Expression of kinesin superfamily genes in cultured hippocampal neurons.

The nature of the different kinesin family members that function in a single, specific neuron type has not been systematically investigated. Here, we used quantitative real-time PCR to analyze the developmental expression patterns of kinesin family genes in cultured mouse hippocampal neurons, a highly homogeneous population of nerve cells. For purposes of comparison, we also determined the set of kinesins expressed in embryonic and adult hippocampal tissue. Twenty kinesins are expressed at moderate-to-high levels in mature hippocampal cultures. These include 9 plus-end directed kinesins from the Kinesin-1, -2, and -3 families that are known to mediate organelle transport and 6 other members of the Kinesin-3 and -4 families that are candidate organelle motors. Hippocampal cultures express high levels of a Kinesin-13, which regulates microtubule depolymerization, and moderate-to-high levels of Kinesin-9 and -14 family members, whose functions are not understood. Twelve additional kinesins, including 10 known mitotic kinesins, are expressed at moderate levels in embryonic hippocampus but at very low levels in mature cultures and the adult hippocampus. Collectively, our findings suggest that kinesins subserve diverse functions within a single type of neuron.

Motifs that mediate dendritic targeting in hippocampal neurons: a comparison with basolateral targeting signals.

One model for dendritic protein sorting in neurons is based on parallels with basolateral targeting in Madin-Darby Canine Kidney (MDCK) epithelial cells. The goal of this study was to further evaluate this model by analyzing the neuronal targeting of several proteins that contain well-defined basolateral sorting motifs. When we expressed FcRgammaII-B2 and CD44, two basolateral markers whose sorting depends on dihydrophobic motifs, they were unpolarized in hippocampal neurons. We also assessed the localization of the Epidermal Growth Factor Receptor (EGFR), a basolateral protein whose sorting signal contains a proline-rich motif and two dihydrophobic motifs. EGFR was restricted to the dendrites in neurons and relied on the same sorting signal for proper targeting. These results show that the dendritic sorting machinery in neurons does not recognize dihydrophobic-based basolateral sorting signals. In contrast, the sorting signal present in EGFR directs both basolateral and dendritic targeting and defines a novel dendritic targeting motif.

Examination of oxidized cellulose as a macromolecular prodrug carrier: preparation and characterization of an oxidized cellulose-phenylpropanolamine conjugate.

The purpose of this study was to investigate the use of 6-carboxycellulose (OC), a biocompatible and bioresorbable polymer, as a prodrug carrier for amine drugs. Phenylpropanolamine hydrochloride (PPA.HCl) was used as a model drug. OC and PPA were reacted in dimethylformamide (DMF) in the presence of 1,3-dicyclohexylcarbodiimide (DCC) for 2.5 days at room temperature. Filtration, followed by washing with methanol, and subsequent drying under vacuum, produced the conjugate in 65-78% yield. The amount of PPA in the product, determined from the difference in the carboxylic content before and after the reaction, was 24.2% (w/w), corresponding to a degree of substitution (DS) value of 0.7. The Fourier transform-infra red (FT-IR) spectrum of the conjugate, compared with that of OC and PPA.HCl, showed a new band at about 1533 cm(-1) attributable to a C = O (amide II) stretching and N single bond H (amide I and amide II) bending vibrations, a decrease in intensity of the characteristic free carboxylic acid carbonyl stretching band at about 1748 cm(-1), and a strong band at 1663 cm(-1) due to C = O (amide I) stretching vibration, suggesting that the OC is linked to PPA via an amide bond. The solid-state carbon-13 cross polarization/magic angle spinning nuclear magnetic resonance ((13)CCP/MAS NMR) spectrum of the conjugate was also consistent with this structure. The release studies performed in pH 4.5, 5.5, and 7.4 buffer solutions and in rat liver homogenate (pH 7.4), showed the conjugate to be more susceptible to hydrolysis at a lower pH and in the presence of rat liver homogenate. In conclusion, the results presented show that OC can be covalently linked to amine drugs via an amide bond in DMF using DCC as a coupling agent, and provide a macromolecular prodrug delivery system.

