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IQGAP1 is a scaffolding protein that regulates spine number. We now show a differential role for IQGAP1 domains in spine morphogenesis, in which a region of the N-terminus that promotes Arp2/3-mediated actin polymerization and branching stimulates spine head formation while a region that binds to Cdc42 and Rac is required for stalk extension. Conversely, IQGAP1 rescues spine deficiency induced by expression of dominant negative Cdc42 by stimulating formation of stubby spines. Together, our observations place IQGAP1 as a crucial regulator of spine number and shape acting through the N-Wasp Arp2/3 complex, as well as upstream and downstream of Cdc42. Fil: Jausoro, Ignacio. Consejo Nacional de Invest.cientif.y Tecnicas. Centro Cientifico Tecnol.conicet - Cordoba. Instituto de Inv. Medicas Mercedes y Martin Ferreyra; Universidad Nacional de Cordoba; Fil: Mestres Lascano, Ivan. Consejo Nacional de Invest.cientif.y Tecnicas. Centro Cientifico Tecnol.conicet - Cordoba. Instituto de Inv. Medicas Mercedes y Martin Ferreyra; Universidad Nacional de Cordoba; Fil: Quassollo Infanzon, Gonzalo Emiliano. Consejo Nacional de Invest.cientif.y Tecnicas. Centro Cientifico Tecnol.conicet - Cordoba. Instituto de Inv. Medicas Mercedes y Martin Ferreyra; Universidad Nacional de Cordoba; Fil: Masseroni, Maria Luján. Consejo Nacional de Invest.cientif.y Tecnicas. Centro Cientifico Tecnol.conicet - Cordoba. Instituto de Inv. Medicas Mercedes y Martin Ferreyra; Universidad Nacional de Cordoba; Fil: Heredia, María Florencia. Consejo Nacional de Invest.cientif.y Tecnicas. Centro Cientifico Tecnol.conicet - Cordoba. Instituto de Inv. Medicas Mercedes y Martin Ferreyra; Universidad Nacional de Cordoba; Fil: Caceres, Alfredo Oscar. Consejo Nacional de Invest.cientif.y Tecnicas. Centro Cientifico Tecnol.conicet - Cordoba. Instituto de Inv. Medicas Mercedes y Martin Ferreyra; Universidad Nacional de Cordoba

On the basis of experimental data obtained in vitro, we propose that differential segregation of actin and tubulin in the cytoplasm may be a regulatory mechanism of metabolic fluxes. The results presented point out that the same enzymes may be differentially modulated at different locations in the cytoplasm, depending on the cytoskeletal protein present at that location, its concentration, polymeric status, or geometric arrangement. Essentially, actin or microtubular protein would exert their effect on enzymatic catalysis through displacement of the equilibrium of enzyme oligomers either to active or less active species. The latter was corroborated by mathematical modeling of the dynamic coupling between microtubular protein assembly-disassembly and pyruvate kinase activity. From these results, a precise biochemical meaning can be given to the putative linkage existing between the mechanisms by which cells rearrange their cytoplasmic architecture and the dynamics of biochemical reactions taking place concomitantly. © 1996 Wiley-Liss, Inc.

In sexually segregated cultures of dissociated neurons taken from ventromedial hypothalamus of rat fetuses at embryonic day 16 (E16), it is demonstrated that only neurons from males respond with increased axonal growth to the addition of 17-β-estradiol 100 nM (E2) to the culture medium. Moreover, this response is contingent upon co-culture with heterotopic glia from a target region (amygdala), whereas in the presence of homotopic glia or in cultures without glia, E2 has no effect. It is concluded that before neurons are exposed to gonadal steroids in utero there is a sexual difference in the response to E2, probably explained by earlier maturation of neurons from males as compared to females. The possibility that the observed axogenic effect may be the consequence of an interaction among E2, cells equipped with specific receptors, and glia-producing trophic factors is discussed. © 1995 Wiley-Liss, Inc.

Primary cilia undergo cell-cycle-dependent assembly and disassembly. Emerging data suggest that ciliary resorption is a checkpoint for S phase reentry and that the activation of phospho(T94)Tctex-1 couples these two events. However, the environmental cues and molecular mechanisms that trigger these processes remain unknown. Here, we show that insulin-like growth-1 (IGF-1) accelerates G1-S progression by causing cilia to resorb. The mitogenic signals of IGF-1 are predominantly transduced through IGF-1 receptor (IGF-1R) on the cilia of fibroblasts and epithelial cells. At the base of the cilium, phosphorylated IGF-1R activates an AGS3-regulated Gβγ signaling pathway that subsequently recruits phospho(T94)Tctex-1 to the transition zone. Perturbing any component of this pathway in cortical progenitors induces premature neuronal differentiation at the expense of proliferation. These data suggest that during corticogenesis, a cilium-transduced, noncanonical IGF-1R-Gβγ-phospho(T94)Tctex-1 signaling pathway promotes the proliferation of neural progenitors through modulation of ciliary resorption and G1 length. Fil: Yeh, Celine. Cornell University; Estados Unidos Fil: Li, Aiqun. Cornell University; Estados Unidos Fil: Chuang, Jen Zen. Cornell University; Estados Unidos Fil: Saito, Masaki. Cornell University; Estados Unidos. Tohoku University; Japón Fil: Caceres, Alfredo Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina Fil: Sung, Ching Hwa. Cornell University; Estados Unidos

In this article, we review our current understanding of the biology of IQ domain-containing GTPase-Activating Protein 1, IQGAP1, a scaffolding protein with multiple binding partners, which is widely expressed among different cell types, including neurons, and capable of linking Rho-GTPase signaling with cytosleletal elements and environmental cues. Interestingly, a series of recent studies suggest that IQGAP family members have an important role in neuronal development, synaptic plasticity and nervous system disorders involving alterations in spine density.

In a biological sense, polarity refers to the extremity of the main axis of an organelle, cell, or organism. In neurons, morphological polarity begins with the appearance of the first neurite from the cell body. In multipolar neurons, a second phase of polarization occurs when a single neurite initiates a phase of rapid growth to become the neuron's axon, while the others later differentiate as dendrites. Finally, during a third phase, axons and dendrites develop an elaborate architecture, acquiring special morphological and molecular features that commit them to their final identities. Mechanistically, each phase must be preceded by spatial restriction of growth activity. We will review recent work on the mechanisms underlying the polarized growth of neurons. © 2012 Elsevier Ltd., Flanders Fund for Scientific Research (FWO G 0.666.10N); Federal Office for Scientific Affairs (IUAP p6/43); the Flemish Government Methusalem Grant; Spanish Ministry of Science and Innovation Ingenio-Consolider (CSD2010-00064 and SAF2010-14906); Agencia Nacional Promocion Cientifica y Tecnica (Argentina); CONICET y Agencia Cordoba Ciencia; National Institutes of Health (R00MH080599 and R01MH091186); the Whitehall Foundation; Pew Charitable Trusts

The highly dynamic remodeling and cross talk of the microtubule and actin cytoskeleton support neuronal morphogenesis. Small RhoGTPases family members have emerged as crucial regulators of cytoskeletal dynamics. In this review we will comprehensively analyze findings that support the participation of RhoA, Rac, Cdc42, and TC10 in different neuronal morphogenetic events ranging from migration to synaptic plasticity. We will specifically address the contribution of these GTPases to support neuronal polarity and axonal elongation.

