
==== Front
Mol Ther Methods Clin Dev
Mol Ther Methods Clin Dev
Molecular Therapy. Methods & Clinical Development
2329-0501
American Society of Gene & Cell Therapy

S2329-0501(24)00128-1
10.1016/j.omtm.2024.101312
101312
Original Article
Combination AAV therapy with galectin-1 and SOD1 downregulation demonstrates superior therapeutic effect in a severe ALS mouse model
Baird Megan C. 124
Likhite Shibi B. 14
Vetter Tatyana A. 13
Caporale Joseph R. 1
Girard Holly B. 1
Roussel Florence S. 1
Howard Abigail E. 1
Schwartz Maura K. 12
Reed Addison R. 1
Kaleem Abuzar 1
Zhang Xiaojin 1
Meyer Kathrin C. kathrin.meyer@nationwidechildrens.org
123∗
1 Center for Gene Therapy, Abigail Wexner Research Institute at Nationwide Children’s Hospital, Columbus, OH 43205, USA
2 Biomedical Sciences Graduate Program, The Ohio State University, Columbus, OH 43210, USA
3 Department of Pediatrics, The Ohio State University, Columbus, OH 43210, USA
∗ Corresponding author: Kathrin C. Meyer, Center for Gene Therapy, Abigail Wexner Research Institute at Nationwide Children’s Hospital, 700 Children’s Drive, Columbus, OH 43205, USA. kathrin.meyer@nationwidechildrens.org
4 These authors contributed equally

06 8 2024
12 9 2024
06 8 2024
32 3 1013127 4 2023
2 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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 SOD1G93A 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 SOD1G93A mice over SOD1 suppression alone. These results highlight the importance of targeting inflammatory microglia as a critical component in future therapeutic development.

Graphical abstract

Baird and colleagues demonstrate that the efficacy of a previously characterized AAV9 SOD1 shRNA gene therapy can be significantly potentiated by co-expression of Gal1 in the same vector. Gal1 is secreted by transduced cells and reduces microglia inflammation, an important aspect of ALS progression that is otherwise not targeted with AAV9.

Keywords

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

Amyotrophic lateral sclerosis (ALS) is a complex and devastating neurodegenerative disease for which there is no cure. With 6,000 new cases diagnosed in the United States each year, the prevalence is believed to be 15,000–21,000 Americans living with ALS at any one time.1 ALS is characterized by progressive and fatal loss of motor neurons in the brain and spinal cord. The loss of this cell population manifests as symptoms of insidious progressive paralysis and usually leads to death within 2–5 years of symptom onset. Approximately 90% of ALS cases are sporadic with no known etiology; up to 10% of ALS patients have a positive family history. Mutations in the superoxide dismutase 1 (SOD1) gene account for approximately 20% of familial cases of ALS, with more than 150 mutations in this gene identified.2,3 Multiple mechanisms have been attributed to SOD1 mutation-related pathogenesis in ALS. It is widely accepted that mutations in SOD1 incur a toxic gain of function and that a proportion of SOD1 is misfolded and accumulated in aggregates, a histological hallmark of SOD1-mediated ALS.4,5 In addition to its involvement in familial ALS, SOD1 aggregates and inclusions have been implicated in sporadic ALS, alluding to a potentially larger patient population that might benefit from treatments targeting SOD1.

Although ALS is characterized as a motor neuron disease, glial cells have been shown in many studies to be directly affected by the disease and to contribute to motor neuron death. For example, selective gene expression studies have shown that the presence of mutant SOD1 in motor neurons alone is sufficient to trigger the onset and early progression of ALS, while reduction of SOD1 expression selectively in astrocytes and microglia leads to extended survival via slowing disease progression, implicating glial cells as a driving cell type of ALS progression.6,7,8 Several in vitro studies demonstrate ALS patient-derived astrocytes to be toxic to healthy motor neurons in co-cultures, indicating a glial role in motor neuron death.9,10,11,12,13,14

Microglia, the innate immune cells of the CNS, are critical mediators of neuroinflammatory responses. In the specific context of ALS, microglia initially might play a neuroprotective role, secreting anti-inflammatory cytokines and neurotrophic factors and decreasing neuronal stress.15,16 At later stages of the disease, however, the balance shifts toward a neurotoxic microglial phenotype, with inflammatory microglia accelerating disease progression.15,16,17,18,19,20 Multiple therapeutic approaches have aimed to target inflammatory genes and mechanisms, but have mostly failed to show a beneficial effect. In some cases, these treatments have even led to a hastening of disease progression.21 We hypothesized that the most successful therapeutic approach must not only target neuroinflammation in ALS, but also target and correct motor neuron and astrocyte pathology, as treatment of individual cell types may not be as effective as targeting all major disease-driving cell types. To test this hypothesis, we designed an adeno-associated gene therapy vector carrying our previously established SOD1 short hairpin RNA (shRNA) in combination with a galectin-1 (Gal1) expression cassette.22,23,24,25 Galectins are widely expressed at sites of inflammation and play an active role in amplification or resolution of inflammatory responses.26 Gal1 counteracts synthesis of pro-inflammatory cytokines, displaying broad anti-inflammatory properties. Gal1 has been shown to attenuate microglial activation by shifting neurotoxic microglia to a neuroprotective phenotype in experimental autoimmune encephalomyelitis (EAE).27

Previous studies have shown adeno-associated virus (AAV)-mediated knockdown of mutant SOD1 in motor neurons and astrocytes significantly extends survival and preserves motor function in multiple mouse models of ALS.22,23,24,25,28,29,30 However, current AAV gene therapy vectors do not effectively target microglia, or target only microglia without being able to reach all three cell types in need.31 To address this limitation, we developed a combination therapy approach for the treatment of ALS, wherein AAV9-mediated delivery is used to target motor neurons and astrocytes for the knockdown of mutant SOD1 with concurrent expression of Gal1 and its secretion from astrocytes to indirectly target microglia, thus decreasing inflammation. We first validated the modulatory effect of Gal1 on ALS microglia in vitro, demonstrating successful decrease in inflammatory cytokine expression in Gal1-treated SOD1G93A microglia. We then generated a dual expression AAV9 vector expressing both Gal1 cDNA and our previously developed well-characterized SOD1.shRNA and tested its efficacy in vivo using both single expression AAVs as controls.22,23,24,25 Importantly, combining AAV-mediated knockdown of SOD1 with Gal1 expression shows a highly synergistic effect with significantly extended survival in the severe SOD1G93A mouse model, along with significant improvements in the motor phenotype and neuromuscular junction (NMJ) innervation. Our results underline the importance of combination therapeutic approaches for complex neurodegenerative disorders, including ALS, to achieve optimal outcomes.

