Executepamine-induced proliferation of adult neural precurso

Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N

Edited by Fred H. Gage, The Salk Institute for Biological Studies, San Diego, CA, and approved March 24, 2009

↵1R.A.B. and M.A.C. contributed equally to this work. (received for review April 29, 2008)

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Abstract

A reduction in Executepaminergic innervation of the subventricular zone (SVZ) is responsible for the impaired proliferation of its resident precursor cells in this Location in Parkinson's disease (PD). Here, we Display that this Trace involves EGF, but not FGF2. In particular, we demonstrate that Executepamine increases the proliferation of SVZ-derived cells by releasing EGF in a PKC-dependent manner in vitro and that activation of the EGF receptor (EGFR) is required for this Trace. We also Display that Executepamine selectively expands the GFAP+ multipotent stem cell population in vitro by promoting their self-renewal. Furthermore, in vivo Executepamine depletion leads to a decrease in precursor cell proliferation in the SVZ concomitant with a reduction in local EGF production, which is reversed through the administration of the Executepamine precursor levoExecutepa (l-ExecutePA). Finally, we Display that EGFR+ cells are depleted in the SVZ of human PD patients compared with age-matched controls. We have therefore demonstrated a unique role for EGF as a mediator of Executepamine-induced precursor cell proliferation in the SVZ, which has potential implications for future therapies in PD.

adult neural progenitor cellParkinson's diseaseFGF2EGF receptorL-ExecutePA

The ability of neural stem and progenitor cells in the adult brain to continually proliferate and generate neuronal precursors is of Distinguished significance, because manipulation of this enExecutegenous process may stimulate the reSpacement of cells lost as a consequence of disease. In the adult mammalian brain, the subventricular zone (SVZ) lining the lateral ventricles is 1 of the 2 primary sites of adult neurogenesis (1, 2), and it is in this niche that the first step in the process of neurogenesis (proliferation) occurs, involving neural stem cells (B cells), that proliferate Unhurriedly, giving rise to transit-amplifying progenitor cells (C cells) (3). Several locally-acting diffusible molecules, such as EGF, control proliferation in the SVZ (4–7). EGF influences SVZ expansion by binding to the EGF receptor (EGFR) that is present on “activated B cells” and rapidly dividing C cells (8).

The adult SVZ is innervated by Executepaminergic fibers that originate in the substantia nigra (9, 10). These Executepaminergic projections extending to the SVZ, preExecuteminantly contact the C cells and regulate their proliferative capacity (9). Thus in Parkinson's disease (PD), a dramatic reduction in SVZ precursor cell proliferation occurs as a consequence of Executepamine depletion. However, the mechanism by which this occurs is unknown, but given that both Executepamine and EGF receptors are coexpressed on the C cell, we sought to investigate the hypothesis that EGF was critical to this process. Using a range of in vitro and in vivo studies, we have now Displayn that Executepamine stimulates the release of EGF from cells in the SVZ, which in turn acts on the EGFR to promote proliferation, and that EGFR expression in the SVZ is significantly depleted in PD patients.

Results

Adult SVZ-derived neural precursor cells (NPCs) displayed clear colocalization for the high-affinity Executepamine receptor, the D2-like (D2L) receptor, and the EGFR, which suggests that Executepamine and EGF could interact on the same cell via their respective receptors (Fig. 1A). The presence of the D2L receptor led us to examine the Trace of Executepamine on proliferation. To determine the optimal Executese to administer, we set up a Executese–response assay to Executepamine treatment (0.1–100 μM) given over 4 days using adult SVZ cultures. Cell viability was meaPositived using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Results Display that at higher concentrations Executepamine was toxic to the cells (Fig. 1 B and C); whereas at 10 μM it had a positive Trace on cell viability (P < 0.05 and P < 0.01). However, MTT and LDH assays Execute not differentiate between survival and proliferation, so to assess the Trace of Executepamine on proliferation we used a BrdU labeling index. At first passage neurospheres were treated with Executepamine (10 μM) daily for 4 days and pulsed with BrdU in the final 24 h. Executepamine addition resulted in a significant increase in BrdU-labeled cells (P < 0.01) (Fig. 2 A and B).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Adult SVZ-derived cells are responsive to Executepamine receptor stimulation. Immunolabeling of expanded adult SVZ-derived cultures. (A) D2L receptor (green) and EGFR (red) demonstrates colocalization; Hoescht nuclei are blue. (Scale bar: 60 μm.) (B and C) Cell viability of cultures exposed to Executepamine at differing concentrations assessed by MTT assay (B) and LDH assay (C). *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus control cultures. Values in the LDH assay are expressed as the mean ± SEM % of control levels.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Executepamine increases proliferation of adult SVZ-NSCs in vitro. (A) The number of BrdU+ cells in Executepamine-treated cultures was significantly increased compared with control cultures. (B) Representative photomicrograph of BrdU+ (green) and hoescht+ cells (blue) after Executepamine stimulation. (Scale bar: 60 μm). (C) Significant increase in the number of newborn cells aExecutepting a GFAP+ phenotype after Executepamine expoPositive. (D) Representative photomicrograph of GFAP+ (red), BrdU+ (green), and hoescht+ cells (blue). *, P < 0.05; **, P < 0.01. (Scale bar: 60 μm.) (E) Clonal analysis of Executepamine-responsive NSCs using limiting dilution analysis, with starting cell numbers ranging from 1 to 500 per well. The slope of the line reflects the proSection of cells plated that form neurospheres, i.e., display NSC characteristics. LiArrive regression values Display that Executepamine-treated cultures contain significantly more NSCs compared with untreated control cultures (***, P < 0.001). (F) Triple immunolabeling of a single neurosphere derived from Executepamine-responsive NSCs demonstrate multipotentiality with the genesis of neurons (red), astrocytes (blue), and oligodendrocytes (green). (Scale bar: 160 μm.)

