Molecular targets for endogenous glial cell line-derived neurotrophic factor modulation in striatal parvalbumin interneurons

Molecular targets for endogenous glial cell line-derived neurotrophic factor modulation in striatal parvalbumin interneurons Daniel Enterrı´a-Morales, 1,2 Natalia Lo´pez-Gonza´lez del Rey, 3 Javier Blesa, 3,4 Ivette Lo´pez-Lo´pez, 1 Sarah Gallet, 5 Vincent Pre´vot, 5 Jose´Lo´pez-Barneo 1,2,4 and Xavier d’Anglemont de Tassigny 1,2,4 Administration of recombinant glial cell line-derived neurotrophic factor into the putamen has been tested in preclinical and clinical studies to evaluate its neuroprotective effects on the progressive dopaminergic neuronal degeneration that characterizes Parkinson’s disease. However, intracerebral glial cell line-derived neurotrophic factor infusion is a challenging therapeutic strategy, with numerous potential technical and medical limitations. Most of these limitations could be avoided if the production of endogenous glial cell linederived neurotrophic factor could be increased. Glial cell line-derived neurotrophic factor is naturally produced in the striatum from where it exerts a trophic action on the nigrostriatal dopaminergic pathway. Most of striatal glial cell line-derived neurotrophic factor is synthesized by a subset of GABAergic interneurons characterized by the expression of parvalbumin. We sought to identify molecular targets specific to those neurons and which are putatively associated with glial cell line-derived neurotrophic factor synthesis. To this end, the transcriptomic differences between glial cell line-derived neurotrophic factor-positive parvalbumin neurons in the striatum and parvalbumin neurons located in the nearby cortex, which do not express glial cell line-derived neurotrophic factor, were analysed. Using mouse reporter models, we have defined the genomic signature of striatal parvalbumin interneurons obtained by fluorescenceactivated cell sorting followed by microarray comparison. Short-listed genes were validated by additional histological and molecular analyses. These genes code for membrane receptors (Kit,Gpr83,Tacr1,Tacr3,Mc3r), cytosolic proteins (Pde3a,Crabp1,Rarres2, Moxd1) and a transcription factor (Lhx8). We also found the proto-oncogene cKit to be highly specific of parvalbumin interneurons in the non-human primate striatum, thus highlighting a conserved expression between species and suggesting that specific genes identified in mouse parvalbumin neurons could be putative targets in the human brain. Pharmacological stimulation of four G-protein-coupled receptors enriched in the striatal parvalbumin interneurons inhibited Gdnf expression presumably by decreasing cyclic adenosine monophosphate formation. Additional experiments with pharmacological modulators of adenylyl cyclase and protein kinase A indicated that this pathway is a relevant intracellular route to induce Gdnf gene activation. This preclinical study is an important step in the ongoing development of a specific pro-endo-glial cell line-derived neurotrophic factor pharmacological strategy to treat Parkinson’s disease. 1 Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocı´o, CSIC, Universidad de Sevilla, Seville, Spain 2 Departamento de Fisiologı´a Me´dica y Biofı´sica, Facultad de Medicina, Universidad de Sevilla, Seville, Spain 3 HM CINAC, Hospital Universitario HM Puerta del Sur, Mo´stoles, Spain 4 Centro de Investigacio´n Biome´dica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain 5 Univ. Lille, Inserm, CHU Lille, Lille Neuroscience & Cognition, Laboratory of Development and Plasticity of the Neuroendocrine Brain, UMR-S 1172, Lille, France Correspondence to: Xavier d’Anglemont de Tassigny Instituto de Biomedicina de Sevilla (IBiS) Campus Hospital Universitario Virgen del Rocı´o, Avenida Manuel Siurot s/n, 41013 Seville, Spain E-mail: [email protected] Received April 12, 2020. Revised June 5, 2020. Accepted June 23, 2020. Advance Access publication July 15, 2020 V CThe Author(s) (2020). Published by Oxford University Press on behalf of the Guarantors of Brain. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] B BR AIN COMMUNICATIONS AIN COMMUNICATIONS doi:10.1093/braincomms/fcaa105 BRAIN COMMUNICATIONS 2020: Page 1 of 18 |1 Downloaded from https://academic.oup.com/braincomms/article/2/2/fcaa105/5871916 by Universidad de Sevilla user on 11 July 2023 Keywords: Parkinson’s disease; pro-endo-GDNF pharmacology; parvalbumin interneurons; striatum; gene expression Abbreviations: AC ¼adenylyl cyclase; AMP ¼adenosine monophosphate; cAMP ¼cyclic adenosine monophosphate; CRABP1 ¼cellular retinoic acid-binding protein 1; CRE ¼cAMP response element; CREB ¼cAMP response element-binding protein; CTX ¼cortex; DA ¼dopamine; DE ¼differentially expressed; FACS ¼fluorescence-activated cell sorting; FC ¼fold-change; FSK ¼forskolin; GDNF ¼glial cell line-derived neurotrophic factor; GO ¼gene ontology; GPCR ¼G-protein-coupled receptor; GPR83 ¼ G-protein-coupled receptor 83; LHX8 ¼LIM homeobox 8; MC3R ¼melanocortin receptor 3; NK1R ¼neurokinin receptor 1; NK3R ¼neurokinin receptor 3; PDE3A ¼phosphodiesterase 3A; PKA ¼protein kinase A; PV ¼parvalbumin; RA ¼retinoic acid; RARRES2 ¼retinoic acid receptor responder 2; RET ¼rearranged during transfection; SNpc ¼substantia nigra pars compacta; SPATA18 ¼spermatogenesis-associated 18; ST ¼striatum; XGal ¼5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside. Introduction Parkinson’s disease, the second most prevalent neurodegenerative disorder, is characterized by the progressive loss of mid-brain dopamine (DA) neurons (Surmeier et al., 2017). Currently, there are no therapies available to prevent damage to dopaminergic nigrostriatal neurons or promote regeneration of their axons, with existing treatments addressing symptom relief only (Pires et al., 2017). Glial cell line-derived neurotrophic factor (GDNF) has long been known for its potent trophic action on mesencephalic DA neurons (Lin et al., 1993;Kordower and Bjorklund, 2013). GDNF binds to GDNF family receptor a1 (GFRa1), and the resulting complex activates the RET receptor tyrosine kinase and subsequent downstream signals. Both receptors are expressed by DA neurons and their selective deletion in mice is associated with progressive loss of the nigrostriatal pathway (Kramer and Liss, 2015). Numerous preclinical studies involving rodent and non-human primate models have established the efficacy of cell-derived or recombinant GDNF treatment in protecting and restoring substantia nigra pars compacta (SNpc) DA neurons (Sauer et al., 1995;Kirik et al., 2004;Gash et al., 2005;Mu~ noz-Manchado et al., 2013). The clinical effect of GDNF treatment delivered directly to the brain via intracerebral infusion devices has also been tested in Parkinson’s disease patients (d’Anglemont de Tassigny et al., 2015). Although safety has been shown for direct intraparenchymal injection of recombinant GDNF (Gill et al., 2003) or viral-mediated strategy (Heiss et al., 2019), clinical evidences have not fully demonstrated the efficacy of these approaches (Slevin et al., 2005;Lang et al., 2006). Recently, GDNF therapy in Parkinson’s disease has regained attention due to the publication of a novel, intermittent intraputamenal delivery strategy to optimize GDNF diffusion (Whone et al., 2019). Although the criteria for clinical improvement were not met after 40 weeks of GDNF delivery, this new approach resulted in significant progress on PET images. Indeed, there was a significant increase in 18 F-DOPA putamenal uptake compared to the placebo group in patients that received GDNF (Whone et al., 2019). These data, suggesting dopaminergic sprouting in the putamen, pointed to a regenerative effect of GDNF, an outcome that had previously been observed only in a single-case report (Love et al., 2005). The use of exogenous GDNF as restorative therapy for Parkinson’s disease is faced with serious limitations (e.g. major surgery, outpatient care or intraputamenal delivery), which highlight the importance of the further Graphical Abstract 2|BRAIN COMMUNICATIONS 2020: Page 2 of 18 D. Enterrı´a-Morales et al. Downloaded from https://academic.oup.com/braincomms/article/2/2/fcaa105/5871916 by Universidad de Sevilla user on 11 July 2023 investigation to explore new options for GDNF-based neuroprotection. In this regard, new GDNF mimetic compounds, such as the brain penetrant RET receptor agonist BT13 (Mahato et al., 2020), are also being tested with a good prospect. Interestingly, GDNF is naturally produced in the adult mammalian brain, particularly in the striatum (ST) (Springer et al., 1994;Trupp et al., 1997; Mogi et al., 2001;Ba¨ckman et al., 2006) and provides trophic signals to distantly located somas of catecholaminergic neurons by retrograde transport along the axon (Ito and Enomoto, 2016). For instance, noradrenergic neurons of the locus coeruleus depend on GDNF expressed in the septo-thalamic regions (Arenas et al., 1995;Zaman et al., 2003;Pascual et al., 2008;Enterrı´a- Morales et al., 2020). Likewise, GDNF produced in the ST seems to promote the survival of DA neurons located in the SNpc by stimulating their broad striatal axonal network. Although the physiological effects of striatal GDNF on DA nigrostriatal neurons are a matter of debate (Kopra et al., 2015;Pascual and Lopez-Barneo, 2015;Enterrı´a-Morales et al., 2020), it is well established that heterozygous GDNF þ/ mice show accelerated impairment of the nigrostriatal pathway and decline of motor coordination with age (Boger et al., 2006). In addition, conditional deletion of the Gdnf gene in adult mice results in a progressive degeneration of the nigrostriatal dopaminergic pathway when GDNF levels are sufficiently low (Pascual et al., 2008;Enterrı´a-Morales et al., 2020). Importantly, the main source of striatal GDNF has been identified as a subclass of GABAergic interneurons containing parvalbumin (PV), a calcium-binding protein (Hidalgo-Figueroa et al., 2012). PV neurons are uniformly distributed throughout the ST, therefore providing a homogenous trophic support to the profusely ramified axons of nigrostriatal DA neurons. Genetically, modified PV neurons producing twice the normal amount of GDNF protect DA neurons from neurotoxic insults (Kumar et al., 2015). Similarly, recent data obtained with SINEUPs, a new class of antisense long non-coding RNA targeting Gdnf mRNA, provided evidence that a 2-fold increase in GDNF is sufficient to significantly ameliorate motor deficits and prevent degeneration of DA neurons in a Parkinson’s disease neurochemical mouse model (Espinoza et al., 2020). These observations support the view that the pharmacological modulation of endogenous GDNF production in the ST, which is normally expressed at very low concentrations, could be a therapeutic approach to Parkinson’s disease complementary to other treatments. Identifying molecular targets specific to striatal PV interneurons is therefore a critical step to stimulate GDNF production. Here, we report the differential molecular characteristics of GDNF-synthesizing striatal PV interneurons and their potential role in endogenous GDNF modulation. We performed a detailed transcriptomic analysis of two PV interneuron populations with the same embryological origin (Marı´n et al., 2000; Xu et al., 2004) and sharing several neurochemical and electrophysiological properties (Hu et al., 2014) but differing in their ability to express GDNF [GDNF negative in the cortex (CTX) and GDNF positive in the ST] (this work). The specific striatal PV interneuron transcriptomic profile, obtained by using fluorescence-activated cell sorting (FACS) followed by microarray comparison, was validated by additional histological and molecular analyses, with some of the studies in mice extended to striatal PV interneurons in non-human primates. Finally, we evaluated the impact on GDNF synthesis of pharmacological modulation of PV interneuron membrane receptors and the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) intracellular pathway. Materials and methods Mice Adult heterozygous Gdnf LacZ mice were used for the detection of GDNF expression pattern (Sanchez et al.,1996; Hidalgo-Figueroa et al.,2012). The PV-tdTomato mice were obtained from The Jackson Laboratory (C57BL/6- Tg(Pvalb-tdTomato)15Gfng/J – JAX no. 027395), originally published in (Kaiser et al., 2016). The PV-Cre; tdTomato mice were obtained by crossing PV-Cre mice (B6; 129P2-Pvalb tm1(cre)Arbr /J - JAX Stock no. 008069) (Hippenmeyer et al.,2005)withtdTomatoreportermice (B6.Cg-Gt(ROSA)26Sor tm27.1(CAGCOP4*H134R/tdTomato)Hze /J - JAX Stock no. 012567). All transgenic mice were bred in our animal facility and housed under a 12:12 h lighting schedule (lights on at 7:00 a.m.) with ad libitum access to food and water. The wild-type C57BL/6J mice for ex vivo experiments were purchased from Charles River France. Mice were sacrificed on postnatal Day 30 (P30) or at 2 months of age by thiobarbital overdose. All experiments were performed according to European Community guidelines (Council Directive 86/609/EEC) and were approved by the Ethics Committee of the ‘Virgen de la Macarena’ and “Virgen del Rocı´o’ hospitals (Seville, Spain) under project license no. 27-05-15-255 provided by the Government of Andalusia. Monkeys We analysed brain tissue sections stored in our brain bank and obtained from control monkeys (Macaca fascicularis) used in previous publications (Blesa et al., 2012; Jime´nez-Sa´nchez et al., 2020;Monje et al., 2020). Nonhuman primate studies were performed according to European and Spanish guidelines (86/609/EEC and 2003/ 65/EC European Council Directives; and Spanish Government) and were approved by the research ethics committees of the universities of Navarra and Auto´noma de Madrid. Molecular targets for striatal GDNF modulation BRAIN COMMUNICATIONS 2020: Page 3 of 18 |3 Downloaded from https://academic.oup.com/braincomms/article/2/2/fcaa105/5871916 