Therapeutic Strategies for SCA1

SCA1 transgenic mice
Rotarod performance is used as a measure of ataxia severity [2]

Current research on SCA1 presents evidence on several features of the mutant ataxin-1 protein (ATXN1) that are critical in determining disease severity, toxicity and neuropathology. These features are an expanded polyglutamine (polyQ) tract, a nuclear localization signal (NLS), an AXH domain for protein-protein interactions, and the phosphorylation of Serine residue 776 [1]. Similar to other polyglutamine disorders, mutant ATXN1 also forms protein aggregates, and its toxicity is mediated by a gain-of-function mechanism [1]. Many of these pathological and characteristic features can be targeted by various therapeutic strategies through the use of transgenic and knock-in mouse models. Potential treatments include mutant gene silencing via RNAi mechanisms, mesenchymal stem cell therapy, interfering with mutant protein interactions, increasing mutant ATXN1 degradation, and using protein kinase inhibitors.

1 Recovery Potential

Since SCA1 is a progressive degenerative disease, it is important to have an understanding of the critical treatment period, that is, the period during which neuropathology may be completely or partially reversed, and normal function completely or partially recovered. Zu et al make use of the tetracycline-controlled transcriptional activation system to create a conditional SCA1 (82Q) disease model [3]. When the tetracycline transactivator protein (tAT) binds to the TetO operator sequences, the downstream gene is expressed [3]. When the antibiotic doxycycline is added, it interferes with the tAT-TetO binding and there is gene suppression [3]. SCA1 (82Q) gene expression was turned off at 6 weeks, 12 weeks and 32 weeks to represent treatment at an early stage, middle stage and late stage of the disease [3]. At 6 weeks, stopping gene expression led to complete recovery in rotarod performance, in molecular layer thickness and in dendritic branching [3]. At both 12 weeks and 32 weeks, there is some recovery of motor performance and Purkinje cell morphology, but only after an extended period of dox treatment (gene-off) [3].

2 SCA1 Mouse Model

Animal models are critical in research because they provide behavioral and genetic insight into various cellular, molecular and physiological processes. Animal models of disease further provide the framework on which therapeutic treatments can be developed and improved for human clinical implementation. Good SCA1 models must not only imitate the natural disease progression, neuropathology and motor deficits, but must also demonstrate these characteristics in a reasonable time frame.

2.1 B05

Ectopic cells
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Left image shows abnormal (ectopic) cell layering in B05 mice.
Right image is wild-type, normal cell morphology [3].

The B05 mouse model, designed by Burright et al in 1995, is the first SCA1 mouse model [4]. The transgene construct is composed of the murine Pcp2 gene, whose 5’ regulatory sequence is able to target gene delivery to Purkinje cells, and the human SCA1 gene with an expanded 82 glutamine repeats [4]. The B05 transgene RNA levels are expressed at a level 100-fold greater than endogenous SCA1 RNA [4]. Heterozygous B05 mice develop ataxia at 12 weeks of age, the earliest of the mutant transgenic lines [4]. B05 mice also develop severe morphological abnormalities such as Purkinje cell loss, thinning of the cerebellar molecular layer, and ectopic cell distribution [4].

2.2 154Q Knock-in Model

Morris water maze assessment
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SCA1 (154Q) knock-in mice took longer to locate the hidden platform
suggesting impaired spatial learning and memory [5].

Although the B05 mouse model is able to develop Purkinje cell pathology and motor deficits within a reasonable time frame, it is limited because there is no development of cognitive impairments or a shorter lifespan as seen in human SCA1 patients [5]. A 154Q knock-in model has 154 glutamine repeats inserted directly into the mouse endogenous SCA1 locus which ensures more accurate spatial and temporal expression patterns [5]. SCA1 (154Q) mice have reduced rotarod performance as early as 5 weeks of age, muscle wasting and ataxia by 20 weeks, progressive weight loss and premature death [5]. In addition, spatial memory was assessed using the Morris water maze and found to be impaired as mutant SCA1 (154Q) mice took more time to find the hidden platform [5].

