Minor splicing pathway is not minor any more: Implications for the pathogenesis of motor neuron diseases



To explore the molecular pathogenesis of amyotrophic lateral sclerosis (ALS), the nuclear function of TAR-DNA binding protein 43 kDa (TDP-43) must be elucidated. TDP-43 is a nuclear protein that colocalizes with Cajal body or Gem in cultured cells. Several recent studies have reported that the decreasing number of Gems accompanied the depletion of the causative genes for ALS, TDP-43 and FUS. Gems play an important role in the pathogenesis of spinal muscular atrophy. Gems are the sites of the maturation of spliceosomes, which are composed of uridylate-rich (U) snRNAs (small nuclear RNAs) and protein complex, small nuclear ribonuclearprotein (snRNP). Spliceosomes regulate the splicing of pre-mRNA and are classified into the major or minor classes, according to the consensus sequence of acceptor and donor sites of pre-mRNA splicing. Although the major class of spliceosomes regulates most pre-mRNA splicing, minor spliceosomes also play an important role in regulating the splicing or global speed of pre-mRNA processing. A mouse model of spinal muscular atrophy, in which the number of Gems is decreased, shows fewer subsets U snRNAs. Interestingly, in the central nervous system, U snRNAs belonging to the minor spliceosomes are markedly reduced. In ALS, the U12 snRNA is decreased only in the tissue affected by ALS and not in other tissues. Although the molecular mechanisms underlying the decreased U12 snRNA resulting in cell dysfunction and cell death in motor neuron diseases remain unclear, these findings suggest that the disturbance of nuclear bodies and minor splicing may underlie the common molecular pathogenesis of motor neuron diseases.


Motor neuron system selectivity is a major mystery of motor neuron diseases. Although research has shown that the pathology is not restricted to motor neurons but also extends into other neurons as well as glial cells, the selective vulnerability of motor neurons is a characteristic feature of amyotrophic lateral sclerosis (ALS). However, the molecular mechanism underlying the vulnerability of the motor neuron system has not been fully explained. To clarify this issue, researchers must clarify what distinguishes the motor neuron.

Researchers have identified several molecular markers and physiological characters that distinguish motor neurons from others.[1] However, the morphology and location of the cell have been used as the most significant signature for identifying motor neurons in tissues. The cells of the CNS are diverse and complex, and they are mostly defined by their shape, size and location in the tissues. The complexity of the cells reflects the complexity of the cells’ RNAs. The diversity of RNAs results in part from the methylation of DNA, but studies have shown that other mechanisms also control cell-specific RNA diversity.

A higher structure of the nucleus, chromatin, and nuclear bodies, is another mechanism that regulates the cell-specific RNA diversity. Recent findings have revealed that chromatin has a unique structure and location in the nucleus in each type of cell. The chromatin structure is strongly associated with the diversity of RNA.[2] Moreover, the other intranuclear structures also play an important role in maintaining cell function and cell survival. Thus, the intracellular location or character of nuclear bodies may also differ in each cell type.

In 1906, Ramón y Cajal won the Nobel Prize for his description of the intranuclear fine structure.[3] In the nucleus, he identified several distinct structures, including the Cajal body. It has taken a long time to understand the functions of these intranuclear structures. However, little research has been conducted to clarify the differences of nuclear bodies in each cell type or in healthy versus pathogenic conditions. To clarify the molecular mechanisms underlying the systemic pathology of neurodegenerative disorders, we must investigate the nucleus structure and related functions, which might help us to determine the unique characteristics of motor neurons. In this review, we first focus on the alteration of nuclear bodies in ALS and then discuss the association between a disturbance of uridylate-rich (U) small nuclear (sn)RNA and motor neuron diseases.