by Universidad de Sevilla user on 11 July 2023 Histology and immunohistochemistry procedures For histological detection of GDNF expression in adult mouse coronal brain sections, we revealed b-galactosidase activity with 5-bromo-4-chloro-3-indolyl- b-D-galactopyr- anoside (XGal) staining. This step was followed by immunohistochemical detection of PV interneurons following the procedures detailed in the Supplementary material. To reveal PV-Cre activity and PV-tdTomato fluorescence in the PV neurons, we followed the procedures detailed in the Supplementary material (see also Enterrı´a-Morales et al., 2020). Co-expression analyses of cKit and PV in the mouse or monkey brain were performed as indicated in the Supplementary material. Tissue dissociation and cell preparation Motor and somatosensory CTX or dorsal ST brain regions were dissociated into single-cell suspensions using a protocol modified from Zeisel et al. (2015). See Supplementary material for extended protocol. Fluorescence-activated cell sorting After dissociation of CTX and ST cells from PV-Cre; tdTomato or PV-tdTomato mice, tdTomato-positive cells were sorted using a FACS Jazz cell sorter (BD Biosciences). The tdTomato-positive cells were collected in RNAlater solution (Thermo Fisher Scientific) and immediately frozen at 80C until RNA isolation. The yield obtained from two PV-Cre; tdTomato brains was 2000 cells/CTX and 250–1000 cells/ST. The yield from PV-tdTomato brains was 5000 cells/CTX and 2000 cells/ST. When necessary, cells from two or three sorting experiments were pooled to bring the total number to 2000 tdTomato-positive cells per replicate, which we estimated to be the minimum number required for microarray analysis (Supplementary Fig. 1C and D). CTX and ST whole tissue preparation As described in the ‘Tissue dissociation and cell preparation’ section in Supplementary material, mice were euthanized at P30 by thiobarbital overdose, and intracardially perfused with ice-cold oxygenated working solution (ICOWS). The brain was extracted rapidly, placed in O 2 - saturated ICOWS, and sectioned (thickness 300 mm) on a VT1200S vibratome (Leica). Next, brain slices were placed in oxygenated working solution at 37C for 45 min and areas of interest (motor CTX; and dorsal ST) then dissected from each slice, snap frozen in liquid nitrogen, and stored at 80C until used for RNA isolation. RNA preparation, quality control and quantitation Total RNA from cortical and striatal whole tissue samples and FACS-captured cells was isolated using the TRIzol method (Thermo Fisher) following the manufacturer’s instructions with minor modifications. Quantification and quality control were performed using a Nanodrop 2000 spectrophotometer (Thermo Fisher) for tissue RNA, or with an Agilent 2100 Bioanalyzer system for RNA obtained from FACS-captured PV cells. See Supplementary material for detailed protocol. Microarray analysis RNA from FACS-captured cells was amplified and labelled using Affymetrix GeneChip WT PLUS Reagent Kit (Thermo Fisher). Amplification was performed with 5 ng of total RNA as per procedures described in the WT PLUS Reagent Kit user manual. The amplified cDNA was quantified, fragmented and labelled in preparation for hybridization to an Affymetrix GeneChip Mouse Transcriptome 1.0 Array (Thermo Fisher) using 5.5 lgof single-stranded cDNA product and following protocols outlined in the user manual. Washing, staining (GeneChip Fluidics Station 450, Affymetrix) and scanning (GeneChip Scanner 3000, 10 Affymetrix) were performed as per protocols outlined in the user manual for cartridge arrays. Data were processed for gene-level background subtraction, normalization and signal summarization (SST-RMA, signal space transformation-robust multiarray average) using the BioConductor oligo package (R version 3.2.3) (Carvalho and Irizarry, 2010). To assess the overall similarity among samples, hierarchical clustering analysis of full expression profiles and principal component analysis were then performed. For hierarchical clustering analysis, the Pearson correlation metric and complete agglomeration methods were used. Differential expression analysis was performed using the R Limma (Linear Models for Microarray Analysis) package. This analysis applies a t-test empirically adjusted by a Bayes test (Huber et al., 2015). Changes in gene expression between groups were considered significant with false discovery rate (FDR) <0.05 and fold change >2or<2. Heatmap figures (hierarchical clustering) were obtained using the Transcriptome Analysis Console 3.0 Software (TAC 3.0) from Affymetrix. Real-time quantitative RT-PCR Real-time quantitative RT-PCR (qPCR) was run to validate the microarray results obtained from the FACS-cap- tured PV-positive cells, as well as to compare transcriptomic differences between the PV cells and whole tissue from which they were isolated. See Supplementary Table 1 and Supplementary material for the full protocol. 