PTP1B is an endoplasmic reticulum (ER) anchored enzyme whose access to substrates is partly dependent on the ER distribution and dynamics. One of these substrates, the protein tyrosine kinase Src, has been found in the cytosol, endosomes, and plasma membrane. Here we analyzed where PTP1B and Src physically interact in intact cells, by bimolecular fluorescence complementation (BiFC) in combination with temporal and high resolution microscopy. We also determined the structural basis of this interaction. We found that BiFC signal is displayed as puncta scattered throughout the ER network, a feature that was enhanced when the substrate trapping mutant PTP1B-D181A was used. Time-lapse and co-localization analyses revealed that BiFC puncta did not correspond to vesicular carriers; instead they localized at the tip of dynamic ER tubules. BiFC puncta were retained in ventral membrane preparations after cell unroofing and were also detected within the evanescent field of total internal reflection fluorescent microscopy (TIRFM) associated to the ventral membranes of whole cells. Furthermore, BiFC puncta often colocalized with dark spots seen by surface reflection interference contrast (SRIC). Removal of Src myristoylation and polybasic motifs abolished BiFC. In addition, PTP1B active site and negative regulatory tyrosine 529 on Src were primary determinants of BiFC occurrence, although the SH3 binding motif on PTP1B also played a role. Our results suggest that ER-bound PTP1B dynamically interacts with the negative regulatory site at the C-terminus of Src at random puncta in the plasma membrane/substrate interface, likely leading to Src activation and recruitment to adhesion complexes. We postulate that this functional ER/plasma membrane crosstalk could apply to a wide array of protein partners, opening an exciting field of research.

The distribution of membrane-bound organelles was studied in cultured hippocampal neurons after antisense oligonucleotide suppression of the kinesin-heavy chain (KHC). We observed reduced 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)) fluorescent staining in neurites and growth cones. In astrocytes, KHC suppression results in the disappearance of the DiOC6(3)-positive reticular network from the cell periphery, and a parallel accumulation of label within the cell center. On the other hand, mitochondria microtubules and microfilaments display a distribution that closely resembles that observed in control cells. KHC suppression of neurons and astrocytes completely inhibited the Brefeldin A-induced spreading and tubulation of the Golgi-associated structure enriched in mannose-6-phosphate receptors. In addition, KHC suppression prevents the low pH-induced anterograde redistribution of late endocytic structures. Taken collectively, these observations suggest that in living neurons, kinesin mediates the anterograde transport of tubulovesicular structures originated in the central vacuolar system (e.g., the endoplasmic reticulum) and that the regulation of kinesin-membrane interactions may be of key importance for determining the intracellular distribution of selected organelles.

Low density dissociated cultures of embryonic rat hippocampal cells were used to study the effects of neurotrophin-3 (NT-3) on neuronal morphogenesis. The results obtained indicate that NT-3 enhances neurite outgrowth and branching; this is a dose-dependent effect, detected in approximately 50% of the neurons, and prevented by K-252a, an inhibitor of the trk family of receptor protein kinases. NT-3 also accelerates the development of neuronal polarity, a phenomenon preceded by a dramatic accumulation of bundles of looped microtubules within axonal growth cones; these microtubule bundles contain tyrosinated, detyrosinated, and acetylated α-tubulin. Taken collectively, our data suggest that even though the basic shape of hippocampal neurons may be endogenously determined, critical aspects of their morphological development may be modulated by trophic factors such as NT-3. In addition, our observations suggest that at least some of the neuritogenic effects of NT-3 involve a stimulation of microtubule assembly and/or transport. Copyright © 1994 Wiley-Liss, Inc.

The fluxes through HK/G6PDH and PK/LDH coupled-enzymatic reactions were quantified in the presence of physiological concentrations (1-15 microM) of polymerized or non-polymerized microtubular protein (MTP) from rat brain and in a permeabilized yeast cell system. In vitro enzymatic fluxes were increased by either polymerized or nonpolymerized brain MTP mainly in the lower range of MTP concentration. At fixed MTP concentrations in the flux stimulatory range of HK/G6PDH (1 mg/ml MTP) or PK/LDH (0.4 mg/ml MTP), a hyperbolic and sigmoidal response to NADP and PEP, respectively, was detected. That dependence varied according to the polymeric status of MTP. The specificity of the phenomenon observed in vitro, was tested for the PK/LDH and HK/G6PDH enzymatic couples in the presence of neutral polymers such as glycogen (< or = 10 mg/ml), poly(ethylene glycol) (up to 10% w/w) or G-actin (< or = 1 mg/ml). In permeabilized Saccharomyces cerevisiae cells, the PK-catalyzed flux was sensitive to microtubule disruption by nocodazole (15 micrograms/ml). The HK/G6PDH system was not affected by nocodazole showing values of kinetic parameters close to those obtained in vitro in the presence of polymerized brain MTP. Indirect immunofluorescence with specific antibodies against tubulin allowed to confirm the microtubules disruption in the presence of nocodazole in permeabilized yeast cells under the same conditions in which enzymes were assayed intracellularly. The experimental evidence is in agreement with the observed phenomenon of increase in fluxes in the enzymatic reactions assayed to be specifically induced by MTP either in vitro or in situ. The results presented are discussed in terms of the assembly of large supramolecular structures as a supraregulatory mechanism of synchronization of systemic cellular processes such as metabolic fluxes.

We report here a novel intracellular localization and function of Tau proteins in cultured cerebellar neurons. Immunofluorescence staining of detergent-extracted cytoskeletons with antibodies specific for Tau proteins revealed intense labeling of growth cone microtubules. Besides, suppression of Tau by antisense oligonucleotide treatment results in the complete disappearance of antigen 13H9, a specific growth cone component with properties of microfilament- and microtubule-associated protein [Goslin et al., 1989: J. Cell Biol. 109:1621–1631], from its normal intracellular location. This phenomenon is unique to neurite-bearing cells, is not associated with the disappearance of microtubules from growth cones, and is not reversed by taxol, a microtubule-stabilizing agent. In addition, Tau-suppressed neurons display a significant reduction in growth cone area and fillopodial number; on the contrary, fillopodial length increases significantly. The alterations in growth cone morphology are accompanied by considerable changes in the phalloidin staining of assembled actin. Taken together, the present results suggest that in developing neurons Tau proteins participate in mediating interactions between elements of the growth cone cytoskeleton important for maintaining the normal structural organization of this neuritic domain. © 1994 Wiley-Liss, Inc.

