CSPG-induced retrograde cell death and inhibition of regeneration after SCI

 描述（由申请人提供）：人类脊髓损伤（SCI）导致永久性瘫痪，因为受损的神经纤维（轴突）不能再生。其中一个原因是损伤附近的细胞分泌硫酸软骨素蛋白聚糖（CSPG）。CSPG与蛋白酪氨酸磷酸酶（RPTP）家族的受体PTPσ和LAR结合，并抑制轴突生长。脊髓损伤后的再生在七鳃鳗中更成功，但一些已鉴定的网状脊髓（RS）神经元是不良的再生者，当它们的轴突受损时，这些神经元经历非常延迟的程序性细胞死亡（凋亡）。我们将确定CSPG是否通过PTPσ和/或LAR起作用，既抑制真正的轴突再生（与未损伤轴突的发芽相反），又触发SCI后的逆行凋亡。我们将测试用软骨素酶（ChABC）消化CSPGs是否会增加脊髓投射神经元的存活和/或其轴突的再生，以及添加外源性CSPGs是否具有相反的效果。体外证据表明，CSPG通过LAR激活小GT3 RhoA和Akt。这两种信号都有下游效应，可以抑制轴突生长。通过阻断RhoA的合成或激活并观察对Akt活性的影响，我们将确定对Akt的影响是否在RhoA的下游，或者这两条通路是否是独立触发的，也许是由不同的RPTP触发的。将吗啉代反义寡核苷酸（MOs）从损伤部位逆行递送至RS神经元将抑制RhoA的合成。RhoA的激活将被C3转移酶阻断。将确定对细胞凋亡标志物和轴突再生的影响。RPTP的合成也将被MO抑制，以确定哪种受体介导CSPG的哪种负面作用。为了测试七鳃鳗中的效应是否也适用于哺乳动物神经元，我们将对出生后和成年哺乳动物原代神经元培养物进行平行实验。我们还将在小鼠视神经挤压模型中测试RPTP的遗传敲除或药理学抑制是否减少逆行神经元死亡并增强体内视网膜神经节细胞的轴突再生。了解介导CSPG对细胞存活和轴突再生的抑制作用的细胞内途径可能会导致人类SCI治疗的发展。

 DESCRIPTION (provided by applicant): Spinal cord injury (SCI) in humans leads to permanent paralysis because injured nerve fibers (axons) do not regenerate. One reason for this is the secretion of chondroitin sulfate proteoglycans (CSPGs) by cells near the injury. CSPGs bind to receptors of the protein tyrosine phosphatase (RPTP) family, PTPσ and LAR, and inhibit axon growth. Regeneration after SCI is more successful in lampreys, but some identified reticulospinal (RS) neurons are bad regenerators, and when their axons are injured, these undergo very delayed programmed cell death (apoptosis). We will determine whether, acting through PTPσ and/or LAR, CSPGs both inhibit true axon regeneration (as opposed to sprouting by uninjured axons) and trigger retrograde apoptosis after SCI. We will test whether digesting CSPGs with chondroitinase (ChABC) increases survival of spinal-projecting neurons and/or regeneration of their axons, and whether adding extrinsic CSPGs has the opposite effects. Evidence in vitro suggests that CSPGs, acting through LAR activate the small GTPase RhoA and inactivate Akt. Both signals have downstream effects that could inhibit axon growth. By blocking RhoA synthesis or activation and observing the effect on Akt activity, we will determine whether the effect on Akt is downstream of RhoA, or whether the two pathways are triggered independently, perhaps by different RPTPs. RhoA synthesis will be inhibited with morpholino antisense oligonucleotides (MOs) delivered to RS neurons retrogradely from the injury site. Activation of RhoA will be blocked with C3 transferase. The effect on apoptosis markers and axon regeneration will be determined. Synthesis of the RPTPs also will be inhibited with MOs, to determine which receptor mediates which of the negative effects of CSPGs. To test whether effects in lampreys also apply to mammalian neurons, we will perform parallel experiments on postnatal and adult mammalian primary neuronal cultures. We also will test in a mouse optic nerve crush model, whether genetic knockdown or pharmacological inhibition of RPTPs reduces retrograde neuronal death and enhances axonal regeneration in retinal ganglion cells in vivo. Understanding the intracellular pathways that mediate the inhibitory effects of CSPGs on cell survival and axon regeneration could lead to development of therapies for human SCI.