In: Horizons in Neuroscience Research ISBN: 978-1-68507-509-5

Editors: A. Costa and E. Villalba © 2022 Nova Science Publishers, Inc.

THE JANUS FACE OF -SITOSTEROL- -D-

GLUCOSIDE (BSSG): HEALTH PROMOTER
AND NEURODEGENERATION INDUCER

Luis O. Soto-Rojas1, , , PhD, MD,

Daniel Martinez-Fong , PhD, MD,
Irma A. Martinez-Davila , PhD,
Alberto Santiago-Balmaseda1, MD,
Yazmin M. Flores-Martinez4, PhD,
M E. Gutierrez-Castillo5, PhD,
Irais E. Valenzuela-Arzeta2,
Paola A. Martínez-Gómez1, MD
and Claudia Luna-Herrera6, PhD
1
Facultad de Estudios Superiores Iztacala,
Universidad Nacional Autónoma de México, Edo. de México, México

-mail: oskarsoto123@unam.mx.
These authors have contributed equally to this work.

Complimentary Contributor Copy

76 L. O. Soto-Rojas, D. Martinez-Fong, I. A. Martinez-Davila et al.

2
Departamento de Fisiología, Biofísica y Neurociencias,
Centro de Investigación y de Estudios Avanzados,
Ciudad de México, México
3
Programa de Doctorado en Nanociencias y Nanotecnología,
Centro de Investigación y de Estudios Avanzados,
Ciudad de México, México
4
Programa Institucional de Biomedicina Molecular,
Escuela Nacional de Medicina y Homeopatía,
Instituto Politécnico Nacional, Ciudad de México, México
5
Departamento de Biociencias e Ingeniería,
Centro Interdisciplinario de Investigaciones y Estudios sobre Medio
Ambiente y Desarrollo, Instituto Politécnico Nacional,
Ciudad de México, México
6
Departamento de Fisiología, Escuela Nacional de Ciencias
Biológicas, Instituto Politécnico Nacional, Ciudad de México, México

ABSTRACT

-Sitosterol -D-Glucoside (BSSG) belongs to the family of -

sitosterols, which are major phytosterols derived dietarily from plant
products, including fruit, vegetables, nuts, and seeds. Despite these
chemicals being functionally and structurally related to cholesterol, they
are firmly bound to plant fibers and therefore poorly absorbed during
intestinal transit in humans, resulting in 800-1000 times lower plasma
concentrations than cholesterol. Several studies have shown that BSSG
exerts beneficial effects on metabolic parameters (lipid-lowering and
normoglycemic), cellular responses (immunomodulatory and
neurotrophic), and disease prevention (reduced incidence of
cardiovascular disease and diabetes; antitumor effects when used as an
adjuvant). Conversely, sustained consumption of BSSG can also exert
severe neurotoxic effects, as first demonstrated by the association of the
amyotrophic lateral sclerosis-parkinsonism-dementia complex (ALS-
PDC) syndrome with the daily intake of nutrients derived from cycad
(Cycad circinalis) seeds. Upon identifying BSSG as the main
neurotoxic component of cycad seeds, multiple studies have
demonstrated its neurotoxic effect and studied the underlying
mechanism of action by which BSSG causes toxicity. The first studies

Complimentary Contributor Copy

 -Sitosterol- -D-Glucoside (BSSG) 77

suggested that high concentrations of BSSG trigger cell death, possibly

through
rough NMDA receptor-mediated excitotoxicity and glutathione
depletion. Subsequent experiments in Sprague-Dawley rats
demonstrated that long-term feeding of BSSG-enriched pellets evokes

progressive loss of dopaminergic neurons, motor and non-motor

alterations, neuroinflammation, and Lewy pathology, characterized by
the progressive aggregation and spreading of pathological -synuclein.
To shorten the long period of continuous administration of a high BSSG
dose in the above-mentioned PD model, our group injects unilaterally a
single microdose of BSSG in the Substantia Nigra pars compacta
(SNpc) of Wistar rats. Interestingly, Lewy pathology begins in the
injected SNpc and then spreads in a prion-like manner to the whole
brain, eliciting motor and non-motor alterations. Furthermore, the
pathological -synuclein aggregates closely correlate with
neuroinflammation and neurodegeneration. This chapter aims to discuss
both the beneficial and neurotoxic effects of BSSG, highlighting the
usefulness of the stereotaxic BSSG model to understand the
pathophysiological mechanisms of PD and assess new therapeutic
approaches for this presently incurable disease.

1. INTRODUCTION

Plant sterols include -Sitosterol -D-Glucoside (BSSG), which

-sitosterols, present in several fruits,
vegetables, nuts, and seeds [1, 2]. BSSG has a high structural and
functional similarity to the cholesterol molecule [3, 4] -
Sitosterols are not synthesized endogenously in humans, and their
intestinal absorption is minimal for various reasons, including limited
overall consumption in the total diet and chemical affinity to non-
processed plant fibers [5-7]. Besides, they can be rapidly secreted by the
bile, released internally within the duodenum, and subsequently
incorporated in the feces [8].
Since the twentieth century, BSSG was recognized as a bioactive and
health-promoting molecule because it has several medicinal effects such
as anti-inflammatory, anxiolytic, immunomodulatory, antitumor, lipid-
lowering, and normoglycemic [9]. Therefore, BSSG has been considered
as a suitable therapeutic candidate for different chronic-degenerative

Complimentary Contributor Copy

78 L. O. Soto-Rojas, D. Martinez-Fong, I. A. Martinez-Davila et al.

diseases, including diabetes mellitus, obesity, dyslipidemia, metabolic

syndrome, chronic inflammatory diseases, adjuvant in certain types of
cancer, autoimmune/immunosuppressive, and suggestively for
neurodegenerative diseases [3, 4, 9-20].
Conversely, BSSG was discovered as one of the Cycad circinalis
seed compounds, with which the Chamorro population (belonging to
western Pacific islands of Guam and Rota) elaborated their flour, causing
the neurodegenerative disorder called amyotrophic lateral sclerosis (ALS)
with parkinsonism and dementia complex [ALS / PDC] [21-24].
Therefore, BSSG has also been considered a neurodegeneration inducer
since it was associated with glutamate excitotoxicity, misfolding of the -
synuclein -syn) protein, neuroinflammation, oxidative/nitrosative
stress, and lipid dyshomeostasis [25-32]. As we discuss below, the stark
contrast between the two outcomes of exposure to BSSG could be related
to dose and route of administration.
Subsequent studies showed that oral or local BSSG administration in
rodents developed the classic hallmarks of (PD). PD
is a neurodegenerative disorder estimated to have a worldwide prevalence
of 1 to 3 per 1000 in unselected populations and affects about 1% of the
population older than 60 [33]. The Global Burden of Disease Study
indicates that by 2040 there will be 13 million PD patients worldwide
[34]. Clinically, this disorder presents both motor and non-motor
alterations, associated with the gradual and progressive loss of
dopaminergic (DA) neurons in the Substantia Nigra pars compacta
(SNpc) and -syn misfolding [35-40]. Several experimental models have
been developed to study PD, including genetically modified organisms or
neurotoxin administration. However, most of these models fail to develop
a chronic onset of symptoms and only partially reproduce the classical
hallmarks of PD, namely its characteristic behavioral alterations,
nigrostriatal neurodegeneration, neuroinflammation, and -syn
misfolding
sfolding that leads to Lewy body formations [41]. Interestingly, both
systemic and local (into SNpc) BSSG administration produce these
characteristic hallmarks. Remarkably, local BSSG administration
reproduces all hallmarks of PD in a short time and with lower cost

Complimentary Contributor Copy

 -Sitosterol- -D-Glucoside (BSSG) 79

(because of the single dose of drug administration required) compared to

systemic administration [28-32]. Furthermore, the BSSG-injected
animals develop prion-like -syn spreading in neuroanatomically
interconnected areas [28-32]. Consequently, BSSG administration has
been proposed as a suitable model for understanding the
pathophysiological mechanisms involved in PD.
In this chapter, we will describe and discuss the biochemical
properties of BSSG, its dual effects depending on the study model,
dose/concentration, and route of administration, as well as its
advantageous use for generating a suitable animal model of PD. We will
also discuss how studies with BSSG have contributed to neuroscience by
providing insight into the mechanisms associated with
neurodegeneration, neuroinflammation, and -syn
misfolding/propagation. The practical outcome of these studies is the
identification and development of neuroprotective, neuroregenerative,
anti-inflammatory, and anti- -syn aggregation therapies for PD.