Neurodegenerative diseases encompass a broad variety of motor and cognitive disorders that are accompanied by death of specific neuronal populations or brain regions. Cellular and molecular mechanisms underlying these complex disorders remain largely unknown. In a previous work we searched for novel Drosophila genes relevant for neurodegeneration and singled out enabled (ena), which encodes a protein involved in cytoskeleton remodeling. To extend our understanding on the mechanisms of ENA-triggered degeneration we now investigated the effect of silencing ena ortholog genes in mouse hippocampal neurons. We found that ENA/VASP downregulation led to neurite retraction and concomitant neuronal cell death through an apoptotic pathway. Remarkably, this retraction initially affected the axonal structure, showing no effect on dendrites. Reduction in ENA/VASP levels blocked the neuritogenic effect of a specific RhoA kinase (ROCK) inhibitor, thus suggesting that these proteins could participate in the Rho-signaling pathway. Altogether these observations demonstrate that ENA/VASP proteins are implicated in the establishment and maintenance of the axonal structure and that a change on their expression levels triggers neuronal degeneration. Fil: Franco, Diana Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina Fil: Rezaval, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina Fil: Caceres, Alfredo Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Instituto de Investigaciones Médicas Mercedes y Martín Ferreyra; Argentina Fil: Schinder, Alejandro Fabian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina Fil: Ceriani, Maria Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina

Cultured neurons obtained from MAP1B-deficient mice have a delay in axon outgrowth and a reduced rate of axonal elongation compared with neurons from wild-type mice. Here we show that MAP1B deficiency results in a significant decrease in Rac1 and cdc42 activity and a significant increase in Rho activity. We found that MAP1B interacted with Tiam1, a guanosine nucleotide exchange factor for Rac1. The decrease in Rac1/cdc42 activity was paralleled by decreases in the phosphorylation of the downstream effectors of these proteins, such as LIMK-1 and cofilin. The expression of a constitutively active form of Rac1, cdc42, or Tiam1 rescued the axon growth defect of MAP1B-deficient neurons. Taken together, these observations define a new and crucial function of MAP1B that we show to be required for efficient cross-talk between microtubules and the actin cytoskeleton during neuronal polarization., This work was supported by FONDECYT (PICT 05-01697) and grants from FONCyT (PICT 815), Agencia Cordoba Ciencia, and HHMI to A.C. J.A. was supported by a Plan National Grant (SAF 2006-02424), CIBERNED and Comunidad de Madrid (SAL 0202-2006). C.G.-B. was supported by Fondecyt 1095089 and ICM P05-001-F.

Dissociated cell cultures from embryonic rat medial amygdala were studied using sequential photography and immunocytochemical staining for cytoskeletal proteins and substance P (sP). Cultures were seeded with cells taken from fetuses grouped by sex; experimental cultures were raised in medium containing 17-beta-estradiol (E2). Forty-eight hours after plating a few neurons begin to define their morphological polarity by the differentiation of an axon-like process; at 5 days in vitro (DIV) almost all neurons had developed an axon. Tapering, daughter branch ratio and branch power coefficient coincided with identification of dendrites which could be confirmed by retrospective analysis of immunocytochemically stained cultures: at 5 DIV MAP-2 was restricted to dendrites whereas Tau immunoreactivity was differentially localized with a clear predominance in the axon. At 21 DIV neuronal shape parameters were strikingly like those of amygdaloid neurons in vivo. It was demonstrated in living neurons that E2 increased total dendritic length and that this is due to increased ramification of third or higher order dendritic segments whose individual lengths are not different from controls. Densitometric measurement of MAP-2 stained neurons showed a highly significant increase of immunoreactive material in cells grown in the presence of E2; readings for alpha-tubulin were not different between controls and E2 treated cultures. The effect of E2 on dendritic length was just as manifest in sP-positive as in sP-negative neurons. No sexual differences in morphological parameters, growth characteristics or effects of E2 were found in neurons taken from female fetuses versus neurons from male fetuses. The significance of these results for the generation of sexual differences in the amygdala in vivo is discussed and contrasted with reported results on the effects of E2 in cultures of different neural regions. © 1992 Wiley-Liss, Inc.

Cultures of dissociated hypothalamic cells taken from rat fetuses of 19 days of gestation were studied using time-lapse recording and sequential microphotography from 1 to 5 days in vitro (DIV) and at 7 and 21 DIV. Cultures were seeded with cells taken from fetuses grouped by sex or sexually mixed; experimental cultures were raised in medium containing 17-beta-estradiol 100 nM (E2). Cells were plated on poly-D-lysine-coated coverslips at a culture density of approximately 4,000 cells/cm2. Immunocytochemistry of cell cultures was performed using a Tau monoclonal antibody (clone Tau-1 PC1C6) and a monoclonal antibody against MAP-2 (clone AP-20). Cells started to produce lamellipodia and neuritic processes approximately 4 hr after plating. Forty-eight hours later a few neurons had defined their morphological polarity by the differentiation of an axon-like process that grows faster than the others; at 5 DIV almost all neurons had defined their axons. At this time, monoclonal antibody against MAP-2 clearly stained soma and dendrites, but not axons. Tau immunoreactivity (lots CCA101 and CCA101N from Boeringher Mannheim) was differentially distributed, with a clear predominance in axon and soma. Results on the morphometric analysis of control and E2 treated neurons provide direct evidence for the existence of sex related differences in the neurite outgrowth response of hypothalamic neurons, since cultured neurons taken from female fetuses differentiated axons later and had fewer primary neurites and shorter dendrites than neurons taken from male fetuses or sexually mixed cultures. Also, it was demonstrated in living neurons that E2 effectively enhances outgrowth and elongation in axons. The frequency distribution curves of axonal length for control and E2 treated cultures was unimodal, suggesting that the effect of E2 was a uniform increase in the axonal length of all neurons. The structural differences between neurons from both sexes and the changes induced by E2 may contribute to explain the differences in brain function found between the sexes.

In this study, we have used a combination of biochemical and molecular biology techniques to demonstrate that the C-terminal tail domain of KIF4 directly interacts with P0, a major protein component of ribosomes. Besides, in dorsal root ganglion neurons, KIF4 and P0, as well as other ribosomal constituents, colocalize in clusters distributed along axons and neuritic tips. RNA interference suppression of KIF4 or expression of KIF4 variants lacking the tail domain or mutations of the ATP-binding site result in accumulation of P0 and other ribosomal proteins at the cell body and in their disappearance from axons. Our results also show one additional function for KIF4 involving an Ezrin-Radixin-Moesin-like domain in the second coiled-coiled region of KIF4. Expression of a KIF4 mutant lacking this domain abolishes the clustering of ribosomal constituents and prevents the anterograde translocation of the cell adhesion molecule L1. Taken together, the present results suggest that by binding to P0 through its tail domain and by using its motor activity, KIF4 is involved in the anterograde trafficking of ribosomal constituents to axons and that by means of its Ezrin-Radixin-Moesin-like domain interacts and transports L1.

