Mitochondrial Imaging

#### **Chapter 3**

## PET Imaging of Mitochondrial Function in the Living Brain

*Hideo Tsukada*

#### **Abstract**

In the last two and half decades, we have conducted research on brain functional imaging in nonhuman primates using animal positron emission tomography (PET) scanners with high spatial resolution. We recently designed and synthesized the novel PET probe [18F]BCPP-EF to quantitatively image mitochondria complex-I (MC-I) activity in the living brain. Brain MC-I activity, measured using [18F] BCPP-EF, was significantly lower in aged monkeys than that in young animals, while no significant reduction was observed in SV2A activity, a synaptic-specific parameter that was measured using [11C]UCB-J. Some aged monkeys exhibited increased amyloid-β deposition in the brain, measured using [11C]PiB, which induced neuroinflammation. A positive correlation was noted with neuroinflammation, measured using [11C]DPA-713 and a negative correlation with MC-I activity. Furthermore, a monkey model of Parkinson's disease prepared by the chronic administration of MPTP revealed suppressed MC-I activity not only in the nigrostriatal dopamine pathway, measured using [11C]PE2I and [11C]6Me*m*Tyr, but also in cortical serotonergic neurons, measured using [11C]DASB. This review introduces the translational application of a novel PET probe for noninvasive MC-I imaging from preclinical to clinical PET measurements.

**Keywords:** brain, mitochondria, aging, neurodegeneration, PET

#### **1. Introduction**

The brain has the most complex system among all human organs and plays a central role in physiological, neurological, and metabolic regulation throughout the body. The brain is very metabolically active in proportion to its volume, consuming 20% of total oxygen and 25% of total glucose, and each cell relies on mitochondria to produce energy as adenosine triphosphate (ATP). The brain consists of neuronal and glial cells, each of which uses the different metabolic pathways to produce ATP; astrocytes are highly glycolytic, while neurons depend on oxidative phosphorylation (OXPHOS) in the mitochondria [1]. The electron transport chain (ETC) for OXPHOS in mitochondria consists of five types of complexes from I to V, with complex-I (MC-I; NADH–ubiquinone oxidoreductase, EC 1.6.5.3) forming the first and rate-limiting steps of overall respiratory and OXPHOS.

Neuronal death is regarded the dominant cause of brain aging. The "Mitochondrial Free Radical Theory of Aging" considers mitochondria to be the main drivers of aging due to the generation of reactive oxygen (ROS) and nitrogen (RNS) species through the ETC, and the cumulated impairment of mitochondria by oxidative stress in mitochondrial lipids, proteins, and DNA leads to neuronal

death in the brain [2]. Neuronal damage related to impaired mitochondrial function is a hallmark for several neurodegenerative diseases, including Alzheimer's (AD), Parkinson's (PD), Huntington's diseases (HD), as well as amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS).

The etiology of AD, the most prominent age-related neurodegenerative disease, is multifactorial and associated with various environmental and genetic factors that play a role in its pathogenesis. The deposition of amyloid-β (Aβ) in the brain has been suggested to enhance neurodegenerative damage. Structural disruption and loss of neuronal cells induced by Aβ, leading to neuronal network dysfunction and synaptic loss in the hippocampus and cerebral cortex, is associated with cognitive impairment in AD patient [3]. Although the molecular mechanisms underlying synaptic dysfunction induced by Aβ have not been fully elucidated, mitochondrial dysfunction caused by Aβ is associated with synaptic functional alterations in the living brain [3].

PD is the second most prominent neurodegenerative disease and is characterized by the progressive degradation of the nigrostriatal pathway with the selective loss of dopamine (DA) neurons, resulting in movement abnormalities such as resting tremor, rigidity, akinesia, and impaired postural reflexes. A hallmark of idiopathic PD is the deposition of Lewy bodies containing insoluble and aggregated α-synuclein in the cytoplasmic fraction of nigrostriatal neurons in the DA pathway [4]. The loss of MC-I catalytic activity in the ETC has been reported in tissues obtained from sporadic PD patients [5] with increased oxidative stress [6].

Regarding the noninvasive assessment of mitochondrial function in the living brain, we recently developed a novel probe, [18F]2-tert-nutyl-4-chloro-5-{6-[2-(2 fluoroethyl)-ethoxyl-pyridin-3-3-ylmetoxy]-2H-pyrisazin-3-one ([18F]BCPP-EF), for positron emission tomography (PET) [7]. The effects of physiological aging, aging-related Aβ deposition, and chronic MPTP treatments on cortical MC-I function were assessed in conscious monkeys using [18F]BCPP-EF, with the aim of translating clinical PET research to AD and PD patients.

#### **2. Effects of aging on MC-I and SV2A function in the living brain**

Mitochondria from aged brains consume less oxygen and, thus, produce lower amounts of ATP [8]. Different types of neurons show differences in topologies with various numbers of synaptic connections. Since synaptic and non-synaptic mitochondria have different protein compositions and respiratory and ROS production rates, synaptic mitochondria are more vulnerable to oxidative damage during the aging process than non-synaptic mitochondria [9]. In quantitative assessments of the effects of physiological aging on mitochondria and synaptic function in the living brain, the activities of MC-I and synaptic vesicle glycoprotein 2A (SV2A), an ideal biomarker for synaptic density, were comparatively evaluated in the living brains of young and aged rhesus monkeys.

#### **2.1 Methods**

Six young male (3–5 years old) and eight aged male (20–24 years old) rhesus monkeys (*Macaca mulatta*) were examined in the PET study using [18F]BCPP-EF for MC-I and (*R*)-1-((3-[11C]methyl-[11C])pyridine-4-yl)methyl)-4-(3,4,5-trifluorophenyl)pyrrolidin-2-one ([11C]UCB-J) for SV2A [10]. MRI was performed on each monkey using a 3.0 T MR imager (Singna Excite HDxt 3.0 T, GE Healthcare) using a 3D spoiled gradient echo (SPGR) sequence under pentobarbital anesthesia. To avoid anesthetic effects on brain function as well as PET probe kinetics, PET scans were

#### *PET Imaging of Mitochondrial Function in the Living Brain DOI: http://dx.doi.org/10.5772/intechopen.86492*

conducted under conscious condition using high-resolution animal PET scanners (SHR-7700 and 38,000, Hamamatsu Photonics) as reported previously [11–14].

[ 18F]BCPP-EF was prepared by the nucleophilic [18F] fluorination of the corresponding precursor, as reported previously [9]. Radiochemical purity was more than 99%, and specific radioactivity was 47.8 ± 12.7 GBq/μmol. [11C]UCB-J was labeled via the Suzuki cross-coupling of [11C]methyl iodide with a boronate precursor, as reported previously [10]. Radiochemical purity was more than 98%, and specific radioactivity was 62.3 ± 25.1 GBq/μmol.

After overnight fast, a venous cannula for the PET ligand injection and an arterial cannula for blood sampling were inserted into both inferior limbs. The animal's head was rigidly fixed to a monkey chair using an acrylic head-restraining device surgically attached on the skull. The monkey sitting in the chair was placed at a position in the PET gantry with stereotactic coordinates aligned parallel to the orbito-metal (OM) plane. After a transmission scan for 30 min using a [68Ge]- [ 68Ga] rotation rod source, a dynamic emission scan with [18F]BCPP-EF or [11C] UCB-J was conducted for 90 min after the PET probe injection as a bolus.

To assess the specificity of [18F]BCPP-EF binding to MC-I, which was previously examined in rats [15], rotenone, an MC-I inhibitor, at a dose of 0.1 mg/kg was infused into young monkeys through a vein cannula for 1 h, followed by [18F] BCPP-EF injection.

PET data were reconstructed by the dynamic row action maximum likelihood algorithm (DRAMA) method using a 2.0-mm Gaussian post filter [16], with attenuation correction using transmission scan data. Individual PET and MRI images were co-registered. Volumes of interest (VOIs) in brain regions were drawn manually on MRI, and the time activity curve (TAC) of each PET probe was taken for kinetic analyses. These imaging annalistic processes were performed using PMOD software (PMOD Technologies Ltd.).

In a quantitative analyses of [18F]BCPP-EF and [11C]UCB-J, arterial blood samples were frequently obtain after the PET probe injection, centrifuged to separate plasma, and ethanol was added to some plasma samples, followed by centrifugation. The supernatants obtained were developed with thin layer chromatography with a mobile phase of ethyl acetate for [18F]BCPP-EF and chloroform/methanol = 9:1 for [11C]UCB-J, respectively. The ratio of the unmetabolized fraction was assessed using a phosphoimaging plate analyzed by a bioimaging analyzer (FLA-7000, GE Healthcare).The arterial input function of unmetabolized [18F]BCPP-EF or [11C]UCB-J was calculated using data obtained by a correction of the radioactivity ratio in the unmetabolized fraction to total fraction. A kinetic analysis of [18F] BCPP-EF or [11C]UCB-J was performed to calculate distribution volume (VT) using a Logan graphical analysis [17] with PMOD software (PMOD Technologies Ltd.).

#### **2.2 Results**

Brain TACs of [18F]BCPP-EF in young animals peaked at approximately 15 min after the injection, except in the occipital cortex, which peaked at 40 min, and TACs then were gradually eliminated with time under conscious condition. The pre-administration of rotenone resulted in a faster elimination rate of [18F]BCPP-EF from the brain than that in normal. In aged animals, the peak time of TACs shifted to slightly later after the injection and showed significant lower [18F]BCPP-EF uptake levels than those in young animals.

The washout of [18F]BCPP-EF-related radioactivity and its metabolites rate in plasma were very rapid, with only 10% of the non-metabolized parent probe remaining 60 min after the injection. These kinetic patterns of [18F]BCPP-EF were almost identical among control young, rotenone-treated young, and aged animals. A metabolic analysis confirmed that its metabolites were very polar, indicating no uptake of metabolites into the brain through the blood–brain barrier. These results confirmed that this PET probe has ideal properties for the quantitative analysis of MC-I activity using a metabolite-corrected arterial input function.

As shown in the upper panel of **Figure 1**, VT-PET images of young brains indicated that the binding of [18F]BCPP-EF was highest in the occipital cortex, higher in the striatum, intermediate in the frontal and temporal cortices and cerebellum, and lowest in the hippocampus. The pre-administration of rotenone at a dose of 0.1 mg/ kg/h significantly reduced the uptake of [18F]BCPP-EF into the young monkey brain, specifically in the frontal and temporal cortices and striatum. When aging effects on VT-PET images were assessed, the binding of [18F]BCPP-EF throughout the brain was significantly lowered in aged animals than in young animals, as shown in the lower panel of **Figure 1**.

The TAC of [11C]UCB-J in young animals exhibited slower kinetics than [18F] BCPP-EF throughout the brain regions, peaking approximately 30 min after the injection, and TACs gradually decreased with time under conscious conditions. TACs in aged animals peaked 30 min after the injection and showed slightly lower [11C] UCB-J uptake levels than those in young animals. The washout of [11C]UCB-J-related radioactivity and metabolic rates in plasma were slower than those of [18F]BCPP-EF, with 25% of the non-metabolized parent probe remaining 60 min after the injection. These kinetic patterns of [18F]BCPP-EF were not significantly different between young and aged animals. A metabolic analysis confirmed that its metabolites were very polar, suggesting no uptake of the metabolites into the brain through the blood– brain barrier. These results confirmed that this PET probe is useful for the quantitative analysis of SV2A activity using a metabolite-corrected arterial input function.

As shown in the upper panel of **Figure 2**, VT-PET images of the young brain revealed the highest binding of [11C]UCB-J in the frontal, temporal, and occipital

#### **Figure 1.**

*Effects of aging on PET images of MC-I in living brains of conscious monkeys. PET scans were conducted for 91 min using [18F]BCPP-EF for MC-I, and VT images of [18F]BCPP-EF were created using a Logan graphical plot analysis with metabolite-corrected plasma input.*

*PET Imaging of Mitochondrial Function in the Living Brain DOI: http://dx.doi.org/10.5772/intechopen.86492*

**Figure 2.**

*Effects of aging on PET images of SV2A in living brains of conscious monkeys. PET scans were conducted for 91 min using [11C]UCB-J for SV2A, and VT images of [11C]UCB-J were created using a Logan graphical plot analysis with metabolite-corrected plasma input.*

cortices, higher in the striatum, intermediate in the hippocampus, and the lowest in the cerebellum. Thus, the distribution pattern of [11C]UCB-J resembled to that of [ 18F]BCPP-EF throughout the brain of young animal.

However, in contrast to [18F]BCPP-EF, when the VT of [11C]UCB-J was compared between young and aged monkeys, no significant age-related changes in cerebral SV2A activity were detected except the olfactory bulb, as shown in the lower panel of **Figure 2**.

#### **2.3 Discussion**

The present results showed age-related reduction in mitochondria function assessed by MC-I activity using [18F]BCPP-EF, as reported previously [11], while no significant alterations in synaptic density were observed based on SV2A activity using [11C]UCB-J.

We evaluated [18F]BCPP-EF by in vitro (living brain slices), ex vivo (dissected tissues), and in vivo (living rat brains) assessments in comparison with the conventional MC-I PET probe, [18F]BMS-747158-02 ([18F]BMS), which was developed as a myocardial perfusion PET imaging probe [18], and the results obtained suggested that [18F]BCPP-EF is a more suitable PET probe than [18F]BMS for MCI assessments in the living brain [7, 15]. [18F]BCPP-EF was characterized by (1) high specific binding with moderate affinity, (2) a proper logD7.4 value, (3) long metabolic stability with fast clearance in plasma, (4) sufficient brain uptake with a proper elimination rate from the brain, and (5) low dependency to cerebral blood flow changes [19]. As a PET probe, these properties of [18F]BCPP-EF may contribute for a noninvasive and quantitative analysis of MC-I in the living brain [11], thereby allowing for the high detectability of age-related reductions in MC-I activity in the living monkey brain. Although several postmortem in vitro studies previously reported that (1)

age-related brain MC-I impairments were associated with a decline in mitochondria respiration [20, 21] and (2) the activities of MC-I and -IV, but not MC-II, III, or V, decreased with aging in the brains of rodents [22] and humans [20, 21], this study was the first to successfully confirm age-related reductions in MC-I activity in the living brain of nonhuman primates [11].

As described above, [18F]BCPP-EF detected age-related reduction in MC-I activity; however, this PET probe was unable to discriminate the subdomain (synapse, dendrites, or soma) of MC-I that was exclusively damaged due to the limitation of the spatial resolution of PET. In attempts to answer this question, [11C]UCB-J, a novel PET probe for SV2A [10], was applied to the same subject groups on the assumption that if this MC-I reduction mainly reflected the presynaptic domain, the binding of [11C]UCB-J to SV2A will also show a similar age-related reduction pattern in the brain. However, contrary to this assumption, no significant changes in [11C]UCB-J binding to SV2A were observed, except the olfactory bulb, from those in the same young and aged animals applied in the assessment using [18F]BCPP-EF.

Several postmortem in vitro studies have suggested age-related reductions in spine density; however, these findings remain controversial. One study using rat brains revealed that the synaptic vesicle density in axospinous synapse, a major population of synapses, rapidly increased until 3 weeks old and then decreased to the adult level, followed by no changes in senescence [23]. However, another study on mouse brain showed that age-related deficits in sensory perception were not associated with synaptic loss in the somatosensory cortex but were related to alterations in the size and stability of spine buttons [24], which might not be detected as the changes in SV2A activity.

The discrepancy in age-related effects between [11C]UCB-J and [18F]BCPP-EF suggest that MC-I dysfunction detected by [18F]BCPP-EF mainly reflect neuronal damage in dendrite and soma domains and are not specific to presynaptic domain. Although difficulties are associated with assessing mitochondrial distribution in neuronal cells, it was demonstrated in the cultured neuron that more than 90% of mitochondria were confirmed to be in the dendrite shaft overlapping in soma and large-diameter proximal dendrites [25]. This localized distribution pattern of the mitochondria suggests that even if mitochondria in the presynaptic domain are specifically impaired, difficulties may be associated with detecting changes in the minor subdomain using [ 18F]BCPP-EF. The present results also suggested that in the early stage of the aging process, at least, in nonhuman primates, neuronal damage in the synaptic domain, if any, may be too subtle to be detected as decreased [11C]UCB-J binding to SV2A.

