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PTBP-dependent PSD-95 and CamKIIα alternative splicing in the lens.

Frederikse P, Nandanoor A, Kasinathan C - Mol. Vis. (2014)

Bottom Line: Intriguingly, associated modes of molecular regulation of these genes that had been thought to distinguish neurons have been identified in the lens as well.During neurogenesis, miR-124 is expressed that suppresses PTBP1 and promotes these splicing events. miR-124 is also expressed in mammalian lenses and upregulated during lens regeneration in amphibians, consistent with previous demonstrations of PTBP1,2 and PTBP-dependent PTBP2 exon 10 splicing in rodent lenses.Findings of this dendritic spine scaffold protein and conservation of its key mode of molecular regulation in the lens provides further evidence that key aspects of the neuron morphogenetic program are shared with the lens.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Biology, Rutgers SDM, Newark, NJ ; Department of Pharmacology & Physiology, Rutgers BHS.

ABSTRACT

Purpose: Parallels described between neurons and lens fiber cells include detailed similarities in sub-cellular structures that increasingly show shared expression of genes involved in the construction and function of these structures in neurons. Intriguingly, associated modes of molecular regulation of these genes that had been thought to distinguish neurons have been identified in the lens as well. Both elongated cell types form membrane protrusions with similar size, shape, and spacing that exclude microtubules, contain F-actin, and are coated with the clathrin/AP-2 adaptor. Lenses express glutamate and gamma-aminobutyric acid (GABA) receptors with signaling and channel proteins shown to act together at neuronal membranes. Postsynaptic density protein 95 (PSD-95) and Ca(2+)/calmodulin-dependent protein kinase (CaMKIIα) expression and functions illustrate the integration of aspects of neuronal molecular and cell biology and were investigated here in the lens.

Methods: Immunofluorescence, immunoblot, and RT-PCR methods were used to assess protein expression and alternative transcript splicing.

Results: We showed the essential dendritic spine scaffold protein PSD-95 is expressed in lenses and demonstrated lens PSD-95 transcripts undergo polypyrimidine tract binding protein (PTBP)-dependent alternative splicing of its pivotal exon 18 required to avoid nonsense-mediated decay, and showed PTBP-dependent alternative splicing of CaMKIIα transcripts in the lens. The PSD-95 protein was observed at fiber cell membranes overlapping with N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate and GABA receptor proteins, tyrosine phosphatase STEP, CaMKIIα, the Ca(V)1.3 calcium channel, and clathrin, which were previously identified at lens fiber cell membranes. During neurogenesis, miR-124 is expressed that suppresses PTBP1 and promotes these splicing events. miR-124 is also expressed in mammalian lenses and upregulated during lens regeneration in amphibians, consistent with previous demonstrations of PTBP1,2 and PTBP-dependent PTBP2 exon 10 splicing in rodent lenses.

Conclusions: Findings of this dendritic spine scaffold protein and conservation of its key mode of molecular regulation in the lens provides further evidence that key aspects of the neuron morphogenetic program are shared with the lens.

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Related in: MedlinePlus

Post-synaptic protein 95 (PSD-95) and polypyrimidine PTBP2 (nPTB) proteins are expressed in approximately 4-month-old rabbit and 28-day-old mouse lenses. A: Immunoblot detection of about 100 kDa PSD-95 in two individual rabbit lens samples. B: Detection of PSD-95 in two mouse lens samples of four lenses. C: Detection of about 50 kDa PTBP2 (nPTB) in rabbit lens. D: In situ detection of PTBP2 in rabbit and mouse lens. Photos show peripheral lens fibers at the lens border: I) indirect immunofluorescence detecting PTBP2 in rabbit lens, II) no primary antibody control in rabbit lens, III) anti-PTBP2 mouse lens, and IV) no primary control in mouse lens (1:200 original magnification; asterisk adjacent to lens epithelial cells beneath capsule at the anterior surface).
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f1: Post-synaptic protein 95 (PSD-95) and polypyrimidine PTBP2 (nPTB) proteins are expressed in approximately 4-month-old rabbit and 28-day-old mouse lenses. A: Immunoblot detection of about 100 kDa PSD-95 in two individual rabbit lens samples. B: Detection of PSD-95 in two mouse lens samples of four lenses. C: Detection of about 50 kDa PTBP2 (nPTB) in rabbit lens. D: In situ detection of PTBP2 in rabbit and mouse lens. Photos show peripheral lens fibers at the lens border: I) indirect immunofluorescence detecting PTBP2 in rabbit lens, II) no primary antibody control in rabbit lens, III) anti-PTBP2 mouse lens, and IV) no primary control in mouse lens (1:200 original magnification; asterisk adjacent to lens epithelial cells beneath capsule at the anterior surface).

