vehicle accidents, wars, and sports [1]. The mortality rates worldwide are assessed to be between 7% and 23%, 90% of which are in developing countries [2]. TBI studies in the Middle East are limited; however, it was predicted that the incidence in the region is 45 per 100,000 [2]. Survivors suffering from disabilities endure major socioeconomic burdens as well [3]. TBI leads to neurological complications through two major events, dynamic and overlapping, denoted as primary and secondary injuries. Primary injury defines the severity of TBI and therefore represents the major prognostic factor. Damaged neural cells undergo ionic imbalance characterized by an influx of calcium (Ca2+), potassium (K+), and glutamate [4]. The resultant excitotoxicity leads to an energetic crisis and oxidative stress by increasing reactive oxygen species (ROS) [5]. These can then directly stimulate the release of cytokines and pro-inflammatory factors, contributing to an elevated inflammatory state [6,7]. Following this, secondary injury is triggered, initiating a cascade of metabolic events that include further excitotoxicity, neuroinflammation, disruption of the blood–brain barrier (BBB), and cell death [8]. Notably, mitochondrial dysfunction, characterized by their membrane disruption and fission/fusion imbalance, plays an important role in the pathology of TBI via the excessive production of ROS, eventually leading to apoptotic cell death [9].

Finding a treatment for TBI remains a major challenge as there are currently no FDAapproved drugs with present approaches mainly targeting the symptoms. Depending on the severity of the injury, these can range from anti-seizure drugs and diuretics to comainducing drugs. Anti-seizure prophylaxis is administered to prevent the development of epileptic episodes post-TBI; however, due to the shortage of evidence on its effects in the long term, it is usually discontinued a week after injury [10]. Intracranial pressure is also monitored when individuals experience TBI, and in the case of its elevation, pressure is released by medically inducing venous flow and removing cerebrospinal fluid (CSF) from the intracranial regions [11].

Current animal studies on possible therapies have explored multiple candidates such as calcium channel blockers, erythropoietin, and hypothermia. Calcium channel blockers such as nimodipine and ziconotide have been considered as possible agents to enhance mitochondrial function and improve outcomes in patients with spontaneous subarachnoid hemorrhage [12]. However, the former was later revealed to have no effects on mortality and morbidity in TBI patients, and the latter was shown to lead to numerous side effects such as hypotension [13,14]. On the other hand, other suggested treatments target oxidative stress since antioxidants have been shown to ameliorate the pathology of TBI [15,16]. An example of these is the mitochondria-targeted drug mitoquinone (MitoQ). It is synthesized by conjugating a ubiquinone moiety to a triphenylphosphonium cation (TPP+) [17]. TPP+ is a lipophilic cation that directs the ubiquinone moiety to the inner mitochondrial membrane (IMM) and enables its accumulation due to the high electrochemical potential [18]. Accumulated MitoQ in the IMM is reduced by complex II into its antioxidative quinol form [19], noting that almost all molecules that enter the mitochondria are adsorbed to the matrix surface of the IMM where they are constantly recycled [20]. MitoQ can easily cross the BBB [21] and has been shown to act by activating the nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf2) pathway (Nrf2-ARE) [22]. Previous studies from our laboratory (currently under review) have shown that MitoQ improved the outcomes of closed head repetitive mild TBI in mice at acute, subacute, and chronic time points. Our current study aimed at assessing MitoQ's neuroprotective effects on the long-term behavioral, cognitive, and molecular outcomes in a moderate open head TBI mouse model. We hypothesized that improved outcomes are carried out by MitoQ's ability to dampen the state of oxidative stress, decrease cellular injury, and ameliorate neuroinflammation. We were able to show that MitoQ indeed exhibits neuroprotective properties in a moderate controlled cortical impact (CCI) mouse model, with effects translating to the molecular and behavioral/cognitive levels.

#### **2. Materials and Methods**

#### *2.1. Animals*

Thirty-four C57BL/6 male mice were housed in a controlled environment (12 h light/dark cycles, 22 ± 2 ◦C). The study was carried out at the Animal Care Facility of the American University of Beirut (AUB) and all animal experiments were performed in compliance with the AUB Institutional Animal Care and Use Committee (IACUC) guidelines with the reference number 17-01-458, 1 February 2019. All animals were handled under pathogen-free conditions and fed chow diet ad libitum. 7–8 weeks old male mice were divided into three groups: Sham, CCI, and CCI + MitoQ. The mice in the CCI and CCI + MitoQ groups were subjected to TBI using a controlled cortical impact machine (CCI). Thirty minutes after TBI, mice in the CCI + MitoQ group were injected intraperitoneally (IP) with MitoQ at a dosage of 8 mg/kg administered three times per week over 30 days, starting at 30 min post-injury. The dose was adopted from a previous study on MitoQ and TBI [22].

Keeping in mind that the test most prone to variability is the Morris water maze, we used it to determine the number of mice required for cognitive and behavioral tests. To achieve 90% power to detect a difference with 95% confidence, 9 animals were required. Therefore, we assigned 11–12 mice per group for behavioral, cognitive, and neurological tests: group 1: Sham (*n* = 10); group 2: CCI (*n* = 10); and group 3: CCI + MitoQ (*n* = 10). Animals were assigned to experimental groups by randomly selecting which animals received the treatment from those that underwent the injury. Animals that showed weak health post-surgery and exhibited any disease condition were not included in the study as per the IACUC regulations. Following sacrifice, four animals per group (*n* = 4) were used to perform immunofluorescence and another four animals (*n* = 4) per group for the RT-qPCR.

#### *2.2. Stereotaxic Surgery: Controlled Cortical Impact*

Each mouse was anesthetized with a ketamine/xylazine mixture (50 mg/kg and 15 mg/kg, respectively) administered intraperitoneally. Each mouse was then fixed on a stereotaxic frame, and a longitudinal skin incision was made in the middle of the mouse's head by a surgical scalpel to expose the skull. An ointment (Xailin®,, Nicox, France) was applied to the eyes to protect vision during surgery. Using a drill and forceps, the part of the skull above the somatosensory area of the parietal lobe was removed to expose the brain. The injury was created using the Leica Impact One Angle Controlled Cortical Impact (CCI) machine. The center of the impactor was placed above the somatosensory area of the parietal cortex of the brain using the following coordinates: +1.0 mm AP, +1.5 mm ML, and −2 mm DV. The duration of impact was kept constant with a dwell time of 1 s, at a velocity of 4 m/s. The depth of the injury was set to 1.5 mm. The tip of the impactor was 1 mm in diameter. The size of the bone flap was 1.7 mm in diameter, which was carefully removed using a manual trephine to expose the dura matter without damaging it. The wound was closed using silk sutures (MERSILK™-W587H, Ethicon, OH, USA). Each mouse was placed on a heating pad to maintain the body's temperature. The CCI + MitoQ group received the first MitoQ injection 30 min following injury. For the animals in the Sham group, drilling was performed to remove confounding factors.

#### *2.3. Mitoquinone Supplementation*

Mitoquinone (Focus Biomolecules, 10-1363, MW = 663.64) was prepared by dissolving 25 mg in 10% dimethylsulfoxide (DMSO) (100 μL in 900 μL phosphate-buffered saline (PBS)). Further dilutions using PBS were carried out to obtain working solutions of 1 mg/mL. MitoQ was intraperitoneally (IP) supplemented at a dose of 8 mg/kg three times per week over 30 days, starting at 30 min post-injury. The experimental timeline is presented below in Figure 1A.

**Figure 1.** Experimental timeline of the study. (**A**) Mice were divided across three groups: Sham, CCI, and CCI + MitoQ. MitoQ injections were supplemented starting 30 min after the first injury via IP injections at a dose of 8 mg/kg every 3 days for 30 days. Behavioral and cognitive testing commenced after day 21 of the experimental timeline and continued until day 30. (**B**) A coronal section showing the site of injury and the brain regions (green and blue) that were investigated for quantification parameters. The section was obtained from the Allen Mouse Brain Atlas and Allen Reference Atlas—Mouse Brain [23].

#### *2.4. Garcia Neuroscore*

The neuroscore was adopted from Garcia et al. to assess the integrity of neurological function via different criteria [24]. Every group was tested 3 days before CCI and then on days 3, 7, and 30 after the injury. The animals underwent six different evaluations on every test day, each being scored from 0 to 3, according to Table 1.

**Table 1.** Evaluation criteria for Garcia neuroscore.

#### *2.5. Grip Strength Test*

This grip strength test was performed to assess endurance of motor skills and muscular dysfunction using the 4700-grip strength meter (UGO BASILE®, Gemonio, Italy). Each animal was held by its tail and was allowed to catch a trapeze-shaped metal object with both of its paws. A total of three trials were conducted for each animal. Muscle strength in gram force (gf) was recorded along with the total time of grip and the time the mouse applied the force. For analysis, the average of muscle strength, in gf, in the three trials, was normalized to the mouse weight [25].

#### *2.6. Pole Climbing Test*

The pole climbing test was conducted to evaluate motor coordination. The apparatus consisted of a metal pole with a length of 60 cm and a diameter of 1 cm, set perpendicularly in a big cage. The pole was wrapped with tape to facilitate the animal's grip on the apparatus. Animals were habituated to descend on the pole for three trials before the recorded testing. The mice were placed on top of the pole with their heads directed upwards and allowed to descend freely [26]. The time needed for each mouse to reach the bottom of the pole (total time) was recorded. For each animal, a total of three testing trials were conducted on the testing day.

#### *2.7. Adhesive Removal Test*

This test was performed by placing a rectangular adhesive tape strip on each mouse's nostril to assess sensorimotor function. Then, the animal was placed back in its cage. Two different values were recorded using two stopwatches: (i) the time needed to make the first contact with the tape, which represents nose sensitivity, and (ii) the time needed to remove the tape, which represents dexterity.

#### *2.8. Forced Swim Test*

This test was used to assess despair and depression-like behavior. Animals were placed in a glass cylinder (20 (height) × 11 cm (diameter)) filled with 25 ◦C water to a depth of 15 cm for 6 min. The 6 min included a 6 min acclimation period at the beginning of the test, followed immediately by 5 min of testing. Depression was reflected by the time mice spent floating on the surface of the water without moving. Animals were then dried with a towel and returned to their heated cage. The behavior of the mice was recorded using a camera, and immobility time was analyzed later during the last five-minute period of the test. Immobility time was defined as the time a mouse spent floating and only making necessary movements to keep its body balanced and head above the water.

#### *2.9. Morris Water Maze Test*

The Morris water maze (MWM) was used to study spatial learning and memory. The apparatus consisted of a circular pool (110 cm diameter, 55 cm depth) half-filled with water and maintained at 22 ◦C. A 10 cm-diameter platform was placed in the water. Three extra-maze cues were located around the pool to spatially guide the mice. Black and white cues were used to omit any variance in color discrimination among mice. Non-toxic white paint was added to the water surface to make it opaque and ensure accurate tracking. The ANY-maze 5.2 software (Stoelting Co., Wood Dale, IL, USA) was used to track the movement of mice and record the different parameters to be evaluated during the five days of testing.

On the first day, the platform was made visible by mounting a flag for cued trials. Four trials were carried out for each animal where the position of the platform and the starting position of animals changed among trials. On days two through four, which are the acquisition days, the flag was removed, and the platform was fixed in the northeast (NE) quadrant submerged in the water. However, the starting position of the animals varied among trials where three trials were carried out. The maximum time of a trial was set to one minute. If the animal failed to find the platform during this time, it was guided to the platform by the experimenter, where the animals were allowed to sit on the platform for 20 s for memory consolidation. If the animals found the platform before this time, the test was considered completed, and the animals were kept on the platform for five seconds. On day five, or the probe trial day, the platform was removed, and the mice were allowed to

swim for 1 min for one trial only. After each trial on all days, the mice were dried with a towel and allowed to rest in a heated cage.

#### *2.10. Novel Object Recognition Test*

Novel object recognition depends on the innate capacity of rodents to discriminate a novel object from a familiar object (previously encountered). Mice were placed in the center of an open field at the beginning of each trial and freely explored the open field and objects. At the end of each trial (5 min), mice were removed from the open fields and placed in their home cages next to each testing area for the inter-trial interval (ITI—5 min). The open fields and objects were cleaned with 70% ethanol during the ITI. During Trial 1 (learning trial), the animals explored an open field with two identical objects. During trial 2 (testing trial), one object was kept in the testing field, while the other was replaced by a novel object for exploration. All data were recorded by a video camera that was connected to image analyzer software Any-Maze 5.2 (Stoelting Co., Wood Dale, IL, USA). The zones were located 5 cm around each object on the software, and the software recorded the time the animal spent in each zone.

#### *2.11. RT-qPCR*

RNA was extracted using Trizol (T9424-100ML, Sigma Aldrich, St. Louis, MO, USA), and then any contaminating genomic DNA was removed using TURBO DNA-free™ Kit (AM1907, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. An amount of 1.5 μg of total RNA was reverse transcribed using the iScript™ cDNA Synthesis Kit (1708890, Bio-Rad, Hercules, CA, USA). Quantitative real-time PCR was applied to 1 μL of the obtained cDNA using the Quantifast® SYBR® Green PCR Master Mix (204054, Qiagen, Hilden, Germany) with 10 μM of each of the reverse and forward primers. Cycling conditions were as follows: 95 ◦C for 10 min for one cycle, then 95 ◦C for 10 s followed by 60 ◦C for 30 s and 72 ◦C for 30 s for 40 cycles, and, finally, 72 ◦C for 5 min for one cycle. The primers used are listed in Table 2.