3 RNAi Gene Silencing

RNAi
The cell's natural gene regulatory mechanism [6]

RNAi or RNA interference molecules are the cell’s natural gene regulatory mechanism and can be divided into two subclasses: microRNA (miRNA) and small interfering RNA (siRNA). Both these molecules function by binding to complementary mRNA sequences, preventing their translation, and eventually signaling for their degradation.

In laboratory applications, short hairpin RNA sequences (shRNA) or artificial microRNAs (miRNA) are designed to target gene suppression via the RNAi mechanism. More recent studies have found that shRNAs are less safe than miRNAs and may induce cellular toxicity [7]. This toxicity is mediated by excessive shRNA expression and inefficient precursor processing which interferes with endogenous miRNA biogenesis and function [7]. Safety concerns also arise over the problem of “off-targeting” in which a non-targeted but similar gene sequence is also repressed. A computational study estimates that this occurs about 10% of the time [8]. Viral delivery of the RNAi molecule is another area of concern. Although much work has been put into improving the specificity, efficiency and safety of viral vectors, the risk of a harmful gene insertion event or an adverse immune system reaction still persists [9].

Even with the risks, the ability of RNAi to selectively target and manipulate gene expression has high therapeutic potential in a variety of medical fields including hematology, oncology, infectious diseases and neurodegenerative diseases. [10] [11]. In a knock-in model of SCA1, miRNA targeting the ataxin-1 gene was virally delivered into the deep cerebellar nuclei and shown to reduce mutant ataxin-1 levels, improve gait and rotarod performance and alleviate molecular thinning [12].

4 Mesenchymal Stem Cell Therapy

Stem cells have the ability to differentiate and undergo self-renewal. These properties impart high therapeutic potential in tissue regeneration and injury recovery. In neurodegenerative diseases, there is the progressive and selective loss of certain neuronal populations. In SCA1, the cerebellar Purkinje neurons as well as some basal ganglia nuclei are targeted. Multiple studies have investigated whether insertion of stem cells into the damaged area is able to reverse disease pathology and recover regular neuronal function. There are multiple mechanisms by which MSCs are proposed to exert their therapeutic effects. First, there is the homing efficiency which describes the ability of MSCs to migrate to inflammatory sites [13]. At the injury site, MSCs release growth factors which promote tissue repair [13]. They also play a crucial role in immunomodulation by releasing factors that suppress the effects of toxic cytokines and other immune factors [13]. These mechanisms are by no means exclusive and though there is currently no agreement on the exact mechanism, it is generally accepted that MSC therapy involves a complex integration of multiple cellular and molecular processes.

Matsuura et al injected adult mesenchymal stem cells found in bone marrow into SCA1 transgenic mice and found reduced Purkinje cell and dendritic atrophy as well as suppression of motor deficit progression [14]. In a comparison of neural precursor cells (NPCs) with mesenchymal stem cells (MSCs), NPC treatment was not effective in young transgenic mice, treated at 5 or 13 weeks, while MSCs injected in 5 week old transgenic mice displayed complete motor recovery [14]. This suggests that MSCs are more suitable for early treatment of disease [14].

5 Interference of Mutant ATXN1-Capicua Interactions

Mutant ataxin-1 toxicity is mediated by its protein interactions some of which include the transcriptional corepressor SMRT, the RNA-splicing factor RBM17 and the transcriptional repressor Capicua [1]. Interfering with transcription and translation regulatory mechanisms has negative effects on downstream gene expression or suppression as well as protein folding. Targeting the restoration of normal protein-protein interactions is likely to significantly mitigate disease toxicity.

5.1 ATXN1-like/BOAT

BOAT
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BOAT (human) also contains the conserved AXH domain.
Sequence similarity in the domain is 66% [15].