#### **3. Effects of amyloid-β deposition on MC-I function in the living brain**

Patients of AD, the most prominent age-related neurodegenerative disease, are characterized by the deposition of fibrillary Aβ into senile plaques and hyperphosphorylated tau (P-tau) into neurofibrillary tangle (NFT) in the brain. Monomer Aβ has been implicated in normal developmental synaptic plasticity, for example, in the olfactory bulb under physiological conditions [26]. However, the aggregation of Aβ switches its physiological role into a pathologically toxic function; thus dense plaques damage the surrounding brain tissues [27], causing synaptic elimination and impaired synaptic function [28]. Although the molecular mechanisms underlying neuronal damage induced by Aβ have not been fully elucidated, mitochondrial dysfunction may be associated with Aβ-induced synaptic dysfunction [3] and also with neuroinflammation [29], resulting in cognitive impairment in AD patients. In order to clarify the relationship between Aβ deposition, neuroinflammation, and mitochondrial function, [11C]PiB for Aβ [30], [11C]DPA-713 for translocator protein (TSPO) [31], and [18F]BCPP-EF for MC-I were comparatively evaluated in the living brains of aged rhesus monkeys. Furthermore, as a gold standard parameter of cerebral metabolism, [18F]fluoro-2-deoxy-D-glucose ([18F]FDG) was also applied to compare the diagnostic and prognostic usefulness with [18F]BCPP-EF.

#### **3.1 Methods**

Twenty aged male (20–24 years old) rhesus monkeys (*Macaca mulatta*) were investigated in the PET study using [11C]PiB for Aβ, [11C]DPA-713 for TSPO, and [ 18F]BCPP-EF for MC-I under conscious conditions using a high-resolution PET scanner as described in 2.1.

[ 11C]PiB was synthesized by the *N*-methylation of nor-compound *N*-desmethyl-PiB with [11C]methyl triflate [30]. Radiochemical purity was more than 99%, and specific radioactivity was 36.7 ± 10.1 GBq/μmol. [11C]DPA-713 was synthesized by the *N*-methylation of nor-compound *N*-desmethyl-DPA with [11C] methyl triflate [31]. Radiochemical purity was more than 99%, and specific radioactivity was 99.3 ± 32.2 GBq/μmol. [18F]BCPP-EF was radiolabeled as shown in 2.1. Radiochemical purity was more than 99%, and specific radioactivity was 139.6 ± 37.0 GBq/μmol. [18F]FDG was produced by the nucleophilic [ 18F]fluorination of mannose triflate following the basic hydrolysis of 2-[18F] fluoro-1.3.4.6-tetra-*O*-acetyl-D-glucose.

In the analysis of [11C]PiB, standard uptake value (SUV) images from 60 to 90 min were created, VOIs were set on each SUV images, and the SUV ratios (SUVR) of each region (SUVreg) against SUV in the cerebellum (SUVcereb) were calculated [32]. In the analysis of [11C]DPA-713, SUV images from 40 to 60 min were created, and VOIs were set on each SUV image. Since any cerebral regions were not applicable as the reference region with negligible TSPO levels [33], the SUV, not SUVR, was assessed. A quantitative analysis of [18F]BCPP-EF was performed as described in 2.1. During PET scanning with [18F]FDG, continuous arterial sampling was conducted, and PET images from 40 to 60 min after the injection were obtained to calculate the regional cerebral metabolic rate of glucose (rCMRglc) using an autoradiographic method. The rCMRglc ratios of each region (rCMRglcregion) against rCMRglc in the cerebellum (rCMRglc-cere) were calculated [34].

#### **3.2 Results**

Since the cortical VT values of [18F]BCPP-EF in aged monkeys were previously shown to have a higher CV value of ca. 25% than those in young ones of ca. 7% [11], the reasons for the lager variation of MC-I activity were elucidated using PET imaging with [11C]PiB for Aβ deposition and [11C]DPA-713 for TSPO in 20 aged monkeys. **Figure 3** shows the PET/MRI images of aged monkeys with the lowest (A) and highest (E) [11C]PiB binding, with high SUV not only being detected in cortical but also subcortical regions. PET results obtained with [11C]PiB were supported by an immunohistochemical assessment conducted after PET assessments [12]. The images of [11C]DPA-713 (B and F) corresponding to each monkey (A and E) revealed that monkeys with the lowest (A) and highest (E) [11C]PiB binding showed the lower (B) and higher (F) [11C]DPA-713 binding. In contrast, the images of [18F] BCPP-EF corresponding to each monkey with the lowest (A) [11C]PiB binding exhibited higher [18F]BCPP-EF uptake (C), while those with the highest [11C]PiB binding (E) showed the lower [18F]BCPP-EF uptake (G).

As shown in **Figure 4A**, when the SUV values of [11C]DPA-713 were plotted against the SUVR of [11C]PiB in the cortical VOIs of all animals, the results obtained revealed a significant positive correlation between each parameter, indicating that Aβ deposition-induced neuroinflammation in the living brains of aged monkeys. In contrast, the plotting of VT of [18F]BCPP-EF against SUVR of [11C]PiB showed a reverse correlation in the cortical VOIs of all animals, suggesting that Aβ deposition-induced MC-I impairments in the brains of aged monkeys (**Figure 4B**).

Based on **Figures 3H** and **4C**, it is important to note that a correlation was not observed between glucose metabolism assessed as the SUVR of [18F]FDG and Aβ deposition measured as the SUVR of [11C]PiB, and this may have been due to [18F] FDG uptake into the neuroinflammation-related activated microglial cells.

**Figure 3.**

*Effects of Aβ deposition on PET images of [11C]PiB (A and E), [11C]DPA-713 (B and F), [18F]BCPP-EF (C and G), and [18F]FDG (D and H) in living brains of conscious monkeys. PET scans were conducted in monkey with the lowest (A–D) and highest (E-H) Aβ deposition among 20 aged monkeys.*

**Figure 4.**

*Correlations between the SUV of [11C]DPA-713 (A), VT of [18F]BCPP-EF (B), and rCMRglc ratio of [18F] FDG (C) against the SUVR of [11C]PiB in cortical and hippocampal regions of living brains of aged monkeys.*

#### **3.3 Discussion**

The present results demonstrated the potential of MC-I impairment assessed using [18F]BCPP-EF as a useful biomarker for Aβ deposition-related neurodegeneration, measured using [11C]PiB, and neuroinflammation, measured using [11C] DPA-713. In contrast, the assessment of rCMRglc using conventional [18F]FDG was not as sensitive for detecting the neurodegenerative damage associated with inflammation in the early stage of disease onset because of [18F]FDG uptake into not only normal neuronal cells but also into inflammatory cells, such as activated microglia in the brain [15, 19, 29]. Since activated microglia dominantly facilitate glycolysis, not ORPHOS system through mitochondria, to produce ATP, more glucose is required in microglia than in normal neuron, resulting in higher [18F]FDG uptake than in normal cells. Thus, MC-I assessed using [18F]BCPP-EF has a potential as a biomarker to assess neurodegeneration more accurately without being affected by inflammation in the living brain.

The present PET images obtained using [11C]PiB did not reveal as prominent Aβ deposition in aged monkey brains as that in the brains of PD patients. Previous studies suggested that no species other than humans exhibited marked neuron loss or cognitive impairment observed as clinical grade AD in humans, and aged monkeys did not exhibit as high Aβ deposition as seen in AD patients [35] or [11C] PiB binding [36] as that in AD patients. Therefore, we assumed that the aged monkey model exhibited a similar mild cognitive impairment (MCI)-like, not AD-like, state to that in humans. In the AD state after a lag period of MCI, Aβ deposition level reached a plateau; therefore no correlation was observed between [ 11C]PiB uptake and cognitive levels or metabolic dysfunction assessed using [ 18F]FDG [37]. In contrast, [11C]PiB may function as a quantitative and sensitive biomarker for Aβ-related neuronal damage in aged monkey brains resembling the MCI-like state in humans [38]. Furthermore, a recent study reported the primary role of non-deposited, non-fibrillar assembles of Aβ peptides, and, thus, they may become precursors for fibrillogenesis, which lead to oxidative neurotoxicity by ROS in the brains of very early stage of AD patients [39]. ROS-related mitochondrial OXPHOS failure in the brain has been implicated in neurodegenerative disorders [40]. Mitochondria are the main intercellular source of ROS and also the main target of oxidative damage, leading to the significant disruption of brain function. A recent study demonstrated the direct effects of Aβ on mitochondria; thus, in addition to extracellular deposition, Aβ was detected in cytoplasmic mitochondria compartments [41], leading to dysfunction with the suppressed availability of nucleus-encoded proteins, a decreased rate of NABH reduction, and enhanced ROS generation [42]. Since we previously confirmed a positive correlation between the VT of [18F]BCPP-EF and rCMRO2, a gold standard for brain activation [19], the Aβ deposition-related MC-I functional impairment observed in the present study may reflect diminished activity and/or the loss of neurons with neuroinflammation. The present study revealed a correlation between [11C]PiB binding and [18F]BCPP-EF uptake, demonstrating the usefulness of an assessment of MC-I for the diagnostic staging of MCI and early stage AD.

In the present study, TSPO activity evaluated using [11C]DPA-713, an index of neuroinflammation, was stronger in the aged monkey brain with higher [11C]PiB binding to Aβ. Furthermore, [11C]DPA-713 binding to TSPO and rCMRglc measured using [18F]FDG revealed a correlation (data not shown) [12]. These results suggest Aβ deposition-induced neuroinflammation with the activation of microglial cells.

Therefore, the detectability of MC-I impairment using [18F]BCPP-EF for neuronal damage in AD appears to be superior to the assessment of glucose metabolism measured using conventional [18F]FDG.

#### **4. Effects of MPTP-induced parkinsonism on MC-I in the living brain**

Patients with PD show the progressive degradation of the nigrostriatal pathway with the selective loss of DA neurons in the substantia nigra pars compacta (SNc), resulting in movement disorders, which are induced after the loss of ca. 50% of neurons in the SNc and a reduction in DA to ca. 20% of normal levels in the striatum [43]. A pathological hallmark of PD is Lewy bodies and Lewy neurites, containing intracytoplasmic insoluble and aggregated protein of α-synuclein [4]. The spread of these pathologies closely correlates with disease progression. Furthermore, an intracerebral injection of insoluble α-synuclein converted normal α-synuclein into an abnormal form, which then propagated throughout the brains of monkeys [44] and common marmosets [45]. The soluble, β sheet-rich oligomers of α-synuclein induced mitochondrial dysfunction by inhibiting MC-I, enhancing ROS production, and activating the mitochondrial permeability transition pore (PTP), leading to mitochondrial swelling and neuronal death [46].

Since exposure to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces a syndrome mimicking PD symptoms accompanied by selective DA damage in the nigrostriatal pathway [47], we developed a PD model of monkeys by systemic and repeated MPTP administration [48, 49] and evaluated serotonin (5-HT) transporter (SERT) availability using [11C]-3-amino-4-(2-dimethylaminomethylphenylsulfanyl)-benzonitrile ([11C]DASB) [50] 5-HT 1A receptor (5-HT1AR) binding using 4-(2′-methoxyphenyl)-1-[2′-(*N*-2″-pyridinyl)-*p*-[18F]fluoro-benzamido]ethylpiperazine ([18F]MPPF) [51] in parallel with DA parameters for presynaptic DA synthesis (DAS) using 6-[11C]methyl-*m*-tyrosine ([11C]6Me*m*Tyr) [52, 53], DA transporter (DAT) using [11C]*N*-(3-iodoprop-2E-enyl)-2β-carbomethoxy-3β-(4-methyl-phenyl) nortropane ([11C]PE2I) [54], postsynaptic DA D2R using [ 11C]Raclopride, and MC-I activity using [18F]BCPP-EF in the living brains of MPTP-treated monkeys [13].

#### **4.1 Methods**

Young adult male rhesus monkeys (*Macaca mulatta*) were used to prepare the PD model, as reported previously [48, 49]. MPTP at doses ranging between 0.2 and 0.4 mg/kg in phosphate-buffered saline was injected intravenously over a 4-month period until stable Parkinsonian syndrome developed. MPTP-treated monkeys were subjected to PET scans using specific PET probes for DA, 5-HT neuronal systems, and MC-I under conscious condition using a high-resolution PET scanner, as described in 2.1. In order to avoid the potential for spontaneous recovery as well as direct inhibition of [18F]BCPP-EF binding to MC-I by MPTP, all PET measurements were started at least after 2 months after the last treatment with MPTP.

[ 11C]PE2I was radiolabeled by the *O*-[11C]methylation of its nor-compound with [ 11C]methyl triflate. Radiochemical purity was more than 98% and specific radioactivity of 117.1 ± 42.9 GBq/μmol. [11C]6Me*m*Tyr was developed in our laboratory using rapid Pd(0)-mediated cross-coupling between [11C]methyl iodide and its boronate precursor [52, 53], showing radiochemical purity of more than 99% and specific radioactivity of 71.6 ± 37.4 GBq/μmol. [11C]Raclopride was radiolabeled by *N*-[11C]methylation of its nor-compound with [11C]methyl triflate. Radiochemical purity was more than 98% and specific radioactivity of 65.4 ± 15.8 GBq/μmol. [ 18F]BCPP-EF was radiolabeled as shown in 2.1. Radiochemical purity was more than 99%, and specific radioactivity was 58.9 ± 7.9 GBq/μmol. [11C]DASB was prepared by the *N*-[11C]methylation of its nor-compound with [11C]methyl triflate [50] with radiochemical purity of more than 99% and specific radioactivity of

47.6 ± 11.1 GBq/μmol. [18F]MPPF was synthesized by the nucleophilic [18F]fluorination of its nitro precursor [51] with radiochemical purity of more than 99% and specific radioactivity of 90.4 ± 25.6 GBq/μmol.

Quantitative analyses of [11C]PE2I, [11C]Raclopride, [11C]DASB, and [18F] MPPF were performed with a simplified reference tissue model to calculate non-displaceable binding potential (BPND) [55] using PMOD software (PMOD Technologies Ltd., Zurich, Switzerland). As an indirect input function, TAC in the cerebellum was applied. A quantitative analysis of [18F]BCPP-EF was performed as described in 2.1. A quantitative analysis of [11C]6Me*m*Tyr to calculate the Ki value was performed using a multiple-time graphical analysis [56] using PMOD software (PMOD Technologies Ltd., Zurich, Switzerland) applying the TAC in the occipital cortex as an input function.

#### **4.2 Results**

The effects of chronic MPTP treatments on the DA neuronal system in the brain were revealed in **Figure 5**. PET measurements using [11C]PE2I for DAT (A), [ 11C]6Me*m*Tyr for DAS (B), and [11C]Raclopride for D2R (C) clearly imaged the striatum of the normal monkey brain. After the repeated treatment with MPTP, presynaptic DAT availability assessed using [11C]PE2I was significantly lower in the caudate (4.0% of normal), putamen (4.9% of normal), and SNc (18.6% of normal) (E) of the MPTP-treated monkey brain than in the normal monkey (A). Another presynaptic parameter of DAS assessed using [11C]6Me*m*Tyr was also markedly lower in the caudate (13.6% of normal), putamen (12.3% of normal), and SNc (41.1% of normal) (F) of MPTP monkey brain than in normal animal monkey brain (B). In contrast, no significant changes in [11C]Raclopride binding to postsynaptic D2R were observed in the striatum or SNc between normal (C) and MPTP-treated animals (G).

The effects of the MPTP treatments on PET images of MC-I in the brain are shown in **Figure 5D** and **H**. The cerebral uptake of [18F]BCPP-EF in normal monkeys showed homogeneous and symmetric patterns in the both hemispheres with clear separation of the cortical and basal ganglion regions (D). After the repeated treatment with MPTP, the VT of [18F]BCPP-EF exhibited significant reductions ranging between 60 and 70% of normal levels not only in the nigrostriatal pathway with abundant DA neurons but also in extra-striatal non-DA regions such as the cortex (H). No significant decreases in the VT of [18F]BCPP-EF were observed in the cerebellum.

