TY - JOUR
AU - Matsumoto, Mitsuyuki
AB - Abstract In the postnatal hippocampus, newly generated neurons contribute to learning and memory. Disruptions in neurogenesis and neuronal development have been linked to cognitive impairment and are implicated in a broad variety of neurological and psychiatric disorders. To identify putative factors involved in this process, we examined hippocampal gene expression alterations in mice possessing a heterozygous knockout of the calcium/calmodulin-dependent protein kinase II alpha heterozygous knockout gene (CaMK2α-hKO), an established model of cognitive impairment that also displays altered neurogenesis and neuronal development. Using this approach, we identified gastrin-releasing peptide (GRP) as the most dysregulated gene. In wild-type mice, GRP labels NeuN-positive neurons, the lone exception being GRP-positive, NeuN-negative cells in the subgranular zone, suggesting GRP expression may be relevant to neurogenesis and/or neuronal development. Using a model of in vitro hippocampal neurogenesis, we determined that GRP signaling is essential for the continued survival and development of newborn neurons, both of which are blocked by transient knockdown of GRP’s cognate receptor (GRPR). Furthermore, GRP appears to negatively regulate neurogenesis-associated proliferation in neural stem cells both in vitro and in vivo. Intracerebroventricular infusion of GRP resulted in a decrease in immature neuronal markers, increased cAMP response element-binding protein (CREB) phosphorylation, and decreased neurogenesis. Despite increased levels of GRP mRNA, CaMK2α-hKO mutant mice expressed reduced levels of GRP peptide. This lack of GRP may contribute to the elevated neurogenesis and impaired neuronal development, which are reversed following exogenous GRP infusion. Based on these findings, we hypothesize that GRP modulates neurogenesis and neuronal development and may contribute to hippocampus-associated cognitive impairment. Stem Cells 2014;32:2454–2466 Hippocampus, Neurogenesis, Gastrin-releasing peptide, Neural stem cell, Depression, Alzheimer’s disease, Epilepsy, Schizophrenia, Bipolar disorder Introduction Many psychiatric and neurological diseases are characterized by varying degrees of cognitive impairment. The hippocampus is a key brain structure associated with cognition (including learning and memory), which functions through a tri-synaptic cellular circuit spanning several cell types that contribute uniquely to ongoing cognitive processing [1-3]. A particularly dynamic portion of this circuit exists within the dentate gyrus (DG), which contains neural stem/progenitor cells (NSCs) that perpetually generates new neurons. A growing body of evidence suggests that new neurons have the capability to modify hippocampal neural circuitry [4] and that alterations in ongoing neurogenesis have the capability to impair hippocampal function [5] and induce cognitive impairment. To this end, a significant number of studies have reported alterations in neurogenesis in several neurological and psychiatric disorders, including epilepsy [6], autism [7], schizophrenia, Alzheimer’s disease, and depression [8, 9], among others. As such, identification of common factors involved in neurogenesis and/or neuronal development within the hippocampus (particularly those associated with psychiatric/neurological diseases) remains of high interest. Using a mouse model possessing traits common to psychiatric disease and cognitive dysfunction, we sought to identify novel factors that govern hippocampal neurogenesis and/or neuronal development. For these studies, we selected calcium/calmodulin-dependent protein kinase II alpha heterozygous knockout (CaMK2α-hKO) mutant mice as our primary model system. These animals are a well-established model of cognitive impairment [10, 11] and also display hyperactive neurogenesis and impaired neuronal development within the hippocampus [12]. Furthermore, these animals are regarded as a model for schizophrenia and bipolar disorder [12], both diseases characterized by general cognitive impairment. Gastrin-releasing peptide (GRP) is an endogenous ligand for a G-protein coupled receptor family member belonging to the bombesin receptor family that functions via Gq signaling [13]. It is a key member of the bombesin pathway, with multiple roles in the body (including digestion, cancer, cell growth/proliferation, lung development, and injury) [14]. In the central nervous system (CNS), GRP regulates cell growth/proliferation during development, circadian rhythm, emotional responses, and memory [15]. Several overlapping members of the bombesin family (including GRP and neuromedin B [NMB]) exist, and receptors for both ligands are enriched in the human and rodent CNS (Supporting Information Table S1). GRP transcriptional alterations have previously been shown in the hippocampus of a number of animal models for psychiatric disease, including CaMK2α-hKO mice [12], syntenic deletion of human chromosome 22 deletion mice [16], Schnurri-2 knockout (KO) mice [17], synaptosomal-associated protein 25 knock-in mice [18], as well as SSRI-treated mice [19] and at least one mouse model of epilepsy [20]. Many of these animal models possess common hippocampal features, including altered neurogenesis, a conserved immaturity of neurons within the DG (the so-called immature DG [iDG] reviewed in [21]) and frequently display alterations in hippocampus-related function, most notably spatial memory [12, 21]. Correlates of iDG have also been identified in the DG of human psychiatric patients [22]. Based on the reports of conserved altered GRP expression in these groups, we sought to examine this factor for a putative role in neurogenesis and development of hippocampal neurons. Using in vivo and in vitro studies of hippocampal neurogenesis and maturation, we identified an inverse relationship between