Fig. 1
Schematic representation of the TSC1 and TSC2 proteins, showing phosphorylation sites and important structural and functional features [15]. Numbers are according to the human amino acid sequences.
3 Phylogenetic Distribution of Components of the Signaling Pathway
Homologues of TSC1 and TSC2 as well as other components of the TOR signaling pathway have been reported from a number of eukaryotes, including Drosophila melanogaster, the fission yeast Schizosaccharomyces pombe, mice, and rats. Presumably based on the (incorrect) belief that, because S. pombe is unicellular and humans and D. melanogaster are multicellular animals, S. pombe, D. melanogaster and mammals are representative of eukaryotes as a whole, the presence of the pathway in these organisms led to the view that the pathway, including TSC1/TSC2, was widespread throughout eukaryotes (e.g., [23, 27]). In fact, S. pombe (as well as Saccharomyces cerevisiae and other fungi) together with animals, belongs to just one of several eukaryotic groups, the Opisthokonta [28]. The other groups are the Amoebozoa (e.g., the slime mold Dictyostelium and the pathogen Entamoeba), the Archaeplastida (e.g., land plants, green and red algae), the Excavata (e.g., Giardia, Leishmania, Naegleria, Trichomonas, and Trypanosoma), the Rhizaria (including a number of poorly characterized protozoa), and a complex group, including brown algae (e.g., diatoms, kelps), Plasmodium, and Toxoplasma that may not be monophyletic, but is often referred to as the chromalveolates [28]. Thus, the presence of a given pathway in S. pombe and animals cannot be taken as a reliable indication of its presence throughout eukaryotes. Given this, Serfontein et al. carried out a bioinformatic survey for components of the TSC1/TSC2-TOR across representatives of these groups with sequenced genomes [29]. This study concluded that, although a rudimentary version of the pathway, including TOR, Raptor, AMPK and S6K was present throughout eukaryotes, the full pathway, including TSC1 and TSC2 was not. The basic pathway appeared to be sufficient to allow the coupling of cell growth to energy levels, and given the fact that it appeared to be absent from bacteria, it was suggested [29] that the pathway evolved at the root of the eukaryotic tree (i.e., early in the evolution of eukaryotes), perhaps as a result of the origin of mitochondria [30]. Additional elements of the pathway had been “bolted on” in different lineages. Rictor appeared to be present in the Chromalveolata, Excavata, Amoebozoa, and Opisthokonta (i.e., all groups apart from the Archaeplastida), with Akt and 4E-BP in the Excavata, Amoebozoa, and Opisthokonta. There was some evidence for a TSC2 homologue in the Amoebozoa as well as in the Opisthokonta (but not elsewhere) and TSC1 appeared to be present only in the Opisthokonta (Fig. 2). A subsequent study by van Dam et al. [31] argued that TSC2 was more widely distributed than reported by Serfontein et al. [29]. However, the conclusion of van Dam et al. was based on the widespread distribution of the GAP domain, without reference to other features of the TSC2 protein. Noting that many proteins with different functions contain GAP domains, the database searches used by Serfontein et al. were more conservative, requiring the presence of other features for a homologue to be recorded as TSC2. Thus, although GAP-containing proteins are widely distributed, proteins containing other features of TSC2 appear to follow the distribution reported in ref. [29]. The distribution of TSC1 described by van Dam et al. was consistent with that previously reported. So although organisms such as Dictyostelium may have many attractions as models for some aspects of human biology (e.g., [32]), and understanding of TOR signaling in Dictyostelium may help us understand the process in other organisms (e.g., [33]), the most appropriate models for TSC1/TSC2-signaling will come from within the Opisthokonta.
