Parkinson’s disease (PD) is a chronic, progressive neurodegenerative disorder.
PD symptoms result from the loss of dopaminergic neurons in the midbrain
substantia nigra (SN) pars compacta, culminating in the depletion of dopamine in
the striatum . The degenerative changes in PD are not only limited to the
nigrostriatal pathway; other sites throughout the nervous system, e.g., the
spinal cord (SC), have been shown to also play a critical role in the
pathophysiology of the disease [2, 3, 4]. Neuron loss manifests symptomatically as
tremors, bradykinesia, limb rigidity, and gait/balance abnormalities. The cause
of PD is not completely understood, therefore noncurative therapies aim to
alleviate symptoms and improve patients’ quality of life . Systemic
administration of levodopa (L-DOPA), the amino acid precursor of dopamine, is
commonly used for therapeutic treatment of PD . However, L-DOPA only offers
temporary relief and can lead to adverse side effects such as motor fluctuations
and L-DOPA-induced dyskinesias . Hence, current studies are focused on the
identification of potential therapeutic targets for both the SN and SC and
development of more effective therapeutic treatments for PD.
It is well known inflammation plays a crucial role in PD’s pathology through
destruction of dopaminergic neurons as a result of Lewy body (LB) formation,
glial cell activation, and perpetual inflammatory cycles in the SN [1, 7, 8, 9].
Recently, it has been shown these same mechanisms occur in the SC, destroying
motor neurons through abnormal -synuclein (-syn) aggregation
and LB formation. LB’s are cytoplasmic, proteinaceous, lipid-rich inclusions
composed of -syn aggregates and other biological components such as
lipid membrane fragments and distorted organelles [10, 11, 12]. LB’s serve as
hallmarks of the pathogenesis in PD and other neurodegenerative disorders [10, 11]. Studies have shown an association exists between the mechanism driving
-syn fibril assembly and the recruitment of other proteins and
organelles with the formation of LB . -Syn aggregation may occur as
a result of glial cell dysregulation .
Glial cells in the central nervous system (CNS) are shown to be activated in PD
patients. These glial cells are activated by 1-methyl-4-phenylpyridinium (MPP+),
rotenone, and 6-hydroxydopamine (6-OHDA) in both the SN and SC [14, 15]. Studies
have focused on microglia and astrocytes, the most abundant glia in the CNS
responsible for homeostasis maintenance and other metabolic functions [7, 16, 17, 18].
Upon activation, microglia proliferate and differentiate, altering their number
and morphology; they contribute to neuronal death through a variety of mechanisms
including inflammation, -syn accumulation, oxidative stress, and damage
to the blood brain barrier (BBB) [9, 19, 20]. Microglial activation forms a
positive feedback loop, where dead neurons subsequently activate astrocytes,
perpetuating an inflammatory cycle [7, 21]. Astrocytes have a variety of
functions, some of which are critical for neuronal health including: structural
and metabolic support, synaptic transmission regulation, BBB support, production
of neurotrophic factors, etc. [22, 23]. One such neurotrophic factor is
glial-derived neurotrophic factor (GDNF), which is essential for the development
and survival of dopaminergic neurons . In healthy individuals, after
microglial initiation of an immune response, astrocytes migrate to the damaged
area and form a barrier to prevent the spread of toxic compounds [22, 23]. In PD
patients, however, the function of astrocytes is disrupted, resulting in the
degeneration of neighboring neurons in the CNS. It is believed astrocytes may
function in a neuroprotective manner in PD due to the effects of astrocyte
dysfunction on surrounding neurons in the SN and SC [22, 23]. Investigations into
the neuroprotective effects of astrocytes need to be conducted to fully
understand the capabilities of astrocytes as therapeutic mechanisms.
Other contributors to the inflammatory cycle in PD are T helper (Th) cells,
specifically Th1 and Th17 cells . Th cells are activated by various immune
substrates and participate in the inflammatory response implicated in the
pathogenesis of PD [25, 26, 27]. Injured neurons and immune cells in the CNS release
various cytokines, for example high mobility group box (HMGB1), that induce T
cell differentiation into Th17 cells. Th17 cells are the most inflammatory Th
cell phenotype; they exert cytotoxic effects through cytokine and chemokine
release . Interleukin-17A (IL-17A), an inflammatory cytokine released by Th17
cells, is cytotoxic to neurons in the CNS, and when released, causes neuronal
cell death in the SN and SC [26, 29].
