biomolecules

Review
Stalling the Course of Neurodegenerative Diseases: Could
Cyanobacteria Constitute a New Approach toward Therapy?
Vitória Ramos 1 , Mariana Reis 2 , Leonor Ferreira 2,3 , Ana Margarida Silva 1 , Ricardo Ferraz 1,4 ,
Mónica Vieira 1,5 , Vitor Vasconcelos 2,3 and Rosário Martins 1,2, *

1 School of Health, Polytechnic Institute of Porto (ESS/P.PORTO), Rua Dr. António Bernardino de Almeida 400,
4200-072 Porto, Portugal; mvitorianetor@gmail.com (V.R.); agl@ess.ipp.pt (A.M.S.); rferraz@ess.ipp.pt (R.F.);
mav@ess.ipp.pt (M.V.)
2 Interdisciplinary Centre of Marine and Environmental Research, University of Porto (CIIMAR/CIMAR),
Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos s/n, 4450-208 Matosinhos, Portugal;
mreis@ciimar.up.pt (M.R.); lferreira@ciimar.up.pt (L.F.); vmvascon@fc.up.pt (V.V.)
3 Department of Biology, Faculty of Sciences, University of Porto (FCUP), Rua do Campo Alegre, Edifício FC4,
4169-007 Porto, Portugal
4 Associated Laboratory for Green Chemistry—Network of Chemistry and Technology (LAQV-REQUIMTE),
Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Rua do Campo
Alegre 687, 4169-007 Porto, Portugal
5 Center for Translational Health and Medical Biotechnology Research (TBIO/ESS/P.PORTO), Rua Dr. António
Bernardino de Almeida 400, 4200-072 Porto, Portugal
* Correspondence: mrm@ess.ipp.pt; Tel.: +351-222-061-000; Fax: +351-222-061-001

Abstract: Neurodegenerative diseases (NDs) are characterized by progressive and irreversible neu-
ronal loss, accompanied by a range of pathological pathways, including aberrant protein aggregation,
altered energy metabolism, excitotoxicity, inflammation, and oxidative stress. Some of the most
common NDs include Alzheimer’s Disease (AD), Parkinson’s Disease (PD), Multiple Sclerosis (MS),
Amyotrophic Lateral Sclerosis (ALS), and Huntington’s Disease (HD). There are currently no available
cures; there are only therapeutic approaches that ameliorate the progression of symptoms, which
Citation: Ramos, V.; Reis, M.;
makes the search for new drugs and therapeutic targets a constant battle. Cyanobacteria are ancient
Ferreira, L.; Silva, A.M.; Ferraz, R.;
prokaryotic oxygenic phototrophs whose long evolutionary history has resulted in the production of
Vieira, M.; Vasconcelos, V.; Martins, R.
a plethora of biomedically relevant compounds with anti-inflammatory, antioxidant, immunomodu-
Stalling the Course of
latory, and neuroprotective properties, that can be valuable in this field. This review summarizes
Neurodegenerative Diseases: Could
Cyanobacteria Constitute a New
the major NDs and their pathophysiology, with a focus on the anti-neurodegenerative properties of
Approach toward Therapy? cyanobacterial compounds and their main effects.
Biomolecules 2023, 13, 1444. https://
doi.org/10.3390/biom13101444 Keywords: cyanobacteria; spirulina; neurodegenerative diseases; natural products

Academic Editor: Antonio

Di Stefano

Received: 30 August 2023 1. Introduction

Revised: 20 September 2023 Neurodegenerative diseases (NDs) are a broad category of neurological ailments that
Accepted: 22 September 2023
induce progressive and irreversible neuronal loss in the central and peripheral nervous
Published: 25 September 2023
system (CNS and PNS, respectively) [1]. The loss of neurons, which are unable to efficiently
regenerate owing to their terminally differentiated nature, promotes the collapse of func-
tional neuronal networks and the loss of synaptic plasticity, impairing brain and nerve
Copyright: © 2023 by the authors.
function. This results in a wide and often overlapping spectrum of symptoms typical of
Licensee MDPI, Basel, Switzerland.
these disorders, such as impaired memory, cognition, behavior, sensory, and/or motor
This article is an open access article function [2].
distributed under the terms and Common NDs include Alzheimer’s Disease (AD), Parkinson’s Disease (PD), Multiple
conditions of the Creative Commons Sclerosis (MS), Amyotrophic Lateral Sclerosis (ALS), and Huntington’s Disease (HD).
Attribution (CC BY) license (https:// Each disease differs in clinical presentation and underlying physiology but they all share
creativecommons.org/licenses/by/ converging neurodegenerative pathways that lead to neuronal death, such as aberrant
4.0/). protein aggregation, neuroinflammation, oxidative stress, altered energy metabolism, and

Biomolecules 2023, 13, 1444. https://doi.org/10.3390/biom13101444 https://www.mdpi.com/journal/biomolecules

Biomolecules 2023, 13, 1444 2 of 34

excitotoxicity [1]. Although the etiology of these diseases is multifactorial, aging is the
primary risk factor because it is a natural process involving the dysregulation of multiple
pathways implicated in neurodegeneration. However, environmental factors, genetic
makeup, and other medical disorders, such as metabolic diseases, can all play a role [3].
NDs place significant health, social, and economic burdens on patients and caregivers
and represent a serious public health concern. Millions of individuals are affected world-
wide and this number is predicted to escalate rapidly as the population and life expectancy
increase, making it a leading cause of mortality and morbidity [4].
NDs are complex diseases with multiple factors involved in their origin and progres-
sion. Despite extensive research, most attempts to develop effective treatments have been
unsuccessful, many due to adverse side effects such as nausea, diarrhea, fatigue, hepatotox-
icity, bradycardia, and secondary autoimmune adverse effects [2,5]. Currently, there are no
therapeutic options to reverse the onset of NDs. Most of the few approved drugs such as the
acetylcholinesterase (AChE) inhibitors donepezil, rivastigmine, and galantamine, and the
N-methyl-D-aspartate (NMDA) receptor antagonist memantine for AD [6]; dopaminergic
drugs such as levodopa for PD [7]; riluzole and edaravone for ALS [8]; and tetrabenazine
and deutetrabenazine to reduce chorea in HA [9] only provide symptom management,
while disease-modifying drugs are still in their infancy. Therefore, most conditions progress
without remission and are ultimately fatal. Given the gravity and rising prevalence of NDs,
it is imperative to identify new and effective pharmacological candidates and targets [5].
Although compounds produced naturally by our body are considered promising in
the treatment of NDs, such as melatonin and the immunosuppressive cytokine IL-10 [10,11],
natural products derived from plants, algae, macrofungi, invertebrates, and microorgan-
isms have traditionally been key contributors to drug development due to their great
diversity and structural complexity [12]. Natural compounds, synthetic derivatives, and
pharmacophore-inspired drugs account for more than 60% of all approved drugs [13].
Cyanobacteria are primitive prokaryotes that produce several bioactive metabolites
with diverse pharmacological properties, such as being neuroprotective, antioxidant, anti-
inflammatory, and immunomodulatory [14–16], which can be an asset in the treatment of
NDs.
Given the ubiquity of NDs and the potential of cyanobacteria in innovative treatment
options, the purpose of this review is to compile existing evidence on the potential of
cyanobacteria-derived products to combat neurodegeneration and the major NDs.

2. Cyanobacteria
Cyanobacteria, also known as green–blue algae, are a diverse phylum of gram-negative
microorganisms that are unique in their ability to perform oxygenic photosynthesis, setting
them apart from other prokaryotes [17]. Cyanobacteria were among the first species to
live on Earth, with more than 3.5 billion years of fossil records. These organisms are
key oxygen producers and nitrogen fixers that play important roles in ecosystems and in
shaping the biosphere [18]. Cyanobacteria exhibit diverse morphologies, ranging from
single cells to colonies and filaments, and can be present at high densities, such as in
crusts or blooms. They thrive in a wide range of environments, including freshwater,
marine, and terrestrial ecosystems, even those deemed hostile to life [17]. Their ability
to adapt and survive is a result of their metabolic diversity, flexibility, and reactivity,
which involves unique biochemical pathways that yield a variety of metabolites including
proteins, essential fatty acids, vitamins, minerals, flavonoids, carotenoids, chlorophylls, and
phycobiliproteins [18,19]. Cyanobacteria also offer economic and sustainable advantages as
they have a fast-growing potential with high yields without the need for many resources,
making them an appealing option for biomedical research [20].
Cyanobacteria’s health benefits have long been documented as Nostoc species have
been used to treat gout, fistulas, and cancer since 1500 B.C. and as Aztecs employed Spirulina
strains as a food source [20,21]. Spirulina remains one of the most extensively studied genera
of cyanobacteria and is widely used as a dietary supplement due to its impressive health
Biomolecules 2023, 13, 1444 3 of 34

benefits and nutritional makeup, which includes a high protein content (60–70% of dry
weight), vitamin B12, essential fatty acids, polysaccharides, and various pigments such as
β-carotene and phycocyanin, one of the most biologically active components [20].
Several cyanobacteria-derived metabolites have been identified, exhibiting anti-cancer,
anti-viral, anti-bacterial, and anti-diabetic properties, among others. Some of these, such
as the anticancer drug AdcetrisTM , are in commercial use, whereas others are undergoing
preclinical and clinical trials [20,22,23].
Regarding neuroprotection, cyanobacteria produce several neuroactive compounds
that have been linked to ecological roles, such as enhancing competitiveness in grazing
defenses by reducing palatability and repelling predators [24]. However, the effects of
cyanobacteria-derived products can vary widely, from the medicinal potential of phyco-
cyanin to lethal cyanotoxins like microcystins, nodularin, and β-N-methylamino-L-alanine
(BMAA), whose exposure has been associated with the onset of NDs [24,25].