Since the chronic treatment with MPTP, known as a DA specific toxin, unexpectedly induced decreases in MC-I activity in extra-striatal non-DA regions, another monoaminergic neuronal system of 5-HT was further evaluated. In the normal monkey brain, [11C]DASB binding to SERT was high in the striatal and midbrain regions (**Figure 6A**), while [18F]MPPF binding to 5-HT1AR was high in the cortical regions (B). The quantitative analysis of [11C]DASB revealed significantly lower SERT availability throughout the brain in MPTP-treated monkey, except the raphe nucleus (**Figure 6C**), than in normal monkeys (A). The reduction in [11C] DASB availability to SERT was the greatest in the occipital cortex (19.7% of normal); intermediate in the frontal, parietal, and temporal cortices (50.0, 40.7, and 51.6% of normal); smaller in the caudate (60.3% of normal); and the smallest in the putamen and SNc (66.0 and 67.7% of normal). In contrast, no significant changes were observed in [18F]MPPF binding to 5-HT1AR throughout the brain between normal (**Figure 3B**) and MPTP-treated monkeys (D).

In order to assess the relationship between MC-I and DA and 5-HT neuronal systems in MPTP-treated monkey brains, the degrees of reductions in DAT measured using [11C]PE2I, DAS measured using [11C]6Me*m*Tyr in the nigrostriatal pathway (caudate, putamen, and SNc), and SERT measured using [11C]DASB in

#### **Figure 5.**

*Effects of the MPTP treatment on PET images of DA and MC-I in living brains of conscious monkeys. PET scans were conducted for 91 min using [11C]PE2I for DAT (A and E) and [18F]BCPP-EF for MC-I (D and H) and for 60 min using [11C]6Me*m*Tyr for DAS (B and F) and [11C]Raclopride for D2R (C and D). VT images of [18F]BCPP-EF (D and H) were created using a Logan graphical plot analysis with metabolite-corrected plasma input. BPND images of [11C]PE2I (A and E) and 11C-Raclopride (C and G) and multiple-time graphical analysis Ki images of [11C]6Me*m*Tyr (B and F) were created using the corresponding TACs in the cerebellum as input functions.*

#### **Figure 6.**

*Effects of the MPTP treatment on PET images of 5-HT in living brains of conscious monkeys. PET scans were conducted for 91 min with [11C]DASB for SERT (A and C) and [18FMPPF for 5-HT1AR (B and D). BPND images of [11C]DASB (A and C) and [18F]MPPF (B and D) were created using the corresponding TACs in the cerebellum as input functions.*

the nigrostriatal and cortical (frontal, occipital, temporal, and parietal cortices) regions were plotted against those in MC-I measured using [18F]BCPP-EF. Positive correlations were observed between the ΔBPND of [11C]PE2I and ΔVT of [18F] BCPP-EF and between ΔKi of [11C]6Me*m*Tyr and ΔVT of [18F]BCPP-EF in the nigrostriatal regions. In addition to the DA system, a positive correlation was noted between the ΔBPND of [11C]DASB and ΔVT of [18F]BCPP-EF in the cortical regions, while no correlation was found between ΔBPND of [11C]DASB and ΔVT of [18F] BCPP-EF in the nigrostriatal regions.

#### **4.3 Discussion**

The administration of MPTP causes the slow progressive loss of DA neurons over a period of several months, and the decrease in nigrostriatal DA levels is responsible for the motor symptoms of MPTP-treated animals, resembling clinical symptoms in PD patient. Thus, impaired ETC for OXPHOS due to a MC-I deficiency may account for neuronal cell death in PD. Alternatively, the MC-I deficiency in nigrostriatal DA system of PD patients may be secondary to mitochondrial damage due to oxidative stress. MC-I is a site of ROS production and is particularly vulnerable to oxidative damage [57]. The results of the present study showing a deficiency in presynaptic DA activity in the nigrostriatal pathway were consistent with the conventional theory shown above.

In contrast, the present study applying [18F]BCPP-EF, a novel PET probe for MC-I, demonstrated that systemic and chronic MPTP treatments induced neuronal damage not only in the nigrostriatal DA pathway but also in the 5-HT neuronal system in the cortex of the monkey brain. Recent clinical studies suggested that in addition to motor symptoms, which are exclusively related to the nigrostriatal DA system, PD is a disease associated with non-motor symptoms, such as depression and cognitive deficits, which may be related to changes in other monoamines (noradrenaline and serotonin) in extra-striatal regions [58]. Lewy body and neurite deposition, a pathological hallmark of PD, occurs within the raphe nucleus containing 5-HT neurons of the caudal brainstem [59]. Decreases in 5-HT concentrations have been reported in the cortical regions of the postmortem brains of PD patients [60]. Furthermore, PET imaging on non-depressed PD patients revealed diffuse reductions in SERT availability throughout the brain [61] and decreased 5-HT1AR binding in the brains of non-depressed and depressed PD patients [62]. The present results obtained in MPTP-treated monkeys were consistent with the recent clinical observations on 5-HT abnormalities in PD patients, which cannot be diagnosed by PET measurements of dopaminergic parameters only.

#### **5. Conclusion**

This chapter introduced the potential of [18F]BCPP-EF as a PET probe for the noninvasive and quantitative imaging of MC-I activity in the living brain. The detectability of MC-I impairments using [18F]BCPP-EF for neuronal damage in AD appears to be superior to the assessment of glucose metabolism measured using conventional [18F]FDG. Furthermore, impaired serotonergic neuronal function in cortical regions suggests a relationship with depressive syndrome in PD patients. PET imaging of mitochondria function using [18F]BCPP-EF will provide novel insights into the pathology, diagnosis, and treatment efficacy assessments of a wide range of neurodegenerative diseases, including AD, PD, HD, ALS, and MS.

### **Acknowledgements**

We gratefully acknowledge the technical assistant of the members of the PET research group of Central Research Laboratory, Hamamatsu Photonics for PET studies.

### **Conflict of interest**

All authors are employees of Hamamatsu Photonics K.K., and this research was supported by the company's budget. The authors declare that they have no competing interests.

### **Author details**

Hideo Tsukada Central Research Laboratory, Hamamatsu Photonics K.K., Hamamatsu, Shizuoka, Japan

\*Address all correspondence to: tsukada@crl.hpk.co.jp

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*PET Imaging of Mitochondrial Function in the Living Brain DOI: http://dx.doi.org/10.5772/intechopen.86492*

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Section 4

## Mitochondrial Function and Dysfunction

#### **Chapter 4**

## Mitochondrial Proteomic and Molecular Network Alterations in Human Ovarian Cancers

*Xianquan Zhan and Na Li*

#### **Abstract**

Mitochondrion is a multi-functional organelle, which plays important role in human ovarian cancers. Mitochondrial quantitative proteomics was used to detect, identify, and quantify proteins from mitochondrial samples prepared from ovarian cancer and normal control ovary tissues. A total of 5115 mitochondrial proteins and 1198 mitochondrial differentially expressed proteins (mtDEPs) were identified in human ovarian cancer compared to control tissues. Pathway network analysis revealed multiple pathway network changes to involve those mitochondrial proteins and mtDEPs. These findings provide the scientific data about the role of mitochondria plays in ovarian cancer, and offer the source for discovery of mitochondrial biomarker for ovarian cancers.

**Keywords:** mitochondrial proteome, proteomics, molecular networks, biomarker, ovarian cancer

#### **1. Introduction**

Mitochondrion is a multi-functional organelle, which is the center of cell energy metabolism, cell signaling, and oxidative stress [1, 2]. Mitochondrial dysfunction is a hallmark in human ovarian cancers, and plays important roles in ovarian carcinogenesis, which has been looked as the cause, biomarker, and therapeutic target for ovarian cancers [3–5]. First, a study finds mitochondrial morphology is significantly changed in ovarian cancers compared to controls. Electron microscopy morphology study shows that mitochondria are abundant and large volume in ovarian cancer cells and tissues [6, 7]. Second, mitochondrial ribosomal proteinencoding genes might be the anti-oncogenes to serve as new biomarkers and therapeutic targets. For example, bcl-2-interacting mitochondrial ribosomal protein L41 (MRPL41) is differentially expressed in carcinomas to associate with various epigenetic states [8]. Mitochondrial ribosomal protein S23 (MRPS23) is involved in cancer cell proliferation, which might serve as the therapeutic target [9]. MRPS15 is significantly upregulated in epithelial breast cells and tissues [10]. Mitochondrial COX1 is expressed abnormally in multiple cancers [11–13]. Many cancer-relevant communication signaling pathways are linked to mitochondrial proteins. Third, mitochondria are the center of oxidative stress, which might be the 'fuel' center for a cancer metabolism [10]. The abnormal energy metabolism, namely the Warburg and reverse-Warburg effects, is the important characteristics in cancers [14].

Therefore, mitochondria play important roles in tumorigenesis, proliferation, angiogenesis, invasiveness, and metastasis of cancer cells [14, 15]. Proteins are the important performer in maintaining mitochondrial morphology and functions. It emphasizes the important scientific merits of mitochondrial proteomics in ovarian cancer research and clinical practice [16–22]. Mitochondrial proteins function in mutually interacted molecular pathway network system, which fits the real situation of ovarian cancer that is a multi-cause, multi-process, and multi-result disease [23–25]. It is very difficult to use single-parameter biomarker to predict, diagnose, and prognostic assess ovarian cancer, thus multi-parameter biomarkers or molecule pattern biomarker is necessary for ovarian cancer prediction, prevention, and treatment [26, 27]. Mitochondrial proteomics is an effective approach to systematically investigate the role of mitochondria in ovarian cancer for discovery of reliable mitochondrial protein biomarkers to insight into the molecular mechanism and determination of therapeutic target to mitochondria for ovarian cancers. Quantitative proteomic methods commonly include two-dimensional gel electrophoresis (2DGE) [28, 29] or two-dimensional difference in-gel electrophoresis (2D DIGE) [30] comparative proteomics, and gelfree-based quantitative proteomics [14, 15], for example, isobaric tags for relative and absolute quantification (iTRAQ ) [31, 32], tandem mass tag (TMT) [33], or label-free-based quantitative proteomics [34, 35], with different advantages and disadvantages, respectively. Those quantitative proteomic methods can achieve a high-throughput and high-sensitive identification of mitochondrial proteins and post-translational modifications. Currently, stable isotopic labeled large-scale 2DGE coupled with high-sensitivity liquid chromatography-tandem mass spectrometry (LC-MS/MS) is able to detect, identify, and quantify up to least 500,000 protein proteoforms in human tissue proteoforms [36, 37]. iTRAQ, TMT, or label-free is commonly coupled with two-dimensional LC-MS/MS (2DLC-MS/MS), which enables detect, identify, and quantify up to several thousands of proteins and PTMs, even though these gel-free methods are unable to discriminate proteoforms and homolog proteins [38].

Ovarian cancer is a malignant cancer with high morbidity and mortality [39, 40] and without clear molecular mechanisms and effectively reliable biomarkers for its early-stage diagnosis to improve its prognosis. This book chapter used iTRAQ-labeled strong cation exchange chromatography (SCX)-LC-MS/MS method to detect, identify, and quantify mitochondrial proteins and mitochondrial differentially expressed proteins (mtDEPs) between human ovarian cancer and control ovary tissues. The identified mitochondrial proteins and differentially expressed proteins were subject to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway network analysis for revealing pathway network alteration in ovarian cancers compared to controls. Those findings provide the scientific data to establish mitochondrial proteomic reference map of ovarian cancer, mtDEP profile and the corresponding pathway network alterations to link with ovarian cancer pathogenesis, which is the resource for discovery of potential biomarkers and mitochondria-targeting drug targets for ovarian cancers.

#### **2. Methods**

#### **2.1 Ovarian cancer tissues and preparation of mitochondria protein samples**

Seven ovarian cancer tissues and eleven control ovaries with benign gynecologic disease were used in this study. Mitochondria were isolated and purified from ovarian cancer and control tissues with differential-speed centrifugation and

*Mitochondrial Proteomic and Molecular Network Alterations in Human Ovarian Cancers DOI: http://dx.doi.org/10.5772/intechopen.86493*

Nycodenz density gradient centrifugation. The purified mitochondria were verified with electron microscopy, and Western blot with different antibodies specific to different subcellular organelles, including COX4I1 (mitochondrion), flotillin-1 (cytomembrane), GM130 (Golgi apparatus), catalase (peroxisomes), cathepsin B (lysosome), and lamin B (cell nucleus). The proteins were extracted from purified mitochondrial samples for iTRAQ-labeled quantitative proteomic analysis. The detailed procedure was described in our previous publications [14, 15].

#### **2.2 iTRAQ-based quantitative proteomics analysis**

The prepared mitochondrial proteins (200 μg/each sample) were treated with *N*-hydroxysuccinimide (SDT), followed by reduction, alkylation, digestion with trypsin, and desalination. The tryptic peptide (100 μg/each sample) was labeled with iTRAQ reagent, and each sample was labeled three times. The six labeled tryptic peptide samples were mixed, followed by peptide fractionation with strong cation exchange (SCX) chromatography. Each SCX-fractionated sample was subject to LC-MS/MS analysis on a Q Exactive mass spectrometer (Thermo Scientific) within a 60-min LC separation gradient to obtain MS/MS data. The MS/MS data were used for identity of proteins with MASCOT search engine. The iTRAQ reporter-ion intensities were used to quantify each protein and determine each mtDEPs. The detailed procedure was described in our previous publications [14, 15].

#### **2.3 Bioinformatics and pathway network analysis**

The identified proteins and DEPs in mitochondrial samples were subject to GO and KEGG pathway enrichment analysis with Cytoscape, and DAVID online software (https://david.ncifcrf.gov/home.jsp). Multiple Experiment Viewer (https://sourceforge.net/projects/mev-tm4/files/mev-tm4/) was used to make heat map. GO analysis included cellular component (CC), molecular function (MF), and biological process (BP). PANTHER (http://www.pantherdb.org/) was used to further enrich GO CC.

#### **2.4 Validation of mtDEPs and molecular networks in cell models and mitochondrial tissues**

Ovarian cancer cells TOV-21G and control cells IOSE80 were used to extract RNAs and proteins. Quantitative real-time PCR (qRT-PCR) was used to measure the mRNA expression levels of GLDC, PCK2, IDH2, CPT2 and HMGCS2 in TOV-21G cells compared to IOSE80 cells. Western blot was used to measure the protein expression levels of GLDC, PCK2, IDH2, CPT2 and HMGCS2 in TOV-21G cells compared to IOSE80 cells, and in ovarian cancer mitochondrial samples compared to control mitochondrial samples; and β-actin was used as internal standard for Western blot analysis.

#### **2.5 Statistical analysis**

For GO and KEGG enrichment analyses, p values were corrected with Benjamini-Hochberg (FDR) for multiple testing. For qRT-PCR and Western blot, the student's t-test was used to measure between-group difference with SPSS software 13.0, and data was presented as the mean ± SD with p < 0.05. Each experiment was repeated at least three times.

### **3. Results and discussion**

### **3.1 Mitochondrial proteomic profile in human ovarian cancer tissue**

iTRAQ-labeling coupled with SCX-LC-MS/MS identified 5115 proteins in mitochondrial samples prepared from human ovarian cancer and control ovary tissues, with at least one peptide sequence matches (PSMs). All of identified proteins was collected in the supplemental Table 1 in our previous publication [15]. Those 5115 proteins mainly distributed within pI 3.81–12.25 and molecular weight (MW) 2.6–1158.2 kDa, and in multiple cell components including cell junction (0.8%), cell part (42.7%), extracellular matrix (0.6%), macromolecular complex (17.8%), organelle (28.2%), and synapse (0.3%) (**Figure 1**). Of them, 2565 (50.14%) were increased, and 2550 (49.86%) were decreased in the abundance in ovarian cancers compared to control ovaries. Furthermore, statistical significance analysis revealed 1198 mtDEPs in human ovarian cancers compared to control ovaries, including 523 (43.66%) upregulated proteins and 675 (56.34%) downregulated proteins, with fold-change ≥1.5 or ≤−1.5, and p < 0.05. Those 1198 mtDEPs were collected in the supplemental Table 1 in our previous publication [14]. Those mtDEPs might be directly linked to ovarian cancer pathogenesis, and the potential resource for biomarkers. From a systemic molecular network angle, one must realize that those non-significant difference proteins might be also important in ovarian cancer pathogenesis because they might be the hub-molecule in a network, because some studies have found that some hub-molecules changed smaller than those boundary molecules in a molecular network in a given condition.