Mentions: PSD-95 expression and functions in neurons illustrate the integration of basic aspects of neuronal molecular and cell biology. We began our study by determining whether the PSD-95 protein is expressed in mammalian lenses. Immunoblots identified expression of PSD-95 in rabbit and mouse lenses (Figure 1A,B). We next confirmed that PTBP2 (nPTB) is expressed in rabbit lenses (Figure 1C), which agrees with our previous identification of PTBP2 in mouse lenses [9,33]. Expression of the PSD-95 protein indicated PSD-95 exon 18 alternative splicing occurs in the lens, consistent with the PTBP2 exon 10 alternative splicing we showed previously in lenses [33], which is also necessary to produce a coherent full-length transcript for productive protein translation [22]. Lenses are comprised of cuboidal lens epithelial cells that cover their anterior surface. At the anterior/posterior equator, these cells exit the cell cycle and begin a process of pronounced elongation as they move into the lens interior. Examination of PTBP2 protein distribution in rabbit and mouse lens sections (Figure 1D) identified PTBP2 in peripheral differentiating fiber cells (lens cortex) in rabbit and mouse lenses, with less signal in mitotic lens epithelial cells below the lens capsule at the anterior surface, or terminally differentiated mature fiber cells in the center of the lens in the interior (lens nucleus), and agreed with our earlier examination of mouse lenses [2,9].


PTBP-dependent PSD-95 and CamKIIα alternative splicing in the lens.

Frederikse P, Nandanoor A, Kasinathan C - Mol. Vis. (2014)

Post-synaptic protein 95 (PSD-95) and polypyrimidine PTBP2 (nPTB) proteins are expressed in approximately 4-month-old rabbit and 28-day-old mouse lenses. A: Immunoblot detection of about 100 kDa PSD-95 in two individual rabbit lens samples. B: Detection of PSD-95 in two mouse lens samples of four lenses. C: Detection of about 50 kDa PTBP2 (nPTB) in rabbit lens. D: In situ detection of PTBP2 in rabbit and mouse lens. Photos show peripheral lens fibers at the lens border: I) indirect immunofluorescence detecting PTBP2 in rabbit lens, II) no primary antibody control in rabbit lens, III) anti-PTBP2 mouse lens, and IV) no primary control in mouse lens (1:200 original magnification; asterisk adjacent to lens epithelial cells beneath capsule at the anterior surface).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4265770&req=5