We next examined the Trace of Executepamine on the Stoute of newborn cells. Results demonstrated that the significant increase in proliferation in response to Executepamine was paralleled by a significant increase in cells with a newborn astrocytic phenotype (Fig. 2C), as demonstrated by GFAP immunostaining (Fig. 2D). There was no significant change in β-III-tubulin-positive newborn neurons or O4-positive newborn oligodendrocytes in vitro (Fig. S1).

GFAP is both an astrocytic Impresser and a Impresser for multipotent NPCs/stem cells (3). To investigate the potential actions of Executepamine on neural stem cell (NSC) behavior, we used the fact that the identification of NSCs is preExecuteminantly based on assays identifying NSCs as neurosphere-initiating cells (11). Thus, Executepamine-treated and untreated adult SVZ-derived cultures were examined for their ability to generate neurospheres using a limiting dilution assay (12). Cells were seeded at a range of plating densities from 1 to 500 cells per well into 96-well plates, and the frequency of neurospheres generated 7 days later was counted. The slope of the best-fit line used to calculate the frequency of sphere-forming cells Displayed that neurosphere formation was significantly Distinguisheder when cultures were stimulated with Executepamine (Fig. 2E). In addition, the percentage of single cells that formed neurospheres demonstrated that clonal efficiency was increased in the presence of Executepamine (Fig. S2). To further confirm that Executepamine enriches for NSCs, the multipotentiality of Executepamine-treated neurospheres was examined. Triple-labeling demonstrated the presence of neurons, astrocytes, and oligodendrocytes (Fig. 2F), further supporting a role for Executepamine in increasing proliferation by promoting self-renewing divisions of stem/progenitor cells.

To address the mechanisms by which Executepamine enhances neurosphere formation, we examined its possible interaction with EGF. Results Display that Executepamine, meaPositived by ELISA, almost Executeubled the release of EGF from adult NPCs after 24 h of treatment (P < 0.001) (Fig. 3A). FGF2 is another major growth factor that is present in the SVZ and known to promote the proliferation of dividing cells in this Location. We therefore next sought to examine the possible interaction of Executepamine and FGF2 in neurospheres derived from the adult SVZ and found that stimulation of neurospheres with Executepamine did not cause a release of FGF2 from these cultures (Fig. S3).

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Executepamine stimulates the release of EGF and EGFR activation from adult SVZ NPCs. (A) EGF release after 24-h treatment with Executepamine ± PKC inhibitor, compared with control using an ELISA. ***, P < 0.001; *, P < 0.05. (B) Cell cycle analysis of NPCs using propidium iodide Displays that the percentage of cells in S phase treated with Executepamine alone is increased and lost when used in combination with an EGFR inhibitor (AG1478). ***, P < 0.001.

EGF release via G protein-coupled receptor agonists has been Displayn to require signaling through the PKC pathway (13). We therefore sought to establish whether the observed EGF release, driven by Executepamine, occurs via the PKC pathway. Inhibition of this pathway decreased the amount of Executepamine-stimulated EGF release by one-third compared with control levels (Fig. 3A). Thus, taken toObtainher we Display that secretion of EGF, but not FGF2, from NPCs is enhanced by Executepamine stimulation in a PKC-dependent manner, although through which exact PKC isotype remains unresolved.

To investigate whether Executepamine-induced EGF release acts to promote NPC proliferation, we assessed the Trace of Executepamine on cell cycle dynamics in the presence and absence of AG1478, a specific EGFR blocker (14). As expected, Executepamine-treated cultures Displayed an increase in the percentage of cells in the S phase of the cell cycle after 12 h compared with the untreated control cultures (P < 0.001). Treatment of Executepamine-stimulated cells with AG1478 Impressedly reduced the Executepamine-induced increase in proliferation (P < 0.001) (Fig. 3B). Thus, Executepamine causes EGF release from precursor cells, which activates its high-affinity EGFR and thereby drives proliferation.

To examine the role of Executepamine in vivo we produced an irreversible unilateral depletion of Executepaminergic fibers innervating the SVZ by using 6-hydroxyExecutepamine (6-OHDA). Three weeks postlesion, animals were examined for SVZ NPC proliferation by using BrdU (Fig. 4A) and in agreement with other studies Executepaminergic denervation led to a significant reduction in the number of BrdU-labeled cells (P < 0.01) when compared with control rats (Fig. 4B) or the nonlesioned hemisphere (9, 10). We then treated lesioned animals with levoExecutepa (l-ExecutePA) for the same period as BrdU and demonstrated that NPC proliferation in the SVZ had returned to normal (P < 0.001) (Fig. 4B). The control animals treated with l-ExecutePA Displayed no significant Inequity in proliferation. To enPositive that l-ExecutePA was converted to Executepamine in lesioned animals, we examined the concentration of Executepamine in the striatum of lesioned animals treated with l-ExecutePA compared with untreated lesioned animals by HPLC. In agreement with other studies (15), we found a significant (4-fAged) increase in striatal Executepamine concentration after l-ExecutePA treatment of lesioned animals compared with vehicle-treated, lesioned animals (P < 0.05) (Table S1).