1.1. BSSG Biochemical and Pharmacological Properties

BSSG is mainly found in leaves, rhizomes, flowers, seeds, and fruits

of higher plants (Figure 1 step 1). It is a secondary metabolite with
regulatory functions in maintaining cell membrane homeostasis in
response to biotic and abiotic stresses and defense against herbivores. In
addition, its chemical structure enables an extensive array of biological
actions and bioavailability (uptake) in other organisms. Consequently, the
extraction/purification from natural resources or commercial synthesis
has acquired particular importance because it has been reported to display
a broad spectrum of biological activities in animals and humans (1, 2).
Table 1 summarizes the physical and chemical properties of BSSG,
along with identifiers and molecular descriptors, surrogate properties by
in silico computational studies. The physicochemical profile of BSSG
influences its pharmacokinetics (absorption, distribution, metabolism,
and excretion) and toxicity.

Complimentary Contributor Copy

80 L. O. Soto-Rojas, D. Martinez-Fong, I. A. Martinez-Davila et al.

Table 1. General, physical and chemical characteristics of BSSG

Property/molecular Value Ref.
descriptor
CAS number 474-58-8 [42]
IUPAC name (2R,3R,4S,5S,6R)-2-[[(3S,8S,9S,10R,13R,14S,17R)-17-[(2R,5R)-5- [42]
ethyl-6-methylheptan-2-yl]-10,13-dimethyl
2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]
phenanthren-3-yl]oxy]-6-(hydroxymethyl) oxane-3,4,5-triol
Synonyms D- -stigmast-5-en-3- -Sitosterol -D- [42]
glucoside -sitosterol-D-glucoside, b- -sitosterol 3-O- -
D- -sitosteryl- -D-glucopyranoside, BSSG,
-Sitosteryl glucoside, 3- -D-glucosylsitosterol.
Appearance White crystals [42]
Molecular Formula C35H60O6
Chemical structure [43]

Molecular Weight 576.8 g/mol [42]

Melting point 275 to 277°C
DMSO Solubility 3.33 g/L
Value Acceptable range
PSA* 97.958 PSA [43]
TPSA* is often needed for penetrating the BBB
Oral absorption prediction
Water solubility QPlogS -7.225 Predicted aqueous solubility: -6.0 to 0.5
Percent Human Oral 74.699% 25 to 80
Absorption on 0 to 100% (more than 80% is great)
scale.
Plasma protein binding prediction
QPlogKhsa 0.937 Prediction of binding to HSA: -1.5 to 1.5
Metabolism prediction
#metab 6 Number of likely metabolic reactions: 1 to 8 [43]
Excretion prediction
Lipophilicity QPlogPo/w 5.1 Predicted octanol/water partition coefficient: -2.0 to 6.5. [43]
QPpolrz 60.03 Predicted polarizability
QPlogPC16 17.64 Predicted hexadecane/gas partition coefficient: 4 to 18.
QPlogPoct 31.21 Predicted octanol/gas partition coefficient: 8 to 35.
QPlogPw 16.18 Predicted water/gas partition coefficient: 4 to 45.
Cardiotoxicity prediction
QPlogHERG -5.216 The predicted IC50 value for hERG K+ channels blockage. [43]
Not below. -5.0
Abbreviations: BBB, blood-brain barrier; CAS, Chemical Abstracts Service; DMSO, dimethyl sulfoxide; hERG,
The human ether-a-go-go related gene; HSA, human serum albumin; IUPAC, International Union of Pure
and Applied Chemistry; PSA, Van der Waals surface area of polar nitrogen and oxygen atoms; TPSA,
Topological Polar Surface Area. *Predicted Property in silico by QikProp.

Complimentary Contributor Copy

 -Sitosterol- -D-Glucoside (BSSG) 81

The absorption, distribution, metabolism, excretion, and toxicity

profiles for BSSG were reported by Dawood et al. (2020), who showed
that this molecule has the ability to permeate cells, with a reduced
aptitude to cross the blood-brain barrier (BBB), moderate human oral
bioavailability and intestinal absorption. Moreover, BSSG binds to
plasma proteins reducing the concentration of free compounds available
to diffuse to target organs. Also, its metabolic turnover occurs before
reaching the targeted site of action, resulting in efficient elimination from
the body [43]. Although some potential for cardiotoxicity has been
discussed [43], further in-vitro and in vivo, pharmacokinetic,
pharmacodynamic, and toxicity studies are necessary to obtain a
consistent view of the absorption, distribution, metabolism, excretion,
and toxicity profiles of BSSG.

1.2. BSSG as a Health Promoter

BSSG was recognized early in the 20th century as a consistent

obligatory component present in plant extracts with medicinal properties
[9]. Among other phytosterols, BSSG is one of the few food constituents
that have been appointed an approved health claim by the Food and Drug
Administration (FDA) and European Food Safety Authority [EFSA]
[4, 13]. Demonstrated BSSG pharmaceutical properties include
hypocholesterolemic, anti-inflammatory, and anti-carcinogenic effects [4]
and anti-pyretic and immune-modulating properties [44]. The positive
effects of BSSG on human health will be described and discussed in this
section.
As already mentioned, BSSG is structurally and functionally similar
to cholesterol. Interestingly, BSSG consumption interferes with intestinal
absorption of cholesterol through the displacement of cholesterol
micelles (Figure 1 step 2) and competition for cholesterol-binding
proteins [4]. As a result, low-density lipoprotein (LDL) cholesterol levels
decrease, while high-density lipoprotein (HDL) cholesterol and

Complimentary Contributor Copy

82 L. O. Soto-Rojas, D. Martinez-Fong, I. A. Martinez-Davila et al.

triglyceride levels remain within normal ranges [4, 10], thus lowering the
risk for ischemic heart disease [IHD] (Figure 1 step 2).

Figure 1. Schematic representation of beneficial BSSG properties. Abbreviations:

BPH, benign prostatic hyperplasia -sitosterol- -D-glucoside; HDL, high-
density lipoprotein cholesterol; HIV, human immunodeficiency virus; HPV, human
papillomavirus; IGF1, insulin-like growth factor 1; IHD, ischemic heart disease;
LDL, low-density lipoprotein cholesterol; NK, natural killers cells; NSC, neural stem
cell; PI3K, phosphoinositide 3-kinase; TB, pulmonary tuberculosis; Th1, T helper
type 1 lymphocyte. This figure was created with BioRender.

Complimentary Contributor Copy

 -Sitosterol- -D-Glucoside (BSSG) 83

Figure 2. Schematic representation of pathological brain mechanisms triggered by

local or systemic BSSG administration in animal models. Abbreviations: BBB,
blood-brain barrier; BSSG -sitosterol- -D-glucoside. This figure was created with
BioRender.

The nutraceutical BSSG effects have been demonstrated in several

studies [11]; oral BSSG treatment increases fasting plasma insulin levels,
decreases fasting glycemia (Figure 1 step 3), and, simultaneously,
improves oral glucose tolerance. These effects could be explained by an
increase in insulin secretion [12], as well as by direct insulin-mimetic
activity of BSSG, favoring glucose uptake in adipocytes and promoting
adipogenesis (Figure 1 step 4) [11]. Increased glucose uptake has also
been demonstrated in muscle cells, presumptuously due to an increase in
phosphorylation of the AMP-activated protein kinase (AMPK) and
acetyl-CoA carboxylase [45], leading to translocation and expression of
glucose transporter 4 [46]. On the other hand, BSSG has also been shown

Complimentary Contributor Copy

84 L. O. Soto-Rojas, D. Martinez-Fong, I. A. Martinez-Davila et al.

to stimulate lipolysis, not regulated through insulin activity, but rather by

downregulating the expression of the Protein kinase B (Akt) and
phosphoinositide 3-kinase (PI3K) genes (Figure 1 step 4) [11]. Adding to
the above, BSSG acts as a free radical scavenger and a membrane
stabilizer, turning it into an excellent therapeutic candidate drug for
diabetes and obesity (Figure 1 step 3, 4) [13].
BSSG has been used as an adjuvant in the therapy of benign prostatic
hyperplasia (BPH), pulmonary tuberculosis, human immunodeficiency
virus (HIV), and human papillomavirus (HPV) anogenital warts patients
due to its immunomodulatory qualities (Figure 1 step 5) [3, 9, 14, 15].
Studies in vitro and in vivo demonstrate that BSSG enhances T-cell
proliferation in the presence of an antigenic stimulus [9, 44]. This effect
could result from several mechanisms, such as the acquisition of surface
activation antigens (CD25, HLA-DR) and the up-regulation of
transcription factors involved in the growth and proliferation of T-cells
[9]. Moreover, it has been suggested that BSSG enhances the secretion of
interleukin 2 (IL-2) and interferon-gamma (INF- -4
secretion. Therefore, it promotes T helper type 1 (Th1) lymphocyte
differentiation (Figure 1 step 5) over the TH2 phenotype [3, 9],
downregulating antibody production [47]. Also, BSSG increases the
natural killer (NK) cell-mediated lysis of marked cancer cells [3, 9].
Lastly, BSSG decreases cortisol and IL-6 secretion while increasing
dehydroepiandrosterone (DHEA) secretion upon physical stress [14].
Thus, the therapeutical role of BSSG is also under consideration in
chronic inflammatory diseases.
Epidemiological and experimental studies have reported antitumoral
properties for BSSG (Figure 1 step 6), particularly in colon cancer [48].
BSSG decreases the rate of colonic epithelial cell proliferation and

expression of the altered cancerous genome. It has been hypothesized that

these effects are due to BSSG decreasing cholesterol levels and bacterial
metabolism of cholesterol and/or bile acids, etiological factors in colon
cancer [16, 17]. However, specific antitumoral mechanisms by which the
BSSG acts have not been elucidated. Proposed mechanisms include that