Results

Gal1 conditioning of SOD1G93A microglia decreases microglia mediated motor neuron toxicity by modulation of microglial activation in vitro

To determine the effect of Gal1 on ALS microglia, HEK293 cells were transfected with plasmid containing Gal1 as previously described.32 A red fluorescent protein (RFP) reporter plasmid was used as a control. Supernatant was collected from transfected cells every 24 h up to 72 h after transfection. After the last supernatant collection, HEK293 cells were fixed and overexpression of Gal1 was confirmed by immunocytochemistry (Figure 1A). A corresponding increase in secretion of Gal1 from HEK293 cells was confirmed via ELISA on collected HEK293 supernatants (Figure S1). Cultured microglia isolated from endpoint SOD1G93A mice and age-matched littermate controls were then conditioned with Gal1 or RFP containing supernatant mixed with microglia culture medium for 3 days before co-culture with GFP-positive mouse motor neurons (Figure 1B). For co-culture analysis, motor neurons expressing GFP under the HB9 promoter were differentiated from mouse embryonic stem cells in vitro and FACS sorted for enrichment of GFP+ cells. GFP+ motor neurons were plated at a density of 6,000 cells per well and microglia conditioned with either Gal1 or RFP were added to motor neuron cultures at a density of 35,000 cells per well. Co-culture plates were imaged daily using IN Cell Analyzer 6000 (GE Healthcare). After 72 h in co-culture, a significant reduction in motor neuron survival was observed in neurons in contact with SOD1G93A microglia compared with wild-type (WT) control microglia (Figure 1C). This result is consistent with our other studies that show ALS microglia to be highly toxic to motor neurons in culture.17 However, Gal1 preconditioning of SOD1G93A microglia rescued motor neuron death (Figures 1D and 1E).Figure 1 Gal1 conditioning of SOD1G93A 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 SOD1G93A microglia co-cultured with motor neurons. (C) Representative images of motor neurons co-cultured with untreated and Gal1 conditioned SOD1G93A 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.

To determine the mechanism by which Gal1 conditioning of SOD1G93A microglia enhanced motor neuron survival in vitro, we investigated the neuroinflammatory state of conditioned and untreated SOD1G93A microglia. As microglia-mediated motor neuron toxicity has been attributed to secretion of proinflammatory cytokines, we tested co-culture supernatants for tumor necrosis factor (TNF)-α and nuclear factor (NF)-κB activation. ELISA-based quantification of TNF-α levels in co-culture supernatants showed nearly 2-fold decreased expression of TNF-α in SOD1G93A and WT microglia as a result of Gal1 conditioning (Figures 2A and 2B). Next, we examined the effect of Gal1 conditioning on NF-κB activation via quantification of phospho-p65 and total p65.17 p65 is an NF-κB subunit and, along with p50, forms the most abundant heterodimer in the NF-κB pathway.33 Upon activation via proinflammatory cytokines, p65 is phosphorylated, which induces a confirmational change, subsequent translocation of the complex to the nucleus, and promotion of NF-κB activation.33 ELISA-based quantification of phospho-p65 and total p65 in microglia protein extracts showed a significantly decrease phospho-p65/total p65 ratio in conditioned microglia compared with unconditioned microglia (Figures 2C and 2D).Figure 2 Gal1 conditioning of SOD1G93A 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.

Last, we investigated the expression of common M1/M2 microglial markers to determine Gal1 conditioned and unconditioned microglia neurotoxic vs. neuroprotective state. qRT PCR analysis showed very little change in the expression of M1 markers CD68/CD86 (Figures S2A and S2B) and M2 marker IL-10 (Figure S2C) in Gal1-conditioned SOD1G93A microglia. However, the expression of arginase-1, an M2 marker indicative of alternative, anti-inflammatory activation, was greatly increased with Gal1 conditioning (Figure S2D). Taken together, these data show that, while Gal1 may not completely resolve classical microglial activation, it decreases the inflammatory profile and might maintain a better balance between neurotoxic and neuroprotective activation of ALS microglia.

Combination therapy using AAV9.SOD1.shRNA.Gal1 synergistically enhances survival of SOD1G93A mice compared with AAV9.Gal1- or AAV9.SOD1.shRNA-only treatments

Since we established that Gal1 treatment of SOD1G93A microglia was able to enhance motor neuron survival and modulate microglial activation in vitro, we tested the hypothesis that a combination therapy targeting microglial activation in tandem with the suppression of mutant SOD1 in motor neurons and astrocytes would be superior to either single treatments alone. We used our previously developed AAV9.SOD1.shRNA vector and developed 2 additional AAV vectors, one containing the Gal1 expression cassette only under the cytomegalovirus (CMV) enhancer/chicken β-actin promoter (AAV9.Gal1), and a combination vector containing the SOD1.shRNA under H1 promoter and Gal1 expression cassette (AAV9.SOD1.shRNA.Gal1) (Figure 3A). Importantly, all vectors were manufactured with the same process and the same titration assay (AAV2 inverted terminal repeat [ITR]-based digital droplet PCR [ddPCR]) was used to determine the vector concentrations. Before vector manufacturing, we verified plasmid expression in HEK293 cells (Figure S3). We ensured the proper packaging of the new viral vectors (AAV9.Gal1 and AAV9.SOD1.shRNA.Gal1) by confirming the presence of the Gal1 expression cassette in corresponding vectors (Figure S4B). As we have extensively assessed SOD1 knockdown using this exact SOD1 shRNA, an extensive expression and knockdown analysis was not necessary in this present study.22,24,25,34 However, we dosed a small cohort of mice with our new AAV construct expressing both the SOD1 shRNA and Gal1 cDNA to ensure that the SOD1 shRNA was working properly in the new vector in vivo. Neonatal SOD1G93A and WT mice were dosed via intracerebroventricular (ICV) injection with AAV9.SOD1.shRNA.Gal1 at 7.5e10 vector genomes (vg)/mouse (n = 1 uninjected SOD1G93A, n = 2 AAV9.SOD1.shRNA.Gal1 SOD1G93A, n = 1 uninjected WT). Treated and uninjected control animals were sacrificed 1 month after injection and lumbar spinal cords were harvested. Western blot analysis of lumbar spinal cord showed a decrease in SOD1 expression in injected SOD1G93A mice and an increase in Gal1 expression (composed of endogenous Gal1 and vector-derived Gal1) in injected SOD1G93A and WT mice as compared with the uninjected controls (Figures S4C and S4D).Figure 3 Combination of Gal1 and SOD1.shRNA therapies synergistically increases survival and preserves motor function of SOD1G93A 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.

After preliminary vector confirmation, we initiated efficacy studies. All animals were injected on postnatal day (p) 1 via ICV injection to deliver the AAV into the cerebrospinal fluid (CSF) with one of our viral vector constructs. Untreated SOD1G93A littermates served as age-matched controls. Based on our previous studies with the AAV9.SOD1.shRNA, we chose the suboptimal dose of 7.5e10 vg (based on AAV-ITR ddPCR titration method) per animal so that differences in efficacy would be easier to identify. A cohort containing a minimum of 20 animals (10 males and 10 females) per group were followed to their humane endpoint (as defined in Methods) to establish survival. Survival study animals underwent behavioral testing such as body weight monitoring, performance on accelerating rotarod, and hindlimb grip strength measures beginning at 60 days of age (Figures 3 and S5A). A cohort of treated SOD1G93A mice were sacrificed at 130 days (n = 8 per group) to assess disease status at an age correlating to the approximate median survival of untreated SOD1G93A mice (Figures 6, S7, and S9).

