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Keywords:

  • diabetes;
  • galanin;
  • GalR3;
  • neural stem cells;
  • neuroprotection

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
Thumbnail image of graphical abstract

Type 2 diabetes impairs adult neurogenesis which could play a role in the CNS complications of this serious disease. The goal of this study was to determine the potential role of galanin in protecting adult neural stem cells (NSCs) from glucolipotoxicity and to analyze whether apoptosis and the unfolded protein response were involved in the galanin-mediated effect. We also studied the regulation of galanin and its receptor subtypes under diabetes in NSCs in vitro and in the subventricular zone (SVZ) in vivo. The viability of mouse SVZ-derived NSCs and the involvement of apoptosis (Bcl-2, cleaved caspase-3) and unfolded protein response [C/EBP homologous protein (CHOP) Glucose-regulated protein 78/immunoglobulin heavy-chain binding protein (GRP78/BiP), spliced X-box binding protein 1 (XBP1), c-Jun N-terminal kinases (JNK) phosphorylation] were assessed in the presence of glucolipotoxic conditions after 24 h. The effect of diabetes on the regulation of galanin and its receptor subtypes was assessed on NSCs in vitro and in SVZ tissues isolated from normal and type 2 diabetes ob/ob mice. We show increased NSC viability following galanin receptor (GalR)3 activation. This protective effect correlated with decreased apoptosis and CHOP levels. We also report how galanin and its receptors are regulated by diabetes in vitro and in vivo. This study shows GalR3-mediated neuroprotection, supporting a potential future therapeutic development, based on GalR3 activation, for the treatment of brain disorders.

Adult neurogenesis impairment in diabetes could play a role in the development of neurological complications. GalR3 activation counteracts glucolipotoxicity in adult neural stem cells (NSCs) in the subventricular zone (SVZ) by decreasing apoptosis. At least part of the protective effect mediated by GalR3 activation occurs through modulation of the unfolded protein response (UPR) signaling in the endoplasmic reticulum. The data support a potential therapeutic development for treatment of diabetic brain disorders, based on increased neurogenesis by GalR3 activation. CB, cerebellum; LV, lateral ventricle; OB, olfactory bulb.

Abbreviations used
AD

Alzheimer's disease

EGF

epidermal growth factor

ER

endoplasmic reticulum

GalR

galanin receptor

NSCs

neural stem cells

PD

Parkinson's disease

SVZ

subventricular zone

T2D

Type 2 diabetes

UPR

unfolded protein response

New neurons are continuously born from a proliferating population of neural stem cells (NSCs) throughout adulthood via a mechanism known as adult neurogenesis. This process occurs mainly in two regions of the brain, the subgranular zone of the hippocampal formation, and the subventricular zone (SVZ) of the lateral ventricle wall (Alvarez-Buylla and Lim 2004). However, neurogenesis has also been reported in brain regions outside the subgranular zone and SVZ (Gould 2007), including recent data on hypothalamic neurogenesis (Kokoeva et al. 2005). Hippocampal neurogenesis contributes to cognitive plasticity in the rodent (Sahay et al. 2011), and it has been shown to occur at rates comparable in humans and mice, suggesting a similar function in both species (Spalding et al. 2013). Newly born neuroblasts in the SVZ migrate to the olfactory bulb through the rostral migratory stream, where they terminally differentiate into neurons (Whitman and Greer 2009). This process seems related to the acquisition or discrimination of new odors that are important for survival (Whitman and Greer 2009). However, the SVZ in rodents and human presents organizational differences which could also reflect functional differences (Gonzalez-Perez 2012). Hippocampal and SVZ neurogenesis is dysregulated in Huntington's, Parkinson's (PD), and Alzheimer's (AD) diseases (Winner et al. 2011), suggesting that this process could play a functional role in the development and/or response to neurodegeneration. In stroke, neural precursor cells from the SVZ can divert their migration from their normal route along the rostral migratory stream and instead migrate to the site of neural damage (Arvidsson et al. 2002). Similarly, this process has been shown to also occur in the human brain (Marti-Fabregas et al. 2010).Whether these cells can correctly integrate in brain damage areas and contribute to recovery remains to be evaluated. Diabetes and obesity are strong risk factors for pre-mature stroke (Sander and Kearney 2009) and neurodegenerative diseases such as AD (Kalaria 2009) and PD (Vanitallie 2008). Interestingly, recent studies have shown that adult neurogenesis is impaired in obese and diabetic animal models in vivo and also by a diabetic milieu in vitro (Suh et al. 2005; Zhang et al. 2008; Alvarez et al. 2009; Guo et al. 2010; Park et al. 2010; Mansouri et al. 2012).