Complimentary Contributor Copy

 -Sitosterol- -D-Glucoside (BSSG) 85

BSSG could trigger inhibition of carcinogen production, cancer cell

growth and multiplication, invasion, metastasis, induction of cell cycle
arrest, and apoptosis [18]. Consistent with the latter proposal (BSSG as a
pro-apoptotic factor), it has been shown to cause depolarization of
mitochondrial membrane potential and increase in: i) Bcl-2 associated X-
protein (BAX)/ B-cell lymphoma 2 (Bcl-2) ratio; ii) Fas cell surface
death receptor (FAS) protein levels; iii) caspase-3 and caspase-9 activity;
iv) expression of caveolin-1, p53, and p21 proteins [18, 19].
Finally, several studies have demonstrated anxiolytic, sedative,
analgesic, antimicrobial, hepatoprotective, wound healing [13, 17],
angiogenic [11], and neurotrophic [20] effects of BSSG. Concerning the
neurotrophic effect, BSSG could increase hippocampal neural stem cell
proliferation due to the regulation of several genes, especially inducing
insulin-like growth factor 1 (IGF-1), suggesting BSSG as an alternative
growth factor therapy [20]. However, future research in different
experimental models is required to elucidate the neurotrophic
mechanisms and determine the therapeutic doses of BSSG (Table 2a and
Figure 1 step7).
In conclusion, the mechanisms by which BSSG confers beneficial
health effects remain under study, but the following effects have been
demonstrated (Figure 1 and Table 2a): i) hypolipemiant and antioxidant
effects, decreasing the risk factor of IHD; ii) nutraceutical effects
increasing the fasting plasma insulin levels, decreasing fasting glycemia,
and simultaneously, improving oral glucose tolerance, adjuvant in the
treatment of diabetes and obesity; iii) immunomodulatory effects, and
therefore very useful in the treatment of BPH, pulmonary tuberculosis,
HIV and HPV; iv) antitumoral effects, therapeutic adjuvant against
several types of cancer; and v) neurotrophic effects.

1.3. BSSG as a Neurodegeneration Inducer

Having emphasized in the previous section the beneficial BSSG

effects, in this section, we contrast these observations against findings

Complimentary Contributor Copy

86 L. O. Soto-Rojas, D. Martinez-Fong, I. A. Martinez-Davila et al.

that BSSG also triggers neurodegeneration. The neurodegeneration

process involves gradual and progressive neuronal death, which causes
dysfunction of the nervous system [49]. The discovery of BSSG as a
neurodegeneration inducer was made when it was demonstrated that
BSSG is the main neurotoxin causing a neurological pathology called
ALS / PDC. This neurological disorder, first described in the Western
Pacific islands of Guam and Rota, combines skeletal muscle atrophy with
motor neuron degeneration. This complex pathology has periodically
affected a significant part of the indigenous Chamorro population of
these islands, thus attracting scientific attention over the past century (for
reviews see [21-24]). Efforts to identify the causal agent for the disorder
and its mechanism of action were motivated by the idea that such
information would provide insight into developing therapies against
neurodegenerative disease more generally. After excluding genetic and
viral causes and following leads from the local population, attention was
paid to seed processing of Cycad circinalis [23, 50, 51]. Indeed, the
locals would feed chicken with the final seed washed to ensure no
residual toxicity [50]. These studies culminated with the identification of
BSSG as a potent neurotoxin able to activate N-methyl-D-aspartate
(NMDA) brain receptors (Figure 3 C), leading to glutamate-like
excitotoxicity [25, 26]. Christopher Shaw and colleagues isolated BSSG
and proposed the glutamate excitotoxicity hypothesis as a mechanism of
action for its toxicity (Figure 3A). Their group had previously worked
with methionine sulfoximine (MSO), a byproduct of former industrial
processing of flour known as agenization and inhibitor of glutamine
synthase and gamma-glutamylcysteine synthetase. They showed that
MSO led, likely indirectly through the loss of glutathione (GSH) and
glutamate accumulation, to glutamate release and NMDA activation [52,
53]. BSSG-containing extracts exhibited similar pharmacological
properties to MSO, causing neuronal death in a manner that could be
inhibited by NMDA antagonists, also involving oxidative stress [26, 53] 53].
It is important to emphasize that the previous data were obtained by
incubating brain sections with purified extracts of the compound.

Complimentary Contributor Copy

 -Sitosterol- -D-Glucoside (BSSG) 87

Figure 3. Presumptive pathological mechanisms by which BSSG can trigger

neurodegeneration. (A) BSSG could cause several pathological brain events as
neuroinflammation, ferroptosis, excitotoxicity, and -synucleinopathy. Gene
expression of the putative BSSG brain receptors, LXRB (B), and GluN2 NMDA
receptor (C), in several neuroanatomic areas: 1. Amg, amygdala; 2. ACC, anterior
cingulate cortex; 3. Cd, caudate nucleus; 4. Cbh, cerebellum hemisphere; 5. Cb,
cerebellum; 6. Cx, cortex; 7. FroCx, frontal cortex; 8. HiF, hippocampal formation;
9. Hy, hypothalamus; 10. Acb, nucleus accumbens; 11. Pu, putamen; 12. SC, spinal
cord; 13. SN, substantia nigra -sitosterol- -D-glucoside;
ER, endoplasmic reticulum; GluN2, glutamate receptor, ionotropic, N-methyl D-
aspartate 2A; GPX4, glutathione peroxidase 4; GSH, glutathione; LXRB, liver X
receptor B; mt, mitochondria; NKA, Na/K-ATPase; NMDA, N-methyl-D-aspartate
receptors; ROS reactive oxygen species; TPM, transcripts per million; -syn, -
synuclein. The figure was created with BioRender. The GTEx Analysis Release V8
(dbGaP Accession1056 phs000424.v8.p2) was used to obtain the graphs of gene
expression data in several brain areas.

Complimentary Contributor Copy

88 L. O. Soto-Rojas, D. Martinez-Fong, I. A. Martinez-Davila et al.

Table 2. Dual effects of BSSG, depending on study models

and dose/concentration

Model of Treatment Beneficial/Pathological Proposed molecular mechanism Ref.

study manifestations
a) Beneficial effects
In vivo Daily phytosterols in plasma LDL cholesterol absorption through [69]
(humans). intake (0.6-3.3 g concentrations. the displacement of cholesterol
In vivo human and in plasma from micelles. [10]
(murine 400mg/ kg/ day, cholesterol levels.
model). for 38 days in
murine model).
In vitro (rat -Sitosterol 0.1- in fasting glycemia adipocytes glucose uptake by [11]
adipocytes). 1000 µM. levels. translocation of GLUT4.
adipogenesis. differentiating preadipocytes.
lipolysis. Downregulation of AKT and PI3K
gene expression. cellular cAMP
and activates PKA.
In vivo Oral BSSG (20-30 fasting glycemia insulin secretion (insulin [12]
(murine mg/kg) levels and oral secretagogue)
model) glucose tolerance.
In vitro (L6 -Sitosterol 20 glucose uptake and phosphorylation of the AMPK [45]
myotube µM triglycerides and and ACC.
cells) cholesterol in muscle GLUT4 translocation to the
cells. plasma membrane
In vivo Daily phytosterols fasting glycemia the expression and translocation [46]
(humans) intake (1-2 g). and HbA1c levels. of GLUT4 in several tissues.
In vivo -Sitosterol 20 Normalize the glycemic Restores the insulin receptors and [13]
(murine mg/kg orally for levels and restores the GLUT4 levels. Act as a radical
model) 30 days. lipid profile, oxidative scavenger and membrane stabilizer.
stress markers, and
antioxidant enzymes.
In vitro BSS:BSSG the proliferative the CD25 expression and HLA- [9]
(human (100:1) 10 ng/ml. response of T-cells. Dr activation antigens.
peripheral BSS:BSSG TH1 lymphocyte the secretion of IL-2 and INF-
blood (100:1) 1 µg/ml. differentiation.
lymphocytes)
n/a
NK-cells mediated
lysis of marked cancer
cells.
Ex vivo BSS:BSSG (20 the proliferative the expression of CD25 and
(mononuclear mg: 0.2mg) 3 response of T-cells. HLA-Dr activation antigens.
cells) times a day for 4
weeks

