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Abstract

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Table of Contents
Abstract ........................................................................................................................................................ 2
1. Introduction ............................................................................................................................................. 5
1.1 Etiology and Pathogenesis ............................................................................................................ 5
1.2 Symptoms and treatment ............................................................................................................... 7
1.3 Parkinson’s disease stages ........................................................................................................... 8
1.3.1 Clinical stages ........................................................................................................................... 8
1.3.2 Pathological stages .................................................................................................................. 8
1.4 Mechanisms of neuronal death ..................................................................................................... 9
1.5. Role of the adaptive immune system in the onset of Parkinson’s disease ....................... 11
1.5.1 Genetic and environmental risk factors.............................................................................. 11
1.5.2 Clinical and postmortem evidence ...................................................................................... 11
1.6 Submandibular gland as an accessible organ to study the pathogenesis of Parkinson’s
disease.................................................................................................................................................... 13
1.7 Digital pathology and artificial intelligence............................................................................... 14
2. Objectives .............................................................................................................................................. 14
3. Materials and methods ........................................................................................................................ 15
3.1 Human post-mortem brain and submandibular gland tissue ................................................ 15
3.2 Immunohistochemistry ................................................................................................................. 15
3.3 Algorithm training (deep learning artificial intelligence) ........................................................ 16
3.4 Quantitative analysis ..................................................................................................................... 17
3.4.1 Neuromelanin-containing dopaminergic neurons density in the substantia Nigra
pars compacta ................................................................................................................................... 17
3.4.2 CD8+ T cells density in the substantia nigra pars compacta ......................................... 17
3.4.3 Adipose tissue density in the submandibular gland ........................................................ 17
3.4.4 Tyrosine Hydroxylase-positive fibers density in the submandibular gland ................ 17
3.4.5 CD8+ T cell density in the submandibular gland .............................................................. 17
3.5 Statistical analysis ......................................................................................................................... 18
4. Results ................................................................................................................................................... 18
4.1 Algorithm development................................................................................................................. 18
4.2 Quantification of the neuromelanin-containing dopaminergic neurons density in the
SNpc using OlyVIA algorithm............................................................................................................. 19
5. Discussion ............................................................................................................................................. 20
6. Conclusions .......................................................................................................................................... 20
References ................................................................................................................................................. 21

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Parkinson’s Disease

1. Introduction

1.1 Etiology and Pathogenesis

Parkinson’s disease (PD) is a progressive neuronal disorder that primarily affects the elderly
population and is known as the second most frequent form of neurodegenerative disease
after Alzheimer’s disease (1). Although it is widely accepted that PD is mainly caused by the
loss of dopaminergic (DA) neurons of the substantia nigra pars compacta (SNpc) in the
midbrain, its etiology remains unknown(2). DA neurons accumulate a brownish pigment
called neuromelanin (NM) that allows seeing the loss of these neurons macroscopically in
postmortem brain tissue of individuals with PD (3). NM accumulation increases with age and
subsequently makes the individual more susceptible to neuronal loss ((4,5). Other vulnerable
brain regions in PD include locus coeruleus (LC), nucleus basalis of Meynert (NBM), dorsal
motor nucleus of the vagus (DMV), raphe nuclei (RN), pedunculopontine nucleus (PPN),
ventral tegmental area (VTA) and the olfactory bulb (Ob) (6,7).

One of the neuropathological hallmarks of PD is the presence of intraneuronal α-synuclein

aggregates in the form of Lewy bodies (LB) and Lewy neurites (LN) in the SNpc (8). α-
synuclein is a 140 amino-acidic protein encoded by a gene called SNCA, located on
chromosome 4 (4q21) which is mainly expressed the presynaptic nerve terminals in the CNS,
close to the synaptic vesicles (9–12). It seems to be involved in neurotransmitter release and
vesicle trafficking (9). Normally in neurons, there is a balance between the cytosolic (unfolded
monomers) and the membrane-bound (helical multimeric structure) states (13,14). On the
contrary, the α-synuclein monomers conformationally can turn into β-sheet and crossed-b
structures, forming amyloid-like fibrils that finally appear as pathologic Lewy inclusions and
neurites (13,14). While spindle-shaped Lewy neurites appear in the neuronal branches, the
eosinophilic Lewy bodies are mostly detected in the cytoplasm. These amyloid-like
characteristics prevent their degradation via autophagy or other endogenous cellular removal
mechanisms (14,15). Regarding this, LNs seem to be the first detectable marks of
synucleinopathy (14).

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Up to now, the exact role of these α-synuclein aggregates, whether they appear as a
neuroprotective response to a pathological threat or they actually contribute to
neurodegeneration by disturbing the normal cell functioning, is unclear (15). Moreover, there
are many different posttranslational modifications (PTMs) that might be associated with α-
synuclein toxicity such as α-synuclein phosphorylation, glycation, glycosylation,
glutathionylation acetylation, ubiquitination, nitration, SUMOylation and truncation (16,17).

Over the course of PD progression, in addition to brain regions like SNpc, LC, hypothalamus
and subnuclei of the thalamus, amygdala and Ob, α-synucleinopathy can affect the peripheral
nervous system (PNS), enteric nervous system (ENS), and the autonomic nervous system
(ANS) including the vagus nerve, colon, paraspinal sympathetic ganglia, skin, submandibular
gland (SG) and lower esophagus. It can also be found in biological fluids of PD patients
including their serum, saliva and cerebrospinal fluid (CSF) (18–23)The accumulation of α-
synuclein has been shown to somehow be associated with DA neurodegeneration in PD,
which seems to precede the appearance of the cardinal motor symptoms by 7-10 years (20).
Moreover, the SG and lower esophagus reportedly contain higher densities of α-synuclein
aggregation among other PNS sites. A rostro-caudal gradient of α -synucleinopathy in the
gastrointestinal tract (GI) has also been detected (24,25). However, there is no consensus
on whether this synucleinopathy first appears in the brain or within the organs innervated by
the PNS.

Even though the cause of DA cell loss is still unexplained, it seems that having a genetic
background increases the chances of PD manifestation later in life (26).The idiopathic form
of PD occurs as a result of both genetic and environmental risk factors and comprises the
majority of PD cases (1). However, low percentage of PD cases are familial, which has been
found to be related to monogenic mutations such as SNCA, LRRK2, VPS35, PINK1, Parkin
and DJ-1 (27–29). Reportedly, GBA genetic mutations are one of the genetic risk factors for
PD development (Do et al., 2019). It is well known that mutations in SNCA were the first ones
to be linked to familial PD (12). It seems that in addition to point mutations in the SNCA gene
that encodes -synuclein, there are duplications and triplications of this gene that might be
associated with the age of onset and severity of the disease in familial PD (30). Point
mutations in α-synuclein including A53T, A30P, and E46K can somehow lead to an enhanced
expression of α-synuclein which is related to a higher risk for sporadic PD (31).

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Among all the environmental risk factors, age seems to be the most important one in
idiopathic PD. For about one fourth of PD patients the age of onset is younger than 65 years
old and for 5%-10% of them, it is before 50 years old (32). Moreover, the frequency seems to
be higher in men with a ratio from 1.3 to 2 (9). Among the environmental risk factors, exposure
to pesticides, head injury, cancer, methamphetamine and consumption of dairy products are
known to be associated with an increased risk of PD development whereas tobacco and
nicotine consumption, coffee intake, antioxidants like urate, physical activity, and non-
steroidal anti-inflammatory drugs (NSAIDS) are suggested as protective factors (27,33). In
addition to autoimmune diseases, a link between PD and various infections including
measles, hepatitis C, influenza, Helicobacter pylori and infections of the CNS have been
reported (33).

1.2 Symptoms and treatment

PD patients suffer from an excessive reduced quality of life. DA depletion is the underlying
cause of the cardinal motor symptoms observed in PD patients including resting tremors,
postural and gait instability, rigidity and bradykinesia (9,19,34). The degeneration of DA
neurons of the SNpc whose axonal projections are part of the nigrostriatal pathway from the
SNpc to the putamen and caudate nuclei (known as striatum) of the basal ganglia, an
important area for muscle control and movement, leads to motor dysfunction (3,35–37).

The prodromal phase which occurs up to 15-20 years before the onset of motor dysfunction,
is accompanied by manifestations such as olfactory dysfunction (hyposmia and anosmia),
rapid eye movement sleep disorder (RBD), depression, gastrointestinal problems especially
constipation and autonomic dysfunction (9,19). Several other non-motor symptoms including
psychosis, hypersialorrhea or drooling, pain, cognitive impairment and dementia might
appear along with the motor symptoms (9,19,38). Furthermore, about 80% of PD patients
experience dysphagia (i.e. having difficulty while swallowing food) and hypersialorrhea as a
result of hyposalivation (39–41).

Up to date, the best drug treatment for PD patients is Levodopa (L-3,4-

dihydroxyphenylalanine; L-DOPA), a dopamine precursor that unlike dopamine itself, can
pass through the blood-brain barrier (BBB) (37,42). However, there are patients who develop
resistance to L-DOPA after a period of time (42). Several other complications such as
dyskinesia, psychosis and fluctuations can occur as a result of chronic dopaminergic

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treatment (9,19). Administration of anticholinergics, monoamine oxidase inhibitors (MAOIs),
dopamine agonists or less commonly, amantadine has also been suggested (9,19). Other
types of surgical and medical interventions include deep brain stimulation, gene therapy and
cell transplantation (19,43–46). A PD diagnosis is mainly based on the appearance of the
cardinal motor symptoms (47), and it is further confirmed by a positive symptomatic response
to the administration of L-DOPA (24,26).

1.3 Parkinson’s disease stages

1.3.1 Clinical stages
The motor and non-motor features of PD led to the introduction of clinical staging systems
that help clinicians have a classification according to the severity and symptoms manifested
in each patient. The standard staging reference for PD is the Hoehn and Yahr (HY) scale,
even though it has limitations of mixing impairment and disability, nonlinear character
between the stages and its tendency towards postural instability. HY correlates with
neuroimaging studies of DA cell loss (48). Unified Parkinson’s disease Rating Scale (UPDRS)
is another staging system that exclusively evaluates the main clinical symptoms of PD and
monitors the severity of the disease (49). Later on, the modified UPDRS by Movement
Disorder Society Task Force on Rating Scales (MDS-UPDRS) was introduced which includes
the non-motor symptoms in addition to the previous ones and shows a higher sensitivity in
PD diagnosis (48,50).

1.3.2 Pathological stages

The prodromal phase of PD can be explained according to the neuropathological events
involved. Braak was the first person who suggested a staging system for PD depending on
when the synucleinopathy first emerges in different regions of the brain (51). He defined this
staging system by including cases with synucleinopathy that displayed symptoms and cases
who lacked them. The following classification was the result including 6 stages: stage 1,
medulla oblongata; stage 2, medulla oblongata and pontine tegmentum; stage 3, midbrain
particularly SNpc and the basal nucleus of Meynert; stage 4, basal prosencephalon and
mesocortex; stage 5, neocortex and prefrontal neocortex; stage 6, premotor and sensory
association areas of the neocortex. Based on Braak’s staging system, synucleinopathy in
brainstem was a representation of the early stages of PD (51,52). Braak was also a pioneer
in including the Ob as one of the earliest areas affected by synucleinopathy in PD (51). He

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suggested the vagus nerve as another initially affected area by synucleinopathy which
remains dubious because reportedly, patients who lack -synuclein aggregation in the brain
do not seem to show synucleinopathy in the vagus nerve (53).

Subsequently, the Unified Staging System for Lewy Body Disorders (USSLBD) was proposed
by Beach and his colleagues to improve the Braak’s staging system which can be applied to
all the Lewy body disorders (PD, DLB iLBD and AD) . USSLBD suggests the next model
which is determined by the α-synucleinopathy density score in the examined brain regions: I.
Olfactory bulb only; IIa. Brainstem Predominant; IIb. Brainstem Predominant; III. Brainstem
and limbic; IV. Neocortical (23). According to USSLBD, the presence of -synuclein
aggregates in the Ob increases the chances of developing Lewy body disorders later in life
(23). Regarding that, clinical observations and postmortem evidence coincide in the olfactory
problems being an initial sign of the disease (23). These classifications help us better
understand the prodromal stages of PD, known as incidental Lewy body disease (iLBD) cases
who probably were in the initial phase and were not clinically diagnosed before passing away
(23,54). Tyrosine Hydroxylase (TH) levels, an essential enzyme in dopamine biosynthesis
(55), have been found to be lower in the iLBD cases than in PD patients, which is a proof of
dissimilarity between them and healthy controls (56,57). Moreover, it has been reported that
during the first two stages of Braak’s staging system when no trace of -synuclein
aggregations is apparent yet, there are lower numbers of TH-containing neurons as well as
more neuronal loss in the SNpc (58), therefore suggesting that neuronal loss probably
predates synucleinopathy.

1.4 Mechanisms of neuronal death

The main reason why DA neurons are pushed toward death in PD is not known yet. Different
cell death mechanisms including NM accumulation, oxidative stress and mitochondrial
dysfunction, -synuclein aggregation, lysosomal dysfunction and innate and adaptive
immune response have been suggested to be associated with this neuronal loss, which either
triggers the initiation of apoptosis or promotes necrosis (5,9). Understanding these
pathophysiological mechanisms will help us both in disease prevention and treatment.
However, there is no universal consensus on whether these mechanisms independently or
together are provoking the detrimental events in the brain (9).

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By using an animal model overexpressing NM, an association between neuronal loss and
age-related NM production in the DA neurons of the SNpc has been found. This has led to
the definition of a threshold for NM accumulation inside these cells. Interestingly, while it
seems that in PD patients and iLBD cases the amount of intracellular NM might cross this
threshold, in healthy controls it does not reach that. This points out to the possible role of the
age-dependent accumulating NM inside the DA neurons of the SNpc as an element that
makes them more vulnerable to degeneration (5).

Oxidative stress, as a result of an unbalanced production of Reactive oxygen species (ROS)

whose accumulation can cause oxidative damage to proteins, DNA and membrane lipids, can
be a cause of deregulated activity of complex I (3,9,59). Mitochondrial dysfunction causes a
chronic cycle of ROS production (60). It has been shown that the impairment of the complex
I might increase the chances of neurodegeneration by making the DA neurons more
susceptible to neurotoxin exposure (60–62). The presence of a defective complex I as a result
of mitochondrial dysfunction in the SNpc of PD patients has been reported (63). Inhibition of
complex I can reversely increase the production of ROS, especially in the DA neurons of the
SNpc that seems to be more sensitive to this mechanism due to auto-oxidation of NM and
dopamine catabolism (3,7).

Abnormal -synuclein misfolding has been reported to be toxic for DA neurons which can be
one of the underlying causes of neuronal loss in PD (9). Oxidative stress and gene mutations
in PD seem to affect these conformational changes of -synuclein (7). Moreover,
mitochondrial damage has been found to be a cause of reduced glucocerebrosidase and
increased accumulation of oxidized dopamine products (9,64). Since recycling the -
synuclein aggregates is primariliy dependent on the function of lysosomes, lysosomal
dysfunction is another factor that can contribute to neuronal death by decreasing the turnover
of these aggregates and therefore, increasing their intracellular accumulation (65). On the
other hand, the adaptive immune response, either to endogenous antigens (aforementioned
mechanisms) hence leading to autoimmunity or to exogenous antigens such as viral particles
(9,53).

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1.5. Role of the adaptive immune system in the onset of Parkinson’s
disease
1.5.1 Genetic and environmental risk factors
There are various genetic and environmental risk factors involved in PD pathogenesis that
might also contribute to this altered immune response (32). In familial PD, mutations in genes
such as SNCA, LRRK2, DJ1 and PINK1, which seem to play an important role in regulation
and homeostasis of the immune response (especially T cells), can also contribute to PD
development (66). Environmental risk factors including exposure to chemicals, infectious
organisms (e.g. bacteria or viruses) and more importantly, age-related physiological changes
of the immune system, immunosenescence and inflammaging can provide us with an
explanation for the underlying cause of this altered immune response (32,67,68).

Accumulating evidence supports the involvement of the adaptive immune system in the
etiopathogenesis of PD (69). Genome-wide association studies (GWAS) analyses have
shown a clear association between PD and other autoimmune disorders due to common
genetic risk factors, which sheds light on the possible role of autoimmunity in PD
pathogenesis (70,71). Moreover, various single nucleotide polymorphisms (SNPs) such as
SNPs of some genes related to the lymphocyte function, T cell receptor (TCR) signaling and
regulatory variants in the HLA region were identified as the possible genetic risk factors (72–
75). Additionally, various genetic modifications in MHCII and LRRK2 have been shown to
mutually be associated with an increased immune response and a higher risk of PD
development (71,76).

1.5.2 Clinical and postmortem evidence

There is increasing evidence supporting the crucial role of the adaptive immune system,
specifically CD8+ T lymphocytes, in PD pathogenesis (2). Increased infiltration of T cells into
the brain parenchyma of PD patients, especially during the early phases of the disease in the
absence of nigral synucleinopathy has been reported (2,77). This sheds light on the role of
the immune system not only in the disease initiation but also in its progression.

Brochard et al. (2009) described the CD4+ T cells as the responsible components of the
adaptive immune system in MPTP animal models and PD patients, even when the CD8+ T
cell infiltration preponderated (78). The results of a recent study done by our team clearly
demonstrate the importance of the nigral cytotoxic attack of the CD8+ T cells after infiltration,

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which precedes the α-synuclein aggregation and neurodegeneration during the premotor
phases (2). We assessed both CD4+ and CD8+ T cell densities in the parenchyma and the
perivascular space of PD and iLBD cases. We could only find the infiltration of CD8+ T cells
in the parenchyma of PD patients and perivascular CD4+ T cells. A positive correlation
between neurodegeneration and CD8+ T cell densities was observed whereas no such thing
was seen with CD4+ T cells (2). Our findings demonstrate that the CD8+ T cells were in close
proximity with the NM-containing neurons (2). Accordingly, this cytotoxic attack could be
triggering the initiation of this deadly cascade of neurodegeneration as well as its progression
(2). These results can explain the role of the adaptive immune system in the PD
pathogenesis. During the first stage, which happens almost concomitantly with the first stage
of Braak’s staging system, there is a large number of CD8+ T cells infiltrating the SNpc. Then,
following a sudden reduction in the infiltrating cells, another wave of infiltration occurs which
also is accompanied with the appearance of Lewy bodies, Lewy neurites and DA
neurodegeneration (2). This somehow implies that CD8+ T cells are not cytotoxic at the
beginning (2). Nonetheless, whether this α-synuclein accumulation acts as a neuroprotective
shield or has a detrimental role in PD etiopathogenesis remains enigmatic (2).

Moreover, microglial hyperactivation in the SNpc is considered as one of the pathological

characteristics of PD (31), in relation to which, animal model studies have reported an
abundance of reactive human leukocyte antigen-DR (HLA-DR)–positive microglial cells in
that region (79). Although the process of neuroinflammation in PD is not clear yet, there is an
increased expression of proinflammatory cytokine levels such as TNF-α, IL-1β, and IFN-γ by
the microglia in the striatum of the postmortem brain and CSF compared with age-matched
controls (31,80).

The clinical symptoms present in PD patients together with the presence of synucleinopathy
in the periphery brought up the hypothesis that a neurotropic pathogen, probably a viral agent,
might enter the brain either through the nasal cavity or after ingesting the nasal secretions in
saliva (81). Having in mind both exposure to a neurotropic pathogen and the self-antigens of
the individual that might be triggering an autoimmune response, these results support the
hypothesis that there might have be an infection or an autoimmune response that leads to
the following deleterious events.

In PD and other neurodegenerative diseases in general, having samples from the brain tissue
would be the ideal condition in order to study the etiology of the disease. However, since that

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is not possible until we have access to the postmortem tissue, finding adequate substitutes
would be really helpful. This is why we are interested in studying the possible association
between the immunological responses, mainly CD8+ T cell infiltration, and α-synucleinopathy
in the SG which may be mirroring the immunologic events that occur in the brain. Moreover,
this might help us in the future to design inidividualized therapies for each patient knowing
the little differences that exist during the early stages of PD.

1.6 Submandibular gland as an accessible organ to study the

pathogenesis of Parkinson’s disease
The submandibular gland (SG) is a seromucous salivary gland that is located subcutaneously
in the posterior part of the submandibular triangle, under the mandible which is known as the
second largest salivary gland after the parotid gland (82,83). The paired SGs are responsible
for 70% of saliva production in unstimulated conditions (82).

Histologically, the SG is made up of parenchymal (secretory acini and ducts) and stromal
components (84). Moreover, the SG is divided into superficial and deep lobes by the
mylohyoid muscle, with the superficial part lying beneath the deep cervical fascia that forms
a capsule of connective tissue wrapping around the gland (82). This division further arranges
a series of lobules inside the gland that contain the ducts and provide the blood supply and
innervation of the tissue (84). There are serous and mucous acini with serous cells being
predominantly distributed throughout the SG tissue (83). The produced saliva is excreted
through the intercalated, striated and excretory ducts towards the Wharton duct which opens
into the oral cavity (82,84).

The parasympathetic (cholinergic) post ganglionic fibers innervate the SG coming from the
submandibular ganglion, while the sympathetic (adrenergic) innervation is extended through
the branches of the external carotid artery, originating from the superior cervical ganglion
(82,85). The parasympathetic nerves mainly stimulate the serous end pieces that are
distributed predominantly in the parenchyma, whereas the sympathetic ones are mostly
involved in protein secretion (82,83).

Beach et al. (2016) confirmed the possible detection of Lewy pathology in the SG by showing
the presence of α-synuclein aggregations in 91% of PD subjects and 11% of iLBD subjects
(18). This suggests that in a high percentage of PD cases show α-synucleinopathy in this
gland. It seems that Lewy pathology initially affects the peripheral tissues of the ANS, even

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before the involvement of the SNpc (86). Moreover, it has been reported that α-
synucleinopathy in the SG exists concomitantly with its presence in the brain (18). During the
advanced stages of disease, the SG showed higher sensitivity and specificity in α-synuclein
detection compared with colon or skin (21). Regarding this, the SG has been suggested as a
good biopsy site to look for synucleinopathy. While it is an accessible and safe-to-be-biopsied
organ, some authors believe that it increases the possibility of having a more accurate
diagnosis while the individual is still alive (18).

1.7 Digital pathology and artificial intelligence

Digital pathology is an almost recent phenomenon in pathology laboratory, which aids the
pathologists and pathology researchers to not only obtain full-coverage scans of the sections
but also have access to this more detailed information anytime they need it (87). One of the
important characteristics of this digital tool is that in addition to giving us high-quality digitized
images, it encompasses both machine learning and deep learning, both of which help us
create algorithms that help us analyze the data we have on the sections (88,89).

An open access software such as QuPath and a closed-access one like OlyVIA are great
examples that incorporate useful tools for annotating and viewing in both bright-field and
fluorescence images. They make it possible to train algorithms that are beneficial for a great
variety of tasks such as quantitative analyses and cell counting. More features include cell
segmentation, tissue microarray dearraying, batch processing and stain estimation and cell
detection tools (68,89). This highly potential technology that we use in this study, accelerates
the process of sample evaluation and also increases the quantitative accuracy by decreasing
the risk of bias (89,90).

2. Objectives
We hypothesize that, there is a CD8+ T cell infiltration in PNS regions affected by
synucleinopathy such as the submandibular gland in PD patients, which supposedly is
mirroring the pathogenic events observed in the brain. We hypothesize that this infiltration
precedes the α-synuclein aggregation just as it does in the SNpc. Moreover, we advance the
hypothesis that there is a denervation of TH+ fibers in the SG. Therefore, we propose the SG
as an organ for studying the etiopathogenesis of PD.

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The main goal of this study is to determine whether there are similar pathological events
occurring in the SG and the SNpc regarding the CD8+ T cell infiltration in FFPE postmortem
tissue sections. To accomplish this main goal, we define the following specific objectives:

1. To train two deep learning algorithms in OlyVIA software to automatically count the
CD8+ T cells and to assess the TH+ fibers in human postmortem SG tissue sections.
2. To train an algorithm in OlyVIA to quantify the NM-containing neurons in human
postmortem SNpc tissue sections and thus, assess the DA neuronal density in Healthy
controls, iLBD1, iLBD2 and PD groups.
3. To ascertain whether there is an increase of CD8+ T cell infiltration in the SG of PD
patients and if so, whether it precedes the synucleinopathy.
4. To determine if there is neuronal loss in the SG of PD patients.
5. To determine whether there is a positive correlation between the CD8+ T cell densities
in the SG and SNpc.

3. Materials and methods

3.1 Human post-mortem brain and submandibular gland tissue

Formalin-Fixed Paraffin-Embedded (FFPE) SNpc and SG sections (thickness 5 µm) from
deceased PD patients with or without synucleinopathy in the SNpc (n= 34) and SG (n= 24)
and age-matched healthy control individuals were provided by Banner Sun Health Research
Institute (BSHRI), Sun City, Arizona. The Brain and Body Donation Program operates with
the approval of Western Institutional Review Board (Puyallup, WA). Cases were selected
from individuals of both sexes, who had volunteered for the BSHRI Brain and Body Donation
Program (BBDP), and who had full autopsy reports, clinical history and neuropathological
information, including the SG. Two cases were obtained from the Neurological Tissue
BioBank at IDIBAPS-Hospital Clinic (Barcelona). For each case, three sections were used.

3.2 Immunohistochemistry
Briefly, tissues were deparaffinized at 60°C for 30 min. Sections were then rehydrated in two
solutions of xylene (5 min each) and then transferred to 100%, 95% ethanol (10 min each),
70% ethanol and distilled water (5 min each), respectively. Then sections were rinsed in 0.1
M Tris buffered saline (1x TBS) 100 mM pH 7.4 (Ref. S3014; Trizma base, Sigma-Aldrich

15
Ref. T6066; NaCl, Sigma-Aldrich) between each incubation period. An antigen retrieval
protocol was performed using sodium citrate (10 mM; pH 6) in a water bath at 95°C for 20
min to unmask the epitopes. Afterwards, the slides were immersed in 10% methanol (vol/vol)
and 3% hydrogen peroxide (vol/vol) for 10 min to inhibit the endogenous peroxidase activity.
A permeabilization step was performed by washing the sections in 1x TBS + 0.3% Triton TM X-
100 two times (5 min each) and Normal Goat serum (Ref. S-1000-20; NGS, Vector) was used
to block for 1h at room temperature. An additional incubation with Avidin/Biotin blocking kit
was also performed on the SG sections (Ref. SP-2001; Vector). Sections were then incubated
overnight with primary antibodies at 4°: rat anti-human CD8α (Supernatant; Ref. NOR132;
CNIO) and rabbit anti-human tyrosine hydroxylase (1:1000; Ref. 657012; Sigma-aldrich). The
following day, appropriate biotinylated secondary antibodies were applied to the sections for
a 1-hour incubation at room temperature. ABC kit (Ref. 32050; Thermo Fischer Scientific),
Vector SG (Ref. SK-4700; Vector), DAB (Ref. D3939-1SET; Sigma) were then applied to
sections to detect the antigen-antibody reaction. The SG tissue sections were counterstained
with Hematoxylin (Ref. GHS316-500ML; Sigma-Aldrich) & Eosin (Ref. HT110116-500ML;
Sigma-Aldrich). Sections were finally dehydrated through 70% and 95% (1 min each), 100%
ethanol (2 min) and finally in two changes of xylene (5 min each). Tissue sections were then
coverslipped using DPX non-aqueous mounting medium (Ref. 1.005.790.500; Sigma-
Aldrich). All antibodies were previously tested in positive control tissue to optimize
immunohistochemistry conditions.

In order to train the NM algorithm, previously immunostained SNpc tissue sections were used
including rabbit anti-human CD4 (1:100; Ref. HPA004252; Sigma-Aldrich), rat anti-human
CD8 α (Supernatant; Ref. NOR132; CNIO), rabbit anti-human GFAP (2μg/mL; Ref.
HPA056030, Sigma-Aldrich) and mouse anti-human pS129 a-syn (1:100; Ref. 015-25191,
Wako).

3.3 Algorithm training (deep learning artificial intelligence)

Part of this project was dedicated to the development of different algorithms for the
automatized quantification of different antigens in both substantia nigra pars compacta
(SNpc) and submandibular gland (SG). In this regard, all sections were scanned with the
Slide scanner Olympus SV200 using a multi-layer image with a 20x objective scan mode with
EFI 1.5. All images were focused semi-automatically and manually corrected before saving.

16
Digitized images were then processed with Olympus SV200 desktop software for algorithm
training and further analysis.

3.4 Quantitative analysis

3.4.1 Neuromelanin-containing dopaminergic neurons density in the substantia
Nigra pars compacta
In order to evaluate the neuronal degeneration in the SNpc, the area was drawn manually in
HC (n=6), iLBD1 (n=8), iLBD2 (n=5) and PD (n=15) by using Olympus SV200 desktop
software. The NM algorithm was run in the regions of interest and the NM-containing neurons
were quantified using the count and measure tool (FIGURE). Finally, the density values were
assessed by dividing the total number of neurons by the delimitated area of SNpc (cells/mm2).

3.4.2 CD8+ T cells density in the substantia nigra pars compacta

CD8+ T cell densities were quantified using X sections per case, on the same tissue sections
used in section 3.4.1. These sections were immunohistochemically stained for CD8+ T cells
detection. Quantification was performed manually using QuPath software, differentiating
between parenchymal and perivascular CD8+ T cells (FIGURE). Afterwards, the number of
CD8+ T cells was divided by the area of the SNpc that was calculated for the DA neuron
quantification (CD8+ T cells/mm2).

3.4.3 Adipose tissue density in the submandibular gland

QuPath software was used to manually determine SG and adipose tissue areas, as fatty
infiltration is an age-associated phenomenon (91). The SG tissue sections used here were
counterstained with Hematoxylin & Eosin (H&E). In order to compare the cases, the sections
were normalized.

3.4.4 Tyrosine Hydroxylase-positive fibers density in the submandibular gland

Quantification of TH+ fibers in the SG was calculated by dividing the area occupied by the
TH+ fibers obtained with the TH algorithm by the total area of the SG (TH+ area/mm2).

3.4.5 CD8+ T cell density in the submandibular gland

The SG total areas were drawn manually using QuPath software. The density was further
measured by dividing the number of CD8+ T cells detected by the algorithm by the total area
of the SG (cell counts/mm2).

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3.5 Statistical analysis
All statistical analyses were performed using GraphPad Prism 6 software (version 6,
GraphPad Software, USA). We analyzed the obtained data for normal distribution using the
Shapiro-Wilks test. For parametric analyses, One-way ANOVA with Tukey's post-hoc multiple
comparisons analysis was performed. For our non-parametric analysis, Kruskal-Wallis with
Dunnet post-hoc multiple comparisons analysis was performed. P < 0.05 is considered
significant. Error bars in all graphs are represented as ± standard deviation (SD). Correlations
between two numerical variables were done using the Pearson correlation test, where results
are considered significant for P value < 0.05.

4. Results

4.1 Algorithm development

One of the main goals of our study was to train an algorithm that could count our target cells,
in our case the NM-containing neurons in the SNpc, the CD8+ T cells in the SG and the TH+
fibers of the SG. Before that, this quantification was performed manually and from now on,
the possibility of using machine learning algorithms to automatically count the cells exists.

The NM algorithm was designed and trained in order to count dopaminergic neurons and
evaluate neuronal cell death. To train the algorithm, a total number of 24 SNpc sections that
were previously undergone immunohistochemical staining for CD8, CD4, GFAP and
phosphorylated-synuclein were selected (6 sections per group). DA-neurons are identified by
the presence of unstained neuromelanin brown pigment in SNpc, which are easy to
distinguish from the background without any counterstaining. Therefore, two label classes
(NM and background) were added in these sections. About 100 NM object annotations and
50 annotations marking the background were drawn manually in the regions of interest
(FIGURE).

The selection of four groups for the training helped us improve the accuracy of the algorithm
by introducing different combinations of NM-containing neurons and the background. For SG,
two algorithms were designed independently. On one hand, CD8+ T cell SG algorithm was
designed using X images with X object annotations named as CD8+ T cells and background
(FIGURE). On the other hand, for TH+ fiber quantification the TH+ algorithm was made, using
a total of X images with X object annotations drawn named as TH+ fiber and background

18
(FIGURE).. All algorithms were trained using a non-iteration limit (accepting as good when
the algorithm reached a checkpoint value of at least 0.75).

Thus, the algorithms were created at checkpoints, X (NM algorithm), X (CD8+ T cell SG
algorithm) and X (TH+ algorithm). Once the algorithms were created, they were manually
checked and if necessary adjusted. CD8+ T cell algorithm as it had a huge amount of CD8+
T cells made some mistakes when counting CD8+ T cells that were close to one another
identifying them as just one. This error was corrected by applying the “automatically split
objects” tool. Finally, each algorithm was validated by being compared with manually
counts of the same sections, to make sure that the algorithm is counting correctly.

4.2 Quantification of the neuromelanin-containing dopaminergic

neurons density in the SNpc using OlyVIA algorithm
Based on the presence of -synuclein aggregations in the brain, four groups were defined as
HC, iLBD1, iLBD2 and PD. Individuals in the iLBD1 group represent the earliest stage of PD
that lack synucleinopathy in the SNpc, whereas the iLBD2 group shows synucleinopathy in
the SNpc. After the data analysis, a significantly less density of DA neurons was observed
in the PD group (number of neurons/mm2=?) compared with that of the HC (number of
neurons/mm2=?) and the iLBD groups (number of neurons/mm2=?) (p=?). Our results
demonstrate that the loss of NM-containing neurons severely affects the SNpc during the
advanced stages of the disease. Moreover, an increased tendency towards
neurodegeneration is observed as the disease proceeds (FIGURE X).

19
5. Discussion

6. Conclusions

20
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