validation
PhD Program Director
Prof. Stefano Del Prato
Tutor:
Prof. Anna Solini
PhD Candidate:
Dr.ssa Chiara Rossi
1 Introduction 4
2 Materials and Methods 12
2.1 Human studies . . . 12
2.1.1 Participants . . . 12
2.1.2 Biochemistry . . . 13
2.1.3 Mononuclear cells isolation . . . 13
2.1.4 Cytokine levels . . . 15
2.2 Animal studies . . . 17
2.2.1 WT and P2X7 receptor (P2X7R) KO mice . . . 17
2.2.2 Biochemical parameters . . . 18
2.2.3 Behavioural test . . . 18
2.2.4 Anatomy . . . 22
2.2.5 Substantia Nigra analysis . . . 22
2.3 Molecular and cellular Biology . . . 24
2.3.1 RNA . . . 24 2.3.2 Quantitative real-time PCR . . . 25 2.3.3 Circulating miRNAs . . . 27 2.3.4 Western Blot . . . 29 2.4 Imaging . . . 33 I
2.4.1 Immunofluorescence . . . 33
2.4.2 Quantitative analysis of immunofluorescence images 34 2.5 Statistical analysis . . . 36
3 Results 37 3.1 Human studies . . . 37
3.2 Animal Studies . . . 46
3.2.1 Metabolic overview . . . 46
3.2.2 Substantia Nigra analysis . . . 48
4 Conclusions 53 5 Future perspectives 60 5.1 Hippocampus analysis . . . 60 5.1.1 Behavioural test . . . 62 5.1.2 Molecular analysis . . . 65 Bibliography 70
Background
Neuro, and likely systemic inflammation, with abnormal α-synuclein deposi-tion, play a key role in the development of Parkinson disease (PD). The P2X7 receptor/NLRP3 inflammasome complex is upregulated in the brain of PD patients. Aim of this study was to explore whether the systemic activation of such complex might participate in the pathogenesis of PD.
Subjects and Methods
Twenty-five newly diagnosed, treatment-naive PD referring in the years 2015-2016 to the Centre for PD, Neurology Unit, University Hospital in Pisa, Italy were enrolled and compared with 25 healthy controls. Expression and functional activity of the P2X7 receptor-NLRP3 inflammasome was measured in circulating lymphomonocytes, relating it to circulating levels of 1β, IL-18 and α-synuclein. The intracellular signalling involved in such activation (by measuring ERK 1/2, p38 MAPK, JNK and their relative phosphorylated isoforms) and its epigenetic regulation (by measuring circulating miRNA7 and miRNA30, likely involved in the pathogenesis of PD) was also evaluated. A putative mechanistic explanation of results obtained in humans was explored in a murine model of neuroinflammation induced in WT and P2X7 receptor KO mice, where we quantified NLRP3, Parkin and α-synuclein gene and
protein expression by realtime PCR and immunostaining at the level of the substantia nigra.
Findings
PD displayed an almost two-fold higher P2X7R gene expression; accord-ingly NLRP3, and Caspase-1 were more expressed in PD. These data were corroborated by those obtained in immunohistochemistry, with P2X7R and NLRP3 proteins abundantly represented in lymphomonocytes from PD pa-tients; the activation of the P2X7R-NLRP3 inflammasome, however, does not translate in different plasma levels of IL-1β and IL-18. To gain insights of the mechanism linking P2X7R to PD, we explored α-synuclein protein expression in lymphomonocytes and, as a putative negative control, in red blood cells, also measuring its circulating levels. PD, but not CTL lym-phomonocytes, were strongly enriched by α-synuclein, while erythrocytes displayed the same amount of α-synuclein in the two groups. In face of that, circulating α-synuclein levels did not differ between CTL and PD. The lym-phomonocyte expression of total and phosphorylated isoforms of extracellular signal-regulated kinases (ERK 1/2), p38 MAP kinase and c-Jun N-terminal kinase (JNK) revealed that JNK phosphorylation was reduced by approxi-mately 60% in PD (p=0.03). Searching for a putative epigenetic regulation of such complex scenario, we also measured miR-7 and miR-30, involved in the post-transcriptional control of α-synuclein and NLRP3 expression. Cir-culating levels of these miRNA were both increased in PD patients vs CTL. To better explore such complex system in the brain, we treated WT and P2X7R KO mice with high fat diet (HFD), a stimulus able to induce an inflammatory damage at the level of substantia nigra. We measured gene and protein expression of NLRP3, α-synuclein and parkin (protective toward
the synaptic dysfunction related to the early symptom of PD). HFD induced in both strains a slightly expression of α-synuclein in the substantia nigra. Parkin expression did not vary in WT animals treated with HFD, while it was strongly increased in P2X7R KO mice, while NLRP3 inflammasome followed exactly an opposite trend, being upregulated in the substantia nigra of WT animals treated with HFD. In WT animals, in the same brain area, P2X7R and NLRP3 proteins were upregulated, reinforcing the hypothesis of their participation in the neuroinflammatory process.
Interpretation
Newly-diagnosed PD subjects display a systemic hyper-expression of the P2X7R-NLRP3 inflammasome platform that seems to modulate circulating and lymphomonocyte α-synuclein; a reduced JNK phosphorylation might be the intracellular signalling mediating this effect, undergoing an epigenetic regulation by miR-7 and miR-30. In P2X7R KO mice, a neuroinflammatory stimulus induces a strong expression of parkin, a protective protein, suggest-ing that a P2X7R activation might participate in the inflammatory damage occurring in specific brain areas also by inhibiting parkin expression.
Introduction
Neuroinflammation
Neuroinflammation is a common pathological treat of neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), amy-otrophic lateral sclerosis (ALS) and dementia, occurring primarily in the later stage of life; in these disorders memory, learning, coordination, and mobil-ity are progressively lost and their pathophysiological hallmarks are protein aggregates within neurons (i.e. neurofibrillary tangles in AD e Lewy bodies in PD), and out of neurons (i.e. extracellular amyloid deposits in AD and PD with dementia). Neurodegeneration, by definition, is an impairment of neuron structure or survival, caused mainly by cell-autonomous processes. In the last years, a body of evidence indicates that also non-cell-autonomous processes take part in these dysfunctions, and the aforementioned mechanism might be promoted by central nervous system inflammation [1]. After all, in AD and PD it was demonstrated that glial activation and pro-inflammatory cytokines production are not only secondary to neuronal insult, but they also contribute to the pathogenesis of the disease [2, 3].
In CNS, IL-1β and IL-18 are the main pro-inflammatory cytokines con-4
tributing to neuronal injury and cell death [4, 5], but they are also involved in important physiological functions, as cognition, learning, and memory [6]. Their production can be tightly controlled, and this process is under control of inflammasomes [7].
The fundamental structure of these cytosolic multiprotein complexes is formed by a “sensor”, and an “effector” constituted by an enzymatic com-ponent (caspase-1), frequently joined by an adaptor molecule (ASC) (Fig. 1.1).
Figure 1.1
The “sensors” are different kinds of receptor, belongings to PRRs (Pat-tern Recognition Receptors), and they dictate the inflammasome assembly in response to danger signals.
groups: “nucleotide-binding domain-like receptors” family (NLRs), generi-cally activated by pathogassociated molecular pattern (PAMPs) or en-dogenous danger signals in the cytosol (DAMPs); ALRs family, constituted by AIM2 “absent in melanoma 2-like receptos”, the only one binding cytoso-lic double-stranded DNA; and the recently identified pyrin, that induces a pro-inflammatory form of cell death, so-called pyroptosis.
In contrast to other inflammasome receptors, NRLP3 requires a two-steps activation (Fig. 1.2).