Galanin is a 29 (rodents) or 30 (human) amino acid peptide (Tatemoto et al. 1983) with a wide-range of biological effects in both the CNS and PNS (Bartfai et al. 1993; Gundlach et al. 2001; Lang et al. 2007; Ogren et al. 2010). Galanin is involved in metabolism and reproduction (Barson et al. 2010) (Merchenthaler 2010), survival, regeneration (Hobson et al. 2010) and cognition (Crawley 1999; Ogren et al. 2010). In addition, several studies have shown that galanin plays a role in pathological conditions such as pain (Liu and Hokfelt 2002; Xu et al. 2010), AD (Counts et al. 2010), addiction (Picciotto 2010), and epilepsy (Lerner et al. 2010).

Galanin signals through three G-protein coupled receptors (GalR1, -R2 and -R3) (Lang et al. 2007; Mitsukawa et al. 2008). Both GalR1 and GalR2 are widely expressed in the rat brain (Smith et al. 1997; O'Donnell et al. 1999; Burazin et al. 2000; Waters and Krause 2000). In contrast, the GalR3 expression pattern has a more restricted distribution in rodents with transcript levels most abundant in the hypothalamus (Mennicken et al. 2002). Individual galanin receptors have been associated with certain functions, in particular GalR1 and –R2 (Mitsukawa et al. 2010; Webling et al. 2012), for example GalR2 showing neuroprotection (Elliott-Hunt et al. 2004; Pirondi et al. 2010). The physiological and pathological role of GalR3 in the brain is less well-characterized (Barreto et al. 2011; Webling et al. 2012).

The mRNA and proteins for galanin and its receptors are high in rodent neurogenic areas (Elliott-Hunt et al. 2004; Shen et al. 2005) and in rodent and human embryonic stem cells (Anisimov et al. 2002; Assou et al. 2007). In addition, a not clearly defined GalR2/GalR3 activation has been suggested to regulate the proliferation of adult hippocampal-derived NSCs (Abbosh et al. 2011). Finally, a recent study in mice has shown that galanin receptors (GalRs) are highly expressed in NSCs from the SVZ and that GalR2 and/or GalR3 activation can regulate NSC differentiation (Agasse et al. 2013).

The aim of this study was firstly to determine the potential role of galanin and its receptors to protect NSCs in response to a diabetic glucolipotoxic milieu in vitro. Furthermore, we studied apoptosis and unfolded protein response (UPR) signaling as potential mechanisms mediating such a protective effect. In addition, we performed quantitative studies on NSC regulation of galanin and GalRs in response to diabetes-like conditions in vitro and in vivo.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Animal experiments

All experiments were conducted according to the regional ethics committees with permissions from the local ethical committee at the Southern Hospital (S-7709, S-7212) and from Stockholm's northern ethical committee for animal experiments (N172/11). The animal experimentation conformed to the ‘Guide for the Care and Use of Laboratory Animals’ published by U.S. National Institutes of Health (NIH publication # 85-23, revised 1985). The C57 BL6/J mice were imported from Nova-SCB, Stockholm, Sweden. The ob/ob mice or their lean litter mates were taken from our local breed in house. The ARRIVE guidelines have been followed.

Cell cultures

The SVZ of the lateral brain ventricles of adult male mice 5–7 weeks of age (five C57 BL/6J mice in each experiment) was micro-dissected and enzymatically dissociated and the cells were isolated and expanded (see Supporting information). Neurospheres were split every 5 days for 4 weeks and all experiments were performed between passage 2 and 5.

High glucose and fatty acid-enriched media

To mimic a hyperglycemic and hyperlipidemic milieu in vitro, Dulbecco's modified Eagle's medium/F12 (19 mM glucose) and sodium palmitate (Sigma-Aldrich, St. Louis, MO, USA) at the concentration of 0.2–0.3 mM were used (see Supporting information for details).

ATP assay

Previous reports have demonstrated that intracellular ATP levels correlate to cell numbers (Crouch et al. 1993). Moreover, this assay has been shown to be more sensitive in detecting cell number than the 3-[4,4-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (Petty et al. 1995). To measure NSC viability, NSCs were seeded as single cells into 96-well plates (Corning B.V. Life Sciences, Amsterdam, Netherlands) at the final concentration of 50 000 cells/well in Dulbecco's modified Eagle's medium/F12 supplemented with B27 and in 0.01 × 10−3 g/L of epidermal growth factor (EGF). PACAP 38 (Phoenix Pharmaceuticals, Burlingame, CA, USA), native galanin, AR-M 1896 (gal 2-11; Tocris Biosciences, Bristol, UK), SNAP-3788 (KO Key Organics, London, UK), M871 (kindly given by Ulo Langel, Stockholm University), and 4-phenylbutyrate (Sigma-Aldrich) were added to NSCs 15 min prior to exposing the cells to palmitate at the concentrations shown in the Results section. The treatments were maintained for 24 h. To block the effect of AR-M1896, the antagonists M871 and SNAP-3788 were given 15 min prior to AR-M1896 with a 100 times higher concentration.

After 24 h of incubation at 37°C (5% CO2, 98% humidity), intracellular ATP levels were measured using the Cellular ATP Kit HTS according to the manufacturer's instructions (BioThema, Stockholm, Sweden). In these experiments, the effect of each treatment at a certain concentration was determined in quadruplicate or octo-duplicate in three to five different sets of experiments.

3H-Thymidine incorporation

See Supporting information for the protocol.

Western blotting

NSCs were plated as single cells and expanded in a 10-cm Petri dish with EGF/basic fibroblast growth factor (bFGF) (see under cell cultures) for 3–4 days. When neurospheres were formed, the different treatments were added for 0, 6, 12, and 24 h. See Supporting information for the details of the protocol of Western blot experiments.

RT-PCR

To quantify the mRNAs from NSCs in vitro, adult brain of 5- to 6-week-old C57BL6/SCA mice was isolated. NSCs were grown in EGF/bFGF at passage 2–4 in presence or absence of palmitate or the different treatments for 24 h. In the in vivo quantitative experiments, tissue from the SVZ of the lateral ventricle (less than 1 mm of tissue facing the lateral ventricle and including the SVZ) was isolated for analysis by using a micro-dissector scissor under the microscope. In this set of experiments, the SVZ of 5-week-old (young adult) and 36-week-old (middle-aged) ob/ob mice plus their lean litter mates was isolated. Five to twelve brains were pooled in each experiment. See Table S1 for the details of the RT-PCR protocol and primers sequence.

Measurement of XBP1 mRNA splicing

See Supporting information for the protocol.

Statistical analysis

For both in vivo and in vitro experiments, data are presented as mean ± SEM. Student's t-test was used when comparing the difference between two groups, while multiple comparisons were made by one-way anova followed by post hoc Fisher LSD test (Sigma Plot v.11 software: Systat Software Inc, San Jose, CA, USA). p < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Galanin increases NSC viability by GalR3 activation

To determine the potential effect of the (native) galanin 1-29 on adult NSC viability, intracellular ATP levels were assessed after 24 h in culture, in presence of EGF, with or without galanin 1-29. The results show that galanin 1-29 at 10 nM significantly increased NSC viability, while lower doses of the galanin 1-29 (0.1 nM, 1 nM) did not show any effect (Fig. 1a). To determine the receptor subtype involved, we took advantage of the equally affinity agonist for GalR2/3, AR-M 1896 (Lu et al. 2005). The results show that AR-M 1896 at 10 nM (a concentration not targeting GalR1) significantly increased NSC viability after 24 h (Fig. 1b). As for galanin, lower doses of AR-M1896 had no effect on NSC viability (Fig. 1b).