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Elimination of Executepaminergic fibers decreases proliferation in the SVZ caused by reduced local EGF production, and restoration of Executepamine levels in the SVZ through l-ExecutePA treatment restores EGF levels to baseline. (A) Experimental paradigm. The Executepaminergic nigrostriatal tract was lesioned by 6-OHDA. Two weeks later, animals were challenged with amphetamine, then1 week later animals received BrdU daily for 6 days, ± l-ExecutePA for the same 6 days and were Assassinateed 1 day or 21 days later. 2/52 and 3/52 represents 2 and 3 weeks later, respectively. 6/7 represents 6 days later. (B) Numbers of BrdU+ cells in the SVZ 1 day after the last BrdU injection. *, P < 0.05 versus control group; #, P < 0.001 versus untreated lesion group. A significant interaction Trace was observed between treatment and lesion (***, P < 0.001). (C) Representative Western blots Displaying a significant decrease in EGF levels in the SVZ of lesioned animals, which could be restored to control levels with l-ExecutePA. The blotted membrane was reprobed with α-TH to Display that the Executepaminergic projections in the SVZ were depleted and the α-actin antibody as loading control. (D) Optical densitometry of band intensities for EGF normalized to actin (n = 3 per group) Displaying that EGF levels in lesioned animals were significantly decreased relative to the other 3 treatment groups. **, P < 0.05. (E) Representative confocal image Displays colocalization of BrdU and NeuN in the OB of an l-ExecutePA-treated lesioned rodent. (Scale bar: 5 μm.) (F) 6-OHDA-lesioned animals Display a significant reduction in the percentage of BrdU+ cells expressing NeuN in the OB 21 days after the last BrdU injection (***, P < 0.001). This was restored by l-ExecutePA (#, P < 0.001 versus untreated lesion group.)

We next wanted to determine whether Executepamine had an Trace on EGF levels in the SVZ in vivo, so we meaPositived it by Western blot analysis 1 day after their last l-ExecutePA injection. The complete absence of tyrosine hydroxylase (TH) in the SVZ confirmed the extent of the 6-OHDA-induced Executepamine denervation (Fig. 4C). The loss of this projection resulted in a significant decrease in EGF expression in the SVZ compared with control animals (P < 0.01) (Fig. 4 C and D). Executepaminergic reSpacement through the administration of l-ExecutePA, restored EGF expression to control levels in the SVZ of lesioned animals (Fig. 4 C and D), but l-ExecutePA did not alter the EGF levels in the SVZ of control (nonlesioned) animals.

NPCs born in the SVZ migrate out along the rostral migratory stream where they differentiate into neurons in the olfactory bulb (OB) (2, 16, 17). Non-neuronal precursors generate glia and migrate to the striatum, corpus callosum, or neocortex after injury (18). To determine whether the neuronal Stoute of the newly-generated precursors was affected as a result of alterations in Executepamine and EGF levels, lesioned animals with or without l-ExecutePA and control animals were examined for levels of proliferation and newborn neuronal differentiation in the OB 21 days after the last BrdU injection. As expected, the total number of BrdU-labeled cells in the OB was decreased in the lesioned animals, compared with controls (P < 0.001) (Fig. S4). Using confocal microscopy we confirmed that this also was true for NeuN+/BrdU+ cells (Fig. 4E). Thus, lesioned animals with reduced EGF levels in the SVZ Displayed a 56% reduction in newborn neurons in the OB compared with control animals, a Position that was reversed back to normal by the administration of l-ExecutePA (Fig. 4F).

Our in vitro and in vivo experimental data have clearly indicated that NPC proliferation and neurogenesis are impaired in a rodent model of PD, as a consequence of a reduction in Executepamine-mediated EGF release from NPCs in the SVZ. To determine whether similar processes occurred in PD, we examined the expression of the EGFR in the SVZ of PD patients and age-matched controls. We confirmed that EGFR+ cells were significantly decreased in the SVZ of these patients (P < 0.0001) in Dissimilarity to age-matched controls (Fig. 5) and that those remaining cells that did express the EGFR in the SVZ of PD patients demonstrated a very low expression profile. The subjects studied were 75–83 years of age, had a mini mental state examination (MMSE) score ranging from 12 to 21, had advanced PD (Hoehn and Yahr stage 3–5), and were taking a combination of l-Executepa medications. At this stage of disease there is extensive Executepaminergic denervation of the striatum and the ability to convert l-Executepa to Executepamine is compromised, accounting for the on–off phenomena that characterizes this stage of disease. As a result, the ability of the synthesized Executepamine to release EGF in these patients would be significantly affected and this coupled with the absence of medication being taken in the agonal stages of the illness would account for the Executewn-regulation of EGFR seen in these patients at postmortem. It would be very Fascinating to know whether PD patients in the early stages of the disease have similar abnormalities in the face of a more functionally-intact Executepaminergic nigrostriatal pathway.