A first signal (priming signal) through PRRs receptors (Toll-like and C-type lectin receptors) operates on NF-kB transcription factor, that promotes transcription of all proteic elements (pro-IL-1β, pro-IL-18 and NRLP3) re-quired for the complex formation. A second signal activates this complex, by facilitating the oligomerization of inactive NRLP3 with ASC and pro-caspase-1: the activated caspase-1 is now able to cleave pro-IL-1β and pro-IL-18 in their mature forms, which can be released. While PAMPs derive from micro-organisms infection (virus, fungi and bacteria), DAMPs encompass molecules normally present inside the cells, released in response to invasion of microor-ganism (septic inflammation) or to metabolic stress (sterile inflammation) [8]. Among DAMPs, extracellular ATP and other nucleotides play an undis-puted role in inflammasome activation: for this reason purinergic receptors, chiefly P2X7 receptor (P2X7R), have been extensively studied.
P2X7R belongs to the family of ionotropic ATP-gated P2X receptors: its activation within milliseconds results in a reversible plasma membrane per-meability to small cations (Na, K, Ca); a repeated or prolonged activation (within seconds), instead, induces the opening of a larger pore, which allows big molecules permeation (up to 900 Da)[9]. For these atypical pharmaco-logical and structural features, P2X7R is also one of the most controversial purinergic receptor; the pore forming related to P2X7R activation is, so far, a debated issue, with two alternative hypothesis: a progressive dilation of the P2X7R-gated channel, or the recruitment of additional pore-forming proteins (Panx1)[10].
P2X7R, as mentioned above, is extensively studied for its involvement in many inflammatory conditions depending from an inflammasome com-plex [11, 12]. However, it is now accepted that P2X7R also plays a pivotal role in central nervous system (CNS) diseases. It is expressed in several
brain cell types (neurons, astrocytes, microglia), and it is upregulated in var-ious pathological conditions: it may affect neuronal cell death by regulating the processing and release of IL-1β, a key mediator in neurodegeneration, chronic inflammation, and chronic pain; activation of this receptor provides an inflammatory stimulus, and P2X7R-deficient mice display a marked at-tenuation of inflammatory responses, including models of neuropathic and chronic inflammatory pain [13]. For these reasons, P2X7R antagonists are driving an increasing attention in the treatment of human neurodegenerative diseases; in this context, P2X7R has been recently quantified in the brain of Parkinson’s patients by whole-body PET/CT [14].
Stress signals, such as metabolic alterations, mechanical injuries, and bacterial/chemical toxins elicit the P2X7R activation in neurons and neigh-bouring cells (Fig. 1.3). The metabolic pathways involved in response to P2X7R activation are various [10]:
- excitotoxicity induced by glutamate release from neurons and astrocytes - neuroinflammation produced by IL-1β and other cytokines release
- enhance reactive oxygen species (ROS) production that aggravates protein misfolding and neuronal damage
- direct or indirect cell death
- downregulation of brain-derived neurotrophic factor (BDNF) and relative neuroplasticity.
These key mechanisms could contribute to neuronal disorders with differ-ent etiology, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS) and psychiatric disorders.
Obesity and inflammation
As known, obesity is a risk factor for multiple pathological conditions (T2D, hypertension, stroke ..) and many types of cancer [15]; even more, in the last decade, clinical studies related obesity and high fat diet consumption to cognitive functions decline [16] and dementias [17]. The connection between obesity and low-grade inflammation in peripheral tissues has been well es-tablished since many years, but when De Souza and colleagues [18] in 2005 demonstrated for the first time that high fat diet in rats resulted in brain inflammation, it was hypothesized that systemic and central inflammation induced by obesity may converge towards cognitive dysfunction [19]. Addi-tionally, growing evidence indicate that neurodegenerative disease, as AD and
PD, are linked to perturbation of energy metabolism [20]: oxidative stress, mitochondrial dysfunction and impaired glucose uptake may render neurons vulnerable to excitotoxicity and apoptosis. Recently, metabolic syndrome, common in patients with obesity, has been associated to neurodegenerative diseases [21]: a longitudinal population study performed in South Korea in-dicated that incidence and risk of PD increased gradually with the number of metabolic syndrome components, suggesting that neurodegenerative disease and metabolic abnormalities could share pathophysiological mechanisms.
Many experiments in animal models clarified the mechanism underpinning this relation: rodents fed with high fat diet showing memory and learning failure, presented a reducted synaptic plasticity in the hippocampus [22, 23] and an increased neuronal apoptosis in hippocampus and hypothalamus [24, 25].
The present study has been designed to address the role of the systemic P2X7R-inflammasome complex in neurodegenerative dis-eases, and in particular its translational validation in Parkinson’s Disease.
Parkinson disease (PD) is a chronic degenerative disease characterized by a progressive loss of dopaminergic neurons in the substantia nigra (SN). The reduction of striatal dopamine accounts for classical motor symptoms, as bradykinesia, rigidity, and tremor; but, during the evolution of the disease, PD patients also show olfactory disfunction and sleep disorders, arising from later non-dopaminergic neurons dysregulation [26]. Its pathophysiological mechanisms are still partially unknown; a main role seems to be played by chronic neuroinflammation [3], with an excess of microglia and astrocytes ac-tivation and increased expression of pro-inflammatory mediators like TNF-α, IL-1β, IL-6, and interferon-γ [27, 28] able to rapidly induce neuronal
degen-eration. Among these mediators, IL-1β appears of interest: it is abundant in the microglia surrounding Lewy bodies in experimental models and in PD patients [29]; however, it should be pointed out that, in PD, Lewy bodies are not limited to substantia nigra, being also located in other sites of central and peripheral nervous system. The first gene associated to PD was SNCA, encoding for α-synuclein (α-Syn), main component of Lewy bodies in several degenerative disorders [30]; such protein can also form aggregates able to damage pre-synaptic terminations and determining a sort of “synaptotoxic-ity” by interfering with mitochondrial or microtubular function and axonal protein transport [31]. Recently, a role of fibrillar α-Syn, through interaction with Toll-like receptor 2 (TLR2) has been reported [32, 33]. A few reports have addressed the possible involvement of the inflammasome in PD, just describing the protective effect of P2X7R blockers in murine models of the disease [34, 35] and in microglial cells, where NLRP3 is activated by α-Syn, triggering a neuroinflammation that contributes to degeneration of dopamin-ergic neurons [36, 37]. It is still unclear whether, in addition to the increased brain expression and function of the NLRP3 inflammasome platform, a sys-temic activation of such complex might participate in the pathogenesis of PD, which could be the role of the P2X7R at the onset of the disease, and whether such patterns undergo any specific epigenetic regulation.
To gain insight in the molecular mechanisms underpinning this scenario, we induce in a mouse model a chronic low grade inflammation by using a high fat diet, and we analyze the putative protective effects of P2X7R ablation in P2X7R KO mice.
Materials and Methods
2.1
Human studies
2.1.1
Participants
This part of my project was conducted in collaboration with the Unit of Neurology-Neurophysiopathology - AUOP (prof. R. Ceravolo). The study consecutively enrolled 25 newly diagnosed, treatment-naive PD individuals among those referring in the years 2015-2016 to the Centre for PD, Neu-rology Unit, University Hospital in Pisa, Italy. Inclusion criteria were onset of suggestive symptoms not later than 3 months, age<80 years, no previous specific treatment, no previous personal history of any neurological disease, including ischemic stroke, no anti-inflammatory drugs assumed in the three months preceding the enrolment. Diagnosis matched the UK Parkinson’s Disease Society Brain Bank [38], and was confirmed by nuclear magnetic res-onance (NMR). An age- and gender-matched control group (CTL, n = 25) was formed, on a volunteer basis, by the spouse of the probands partici-pating in the study. The protocol was approved by the Ethics committee of
the University of Pisa (#14282) and registered on ClinicalTrials.gov platform (NCT03918616); all participants signed an informed consent.