image

Figure 1. The native galanin peptide increases neural stem cell (NSC) viability via galanin receptor (GalR)3 activation through protection. (a–c) NSCs were plated as single cells and exposed to 0.1 nM, 1 nM, and 10 nM Gal 1-29 or 0.1 nM, 1 nM, and 10 nM AR-M 1896 and 100 nM PACAP. Treatments were maintained for 24 h. NSC viability was determined by measuring intracellular ATP levels. [3H]thymidine incorporation was assessed to measure NSC proliferation. (d, e). NSCs were plated as single cells and pre-treated with GalR2 antagonist M871 (1 μM) and GalR3 antagonist SNAP 37889 (1 μM) before being exposed to AR-M 1896 (10 nM) and PACAP (100 nM). Treatments were maintained for 24 h. NSC viability was determined by measuring intracellular ATP levels. Data are shown as mean ± SEM (a, n = 10; b, n = 8; c, n = 12; d, n = 18; e, n = 30). *p < 0.05 compared with control.

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To determine whether enhanced NSC viability by GalR2/3 activation was because of increased proliferation, we assessed [3H]thymidine incorporation in NSCs treated with or without 10 nM of AR-M1896 for 24 h. In this set of experiments, no difference in NSC proliferation between control and AR-M1896 was observed, contrasting the significantly enhanced NSC proliferation by the neuropeptide PACAP (Fig. 1c).

To determine whether the protective effect by AR-M1896 is mediated through GalR2, GalR3 or both receptors, we took advantage of the preferring and the specific antagonist for GalR2 and GalR3, M871 (Sollenberg et al. 2006) and SNAP 37889 (Swanson et al. 2005), respectively. NSCs were shortly pre-treated with M871 (1 μM) or SNAP 37889 (1 μM) before exposure to AR-M1896 for 24 h. The results clearly show that blockade of GalR3, but not of GalR2, completely abolished the protective effect mediated by AR-M1896 (Fig. 1d and e).

GalR3 activation counteracts the impaired NSC viability induced by a diabetic milieu

To study the effect of diabetic glucolipotoxicity on NSC viability, we recently established an in vitro cell assay simulating a diabetic milieu by high glucose and palmitate with a negative impact on NSC viability (Mansouri et al. 2012). Thus, next we determined whether the activation of GalR3 could exert a protective effect against such glucolipotoxicity. To this end, NSCs were pre-treated with AR-M 1896 (10 nM) 15 min prior to exposure to glucolipotoxic conditions for 24 h. The results show that AR-M1896 significantly counteracted the negative glucolipotoxic effect (Fig. 2a). PACAP, as previously shown significantly counteracted glucolipotoxicity in this assay (Mansouri et al. 2012).

image

Figure 2. Galanin receptor (GalR)3 activation by AR-M 1896 counteracts glucolipoapoptosis in neural stem cell (NSC) in vitro. (a, d) NSCs were plated as single cells and exposed to a 15 min pre-treatment of AR-M 1896 (10 nM), PACAP (100 nM) or SNAP 37889 (1 μM) prior to 0.2 mM palmitate (P2) addition. Treatments were maintained for 24 h. NSC viability was determined by measuring intracellular ATP levels. (b, c) NSCs grown in epidermal growth factor (EGF)/bFGF were treated for 24 h with/without 0.3 mM palmitate (P3) and 15 min pre-treatment of AR-M 1896 (10 nM), PACAP (100 nM) after which western blots experiments were performed. To obtain quantitative measurements, Bcl-2 and cleaved caspase 3 protein levels were normalized against Coomassie blue. Data are shown as mean ± SEM (a, n = 30; b, n = 4–5; c, n = 3–5; d, n = 30). *p < 0.05 compared with control, #p < 0.05 compared with palmitate.