**Table 2.** Different primer sequences used in RT-qPCR.

#### *2.12. Immunofluorescence Staining*

The animals allocated for immunofluorescent staining underwent perfusion right after terminal tissue collection. Prior to surgery, the mice were anesthetized via IP injection of ketamine and xylazine solution (50 mg/kg and 15 mg/kg, respectively). A lateral incision was made through the integument and abdominal wall beneath the rib cage. The diaphragm was then incised, and the pleural cavity was exposed. The sternum was then lifted and any tissue connecting it to the heart was cut. A perfusion needle was introduced into the left ventricle without reaching the aorta, and an incision was made to the animal's right atrium. PBS was firstly perfused into the animals until the fluids were running clear and the liver was clear as well. When this occurred, PBS was switched with PFA until fixation tremors were observed. Brain tissue was placed in PFA for 48 h before being transferred into 30% sucrose solution for further preservation.

In preparation for immunofluorescence staining, brain tissue underwent sectioning using a microtome and was stored as free-floating sections in sodium azide. Free-floating brain sections (40 μm thick) were washed with PBS followed by PBST (0.1% Triton in PBS) and then incubated for 1.5 h in a blocking solution of 10% heat-inactivated fetal bovine serum (FBS) in PBST. Tissues were then incubated overnight at 4 ◦C with primary antibodies, diluted in 1% FBS solution. The primary antibodies used were anti-GFAP (1:1000 dilution; MCA-5C10, Encor Biotechnology, Gainesville, FL, USA) as an astrocyte marker; anti-NeuN (1:1000 dilution; RPCA-FOX3-AP, Encor Biotechnology) as a marker of mature neurons; anti-Iba-1 (1:1000 dilution; 019-19471, Fujifilm Wako Chemicals, Richmond, WV, USA) as a microglia marker; anti-MBP (1:1000 dilution; MCA-7D2, Encor Biotechnology) as an axonal marker. Then, sections were rinsed in PBST and incubated with the appropriate fluorochrome-conjugated secondary antibody (1:1000 dilution) for 1 h at room temperature, followed by three washes in PBST. The secondaries used were Donkey Anti-Mouse IgG H&L Alexa Fluor® 488 (ab150105, Abcam, MA, USA) and Goat Anti-Rabbit IgG H&L (Alexa Fluor® 568) (ab175696, Abcam, MA, USA). Finally, all sections were counterstained in 1 μg/mL of Hoechst (Sigma Aldrich), diluted in PBS, and mounted using Fluoromount (F4680-25ML, Sigma Aldrich). Microscopic imaging was conducted using a Zeiss LSM 710 confocal microscope. Images were acquired as tile scans with 40× oil objectives and analyzed using the Zeiss ZEN 2009 image analysis software and NIH ImageJ program. Images for the different experimental interventions were acquired under the same laser and microscopic parameters for the purpose of consistency. At least three sections per mouse were used for quantification with different levels to cover the hippocampus and cortex areas. For the hippocampus, quantification was conducted along the CA3 and the dentate gyrus (DG). For the cortex, the entire area around the injury in the somatosensory cortex was captured via tile scanning (Figure 1B). The fields from all three sections were averaged for every animal.

#### *2.13. Statistical Analysis*

Statistical analysis was conducted using GraphPad Prism 9.0 (La Jolla, CA, USA). The Kruskal–Wallis test was performed to compare the datasets that contained more than two groups. Then, Dunn's test was used to compare the three groups simultaneously. All results were considered significant at a *p*-value of < 0.05: \*\*\* (*p* < 0.001), \*\* (*p* < 0.01), \* (*p* < 0.05).

#### **3. Results**

#### *3.1. MitoQ Improves General Neurological Function by Ameliorating Motor Coordination, Muscle Strength, and Sensorimotor Function*

We investigated the general neurological function of TBI mice using the modified Garcia neurological score test. The experiment was first conducted 3 days before the injury to make sure that all animals (*n* = 10 per group) had a similar basal score and no prior neurological dysfunction was present (Figure 2A). As expected, all animals had a perfect score, showing that they were eligible to be included in the study. The test was then repeated at 3, 7, and 30 days post-injury (DPI). On day 3 post-injury, the difference between the CCI + MitoQ and CCI groups was not significant. However, at 7 and 30 days post-injury, the CCI + MitoQ group performed significantly better compared to the CCI group (*p* < 0.05). This first step showed us that MitoQ improved overall neurological performance in mice. However, to be able to further assess this, additional testing was performed, from 21 to 23 DPI, to evaluate specific sensorimotor deficits and observe overall motor coordination.

To start with, the adhesive removal test was performed to assess sensorimotor function by observing sensitivity and dexterity. The CCI + MitoQ group performed significantly better than the CCI group by establishing first contact with the adhesive faster (*p* < 0.05) (Figure 2B,C). This showed improved sensitivity. Additionally, the CCI + MitoQ group required less time to remove the adhesive (*p* < 0.05), indicating better dexterity.

Then, motor function was investigated by exploring motor coordination via the pole climbing test, and muscle strength using the grip strength test. The CCI + MitoQ group was able to perform better by taking less time to descend the pole (*p* < 0.05) (Figure 2E) and revealed increased muscle strength (*p* < 0.001) (Figure 2D) compared to the CCI group. Therefore, MitoQ administration helped improve gross motor function. It is noteworthy

here that the data show no significant difference between the CCI and Sham groups in the pole climbing test due to increased variability.

**Figure 2.** MitoQ ameliorates neurological deficits post-CCI. (**A**) Garcia neuroscore allowed testing for neurological function in mice and showed that MitoQ improved overall neurological performance 7 and 30 days post-CCI. Further testing was conducted to assess sensorimotor function via the adhesive removal test. Mice that received MitoQ took less time to establish contact (**B**) and were able to remove the adhesive faster (**C**) than the CCI group. Additionally, gross motor function was investigated by two tests. (**D**) The grip strength test showed that MitoQ improved overall muscle strength, and the pole climbing test (**E**) showed that CCI + MitoQ had better motor coordination. All tests were executed on Sham (*n* = 10), CCI (*n* = 10), and CCI + MitoQ (*n* = 10). \* (*p* < 0.05); \*\* (*p* < 0.01); \*\*\* (*p* < 0.001).

#### *3.2. Learning Deficits and Recognition Memory Dysfunction Are Alleviated by MitoQ*

MitoQ's ability to potentially alleviate cognitive deficits that result from CCI was subsequently evaluated using the Morris water maze (MWM) test. No differences in latency to the platform were found among groups on day 1 of the test, which was performed 25 DPI. During the acquisition days of the test, CCI mice took more time to reach the hidden platform as compared to the Sham and CCI + MitoQ groups. This delay was significant on days 2 (*p* < 0.05), 3 (*p* < 0.001), and 4 (*p* < 0.05) of the acquisition days (Figure 3A). The data indicate that CCI results in a cognitive learning deficit reversed upon MitoQ administration. On the probe trial day of the test, there were differences in the latency to reach the target quadrant by the CCI group, indicating that retention deficits are caused by CCI in the model (Figure 3B,C). However, this was shown to be attenuated by MitoQ since the CCI + MitoQ group required less time to reach the target quadrant (*p* < 0.05) and spent more time in it (*p* < 0.05) (Figure 3B,C).

**Figure 3.** MitoQ improves special learning and recognition memory. Spatial learning and memory in mice from the three groups were evaluated using the Morris water maze. (**A**) shows the time taken for the first arrival at the platform by mice from the three study groups on the acquisition days where the mice learned how to reach the platform based on visual cues. (**B**) shows the time to reach the target quadrant (NE) on the probe trial day. (**C**) shows the percentage of time spent in the target quadrant. (**D**,**E**) show the representative heat maps and trace plots on the probe trial day, respectively. (**H**,**I**) show the representative heat maps and trace plots on the testing day of the object recognition

test. For further investigation of memory acquisition, the novel object recognition test was performed. (**F**) Exploration time for all three groups was recorded, and (**G**) the discrimination index was calculated, showing that recognition memory was enhanced in the CCI + MitoQ group. Data represent the mean ± SEM (*n* = 10 per group). \* (*p* < 0.05), \*\* (*p* < 0.01), \*\*\* (*p* < 0.001).

The novel object recognition (OR) test was conducted for further learning assessment and recognition memory testing. On the testing day, after carrying out habituation and training for the mice in the platform, the total exploration time for all three groups showed no significant difference. This ensured that all animals had an equal chance of exploring the platform and thus ensured the viability of the test (Figure 3F). During the testing trials, the CCI group exhibited no differentiation between familiar and novel objects (*p* < 0.01). This was not the case in the CCI + MitoQ group, which spent more time exploring the novel object, resulting in a positive differentiation index (*p* < 0.05) (Figure 3G).

#### *3.3. MitoQ Decreases Depressive-like Behavior*

To understand the effects of MitoQ on depressive-like behavior, the forced swim test allowed quantifying hopelessness in all study groups by measuring the immobility time. The CCI-only group showed an increased immobility time when compared to Sham (*p* < 0.001). Of interest, the CCI + MitoQ group demonstrated a decrease in the immobility time compared to the CCI group (*p* < 0.05) (Figure 4). Therefore, MitoQ administration helped in reducing depressive-like behavior in mice.

**Figure 4.** Depressive-like behavior is reduced by MitoQ. Animals were placed in a water container during the forced swim test to measure the immobility time as a sign of hopelessness. The CCI + MitoQ group performed better than the CCI group, with a decreased immobility time. Data represent the mean ± SEM (*n* = 10 per group). \*\* (*p* < 0.01), \*\*\* (*p* < 0.001).

#### *3.4. MitoQ Upregulates Antioxidative Enzymes*

The expression level of Nrf2 and downstream antioxidant enzymes, particularly CAT and SOD2, was quantified by RT-qPCR to draw similarities between previous studies and make sure MitoQ acted on the same axis in this experiment. After collecting ipsilateral cortices 30 DPI, the experiments were performed on total mRNA obtained from all three groups (*n* = 4). For all three genes of interest, MitoQ treatment resulted in a significant

improvement in expression as compared to CCI (*p* < 0.05) (Figure 5). This means that MitoQ increased the expression of antioxidative enzymes via the Nrf2–ARE axis.

**Figure 5.** MitoQ improves expression of Nrf2, SOD2, and CAT in the cortex post-CCI. The mRNA levels of Nrf2, CAT, and SOD2 were determined using RT-qPCR in Sham (*n* = 4), CCI (*n* = 4), and CCI + MitoQ (*n* = 4). Data were normalized to β-actin. \* (*p* < 0.05).

#### *3.5. MitoQ Diminishes Chronic Activation of CNS Inflammatory Cells, Neuronal Death, and Axonal Damage*

We attempted to assess if the decreased state of oxidative stress could be translated into ameliorated cellular recovery post-CCI. For this reason, immunofluorescence staining was performed to examine the activation of glial cells, axonal damage, and neuronal loss (*n* = 4 for every group, three fields were quantified for every *n* and averaged). Ionized calciumbinding adaptor protein-1 (Iba-1), an actin-binding protein, was used to stain activated microglia using immunofluorescence. The results show that MitoQ administration significantly attenuated the number of activated microglia both in the ipsilateral cortex (*p* < 0.05) and hippocampus (*p* < 0.05) compared to that of the CCI group (Figure 6A,B). Quantifying the number of microglia in both brain regions was not enough without examining the morphology of these cells. The CCI group exhibited amoeboid and elongated microglia with thick extensions, representing an activated phenotype [27] (Figure 6C). This, however, was not the case in the Sham and CCI + MitoQ groups, where microglia retained their ramified shape with extended ramification, indicative of the resting surveillant phenotype. Taken together, these data show that MitoQ reduces microgliosis following CCI, which is indicative of decreased chronic inflammation. Further investigation was conducted by assessing astrogliosis using the astrocytic activation marker GFAP.

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein used as a marker of reactive astrocytes. The intensity of its expression was used as a marker of active astrocytes. GFAP expression was significantly higher in the ipsilateral hippocampus and cortex of the CCI group compared to that of the Sham group (*p* < 0.05). It was noticeable that activated astrocytes congregated around the injury site and were absent in the contralateral side of the cortex of the injured group (Figure 7A). Such a result conforms with the focal nature of the injury. Upon quantification, our data show that this state of increased GFAP expression was significantly improved by MitoQ administration in the cortex (*p* < 0.05) and the hippocampus (*p* < 0.05) (Figure 7B,C). Likewise, the morphology of activated astrocytes was noticeable through Z-stack imaging of the dentate gyrus (DG), where they exhibited abundant arborization exclusively in the CCI group and retained an inactive morphology in the CCI + MitoQ group (Figure 7D).

**Figure 6.** MitoQ decreases microglial activation in the cortex and the hippocampus. Microgliosis was assessed by immunofluorescence staining for Iba-1 (blue/green). (**A**) shows Iba-1-positive cells (arrowheads) across the three experimental groups in the ipsilateral cortex. (**B**) shows the same as (**A**), but in the ipsilateral hippocampus. Scale bar = 100 μm. (**C**) exhibits a depth-coded Z-stack of individual microglial cells to showcase different morphologies across the three groups. (**D**,**E**) are bar graphs showing quantification of Iba-1-positive cells per mm2. Iba-1-positive cells were counted, and then the obtained number was divided by the area of the selected field in an average of three fields per animal, four animals per group. \* (*p* < 0.05); \*\* (*p* < 0.01); \*\*\* (*p* < 0.001).

**Figure 7.** MitoQ ameliorates astrocytosis in the cortex and hippocampus post-CCI. Astrocytosis was

assessed by immunofluorescent staining of GFAP (red). (**A**) shows the assessment of GFAP positivity in the entire brain section of all three groups. (**B**) shows GFAP-positive cells in the cortex and (**C**) in the hippocampus. Hoechst was used as a nuclear counterstain. (**C**) represents Z-stacks of sections in the hippocampal DG from all three groups to show different astrocytic morphologies. Scale bar = 100 μm. (**D**) shows a Z-stack of GFAP-positive cells in the hippocampus across the three groups. (**E**,**F**) are bar graphs showing the quantification of GFAP intensity per mm2. GFAP mean fluorescence intensities (MFI) of 3 fields per animal and 4 animals per condition were measured using the NIH ImageJ 1.41 software. Bar graphs display averages <sup>±</sup> SEMs of GFAP MFI/mm2.\*(*<sup>p</sup>* < 0.05); \*\* (*<sup>p</sup>* < 0.01).

