Quinolinic acid induces alterations in neuronal subcellular compartments, blocks autophagy flux and activates necroptosis and apoptosis in rat striatum.

doi:10.21203/rs.3.rs-1443129/v1

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Quinolinic acid induces alterations in neuronal subcellular compartments, blocks autophagy flux and activates necroptosis and apoptosis in rat striatum.

https://doi.org/10.21203/rs.3.rs-1443129/v1

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Quinolinic acid (QUIN) is an agonist of N-methyl-D-aspartate receptor (NMDAr) used to study excitotoxicity in animal models. Striatal QUIN administration increases intracellular Ca²⁺ and oxidative stress, induces cellular damage and activates different mechanisms of cellular death. There is evidence indicating that impairment in autophagy at early times contributes to cellular damage in excitotoxicity; however, the status of autophagy in QUIN model at 7 days remain unexplored. In this study, the ultrastructural analysis of subcellular compartments and the status of autophagy in the striatum of rats administered with QUIN (120 and 240 nmol) was performed at 7 days. QUIN induced motor behavior impairment, tissue neurodegeneration and cellular damage; moreover, promoted swollen mitochondrial crests, spherical-like morphology and mitochondrial fragmentation. QUIN decreased ribosomal density in the rough endoplasmic reticulum and myelin sheaths discontinuity in axons with separation of the compact lamellae. Furthermore, QUIN induced increase and decrease in ULK1 and p-70-S6K phosphorylation, respectively, suggesting autophagy activation; however, the increased microtubule-associated protein 1A/1B-light chain 3-II (LC3-II) and sequestosome-1/p62 (SQSTM1/p62), and decreased in Beclin 1 and mature cathepsin D also indicates a blockage in autophagy flux. Additionally, QUIN administration increased tumor necrosis factor alpha (TNFα) and receptor-interacting protein kinase 3 (RIPK3) as well as decreased B-cell lymphoma 2 (Bcl-2) and increased in Bcl-2-associated X protein (Bax) levels and c-Jun N-terminal kinase (JNK) phosphorylation, suggesting an activation of necroptosis and apoptosis, respectively. These results indicate that QUIN caused subcellular compartments alteration, a blockage autophagy flux and activated necroptosis and apoptosis at 7 days.

Quinolinic acid

excitotoxicity

autophagy impairment

necroptosis

apoptosis

Excitotoxicity is a common event in the physiopathology of a variety of neurological disorders like Alzheimer, Parkinson and Huntington diseases [1, 2]. This event involves overactivation of N-methyl-D aspartate (NMDA) receptors by agonists such as glutamate, NMDA and quinolinic acid (QUIN), increasing intracellular flux of Ca²⁺ from extracellular space, resulting in an imbalance in Ca²⁺ homeostasis. This imbalance triggers energetic depletion, activates different enzymes involved in reactive oxygen species (ROS) production, inducing an oxidative stress state in the cells, and activates proteases, lipases and endonucleases, leading to damage and cellular death [1, 3]. Moreover, there is increasing evidence supporting an alteration in autophagy pathway induced by excitotoxicity, which is associated with brain tissue damage [4, 5]; however, there is a great discrepancy about how this impairment causes tissue damage. The evidence supports an association of brain tissue damage and overactivation of autophagy [6], but also supports that a decrease in autophagy leads to brain tissue damage [5].

Macroautophagy is a degradation pathway of cellular components, where long-lived proteins and organelles are sequestered through the formation of a double-membrane (autophagosome) and then fused with lysosomes, forming an autolysosome, where the material is degraded [7].

The macroautophagy, hereafter autophagy, is a complex pathway that initiates with the formation of phagophore, induced by the activation of Unc51-like autophagy activating kinase 1 (ULK1) protein kinase complex, constituted by ULK1/2, autophagy related protein 13 (ATG13), focal adhesion kinase family interacting protein of 200 kDa (FIP200) and ATG101 proteins, and its translocation to the initiation sites, such as endoplasmic reticulum membrane [8]. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) phosphorylates ULK1 protein in Ser³¹⁷, activating the ULK1 complex whereas mammalian target of rapamycin complex 1 (mTORC1) kinase phosphorylates to ULK1 in Ser⁷⁵⁷ inhibiting the ULK1 kinase complex [9]. The activated ULK1 complex recruits the class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) lipid kinase complex, integrated by vacuolar protein sorting 34 (VPS34), VPS15, Beclin 1, autophagy related protein 14-like (ATG14L), and nuclear receptor binding factor 2 (NRBF2) proteins, and phosphorylates the Ser¹⁵ on Beclin 1 and Ser²⁴⁹ on VPS34, activating the PI3KC3 complex, promoting the formation of phosphatidylinositol 3 phosphate (PtdIns3P), the recruitment of some proteins to the phagophore membrane [10] and the maturation of phagophore to autophagosome. Additionally, the sequestosome-1 SQSTM1/p62 (p62) protein, an autophagy receptor, binds to ubiquitinylated proteins and organelles that will be degraded by autophagy, and directs to the autophagosome through its interaction with microtubule-associated protein 1A/1B-light chain 3-II (LC3-II) protein, recruiting the material into the autophagosome [reviewed in 11]. Autophagosome binds to the lysosomes, which contain degradative proteases such as cathepsin D, an aspartic lysosomal protease, promoting protein degradation in autolysosomes [12].