3. Neurodegeneration
Neurodegeneration is a complex process characterized by the progressive structural
and functional loss of neuronal cells in the CNS and PNS; it is the primary pathologic
feature of NDs. Several pathways, including abnormal protein aggregation, oxidative stress,
neuroinflammation, excitotoxicity, mitochondrial dysfunction, and apoptosis, have been
implicated in the pathogenesis of neurodegeneration [1]. In this context, cyanobacterial com-
pounds exhibit a variety of properties that can aid in the battle against neurodegenerative
processes. This section provides a brief overview of the key hallmarks of neurodegeneration
and how cyanobacterial natural products can help ameliorate them.
Pathological protein aggregation is a typical trait of NDs and contributes to their
diagnosis and categorization. Many NDs are proteinopathies caused by the abnormal
aggregation of proteins, such as β-amyloid (Aβ) and tau in AD, α-synuclein in PD, or TAR
DNA-binding protein 43 (TDP-43) in ALS [26]. Protein misfolding and oligomerization
lead to extracellular or intracellular aggregates, which can appear as oligomers, amorphous
assemblies, or highly structured amyloid fibrils and plaques. This is often favored by
gene mutations, post-translational modifications, or inadequate proteostasis and protein
quality control [27]. Protein aggregates spread in a prion-like manner, with a protein seed
enlisting normally folded molecules to adopt abnormal conformations [28]. Aggregate
toxicity is mostly mediated by gain-of-function, resulting in cellular dysfunction, synaptic
loss, and brain injury [26,27]. Cyanobacterial natural products have shown the potential to
alleviate proteotoxicity. For example, the patented Klamin® extract from Aphanizomenon
flos-aquae, rich in phenylethylamine, interferes with Aβ aggregation kinetics on a cellular
model [29] and phycocyanin from Leptolyngbya sp. N62DM reduces the polyglutamine
(polyQ) aggregation in a worm model of HD [30].
Most NDs are also linked to elevated levels of oxidative stress markers. Oxidative
stress is caused by an imbalance between the production of reactive oxygen and nitro-
gen species (ROS and RNS) and the antioxidant defense system. The CNS is particularly
vulnerable to oxidative stress because of its high metabolic rate and oxidizable substrate
content [31]. A pro-oxidant state promotes lipid, protein, and DNA damage as well as cel-
lular injury and mitochondrial malfunction, all of which contribute to neurodegeneration.
In a complex and reciprocal interplay, oxidative stress promotes many traditional neu-
rodegenerative pathways while also being aggravated by events such as aberrant protein
aggregation and metal homeostasis loss [32]. There is substantial evidence that Spirulina
and other cyanobacteria have strong antioxidant capacity, enhancing the antioxidant de-
fense system, scavenging ROS, inhibiting lipid peroxidation, and modulating genes related
to the oxidative stress response [33–37].
Another common feature in NDs is chronic neuroinflammation. The inflammatory
response in the brain is mediated by microglia and astrocytes. Harmful stimuli, such as
protein aggregation and oxidative stress, activate glial cells causing their phenotype to shift
from neuroprotective to pro-inflammatory. While decreasing their phagocytic function,
 enhancing the antioxidant defense system, scavenging ROS, inhibiting lipid peroxidation,
and modulating genes related to the oxidative stress response [33–37].
Another common feature in NDs is chronic neuroinflammation. The inflammatory
Biomolecules 2023, 13, 1444 response in the brain is mediated by microglia and astrocytes. Harmful stimuli, such 4 of as
34
protein aggregation and oxidative stress, activate glial cells causing their phenotype to
shift from neuroprotective to pro-inflammatory. While decreasing their phagocytic
function, activated
activated microgliamicroglia release pro-inflammatory
release pro-inflammatory mediators mediators
such as suchtumor as necrosis
tumor necrosis
factor
(TNF)-α, interleukin (IL)-1β, IL-16, nitric oxide (NO), and chemokines [38]. These[38].
factor (TNF)-α, interleukin (IL)-1β, IL-16, nitric oxide (NO), and chemokines These
mediators
mediatorsastrocytes
stimulate stimulate to astrocytes to activate
activate further further
reactions thatreactions
can impair thatsynaptic
can impair synaptic
function, the
function, thebarrier,
blood–brain blood–brain barrier,
metabolic metabolic
function, and function,
glutamateand glutamatefurther
metabolism, metabolism, further
exacerbating
exacerbating neurodegeneration
neurodegeneration [38,39]. Cyanobacteria
[38,39]. Cyanobacteria possess strong possess strong anti-inflammatory
anti-inflammatory properties
properties
that thatshown
have been have been shown
to impact to impact
microglial microglial
activation andactivation
response,and response,
decrease decrease
inflammatory
inflammatory mediators, and modulate
mediators, and modulate inflammatory genes [40–44]. inflammatory genes [40–44].
Excitotoxicityisisan
Excitotoxicity anabnormal
abnormal process
process of neuronal
of neuronal deathdeath
causedcaused by pathologically
by pathologically high
high levels
levels of excitatory
of excitatory neurotransmitters,
neurotransmitters, primarily primarily
glutamate. glutamate. This amplifies
This amplifies or prolongs or
prolongs
the the of
activation activation
glutamate of receptors,
glutamatecausing
receptors,rapidcausing rapid and
and prolonged prolonged
calcium (Ca2+calcium
) influx
(Ca2+neurons,
into ) influx into neurons,
which triggerswhich triggers
several several Ca2+-dependent
Ca2+ -dependent enzymes that enzymes
initiate that initiate a
a neurotoxic
neurotoxic
cascade [45].cascade
This has[45]. This implications
negative has negative suchimplications
as mitochondrial such malfunction,
as mitochondrial ROS
malfunction, ROS overproduction, and the release of pro-apoptotic proteins, among
overproduction, and the release of pro-apoptotic proteins, among others. Mitochondria are
others. Mitochondria
particularly are particularly
sensitive because sensitive
they capture excessbecause
cytosolicthey
Cacapture
2+ , causingexcess cytosolic Ca2+,
the mitochondrial
causing the transition
permeability mitochondrialpore to permeability
open, resultingtransition
in energypore to open, and
malfunction resulting in energy
the activation of
malfunction
apoptotic celland
deaththepathways
activation of In
[45]. apoptotic cell death
this context, pathways [45]. In
cyanobacteria-derived this context,
products have
cyanobacteria-derived
shown products
promise. For instance, have shown
phycocyanin frompromise.
SpirulinaForsp.instance,
inhibits phycocyanin
cellular glutamate from
Spirulina sp. inhibits cellular glutamate excitotoxicity [46]; biochanin A (1) (Figure 1),
excitotoxicity [46]; biochanin A (1) (Figure 1), which has been identified in cyanobacterial
which has
blooms, been identified
prevents in cyanobacterial
mitochondrial dysfunction blooms, prevents
and related mitochondrial
cellular apoptosis dysfunction
[47,48] and
2+ in neurons as
and related cellular apoptosis [47,48] and kalkitoxin (2) (Figure 1) from Lyngbya majuscula
kalkitoxin (2) (Figure 1) from Lyngbya majuscula inhibits the elevation of Ca
inhibits
it the elevationion
is a voltage-gated of channel
Ca2+ in neurons
inhibitoras[49].
it is a voltage-gated ion channel inhibitor [49].

Figure 1.
Figure 1. Structure
Structure of of biochanin-A (1), aa phytoestrogen
biochanin-A (1), that prevents
phytoestrogen that mitochondria dysfunction,
prevents mitochondria dysfunction, and
and
kalkitoxin (2),
kalkitoxin (2), aa lipopeptide
lipopeptide that
that interacts
interacts with
with voltage-sensitive
voltage-sensitive sodium
sodiumchannels.
channels.

Neurodegeneration is
Neurodegeneration is aa complex
complex process
process and
and different
different pathological
pathological pathways
pathways maymay
play varying
play varying roles
roles in
in the
the development
development of of each
each ND.
ND. Since
Since these
these processes
processes are intertwined,
are intertwined,
addressing many
addressing many modes
modes of of action
action through
through combinatorial
combinatorial multi-target
multi-target therapy,
therapy, such
such as
as the
the
use of
use of cyanobacteria,
cyanobacteria, is
is aa promising
promising strategy
strategy for
for ND
ND prevention
prevention and
and treatment
treatment[1].
[1].

4.
4. Cyanobacteria
Cyanobacteria Potential
Potential against
against Neurodegenerative
Neurodegenerative Diseases
Diseases
There
There are numerous examples in the literature of cyanobacteria’s potential
are numerous examples in the literature of cyanobacteria’s potential as
as aa source
source
of compounds or extracts with potential in ND therapy. The following section
of compounds or extracts with potential in ND therapy. The following section reviews reviews
the
the
mainmain characteristics
characteristics of the
of the major
major NDsNDsandandthe
thetherapeutic
therapeuticpotential
potential of
of cyanobacteria-
cyanobacteria-
derived
derived compounds
compounds or or extracts.
extracts.
4.1. Cyanobacteria against Alzheimer’s Disease
4.1. Cyanobacteria against Alzheimer’s Disease
Alzheimer’s Disease (AD) is an age-related ND that mostly affects patients aged 65
Alzheimer’s Disease (AD) is an age-related ND that mostly affects patients aged 65
years and older [6]. It is the leading cause of dementia, accounting for 60–70% of the
years and 50
estimated older [6]. It
million is the
total leading
cases [50]. Itcause of dementia,
is characterized byaccounting for 60–70% of the
two main neuropathological
estimated 50 million total cases [50]. It is characterized by two main neuropathological
features in the brain: the extracellular deposition of senile plaques composed of Aβ-peptide
features
and in the brain: ofthe
the accumulation extracellular
intracellular deposition of seniletauplaques
hyperphosphorylated proteincomposed of Aβ-
in neurofibrillary
tangles. These, along with other pathological processes such as acetylcholine deficiency,
vascular damage, oxidative stress, inflammation, and mitochondrial dysfunction, lead to
neuronal death and atrophy, primarily in the entorhinal cortex and hippocampus, resulting
in severe cognitive impairment, memory loss, and behavioral changes [51]. AD can have
multiple causes, such as genetic mutations, mainly in the amyloid precursor protein (APP),
 peptide and the accumulation of intracellular hyperphosphorylated tau protein in
neurofibrillary tangles. These, along with other pathological processes such as
acetylcholine deficiency, vascular damage, oxidative stress, inflammation, and
mitochondrial dysfunction, lead to neuronal death and atrophy, primarily in the
Biomolecules 2023, 13, 1444 entorhinal cortex and hippocampus, resulting in severe cognitive impairment, memory 5 of 34
loss, and behavioral changes [51]. AD can have multiple causes, such as genetic mutations,
mainly in the amyloid precursor protein (APP), presenilin-1 (PSEN-1), presenilin-2
(PSEN-2), and apolipoprotein E (ApoE) genes; lifestyle and environmental factors; and
presenilin-1 (PSEN-1), presenilin-2 (PSEN-2), and apolipoprotein E (ApoE) genes; lifestyle
other medical issues [6].
and environmental factors; and other medical issues [6].
The two main pharmacological classes used in AD are AChE inhibitors, namely
The two main pharmacological classes used in AD are AChE inhibitors, namely
donepezil, rivastigmine, and
donepezil, rivastigmine, andgalantamine,
galantamine, and and
the NMDAthe NMDA receptorreceptor
antagonist antagonist
meman-
memantine. However, these options only provide temporary
tine. However, these options only provide temporary symptom relief, failing symptom relief, failing
to halt to
halt or regress the progression of the disease [52]. Other potential
or regress the progression of the disease [52]. Other potential treatment targets can in- treatment targets can
include immunotherapy,small-molecule
clude immunotherapy, small-moleculeinhibitors,
inhibitors,antioxidants,
antioxidants, and and anti-inflammatory
anti-inflammatory
drugs
drugs [52,53].
[52,53].
The
The potential
potential of of cyanobacteria
cyanobacteria against against AD AD isis vast,
vast, asas reviewed
reviewed by by Castaneda
Castaneda et et al.
al.
(2021) [24]. Recent studies have reinforced this hypothesis
(2021) [24]. Recent studies have reinforced this hypothesis (Table 1). (Table 1).
One
One of of the
the most
mostexplored
exploredtreatment
treatmentapproaches
approaches forfor
ADAD is restoring
is restoring cholinergic
cholinergic sig-
signaling.
naling. In In ADAD patients,
patients, lowlow levels
levels of the
of the neurotransmitters
neurotransmitters acetylcholine
acetylcholine (ACh)(ACh) andand bu-
butyrylcholine
tyrylcholine (BCh) (BCh) andand
high high expression
expression of AChE
of AChE and butyrylcholinesterase
and butyrylcholinesterase (BChE) (BChE)
were
were reported
reported [54].strategy
[54]. The The strategy of inhibiting
of inhibiting thesethese enzymes,
enzymes, whichwhich hydrolyze
hydrolyze ACh AChand BCh, and
BCh, increases their concentration in the synaptic cleft and thus
increases their concentration in the synaptic cleft and thus reduces symptoms [54]. reduces symptoms [54].
Cyanobacteria-derived
Cyanobacteria-derivedAChE AChEand andBChEBChEinhibitors
inhibitors were
were reported.
reported. Anatoxin-a(S)
Anatoxin-a(S) (3)
(Figure 2) from
(3) (Figure Anabaena
2) from flos-aquae
Anabaena is an is
flos-aquae irreversible AChEAChE
an irreversible inhibitor but it is
inhibitor butalso
it aispotent
also a
neurotoxin that canthat
potent neurotoxin cause
cansevere
causecholinergic poisoning
severe cholinergic when administered
poisoning when administeredto rats (0.1–1.0
to rats
mg/kg)
(0.1–1.0[55].
mg/kg)Nostocarboline (4) (Figure(4)
[55]. Nostocarboline from Nostoc
2) (Figure 2) fromis anNostoc
inhibitoris anofinhibitor
AChE and of BChE,
AChE
with half-maximal
and BChE, inhibitory concentration
with half-maximal (IC50) values (IC
inhibitory concentration of 5.3
50 ) µM [56]
values and
of 5.3 13.2
µM µM
[56] [57],
and
respectively.
13.2 µM [57],However,
respectively.it is However,
also a neurotoxin,
it is alsoshowing moderate
a neurotoxin, showingtoxicity when tested
moderate toxicity in
crustaceans
when tested[56]. Although described
in crustaceans [56]. Although as a potent neurotoxin
described as a potent produced by cyanobacteria,
neurotoxin produced by
anatoxin-a(S)
cyanobacteria,isanatoxin-a(S)
also one of the least
is also oneunderstood
of the leastand monitored
understood and[58]. In fact, [58].
monitored as recently
In fact,
reviewed, studies involving
as recently reviewed, cyanobacteria
studies involving neurotoxins
cyanobacteria neurotoxinssuch such as anatoxin-a
as anatoxin-a(S) (S) in
in
standardized neuronal cell
standardized neuronal celllines
linesandandmammals
mammalsare arestill
stillscarce
scarceandandresults
resultsareare inadequate
inadequate to
to confirm
confirm itsits real
real toxicity
toxicity [59].
[59].

Figure 2. Structure
Figure 2. Structure of
of anatoxin-a(S)
anatoxin-a(S) (3) and nostocarboline
(3) and nostocarboline (4),
(4), cyanobacteria-derived
cyanobacteria-derived AChE
AChE and
and
BChE inhibitors.
BChE inhibitors.