Fig. 2
Evolution of the TSC1/TSC2-TOR signaling pathway as proposed in ref. [5]. The names at the right hand side are the major eukaryotic groups. The letters associated with internal regions of the tree indicate the points at which particular components of signaling pathways originated. TSC2* indicates a “rudimentary” form of TSC2 found in Dictyostelium
4 Models Within the Opisthokonta
4.1 Fungi
Within the Opisthokonta the organisms most widely used for studying TSC1/TSC2 signaling are S. pombe, Candida albicans, D. melanogaster, Danio rerio, Mus musculus, and Rattus norvegicus. The budding yeast Saccharomyces cerevisiae does not have homologues of TSC1 and TSC2, although interestingly both S. pombe and Saccharomyces cerevisiae contain two homologues of TOR, which have differing functions [34]. C. albicans has a single TOR homologue [35]. In S. pombe, the best-studied fungus in this regard, the TSC1/TSC2 complex regulates the activity of Tor, as in humans, through the G-protein Rheb. The Tor proteins form two distinct complexes, one containing Tor1 and the other Tor2. Cells deficient in Tor1 are viable, but have abnormalities, such as sterility. Cells deficient in Tor2 are not viable, but can be studied using temperature-sensitive conditional mutants. The Tor2 complex appears to be responsible for the cells’ response to nitrogen starvation. Inactivation of tsc1 and tsc2 in S. pombe by targeted gene disruption causes impairment in conjugation and the ability to take up amino acids, as well as changes in the pattern of gene expression in response to nitrogen starvation [36–39]. Studies with C. albicans also using targeted deletions demonstrated a role in response to nitrogen starvation, including starvation-induced filamentation [40].
4.2 Drosophila
The importance of Tsc1 and Tsc2 in signaling in Drosophila was first indicated by genetic screens showing that mutations in Tsc1 and Tsc2 led to disregulation of eye growth and cell size ([41–44], reviewed in [23]). Further genetic studies allowed the dissection of much of the TSC1/TSC2-TOR signaling pathway, for example through the demonstration that the effects of Tsc1 and Tsc2 mutations could be suppressed by mutations affecting the genes for TOR and S6kinase [45, 46]. Drosophila has also been used as a biochemical model as well as a genetic model for elucidating the signaling pathway, for example in analysis of associated phosphorylation events [47]. A more detailed description of the use of Drosophila as a model system for the biochemistry and molecular biology of TSC1/TSC2-TOR signaling is beyond the scope of this chapter, and it has been well reviewed elsewhere (e.g., [23]). In general, however, genetic analysis with Drosophila has relied on a variety of mutations including in-frame deletions and premature termination codons in a range of positions in the genes, as well as RNAi to knock down levels of expression, and overexpression studies.
4.3 Zebra Fish
The biology of TSC1/TSC2 signaling has also been studied in the zebra fish. This has two TSC1 genes, designated tsc1a and tsc1b, although the rest of the pathway is not duplicated [48]. Given an association between ciliary function and kidney cyst formation in polycystic kidney disease, and the formation of kidney cysts in TSC, diBella et al. [48] tested for effects of knockdown of Tsc1a and Tsc1b expression on ciliary function by injection of antisense morpholino oligonucleotides to regions of the Tsc1a and 1b coding sequences. (Morpholino oligonucleotides are oligonucleotides with six-membered sugar rings, rather than five-membered ones and modified linkages between nucleotides. These features confer greater stability on morpholino oligonucleotides in the target cell.) Injection of the Tsc1a morpholino oligonucleotides led to significant reduction, although not complete loss, of the Tsc1a mRNA. Both Tsc1a and Tsc1b morphants (individuals with levels of expression reduced by morpholino oligonucleotide injection) developed ventral-directed body curvature in the embryos. In addition, the Tsc1a morphants developed kidney cysts, showed abnormalities in the establishment of left–right body plan asymmetry and abnormally long cilia [48]. In a different approach to isolation of mutants, Kim et al. [49] used N-ethyl-N-nitrosourea mutagenesis followed by a screening technique known as TILLING [50] to screen for lines with mutations in the Tsc2 gene. They identified a single line with a C to A transversion converting a tyrosine codon at position 1031 (position 1033 in the reference human sequence) to a nonsense codon, which would generate a protein truncated well before the GAP domain. Heterozygous fish appeared fully normal. Mutant homozygotes all died by 11 days post fertilization, showing liver enlargement and deflated swimbladders. The relatively late expression of developmental defects was interpreted as resulting from the deposition of maternal tsc2 mRNA in egg cells. The homozygotes showed enlarged hepatocytes and neurons, and brain abnormalities. At the biochemical level, they showed significant upregulation of TOR signaling, indicated for example by increased phosphorylation of ribosomal protein S6. Heterozygotes appeared normal, but interestingly also showed some evidence of upregulation of TOR signaling. Immunochemical analysis showed the presence of mutant protein, indicating that the corresponding mRNA had not been destroyed by nonsense-mediated decay (NMD). Abnormalities were also observed when wild-type one cell embryos were injected with mRNA synthesized from the mutant allele, or from an additional template encoding a deletion construct. This study also used antisense morpholino oligonucleotides to reduce levels of Tsc2.
