Coffin-Lowry syndrome

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Coffin-Lowry syndrome
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Drosophila genes associated with
Coffin-Lowry syndrome

S6kII

Related terms

Learning and memory behavior

Overview of the disease

Coffin-Lowry syndrome (CLS) is a rare X-linked disorder, with an estimated incidence of 1:50,000 to 1:100,000. Affected males present with facial abnormalities and severe intellectual disabilities, with IQ scores ranging from 15 to 60. CLS is caused by inactivating mutations in the protein kinase RSK2, which acts as a regulator and mediator of the mitogen-activated protein kinase (MAPK) signaling pathway. This pathway has essential roles in cellular proliferation and differentiation, but the absence of major brain abnormalities in individuals with CLS suggests an additional involvement at the neurophysiological level. RSK2 is predominantly expressed in brain regions involved in learning and memory; however, the exact functions of RSK2 remain poorly understood. Behavioral defects are observed in RSK2 knockout mice and in Drosophila upon knockout of RSK, the single fly ortholog of vertebrate RSK proteins (Beck, 2015 and references therein).

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Relevant studies of Coffin-Lowry syndrome

Beck, K., Ehmann, N., Andlauer, T.F., Ljaschenko, D., Strecker, K., Fischer, M., Kittel, R.J. and Raabe, T. (2015). Loss of the Coffin-Lowry syndrome-associated gene RSK2 alters ERK activity, synaptic function and axonal transport in Drosophila motoneurons. Dis Model Mech 8: 1389-1400. PubMed ID: 26398944

Abstract
Plastic changes in synaptic properties are considered as fundamental for adaptive behaviors. Extracellular-signal-regulated kinase (ERK)-mediated signaling has been implicated in regulation of synaptic plasticity. Ribosomal S6 kinase 2 (RSK2) acts as a regulator and downstream effector of ERK. In the brain, RSK2 is predominantly expressed in regions required for learning and memory. Loss-of-function mutations in human RSK2 cause Coffin-Lowry syndrome, which is characterized by severe mental retardation and low IQ scores in affected males. Knockout of RSK2 in mice or the RSK ortholog in Drosophila results in a variety of learning and memory defects. However, overall brain structure in these animals is not affected, leaving open the question of the pathophysiological consequences. Using the fly neuromuscular system as a model for excitatory glutamatergic synapses, this study shows that removal of RSK function causes distinct defects in motoneurons and at the neuromuscular junction. Based on histochemical and electrophysiological analyses, it was found that RSK is required for normal synaptic morphology and function. Furthermore, loss of RSK function interferes with ERK signaling at different levels. Elevated ERK activity is evident in the somata of motoneurons, whereas decreased ERK activity is observed in axons and the presynapse. In addition, a novel function of RSK in anterograde axonal transport was uncovered. These results emphasize the importance of fine-tuning ERK activity in neuronal processes underlying higher brain functions. In this context, RSK acts as a modulator of ERK signaling (Beck, 2015).

Highlights

Localization of RSK at the presynapse.
Loss of RSK affects ERK activity and subcellular localization in motoneurons.
Loss of RSK affects NMJ size, synapse numbers and function.

Discussion
One emerging common picture from studies in animal models for CLS is that loss of RSK2 function in neurons is associated with deregulation of ERK signaling and synaptic properties. In general, ERK signaling is not only required for cell proliferation, differentiation and survival, but also for synaptic plasticity and memory. The molecular functions of RSK proteins as an interaction partner of ERK proteins are discussed ambivalently in the literature. On the one hand, RSK2 mediates ERK signaling by phosphorylation of numerous targets; on the other hand, it is described as a negative regulator of ERK. This ambivalent picture is also reflected by genetic interaction experiments between RSK and ERK mutants, which do not provide a conclusive answer about the relationship between RSK and ERK with respect to pre- and postsynaptic functions. Further complexity is added because subcellular localization of pERK is changed in RSKΔ58/1 motoneurons, with elevated pERK levels in the somata and strongly decreased levels at the NMJ. Thus, even in a single cell, opposing effects with respect to ERK targets in different subcellular compartments can be expected (Beck, 2015).

