Neuritin 1 promotes retinal ganglion cell survival and axonal regeneration following optic nerve crush

Neuritin 1 (Nrn1) is an extracellular glycophosphatidylinositol-linked protein that stimulates axonal plasticity, dendritic arborization and synapse maturation in the central nervous system (CNS). The purpose of this study was to evaluate the neuroprotective and axogenic properties of Nrn1 on axotomized retinal ganglion cells (RGCs) in vitro and on the in vivo optic nerve crush (ONC) mouse model. Axotomized cultured RGCs treated with recombinant hNRN1 significantly increased survival of RGCs by 21% (n=6–7, P<0.01) and neurite outgrowth in RGCs by 141% compared to controls (n=15, P<0.05). RGC transduction with AAV2-CAG–hNRN1 prior to ONC promoted RGC survival (450%, n=3–7, P<0.05) and significantly preserved RGC function by 70% until 28 days post crush (dpc) (n=6, P<0.05) compared with the control AAV2-CAG–green fluorescent protein transduction group. Significantly elevated levels of RGC marker, RNA binding protein with multiple splicing (Rbpms; 73%, n=5–8, P<0.001) and growth cone marker, growth-associated protein 43 (Gap43; 36%, n=3, P<0.01) were observed 28 dpc in the retinas of the treatment group compared with the control group. Significant increase in Gap43 (100%, n=5–6, P<0.05) expression was observed within the optic nerves of the AAV2–hNRN1 group compared to controls. In conclusion, Nrn1 exhibited neuroprotective, regenerative effects and preserved RGC function on axotomized RGCs in vitro and after axonal injury in vivo. Nrn1 is a potential therapeutic target for CNS neurodegenerative diseases.Central nervous system (CNS) trauma and neurodegenerative disorders trigger a cascade of intrinsic and extrinsic cellular events resulting in regenerative failure and subsequent damage to neurons.^{1, 2, 3, 4, 5} The intrinsic factors include deregulation in growth-promoting factors, apoptotic factors, intracellular signaling molecules and trophic factors.⁶ Similarly, the extrinsic factors correlate to growth inhibition due to inhibitory cues^{3, 7, 8, 9, 10, 11, 12, 13} that include myelin and myelin associated inhibitors, glial scarring,^{5, 14} slow clearance of axonal debris,⁷ incorrect development of neuronal projections⁶ and CNS inflammation.^{15, 16} Progressive degeneration of mature retinal ganglion cells (RGCs) has been associated with loss of trophic support,^{8, 9} detrimental inflammatory processes/immune regulation^{10, 11} and apoptotic effectors.^{9, 12, 13, 15, 17}After injury, mammalian RGC axons show only a short-lived sprouting response but no long-distance regeneration through the optic nerve (ON).¹⁶ Glial responses around the affected area are initiated by injured CNS axons.¹⁸ Axons undergoing Wallerian degeneration are surrounded by astrocytes that upregulate glial fibrillary acidic protein (Gfap) expression and these reactive astrocytes contribute to trauma-induced neurodegeneration.¹⁹ Glial scarring inhibits axonal transport after ON crush (ONC)^{5, 14} decreasing transport of proteins involved in neuroprotection and synaptic plasticity. Regenerative failure is a critical endpoint of these destructive triggers culminating in neuronal apoptosis^{3, 20, 21} and inhibition of functional recovery. Intrinsic factors affecting axonal regeneration after CNS injury are crucial for recovery and thus, dysregulation of genes involved in axonal plasticity and outgrowth can prove detrimental to the neuronal recovery.^{22, 23, 24}Current neuroprotection approaches include promoting survival of RGCs by intraocular injections of recombinant factors like ciliary neurotrophic factor (CNTF) and peripheral nerve (PN) transplantations in vitro²⁵ and in vivo after injury.²⁶ Studies performed with glial cell-line-derived neurotrophic factor and neurturin protect RGCs from axotomy-induced apoptosis.²⁷ Further, in the ON injury model, RGC survival was promoted after deletion of CCAAT/enhancer binding protein homologous protein²⁸ and enhanced regeneration observed with co-deletion of kruppel-like factor 4 (Klf4) and suppressor of cytokine signaling 3 (Socs3).