Complimentary Contributor Copy

 -Sitosterol- -D-Glucoside (BSSG) 89

Model of Treatment Beneficial/Pathological Proposed molecular mechanism Ref.

study manifestations
In vitro (in -Sitosterol 10- viable PBMC levels. Dendritic cell activation [44]
pig PBMC 100 µg/ml. mediated by MHC-II and
and BMDC)
the secretion of IFN- .
In vivo -Sitosterol (2 kg the proliferative n/a
(fattener per ton of final response of T-cells and
pigs) feed) for 8 weeks. apolipoprotein A1
plasma concentrations.
In vivo BSS:BSSG (20 As an adjuvant plasma viral loads. [15]
(humans) mg: 0.2mg) 3 stimulates regression of
CD4 cells activity by
times a day for 3 genital warts in HPV
maintaining the TH1 phenotype.
months. patients.
In vivo BSS:BSSG (20 the inflammatory Improve neutrophilia, lymphopenia, [14]
(humans) mg: 0.2mg) 3 response and and leukocytosis, and IL-6
times a day for 4 immunosuppression plasma levels.
weeks. after physical stress.
Inhibits the hormonal cortisol: DHEA-S ratio.
stress response.
In vivo Control diet the rate of colonic cholesterol absorption and [16]
(cancer containing 0.3% - epithelial cell suppress the bacterial metabolism
murine Sitosterol. proliferation and the of cholesterol and/or secondary bile
model acids in the colon.
induced by compartment.
MNU)
In vivo BSSG 50-100 - Antitumoral activity the expression of apoptotic [19]
(Ehrlich mg/kg i.p. once by inducing apoptosis. protein p53, p21, and Bax and
ascites daily for 14 days down-regulation of Bcl-2 protein.
murine the activity of caspase-9 and
carcinoma caspase-3.
model)
In vitro BSSG 1.25-100 hippocampal NSC Enhances the expression of [20]
(NSC µM proliferation and upregulation genes (333) involved
culture) inhibits cell in the mitotic cell cycle. IGF1
differentiation. protein levels. The expression of
downregulation genes (627)
involved in cell differentiation.
b) Toxic effects
Ex vivo (Rat 1:100 dilution of NMDA activation Glutamate excitotoxicity [53]
brain slices) cycad flour extract and GSH depletion
Ex vivo (Rat Methanol Glutamate excitotoxicity and [25]
brain slices) extracted cycad oxidative stress
flour

Complimentary Contributor Copy

90 L. O. Soto-Rojas, D. Martinez-Fong, I. A. Martinez-Davila et al.

Table 2. (Continued)

Model of Treatment Beneficial/Pathological Proposed molecular mechanism Ref.

study manifestations
In vitro 50 µM of BSSG Apoptosis Upregulation of Hsp70 [70]
(NSC-34) for three days
In vivo Fed on 0.5 g Neurodegeneration Glutamate excitotoxicity and [26]
(CD-1 washed cycad associated with motor oxidative stress
mouse) flour pellet per day and cognitive deficits
Glutamate transporters Glutamate excitotoxicity [27]
unchanged mechanism questioned
In vivo Fed on 10, 100, Motor and DA Serum glucosides were associated [70]
(CD-1 and 1000 µg BSSG per neurodegeneration. with astrocyte activation and
C57/BL6 day Astrocyte activation. glutamate excitotoxicity
mice).
In vivo Fed on 1.25 g MM, DA neurons loss LXRB signaling as a response to [63]
(Sprague washed cycad in SN and, -syn altered sterols.
Dawley rat) flour pellet per day aggregates.
Fed on 3 mg Neuroinflammation, Ginseng extract was protective. [29,
BSSG per day DA cell loss, -syn Lewy body-like synuclein 30]
aggregates, and MM. aggregates.
In vivo Intranigral BSSG A1 reactive Nitric oxide production is [28]
(Wistar rat) administration astrocytes. associated with astrogliosis.
(6µg/µL) Shows Lewy body-like DA neurodegeneration is caused by [31]
synuclein aggregates senescence and apoptosis.
and their spreading.
Proposed in this chapter Ferroptosis Lipid peroxidation and GSH [54-
depletion. 56]
NKA Structural similarity between BSSG [64-
inhibition/signaling and ouabain. 67]
Abbreviation: ACC, acetyl-CoA carboxylase; Akt, Protein kinase B; AMPK, AMP-activated protein kinase;
Bax, Bcl-2 associated X-protein; Bcl-2, B-cell lymphoma 2 protein; BMDC, bone marrow dendritic
cells; BSSG -Sitosterol- -D-glucoside; cAMP, cyclic Adenosine Monophosphate; CD4, cluster of
differentiation 4; CD25, surface activation antigens; DA, dopaminergic/dopamine; DHEA-S,
dehydroepiandrosterone sulfate; GLUT 4, glucose transporter 4; GSH, glutathione; HbA1c,
glycosylated hemoglobin; HLA-Dr, Human Leukocyte Antigen DR isotype; Hsp70, 70 kilodalton
heat shock protein; i.p., intraperitoneal; IFN- alfa; INF- -gamma; IGF1,
insulin-like growth factor 1; IL-2, interleukin-2; LDL, low-density lipoprotein cholesterol; LXRB,
liver X receptor B; MHC-II, major histocompatibility complex class II; MM, motor manifestations;
MNU, 1-methyl-1-nitrosourea; n/a, not assessment; NKA Sodium Potassium ATPase; NMDA, N-
methyl-D-aspartate receptors; NSC, neural stem cells; NSC-34, neuroblastoma hybrid cells; PBMC,
peripheral blood mononuclear cells; PI3K, phosphoinositide 3-kinase; PKA, protein kinase A; SN,
substatia nigra; TB, tuberculosis; Th1, T helper type 1. Arrow up = Stimulate/ increase/ improve;
arrow down= reduction/decrease.

Complimentary Contributor Copy

 -Sitosterol- -D-Glucoside (BSSG) 91

In contrast, results in the BSSG-fed mice questioned the excitotoxic

mechanism for neuronal loss [27]. Reduced GSH depletion has been
associated with ferroptosis [54], a type of neuronal death with similar
characteristics to the one suffered by neurons subjected to diverse toxins,
including glutamate excitotoxicity [55]. Transition metal-catalyzed lipid
peroxidation is the hallmark of ferroptosis cell death [54-56].
Interestingly, BSSG administered in the SNpc triggers lipid peroxidation
[28], suggesting that ferroptosis is a possible neuronal death mechanism
of BSSG in the central nervous system (Figure 3A).
Neuroinflammation has been demonstrated in the chronic oral BSSG
administration model [29, 30], which possibly involved microglial and
astrocyte activation through BSSG-dependent interruption of liver X
receptor B (LXRB) signaling (Figure 3 A and B) [41, 57]. Likewise,
pathological -syn accumulation is another mechanism of DA cell death
associated with neuroinflammation triggered by oxidative stress and
direct activation of microglial cells [58-60]. Chronic oral or intranigral
administration of BSSG triggers Lewy body- -syn aggregates
(Figure 2, step 6) in rats [30-32], correlating with the death of DA
neurons [31]. Furthermore, nitric oxide production during BSSG-induced
neuroinflammation was correlated with neuronal cell death (28),
potentially mediating glutamate neurotoxicity [61, 62].
As an important side note, consumption of washed cycad flour
induces ALS/PDC in mice [26]. However, as already discussed, ingestion
of the same diet produces characteristic features of parkinsonism in rats
[63]. The reasons why the mouse and rat respond differentially to BSSG
remain unclear [30, 62].
Due to the structural similarity between BSSG and ouabain (a cardiac
glucoside), another potential target for BSSG could be the Na/K-ATPase
(NKA), a well-established target of ouabain [64]. Although early
experiments appeared to dismiss this possibility, it should be noted that
the assay used depended on activation of NMDA receptors and was,
therefore, indirect [53]. In this context, it was recently shown that
inhibition of NKA activity aggravated -syn pathology in mice [65] [65].
Furthermore, ouabain injection in the rat SNpc and striatum caused