Next, we investigated the neuronal cell count to assess cell death. Immunofluorescence staining for NeuN, a nuclear protein found in the nuclei and perinuclear cytoplasm of neurons, was used to assess the number of mature neurons in a semi-quantitative manner [28]. Our results show a significant decrease in the neuronal cell count in CCI as compared to Sham in the cortex and the hippocampus (*p* < 0.05) (Figure 8), with significant improvement in the cortex (*p* < 0.05), but not in the hippocampus, in the CCI + MitoQ group.

To measure axonal integrity, which has been shown to correlate with cognitive and behavioral deficits [29], we investigated myelin basic protein (MBP). Used as a marker for myelination, MBP is responsible for the adhesion of myelin to the cytosolic surfaces of cells [30]. Prominent axonal damage was observed in the cortex of the CCI group, with characteristic axonal shearing and blebbing (Figure 9B). However, in the CCI + MitoQ group, the axons exhibited a more refined integrity similar to that shown in the Sham group. Quantification of the MBP intensity in the cortex showed a significant decrease in the CCI group compared to the Sham group. This was significantly increased upon MitoQ administration (Figure 9D,E). Unlike the cortex, the hippocampus did not exhibit any differences in the integrity of MBP between all three experimental groups, retaining the focal nature of the injury.

**Figure 8.** MitoQ decreases neuronal cell loss in the cortex. NeuN staining (red) was performed to assess the number of mature neurons. (**A**) shows representative fluorescent images of NeuN staining in the cortex from the different study groups. NeuN-positive cells were counted, and then the number was divided by the area of the field. (**B**) shows the same as (**A**), but in the hippocampus. GFAP mean fluorescence intensities (MFIs) of 3 fields per animal and 4 animals per condition were measured using the NIH ImageJ software. Scale bar = 100 μm. (**C**,**D**) are bar graphs displaying average MFIs <sup>±</sup> SEMs of NeuN per mm2.\*(*<sup>p</sup>* < 0.05).

**Figure 9.** MitoQ decreases axonal shearing in the cortex after CCI. Representative fluorescent images of MBP staining (green) that were used to assess axonal morphology in the cortex and hippocampus for Sham, CCI, and CCI + MitoQ in (**A**–**C**), respectively. Major axonal blebbing and

shearing are highlighted with arrowheads. Scale bars = 500, 100, 50 μm. (**D**,**E**) are bar graphs showing the quantification of the MBP intensity per mm2 in the cortex and hippocampus. MBP mean fluorescence intensities (MFIs) of 3 fields per animal and 4 animals per condition were measured using the NIH ImageJ software. (**D**,**E**) are bar graphs displaying averages <sup>±</sup> SEMs of MBP MFI/mm2. \* (*p* < 0.05).

#### **4. Discussion**

TBI involves a plethora of pathological pathways that contribute to its heterogenic nature, making it extremely difficult to understand and treat. This explains the lack of FDA-approved drugs against TBI, exacerbating the need to investigate potential therapeutic approaches. For that purpose, this study was conducted to assess the potential protective effects of MitoQ in a CCI mouse model. Previously, MitoQ was demonstrated to ameliorate pathological effects of TBI by decreasing neuronal apoptosis, improving neurobehavior, and enhancing antioxidative capacity in the cortices of mice subjected to mild TBI (mTBI) [22]. Other studies exploring the neuroprotective properties of MitoQ in animal models of Huntington's disease, Alzheimer's disease (AD), and anterolateral sclerosis (ALS) also showed it can improve memory, enhance motor function, and decrease amyloid-beta (Aβ) accumulation [31–33]. In compliance with this, results from our laboratory further support the claim that MitoQ can decrease neurological and cognitive deficits in mTBI at a chronic time point (paper in publication).

In our study, MitoQ was supplemented at a concentration of 8 mg/kg, adapted from Zhou et al., who investigated the effects of MitoQ in experimental traumatic brain injury for the first time in vivo [22]. During the study, different doses of MitoQ were administered to compare dose-dependent effects. The injured mice that received MitoQ at 8 mg/kg exhibited improved neurological severity scores compared to the vehicle-treated group, as well as decreased edema. There was a lack of any improvement in both parameters when the mice received a dose of 2 mg/kg. This study showed that administering MitoQ at 8 mg/kg was safe and revealed neuroprotective effects in a mouse model of weight drop TBI. Similarly, in another study by Xiao et al., MitoQ was administered in mice at a concentration of 5 mg/kg IP to investigate its effects through the Nrf2 pathway [34]. This concentration yielded significant results, where MitoQ was able to provide protective effects in tubular injury via mitophagy.

Any concerns on the side effects of MitoQ, especially its administration for long periods (30 days in our case), have also been addressed in previous studies. There is a comprehensive body of literature showing this in both clinical and pre-clinical trials. Gane et al. showed that MitoQ helped decrease liver damage in hepatitis C patients with no adverse side effects over 28 days [21]. More recently, Rossmann et al. studied the effect of MitoQ on vascular function in adults. The study was conducted over six weeks and showed no significant adverse side effects following administration [35]. Similarly, Rodriguez-Cuenca et al. showed that long-term administration of MitoQ on wild-type mice for up to 28 weeks was safe and did not act as a pro-oxidant or cause damage over this period [36].

Having said that, the first step in our study was to observe the effects of MitoQ on a macroscale and thus investigate its potential in improving cognitive and neurological functions post-CCI. To start with, general neurological testing was carried out via the modified Garcia neurological score test. This allowed scoring the animals on a scale from 0 to 18 to assess general sensorimotor function, motor skill, and coordination-based functions [24]. In this study, MitoQ was shown to improve overall neurological integrity by resulting in better scores at 7 and 30 DPI, which represented the subacute and chronic phases of injury. With these results, we decided to investigate which aspects of the neurological functions were affected by CCI and which were improved by MitoQ.

The adhesive removal test showed that the current CCI model induced sensorimotor deficits in mice, where they took longer to establish contact with and remove tape that was placed on their noses. However, MitoQ was able to shorten this time, showing that it improved sensorimotor function at a chronic time point. To be more specific, this test provided us with the insight that MitoQ improved both dexterity and sensitivity. On the other hand, during the pole climbing test and grip strength test, the current CCI model displayed decreased muscle strength and adversely affected motor coordination, both of which were improved by MitoQ. Although there was a significant difference between the CCI and CCI + MitoQ groups in the pole climbing test, there was no significance between the CCI and Sham groups. This could either be due to high variability in the test scores or the injury model not being sufficient to induce motor coordination dysfunction. In either case, further investigation is warranted. In addition to this, MitoQ demonstrated a positive impact on depressive-like behavior during the forced swim test, revealing that it helped with the notion of hopelessness. This is especially important as TBI has been linked to many neuropsychiatric sequelae in the long run [37,38].

Moreover, CCI resulted in learning deficits and impairment of recognition memory, as seen in the MWM and OR tests. Firstly, during the former, the CCI group displayed latency to reach the platform on the learning days and a tendency towards worsened memory retention, as seen by the latency to reach the target quadrant and the time spent in it on the probe trial day. During the latter, the CCI group showed no differentiation between the familiar and novel objects, revealing recognition memory dysfunction. These findings align with other studies describing increased latency to reach the hidden platform in mice subjected to TBI [39–41]. However, when animals received MitoQ for 30 days, they exhibited improved learning and memory retention. Their ability to recognize and differentiate novel from familiar objects was also significantly improved, showing enhanced recognition memory. Therefore, we were able to demonstrate two things in this part of the study: (a) the animal model used was sufficient to induce cognitive, neurological, and behavioral dysfunctions, in compliance with the previous literature [42–45]; and (b) MitoQ administration showed exciting potential in improving cognitive dysfunctions in the mice at a chronic time point.

Having seen these data on the macroscale, it was important to assess how MitoQ could affect different pathways involved in TBI pathology at a molecular level. We needed to investigate this in the cortex since it retained the injury, and the hippocampus since it is involved in emotions, memory, and other functions impaired in TBI and the CCI model here [46–48]. The observed alterations in cognitive functions translate to damaged neuronal structures in the cortex and hippocampus [44,49]. Due to synapse disruption and other pathological effects such as chronic inflammation, oxidative stress, and glutamate altercations, circuit alterations have been linked to memory impairment in TBI and other neurological deficits [50–52]. We were able to see in the previous data that MitoQ treatment following CCI resulted in enhanced learning ability and recognition memory, pointing at its potential effects on oxidative stress, chronic inflammation, and neuronal circuitry disruption. This is especially true when referring to a previous study by Zhou et al., which showed MitoQ's potential in decreasing neuronal apoptosis and enhancing Nrf2 nuclear localization to induce antioxidative effects [22]. For this reason, we performed RT-qPCR analysis to observe how effective MitoQ was, in the current animal model, in acting on the previously described Nrf2-ARE pathway [22]. In our experiment, MitoQ significantly increased the expression of the Nrf2 transcription factor and its downstream antioxidative enzymes SOD2 and CAT. This showed us that it worked on the antioxidative pathway involving Nrf2 and thus exerted its antioxidative effect as expected. After examining the effect of MitoQ on antioxidative enzymes, it was important to investigate other parameters that might be related to improved cognition and neurological function. This was conducted via immunofluorescence.

Previous studies have shown that TBI was followed by microglia and astrocyte activation, leading to several neurological and cognitive dysfunctions [53–57]. This is accompanied by neuronal cell loss via activation of the cell death machinery [58–60]. The described CCI model exhibited results that are in line with these previous studies. There was an increased expression of GFAP and Iba-1, showing the presence of chronic activation of astrocytes and microglia, indicative of an active inflammatory state [54,57]. The injury also led to a decreased neuronal count, as examined using the neuronal marker NeuN. This pathology in the cortex and the hippocampus is reflected in the depressive-like state exhibited by the mice, and by the disrupted neurological function and cognition. It must also be highlighted that prominent axonal damage and shearing were observed in the cortex with decreased MBP reactivity due to the breakdown of the protein, indicating disrupted circuitry. However, MitoQ was able to help decrease these effects, where astrocytes and microglia showed dampened activation in the cortico-hippocampal system, and there was reduced neuronal cell loss in the cortex. This decreased activation was apparent in the morphology of both glial cells in the ipsilateral cortices and hippocampi of the mice that received the drug. The used CCI model induced a prominent presence of activated intermediate microglia denoted as bushy microglia, characteristic of their activated morphology [27].

The mitigated neuroinflammatory profile may be related to the antioxidant activity of MitoQ that enhances oxidative enzyme production and therefore prevents excessive production of ROS. Enhanced ROS production is known to directly stimulate the expression of the pro-inflammatory interleukins IL-1α and IL-1β within hours after injury [6,7]. Secreted pro-inflammatory cytokines subsequently contribute to the activation of Ca2+-dependent proteolytic enzymes that activate the cell death machinery and induce a pro-inflammatory state [61]. However, these protective effects could also pertain to unexplored pathways that require further investigation. Therefore, this cycle of oxidative stress, neuroinflammation, and neuronal cell death holds potential for many questions that need to be addressed in MitoQ administration. The proposed mechanisms of MitoQ's effect are summarized in Figure 10.

There are a number of limitations that should be addressed. In this work, only male mice were used, which ignores the female hormonal system that has been shown to have effects in TBI [62]. Additionally, between the animal groups, there was no vehicle-only group because MitoQ was injected in PBS, and previous data from our laboratory showed no difference between the vehicle and Sham groups.

**Figure 10.** Summary of proposed MitoQ neuroprotective activity post-CCI. When MitoQ is administered, it exerts its effects via several pathways that eventually translate into improved neurological and cognitive functions. (**A**) MitoQ helps in increasing Nr2 expression and translocation into the nucleus by affecting its sequestration by the Kelch-like ECH-associated protein 1 (Keap1) protein. This allows Nrf2 to bind Maf proteins and interact with its antioxidant response element (ARE), leading to increased expression of antioxidative enzymes such as SOD2 and CAT. (**B**) Neurons are prone to increased apoptosis and axonal shearing due to the hostile environment created by the primary and secondary injuries. MitoQ helps decrease neuronal loss and maintains axonal integrity, resulting in improved circuitry in the brain. These effects translate into enhanced cognitive and neurological functions. (**C**) MitoQ helps decrease the activation of both microglia and astrocytes and therefore might help in decreasing chemical signals produced by both cells such as tumor necrosis factor α (TBFα), interleukin 1β (IL-1β), interleukin 6 (IL-6), cytokines, damage-associated molecular proteins (DAMPs), and alarmins. During a chronic inflammatory state, these would lead to damage to brain tissue and neurodegeneration.

#### **5. Conclusions**

This study shows that MitoQ administration appears to efficiently reduce the deleterious consequences associated with moderate open head TBI at both the molecular and behavioral/cognitive levels. This paves the way towards identifying a potential therapy for TBI that may improve the quality of life for those affected by it. Moreover, the obtained data

provide an additional line of evidence on the potential role of oxidative stress and chronic inflammation in the pathology of brain disorders by mechanisms that need to be unraveled. This also opens more questions on the effects of this supplement on the mitochondrial complexes and neuronal regeneration. This emphasizes two important next steps that need to be addressed. The first is exploring further pathways implicated in the activity of MitoQ in moderate TBI. The second is conducting further assessment of biomarkers of neurodegenerative disorders to check the extent of MitoQ's protective effects at chronic time points.

