High sphingomyelin levels induce lysosomal damage and autophagy dysfunction in Niemann Pick disease type A.

Niemann Pick disease type A (NPA), which is caused by loss of function mutations in the acid sphingomyelinase (ASM) gene, is a lysosomal storage disorder leading to neurodegeneration. Yet, lysosomal dysfunction and its consequences in the disease are poorly characterized. Here we show that undegraded molecules build up in neurons of acid sphingomyelinase knockout mice and in fibroblasts from NPA patients in which autophagolysosomes accumulate. The latter is not due to alterations in autophagy initiation or autophagosome-lysosome fusion but because of inefficient autophago-lysosomal clearance. This, in turn, can be explained by lysosomal membrane permeabilization leading to cytosolic release of Cathepsin B. High sphingomyelin (SM) levels account for these effects as they can be induced in control cells on addition of the lipid and reverted on SM-lowering strategies in ASM-deficient cells. These results unveil a relevant role for SM in autophagy modulation and characterize autophagy anomalies in NPA, opening new perspectives for therapeutic interventions.

The XLMR gene ACSL4 plays a role in dendritic spine architecture.

ACSL4 is a gene involved in non-syndromic X-linked mental retardation. It encodes for a ubiquitous protein that adds coenzyme A to long-chain fatty acids, with a high substrate preference for arachidonic acid. It presents also a brain-specific isoform deriving from an alternative splicing and containing 41 additional N-terminal amino acids. To start to unravelling the link between ACSL4 and mental retardation, we have performed molecular and cell biological studies. By retro-transcription polymerase chain reaction analyses we identified a new transcript with a shorter 5'-UTR region. By immunofluorescence microscopy in embryonic rat hippocampal neurons we report that ACSL4 is associated preferentially to endoplasmic reticulum tubules. ACSL4 knockdown by siRNAs in hippocampal neurons indicated that this protein is largely dispensable for these cells' gross architectural features (i.e. axonal and dendritic formation and final length) yet it is required for the presence of normal spines. In fact, reduced levels of ACSL4 led to a significant reduction in dendritic spine density and an alteration in spine/filopodia distribution. The possible mechanisms behind this phenotype are discussed.

Plasma membrane ganglioside sialidase regulates axonal growth and regeneration in hippocampal neurons in culture.

It has been long recognized that the ganglioside GM1 plays a role in axonal growth and neuronal differentiation. However, the involvement of plasma membrane GM1 has been difficult to elucidate. This is possible now thanks to the recent cloning of plasma membrane ganglioside sialidase (PMGS), the enzyme responsible for the localized hydrolysis of oligosialogangliosides into GM1. In this work we show that PMGS mRNA and protein levels are high at early developmental stages of the hippocampus and low in adulthood both in vivo and in vitro. We also demonstrate that inhibition of PMGS activity blocks axonal elongation, whereas the increase in PMGS activity dramatically enhances axon growth and accelerates the polarization of cytoskeletal proteins. Finally, we show that axotomy close to the cell body in PMGS overexpressing neurons results in the regrowth of the original axon instead of randomly, as is the case in control neurons. In all, these results imply that PMGS activity through the modulation of GM1 surface levels is an important component of the machinery controlling axonal growth. We hypothesize that increasing PMGS activity in the adult nervous system may be useful to improve regeneration after nerve damage.

The Ras-like GTPase Gem is involved in cell shape remodelling and interacts with the novel kinesin-like protein KIF9.

Gem belongs to the Rad/Gem/Kir (RGK) subfamily of Ras-related GTPases, which also comprises Rem, Rem2 and Ges. The RGK family members Ges and Rem have been shown to produce endothelial cell sprouting and reorganization of the actin cytoskeleton upon overexpression. Here we show that high intracellular Gem levels promote profound changes in cell morphology and we investigate how this phenotype arises dynamically. We also show that this effect requires intact microtubules and microfilaments, and that Gem is associated with both cytoskeletal components. In order to investigate the mechanisms of Gem recruitment to the cytoskeleton, we performed a yeast two-hybrid screen and identified a novel kinesin-like protein, termed KIF9, as a new Gem interacting partner. We further show that Gem and KIF9 interact by co-immunoprecipitation. Furthermore, Gem and KIF9 display identical patterns of gene expression in different tissues and developmental stages. The Gem- KIF9 interaction reported here is the first molecular link between RGK family members and the microtubule cytoskeleton.

Endocytosis of NBD-sphingolipids in neurons: exclusion from degradative compartments and transport to the Golgi complex.

