In the present study, we investigated neurite elongation in primary cultures of rat hippocampal and cortical neurons and the differentiation of neuroblastoma cells exposed to low concentrations of DNP. The results indicate that DNP promotes neuritogenesis by increasing neuronal cAMP and tau levels, and raise the possibility that DNP can be useful in preventing neurite retraction and synaptic dysfunction in AD and in other neurodegenerative diseases.

We recently reported that 2,4‐dinitrophenol (DNP) blocks the formation of both soluble oligomers and insoluble amyloid fibrils from the β‐amyloid peptide (Aβ), the main neurotoxin involved in the pathogenesis of Alzheimer's disease (AD) ( 17 , 18 ). Furthermore, DNP blocks the neurotoxicity of Aβ to cultured rat hippocampal neurons. In an earlier study ( 17 ) we noticed that neuronal cultures treated with low concentrations of DNP appeared to exhibit longer neurite extensions than control cultures. AD has been described as a synaptic failure disease ( 19 , 20 ), which may explain the clinical observation of memory loss and cognitive impairment even before massive neuronal death takes place. Moreover, neurite dystrophy is a characteristic pathological finding in AD. Therefore, strategies aimed at preserving, reinforcing, and/or restoring the neurite network and synaptic connections might be beneficial in treating neurodegenerative diseases such as AD ( 21 ).

The best‐characterized pathway controlling neurite extension involves signaling through neurotrophin receptors to a Ras‐dependent, mitogen‐activated protein kinase (MAPK) cascade ( 3 – 6 ). In some cases, such as in nerve growth factor (NGF) ‐responsive cells, the neuritogenic signal transduction pathways have been extensively analyzed whereas the precise signaling pathways involved in the actions of other neuritogenic factors (e.g., glial‐derived factors, extracellular matrix proteins, cAMP, serum deprivation, etc.) have not been completely elucidated. For example, cAMP may activate distinct pathways under certain conditions participating in NGF‐triggered signals ( 7 , 8 ) and, in other instances, modulating neuronal responses to axonal guidance molecules ( 9 ). In any case, it is well known that cAMP plays important roles in both neuronal survival and plasticity (10; for recent reviews, see refs 11 , 12 ). In addition, recent studies have proposed that activation of cAMP signaling may constitute a new strategy for the promotion of axonal growth and functional nerve recovery ( 13 – 16 ).

The specificity of neuronal connections in the central nervous system (CNS) requires an accurate patterning of neurite extension. Neurite elongation and maturation allow the interconnection of different CNS areas and proper function of the brain. The formation and consolidation of neurites in response to extracellular signals depend on the assembly of the neuronal cytoskeleton ( 1 , 2 ). Neuronal function thus is tightly coupled to morphological changes that are determined mostly by regulation of the cytoskeleton.

Mitochondrial generation of reactive oxygen species (ROS) was assessed using the fluorescent dye CM‐H 2 DCFDA (Molecular Probes) according to manufacturer's instructions. Cortical neurons in culture were exposed to 20 µM DNP and the dye for 45 min; control cultures not exposed to DNP were also assayed. After treatment, fluorescence microscopy images of neurons in both control and DNP‐treated cultures were acquired.

The mitochondrial respiratory rates in intact N2A cells in the absence or presence of DNP were measured using a Clark‐type oxygen electrode ( 26 ). Briefly, 6.5 × 10 5 cells were suspended in 1.5 mL of DMEM and placed in the electrode cell. Oxygen consumption was measured as a function of time and DNP was added (at the concentrations indicated in Results) at different time points. Mitochondrial function was also evaluated using the mitochondrial membrane potential fluorescent indicator JC‐1 (Molecular Probes, Eugene, OR, USA) following manufacturer's instructions. In this case, cortical neurons were loaded with the dye and fluorescence microscopy images were acquired for cultures maintained in the absence or presence of 20 µM DNP.

cAMP was measured according to the competitive binding assay described by Gilman (1970) ( 25 ). Cells were preincubated for 10 min at 37°C in Eagle's basal medium, pH 7.4, containing 0.5 mM isobutylmethylxanthine and stimulated for 15 min with either 20 µM DNP or 10 µM forskolin, an adenylyl cyclase activator known to increase intracellular cAMP levels (used as a positive control). The reaction was stopped with trichloroacetic acid, the cells were freeze‐thawed and centrifuged for 5 min, and the supernatant was applied to an ion exchange column (Dowex 50) to remove trichloroacetic acid and nucleotides. The column eluate was then used in a competitive binding assay with the regulatory subunit of protein kinase A, as referenced above, with the addition of a fixed trace amount of [ 3 H] cAMP.

