https://doi.org/10.1093/brain/awac445 BRAIN 2023: 146; 1040–1052 | 1040 In vivo reduction of age-dependent neuromelanin accumulation mitigates features of Parkinson’s disease Marta Gonzalez-Sepulveda,1,2,† Joan Compte,1,2,† Thais Cuadros,1,2 Alba Nicolau,1,2 Camille Guillard-Sirieix,1,2 Núria Peñuelas,1,2 Marina Lorente-Picon,1,2 Annabelle Parent,1,2 Jordi Romero-Giménez,1 Joana M. Cladera-Sastre,1,2 Ariadna Laguna1,2,3 and Miquel Vila1,2,3,4,5 † These authors contributed equally to this work. Humans accumulate with age the dark-brown pigment neuromelanin inside specific neuronal groups. Neurons with the highest neuromelanin levels are particularly susceptible to degeneration in Parkinson’s disease, especially dopa­ minergic neurons of the substantia nigra, the loss of which leads to characteristic motor Parkinson’s disease symp­ toms. In contrast to humans, neuromelanin does not appear spontaneously in most animals, including rodents, and Parkinson’s disease is an exclusively human condition. Using humanized neuromelanin-producing rodents, we re­ cently found that neuromelanin can trigger Parkinson’s disease pathology when accumulated above a specific patho­ genic threshold. Here, by taking advantage of this newly developed animal model, we assessed whether the intracellular build-up of neuromelanin that occurs with age can be slowed down in vivo to prevent or attenuate Parkinson’s disease. Because neuromelanin derives from the oxidation of free cytosolic dopamine, we enhanced dopamine vesicular encapsula­ tion in the substantia nigra of neuromelanin-producing rats by viral vector-mediated overexpression of vesicular monoamine transporter 2 (VMAT2). This strategy reduced the formation of potentially toxic oxidized dopamine spe­ cies that can convert into neuromelanin and maintained intracellular neuromelanin levels below their pathogenic threshold. Decreased neuromelanin production was associated with an attenuation of Lewy body-like inclusion for­ mation and a long-term preservation of dopamine homeostasis, nigrostriatal neuronal integrity and motor function in these animals. Our results demonstrate the feasibility and therapeutic potential of modulating age-dependent intracellular neuro­ melanin production in vivo, thereby opening an unexplored path for the treatment of Parkinson’s disease and, in a broader sense, brain ageing. 1 Neurodegenerative Diseases Research Group, Vall d’Hebron Research Institute (VHIR)-Center for Networked Biomedical Research on Neurodegenerative Diseases (CIBERNED), 08035 Barcelona, Spain 2 Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA 3 Institut de Neurociències (INc-UAB), Autonomous University of Barcelona, 08193 Bellaterra, Barcelona, Spain 4 Department of Biochemistry and Molecular Biology, Autonomous University of Barcelona, 08193 Bellaterra, Barcelona, Spain 5 Catalan Institution for Research and Advanced Studies (ICREA), 08010 Barcelona, Spain Received March 08, 2022. Revised October 10, 2022. Accepted November 08, 2022. Advance access publication January 30, 2023 © The Author(s) 2023. 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-NonCommercial License (https://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 journals.permissions@oup.com In vivo modulation of neuromelanin BRAIN 2023: 146; 1040–1052 | 1041 Correspondence to: Miquel Vila Vall d’Hebron Research Institute (VHIR), Neurodegenerative Diseases Passeig Vall d’Hebron, 119-129 08035 Barcelona, Spain E-mail: miquel.vila@vhir.org Keywords: Parkinson’s disease; neuromelanin; VMAT2; dopamine; substantia nigra Introduction VMAT2 mRNA is decreased in circulating platelets from PD patients, suggesting a systemic VMAT2 deficiency16; and (iv) In Parkinson’s disease (PD), there is a preferential degeneration of gain-of-function VMAT2 variants in humans have been asso­ neurons that contain the pigment neuromelanin (NM), especially ciated to decreased likelihood of developing PD.17,18 By taking ad­ dopamine (DA)-producing neurons of the substantia nigra pars vantage of our newly developed NM-producing animal model, compacta (SNpc), the loss of which leads to a depletion of nigral here we assessed whether enhancement of DA vesicular encap­ and striatal DA that underlies classical PD motor symptoms. NM sulation by VMAT2 overexpression may provide therapeutic is a synthetic byproduct of DA metabolism that progressively accu­ benefit in the context of PD by maintaining DA homeostasis, de­ mulates with age, with neurons reaching the highest levels of NM creasing potentially toxic DA oxidized species and preventing being the most susceptible to PD degeneration.1–3 In contrast to hu­ age-dependent NM accumulation above its pathogenic mans, NM does not appear spontaneously in most animal species, threshold. including rodents.4 However, we recently developed the first ex­ perimental rodent model exhibiting age-dependent production and accumulation of NM within PD-vulnerable DA nigral neurons, Materials and methods at levels up to those reached in elderly humans. This model is based The collection of detailed protocols was deposited in protocols.io: on the viral vector-mediated expression of melanin-producing en­ https://www.protocols.io/view/in-vivo-reduction-of-age-dependent- zyme tyrosinase (TYR) in rat SNpc.5 Using this humanized neuromelanin-ac-rm7vzbno2vx1/v1. NM-producing animal model, we revealed that age-dependent pro­ gressive intracellular build-up of NM ultimately compromised neuronal function and viability when allowed to accumulate above Study design a specific threshold. As a consequence, these animals eventually In this study we aimed to determine whether VMAT2 overexpres­ developed major PD-like features, including motor deficits, Lewy sion could decrease age-related NM production in vivo and pre­ body (LB)-like pathology, nigrostriatal neurodegeneration and neu­ vent/attenuate PD pathology. This question was addressed by roinflammatory changes.5 Relevant to humans, intracellular NM le­ taking advantage of the only currently available rodent model of vels reach this pathogenic threshold in PD patients and pre-PD NM production, which we have recently developed, based on the subjects.5 These results indicate that an excessive production of unilateral viral vector-mediated overexpression of TYR in the rat NM within neurons can compromise neuronal function and trigger SNpc.5 In parallel to NM accumulation, these animals develop a PD pathology. Accordingly, strategies to slow down age-dependent progressive PD-like phenotype characterized by motor deficits, NM production/accumulation could potentially prevent or delay PD LB-like inclusion formation, nigrostriatal neurodegeneration, onset and disease progression. extracellular NM released from dying neurons and neuroinflamma­ DA oxidation to [o-]quinones is an essential event required for tion. Adult male Sprague–Dawley rats were randomly distributed NM synthesis.6–9 In particular, NM is formed by oxidation of excess into three different experimental groups