The Drosophila tracheal (respiratory) system is a tubular epithelial network that delivers oxygen to internal tissues. Sprouting of the major tracheal branches is stereotyped and controlled by hard-wired developmental cues. Here we show that ramification of the fine terminal branches is variable and regulated by oxygen, and that this process is controlled by a local signal or signals produced by oxygen-starved cells. We provide evidence that the critical signal is Branchless (Bnl) FGF, the same growth factor that patterns the major branches during embryogenesis. During larval life, oxygen deprivation stimulates expression of Bnl, and the secreted growth factor functions as a chemoattractant that guides new terminal branches to the expressing cells. Thus, a single growth factor is reiteratively used to pattern each level of airway branching, and the change in branch patterning results from a switch from developmental to physiological control of its expression.

Spinal Muscular Atrophy (SMA) is caused by diminished Survival of Motor Neuron (SMN) protein, leading to neuromuscular junction (NMJ) dysfunction and spinal motor neuron (MN) loss. Here, we report that reduced SMN function impacts the action of a pertinent microRNA and its mRNA target in MNs. Loss of the C. elegans SMN ortholog, SMN-1, causes NMJ defects. We found that increased levels of the C. elegans Gemin3 ortholog, MEL-46, ameliorates these defects. Increased MEL-46 levels also restored perturbed microRNA (miR-2) function in smn-1(lf) animals. We determined that miR-2 regulates expression of the C. elegans M2 muscarinic receptor (m2R) ortholog, GAR-2. GAR-2 loss ameliorated smn-1(lf) and mel-46(lf) synaptic defects. In an SMA mouse model, m2R levels were increased and pharmacological inhibition of m2R rescued MN process defects. Collectively, these results suggest decreased SMN leads to defective microRNA function via MEL-46 misregulation, followed by increased m2R expression, and neuronal dysfunction in SMA.

Background:Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by the degeneration of alpha motor neurons in the spinal cord, leading to muscular atrophy. SMA is caused by deletions or mutations in the survival motor neuron 1 gene (SMN1). In humans, a nearly identical copy gene, SMN2, is present. Because SMN2 has been shown to decrease disease severity in a dose-dependent manner, SMN2 copy number is predictive of disease severity.Objective:To develop a treatment algorithm for SMA-positive infants identified through newborn screening based upon SMN2 copy number.Methods:A working group comprised of 15 SMA experts participated in a modified Delphi process, moderated by a neutral third-party expert, to develop treatment guidelines.Results:The overarching recommendation is that all infants with two or three copies of SMN2 should receive immediate treatment (n = 13). For those infants in which immediate treatment is not recommended, guidelines were developed that outline the timing and appropriate screens and tests to be used to determine the timing of treatment initiation.Conclusions:The identification SMA affected infants via newborn screening presents an unprecedented opportunity for achievement of maximal therapeutic benefit through the administration of treatment pre-symptomatically. The recommendations provided here are intended to help formulate treatment guidelines for infants who test positive during the newborn screening process.

Spinal muscular atrophy (SMA) is caused by deletion or mutation of both copies of the SMN1 gene which produces an essential protein known as SMN. The severity of SMA is modified by variable copy number of a second gene, SMN2 that produces an mRNA that is incorrectly spliced with deletion of the last exon. We described previously the discovery of potent C5-substituted quinazolines that increase SMN2 gene expression by two-fold. Discovery of potent SMN2 promoter inducers relied on a cellular assay without knowledge of the molecular target. Using protein microarray scanning with a radiolabeled C5-quinazoline probe, we identified the scavenger decapping enzyme, DcpS as a potential binder. We show that the C5-quinazolines potently inhibit DcpS decapping activity, and that the potency of inhibition correlates with potency for SMN2 promoter induction. Binding of C5-quinazolines to DcpS holds the enzyme in an open, catalytically incompetent conformation. DcpS is a nuclear shuttling protein that binds and hydrolyzes the m7GpppN mRNA cap structure and a modulator of RNA metabolism. Therefore DcpS represents a novel therapeutic target for modulating gene expression by a small molecule.

Proximal spinal muscular atrophy (SMA) is a common motor neuron disorder caused by mutation of the telomeric survival of motor neuron gene SMN1. The centromeric survival of motor neuron SMN2 gene is retained in all SMA patients but does not produce sufficient SMN protein to prevent the development of clinical symptoms. The SMN1 and SMN2 genes differ functionally by a single nucleotide change. This change affects the efficiency with which exon 7 is incorporated into the mRNA transcript. Thus, SMN2 produces less full-length mRNA and protein than SMN1. We have screened a library of compounds in order to identify ones that can alter the splicing pattern of the SMN2 gene. Here, we report that the compound aclarubicin increases the retention of exon 7 into the SMN2 transcript. We show that aclarubicin effectively induces incorporation of exon 7 into SMN2 transcripts from the endogenous gene in type I SMA fibroblasts as well as into transcripts from a SMN2 minigene in the motor neuron cell line NSC34. In type I fibroblasts, treatment resulted in an increase in SMN protein and gems to normal levels. Our results suggest that alteration of splicing pattern represents a new approach to modification of gene expression in disease treatment and demonstrate the feasibility of high throughput screens to detect compounds that affect the splicing pattern of a gene.