**Author Contributions:** Conceptualization, F.K., K.W. and R.E.K.; methodology, M.A.H., Z.S. and C.B.; validation, R.E.K., K.W. and Y.M.; formal analysis, M.A.H. and Z.S.; investigation, M.A.H.; writing—original draft preparation, M.A.H., Z.S., M.T. and S.M.; writing—review and editing, M.G., D.S. and J.D.P.; supervision, F.K. and H.D.; project administration, H.D.; funding acquisition, M.T. and H.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work is partially funded from the American University of Beirut (AUB); Medical Practice Plan (MPP) grant PI: Riyad ElKhoury and Co-PI: Firas Kobeissy.

**Institutional Review Board Statement:** This study was carried out at the Animal Care Facility of the American University of Beirut (AUB), and all animal experiments were performed in compliance with the AUB Institutional Animal Care and Use Committee (IACUC) guidelines, with the reference number: (17-01-458).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

**Acknowledgments:** We would like to thank Yara Yehya and Leila Nasrallah for their technical contribution to the study.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

## **Neuroprotection: Targeting Multiple Pathways by Naturally Occurring Phytochemicals**

**Andleeb Khan 1,\*, Sadaf Jahan 2, Zuha Imtiyaz 3, Saeed Alshahrani 1, Hafiz Antar Makeen 4, Bader Mohammed Alshehri 2, Ajay Kumar 5, Azher Arafah <sup>6</sup> and Muneeb U. Rehman <sup>6</sup>**

Received: 15 June 2020; Accepted: 5 August 2020; Published: 12 August 2020

**Abstract:** With the increase in the expectancy of the life span of humans, neurodegenerative diseases (NDs) have imposed a considerable burden on the family, society, and nation. In defiance of the breakthroughs in the knowledge of the pathogenesis and underlying mechanisms of various NDs, very little success has been achieved in developing effective therapies. This review draws a bead on the availability of the nutraceuticals to date for various NDs (Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, Huntington's disease, vascular cognitive impairment, Prion disease, Spinocerebellar ataxia, Spinal muscular atrophy, Frontotemporal dementia, and Pick's disease) focusing on their various mechanisms of action in various in vivo and in vitro models of NDs. This review is distinctive in its compilation to critically review preclinical and clinical studies of the maximum phytochemicals in amelioration and prevention of almost all kinds of neurodegenerative diseases and address their possible mechanism of action. PubMed, Embase, and Cochrane Library searches were used for preclinical studies, while ClinicalTrials.gov and PubMed were searched for clinical updates. The results from preclinical studies demonstrate the efficacious effects of the phytochemicals in various NDs while clinical reports showing mixed results with promise for phytochemical use as an adjunct to the conventional treatment in various NDs. These studies together suggest that phytochemicals can significantly act upon different mechanisms of disease such as oxidative stress, inflammation, apoptotic pathways, and gene regulation. However, further clinical studies are needed that should include the appropriate biomarkers of NDs and the effect of phytochemicals on them as well as targeting the appropriate population.

**Keywords:** neurodegenerative diseases; phytochemicals; natural products; neuroprotection

#### **1. Introduction**

Various conditions affecting nerve cells and the nervous system due to the loss of neurons and their connecting networks are described under the superordinate phrase "Neurodegenerative diseases". They lead to disability due to gradual neuronal death in both the central nervous system (CNS) and the peripheral nervous system (PNS). Several diseases are genetic with few coming being caused due to the exposure to various toxins and chemicals. The main symptoms associated with these disorders are related to movements (ataxia) or mental functioning (dementia) or both causing morbidity

and death. This fostered the profound social and economic implications [1]. The most common NDs include cognitive and behavioral disorders, Alzheimer's disease (AD), Parkinson's disease (PD), Amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), Spinocerebellar ataxia (SCA), Spinal muscular atrophy (SMA), Vascular cognitive impairment Prion disease, Frontotemporal disease, Pick's disease, etc. The treatment available for these disorders gives only symptomatic relief to the patient by extending the lifespan to a few years [2]. Still, a lot of research is in progress to find the therapeutic markers in these diseases [3].

There has been an extensive approach to non-pharmacological therapies to combat the ill effects of NDs. Many of them include disease-modifying therapies, non-invasive brain stimulation techniques, physical exercise, adaptive physical activity, complementary and alternative medicine, and several nutraceutical compounds. In a meta-analysis, it was reported that repetitive transcranial magnetic stimulation (rTMS) therapy with Parkinson's patients resulted in mild to moderate improvement of motor activities [4]. In another study, it was reported that both rTMS and transcranial direct current stimulation (tDCS) have shown improvement in the cognitive performance of Alzheimer's patients [5]. Fisicaro and colleagues stated in their review that rTMS can be effectively used as a non-pharmacological tool for various motor and non-motor neurorehabilitation. They suggested that this technique along with other conventional rehabilitative modalities can be effectively used in clinics but still the regime is not clear [6]. Physical exercise has been a promising therapy for motor deficits for years. It could have therapeutic potential for the prevention of mental disorders and neurodegeneration. Extensive research to explain different molecular pathways involved in this therapy is under progress, suggesting multiple pathways may be involved together in neuroprotection [7,8]. The major cornerstones in managing ataxic patients are physiotherapy and kinesitherapy in the present scenario. Studies confirm that customized programs for various exercises of coordination, balance, cognitive skills, and posture maintenance have shown extensive improvement along with the conventional pharmacological therapeutics [9]. Complementary and alternative medicines have always attracted various disease prevention including NDs [10,11]. In addition to these, the various nutraceuticals [12] and phytochemicals multi-target approaches have proved to be a promising adjunct to the present treatment [13].

The large number of pharmacological or biological activities of the phytochemicals have made them appropriate candidates for the treatment of the NDs [14]. They exhibit antioxidant (resveratrol, zingerone), anti-inflammatory property (cineole, thymoquinone), inhibitors of gamma-aminobutyric acid (GABAA) receptors (diterpenes and cyclodepsipeptides), and Monoamine oxidase-B (MAO-B) receptors (selegiline rasagiline), although not proven clinically. Combating disease symptoms by phytochemicals and herbal nutraceutical is known in traditional medicines, and several new research for preventing NDs by phytochemicals are under progress [15]. In this review, our focal point is to discuss the recent advancements in the field of neuroprotection related to the NDs by various phytochemicals and to elicit their potential mechanism of actions. In particular, we discuss phytochemicals used in the prevention of all kinds of NDs and related signaling pathways and mechanism of recovery highlighting the role of phytochemicals. The basic structures of most the common neuroprotective phytochemicals are given in Figure 1.

**Figure 1.** Chemical structures of some common phytochemicals used in the prevention of neurodegenerative diseases (NDs).

#### **2. Methodology**

A Medline (PubMed), Cochrane Library and Embase based literature survey was performed by using keywords of "neurodegenerative diseases, prevention, non-pharmacological therapies, and neurodegenerative diseases, phytochemicals, Alzheimer's disease natural products, Parkinson's disease (PD) phytochemicals, Huntington's disease phytoprotection, natural product amyotrophic lateral sclerosis, phytochemicals cerebral ischemia, phytochemicals prion disease, spinocerebellar ataxia prevention by natural products, natural product and spinal muscular atrophy, frontotemporal dementia natural products, pick's disease, and natural products" till June 2020.

Two independent authors (A.K. and S.J.) screened all the titles and abstracts of the retrieved data, disagreements were resolved by the consensus of a third author (Z.I.). Duplicated entries, retracted publications, studies on other diseases or conditions different from NDs or its subtypes, studies without statistical analysis, non-English written papers, publications that are not research studies (i.e., commentaries, letters, editorials, reviews, meta-analysis), and any other article that did not fit within the scope of this review, were excluded. Articles listed in the references were also reviewed in search of more data.

A total of 873 results were retrieved and screened with the above keywords. Out of these 115 publications were selected and eventually used for qualitative analysis (Figure 2) and the same are summarized in Table 1. In more detail, 28 studies work with AD, 17 with PD, 7 with ALS, 8 with HD, 20 with VCI and stroke, 4 with prion, 7 with FTD, 2 with SMA, and 1 with SCA and others on non-pharmacological therapy and neurodegenerative diseases.

**Figure 2.** Flow diagram showing the search strategy, the number of records identified, and the included/excluded studies [16].

#### **3. Multifunctional Phytochemicals as Novel Therapeutic Agents for Neurodegenerative Disorders**

#### *3.1. Alzheimer's Disease and Related Dementias*

AD is a multifactorial, pernicious disease with genetic and environmental factors leading to irreversible cognitive impairment [17]. The main neuropathological features of AD comprises of neuronal loss, amyloid-β plaques, and neurofibrillary tangles in limbic and neocortical regions [18]. Various mechanisms are associated with the progression of the disorder which includes amyloid deposition, tau hyperphosphorylation and aggregation, loss of cholinergic system, oxidative stress, inflammation, apoptosis, glutamatergic excitotoxicity, and decrease in neurogenesis and neurotrophic factors [17,19,20]. In recent years, approved drugs for AD are only modulators of cholinergic and glutamatergic systems, which can only delay the symptoms to some extent [21]. Divergent antioxidant and anti-inflammatory medications are also approved for AD patients [22]. There are recent reports that herbal products and nutraceuticals can act as a multi-target approach as suggested in the traditional system of medicines [23]. Recently extensive research has been conducted which supports their potential in in vitro, in vivo, and clinical studies. In the list of natural compounds, curcumin is considered on the top because of its miraculous qualities. In this section, the mechanistic approach of the curcumin and other important natural compounds will be discussed. Curcumin is a polyphenolic compound and a natural herb which is an integrative part of every Indian kitchen. In literature, many beneficial properties are reported like anti-inflammatory, anti-cancerous, anti-apoptotic, antioxidative, neuroprotective [24]. Apart from that curcumin is also reported as a therapeutic agent to treat rheumatoid disorders, cough, and neurological disorders. Fortunately, it has intensive effects and have multiple sites of action [25]. The relationship between curcumin consumption and a lower prevalence of AD has been observed. In one study, curcumin emulates the improvement of the spatial learning and memory in the rat model and it is reported as an inhibitor of BAC1 in in vitro study. Curcumin has a promising effect in neuroblastoma SHSY5Y cell lines and also has a therapeutic effect on the AD-induced mechanisms [26]. Acrolein exposed HT22 murine hippocampal neuronal cells were protected by curcumin by following the BDNF/TrkB signaling [27]. Orally administrated curcumin downregulates the level of GSK3β and also inhibit the hippocampus Aβ plaques with the improved cognitive impairment and improved maze test in in vivo study [28]. Curcumin effectively counteracted the p25-mediated glial activation and pro-inflammatory chemokines/cytokines production in p25Tg mice. Moreover, this curcumin-mediated suppression of neuro-inflammation reduced the progression of p25-induced tau/amyloid pathology and in turn ameliorated the p25-induced cognitive impairments [29]. Therefore, through the series of research, curcumin is found to be promising phytochemicals against Aβ accumulation and would be a determined remedy against AD. Pilot study with curcumin and *Ginkgo* on 34 possible or probable AD patients was carried out with curcumin and *Ginkgo* extract. It was found that no side effects of curcumin along with that mini-mental examination scores or plasma amyloid beta40 level between 0 and 6 months [30]. AD-related pathology in the brain is well documented, the disease has also been reported to affect the retina, a developmental outgrowth of the brain, which is more accessible for imaging. Early detection of AD, utilizing curcumin fluorescence imaging to highlight Aβ aggregates in the retina. Results show that retinal fluorescence imaging is justified with brain plaque burden from PET scan [31]. Apigenin, a known phytochemical also targets the AD by its antioxidative effects. In plants, Apigenin is found as aglycon and more often in glycoside form. Apigenin inhibits the copper-mediated β-amyloid neurotoxicity (copper-induced) via antioxidative mechanism followed by MAPK signal inactivation in an AD cell model [32]. The Apigenin follows the ERK/CREB/BDNF pathway for neuroprotection [33]. On the list of phytochemical-induced mechanistic pathways, genistin also has an important role in neuroprotection. It is a component of the soya bean meal. The effect of genistin (100 μM) was studied on the in vitro culture of rat neurons [34]. It was observed that the amyloid-beta peptide instigated the damage of mitochondrial membrane potential, nuclear DNA damage, high expression of *Bcl-2* associated X protein (BAX), p53, Caspases and lower expression of B-cell lymphoma-2 (Bcl-2) [35]. The 100 μM dose of genistin was found to be efficient against the deleterious effects of AD and restores the neuronal activity by following the caspase signaling pathways [36]. It is also reported to lower calcium overload and reverse the low fluidity of the neuronal membranes in the AD model. Free radicles were also reduced with the use of genistin and thus it is suggested for the targeting of anti-ROS (reactive oxygen species) pathway and protect the cells from death. Astrocytes are also studied for the efficiency of genistin [37]. Pretreatment of 5 μM concentration of genistin was used against astrocytes and it protects by working on interleukin 1 beta (IL-1β), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) cascade, and reversing the effect of NDs in the AD model [38]. In another study, 50 μM concentration of genistin was effective and initiate IL-1β /TLR4/NF-κB/ IκB-α cascade against neuroinflammation induced by Aβ in the secondary culture of C6 [39]. It can downregulate TNF-α, IL-1β, TLR4, and NF-κB and promote the upregulation of IκB-α. Other phytochemicals are also having miraculous properties against many NDs specifically AD [40]. Genistein protects Aβ-induced neurotoxicity by inducing the PKC signaling pathway, which further regulates the activities of α- and β-secretase and thereby inhibits the formation and toxicity of Aβ [41]. Genistein also protected the hippocampal neurons against injury by calcium/calmodulin dependent protein kinase IV protein levels in AD model [42]. *Ginkgo biloba* is also considered as a neuroprotective agent. Ginkgolide A inhibits Aβ-induced neuronal degradation via JNK phosphorylation and NMDA/AMPA-induced depolarization, thus suppress the activity of plaque [43]. EGb 761 increases the neurogenesis in the adult hippocampus region and CREB phosphorylation in the transgenic mouse model of AD [44]. In one study, *G. biloba* demonstrated protection against a high dose of Bisphenol A (BPA) and played a role in the improvement of cognitive deficits [45]. A flavonoid, luteolin, would be discussed against the AD model as a possible therapeutic agent. In an in vivo study (rat model), it protects against the cognitive dysfunction which was induced by chronic cerebral hypoperfusion [46]. It is also found to be protective against high-fat diet-induced cognitive abnormalities. GSK 3α isoform is involved in the mechanistic signaling. The inactivation of GSK 3α isoform phosphorylates the PS1 which is the catalytic core of the γ-secretase complex causing a decrease in PS1-APP interaction and generation of Aβ. In literature, the role of luteolin against zinc-induced hyperphosphorylation of the protein τ in SH-SY5Y secondary cell culture is reported [47]. Luteolin is suggested as a promising candidate because of its antioxidative properties as well. In the AD animal model, luteolin slows down the escape latency and traveled distance parameters in the morris water maze while increasing the time spent in the target quadrant [48]. Melatonin modulates the proteins (i.e., GSTP1 and CPLX1) that are involved in depression and anxiety. The modulation improves the neuropsychiatric behaviors in the AD model [49]. Melatonin improves anxiety and depression-like behaviors and targets the proteomic changes in the triple transgenic AD mice model [50]. A multicenter, randomized, placebo-controlled clinical trial of 2 dose formulations of oral melatonin coordinated by the National Institute of Aging-funded Alzheimer's Disease Cooperative Study, and it was found that melatonin was effective against AD [51]. Naringenin protects Aβ25-35-caused damage via ER and PI3K/Akt-Mediated Pathways [49,52]. Naringenin also protects AlCl3/D-galactose-induced neurotoxicity in the rat model of AD via inhibition of attenuation of acetylcholinesterase levels and attenuation of oxidative stress [53]. A new study suggested the therapeutic effects of piperine against oxidative insult, neuroinflammation, and neurochemical changes induced by ICV-STZ infusion in mice [54]. In the human brain, microvascular endothelial cells from fibrillar β-amyloid are protected by quercetin [55]. The flavonoid quercetin improves Alzheimer's disease pathology and protects cognitive and emotional function in aged triple transgenic Alzheimer's disease model [56]. in vitro AD model, resveratrol works on oxidative damage via activating mitophagy [57]. Resveratrol is found to be active against the harmful process that occurs in AβPP/PS1 mouse hippocampus and inhibit memory loss [58]. For the AD model, resveratrol is studied in randomized, double-blind, placebo-controlled, phase II trial. Resveratrol was found to be safe, effective, and can protect the BBB integrity with CNS response [59]. Thymoquinone inhibits the Aβ-induced neurotoxicity by blocking the mitochondrial dysfunction and oxidative stress [60]. S-allyl cysteine also targets the oxidative stress-induced cognitive neuronal impairment and provide possible rescue from the diseased [61]. In mice model treated with D-galactose, S-allyl cysteine involves in the alleviation of β-Amyloid, oxidative damage [62]. Eucalyptol ameliorates inflammation induced by Aβ(25–35) in differentiated PC12 cells by following the NOS-2, COX-2, and NF-κB associated pathways [63]. Silibinin and silymarin administration could recover memory impairment and could decrease Aβ plaque in the brain of APP/PS1 mice [64]. By following the BDNF/TrkB pathway, Silibinin ameliorates anxiety/depression-like behaviors in amyloid β-treated rats [65]. From all the above discussion of the phytochemicals, it is imminent that

