**3.1. The role of mitochondrial dysfunction in Parkinson's disease etiology**

Mitochondrial dysfunction relevance in PD was first documented when 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was associated with parkinsonian syndrome in humans [147]. MPTP is able to cross the blood-brain barrier, is metabolized to 1-methyl-4-phenylpyridinium (MPP+ ) and is uptaken by dopaminergic neurons, inhibiting mitochondrial respiration at complex I [148]. Complex I activity was shown to be decreased in PD brain samples [149], in peripheral tissues namely platelets and lymphocytes [150] and in PD cybrids [151]. The inhibition of complex I, with MPTP and rotenone, is widely used as *in vitro* and *in vivo* models of PD since these recapitulate the main features of the disease [152–154]. Mitochondrial dysfunction in PD tissues and models is also characterized by a decrease in ∆Ψmit [52, 155, 156]. Accordingly, at a functional level, brain bioenergetics is compromised in PD where PET scans showed glucose utilization are decreased in PD individuals in the occipital cortex compared to control individuals [157].

the other hand,systemic inflammation may be induced from several chronic diseases [135], such as obesity and T2D, all characterized by CNS inflammation and microglia activation [136, 137].

PD is the most common movement neurodegenerative disorder characterized by numerous motor symptoms, including tremor, bradykinesia, rigidity and postural instability [138]. PD is twice as common in men than in women, and about 2% of the population above the age of 60 is affected by the disease [139]. PD is characterized by the severe loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and by the presence of intracytoplasmatic proteinaceous inclusions called Lewy bodies, which are primarily composed of fibrillary α-synuclein

Several evidences from autopsy studies showed that multiple processes are involved in cell death, including oxidative stress, mitochondrial dysfunction, neuroinflammation, excitotoxicity and accumulation of misfolded proteins due to proteasomal and autophagic impairment [142]. Data show that mitochondrial deficits occur in PD patient's brain neurons, platelets and lymphocytes [139],which play a critical role in the loss of dopaminergic neurons [143]. Furthermore, data suggest that mitochondrial dysfunction can be potentiated by defects in mitochondrial biogenesis caused by the deregulation of transcription factors, such as peroxisome proliferatoractivated receptor gamma coactivator1-alpha (PGC-1α) [144], which levels are decreased in postmortem brains of PD and in white blood cells [139]. Recent studies in post-mortem PD brain tissue showed that nigrostriatal axon terminals are dysfunctional, which can alter normal axonal transport. Also, the generation of ROS induces the damage of complexes I and III and protein oxidation in mitochondria and in cytoplasmic proteins, leading to mitochondrial dysfunction [145]. Several studies obtained in post-mortem PD brain tissue, human clinical imaging and fluid biomarker have demonstrated that neuroinflammation is a salient feature and probably an essential contributor to PD pathogenesis [145]. Inflammation associated with oxidative stress and cytokine-dependent toxicity has been described and can lead to both innate and adaptive immune responses. Immune responses can act a secondary response to cellular damage and/ or neuronal loss in the affected regions of the nervous system. These mechanisms imply not only a complex crosstalk between the CNS and the peripheral immune system but also interactions between the brain resident immune cells (microglial cells) and other brain cells (neurons, astrocytes, endothelial cells) [146]. Indeed, it has been described that PD brains show microglial activation and lymphocyte infiltration in the areas of degeneration and an increased expression of inflammatory cytokines with alterations in the composition of periph-

(SNCA), and ubiquitinated proteins within some remaining nigral neurons [140, 141].

eral immune cells, suggesting the key role of neuroinflammation in PD.

**3.1. The role of mitochondrial dysfunction in Parkinson's disease etiology**

Mitochondrial dysfunction relevance in PD was first documented when 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was associated with parkinsonian syndrome in humans [147].

Therefore, in AD, neuroinflammation can cause and drive pathogenesis [22].

**3. Parkinson's disease hallmarks**

144 Mitochondrial Diseases

Oxidative damage driven by mitochondria malfunctioning is a prominent aspect in PD [158]. Mitochondrial complex I is one of the most important sites of ROS production in the cell, primarily O2 – [159]. The consequences of oxidative damage are such in PD that oxidative stress was proposed as the cause for dopaminergic neurons death in the SNpc [160, 161]. The same authors found in post-mortem samples from PD subjects increased lipid peroxidation whereas glutathione pathway, an antioxidant defense, is impaired [160]. Mitochondria are the main producers and are also the primary targets of ROS. PD brain biopsies revealed complex I itself is oxidatively damaged, which prevents its proper assembly and function [149]. Although it is incontestable that oxidative stress contributes to PD pathology, it is now generally accepted that ROS are a by-product of mitochondrial dysfunction that contributes to worsen cell demise [162].