For specific determination of the levels of phosphorylated tau, cortical cultures were exposed to DNP and Western blot was carried out exactly as described above, except that P‐404 antibody (overnight incubation; 1:1,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used. This antibody recognizes the Ser404 phosphoepitope of tau, which is characteristically hyperphosphorylated in AD and in other tauopathies.

Primary cultures of cortical and hippocampal neurons (or, alternatively, N2A cells) were maintained for different periods in the absence or presence of different concentrations of DNP (see Results). The cultures were then rinsed with PBS and lysed in buffer containing 100 mM Tris‐HCl, pH 6.8, 4% SDS, 2 mM EDTA, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM sodium pyrophosphate, and a cocktail of protease inhibitors (10 µg/mL each of aprotinin, leupeptin, and pepstatin). The lysate was incubated in a boiling water bath for 5 min. Protein content in the samples was measured using BSA as a standard ( 23 ). Samples (30 µg of total protein applied per lane) were resolved bySDS‐PAGE on 12% gels. As an additional loading control, half of the gel was stained with Coomassie blue to allow direct comparison between the total protein mass applied to different lanes. Protein bands on the other half of the gel were transferred to nitrocellulose membranes ( 24 ). The membranes were blocked with 5% nonfat milk in Tris‐buffered saline/Tween 20 (TBS‐T) (10 mM Tris, pH 7.2, 150 mM NaCl, 0.1% Tween 20), followed by incubation for 2 h with the phosphorylation‐independent anti‐tau antibody (see above; 1:3000 dilution) at 4°C. After washing with TBS‐T, immunoreactive proteins were visualized using peroxidase‐conjugated anti‐rabbit IgG secondary antibody (overnight incubation; 1:1,000 dilution; Sigma) and enhanced chemiluminescence detection (ECL Plus kit, Amersham, Buckinghamshire, England). Densitometric scanning and quantification of the intensities in Western blot bands were carried out using Image J (NIH; Windows version).

Quantitative analysis of the immunofluorescence data was carried out by histogram analysis of the fluorescence intensity at each pixel across the images using a program developed in LabVIEW (O. Holub and S. T. Ferreira, to be published elsewhere). Briefly, the program converts an RGB bitmap image into an intensity linear grey scale image according to predefined conversion coefficients. This grey scale image is subsequently analyzed by plots of the intensity histogram and probability distribution of brightness and by calculation of several statistical parameters, including average brightness and standard deviation. The results of the analysis of multiple images acquired under each experimental condition were then combined to allow quantitative estimates of changes in neuronal tau levels induced by DNP.

Hippocampal and cortical neurons were incubated for 24 h in the absence or presence of 20 µM DNP and fixed for 20 min with 4% paraformaldehyde in 0.01 M PBS followed by blocking and permeabilization in 2.5% BSA, 0.3% Triton X‐100 in PBS. After 30 min, cells were incubated for 4 h with rabbit anti‐human tau primary antibody (1: 500 dilution; Dako, Glostrup, Denmark). This antibody recognizes both nonphosphorylated and phosphorylated forms of tau, and thus permits detection of the total level of intraneuronal tau. After washing, the cells were incubated for 2 h with anti‐rabbit IgG conjugated to Cy3. The cells were again washed and mounted for fluorescence microscopy on a Nikon Eclipse TE300 microscope.