Sorting and directed transport of membrane proteins during development of hippocampal neurons in culture.

Hippocampal neurons in culture develop morphological polarity in a sequential pattern; axons form before dendrites. Molecular differences, particularly those of membrane proteins, underlie the functional polarity of these domains, yet little is known about the temporal relationship between membrane protein polarization and morphological polarization. We took advantage of viral expression systems to determine when during development the polarization of membrane proteins arises. All markers were unpolarized in neurons before axonogenesis. In neurons with a morphologically distinguishable axon, even on the first day in culture, both axonal and dendritic proteins were polarized. The degree of polarization at these early stages was somewhat less than in mature cells and varied from cell to cell. The cellular mechanism responsible for the polarization of the dendritic marker protein transferrin receptor (TfR) in mature cells centers on directed transport to the dendritic domain. To examine the relationship between cell surface polarization and transport, we assessed the selectivity of transport by live cell imaging. TfR-green fluorescent protein-containing vesicles were already preferentially transported into dendrites at 2 days, the earliest time point we could measure. The selectivity of transport also varied somewhat among cells, and the amount of TfR-green fluorescent protein fluorescence on intracellular structures within the axon correlated with the amount of cell surface expression. This observation implies that selective microtubule-based transport is the primary mechanism that underlies the polarization of TfR on the cell surface. By 5 days in culture, the extent of polarization on the cell surface and the selectivity of transport reached mature levels.

Compression, compaction, and disintegration properties of low crystallinity celluloses produced using different agitation rates during their regeneration from phosphoric acid solutions.

The tabletting characteristics of low crystallinity celluloses (LCPC)-LCPC-700, LCPC-2000, and LCPC-4000-prepared using agitation rates of 700, 2000, and 4000 rpm, respectively, during their regeneration from phosphoric acid, were evaluated and compared with those of Avicel PH-102 and Avicel PH-302. The mean deformation pressure values calculated from the linear region of the Athy-Heckel curves indicated LCPC-4000 to be the most ductile material. The area under the Athy-Heckel curve for LCPC-4000 was 330 MPa, whereas LCPC-700 and LCPC-2000 showed a corresponding value similar to that of Avicel PH-102 and Avicel PH-302 (192-232 MPa). The tensile strength of LCPC and Avicel compacts increased linearly with increasing applied pressures. A comparison of the area under the tensile strength-compression pressure curves indicated that LCPC-4000 formed the strongest tablets. The strengths of LCPC-700 and LCPC-2000 compacts, in contrast, were slightly lower than that of Avicel PH-302 and Avicel PH-102, respectively. The compacts of both LCPC-4000 and Avicel PH-102 were intact in water for 6 hours, whereas LCPC-2000 and Avicel PH-302 compacts disintegrated in 4 minutes and 2 minutes, respectively. In conclusion, LCPC-4000 was the most ductile material and exhibited the highest compression and compaction characteristics. The corresponding properties of LCPC-700 and LCPC-2000, in contrast, were comparable to that of Avicel PH-102 or Avicel PH-302.

The role of selective transport in neuronal protein sorting.

To assess whether selective microtubule-based vesicle transport underlies the polarized distribution of neuronal proteins, we expressed green fluorescent protein- (GFP-) tagged chimeras of representative axonal and dendritic membrane proteins in cultured hippocampal neurons and visualized the transport of carrier vesicles containing these proteins in living cells. Vesicles containing a dendritic protein, transferrin receptor (TfR), were preferentially transported into dendrites and excluded from axons. In contrast, vesicles containing the axonal protein NgCAM (neuron-glia cell adhesion molecule) were transported into both dendrites and axons. These data demonstrate that neurons utilize two distinct mechanisms for the targeting of polarized membrane proteins, one (for dendritic proteins) based on selective transport, the other (for axonal proteins) based on a selectivity "filter" that occurs downstream of transport.