ALS is a TDP-43 proteinopathy

Disease-specific intra- and extracellular inclusions serve as the diagnostic signature for each neurodegenerative disorder. In particular, the identification of the component proteins has changed our concepts about several neurodegenerative disorders. For example, the common identification of synuclein in several types of neurodegenerative diseases has led them to be known as synucleinopathy, including olivopontocerebellar degeneration, striatonigral degeneration, Parkinson disease and diffuse Lewy body disease. Recently, the identification of trans-activation response DNA protein 43 (TDP-43) as a component protein in ubiquitin-positive inclusions in ALS and frontotemporal lobar degeneration, has led to the classification of TDP-43 proteinopathy.[4, 5] The identification of the TARDBP gene for TDP-43 mutation in both familial and sporadic ALS patients whose neuropathological findings are identical to those in sporadic ALS indicates that TDP-43 plays a fundamental role in the pathogenesis of not only ALS with TARDBP mutation but also that of sporadic ALS.[6-8]

In healthy cells, TDP-43 is a ubiquitously expressed nuclear protein that forms some bodies in the nucleus.[9, 10] Under stress conditions, some TDP-43 moves to stress granules in the cytoplasm.[11] In ALS, TDP-43 forms cytoplasmic inclusions, which are phosphorylated, and then disappear from the nucleus.[12-14] These characteristic pathological findings may underlie the molecular pathogenesis of ALS. Although the molecular mechanism of the transport of TDP-43 to cytoplasm and the formation of inclusions is unclear, researchers have speculated that the disappearance of nuclear TDP-43 might precede the formation of visible cytoplasmic inclusions or abnormal modification, phosphorylation or ubiquitination of TDP-43.[13-15] These findings raise two possibilities regarding the pathogenesis of ALS: (i) the obtaining of toxic function by cytoplasmic inclusions; or (ii) the loss of the normal nuclear function of TDP-43.[14, 15] The model animals deleting TDP-43 are embryonically lethal, indicating that TDP-43 is a fundamental protein in the maintenance of cell function and survival.[16] Therefore, the loss of nuclear function in affected neurons in ALS might cause cell dysfunction or cell loss. However, it is not clear how the loss of TDP-43 results in cell dysfunction or cell loss.

RNA metabolism and TDP-43

TDP-43 was first identified as a protein that binds to DNA, and it is now considered to regulate RNA metabolism.[17] Using a method that identifies the mRNA binding to a specific protein, many RNAs that might be regulated by TDP-43 have been identified.[18, 19] These studies have shown that TDP-43 binds to long mRNA molecules with large introns and regulates the splicing and amounts of mRNA in several ways.[18, 19] Consequently, the depletion of TDP-43 might alter pre-mRNA metabolism. Indeed, the alteration of RNA profiles has been reported from cultured cells and model animals with depleted TDP-43.

In ALS, alterations of mRNA expression profiles have been reported,[20-22] although the association between TDP-43 and these alterations of mRNA observed in ALS remain to be clarified. To our knowledge, POLDIP3 is the only gene in which the splicing is directly regulated by TDP-43 and is altered in spinal motor neurons with ALS but not in brain with frontotemporal lobar degeneration.[23, 24] In addition, immunohisotochemical analysis indicated that several genes processed by TDP-43 express key molecules for function or survival of spinal motor neurons and show decreasing amounts of products.[25] However, it is unclear how the function of TDP-43 correlates with the depletion of these products. Thus, the specific functions of TDP-43 have not been fully evaluated in vitro or in ALS patients. These disturbances of RNA metabolism might not be explained simply by the loss of TDP-43 function on pre-mRNA. Therefore, some researchers have speculated that TDP-43 serves another function associated with RNA metabolism.[26] TDP-43 forms foci in the nucleus and associates with several nuclear bodies, suggesting that TDP-43 plays a role in the functioning of nuclear bodies.

Cajal bodies and Gems

Nuclear bodies are classified and identified by their unique protein components.[27] In addition, most of these bodies are tightly associated with a unique RNA and regulate that particular RNA metabolism.[28, 29] In contrast to cytoplasmic organelles, nuclear bodies do not have a membranous structure that separates their contents from nucleoplasm. Thus, the components of nuclear bodies are frequently exchanged between the bodies and the nucleoplasm. The dynamism of the components is a unique characteristic of nuclear bodies. The protein components decrease their mobility in nuclear bodies as compared to that in nucleoplasm. Thus, the bodies are recognized based on the increased concentration of the component protein.

