SCA1 Gene and Protein Biochemistry

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This diagram depicts the progressive degeneration of Purkinje cells
in the cerebellum of transgenic mice with SCA1 over a 40 week period [3].

Mutations in the ATXN1 gene have long been known to cause Spinocerebellar Ataxia I (SCA1), a neurodegenerative disease that particularly affects Purkinje neurons in the cerebellum, leading to motor function problems [1]. Not only is the gene itself responsible for expression of neuropathology, but the subsequently transcribed mRNA also appears to play a role in expression of symptoms, as a result of the formation of secondary structures [2]. This transcript, when translated into the Ataxin-1 protein, causes protein aggregates to form because of certain domains and amino acid residues present in it [3]. It is important to study the gene and protein biochemistry of ATXN1 because it offers insights into treatment options for SCA1.

Genetic mechanism at DNA level

The effect of size of CAG nucleotide expansions on disease phenotype

One possible genetic mechanism at the level of DNA that has been shown to induce SCA1 has been trinucleotide expansions, which also appear to play a role in amyotrophic lateral sclerosis (ALS) [1]. In SCA1 specifically, CAG expansions in the coding region of the mutant ATXN1 gene seem to be responsible for SCA1 neuropathology [1]. CAG normally codes for amino acid glutamine. In the wild type gene, the number of repeats range from 3 to 43, but with 0 to 3 CAT nucleotide interruptions, which break the continuity of CAG repeats. In the mutated form of the gene, there are no CAT interruptions [1].

In an experiment by Burright et al., transgenic mice with either 32 or 82 CAG repeats in the ATXN1 gene were tested for the SCA1 phenotype [1]. Compared to the mouse lines without the trinucleotide expansion, the transgenic mice containing the CAG expansions developed SCA1, which was quantified using measures of the loss of Purkinje neurons [1]. The 6 lines of mice with the CAG expansion gene ended up getting SCA1, whereas none of the 6 lines of mice without the mutation developed SCA1 [1].

Transgenic mouse with SCA1
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Figure 1. This pictures depicts the mouse with SCA1 attempting to walk,
but falling over to the side due to imbalance [1].

Long trinucleotide expansions, such as those found in the mutant ATXN1 gene, appear to have higher binding affinities for certain ligands than the shorter CAG repeats in the wild type gene [4]. This trend especially holds true for trinucleotide expansion tracts with more than 36 repeats, which is the threshold for the expression of neuropathology [4]. This could be because the increase in length of expansion is resulting in a greater number of binding sites for ligands, such as monomeric or multimeric proteins, including Capicua and RBM17 [4]. In the future, this information could be used to design drugs that target the binding affinity of the mutant ATXN1 gene [4].

One limitation of transgenic mice studies is that diseases involving trinucleotide expansions have not been observed in organisms besides human beings, but the presence of trinucleotide expansions still seemed to result in the expression of disease phenotype in transgenic mice with the mutant ATXN1 gene in the aforementioned experiment [1]. Another limitation of this mouse model is that in humans, the number of CAG repeats is not intergenerationally stable, meaning that a paternal mutant allele for the SCA1 gene consisting of 53 CAG repeats could result in 82 repeats in the next generation [1]. In mice, the number of CAG repeats was found to be stable between generations [1].

Problem in the ATXN1 RNA transcript

Not only are problems present in the gene sequence itself, but also in the transcript. One problem is the formation of stable hairpin structures, as depicted in Figure 2, which appear to be contributing to SCA1 pathology [2]. Previous experiments have shown that the transcripts of trinucleotide repeats form thermodynamically stable hairpin structures, and sequester RNA-binding proteins that should be binding other RNA transcripts [1].

SCA1 Hairpin Structures
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Figure 2. This diagram, adapted from Sobczak et al. [2], shows the comparison between
normal ATXN1 transcripts (right) and transcripts resulting from the mutant (left)

When mutant ATXN1 is transcribed, the transcript tends to form a hairpin with a stem that contains a C base paired with a G, a G paired with a C, as well as a mismatched A and A pair per CAG repeat [2]. This motif continues, depending on the number of repeats. A similar hairpin structure has also been elucidated in the wild type ATXN1 transcript [2]. The difference between the wild type and mutant transcripts only arises when considering the size and stability of the hairpin stem as well as trinucleotide interruptions [2].

Recall CAT interruptions that disrupt CAG repeats in the normal allele of the ATXN1 gene. These decrease the stability of hairpin structures formed by the ATXN1 transcript in three ways. The first is by making the stem loop of the hairpin larger [2]. When the CAT repeats (or CAU repeats in RNA) are present, instead of having a G base paired to a C, U will be paired to C [2]. Since these interruptions occur in the middle of the CAG repeats, and the C-U base pair is not favourable, the stem loop is enlarged to include the CAU interruption [2]. The second way in which hairpin stability is decreased is when the CAU interruption happens to be far away from the stem loop [2]. As a result, a smaller loop structure will be formed within the stem to accommodate the interruption. The third way in which CAU interruptions decrease stability of the hairpin is by causing it to break off into two separate hairpins [2]. On the other hand, uninterrupted hairpins are quite stable and as a result, they interact with CAG repeat-binding proteins, resulting in the expression of SCA1 pathology [2].