4|BRAIN COMMUNICATIONS 2020: Page 4 of 18 D. Enterrı´a-Morales et al. Downloaded from https://academic.oup.com/braincomms/article/2/2/fcaa105/5871916 by Universidad de Sevilla user on 11 July 2023 RNAscope We carried out in situ hybridization using the protocol from Advanced Cell Diagnostics (ACDbio), following the manufacturer’s instructions (RNAscope V R Fluorescent Multiplex kit). This technique is used to visualize single RNA molecules in tissue samples on glass slides (Wang et al., 2012). See Supplementary material for extended protocol and analysis. Ex vivo pharmacological experiments We used 250 mm-thick coronal slices containing the ST (hemibrain) prepared from P30 male mice. The slices were kept in oxygenated artificial cerebrospinal fluid (aCSF) at 36C and challenged with different pharmacological compounds for 5 or 12 h. At the end of incubation, striatal regions were dissected from each slice of the same hemibrain, placed in RNAlater and stored at 80C until RNA extraction. Survival tests consisting of amperometric measurements of DA release in response to high potassium were performed to validate this ex vivo protocol. See Supplementary material for detailed procedures and pharmacological compounds used in this study. In vivo stereotaxic experiments Detailed procedures for administering stereotaxic injections into 2-month-old f male mice are available in the Supplementary material. Statistical analysis No statistical methods were used to predetermine sample size. For qPCR analyses, a minimum of two technical replicates were performed, with mean values used in calculations. Statistical analysis was performed using Prism software version 6.0 (GraphPad). Normality was tested with the Shapiro–Wilk test. We applied ordinary one-way analysis of variance with Tukey’s multiple comparison tests for a parametric set of data, or one-way ANOVA on ranks (Kruskal–Wallis) for non-parametric tests. A two-tailed Student’s t-test was performed for pairwise data. Data were presented as the mean 6SEM with the number (N) of experiments indicated in the figures. A value of P<0.05 was considered statistically significant. Significance thresholds are indicated with asterisks in the figures as P<0.05, P<0.01, P<0.001 and P<0.0001, and standard errors of the mean are indicated by error bars. The statistical analysis specific to the microarray data is described in the ‘Microarray analysis’ section. Data availability The gene expression datasets generated in this study are available through NCBI Gene Expression Omnibus (GEO) with the series accession number (GEO: GSE100300). All other data generated or analysed during this study are included in this published article (and Supplementary material files). Results Differential GDNF expression in PV interneuron populations We studied neural GDNF expression by histological XGal staining of coronal brain sections from adult Gdnf LacZ mice, which showed b-galactosidase activity resulting from the LacZ insert within exons 1 and 2 of the Gdnf gene. Dual XGal and PV immunohistochemical staining showed that, as reported previously (Hidalgo-Figueroa et al., 2012), GDNF is expressed in more than 95% of striatal PV-positive interneurons distributed uniformly over the ST (Fig. 1A and B). Remarkably, XGal staining, indicative of GDNF expression, was never seen in the PV neurons of the nearby somatosensory CTX or external globus pallidus (Fig. 1B). Moreover, the qPCR analysis confirmed the XGal data, as Gdnf mRNA expression levels were much higher in the ST than in the CTX of the mouse brain (P<0.01, N¼4) (Fig. 1C). Indeed, Gdnf mRNA was barely amplified in cortical tissue. These data indicate that GDNF is selectively expressed by the striatal subclass of brain PV interneurons. Reporter mouse models and PV cell sorting To capture PV-positive cells by FACS, we bred two mouse models expressing the red fluorescent protein variant tdTomato (Fig. 2A and Supplementary Fig. 1). We first used Cre-inducible tdTomato mice (PV-Cre; tdTomato), which harbour a loxP-flanked STOP cassette preventing the transcription of a CAG promoter-driven channelrhodopsin-2 (ChR2)/tdTomato fusion protein. Following exposure to Cre recombinase, the ChR2/ tdTomato fusion protein is expressed with a predominant membrane location (Supplementary Fig. 2A). Only about half of PV