Injured retinal ganglion cell (RGCs) axons do not regenerate spontaneously, causing loss of vision in glaucoma and after trauma. Recent studies have identified several strategies that induce long distance regeneration in the optic nerve. Thus, a pressing question now is whether regenerating RGC axons can find their appropriate targets. Traditional methods of assessing RGC axon regeneration use histological sectioning. However, tissue sections provide fragmentary information about axonal trajectory and termination. To unequivocally evaluate regenerating RGC axons, here we apply tissue clearance and light sheet fluorescence microscopy (LSFM) to image whole optic nerve and brain without physical sectioning. In mice with PTEN/SOCS3 deletion, a condition known to promote robust regeneration, axon growth followed tortuous paths through the optic nerve, with many axons reversing course and extending toward the eye. Such aberrant growth was prevalent in the proximal region of the optic nerve where strong astroglial activation is present. In the optic chiasms of PTEN/SOCS3 deletion mice and PTEN deletion/Zymosan/cAMP mice, many axons project to the opposite optic nerve or to the ipsilateral optic tract. Following bilateral optic nerve crush, similar divergent trajectory is seen at the optic chiasm compared to unilateral crush. Centrally, axonal projection is limited predominantly to the hypothalamus. Together, we demonstrate the applicability of LSFM for comprehensive assessment of optic nerve regeneration, providing in-depth analysis of the axonal trajectory and pathfinding. Our study indicates significant axon misguidance in the optic nerve and brain, and underscores the need for investigation of axon guidance mechanisms during optic nerve regeneration in adults.

Lack of axon growth ability in the central nervous system poses a major barrier to achieving functional connectivity after injury. Thus, a non-transgenic regenerative approach to reinnervating targets has important implications in clinical and research settings. Previous studies using knockout (KO) mice have demonstrated long distance axon regeneration. Using an optic nerve injury model, here we evaluate the efficacy of viral, RNAi and pharmacological approaches that target the PTEN and STAT3 pathways to improve long distance axon regeneration in wild type (WT) mice. Our data show that adeno-associated virus (AAV) expressing short hairpin RNA (shRNA) against PTEN (shPTEN) enhances retinal ganglion cell axon regeneration after crush injury. However, compared to the previous data in PTEN KO mice, AAV-shRNA results in a lesser degree of regeneration, likely due to incomplete gene silencing inherent to RNAi. In comparison, an extensive enhancement in regeneration is seen when AAV-shPTEN is coupled to AAV encoding ciliary neurotrophic factor (CNTF) and to a cyclic adenosine monophosphate (cAMP) analogue, allowing axons to travel long distances and reach their target. We apply whole tissue imaging that facilitates three-dimensional visualization of single regenerating axons and document heterogeneous terminal patterns in the targets. This shows that some axonal populations generate extensive arbors and make synapses with the target neurons. Collectively, we show a combinatorial viral RNAi and pharmacological strategy that improves long distance regeneration in WT animals and provide single fiber projection data that indicates a degree of preservation of target recognition.

Growing axons in the central nervous system (CNS) often migrate along specific pathways to reach their targets. During embryonic development, this migration is guided by different types of cell adhesion molecules (CAMs) present on the surface of glial cells or other neurons, including the neural cadherin (NCAD). Axons in the adult CNS can be stimulated to regenerate, and travel long distances. Crucially, however, while a few axons are guided effectively through the injured nerve under certain conditions, most axons never migrate properly. The molecular underpinnings of the variable growth, and the glial CAMs that are responsible for CNS axon regeneration remain unclear. Here we used optic nerve crush to demonstrate that NCAD plays multifaceted functions in facilitating CNS axon regeneration. Astrocyte-specific deletion of NCAD dramatically decreases regeneration induced by PTEN ablation in retinal ganglion cells (RGCs). Consistent with NCAD's tendency to act as homodimers, deletion of NCAD in RGCs also reduces regeneration. Deletion of NCAD in astrocytes neither alters RGCs' mTORC1 activity nor lesion size, two factors known to affect regeneration. Unexpectedly however, we find that NCAD deletion in RGCs reduces PTEN-deletion induced RGC survival. We further show that NCAD deletion, in either astrocytes or RGCs, has negligible effects on the regeneration induced by ciliary neurotrophic factor (CNTF), suggesting that other CAMs are critical under this regenerative condition. Consistent with this notion, CNTF induces expression various integrins known to mediate cell adhesion. Together, our study reveals multilayered functions of NCAD and a molecular basis of variability in guided axon growth. 3 Significance Statement Growing axons often travel long distances and migrate along explicit pathways to reach their targets. Cell adhesion molecules (CAMs) including cadherins play vital roles in these processes during development. However, it remains unclear whether the same factors are involved for the adult axons after injury. This study used knockout mice to demonstrate that ablation of NCAD in astrocytes or retinal ganglion cells, prevents regeneration and cell survival induced by PTEN deletion. In contrary, NCAD deletion has negligible effects on the regeneration and survival induced by cytokines, suggesting that distinct CAMs control axon adhesion and growth under different regenerative conditions. Together, our study illustrates cadherins' versatile functions in the injured CNS, and points to distinct mechanisms that shape directed axon growth.

Background Emerging evidence indicates that long noncoding RNAs (lncRNAs) are important regulators of various biological processes, and their expression can be altered following certain pathological conditions, including central nervous system injury. Retinal ganglion cells (RGCs), whose axons form the optic nerve, are a heterogeneous population of neurons with more than 40 molecularly distinct subtypes in mouse. While most RGCs, including the ON-OFF direction-selective RGCs (ooDSGCs), are vulnerable to axonal injury, a small population of RGCs, including the intrinsically photosensitive RGCs (ipRGCs), are more resilient. Results By performing systematic analyses on RNA-sequencing data, here we identify lncRNAs that are expressed in ooDSGCs and ipRGCs with and without axonal injury. Our results reveal a repertoire of different classes of lncRNAs, including long intergenic noncoding RNAs and antisense ncRNAs that are differentially expressed between these RGC types. Strikingly, we also found dozens of lncRNAs whose expressions are altered markedly in response to axonal injury, some of which are expressed exclusively in either one of the types. Moreover, analyses into these lncRNAs unraveled their neighboring coding genes, many of which encode transcription factors and signaling molecules, suggesting that these lncRNAs may act in cis to regulate important biological processes in these neurons. Lastly, guilt-by-association analysis showed that lncRNAs are correlated with apoptosis associated genes, suggesting potential roles for these lncRNAs in RGC survival. Conclusions Overall, the results of this study reveal RGC type-specific expression of lncRNAs and provide a foundation for future investigation of the function of lncRNAs in regulating neuronal type specification and survival.