The murine neuroblastoma cell line N2A (# CR098, purchased from the Rio de Janeiro Cell Bank, Federal University of Rio de Janeiro, Brazil) was grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Cultilab, Campinas, São Paulo, BR), 100 U/mL penicillin, and 100 µg/mL streptomycin at 37°C in a humidified atmosphere with 5% CO 2 . For morphological analyses, cells were plated at a density of 1.5 × 10 2 cells/mm 2 . After 24 h in culture, the medium was replaced by either fresh medium (for control cultures) or medium containing DNP or other additions, as described in Results. Cultures were further incubated for 24 h and evaluated for morphological changes under bright‐field illumination on a Nikon Eclipse TE300 microscope. Undifferentiated N2A cells exhibit rounded cell bodies and no processes (see Fig. 4 A ); control cultures predominantly contain such undifferentiated cells (Fig. 4 D ). Exposure of the cultures to DNP led to cellular differentiation, as indicated by star‐shaped cell bodies and the appearance of processes (Fig. 4 B, C ). These morphological criteria were used to define differentiated cells in this study. Five randomly chosen fields were photographed and examined in each of three coverslips for each experimental condition. Approximately 400 cells were scored in each condition. Similar results were obtained in three to five independent experiments using different cell cultures.

For morphometric analysis, neurons were plated at a density of 1.25 × 10 3 cells/mm 2 , stained with anti‐tau antibody (see below), and photographed in a Nikon Eclipse TE300 microscope. The number of neurites per cell and the lengths of individual neurites were analyzed using Sigma Scan Pro software (Jandel Scientific, San Rafael, CA, USA). Three experiments with neurons from different animals were performed and ∼100 neurons were analyzed in each experiment.

Hippocampal and cerebral cortical neurons (from 18‐ or 14‐day‐old rat embryos, respectively) were dissected and cultured as described previously ( 22 ). Briefly, cells were plated at a final density of 2.5 × 10 3 cells/mm 2 on glass coverslips precoated with 10 µg/mL poly‐d‐lysine in Neurobasal‐B27 medium supplemented with 1 mM glutamine. After 2 days in culture, the cells were further incubated for periods ranging from 24 to 96 h (see Results) in the absence or presence of DNP at final concentrations of 1, 3, 10, or 20 µM (from a stock solution freshly prepared in water). The cells were kept at all times at 37°C in a humidified atmosphere with 5% CO 2 .

The possibility that DNP could interfere with mitochondrial generation of reactive oxygen species (ROS) was also investigated. The fluorescence of DCFDA‐loaded cortical neurons ( 31 ) exposed to 20 µM DNP was virtually indistinguishable from that of control neurons (data not shown), indicating a lack of interference with mitochondrial ROS production under these conditions. Impairment of ROS generation was detected only in neurons exposed to higher (1 mM) concentrations of DNP.

DNP is a known uncoupler of mitochondrial oxidative phosphorylation. Therefore, it was important to determine whether, at the low concentrations used in our experiments (up to 20 µM), DNP induced any changes in mitochondrial functions. No changes in respiratory rate (oxygen consumption) of N2A cells treated with 20 µM DNP were detected using a Clark electrode ( Fig. 7 ), although mitochondrial uncoupling could be clearly detected in the presence of higher (1 mM) concentrations of DNP. Furthermore, no changes in mitochondrial membrane potential of cortical neurons exposed to 20 µM DNP were noted using the potential‐sensitive fluorescent dye JC‐1 ( 30 ) (data not shown). These results indicate that, under the experimental conditions we used, DNP had no detectable effect on mitochondrial respiration of primary neurons or N2A cells in culture.

Among other possible effects, elevation of cAMP levels may lead to the phosphorylation and activation of the extracellular signal‐regulated kinase (ERK). To investigate whether the ERK pathway was involved in the differentiation of N2A induced by DNP, we used an inhibitor of ERK1/2 phosphorylation, U0126. N2A cultures treated with DNP in the presence of U0126 exhibited only 32% of cells with differentiated morphology (Fig. 6 B ), showing a complete blockade of DNP‐induced differentiation. U0126 alone had no effect on the morphology of N2A cultures. These results implicate ERK in the signaling pathway involved in DNP‐induced N2A differentiation.

Involvement of cAMP and ERK in the differentiation of N2A cells induced by DNP. A ) Intracellular cAMP levels in control and DNP‐treated N2A cells. *A statistically significant ( P <0.01) increase in cAMP level compared with control. B ) N2A cultures were maintained in culture for 24 h in the presence of 20 µM DNP, 10 µM forskolin, 100 µM RpcAMPS, 10 µM UO126. The percentages of N2A cells exhibiting differentiated morphology (see Materials and Methods) under different conditions are shown. Bars represent means ± sd from 2–3 independent experiments in each condition (2–3 coverslips/experiment).

