Cell Mol Neurobiol (2015) 35:1127–1136 DOI 10.1007/s10571-015-0206-6 ORIGINAL RESEARCH The Docosanoid Neuroprotectin D1 Induces TH-Positive Neuronal Survival in a Cellular Model of Parkinson’s Disease 1 1 1 Jorgelina M. Calandria • Michelle W. Sharp • Nicolas G. Bazan Received: 6 March 2015 / Accepted: 5 May 2015 / Published online: 6 June 2015 Ó The Author(s) 2015. This article is published with open access at Springerlink.com Abstract Parkinson’s disease (PD) does not manifest partially reverted the dendrite retraction caused by MPP? clinically until 80 % of striatal dopamine is reduced, thus and MPTP. These results suggest that the apoptosis most neuronal damage takes place before the patient pre- occurring in mesencephalic TH-positive neurons is not a sents clinical symptoms. Therefore, it is important to direct consequence of mitochondrial dysfunction alone and develop preventive strategies for this disease. In the that NPD1 signaling may be counteracting this damage. experimental models of PD, 1-methyl-4-phenylpyridinium These findings lay the groundwork for the use of the ion (MPP?) and rotenone induce toxicity in dopaminergic in vitro model developed for future studies and for the neurons. Neuroprotectin D1 (NPD1) displays neuropro- search of specific molecular events that NPD1 targets to tection in cells undergoing proteotoxic and oxidative stress. prevent early neurodegeneration in PD. In the present report, we established an in vitro model using a primary neuronal culture from mesencephalic embryonic mouse tissue in which 17–20 % of neurons Keywords Neuroprotectin D1 Á TH-positive neurons Á were TH-positive when differentiated in vitro. NPD1 Parkinson’s disease Á Neuroprotection Á MPP? (100 nM) rescued cells from apoptosis induced by MPP? and rotenone, and the dendritic arbor of surviving neurons was examined using Sholl analysis. Rotenone, as well as MPP? and its precursor 1-methyl-4-phenyl-1,2,3,6- Introduction tetrahydropyridine (MPTP), severely promoted retraction of dendritic arbor distal segments, thus decreasing the Parkinson’s disease (PD) does not manifest clinically until maximum branch order reached. On average, NPD1 80 % of the striatal dopamine has been lost (Betarbet et al. counteracted the effects of MPP? on the dendritic 2002). Therefore, it is vital to develop strategies that pre- arborization, but failed to do so in the rotenone-treated vent the loss of dopamine neurons. neurons. However, rotenone did decrease the Sholl inter- Until recently, the significance of the selective enrich- section number from radii 25 to 125 lm, and NPD1 did ment of omega-3 essential fatty acids (docosahexaenoyl— restore the pattern to control levels. Similarly, NPD1 DHA—chains of membrane phospholipids, 22C and 6 double bonds) in the nervous system had remained incom- pletely understood (Bazan et al. 2011; Bazan 2014). We now Electronic supplementary material The online version of this know that an integral membrane protein is engaged in DHA article (doi:10.1007/s10571-015-0206-6) contains supplementary retention in photoreceptors/retinal pigment epithelial cells as material, which is available to authorized users. a necessary event for the function of these cells (Rice et al. & Nicolas G. Bazan 2015). By studying early mechanisms of cell survival, we [email protected] found that a docosanoid synthesized from docosahexaenoic acid (DHA) by 15-lipoxygenase-1 (15-LOX-1), neuropro- 1 Neuroscience Center of Excellence, School of Medicine, Louisiana State University Health Sciences Center, 2020 tectin D1 (NPD1; 10R,17S-dihydroxy-docosa-4Z,7Z,11E,13 Gravier Street, Suite D, New Orleans, LA 70112, USA E,15E,19Z hexaenoic acid) (Bazan et al. 2010;Calandria 123 1128 Cell Mol Neurobiol (2015) 35:1127–1136 et al. 2009; Serhan et al. 2015), displays neuroprotective modifications. Briefly, 1 mm3 of the ventricular mesen- bioactivity. Moreover, NPD1 prevents apoptosis induced by cephalic region was obtained from Sprague–Dawley rat oxidative stress (Calandria et al. 2009, 2012) involving embryos (embryonic day 15). Two incisions were made, transcriptional regulation (Calandria et al. 2015). Previously, one in the forebrain/mesencephalic limit and the other it has been shown in experimental models of PD that MPP? between the mesencephalon and the hindbrain. Once dis- and rotenone inhibit the Complex I of the mitochondrial sected from the meninges, the tissue was disaggregated electron chain and causes specific toxicity to dopaminergic using trypsin and DNaseI. Twenty thousand cells were neurons in the substantia nigra pars compacta (SNpc) (Be- plated in each well of an 8-well chamber slide with B-27/ tarbet et al. 2000; Davis et al. 1979;Langstonetal.1983; N2 medium containing 2.5 % serum. GDNF, TGF-b, and Richardson et al. 