Even though the mutations that underlie SMA, a serious childhood genetic disease of motor neurons, are now well known, the disease is still not completely understood. In particular, why motor neurons die selectively when SMN levels decrease below a certain threshold remains unclear, also providing uncertainty as to whether the functional protein in motor neurons is the SMN found in nuclear gems or, for example, in the axon. However, what does seem clear is that the severity of the disease diminishes as the number of copies of the SMN2 gene increases. That further suggests that higher levels of functional protein will also lead to improvement in the course of the disease. Therefore, we carried out a cell-based screen designed to identify compounds that increase SMN anywhere within cells, rather than just in gems. In addition, we established an additional assay in which SMN-elevating compounds could be tested for their ability to correct a phenotypic defect associated with the disease. In experiments included here, we chose motor neuron death caused by lentiviral knockdown of Smn.
We tested collections of annotated bioactive molecules, rather than diverse compound sets. Our rationale for doing this is that one of our main objectives was to identify SMN regulatory pathways that might lead us to the identification of receptors or enzymes that could be targeted to treat SMA. Our image-based screening was probably more difficult to carry out than a standard reporter gene assay, but, nonetheless, we identified numerous reproducible hits that increased SMN levels quite significantly in SMA patient fibroblasts. Hits from our screen fell into a variety of classes, some of which are described in more detail in this paper. We found that relatively low concentrations of various Na,K-ATPase inhibitors scored consistently. Part of the effect of this compound class could be attributed to the increased intracellular Na+ and Ca2+ that accompanies inhibition of that membrane transporter. However, part of the effect of Na,K-ATPase inhibitors may also be explained by the ability of cardiac glycosides such as ouabain to activate intracellular signaling pathways downstream of RTKs. To probe these pathways, we treated fibroblasts with different RTK ligands and found many of them to be active as well, with PDGF-BB producing the greatest level of increase. SMN levels were very responsive to changes in PDGF concentrations in the cell culture medium. PDGF addition elevated SMN, while resting levels of SMN could be decreased significantly by blocking endogenous PDGF in serum (or simply reducing the amount of serum in the cell culture medium). This suggests that RTK ligands, such as PDGF, might control SMN levels under normal cell growth conditions.
Since several growth factors increased cell numbers in addition to SMN levels, there is some chance that those two processes are inextricably linked. That is, perhaps SMN levels simply increase when cells are subjected to a mitogenic stimulus. However, this cannot be the entire explanation for our results since many of the molecules that induce cells to produce more SMN actually decrease cell numbers rather dramatically. For example, this is readily seen with both HDAC inhibitors and proteasome inhibitors, which are well known to have cytostatic or anti-proliferative effects. Furthermore, some of the hits from this fibroblast screen are also effective on motor neurons, which are clearly incapable of proliferating. Thus, there is no absolute connection between SMN levels and the cell cycle. Nonetheless, it is also true that we and others have found that C2C12 cells42
proliferate relatively slowly, while neurospheres with low levels of SMN proliferate faster43
. So, under certain circumstances elevated SMN levels may play a role in, or be responsive to, the cell division process.
Based on a phosphoproteomic analysis, PDGF was seen to be associated with activation of PI3K/AKT/GSK-3-mediated signaling, with some activation of RSK. The cells also appeared to have activated p38, normally considered to be regulated by stress44
, rather than by growth factors like PDGF. We did find that anisomycin, a p38 activator, scored in our screen, suggesting that this pathway can modulate SMN levels. A recent report also found anisomycin could rapidly increase SMN levels, apparently by stabilizing and increasing mRNA levels45
. However, we found that both ERK and p38 inhibitors had a relatively minor ability to block the increase in SMN that followed PDGF treatment. In contrast, PI3K antagonists had a strong inhibitory effect, suggesting that this is a major arm of the SMN regulatory pathway. Consistent with that was our demonstration that PDGF addition also led to an increase in Ser-9 and Ser21 phosphorylation of GSK-3α and β in our cells, thereby inhibiting that enzyme. Treating cells with a variety of chemical GSK-3β inhibitors produced an elevation of cellular SMN. We confirmed that some of the activity of the inhibitors is likely to be due to inhibition of GSK-3. Reducing levels of GSK-3α and β individually or together using shRNAs produced an extremely impressive increase in SMN levels in patient fibroblasts. This suggests that chemical enzyme inhibitors more potent or specific than the ones we used might also produce even a larger increase in SMN.