4.4 Mouse
Several different mouse genetic models are available for TSC. These are reviewed in detail in this book and elsewhere [51, 52], so will be summarized only briefly here. For Tsc1, knockout mice were developed by the standard approach of replacing endogenous genes in embryonic stem cells with disrupted copies. Thus, Kobayashi et al. [53] replaced exons 6–8 with a selectable cassette, resulting in the splicing of exons 5–9 and a frameshift. Mice were embryo lethal as homozygotes, and as heterozygotes showed a number of abnormalities, such as tumor development. Knockout lines have been developed with deletions at a range of positions and in some cases incorporating reporter genes to indicate Tsc1 gene expression (e.g., [54, 55]). In order to study mutations that are homozygous lethal in the germ line, conditional knockouts have been developed in which deletions are generated in specific tissues such as astrocytes by (1) flanking regions of the Tsc1 gene with a recombination target and (2) arranging for tissue-specific expression of the corresponding recombinase. This results in a targeted deletion in the particular cell-types [56]. Similar approaches have been taken with Tsc2, with generation of knockout and conditional knockout lines (e.g., [57–60]). Some mutations show more subtle effects on the TSC2 protein. Deletion of exon 3, encoding 37 amino acids, resulted in a hypomorphic allele, with low, but detectable, levels of expression of mutant protein. Heterozygotes showed a less severe kidney tumor burden than individuals heterozygous for null alleles, and homozygous embryos survived longer than homozygous null ones [61]. There are also transgenic mice expressing modified forms of TSC2 under strong promoters. These include the ∆RG dominant negative modified form of TSC2, which has a deletion of residues 1617–1655 in the GAP domain of the protein, and a substitution (resulting from a splice variant) of residues 1679–1742 in the rabaptin-5 binding domain, and the ∆RL form, which combines a deletion of residues 81–102 (corresponding to the leucine zipper region) and the same rabaptin-5 binding domain substitution [62–64].
4.5 Rat
There is also a widely used rat model for TSC2, known as the Eker rat, after its description by Eker as a rat line heritably subject to renal adenomas [65]. The susceptibility was determined by a dominant mutation [66]. The mutation was subsequently recognized as being in a tumor suppressor gene, and at the molecular level was shown to be due to an insertion of approximately 5 kbp in the Tsc2 gene at the end of exon 30 [67]. This resulted in a change in the transcript pattern from the locus, and a premature termination codon.
5 Appropriateness of Existing Animal Models
There are three main criteria to be considered when we assess the appropriateness of models of tuberous sclerosis complex based on Tsc1 and Tsc2 mutations in other organisms. We refer to these as “mutational equivalence,” “structural and functional equivalence,” and “phenotypic equivalence.” The first is whether the particular types of mutations studied, such as deletions or nonsense mutations, are appropriate models for the actual mutations seen in humans with TSC. The second is whether the structural and functional features of the human TSC1 and TSC2 proteins are also present in the model species, i.e., whether the nonhuman proteins are appropriate models for the human ones. The third is whether the particular pathological features of tuberous sclerosis complex have appropriate phenotypic equivalents in the organism used.
