Molecular Pathogenesis and Neuroinflammation in Parkinson Disease

beyond Alfa-synuclein: A Current Overview

Authors

Marcos Altable, M.D.¹*, Alfonso Cruzado, MSc2

1. Private Practice of Neurology. Neuroceuta. (Virgen de Africa Clinic). Ceuta, Spain

2. Psychologist at Ability. Ceuta. Spain

Abstract

Various mechanisms play an important role in the pathogenesis of Parkinson's disease,

including a disruption of the cellular energy balance (mitochondrial dysfunction) and

oxidative stress, as well as a disruption of protein breakdown (lysosomal and proteasomal

dysfunction). The protein α-synuclein plays a central role in the pathogenesis of

Parkinson's disease and is involved in many intracellular functions or their disruption in

Parkinson's disease (Lashuel et al. 2013). Several important cellular processes are

inhibited by aggregated α-synuclein. The disruption of these processes in turn leads to an

increased aggregation of α-synuclein. Protein misfolding, aggregation, and accumulation

of aggregated α-synuclein is a key feature of Parkinson's disease. In the course of the

disease there is an inflammatory reaction in the brain (neuroinflammation), in which

mainly microglial cells are involved. The exact triggers of neuroinflammation are not

known. However, it is known that aggregated α-synuclein and neuromelanin can activate

microglia (Wilms et al. 2009). During neuroinflammation, there is a change in the

expression of toll-like receptors (TLR) on the microglial cells (Beraud and Maguire-Zeiss

2012). Hence, in Parkinson's disease, both the aggregation of proteins, primarily α-

synuclein, and one that occurs during the disease play a role neuroinflammation with

microglial activation in the brain of Parkinson's patients play a role in the pathogenesis

of the disease.

Parkinson's Disease

Parkinson's disease is the second most common neurodegenerative disease and the most

common hypokinetic motor disorder worldwide (Hughes et al. 1992). Parkinson's disease

was named after the English doctor James Parkinson (1755-1824), who first described

the disease in 1817 in his monograph "Essay on the Shaking Palsy". The slow

development of the clinical picture and unspecific early symptoms make an early

diagnosis difficult.

A relatively reliable clinical diagnosis can be made according to the UK Brainbank

Criteria if there is slow movement up to immobility (bradykinesia or akinesia) and at least

one other of the following symptoms: muscle stiffness (rigidity), a resting tremor with a

frequency of 4-6 Hz or postural instability (Hughes et al. 1992, Fahn 2003).

The muscle tremors (tremor) are a core symptom, but not found in all patients. In addition,

there are often several vegetative and psychological symptoms. Over 40% of patients

suffer from depression and constipation.

The disease is sporadic in more than 85% of Parkinson's patients and the cause of the

disease is not yet fully understood. In these cases, however, a genetic complexity due to

the interaction of several genes and environmental factors is suspected. In less than 15%

of patients, the disease is of monogenic origin, i.e., caused by a change in a gene and,

according to Mendel's inheritance rules, is usually inherited as an autosomal dominant or

an autosomal recessive trait (Bekris et al. 2010). Parkinson's disease predominantly

occurs in old age. The age of onset of the disease is usually between the ages of 50 and
70, with 1.4% of the 55-year-olds and 3.4% of the 75-year-old population being affected

(de Rijk et al. 1997).

Neuropathological and neurophysiological characteristics

Pathophysiologically, Parkinson's disease is characterized by an initially largely selective

loss of dopaminergic neurons in the substantia nigra (SN) (Forno 1996). Later other brain

areas are also affected by the disease. The loss of dopaminergic neurons causes a reduced

dopamine concentration at the striatal dopamine receptors. Only when about 60-70% of

all dopaminergic cells in the SN have perished do clinical symptoms of Parkinson's

disease appear. At this point in time, the striatal dopamine content has already been

reduced by almost 80% (Becker et al. 2002). Pathognomonic for Parkinson's disease are

cytoplasmic inclusions in the nerve cells that are still alive, the so-called Lewy bodies

(Duffy and Tennyson 1965). These are characterized by an eosinophilic nucleus and a

pale halo (“halo”) under the light microscope and consist of various proteins, among

which α-synuclein, ubiquitin, neurofilaments and heat shock proteins are present in

particularly high concentrations (Spillantini et al. 1997).

Oxidative and nitrosative stress

Oxygen is essential for human life and the life of all aerobic organisms. Almost all the

molecular oxygen is in the respiratory chain converted into energy in the mitochondria

(DiMauro and Schon 2003). This also creates reactive oxygen species (ROS) as a by-

product. Reactive oxygen species are toxic forms of oxygen that include the superoxide

radical (O2 · -), the highly reactive hydroxyl radical (HO ·), and more stable oxidants

such as hydrogen peroxide (H2O2). Oxidative stress is an imbalance between the

formation and breakdown of ROS. This imbalance leads to the oxidation of lipids and

proteins as well as the degradation of the DNA (Trachootham et al. 2008). The healthy
body has anti-oxidative systems such as glutathione peroxidases (GPx) and superoxide

dismutases (SOD). These form an anti-oxidative system for the breakdown of ROS and

thus reduce the oxidative modification of biomolecules (Dringen 2000).

Nitrosative stress is a special form of oxidative stress and describes the conversion of

nitrogen compounds and reactive oxygen compounds to reactive nitrogen-oxygen

compounds (reactive nitrogen species, RNS). The reactive nitrogen species include the

free radical nitrogen monoxide (NO), but above all its secondary product peroxynitrite.

Peroxinitrite is produced by the reaction of superoxide (O2-) and nitrogen monoxide and

is, due to its high redox potential, much more aggressive than its precursor molecules.

Like ROS, RNA proteins can also modify the mitochondrial respiratory chain and damage

DNA (Gille and Sigler 1995, Halliwell 2007).

Increased oxidative stress plays a role in the pathogenesis of various neurodegenerative

diseases, but especially in Parkinson's disease. The expression of glutathione peroxidases

is reduced in the SN of Parkinson's patients and an increased oxidative modification of

lipids and proteins could be shown (Sian et al. 1994, Pearce et al. 1997). In the brain of

Parkinson's patients there is an immune reaction with activation of microglial cells.

Microglial cells are part of the innate immune system in the central nervous system

(CNS), the activation of which leads to the overexpression of the inducible NO synthase

(iNOS). The activity of the iNOS leads to the oxidation of L-arginine on a nitrogen atom,

because of which large amounts of nitric oxide are synthesized (Wiesinger 2001). In

addition to the inducible NOS in microglial cells, other isoforms of this enzyme are

known. These are located on the inside of endothelial cells (eNOS) or act in the neurons

in the CNS (nNOS). All three isoforms are always present in the body. However, iNOS

is extremely highly expressed in inflammatory reactions and is involved in the unspecific,

innate defense reaction. The expression of iNOS is induced, among other things, by
bacterial products (LPS) (Arizono et al. 1995) and by the proinflammatory cytokines

(TNF-α, INF-γ, IL-1β). The activity of the iNOS is also determined by the substrate

available (L-arginine) (Marletta et al. 1998).

Protein quality control in eukaryotic cells

The following describes the importance of protein quality control in the pathogenesis of

Parkinson's disease. Protein quality control is a cellular protective mechanism of

eukaryotic cells. Proteins are made up of linear polypeptide chains of amino acids. The

cellular function of proteins is essentially defined by their secondary and tertiary

structure, which in turn is created by folding the linear polypeptide chains into three-

dimensional structures. Protein folding is a complex process that is highly prone to error

and can lead to the formation of misfolded proteins. Cytoplasmic α-synuclein sometimes

does not have a strictly defined fold and is therefore referred to as a “native unfolded

protein”. The oligomerization of several α-synuclein molecules results in oligomeric

dysfunctional protein complexes (Breydo et al. 2012). The oligomers are intermediate

products and can then grow into fibrils (Rochet and Lansbury 2000). Natively unfolded

proteins are particularly predisposed to aggregation for reasons discussed in Section 1.2.

Causes of the aggregation of proteins are e.g., a changed primary structure, but also

oxidative and nitrosative stress (Jahn and Radford 2008). Changes in the primary

structure are triggered by mutations, RNA modifications or translational errors (Kopito

2000). The cell reacts to misfolded proteins by activating the protein quality control

system. The protein quality control system can distinguish correctly folded from

incorrectly folded proteins and degrade the latter (Jahn and Radford 2008). The protein

quality control system includes different groups of proteins, such as those of the group of

chaperones which also include members of heat shock protein family (Hsp70 / Hsp60).

Incorrectly folded proteins can be broken down via the ubiquitin proteasome system
(UPS) or by lysosomal breakdown, which ultimately also represents the end of autophagy,

with the help of which larger protein aggregates can also be broken down.

The Ubiquitin Proteasome System (UPS)

As part of the degradation of misfolded proteins by the ubiquitin proteasome system

(UPS), the misfolded protein is ubiquitinylated and then bound by the proteasome.

Ubiquitinylation is essential for proteosomal degradation and is mediated by three

different enzymes: the ubiquitin-activating enzyme (E1), the ubiquitin-conjugating

enzyme (E2) and the ubiquitin protein ligase (E3). Ubiquitin monomers are activated by

E1 in an ATP-dependent reaction and transferred to E2. Finally, the activated ubiquitin is

transferred to the target protein by the E3 ligase (Pickart and Eddins 2004).

Native proteins can be distinguished from misfolded proteins by means of

ubiquitinylation. The proteasome binds ubiquitinylated proteins and degrades them

through ATP-dependent proteolysis. The ubiquitin molecules are recycled and reused in

further degradation processes. A disturbance in this system leads to cell death due to

protein accumulations in the cell (McNaught et al. 2003). In Parkinson's disease,

inhibition of the UPS leads to an accumulation of incorrectly folded α-synuclein and

ubiquitin-containing inclusion bodies, as well as to the loss of dopaminergic neurons

(Rideout et al. 2004). Disturbances of the UPS promote the formation of α-synuclein

aggregates, which play an important role in various neurodegenerative diseases (Recchia

et al. 2004).

Autophagy and lysosomal protein degradation

Autophagy was first described by Christian De Duve in 1963 as a cellular process in

which a vesicle with a double membrane, the autophagosome, supplies cytoplasmic

components for degradation and recycling to the lysosomes (De Duve 1963). Autophagy
can be divided into three main processes: macroautophagy, microautophagy and

chaperone-mediated autophagy (CMA).

Macroautophagy is the main breakdown pathway of large protein aggregates and

damaged organelles. This also breaks down α-synuclein aggregates (Banerjee et al. 2010).

Microautophagy involves pinocytosis of the smallest amounts of fluid and the substances

dissolved therein. In chaperone-mediated autophagy, a chaperone protein specifically

binds proteins to be broken down and promotes their translocation through the

membranes of the lysosomes. Most of the α-synuclein monomers are broken down in this

way (Vogiatzi et al. 2008).

Protein aggregation and proteasomal malfunction

The appearance of protein aggregates and thus a disruption of protein homeostasis is

characteristic of Parkinson's disease. The end products of the aggregation process are

probably not toxic, but oligomeric intermediates that occur as precursors of the so-called

mature aggregates and are in dynamic equilibrium with these and with the α-synuclein

(Rochet and Lansbury 2000). In Parkinson's disease, the mature α-synuclein aggregates

represent a major component of the cytosolic Lewy bodies. Numerous proteins

aggregated in Lewy bodies are oxidatively or nitrosatively modified (Schulz-Schaeffer

2010). The breakdown of intracellular proteins by the UPS and the lysosome is a complex

and strictly regulated process that is essential for the cell. By inhibiting the ubiquitin C-

terminal hydroxylase (UCH), the formation of Lewy bodies could be induced

experimentally (McNaught and Olanow 2006).

Alfa-synuclein

α-Synuclein belongs to the synuclein family, which in humans comprises the three

isoforms α-, β- and γ-synuclein. The name was given due to the localization in the vicinity
of the synapses and the nucleus of neurons (Maroteaux et al. 1988), but it was later shown

that physiologically α-synuclein is essentially found in the presynaptic terminus in

neurons (Jakes et al. 1994). The human form of the α-synuclein is encoded by the SNCA

gene, which is located on chromosome 4q21. It consists of seven exons, five of which are

translated (Spillantini et al. 1995, Davidson et al. 1998, Mizuno et al. 2001). The

canonical isoform of the protein consists of 140 amino acids and has a molecular mass of

approximately 14 kDa (George 2002). The amino acid sequence can be divided into three

different sections. The amphipathic N-terminus of the α-synuclein extends from amino

acid 7 to 87. It contains seven not completely identical repetitions of a motif consisting

of eleven amino acid residues with the sequence XKTKEGVXXXX (Bisaglia et al. 2009).

This sequence forms an alpha-helix structure, especially when it binds to membranes tur

(Clayton and George 1998). The middle part of the α-synuclein is characterized by

hydrophobic side chains of the amino acids and contains the non-amyloid-β component

(NAC) domain (position 61-95), as well as the so-called GAV motif (position 68-76)

within the NAC domain. The NAC domain and in particular the GAV motif are essential

for the aggregation and formation of protofibrils of the α-synuclein (el-Agnaf and Irvine

2002, Du et al. 2003). The protofibrils, which form a β-sheet structure, are initially still

water-soluble. In the further course, mature α-synuclein fibrils develop. These are

insoluble in water and resistant to proteasomal degradation (Goedert et al. 1998, Serpell

et al. 2000, Conway et al. 2001). The mature α-synuclein fibrils show essential features

of a pathological protein structure, the so-called amyloid. The core characteristics of the

amyloid are antiparallel, often repeated β-sheet structures. Amyloidogenic misfolding can

have a pathogenic effect not only on α-synuclein, but also on numerous other proteins. In

the field of neurodegenerative diseases, this includes the amyloid precursor protein

(APP), which plays an important role in the development of Alzheimer's disease. The
third region of the α-synuclein is a strongly negatively charged C-terminus, which mainly

consists of the acidic aminoacids glutamic and aspartic acid. The C-terminal region of the

protein defines the solubility and has some phosphorylation sites (George 2002, Recchia

et al. 2004, Bisaglia et al. 2009). Deletion of the C-terminal region of α-synuclein reduces

its solubility and increases the tendency to aggregate.

α-Synuclein is a so-called native unfolded protein, i.e., it does not have a defined

secondary structure in the cytoplasm of the cell. Natively unfolded proteins often show a

tendency to aggregate spontaneously. The increased aggregation of α-synuclein is, in

addition to the loss of neurons, a main pathological characteristic of Parkinson's disease.

The aggregation of α-synuclein takes place via neurotoxic oligomeric intermediate stages

and can lead to neurodegeneration (Uversky and Eliezer 2009). Aggregated α-synuclein

is also the main component of the so-called Lewy bodies, intraneuronal protein deposits,

which are the main pathological characteristic of Parkinson's disease (Houlden and

Singleton 2012).

In addition to the reduced breakdown of α-synuclein through inhibition of the UPS

system, point mutations of the SNCA gene can also be the sole cause of the disease and

cause an increased aggregation of α-synuclein (Ikebe et al. 1995). The Park1 and Park4

mutations in the SNCA gene lead to forms of familial Parkinson's disease (Krüger et al.

2007). Five point mutations in the SNCA gene are already known. First, the point

mutation A53T, which shows an exchange from alanine to threonine at position 53, was

identified in an Italian and a Greek family (Polymeropoulos et al. 1997). The second

mutation A30P was detected in a German family. In this case, alanine is exchanged for

proline at position 30 (Kruger et al. 1998). The third mutation E46K with an exchange of

glutamic acid to lysine at position 46 could be detected in a Spanish family (Bisaglia et

al. 2009, Gasser 2009). Two more familial mutations (G51D and H50Q) have recently
been identified. The H50Q mutation, where histidine is exchanged for glutamine at

position 50, is associated with a late onset of Parkinson's disease (Khalaf et al. 2014),

whereas the G51D mutation with an exchange of glycine to aspartic acid at position 51

with an early onset of the disease is associated (Lesage et al. 2013). Since duplications

and triplications of the SNCA gene - referred to as PARK4 - also cause monogenic

Parkinson's disease, it becomes clear that even a relatively low overexpression of α-

synuclein in the range of 150 - 200% of the normal expression level can cause Parkinson's

disease (Farrer et al. 2004).

The isoforms of α-synuclein

Alternative splicing of the SNCA gene generates at least four alternative isoforms of α-

synuclein. The main isoform consists of 140 amino acids. The three other isoforms with

126, 112 or 98 amino acids are missing exon 3 (aa 40-55), exon 5 (aa 103-131) or exon 3

and exon 5 (Beyer 2006) (Fig. 3). Since these exons are in the protein-coding region of

SNCA, the absence of one or more exons leads to a loss of structure information. The

third exon contains, for example, parts of the information for the N-terminal sequence

segment of the α-synuclein and three of the five known Parkinson's-causing missense

mutations. This section also forms the α-helices through which the protein binds to the

membrane (Bisaglia et al. 2006). The C-terminus, mostly coded by exon 5, on the other

hand, has anti-aggregatory properties (Beyer 2006).

Proneuroinflammatory, resident cells of the CNS

Reactive microglia were detected early in the SN of Parkinson's patients. McGeer

emphasizes the presence of reactive microglia in Parkinson's disease 1988 and sees

reactive microglia as an important part of the pathomechanism of Parkinson's disease,

which is correlated with the intensity of the disease process (McGeer et al. 1988).
Dopaminergic neurons of the SN are particularly sensitive to oxidative stress and

inflammatory processes. Glial cells, which normally play neuroprotective roles, can

contribute to harmful, chronic inflammation after stimulation (McGeer and McGeer

2008). This shows that not only cells of the immune system actively participate in the

inflammation, but that cells of the CNS also play a fundamental role in this process.

Neurons and astrocytes, but especially microglia, can produce a broad spectrum of

immunologically relevant and inflammatory molecules (McGeer and McGeer 2001,

Chavarria and Alcocer-Varela 2004).

The relationship between α-synuclein and microglia in Parkinson's disease

In many neurodegenerative diseases, the α-synuclein-mediated activation of the microglia

leads to a disruption of neuronal homeostasis and thus to the development of pathological

conditions (Su et al. 2008). The activation of the microglia includes changing the shape

from ramified to amoeboid (Sanchez-Lopez et al. 2014). More recent studies in the

murine model also show that overexpression of α-synuclein leads to increased microglial

activation and, subsequently, to increased death of nerve cells (Beraud et al. 2011).

Furthermore, several in vitro studies have focused on the effect of α-synuclein on primary

microglia. These have shown that extracellular α-synuclein causes proinflammatory

factors, including TNF-α (Couch et al. 2011) and iNOS (Lee et al. 2009). Other working

groups have investigated the effect of oligomeric or aggregated / fibrillar α-synuclein on

primary microglia and the same increase in TNF-α (Beraud et al. 2011) and oxidative /

reactive stress (Zhang et al. 2005, Thomas et al. 2007).

The glial cells of the central nervous system are divided into two main groups. These are

the group of macroglial cells with astrocytes and oligodendrocytes, as well as the group

of microglial cells (Benveniste 1997). Microglial cells are the resident macrophages of

the CNS and are considered the antigen presenting cell type. Microglial cells react very
sensitively to the smallest changes in the physiological environment in their environment

with the expression of certain surface molecules and morphological changes (Kreutzberg

1996). This process is known as microglia activation. There are three main stages of

microglial cells: (I) the dormant, ramified microglial cells, as they occur in the normal

mature CNS, (II) the activated, non-phagocytotic microglial cells, as they occur in areas

of incipient CNS inflammation and (III) the reactive, phagocytic microglial cells that

occur during infections and other immune processes (Minagar et al. 2002).

During damage to the tissue of the CNS through inflammation, microglial cells control

and regulate the condition of their environment (Neumann 2001). To do this, they need

receptors and ion channels through which they can detect the appearance, unusual

concentrations or changed forms of factors and thus register damage or disturbances. The

interaction between the individual cells is essentially based on the signaling pathways of

cytokines and chemokines (Hanisch 2002).

Inflammatory messenger substances - cytokines and chemokines

A wide range of cytokines are released during inflammation. These include interleukins,

TNF-α and interferons (INF). Activated microglial cells present the foreign antigen in

conjugation with class II MHC molecules to the CD4 + T helper cells. These are activated

and secrete cytokines like INF-γ, which in turn cause further activation of the microglial

cells. The TNF-α and nitrogen oxide they then produce can lead to oligodendrocyte

damage here (Kreutzberg 1996).

α-synuclein and toll-like receptors

In the last years ago, i.e., during the preparation of this work, it was shown that α-

synuclein can induce microglia-mediated neuroinflammation, which accompanies the

development of Parkinson's disease and possibly contributes to the progression of the

disease. The microglial cells are stimulated directly by α-synuclein in a classic activation

path that includes changes in the expression of the toll-like receptors.

α-Synuclein is released by neurons or presynaptic ends (e.g., activity-dependent release

or because of sustained neurodegeneration) and leads to the activation of microglia, which

is often associated with a change in the morphology of highly branched cells to rounded

cells with less branching. This α-synuclein-mediated activation of the microglia leads to

an increase in the expression of various toll-like receptors (Parks et al. 2007). The increase

in TLR expression influences downstream signaling pathways, which among other things

leads to translocation of the transcription factor NF-κB into the nucleus (Kobayashi et al.

1993). The translocated NF-κB finally induces the expression of various proinflammatory

molecules, which then have a neurodegenerative effect on dopaminergic neurons.

Microglia react continuously to their microenvironment and can be specifically activated

by pattern-recognizing receptors (PRRs), which are specific for pathogen-associated

molecular structures (PAMPs) (Block et al. 2007). These receptors are located on

microglial membranes and in intracellular compartments. The toll-like receptor family is

one of them (Beraud et al. 2011).

The immune response in the central nervous system

Cells of the innate immune system, such as microglial cells, recognize pathogen-

associated molecular structures of pathogens (pathogen-associated molecular patterns,

PAMPs) (Medzhitov and Janeway 1997). PAMPs are, for example, molecules such as

lipopolysaccharides (LPS), peptidoglycans (PGN) or double-stranded RNA, which occur

in many classes of pathogens and are important for the pathogen. These structures have

several things in common: (I) they are only formed by microorganisms and not by the

host itself, (II) they are essential for the pathogenicity of the microorganism and (III) they

occur in many pathogens in a group (Medzhitov and Janeway 2000). PAMPs are
recognized by pattern recognition receptors (PRRs) on the surface of cells of the innate

immune system. In contrast, B and T lymphocytes play an important role in adaptive

immunity. Each unimprinted B or T cell only carries receptors of a single, narrowly

defined specificity, which are determined by a unique genetic mechanism during the

development of the cell.

Pattern Recognizing Receptors (PRRs)

PRRs are expressed both intracellularly and on the surface of various cells. They can also

be secreted in blood or tissue fluids. Neutrophils and eosinophils, killer cells,

macrophages and dendritic cells have PRRs. They are involved in various activities such

as opsonization, the activation of pro-inflammatory signaling pathways and the induction

of apoptosis (Medzhitov and Janeway 2000).

The secreted PRRs include the LPS-binding protein (LBP). LBP is involved in the

detection of bacterial LPS by forming a complex with LPS, which is then transferred to

the LPS receptor protein CD14 (Medzhitov and Janeway 2000). CD14 is a glycoprotein

and belongs to the group of PRRs that occur on the cell surface. It gets on

myelomonocytic cells are expressed where it is bound to the cell surface via a

phosphoinositol glycolopid tail (Kaisho and Akira 2002).

Toll-like receptors (TLR)

One group of the PRRs are the toll-like receptors. Due to their immune-stimulating and

immunomodulating properties, they play a central role in the innate immune response

(Medzhitov et al. 1997). Toll genes were first discovered in Drosophila melanogaster in

connection with embryonic development (Anderson et al. 1985).

Currently 13 members (TLR1-13) have been identified and characterized (Tabeta et al.

2004). There are differences between the various receptors, both in their structural design
and in their affinity for certain ligands. For example, peptidoglycan, as a component in

the cell wall of bacteria, binds to TLR2 as an agonist (Takeuchi et al. 1999). Double-

stranded viral RNA interacts with TLR3 (Sarkar et al. 2003), using polyinosinic-

polycytidylic acid (poly (I: C)) as a synthetic analogue to double-stranded rRNA serves

(Alexopoulou et al. 2001). TLR4, on the other hand, shows an affinity for

lipopolysaccharides (LPS), which occur in the cell wall of Gram-negative bacteria

(Tapping et al. 2000). Toll-like receptors are transmembrane proteins that have a TIR

domain (Toll / IL-1R domain) in their cytoplasmic portion and a leucine-rich region

(leucine-rich repeat, LRR) in the extracellular domain (Medzhitov et al. 1997). Proteins

with an LRR domain are involved in numerous important biological processes. A major

function appears to be the mediation of protein-protein interactions, such as the

recognition and binding of ligands, and signal transmission (Kobe and Kajava 2001). A

glycoprotein with an LRR domain is, for example, the LPS receptor cCD14 / CD14,

which occurs freely in the plasma or on the surface of myelomonocytic cells and, together

with TLR4, plays a central role in the detection of LPS gram-negative bacteria (Takeuchi

and Akira 2001). Regarding the α-synuclein-induced cytotoxicity, Stefanova and

colleagues were able to show that a lack of TLR4 in a transgenic mouse model, which

overexpresses human α-synuclein in the oligodendrocytes, leads to increased dopamine-

dependent neurodegeneration. The observed increase in neuronal cell death was

associated with an increased α-synuclein content, a reduced phagocytic ability and an

increased TNF-α-mediated, pro-inflammatory reaction. On the one hand, this indicates a

TLR4-mediated, phagocytotic uptake of α-synuclein, but a reduction in the TLR4 content

could not completely eliminate the inflammatory reaction, which points to a parallel,

TLR4-independent mechanism (Stefanova et al. 2011).

Pathogen-Associated Molecular Structures (PAMPs)

Pathogen-associated molecular patterns are highly conserved structural motifs in

evolution. With their help, cells of the innate immune system can recognize the invasion

of pathogens and activate defense mechanisms. PAMPs of a certain group of

microorganisms are very similar to each other, so

E.g., all gram-negative bacteria have LPS in their outer cell wall and this can be

recognized by the biological structure of the lipid A part. Therefore, through innate

immunity, it is possible to recognize and repel a large number of pathogens with a

relatively small number of germline-encoded receptors (Dudley 1992).

Lipopolysaccharides (LPS)

LPS are contained in the outer membrane of gram-negative bacteria. The cell envelope

has a multilayer structure. In addition to a thin layer of murein, it has an outer membrane,

in the outer half of which lamellae LPS occurs as an integral component. LPS are

glycolipids with three sub-areas. Lipid A forms the inner area of LPS, of which lipid A

represents the biologically active part and is also responsible for anchoring it in the

membrane. The core region, which consists of a heterooligosaccharide, is bound to lipid

A. Different serotypes can differ in their core components. The third, outer area connects

to the core region. This part, which consists of polysaccharides, consists of 1-8 different

saccharide residues, which are strung together in different order. This polysaccharide is

called O-specific polysaccharide because this part defines the O-antigen of the bacterium

(Rietschel et al. 1991).

As an endotoxin, LPS is a potent antigen and can trigger an inflammation reaction within

a short time. When macrophages are stimulated with LPS, they produce cytokines such

as TNF-α, IL-1, IL-6, IL-8 and IL-10. LPS occurring free in the blood is immediately
bound by the LPS-binding protein and transported to the surface receptor CD14 or its

soluble form sCD14. TLR4 and the secreted MD-2 protein bound to the extracellular

domain of TLR4 binds the LPS / LBP / CD14 complex. CD14 and MD-2 have no

transmembrane domains, so that signal transduction only takes place over the complete

complex with TLR4 (Guha and Mackman 2001, Akira and Hemmi 2003).