Sphingolipids are abundant constituents of neuronal membranes that have been implicated in intracellular signaling, neurite outgrowth and differentiation. Differential localization and trafficking of lipids to membrane domains contribute to the specialized functions. In non-neuronal cultured cell lines, plasma membrane short-chain sphingomyelin and glucosylceramide are recycled via endosomes or sorted to degradative compartments. However, depending on cell type and lipid membrane composition, short-chain glucosylceramide can also be diverted to the Golgi complex. Here, we show that NBD-labeled glucosylceramide and sphingomyelin are transported from the plasma membrane to the Golgi complex in cultured rat hippocampal neurons irrespective of the stage of neuronal differentiation. Golgi complex localization was confirmed by colocalization and Golgi disruption studies, and importantly did not result from conversion of NBD-glucosylceramide or NBD-sphingomyelin to NBD-ceramide. Double-labeling experiments with transferrin or wheat-germ agglutinin showed that NBD-sphingolipids are first internalized to early/recycling endosomes, and subsequently transported to the Golgi complex. The internalization of these two sphingolipid analogs was energy and temperature dependent, and their intracellular transport was insensitive to the NBD fluorescence quencher sodium dithionite. These results indicate that vesicles mediate the transport of internalized NBD-glucosylceramide and NBD-sphingomyelin to the Golgi complex.

Differentiated neurons retain the capacity to generate axons from dendrites.

Cutting the axon of a morphologically polarized neuron (stage 3) close to the cell body causes another neurite to grow as an axon [1-3]. Stage 3 neurons still lack molecular segregation of axonal and dendritic proteins, however. Axonal and dendritic compartments acquire their distinct composition at stage 4 (4-5days in culture), when proteins such as the microtubule-associated protein 2 (MAP-2) and the glutamate receptor subunit GluR1 localize to the dendrites and disappear from the axon [4,5]. We investigated whether cultured hippocampal neurons retained axon/dendrite plasticity after axons and dendrites have created their distinct cytoskeletal architecture and acquired their specific membrane composition. We found that axotomy of stage 4 neurons transformed a dendrite into an axon. Using axonal and dendritic markers, we tested whether cytoskeletal changes could cause similar transformations, and found that actin depolymerization induced multiple axons in unpolarized neurons. Moreover, depletion of actin filaments from both morphologically and molecularly polarized cells also resulted in the growth of multiple axons from pre-existing dendrites. These results imply that dendrites retain the potential to become axons even after molecular segregation has occurred and that the dendritic fate depends on the integrity of the actin cytoskeleton.

Microtubule-associated protein-2 located in growth regions of rat hippocampal neurons is highly phosphorylated at its proline-rich region.

Neuronal morphogenesis is regulated, among other factors, by microtubule-associated proteins (MAPs). A family of these proteins, MAP2, which is very abundant in the mammalian nervous system, has been associated with the formation of neurites at early developmental stages and with the dendritic scaffold upon maturation. The function of MAP2 is regulated by its phosphorylation state. One of the phosphorylation sites that has been described is located in the proline-rich region of the protein. It comprises of the residues 1616-1626 and is specifically recognized by the antibody 305. However, little is known about the functional consequences of its modification in vivo. To gain insight into this, we have analysed the expression levels and intracellular distribution of MAP2 phosphorylated at this site (MAP2-P), in primary cultures of rat hippocampal neurons at different developmental stages. Western blot analysis of hippocampal neuron protein extracts revealed that the ratio of MAP2-P:MAP2 was 4:1 at early developmental stages and became 1:4 at later developmental stages, suggesting a role of such phosphorylated forms of the protein in neuritogenesis. Consistent with this view, immunofluorescence microscopy analysis showed that the ratio MAP2-P:MAP2 was 2 in the neurite growth cones, sites where net elongation takes place. A higher presence of phosphorylated MAP2 was observed in growth regions with higher levels of microfilaments, which may be related with the growth region stability. Indeed, when growth-cone collapse was induced in hippocampal neurons after cytochalasin D treatment, which depolymerizes microfilaments, the ratio MAP2-P:MAP2 in these growing regions decreased down to 1. Finally, acceleration of neuronal maturation induced by the activation of glutamate-receptors triggered a dramatic decrease in the phosphorylation of MAP2 at the site recognized by antibody 305. From these results we suggest that the phosphorylation of MAP2 at its proline-rich region is an important event during neuritogenesis.

Establishment of neuronal polarity: lessons from cultured hippocampal neurons.

