From the Cover: Alterations in Optineurin Expression and Localization in Pre-clinical Parkinson’s Disease Models

Abstract

Parkinson’s disease (PD) is a progressive neurodegenerative disease that affects ∼5 million people around the world. PD etiopathogenesis is poorly understood and curative or disease modifying treatments are not available. Mechanistic studies have identified numerous pathogenic pathways that overlap with many other neurodegenerative diseases. Mutations in the protein optineurin (OPTN) have recently been identified as causative factors for glaucoma and amyotrophic lateral sclerosis. OPTN has multiple recognized roles in neurons, notably in mediating autophagic flux, which has been found to be disrupted in most neurodegenerative diseases. OPTN⁺aggregates have preliminarily been identified in cytoplasmic inclusions in numerous neurodegenerative diseases, however, whether OPTN has a role in PD pathogenesis has yet to be tested. Thus, we chose to test the hypothesis that OPTN expression and localization would be modulated in pre-clinical PD models. To test our hypothesis, we characterized midbrain OPTN expression in normal rats and in a rat rotenone PD model. For the first time, we show that OPTN is enriched in dopamine neurons in the midbrain, and its expression is modulated by rotenone treatment in vivo. Here, animals were sampled at time-points both prior to overt neurodegeneration and after severe behavioral deficits, where a lesion to the nigrostriatal dopamine system is present. The effect and magnitude of OPTN expression changes are dependent on duration of treatment. Furthermore, OPTN colocalizes with LC3 (autophagic vesicle marker) and alpha-synuclein positive puncta in rotenone-treated animals, potentially indicating an important role in autophagy and PD pathogenesis.

Parkinson’s disease (PD) pathology is characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra (SN) and the formation of Lewy bodies in these and other neurons (Braak et al., 2003; Fornai et al., 2003; Forno, 1996; Gai et al., 2000; Spillantini et al., 1997). Lewy bodies are intracellular inclusions largely composed of aggregated proteins, notably α-synuclein. Despite significant advancements in understanding the genes, proteins, and mechanisms involved, the etiology of PD remains largely unknown. Of note, most data from animal studies is obtained from end-stage models, where significant cell loss and pathology has already occurred. Given that PD is not diagnosed until significant dopaminergic cell loss occurs, there is a critical need to discover molecular pathways affected in the earliest stages of disease (Bernheimer et al., 1973; Fearnley and Lees, 1991).

Whereas the role of Lewy bodies in pathogenesis remains unclear, it is evident that α-synuclein positive inclusions form prior to neuronal loss, suggesting that aberrant protein degradation is a critical early event (Braak et al., 2003). Protein aggregates form by an accumulation of misfolded or damaged proteins that would normally be degraded by autophagy, but autophagy is clearly impaired in PD (Anglade et al., 1997; Higashi et al., 2011; Zhu et al., 2003). This pathway is also responsible for clearing damaged or dysfunctional mitochondria, which are also prevalent in PD (Zhu et al., 2003). Both dysfunctional mitochondria and protein aggregates induce oxidative stress, which may be detrimental to autophagy in conditions of chronic oxidative stress (Dagda et al., 2013). Given that DA neurons of the substantia nigra are characterized by a high basal rate of oxidative stress (Pacelli et al., 2015), it is very likely that impaired autophagy in these neurons is a critical factor in selective sensitivity to genetic and neurotoxic insults.

