Nose ‐ To ‐ Brain Delivery of Mitochondria for Treatment of Parkinson’s Disease Model Rats With 6-Hydroxydopamine

Feasibility of mitochondrial organelle transport via the olfactory bulb route for Parkinson's disease (PD) therapy was evaluated in the unilateral 6-OHDA-lesioned rats due to the distinct difference based on payload properties.


Background
The intranasal route of drug administration promises a new approach to treating brain/central nervous system (CNS) disorders, including Parkinson's disease (PD), Alzheimer disease, Huntington's disease, epilepsy, etc. [1] It accelerates drug development because therapeutic substances traditionally delivered via intravenous, intraperitoneal, and oral routes are hampered from crossing the blood-brain barrier (BBB) to reach targets in the CNS. Nasal spray, nose infusion, or nasal inhalation are possible routes for nasal administration of drugs. However, the e ciency of drug absorption must be considered, depending upon the target to be treated. For example, a portion of a therapeuric agent administered via a nasal spray or inhaler enters the pulmonary circulation, reducing delivery to the brain through olfactory or trigeminal nerves, a shortcut for nose-to-brain targeting [2]. The intranasal delivery route has advantages and disadvantages [3]. In addition to the rapid onset of action due to delivery of drugs to the CNS within minutes, e cacy of neurotherapeutics is enhanced and side effects are decreased due to direct delivery of drugs to the brain without systemic absorption. In contrast, drug properties (excluding conventional nasal medications), volume of solution, and molecular masses of drugs are restricted, and absorption via the nasal mucosa and brain-targeting pathways of drugs also vary depending upon pathological conditions of the individual and drug chemistry.
Mitochondria are vital, nano-sized organelles. Mitochondrial replacement therapy has been employed to treat mitochondrial diseases in clinical trials [4,5]. Thus, based on a similar treatment concept, mitochondrial transplantation has been extensively developed to cure diverse diseases in recent years [6].
Parkinson's disease (PD), a common neurodegenerative disease with no cure, is associated with mitochondrial dysfunction, damaging dopaminergic neurons in the substantia nigra (SN) of the midbrain [7], but mitochondrial transplantation raises the prospect of new treatments for PD. In situ or intravenous injection of naked mitochondria isolated from healthy cells has successfully restored mitochondrial function, with subsequent improvement of mobility in neurotoxin-induced rat models of PD [8,9], but the feasibility and e cacy of intranasal adminstration of mitochondria, a relatively fast, safe approach are unknown. However, studies have demonstrated that stem cells delivered intranasally entered the CNS [10] and have been used in treatment of PD [11] and malignant brain tumours [12]. Treatment e cacy for mitochondrial transplantation depends on several variables, including how many can be taken up by cells in a nite period, because mitochondria not internalized by cells gradually lose activity [13,14], and different delivery routes have different e ciencies of mitochondrial intake [13].
Mitochondrial transplantation via appropriate approaches may accelerate active, instead of passive uptake via interstitial pressure, osmosis, or endocytosis [13]. It is especially suitable for diseases involving chronic neuroin ammation or pathogenic disruption of cytoskeletal function, decreasing the probability of mitochondrial intake via actin-dependent endocytosis [15,16]. Pep-1, a member of the cellpenetrating peptide (CPP) family, has been approved to increase importation of foreign mitochondria by damaged cells, so as to restore mitochondrial function and to reduce oxidative stress without prior chemical modi cations for treatment of mitochondrial diseases such as Myoclonic Epilepsy Associated with Ragged-Red Fibers (MERRF) and mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke (MELAS)] [14,15,17,18] in vitro, or PD in vivo [8]. Unlike our previous study, which employed local injections of mitochondria for treatment of PD model rats lesioned with 6-hydroxydopamine, in the present study, we explored the feasibility of intranasal delivery of mitochondria for brain targeting and compared it to mitochondrial transplantation with or without Pep-1 pre-modi cation in same animal model. In support of direct nose-to-brain drug/cell delivery for more effective PD treatment [19,20], we propose that mitochondria may be a useful intranasal delivery system to allow non-invasive clinical treatment in practice.

Mitochondria conjugated with Pep-1
Detailed procedures for Pep-1 conjugation have been described previously [22]. 200 μg allogeneic mitochondria suspended in respiration buffer were conjugated with 0.11 mg Pep-1 diluted with sterile water (Anaspec, San Jose, CA, USA). Incubation for 10 min at room temperature was performed to ensure complex assembly.

