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Dopaminergic Cell Loss and Alpha- expression in TLR4-deficient Mice

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Published: 8th Feb 2020 in Biology

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Role of Misfolded α-synuclein and Toll-like Receptors 4in Mediating Dopaminergic Cell Loss and Alpha-Synuclein Expression in the Acute MPTP Mouse Model of Parkinson’s Disease



Many neurodegenerative disorders, such as Parkinson’s disease (PD), Alzheimer’s disease (AD), and Huntington disease (HD) are characterized by a neuronal loss in different regions of the central nervous system. Parkinson’s disease (PD) is distinguished by the progressive loss of dopaminergic neurons of the substantia nigra pars compacta (SNpc) of the brain, accompanied by accumulation of proteinaceous inclusions of α-synuclein (Lewy bodies)- oxidatively modified molecules, neurites, neuroinflammation, and activated microglia. There is growing evidence that misfolded α-synuclein directly activates microglia, promoting inflammation, oxidative stress and altering the expression of Toll-like receptors (TLRs). The Toll-like receptors play a prominent role in the regulation of host innate immune system by detecting and responding to pathogens. TLRs are pattern recognition receptors that recognize damage-associated molecular patterns and pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide (LPS) from a gram-negative bacterium. Activation of TLR4 pathways may result in proinflammatory pathway initiation; where activated microglia contribute to oxidative stress through the release of molecules such as cytokines, nitric oxide, and other reactive oxygen species, which adversely impact adjacent neurons. A significant goal of Parkinson’s disease research is the development of disease-modifying drugs that slows or stops the neurodegenerative process. Current medications for PDs are limited and focuses only on treating the symptoms. Drugs that enhance the intracerebral dopamine concentrations or stimulate dopamine receptors remains the centerpiece treatment for PD. This study aimed to show that microglia are directly activated by α-synuclein in a classical activation pathway that includes alterations in the expression of toll-like receptors.  Also, define the role of TLR in mediating dopaminergic cell in the substantia nigra (SN) in a transgenic mouse model. Our results should provide a novel insight into the mechanisms of α-synuclein-induced microglial activation which will aid in further improving the therapeutic drugs.


Keywords: Neurodegenerative disorders, Microglia, misfolded α-synuclein, Toll-like Receptors, Inflammation, Oxidative stress, Dopaminergic neurons, Substantia nigra.



To determine the role of misfolded α-synuclein and TLR4 in mediating dopaminergic cell loss that will ultimately allow us to develop a therapeutic drug that will specifically target TLR4 to help delay or prevent degeneration of dopaminergic (DA) neurons in the substantia nigra (SN) in Parkinson disease patients. By conducting further researches on Toll-like receptors, microglia, and α-synuclein, it will be possible to prevent microglial activation that eventually alters toll-like receptors, causing the death of DA neurons. The Toll-like receptors play a prominent role in the regulation of host innate immune system by detecting and responding to pathogens via pathogen-associated molecular patterns PAMPs, but its role in neurodegeneration is still unknown. Understanding both the cellular and molecular biology of TLRs in the healthy and dead nervous system may lead to novel approaches for preventing neuronal degeneration and promoting recovery of function in an array of neurodegenerative conditions (Owens, 2009).

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 In this paper, we will investigate the dopaminergic cell loss and alpha- expression in TLR4-deficient mice (TLR4−/−), acutely exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a PD animal model (Mariucci et al., 2018). Recent studies demonstrated that TLR-4 plays an initiating role in Parkinson’s disease pathogenesis by inducing inflammation in cells to control infections (Mariucci et al., 2018). The expression of TLR4 increased in blood and brain in PD patients, suggesting clinical relevance to TLR4 in PD (Drouin Ouellet et al.2015). Fellner et al. showed that α-SYN-dependent microglial activation required TLR4, phagocytic activity, and release of pro-inflammatory cytokine and ROS (Fellner et al.2013). Moreover, in another study, it was found that TLR4 mediated cell death in an MPTP model of PD (Noelker et al. 2013) and its upregulation in microglia is a natural mechanism to boost the clearance of extracellular AS in ASPs (Stefanova et al. 2011). We recently found that TLR4 contributes to MPTP-induced nigral dopamine loss and influences the MPTP-induced biochemical changes in a brain region-specific manner (Conte et al.2017). Hence, in this study, by using a well-characterized acute MPTP mouse model of PD, we will further investigate the contribution of TLR4 to the brain dopaminergic cell loss and analyze for the first time the α-SYN expression in WT and TLR4−/−mice.



Parkinson’s disease (PD) is the second most common age-related neurodegenerative disorder after Alzheimer’s disease (AD). Characterized by slow and progressive degeneration of dopaminergic neurons (DA) in the substantia nigra pars compacta (SNpc) due to the accumulation of proteinaceous inclusions of α-synuclein (Lewy bodies) (Sohn et al., 2014). DA neurons are associated with motor functions, and the loss of these neurons in PD patients could lead to symptoms such as tremor, bradykinesia, rigidity, and akinesia (Miller et al., 2009). Microglia are highly conserved and serve immune-like functions in the brain, continuously monitoring and reacting to their microenvironment recognizing and responding to foreign material. Engagement of microglia with foreign substances results in activation mediated by pattern recognition receptors (PRRs) found on the cell surface as well as on endosomal membranes (Béraud et al. 2012). Studies suggest that the pathological role of α-synuclein is linked to this ability to misfold and self-assemble into higher-order structures. In cell culture models, α-synuclein-induced cell death has been associated with the formation of oligomeric α-synuclein, increased cell membrane conductance, mitochondrial, lysosomal and proteasomal dysfunction, and microglial activation (Béraud et al. 2012). Recent studies have shown that neuroinflammation plays a critical role in the progression of neurodegenerative disease. The presence of activated T-lymphocytes and microglia proximal to the SN has been observed postmortem in PD patients (González, 2014). These cell populations are believed to contribute to the process of neurodegeneration and the release of pro-inflammatory and cytotoxic factors, including interleukin-1β, tumor necrosis factor-α, nitric oxide, and reactive oxygen intermediates (González, 2014), all of which are highly neurotoxic when released in high doses by activated microglia (González, 2014).  Several studies have shown that stimulation of toll-like receptors (TLRs) evoke microglial activation through the aggregated proteins in the central nervous system (CNS) of individuals with neurodegenerative diseases, as well as in animal models (González, 2014).

Neuroinflammation of the central nervous system (CNS), is recognized as a prominent hallmark of many pathological conditions (Glass et al., 2010). Inflammation is a host defense mechanism against invading pathogens, by engulfing the pathogen and triggering tissue repair. Lipopolysaccharide (LPS), is a principal constituent of the gram-negative bacterial cell wall. It is a potent inducer of immune responses such as the release of proinflammatory mediators, including cytokines, nitric oxide (NO), and reactive oxygen species (ROS) (Panaro et al., 2008). LPS effects are mediated through the interactions with several receptors such as the Toll-like receptors for microbial products. Toll-like receptors (TLRs) are a group of transmembrane proteins that function as pattern-recognition receptors (PRR) for detecting and responding to microbial ligands denominated pathogen-associated molecular patterns (PAMPs), present on bacteria, and bacterial products (Panaro et al., 2008). There are at least 13 mammalian TLRs which are integral membrane proteins with a leucine-rich extracellular domain, and a cytoplasmic domain like that of the interleukin-1 receptor which initiates downstream signaling through kinases to activate transcription factors such as AP-1 and NFκB (Lucas et al., 2008). TLRs are activated in glial cells such as the microglia, astrocytes, and oligodendrocytes and lymphocytes that invade the nervous system in response to inflammation caused by infectious agents, tissue injury or autoimmune conditions. By inducing the production of pro-inflammatory cytokines and cell adhesion molecules in immune cells, TLRs may indirectly damage neurons in conditions such as ischemic stroke and multiple sclerosis (Lucas et al., 2008). TLRs are involved in the development and homeostasis of the nervous system, and notably in several neurodegenerative diseases. The most notable TLRs are TLR2 and TLR4, which are involved in neuronal apoptosis (Béraud et al., 2011). LPS is a potent stimulator of both peripheral immune cells and CNS glial cells and does not have a direct effect on neurons, probably because of lack of functional TLR4 expression (Kielian, 2006).

Experimental design and Methods


For the study, we will use flow cytometry and western blots to detect toll-like receptor four expressions in blood and brain samples of Parkinson’s disease patients and mice overexpressing human α-synuclein. For the experiment, 6-8 weeks old TLR4- deficient male mice will be used. TLR4-/- and WT mice will each receive four injections of MPTP-HCl at the two-hour interval. The mice will be sacrificed by cervical dislocation seven days later after MPTP intoxication. The brains from WT and TLR4-/- will be collected to isolate a cerebellum, hippocampus, striatum, and midbrain. The parts of the brain will be then stored at −80 °C until further processing. To asses differentially expressed proteins in the different parts of the brain, immunohistochemistry (IHC) process will be used. Tyrosine hydroxylase immunoreactivity is used as a phenotypic marker for dopaminergic neurons.

