Pamela J. Yao

Evidence for CALM in directing VAMP2 trafficking.

Clathrin assembly lymphoid myeloid leukemia protein (CALM) is a clathrin assembly protein with a domain structure similar to the neuron‐specific assembly protein AP180. We have previously found that CALM is expressed in neurons and present in synapses. We now report that CALM has a neuron‐related function: it facilitates the endocytosis of the synaptic vesicle protein VAMP2 from the plasma membrane. Overexpression of CALM leads to the reduction of cell surface VAMP2, whereas knockdown of CALM by RNA interference results in the accumulation of surface VAMP2. The AP180 N‐terminal homology (ANTH) domain of CALM is required for its effect on VAMP2 trafficking, and the ANTH domain itself acts as a dominant‐negative mutant. Thus, our results reveal a role for CALM in directing VAMP2 trafficking during endocytosis.

Plasma membrane ion permeability induced by mutant α-synuclein contributes to the degeneration of neural cells

Mutations in α‐synuclein cause some cases of familial Parkinsons disease (PD), but the mechanism by which α‐synuclein promotes degeneration of dopamine‐producing neurons is unknown. We report that human neural cells expressing mutant α‐synuclein (A30P and A53T) have higher plasma membrane ion permeability. The higher ion permeability caused by mutant α‐synuclein would be because of relatively large pores through which most cations can pass non‐selectively. Both the basal level of [Ca2+]i and the Ca2+ response to membrane depolarization are greater in cells expressing mutant α‐synuclein. The membrane permeable Ca2+ chelator BAPTA‐AM significantly protected the cells against oxidative stress, whereas neither l‐type (nifedipine) nor N‐type (ω‐conotoxin‐GVIA) Ca2+ channel blockers protected the cells. These findings suggest that the high membrane ion permeability caused by mutant α‐synuclein may contribute to the degeneration of neurons in PD.

Clathrin Assembly Protein AP180 and CALM Differentially Control Axogenesis and Dendrite Outgrowth in Embryonic Hippocampal Neurons

Emerging data suggest that, much like epithelial cells, the polarized growth of neurons requires both the secretory and endocytic pathways. The clathrin assembly proteins AP180 and CALM (clathrin assembly lymphoid myeloid protein) are known to be involved in clathrin-mediated endocytosis, but their roles in mammalian neurons and, in particular, in developmental processes before synaptogenesis are unknown. Here we provide evidence that AP180 and CALM play critical roles in establishing the polarity and controlling the growth of axons and dendrites in embryonic hippocampal neurons. Knockdown of AP180 primarily impairs axonal development, whereas reducing CALM levels results in dendritic dystrophy. Conversely, neurons that overexpress AP180 or CALM generate multiple axons. Ultrastructural analysis shows that CALM affiliates with a wider range of intracellular trafficking organelles than does AP180. Functional analysis shows that endocytosis is reduced in both AP180-deficient and CALM-deficient neurons. Additionally, CALM-deficient neurons show disrupted secretory transport. Our data demonstrate previously unknown functions for AP180 and CALM in intracellular trafficking that are essential in the growth of neurons.

Alteration in calcium channel properties is responsible for the neurotoxic action of a familial frontotemporal dementia tau mutation

Tau, a microtubule binding protein, is not only a major component of neurofibrillary tangles in Alzheimers disease, but also a causative gene for hereditary frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP‐17). We show here that an FTDP‐17 tau mutation (V337M) in SH‐SY5Y cells reduces microtubule polymerization, increases voltage‐dependent calcium current (ICa) density, and decreases ICa rundown. The reduced rundown of ICa by V337M was significantly inhibited by nifedipine (L‐type Ca channel blocker), whereas ω‐conotoxin GVIA (N‐type Ca channel blocker) showed smaller effects, indicating that tau mutations affect L‐type calcium channel activity. The depolarization‐induced increase in intracellular calcium was also significantly augmented by the V337M tau mutation. Treatment with a microtubule polymerizing agent (taxol), an adenylyl cyclase inhibitor, or a protein kinase A (PKA) inhibitor, counteracted the effects of mutant tau on ICa. Taxol also attenuated the Ca2+ response to depolarization in cells expressing mutant tau. Apoptosis in SH‐SY5Y cells induced by serum deprivation was exacerbated by the V337M mutation, and nifedipine, taxol, and a PKA inhibitor significantly protected cells against apoptosis. Our results indicate that a tau mutation which decreases its microtubule‐binding ability augments calcium influx by depolymerizing microtubules and activating adenylyl cyclase and PKA.

Synaptic distribution of the endocytic accessory proteins AP180 and CALM

Clathrin‐coated vesicles mediate a variety of endocytosis pathways in cells, including endocytic events at synapses. AP180 and clathrin assembly lymphoid myeloid leukemia protein (CALM) are clathrin accessory proteins that promote the formation of clathrin‐coated vesicles. Both proteins bind to membrane lipids through their epsin N‐terminal homology domains and interact with clathrin and related protein components through their carboxyl‐terminal peptide motifs. We examine their neuronal expression and synaptic distribution. We show that both proteins are detected in synapses but demonstrate different distribution patterns. AP180 is located predominantly in presynaptic profiles, whereas CALM is found nonselectively in pre‐ and postsynaptic profiles and also in perisynaptic processes. These observations reveal an unexpected relationship between AP180 and the presumed non‐neuronal homologue CALM. We propose that both AP180 and CALM function as endocytic accessory proteins at synapses, but each may regulate distinct clathrin pathways. J. Comp. Neurol. 481:58–69, 2005. Published 2004 Wiley‐Liss, Inc.