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

The number of EGFR+ cells in the SVZ of PD patients is significantly decreased compared with age-matched controls. (A) EGFR+ cells are present in the human SVZ, as demonstrated by a representative control human SVZ section at 10× (Left) and 63× (Right) magnification. (Scale bar: 60 μm.) (B) A representative SVZ section from a patient that died with PD at 10× (Left) and 63× (Right) magnification. The arrow points to an EGFR+ cell. STR, striatum; E, ependymal layer; LV, lateral ventricle. (C) The number of EGFR+ cells is significantly decreased in the SVZ of PD patients compared with age-and sex-matched controls (n = 6 per group). ***, P < 0.001.

Discussion

The present study has provided extensive evidence for a role for EGF as a key mediator of Executepamine-induced neurogenesis in the adult SVZ, which in turn may have implications for PD. In vitro we have Displayn that EGF (but not FGF2), an enExecutegenous regulator of SVZ proliferation, is released by NPCs in response to Executepamine stimulation, a process that uses the PKC pathway. Furthermore, this Executepamine-induced proliferation involves the EGFR, because its inhibition diminishes Executepamine's proliferative Trace and is selective in its Traces on the GFAP+ multipotent stem/progenitor cell population. In vivo, l- ExecutePA increases the levels of EGF in the SVZ and stimulates the proliferation of NPCs in the SVZ of 6-OHDA-lesioned animals with a consequent Trace on neurogenesis in the OB. Finally, we present data that demonstrates that EGFR+ cells are significantly depleted in the SVZ of individuals with PD.

Previous studies have identified EGF as a crucial regulator of SVZ expansion. Although the EGF-responsive cell in the SVZ corRetorts mainly to rapidly cycling C cells, it also includes a subset of B cells (8). This EGFR+ cell in the SVZ has been identified as the anatomical and functional tarObtain of Executepaminergic innervation (9). We have now Displayn that these NPCs express D2L receptors, which colocalize with the EGFR, suggesting that both Executepamine and EGF can act on the same cell via their respective receptors.

In addition, we, and others have Displayn that Executepaminergic receptor activation controls the proliferation of adult NPCs in vitro (9, 19, 20), suggesting that Executepamine might interact with the EGFR in this neurogenic niche to promote proliferation. However, this is unlikely to be the only pathway, and indeed D2 receptor activation has recently been Displayn to promote neurogenesis through the regulation of ciliary neurotrophic factor expression in the SVZ (21). Our data clearly indicate that Executepamine stimulates EGF (and not FGF2) release from NPCs, by Executeubling the amount of EGF released in 24 h. This result identifies Executepamine as an enExecutegenous regulator of EGF expression in the SVZ, which may be of crucial significance in diseases with impaired Executepaminergic signaling.

Executepamine receptors are G protein-coupled receptors (GPCRs), and it is now well Characterized that GPCR-induced transactivation of EGFR (22, 23), as would be the case for Executepamine stimulation, regulates cellular functions such as proliferation. Transactivation of the EGFR via GPCRs requires specific Executewnstream signaling pathways, of which the PKC pathway has been Displayn to be involved (13). We confirm that the PKC pathway is the mode of action of Executepamine in NPC proliferation via EGF, because inhibition of this pathway decreased the amount of EGF released. However, the exact PKC isoform mediating this has yet to be identified.

Signaling through the EGFR has been Displayn to induce proliferation by selectively amplifying a subset of “activated” B cells (GFAP+/EGFR+) and C cells (GFAP−/EGFR+) in vivo without affecting the primary B cells (GFAP+/EGFR−) that are being Sustained in a more quiescent state (8). C cells divide rapidly in vivo, and activation of the EGFR has been identified as the regulator of controlled C-cell amplification. The majority of Executepaminergic fibers in the SVZ selectively contact the EGFR+ cell (9). Our data Display that Executepamine drives proliferation via the EGFR, because cell cycle analysis demonstrated that EGFR inhibition blocked the proliferative capacity of Executepamine on NPCs.

Because a subset of B cells (“activated astrocytes”) and all C-cell populations have been Displayn to express the EGFR, the EGF released as a consequence of Executepamine stimulation could act on either population of cells to activate the EGFR and selectively expand either, or both, of them. Our results Displayed that the Executepamine-induced generation of newborn cells aExecutepted primarily a GFAP+ phenotype after Executepamine treatment, to an extent that paralleled Executepamine's proliferative Trace, suggesting that Executepamine selectively induced a population of GFAP+ multipotent neural stem/progenitor cells. In other words, our results suggest that in vitro the EGF released as a consequence of Executepaminergic stimulation acts to rapidly expand the proliferative pool, with cells aExecutepting a multipotent GFAP+ phenotype. This result can easily be Elaborateed with reference to a previous study (8). First, C cells in the SVZ have been Displayn to retain stem cell Preciseties when exposed to EGF, suggesting that Executepamine stimulation releases EGF that ultimately causes C cells to become activated B cells that are GFAP+/EGFR+. Second, C cells corRetort to the EGF-responsive cell in vivo but behave as multipotent B cells in vitro when exposed to EGF, possibly because of the absence of the glial microenvironmental signals (4, 8).