Neuronal calcium activates a Rap1 and B-Raf signaling pathway via the cyclic adenosine monophosphate-dependent protein kinase.

Activity-dependent regulation of neuronal events such as cell survival and synaptic plasticity is controlled by increases in neuronal calcium levels. These actions often involve stimulation of intracellular kinase signaling pathways. For example, the mitogen-activated protein kinase, or extracellular signal-regulated kinase (ERK), signaling cascade has increasingly been shown to be important for the induction of gene expression and long term potentiation. However, the mechanisms leading to ERK activation by neuronal calcium are still unclear. In the present study, we describe a protein kinase A (PKA)-dependent signaling pathway that may link neuronal calcium influx to ERKs via the small G-protein, Rap1, and the neuronal Raf isoform, B-Raf. Thus, in PC12 cells, depolarization-mediated calcium influx led to the activation of B-Raf, but not Raf-1, via PKA. Furthermore, depolarization also induced the PKA-dependent stimulation of Rap1 and led to the formation of a Rap1/B-Raf signaling complex. In contrast, depolarization did not lead to the association of Ras with B-Raf. The major action of PKA-dependent Rap1/B-Raf signaling in neuronal cells is the activation of ERKs. Thus, we further show that, in both PC12 cells and hippocampal neurons, depolarization-induced calcium influx stimulates ERK activity in a PKA-dependent manner. Given the fact that both Rap1 and B-Raf are highly expressed in the central nervous system, we suggest that this signaling pathway may regulate a number of activity-dependent neuronal functions.

Aligned microcontact printing of micrometer-scale poly-L-lysine structures for controlled growth of cultured neurons on planar microelectrode arrays.

We describe a method for producing high-resolution chemical patterns on surfaces to control the attachment and growth of cultured neurons. Microcontact printing has been extended to allow the printing of micron-scale protein lines aligned to an underlying pattern of planar microelectrodes. Poly-L-lysine (PL) lines have been printed on the electrode array for electrical studies on cultured neural networks. Rat hippocampal neurons showed a high degree of attachment selectivity to the PL and produced neurites that faithfully grew onto the electrode recording sites.

Bone morphogenetic protein-7 enhances dendritic growth and receptivity to innervation in cultured hippocampal neurons.

Members of the bone morphogenetic protein (BMP) family of growth factors are present in the central nervous system during development and throughout life. They are known to play an important regulatory role in cell differentiation, but their function in postmitotic telencephalic neurons has not been investigated. To address this question, we examined cultured hippocampal neurons following treatment with bone morphogenetic protein-7 (BMP-7, also referred to as osteogenic protein-1). When added at the time of plating, BMP-7 markedly stimulated the rate of dendritic development. Within 1 day, the dendritic length of BMP-7-treated neurons was more than twice that of controls. By three days the dendritic arbors of BMP-7-treated neurons had attained a level of branching similar to that of 2-week-old neurons cultured under standard conditions. Several findings indicate that BMP-7 selectively enhances dendritic development. While dendritic length was significantly increased in BMP-7-treated neurons, the length of the axon was not. In addition, the mRNA encoding the dendritic protein MAP2 was significantly increased by BMP-7 treatment, but the mRNA for tubulin was not. Finally, BMP-7 did not enhance cell survival. Because dendritic maturation is a rate-limiting step in synapse formation in hippocampal cultures, we examined whether BMP-7 accelerated the rate at which neurons became receptive to innervation. Using two separate experimental paradigms, we found that the rate of synapse formation (assessed by counting synapsin I-positive presynaptic vesicle clusters) was increased significantly in neurons that had been exposed previously to BMP-7. Because BMP-7 and related BMPs are expressed in the hippocampus in situ, these factors may play a role in regulating dendritic branching and synapse formation in both development and plasticity.

Differential effects of NgCAM and N-cadherin on the development of axons and dendrites by cultured hippocampal neurons.