#### **3.2 Pathway networks involved in mitochondrial proteins in ovarian cancer**

KEGG pathway network analysis revealed 52 statistically significant pathways to involve mitochondrial proteins including mtDEPs in ovarian cancers compared to

#### **Figure 1.**

*Subcellular location of 5115 proteins analyzed with PANTHER. Modified from Li et al. [15], with permission from Bioscientifica Ltd., copyright 2018.*


*Mitochondrial Proteomic and Molecular Network Alterations in Human Ovarian Cancers DOI: http://dx.doi.org/10.5772/intechopen.86493*


*Mitochondrial Proteomic and Molecular Network Alterations in Human Ovarian Cancers DOI: http://dx.doi.org/10.5772/intechopen.86493*


#### **Table 1.**

*52 statistically significant KEGG pathways enriched from 5115 proteins in ovarian cancers.*


#### **Figure 2.**

*52 statistically significant KEGG pathways enriched from 5115 proteins in ovarian cancers. Modified from Li et al. [15], with permission from Bioscientifica Ltd., copyright 2018.*

control ovaries (**Table 1** and **Figure 2**), including phagosome, peroxisome, valine, leucine and isoleucine degradation, lysosome, fatty acid metabolism, citrate cycle (TCA cycle), oxidative phosphorylation, glycolysis/gluconeogenesis, metabolic


*PSMs = peptide sequence matches; MW = molecular weight; Ratio (T/N) = ratio of tumors to normal controls. Reproduced from Li et al. [15], with permission from Bioscientifica Ltd., copyright 2018.*

#### **Table 2.**

*Mitophagy adaptors and regulatory molecules involved the identified proteins in ovarian cancer biological system.*

pathways, carbon metabolism, glyoxylate and dicarboxylate metabolism, glutathione metabolism, propanoate metabolism, sulfur metabolism, 2-oxocarboxylic acid metabolism, pyruvate metabolism, porphyrin and chlorophyll metabolism, beta-alanine metabolism, butanoate metabolism, tryptophan metabolism, arginine and proline metabolism, ribosome, protein processing in endoplasmic reticulum, biosynthesis of amino acids, aminoacyl-tRNA biosynthesis, proteasome, protein

*Mitochondrial Proteomic and Molecular Network Alterations in Human Ovarian Cancers DOI: http://dx.doi.org/10.5772/intechopen.86493*

#### **Figure 3.**

*Energy metabolism pathway changed in ovarian cancer. Reproduced from Li et al. [14], with permission from Elsevier Inc., copyright 2018.*

digestion and absorption, ECM-receptor interaction, focal adhesion, protein export, signaling pathway, complement and coagulation cascades, platelet activation, PPAR pentose phosphate pathway, fatty acid degradation, vasopressinregulated water reabsorption, and regulation of actin cytoskeleton. Those pathway systems provided an overall molecular network changes in ovarian cancers, which might be important in ovarian cancer pathogenesis.

Among those altered pathway systems, especially interested is that mitophagy pathway and energy metabolism pathway were significantly changed in ovarian cancers compared to controls. The changed mitophagy pathway in ovarian cancer included phagosome, peroxisome, valine, leucine and isoleucine degradation, lysosome, and fatty acid metabolism pathways [15]. Mitophagy is to engulf any


#### **Table 3.**

*Differentially expressed glycolysis/Kreb's cycle/mitochondrial respiratory chain/RNA binding proteins in EOC.*

material in autophagosome, and subsequently fuses with lysosomes to release high-energy substance such as fatty acid and amino acid. Autophagosome also commonly contains mitochondria, proteins, or peroxisome. Mitophagy processes are involved in autophagy machinery, mitophagy adaptors, and regulatory molecules such as Bcl2-L12, p62, OPTN, prohibitin 2, OPA1, CK, PGAM5, BNIP3L(NIX), and FUNDC1 (**Table 2**). These findings were consistent with previous studies. The changed energy metabolism pathway in ovarian cancers included citrate cycle (TCA cycle), oxidative phosphorylation, and glycolysis (**Figure 3**) [14], and the important molecules were significantly changed in three energy metabolism pathways, including PFKM, PKM, PDHB, CS, and IDH2 (**Table 3**). It clearly demonstrated the Warburg and reverse-Warburg effects coexisted in ovarian cancers.

*Mitochondrial Proteomic and Molecular Network Alterations in Human Ovarian Cancers DOI: http://dx.doi.org/10.5772/intechopen.86493*

#### **Figure 4.**

*Validation of potential biomarkers (GLDC, PCK2, IDH2, CPT2 and HMGCS2) in ovarian cancer cell model with qRT-PCR (A) and Western blot (B), and in human mitochondrial samples with Western blot (C). β-actin was used as internal standard. Reproduced from Li et al. [15], with permission from Bioscientifica Ltd., copyright 2018.*

#### **3.3 Potential biomarkers for ovarian cancers**

Those 5115 mitochondrial proteins including 1198 mtDEPs were the resource of potential biomarkers for ovarian cancers. For example, mtDEPs in mitophagy pathway and energy metabolism pathway might be effective biomarkers and therapeutic targets for ovarian cancer. Five mtDEPs, including GLDC, PCK2, and IDH2 in peroxisome pathway, CPT2 in fatty acid degradation pathway, and HMGCS2 in the valine, leucine and isoleucine degradation pathway were further validated by qRT-PCR and Western blot in ovarian cancer cells compared to normal control cells (**Figure 4A** and **B**), and by Western blot in the ovarian cancer tissue mitochondrial samples (**Figure 4C**). These results also confirmed the results of iTRAQ quantitative proteomics.

#### **4. Conclusions**

iTRAQ-labeled SCX-LC-MS/MS quantitative proteomics was an effective method to detect, identify, and quantify mitochondrial proteins and mtDEPs in mitochondrial samples prepared from human ovarian cancer and control ovary tissues. Totally 5115 mitochondrial proteins including 1198 mtDEPs were identified in ovarian

cancers, and 52 statistically significant pathways were identified to involve those mtDEPs. More interested is that this study found mitophagy pathway (phagosome, peroxisome, valine, leucine and isoleucine degradation, lysosome, and fatty acid metabolism), and energy metabolism pathways (citrate cycle, oxidative phosphorylation, and glycolysis) were significantly changed in ovarian cancers. The important molecules Bcl2-L12, p62, OPTN, prohibitin 2, OPA1, CK, PGAM5, BNIP3L(NIX), and FUNDC1 in mitophagy pathway, and PFKM, PKM, PDHB, CS, and IDH2 in energy metabolism pathways were significantly changed. It clearly demonstrated the changed mitophagy and energy metabolism pathways played important roles in ovarian cancers. These findings provide the large-scale proteomic variation profiles and molecular network alterations for ovarian cancer, which are the important scientific data to insight into the roles of mitochondrial dysfunction in ovarian cancer.

#### **Acknowledgements**

The authors acknowledge the financial supports from the Hunan Provincial Hundred Talent Plan (to X.Z.), National Natural Science Foundation of China (Grant no. 81572278 and 81272798 to X.Z.), China "863" Plan Project (Grant No. 2014AA020610-1 to X.Z.), the Hunan Provincial Natural Science Foundation of China (Grant No. 14JJ7008 to X.Z.), and the Xiangya Hospital Funds for Talent Introduction (to X.Z.).

#### **Conflict of interest**

We declare that we have no financial and personal relationships with other people or organizations.

#### **Author's contributions**

X.Z. conceived the concept, designed the manuscript, wrote and critically revised the manuscript, coordinated and was responsible for the correspondence work and financial support. N.L. participated in the literature analysis, data analysis, and prepared figures.

#### **Acronyms and abbreviations**


*Mitochondrial Proteomic and Molecular Network Alterations in Human Ovarian Cancers DOI: http://dx.doi.org/10.5772/intechopen.86493*

#### **Author details**

Xianquan Zhan\* and Na Li Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital, Central South University, Changsha, China

\*Address all correspondence to: yjzhan2011@gmail.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 5**

## Mitochondrial Dysfunction as a Key Event during Aging: From Synaptic Failure to Memory Loss

*Claudia Jara, Angie K. Torres, Margrethe A. Olesen and Cheril Tapia-Rojas*

#### **Abstract**

Mitochondria are important cellular organelles with key regulatory functions in energy production, oxidative balance, and calcium homeostasis. This is especially important in the brain, since neurons require a large number of functional mitochondria to supply their high energy requirement, mainly for synaptic processes. A decrease in the activity and quality of mitochondria in the brain, particularly in the hippocampus, is associated with normal aging and a large number of neurodegenerative diseases compromising memory function. Although synaptic and cognitive dysfunction is multifactorial, growing evidence demonstrates that mitochondria play a key role in these processes and suggests that maintaining mitochondrial function could prevent these age-dependent alterations. In this chapter, we will discuss the hippocampal mitochondrial dysfunction present in aging and how these defects promote age-associated synaptic damage and cognitive impairment. We will summarize evidence that shows how neurodegeneration can be accelerated or attenuated during aging by modulating mitochondrial function.

**Keywords:** aging, mitochondria, oxidative stress, synapses, memory

#### **1. Introduction**

Aging is an extensively studied process, identifying a growing interest in how and why cognitive processes are affected from a neurobiological approach [1]. Aging is a multifactorial biological process, characterized by deterioration of physiological and cellular functions including brain function [2], where age is the main risk factor for the development of pathologies such as cancer, diabetes, cardiovascular disorders, and neurodegenerative diseases [3]. Cognitive deterioration occurs during aging, where reasoning, attention, and memory, among other processes, decrease gradually with the age [4]. Cellular senescence and alterations to mitochondria and in proteolytic systems are considered hallmarks of aging [3], where one of the most studied is the mitochondria [5]. In fact, mitochondrial dysfunction has been directly associated with the aging phenotype and the majority of diseases that lead to cognitive damage.

Over the last decades, a great interest has arisen regarding mitochondrial structure and function due to its relation with the aging brain [5]. Mitochondria are organelles essential for energy production, whose size is usually 0.5–1 μm, composed by two membranes, forming the intermembranous space and the mitochondrial matrix [6]. The outer membrane contains many copies of the transport protein porin (or voltage-dependent anion-selective channels (VDAC)), which allows the passage of molecules with a maximum weight of 5 KDalton (KDa), and the inner membrane forms numerous invaginations, tubular structures, called cristae [6]. Mitochondria are capable of remodeling their architecture through fission and fusion processes, allowing morphological adaptation to different situations [6]. Fission is essential for mitochondrial duplication and is necessary for mitophagy, allowing dysfunctional mitochondrial sections to be recycled. Fusion allows mitochondria to interconnect, allowing damaged mitochondria to maintain their function. However, fission-fusion processes are interrupted during aging, generating damaged mitochondria [7].

Mitochondria have a small circular genome called mtDNA, which encodes 22 tRNAs, 2 mitochondrial rRNAs, and 13 subunits of the electron transport chain (ETC) [8]. mtDNA can be damaged by exposure to reactive oxygen species (ROS), chemical carcinogens, and ionizing radiation affecting the mitochondrial function; changes are also observed during aging [9]. The internal mitochondrial membrane contains the ETC, responsible for generating ATP. ETC is formed by five protein complexes; complex I (NADH dehydrogenase) receives electrons of NADH which pass through the ETC via oxidation-reduction reactions forming an electrochemical gradient that allows the formation of ATP. In addition, FADH2 donates its electrons to complex II (succinate dehydrogenase) performing the same action for ATP generation but at lower production levels [10]. As a secondary product, the ETC forms ROS, specifically by complexes I and III, but its production is controlled by antioxidant enzymes [11]. Therefore, in normal conditions ROS production is moderate, providing certain physiological roles [11]; however, during aging ROS accumulation causes biological damage known as "oxidative stress" [12].

In the past, mitochondria have always been highlighted for its role in ATP production; however, another key function is to maintain intracellular calcium homeostasis [13]. The outer mitochondrial membrane is permeable to ions and ~5 KDa metabolites because its lipid bilayer has transmembrane proteins that form the mitochondrial permeability transition pore (mPTP). mPTP opening and closing dynamics regulates the concentration of calcium [13]. However, in conditions of high calcium concentrations, permanent mPTP opening generates massive transport of ions and small molecules <1.5 KDa through the membrane, causing increased ROS production, inhibition of the ETC, and mitochondrial swelling, which finally results in the release of pro-apoptotic factors and cell death [14].

In this chapter we will discuss the mitochondrial alterations observed in the brain during aging, focusing on mitochondrial functions including redox balance, bioenergetics, and calcium homeostasis, and its implications in the aging process. In addition, we will discuss the contribution of mitochondrial dysfunction to synaptic failure and cognitive impairment. Finally, we will summarize potential treatments that have been proposed to prevent or attenuate the loss of mitochondrial function that could be used as potential antiaging treatment.

#### **2. Oxidative stress: the main characteristic of normal aging**

Aging is a complex process that involves both intrinsic and extrinsic factors [3]. Several researches showed that the reduction of synaptic function during aging could be related to increased oxidative stress and mitochondrial dysfunction [15, 16]. The latter involves decreased production of energy and redox balance, activation of nitric oxide synthase, and an abundant generation of free radicals; meanwhile increased ROS production impairs neuronal function at advanced ages [17].

#### *Mitochondrial Dysfunction as a Key Event during Aging: From Synaptic Failure to Memory Loss DOI: http://dx.doi.org/10.5772/intechopen.88445*

Mitochondria have a pivotal role in ROS production; they are the main organelle producer of species such as hydrogen peroxide (H2O2), superoxide anion (O2<sup>−</sup>), and hydroxyl radicals (OH<sup>−</sup>) [18]. ROS comprise all molecules derived from oxygen, can exist independently, and contain one or more unpaired electrons in their orbitals [19]. Normally, mitochondria generate ROS as a result of adequate function of the ETC by complexes I and III in OXPHOS to produce ATP. Likewise, electrons that escape mitochondrial ETC can reduce oxygen to form O2<sup>−</sup> [20]. Additionally, H2O2 is more stable than O2<sup>−</sup> and can diffuse freely through the membranes to the cytosol or nucleus, causing oxidative damage to many cellular compartments [21].

The mitochondrial ROS produced in normal conditions have important physiological roles in maintaining cell homeostasis, participate as signaling molecules, and are also related to the regulation of cell survival [22]. In contrast, excessive ROS production promotes cellular damage [23]. For example, recent evidence suggests that higher mitochondrion-derived ROS result in enhanced formation of Aβ, an effect that is prevented with the use of antioxidants that rescue mitochondrial function in cellular and animal models of Alzheimer's disease (AD) [24]. This suggests that higher ROS generation mediated by mitochondria is involved in early stages of age-associated diseases and during aging [25].

Cells maintain a balance between free radicals by the action of antioxidants molecules, which neutralize or remove them [22]. Cells are equipped with a variety of defense mechanisms to remove ROS, including antioxidant enzymes that facilitate antioxidant reactions and decompose ROS [26]. Among the antioxidant enzymes are glutathione reductase (GR), glutathione peroxidase (GPx), and catalase (CAT). In addition, superoxide dismutases (SODs), such as copper-zinc-superoxide dismutase (CuZnSOD) and manganese superoxide dismutase (MnSOD), help the dismutation of superoxide radicals to generate H2O2, which is further removed by CAT and GPx enzymes [26]. Altogether, these antioxidant defenses regulate the amount of ROS, preventing accumulation and oxidative stress [27].

