Biomedical subjects

Carlos G Dotti

Publications and source records attributed to Carlos G Dotti.

At least 19 recordsLinked to original sources

PubMed2007

Transmitting on actin: synaptic control of dendritic architecture.

Excitatory synaptic transmission in the central nervous system mainly takes place at dendritic spines, highly motile protrusions on the dendritic surface. Depending on the stimuli received, dendritic spines undergo rapid actin-based changes in their morphology. This plasticity appears to involve signaling through numerous proteins that control the organization of the actin cytoskeleton (actin regulators). At least in part, recruitment and activation of these depends on neurotransmitter receptors at the post-synapse, which directly link neurotransmission to changes in dendritic spine architecture. However, other, non-neurotransmitter-receptors present at dendritic spines also participate. It is likely that several receptor types can control the activity of a single actin-regulatory pathway and it is the complex integration of numerous signals that determines the overall architecture of a dendritic spine.

Actins↗

PubMed2006

Amyloid excess in Alzheimer's disease: what is cholesterol to be blamed for?

A link between alterations in cholesterol homeostasis and Alzheimer's disease (AD) is nowadays widely accepted. However, the molecular mechanism/s underlying such link remain unclear. Numerous experimental evidences support the view that changes in neuronal membrane cholesterol levels and/or subcellular distribution determine the aberrant accumulation of the amyloid peptide in the disease. Still, this view comes from rather contradictory data supporting the existence of either high or low brain cholesterol content. This is of particular concern considering that therapeutical strategies aimed to reduce cholesterol levels are already being tested in humans. Here, we review the molecular mechanisms proposed and discuss the perspectives they open.

Alzheimer Disease↗

PubMed2006

gamma-cleavage-independent functions of presenilin, nicastrin, and Aph-1 regulate cell-junction organization and prevent tau toxicity in vivo.

Genetic analysis of familial Alzheimer's disease has revealed that mutations in the gamma-secretase enzyme presenilin promote toxic Abeta secretion; however, presenilin mutations might also influence tau hyperphosphorylation and neurodegeneration through gamma-secretase-independent mechanisms. To address this possibility and determine whether other components of the gamma-secretase complex possess similar regulatory functions, we analyzed the roles of presenilin, nicastrin, and aph-1 in a Drosophila model for tau-induced neurodegeneration. Here, we show that presenilin and nicastrin prevent tau toxicity by modulating the PI3K/Akt/GSK3beta phosphorylation pathway, whereas aph-1 regulates aPKC/PAR-1 activities. Moreover, we found that these transmembrane proteins differentially regulate the intracellular localization of GSK3beta and aPKC at cell junctions. Inhibition of gamma-secretase activity neither interfered with these kinase pathways nor induced aberrant tau phosphorylation. These results establish new in vivo molecular functions for the three components of the gamma-secretase complex and reveal a different mechanism that might contribute to neuronal degeneration in Alzheimer's disease.

Alzheimer Disease↗

PubMed2006

Rac3-induced neuritogenesis requires binding to Neurabin I.

Rac3, a neuronal GTP-binding protein of the Rho family, induces neuritogenesis in primary neurons. Using yeast two-hybrid analysis, we show that Neurabin I, the neuronal F-actin binding protein, is a direct Rac3-interacting molecule. Biochemical and light microscopy studies indicate that Neurabin I copartitions and colocalizes with Rac3 at the growth cones of neurites, inducing Neurabin I association to the cytoskeleton. Moreover, Neurabin I antisense oligonucleotides abolish Rac3-induced neuritogenesis, which in turn is rescued by exogenous Neurabin I but not by Neurabin I mutant lacking the Rac3-binding domain. These results show that Neurabin I mediates Rac3-induced neuritogenesis, possibly by anchoring Rac3 to growth cone F-actin.

Actins↗

PubMed2006

Localized recruitment and activation of RhoA underlies dendritic spine morphology in a glutamate receptor-dependent manner.