Familial forms of PD bearing mutations in mitochondrial proteins reinforced the involvement of mitochondrial dysfunction in PD etiology and shed light into the mechanisms leading to neuronal death, unifying both familial and sporadic cases. Rare mutations causing juvenile PD are related to mitochondrial degradation by mitophagy created an opportunity for clarification of the disease mechanisms. The first identified mutation in *PARK2* (Parkin), an E3 ubiquitin ligase, cause early onset PD [163]. The second mutation was identified in *PARK6,* PTEN-induced kinase 1 (PINK1) and a mitochondrial kinase [164]. PINK1 and Parkin act together in a tightly regulated process to target dysfunctional mitochondria for degradation, named mitophagy. This process is crucial for the maintenance of a healthy pool of mitochondria, potentially protecting cells in early stages of mitochondrial dysfunction [165]. In healthy mitochondria, PINK1 levels are maintained low as this protein is degraded within mitochondrial matrix after its import from cytosol [166]. When mitochondria lose their membrane potential, PINK1 is stabilized at their surface recruiting Parkin that, in turn, ubiquitinates and targets mitochondria to undergo mitophagy [167–169]. PD caused by PINK1 and Parkin mutations is not clinically differentiated from idiopathic PD [170]. Morphologically, PINK1 mutations have drastic repercussions in mitochondria from *Drosophila melanogaster* to mouse models, with larger, swollen and disrupted cristae [171, 172]. In cybrids from sporadic PD subjects, mitochondria also present abnormal structure with enlarged and scarce cristae [52, 173]. Mitochondrial network images show that in PD models it presents a fragmented structure. From PD cybrids [174] to dopaminergic neurons treated with MPTP [175], a number of models show early mitochondrial fragmentation that precedes cell death. Although DRP1 has been implicated in the fragmentation of mitochondria in PD [174], studies point to SNCA directly interacting with mitochondria inducing fragmentation, in a process that does not require DRP1 [176]. Recently, it was found a common mechanism for mitophagy failure, besides Pink1-Parkin axis, that is shared by familial and sporadic PD, with potential of an early biomarker [177]. In fibroblasts isolated from patients that carry PD mutations and idiopathic PD subjects, it was found an impairment in RHOT1 degradation that in turn delays mitochondria immobilization and consequent degradation [178]. RHOT1 is a mitochondrial kinesin adaptor protein that, upon mitochondrial damage, interacts with PINK1 and Parkin to target mitochondria for proteasomal degradation [179]. Consequently, abnormal levels of autophagy markers were found in brain tissue preparations from PD patients, both sporadic and early onset [180, 181]. This impairment in autophagy has been related to the decreased transport along microtubules and fusion of autophagic vesicles with the lysosomes rather than a defect in cell waste recognition by autophagy machinery [173]. Mitochondrial dysfunction is intimately connected to microtubule instability and, thus, autophagy impairment in PD models. In PD cybrids, intracellular transport of autophagosomes and mitochondria is compromised [173]. Accordingly, MPP+ -treated cells have disrupted microtubule network and a decrease in mitochondrial trafficking [182]. Also, there are some data pointing that Parkin can bind to microtubules contributing to their stabilization, whereas ablation of Parkin causes reduced microtubule mass [183, 184]. Accumulation of non-degraded mitochondria and other autophagic substrates, such as SNCA aggregates, increments cell demise and contributes to Lewy body-like structure formation. Oxidative stress provoked by mitochondrial malfunctioning is able to induce proteasomal subunit disassembly, leading to the accumulation of degrading substrates, such as ubiquitin [185], contributing to Lewy body formation and cell death. In fact, ubiquitin accumulation, impaired ubiquitin proteasome system (UPS) function and mitochondrial dysfunction have been proposed to be intimately associated [186].