Oxidative stress occurs when the antioxidant defense mechanisms are unable to neutralize free radicals in the cell. This imbalance between the production of oxidative molecules and the antioxidant defense leads to an accumulation of ROS, which oxidize and produce damage to lipids, proteins, and DNA molecules. Similarly, ROS could alter many cell compartments, for example, promoting peroxidation of lipid membranes and inactivation of enzymes by oxidation [28].

In oxidative stress conditions, the concentration of ROS increases transiently or chronically, altering the cellular metabolism and its regulation [23]. Interestingly, these elements are implicated in the aging process, the mitochondrial free radical theory of aging (MFRTA) being the most accepted theory to explain the age-associated degeneration [29]. This theory exposed by Harman proposes that mitochondria play a central role in aging and indicates that aging is the product of accumulated damage caused by mitochondrial ROS in the cells and tissues of organisms [30, 31]. Nevertheless, this theory has also been questioned, since aging is a multifactorial biological process and not just the consequence of a unique factor [32]. Thus, the mitochondrial theory of aging is relevant since these organelles are energy sources for cells and coordinate important processes such as apoptosis. During aging, accumulation of mtDNA mutations is increased, the mitochondrial genes related to energy production become progressively less active, and the mitochondria are observed as fragmented, producing less energy [33]. The brain is particularly susceptible to oxidative damage being the most aerobically active organ in the body due to its high metabolism [34]. The brain is generally in a redox balance; however, the high production and accumulation of ROS accompanied by a reduction in the antioxidant defense system plays a key role in aging, causing damaging effects due to the large number of potential harmful intermediates that cause neuronal

dysfunction [3, 35]. In turn, increased oxygen radical-induced oxidative damage during aging leads to significant changes in brain mitochondrial function [29]. Therefore, oxidative stress is implicated in aging and a wide range of age-related pathologies, such as AD and Parkinson's disease (PD), among others [3, 16, 36].

In the aged brain, a reduction in normal antioxidant defense machineries is observed, which increases the brain's susceptibility to the harmful effects of oxidative molecules [27, 37]. In addition to this, mitochondrial dysfunction contributes to ROS overproduction. It is important to emphasize that a decrease in the activity of oxidative enzymes accompanied by excessive production of oxidant molecules during aging is the main toxic mechanism that explains the neurodegeneration observed at an advanced age [27, 37]. Since mitochondria are the main source of ROS production, they are in turn more exposed to oxidative damage at a faster and stronger rate than other organelles and cell compartments [16]. Moreover, the mitochondrial antioxidant system is less active than the antioxidant systems of other organelles, a feature that increases with age [5]. These mitochondrial "defects" can greatly affect several cellular processes that contribute to the aging phenotype [38]. Therefore, age is an important risk factor that increases the susceptibility of mitochondria, making them more vulnerable to oxidative stress, resulting in a vicious cycle of mitochondrial dysfunction and more oxidative damage [5, 33]. Mitochondria should be considered as a key factor in the development of agerelated neurodegeneration, and therefore therapeutic strategies such as mitochondrial protectors or antioxidants that improve mitochondrial function could be used to prevent or delay aging.

#### **3. Bioenergetic failure during aging**

One of the main functions of mitochondria is energy production in the form of ATP through OXPHOS [39]. The main substrate of neurons is glucose. Through the glycolytic pathway, the cell generates only two ATP molecules per glucose molecule; however in this pathway two pyruvate molecules are produced. These molecules then enter the mitochondria to be oxidized in the tricarboxylic acid (TCA) cycle producing NADH and FADH2, which in turn enter the ETC to produce high amounts of ATP by OXPHOS [40]. Electrons from NADH and FADH2 are transferred through four complexes to molecular oxygen, pumping protons to the intermembrane space, which form a proton gradient that generates the mitochondrial membrane potential (Δψm). This Δψm is fundamental for adequate mitochondrial function, mainly for ATP synthesis by the ATPase complex [39].

Due to the importance of mitochondrial energy production, failures in mitochondrial bioenergetics are related to several neurological diseases such as amyotrophic lateral sclerosis (ALS) [41] and several age-associated neurodegenerative disorders such as AD [42]. A mitochondrial failure can be caused by either a dysfunction in the OXPHOS complexes or by mutations in the mtDNA, which encodes for 13 proteins that makes the different subunits of the ETC complexes. Interestingly, mtDNA mutations produce a group of pathologies known as primary mitochondrial disorders, characterized by neurological alterations. Thus, neurodegeneration is often related to mitochondrial dysfunction as a primary or secondary target, mediating the pathogenic events [43]. Since the main energy source in brain cells is glucose oxidation, the energy obtained from the mitochondrial OXPHOS system is vital to fulfilling their high basal energy requirement, including maintenance of the membrane potential for the propagation of electric signals, reestablishment of the ion balance after the action potential (Na+/K+ ATPase activity), vesicle recycling, and neurotransmitter release [44].

#### *Mitochondrial Dysfunction as a Key Event during Aging: From Synaptic Failure to Memory Loss DOI: http://dx.doi.org/10.5772/intechopen.88445*

Therefore, a deficit in ATP production can lead to neuronal damage and finally cell death, producing diverse defects in brain functions as occurs during normal aging, considered an important risk to the development of neurodegenerative disease [45]. An example of this is the glutamate-glutamine cycle, an essential process for the release of glutamate from the presynaptic terminal. Glutamate uptake occurs in astrocytes, where it is converted to glutamine and then transferred back to glutamatergic neurons. For these processes to occur, the neuron requires ATP [46], as well as for the accumulation of glutamate in synaptic vesicles [47]. Therefore lower ATP levels will result in reduced glutamate release leading to decreased excitatory synapsis and consequently decreased synaptic plasticity as a result of altered longterm potentiation (LTP) and long-term depression (LTD) [48].

In addition, AD is a neurodegenerative pathology where mitochondrial deficiency is observed in oxidative phosphorylation, with defective OXPHOS enzymes [49]. Several studies showed decreased cytochrome c oxidase activity but an increase in mitochondrial mRNA for complex IV, which may be a compensatory response for the reduced cytochrome c oxidase activity [50, 51]. Also, there is a decrease in NADH dehydrogenase expression and an increase in complex III mRNA in AD patients [51]. These defects in the OXPHOS complex impede correct ATP production and increase ROS production [52], which could generate damage to mitochondrial proteins, activate the mPTP, and mutagenize the mtDNA, leading to defective OXPHOS. All these mitochondrial defects can ultimately contribute to the characteristic synapse loss in the neocortex and hippocampus of AD patients [53], which correlates with cognitive impairment and memory loss. Similarly, critical mitochondrial dysfunction has been associated with PD. Several mutations in proteins that can directly or indirectly regulate mitochondrial activity and morphology have been described. Examples of this are PTEN-induced kinase 1 (PINK1) which induced mitochondrial autophagy during stress [54] and protein deglycase (DJ-1) which is a multifunctional protein that reacts against anti-oxidative stress [55]. These two proteins are localized in the mitochondria, while parkin, another protein that degrades dysfunctional mitochondria, translocates to damaged mitochondria [54]. The first evidence that mitochondria could be related to PD was published by Langston et al. in 1983 where they showed that the mitochondrial complex I inhibitor, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) can cause acute and irreversible parkinsonian symptoms in humans [56]. Later, other mitochondrial toxins such as rotenone, which induces similar symptoms to MPTP, were described, leading to the development of a rotenone rodent model of Parkinson's disease [57]. Other studies of postmortem tissue from PD patients showed defects in complex I, NADH dehydrogenase in the substantia nigra [58].

Interestingly, similar bioenergetic deficits present in neurodegenerative disease are also seen in normal aging. In this natural process, it is well known that a large number of mutations accumulate in the mtDNA in different tissues, such as the brain and the muscle, the tissues with most accumulation of mutations, possibly due to the higher energetic demand [59]. For example, in a study performed with humans between 49 and 92 years old, they showed an increase by 25% in the muscle fibers that exhibit abnormalities in mitochondrial ETC in 92-year-old individuals compared to 49 year olds [60]. Another study showed that a mouse with a deficiency for mitochondrial DNA polymerase γ (POLG) had an impaired proofreading ability, accumulated mtDNA mutations, and presented a premature-aged phenotype (hair loss, graying, kyphosis, reduced survival percentage, loss of bone mass, etc.) at the age of 9 months [61]. These mutations can lead to mitochondrial dysfunction associated with a deficient respiratory chain and a decrease in ATP production [62]. There is evidence that the brain of aged mice (26 months old) contains mutations in protein-coding regions that result in significant changes

in the complex I subunit ND5 and complex III subunit CytB [62]. These mutations may limit correct assembly of these complexes, which correlates with their decreased activity during aging [62]. Other studies also showed downregulation of several genes coding for mitochondrial proteins in heart tissue, which correlated with a significant decrease in the respiratory capacity of mitochondria to oxidize substrates [63]. In liver tissue, there is a decrease in the respiratory control ratio and in ADP/oxygen (an index of ATP synthesis efficiency) in senescence-accelerated mice (SAMP8) mitochondria [64], suggesting that at 18 months of age, there is insufficient ATP for normal cell metabolism, which may be due to a dysfunctional energy transfer mechanism.

In the brain, it is widely known that with aging there is a decrease in the electron transfer activity accompanied by a decrease mainly in complexes I and IV [65, 66]. Several studies showed that complex IV activity is decreased in substantia nigra, hippocampal dentate gyrus, frontal cortex, and cerebellum during aging [67, 68]. A study performed with aged CD1 Swiss mice showed that NADH-cytochrome c reductase (complexes I and III) activity is the most affected during aging, decreasing by 48% in old mice (18 months), while succinate-cytochrome c reductase activity (complexes II and III) remain unmodified with age, indicating selective impairment of NADH dehydrogenase activity (complex I) during normal aging [69]. Likewise, cytochrome oxidase (complex IV) activity is decreased by 13% in old animals [69]. Additionally, there is evidence of increased expression of mitochondrial genes for complexes I, III, IV, and V in 18-month-old mice in the hippocampus, medial prefrontal cortex, and striatum [70], suggesting a compensatory mechanism that could induce overproduction of ETC proteins. However, this increased mRNA expression is not sustained over time, since 24-month-old mice have decreased expression of ETC complexes [70].

Another parameter that is altered during aging is the Δψm due to increased H<sup>+</sup> permeability of the inner mitochondrial membrane and a consequent failure in maintaining the H+ electrochemical gradient [71]. There is evidence that there is a decrease in the membrane potential in the cortical and striatal mitochondria of 33-month-old rats [72]. In the same way, a study in primary cultures of glial cells from the brain of young (4–6 months) and old (26–29 months) mice shows a decay in the Δψm in astrocytes [73].

Thus, mitochondrial bioenergetic failure is a hallmark of different diseases including neurodegenerative disorders. Interestingly, these same patterns of decreased ATP production, OXPHOS failure, and depolarization of the mitochondrial membrane are seen during aging, a natural process of everybody's life. This makes the mitochondria an important player in all neurological degeneration related with aging, such as synaptic failure and cognitive impairment.

#### **4. Age-associated calcium dysregulation**

Calcium (Ca+2) is an ion that participates in a wide variety of functions in the cells of organisms, being an intracellular regulator of many physiological processes [74]. Intracellular calcium signals participate in the regulation of a large number of processes, which include gene expression, cell cycle stages, control of muscle contraction, autophagy, and cell death, among other functions, being a second intracellular messenger [74, 75].

In the central nervous system (CNS), Ca+2 plays a very important role in the neuronal synapse, mainly promoting exocytosis of the synaptic vesicles in the presynaptic region, meanwhile in the postsynaptic site is important for regulating the morphology of dendritic spines and spinogenesis [76, 77]. Calcium homeostasis

#### *Mitochondrial Dysfunction as a Key Event during Aging: From Synaptic Failure to Memory Loss DOI: http://dx.doi.org/10.5772/intechopen.88445*

is fundamental for correct cellular function, and the mitochondria are structures important for maintaining the intracellular calcium concentrations [78]. They participate in the local regulation of cellular Ca+2 homeostasis, since it captures Ca+2 from the cytosol in response to ion fluxes through channels in both the inner and outer plasma membranes or by release of Ca+2 from the endoplasmic reticulum (ER) [79]. Thus, when the cytosolic concentration of calcium increases, mitochondria capture and accumulate large amounts of the ion in order to control intracellular concentrations. Therefore, for appropriate neuronal functioning, adequate parameters of intracellular calcium concentrations must be maintained [80]. Interestingly, aged brain neurons are incapable of regulating intracellular calcium, mainly due to dysfunctional mitochondria and increased oxidative stress [78, 81].

Mitochondrial dysfunction substantially contributes to biological aging [78]. In aging, oxidative stress affects mitochondrial function and, therefore, its role in Ca+2 homeostasis [78, 82]. When calcium homeostasis is altered, it has a detrimental role on the aging brain and is also associated with the development of neurodegenerative diseases [83]. Several studies showed that an increase of the Ca+2 affects synaptic communication, neurotransmitter release, and signal transduction, all this generating excitotoxicity and neuronal loss [84]. In addition, these alterations could also contribute to memory impairment [16, 84].

In this way, since the aging is associated with a marked cognitive decline, the calcium imbalance hypothesis proposed by Khachaturian turns out to be well accepted [85, 86]. This hypothesis proposes that changes in calcium regulation gradually modulate normal brain aging and, at the same time, increase their vulnerability to neurodegenerative diseases such as AD [87]. Calcium signaling depends of the transient elevation of its intracellular concentration. In brain cells, reduced regulation of calcium homeostasis is an early event during aging, altering multiple signaling pathways and affecting various molecular and cellular functions [88].

Due to their high buffering capacity, mitochondria are an essential component for maintaining calcium homeostasis, due to their involvement in the regulation of intracellular calcium signaling [89]. Also, other mitochondrial characteristics that facilitate its role in the regulation of calcium signaling is its structural plasticity produced by fusion and fission processes in the mitochondrial network, as well as its distribution within the neuron [90]. Aging affects mitochondrial dynamics leading to mitochondrial fragmentation and alterations to these functions [90]. Studies in mitochondria isolated from the cortex of aged animals exhibited more ROS production and mitochondrial swelling after increased Ca+2 loading than that of young animals [78]. Therefore, these findings suggest that the aging increased the sensitivity of the mitochondria to calcium overload, generating mitochondrial swelling [81]. Mitochondrial swelling results in the opening of the mPTP [91], and in aged animals, mPTP opening occurs prematurely, indicating reduced Ca+2 buffering during aging [78, 81].

The mPTP is a large nonselective channel located in the inner mitochondrial membrane and communicates the mitochondrial matrix directly with the cytoplasm [81]. Their opening is activated by Ca+2, phosphate, ROS, increased pH, and magnesium (Mg+2) [92]. Transitory opening of mPTP allows the release of excessive calcium ions that accumulate in the mitochondria, but prolonged opening leads to the movement of ions and small molecules generating depolarization of the mitochondrial membrane and in turn releasing pro-apoptotic factors, which results in a reduction of ATP and finally causes cell death [81, 91].

The structure of mPTP is not completely clear. Experimental approaches have distinguished several protein components such as VDAC, the adenine nucleotide translocase protein (ANT), and the mitochondrial matrix protein cyclophilin D (Cyp-D) [91]. Recent research incorporates the F1FO subunits of ATP synthase, a key enzyme of the OXPHOS complex, which participates in ATP production and maintenance of the membrane potential [91]. Interestingly, deregulation of this enzyme associated with aging has been reported, showing decreased expression of OSCP and in F1FO ATP synthase activity [81, 91]. These changes have also been observed in imaged brains that present age-related neurodegenerative pathologies [81, 91]. Cyp-D is a specific mitochondrial protein and generally considered to be a critical component of mPTP formation [91]. Several studies indicate that Cyp-D is the most important component facilitating mPTP formation, thus leading to decreased ATP production, increasing ROS generation, and eventually causing cell death [81, 91], although it is not yet completely clear how Cyp-D triggers mPTP formation [92]. The opening of mPTP dissipates Δψm, uncoupling the mitochondria and causing swelling [91]. The expression of Cyp-D increases with age and is related to several age-associated neurological diseases such as AD [91, 93]. For example, Gauba et al. have reported that Cyp-D promotes the dysfunction of ATP synthase F1FO, in the mitochondria of aged brains, observing a significant increase in the expression of this protein with age [91]. In contrast, it has been observed that deletion of Cyp-D improves cognitive and mitochondrial functions in both aging and in neurodegenerative diseases [91, 93].