We have used the monoclonal antibody YL 1/2 (Tyr) specific for tyrosinated tubulin, and a polyclonal antibody (Glu) specific for detyrosinated tubulin to visualize the distribution of microtubules and microtubule assembly sites during axonal outgrowth. Cerebellar macroneurons growing in culture initially extend several short and thin neurites which have the potential to differentiate either as axons or dendrites (Ferreira and Caceres: Developmental Brain Research 49:205-213, 1989). At the onset of axonal outgrowth the Tyr antibody labels the minor neurites, the axon, and its growth cone, while the Glu antibody only shows immunoreactivity in the axonal shaft. After nocodazole treatment, the Tyr staining disappears, whereas that produced by the Glu antibody remains practically unchanged. When nocodazole was removed, tyrosinated microtubules reappeared first at the tip of the axon, in a more distal region than that occupied by detyrosinated microtubules; another focal site of tyrosinated tubulin incorporation was detected in the cell body. Incorporation of tyrosinated tubulin into growing axons was also studied after taxol treatment. After long incubation periods in the presence of taxol, the Tyr staining disappeared from the axon but remained in the cell body; however, immunoreactivity in this site was negative when the cells were preincubated in the presence of protein synthesis inhibitors. Release from taxol results in the reappearance of Tyr immunoreactivity at the distal end of the axon. Taken collectively, the present results indicate 1) that in cerebellar macroneurons axonal differentiation is accompanied by a temporal and spatial differentiation of microtubules and 2) that there is an active site of tyrosinated tubulin assembly at the tip of axonal processes, and they suggest that the highly tyrosinated domain in this region is a consequence of rapid microtubule turnover and tubulin tyrosine ligase activity.

Delta-catenin is a synaptic adherens junction protein pivotally positioned to serve as a signaling sensor and integrator. Expression of delta-catenin induces filopodia-like protrusions in neurons. Here we show that the small GTPases of the Rho family act coordinately as downstream effectors of delta-catenin. A dominant negative Rac prevented delta-catenin-induced protrusions, and Cdc42 activity was dramatically increased by delta-catenin expression. A kinase dead LIMK (LIM kinase) and a mutant Cofilin also prevented delta-catenin-induced protrusions. To link the effects of delta-catenin to a physiological pathway, we noted that (S)-3,5-dihydroxyphenylglycine (DHPG) activation of metabotropic glutamate receptors induced dendritic protrusions that are very similar to those induced by delta-catenin. Furthermore, delta-catenin RNA-mediated interference can block the induction of dendritic protrusions by DHPG. Interestingly, DHPG dissociated PSD-95 and N-cadherin from the delta-catenin complex, increased the association of delta-catenin with Cortactin, and induced the phosphorylation of delta-catenin within the sites that bind to these protein partners.

In this study, we have examined the effects of rotenone in primary cultures of hippocampal and dopaminergic neurons in order to obtain insights into the possible mechanisms underlying the neurotoxic effects of this pesticide. The results obtained indicate that a 48-h exposure to rotenone (0.1 microM) produces a complete and selective suppression of axon formation. This effect was dose dependent, not accompanied by changes in microtubule organization, and reversible after washout of the agrochemical from the tissue culture medium. Interestingly, pull-down assays revealed that rotenone decreases Cdc42 and Rac activities, whereas increasing that of Rho. In accordance with this, treatment of neuronal cultures with cytochalasin D, an actin-depolymerizing drug, or with the Rho-kinase inhibitor Y27632, or overexpression of Tiam1, a guanosine nucleotide exchange factor for Rac, reverts the inhibitory effect of rotenone on axon formation. Taken together, our data suggest that at least some of the neurotoxic effects of rotenone are associated with an inhibition of actin dynamics through modifications of Rho-GTPase activity.

The role of growth factors in the pathogenesis of Alzheimer disease is unknown. The beta-amyloid protein accumulates abnormally in the brain in Alzheimer disease and is neurotoxic to differentiated hippocampal neurons in culture. Nerve growth factor (NGF) increased the neurotoxic potency of a beta-amyloid polypeptide by a factor of approximately 100,000, which resulted in a reduction of the beta-amyloid neurotoxic EC50 from 0.1 microM to 1 pM. This potentiating effect of NGF was reversed by a monoclonal antibody against NGF and was not observed for a variety of other neurotrophic growth factors. Exposure of hippocampal neurons to very low concentrations of beta amyloid alone resulted in a marked induction of immunoreactive NGF receptors. Addition of NGF with beta amyloid resulted in the appearance of neurodegenerative changes in NGF receptor-positive neurons. The early and profound degeneration of hippocampal and basal forebrain cholinergic neurons that occurs in Alzheimer disease may result from a neurotoxic interaction of beta amyloid with NGF.

Tctex-1, a light-chain component of the cytoplasmic dynein motor complex, can function independently of dynein to regulate multiple steps in neuronal development. However, how dynein-associated and dynein-free pools of Tctex-1 are maintained in the cell is not known. Tctex-1 was recently identified as a Gbetagamma-binding protein and shown to be identical to the receptor-independent activator of G protein signaling AGS2. We propose a novel role for the interaction of Gbetagamma with Tctex-1 in neurite outgrowth. Ectopic expression of either Tctex-1 or Gbetagamma promotes neurite outgrowth whereas interfering with their function inhibits neuritogenesis. Using embryonic mouse brain extracts, we demonstrate an endogenous Gbetagamma-Tctex-1 complex and show that Gbetagamma co-segregates with dynein-free fractions of Tctex-1. Furthermore, Gbeta competes with the dynein intermediate chain for binding to Tctex-1, regulating assembly of Tctex-1 into the dynein motor complex. We propose that Tctex-1 is a novel effector of Gbetagamma, and that Gbetagamma-Tctex-1 complex plays a key role in the dynein-independent function of Tctex-1 in regulating neurite outgrowth in primary hippocampal neurons, most likely by modulating actin and microtubule dynamics.

Exocytotic incorporation of plasmalemmal precursor vesicles (PPVs) into the cell surface is necessary for axonal outgrowth and is known to occur mainly at the nerve growth cone. We have demonstrated recently that plasmalemmal expansion is regulated at the growth cone by IGF-1, but not by BDNF, in a manner that is quasi independent of the neuron's perikaryon. To begin elucidating the signaling pathway by which exocytosis of the plasmalemmal precursor is regulated, we studied activation of the IRS/PI3K/Akt pathway in isolated growth cones and hippocampal neurons in culture stimulated with IGF-1 or BDNF. Our results show that IGF-1, but not BDNF, significantly and rapidly stimulates IRS/PI3K/Akt and membrane expansion. Inhibition of PI3K with Wortmannin or LY294002 blocked IGF-1-stimulated plasmalemmal expansion at the growth cones of cultured neurons. Finally, our results show that, upon stimulation with IGF-1, most active PI3K becomes associated with distal microtubules in the proximal or central domain of the growth cone. Taken together, our results suggest a critical role for IGF-1 and the IRS/PI3K/Akt pathway in the process of membrane assembly at the axonal growth cone.

