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GPR49 is implicated in motor neuron specification in nervous system
Neuroscience Letters 584 (2015) 135–140
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
LGR5/GPR49 is implicated in motor neuron specification in nervous system Shao-jun Song a,b,∗,1 , Xing-gang Mao c,∗∗,1 , Chao Wang d , An-guo Han a , Ming Yan e , Xiao-yan Xue f a
Department of Neurosurgery, PLA 254 Hospital, Tianjin, China Department of Neurosurgery, Hinan Branch of Chinese PLA General Hospital, Haitang Harbor, Sanya, Hainan Province 572013, China c Department of Neurosurgery, Xijing Hospital, Fourth Military Medical University, Xi’an, Shaanxi Province, China d Department of Neurosurgery, Tangdu Hospital, Fourth Military Medical University, Xi’an, Shaanxi Province, China e Department of Orthopaedic Surgery, Xijing Hospital, Fourth Military Medical University, Xi’an, Shaanxi Province, China f Department of Pharmacology, School of Pharmacy, Fourth Military Medical University, Xi’an, Shaanxi Province, China b
h i g h l i g h t s • • • • •
The biological role of LGR5 is closely related with neuron development and functions. LGR5 is specifically highly expressed in projection motor neurons in nervous system. Notch signal inhibited neurogenic potential and LGR5 expression in neural stem cells. Knockdown of LGR5 inhibited neurogenic potential of neural stem cells. LGR5 regulated the expression of key factors programing the identity of motor neurons.
a r t i c l e
i n f o
Article history: Received 4 June 2014 Received in revised form 3 September 2014 Accepted 29 September 2014 Available online 16 October 2014 Keywords: GPR49 Motor neuron differentiation Neural stem cell Notch WNT Bioinformatics
a b s t r a c t The biological roles of stem cell marker LGR5, the receptor for the Wnt-agonistic R-spondins, for nervous system are poorly known. Bioinformatics analysis in normal human brain tissues revealed that LGR5 is closely related with neuron development and functions. Interestingly, LGR5 and its ligands R-spondins (RSPO2 and RSPO3) are specifically highly expressed in projection motor neurons in the spinal cord, brain stem and cerebral. Inhibition of Notch activity in neural stem cells (NSCs) increased the percentage of neuronal cells and promoted LGR5 expression, while activation of Notch signal decreased neuronal cells and inhibited the LGR5 expression. Furthermore, knockdown of LGR5 inhibited the expression of neuronal markers MAP2, NeuN, GAP43, SYP and CHRM3, and also reduced the expression of genes that program the identity of motor neurons, including Isl1, Lhx3, PHOX2A, TBX20 and NEUROG2. Our data demonstrated that LGR5 is highly expressed in motor neurons in nervous system and is involved in their development by regulating transcription factors that program motor neuron identity. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Abbreviations: ALS, amyotrophic lateral sclerosis; ARACNe, Algorithm for the Reconstruction of Accurate Cellular Networks; CNS, central nervous system; DAPT, N-[N-(35-difluorophenacetyl)-l-alanyl]-S- phenylglycine t-butyl ester; DPI, data processing inequality; GSC, glioblastoma stem cells; LGR5-DIGs, directly interacting genes of LGR5; NODRN, neuronal and oligodendrocyte-differentiation related network; NSC, neural stem cell; qPCR, quantitative real time PCR; SFM, serum free medium. ∗ Corresponding author. ∗∗ Corresponding author. Tel.: +86 02984771031. E-mail addresses: [email protected] (S.-j. Song), [email protected] (X.-g. Mao). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.neulet.2014.09.056 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
LGR5 (leucine-rich repeat-containing G protein-coupled receptor 5 also known as GPR49) was recently identified as a stem cell marker for several tissues and cancers, including intestine and colon normal and cancer stem cells, hair follicle stem cells, etc. [1–4]. More recently, LGR5 was reported to be expressed in glioblastoma stem cells (GSCs), and contribute to the maintenance of GSCs [5,6]. However, the biological functions of LGR5 for stem cells are poorly acknowledged, even for the established stem cells that it marks, such as intestine and colon stem cells. The potential of LGR5 to identify stem cells in different kinds of tissues implies it may be a more universal stem cell marker, raising the interesting question that what is its biological function in nervous system?
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By taking advantage of bioinformatics analysis, our previous study demonstrated that LGR5 is involved in the process of neurogenesis [6]. Biological experiments further confirmed that LGR5 regulate a cohort of genes involved in the network of neuronal and oligodendrocyte differentiation (NODRN) [6]. Interestingly, a positive feedback between LGR5 and OLIG2 was identified [6]. In addition, LGR5 is strongly regulated by neurogenin 1 [7], a proneural basic helix-loop-helix (bHLH) transcription factor that is important for neural stem cell (NSC) differentiation. All of these results imply that LGR5 may play a role in the neuronal differentiation and maturation. Here, we further studied the biological role of LGR5 in nervous system, and revealed that LGR5 is involved in the process of neuronal differentiation and maturation, especially in the development of motor neurons in central nervous system (CNS). 2. Methods and materials
of Notch2 (pcDNA3.1-NICD2). Then the plasmid pcDNA3.1-NICD2 was transfected into the cells with LipofectamineTM 2000 according to the manufacturer’s instructions. Vector pcDNA3.1 was used as control. 2.4. siRNA transfection LGR5 knockdown by siRNA was performed as described previously [6]. Briefly, the NSC spheres were dissociated into single cells and plated on poly-l-ornithine (50 mg/ml, P3655, Sigma Chemical) coated wells (50,000 cells per 24–well platewell) to attach for 24 h. Then the cells were transfected by Stealth small interfering RNAs (siRNA) targeting LGR5 or non-targeting RNA sequence (Invitrogen Lifetechnologies, USA) with OligofectamineTM RNAiMAX Reagent according to the manufacturers protocol (Invitrogen). After 24 h of incubation, the cells were washed and detached from the wells and placed back to SFM medium. The efficiency of siRNA interference was evaluated by qPCR after 72 h of transfection.