Similar to the result obtained with primary neuronal cultures, treatment of N2A with DNP led to an increase in intracellular cAMP levels ( Fig. 6 A ). To test the hypothesis that the morphological changes induced by DNP were mediated by cAMP accumulation, we used RpcAMPS, a cAMP structural analog known to block the effects of cAMP ( 28 ). Figure 6 B shows the percentages of N2A cells exhibiting differentiated morphology in cultures maintained for 24 h in the presence of DNP alone, forskolin (known to promote cAMP‐dependent differentiation of NG105‐15 neuroblastoma) ( 29 ), Rp‐cAMPS, or DNP + RpcAMPS. N2A cultures treated with DNP or forskolin exhibited 56% and 68% of differentiated cells, respectively. By contrast, cultures treated with DNP in the presence of RpcAMPS exhibited only 26% of differentiated cells (very similar to the percentage found in control cultures), indicating blockade of DNP‐induced differentiation. This result indicates that DNP leads to cell differentiation via a cAMP‐dependent signaling pathway. RpcAMPS by itself had no effect on N2A differentiation.

The effects of DNP on the morphology of mouse neuroblastoma N2A cells were investigated next. Most untreated N2A cells in serum‐containing medium exhibited an undifferentiated morphology, with rounded cell bodies and an absence of processes ( Fig. 5 A ). On the other hand, N2A cells treated with DNP for 24 h exhibited a differentiated morphology characterized by pyramidal shaped cell bodies projecting several neurites (Fig. 5 B ) or by the presence of occasional bipolar neurites (Fig. 5 C ). Quantitative morphological analysis of N2A cultures showed that the percentage of cells exhibiting differentiated morphology increased from 31% in control cultures to 56% in cultures exposed to DNP (Fig. 5 D ).

DNP increases cAMP levels in cortical and hippocampal neurons. Neurons were maintained in culture for 2 days prior to any treatments. Cortical ( A ) or hippocampal ( B ) neuronal cultures were then exposed to 20 µM DNP for 15 min and the intracellular levels of cAMP were measured as described in Materials and Methods. Bars represent means ± sd from 5 independent determinations. *A statistically significant ( P <0.01) increase compared with control cultures.

Since cAMP is known to induce neuritogenesis (for a recent example, see ref 27 ), we measured the intracellular levels of cAMP in cortical and hippocampal neurons in culture. Forskolin, an activator of adenylyl cyclase, was used as a positive control and evoked a 7‐fold increase in cAMP (not shown). Under the same conditions, cortical and hippocampal neurons treated with DNP exhibited 3.6‐ and 2.6‐fold increases in intracellular cAMP levels ( Fig. 4 A , B , respectively). These results suggested that the neurite outgrowth promoted by DNP in primary neuronal cultures could be due to cAMP‐dependent pathways.

Hyperphosphorylation of tau at specific serine or threonine residues is characteristic of a number of tauopathies and results in the impairment of its ability to act as a microtubule‐associated protein. This raised the question of whether the increase in tau levels induced by DNP might be accompanied by an increase in tau phosphorylation. To investigate this issue, we measured levels of phosphorylated tau in cultures exposed to DNP using an antibody that recognizes tau phosphorylated at Ser404, an epitope that becomes phosphorylated in tauopathies. Figure 3 B shows that the level of Ser404 phosphorylated tau was unaffected by DNP (up to 20 µM) in cultures treated for 24 h. For longer incubation periods (48 or 96 h), the level of p‐Ser404 tau appeared to be reduced by ∼20–30% at higher DNP concentrations.

Dose‐response curve and time dependence for the increase in tau levels induced by DNP. Cortical neuronal cultures were exposed to the indicated concentrations of DNP for the periods shown. The immunoreactivities for both total tau ( A ) and phosphorylated tau ( B ) were determined in neuronal culture lysates as described in Materials and Methods. Bars represent average values from densitometric analysis (using ImageJ software; NIH Windows version) of the bands obtained in 2 separate experiments that yielded very similar results. Identical protein amounts (30 µg) were applied to each lane; protein loading in the gels was further controlled by Coomassie blue staining of lanes run in parallel.