Additional to the impairment in autophagy pathway due to excitotoxic process, there is evidence supporting the activation of necroptosis as a consequence of excitotoxicity, inducing cellular damage and neuronal death [13, 14]. Necroptosis is a regulated cellular death variant of necrosis that is activated by specific death receptors such as tumor necrosis factor receptor 1 (TNFR1), inducing assemble of necrosome, composed by proteins such as receptor-interacting protein kinase 1 (RIPK1) and receptor interacting protein kinase 3 (RIPK3). The binding of TNFR1 to tumor necrosis factor alpha (TNF-α), its ligand, promotes the auto-phosphorylation of RIPK1 that phosphorylates to RIPK3, which induces mixed lineage kinase domain-like protein (MLKL) phosphorylation. Phosphorylation of MLKL translocates it to cellular membrane forming a pore, inducing cell lysis and morphological characteristics of necrotic cell death. Necroptosis is observed in different neurological disorders like Alzheimer, Parkinson and cerebral ischemia [13, 15]. Furthermore, there is evidence indicating that necroptosis participates in axonal degeneration [13], neuronal death in excitotoxic models [14] and impairment in autophagy pathway, due to the translocation of MLKL to autolysosomal membranes, decreasing degradation [16].

QUIN is an active metabolite produced in the kynurenine pathway, the major route of tryptophan catabolism [17]. Under physiological conditions, QUIN is produced as an intermediate metabolite during the nicotinamide adenine dinucleotide (NAD⁺) synthesis; however, as part of the neuroinflammatory processes, it is produced and released by infiltrating macrophages and activated microglia [18]. The extracellular presence of QUIN leads to an excessive activation of NMDA receptor inducing excitotoxicity [19], and an inhibition of glutamate uptake by astrocytes [20], extending the excitotoxic damage [21]. Striatal and intracerebroventricular administration of QUIN have been used as model for the study of the underlying mechanism of excitotoxicity in the striatum and hippocampus [22, 23].

There is evidence indicating than QUIN administration activates autophagy at early times (12 h after its administration) in vivo [24]. In contrast, when QUIN was incubated by 24 h in vitro, in primary neuron and astrocyte culture, the expression of Beclin 1 decreases and that of cathepsin D increases [12], suggesting inhibition [5] and activation [25] of autophagy at the same time. However, at 7 days, the role of QUIN on autophagy and the contribution of impaired autophagy to brain tissue damage remains unexplored. Additionally, to the impairment of autophagy due to QUIN administration at early times, there is evidence indicating that QUIN induces other types of cellular death such as apoptosis and necrosis. Morphological analyses in vivo suggest that QUIN induces a rapid cytoplasmic disintegration, characteristic of death by necrosis in the lesion core [26], but apoptotic cells are observed in areas surrounding the lesion core [27]. However, other types of cellular death such as necroptosis in brain tissue, after QUIN administration are still poorly studied. In this study we explore the effect of QUIN on morphological alterations in subcellular compartments, on the autophagy flux and necroptosis and apoptosis activation at 7 days in the rat striatum to determine if these pathways are involved in a secondary damage generating by QUIN administration.

Animals

Forty-five male Wistar rats (280-320 g) from the bioterium of Faculty of Medicine of the National Autonomous University of Mexico were kept under controlled conditions of temperature (25±3°C), humidity (50±10%), and lighting (12 h light/dark cycles). Animals had free access to standard commercial rat chow diet (Laboratory rodent diet 5001; PMI Feeds Inc., Richmond, IN, USA) and water ad libitum.

All procedures with animals were carried out strictly according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and to the Norma Oficial Mexicana NOM-062-ZOO-1999. The experimental procedures were approved by the Institutional and Local Committee for the Care and Use of Laboratory Animals on the Ethical Use of Animals from Instituto Nacional de Neurología y Neurocirugía Manuel Velasco Suárez, INNN project 44/15. During the experiments, all efforts were made to minimize animal suffering.

Reagents

QUIN, apomorphine, paraformaldehyde (PAF), glutaraldehyde, osmium tetroxide, sodium cacodylate, lead citrate, uranyl acetate, acrylamide, bis-acrylamide, ammonium persulfate, glycine, sodium dodecyl sulfate, phenylmethylsulfonyl fluoride (PMSF), pepstatin A, aprotinin, leupeptin, Triton® X-100, Folin & Ciocalteu’s reagent, bovine serum albumin (BSA), rabbit anti-p62 antibody (P0067) and mouse anti-α-tubulin antibody (T9026) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rabbit anti-p62 (ab101266), rabbit anti-RIP3 (RIPK3, ab56164) and rabbit anti-RIP3 (RIPK3, ab62344) antibodies were obtained from Abcam (Cambridge, MA, USA). Mouse anti-MAP LC3β (LC3-I/LC3-II, G-9; sc-376404), mouse anti-TNFα (52B83; sc-52746), mouse anti-RIP (RIPK1, C-12; sc-133102), mouse anti-BECN1 (Beclin 1, E-8; sc-48341) and mouse anti-cathepsin D (C-5; sc-377124) antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Rabbit anti-p-ULK1 Ser317 (12753), rabbit anti-ULK1 (8359), mouse anti-p-p70-S6K Thr389 (9206), rabbit anti-p70-S6K (9202), rabbit anti-p-JNK Thr183/Tyr185 (4671) and rabbit anti-JNK (9252) were obtained from Cell Signaling Technology (Danvers, MA, USA). Rabbit anti-Bax (GTX61026) was from Gene Tex (Irvine, CA, USA). Mouse anti-B-cell lymphoma 2 (Bcl-2, 14-6992) was from eBioscience Inc. (San Diego, CA, USA). Donkey-anti mouse IgG horseradish peroxidase-conjugate (715-035-150) and donkey-anti rabbit IgG horseradish peroxidase-conjugate (711-035-152) antibodies were from Jackson ImmunoResearch Laboratories Inc. (Jennersville, PA, USA). Fluoro-Jade B, polyvinylidene fluoride (PVDF) membrane, Immobilon Western kit and DPX mounting medium were obtained from Millipore (Bedford, MA, USA) and Universal L Kit SAB-System horseradish peroxidase (HRP) was from DAKO (Carpinteria, CA, USA). All the others reagents were obtained from known commercial sources.

