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

 描述(申请人提供)：人类脊髓损伤(SCI)会导致永久性瘫痪，因为受伤的神经纤维(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多糖(CSPGs)。CSPGs与蛋白酪氨酸磷酸酶家族的受体、蛋白酪氨酸磷酸酶σ和蛋白酪氨酸磷酸酶受体结合，抑制轴突生长。七鳃鳗脊髓损伤后的再生更为成功，但一些已发现的网状脊髓(RS)神经元再生能力较差，当其轴突受损时，这些神经元会经历非常延迟的程序性细胞死亡(细胞凋亡)。我们将确定CSPG是否通过PTPσ和/或LAR作用，既抑制真正的轴突再生(而不是由未损伤的轴突萌发)，又触发脊髓损伤后的逆行凋亡。我们将测试用软骨素酶(ChABC)消化CSPG是否增加脊髓投射神经元的存活率和/或轴突的再生，以及添加外源性CSPG是否具有相反的效果。体外证据表明，CSPG通过LAR激活小GTP酶RhoA，并使Akt失活。这两种信号都有下游效应，可能会抑制轴突生长。通过阻断RhoA的合成或激活并观察其对Akt活性的影响，我们将确定对Akt的影响是否位于RhoA的下游，或者这两条途径是否独立触发，可能是由不同的RPTP触发的。从损伤部位向RS神经元逆行递送的吗啡反义寡核苷酸将抑制RhoA的合成。RhoA的激活将被C3转移酶阻断。对凋亡标志物和轴突再生的影响将被确定。MOS也将抑制RPTP的合成，以确定哪个受体介导了CSPG的哪些负面影响。为了测试七鳃鳗的影响是否也适用于哺乳动物的神经元，我们将在出生后和成年哺乳动物的原代神经元培养上进行平行实验。我们还将在小鼠视神经挤压模型中测试RPTPs的基因敲除或药物抑制是否减少体内视网膜神经节细胞的逆行神经元死亡和促进轴突再生。了解CSPGs抑制细胞存活和轴突再生的细胞内通路有助于开发治疗人类脊髓损伤的方法。

 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.