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Previous results from our group show that a diabetic milieu induces apoptosis in NSCs (Mansouri et al. 2012). To determine whether AR-M 1896 counteracted apoptosis induced in this model, Bcl-2 and c-caspase 3 expression was assessed by Western blot analysis after 24 h in the presence of palmitate, with or without AR-M 1896. The results in Fig. 2b and c show that AR-M1896 was able to increase Bcl-2 and to decrease c-caspase 3 protein expression, similarly to PACAP. To confirm that even under diabetic conditions the protective role mediated by AR-M 1896 occurred via GalR3 and not by GalR2 activation, the same type of experiment as illustrated in Fig. 1b was carried out by using the GalR3 antagonist SNAP 37889. The results in Fig. 2d show that the protective effect mediated by AR-M 1896 was abolished by SNAP 37889, indicating that it is indeed GalR3-mediated.

A diabetic milieu impairs NSC viability via UPR activation

Emerging evidence suggests that endoplasmic reticulum (ER) stress may play a pivotal role in the development and pathology of both certain forms of diabetes and neurodegenerative diseases. In response to such conditions, cells initiate pro-survival signaling pathways collectively known as the UPR (Lindholm et al. 2006; Ortsater and Sjoholm 2007). To study whether diabetic glucolipotoxicity induces UPR signaling, we treated NSCs with/without palmitate for 24 h before performing RT quantitative PCR studies on two markers for UPR activation: CHOP and BIP (Hotamisligil 2010). Results show that the ER stress markers were significantly up-regulated in the presence of glucolipotoxic conditions in comparison with control (Figure S1a and b). The ER stress-inducer salubrinal (Cnop et al. 2007) was used as a positive control in this series of experiments. ER stress activation was also confirmed at protein levels (Figure S1d), since CHOP expression was increased at 24 h in response to a diabetic milieu. Our results also show that high glucose and palmitate induced inositol-requiring enzyme 1 pathway activity, as evidenced by enhanced alternative splicing of XBP1 and increased phosphorylation of JNK (Figure S1c, e).

GalR3 activation increases NSC viability in response to a diabetic milieu in correlation with UPR signaling modulation

We next addressed whether the GalR3 protective effect on NSCs against a diabetic milieu correlated to the expression of the ER stress markers CHOP and BIP. To do so, NSCs were pre-treated with 10 nM of AR-M1896 before exposure to palmitate for 24 h. The results show that the activation of GalR3 by AR-M1896 modulated the UPR signaling by decreasing the ER stress marker CHOP. On the other hand, no effect on BIP mRNA levels was recorded (Fig. 3a and b). These data indicate that activation of GalR3 reduced ER stress-associated apoptosis without modulating chaperone expression.

image

Figure 3. Activation of galanin receptor (GalR)3 in response to palmitate leads to CHOP down-regulation in neural stem cell (NSC) as quantified by mRNA levels. (a, b) NSCs were pre-treated with/without 10 nM AR-M 1896 before exposed to 0.3 mM palmitate (P3) for 24 h. RNA was then extracted and quantitative PCR experiments were performed by using primer pairs specific for CHOP (a) and BIP (b) genes (See also Table S1 for primers sequence). Data are shown as mean ± SEM (a, n = 4; b, n = 5). *p < 0.05 compared with control, #p < 0.05 compared with palmitate.

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Regulation of galanin and GalR expression in diabetes-like conditions in vitro and in vivo

To study whether expression of galanin and its receptor subtypes are regulated in diabetes, we performed both in vitro and in vivo studies. In the former, NSCs were exposed to a diabetic milieu for 24 h before RT quantitative PCR studies on galanin 1-29 and GalR expression were carried out. Figure 4a, c shows that galanin 1-29 and GalR2 were up-regulated by glucolipotoxic conditions compared with control. The results also show a trend for GalR1 (= 0.08), while GalR3 was not affected (Fig. 4b, d).

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Figure 4. Regulation of galanin and galanin receptor (GalR) expression in response to a diabetic milieu in vitro. (a–d) neural stem cells (NSCs) were grown with/without 0.3 mM palmitate (P3) for 24 h before extracting RNA. Primer pairs specific for native galanin (a), GalR1 (b), GalR2 (c), and GalR3 (d) genes were used to quantify the mRNA expression levels by quantitative PCR. (See also Table S1 for primers sequence). Data are shown as mean ± SEM (a, n = 4; b, n = 4; c, n = 4; d. n = 4). *p < 0.05 compared with control.