f1: Post-synaptic protein 95 (PSD-95) and polypyrimidine PTBP2 (nPTB) proteins are expressed in approximately 4-month-old rabbit and 28-day-old mouse lenses. A: Immunoblot detection of about 100 kDa PSD-95 in two individual rabbit lens samples. B: Detection of PSD-95 in two mouse lens samples of four lenses. C: Detection of about 50 kDa PTBP2 (nPTB) in rabbit lens. D: In situ detection of PTBP2 in rabbit and mouse lens. Photos show peripheral lens fibers at the lens border: I) indirect immunofluorescence detecting PTBP2 in rabbit lens, II) no primary antibody control in rabbit lens, III) anti-PTBP2 mouse lens, and IV) no primary control in mouse lens (1:200 original magnification; asterisk adjacent to lens epithelial cells beneath capsule at the anterior surface).
Mentions: PSD-95 expression and functions in neurons illustrate the integration of basic aspects of neuronal molecular and cell biology. We began our study by determining whether the PSD-95 protein is expressed in mammalian lenses. Immunoblots identified expression of PSD-95 in rabbit and mouse lenses (Figure 1A,B). We next confirmed that PTBP2 (nPTB) is expressed in rabbit lenses (Figure 1C), which agrees with our previous identification of PTBP2 in mouse lenses [9,33]. Expression of the PSD-95 protein indicated PSD-95 exon 18 alternative splicing occurs in the lens, consistent with the PTBP2 exon 10 alternative splicing we showed previously in lenses [33], which is also necessary to produce a coherent full-length transcript for productive protein translation [22]. Lenses are comprised of cuboidal lens epithelial cells that cover their anterior surface. At the anterior/posterior equator, these cells exit the cell cycle and begin a process of pronounced elongation as they move into the lens interior. Examination of PTBP2 protein distribution in rabbit and mouse lens sections (Figure 1D) identified PTBP2 in peripheral differentiating fiber cells (lens cortex) in rabbit and mouse lenses, with less signal in mitotic lens epithelial cells below the lens capsule at the anterior surface, or terminally differentiated mature fiber cells in the center of the lens in the interior (lens nucleus), and agreed with our earlier examination of mouse lenses [2,9].

Bottom Line: Intriguingly, associated modes of molecular regulation of these genes that had been thought to distinguish neurons have been identified in the lens as well.During neurogenesis, miR-124 is expressed that suppresses PTBP1 and promotes these splicing events. miR-124 is also expressed in mammalian lenses and upregulated during lens regeneration in amphibians, consistent with previous demonstrations of PTBP1,2 and PTBP-dependent PTBP2 exon 10 splicing in rodent lenses.Findings of this dendritic spine scaffold protein and conservation of its key mode of molecular regulation in the lens provides further evidence that key aspects of the neuron morphogenetic program are shared with the lens.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Biology, Rutgers SDM, Newark, NJ ; Department of Pharmacology & Physiology, Rutgers BHS.

ABSTRACT

Purpose: Parallels described between neurons and lens fiber cells include detailed similarities in sub-cellular structures that increasingly show shared expression of genes involved in the construction and function of these structures in neurons. Intriguingly, associated modes of molecular regulation of these genes that had been thought to distinguish neurons have been identified in the lens as well. Both elongated cell types form membrane protrusions with similar size, shape, and spacing that exclude microtubules, contain F-actin, and are coated with the clathrin/AP-2 adaptor. Lenses express glutamate and gamma-aminobutyric acid (GABA) receptors with signaling and channel proteins shown to act together at neuronal membranes. Postsynaptic density protein 95 (PSD-95) and Ca(2+)/calmodulin-dependent protein kinase (CaMKIIα) expression and functions illustrate the integration of aspects of neuronal molecular and cell biology and were investigated here in the lens.

Methods: Immunofluorescence, immunoblot, and RT-PCR methods were used to assess protein expression and alternative transcript splicing.

Results: We showed the essential dendritic spine scaffold protein PSD-95 is expressed in lenses and demonstrated lens PSD-95 transcripts undergo polypyrimidine tract binding protein (PTBP)-dependent alternative splicing of its pivotal exon 18 required to avoid nonsense-mediated decay, and showed PTBP-dependent alternative splicing of CaMKIIα transcripts in the lens. The PSD-95 protein was observed at fiber cell membranes overlapping with N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate and GABA receptor proteins, tyrosine phosphatase STEP, CaMKIIα, the Ca(V)1.3 calcium channel, and clathrin, which were previously identified at lens fiber cell membranes. During neurogenesis, miR-124 is expressed that suppresses PTBP1 and promotes these splicing events. miR-124 is also expressed in mammalian lenses and upregulated during lens regeneration in amphibians, consistent with previous demonstrations of PTBP1,2 and PTBP-dependent PTBP2 exon 10 splicing in rodent lenses.

Conclusions: Findings of this dendritic spine scaffold protein and conservation of its key mode of molecular regulation in the lens provides further evidence that key aspects of the neuron morphogenetic program are shared with the lens.

Show MeSH
Related in: MedlinePlus