The different strains of microtubule-associated protein (MAP)-1B-deficient mice that have been generated appear to express different phenotypes. This variability could be the consequence of the distinct genetic backgrounds of the animals used to generate these lines. Certain proteins might be able to complement the deficit of MAP1B function in these mice. Therefore, we examined whether the concentrations of potential compensatory proteins varied among these mutant strains. In this way, we identified significant differences in the amounts of the microtubule-associated EB1 protein between two of these strains. Furthermore, in transfection studies, we demonstrated that the overexpression of end binding protein-1 (EB1) could facilitate axonogenesis in MAP1B-/- cells in which EB1 is normally weakly expressed. Thus, we suggest that EB1 could complement MAP1B function during neural development.

SummaryCoordinated microtubule and microfilament changes are essential for the morphological development of neurons; however, little is know about the underlying molecular machinery linking these two cytoskeletal systems. Similarly, the indispensable role of RhoGTPase family proteins has been demonstrated, but it is unknown how their activities are specifically regulated in different neurites. In this paper, we show that the cytoplasmic dynein light chain Tctex-1 plays a key role in multiple steps of hippocampal neuron development, including initial neurite sprouting, axon specification, and later dendritic elaboration. The neuritogenic effects elicited by Tctex-1 are independent from its cargo adaptor role for dynein motor transport. Finally, our data suggest that the selective high level of Tctex-1 at the growth cone of growing axons drives fast neurite extension by modulating actin dynamics and also Rac1 activity.

Microtubule-associated protein 1B is the first MAP to be expressed during the development of the nervous system. Several different approaches have revealed that MAP1B function is associated with microtubule and actin microfilament polymerization and dynamics. In recent years, the generation of molecular models to inactivate MAP1B function in invertebrates and mammals has sparked some controversy about the real role of MAP1B. Despite discrepancies between some studies, it is clear that MAP1B plays a principal role in the development of the nervous system. In this article, we summarize the evidence for MAP1B function in a wide variety of cellular processes implicated in the proper construction of the nervous system. We also discuss the role of MAP1B in pathological processes., Contract grant sponsor: Spanish CICYT. Contract grant sponsor: Comunidad de Madrid. Contract grant sponsor: Fundacion Lilly. Contract grant sponsor: NeuroPharma.

OBJETIVOS: El flujo vaginal y los síntomas asociados son consultas frecuentes, especialmente en mujeres jóvenes, y la candidiasis es una de las causas. Este estudio investiga cuáles son las diferentes especies de Candida que se aíslan en secreciones vaginales así como sus manifestaciones clínicas. MATERIAL Y MÉTODOS: Se estudió a 100 pacientes que concurrieron al consultorio externo de Ginecología del Hospital Arzobispo Loayza por presentar flujo vaginal y cuyas edades fluctuaron entre 23 y 47 años de edad. Se tomó muestras de la secreción y se realizó estudios microbiológicos en fresco, coloraciones, cultivos y pruebas de fermentación y asimilación de carbohidratos para la identificación de especies. RESULTADOS: A 24 se les aisló Candida que fueron identificados 12 como C. albicans, 6 C. tropicalis, 4 C. famata, 1 C. glabrata y 1 C. kruseii. No encontramos diferencias clínicas entre los síndromes causados por diferentes especies de Candida. CONCLUSIONES: Las características clínicas y del flujo vaginal no son diagnósticas en cuanto a la especie de Candida. C. albicans es la especie más frecuentemente aislada como causante de vulvovaginitis, siendo el cultivo en suero humano un método rápido para el diagnóstico, con pocas excepciones. Las especies de Candida, cuando son los únicos patógenos, están asociadas al bacilo de Doderlein. Gardnerella vaginalis es el patógeno asociado con más frecuencia a la infección por Candida spp.

OBJECTIVES: To determine if presence of Enterococcus is related to pathological vaginal discharge (VD), as well its frecuency in VD, and its antimicrobial-resistance levels. MATERIALS AND METHODS: In a 4-month period were selected 98 women who attend to gynecological outpatient service due to VD; in samples of these VD leukocytes search, and isolation and culture procedures for Enterococcus and other female-genital-tract-associated microorganisms were performed. RESULTS: Enterococcus species, mainly E. faecalis were isolated from 39% of patients. Gardnerella vaginalis was identified in 44% of patients. Presence of enterococcus was associated with neither pathogeny nor anal intercourse. There was no penicillin-resistant Enterococcus strain. CONCLUSIONS: In the studied VD patients Enterococcus would not exert pathogenic activity, and was ranked 2th in frecuency after G. vaginalis. There was no in vitro penicillin-resistance among the 38 strains studied. OBJETIVOS: Determinar si la presencia de Enterococcus se asocia a flujo vaginal patológico, su frecuencia en éste y los niveles de resistencia a antimicrobianos. MATERIAL Y MÉTODOS: En un período de 4 meses se seleccionó 98 pacientes que acudieron a consulta externa ginecológica con flujo vaginal, en las cuales se buscó tanto la presencia de leucocitos como aislamiento para Enterococcus y otros microorganismos. RESULTADOS: En el 39% de las pacientes se aisló Enterococcus, principalmente E.faecalis. Gardnerella vaginalis fue identificada en el 44% de las pacientes. No se encontró relación entre la presencia de Enterococcus y signos de patogenicidad, ni con relaciones sexuales contranatura. No hubo Enterococcus resistente a la Penicilina. CONCLUSIONES: Enterococcus no ejercería acción patógena en las pacientes estudiadas, aun cuando ocupó el segundo lugar en frecuencia entre las pacientes con flujo vaginal, luego de Gardnerella vaginalis. No hubo resistencia in vitro a la penicilina en las 38 cepas estudiadas.

We show here that an active Cdk5-p35 kinase is present in Golgi membranes, where it associates with a detergent-insoluble fraction containing actin. In addition, Cdk5-p35-dependent phosphorylation of alpha-PAK immunoreactive protein species was detected in Golgi membranes, as well as an interaction with the small GTPase, Cdc42. Moreover, antisense oligonucleotide suppression of Cdk5 or p35 in young cultured neurons, as well as inhibition of Cdk5 activity with olomoucine, blocks the formation of membrane vesicles from the Golgi apparatus. Taken together, these results show a novel subcellular localization of this kinase and suggest a role for Cdk5-p35 in membrane traffic during neuronal process outgrowth.

In cultured neurons, axon formation is preceded by the appearance in one of the multiple neurites of a large growth cone containing a labile actin network and abundant dynamic microtubules. The invasion-inducing T-lymphoma and metastasis 1 (Tiam1) protein that functions as a guanosine nucleotide exchange factor for Rac1 localizes to this neurite and its growth cone, where it associates with microtubules. Neurons overexpressing Tiam1 extend several axon-like neurites, whereas suppression of Tiam1 prevents axon formation, with most of the cells failing to undergo changes in growth cone size and in cytoskeletal organization typical of prospective axons. Cytochalasin D reverts this effect leading to multiple axon formation and penetration of microtubules within neuritic tips devoid of actin filaments. Taken together, these results suggest that by regulating growth cone actin organization and allowing microtubule invasion within selected growth cones, Tiam1 promotes axon formation and hence participates in neuronal polarization.