Remarkably, epidemiological studies showed that non-steroidal anti-inflammatory drugs, such as ibuprofen lowers the risk of PD further supporting the contribution of inflammation to disease process [192–194]. Interestingly, the SNpc (main area affected in PD) exhibit high sensitivity to proinflammatory compounds, whereas the hippocampus appears to be more resistant, which can be explained due to the differences in the number of microglial cells between both areas [195]. In fact, numerous evidences that came from experimental PD models suggest that dopaminergic neurons are extremely vulnerable to inflammatory challenge [196, 197]. Moreover, stereotaxic injection of lipopolysaccharide (LPS, a Gram-negative bacteriotoxin that activates microglial cells) into the SNpc induced degeneration of dopaminergic neurons while sparing GABAergic and serotonergic neurons, suggesting selective dopami-

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147

There are several factors that may be underlying this selectivity. Dying neurons release substances that are recognized by glial cells, activating them, such as dopamine, neuromelanin and SNCA [199]. Dopamine seems to play a role in the inflammatory response induced by LPS, since depletion of this neurotransmitter prevents gliosis and reduces peripheral macrophages infiltration and dopaminergic neuronal death induced by 6-hydroxydopamine (6-OHDA) [200]. Recently, Dominguez-Meijide and colleagues observed that the decrease in dopamine levels observed in early stages of PD promotes neuroinflammation and disease progression via glial renin-angiotensin system exacerbation [201]. Neuromelanin is able to activate microglia cells leading to neuroinflammatory processes and degeneration of dopaminergic neurons [202, 203]. Extracellular and misfolded SNCA prompts microglia activation

Further support for a role of innate immunity activation in PD pathogenesis come from genetic studies showing that polymorphisms in some proinflammatory cytokines may influence the risk of developing PD. Indeed, there is an association between genetic variations in the human leukocyte antigen (HLA) region and sporadic PD [207, 208]. HLA isalso called human MHC molecules, which presentation activates CD+4 T cells and CD+8 cytotoxic lymphocytes. Remarkably, in a GWA study, several susceptibility loci have been identified as strong risk factors that are related to both innate and adaptive immune functions [209]. Moreover, PD-linked genes such as LRRK2 and SNCA are also known to stimulate inflammatory responses and immunological regulation [210]. In fact, Harms and colleagues reported that accumulation of pathological SNCA in PD brain leads to T cell infiltration, microglial activation and increased production of inflammatory cytokines and chemokines [211]. Furthermore, transgenic mice with overexpression of wild-type or mutated SNCA showed an early microglial activation [212, 213]. Beraud and colleagues demonstrated that misfolded SNCA directly activates microglia, inducing production and release of TNFα and increasing expression of Nfr2-dependent antioxidant enzymes [214]. Aggregated and nitrated SNCA also stimulates microglia activation triggering innate and adaptive immune responses [215]. Intranigral injection of SNCA resulted in the upregulation of mRNA expression of proinflammatory cytokines and the expression of endothelial markers of inflammation and microglial activation [216, 217]. Multiple immune cells show high levels of LRRK2 expression [218, 219]. R1441G LRRK2 mutation was shown to increase proinflammatory cytokine release from activated microglial cells [220, 221]. Moreover, LPS-mediated neuroinflammation is attenuated

nergic neurons vulnerability to PAMPs [198].

and production of proinflammatory molecules [204–206].

in murine *lrrk2*-knockdown brain microglia [222].

#### **3.2. Immune response in Parkinson's disease**

Despite PD is characterized by a slow and progressive degeneration of dopaminergic neurons in the SNpc, the cause of this neuronal loss is still poorly understood. Nevertheless, neuroinflammatory mechanisms, such as microglial activation, astrogliosis and lymphocytic infiltration have been postulated to contribute to the cascade of events leading to neuronal degeneration [187].

A growing body of evidence suggests a role of autoimmune and neuroinflammatory mechanisms in the etiopathogenesis of PD [188]. Peripheral immune responses can trigger inflammation and exacerbate neurodegeneration in several neurodegenerative disorders including PD. Indeed, peripheral inflammation in early stages of disease appears to accompany the development of preclinical non-motor symptoms, including olfactory and gastrointestinal dysfunction, providing a possible association between autoimmunity and PD [189]. Strikingly, chronic constipation, which occurs many years before the first motor symptoms of PD, is casually linked to peripheral inflammation [190].