BackgroundThe clinical features of SMA, which range along a spectrum of severity, are relatively well described. In contrast, the literature on how individuals with SMA and their families experience this condition is limited. To address this gap, we undertook a qualitative study with individuals affected by SMA Types I, II and III, parents of those affected, and clinicians.MethodsWe completed 16 focus group sessions and 37 interviews in the US with 96 participants including: 21 with individuals with SMA; 64 parents of individuals affected by SMA; and 11 clinicians who specialize in the care of SMA patients.ResultsThe Diagnostic Journey: Families reported substantial diagnostic delays owing to: 1) lack of awareness and knowledge about SMA; 2) the difficulty of distinguishing normal from abnormal development; and 3) the challenge of differential diagnosis. Lack of sensitivity in how clinicians communicated this potentially devastating diagnosis compounded parents’ negative impressions.Newborn Screening: Parents generally held positive views about adding SMA to newborn screening panels. For example, it would: 1) enable earlier access to care; 2) shorten the diagnostic journey; and 3) give families more time to prepare to care for a disabled child. Some noted negative outcomes such as prematurely affecting a parent’s relationship with a child before symptoms are evident.The Psychosocial Impact of Living with SMA: Ten thematic areas characterized the impact: 1) confronting premature death; 2) making difficult treatment choices; 3) fearing the loss of functional ability; 4) coming to terms with lost expectations; 5) loss of sleep and stress; 6) stigma; 7) limitations on social activities; 8) independence; 9) uncertainty and helplessness; and 10) family finances.ConclusionsThe results of this study suggest high levels of burden experienced by individuals with SMA and their families. The difficulties of living with SMA begin with the long and often arduous process of finding a diagnosis for their child. Newborn screening for SMA is seen as an important step toward shortening this journey. The psychosocial effects of coping with SMA are substantial and wide ranging both for the individual living with this condition and family members of affected individuals.

We have exploited the existence of a second copy of the human SMN gene (SMN2) to develop a high-throughput screening strategy to identify potential small molecule therapeutics for the genetic disease spinal muscular atrophy (SMA), which is caused by the loss of the SMN1 gene. Our screening process was designed to identify synthetic compounds that increase the total amount of full-length SMN messenger RNA and protein arising from the SMN2 gene, thereby suppressing the deleterious effects of losing SMN1. A cell-based bioassay was generated that detects SMN2 promoter activity, on which greater than 550,000 compounds was tested. This resulted in the identification of 17 distinct compounds with confirmed biological activity on the cellular primary assay, belonging to nine different structural families. Six of the nine scaffolds were chosen on the basis of their drug-like features to be tested for their ability to modulate SMN gene expression in SMA patient-derived fibroblasts. Five of the six compound classes altered SMN mRNA levels or mRNA splicing patterns in SMA patient-derived fibroblasts. Two of the compound classes, a quinazoline compound series and an indole compound, also increased SMN protein levels and nuclear gem/Cajal body numbers in patient-derived cells. In addition, these two distinct scaffolds showed additive effects when used in combination, suggesting that they may act on different molecular targets. The work described here has provided the foundation for a successful medicinal chemistry effort to further advance these compounds as potential small molecule therapeutics for SMA.

BackgroundThis qualitative study examined how individuals with Spinal Muscular Atrophy (SMA), their caregivers, and clinicians defined meaningful change, primarily in the Type II and non-ambulant type III patient populations, associated with treatment of this condition. In addition, we explored participants’ views about two measures of motor function routinely used in clinical trials for these SMA subtypes, namely the expanded version of the Hammersmith Functional Motor Scale (HFMSE) and the Upper Limb Module (ULM).MethodsThe 123 participants (21 with SMA, 64 parents, and 11 clinicians), recruited through SMA advocacy organizations, participated in one of 16 focus groups or 37 interviews. The sessions were audio-recorded, and verbatim transcripts were analyzed using a grounded theory approach.ResultsFor the participants, meaningful change was relative to functional ability, and small changes in motor function could have an important impact on quality of life. Because patients and families feared progressive loss of functional ability, the participants saw maintenance of abilities as a meaningful outcome. They believed that measures of motor function covered important items, but worried that the HFMSE and ULM might not be sensitive enough to capture small changes. In addition, they felt that outcome measures should assess other important features of life with SMA, including the ability to perform daily activities, respiratory function, swallowing, fatigue, and endurance.ConclusionsGiven the heterogeneity of SMA, it is important to expand the assessment of treatment effects to a broader range of outcomes using measures sensitive enough to detect small changes.