Pharmacological inhibition or genetic deletion of a neurotoxin found abundantly at sites of spinal cord injury will neuroprotect and improve outcome.

背景：脊髓损伤或脑损伤每年使数百万人致残，给国民经济造成的损失高达数百亿英镑。减少损伤后神经细胞死亡程度和改善幸存者预后的治疗方法是迫切需要的。创新：我们发现了一种神经毒性分子，这种分子被白细胞（包括中性粒细胞）释放后，在人类、大鼠和小鼠的神经创伤部位大量存在。每个分子的神经毒性是谷氨酸的700倍（谷氨酸是一种从受伤的神经元中释放出高浓度毒性的分子）。制药公司已经花费了数亿英镑试图开发抑制谷氨酸等毒素的药物；鉴于我们在神经创伤部位发现了这种更强效的神经毒素，它值得紧急关注，可能是一个非常有价值的目标。令人惊讶的是，这种神经毒素在神经损伤后基本上没有被研究过。令人兴奋的是，我们已经鉴定出一种多克隆抗体，可以在体外至少48小时内完全阻断该分子杀死中枢神经系统神经元的能力。目的1：我们希望开发抑制这种神经毒素的治疗性“单克隆抗体”。“单克隆抗体”是一类治疗药物，可以非常有效地抑制特定的分子靶点；它们适用于特定特性的工程设计（例如，尺寸，身体寿命，安全性），并已在全球范围内提供健康和商业效益。我们现在寻求开发和评估新的单克隆抗体，以提高在培养皿（目的1和2）或体内（目的3）暴露于该神经毒素的人类和啮齿动物中枢神经系统神经元的存活率。目的2：我们还发现，脊髓损伤48小时内通过腰椎穿刺获得的脑脊液对培养皿中培养的啮齿动物中枢神经系统神经元有毒性；我们现在希望通过应用我们的新治疗性抗体，不含或与其他毒素抑制剂（如谷氨酸和活性氧）结合，最大限度地提高培养皿中受伤的人类神经元的存活率。我们将通过将脑脊液分离成组成部分（例如，根据分子电荷或大小）来分析任何其他残留的有毒分子，以便使用现代生化方法进行鉴定，包括但不限于蛋白质组学。目的3：我们希望测试这样一种观点，即在医学上可行的时间框架内，通过腰椎穿刺（注入脑脊液）在小鼠中注射这些治疗性单克隆抗体，可以改善临床相关的挫伤脊髓损伤模型的预后。我们预测，治疗性单克隆抗体的短期治疗将中和这种神经毒素，将提高人类中枢神经系统神经元的存活率，并将改善长期的感觉运动结果（如行走）。另外，我们将评估腰椎注射已知的这种毒素的人类蛋白抑制剂是否可以改善结果。目的4：最后，我们希望确定缺乏小鼠等量神经毒素的小鼠是否在相同的临床相关的挫伤脊髓损伤模型中表现出更好的中枢神经系统细胞存活和改善的恢复。这将使我们能够确认我们的治疗性单克隆抗体的特异性，以及它们的作用机制，这反过来将帮助我们优化我们的治疗以获得最大的收益。临床重要性：这些实验很重要，因为单克隆抗体可以在损伤后几小时内通过腰椎穿刺直接给予，以减少脊髓损伤后的残疾数量，也可能在中风或创伤性脑损伤后。这些实验将帮助我们将这种潜在的疗法向临床试验迈进一步。

Background:Spinal cord injuries or brain injuries disable millions of people each year, and the cost to national economies run into tens of billions of pounds. Therapies which reduce the extent of neural cell death after injury and that improve survivor outcomes are badly needed.Innovation: We have discovered a neurotoxic molecule that is found abundantly at sites of neurotrauma in humans, rats, and mice after it is released by white blood cells (including neutrophils). Per molecule, it is up to 700 times more neurotoxic than glutamate (a molecule which is toxic at high concentrations when released from injured neurons). Pharmaceutical companies have spent hundreds of millions of pounds trying to develop medicines that inhibit toxins like glutamate; given our discovery of this even more potent neurotoxin at sites of neurotrauma, it merits urgent attention and could be a highly valuable target. Surprisingly, this neurotoxin remains essentially unstudied after neurological injury. Excitingly, we have identified a polyclonal antibody which completely blocks this molecule's ability to kill CNS neurons for at least 48 hours in vitro.Aim 1: We wish to develop therapeutic "monoclonal antibodies" that inhibit this neurotoxin. "Monoclonal antibodies" are a class of therapeutic that can be extraordinarily effective at inhibiting defined molecular targets; they are amenable to engineering for specific properties (e.g., size, longevity in the body, safety profile) and already provide health and commercial benefits worldwide. We now seek to develop and evaluate novel monoclonal antibodies that improve survival of human and rodent CNS neurons exposed to this neurotoxin in Petri dishes (Aim 1 and 2) or in vivo (Aim 3). Aim 2: We have also discovered that cerebrospinal fluid obtained by lumbar puncture from humans within 48 hours of spinal cord injury is toxic to rodent CNS neurons cultured in Petri dishes; we now wish to maximise survival of injured human neurons in Petri dishes by applying our new therapeutic antibodies without, or combined with, inhibitors of other toxins (e.g., glutamate and reactive oxygen species). We will analyse any other residual toxic molecule(s) by separating cerebrospinal fluid into component parts (e.g., based on molecular charge or size), for identification using modern biochemical methods, including but not limited to proteomics. Aim 3: We wish to test the idea that injection of these therapeutic monoclonal antibodies by lumbar puncture (into the cerebrospinal fluid) in mice would improve outcome in a clinically relevant model of contusive spinal cord injury when given in a medically feasible time frame. We predict that short-term treatment with the therapeutic monoclonal antibodies will neutralize this neurotoxin, will improve survival of human CNS neurons, and will improve sensorimotor outcomes (e.g., walking) in the long-term. Alternatively, we will evaluate whether lumbar injection of a known human protein inhibitor of this toxin can improve outcome. Aim 4: Finally, we wish to determine whether mice that lack the mouse equivalents of this neurotoxin show better CNS cell survival and improved recovery in the same clinically relevant model of contusive spinal cord injury. This will enable us to confirm the specificity of our therapeutic monoclonal antibodies, and their mechanisms of action, which in turn will help us optimise our therapy for maximum benefit.Clinical importance: These experiments are important because monoclonal antibodies could be given by straightforward lumbar puncture, within hours of injury, to reduce the amount of disability after spinal cord injury, and potentially also after stroke or traumatic brain injury.These experiments will help us take this potential therapy one step closer to clinical trials.