receiving, respectively, cytosolic DA that is not accumulated into synaptic vesicles by the unilateral intranigral injections of either adeno-associated viral vesicular monoamine transporter-2 (VMAT2; SLC18A2), an vector (AAV)-TYR, AAV-VMAT2 or a combination of both. After con­ H+-ATPase antiporter found on the membrane of secretory vesicles firming that the combination of both vectors did not interfere with of presynaptic neurons that rapidly internalizes into synaptic vesi­ the expression of one another, we evaluated in all groups behav­ cles both recently-synthesized DA and extracellular DA reuptaken ioural (motor asymmetry), histological (intracellular NM levels, by the dopamine transporter (DAT).10–12 VMAT2-mediated DA ves­ LB-like inclusion formation, TH and VMAT2 downregulation, ni­ icular uptake is essential to prepare DA for subsequent release and grostriatal degeneration, extracellular NM, neuroinflammation) to prevent its cytosolic accumulation and oxidation into potentially and metabolic (DA vesicular uptake, DA levels, DA metabolism, toxic [o-]quinones, the latter being rapidly transformed into eume­ DA oxidation) changes. Based on our previous observations, two lanic or pheomelanic intermediates that constitute the melanic different experimental time-points were selected for these evalua­ component of NM.6–9 In line with this, there is an inverse relation­ tions: (i) at the onset of NM-linked neuronal dysfunction but before ship between NM content and VMAT2 immunoreactivity in human neurodegeneration (i.e. 2 months post-AAV), equivalent to pro­ midbrain DA neurons,13 with neurons exhibiting the highest dromal PD; and (ii) once nigrostriatal neurodegeneration is fully es­ VMAT2 levels being the ones with the lowest NM levels, and the tablished in these animals (i.e. 6 months post-AAV), equivalent to least vulnerable to Parkinson’s disease-linked neurodegeneration. established PD.5 Two types of controls were used for the different Conversely, the most vulnerable ventral SNpc neurons accumulate quantifications: (i) the unaffected contralateral (non-AAV injected) the highest NM levels and exhibit the lowest VMAT2 levels.13 hemisphere of each animal, representing an internal control/base­ Supporting a potential pathogenic perturbation of VMAT2 in PD: line; and (ii) a group of rats unilaterally injected with AAV-VMAT2 (i) post-mortem PD brains exhibit decreased VMAT2 expression in in the SN, which serve as a control of the potential effects of nigrostriatal DA terminals14; (ii) VMAT2-dependent DA encapsula­ VMAT2 overexpression by itself. In addition, we had previously re­ tion within synaptic vesicles is defective in early PD cases15; (iii) ported that unilateral nigral injections of the corresponding 1042 | BRAIN 2023: 146; 1040–1052 M. Gonzalez-Sepulveda et al. AAV-empty vector (EV) or vehicle/sham does not produce any ni­ with a sliding microtome (Leica) at 5-µm thickness. For ultra- gral pathology in these animals in contrast to AAV-TYR injections.5 performance liquid chromatography-tandem mass-spectrometry Therefore, AAV-EV or vehicle/sham injections were not used in this (UPLC-MS/MS) analyses, contralateral and ipsilateral striata (Str) study, to minimize the number of animals according to internation­ were dissected, frozen on dry ice, and stored at −80°C until use. In al regulations on the protection of animals used for experimental an additional set of animals euthanized at two months, Str and ven­ purposes. All researchers were blinded to the experimental groups tral midbrain (vMB) were dissected, frozen on dry ice, and stored at analysed. −80°C until analysed by UPLC-MS/MS. Two additional sets of animals were euthanized at one month post-AAV injection and their brains Animals processed for either histology or qPCR, respectively, to assess trans­ duction efficiency of the viral vectors, alone or in combination. Adult male Sprague–Dawley rats (RRID: MGI:5651135, Charles River), 225–250 g at the time of surgery, were housed two to three per cage with ad libitum access to food and water during a 12 h Immunohistochemistry light/dark cycle. All the experimental and surgical procedures Deparaffinized rat brain sections were quenched for 10 min in 3% were conducted in accordance with the European (Directive 2010/ H2O2-10% (vol/vol) methanol. Antigen retrieval in paraffin sections 63/UE) and Spanish laws and regulations (Real Decreto 53/2013; was performed with a 10 mM citric acid solution at pH 6.0 in a Generalitat de Catalunya Decret 214/97) on the protection of ani­ microwave for 20 min. Sections were rinsed three times in 0.1 M mals used for experimental and other scientific purposes, and ap­ Tris-buffered saline (TBS) between each incubation period. proved by the Vall d’Hebron Research Institute (VHIR) Ethical Blocking for 1 h with 5% (vol/vol) normal goat serum (NGS, Vector Experimentation Committee, to ensure the use of the minimum ne­ Laboratories) was followed by incubation with the appropriate pri­ cessary number of animals and the application of protocols that mary antibody at 4°C for 48 h in 2% (vol/vol) serum. Details about cause the least pain, suffering or distress to animals. Rats were ran­ primary antibodies used for immunohistochemistry can be found domly distributed into the different experimental groups and con­ in Table 1. Sections were then incubated with the corresponding trol and experimental groups were processed at once to minimize secondary biotinylated antibody (Vector Laboratories), visualized bias. by incubation with avidin-biotin-peroxidase complex (Immunopure ABC Peroxidase staining kit; Thermo Fisher Scientific), using the Stereotaxic infusion of viral vectors VectorSG Peroxidase Substrate Kit (Vector Laboratories) as a chromo­ Recombinant AAV vector serotype 2/1 expressing the human tyro­ gen, and mounted and coverslipped with DPX mounting medium sinase cDNA driven by the cytomegalovirus (CMV) promoter (Sigma-Aldrich). (AAV-TYR; concentration 2.6 × 1013 gc/ml; #UPV2337) and AAV sero­ type 2/9 containing the human VMAT2 cDNA fused to three Flag Immunofluorescence epitopes under control of the CMV promoter (AAV-VMAT2; concen­ tration 7.2 × 1012 gc/ml; #UPV2637) were produced at the Viral The immunofluorescence procedure was similar to the previously Vector Production Unit of the Autonomous University of reported immunohistochemistry protocol without the quenching Barcelona (UPV-UAB, Spain). Surgical procedures were performed step. Blocking was performed with 5% (vol/vol) NGS and 0.1% (vol/ with the animals placed under general anesthesia using vol) Triton X-100 (Sigma-Aldrich) in PBS solution. Corresponding isoflurane (5% for the induction phase and 2% for the maintenance primary antibodies were incubated together overnight at 4°C in phase) (Baxter). Vector solutions were injected using a 10 μl 2% (vol/vol) serum. Details about primary and secondary antibodies Hamilton syringe fitted with a glass capillary (Hamilton used for immunofluorescence can be found in Table 