Numerous pathways and pathologies overlap in major neurodegenerative diseases (Cannon and Greenamyre, 2011). Thus, there has been significant effort to identify common pathways that lead to risk for neurological diseases. Optineurin (OPTN) is a 577 amino acid protein that is a known genetic link to amyotrophic lateral sclerosis (ALS) and primary open angle and normal tension glaucoma (Maruyama et al., 2010; Sarfarazi and Rezaie, 2003; Ying and Yue, 2012). Of note is that PD patients have a higher risk for the development of glaucoma (Nucci et al., 2015). The OPTN protein contains several putative domains, including a LC3 interacting domain, bZIP motif, 2 leucine zippers, multiple coiled-coil motifs, ubiquitin binding domain, and a C-terminal C₂H₂ type of zinc finger, with numerous binding partners that include LC3, Rab8, Huntingtin, myosin VI, TANK binding kinase 1 (TBK1), ring finger protein 11, serine/threonine kinase receptor interacting protein 1 (RIP1), etc., indicating diverse cellular functions (Chibalina et al., 2008; Swarup and Nagabhushana, 2010; Ying and Yue, 2012). Given the repeatedly identified alterations to autophagy in PD, OPTN’s role in this process is of particular interest. To date, the presence of OPTN has been reported to occur in protein inclusions of many neurodegenerative diseases, including ALS, Alzheimer’s disease, Huntington’s disease, Creutzfeld-Jakob disease, multiple system atrophy, Pick disease, and frontotemporal lobar degeneration (Fujita et al., 2011; Hortobagyi et al., 2011; Mori et al., 2012; Osawa et al., 2011; Schwab et al., 2012). OPTN is also reported to occur in Lewy bodies in PD, although there are major limitations (Osawa et al., 2011). In that study, no control tissue was examined, a single high magnification image of a single cell was shown, and no counterstain was utilized to confirm cell type or that immunoreactivity was due to protein aggregation.

In the present study, we wished to test the hypothesis that based upon known cellular roles, OPTN expression and modulation would be altered in early-stage PD models. For this study we utilized the rat rotenone model, which has been extensively utilized to study PD (Betarbet et al., 2000; Cannon et al., 2009). Rotenone exposure has also been identified as a risk factor for PD (Tanner et al., 2011). Here, for the first time, we have examined OPTN expression in the mammalian midbrain and in pre-clinical PD models. These studies are expected to be a critical first step to understanding how OPTN may mediate neurodegeneration in PD.

METHODS AND MATERIALS

Animals

All animals were male wild-type Lewis rats purchased from Harlan (now Envigo; Indianapolis, IN) and were between the ages of 31–42 weeks when euthanized. Rats were housed under standard 12 h light cycle, fed ad libitum. All studies were approved by the Purdue University Animal Care and Use Committee.

Chemicals and reagents

Primary antibodies included: rabbit anti-OPTN (ab23666; Abcam, Cambridge, Massachusetts); rabbit anti-OPTN (100000; Cayman Chemical, Ann Arbor, Michigan); mouse anti-LC3 (M152-3; MBL International Corporation, Woburn, Massachusetts); sheep and mouse anti-tyrosine hydroxylase (AB1542, MAB318) were purchased from EMD Millipore (Billerica, Massachusetts); and mouse anti-alpha-synuclein (610786; BD Biosciences, San Jose, California). Secondary antibodies Biotin-SP anti-rabbit (711-065-152), Alexa Fluor 647 anti-sheep (713-605-147), Alexa Fluor 488 anti-rabbit (711-545-152), Cy3 anti-mouse (715-165-151) were purchased from Jackson ImmunoResearch Laboratories, Inc (West Grove, Pennsylvania); IR 800CW anti-mouse (926-32212) was purchased from Licor (Lincoln, Nebraska). Vectastain Elite ABC kit (PK-1600) and DAB Peroxidase (HRP) Substrate kit (SK-4100) were purchased from Vector Laboratories (Burlingame, California). Rotenone (R8875) and glycerol (G5516) were purchased from Sigma-Aldrich (St. Louis, Missouri). DPX mountant was purchased from VWR (100503834, Chicago, Illinois). Triton X-100 was purchased from Fisher BioReagents (BP151-500, Fair Lawn, New Jersey). Histomount was purchased from National Diagnostics (HS-103, Atlanta, Georgia).

Rotenone administration

Rotenone was administered at 3 mg/kg/day by intraperitoneal injection as previously described (Cannon et al., 2009). This model has been extensively characterized in terms of the temporal development of behavioral, neurochemical, and neuropathological alterations in the nigrostriatal dopamine system that are relevant to PD (Cannon and Greenamyre, 2010; Cannon et al., 2009, 2013; Tapias et al., 2010, 2014; Zharikov et al., 2015). Of note, the dose and/or time of exposure can be adjusted to produce a pre-clinical model, where early stage pathogenesis can be studied, prior to overt cell death (Drolet et al., 2009; Sanders et al., 2014a, b). In the present study, we chose to sample animals at both pre-clinical time-points, where overt motor phenotypes were not yet present and at end-stage, where postural instability, rigidity, and bradykinesia were present. These deficits have been well described in the rotenone model at this dose (Cannon et al., 2009). Thus, animals were euthanized after 24 h (n = 2), 5 days (n = 6), or at end-stage (n = 8) (typically at 6-9 days). In this model, overt behavioral deficits are associated with a lesion to the nigrostriatal dopamine system (Betarbet et al., 2000; Cannon et al., 2009), whereas sampling at 5 days or before is prior to the development of a detectable lesion (Sanders et al., 2014a,b).

