Discrete encounter kw to Rockford

Added: Sheron Birnbaum - Date: 25.02.2022 08:49 - Views: 28518 - Clicks: 3451

Try out PMC Labs and tell us what you think. Learn More. The primary mediators of cell migration during development, wound healing and metastasis, are receptors of the integrin family. In the developing and regenerating nervous system, chondroitin sulfate proteoglycans CSPGs inhibit the integrin-dependent migration of neuronal growth cones. Here we report that embryonic sensory neurons cultured on the growth-promoting molecule laminin in combination with the inhibitory CSPG aggrecan rapidly adapt to inhibition. Directly increasing integrin expression by adenoviral infection is sufficient to eliminate the inhibitory effects of aggrecan, indicating that upregulation of integrin receptors may Discrete encounter kw to Rockford neuronal regeneration in the presence of inhibitory matrix components.

Many matrix components are considered either growth promoting or inhibiting based on the ability of purified protein to support or block neurite outgrowth in culture. It is becoming increasingly evident, however, that the precise nature of a neuron's response to individual matrix proteins depends on both the context in which they are encountered and the past history of the individual neuron Snow and Letourneau, ; Condic and Letourneau, Recent work has shown that a of molecules can have either an attractive or a repulsive effect on neuronal outgrowth, depending on the levels of cyclic nucleotides present in the growth cone Song et al.

These suggest that the history of a growth cone or the precise balance of molecules that it encounters can greatly influence the growth response. Luo and Raper distinguish between two types of inhibitory elements for growth cones: molecules that directly impair intrinsic motility and molecules that interfere with substratum adhesion. Chondroitin sulfate proteoglycans CSPGs are a major class of inhibitory matrix proteins that have been proposed to act either as adhesion-inhibiting molecules or through receptors to directly affect cell motility Luo and Raper, ; Faissner and Steindler, ; Hoke and Silver, In the adult CNS, the upregulation of CSPGs after injury is correlated with the failure of axons to Discrete encounter kw to Rockford despite the continued presence of growth-promoting molecules such as laminin for review, see Hoke and Silver,suggesting that elevated expression of CSPGs contributes to the inability of neurons to regenerate after injury.

Aggrecan is inhibitory to the extension of retinal and sensory neurons in culture Snow and Letourneau, ; Challacombe et al. Embryonic neurons can adapt to the inhibitory effects of aggrecan over time when aggrecan is presented with growth-promoting molecules such as laminin Snow et al.

Adaptation could be accomplished either through the upregulation of receptors for growth-promoting ligands such as laminin or through the downregulation of neuronal receptors for aggrecan. Although the effects of aggrecan are likely to be mediated in part through a receptor-based mechanism Snow et al.

Neuronal outgrowth is mediated predominantly by receptors of the integrin family Letourneau et al. In sensory neurons, neurite outgrowth on ECM proteins such as laminin is entirely dependent on integrin function Tomaselli et al. In this work, we have examined the expression of integrins in response to aggrecan to determine the role of integrin regulation in neuronal adaptation to inhibitory proteoglycans.

Cell culture and substratum preparation. Neurons in suspension were harvested, rinsed in PBS, and cultured overnight on glass coverslips in serum-free media as described Gomez and Letourneau, For overnight cultures, glass coverslips were coated for 1 hr at room temperature with Engelbreth-Holm-Swarm tumor laminin gift of Dr.

Aggrecan-containing substrata for culture were prepared by coating coverslips with chick embryonic limb-bud aggrecan gift of Dr. These concentrations are likely to be above those predicted to form a molecular monolayer. Metabolically labeled chick embryonic aggrecan was obtained from Dr. David Corrino. Laminin was labeled with 3 H as ly described Snow and Letourneau, RNA and protein analysis. Immunoprecipitated proteins were size-fractionated under reducing conditions on acrylamide gels and electrophoretically transferred to nitrocellulose membrane using standard protocols.

