A potential treatment strategy for SMA is the upregulation of SMN protein originating from SMN2
, a gene that is present in all affected SMA individuals, compensating in part for the absence of a functional SMN1
). One of a number of pathways that increase SMN is STAT5 (23
), and one of the more potent known inducers of STAT5 is PRL. The facts that PRL passes the blood brain barrier (BBB) and that its receptor is expressed throughout the central nervous system (26
) increase its potential as an SMA therapeutic.
We document here a PRL-mediated increase in both SMN
mRNA and protein levels in the human neuronal cell line and murine motor neuron MN-1 cells. We also show an associated rapid increase in both phospho and total STAT5 levels upon PRL treatment. SMN
mRNA induction is also seen when cells are treated with the small molecule STAT5 activator ATA (28
). Conversely, an attenuation of PRL-mediated SMN induction was observed when cells were pretreated with STAT5 RNAi. An earlier report showing in vitro STAT5 conferred SMN induction revealed the induction to be transcriptional in nature (23
) (rather than transcript stabilization or altered splicing); in keeping with this, we have found that actinomycin D (a transcription inhibitor) treatment effectively abrogated the SMN induction in NT2 cells (data not shown).
Treatment with different doses of PRL in WT mice next revealed, as with cell culture, a sustained induction of SMN protein in brain and, to a lesser degree, in spinal cord. An increased and optimized PRL dose in SMA mice resulted in a significant and sustained increase in SMN
mRNA and protein levels, surpassing that observed in previously identified SMN-inducing agents (23
). Immunohistochemical analysis revealed a significant SMN induction in motor neurons. The second site of profound SMN upregulation appeared to be capillaries and endothelial cells. Although recent work suggests a central nervous system–mediated role in this phenomenon (35
), whether the modulation of SMN in the endothelium also has implications for the peripheral necrosis recently observed in both mouse model of the disease (30
) and in patients (36
) is an open question.
Although the mSmn–/–;SMN2+/+
mouse (null for mouse Smn
gene rescued with 2 transgenes; human SMN2
and an SMN2
cDNA deleted for exon 7), with a phenotype that closely resembles type I SMA in humans, is one of the most widely used in preclinical assessment of SMA therapeutics (29
), interlaboratory comparisons of the impact of drug or other intervention poses a challenge. The sole sure commonality, in addition to its being the same species, is the absence of endogenous mouse Smn
and presence of presumably identical human SMN2
transgenes. Otherwise the genetic background, housing conditions (e.g., temperature, dark/light cycle), and not least, feeding conditions can vary widely as can be seen by survivals ranging from 12 to 17 days. Thus, we find that taking the ratio of median survival of treated to nontreated is a useful metric by which to assess effectiveness of a given intervention, irrespective of which laboratory is conducting the analysis. We have achieved a ratio of 21 d/14 d or 1.6, a number that compares favorably with the 1.2 (19 d/16 d) observed with TSA (albeit P1 PRL initiation versus P5 TSA initiation) and 1.3 (12.9 d/9.9 d) seen with SAHA (32
In addition to apparently being more effective than the comparatively toxic TSA treatment, the phenomenon of responder and nonresponder mice observed with TSA treatment (33
) was not observed with PRL treatment, as the hormone appeared to confer roughly consistent benefit on all treated SMA pups. Another compound that has shown promise for the treatment of SMA is an antisense oligonucleotide that prevents alternative splicing of SMN2 transcript, resulting in more full-length transcript (30
). The most encouraging report so far in the field of SMA therapeutics is on the use of self-complementary AAV9 gene therapy with SMN as a payload, which resulted in an extension in longevity of SMA mice from 2 weeks to 250 days plus (38
). Similar results have been seen by other groups who used similar gene therapy approaches to treat SMA mice (39
). However, the clinical introduction of this treatment for SMA must await resolution of the issues of clinical safety, a potential species barrier including immune response, the ability to prepare an adequate quantity of GMP-grade virus, and overall cost (42
We were struck by the dramatically greater SMN induction observed in SMA mice when compared with WT mice; we also noted that SMN induction in human cell lines surpassed that observed in murine cell lines. The sole sources of SMN protein in the SMA mice are human SMN
genes. This prompted an analysis of the putative STAT5-binding sites in murine Smn
and human SMN
genes. In the original STAT5 SMN paper, promoter sequence analysis of both murine and human SMN
genes showed 2 conserved Stat5-binding sites (TTCNNNGAA/TTCNNNTAA) in the murine SMN promoter (NCBI AF027668) but none in the human SMN2
promoter (NCBI AF027688). Three similar CTCNNNTAA elements were detected uniquely in the SMN2
promoter (–413 to –409 bp, –2338 to –2330 bp, and –3881 to –3873 bp downstream of the SMN2
start codon [+1]; ref. 23
). However, our further analysis using the less stringent online database known as DECODE (the Champion ChiP Transcription Factor Search Portal based on SABiosciences’ proprietary database;
) revealed a total of twelve STAT5-binding sites for the human SMN2
and none for mouse Smn
promoter. We believe that this difference may account for the profound SMN induction we see when SMN2 is the source of SMN protein.