SOD1G93A mice treated with AAV9.Gal1 vector had no improvement in survival compared with untreated SOD1G93A mice (Figure 3A), with a median survival of 138 days, while SOD1G93A mice treated with AAV9.SOD1.shRNA had a significantly increased median survival of 190 days, consistent with our previous studies using this viral vector. Importantly, SOD1G93A mice treated with AAV9.SOD1.shRNA.Gal1 showed an impressive further extension of survival by an additional 38 days over AAV9.SOD1.shRNA-treated mice. AAV9.SOD1.shRNA.Gal1-treated SOD1G93A mice had a median survival of 228 days, an extension of 89 days over untreated controls (Figures 3B and 3C; untreated: 139 ± 1.7; AAV9.Gal1: 138 ± 1.5; AAV9.SOD1.shRNA: 190 ± 4.5; AAV9.SOD1.shRNA.Gal1: 228 ± 4.8; median ± SEM; one-way ANOVA; p < 0.0001). In addition to median survival, we also analyzed the survival distribution across treatment groups. All of untreated animals survived to less than 150 days of age, whereas 19% of AAV9.Gal1-treated SOD1G93A mice survived just past 150 days of age. Eighty-one percent of the AAV9.SOD1.shRNA-treated animals survived longer than 150 days of age and 19% survived in excess of 200 days. Finally, 90% of AAV9.SOD1.shRNA.Gal1-treated SOD1G93A mice survived more than 200 days of age, with 20% surviving in excess of 250 days of age (Figure 3D).

Changes in body weight (Figures S5B and S5C) as well as motor performance on accelerating rotarod and hindlimb grip strength were also evaluated in the survival cohorts of mice (Figures 3E and 3F). Treatment with AAV9.Gal1 was insufficient to improve either body weight or motor function over untreated controls. However, SOD1G93A mice treated with AAV9.SOD1.shRNA and AAV9.SOD1.shRNA.Gal1 maintained body weight and performance on accelerating rotarod and hindlimb grip strength until just before reaching humane endpoint. We did not see an overt benefit of AAV9.SOD1.shRNA.Gal1 treatment over AAV9.SOD1.shRNA treatment alone for these measures. In summary, with combination treatment via AAV9.SOD1.shRNA.Gal1, we profoundly extended the median survival of SOD1G93A mice by 27% over AAV9.SOD1.shRNA therapy alone and 64% over untreated controls and saw a preservation of motor function until humane endpoint, indicating improvement in quality of life for AAV9.SOD1.shRNA- and AAV9.SOD1.shRNA.Gal1-treated SOD1G93A mice.

AAV9.SOD1.shRNA.Gal1 treatment decreases mutant SOD1 expression, preserves motor neuron survival, and decreases astrocytosis in the lumbar spinal cord of SOD1G93A mice

Treatment with AAV9.SOD1.shRNA.Gal1 effectively decreased SOD1 expression in the lumbar spinal cord of SOD1G93A mice sacrificed at their respective endpoints, similar to AAV9.SOD1.shRNA treatment, as indicated by immunohistochemistry (IHC) and as experienced with this SOD1.shRNA construct in other studies (Figures 4A–4D). The effect of treatment on motor neuron survival was examined via immunohistological automated image quantification. We quantified choline-acetyltransferase (ChAT)-positive motor neurons in the lumbar spinal cord of SOD1G93A mice at respective endpoints for each treatment group via automated motor neuron detection and quantification (Figure S6). We observed approximately 2-fold more motor neurons in sections of the lumbar spinal cord of AAV9.SOD1.shRNA- and AAV9.SOD1.shRNA.Gal1-treated mice compared with untreated mice (Figures 4E–4H and 4Q). Accordingly, we also found the motor neuron area to be higher across lumbar spinal cord sections of AAV9.SOD1.shRNA and AAV9.SOD1.shRNA.Gal1 treated animals, (Figures 4E–4H, and 4R). Next, we evaluated the SOD1 signal intensity in ChAT-positive motor neurons identified during automated analysis (Figure S4). We found that the average intensity of SOD1 signal in motor neurons was decreased by approximately 60% in mice treated with AAV9.SOD1.shRNA and AAV9.SOD1.shRNA.Gal1 vectors (Figures 4I–4L, and 4S). Reduction of SOD1 expression in the lumbar spinal cord of endpoint SOD1G93A mice was also confirmed by western blot analysis. At the respective humane endpoints, SOD1 expression was decreased approximately 40% in AAV9.SOD1.shRNA-only treated animals and by approximately 50% in AAV9.SOD1.shRNA.Gal1-treated animals (Figures 4U and 4V). Additionally, we found that treatment with AAV9.SOD1.shRNA and AAV9.SOD1.shRNA.Gal1 led to significantly lower GFAP signal intensity, indicating a reduction in astrocytosis (Figures 4M–4P and 4T).Figure 4 AAV9.SOD1.shRNA.Gal1 treatment decreases expression of SOD1 and increases motor neuron survival in the lumbar spinal cord of endpoint male SOD1G93A mice

(A–P) Representative deconvolved EDF images of spinal cord ventral horns from SOD1G93A 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 SOD1G93A mice sacrificed at respective endpoint (n = 3 per group). Error bars represent ±SEM. ∗∗∗∗p < 0.0001, ∗p < 0.05.

At p130, untreated and AAV9.Gal1-treated SOD1G93A mice have severe hindlimb weakness, resulting in extensive hindlimb paralysis and begin to approach humane endpoint. However, at this time point, AAV9.SOD1.shRNA and AAV9.SOD1.shRNA.Gal1-treated mice seem to be phenotypically normal (Figures 3B–3F). To determine molecular differences between groups at this critical time point, a cohort of treated and untreated SOD1G93A mice were sacrificed.

Similar to endpoint tissues, p130-untreated and AAV9.Gal1-treated mice have high SOD1 expression throughout the lumbar spinal cord and significantly higher SOD1 intensity, specifically in motor neurons compared with AAV9.SOD1.shRNA- and AAV9.SOD1.shRNA.Gal1-treated mice (Figures S7A–S7L and S7S). Correlating with SOD1 intensity per motor neuron, untreated and AAV9.Gal1-treated SOD1G93A mice have significantly fewer motor neurons and reduced motor neuron area compared with AAV9.SOD1.shRNA- and AAV9.SOD1.shRNA.Gal1-treated mice (Figures S7Q and S7R). Western blot analysis of lumbar spinal cord showed approximately 40% and 50% reduction in SOD1 expression with AAV9.SOD1.shRNA and AAV9.SOD1.shRNA.Gal1 treatments, respectively (Figures S7U and S7V). Treatment with AAV9.SOD1.shRNA and AAV9.SOD1.shRNA.Gal1 also decreased astrocytosis in the lumbar spinal cord of SOD1G93A mice, as indicated by reduced intensity of GFAP staining (Figures S7M–S7P and S7T). Taken together, these data suggest the lack of phenotype in AAV9.SOD1.shRNA- and AAV9.SOD1.shRNA.Gal1-treated SOD1G93A mice at this critical time point in disease state can be attributed to reduction of mutant SOD1 and subsequent preservation of motor neurons, as well as reduction of astrocytosis in the lumbar spinal cord.