Immunohistochemistry and silver staining

Rats were put under deep anesthesia with pentobarbital (>50 mg/kg) (Beuthanasia-D Special, Schering-Plough Animal Health Corp, Union, New Jersey), then transcardially perfused with 100–150 ml PBS followed by 250–300 ml 4% buffered paraformaldehyde (PFA). Brains were surgically removed, post-fixed in 4% PFA for approximately 24 h, and then saturated with 30% sucrose at 4 °C for at least 5–7 days until sinking. Each brain was coronally sectioned on a frozen sliding microtome (Microm HM 450, Thermo Scientific) at a 35 μm thickness and stored in cryoprotectant at −20 °C until used for staining. Midbrain sections containing the substantia nigra pars compacta were randomly selected, rinsed in PBS for 10 min, 6 times, at room temperature (RT) on an open air platform shaker.

For colorimetric staining, sections were then quenched in 3% hydrogen peroxide (diluted from 30% hydrogen peroxide, cat. # 5240-05; Macron Fine Chemicals, Center Valley, Pennsylvania); rinsed in PBS again 3 times at RT, 10 min each time (rinsed); blocked in 10% normal donkey serum (NDS, cat. # 017-000-121) in PBS with 0.3% Triton X-100 (PBS-T) for 90 min at RT; incubated with primary antibodies for ∼48 h in PBS-T with 1% NDS at 4 °C. Primary antibody concentrations were as follows: rabbit anti-optineurin [Abcam (1:5000) or Cayman (1:100)]. Optimal primary antibody concentrations were determined in pilot studies using graded antibody concentrations. The sections were then rinsed 3 times with PBS at RT for 10 min each time; incubated with secondary antibodies Biotin-SP anti-rabbit (1:200) in PBS-T with 1% NDS for 90 min at RT; rinsed 3 times with PBS at RT for 10 min each time; incubated in ABC reaction mixture for 60 min at RT; rinsed 3 times with PBS at RT for 10 min each time; incubated in DAB stain until color was visually quenched (typically ∼30 s); and rinsed 6 times with PBS at RT for 10 min each time before mounting on slides. DAB-stained sections were mounted on slides and allowed to dry overnight, then dehydrated in graded alcohols and histoclear, and coverslipped using histomount.

For striatal DA terminal density staining; sections were initially rinsed 6 times with PBS at RT for 10 min each time; blocked in 10% normal donkey serum in PBS-T for 90 min at RT; incubated with mouse anti-tyrosine hydroxylase (1:2000) for ∼48 h in PBS-T with 1% NDS in PBS-T; rinsed 3 times with PBS at RT for 10 min each time; incubated with IR800 anti-mouse (1:20 000) with 1% NDS in PBS-T for 90 min at RT; and rinsed 6 times with PBS at RT for 10 min each time before mounting on slides. Mounted sections were allowed to dry overnight, then dehydrated in graded alcohols and histoclear, and coverslipped with DPX mountant.

Silver staining was conducted to identify degenerating neurons (Switzer and Butt, 2011). Staining was conducted using FD NeuroSilver Kit II (PK301A; FD NeuroTechnologies, Inc, Columbia, Maryland) from coronal brain sections obtained as described above. Brain tissue was processed according to manufacturer instructions.

