Elsevier

Brain Research

Volume 1706, 1 March 2019, Pages 135-146
Brain Research

Research report
Abnormal Golgi morphology and decreased COPI function in cells with low levels of SMN

https://doi.org/10.1016/j.brainres.2018.11.005Get rights and content

Highlights

  • SMA fibroblasts have abnormal Golgi apparatus morphology which can be rescued by restoring SMN protein levels to normal.

  • Knockdown of the SMN binding partner α-COP produces a similar diffuse Golgi morphology.

  • α-COP over-expression restores normal Golgi morphology in SMA fibroblasts.

  • COPI-dependent intracellular trafficking is altered in cells after knockdown of SMN.

Abstract

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.

Introduction

Spinal Muscular Atrophy (SMA) is a progressive neurodegenerative disease characterized by loss of alpha motor neurons. Understanding the basic cellular processes that underlie this neuronal loss opens up potential avenues for novel therapeutic interventions in SMA as well as other motor neuron diseases. We have focused our recent studies on the interaction between SMN and the alpha subunit of the COPI coatomer. As we previously reported, the alpha-COP and SMN proteins interact and co-migrate in axons of cultured neurons (Peter et al., 2011). In cells depleted of alpha-COP, there was decreased delivery of SMN protein to the leading edge of the cell. We also showed that in motor neuron-like cells, depletion of SMN resulted in poor neurite outgrowth, which could be rescued by expressing SMN but not SMN mutants that do not bind to alpha-COP (Custer et al., 2013). Interestingly, heterologous expression of alpha-COP in these SMN depleted cells produced a robust rescue of neurite outgrowth comparable to that generated by expression of human SMN. NSC-34 cells expressing shRNA against murine alpha-COP were unable to produce neurites. This phenotype could be rescued by expression of wild type alpha-COP but not by a point mutant of alpha-COP that no longer bound SMN (Li et al., 2015). These studies demonstrate the biological relevance of the interaction between SMN and alpha-COP.

For the research discussed here, we sought to assess the function of the COPI coatomer in cells depleted of SMN. In mammals, coatomer function appears to be important for maintenance of neuronal health. The mdf mutant mouse develops muscle wasting, motor neuron loss and cerebellar degeneration as a result of mutations in Scyl1, a protein that binds to the COPI coat complex and regulates Golgi-ER retrograde trafficking (Schmidt et al., 2007). Mice lacking Scyl1 develop a motor neuron disease similar to amyotrophic lateral sclerosis (ALS) (Pelletier et al., 2012). Mice with a mutation in delta-COP/archain 1 develop Purkinje neuron degeneration and cerebellar atrophy (Xu et al., 2010). A family with autosomal dominant mutations in alpha-COP developed exercise-induce dyskinesia along with a constellation of autoimmune issues (Watkin et al., 2015). These findings underscore the importance of COPI function in maintaining neuronal health, especially in large neurons with heavy transport burdens such as Purkinje cells and alpha motor neurons. The majority of COPI is resident at the Golgi apparatus, and it regulates intracellular trafficking between the endoplasmic reticulum (ER) and the Golgi (Szul and Sztul, 2011). COPI coatomer function is particularly important for maintenance of normal Golgi morphology both in resting cells and in cells entering mitosis. In HeLa cells, abnormal Golgi morphology was observed after depletion of alpha, beta or beta’-COP (Razi et al., 2009). Abnormal Golgi morphology has been observed in numerous models of motor neuron diseases including ALS caused by mutations in superoxide dismutase 1 (SOD1) (van Dis et al., 2014), TDP-43, FUS and optineurin (OPTN) (Fifita et al., 2017, Soo et al., 2015). It has also been reported in various mouse models of motor neuron disease including the pmn mouse (progressive motor neuropathy) (Bellouze et al., 2014). Golgi fragmentation has been described in postmortem studies of Alzheimer’s (Ayala and Colanzi, 2017) and Parkinson’s disease brains (Fujita et al., 2006).