Inflammation is a defense mechanism aimed at counteracting with diverse insults. In neurodegenerative disorders, such as PD, inflammation could results from the activation of innate immunity by PAMPs; DAMPs or protein aggregates. Other than the activation of inflammatory responses, there is also the ability of the immune system to detect harmful agents. Mounting evidence indicates that dopaminergic cell death is influenced by the innate immune system and neuroinflammatory processes in PD. Soreq and coworkers described an altered expression of neuroimmune signaling-related transcripts in early stages of PD [191]. Remarkably, epidemiological studies showed that non-steroidal anti-inflammatory drugs, such as ibuprofen lowers the risk of PD further supporting the contribution of inflammation to disease process [192–194]. Interestingly, the SNpc (main area affected in PD) exhibit high sensitivity to proinflammatory compounds, whereas the hippocampus appears to be more resistant, which can be explained due to the differences in the number of microglial cells between both areas [195]. In fact, numerous evidences that came from experimental PD models suggest that dopaminergic neurons are extremely vulnerable to inflammatory challenge [196, 197]. Moreover, stereotaxic injection of lipopolysaccharide (LPS, a Gram-negative bacteriotoxin that activates microglial cells) into the SNpc induced degeneration of dopaminergic neurons while sparing GABAergic and serotonergic neurons, suggesting selective dopaminergic neurons vulnerability to PAMPs [198].

of an early biomarker [177]. In fibroblasts isolated from patients that carry PD mutations and idiopathic PD subjects, it was found an impairment in RHOT1 degradation that in turn delays mitochondria immobilization and consequent degradation [178]. RHOT1 is a mitochondrial kinesin adaptor protein that, upon mitochondrial damage, interacts with PINK1 and Parkin to target mitochondria for proteasomal degradation [179]. Consequently, abnormal levels of autophagy markers were found in brain tissue preparations from PD patients, both sporadic and early onset [180, 181]. This impairment in autophagy has been related to the decreased transport along microtubules and fusion of autophagic vesicles with the lysosomes rather than a defect in cell waste recognition by autophagy machinery [173]. Mitochondrial dysfunction is intimately connected to microtubule instability and, thus, autophagy impairment in PD models. In PD cybrids, intracellular transport of autophagosomes and mitochondria is com-

a decrease in mitochondrial trafficking [182]. Also, there are some data pointing that Parkin can bind to microtubules contributing to their stabilization, whereas ablation of Parkin causes reduced microtubule mass [183, 184]. Accumulation of non-degraded mitochondria and other autophagic substrates, such as SNCA aggregates, increments cell demise and contributes to Lewy body-like structure formation. Oxidative stress provoked by mitochondrial malfunctioning is able to induce proteasomal subunit disassembly, leading to the accumulation of degrading substrates, such as ubiquitin [185], contributing to Lewy body formation and cell death. In fact, ubiquitin accumulation, impaired ubiquitin proteasome system (UPS) function

and mitochondrial dysfunction have been proposed to be intimately associated [186].

Despite PD is characterized by a slow and progressive degeneration of dopaminergic neurons in the SNpc, the cause of this neuronal loss is still poorly understood. Nevertheless, neuroinflammatory mechanisms, such as microglial activation, astrogliosis and lymphocytic infiltration have been postulated to contribute to the cascade of events leading to neuronal

A growing body of evidence suggests a role of autoimmune and neuroinflammatory mechanisms in the etiopathogenesis of PD [188]. Peripheral immune responses can trigger inflammation and exacerbate neurodegeneration in several neurodegenerative disorders including PD. Indeed, peripheral inflammation in early stages of disease appears to accompany the development of preclinical non-motor symptoms, including olfactory and gastrointestinal dysfunction, providing a possible association between autoimmunity and PD [189]. Strikingly, chronic constipation, which occurs many years before the first motor symptoms of

Inflammation is a defense mechanism aimed at counteracting with diverse insults. In neurodegenerative disorders, such as PD, inflammation could results from the activation of innate immunity by PAMPs; DAMPs or protein aggregates. Other than the activation of inflammatory responses, there is also the ability of the immune system to detect harmful agents. Mounting evidence indicates that dopaminergic cell death is influenced by the innate immune system and neuroinflammatory processes in PD. Soreq and coworkers described an altered expression of neuroimmune signaling-related transcripts in early stages of PD [191].


promised [173]. Accordingly, MPP+

146 Mitochondrial Diseases

**3.2. Immune response in Parkinson's disease**

PD, is casually linked to peripheral inflammation [190].

degeneration [187].