Animals and intranasal administration of mitochondria
Adult female Sprague-Dawley rats (8 weeks old, weighing 250-300xg) were purchased from BioLASCO (Taipei, Taiwan) and were maintained under standard laboratory conditions with free access to food and tap water in the Laboratory Animal Center, Changhua Christian Hospital, Changhua, Taiwan. All experimental procedures of animals were approved by the Animal Experiments and Ethics Committee of Changhua Christian Hospital (approval number CCH-AE-105-018). A detailed procedure for creating a neurotoxic rat model of Parkinson's disease using a unilateral injection of 6-OHDA into the medial forebrain bundle (MFB) has been described previously [8]. Successful induction of PD rats after three weeks was validated by a rotational behavior test, described below. PD rats were assigned randomly to ve groups of six rats: 1) control group receiving no treatment (WT), 2) intranasal infusion of vehicle only (Sham), 3) mitochondria alone (Mito), 4) Pep-1-labelled mitochondria (P-Mito) and 5) Pep-1 alone (Pep-1) . Rats received a unilateral, intranasal infusion (ipsilateral side relative to the lesion) of 200 μg Mito or P-Mito in a total of 50 µL MiR05 respiration buffer, and treatment once a week for three months. Experiments were designed to minimize the number of animals used and their suffering.

Rotational behavior test
Motor imbalance of 6-OHDA-lesioned animals was assayed by methamphetamine (3 mg/kg, i.p.)-induced rotation. Rotational asymmetry was assessed using an automated rotometer system (AccuScan Instruments, Columbus, OH, USA) based on the design of Ungerstedt & Arbuthnott [23] to record the counterclockwise rotation with full-body turns. Rats that exhibited more than 360 rotations within 60 min were randomly assigned for experimental use. During the period of the experiment, the rotating test was reperformed for 3 or 4 weeks post-treatment.

Open eld test
To assess general motor behavior, an open eld test was employed 3 months after mitochondrial transplantation. Animals were placed in a 50 cm x 50 cm white plexiglass box and allowed an adaptation period of 30 min prior to being analyzed. Activity was recorded for two consecutive sessions, each lasting 15 min, using a ceiling-mounted video camera. Ethovision software (Noldus, Leesburg, VA, USA) was used to measure distance, velocity, total number of zone boundaries crossed, and duration of movement (seconds). Locomotion frequencies were assessed by dividing the oor of the box into four quadrats and by counting the number of quadrats entered. Entry was counted when a rat entered a new quadrat with all four paws. The apparatus was washed with 5% ethanol between tests to eliminate possible bias due to odors left by previous rats.

Histological and immunohistochemical staining
Rats that survived 3 months after transplantation were sacri ced with an overdose of chloral hydrate (800 mg/kg i.p.) and xed by intracardiac perfusion with 300 mL of saline followed by 300 mL of 4% paraformaldehyde (Sigma-Aldrich). Brains were removed, post xed in 4% paraformaldehyde for 4 h, and cryoprotected in 30% w/v sucrose in PBS for 20 h. Brains were then frozen and embedded in OCT medium (Tissue-Tek, Sakura Finetek, USA) and sectioned at 5-10-μm. For Nissl staining, sections mounted on glass slides were dried overnight. Slides were immersed in 0.025% cresyl violet (Sigma-Aldrich) in 90 mM acetic acid (Merck, Darmstadt, Germany) and 10 mM sodium acetate (Sigma-Aldrich) for 3 h, followed by dehydration in ascending ethanol and xylene series. Slides were then coverslipped with Histochoice mounting media (AMRESCO). For immunohistochemical staining (IHC), xed sections were subjected to heat-induced epitope retrieval in 10mM citrate buffer pH 6.0 for 25 min at 100°C . Sections stained with BrdU were additionally treated with 2 N HCl for 30 min at 37°C. After washing and blocking non-speci c sites with blocking buffer (5% BSA and 0.5% Tween-20 in PBS, pH7.4) for 30 min at room temperature, sections were incubated at 4°C overnight with a primary antibody, anti-Tyrosine

Statistical analysis
All analyses were performed in triplicate or quadruplicate in each group of experiments. Biochemical data are presented as means ± standard deviations, except results of the animal behavior test, presented as means ± standard errors of the means. There were six animals per group. The two mitochondrial treatments were evaluated using paired Student's t-tests and differences with p < 0.05 were considered statistically signi cant.