Cultured neurons obtained from a hypomorphous MAP1B mutant mouse line display a selective and significant inhibition of axon formation that reflects a delay in axon outgrowth and a reduced rate of elongation. This phenomenon is paralleled by decreased microtubule formation and dynamics, which is dramatic at the distal axonal segment, as well as in growth cones, where the more recently assembled microtubule polymer normally predominates. These neurons also have aberrant growth cone formation and increased actin-based protrusive activity. Taken together, this study provides direct evidence showing that by promoting microtubule dynamics and regulating cytoskeletal organization MAP1B has a crucial role in axon formation., This work was supported by grants from the Spanish Direccion General de Investigacion Cientifica y Tecnica, Comunidad de Madrid, European Union, and an institutional grant from Formation de Recherche Associee to J.A. It was also supported by grants from Consejo Nacional de Investigaciones Cientificas y Tecnicas de Argentina (PICT-PIP 4906), FONCyT (PICT 05-00000-00937 and 99-5-6179), CONICOR, and a Howard Hughes Medical Institute Grant (HMMI 75197-553201) awarded under the International Research Scholars Program to A.C. An A.E.C.I./I.C.I. predoctoral fellowship was awarded to C.G.-B. The MAP1B mutant mouse was generated with financial support from Amgen and the Max Planck Society in the laboratory of Prof. P. Gruss.

2,4-Dichlorophenoxyacetic acid (2,4-D) is a potent neurotoxic herbicide widely used in agriculture. The basic mechanisms by which 2,4-D produces cell damage have not yet been determined. In this study we have examined the effects of 2,4-D in primary cultures of cerebellar granule cells in order to obtain insights into the possible mechanisms underlying the toxic effects of this herbicide. The results obtained indicate that a 24-hour exposure to 2,4-D produces a striking and dose-dependent inhibition of neurite extension. This phenomenon is paralleled by a significant reduction in the cellular content of both dynamic and stable microtubules, a disorganization of the Golgi apparatus, and an inhibition in the synthesis of complex gangliosides. Interestingly, 2,4-D inhibits the in vitro polymerization of purified tubulin. Taken together, the present observations raise the possibility that at least one basic mechanism underlying 2,4-D neurotoxicity involves an inhibition of microtubule assembly. That event may cause a decreased neurite outgrowth response, and could also explain the observed differences in the pattern of ganglioside biosynthesis and/or the disorganization of the Golgi apparatus.

In this study we present evidence about the cellular functions of KIF4. Using subcellular fractionation techniques and immunoisolation, we have now identified a type of vesicle that associates with KIF4, an NH2-terminal globular motor domain kinesin-like protein. This vesicle is highly concentrated in growth cones and contains L1, a cell adhesion molecule implicated in axonal elongation. It lacks synaptic vesicle markers, receptors for neurotrophins, and membrane proteins involved in growth cone guidance. In cultured neurons, KIF4 and L1 predominantly localize to the axonal shaft and its growth cone. Suppression of KIF4 with antisense oligonucleotides results in the accumulation of L1 within the cell body and in its complete disappearance from axonal tips. In addition, KIF4 suppression prevents L1-enhanced axonal elongation. Taken collectively, our results suggest an important role for KIF4 during neuronal development, a phenomenon which may be related to the anterograde transport of L1-containing vesicles.

The key target of this study was the tau protein kinase II system (TPK II) involving the catalytic subunit cdk5 and the regulatory component p35. TPK II is one of the tau phosphorylating systems in neuronal cells, thus regulating its functions in the cytoskeletal dynamics and the extension of neuronal processes. This research led to demonstration that the treatment of rat hippocampal cells in culture with fibrillary beta-amyloid (Abeta) results in a significant increase of the cdk5 enzymatic activity. Interestingly, the data also showed that the neurotoxic effect of 1-20 microM Abeta on primary cultures markedly diminished with co-incubation of hippocampal cells with the amyloid fibers plus the cdk5 inhibitor butyrolactone I. This inhibitor protected brain cells against Abeta-induced cell death in a concentration dependent fashion. Moreover, death was also prevented by a cdk5 antisense probe, but not by an oligonucleotide with a random sequence. The cdk5 antisense also reduced neuronal expression of cdk5 compared with the random oligonucleotide. The studies indicate that cdk5 plays a major role in the molecular path leading to the neurodegenerative process triggered by the amyloid fibers in primary cultures of rat hippocampal neurons. These findings are of interest in the context of the pathogenesis of Alzheimer's disease.

When cultured cerebellar macroneurons develop attached to a laminin-containing substrate or after the acute addition of laminin to the tissue culture medium, there is an acceleration in the rate and extent of axonal elongation. Furthermore, laminin is capable of inducing axonal formation and microtubule stabilization in neurons arrested at stage II of neuritic development by tau suppression (Caceres and Kosik, 1990; Caceres et al., 1991). Laminin-enhanced or induced axonal extension is paralleled by a selective and dramatic incorporation of phosphorylated MAP-1b into axonal microtubules. Axonal formation in neurons growing in the presence of laminin is prevented by treatment of the cultures with a mixture of MAP-1b and tau antisense oligonucleotides, but not by the single suppression of any one of these MAPs. However, suppression of MAP-1b, but not of tau, greatly reduces the increase in the rate and extent of axonal elongation induced by laminin. No such effects are elicited by MAP-1b antisense oligonucleotides in neurons growing in the absence of laminin, e.g. polylysine alone, where most of the MAP-1b present in the cells is dephosphorylated and not associated with the cytoskeleton. Taken collectively, these data suggest that, with regard to axonal elongation, MAP-1b and tau can be functionally substituted, and that extracellular matrix molecules, such as laminin, affect axonal extension by promoting the in vivo utilization of MAP-1b.

The amyloid precursor protein (APP) is the parent molecule from which beta-amyloid protein is cleaved and deposits as amyloid fibrils in the senile plaques of Alzheimer's disease. Its primary structure resembles a receptor; however, no ligand has been identified. In growing hippocampal neurons APP is localized to growth cones. APP immunoreactivity was highly enriched in the axons of mature cultured neurons, where it appears as a specialization of the axonal membrane. Its anterograde translocation occurs via a kinesin-based motor. Following cytosolic acidification, APP colocalizes with late endosomes that get redistributed from the neuronal cell body to the processes. APP colocalizes in cultured hippocampal neurons to clathrin- immunoreactive clusters of vesicular-like structures. The finding lends additional credence to the possibility that APP could function as a receptor.