Experimental design

Animals were randomly divided into three groups (n = 3-4) as follows: 1) SHAM group; 2) 120 nmol of QUIN (QUIN120); and 3) 240 nmol of QUIN (QUIN240). Animals from SHAM group received a unilateral striatal injection of isotonic saline solution (ISS) whereas the animals from QUIN120 and QUIN240 groups received a unilateral striatal injection of QUIN equivalent to 120 and 240 nmol, respectively. All animals were sacrificed at 7 days after ISS or QUIN administration and samples (striatum or perfused whole brain) were collected.

Four independent groups of animals were used for the evaluation of: 1) rotation behavior by apomorphine administration, neurodegeneration by Fluoro-Jade B staining and cellular and histological damage by hematoxylin and eosin (H&E) staining; 2) subcellular compartment alterations by transmission electron microscopy (TEM); 3) protein localization by immunohistochemistry; and 4) protein levels by Western blot (Fig 1).

Striatal administration

Rats were deeply anesthetized with sodium pentobarbital (40 mg/kg, i.p.), placed in a stereotaxic equipment (Stoelting Co., Woo Dale, IL, USA) and administered in the right striatum with 1 μL of ISS or QUIN (equivalent to 120 or 240 nmol) using a 10 μL Hamilton microsyringe (Hamilton Co., Reno, NV, USA). The administration was carried out according the following stereotaxic coordinates: +0.5 mm anterior to bregma, -2.6 mm lateral to bregma and -4.5 mm ventral to dura [28].

Rotation behavior

Six days after ISS or QUIN injection, animals were administered with apomorphine (1 mg/kg, s.c.) and placed in individual acrylic box cages. Five min later, the number of ipsilateral rotations was counted for 1 h. One rotation was defined as a complete 360º turn. Data were expressed as the total number of turns in 1 h.

Samples for histological analysis

Seven days after ISS or QUIN injection, animals were euthanized with sodium pentobarbital (100 mg/kg, i.p.) and perfused transcardially with ISS plus heparin (5 U/mL) followed by 4% PFA in 0.2 M phosphate buffer (pH 7.4). Brains were removed and post-fixed in 4% PFA for 48 h and subsequently dehydrated in ethanol and xylene solutions and embedded in Paraplast plus paraffin (McCormick Scientific, St Louis, MO, USA). Coronal sections of 5 µm were obtained in a HM 325 rotatory microtome (Thermo Fisher Scientific Inc., Waltham, MA, USA) every 100 µm, covering a total distance of 300 µm. These sections were used in Fluoro-Jade B, H&E and immunohistochemistry assays.

Fluoro-Jade B staining

Neurodegeneration was evaluated using Fluoro-Jade B staining. Sections were deparaffinized in xylene for 15 min and placed in absolute ethanol for 2 min, followed by 1% NaOH-80% ethanol solution for 5 min, 70% ethanol solution for 2 min and rinsed with distilled water for 2 min. Sections were immersed in 0.06% KMNO₄ solution for 10 min and rinsed with distilled water for 2 min. Finally, samples were stained with 0.0004% Fluoro-Jade B for 20 min, washed with distilled water, dried at 50ºC for 15 min, immersed in xylene and mounted with DPX mounting medium. Five fields in the striatum localized in areas surrounding the lesion core of the injection were counted with a Leica microscope (Cambridge, UK) using a 40X objective. Results were expressed as the number of positive cells to Fluoro-Jade B per field.

H&E staining

Cellular and histological damage was evaluated using H&E staining. Sections were deparaffinized in xylene and hydrated in solutions of decreasing ethanol concentration and distilled water. Sections were stained with hematoxylin for 1 min, then rinsed with distilled water and stained with eosin for 1 min. Finally, samples were dehydrated in solutions of increasing ethanol concentration and xylene, and mounted with DPX mounting medium. Five fields in the striatum localized in areas surrounding the lesion core of the injection were counted with a Leica microscope (Cambridge, UK) using a 40X objective. Results were expressed as the percent of damaged cells per field.

