IU Indianapolis ScholarWorks :: Browsing by Author "Custer, Sara K."

Browsing by Author "Custer, Sara K."

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Abnormal Golgi morphology and decreased COPI function in cells with low levels of SMN
(Elsevier, 2019-03) Custer, Sara K.; Foster, Joycelynn N.; Astroski, Jacob W.; Androphy, Elliot J.; Dermatology, School of Medicine
We report here the finding of abnormal Golgi apparatus morphology in motor neuron like cells depleted of SMN as well as Golgi apparatus morphology in SMA patient fibroblasts. Rescue experiments demonstrate that this abnormality is dependent on SMN, but can also be rescued by expression of the COPI coatomer subunit alpha-COP. A motor neuron-like cell line containing an inducible alpha-COP shRNA was created to generate a parallel system to study knockdown of SMN or alpha-COP. Multiple assays of COPI-dependent intracellular trafficking in cells depleted of SMN demonstrate that alpha-COP function is suboptimal, including failed sequestration of plasma membrane proteins, altered binding of mRNA, and defective targeting and transport of Golgi-resident proteins.
Altered mRNA Splicing in SMN-Depleted Motor Neuron-Like Cells
(Public Library of Science (PLoS), 2016) Custer, Sara K.; Gilson, Timra D.; Li, Hongxia; Todd, A. Gary; Astroski, Jacob W.; Lin, Hai; Liu, Yunlong; Androphy, Elliot J.; Department of Dermatology, School of Medicine
Spinal muscular atrophy (SMA) is an intractable neurodegenerative disease afflicting 1 in 6-10,000 live births. One of the key functions of the SMN protein is regulation of spliceosome assembly. Reduced levels of the SMN protein that are observed in SMA have been shown to result in aberrant mRNA splicing. SMN-dependent mis-spliced transcripts in motor neurons may cause stresses that are particularly harmful and may serve as potential targets for the treatment of motor neuron disease or as biomarkers in the SMA patient population. We performed deep RNA sequencing using motor neuron-like NSC-34 cells to screen for SMN-dependent mRNA processing changes that occur following acute depletion of SMN. We identified SMN-dependent splicing changes, including an intron retention event that results in the production of a truncated Rit1 transcript. This intron-retained transcript is stable and is mis-spliced in spinal cord from symptomatic SMA mice. Constitutively active Rit1 ameliorated the neurite outgrowth defect in SMN depleted NSC-34 cells, while expression of the truncated protein product of the mis-spliced Rit1 transcript inhibited neurite extension. These results reveal new insights into the biological consequence of SMN-dependent splicing in motor neuron-like cells.
Autophagy dysregulation in cell culture and animals models of Spinal Muscular Atrophy
(Elsevier, 2014-07) Custer, Sara K.; Androphy, Elliot J.; Department of Dermatology, IU School of Medicine
Abnormal autophagy has become a central thread linking neurodegenerative diseases, particularly of the motor neuron. One such disease is spinal muscular atrophy (SMA), a genetic neuromuscular disorder caused by mutations in the SMN1 gene resulting in low levels of Survival Motor Neuron (SMN) protein. Despite knowing the causal protein, the exact intracellular processes that are involved in the selective loss of motor neurons remains unclear. Autophagy induction can be helpful or harmful depending on the situation, and we sought to understand the state of the autophagic response in SMA. We show that cell culture and animal models demonstrate induction of autophagy accompanied by attenuated autophagic flux, resulting in the accumulation of autophagosomes and their associated cargo. Expression of the SMN-binding protein a-COP, a known modulator of autophagic flux, can ameliorate this autophagic traffic jam.