There are several factors that may be underlying this selectivity. Dying neurons release substances that are recognized by glial cells, activating them, such as dopamine, neuromelanin and SNCA [199]. Dopamine seems to play a role in the inflammatory response induced by LPS, since depletion of this neurotransmitter prevents gliosis and reduces peripheral macrophages infiltration and dopaminergic neuronal death induced by 6-hydroxydopamine (6-OHDA) [200]. Recently, Dominguez-Meijide and colleagues observed that the decrease in dopamine levels observed in early stages of PD promotes neuroinflammation and disease progression via glial renin-angiotensin system exacerbation [201]. Neuromelanin is able to activate microglia cells leading to neuroinflammatory processes and degeneration of dopaminergic neurons [202, 203]. Extracellular and misfolded SNCA prompts microglia activation and production of proinflammatory molecules [204–206].

Further support for a role of innate immunity activation in PD pathogenesis come from genetic studies showing that polymorphisms in some proinflammatory cytokines may influence the risk of developing PD. Indeed, there is an association between genetic variations in the human leukocyte antigen (HLA) region and sporadic PD [207, 208]. HLA isalso called human MHC molecules, which presentation activates CD+4 T cells and CD+8 cytotoxic lymphocytes. Remarkably, in a GWA study, several susceptibility loci have been identified as strong risk factors that are related to both innate and adaptive immune functions [209]. Moreover, PD-linked genes such as LRRK2 and SNCA are also known to stimulate inflammatory responses and immunological regulation [210]. In fact, Harms and colleagues reported that accumulation of pathological SNCA in PD brain leads to T cell infiltration, microglial activation and increased production of inflammatory cytokines and chemokines [211]. Furthermore, transgenic mice with overexpression of wild-type or mutated SNCA showed an early microglial activation [212, 213]. Beraud and colleagues demonstrated that misfolded SNCA directly activates microglia, inducing production and release of TNFα and increasing expression of Nfr2-dependent antioxidant enzymes [214]. Aggregated and nitrated SNCA also stimulates microglia activation triggering innate and adaptive immune responses [215]. Intranigral injection of SNCA resulted in the upregulation of mRNA expression of proinflammatory cytokines and the expression of endothelial markers of inflammation and microglial activation [216, 217]. Multiple immune cells show high levels of LRRK2 expression [218, 219]. R1441G LRRK2 mutation was shown to increase proinflammatory cytokine release from activated microglial cells [220, 221]. Moreover, LPS-mediated neuroinflammation is attenuated in murine *lrrk2*-knockdown brain microglia [222].

The first evidence for a neuroinflammatory processes in PD came in 1988 when McGeer and co-workers observed the presence of activated microglial cells and inflammatory macrophages, as well as, proinflammatory cytokines in post-mortem brain samples of the SNpc of PD patients [223]. Similarly, Langston and coworkers reported an accumulation of activated microglia around dopaminergic neurons in post-mortem human brains with MPTP-induced parkinsonism [224]. Later, several authors corroborated this result and further observed the presence of other markers such as HLA-DP, HLA-DQ, HLADR (CR3/43), CD68 (EBM11, a low-density lipoprotein binding glycoprotein, equivalent to macrosialin in mice) and ferritin in the SNpc and putamen [225–227]. In addition, intercellularadhesion molecule-1-positive glia levels are also increased in the SNpc of PD brains, indicating activation of cells of the innate immune system, in particular, in areas with neuronal loss and extracellular melanin accumulation [228]. Furthermore, Damien and colleagues used glutathione peroxidase as an astrocytic marker and observed that the density of astrocytes in the SNpc is low when compared to the ventral tegmental area. This indicates that vulnerable neurons in patients with PD have less surrounding astroglial cells and as a result reduced detoxification of oxygen-free radicals by glutathione peroxidase [229]. McGeer and colleagues described for the first time the presence of cytotoxic T lymphocytes (CD8+) in the substantia nigra from one patient with PD [223]. Moreover, several reports found alterations in the population of blood T lymphocytes in PD patients [230–232]. In addition, cytotoxic infiltration of CD8+ and CD4+ T cells into the brain parenchyma of both post-mortem human PD specimens and in the MPTP mouse model of PD was described during the course of neuronal degeneration [233, 234]. Interestingly, these markers were not detected in the red nucleus suggesting that this infiltration is selective for the injured brain areas. Furthermore, these cells were in close contact with blood vessels and near to melanized dopaminergic neurons. These data indicate that cells migrate from the bloodstream and suggest an interaction between the lymphocytes and the dopaminergic neurons during the neurodegenerative process. Hence, alterations in the BBB might occur in the brains of PD patients. Not only during aging but also in PD, a BBB disruption can occur, leading to an invasion of immune cells, peripheral mediators, toxins and elements of adaptive immunity to the brain parenchyma potentiating the degenerative process [235]. Additionally, PD patients have increased permeability of the intestinal epithelial barrier and a chronic gut inflammation characterized by increased expression levels of proinflammatory cytokines and inflammatory markers [236, 237]. Moreover, several studies reported increase in TNFα, β2-microglobulin, epidermal growth factor (EGF), transforming growth factor α (TGFα), TGFβ1 and interleukins 1β, 6 and 2 levels in the striatum of PD patients and increasein TNFα, interleukin 1β and interferon γ levels in the SN of PD patients [238–243]. Interestingly, dopaminergic neurons express the receptors for these cytokines, suggesting that they are sensitive to these cytokines [244, 245]. Proinflammatory cytokines, such as TNFα, interleukin 1β, and interferon γ, can induce the expression of the inducible form of nitric oxide synthase (iNOS) or cyclo-oxygenase 2 (COX2), which are known to produce toxic reactive species. To corroborate the previous studies, a CD23-mediated increase in iNOS in the SN of PD patients was found. Furthermore, enzymes that are involved in neuroinflammatory processes mediated by oxidative stress, such as NADPH oxidase, COX2 and myeloperoxidase, are also increased in PD patients [239, 246, 247]. This may indicate that the inflammation-derived oxidative stress could contribute to dopaminergic neuronal degeneration.