**Figure 1.** *The mitochondrial functions are impaired during aging.*

#### *Mitochondrial Dysfunction as a Key Event during Aging: From Synaptic Failure to Memory Loss DOI: http://dx.doi.org/10.5772/intechopen.88445*

The increased life expectancy and the high incidence of neurodegenerative diseases require a better understanding of the aging processes and the mechanisms associated with it. Thus, comprehension of the interactions between calcium homeostasis and calcium-dependent processes during aging can help in the design of more effective therapeutic strategies. Maintaining calcium homeostasis and controlling the opening of mPTP are important factors that can be considered as a potential therapeutic objective to maintain the quality of life during aging and prevent mitochondrial damage and progressive cognitive deterioration associated with age that contribute to the development of neurodegenerative diseases.

**Figure 1** is a schematic representation of the main mitochondrial functions affected during normal aging. It shows increased oxidative stress as a result of a REDOX imbalance due to decreased activity of antioxidant enzymes and increased reactive oxygen species. It also shows the characteristic bioenergetics failure, as a consequence of diminished OXPHOS functioning, specifically by decreased activity of complexes I and IV of the ETC, which lead to reduced ATP production and, finally, calcium dysregulation, which leads to mitochondrial swelling due to a permanent opening of the mitochondrial permeability transition pore.

#### **5. Mitochondrial dysfunction and cognitive impairment**

Synaptic plasticity in the adult nervous system is a response to changes in the environment and synaptic activity, involving dendritic spine growth or retraction and synaptogenesis, which are believed to be responsible for learning and memory [94]. In the hippocampus, one form of synapse plasticity is LTP, which produces a stronger transmission for consolidation of long-term memory [95]. For this process, neurons need to synthesize proteins de novo at the dendritic spines where different neurotrophic factors play a key role [96]. An impairment in this process leads to neurodegeneration, as a result of the initial loss of synaptic structure and function and finally cell death [96].

The previously described mitochondrial dysfunction could be an important factor in synapse loss associated with cognitive decline observed during aging and in neurodegenerative disease [97]. Since mitochondria are present in axonal terminals and dendrite spines playing a critical role in calcium flux, ROS homeostasis, and ATP production in the synapses, this organelle is a key element for neuronal plasticity [94]. In addition, mitochondrial transport to the synaptic regions is essential for the correct function of this neuronal network [98].

Multiple pathological conditions present cognitive impairment related with a mitochondrial dysfunction. For example, chronic kidney disease (CKD) patients with cognitive damage have increased oxidative stress and decreased antioxidant enzymes (SOD, CAT, GPx, and GSH levels in plasma) compared to CKD patients without cognitive impairment [99]. Also, patients with hypoxia, ischemia induced by a traumatic brain injury (TBI), and diabetes showed cognitive decline and different signs of mitochondrial impairment such as glutamate excitotoxicity, calcium overload, opening of mPTP (which dissipates the mitochondrial electrochemical gradient leading to cell death), and increased ROS levels [100–102]. Interestingly, pyramidal neurons in the cerebral cortex and hippocampus are more susceptible to this type of injury [103], suggesting that these cellular defects may affect mainly synaptic plasticity, learning, and memory.

In the context of neurodegenerative disease, mitochondrial impairment and oxidative stress are the target of Aβ neurotoxicity, promoting cognitive impairment in AD [104]. The degree of cognitive impairment in AD has been related to the amount of Aβ accumulated in mitochondria [105], resulting in a loss of the Δψm in

synaptic regions and ultimately leading to the characteristic synaptic loss observed in AD [106]. It has been suggest that Cyp-D can interact with Aβ contributing to synaptic perturbations. A Cyp-D deficiency can notably improve synaptic function and therefore improve learning and memory in an AD mouse model [107]. It was recently proposed that tau protein can regulate synaptic activity, affecting mitochondrial function and axonal transport [108], and post-transductionally modified tau can induce mitochondrial damage, leading to synaptic dysfunction [109]. In fact, hyper-phosphorylated tau impairs mitochondrial respiratory chain function, increases ROS levels, decreases the activity of detoxifying enzymes, and produces Δψm dissipation [108]. Thus, the accumulation of tau can lead to synaptic deficits and cognitive impairment [110].

Interestingly, cognitive decline is not only characteristic of disease and injury, since cognitive impairment is also observed during aging. During normal aging, it is well established that there is a reduction in the surface area and cortical thickness, resulting in a volume loss in the whole brain, being the non-cortical regions, such as the hippocampus and striatum, more vulnerable to this age-related atrophy [111]. In this context, a study performed with Sprague-Dawley rats of 14, 18, 23, and 27 months of age showed changes in the volume of different brain parts using magnetic resonance imaging (MRI) [112]. In that study they showed enlargement of lateral ventricles and a decrease in the volume of the medial prefrontal cortex, hippocampus, and striatum in 27-month-old rats, which correlates with cognitive deficiency. Twenty-three- and twenty-seven-month-old rats have decreased recognition memory and decreased spatial learning and memory [112]. Another common symptom of aging is cognitive fatigue (CF) characterized by an increase in the facility of becoming tired, lack of energy, and failure to sustain attention when performing cognitively demanding tasks with a high mental effort [113]. There is evidence of a correlation between the decreased connectivity strength of the neuronal network established between the cortical and the striatum areas and a higher CF at an advanced age, suggesting that the cortical-striatal network plays a crucial role in the CF phenomenon [114].

In humans, similar cognitive decline is observed during aging, present as a deficit in episodic or declarative memory, spatial learning, working memory, and attention [115]. These processes are mainly dependent on an adequate function of the hippocampus. Structural and functional changes in the hippocampus are related to the severity and development of neurodegenerative disorders associated with cognitive decline. In fact, many of the cognitive deficits seen with aging can be replicated in animal models with bilateral hippocampal damage [116]. The connection between dentate gyrus (DG) and the CA3 area of the hippocampus is responsible for the formation of new memories, and this is naturally decreased in the aged brain [117], with different biochemical modifications that affect its ability to generate and consolidate LTP [117]. Diverse studies have shown that during aging, the autoassociative network of CA3 is strengthened, and the processing of new information coming in from the entorhinal cortex is weaker [118]. Thus, the stored information becomes dominant in contrast to the ability to encode new information [118]. Also, there is a decrease in gray matter volume, where age-related changes in the temporal lobe involve mainly changes in the hippocampus [4].

Also, different studies show high levels of tau in cerebrospinal fluid (CSF) during aging [119, 120]. For example, the characteristic hearing loss (HL) present in aging influences neurodegeneration by promoting tau pathology in CSF [120], which produces cognitive impairment via synapsis dysfunction and neuronal loss [110]. Other studies evidence the age-related impairment of executive functions, verbal and nonverbal cognitive switching (independent of gender, education, and IQ ), and the ability to focus attention and/or multitask [121, 122]. Studies have

#### *Mitochondrial Dysfunction as a Key Event during Aging: From Synaptic Failure to Memory Loss DOI: http://dx.doi.org/10.5772/intechopen.88445*

shown that during aging, there is a reduction in inhibitory mechanisms in the CA3, where short-term plasticity and LTP are compromised [123]. Interestingly, mitochondria play a central role in LTP, enhancing mitochondrial gene expression [124], satisfying the ATP demand by producing changes in mitochondrial energy production, and regulating calcium homeostasis by increasing calcium pump activity [125]. Also, there is evidence that mitochondrial dysfunction can lead to failure in connectivity of brain cortex producing cognitive impairment [126]. Thus, mitochondrial dysfunction during aging can be related with degeneration of synapses, triggering cell death.

In this mitochondrial context of aging, it is interesting that in brain regions highly associated with cognitive function, such as the hippocampus and cortex, there is a high amount of impaired mitochondria, with dysfunctional respiration, excessive ROS production, loss of Δψm, and decreased cytochrome c oxidase activity. Meanwhile mitochondria are less affected in areas of the brain that are less involved in cognitive abilities [105]. Therefore, mitochondrial function is a key component in cognition. It allows proper information processing through the brain network, being an important player in synaptic transmission. Mitochondrial dysfunction generates deficits in synapsis that trigger cognitive impairment in

**Figure 2.** *A synaptic failure leads to cognitive impairment in aging.*

neurodegenerative disease but also in natural aging. Thus, the understanding of these processes may be critical in these times where the aged population is increasing; therefore, improving their quality of life is a priority.

**Figure 2** above shows the synaptic effects of mitochondrial failure. In the presynaptic region, decreased mitochondrial activity diminishes ATP content, altering the exocytosis of synaptic vesicles. Also, increased ROS production induces lipid peroxidation, affecting glutamate and glucose transport. In the postsynaptic region, decreased mitochondrial activity disrupts calcium homeostasis, altering postsynaptic signaling. Besides, the increased ROS production and consequent lipid peroxidation impaired ion-motive ATPases. **Figure 2** below schematizes that mitochondrial dysfunction at the CA1 of the hippocampus impaired synaptic transmission resulting in cognitive impairment.

#### **6. Mitochondrial therapies as an antiaging treatment**

Since mitochondrial dysfunction is a key event promoting aging, interventions that focus on maintaining or restoring the correct functioning of the mitochondria seem fundamental. For this purpose, two different experimental strategies could be used [127], physiological approaches or pharmacological approximations, which will be briefly summarized in this section.

From the physiological point of view, maintaining a lifestyle that includes recurrent physical exercise preserves mitochondrial function [127]. During aging a loss of age-associated muscle mass is directly related with decreased mitochondria-dependent metabolic capacity, as well as with reduced mitochondrial biogenesis [128]. Biogenesis of new mitochondria is regulated by the transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), which also regulates redox balance and energetic function [129]. Interestingly, PGC-1α expression is decreased in aging reducing its signaling pathway and gene target [129], an effect that could be counteracted by exercise, demonstrating that exercise also increases mtDNA content in the muscle of aged rats [130]. In addition, exercise increases the expression of the CAT enzyme, reducing ROS levels [131]. Similarly, exercise promotes both fission and fusion events as indicated by upregulated levels of Fis1 and Mfn1 protein in the muscle tissue of old animals and by increased Mfn2 and Drp1 mRNA in the skeletal muscle of older women [132]. Finally, exercise could contribute to restoration of mitochondrial Ca+2 homeostasis, increasing the protein levels of mitochondrial Ca+2 uniporter (MCU) [132]. Thus, exercise during aging could promote the genesis of new mitochondria or could attenuate the mitochondrial dysfunction observed at an advanced age.

A second physiological approximation important for simulating mitochondrial function is caloric restriction, which has been demonstrated in different models that are able to reduce the age-related phenotype and to increase lifespan [133]. The beneficial effects of caloric restriction are directly associated with the bioenergetic defects observed in aging, activating ATP production through fatty acid metabolism [134]. Mechanistically, caloric restriction increases the activity of complexes I, III, and IV of the ETC, as well as MnSOD, which results in increased ATP and reduced ROS levels [135]. Likewise, caloric restriction enhances Ca+2 mitochondrial buffering, decreasing Cyp-D levels [135]. Therefore, regulating caloric ingestion is possible for maintaining mitochondrial activity during aging.

Mitochondrial function can also be regulated pharmacologically, for example, through the administration of polyphenols such as resveratrol, green tea, and red

#### *Mitochondrial Dysfunction as a Key Event during Aging: From Synaptic Failure to Memory Loss DOI: http://dx.doi.org/10.5772/intechopen.88445*

wine [127, 136]. Specifically, they act by promoting mitochondrial OXPHOS and activating cellular antioxidant mechanisms [137]. Another possibility is the use of antioxidant compounds such as MitoQ, an electron scavenger that prevents the formation of mitochondrial free radicals [138]. Similarly, α-tocopherol (MitoVitE), α-phenyl-tert-butylnitrone (MitoPBN), the piperidine nitroxide MitoTEMPOL, the antioxidant SkQ1, and elamipretide (SS-31) enter and accumulate in the mitochondria preventing oxidative stress and preserving mitochondrial function [139, 140]. The numerous studies probing the clinical efficacy of these compounds validate the importance of mitochondria in aging [139]. To promote the natural antioxidant effect in the cell, treatment with N-acetylcysteine, vitamin, C and other physiological antioxidant molecules have also been shown to be effective as palliative treatment of senescence [141, 142].

It is also important to highlight the positive effects induced by the direct administration of fatty acids including omega-3 fatty acid α-linolenic acid, due to studies in vivo that have shown its capacity to extend lifespan [143]. This could be a consequence of increased β-oxidation, which results in higher mitochondrial energy production, by increasing mitochondrial biogenesis or by reducing oxidative stress [144]. Finally, we will mention the effects of Metformin, a drug commonly used for the treatment of type 2 diabetes. Metformin has a hypoglycemic effect in the plasma and promotes increased insulin sensibility by a mechanism that remains unclear [145]. However, favorable effects have been observed, where DNA damage and inflammation are prevented, impeding cellular damage by reducing ROS production [146].

Therefore, these approaches highlight the key role that mitochondrial function play during aging, where correct mitochondrial activity could extend lifespan, whereas metabolic alterations could compromise mitochondrial function, accelerating the aging phenotype.

#### **7. Future directions: importance of synaptic mitochondrial dysfunction in aging**

It is now known that the mitochondria have a fundamental role during the aging process. In neurons, the mitochondria are classified into two groups according to their localization, such as synaptic and non-synaptic mitochondria. Non-synaptic mitochondria are distributed throughout the cell body and in the neural prolongations, meanwhile synaptic mitochondria are exclusively found in synapses, both at the pre- and postsynaptic level [147]. Thus, it is not surprising that synaptic mitochondria, which have a higher energy requirement in order to sustain synaptic activity, present functional differences compared to non-synaptic mitochondria. For example, synaptic mitochondria have higher peroxide production than non-synaptic ones [148]. During aging, it seems that these differences are accentuated between these two mitochondrial populations. Aged cortical synaptic mitochondria present decreased oxidative capacity and higher susceptibility to calcium overload, in contrast to non-synaptic mitochondria that preserve their respiratory capacity [16]. Similarly, we observed that hippocampal synaptic mitochondria fail previous to non-synaptic mitochondria during aging and suffer premature mitochondrial swelling with age, contributing hippocampus-dependent memory loss (manuscript in preparation). Thus, maintaining adequate function of synaptic mitochondria seems to be the new challenge in order to attenuate the aging phenotype, reducing the synaptic and cognitive failure characteristics of older individuals.

### **8. Conclusions**

Taken together, the evidence presented in this chapter strongly suggests a close relationship between mitochondrial function and a wide range of processes associated with aging. In general, it is possible to propose an age-dependent decline observed in several organs such as the brain correlated with a loss of mitochondrial activity, generating a bioenergetic deficit and redox imbalance that promote oxidative stress. This promotes additional mitochondrial fail, affecting cellular calcium homeostasis, critical for neurons due to its important roles in the synapses. Thus, synaptic defects conduce to cognitive impairment. Finally, we propose that the synaptic mitochondria are a critical mitochondrial pool to preserve synaptic communication despite the passing of the years.

### **Acknowledgements**

This work was supported by FONDECYT N°11170546 and CONICYT PAI N°77170091 to CTR.

### **Author details**

Claudia Jara† , Angie K. Torres† , Margrethe A. Olesen and Cheril Tapia-Rojas\* Laboratory of Neurobiology of Aging, Centro de Biología Celular y Biomedicina (CEBICEM), Facultad de Medicina y Ciencia, Universidad San Sebastián, Chile

\*Address all correspondence to: cheril.tapia@uss.cl

† Both authors contributed equally to this work.