Actin is the major cytoskeletal source of dendritic spines, which are highly specialized protuberances on the neuronal surface where excitatory synaptic transmission occurs (Harris, K.M., and S.B. Kater. 1994. Annu. Rev. Neurosci. 17:341-371; Yuste, R., and D.W. Tank. 1996. Neuron. 16:701-716). Stimulation of excitatory synapses induces changes in spine shape via localized rearrangements of the actin cytoskeleton (Matus, A. 2000. Science. 290:754-758; Nagerl, U.V., N. Eberhorn, S.B. Cambridge, and T. Bonhoeffer. 2004. Neuron. 44:759-767). However, what remains elusive are the precise molecular mechanisms by which different neurotransmitter receptors forward information to the underlying actin cytoskeleton. We show that in cultured hippocampal neurons as well as in whole brain synaptosomal fractions, RhoA associates with glutamate receptors (GluRs) at the spine plasma membrane. Activation of ionotropic GluRs leads to the detachment of RhoA from these receptors and its recruitment to metabotropic GluRs. Concomitantly, this triggers a local reduction of RhoA activity, which, in turn, inactivates downstream kinase RhoA-specific kinase, resulting in restricted actin instability and dendritic spine collapse. These data provide a direct mechanistic link between neurotransmitter receptor activity and the changes in spine shape that are thought to play a crucial role in synaptic strength.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed2006

The role of seladin-1/DHCR24 in cholesterol biosynthesis, APP processing and Abeta generation in vivo.

The cholesterol-synthesizing enzyme seladin-1, encoded by the Dhcr24 gene, is a flavin adenine dinucleotide-dependent oxidoreductase and regulates responses to oncogenic and oxidative stimuli. It has a role in neuroprotection and is downregulated in affected neurons in Alzheimer's disease (AD). Here we show that seladin-1-deficient mouse brains had reduced levels of cholesterol and disorganized cholesterol-rich detergent-resistant membrane domains (DRMs). This was associated with inefficient plasminogen binding and plasmin activation, the displacement of beta-secretase (BACE) from DRMs to APP-containing membrane fractions, increased beta-cleavage of APP and high levels of Abeta peptides. In contrast, overexpression of seladin-1 increased both cholesterol and the recruitment of DRM components into DRM fractions, induced plasmin activation and reduced both BACE processing of APP and Abeta formation. These results establish a role of seladin-1 in the formation of DRMs and suggest that seladin-1-dependent cholesterol synthesis is involved in lowering Abeta levels. Pharmacological enhancement of seladin-1 activity may be a novel Abeta-lowering approach for the treatment of AD.

Alzheimer Disease↗

PubMed2005

Proper axonal distribution of PrP(C) depends on cholesterol-sphingomyelin-enriched membrane domains and is developmentally regulated in hippocampal neurons.

Defects in cellular localization and trafficking seem to facilitate the conversion of PrP(C) into the disease-associated form, PrP(Sc). Still, it is not clear to which membrane compartments PrP(C) localizes in hippocampal neurons a population most affected in the prion disease. We here show that in developing hippocampal neurons in culture PrP(C) is equally distributed to all neurites yet enriched in growth cones. By contrast, in fully mature neurons PrP(C) is restricted to axons. The axonal distribution in mature stages is paralleled by the increased partitioning of PrP(C) into detergent-resistant cholesterol-sphingolipid-rich domains (DRMs). Consistent with a cause-effect mechanism, disruption of DRMs by sphingolipid or cholesterol depletion leads to the non-polarized distribution and impaired endocytosis of PrP(C). These results indicate that DRMs are essential for proper trafficking and distribution of PrP(C) at late stages of neuronal differentiation and that its function, at least in hippocampus, is restricted to the axonal domain.

Animals↗

PubMed2005

Centrosome localization determines neuronal polarity.