1. Adequate model Cat. No. #701). Animals received 2 μl of a 0.65:1.35 mixture Alexa 488 and/or 647-conjugated secondary antibodies were incu­ of AAV-TYR+AAV-Flag-VMAT2, AAV-TYR+vehicle or vehicle bated simultaneously for 1 h at room temperature in 2% (vol/vol) +AAV-Flag-VMAT2 to achieve a final titration of 1.7 × 1013 gc/ml serum. Nuclei were stained with Hoechst 33342 (1:2000, Thermo for AAV-TYR and 9.7 × 1012 gc/ml for AAV-VMAT2. In the TYR/ve­ hicle and VMAT2/vehicle groups, viral vectors were diluted with ve­ Table 1 Antibodies used in immunohistochemistry/ hicle (PBS-MK/40% Iodixanol). Infusion was performed at a rate of immunofluorescence experiments 0.4 μl/min and the needle was left in place for an additional 4 min period before it was slowly retracted. Injection was carried out uni­ Antibody Manufacturer Dilution laterally on the right side of the brain at the following coordinates Primary (flat skull position), right above the SNpc: antero-posterior: Anti-TH Calbiochem (657012) 1:40 000 (IHC) −5.29 mm; medio-lateral: −2 mm; dorso-ventral: −7.6 mm below 1:1000 (IF) dural surface, calculated relative to bregma according to the stereo­ Anti-Flag Sigma (F3165) 1:1000 (IHC) taxic atlas of Paxinos and Watson.19 Anti-VMAT2 Progen (16085) 1:500 (IHC) Anti-TYR Epredia (MS-800-P1) 1:750 (IHC) Brain processing for UPLC-MS/MS, qPCR and Anti-CD68 Serotec (MCA341R) 1:100 (IHC) Anti-GFAP Sigma (G3893) 1:1000 (IHC) histological analyses Anti-Iba-1 Wako (019–19741) 1:1000 (IHC) Two or six months after injection of viral vectors animals were eu­ Anti-p62 Progen (GP62-C) 1:500 (IF) thanized and the brains quickly removed and placed over a cold Secondary plate. For histological analyses, the posterior portion of the brain Anti-Guinea Thermo Fisher Scientific (A11073) 1:1000 (IF) Pig- 488 was embedded in ice-cold formaldehyde solution 4% phosphate buf­ Anti-Rabbit-647 Thermo Fisher Scientific (A21443) 1:1000 (IF) fered (Panreac) for 24 h and subsequently processed for paraffin em­ bedding following standard procedures. Sectioning was performed a IF = immunofluorescence; IHC = immunohistochemistry. In vivo modulation of neuromelanin BRAIN 2023: 146; 1040–1052 | 1043 Fisher Scientific, ref: H3570) in 1× PBS for 10 min. Sections were cov­ Desktop 3.3 software (https://www.olympus-lifescience.com/en/ erslipped using the Dako Cytomation Fluorescent Mounting solutions-based-systems/vs200/). Adjacent serial 5-µm thick paraf­ Medium (Dako). fin sections covering the entire SNpc were included in the counting Immunofluorescent images were taken with a LSM 980 with procedure (every 17th section for a total of 10–12 sections analysed/ Airyscan 2 confocal microscopeand were analysed with ZEN 3.1 animal; Supplementary Fig. 1). Each automatized counting was software (RRID: SCR_013672; http://www.zeiss.com/microscopy/ manually revised by an investigator blinded to the experimental en_us/products/microscope-software/zen.html#introduction). The groups. total number of p62-positive cytoplasmic inclusions was manually determined in a selected SNpc middle section/animal exhibiting Quantification of neuroinflammation parameters high numbers of nigral neurons, including NM-containing neurons, from two different experimental groups: TYR (n = 8) and TYR Quantification of Iba-1, CD68 and GFAP-positive cells was per­ +VMAT2 (n = 7), at 2 months post-AAV injections. Equivalent ana­ formed in SNpc sections adjacent to those used for stereological tomical levels (based on Paxinos and Watson19) were analysed cell counts. Serial 5-µm thick paraffin sections covering the entire across animals. All quantifications were performed by an investiga­ SNpc (every 17th section for a total of 10–12 sections analysed/ani­ tor blinded to the experimental groups. mal; Supplementary Fig. 1) were scanned using the Panoramic Midi II FL, HQ SCIENTIFIC 60x scanner and section images were acquired with CaseViewer software (RRID:SCR_017654; https://www. Cell counting 3dhistech.com/caseviewer). For quantification of Iba-1, CD68 and Assessment of the total number of TH-positive neurons, the GFAP-positive cells, specific AI-based algorithms were implemen­ number of NM-laden neurons (with or without TH) and extracellu­ ted using the Aiforia 5.3 platform (RRID:SCR_022739, https://www. lar NM aggregates in the SNpc was performed by stereology aiforia.com). Iba-1-positive cells were counted separately in two according to the fractionator principle, using the MBF Bioscience different groups according to their activation state: non-reactive StereoInvestigator 11 (64 bits) Software (RRID: SCR_004314; https:// (branched) and reactive (amoeboid). CD68 and GFAP-positive cells www.mbfbioscience.com/help/si11/Default.htm) coupled to a were counted individually. Data are presented as the number of Zeiss Imager.D1 microscope with an AxioCam MRc camera positive cells per quantified area (in mm2). All quantifications (Zeiss). Serial 5-µm thick paraffin sections covering the entire were performed by an investigator blinded to the experimental SNpc were included in the counting procedure (every 17th section groups. For illustration purposes, high resolution micrographs for a total of 10–12 sections analysed/animal; Supplementary Fig. were acquired with the Olympus Slideview VS200 slide scanner 1). The following sampling parameters were used: (i) a fixed count­ and the Olyvia 3.3 software. ing frame with a width and length of 50 μm; (ii) a sampling grid size of 115 × 70 μm; and (iii) a multiplication factor of 17. The counting Intracellular NM quantification frames were placed randomly by the software at the intersections Intracellular NM levels were quantified in both AAV-TYR and of the grid within the outlined structure of interest. Objects in AAV-TYR+AAV-VMAT2-injected animals at two months both brain hemispheres were independently counted following post-AAV injections in 5-µm thick paraffin-embedded haematoxy­ the unbiased sampling rule using a 100× lens and included in the lin and eosin (H&E)-stained sections covering the whole SNpc for measurement when they came into focus within the dissector. A each animal. In these sections, SNpc dopaminergic neurons were coefficient of error of <0.10 was accepted. Data for the total num­ identified by the visualization of unstained NM brown pigment. bers of TH-positive neurons and NM-containing neurons are ex­ Midbrain sections were scanned using the Pannoramic Midi II FL, pressed as absolute numbers for each hemisphere. The total HQ SCIENTIFIC 60× and section images were acquired with number of SNpc DA neurons was calculated by considering all CaseViewer software at an objective magnification of 63×. For illus­ TH+NM+, TH−NM+ and TH+NM− neurons. The percentage of TH tration purposes, high resolution micrographs were acquired with downregulation was calculated by considering the total number an Olympus Slideview VS200 slide scanner and the Olyvia 3.3 soft­ of TH+NM+ and the total number of TH−NM+ with respect to the to­ ware. All NM-positive neurons