In recent years we have learned a great deal about the molecular mechanisms underlying axonal elongation and navigation and the manner in which extracellular signals modify a growth cone's course of action. Yet, the mechanisms responsible for the earlier events of axonal and dendritic generation are just beginning to be understood. The recent advances in this exciting field highlight the importance of studies of cell migration and axonal elongation for our current understanding of the establishment of neuronal polarity.

EEA1, a tethering protein of the early sorting endosome, shows a polarized distribution in hippocampal neurons, epithelial cells, and fibroblasts.

EEA1 is an early endosomal Rab5 effector protein that has been implicated in the docking of incoming endocytic vesicles before fusion with early endosomes. Because of the presence of complex endosomal pathways in polarized and nonpolarized cells, we have examined the distribution of EEA1 in diverse cell types. Ultrastructural analysis demonstrates that EEA1 is present on a subdomain of the early sorting endosome but not on clathrin-coated vesicles, consistent with a role in providing directionality to early endosomal fusion. Furthermore, EEA1 is associated with filamentous material that extends from the cytoplasmic surface of the endosomal domain, which is also consistent with a tethering/docking role for EEA1. In polarized cells (Madin-Darby canine kidney cells and hippocampal neurons), EEA1 is present on a subset of "basolateral-type" endosomal compartments, suggesting that EEA1 regulates specific endocytic pathways. In both epithelial cells and fibroblastic cells, EEA1 and a transfected apical endosomal marker, endotubin, label distinct endosomal populations. Hence, there are at least two distinct sets of early endosomes in polarized and nonpolarized mammalian cells. EEA1 could provide specificity and directionality to fusion events occurring in a subset of these endosomes in polarized and nonpolarized cells.

Involvement of the proximal C terminus of the AMPA receptor subunit GluR1 in dendritic sorting.

Studies on dendritic sorting of transmembrane proteins in hippocampal neurons in culture have shown that these cells use similar mechanisms as epithelial cells to sort transmembrane proteins to the basolateral membrane domain. However, information is still scarce with regard to which amino acidic sequences are required for dendritic sorting in neurons. The glutamate receptor 1 (GluR1) subunit of the AMPA receptor is present on the dendritic compartment of hippocampal neurons in culture. To identify the GluR1 sorting signal responsible for dendritic targeting, we have expressed the wild-type GluR1, a deletion mutant in the C-terminal cytoplasmic tail, and chimeric GluR1 proteins in hippocampal neurons using a calcium phosphate transfection method. The recombinant full-length GluR1 is polarized to the dendritic domain. Truncated GluR1 with a deletion of the C-terminal cytoplasmic tail is still delivered to the somatodendritic domain. However a chimeric protein made of the luminal and transmembrane domain of the influenza virus hemagglutinin (HA) fused to the GluR1 C-terminal cytoplasmic tail (HaemR1) is detected in the somatodendritic domain. This finding indicates that the GluR1 C-terminal cytoplasmic tail contains a dendritic sorting signal, which redirects the axonal or axonal-dendritic protein HA to the dendritic compartment exclusively. Deletion analysis of HaemR1 shows that the proximal segment of the GluR1 C-terminal cytoplasmic tail contains a novel dendritic sorting signal.

Axonal membrane proteins are transported in distinct carriers: a two-color video microscopy study in cultured hippocampal neurons.

Neurons transport newly synthesized membrane proteins along axons by microtubule-mediated fast axonal transport. Membrane proteins destined for different axonal subdomains are thought to be transported in different transport carriers. To analyze this differential transport in living neurons, we tagged the amyloid precursor protein (APP) and synaptophysin (p38) with green fluorescent protein (GFP) variants. The resulting fusion proteins, APP-yellow fluorescent protein (YFP), p38-enhanced GFP, and p38-enhanced cyan fluorescent protein, were expressed in hippocampal neurons, and the cells were imaged by video microscopy. APP-YFP was transported in elongated tubules that moved extremely fast (on average 4.5 micrometer/s) and over long distances. In contrast, p38-enhanced GFP-transporting structures were more vesicular and moved four times slower (0.9 micrometer/s) and over shorter distances only. Two-color video microscopy showed that the two proteins were sorted to different carriers that moved with different characteristics along axons of doubly transfected neurons. Antisense treatment using oligonucleotides against the kinesin heavy chain slowed down the long, continuous movement of APP-YFP tubules and increased frequency of directional changes. These results demonstrate for the first time directly the sorting and transport of two axonal membrane proteins into different carriers. Moreover, the extremely fast-moving tubules represent a previously unidentified type of axonal carrier.

Chemical stimulation of synaptosomes modulates alpha -Ca2+/calmodulin-dependent protein kinase II mRNA association to polysomes.