2.1.2
Biochemistry
The day of the study (T0) patients underwent a complete clinical evalua-tion and a blood routine analysis. The Unified Parkinson’s Disease rating scale was administered and the PD was staged according to the HoehnYahr scale [39]. Blood samples were collected from an antecubital vein to assess serum/plasma aliquots (frozen at -20◦C until required for quantitation) and circulating lymphomonocytes isolation. Biochemical parameters were mea-sured by standard methods in the biochemistry laboratory of the University Hospital in Pisa; the evaluated parameters were:
• fasting glucose • lipid profile • serum creatinine • uric acid.
2.1.3
Mononuclear cells isolation
Lymphomonocytes were isolated from fresh blood samples by density gradi-ent cgradi-entrifugation. The method is based on the use of Ficoll sodium dia-trizoate solution (Ficoll-Paque PLUS, GE Healthcare, Uppsala Sweden): it is an aqueous solution of density 1.077 ± 0.001 g/ml containing Ficoll 400, a synthetic polymer of sucrose, and sodium diatrizoate, that provides the optimal density and osmolarity necessary for the efficient removal of other cells from the lymphocytes. On centrifugation, cells in the blood sample sediment towards the blood/Ficoll-Paque PLUS interface: red blood cells
are efficiently aggregated by the agents present in Ficoll, increasing their rate of sedimentation, which rapidly collect as a pellet at the bottom of the tube; granulocytes also sediment to the bottom facilitated by an increase in their densities caused by the slightly hypertonic Ficoll-Paque PLUS medium. Thus, on completion of centrifugation, both granulocytes and red blood cells are found at the bottom of the tube, at the bottom of the density gradient.
Lymphocytes, monocytes, and platelets are not dense enough to pene-trate into the Ficoll-Paque PLUS layer. These cells therefore collect as a concentrated band at the interface between the original blood sample and the high density solution.
For lymphocyte separation, 12 ml anticoagulant-treated blood was diluted with an equal volume of balanced salt solution (D-PBS) and layered carefully over Ficoll-Paque PLUS (without intermixing) in a centrifuge tube. After a short centrifugation at room temperature (typically at 400 xg for 30–40 min) lymphocytes, together with monocytes and platelets, were harvested from the interface between the Ficoll-Paque PLUS and sample layers. This material was then centrifuged twice in D-PBS (at 100 xg for 10 min) to wash the lymphocytes and to remove the platelets. The lymphocytes were
immediately frozen for RNA/protein extraction (see section 2.3.1) or were plated on slides for immunofluorescence experiments (see section 2.4.1)
2.1.4
Cytokine levels
Plasma levels of IL-1β, IL-18 and α-synuclein were measured by high sen-sitivity Quantikine enzyme-linked immunosorbent assay (ELISA) Kits (R&D Systems, Inc, Minneapolis, MN, USA) following manufacturer’s instructions. This assay employs the quantitative sandwich enzyme immunoassay tech-nique. A monoclonal antibody specific for the human molecules above re-ported has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any specific molecule present is bound by the im-mobilized antibody. After washing away any unbound substances, an
HRP-labeled antibody (detection antibody) specific for each human molecule is added to the wells. Following a wash to remove any unbound antibody, a substrate solution (Tetramethylbenzidine, TMB) is added to the wells and color develops in proportion to the amount of analyte bound in the initial
step. The color development is stopped and the intensity of the color is measured at 450 nm. The sensitivity of the assay was <1 pg/ml−1, with an interassay variability of 4.5%.
2.2
Animal studies
2.2.1
WT and P2X7 receptor (P2X7R) KO mice
Eight WT (strain C57BL6J, ENVIGO, Udine, Italy) and eight P2X7R KO mice (Jackson Laboratory, through Charles River, Lecco, Italy), housed in a germ-free stabularium in accordance with the principles of Italian Minister for Health(protocol authorization number #943/2015-PR), were maintained under controlled ambient illumination on a 12 h light/dark cycle and free access to food. Each mouse strain was divided in two groups and treated for
16 weeks with a normal fat diet (NFD, a routine diet used in stabulation) and a high fat diet (HFD) (PF4215, Research Diets Mucedola, Settimo Milanese, Italy): the composition of this diet was characterized by 60% of total calories from fat, and it is already shown to be able to induce PD [40, 41].
2.2.2
Biochemical parameters
During the treatment with different diet, the following metabolic parameters were measured:
s weight s glycemia s triglycerides s cholesterol
s AST (Aspartate Aminotransferase).
While the weight was monitored weekly, the other parameters were evalu-ated before the treatment (basal) and after one, two, and four months. The quantitative determination of glucose, cholesterol and triglycerides was per-formed by a digital multimeter (multiCare-in): this instrument, requiring just one drop of blood (obtained from the mouse tail in non-invasive way), can be used to monitor more times the biochemical parameters of mice. The AST activity was measured by an enzymatic rate method in a Synchron Clinical System (Beckman Coulter).
2.2.3
Behavioural test
In behavioral neuroscience there are several test applied for studying the physiological processes and neural mechanism of spatial learning and mem-ory; they can be used to evaluate dysfunctional behaviour or cognitive im-pairment induced by an experimental treatment, as a High Fat Diet.
At the end of dietary treatment, six P2X7R KO and six WT mice carried out two behavioural test, allowing them to recover for two days between the different tasks.
Morris Water Maze
The Morris Water Maze (MWM) is designed to test spatial memory and long term memory by recording various parameters during the time that rodent spend in a water tank. The typical equipment consists of a circular pool (150 cm diameter) filled with opaque water at around 21◦C , a temperature sufficiently stressful to motivate the animals to escape but not excessively low to inhibit learning. Being used brown mice, non-toxic white paint was added into the water until it became opaque, and in the same time it allowed a good contrast for the images recording.
A 10 cm diameter platform was placed about 1.5 cm below the water surface and the water tank was surrounded by 3 visual cues useful to create a spatial map (the ambient around the pool was made neutral covering the objects present); the platform remained in a fixed location relative to the cues. The subjects were monitored by a video tracking system placed directly above the water tank and the following parameters were measured
using a Video Tracking Software (Panlab):
s Escape Latency (EL), time that animal needs to find the hidden platform s distance moved, length of distance travelled
s swim-speed
s time in quadrant, time that animal spends in each quadrant of the pool
The procedure consisted of five days trial, four training per day. In each trial the animal was placed into the water at different start positions, and it allowed to swim for 60 seconds: if the mouse did not find the platform in this time, it was placed manually on the platform; in any case it stayed there for 20 seconds before returning to its cage.
1 2 3 4 5 6 Days Esc ap e La tenc y (sec ) 0 40 20 60
Day 1 Day 3 Day 5
Training period Probe trial
Day 6
The animals quickly learned the correct location of hidden platform: dur-ing the five days of traindur-ing, the escape latencies decreased and the swim path became more direct (see “Day 5” in the figure above). In the last day (probe trial) the hidden platform was removed from the pool, and the animal
allowed to swim for 60 sec: typically, in a normal condition, mouse swim to the target quadrant of the pool and repeatedly across the former location of the platform, spending more time in this quadrant respect to the other.
Y maze
It is a spontaneous alternation behavioural test to measure spatial working memory. The test is executed in a compartment made of three arms arranged in Y-shape, each arm being 35 cm long and 7 cm wide. The test is based on rodent preference to explore new ambient to find an escape: over the course of multiple arm entries, the subject should show a tendency to enter a less recently visited arm.
Each mouse was placed in the center of the maze, and it was free to explore the arena for 8 min. The number of arm entries and the number of triads were recorded by a video tracking system: an entry occurred when all four limbs were within the arm. Video Tracking Software (Panlab) calculate the percentage of correct alternation.
2.2.4
Anatomy
At the end of the treatment period, mice were deeply anaesthetized with sevoflurane; brains were removed from the skull, and sagittally cut in two part. Half part was immediately frozen on dry ice for RNA isolation. The other half brain was fixed by immersion in 10% Neutral Buffered Formalin for 24 h at 4◦C : after the washing step in phosphate buffer (PBS), the brains were left overnight at 4◦C in a cryoprotection solution, containing 30% sucrose in PBS; the next day, they were frozen in H-OCT compound (Histo-Line Laboratories).
Solutions
10% Neutral Buffered Formalin (formaldehyde 4%)
formalin stock solution (formaldehyde 37-40%) 10%
NaH2PO4 30 mM
Na2HPO4 45 mM
PBS (0.01 M pH 7.4)
KCl 22.7 mM
NaCl 137 mM
2.2.5
Substantia Nigra analysis
Coronal brain sections cutted in the cryostat and mounted on non-charged slides were immediately observed with a microscope and sections containing
midbrain were established on the base of mouse brain atlas.
In the case of half brain frozen, the ventral midbrain was dissected out from each section using a needle and the tissue was used for the subsequent RNA extraction; in the case of half brain fixed, the sections containing SN were recovered in PBS for free-floating immunostaining.
Real Time PCR
NLRP3, Parkin and α-synuclein gene expression in murine SN were measured by real-time PCR (see section 2.3.1).
Free-floating immunostaining
TH, P2X7, NRLP3, Iba1, GFAP protein expression was evaluated by im-munofluorescence: coronal sections (50 µm thick) through the SN were cut on a cryostat and collected in PBS; after a post-fixation step (1h at room temperature) in PFA 4% and three washes with PBS, serial sections were treated with specific antibodies (see section 2.4.1).
2.3
Molecular and cellular Biology
2.3.1
RNA
Extraction
RNA extraction from human cells and animal tissues was performed with QIAcube (Qiagen, Hilden, Germany), a robotic workstation for automated purification of nucleic acids, using RNeasy mini kit (Qiagen) and following the manufacturing protocol. The starting material was:
- 6x50 µm brain sections or
- human lymphomonocytes pellets
they were lysed in 350 µl of RLT buffer with β-mercaptoethanol using the TissueLyser (Qiagen): in this instrument disruption and homogenization are achieved through the beating and grinding effect of steel beads on the sample, during a shacking step at 30 Hz for 2 min. After a centrifugation step to remove insoluble material, the supernatant was carefully transferred in new tube and loaded in QIAcube instrument.
Retrotranscription
After a spectrophotometric quantification (by NanoDrop 2000c, Thermo Fisher), the following amount of total RNA
- 250 ng from brain sections or
- 750 ng from lymphomonocytes pellets
were reverse transcripted (RT) to single-stranded cDNA by High Capacity cDNA Reverse Transcription kit (Applied Biosystems). The standard reaction
used and the relative thermal conditions are listed below:
Standard reaction
10X RT buffer 2 µl
25X dNTP mix 0.8 µl
MultiScribe Reverse Transcriptase 1 µl RNase Inhibitor 1 µl
RNA+H2O 13.2 µl
Thermal condition
2.3.2
Quantitative real-time PCR
Gene expression analysis was executed in real-time PCR using the TaqMan Assays (TermoFisher-LifeTechnologies): they consist of a target specific-probe flanked by a pair of PCR primers, and the specific-probe is conjugated with
a fluorescence-dye on the 5’-end and a quencher on the 3’-end. Triplicate reactions were prepared for each sample according to the TaqMan standard protocol (for the TaqMan assay codes view tab):
Standard reaction
2X iTaq Universal Probes Supermix (Bio-Rad) 10 µl 20X TaqMan Gene Expression Assays 1 µl
cDNA diluted 1:10 2 − 4 µl
H2O to 20 µl
Real Time PCR was performed on an Eco real time instrument (Illumina Inc., San Diego, CA, USA) following this thermal profile:
TaqMan⃝ assaysR
GENES human mouse
P2X7R Hs00175721_m1 NRLP3 Hs00918082_m1 Mm00840904_m1 Caspase1 Hs00354832_m1 NF-kB Hs00765730_m1 Parkin Mm01323528_m1 α-synuclein Mm01188700_m1 GAPDH Hs02758991_g1 Mm99999915_g1
Amplifications were normalized by housekeeping gene and quantitation of gene expression was performed using the ∆∆Ct calculation, where Ct is the threshold cycle. The amount of the target gene, normalized to GAPDH and relative to the calibrator (sample not treated or wt animal) is given as 2−∆∆Ct.
2.3.3
Circulating miRNAs
Circulating miRNA7 and miRNA30, likely involved in the pathogenesis of PD [36, 37] were isolated by the robotic workstation QIACUBE (Qiagen) loaded with miRNeasy Serum/Plasma Kit (cat. 217184, Qiagen, Hilden, Germany). After an equilibration period at room temperature, samples were centrifuged to remove cryoprecipitates, and 200 µl of thawed serum were processed following the manufacturer’s instructions. For each patient, the cDNA templates was assessed from 2 µl of sample eluent; we used the Taq-Man Advanced miRNA cDNA Synthesis kit (A28007, Applied Biosystems, Foster City, CA, USA), because it enables to analyse multiple miRNAs from a
single amplified sample, very useful with samples limited in quantity as serum. The complete procedure requires 5 steps (for details, refer to TaqMan⃝ Ad-R vanced miRNA Assays USER GUIDE); briefly, mature miRNAs from total RNA are modified by:
1. poly(A)-tail addition to the 3’-end of the mature transcript 2. adaptor ligation to the 5’-end of the mature transcript 3. universal reverse transcription of modified miRNAs
4. total amplification to increase uniformly the amount of cDNA for all miRNAs (miR-Amp reaction)
5. PCR amplification of the cDNA template for miR of interest
In our experimental protocol, miR7 and miR30 expression was measured by TaqMan Advanced MicroRNA Assays (cat. A25576, Applied Biosystems), following this reaction composition:
Standard reaction
2X TaqMan⃝ Fast Advanced Master MixR 10 µl 20X TaqMan⃝ Advanced miRNA AssayR 1 µl
miR-Amp diluted 1:10 3 − 5 µl
H2O to 20 µl
PCR reactions were run in triplicate using an Eco real time instrument (Illu-mina Inc., San Diego, CA, USA) according to the recommended procedure.
Thermal condition
miRNAs levels were expressed as 2−∆Ctusing two references miRNAs
(miR-484 and miR-191-5p) selected on the basis of the scientific literature and checking their low variability in our samples.
2.3.4
Western Blot
Total proteins were extracted from lymphomonocytes of a subset of PD (n=2) and CTL (n=3) individuals to measure total and phosphorylated ERK 1/2, p38 and JNK by Western Blot analysis. Each lymphocytes pellet was homogenated with 200 µl of Lysis Buffer (based on NP40 mix, Invitrogen), modified to protect the protein phosphate groups. After a centrifugation step at 14000 rpm 4◦C to precipitate cellular debris, the protein content of super-natant was evaluated by a colorimetric assay (Quick Start Bradford Protein Assay, BioRad). This method involves the binding of a dye (Coomassie
Bril-liant Blue G-250) to basic and aromatic amino acid residues: when the dye binds protein, it is converted to a stable blue form, detected at 595 nm with a spectrophotometer. The sample protein concentration was determined by interpolation of a standard curve, generated treating several dilution of a standard protein (in our case BSA) with the same colorimetric assay.
For the WB experiments, 20 µg of total protein were diluted in SDS-loading buffer and heated at 100◦C for 5 min. Samples were separated on precast gels (Any kD Mini-Protean TGX gels, Bio-Rad), run at voltage constant in a vertical electrophoresis apparatus, and subsequently transferred to a polyvinyl difluoride (PVDF) membrane (Millipore, Billerica, MA, USA) with a semi-dry blotting process (TransBlot Turbo, Bio-Rad).
After a treatment (1h at room temperature) with Blocking Solution to saturate aspecific binding sites, blots were incubated overnight at 4◦C with primary antibodies diluted 1:1000 (all of them Cell Signaling, see tab for cat-alog number). The next day, specific bands identification was performed by a chemiluminescence reaction: after 1h incubation with secondary antibody conjugated with HRP (Horseradish Peroxidase), the membrane was covered with a solution containing the chemiluminescent substrate (Clarity Western ECL, Bio-Rad); in the presence of H2O2, HRP catalyzes the oxidation of
luminol, which then generates light only where is the target protein. The lighted bands, detected by a digital camera (Versadoc instrument, Bio-Rad), were quantified: their optical density were evaluated by ImageJ (Software); they were normalized with GAPDH bands (mab374, Millipore)
Solutions
Lysis Buffer
NP40 Mix
Protease Inhibitor Cocktail (Sigma) 10% Sodium pyrophosphate 20 mM PMSF (phenylmethylsulfonyl fluoride) 1 mM
Pepstatin 5 µM
4X Laemmli Sample Buffer
Tris-HCl pH 6.8 277.8 mM
Glycerol (v/v) 44.4%
SDS (sodium dodecyl sulphate) 4.4%
Bromophenol Blue 0.02%
Running Buffer
Tris 25 mM
Glycine 192 mM
SDS (sodium dodecyl sulphate) (w/v) 0.1% pH 8.3
TBS
Tris 50 mM
Blocking Solution BSA 5% Triton X-100 0.3% in TBS Antibody Solution BSA 1% Triton X-100 0.3% in TBS Antibody 1:1000
Antibodies Cell Signaling codes
form total phosphorylated
ERK 1/2 9102 9101
p38 9212 9211
2.4
Imaging
2.4.1
Immunofluorescence
The immunofluorescence experiments in human lymphomonocytes (l) and mouse brain tissues (b) were performed following these general steps:
1. cells or tissues reacted 1-2h at room temperature with a blocking so-lution to saturate aspecific binding sites
2. samples were incubated overnight at 4◦C with specific 1◦ antibodies (see TAB for details)
3. after 3 washing steps in PBS, immunoreactivity was revealed using 2◦ antibodies conjugated with fluorescence molecules: depending of the 1◦ antibody host species, the 2◦ antibodies were against mouse or rabbit antigen (Alexa Fluor 594-goat@rabbit and 488-goat@mouse antibodies,Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA).
Details on Antibodies
host dilution tissues
P2X7R (APR-004 Alomone) rabbit 1:100 l NRLP3 (AG-20B-0014 Adipogene) mouse 1:200 l,b α-synuclein (ab212184 abcam) rabbit 1:100 l
GFAP (ab7260 abcam) rabbit 1:500 b
Iba1 (sc-32725 S.Cruz) mouse 1:100 b
For the immunofluorescence experiment in lymphomonocytes, 400x103 cells were plated on two-chamber slides and left to adhere for 1h in incubator; after a fixation step (10 min) in PFA 4% and 3 wash with PBS, the cells were reacted with the above-reported antibodies.
For the immunofluorescence experiment in mouse brain, coronal sections (50 µm thick) through the SN were cut on a cryostat and collected in PBS; after a post-fixation step (1h room temperature) in PFA 4% and 3 wash with PBS, serial sections (one out of three) were treated in “free-floating” way with the antibodies above reported.
2.4.2
Quantitative analysis of immunofluorescence
im-ages
The images used for the quantitative analysis of immunofluorescence were acquired with a Leica TCS SP8 confocal microscope. After a preliminary analysis of different samples to establish the best conditions for the instru-ment, the confocal setting was held constant within all experimental sessions. All image analyses were performed using ImageJ (public domain software de-veloped at the NIH).
Human Lymphomonocytes
For the human lymphomonocytes, stacks of optical sections were collected in three different fields for each patients, and 30 immuno-positive cells were assessed for the quantification. Each single cell area was traced (avoiding the background) and the mean value of signal intensity was calculated; one hundred immuno-positive cells were assessed for each experimental group.
Mouse brain
In the mouse brain, similar stacks of optical sections were acquired for each animal. Threshold area was established on the base of average background signal. Briefly, for each image transformed in grayscale, the grey value in 3
different not-signal area was measured and the average value multiply for an arbitrary number (in this case 3) was used to set the threshold; in the binary image obtained, the percentage area of positive pixel was calculated. In the case of not specific green signal in vessels, these parts were masked before calculating the threshold area.
2.5
Statistical analysis
Results are expressed as mean ± SD. Statistical analysis was carried out using one-way analysis of variance with post-hoc Bonferroni correction, the Kruskal-Wallis test for non-parametric data and paired t-test for comparison between groups. A value of p <0.05 was considered statistically significant.
Results
3.1
Human studies
Clinical characteristics of the participants are shown in Table below. Controls (CTL) and Parkinson disease patients (PD) resulted adequately matched for age gender and prevalence of chronic comorbidities.
PD (n=25) CTL (n=25) Age (yrs) 69.2±6.9 69.1±7.7 Sex (m/f) 14/11 10/15 Diabetes (n;%) 7;28 6;24 Hypertension (n;%) 15;60 13;52 Hypercholesterolemia 10;40 11;44 Fasting glucose (mg/dl) 92±10 == Total cholesterol (mg/dl) 189±12 == HDL-cholesterol (mg/dl) 52±5 == Triglycerides (mg/dl) 116±14 == Creatinine (mg/dl) 0.90±12 == 37
Figure 3.1 shows the expression of P2X7R and NLRP3 inflammasome components in lymphomonocytes of PD and CTL. PD displayed an almost two-fold higher P2X7R gene expression; accordingly NLRP3, and Caspase-1 were more expressed in PD. These data were corroborated by those obtained in immunohistochemistry: as shown in Figure 3.2, P2X7R and NLRP3 pro-teins were abundantly represented in lymphomonocytes from PD patients,and their expression in cells from CTL was negligible.
The activation of the P2X7R-NLRP3 inflammasome is expected to drive the release of IL-1β and IL-18, pro-inflammatory markers of PD [42]. There-fore, we measured plasma levels of these cytokines in CTL and PD patients, and we found that both were within the normal range, and no difference emerged between the two groups (Figure 3.3 a and b). Also, circulating α-synuclein (the main constitutive factor of plaques accumulating in the substantia nigra) did not differ between CTL and PD (Figure 3.3 c). How-ever, a weak direct correlation was found between P2X7R expression and IL-18 levels in the whole study population (R=0.279, p=0.05).
Activation of the P2X7R/NF-kB signalling stimulates the expression of the upstream IKK gene and the p65 subunit of NF-kB, both of which play a role in inflammatory responses and form an alternative pathway able to trigger IL-1β release; therefore, to confirm the P2X7R-NLRP3 axis as main pathway mediating this cytokine release, we also measured the expression of NF-kB. No difference emerged between PD and CTL in the expression of such gene (Figure 3.4)
To gain insights of the mechanism linking P2X7R to PD, we explored by immunofluorescence α-synuclein (the main constitutive factor of plaques accumulating in the substantia nigra) protein expression in lymphomonocytes from PD and CTL. Despite of no difference in free α-synuclein (Figure 3.3c),
Figure 3.1: P2X7 receptor, NLRP3 and Caspase-1 gene expression in lymphomonocytes
of twenty-five CTL (white plots) and twenty-five PD patients (gray plots). * p=0.013 for P2X7R; p=0.0086 for NLRP3; p=0.045 for Caspase-1.
Figure 3.2: P2X7R and NLRP3 protein expression in lymphomonocytes of CTL and PD
patients. A representative immunofluorescence image shows as both proteins are highly expressed, and colocalize, in PD lymphomonocytes, while they are virtually absent in CTL cells. Graphs show the quantification of immunoreactivity, expressed as mean±SD of all study subjects. *p < 0.005 vs CTL; §p < 0.01 vs CTL
Figure 3.3: Circulating IL-1β, IL-18 and α-synuclein levels in all study participants
Figure 3.4: NF-kB gene expression in lymphomonocytes of CTL (white plots) and PD
patients (grey plots). p=0.85
PD patients showed for this molecule a different pattern of expression in circulating cells: erythrocytes displayed a quite similar amount of α-synuclein in the two groups, while PD lymphomonocytes were strongly enriched by the protein, with a main localization in the perinuclear area (Figure 3.5a); α-synuclein accumulation was scarce in immune cells from CTL (Figure 3.5b). Interestingly, a significant direct correlation between P2X7R and α-synuclein levels was observed in PD subjects (R=0.400, p=0.043), but not in CTL (R=0.198, p=0.344).
We next tried to possibly identify the intracellular signalling supporting the activation of the P2X7R-inflammasome complex in lymphomonocytes, and to verify a possible epigenetic regulation of such pathway. To this aim, we compared the lymphomonocyte expression of total and phosphorylated isoforms of extracellular signal-regulated kinases (ERK 1/2), p38 MAP kinase and c-Jun N-terminal kinase (JNK). As shown in Figure 3.6, we found that JNK phosphorylation was reduced by approximately 60% in PD (p=0.03); a coherent trend was also observed for p38.
Figure 3.5: Panel a: Intracellular α-synuclein protein in erythrocytes and
lymphomono-cytes of CTL and PD patients. Upper panels: representative immunofluorescence image; lower panels: magnification of the areas of interest (white squares). Red signal: α-synuclein ; blue signal: DAPI staining for nuclei. In red blood cells (dashed white arrows), the immunoreactivity for α-synuclein protein is the same in CTL and PD, while it is much higher in lymphomonocytes of PD (continuous white arrows). Panel b: quantification of α-synuclein immunoreactivity in all study subjects (mean±SD). *p < 0.001 vs CTL
Figure 3.6: Total and phosphorylated isoform expression of ERK, JNK and p38 MAPK
in CTL and PD. Representative immunoblots of two CTL and three PD are shown on the left; on the right, quantification of all determinations are reported. * p<0.01 vs CTL
scenario, we explored two miRNAs (miR-7 and miR-30) involved in the post-transcriptional control of α-synuclein and NLRP3 expression. We found that circulating levels of these miRNA are both increased in PD patients vs CTL (Figure 3.7).
Figure 3.7: Quantification of circulating miR-7 and miR-30 in CTL (white plots) and
3.2
Animal Studies
The second part of our study design was planned to better explore such complex system in the brain, in the attempt to link our results to an organ-and tissue- specific damage. To this aim we used an animal model, i.e. WT and P2X7R KO mice treated with normal chow and a high fat diet (HFD), a stimulus able to induce an inflammatory damage at the level of substantia nigra, the brain area which is considered the anatomical target of PD.
3.2.1
Metabolic overview
During the dietary treatment along 16 weeks, the animal weight were moni-tored weekly, while other biochemical parameters were evaluated before the treatment and after one, two, four months. A metabolic stimulus like HFD, as expected, induced a higher weight in all animals (Figure 3.8), but it didn’t affect their clinical parameters: triglycerides, cholesterol, and AST at the end of the treatment have similar value in wt and KO mice; the glucose levels, instead, result slightly higher in the animal treated with HFD.
3.2.2
Substantia Nigra analysis
Substantia Nigra (SN), is the area characterized by a massive loss of dopamin-ergic neurons in PD. Here, I analyzed two molecules important for the patho-genesis of PD, α-synuclein and Parkin. α-synuclein expression levels resulted tendentially higher in both HFD experimental group (Figure 3.10 a), and sim-ilar in the two strains. Conversely, Parkin showed a very intriguing expression profile: its expression did not vary in WT animals treated with HFD, while it was strongly increased in P2X7R KO mice (Figure 3.10 b). Of note, the NLRP3 inflammasome followed exactly an opposite trend, being upregulated in the substantia nigra of WT animals treated with HFD, and not varying in KO mice (Figure 3.10 c).
To confirm SN as target of the specific inflammatory damage induced by HFD, we also looked at tyrosine hydroxylase (TH)-positive neurons. In WT treated with HFD, these dopaminergic cells showed a reduced diameter, and the immunoreactivity signal was also less intense (Figure 3.11).
Figure 3.11: Panel a: Immunofluorescence images of tyrosine hydroxylase (TH)-positive
neurons in the substantia nigra. In WT animals, TH cell diameter is significantly reduced by HFD (white arrows), while it is relatively preserved in P2X7R KO animals. Panel b: quantification of IF in the whole study group. *p < 0.001 vs WT NFD
Double staining experiments were performed in all experimental group, and the confocal images were acquired in the white square area indicated in Figure 3.12. First of all, these experiments showed the activated microglia (Iba1-positive cells) mostly expressed in the SN of WT mice with HFD, while in P2X7R KO mice, HFD did not induce any difference (Figure 3.13).
Figure 3.12: Representative section of murine half brain showing TH-immunostaining
(red) that identifies the substantia nigra. The immunofluorescence experiments executed in serial sections were performed with the indicated antibody. White square indicates the area where the confocal images were acquired.
In WT animals, in the same brain area, P2X7R and NLRP3 proteins were upregulated (figure 3.14 a,b), reinforcing the hypothesis of its participation in the neuroinflammatory process. Interestingly, P2X7R co-localizes with microglia (identified by Iba1 positive cells), and NLRP3 does not colocalize with astrocytes (identified by GFAP) (Figure 3.14 c,d).
Figure 3.13: Panel a Confocal image of WT mouse brain treated with HFD: red staining
labels TH neurons; activated microglia (green) is labeled with Iba1 antibody; nuclei are blue (DAPI). Panel b Quantification of Iba1 immunoreactivity. *p < 0.001 vs WT NFD
Figure 3.14: Quantification of P2X7R (a) and NRLP3 (b) immunoreactivity in WT mice
treated with normal (NFD) and high fat diet (HFD). c representative image of P2X7R (red) and Iba1 (green) colocalization; nuclei are blue (DAPI). d representative image of NRLP3 (green) and GFAP (red) double staining, that does not show any colocalization; nuclei are blue (DAPI). Both inserts are single focal planes of enlarged area.
Conclusions
This work offers a novel, relevant contribution in clarifying the role played by P2X7R in the pathogenesis of PD. We show here for the first time:
1. a systemic hyper-expression of the P2X7R-NLRP3 inflammasome plat-form in neo-diagnosed, treatment-naïve carriers of the disease;
2. that such complex presents an association with circulating α-synuclein, whose deposits represent the main pathogenetic factor of PD;
3. that a reduced JNK phosphorylation might be the intracellular sig-nalling mediating this effect;
4. that circulating levels of miR-7 and miR-30, epigenetic modulators of neuroinflammation responses, are enhanced in PD patients;
5. that in P2X7R KO mice, a neuroinflammatory stimulus induces a strong expression of parkin, a protective protein, suggesting that a P2X7R activation might participate in the inflammatory damage oc-curring in specific brain areas also by inhibiting parkin expression.
P2X7R is expressed in dopaminergic areas affected by PD, where its acti-vation promotes death of nigrostriatal dopaminergic neurons, and its inhibi-tion is neuroprotective in rat models of PD [43, 34, 35]. On the other hand, the components of the NLRP3 inflammasome (NLRP3, apoptosis-associated speck-like protein containing a caspase activating recruitment domain [ASC] and Caspase-1) assembly to react to several stimuli and promote secretion of IL-1β and IL-18, key cytokines in the neuroinflammation process [44]. Such assembly is known to occur via TLR4/NF-κB activation, as shown in astrocytes and in microglia [45, 46, 47]. We show here an increased mRNA and protein expression of the P2X7R-NLRP3 inflammasome in lymphomono-cytes of PD patients, suggesting that extra-neural P2X7R might be relevant in the early phase of the disease, while systemic NF-κB activation does not, indicating the involvement of such intracellular pathway as relevant mainly in neuronal cells.
This potentiated system does not translate, in our study, in different level of systemic inflammation, as shown by the similar IL-1β and IL-18 levels in PD and CTL, even in the presence of a trend for IL-18. This resonates with the recent publication by White et al [48], even in a stimulated setting, and could be due to the short duration of the disease of our patients, the very good matching of our controls (same age and biochemical profile), or to the relatively small number of studied subjects. Matter of fact that a weak but significant correlation between IL-18 and P2X7R was found. Alternatively, we might hypothesize that the systemic activation of the P2X7R-NLRP3 complex has much more to do with other signal pathways.
In PD, NLRP3 has been previously related to α-synuclein [32, 49]; P2X7R also participates in microglial activation by extracellular α-synuclein, thus inducing oxidative stress and accelerating PD [50]. However, this piece of
knowledge comes from studies performed in cell and animal models. We con-firm the functional link between the two molecules in human beings, firstly by the finding of higher lymphomonocyte levels of α-synuclein in PD than in CTL, so far described only in the cerebrospinal fluid and in brain plaques [51, 52], and secondly by the strict linear correlation between P2X7R and α-synuclein expression only in PD subjects. Even more, the different enrich-ment in α-synuclein of circulating immune cells suggests for the first time a participation of infiltrating macrophages in the process of α-synuclein accu-mulation in the CNS, never described before. On the other hand, red blood cells, recognized as the main source of circulating α-synuclein [53], appear similar at immunofluorescence, thus likely explaining the lack of difference occurring in plasma levels between PD and CTL.
Lymphomonocyte phosphorylation of JNK appears significantly reduced in PD; a trend was evident also for p38. Intriguingly, this could be linked to the increased α-synuclein expression, as already reported in some neuronal cell lines [54, 55]. Several studies have pointed out as α-synuclein might either induce toxic or neuroprotective effects, depending on its expression levels and its conformational structure [56, 57]; we cannot exclude that α-synuclein acts as a competitive substrate for such kinase, whose activation is required for calcium-dependent dopamine release, thus influencing dopamin-ergic neurons degeneration [58]. Taken as a whole, our results suggest that, at least in an early phase of the disease, α-synuclein might exert a sort of systemic compensatory role, by counteracting inflammation via JNK inhibi-tion. This is indirectly confirmed by the same level of systemic inflammation (as from IL-1β e IL-18 plasma levels) in PD and CTL.
Searching for putative mechanisms at the level of CNS supporting this role of P2X7R in the pathophysiology of PD, we explored such pathways in
WT and P2X7 KO mice treated with high fat diet, able to induce neuroin-flammation specifically at the level of dopaminergic neurons, as shown by their morphologic alterations. To this aim, beside α-synuclein, we evaluated the effect of such pro-inflammatory stimulus on parkin, whose main function is to ligate ubiquitin to lysine residues, an essential post-translational mod-ification involved in numerous cellular pathways. Mutations of parkin gene have been related to familial and sporadic forms of PD [59, 60]. In our murine model of P2X7R KO, we describe a marked increase in brain parkin expres-sion induced by the neural inflammatory stimulus (high fat diet); at the same time, as expected on the basis of our working hypothesis of a functional link between P2X7R and NLRP3, the latter is upregulated in WT animals where P2X7R is present and is likely promoting inflammation. Such relationship between P2X7R and parkin has been never described before, even though parkin was shown to potentiate ATP-induced currents that result from acti-vation of P2X receptors [61], suggesting a relationship between parkin and neurotransmitter receptors involved in synaptic activity. Differently, an in-verse relation between parkin and NLRP3 inflammasome has been already described, with the former inhibiting the latter via NF-kB [62, 63]. It might be hypothesized that this increased parkin expression induced by high fat diet in P2X7R KO mice might protect toward the formation of α-synuclein aggre-gates, rather than on the α-synuclein amount, that - in fact - does not differ in the substantia nigra of WT and P2X7R KO animals. Such shield is lack in WT animals, in which P2X7R is hyper-expressed and hyper-functioning. We should also point out that high fat diet induces specific structural alterations in dopaminergic neurones.
Lastly, searching for a putative epigenetic regulation of such complex scenario, we explored miR-7 (regulating neuro-inflammation in animal models
of Parkinson’s disease by repressing α-synuclein expression) and miR-30, that post-transcriptionally controls NLRP3 activation [64, 37]: we found as circulating levels of both these markers are increased in PD. Such results are at odds to that observed in the brain of animal models, and even of PD patients, where a decreased miR-7 has been described [65]; we may hypothesize that, at the onset of the disease, these two markers could be released in excess by the injured brain, where they might be abundantly synthetized in the attempt to counter the onset of the disease.
In conclusion, neo-diagnosed PD displays a systemic hyper-expression of the P2X7R-NLRP3 inflammasome platform that seems to modulate lym-phomonocyte α-synuclein; a reduced JNK phosphorylation might be the in-tracellular signalling mediating this effect, undergoing to an epigenetic regu-lation by miR-7 and miR-30. A role of such inflammasome in the moduregu-lation of the balance α-synuclein/parkin in PD can be reasonably hypothesized.
Potentially Translational Value
Evidence before this study
The purinergic signalling plays a relevant role in the pathogenesis of sev-eral diseases of the central nervous system including Parkinson’s disease. The P2X7 receptor (P2X7R), an adenosine triphosphate (ATP)-gated ion channel predominantly expressed on activated microglia, triggers the release of proinflammatory cytokines like IL-1β and IL-18 by promoting the NLPR3 inflammasome assembly. The use of P2X7R antagonists is driving an in-creasing attention in the field of the treatment of neurodegenerative diseases, and in this prospective, P2X7R has been recently quantified in the brain of Parkinson’s patients and controls by a whole-body PET/CT. A few reports have addressed the possible involvement of the inflammasome in Parkinson’s disease, just describing the protective effect of P2X7R blockers in murine models of the disease and in microglial cells, where NLRP3 is activated by α-Synuclein, triggering a neuroinflammation that contributes to degeneration of dopaminergic neurons. Additionally, the inflammasome activation might undergo an epigenetic regulation by short, non-coding RNA species, poten-tially influencing several genes involved in neuroinflammation and chronic neurodegenerative diseases. No data are so far available on the presence, the activation and the putative role of such platform in the periphery, and whether or not it might configure a marker of disease progression and/or could be related to the disease mechanisms.
Added value of this study
This study shows as, in addition to the increased brain expression and function of the NLRP3 inflammasome platform, a systemic activation of such complex might participate in the pathogenesis of Parkinson’s disease. We demonstrate not only as this system is activated in an early phase of
the disease, but also that such activation undergoes an epigenetic regulation mediated by the same miRNAs identified in experimental models of the dis-ease, but of the opposite sign. A putative intracellular signalling responsible for this is also identified. The role of the P2X7R acquires solidity because we propose a possible mechanism occurring in the brain of wild type and P2X7R KO mice treated with a pro-inflammatory diet.
Implications of all the available evidence
These results suggest a future use of the systemic evaluation of the P2X7R/NLRP3 platform in an integrate view of an early diagnosis and a correct identification of the main pathophysiologic mechanisms. This rela-tively easy clinical approach, starting from a blood rather than a cerebrospinal fluid sample (lower cost and time-effectiveness), highlights the possibility to look at peripheral biomarkers that could correlate with brain lesions and dis-ease progression. Such approach might also represent a future, novel tool to monitor the response to treatments.
Future perspectives
5.1
Hippocampus analysis
It is well known that an incorrect diet and obesity can lead to different disorders, such as type 2 diabetes, metabolic syndrome, and cardiovascu-lar diseases. However, in human epidemiological studies, it has been shown that a high fat diet and/or obesity is associated with worse performance on a cognitive task, as well as risk of dementia. The most common form is Alzheimer’s disease (AD). The biological mechanisms underlying this cogni-tive impairment, explored in animal models, regard especially oxidacogni-tive stress, insulin resistance, inflammation, and vascularization/brain blood barrier in-tegrity [66].
Another important point to considerer is that pro-inflammatory cytokines are able to across the blood-brain barrier, but they are also produced by resident cells in CNS (microglia, astrocytes, endothelial cells). It is now evi-dent that inflammation has a pathogenetic role in several neurodegenerative diseases: for example, in AD neuroinflammation is now considered not a consequence of β-amyloid aggregates, and neurofibrillary tangles, but rather
a contributor to the pathogenetic processes themselves [2].
The main pathological features characterizing AD (extracellular β-amyloid aggregates, and intracellular neurofibrillary tangles) are also associated with loss of synapses and neurons, inflammatory activation of microglia and as-trocytes, as well as impairment in glucose metabolism and PIK3-Akt brain signaling. On the other hand, type 2 diabetes, characterized by increased blood glucose and insulin levels, insulin resistance, and chronic inflammation, increases two-to-fourfold the risk of AD [67].
Hippocampus is a brain area in which neuroinflammation can be con-sidered a pathophysiological mechanisms of several neurodegenerative dis-eases. Extensive literature based on animal models supports the link between neuroinflammation-obesity and cognitive impairment. Rats becoming over-weight and insulin-resistant after a diet treatment, show poorer performances than control animals in spatial learning ability task, and a reduced hippocam-pal plasticity [68, 23]. Mice fed with high fat/high cholesterol diet showed impaired working memory, associated to a hippocampus dysregulation: here were reported an activation of microglia and astrocytes, and an increased mRNA expression of various pro-inflammatory cytokines/mediators (TNF-α, IL-1β, IL-6) [69]. In an other experimental setting, mice fed with high fat diet display an impaired cognition, as measured by the Stone T-maze, ac-companied by brain inflammation and BDNF reduction. Interestingly, only the diet characterized by 60% energy from fat produces these effects; an alternative diet consisting in 41% energy from fat and 29% energy from sucrose did not enable to induce alterations [70].
For these reasons, I decided to analyse also the hippocampus in our mouse model; preliminary results concern the functionality of this brain area, evaluated by behavioural test, and the molecular expression of important
metabolic pathways.
5.1.1
Behavioural test
In behavioural neuroscience there are several test applied for studying the physiological processes and neural mechanism of spatial learning and mem-ory; they can be used to evaluate dysfunctional behaviour or cognitive im-pairment induced by an experimental treatment, as a High Fat Diet.
At the end of dietary treatment (see section 2.2.1), six P2X7R KO and six WT mice carried out the following behavioural test, allowing them to recover for two days between the different tasks.
Y-maze
This test evaluates the spatial working memory by measuring a spontaneous alternation behaviour: it is based on rodent preference to explore new envi-ronment to find an escape, then over the course of multiple arm entries, the animal should alternate the visited arms. A video tracking system records animals movement and calculate the number of total entries in the arms and the correct alternation triplets. We did not observe significative differ-ences between groups (WT/KO mice) or treatment (NFD/HFD) for these parameters (figure 5.1).
Morris Water Maze
This behavioural test is widely used to study learning and spatial memory, evaluating numerous parameters calculated on the base of videos recorded during the experiments. Here, I considered two parameters: Escape Latency (EL) and Time in Zone. EL is the time that the animals need to find out the hidden platform in the pool: during the training period along 5 days, in WT
group the feeding with HFD resulted in a poorer performance respect to NFD (Figure 5.2 a); conversely, P2X7R KO mice treated with HFD achieve the same performance of their littermate with NFD after only 2 days of training (Figure 5.2 b). In the last day of the experiment, executed without the platform in the pool, the software recorded the time that each animal spent in the area of the pool where the platform was placed during the training. In this experiment, P2X7R KO mice remained the same time in this area, independently of the treatment (Figure 5.2 d); conversely, WT animals fed with HFD spent less time in this quadrant with respect to their littermate treated with a normal diet (Figure 5.2 c).
Figure 5.1: Results of Y-maze experiment are reported: the upper graph shows the
percentage of correct alternation triplets, the lower reports the number of total entries. We did not observe significative differences in both parameters.
Figure 5.2: Upper graphs show for each day of the trial (1◦-5◦) the Escape Latency; in the final day (6◦) when the experiment was performed without platform, they report the time that animal required to cross the point where the platform was placed (a, b). Lower graphs report Time in Zone, i.e. the time spent in each quadrant of the pool in day 6◦ (c, d).
5.1.2
Molecular analysis
In the hippocampus, an area involved in the loss of cognitive function in neurodegenerative diseases (like Alzheimer disease), I focused my attention on the expression of insulin and IGF-1, as well as of their respective recep-tors (figure 5.3). In two mouse strains (WT and P2X7R KO mice), insulin pathway showed a relevant differential expression: while the insulin receptor was expressed at the same levels in all animals, insulin gene resulted over-expressed only in KO mice. For IGF-1, the picture was different: beside an equal expression of growth factor, the respective receptors seemed to be influenced by the diet, both resulting over-expressed in WT and P2X7R KO mice treated with HFD.
Figure 5.3: Hippocampal mRNA quantification by Real Time-PCR of insulin, IGF and
their respective receptors expression in mice treated with normal (NFD) and high fat diet (HFD). Note the different scale in the graphs.
Cerebral insulin signaling and action are impaired in Alzheimer disease and in other forms of dementia. Insulin regulates a variety of downstream signaling proteins, including PI3K, glycogen synthase kinase-3β (GSK-3β), and mTOR, that drive neuronal death and survival. Particularly, GSK-3β in-hibits α-secretase, the enzyme that cleaves amyloid precursor protein (APP) in non-amyloidogenic way: P2X7R inhibition or deletion, by promoting a selective GSK-3β block at the level of the phosphorylation of serine 9, pro-duces positive effects, avoiding plaques accumulation [71]. For this reason, I evaluated GSK-3β expression in our animal model. While hippocampus revealed the same mRNA level in each experimental group (Figure 5.4 a), at the protein level (Figure 5.4 b) the ratio of GSK and its phosphorylated form was reduced in the WT animals treated with HFD.
Regarding the intracellular signaling activated by insulin receptors, in WT animals HFD seemed to reduce both pathways related to insulin: the glucose metabolism pathway regulated by PI3-AKT (“the metabolic signaling”), and the cellular division and differentiation pathway controlled by JNK (“the growth signaling”); conversely, in P2X7R KO mice the pAKT/AKT and pJNK/JNK ratio did not change with the different diets. On the other hand, HFD modified ERK protein expression: WT and KO mice displayed a specular expression pattern of pERK/ERK ratio (Figure 5.5).
Figure 5.4: G SK − 3β: Panel a mRNA expression evaluated by Real Time PCR in each experimental group; Panel b western blot analysis and relative quantification of G SK − 3β and its phosphorylated form.
Figure 5.5: Panel a Representative images of Western blot analysis of three intracellular
In conclusion, the P2X7R, a key receptor involved in several inflammatory conditions, seems to take part in cognitive impairment linked to metabolic diseases. In fact, its absence in P2X7R KO mice modulates different path-ways:
- KO mice perform better that control animals in behavioural test (as from evaluation of learning and spatial memory)
- HFD doesn’t affect G SK −3β phosphorylation (enzyme involved in amyloid aggregation)
- KO mice show an increased brain insulin expression and function; this favor-able metabolic condition might also activate other downstream enzymes, like AKT, JNK, ERK
Therefore, we might hypothesize that a modulation of these pathways, blocking P2X7R and/or downstream molecules, might attenuate cerebral insulin resistance/deficiency, that plays a major role in cognitive impairment occurring during the course of diabetes and obesity.