Improvement of rotational and locomotor behavior
Behavior of PD rats was assessed in terms of apomorphine-induced rotations (Fig. 1A) and performance in the open eld test (Fig. 1B). Rotation data for the WT group are not presented due to the extremely low frequency of apomorphine-induced turning in normal rats (Fig. 1A). The Sham group showed a dramatic increase of apomorphine-induced rotational activity relative to the inactivity of the WT group (Fig. 1). Unilateral intranasal infusion (ipsilateral to the lesioned side) of Mito or P-Mito caused a signi cant decrease in rotational activity 11 weeks post-treatment (Fig. 1A) and recovery of normal locomotor activity at 12 weeks, as measured by mean velocity, mobility duration, frequency of crossed zones, and traveling distance, compared to the Sham group ( Figure 1B). There was no signi cant difference in behavior between the Sham and Pep-1 groups, or between the Mito and P-Mito groups (Fig. 1).

Support of nigral dopaminergic neurons against lesion-induced cell death in SN and ST
Neuron survival in the substantia nigra (SN) was examined by Nissl staining 3 months post-treatment ( Fig. 2A, B) and survival of dopaminergic (DA) neurons was further con rmed by TH immuno uorescence staining (Fig. 2C, D). Obvious asymmetry was consistently apparent in both Nissl-stained ( Fig. 2A) and DA neurons (Fig. 2C) between the left (intact) and right (6-OHDA-lesioned) halves of the brain in the SN of the Sham group versus the WT group. In the sham group, there was a signi cant loss of both signals in the lesioned side of the SN, with an average survival of ~18.5% relative to the intact side (Fig. 2B, D). In contrast, a signi cant increase in survival of SN (Fig. 2B) and DA neurons (Fig. 2D) in the lesioned side of the SN was found in the Mito (µ = 68.4%) and P-Mito (µ = 73.2%) groups, with similar levels of performance indicating the effectiveness of mitochondrial treatment for neuron survival. No effect was seen in the Pep-1 group (Fig. 2).
Furthermore, immunohistochemical TH staining also showed consistent results in the striata (ST) of the Mito and P-Mito groups, which were comparable to those of the sham and Pep-1 groups (Fig. 3A). The signi cant loss of DA neurons that project to the ST, disrupting the motor circuit of the basal ganglia, was restored by Mito and P-Mito treatments, but not in the sham or Pep-1 groups (Fig. 3B). The difference of TH performance between lesioned and intact sides diminished signi cantly in the P-Mito and Mito groups (Fig. 3B), though there was no signi cant difference between them (p=0.08).

Restoration of mitochondrial function in SN and modulation of plasma in ammatory cytokine responses
Mitochondrial function in SN neurons was examined by analyzing expression of proteins of the electron transport chain and oxidative damage using 8-hydroxy-2-deoxyguanosin (8-OHdG) staining. Western blots showed an obvious loss of complex I (CI) in the SN of sham and Pep-1 groups compared with the WT group. In contrast, complexes II-IV (CII-CIV) showed a signi cant increase in band intensity and obvious shifts in molecular weights (Fig. 4A). Both the Mito and P-Mito groups showed patterns of mitochondrial complex proteins like those of WT, documenting a signi cant recovery of CI and explaining the performance normalization of CII-CIV relative to the sham group (Fig. 4A). The quanti cation revealed that CI-CIV substantially returned to normal in the Mito and P-Mito groups and that the adjustment in CIV expression in the P-Mito group was more signi cant than that in the Mito group (Fig.  4B). Meanwhile, IHC staining of nuclei in SN neurons exhibited strong 8-OHdG signals in the Sham group compared to the WT group, whereas nuclei were signi cantly less stained in both the Mito and P-Mito groups, though their staining levels were still higher than those observed in the WT group (Fig. 4C) .
There was no signi cant difference in plasma levels of in ammatory cytokines between WT and Sham groups, except for Interleukin (IL)-1alpha (IL-1α), which showed a signi cant decrease in the Sham group (Fig. 5). Compared to the Sham group, Mito treatment evoked a strong increase of in ammatory cytokines, especially IL-1α, IL-1beta (IL-1β), IL-10 and IL-17A. P-Mito treatment not only decreased expression of IL-1α and IL-17A as in the Mito group, but also suppressed IL-12 levels (Fig. 5). Thus, Pep-1 conjugation of infused mitochondria further attenuated the plasma in ammatory cytokine response.
Presence of allogeneic mitochondria in nigral dopaminergic neurons innervating the striatum through the rostral migratory stream BrdU IHC images of brain sagittal sections (lesioned side) visualized the uptake of intranasally infused allogeneic mitochondria labelled with BrdU in different parts of brain, including the corpus callosum (CC) and ST, accessory olfactory bulb (AOB), rostral migratory stream (RMS) track, and glomerular layer (GL) layer of the main olfactory bulb (Fig. 6A). Compared to the untreated WT group, even despite having a mild antibody background around the AOB area (asterisks), mitochondrial treatment groups clearly revealed a marked presence of allogeneic mitochondria in the region of an RMS-like track penetrating into the AOB. They also diffused into parts of the CC (Fig. 6A), as well as the ST (Fig. 6B). Expression of BrdU signals, excluding background noise, was relatively less visible in the GL layer olfactory bulb network and was hardly discernible in deeper layers of the external plexiform cells (EPC) and mitral cells (MCL), than in the GL layer (Fig. 6A). For further con rmation of mitochondrial delivery via RMS neurons, double immuno uorescence labeling of migrating neuroblasts (doublecortin (DCX)-and BrdU-labeled mitochondria) showed exogenous mitochondria in DCX-positive neurons of the RMS in the area of the AOB / CC/ST interface (Fig. 6C) in the Mito and P-Mito groups, but not in the WT group. Colocalization of the BrdU and DCX signals revealed by Z-stack sections con rmed internalization of allogeneic mitochondria in RMS neurons around the CC/ST area (Fig. 6D).