TNFα, TNFR1 and RANTES is increased [248–254]. Interestingly, RANTES levels were correlated with the severity and duration of the disease [255]. Additionally, studies analyzing CSF from PD patients reported proinflammatory changes such as the presence of TNFα [238] and interleukin 1β [225, 256, 257] and osteopontin (a member of the integrins family) [258]. Moreover, PET scan analysis also reported the presence of PK-11195 in PD samples, which is indicative of microglia activation [259, 260]. PET analysis using radioligand 11C-PK-11195 corroborated these results in the SNpc of sporadic PD patients within a year from clinical onset [261]. More recently, microglial activation in PD has been observed with PET by using [18F]-FEPPA [262]. Moreover, it was found a significantly increase numbers of T-helper 17 cells and myeloid-derived suppressor cells in peripheral circulation in PD patients compared with controls [263]. This suggests that

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149

It has also been demonstrated that mitochondrial toxins, such as 6-OHDA, MPTP and rotenone, trigger an immune reaction in the striatum and SNpc suggesting that a primary damage to the mitochondrial respiratory chain represents, per se, a trigger for microglial activation and neuroinflammatory processes [264–267]. This reaction includes activation of microglia and infiltration of CD4+ and CD8+ T cells. Rotenone administration was shown to cause microglial activation not only in rodent models [268] but also in human microglial cell lines [269]. Similarly, a significant increase in the number of activated microglial cells was detected in the brain of 6-OHDA rats, at both nigral and striatal areas [233, 270]. Moreover, in the same model CD+3, CD+4 and CD+8 T cells were abundant and migrated from blood vessels into the SNpc [271]. Additionally, in the brains of both monkeys and mice after systemic injection of MPTP, an activated microglia and infiltration of T-lymphocytes has been observed [197]. Microglial activation was also observed in mice that overexpress SNCA [213], in the SNpc and

Interestingly, intranigral or systemic injection of LPS in animals can selectively kill dopaminergic neurons [200, 275–279]. Furthermore, injection of LPS into pregnant female rats led to offspring with less and abnormal dopaminergic neurons and increased levels of TNFα in the striatum when compared to the controls [280]. Remarkably, the offspring in adulthood were also more susceptible to the effects of parkinsonian toxins than were the controls [281, 282]. Furthermore, the injection of other proinflammatory compounds such as thrombin within the SNpc also induced the death of dopaminergic neurons [283, 284]. These studies suggest that