in a representative SNpc middle sec­ tal number of neurons containing NM. All quantifications were per­ tion exhibiting high numbers of NM-containing neurons were formed by an investigator blinded to the experimental groups. For analysed by means of optical densitometry using ImageJ software the assessment of the number of VMAT2-positive neurons, a simi­ (RRID:SCR_003070; https://imagej.net/) to quantify the intracellular lar approach was applied to a selected SNpc middle section/animal density of NM pigment, as previously reported.5 The pixel bright­ exhibiting high numbers of nigral neurons, including ness values for all individual NM-positive cells (excluding the nu­ NM-containing neurons. Equivalent anatomical levels (based on cleus) in all acquired images were measured and corrected for Paxinos and Watson19) were analysed across animals. The percent­ non-specific background staining by subtracting values obtained age of ipsilateral VMAT2 downregulation was calculated by consid­ from the neuropil in the same images. Equivalent anatomical levels ering the number of VMAT2−NM+ neurons versus total SNpc DA (based on Paxinos and Watson19) were analysed across animals. All neurons (including VMAT2+NM+, VMAT2+NM− and VMAT2−NM+ quantifications were performed by an investigator blinded to the neurons). experimental groups. For the quantification of VMAT2-Flag and TYR transduction efficiency, high resolution micrographs were acquired with Chromatographic determination of dopaminergic an Olympus Slideview VS200 slide scanner and the Olyvia 3.3 soft­ metabolites in brain samples ware (RRID:SCR_016167; https://www.olympus-lifescience.com/en/ support/downloads/#dlOpen=%23detail847249644). A specific arti­ UPLC-MS/MS analysis of rat brain regions was performed using our ficial intelligence (AI)-assisted algorithm was implemented for the previously validated method20 with some modifications. Each sample quantification of vector SG immunostained neurons (VMAT2-Flag, was injected three times into the UPLC-MS/MS system to analyse dif­ TYR or TH; Supplementary Fig. 2) using the Olympus V200 ferent sets of compounds i.e. MIX1, MIX2 and MIX3. MIX1 included DA, 1044 | BRAIN 2023: 146; 1040–1052 M. Gonzalez-Sepulveda et al. Table 2 MRM acquisition settings Analyte Transition (m/z) Retention time (min) D (ms) CV (V) CE (eV) CpV (kV) DA4d (IS) 157.83 > 94.8 1.44 52 10 20 0.5 DA 153.93 > 90.57 1.46 52 10 20 0.5 L-DOPA 198.1 > 152.1 1.48 52 15 15 0.5 5SCDA 273.1 > 166.9 1.73 58 20 20 0.5 5SCDA 317 > 154.86 2.01 58 24 30 0.5 3MTa 150.7 > 90.96 3.09 52 35 20 0.5 AC 149.61 > 121.91 3.36 52 25 25 0.5 DOPACb 166.99 > 122.82 3.72 56 18 22 2 CE = collision energy; CV = cone voltage; CpV = capillary voltage; D = dwell time. a Parent mass after loss of water. b Detected in negative mode. 3-methoxytyramine (3MT), 3,4-dihydroxyphenylalanine (L-DOPA) Mm02601633_g1 and Ppia Mm02342430_g1). Water was included and aminochrome (AC); MIX2 included 3,4-dihydroxyphenylacetic in the reaction as a non-template (negative) control. The relative acid (DOPAC) and MIX3 included 5-S-cysteinyldopa (5SCD) and expression was calculated with the ΔCt-method. 5-S-cysteinyldopamine (5SCDA). IS was added to every set of com­ pounds. 5SCD and 5SCDA standards were kindly donated by Statistical analysis Professor Kazumasa Wakamatsu and Professor Shosuke Ito at the Fujita Health University, Aichi, Japan. Aminochrome standard Statistical analyses were performed with GraphPad Prism v6 soft­ (0.5 mM) was freshly prepared as previously described.20,21 Multiple ware (RRID:SCR_002798; http://www.graphpad.com/). No statistical Reaction Monitoring (MRM) acquisition settings for the targeted meta­ methods were used to predetermine sample size but our sample bolites are summarized in Table 2. Samples with a concentration be­ sizes were equivalent to those reported in previous similar publica­ tween LOD and LOQ or bigger than LOQ were considered acceptable; tions.5 Outlier values were identified by the ROUT test and excluded samples with a concentration lower than LOD were considered as from the analyses when applicable. Selection of the pertinent data the LOD value. Data were normalized by the protein concentration representation and statistical test for each experiment was deter­ and presented as the percentage of the contralateral concentration mined after formally testing for normality with the Shapiro–Wilk or ratio. normality test. Accordingly, differences among means or medians were analysed either by one- or two-way ANOVA, Kruskal–Wallis ANOVA on ranks or Mann–Whitney rank sum test, as appropriate Cylinder behavioural test and indicated in each figure legend. When ANOVA showed signifi­ Rats were tested for left and right forepaw use with the cylinder test cant differences, pairwise comparisons between means were sub­ 1 week before surgery and at 2 and 6 months after viral injection. jected to Tukey’s post hoc testing for multiple comparisons. Values The number of animals for each condition was VMAT2 (n = 7), are expressed either as mean ± standard error of the mean (SEM) TYR (n = 8) and TYR+VMAT2 (n = 7–8). For the performance of the cy­ or presented as box plots, with minimum, maximum and median linder test, rats were first allowed to habituate to the experimental indicated, depending on the performance of parametric or non- room for at least 1 h before each test. Then, rats were put in a glass parametric analyses, respectively. In all analyses, the null hypoth­ cylinder and the total number of left and right forepaw touches per­ esis was rejected at the 0.05 level. formed within 5 min was counted. Data are presented as the per­ centage of the contralateral paw usage. Behavioural equipment Data availability was cleaned with 70% ethanol after each test session to avoid olfac­ tory cues. All behavioural tests were performed during the light cy­ Full datasets can be found at Zenodo repository (https://doi.org/10. cle by an investigator blinded to the experimental groups. 