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Mitochondrial Dysfunction as a Key Event during Aging: From Synaptic Failure to Memory Loss DOI: http://dx.doi.org/10.5772/intechopen.88445*

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#### **Chapter 6**

## Coenzyme Q10 and L-Carnitine Disturbances in Children with Mitochondrial Diseases

*Ekaterina A. Nikolaeva, Ilgar S. Mamedov and Irina V. Zolkina*

#### **Abstract**

Coenzyme Q10 (CoQ10) and L-carnitine are very important biologically active compounds involved in energy metabolism. L-carnitine and coenzyme Q10 disturbances in mitochondrial diseases require the correction. Patients and methods: The levels of coenzyme Q10 and L-carnitine (total carnitine, free carnitine, and acylcarnitines) were determined in children with mitochondrial diseases (25 children and 16 children, respectively). High-performance liquid chromatography with UV detection (chromatograph Shimadzu Nexera LC-30) and chromatography-mass spectrometry (Agilent 6410 QQQ, USA) were used. As an additional parameter of possible coenzyme Q10 and carnitine insufficiency, the coenzyme Q10/cholesterol ratio and acylcarnitines/free carnitine ratio were calculated. Results: A significantly low ratio of coenzyme Q10/cholesterol in children with mitochondrial diseases was revealed—0.10 ± 0.01 vs. 0.19 ± 0.01 in the control group (p < 0.001). A lower absolute level of coenzyme Q10 and tendency toward a more pronounced decrease in the Q10/cholesterol ratio in older patients (6–16 years) were shown. The free carnitine blood level was within the normal range and averaged at 29.8 ± 2.6 μmol/l; however, the level was lower than that in the control group (44 ± 5.2 μmol/l, p < 0.05). A pronounced significant increase in the acylcarnitines/free carnitine ratio was determined—1.5 ± 0.05 (the normal range < 0.6).

**Keywords:** children, mitochondrial diseases, coenzyme Q10, carnitine, treatment

#### **1. Introduction**

Mitochondrial diseases are a large heterogeneous group of pathological conditions caused by genetically determined defects in the mitochondria's structure and function in the release of the energy of organic substances and its accumulation in the form of macroergic phosphate compounds by the generation of adenosine triphosphate [1]. These disorders can be due to mutations in mitochondrial DNA or due to mutations in nuclear DNA. Some mitochondrial diseases are rare. However, in general, mitochondrial encephalopathy is the most common neurometabolic disorder [2]. Defects in the respiratory chain and oxidative phosphorylation are the basis of the pathogenesis of these severe diseases.

Mitochondrial diseases have a wide range of clinical presentations with a generally poor prognosis: failure to thrive, encephalopathy, respiratory insufficiency,

hypotonia, ataxia, seizures, cardiac involvement, hepatopathy and nephropathy, sensorineural hearing loss, retinopathy, lesions of the basal ganglia, and others. The common laboratory signs are lactic acidosis, hypo- or hyperglycemia (diabetes), elevated creatine kinase and aminotransferases, and urine organic acid/amino acid abnormalities [3].

Current therapies are frequently inefficient and mostly palliative. The treatment strategy for mitochondrial diseases is to improve the efficiency of biological processes in the respiratory chain and oxidative phosphorylation. Patients are prescribed complex treatment, including drugs that affect different stages of energy metabolism. This treatment approach shows a higher positive effect than monotherapy [4]. Coenzyme Q10 and L-carnitine are very important biologically active substances involved in energy metabolism. So, coenzyme Q10 and L-carnitine are often recommended for the treatment of mitochondrial diseases [5]. However, some authors acknowledge the lack of rationale behind these recommendations since the data from randomized clinical trials are still lacking.

#### **2. Functions of CoQ10 and its biological role**

Coenzyme Q10 is the most common ubiquinone in the human body. Its structure contains a quinoid ring and 10 isoprenyl groups. Coenzyme Q10 is structurally similar to vitamins E and K. Coenzyme Q10 exists in oxidized (ubiquinone) and reduced (ubiquinol) forms and is known to be a constituent of the biological membranes [6]. Coenzyme Q10 is one of the main components of the electron transport chain of mitochondria. In the form of ubiquinone, it acts as an electron transporter from Complex I and Complex II to Complex III. In this process, the formation of the reduced form—ubiquinol—occurs. Ubiquinol is a powerful antioxidant, which has a protective effect on biological membranes, regulates their permeability, inhibits peroxidation of plasma lipoproteins, and provides a recovery of tocopherol activity [6, 7]. According to the recent data, coenzyme Q10 is reported to be involved in the regulation of some gene expression and inflammatory mediators, in particular, by influencing the transcription factor NFkappaB1; its participation in DNA replication and repair was shown [8, 9].

In mammals, the largest amount of coenzyme Q10 is found in the heart and skeletal muscles. In the peripheral blood, coenzyme Q10 is bound to lipoproteins, and its level is positively correlated with total cholesterol [10, 11].

Most of the body's daily coenzyme Q10 requirement is derived from endogenous synthesis; small amounts of coenzyme Q10 are obtained from foods such as meat, fish, and nuts. Biosynthesis is a multi-step process, taking place on the inner mitochondrial membrane. Vitamins B2, B3, B6, B12, and C and folic and pantothenic acids are known to participate in the coenzyme Q10 biosynthetic pathway under the control of a dozen genes. The intensity of biosynthesis declines substantially with age [12, 13].

#### **3. Coenzyme Q10 deficiency in disorders: the possibility of diagnosis**

Primary coenzyme Q10 deficiency is due to a defect in its biosynthesis. These diseases form a separate group of mitochondrial diseases and are associated with mutations in multiple genes including PDSS1, PDSS2, CoQ2, CoQ6, CoQ9, and ADCK3. These diseases are characterized by a decrease in the level of coenzyme Q10 in tissues and in fibroblasts whereas the blood levels can be normal [11, 14, 15]. Secondary coenzyme deficiency with low plasma and tissue coenzyme Q10 levels

#### *Coenzyme Q10 and L-Carnitine Disturbances in Children with Mitochondrial Diseases DOI: http://dx.doi.org/10.5772/intechopen.87950*

can occur in patients taking anticancer agents and statins. The hypocholesterolemic effect of statins is due to the inhibition of 3-hydroxy-3-methylglutaryl-COA reductase and a disruption of the synthesis of mevalonic acid, which is one of the precursors of not only cholesterol but also ubiquinone. Approximately a half of patients receiving statins show a decrease in coenzyme Q10 in the muscle tissue; myalgia and myoglobinuria may also be observed [16, 17].

Secondary coenzyme Q10 deficiency and low levels of coenzyme Q10 in plasma and tissues are found in certain diseases of older age (Parkinson's and Alzheimer's disease, atherosclerosis, diabetes mellitus, etc.) [13, 18, 19], in some hereditary diseases, including inborn errors of metabolism— mevalonic aciduria, phenylketonuria, glutaric acidemia II, ataxia-oculomotor apraxia 1, and cardiofaciocutaneous syndrome [11, 20]. Coenzyme Q10 deficiency in mevalonic aciduria and in phenylketonuria can be explained by insufficient cholesterol production: the decrease in the activity of mevalonate kinase and the inhibition of 3-hydroxy-3-methylglutaryl-COA reductase with high levels of phenylalanine, respectively. In phenylketonuria, ubiquinone deficiency may well be exogenous due to dietary restrictions such as avoidance of animal products [10].

A decrease in coenzyme Q10 was reported in the peripheral blood and muscles in some (20–40%) patients with mitochondrial pathology associated with mutations and depletion of mitochondrial DNA [21, 22]. Of interest, in children with myopathy due to other causes, there were no changes in the content of coenzyme Q10 in the muscles, except for the patients with Duchenne muscular dystrophy [23].

The coenzyme Q10 deficiency can be detected in biological fluids (plasma or serum), fibroblasts, and muscle tissue. However, the blood level of coenzyme Q10 is not considered as a reliable indicator of its state in the body. There is no clear correlation between the levels of ubiquinone in plasma and muscle tissue. This parameter is influenced by the lipid intake from foods and the blood levels of cholesterol and low-density lipoproteins [7, 10, 11]. Therefore, the ratio of coenzyme Q10 to cholesterol and low-density lipoproteins is proposed for clinical use. Apparently, the measurement of coenzyme Q10 in the peripheral blood mononuclear cells appears to be a promising detection method.

#### **4. Low blood level of coenzyme Q10 as a diagnostic marker of mitochondrial encephalomyopathy and the rationale for therapy**

In the Research and Clinical Institute of Pediatrics, an examination of 16 children (group 1) aged 1–16 years (average age 8.3 ± 1.5 years) with mitochondrial diseases was performed.

In nine children, the disease was caused by deletions or point mutations of mitochondrial DNA: Kearns-Sayre syndrome (common deletion of mitochondrial DNA) in three; mitochondrial encephalomyopathy with pyramidal-extrapyramidal syndrome (*MTND1* mutation) in two; mitochondrial encephalomyopathy with cardiomyopathy (*MTTK* mutations) in two; mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (*MTTL1* mutation) in 1; and maternal inherited Leigh syndrome (*MTND3* mutation) in 1.

Seven children were diagnosed with mitochondrial diseases of nuclear origin: leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation (*DARS2* mutations) in four and Leigh syndrome (*SURF1* mutations) in three.

The comparison group (group 2) consisted of 13 children with neurodegenerative diseases aged 1–15 years (mean age 6.4 ± 1.3 y): 6 children with neuronal ceroid lipofuscinosis 2 (*TPP1* mutations), 2 with sialidosis type 2 (*NEU1* mutations), and 5 with progressive ataxia not genetically confirmed. The control group (group 3) consisted of 29 healthy children aged 1–16 years (mean age 7.4 ± 0.8 y) who underwent a routine examination at the health center (an informed consent of the parents was obtained) [24]. The ratio of boys:girls is 16:13. There was no statistically significant difference in the average age between the three groups.

A detailed work-up of the patients of the first two groups included clinical (pedigree, neurological, cardiological, and other examinations) examination and biochemical tests. In all children, the plasma level of coenzyme Q10 was determined by high-performance liquid chromatography with UV detection (chromatograph Shimadzu Nexera LC-30). The blood cholesterol level was determined photometrically (analyzer Konelab Prime 60i), followed by the calculation of the coenzyme Q10/cholesterol ratio. The DNA diagnosis of diseases was carried out in the laboratory of inborn errors of metabolism of Research Centre for Medical Genetics (Moscow, Russia).

Statistical data processing was carried out by methods of variation statistics and correlation analysis (Statistica, Excel 7.0). Student's t-test was used to assess the statistical significance of the data, and the differences were considered statistically significant at p < 0.05.

The blood levels of coenzyme Q10 in children with mitochondrial diseases (group 1) were 0.56 ± 0.05 μmol/l (**Table 1**) and did not differ from that in the control group but was significantly lower (p < 0.01) than that in children with neurodegenerative diseases (group 2). The blood level of coenzyme Q10 in children with neurodegenerative diseases was 1.53 ± 0.23 μmol/l and significantly exceeded that in healthy children (p < 0.01).

The blood cholesterol level in patients of the first and second groups was significantly higher than that in healthy children (p < 0.01). The ratio of coenzyme Q10/cholesterol was severely impaired compared with that in healthy children (see **Table 1**). This parameter was significantly decreased in patients with mitochondrial diseases (0.10 ± 0.01 vs. 0.19 ± 0.01, p < 0.001) but increased in those with neurodegenerative diseases (0.31 ± 0.04, p < 0.002).

For the subgroup analysis, children aged 1–5 and 6–16 years were analyzed separately within each group (**Table 2**). In healthy children (group 3), there were no age-related differences.

In the group of older patients with mitochondrial diseases, the blood level of coenzyme Q10 was significantly lower than that in the younger subgroup (p < 0.05) and was significantly different from its level in children in the second


#### **Table 1.**

*Coenzyme Q10 and cholesterol levels (M ± m) in the blood of the examined children.*


*Coenzyme Q10 and L-Carnitine Disturbances in Children with Mitochondrial Diseases DOI: http://dx.doi.org/10.5772/intechopen.87950*

*\*Significant difference from the older age subgroup; р < 0.05. 1-2p, 1-3p, and 2-3p - significance of differences between groups (1-2, 1-3, and 1-3)*

#### **Table 2.**

*Coenzyme Q10 and cholesterol levels (M ± m) in the blood in children of different age subgroups.*

(p < 0.001) and third (p < 0.05) groups of the same age. In addition, older patients (6–16 years) tend to have lower cholesterol levels and lower coenzyme Q10/cholesterol ratios than younger patients (1–5 years). In the group of children with mitochondrial encephalomyopathies, there was a negative correlation between the blood level of coenzyme Q10 and cholesterol and the patient's age (r = −0.54 and r = −0.48, respectively; p < 0.05). There were no differences in the studied parameters between children with mitochondrial diseases caused by mutations of mitochondrial (n = 9) and nuclear (n = 6) DNA. By age, these subgroups were not different.

In the group of children with neurodegenerative diseases, the opposite tendency was observed toward higher rates of coenzyme Q10, cholesterol, and their ratios in patients of the older subgroup (see **Table 2**); unreliability of differences seems to be associated with a small number of patients. A positive correlation between the blood levels of coenzyme Q10 and cholesterol (r = 0.66; p < 0.05) was established. These parameters correlated positively with the age of the patients (r = 0.37 and r = 0.41, respectively; p < 0.05).

To sum up, our study demonstrated that the average level of coenzyme Q10 in patients with mitochondrial diseases did not differ from that in healthy children. However, these patients had a higher cholesterol level and, as a result, a reduced ratio of coenzyme Q10/cholesterol. In patients of the older subgroup (6–16 years), the changes were more pronounced: significantly lower levels of coenzyme Q10 and a tendency toward a lower Q10/cholesterol ratio than the younger subgroup (1–5 years).

In children with neurodegenerative diseases (not caused by primary mitochondrial dysfunction), the level of cholesterol (as well as in the first group) was higher than that in healthy children. Other test results differed considerably from those in patients with mitochondrial pathology. High blood level of coenzyme Q10 and an

increase in the coenzyme Q10/cholesterol ratio were revealed, and these changes increased with age.

Due to a small sample size, the obtained data were considered as preliminary. Nevertheless, our results revealed a coenzyme Q10 deficiency in children with mitochondrial diseases and emphasized the difference in the pathogenesis of primary mitochondrial diseases and neurodegenerative diseases of non-mitochondrial origin. In both patient groups, the age-related aggravation of these changes was noted to correspond to a progressive disease course. The similarity of clinical manifestations underlies the difficulties in the differential diagnosis between mitochondrial encephalomyopathies and neurodegenerative diseases. The detection of the plasma coenzyme Q10 level can be helpful in the differential diagnosis between these conditions.

In addition, a low coenzyme Q10/cholesterol ratio and a tendency toward a decrease in the coenzyme Q10 levels with age suggest its insufficiency in patients with progressive mitochondrial encephalomyopathy. This provides a rationale for the use of coenzyme Q10 in the treatment of patients with mitochondrial diseases.

#### **5. Functions of carnitine and its biological role**

Carnitine (β-hydroxy-γ-trimethylaminobutyric acid) is a low molecular weight compound, which is crucial for energy metabolism in the human body. As an L-stereoisomer, carnitine is present in various tissues. Endogenous carnitine formation occurs in the liver, kidney, and brain cells through the transformation of the amino acids lysine and methionine, with a glycine involvement. Vitamins C, B6, and B3 and iron ions are the cofactors for carnitine biosynthesis. The biosynthesis steps are under the control of mitochondrial enzymes (trimethyl-lysine deoxygenase, 3-OH-trimethyl-lysine aldolase, 4-trimethylaminobutanal dehydrogenase, butyrobetaine dioxygenase) [25]. However, biosynthesis provides only a part of carnitine daily requirements, and the main source of its intake is animal foods including red meat, fish, and dairy products. Carnitine absorption in the gastrointestinal tract, tubular reabsorption, and delivery to the tissue are provided by transport proteins, with OCTN2 being the main carnitine transporter. Carnitine is transported to the skeletal muscles and myocardium—these tissues contain the main reserves of carnitine, due to their high activity of lipid metabolism [26]. The importance of carnitine for the body is evidenced by its almost complete reabsorption in the renal tubules.