We show here that antisense MAP2 oligonucleotides inhibit neurite outgrowth in cultured cerebellar macroneurons. Unlike control neurons, which first extend a lamellipodial veil followed by a consolidation phase during which the cells extend minor neurites, MAP2-suppressed cells persist with lamellipodia and later become rounded. The induction of microtubules containing tyrosinated tubulin, which parallels neurite outgrowth in control neurons, was blocked under antisense conditions. The small but significant increase in acetylated microtubules was not affected. In contrast, the suppression of tau, which selectively blocks axonal elongation, completely prevented the increase of acetylated microtubules, but did not modify the induction of labile microtubules. These results suggest that MAP2 and tau have different functions: the initial establishment of neurites depends upon MAP2, whereas further neurite elongation depends upon tau and microtubule stabilization.

The expression of the class III beta-tubulin isotype was studied in cultured brain neurons by means of a monoclonal antibody (TuJ1). The results obtained indicate that during early axonal outgrowth most of the class III beta-tubulin is not incorporated into microtubules, a phenomenon which is also observed under conditions which alter the rate and extent of the neurite outgrowth response. On the other hand, a dramatic increase in its incorporation into microtubules is observed after the neurons have differentiated their neurites as axons and dendrites. In addition, the appearance of colchicine-resistant microtubules containing this isotype, a phenomenon which occurs late in neurite development, is highly coincident with the appearance of stable microtubules containing high molecular weight microtubule-associated proteins (MAPs). This pattern is different from that of the accumulation and incorporation of other beta-tubulin isotypes into microtubules. Taken collectively, our results indicate that differences exist in the in vivo utilization of tubulin isotypes in developing brain neurons and suggest that the class III beta-tubulin isotype is not a primary factor involved in the regulation of microtubule assembly during early neurite outgrowth, but that it may be important for maintaining further neurite elongation and/or determining some unique binding property of MAPs to specific microtubule subsets.

Tau, a microtubule-associated protein (MAP) enriched in axons, may have a role in the generation and maintenance of an axonal morphology. Neurons from embryonic day 15 rat cerebellum in culture elaborate two morphologically distinct neurite populations--one with nontapering, elongated axonlike neurites and the other with tapered dendritelike neurites that branch frequently and are selectively stained with antibodies to MAP2. Tau antisense oligonucleotides were utilized in two ways: (1) continuous application of antisense every 24 hr for variable periods of time or (2) application of antisense that was delayed until neurite differentiation was underway. In both cases, 24 hr after the administration of the antisense, tau protein was not detected immunocytochemically. When the antisense was given continuously directly after plating, the neurites persisted as simple minor outgrowths. When antisense was added 72 hr after plating, axonlike neurites were lost, while the remaining neurites continued to grow and increase in complexity. We concluded that the initial establishment of an elongated axonlike neurite is a prerequisite for further neurite maturation; however, once the axon is established, the remaining neurites are able to grow independently and assume a tapered dendritelike appearance.

Summary Dissociated neuronal cultures from several regions of the nervous system elaborate two populations of neurites which have features of axons and dendrites. The microtubule-associated protein tau appears to segregate to the axon in some of these culture systems, however it does not do so until after the development of morphological polarity. Despite this observation, tau very likely has some role in the development of polarity because in cultured cerebellar macroneurons taken from the rat embryonic day 15 primordial cerebellum, the inhibition of tau expression by antisense techniques resulted in the failure of a single minor neurite to elongate and form an axon-like neurite. Tau antisense given continuously for up to 72 h kept neurons locked in a stage with minor neurites only; however when released from the effects of the antisense they fully recovered. The administration of tau antisense after the development of polarity resulted in the loss of the axon-like neurite, while dendrite-like neurites continued to grow. Together these results suggest that dendritic differentiation in cerebellar macroneurons requires the prior elaboration of an axon-like structure.

Neurons in culture can have fundamentally distinct morphologies which permit their cytological identification and the recognition of their neurites as axons or dendrites. Microtubules may have a role in determining morphology by the selective stabilization of spatially distinct microtubule subsets. The plasticity of a neurite correlates inversely with the stability of its component microtubules: microtubules in growth cones are very dynamic, and in initial neurites there is continuous incorporation of labelled subunits, whereas in mature neurites, microtubules are highly stabilized. The binding of microtubule-associated proteins to the microtubules very probably contributes to this stability. Cerebellar neurons in dissociated culture initially extend exploratory neurites and, after a relatively constant interval, become polarized. Polarity becomes evident when a single neurite exceeds the others in length. These stable neurites cease to undergo the retractions and extensions characteristic of initial neurites and assume many features of axons and dendrites. We have now studied the role of the neuronal microtubule-associate protein tau in neurite polarization by selectively inhibiting tau expression by the addition of antisense oligonucleotides to the culture media. Although the extension of initial exploratory neurites occurred normally, neurite asymmetry was inhibited by the failure to elaborate an axon.

The expression of gangliosides of the lactosylceramide (LC) and of the gangliotertrasylceramide (GTC) series on the surface of cells from rat embryonic cerebellar tissue was investigated by double-color indirect immunofluorescence. GD3 was assumed to be representative of LC and was detected using a specific monoclonal antibody. GM1 was assumed to be representative of GTC and was detected using the binding of cholera toxin followed by the binding of cholera toxin antibodies. The expression of polysialosylated GTC (polysialosy-GTC) was detected using the cholera toxin-cholera toxin antibody experimental approach after conversion of polysialosyl-GTC to GM1 by treatment of the cells with neuraminidase. To distinguish the major neural cell types present in the cultures the expression of the following cell type-specific markers was investigated: neurons-specific markers was investigated: neuron-specific enolase and microtubule-associated protein-2 (MAP-2) as probes for neuronal cells and the intermediate filament protein glial fibrillar acidic protein (GFAP) as a probe for astroglial cells. More than 80% of cells dissociated from cerebellar tissue of 15-day-old rat embryos (E15) are positive for the expression of GD3 and about 50% for the expression of GM1 and polysialosyl-GTC, but most are negative for the expression of neuron-specific enolase, MAP-2, and GFAP. After culturing for 4 days (E15+4) most cells that show characteristics of neuronal cells are positive for the expression of polysialosyl-GTC and “inactivate” the expression of GD3. Most cells with characteristics of radial and stellate glial cells are also positive for the expression of polysialosyl-GTC, but unlike neuron-like cells, they do not “inactivate” the expression of GD3.