2.1. ARACNe network reconstruction and bioinformatics analysis 2.5. Immunofluorescence ARACNe (Algorithm for the Reconstruction of Accurate Cellular Networks), an information-theoretic algorithm for inferring transcriptional interactions [8], was used to identify a repertoire of candidate transcriptional regulators of interesting genes, as described previously [9]. Expression profiles of normal brain tissue used in the analysis were from GSE15745 (Gene expression profiles derived from frozen tissue samples of the cerebellum (CRBLM), frontal cortex (FCTX), caudal pons (PONS) and temporal cortex (TCTX) obtained from 150 neurologically normal subjects) [10] and GSE11882 (Gene expression profiles derived from frozen tissue samples in the hippocampus (HC), entorhinal cortex (EC), superior frontal gyrus (SG), and postcentral gyrus (PCG) across the lifespan of 63 cognitively intact individuals from 20–99 years old) [11]. First, candidate interactions between a transcription factor (TF, x) and its potential target (y) are identified by computing pairwise mutual information, MI[x; y], using a Gaussian kernel estimator and by thresholding the MI based on the null-hypothesis of statistical independence (p < 0.05, Bonferroni corrected for the number of tested pairs). Then, indirect interactions are removed using the data processing inequality (DPI) analysis with a tolerance of 20%, a well-known property of the mutual information. 2.2. Culture of primary neural stem cells (NSCs) Culturing of human fetal cortical NSCs were isolated from two spontaneous aborted fetuses (8–12 weeks) as described previously [9,12]. The study protocol was approved by Institutional Review Committee of Xijing Hospital of the Fourth Military Medical University. Written informed consent was obtained from patients. Briefly, samples were dissociated to a single cell suspension using a fire-polished Pasteur pipette and cultured in serum free medium (SFM) consisting of DMEM-F12 medium, EGF (20 ng/mL; Invitrogen, Carlsbad, CA), bFGF (20 ng/mL; Invitrogen) and B27 (1:50; Invitrogen). 2.3. Inhibition and activation of Notch signal The highly active ␥-secretase inhibitor, N-[N-(3, 5difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT), purchased from Sigma–Aldrich Corporation (USA), was used to inhibit the Notch signal activity. To activate Notch signal, a truncated, constitutively active form of the NOTCH2 receptor (NICD2) was subcloned to adenovius. Breifly, cytoplasmic portion of the human Notch2 receptor (amino acids 1747–2531) was inserted into a mammalian expression vector pcDNA3.1 (Invitrogen) to create a constitutively active form
Immunofluorescence was performed on cultured and differentiated NSCs as described previously [12]. Briefly, undifferentiated neural spheres were plated onto poly-l-lysine coated glass coverslips for 4 h, fixed with 4% paraformaldehyde, and incubated with NeuN (1:1000, mouse monoclonal IgG1; Chemicon, Temecula, CA), GFAP (1:5000, rabbit polyclonal; Abcam), and MAP2 (1:1000, Santa Cruz) antibody overnight at 4 ◦ C. Texas Red goat anti-rabbit (Molecular Probes, Invitrogen, Carlsbad, CA) was used as secondary antibody. Counterstaining with Hoeschst 33342 (Sigma, St. Louis, MO) was additionally performed to permit counting of cell nuclei. 2.6. qPCR qPCR was performed as described previously [9,13]. briefly, RNA was extracted from cultured cells using Trizol Reagent (Invitrogen), and reverse transcribed into cDNA. qPCR analysis was performed on an ABI7700 using SYBR Green PCR Core Reagents in 20 L volume (Applied Biosystems, Warrington, UK). Primer sequences for qPCR studies are shown in Supplementary Table 1. All samples, including template-free controls, were assayed in triplicate and the relative amount of target transcripts normalized to the number of human actin transcripts found in the same sample. Specificity was verified by melting curve analysis and agarose gel electrophoresis. Relative fold changes were calculated using the Ct method by using the threshold cycle values of each sample. 2.7. Western blotting Cultured cells were lysed in SDS sample buffer, and 20 g proteins were run on 6% SDS-PAGE gel and transferred to a nitrocellulose membrane. Blots were blocked in PBS containing 5% nonfat dry milk powder and incubated overnight at 4 ◦ C with primary antibody of LGR5 (1:1000, Abcam) or GAPDH (1:500,000; Abcam). Blots were then washed with PBS containing 0.1% Tween 20 (PBST) and incubated in secondary antibodies coupled to peroxidase. After washing in PBST, blots were developed with chemiluminescence according to the manufacturer’s instructions (enhanced chemiluminescence, Amersham Biosciences, GE Healthcare, France). All Western blot analyses were done in duplicates. 2.8. Statistical analysis Statistical analyses were performed using Student’s t-tests and one-way analysis of variance with least squared difference post hoc tests, as appropriate. All P values are two-tailed. A value of
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Table 1 David analysis of LGR5-DIGs inferred from the 2 normal brain datasets. The representative enriched terms in the top 20 clusters were shown. Category
Term
N*
% of involved genes/total genes
Modified Fisher Exact P-Value
Benjamini
GOTERM MF FAT GOTERM MF FAT SP PIR KEYWORDS GOTERM BP FAT SP PIR KEYWORDS GOTERM BP FAT GOTERM BP FAT GOTERM BP FAT GOTERM BP FAT KEGG PATHWAY SP PIR KEYWORDS GOTERM BP FAT GOTERM BP FAT UP SEQ FEATURE GOTERM BP FAT GOTERM BP FAT
Transcription factor activity Sequence-specific DNA binding Homeobox Neurological system process glycoprotein Sensory perception Cognition Neuron differentiation Metencephalon development Olfactory transduction g-protein coupled receptor Neuron development Neuron projection Morphogenesis Domain:Leucine-zipper Hindbrain development Regulation of transcription from RNA polymerase II promoter Cerebellum development Extracellular region Sensory organ development Transcription from RNA Polymerase II promoter Mesenchymal cell development
28 20 11 25 53 18 19 12 4 11 15 9 7 5 4 13
18.18 12.99 7.14 16.23 34.42 11.69 12.34 7.79 2.60 7.14 9.74 5.84 4.55 3.25 2.60 8.44
2.70E-07 3.60E-06 2.30E-05 1.20E-04 5.90E-04 7.10E-04 9.60E-04 1.60E-03 3.50E-03 3.80E-03 5.30E-03 1.00E-02 1.10E-02 1.20E-02 1.60E-02 2.60E-02
7.7E-5 3.5E-4 3.0E-3 6.5E-2 3.8E-2 1.8E-1 2.0E-1 2.3E-1 3.3E-1 2.6E-1 1.2E-1 5.9E-1 6.0E-1 8.2E-1 6.8E-1 7.8E-1
3 23 6 6
1.95 14.94 3.90 3.90
2.80E-02 5.00E-02 5.20E-02 5.60E-02
7.9E-1 1.0E0 8.7E-1 8.8E-1
3
1.95
7.40E-02
9.2E-1
GOTERM GOTERM GOTERM GOTERM
BP CC BP BP
FAT FAT FAT FAT
GOTERM BP FAT *
N: number of LGR5-related genes involved in the annotation category.
