Amyotrophic Lateral Sclerosis: treating the circuit behind the disease

肌萎缩侧索硬化症(ALS)是一种不治之症，其特征是运动神经元(MNS)的丧失，运动神经元是直接控制运动的神经细胞，因为它们连接到身体周围的肌肉。因此，MN可以被认为是大脑的输出；然而，它们是由脊髓中发现的复杂的神经细胞电路激活的，该电路解码来自大脑的信息，并以同步的方式激活MN。这些解码神经元(也称为中间神经元)可以兴奋或抑制MN，这取决于需要连接电路的哪一部分来执行所需的运动。我们最近发现，在ALS的小鼠模型中，一组抑制性中间神经元在疾病早期失去了与MNS的联系，并且不能再正确地激活它们。连通性的这些变化可能会导致MNS的失调和退化。我们还看到，失去连接会导致类似于患者身上观察到的症状，其中包括运动步幅和速度的变化(Alaldi等人，2021，自然交流)。在一项新的研究(Mora等人，2022)中，我们使用了一种使用病毒感染来传递基因作为治疗的方法，称为基因治疗。我们传递了一种基因，可以自然地刺激神经细胞之间的连接，我们提高了这种基因在抑制中间神经元中的水平。这种方法使我们能够稳定抑制的中间神经元和MN之间的连接，因此我们提高了MN的存活率，并改善了小鼠的ALS症状。然而，到目前为止，我们的结果是从携带SOD1突变的小鼠模型中获得的，该突变已知会导致家族性ALS，仅占ALS病例的2%。出于这个原因，我们现在计划将我们的研究扩大到其他导致ALS的基因突变，并澄清抑制性中间神经元和MN之间的连接丧失是否在ALS病理中是常见的事件。如果是这样的话，我们的新基因疗法可能会在未来应用于更多的ALS病例。在这个项目中，我们将调查另外两个携带TDP-43和FUS突变的小鼠模型中的抑制性中间神经元是否受到影响，利用一种方法，我们可以可视化中间神经元和MN之间的联系，并对它们进行量化。这种方法以前是在实验室中建立的(阿洛迪等人，2021年，自然通信)，并将帮助我们识别潜在的连接丢失。其次，我们将研究抑制性中间神经元是否也在散发性ALS中受到影响。由于我们与丹麦BjSpebJerg脑库的合作，我们可以分析21名被诊断为散发性肌萎缩侧索硬化症的捐赠者的身体组织。在这里，抑制性中间神经元将被量化，以阐明它们在散发性ALS病例中的潜在变性。抑制性中间神经元将被计算在内，而不是它们的连接，因为人类死后组织的变性程度很高，我们预计会失去大量的连接。最后，我们将产生一种改进的基因疗法，以传递自然刺激人类连通性的基因。尽管结果令人振奋，但我们目前的方法(Mora等人2022)存在翻译限制，因为我们使用的遗传策略不适用于人类。然而，抑制性中间神经元可以使用特定的DNA序列(如条形码)作为靶点，并在小鼠、黑猩猩和人类中保留。该序列可用作增强剂。增强子将只针对特定的抑制性中间神经元，并迫使我们的治疗在细胞中表达。重要的是，这种新的基因疗法可以通过静脉注射进行，因此不需要侵入性治疗。我们希望这一策略将减缓抑制性中间神经元失去联系和MN退化的速度。

The incurable disease Amyotrophic lateral sclerosis (ALS) is characterized by loss of motor neurons (MNs), which are the nerve cells directly controlling movements since they connect to the muscles in the periphery of the body. So, MNs can be considered the output of the brain; however, they are activated by a complex circuit of nerve cells found in the spinal cord that decodes the information coming from the brain and activates the MNs in a synchronised manner. These decoding neurons (also called interneurons) can either excite or inhibit the MNs depending on which part of the circuit needs to be engaged to perform the desired movement. We recently discovered that, in a mouse model of ALS, a group of inhibitory interneurons loses its connection to the MNs early in disease, and cannot activate them properly anymore. These changes in connectivity can contribute to MNs dysregulation and degeneration. We also saw that loss of connectivity led to symptoms resembling the ones observed in patients, which included changes in the stride and speed of locomotion (Allodi et al 2021, Nature Communication). In a new study (Mora et al 2022), we used an approach which uses viral infection to deliver genes as therapy, called gene therapy. We delivered a gene that naturally stimulates connections between nerve cells, and we increased the levels of this gene specifically in the inhibitory interneurons. This approach allowed us to stabilise the connectivity between the inhibitory interneurons and the MNs, and as a consequence we increased MN survival and ameliorated ALS symptoms in mice. However, to date, our results are obtained from a mouse model carrying the SOD1 mutation known to cause familial ALS, which accounts only for the 2% of the ALS cases. For this reason, we are now planning to broaden our investigations also to other ALS-causing genetic mutations and to clarify if the loss of connectivity between inhibitory interneurons and MNs is a common event in ALS pathology. If this happen to be the case, our new gene therapy could be apply to a wider number of ALS cases in the future. In this project, we will investigate if the inhibitory interneurons are affected in two other mouse models carrying the TDP-43 and the FUS mutations, utilising an approach that allows us to visualise the connections between interneurons and MNs, and to quantify them. This approach was previously established in the lab (Allodi et al 2021, Nature Communication) and will help us identifying the potential loss of connectivity. Secondly, we will investigate if inhibitory interneurons are also affected in sporadic ALS. Thanks to our collaboration with the Bjspebjerg Brain Bank in Denmark, we can analyse post-mortem tissue from 21 donors which were diagnosed with sporadic ALS. Here, inhibitory interneurons will be quantified to elucidate their potential degeneration in sporadic ALS cases. The inhibitory interneurons will be counted instead of their connections, because the level of degeneration in the human post-mortem tissue is high and we expect a lot of the connectivity to be lost. Finally, we will generate an improved gene therapy to deliver the gene that naturally stimulates connectivity in humans. Despite the promising results, our current approach (Mora et al 2022) has translational limitations because uses a genetic strategy not applicable in humans. However, the inhibitory interneurons can be targeted using a DNA sequence that is specific (like a barcode) and conserved in mouse, chimps, and humans. This sequence can be used as an enhancer. The enhancer will target only the specific inhibitory interneurons and force the expression of our treatment in the cells. Importantly, this new gene therapy can be administered by intravenous injection, so it does not require invasive treatments. We hope that this strategy will slow down inhibitory interneurons from losing their connections and MNs from degeneration.