A phytosterol-richextract
A phytosterol-rich extractofof Phormidium
Phormidium autumnale
autumnale obtained
obtained through
through supercritical
supercritical fluid
fluid extraction with ethanol (SFE-EtOH) revealed moderate to high inhibitory activity
extraction with ethanol (SFE-EtOH) revealed moderate to high inhibitory activity against
against
AChE (IC AChE (IC50 =µg/mL)
50 = 65.80 65.80 µg/mL) and lipoxygenase
and lipoxygenase (IC50 = (IC 50 =µg/mL)
58.20 58.20 µg/mL) while showing
while showing a high
aantioxidant
high antioxidant
capacity capacity
(IC50 = 7.40(ICµg/mL).
50 = 7.40 Theµg/mL). Theofpresence
presence of the stigmasterol
the phytosterol phytosterol
stigmasterol
(5) (Figure 3)(5)in(Figure 3) in the
the extract extract significantly
significantly correlates correlates
with AChE with AChE inhibition
inhibition as it showedas it
showed interactions
interactions withAChE
with several severalbinding
AChE binding sites in molecular
sites in molecular docking docking assays [60].
assays [60].
Refaay
Refaay et al. (2022)
et al. (2022) [61]
[61] found that fraction
found that fraction 77 of
of the Anabaena variabilis
the Anabaena variabilis methylene
methylene
chloride/methanol (1:1) extract effectively reduced AChE activity (73.6%). This can
chloride/methanol (1:1) extract effectively reduced AChE activity (73.6%). be can
This due be
to
the presence of two aromatic compounds, the flavonoid 5,7-dihydroxy-2-phenyl-4H-chrome-
due to the presence of two aromatic compounds, the flavonoid 5,7-dihydroxy-2-phenyl-
4H-chrome-4-one (6) and the alkaloid 4-phenyl-2-(pyridin-3-yl)-quinazoline (7), shown
in Figure 3, which interact with the allosteric binding site of AChE in molecular docking
studies.
In another in vitro experiment, a crude methylene chloride/methanol (1:1) extract of
Oscillatoria sancta lowered AChE activity by 60.7% [62]. The ethanolic extract of Nostoc sp.
also showed significant inhibitory action against AChE (69.9%) at 3 mg/mL and against
BChE (72.7%) at 5 mg/mL, as well as a high radical scavenging ability [63].
Biomolecules 2023, 13, x 6 of 32

Biomolecules 2023, 13, 1444 6 of 34

4-one (6) and the alkaloid 4-phenyl-2-(pyridin-3-yl)-quinazoline (7), shown in Figure 3, which
interact with the allosteric binding site of AChE in molecular docking studies.

Figure 3.
Figure 3. Structure of stigmasterol
Structure of stigmasterol (5),
(5), 5,7-dihydroxy-2-phenyl-4H-chrome-4-one
5,7-dihydroxy-2-phenyl-4H-chrome-4-one(6),
(6),and
and4-phenyl-2-
4-phenyl-
2-(pyridin-3-yl)-quinazoline (7), which interact with AChE in silico.
(pyridin-3-yl)-quinazoline (7), which interact with AChE in silico.

In another
Other in vitro
possible experiment,
therapeutic targets a crude
include methylene
lowering chloride/methanol
the Aβ load, which(1:1) can beextract
accom- of
Oscillatoria
plished sancta lowered
by hindering AChE activity
Aβ formation [64]. Luoby 60.7%
and Jing [62]. The [65]
(2020) ethanolic
showed extract of Nostoc sp.
that phycocyanin
also showed
(0.5–50 µg/mL) significant inhibitory
from Spirulina sp. action against AChE
spontaneously inhibits(69.9%)
the Aβat 3formation
mg/mL and against
process of
BChE (72.7%)
bovine at 5 mg/mL,
serum albumin (BSA) asbywell as a highinradical
interacting scavenging
a gomphosis abilityAnother
structure. [63]. study found
Other possible
that phycocyanin at atherapeutic targets include
5:1 (Aβ: phycocyanin) molarlowering
ratio hadthe Aβ load, which activity,
anti-amyloidogenic can be
accomplished
as by hindering
seen by its ability to inhibitAβ formation
Aβ40/42 [64]. Luo
fibrillation [66].and Jing (2020) [65] showed that
phycocyanin (0.5–50ofµg/mL)
The inhibition from Spirulina
the amyloidogenic sp. spontaneously
pathway enzymes is inhibits the Aβstrategy
an important formationfor
process of bovine serum albumin (BSA) by interacting in a gomphosis structure. Another
reducing Aβ-peptide synthesis. This stops the conversion of APP into Aβ-peptide via
study found
sequential that phycocyanin
proteolytic cleavages byatβ-secretase
a 5:1 (Aβ: phycocyanin)
1 (BACE-1) molar ratio
and γ-secretase had anti-
enzymes [64].
amyloidogenic activity, as seen by its ability to inhibit Aβ40/42 fibrillation [66].
BACE-1 inhibitors derived from cyanobacteria have been identified, such as tasiamide B (8)
(FigureThe4)inhibition of the
isolated from amyloidogenic
Symploca sp. [67,68] pathway enzymes
and its analog is an important
tasiamide strategy
F (9) (Figure for
4) from
reducingsp.
Lyngbya Aβ-peptide synthesis.
[69]. Tasiamide B (IC50This= 80stops
nM) is the conversion
eight times more of APP intothan
effective Aβ-peptide
tasiamide via F
sequential proteolytic cleavages by β-secretase 1 (BACE-1) and γ-secretase enzymes [64].
(IC 50 = 690 nM) due to modifications in the residues that engage in hydrophobic interactions
BACE-1
with the inhibitors
receptor’s derived
pocket and from cyanobacteria
provide have been
the inhibitory effectidentified,
[69]. Thesesuchcan as
be tasiamide
the starting B
point for the design of more potent and selective BACE-1 inhibitors
(8) (Figure 4) isolated from Symploca sp. [67,68] and its analog tasiamide F (9) (Figure 4) [67,68].
fromPhycobiliproteins
Lyngbya sp. [69].from cyanobacteria
Tasiamide B (IC50also = 80have
nM) potential
is eightastimes
BACE-1 moreinhibitors.
effectiveMolec-
than
ular docking studies show that phycocyanin from
tasiamide F (IC50 = 690 nM) due to modifications in the residues that Leptolyngbya sp. N62DM interacts
engage with in
BACE-1 in an energetically favorable manner [70]. In the same
hydrophobic interactions with the receptor’s pocket and provide the inhibitory effect [69].study, an experiment was
conducted
These can using be theCaenorhabditis
starting pointelegans for theCL4176,
designa of transgenic
more potentmodeland of AD that expresses
selective BACE-1
Aβ in its muscle
inhibitors [67,68].
1–42 cells. It was found that phycocyanin administered through the medium
(100 µg/mL) was able to rescue paralysis worms [70]. Similarly, Chaubey et al. (2019) [71]
Phycobiliproteins from cyanobacteria also have potential as BACE-1 inhibitors.
found that phycoerythrin from Lyngbya sp. A09DM exhibited significant interaction and
Molecular docking studies show that phycocyanin from Leptolyngbya sp. N62DM interacts
binding affinity with BACE-1 in molecular docking studies and protein–protein interac-
with BACE-1 in an energetically favorable manner [70]. In the same study, an experiment
tions in vitro. These results were also further supported by in vivo experiments on C.
was conducted using Caenorhabditis elegans CL4176, a transgenic model of AD that
elegans CL4176, where treatment with phycoerythrin (100 µg/mL) led to a reduction in Aβ
expresses Aβ1–42 in its muscle cells. It was found that phycocyanin administered through
deposition and senile plaque formation.
the medium (100 µg/mL) was able to rescue paralysis worms [70]. Similarly, Chaubey et
A study looked at the effects of oral pre-treatment with a 70% ethanol extract of Spir-
al. (2019) [71] found that phycoerythrin from Lyngbya sp. A09DM exhibited significant
ulina maxima (SM70EE) on rats with cognitive impairment caused by intracerebroventricular
interaction and binding affinity with BACE-1 in molecular docking studies and protein–
injection of Aβ1–42 . The extract (150 and 450 mg/kg/day) decreased the levels of APP and
protein interactions in vitro. These results were also further supported by in vivo
BACE-1, thereby reducing APP processing and lowering Aβ accumulation in the hippocam-
experiments on C. elegans CL4176, where treatment with phycoerythrin (100 µg/mL) led
pus. It also improved cognition, reduced AChE activity, and suppressed hippocampal oxida-
to a stress
tive reduction in Aβ deposition
by improving and senile
the antioxidant plaque
system. Theformation.
treatment stimulated the brain-derived
neurotrophic factor (BDNF)/phosphatidylinositol-3 kinase (PI3K)/serine/threonine pro-
tein kinase (Akt) signaling pathway, which reduced glycogen synthase kinase-3 (GSK3β)
phosphorylation, contributing to BACE-1 suppression [72].
Biomolecules2023,
Biomolecules 13,1444
2023,13, x 77 of 32
of 34

Figure 4.
Figure 4. Structure
Structure of
of the
the cyanobacterial
cyanobacterial BACE-1
BACE-1 inhibitor
inhibitor tasiamide
tasiamide BB (8)
(8) and
and its
its analog
analog tasiamide
tasiamide
F (9).
F (9).
® , a supplement derived from
A study
Galizzi et looked
al. (2023) at [73]
the studied
effects oftheoral
effectspre-treatment
of KlamExtrawith a 70% ethanol extract of
®
Spirulina maxima
Aphanizomenon (SM70EE)
flos-aquae, on diet
in a high-fat ratsrodentwithmodel
cognitive impairment KlamExtra
of neurodegeneration. caused by
® ® ®
intracerebroventricular
is a combination of the patentedinjection of Aβ1–42. Klamin
extracts The extractand (150 and 450 mg/kg/day)
AphaMax decreased
. Klamin contains a
the levels of APP and BACE-1, thereby reducing APP processing and lowering Aβ
concentrated dose (15–18 mg) of phenylethylamine (10) (Figure 5), a compound that modu-
accumulation
lates in the hippocampus.
both the nervous It also improved
and immune systems, as well ascognition,
phycocyanins,reduced AChE activity,
mycosporine-like
amino-acids,
and suppressed hippocampal oxidative stress by improving the antioxidant monoamine
and AFA-phytochrome, which are neuroprotectants and selective system. The
oxidase
treatment B inhibitors
stimulated[74]. AphaMax® isfactor
Additionally, neurotrophic
the brain-derived rich in(BDNF)/phosphatidylinositol-
phycocyanins (25–30%) and
polyphenols, which are powerful
3 kinase (PI3K)/serine/threonine antioxidants,
protein kinase (Akt)and signaling
anti-inflammatory
pathway, molecules
which reduced[75].
Specifically, polyphenols were also found
glycogen synthase kinase-3 (GSK3β) phosphorylation, to be involved in the regulation of autophagy
contributing to BACE-1
in various NDs ®
suppression [72].[76]. Treatment with KlamExtra (0.9 mg/mouse) induced a pattern of
decreased
Galizzi BACE-1 and PSEN-1
et al. (2023) expression,
[73] studied the effects resulting in reduced
of KlamExtra APP processing
®, a supplement andfrom
derived the
accumulation of Aβ. It also safeguarded neural function and synaptic transmission by ele-
Aphanizomenon flos-aquae, in a high-fat diet rodent model of neurodegeneration.
vating synaptophysin levels and maintaining normal neuronal morphology. Furthermore,
KlamExtra® is a combination of the patented extracts Klamin® and AphaMax®. Klamin®
the extract improved the levels of metabolic markers related to glucose metabolism and
contains a concentrated dose (15–18 mg) of phenylethylamine (10) (Figure 5), a compound
showed anti-inflammatory properties by increasing IL-10 and modulating the astrocyte
that modulates both the nervous and immune systems, as well as phycocyanins,
and microglia activation, with a decrease in the astrocyte marker glial fibrillary acid pro-
mycosporine-like amino-acids, and AFA-phytochrome, which are neuroprotectants and
tein (GFAP) and an increase in soluble triggering receptor expressed on myeloid cells-2
Biomolecules 2023, 13, x selective monoamine oxidase B inhibitors [74]. Additionally, AphaMax® is rich 8 of in
32
(sTREM-2) [73]. Particularly, the increase in the immunosuppressive cytokine IL-10 has
phycocyanins (25–30%) and polyphenols, which are powerful antioxidants, and anti-
been described as promising in ND therapeutics, as recently reviewed [11].
inflammatory molecules [75]. Specifically, polyphenols were also found to be involved in
the regulation of autophagy in various NDs [76]. Treatment with KlamExtra® (0.9
mg/mouse) induced a pattern of decreased BACE-1 and PSEN-1 expression, resulting in
reduced APP processing and the accumulation of Aβ. It also safeguarded neural function
and synaptic transmission by elevating synaptophysin levels and maintaining normal
neuronal morphology. Furthermore, the extract improved the levels of metabolic markers
related to glucose metabolism and showed anti-inflammatory properties by increasing IL-
10 and modulating the astrocyte and microglia activation, with a decrease®in the astrocyte
Figure 5.
Figure 5. Structure
Structure of of phenethylamine,
phenethylamine, one one ofof the
the main
main components
componentsof ofthe Klamin® extract.
theKlamin extract.
marker glial fibrillary acid protein (GFAP) and an increase in soluble triggering receptor
expressed on myeloid cells-2
Neurofibrillary (sTREM-2) [73]. Particularly, the increase in are
the
Neurofibrillarytangles,tangles,which
whichare arecomposed
composed of of
hyperphosphorylated
hyperphosphorylated tau tau
protein,
protein,
immunosuppressive
also a hallmark of cytokine
AD. Kinases, IL-10 has GSK3β,
mainly been described
are as promising
responsible for in phosphorylation
tau ND therapeutics,
are also a hallmark of AD. Kinases, mainly GSK3β, are responsible for tau
as recently
and reviewed
thus, reducing [11].
enzymatic activityenzymatic
can reduce tau load
phosphorylation and thus, reducing activity can[77]. In atau
reduce studyloadwith
[77].Wistar rats
In a study
treated with nicotine, a daily intraperitoneal injection with S. platensis-lipopolysaccharides
with Wistar rats treated with nicotine, a daily intraperitoneal injection with S. platensis-
lipopolysaccharides (100 µg/kg) provided neuroprotection by suppressing the up-
regulation of phosphorylated-tau ratio expression by two fold, while showcasing
antioxidant, anti-inflammatory, and anti-apoptotic activities [78]. Dietary
supplementation of 1% and 2% Spirulina platensis dry powder in high-fat diet mice
 Neurofibrillary tangles, which are composed of hyperphosphorylated tau protein,
are also a hallmark of AD. Kinases, mainly GSK3β, are responsible for tau
phosphorylation and thus, reducing enzymatic activity can reduce tau load [77]. In a study
Biomolecules 2023, 13, 1444 with Wistar rats treated with nicotine, a daily intraperitoneal injection with S. platensis- 8 of 34
lipopolysaccharides (100 µg/kg) provided neuroprotection by suppressing the up-
regulation of phosphorylated-tau ratio expression by two fold, while showcasing
antioxidant,
(100 µg/kg) providedanti-inflammatory,
neuroprotection and anti-apoptotic
by suppressing activitiesof phosphorylated-
the up-regulation [78]. Dietary
supplementation of 1% and 2% Spirulina platensis dry powder
tau ratio expression by two fold, while showcasing antioxidant, anti-inflammatory, in high-fat diet mice
and
lowered the tau burden by reducing both phosphorylated-tau and phosphorylated-GSK
anti-apoptotic activities [78]. Dietary supplementation of 1% and 2% Spirulina platensis dry
levels, while
powder it also
in high-fat dietdecreased
mice lowered Aβ1–42
theconcentrations, APP, and
tau burden by reducing bothBACE-1 levels in the
phosphorylated-tau
hippocampus
and [79].
phosphorylated-GSK levels, while it also decreased Aβ1–42 concentrations, APP, and
BACE-1AD levels
has also been
in the linked to mitochondrial
hippocampus [79]. dysfunction and endoplasmic reticulum
stress. Santacruzamate A (11) (Figure
AD has also been linked to mitochondrial dysfunction 6), a compoundand produced
endoplasmic by reticulum
a marine
cyanobacterium cf. Symploca sp., has shown therapeutic potential
stress. Santacruzamate A (11) (Figure 6), a compound produced by a marine cyanobac- in vitro and in vivo. It
inhibited the Aβ 25–35 -induced apoptosis in PC12 cells (2 µM STA)
terium cf. Symploca sp., has shown therapeutic potential in vitro and in vivo. It inhibited by reversing the
endoplasmic
the reticulum
Aβ25–35 -induced and in
apoptosis unfolded
PC12 cells protein
(2 µM response stress. Ittheregulated
STA) by reversing endoplasmic the
endoplasmic
reticulum andreticulum
unfolded proteinretention signal (KDEL)
response stress. Itreceptor,
regulatedwhich increased chaperone
the endoplasmic reticulum
luminal retention.
retention signal (KDEL) Compound
receptor,11which
also increased
restored the mitochondrial
chaperone luminal intermembrane
retention. Compound space
assembly
11 pathway
also restored and regulatedintermembrane
the mitochondrial the expressionspace of the mitochondrial
assembly pathwayintermembrane
and regulated
space
the assemblyofprotein
expression 40 (Mia40) and
the mitochondrial the augmenter
intermembrane of the
space liver regeneration
assembly (ALR)
protein 40 (Mia40)
system,
and the resulting
augmenter in aofreduction
the liver in the mitochondrial
regeneration (ALR) fission
system,and apoptosis
resulting in apathways
reduction [80].
in
Thismitochondrial
the was confirmed by inand
fission vivoapoptosis
studies inpathways
APPswe/PS1dE9
[80]. Thismice,
wasaconfirmed
common AD mouse
by in vivo
model bearing
studies mutant transgenes
in APPswe/PS1dE9 of the amyloid
mice, a common AD mouse precursor proteinmutant
model bearing and presenilin-1,
transgenes
which
of lead to precursor
the amyloid an early-onset proteinincrease in parenchymal
and presenilin-1, which leadAβ-levels and otherincrease
to an early-onset clinicallyin
relevant AD-like
parenchymal symptoms
Aβ-levels [81]. clinically
and other Treatment with AD-like
relevant santacruzamate
symptoms A [81].
(11) (5 and 10
Treatment
mg/kg/day)
with promotedAmemory
santacruzamate (11) (5 andperformance
10 mg/kg/day) in behavioral
promoted tests and enhanced
memory performanceKDELR in
and Mia40-ALR functions in the brain tissue [80].
behavioral tests and enhanced KDELR and Mia40-ALR functions in the brain tissue [80].

