The failure of mammalian CNS neurons to regenerate their axons derives from a combination of intrinsic deficits and extrinsic obstacles. Following injury, chondroitin sulfate proteoglycans (CSPGs) accumulate within the glial scar that forms at the lesion site in response to the insult. CSPGs inhibit axonal growth and regeneration, an action mediated by their sulfated glycosaminoglycan (GAG) chains, especially those with 4-sulfated (4S) sugars. Arylsulfatase B (ARSB) selectively cleaves 4S groups from the non-reducing ends of GAG chains without disrupting other, potentially growth-permissive motifs. In this thesis, "Modifying Chondroitin Sulfation Enhances Retinal Ganglion Cell Axon Regeneration," I, Craig Pearson, seek to determine the time course and spatial distribution of CSPG accumulation in the glial scar following acute injury, and then to demonstrate that ARSB is effective in reducing the inhibitory actions of CSPGs. I examine the effects of ARSB in an in vitro model of the glial scar and in vivo, using optic nerve crush (ONC) in adult mice. ARSB is clinically approved for replacement therapy in patients with mucopolysaccharidosis VI and therefore represents an attractive candidate for translation to the human CNS. My findings illustrate the importance of CSPGs as a barrier to axon extension following injury, and show compelling evidence that selective modification of the sulfation pattern on GAG chains results in significant enhancement of RGC axonal regeneration. Finally, I combine ARSB treatment with a host of intrinsic pro-regenerative stimuli and show robust, long-distance regeneration of RGC axons through the optic chiasm and into the optic tract. Taken together, the results of this thesis argue for the therapeutic potential of modifying the extracellular matrix to promote regeneration of axons in the CNS.

The murine eye naturally undergoes post-natal changes in eye size. This dissertation quantifies longitudinal structural and functional changes in control mice (C57BL/6J (B6), D2-Gpnmb+/SjJ) and in DBA/2J (D2) mice, which spontaneously develop elevated intraocular pressure (IOP). IOP elevation results in abnormal eye elongation, retinal nerve fiber layer (RNFL) thickness thinning and retinal ganglion cell (RGC) dysfunction and demise resembling human glaucoma. I measured structural changes with Optical Coherence Tomography (OCT), and RGC function with Pattern Electroretinogram (PERG). I also developed and refined provocation approaches (IOP elevation with changes in body posture; metabolic load with flickering light) to probe susceptibility of RGC function in D2 mice prone to glaucoma. Finally, I developed a novel system for recording, simultaneously but independently, the PERG from both eyes using asynchronous visual stimuli and deconvolution analysis. Simultaneous PERG recording from each eye was hitherto impossible due to the interocular cross-talk of the PERG signal. Altogether, the combination of these measures (OCT, PERG) and provocative conditions may represent powerful tools for glaucoma research using mouse models.

Mammalian central nervous system (CNS) neurons lose their ability to regenerate their axons after injury during development. For example, optic nerve injury studies in hamsters have shown that optic nerve axons injured around the time of birth retain the ability to regenerate to their target, but this ability is lost during development (So et al., 1981). The development of an inhibitory CNS environment has been implicated in the inability of the adult CNS to regenerate, however there is also support for this loss being a result of changes in developmental programs intrinsic to the neurons themselves (Goldberg et al., 2002a; Goldberg, 2004). While some molecules have been identified as being involved in intrinsic mechanisms controlling axon growth, there is still much to be discovered. Using genes shown to be regulated in retinal ganglion cells (RGCs) during development (Wang et al., 2007), I performed an overexpression screen in embryonic primary neurons measuring changes in neurite growth. Of these genes, the most significant effect in neurite growth was seen with overexpression of Krüppel-like factor 4 (KLF4), resulting in a greater than 50% decrease in growth. KLF4 is a member of the KLF family of transcription factors which all possess a DNA binding domain containing 3 zinc finger motifs. Outside of the nervous system, KLF4 has been implicated in cancer (Black et al., 2001; Rowland and Peeper, 2006), mitotic growth arrest (Shields et al., 1996) and most recently in the induction of pluripotency (Yamanaka, 2008; Zhao and Daley, 2008). In the CNS, KLF4 has recently been implicated in increasing the sensitivity of cortical neurons to NMDA insult (Zhu et al, 2009), though no effect of KLF4 on neurite growth or regeneration has yet been described. I found that KLF4 overexpression in RGCs results in decreased neurite growth and neurite initiation. KLF4 overexpression also leads to decreases in polarity acquisition in hippocampal neurons, though even when they acquire polarity, they still display decreased neurite growth. Additionally, KLF4 knockout targeted to RGCs leads to an increased neurite growth ability and increased neurite initiation in vitro. In vivo, KLF4 knockout increases RGC axon regeneration after optic nerve injury. Interestingly, KLF4 is one of 17 members of the KLF family, known for their ability to act redundantly and competitively amongst family members for their binding sites. Therefore, we looked to see if other KLFs could affect neurite growth ability. 15 of 17 KLF family members are expressed in RGCs, and their overexpression results in differential effects on neurite growth in both cortical neurons and RGCs. Additionally, many of the family members are developmentally regulated in a manner that typically correlates with their ability to affect neurite growth. For example, KLF6 and -7, whose expression decreases during development, when overexpressed, increase neurite growth, whereas KLF9, whose expression increases developmentally, when overexpressed, decreases neurite growth. Surprisingly, there are multiple KLFs expressed in RGCs that are neurite growth-suppressors, and further study has revealed that the combination of KLF growth enhancers with KLF growth suppressors results in a suppressive or neutral phenotype (Moore et al., 2009), suggesting that to further enhance regeneration after injury in vivo, we will need to additionally remove the growth suppression from other KLF family members. Taken together, these data suggest that KLFs may play an important role in the intrinsic loss of axon growth and regeneration seen during development. Further characterization of downstream targets of KLF4 and other KLF family members may reveal specific neuronal gene targets that could mediate the phenotypic effects of these transcription factors. It is my hope that by determining the developmental programs that underlie the loss of intrinsic axon growth ability of CNS neurons, we may ultimately determine how to revert adult CNS neurons to their embryonic axon growth ability.