Communication breakdown: The impact of ageing on synapse structure

Impaired synaptic plasticity is implicated in the functional decline of the nervous system associated with ageing. Understanding the structure of ageing synapses is essential to understanding the functions of these synapses and their role in the ageing nervous system. In this review, we summarize studies on ageing synapses in vertebrates and invertebrates, focusing on changes in morphology and ultrastructure. We cover different parts of the nervous system, including the brain, the retina, the cochlea, and the neuromuscular junction. The morphological characteristics of aged synapses could shed light on the underlying molecular changes and their functional consequences.

Aging and longevity in the simplest animals and the quest for immortality

Here we review the examples of great longevity and potential immortality in the earliest animal types and contrast and compare these to humans and other higher animals. We start by discussing aging in single-celled organisms such as yeast and ciliates, and the idea of the immortal cell clone. Then we describe how these cell clones could become organized into colonies of different cell types that lead to multicellular animal life. We survey aging and longevity in all of the basal metazoan groups including ctenophores (comb jellies), sponges, placozoans, cnidarians (hydras, jellyfish, corals and sea anemones) and myxozoans. Then we move to the simplest bilaterian animals (with a head, three body cell layers, and bilateral symmetry), the two phyla of flatworms. A key determinant of longevity and immortality in most of these simple animals is the large numbers of pluripotent stem cells that underlie the remarkable abilities of these animals to regenerate and rejuvenate themselves. Finally, we discuss briefly the evolution of the higher bilaterians and how longevity was reduced and immortality lost due to attainment of greater body complexity and cell cycle strategies that protect these complex organisms from developing tumors. We also briefly consider how the evolution of multiple aging-related mechanisms/pathways hinders our ability to understand and modify the aging process in higher organisms.

Stromal factors SDF1α, sFRP1, and VEGFD induce dopaminergic neuron differentiation of human pluripotent stem cells.

Human embryonic stem cell (hESC)‐derived dopaminergic (DA) neurons hold potential for treating Parkinsons disease (PD) through cell replacement therapy. Generation of DA neurons from hESCs has been achieved by coculture with the stromal cell line PA6, a source of stromal cell‐derived inducing activity (SDIA). However, the factors produced by stromal cells that result in SDIA are largely undefined. We previously reported that medium conditioned by PA6 cells can generate functional DA neurons from NTera2 human embryonal carcinoma stem cells. Here we show that PA6‐conditioned medium can induce DA neuronal differentiation in both NTera2 cells and the hESC I6 cell line. To identify the factor(s) responsible for SDIA, we used large‐scale microarray analysis of gene expression combined with mass spectrometric analysis of PA6‐conditioned medium (CM). The candidate factors, hepatocyte growth factor (HGF), stromal cell‐derived factor‐1 α (SDF1α), secreted frizzled‐related protein 1 (sFRP1), and vascular endothelial growth factor D (VEGFD) were identified, and their concentrations in PA6 CM were established by immunoaffinity capillary electrophoresis. Upon addition of SDF1α, sFRP1, and VEGFD to the culture medium, we observed an increase in the number of cells expressing tyrosine hydroxylase (a marker for DA neurons) and βIII‐tubulin (a marker for immature neurons) in both the NTera2 and I6 cell lines. These results indicate that SDF1α, sFRP1, and VEGFD are major components of SDIA and suggest the potential use of these defined factors to elicit DA differentiation of pluripotent human stem cells for therapeutic intervention in PD.

Sonic hedgehog regulates presynaptic terminal size, ultrastructure and function in hippocampal neurons

Summary Sonic hedgehog (Shh) signaling is essential to the patterning of the embryonic neural tube, but its presence and function in the postmitotic differentiated neurons in the brain remain largely uncharacterized. We recently showed that Shh and its signaling components, Patched and Smoothened, are expressed in postnatal and adult hippocampal neurons. We have now examined whether Shh signaling has a function in these neurons. Using cultured hippocampal neurons as a model system, we found that presynaptic terminals become significantly larger in response to the application of Shh. Ultrastructural examination confirmed the enlarged presynaptic profiles and also revealed variable increases in the size of synaptic vesicles, with a resulting loss of uniformity. Furthermore, electrophysiological analyses showed significant increases in the frequency, but not the amplitude, of spontaneous miniature excitatory postsynaptic currents (mEPSCs) in response to Shh, providing functional evidence of the selective role of Shh in presynaptic terminals. Thus, we conclude that Shh signaling regulates the structure and functional properties of presynaptic terminals of hippocampal neurons.

Heterogeneity of endocytic proteins: distribution of clathrin adaptor proteins in neurons and glia

Clathrin adaptor protein (AP)180 is a synaptic protein that regulates the assembly of clathrin-coated vesicles. Several endocytic proteins including AP2, CALM, and epsin 1 have functions or molecular structures similar to AP180. We determined if AP180 associates with functional synapses in cultured hippocampal neurons. We also compared the expression pattern of AP180 with the other endocytic proteins. The distribution of AP180 corresponded with the synaptic vesicle-associated protein synapsin I, and with functional presynaptic terminals labeled with the styryl dye FM1-43. Synaptic AP2 colocalized with AP180, but the distribution of AP2 was not limited to synapses of neurons and it was also expressed in glia. CLAM and epsin 1 immunoreactivities were also detected in both neurons and glia. Unlike AP180, the neuronal immunoreactivity of CALM was not intense in the synaptic puncta. Epsin 1 immunoreactivity was found in both synaptic and extrasynaptic sites, and its synaptic distribution only partially overlapped with that of AP180. These results support roles for AP180 in synaptic function in neurons. The findings also provide information on the distribution of AP2, CALM, and epsin 1 in cells of the nervous system that suggest different roles for these endocytic proteins in the biology of these cells.