The presence of specific mRNAs in dendrites and at synapses is well established, but a direct and reliable demonstration that they are associated with polysomes is still missing. To address this point we analyzed the polysomal association of the mRNAs for the alpha-subunit of Ca(2+)/calmodulin-dependent protein kinase II (alpha-CaMKII), for type 1 inositol 1,4,5-trisphosphate receptor (InsP3R1) and for the activity-regulated cytoskeleton-associated protein (Arc) in a synaptosomal preparation devoid of contaminating material from neuronal and glial perikarya. We show that a fraction of alpha-CaMKII, InsP3R1, and Arc mRNAs present in synaptosomes is indeed associated with polysomes. Moreover, we show that polysomal association of alpha-CaMKII mRNA, but not InsP3R1 and Arc mRNAs, increases with depolarization of the synaptosomal membrane. Finally, we show that the synthesis of alpha-CaMKII protein increases with stimulation. Dendritic mRNA recruitment onto polysomes in response to synaptic stimulation might represent one of the mechanisms underlying the processes of learning and memory.

Polarized distribution of glycine transporter isoforms in epithelial and neuronal cells.

Asymmetrical distribution of Na(+)- and Cl(-)-dependent neurotransmitter transporters on the cell surface of polarized cells seems to be a generalized feature in this gene family. In the present study we analyzed the subcellular distribution of the various isoforms of the glycine transporters GLYT1 and GLYT2 after heterologous expression in polarized MDCK cells and in hippocampal neurons. Our results indicate that glycine transporters are asymmetrically distributed in an isoform- and cell-type-specific manner. GLYT1b is localized in the basolateral and somatodendritic domains of MDCK cells and neurons, respectively. However, GLYT1a is somatodendritic in neurons but is predominantly expressed in the apical surface of MDCK cells. The two isoforms of GLYT2 (GLYT2a and GLYT2b) are found at the apical surface in epithelial cells but are uniformly distributed in neurons. By using site-directed mutagenesis we have been able to identify signals for basolateral/somatodendritic localization in the amino-terminal region of GLYT1 and in two dileucine motifs located in the carboxyl tail of this protein. These results contribute to defining the mechanisms of asymmetrical distribution of transporters on the cell surface of polarized cells.

Changes in membrane trafficking and actin dynamics during axon formation in cultured hippocampal neurons.

Neurons begin to polarize when one of the neurites becomes the axon. Hippocampal neurons in cell culture have a sharp transition between their unpolarized and polarized stage revealed by the rapid growth of the future axon. Recent progress shows that both a cytoplasmic membrane flow and actin dynamics govern axon formation, and thereby initial neuronal polarization. We here review these mechanisms, evaluate their physiological role, and show similarities to the transient polarization of migrating fibroblasts. Finally, we present a model how actin dynamics and vectorial membrane flow may interact to achieve axon formation.

Brain plasmin enhances APP alpha-cleavage and Abeta degradation and is reduced in Alzheimer's disease brains.

The proteolytic processing of amyloid precursor protein (APP) has been linked to sphingolipid-cholesterol microdomains (rafts). However, the raft proteases that may be involved in APP cleavage have not yet been identified. In this work we present evidence that the protease plasmin is restricted to rafts of cultured hippocampal neurons. We also show that plasmin increases the processing of human APP preferentially at the alpha-cleavage site, and efficiently degrades secreted amyloidogenic and non-amyloidogenic APP fragments. These results suggest that brain plasmin plays a preventive role in APP amyloidogenesis. Consistently, we show that brain tissue from Alzheimer's disease patients contains reduced levels of plasmin, implying that plasmin downregulation may cause amyloid plaque deposition accompanying sporadic Alzheimer's disease.

Axonal transport of synucleins is mediated by all rate components.

Synucleins are abundant nerve terminal proteins of hitherto unknown function. In diseases with Lewy bodies, human alpha-synuclein concentrates in these lesions in the cell body and mutations in alpha-synuclein lead to heritable Parkinson's disease with Lewy bodies. This indicates that changes in the normal metabolism and axonal transport of alpha-synuclein is perturbed in these diseases. To investigate the normal axonal transport of synucleins we studied the rat visual system by nerve crush operations and metabolic labelling of the retinal ganglion cells followed by immunoprecipitation of nerve segments. We found by immunofluorescence microscopy of the crush-operated nerves that synucleins are transported by fast antero- and retrograde transport and colocalize with synaptophysin and SNAP-25 around the lesion. The metabolic labelling studies demonstrated that synucleins were moved through the nerve with all the rate components, the fast component and the slow components a and b, with component b predominating. Two-dimensional gel electrophoresis revealed that both alpha- and beta-synuclein migrate through the nerve by slow component b in a ratio of 2:1.