Figure 6.
Figure 6. Structure
Structure of
of santacruzamate
santacruzamate A
A (11),
(11), aa carbamate
carbamate derivative
derivative with
with neuroprotective
neuroprotective activity.
activity.

Another pathological
Another pathological aspect
aspect of
of AD
AD is
is heavy
heavy metal
metal bioaccumulation
bioaccumulation and and reversing
reversing its
its
toxicity can
toxicity canimprove
improvedisease
diseaseoutcomes.
outcomes.InInWistar
Wistar rats,
rats, tablets
tablets S. platensis
of platensis
of S. (1500
(1500 mg/kg)
mg/kg) re-
revealed
vealed neuroprotective
neuroprotective potential
potential against
against brain brain degeneration
degeneration inducedinduced by aluminum
by aluminum chloride
chloride
(AlCl 3 ). (AlCl3). While lowering the number of illuminated Aβ protein aggregates,
While lowering the number of illuminated Aβ protein aggregates, the the
treat-
treatment also reduced histopathological alterations in the cerebral cortex and
ment also reduced histopathological alterations in the cerebral cortex and hippocampus,
hippocampus,
with with close
close to normal neuronto morphology
normal neuron andmorphology and fewer neurodegenerative
fewer neurodegenerative features. It also
features. Itmetabolic
improved also improved metabolic
indices indices and demonstrated
and demonstrated anti-inflammatoryanti-inflammatory activity
activity through the
through the
reduction reduction
in TNF-α. Theintablets
TNF-α. The tablets
showed strong showed
antioxidant strong antioxidant
potential potentialthio-
by decreasing by
decreasing thiobarbituric acid reactive substances (TBARS) levels and restoring
barbituric acid reactive substances (TBARS) levels and restoring glutathione (GSH) levels,
glutathione
thiol content,(GSH) levels,
and total thiol content,
antioxidant and (TAC)
capacity total antioxidant
[82]. capacity (TAC) [82].
In a study by Abdelghany et al. (2023) [83], an S. platensis-loaded niosome (SPLN)
formulation was explored as a drug delivery system in an AlCl3 -induced AD rat model.
The use of nanoparticles enables more effective, controlled, and targeted brain treatment. S.
platensis-loaded niosome (300 mg/kg) improved recognition and working memory and
demonstrated neuroprotective activity by maintaining normal morphology in hippocampal
brain tissue. Additionally, it restored AChE activity, ACh, and monoamine levels in the
brain and also improved the oxidative state as it lowered the malondialdehyde (MDA)
levels and TAC [83].
Growing data suggest that AD is associated with dysbiosis of the human gut micro-
biota via neuroinflammatory processes across the microbiota–gut–brain axis, suggesting
that modifying the gut microbiota could be a strategy for treating the condition [84]. Ac-
cording to Zhou et al. (2021) [79], dietary supplementation with 1% and 2% S. platensis
dry powder in high-fat diet mice alleviated cognitive impairment and restored gut mi-
crobial dysbiosis by increasing the Shannon, ACE, and Chao indices while decreasing
the Simpson index, indicating enhanced microbial community richness and diversity. It
Biomolecules 2023, 13, 1444 9 of 34

improved the intestinal environment by balancing microbiota and increasing the abun-
dance of beneficial microorganisms, such as Verrucomicrobia, while reducing the presence
of harmful microorganisms, like Firmicutes. Supplementation also lowered inflammatory
lipopolysaccharide levels in the feces and serum and raised fecal levels of short-chain fatty
acids, which improves neuronal homeostasis. Furthermore, it showed anti-inflammatory
benefits by lowering inflammatory markers such as GFAP, TNF-α, IL-1β, IL-6, and ionized
calcium-binding adapter molecule 1 (IBA-1) in the hippocampus [79].
Aside from the modes of action outlined above, cyanobacteria, particularly Spirulina
and its component phycocyanin, largely work through gene modulation.
In a study conducted in rodents intracerebroventricularly injected with Aβ25–35 , the
oral pre-treatment with a proteolysis product of phycocyanin (EDPC) from S. platensis
(750 mg/kg) improved cognitive impairment in a Y maze spontaneous alternation test and
modulated de gene expression profile in a DNA microarray analysis. It counteracted the
aberrant expression of 35 genes, including Prnp, Cct4, Vegfd, Map9, Pik3cg, Zfand5, Endog,
and Hbq1a, which are directly linked to AD or other neurological diseases [85].
In C57BL/6 mice injected with oligomeric Aβ1–42 , treatment with phycocyanin
(200 mg/kg) from S. platensis improved spatial memory and reversed the epigenetic dysreg-
ulation. It restored the expression of the regulatory miRNA-335, which was downregulated
by 76%, and the expression of the BDNF gene, which was reduced to 24% in Aβ-mice.
On the other hand, it downregulated the histone deacetylase 3 (HDAC3) gene, whose
expression was amplified three fold in Aβ-mice. The treatment also showed anti-apoptotic
and anti-inflammatory effects, by restoring Bax/Bcl-2 equilibrium, decreasing caspase-3
and caspase-9 release, and lowering inflammatory cytokine levels (IL-6 and IL-1β) [86].
Agrawal et al. (2020) [87] demonstrated that phycocyanin administration (100 mg/kg)
in an intracerebroventricular streptozotocin-induced AD-mice model improved spatial
memory and reduced memory impairment in behavioral tests. It improved metabolic
parameters, by restoring the gene expression of insulin signaling molecules such as the
insulin (INS) gene, insulin receptor substrate 1 (IRS-1), PI3K, and Akt. Thereby, it increased
the activation of the insulin-PI3K-Akt pathway while it lowered the expression of one
of its inhibitors, the phosphatase and tensin homolog (PTEN) gene. In addition, the
treatment upregulated the anti-apoptotic marker Bcl-2 whereas the pro-apoptotic marker
Bax was downregulated. It also altered acetylcholine metabolism by lowering AChE activity
while increasing choline acetyltransferase (ChAT) in the hippocampus and mitigated
neuroinflammation by reducing TNF-α and nuclear factor (NF)-kβ levels [87].
In another study, treatment with S. platensis-loaded niosome (300 mg/kg) modulated
gene expression, restoring the mRNA levels of the enzymes AChE and monoamine oxi-
dase and reversing both the AlCl3 -induced decrease in the anti-apoptotic protein B-cell
lymphoma-2 (Bcl-2) and increase in the pro-apoptotic protein Bcl-2 associated X-protein
(Bax) mRNA levels [83].
Biomolecules 2023, 13, 1444 10 of 34

Table 1. Cyanobacteria-derived products/extracts studied in AD disease models.

Strain Compound/Extract Effect In Vitro Assays In Vivo Assays Reference

Anabaena flos-aquae AChE and BChE inhibition
Anatoxin-a(s) (3) AChE and BChE inhibition [55]
NRC-525-17 assay
AChE and BChE inhibition
Nostoc 78-12A Nostocarboline (4) BChE inhibition [57]
assay
AChE inhibition assay.
AChE and LOX inhibition.
Phormidium autumnale SFE-EtOH extract LOX inhibition assay. ORAC [60]
Antioxidant.
assay.
Methylene chloride/
Anabaena variabilis AChE inhibition AChE inhibition assay [61]
methanol extract (Fraction 7)
Methylene chloride/
Oscillatoria sancta AChE inhibition AChE inhibition assay [62]
methanol (1:1) extract
AChE and BChE inhibition
AChE and BChE inhibition.
Nostoc sp. Ethanolic Extract assay. [63]
Antioxidant.
DPPH assay.
Fluorimetric assay. Kinetic
Inhibition of Aβ
Spirulina sp. Phycocyanin analysis. [65]
formation
Circular dichroism analysis.
Fibrillar and amorphous
Inhibition of Aβ40/42 aggregation assays.
Spirulina sp. Phycocyanin [66]
amyloid fibrillation Transmission
electron microscopy imaging.
Symploca sp. Tasiamide B (8) BACE-1 inhibition BACE-1 inhibition assay [67]
Lyngbya sp. Tasiamide F (9) BACE-1 inhibition BACE-1 inhibition assay [69]
Caenorhabditis elegans CL4176
Leptolyngbya sp. Protein-complex interface
Phycocyanin BACE-1 inhibition transgenic AD-model: [70]
N62DM identification
Paralysis assay
Biomolecules 2023, 13, 1444 11 of 34

Table 1. Cont.

Strain Compound/Extract Effect In Vitro Assays In Vivo Assays Reference

Surface plasmon resonance.
Isothermal titration Caenorhabditis elegans CL4176
Lyngbya sp.
Phycoerythrin BACE-1 inhibition calorimetry. transgenic AD-model: [71]
A09DM
Enzyme activity by kinetic Thioflavin-T staining assay
parameters.
Wistar albino rats exposed to nicotine:
Downregulation of p-tau
Biochemical assessments (Oxidative
Spirulina platensis Lipopolysaccharide expression. Antioxidant. [78]
and inflammatory markers).
Anti-inflammatory.
RT-PCR. Western Blot (p-tau).
AChE inhibition. Reduced ICR mice injected with Aβ1–42 :
Aβ, APP, and BACE-1 levels. Passive Avoidance Test. Morris
Spirulina maxima 70% ethanol extract BDNF/PI3K/Akt pathway WaterMaze Test. Biochemical Analysis [72]
activation. Antioxidant. (Aβ1–42 , GSH, BDNF, AChE). Western
Improved cognition. Blot.
Reduced Aβ, APP and High-Fat Diet C57BL/6J mice:
BACE-1 Metabolic parameters analysis. Western
levels. Anti-inflammatory Blot (IR, Akt, PSEN-1, BACE-1, PSD-95,
and anti-gliosis. synaptophysin, TNF-α, GFAP, IL-10,
Aphanizomenon flos-aquae KlamExtra® [73]
Improved metabolic TREM-2). Histopathology and
parameters. Protection of Immunohistochemistry (GFAP, TREM-2,
neuronal Aβ). Thioflavin T staining. TUNEL
morphology and synapses. assay.
Decreased Aβ1–42 , APP,
BACE-1, p-tau, and p-GSK High-Fat Diet C57BL/6J mice:
levels. Anti-inflammatory. Barnes Maze test. Morris Water Maze
Improved microbiota test. ELISA (Aβ1–42 , TNF-α, IL-1β, IL-6,
Spirulina platensis Diet supplementation [79]
dysbiosis. Improved LPS). RT-PCR. Western Blot (APP,
metabolic parameters. BACE-1, p-tau, p-GSK, IBA-1). Microbial
Improved locomotor and diversity analysis. GC (SCFAs).
cognitive function.
Biomolecules 2023, 13, 1444 12 of 34

Table 1. Cont.

Strain Compound/Extract Effect In Vitro Assays In Vivo Assays Reference

PC12 cells:
Cell viability and apoptosis
assays.
Anti-apoptotic. Anti-UPR
Electrophysiological
and ER stress. Improvement APPswe/PS1dE9 mice:
recordings.
of the mitochondrial fission Open-Field test.
cf. Symploca sp. Santacruzamate A (11) Immunoblot analyses. [80]
pathway. Modulation of Morris Water Maze test.
Measurement
KDELR and Mia40-ALR. RT-PCR (Mia40, KDEL).
of mitochondrial permeability
Memory improvement.
transition pore. Opening and
mitochondrial membrane
potentials.
Protection of neuronal
Wistar rats treated with AlCl3 :
morphology. Reduction in
TBARS assay. GSH content assay. Total
Aβ
Diet supplementation thiol content assay. TAC assay. GPx, GST,
Spirulina platensis accumulation. Improvement [82]
(tablets) SOD activity assay. Lipid profile
of metabolic parameters.
determination. ELISA (TNF-α).
Antioxidant.
Histology. Immunofluorescence (Aβ).
Anti-inflammatory.
Protection of
Wistar rats treated with AlCl3 :
neuronal morphology.
Novel object recognition test. Y-maze
Restored
S. platensis- test. TAC assay. MDA assay.
Spirulina platensis levels of AChE and ACh. [83]
loaded niosome AChE assay. Histology.
Gene modulation.
HPLC (ACh, NE, 5HT, DA, DOPAC).
Recognition and working
qPCR (Bax, Bcl-2, AChE, MAO).
memory improvement.
Cognitive function Male Slc:ddY SPF mice
Enzyme Digested
Spirulina platensis improvement. injected with Aβ25–35 : [85]
Phycocyanin (EDPC)
Gene modulation. Y Maze test. DNA microarray.
Biomolecules 2023, 13, 1444 13 of 34

Table 1. Cont.

Strain Compound/Extract Effect In Vitro Assays In Vivo Assays Reference

Male C57BL/6 mice injected
Gene and miRNA with oligomeric Aβ1–42 :
modulation. Eight-arm radial maze. RT-PCR
Spirulina platensis Phycocyanin Anti-inflammatory. (caspase-3, caspase-9, miR-335). Western [86]
Anti-apoptotic. Memory Blot (HDAC3, Bcl-2, Bax, IL-6, IL-1β).
improvement. Immunohistochemistry (Bcl-2, Bax).
Immunofluorescence (BDNF, HDAC3).
Female Wistar Rats injected with STZ:
AChE inhibition. ChAT Morris Water Maze. Memory
activity increase. Gene consolidation test. Novel object
modulation. Increased recognition test. Open field test. AChE
Spirulina platensis Phycocyanin [87]
PI3K/Akt pathway. and ChAT activity assays. ELISA
Anti-inflammatory. (TNF-α, NF-kB p56, Bcl-2, Bax, BDNF,
Memory improvement. IGF-1). qRT-PCR
(IRS-1, INS, PI3K, Akt, PTEN).
Randomized, double-blind, and placebo-
70% ethanolic extract Memory and vocabulary controlled clinical trial. Visual learning,
Spirulina maxima [88]
(SM70EE) pills improvement. visual working memory, and verbal
learning tests.
Biomolecules 2023, 13, 1444 14 of 34

Table 1. Cont.

Strain Compound/Extract Effect In Vitro Assays In Vivo Assays Reference

Randomized, double-blind, and placebo.
-controlled clinical trial. Mini-mental
Improved cognitive function.
Spirulina platensis Dietary supplementation state exam. ELISA (hs-CRP, Insulin). [89]
Improved metabolic status.
Biochemical analysis (NO, TAC, GSH,
MDA, FPG, lipid profile).
Abbreviations: AChE—Acetylcholinesterase. BChE—Butyrylcholinesterase. SFE-EtOH—Supercritical Fluid Extraction with Ethanol. LOX—Lipoxygenase. ORAC—Oxygen Radical
Absorbance Capacity. DPPH—2,2-diphenyl-1-picrylhydrazyl. BACE-1—Beta Secretase 1. AD—Alzheimer’s Disease. Aβ—Beta-amyloid peptide. APP—Amyloid-beta Precursor Protein.
BDNF—Brain-derived Neurotrophic Factor. PI3K—Phosphoinositide 3-kinase. Akt—Protein kinase B. TBARS—Thiobarbituric Acid Reactive Substances. GSH—Total Glutathione.
IR—Insulin receptor. PSEN-1—Presenilin-1. PSD-95—Postsynaptic density protein 95. TNF-α—Tumor Necrosis Factor α. GFAP—Glial fibrillary acidic protein. IL-10—Interleukin.
TREM-2—Triggering receptors expressed on myeloid cells-2. p-tau—Phosphorylated Tau. p-GSK—Phosphorylated Glycogen Synthase. LPS—Lipopolysaccharide. RT-PCR—Reverse
Transcription Polymerase Chain Reaction. Iba-1—Ionized calcium-binding adaptor molecule 1. GC—Gas Chromatography. SCFAs—Short-chain fatty acids. UPR—Unfolded
Protein Response. ER—Endoplasmic Reticulum. KDELR—Endoplasmic Reticulum Protein Retention Receptor. Mia40—Mitochondrial Intermembrane Space Assembly Protein
40. ALR—Augmenter of the Liver Regeneration. AlCl3 —Aluminum Chloride. TAC—Total Antioxidant Capacity. GPx—Glutathione Peroxidase. GST—Glutathione S-transferase.
SOD—Superoxide Dismutase. ELISA—Enzyme-Linked Immunosorbent Assay. MDA—Malondialdehyde. HPLC—High-Performance Liquid Chromatography. ACh—Acetylcholine.
NE—Norepinephrine. 5HT—Serotonin. DA—Dopamine. DOPAC—3,4-Dihydroxyphenylacetic acid. Bcl-2—B-cell Lymphoma-2. Bax—Bcl-2 Associated X-protein. MAO—Monoamine
oxidase. HDAC3—Histone deacetylase 3. STZ—Streptozotocin. ChAT—Choline acetyltransferase. NF-kβ—Nuclear Factor Kappa β. IGF-1- Insulin-like growth factor 1. IRS-1—Insulin
receptor substrate 1. INS—Insulin Gene. PTEN—Phosphatase and Tensin Homolog. hs-CRP—High sensitivity C-reactive protein. NO—Nitric Oxide. FPG—Fasting Plasma Glucose.
Human clinical trials have also validated the potential of Spirulina as a nutraceutical. Patients with mild cognitive impairment who consumed 1 g/day of S. maxima 70% ethanol
extract (SM70EE) capsules experienced statistically significant gains in visual learning and working memory according to a randomized, double-blind, and placebo-controlled clinical
trial [88]. Another randomized, double-blind, and controlled clinical trial investigated the cognitive and metabolic status of patients with AD who took S. platensis capsules twice daily
(500 mg/day). Supplementation considerably improved the Mini-Mental State Examination score, indicating an enhancement in cognitive function. It also had a favorable impact on the
metabolic status by lowering C-reactive protein, fasting glucose, insulin levels, and insulin resistance while increasing insulin sensitivity [89].
Biomolecules 2023, 13, 1444 15 of 34

4.2. Cyanobacteria against Parkinson’s Disease

Parkinson’s Disease (PD) is a neurodegenerative movement disorder whose incidence
and prevalence increase with age. It is distinguished by the presence of Lewy bodies, which
are intracellular protein aggregates of misfolded α-synuclein (α-Syn) protein, as well as the
gradual loss of dopaminergic nigrostriatal neurons in the midbrain substantia nigra pars
compacta [90]. Other features of PD include decreased dopamine metabolism impaired
mitochondrial function, autophagy failure, oxidative stress, inflammation, and accelerated
apoptosis. These lead to symptoms like decreased motor function, bradykinesia, postural
instability, and muscle rigidity [91,92]. PD can be caused by many factors, including
environmental exposure and genetics/epigenetics, with the most common mutations being
in SNCA, LRRK2, PRKN, PINK1, and GBA genes [90].
There is no cure for PD but some treatments do exist to manage its symptoms. Most
options aim to increase dopamine levels, including dopaminergic drugs such as levodopa,
and enzyme inhibitors of monoamine oxidase B (MAO-B) and catechol-O-methyltransferase
(COMT). Antioxidants, anti-inflammatory drugs, gene therapies, stem cell treatments, and
protein aggregation inhibitors can be future therapeutic approaches [7].
Regarding cyanobacteria, the most compelling evidence for its use against PD comes
from research on Spirulina and phycobiliproteins, namely phycocyanin (Table 2). This
molecule can reduce the synucleinopathy typical of PD. At a molar ratio of 2:1 (α-Syn: phy-
cocyanin), it was found to be an efficient inhibitor of A53Tα-synuclein amyloid fibrillation
in silico. Interactions between phycocyanin and α-Syn were unstable, implying that brief
interactions may limit fibril formation [66].
Macedo et al. (2017) [93] studied the effects of phycocyanin from S. platensis in a
yeast model of PD transformed with a plasmid carrying the human gene of α-Syn. The
phycocyanin-supplemented medium (48 mg/mL) promoted cell viability while drastically
decreasing the fraction of cells exhibiting αSyn-GFP inclusions. It significantly reduced
oxidative stress by lowering superoxide levels and lipid peroxidation, while enhancing
thiol levels and catalase activity. Phycocyanin also displayed gene modulation properties.
It ameliorated the oxidative stress response by modulating SOD1, SOD2, HAP4, and
GLR1 genes and improved proteostasis by restoring RPN4 and ATG8 transcript levels
while decreasing HSP26 mRNA levels, all of which are key players in proteosomal and
autophagic activity.
Another distinguishing aspect of PD is low dopamine (DA) levels. To ameliorate
the disease severity and reduce typical motor symptoms, it is vital to find therapeutic
solutions that safeguard the levels of DA and its metabolites in the synaptic cleft [94].
Oral pre-treatment with S. maxima (150 mg/kg) partially protected dopamine depletion by
51%, and blocked lipid peroxidation by 100% in C-57 mice subjected to 1-methyl-4-phenyl-
1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity, which is a common chemically induced
PD animal model [95].
According to Tóbon-Velasco et al. (2013) [96], rats injected with 6-hydroxydopamine
(6-OHDA), another common animal model of chemically induced PD, and fed a diet
supplemented with S. maxima (700 mg/kg/day) showed partial protection in the levels
of DA (31%), homovanillic acid (47%), and 3,4-dihydroxyphenylacetic acid (23%) in the
striatum. The treatment improved locomotor function, including greater use of both
forelimbs and decreased circling behavior. It also enhanced antioxidation by reducing
ROS production by 112% and nitrite levels by 77%, as well as considerably lowering lipid
peroxidation and mitochondrial reduction activity [96].
In another study using a 6-OHDA-induced PD rat model, treatment with an aqueous
freeze-dried extract of Spirulina fusiform (500 mg/kg, twice daily) resulted in a positive
response in behavioral and motor tests, DA levels, and oxidative state. Moreover, the
treatment in conjunction with amantadine, a medication commonly used to treat dyskinesia
in PD patients, resulted in a significant increase in DA levels, a recovery of glutathione
levels, and a reduction in TBARS content by 73% [97].
Biomolecules 2023, 13, 1444 16 of 34