5281/zenodo.7075026). Human tyrosinase gene expression Results Total RNA was extracted from dissected ipsilateral and contralat­ VMAT2 overexpression reduces catechol oxidation, eral vMB from AAV-TYR-injected rats using mirVana PARIS RNA NM production and LB-like inclusion formation in and Native Protein Purification Kit (Thermo Fisher Scientific # humanized rats AM1556). RNA concentration was determined using a NanoDrop ND-1000 Spectrophotometer. Total RNA (0.5 µg) was retrotran­ To determine first whether VMAT2 overexpression was able to scribed using a High-Capacity cDNA Reverse Transcription Kit boost DA vesicular uptake in vivo, adult rats received a single unilat­ (Thermo Fisher Scientific # 4368814). qPCR was performed with eral stereotaxic injection of an AAV expressing flagged human 10 ng of cDNA per well in technical triplicates mixed with VMAT2 (AAV-VMAT2) above the right SNpc (Fig. 1A). In these ani­ Taqman Gene Expression Master Mix (Applied Biosystems, # mals, conspicuous exogenous VMAT2 expression was observed in 4369016) and Taqman gene expression assays [human TYR ipsilateral SNpc and striatum at 1 month post-AAV injection, as as­ (Hs00165976_m1, Applied Biosystems)] using standard procedures sessed by Flag immunohistochemistry (Fig. 1B). Overexpressed in a 7900HT Fast Real Time Instrument (Applied Biosystems). VMAT2 proved to be functionally active, as demonstrated by (i) an Thresholds cycles (Cts) for each target gene were normalized to enhanced striatal DA storage accompanied by a decreased DA me­ an endogenous reference gene (Gapdh Mm99999915_g1, Rpl19 tabolism22; and (ii) a ∼2-fold increase in striatal DA:L-DOPA ratio, an In vivo modulation of neuromelanin BRAIN 2023: 146; 1040–1052 | 1045 Figure 1 Overexpression of functional VMAT2 in the nigrostriatal pathway of rats. (A) Schematic representation of the site of AAV-VMAT2 unilateral stereotaxic injection above the SN of the rat brain. (B) Left: Representative images of nigral (top) and striatal (bottom) Flag-VMAT2 immunolabelling ip­ silateral to the injection in AAV-VMAT2-injected rats at 1 month post-AAV injection. Right, representative images of TH-Flag colocalization in ipsilat­ eral SNpc of AAV-VMAT2-injected rats at 1 month post-AAV injection. Scale bar = 50 µm (top), 10 µm (bottom). (C) Quantification of striatal DA vesicular uptake by UPLC-MS/MS in AAV-VMAT2-injected rats at 2 months post-AAV injection, as calculated by the ratio between striatal DA:L-DOPA levels.23 Values are mean ± SEM. *P < 0.05 versus contralateral (non-injected) side; Two-tailed t-test. n = 8 animals per group. Drawing in A was created with BioRender.com. index of DA vesicular uptake,23 measured by UPLC-MS/MS at levels below the pathogenic NM threshold previously identified in 2 months post-AAV injection (Fig. 1C and Supplementary Table 1). NM-producing animals5 (Fig. 2F, red dotted line). Above this thresh­ After confirming that we are able to overexpress functional old, in addition to the functional defects reported earlier, NM-laden VMAT2 in vivo, we next assessed whether nigrostriatal VMAT2 neurons develop LB-like inclusions (Fig. 2G and Supplementary overexpression in NM-producing rats could increase DA vesicular Table 3), a hallmark of PD pathology closely associated to NM accu­ uptake and reduce the rate of non-encapsulated DA oxidation mulation.5,26,27 LB-like inclusions in NM-producing rats are immu­ into NM precursors. To address this question, AAV-VMAT2 and nopositive for p62, a common component of LBs in PD brains,28 and AAV-TYR were co-injected above the rat SNpc (Fig. 2A, were previously reported to peak at 2 months post-AAV-TYR injec­ Supplementary Fig. 2 and Supplementary Table 2), with additional tion, thus coinciding with functional alterations and preceding groups of animals receiving equivalent amounts of either AAV-TYR neurodegeneration in these animals.5 Importantly, the reduction or AAV-VMAT2 separately. In AAV-TYR-injected rats, we have pre­ of intracellular NM levels by VMAT2 overexpression was accom­ viously reported that intracellular NM starts reaching pathological panied with attenuated LB-like inclusion formation in these neu­ levels at 2 months post-AAV injection, at which time these animals rons (Fig. 2G and Supplementary Table 3). Taken together, these exhibit early functional defects, including impaired DA release and results demonstrate that VMAT2 overexpression is able to restore motor deficits, prior to degeneration.5 Here we found, similar to PD DA homeostasis, reduce NM production/accumulation and attenu­ patients,15,24 that early functional changes in AAV-TYR melanized ate LB-like pathology in vivo. rats are accompanied by a downregulation of endogenous vmat2 le­ vels, as shown by a reduction of vmat2 expression within VMAT2 overexpression prevents NM-linked NM-containing neurons (Fig. 2A and Supplementary Table 3), and neurodegeneration alterations in nigrostriatal DA homeostasis, such as reduced DA vesicular uptake, decreased DA levels, increased DA metabolism In NM-producing rats, early functional defects observed at and enhanced catechol oxidation (Fig. 2B–E, Supplementary Fig. 3 2 months are followed by progressive nigrostriatal neurodegenera­ and Supplementary Tables 4 and 5). Remarkably, all these patho­ tion beginning at 4 months post-AAV-TYR injection.5 To determine logical changes were prevented by overexpressing VMAT2 in these whether decreased DA oxidation and subsequent reduction in NM animals (Fig. 2A–E and Supplementary Tables 4 and 5). accumulation achieved by VMAT2 overexpression were associated When oxidized, tyrosine, L-DOPA or DA produce dopaquinone or with preserved neuronal integrity, we assessed the long-term ef­ dopamine-o-quinone that will act as precursors for either the eu­ fects of VMAT2 overexpression in NM-producing rats at 6 months melanin (brown) and/or the pheomelanin (reddish) melanic com­ post-AAV-TYR injection, once nigrostriatal degeneration is well- ponents of NM (Supplementary Fig. 3). Thus, we next assessed established in this animal model.5 By this time, NM-producing whether attenuated catechol oxidation in VMAT2/TYR co-injected rats exhibited a ∼90% reduction in ipsilateral SNpc TH-positive neu­ animals was associated to a reduced NM production. rons that was markedly attenuated by concomitant VMAT2 overex­ Quantification of intracellular NM optical density within individual pression (Fig. 3A and Supplementary Table 6). Similar to PD brains, SNpc neurons, which reflects NM levels,3,25 revealed that intracel­ NM-producing rats also exhibited a phenotypic loss of TH expres­ lular NM density was significantly decreased in SNpc neurons of sion within NM-laden neurons, which reflects neuronal dysfunc­ TYR+VMAT2 rats, compared to AAV-TYR-injected animals tion at early stages of neurodegeneration,1 as shown by an (Fig. 2F). While intracellular NM optical density could be influenced increased percentage of TH-immunonegative neurons within the by potential changes in NM composition, independently of the ac­ total population of NM-containing neurons (Fig. 3B and tual levels of NM, we also observed a significant reduction in the Supplementary Table 6). Interestingly, VMAT2 overexpression percentage of cytosolic neuronal area occupied by NM in TYR greatly attenuated TH downregulation occurring within pigmented +VMAT2 animals compared to their TYR counterparts (Fig. 2F), indi­ neurons, indicating a functional preservation of these neurons cating an actual decrease of NM levels in these animals. (Fig. 3B and Supplementary Table 6). To distinguish between the ef­ Importantly, VMAT2 overexpression reduced intracellular NM to fects of VMAT2 on TH downregulation and on actual cell death, we 1046 | BRAIN 2023: 146; 1040–1052 M. Gonzalez-Sepulveda et al. Figure 2 Reduced catechol oxidation, NM accumulation and LB-like pathology by VMAT2 overexpression in NM-producing rats. (A) Left: Schematic representation of the site of AAV-VMAT2, AAV-TYR or AAV-VMAT2+AAV-TYR unilateral stereotaxic injection above