GRP and acute neurogenesis-associated proliferation. GRP also appears to support neuronal maturation and survival, both during development and throughout life. Animal models of cognitive dysfunction display altered levels of GRP peptide, and several hallmark deficits in these animals are reversed by GRP infusion. Based on these data, GRP expression may play a role in directly modulating neurogenesis and neuronal development and may contribute to hippocampal circuit function. Materials and Methods Animal Handling Young (P5, 14 or 28) and adult (P90) C57/Bl6 mice were used. CaMK2α-hKO mice were generated and described previously [10]. Mice were single-housed following weaning and maintained under standard housing conditions. All protocols involving animals were developed and conducted in accordance with institutional IACUC protocols. Animals were deeply anesthetized with ketamine–xylazine (80 and 5 mg/kg, respectively) and killed before tissue collection. For immunohistochemistry (IHC) studies, cardiac perfusion was performed using ice-cold 4% paraformaldehyde. Brains were removed and placed in 4% paraformaldehyde overnight, then transferred to 30% sucrose solution. For 5-bromodeoxyuridine (BrdU) incorporation studies, 100 mg/kg BrdU (Sigma, St Louis, MO) was injected and animals were killed 1 day later. For NSC isolation, killed animals were rapidly decapitated and brains were processed as described. Drug Infusion C57BL/6 mice (n = 5/6 per group) were stereotaxically implanted with permanent unilateral guide cannulae (24 gauge) under a mixture of ketamine (80 mg/kg, intraperitoneally [i.p.]) and xylazine (10 mg/kg, i.p.) anesthesia. Cannulae for the lateral ventricle were inserted according to the following coordinates: 0.4 mm posterior to bregma, 1.1 mm lateral to the midline, and 1.75 mm ventral to the skull surface with no angle. Each cannula was subsequently anchored to the skull by four stainless steel screws and dental acrylic. While the injection cannula (31 gauge), which extended 0.75 mm beyond the tip of the guide, was not in use, a 28-gauge dummy stylet maintained the patency of the guide cannula. Human GRP (Tocris) was administered in a constant volume of 5 µl over 5 minutes and administered on a dosing curve ranging from 0.05567 to 1.67 mg/kg (corresponding to 1–30X administration levels). Porcine NMB (Tocris) was given in a 5-µl volume at 0.066 mg/kg (stoichastically equivalent to 3X GRP). Four days after surgery, GRP, NMB, or vehicle was infused directly into the ventricle once a day for 10 days. After the last infusion, a single BrdU injection (100 mg/kg, i.p.) was administered. Animals were killed 24 hours later, and their brains were processed for IHC as described. Quantitative IHC Expression of calretinin, calbindin, GRP 1–27, phospho-CREB (pCREB), doublecortin (DCX), NeuN, polysialated neural cell adhesion molecule (PSA-NCAM), and Tuj1 was measured in the DG or hippocampus of C57/Bl6 mice treated with GRP 1–27, NMB, or vehicle. Mice (n = 5/6 per group) were infused as described, killed, and their brains sectioned and assayed for protein expression using IHC as described. Every fourth section was sampled for quantification. For in vitro cell analysis, nine fields were photographed per condition, with three technical replicates per experimental condition. Antibody immunoreactivity was measured using ImageJ (NIH; Bethesda, MD) and quantified as intensity/area (for sections) or intensity/total 4′,6-diamino-2-phenylindole (DAPI) cell number (for cells). Individual experimental groups were compared using paired t-test. Running Wheel Analysis Young adult wild-type (WT) and CamK2α-hKO mice (n = 6/group) were singly housed for 14 days in standard housing containing wireless running wheels (Med Associates; St. Albans, VT) or as sedentary controls. Following 2 weeks of voluntary running, running mice and sedentary controls were given a single injection of BrdU (10 mg/kg) and killed 24 hours later. The brains of one cohort were processed for IHC as described, while DG punches were taken from a second cohort for gene expression studies. Quantification of BrdU and GRP was performed on every third section: BrdU was counted manually and expressed as cells/DG section, while GRP labeling was quantitated in the DG using matched-intensity, whole-DG exposures (at 20X magnification), and proprietary quantitative software (Keyence; Elmwood Park, NJ). For GRP/BrdU coexpression studies, four sections per animal were randomly selected and BrdU/GRP coexpression was assessed using confocal microscopy. Real-Time PCR Hippocampal tissue was isolated from the proximal intersection of the dorsal and ventral blades of the DG. 500 µm coronal sections were placed under a dissection microscope. Tissue was punch-sampled using a 300 µl pipet. Four punches were isolated from serial sections and homogenized in RLT buffer (Qiagen; Germantown, MD). Cell cultures were washed with PBS and lysed in RLT buffer. RNA was isolated and purified using the RNAeasy Micro Kit (Qiagen) and reverse transcribed using the Superscript III kit (Invitrogen; Grand Island, NY) using random hexamers. cDNA quality/quantity was verified on a Nanodrop 1000 (Thermo Scientific; Waltham, MA). Human mRNA was obtained from Clontech (Mountain View, CA). Real-time quantitative polymerase chain reaction (PCR) was performed on selected genes using a Viaa 7 analyzer (Applied Biosystems; Carlsbad, CA) using Fast Sybr Green reagent (Invitrogen). Genes of interest were analyzed in duplicate technical replicates, quantified on a standard curve of genomic DNA and normalized to GAPDH or 18s mRNA. Data analysis was performed using Viaa7 RUO software (Applied Biosystems) and statistical significance calculated using Graphpad Prism (Graphpad; La Jolla, CA). NSC Isolation and Differentiation For isolating, expanding, and differentiating NSC/progenitor cells from the