy psychologie   MeSH
• poruchy senzitivity etiologie   psychologie   MeSH
• potkani inbrední LEW MeSH
• potkani Long-Evans MeSH
• potkani Sprague-Dawley MeSH
• potkani Wistar MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Adult mammalian central nervous system axons have intrinsically poor regenerative capacity, so axonal injury has permanent consequences. One approach to enhancing regeneration is to increase the axonal supply of growth molecules and organelles. We achieved this by expressing the adaptor molecule Protrudin which is normally found at low levels in non-regenerative neurons. Elevated Protrudin expression enabled robust central nervous system regeneration both in vitro in primary cortical neurons and in vivo in the injured adult optic nerve. Protrudin overexpression facilitated the accumulation of endoplasmic reticulum, integrins and Rab11 endosomes in the distal axon, whilst removing Protrudin's endoplasmic reticulum localization, kinesin-binding or phosphoinositide-binding properties abrogated the regenerative effects. These results demonstrate that Protrudin promotes regeneration by functioning as a scaffold to link axonal organelles, motors and membranes, establishing important roles for these cellular components in mediating regeneration in the adult central nervous system.

• MeSH
• axony metabolismus   fyziologie   MeSH
• centrální nervový systém fyziologie   MeSH
• endoplazmatické retikulum genetika   metabolismus   MeSH
• endozomy metabolismus   MeSH
• fosforylace MeSH
• integriny metabolismus   MeSH
• krysa rodu rattus MeSH
• kultivované buňky MeSH
• lidé MeSH
• mutace MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• neurony metabolismus   fyziologie   MeSH
• neuroprotektivní látky aplikace a dávkování   MeSH
• poranění nervus opticus farmakoterapie   metabolismus   patologie   MeSH
• potkani Sprague-Dawley MeSH
• proteinové domény MeSH
• regenerace nervu * účinky léků   MeSH
• retina účinky léků   fyziologie   MeSH
• vezikulární transportní proteiny aplikace a dávkování   chemie   genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH

Axon regeneration in the CNS is inhibited by many extrinsic and intrinsic factors. Because these act in parallel, no single intervention has been sufficient to enable full regeneration of damaged axons in the adult mammalian CNS. In the external environment, NogoA and CSPGs are strongly inhibitory to the regeneration of adult axons. CNS neurons lose intrinsic regenerative ability as they mature: embryonic but not mature neurons can grow axons for long distances when transplanted into the adult CNS, and regeneration fails with maturity in in vitro axotomy models. The causes of this loss of regeneration include partitioning of neurons into axonal and dendritic fields with many growth-related molecules directed specifically to dendrites and excluded from axons, changes in axonal signalling due to changes in expression and localization of receptors and their ligands, changes in local translation of proteins in axons, and changes in cytoskeletal dynamics after injury. Also with neuronal maturation come epigenetic changes in neurons, with many of the transcription factor binding sites that drive axon growth-related genes becoming inaccessible. The overall aim for successful regeneration is to ensure that the right molecules are expressed after axotomy and to arrange for them to be transported to the right place in the neuron, including the damaged axon tip.

• MeSH
• axonální transport fyziologie   MeSH
• axony fyziologie   MeSH
• centrální nervový systém cytologie   fyziologie   MeSH
• lidé MeSH
• nervový útlum fyziologie   MeSH
• neurogeneze fyziologie   MeSH
• proteosyntéza fyziologie   MeSH
• regenerace nervu fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Peripheral nervous system (PNS) neurons support axon regeneration into adulthood, whereas central nervous system (CNS) neurons lose regenerative ability after development. To better understand this decline whilst aiming to improve regeneration, we focused on phosphoinositide 3-kinase (PI3K) and its product phosphatidylinositol (3,4,5)-trisphosphate (PIP3 ). We demonstrate that adult PNS neurons utilise two catalytic subunits of PI3K for axon regeneration: p110α and p110δ. However, in the CNS, axonal PIP3 decreases with development at the time when axon transport declines and regenerative competence is lost. Overexpressing p110α in CNS neurons had no effect; however, expression of p110δ restored axonal PIP3 and increased regenerative axon transport. p110δ expression enhanced CNS regeneration in both rat and human neurons and in transgenic mice, functioning in the same way as the hyperactivating H1047R mutation of p110α. Furthermore, viral delivery of p110δ promoted robust regeneration after optic nerve injury. These findings establish a deficit of axonal PIP3 as a key reason for intrinsic regeneration failure and demonstrate that native p110δ facilitates axon regeneration by functioning in a hyperactive fashion.

• MeSH
• axony * MeSH
• centrální nervový systém MeSH
• dospělí MeSH
• fosfatidylinositol-3-kinasy * MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• myši MeSH
• neurony MeSH
• regenerace nervu MeSH
• zvířata MeSH
• Check Tag
• dospělí MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The perineuronal net (PNN) is a mesh-like proteoglycan structure on the neuronal surface which is involved in regulating plasticity. The PNN regulates plasticity via multiple pathways, one of which is direct regulation of synapses through the control of AMPA receptor mobility. Since neuronal pentraxin 2 (Nptx2) is a known regulator of AMPA receptor mobility and Nptx2 can be removed from the neuronal surface by PNN removal, we investigated whether Nptx2 has a function in the PNN. We found that Nptx2 binds to the glycosaminoglycans hyaluronan and chondroitin sulphate E in the PNN. Furthermore, in primary cortical neuron cultures, the addition of NPTX2 to the culture medium enhances PNN formation during PNN development. These findings suggest Nptx2 as a novel PNN binding protein with a role in the mechanism of PNN formation.

• MeSH
• C-reaktivní protein metabolismus   MeSH
• krysa rodu rattus MeSH
• kultivované buňky MeSH
• nervová síť chemie   cytologie   metabolismus   MeSH
• neurony chemie   metabolismus   MeSH
• neuroplasticita fyziologie   MeSH
• perineuronální satelitní buňky chemie   metabolismus   MeSH
• potkani Sprague-Dawley MeSH
• proteiny nervové tkáně metabolismus   MeSH
• vazba proteinů fyziologie   MeSH
• zrakové korové centrum chemie   cytologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Perineuronal nets (PNNs) are extracellular matrix (ECM) chondroitin sulfate proteoglycan (CSPG)-containing structures that surround the soma and dendrites of various mammalian neuronal cell types. PNNs appear during development around the time that the critical periods for developmental plasticity end and are important for both their onset and closure. A similar structure - the perinodal ECM - surrounds the axonal nodes of Ranvier and appears as myelination is completed, acting as an ion-diffusion barrier that affects axonal conduction speed. Recent work has revealed the importance of PNNs in controlling plasticity in the CNS. Digestion, blocking or removal of PNNs influences functional recovery after a variety of CNS lesions. PNNs have further been shown to be involved in the regulation of memory and have been implicated in a number of psychiatric disorders.

• MeSH
• duševní poruchy patofyziologie   MeSH
• extracelulární matrix fyziologie   MeSH
• lidé MeSH
• modely neurologické MeSH
• nemoci centrálního nervového systému patofyziologie   MeSH
• neurony fyziologie   MeSH
• neuroplasticita fyziologie   MeSH
• paměť fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH