Even though oxidative stress, inflammation,
excitotoxicity, mitochondrial dysfunction, and hereditary and environmental factors
have all been implicated in the pathogenesis of PD, the exact cause of the loss
of dopaminergic neurons remains obscure (Hattori and Mizuno, 2004: Eriksen et al., 2005).
The first familial PD (FPD)-linked
gene that was identified is alpha-synuclein (?-syn)
(Polymeropoulos et al.,
1999). Later, four more genes have been identified and they are Parkin, DJ-1, PINK1 and LRRK2. These genes play important role in mitochondrial
function or in the handling of misfolded proteins. Mutations in PINK1, DJ-1 and Parkin
cause early onset autosomal
recessive form of PD whereas mutations in LRRK2, like ?- syn, cause autosomal dominant PD (Savitt et
al., 2006). ROS and RNS are known to
cause protein misfolding and aggregation. Hence oxidative and nitrosative
stress play important roles in affecting the normal function of genes which
leads to the formation of toxic aggregates and ultimately induce
neurodegeneration in PD (Jenner et al., 1992). This hypothesis is best
demonstrated by the oxidative stress induced aggregation of ?-syn
which is the major component of Lewy body (Spillantini et al.,
1997). This notion is supported
by the observation that nitrated or oxidative damaged protein aggregates are
prominent in brain tissues from PD patients (Danielson, Andersen, 2008).Another important cause of PD
is lysosomal impairments due to mutations in a gene encoding for the lysosomal P-type
ATPase named ATP13A2 which
results in juvenile and early-on set form of parkinsonism (Ramirezetal.,
2006). Later it was found that there is patho-physiological link between lysosomal
dysfunction and a-synuclein
in dopaminergic neurodegeneration.
Peroxynitrite and its effect on PD gene expression
A common feature of
neurodegenerative diseases, such as Parkinson’s disease, Alzheimer’s disease,
MS, and ALS is extensive evidence of oxidative and nitrosative stress via ROS
and RNS, which might be responsible for the neuronal dysfunction and death
contributing to pathogenesis of these disorders (Bar-Shai and Reznick, 2006:
Ischiropoulous and Beckman, 2003: Moreira et
al., 2005). Because neurons are incapable of division, the consequences of
severe oxidative stress killing neurons makes sustained damage irreversible.
Peroxynitrite, one of the well known reactive nitrogen species, formation has
been implicated in Parkinson’s disease and other neurodegenerative diseases. Once
formed in the diseased brain, peroxynitrite may exert its toxic effects through
multiple mechanisms, including lipid peroxidation, mitochondrial damage,
protein nitration and oxidation, depletion of antioxidant reserves (especially
glutathione), activation or inhibition of various signaling pathways, and DNA
damage followed by the activation of the nuclear enzyme PARP (Ischiropoulous
and Beckman, 2003: Sarchielli et al., 2003). Mitochondria are particularly
vulnerable targets of oxidative stress and protein nitration in
neurodegeneration (Schulz et al.,
1997). Peroxynitrite also readily damages complex I in the mitochondria (Murry et al., 2003) and also inactivate mitochondrial
Mn-SOD which might further amplify injury (MacMillan-Crow and Cruthirds, 2001). In vivo, nitration of Mn-SOD has been
detected in cerebrospinal fluid of patients with ALS, Alzheimer’s disease, and
Parkinson’s disease (Aoyama et al., 2000). The inactivation of Mn-SOD
by peroxynitrite will make mitochondria more vulnerable in neurodegeneration. Nitration
of tyrosine residue in protein represents a major cytotoxic pathway in the
nervous system, possibly contributing to neurodegenerative disorders.
Nitrotyrosine immunoreactivity has been found in early stages of the disease in
human autopsy samples as well as in experimental animal models. Peroxynitrite
may induce nitration of tyrosine hydroxylase, the initial and rate-limiting
enzyme in the biosynthesis of dopamine, leading to inhibition of enzyme
activity and consequent failure in the synthesis of dopamine (Ara et
al., 1998)
which leads to loss of dopaminergic neuron which is hallmark of PD pathogenesis.