Complimentary Contributor Copy

92 L. O. Soto-Rojas, D. Martinez-Fong, I. A. Martinez-Davila et al.

neurodegeneration [66]. Ouabain toxicity may not be directly related to

its classical action as an NKA inhibitor but seems to be associated to
signal transduction initiated from NKA with GSH protecting from
ouabain-induced cell death [67]. Based on this, we hypothesize that the
local or systemic BSSG administration could inhibit NKA, thus
triggering the pathological -syn aggregation and subsequently
neurodegeneration (Figure 3A, Table 2b). Indeed, pharmacological
activation of NKA could represent a new therapeutic strategy for PD
[65].
In conclusion, the mechanisms by which BSSG can cause
neurodegeneration are unknown, but the following (non-mutually
exclusive) possibilities have been considered depending on the model
study, concentration, and route of administration (Figure 3, Table 2b): i)
glutamate excitotoxicity, ii) glutathione depletion, iii) oxygen and
nitrogen reactive species production, iv) lipid dyshomeostasis, v) and
presumptively the NKA inhibition/signaling. We further propose that
BSSG could drive ferroptosis if it is found to deplete intracellular GSH
pools (as is the case for MSO) or if it causes the accumulation of
polyunsaturated fatty acids through disturbances in cholesterol and/or
sphingolipid metabolism, through LXRB (Figure 3B), in conjunction
with oxidative stress already documented. There is increasing evidence
that changes in the lipid composition of membranes affect receptor-
mediated signaling [68]. Furthermore, BSSG could cause neurotoxicity
due to the expression of LXRB and NMDA receptors in several brain
areas (Figure 3A, B). Last, future experiments should evaluate the
chemical affinity of BSSG to NKA, whose ionic exchange and cell
signaling activities are required for neuronal viability. The outcome of
these studies will provide new targets for PD treatment.

1.4. Main Characteristics of PD

PD belongs to a group of neurological ailments classified as

movement disorders and presents four pathognomonic hallmarks [33,

Complimentary Contributor Copy

 -Sitosterol- -D-Glucoside (BSSG) 93

36]: 1) movement disorders (MDs) and non-movement disorders

(NMDs); 2) DA neurodegeneration (slowly progressive neuronal death)
in the SNpc; 3) chronic neuroinflammation; and 4) Lewy bodies
pathology -syn neuronal inclusions).
The telltale MDs are tremor, rigidity, akinesia, bradykinesia, and
postural instability resulting from the neurodegeneration of the
nigrostriatal pathway. This pathological event involves the progressive
loss of DA neurons in the SNpc and their projections to not only the
striatum [38]. PD patients also show NMDs, such as hyposmia, anosmia,
visual disturbances, depression, cognitive deficits, dementia, sleep
disorders, and intestinal dysbiosis, inclusive several years before the
appearance of MDs [35-37]. These NMDs are caused, in part, by the DA
innervation loss to cerebral cortex areas and local neurodegeneration
-syn neuronal aggregates [71]. Unfortunately, MDs in
PD patients only appear when a high percentage (around 80%) of DA
neurons in the SNpc have degenerated [39, 40], preventing early
diagnosis and limiting therapeutic duration.
Based on postmortem analysis of PD patients and experimental
results in parkinsonian animal models, it has been postulated that the
neuroinflammatory environment may precede and trigger the DA
neurodegeneration process [72-74]. Neuroinflammation is characterized
by the activation of microglia and reactive astrocytes. These cells
synthesize and release immunomodulatory molecules such as cytokines,
chemokines, cell adhesion molecules, reactive oxygen, and nitrogen
species (ROS and RNS), among others [75]. These secreted factors
activate more microglia and reactive astrocytes, disrupt BBB, and recruit
professional immune cells [76, 77]. This hazardous positive feedback
converts the neuroinflammatory environment into a vicious, self-
perpetuating cycle.
The last pathognomonic hallmark of PD is the misfolding of the
small (14 kDa) presynaptic nerve -syn [78]. The
-syn include: a) maintenance of
synaptic vesicle delivery; b) DA release and transport; c) regulation and
interaction with the cell membrane; d) protein degradation; e)

Complimentary Contributor Copy

94 L. O. Soto-Rojas, D. Martinez-Fong, I. A. Martinez-Davila et al.

mitochondrial regulation through the autophagy-lysosome pathway [79,

80]. Mutations in the SNCA -syn) have been
closely linked to genetic/familial PD [81, 82]. The pathological
-syn misfolding is that, apart from altering its
physiological roles, it promotes neuronal death in many
neurodegenerative diseases -
synucleinopathies with Lewy bodies, PD with
dementia, and multiple system atrophy [80, 83]. The common mark of
these diseases -syn inclusions [84], which
in neurons are collectively known as Lewy pathology -syn
inclusions have also been observed in non-neuronal cell types, known as
microglial or astrocytic inclusions and glial bodies of Papp-Lantos in
oligodendrocytes [85-88].

1.5. Animal Models for the Study of PD

Many animal models have been developed to understand the

pathophysiology of PD and test therapeutic strategies (Table 3 and 4).
However, not all of them reproduce all the pathognomonic hallmarks of
the disease required to be qualified as a suitable animal model of PD [41,
89]. In this chapter, we have classified the available rodent PD models
into two groups: Neurotoxin-based models (Table 3) and transgenic
models
(Table 4).
This latter group is further divided into a) genetically engineered
rodent model, b) Bacterial Artificial Chromosomes (BACs) transgenic
mice, and c) viral vector-transduced adult neuronal cells (Table 4).
The main drawbacks of neurotoxin-based animal models are that the
neurodegeneration is acute, lack Lewy pathology, and the
neuroinflammation mechanism is different from chronic
neuroinflammation (Table 3). The classical and best-characterized model
is the intrastriatal lesion with 6-hydroxydopamine (6-OHDA).

Complimentary Contributor Copy

 Table 3. Neurotoxin-based PD animal models

Animal model Behavioral alterations Neurodegeneration Pathological molecular events Lewy pathology Ref.
6-OHDA MM: gait disturbance and There is a decrease of around 80- Microglial and astrocytic Does not induce [90,
Unilateral single (6-20 rotational behavior induced by 90% in striatal innervation and 60- activation in SNpc and Str. Lewy pathology. 92-
Complimentary Contributor Copy

amphetamine*. 80% in TH-positive neurons in Increase of TNF- , IL- - 101]

administration in the Str NMM: Gastro-intestinal and SNpc. Neuronal death is due to 6.
of rats. spatial memory dysfunction, apoptosis and occurs progressively Increase of ROS, iNOS, and nitrite
DLB but in a short time. levels.
Rotenone MM: postural instability, No significant decrease of TH Microglial and astrocytic -syn [102-
i.p. injection unsteady gait, rigidity, staining in SNpc and a nerve activation in the Str, HiF, and Cx. aggregates in DA 108]
1.5-3 mg/kg/day for 21- bradykinesia. terminal Str degeneration. Induces high levels of TNF- , IL- neurons, HiF, Cx,
28 days or systemic NMM: cognitive, sleep, and Apoptosis has been suggested as a and ENS.
infusion of mice and gastrointestinal dysfunction. cell death mechanism.
rats
MPTP MM: In NHPs; akinesia, In NHPs: DA neurons decrease Microglial and astroglial -syn [109-
NHPs: 0.01-0.175 rigidity, postural instability. In (around 95%) in SNpc and DA activation. aggregation in SNpc. 114]
mg/kg (i.v.) for 2 weeks rodent models, the treatment levels in Str (80 %). Oxidative stress is present in the Mice do not induce
or 1 mg/kg for 3 days. did not induce severe MM In mice: decrease of TH-positive neurons of SNpc. Lewy pathology.
Mice: 20-25 mg/kg NMM: Cognitive deficits in neurons (30%) in SNpc, in striatal
(s.c.) one dose each 24 h NHPs. innervation (17%), and the Nurr1
for 5-7 days. mRNA levels (44%).
LPS MM: Increase in drug-induced There is a decrease of around 50- Microglial and astroglial Cytoplasmic [72,
Rats: Intranigral single rotational behavior and poor 80% of TH-positive cells and 34 activation. Increases of pro- -syn 115-
administration performance in the rotarod 50% DA levels in STR. inflammatory cytokines such as accumulation in the 122]
(0.6 16 g) test. NMM: impaired memory, Increase of Pro-apoptotic proteins TNF- , IL-1 , IL-6, and TGF- 2. SNpc neurons. Only
anxiety, DLB, and gastric such as BAX, BAD, and caspase- Increase of ROS, iNOS, nitrite, in mice, but not in
dysmotility. 3. lipid, and protein oxidation levels. rats or NHPs.
 Table 3. (Continued)

Animal model Behavioral alterations Neurodegeneration Pathological molecular events Lewy pathology Ref.
Paraquat MM: In rodent models, there The low dose reduced the density Microglial and astroglial activation Intracellular [123-
i.p. administration low are no clear motor deficits. of striatal DA terminals by 87%, in the ventral midbrain, frontal Cx, -syn 128]
Complimentary Contributor Copy

dose 5 mg/kg and high NMM: Cognitive deficits and and 18% neurons in SNpc. and CB. Increased levels of pro- aggregates in DA
dose 10 mg/kg in mice anxiety. The high dose decrease around inflammatory cytokine GM-CSF. neurons of the SNpc.
one dose for the week 94% striatal DA terminals and Elevated ROS levels and oxidative
for 3 weeks. 28% neurons in SNpc. stress.
Maneb MM: decrease locomotor Reduction of DA levels in Str and Microglial and astroglial - [129-
i.p. administration30 activity loss of TH immunoreactivity in Str activation, and increase of iNOS syn aggregates. 132]
mg/kg NMM: Impairs learning at days 7 and 14 post-treatment, levels (Mn-EBDC in vitro assays).
in mice for 7 days. 50 ability and reduces respectively with Mn-EBDC.
µM Mn-EBDC in vitro exploratory activity.
for 2 hrs.
Abbreviations: 6-OHDA, 6-hydroxydopamine; CB, cerebellum; Cx, cortex; DA, dopamine or dopaminergic; DLB, depressive-like behavior; ENS, enteric nervous system;
GFAP, glial fibrillary acidic protein; GM-CSF, granulocyte-macrophage colony-stimulating factor; HiF, hippocampal formation; i.p., intraperitoneal; i.v..intravenous;
IL, interleukin; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; MM, motor manifestations; Mn-EBCD, manganese ethylene-bis-dithiocarbamate- is
the major active element of maneb; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; n/a, not assessment; NHPs, non-human primates; NMM, non-motor
manifestations; Nurr1, nuclear receptor related-1 protein; ROS, reactive oxygen species; s.c., subcutaneous; SNpc, substantia nigra pars compacta; Str, striatum; TGF-
, Transforming growth factor- TH, tyrosine-hydroxylase; TNF- , tumor necrosis factor- -syn -synuclein. * The amphetamine rotation test is used to
determine the degree of motor impairment induced by neurotoxic or viral vectors, it has also been used to demonstrate functional recovery-induced neuroprotective
therapies aimed at preserving/restoring the DA neuronal function.
 -synuclein-based transgenic animal models

-synuclein mutation Behavioral alterations Neurodegeneration Pathological molecular Lewy pathology Ref.
events
-synuclein
Complimentary Contributor Copy

A30P mutation MM: locomotor deterioration. No significative TH-positive Neuroinflammation or - [133-

Under the Mu Thy1 NMM: impairments in cell loss in SNpc and Str. oxidative stress: n/a. syn in the AMG, Cx, HiF, 135]
promoter in the rat and cognition and fear SC, SNpc, and Str.
mouse. conditioning.
A53T mutation MM: locomotion deficits. 60% apoptotic cells in Neuroinflammation or -syn expression in the Cx, [136-
Under the Mu Thy1 NMM: Reduced anxiety and SNpc, Cx, and Str. Early oxidative stress: OB, Str, brainstem, CB, and 138]
promoter sleep disturbances. apoptotic cell loss in the CB. n/a. SC.
In the rat and mouse.
A53T mutation MM: severe motor Degeneration of nigrostriatal Neuroinflammation: Incl -syn in Cx, [l39-
Under the Mu prion impairments. NMM: anxiety pathway and sciatic nerve. astrogliosis (evaluated with SC, and neocortex. 141]
promoter and depressive-like behavior. Wallerian degeneration in the GFAP).
in the mouse. SC.
Y39C mutation Without MM Increase the apoptotic cell Neuroinflammation or Pser129 -syn and -syn [142]
Under the Mu Thy1 NMM: death in the Cx, SN, Str, oxidative stress: n/a. inclusions
promoter in the mouse. Cognitive deficits. evaluated by the TUNEL in Cx, SN, Str, and SC.
assay.
E57K mutation MM: locomotor decrease 50% decrease in the density of Neuroinflammation: astroglial -syn in the neuropil, [143,
Under the Mu Thy1 (evaluated by rotarod test). positive TH terminals in Str, reactivity (identified with neocortex, limbic system, 144]
promoter in the mouse. NMM: Cognitive deficits with damage to Cx and HiF. basal ganglia, and SN.
markers).
 Table 4. (Continued)

-synuclein mutation Behavioral alterations Neurodegeneration Pathological molecular Lewy pathology Ref.
events
-synuclein using viral vectors
Complimentary Contributor Copy

WT or A53T mutation MM: Increase in rotation Progressive nigral DA neuron Neuroinflammation or -syn in SNpc, [145,
Under the CBA behavior induced by loss (30-80%). oxidative stress: n/a reticular formation, and 146]
promoter in the mouse apomorphine test NMM: n/a. Decrease of DA levels and TH VTA.
and rat. activity in Str and SNpc.
A30P mutation MM were not detected. Around 50% neuronal cell loss Neuroinflammation: -syn expression in the [135,
Under the CBA NMM: n/a in SN and dystrophic neurites Astrocityc evaluation to SNpc, SNpr, and striatal 147]
promoter in mouse and in SN and Str. evidence -syn aggregates. axons
rat
WT, A53T, A30P MM: n/a Around 30% of selective DA Neuroinflammation or -syn expression in SNpc [148]
mutation Under the NMM: n/a neurons decrease in SN. oxidative stress: n/a and the Str.
PGK promoter in the
rat.
BAC transgenic rodents -synuclein
A30P mutation in the Without MM No significative TH-positive Neuroinflammation or Expression of human -syn [149-
mouse and rat. NMM: exhibit an anxiety-like cell loss in the SNpc. oxidative stress: n/a in the Cx, HiF, Str, VTA, 151]
behavior. and SNpc neurons.
A53T mutation in the Without MM DA nigral degeneration Neuroinflammation or Lewy bodies in OB, Cx, [151,
mouse and rat. NMM: REM sleep behavior (around 17%). oxidative stress: n/a Str, SN, DMV, and DRN. 152]
disorder and hyposmia.
Abbreviations: AMG; amygdala; BAC, bacterial artificial chromosome; CB, cerebellum; CBA, chicken -actin; Cx, cortex; DA, dopamine/dopaminergic; DMV, the dorsal
motor nucleus of the vagus; DRN, dorsal raphe nucleus; GC, galactocerebroside; GFAP, glial fibrillary acidic protein; HiF, hippocampal formation; MM, motor
manifestations; n/a, not assessment; NMM, non-motor manifestations; OB, olfactory bulb; PGK, phosphoglycerate kinase 1; Pser129, phospho- -synuclein in serine
129; REM, rapid eye movement -binding protein ; SC, spinal cord; SN, substantia nigra; SNpc, substantia nigra pars compacta; SNpr,
substantia nigra pars reticulata; Str, striatum; TH, tyrosine-hydroxylase; Thy-1, cell surface antigen; VTA, ventral tegmental area; -syn -synuclein.
 -Sitosterol- -D-Glucoside (BSSG) 99

This animal model has been helpful to understand possible

pathological mechanisms at the cellular level, such as inhibition of the
mitochondrial complex I and subsequent ROS generation culminating in
DA neurodegeneration through apoptosis [89, 90]. Like the 6-OHDA
model, the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model
is also widely used [89].
MPTP is more practical because it crosses BBB but is only effective
in mice and monkeys and elicits nonspecific effects in other tissues [89]
[89].
Unlike the two previous models, neurodegeneration induced with the
insecticide -syn aggregation in
different brain areas [91]. Nevertheless, rotenone effects are highly toxic
and unspecific, affecting the DA system, other neuronal systems, and
organs. The high mortality rate of rotenone in cells and experimental
animals comes from crossing the cell membranes because of its high fat-
solubility [91]. Additional details of neurotoxin-based PD animal models
are depicted in Table 3.
In addition to all those disadvantages, the correlation of behavioral,
neuroanatomical, and biochemical alterations has been established with
the rapid onset of a non-progressive DA neuronal death (Table 3).
Therefore, it is a complex task translating findings from these models to
the human condition characterized by slow onset and progression. The
current tendency is to understand the pathophysiological PD mechanisms
and propose neuroprotective or neurorestorative therapeutic approaches
at critical time points of the disease in an animal model that closely
resembles PD.
The main advantage of transgenic models is that they generate the
Lewy pathology (Table 4), making it possible to study the
pathophysiological and propagation -syn and
propose anti- -syn therapies. However, it is necessary to emphasize that
there is no evident degeneration of the nigrostriatal pathway in these
animal models and, consequently, no behavioral alterations, suggesting
-syn expression is not toxic in these animal models. Besides,
the results are variable and contradicting due to unspecific viral vector

Complimentary Contributor Copy

100 L. O. Soto-Rojas, D. Martinez-Fong, I. A. Martinez-Davila et al.

transduction, different transcriptional promoters, and diverse viral vectors

(Table 4).

1.6. BSSG Administration Provides a More Faithful PD Model

Remarkably, all the pathognomonic PD hallmarks with a slow and

progressive onset are developed in rats by the oral or local administration
of BSSG. Therefore, this model is more suitable for clarifying the
mechanism of neuroinflammation and neurodegeneration underpinning
PD and providing more timely therapies [28-32, 41]. The oral BSSG
administration in rats provided the first model that faithfully reproduced
both the behavioral and the cellular characteristics observed in human PD
brains [29, 30, 41]. Pioneer experiments in mice led to the development
of this model. Tabata et al. confirmed that the consumption of chow
pellets containing BSSG was neurotoxic in mice, as first demonstrated
with the consumption of cycad flour [25, 26]. Besides, it was
demonstrated that BSSG induces the same pathological effects of ALS-
parkinsonism dementia complex, namely, motor deficits and motor
neuron loss in the lumbar and thoracic spinal cord, along with decreased
glutamate transporter labeling and increased glial fibrillary acid protein
(GFAP, as an astrocytic activation marker) reactivity [70]. Importantly,
decreased tyrosine-hydroxylase (TH, as a DA neuron marker) labeling in
the SNpc was observed, along with evidence of neuronal apoptosis [70].
These results were accompanied by in vitro experiments that implicated
sterol glucosides as one of the causal factors in the motor neuron
pathology associated with cycad consumption toxicity and ALS-PDC
[70].
Subsequently, Shen et al. exposed Sprague-Dawley male rats to
washed cycad flour pellets and observed progressive parkinsonism with
no spinal cord or muscle pathology [63]. Specifically, cycad-fed rats
displayed spontaneous unilateral rotation, shuffling gait, and other motor
disabilities after 2 to 3 months of treatment. An initial decrease in the DA
levels was observed in the STR, followed by DA neurodegeneration in

Complimentary Contributor Copy

 -Sitosterol- -D-Glucoside (BSSG) 101

the SNpc. Remarkably, pathological -syn aggregates were detected in

different brain regions. These latter results were reproduced ex vivo. The
application of an organic extract of cycad to an organotypic slice culture
of the rat SNpc and STR caused selective DA neuronal loss and
pathological -syn aggregates [63]. As mentioned above, the reasons
why the spinal cord is protected in the rat model remain unclear [30].
The logical next step was to use this BSSG model, which closely
resembles PD, to test therapeutics. The first approach was by Van
Kampen et al. using an extract (G115) of the traditional Chinese
medicinal plant Panax ginseng [29]. Oral administration of this extract to
rats chronically exposed to BSSG reduced DA cell loss, microgliosis, and
-syn aggregates. It also prevented the development of
locomotor deficit, suggesting that the ginseng extract could be a potential
neuroprotective agent for the PD treatment [29].
Afterward, the same authors evaluated the therapeutic efficacy of
levodopa, the first line of therapy for PD [30], in the early parkinsonism
state induced by BSSG. Acute levodopa treatment (20 mg/kg,
intraperitoneal injection), preceded (30 minutes) by the decarboxylase
inhibitor benserazide (15 mg/kg, subcutaneous injection), reversed the
behavioral alterations in the BSSG-induced early parkinsonism [30]. In
this study, Van Kampen et al. described additional attractive features of
the long-lasting BSSG exposure model (chronic oral administration),
including MDs and NMDs (Table 5 and Figure 2). The MDs developed
gradually over time (a PD characteristic absent in other models) and were
also reversed by the levodopa treatment. Besides, the long-lasting BSSG
administration caused progressive DA neurodegeneration and Lewy
pathology in several subcortical and cortical brain regions. The slowly
progressive nature of behavioral and cellular alterations that closely
approximate those observed in patients (Table 5) makes it a good
candidate model for screening new neuroprotective therapies for PD [30].

Complimentary Contributor Copy

102 L. O. Soto-Rojas, D. Martinez-Fong, I. A. Martinez-Davila et al.

2. ABOUT OUR RESEARCH

2.1. Intranigral BSSG Administration Offers Additional

-Synucleinopathies

Recently, our working group evaluated the effectiveness of a single

unilateral intranigral BSSG injection in causing a PD-like phenotype in
Wistar rats [28, 31, 32]. It was interesting that stereotaxic BSSG
administration also reproduced the progressive PD hallmarks, requiring
less time and significantly lower cost (because of the unilateral single
dose) than the systemic administration model described in the previous
section. Table 5 compares the pathognomic PD hallmarks between
systemic and local BSSG administration.
Surprisingly, sensorimotor alterations and neurodegeneration of the
nigrostriatal pathway were observed in both hemispheres (Table 5),
despite the BSSG administration having been applied only on the left
SNpc [32]. We propose that these effects come from the development of
Lewy pathology, which begins at the site of the BSSG administration site
and spreads to the contralateral hemisphere and neuroanatomically
interconnected regions (Table 5). Rats administered with BSSG orally
also develop Lewy pathology in several brain nuclei. However, since the
oral administration leads to a systemic distribution of BSSG, this model
does not support the spread (prion effect) of -syn. The hypothesis that
-syn aggregates can spread in a prion-like
fashion through cell-to-cell
cell- -cell transmission between anatomically
interconnected areas has been proposed previously [153]. Such spreading
leads to cell death propagation and explains the MDs and NMDs
observed in the stereotaxic model (Figure 2, step 6).
-syn aggregates trigger
neuronal death remain unknown (Figure 3A). However, it has been
suggested that pathological -syn aggregates can alter mitochondrial
respiration and subsequently generate ROS release. The resulting drop in
ATP levels and dysfunction of the dendritic neural network can lead to

Complimentary Contributor Copy

 -Sitosterol- -D-Glucoside (BSSG) 103

caspase-1- mediated neuronal death [154]. In the stereotaxic BSSG

model, we further demonstrated a correlation between the pathological -
syn aggregates with senescence and apoptosis of DA neurons (Table 5,
Figure 2, step 4), supporting their mediation in the BSSG-induced cell
death [31]. However, the possibility of neurodegeneration via ferroptosis
remains to be explored (see above). Besides, we found senescence and
apoptosis markers in activated microglia and reactive astrocytes, astrocytes
supporting that these cells also die in the advanced stage of the disease.
This finding suggests that glial cell death is also implicated in PD
pathology (Figure 2, step 4) [31]. The coincidence of our data with
previous reports supports the hypothesis -syn
accumulation can trigger DA cell loss by activating the innate and
adaptive immune system [155] -
syn accumulation is the direct cause of the glial cell activation, the
increased oxidative stress, and the peripheral immune cell infiltration
[58-60, 156].
Based on this evidence, we propose that the BSSG intranigral
administration causes neuroinflammation (Table 5) as occurs in PD
brains [28], as further supported by the following findings: 1) increased
levels of pro-inflammatory cytokines (Figure 2, step 3); 2) oxidative and
nitrative stress; 3) microglial activation (Figure 2, step 4); 4) A1 reactive
astrocyte (neurotoxic) in the SNpc (Figure 2, step 4); 5) disruption of the
BBB permeability; and 6) leukocytes infiltration (Figure 2, step 5). In this
kind of neuroinflammation, the source of pro-inflammatory molecules
could be both microglial/astrocytic cells and infiltrated leukocytes. Thus,
these cell types also contribute to the process and severity of BSSG-
induced neurodegeneration (Figure 2, step 3-5). Future research on the
BSSG stereotaxic model is required to determine whether the
neuroinflammatory environment dependent on Lewy pathology promotes
neurodegeneration in anatomically interconnected neuronal regions.