The ataxin-1-like protein, also named Brother of ataxin-1 (BOAT) by Mizutani et al, shares a conserved AXH domain with the ataxin-1 protein [15]. Since the AXH domain mediates protein interactions, both ataxin-1 and ataxin-1-like have similar binding partners, including forming protein complexes with Capicua [16]. Bowman et al duplicated the ataxin-1-like allele in the SCA1 (154Q) knock-in mouse model which effectively raises the level of the ataxin-1-like protein [16]. They found suppression of the ataxia phenotype, partial recovery of fine dendritic branching and improved survival of the transgenic mice [16]. Results also show that the higher the level of ataxin-1-like, the less interaction there is between mutant ataxin-1 and Capicua [16]. This suggests that ataxin-1-like confers its protective properties through outcompeting mutant ataxin-1 for binding to Capicua, thus reducing the association between mutant ataxin-1 and Capicua [16].

5.2 Exercise

The benefits of exercise have been studied in a whole range of contexts, from its promotion of neurogenesis, to its antidepressant effects to its ability to improve cognitive function [17]. Exercise is even implicated in improving the mild cognitive impairment of Alzheimer’s patients [18]. Fryer et al implemented a mild exercise treatment on SCA1 (154Q) mice and found a highly significant improvement in lifespan [19]. A study of the brain tissue one week after the last exercise trial also determined an increase in the levels of epidermal growth factor (EGF) and a reduction in the levels of Capicua [19]. Further analysis suggests that mutant ataxin-1 may confer a ‘hyper-repressive’ property on Capicua, so when exercise reduces Capicua, it also relieves the repressive pressure. In a sub-study, SCA1 (154Q) transgenic mice with only a single functional copy of Capicua displayed improvements in motor function, learning and memory when compared to transgenic mice with both functional copies of Capicua [19]. While exercise did not lead to any significant improvements in rotarod performance, it did extend lifespan by more than when only Capicua levels were decreased, suggesting that exercise confers its beneficial properties through other molecular pathways as well.

Effects of Exercise
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From left to right, exercise increases lifespan, increases levels of EGF and decreases levels of Capicua [19].

6 Protein Degradation

Protein aggregation is a common feature of neurodegenerative disease [20]. SCA1 aggregates, or inclusion bodies, not only stain positive for the mutant ataxin-1 protein, but also for elements of the protein degradation machinery, including ubiquitin ligases, proteasomes and molecular chaperones [20]. This suggests that the mutant protein interferes with the natural mechanism for protein refolding or degradation, leading to the accumulation of toxic misfolded protein. Therapeutic strategies could focus on reducing the amount of toxic protein by targeting the overexpression of the ubiquitin-proteasome system. This approach was studied by Al-Ramahi et al whose group looked specifically at the E3 ligase, CHIP (C terminus of Hsc-70 interacting protein) [21]. CHIP was found to co-localize with 100% of the ataxin-1 nuclear inclusions in the brain tissue of SCA1 patients [21]. Overexpression of CHIP was found to reduce levels of mutant ataxin-1 and suppress the disease phenotype in a Drosophila model of SCA1 [21].

7 Kinase Inhibitors

The phosphorylation of serine residue 776 is a crucial modulator of disease toxicity and pathogenesis. Transgenic mice with an expanded polyglutamine tract but with S776 replaced with A776 (alanine) still had nuclear localization of the mutant protein in Purkinje cells, but showed no ataxia in home cage behavior, no rotarod performance deficiency, and reduced dendritic thinning and atrophy in Purkinje cells compared to the same-age B05 mouse [22]. Phosphorylation of S776 has been shown to be involved in protein-protein interactions that may stabilize the mutant protein and interfere with downstream pathways and effective degradation [23].

Phosphorylation dysfunction is implicated in many diseases including Alzheimer’s Disease, tauopathies and various cancers. [24] [25]. Multiple drugs have been developed as kinase inhibitors in order to recover homeostatic phosphorylation rates [26]. Park et al used both a Drosophila and human-cell-based genetic screen to find ten genes that modify ataxin-1 toxicity, eight of which are either direct components or regulators of the MAPK pathway [27]. The study further narrows the modifier genes to a single kinase MSK1 which is involved in the phosphorylation of S776. Inhibition of the MAPK pathway reduced mutant ataxin-1 levels, and reducing MSK1 levels in transgenic mice led to improved rotarod performance and less Purkinje cell degeneration. Protein Kinase A is another kinase implicated in S776 phosphorylation that could also serve as a target for inhibitory drugs [28].

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