rodent hippocampus, we used a protocol modified from Scheffler et al. [23] and Babu et al. [24] and described in Supporting Information Methods section Immunohistochemistry IHC/ICC protocols and antibody information is described in Supporting Information Methods section. Results Characterization of GRP Expression In Vivo and In Vitro We first sought to identify the regional and cellular distribution of GRP in the brain. IHC analysis examining GRP coexpression in a variety of cell type-specific CNS markers (including GFAP, Cd11b, MBP, and NeuN) revealed the somatic expression of GRP in mature (NeuN-positive) neurons throughout the brain, including cortex layers I-VI (Fig. 1A–1C) and throughout the DG (Fig. 1D–1F; CA1 not shown). GRP was not observed to coexpress with non-neuronal lineage markers (Supporting Information Fig. S1 and data not shown). The only exception to the exclusive expression of GRP in mature neurons is GRP-positive, NeuN-negative cells in the hippocampal subgranular zone (SGZ) (Fig. 1G–1I). From this data, we suspected GRP expression in neurons may begin following their specification from NSCs. Open in new tabDownload slide Gastrin-releasing peptide (GRP) protein is expressed primarily in neurons throughout the rodent brain. Peptidyl GRP expression occurs in NeuN-positive cells in the cortex (A–C), dentate gyrus/CA3 (D–F), and subgranular zone (G–I). GRP-positive and NeuN-negative cells are occasionally expressed in the subgranular zone (arrows, G–I). Scale bar = 100 µm (A–F) and 70 µm (G–I). Abbreviations: DG, dentate gyrus; GRP, gastrin-releasing peptide; NeuN (Fox-3), subgranular zone. Open in new tabDownload slide Gastrin-releasing peptide (GRP) protein is expressed primarily in neurons throughout the rodent brain. Peptidyl GRP expression occurs in NeuN-positive cells in the cortex (A–C), dentate gyrus/CA3 (D–F), and subgranular zone (G–I). GRP-positive and NeuN-negative cells are occasionally expressed in the subgranular zone (arrows, G–I). Scale bar = 100 µm (A–F) and 70 µm (G–I). Abbreviations: DG, dentate gyrus; GRP, gastrin-releasing peptide; NeuN (Fox-3), subgranular zone. To further determine the relative regional expression of GRP, we compared GRP mRNA expression in whole brain, whole hippocampus, and the medial DG (using a punch sampling technique described in [25]) of C57/Bl6 mice. Using this method, we detected progressively higher GRP expression in the hippocampus and DG (Supporting Information Fig. S2A), matching a previously reported pattern of GRP mRNA expression (Allen Brain Atlas, [26]) and suggesting that GRP expression correlates with increasing neuronal density. Based on the observed expression of GRP in NeuN-negative cells in the SGZ, we hypothesized that GRP expression/signaling may be involved in neurogenesis and subsequent neuronal development. To determine the temporal expression of GRP during neuronal development, we measured GRP mRNA levels in differentiating NSCs isolated from the hippocampus. Under these conditions, NSCs undergo immediate, terminal differentiation mirroring organotypic generation of new neurons observed in vivo [23, 27]. Undifferentiated NSCs initially express GRP mRNA at low levels, but increase expression as newly generated neurons progressively mature (Supporting Information Fig. S2B, compared with isolated primary hippocampal neurons). GRP peptide was expressed in a diffuse pattern in undifferentiated NSCs (Supporting Information Fig. S3A) and is increasingly expressed within the soma of developing neuroblasts and postmitotic neurons (Supporting Information Fig. S3B, S3D; GRP expression in primary neurons, Supporting Information Fig. S4A–S4F) but not astrocytic glial cells (Supporting Information Fig. S3C, S3E). These data support the notion that GRP expression gradually increases in developing NSC-derived hippocampal neurons and is maintained in the mature state. GRP Effects on Neurogenesis and Neuronal Morphology From these data, we hypothesized that GRP contributed to hippocampal function, possibly through regulating neurogenesis-associated proliferation and subsequent maturation. To explore this possibility, we induced isolated hippocampal NSCs to differentiate under the influence of exogenous GRP at several points corresponding to proliferative neurogenesis (0–3 days after differentiation), early neuronal development (3–7 days after differentiation) and subsequent neuronal development/maturation (7–14 days after differentiation, Fig. 2A). Following GRP administration, total neuronal number and neuronal complexity (including dendrite length and axonal branching, 7–14 days period only, shown in Supporting Information Fig. S5) were measured. Exogenous GRP administration had minimal effect on the initial generation of neurons (Fig. 2B). However, GRP treatment of 7-day-old neurons resulted in increased survival and increased morphological maturity when examined at 14 days of age (Fig. 2C, 2D). Open in new tabDownload slide Gastrin-releasing peptide (GRP) decreases neurogenesis-associated proliferation and increases neuronal survival and morphological complexity in developing neurons. (A): Differentiating neural stem cells were treated (+) with GRP and/or siRNA against GRPR (blue line) during developmental periods (dashed red line). (B): Neuronal survival (Tuj1-positive neurons/field) is enhanced in 7-day-old neurons treated with GRP. This effect is reversed with knockdown of GRPR (B). GRP treatment of 7-day-old neurons increased neuronal complexity, as measured by total process length (C) and branch points (D) per Tuj1-positive neuron (analysis in Supporting Information Methods section). (E): GRP treatment decreases early neurogenesis-associated proliferation, which is restored by GRPR knockdown. n = 3 technical replicates from six pooled samples for (B) and (E). *, p < .05, Student’s t-test. Abbreviations: BrdU, 