COPI coatomer subunit α-COP interacts with the RNA binding protein Nucleolin via a C-terminal dilysine motif
(Oxford University Press, 2023) Custer, Sara K.; Gilson, Timra; Astroski, Jacob W.; Nanguneri, Siddarth R.; Iurillo, Alyssa M.; Androphy, Elliot J.; Dermatology, School of Medicine
The COPI coatomer subunit α-COP has been shown to co-precipitate mRNA in multiple settings, but it was unclear whether the interaction with mRNA was direct or mediated by interaction with an adapter protein. The COPI complex often interacts with proteins via C-terminal dilysine domains. A search for candidate RNA binding proteins with C-terminal dilysine motifs yielded Nucleolin, which terminates in a KKxKxx sequence. This protein was an especially intriguing candidate as it has been identified as an interacting partner for Survival Motor Neuron protein (SMN). Loss of SMN causes the neurodegenerative disease Spinal Muscular Atrophy. We have previously shown that SMN and α-COP interact and co-migrate in axons, and that overexpression of α-COP reduced phenotypic severity in cell culture and animal models of SMA. We show here that in an mRNA independent manner, endogenous Nucleolin co-precipitates endogenous α-COP and ε-COP but not β-COP which may reflect an interaction with the so-called B-subcomplex rather a complete COPI heptamer. The ability of Nucleolin to bind to α-COP requires the presence of the C-terminal KKxKxx domain of Nucleolin. Furthermore, we have generated a point mutant in the WD40 domain of α-COP which eliminates its ability to co-precipitate Nucleolin but does not interfere with precipitation of partners mediated by non-KKxKxx motifs such as the kainate receptor subunit 2. We propose that via interaction between the C-terminal dilysine motif of Nucleolin and the WD40 domain of α-COP, Nucleolin acts an adaptor to allow α-COP to interact with a population of mRNA.
Dilysine motifs in exon 2b of SMN protein mediate binding to the COPI vesicle protein α-COP and neurite outgrowth in a cell culture model of spinal muscular atrophy
(Oxford Journals, 2013-10-15) Custer, Sara K.; Todd, Adrian G.; Singh, Natalia N.; Androphy, Elliot J.; Department of Dermatology, School of Medicine
Spinal muscular atrophy (SMA) is a devastating neuromuscular disorder that stems from low levels of survival of motor neuron (SMN) protein. The processes that cause motor neurons and muscle cells to become dysfunctional are incompletely understood. We are interested in neuromuscular homeostasis and the stresses put upon that system by loss of SMN. We recently reported that α-COP, a member of the coatomer complex of coat protein I (COPI) vesicles, is an SMN-binding partner, implicating this protein complex in normal SMN function. To investigate the functional significance of the interaction between α-COP and SMN, we constructed an inducible NSC-34 cell culture system to model the consequences of SMN depletion and find that depletion of SMN protein results in shortened neurites. Heterologous expression of human SMN, and interestingly over-expression of α-COP, restores normal neurite length and morphology. Mutagenesis of the canonical COPI dilysine motifs in exon 2b results in failure to bind to α-COP and abrogates the ability of human SMN to restore neurite outgrowth in SMN-depleted motor neuron-like NSC-34 cells. We conclude that the interaction between SMN and α-COP serves an important function in the growth and maintenance of motor neuron processes and may play a significant role in the pathogenesis of SMA.
Identification of a resilient mouse facial motoneuron population following target disconnection by injury or disease
(IOS, 2018) Setter, Deborah O.; Haulcomb, Melissa M.; Beahrs, Taylor; Meadows, Rena M.; Schartz, Nicole D.; Custer, Sara K.; Sanders, Virginia M.; Jones, Kathryn J.; Anatomy and Cell Biology, IU School of Medicine
Background: When nerve transection is performed on adult rodents, a substantial population of neurons survives short-term disconnection from target, and the immune system supports this neuronal survival, however long-term survival remains unknown. Understanding the effects of permanent axotomy on cell body survival is important as target disconnection is the first pathological occurrence in fatal motoneuron diseases such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). Objective: The goal of this study was to determine if facial motoneurons (FMN) could survive permanent target disconnection up to 26 weeks post-operation (wpo) after facial nerve axotomy (FNA). In addition, the potentially additive effects of immunodeficiency and motoneuron disease on post-axotomy FMN survival were examined. Methods: This study included three wild type (WT) mouse strains (C57BL/6J, B6SJL, and FVB/NJ) and three experimental models (RAG-2-/-: immunodeficiency; mSOD1: ALS; Smn-/-/SMN2+/+: SMA). All animals received a unilateral FNA, and FMN survival was quantified at early and extended post-operative timepoints. Results: In the C57BL/6J WT group, FMN survival significantly decreased at 10 wpo (55 ± 6%), and then remained stable out to 26 wpo (47 ± 6%). In the RAG-2-/- and mSOD1 groups, FMN death occurred much earlier at 4 wpo, and survival plateaued at approximately 50% at 10 wpo. The SMA model and other WT strains also exhibited approximately 50% FMN survival after FNA. Conclusion: These results indicate that immunodeficiency and motoneuron disease accelerate axotomy-induced neuron death, but do not increase total neuron death in the context of permanent target disconnection. This consistent finding of a target disconnection-resilient motoneuron population is prevalent in other peripheral nerve injury models and in neurodegenerative disease models as well. Characterization of the distinct populations of vulnerable and resilient motoneurons may reveal new therapeutic approaches for injury and disease.