Immunohistochemistry

Histological localization of p62, MAP LC3 β (LC3), and RIPK3 was evaluated by immunohistochemistry. Sections were deparaffinized in xylene and hydrated in solutions of decreasing ethanol concentration and distilled water. Sections were permeabilized with phosphate buffered saline (PBS) plus 0.2% Triton® X-100 for 1 h, boiled in 10 mM sodium citrate buffer (pH 6.0) plus 0.2% Triton® X-100 for 1 h and cooled at room temperature for 1 h. The peroxidase endogenous activity was inactivated with 1% hydrogen peroxide (H₂O₂) for 15 min. Sections were blocked with 2.5% BSA for 1 h at room temperature and then were incubated with rabbit anti-p62 (1:500; ab101266) antibody overnight and mouse anti-MAP LC3β (1:25) and rabbit anti-RIP3 (1:200; ab62344) antibodies during 48 h at room temperature. Sections were incubated with Universal L Kit SAB-System HRP according to the manufacturer’s instructions. Finally, sections were incubated with 3,3’-diaminobenzidine, counterstained with hematoxylin and covered with DPX mounting medium. Sections in the striatum area were visualized in a Nikon E 200 microscope (Nikon, Melville, NY, USA) using 40X objective. Results were expressed as representative micrographs of each experimental group.

TEM

Subcellular compartment alterations were evaluated by TEM. Seven days after QUIN injection, rats (n=3 per group) were deeply anesthetized with pentobarbital (100 mg/kg, i.p.), and transcardially perfused with cool ISS followed by 4% PFA and 1.5% glutaraldehyde in Sörensen buffer (46 mM NaH₂PO₄, 154 mM Na₂HPO₄, 154 mM NaCl, pH 7.2). Brains were removed, the areas of interest dissected and fragmented, post-fixed in the same solution for 24 h, immersed in 1% osmium tetroxide in 0.1 M cacodylate buffer (pH = 7.2) and subsequently embedded in epoxy resin. Ultrathin sections (90 nm) of the striatal regions were obtained, contrasted with 3% lead citrate Reynolds and 1% uranyl acetate, and examined with a transmission electron microscope (FEI-Tecnai BioTWIN, Hillsboro, OR, USA). The ultrastructural analysis was carried out in neurons localized in the areas surrounding the lesion core of the injection.

Western blot

Protein levels of p-ULK1, ULK1, p-p70-S6K, p70-S6K, Beclin 1, preprocathepsin D, procathepsin D, cathepsin D, MAP LC3 β (LC3-I/LC3-II), p62, Bcl2, Bax, p-JNK, JNK, TNFα, RIPK1, RIPK3 were evaluated by Western blot. Seven days after ISS or QUIN administration, the right striatum of animals was collected and homogenized in 500 µL of cold lysis buffer (10 mM Tris HCl (pH 7.6), 15 mM NaCl, 0.25 mM sucrose, 1 µg/mL aprotinin, 1 µg/mL leupeptin, 1 µg/mL pepstatin A, 1 mM PMSF and 1% Triton® X-100) and centrifuged at 10,621 x g for 20 min at 4ºC. The supernatant was collected and the protein concentration was measured by Lowry method [29]. Fifty micrograms of supernatant were loaded in 10, 12 or 15% of polyacrylamide gel and proteins were separated and transferred to PVDF membranes. Membranes were blocked with 5% nonfat milk and incubated with primary mouse antibody anti-TNFα (1:500), mouse anti-RIP (1:500), rabbit anti-RIP3 (1:500; ab56164), mouse anti-BECN1 (1:500), mouse anti-cathepsin D (1:500), mouse anti-MAP LC3β (1:500), rabbit anti-p62 (1:1000; P0067), mouse anti-Bcl-2 (1:600), rabbit anti-Bax (1:600), rabbit anti-p-ULK1 (1:1000), rabbit anti-ULK1 (1:1000), mouse anti-p-p70-S6K (1:1000), rabbit anti-p70-S6K (1:1000), rabbit anti-p-JNK (1:1000), rabbit anti-JNK (1:1000) and mouse anti-⍺-tubulin (1:10000) overnight followed by the incubation of the secondary donkey antibody anti-mouse IgG horseradish peroxidase-conjugated (1:10000) or donkey antibody anti-rabbit IgG horseradish peroxidase-conjugated (1:10000). Membranes were revealed using an Immobilon Western kit (Millipore Co, Billerica, MA, USA) and images were obtained with a photodocumenter (Vilber Lourmat, Eberhardzell, Deutschland). Images were analyzed using the ImageJ software (ImageJ v1.52a, NIH, Bethesda, MD, USA) and the data were expressed as relative optical density (OD) of the protein/⍺-tubulin.

Statistical analysis

All data are presented as the mean±SEM. Data were analyzed by one-way ANOVA and post hoc Tukey’s test except for results of apoptosis that were analyzed by t-Student test using Prism 5.0 software (GraphPad, San Diego, CA, USA). Values of P<0.05 were considered to be statistically significant.

QUIN induces rotation behavior impairment and brain tissue damage

QUIN is an endotoxin that induces excitotoxicity and increases oxidative stress, leading to tissue and cellular damage. QUIN120 tends to increase the number of ipsilateral turns per hour at day 6, however only QUIN240 increases significantly the number of ipsilateral turns compared to SHAM group (Fig. 2).