²⁹ Intraocular administration of neurotrophin-4 (NT-4) and brain-derived neurotrophic factor (BDNF) after ON transection has also exerted neuroprotective effects on axotomized RGCs. In addition, PNs transplanted adjacent to ONs, ex vivo PN grafts with lenti-viral transduced Schwann cells, and stimulation of inflammatory processes have strong pro-regenerative effects on injured RGCs.^{26, 30, 31, 32, 33}In addition, using adeno-associated-virus (AAV) therapy, AAV mediated expression of CNTF in bcl2 overexpressing transgenic mice increases cell viability and axonal regeneration,³⁴ whereas BDNF promotes survival of RGCs.³⁵ Likewise, experiments with AAV–BDNF, –CNTF and –growth-associated protein 43 (GAP43) have shown that AAV–CNTF was the most crucial for promoting both long-term survival and regeneration.³⁶ The positive effects of CNTF are observed mainly through simultaneous deletion of both PTEN and SOCS3³⁷ and the concurrent activation of mTOR and STAT3 pathways.³⁸ Although CNTF shows robust increase and sustained axon regeneration in injured ONs of rodents, it causes axonal misguidance and aberrant growth.³⁹ Furthermore, it has been shown that CNTF acts as a chemoattractant. CNTF administration onto autologous PN grafts transplanted within transected ON increased regeneration, but these effects were significantly reduced after removal of macrophages from this site.⁴⁰ In addition, the effects of CNTF using PN grafts at ON transection sites are further subject to debate, as previously it has been shown that Ad-CNTF injections preserved RGC axons but did not induce regeneration of axotomized RGCs.⁴¹ Thus, other studies have addressed RGC survivability and axonal regeneration with CNTF and other growth factors,^{35, 36} but most trophic factors affect neuronal survival and regeneration differentially.Previous studies targeting neuronal apoptosis by overexpressing intrinsic growth factors, inhibiting apoptosis and enhancing regeneration in CNS trauma models have established that a multifactorial approach is required for successful and long-lasting therapeutic outcomes.^{6, 36} Current gaps still exist for a key gene that could effectively target neuroprotection, enhance neuron regeneration and sustain neuronal function.One key gene implicated in neuronal plasticity is Neuritin 1 (Nrn1), also known as candidate plasticity gene 15. It has multiple functions and was first identified and characterized when screening for candidate plasticity genes in the rat hippocampal dentate gyrus activated by kainate.^{42, 43, 44} Nrn1 is highly conserved across species⁴⁵ and translates to an extracellular, glycophosphatidylinositol-linked protein (GPI-linked protein), which can be secreted as a soluble form. Nrn1 stimulates axonal plasticity, dendritic arborization and synapse maturation in the CNS.⁴⁶ During early embryonic development, Nrn1 promotes the survival of neural progenitors and differentiated neurons,⁴⁷ while later in development it promotes axonal and dendritic growth and stabilization, allowing maturation and formation of synapses.^{43, 46, 48} In the adult brain, Nrn1 has been correlated with activity-dependent functional plasticity^{45, 49} and is expressed in post mitotic neurons.Nrn1 may be a crucial gene for neuroprotection and regeneration because growth factors such as nerve growth factor (NGF), BDNF and NT-3 as well as neuronal activity can potentiate the expression of Nrn1.^{44, 50} In addition, we reported that Nrn1 mRNA expression appears to be biphasic after ON axonal trauma, indicating a transient attempt by RGCs at neuroprotection/neuroregeneration in response to ONC injury.⁵¹ The dynamic regulation of Nrn1 coupled with neurotrophic effects may promote axonal regeneration in the CNS. To overcome CNS trauma, a new therapy geared towards neuroprotection and effective axonal regeneration is required to enhance a future multifactorial approach. The purpose of this study is to evaluate the therapeutic effects of Nrn1 in mouse RGC cultures as well as in the mouse ONC model. We have identified a distinct neuroprotective and regenerative strategy that prevents neurodegeneration after ON injury. AAV2–hNRN1 expression vectors partially rescued RGCs from apoptosis, maintained RGC function, and initiated regeneration of injured axons.