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To study the regulation of galanin/GalRs in a young pre-diabetic and middle-aged diabetic, we used 5-week old and 36-week old spontaneously mild diabetic obese mice (ob/ob) (Lindstrom 2007), in comparison with their aged-matched lean littermates. Here, we focused our analysis on the SVZ region, from which NSCs are derived; tissue was isolated and prepared for RT quantitative PCR. The results in Fig. 5a–c show an expression pattern in young-adult ob/ob mice versus their lean littermates partly similar to the in vitro data (Fig. 4): Galanin and GalR2 were significantly increased compared to their lean littermates, with no significant change in GalR3 expression (Fig. 5a–d). However, in the middle-aged ob/ob mice, while GalR2 were significantly up-regulated (Fig. 6c), GalR3 was significantly down-regulated compared with their lean littermates (Fig. 6d). Galanin and GalR1 expression did not change (Fig. 6a, b).

image

Figure 5. In vivo expression of galanin and galanin receptor (GalR) in 5-week-old lean versus ob/ob pre-diabetic mice. (a–d) subventricular zone (SVZ) was dissected from the mouse brain of lean or ob/ob 5-week-old mice. Quantitative PCR experiments were performed on RNA prepared from the isolated tissue by using specific primer pairs (see Table S1 for primers sequence). Data are shown as mean ± SEM (a, n = 4–5; b, n = 4–5; c, n = 4; d. n = 4–5). *< 0.05 compared with their lean litter mates.

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image

Figure 6. In vivo expression of galanin and galanin receptor (GalR) in 36-week-old lean versus ob/ob diabetic mice. (a–d) subventricular zone (SVZ) was dissected from the mouse brain of 36-week-old lean or ob/ob mice. Quantitative PCR were performed on RNA prepared from the isolated tissue using specific primer pairs (see Table S1 for primers sequence). Data are shown as mean ± SEM (a, n = 10–11; b, n = 10–12; c, n = 10–11; d. n = 10–11). *< 0.05 compared with their lean litter mates.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The incidence of type 2 diabetes (T2D) and neurodegenerative disorders is increasing in the western world accompanying the growing number of obese and elderly people. Stroke is over-represented within the diabetic population, where also the risk of developing stroke is increased 2- to 6-fold (Sander and Kearney 2009). Furthermore, there is a strong co-morbidity between T2D and AD (Kalaria 2009) sometimes referred to as T3D, the reasons for which are largely unknown. In T2D, there is a plethora of changes that alone or together can impair brain metabolism and affect neuronal viability. These include glucose toxicity, hyperlipidemia, hypertension, increased inflammation and oxidative stress and insulin resistance (Sims-Robinson et al. 2010). These events may lead to damage of cerebral microvasculature and/or neural tissue, which in turn could pre-dispose to brain disorders, including neurodegeneration.

On top of these well-characterized mechanisms, deranged adult neurogenesis in hippocampus has been proposed to play a role. In fact, pre-clinical data show dysfunctional hippocampal neurogenesis in diabetic and obese animal models (Zhang et al. 2008; Alvarez et al. 2009; Park et al. 2010) that could result in cognitive impairment (Stranahan et al. 2008). In addition, recent pre-clinical reports have indicated a role of hypothalamic adult neurogenesis in maintaining energy balance in response to environmental and physiologic insults (Pierce and Xu 2010). Thus, hypothalamic neurogenesis may represent one of the adaptive mechanisms used by the brain to limit functional impairment resulting from obesity. With regard to SVZ-neurogenesis, in vivo and in vitro experimental models have shown that this process is severely impaired by diabetes (Lang et al. 2009; Guo et al. 2010; Mansouri et al. 2012). SVZ-derived neuroblasts can, after stroke in the normal non-diabetic brain, integrate into the stroke-damaged striatum and might play a role in functional recovery (Arvidsson et al. 2002). If so, SVZ neurogenesis impaired by diabetes could decrease the endogenous brain restorative response after stroke. To understand if this hypothesis has clinical relevance, more research is needed to better characterize the role of adult SVZ neurogenesis in neurological disorders in the normal and diabetic brain as well as to understand whether functional differences between humans and rodents exist. Collectively, these results suggest that impaired adult neurogenesis could play an important role linking metabolic disorders to central neurological complications. Conversely, such a defect is an attractive target in the quest of identifying factors capable of normalizing impaired neurogenesis and, hence, preventing or limiting diabetic CNS complications.