AAV9.SOD1.shRNA.Gal1 treatment decreases microglial activation in the lumbar spinal cord of SOD1G93A mice

To determine the effect of treatment on microglial activation, we examined expression of common microglia markers in the lumbar spinal cord of SOD1G93A mice sacrificed at their respective endpoints. Iba1 is a marker for microglia, expressed in both resting and activated microglia. Upon activation, microglia upregulate their Iba1 expression. Via immunohistochemical staining, we found a significant decrease in Iba1 intensity in the lumbar spinal cord of mice treated with AAV9.SOD1.shRNA and AAV9.SOD1.shRNA.Gal1 (Figures 5A–5D and 5M). Reduction of Iba1 expression in the lumbar spinal cord of AAV9.SOD1.shRNA- and AAV9.SOD1.shRNA Gal1-treated SOD1G93A mice was also confirmed by western blot analysis (Figures 5P and 5Q). We next examined expression of CD68, a marker commonly used to identify inflammation associated with microglial activation, and found lower intensity of CD68 in the lumbar spinal cord of SOD1G93A mice treated with both AAV9.SOD1.shRNA and AAV9.SOD1.shRNA.Gal1 (Figures 5E–5H and 5N). We also investigated the overlap area of Iba1 and CD68, with Iba1+ and CD68+ areas to be indicative of highly activated microglia. We found significantly higher overlap of Iba1 and CD68 staining in the untreated and AAV9.Gal1-treated mice compared with AAV9.SOD1.shRNA- and AAV9.SOD1.shRNA.Gal1-treated mice (Figures 5I–5L and 5O). Additionally, we observed a decrease in ameboid morphology of microglia with AAV9.Gal1 and AAV9.SOD1.shRNA.Gal1 treatment, indicating a decrease in microglia activation (Figure S8). Although we did not see a decrease in CD68 expression with Gal1 administration alone, this is consistent with our in vitro study, which also did not show a significant reduction of CD68 in SOD1G93A microglia conditioned with Gal1 media.Figure 5 AAV9.SOD1.shRNA.Gal1 treatment reduces Iba1 and CD68 expression in lumbar spinal cord of endpoint male SOD1G93A mice

(A–L) Representative deconvolved EDF images of spinal cord ventral horns from SOD1G93A 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 SOD1G93A mice sacrificed at respective endpoint (n = 3 per group). Error bars represent ±SEM. ∗∗∗∗p < 0.0001, ∗p < 0.05.

Next, we examined microgliosis in the lumbar spinal cord of p130 SOD1G93A mice. While AAV9.Gal1 treatment did not have an effect on Iba1 intensity compared with controls, we observed a significant decrease in CD68 intensity in the lumbar spinal cord of p130 AAV9.Gal1-treated SOD1G93A mice (Figure 6). This correlated to a significantly lower Iba1+ CD68+ overlap area (Figure 6O). Surprisingly, though, there were no significant differences in Iba1 or CD68 intensity between AAV9.SOD1.shRNA- and AAV9.SOD1.shRNA.Gal1-treated animals, there was a marked decrease of Iba1+ CD68+ overlap area in AAV9.SOD1.shRNA.Gal1-treated SOD1G93A mice compared with AAV9.SOD1.shRNA treated mice (Figure 6O). Western blot analysis showed a decrease in Iba1 expression with AAV9.SOD1.shRNA and AAV9.SOD1.shRNA.Gal1 treatment, consistent with IHC results (Figures 6P and 6Q). However, western blot analysis also showed a decrease in Iba1 expression of AAV9.Gal1-treated SOD1G93A mice compared with untreated controls (Figures 6P and 6Q). This significant difference was not observed with our IHC staining or quantification, possibly due to variations between overall intensity analysis with IHC vs. expression analysis via western blot.Figure 6 Treatment with AAV9.SOD1.shRNA.Gal1 reduces microglial inflammation in lumbar spinal cord of male p130 SOD1G93A mice

(A–L) Representative deconvolved EDF images of spinal cord ventral horns from SOD1G93A 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 SOD1G93A mice. Error bars represent ±SEM. ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001.

These data suggest a significant reduction in microgliosis in the lumbar spinal cord of p130 SOD1G93A mice with Gal1 treatment. At respective endpoint, however, this reduction of microgliosis with Gal1 treatment is no longer observed. There is no significant reduction of CD68 intensity or Iba1+ CD68+ overlap area with Gal1 treatment compared with endpoint untreated or endpoint AAV9.SOD1.shRNA-treated SOD1G93A mice (Figure 5O). This change could indicate a drastic activation of microglia in all treatment conditions at endpoint in SOD1G93A mice. Additionally, at respective endpoint, there are no longer significant differences in Iba1 or CD68 intensities or Iba1 CD68 overlap area between AAV9.SOD1.shRNA- and AAV9.SOD1.shRNA.Gal1-treated SOD1G93A mice, also indicating increases in microglial activation at respective endpoints. Thus, at respective endpoint, the neuroinflammatory environment skews heavily toward pro-inflammatory state and Gal1 treatment may no longer be sufficient to shift the balance back toward an anti-inflammatory state.

Gal1 expression is increased in the lumbar spinal cord of untreated and AAV9.Gal1-treated SOD1G93A mice

To determine the pattern of Gal1 expression in the lumbar spinal cord of endpoint treated and untreated SOD1G93A mice, we performed immunohistochemical staining. Surprisingly, we found higher Gal1 expression in the lumbar spinal cord of endpoint untreated and AAV9.Gal1-treated SOD1G93A mice as compared with AAV9.SOD1.shRNA- and AAV9.SOD1.shRNA.Gal1-treated mice as indicated by staining and western blot analysis (Figures 7A–7D and 7P–7Q). With regard to Gal1 expression specifically in motor neurons, we found no significant difference in Gal1 intensity within ChAT-positive motor neurons in the ventral horn of the lumbar spinal cord (Figures 7I–7L and 7N). Non-neuronal Gal1 intensity was higher in the ventral horn of the lumbar spinal cord in untreated and AAV9.Gal1-treated endpoint SOD1G93A mice (Figure 7O). When we analyzed AAV9.Gal1- or AAV9.SOD1.shRNA.Gal1-treated animals at their respective endpoints, we did not see an increase in Gal1 expression compared with untreated and AAV9.SOD1.shRNA-treated mice. However, the comparison is made between the respective endpoints, which were significantly different between groups.Figure 7 Gal1 expression is increased in the lumbar spinal cord of endpoint untreated and AAV9.Gal1 treated SOD1G93A mice

(A–L) Representative deconvolved EDF images of spinal cord ventral horns from SOD1G93A 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 SOD1G93A mice sacrificed at respective endpoint (n = 3 per group). Error bars represent ±SEM. ∗∗∗∗p < 0.0001, ∗∗p < 0.01.

We also examined Gal1 expression in the lumbar spinal cord of treated and untreated SOD1G93A mice sacrificed at p130 to determine if the decrease in microgliosis observed at the p130 time point could be correlated to Gal1 expression. Results were consistent with endpoint data, with the highest expression of Gal1 observed in the untreated and AAV9.Gal1-treated groups (Figure S9).

AAV9.SOD1.shRNA.Gal1 treatment preserves innervation of NMJs in SOD1G93A mice

To assess the impact of motor neuron degeneration, we next examined NMJ integrity in the quadriceps muscle of endpoint SOD1G93A mice. We evaluated NMJs as fully innervated or partially innervated (fully or partially intact pre-synaptic and post-synaptic components and a complete contour overlap of axon and endplate) (Figures 8A–8C), and denervated NMJs (only postsynaptic components with no overlap of pre- and post-synaptic components) (Figures 8G–8I). Untreated and AAV9.Gal1-treated SOD1G93A animals had significantly disrupted NMJs, as approximately 90% of the NMJs observed in these groups were denervated (Figure 8J). Treatment with AAV9.SOD1.shRNA and AAV9.SOD1.shRNA.Gal1, however, resulted in incomplete, albeit improved, innervation of NMJs, as the majority of NMJs observed in these groups were either fully or partially innervated (Figures 8A–8F). Thus, enhanced preservation of motor neurons may have led to a corresponding preservation of peripheral synaptic terminals in the quadriceps muscle of AAV9.SOD1.shRNA- and AAV9.SOD1.shRNA.Gal1-treated SOD1G93A mice.Figure 8 AAV9.SOD1.shRNA.Gal1 treatment preserves NMJ integrity in the quadriceps muscle compared with untreated and AAV9.Gal1 treated SOD1G93A mice

(A–I) SOD1G93A mice were sacrificed at their respective humane endpoints and quadriceps were harvested. (A–I) Representative deconvolved EDF images of quadriceps NMJs from SOD1G93A 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).