p < 0.05 was considered statistically significant. Statistical analyses were done using SPSS v.13.0.0 (SPSS, Inc., Chicago, IL). 3. Results and discussion 3.1. The biological roles of LGR5 in CNS is closely related with neuron functions Bioinformatics analysis revealed that LGR5 is more closely related with neuron functions, especially with the maturation and projection of neurons, such as synapse function, ion channel, and axonal formation [6]. In addition, LGR5 is regulated by OLIG2, an important factor associated with neuron and oligodendrocyte differentiation [6]. To further dissect its functional role in normal CNS, we then performed bioinformatics analysis in two normal brain transcriptome datasets (GSE15745 [10] and GSE11882 [11]) by using ARACNE algorithm [6,8,9]. As a result, we got two lists of potential LGR5 direct interacting genes (LGR5-DIGs) in normal CNS. There are totally 171 genes that are overlapped in the two lists of LGR5-DIGs (Supplementary Table 2), and we considered these genes as the most reliable genes that may interact with LGR5 in normal CNS. Next, we performed DAVID analysis to explore the functional roles of these genes. The top 20 functional categories in the Annotation Clusters sorted by Enrichment Score revealed that, beside non-specific functional roles such as DNA binding, transcription binding, glycoprotein, etc., the most important potential functions of LGR5-DIGs are related to neuron development and function (Table 1). The genes related to neuron include ISL1, LHX5, RORA, DMD, GBX2, GAS7, NRL, PAX2, PHOX2A, PSEN1, UNC5C and ERBB3. A portion of these genes is also related to axonogenesis, such as ISL1, GBX2, PAX2, UNC5C, ERBB3. Noticeably, LGR5-DIGs include several genes that are critical for the differentiation of motor neuron in spinal cord and cortex, including ISL1, PHOX2A [14]. 3.2. LGR5 is specifically expressed in projection and motor neurons in the CNS Bioinformatics analysis suggested that LGR5-DIGs are most likely to relate to neuron development and, specifically, motor neuron specification. We then explored the LGR5 expression pattern in
CNS. By taking advantage of another dataset (GSE13379) of singe cell transcriptome in CNS (gene profiles derived from genetically defined cell populations in translating ribosome affinity purification (TRAP) transgenic mice) [15], we demonstrated that LGR5 is specifically highly expressed in a spectrum of neurons including Cholinergic neurons in spinal cord, motor neurons in brain stem, and neurons in Layer 5a and 6 in cortex (Fig. 1). Interestingly, these types of neurons are projection motor neurons in different parts of the CNS that innervate terminal muscles. Cholinergic neurons in spinal cord are primarily motor neurons in the ventricolumna which project to innervate muscle. Layer 5 and 6 in the cortex contain motor neurons that project to distant areas of the CNS, including neostriatum, brainstem, and spinal cord, etc. [16]. Brain stem motor neurons primarily project cranial nerves. In addition, analysis of the expression profile of LGR5 ligands R-spondin (RSPO) family (RSPO1-RSPO4) revealed that RSPO2 and RSPO3 also showed similar expression pattern to LGR5, which are primarily highly expressed in projection and motor neurons (Fig. 1). Together with the bioinformatics analysis results that LGR5-DIGs are mainly related with motor neuron development, these data suggested that LGR5 is closely involved in the development of projection neuron in CNS, especially motor neurons in the spinal cord and brain stem. 3.3. LGR5 is positively regulated by neurogenic signals To more directly address that LGR5 is involved in neuron development, we investigated the expression of LGR5 affected by neurogenic signals. It has been reported that LGR5 was positively regulated by proneural signal OLIG2 and Neurogenin 1 [6,7]. We further evaluated the impact of signals that inhibit neuronal diferentiion on the expression of LGR5. It has been demonstrated that Notch inhibits neurogenesis while promotes glial differnetiation of NSCs [17,18]. To explore the effects Notch signaling in LGR5 expression, we first used a ␥-secretase inhibitor DAPT to inhibit the Notch signal pathway in a human NSC cell line. After DAPT treatment, the expession of the Notch target genes Hes1 and Hey1 were decreased dose dependantly (Fig. 2A), and the neuronal markers TUJ1 and MAP2 were induced (Fig. 2B), indicating increased neuronal differentiation of the NSCs. As a result, the expression level of LGR5 was also increased in the NSCs after DAPT treatment dose dependantly as revelaed by qPCR and Western Blot (Fig. 2C
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Fig. 1. Analysis of a dataset (GSE13379) revealed that LGR5 is specifically expressed in a spectrum of neurons including motor neurons in the spinal cord and cortex. SC, spinal cord; BS, brain stem; Ctx, Cortex; CB, cerebellum; BF, basal forebrain; CS, corpus striatum; OG, oligodendrocyte; Ste/Bas neurons, Stellate and Basket neurons; Cho, cholinergic; Pnoc, prepronociceptin; CCK, cholecystokinin; Cort, cortistatin; Drd1, dopamine receptor D1. Relative mRNA levels were normalized with GCRMA and filtered to remove probesets with low signal and those identified as background [15].