researchers would be able to hit the specific targets for providing promising therapeutic relief against AD Figure 3. The details of several phytochemicals in AD protection are enlisted in Table 1.

**Figure 3.** Schematic representation of the several mechanisms associated with Alzheimer's Disease (AD) and their possible prevention by some phytochemicals. The main hallmarks of AD; amyloid plague and NFT are prevented by curcumin and luteolin via GSK 3β pathway. Curcumin, genistin, and luteolin decreases the oxidative stress in the neuron. Neuroinflammation is also inhibited by genistin and apigenin. The pathway of apoptosis, activated in AD is blocked by the use of apigenin. Apigenin also activates BDNF/ERK/CREB pathway leading to neuronal survival.

deoxynucleotidyl

 TLR2: Toll-like receptor 2, TLR4:

transferase-mediated

 dUTP nick-end labelling+,

Thiobarbituric

Neuron-specific

Gamma-glutamyl

 transferase.

 Class III β-tubulin, TUNEL+: terminal

 acid reactive substances, TH+: Tyrosine hydroxylase,

**Natural**

**In-Vitro**/**In-Vivo**

type information

Toll-like receptor 4, TrkB: Tropomyosin TWEAK: TNF-related weak inducer of apoptosis, γ-GT:

 regulation 2 homolog 1, SNCA: Synuclein Alpha, TBARS:

 receptor kinase B, Tuj-1:

#### *3.2. Parkinson's Disease and Parkinsonism*

PD is the second most prevalent NDs of the elderly after AD, characterized by dopaminergic neuronal loss in substantia nigra pars compacta region of the brain, the striatum, locus ceruleous, raphe nuclei, nucleus basalis of meynert, and hippocampus. The main sign and symptoms of PD patients are resting tremors, rigidity, bradykinesia, muscular impairment, postural instability, difficulty in walking, soft speech, sleep problems, slow handwriting [127].  The treatment of PD mainly rotates around the enhanced activities of dopaminergic neurons or inhibition of cholinergic effects [131]. Several mechanisms associated with PD accounts for oxidative stress, mitochondrial dysfunction, apoptotic pathway activation etc. [132,133] There is no cure for PD but drugs available in the market increases the life expectancy with relief in the symptoms [131]. Besides, the drug therapy demonstrates many side effects in patients [134,135]. Therefore, researchers have to treat PD at molecular and cellular levels, and use such treatments that have a multifactorial approach. Several scientists have reported plant compounds to be effective in the treatment of PD. After dissecting the oxidative stress role in the pathophysiology of PD, the phytochemicals having antioxidative properties are drawing the attention of researchers [136]. This section deals with the phytochemicals which are generally considered as antioxidative agents. In many fruits and vegetables (apples, broccoli, and onions), a polyphenolic compound is detected named quercetin. It restricts the hippocampal neuronal damage resulting in improved memory and learning (by maze test) in the rat model [137]. In one study, when quercetin was administrated to the PD model, it reversed the neuronal impairment via antioxidative pathways [138]. In PD MitoPark transgenic mouse model, quercetin protects against mitochondrial dysfunction and progressive dopaminergic neurodegeneration [66]. Quercetin also plays a specific role in the upregulation of cognitive effects in the 6-hydroxydopamine (6-OHDA) rat PD model [67]. An isoflavone, genistin is also having several health benefits including nourishment of neurons [139]. It is known as antioxidative, anti-inflammatory, and neuroprotective agent. Genistin (oral dose 10, 50, or 100 mg/kg for one week) acts as a therapeutic agent against LPS-induced cognitive impairment. These dose-dependent treatments of genistin determines to lessen memory deficits, decrease the malondialdehyde levels in the hippocampus region, increases the level of superoxide dismutase, catalase, and glutathione [140]. Moreover, genistin reformed the level of AChE activity in the hippocampus against LPS insult in in vivo model. It targets the modulation of Nrf2/NF-κB/IL-6/TNFα/COX2/iNOS/TLR4/GFAP and thus promote neuronal protection via inhibiting the neuronal inflammation and improve cognitive deficits [140]. Genistein shows the neuroprotection in the A53T mutant α-synuclein overexpressed SH-SY5Y cells [68]. Genistin protects the Transgenic *Drosophila* Model of Parkinson's Disease against oxidative stress [69].

Other phytochemical Naringin was studied and was found to be involved in the neuroprotection by triggering the Nrf2/ARE signaling against 6-OHDA-induced neurotoxicity [141]. In in vitro study (neuroblastoma SH-SY5Y cells), it is reported to show neuronal protection by blocking the c-Jun N-terminal kinase (JNK) phosphorylation and regulation of BAX in the rotenone-induced cell line [142]. Additionally, it also inhibits the caspase-3 and PARP cleavage [143]. Naringin can lead to the anti-inflammatory pathway and provide neuroprotection in PD [70]. In in vitro PD model of SH-SY5Y cells, curcumin imparts a role in neuroprotection against toxic insult via controlling the HSP-90 [71]. Curcumin was studied in Pheochromocytoma Cell Line 12 (PC12) cells against 1-methyl-4-phenylpyridinium (MPP+) toxicity and fixation of mitochondrial membrane potential was found against enhancement of intracellular ROS which leads to the instigation of iNOS, and over-expression of Bcl-2 [144]. Curcumin leads to the restoration of mitochondrial membrane potential (MMP) which causes the up-gradation in Cu/Zn SOD and modulates NF-κB nuclear translocation by inhibition of interleukin-6 (IL-6) and TNF-α [145]. It was further reported that it lowers the iNOS mRNA levels and inhibits the induction of NF-κB. It is found to be effective against oxidative stress via following cytochrome c release or caspase-3 activation. In in vitro study, it is reported to act on the aggregation of synuclein inhibition and aggregation of the PD associated, A53T mutant-synuclein

in SH-SY5Y cells in a dose-dependent manner [146]. By inhibiting the α-synuclein aggregation in lipopolysaccharide-induced PD model curcumin plays a role as a promising therapeutic agent [72]. Epigallocatechin-3-gallate (EGCG) is also an important bioactive component with neuroprotective potential. The mode of action of EGCG is on neuroinflammation, oxidative stress, and autophagy. Likewise curcumin, EGCG also ameliorates the mitochondrial restoration by antioxidative properties and associated signaling pathways i.e., via blocking the expression of COX-2, iNOS, and all other enzymes which causes the generation of pro-inflammatory mediators [147]. EGCG protects by antioxidative activity against 6-OHDA administration in SH-SY5Y cells (in vitro dopaminergic model) [148]. EGCG also functions as antagonistic against the NF-κB transcription factor and participates to remove the poisonous accumulation of amyloidogenic α-synuclein in many in vitro models. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treated mice also show improvement when EGCG is administrated orally [149]. EGCG also inhibits the oxidative stress in rotenone-induced in vitro model of SH-SY5Y cells [73]. In the MPTP-induced mouse model of PD EGCG helps in the modulation of peripheral immunity [74]. A natural polyphenolic compound, resveratrol, is involved in the mitochondrial biogenesis by initiating the antioxidative cascade mechanism. It also protects neurotoxic chemicals i.e., 6-OHDA and MPTP in in vitro models [150]. This natural compound shows the promising cure against cell toxicity induced by dopamine (DA) and reverse the neuronal impairment by following the cAMP/PKA pathway and thus inhibit the α-synuclein aggregation [151]. Further study also points out the SIRT1/AMPK/PGC1-α axis as a key neuroprotective pathway and provides a rationale for exploring the therapeutic potential of resveratrol in delaying PD progression [152]. By leading the CXCR4 signaling pathway, resveratrol helps in the restoration of the 6-OHDA-induced damage in the PC 12 cells [75]. Resveratrol attenuates the PD like condition via inhibition of programmed cell death through inducing MALAT1/miR-129/SNCA signaling pathway [76]. A total of 119 participants were randomized to placebo or pure synthetic resveratrol 500 mg orally once daily with a dose-escalation by 500-mg increments every 13 weeks until a final dose of 1000 mg twice daily was reached for the final 13 weeks. Visits occurred at screening, baseline, and every 6 weeks with a total resveratrol exposure of 52 weeks. Compliance with resveratrol and with placebo was confirmed by mass spectrometry of blood samples. Resveratrol may have many other molecular effects, including anti-inflammatory, antioxidant, and anti-Aβ aggregation. 480 PD patients, randomized, double-blind three dosage groups of green tea polyphenol and one placebo control group. Delay in motor function progression, increased cognition, mood, and quality of life was reported [153]. 20 double-blind, crossover, randomized, placebo-controlled phase 1 study BIA 6-512 (trans-resveratrol) 25 mg dose, 50 mg dose, 100 mg dose To study BIA 6-512 effect on levodopa pharmacokinetics when administered with levodopa/benserazide [154]. Rutin, another phytochemical act as an anti-inflammatory agent for neuroprotection which is associated with the involvement of microglia response, cytokines level, and iNOS activation [155]. Rutin facilitates protection in 6-OHDA-induced neurotoxicity in rat pheochromocytoma (PC-12) cells [77]. Rutin also targets the oxidative stress and protects dopaminergic neurons in the animal model of PD [78]. Hesperidin can be a possible promising guide for the designing of inhibitors of mutant SOD1 for therapeutic purposes. Antioxidative and anti-inflammatory properties make hesperidin reliable herbal compound against NDs. ERK/Nrf2 are key signaling molecules for following the neuroprotective pathway [156]. Neurotoxicity induced by 6-OHDA can be suppressed by the use of hesperidin in the mice model of PD [79]. Piperine treatment attenuated rotenone-induced motor deficits, and rescued the loss of dopaminergic neurons in the substantia nigra [157]. Piperine stimulates the anti-apoptotic and anti-inflammatory pathway and shows protection in 6-OHDA-induced PD rat model [80]. PD is known as the second rank on the list of NDs with poor availability of effective treatments still, the studied phytochemicals have given hope for promising and long-lasting therapy. Figure 4. The briefing of phytochemicals and their therapeutic effects on PD are listed in Table 1.

**Figure 4.** Diagrammatic presentation of Parkinson's disease, cause, and role of phytochemicals to reverse the sign of PD. Mitochondrial dysfunction, oxidative stress, and uncontrolled cell death leads to the accumulation of Lewy bodies and resultant neuronal damage. Phytochemicals function with multiple targets. Curcumin targets the inducible nitric oxide synthase (iNOS) and regulates the ratio of BCL-2/BAX which leads to the restoration of mitochondrial function. Genistin increases the activity of SOD, Catalase, and GSH and protect the neurons from damage ignited by oxidative stress. Resveratrol inhibits the formation of Lewy body (aggregation of α-syn protein-a major cause of PD). Rutin protects the neuron by leading the anti-inflammatory pathway and EGCG inhibits the transcriptional factor NFκB and restores the normal function of mitochondria. EGCG also plays a role to inhibit DNA fragmentation. All over the activities of phytochemicals escape neurons from damage.