The dose‐response and time dependence of DNP‐induced increase in intraneuronal tau levels were investigated in more detail in cortical neuronal cultures. To this end, cultures were exposed for different periods of time (24, 48, or 96 h) to 1, 3, 10, or 20 µM DNP. Figure 3 A shows immunoblot analysis of total neuronal tau levels under different conditions. A clear dose‐response curve was determined when DNP treatment was carried out for 24 h: 1 µM DNP had almost no effect on tau levels but, consistent with the results described above, a 1.6‐fold increase in tau level was induced by exposure to 20 µM DNP. Treatment of the cultures for 48 h with DNP brought about a shift in the dose‐response curve to lower concentrations, with a plateau reached at 3 µM DNP. A significantly higher increase in tau level (∼2.4‐fold) was observed in cultures treated for 48 h with 3 µM DNP. Cultures exposed to DNP for 96 h exhibited a 2‐fold increase in tau level even with as little as 1 µM DNP and a plateau at 3 µM DNP corresponding to a 3.5‐fold increase in total tau

DNP increases the levels of tau in cortical and hippocampal neurons. Control ( A, B ) or DNP‐treated ( C, D ) hippocampal and cortical neurons ( A, C and B, D , respectively) were immunostained for tau as described in Materials and Methods. To allow direct comparison of tau imunnofluorescence, identical conditions and exposure times for image acquisition were used for control and DNP‐treated cultures. This explains the faint appearance of the images of control cultures ( A, B ). Scale bar corresponds to 25 µm. E ) Immunoreactivity for tau in cortical and hippocampal culture lysates. Bars represent average values from densitometric analysis of the bands obtained in 2 separate experiments using ImageJ software (NIH Windows version). Identical protein amounts (60 µg) were applied to each lane; protein loading in the gels was further controlled by Coomassie blue staining of lanes run in parallel.

Changes in intracellular levels of tau were also investigated by immunoblot analysis of lysates of neuronal cultures. This showed that the immunoreactivities for tau were significantly increased (1.9‐fold and 1.7‐fold, respectively) by treatment with DNP in cortical and hippocampal cultures (Fig. 2 E ). Immunoblot analysis also showed that intracellular tau levels were increased by ∼1.8‐fold in the murine neuroblastoma N2A cell line (not shown).

Because a large body of evidence indicates that the cytoskeleton, and the microtubular system in particular, is a key determinant of neurite elongation in central neurons, we examined the possibility that DNP modulates the neuronal levels of tau, a microtubule‐associated protein that plays a major role in microtubule assembly and stability and in neurite outgrowth. In both cortical and hippocampal neurons in culture, treatment for 24 h with DNP induced a significant increase in immunolabeling for tau compared with control cultures ( Fig. 2 A–D ). Quantitative analysis of the immunofluorescence data was carried out as described in Materials and Methods and indicated that DNP increased the levels of tau in cortical and hippocampal neurons by ∼1.5‐ and 2‐fold, respectively ( Table 1 ).

The results also show an increase in the number of primary neurites per neuron in DNP‐treated cultures. Neurons treated with DNP presented multiple filopodial spines along their dendritic axes, whereas most of the neurites in control cultures appeared devoid of or exhibited very few spines (Fig. 1 E ). Qualitatively similar results were obtained with hippocampal neurons treated with DNP (data not shown).

DNP promotes neurite outgrowth in cortical neurons. After 2 days in culture, 20 µM DNP ( B ) or an equivalent volume of water ( A ) were added and the cultures were fixed after 24 h. Neuronal morphology was examined by immuno‐staining with an anti‐tau polyclonal antibody. Scale bar corresponds to 50 µm. C ) Distribution of number of neurites per cell in control (white bars) or DNP‐treated cultures (black bars). D ) Distribution of neurite lengths in control (white bars) or DNP‐treated cultures (black bars). E ) Percentages of neurons exhibiting filopodial spines in their dentritic axes in control (white bar) or DNP‐treated cultures (black bar).