The impairment in rotation behavior in QUIN groups could be related with an increase in the neurodegeneration and tissue and cellular damage induced by QUIN at day 7. To explore this, we evaluated neurodegeneration and histological damage. SHAM group shows a mean of 4.06 positive cells per field to Fluoro-Jade B (Fig. 3a upper panel and 3b) and 23.23% of damaged cells (Fig. 3a, lower panel and 3c), that could be associated with the administration of ISS or the mechanical damage induced by the syringe during the striatal administration.

QUIN increases the number of cells in neurodegeneration (positive cells to Fluoro-Jade B) in a dose-response way with respect to SHAM group (Fig. 3a, upper panel). The number of positive cells to Fluoro-Jade B in QUIN120 and QUIN240 groups were 45.68 and 57.17, respectively (Fig. 3b). Besides, QUIN increases the percent of damaged cells per field in a dose-response way at 7 days (Fig. 3a, lower panel) to 68.84% and 86.56% in QUIN120 and QUIN240 groups, respectively (Fig. 3c). Furthermore, interstitial edema and the loss of neuropil integrity can be observed with the dose of 120 of QUIN, but is more evident in the QUIN240 group (Fig. 3a, lower panel).

QUIN induces damage in mitochondria and rough endoplasmic reticulum (RER)

We examined the effect of QUIN (120 and 240 nmol) on cellular organelles at 7 days post-lesion. Neurons from the SHAM group show well-preserved mitochondria, with intact crests (Fig. 4a and 4b). Nonetheless, in neurons of animals from the QUIN groups, the organelles are disorganized in the cytoplasm. QUIN120 produces neuronal swelling and dilatation of mitochondrial cristae compared to SHAM group (Fig. 4c and Fig. 4d). Moreover, QUIN240 increases the subcellular abnormalities compared with QUIN120 group; small and spherical-like morphology of mitochondria with extensive cristae dilatation are observed (Fig. 4e and 4f). Likewise, QUIN240 induces complete effacement of cristae and absence of intermembranal space of mitochondria with matrix effacement (Fig. 4f).

Otherwise, in the perinuclear cytoplasm of SHAM group, abundant well-developed RER and free ribosomes are observed (Fig. 5a and 5b), a characteristic consistent with its high protein synthesis activity. However, QUIN administration induces morphological alterations of RER in striatal neurons such as fragmentation and thinning of RER membrane, as well as a decrease of ribosomes (Fig. 5c-5f). Rats of QUIN120 group show fewer free ribosomes and lesser ribosomes in RER, as well as fragmented RER (Fig. 5c and 5d), and QUIN240 induces more accentuated subcellular abnormalities such as fragmented and thinner RER cisternae with scarce attached ribosomes (Fig. 5e and 5f).

QUIN induces axonal damage

We found that QUIN induces cellular damage as well as mitochondrial and RER damage, for that reason we evaluated the effect of QUIN on the axons of striatal neurons. In the SHAM group we observed intact axons with well-preserved myelin sheaths and axoplams some normal mitochondria and numerous microtubules (Fig. 6a, 6b and 6g).

QUIN induces alteration in the axon morphology and components, such as myelin layer discontinuity, separation of the compact myelin sheaths, and abundant fragmentation with disappearance of neurofilaments and microtubules. Rats of QUIN120 group show axon dilatation, neurofilaments disappearance, separation of myelin layer and disruption, producing vacuoles and total disruption (Fig. 6c and 6d) and QUIN240 produces even more axonal damage, many axons show thinner myelin layer and extensive disruption and fragmentation. Myelin layer exhibits extensive vacuolization and disruption (Fig. 6e and 6f).

QUIN blocks autophagy flux

There is evidence indicating that excitotoxicity impairs autophagy pathway and since we observed that QUIN induced mitochondrial damage, which could be associated to impairment in autophagy, we explore the status of autophagy flux. First, we performed an immunohistochemical analysis of p62 and LC3. In SHAM group, the detection of p62 and LC3 are low in striatal cells (Fig. 7). QUIN120 increases the cytoplasmic immunostaining of p62 and LC3 compared to SHAM group, Additionally, QUIN240 increased even more the staining of p62 and LC3. Interestingly, the positive labeling to p62 was commonly observed in the nucleus of neuronal cells in the three groups, particularly in the QUIN240 group. In both groups, QUIN120 and QUIN240, the immunostaining was more frequently in shrink neurons with pyknotic nuclei, and numerous swollen cells also showed strong immunostaining (Fig. 7).

The increase in p62 and LC3 levels could be an indicative in autophagy impairment, for that reason, we evaluated the autophagy pathway at different levels by western blot. Phosphorylation state of ULK1 in Ser³¹⁷ and p-70-S6K in Thr³⁸⁹ was measured as indicative of activation of autophagy pathway. An increase in ULK1 phosphorylation and a decrease in p70-S6K phosphorylation levels in QUIN120 and QUIN240 groups was observed compared to SHAM group, suggesting an increase in autophagy (Fig. 8a and 8b). Surprisingly, Beclin 1 levels decrease after QUIN administration (120 and 240 nmol) compared with SHAM group (Fig. 8c). Moreover, p62 levels and in LC3-II/LC3-I ratio increase only in QUIN240 group (Fig. 8d and 8e). Finally, the levels of procathepsin D were unchanged after QUIN treatment (Fig. 8f); however, a decrease in the maturation of cathepsin D was observed, since the levels of preprocathepsin D show a decrease in the QUIN240 group (Fig. 8g) and the levels of cathepsin D decrease with both doses of QUIN (Fig. 8h), suggesting a decrease in autophagy flux. These results suggest that QUIN240 (mainly), increases the activation of autophagy at early stages, but it promotes an autophagy flux blockage at late states on the pathway.