5-bromodeoxyuridine; GRP, gastrin-releasing peptide; GRPR, GRP’s cognate receptor; siRNA, small interfering RNA; WT, wild-type. Open in new tabDownload slide Gastrin-releasing peptide (GRP) decreases neurogenesis-associated proliferation and increases neuronal survival and morphological complexity in developing neurons. (A): Differentiating neural stem cells were treated (+) with GRP and/or siRNA against GRPR (blue line) during developmental periods (dashed red line). (B): Neuronal survival (Tuj1-positive neurons/field) is enhanced in 7-day-old neurons treated with GRP. This effect is reversed with knockdown of GRPR (B). GRP treatment of 7-day-old neurons increased neuronal complexity, as measured by total process length (C) and branch points (D) per Tuj1-positive neuron (analysis in Supporting Information Methods section). (E): GRP treatment decreases early neurogenesis-associated proliferation, which is restored by GRPR knockdown. n = 3 technical replicates from six pooled samples for (B) and (E). *, p < .05, Student’s t-test. Abbreviations: BrdU, 5-bromodeoxyuridine; GRP, gastrin-releasing peptide; GRPR, GRP’s cognate receptor; siRNA, small interfering RNA; WT, wild-type. We hypothesized that the lack of GRP sensitivity in young (<7-day-old) neurons may be related to low expression of GRP’s cognate receptor. Using matched cultures, we examined GRPR expression in naïve and differentiating NSCs and their progeny. Undifferentiated NSCs and astrocytes (defined by nestin and GFAP expression, respectively) coexpress moderate levels of GRPR (Supporting Information Fig. S6A, S6B). GRPR was also strongly expressed in more mature (≥7-day-old) neurons (Supporting Information Fig. S6A, S6D). However, newly generated (PSA-NCAM-positive) neuroblasts/early neurons express minimal GRPR (Supporting Information Fig. S6A, S6C). From these data, we believe that only naïve NSCs and postmitotic (≥7-day-old) neurons are directly sensitive to GRP, while intermediate neuroblasts are likely largely insensitive. The expression of GRPR in actively neurogenic cells is not definitive, however, as significant fraction of GRPR-positive cells that are not directly involved in neurogenesis (i.e., lineage undefined cells) are present throughout neurogenesis (Supporting Information Fig. S6A). To confirm the specificity of GRPR-mediated activity of GRP, we treated differentiating NSCs with commercial small interfering RNA (siRNA) directed against GRPR or scrambled siRNA (Fig. 2B). Selective knockdown of GRPR before 7 days after differentiation had minimal effect on neuronal generation (Fig. 2B). However, GRPR knockdown in maturing (≥7-day-old) postmitotic neurons resulted in decreased numbers of total surviving neurons (Fig. 2B) as well as reduced neuronal complexity (Fig. 2C, 2D). From these data, it appears that developing (but not newly generated) hippocampal neurons rely on GRPR-mediated signaling for continued survival and development. GRPR was expressed in a significant fraction of astrotypic/stem cells (Supporting Information Fig. S6A, S6B), indicating GRP signaling may play a significant role in early neurogenesis-associated proliferation. To examine this possibility, we measured cell division in differentiating NSCs treated with GRP and/or GRPR or scrambled siRNA. Proliferation was measured via thymidine analog incorporation in nonoverlapping 24-hour periods. Previous studies of differentiation indicate maximal levels of neurogenesis occur 3–4 days following differentiation [28]. GRP treatment significantly reduced proliferation during this period (Fig. 2E), an effect reversed by GRPR knockdown (Fig. 2E). We next evaluated the effect of GRP on neurogenesis and neuronal development within the DG by infusing 0.056–0.56 mg/kg of GRP1-27 into the lateral ventricle for 10 days. GRP infusion resulted in a significant reduction in the early neuronal markers DCX (Fig. 3A) and calretinin (Fig. 3B) in a dose-dependent manner. Interestingly, expression of pCREB, an established factor in neuronal development, was marginally (albeit significantly) increased following GRP infusion (Fig. 3C). GRP did not significantly alter mature neuronal markers (including calbindin and NeuN; data not shown). Open in new tabDownload slide In vivo administration of gastrin-releasing peptide (GRP) decreases markers of early neurons in wild-type mice. Exogenous GRP administration decreased doublecortin (A) and calretinin (B) immunoreactivity (n = 6 animals/condition for all markers, compared with vehicle-treated animals) in a dose-dependent manner. (C): GRP treatment also resulted in a small increase in CREB phosphorylation (pCREB(Ser133); (D) dentate gyrus proliferation was significantly decreased following GRP (but not neuromedin B) treatment. Scale bar = 50 µm. *, p < .05, Student’s t-test. Abbreviations: BrdU, 5-bromodeoxyuridine; GRP, gastrin-releasing peptide; NMB, neuromedin B; WT, wild-type. Open in new tabDownload slide In vivo administration of gastrin-releasing peptide (GRP) decreases markers of early neurons in wild-type mice. Exogenous GRP administration decreased doublecortin (A) and calretinin (B) immunoreactivity (n = 6 animals/condition for all markers, compared with vehicle-treated animals) in a dose-dependent manner. (C): GRP treatment also resulted in a small increase in CREB phosphorylation (pCREB(Ser133); (D) dentate gyrus proliferation was significantly decreased following GRP (but not neuromedin B) treatment. Scale bar = 50 µm. *, p < .05, Student’s t-test. Abbreviations: BrdU, 5-bromodeoxyuridine; GRP, gastrin-releasing peptide; NMB, neuromedin B; WT, wild-type. To determine whether GRP’s activity was shared by other bombesin pathway members, we infused NMB, a bombesin family member that possesses an affinity for the GRPR approximately 60-fold lower than GRP [13]. We administered NMB for 10 days at an equimolar dose to the median GRP dose tested. Unlike