Studies have established the value of carnitine for the processes of biological oxidation and maintenance of mitochondrial functions in the human body. Carnitine is crucial in conditions of a high energy consumption. These conditions such as intercurrent diseases, increased physical activity, starvation, etc. are characterized by increased catabolism. After depletion of carbohydrate reserves, lipids become the main sources of the ATP synthesis in the body.

One of the main vital functions of carnitine is bioenergetics. Carnitine is involved in lipid catabolism, providing its initial stages—an activation and a transfer of long-chain fatty acids from the cytoplasm through the outer and inner mitochondrial membranes to the mitochondrial matrix, thereby making them available for subsequent β-oxidation to form acetyl-CoA. In addition, fatty acid oxidation is the main pathway of ketogenesis, and ketone bodies are an additional energy substrate for peripheral tissues and the brain [26, 27].

The effect of carnitine on fat metabolism occurs through its participation in the cytoplasmic synthesis of fatty acids. Carnitine provides the reverse transfer of acetyl groups of mitochondrial acetyl-CoA through the mitochondrial membrane into the cytoplasm.

#### *Coenzyme Q10 and L-Carnitine Disturbances in Children with Mitochondrial Diseases DOI: http://dx.doi.org/10.5772/intechopen.87950*

An important function of carnitine is due to its ability to bind acyl radicals. Thus, carnitine regulates tissue energy metabolism, affecting the ratio of acyl-CoA/free CoA in the mitochondria. Likewise, the detoxifying role of carnitine is achieved through binding of organic acid derivatives (intermediates in oxidative processes) and its excretion from the cell. These organic acid derivatives, accumulating in the mitochondria and cytoplasm, have an adverse effect, by inhibiting the enzyme activity.

Carnitine appears to play an important role in the permeability of mitochondrial membranes. The protective impact of carnitine relies on the prevention of a negative membranotropic action of toxic agents, inhibitors of complexes of a mitochondrial respiratory chain (3-nitropropionic acid, methylphenylpyridine, and others), and inducers of apoptosis (long-chain fatty acid radicals). Carnitine supplementation in experimental animals prevents these disorders or significantly reduces their severity and prevents a degenerative damage to the nervous tissue [28–31].

In addition, a favorable impact of carnitine on the cytokine production and on vascular endothelium was revealed. Carnitine is capable of restoring endothelial function and preventing of vascular remodeling caused by a decrease in nitric oxide production [25, 32, 33]. Apparently, the carnitine functions require further research.

#### **6. Carnitine deficiency in diseases**

There are primary and secondary carnitine deficiencies. The primary deficiency is due to an autosomal recessive defect of the gene SLC22A5, which is expressed in the skeletal muscles, heart, and kidneys. The gene SLC22A5 encodes a transport protein OCTN2, the sodium-dependent organic cation transporter. The genetic defect disrupts the transport of carnitine into the tissues and reabsorption in the renal tubules. The clinical manifestations of the disease include cardiomyopathy, skeletal myopathy, fatty liver, and kidney dystrophy [34].

The causes of secondary carnitine deficiency are diverse and associated with an interruption of endogenous synthesis and disturbance of absorption from food and of retention in the body, as well as with an increased excretion through the kidneys or gastrointestinal tract [35]. The activity of endogenous carnitine biosynthesis depends on the function of the liver and kidneys. Biosynthesis decreases with malnutrition due to a protein deficiency. Carnitine removal is enhanced in stress, intercurrent diseases, and impaired renal tubular function. Low blood carnitine level is determined in children with epilepsy treated with valproate, in patients with heart failure, and in patients on hemodialysis [36–38].

Secondary insufficiency occurs in inborn errors of metabolism. In particular, secondary carnitine insufficiency is characteristic for a large group of hereditary diseases of organic and fatty acid metabolism. In these diseases, low levels of carnitine in the peripheral blood and tissues result from the accumulation of acylcarnitines and their enhanced renal excretion [34, 39].

#### **7. The carnitine insufficiency in children with mitochondrial encephalomyopathies**

Some patients with mitochondrial diseases (about one-fourth patients) were reported to have a decrease in carnitine levels in the peripheral blood [40, 41]. Our study was designed to diagnose and treat carnitine insufficiency in patients with mitochondrial diseases; to achieve this, we analyzed the clinical parameters and

laboratory findings of 40 children aged 2–15 years: 25 with mitochondrial encephalomyopathies (group 1) and 15 with congenital myopathies (group 2). Group 1 consisted of 10 children with Kearns-Sayre syndrome (common deletion of mitochondrial DNA); 2 with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MTTL1 mutation); 1 with myoclonic epilepsy associated with ragged-red fibers (MTTK mutation); 5 with leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation (DARS2 mutations); 1 with Barth syndrome (TAZ mutation); 3 with Leigh syndrome (SURF1 mutations); and 3 with POLG-related diseases (mitochondrial recessive ataxia syndrome in 2 and autosomal recessive progressive external ophthalmoplegia in 1). Group 2 consisted of 10 children with central core disease and 5 with minicore myopathy. The control group included 10 children without mitochondrial diseases or congenital myopathies, who attended the clinic.

The clinical examination included pedigree, neurological, cardiological, and other examinations. In all children, acid–base balance of the blood and the level of lactic and pyruvic acids were determined. The level of L-carnitine—total carnitine, free carnitine (C0), and acylcarnitines (AC)—in dry blood spots was measured by chromatography-mass spectrometry (Agilent 6410 QQQ, USA). As an additional parameter of a possible carnitine insufficiency, the ratio AC/C0 was calculated. The DNA diagnosis of mitochondrial diseases was carried out in the laboratory of inborn errors of metabolism of the Research Centre for Medical Genetics (Moscow, Russia). Structural myopathy was diagnosed using a morphological study of muscle tissue (light and electronic microscopy, histochemical methods).

Statistical data processing was carried out by methods of variation statistics and correlation analysis (Statistica, Excel 7.0). Student's t-test was used to assess the statistical significance of the data, and the differences were considered statistically significant at p < 0.05.

In children with mitochondrial diseases, the clinical presentations of the nervous and muscular system involvement prevailed: fast fatigability, low exercise tolerance, muscle weakness and hypotension, and a development delay. Severe psychomotor retardation was noted in three children with Leigh syndrome. Most patients had ataxia, ophthalmoplegia, headaches and vomiting, and heart damage (atrioventricular blockade, cardiomyopathy). Some patients had a short stature, seizures, retinitis pigmentosa, a hearing loss, and an impaired liver function with a moderate increase in blood aspartate and alanine aminotransferase.

Compensated metabolic acidosis was detected in 12 of 25 children. Elevated blood lactate levels were noted within the range 2.9–6.7 mmol/l in 20 children (the normal range 1.0–1.7 mmol/l). In 11 patients, the pyruvate level was elevated (0.19–0.39 mmol/l, norm 0.09–0.12 μmol/l).

In children with mitochondrial diseases, the total carnitine blood levels (**Figure 1**) ranged from 39.1 to 95.3 μmol/l, averaging (M ± m) 75.8 ± 6.2 μmol/l (in the control group—from 41.1 to 119.9 μmol/l; 73.7 ± 9.7 μmol/l). There was a negative correlation between the total carnitine and lactate in the peripheral blood of patients with mitochondrial diseases (r = −0.65; p < 0.05).

The free carnitine blood level was within the normal range (19–60 μmol/l) and averaged at 29.8 ± 2.6 μmol/l; however, the level was lower than that in the control group (44 ± 5.2 μmol/l, p < 0.05). In six patients, the free carnitine blood level was at the lower limit of the normal range, not exceeding 25 μmol/l.

The average acylcarnitine level was 44.5 ± 3.7 μmol/l (see **Figure 1**); 87% of acylcarnitines were represented by acetylcarnitine (C2). Acetylcarnitine level was 38.7 ± 3.9 μmol/l (higher than that in control group; p < 0.05); its level in four children went beyond the limit of the normal range (56.3, 60.2, 64.5, 65.7 μmol/l at a rate of up to 50 μmol/l).

*Coenzyme Q10 and L-Carnitine Disturbances in Children with Mitochondrial Diseases DOI: http://dx.doi.org/10.5772/intechopen.87950*

#### **Figure 1.**

*Comparison of carnitine levels (μmol/l) in dry blood spots in children of three groups. \* significant difference (p < 0.05) with groups 2 and 3. \*\* significant difference (p < 0.05) with group 3.*

About a half (13) of the patients had elevated levels of other acylcarnitines: methylmalonyl-(C4DC), tiglyl-(C5: 1), glutaryl-(C5DC), hydroxybutyryl-(C4OH), hydroxyisovaleryl-(C5OH), and hexanoyl-(C6); there was an increase in the blood levels of alanine, glycine, and leucine. These metabolic changes are likely to be associated with the activation of ketogenesis, an impaired metabolism of lactate and pyruvate in mitochondrial diseases.

On average, the proportion of free carnitine was merely 39% of the total blood carnitine (the normal range is 70–80%). The proportion of acylcarnitines was 61%, significantly exceeding that of healthy subjects (20–30%).

The ratio of acylcarnitines and free carnitine AC/C0 was markedly increased to 1.3–1.8, reaching an average of 1.5 ± 0.05 (the normal range < 0.6). The increase in this ratio suggests the relative insufficiency of free carnitine and confirms the accumulation of esterified forms in the total carnitine pool.

In the patients with congenital myopathies, the clinical presentations predominantly included myopathic manifestations, impaired motor development, and increased fatigue. Moderately elevated blood lactate levels were found in eight children (1.9–2.8 mmol/l, the normal range 1.0–1.7 mmol/l). In 10 patients, the pyruvate level was increased (0.17–0.5 mmol/l, norm 0.09–0.12 mmol/l).

The total carnitine blood levels in children ranged from 46.7 to 106.1 μmol/l, averaging at (M ± m) 71.4 ± 3.5 μmol/l. There was no difference in total carnitine level between the patients with congenital myopathies and the control group.

The mean free carnitine level of 35.0 ± 1.9 μmol/l was within the normal range, but it was lower than that in the control group (p < 0.05). In two children with structural myopathy, free carnitine level was at the lower limit of the normal range.

The mean level of acylcarnitines was normal (36.9 ± 2.4 μmol/l). Likewise, the level of acetylcarnitine is not elevated. Of note, levels of other acylcarnitines were moderately elevated in three patients.

The proportion of free carnitine in the total carnitine was reduced—49% (the normal range 70–80%). The proportion of the esterified forms accounted for 51% of the total carnitine, which significantly exceeded the corresponding values of healthy individuals (20–30%). The ratio of acylcarnitines and free carnitine AC/C0 was increased to 0.9–1.7, averaging at 1.1 ± 0.08 (norm <0.6), which was consistent with carnitine insufficiency. In the control group of conditionally healthy children,

the ratio of AC/C0 was 0.66 ± 0.03 and was significantly different from that in groups 1 and 2 (p < 0.01).

The comparison of the carnitine parameters in patients with mitochondrial encephalomyopathies and congenital myopathies showed that in mitochondrial diseases, carnitine deficiency was more pronounced. Although the levels of acylcarnitines, free and total carnitine, were not significantly different, in the group of children with mitochondrial diseases, there was a tendency for an accumulation of bound carnitine and a lower level of free carnitine. Additionally, an acetylcarnitine level was significantly higher, and the impairment of total carnitine composition was more pronounced in children with mitochondrial diseases than in those with congenital myopathies. This was confirmed by a higher ratio of AC/C0 (1.5 ± 0.05 vs. 1.1 ± 0.08; p < 0.01).

The detection of carnitine deficiency underscores the need for L-carnitine therapy. L-carnitine per os in a dose of 30–50 mg/kg/day (depending on age) was included in the complex of energy treatment (coenzyme Q10, succinates, vitamin B) of patients with mitochondrial diseases. Within 1 year, three courses of therapy (for 2 months) were prescribed with a period off treatment for 1–2 months.

After 10–12 months of a follow-up of 18 children, a distinct improvement in one third of the patients was demonstrated: a reduction of fatigue, an improvement of exercise tolerance, and a reduction in the frequency of headache and nausea attacks. In one half cases, a stabilization with minimal positive dynamics was observed. These 15 children showed a decrease in the blood lactate level to 1.4–2.3 mmol/l. At the same time, in three children with Leigh syndrome, despite the treatment, a moderate progression of the disease with a persistent lacticidemia was observed.

The total carnitine remained at the same level (70.5 ± 5.1 μmol/l); a tendency (p > 0.05) toward an increase in the free carnitine level (36.2 ± 2.9 μmol/l) and a decrease in the acylcarnitine level (34.3 ± 4.1 μmol/l), including acetylcarnitine, was noted. A significant improvement in the AC/C0 ratio was revealed—a decrease to 0.9 (p < 0.001); the proportion of free carnitine in total carnitine increased significantly to 52% (p < 0.01). In general, the data indicate favorable changes: a reduction in carnitine deficiency, an improvement of its function, and a reduction in ketogenesis and in the severity of lactate metabolism disorders.

#### **8. Conclusion**

Coenzyme Q10 and carnitine are important components of energy metabolism that are involved in many biological processes in the human body. Our data suggest that there is an insufficiency of these compounds in patients with mitochondrial diseases. Our studies have not revealed the severe deficiency of these substances, while the evidence for a relative insufficiency were found. According to our laboratory data, coenzyme Q10 deficiency is manifested by a significantly low ratio of coenzyme Q10/cholesterol. Lower absolute level of coenzyme Q10 and tendency toward a more pronounced decrease in the Q10/cholesterol ratio in older patients (6–16 years), in our opinion, are consistent with the progressive course of mitochondrial pathology.

Carnitine deficiency is manifested by a tendency toward a decrease in the free carnitine blood level, a pronounced decrease in its proportion in total carnitine, and a significant increase in the ratio of bound and free carnitines.

The coenzyme Q10 and free carnitine insufficiency certainly adversely affects the course of the disease. The causes for these disorders remain unclear. Perhaps, defective mitochondria are not able to provide adequate biosynthesis of coenzyme Q10. Depletion of free carnitine is likely to occur as a result of the activation of

#### *Coenzyme Q10 and L-Carnitine Disturbances in Children with Mitochondrial Diseases DOI: http://dx.doi.org/10.5772/intechopen.87950*

conjugation of acyl radicals, which accumulate in the disorders of respiratory chain and oxidative phosphorylation.

Given a crucial role of carnitine and coenzyme in mitochondrial energy processes, the insufficiency of these compounds should be treated. Clinical heterogeneity of mitochondrial diseases justifies further research in homogeneous patient groups in order to develop evidence-based recommendations and ensure higher treatment efficacy. Furthermore, a moderate carnitine deficiency in congenital structural myopathies provides indications for carnitine supplementation.

### **Author details**

Ekaterina A. Nikolaeva1 \*, Ilgar S. Mamedov2 and Irina V. Zolkina<sup>2</sup>

1 Research and Clinical Institute of Pediatrics named after Yuri Veltischev, Pirogov Russian National Research Medical University, Russian Ministry of Health, Moscow, Russian Federation

2 LLC "Laboratory of Chromatographic Systems", Moscow, Russian Federation

\*Address all correspondence to: kate\_nikolaeva09@mail.ru

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### *Edited by Stavros Baloyannis*

The mitochondrion is a unique and ubiquitous organelle that contains its own genome, encoding essential proteins that are major components of the respiratory chain and energy production system. Mitochondria play a dominant role in the life and function of eukaryotic cells including neurons and glia, as their survival and activity depend upon mitochondrial energy production and supply. Besides energy production, mitochondria also play a vital role in calcium homeostasis and may induce apoptosis by excitotoxicity. Mitochondrial dysfunction is related to common neurological diseases, such as Parkinson's disease, Alzheimer's disease, Friedreich's ataxia, Huntington's disease, and Multiple Sclerosis. An efficient treatment of mitochondrial dysfunction would open new horizons in the therapeutic perspectives of a substantial number of inflammatory and degenerative neurological disorders.

Published in London, UK © 2020 IntechOpen © Sinhyu / iStock

Mitochondria and Brain Disorders

Mitochondria

and Brain Disorders

*Edited by Stavros Baloyannis*