Dysregulation of innate immune signaling in animal models of Spinal Muscular Atrophy

Abstract

Background: Spinal Muscular Atrophy (SMA) is a devastating neuromuscular disease caused by hypomorphic loss of function in the Survival Motor Neuron (SMN) protein. SMA presents across broad spectrum of disease severity. Unfortunately, vertebrate models of intermediate SMA have been difficult to generate and are thus unable to address key aspects of disease etiology. To address these issues, we developed a Drosophila model system that recapitulates the full range of SMA severity, allowing studies of pre-onset biology as well as late-stage disease processes.

Results: Here, we carried out transcriptomic and proteomic profiling of mild and intermediate Drosophila models of SMA to elucidate molecules and pathways that contribute to the disease. Using this approach, we elaborated a role for the SMN complex in the regulation of innate immune signaling. We find that mutation or tissue-specific depletion of SMN induces hyperactivation of the Immune Deficiency (IMD) and Toll pathways, leading to overexpression of antimicrobial peptides (AMPs) and ectopic formation of melanotic masses in the absence of an external challenge. Furthermore, knockdown of downstream targets of these signaling pathways reduced melanotic mass formation caused by SMN loss. Importantly, we identify SMN as a negative regulator of an ubiquitylation complex that includes Traf6, Bendless and Diap2, and plays a pivotal role in several signaling networks.

Conclusions: In alignment with recent research on other neurodegenerative diseases, these findings suggest that hyperactivation of innate immunity contributes to SMA pathology. This work not only provides compelling evidence that hyperactive innate immune signaling is a primary effect of SMN depletion, but it also suggests that the SMN complex plays a regulatory role in this process in vivo. In summary, immune dysfunction in SMA is a consequence of reduced SMN levels and is driven by cellular and molecular mechanisms that are conserved between insects and mammals.

Keywords: NF-kB; Neuromuscular disease; Toll like receptors, TLR; Traf6; Tumor necrosis factor signaling, TNF; Ubc13.

Figure 1.. The proteomes and transcriptomes of Drosophila Smn hypomorphs display overlapping evidence for innate immune activation.

A) A rectangular cartoon and an AlphaFold model of the relative positions of conserved domains of the Drosophila SMN protein and the location of the patient-derived missense mutations used here. B) Principal component analysis of total protein abundances in the Smn transgenic lines. Smn lines: WT (Smn^Tg:WT;X7/X7); T205I (Smn^{Tg:T205I;X7/X7}), Tyrosine (T) to Isoleucine (I); and V72G (Smn^{Tg:T205I;X7/X7}), Valine (V) to Glycine (G). C) Venn diagram of overlapping protein differences in T205I and V72G relative to WT. D) Volcano plot of protein differences in the T205I line relative to WT. Proteins associated with innate immunity are indicated by larger dots. E) Volcano plot of protein differences in the V72G line relative to WT, and proteins associated with innate immunity are labeled as in (D). Dashed vertical bars in (D) and (E) indicate a Log2 FC ratio of +/− 0.58, and the horizontal dashed line corresponds to q-value = 0.05. F) Comparison of T205I proteome (y-axis) with T205I transcriptome (x-axis). The proteome and transcriptome are relative to the WT genotype. G) Comparison of V72G proteome (y-axis) with V72G transcriptome (x-axis). As in (F), the proteome and transcriptome are relative to WT. H) V72G proteome (y-axis) versus Smn^X7/D null transcriptome (x-axis). The differential gene expression of the Smn^X7/D transcriptome is relative to Oregon-R.

Figure 2.. Proteins involved in Drosophila humoral and melanization defense responses are elevated in Smn mutant proteomes.