Biotin-labeled proteins were detected using streptavidin conjugated to HRP and a chemiluminescent reagent Pierce, Rockford, IL followed by exposure to film. Ratios of protein determined from quantitation of film were comparable to those obtained by direct phosphoimaging of chemiluminescently detected proteins data not shown. Cell adhesion assays. Cells were cultured overnight on either laminin, low laminin, or laminin in combination with low levels of aggrecan see Cell culture and substratum preparation.

On the basis of binding of isotopically labeled proteins, these concentrations of applied protein resulted in surface densities of bound protein similar to those used for culturing data not shown. ANorthern analysis of integrin total RNA. BWestern analysis of biotinylated cell-surface integrin protein. To determine the effect on neuronal adhesion of applying proteins to the substrata in different order see Fig.

Aggrecan and laminin binding to the substrata were determined for all applied concentrations by inclusion of isotopically labeled protein see Cell culture and substratum preparation. Plates were prepared as follows.

At the highest concentrations tested, aggrecan is more inhibitory when applied before laminin, supporting the idea that interactions of soluble laminin with aggrecan result in greater inhibition. Adhesion is given as a percentage of adhesion to control laminin only substrata.

When error bars do not appear, they are smaller than the size of the symbol. Cell outgrowth assays. To determine neurite initiation see Fig. The total of cell bodies and neurites greater than one cell diameter in length were counted for each field and expressed as the average of neurites per cell. Dishes were allowed to equilibrate for 30 min, then images were captured for 1 hr, one image every 5 min. Images were captured every 5 min to confirm that the path of the neuron was linear over the culture period, so the average rate of growth cone advance could be determined from the linear distance traversed.

Discrete encounter kw to Rockford

The rate of axon extension was determined from the distance traversed by the growth cone in 1 hr as measured between the positions of the leading edge of the growth cone at the beginning and end of the period of image acquisition using Image-1 software. To measure outgrowth of neurons with different levels of integrin expression see Fig.

Neurons cultured on substrata containing a low concentration of the inhibitory proteoglycan aggrecan adapt to inhibition and extend neurites. ANeurons were cultured on substrata containing either laminin alone left or laminin in combination with aggrecan right. After 16—20 hr in culture, neurons have extended axons on both substrata. Outgrowth was less profuse and axons were more fasciculated on substrata containing aggrecan relative to outgrowth on laminin alone. BThe initiation of neurites is delayed on substrata containing aggrecan.

By 6 hr in culture, the of neurites initiated on aggrecan-containing substrata has greatly increased, suggesting that the neurons have adapted to inhibition. Increased expression of laminin receptors at the cell surface is associated with increased neuronal adhesion and outgrowth. Adenoviral infection.

Replication-deficient adenoviral constructs were obtained from the laboratory of Dr. Clayton Buck Wistar Institute. The integrin-expressing adenoviral constructs were prepared using standard methods Graham and Prevec, This promoter has been shown to yield efficient expression of transgenes in chick embryonic neurons Yamagata et al. NIH cells were cotransfected with linearized pAd. CMV-link plasmid with insert and the replication-deficient sub or dl adenoviral backbone.

Recombinant virus was collected from plaques, and the inserts were confirmed by PCR. The virus was subjected to three rounds of plaque purification to ensure that a single recombinant was selected and further purified by centrifugation on a cesium gradient. The titer of the purified recombinant virus was determined with plaque assay. After 16 hr, the virus was removed, and neurons were cultured for an additional 48 hr to insure strong expression of the transgene.

Arrows indicate the positions of neuronal cells. Infected neurons were cultured on LM substrata, where endogenous integrin expression is low see Materials and Methods. At this time in culture, endogenous increases in integrin expression have not yet occurred. Embryonic neurons purified from dissected dorsal root ganglia Barres et al.

Neurons were able to adhere to the substratum and extend axons after 20 hr in culture Fig. However, the outgrowth of neurons cultured in the presence of aggrecan right panel was less profuse than that of cells cultured on laminin alone left panelsuggesting that aggrecan affects either the initiation of neurites or the rate of neurite extension once initiated. By 20 hr in culture, however, neurons grown on substrata containing aggrecan had ificantly increased their rate of growth such that the rates of axon extension in the presence or absence of low levels of aggrecan were identical Discrete encounter kw to Rockford.