Peptidoglycan (PGN)

The cell envelope of gram-positive and gram-negative bacteria is essentially composed

of peptidoglycan (PGN) or murein. In gram-positive bacteria there is a multilayered

peptidoglycan layer, whereas in gram-negative bacteria there is only a single-layer

peptidoglycan layer between the cell membrane and the outer membrane. The PGN of the

bacterial cell wall is made up of two glycosidically linked strands of N-acetylglucosamine

and N-acetylmuramic acid. The sugar chains are also cross-linked with oligopeptides and

the resulting network is responsible for the shape and stability of the bacterial cell. In

addition to the number of peptidoglycan layers, gram-positive and gram-negative bacteria

also differ in their oligopeptides. The PGN gram-negative bacteria consists of muramyl

tripeptides and the PGN of gram-positive bacteria consists of muramyl dipeptides.

Stimulation with PGN induces an increase in TNF-α in macrophages, mediated via the

TLR2 receptor (Philpott and Girardin 2004).

Polyinosinic-polycytidylic acid (Poly (I: C))

Poly (I: C) is structurally identical to double-stranded viral RNA, which functions as a

viral PAMP and is produced in the course of a viral infection. TLR3 and the adapter

molecule TIR-domain-containing adapter-inducing interferon-β (TRIF) recognize viral

double-stranded RNA or poly (I: C) in the endolysosome (Kato et al. 2005). The double-

stranded RNA or poly (I: C) then binds to the N-terminal and the C-terminal ends of the
LRR region. Activation of the TLR leads to the release of proinflammatory cytokines

such as TNF-α or IL-6 in macrophages (Kumar et al. 2006).

Transcription Factors

Transcription factors are constitutionally active or inducible DNA binding proteins that

are involved in the control of transcription. These can bind directly to so-called promoter

regions of the DNA, whereby the binding of the transcription machinery and thus the

transcription of the DNA into mRNA is made possible. Transcription factors usually have

at least three domains. The first domain acts as a recognition area, which is used to bind

the active ligands. Another domain is used for binding to the promoter region of the genes,

whose transcription by the respective factor is activated and at least one other domain is

used for stabilization. After activation by ligand binding, usually in the cytoplasm, they

translocate into the cell nucleus to bind to the associated promoter region of the DNA and

to induce the transcription of specific target genes (Schulze-Luehrmann and Ghosh 2006).

Extracellular α-synuclein protofibrils mediate the induction of various signaling

pathways, including NF-κB in vitro, and mediate microglial activation and subsequent

cell death in a rat model in vivo. The results of this study support the model that

protofibrils represent the responsible element for neuronal cell death (Manning-Bog et al.

2003).

The nuclear factor kappa B (NF-κB)

The nuclear factor kappa B (NF-κB) was first described by Sen and Baltimore in 1986 a,

where it was initially identified as an activator of the immunoglobulin κ light chain gene

(Sen and Baltimore 1986) b. A short time later it turned out that NF-κB functions as a

transcription factor ubiquitous in the organism, which plays an important role in

inflammatory processes. It serves as a regulator that can be activated quickly in the

context of various biological defense processes (Barnes 1997). Rosenstiel and colleagues

were able to show that various signaling pathways, such as that of NF-κB, are activated

in microglial cells by extracellular, protofibrillary α-synuclein. NF-κB also regulates the

expression of various proinflammatory cytokine genes such as TNF-α, IL-1β and IL-6.

Thus, α-synuclein can be seen as an important mediator of microglial activation, with the

DNA binding activity of NF-κB appearing to function as an essential key element

(Rosenstiel et al. 2001, Wilms et al. 2009). The genes regulated by NF-κB thus play an

important role in controlling the immune response and are involved in the regulation of

cell proliferation and apoptosis (Barkett and Gilmore 1999, Tak and Firestein 2001). In

most cells, NF-κB is present in the cytoplasm as a complex with inhibitory IκB proteins,

as a result of which NF-κB is kept in an inactive state. Only the cleavage of IκB finally

enables the translocation of NF-κB into the cell nucleus. The nuclear translocation of NF-

κB can therefore be used as a surrogate marker for NF-κB activity for most cell lines.

Only in antibody-producing B cells, activated T cells and macrophages is NF-κB

basically in its activated form in the cell nucleus (Sen and Baltimore 1986a, Baeuerle and

Baltimore 1988a, Baeuerle and Baltimore 1988b).

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