A fundamental step in neuronal development is the acquisition of a polarized form, with distinct axons and dendrites. Although the ability to develop a polarized form appears to be largely an intrinsic property of neurons, it can be influenced by environmental cues. For example, in cell cultures substrate and diffusible factors can enhance and orient axonal development. In this study we examine the effects of growth on each of two cell adhesion molecules (CAMs), NgCAM and N-cadherin, on the development of polarity by cultured hippocampal neurons. We find that although the same pattern of development occurs on control substrates and the CAMs, the CAMs greatly accelerate the rate and extent of development of axons-axons from sooner and grow longer on the CAMs than on the control substrate. In contrast, the CAMs have opposite effects on dendritic development-N-cadherin enhances, but NgCAM reduces dendritic growth compared to control. These results provide further evidence that the development of polarity is largely determined by a cell-autonomous program, but that environmental cues can independently regulate axonal and dendritic growth.

Gangliosides GM1 and GD1b are not polarized in mature hippocampal neurons.

Analysis of the binding of cholera toxin to ganglioside GM1 in both living and fixed neurons, and comparison with the distribution of defined axonal and dendritic proteins, demonstrates that ganglioside GM1 is distributed in a non-polarized manner over the axonal and dendritic plasma membranes of mature, cultured hippocampal neurons. Likewise, ganglioside GD1b is also distributed in a non-polarized manner. These results suggest that a recent report [Ledesma, M.D. et al. EMBO J. 18 (1999) 1761-1771] proposing that ganglioside GM1 is highly enriched on the axonal versus dendritic membrane of hippocampal neurons may need to be re-evaluated.

Local presentation of substrate molecules directs axon specification by cultured hippocampal neurons.

Axon specification is a crucial, early step in neuronal development, but little is known about how this event is controlled in vivo. To test the hypothesis that local presentation of growth-promoting molecules can direct axon specification, we cultured hippocampal neurons on substrates patterned with stripes of poly-L-lysine and either laminin (LN) or the neuron-glia cell adhesion molecule (NgCAM). Although undifferentiated neurites contacted both substrates equally, axons formed preferentially on LN or NgCAM. Time-lapse studies revealed that changes in the growth pattern of a cell indicative of axon specification began almost immediately after the growth cone of one of the neurites of the cell contacted LN or NgCAM. When cells were plated on alternating stripes of LN and NgCAM, cells with their somata on LN usually formed axons on NgCAM, whereas those with somata on NgCAM preferentially formed axons on LN. This suggests that the change from one axon-promoting substrate to another also provides a signal sufficient to specify the axon. These results demonstrate that contact with preferred substrate molecules can govern which neurite becomes the axon and thus direct the development of neuronal polarity.

Role of moving growth cone-like "wave" structures in the outgrowth of cultured hippocampal axons and dendrites.

Hippocampal neurons exhibit periodically recurring growth cone-like structures, referred to as "waves," that emerge at the base of neurites and travel distally to the tip. As a wave nears the tip, the neurite undergoes retraction, and when it reaches the tip, the neurite undergoes a burst of growth. At 1 day in culture, during early axon outgrowth, axons undergo an average 7.5-microm retraction immediately preceding wave arrival at the tip followed by 12-microm growth immediately after arrival (an average net growth of 4.5 microm). In branched axons, waves often selectively travel down one branch or the other. Growth selectively occurs in the branch chosen by the wave. In dendrites, which grow much slower on average, wave-associated retractions are much greater, resulting in less net growth. In the presence of Brefeldin A, which disrupts membrane traffic through the Golgi apparatus and leads to retraction of the axon, axonal waves continue to be associated with both growth spurts and retractions. The magnitude of the growth spurts is not significantly different from untreated axons, but wave-associated retractions are significantly increased. The close association between waves and cyclical elongation suggests that waves may act to bring about this pattern of growth. Our results also show that modulation of regularly occurring retraction phases plays a prominent role in determining average outgrowth rates.

Activity-dependent regulation of dendritic BC1 RNA in hippocampal neurons in culture.