#### *3.3. Amyotrophic Lateral Sclerosis*

ALS also called motor neuron disease (MND) or Lou Gehrig's disease is another NDs that is heterogeneous and both upper and lower motor neurons are degenerated causing motor symptoms. The initial symptoms vary between individuals, some exhibiting spinal-onset disease with muscle weakness of limbs, and others present with bulbar-onset disease in which they have dysarthria and dysphagia [158]. The preponderance of patients face death within 3–5 years after the first sign of disease due to respiratory failure [159]. The cause of this diseases is unknown but few cases are linked to familial history with gene mutations as a major contributor [160] and associated mechanisms of oxidative stress [161], calcium toxicity [162], inflammation [163], chronic viral infections [164], and excitotoxicity [165]. Other symptoms are spasticity, sialorrhoea, pain, muscular cramps, liver problems, deep venous thrombosis, mood alterations, respiratory insufficiency, fever, vomiting, fatigue, and nausea. In the case of ALS, the ability of the brain to convert the toxic radicals into non-toxic substances gets compromised because of mutations in *SOD1* [166]. Although, this kind of mutation does not lead to the progression of the disease but the mutation makes the *SOD1* more prone to the misfolding which causes the accumulation of protein and thus involves the reduction in the activity of motor neurons. The treatment of ALS is often symptomatic with Riluzole (Rilutek) and Edaravone (Radicava) as the only drugs approved by the FDA. These drugs can only increase the life expectancy by three to six months. Much of the efforts of physicians and other health workers is to make the quality of life (QOL) better for ALS patients. Several studies on natural plants and their active compounds are

in progress to combat the dreadful symptoms of this disease. The molecular mechanisms and potential therapeutic role of the plant compounds have to be studied and implemented.

Few phytochemicals and their possible known mechanism of actions will be discussed in this section. Ginseng root shows the protective effect in *SOD-1* (G93A) transgenic mice [81]. An isoflavone, genistin can easily cross the blood–brain barrier (BBB) and attributes its role by anti-inflammatory activity via extracellular-signal-regulated kinase (ERK) and microtubule-associated protein kinase (MAPK) signaling in the microglia which leads to the neuronal inflammation. The neuroprotective role of genistin was detected in the SOD1-G93A mice [82]. Ginseng roots inhibit the onset and increase the survival of affected cells delays the onset and prolongs the survival of affected cells [167]. The most promising phytochemical, curcumin has a protective role in ALS too. It acts for mitochondrial biogenesis leading the AMP/PGC-1α and/or Nfr2 pathway [83]. The clinical studies show the effectiveness of curcumin for slowing down the progress of the disease. Curcumin helps in the improvement of aerobic metabolism and oxidative stress. The antioxidative mechanism is responsible for leading the protective effect against ALS [83]. A studied phytochemical named withaferin A shows an anti-inflammatory effect by working as antagonistic of NF-κB and is found to be effective for the improved motor activity in transgenic mice model [84]. ALS mouse model generated by the point mutation in SOD1 was studied and it was observed that by the withaferin treatment there was a late progression of the disease and more survival time in comparison to the non-treated ALS mice. The root extract of *Withania* was also tested against mutated transgenic mice (TDP-43A315T mice) and manifested with removal of abruptly cytoplasmic motor neurons, reduction in inflammatory markers, and improvement in cognitive performance [168]. It is reported that ALS is following numerous pathways therefore, combined therapy could be more efficient for targeting more than one signaling pathway within a single attempt. EGCG has a power of antioxidation, hampered activation of caspase-3, NF-κB and thus reduces microglial activation resulting in decreased neuroinflammation and increased neuronal survival [169]. EGCG stimulates the iNOS/NF-κB/ Caspase-3 and shows the increased number of neuronal cells, lower microglial activation in transgenic mice model [85]. One more phytochemical named celastrol also reveals the anti-inflammatory, antioxidative property. Celastrol exhibits an antioxidative effect in SOD1G93ANSC34 cells by leading MEK/ERK and PI3K/Akt signaling pathways [86] Celastrol protects the H2O2-induced oxidative stress model by activating the antioxidative pathway in ALS model [86]. The bran oil treatment helps in the significant enhancement of antioxidative enzymes and suppress the effect of ROS and malondialdehyde (MDA) in rotenone-induced ALS model [170].

ALS study was also conducted in bone marrow-derived mesenchymal stem cells (MSCs) by activating the SIRT1/AMPK pathway and it is noted a significant decrease in the ALSMSCs compared to normal healthy control originated BM-MSCs by using resveratrol. Neuro-progenitor markers were increased in resveratrol treated ALS-MSCs [87]. All these phytochemicals along with the ones enumerated in the table could be promising therapeutic agents which can be the best alternative of commercial drugs with negligible side effects Table 1.

#### *3.4. Huntington's Disease*

HD is an annihilative, inherited, and familial illness, with progressive loss of brain and muscle functions. This is a single-gene disorder in which there is programmed degeneration of neurons within different regions of the brain. In wild type gene, there are 6-35 CAG repeats in exon 1 of the *Huntingtin (Htt)* gene on chromosome number 4 whereas, in the affected persons, this repeat increases to more than 36 [171]. As a result of this, there is an accumulation of Huntingtin protein in the neuron causing its death. Almost 95% of GABAergic medium spiny neurons (MSNs) projecting to substantia nigra and globus pallidus are lost causing atrophy of cortex, thalamus, and hypothalamic nuclei. The symptoms include concentration problems, short-term memory, tumbling, lack of focus, clumsiness, depression weight loss, feeding problems, difficulty in speech, uncontrolled face movements, itching etc. [172]. There is a lot of research going on in the field of HD prevention and treatment. Many experimental models have been experimentally validated to test the efficacy of different drugs. Hitherto, there is no cure for the disease, drugs available only give symptomatic relief. There have always been natural compounds linked to various diseases and a lot of them have shown promising effects in preclinical studies.

One of the major factors for the progression of HD is the abrupt function of mitochondria [173]. The activity of complex II of the respiratory chain gets reduced in the affected brain region. Protopanaxtriol limits the overproduction of free radicals and attenuates the expression of heat shock protein 70 (Hsp-70), along with the restoration of SOD activity inhibiting the formation of free radicals. Protopanaxtriol enhances the Nrf2 entry to the nucleus and increases the expression of Heme oxygenase-1 (HO-1) and NAD(P)H dehydrogenase [quinone]1(NQO1) [88]. Melatonin also has been shown to reduce neural damage in PD, AD as well as in ischemia-reperfusion injury against d-aminolevulinic acid and a variety of other neural toxins. In the case of mitochondrial injury, induced by 3-nitropropionic acid there was a restoration by the treatment with melatonin by leading the antioxidative pathway [88,174]. Melatonin also rescues the HD animal model from the oxidative stress induced by 3-nitropropionic acid [89]. Resveratrol attenuates the expression of ATG4 and permits the lipidation of LC3 and promotes the mortification of the polyQ-Htt accumulation and saves the neuronal cells against dopamine-induced toxicity [90]. EGCG inhibits the accumulation of misfolded proteins and oligomerization of mutant Htt exon 1 *protein* in vitro, which indicates the early interference of EGCG in the misfolded protein accumulation [175]. In another study, EGCG reduced the cytotoxicity and polyQ-mediated htt protein aggregation in a yeast model of HD [176]. EGCG attenuates the adverse effect of misfolded huntingtin protein in early-stage in HD model [91].

One more phytochemical, trans-ε-Viniferin (viniferin), was also found to be effective against mutant *Htt*-induced depletion of SIRT3 and thus save the cells from a mutant protein [177]. Viniferin also plays a crucial role in reducing the level of free radicals and thus participate in the protection of MMP in the cells having mutant *Htt*. The presence of mutant *Htt* indicates the lower deacetylase activity of SIRT3 which leads to decreased cellular NAD levels with the biogenesis of mitochondrial cells [178]. Viniferin enhances the mitochondrial biogenesis by activation of AMP-activated kinase [92]. It was reported that systemic 3-NP administration significantly enhances the level of lipid peroxidation (LPO), nitrite and lactate dehydrogenase (LDH), downregulate the antioxidant enzyme (superoxide dismutase and catalase) levels, and inhibit the synthesis of ATP by blocking the activity of mitochondrial complex in the different regions (striatum and cortex) of the brain [179]. Figure 5. The root extract of *Withania somnifera* is used against 3-NP-induced gait abnormalities, mitochondrial dysregulation, and oxidative stress, in striatum and cortex of the rat brain, in vivo model [93]. The detailed functions of phytochemicals are well explained in Table 1.

#### *3.5. Vascular Cognitive Impairment*

Vascular cognitive impairment is distinguished by a specific cognitive profile involving preserved memory with impairments in attentional and executive functioning [180]. Vascular dementia should be broadened to recognize the important part cerebrovascular disease plays in several cognitive disorders, including the hereditary vascular dementia, multi-infarct dementia, post-stroke dementia, subcortical ischemic vascular disease and dementia, mild cognitive impairment, and degenerative dementias (including Alzheimer's disease, frontotemporal dementia, and dementia with Lewy bodies). Alzheimer's disease (AD) and vascular dementia (VD) share key pathologies including oxidative damage, oral supplement of phytochemical medicines, which are well-known for their antioxidant properties, can be a viable therapy for both types of dementia. In this study, the therapeutic potential of the *Aster ageratoides* extract (AAE), was found to be effective in experimental rat models of AD and VD. These results provided evidence that AAE supplements can exert anti-AD and -VD efficacies and suggested that AAE might be used as an edible phytotherapeutic for the two major types of dementia [94]. Another study suggested that bilobalide (BB) protected against learning and memory impairments, neuronal apoptosis, and oxidative stress in a rat model of AD induced by Aβ25–35 peptide. Additionally, the inhibition of TNF-α and Aβ1–40 expression is also involved in the action mechanism of

BB in this experimental model. The results indicated that BB has protective effects on Aβ25–35-induced learning and memory impairments and neuronal oxidative damage [95]. *Ginkgo biloba* extract (EGb 761) is widely used to treat cerebral disorders. EGb761 increases proliferation of neural stem cells in the subventricular zone and dentate gyrus, and significantly improves learning and memory in rats with vascular dementia. Studies reported the effects on endogenous NSCs in rat models of vascular dementia and NSCs in the mouse cochlear. *G. biloba* seemed to slow down the cognitive deterioration in patients with VCI, but the effect was shown in only one of the four neuropsychological tests administered [96]. In a randomized, double-blind, placebo-controlled clinical trial *G. biloba* standardized extract showed effectiveness against vascular cognitive impairment [97]. Huperzine A is a new type of *Lycopodium* alkaloid monomer isolated from *Huperzia serrata*. It inhibits acetylcholinesterase activity and increases levels of acetylcholine in the brain thereby improving cognitive function in patients with dementia [98]. A total of 92 participants were included in the two studies, with 46 in the Hup A group and 46 in the control group. The number of patients in the individual studies ranged from 14 to 78, and the duration of trial ranged from 12 to 24 weeks [99].

**Figure 5.** Diagrammatic presentation of Huntington Disease progression and protection of neurons with phytochemicals: 6-35 CAG repeats lead to the formation of Huntingtin protein which consequently accumulates in the neurons and damage motor neurons. In the early stage, damaged motor neurons result in short term memory while on a later stage, this condition changes into depression, weight loss, and many more. The studied phytochemicals generally work against free radicals and restrict the accumulation of Huntingtin protein in the cells. This inhibition further leads to neuronal survival. Resveratrol works on LC3 lipidation while protopanaxtriol and viniferin leads to the antioxidative pathway and restore the function of mitochondria to protect the neurons and stop further accumulation of Huntingtin protein in neurons.

Stroke is itself not a neurodegenerative disease but can lead to a secondary neurodegeneration. It has been known to be a major cause of VCI. It is the third major cause of death in brain disorders worldwide [181]. There is a sudden cessation of transient or permanent reduction of the blood flow to a particular area in the brain leading to deficiency of oxygen, glucose, and energy. The main region affected in the case of Ischemia is cornu ammonis 1 (CA1) of the hippocampus [182]. There is the death of pyramidal neurons of the CA1 region, delayed to 4 to 5 days of ischemia, hence the name is

"delayed neuronal death" (DND). It is followed by a series of pathological processes working together including excitotoxicity, oxidative stress, apoptosis, inflammation, gliosis of astrocytes, and microglia in the CA1 region after ischemia/reperfusion. There is a huge burden of the depletion of the endogenous antioxidants and loss of mitochondrial function engendering neuronal death via intrinsic programmed cell death causing behavioral and histological alterations [183]. All this is followed by the breakdown of BBB causing edema. Edema activates the secretion of proinflammatory cytokines like TNF-α, IL-1β, and IL-6 by activated immune cells [184]. The oxidative stress pathway along with the inflammatory and apoptotic pathway causes further loss of neurons and brain tissues expanding the infarct area [184]. The treatment of this dreaded disease may lie in the science of phytochemicals. Many studies showing prevention against stroke have been extensively published, some of them are discussed below.