These data indicate that the different substrata are not selecting different neuronal subpopulations with differing abilities to adhere to and extend processes on aggrecan. If neurons that are constitutively able to adhere and extend processes in the presence Discrete encounter kw to Rockford aggrecan were being preferentially selected by aggrecan-containing substrata, then there would be no difference between the behavior of cells measured at 3 versus 20 hr in culture.

Discrete encounter kw to Rockford

Rather, the improved performance of neurons on aggrecan-containing substrata over time indicates that neurons are able to adapt to the inhibitory effects of aggrecan and extend axons under conditions that initially suppress neurite outgrowth. This adaptation could be mediated either through the upregulation of receptors for growth-promoting molecules that are also present in the substrata or through the downregulation of neuronal receptors for aggrecan in response to low levels of the inhibitor. The inhibitory effects of aggrecan on neurite initiation and the rate of neurite outgrowth were confirmed by the observation that aggrecan inhibits neuronal cell adhesion to laminin in a dose-dependent manner Fig.

In the presence of constant amounts of bound laminin, increasing concentrations of aggrecan ificantly reduced the attachment of neuronal cells. It is not currently known how aggrecan inhibits neuronal attachment and outgrowth when co-presented with ECM molecules that promote both neuronal adhesion and outgrowth, such as laminin. If aggrecan blocks access of neurons to laminin, the inhibitory effect of aggrecan on outgrowth should not be strongly influenced by the order of application of the two proteins to the substratum, as long as comparable amounts of both molecules are bound in all conditions.

However, we observed that the inhibitory potency of aggrecan was ificantly affected by the order of application of aggrecan and laminin to the substrata Fig. Aggrecan orbed to the substrata in the presence of soluble laminin was ificantly more inhibitory than comparable amounts of proteoglycan bound either before or after the application of laminin.

For the highest concentrations of proteoglycan tested, aggrecan bound after laminin the condition in which simple masking of laminin should be, if anything, most pronounced was less inhibitory than aggrecan bound before laminin. Tomaselli et al. This increase in integrin cell-surface expression was associated with an increase in integrin mRNA Fig. These suggest that aggrecan-associated increases in integrin expression are attributable to either transcriptional upregulation or an increase in the stability of the integrin message. Both of these mechanisms are distinct from the posttranslational regulation of integrin expression observed in neurons experiencing low availability of laminin Condic and Letourneau,where neurons with increased surface expression of integrins show a concomitant decrease in the levels of integrin mRNA and total protein.

The ratios of surface integrin protein and integrin RNA were calculated from densitometry of films derived from at least three experiments see Materials and Methods. Interestingly, for cells cultured on substrata containing fibronectin and Discrete encounter kw to Rockford Fig. This conclusion is supported by the fact that neurons cultured on substrata containing low levels of aggrecan and laminin adapt to inhibition Fig.

If alterations in integrin expression play a role in the adaptation of neurons to aggrecan, then increased integrin expression must be associated with changes in neuronal behaviors that are important to neurite outgrowth.

To manipulate surface integrin levels, cells were cultured overnight on laminin, low laminin, or laminin in combination with low levels of aggrecan see Cell culture and substratum preparation. These data indicate that increased cell surface integrin expression allows neurons to overcome inhibition by aggrecan and both attach and extend neurites, despite the presence of inhibitory matrix components in the extracellular environment. On this substrata, endogenous laminin receptors are expressed at low levels Fig.

Consequently, altering the expression of this receptor would be predicted to have the greatest effect on neuronal outgrowth on laminin. The levels and time course of infection were manipulated to yield an approximately twofold increase 2. This increase was similar in magnitude to that which occurs naturally in neurons that have adapted to aggrecan Fig. These demonstrate a causal link between integrin modulation and neuronal adaptation, indicating that increased integrin expression is sufficient to promote the outgrowth of neurons in the presence of aggrecan.