The reason U-C base pair mismatches have been selected for by nature to be part of the CAU interruptions is because this non-Watson-Crick base pair is less stable when in the duplex conformation than, for instance, the U-U pair [2]. This reduced stability of the U-C base pair translates to reduced hairpin stability, resulting in ameliorated SCA1 pathology [2].

Ataxin-1 Protein

AXH domain of Ataxin-1 protein

The AXH domain of the Ataxin-1 protein appears to be responsible for self assembly of this protein, which could lead to expression of SCA1 neuropathology [5]. Figure 3 depicts the crystal structure of this domain [5]. It is similar to the HBP-1 protein [6], a transcriptional repressor which is homologous to the AXH domain. The crystal structure reveals an OB fold, which suggests it can also bind RNA based on previous studies [7]. In addition, this domain is very plastic because it can be seen in various conformations unlike HBP1, even though both domains have the same secondary structure [7]. The reason behind the plasticity in the AXH domain is a topic for future research.

The AXH Domain
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Figure 3. This is the crystal structure for the AXH domain,
which is part of the ataxin-1 protein [5].

One important finding from Chiara et al.’s experiments was that when the AXH domain is deleted, there is less aggregation of ataxin-1 within the cell [7]. Aggregation is also decreased when AXH is replaced with HBP1, which does not have the propensity to aggregate [6]. Previous studies have also shown that when the AXH domain is isolated from the rest of the protein, it misfolds and results in the formation of amyloid fibres, similar to the beta-amyloid found in the brain of patients with Alzheimer's Disease [7]. This was found to be the case even when no conditions that reduce stability are induced, such as denaturants or increased temperature [7]. This suggests that the AXH domain by itself is enough to cause aggregation in vitro [7].

Another important finding was that the wild type AXH domain existed as a dimer in solution in equilibrium with monomers [7]. The dimerization interaction could be stopped if proteins that interact with this domain were introduced [7]. This suggests that this competition between dimerization and protein interactions can be used to reduce the self assembly of ataxin-1 in disease interventions [7].

Lysine residue 772 of Ataxin-1 protein

A lysine residue is present at position 772 in the amino acid sequence of the ataxin-1 polypeptide [2]. When site-directed mutagenesis of this residue was carried out in mutant ATXN1 mice, replacing it with threonine, this resulted in a loss of nuclear localization ability due to the loss of the nuclear localization signal (NLS) [2].

The NLS found at the C-terminus end of the protein is what allows the Purkinje cell to recognize it, facilitating its transport into the nucleus [9]. In the mutagenesis experiments, the threonine version of the protein was found to be localized mainly in the cytoplasm, unlike the original protein containing lysine at position 772, which localized to both the cytoplasm as well as the nucleus of Purkinje cells [2]. This suggests that nuclear localization of ATXN1 is a major indicator of the disease because disease neuropathology was not expressed to a great degree in the ATXN1-threonine double mutant, unlike the single ATXN1 mutant [2].

Additionally, the mice expressing the double mutant did not develop ataxia, and did not show symptoms either [2]. The mice did not display signs of cerebellar pathogenesis for up to 1 year, even though they should have expressed neuropathology by that point based on previous studies on mice with SCA1 [2]. This demonstrates that mutant ataxin-1 protein must be present in the nucleus for the expression of neuropathology to occur, which suggests that nuclear function of ataxin-1 is lost in SCA1 [2]. One lost function could be interactions of ataxin-1 with the protein, Capicua, which occur in the nucleus [2].

Protocol Explanation
This video briefly explains the site-directed mutagenesis procedure [8].

Serine residue 776 of Ataxin-1

Site directed mutagenesis

A serine residue is present at position 776 in the amino acid sequence of the Ataxin-1 polypeptide [10]. Site-directed mutagenesis experiments reveal that when serine is replaced with alanine in mice with the mutant ATXN1 gene, it resulted in the inability to form protein complexes of high molecular weight or in other words, protein aggregates [10]. This could be because in the double mutant, the ataxin-1 proteins did not clump together, and were more diffuse and soluble. As a result, SCA1 symptoms were significantly diminished in these transgenic mice [10].

The role of phosphorylation

The serine residue at position 776 in the mutant ataxin-1 protein is normally phosphorylated on its hydroxyl group (Figure 4) [10]. In past experiments, when phosphorylated, ataxin-1 protein displayed the tendency to localize to the nucleus, which led researchers to speculate that phosphorylation plays a role in nuclear transport [10]. However, phosphorylation does not appear to be required for protein transport to the nucleus to occur because the mutagenesis showed that nuclear localization was not hindered in the SCA1-alanine double mutant [10]. Instead, phosphorylation appears to facilitate interactions of ataxin-1 with the protein, 14-3-3 [12], an interaction that contributes to neuropathology. The reduced SCA1 symptoms in the SCA1-alanine double mutant can be applied to find treatment options for the expression of neuropathology in patients with SCA1 [13].

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Figure 4. This is the structure of the amino acid, serine, containing
a hydroxyl group in the side chain that facilitates phosphorylation [14].
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