Here we report finding of abnormal Golgi morphology in both SMN depleted NSC-34 cells and fibroblasts from Type I and Type II SMA patients, indicating that perhaps the COPI coatomer function is impaired under conditions of SMN depletion. To assay the impact of impaired COPI function on neuronal cells, we studied two doxycycline inducible shRNA cell lines, our previously published alpha-COP knockdown SH-SY5Y cell line (1-15d) (Peter et al., 2011) and a newly generated line in NSC-34 cells using the same alpha-COP shRNA. These cells were derived from the same parental NSC-34 line as our inducible shRNA knockdown of SMN for a direct comparison of SMN depletion and alpha-COP depletion.

We demonstrate Golgi abnormalities in both NSC-34 cells and SMA patient fibroblasts can be rescued by over-expression of SMN and that its interaction with alpha-COP was required for this rescue. Expression of alpha-COP also restored normal Golgi morphology in these cultures but this rescue was not dependent on the ability to bind SMN. We assayed a number of intracellular trafficking pathways controlled by COPI coatomer in both alpha-COP and SMN depleted cells to determine if the COPI coatomer function was intact under conditions of low SMN. We find that there is a mild defect in COPI-dependent trafficking of the Kainate receptor subunit (KA2) to the plasma membrane in SMN depleted cells, which was not detected in cells expressing mutant SOD1. SMN depleted cells are also unable to properly localize the COPI target KDEL receptor. Finally, despite their abnormal Golgi morphology, SMN depleted cells did not exhibit increased ER stress as evidenced by their lack of sensitivity to thapsigargin. We conclude that although COPI-dependent intracellular trafficking appears dysregulated after SMN knockdown, the ER-Golgi system is not sufficiently altered to induce ER stress.

Section snippets

Alpha-COP is required for SMN localization to the growth cone of NSC-34 cells

We had previously demonstrated that depletion of alpha-COP in SH-SY5Y cells resulted in a failure of SMN protein to traffic to the leading edge of filipodia (Peter et al., 2011). We found a similar result in the motor neuron-like NSC-34 cells. NSC-34 cells were grown in differentiation conditions for 48 h to induce neurite outgrowth followed by infection with lentiviral particles expressing shRNA against murine alpha-COP or control shRNA against green fluorescent protein (GFP). Western blotting

Discussion

We have shown in several model systems that expression of alpha-COP can compensate for low levels of SMN to restore normal neurite outgrowth. Here we report the finding that in both NSC-34 motor neuron-like cells and human fibroblasts derived from Type I and Type II SMA patients, under conditions of low SMN, there is an increased incidence of abnormal Golgi apparatus morphology, specifically a diffuse, punctate pattern visualized with the cis-Golgi marker GM130. This pattern has been reported

Cell culture

NSC-34 cells were maintained in DMEM with 10% fetal bovine serum (FBS) and Pen/Strep. SH-SY5Y cells were maintained in DMEM/F12 with 10% FBS and Pen/Strep. For transient knockdown of alpha-COP in NSC-34 cells, cultures were infected with lentivirus expressing shRNA against murine alpha-COP (TRNC0000313321) or control lentivirus expressing shRNA against GFP (Sigma SHC005). To generate the inducible alpha-COP knockdown NSC-34 cells, NSC-34 clone #4 (TetR driver alone) were transfected with

Acknowledgements

Thanks to Robert Kalb for SOD1-expressing NSC-34 cells, to Geoffrey Swanson for the myc-KA2 expression vector, P. Vivithanaporn for Myc-tagged KA2, Xue-Jun Li for EF1a-driven FLAG-hSMN lentivirus and Victor Hsu for the YFP-VSVGts045-KDELR reporter construct. The research reported in this publication was supported by the CureSMA Emerging Investigator Award (Custer) and by the National Institute of Neurological Disorders and Stroke under R01NS082284 and R01NS082284-S1 (Androphy). The content is

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