GRP, infusion of NMB was ineffective in altering SGZ proliferation (Fig. 3D) or decreasing immature neuronal markers (Supporting Information Fig. S7) in the DG and did not alter mature neuronal marker expression (NeuN and calbindin, Supporting Information Fig. S7A and data not shown). We measured the effect of GRP and NMB infusion on neurogenesis-associated proliferation by measuring DG cell division in WT animals receiving equimolar GRP and NMB dosing. After 9 days of treatment, a single injection of thymidine analog was administered 24 hours before termination of drug dosing. GRP—but not NMB—treatment resulted in a significant drop in labeled cell number (Fig. 3D), suggesting GRP is unique among bombesin family members in its ability to regulate DG proliferation. To further confirm the relationship between GRP and neurogenesis-associated proliferation, we modulated neurogenesis via free access to running wheels, an approach shown to elicit increases in hippocampal neurogenesis [29]. Running mice exhibited significantly increased levels of DG proliferation (Fig. 4A) and also decreased hippocampal GRP mRNA levels by an average of 96..5% (Fig. 4B). Similarly, increased numbers of BrdU-labeled cells (Fig. 4A) were accompanied by decreases in the total intensity of GRP labeling in the DG ((Fig. 4C). BrdU/GRP double-labeled cells were less prevalent in exercising mice (Fig. 4D). When same-section DG GRP expression and BrdU incorporation are compared, there is a significant inverse correlation between DG GRP expression and BrdU incorporation in both sedentary and exercising animals (Fig. 4E). Open in new tabDownload slide (A): Wild-type (WT) mice express higher levels of dentate gyrus (DG) proliferation (via 24-hour 5-bromodeoxyuridine [BrdU]-labeling, n = 6 animals/condition) after 14 days of voluntary running wheel access. These animals decrease gastrin-releasing peptide (GRP) mRNA expression (B), whole-hippocampus level of GRP immunoreactivity (measured as a fraction of WT GRP expression, (C) and the frequency of BrdU/GRP double-labeled cells (D). (E): Both exercising and sedentary mice display a significant non-zero relationship between GRP immunoreactivity and BrdU-labeled cells in the DG. There is an inverse correlation between DG GRP immunoreactivity and the total number of DG BrdU-positive cells (control/sedentary [gray] = p = .05; runner [red]: p = .004). Calcium/calmodulin-dependent protein kinase II alpha heterozygous knockout mice also downregulate mRNA for GRP in response to increased activity (B), but do not alter subgranular zone proliferation (A) or GRP peptide levels (data not shown). ***, p < .001, *, p < .05, Student’s t-test. Abbreviations: BrdU, 5-bromodeoxyuridine; DG, dentate gyrus; GRP, gastrin-releasing peptide; hKO, heterozygous knockout; WT, wild-type. Open in new tabDownload slide (A): Wild-type (WT) mice express higher levels of dentate gyrus (DG) proliferation (via 24-hour 5-bromodeoxyuridine [BrdU]-labeling, n = 6 animals/condition) after 14 days of voluntary running wheel access. These animals decrease gastrin-releasing peptide (GRP) mRNA expression (B), whole-hippocampus level of GRP immunoreactivity (measured as a fraction of WT GRP expression, (C) and the frequency of BrdU/GRP double-labeled cells (D). (E): Both exercising and sedentary mice display a significant non-zero relationship between GRP immunoreactivity and BrdU-labeled cells in the DG. There is an inverse correlation between DG GRP immunoreactivity and the total number of DG BrdU-positive cells (control/sedentary [gray] = p = .05; runner [red]: p = .004). Calcium/calmodulin-dependent protein kinase II alpha heterozygous knockout mice also downregulate mRNA for GRP in response to increased activity (B), but do not alter subgranular zone proliferation (A) or GRP peptide levels (data not shown). ***, p < .001, *, p < .05, Student’s t-test. Abbreviations: BrdU, 5-bromodeoxyuridine; DG, dentate gyrus; GRP, gastrin-releasing peptide; hKO, heterozygous knockout; WT, wild-type. To examine a putative lifelong role of GRP in mice, we next sought to determine whether GRP levels correlate with long-term changes in neurogenesis. In this case, we examined age-related changes in GRP levels in young and adult WT mice. As older rodents have progressively lower levels of neurogenesis [30, 31], we predicted increased GRP expression in aging animals. We first examined 5-day-old neonates and young adult (3-month-old) WT mice for mRNA levels of calretinin and DCX, both markers for recently generated hippocampal neurons. Messenger levels of both genes decreased with age (Fig. 5A, 5B), while GRP mRNA was significantly increased over the same period (Fig. 5C). To further confirm this, we measured GRP peptide expression in 1-, 2-, 4-, and 12-week-old animals by comparing fluorescent antibody labeling intensity. GRP expression gradually increases with progression into adulthood (Fig. 5D), suggesting a long-term inverse relationship between GRP expression and neurogenesis. Open in new tabDownload slide Gastrin-releasing peptide (GRP) inversely correlates with neurogenesis in aging. Comparison of whole-brain doublecortin (A), calretinin (B), and GRP (C) messenger in neonates (PD5) and adult (PD90) mice (n = 6 animals/group). (D): Dentate gyrus peptidyl GRP levels increase with age in wild-type animals, but are progressively reduced in calcium/calmodulin-dependent protein kinase II alpha heterozygous knockout mice beginning at 2 weeks of age. *, p < .05, Student’s t-test. Abbreviations: GRP, gastrin-releasing peptide; hKO, heterozygous knockout; WT, wild-type. Open in new tabDownload slide Gastrin-releasing peptide (GRP) inversely correlates with neurogenesis in aging. Comparison of whole-brain doublecortin (A), calretinin (B), and GRP (C) messenger in neonates (PD5) and adult (PD90) mice (n = 6 animals/group). (D): Dentate gyrus peptidyl