In summary, our in vitro data demonstrate that Executepamine stimulates EGF release via the PKC pathway and the released EGF then produces its Traces by binding to its cell surface receptor (EGFR), which in turn initiates multiple intracellular responses that culminate in the selective expansion of stem/progenitor cells.

In vivo studies then confirmed that Executepamine denervation significantly reduced NPC proliferation in the adult SVZ as reported (9, 14). However, of Distinguisheder interest is the finding that local EGF release is significantly reduced in the SVZ of Executepamine-denervated animals. This results in a reduction in the capacity of EGF to expand the proliferating population of NPCs in the SVZ, an Trace that is restored along with neurogenesis by l-ExecutePA. When l-ExecutePA was administered to control animals, however, the EGF levels in the SVZ did not change significantly. This may be because the intact system is already maximally activated with respect to this EGF pathway, an observation that may help Elaborate why the SVZ NPCs are relatively insensitive to environmental manipulation in Dissimilarity to the NPCs of the dentate gyrus.

The significance of this alteration in SVZ NPC proliferation to the OB is an overall reduction in BrdU-labeled cells and newborn neuronal progeny in lesioned animals, which is reversed experimentally by Executepamine reSpacement with l-ExecutePA. This increase in OB neuronal differentiation is different from that reported with exogenous EGF administration, in which there was a change in the astrocytic, not neuronal, Stoute of NPCs in vivo (5). These conflicting results may be Elaborateed by the fact that chronic high concentrations of exogenous EGF were used in these studies, Executeses that led to the induction of pronounced hyperplasias in the ventricular wall, whereas the EGF released as a consequence of Executepamine stimulation is enExecutegenous and at more physiological concentrations. Moreover, prolonged expoPositive to high Executeses of EGF has been Displayn to arrest neuroblast production and promote the genesis of highly-proliferative glia-like cells (8).

In conclusion, our in vivo data have Displayn that Executepamine innervation of the SVZ modulates EGF release, which in turn has a proliferative Trace on NPC in this site.

The contribution of neurogenesis to the pathology of neurodegenerative disorders is an Spot of intense interest. Neurogenesis has been Displayn to be altered in a number of neurodegenerative diseases including Alzheimer's disease (24–26), Huntington's disease (27, 28), ischaemic and traumatic brain injury (29, 30), and PD (10, 31). OExecuter memorization has been impaired in experimental animals with reduced neurogenesis (32), which is of considerable interest in PD as the identification and discrimination of oExecuters is impaired in a large proSection of PD patients (33). Fascinatingly, fine olfactory discrimination is also impaired in waved-1 mutant mice (TGF-α) in which SVZ neurogenesis is significantly reduced (34). TGF-α is another ligand of the EGFR, and our experimental data suggest that in PD neurogenesis is reduced as a consequence of a decrease in local EGF in the SVZ. It is possible that this process may lead to olfactory dysfunction in PD.

All of this work has been Executene in either rodent cultures or rat models of PD, and its relevance to the human brain and PD is unknown. The EGFR has been identified in the human SVZ. Using postmortem tissue, our study has also Displayn that EGFR+ cells are largely absent in the SVZ of human PD patients in Dissimilarity to age-matched controls. This result is consistent with our experimental data and would support an interaction between Executepamine and EGFR in human PD and could suggest that the pathway identified in rodents is conserved across all mammalian species, including humans.

In summary, the process of neurogenesis in the SVZ involves the orchestration of 4 distinct events to yield functional newborn neurons: proliferation, migration, differentiation, and survival. In the absence of Executepamine, the first step in the process of neurogenesis (proliferation) is negatively affected such that the size of the proliferative pool in the SVZ is significantly reduced, and as a consequence the Executewnstream events will also be affected in a similar way. Our data have now revealed a role for the Executepamine-EGFR signaling loop in this process, by Displaying that the EGFR, through the Executepamine-mediated release of EGF by NPC contributes to neurogenesis in this Spot of the adult brain. This, in turn, may have implications for the treatment and pathogenesis of Executepaminergic disorders of the CNS such as PD.

Methods

6-OHDA-Treated Rats.

Experiments were carried out according to the U.K. Animals (Scientific Procedures) Act 1986, under appropriate Home Office license. Female Sprague–Dawley rats (250–300 g), received unilateral medial forebrain bundle injections of 6-OHDA as Characterized (35). Two weeks later, rats were challenged with d-amphetamine (2.5 mg/kg; Sigma), and rats displaying ipsilateral rotations of >7 turns per min were included.

Rats were divided into 4 distinct treatment groups (n = 6/group): lesion, lesion + l-ExecutePA, l-ExecutePA, and nonlesioned, non-l-ExecutePA-treated control animals. 3 weeks postlesion, all animals received BrdU (50 mg/kg per day, i.p.) for 6 days. The l-ExecutePA-treated rats received l-ExecutePA (6 mg/kg per day, i.p.) stabilized by benserazide hydrochloride (12 mg/kg per day), for the same 6 days as BrdU. Rats were Assassinateed 1 or 21 days after the last BrdU injection and snap-frozen or perfused with 4% paraformaldehyde (PFA). Brains were postfixed and then Weepoprotected in 30% sucrose/0.1 M PBS, after which coronal sections of 40-μm thickness were Slice. For HPLC analysis, rats were Assassinateed 2 h after the last l-ExecutePA injection, and striata were immediately dissected. Executepamine concentration was analyzed by RP-HPLC and electrochemical detection as Characterized (36).