the SN of the rat brain. Middle left: Quantification of contralateral and ipsilateral SNpc neurons immunopositive for VMAT2 (including VMAT2+NM+ and VMAT2+NM−) in AAV-VMAT2, AAV-TYR and AAV-TYR+AAV-VMAT2-injected rats at 2 months post-AAV injections. Middle right: Representative contralateral and ipsi­ lateral VMAT2-immunostained SNpc sections from these animals are shown in micrographs. Scale bar = 20 µm. Right: Ipsilateral VMAT2−NM+ neurons versus total DA neurons (including VMAT2+NM+, VMAT2+NM− and VMAT2−NM+ neurons) as an index of VMAT2 downregulation. (B–E) UPLC-MS/MS quantification of: (B) DA vesicular uptake, calculated as DA:L-DOPA; (C) DA levels; (D) DA metabolism, calculated as (3MT + DOPAC):DA; (E) catechol oxi­ dation, calculated as [5SCD:L-DOPA] + [(5SCDA + AC):DA] in vMB and striatal samples from AAV-VMAT2, AAV-TYR and AAV-TYR+AAV-VMAT2-injected animals at 2 months post-AAV injections. Results represent ipsilateral levels shown as the percentage of the contralateral (non-injected) side. Values are mean ± SEM. (F) Left: Quantification of intracellular NM levels by optical densitometry in ipsilateral SNpc DA neurons of AAV-TYR and AAV-TYR +AAV-VMAT2-injected rats at 2 months post-AAV injections. Dotted red line indicates the previously reported pathogenic threshold of intracellular NM accumulation.5 Middle: Representative images of 5- µmthick H&E-stained nigral sections from these animals (unstained NM in brown). Scale bar = 10 µm. Right: Percentage of the neuronal cytosolic area occupied by NM in AAV-TYR and AAV-TYR+AAV-VMAT2-injected rats at 2 months post-AAV injections. Values are median + min to max. (G) Left: Quantification of the number of NM-filled neurons exhibiting p62-positive LB-like cyto­ solic inclusions in AAV-TYR and AAV-TYR+AAV-VMAT2-injected rats at 2 months post-AAV injections. Values are median + min to max. Right: Representative confocal images of a melanized TH+ neuron with a LB-like inclusion immunopositive for p62 in the SNpc of a AAV-TYR-injected rat. BF = brightfield. Scale bar = 10 µm. In A, middle left *P < 0.05 versus respective contralateral side; $P < 0.05 versus ipsilateral VMAT2; two-way ANOVA with Tukey’s post hoc test; middle right #P < 0.05 versus TYR; two-tailed t-test n = 7 (VMAT2), n = 5 (TYR), n = 8 (TYR+VMAT2) animals. In B–E, *P < 0.05 ver­ sus VMAT2; #P < 0.05 versus TYR; one-way ANOVA with Tukey’s post hoc test. n = 6–8 (VMAT2), n = 6–8 (TYR), n = 6–8 (TYR+VMAT2) animals. In F–G, *P < 0.05 versus TYR; Mann–Whitney test. n = 594 neurons from n = 8 AAV-TYR-injected animals and n = 180 neurons from n = 8 AAV-TYR +AAV-VMAT2-injected animals. The drawing in A was created with BioRender.com. assessed the number of total SNpc DA neurons (including Attenuated neurodegeneration by VMAT2 TH-immunopositive and TH-immunonegative pigmented SNpc overexpression is associated to decreased neurons), which confirmed the ability of VMAT2 overexpression extracellular NM debris and inflammation to prevent NM-linked SNpc DA neurodegeneration in NM-producing rats (Fig. 3C and Supplementary Table 6). Overall, Concomitant with SNpc pigmented cell death, both PD brains and these results indicate that VMAT2 overexpression is able to prevent NM-producing rats exhibit abundant extracellular NM released neuronal dysfunction and degeneration linked to excessive NM from dying neurons, some of which is surrounded by, or contained production/accumulation. within activated microglia, indicative of an active, ongoing In vivo modulation of neuromelanin BRAIN 2023: 146; 1040–1052 | 1047 Figure 3 VMAT2 overexpression prevents SNpc neurodegeneration in NM-producing rats. Stereological nigral cell counts of (A) TH+ neurons, (B) TH−NM+ neurons versus total NM+ neurons (including TH+NM+ and TH−NM+ neurons) as an index of TH downregulation and (C) total DA neurons (in­ cluding TH+NM+, TH+NM− and TH−NM+ neurons) in AAV-VMAT2, AAV-TYR and AAV-TYR+AAV-VMAT2-injected rats at 6 months post-AAV injections. (D) Representative images of TH-positive neurons (TH in blue, unstained NM in brown), in which TH−NM+ (black arrowhead) and TH+NM+ (white arrow­ head) neurons are indicated for identification purposes. Scale bars = 200 µm and 20 µm (inset). Values are mean ± SEM. *P < 0.05 versus ipsilateral VMAT2; #P < 0.05 versus ipsilateral TYR; $P < 0.05 versus respective contralateral side; &P < 0.05 versus TH+NM+; Two-way ANOVA with Tukey’s post hoc test. n = 7 (VMAT2), n = 8 (TYR) and n = 7 (TYR+VMAT2) animals. neurodegenerative process (i.e. neuronophagia).5,29 In this context, observed in NM-producing rats included increased astrocyte re­ the marked neuroprotective effect provided by VMAT2 overexpres­ activity, widely distributed within the SNpc, which was also mark­ sion in NM-producing rats at 6 months post-AAV injection was as­ edly attenuated by VMAT2 overexpression (Fig. 4D). Taken sociated to a drastic reduction of extracellular NM in these animals together, these results reveal that, by reducing cell death and sub­ (Fig. 4A). Microglial cells are the main players in the recognition, en­ sequent NM release from dying neurons, VMAT2 overexpression is gulfment and clearance of extracellular NM.29,30 In agreement with able to prevent the overall PD-like inflammatory response linked to this, the number of microglial cells with non-reactive (ramified) and the accumulation of extracellular NM. phagocytic/reactive (large amoeboid de-ramified) morphology was markedly increased in AAV-TYR-injected rats in association to Preservation of striatal DA levels and metabolism extracellular NM and concomitant to cell death (Fig. 4B and by VMAT2 overexpression prevents motor deficits Supplementary Table 6). By attenuating neurodegeneration and in NM-producing rats subsequent accumulation of extracellular NM, VMAT2 overexpres­ sion was associated to a diminished microglial activation in these We next determined whether the neuroprotective effects of animals (Fig. 4B and Supplementary Table 6). Similarly, VMAT2 VMAT2 overexpression on SNpc DA neuron cell bodies were ac­ overexpression also precluded the recruitment of CD68-positive companied by a preservation of striatal DA levels in cells occurring in NM-producing animals, corresponding to NM-producing rats. At 6 months post-AAV-TYR injections, con­ tissue-resident or blood-borne macrophages with phagocytic activ­ comitant to SNpc neurodegeneration, NM-producing rats exhib­ ity that are found in close association with extracellular NM (Fig. 4C ited a marked reduction of striatal DA levels as assessed by and Supplementary Table 6). Additional inflammatory changes UPLC-MS/MS (Fig. 5A and Supplementary Table 7). Consistent 1048 | BRAIN 2023: 146; 1040–1052 M. Gonzalez-Sepulveda et al. Figure 4 VMAT2 overexpression reduces extracellular NM debris and inflammation in the SNpc of NM-producing rats. Quantification of (A) extracel­ lular NM clusters; (B) Iba1+ non-reactive (left) and reactive (right) microglia; (C) CD68+ cells; (D) GFAP+ astrocytes, in AAV-VMAT2, AAV-TYR and