Complimentary Contributor Copy

104 L. O. Soto-Rojas, D. Martinez-Fong, I. A. Martinez-Davila et al.

Table 5. Comparison between the oral and intranigral BSSG model

Features Oral BSSG administration [29, Intranigral (stereotaxic) BSSG

30] administration [28, 31, 32]
General features
Animal strain Sprague-Dawley rats Wistar rats
Dose/ Flour pellets of BSSG (3 mg/day
concentration five days a week for 4 months) of DMSO).
Times At 4, 6, 8, and 10 Mos following At 15, 30, 60, and 120 days after the single
evaluated initial BSSG exposure. intranigral administration.
Behavioral alterations
Non-motor Hyposmia (from 3 months AITE) Progressive depressive-like behavior (from
alterations and cognitive impairment (10 60 days AI) and hyposmia (30 days AI).
months AITE).
Motor Progressive locomotor asymmetry Progressive sensorimotor dysfunction,
alterations (4 months AITE), and a decrease postural instability, claudications, and
of locomotor activity and uncoordinated gait (from 15 days AI), and
coordination (6 months AITE). locomotor activity decrease (30 AI).
Neurodegenerative process
Slow and Progressive DA neuron loss in theProgressive DA neuron loss in the injured
progressive SNpc evidenced by TH marker SNpc, control SNpc, and VTA evidenced by
loss of DA (from 6 months AITE) and Nissl TH marker (from 15 days AI), showing a
neuronal and staining (8 months AITE), maximum decrease of 70-80% TH+ cells in
its branches. showing a maximum decrease of injured SNpc (4 months AI). Bilateral
70-80% TH+ cells (10 months progressive neurodegeneration showed by F-
AITE). Progressive loss of DA J C staining (15 days AI). Progressive loss of
terminals in STR evidenced by DA terminals in injured and control STR (15
DAT immunolabeling (4 months days AI), and injured (30 days AI) and
AITE). control (60 days AI) SNpr evaluated by TH
marker. MSN atrophy of both STR (15 days
AI).
Other A significant decrease of -III
neuronal synaptophysin as a synaptic tubulin (neuronal cytoskeleton marker, from
markers marker (until the end of the study- 30 days AI) and NTSR1 (non-DA phenotype
10 months AITE) marker, 15 days AI).
Mechanism Progressive apoptosis of SNpc Bilateral apoptosis (evidenced by caspase-3
of cellular neurons, analyzed by caspase-3 marker, from 60 days AI) and senescence
death immunostaining and TUNEL assay (evidenced by -Galactosidase staining, from
(from 6 months AITE). 30 days AI) of neurons, astrocytes, and
microglia cells in the SNpc.
Neuroinflammation environment
Astrocyte A1 n/a The A1 reactive astrocyte in SNpc with a
(neurotoxic) maximum expression at 15 and 30 days AI,
reactivity and associated with neurodegeneration.

Complimentary Contributor Copy

 -Sitosterol- -D-Glucoside (BSSG) 105

Features Oral BSSG administration [29, Intranigral (stereotaxic) BSSG

30] administration [28, 31, 32]
Microglia A significant increase of iba1+ The microglial activation in SNpc was
activation cells as an activated microglia evaluated with OX42 and Iba1 markers, with
marker (from 4 months AITE) in a maximum expression at 15 and 30 days,
SNpc. and associated with neurodegeneration.
Infiltrated n/a Increased the BBB permeability and
immune cells infiltrated leukocytes (assessed by CD45
marker) in SNpc with a maximum expression
at 15 and 30 days AI.
Pro- n/a Increased TNF- , IL- -6 pro-
inflammatory inflammatory cytokines levels in the SNpc
mediators with a maximum expression at 15 and 30
days AI.
Oxidative Increase of Grp78 as an ER stress Increase in levels of NO (with a maximum
/nitrative marker in SNpc and ST (until 8 expression at 15 and 30 days AI), and
stress months AITE). MDA/4-HAE (until 120 days AI).
Lewy pathology
Begins the At 4 months AITE increases At 15 days AITE increases pathological -
Lewy pathological -syn aggregates and syn aggregates and begins in the SNpc.
pathology. begins in the OB.
Brain nuclei OB, STR, SNpc, M1Cx, EC, CA1, OB, HiF, Cx, M1Cx, LC, SNpc, and STR.
with Lewy DG.
pathology.
Spreading of Future experiments are required to Yes.
-syn. determine it.
Abbreviations: 4-HAE, 4-hydroxyalkenals; AI, after injury; AITE, after initial toxin exposure; BBB, blood-
brain barrier; BSSG -sitosterol- -D-glucoside; C3, complement component 3; CA1, hippocampal
cornu ammonis; CD45, lymphocyte common antigen; Cx cortex; DA, dopaminergic, dopamine; DAT,
dopamine transporter; DG, dentate gyrus; DMSO, dimethyl sulfoxide; EC, entorhinal cortex; ER,
endoplasmic reticulum; F-J C, Fluro-Jade C; GFAP, glial fibrillary acidic protein; Grp78, Glucose-
regulated protein 78 kDa; HiF, hippocampal formation; Iba1, ionized calcium-binding adaptor
molecule 1; IL, Interleukin; LC, locus coeruleus; M1Cx, motor cortex; MDA, malondialdehyde;
MSN, medium spiny neurons; NO, nitric oxide; NO, nitric oxide; NTSR1, neurotensin Receptor 1;
-binding protein B; SNpc, substantia nigra pars compacta;
SNpr, substantia nigra pars reticulate; STR, striatum; TH, tyrosine hydroxylase; TNF- , tumor
necrosis factor-alpha; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; VTA,
ventral tegmental area.

The recent discovery of neurotoxic A1 reactive astrocytes has

demonstrated that neuroinflammation in neurodegenerative diseases uses
mechanisms different from innate immune response inflammation.
inflammation
Liddelow et al. designated A1 astrocytes to those reactive astrocyte
subtypes capable of inducing neuronal death in several neurodegenerative
diseases, including PD [117]. These researchers demonstrated that

Complimentary Contributor Copy

106 L. O. Soto-Rojas, D. Martinez-Fong, I. A. Martinez-Davila et al.

activated microglia with an M1 pro-inflammatory phenotype induce A1

reactive astrocytes through the release of interleukin 1- (IL-
necrosis factor (TNF), and complement component 1q (C1q). In turn, the
A1 reactive astrocyte, releasing molecules not yet identified, interrupts
functions of the brain homeostasis, such as neuronal survival,
synaptogenesis, and phagocytosis, while inducing the death of neurons
and oligodendrocytes [117]. The importance of neurotoxic A1 reactive
astrocytes in neurodegenerative diseases has opened new research
avenues to know their role in the pathogenesis of PD and develop
targeted therapies against neurotoxic A1 reactive astrocytes to reduce DA
neuronal death [28].
In summary, the stereotaxic BSSG animal model recapitulates all the
pathognomonic PD hallmarks, including Lewy pathology, which makes it
also suitable for studying the pathogenesis of -synucleinopathies.
In particular, the BSSG model is a valuable tool to evaluate the following
cell events underling misfolded -syn neurotoxicity: 1) Prion-like
spreading to the central nervous system and peripheral organs; 2)
mitochondrial dysfunction; 3) cell death mechanisms; 4) involvement of
the neurovascular unit and the BBB dysfunction; 5) peripheral immune
cell infiltration to the central nervous system; 6) molecular
intercommunication between A1 reactive astrocytes and M1 microglia.
Clarifying those pathophysiological mechanisms will settle the bases for
appropriate therapies targeting pathological -syn and neurotoxic A1
reactive astrocytes to develop new therapies.

CONCLUSION

While BSSG offers diverse clinical benefits, its neurodegenerative

potential on the dopaminergic system outweighs its health promotion
actions. Furthermore, contrary to studies on its beneficial potential, the
studies in humans and rodents have rigorously correlated BSSG chronic
intake with PD development. Since the boundary between the good and

Complimentary Contributor Copy

 -Sitosterol- -D-Glucoside (BSSG) 107

bad sides of BSSG consumption is unknown, it is best to avoid its use

because there are other therapeutic alternatives on the conditions it acts.
Since BSSG reproduces all PD hallmarks in the rat, it has become an
invaluable tool to untangle the intricate physiopathological mechanisms
of PD and select the more effective antiparkinsonian therapies. Between
the oral and stereotaxic BSSG administration, the latter procedure
endows more competitive advantages for experimental studies on
mechanisms of DA neurodegeneration and its reversion by
neuroregenerative therapies. The stereotaxic BSSG administration is less
expensive and elicits the PD hallmarks soon, and it is mainly adequate to
clarify the mechanisms of pathological -syn spreading and A1 reactive
astrocyte neurotoxicity. The new antiparkinsonian therapies aim to block
pathological -syn spreading and neurotoxic A1 reactive astrocyte
activation and promote neural regeneration. These therapeutic objectives
can be achieved in a timely and significant manner using the stereotaxic
BSSG model of PD.

ACKNOWLEDGMENTS

This work was supported by UNAM-PAPIIT (IA202021).

REFERENCES

[1] Lopez-Salazar H, Camacho-Diaz BH, Avila-Reyes SV, Perez-

Garcia MD, Gonzalez-Cortazar M, Arenas Ocampo ML, et al.
Identification and Quantification of beta-Sitosterol beta-d-
Glucoside of an Ethanolic Extract Obtained by Microwave-
Assisted Extraction from Agave angustifolia Haw. Molecules.
2019;24(21).

Complimentary Contributor Copy