The distribution of acetylated microtubules in cerebellar macroneurons which develop in culture was studied with 6-11B-1, a monoclonal antibody specific for acetylated alpha-tubulin. In these neurons 6-11B-1 helps to define a subset of stable, colchicine-resistant, microtubules that during early neuronal development are exclusively localized in the neuron's axon. On the other hand, in mature neurons acetylated microtubules display a widespread distribution being localized in the axon and thick dendritic trunks, a phenomenon correlated with an increase in the levels of acetylated alpha-tubulin and colchicine-resistant microtubules. Taken collectively, these observations suggest that the acetylation of alpha-tubulin is important for differentiating microtubules during neurite growth; in young neurons this post-translational modification may contribute to determine a selective stabilization of microtubules accompanying axonal differentiation.

n dissociated-cell cultures prepared from the embryonic rat hippocampus, neurons establish both axons and dendrites, which differ in geometry, in ultrastructure, and in synaptic polarity. We have used immunocytochemistry with monoclonal antibodies to study the regional distribution of beta-tubulin and micro-tubule-associated protein 2 (MAP2) in hippocampal cultures and their localization during early stages of axonal and dendritic development. After development for a week or more in culture, when axons and dendrites were well- differentiated, the distribution of these two proteins was quite different. Beta-tubulin was present throughout the nerve cell, in soma, dendrites, and axon. It was also present in all classes of non-neuronal cells, astrocytes, fibroblasts, and a presumptive glial progenitor cell. In contrast, MAP2 was preferentially localized to nerve cells; within neurons, MAP2 was present in soma and dendrites, but little or no immunostaining was detectable in axons. Both beta-tubulin and MAP2 were present in nerve cells at the time of plating. From the earliest stages of process extension, beta-tubulin was present in all neuronal processes, both axons and dendrites. Surprisingly, MAP2 was also initially present in both axons and dendrites, extending as far as the axonal growth cone. With subsequent development, MAP2 staining was selectively lost from the axon so that after 1 week in vitro little or no axonal staining remained. Taken together with earlier results (Caceres et al., 1984a), these data indicate that the establishment of neuronal polarity, as manifested by the molecular differentiation of the axonal and dendritic cytoskeleton, occurs largely under endogenous control, even under culture conditions in which cell interactions are greatly restricted.(ABSTRACT TRUNCATED AT 250 WORDS)

The distribution and subcellular localization of calmodulin in adult and developing cerebellum was studied in rats by immunocytochemistry. Calmodulin immunoreactivity was found both in neurons and in glial cells. Within neurons the staining was particularly intense in the cell nucleus and in dendrites, the cytoplasm of the cell body was more lightly stained than the nucleus, and light immunoreactivity was observed in axons. Electron microscopic analysis confirmed the association of calmodulin with the nuclear chromatin, while the nucleolus remained unstained. The reaction product was also found overlying the membranes of several organelles, in postsynaptic densities and decorating both dendritic and axonal microtubules. In developing Purkinje cells, calmodulin immunoreactivity was found as early as 5 days after birth. During the initial phases of dendritic development (5–10 days post-natal), the reaction product was associated with the organelles of the apical cone, while little or no staining was observed in the elongating dendrites or in the cell nucleus. Later in development, calmodulin was found in primary and secondary dendrites, and by 20 days after birth immunoreactivity appeared in the cell nucleus, and in the postsynaptic densities of immature spines located in dendrites. The presence of calmodulin in the apical cone suggests the possibility that this protein may participate in the regulation of microtubule formation during the initial stages of dendritic development. Its presence in dendrites at later stages (during the period of synaptogenesis) may indicate that it also participates in the formation of synapses between the parallel fibres and dendritic spines.

A monoclonal antibody against the microtubule-associated protein (MAP) Tau was used to examine the fate of this molecule during post-lesion afferent reorganization in the hippocampus of adult rats. An immunocytochemical analysis was carried out in the dentate gyrus after unilateral destruction of the entorhinal cortex (EC). In the non-denervated hippocampus, Tau immunoreactivity was detected in parallel axons and mossy fibers; no staining was present in neuronal cell bodies and dendrites. A significant decrease in Tau immunoreactivity was detected in the outer 23 of the ipsilateral dentate gyrus molecular layer (ML) 2 days after an EC lesion, whereas staining in the inner 13 of the same ML increased considerably. This was followed by a very rapid recovery of Tau immunoreactivity in the outer 23 of the denervated ML, which by 10 days post-lesion was almost identical to that of the contralateral non-denervated ML. A similar phenomenon was observed in other regions of the hippocampus denervated by the EC lesion. The modifications in Tau immunoreactivity in the denervated hippocampus were also accompanied by changes in the polypeptide composition of this heterogeneous group of MAPs, as revealed by immunoblot analysis of hippocampal extracts obtained at different post-lesion intervals; these changes involved a rapid and significant increase in low molecular weight migrating Tau-immunoreactive polypeptides. The present observations indicate that important modifications in Tau proteins occur in the deafferented hippocampus, a phenomenon that may well be related with the regulation of microtubule polymerization during post-lesion axonal growth.

The modifications in the localization of tubulin and the high molecular weight microtubule-associated protein 2 were studied in the cerebellum after partial denervation. Both proteins were localized in 40 μm sections using monoclonal antibodies against β-tubulin (clones Tu9B and Tu 12) or microtubule-associated protein 2 (clones AP9 and AP13), and polyclonal antisera against α- and β-tubulin or microtubule-associated protein 2, visualized with the immunoperoxidase method of Sternberger [ Sternberger (1979) Immunocytochemistry; Sternberger and Sternberger (1983) Proc. natn. Acad. Sci. U.S.A.80 6126–6130] or a biotin-avidin system. The destruction of the inferior olive was performed in adult male rats by electrocoagulation or by intraperitoneal administration of 3-acetylpyridine. One day after chemical destruction of the inferior olive, anti-microtubule-associated protein 2 staining with either of the monoclonal antibodies or with the polyclonal antiserum was almost identical to that observed in the cerebellum of non-denervated animals. Specific staining was intense in the cell somata and dendrites and absent in myelinated tracts and in parallel fibers. However, 3 days after the lesion anti-microtubule-associated protein 2 staining showed a clear decrease, both in the proximal and the distal portions of thick secondary and tertiary dendritic trunks of the Purkinje cell. The intensity of the staining was also considerably reduced in the fine dendritic ramifications. By 8 days post-lesion, microtubule-associated protein 2 immunoreactivity began to increase, but only in the portions of the dendrites deprived of the climbing fibre; on the contrary, low immunoreactivity was found in the fine dendritic ramifications which are contacted by normal parallel fibers; microtubule-associated protein 2 immunoreactivity increased considerably by 11 days post-lesion, giving a pattern quite similar to that of non-denervated Purkinje cells. The alterations in microtubule-associated protein 2 immunoreactivity were also accompanied by a dramatic decrease in the immunostaining for tubulin, beginning on day-3 post-lesion and lasting until day-15 post-lesion. These changes were observed with either the monoclonal antibodies against β -tubulin or with the polyclonal antiserum against α- and β-tubulin. The changes in both molecules were also observed in animals in which the inferior olive was destroyed by electrocoagulation, ruling out the possibility of a direct action of 3-acetylpyridine on dendritic microtubular proteins. The present observations provide evidence indicating that the afferent systems play an important role in maintaining the cytoskeleton of postsynaptic dendrites. They also show that the alterations are reversible, a phenomenon that may well be associated with, or consequent to, post-lesion afferent reorganization.