Interaction between alpha-COP and SMN ameliorates disease phenotype in a mouse model of spinal muscular atrophy
(Elsevier, 2019-05-03) Custer, Sara K.; Astroski, Jacob W.; Li, Hong Xia; Androphy, Elliot J.; Dermatology, School of Medicine
We report that expression of the α-COP protein rescues disease phenotype in a severe mouse model of Spinal Muscular Atrophy (SMA).. Lentiviral particles expressing α-COP were injected directly into the testes of genetically pure mouse strain of interest resulting in infection of the spermatagonial stem cells. α-COP was stably expressed in brain, skeletal muscle, and spinal cord without altering SMN protein levels. SMA mice transgenic for α-COP live significantly longer than their non-transgenic littermates, and showed increased body mass and normal muscle morphology at postnatal day 15. We previously reported that binding between SMN and α-COP is required for restoration of neurite outgrowth in cells lacking SMN, and we report similar finding here. Lentiviral-mediated transgenic expression of SMN where the dilysine domain in exon 2b was mutated was not able to rescue the SMA phenotype despite robust expression of the mutant SMN protein in brain, muscle and spinal cord. These results demonstrate that α-COP is a validated modifier of SMA disease phenotype in a mammalian, vertebrate model and is a potential target for development of future SMN-independent therapeutic interventions.
Mutations in the COPI coatomer subunit α-COP induce release of Aβ-42 and amyloid precursor protein intracellular domain and increase tau oligomerization and release
(Elsevier, 2021) Astroski, Jacob W.; Akporyoe, Leonora K.; Androphy, Elliot J.; Custer, Sara K.; Dermatology, School of Medicine
Understanding the cellular processes that lead to Alzheimer's disease (AD) is critical, and one key lies in the genetics of families with histories of AD. Mutations a complex known as COPI were found in families with AD. The COPI complex is involved in protein processing and trafficking. Intriguingly, several recent publications have found components of the COPI complex can affect the metabolism of pathogenic AD proteins. We reduced levels of the COPI subunit α-COP, altering maturation and cleavage of amyloid precursor protein (APP), resulting in decreased release of Aβ-42 and decreased accumulation of the AICD. Depletion of α-COP reduced uptake of proteopathic Tau seeds and reduces intracellular Tau self-association. Expression of AD-associated mutant α-COP altered APP processing, resulting in increased release of Aβ-42 and increased intracellular Tau aggregation and release of Tau oligomers. These results show that COPI coatomer function modulates processing of both APP and Tau, and expression of AD-associated α-COP confers a toxic gain of function, resulting in potentially pathogenic changes in both APP and Tau.
α-COP binding to the survival motor neuron protein SMN is required for neuronal process outgrowth
(Oxford University Press, 2015-12-20) Li, Hongxia; Custer, Sara K.; Gilson, Timra; Hao, Le Thi; Beattie, Christine E.; Androphy, Elliot J.; Department of Dermatology, IU School of Medicine
Spinal muscular atrophy (SMA), a heritable neurodegenerative disease, results from insufficient levels of the survival motor neuron (SMN) protein. α-COP binds to SMN, linking the COPI vesicular transport pathway to SMA. Reduced levels of α-COP restricted development of neuronal processes in NSC-34 cells and primary cortical neurons. Remarkably, heterologous expression of human α-COP restored normal neurite length and morphology in SMN-depleted NSC-34 cells in vitro and zebrafish motor neurons in vivo. We identified single amino acid mutants of α-COP that selectively abrogate SMN binding, retain COPI-mediated Golgi-ER trafficking functionality, but were unable to support neurite outgrowth in cellular and zebrafish models of SMA. Taken together, these demonstrate the functional role of COPI association with the SMN protein in neuronal development.

Browsing by Author "Custer, Sara K."

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