Alfredo Cáceres

Suppression of MAP2 in cultured cerebeller macroneurons inhibits minor neurite formation

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.

Inhibition of tau phosphorylating protein kinase cdk5 prevents β‐amyloid‐induced neuronal death

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 β‐amyloid (Aβ) results in a significant increase of the cdk5 enzymatic activity. Interestingly, the data also showed that the neurotoxic effect of 1–20 μM Aβ 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 Aβ‐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 Alzheimers disease.

Microtubule formation and neurite growth in cerebellar macroneurons which develop in vitro: evidence for the involvement of the microtubule-associated proteins, MAP-1a, HMW-MAP2 and Tau

The relationship between the expression of microtubule-associated proteins (MAPs) and microtubule formation was studied in embryonic cerebellar macroneurons maintained in culture. The results obtained suggest that in these neurons high molecular weight-MAP2 (HMW-MAP2) acts as a promoter of tubulin assembly since its induction and pattern of distribution are highly correlated with the increase in microtubule mass which parallels axonal and dendritic growth; MAP-1a may have a similar role but restricted to the assembly of dendritic microtubules. On the other hand, Tau expression and accumulation follows a time course identical to that of the induction of stable microtubules; besides, at all stages of neurite differentiation and growth this protein seems to be preferentially associated with this subset of microtubules as opposed to the other MAPs, observations which suggest an important role for this protein in determining microtubule stability during axonal and dendritic elongation. Finally, the present results show that environmental stimuli are capable of regulating the expression of these MAPs; the induction of each of them varies as a function of the type of signal. Thus, while diffusable substances are able to dramatically induce HMW-MAP2, MAP-1a and Tau inductions depend on cell substrate attachment and/or cell-cell interactions.

IGF-1 receptor is essential for the establishment of hippocampal neuronal polarity

How a neuron becomes polarized remains largely unknown. Results obtained with a function-blocking antibody and an siRNA targeting the insulin-like growth factor-1 (IGF-1) receptor suggest that an essential step in the establishment of hippocampal neuronal polarity and the initiation of axonal outgrowth is the activation of the phosphatidylinositol 3-kinase (PI3k)-Cdc42 pathway by the IGF-1 receptor, but not by the TrkA or TrkB receptors.

An immunocytochemical analysis of the ontogeny of the microtubule-associated proteins MAP-2 and Tau in the nervous system of the rat

The developmental distribution patterns of beta-tubulin and the microtubule-associated proteins, MAP-2 and Tau, were studied by immunocytochemistry with monoclonal antibodies. The analysis of the in situ distribution of these proteins in embryonic brain tissue revealed intense immunoreactivity for beta-tubulin in proliferative and migrating neuroblasts. On the contrary, no immunoreactivity for MAP-2 or Tau was detected in this neuroepithelium; specific immunostaining for these MAPs was only present in those neuroblasts which have reached their final destination within a developing brain area, and have initiated terminal differentiation, i.e. the sprouting of axons and dendrites. During the initial stages of neuritic outgrowth both MAPs were detected in the somatodendritic compartment of developing brain neurons; Tau was also present in axons. While the distribution of MAP-2 remained essentially the same throughout development, Tau was progressively lost from cell bodies and dendrites. This pattern of compartmentation was observed in pyramidal neurons of the cerebral cortex and hippocampus, as well as in cells of other brain regions (e.g. thalamus, hypothalamus, cerebral amygdala and tectum). It was not detected in cerebellar Purkinje cells which compartmentalize Tau to axons from the outset of neuritic differentiation, and in neurons of the Gasser ganglion which transiently express MAP-2 in axons. The expression and distribution of these MAPs was also analyzed in embryonic cerebellar and hippocampal pyramidal neurons grown in culture. Both MAPs were found in these cells as soon as 6 h after plating; they were also present in all of the neurites, axons and dendrites, that these cells extend after development in vitro for several days. With subsequence development (more than 4 days in vitro) MAP-2 was lost from axons, while Tau remained homogeneously distributed in both types of neurites. Taken collectively, the present results indicate that the development of the compartmentalized distribution of MAP-2 and Tau follows a complex pattern which is specific for each of these MAPs, and which varies as a function of the neuron type and the conditions under which the cell develops. In addition, the complex variations in the distribution of both MAPs during in situ and in vitro development make it unlikely that these proteins have a role in determining the fate of a neurite as an axon or a dendrite.