For immunofluorescence staining to be analyzed by confocal microscopy, sections were initially rinsed 6 times with PBS at RT for 10 min each time; blocked in 10% NDS in PBS-T for 90 min at RT; incubated with primary antibodies for ∼48 h in PBS-T with 1% NDS at 4 °C; rinsed 3 times with PBS at RT for 10 min each time; incubated with secondary antibodies in PBS-T with 1% NDS for 90 min at RT; and rinsed 6 times with PBS at RT for 10 min each time before mounting on slides and coverslipped as a wet mount using 50/50 glycerol:PBS solution. Primary antibody dilutions were: mouse anti-LC3 (1:1000); rabbit anti-optineurin (1:5000); sheep anti-tyrosine hydroxylase (1:1000); mouse anti-alpha synuclein (1:2000). Secondary antibody dilutions were: Alexa Fluor 647 anti-sheep, Alexa Fluor 488 anti-rabbit, or Cy3 anti-mouse (1:500).

Microscopy and image analysis

Light microscopy images were captured on an Olympus System Microscope Model BX53 with an Olympus digital camera model DP72 (12.8 megapixel, color), and using the Olympus cellSens Dimensions software (ver. 1.14).

To verify the expected temporal development of the rotenone-induced lesion to the nigrostriatal dopamine system, we qualitatively analyzed DA striatal terminal density by fluorescently detecting tyrosine hydroxylase (TH) as previously described (Cannon et al., 2011; Lee et al., 2015; Tapias et al., 2014). For this experiment, 1 in 12 striatal sections from each animal were stained using mouse anti-TH primary antibody and IR 800CW anti-mouse secondary antibody. Immunolabeled sections were scanned at 800 nm on an Odyssey Infrared Imaging System (Licor Inc., Lincoln, Nebraska) at a resolution of 42 μm.

Confocal images were captured on an inverted Nikon fully motorized Ti-E microscope equipped with ×10, ×20 and ×60 high NA oil immersion objectives. Region of interest analysis was then conducted on images using the NIS-Elements software ver. 4.30 to measure signal intensity. High magnification images (×60) were analyzed for puncta analysis; puncta were considered bright circular dots with diameters ranging from 0.5 to 1.5 μm, as has been previously described for autophagosome diameters (Klionsky et al., 2016).

Statistical analysis

Statistical analysis was conducted using GraphPad PRISM, ver. 6. Data for intensity and puncta analyses were found not to have a Gaussian distribution by the D'Agostino & Pearson omnibus normality test. Thus, non-parametric analysis was conducted using Kruskal–Wallis non-parametric ANOVA, followed by post-hoc analysis using the Dunn’s test. P < .05 deemed significant for all tests.

RESULTS

OPTN in the Rat Midbrain and Striatum

Immunostaining with colorimetric development in untreated rats clearly shows that in the midbrain, OPTN is enriched in the pars compacta region of the substantia nigra (primary antibody from Abcam as noted in Methods) (Figs. 1A–C). In the midbrain specifically, expression appears to be far higher in the substantia nigra than in surrounding nuclei. Expression also appears to be high in pyramidal neurons of the hippocampus and in cortical layers 1 and 2. Colocalization of OPTN with TH by immunofluorescence using confocal microscopy clearly shows high expression in DA neurons, with limited expression in non-dopaminergic neurons (Figs. 1D–F). Expression is also evident in the medial DAergic ventral tegmental area. Additional stainings conducted with a second primary antibody generated from a different epitope and supplier revealed a similar staining pattern (supplied by Cayman Chemical as noted in the “Materials and Methods” section) (data not shown).

FIG. 1.

OPTN expression is enriched in dopaminergic neurons of the substantia nigra. (A–C) Untreated rat coronal midbrain section stained for OPTN at × 10, ×20, and ×40, respectively (scale bars = 500, 50, 20 μm, respectively). (D-F) Double immunofluorescent stained section with colocalization of tyrosine hydroxylase (TH) (DA neurons) and OPTN within the substantia nigra validate OPTN expression is in DA neurons; OPTN in red, TH in green, colocalized in yellow. Scale bar = 25 μm.

OPTN staining in the striatum showed an overall diffuse expression (Figure 2A). Axon terminals within the striatum do not overtly express OPTN (Figure 2B), although some interneurons exhibit expression. Cellular expression was also noted in the cortex (Figure 2C) and lateral septum (Figure 2D), though enrichment was not nearly as striking as in dopaminergic neurons in the substantia nigra (Figure 1).

FIG. 2.

OPTN expression in the striatum. (A) Low magnification coronal section of striatum stained for OPTN (bar = 500 μm). (B-C) High magnification images of dorsolateral striatum (B), cortex (C), and lateral septum (D). Scale bar = 25 μm.