PRL treatment in SMA mice has revealed a significant in vivo induction of SMN protein, which correlates with an overall improvement in the phenotype of the disease. PRL treatment attenuated the weight loss and improved motor neuron function considerably; it also resulted in an approximately 70% increase in life span in SMA mice. However, the degree of SMN induction, greater than that observed in heterozygote mice that have a normal life span (Supplemental Figures 4 and 5), is at odds with the significant but in contrast comparatively modest improvement in longevity. Four obvious sources of this disconnect are as follows: (a) notwithstanding the P1 inception of treatment, a delay in SMN induction in the target motor neuron; (b) a role for SMN in other neuronal cells besides motor neurons refractory to PRL-mediated induction; (c) a role for SMN in other tissues refractory to PRL-mediated induction; and (d) inconsistent SMN induction. Given the small size of P1 and P2 pups, there can be technical challenges in ensuring complete administration of the total PRL dose in the first few days of life. However, the recent AAV 9 SMN
gene therapy rescue of the same mouse model work has shown that motor neuron transduction as late as 5 days still results in survival extension to 40 days, suggesting that a failure to induce SMN in other neuronal cells or in tissues other than the motor neuron may be the more likely source of the mortality (38
). In this regard, the recent observation of significant cardiac pathology in this mouse model (43
) combined with the lack of SMN induction we observed in the myocardium tissue (Supplemental Figure 2) suggest that cardiac failure may underlie the early mortality. However, it has been shown that induction of SMN in heart tissue itself does not rescue SMA mice, whereas neuronal-specific transgenic expression of SMN ameliorates disease phenotype with an increase in the survival of SMA mice (46
). It may be that SMN levels higher than those induced by Prl are uniquely required in the neurons that innervate heart tissue.
Clinical experience with PRL is limited, although a recent study has demonstrated the safe and effective use of recombinant PRL for mothers with lactation insufficiency (47
). Moreover, PRL has been shown to regulate oligodendrocyte precursor proliferation and mimic the regenerative effects of pregnancy. PRL’s striking ability to repair demyelination identifies it as a potential therapeutic agent in multiple sclerosis (48
). There could be some potential side effects of higher levels of PRL. A condition called hyperprolactinaemia (resulting from higher levels of blood PRL) can lead to hypoestrogenism, which may result in infertility and osteoporosis (49
). Higher levels of PRL also decrease dopamine release; therefore, some antipsychotic and antidepressant drugs can elevate PRL levels as well.
Our results demonstrate a clear promise for PRL use in clinical trial studies: amelioration of disease phenotype in a mouse model, BBB penetration, and safe and FDA-approved status. Recent work has shown that the maximum treatment benefit for murine SMA is contingent upon early timing of SMN
gene therapy. An early diagnosis followed by a prompt initiation of PRL treatment in SMA newborns may therefore be key to an optimal outcome in humans. We hope that SMA type II and III patients will also benefit from PRL treatment, as we hope an increase in SMN levels upon treatment will ameliorate disease progression and will improve the function of the remaining motor neurons. It will also be interesting to combine the effect of PRL with SMN2 transcript stabilizers (50
) and/or neuroprotective compounds such as Y-27632 (Rho kinase inhibitor) (51
Presently there is no cure for SMA. This study provides a good mechanistic insight into how SMN protein is regulated through PRL via the STAT5 pathway (Figure ) and its effect on the phenotype of the disease as well its potential for future therapeutic use for the treatment of SMA.
Proposed model for PRL-mediated induction of SMN in motor neurons.