and D). Next, we increasd the Notch activity in the NSC cells by introducing a truncated, constitutively active form of the NOTCH2 receptor (NICD2) with pcDNA3.1 plasmid (pcDNA3.1-NICD2). Following NICD2 transfection, the mRNA levels of NOTCH2 and the target genes Hey1 and HES1 were increased (Fig. 2E). Immuno flourecence stainig revealed that MAP2+ neuronal cells was significantly reduced and the GFAP+ cells was increased (Fig. 2F), indicating the neurogenic potential of the NSCs was inhibited. Interestingly, the mRNA and protein levels of LGR5 were also significantly decreased as revealed by qPCR and Western Blot (Fig. 2G and H). These results imply that the level of LGR5 expression was concordant with the neurogenic potential of NSCs after treatment by Notch inhibitor or activitor.
3.4. LGR5 is involved in neuron differentiation We then examined whether LGR5 may influence neuron differentiation of NSCs. we used siRNA to knockdown LGR5 expression in the NSC cell line. The efficiency of LGR5 knockdown in NSCs detected by qPCR was about 70%. As a result, neuronal marker genes, including the ones that are intimately associated with motor neuron function such as GAP43, MAP2, SYP and CHRM3 were reduced after LGR5 knockdown (Fig. 3A). Immuno flourecence staining demonstrated that the percentage of NeuN, MAP2 positive neurons were significantly reduced (Fig. 3B and C), implying the neurogenic potential of NSCs was inhibited after LGR5 knockdown.
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Fig. 2. Inhibition of Notch activity (A–D) in NSCs by the ␥-secretase inhibitor DAPT decreased the expression of Notch target genes Hes1 and Hey1 (A) dose dependently. After Notch activity inhibition, the expression of neuronal markers Tuj1 and MAP2 (B) and LGR5 were increased (C). (D) Western blot showed that LGR5 protein level was increased after DAPT treatment. In line with this, expression of constitutive Notch2 intracellular domain in NSCs increased the expression of Notch2 and Notch target genes such Hes1 and Hey1 (E), while MAP2 neuronal cells were decreased and GFAP astrocytic cells were increased (F). In addition, LGR5 expression was inhibited after Notch activation as revealed by qPCR (G) and Western blot (H). * p < 0.05, ** p < 0.001 compared to control group (n = 3).
Fig. 3. Knockdown of LGR5 in NSCs by siRNA inhibited the expression of neuronal markers GAP43, MAP2, SYP and CHRM3 (A). Immunofluorescence staining of neuronal markers NeuN and MAP2 (B) revealed that the percentage of neuronal cells were decreased after LGR5 knockdown (LGR5 KD) (C). In addition, the expression levels of genes that control the motor neuron development were reduced, including Isl1, Lhx3, PHOX2A, TBX20, NEUROG2 (C). * p < 0.05, ** p < 0.001 compared to control group (n = 3).
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3.5. LGR5 regulated genes that are critical for the development of motor neurons The above results revealed that LGR5 is involved in neurogenic potential in CNS, especially the motor neuron differentiation. We next explored the possible mechanisms of LGR5 regulated motor neuron differentiation. It has been demonstrated that motor neuron specification was controlled by key transcriptional factors. Isl1, Lhx3 and NEUROG2 are critical for spinal motor neuron identity on spinal progenitors derived from embryonic stem cells [14,19], while Isl1, Phox2a/2b and Tbx20 were important for the differentiation of cranial motor neurons located in the ventral midbrain, hindbrain and cervical spinal cord [20,21]. Our bioinformatics analysis revealed that LGR5 may interact with these factors such as ISL1, PHOX2A. We then explored the impact of LGR5 on the expression of these genes. Intriguingly, the mRNA levels of Isl1, Lhx3, PHOX2A/B, TBX20, NEUROG2 were reduced after LGR5 knockdown in NSCs as revealed by RT-PCR. These results imply that LGR5 may influence the motor neuron differentiation of NSCs by regulating these factors.
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4. Discussion The control of neuron differentiation of NSCs, especially the development of motor neurons, remained challenges for long time in regenerative medicines such as spinal cord injury, brain injury, stroke, and amyotrophic lateral sclerosis (ALS) [16,22–24], mainly due to that the mechanisms underlying neuron specification is not fully acknowledged. Although some of the molecular regulators were reported, ideal targets to effectively control neuronal differentiation and maturation are lacking [14,25,26]. Therefore, it is important and urgent to discover novel potential molecular targets that may promote the neurogenic potential, especially the specification of motor neurons. Our results firstly demonstrated that LGR5 is involved in the differentiation and specification of motor neurons in CNS including spinal cord and brain. Phenotypically, bioinformatics analysis revealed that LGR5-DIGs mainly contained genes that are involved in neuronal differentiation and functions, including factor genes control the motor neuron development. In addition, LGR5 was more highly expressed in projection and motor neurons in CNS, especially in motor neurons in spinal cord and brain stem. Functionally, LGR5 was positively regulated by neurogenic signals such as Neurogenin 1 and OLIG2 [6,7], and was negatively regualted by neuroginc inhibiting signals such as Notch pathway. Importantly, LGR5 regulated the neurogenic potential of NSCs probably by interacting with transcriptional factors that control the motor neuron identity. Consisting with the bioinformatics analysis, LGR5 regulated the expression of ISL1, PHOX2A, which are critical factors controlling the motor neuron specification. Taken together, our results provide new insight into the biological roles of LGR5 in nervous system, and, importantly, potential novels targets that may promote motor neuron specification in CNS.