AAV9.Gal1 and AAV9.SOD1.shRNA.Gal1 treatments are well tolerated and have no effect on body weight or survival of WT mice

We injected a small cohort of WT mice with the same vectors at the same dose and monitored them 1 year after injection (n = 3 AAV9.Gal1; n = 3 AAV9.SOD1.shRNA; n = 3 AAV9.SOD1.shRNA.Gal1). We found that treatment with Gal1-containing vectors did not adversely affect behavior or motor function of WT mice. Injected mice seemed to be normal and indistinguishable from uninjected WT mice with respect to behavior monitoring, body weight, and performance on accelerating rotarod and hindlimb grip strength meter (Figure S10).

In this study, we show for the first time the effect of combination therapy using SOD1 downregulation and Gal1 on motor function and survival of SOD1G93A mice. We developed two novel AAV vectors (AAV9.Gal1 and AAV9.SOD1.shRNA.Gal1) and tested our combination therapy against both single construct vectors as controls. Treatment with AAV9.Gal1 alone was unable to rescue motor function or survival of SOD1G93A mice. When paired with SOD1 downregulation, however, the addition of Gal1 remarkably extended survival of SOD1G93A mice. With AAV9.SOD1.shRNA treatment, we increased the median survival of SOD1G93A mice from 139 days to 228 days, an impressive 89-day and 64% extension over untreated controls. Importantly, we improved upon our previously established and highly effective AAV9.SOD1.shRNA therapy, increasing survival an additional 38 days when administered in tandem with Gal1. This profound effect on survival highlights the utility of combination therapies for complex diseases with a hallmark of non-cell autonomous toxicity.

Discussion

ALS is the most common adult-onset neurodegenerative disease. Onset typically occurs between the ages of 55 and 65 years and, as with many other neurodegenerative diseases, age is a major risk factor for ALS.35 Globally, cases of ALS are expected to increase by 69% by 2040, predominantly due to the aging of the population.36 There are only three medications approved by the US Food and Drug Administration (FDA) for the treatment of ALS, a fatal aggressive degenerative disease. These include riluzole, edaravone, and the newly approved phenylbutyrate/ursodoxicoltaurine (Relyvrio) (AMX0035), the latter of which is the only FDA-approved medication in the last 5 years.37,38 These drugs slow the rate of motor decline and provide modest survival benefit via reduction of glutamate excitotoxicity, reduction of oxidative stress, and regulation of mitochondria/endoplasmic reticulum stress, respectively.37,38,39,40 Treatments targeting mutant SOD1, a gene with more than 150 mutations identified leading to 20% of familial ALS cases, have been a focus of numerous potential therapeutic approaches. These therapies result in increased survival, delayed disease onset, and preserved motor function in rat and mouse models of ALS.22,23,25,29,30,41 In addition, clinical trials with antisense oligonucleotide (ASO) and small interfering RNAs targeting SOD1 have shown reduction of SOD1 in patient CSF and spinal cord. Tofersen, an ASO targeting SOD1, which was recently FDA approved for the treatment of ALS, showed up to 36% reduction in CSF SOD1 concentration in the highest-dose group.42,43 Another recent study using AAV rh10 containing an anti-SOD1 microRNA (AAV-miR-SOD1) showed 90% reduced levels of SOD1 expression in the postmortem lumbosacral spinal cord of a patient in a phase I/II clinical trial.44 In both of these trials, patients did not have significant clinical improvement associated with the knockdown of SOD1 expression. However, the primary outcome for these studies was safety, and clinical efficacy outcomes were exploratory. Both studies cite the small number of participants as study limitations and suggest additional studies to determine clinical efficacy of this approach.42,44

A striking hallmark of ALS and other neurodegenerative disorders is neuroinflammation, which involves an intricate balance of anti-inflammatory and pro-inflammatory mediators. Microglia, the innate immune cells of the CNS, are critical mediators of these neuroinflammatory responses. These cells adopt phenotypes along the continuum of alternatively activated (M2) and classically activated (M1) microglia.15,16 It is thought that, initially, these cells are neuroprotective and provide support and growth factors to failing motor neurons.45 At late stages of the disease, the balance shifts toward a neurotoxic microglial phenotype and these cells become hyper-reactive to inflammatory stimuli, thereby accelerating disease progression.17,46,47

Several previous studies have demonstrated the potential of AAV delivery of molecules that modulate glial inflammation such as insulin-like growth factor-1, vascular endothelial growth factor, granulocyte colony stimulating factor, and hepatocyte growth factor.48,49,50,51,52 In all cases, these treatments led to preserved motor function and increased survival of ALS mice. A clinical trial testing efficacy of trehalose, a compound which activates autophagy, is currently recruiting (HEALY ALS Platform Trial, NCT05136885).53 In preclinical studies, trehalose-induced autophagy activation reduced SOD1 accumulation in microglia, thereby reducing microglial activation and subsequent neurotoxicity.54 These studies highlight the importance of glial modulation in the treatment of ALS. Several drugs have been used to target neuroinflammation in ALS. A clinical trial of NP001, a drug that targets neuroinflammation via regulation of macrophage activation, was unsuccessful in improving disease severity (NCT02794857).55,56 Researchers did, however, see a decrease in progression in a subgroup of patients with high inflammation.57 Minocycline is an antibiotic that has been shown to have anti-apoptotic and anti-inflammatory effects. While preclinical studies showed a reduction of cell death and extended survival of ALS animal models, use in human ALS patients accelerated decline on the ALS functional rating scale and forced vital capacity measures (NCT00047723).58,59 These studies allude to the fact that modulation of neuroinflammation in ALS is highly complex and treatment of neuroinflammation alone may not be sufficient to ameliorate ALS phenotype. Combining neuroinflammatory modulators with therapies that address additional underlying mechanisms of ALS may provide additional benefit over the treatment of neuroinflammatory mechanisms alone. Here, we use our previously developed AAV9.SOD1.shRNA vector to decrease mutant SOD1 in the central nervous system and combine it with a therapy to address neuroinflammation.

Galectins, soluble β-galactoside-binding proteins, are widely expressed at sites of inflammation and play an active role in amplification or resolution of inflammatory responses.26 Gal1, a glycan-binding protein, counteracts the synthesis of pro-inflammatory cytokines and displays broad anti-inflammatory properties. Gal1 has been shown to attenuate microglial activation by shifting neurotoxic microglia toward a neuroprotective M2 phenotype in mouse models of EAE and spinal cord injury.27,60 Importantly, intramuscular administration of oxidized Gal1 showed a beneficial effect on SOD1H46R mice, a milder mouse model than the severe SOD1G93A mouse.61 The treatment resulted in delayed onset, improved motor performance, and extended survival, indicating the viability of Gal1 as a therapeutic candidate for the treatment ALS. In the CNS, astrocytes are the main source of Gal1, with astrocyte stimulation resulting in increased expression and secretion of Gal1. Gal1 plays a crucial role in maintaining the homeostasis of inflammatory processes, as it modulates initiation and resolution of neuroinflammatory cascades,22,23,24,25,26

Here, we show the effect of Gal1 conditioning of ALS microglia in vitro. Gal1 conditioning of ALS microglia reduced microglia mediated motor neuron toxicity in microglia/motor neuron co-cultures. In addition, we found Gal1 conditioning reduced TNF-α and NF-κB activation, reduced neurotoxic markers CD68 and CD86 and increased neuroprotective markers IL-10 and Arg1. Thus, conditioning with Gal1-containing media shifted SOD1G93A microglia from a neurotoxic toward a neuroprotective phenotype. After initial confirmation of the effect of Gal1 on ALS microglia in vitro, we generated two novel AAV vectors containing Gal1 expression cassette alone or in combination with SOD1.shRNA (AAV9.Gal1 and AAV9.Gal1.SOD1.shRNA) for the treatment of SOD1G93A mice in vivo.

