Multiplexed chemogenetics in astrocytes and motoneurons restore blood–spinal cord barrier in ALS

Abstract

Blood–spinal cord barrier (BSCB) disruption is thought to contribute to motoneuron (MN) loss in amyotrophic lateral sclerosis (ALS). It is currently unclear whether impairment of the BSCB is the cause or consequence of MN dysfunction and whether its restoration may be directly beneficial. We revealed that SOD1G93A, FUSΔNLS, TDP43G298S, and Tbk1+/− ALS mouse models commonly shared alterations in the BSCB, unrelated to motoneuron loss. We exploit PSAM/PSEM chemogenetics in SOD1G93A mice to demonstrate that the BSCB is rescued by increased MN firing, whereas inactivation worsens it. Moreover, we use DREADD chemogenetics, alone or in multiplexed form, to show that activation of Gi signaling in astrocytes restores BSCB integrity, independently of MN firing, with no effect on MN disease markers and dissociating them from BSCB disruption. We show that astrocytic levels of the BSCB stabilizers Wnt7a and Wnt5a are decreased in SOD1G93A mice and strongly enhanced by Gi signaling, although further decreased by MN inactivation. Thus, we demonstrate that BSCB impairment follows MN dysfunction in ALS pathogenesis but can be reversed by Gi-induced expression of astrocytic Wnt5a/7a.

Side-by-side comparison of the effects of Gq- and Gi-DREADD-mediated astrocyte modulation on intracellular calcium dynamics and synaptic plasticity in the hippocampal CA1

“…Comprehensive knowledge of astrocytic GPCR downstream signalling will help unveil the various roles of the excitatory and inhibitory neuro- and gliotransmitters in the hippocampal network and the resulting effects on brain function. Moreover, recent research has put astrocytic DREADD modulation forward as a potential therapeutic strategy in the treatment of CNS diseases [ 49 , 73 , 74 ] and understanding the relevant signalling cascades in physiological conditions will be the first step towards translation to the clinic.…”

Acute TBK1/IKK-ε Inhibition Enhances the Generation of Disease-Associated Microglia-Like Phenotype Upon Cortical Stab-Wound Injury

“…Male mice originated from the breeding of wild-type male and female B6SJL/F1 mice (from Jackson labs 1 ) were used for the present study at the age of 55–65 days. Mice were maintained at 22°C with a 12/12 h light/dark cycle and had food and water ad libitum as previously reported ( Alami et al, 2020 ). The present study has been authorized by Ulm University Animal Experimentation oversight service and by the Regierungspräsidium Tübingen under the permit no.…”

“…Protein concentrations in the samples were determined using the BiCinchoninic Acid (BCA) assay kit (Thermo Fisher Scientific). The Protein samples (30 μg) were then run on an 10% gel for 2 h at 60V and transferred to a nitrocellulose membrane using semidry blotting method (Ouali- Alami et al, 2020 ). The membranes were blocked for 1 h at room temperature with 5% skim milk powder or 5% BSA dissolved in Tris-Buffered Saline-Tween (TBS-T), followed by an overnight incubation of primary antibodies at 4°C: rabbit anti NAK/TBK1, 1:2,000 (Abcam); rabbit anti NAK/TBK1 (Ser 172), 1:2,000 (Abcam); mouse anti-β-Actin, 1:5,000 (CST); rabbit anti SQSTM1/p62, 1:2,000 (Abcam); rabbit anti phospho-SQSTM1/p62 (Ser403), 1:1,000 (GeneTex); rabbit anti STING, 1:1,000 (Cell signaling); rabbit anti phospho-STING (Ser365), 1:1,000 (Cell signaling), mouse anti PSD95, 1:1,200 (abcam) rabbit anti NR1, 1:1,000 (Sigma).…”

“…In Tbk1 ± mice, the transcriptional profile characteristic of age-associated pro-inflammatory state appears earlier and progresses faster than in wild-type littermates ( Bruno et al, 2020 ), without overt neurodegeneration. Likewise, blood-brain-barrier permeability is disrupted in Tbk1 ± mice ( Alami et al, 2020 ) and conditional myeloid loss of Tbk1 results in a pro-inflammatory state and cellular infiltrate in the spinal cord and other organs ( Duan et al, 2021 ). Moreover, loss of Tbk1 has been shown to synergize with TAK1 downregulation in bringing about the age-associated activation of Receptor-interacting serine/threonine-protein kinase-1 (RIPK1) and related inflammaging phenotype ( Xu et al, 2018 ).…”

Reply to Dr. Héroux

“…Similar experimental approaches to ours are widely used in studies of neuronal plasticity, ranging from classical studies (3) to more recent ones (4). Indeed, it is not uncommon to use multiple data points recorded from a single animal (5,6). As such, we understand that the statistical concerns raised by Dr. H eroux are not limited to our particular paper but should be applied to the majority of motoneuron studies and the substantial part of neuroscience literature.…”