Combination AAV therapy with galectin-1 and SOD1 downregulation demonstrates superior therapeutic effect in a severe ALS mouse model

Abstract

Neuroinflammation is a miscreant in accelerating progression of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). However, treatments targeting neuroinflammation alone have led to disappointing results in clinical trials. Both neuronal and non-neuronal cell types have been implicated in the pathogenesis of ALS, and multiple studies have shown correction of each cell type has beneficial effects on disease outcome. Previously, we shown that AAV9-mediated superoxide dismutase 1 (SOD1) suppression in motor neurons and astrocytes significantly improves motor function and extends survival in ALS mouse models. Despite neuron and astrocyte correction, ALS mice still succumb to death with microgliosis observed in endpoint tissue. Therefore, we hypothesized that the optimal therapeutic approach will target and simultaneously correct motor neurons, astrocytes, and microglia. Here, we developed a novel approach to indirectly target microglia with galectin-1 (Gal1) and combined this with our previously established AAV9.SOD1.short hairpin RNA treatment. We show Gal1 conditioning of SOD1 ^G93A microglia decreases inflammatory markers and rescues motor neuron death in vitro. When paired with SOD1 downregulation, we found a synergistic effect of combination treatment in vivo and show a significant extension of survival of SOD1 ^G93A mice over SOD1 suppression alone. These results highlight the importance of targeting inflammatory microglia as a critical component in future therapeutic development.

Keywords: AAV; SOD1G93A mouse; amyotrophic lateral sclerosis; combination therapy; galectin; gene therapy; microglia; motor neuron disease; neuroinflammation; non-cell autonomous toxicity.

Graphical abstract

Figure 1

Gal1 conditioning of SOD1^G93A microglia reduces microglia-mediated motor neuron toxicity (A) Transfection of HEK293 cells with Gal1 containing plasmid leads to increase of Gal1 expression in vitro. (B) Experimental design of HEK293 transduction and Gal1-conditioned SOD1^G93A microglia co-cultured with motor neurons. (C) Representative images of motor neurons co-cultured with untreated and Gal1 conditioned SOD1^G93A microglia. Quantification of number of GFP⁺ motor neurons per well (D) and percent of surviving motor neurons (E). For graphs d and e, n = 2 WT microglia + RFP, n = 4 WT microglia + Gal1, n = 3 ALS microglia + RFP, n = 4 ALS microglia + Gal1. Error bars represent ±SEM. ∗∗p < 0.01, ∗p < 0.05.

Figure 2

Gal1 conditioning of SOD1^G93A microglia modulates microglial inflammation (A) ELISA quantification of TNF-α activation (∗∗∗∗p < 0.0001, ∗∗∗p = 0.0001, ∗p = 0.0225) and (B) fold change (∗∗∗∗p < 0.0001, ∗∗∗p = 0.0005). (C) ELISA quantification of NF-κB activation and (D) fold change. For all graphs, n = 2. Error bars represent ±SEM.

Figure 3

Combination of Gal1 and SOD1.shRNA therapies synergistically increases survival and preserves motor function of SOD1^G93A mice (A) Viral vector schematics. (B) Kaplan-Meier survival percent survival plot. Survival log rank (Mantel-Cox) test, p < 0.0001. (C) Box-whisker plot of median survival (min/max, 25th to 75th percentiles). Median survival untreated: 139 days, AAV9.Gal1: 138 days, AAV9.SOD1.shRNA: 190 days, AAV9.SOD1.shRNA: 228 days. One-way ANOVA performed, p < 0.0001. (D) Stacked bars graph indicating survival distribution across treatment groups (days of life). (E) Latency to fall on accelerating rotarod plotted from day 60 to endpoint. (F) Performance on hindlimb grip strength meter plotted from day 60 to endpoint. Line of best fit for WT mouse data across the same time period indicated by gray dotted line (full data for WT animals presented in Figure S10). For all graphs, n = 23 untreated, n = 21 AAV9.Gal1, n = 21 AAV9.SOD1.shRNA, n = 20 AAV9.SOD1.shRNA.Gal1. Error bars represent ±SEM.

Figure 4

AAV9.SOD1.shRNA.Gal1 treatment decreases expression of SOD1 and increases motor neuron survival in the lumbar spinal cord of endpoint male SOD1^G93A mice (A–P) Representative deconvolved EDF images of spinal cord ventral horns from SOD1^G93A mice sacrificed at their respective endpoint, 20×, scale bar, 100 μm. (A–D) SOD1 expression (green). (E–H) ChAT⁺ motor neurons (red). (I–L) SOD1/ChAT channels merged, yellow indicates presence of SOD1 in ChAT⁺ motor neurons. (M–P) GFAP expression, indicative of astrocytosis (purple). (Q–T) Quantification of 10× EDF images, one-way ANOVA performed. (Q) Quantification of the number of motor neurons per section (n = 3 per group, 12 sections per sample). (R) Quantification of total area of motor neurons (n = 3 per group, 12 sections per sample). (S) Quantification of SOD1 intensity per motor neuron, normalized to control (n = 3 per group, 4 sections per sample). (T) Quantification of GFAP intensity (n = 3 per group, 4 sections per sample). (U) Representative western blot image showing SOD1 expression and (V) quantification of SOD1 expression in lumbar spinal cord of SOD1^G93A mice sacrificed at respective endpoint (n = 3 per group). Error bars represent ±SEM. ∗∗∗∗p < 0.0001, ∗p < 0.05.