To better differentiate effects of treatment, we used a suboptimal dose of 7.5e10vg (ITR ddPCR titer), which is significantly lower compared with what we used in previous studies with AAV9.SOD1.shRNA.22,23,24,25 Importantly, our goal in the current study was not to compare survival with the previous studies, but to perform a side-by-side comparison between the two AAV vector versions. It is, therefore, likely that the survival of SOD1G93A mice could be further extended if treated with the optimal dose. We found that Gal1-only treatment was insufficient to rescue survival or motor phenotype of SOD1G93A mice. While AAV9.SOD1.shRNA treatment significantly extended survival of SOD1G93A mice over untreated and Gal1-only treated mice, combined treatment led to a highly potentiated effect of SOD1.shRNA therapy, extending survival an additional 38 days over AAV9.SOD1.shRNA-treated mice while maintaining grip strength and weight, two potential indicators of quality of life, up until the endpoint. Although we have greatly extended survival and preserved motor function with AAV9.SOD1.shRNA.Gal1 treatment, these treated SOD1G93A mice experience severe kyphosis, lethargy, and rapid weight loss as they approach a humane endpoint. It is unclear precisely why these animals still succumb to death, although other studies using SOD1 silencing in this model have shown an incomplete rescue of pulmonary mechanics after treatment, suggesting respiratory dysfunction as a probable cause of death.30 While we did see increased Gal1 expression with Gal1 treatment at 1 month after injection, when we compared the levels at the respective endpoints of each cohort, we did not find higher Gal1 levels over untreated controls. Of note, we performed this comparison at end stage for each treatment group and the age of the animals, thus, was vastly different at time of analysis since the double-treated animals survived much longer. It is possible that at end stage, other inflammatory factors become too prominent for modulation and could lead to downregulation of Gal1. Further analysis is needed to unravel these mechanisms and to test expression at earlier time points. Regardless, our results demonstrate a profound effect of combination treatment over SOD1 shRNA alone and prominently advocates for combinatory treatment for this and other complex neurodegenerative disorders.

Ideally, treatment regimens would target all the major players of non-cell autonomous toxicity in ALS, thereby imparting an optimal therapeutic effect. Since neuroinflammation is an important aspect of disease in many neurodegenerative disorders including ALS, Alzheimer’s disease, Parkinson’s disease, and multiple sclerosis, our findings for the potential of Gal1 as combination therapeutic agent might also translate to additional disorders.

Materials and Methods

Animals

All procedures performed were in accordance with the National Institute of Health Guidelines and approved by the Institutional Animal Care and Use Committee (IACUC) at the Abigail Wexner Research Institute at Nationwide Children’s Hospital. High copy number SOD1G93A male mice were obtained from Jackson Laboratories (Catalog #: 002726) and bred in the Meyer laboratory. Animals were genotyped to identify SOD1G93A-expressing mice before treatment. Both male and female mice were included in the SOD1G93A and WT mouse experiments. To obtain statistically meaningful results for behavior and survival analysis, a power analysis was performed with data from a previous pilot study; this analysis showed a minimum of eight animals per sex per group were required. Group sizes were set to at least 10 males and 10 females per treatment group to allow for eventual random non-related health issues of mice without losing statistical power. An additional five males and five females per treatment group were added for immunohistochemical and expression analysis at various time points. All mice were housed in rooms with a diurnal cycle of 12 h light, 12 h dark (06:00–18:00) and provided food and water ad libitum.

Vectors

AAV9.SOD1.shRNA has been described previously.22,23,24,25 Briefly, shRNA construct targeting human SOD1 is expressed under the human H1 promoter along with a stuffer sequence optimized for efficient packaging of the AAV vector. In addition, an AAV vector expressing GAL1 cDNA under the CMV enhancer/chicken β-actin promoter was generated. A novel vector combining SOD1 shRNA and GAL1 cDNA, AAV9.SOD1.shRNA.GAL1, was designed and used for this study. All constructs were packaged into self-complementary AAV9 vector using a triple DNA plasmid transfection into NCH HEK 293 cells at NCH Viral Vector Core with respective AAV2-ITR based transgene constructs, an AAV9 helper plasmid containing the AAV rep2 and cap9 genes, and the helper adenovirus plasmid pHELP. The purified vectors were titered using AAV2-ITR based ddPCR assay.

Cells

HEK-293 cells were maintained in Iscove’s modified Dulbecco’s media containing 10% fetal bovine serum (FBS), 1% L-glutamine, and 1% penicillin/streptomycin. Upon reaching approximately 60% confluence, HEK-293 cells were transfected with pBOB-Gal1 or pBOB-RFP plasmids in Iscove’s modified Dulbecco’s media containing 10% FBS, 1% l-glutamine, and 1% penicillin/streptomycin. At 24 h post transfection, the medium was changed to Iscove’s modified Dulbecco’s media containing 2% FBS, 1% l-glutamine, and 1% penicillin/streptomycin. The supernatant was collected every 24 h for 3 days post transfection. Supernatant was filtered through 0.2-μ filter and stored frozen at −80°C. Microglia were pre-incubated with HEK-293 supernatants mixed with microglia medium in a ratio of 1:1, for 3 days before the co-culture.

Isolation and culture of adult mouse primary microglia

Adult microglia were isolated from brains of SOD1G93A and WT littermates as previously described with minor modifications.62 The 4-month-old SOD1G93A and WT littermate mice were deeply anesthetized and perfused transcardially with ice-cold Ringers solution (Thermo Fisher Scientific). Brains that appeared to not be fully exsanguinated were discarded. Brains were fragmented with a scalpel and incubated with an enzymatic solution containing papain for 60 min at 37°C, 5% CO2. The papain solution was quenched with 20% FBS in HBSS and centrifuged for 4 min at 200×g. The pellet was resuspended in 2 mL 0.5 mg/mL DNase I (Worthington Biochemical) in HBSS and incubated for 5 min at room temperature. The brain tissue was gently disrupted with fire-polished Pasteur pipettes and then filtered through a 70-μ cell strainer (Thermo Fisher Scientific) and centrifuged at 200×g for 4 min. The resulting pellet was then resuspended in 20 mL 20% isotonic Percoll (GE Healthcare) in HBSS. We carefully laid 20 mL pure HBSS on top the Percoll layer and centrifugation was performed at 200×g for 20 min with slow acceleration and no brake. The interphase layer containing myelin and cell debris was discarded, and the pellet containing the mixed glial cell population was washed once with HBSS and suspended in Dulbecco’s modified Eagle’s/F12 medium with GlutaMAX (DMEM/F12) supplemented with 10% heat inactivated FBS, antibiotic-antimycotic (all from Life Technologies) and 5 ng/mL carrier-free murine recombinant granulocyte and macrophage colony stimulating factor (GM-CSF) (R&D Systems). The cell suspension from four mouse brains was plated on a 15 cm2 plate (Corning) coated with poly-L-lysine (Sigma) and maintained in culture at 37°C in a 95% air/5% CO2. The medium was replaced every 3 days until the cells reached confluency (after approximately 2 weeks). After the glial layer becomes confluent, microglia form a non-adherent, floating cell layer that can be collected, replated, and cultured for an extended period. After collecting the floating layer, microglia were incubated for 3 days without GM-CSF before re-plating for co-culture with MNs.