The nucleolus and Cajal bodies are the most well-known nuclear bodies. The nucleolus is the center for maturation of rRNA, whereas Cajal bodies are sites for the maturation of U snRNAs and consist of coilin.[30-33] In addition, the causative protein for spinal muscular atrophy (survival of motor neuron: SMN) forms a body beside the coilin immunopositive body. Therefore, the foci stained by anti-SMN antibody have been designated as Gemini of the Cajal body, or Gems. However, coilin and SMN are colocalized in most of the cell. Therefore, these bodies are indistinguishable in most cell types.[30]

Decreasing the number of Gems in spinal motor neurons with ALS

It has been reported that Gems are partly colocalized with TDP-43 bodies in cultured cells.[9] In human spinal motor neurons, some Gems are stained with TDP-43, but not all of them.[34] In addition, the depletion of TDP-43 decreases the number of Gems in HeLa cells and mouse spinal motor neurons.[34, 35] A decrease in the number of Gems is also observed in spinal muscular atrophy.[36] Thus, we hypothesized that the loss of nuclear TDP-43 may result in a decrease in the number of Gems in spinal motor neurons with ALS as well. Indeed, our group and others have found that the number of Gems decreased in spinal motor neurons with ALS.[34, 37]

However, surprisingly we found that the number of Gems was decreased in spinal motor neurons that still contained nuclear TDP-43.[34] This result raises the possibility that the decreasing number of Gems precedes the alteration of TDP-43. However, in spinal motor neurons with spinal muscular atrophy, no alteration of TDP-43 has been reported, suggesting that the alteration of TDP-43 precedes the decrease in the number of Gems. Therefore, we propose that disturbance of a function of TDP-43 associated with the formation of Gems precedes the disappearance of TDP-43 from the nucleus (Fig. 1a–c). Accumulating evidence suggests that the disappearance of nuclear TDP-43 precedes the inclusion formation of TDP-43 (Fig. 1d,e).[14] Although the mechanism for the disappearance of nuclear TDP-43 is unclear, the dysfunction of TDP-43 might precede their disappearance from the nucleus. Research has shown that TDP-43 regulates its own amounts of product by affecting its own mRNA.[18, 38] Thus, the decreasing amount of nuclear TDP-43 should induce the production of more TDP-43. However, in spinal motor neurons with ALS, nuclear TDP-43 disappears. Therefore, these cells lose TDP-43 function, which is associated with pre-mRNA splicing, including the autoregulation mechanism (Fig. 1a–g).

Figure 1.

Splicing alteration and nuclear pathology in ALS. (a–e) TAR-DNA binding protein 43 kDa (TDP-43) and Gem pathology in ALS. (a) Normal neurons show (TDP-43 immunoreactivity (shown in red) with Gems (green circles). (b) TDP-43 alters its property and loses its normal functions of splicing pre-mRNAs and facilitating the formation of Gems (altered TDP-43 is shown in brown). (c) Gems disappear (gray circles). (d) So-called pre-inclusions, which consist of granular cytoplasmic aggregates that are positive for phospho-TDP43 epitopes (p409 and p410), are formed (brown circles), and nuclear TDP-43 disappears. (e) Skein-like inclusions in cytoplasm (brown lines). (f–h) Alteration in the splicing at each stage. (f) Genes in which splicing is directly regulated by TDP-43 (blue), genes containing U12 type introns (green), and the other genes (red). Boxes represent exons and lines represent introns. Dashed lines represent splicing. (g) In neurons with altered TDP-43 (b), some of the splicing regulated by TDP-43 is disturbed (red dashed lines for blue exon and introns). (h) In neurons that lose Gems (c–e), some of the splicing of U12-type introns is also disturbed (red dashed lines for green exons and introns).

We must consider the possibility that the decreasing number of Gems results from the decreasing number of large motor neurons in ALS, because the number of Gems is positively correlated with the size of the cell.[39, 40] Moreover, large motor neurons are more vulnerable to ALS than small ones.[41] To rule out this possibility, multiple regression analysis should be conducted to investigate whether ALS is an independent factor determining the number of Gems regardless of cell size.

The molecular mechanism for the decrease in the number of Gems

If our hypothesis is correct, the next question is whether the decreasing number of Gems results from a direct or indirect function of TDP-43. The number of Gems also declines due to decreasing transcriptional activity.[39, 42, 43] Therefore, it is possible that the alteration of TDP-43 suppresses the transcriptional activity, resulting in the decreasing number of Gems. However, in contrast to ALS, the number of Gems does not decrease in the spinal motor neurons in other motor neuron diseases.[34] Thus, in human spinal motor neurons, the nonspecific alteration of Gems resulting from the suppression of transcriptional activity is less likely. Therefore, we speculate that the alteration of TDP-43 directly decreases the number of Gems.