Figure 5

AAV9.SOD1.shRNA.Gal1 treatment reduces Iba1 and CD68 expression in lumbar spinal cord of endpoint male SOD1^G93A mice (A–L) Representative deconvolved EDF images of spinal cord ventral horns from SOD1^G93A mice sacrificed at their respective endpoint, 20x, scale bar, 100 μm. (A–D) Iba1 expression (green). (E–H) CD68 expression (red). (I–L) Iba1/CD68 channels merged, yellow indicates colocalization of Iba1 and CD68. (M–O) Quantification of 10× EDF images, One-way ANOVA performed. (M) Quantification of Iba1 intensity (n = 3 per group, 4 sections per sample). (N) Quantification of CD68 intensity (n = 3 per group, 4 sections per sample). (O) Quantification of Iba1⁺ CD68⁺ overlap area (n = 3 per group, 4 sections per sample). (P) Representative western blot image showing Iba1 expression and (Q) quantification of Iba1 expression in lumbar spinal cord of SOD1^G93A mice sacrificed at respective endpoint (n = 3 per group). Error bars represent ±SEM. ∗∗∗∗p < 0.0001, ∗p < 0.05.

Figure 6

Treatment with AAV9.SOD1.shRNA.Gal1 reduces microglial inflammation in lumbar spinal cord of male p130 SOD1^G93A mice (A–L) Representative deconvolved EDF images of spinal cord ventral horns from SOD1^G93A mice sacrificed at their respective endpoint, 20×, scale bar, 100 μm. (A–D) Iba1 expression (green). (E–H) CD68 expression (red). (I–L) Iba1/CD68 channels merged, yellow indicates colocalization of Iba1 and CD68. (M–O) Quantification of 10× EDF images, One-way ANOVA performed. (M) Quantification of Iba1 intensity (n = 3 per group, 4 sections per sample). (N) Quantification of CD68 intensity (n = 3 per group, 4 sections per sample). (O) Quantification of Iba1⁺ CD68⁺ overlap area (n = 3 per group, 4 sections per sample). (P) Representative western blot image showing Iba1 expression and (Q) quantification of Iba1 expression in lumbar spinal cord of p130 SOD1^G93A mice. Error bars represent ±SEM. ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001.

Figure 7

Gal1 expression is increased in the lumbar spinal cord of endpoint untreated and AAV9.Gal1 treated SOD1^G93A mice (A–L) Representative deconvolved EDF images of spinal cord ventral horns from SOD1^G93A mice sacrificed at their respective endpoint, 20×, scale bar, 100 μm. (A–D) Gal1 expression (green). (E–H) ChAT⁺ motor neurons (red). (I–L) Gal1/ChAT channels merged, yellow indicates presence of Gal1 in motor neurons. (M and N) Quantification of 10x EDF images, One-way ANOVA performed. (M) Quantification of Gal1 intensity (n = 3 per group, 4 sections per sample). (n) Quantification of Ga1 intensity in motor neurons (n = 3 per group, 4 sections per sample). (O) Quantification of non-neuronal Gal1 signal intensity in lumbar spinal cord ventral horn (n = 3 per group, 4 sections per sample). (P) Representative Western Blot image showing Gal1 expression and (Q) quantification of Gal1 expression in lumbar spinal cord of SOD1^G93A mice sacrificed at respective endpoint (n = 3 per group). Error bars represent ±SEM. ∗∗∗∗p < 0.0001, ∗∗p < 0.01.

Figure 8

AAV9.SOD1.shRNA.Gal1 treatment preserves NMJ integrity in the quadriceps muscle compared with untreated and AAV9.Gal1 treated SOD1^G93A mice (A–I) SOD1^G93A mice were sacrificed at their respective humane endpoints and quadriceps were harvested. (A–I) Representative deconvolved EDF images of quadriceps NMJs from SOD1^G93A mice sacrificed at their respective endpoint, 40x water immersion, scale bar, 20 μm. (A–C) Fully innervated NMJ, (D–F) partially innervated NMJ, and (G–I) denervated NMJ as indicated by overlap of presynaptic elements (synapsin/NF200 – red) and postsynaptic elements (α-bungarotoxin – green). (J) Quantification of fully innervated, partially innervated, and denvervated NMJs (n = 3 per group, 70–100 NMJs analyzed per sample).