AAV-TYR+AAV-VMAT2-injected rats at 6 months post-AAV injections. Photomicrographs correspond to representative images of (A) H&E-stained SNpc sections (unstained NM in brown) and (B–D) immunostained SNpc sections for Iba1, CD68 or GFAP (in blue, unstained NM in brown). Scale bars = 10 µm (A, insets in B–D), 200 µm (B–D). In A, values are median + min to max. In B–D, values are mean ± SEM. *P < 0.05 versus ipsilateral VMAT2; #P < 0.05 versus ipsilateral TYR; $P < 0.05 versus contralateral side; Mann–Whitney test (A) or two-way ANOVA with Tukey’s post hoc test (B–D). n = 7 (VMAT2), n = 8 (TYR), n = 7 (TYR+VMAT2) animals. with its effect at preserving DA nigral cell bodies, VMAT2 overex­ therapeutic purposes in vivo. Indeed, until the recent introduction pression also prevented the loss of striatal DA in these animals of the first rodent model of NM production,5 NM had not been taken (Fig. 5A and Supplementary Table 7). The preservation of striatal into account in experimental PD in vivo modelling, despite the fact DA levels by VMAT2 overexpression was associated to a re- that this pigment has long been established as a major vulnerability established striatal DA vesicular uptake, reduced DA metabolism factor for PD-linked neurodegeneration.34 This new melanized ani­ and decreased catechol oxidation in these animals (Fig. 5B–D and mal model revealed that continuous, age-dependent intracellular Supplementary Table 7). Confirming a functional preservation of build-up of NM within autophagic structures ultimately leads to a the nigrostriatal circuit, VMAT2 overexpression prevented general failure of cellular proteostasis associated to major PD-like contralateral forepaw hypokinesia occurring in NM-producing features, including motor deficits, LB-like inclusion formation and rats, both at 2 and 6 months post-AAV injections (Fig. 5E). nigrostriatal neurodegeneration.5 Here we show that all these Collectively, these results demonstrate the ability of VMAT2 over­ pathological PD-like features can be prevented or attenuated in expression to provide a morphological and functional long-term these animals when lowering intracellular NM to levels below its preservation of the nigrostriatal pathway in vivo in a humanized pathogenic threshold by VMAT2 overexpression (see summary PD model. Supplementary Fig. 4). Relevant to PD, it has been reported in post- mortem human brains that midbrain DA neurons with the highest VMAT2 protein expression exhibit the lowest NM levels and are the Discussion least vulnerable to PD-linked neurodegeneration.13 Conversely, Modulation of VMAT2 activity has been in the spotlight as a poten­ the most vulnerable human midbrain DA neurons accumulate tial therapeutic strategy for a wide range of diseases involving dis­ the most NM and have the lowest VMAT2 levels.13 turbances of DA metabolism and/or signalling, from addiction, Because VMAT2 overexpression reduces intracellular NM levels schizophrenia or depression to neurodegenerative diseases such by decreasing free cytosolic DA that can subsequently oxidize into as PD.6,31,32 However, while some well-characterized pharmaco­ NM, the beneficial effects of VMAT2 could be related to reduced le­ logical inhibitors of VMAT2 are available, such as reserpine and tet­ vels of potentially toxic oxidized DA species that serve as NM pre­ rabenazine, positive modulators of VMAT2 function are currently cursors. In particular, tyrosine, L-DOPA or DA-derived [o-] lacking.33 Here, we found that viral vector-mediated VMAT2 over­ quinones, which are rapidly transformed into NM, have long been expression provides therapeutic benefit in parkinsonian proposed as potential pathogenic factors in PD.7 In fact, NM synthe­ NM-producing rats by maintaining DA homeostasis, reducing po­ sis is regarded as a protective antioxidant mechanism by trapping tentially toxic DA oxidized species and preventing excessive age- cytosolic quinones and semiquinones in lysosome-associated or­ dependent NM accumulation. The latter, in particular, represents ganelles so that they are no longer reactive with cytosolic compo­ the first demonstration that age-dependent NM production and nents.35 However, while high concentrations of DA can be acutely subsequent intracellular buildup can be slowed down for toxic in vitro,36 the chronic enhancement of DA levels and oxidation In vivo modulation of neuromelanin BRAIN 2023: 146; 1040–1052 | 1049 Figure 5 VMAT2 overexpression preserves striatal DA levels and motor function in NM-producing rats. UPLC-MS/MS quantification of striatal (A) DA levels; (B) DA vesicular uptake, calculated as DA:L-DOPA ratio; (C) DA metabolism, calculated as (3MT + DOPAC):DA ratio; (D) catechol oxidation, calcu­ lated as [5SCD:L-DOPA] + [(5SCDA + AC):DA] ratio, in AAV-VMAT2, AAV-TYR and AAV-TYR+AAV-VMAT2-injected animals at 6 months post-AAV injec­ tions. Results represent ipsilateral levels shown as the percentage of the contralateral (non-injected) side. (E) Contralateral forepaw use as assessed with the cylinder asymmetry test at 2 and 6 months post-AAV injections. Values are mean ± SEM. *P < 0.05 versus VMAT2; #P < 0.05 versus TYR, one-way ANOVA with Tukey’s post hoc test. n = 7 (VMAT2), n = 7–8 (TYR), n = 7–8 (TYR+VMAT2) animals. does not cause DA nerve terminal or cell body loss in vivo.37–39 For and dysmorphic organelles.27 In agreement with this, here we instance, chronic L-DOPA treatment37,38 or genetic enhancement found that decreased intracellular NM levels by VMAT2 overexpres­ of TH activity39 in rodents, both resulting in increased levels of sion was associated to reduced PD-like inclusion formation in vivo. DA and oxidized DA species, are not toxic to regular, wild-type In any case, any putative contribution of these species to NM-linked animals but only to animals displaying additional PD-related al­ PD pathology would be therapeutically targeted by VMAT2 overex­ terations, such as DJ-1 deficiency37 or A53T α-synuclein overexpres­ pression, as the latter reduces both oxidized DA species and age- sion.39 Relevant to humans, there is no clinical evidence for L-DOPA dependent NM accumulation. being neurotoxic and detrimental to the progression of PD symp­ The regulation of the balance of cytosolic DA levels is complex toms.40 In addition, antioxidant strategies have systematically and involves an interplay between different processes. From one failed to provide any therapeutic benefit in PD clinical trials.41 hand, VMAT2-mediated DA encapsulation into synaptic vesicles Even if NM synthesis per se was initially neuroprotective, its long- and MAO-mediated DA metabolism decrease cytosolic DA levels.46 term accumulation until occupying most of the neuronal cyto­ On the other hand, DAT-mediated reuptake of extracellular plasm has deleterious consequences by physically interfering DA47–49; DA leaking from synaptic vesicles46 and TH-mediated DA with intracellular communication42–44 and impairing proteosta­ synthesis increase cytosolic DA levels. While TYR can mediate sis.5,34 