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 a broad spectrum of disease severity. Unfortunately, genetic models of intermediate SMA have been difficult to generate in vertebrates 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, the 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 a 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: Neuromuscular disease; Traf6; Ubc13; NF-kB; Toll-like receptors; TLR; Tumor necrosis factor signaling; TNF; Innate immunity.

Fig. 1

The proteomes and transcriptomes of Drosophila Smn hypomorphs provide 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^X7/X7,Flag-Smn^Tg:WT); T205I (Smn^X7/X7,Flag-Smn^Tg:T205I), Tyrosine (T) to Isoleucine (I); and V72G (Smn^X7/X7,Flag-Smn^Tg:T205I), 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. Note that the total (Ribo-minus) RNA-seq data [18] on the Smn hypomorphs were originally generated with the intent to measure non-coding RNA levels (specifically, spliceosomal snRNAs) and are therefore not as deep as one might like to use for measuring mRNAs, particularly the lowly-expressed ones. The Smn null datasets were polyA-selected and are thus better able to detect changes in mRNA levels

Fig. 2

Proteins involved in Drosophila humoral and melanization defense responses are elevated in Smn mutant proteomes. A Gene Ontology (GO) 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 pathway (panel C, Wikipathways, WP3660) or the Melanization Cascade pathway (panel D) and shaded according to their respective keys

Fig. 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 MMs in animals expressing Smn missense mutations. Bars show the mean, and error bars show the standard error of the mean. Asterisks indicate p-values relative to WT: * < 0.05; ** < 0.01; and *** < 0.001. E Graph showing correlation between the overall phenotypic severity of SMA-causing Smn missense mutations and the number of MMs per animal (from panel B). SMA-like phenotypic severity scores were assigned for each allele (zero being the mildest) based on previously published viability and locomotor assays [20]. Dotted line shows linear regression between data points along with a goodness-of-fit coefficient (R ²)

Fig. 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 two different RNAi transgenes, UAS-Smn^JF (P|TRiP.JF02057|attP2) or UAS-Smn^HM (P|TRiP.HMC03832|attP40). These lines were used 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 for knockdown in both neurons and muscles [53]; and Cg (Collagen 4a1 gap), for knockdown in the fat body, hemocytes, and the larval lymph gland [54]. OreR is the control strain. A plus sign ( +) indicates a wild-type chromosome. B Representative image of wild-type control and MMs in a larva with SMN depleted in the fat body, hemocytes, and lymph gland (Cg-Gal4 > UAS-Smn^JF) or only in the hemocytes (Hml-Gal4 > UAS-Smn^JF). C Number of MMs per animal with and without SMN depletion, as in A

Fig. 5

Innate immune signaling pathways contribute to MMs upon SMN depletion. A Diagram summarizing the features and interconnections between innate immune signaling pathways in Drosophila. Bendless/Ubc13 (Ben) is an E2 ubiquitin conjugase that heterodimerizes with Uev1a and functions in a complex (boxed in gray) with Effete/Ubc5 (another E2) and two different E3 ligases (Traf6 for TLR/Toll or TNF/Wgn, and Diap2 for the Imd/PGRP pathway). The Immune Deficiency protein (Imd) serves not only as a receptor-proximal signaling factor, but also as a secondary substrate for K63-linked polyubiquitylation via Ben•Uev1a. Bendless thus sits at a node that connects many different signaling pathways and cellular processes. B Mutations in the IMD and Toll signaling pathways suppress the number of MMs per animal in Smn RNAi lines. Reduced dosage of Protein Arginine Methyltransferase 5 (PRMT5) also suppresses MMs upon depletion of SMN. C MMs per animal were measured following co-expression of an Smn RNAi transgene together with the indicated RNAi lines targeting selected members of the Toll and IMD pathways, as well as to genes encoding the Jumonji domain containing 6 (JMJD6), Gemin 2 (Gem2), and refractory to sigma P (ref(2)P) proteins. Co-expression of UAS:NLS-GFP was used as a Gal4 negative control (see text for details). D Pie chart of the identified enhancers and suppressors of MM formation, resulting from Smn RNAi depletion using the Cg-Gal4 driver. See Table 1 for details.