Motor neuron differentiation

Mouse embryonic stem cells expressing GFP driven by the Hb9 promoter (HBG3 cells, kind gift from Tom Jessell) were cultured on primary mouse embryonic fibroblasts (Millipore) and differentiated to MNs with the addition of 2 μM retinoic acid (Sigma) and 2 μM purmorphamine (Calbiochem). After 5 days of differentiation, the embryoid bodies were dissociated and sorted for GFP on a FACSVantage/DiVa sorter (Becton Dickinson).

Microglia and motor neuron co-culture

To obtain Gal1 cDNA, total RNA from mouse lumbar spinal cord tissue was obtained using RNAeasy kit. The RNA was converted to cDNA using Qiagen RT first strand synthesis kit. Mouse Gal1 was PCR amplified from the lumbar spinal cord cDNA and further cloned into a lentiviral vector pBOB-empty under CMV promoter using Xba1 site. Cultured adult mouse microglia were conditioned with supernatants of HEK-293 cells transfected with pBOB-Gal1 or pBOB-RFP mixed with 1:1 with fresh microglia medium, for 3 days before the co-culture. Hb9-GFP+ MNs were plated in 96-well plates coated with poly-ornithin (10 μg/mL, Sigma) and laminin (5 μg/mL, Invitrogen) at a density of 6,000 cells per well in 100 μL MN medium containing DMEM:F12 (Invitrogen), 5% horse serum, 2% N2 (Invitrogen), 2% B27 (Invitrogen) + glial cell line-derived neurotrophic factor (10 ng/mL, Invitrogen), brain-derived neurotrophic factor (10 ng/mL, Invitrogen), and ciliary neurotrophic factor (10 ng/mL, Invitrogen). The day after, pre-conditioned microglia were plated on top of MNs at a density of 35,000 cells per well in 100 μL MN media. The co-culture plate was imaged each day by the IN Cell Analyzer 6000 (GE Healthcare), which detects GFP+ motor neurons via automated fluorescent microscopy. Automated data analysis was performed using IN Cell Developer Toolbox 1.9 and IN Cell Analyzer Workstation 3.7 software (GE Healthcare) to quantify number of surviving GFP+ MNs per well, as defined by neurons with neurite outgrowth greater than 50 μm. For each independent experiment, MN count was normalized to the average of WT controls to determine percent survival. Depending on the assay, culture medium or cell lysates were prepared after 3-day co-culture.

Injections

For neonatal mouse injections, p1-p2 SOD1G93A pups were anesthetized on ice for 10 min. The AAV9 vectors were diluted in PBS to obtain correct doses. A total volume of 5 μL containing 7.5e10 vg was injected into the lateral ventricle via ICV injection with a 33G needle and Hamilton syringe as previously described.22,63 This dose is based on previous experiments and allows widespread targeting of cells throughout the entire brain and spinal cord. After injection, pups were warmed and returned to their cage.

Behavior testing and survival analysis

Treated and control SOD1G93A mice, as well WT littermates, were monitored for changes in body mass twice a week. Motor coordination was monitored using an accelerating rotarod instrument (Columbus Instruments) twice per week. Each behavior session consisted of three trials on the accelerating rotarod, beginning at 5 rpm/min, and the time each mouse remained on the rod was recorded. Hindlimb grip strength was also assessed twice per week using a grip strength meter (Columbus Instruments). Each session consisted of three tests per animal. Humane endpoint is defined as an artificial death point when animals can no longer right themselves within 20 s after being placed on their back and/or have severe urinary incontinence and scalding. Survival analysis was performed using Kaplan-Meier survival analysis.

IHC and imaging

Spinal cords were fixed in 4% PFA then removed from vertebrae. Lumbar spinal cords were embedded in agarose and sectioned at 40 μm on a vibratome (Leica) then stained as floating sections. Briefly, tissues were washed three times for 10 min each in PBS, followed by permeabilization and blocking in 10% normal donkey serum in PBS with 0.1% Triton X-100 for 2 h at room temperature. Primary antibodies used were as follows: rabbit anti-SOD1 (1:200; Cell Signaling), goat anti-ChAT (1:50; Millipore chicken anti-GFAP (1:400; Abcam), rat anti-CD68 (1:500; Abcam), rabbit anti-Iba1 (1:200; Wako), and rabbit anti-Gal1 (1:200; Abcam). Tissues were incubated in primary solution for 48 h at 4°C, then washed three times for 10 min each with PBS. After washing, sections were incubated in appropriate fluorescein isothiocyanate-, Cy3-, or Cy5-conjugated secondary antibodies (1:200; Jackson Immunoresearch) and DAPI (1:1,000; Invitrogen). Tissues were then washed three times for 10 min each in PBS, serially mounted on slides from rostral to caudal, and cover slipped with ProLong Gold Antifade Mountant (Invitrogen).

Quadriceps were fixed for 20 min in 4% PFA, washed three times for 10 min each in PBS, then cryoprotected in 30% sucrose. Tissues were then embedded in OCT (TissueTek) and sectioned on a cryostat at 30 μm. Tissues were permeabilized and blocked with 10% normal goat serum, 4% BSA, and 3% Triton X-100 in PBS for 2 h at room temperature in a humidity chamber. Sections were stained with the following primary reagents: chicken anti-NF200 antibody (1:5,000; Abcam), and rabbit anti-Synapsin antibody (1:200; Cell Signaling). Tissues were incubated in primary antibody solution overnight at 4°C in a humidity chamber. Tissues were washed three times for 10 min each in TBS. After washing, sections were incubated in secondary solution containing α-bungarotoxin-488 (1:1,000, Invitrogen), Alexa Fluor 594 goat anti-chicken (1:1,000), Alexa Fluor 546 goat anti-rabbit (1:1,000, Invitrogen), and DAPI (1:1,000; Invitrogen), for 2 h at room temperature, then washed three times for 10 min each in TBS. Slides were cover slipped with ProLong Gold Antifade Mountant (Invitrogen).

Images of whole spinal cord sections were captured as overlapping Z-stacks on a Nikon Eclipse Ti2-E fluorescence microscope with a Hamamatsu ORCA Fusion camera and Nikon D-LEDI light source. The Nikon 10x Plan Apo Lambda D objective was used with a Z-step of 2.5 μm and a final resolution of 0.65 μm/pixel. Additional spinal cord images were captured using a Nikon 20x Plan Apo Lambda objective with a Z-step of 0.9 μm and a final resolution of 0.32 μm/pixel. Representative ventral horn figure images were processed from Z-stacks using automatic three-dimensional (3D) deconvolution and flattened using extended depth of focus (EDF) to a two-dimensional (2D) image in Nikon NIS-Elements software (versions 5.31 and 5.42). Quadriceps NMJ images were captured as Z-stacks using a Nikon AX R confocal in resonant mode on a Ti2-E base with the 40× Apo LWD water immersion objective with a Z-step of 0.31 μm and a final resolution of 0.14 μm/pixel.

