Abstract
C5-substituted 2,4-diaminoquinazolines (2,4-DAQs) ameliorate disease severity in SMA mice. It is uncertain, however, that these compounds increase SMN protein levels in vivo even though they were identified as activators of the SMN2 promoter. These compounds also regulate the expression of other transcripts in neuroblastoma cells. In this study, we investigate the mechanism by which the 2,4-DAQs regulate the expression of SMN2 as well as other targets. D156844, D158872, D157161 and D157495 (RG3039) increased SMN2 promoter-driven reporter gene activity by at least 3-fold in NSC-34 cells. These compounds, however, did not significantly increase SMN2 mRNA levels in type II SMA fibroblasts nor in NSC-34 cells, although there was a trend for these compounds increasing SMN protein in SMA fibroblasts. The number of SMN-containing gems was increased in SMA fibroblasts in response to 2,4-DAQ treatment in a dose-dependent manner. ATOH7 mRNA levels were significantly lower in type II SMA fibroblasts. 2,4-DAQs significantly increased ATOH7, DRNT1 and DRTN2 transcript levels in type II SMA fibroblasts and restored ATOH7 levels to those observed in healthy fibroblasts. These compounds also increase Atoh7 mRNA expression in NSC-34 cells. In conclusion, 2,4-DAQs regulate SMN2 by increasing protein levels and gem localization. They also increase ATOH7, DRNT1 and DRNT2 transcript levels. This study reveals that the protective effects of 2,4-DAQs in SMA may be independent of SMN2 gene regulation. These compounds could be used in concert with a proven SMN2 inducer to develop a multi-faceted approach to treating SMA.
Highlights
Introductionspinal muscular atrophy (SMA) can be classified into five clinical grades based on age of onset and the highest achieved motor milestone
As the 2,4-DAQ D156844 has been previously shown to increase the number of SMN-positive gems within the nuclei of spinal muscular atrophy (SMA) cells [19], we examined the effects of three other C5-substituted 2,4-DAQs—D158872, D157161 and D157495—on gem localization in GM03813 type II SMA fibroblasts
To determine whether or not these observations were unique to this fibroblast line, we examined the effects of the C5-substituted 2,4-DAQs on SMN mRNA levels in two additional type II SMA fibroblast lines, GM22592 and AIDHC-SP22, that possess the same SMN2 copy number as GM03813 cells [31]
Summary
SMA can be classified into five clinical grades based on age of onset and the highest achieved motor milestone. Regardless of clinical grade, proximal SMA results from the loss or mutation of SMN1 (survival motor neuron 1) on chromosome 5q13 [5]. SMN is implicated in stabilizing messenger ribonucleoprotein (mRNP) complexes in axons [6]. In addition to these well characterized functions, SMN is involved in many other intracellular processes [7]
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