QUIN induces apoptosis

It has been reported, that the blockage in autophagy flux could be related to an increase in apoptosis or necroptosis [16, 30]. To explore the participation of apoptosis after QUIN treatment, we measured the levels of pro-apoptotic protein Bax, the anti-apoptotic protein Bcl-2, the Bax/Bcl-2 ratio and the activation (phosphorylation at Thr¹⁸³/Tyr¹⁸⁵) of JNK. QUIN240 decreases Bcl-2 levels and increases Bax levels, compared to SHAM group (Fig. 9a and 9b). Moreover, the Bax/Bcl-2 ratio (Fig. 9c) and the activation of JNK (Fig. 9d) increase in the QUIN240 group suggesting an activation of apoptosis pathway after 7 days. This was in agreement with the electron microscopy study that showed numerous nervous cells with condensed nucleus and almost preserved cytoplasm that correspond to apoptotic cells (Fig. 9e-g).

QUIN induces necroptosis

Also, in this work we explored the activation of necroptosis pathway as mechanism of tissue damage since alterations in axons and blockage in autophagy flux was observed after QUIN injection. The expression of RIPK3 protein, one of the principal indicators of necroptosis, was evaluated at 7 days. In SHAM group few cells showed slight immunostaining while QUIN treated group shower numerous positive cells (Fig. 10a). In QUIN120 group the main positive mark to RIPK3 is located in the cytoplasm and axons of shrink and swollen neurons. In QUIN240 group there are more RIPK3 immunostained in damaged nervous cells with cytoplasmic and nuclear immune-reactivity, endothelial cells also showed intense immunostaining (Fig. 10a).

The electron microscopy study confirms the extensive cellular damage and cell death, showing necrotic cells with pyknotic nucleus and shrinked cytoplasm or swollen neurons with chromatin disappearance and dilated cytoplasm with few and damaged organelles (Fig. 9f). The activation of the necroptotic pathway starts with the binding of TNFα to its cellular receptor, TNFR1, which promotes the phosphorylation of RIPK1, when caspase 8 is inhibited. This phosphorylation activates RIPK1 and induces the subsequent activation of RIPK3. To evaluate with more detail the necroptotic pathway, we measured the levels of TNFα, RIPK1 and RIPK3 by western blot, with the aim to support our finding in RIPK3 immunohistochemistry and determined whether necroptosis pathway could be participating in neuronal damage after QUIN administration. TNFα levels tend to increase in the QUIN120 group, but only QUIN240 increases TNFα levels compared to SHAM group (Fig. 10b). QUIN does not modify RIPK1 levels, nonetheless a tendency to increase is observed with QUIN240 (Fig. 10c). Finally, QUIN240 increases the levels of RIPK3 compared to SHAM group, while QUIN120 only show a tendency to increase RIPK3 levels (Fig. 10d).

Excitotoxicity is a common mechanism of cellular damage in neurological diseases and its study using molecules such as QUIN is an important tool for the description of different molecular events involved in cellular damage and death as well as in the search of potential therapeutic targets in these disorders. Excitotoxicity occurs when NMDA receptors are overactivated by agonist such as QUIN, inducing an increase in Ca²⁺ intracellular levels, promoting ROS production, and leading to cellular death [3].

In this work, QUIN caused impairment in rotation behavior of animals after apomorphine administration in a dose-response manner. Apomorphine is an agonist of dopamine receptors and its administration in animals with unilateral striatum lesion induces an ipsilateral rotation behavior, due to a decrease in dopamine receptor activity and the size of lesion is proportional to the speed of rotation [31]. The impairment in rotation behavior correlates with the extension of tissue damage, since QUIN increased neurodegeneration and cellular damage in a dose-response manner.

The cellular damage observed after QUIN administration (120 and 240 nmol) could be related with the increase in oxidative stress. Previous reports show that QUIN increases Ca²⁺ uptake from 30 min until 24 h after injection [32], generating an overproduction of hydroxyl radical and superoxide anion, at 50 min and 2 h, respectively [33, 34]. Also, the increase in ROS production induces lipoperoxidation at 2 h [35], decreases glutathione levels and increases the protein oxidation at 4 h after QUIN administration [36, 37]. Moreover, Colín-González et al. [37] reported that the inhibition of hemeoxygenase-1 exacerbates the damage induced by QUIN240 [37]. It has been reported that oxidative stress induced by QUIN causes damage in subcellular compartments such as mitochondria and RER in neurological diseases [38]. Here, QUIN induced in a dose-response way, dilatation of cristae and small and round mitochondrial morphology. These mitochondrial alterations could be a consequence of Ca²⁺ influx and/or by the overproduction of ROS, since mitochondria is involved in Ca²⁺ homeostasis and is the main source of ROS generation [39]. Also, as a consequence of the oxidative stress induced by QUIN, signaling pathways that promote cell death such as JNK pathway, could be activated at long times. Santana-Martínez et al. [40] reported an increase in p-JNK (active JNK) levels in mitochondria, which could be associated with apoptosis induction. Additionally, there are reports in vivo indicating that QUIN produces alterations in mitochondrial complexes [41], and mitochondrial dysfunction at 24 h [42] and 21 days post-lesion [41], which could be associated with the morphological alterations observed in mitochondria.