Expression of the CDH1-associated form of the anaphase-promoting complex in postmitotic neurons.

The anaphase-promoting complex/cyclosome (APC) is a tightly cell cycle-regulated ubiquitin-protein ligase that targets cyclin B and other destruction box-containing proteins for proteolysis at the end of mitosis and in G1. Recent work has shown that activation of the APC in mitosis depends on CDC20, whereas APC is maintained active in G1 via association with the CDC20-related protein CDH1. Here we show that the mitotic activator CDC20 is the only component of the APC ubiquitination pathway whose expression is restricted to proliferating cells, whereas the APC and CDH1 are also expressed in several mammalian tissues that predominantly contain differentiated cells, such as adult brain. Immunocytochemical analyses of cultured rat hippocampal neurons and of mouse and human brain sections indicate that the APC and CDH1 are ubiquitously expressed in the nuclei of postmitotic terminally differentiated neurons. The APC purified from brain contains all core subunits known from proliferating cells and is tightly associated with CDH1. Purified brain APC(CDH1) has a high cyclin B ubiquitination activity that depends less on the destruction box than on the activity of mitotic APC(CDC20). On the basis of these results, we propose that the functions of APC(CDH1) are not restricted to controlling cell-cycle progression but may include the ubiquitination of yet unidentified substrates in differentiated cells.

Microtubule-dependent recruitment of Staufen-green fluorescent protein into large RNA-containing granules and subsequent dendritic transport in living hippocampal neurons.

Dendritic mRNA transport and local translation at individual potentiated synapses may represent an elegant way to form synaptic memory. Recently, we characterized Staufen, a double-stranded RNA-binding protein, in rat hippocampal neurons and showed its presence in large RNA-containing granules, which colocalize with microtubules in dendrites. In this paper, we transiently transfect hippocampal neurons with human Staufen-green fluorescent protein (GFP) and find fluorescent granules in the somatodendritic domain of these cells. Human Stau-GFP granules show the same cellular distribution and size and also contain RNA, as already shown for the endogenous Stau particles. In time-lapse videomicroscopy, we show the bidirectional movement of these Staufen-GFP-labeled granules from the cell body into dendrites and vice versa. The average speed of these particles was 6.4 microm/min with a maximum velocity of 24. 3 microm/min. Moreover, we demonstrate that the observed assembly into granules and their subsequent dendritic movement is microtubule dependent. Taken together, we have characterized a novel, nonvesicular, microtubule-dependent transport pathway involving RNA-containing granules with Staufen as a core component. This is the first demonstration in living neurons of movement of an essential protein constituent of the mRNA transport machinery.

Maturation of the axonal plasma membrane requires upregulation of sphingomyelin synthesis and formation of protein-lipid complexes.

Neuronal maturation is a gradual process; first axons and dendrites are established as distinct morphological entities; next the different intracellular organization of these processes occurs; and finally the specialized plasma membrane domains of these two compartments are formed. Only when this has been accomplished does proper neuronal function take place. In this work we present evidence that the correct distribution of a class of axonal membrane proteins requires a mechanism which involves formation of protein-lipid (sphingomyelin/cholesterol) detergent-insoluble complexes (DIGs). Using biochemistry and immunofluorescence microscopy we now show that in developing neurons the randomly distributed Thy-1 does not interact with lipids into DIGs (in fully developed neurons the formation of such complexes is essential for the correct axonal targeting of this protein). Using lipid mass spectrometry and thin layer chromatography we show that the DIG lipid missing in the developing neurons is sphingomyelin, but not cholesterol or glucosylceramide. Finally, by increasing the intracellular levels of sphingomyelin in the young neurons the formation of Thy-1/DIGs was induced and, consistent with a role in sorting, proper axonal distribution was facilitated. These results emphasize the role of sphingomyelin in axonal, and therefore, neuronal maturation.

The role of local actin instability in axon formation.

The role of localized instability of the actin network in specifying axonal fate was examined with the use of rat hippocampal neurons in culture. During normal neuronal development, actin dynamics and instability polarized to a single growth cone before axon formation. Consistently, global application of actin-depolymerizing drugs and of the Rho-signaling inactivator toxin B to nonpolarized cells produced neurons with multiple axons. Moreover, disruption of the actin network in one individual growth cone induced its neurite to become the axon. Thus, local instability of the actin network restricted to a single growth cone is a physiological signal specifying neuronal polarization.