interneurons are tdTomato-positive in the ST and CTX of this model due to a relatively low Cre-medi- ated recombination (Enterrı´a-Morales et al., 2020). This resulted in few FACS-captured cells (see below) and, therefore, several sorting experiments were pooled to reach the critical threshold of 2000 cells required to guarantee good-quality RNA (Supplementary Fig. 1C and D). The second mouse strain studied (transgenic PV- tdTomato) was free of Cre recombinase and constitutively expressed tdTomato in PV interneurons. Soluble tdTomato fluorescence was easily detected in 30 mm-thick brain cross-sections (Fig. 2A). Consistent with the original characterization of this model (Kaiser et al., 2016), we observed that most PV-immunopositive cells were Molecular targets for striatal GDNF modulation BRAIN COMMUNICATIONS 2020: Page 5 of 18 |5 Downloaded from https://academic.oup.com/braincomms/article/2/2/fcaa105/5871916 by Universidad de Sevilla user on 11 July 2023 tdTomato-positive in CTX and striatal regions (Supplementary Fig. 2B). We set up a FACS protocol to specifically capture PV interneurons from striatal and cortical tissue preparations (Fig. 2B and C). In each model, dispersed cells from tdTomato-negative mice were used to detect non-specific fluorescence and to set up the gate to collect selectively tdTomato-positive cells. In a typical experiment, brain tissues were pooled from two PV-Cre; tdTomato mice and 1000–4000 (CTX) and 250–1000 (ST) tdTomato-positive cells were harvested, accounting for 3% and 0.5% of the total cell number, respectively. A typical harvest from two PV-tdTomato brains was 4000–10 000 cells from the CTX and 1000–2500 cells from the ST, representing 2.5% and 1% of the total cell number, respectively. Figure 2B shows representative FACS plots from PV; tdTomato striatal tissue; additional FACS plots for the two mouse models used are presented in Supplementary Fig. 3. To illustrate FACS selectivity, cells collected from the positive gate were red fluorescent under 568 nm excitation wavelength, whereas cells harvested from the negative gate were non-fluorescent (Fig. 2D). PV neuron enrichment was also confirmed by qPCR. Pvalb expression was high in captured tdTomato-positive cells from both the ST and CTX compared to cells harvested from the negative gate. In addition, only striatal tdTomato-positive cells exhibited high Gdnf mRNA levels (Fig. 2E). ST- and CTX-derived tdTomato-positive cells were then processed for RNA isolation and microarray analysis (Fig. 2F). Transcriptomic differences between ST- and CTX-derived PV interneurons The expression profiles of 24 334 genes of FACS-cap- tured PV neurons were compared between CTX and ST Figure 1 Selective expression of GDNF in mouse striatal PV interneurons. (A) Schematic illustration of the CTX, STand globus pallidus in a coronal section of the mouse brain. (B) Immunostaining of PV interneurons (brown) and selective expression of GDNF, reported by positive XGal-positive staining (blue dots), in the ST. (a), (b) and (c) are magnified fields showing details such as XGal blue dots only visible in (b). (C) Comparison of Gdnf mRNA expression (measured by qPCR) in the STand CTX (N¼4, ** P<0.01, two-tailed Student’s t-test). 6|BRAIN COMMUNICATIONS 2020: Page 6 of 18 D. Enterrı´a-Morales et al. Downloaded from https://academic.oup.com/braincomms/article/2/2/fcaa105/5871916 by Universidad de Sevilla user on 11 July 2023 Figure 2 Experimental workflow for cell sorting from PV-tdTomato mice and microarray analysis. (A) Left, gene construction of PV- tdTomato mice and schematic illustration of CTX (CTX) and ST tissue samples obtained for cell dispersion. Right, histological examples of fluorescent cells (tdTomato-positive) in CTX and ST. (B) Flow cytometry dot plot graphical analysis of dispersed cells from tdTomato-negative (þ/þ) or positive (tdTomato/þ) ST. The sorting gate (red line) was defined to separate the tdTomato-positive cells (pos) from other cells (see Supplementary Fig. 3 for extended plots). A negative gate was set to collect tdTomato-negative cells (white square). (C) 2000 tdTomato-positive cells (pos) were collected for experiment. (D) Phase contrast and fluorescence (568 nm filter) composite microphotographs of floating FACS- captured striatal cells. Left, negative gate-captured cells. Right, positive gate-captured cells. (E) Qualitative comparison of Gdnf and Pvalb mRNA expression (measured by qPCR) in tdTomato-positive and negative cells obtained from cortical and striatal cell preparations (N¼1). (F) Schematic diagram illustrating the experimental workflow after FACS capture of tdTomato-positive