Synapsin and synaptic vesicle protein expression during embryonic and post-natal lens fiber cell differentiation.

PURPOSE: Reorganization of cytoskeleton and membrane biogenesis are dynamically coordinated during lens fiber cell differentiation and development to produce an organ with precise dimensions and optical properties. Cargo vesicle trafficking is fundamental to cell elongation and has also been implicated in degenerative disease mechanisms. Alzheimer precursor protein (AbetaPP) acts with kinesin, synapsin, and synaptic vesicle proteins to mediate cargo vesicle transport and membrane fusion in neurons. In our previous studies we demonstrated that AbetaPP is also a key element in lens fiber cell formation, and in early-onset cataract that occurs along with early-onset Alzheimer disease in Down syndrome. In the present study we examine lens expression and regulation of a complement of genes associated with cargo and synaptic vesicle transport in neurons. METHODS: RT-PCR, immunoblot, and immunohistochemical methods were used to characterize expression of AbetaPP and kinesin associated motor proteins, synapsins, and synaptic vesicle proteins in mouse and rat embryonic, post-natal, and adult lenses. Phospho-specific anti-synapsin antibodies were used to determine the distributions of site-1 phosphorylated and dephosphorylated synapsin protein. RESULTS: We demonstrate that a substantial complement of cargo and synaptic vesicle proteins involved in AbetaPP mediated vesicle transport are expressed in lenses along the anterior-posterior axis of fiber cells in embryonic and adult lenses, consistent with vesicles, actin filaments, and neuron-like arrangement of microtubules in lenses shown by others. We identify temporal regulation of synapsins I, II, and III during embryonic and post-natal lens development consistent with their roles in neurons. Regulation of vesicle cytoskeleton attachment, actin polymerization, and the capacity to stimulate cell differentiation by synapsins are governed in large part by phosphorylation at a conserved Ser9 residue (site-1). We demonstrate discrete distributions of Ser9 phospho- and dephospho-synapsins along the axial length of rapidly elongating embryonic lens fiber cells, and decreased levels of site-1 phosphorylated synapsins in adult lenses. CONCLUSIONS: The present findings demonstrate several fundamental parallels between lens and neuron vesicle trafficking cell biology and development, and suggest that more extensive AbetaPP related vesicle trafficking disease mechanisms may be shared by lens and brain.

Beta-amyloid secretases and beta-amloid degrading enzyme expression in lens.

PURPOSE: Beta- and gamma-secretases are proteases involved in the processing of the Alzheimer precursor protein (AbetaPP) that releases the transmembrane beta-amyloid fragment (Abeta), associated with age-dependent disease in lens and brain. Gamma-secretase is a protein complex containing Presenilin and Nicastrin proteins, which also processes Notch and other receptors involved in the eye and lens development. Neprilysin (NEP), a major protease involved in degrading Abeta, acts with beta- and gamma-secretases to regulate steady-state levels of Abeta. Previously, we demonstrated AbetaPP and Presenilin expression and processing in the lens and demonstrated cell degeneration in classic Alzheimer disease (AD) transgenic and systemic oxidative stress animal models, suggesting that additional AbetaPP processing proteins are also present in the lens. Here we investigate lens expression of beta-secretases, nicastrin and NEP proteins, and compare their protein distribution to Notch and Presenilin in lens. METHODS: RT-PCR was used to analyze mRNA transcripts. Immunoblots and immunohistochemistry were used to examine the protein expression and distribution of secretase and Abeta degrading proteins, as well as Presenilin and Notch proteins in mouse lenses. RESULTS: Beta-acting cleaving enzymes, BACE (BACE1) and BACE2, Nicastrin, Presenilins, Notch and NEP are expressed in the lens. In situ examination of protein distribution in lens indicates expression of each of these proteins is upregulated in peripheral elongating fiber cells at the lens equatorial margin and overlaps with Notch and Presenilin proteins, and also with the distribution of AbetaPP and Abeta proteins demonstrated in a previous study. Neprilysin exon 1-4 splicing, previously described as diagnostic for neuronal expression, also occurs in lens. CONCLUSIONS: BACE, BACE2, Nicastrin and NEP are expressed primarily in elongating peripheral fiber cells, overlapping with Notch, Presenilin, and AbetaPP protein distribution in lens, consistent with their role in regulating Notch and AbetaPP ectodomain shedding in lens. Lens expression of beta- and gamma-secretases together with NEP suggests these proteins may also regulate Abeta turnover in the lens. The presence of Abeta processing and degrading proteases in lens provides further evidence that Alzheimer-related cell biology is fundamentally involved in lens development, and provides additional evidence that mechanisms of Alzheimer pathophysiology can contribute to lens degeneration, suggesting further that therapeutics targeting Abeta proteases may be applicable to lens degenerative disease.