Mitochondrial delivery to dopaminergic neurons of the contralateral striatum via interhemispheric commissures
Double staining of brain cross-sections with BrdU and TH con rmed the internalization of allogeneic mitochondria in nerve terminals of nigral DA neurons in the ST of Mito and P-Mito groups (Fig. 7A). Some TH-positive DA neurons possessed exogenous mitochondria that could only be seen at higher magni cation (Fig. 7A). Notably, mitochondria from unilateral intranasal infusion were not only observed in ipsilateral ST of the lesioned side (Fig. 7A), but also in the contralateral side of the intact hemisphere (Fig. 7A). The latter was visibly higher in the P-Mito group than in the Mito group, as revealed by denser BrdU signals, but exhibited similar uorescence in the lesioned side (Fig. 7A,B). To explain allogeneic mitochondrial internalization in both cerebral hemispheres, double immuno uorescence staining (DCX/BrdU) of migrating neuroblasts was performed in coronal brain sections of the decussation of the anterior commissure (AC) (Fig. 7C). BrdU immunoreactivity in the Mito and P-Mito groups revealed expression of foreign mitochondria in neurons of the AC between the ipsilateral and contralateral hemispheres. Mitochondrial transmission in AC neurons from the treated side to the contralateral (intact) side was also more obvious in the P-Mito group than in the Mito group (Fig. 7C), which was consistent with BrdU expression on the contralateral side in the ST (Fig. 7A). The Z-stack for 3D reconstruction at high-magni cation merged confocal uorescence images of DCX-positive cells on the lesioned side AC (Fig. 7D). Co-expression of DCX and BrdU signals in merged images showed mitochondrial internalization in DCX cells with a typical fusiform morphology.