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The authors have no conflicts of interest to disclose.
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LGR5/GPR49 is implicated in motor neuron specification in nervous system Shao-jun Song a,b,∗,1 , Xing-gang Mao c,∗∗,1 , Chao Wang d , An-guo Han a , Ming Yan e , Xiao-yan Xue f a
Department of Neurosurgery, PLA 254 Hospital, Tianjin, China Department of Neurosurgery, Hinan Branch of Chinese PLA General Hospital, Haitang Harbor, Sanya, Hainan Province 572013, China c Department of Neurosurgery, Xijing Hospital, Fourth Military Medical University, Xi’an, Shaanxi Province, China d Department of Neurosurgery, Tangdu Hospital, Fourth Military Medical University, Xi’an, Shaanxi Province, China e Department of Orthopaedic Surgery, Xijing Hospital, Fourth Military Medical University, Xi’an, Shaanxi Province, China f Department of Pharmacology, School of Pharmacy, Fourth Military Medical University, Xi’an, Shaanxi Province, China b
h i g h l i g h t s • • • • •
The biological role of LGR5 is closely related with neuron development and functions. LGR5 is specifically highly expressed in projection motor neurons in nervous system. Notch signal inhibited neurogenic potential and LGR5 expression in neural stem cells. Knockdown of LGR5 inhibited neurogenic potential of neural stem cells. LGR5 regulated the expression of key factors programing the identity of motor neurons.
a r t i c l e
i n f o
Article history: Received 4 June 2014 Received in revised form 3 September 2014 Accepted 29 September 2014 Available online 16 October 2014 Keywords: GPR49 Motor neuron differentiation Neural stem cell Notch WNT Bioinformatics
a b s t r a c t The biological roles of stem cell marker LGR5, the receptor for the Wnt-agonistic R-spondins, for nervous system are poorly known. Bioinformatics analysis in normal human brain tissues revealed that LGR5 is closely related with neuron development and functions. Interestingly, LGR5 and its ligands R-spondins (RSPO2 and RSPO3) are specifically highly expressed in projection motor neurons in the spinal cord, brain stem and cerebral. Inhibition of Notch activity in neural stem cells (NSCs) increased the percentage of neuronal cells and promoted LGR5 expression, while activation of Notch signal decreased neuronal cells and inhibited the LGR5 expression. Furthermore, knockdown of LGR5 inhibited the expression of neuronal markers MAP2, NeuN, GAP43, SYP and CHRM3, and also reduced the expression of genes that program the identity of motor neurons, including Isl1, Lhx3, PHOX2A, TBX20 and NEUROG2. Our data demonstrated that LGR5 is highly expressed in motor neurons in nervous system and is involved in their development by regulating transcription factors that program motor neuron identity. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Abbreviations: ALS, amyotrophic lateral sclerosis; ARACNe, Algorithm for the Reconstruction of Accurate Cellular Networks; CNS, central nervous system; DAPT, N-[N-(35-difluorophenacetyl)-l-alanyl]-S- phenylglycine t-butyl ester; DPI, data processing inequality; GSC, glioblastoma stem cells; LGR5-DIGs, directly interacting genes of LGR5; NODRN, neuronal and oligodendrocyte-differentiation related network; NSC, neural stem cell; qPCR, quantitative real time PCR; SFM, serum free medium. ∗ Corresponding author. ∗∗ Corresponding author. Tel.: +86 02984771031. E-mail addresses: [email protected] (S.-j. Song), [email protected] (X.-g. Mao). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.neulet.2014.09.056 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
LGR5 (leucine-rich repeat-containing G protein-coupled receptor 5 also known as GPR49) was recently identified as a stem cell marker for several tissues and cancers, including intestine and colon normal and cancer stem cells, hair follicle stem cells, etc. [1–4]. More recently, LGR5 was reported to be expressed in glioblastoma stem cells (GSCs), and contribute to the maintenance of GSCs [5,6]. However, the biological functions of LGR5 for stem cells are poorly acknowledged, even for the established stem cells that it marks, such as intestine and colon stem cells. The potential of LGR5 to identify stem cells in different kinds of tissues implies it may be a more universal stem cell marker, raising the interesting question that what is its biological function in nervous system?
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By taking advantage of bioinformatics analysis, our previous study demonstrated that LGR5 is involved in the process of neurogenesis [6]. Biological experiments further confirmed that LGR5 regulate a cohort of genes involved in the network of neuronal and oligodendrocyte differentiation (NODRN) [6]. Interestingly, a positive feedback between LGR5 and OLIG2 was identified [6]. In addition, LGR5 is strongly regulated by neurogenin 1 [7], a proneural basic helix-loop-helix (bHLH) transcription factor that is important for neural stem cell (NSC) differentiation. All of these results imply that LGR5 may play a role in the neuronal differentiation and maturation. Here, we further studied the biological role of LGR5 in nervous system, and revealed that LGR5 is involved in the process of neuronal differentiation and maturation, especially in the development of motor neurons in central nervous system (CNS). 2. Methods and materials
of Notch2 (pcDNA3.1-NICD2). Then the plasmid pcDNA3.1-NICD2 was transfected into the cells with LipofectamineTM 2000 according to the manufacturer’s instructions. Vector pcDNA3.1 was used as control. 2.4. siRNA transfection LGR5 knockdown by siRNA was performed as described previously [6]. Briefly, the NSC spheres were dissociated into single cells and plated on poly-l-ornithine (50 mg/ml, P3655, Sigma Chemical) coated wells (50,000 cells per 24–well platewell) to attach for 24 h. Then the cells were transfected by Stealth small interfering RNAs (siRNA) targeting LGR5 or non-targeting RNA sequence (Invitrogen Lifetechnologies, USA) with OligofectamineTM RNAiMAX Reagent according to the manufacturers protocol (Invitrogen). After 24 h of incubation, the cells were washed and detached from the wells and placed back to SFM medium. The efficiency of siRNA interference was evaluated by qPCR after 72 h of transfection.