5.1 Mutational Equivalence
A wide range of mutations have been identified in individuals with tuberous sclerosis complex [68, 69]. For TSC1 the majority of mutations, where identified, are nonsense mutations or frameshift mutations that will lead to premature termination of the protein. There are also splice-site mutations, but only about 1 % of mutations are missense mutations or short in-frame insertions or deletions (reviewed in [18]). In TSC2 by contrast, about 25 % are missense mutations or short in-frame insertions or deletions. Although it was typically supposed that truncating mutations would lead to a complete loss of protein, whether by instability or by nonsense-mediated decay (NMD, a process by which mRNAs containing premature termination codons are detected and degraded), there is little direct evidence for this in human TSC. In fact nonsense-mediated decay does not lead to complete loss of the affected transcripts for TSC1 [53, 70]. Although prematurely truncated proteins produced from transcripts that escape NMD may have reduced stability compared to full-length ones, the degree of reduction in stability depends on the position of the truncation [16–18]. Thus, it seems likely that some truncations may lead to residual levels of proteins with a dominant negative effect, which may not be adequately modelled by, say, knockdown of wild-type proteins in model organisms. It has been proposed [11] that the range of effects of TSC seen in different individuals may reflect the particular mutations, and evidence is accumulating from genotype–phenotype studies to support this (e.g., refs. [71–73]). Thus, a given model will be representative of some individuals with TSC with similar mutations, but not necessarily all. The clinical manifestations of TSC in individuals may also be influenced by modifier genes [11], and mouse models have shown some differences in effects according to the genetic background used [55, 58]. Thus, possible modifier effects dependent on the genetic background should also be borne in mind when interpreting the results of nonhuman TSC models. Other factors such as loss of heterozygosity and somatic mosaicism (although high throughput sequencing suggests mosaicism may be relatively uncommon [74]) may modify the effects seen in particular individuals. Finally, although tissue-specific or cell-type specific conditional knockouts have been used as an attractive way of studying the effects of mutations, especially those that are embryo lethal in homozygotes (e.g., [56, 75]), the specific ablation of a gene in an otherwise wild-type background is not necessarily a realistic reflection of the situation in patients with TSC, who are likely to be heterozygous throughout.
5.2 Structural and Functional Equivalence
Serfontein et al. [15] raised the question whether key structural and functional elements of human TSC1 and TSC2 were also seen in the homologues from other species. If a particular feature were absent from another organism, then that species would not be appropriate for mutations affecting that feature in humans. Serfontein et al. considered the homologues in rat (Rattus norvegicus), mouse (Mus musculus), zebra fish (Danio rerio), fruit fly (Drosophila melanogaster), sea urchin (Strongylocentrotus purpuratus), fission yeast (S. pombe), and slime mold (Dictyostelium discoideum). The conclusions of this study are summarized in Tables 1 and 2.
Table 1
Summary of the conservation of phosphorylation sites and other features of TSC1 across model organisms (from ref. [15])
Rat | Mouse | Zebra fish | Fruit fly | S. pombe | |
|---|---|---|---|---|---|
TSC1 phosphorylation sites | |||||
IKKβ | Strong | Strong | Weak | Weak | Very weak/absent |
CDK | Strong | Strong | Weak | Weak | Very weak/absent |
GSK3β | Strong | Strong | Moderate | Weak | Very weak/absent |
TSC1 other features | |||||
Coiled-coil | Present | Present | Present | Present | Present |
ERM-binding | Present | Present | Present | Not clear | Not clear |
Rho-activating domain | Present | Present | Present | Present | Present (low) |
TSC1/TSC2 interaction | Present | Present | Present | Present | Present (low) |
Table 2
Summary of the conservation of phosphorylation sites and other features of TSC2 across model organisms (from ref. [15])
Rat | Mouse | Zebra fish | Fruit fly | S. pombe | |
|---|---|---|---|---|---|
TSC2 phosphorylation sites | |||||
Akt/RSK | Strong | Strong | Moderate | Moderate to strong | Very weak/absent |
ERK | +/+ | +/+ | +/− | −/− | +/− |
AMPK | +/+ | +/+ | +/+ | +/− | −/− |
GSK3β | Strong | Moderate | Moderate to strong | Weak to strong | Absent |
MK2 | + | + | + | − | − |
Unknown | Strong | Strong | Moderate | Moderate
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