Another important question is how TDP-43 is associated with the formation of Gems. Two hypotheses have been proposed for the formation of nuclear bodies: (i) ordered assembly of the component proteins; or (ii) stochastic assembly, in which component proteins accumulate in an unordered manner at specific RNA or the complex of core proteins.[27-29, 44, 45] Although the process of how nuclear bodies are formed remains unclear, there are several indispensable component proteins in each body. Thus, two possible molecular mechanisms exist for decreasing the number of Gems by depletion of TDP-43: (i) the depletion of TDP-43 alters the mRNA of the component proteins of Gem; or (ii) TDP-43 directly contributes to the formation of Gems, such that its depletion results in fewer Gems.

With regard to the first possibility, it has been reported that TDP-43 regulates the alternative splicing of SMN. The depletion of TDP-43 increased the SMN splicing variant excluding exon 7 in a reporter system.[46] The SMN excluding exon 7 is less stable than SMN with exon 7, resulting in less SMN product.[47] Indeed, we found that the amount of SMN proteins decreased due to the depletion of TDP-43.[34] However, we were unable to confirm the increase in the SMN splicing variants excluding exon 7 in intrinsic SMN mRNA by depletion of TDP-43. Instead of the alteration of splicing variants, we found that the SMN mRNA decreased in the cells with depleted TDP-43, suggesting that the depletion of TDP-43 induces additional splicing, and the splicing isoform may be less stable than canonical SMN mRNA. However, we were unable to detect the additional splicing variants, which may contribute to the reduced amount of SMN mRNA. Moreover, researchers have not fully evaluated whether the SMN protein or mRNA are reduced in tissues affected with ALS.[48] Therefore, although the intrinsic SMN protein is reduced in cultured cells with the depletion of TDP-43, it is not clear that this is the mechanism underlying the reduction of SMN in tissue affected by ALS.

Next, we hypothesized that TDP-43 binds to the component proteins of Gem and increases their stability. Indeed, the protein–protein interaction between TDP-43 and SMN has been reported in a forced expression system,[9, 37, 49] although the result is controversial.[34] In addition, comprehensive analysis of binding proteins to TDP-43 using mass spectrometry failed to identify SMN or other component proteins of Gem.[50] Therefore, if an association exists between TDP-43 and Gems, it might be temporary, as is common in the components of nuclear bodies. Because these intranuclear structures do not have a membrane, the components of nuclear bodies and nuclear structures can rapidly interact. Many components of nuclear bodies change quickly, and an increased retention time of each component at a place represents foci.[27, 51] Therefore, the interaction should be regulated temporally and rapid dissociation depends on the circumstance.

Finally, we examine the possibility that TDP-43 directly contributes to the formation of Gems. In TDP-43-depleted cells, a substantial number of Gems were still observed, whereas TDP-43 was not detected in the nucleus or Gems.[34] In addition, not all Gems include TDP-43 in cultured cells and normal spinal motor neurons.[34] Moreover, the size of each Gem was similar between control and ALS cells.[34] These results clearly indicate that TDP-43 is not a necessary component for all types of Gems. Thus, we propose two possibilities regarding the contribution of TDP-43 in the formation of Gems: (i) TDP-43 contributes to the formation of Gems only at a specific stage during their maturation (Fig. 2a); or (ii) TDP-43 is associated with only a subtype of Gems, but not all Gems (Fig. 2b). Interestingly, the overexpression of TDP-43 also decreased the number of Gems in the cultured cells,[34] indicating that the proper amount of each component is important for maintaining the number of Gems.

Figure 2.

Mechanisms of how TAR-DNA binding protein 43 kDa (TDP-43) contributes to Gem assembly. (a) TDP-43 contributes to the formation of Gems only at a specific stage during their maturation. TDP-43 may facilitate the formation of a Gem or associate with the maturation of a Gem. Thus TDP-43 temporarily binds to the Gem components. After Gems are formed, TDP-43 exits the complex. (b) The components differ among Gems, and TDP-43 is a component in only a subset of Gems. Gem (red dashed circle), TDP-43 (orange circle), survival of motor neuron (SMN) (green circle), other components (blue circle).

What is the consequence of the decreasing number of Gems?