According to Xu et al. (2023) [98], three novel peptides (MAAAHR, MPQPPAK, and
MTAAAR), derived from phycocyanin from S. platensis, showed significant neuroprotec-
tive activity in MPTP-induced PD zebrafish. The peptides (12.5 µg/mL, 25 µg/mL, and
50 µg/mL) relieved locomotion constraints and reversed the DA neuron degeneration
and neural vasculature disorganization. Furthermore, they increased antioxidant enzyme
activity (SOD, CAT, and GSH-Px) while decreasing ROS and protein carbonyl levels. They
also had anti-apoptotic effects, lowering the number of apoptotic brain cells and the activity
of AChE, which is involved in apoptotic pathways. The observed effects can be attributed
to the modulation of gene expression as they upregulated oxidative stress response genes
(nrf2, ho-1, nqo-1, gclc, and gclm) and downregulated genes linked to autophagy (α-syn,
parkin, beclin1, atg5, map1lc3b, and atg3) and apoptosis (caspase-1, caspase-3, caspase-8,
caspase-9, and Bax) [98].
Drosophila melanogaster flies are a common animal model of PD [99]. Treatment with
S. platensis methanolic extract (120 µg/mL) in D. melanogaster subjected to FeSO4 toxicity,
improved the survival rate and locomotor ability of the flies. It promoted an increase in
DA levels and showed strong antioxidant activities by scavenging DPPH free radicals
(IC50 = 64.55 µg/mL) and reducing MDA levels [100]. Another study explored the ef-
fects of Spirulina supplementation (5% or 10%) in DJ-1β∆93 flies exposed to chemically
induced oxidative stress using paraquat. This is a transgenic model of PD, in which the
loss of the DJ-1β ortholog gene improves vulnerability to oxidative stress and leads to
dopaminergic neuronal degeneration [99]. The mixed diet with Spirulina significantly
increased the locomotor capacity and the lifespan of the flies and improved the antioxidant
state by downregulating the SOD/CAT activity. The authors also studied the effects of a
phycocyanin-mixed diet (1 or 2 µg/mL). Both Spirulina and phycocyanin reduced cellular
stress, as evidenced by a decrease in the expression of heat shock protein 70 (HSP70) and
Jun-N-terminal kinase (JNK) expression [101].
Some of the studies present in the literature refer to the effect of cyanobacteria on the
tyrosine hydroxylase (TH) levels, since this is the limiting enzyme in dopamine synthesis,
and on the DA transporter (DAT), which regulates DA reuptake. Both TH and DAT
expression is reduced in PD [102].
A study directed to evaluate the pre-treatment with intraperitoneal injections of a
polysaccharide derived from S. platensis (800 mg/kg) in MPTP-treated mice revealed a
significant increase in the DAT binding ratio and the TH-immunoreactive neurons in the
substantia nigra pars compacta, along with their mRNA expression. It also showed strong
antioxidant capacity, with increased serum levels of superoxide dismutase (SOD) and
glutathione peroxidase (GSH-Px) [103]. Also, in rats subjected to an intrastriatal injec-
tion of 6-OHDA, treatment with a protein-enriched fraction of S. platensis (10 mg/kg)
improved behavioral assessments. It also promoted the reversal of the 6-OHDA-induced
decrease in striatal dopamine and 3,4-dihydroxyphenylacetic acid levels, while it signifi-
cantly protected the striatal expression of TH and DAT. By lowering brain nitrite levels and
lipid peroxidation, as well as the expression of GFAP, hippocampus inducible nitric oxide
synthase (iNOS), and cyclooxygenase-2 (COX-2) enzymes, the fraction demonstrated an-
tioxidant and anti-inflammatory potential [104]. Similarly, Lima et al. (2017) [105] showed
that treatment with 10% (w/v) aqueous extract of S. platensis at 25 mg/kg or 50 mg/kg
in rats subjected to 6-OHDA-induced parkinsonism is neuroprotective. Both treatments
improved apomorphine-induced rotational behavior, reversed the reduction in dopamine
and 3,4-dihydroxyphenylacetic acid levels in the striatum, and showed antioxidant activity
by reducing nitrite levels and inhibiting lipid peroxidation. Treatment at 50 mg/kg par-
tially blocked the decrease in TH (42%) and DAT immunoreactivity (55%) and exhibited
anti-inflammatory activities, as seen by the decrease in iNOS and COX-2 immunoreactivity,
two enzymes related to inflammation [105].
Pre-treatment with a 0.1% Spirulina-supplemented diet in a rat model of PD inocu-
lated with an adeno-associated virus for α-Syn protected against neuronal loss, as seen
by the increase in TH-positive and NeuN-positive neurons in the substantia nigra pars
Biomolecules 2023, 13, 1444 17 of 34

compacta. It also showed anti-inflammatory activity, with a decrease in the number of

activated microglial cells, as evaluated by a reduction in OX-6-(MHC class II)-positive
immunoreactivity and a significant increase in the expression of the fractalkine receptor
(CX3CR1) in microglia that, when stimulated, promotes anti-inflammatory activities [106].
On the same note, Strömberg et al. (2005) [107] showed that a diet enriched with 0.1%
Spirulina fed to rats injected with 6-OHDA promoted the recovery of striatal dopamine
innervation and positive TH nerve fibers, driven by an early and temporary increase in
OX-6-(MHC class II) positive microglia that induce remodulation.
In PD, a strong association between DA neurodegeneration and inflammation has
been described with the involvement of several inflammatory mediators and cells, such
as peripheral immune cells. Hence, the increase in TH and DAT induced by Spirulina,
as described before, might not be due to a direct effect on the production of the enzyme
and DAT but instead due to anti-inflammatory and antioxidant responses. In fact, several
results support that in CNS, DA depletion modulates peripheral immunity and expression
of the dopaminergic markers TH and DAT on peripheral immune cells [99–106].
Biomolecules 2023, 13, 1444 18 of 34

Table 2. Cyanobacteria-derived products/extracts studied in PD disease models.

Strain Compound/Extract Effect In Vitro Assays In Vivo Assays Reference

Fibrillar and amorphous
Inhibition of
aggregation assays.
Spirulina sp. Phycocyanin A53Tα-synuclein [66]
Transmission
amyloid fibrillation
electron microscopy imaging.
BY4741 Yeast transformed
with p42FAL-αsyn-GFP:
Reduction in α-synuclein Spot assay. Fluorescence
inclusions. Gene modulation. microscopy. Western Blot (α-syn).
Spirulina platensis Phycocyanin [93]
Antioxidant. Flow cytometry. TBARS assay.
Improved proteostasis. CAT activity. Total thiols assay.
qRT-PCR (SOD1, SOD2, HAP4, LHS1,
HRD1, GSH1, GLR1, RPN4, ATG8).
MPTP-induced parkinsonism
Improved locomotion. in transgenic zebrafish:
Phycocyanin
Neuronal Fluorescence Microscopy. Behavioral
derived peptides
Spirulina platensis protection. Antioxidant. tests. Fluorescence ROS determination. [98]
(MHLWAAK, MAQAAEYYR,
Anti-apoptosis. Gene Biochemical analysis
MDYYFEER)
modulation. (SOD, CAT, GSH-Px, CO, AChE).
Acridine orange staining. qRT-PCR.
MPTP-induced parkinsonism
Protection of DA and HVA
in male C-57 rats:
Spirulina maxima Diet supplementation content. Blockage of lipid [95]
HPLC (DA, HVA, 5-HIAA, 5-HT).
peroxidation.
TBARS Assay.
6-OHDA-induced parkinsonism
Improved locomotion.
in male Wistar rats:
Recovery
Turn-behavior test. Closed-field test.
of mitochondrial activity.
Spirulina maxima Diet supplementation Cylinder test. Fluorescence ROS [96]
Protection of DA, DOPAC,
determination. Griess reaction.
and
TBARS assay. MTT assay.
HVA levels. Antioxidant.
HPLC (DA, DOPAC, HVA).
Biomolecules 2023, 13, 1444 19 of 34

Table 2. Cont.

Strain Compound/Extract Effect In Vitro Assays In Vivo Assays Reference

6-OHDA-induced parkinsonism
in male Wistar albino rats:
Aqueous freeze-dried extract Improved behavior and
Amphetamine- and Apomorphine
Spirulina fusiform suspended locomotion. Protection of DA [97]
-induced rotations. Locomotor activity.
in olive oil levels. Antioxidant.
Rota rod. TBARS assay. Reduced
glutathione content assay. HPLC (DA).
Drosophila Melanogaster
exposed to FeSO4 :
Increased lifespan and
Total phenol Content. DPPH radical
Spirulina platensis Methanolic extract locomotion. Antioxidant. [100]
scavenging activity. Survival rate.
Protection of DA content.
Negative Geotaxis assay. Lipid
Peroxidation Assay. DA content assay.
DJ-1β∆93 Drosophila Melanogaster
exposed to paraquat:
Increased lifespan and
Survival assay. Locomotor assay.
Spirulina platensis Diet supplementation locomotion. Antioxidant. [101]
PCR (HSP70). SOD and CAT
Reduced cellular stress.
enzymatic assays. Immunostaining
(Hsp70 and JNK).
MPTP-induced parkinsonism
Increased TH and DAT in male C57BL/6J mice:
Spirulina platensis Polysaccharide [103]
expression. Antioxidant. Immunohistochemistry and RT-PCR
(TH, DAT). SOD and GSH-Px assays.
Improved behavior. 6-OHDA-induced hemiparkinsonism
Protection of in male Wistar rats:
DA and DOPAC levels. Apomorphine-induced rotational
Protein-rich Increased test. Open-field test. Forced swim
Spirulina platensis [104]
fraction (SPF) TH and DAT expression. test. HPLC (DA, DOPAC).
Reduced iNOS, COX-2, and Griess Reaction. TBARS assay.
GFAP Immunohistochemistry
expression. Antioxidant. (TH, DAT, iNOS, GFAP, COX-2)
Biomolecules 2023, 13, 1444 20 of 34

Table 2. Cont.

Strain Compound/Extract Effect In Vitro Assays In Vivo Assays Reference

6-OHDA-induced parkinsonism
Improved behavior.
in male Wistar rats:
Protection
Apomorphine-induced rotational
10% (w/v) of DA and DOPAC levels.
Spirulina platensis test. HPLC (DA, DOPAC). [105]
aqueous extract Protection of TH and DAT
Griess Reaction. TBARS assay.
expression. Decreased iNOS
Immunohistochemistry
and COX-2. Antioxidant.
(TH, DAT, iNOS, COX-2).
F344 rats treated with AAV9α-synuclein:
Increase in TH+ and NeuN+ Immunohistochemistry (TH,
Spirulina Diet supplementation [106]
neurons. Anti-inflammatory. α-synuclein, OX-6, NeuN). Stereology.
Western Blot (CX3CR1).
Recovery of striatal 6-OHDA-induced parkinsonism
dopamine in F344 male rats:
Spirulina Diet supplementation [107]
innervation. Increased TH+ Immunohistochemistry (TH, OX-6,
fibers. Anti-inflammatory. Iba1, GFAP). Cell counting.
Abbreviations: α-syn—α-synuclein. GFP—Green fluorescent protein. TBARS—Thiobarbituric acid reactive substances. CAT—Catalase. RT-PCR -Reverse transcription polymerase
chain reaction. SOD1—Copper-zinc superoxide dismutase. SOD2—Manganese superoxide dismutase. HAP4—Glucose-repressed regulated subunit of the HAP transcriptional
complex. LHS1—Heat shock protein 70 homolog. HRD1—E3 ubiquitin-protein ligase. GSH1—Gamma-glutamylcysteine synthetase. GLR1—Glutathione reductase. RPN4—Zinc-
coordinating proteasomal transcription factor. ATG8—Autophagy-related protein 8. DA—Dopamine. HVA—Homovanillic acid. MPTP—1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.
HPLC—High-performance liquid chromatography. 5-HIAA—5-Hydroxyindoleacetic acid. 5-HT—5-hydroxytryptamine. DOPAC—3,4-Dihydroxyphenylacetic acid. 6-OHDA—6-
hydroxydopamine. ROS—Reactive oxygen species. MTT—3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide. TH—Tyrosine hydroxylase. DAT—Dopamine transporter.
SOD—Superoxide dismutase. GSH-Px—Glutathione peroxidase. iNOS—Inducible nitric oxide synthase. COX-2—Cyclooxygenase 2. GFAP—Glial fibrillary acidic protein. NeuN—
Neuronal nuclear protein. OX-6—Major histocompatibility complex (MHC) class II antigen. CX3CR1—Fractalkine receptor. Iba1—Ionized calcium-binding adaptor. FeSO4—Ferrous
sulfate. DPPH—2,2-diphenyl-1-picrylhydrazyl. HSP70—70 kilodalton heat shock protein. JNK—c-Jun N-terminal cinase. CO—Protein carbonyl. AChE—Acetylcholinesterase.
Biomolecules 2023, 13, 1444 21 of 34