Resveratrol has been used in one of the studies where it is reported that pretreatment of resveratrol improved the neuronal functions by lowering the chances of ischemic injury in in vivo model. Additionally, it also inhibits axonal deterioration and promotes neuronal growth and synaptic formation. In in vitro study, it is found to be effective against oxygen-glucose deprivation/reoxygenation (OGD/R) injury and promoted neural stem cell (NSC) proliferation [100]. SIRT 1 is involved in the Shh signaling arbitrated the effects of resveratrol on the OGD/R injury neuronal model [100]. Its administration leads to neuronal survival, reticent the apoptosis, and synaptic formations with the upregulation of SIRT1. In rat stroke model, resveratrol slows down the ischemic injury and upgraded the neuronal functions by Sonic Hedgehog Signalling [101]. Ginkgolide B stimulated the proliferation and differentiation of neural stem cells in the in vivo models of NSCs in the rat brain following cerebral ischemia, thereby increasing the proportion of neurons to improve neurological outcomes [102]. Ginkgolide B is a potential neuro-protectant and in a study, the cellular and molecular mechanisms were dissected out by following the BDNF, EGF, and Suppressor of cytokine signaling 2 (SOCS2) [185]. Another phytochemical, curcumin, extensively studied shows beneficial effect in case of stroke too. The role of inflammation is the key factor in raising the problem of the ischemic stroke. Curcumin has shown the neuroprotective effect in the in vivo model of brain ischemia by showing anti-inflammatory properties [186]. It has also shown protection against ischemic injury in N2a cells and mouse brain with stroke [103]. Curcumin plays a significant role in neuronal survival and proliferation in the model of global brain ischemia [104]. EGCG, another phytochemical, also can remove the free radicals by increasing the antioxidative enzyme level and also downregulating the MMP-9. It inhibits the neuronal damage in transient focal cerebral ischemia instigated by middle cerebral artery occlusion (MCAO) in mice. It follows the mTOR-AMPK pathway in stressed ER and impaired the oxidative stress responses and enhance autophagy-dependent survival. It also promotes cell multiplication, differentiation, neovascularization, tube formation, and cell homing by associating the vascular endothelial growth factor (VEGF), regulation of BAX, Bcl-2, LC3B, caspase-3, mTOR and Beclin-1 [187]. It has also protected HBMVECs from Ischemia/Reperfusion injury by ameliorating apoptosis and autophagy and promoting neurovascularization [105]. EGCG has a protective effect on rat brain injury induced by MCAO, possibly by modulating the PI3K/Akt/eNOS signaling pathway [106]. Baicalein also has shown a promising effect in the subacute phase of cerebral Ischemia/reperfusion (I/R) injury in a rat model of ischemia-induced by occlusion of the middle cerebral artery (MCA). Furthermore, it is showed that baicalein inhibits the expression of NF-κB by reducing the IκBα phosphorylation and nuclear translocation of NF-κB/p65, this cascade of signaling molecules is associated with the down-regulation of the pro-inflammatory factors IL-6, IL-18, and TNF-α [107]. Additionally, baicalein inhibits the phosphorylation of ERK, p38, and JNK. These key molecules were involved with the modulation of microglia/macrophage M1/M2 polarization. Studies show the antioxidative activity by decreasing the expression of caspase-3 along with increasing the Bcl-2/Bax ratio, thus protecting the neurons. Baicalein follows the PI3K/Akt/mTOR signaling pathway via decreasing the LC3-II/ LC3-I ratio [188]. Figure 6. *Ligusticum chuanxiong* is used as a therapy for cerebrovascular and neuronal diseases. It works against OGD-reoxy-induced injury and can induce protective HSP-70 expression via the activation of the MAPK pathway [108]. Sinomenine, a natural

phytochemical shows the neuroprotection by behaving as an anti-inflammatory and anti-apoptotic agent in a mouse model of middle cerebral artery occlusion (MCAO) and astrocytes/microglia treated with oxygen-glucose deprivation (OGD). It was observed that OGD downregulates the p-AMPK and at the same time activates the inflammasome NLRP3 in mixed glial culture (in vitro model) [109,189]. Honokiol, the main biphenyl neolignan, also shows the antioxidative effects, anti-inflammatory, anti-apoptotic activity. It helps to reduce the cerebral infarction [190]. Honokiol attenuates the inflammatory reaction during cerebral ischemia-reperfusion via inhibiting the by NF-κB activation and cytokine production of glial cells [110]. Zingerone targets the oxidative stress and provide protections in focal transient ischemic rats model [111]. Zingerone (4-(4-hydroxy-3-methoxyphenyl)-2-butanone) is an innocuous, easily available, and economically cost-effective compound with promising therapeutic effects. Having anti-inflammatory, antispasmodic, antioxidative properties, zingerone protects against oxidative stress and damage. It leads to the antioxidative pathway for protection, thus help against ischemic insult [111]. Perillyl alcohol ameliorates the condition of ischemia-reperfusion injury via inhibition of the oxidative damage by following the NF-κB, COX-2, and NOS-2 associated pathway in in vivo Rat model [112]. It is also reported that piperine vanquishes the cerebral ischemia–reperfusion-induced inflammation anti-inflammatory pathway i.e., NOS-2, COX-2, and NF-κB [113]. The list of phytochemicals and the associated mechanisms are explained in Table 1.

**Figure 6.** Overall pathogenesis of neuronal loss in ischemic stroke and their prevention by phytochemicals. Oxidative stress, found in the neurons of the ischemic brain is prevented by resveratrol and curcumin. These phytochemicals also prevent the inflammation and apoptosis induced by oxidative stress. Baicalein prevents the formation of inflammasome reducing inflammation and edema. Resveratrol activates SMO/GLI pathway leading to the development of hippocampal neuronal circuit. It also activates the PI3K/Akt/mTOR pathway leading to cell survival. Another phytochemical ginkgolide B acts on MEK/ERK/SOCS2 pathway causing inhibition of inflammation in neurons.

#### *3.6. Prion Disease*

Prion disease is also known as transmissible spongiform encephalopathies (TSE) and is the only naturally occurring infectious protein misfolding disorder. Human prion disease is caused by the unnatural conversion of normal prion protein (PrPc) into an abnormal form of a protein called prions (PrPsc), it stands for pertinacious infectious particles [191]. PrPc is a surface glycoprotein expressing in multiple tissues, however, it is highly expressed in the CNS [192]. The onset and progression of the prions disease are explained by the "protein only-hypothesis", wherein it is understood how a protein self-replicates without a nucleic acid. It claims that PrPc itself is the infectious agent [193]. During the progression of prion disease, the infectious protein PrPsc aggregates and becomes resistant to proteinase digestion. This is followed by PrPsc-seeded conversion of PrPc into PrPsc, therefore, resulting in the high accumulation of PrPsc in an individual, which leads to neurodegeneration and the clinical manifestation of the disease [194]. This disease is classified as rare both in the progression and manifestation [195].

Several studies have been done to identify the potent agents for drug development for Prion disease. In a recent study, synthetic human prion protein (PrP 106-126) was used as an inducer to manifest prion diseases like signaling. Baicalein from *Scutellaria biacalenesis* attenuated ROS production and also inhibited ROS-induced mitochondrial dysfunction in PrP 106-126-induced-SH-SY5Y. JNK plays an important role in PrP 106-126-induced neuronal apoptosis in SK-N-SH cells, however, baicalein inhibited the JNK signaling in SK-N-SH cells [114]. Resveratrol, a flavonoid with several bioactivities is also known to prevent the PrP 106-126-induced neurotoxicity in SH-SY5Y and SK-N-SH cells by activating autophagy. Thus preventing the mitochondrial dysfunction, apoptosis, and neuronal cell death [115]. Autophagy is a cellular mechanism opted by several phytochemicals to protect against PrP-106-126-induced neurotoxicity, hinokitiol is a monoterpene that activates autophagy and it inhibits apoptosis through stabilization of hypoxia-inducible factor (HIF)-1α, which is important for the regulation of oxygen homeostasis [116]. Rutin has been found to prevent the PrP 106-126-induced neurotoxicity in mouse hippocampal cell line (HT22). Their results showed that rutin was able to increase the expression levels of neurotrophic factors like BDNF, glial cell-derived neurotrophic factor (GDNF), and nerve growth factor (NGF). They also observed the inhibition in apoptosis through suppression of FASL, FAS, caspase 8, and by inhibiting the activity of caspase 3 [117]. Table 1 enlist various neuroprotectant phytochemicals used in the case of Prion disease.

#### *3.7. Frontotemporal Dementia (FTD)*

FTD is a term that encompasses a broad group of heterogeneous progressive neurological syndromes. These are non-Alzheimer dementias characterized by the atrophy in the frontal and temporal lobe [196]. As frontal and temporal lobes are highly affected in this disease, the functions controlled by these areas are the outcome of FTD such as behavior and language. Some people with FTD have drastic behavioral and personality changes that make them socially inappropriate and impulsive whereas some people lose the ability to use language [197]. Some other common symptoms include lack of inhibition, apathy and judgment, repetitive compulsive behavior, decline in personal hygiene, eating the inedible objects, and loss of sensitivity towards another person's feelings [198]. FTD can be categorized into three common variant syndromes; behavioral variant FTD (bvFTD), non-fluent/agrammatic variant primary progressive aphasia (nfvPPA), semantic variant PPA (svPPA) [199]. A subtype of FTD is Pick's disease (PiD) which is a neurodegenerative condition that results in irreversible dementia where an excessive amount of tau inclusions known as Pick bodies is a key pathological feature [200]. Pick bodies are round argyrophilic neuronal inclusions. PiD causes cortical atrophy affecting frontal and temporal poles including limbic systems, neocortex, and dentate granular cells of the hippocampus. Such neuronal degenerations result in behavioral changes/alterations and personality disorders [201]. The early signs for PiD are apragmatism and mutism. Some studies refer to PiD as Niemann-Pick disease (NPD) based on the name of the scientist who reported the first case [200].

Not many phytochemicals have been explored for their effect on FTD. Nicotine, a plant alkaloid has been found to have a correctional effect on the behavior of *Grn*-/- (progranulin gene) mice. Haploinsufficiency of progranulin levels is the result of any mutation in the Grn gene, which is one of the cases of FTD [118]. Mice with insufficient amounts of progranulin secrete inflammatory cytokines at elevated levels and develop microgliosis [202,203]. This study reported that daily intervention

of nicotine reversed the sociability and improved the behavioral change in the *Grn*-/- mice [202]. A traditional Chinese herbal medicine known as Yi-Gan San also known as Yokukansan in Japan containing a mixture of seven dried herbs *Atractylodes lancea, Cnidium o*ffi*cinale, Uncaria rhynchophylla, Angelica acutiloba, Poria cocos, Bupleurum falcatum*, and *Glycyrrhiza uralensis*in a ratio of 4:3:3:3:4:2:1.5 [204] was found to alleviate behavioral changes in patients with FTD [205]. Extracts of *Piper nigrum* (fruit) and *Curcuma longa* (rhizome) also inhibited the AChE significantly which is considered one of the key targets for therapeutic strategies against dementia [119]. The nanoformulation of curcumin can be a promising agent against Niemann-Pick disease type C astrocytes [120]. Curcumin has been reported to have several medicinal properties, wherein it was observed that nanoformulations of curcumin were able to increase cytosolic calcium ion levels and cell viability in astrocytes [120]. Another compound δ-tocopherol was found to decreased cholesterol levels and also lipid accumulation, it also reduced lysosomal enlargement. It was also reported to increase intracellular Ca2<sup>+</sup> levels [121]. A study by Nekohashi et al., reported that quercetin and luteolin significantly decreased the cholesterol levels as cholesterol [122] as cholesterol is one of the key characteristics of PiD. The details of phytochemicals are given in Table 1.

#### *3.8. Spinocerebellar Ataxia (SCA)*

SCA is a term used to refer to hereditary (autosomal dominant) ataxias, these are neurodegenerative conditions affecting the cerebellum (movement controlling) part of the brain and the spinal cord [206]. It comprises a large group of neurodegeneration characterized by cerebellar ataxia with oculomotor dysfunction, dysarthria, also affecting the brain stem. The resulting ataxia is an outcome of atrophy in the cerebellum [207]. All types of SCA are associated with the loss of coordination in eye and hand movements including speech. SCA usually has an adult-onset and the progression is gradual, therefore worsening with time [208]. To date, 44 types of SCA have been identified and classified as SCA1 through SCA44. Many SCAs are an outcome of CAG nucleotide repeat expansion encoding polyglutamine, resulting in the involvement of toxic polyglutamine protein (polyQ). The most recent genetic cause of SCA44 reported in 2017 [209].

The focus on nutraceuticals against this disease is not that much, however, trehalose is a natural compound abundantly found in invertebrates, plants, and microorganisms. Studies have shown that it has a neuroprotective effect exhibited through interaction with protein folding. Analog of trehalose, melibiose has been reported the combat neurotoxicity/neurodegeneration by upregulating autophagy [123]. Melibiose significantly inhibited the aggregation of polyQ decreased ROS levels. It also displayed potential in decreasing SCA17TBP/Q79 and significant inhibition to Purkinje cell aggregation in SCA17 transgenic mice [124] Table 1.

#### *3.9. Spinal Muscular Atrophy (SMA)*

SMA is a genetic disorder characterized by the atrophy in skeletal muscle. The onset of SMA takes place due to the loss of motor neurons that regulate muscle movement. It results in weakness and makes the movement or any physical activity challenges, this condition worsens with time [210]. This disease is caused by the inadequate production of survival motor neuron (SMN) which is mainly expressed by gene SMN1. SMN2 is also responsible for the production of SMN but relatively far less. [211]. Mutations in SMN1 such as alter or loss results in observed in patients with SMA [212]. The presence of additional third or more copies of the SMN2 gene partially compensates for the damage caused due to the SMN1 gene and milder symptoms are seen in patients [213]. It mainly affects infants and restrict physical movement. SMA is categorized into five types (0-IV) based on the age of the patient and the severity of the disorder [214].