One outcome of a decrease in the number of Gems can be speculated based on the molecular mechanism underlying spinal muscular atrophy. Gems are the sites of assembly and maturation of snRNP.[29, 31, 52] In the assembly of snRNP, SMN first forms a dimer and directly binds to Gemin 2, 3 and 8 and indirectly binds to Gemin 4, 5, 6 and 7 and unrip.[53] This SMN complex then binds to the Sm complex and U snRNA and transports them into the nucleus.[47] At the Gems, additional proteins are assembled to snRNPs and U snRNAs are modified, consequently forming a spliceosome, which functions for pre-mRNA splicing. In addition, Gems accumulate at most U snRNA genes.[30] These findings suggest that the Gems may regulate the quality as well as the quantity of the U snRNA. Therefore, researchers have speculated that the depletion of SMN or Gems may result in decreasing amounts of SMN complex, snRNPs and U snRNAs. Indeed, Gemin 2, 3 and 8 are decreased in SMN-depleted cells and tissues.[54, 55] In addition, the assembly of snRNP is also disrupted in these cells and tissues. Furthermore, a subset of U snRNA is decreased in the affected tissues in spinal muscular atrophy.[47, 54] The U snRNAs are involved in the splicing machinery, the spliceosome, and are categorized into major and minor classes depending on the consensus sequences of the donor and acceptor splice sites of the introns.[56] Most of the splicing is regulated by major spliceosomes, whereas less than 1% is regulated by minor spliceosomes. In spinal muscular atrophy, U snRNA of the minor spliceosomes is markedly decreased in the CNS but not in other organs.[47, 54]

Because we found decreased amounts of SMN in TDP-43-depleted cultured cells and fewer Gems in the spinal motor neurons with ALS, we speculated that the amounts of SMN complex, snRNPs and U snRNAs were decreased in TDP-43-depleted cells and tissues affected with ALS. As expected, a subset of Gemins were decreased in TDP-43-depleted cells and a subset of U snRNA was decreased in a subtype of cultured cells.[34] Among them, U12 snRNA, belonging to the minor spliceosome class, was decreased in the tissue with TDP-43 pathology but not in tissue without TDP-43 pathology. The repertoires of U snRNAs are not identical between cultured cells depleted of SMN and TDP-43, indicating that the contribution of each protein to the maturation of U snRNAs is different. Finally, immunohisotochemical analysis revealed that the amounts of snRNPs belonging to minor spliceosomes decreased in spinal motor neurons with ALS. These findings are consistent with the previous results obtained using a SMN-reduced mouse model.[54, 55] However, another group reported that increased subtypes of U snRNAs and snRNPs accompanied the decreasing number of Gems in tissues affected with ALS.[37] Therefore, it is still unclear what type of alteration in U snRNA and snRNPs occurs in ALS.

The vulnerability of U snRNA belonging to the minor spliceosome class might be explained by the difference in the number of genes between U snRNAs belonging to major versus minor spliceosomes.[57] The genes for major spliceosomes are multicopy genes, whereas most of the genes encoding minor spliceosome U snRNAs have only a single copy. Therefore, because Gems contribute to the transcription and maturation of U snRNA, a decreasing number of Gems would have a proportionally greater effect on the expression of U snRNA belonging to the minor spliceosome class.

However, the specific decline of U snRNA in spinal muscular atrophy cannot be explained simply by the number of genes for U snRNA. Because the amount of SMN, which is a ubiquitously expressed protein, is decreased in all tissues in a spinal muscular atrophy model mouse, the minor spliceosome U snRNA is decreased selectively in the spinal cord.[54] Moreover, the disturbance of the repertoires of U snRNA differs depending on the cell type and tissues.[54] These results clearly indicate that the contribution of SMN to the regulation of U snRNA differs among cell types. These findings suggest that the maturation system for minor spliceosome snRNP is more vulnerable to the depletion of SMN in cells of the motor neuron system as compared to other systems.

Does disturbance of U snRNA cause degeneration in the selective system?