4.3. Cyanobacteria against Multiple Sclerosis

Multiple Sclerosis (MS) is a chronic and inflammatory ND with an autoimmune origin
that affects the CNS of more than two million people worldwide [108]. The main pathologi-
cal hallmark is focal plaques, which are areas of immune cell infiltration and demyelination
in the white and grey matter, that can be found in the brain, optic nerve, and spinal cord.
These contribute to axon loss, myelin sheath destruction, and neuronal impairment. Other
pathological processes include immune dysfunction, blood–brain barrier permeability, mito-
chondrial dysfunction, and oxidative injury. This can result in several symptoms, including
visual loss, muscle weakness, balance problems, and cognitive impairment [109,110]. Al-
though the specific causes of MS are unknown, genetic polymorphisms, particularly in the
genes encoding human leukocyte antigen (HLA), lifestyle, and environmental factors are
considered to play a role [110].
There is no cure for MS and most treatments revolve around immunomodulation, like
interferon-beta. Research is being conducted to uncover new immune modulation targets
and other strategies, such as remyelinating and cell-based therapies [108]. Preserving the
normal functioning of the immune and inflammatory systems, decreasing oxidative stress,
and maintaining neuronal integrity are crucial objectives in MS treatment. In this context,
Spirulina-derived compounds such as phycocyanin and its tetrapyrrolic prostate group,
phycocyanobilin, have been thoroughly studied [14] (Table 3).
Most research is conducted in experimental autoimmune encephalomyelitis (EAE)
rodent models. EAE is an induced inflammatory disease of the CNS, where the immune
system becomes activated in response to self-antigens, resulting in a pathology that is
similar to that of MS [111]. Pentón-Rol et al. (2011) [112] investigated the prophylactic
and therapeutic effects of phycocyanin (25 mg/kg/day) from S. platensis in an EAE
model. The prophylactic schedule prevented disease development and both schedules
ameliorated the mean cumulative score. The treatments provided neuronal protection as
rats showed compressed, solid, and squashed myelin and no signs of axonal breakdown.
It also attenuated protein and lipid damage, as evidenced by the reduced levels of MDA,
advanced oxidation protein products (AOPP), peroxidation potential (PP), and ferric-
reducing ability of plasma (FRAP) [112]. In peripheral blood mononuclear cells (PBMC)
from MS patients, stimulation with phycocyanin induced a regulatory T cell (Treg) response,
by increasing the expression of all Treg cell markers, including CD25, Foxp3, TGF-β, and
IL-10, and the number of CD4+ CD25high Foxp3+ T cells, indicating an ability to induce the
Treg subset, which is reduced in MS patients [112].
In another study, the effects of phycocyanin from S. platensis and its tetrapyrrolic
prostate group, phycocyanobilin (12) (Figure 7), were investigated. In EAE-Lewis rats,
the prophylactic regimen of oral phycocyanin administration (200 mg/kg) eliminated
disease symptoms, whereas the therapeutic regimen (200 mg/kg) significantly reduced
the maximum clinical score and delayed disease onset. Both regimens produced a positive
effect on motor impairment. Phycocyanin exhibited antioxidant activity by lowering MDA,
PP, and FRA levels in serum and preserved myelin integrity, as evidenced by transmission
electron microscopy, which revealed that rats treated with phycocyanin had compressed,
solid, and squashed myelin. In the same study, oral phycocyanobilin (5 mg/kg) treatment
in EAE-C57BL/6 mice improved clinical progression and reduced neuroinflammation by
significantly lowering brain expression of IL-6 and IFN-γ, which are pro-inflammatory
cytokines implicated in MS pathology [113].
A therapeutic regimen of phycocyanin (4 or 8 mg/kg) from S. platensis decreased
disease severity and improved clinical performance in an EAE-mice model. It protected
against demyelination and axonal degeneration, as evidenced by a lesser extent of demyeli-
nation in the spinal cord and a decrease in the density of APP-positive axons in white
matter. It also demonstrated anti-inflammatory activity as it reduced inflammatory foci in
the spinal cord, decreased Mac-3 activated microglia and CD3-positive T cells in lesions,
and down-regulated Foxp3, a regulatory T cell marker, in the brain. The treatment also
reduced the expression of IL-17 mRNA in the brain and serum, the main effector cytokine
 severity. The treatment regulated gene expression by down-regulating the TNF-α,
LINGO1, and NOTCH1 genes, which mediate myelin damage, and by up-regulating the
CXCL12, MOG, NKX2-2, OLIG1, and MAL genes, which mediate myelin protection.
Phycocyanobilin reduced demyelination in the white matter of the spinal cord and
showed anti-inflammatory properties as it decreased the levels of the pro-inflammatory
Biomolecules 2023, 13, 1444 22 of 34
cytokines IL-17A and IL-6 and the number of microglia/macrophages, marked by the
diminution of Mac-3 and CD3 expression. It also increased the number of oligodendrocyte
precursor cells and mature oligodendrocytes as shown by a significant increase in both
oligodendrocyte
in markers
MS, as well as other (Olig2 and TPPP/p25)
pro-inflammatory while
cytokines decreasing
(IL-6). APP
It was also levels, in
efficient a marker
lowering of
axonal damage [116].
oxidative stress, as measured by lower levels of MDA, PP, and the CAT/SOD ratio [114].

Figure 7.
Figure 7. Structure
Structureof
ofphycocyanobilin
phycocyanobilin(12),
(12),a alinear
lineartetrapyrrole
tetrapyrrole chromophore
chromophore covalently
covalently attached
attached to
to protein subunits of phycocyanin.
protein subunits of phycocyanin.

Phycocyanin’s therapeutic effects can be linked to its gene-modulatory properties.

In the same study by Pentón-Rol et al. (2016) [114], phycocyanin (8 mg/kg) modulated
the expression of 918 genes, with prominence in the upregulation of genes involved in
remyelination, gliogenesis, and axon–glia interactions (Mal, Mog, Mobp, Nkx6-2, Nkx2-
2, Bmpa, and the transcription factor Olig1) while decreasing the expression of genes
implicated in demyelination, such as CD44 and PPARMal. The authors also compared the
effects of phycocyanin treatment to those of the standard treatment for MS, interferon-beta
(IFN-β). While both had antioxidant and anti-inflammatory effects and modulated some
of the same genes, they differed in several specific biological processes, implying that the
combined treatment may provide additional benefits [114].
Gardón et al. (2022) [115] investigated the effects of phycocyanobilin (12) on gene
modulation in vitro in a SH-SY5Y cell line model of glutamate-induced excitotoxicity. Pre-
stimulation with phycocyanobilin (0.1 M) promoted a significant downregulation of the
CYBB, HMOX-1, and HIF1A genes, all of which are linked to neurodegenerative diseases.
On the other hand, it led to an increase in the expression levels of the genes encoding the
detoxifying enzymes SOD2 and CAT, the antioxidant gene GPX1, the apoptosis-related
proteins Bax and Bcl-2, and the transcription factor NFBK1. They also investigated the
effects of oral phycocyanobilin treatment (1 mg/kg) in an EAE-induced MS rat model,
which showed a tendency to reduce clinical signs as well as significantly lower the levels of
pro-inflammatory cytokines (IL-17, IFN-γ, and IL-6) [115].
Similarly, in a MOG35–55 induced EAE rat model, treatment with phycocyanobilin (12)
(0.5 and 1 mg/kg) showed protection against MS, delaying the disease’s symptoms and
severity. The treatment regulated gene expression by down-regulating the TNF-α, LINGO1,
and NOTCH1 genes, which mediate myelin damage, and by up-regulating the CXCL12,
MOG, NKX2-2, OLIG1, and MAL genes, which mediate myelin protection. Phycocyanobilin
reduced demyelination in the white matter of the spinal cord and showed anti-inflammatory
properties as it decreased the levels of the pro-inflammatory cytokines IL-17A and IL-6
and the number of microglia/macrophages, marked by the diminution of Mac-3 and CD3
expression. It also increased the number of oligodendrocyte precursor cells and mature
oligodendrocytes as shown by a significant increase in both oligodendrocyte markers (Olig2
and TPPP/p25) while decreasing APP levels, a marker of axonal damage [116].
Biomolecules 2023, 13, 1444 23 of 34

Table 3. Cyanobacteria-derived products studied in MS disease models.

Strain Compound/Extract Effect In Vitro Assays In Vivo Assays Reference

Decreased the mean
PBMCs:
cumulative EAE induction in male Lewis rats:
RT-PCR (TGF-β, IL-10,
score. Neuronal Morphology MDA assay. PP assay. TOP assay. AOPP
Spirulina platensis Phycocyanin CD25, Foxp3). Flow [112]
Protection. Antioxidant. assay. FRAP assay. Transmission electron
cytometry
Anti-inflammatory. Treg microscopy studies.
(CD4, CD25, CD69).
induction.
EAE induction in male Lewis rats
Improvement in disease onset
and female C57BL/6 mice:
and locomotion. Neuronal
Rotarod test. MDA assay.
Spirulina platensis Phycocyanin Morphology Protection. [113]
PP assay. FRA assay. Transmission electron
Antioxidant.
microscopy studies.
Anti-inflammatory.
ELISA (IL-17, IL-6, IFN-γ).
Improvement in disease
EAE induction in C57BL/6 mice:
onset.
Immunohistochemistry (CD3, Mac-3, APP).
Antioxidant.
Morphometric Analysis. MDA assay. PP
Spirulina platensis Phycocyanin Anti-inflammatory. [114]
assay. SOD, CAT, and GSH
Anti-demyelination.
assays. IL-17 quantification. RT-PCR.
Neuronal
Microarray Analysis.
Protection. Gene Modulation.
Improvement in disease EAE induction in C57BL/6 mice:
Human SHSY5Y cells:
onset. Anti-inflammatory. ELISA (IL-17, IL-6, IFN-γ). Transmission
Spirulina platensis Phycocyanobilin (12) RT-PCR. Gene expression [115]
Antioxidant. Anti-apoptosis. electron microscopy. Immunohistochemistry
profile analysis.
Gene modulation. (caspase-3, CD11).
Improvement in disease EAE induction in C57BL/6 mice:
onset. Immunohistochemistry (CD3,
TMBP-GFP cells:
Anti-inflammatory. Mac-3, APP, TPPP/p25, Olig2).
Spirulina Phycocyanobilin (12) Proliferation assay. [116]
Anti-demyelination. Morphometric analysis. ELISA
Fluoresce microscopy.
Neuronal protection. (IL-17A, IL-6, and IL-10).
Gene modulation. qPCR. Flow cytometry.
Abbreviations: PBMCs—Human peripheral blood mononuclear cells. RT-PCR—Reverse transcription polymerase chain reaction. TGF-β—Transforming growth factor beta. CD25—
Cluster of differentiation 25. FOXP3—Forkhead box P3. CD4—Cluster of differentiation 4. CD69—Cluster of Differentiation 69. EAE—Experimental autoimmune encephalomyelitis.
MDA—Malondialdehyde. PP—Lipid peroxidation. TOP—Total organoperoxides. AOPP—Advanced Oxidation of Protein Products. FRAP—Ferric Reducing Ability of Plasma.
FRA—Ferric Reducing Ability. IL—Interleukin. IFN-γ—Interferon gamma. ELISA—Enzyme-Linked Immunosorbent Assay. CD3—Cluster of Differentiation 3. APP—Amyloid-beta
precursor protein. SOD—Superoxide dismutase. CAT—Catalase. GSH—Total Glutathione. CD11—Cluster of Differentiation 11. TPPP/p25—Tubulin polymerization-promoting protein.
Olig2—Oligodendrocyte transcription factor.
Biomolecules 2023, 13, 1444 24 of 34

4.4. Cyanobacteria against Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease, is a rare and
fatal ND that affects motor neurons, with an estimated global incidence of 2 per 100,000
person each year [117]. It is characterized by the degeneration of upper motor neurons
in the brain’s motor cortex and the loss of lower motor neurons in the brainstem and
spinal cord. The most significant neuropathological findings are intracellular cytoplasmic
aggregates of eosinophilic Bunina bodies and ubiquitinated TDP-43 protein. Excitotoxicity,
mitochondrial dysfunction, oxidative stress, inflammation, decreased axonal transport,
and faulty RNA and DNA metabolism are all implicated in ALS pathophysiology. It
causes muscle weakening, spasticity, and atrophy, which results in movement, speech, and
breathing difficulties, finally leading to paralysis and death from respiratory failure within
two and three years [118,119].
Although the cause of ALS is unknown, genetic mutations, especially in the SOD1,
C9orf72, TARDB, and FUS genes, or environmental factors, can be involved [118,119].
There is currently no cure for ALS and the available treatments merely provide symp-
tomatic alleviation. Medications like riluzole and edaravone can delay disease progression
and improve survival but they cannot reverse the damage. Gene therapy, stem cells, or
antibodies may be explored as future treatments [120].
However, to a much lesser extent, cyanobacteria-derived compounds have also been
shown to be beneficial in the treatment of ALS (Table 4).
De Paola et al. (2012) [121] investigated the in vitro and in vivo effects of VB3323,
a highly (95%) purified form of cyanobacterial LPS-like molecule (CyP) isolated from
Oscillatoria planktothrix sp. Co-treatment with VB3323 (20 µg/mL) after LPS inhibited
cell activation and morphological changes to the reactive phenotype in purified cultures
of microglia. This molecule also significantly reduced the release of pro-inflammatory
cytokines (TNF-α, IL-1β, and IL-6) induced by LPS in co-cultures of motor neuron cells and
microglia and restored motor neuron viability (91.3%), counteracting LPS and its bioactive
form lipid A toxicity. Using an in vivo model of motor neuron degeneration (Wobbler
mice), the treatment with VB3323 intraperitoneally injected three times a week (0.5 mg/mL)
slowed the disease progression and improved motor behavioral scores. It also reversed
the morphological changes in motor neurons while reducing GFAP immunoreactivity and
TNF-α expression in the ventral horn of the cervical spinal cord [121].
In an in silico experiment, β-carotene, chlorophyll-a, chlorophyll-b, phycoerythrin,
and phycocyanin, which are the main natural pigments in cyanobacteria, were docked
against the p75 neurotrophin receptor, the EphA4 receptor, and the HDAC receptor, which
are promising therapeutic targets in ALS. It was discovered that β-carotene, phycoerythrin,
and phycocyanin had high binding energies to the targets, indicating possible antagonistic
activity [122].
A study explored the effects of a diet supplemented with 0.1% Spirulina for 10 weeks
in a SOD1G93A mice model of ALS. This model overexpresses the human SOD1 protein con-
taining the G93A mutation, which is common in human familial ALS, and exhibits similar
clinical and neuropathological findings of ALS [123]. The supplementation with Spirulina
promoted extension reflex maintenance, particularly in the right hindlimbs, delaying the
development of symptoms. It reduced motor neuron degeneration in the lumbar spinal
cord, with fewer FluorJade-labeled neurons, a marker of degeneration, and fewer activated
astrocytes marked by GFAP. It also had anti-inflammatory properties, reducing the levels
of pro-inflammatory cytokines like IL-1β and TNF-α in the brainstem [124].
Biomolecules 2023, 13, 1444 25 of 34

Table 4. Cyanobacteria-derived products/extracts studied in ALS disease models.