Neuronal polarization occurs shortly after mitosis. In neurons differentiating in vitro, axon formation follows the segregation of growth-promoting activities to only one of the multiple neurites that form after mitosis. It is unresolved whether such spatial restriction makes use of an intrinsic program, like during C. elegans embryo polarization, or is extrinsic and cue-mediated, as in migratory cells. Here we show that in hippocampal neurons in vitro, the axon consistently arises from the neurite that develops first after mitosis. Centrosomes, the Golgi apparatus and endosomes cluster together close to the area where the first neurite will form, which is in turn opposite from the plane of the last mitotic division. We show that the polarized activities of these organelles are necessary and sufficient for neuronal polarization: (1) polarized microtubule polymerization and membrane transport precedes first neurite formation, (2) neurons with more than one centrosome sprout more than one axon and (3) suppression of centrosome-mediated functions precludes polarization. We conclude that asymmetric centrosome-mediated dynamics in the early post-mitotic stage instruct neuronal polarity, implying that pre-mitotic mechanisms with a role in division orientation may in turn participate in this event.

Animals↗

PubMed2005

Tissue plasminogen activator mediates amyloid-induced neurotoxicity via Erk1/2 activation.

Tissue plasminogen activator (tPA) is the main activator of plasminogen into plasmin in the brain where it may have beneficial roles but also neurotoxic effects that could be plasmin dependent or not. Little is known about the substrates and pathways that mediate plasmin-independent tPA neurotoxicity. Here we show in primary hippocampal neurons that tPA promotes a catalytic-independent activation of the extracellular regulated kinase (Erk)1/2 signal transduction pathway through the N-methyl-D-aspartate receptor, G-proteins and protein kinase C. This results in GSK3 activation in a process that requires de novo synthesis of proteins, and leads to tau aberrant phosphorylation, microtubule destabilization and apoptosis. Similar effects are produced by amyloid aggregates in a tPA-dependent manner, as demonstrated by pharmacological treatments and in wt and tPA-/- mice neurons. Consistently, in Alzheimer's disease (AD) patients' brains, high levels of tPA colocalize with amyloid-rich areas, activated Erk1/2 and phosphorylated tau. This is the first demonstration of an intracellular pathway by which tPA triggers kinase activation, tau phosphorylation and neurotoxicity, suggesting a key role for this molecule in AD pathology.

Alzheimer Disease↗

PubMed2005

Asymmetric membrane ganglioside sialidase activity specifies axonal fate.

Axon specification triggers the polarization of neurons and requires the localized destabilization of filamentous actin. Here we show that plasma membrane ganglioside sialidase (PMGS) asymmetrically accumulates at the tip of one neurite of the unpolarized rat neuron, inducing actin instability. Suppressing PMGS activity blocks axonal generation, whereas stimulating it accelerates the formation of a single (not several) axon. PMGS induces axon specification by enhancing TrkA activity locally, which triggers phosphatidylinositol-3-kinase (PI3K)- and Rac1-dependent inhibition of RhoA signaling and the consequent actin depolymerization in one neurite only. Thus, spatial restriction of an actin-regulating molecular machinery, in this case a membrane enzymatic activity, before polarization is enough to determine axonal fate.

Actins↗

PubMed2005

Gamma-BAR, a novel AP-1-interacting protein involved in post-Golgi trafficking.

A novel peripheral membrane protein (2c18) that interacts directly with the gamma 'ear' domain of the adaptor protein complex 1 (AP-1) in vitro and in vivo is described. Ultrastructural analysis demonstrates a colocalization of 2c18 and gamma1-adaptin at the trans-Golgi network (TGN) and on vesicular profiles. Overexpression of 2c18 increases the fraction of membrane-bound gamma1-adaptin and inhibits its release from membranes in response to brefeldin A. Knockdown of 2c18 reduces the steady-state levels of gamma1-adaptin on membranes. Overexpression or downregulation of 2c18 leads to an increased secretion of the lysosomal hydrolase cathepsin D, which is sorted by the mannose-6-phosphate receptor at the TGN, which itself involves AP-1 function for trafficking between the TGN and endosomes. This suggests that the direct interaction of 2c18 and gamma1-adaptin is crucial for membrane association and thus the function of the AP-1 complex in living cells. We propose to name this protein gamma-BAR.

Adaptor Protein Complex 1↗

PubMed2005

The conflicting role of brain cholesterol in Alzheimer's disease: lessons from the brain plasminogen system.