Parkinson’s
disease
was first described by James Parkinson in early 1800s as “shaking palsy”. Parkinson’s disease is
the second most common neurodegenerative disorder after Alzheimer’s, disease (AD),
affecting an estimated 10 million people worldwide. More than 1% in
those over 60 years suffers from PD and the frequency of the disease in general
population is about 0.3%. Parkinson’s
disease is more prevalent in males (1729 per 100,000, >65 years) than in
females (1644 per 100,000) (Cacabelos, 2017; Riedel et al., 2016).
PD is mostly associated with
loss of dopaminergic neurons in the substantia nigra that coordinate motor
movements, along with intracellular inclusions of abnormal proteins called Lewy
bodies. Lewy bodies in the diseased neurons are identified as eosinophilic inclusions,
and are considered to be the hallmark of PD and its progression. Distribution
of Lewy bodies is seen in several regions of the brain, accounting for the
severe neuronal loss in affected areas. Symptoms commonly found in PD are bradykinesia (slowness
in movements), rigidity, and tremor that progressively worsen with age,
ultimately leading to near total immobility. AD can be detected with
approximately 85–90% accuracy but PD with approximately 75% accuracy. PD is heterogeneous
caused by both environmental factor and genetic factor. Mutations in these genes SNCA, PARKIN, DJ-1, PINK1,
LRRK2, ATP13A2, PLA2G6,
FBXO, PLA2G6, and VPS35 are responsible for
PD and are
found to be associated with its inheritance. Free radicals have a
significant role in the pathogenesis of neurodegenerative diseases, including
PD. Interesting facts have been revealed about mitochondrial dysfunction and
its role in neurodegenerative diseases as well. Because of the
known association between reductions in dopamine content within the neostriatum
and the onset of symptoms of Parkinson’s disease, several lines of treatment
therapies have been developed. One form of treatment involves the
administration of the drug, L-3,
4-hydroxyphenylalanine (L-DOPA). The drugs currently available for PD treat the disease
symptomatically, either by mimicking dopaminergic actions or by enhancing the
duration of action of dopamine. Administration of glial-derived neurotrophic
factor has shown promising improvement in restoring dopaminergic neurons in
animal models of PD. Till now much advancement has been made for the treatment
of PD, but treating with L-Dopa is still the gold standard.
Parkinson’s
disease (PD)
(Source: International Journal of
Nanomedicines Review, Neurological and therapeutic targeted to surmount the
Blood brain barrier by Jagat R Kanwar, Bhasker Sriramoju and Rupinder K Kanwar;
2012)
(C)
Mitocondrial dysfunction due to old age or toxins
generates free radicals that fragment DNA.
(B)
Excitotoxicity is another major cause whereby the
N-methyl-D-aspartate (NMDA) receptor is excessively activated by the endogenous
ligand, glutamate. This drives the influx the extracellular calcium
intracellularly, activating caspases which in turn destroy the neuclic acids
mediating cell death.
(A)
Apoptosis, programmed cell death that allow
clearing out of old cells by inducing their death. However if the same
mechanism dysregulated as a result of mutation, healthy cells also die, leading
to neuronal loss followed by disease symptoms.
Fig. E. Various causes of
neurodegeneration
c.
Mitochondrial dysfunction due to old age or toxins
that generates free radicals which in turn causes DNA fragments and leads to
neuronal death (Kanwar et al., 2012).
b.
Apoptosis, or programmed cell death,
has a convincing role in mediating neurodegenerative diseases, particularly
Parkinson’s disease. Neurons that fail to complete this apoptotic phase become
necrotic and inflamed which, in turn, leads to neurodegeneration (Waldmeier,
2003).
a.
Excitotoxicity is mediated via glutamate,
the major excitatory amino acid in the central nervous system. Excitotoxicity
is mediated by repeated stimulation of glutamate receptors, leading to
neuronal damage (Relja, 2011).
Till now the precise causes and mechanisms of neurodegeneration
are unknown. Individuals with a family history of neurodegeneration are at
higher risk of neurodegenerative disease (Familial type of neurodegeneration),
suggesting a role for genes in its initiation. Excitotoxicity, apoptosis and
mitochondrial (Mt) dysfunctions have been reported as pathological cause for
aging and neurodegenerative diseases such as Parkinson’s disease (PD),
Alzheimer’s disease (AD), Multiple sclerosis (MS) and Amyolotrophic lateral
sclerosis (ALS) (Fig. E).