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 Western blot analysis will be utilized to determine the level of nigra TH and α-SYN protein from WT and TLR4−/− mice. Each brain sections will be deparaffinized by xylene and hydrated through graded alcohols. After each incubation, the tissues will be pelleted for 4 minutes. Then they will be weighed and resuspended in a 20mH Tris-HCl, pH 8.8, 2% SDS, 200 mM DTT extraction buffer. The proteins from the pellets will then resolved on a 10% SDS-PAGE for TH, GATA, and 15% for α-SYN and blotted onto nitrocellulose sheets. The blots will be incubated overnight at 4°C with rabbit monoclonal anti-TH antibodies. The membranes will be washed and then exposed to anti-mouse secondary antibodies conjugated to horseradish peroxidase and visualized with enhanced chemiluminescence. The blots will be analyzed using ImageJ software.

 Pool RNA from the cerebral cortex, striatum, hippocampus, and cerebellum according to the manufacturer’s protocol. Real-time PCR will be performed in a Light Cycler instrument using a Fast Start DNA SYBR Green Master kit. For α-SYN gene amplification, a forward and reverse primers will be used. PCR amplification will be carried out in 96-well plate in a 25μ l reaction volume containing 12.5μ l of 2× FastStart DNA SYBR Green Master Mix, 1μ l of each forward and reverse primer (400 NM), 8.5μ l water PCR grade and 2μl of cDNA. Each sample had three replicates, and the thermal profile was at 95 °C for 10 min, followed by 45 cycles of denaturation for 10 s at 95 °C, a 20-s annealing step at 60 °C, and 1 min extension at 72 °C. The results will be analyzed by LDCA software supplied with the machine, and the fluorescence signal was detected at the end of each cycle. Melting curve analysis will be used to confirm the specificity of the products.

The behavioral experimenter will be conducted to test the locomotor disability of mice. Mice were tested in a pole test and an open field test. In the first paradigm, we will evaluate the ability of each mouse to climb down a vertical wooden pole with a rough surface, 1 cm wide and 50 cm high, as a measure of bradykinesia, balance, and coordination. Each mouse will be placed at the top of the pole, and the time for turning downwards and the total time for climbing down the pole until the mouse reached the floor with the four paws will be taken in five trials. The best performance of each mouse will be used for the statistical analysis. In the second paradigm, spontaneous locomotor activity within a 15-minute interval in an open field arena will be measured by Flex Field Activity System (San Diego Instruments, San Diego, CA), which allows monitoring and real-time counting of horizontal and vertical locomotor activity by 544 photo-beam channels. Mice will be placed in the center of the open field and always tested at the same time of the day. The tests will be conducted in a dark room that is completely isolated from external noises and light during the test period

Expected Results and possible interpretations


Our results should show elevated levels of TH immunoreactivity in WT animals than in TLR4 -/- littermates. TH protein levels will be further determined using western blot. The absence of TLR 4 should modify the MPTP-induced dopaminergic neuronal loss. Western blot analysis of α-SYN protein content in the midbrain region of the mice should exhibit elevated levels in TLR4 -/- mice than the WT group. MPTP injection should not induce any significant changes in both the genotypes even if increased expression of the protein is observed in TLR4 -/- mice. Results from real-time PCR should show the mRNA expression levels of α-SYN. TLR4 -/- mice should show higher α-SYN mRNA levels when compared to WT group after MPTP treatment. The behavioral test of the mice should show if the lack of TLR4 -/- has any effect on the motor disability. The upregulation of TLRs has been demonstrated in PD and all α-synucleinopathies. The TLR4 signaling pathway is known for its crucial role in different aspects of neuropathology that include PD and other neurodegenerative disorders.

Our results should show that when α-SYN along with MPTP will cause the brain to undergo some characteristic changes. Our results should confirm that TLR4 influences MPTP- induced nigral degeneration through TH expression level, the critical enzyme protein in dopamine biosynthesis pathway of dopaminergic neurons and their terminals. TLR4 expression affects the MPTP-induced biochemical changes in a region-specific brain. Although the precise nature of the pathogenic α-SYN species is still under debate, the intravesical portion seems highly prone to aggregation than the cytosolic form, and the aggregates are secreted from the cells (Trotta et al. 2014 ). The brain accumulation of α-SYN may lead to microglial activation, inflammation, and ultimately to neurodegeneration by the toxic effect on dopamine neurons (Zhang et al.2005). In this scenario, TLR4 is required for α-SYN-dependent microglial and astroglial activation resulting in the production and release of proinflammatory cytokines (Fellner et al.2013). The investigation of precise underlying mechanisms of α-SYN accumulation in PD will be useful to reveal potential targets for therapeutic modulation and deserve to be further explored.




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