Since SMA is a genetic disorder, the plant products that can reverse or correct the damage are to be identified. *Brucea javanica* and its major compound as found to be bruceine D were found to have SMN2 splicing-correcting property. This study showed that they corrected the splice defect and also increased the activity of SMN2 significantly. *B. javanica* was able to ameliorate the muscle defects [125]. Triptolide is another phytochemical that showed a significant effect in combating SMA in SMA-like mice. The results showed that triptolide isolated from *Tripterygium wilfordii* elevated the production of SMN protein and transcription of SMN2 both in vivo and in vitro experimental models. Hsu et al. concluded that triptolide is a modulator of SMN expression. Triptolide enhances the expression of transcript and protein levels of survival motor neurons in human SMA fibroblasts and improves survival in SMA-like mice [126] Table 1.

#### **4. Conclusions**

This review focuses on the use of phytochemicals as a preventive approach to neurodegenerative disorders as they are cost-effective, readily applicable, and easily accessible compounds with a promise in future use. On the other hand, there have always been limitations to the clinical use of these phytochemicals. The main issue focuses on the bioavailability and digestibility of these natural compounds in the body of the patients. Blood–brain barrier is another hurdle in the use of phytochemicals for problems related to the nervous system. Moreover, it is time-consuming therapy for effectiveness against the disease.

Natural products possess variable neuroprotective properties, which can be exploited to target ND therapeutically. Phytochemicals have multi-targeted applications with numerous advantages over currently targeted drugs on the market and have limited adverse reactions. They can also help in developing food supplements and tonics from the edible plants possessing neuroprotective activity. However, given the complexities and heterogeneity of phytochemicals, a cautious approach is necessary to define herbal mixtures and compositions of different compounds for treatment purposes. Keeping this in mind, more preclinical and clinical studies are being conducted. Finally, these plants and other remaining body of studies can very well be executed in not only designing safer drugs to combat the NDs but also preventing the onset. Consequently, this review will abet the researchers for further extensive study in this field.

**Author Contributions:** Conceptualization: A.K. (Andleeb Khan), M.U.R.; writing—Original draft preparation: A.K. (Andleeb Khan), S.J., and Z.I.; writing—Review and editing: S.A., H.A.M., B.M.A., A.K. (Ajay Kumar), A.A. and M.U.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**

of Different Flavonoids in Activating Estrogenic Effect and in Preventing β-Amyloid-Induced Cell Death. *J. Agric. Food Chem.* **2007**, *55*, 2438–2445. [CrossRef]

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### **Effects of Treadmill Exercise on Neural Mitochondrial Functions in Parkinson's Disease: A Systematic Review of Animal Studies**

**Nguyen Thanh Nhu 1,2, Yu-Jung Cheng <sup>2</sup> and Shin-Da Lee 2,3,4,\***

**Abstract:** This systematic review sought to determine the effects of treadmill exercise on the neural mitochondrial respiratory deficiency and neural mitochondrial quality-control dysregulation in Parkinson's disease. PubMed, Web of Science, and EMBASE databases were searched through March 2020. The English-published animal studies that mentioned the effects of treadmill exercise on neural mitochondria in Parkinson's disease were included. The CAMARADES checklist was used to assess the methodological quality of the studies. Ten controlled trials were included (median CAMARADES score = 5.7/10) with various treadmill exercise durations (1–18 weeks). Seven studies analyzed the neural mitochondrial respiration, showing that treadmill training attenuated complex I deficits, cytochrome c release, ATP depletion, and complexes II–V abnormalities in Parkinson's disease. Nine studies analyzed the neural mitochondrial quality-control, reporting that treadmill exercise improved mitochondrial biogenesis, mitochondrial fusion, and mitophagy in Parkinson's disease. The review findings supported the hypothesis that treadmill training could attenuate both neural mitochondrial respiratory deficiency and neural mitochondrial quality-control dysregulation in Parkinson's disease, suggesting that treadmill training might slow down the progression of Parkinson's disease.

**Keywords:** treadmill exercise; Parkinson's disease; neural mitochondrial functions

#### **1. Introduction**

Parkinson's disease (PD) is the second most common neurodegenerative disorder, causing a considerable number of disabilities globally [1,2]. The underlying mechanisms of PD are unclear, making it difficult to find efficient targeted therapies [3]. The current treatment of PD addresses symptomatic improvement only because none of the available treatment strategies have been confirmed to slow down PD progression [2].

Recently, neural mitochondrial respiratory deficiency has emerged as a central hallmark of Parkinsonian etiology [4,5]. In PD, the electron transport system of the neural mitochondria is impaired, mainly characterized by mitochondrial complex I deficit, cytochrome *c* release, and ATP depletion [6]. Impaired mitochondrial respiration in the brain increases oxidative stress and neuron loss, thereby augmenting PD progression [4].

Studies have also linked neural mitochondrial respiratory deficiency to neural mitochondrial quality-control dysregulation in PD [7,8]. In the physiological condition, neural mitochondrial quality-control involves a balance among biogenesis, dynamics (fusion/fission), and mitophagy (autophagy of mitochondria) [7]. Mitochondrial biogenesis produces the new mitochondria and mitochondrial content, accompanied by mitochondrial fusion to maintain a healthy mitochondrial network [9]. Meanwhile, mitochondrial fis-

**Citation:** Nhu, N.T.; Cheng, Y.-J.; Lee, S.-D. Effects of Treadmill Exercise on Neural Mitochondrial Functions in Parkinson's Disease: A Systematic Review of Animal Studies. *Biomedicines* **2021**, *9*, 1011. https://doi.org/10.3390/ biomedicines9081011

Academic Editor: Kumar Vaibhav

Received: 16 July 2021 Accepted: 12 August 2021 Published: 13 August 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

sion segregates the damaged mitochondria and provides those for the mitophagy process, preventing the accumulation of dysfunctional mitochondria in the brain [10].

In PD, the biogenesis regulators and import machinery of neural mitochondria are reduced, leading to inhibition of mitochondrial biogenesis [11]. Additionally, PD has been confirmed to induce an imbalance of neural mitochondrial dynamics (fusion/fission) [4]. Moreover, the mitophagy process has been proven to be impaired with reductions of lysosomal activities in PD [12]. Disorders of biogenesis, fusion/fission, and mitophagy reduce the quantity and quality of neural mitochondria, leading to a neural mitochondrial respiratory deficiency in PD [4,7].

Treadmill exercise (TE) has been widely applied in PD rehabilitation [13,14]. Previous evidence indicated that TE training improved both the symptoms and the quality of life in PD patients [13]. In addition, a previous study showed that TE training improved gait functions by modulating neural mitochondrial dynamics in a PD rats model [15]. Another study reported that TE training reduced both neuron loss and behavioral disorders by improving mitochondrial respiration in a PD mouse model [16]. Those data suggested that TE training not only improves symptoms but also delays PD progression by attenuating PD-induced neural mitochondrial damage. However, the various approaches of the individual studies make it difficult to comprehensively understand the effects of TE on neural mitochondria in PD. Although several systematic reviews have been carried out to summarize the neuroprotective effects of TE training on PD, none of them specifically analyzed neural mitochondrial respiratory deficiency and neural mitochondrial quality-control dysregulation.

Therefore, we conducted a systematic review of animal studies to summarize the effects of TE training on mitochondrial functions in PD, focusing on the following objectives: (1) The effects of TE training on neural mitochondrial respiratory deficiency in PD; and (2) The effects of TE training on neural mitochondrial quality-control dysregulation in PD, including neural mitochondrial biogenesis, neural mitochondrial dynamics (fusion/fission), and neural mitophagy.

#### **2. Materials and Methods**

#### *2.1. Protocol and Registration*

The protocol of this systematic review was registered on PROSPERO with the registration number: CRD42020164122. We followed "the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist" [17] and "the PRISMA for abstract checklist" [18] to conduct and report this systematic review.

#### *2.2. Eligibility Criteria*

*Types of study designs:* controlled-trial animal studies with separate experimental groups. The studies were English publications without any restriction of publication date. Protocol articles, case reports, reviews, and conference abstracts were excluded from this systematic review.

*Types of animal models:* animal models of PD. Sex, age, and species of the subjects were not restricted. Studies were excluded if they did not provide sufficient data about the animal species or the PD induction (model types and timing).

*Types of intervention:* treadmill exercise (TE) training without any restriction of the protocol. The information about timing, duration, and frequency must be provided. Studies that evaluated the effects of TE in combination with the other therapies were excluded.

*Type of comparators:* studies that at least reported a comparison among a normal group, a sedentary PD group, and a TE-trained PD group. The normal control group must not be treated with any other therapeutic methods.

*Type of outcomes:* for the effects of TE training on neural mitochondrial respiratory deficiency in PD (the first objective), our outcomes were the components of the electron transport system (complexes I–V, cytochrome c, coenzyme Q10) and ATP production. For the effects of TE training on neural mitochondrial quality-control dysregulation in PD (the second objective), our outcomes included neural mitochondrial biogenesis, neural mitochondrial dynamics (fusion/fission), and neural mitophagy. The neural mitochondrial biogenesis outcome was measured through biogenesis regulators and translocase factors. The neural mitochondrial dynamic outcome was measured through fusion factors and fission factors. The neural mitophagy outcome was measured through dysfunctional mitochondria detectors, autophagosomal factors, and lysosomal factors.

#### *2.3. Information Sources and Search Strategy*

Relevant studies were identified by keywords searching on PubMed, Web of Science and EMBASE databases through March 2020, with a combination of the terms: (*"Parkinson" OR "Parkinsonism" OR "Parkinsonian"*) *AND "treadmill" AND* ("*mitochondria*" *OR* "*mitochondrial*" *OR* "*mitochondrion*" *OR* "*mitophagy*" *OR* "*ATP*" *OR* "*SIRT*" *OR* "*AMPK*" *OR* "*PGC-1α*" *OR* "*TFAM*" *OR* "*NRF*" *OR* "*mito-fusion*" *OR* "*mito-fission*"). We also reviewed references of the included studies to find other eligible papers. Briefly, the titles and abstracts of studies were screened to exclude duplications and irrelevant studies that did not mention the relevant information of Parkinson's disease and exercise in their abstracts. Then, full texts were assessed and read to see if they met the eligibility criteria. Two independent assessors conducted the process of study selection. When a disagreement occurred, two assessors discussed with a third consultant to make the final decision.

#### *2.4. Data Collection Process*

Two independent reviewers extracted the data (including study characteristics and outcomes) by reading the text, graphs, and tables of the included studies. If the data were not available, we contacted the corresponding authors to request it. For study characteristics, we extracted the data of the first author's name, published year, PD model (type, species, and sex), and TE protocol (timing, frequency, duration, and speed). For the outcome extraction, we extracted the data of neural mitochondrial respiration, neural mitochondrial biogenesis, neural mitochondrial dynamics (fusion/fission), and neural mitophagy.

#### *2.5. Study Quality Evaluation*

The study quality was evaluated by using the "Collaborative Approach to Meta-Analysis and Review of Animal Data from Experimental Studies" (CAMARADES) checklist with ten items [19]. Two authors independently evaluated and filled in the predesigned datasheets of the CAMARADES checklist, then disagreements were resolved by discussing among the two evaluators and the third consultant.

#### *2.6. Data Synthesis and Presentation*

The results of the search strategy are shown in the PRISMA flowchart and the narrative synthesis. Text and tables were used to present the study characteristics and the outcomes. For presentation of study characteristics, the summaries of the PD models, TE protocols, and types of outcomes are provided. For presentation of the outcomes, the effects of TE on neural mitochondrial respiratory deficiency and mitochondrial quality-control dysregulation in PD are described in the comparison among the normal control group, the PD group, and the TE-trained PD group.

#### **3. Results**

#### *3.1. Search Results*

A total of 76 articles were found from PubMed (n = 15), Web of Science (n = 28), and EMBASE (n = 33) (Figure 1). After title and abstract screening, we removed 50 records: 31 duplications and 19 irrelevant studies. After full-text reviewing, we excluded 15 studies, including 1 erratum paper, 2 reviews, 4 conference abstracts, and 1 ex-vivo study. In addition, we excluded 2 studies that did not use TE, 4 studies that did not mention our outcomes, and 1 study that was missing data. There were two publications that came from

one group of authors, conducting the same protocol and reporting the same outcome in one year. We considered them as one study, therefore reported one study and excluded the other. Besides, the studies that came from the same group of authors and conducting the same protocol, but reported different outcomes were considered as separated publications to discuss. Finally, ten publications were included in the current systematic review. No additional articles were found by reading the reference lists of the included studies.

**Figure 1.** PRISMA flow chart for the selected protocol.

#### *3.2. Study Characteristics*

*Types of PD models:* rats and mice were used with two models, including the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model (n = 6) and the 6-hydroxydopamine (6-ODHA) model (n = 4). Eight studies used young animals (7–8 weeks old), whereas two studies used middle-aged animals (6–10 months old) (Table 1).

**Table 1.**

Characteristics

 and outcomes of the included studies.

**Table 1.**

 *Cont.*

**Table 1.** *Cont.*

subunit I & IV; PGC-1α = peroxisome

A; SIRT3 = sirtuin-3; AMPK =

heat shock protein 70; PINK1 =

membrane protein 2; i.p =

proliferator-activated

AMP-activated

PTEN-induced

intraperitoneal

 injection; s.c =

 kinase-1; OPA1 = Dynamin-like

 120 kDa protein; MFN = Mitofusin-2; Dpr1 =

subcutaneous

 injection.

 protein kinase; SIRT1 = sirtuin-1; TOM = translocase of outer membrane; TIM = translocase of inner membrane; mtHSP70 =

 receptor gamma coactivator 1-alpha; NRF-1,2 = nuclear respiratory factor 1 and 2; TFAM =

mitochondrial

Dynamin-related

 protein-1; LAMP2 =

 transcription

 factor mitochondrial

Lysosome-associated

*Type of TE training protocol:* there were three types of TE protocol: preventive training (n = 2), treatment training (n = 6), and both preventive and treatment training (n = 2). The exercise duration ranged from 1 to 18 weeks. TE training was conducted 3–7 days/week, 20–60 min for each session. The speed of the TE ranged between 10 m/min to 21 m/min (Table 1).