Retrospective clinical studies indicate that individuals chronically treated with cholesterol synthesis inhibitors, statins, are at lower risk of developing AD (Alzheimer's disease). Moreover, treatment of guinea pigs with high doses of simvastatin or drastic reduction of cholesterol in cultured cells decrease Abeta (beta-amyloid peptide) production. These data sustain the concept that high brain cholesterol is responsible for Abeta accumulation in AD, providing the scientific support for the proposed use of statins to prevent this disease. However, a number of unresolved issues raise doubts that high brain cholesterol is to blame. First, it has not been shown that higher neuronal cholesterol increases Abeta production. Secondly, it has not been demonstrated that neurons in AD have more cholesterol than control neurons. On the contrary, the brains of AD patients show a specific down-regulation of seladin-1, a protein involved in cholesterol synthesis, and low membrane cholesterol was observed in hippocampal membranes of ApoE4 (apolipoprotein E4) AD cases. This effect was also evidenced by altered cholesterol-rich membrane domains (rafts) and raft-mediated functions, such as diminished generation of the Abeta-degrading enzyme plasmin. Thirdly, numerous genetic defects that cause neurodegeneration are due to defective cholesterol metabolism. Fourthly, in female mice, the most brain-permeant statin induces neurodegeneration and high amyloid production. Altogether, this evidence makes it difficult to accept that statins are beneficial through acting as brain cholesterol-synthesis inhibitors. It appears more likely that their advantageous role arises from improved brain oxygenation.

Alleles↗

PubMed2004

Neuronal membrane cholesterol loss enhances amyloid peptide generation.

Recent experimental and clinical retrospective studies support the view that reduction of brain cholesterol protects against Alzheimer's disease (AD). However, genetic and pharmacological evidence indicates that low brain cholesterol leads to neurodegeneration. This apparent contradiction prompted us to analyze the role of neuronal cholesterol in amyloid peptide generation in experimental systems that closely resemble physiological and pathological situations. We show that, in the hippocampus of control human and transgenic mice, only a small pool of endogenous APP and its beta-secretase, BACE 1, are found in the same membrane environment. Much higher levels of BACE 1-APP colocalization is found in hippocampal membranes from AD patients or in rodent hippocampal neurons with a moderate reduction of membrane cholesterol. Their increased colocalization is associated with elevated production of amyloid peptide. These results suggest that loss of neuronal membrane cholesterol contributes to excessive amyloidogenesis in AD and pave the way for the identification of the cause of cholesterol loss and for the development of specific therapeutic strategies.

Alzheimer Disease↗

PubMed2004

Hippocampal neurons and recombinant galectins as tools for systematic carbohydrate structure-function studies in neuronal differentiation.

Membrane glycoconjugates play a central role in neuronal interactions and regulation. To define the precise links between membrane polysaccharides and neuronal functions, two main requirements must be fulfilled: (1) the availability of molecular tools able to finely discriminate among carbohydrate structures and (2) the use of an experimental system suitable for systematic and quantitative studies of particular neuronal processes. In this work, we used two chicken proto-type galectins, i.e., monomeric CG-14 and dimeric CG-16, with very similar carbohydrate affinities, and rat hippocampal neurons in culture to quantitatively measure the involvement of carbohydrate-protein interaction in axonal growth and directionality, neurite sprouting and axon regenerative capacity after section. CG-16 potently stimulated axonal growth and guidance. Neurite sprouting was enhanced by immobilized CG-16 and, notably, reduced by lectin in solution. Overall, cross-linking CG-16 invariably excelled CG-14 in these functional assays, although none of them were able to improve axon regenerative capacity when compared to mammalian galectin-1. Our results demonstrate the potential of the experimental set-up to perform a systematic study of galectin functionality in neuronal differentiation. In view of the concept of the sugar code, the presented results indicate that biological effects triggered by glycan binding engaging an endogenous lectin can be modulated by carbohydrate affinity and/or by other factors like differential cross-linking capacity.