Temporal Development of a Lesion to the Nigrostriatal Dopamine System

We confirmed that our 24 h and 5-day sampling points did not exhibit an overt lesion to the nigrostriatal DA system, as expected based on the literature and our extensive experience with this model. Low magnification immunofluorescent images of coronal sections from the striatum that were stained for TH show no overt lesion in controls or animals treated with rotenone for 24 h or 5 days. However, an overt lesion of DA terminals is clearly evident in animals treated until the emergence of severe behavioral deficits (Figure 3). Further, silver staining shows that argyrophilic cells are notable after 5 days of rotenone treatment (Figs. 3E–H) and upon the emergence of end-stage behavioral deficits, there is an apparent loss of large, angular cells, with a morphology characteristic of DA neurons. These findings are highly consistent with numerous published detailed pathological and mechanistic reports in this model, where lesion development has been extensively quantified (Cannon et al., 2009; Sanders et al., 2014a, b; Tapias et al., 2010, 2014).

FIG. 3.

An overt lesion to the nigrostriatal dopamine system does not occur until the emergence of behavioral deficits. (A–D) Representative images of striatal TH terminal densities indicate significant striatal lesion in end-stage models and with no visual lesion in pre-clinical models; (A) control treated with DMSO, (B) 24 h rotenone, (C) 5-day rotenone, and (D) treatment until end-stage behavioral phenotype presented. Scale bar = 500 μm. (E-H) Silver staining in the pars compacta region of the substantia nigra showing limited affinity in (E) control and (F) 24 h rotenone. Cells become argyrophilic (arrowheads show examples) after (G) 5-day rotenone, and (H) upon treatment until end-stage behavioral phenotype emerges, there is an expected decrease of large, angular neurons consistent with dopaminergic neuron morphology. Scale bar = 50 μm.

Rotenone Alters OPTN Expression in Relation to Autophagic Activity and alpha-Synuclein Puncta

Given OPTN’s known roles in autophagy in neurodegenerative diseases (eg, glaucoma, ALS, Huntington’s disease) and autophagy’s roles in PD, we examined a potential role for OPTN in PD-relevant autophagic dysfunction. Initially, we examined whether overt changes in OPTN expression were temporally related to nigrostriatal DA lesion development. Figure 4 shows DAB-immunostained OPTN in each stage. Colorimetric staining indicated that OPTN expression increased with rotenone exposure and that localization appeared to shift into puncta. This finding suggested that quantitative analyses of both whole cell OPTN expression (conducted in Figure 5E) and puncta localization (conducted in Figs. 5 G–I) was warranted.

FIG. 4.

OPTN expression in the substantia nigra increases in pre-clinical and end-stage PD models. Representative images of nigral dopamine neurons stained for OPTN in Lewis rats treated with DMSO (control; A, E, F) or 3.0 mg/kg/day rotenone i.p. for 24 h (B, G, H), 5 days (C, I, J), or until end-stage behavioral phenotype (D, K, L) to model different stages of PD, respectively. (A–D) Low magnification (×20) representative images of OPTN-positive cells in the substantia nigra pars compacta of rats from each stage. (E–L) High magnification (×60) representative images of OPTN-positive cells in the substantia nigra pars compacta region of rats from each stage. Scale bar = 50 μm (A–D), 10 μm (E–L).

FIG. 5.

OPTN expression, aggregation, and colocalization with LC3 increases in pre-clinical and end-stage PD models. (A–D) Representative images of nigral dopamine cells co-stained with OPTN, LC3 and TH from animals treated as control, 24 h, 5 days, and end stage, respectively; TH in blue, OPTN in green, LC3 in red, colocalized OPTN/LC3 in yellow. Scale bar = 10 μm. (E–F) Quantitative analysis of OPTN and LC3 expression, respectively, measured by relative intensity of total cellular expression (relative to control); (G–I) mean number of puncta per cell for OPTN, LC3, or colocalized puncta, respectively, across stages; data are presented as mean ± SEM; *P < .05 from control, Dunn’s multiple comparison test after significant Kruskal–Wallis test.