Damage to visual pathways can lead to irreversible blindness. Posterior visual pathways, located within a watershed area, are predisposed to hypoperfusion/reperfusion injury. In a novel rat model of bilateral common carotid artery occlusion (BCCAO), oxidative injury to the superior colliculus (SC), a major visual center within the watershed area was evaluated, in addition to its effects on retinal ganglion cells (RGCs). Nitrotyrosine, a footprint of peroxynitrite-mediated oxidative injury in the SC, and microtubule-associated protein 2, a dendrite marker in the retina, were assessed using immunofluorescence and confocal microscopy. Nitrotyrosine-immunoreactivity in the SC was increased 2 weeks after BCCAO compared to controls. Microtubule-associated protein 2-immunoreactivity in the central inner plexiform layer was reduced 3 weeks after BCCAO compared to controls. Global incomplete cerebral hypoperfusion/reperfusion induced oxidative injury in the SC and retrograde RGC dendritic changes. This suggests that cerebrovascular injury affecting the posterior visual pathways may contribute to vision loss in patients.

Damage to visual pathways can lead to irreversible blindness. Posterior visual pathways, located within a watershed area, are predisposed to hypoperfusion/reperfusion injury. In a novel rat model of bilateral common carotid artery occlusion (BCCAO), oxidative injury to the superior colliculus (SC), a major visual center within the watershed area was evaluated, in addition to its effects on retinal ganglion cells (RGCs). Nitrotyrosine, a footprint of peroxynitrite-mediated oxidative injury in the SC, and microtubule-associated protein 2, a dendrite marker in the retina, were assessed using immunofluorescence and confocal microscopy. Nitrotyrosine-immunoreactivity in the SC was increased 2 weeks after BCCAO compared to controls. Microtubule-associated protein 2-immunoreactivity in the central inner plexiform layer was reduced 3 weeks after BCCAO compared to controls. Global incomplete cerebral hypoperfusion/reperfusion induced oxidative injury in the SC and retrograde RGC dendritic changes. This suggests that cerebrovascular injury affecting the posterior visual pathways may contribute to vision loss in patients.

The retina contains neural circuits that carry out computations as complex as object motion sensing, pattern recognition, and position anticipation. Models of some of these circuits have been recently discovered. A remarkable outcome of these efforts is that all such models can be constructed out of a limited set of components such as linear filters, instantaneous nonlinearities, and feedback loops. The present study explores the consequences of assuming that these components can be used to construct models for all retinal circuits. I recorded extracellularly from several retinal ganglion cells while stimulating the photoreceptors with a movie rich in temporal and spatial frequencies. Then I wrote a computer program to fit their responses by searching through large spaces of anatomically reasonable models built from a small set of circuit components. The program considers the input and output of the retinal circuit and learns its behavior without over-fitting, as verified by running the final model against previously unseen data. In other words, the program learns how to imitate the behavior of a live neural circuit and predicts its responses to new stimuli. This technique resulted in new models of retinal circuits that outperform all existing ones when run on complex spatially structured stimuli. The fitted models demonstrate, for example, that for most cells the center--surround structure is achieved in two stages, and that for some cells feedback is more accurately described by two feedback loops rather than one. Moreover, the models are able to make predictions about the behavior of cells buried deep within the retina, and such predictions were verified by independent sharp-electrode recordings. I will present these results, together with a brief collection of ideas and methods for furthering these modeling efforts in the future. / Physics

Purpose: Retinal ganglion cell (RGC) loss is a measure of the progression of many visual disorders. It is important to identify RGCs with good specificity, so RGC numbers can be reliably analyzed. The purpose of this study was to analyze the effectiveness of two current RGC markers: Brn3a immunohistochemistry and the expression of Thy1-CFP in the Thy1-CFP transgenic mouse. Methods: Rhodamine-?-isothiocyanate (RITC) retrograde labeling, immunohistochemistry, wholemount retinal imaging, western blot, cross sectional analysis and cell densities in uninjured control animals and 3, 5, 7 and 14 days post-optic nerve crush (ONC) or transection (ONT) were tabulated. Results: Brn3a positive (Brn3a+) cell density was significantly less than RITC positive (RITC+) cell density in control mice. After ON injury, Brn3a+ cell density did not decrease at the same rate as RITC+ cell density. The density of RGCs that express Brn3a was significantly less than the individual Brn3a+ and RITC+ cell density at all experimental time points. Thy1-CFP positive (Thy1-CFP+) cell density was significantly less than RITC+ in control mice and significantly more than RITC+ cell density 14 days after ON injury. Thy1-CFP co-localized with ChAT positive (ChAT+) cells 7 days after ONT. Conclusion: Brn3a and Thy1-CFP are not reliable markers of RGCs. Retrograde labeling remains one of the most reliable methods of labeling RGCs in mice.

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京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18875号 / 医博第3986号 / 新制||医||1008(附属図書館) / 31826 / 京都大学大学院医学研究科医学専攻 / (主査)教授 森田 智視, 教授 福山 秀直, 教授 大森 治紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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