GRP levels increase with age in wild-type animals, but are progressively reduced in calcium/calmodulin-dependent protein kinase II alpha heterozygous knockout mice beginning at 2 weeks of age. *, p < .05, Student’s t-test. Abbreviations: GRP, gastrin-releasing peptide; hKO, heterozygous knockout; WT, wild-type. GRP Expression in Genetic Models of Cognitive Impairment Finally, we sought to determine whether GRP alterations were present/contribute to the altered neurogenesis and neuronal development present in the hippocampus of CaMK2α-hKO mice (and possibly other iDG models of psychiatric disease). Previous work indicates that GRP mRNA is elevated in the hippocampus of CaMK2α-hKO mice and other iDG models (Fig. 6B and overviewed in Supporting Information Table S2), a finding seemingly at odds with the hyperactive neurogenesis and diminished neuronal maturation reported in these models [21]. As such, we first sought to directly measure GRP mRNA and protein levels in CaMK2α-hKO mutant mice. Open in new tabDownload slide Calcium/calmodulin-dependent protein kinase II alpha heterozygous knockout (CaMK2α-hKO) mice overexpress gastrin-releasing peptide (GRP) mRNA but exhibit lower levels of peptidyl GRP. (A): Major GRP isoforms. (B): GRP mRNA is overexpressed in CaMK2α-hKO mice. Antibodies directed against full-length GRP (C), mature GRP (D), and neuromedin C (E) all reveal peptidyl GRP is decreased in the hippocampus (dotted lines indicate granule cell layer border). (F): Immunoreactivity of mature GRP in dentate gyrus (DG) and CA1/3. (G): Comparison of GRP immunoreactivity in DG and CA1/3 shows no difference in the relative expression of these regions in mutant mice. n = 6 animals/group for all experiments. Scale bar = 50 µm. ***, p < .001, *, p < .05, Student’s t-test. Abbreviations: GRP, gastrin-releasing peptide; hKO, heterozygous knockout; WT, wild-type. Open in new tabDownload slide Calcium/calmodulin-dependent protein kinase II alpha heterozygous knockout (CaMK2α-hKO) mice overexpress gastrin-releasing peptide (GRP) mRNA but exhibit lower levels of peptidyl GRP. (A): Major GRP isoforms. (B): GRP mRNA is overexpressed in CaMK2α-hKO mice. Antibodies directed against full-length GRP (C), mature GRP (D), and neuromedin C (E) all reveal peptidyl GRP is decreased in the hippocampus (dotted lines indicate granule cell layer border). (F): Immunoreactivity of mature GRP in dentate gyrus (DG) and CA1/3. (G): Comparison of GRP immunoreactivity in DG and CA1/3 shows no difference in the relative expression of these regions in mutant mice. n = 6 animals/group for all experiments. Scale bar = 50 µm. ***, p < .001, *, p < .05, Student’s t-test. Abbreviations: GRP, gastrin-releasing peptide; hKO, heterozygous knockout; WT, wild-type. While GRP is reported to encode a single transcript [32], the peptide is processed into multiple active fragments, only some of which are believed to have roles within the CNS [33]. To confirm that GRP transcript encodes CNS-active peptide sequence, we compared the relative mRNA expression using qPCR primers targeting GRP1-27, the primary receptor binding region of GRP in the brain. Using this approach, we confirmed the CNS-active isoform GRP is both present and elevated in the hippocampus of CaMK2α-hKO (Fig. 6B) mice. Next, we examined GRP levels in the hippocampus of CaMK2α-hKO mice. Mutant mice displayed significant reductions in GRP peptide throughout the hippocampus, including DG, CA1, and CA3 (Fig. 6C–6F). To verify this decrease was not an artifact of selective antibody isoform labeling, we compared the relative DG expression of three different antibodies, each directed against discrete CNS-active GRP isoforms ([33], labeled region in Fig. 6A). Levels of all three isoforms of GRP peptide were reduced in mutant mice (Fig. 6C–6E). When quantified by antigen labeling intensity, mutant animals expressed significantly lower GRP1-27 (so-called “mature GRP”) throughout the hippocampus, including DG, CA1, and CA3 (Fig. 6F, although the relative expression of GRP remained constant between these regions [Fig. 6G]). To further confirm these findings, we examined hippocampal GRP mRNA and peptide levels in Schnurri-2 KO mice, a second model of iDG reported to possess increased GRP mRNA, elevated neurogenesis, impaired neuronal development and a similar behavioral phenotype to the CaMK2α-hKO strain [17]. Schnurri-2 KO animals displayed elevated levels of CNS-active hippocampal GRP mRNA (Supporting Information Fig. S8A) and decreased GRP1-27 peptide within the hippocampus (Supporting Information Fig. S8B–S8D). Previous data shows that that iDG pathophysiology (i.e., impaired neuronal development) emerges 2–4 weeks after birth [28]. We hypothesized that mutant mice may exhibit reduced levels of GRP peptide in this developmental time frame. To investigate this possibility, we compared hippocampal GRP expression of CaMK2α-hKO mice to WT littermates at 1-, 2-, 4-, and 12 weeks of age. Mutant mice progressively reduce hippocampal GRP peptide expression, beginning at 2 weeks of age and continuing into adulthood (Fig. 5D). Although only correlative, this finding maintains the possibility that GRP contributes to the development of iDG pathophysiology. Based on these observations, the transcription and translation of GRP may be somewhat uncoupled. To further examine this point, we tested the response of GRP mRNA and peptide levels in CaMK2α-hKO mice subjected to free running. Similar to their WT littermates, CaMK2α-hKO mice display lower GRP mRNA levels following voluntary exercise (Fig. 4B). However, GRP peptide levels in mutant mice were unaffected (data not shown), suggesting that mRNA, but not peptide levels could be altered via exercise. Exercise did not significantly alter total proliferation in the DG of mutant animals (likely due to a ceiling effect, Fig. 4A), nor did exercise reverse iDG deficits