SCA1 is a fatal neurodegenerative disease induced by brain-region-specific cell death and dysfunction, and is characterized by impaired motor balance and coordination . SCA1 is caused by a polyglutamine expansion in ataxin-1 which is involved in gene transcription. Transcriptional regulators including Capicua, Rbm17, SMRT, Gfi-1, and RORa/Tip-60, have been shown to interact with ataxin-1 [6, 7, 11,12,13]. Previous studies have explored how polyglutamine-expanded ataxin-1 affected the normal transcriptional signature in the cerebellum. These studies have found that various biological pathways including calcium signaling, glutamate signaling, and long-term depression are involved in the cerebellum at different stages [12,13,14]. Also, interacting protein partners can affect ataxin-1 stability and function . The best-studied example is 14-3-3 protein, and its interaction with phosphorylated ataxin-1 in the cytoplasm can prevent ataxin-1 dephosphorylation and degradation and inhibit the ataxin-1 nuclear translocation required for its toxicity .
A better understanding of the pathogenesis of SCA1 requires a comprehensive assessment of the ataxin-1 interactome. Ataxin-1-interacting proteins were isolated using co-immunoprecipitation and screened by LC−MS/MS in HEK-293T cells expressing wild-type and mutant ataxin-1. GNAS, MCM2, and TMEM206 were identified as wild-type ataxin-1-interacting proteins using Western blots in HEK-293T cells. No mutant-ataxin-1-interacting proteins were pulled down in HEK-293T cells in our study. GNAS, MCM2, and TMEM206 were not reported as ataxin-1-interacting proteins in a previous study. Certainly, the microenvironment associated with HEK-293T cells might affect ataxin-1 post-translational modifications, ultimately affecting its molecular interactions. Currently, it is not clear whether ataxin-1 interacts with GNAS, MCM2, and TMEM206 in Purkinje cells since phosphorylation of S776 is associated with a stabilization of ataxin-1 . Co-immunoprecipitation using a phospho-mimetic ataxin-1 or phospho-resistant ataxin-1 may be helpful to clarify the ataxin-1-interacting proteins in Purkinje cells.
Three new components of the ataxin-1 interactome required additional interrogation. GNAS mediates receptor-stimulated cAMP signaling which integrates different environmental cues with intracellular responses . Mutations in the GNAS gene result in pseudohypoparathyroidism type 1a, pseudohypoparathyroidism type 1b, pseudopseudohypoparathyroidism, progressive osseous heteroplasia, polyostotic fibrous dysplasia of bone, McCune-Albright syndrome, Albright hereditary osteodystrophy, and several tumors, although the pathogenic mechanisms remain elusive . A previous study reported that the presence of mutant GNAS is critical for a pancreatic tumor which is driven by protein kinase A (PKA)-mediated suppression of salt-inducible kinases . So, we infer the GNAS mutation may affect PKA kinase activity, and these changes may happen in protein–protein interaction networks. Phosphorylation of ataxin-1 at the serine 776 residue plays an essential role in protein toxicity, and PKA is a critical kinase for ataxin-1-pS776 in cerebellar Purkinje cells . Thus, our results suggested that ataxin-1-pS776, which is driven by the PKA pathway, might be mediated by GNAS-PKA interaction networks in neurons. The present study also revealed that RAC-PAK pathway is indeed a target gene of ataxin-1 in the KEGG axon guidance. Our study demonstrates the phosphorylation of ataxin-1 may involve in the pathogenesis of SCA1. These data provide further insight into how RAC-PAK pathway regulates ATXN1 levels in vitro and neurodegeneration in vivo. Together, these findings raise the possibility that GNAS may get involved in the pathogenesis of SCA1.
MCM2 is the highly conserved mini-chromosome maintenance protein involved in recruiting other DNA replication-related proteins and the formation of replication forks . MCM2 forms a complex with MCM4, 6, and 7 and regulates the helicase activity of the complex. MCM2 is phosphorylated by protein kinases CDC2 and CDC7 . A previous investigation revealed that MCM2 promotes cell proliferation, possibly through the regulation of HMGA1 phosphorylation . A role for MCM2 in neurons has not been reported. Thus, the role of the MCM2/ataxin-1 interaction in neurons needs further investigation.
TMEM206 regulates the progression of colorectal cancer by promoting colorectal cancer cell proliferation and controlling colorectal cancer cell migration and invasion . The TMEM206 target may be AKT, which is involved in regulating the biological behaviors of some cancers . TMEM206 is a component of the proton-activated Cl− channel that mediates Cl− influx and is involved in acid-induced cell death . A knockout of TMEM206 in neurons in mice attenuated brain damage after ischemic stroke . We speculated that the pathological mechanism in SCA1 might be partially mediated by TMEM206, which was associated with the expansion of the CAG-repeat in the ATXN1 gene, and led to chlorine influx-induced neuron death. TMEM206 is an unreported ataxin-1-interacting protein, and its molecular function in neurons should be explored in the future. In this study, the polyglutamine expansion in ataxin-1 led to its inability to interact with other partner proteins. This result suggested that the polyglutamine tract of ataxin-1 was essential to allow interactions with its protein partners.