2.1. ARACNe network reconstruction and bioinformatics analysis 2.5. Immunofluorescence ARACNe (Algorithm for the Reconstruction of Accurate Cellular Networks), an information-theoretic algorithm for inferring transcriptional interactions [8], was used to identify a repertoire of candidate transcriptional regulators of interesting genes, as described previously [9]. Expression profiles of normal brain tissue used in the analysis were from GSE15745 (Gene expression profiles derived from frozen tissue samples of the cerebellum (CRBLM), frontal cortex (FCTX), caudal pons (PONS) and temporal cortex (TCTX) obtained from 150 neurologically normal subjects) [10] and GSE11882 (Gene expression profiles derived from frozen tissue samples in the hippocampus (HC), entorhinal cortex (EC), superior frontal gyrus (SG), and postcentral gyrus (PCG) across the lifespan of 63 cognitively intact individuals from 20–99 years old) [11]. First, candidate interactions between a transcription factor (TF, x) and its potential target (y) are identified by computing pairwise mutual information, MI[x; y], using a Gaussian kernel estimator and by thresholding the MI based on the null-hypothesis of statistical independence (p < 0.05, Bonferroni corrected for the number of tested pairs). Then, indirect interactions are removed using the data processing inequality (DPI) analysis with a tolerance of 20%, a well-known property of the mutual information. 2.2. Culture of primary neural stem cells (NSCs) Culturing of human fetal cortical NSCs were isolated from two spontaneous aborted fetuses (8–12 weeks) as described previously [9,12]. The study protocol was approved by Institutional Review Committee of Xijing Hospital of the Fourth Military Medical University. Written informed consent was obtained from patients. Briefly, samples were dissociated to a single cell suspension using a fire-polished Pasteur pipette and cultured in serum free medium (SFM) consisting of DMEM-F12 medium, EGF (20 ng/mL; Invitrogen, Carlsbad, CA), bFGF (20 ng/mL; Invitrogen) and B27 (1:50; Invitrogen). 2.3. Inhibition and activation of Notch signal The highly active ␥-secretase inhibitor, N-[N-(3, 5difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT), purchased from Sigma–Aldrich Corporation (USA), was used to inhibit the Notch signal activity. To activate Notch signal, a truncated, constitutively active form of the NOTCH2 receptor (NICD2) was subcloned to adenovius. Breifly, cytoplasmic portion of the human Notch2 receptor (amino acids 1747–2531) was inserted into a mammalian expression vector pcDNA3.1 (Invitrogen) to create a constitutively active form
Immunofluorescence was performed on cultured and differentiated NSCs as described previously [12]. Briefly, undifferentiated neural spheres were plated onto poly-l-lysine coated glass coverslips for 4 h, fixed with 4% paraformaldehyde, and incubated with NeuN (1:1000, mouse monoclonal IgG1; Chemicon, Temecula, CA), GFAP (1:5000, rabbit polyclonal; Abcam), and MAP2 (1:1000, Santa Cruz) antibody overnight at 4 ◦ C. Texas Red goat anti-rabbit (Molecular Probes, Invitrogen, Carlsbad, CA) was used as secondary antibody. Counterstaining with Hoeschst 33342 (Sigma, St. Louis, MO) was additionally performed to permit counting of cell nuclei. 2.6. qPCR qPCR was performed as described previously [9,13]. briefly, RNA was extracted from cultured cells using Trizol Reagent (Invitrogen), and reverse transcribed into cDNA. qPCR analysis was performed on an ABI7700 using SYBR Green PCR Core Reagents in 20 L volume (Applied Biosystems, Warrington, UK). Primer sequences for qPCR studies are shown in Supplementary Table 1. All samples, including template-free controls, were assayed in triplicate and the relative amount of target transcripts normalized to the number of human actin transcripts found in the same sample. Specificity was verified by melting curve analysis and agarose gel electrophoresis. Relative fold changes were calculated using the Ct method by using the threshold cycle values of each sample. 2.7. Western blotting Cultured cells were lysed in SDS sample buffer, and 20 g proteins were run on 6% SDS-PAGE gel and transferred to a nitrocellulose membrane. Blots were blocked in PBS containing 5% nonfat dry milk powder and incubated overnight at 4 ◦ C with primary antibody of LGR5 (1:1000, Abcam) or GAPDH (1:500,000; Abcam). Blots were then washed with PBS containing 0.1% Tween 20 (PBST) and incubated in secondary antibodies coupled to peroxidase. After washing in PBST, blots were developed with chemiluminescence according to the manufacturer’s instructions (enhanced chemiluminescence, Amersham Biosciences, GE Healthcare, France). All Western blot analyses were done in duplicates. 2.8. Statistical analysis Statistical analyses were performed using Student’s t-tests and one-way analysis of variance with least squared difference post hoc tests, as appropriate. All P values are two-tailed. A value of
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Table 1 David analysis of LGR5-DIGs inferred from the 2 normal brain datasets. The representative enriched terms in the top 20 clusters were shown. Category
Term
N*
% of involved genes/total genes
Modified Fisher Exact P-Value
Benjamini
GOTERM MF FAT GOTERM MF FAT SP PIR KEYWORDS GOTERM BP FAT SP PIR KEYWORDS GOTERM BP FAT GOTERM BP FAT GOTERM BP FAT GOTERM BP FAT KEGG PATHWAY SP PIR KEYWORDS GOTERM BP FAT GOTERM BP FAT UP SEQ FEATURE GOTERM BP FAT GOTERM BP FAT
Transcription factor activity Sequence-specific DNA binding Homeobox Neurological system process glycoprotein Sensory perception Cognition Neuron differentiation Metencephalon development Olfactory transduction g-protein coupled receptor Neuron development Neuron projection Morphogenesis Domain:Leucine-zipper Hindbrain development Regulation of transcription from RNA polymerase II promoter Cerebellum development Extracellular region Sensory organ development Transcription from RNA Polymerase II promoter Mesenchymal cell development
28 20 11 25 53 18 19 12 4 11 15 9 7 5 4 13
18.18 12.99 7.14 16.23 34.42 11.69 12.34 7.79 2.60 7.14 9.74 5.84 4.55 3.25 2.60 8.44
2.70E-07 3.60E-06 2.30E-05 1.20E-04 5.90E-04 7.10E-04 9.60E-04 1.60E-03 3.50E-03 3.80E-03 5.30E-03 1.00E-02 1.10E-02 1.20E-02 1.60E-02 2.60E-02
7.7E-5 3.5E-4 3.0E-3 6.5E-2 3.8E-2 1.8E-1 2.0E-1 2.3E-1 3.3E-1 2.6E-1 1.2E-1 5.9E-1 6.0E-1 8.2E-1 6.8E-1 7.8E-1
3 23 6 6
1.95 14.94 3.90 3.90
2.80E-02 5.00E-02 5.20E-02 5.60E-02
7.9E-1 1.0E0 8.7E-1 8.8E-1
3
1.95
7.40E-02
9.2E-1
GOTERM GOTERM GOTERM GOTERM
BP CC BP BP
FAT FAT FAT FAT
GOTERM BP FAT *
N: number of LGR5-related genes involved in the annotation category.