Several neuronal RNAs have been identified in dendrites, and it has been suggested that the dendritic location of these RNAs may be relevant to the spatiotemporal regulation of mosaic postsynaptic protein repertoires through transsynaptic activity. Such regulation would require that dendritic RNAs themselves, or at least some of them, be subject to physiological control. We have therefore examined the functional regulation of somatodendritic expression levels of dendritic BC1 RNA in hippocampal neurons in culture. BC1 RNA, an RNA polymerase III transcript that is a component of a ribonucleoprotein particle, became first detectable in somatodendritic domains of developing hippocampal neurons at times of initial synapse formation. BC1 RNA was identified only in such neurons that had established synapses on cell bodies and/or developing dendritic arbors. When synaptic contact formation was initiated later in low-density cultures, BC1 expression was coordinately delayed. Inhibition of neuronal activity in hippocampal neurons resulted in a substantial but reversible reduction of somatodendritic BC1 expression. We conclude that expression of BC1 RNA in somatic and dendritic domains of hippocampal neurons is regulated in development, and is dependent upon neuronal activity. These results establish (for the first time to our knowledge) that an RNA polymerase III transcript can be subject to control through physiological activity in nerve cells.

The polarized sorting of membrane proteins expressed in cultured hippocampal neurons using viral vectors.

One model of neuronal polarity (Dotti and Simons, 1990) proposes that neurons and polarized epithelia use similar mechanisms to sort membrane proteins. To explore this hypothesis, we used viral vectors to express proteins in cultured neurons and assessed their distribution using quantitative immunofluorescence microscopy. Basolateral epithelial proteins were polarized to dendrites; more significantly, mutations of sequences required for their basolateral targeting in epithelia also disrupted dendritic targeting. Unexpectedly, apical proteins were not polarized to axons but were expressed at roughly equal amounts in dendrites and axons. These data provide strong evidence that targeting of basolateral and dendritic proteins depends on common mechanisms. In contrast, the sorting of proteins to the axon requires signals that are not present in apical proteins.

Actin-dependent anterograde movement of growth-cone-like structures along growing hippocampal axons: a novel form of axonal transport?

In time-lapse video recordings of hippocampal neurons in culture, we have identified previously uncharacterized structures, nicknamed "waves," that exhibit lamellipodial activity closely resembling that of growth cones, but which periodically emerge at the base of axons and travel distally at an average rate of 3 microm/min. In electron micrographs of identified waves, the cortical region of the axon appears expanded to either side, forming lamellipodia like those at growth cones. No other gross differences were noted in the ultrastructural features of the axon shaft at the site of a wave. Immunocytochemistry revealed that waves contain a marked concentration of F-actin, GAP-43, cortactin, and ezrin or a related protein, constituents that are also concentrated in growth cones. Treatment with the actin-disrupting agent cytochalasin B caused a reversible collapse of lamellipodia and cessation of the forward movement of individual waves along the axon, indicating that their anterograde transport is dependent on intact actin filaments. Treatment with the microtubule-depolymerizing agent nocodazole led to a rapid disorganization of wave structure and a subsequent suppression of wave activity that may reflect a role of microtubules in actin organization. The results suggest that actin and other cytoskeletal components concentrated in growth cones may be transported together as growth-cone-like structures from the cell body to the axon tip via an actin-dependent mechanism.

Selective blockade of axonogenesis in cultured hippocampal neurons by the tyrosine phosphatase inhibitor orthovanadate.