In fact, L-DOPA seems to be toxic in vitro only at doses asso­ L-DOPA synthesis from tyrosine, 46 and thus potentially accelerate ciated with NM formation,36 which has been attributed to an DA production, it is disputed whether TYR is actually present in interference by NM with intracellular neurotrophin signalling, the human brain and, if so, it is in small quantity and with unclear and not to an acute toxic effect of L-DOPA per se.45 Consistent function.50–54 In TYR animals, the overall levels of cytosolic DA are with a macromolecular crowding effect linked to progressive NM not expected to remain significantly increased due to the feedback accumulation, in-depth ultrastructural analyses have revealed inhibition of cytosolic DA on TH22,55 and the transient condition of that Lewy pathology in NM-containing neurons from PD brains cytosolic DA, which is either rapidly metabolized or oxidized. In mostly consists of a crowded environment of vesicular structures fact, in these animals TYR promotes the continuous oxidation of 1050 | BRAIN 2023: 146; 1040–1052 M. Gonzalez-Sepulveda et al. cytosolic DA into NM. In addition, the progressive accumulation of by concomitantly injecting AAV-VMAT2 and AAV-TYR, the produc­ NM was associated to a marked downregulation of VMAT2 and TH tion of NM was slowed down from its initial synthesis, thus being that may contribute to the decreased DA vesicular uptake and ves­ able to prevent or delay disease onset. It remains to be determined icular DA levels56 observed in these animals. Furthermore, we have whether in a PD patient in which intracellular NM has already previously reported that striatal DAT levels are decreased by ∼70% reached pathological levels, VMAT2 would be able to halt or delay in AAV-TYR-injected animals by 2 months post-AAV-TYR injec­ disease progression. Finally, while we have shown a beneficial ef­ tions,5 which concur with neuroimaging in vivo analyses in PD pa­ fect of VMAT2 overexpression up to 6 months post-AAV injection, tients exhibiting reductions in DAT striatal density at early stages we do not know how much longer this beneficial effect would po­ of the disease.57 In this context, it is important to recall that DAT tentially last. Indeed, VMAT2 overexpression may slow down but is also expressed in astrocytes and macrophages, which contribute not completely halt NM production, so NM could continue to stead­ to DA clearance from the synaptic cleft.48,49 The increased number ily accumulate with age despite VMAT2 overexpression, although of CD68- and GFAP-positive cells in AAV-TYR-injected animals at a slower rate. To overcome this limitation, VMAT2 overexpres­ could thus favor an increased DA uptake and metabolism in glial sion could be potentially combined with strategies aimed at elimin­ cells, which is consistent with increased 3MT levels observed in ating intracellular NM once it has already been produced, such as the ventral midbrain of AAV-TYR-injected animals at 2 months by overexpression of autophagy master regulator TFEB, which we post-AAV injection (Supplementary Table 6) and that could restrict have recently shown to promote the clearance of NM-filled autop­ the amount of DA available to be re-uptaken by neuronal DAT. All hagic structures.62 these changes in DA metabolism, combined with the progressive buildup of NM within the neuronal cytosol, may potentially contrib­ ute to the vulnerability of the dopaminergic system in TYR animals Acknowledgements and in PD patients. We are grateful to Dr Iria Carballo-Carbajal for helping prepare the In addition to attenuate nigrostriatal neurodegeneration, AAV-VMAT2, to Professor Kazumasa Wakamatsu and Professor VMAT2 overexpression also mitigated the phenotypic loss of TH ex­ Shosuke Ito (Fujita Health University, Aichi, Japan) for providing pression that occurs within NM-laden neurons at early stages of de­ both SCD and 5SCDA standards and to the U20/FVPR unit of generation. Indeed, despite the general notion that the number of ICTS-NANBIOSIS at the VHIR (Barcelona, Spain) for assistance in DA neurons are reduced in PD, NM-containing neurons from both histological processing. PD patients and parkinsonian NM-producing rats exhibit an early phenotypic loss of TH expression, associated to impaired DA re­ lease and early motor deficits, prior to degeneration.5,58 In agree­ Funding ment with this, genetic disruption of mitochondrial complex I in mice has been recently reported to induce progressive parkinson­ This research was funded in whole or in part by Aligning Science ism in which TH downregulation and defective DA release in both Across Parkinson’s [ASAP-020505] through the Michael J. Fox striatal terminals and SNpc neuronal cell bodies precede neurode­ Foundation for Parkinson’s Research (MJFF). For the purpose of generation.59 The occurrence of neuronal dysfunction before overt open access, the author has applied a CC-BY 4.0 public copyright li­ cell death and its restoration by VMAT2 may have important thera­ cense to all Author Accepted Manuscripts arising from this submis­ peutic implications, as it provides a therapeutic window in which sion. Additional funding sources: La Caixa Foundation, Spain neuronal function can be restored before the actual loss of the (INPhINIT fellowship, code LCF/BQ/DI18/1166063 to J.C.; Junior cell. The preservation of neuronal function by VMAT2 overexpres­ Leader Fellowship LCF/BQ/PR19/11700005 to A.L.; Health Research sion was not limited to a protection of nigrostriatal DA fibres and Grant, ID 100010434 under the agreement LCF/PR/HR17/52150003 a maintenance of TH protein expression but also to a direct effect to M.V.). The Michael J. Fox Foundation for Parkinson’s Research, at maintaining DA homeostasis/metabolism, all of which resulted USA (MJFF-007184 and MJFF-001059 to M.V.). Ministry of Science in an early and long-lasting preservation of the motor function in and Innovation (MICINN), Spain (PID2020-116339RB-I00 to M.V.). NM-producing parkinsonian rats. Importantly, overexpression of EU Joint Programme Neurodegenerative Disease Research (JPND), VMAT2 by itself did not alter the normal motor function in non- Instituto de Salud Carlos III, EU/Spain (AC20/00121 to M.V.). parkinsonian (control) animals. This is in agreement with previous studies in VMAT2-overexpressing transgenic mice.60 Indeed, while VMAT2 overexpression has been reported to increase DA quantal Competing interests size and DA release,61 which could theoretically result in behav­ The authors report no competing interests. ioural changes, VMAT2 overexpression in transgenic mice did not result in any dramatic behavioural phenotype, as measured by mo­ tor, sensory, affective, appetitive, or social assays.60 From a transla­ Supplementary material tional point of view, these observations support the feasibility and safety of increasing VMAT2 function as a potential therapeutic Supplementary material is available at Brain online. strategy in humans. 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