p < 0.05 was considered statistically significant. Statistical analyses were done using SPSS v.13.0.0 (SPSS, Inc., Chicago, IL). 3. Results and discussion 3.1. The biological roles of LGR5 in CNS is closely related with neuron functions Bioinformatics analysis revealed that LGR5 is more closely related with neuron functions, especially with the maturation and projection of neurons, such as synapse function, ion channel, and axonal formation [6]. In addition, LGR5 is regulated by OLIG2, an important factor associated with neuron and oligodendrocyte differentiation [6]. To further dissect its functional role in normal CNS, we then performed bioinformatics analysis in two normal brain transcriptome datasets (GSE15745 [10] and GSE11882 [11]) by using ARACNE algorithm [6,8,9]. As a result, we got two lists of potential LGR5 direct interacting genes (LGR5-DIGs) in normal CNS. There are totally 171 genes that are overlapped in the two lists of LGR5-DIGs (Supplementary Table 2), and we considered these genes as the most reliable genes that may interact with LGR5 in normal CNS. Next, we performed DAVID analysis to explore the functional roles of these genes. The top 20 functional categories in the Annotation Clusters sorted by Enrichment Score revealed that, beside non-specific functional roles such as DNA binding, transcription binding, glycoprotein, etc., the most important potential functions of LGR5-DIGs are related to neuron development and function (Table 1). The genes related to neuron include ISL1, LHX5, RORA, DMD, GBX2, GAS7, NRL, PAX2, PHOX2A, PSEN1, UNC5C and ERBB3. A portion of these genes is also related to axonogenesis, such as ISL1, GBX2, PAX2, UNC5C, ERBB3. Noticeably, LGR5-DIGs include several genes that are critical for the differentiation of motor neuron in spinal cord and cortex, including ISL1, PHOX2A [14]. 3.2. LGR5 is specifically expressed in projection and motor neurons in the CNS Bioinformatics analysis suggested that LGR5-DIGs are most likely to relate to neuron development and, specifically, motor neuron specification. We then explored the LGR5 expression pattern in
CNS. By taking advantage of another dataset (GSE13379) of singe cell transcriptome in CNS (gene profiles derived from genetically defined cell populations in translating ribosome affinity purification (TRAP) transgenic mice) [15], we demonstrated that LGR5 is specifically highly expressed in a spectrum of neurons including Cholinergic neurons in spinal cord, motor neurons in brain stem, and neurons in Layer 5a and 6 in cortex (Fig. 1). Interestingly, these types of neurons are projection motor neurons in different parts of the CNS that innervate terminal muscles. Cholinergic neurons in spinal cord are primarily motor neurons in the ventricolumna which project to innervate muscle. Layer 5 and 6 in the cortex contain motor neurons that project to distant areas of the CNS, including neostriatum, brainstem, and spinal cord, etc. [16]. Brain stem motor neurons primarily project cranial nerves. In addition, analysis of the expression profile of LGR5 ligands R-spondin (RSPO) family (RSPO1-RSPO4) revealed that RSPO2 and RSPO3 also showed similar expression pattern to LGR5, which are primarily highly expressed in projection and motor neurons (Fig. 1). Together with the bioinformatics analysis results that LGR5-DIGs are mainly related with motor neuron development, these data suggested that LGR5 is closely involved in the development of projection neuron in CNS, especially motor neurons in the spinal cord and brain stem. 3.3. LGR5 is positively regulated by neurogenic signals To more directly address that LGR5 is involved in neuron development, we investigated the expression of LGR5 affected by neurogenic signals. It has been reported that LGR5 was positively regulated by proneural signal OLIG2 and Neurogenin 1 [6,7]. We further evaluated the impact of signals that inhibit neuronal diferentiion on the expression of LGR5. It has been demonstrated that Notch inhibits neurogenesis while promotes glial differnetiation of NSCs [17,18]. To explore the effects Notch signaling in LGR5 expression, we first used a ␥-secretase inhibitor DAPT to inhibit the Notch signal pathway in a human NSC cell line. After DAPT treatment, the expession of the Notch target genes Hes1 and Hey1 were decreased dose dependantly (Fig. 2A), and the neuronal markers TUJ1 and MAP2 were induced (Fig. 2B), indicating increased neuronal differentiation of the NSCs. As a result, the expression level of LGR5 was also increased in the NSCs after DAPT treatment dose dependantly as revelaed by qPCR and Western Blot (Fig. 2C
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Fig. 1. Analysis of a dataset (GSE13379) revealed that LGR5 is specifically expressed in a spectrum of neurons including motor neurons in the spinal cord and cortex. SC, spinal cord; BS, brain stem; Ctx, Cortex; CB, cerebellum; BF, basal forebrain; CS, corpus striatum; OG, oligodendrocyte; Ste/Bas neurons, Stellate and Basket neurons; Cho, cholinergic; Pnoc, prepronociceptin; CCK, cholecystokinin; Cort, cortistatin; Drd1, dopamine receptor D1. Relative mRNA levels were normalized with GCRMA and filtered to remove probesets with low signal and those identified as background [15].