The effects of DNP on neurite elongation were initially investigated in rat embryo cortical neurons. After 2 days in culture, DNP (20 µM) was added to the culture medium and the neurons were fixed 24 h later. Visual inspection of the cultures showed that neurons maintained in the presence of DNP exhibited significantly longer neurites than neurons in untreated cultures (Fig. 1 A, B ). Morphometric analysis of a large number of representative fields was carried out, and both the number of neurites per cell and the lengths of individual primary neurites were determined. Plots of the distributions of frequencies of the number of neurites per cell and of the lengths of individual neurites are shown in Fig. 1 C, D , respectively). In control cultures, most of the neurons developed neurites with average lengths between 20 and 80 µm, whereas a significantly larger proportion of the neurons exposed to DNP exhibited neurites longer than 80 µm (Fig. 1 D ). Neurites longer than 200 were virtually absent in control cultures and were observed only in cultures treated with DNP.

DISCUSSION

The present study shows that DNP promotes neuritogenesis in cultured hippocampal and cortical neurons and differentiation in neuroblastoma cells. Neurite elongation induced by DNP is accompanied by increased levels of tau and cAMP in both neuroblastoma and primary neurons.

The cAMP cascade plays important roles in neuronal survival, plasticity, and differentiation (10). For example, cAMP leads to differentiation of cell lines derived from various tumors (32, 33). Modulation of neuronal cAMP levels has been shown to alter growth cone behavior toward a number of axon guidance cues (34–36). In addition, studies with progenitor cells in culture demonstrate that activation of cAMP‐mediated pathways increases neuronal differentiation and neurite outgrowth (37–39).

Neurons in the human central nervous system are generally considered unable to regenerate as a result of an inhibitory environment and their inherent inability to regrow (40). It has been shown that cAMP presentation at the growth cone can influence the turning behavior of nerve growth cones (34), affect guidance toward chemoattractive cues (2), and enable nerve cells from mature animals to extend fibers across inhibitory substrates (36). Thus, the modulation of neuronal cAMP levels by either direct addition of cAMP (40, 15) or the inhibition of phosphodiesterases (13, 14) could lead to promising new strategies for neuroregeneration.

Our results suggest that DNP promotes neuritogenesis in primary neuronal cultures by increasing intracellular cAMP levels (Fig. 4). In N2A cells, treatment with DNP also leads to increased intracellular cAMP levels (Fig. 6A). The cAMP analog RpcAMPS blocked the differentiation of N2A cells induced by DNP, indicating that differentiation depends on the activation of target proteins by cAMP (Fig. 6B). Blockade of DNP‐induced differentiation by U0126 indicates that activation of ERK by MEK is required for effect (Fig. 6B). Therefore, although the complete sequence of events remains to be fully elucidated, our results clearly demonstrate the dependence on cAMP and ERK activation for DNP‐induced differentiation.

Although the present data indicate that increased cAMP levels mediate the signaling of neuritogenesis and neuronal differentiation promoted by DNP, the exact manner by which DNP increases the intracellular level of cAMP is still not completely clear. Accumulation of cAMP could be due to the activation of adenylyl cyclase or the inhibition of phosphodiesterase (PDE) activities by DNP. The latter seems less likely, as all our cAMP measurements were carried out in the presence of IBMX, a phosphodiesterase inhibitor commonly used in such assays to prevent the degradation of cAMP during the measurements. Thus, PDE activity was already routinely inhibited under our experimental conditions, and it seems unlikely that the presence of an additional inhibitor might cause any additional effect on cAMP levels. On the other hand, DNP could lead to the activation of adenylyl cyclase via a direct effect on the enzyme or via its interaction with some G‐protein‐coupled receptor in the neuronal plasma membrane. Because of its amphipathic nature, DNP can permeate through the plasma membrane and may reach an intracellular level comparable to its extracellular concentration (41).

