The BMP signaling pathway at the Drosophila neuromuscular junction and its links to neurodegenerative diseases
Introduction
Neurodegenerative diseases are among the most common diseases and yet probing the pathological mechanisms has remained a challenge. The diseases typically come in both sporadic and hereditary forms, and mutations in numerous genes in familial cases have been identified. To obtain insights into how these genes and their mutations are involved in specific diseases, one of our best strategies is to study these genes and the corresponding mutations in model organisms like Drosophila and mice. The Drosophila larval neuromuscular junction (NMJ) is an especially amenable system to study the role of these genes in invertebrates [1]. As shown in Figure 1, the motor neuron's terminals form close spherical connections with the muscle, called boutons [1]. At each bouton, many synapses, the presynaptic regions of which are known as active zones, form between neuron and muscle. These zones have a T-shaped structure known as a T-Bar where synaptic vesicles (SVs, green) cluster, fuse and release glutamate (red lightning) into the extracellular space. The neurotransmitter is then bound by glutamate receptors (purple cylinders) on the muscle that triggers calcium influx and subsequent muscle contraction [2]. Given that Drosophila permits sophisticated genetic manipulations [3, 4], numerous probing experiments can be performed. For example, in a mutant background, proteins can be expressed presynaptically or postsynaptically to determine where they are required. Electrophysiological experiments on individual nerves targeting specific muscles allow one to derive very valuable information about the ability to release vesicles, endocytose membranes, the function of postsynaptic receptors, etc. [5]. FM1-43 dye uptake experiments allow one to establish how much membrane is taken up during endocytosis and how much is released during exocytosis, thus providing real-time analysis of vesicle trafficking [6]. Antibodies against numerous proteins that are present at the NMJ presynaptically and postsynaptically are available and allow one to assess which proteins may be implicated in the process or pathology that is being studied [7]. These data, combined with Transmission Electron Microscopy and Immuno-EM studies provide very valuable information about the number, distribution and size of SV, and the number and size of active zones [7, 8]. Finally, genetic interactions and epistasis with a vast number of mutants as well as the ability to overexpress specific proteins allow for a detailed analysis of genetic pathways [9]. In summary, no synapses are currently more amenable to a diverse set of manipulations in vivo than the Drosophila NMJ, and by combining the information obtained from different experimental datasets, very valuable biological information can be derived.
The development and growth of the Drosophila NMJ requires an anterograde as well as a retrograde input from the muscle [2]. The anterograde signaling is primarily mediated by the Wnt ligand Wingless (Wg) [10], whereas retrograde signaling occurs mostly by a Bone Morphogenetic Protein (BMP) ligand named Glass bottom boat (Gbb) that is released from the muscle and binds to its tetrameric presynaptic BMP receptors containing Wishful Thinking (Wit), Thickveins (Tkv) and Saxophone (Sax) on the neuronal cell membrane (Figure 1) [11]. Binding of Gbb to its receptor has two parallel effects. The first one involves the activation of the Williams Syndrome-associated kinase LIMK1 to act in the stabilization of the synapse [12]. In the absence of BMP signaling or LIMK1, one sees many ‘synaptic footprint’ or ‘retraction’ sites at the NMJ, in which one still sees clustered postsynaptic proteins like Discs Large (Dlg) but no longer any presynaptic molecules [12]. The other involves receptor-mediated phosphorylation of the Smad family transcription factor Mad (Mothers against decapentaplegic), signaling the neuron to expand the number of synapses [11]. In the absence of key components of the pathway, including Wit and Gbb, small NMJs with reduced neurotransmission develop, and in the absence of negative pathway components such as Daughters against decapentaplegic (Dad), the NMJs overgrow, with characteristic ‘satellite boutons’ that appear disconnected from the other boutons [13]. In addition to the BMP and WNT pathways, a MAP Kinase pathway regulated by Highwire, a putative RING finger E3 ubiquitin ligase, also controls NMJ growth and branching [2].
Here we will review some of the salient recent work related to several genes that cause familial forms of neurodegenerative diseases whose culprits are conserved in flies and have been studied at the Drosophila NMJ. Some of the discussed work was based on forward genetics and only later was it realized that these genes caused or were related to neurological diseases. Other genes were studied specifically because they are known to be mutated in such diseases and the NMJ offers a highly relevant toolkit to study the pathogenesis of neurodegenerative diseases.
Section snippets
Hereditary Spastic Paraplegia
The Hereditary Spastic Paraplegias (HSPs) are a diverse set of diseases that share the primary feature of progressive, severe, lower extremity spasticity, with many causative genes identified so far [14]. Autopsies have revealed, among many other findings, myelin pallor and axonal loss in the corticospinal tracts, and in the brain, a high density of irregular tau-positive neurofibrillary tangles [15]. Mutations, frequently nonsense, in the gene spastin are responsible for almost half of all
Amyotrophic Lateral Sclerosis
Amyotrophic Lateral Sclerosis is a devastating motor neuron disease characterized by late-onset progressive upper and lower motor neuron degeneration. About 90% of the cases are sporadic, but mutations in 7 genes have been identified in familial cases, including VapB [14]. Histologic and ultrastructural studies of ALS patients’ NMJs have provided strong evidence of frequent denervation and reinnervation of the muscle endplates. In addition, synaptic terminals are on average smaller than normal
Spinal Muscular Atrophy
Spinal Muscular Atrophy (SMA) is a disease in which the lower motor nerves degenerate, resulting in progressive muscular atrophy [14]. SMA shares some key features with ALS, although SMA affects lower motor neurons, whereas ALS typically affects both upper and lower motor neurons. SMA Type I is a recessive disease often caused by deletions of Survival Motor Neuron1 (Smn1). Interestingly, abnormal copy numbers (1 or 3) of Smn1 have also been found in 12% of patients with sporadic ALS as compared
Multiple Sclerosis
Multiple Sclerosis is a neuroinflammatory disorder in which patients develop demyelinated plaques in their CNS with corresponding neurological deficits. At least five independent genetic studies have linked polymorphisms in the Clec16A gene to the disease [39]. In a screen designed to identify new genes that regulate synaptic terminal growth, fly mutants of the homologues of Clec16A, endosomal maturation defective (ema), were observed to possess dramatically overgrown synapses (Table 2) [40•].
Huntington's Disease
Huntington's Disease is a late-onset progressive disorder characterized by increasingly jerky and uncoordinated movements, rigidity, and neuropsychiatric symptoms [48]. Gradual degeneration of the basal ganglia is a key feature of the disease, which is caused by polyglutamine expansion of the protein Huntingtin. One of the early primary dysfunctions found in Huntington's Disease is excessive glutamatergic neurotransmission, while later in the disease course, too little glutamatergic
Concluding remarks
In summary, studies at the fly NMJ are rapidly providing a better understanding of the potential pathological mechanisms of fly homologues of human neurodegenerative diseases. The BMP signaling pathway may be playing a central role in many of these diseases. This is important because the BMP signaling pathway is highly conserved in mammals and has been well studied in relation to bone development, angiogenesis and stem cell proliferation. However, there are surprisingly few studies of its role
Competing interests
The authors declare that they have no competing financial interest.
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
V. Bayat has received support from the Edward and Josephine Hudson Scholarship Fund and the Developmental Biology Program training grant (T32 HD055200 07/01/2007 - 08/02/2009). We thank Drs. Yao, Giagtzoglou, Tsuda, Jawaid, Yamamoto, Sandoval, DiAntonio and O’Kane for helpful discussions. HJB is an investigator of the HHMI.
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