cells. Cells were processed for RNA extraction followed by quality check. Samples with an RNA integrity number above 7 were selected for the Affymetrix microarray procedure. Final data were analysed by bioinformatics to compare gene expression between STand CTX parvalbumin interneurons. Molecular targets for striatal GDNF modulation BRAIN COMMUNICATIONS 2020: Page 7 of 18 |7 Downloaded from https://academic.oup.com/braincomms/article/2/2/fcaa105/5871916 by Universidad de Sevilla user on 11 July 2023 in two separate microarray experiments (corresponding to PV-Cre; tdTomato and PV-tdTomato mouse models). As illustrated by volcano plots (Fig. 3A and B), a number of differentially expressed (DE) genes between ST- and CTX-derived PV neurons were distributed in the top left (FDR <0.05 and log2 FC <1) and top right (FDR < 0.05 and log2 FC >1) sections of the plots. In the PV- Cre; tdTomato model, 294 DE genes were identified (Fig. 3A and Supplementary Table 2). In contrast, 199 DE genes were identified in PV cells from PV-tdTomato mice (Fig. 3B and Supplementary Table 3). Two modelcombined differential expression analyses (FDR <0.05, log2 FC >1or<1) yielded 185 DE genes specific to PV-Cre; tdTomato mice, and 90 DE genes specific to the PV-tdTomato-derived PV neurons. Finally, 109 DE genes overlapped in the two models (Fig. 3C). Although Gdnf was not DE in the initial microarray analysis, FDR and P-value indicate enrichment in striatal PV cells (Fig. 3D). Clustering of selected ST-specific DE genes showed that genes such as Gpr83,Mia,Tmem212,Spata18,Mc4r, Pde3a and Prokr2 are shared between the two reporter models (Fig. 3E and F). Other DE genes, such as Kit, were identified in PV-Cre; tdTomato mice but not in PV- tdTomato mice (Fig. 3E). On the other hand, Mc3r, Figure 3 Microarray analysis of transcriptomes from cortical (CTX) and striatal (ST) PV interneurons. (A) Volcano plot illustrating DE genes [DE, magenta, logFC <1or>1, log2(FDR) <0.05] compared to genes without differential expression (non-DE, green) from PV-Cre; tdTomato P30 mice. (B) Volcano plot illustrating DE genes from PV-tdTomato P30 mice. (C) Venn diagram showing ST/CTX gene expression variation present in the two set of arrays. (D) Detection of Gdnf gene transcripts (in log2 scale) in RNA samples from STand CTX PVþcells captured by FACS in the two reporter mouse models. (E) Hierarchical clustering of selected genes from PV-Cre; tdTomato CTX and ST (N¼4). The colour-ratio bar on the side indicates relative intensity (log2 scale) of gene up-regulation (magenta), down-regulation (black) and no change (white). (F) Hierarchical clustering of selected genes from PV-tdTomato CTX and ST. Asterisks indicate selected genes that were found in both arrays. logFC: log2-fold change, log2(FDR): log2 FDR. 8|BRAIN COMMUNICATIONS 2020: Page 8 of 18 D. Enterrı´a-Morales et al. Downloaded from https://academic.oup.com/braincomms/article/2/2/fcaa105/5871916 by Universidad de Sevilla user on 11 July 2023 Tacr1,Tacr3 and Lhx8 were DE only in striatal PV cells of PV-tdTomato mice (Fig. 3F). We next short-listed several DE genes obtained from the two models to carry out qPCR validation. This shortlist was based on positive ST/CTX fold change (i.e. higher expression in striatal PV interneurons) and biological interest (membrane receptors, cellular function and gene regulation). For this step, we included cDNA prepared from whole tissue samples (ST or CTX) in order to discard DE genes that although showed regional specificity was not highly expressed in PV neurons. Figure 4 shows qPCR data from eleven striatal PV-enriched genes (Kit,Gpr83,Mc3r,Tacr1,Tacr3,Crabp1,Rarres2, Moxd1,Pde3a,Spata18 and Lhx8). Other DE genes were discarded because exhibited either a low level of expression in PV neurons or high, non-PV-neuron-selective regional differences (ST versus CTX) (Supplementary Fig. 4). These genes were considered marginally selective and, therefore, less relevant for our study. Histological analysis of PV interneuron-specific gene expression Based on microarray and qPCR data, we examined the anatomical distribution of selected PV neuron-specific genes in the mouse brain with particular attention paid to the ST. We first browsed through the Allen Mouse Brain Atlas in situ hybridization database for pattern similarity (Lein et al.,2007) and found a very similar distribution across the ST between Pvalb and some of our short-listed striatal PV neuron-specific genes (Crabp1,Lhx8,Pde3a, Tacr1,Tacr3) (Supplementary Fig. 5). We next searched for co-expression of selected genes in the mouse brain by histological methods. We chose the Kit gene for immunohistochemical