These results coincide at several points with findings in the vertebrate nervous system. Elevated pERK levels have been observed in the hippocampus of RSK2 knockout mice, resulting in deregulation of ERK-mediated gene transcription. For instance, transcription of the Gria2 gene encoding the GLUR2 subunit of the AMPA receptor is upregulated. Nevertheless, electrophysiological, biochemical and ultrastructural analyses carried out with isolated cortical neurons and in the hippocampus reveals impaired AMPA-receptor-mediated synaptic transmission. This can be explained, at least in part, by the requirement of RSK2 for phosphorylation of postsynaptic PDZ [post synaptic density protein-95 (PSD95), discs large 1 (DLG1), zonula occludens-1 (ZO1)] domain-containing proteins to regulate channel properties. Morphological and electrophysiological data at the NMJ are also consistent with a postsynaptic requirement of RSK for synaptic transmission. In addition, RSK mutants display a number of defects in the presynaptic motoneuron, including upregulation and relocalization of pERK and a reduction in active zone numbers. How do these phenotypes relate to known functions of ERK in Drosophila motoneurons? First, alterations in ERK activity at the NMJ are inversely correlated with levels of the neural cell adhesion molecule Fasciclin II. Given that Fasciclin II has been found to be excluded from pERK-positive spots at synapses, a direct regulatory mechanism at the protein level seems plausible. Thus, it is conceivable that synaptic RSK contributes to Fasciclin II-mediated cell adhesion either directly, by acting as an upstream kinase, or by feedback inhibition of ERK activity. Second, besides RSK, the serine-threonine kinase UNC-51 also acts as a negative regulator of ERK in motoneurons. It could therefore be expected that RSK and UNC-51 mutations display similar synaptic phenotypes. Indeed, NMJ size, number of active zones and eEPSC amplitudes are decreased in both mutants. Interestingly, transgenic rescue experiments for the electrophysiological defects fail to work in both mutants, emphasizing the importance of fine-tuning ERK activity for maintaining normal synaptic functions. However, there are also significant differences between the two mutants. In general, UNC-51 phenotypes are much more pronounced. In the UNC-51 mutant, many postsynaptic GluRs are unapposed to presynaptic BRP, a phenotype that was not observed in the case of loss of RSK. Both mutants show a decrease in eEPSC amplitude, but although this is attributable to defective transmitter release at UNC-51 mutant synapses, no such presynaptic defect is observed in RSK mutants. Instead, the reduced mini amplitude at RSK mutant synapses indicates impaired postsynaptic sensitivity, which in turn is unaltered in UNC-51 mutants. Thus, although RSK and UNC-51 act as negative regulators for ERK, their relative contribution to ERK signaling in different cell types appears to be different. At least in the case of the RSK mutant, hyperactivation of ERK is modest and has no effect on development or viability of the fly, which implies a subtle modulatory function of RSK (Beck, 2015).

Finally, analyses uncovers aberrant axonal BRP and CSP localization and anterograde transport defects of mitochondria. Transport of presynaptic components and their appropriate delivery at synaptic terminals require a complex interplay between motor proteins, the different transported components and local signaling events. In addition, mechanisms must exist to restrain localization of presynaptic components at the nerve terminals. Interfering with these processes in Drosophila motoneurons causes distinct phenotypes. For instance, loss of Liprin-α results in ectopic accumulation of synaptic vesicles and presynaptic cytomatrix proteins in distal axon regions close to the synaptic terminals without affecting mitochondria or motor protein localization. SR protein kinase 79D (SRPK79D) is required to prevent formation of large axonal agglomerates of BRP. Given that axonal transport processes and other synaptic proteins are not affected in SRPK79D mutants, a function of this kinase for site-specific active zone assembly at presynaptic membranes has been suggested. Large organelle-filled axonal swellings have been observed in mutants defective for motor protein components; however, these aggregates do not serve as physical barriers for mitochondrial transport. Local effects caused by changes in axonal transport are seen in dAcsl mutations. Here, mitochondrial transport is unaffected, but an increased velocity of anterograde transport and reduced velocity of retrograde transport of vesicles results in aggregates in distal axon regions. Mutation of the human ortholog ACSL4 (acyl-CoA synthase long chain family member 4) causes non-syndromic X-linked mental retardation (Beck, 2015).

