Safety and efficacy of olesoxime in patients with type 2 or non-ambulatory type 3 spinal muscular atrophy: a randomised, double-blind, placebo-controlled phase 2 trial.

BACKGROUND: Spinal muscular atrophy (SMA) is a progressive motor neuron disease causing loss of motor function and reduced life expectancy, for which limited treatment is available. We investigated the safety and efficacy of olesoxime in patients with type 2 or non-ambulatory type 3 SMA. METHODS: This randomised, double-blind, placebo-controlled, phase 2 study was done in 22 neuromuscular care centres in Belgium, France, Germany, Italy, Netherlands, Poland, and the UK. Safety and efficacy of olesoxime were assessed in patients aged 3-25 years with genetically confirmed type 2 or non-ambulatory type 3 SMA. A centralised, computerised randomisation process allocated patients (2:1 with stratification by SMA type and centre) to receive olesoxime (10 mg/kg per day) in an oral liquid suspension or placebo for 24 months. Patients, investigators assessing outcomes, and sponsor study personnel were masked to treatment assignment. The primary outcome measure was change from baseline compared with 24 months between the two treatment groups in functional domains 1 and 2 of the Motor Function Measure (MFM D1 + D2) assessed in the full analysis population. A shorter, 20-item version of the MFM, which was specifically adapted for young children, was used to assess patients younger than 6 years. Safety was assessed in all patients who received one or more doses of the study drug. The trial is registered with ClinicalTrials.gov, number NCT01302600. FINDINGS: The trial was done between Nov 18, 2010, and Oct 9, 2013. Of 198 patients screened, 165 were randomly assigned to olesoxime (n=108) or placebo (n=57). Five patients in the olesoxime group were not included in the primary outcome analysis because of an absence of post-baseline assessments. The change from baseline to month 24 on the primary outcome measure was 0·18 for olesoxime and -1·82 for placebo (treatment difference 2·00 points, 96% CI -0·25 to 4·25, p=0·0676). Olesoxime seemed to be safe and generally well tolerated, with an adverse event profile similar to placebo. The most frequent adverse events in the olesoxime group were pyrexia (n=34), cough (n=32), nasopharyngitis (n=25), and vomiting (n=25). There were two patient deaths (one in each group), but these were not deemed to be related to the study treatment. INTERPRETATION: Olesoxime was safe at the doses studied, for the duration of the trial. Although the primary endpoint was not met, secondary endpoints and sensitivity analyses suggest that olesoxime might maintain motor function in patients with type 2 or type 3 SMA over a period of 24 months. Based on these results, olesoxime might provide meaningful clinical benefits for patients with SMA and, given its mode of action, might be used in combination with other drugs targeting other mechanisms of disease, although additional evidence is needed. FUNDING: AFM Téléthon and Trophos SA.

Notch activity modulates the responsiveness of neural progenitors to sonic hedgehog signaling.

Throughout the developing nervous system, neural stem and progenitor cells give rise to diverse classes of neurons and glia in a spatially and temporally coordinated manner. In the ventral spinal cord, much of this diversity emerges through the morphogen actions of Sonic hedgehog (Shh). Interpretation of the Shh gradient depends on both the amount of ligand and duration of exposure, but the mechanisms permitting prolonged responses to Shh are not well understood. We demonstrate that Notch signaling plays an essential role in this process, enabling neural progenitors to attain sufficiently high levels of Shh pathway activity needed to direct the ventral-most cell fates. Notch activity regulates subcellular localization of the Shh receptor Patched1, gating the translocation of the key effector Smoothened to primary cilia and its downstream signaling activities. These data reveal an unexpected role for Notch shaping the interpretation of the Shh morphogen gradient and influencing cell fate determination.

Gene regulatory logic for reading the Sonic Hedgehog signaling gradient in the vertebrate neural tube.

Secreted signals, known as morphogens, provide the positional information that organizes gene expression and cellular differentiation in many developing tissues. In the vertebrate neural tube, Sonic Hedgehog (Shh) acts as a morphogen to control the pattern of neuronal subtype specification. Using an in vivo reporter of Shh signaling, mouse genetics, and systems modeling, we show that a spatially and temporally changing gradient of Shh signaling is interpreted by the regulatory logic of a downstream transcriptional network. The design of the network, which links three transcription factors to Shh signaling, is responsible for differential spatial and temporal gene expression. In addition, the network renders cells insensitive to fluctuations in signaling and confers hysteresis--memory of the signal. Our findings reveal that morphogen interpretation is an emergent property of the architecture of a transcriptional network that provides robustness and reliability to tissue patterning.

