Chemogenetic investigation of amyotrophic lateral sclerosis (ALS): controlling signaling in astrocytes and in motoneurons to affect disease manifestations in an ALS mouse model

Background and purpose: Amyotrophic lateral sclerosis (ALS) is among the fatal neurodegenerative diseases that lack widely available effective treatments. Extensive evidence shows that the pathogenic cascade of ALS is associated with a non-cell autonomous mechanism, in which other non-neuron cells such as astrocytes, microglia, and immune cells are comprehensively responsible for the neurodegeneration. To date, there is no effective cure for ALS, and novel therapies are needed for targeting astrocytes or motor neurons (MNs) to prevent disease burdens. The ultimate purpose of this study was to investigate whether manipulation on astrocytes and MNs via chemogenetic approaches at the pre-symptom stage of ALS are able to improve Blood-Spinal cord-Barrier (BSCB) integrity and MNs burdens. Methods: We performed a series of experiments on mutant superoxide dismutase 1 (SOD1-G93A) transgenic mice, double transgenic SOD1-G93A/ChAT-cre mice, and their wild type (WT) littermates. Firstly, we infected astrocytes located in right anterior horns of lumbar spinal cords with AAV8-GFAP::DREADD(Gq) viral vectors expressing designed Gq receptors under control of the glial fibrillary acidic protein (GFAP) promoter. Secondly, we individually infected MNs located in the right anterior horns of lumbar spinal cords with AAV2/9-hSyn::DREADD(Gs) viral vectors under control of the cre recombinase. Thirdly, we infected MNs with mixed AAV2/9-hSyn::DREADD(Gs) and AAV9-CBA::PSAM inhibitor vectors in the right anterior horns of lumbar spinal cords under control of the cre recombinase. We achieved the temporally and spatially control of these cells through intraspinal cord injection. Through immunochemistry staining, we measured Claudin-5 disruption of BSCB, and LC3A and misfolded SOD1 levels of MNs. Effects of these chemogenetics approaches on BSCB integrity and MN disease burdens have been evaluated. Results: Intraspinal cord injection into the anterior horns resulting efficiently infection on local astrocytes or MNs by visualized fluoresce expression, whereas no reporter fluorescence on the left anterior horns (in single injection cases), which suggests the intraspinal cord injection technique can stereotactically control desired cells under the control of the designed promoter or cre system. (1) 7 days after systematic CNO administration, no differences were found in tight-junction disruptions between AAV8-GFAP::eGFP infected anterior horns and uninfected anterior horns in the lumbar spinal cord, which can exclude the impact of the intraspinal cord injection per se on the microvessels of BSCB. Surprisingly, chemogenetic activation of astrocytes in the lumbar spinal cord of SOD1-G93A mice at P20 through AAV8-GFAP::DREADD(Gq) tools improved the tight-junction lesions of BSCB. (2) On the other hand, chemogenetic activation of MNs in the lumbar spinal cord of SOD1-G93A mice at P20 through AAV2/9-hSyn::DREADD(Gs) tools improved the disease burdens of infected MNs. In addition, AAV9-CBA::PSAM inhibitor silencing MNs firing results in increased disease burdens, which can be rescued by AAV2/9-hSyn::DREADD(Gs) activation. Conclusion: Recombinant adeno-associated virus can infect desired cells (astrocytes and MNs in this thesis) efficiently after spatially injected into anterior horns of the lumbar spinal cord. In addition, the direct virus microinjection into the spinal cord has no obvious damage to the local tissue. AAV8-GFAP::DREADD(Gq) significantly improved the Claudin-5 loss of in BSCB, and AAV2/9-hSyn::DREADD(Gs) decreased the disease burdens of infected MNs at the presymptomatic disease stage after activated by CNO. All together, chemogenetic manipulation on astrocytes and MNs can improve disease manifestations in tight-junctions integrity and MN disease burdens of ALS