and D). Next, we increasd the Notch activity in the NSC cells by introducing a truncated, constitutively active form of the NOTCH2 receptor (NICD2) with pcDNA3.1 plasmid (pcDNA3.1-NICD2). Following NICD2 transfection, the mRNA levels of NOTCH2 and the target genes Hey1 and HES1 were increased (Fig. 2E). Immuno flourecence stainig revealed that MAP2+ neuronal cells was significantly reduced and the GFAP+ cells was increased (Fig. 2F), indicating the neurogenic potential of the NSCs was inhibited. Interestingly, the mRNA and protein levels of LGR5 were also significantly decreased as revealed by qPCR and Western Blot (Fig. 2G and H). These results imply that the level of LGR5 expression was concordant with the neurogenic potential of NSCs after treatment by Notch inhibitor or activitor.
3.4. LGR5 is involved in neuron differentiation We then examined whether LGR5 may influence neuron differentiation of NSCs. we used siRNA to knockdown LGR5 expression in the NSC cell line. The efficiency of LGR5 knockdown in NSCs detected by qPCR was about 70%. As a result, neuronal marker genes, including the ones that are intimately associated with motor neuron function such as GAP43, MAP2, SYP and CHRM3 were reduced after LGR5 knockdown (Fig. 3A). Immuno flourecence staining demonstrated that the percentage of NeuN, MAP2 positive neurons were significantly reduced (Fig. 3B and C), implying the neurogenic potential of NSCs was inhibited after LGR5 knockdown.
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Fig. 2. Inhibition of Notch activity (A–D) in NSCs by the ␥-secretase inhibitor DAPT decreased the expression of Notch target genes Hes1 and Hey1 (A) dose dependently. After Notch activity inhibition, the expression of neuronal markers Tuj1 and MAP2 (B) and LGR5 were increased (C). (D) Western blot showed that LGR5 protein level was increased after DAPT treatment. In line with this, expression of constitutive Notch2 intracellular domain in NSCs increased the expression of Notch2 and Notch target genes such Hes1 and Hey1 (E), while MAP2 neuronal cells were decreased and GFAP astrocytic cells were increased (F). In addition, LGR5 expression was inhibited after Notch activation as revealed by qPCR (G) and Western blot (H). * p < 0.05, ** p < 0.001 compared to control group (n = 3).
Fig. 3. Knockdown of LGR5 in NSCs by siRNA inhibited the expression of neuronal markers GAP43, MAP2, SYP and CHRM3 (A). Immunofluorescence staining of neuronal markers NeuN and MAP2 (B) revealed that the percentage of neuronal cells were decreased after LGR5 knockdown (LGR5 KD) (C). In addition, the expression levels of genes that control the motor neuron development were reduced, including Isl1, Lhx3, PHOX2A, TBX20, NEUROG2 (C). * p < 0.05, ** p < 0.001 compared to control group (n = 3).
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3.5. LGR5 regulated genes that are critical for the development of motor neurons The above results revealed that LGR5 is involved in neurogenic potential in CNS, especially the motor neuron differentiation. We next explored the possible mechanisms of LGR5 regulated motor neuron differentiation. It has been demonstrated that motor neuron specification was controlled by key transcriptional factors. Isl1, Lhx3 and NEUROG2 are critical for spinal motor neuron identity on spinal progenitors derived from embryonic stem cells [14,19], while Isl1, Phox2a/2b and Tbx20 were important for the differentiation of cranial motor neurons located in the ventral midbrain, hindbrain and cervical spinal cord [20,21]. Our bioinformatics analysis revealed that LGR5 may interact with these factors such as ISL1, PHOX2A. We then explored the impact of LGR5 on the expression of these genes. Intriguingly, the mRNA levels of Isl1, Lhx3, PHOX2A/B, TBX20, NEUROG2 were reduced after LGR5 knockdown in NSCs as revealed by RT-PCR. These results imply that LGR5 may influence the motor neuron differentiation of NSCs by regulating these factors.
[2]
[3]
[4]
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[9]
4. Discussion The control of neuron differentiation of NSCs, especially the development of motor neurons, remained challenges for long time in regenerative medicines such as spinal cord injury, brain injury, stroke, and amyotrophic lateral sclerosis (ALS) [16,22–24], mainly due to that the mechanisms underlying neuron specification is not fully acknowledged. Although some of the molecular regulators were reported, ideal targets to effectively control neuronal differentiation and maturation are lacking [14,25,26]. Therefore, it is important and urgent to discover novel potential molecular targets that may promote the neurogenic potential, especially the specification of motor neurons. Our results firstly demonstrated that LGR5 is involved in the differentiation and specification of motor neurons in CNS including spinal cord and brain. Phenotypically, bioinformatics analysis revealed that LGR5-DIGs mainly contained genes that are involved in neuronal differentiation and functions, including factor genes control the motor neuron development. In addition, LGR5 was more highly expressed in projection and motor neurons in CNS, especially in motor neurons in spinal cord and brain stem. Functionally, LGR5 was positively regulated by neurogenic signals such as Neurogenin 1 and OLIG2 [6,7], and was negatively regualted by neuroginc inhibiting signals such as Notch pathway. Importantly, LGR5 regulated the neurogenic potential of NSCs probably by interacting with transcriptional factors that control the motor neuron identity. Consisting with the bioinformatics analysis, LGR5 regulated the expression of ISL1, PHOX2A, which are critical factors controlling the motor neuron specification. Taken together, our results provide new insight into the biological roles of LGR5 in nervous system, and, importantly, potential novels targets that may promote motor neuron specification in CNS.
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The authors have no conflicts of interest to disclose.
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