We have shown that integrin expression at the surface of sensory neurons increases in response to low levels of the inhibitory proteoglycan aggrecan, an ECM Discrete encounter kw to Rockford that is not itself a ligand for integrins. The aggrecan-associated increase in integrin expression occurs through a mechanism that is distinct from that seen in response to low availability of laminin Fig. Increased integrin expression is correlated with increased neuronal adhesion and neurite outgrowth in the continued presence of the inhibitor. Manipulation of integrin expression directly with adenoviral constructs indicates that high levels of integrin expression are sufficient to mediate adaptation of neurons to aggrecan.

The ability of embryonic neurons to adapt to aggrecan suggests that modulation of integrin expression contributes to the regenerative capability of embryonic neurons in the presence of inhibitory proteoglycans after injury.

Discrete encounter kw to Rockford

The regulation of integrins by aggrecan is ificant because it demonstrates that proteoglycans can alter expression of receptors that mediate neuronal interactions with other, non-proteoglycan components of the ECM and because it suggests a possible mechanism for promoting the outgrowth of adult neurons in the presence of inhibitory proteoglycans that are expressed after injury to the nervous system.

There have been conflicting reports regarding the role that CSPGs play in regulating neurite outgrowth. CSPG immunostaining in vivo often localizes to regions of embryos that exclude axons Snow et al. In contrast, CSPG immunostaining in vivo is sometimes correlated with tissues that support the growth of axons for review, see Pearlman and Sheppard,and CSPGs in combination with other matrix proteins can constitute a permissive substratum for axon outgrowth under some conditions in vitro Streit et al.

One possible explanation for these conflicting is that different studies have measured the response of neurons to different species of CSPG. Alternatively, it has been suggested that the growth-inhibiting or growth-promoting properties of CS-containing ECM preparations do not reflect CSPG function but are attributable to the presence of differing CS-binding proteins that in turn mediate the observed effects on neurite outgrowth Emerling and Lander, This idea is supported by the fact that proteins known to be inhibitory to neurite outgrowth, including tenascins and thrombospondins, also bind to CS Winnemoller et al.

A third possibility is that CSPGs do affect neurite adhesion and outgrowth, but this effect is modulated by the precise combinations or concentrations of molecules present in the ECM Snow and Letourneau, For example, the CSPGs versican and decorin inhibit adhesion of sensory neurons to fibronectin but not to laminin or collagen Braunewell et al.

A final possibility presented by the current observations is that because inhibitory CSPGs alter the neuronal response to growth-promoting matrix components over time, an individual neuron's response to CSPG can change depending on the length of time it has been in contact with CSPG-containing matrix and the availability of alternative and perhaps more permissive substrata for neurite outgrowth.

Our demonstrate that under conditions in which neurons do not have the option of extending onto more favorable substrata, they can adjust their surface expression of integrins to compensate for their environment and extend neurites, in agreement with reports of limited neurite outgrowth on substrata containing either high amounts of CSPG Snow et al. In short-term assays, aggrecan inhibits neuronal attachment Figs. If only 20 hr cultures had been examined in isolation, none of these effects would have been evident because of the adaptive response of the neurons, and quite different conclusions might have been drawn regarding the properties of aggrecan in this system.

The past experience of a growth cone may ificantly alter the strength and even the nature of a neuron's response Discrete encounter kw to Rockford what is currently present in the extracellular environment.

Discrete encounter kw to Rockford

Sensory neurons do not compensate for the inhibitory effects of aggrecan under all circumstances. When cells are cultured on substrata containing both fibronectin and aggrecan, the rate of neurite extension remains quite low relative to cells grown on fibronectin alone after 10 hr or more in culture Snow et al.

Fibronectin is known to interact with CSPGs including aggrecan.

Discrete encounter kw to Rockford

email: [email protected] - phone:(870) 742-1491 x 3069

Embryonic Neurons Adapt to the Inhibitory Proteoglycan Aggrecan by Increasing Integrin Expression