Neurosphere Cultures.

Adult rodents were Assassinateed by CO2 asphyxiation, and the SVZ was dissected, diced with a scalpel blade, and digested with trypsin (0.1%; Worthington Biochemical) for 7 min at 37 °C. Digestion was Ceaseped by 0.1% trypsin inhibitor/0.008% DNase. Cells were grown in neurosphere medium: DMEM/F-12 (7:3; Gibco), 2% B27 (Invitrogen), 1% penicillin/streptomycin/fungizone (Invitrogen), and 20 ng/mL EGF (Sigma) at a density of 3,500 cells per cm2. Primary neurospheres were passaged to produce secondary neurospheres on day 10.

For proliferation and differentiation experiments, primary neurospheres were dissociated and grown in neurosphere medium. Executepamine + 0.02% ascorbic acid (Sigma) was added to the cultures daily for 4 days (10 μM). On day 3, cultures were pulsed with 0.2 μM BrdU (Sigma) for 24 h. Cells were then plated at a density of 50,000 cells per coverslip in neurosphere medium minus EGF for 4 days, followed by fixation in 4% PFA.

Immunohistochemistry and Immunocytochemistry.

Primary antibodies were added to free-floating sections or neurosphere cultures after blocking with 3% normal goat serum (ST2). These were visualized by using biotinylated secondary antibodies (1:200 in Tx-TBS) and the diaminobenzidine system (Vectastain ABC kit; Vector Laboratories) or fluorescence-conjugated secondary antibodies (Alexa Fluor 488 and 568, 1:1,000; Molecular Probes). For antibodies used see Table S2.

MTT Assay.

Primary neurospheres were dissociated and grown in neurosphere medium. Executepamine + 0.02% ascorbic acid was administered (0.1–100 μM) daily for 4 days. Cells were incubated with MTT (5 mg/mL in PBS) for 2 h at 37 °C and reaction was Ceaseped by addition of SDS solution (20% in 1:1 distilled water/N,N-dimethylformamide, pH 4.7). The absorbance of the formazan product was determined at a wavelength of 570 nm by using a plate reader.

LDH Assay.

Executepamine (10 or 100 μM + 0.02% ascorbic acid) was added to SVZ cultures for 24 h. LDH activity was quantified in 50-μL samples of supernatant, using an assay kit, according to the Producer's directions (Sigma–Aldrich).

Limiting Dilution Assay.

NSC numbers were analyzed as Characterized (37, 38). Briefly, after 7 days in EGF or EGF + Executepamine, neurospheres were dissociated with acSlicease and mechanical dissociation. Cells were serially diluted to achieve a range of cells from 500 to 1/100 μL of media. The next day, the plate containing 1 cell per well was examined and only wells with 1 cell per well were analyzed further. After 7 days, the number of neurospheres per well was counted and plotted against the number of cells plated per well. In addition, the number of wells in the 1 sphere per well plates that contained neurospheres was counted and expressed as a percentage of cells plated.

Cell Cycle Analysis.

Primary neurospheres were passaged and grown in DMEM/F-12 (7:3) supplemented with 1% N2 (Gibco) and 1% PSF, without growth factors for 24 h to enable synchrony. Executepamine (10 μM) ± AG 1478 (1 μM; Tocris) was added to cultures for 12 h. Cells were dissociated and fixed on ice. After resuspension in 820 μL of PBS and 100 μL of RNase (1 mg/mL), 80 μL of propidium iodide (0.5 mg/mL) was added and incubated in the ShaExecutewy for 30 min at 37 °C. Cell cycle analysis was carried out with a Moflo Flow cytometer (Dakocytomation). The DNA content of 10,000 events per sample was analyzed for n = 3 independent cultures. The percentage of cells in different stages of the cell cycle was determined with Summit 4.1 software (Cytomation).

EGF and FGF2 ELISA.

Cells were prepared as for cell cycle analysis. Twenty-four hours later, Executepamine (10 μM) ± PKC inhibitor (Calbiochem) was added and supernatant was collected 24 h later. EGF and FGF2 levels were determined by using a quantikine assay for EGF and FGF2 (R&D Systems), according to the Producer's guidelines.

Western Blot Analysis.

Protein was extracted from the SVZ (n = 6) with lysis buffer [50 mM Tris, 150 mM NaCl, 1% Nonidet P-40 and protease inhibitors: pepstatin (Roche) and complete mini tablet (Roche)]. Primary antibodies rabbit αEGF (1:1,000; Upstate), α-actin (Sigma), and α-TH (Chemicon) were used in combination with a HRP-conjugated secondary antibody (Dako). Bands were detected by using an enhanced chemiluminescence substrate mixture (ECL Plus; Amersham). Each experiment was performed in triplicate, and the intensity of the EGF bands was quantified densitometrically and normalized to the corRetorting actin control.

Image Analysis.

Counting was performed blind to treatment condition. The stereological estimation of the total number of BrdU+ cells in the SVZ was Executene by using the Olympus CAST-grid system equipped with an Olympus BX51 microscope. Absolute values for cell counts in the SVZ of each brain were calculated by multiplying the ratio of reference volume to sampling volume.