We investigated the possibility that OPTN puncta may relate to rotenone-induced changes in autophagy by co-staining nigral tissues of our staged rats with TH, OPTN, and LC3 (autophagy biomarker). Our results clearly show OPTN and LC3 colocalization in puncta 0.5–1.5 μm in diameter (Figs. 5A–D), the typical size range of autophagosomes. We considered changes in expression of each protein by measuring the relative intensity within the cell soma; our analysis indicates an increase in OPTN expression in rotenone groups [100 ± 2.27 vs 136.64 ± 5.89, 121.10 ± 1.97, 141.59 ± 4.25; mean (normalized to control) ± SEM for control, 24 h, 5 day, respectively; P < .05 from control, Dunn’s multiple comparison test after significant Kruskal–Wallis test; n = 131–358 cells analyzed/group from 2 to 5 animals] (Figs. 5A–E). We found that total LC3 expression increases after only a single rotenone dose; we observed an initial increase in LC3 expression after 24 h rotenone with a temporal decrease near control levels at 5 days and end-stage expression in rotenone groups (100 ± 1.23 vs 125.77 ± 3.60, 92.73 ± 0.71, 96.41 ± 1.38 mean (normalized to control) ± SEM for control, for control, 24 h, 5 days, end-stage, respectively; P < .05 from control, Dunn’s multiple comparison test after significant Kruskal–Wallis test) (Figs. 5A–D, F). Further, OPTN was found to be associated with changes in autophagic vesicle number through puncta analysis. Puncta number per cell for OPTN⁺; LC3⁺; OPTN⁺/LC3⁺puncta were quantified (Figs. 5G–I). We observed an increase in the average number of OPTN puncta per cell that became statistically significant in 5 days or end-stage groups (16.70 ± 0.66 vs 19.55 ± 0.59, 19.71 ± 0.49 mean ± SEM for control, for control, 5 days, end-stage, respectively; P < .05 from control, Dunn’s multiple comparison test after significant Kruskal–Wallis test). LC3 puncta showed a biphasic response relative to rotenone treatment, with an increase that was only detectable at 5 days (11.89 ± 0.49 vs 14.33 ± 0.45 mean ± SEM for control, for control and 5 days, respectively; P < .05 from control, Dunn’s multiple comparison test after significant Kruskal–Wallis test). A similar phenomenon was observed for puncta expressing both LC3 and OPTN, where increases were detectable only at 5 days (8.27 ± 0.35 vs 10.81 ± 0.40 mean ± SEM for control, for control and 5 days, respectively; P < .05 from control, Dunn’s multiple comparison test after significant Kruskal–Wallis test).

Alpha-synuclein staining, along with TH and OPTN shows increasing OPTN puncta similar to Figure 5 that largely colocalize with alpha-synuclein positive puncta (Figure 6). Alpha-synuclein positive puncta appear to increasingly colocalize with OPTN as the number of rotenone treatments increase. Representative images were shown from >50 images collected per group.

FIG. 6.

OPTN and alpha-synuclein expression in pre-clinical and end-stage PD models. OPTN and alpha-synuclein positive puncta increase with rotenone treatment. (A–D) Representative images of nigral dopamine cells co-stained with OPTN, alpha-synuclein and TH from animals treated as control, 24 h, 5 days, and end stage, respectively; TH in blue, OPTN in green, alpha-synuclein in red, colocalized OPTN/alpha-synuclein in yellow. Scale bar = 5 μm.

Thus, in pre-clinical and end-stage neurotoxicant-induced PD models, changes in OPTN expression, localization and relation to autophagosomes and alpha-synuclein positive puncta clearly occur, and in many cases prior to lesion formation. These changes occur within DA neurons (TH⁺).

DISCUSSION

There is considerable overlap in many pathological processes between neurodegenerative diseases. Given known roles in cellular processes that are disrupted in neurodegenerative diseases, the protein OPTN is a plausible important effector. Whereas OPTN mutations have been linked to several neurodegenerative diseases, the protein has received minimal attention in PD research. For the first time, we show that OPTN expression is enriched in DA neurons in the midbrain, and that expression is modulated in a neurotoxic PD model. Notably, changes occur prior to an overt lesion. It is expected that these findings will prompt follow-up mechanistic investigations to determine if OPTN is a critical mediator of PD pathogenesis.

