Amyotrophic Lateral Sclerosis: treating the circuit behind the disease

无法治愈的疾病肌萎缩性侧索硬化症（ALS）的特征在于运动神经元（MN）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神经细胞回路激活的，该神经细胞回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）可以激励或抑制MN，这取决于电路的哪一部分需要参与执行所需的运动。我们最近发现，在ALS小鼠模型中，一组抑制性中间神经元在疾病早期失去了与MN的连接，并且无法再正确激活它们。这些连接的变化可能导致MN的失调和退化。我们还发现，连接的丧失导致的症状与在患者中观察到的症状相似，包括步幅和运动速度的变化（Allodi et al 2021，Nature Communication）。在一项新的研究中（莫拉等人，2022），我们使用了一种使用病毒感染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，它可以自然地刺激神经细胞之间的连接，我们特别在抑制性中间神经元中增加了这种基因的水平。这种方法使我们能够稳定抑制性中间神经元和MN之间的连接，因此我们增加了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已知引起家族性ALS的SOD 1突变的小鼠模型中获得的，该突变仅占ALS病例的2%。出于这个原因，我们现在正计划将我们的研究也扩大到其他ALS引起的基因突变，并澄清抑制性中间神经元和MN之间的连接丢失是否是ALS病理学中的常见事件。如果是这样的话，我们的新基因疗法可以在未来应用于更广泛的ALS病例。在这个项目中，我们将研究在携带TDP-43和FUS突变的另外两个小鼠模型中，抑制性中间神经元是否受到影响，利用一种方法，使我们能够可视化中间神经元和MN之间的连接，并对其进行量化。这种方法以前在实验室中建立（Allodi et al 2021，Nature Communication），将帮助我们识别潜在的连接丢失。其次，我们将研究是否抑制性中间神经元也在散发性ALS中受到影响。由于我们与丹麦的Bjspebjerg脑库的合作，我们可以分析来自21名被诊断患有散发性ALS的捐赠者的死后组织。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，而不是它们的连接，因为人类死后组织的退化程度很高，我们预计会失去很多连接。最后，我们将产生一种改进的基因疗法，以提供自然刺激人类连接的基因。尽管结果令人鼓舞，但我们目前的方法（莫拉等人，2022年）具有翻译限制，因为使用了不适用于人类的遗传策略。然而，抑制性中间神经元可以使用在小鼠、黑猩猩和人类中特异性（如条形码）和保守的DNA序列来靶向。该序列可用作增强子。增强剂将仅靶向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给药，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连接和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.