analysis as highly specific antibodies for the cKit receptor are available (May et al.,2018)anddetermined that virtually all PV-immunopositive neurons were also cKit-positive in the mouse ST. On the other hand, only a small proportion of striatal cKit-positive cells were PV-negative (Fig. 5A; Supplementary Fig. 6A). Although cKit-positive cells were observed in the nearby somatosensory CTX, very few expressed both cKit and PV (Supplementary Fig. 6B). These histological results confirm the molecular data regarding Kit gene expression. In the absence of highly specific antibodies, we performed RNAscope V R in situ hybridization analyses to examine Gpr83 and Lhx8 expression in the ST. Gpr83,Lhx8 and Gdnf mRNA were clearly co-labelled in the ST as illustrated in Fig. 5B (and Supplementary Fig. 7). These histological results support the molecular data described above. To evaluate whether the PV neuron-specific genes identified in the mouse ST might be conserved across species, we Figure 4 Differential gene expression between parvalbumin interneurons from STand CTX compared to tissue gene expression level in P30 mice. (A) Schematic illustration of parvalbumin interneurons (in red) within the whole CTX or STregion (yellow) in a coronal section of the mouse brain. Vertical bar graphs show cDNA level (measured by qPCR), as a measure of mRNA, in FACS-captured PV interneurons (red) and in whole tissue preparation (yellow) from ST (ST) and CTX (CTX). Gene expression for each sample was normalized to Actb mRNA. (B) Genes coding for membrane receptors. (C) Genes coding for cell signalling- and metabolism-associated proteins. (D) Gene coding for the transcription factor LHX8. N¼3or4,*P<0.05, **P<0.01, non-parametric one-way ANOVA on ranks (Kruskal–Wallis test). Molecular targets for striatal GDNF modulation BRAIN COMMUNICATIONS 2020: Page 9 of 18 |9 Downloaded from https://academic.oup.com/braincomms/article/2/2/fcaa105/5871916 by Universidad de Sevilla user on 11 July 2023 low basal level of GDNF synthesis and secretion by PV neurons. GDNF homeostasis is likely more complex and involves regulation at several non-transcriptomic levels (d’Anglemont de Tassigny et al.,2015). For instance, Gdnf mRNA degradation is highly regulated by microRNAs (Kumar et al., 2015;Xia et al., 2016;Zhang et al.,2019). However, a low basal level of expression is probably necessary to prevent over-activation of dopaminergic terminals and deleterious effects of GDNF. It is well known that local injection of GDNF produces structural aberrations in SNpc neurons (Kirik et al., 2000a,b); and that the intracerebroventricular injection of GDNF in humans induces a wide variety of side effects (Nutt et al., 2003). Overlapping with this tonic inhibition, selective activation of the cKit and Wnt/b-catenin pathways could result in a controlled production of GDNF as per physiological needs. In sum, the therapeutic potential of PV interneurons as a target is significant since they are the only cells to synthesize GDNF in the ST. These neurons remain functional, and continue producing GDNF after experimental destruction of nigrostriatal dopaminergic neurons in mice (Hidalgo-Figueroa et al., 2012) and monkeys (this work). We have shown that striatal GDNF production can be pharmacologically modulated (increased or decreased) and have also identified several pathways with druggable targets that are potentially relevant for controlling GDNF synthesis and/or release by striatal PV neurons. Development of a specific and safe pharmacology capable of modulating striatal GDNF production in man is a feasible goal that could eventually be of major therapeutic benefit for combating Parkinson’s disease. Supplementary material Supplementary material is available at Brain Communications online. Acknowledgements The authors wish to thank Dr. Patricia Ortega-Saenz for her help with the amperometric measurements, and Dr. Francisco Moro´n-Civanto, Mr. Juan Cordero-Varela and Ms. Maria Jose´ Castro-Pe´rez for their technical assistance. We also thank the IBiS animal facility staff for excellent animal care. Funding This research was supported by the Spanish Ministries of Science and Innovation and Health (SAF2012-39343, SAF2016-74990-R), the Carlos III Health Institute’s Miguel Servet Program (CPII17/00016, CP19/00200), the European Research Council (ERC-ADGPRJ201502629), the Fundacio´n BBVA (Ayudas a Equipos de Investigacio´n Cientı´fica 2015) and the Agence Nationale de la Recherche grants ANR-17-CE16-0015 and the LabEx DistAlz. Competing interests The authors report no competing interests. 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