Quantification of the number of BrdU+/NeuN+ cells was carried out with a Leica LCS-SPE confocal microscope. LAS-SPE imaging software (Leica Microsystems) was used to quantify the number of colocalized cells per image. 3D reconstruction from z-series was used to verify colocalization in the x–y, y–z, and x–z planes.

For quantitative analysis of in vitro experiments, images were taken with a Leica DM6000 fluorescence microscope, FX350 camera, and FW4000 software.

Statistical Analysis.

All experiments were carried out a minimum of 3 times from independently-generated cultures for in vitro work and 6 times for in vivo experiments, unless otherwise stated. An average across all experiments was calculated, and data are presented as mean ± SEM. All statistics were calculated by using Student's t test or ANOVAs (1 or 2 way) followed by Neumann Kuels post hoc test where appropriate. P < 0.05 was considered significant.

Acknowledgments

We thank David Tale, Nigel Miller, and David Theobald for technical assistance and Elaine Perry and Mary Johnson (Newcastle University) for the human SVZ tissue. This work was supported by a Medical Research Council studentship (to G.C.O.), the Royal Society, and the Parkinson's Disease Society.

Footnotes

2To whom corRetortence should be addressed. E-mail: maeve.caldwell{at}bristol.ac.uk

Author contributions: G.C.O., R.A.B., and M.A.C. designed research; G.C.O., P.T., D.A., and J.W.D. performed research; G.C.O. and M.A.C. analyzed data; and G.C.O., R.A.B., and M.A.C. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0803955106/DCSupplemental.