Additionally, in this work we observed alterations on RER morphology such as fragmentation and thinning membrane, as well as a decrease of ribosomes. The damaged observed in RER also follows a dose-response effect. Fernandes et al. [43] reported that QUIN decreases the sarco/ER Ca²⁺-ATPase (SERCA)-mediated Ca²⁺ uptake in microsomal fractions of rat striatum, which could be involved in the morphological alterations observed in RER with QUIN. Furthermore, damage in organelles and a decrease in ER were also observed in cells of rat liver in response to 60 nmol of QUIN [44].

QUIN also induced axonal damage in striatal neurons such as myelin sheaths discontinuity, separation of the compact myelin sheaths, and abundant fragmentation with disappearance of neurofilaments and microtubules. There is evidence indicating that QUIN is involved in demyelinating diseases, such as multiple sclerosis [45] and it is toxic to oligodendrocytes [46]. Moreover, the participation of excitotoxic processes has been observed in axonal damage. NMDA receptors in compact myelin of oligodendrocytes (part of oligodendrocyte that surrounds the axon cylinder of neurons) in optic nerve cells, induces accumulation of Ca²⁺ in myelin sheets during ischemia injury, leading to damage in the myelin sheath [47]. Additionally, the glutamate-induced excitotoxic process causes axonal degeneration through the activation of the necroptotic pathway [13].

There are reports indicating that excitotoxicity and oxidative stress promote disruption in autophagy flux and in consequence promote the accumulation of damaged organelles, increasing cell damage [5, 48]. Previously, the autophagy flux blockage was observed in excitotoxic models at early times in hippocampal neurons [4] and in the striatum of rats administered with glutamate [49]. The blockage in autophagy flux has been associated with the increase in cellular damage and cellular death [4]. In this work, we observe that QUIN240 increased the levels of p62 and LC3-II suggesting a blockage in autophagy flux, this proposal is supported by the reduction in mature cathepsin D and Beclin 1 levels, despite the increase observed in ULK1 phosphorylation and the decrease in p70-S6K phosphorylation.

The initiation of autophagy is regulated by the phosphorylation of ULK1 by mTORC1 and AMPK kinases. mTORC1 phosphorylates the Ser⁷⁵⁷ in ULK1 inhibiting the ULK1 complex, and decreasing the autophagy pathway; on the other hand, phosphorylation of Ser³¹⁷ in ULK1 by AMPK activates the ULK1 complex activating the autophagy pathway [9]. The increase in Ser³¹⁷ phosphorylation on ULK1 suggests an activation of autophagy; moreover, the decreases in Thr³⁸⁹ phosphorylation in p70-S6K, a target of mTORC1, support an activation of autophagy, since indicates a decrease in mTORC1 activity. These results suggest an increase in autophagy at early stages; however, we found a decrease in Beclin1 and accumulation of LC3-II and p62 proteins, suggesting an impairment of this pathway in later stages, possible at phagophore formation level or later step such as lysosomal dysfunction.

Beclin 1 is an important protein involved in the autophagy pathway, it is required in nucleation of phagophore, is part of the PI3KC3 complex and mediates the localization of some proteins important in phagophore formation [11]. Nguyen et al. [30] reported a decrease in Beclin 1 levels induced by its cleavage by calpain in an excitotoxic and oxidative stress dependent mechanisms, according with our results. Furthermore, Bieri et al. [50] reported a decrease in Beclin 1 by its cleavage induced by caspase 3 in patients with Alzheimer disease, hAAP transgenic mouse model and excitotoxic model-induced by kainic acid. The decrease of Beclin 1 inhibit the autophagy, but increase the cell damaged-induced by NMDA administration though excitotoxic process [5]. Excitotoxicity is involved in the cellular damage induced by QUIN [3, 32, 34] and the activation of calpain has been reported after excitotoxic event [51]. The decrease in Beclin 1 levels observed in this work could be associated with the activation of caspase 3, since we observed a decrease in Bcl-2 and an increase in Bax levels and Bax/Bcl-2 ratio, indicating an activation of apoptosis, which suggest the activation of caspase 3. Indeed, we observed many apoptotic cells in our ultrastructural study. Moreover, is important to note that the decrease in Beclin 1 was observed 5 days after kainic acid administration [50], similar to our model (7 days after QUIN injection).

Additionally, the blockage in autophagy flux by QUIN was evident by the accumulation of LC3-II and p62 levels, since these two proteins are degraded in lysosomes during autophagy. This blockage cold be associated with the decrease in mature cathepsin D levels, an aspartic lysosomal protease important in the maintaining of protein degradation by lysosomes or by the activation of necroptosis. Cathepsin D decreases after cerebral ischemia [25, 52] and this decrease promotes lysosomal dysfunction, inducing blockage of autophagy flux, without altering the autophagy initiation [25]; furthermore, there is evidence indicating that knockout mice to cathepsin D presents accumulation of autophagosomes in neurons from brain cortex [53]. Moreover, the decrease of cathepsin D, compared with the decline in other cathepsins involved in lysosomal function such as cathepsin B and L, is the cause of autophagy dysfunction [25, 54] whereas the increase in cathepsin D levels in cerebral ischemia restore the autophagy flux and lead to neuroprotection against the damage in cerebral ischemia [25].