As previously mentioned, microglial cells when activated produce and release toxic oxygenderived and nitrogen-derived products, which rely on the regulated induction of several enzymatic systems such as NADPH oxidase and iNOS.Indeed, the expression of these biocatalytic systems within the SNpc is significantly increased in PD patient's post-mortem samples as well as in PD animal models [239, 247]. Oxygen and nitrogen-derived products such as NO, O2

 can directly cross membranes and enter dopaminergic neurons, which can cause oxidative damage in tyrosine hydroxylase decreasing its enzymatic activity and in SNCA promoting its aggregation [285, 286]. Additionally, activated microglia can release inflammatory cytokines and chemokines, such as TNFα, interleukin 1β and interferon,whichcan induce neurotoxicity via a direct mechanism through receptor binding on dopaminergic neurons or an indirect mechanism through glial-cell activation and expression of inflammatory factors. In fact, chronic adenoviral expression of TNFα in the SNpc of rats can cause time-dependent dopaminergic cell death [287].

− and

microglia-mediated inflammation underlies the neuronal cell death in the SNpc.

a microglial-mediated inflammatory process occurs early in PD process.

striatum of rats exposed to 6-OHDA [272, 273] and to MPTP [274].

ONOO<sup>−</sup>

The results obtained in post-mortem studies were further corroborated by studies carried out in biological fluids (serum or CSF) of patients suffering from PD. Serum samples from PD patients indicated that the expression of certain cytokines such as IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, TNFα, TNFR1 and RANTES is increased [248–254]. Interestingly, RANTES levels were correlated with the severity and duration of the disease [255]. Additionally, studies analyzing CSF from PD patients reported proinflammatory changes such as the presence of TNFα [238] and interleukin 1β [225, 256, 257] and osteopontin (a member of the integrins family) [258]. Moreover, PET scan analysis also reported the presence of PK-11195 in PD samples, which is indicative of microglia activation [259, 260]. PET analysis using radioligand 11C-PK-11195 corroborated these results in the SNpc of sporadic PD patients within a year from clinical onset [261]. More recently, microglial activation in PD has been observed with PET by using [18F]-FEPPA [262]. Moreover, it was found a significantly increase numbers of T-helper 17 cells and myeloid-derived suppressor cells in peripheral circulation in PD patients compared with controls [263]. This suggests that a microglial-mediated inflammatory process occurs early in PD process.

The first evidence for a neuroinflammatory processes in PD came in 1988 when McGeer and co-workers observed the presence of activated microglial cells and inflammatory macrophages, as well as, proinflammatory cytokines in post-mortem brain samples of the SNpc of PD patients [223]. Similarly, Langston and coworkers reported an accumulation of activated microglia around dopaminergic neurons in post-mortem human brains with MPTP-induced parkinsonism [224]. Later, several authors corroborated this result and further observed the presence of other markers such as HLA-DP, HLA-DQ, HLADR (CR3/43), CD68 (EBM11, a low-density lipoprotein binding glycoprotein, equivalent to macrosialin in mice) and ferritin in the SNpc and putamen [225–227]. In addition, intercellularadhesion molecule-1-positive glia levels are also increased in the SNpc of PD brains, indicating activation of cells of the innate immune system, in particular, in areas with neuronal loss and extracellular melanin accumulation [228]. Furthermore, Damien and colleagues used glutathione peroxidase as an astrocytic marker and observed that the density of astrocytes in the SNpc is low when compared to the ventral tegmental area. This indicates that vulnerable neurons in patients with PD have less surrounding astroglial cells and as a result reduced detoxification of oxygen-free radicals by glutathione peroxidase [229]. McGeer and colleagues described for the first time the presence of cytotoxic T lymphocytes (CD8+) in the substantia nigra from one patient with PD [223]. Moreover, several reports found alterations in the population of blood T lymphocytes in PD patients [230–232]. In addition, cytotoxic infiltration of CD8+ and CD4+ T cells into the brain parenchyma of both post-mortem human PD specimens and in the MPTP mouse model of PD was described during the course of neuronal degeneration [233, 234]. Interestingly, these markers were not detected in the red nucleus suggesting that this infiltration is selective for the injured brain areas. Furthermore, these cells were in close contact with blood vessels and near to melanized dopaminergic neurons. These data indicate that cells migrate from the bloodstream and suggest an interaction between the lymphocytes and the dopaminergic neurons during the neurodegenerative process. Hence, alterations in the BBB might occur in the brains of PD patients. Not only during aging but also in PD, a BBB disruption can occur, leading to an invasion of immune cells, peripheral mediators, toxins and elements of adaptive immunity to the brain parenchyma potentiating the degenerative process [235]. Additionally, PD patients have increased permeability of the intestinal epithelial barrier and a chronic gut inflammation characterized by increased expression levels of proinflammatory cytokines and inflammatory markers [236, 237]. Moreover, several studies reported increase in TNFα, β2-microglobulin, epidermal growth factor (EGF), transforming growth factor α (TGFα), TGFβ1 and interleukins 1β, 6 and 2 levels in the striatum of PD patients and increasein TNFα, interleukin 1β and interferon γ levels in the SN of PD patients [238–243]. Interestingly, dopaminergic neurons express the receptors for these cytokines, suggesting that they are sensitive to these cytokines [244, 245]. Proinflammatory cytokines, such as TNFα, interleukin 1β, and interferon γ, can induce the expression of the inducible form of nitric oxide synthase (iNOS) or cyclo-oxygenase 2 (COX2), which are known to produce toxic reactive species. To corroborate the previous studies, a CD23-mediated increase in iNOS in the SN of PD patients was found. Furthermore, enzymes that are involved in neuroinflammatory processes mediated by oxidative stress, such as NADPH oxidase, COX2 and myeloperoxidase, are also increased in PD patients [239, 246, 247]. This may indicate that the inflammation-derived