Motor neuron quantification and image analysis

All image processing and analysis was performed in Nikon NIS-Elements software (v 5.31 and 5.42), and all steps were applied identically and automatically to all images. Overlapping z stack images of each spinal cord section were processed with rolling ball background subtraction (30-μm radius), stitched, and flattened using EDF to a create a single 2D image of each section for analysis.

To identify ChAT-positive motor neurons, an automated analysis algorithm was developed to detect bright objects in the ChAT channel and refine the selections based on size and shape characteristics. This detection was limited to a manually defined rectangular region spanning the ventral horns of each spinal cord. As a part of the analysis algorithm, the ChAT-positive cells in the ventral horns with a defined soma and a minimum size of 20 μm were counted and measured for each spinal cord section, and mean Gal1 and SOD1 signal intensities were measured in the ChAT-positive cells.

An additional analysis algorithm was used to select the whole spinal cord section using DAPI signal, and measure the mean signal intensities for GFAP, Iba1, and CD68 in the whole spinal cord. Automated thresholds were used to segment the Iba1-positive pixels and CD68-positive pixels in the whole spinal cords, and the CD68-positive/Iba1-positive overlap area was calculated.

Quantification of NMJ integrity

At least 30 z stack images per animal, showing a total of 70–100 distinct NMJs per mouse, were analyzed in NIS-Elements to evaluate integrity of presynaptic (labeled with synapsin and NF200) and postsynaptic (labeled with α-bungarotoxin) components of each NMJ. Using 3D analysis with automated thresholds calculated from background signal intensity in each image, the α-bungarotoxin signal was used to quantify the total volume of the postsynaptic NMJ, and the combined signal from synapsin and NF200 was used to quantify the total volume of innervation overlapping with the postsynaptic NMJ. NMJ innervation was calculated as the percentage of the α-bungarotoxin signal overlapping with presynaptic signal that had been dilated to yield a dynamic range from 0% to 100% innervation. A motor endplate/nerve terminal overlap area of at least 60% was considered fully innervated, while a ≥20% and ≤60% overlap area was considered partially innervated. Any motor endplate/nerve terminal overlap area <20% was considered denervated. Total object volume filtering based on the α-bungarotoxin signal was used to ensure that only NMJs large enough to be complete or near complete were quantified.

Western blot analysis

Spinal cord tissue was lysed using tissue protein extraction reagent buffer (Thermo Fisher Scientific) and complete protease inhibitor cocktail (Roche). We separated 10–40 μg protein by SDS-PAGE (4%–12% NuPage Bis-Tris gels, Life Technologies) then transferred onto polyvinylidene fluoride membrane (Millipore). Membrane was blocked using Intercept Blocking buffer (LI-COR Biosciences) and incubated with primary antibodies overnight at 4C. Primary antibodies used were as follows: rabbit anti-SOD1 (1:750; Cell Signaling), rabbit anti-Iba1 (1:500; Wako), rabbit anti-Gal1 (1:1,000; Abcam), and mouse anti-GAPDH (1:5,000; Millipore). The next day, the membrane was washed, followed by incubation using LiCor IRDye secondary antibodies (1:1 TBST+SDS), according to the manufacturer’s protocols. Membranes were imaged using Odyssey CLx (LI-COR Biosciences). After imaging, membranes were reblotted against a housekeeping gene (GAPDH) followed by another incubation with secondary antibodies.

qRT-PCR

RNA from co-cultured microglia was isolated using the RNAqueous Micro Kit (Ambion) according to the manufacturer’s instructions. RNA was then reverse transcribed into cDNA using the RT2 HT First Strand Kit (SABiosciences). We used 12.5 ng RNA in each qPCR reaction and SyBR Green (Invitrogen) to establish the relative quantities of CD68, CD86, arginase 1, and IL-10 transcripts in WT and SOD microglia preconditioned with Gal1- or RFP-containing medium. Each sample was run in triplicate and relative concentration was calculated using ddCt values normalized to endogenous actin transcript.

RNA from the lumbar spinal cord segments were isolated using phenol/chloroform extraction. Briefly, samples were homogenized using TissueLyser (Qiagen, Valencia, CA) then immersed in 1 mL TRIzol reagent solution. After homogenization, 500 μL chloroform was added to isolate the RNA from the organic phase. The RNA quantity and quality was determined by using NanoDrop spectrophotometer. We reverse transcribed 1 μg RNA into cDNA using the RT2 HT First Strand Kit (Qiagen). We used 12.5 ng RNA in each qPCR reaction and SyBR Green (Invitrogen) to establish the relative quantity of desired transgenes expressed in treated animals versus control animals. Each sample was run in triplicate and relative concentration calculated using the ddCt values, normalized to endogenous transcript.

ELISA

To quantify TNF-α concentration in co-culture medium, TNF-α Quantikine ELISA kit (R&D Systems) was used according to manufacturer instructions. Co-culture medium was collected and centrifuged at 200×g for 2 min. We added 50 μL medium to each well for analysis. Phospho-p65 and Total p65 ELISA kits (Cell Signaling) were used according to the manufacturer’s instructions to quantify NF-κB activation in cell lysates. Secretion of Gal1 in HEK293 cells was confirmed by Gal1 Mouse ELISA (in house) on HEK293 supernatant after transfection. All conditions were tested in triplicate.

Statistical analysis

All statistical tests were performed using Prism software (version 9, GraphPad). Survival log rank (Mantel-Cox) test was performed for survival analysis between treatment groups. One-way or two-way analysis of variance was performed followed by a Bonferroni post hoc analysis of mean differences between groups.

Data and code availability

The datasets generated in this study are available from the corresponding author upon reasonable request.

Supplemental information

Document S1. Figures S1–S10

Document S2. Article plus supplemental information

Acknowledgments

The authors would like to thank the Microscopy Core at the Abigail Wexner Research Institute at Nationwide Children’s Hospital (AWRI-NCH), especially Tatyana Vetter, for microscopy resources and support. Thank you to Andelyn Biosciences for their commitment to quality and production of the vectors described in this study. Special thanks to the Animal Resources Core at the AWRI-NCH for maintaining the high quality of care provided to our animals. We thank Julieth Andrea Sierra-Delgado and Kyle Faulconer for assisting with ddPCR and colony genotyping. Funding for the research in this publication was provided by the Office of Trainee Affairs at the AWRI-NCH and the Helping Link Foundation. Figures (graphical abstract, Figures 1B; 3A; Figures S3A; S4A) created with BioRender.com.

Author contributions

M.B. designed experiments, gathered and analyzed in vivo data, constructed the figures, and wrote the manuscript. S.L. conceived the research, designed experiments, performed in vitro experiments, analyzed data, and edited the manuscript. T.V. developed the IHC imaging and quantification protocols, analyzed data, constructed figures, and edited the manuscript. J.C. assisted in executing experiments and edited the manuscript. H.B., F.R., A.H., M.S., A.R., A.K., and X.Z. assisted in executing experiments. K.M. provided intellectual guidance, supervised the study, edited the manuscript, and provided funding for the completion of this study.

Declaration of interests

A patent on AAV gene therapy vectors that contain a therapeutic protein and additionally express an anti-inflammatory protein or peptide has been filed by The Research Institute at Nationwide Children’s Hospital with K.C.M. and S.B.L. as inventors.

Supplemental information can be found online at https://doi.org/10.1016/j.omtm.2024.101312.
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