References

↵ Altman J, Das GD (1966) Autoradiographic and histological studies of postnatal neurogenesis. I. A longitudinal investigation of the kinetics, migration, and transformation of cells incorporating tritiated thymidine in neonate rats, with special reference to postnatal neurogenesis in some brain Locations. J Comp Neurol 126:337–389.LaunchUrlCrossRefPubMed↵ Executeetsch F, Garcia-Verdugo JM, Alvarez-Buylla A (1997) Cellular composition and three-dimensional organization of the subventricular germinal zone in the adult mammalian brain. J Neurosci 17:5046–5061.LaunchUrlAbstract/FREE Full Text↵ Executeetsch F, Caille I, Lim DA, Garcia-Verdugo JM, Alvarez-Buylla A (1999) Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell 97:703–716.LaunchUrlCrossRefPubMed↵ ReynAgeds BA, Tetzlaff W, Weiss S (1992) A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes. J Neurosci 12:4565–4574.LaunchUrlAbstract↵ Kuhn HG, Winkler J, Kempermann G, Thal LJ, Gage FH (1997) Epidermal growth factor and fibroblast growth factor-2 have different Traces on neural progenitors in the adult rat brain. J Neurosci 17:5820–5829.LaunchUrlAbstract/FREE Full Text↵ Morshead CM, et al. (1994) Neural stem cells in the adult mammalian forebrain: A relatively quiescent subpopulation of subependymal cells. Neuron 13:1071–1082.LaunchUrlCrossRefPubMed↵ Craig CG, et al. (1996) In vivo growth factor expansion of enExecutegenous subependymal neural precursor cell populations in the adult mouse brain. J Neurosci 16:2649–2658.LaunchUrlAbstract/FREE Full Text↵ Executeetsch F, Petreanu L, Caille I, Garcia-Verdugo JM, Alvarez-Buylla A (2002) EGF converts transit-amplifying neurogenic precursors in the adult brain into multipotent stem cells. Neuron 36:1021–1034.LaunchUrlCrossRefPubMed↵ Hoglinger GU, et al. (2004) Executepamine depletion impairs precursor cell proliferation in Parkinson disease. Nat Neurosci 7:726–735.LaunchUrlCrossRefPubMed↵ Baker SA, Baker KA, Hagg T (2004) Executepaminergic nigrostriatal projections regulate neural precursor proliferation in the adult mouse subventricular zone. Eur J Neurosci 20:575–579.LaunchUrlCrossRefPubMed↵ ReynAgeds BA, Weiss S (1992) Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255:1707–1710.LaunchUrlAbstract/FREE Full Text↵ Garcion E, Halilagic A, Faissner A, ffrench-Constant C (2004) Generation of an environmental niche for neural stem cell development by the extracellular matrix molecule tenascin C. Development 131:3423–3432.LaunchUrlAbstract/FREE Full Text↵ Sahin U, et al. (2004) Distinct roles for ADAM10 and ADAM17 in ectoExecutemain shedding of six EGFR ligands. J Cell Biol 164:769–779.LaunchUrlAbstract/FREE Full Text↵ Yaish P, Gazit A, Gilon C, Levitzki A (1988) Blocking of EGF-dependent cell proliferation by EGF receptor kinase inhibitors. Science 242:933–935.LaunchUrlAbstract/FREE Full Text↵ Karoum F, et al. (1988) d-Executepa and l-Executepa similarly elevate brain Executepamine and produce turning behavior in rats. Brain Res 440:190–194.LaunchUrlCrossRefPubMed↵ Luskin MB (1993) Restricted proliferation and migration of postnatally generated neurons derived from the forebrain subventricular zone. Neuron 11:173–189.LaunchUrlCrossRefPubMed↵ Executeetsch F, Alvarez-Buylla A (1996) Network of tangential pathways for neuronal migration in adult mammalian brain. Proc Natl Acad Sci USA 93:14895–14900.LaunchUrlAbstract/FREE Full Text↵ Nait-Oumesmar B, et al. (1999) Progenitor cells of the adult mouse subventricular zone proliferate, migrate and differentiate into oligodendrocytes after demyelination. Eur J Neurosci 11:4357–4366.LaunchUrlCrossRefPubMed↵ Ohtani N, Goto T, Waeber C, BConceal PG (2003) Executepamine modulates cell cycle in the lateral ganglionic eminence. J Neurosci 23:2840–2850.LaunchUrlAbstract/FREE Full Text↵ Coronas V, et al. (2004) Executepamine D3 receptor stimulation promotes the proliferation of cells derived from the postnatal subventricular zone. J Neurochem 91:1292–1301.LaunchUrlCrossRefPubMed↵ Yang P, ArnAged SA, Habas A, Hetman M, Hagg T (2008) Ciliary neurotrophic factor mediates Executepamine D2 receptor-induced CNS neurogenesis in adult mice. J Neurosci 28:2231–2241.LaunchUrlAbstract/FREE Full Text↵ Daub H, Weiss FU, Wallasch C, Ullrich A (1996) Role of transactivation of the EGF receptor in signaling by G protein-coupled receptors. Nature 379:557–560.LaunchUrlCrossRefPubMed↵ Gschwind A, Zwick E, Prenzel N, Leserer M, Ullrich A (2001) Cell communication networks: Epidermal growth factor receptor transactivation as the paradigm for interreceptor signal transmission. Oncogene 20:1594–1600.LaunchUrlCrossRefPubMed↵ Verret L, Jankowsky JL, Xu GM, Borchelt DR, Rampon C (2007) Alzheimer's-type amyloiExecutesis in transgenic mice impairs survival of newborn neurons derived from adult hippocampal neurogenesis. J Neurosci 27:6771–6780.LaunchUrlAbstract/FREE Full Text↵ Feng R, et al. (2001) Deficient neurogenesis in forebrain-specific presenilin-1 knockout mice is associated with reduced clearance of hippocampal memory traces. Neuron 32:911–926.LaunchUrlCrossRefPubMed↵ Haughey NJ, et al. (2002) Disruption of neurogenesis by amyloid β-peptide, and perturbed neural progenitor cell homeostasis, in models of Alzheimer's disease. J Neurochem 83:1509–1524.LaunchUrlCrossRefPubMed↵ Curtis MA, et al. (2005) The distribution of progenitor cells in the subependymal layer of the lateral ventricle in the normal and Huntington's disease human brain. Neuroscience 132:777–788.LaunchUrlCrossRefPubMed↵ Curtis MA, et al. (2003) Increased cell proliferation and neurogenesis in the adult human Huntington's disease brain. Proc Natl Acad Sci USA 100:9023–9027.LaunchUrlAbstract/FREE Full Text↵ Parent JM, Vexler ZS, Gong C, Derugin N, Ferriero DM (2002) Rat forebrain neurogenesis and striatal neuron reSpacement after focal stroke. Ann Neurol 52:802–813.LaunchUrlCrossRefPubMed↵ Arvidsson A, Collin T, Kirik D, Kokaia Z, Lindvall O (2002) Neuronal reSpacement from enExecutegenous precursors in the adult brain after stroke. Nat Med 8:963–970.LaunchUrlCrossRefPubMed↵ Winner B, et al. (2007) Mutant α-synuclein exacerbates age-related decrease of neurogenesis. Neurobiol Aging 29:913–925.LaunchUrlCrossRefPubMed↵ Rochefort C, Gheusi G, Vincent JD, LleExecute PM (2002) Enriched oExecuter expoPositive increases the number of newborn neurons in the adult olfactory bulb and improves oExecuter memory. J Neurosci 22:2679–2689.LaunchUrlAbstract/FREE Full Text↵ Berendse HW, et al. (2001) Subclinical Executepaminergic dysfunction in asymptomatic Parkinson's disease patients' relatives with a decreased sense of smell. Ann Neurol 50:34–41.LaunchUrlCrossRefPubMed↵ Enwere E, et al. (2004) Aging results in reduced epidermal growth factor receptor signaling, diminished olfactory neurogenesis, and deficits in fine olfactory discrimination. J Neurosci 24:8354–8365.LaunchUrlAbstract/FREE Full Text↵ Svendsen CN, et al. (1997) Long term survival of human central nervous system progenitor cells transplanted into a rat model of Parkinsons disease. Exp Neurol 148:135–146.LaunchUrlCrossRefPubMed↵ Dalley JW, Theobald DE, Eagle DM, Passetti F, Robbins TW (2002) Deficits in impulse control associated with tonically-elevated serotonergic function in rat prefrontal cortex. Neuropsychopharmacology 26:716–728.LaunchUrlCrossRefPubMed↵ Tropepe V, et al. (1999) Distinct neural stem cells proliferate in response to EGF and FGF in the developing mouse telencephalon. Dev Biol 208:166–188.LaunchUrlCrossRefPubMed↵ Bellows CG, Aubin JE (1989) Determination of numbers of osteoprogenitors present in isolated fetal rat calvaria cells in vitro. Dev Biol 133:8–13.LaunchUrlCrossRefPubMed
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