The decline in mature cathepsin D could be related to the decrease in its maturation process or increase in mature cathepsin D degradation, instead of impairment in its synthesis. We observed that QUIN240 does not alter the pre-cathepsin D levels, while decrease the maturation process, suggesting that the impairment in its synthesis does not occurs. According with our results, Hossain et al. [25] reported that decrease in cathepsin D levels after cerebral ischemia is not associated with decline in its synthesis.

The deficiency of cathepsin D is also associated with an increase in apoptosis in retinal photoreceptor cells [54]. Correlating with this report, we observed an increase in Bax levels and Bax/Bcl-2 ratio and decrease in Bcl-2 levels, as well as an increase in JNK phosphorylation suggesting an active apoptosis pathway, which could be associated with the decrease in mature cathepsin D as mentioned above. Moreover, evidence support that autophagy disruption promotes apoptosis activation [48].

Another possibility involved in blockage of autophagy flux is the lysosomal dysfunction promoted by activation of necroptosis pathway at early times. QUIN induces necrosis and apoptosis death in striatum [26, 27], however there is increasing evidence suggesting the participation of other types of cellular death such as necroptosis, since this pathway is observed in excitotoxic models [13, 14]. Necroptosis initiates with the signaling of TNFα to its receptor, inducing the activation of RIPK1 that activates RIPK3, and in consequence activates its late effector MLKL [55]. The necroptosis pathway is implied in brain damage after cerebral ischemia [55, 56], subarachnoid hemorrhage [57] and contributes to axonal degeneration in excitotoxic models [13]. Its activation occurs at early events in brain damage [57] followed by apoptosis induction [55], and the inhibition of necroptosis decrease cerebral damage [56, 58]. Despite the fact that necroptosis is an event that occurs early in brain damage with a major expression of RIPK3 between 24 to 48 h [ 55, 57], we observed an increase in RIPK3 and TNFα levels at 7 days after QUIN240, suggesting that necroptosis remain active at this time. The levels of RIPK1 did not change compared to SHAM group, however a tendency to increase was observed in QUIN240 group, suggesting that at this time the activation of necroptosis decline. As mentioned above, the activation of necroptosis could be involved in the autophagy disruption observed in QUIN240 group, through to lysosomal dysfunction. Phosphorylated MLKL disrupt autolysosomal membrane, decreasing intralumninal lysosomal acidity, promoting autolysosomal dysfunction, and blocking the autophagy flux [16]. For that reason, the blocking in autophagy flux after QUIN administration could be associated to necroptosis activation at early times.

We propose that QUIN induced excitotoxicity and oxidative stress promoting cellular and subcellular compartment damage, which could be exacerbate by the blockage in autophagy flux, though the activation of necroptosis and apoptosis. Moreover, the axonal damage could be related with the activation of necroptosis (Fig. 11).

QUIN induces rotation behavior, neurodegeneration, and morphological alterations in mitochondria, RER and axons of striatal neurons at 7 days post-lesion, possibly through the impairment in autophagy and increase in necroptosis and apoptosis at early times.

Funding

This work was supported by CONACYT [Grant No. A1-S-21433 to PDM].

Competing interest

The authors have no relevant financial or non-financial interest to disclose.

Author contribution

Conceptualization: Perla D. Maldonado, Carlos Alfredo Silva-Islas; Methodos: Carlos Alfredo Silva-Islas, Ricardo Alberto Santana-Martínez, Juan Carlos León-Contreras; Formal analysis and investigation: Carlos Alfredo Silva-Islas; Writing-original draft preparation: Carlos Alfredo Silva-Islas, Ricardo Alberto Santana-Martínez; Writing-review and editing: Carlos Alfredo Silva-Islas, Diana Barrera-Oviedo, Jose Pedraza-Chaverri, Rogelio Hernández-Pando, Perla D. Maldonado; Funding acquisition: Perla D. Maldonado; Resources: Perla D Maldonado; Supervision: Jose Pedraza-Chaverri, Rogelio Hernández-Pando, Perla D. Maldonado. All authors read and approved the final manuscript.

Data Availability

The datasets generated during and/or analysed during the current study are not publicly available due to we do not have a persistent link to datasets but are available from the corresponding author on reasonable request.

Ethics approval

Not applicable.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Acknowledgements

Not applicable.

Disclosure of potential conflicts of interest

The authors of this manuscript declare that there are no potential conflicts of interest.

Research involving Human Participants and/or Animals

This research only involves animals. All procedures with animals were carried out strictly according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and to the Norma Oficial Mexicana NOM-062-ZOO-1999. The experimental procedures were approved by the Institutional and Local Committee for the Care and Use of Laboratory Animals on the Ethical Use of Animals from Instituto Nacional de Neurología y Neurocirugía Manuel Velasco Suárez, INNN project 44/15. During the experiments, all efforts were made to minimize animal suffering.

Informed consent

Not applicable.

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Quinolinic acid induces alterations in neuronal subcellular compartments, blocks autophagy flux and activates necroptosis and apoptosis in rat striatum.

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QUIN induces rotation behavior impairment and brain tissue damage

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