The axonal phenotypes seen in RSK mutants differ in several respects from these phenotypes. Large axonal swellings are not evident, and the increase in the number of BRP and CSP particles is largely confined to the proximal portion of the nerve (close to the ventral nerve cord). Together with the finding of more stationary mitochondria and fewer mitochondria transported in the anterograde direction, one explanation could be a function of RSK at the level of motor-cargo interaction. Specificity of cargo transport in the anterograde direction is determined at the levels both of individual Kinesins and of cargo-specific adaptor proteins. For example, the catalytic subunit Kinesin-1 in Drosophila (kinesin heavy chain, KHC) recruits mitochondria via the adaptor protein Milton, whereas UNC-76 provides a link to the synaptic vesicle protein Synaptotagmin. Motor-cargo interactions are also regulated in a phosphorylation-dependent manner, as exemplified by the UNC-51 kinase-dependent interaction of UNC-76 with Synaptotagmin. Loss of either UNC-51 or UNC-76 results in accumulations of synaptic vesicles along motoneuron axons. Another example is glycogen synthase kinase 3 (GSK-3), which has been proposed to inhibit anterograde transport by phosphorylating Kinesin light chain and thereby causing dissociation of membrane-bound organelles from KHC. Based on genetic analyses in Drosophila, an alternative model proposes a function of GSK-3 in regulating motor protein activity rather than cargo binding. Interestingly, RSK2 has been reported to inhibit GSK-3 activity in different cellular contexts and is able to phosphorylate GSK-3, at least in vitro. Future studies are required to clarify a function of RSK in GSK-3-mediated control of anterograde transport processes. So far, there is no evidence for a direct or an indirect requirement of RSK for phosphorylation of motor protein components and, if so, whether this might have an impact on their in vivo function (Beck, 2015).

In summary, an emerging common picture from knockout studies in mice and flies as animal models for CLS is a postsynaptic requirement of RSK proteins for efficient synaptic transmission. In addition, this study uncovered changes in the presynaptic neuron; in particular, defects in anterograde axonal transport and changes in localization of activated ERK. Whether these phenotypes reflect independent functions of RSK or whether they are interdependent remains to be determined. Future studies will also have to aim at understanding the function of RSK at central brain synapses in learning and memory processes (Beck, 2015).

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Putz, G., Bertolucci, F., Raabe, T., Zars, T. and Heisenberg, M. (2004). The S6KII (rsk) gene of Drosophila melanogaster differentially affects an operant and a classical learning task. J Neurosci 24: 9745-9751. PubMed ID: 15525759

Abstract
In an attempt to dissect classical and operant conditioning in Drosophila melanogaster, this study isolated the gene for ribosomal S6 kinase II (S6KII). This enzyme is part of a family of serine-threonine kinases that in mammals have been implicated in the MAPK (mitogen-activated protein kinase) signaling cascade controlling (among other processes) synaptic plasticity (long-term potentiation/long-term depression) and memory formation. The human homolog rsk2 has been linked to mental retardation (Coffin-Lowry syndrome). Mutant analysis in Drosophila shows that S6KII serves different functions in operant place learning and classical (pavlovian) olfactory conditioning. Whereas in the null mutant only pavlovian olfactory learning is affected, a P-element insertion mutant reducing the amount of S6KII only affects operant place learning. A mutant lacking part of the N-terminal kinase domain and performing poorly in both learning tasks is dominant in the operant paradigm and recessive in the pavlovian paradigm. The behavioral defects in the pavlovian task can be rescued by the genomic S6KII transgene. Overexpression of S6KII in wild type has a dominant-negative effect on the operant task that is rescued by the null mutant, whereas in the pavlovian task overexpression may even enhance learning performance (Putz, 2004).

Highlights

ignP1 mutant shows a male-specific behavioral phenotype in heat-box conditioning.
Performance of jump-out lines in heat-box conditioning.
Transgenic expression of S6KII interferes with place learning.
Pavlovian olfactory discrimination learning is impaired in the S6KII null mutant but not significantly in ignP1.
Genomic rescue in olfactory conditioning.

Discussion
The Drosophila S6KII is, perhaps surprisingly, encoded by a non-essential gene. Null mutants are viable and normal by morphological criteria and casual behavioral observation. Also, brain structure seems not to be severely affected. As the only apparent defect, Df(1)ignΔ58-1 shows a 40% reduced memory score in pavlovian olfactory learning. Regarding this memory component, the properties of the S6KII mutants are in line with what one might expect from S6KII in vertebrates. If it is the kinase domain in the N-terminal part of the enzyme that is phosphorylating target proteins, deletion of that domain should abolish phosphorylation. Indeed, in the mutant Df(1)ignΔ24-3, lacking part of the N-terminal kinase domain olfactory memory is reduced as much as in the null mutant [Df(1)ignΔ58-1]. The small and the large deletions are recessive, indicating that 50% S6KII enzymatic activity in the heterozygote is sufficient to provide the full function. In the mutant ignP1, again the reduced overall amount of protein is enough to allow for near to normal learning/memory. Also, the transgene in the genetic background of the null mutant or the partial deletion Df(1)ignΔ24-3 provides enough enzyme to fully support the behavioral task. Finally, overexpression of S6KII as a transgene has no deleterious effect. If at all, it enhances memory (Putz, 2004).

