Full recovery from spinal cord injury requires axon regeneration to re-establish motor and sensory pathways. In mammals, the failure of sensory and motor axon regeneration has many causes intrinsic and extrinsic to neurons, amongst which is the lack of adhesion molecules needed to interact with the damaged spinal cord. This study addressed this limitation by expressing the integrin adhesion molecule α9, along with its activator kindlin-1, in sensory neurons via adeno-associated viral (AAV) vectors. This enabled sensory axons to regenerate through spinal cord injuries and extend to the brainstem, restoring sensory pathways, touch sensation and sensory behaviours. One of the integrin ligands in the injured spinal cord is tenascin-C, which serves as a substrate for α9β1 integrin, a key receptor in developmental axon guidance. However, the adult PNS and CNS neurons lack this receptor. Sensory neurons were transduced with α9 integrin (which pairs with endogenous β1 to form a α9β1 tenascin receptor) together with the integrin activator kindlin-1. Regeneration from sensory neurons transduced with α9integrin and kindlin-1 was examined after C4 and after T10 dorsal column lesions with C6,7 and L4,5 sensory ganglia injected with AAV1 vectors. In animals treated with α9 integrin and kindlin-1, sensory axons regenerated through tenascin-C-expressing connective tissue strands and bridges across the lesions and then re-entered the CNS tissue. Many axons regenerated rostrally to the level of the medulla. Axons grew through the dorsal grey matter rather than their normal pathway the dorsal columns. Growth was slow, axons taking 12 weeks to grow from T10 to the medulla, a distance of 4-5 cm. Functional recovery was confirmed through cFos activation in neurons rostral to the injury after nerve stimulation and VGLUT1/2 staining indicating new synapse formation above the lesion. Behavioural recovery was seen in both heat and mechanical sensation, as well as tape removal tests. This approach demonstrates the potential of integrin-based therapies for long distance sensory axon regeneration and functional recovery following thoracic and partial recovery after cervical spinal cord injury.

• Keywords
• AAV vectors, Axon regeneration, Integrins, Kindlin, Sensory testing, Spinal cord injury,
• MeSH
• Axons MeSH
• Dependovirus genetics   MeSH
• Genetic Vectors MeSH
• Rats MeSH
• Disease Models, Animal MeSH
• Mice MeSH
• Sensory Receptor Cells * metabolism   physiology   pathology   MeSH
• Recovery of Function physiology   MeSH
• Spinal Cord Injuries * pathology   physiopathology   metabolism   MeSH
• Rats, Sprague-Dawley MeSH
• Nerve Tissue Proteins metabolism   genetics   MeSH
• Nerve Regeneration * physiology   MeSH
• Tenascin metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Nerve Tissue Proteins MeSH
• Tenascin MeSH

Neurons in the CNS lose regenerative potential with maturity, leading to minimal corticospinal tract (CST) axon regrowth after spinal cord injury (SCI). In young rodents, knockdown of PTEN, which antagonizes PI3K signaling by hydrolyzing PIP3, promotes axon regeneration following SCI. However, this effect diminishes in adults, potentially due to lower PI3K activation leading to reduced PIP3. This study explores whether increased PIP3 generation can promote long-distance regeneration in adults. We used a hyperactive PI3K, PI3Kδ (PIK3CD), to boost PIP3 levels in mature cortical neurons and assessed CST regeneration after SCI. Adult rats received AAV1-PIK3CD and AAV1-eGFP, or AAV1-eGFP alone, in the sensorimotor cortex concurrent with a C4 dorsal SCI. Transduced neurons showed increased pS6 levels, indicating elevated PI3K/Akt/mTOR signaling. CST regeneration, confirmed with retrograde tracing, was evaluated up to 16 weeks post injury. At 12 weeks, ∼100 axons were present at lesion sites, doubling to 200 by 16 weeks, with regeneration indices of 0.1 and 0.2, respectively. Behavioral tests showed significant improvements in paw reaching, grip strength, and ladder-rung walking in PIK3CD-treated rats, corroborated by electrophysiological recordings of cord dorsum potentials and distal flexor muscle electromyography. Thus, PI3Kδ upregulation in adult cortical neurons enhances axonal regeneration and functional recovery post SCI.