in CaMK2α-hKO mice (including elevated neurogenesis/immature neuron markers and decreased calbindin; Fig. 4A and data not shown). The underlying factors that decouple transcription and translation in mutant animals are unknown. Finally, to determine the capacity of GRP to reverse the hallmark deficits in iDG mice, we measured markers of neuronal development following the intracerebroventricular administration of exogenous GRP in CaMK2α-hKO mice. Treatment with GRP reversed the elevated expression of multiple conserved markers of neuronal immaturity in iDG mice, including DCX (Fig. 7A) and calretinin (Fig. 7B). GRP also decreased pCREB expression toward WT levels (Fig. 7C). NMB administration was ineffective in reverting hallmarks of immaturity in mutant mice (Supporting Information Fig. S7B). These findings reveal lowered GRP peptide expression contributes to neuronal development alterations that exist in at least one pathophysiological model of psychiatric disease. Open in new tabDownload slide Administration of exogenous gastrin-releasing peptide (GRP) reverses hallmark immaturity and pCREB hyperphosphoylation in calcium/calmodulin-dependent protein kinase II alpha heterozygous knockout mice. A 10-day infusion of GRP1-27 reversed the overexpression of doublecortin (A) and calretinin (B) and pCREB(Ser133) in mutant animals (n = 6 animals/treatment condition). All values expressed as a ratio of vehicle-treated wild-type animal immunoreactivity. Scale bar = 50 µm. ***, p < .001, Student’s t-test. Abbreviations: GRP, gastrin-releasing peptide; hKO, heterozygous knockout; pCREB, phospho-CREB; WT, wild-type. Open in new tabDownload slide Administration of exogenous gastrin-releasing peptide (GRP) reverses hallmark immaturity and pCREB hyperphosphoylation in calcium/calmodulin-dependent protein kinase II alpha heterozygous knockout mice. A 10-day infusion of GRP1-27 reversed the overexpression of doublecortin (A) and calretinin (B) and pCREB(Ser133) in mutant animals (n = 6 animals/treatment condition). All values expressed as a ratio of vehicle-treated wild-type animal immunoreactivity. Scale bar = 50 µm. ***, p < .001, Student’s t-test. Abbreviations: GRP, gastrin-releasing peptide; hKO, heterozygous knockout; pCREB, phospho-CREB; WT, wild-type. Discussion Our findings indicate that GRP has distinct roles in modulating neurogenesis and neuronal development. Specifically, GRP signaling regulates neurogenesis-associated proliferation. A second role of GRP is the regulation of neuronal development, specifically and the acquisition of phenotypic and morphological complexity. Both functions appear to be mediated primarily through GRP’s cognate receptor. Furthermore, we have identified reduced GRP peptide expression in two mouse models of schizophrenia/bipolar disorder that possess elevated neurogenesis and reduced neuronal maturity within the hippocampus. Treatment with GRP reversed these hallmark indicators, confirming GRP’s role in these processes. Alterations in neurogenesis are widely reported in a variety of psychiatric/neurological diseases, including epilepsy, Alzheimer’s disease, schizophrenia, and depression [8, 9, 34]. Examining the existing literature, we were able to identify transcriptional dysregulation of GRP in the hippocampus of several mouse models of schizophrenia/bipolar disorder and epilepsy. Hypothesizing that GRP represents a potential link between the regulation of neurogenesis, subsequent maturation and ongoing hippocampal circuit construction (which may be altered in multiple CNS diseases), we sought to identify a functional role for GRP in neurogenesis and neuronal development. Our investigations indicated GRP and its major receptor GRPR are highly expressed in the hippocampus (particularly the DG/SGZ) and confirmed its altered expression in multiple models of psychiatric disease. Furthermore, human and rodent hippocampi are enriched for GRPR (but not NMB receptor; Supporting Information Table S1 and Fig. S9), suggesting shared sensitivity to GRP. While the effect of GRP on subventricular NSCs is not known, these cells do express GRPR and may be similarly modulated. Despite its eponymous name, GRP has myriad roles in the CNS, including emotional responses, social interaction, memory, and feeding behavior [15]. We found strong GRP messenger expression in the rodent brain, especially in regions with increased density of granule cell neurons. Indeed, most GRP-positive cells coexpressed NeuN. However, GRP-positive, NeuN-negative cells were observed in the hippocampal SGZ. We isolated hippocampal NSCs, which increased mRNA and peptidyl GRP expression as they differentiate into neurons, confirming the coincidence of increasing GRP expression and neuronal identity. These findings bring into question which parts of neurogenesis are sensitive to GRP-induced signaling. In particular, it was unclear whether this pathway contributes to early neurogenesis (i.e., neurogenesis-associated proliferation), late neurogenesis (i.e., postmitotic development of immature neurons), or the linkage of these phases. GRP is rarely expressed in cells containing glial or NSC lineage markers (Supporting Information Fig. S1/S3), but these cells frequently express GRPR (Supporting Information Fig. S6), suggesting they may be sensitive to other nonlocal cells. For example, GRP-mediated signaling from neurons in other regions has been previously described [35]. Conclusion Using isolated hippocampal NSCs, we examined the effect of exogenous GRP on neurogenesis-associated proliferation. Treatment of differentiating NSCs/early progenitors with exogenous GRP suppressed proliferation. This effect appears mediated by GRP’s primary receptor, as GRPR knockdown was effective in reversing this proliferative deficit. Using a similar approach, we also tested whether GRP was effective in promoting neuronal survival and