2005). dcAMP were added once to increase TH-positive cells The goal of this study was to develop an in vitro model (Supplementary Fig. 1) and the media were changed every of PD where lipid mediators could be studied in a direct 2 days. The percentage of TH-positive cells achieved was manner and to determine the ability of NPD1 to enhance 17–20 %. NPD1 (100 nM) was added to the culture at 7 survival during early toxicity in mesencephalic TH-posi- DIV, 5 min before the addition of 100 nM rotenone, tive neurons. We used a onetime application of 1-methyl-4- 100 lM MPP? or 100 lM MPTP. To decide the time of phenylpyridinium ion (MPP?), 1-methyl-4-phenyl-1,2,3,6- exposure to MPTP and rotenone, 24- and 48-h time points tetrahydropyridine (MPTP), or rotenone, measuring sur- were used (Supplementary Figs. 2, 3). Immunostaining was vival and dendritic architecture modification in the performed following a previously described protocol (Ca- remaining neurons after 24 h. In this manner, we estab- landria et al. 2012). Briefly, cells were fixed with 4 % lished a cellular model to reenact the first toxic event that paraformaldehyde, permeabilized with 1 % Triton and 1 % dopaminergic cells undergo as an early response in normal serum in phosphate buffered saline (PBS) for neurodegeneration. 5 min, and then blocked with 10 % normal serum solution in PBS. The slides were incubated with the primary anti- body overnight and then for 1 h with Alexa fluor-conju- Materials and Methods gated secondary antibodies. Animals and Reagents Image Acquisition, Assessment of the Dendritic Arbor, and Statistics Animals were handled following protocols on animal experimentation approved by the Institutional Animal Images were obtained using a custom-built deconvolution Care and Use Committee (IACUC), Louisiana State microscope and Slidebook 4.0 software (3i-Intelligent University Health Sciences Center (LSUHSC), New Imaging Innovations, Inc., Denver, CO). Images were Orleans. Culture media, additives and other related imported and analyzed using the software Imaris 7.4 (Bit- reagents, including trypsin and B-27/N2, were purchased plane Scientific Software, Ireland, UK) or NeuronJ (Mei- from Life Technologies (Carlsbad, CA). Fetal bovine jering et al. 2004) plugged into ImageJ (Rasband 1997– serum (FBS) was acquired from Tissue Culture Biologi- 2014). The intersection frequencies of Sholl analysis were cals (Long Beach, CA), and glial cell line-derived neu- plotted and analyzed statistically to fit a polynomial regres- rotrophic factor (GDNF) and transforming growth factor sion to assess the statistically significant differences between beta (TGF-b) were from Cell Sciences (Canton, MA). treatments within an experiment (Supplementary Table 1; DNAseI, dbcAMP, MPTP, MPP? and rotenone were Fig. 2q). The Sholl values were used to construct a frequency obtained from Sigma (St. Louis, MO). Anti-tyrosine histogram and compared between treatments within each hydroxylase antibody was purchased from Pel-Freez class interval and between intervals using Student’s t test (Rogers, AR); anti-b-tubulin III was from Sigma (St. (Tables 1, 2). The statistical analysis was performed using Louis, MO) and anti-Map2 from Millipore (Billerica, the SAS software (SAS Institute, Cary, NC). MA). Secondary antibodies conjugated with Alexa Fluor 488 and 555 and nuclei staining DAPI were obtained from Life Technologies (Carlsbad, CA). Results Rat Mesencephalic TH-Positive Neurons: Primary TH-Positive Neuron Cell Death Triggered Culture, Treatment, and Immunocytochemistry by Rotenone and MPP1 is Prevented by NPD1 The cultures were performed following the methods Rotenone and the MPTP metabolite, MPP?, was proposed described previously (Sun et al. 2004) with some to induce toxicity primarily by blocking mitochondrial 123 Cell Mol Neurobiol (2015) 35:1127–1136 1129 Complex I, which results in the production of superoxide 2D Analysis of Dendritic Branching shows radicals and consequently, oxidative stress (Ramachandi- that NPD1 prevents Neurite Retraction at Short ran et al. 2007). To determine the ability of NPD1 to to Medium Radii prevent cell death induced by these three compounds, mesencephalic primary-cultured neurons were exposed to The Sholl number is the frequency by which neurites 100 nM rotenone, 100 lM MPTP, or 100 lM MPP? in the intersect in concentric circles drawn around the soma at presence and absence of NPD1 for 24 h (Fig. 1a–c). MPP? different radii (Fig. 2b). To further visualize spatially the severely decreased the number of TH-positive neurons alterations introduced by MPP? and its precursor MPTP or from 40.3 ± 7.3 in the control to 7.6 ± 1.6; with the rotenone, the total number of Sholl intersections per addition of NPD1, the number of surviving cells was raised treatment