OPTN has numerous structural motifs linked to many roles in the cell including: vesicle trafficking, Golgi structural protein, an autophagic cargo adaptor, transcription factor, and in the innate immune system (Ying and Yue, 2012). OPTN point mutations have been linked to glaucoma or ALS and evidence implicates impaired autophagy and compromised mitochondrial integrity as key pathogenic mechanisms (Chalasani et al., 2014; Sirohi et al., 2015; Wong and Holzbaur, 2014). Autophagy disruptions and mitochondrial dysfunction occur in PD and are likely key pathogenic events in the earliest disease stages (Parker et al., 1989; Tan et al., 2014). PD patients also have a higher risk for glaucoma (Nucci et al., 2015). Further, meta-analysis of genome-wide association studies shows that single nucleotide polymorphisms in OPTN may increase PD risk. Specifically, the M98K mutation, which likely affects OPTN-mediated autophagy, causes glaucoma and was identified as a PD risk modulator (Lill et al., 2012; Sirohi et al., 2015). Thus, mechanistic and genetic data provide a strong rationale for investigation in PD.

To date, there is a single human report on OPTN in PD and no known animal model studies. The human study focused on comparing intracellular OPTN aggregates across several neurodegenerative diseases (Osawa et al., 2011). With respect to PD, there are many limitations in this initial report: (1) no control tissue was stained; (2) a single cell was shown in a high-magnification image; (3) a single possible Lewy neurite was shown, though this image possibly shows a blood vessel; (4) no counterstain was used to confirm that expression was in a neuron. Given known OPTN functions, and relevance to PD, we chose to conduct the first study of OPTN expression in normal animals and in PD models.

In the midbrain, we found OPTN expression to be enriched in the substantia nigra, particularly in the pars compacta region (Figure 1). Expression also appears to be high in pyramidal neurons of the hippocampus and in cortical layers 1 and 2. Whereas expression is not exclusive to the nigra, staining intensity is clearly higher than in surrounding regions. Through immunofluorescence staining and confocal microscopy, we confirmed expression in DA neurons, which appears to be higher than in non-DA neurons. Expression appears to be primarily limited to the soma, as there was not clear evidence of terminal staining in the striatum (Figure 2). Proteins enriched in nigral DA neurons typically have important roles in DA cell survival (Bissonette and Roesch, 2016). Thus, our findings suggest that mechanistic studies to determine how OPTN influences dopamine neuron integrity should be conducted. Further, whole brain, cell-type specific expression analysis should be conducted in the future to characterize expression in other major brain nuclei, particularly those affected in PD.

For toxicant response studies, we utilized the rat rotenone model. Rotenone exposure is a PD risk factor in humans and rotenone has been used extensively to model the disease in animals (Cannon and Greenamyre, 2010; Tanner et al., 2011). Here, rotenone exposure was utilized to produce models of different stages of PD by dosing the rats with 3 mg/kg/day rotenone for 24 h (to model very early stage), 5 days (to model early-mid stage), and until animals exhibited severe motor deficits (typically beginning at ∼7 days) (Cannon et al., 2009). The emergence of qualitative behavioral deficits such as bradykinesia, postural insatiability, and rigidity have been shown to coincide with development of a lesion to the nigrostriatal dopamine system characteristic of late stage PD (Cannon et al., 2009). This specific model has been extensively characterized in terms of the temporal development of quantitative behavioral deficits and the emergence of a lesion to the nigrostriatal DA system (Cannon et al., 2009, 2013; Tapias et al., 2010, 2014; Zharikov et al., 2015). Of note, sampling time can easily be adjusted to obtain a “pre-clinical” model, where endpoints are assessed prior to the emergence of a behavioral phenotype and overt lesion to the nigrostriatal DA system (Cannon and Greenamyre, 2014; Sanders et al., 2014a, 2014b). Thus, this model offered unique advantages with respect to environmental relevance, replication of key pathologies, and availability of sampling time-points akin to pre-clinical time-points in humans. Our findings confirmed expected lesion development with respect to striatal TH terminal density and nigral silver staining—only the end stage animals exhibited an obvious striatal lesion and overt neuronal loss (Figure 3). These findings are highly consistent with our many publications in this model and confirm suitability of early time-points for pre-clinical assessment (Cannon and Greenamyre, 2014). Examination of changes in OPTN expression and localization in other environmental and genetic animal models will strengthen conclusions on a potential role in PD.