Strain Compound/Extract Effect In Vitro Assays In Vivo Assays Reference

Purified microglial cells:
Immunocytochemistry
(CD11b).
TLR4 antagonist. Immunoblotting (CD68).
Improved motor Live cell imaging (GFP) Wobbler Mice:
function Motor neurons/glia Paw abnormality and
Oscillatoria
LPS-like molecule tests. co-culture: grip strength test.
planktothrix [121]
VB3323 Anti-inflammatory ELISA (TNF-α, IL-1β and Immunohistochemistry
sp.
and IL-6). (GFAP, CD11, and
anti-gliosis. Motor neurons/glia TNF-α).
Neuroprotection. coculture
and purified motor neurons:
Motor Neuron Viability
Assay (SMI32).
Maintenance of SOD1G93A mice:
extension Weight and
reflex. measurement. Extension
Diet Anti-inflammatory. Reflex test. Ribonuclease
Spirulina [124]
supplementation Neuroprotection Protection Assay (IL-1α,
against motor IL-1β, IL-6, TNF-α).
neuron Immunohistochemistry
degeneration. (Fluoro-Jade, GFAP)
Abbreviations: IL—Interleukin. TNF-α—Tumor necrosis factor-alpha. GFAP—Glial fibrillary acidic protein.
TLR4—Toll-like receptor 4. CD11—Cluster of Differentiation 11. CD68—Cluster of Differentiation 68. GFP—Green
fluorescent protein. ELISA—Enzyme-Linked Immunosorbent Assay.

4.5. Cyanobacteria against Huntington’s Disease

Huntington’s disease (HD) is a genetic ND that follows an autosomal-dominant in-
heritance pattern, affecting around 10.6–13.7 per 100,000 individuals [125]. It is caused by
the expansion of a cytosine–adenine–guanine (GAG) trinucleotide repeat in the huntingtin
(HTT) gene, resulting in a mutant huntingtin protein (mHTT) with an abnormally long
polyQ tract. This mutation is fully penetrant at 40 or more repeats [126]. The propensity
of these proteins and polyQ N-terminal fragments to aggregate results in the formation
of intranuclear inclusions, which are characteristic of HD. mHTT transcripts are also toxic
through a gain-of-function mechanism. This disrupts cellular functions, with compromised
proteostasis, mitochondrial dysfunction, aberrant immune activation, synaptic excitotoxic-
ity, neuroinflammation, oxidative stress, and defective transcription. The consequence is
neuronal death, particularly in the striatum, where medium-spiny neurons (MSNs) are the
most vulnerable [9]. HD presents as a triad of motor, cognitive, and emotional impairments.
The signature clinical feature is chorea, characterized by involuntary and uncontrolled
movements, but it also encompasses a broad range of neuropsychiatric disturbances, in-
cluding mood disorders and dementia. It typically manifests in adulthood between the
ages of 30 and 50, progressing relentlessly with significant disability and is ultimately fatal,
with an average survival of 18 years [125,126].
There is currently no cure for HD and available treatments are limited to symptomatic
management, such as tetrabenazine and deutetrabenazine to reduce chorea. Disease-
modifying therapies are being investigated, including DNA/gene therapies, RNA modula-
tion, stem cell-based therapies, and immunization against mHTT [9,127].
There is limited evidence suggesting the potential of cyanobacteria-derived products
in treating HD. The primary mechanism of action is through reducing neurotoxicity and
oxidative stress caused by polyQ aggregation (Table 5).
The anti-proteostasis potential of phycocyanin, isolated from Leptolyngbya sp. N62D,
was demonstrated in C. elegans AM141, a model of HD polyQ tract expansion [30]. It
Biomolecules 2023, 13, 1444 26 of 34

expresses polyQ fused to a yellow reporter protein (polyQ::YFP) in muscle cells and
becomes progressively paralyzed with age, mimicking the disease [128]. The treatment
with phycocyanin (100 µg/mL) in the medium, both in the presence and absence of
paraquat, which is a potent oxidative stress inducer, led to a significant decrease in the
formation of polyQ::YFP aggregates by 0.63-fold and 0.53-fold, respectively. The treatment
also markedly increased the survival rate of AM141 worms, whether paraquat was present
or not. Furthermore, phycocyanin (100 µg/mL) demonstrated anti-aging activity in wild-
type C. elegans (N2) by increasing the mean lifespan, the pharyngeal pumping, and the
locomotion rate. It also showed antioxidant potential in vitro, with radical scavenging and
reducing power abilities, as well as in vivo, by enhancing tolerance to oxidative stress and
thermotolerance of C. elegans [30].
Zhong et al. (2021) [129] studied the geroprotective effects of polysaccharides derived
from Nostoc sphaeroides colonies in C. elegans HA759, another model of HD. This transgenic
strain exhibits human polyQ expansions in ASH neurons, replicating the HD phenotype
and displays impaired avoidance behavior. Exposure to oligosaccharides (NOS-HCA and
NOS-TFA) chemically derived from N. sphaeroides polysaccharides, at a concentration of
0.5 mg/mL, improved the chemosensory avoidance index of worms, protecting them
from polyQ-mediated neurotoxicity. They also upregulated genes linked to stress and
proteostasis, namely the glutathione S-transferase gene (gst-4), the catalase gene (ctl-2), and
the heat shock protein genes (hsp-6 and hsp-60). In addition, both polysaccharides and
their derived oligosaccharides possessed in vitro antioxidant activity, as they scavenged
ABTS and DPPH free radicals (2 mg/mL), and in vivo (0.5 mg/mL), by increasing the
survival rate of C. elegans under both oxidative stress and normal condition [129].
Biomolecules 2023, 13, 1444 27 of 34

Table 5. Cyanobacteria-derived products with neuroprotective activity against HD.

Strain Compound/Extract Effect In Vitro Assays In Vivo Assays Reference

N2 Caenorhabditis elegans:
Life span assay. Pharyngeal pumping and
locomotion assays. DCFH-DA fluorescence
Anti-polyQ aggregation. staining. Stress resistance assay.
DPPH assay. FRAP assay.
Leptolyngbya sp. N62DM Phycocyanin Antioxidant. DAF-16::GFP localization [30]
SRSA assay. R-Power assay.
Increased lifespan. Caenorhabditis elegans AM141:
PolyQ aggregation assay. Paraquat
sensitivity assay. Life span assay.
DAF-16::GFP localization
N2 Caenorhabditis elegans:
Oxidative survival assay.
Improved chemosensory
Chemically derived Lifespan assay. qPCR.
behavior. Improved
Nostoc sphaeroides oligosaccharides ABTS assay. DPPH assay. Caenorhabditis elegans HA759: [129]
lifespan. Antioxidant.
(NOS-HCA, NOS-TFA) Oxidative survival assay. Chemosensory
Gene modulation.
behavior assay. Lifespan assay.
qPCR (gst-4, ctl-2, hsp-6, and hsp-6).
Abbreviations: polyQ—Polyglutamine. DPPH—2,2-Diphenyl-1-picrylhydrazyl. FRAP—Ferric reducing ability of plasma. SRSA—Superoxide radical scavenging activity. R-Power—
Reducing power. DCFH-DA—Dichlorodihydrofluorescein diacetate. DAF-16—Forkhead box protein ortholog gene. GFP—Green fluorescent protein. ABTS—(2,20 -azino-bis(3-
ethylbenzothiazoline-6-sulfonic acid)). qPCR—Real-Time Polymerase chain reaction. gst-4—Glutathione S-transferase 4 gene. ctl-2—Peroxisomal catalase 1 gene. hsp-6—Heat shock
protein 6 gene. hsp-60—Heat shock protein 60 gene.
Biomolecules 2023, 13, 1444 28 of 34

5. Conclusions
The increasing burden of NDs on aging populations requires urgent attention. Due
to limited progress in research, there is a high demand for new therapies. Given the
remarkable chemical prolificacy of cyanobacteria and their ability to produce neuroac-
tive compounds, this review aimed to explore the anti-neurodegenerative potential of
cyanobacterial natural products.
The data presented show that multiple in silico, in vitro, and in vivo studies support
the neuroprotective potential of cyanobacteria. This suggests their ability to combat neu-
rodegeneration through various mechanisms, including acting as enzyme and protein
aggregation inhibitors, antioxidants, anti-inflammatories, immunomodulators, or gene
modulators. Given that NDs are associated with multiple cellular malfunctions, a multi-
target drug strategy such as this, as a standalone treatment or as adjuvant therapy, may
prove to be very effective.
The variety of treatment options presented is noteworthy. Several results were credited
to complex extracts or whole cyanobacteria, which contain multiple active components that
may interact to produce additive/synergistic effects. Moreover, there were also isolated
compounds, such as tasiamide B, which highlight the structural and biological diversity
of cyanobacteria. The products showcased a range of delivery methods and formulations,
with an emphasis on dietary supplements, implying a possible use as nutraceuticals.
Furthermore, both regimens of pre-treatment and treatment were investigated, with positive
results in both cases, indicating the importance of prevention in NDs.
Spirulina was found to be the most versatile among the strains of cyanobacteria
mentioned and phycocyanin, which is found in most cyanobacterial strains, was the
most studied compound. However, the potential of cyanobacteria in combating NDs
is still largely untapped. Further investigating other genera such as Nostoc or Lyngbya
and applying high-throughput screening techniques is worthwhile as each strain has the
potential to produce unique sets of compounds that can be valuable. It is also crucial to
expand the research to other therapeutic targets and NDs such as ALS, HD, and prion
diseases, as there is still a paucity of research on this subject.
Despite the evidence presented and the numerous preclinical studies conducted, trans-
lating these insights into clinical applications can be challenging. The assays and animal
models may not fully capture the complexity of NDs and pharmacokinetic issues such as
the bioavailability, efficacy, and safety of the products can also be a concern. In fact, many
cyanobacterial neuroactive compounds are actual neurotoxins. Moreover, the few human
clinical trials that have been conducted mostly focused on behavioral/cognitive improve-
ments in patients rather than evaluating molecular markers specific to NDs, suggesting the
need for further research in this area.
In conclusion, while cyanobacteria demonstrate promise as a potential treatment
option, this field is still in its infancy and further in-depth research is necessary to fully
comprehend and harness the potential of cyanobacteria in battling NDs.

Author Contributions: Conceptualization, V.R. and R.M.; resources, R.M.; data curation, V.R., A.M.S.,
M.R. and L.F.; writing—original draft preparation, V.R. and R.M.; writing—review and editing, M.R.,
L.F., A.M.S., R.F., M.V., V.V. and R.M.; supervision, M.R. and R.M.; funding acquisition R.M. and V.V.
All authors have read and agreed to the published version of the manuscript.
Funding: This work was funded by the European Regional Development Fund (ERDF) through
the Regional Operational Program North 2020, within the scope of Project GreenHealth and digital
strategies in biological assets to improve well-being and promote green health, Norte-01-0145-FEDER-
000042.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Biomolecules 2023, 13, 1444 29 of 34

Acknowledgments: Ana Margarida Silva acknowledges the GreenHealth project through the POR-
TIC/HealthTech/BI/2021/01 grant. Leonor Ferreira acknowledges Fundação para a Ciência e
Tecnologia (FCT) grant 2022.11979.BD. The authors are also grateful to FCT’s financial support to
CIIMAR (UIDB/04423/2020 and UIDP/04423/2020) and to WP9- Portuguese Blue Biobank under the
Blue Economy Pact, Project Nº. C644915664-00000026 co-funded by PRR, The Portuguese Republic,
and the European Union.
Conflicts of Interest: The authors declare no conflict of interest.

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