To identify molecules with such properties, an in vitro system has been developed, where we have shown that a diabetic milieu characterized by glucolipotoxicity impairs NSCs viability (Mansouri et al. 2012). In this study, we demonstrate that galanin and the equally affinity agonist for GalR2/GalR3, AR-M 1896 (galanin 2-11) (Liu et al. 2001; Lu et al. 2005) can counteract the impaired NSC viability induced by such a diabetic milieu. This protective effect does not seem to be restricted to the diabetic challenge, since the limited, naturally occurring cell death in our primary cultures was also improved by AR-M 1896. Furthermore, our results show that the increased NSC viability conferred by AR-M 1896 in our assay occurs entirely through cell protection, since no change in proliferation was observed. Ma et al. have shown that AR-M1896 increases the length of neuritis in SVZ-derived NSCs (Ma et al. 2008), in agreement with studies on injured dorsal root ganglion neurons (Mahoney et al. 2003). However, Abbosh et al. (2011) reported that low concentrations of AR-M 1896 are both trophic and proliferative in adult hippocampal NSCs and that these effects could be blocked by the GalR2 selective antagonist M871. We note that Abbosh et al.'s studies are from hippocampal NSCs (vs. ours being SVZ-derived), and that different exposure times of AR-M 1896 to the cells were used (only 24 h in our assay vs. 5 days in the Abbosh et al. work).

The unselectivity of AR-M 1896 may explain why some previous studies, not only in CNS, have failed to discriminate between GalR2- and GalR3-mediated effects (Lu et al. 2005; Shen et al. 2005; Pirondi et al. 2010; Agasse et al. 2013). Moreover, two recent reports that attempted to address galanin-mediated NSC proliferation and differentiation were not able to clearly distinguish between a GalR2- and/or a GalR3-mediated effect (Abbosh et al. 2011; Agasse et al. 2013). By combining AR-M 1896 with a GalR2 preferring antagonist and a specific antagonist for GalR3, respectively, we now are able to show that the protective effect mediated by AR-M 1896 occurred selectively via GalR3 activation. These results are supported by the recent demonstration of GalR3 protein with immunohistochemistry and Western blot in the mouse SVZ (Agasse et al. 2013) (also see below) and our own demonstration, in vivo and in vitro, of GalR3 mRNA in NSCs and the SVZ region, to our knowledge the first to show cellular protection specifically mediated by GalR3.

Our results also show that the protective effect mediated by galanin against glucolipotoxicity correlates with increased protein levels of Bcl-2 and decreased cleaved-caspase 3, suggesting that the protective effect mediated by GalR3 activation occurs via decreasing apoptosis. ER stress may play a fundamental role in the development and pathology of certain forms of both diabetes and neurodegenerative diseases (Lindholm et al. 2006; Ortsater and Sjoholm 2007). Therefore, in this study we have extended our previous work on the effect of glucolipotoxicity on NSCs (Mansouri et al. 2012) by determining whether ER stress is regulated by a diabetic milieu. Indeed, we show that glucolipotoxicity significantly activates JNK and UPR signaling, by increasing the ER stress markers CHOP, BIP and the alternative spliced form of XBP1. Although similar results were obtained by Li et al. in the C17.2 immortalized cell line derived from cerebellum (Li et al. 2011), our results on the activation of ER stress by a diabetic milieu are, to our knowledge, the first obtained in primary adult NSCs. By assessing the potential role of GalR3 activation on the UPR signaling, we quantified mRNA levels of CHOP after palmitate and AR-M1896 exposure, and we show that the activation of GalR3 decreased the enhanced CHOP levels induced by a diabetic milieu. As activation of GalR3 was without any effect on BIP mRNA levels, our data indicate that stimulation of the GalR3 regulates the cytotoxic pathway of the UPR by a mechanism independent of increased chaperone expression. This suggests that at least part of the protective effect mediated by GalR3 activation occurs through UPR regulation.