In rotenone treated animals, immunohistological staining with chromogenic development (DAB) for OPTN showed possible increases in expression and increased localization in puncta. Specifically, subcellular staining appeared to be more punctate in later PD stage models than in controls (Figure 4). Thus, we chose to conduct quantitative immunofluorescence studies to further identify changes in whole cell expression (Figure 5E) and localization (Figure 5G). Given known roles in autophagy, we suspected that OPTN may localize to autophagosomes. To investigate the OPTN puncta, we conducted fluorescent immunohistochemistry, using antibodies against OPTN and LC3 with TH as a cellular marker of dopaminergic neurons. Here, we found a significant proportion of the OPTN puncta to be colocalized with LC3 (biomarker for macroautophagy) in punctate structures within the known size range for macroautophagy (Figures 5A–D, I) (Klionsky et al., 2016). Furthermore, OPTN and LC3 puncta followed a similar pattern of incidence across PD stage models; puncta increased with later PD stages. Our results show a decrease in LC3 and colocalized puncta from 5-day to end-stage models, but OPTN puncta number remains the same. Further experiments will need to be conducted to determine why this discrepancy occurred, but we can surmise some possible explanations. Possible explanations are that fewer autophagosomes were created in the cells of end-stage animals, which led to fewer colocalized puncta, independent of OPTN mediated autophagy or that severe autophagic stress resulted in cell death. A more detailed examination of the data shows approximately 16% decrease in LC3 puncta and approximately 20% decrease in colocalized puncta—this discrepancy could be in part due to changes in phosphorylation of OPTN. Previous studies have demonstrated phosphorylation of human OPTN on Ser177 (likely Ser188 in rat) is critical for its role in autophagy, blocking this phosphorylation site significantly delays autophagic clearance of cargo (Korac et al., 2013; Rogov et al., 2013; Wild et al., 2011).

OPTN expression is known to be regulated by the autophagy regulator transcription factor EB (TFEB) and the inflammatory cytokine TNF-alpha (Nagabhushana et al., 2011; Visvikis et al., 2014). Both autophagic dysfunction and neuroinflammation occur in PD and are linked to pathogenesis (Macchi et al., 2015; Tan et al., 2014). OPTN itself is typically degraded by the proteasome, and is a known autophagy cargo adaptor (Shen et al., 2011). Thus, our data showing LC3 colocalization strongly suggest puncta stem from autophagic vesicles, instead of aberrant OPTN degradation (Shen et al., 2011). Overall, our findings are consistent with the observations that autophagy is impaired in PD brains and models, where alpha-synuclein accumulation has been intensely investigated (Mader et al., 2012; Tan et al., 2014). Alpha-synuclein is a pivotal PD protein linked to the pathogenesis and progression of sporadic and genetic PD (Braak et al., 2004; Polymeropoulos et al., 1997; Spillantini et al., 1997). Alpha-synuclein increases are replicated in the rat rotenone model (Betarbet et al., 2000). Here, we show evidence (Figure 6) that OPTN localization with alpha-synuclein positive intracellular puncta are increasingly evident in rotenone treated animals. Given that alpha-synuclein can be degraded by the ubiquitin-proteasome pathway, chaperone mediated autophagy, and macroautophagy, targeted studies will be required to understand OPTN and alpha-synuclein interactions (Lopes da Fonseca et al., 2015). Mechanistic in vitro studies will need to be conducted in the future to confirm and understand the role of OPTN in mediating aberrant autophagy and PD-relevant neurotoxicity.

In summary, we provide the first evidence of OPTN in the rodent midbrain and that expression and localization may be modulated in pre-clinical and end-stage environmentally relevant PD models. Our data suggest that OPTN-associated changes in autophagy may be linked to progression of a nigrostriatal dopamine lesion. Future studies are aimed at a more thorough mechanistic examination and whether OPTN may be a therapeutic target.

FUNDING

National Institute of Environmental Health Sciences at the National Institutes of Health [grant numbers R00ES019879, R03ES022819, R01ES025750 to J.R.C.].