Animals↗

PubMed2004

RhoA, Rac1, and cdc42 intracellular distribution shift during hippocampal neuron development.

Differences in cytoskeleton organization are key determinants of the architecture and dynamics of axons and dendrites. This is most clearly illustrated by the diverse pools of microtubule-associated proteins in axons and dendrites. Whether similar polarized organization occurs for actin regulatory proteins remains to be determined. To address this issue, we analyzed the intracellular distribution of the Rho GTPases, RhoA, Rac1, and cdc42 in hippocampal neurons in culture. We report that all three Rho members are evenly distributed during the time of axon and dendrite sprouting. This is not the case in mature neurons, as RhoA enriches in dendrites, Rac1 in axons, and Cdc42 is equally abundant in both domains. Polarized segregation of the actin regulatory machinery in mature neurons might play an important role in axonal and dendritic architectural plasticity.

Actin Cytoskeleton↗

PubMed2004

Plasmin deficiency in Alzheimer's disease brains: causal or casual?

Substantial recent evidence suggests that defects in amyloid peptide degradation can be at the base of cases of sporadic Alzheimer's disease (AD). Among the discovered brain enzymes with the capacity to degrade amyloid peptide, the serine protease plasmin acquires special physiological relevance because of its low levels in areas of AD human brains with a high susceptibility to amyloid plaque accumulation. In this article we comment on a series of observations supporting the fact that plasmin paucity in the brain is not simply a secondary event in the disease but rather a primary defect in certain cases of sporadic AD. We also refer to recent data pointing to alterations in raft membrane domains and diminished membrane cholesterol as the underlying cause. Finally, we discuss the possibility that plasmin deficiency in the brain could lead to AD symptomatology because of amyloid aggregation and the triggering of cell death signaling cascades.

Alzheimer Disease↗

PubMed2003

Raft disorganization leads to reduced plasmin activity in Alzheimer's disease brains.

The serine protease plasmin can efficiently degrade amyloid peptide in vitro, and is found at low levels in the hippocampus of patients with Alzheimer's disease (AD). The cause of such paucity remains unknown. We show here that the levels of total brain plasminogen and plasminogen-binding molecules are normal in these brain samples, yet plasminogen membrane binding is greatly reduced. Biochemical analysis reveals that the membranes of these brains have a mild, still significant, cholesterol reduction compared to age-matched controls, and anomalous raft microdomains. This was reflected by the loss of raft-enriched proteins, including plasminogen-binding and -activating molecules. Using hippocampal neurons in culture, we demonstrate that removal of a similar amount of membrane cholesterol is sufficient to induce raft disorganization, leading to reduced plasminogen membrane binding and low plasmin activity. These results suggest that brain raft alterations may contribute to AD by rendering the plasminogen system inefficient.

Adaptor Protein Complex gamma Subunits↗

PubMed2003

Citron-N is a neuronal Rho-associated protein involved in Golgi organization through actin cytoskeleton regulation.

The actin cytoskeleton is best known for its role during cellular morphogenesis. However, other evidence suggests that actin is also crucial for the organization and dynamics of membrane organelles such as endosomes and the Golgi complex. As in morphogenesis, the Rho family of small GTPases are key mediators of organelle actin-driven events, although it is unclear how these ubiquitously distributed proteins are activated to regulate actin dynamics in an organelle-specific manner. Here we show that the brain-specific Rho-binding protein Citron-N is enriched at, and associates with, the Golgi apparatus of hippocampal neurons in culture. Suppression of the whole protein or expression of a mutant form lacking the Rho-binding activity results in dispersion of the Golgi apparatus. In contrast, high intracellular levels induce localized accumulation of RhoA and filamentous actin, protecting the Golgi from the rupture normally produced by actin depolymerization. Biochemical and functional analyses indicate that Citron-N controls actin locally by assembling together the Rho effector ROCK-II and the actin-binding, neuron-specific, protein Profilin-IIa (PIIa). Together with recent data on endosomal dynamics, our results highlight the importance of organelle-specific Rho modulators for actin-dependent organelle organization and dynamics.

Actin Cytoskeleton↗