Neurodegenerative diseases such
as amyotrophic lateral sclerosis, Alzheimer disease, Parkinson’s disease, Huntington
diseases, ataxia etc. are characterized pathologically by the insidious
accumulation of proteins or peptides in the central nervous system (CNS). These
protein or peptides are normally soluble but in disease condition they form
insoluble and form aggregates in CNS. These filamentous aggregates display the
ultrastructural and tinctorial properties of amyloid. So these diseases can be
grouped together as brain amyloidoses. Each of
these related brain amyloidoses have phenotypic diversity and can be
distinguished by (a) distinct temporal and regional patterns of
deposition of aggregates, (b) varying cellular hosts or extracellular
locales of the aggregates, and (c) different protein constituents of the
aggregates. The aggregates accumulate early in the lifetime of the individual,
but only manifest clinically in middle or late life. Most cases are sporadic
(disease due to environmental conditions), but genetic forms (familial) can be
caused by mutations in the gene encoding the amyloidgenic protein that make it
more prone to misfold and aggregate.
Neurodegenerative
disorders are the most serious health problem and the greatest challenges to
the increasing population age. Hundreds
of millions of people worldwide are affected by neurological disorders. Neurodegenerative
diseases are incurable and progressive resulting from continuous degeneration
and death of nerve cells in different parts of brain. Neurodegenerative
diseases
are
diseases of the central and peripheral nervous system. These disorders include
epilepsy, Alzheimer disease, multiple sclerosis, Parkinson’s disease, and other
dementias, cerebrovascular diseases (stroke, migraine and other headache
disorders), neuro infections, brain tumours, traumatic disorders of the nervous
system such as brain trauma. On the other hand,
mental disorders are “psychiatric illnesses” or diseases which appear
primarily as abnormalities of thought, feeling or behaviour, producing either
distress or impairment of function.
Neurodegenerative diseases
8-NitrodG further reacts with
peroxynitrite to give additional 8-oxodG further react to give plethora of
products. The consequences of these modified
products are different. 8-Oxodg, Iz and Oz are known to cause G T
mutation (Duarte V et al., 1999) with Oz also as a partially blocking lesion (Henderson et al., 2002). 8-NitrodG reacts further with
peroxynitriteor depurinates (Lee et al.,
2002), leaving a potentially mutagenic abasic site (Tretyakova
et al., 2000) while NitroIm induces G T and G C mutations (Niles et al., 2000).
(Source: Biochemistry Peroxynitrite-induced
reaction of synthetic oligo 2′-deoxynucleotides and DNA containing guanine:
formation and stability of a 5-guanidino-4-nitroimidazole lesion by Gu F, Stillwell WG, Wishnok JS, Shallop AJ, Jones RA, Tannenbaum SR; 2002)
Fig.D.
Schematic pathway of product formed Via. The reaction of peroxynitrite and
deoxyguanosine
The
reaction of ONOO_
with dG and 8-oxodG produces a large number of
different base lesions in DNA, the chemistry and proportions of which are strongly
dependent on the CO2 concentration. The reaction with dG (Fig. D)
produces several primary products including 8-nitro-20-deoxyguanosine (8-nitrodG), which rapidly depurinates to 8-nitroG and
an abasic site; 8-oxo-7,8-dihydro-20-deoxyguanosine (8- oxodG); 5-guanidino-4-nitroimidazole (NitroIm);
and 2,2-diamino-4-(2-deoxy-b-D-erythro-pentofuranosyl)
amino-5(2H)-oxazolone (oxazolone; Oz) (Niles et al., 2000: Lee et al.,2002)
The oxidative and nitrative chemistry associated with reactive
nitrogen species is mediated primarily by ONOO_ with additional contributions from NO2_ (Shafirovich et al., 2001). Peroxynitrite is capable of oxidizing and nitrate
almost all the biomolecules like nucleic acid, lipids, proteins etc. DNA is the
one of the sensitive biotarget for peroxynitrite and DNA modifications may be
responsible for a number of pathological conditions and disease (Gu F et al., 2002). The spectrum of DNA damage
by peroxynitrite is much more complex then damage induced by nitric oxide. Peroxynitrite induces both sugar-phosphate backbone damage and
base damage (Tretyakova et
al., 2000). Direct
DNA modification by NO_ is
governed by three chemical processes, deamination, oxidation, and nitration (Gal
et al., 1996). In
addition to DNA strand breaks, ONOO_ reacts primarily with dG in DNA to form a
variety of modifications including 8-nitrodG and 8-oxodG. 8-oxodG was at least 1000-fold more
reactive than the parent dG (Uppu et
al., 1996), because
oxidation of 8-oxodG (E0 ¼ 0:74V vs. NHE) is thermodynamically more favourable than oxidation of
dG (E0 ¼ 1:29V vs. NHE). This observation led us to hypothesize that 8-oxodG in
DNA may be a target for ONOO_, and that the products of this reaction could provide further
insights into the mechanism of ONOO_-induced mutations (Dedon et al.,
2004).