PI3K activation by IGF-1 is essential for the regulation of membrane expansion at the nerve growth cone

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 neurons 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 TC10-exo70 complex is essential for membrane expansion and axonal specification in developing neurons

Axonal elongation is one of the hallmarks of neuronal polarization. This phenomenon requires axonal membrane growth by exocytosis of plasmalemmal precursor vesicles (PPVs) at the nerve growth cone, a process regulated by IGF-1 activation of the PI3K (phosphatidylinositol-3 kinase) pathway. Few details are known, however, about the targeting mechanisms for PPVs. Here, we show, in cultured hippocampal pyramidal neurons and growth cones isolated from fetal rat brain, that IGF-1 activates the GTP-binding protein TC10, which triggers translocation to the plasma membrane of the exocyst component exo70 in the distal axon and growth cone. We also show that TC10 and exo70 function are necessary for addition of new membrane and, thus, axon elongation stimulated by IGF-1. Moreover, expression silencing of either TC10 or exo70 inhibit the establishment of neuronal polarity by hindering the insertion of IGF-1 receptor in one of the undifferentiated neurites. We conclude that, in hippocampal pyramidal neurons in culture, (1) membrane expansion at the axonal growth cone is regulated by IGF-1 via a cascade involving TC10 and the exocyst complex, (2) TC10 and exo70 are essential for the polarized externalization of IGF-1 receptor, and (3) this process is necessary for axon specification.

LIM Kinase 1 and Cofilin Regulate Actin Filament Population Required for Dynamin-dependent Apical Carrier Fission from the Trans-Golgi Network

The functions of the actin cytoskeleton in post-Golgi trafficking are still poorly understood. Here, we report the role of LIM Kinase 1 (LIMK1) and its substrate cofilin in the trafficking of apical and basolateral proteins in Madin-Darby canine kidney cells. Our data indicate that LIMK1 and cofilin organize a specialized population of actin filaments at the Golgi complex that is selectively required for the emergence of an apical cargo route to the plasma membrane (PM). Quantitative pulse-chase live imaging experiments showed that overexpression of kinase-dead LIMK1 (LIMK1-KD), or of LIMK1 small interfering RNA, or of an activated cofilin mutant (cofilin S3A), selectively slowed down the exit from the trans-Golgi network (TGN) of the apical PM marker p75-green fluorescent protein (GFP) but did not interfere with the apical PM marker glycosyl phosphatidylinositol-YFP or the basolateral PM marker neural cell adhesion molecule-GFP. High-resolution live imaging experiments of carrier formation and release by the TGN and analysis of peri-Golgi actin dynamics using photoactivatable GFP suggest a scenario in which TGN-localized LIMK1-cofilin regulate a population of actin filaments required for dynamin-syndapin-cortactin-dependent generation and/or fission of precursors to p75 transporters.

The Cdk5‐p35 kinase associates with the Golgi apparatus and regulates membrane traffic

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 α‐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.

Regulation of membrane expansion at the nerve growth cone

Exocytotic incorporation of plasmalemmal precursor vesicles (PPVs) into the cell surface is necessary for neurite extension and is known to occur mainly at the growth cone. This report examines whether this is a regulated event controlled by growth factors. The Golgi complex and nascent PPVs of hippocampal neurons in culture were pulse-labeled with fluorescent ceramide. We studied the dynamics of labeled PPVs upon arrival at the axonal growth cone. In controls and cultures stimulated with brain-derived neurotrophic factor (BDNF), PPV clusters persisted in growth cones with a half-life (t1/2) of >14 minutes. Upon challenge with IGF-1, however, fluorescent elements cleared from the growth cones with a t1/2 of only 6 minutes. Plasmalemmal expansion was measured directly as externalization of membrane glycoconjugates in resealed growth cone particles (GCPs) isolated from fetal forebrain. These assays demonstrated that membrane expansion could be stimulated by IGF-1 in a dose-dependent manner but not by BDNF, even though intact, functional BDNF receptor was present on GCPs. Because both BDNF and IGF-1 are known to enhance neurite growth, but BDNF did not stimulate membrane expansion at the growth cone, we studied the effect of BDNF on the IGF-1 receptor. BDNF was found to cause the translocation of the growth-cone-specific IGF-1 receptor subunitβ gc to the distal axon, in a KIF2-dependent manner. We conclude that IGF-1 stimulates axonal assembly at the growth cone, and that this occurs via regulated exocytosis of PPVs. This mechanism is affected by BDNF only indirectly, by regulation of the βgc level at the growth cone.