The functional role of galanin on NSCs in the SVZ begins to be uncovered (Shen et al. 2005; Ma et al. 2008), and the recent report by Agasse et al. showed that Gal R1, -R2, and -R3 are expressed in the SVZ not only in tissue samples but also in neurospheres from adult mouse, using qPCR and blotting [(protein data to be confirmed since not validated as outlined by Lu et al. (Lu and Bartfai 2009)] of the adult mouse (Agasse et al. 2013). Here, they promote neuronal differentiation through GalR1 and -R2 activation, but not self-renewal, proliferation or cell death (Agasse et al. 2013). In view of the emerging importance of the galanin system in SVZ neurogenesis, and of the potential relevance at the pharmacological/therapeutic level, we wanted to address whether or not galanin and GalRs expression are impacted by diabetes in this brain area. To do so, we quantified galanin and GalR expression in response to glucolipotoxicity in vitro, and by comparing young pre-diabetic and old diabetic ob/ob mice with aged matched lean littermates in vivo. Our results show that GalR3 expression remained unchanged in a diabetic milieu in vitro as well as in young pre-diabetic ob/ob mice in vivo. However, GalR3 expression was strongly down-regulated in aged diabetic ob/ob mice in comparison with their lean littermates.

Whether or not the decreased expression of GalR3 in diabetes plays a role in the decreased NSC survival and/or proliferative and differentiative capacity remains an interesting hypothesis to be further investigated in vivo. In contrast to GalR3 expression, we also show that galanin, GalR1, and GalR2 were up-regulated by diabetes, both in vitro and in vivo. Furthermore, GalR2 expression remained high in aged diabetic ob/ob mice in comparison with their age-matched lean littermates. Although galanin and GalRs have been previously reported to be highly expressed in the SVZ of the adult rodent brain (Agasse et al. 2013) (Shen et al. 2005), very little is known about their physiological role in this brain area. The fact that the expression of both galanin and its receptors is regulated by diabetic conditions in SVZ provides an impetus for future in vivo research, aimed at understanding their physiological role in this brain area under normal and diabetic conditions.

The regulation of NSCs in the SVZ has been suggested to play a regenerative role in AD, PD, and stroke (Emsley et al. 2005), disorders that are strongly over-represented in the diabetic population (Vanitallie 2008; Kalaria 2009; Sander and Kearney 2009). Our results indicate the possibility that NSC protection via GalR3 agonists may be used to prevent and treat the neurological complications of diabetes, but is so far an attractive hypothesis for further studies.

In conclusion, we found that galanin via GalR3 activation counteracts NSC glucolipotoxicity, which correlates with decreased apoptosis and modulation of the UPR signaling. Furthermore, we show that each of the three GalR subtypes is regulated in response to glucolipotoxicity both in vitro and diabetic milieu in vivo, effects that may serve to regulate neuronal differentiation, proliferation, and survival in diabetes/obesity.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We thank R. Fall, D. Rydholm (Södersjukhuset AB) for skilled animal technical assistance. Support by the Swedish Research Council (04X-2887), by foundations Tornspiran and Gamla Tjänarinnor and Karolinska Institutet funds is gratefully acknowledged. The authors declare to have no conflicts of interest relevant to this article.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
FilenameFormatSizeDescription
jnc12396-sup-0001-TableS1-FigS1.pdfapplication/PDF230K

Table S1. Gene accession number and primer sequence of the RT-PCR products for galanin receptor subtypes, UPR stress markers and β-actin genes are shown.

Figure S1. A diabetic milieu up-regulates NSC mRNA and protein levels of CHOP and BIP and induces IRE1 pathway activity by phosphorylation of JNK and enhancing alternative splicing of XBP1.

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