Discussion
Mitochondrial transplantation has been extensively studied in recent years, with efforts to apply it to treatments for a variety of mitochondrial and non-mitochondrial diseases [21,24]. Based on the pathogenesis of each disease, different invasive approaches have been used for transplantation, including intravascular injection, subcutaneous injection, and in situ injection [6,21,25]. Non-invasive mitochondrial transplantation, however, has rarely been studied due to certain limitations, including the inability to preserve mitochondrial activity after isolation, the lack of speci city in targeted delivery, and the problem of poor delivery e ciency. To the best of our knowledge, this is the rst study to demonstrate the therapeutic feasibility of brain targeting via intranasal administration of allogeneic mitochondria in a neurotoxin-induced PD rat model. In addition, we utilized BrdU for mitochondrial labeling. BrdU is a useful labeling method that does not gradually lose signal strength, as does green uorescent protein. BrdU can be used for long-term mitochondrial tracking and does not affect the morphology or activity of mitochondria [22,26]. In contrast to our previous study using a medial forebrain bundle (MFB) injection of mitochondria for PD treatment [8], the present results showed that when mitochondria were twice administered intranasally, restoration of mitochondrial complex I protein in substantia nigra neurons (~52%) was signi cantly lower than with local injection (~85%) using the same treatment frequencies and intervals. However, intriguingly, the improvement of animal behavior (except for the 40 % lower index of cross-zoom frequency) and the increase in survival of nigra DA neurons were similar. This may be related to differences of mitochondrial uptake in speci c regions of PD brains, due to different interventions. Indeed, neurons, astrocytes and glial cells of the cerebrum differed in mitochondrial internalization e cacy, which was also affected by different transplantation routes in the rescue model of stroke rats [26]. By tracking the signal of BrdU-labelled mitochondria, we found that mitochondria delivered intranasally could only be observed in the ST (the terminal of DA neurons) in contrast to mitochondria injected into the MFB, which were expressed in SN (somata of DA neurons) [8]. Chemogenetic stimulation of striatal projection neurons modulated responses to Parkinson's disease therapy [27] and this supports the notion that while the intranasal route for mitochondrial delivery had lower restoration e cacy for mitochondrial complex I protein of nigra DA neurons, it still had therapeutic effects similar to those of local injections.
Compared to injected MFB mitochondria, which need to be modi ed for uptake into nigra DA neurons [11], nose-to-brain delivery affords easier access to the brain since mitochondria do not need to be modi ed to penetrate the AOB and to enter the ST via the RMS pathway. The RMS pathway provides a "conduit" for intranasal mitochondrial delivery, guiding internalized mitochondria into the ST for restoration of dysfunctional DA neurons in PD. Moreover, naked mitochondria can also enter neurons by perfusion ipsilateral perfusion to the lesioned side and can be delivered to DA neurons of the ST on the contralateral, non-lesioned side via axons of interhemispheric commissures, such as the AC and CC [28]. Although contralateral delivery e ciency of naked mitochondria was not as high as that of peptidemodi ed mitochondria, therapeutic effects were not affected in the unilateral lesion rat model PD used here. Further studies are required to determine whether better contralateral mitochondrial delivery e ciency would enhance therapeutic effects in bilateral SN lesions of PD model rats. It would be bene cial to increase delivery e ciency of mitochondria from nose-to-brain and to simplify the process of transplantation. In addition, studies have shown that proteins and nanoparticles can be delivered intranasally to brain parenchyma, the third and fourth ventricles, midbrain, and hippocampus, via the same pathway (the RMS pathway) in transgenic mouse models of Alzheimer's disease [29]. However, in contrast to these techniques, which could also utilize olfactory and trigeminal routes, mitochondria can only be delivered via the RMS pathway (since no signi cant presence of mitochondria was observed in olfactory neurons and the olfactory-trigeminal pathway) to basal ganglia of the forebrain and lateral ventricles. This may be caused by variation in transport of mitochondria internalized in different types of neurons [29]. Otherwise, the diversity of intracellular signal transduction pathways in response to extracellular guide signals leads to variable cellular uptake of mitochondria [30].
Moreover, we found that expression of plasma in ammatory cytokines was affected by different interventional approaches. Contrary to more serious contraindications resulting from local injections of vehicle relative to PD non-treatment controls [8], high plasma in ammatory cytokine levels of PD rats were signi cantly reduced by intranasal vehicle perfusion, implying that this intervention will not exacerbate the disease during treatment [31]. Saline nasal irrigation is clinically proven for postoperative in ammation and to minimize antibiotic resistance [32] . One explanation for its utility is that the nasal in ammatory mediator, leukotriene C4, is substantially less elevated 2-6 hours after treatment [32]. Clinical studies have shown that intranasal delivery of a placebo (saline) or an antioxidant enzyme (glutathione) alone over a three-month period provided symptomatic improvements in PD patients [33] and anti-in ammatory treatment bene ts for PD [22]. Moreover, our results were consistent with our previous nding that modifying mitochondria with Pep-1 reduces their induction of plasma proin ammatory cytokines [8]. Although this outcome did not signi cantly affect therapeutic e cacy after just three months of treatment, further investigation is necessary to study its long-term postoperative effects. Moreover, while mitochondria delivered via brain injection induced higher levels of proin ammatory cytokines [8], intranasal mitochondrial delivery only induced expression of interleukin (IL)-1α, IL-1β, IL-10, and IL-17A, further demonstrating the immunological safety of intranasal drug delivery.