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. 2013 Nov 26;110(48):19348-53.
doi: 10.1073/pnas.1319280110. Epub 2013 Nov 4.

Dysregulation of synaptogenesis genes antecedes motor neuron pathology in spinal muscular atrophy

Zhenxi Zhang  1 , Anna Maria Pinto, Lili Wan, Wei Wang, Michael G Berg, Isabela Oliva, Larry N Singh, Christopher Dengler, Zhi Wei, Gideon Dreyfuss
Affiliations

Dysregulation of synaptogenesis genes antecedes motor neuron pathology in spinal muscular atrophy

Zhenxi Zhang et al. Proc Natl Acad Sci U S A. .

Abstract

The motor neuron (MN) degenerative disease, spinal muscular atrophy (SMA) is caused by deficiency of SMN (survival motor neuron), a ubiquitous and indispensable protein essential for biogenesis of snRNPs, key components of pre-mRNA processing. However, SMA's hallmark MN pathology, including neuromuscular junction (NMJ) disruption and sensory-motor circuitry impairment, remains unexplained. Toward this end, we used deep RNA sequencing (RNA-seq) to determine if there are any transcriptome changes in MNs and surrounding spinal cord glial cells (white matter, WM) microdissected from SMN-deficient SMA mouse model at presymptomatic postnatal day 1 (P1), before detectable MN pathology (P4-P5). The RNA-seq results, previously unavailable for SMA at any stage, revealed cell-specific selective mRNA dysregulations (~300 of 11,000 expressed genes in each, MN and WM), many of which are known to impair neurons. Remarkably, these dysregulations include complete skipping of agrin's Z exons, critical for NMJ maintenance, strong up-regulation of synapse pruning-promoting complement factor C1q, and down-regulation of Etv1/ER81, a transcription factor required for establishing sensory-motor circuitry. We propose that dysregulation of such specific MN synaptogenesis genes, compounded by many additional transcriptome abnormalities in MNs and WM, link SMN deficiency to SMA's signature pathology.

Keywords: C1q complex; Z+ (neuronal) agrin; transcriptome perturbations.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Isolating MNs and surrounding WM from spinal cord of P1 SMA mice by LCM. (A) Frozen cross-sections of WT mouse lumbar spinal cord were prepared as described in Materials and Methods. Images were taken before and after LCM. (Scale bar, 50 μm.) (B) Purity of LCM samples was confirmed by RT-PCR detecting neuron (Chat and p75NTR) and astrocyte (Gfap) markers, using total RNA isolated from MNs, WM, and Dorsal of WT mice. (C) UCSC Genome Browser view of p75NTR and Gfap displaying RNA-seq mapped reads profile, showing four tracks for MNs and WM samples from each of the WT or SMA mouse, respectively. Gene structures (red boxes for exons and gray lines for introns) are depicted below the tracks. Strong p75NTR expression is observed in all four MNs samples and barely detectable in the WM, but Gfap expression is WM-specific.
Fig. 2.
Fig. 2.
Summary of transcriptome changes identified by RNA-seq analysis in P1 SMA MNs and WM. (A) Venn diagrams showing up-regulated and down-regulated genes in SMA MNs and WM. (B) Pie charts showing splicing changes identified in SMA MNs and WM, classified by types of splicing events.
Fig. 3.
Fig. 3.
Agrin Z exon skipping results in drastically decreased levels of Z+ agrin mRNA and protein in SMA MNs. (A) RT-PCR reactions confirmed agrin Z exon skipping in P1 SMA MNs. Similar results have been obtained from at least another P1 SMA mouse littermate. Spliced isoforms are shown as boxes labeled with the corresponding exon number. Black dots on top of the boxes indicate primer binding sites. The red arrows indicate splicing abnormalities observed in SMA mice. (B) Immunofluorescence staining was performed to detect Z+ agrin in lumbar MNs of P1 SMA mice. Dramatic loss of Z+ agrin was detected in P1 SMA MNs. Similar results have been obtained from at least three P1 SMA mice. Immunostaining of p75NTR was used to identify MNs in the anterior horn region of the lumbar spinal cord. (Scale bar, 25 μm.) (C) Immunofluorescence staining was performed to detect Z+ agrin in lumbar MNs of P3 SMA mice. Z+ agrin was completely undetectable in P3 SMA MNs. (Scale bar, 25 µm.) Similar results have been obtained from at least three P3 SMA mice.
Fig. 4.
Fig. 4.
RT-PCR and RT-qPCR experiments validated additional transcriptome changes occurring in genes important for MN functions. (A) RT-PCR reactions were performed as described in Fig. 3A. The abundance of Gria4 flop splice variant was quantitated by RT-qPCR. The unannotated exon 3a in Dusp22 is shown in red. Alternative 5′ splice sites created two exon 7 variants in Adarb1, indicated by 7L (long exon 7) and 7S (short exon 7). (B) RT-qPCR reactions confirmed differential expressed genes identified by RNA-seq. Each reaction used cDNA produced using MNs, WM, and Dorsal isolated from two WT or two SMA mice. Expression level of each gene was monitored by using validated Taqman gene-expression assay and normalized by Gapdh mRNA levels. Error bars indicate SD (*P < 0.05, Student t test).
Fig. 5.
Fig. 5.
Up-regulation of C1q mRNA in P1 SMA MNs results in dramatically increased level of C1q proteins at P2. (A) RT-qPCR reactions quantitating C1qb mRNA levels were performed as described in Fig. 4B, confirming MN-specific up-regulation. Error bars indicate SD (*P < 0.05, Student t test). (B) Immunofluorescence staining was performed to detect C1q complex in lumbar MNs of P2 SMA mice. Substantial increase of C1q was detected in P2 SMA MNs. Immunostaining of p75NTR was used as MN marker. (Scale bar, 25 µm.) Similar results have been obtained from at least three P2 SMA mice.
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