PDGF and some GSK-3 inhibitors increased SMN levels without yielding a consistent or dramatic increase in mRNA or a change in splicing. Given that proteasome inhibitors, which act to block protein degradation, also increased SMN, one possibility is that PDGF and its downstream mediator GSK-3β increase SMN by blocking its degradation. Interestingly, there is a consensus GSK-3 phosphorylation site on Ser4 of SMN. Using mass spectrometry, we confirmed that this site in phosphorylated in our cells, but a more detailed study will be needed to explore SMN posttranslational modifications quantitatively. Furthermore, recent data suggest that phosphorylation by GSK-3, which has a well-documented role in mediating degradation of β-catenin in the Wnt signaling pathway33
, may have a broader role in regulating turnover of a variety of intracellular proteins37
. Therefore, we hypothesized that RTK signaling inhibits GSK-3, decreasing its phosphorylation of SMN, thereby slowing Smn degradation. This was confirmed using mutagenesis experiments in which we showed that replacing Ser4 with an aspartic acid residue, mimicking a state of chronic phosphorylation, causes a sharp increase in the degradation rate of SMN.
In order to confirm that the hits we discovered had SMA-relevant biological activity, we were interested in establishing an appropriate phenotypic assay. Since SMA is a disease that involves motor neuron dysfunction and death, we focused on setting up a motor neuron assay. Previously, we isolated ES cells from a mouse model of SMA and found that motor neurons produced from these ES cells die soon after differentiation (Sinor-Anderson et al., in preparation). However, for studies described here, we chose a model in which lentiviral shRNA was used to reduce SMN in wildtype ES cell-derived motor neurons. One important issue relates to whether this is a valid phenotypic assay to test SMN-elevating compounds for their potential usefulness. It is well known that all cells require SMN and cannot proliferate or survive if their SMN is reduced sufficiently. Under the conditions of our experiments, motor neurons were selectively infected with the lentiviral vector and hence died preferentially compared to other cells in the culture, but not until their levels of SMN were reduced by approximately 75%. By way of comparison, in cultures in which SMN-deficient mouse ES are induced to differentiate into motor neurons and glial cells, motor neurons die rapidly, as mentioned, but glial cells are relatively unaffected. Therefore, that while the cell death that we observed following lowering of SMN may not be absolutely specific to motor neurons, it can nonetheless form the basis of an assay capable of choosing compounds that can modify the neurodegenerative changes that constitute the pathological basis of SMA.
PDGF itself did not affect either SMN levels or survival of the motor neurons, which lack PDGF receptors46
. However, GSK-3 chemical inhibitors did increase SMN in motor neurons and rescued virtually all of the death that was seen in the motor neurons with lower Smn. In contrast, while HDAC inhibitors and various proteasome inhibitors can elevate SMN levels in ES cell-derived motor neurons, both classes of compounds are relatively toxic over the course of the survival experiments described here and neither is able to provide any phenotypic rescue. Jablonka et al. (2009) also found that the HDAC inhibitor valproic acid had negative effects on motor neurons47
. Some of our data suggest that the most effective compounds, such as alsterpaullone, may inhibit one or more other kinases and this may contribute to their effectiveness. Future work will be directed at identifying these other kinases.
It is worthwhile pointing out that some of the commercially available GSK-3 inhibitors, with several different chemical scaffolds, were quite effective in elevating SMN in fibroblasts and motor neurons and some were not. There are several possible explanations for this. One is that most of these inhibitors are likely to affect more than one kinase48
, and it may be that there are kinases other than GSK-3α and β that play a role in SMN regulation. It will be important to determine which kinase inhibition profile correlates best with the ability to increase SMN and to use that information as part of a chemical optimization campaign to maximize the beneficial effects of using these compounds therapeutically (for example, there are some negative activities associated with alsterpaullone addition to Jurkat cells)49
. It is also interesting to note that GSK-3 has been shown to have anti-apoptotic effects on different neuronal populations50
. Determining whether any part of the survival promoting effect that GSK-3 inhibitors have on motor neurons is independent of SMN levels is essential (although we did not find that they enhanced motor neuron survival in general under the conditions of our experiment), as is investigating whether some of the pro-survival effects on other neurons might, unexpectedly, be modulated, in part, by increased SMN levels.
In summary, we have carried out an image-based screen of annotated collections to find compounds that increase SMN in any intracellular compartment in fibroblasts. We found more than 150 active compounds that fell into different categories. Some, but not all, of these compounds also increase Smn in motor neurons, confirming that it is not absolutely essential to carry out primary screens in motor neurons themselves. Included among the hits were several signal transduction pathways, one of which lies downstream of membrane RTKs. GSK-3 appears to be a particularly important druggable intracellular target and inhibitors of that enzyme not only increase SMN, but rescue motor neuron death. We believe that this is the first time that an SMA screen has produced compounds that have such a striking effect on an important component of the disease. Future work will be directed at testing GSK-3 inhibitors and other modulators of intracellular signaling in mouse models of SMA.