development. GRP administration of postmitotic (7-day-old) neurons resulted in an elevated neuronal survival and increased neuronal complexity. Again, this appears mediated by the GRPR, as knockdown reversed this increased survival and maturation. Interestingly, GRP treatment before this period did not affect neuronal survival (or neuroblast proliferation), a finding we attribute to a lack of GRPR expression in developing neuroblasts. Collectively, these experiments demonstrate that GRP signaling negatively regulates neurogenesis-associated proliferation in NSCs/progenitors and—at later stages of development—improves survival and morphological development of generated neurons. Ventricular infusion of GRP decreased neurogenesis-associated proliferation, matching our findings in vitro. While we were unable to detect quantifiable changes in several mature markers (most likely due to our relatively brief treatment duration or the ubiquity of these markers in DG granule cell layer), GRP treatment significantly reduced expression of hippocampal DCX and calretinin, generally in a dose-dependent manner. This unique set of effects was not shared by other members of the bombesin pathway: infusion of NMB did not result in changes in proliferation or neuronal maturation. This suggests GRP is unique among bombesin members in facilitating neurogenesis/neuronal development. Additionally, the reduced affinity of NMB for GRPR suggests this modulation occurs primarily through GRPR. We examined GRP expression and cell proliferation following modulation of neurogenesis via voluntary exercise. Free running increased neurogenesis while decreasing GRP both within the region and within labeled dividing cells. Numerous sources have documented the decreased neurogenesis (including cell division and immature neurons) that occurs in aging humans [36] and rodents [30]. We noted increased hippocampal GRP expression with increasing age and were able to identify a reciprocal relationship between GRP and DCX/calretinin mRNA. To examine the state of GRP signaling in models of psychiatric disease, we measured mRNA and peptide levels of GRP in two mouse models of schizophrenia reported previously to have elevated levels of neurogenesis [12, 17] and found both to exhibit lower levels of CNS-specific isoforms of this peptide. This is curious, as multiple iDG models of schizophrenia, bipolar disorder, and epilepsy overexpress GRP mRNA. Numerous examples of transcription/translation uncoupling have been reported [37-40] and are attributed to disparate causes, including attempted compensation [40]. CaMK2α-hKO animals receiving exogenous GRP reversed overexpression of immature neuronal markers. Interestingly, phosphorylated CREB was decreased in mutant animals towards WT levels. In NSCs isolated from CaMK2α-hKO mice, pCREB was overexpressed during early phases of differentiation [28]. While the exact mechanism for this regulation remains unknown, GRP may function through stabilizing or dampening CREB phosphorylation. We also examined the GRP-neurogenesis relationship in CaMK2α-hKO models of voluntary exercise and aging. Exercise had similar effects in reducing GRP mRNA levels in mutants, but did not alter peptide levels or ameliorate hippocampal neuronal immaturity present in this mouse model. Aging CaMK2α-hKO mice progressively exhibit decreased GRP peptide beginning at 2 weeks of age. This is noteworthy, as iDG pathophysiology (most notably neuronal immaturity) is reported to emerge at this period [28]. CaMK2α-hKO mice possess lifelong elevated neurogenesis (which is not a result of increased NSCs [Supporting Information Fig. S10]). While the full extent of GRP signaling and neurogenesis remains to be seen, it appears likely that alterations in this pathway contribute to normal short- and long-term alterations, as well as at least one model of psychiatric disease. Several key questions remain in addressing the source and function of GRP signaling. The regional expression of GRPR suggests that GRP targets stem/progenitor cells to achieve its regulation of neurogenesis. It is important to note that there is no definitive long term- or electrophysiological evidence demonstrating GRP’s direct effects on neuron functionality, and the possibility that GRP’s effects are epiphenomenal cannot be discounted. To this end, the presence of undefined GRPR-expressing cell types leaves open the possibility of indirect modulation of neurogenesis from non-neurogenic population(s). Similarly, the maturation of neurons may be regulated by neuronal populations: While at least one study has shown GRP contributes directly to the formation of new memories [35], the hippocampus is regulated by no less than 11 other regions [41], several of which are reported to modulate neurogenesis and development [28, 42-45]. Studies probing these relationships, as well as identification of GRP signaling effects on CREB and other downstream targets, may aid in elucidating a more complete global theory of hippocampal function and regulation. Author Contributions N.M.W., A.d.K., X.X., R.S., and M.M.: designed and performed experiments and wrote the manuscript; Q.C., S.M., and K.T.: provided reagents and technical assistance; J.H.K., K.T., A.K.G., and C.L.H.: provided miscellaneous assistance. N.M.W. and A.d.K. contributed equally to this article. Disclosure of Potential Conflicts of Interest The authors indicate no potential conflicts of interest. References 1 Gold AE , Kesner RP. 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TI - Gastrin-Releasing Peptide Contributes to the Regulation of Adult Hippocampal Neurogenesis and Neuronal Development
JO - Stem Cells
DO - 10.1002/stem.1740
DA - 2014-09-01
UR - https://www.deepdyve.com/lp/oxford-university-press/gastrin-releasing-peptide-contributes-to-the-regulation-of-adult-c8tObTfvMm
SP - 2454
EP - 2466
VL - 32
IS - 9
DP - DeepDyve
ER -