Dynamic assignment and maintenance of positional identity in the ventral neural tube by the morphogen sonic hedgehog.

Morphogens are secreted signalling molecules that act in a graded manner to control the pattern of cellular differentiation in developing tissues. An example is Sonic hedgehog (Shh), which acts in several developing vertebrate tissues, including the central nervous system, to provide positional information during embryonic patterning. Here we address how Shh signalling assigns the positional identities of distinct neuronal subtype progenitors throughout the ventral neural tube. Assays of intracellular signal transduction and gene expression indicate that the duration as well as level of signalling is critical for morphogen interpretation. Progenitors of the ventral neuronal subtypes are established sequentially, with progressively more ventral identities requiring correspondingly higher levels and longer periods of Shh signalling. Moreover, cells remain sensitive to changes in Shh signalling for an extended time, reverting to antecedent identities if signalling levels fall below a threshold. Thus, the duration of signalling is important not only for the assignment but also for the refinement and maintenance of positional identity. Together the data suggest a dynamic model for ventral neural tube patterning in which positional information corresponds to the time integral of Shh signalling. This suggests an alternative to conventional models of morphogen action that rely solely on the level of signalling.

Distinct Sonic Hedgehog signaling dynamics specify floor plate and ventral neuronal progenitors in the vertebrate neural tube.

The secreted ligand Sonic Hedgehog (Shh) organizes the pattern of cellular differentiation in the ventral neural tube. For the five neuronal subtypes, increasing levels and durations of Shh signaling direct progenitors to progressively more ventral identities. Here we demonstrate that this mode of action is not applicable to the generation of the most ventral cell type, the nonneuronal floor plate (FP). In chick and mouse embryos, FP specification involves a biphasic response to Shh signaling that controls the dynamic expression of key transcription factors. During gastrulation and early somitogenesis, FP induction depends on high levels of Shh signaling. Subsequently, however, prospective FP cells become refractory to Shh signaling, and this is a prerequisite for the elaboration of their identity. This prompts a revision to the model of graded Shh signaling in the neural tube, and provides insight into how the dynamics of morphogen signaling are deployed to extend the patterning capacity of a single ligand. In addition, we provide evidence supporting a common scheme for FP specification by Shh signaling that reconciles mechanisms of FP development in teleosts and amniotes.

Pattern formation in the vertebrate neural tube: a sonic hedgehog morphogen-regulated transcriptional network.

Neuronal subtype specification in the vertebrate neural tube is one of the best-studied examples of embryonic pattern formation. Distinct neuronal subtypes are generated in a precise spatial order from progenitor cells according to their location along the anterior-posterior and dorsal-ventral axes. Underpinning this organization is a complex network of multiple extrinsic and intrinsic factors. This review focuses on the molecular mechanisms and general strategies at play in ventral regions of the forming spinal cord, where sonic hedgehog-based morphogen signaling is a key determinant. We discuss recent advances in our understanding of these events and highlight unresolved questions.

Interpretation of the sonic hedgehog morphogen gradient by a temporal adaptation mechanism.

Morphogens act in developing tissues to control the spatial arrangement of cellular differentiation. The activity of a morphogen has generally been viewed as a concentration-dependent response to a diffusible signal, but the duration of morphogen signalling can also affect cellular responses. One such example is the morphogen sonic hedgehog (SHH). In the vertebrate central nervous system and limbs, the pattern of cellular differentiation is controlled by both the amount and the time of SHH exposure. How these two parameters are interpreted at a cellular level has been unclear. Here we provide evidence that changing the concentration or duration of SHH has an equivalent effect on intracellular signalling. Chick neural cells convert different concentrations of SHH into time-limited periods of signal transduction, such that signal duration is proportional to SHH concentration. This depends on the gradual desensitization of cells to ongoing SHH exposure, mediated by the SHH-dependent upregulation of patched 1 (PTC1), a ligand-binding inhibitor of SHH signalling. Thus, in addition to its role in shaping the SHH gradient, PTC1 participates cell autonomously in gradient sensing. Together, the data reveal a novel strategy for morphogen interpretation, in which the temporal adaptation of cells to a morphogen integrates the concentration and duration of a signal to control differential gene expression.

Cooperation between GDNF/Ret and ephrinA/EphA4 signals for motor-axon pathway selection in the limb.

