Reorganization of Cajal bodies and nucleolar targeting of coilin in motor neurons of type I spinal muscular atrophy

Abstract

Type I spinal muscular atrophy (SMA) is an autosomal recessive disorder caused by loss or mutations of the survival motor neuron 1 (SMN1) gene. The reduction in SMN protein levels in SMA leads to degeneration and death of motor neurons. In this study, we have analyzed the nuclear reorganization of Cajal bodies, PML bodies and nucleoli in type I SMA motor neurons with homozygous deletion of exons 7 and 8 of the SMN1 gene. Western blot analysis is is revealed a marked reduction of SMN levels compared to the control sample. Using a neuronal dissociation procedure to perform a careful immunocytochemical and quantitative analysis of nuclear bodies, we demonstrated a severe decrease in the mean number of Cajal bodies per neuron and in the proportion of motor neurons containing these structures in type I SMA. Moreover, most Cajal bodies fail to recruit SMN and spliceosomal snRNPs, but contain the proteasome activator PA28, a molecular marker associated with the cellular stress response. Neuronal stress in SMA motor neurons also increases PML body number. The existence of chromatolysis and eccentric nuclei in SMA motor neurons correlates with Cajal body disruption and nucleolar relocalization of coil in, a Cajal body marker. Our results indicate that the Cajal body is a pathophysiological target in type I SMA motor neurons. They also suggest the Cajal body-dependent dysfunction of snRNP biogenesis and, therefore, pre-mRNA splicing in these neurons seems to be an essential component for SMA pathogenesis.

The SMN Tudor SIM-like domain is key to SmD1 and coilin interactions and to Cajal body biogenesis

“…The efficiency of the siRNA was confirmed by immunoblotting ( (Lemm et al, 2006). This coilin reorganization is consistent with previous studies showing nucleolar accumulations of coilin upon the experimental disruption of CBs (Tapia et al, 2010;Gilder and Hebert, 2011) and with our recent observation that SMN depletion in human motor neurons from SMA patients causes CB loss and nucleolar relocalization of coilin (Tapia et al, 2012).…”

“…Cell culture and transfection assays UR61, MCF7 and 293T cell lines were cultured as described previously (Tapia et al, 2012). Transfections with siRNA directed against the human 39UTR SMN were performed with Lipofectamine-RNAiMAX (Invitrogen), and cells were assayed 48 h after transfection.…”

“…3J), suggesting a defective interaction of SMN with CB-protein partners rather than a defective nuclear import (Narayanan et al, 2004). In this way, a defect in the targeting of SMN to CBs is a cardinal feature of SMA motor neurons (Hebert et al, 2001;Tapia et al, 2012).…”

Cajal bodies in neurons

“…96 Taken together, the depletion of canonical CBs seems to be a hallmark feature of SMA motor neurons. Moreover, most of the remaining coilin-positive CBs detected in SMA motor neurons fail to recruit SMN and snRNPs 96,97 and frequently appear as unstructured mini CBs (Fig. 7H, I, inset), suggesting that they are not involved in snRNP biogenesis.…”

“…7J-L). 96 This coilin redistribution probably reflects the impact of reduced SMN levels on transcription and splicing in motor neurons, resulting in a deficient nucleation of canonical CBs. This interpretation is consistent with experimental observations showing that CB disruption with transcriptional inhibitors (actinomycin D or DRB) causes coilin to relocalize and accumulate in perinucleolar caps.…”