cAMP may present different intracellular targets such as guanine nucleotide exchange factors (GEFs) for Rap1 and Ras and cAMP‐dependent protein kinase (PKA) that can modulate ERK activation through various intracellular pathways. It has been shown that Epac (a Rap1‐specific GEF) synergizes with PKA and NGF to promote neurite extension in PC12 cells (42). However, it has also been reported that Epac and PKA may act in opposite directions, for example, in regulating the protein kinase B signaling cascade (43). In cortical neurons, it has been shown that activation of the cAMP cascade leads to activation of Ras and, consequently, of ERK, and that this effect is at least in part dependent on PKA (44). In hippocampal neurons, similar results have been reported, suggesting a general dependence on Ras for the activation of ERK by cAMP (45). Besides the possible involvement of PKA, a direct connection between cAMP and Ras has also been described (46). Moreover, Lin and co‐workers reported that ERK activation in hippocampal neurons and in PC12 cells expressing 5‐HT 7A receptors was not dependent on PKA but could involve Epac (47). Thus, several pathways (including the PKA, EPAC, and alternative pathways) could be activated by DNP, ultimately leading to increased tau levels and neuritogenesis.

The final differentiation of neurons is characterized by formation of functionally and structurally defined axons and dendrites. The molecular events that underlie neurite initiation and branching are not completely understood. Some of the known steps involve significant rearrangement of microtubules and their associated proteins, such as MAP2 and tau, and actin‐based structures (16). For example, an isoform of MAP was recently reported to enhance neurite initiation by stabilizing microtubules and the actin cytoskeleton (48). Here we show that DNP increases both the frequency of filopodial neurite structures and tau levels in primary neurons in culture (Figs. 1, 2). Under physiological conditions, tau facilitates tubulin assembly (49), nucleates the polymerization, and stabilizes microtubules (50–53). In primary neurons, tau has been shown to be centrally involved in neurite outgrowth (54–56). By contrast, under pathological conditions (such as in Alzheimer's disease and other tauopathies), tau undergoes abnormal hyperphosphorylation, resulting in self‐aggregation and formation of insoluble cytoplasmic inclusions, which appear to be related to the destabilization of microtubules (57). Therefore, the increase in neuronal tau levels induced by DNP may facilitate microtubule growth and/or lead to stabilization of existing microtubules, resulting in increased cellular capacity to extend neurites. An important observation was that the increase in neuronal tau levels induced by DNP was not accompanied by tau hyperphosphorylation (Fig. 3). Abnormal tau hyperphosphorylation has been shown to impair its function as a microtubule binding protein. These results thus show that exposure to DNP induces an increase in functional tau levels in central neurons.

DNP is known to be toxic at high concentrations, an effect that may be related to interference with cellular energy metabolism due to uncoupling of oxidative phosphorylation. However, it is important to note that under our experimental conditions we detected no changes in oxygen consumption (Fig. 7) or in mitochondrial membrane potential of N2A cells or cortical neurons treated with up to 20 µM DNP (data not shown). Furthermore, DNP by itself had no detectable effect on the viability of neuronal primary cultures at the low concentrations used in our studies (17).

Neurite dystrophy is a characteristic pathological finding in AD brains. Indeed, exposure of neuronal cultures to the β‐amyloid peptide (Aβ) leads to marked neurite retraction and neuronal death (e.g.,17). We recently showed that DNP blocks the oligomerization and neurotoxicity of Aβ, and inhibits amyloid deposition in an in vivo rat cerebral model of amyloidosis (17, 18). Results from the present study suggest that one of the mechanisms by which this compound protects neurons in culture from amyloid toxicity may be related to the increase in levels of tau and, ultimately, neurite stabilization and outgrowth. Indeed, a large body of evidence indicates that, in central neurons, neurite elongation is accompanied by extensive remodeling of the cytoskeleton, including the polymerization and stabilization of microtubules driven by the expression of different MAPs (48, 58–65).

There are currently no clinically approved treatments available to prevent neurodegeneration in diseases such as AD, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, and Huntington's disease. An interesting new approach that has recently been proposed is the use of growth factor gene therapy for treating the neurodegeneration observed in such diseases (21, 66). AD has recently been described as a synaptic disease (20, 67), which may explain the early cognitive loss and functional impairment observed in AD patients. Taken together, the effects of DNP in promoting neuritogenesis and neuronal differentiation described in the present work suggest that this compound may be of significant interest to the development of novel therapeutical approaches for AD and other neurodegenerative diseases.