Establishment of limb innervation by motor neurons involves a series of hierarchical axon guidance decisions by which motor-neuron subtypes evaluate peripheral guidance cues and choose their axonal trajectory. Earlier work indicated that the pathway into the dorsal limb by lateral motor column (LMC[l]) axons requires the EphA4 receptor, which mediates repulsion elicited by ephrinAs expressed in ventral limb mesoderm. Here, we implicate glial-cell-line-derived neurotrophic factor (GDNF) and its receptor, Ret, in the same guidance decision. In Gdnf or Ret mutant mice, LMC(l) axons follow an aberrant ventral trajectory away from dorsal territory enriched in GDNF, showing that the GDNF/Ret system functions as an instructive guidance signal for motor axons. This phenotype is enhanced in mutant mice lacking Ret and EphA4. Thus, Ret and EphA4 signals cooperate to enforce the precision of the same binary choice in motor-axon guidance.

Identification of lynx2, a novel member of the ly-6/neurotoxin superfamily, expressed in neuronal subpopulations during mouse development.

We isolated a new gene which shares all the features of the Ly-6/neurotoxin superfamily, from gene organization to predicted 3D structure. As it is preferentially expressed in the nervous system, we called this gene lynx2, by analogy with lynx1, a nAChR modulator. In embryonic and postnatal mouse, lynx2 is expressed in postmitotic central and peripheral neurons. These include subpopulations of motor neurons, sensory neurons, interneurons and neurons of the autonomous nervous system. In addition, lynx2 is transiently expressed around the growing nerves in the limb bud. Comparison of its spatio-temporal expression pattern with that of two other members of this family, lynx1 and ly-6h, shows that these genes are detected both in distinct and overlapping neuron populations.

Synaptopodin and 4 novel genes identified in primary sensory neurons.

We performed differential gene expression profiling in the peripheral nervous system by comparing the transcriptome of sensory neurons with the transcriptome of lower motor neurons. Using suppression subtractive cDNA hybridization, we identified 5 anonymous transcripts with a predominant expression in sensory neurons. We determined the gene structures and predicted the functional protein domains. The 4930579P15Rik gene encodes for a novel inhibitor of protein phosphatase-1 and 9030217H17Rik was found to be the mouse gene synaptopodin. We performed in situ hybridization for all genes in mouse embryos, and found expression predominantly in the primary class of sensory neurons. Expression of 4930579P15Rik and synaptopodin was restricted to craniospinal sensory ganglia. Neither synaptopodin, nor any known family member of 4930579P15Rik, has ever been described in sensory neurons. The identification of protein domains and expression patterns allows further functional analysis of these novel genes in relation to the development and biology of sensory neurons.

Met signaling is required for recruitment of motor neurons to PEA3-positive motor pools.

Motor neurons in the spinal cord are grouped into motor pools, each of which innervates a single muscle. The ETS transcription factor PEA3 is a marker of a few such motor pools. Here, we show that pea3 is first induced by GDNF in a caudal subset of the motor neurons that will constitute the pea3+ population. Expansion of the pea3 domain subsequently occurs by recruitment of neurons from more anterior segments. Signaling by Met, the HGF receptor, is required for the rostral expansion of the pea3 domain, while the onset of pea3 expression is independent of met function. met expression is observed in pioneer neurons but does not precede that of pea3 in recruited neurons. We provide genetic evidence for a non-cell-autonomous function of met during the recruitment process. We propose the presence of a relay mechanism allowing cells induced by peripheral signals to recruit more anterior neurons to adopt the same motor pool-related phenotype.

GDNF acts through PEA3 to regulate cell body positioning and muscle innervation of specific motor neuron pools.

Target innervation by specific neuronal populations involves still incompletely understood interactions between central and peripheral factors. We show that glial cell line-derived neurotrophic factor (GDNF), initially characterized for its role as a survival factor, is present early in the plexus of the developing forelimb and later in two muscles: the cutaneus maximus and latissimus dorsi. In the absence of GDNF signaling, motor neurons that normally innervate these muscles are mispositioned within the spinal cord and muscle invasion by their axons is dramatically reduced. The ETS transcription factor PEA3 is normally expressed by these motor neurons and fails to be induced in most of them in GDNF signaling mutants. Thus, GDNF acts as a peripheral signal to induce PEA3 expression in specific motor neuron pools thereby regulating both cell body position and muscle innervation.