Studies of the central nervous system‐derived CAD cell line, a suitable model for intraneuronal transport studies?

Li, Yongling; Hou, Linda; Aktiv, Annika; Dahlström, Annica

doi:10.1002/jnr.21216

Cited by 23 publications

(19 citation statements)

References 115 publications

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“…We verified that this was also true for SERT by employing the murine CAD cell line, which is derived from the Cath.a tumor cell line (17). These cells are catecholaminergic; they are readily transfected and undergo differentiation upon serum removal, whereupon they develop neurite extensions that express axonal and dendritic markers (3, 5, 17, 18). If wild type SERT was expressed in CAD cells and the cells were subsequently differentiated by serum withdrawal, the transporter was enriched at the tips of neurite extensions (see images in Fig.…”

Section: Resultsmentioning

confidence: 99%

The Serotonin Transporter Is an Exclusive Client of the Coat Protein Complex II (COPII) Component SEC24C

Sučić

El‐Kasaby

Kudlacek

et al. 2011

Journal of Biological Chemistry

112

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The transporters for serotonin (SERT), dopamine, and noradrenaline have a conserved hydrophobic core but divergent N and C termini. The C terminus harbors the binding site for the coat protein complex II (COPII) cargo-binding protein SEC24. Here we explored which SEC24 isoform was required for export of SERT from the endoplasmic reticulum (ER). Three lines of evidence argue that SERT can only exit the ER by recruiting SEC24C: (i) Mass spectrometry showed that a peptide corresponding to the C terminus of SERT recruited SEC24C-containing COPII complexes from mouse brain lysates. (ii) Depletion of individual SEC24 isoforms by siRNAs revealed that SERT was trapped in the ER only if SEC24C was down-regulated, in both, cells that expressed SERT endogenously or after transfection. The combination of all siRNAs was not more effective than that directed against SEC24C. A SERT mutant in which the SEC24C-binding motif (607RI608) was replaced by alanine was insensitive to down-regulation of SEC24C levels. (iii) Overexpression of a SEC24C variant with a mutation in the candidate cargo-binding motif (SEC24C-D796V/D797N) but not of the corresponding mutant SEC24D-D733V/D734N reduced SERT surface levels. In contrast, noradrenaline and dopamine transporters and the more distantly related GABA transporter 1 relied on SEC24D for ER export. These observations demonstrate that closely related transporters are exclusive client cargo proteins for different SEC24 isoforms. The short promoter polymorphism results in reduced SERT cell surface levels and renders affected individuals more susceptible to depression. By inference, variations in the Sec24C gene may also affect SERT cell surface levels and thus be linked to mood disorders.

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Section: Resultsmentioning

confidence: 99%

The Serotonin Transporter Is an Exclusive Client of the Coat Protein Complex II (COPII) Component SEC24C

Sučić

El‐Kasaby

Kudlacek

et al. 2011

Journal of Biological Chemistry

112

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“…To study ciliary biogenesis in neuronal cells, we used CAD (Cath.a-differentiated) cells, a central nervous system (CNS)-derived cell line (Qi et al, 1997; Li et al, 2007), and primary hippocampal neurons, as model systems. Seventy-two hours after seeding cells in Opti-MEM medium without serum, nearly 45% of CAD cells had visible cilia (Fig.…”

Section: Resultsmentioning

confidence: 99%

CPAP is required for cilia formation in neuronal cells

Tang

2012

Biology Open

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SummaryThe primary cilium is a microtubule-based structure protruded from the basal body analogous to the centriole. CPAP (centrosomal P4.1-associated protein) has previously been reported to be a cell cycle-regulated protein that controls centriole length. Mutations in CPAP cause primary microcephaly (MCPH) in humans. Here, using a cell-based system that we established to monitor cilia formation in neuronal CAD (Cath.a-differentiated) cells and hippocampal neurons, we found that CPAP is required for cilia biogenesis. Overexpression of wild-type CPAP promoted cilia formation and induced longer cilia. In contrast, an exogenously expressed CPAP-377EE mutant that lacks tubulin-dimer binding significantly inhibited cilia formation and caused cilia shortening. Furthermore, depletion of CPAP inhibited ciliogenesis and such effect was effectively rescued by expression of wild-type CPAP, but not by the CPAP-377EE mutant. Taken together, our results suggest that CPAP is a positive regulator of ciliogenesis whose intrinsic tubulin-dimer binding activity is required for cilia formation in neuronal cells.

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“…The parental CAD cells (Y Li et al 2007) and their derivatives were transfected with Ptbp1-and/or Ptbp2-specific or control ON-TARGETplus siRNAs (Dharmacon) using Lipofectamine 2000 (Invitrogen) and analyzed 72 h post-transfection. Expression of shRNAs was induced by treating corresponding CAD-A13 cell pools with 2 mg/mL Dox for 72 h as described (Khandelia et al 2011).…”

Section: Rnaimentioning

confidence: 99%

Coordinated regulation of neuronal mRNA steady-state levels through developmentally controlled intron retention

Yap¹,

Lim²,

Khandelia³

et al. 2012

Genes Dev.

268

295

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Differentiated cells acquire unique structural and functional traits through coordinated expression of lineagespecific genes. An extensive battery of genes encoding components of the synaptic transmission machinery and specialized cytoskeletal proteins is activated during neurogenesis, but the underlying regulation is not well understood. Here we show that genes encoding critical presynaptic proteins are transcribed at a detectable level in both neurons and nonneuronal cells. However, in nonneuronal cells, the splicing of 39-terminal introns within these genes is repressed by the polypyrimidine tract-binding protein (Ptbp1). This inhibits the export of incompletely spliced mRNAs to the cytoplasm and triggers their nuclear degradation. Clearance of these introncontaining transcripts occurs independently of the nonsense-mediated decay (NMD) pathway but requires components of the nuclear RNA surveillance machinery, including the nuclear pore-associated protein Tpr and the exosome complex. When Ptbp1 expression decreases during neuronal differentiation, the regulated introns are spliced out, thus allowing the accumulation of translation-competent mRNAs in the cytoplasm. We propose that this mechanism counters ectopic and precocious expression of functionally linked neuron-specific genes and ensures their coherent activation in the appropriate developmental context.

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Studies of the central nervous system‐derived CAD cell line, a suitable model for intraneuronal transport studies?

Cited by 23 publications

References 115 publications

The Serotonin Transporter Is an Exclusive Client of the Coat Protein Complex II (COPII) Component SEC24C

The Serotonin Transporter Is an Exclusive Client of the Coat Protein Complex II (COPII) Component SEC24C

CPAP is required for cilia formation in neuronal cells

Coordinated regulation of neuronal mRNA steady-state levels through developmentally controlled intron retention

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