Ataxin-1 functions as a regulator of transcription in the nucleus, and it regulates different transcription processes in concert with transcriptional modulators . Transcriptional derangements precede the pathologic and behavioral features associated with SCA1 and mutant ataxin-1 causes alterations in gene expression in mouse models . To identify target genes for ataxin-1 in wild-type and mutant conditions, we performed ChIP-seq in HEK-293T cells expressing wild-type and mutant ataxin-1. Our experiment identified a comprehensive set of wild-type ataxin-1 mRNA partners. The top two motifs contained the core, GGAG and AAAT, and were enriched in the ataxin-1-binding targets in HEK-293T cells expressing wild-type ataxin-1. The 1573 protein-coding genes were associated with wild-type ataxin-1 and identified using high-throughput sequencing. SLC6A15, NTF3, KCNC3, and DNAJC6 were functional neuronal genes among the top 15 ataxin-1 binding genes identified using ChIP-seq.
The SLC6A15 gene encodes a member of the solute carrier family 6 protein family, which plays an essential role in amino acid transport in neurons and might be associated with major depression . SLC6A15 expression is specific to the brain and revealed a strong preference for branched-chain amino acids and methionine transport . Amino acids play integral roles in the central nervous system as neurotransmitters, neuromodulators, and regulators of metabolism . PKC activation can reduce the plasma membrane expression of the SLC6A15 protein . Research presented in this study found that the SLC6A15 gene is an interacting mRNA partner of wild-type ataxin-1. Our studies indicated that ataxin-1 might have a potential role in amino acid transport and ataxin-1 dysfunction that alters amino acid transport might contribute to SCA1 onset.
NTF3 is a protein member of the neurotrophin family that controls neuron survival and differentiation . The NTF3 protein belongs to both brain-derived neurotrophic factor and nerve growth factor, which are involved with maintaining the adult nervous system and neuronal development in embryos . NTF3-deficient mice displayed severe movement defects of the limbs . Ataxin-1, a candidate binding protein for the NTF3 gene, might be involved in the movement defect observed in NTF3-deficient mice through protein–DNA interactions.
The physiological function of KCNC3 in the cerebellum is well known . Purkinje cells express KCNC3 in both their soma and dendrites, and KCNC3 plays a critical role in the Purkinje cell spikelet repolarization and the shaping of the complex spike . Mutations in the KCNC3 gene cause cerebellar neurodegeneration and impair auditory processing, termed spinocerebellar ataxia type 13 (SCA13) . Our results determined that ataxin-1 binds the KCNC3 gene, which suggests that the mutant ataxin-1 might contribute to the onset of SCA13 by regulating KCNC3 gene transcription.
DNAJC6 is a brain-specific protein with 970-amino acids that is enriched in presynaptic termini; it belongs to the conserved DNAJ/HSP40 family of proteins, which regulate molecular chaperone activity by stimulating ATPase activity . The DNAJC6 protein has three distinct domains including a conserved 70-amino acid domain at the N terminus that allows for its interaction with Hsc70, a cysteine-rich domain containing four motifs resembling a zinc finger domain, and a glycine/phenylalanine-rich region. In cells depleted of DNAJC6, vesicle trafficking is disrupted between the ER and Golgi, as well as throughout the Golgi . A previous functional study demonstrated that ataxin-2 played a role in endocytic pathways associated with endosomal trafficking . Our study suggested that ataxin-1 might affect vesicle trafficking mediated by DNAJC6.
Axons need to be correctly guided to their target during brain development . Axon guidance allows the formation of intricate neural circuits that control the function of the brain . Faulty disintegration and assembly of these circuits result in disorders of the nervous system. Some studies have demonstrated that axon guidance signaling pathways control gene expression through localized translation and transcription . Among the 1573 protein-coding genes identified by the ataxin-1 by ChIP-seq, twelve were implicated in axon guidance. Axon guidance is mediated by a range of extracellular guidance contacts that include secreted factors and cell adhesion molecules . The importance of axon guidance contacts and their receptors can be revealed based on links between mutations in genes that encode proteins associated with neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis . The GO analysis identified ataxin-1 binding genes that were involved in axon guidance. Thus, we inferred that axon guidance disruption might be involved in the pathogenesis of SCA1. Our results also indicated that mutant ataxin-1 with the polyglutamine expansion nearly completely lost the ability to bind target genes. This result suggested that the normal polyglutamine tract of ataxin-1 was essential for protein–DNA interactions, and an abnormal expansion of polyglutamine led to SCA1.
RNA-binding proteins regulate RNA processing, including pre-mRNA splicing, mRNA transport, 3′end formation, translation, and degradation . Ataxin-1 functions to regulate transcription and RNA processing in the nucleus . In this study, mapping of RNA–protein interactions was performed using RIP-seq of the expression of wild-type and mutant ataxin-1 in HEK-293T cells. The GO analysis confirmed that the top two enriched biological processes were linked to cellular nitrogen compound metabolic processing and biosynthetic processes for wild-type and mutant ataxin-1. We also confirmed that the abnormal polyglutamine expansion did not affect on the ability of ataxin-1 to bind target RNAs. The GO analysis also identified the top two enriched molecular functions, which were linked to RNA binding and ion binding for wild-type and mutant ataxin-1, respectively. However, the most enriched molecular function was RNA binding for wild-type ataxin-1. On the other hand, the most enriched molecular function was ion binding for mutant ataxin-1. These data indicated that the polyglutamine expansion in ataxin-1 had little effect on the ability of ataxin-1 to bind target RNAs.