Protein tyrosine phosphorylation has been implicated in several aspects of neurite outgrowth regulation. To address specific roles in early neuronal morphogenesis, hippocampal neurons in culture were treated with the tyrosine phosphatase inhibitor orthovanadate. This treatment completely suppressed axon formation, yet enhanced formation of minor neurites. The inhibition of axonogenesis was dose dependent and occurred in parallel with a marked increase in cellular phosphotyrosine immunoreactivity, which was especially concentrated within neuritic growth cones and showed partial colocalization with f-actin. Both the blockade of axonogenesis and the elevation of phosphotyrosine were completely reversible. An additional and unexpected effect of orthovanadate was the appearance of many binucleate neurons. Immunoblotting experiments using a phosphotyrosine-specific antibody revealed an orthovanadate-induced reversible hyperphosphorylation of several protein bands, especially of two at 115 and 125 kD. These data suggest a potentially important role for tyrosine phosphatases and their phosphoprotein substrates in axonogenesis.

Inhibition of axonal growth by brefeldin A in hippocampal neurons in culture.

The outgrowth of neuronal processes involves a great increase in the surface area of the cell. The supply of membrane material necessarily must be coordinated with the demands for neurite growth. The selective growth of only one or two neurites at any given time during the development of polarity raises the possibility that the production of materials by the soma is limiting for growth (Dotti and Banker, 1987; Dotti et al., Goslin and Banker, 1990). To examine the role of the availability of membrane components during the development of polarity and axonal elongation, we treated neurons with brefeldin A, an antibiotic that disrupts the trafficking of vesicles from the Golgi complex to the plasma membrane. Treatment with brefeldin A (1 microg/ml) inhibited axonal growth within 0.5 hr; in unpolarized cells it prevented the formation of an axon. These results indicate that the availability of membrane components of Golgi-derived vesicles is required for axonal growth and hence the development of polarity. Inhibitors of protein and RNA synthesis also blocked axonal growth and the development of polarity, but over a much slower time course. This suggests that the full complement of proteins and mRNAs required for the initial development of polarity is present for several hours before polarity is actually established.

Delayed localization of synelfin (synuclein, NACP) to presynaptic terminals in cultured rat hippocampal neurons.

Synelfin is a presynaptic protein of unknown function that is differentially regulated in the avian song control circuit during the critical period for song learning; in humans, it gives rise to an amyloidogenic peptide found in senile plaques of Alzheimer's disease. To gain insight into the potential involvement of synelfin in synapse development, we investigated its expression in neurons cultured from the embryonic rat hippocampus. These neurons express a variety of defined synaptic proteins, and form numerous synaptic connections after several days in culture. Synapsin I, a synaptic vesicle-associated protein, was detected within one day after the neurons were put in culture, but significant immunoreactivity for synelfin was not detected until approximately 5 days in vitro (DIV). By 3 DIV, synapsin-positive puncta (previously shown to correspond to presynaptic specializations) were detected surrounding the soma and proximal dendritic processes, whereas comparable aggregations of synelfin did not appear until several days later. By 14 DIV the punctate concentrations of synelfin and synapsin overlapped completely. Thus synelfin is expressed in these cultured neurons and eventually becomes localized to presynaptic terminals, but it is absent from these specializations when they first form. We conclude that presynaptic terminals can change in molecular composition, and that synelfin is associated with later stages in synaptic development or modulation.

Low density lipoprotein receptor-related protein is expressed early and becomes restricted to a somatodendritic domain during neuronal differentiation in culture.

Low density lipoprotein receptor-related protein (LRP) is a multi-functional receptor which mediates the endocytotic uptake of several ligands implicated in neuronal pathophysiology. In this study, LRP expression and localization, in cultured hippocampal neurons from 18-day-old rats, were examined by immunofluorescence microscopy. LRP was restricted to the cell bodies and dendrites of mature neurons, where it was uniformly distributed on both dendritic shafts and spines. Immunoreactive protein was detected within the first 24 h of culture and acquired a polarized distribution by the end of the first week. Expression of LRP mRNA by the cultured neurons was demonstrated by Northern blot analysis. Binding studies with the LRP ligand, activated alpha2-macroglobulin, confirmed that LRP was present and functional on the hippocampal neuron cell surface. These studies demonstrate that neuronal LRP undergoes selective compartmentation during neuronal maturation and suggest that LRP-mediated endocytosis is largely restricted to the somatodendritic compartment.