How does the disturbance of U snRNAs belonging to the minor spliceosome class cause motor neuron death? The U snRNAs recognize the donor branch site sequence and contribute to pre-mRNA splicing. Although minor spliceosomes are involved in less than 1% of the splicing, they are involved in the splicing of “information function” genes, including genes associated with the replication, repair and translation of DNA: transcription and ion channels.[56] In addition, the splicing regulated by minor spliceosomes is a rate-limiting factor in the gene-splicing process.[56, 58] The speed of splicing alters the splicing as well as the stability of mRNA. Therefore, the disturbance of minor spliceosomes may affect the quality and quantity of many genes (Fig. 1f–h). Indeed, the mutation of U4atac gene, the product of which is a key component of minor spliceosome, contributes to systemic developmental and degenerative disorders,[59-62] indicating that all tissues are vulnerable to the alteration of minor spliceosomes. However, patients with the U4atac gene mutation with a less severe phenotype do not show motor neuron disease.[63] This result clearly indicates that selectivity in the motor neuron system cannot be explained simply by the vulnerability of the motor neuron system to the alteration of minor spliceosomes.

Decreasing U12 snRNA may explain the selectivity in the motor neuron system. Interestingly, mutation of the U2 snRNA gene causes selective granule cell loss in mice.[64] This is surprising for two reasons. First, U2 snRNA is involved in the major spliceosome, which is fundamental machinery for pre-mRNA splicing. Second, although the gene for U2 snRNA is a multicopy, one of the U2 snRNA genes causes selective neurodegeneration. This may explain why the granular cell is more vulnerable to the depletion of U2 snRNA. However, the finding that the each U1 snRNA gene, which is also a multicopy, selectively regulates a subset of targeted genes suggests that each U2 snRNA gene may have a unique property for maintaining a specific type of splicing in specific cells.[65] Indeed, studies using a spinal muscular atrophy Drosophila model suggested that alteration of the splicing of U12 type intron in the specific gene in the intermediate and sensory neurons may result in selective motor neuron death.[66-68] Although the system selectivity in ALS may be explained by the limited TDP-43 pathology, it would be interesting to investigate whether alterations of the specific gene, which is regulated by minor spliceosomes, may underlie the pathogenesis of ALS.


Because the RNA-associated proteins have been identified as causative proteins for ALS as well as spinal muscular atrophy, the disturbance of RNA metabolism may underlie the pathogenesis of motor neuron diseases. In particular, the decline of minor spliceosome U snRNA in spinal muscular atrophy and ALS suggest the existence of a common molecular mechanism in motor neuron diseases. In addition, the evidence of alterations in the nuclear structure in ALS opens a new avenue for the study of neurodegenerative disease. Interestingly, it has been reported that product of FUS, another causative gene for ALS, interacts with SMN, and the number of Gems decreased in cultured cells depleted of FUS.[49] Moreover, the number of Gems decreased in fibroblasts with causative mutations in FUS and TARDBP.[49] Although the significance of the decreasing number of Gems in the affected tissues with FUS mutation has yet to be evaluated, this finding reinforces the importance of Gems in ALS.

The fine structure of the nucleus, including the nuclear bodies, might play an important role in regulating cell-specific RNA metabolism. For example, Hutchinson-Gilford progeria syndrome is caused by a mutation in LMNA.[69] Lamin A, a product of LMNA, is a dense network inside the nucleus and participates in chromatin organization.[70-72] Although the mutated lamin A may disturb the function of the nuclear membrane, the mutated lamin also affects chromatin organization and RNA metabolism, resulting in cell death.[69] In addition, the nuclear bodies have more diversity than expected. The diversity and dynamics of nuclear body components might be investigated more fully in each neuron, and neurons or glial cells in neurodegenerative disorders. In addition, the location of a nuclear body in association with other nuclear bodies may be important in the regulation of RNA metabolism. Little research has been conducted on the differences in the nuclear structure between various types of healthy and pathological cells. Closer investigation of the nucleus may help to elucidate the complex system underlying the regulation of cell identity and clarify the motor neuron system pathology of ALS.


This research was supported through a Grant-in-Aid for Scientific Research (A), Grant for Scientific Research on Innovative Areas (Foundation of Synapse and Neurocircuit Pathology), and a Research Activity Start-up Grant from the Japan Society for the Promotion of Science; a Grant-in-Aid from the Research Committee of CNS Degenerative Diseases and Comprehensive Research on Disability Health and Welfare, Ministry of Health, Labor and Welfare, Japan; a Grant-in-Aid from the Uehara Memorial Foundation; a Grant-in-Aid from the Tsubaki Memorial Foundation; and a Grant-in-Aid for JSPS Fellows from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The authors declare no conflicts of interest.