A) Gene Ontology (GO) term analysis of protein differences in V72G. Adjusted p-values (p.adjust) and number of genes per GO term (Count) are shown at right, which is used to compute a combined score. B) Heat maps of select protein abundance differences from genes within the melanization defense response GO category, known immune-induced peptides, as well as for the NF-kB transcription factors Dorsal-related immunity factor (Dif) and dorsal (dl). C-D) Heatmap illustrations of TMT-MS data from V72G mutants. Log₂-fold change (log2FC) values (mutant/control) for differentially expressed proteins are illustrated within the context of the Humoral Immune Response (Wikipathway WP3660, panel C) or the Melanization Defense Response (panel D) pathway and shaded according to the keys below each pathway.

Figure 3.. Smn missense mutants exhibit elevated melanotic masses.

A-C) Melanotic mass (MM) data for wandering third instar larvae expressing Smn missense mutations. The data in each panel are a different measure of the melanotic mass phenotypes of the same set of larvae. A) Percent of larvae with one or more melanotic mass. Individual data points are the percent of larvae with MMs, 10 larvae per data point. B) The average number of melanotic masses per animal. Data points show the number of MMs in each animal. Number (N) = 50 larvae for each genotype. C) Qualitative size scoring of the largest melanotic mass in each larva. D) Representative images of melanotic masses in animals expressing Smn missense mutations. Bars show the mean, and error bars show standard error of the mean. Asterisks indicate p-values relative to WT: * < 0.05; ** < 0.01; and *** < 0.001.

Figure 4.. Targeted RNAi depletion of Smn in Drosophila immune cells yields melanotic masses and reduced viability.

A) Fraction of larvae that display MMs. RNAi mediated knockdown of SMN was carried out using the Drosophila GAL4/UAS system to drive expression using UAS-Smn^JF (P{TRiP.JF02057}attP2) together with the following GAL4 drivers: da, daughterless (da) for ubiquitous knockdown; C15 (a composite driver that includes elav- (embryonic lethal abnormal vision), sca- (scabrous) and BG57-GAL4 see (Budnik et al. 1996; Brusich et al. 2015) for knockdown in both neurons and muscles; and Cg (Collagen 4a1 gap), for knockdown in the fat body, hemocytes, and the larval lymph gland. OreR is the control strain. A plus sign (+) indicates a wild-type chromosome. B) Representative image of a wild-type control and MMs in a larva with SMN depleted in the fat body, hemocytes, and lymph gland (Cg-GAL4>UAS-Smn^JF). C) Number of MMs per animal with and without SMN depletion, as in (A). Smn^HM (P{TRiP.HMC03832}attP40) is an alternative UAS RNAi line that targets Smn. D and E) Fraction of larvae with MMs (D) and number of MMs per animal (E) using the hemocyte specific Gal4 driver Hml (Hemolectin) together with the UAS-Smn^JF transgene. Control strains as per panel A.

Figure 5.. Innate immune signaling pathways contribute to MMs upon SMN depletion.

A) Mutations in the IMD and Toll signaling pathways suppress the number of MMs per animal in Smn RNAi lines. Mutation of Protein Arginine Methyltransferase 5 (PRMT5) also suppresses MMs upon depletion of SMN. B) Co-depletion of SMN and the indicated RNAi lines for members of the Toll and IMD pathways, Jumonji domain containing 6 (JMJD6), Gemin 2 (Gem2), and refractory to sigma P (ref(2)P). C) Pie chart of the identified enhancer and suppressors of MMs, resulting from Smn RNAi depletion in the fat body, hemocytes, and lymph gland. D) Model summarizing the role of SMN in the homeostatic regulation of the Toll, TNF and IMD signaling pathways in Drosophila larvae. Bendless (Ben) is an E2 ubiquitin conjugase that functions together with two different E3 ligases (Traf6 for Wengen/Toll and Diap2 for the PGRP pathway). The Immune Deficiency protein (Imd) serves not only as an upstream signaling factor, but also as a downstream target for K63-linked polyubiquitylation via Ben•Diap2. Ben thus sits at a node that connects several different signalling pathways and its activity is negatively regulated by SMN. Reduced levels of SMN thereby lead to hyperactivation of downstream targets.