Peroxynitrite mediated DNA oxidation/nitration
Elevated level of ROS or RNS or both in
neurons along with malfunctioning antioxidant defence system causes neuronal
loss which ultimately leads to various neurodegenerative diseases such as
Parkinson’s disease, Alzheimer’s disease, Amyotrophic lateral sclerosis,
Schizophrenia and list goes on.
Fig. C. Molecular targets of RNS. RNS
act on various components and shows its pleiotropic effect and its consequences
(Source: Antioxidants
and Redox signalling. Reactive Nitrogen Species: Molecular mechanism and
potential significance in health and disease by Martinez CM, Andriantsitohaina
R. 2009, 11: 670-689)
Free Radicals
Non-Radicals
Nitric Oxide (NO.)
Nitrogen dioxide (NO.2)
Nitrate (NO3)
Nitrous acid (HNO2)
Nitrosyl cation (NO+)
Nitroxyl anion (NO-)
Dinitrogen tetraoxide (N2O4)
Dinitroge trioxide (N2O3)
Peroxynitrite (ONOO-)
Peroxynitrous acid (ONOOH)
Peroxynitric acid (O2NOOH)
Nitronium (nitryl) cation (NO+2)
Alkyl peroxynitrites (ROONO)
Alkyl peroxynitrates (RO2ONO)
Nitryl Chloride (NO2Cl)
Peroxyacetyl nitrate (CH3(O)OONO2)
Table B. List of Reactive Nitrogen Species. RNS may be radical or non
radical.
Reactive nitrogen species (RNS) are various
nitric oxide derived compounds. RNS possess pleiotropic properties on cellular
targets, mainly DNA, lipids and proteins, after both posttranslational
modifications and interactions with reactive oxygen species (Fig. C). Elevated
level of RNS causes cell injury which leads to cell death by inducing
nitrosative stress.
Reactive Nitrogen species (RNS)
(Source: Experimental Neurobiology. The role of
oxidative stress in neurodegenerative diseases by Geon Ha Kim, Jieun E. Kim,
Sandy Jeong Rhie, Sujung Yoon; 2015)
Free Radicals
Non-Radical
Superoxide(O2.-)
Hydroxyl , OH.
Hypoperoxyl, HO2.
Carbonate, CO.-3
Peroxyl, RO2.
Alkoxyl, RO.
Carbon dioxide, CO2.-
Hydrogen peroxide, H2O2
Hypobromous acid, HOBr
Hypochlorous acid, HOCl
Organic Peroxides, ROOH
Peroxynitrite, ONOO-
Nitrosoperoxycarbonate, ONOOCO2-
Peroxomonocarbonate, HOOCO2-
Table A. List of Reactive oxygen Species. ROS may be radical
or non radical.
Oxygen radicals are involved in many
biochemical activities of cells such as signal transduction, gene transcription
and regulation of soluble guanylate cyclase activity. If the equilibrium between
pro-oxidant/antioxidant homeostasis get disturbed result in generation of ROS
and other oxygen related free radicals those are potentially toxic for cells
which leads to oxidative stress.
Human body produce oxygen free radicals
and other reactive oxygen species as by products through numerous physiological
and biochemical processes.
1.2.1. Reactive
Oxygen Species (ROS)
Free radicals are common outcome of
normal aerobic cellular metabolism (Uttara et
al., 2009).