148 Mitochondrial Diseases

oxidative stress could contribute to dopaminergic neuronal degeneration.

The results obtained in post-mortem studies were further corroborated by studies carried out in biological fluids (serum or CSF) of patients suffering from PD. Serum samples from PD patients indicated that the expression of certain cytokines such as IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, It has also been demonstrated that mitochondrial toxins, such as 6-OHDA, MPTP and rotenone, trigger an immune reaction in the striatum and SNpc suggesting that a primary damage to the mitochondrial respiratory chain represents, per se, a trigger for microglial activation and neuroinflammatory processes [264–267]. This reaction includes activation of microglia and infiltration of CD4+ and CD8+ T cells. Rotenone administration was shown to cause microglial activation not only in rodent models [268] but also in human microglial cell lines [269]. Similarly, a significant increase in the number of activated microglial cells was detected in the brain of 6-OHDA rats, at both nigral and striatal areas [233, 270]. Moreover, in the same model CD+3, CD+4 and CD+8 T cells were abundant and migrated from blood vessels into the SNpc [271]. Additionally, in the brains of both monkeys and mice after systemic injection of MPTP, an activated microglia and infiltration of T-lymphocytes has been observed [197]. Microglial activation was also observed in mice that overexpress SNCA [213], in the SNpc and striatum of rats exposed to 6-OHDA [272, 273] and to MPTP [274].

Interestingly, intranigral or systemic injection of LPS in animals can selectively kill dopaminergic neurons [200, 275–279]. Furthermore, injection of LPS into pregnant female rats led to offspring with less and abnormal dopaminergic neurons and increased levels of TNFα in the striatum when compared to the controls [280]. Remarkably, the offspring in adulthood were also more susceptible to the effects of parkinsonian toxins than were the controls [281, 282]. Furthermore, the injection of other proinflammatory compounds such as thrombin within the SNpc also induced the death of dopaminergic neurons [283, 284]. These studies suggest that microglia-mediated inflammation underlies the neuronal cell death in the SNpc.

As previously mentioned, microglial cells when activated produce and release toxic oxygenderived and nitrogen-derived products, which rely on the regulated induction of several enzymatic systems such as NADPH oxidase and iNOS.Indeed, the expression of these biocatalytic systems within the SNpc is significantly increased in PD patient's post-mortem samples as well as in PD animal models [239, 247]. Oxygen and nitrogen-derived products such as NO, O2 − and ONOO<sup>−</sup> can directly cross membranes and enter dopaminergic neurons, which can cause oxidative damage in tyrosine hydroxylase decreasing its enzymatic activity and in SNCA promoting its aggregation [285, 286]. Additionally, activated microglia can release inflammatory cytokines and chemokines, such as TNFα, interleukin 1β and interferon,whichcan induce neurotoxicity via a direct mechanism through receptor binding on dopaminergic neurons or an indirect mechanism through glial-cell activation and expression of inflammatory factors. In fact, chronic adenoviral expression of TNFα in the SNpc of rats can cause time-dependent dopaminergic cell death [287].