The involvement of the S6KII gene in operant place learning could hardly be more different. In wild-type Drosophila, S6KII is not essential for place learning, because removing the whole gene has no apparent phenotype. An influence of the S6KII gene on heat-box learning is seen only in mutants that leave the gene or part of it intact. The P insertion, the N-terminally truncated gene, and the S6KII transgenes all reduce learning performance. In the P-insertion mutant, the learning defect seems not to be related to the overall amount of S6KII because females learn well and show the same reduced amount of S6KII-like immunoreactivity as males. The heat-box phenotypes need to be studied in detail to find out whether the three kinds of mutations (P insertion, partial deletions, overexpression) interfere with heat-box learning in the same manner. Whether S6KII exerts its various effects in adulthood or during development can now be addressed using conditional expression systems (Putz, 2004).

The S6KII gene is nested in a large intron of a putative gene (CG17602) of unknown function. It should be noted that the mutant phenotypes cannot be assigned to this nearby gene because the genomic transgenes do not contain CG17602 and are inserted far away from the original genomic location. Their phenotypes correspond to and even interact with those of the deficiency mutants (Putz, 2004).

Assuming that it is at the protein level in which S6KII exerts its deleterious effect on heat-box learning in the mutants, a smaller protein must be synthesized from the C-terminal part of the S6KII gene in the small-deletion mutants. If this is true for the small deletions, it may well be true also for the wild type. This hypothetical small isoform might be mostly suppressed by the large isoform in wild type. Kinases with a large and a small isoform are not uncommon, e.g., in the protein kinase C family. It is too early to speculate about mechanisms before a small isoform is found (Putz, 2004).

Both olfactory learning and place learning require cAMP signaling but have their cAMP-dependent memory traces in different neurons. Because it is now well established that the memory trace for olfactory learning is localized in sets of intrinsic neurons (Kenyon cells) of the mushroom bodies, it will be of interest to determine, by the same reconstitution approach, whether the molecular functions of S6KII documented in this study colocalize in the same cells. For operant place learning, the localization of the memory trace is not as clear cut as in olfactory learning because several groups of neurons are still candidate locations for the memory trace. However, using the reconstitution strategy, it can be determined whether the same GAL4 lines that rescue in the case of rutabaga also do so with S6KII-cDNA in Df(1)ignΔ58-1 (Putz, 2004).

Schemes for vertebrates place cAMP upstream of S6KII in the same signaling pathway. This may hold true in Drosophila for pavlovian olfactory conditioning, whereas for operant place learning, S6KII may not lie in the direct pathway (because it is dispensable) but rather in a side branch. With the phenotypes of the mutants and overexpression lines, one is inclined to assume that a close interaction partner or a protein similar to S6KII should be directly involved in place learning. For instance, the signaling pathways for the two learning tasks might diverge at the level of the MAPK that could be blocked by an excess of S6KII and, in particular, by the hypothetical small C-terminal isoform of S6KII. Studies in Xenopus oocytes have already shown that an N-terminally truncated RSK mutant can constitutively interact with ERK (Putz, 2004).

It is not known whether S6KII in Drosophila is, as in vertebrates, part of the Raf-Ras pathway. Mutant studies on the Drosophila gene leonardo encoding a 14-3-3 protein suggest a role for Raf/Ras signaling in olfactory learning and memory. Yet, if S6KII would, as shown in vertebrates, exert its function by phosphorylating CREB or some other transcription factors, it could not be involved directly in synaptic plasticity. The defect in olfactory learning in the S6KII mutants is apparent already within 3 min, much too early to reflect transcriptional control induced by the learning process. This does not prove an involvement in the modulation of synaptic efficacy, though. A role of the Raf-Ras pathway in the formation and decay of synapses has been demonstrated for the Drosophila neuromuscular junction. This may be the way in which the leonardo gene affects olfactory learning and memory. This gene has been shown to be required during adulthood. Yet, the rescue experiments using a heat shock GAL4 driver left a time window of several hours between heat induction and memory test, which would be enough to restore, for instance, the abundance of synapses in a mutant with reduced synaptic density (Putz, 2004).

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