Free radicals are molecules with
unpaired electron in their outermost orbit. They play very important role in origin
of life and biological evolution, leaving beneficial effects on the organisms (Gow and Ischiropoulos H, 2001).
Humans are constantly exposed to free radicals created by electromagnetic radiation
from the manmade environment such as pollutants and cigarette smoke. Natural
resources such as radon, cosmic radiation, as well as cellular metabolisms
(respiratory burst, enzyme reactions) also add free radicals to the
environment. The most common reported cellular
free radicals are hydroxyl (OH·), superoxide (O2–·) and nitric
monoxide (NO·). Even some other molecules like hydrogen peroxide (H2O2)
and peroxynitrite (ONOO–) are not free radicals; they are reported to generate
free radicals through various chemical reactions in many cases (Gilgun-Sherki et al., 2001). These free radicals have
been reported for their great contribution to neuronal loss in cerebral
ischemia, seizure disorders, schizophrenia, Parkinson’s disease and Alzheimer’s
disease.
1.2.
Oxidative
and nitrosative stress
Most of the detrimental effects of
NO have been associated to ONOO_, because this molecule is able to nitrate and oxidize
protein residues, DNA, and lipids, thereby affecting cellular homeostasis (Torreilles
et al., 1999). 3-nitrotyrosine is the
product of tyrosine nitration due to peroxynitrite has been found to be in
increased amount in tissues and has been considered as biomarker for
peroxynitrite induced damage. Like tyrosin nitration in protein, 8-nitroguanine
is considered as biomarker for peroxynitrite induced damage in DNA (Hiraku Y,
2010). Increased levels of protein nitration have been observed in cerebral
infarct zones and Lewy bodies as well as in lesions in multiple sclerosis,
Parkinson’s disease Alzheimer’s disease, Huntington’s disease, and amyotrophic
lateral sclerosis (Torreilles et al.,
1999).
(Source: Free radical Biology and Medicine. Oxidative
chemistry of nitric oxide: The roles of superoxides, peroxynitrite and carbon
dioxide by Squadrito GL and Pryor WA; 1998).
NO found to play important role as
neurotransmitter as we as signalling molecule. NO
formation is highest in the brain and also a major factor contributing to the
loss of neurons in ischemic stroke, demyelinating diseases, and other
neurodegenerative disorders (Heales et
al., 1999). NO can regulate cellular and whole body activity
in a surprisingly versatile way. NO
itself is a free radical, but in combination with superoxide, it forms the very
reactive peroxynitrite anion (ONOO_) (Fig. B) (Meij et al., 2004). Ischiropoulos
et al. were among the first to examine the formation of peroxynitrite from
activated macrophages (Ischiropoulos, 1999). The
formation of peroxynitrite can be regulated by limiting the formation of
peroxynitrite from •NO by lowering the concentration of O2•- using SOD or SOD
mimics (Squadrito and Pryor, 1998).
Other source of NO formation is, in
the trophosphere by the action of lightning (Levine
et al., 1984)
and is found in large amounts in cigarette smoke––up to 800ppm which also
classed it as a toxin (Vleeming et
al., 2002). In contrast, it can also be formed in
the soil by nitrifying bacteria and removed by denitrifying bacteria which form
nitrous oxide from it (Conrad, 1996). So
nitric oxide, along with other oxides of nitrogen, are constantly being
released into the air from the soil as part of cycle involving plants and bacteria,
both of which are capable of producing NO (Bruckdorfer R, 2005).
(Source: Molecular Aspects of
Medicine. The basic bout Nitric oxide by Bruckdorfer R; 2005)
Fig. A. Biosynthesis of nitric
oxide from L-arginine
Nitric
oxide was characterised in early 1980s by
Furchgott, Ignarro and others (Ignarro
et al., 1987a: Ignarro LJ et al., 1987b: Martin W et al.,1985).Nitric oxide is a gas and a free radical which is now recognised to
have very important physiological roles. It is synthesised enzymatically from
the amino acid L-arginine in a number of tissues using the three isoforms of
nitric oxide synthase (Bruckdorfer
R, 2005) (Fig.A).
1.1.
Nitric oxide and
peroxynitrite