• Keywords
• CST, PI3K, axon regeneration, c-Fos, electrophysiology, pS6, signaling, skilled paw reaching, spinal cord, spinal cord injury,
• MeSH
• Axons metabolism   MeSH
• Dependovirus genetics   MeSH
• Class I Phosphatidylinositol 3-Kinases * genetics   metabolism   MeSH
• Phosphatidylinositol 3-Kinases * genetics   metabolism   MeSH
• Genetic Vectors genetics   MeSH
• Rats MeSH
• Disease Models, Animal MeSH
• Neurons metabolism   MeSH
• Spinal Cord Injuries * therapy   metabolism   physiopathology   genetics   MeSH
• Pyramidal Tracts * metabolism   physiology   MeSH
• Nerve Regeneration * genetics   MeSH
• Signal Transduction MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Class I Phosphatidylinositol 3-Kinases * MeSH
• Phosphatidylinositol 3-Kinases * MeSH

Mammalian neurons lose the ability to regenerate their central nervous system axons as they mature during embryonic or early postnatal development. Neuronal maturation requires a transformation from a situation in which neuronal components grow and assemble to one in which these components are fixed and involved in the machinery for effective information transmission and computation. To regenerate after injury, neurons need to overcome this fixed state to reactivate their growth programme. A variety of intracellular processes involved in initiating or sustaining neuronal maturation, including the regulation of gene expression, cytoskeletal restructuring and shifts in intracellular trafficking, have been shown to prevent axon regeneration. Understanding these processes will contribute to the identification of targets to promote repair after injury or disease.

• MeSH
• Axons * physiology   MeSH
• Humans MeSH
• Neurogenesis * physiology   MeSH
• Neurons physiology   MeSH
• Nerve Regeneration * physiology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review 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
• Actins genetics   metabolism   MeSH
• Cytomegalovirus Infections * genetics   metabolism   MeSH
• Dependovirus genetics   metabolism   MeSH
• Genetic Vectors genetics   MeSH
• Humans MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Parvovirinae * genetics   MeSH
• Retinal Ganglion Cells metabolism   MeSH
• Transduction, Genetic MeSH
• Transgenes MeSH
• Green Fluorescent Proteins genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Actins MeSH
• Green Fluorescent Proteins MeSH

Regeneration capacity is reduced as CNS axons mature. Using laser-mediated axotomy, proteomics and puromycin-based tagging of newly-synthesized proteins in a human embryonic stem cell-derived neuron culture system that allows isolation of axons from cell bodies, we show here that efficient regeneration in younger axons (d45 in culture) is associated with local axonal protein synthesis (local translation). Enhanced regeneration, promoted by co-culture with human glial precursor cells, is associated with increased axonal synthesis of proteins, including those constituting the translation machinery itself. Reduced regeneration, as occurs with the maturation of these axons by d65 in culture, correlates with reduced levels of axonal proteins involved in translation and an inability to respond by increased translation of regeneration promoting axonal mRNAs released from stress granules. Together, our results provide evidence that, as in development and in the PNS, local translation contributes to CNS axon regeneration.

• Keywords
• Axon regeneration, Axotomy, Human stem cells, In vitro live imaging, Local translation, Proteomics,
• MeSH
• Axons physiology   MeSH
• Embryonic Stem Cells physiology   MeSH
• Coculture Techniques MeSH
• Humans MeSH
• Protein Biosynthesis physiology   MeSH
• Nerve Regeneration physiology   MeSH
• Cellular Senescence physiology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural 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.

• Keywords
• axon growth, axon regeneration, inter-organelle membrane contact sites, organelles,
• MeSH
• Humans MeSH
• Organelles metabolism   pathology   MeSH
• Spinal Cord Injuries * metabolism   pathology   therapy   MeSH
• Nerve Regeneration * MeSH
• Growth Cones metabolism   pathology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review 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
• Axons metabolism   physiology   MeSH
• Central Nervous System physiology   MeSH
• Endoplasmic Reticulum genetics   metabolism   MeSH
• Endosomes metabolism   MeSH
• Phosphorylation MeSH
• Integrins metabolism   MeSH
• Rats MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Mutation MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Neurons metabolism   physiology   MeSH
• Neuroprotective Agents administration & dosage   MeSH
• Optic Nerve Injuries drug therapy   metabolism   pathology   MeSH
• Rats, Sprague-Dawley MeSH
• Protein Domains MeSH
• Nerve Regeneration * drug effects   MeSH
• Retina drug effects   physiology   MeSH
• Vesicular Transport Proteins administration & dosage   chemistry   genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Intramural MeSH
• Names of Substances
• Integrins MeSH
• Neuroprotective Agents MeSH
• Vesicular Transport Proteins MeSH
• ZFYVE27 protein, human MeSH Browser