Recent studies have also shown PD-related neurotoxins, such as MPTP, activate
calpain the SN and SC in animal models [21, 30]. Calpain is a neutral protease
known to activate glial cells and T-cells in the presence of elevated
intracellular calcium (Ca) concentrations . Calpain activation has been
shown to contribute to the disruption of mitochondrial function and dysregulation
of various immune cells (i.e., microglia and T-cells). Ca activation of
calpain also results in the misfolding of -syn, leading to similar
dopaminergic neuron loss found in PD patients . -Syn aggregation
also causes the disruption of dopamine synthesis through phosphorylation of TH, a
critical enzyme in the synthesis of dopamine from L-DOPA [32, 33]. Decreased
dopamine levels lead to the stimulation of pro-inflammatory immune cells due to
the use of high affinity dopamine receptors (DR), DRD3 and DRD5, which are not
normally active in healthy individuals . These high affinity receptors induce
the Th1 and Th17 cell phenotype, further modulating the inflammatory response
promoting dopaminergic neuronal death [24, 32]. Similar -Syn
aggregation and disruption of motor neurons has also been shown to occur in the
SC upon Calpain activation . Therefore, calpain appears to be a critical
component of the inflammatory response that results in neuronal cell death in PD
patients. Thus, immunomodulatory agents targeting calpain, such as the calpain
inhibitor Calpeptin, have been investigated as potential therapies in PD animal
models [34, 35].
2. LB and -SYN aggregation in PD
One significant feature of PD is the accumulation of LBs in the CNS [11, 36, 37]. LBs are presynaptic neural proteins, formed from abnormal clumps of
-syn, a neuronal protein implicated in a variety of mechanisms
including synaptic vesicle release and recycling, binding of dopamine and
serotonin transporters, and synaptic plasticity regulation . LBs themselves
are relatively non-toxic; however, oligomerization of -syn forms toxic
fibril structures in neuronal plasma membranes (Fig. 1). These toxic fibrils lead
to lipid bilayer penetration, pore formation, perturbation of homeostatic
Ca influx, oxidative stress, and dopamine associated neuronal death in the
brain and SC . -Syn concentrations are thought to be controlled by
microglia through autophagy and lysosomal clearance. When microglial populations
are diminished, however, -syn levels are dysregulated — leading to
increased transfer of -syn between grafted neurons and LB formation
. Aging, a significant risk factor for PD, is also associated with the
decreased ability of microglia to phagocytize -syn . Histological
evaluations of neurons taken from postmortem PD samples and MPTP-treated lesioned
rats likewise reveal gliosis, neurofibrillary tangles, and LB formation in both
the SN and SC [3, 21, 42].
-Syn dysregulation by microglia resulting in
neuroinflammation and neuronal death. Impaired microglial function prevents
normal autophagy and lysosome clearance, leading to aggregation of -syn
monomers into oligomers, proto-fibrils, fibrils, and eventually LB’s. LB, in
conjunction with aggregated -syn, results in lipid bilayer penetration,
Ca ion influx, oxidative stress and damage to the BBB. -Syn
aggregation into LB’s results in the activation of TLR2 and TLR4, which further
activate inflammasomes leading to inflammatory signaling and neurodegeneration.
-Syn aggregation also results in the disruption of dopamine synthesis
through the phosphorylation of TH, a critical enzyme in the synthesis of L-DOPA,
the amino acid precursor to dopamine. Reduced TH and dopamine levels results in
dysfunctional DR signaling leading to further neuroinflammation and
neurodegeneration. -Syn aggregation into oligomers and proto-fibrils
also allows -syn to interact with CD36 and FYN, resulting in further
inflammasome activation, -syn uptake, and neurodegeneration. (Figure
created with BioRender.com).
In healthy individuals, -syn proteins are primarily located in the CNS
and comprise 10% of cytosolic protein . In PD patients, however,
-syn proteins mutate and truncate, leading to harmful aggregation. The
exact mechanisms leading to -syn aggregation are not completely
understood, but the process is critical in the progression of PD due to the
activation of microglia and disruption of Ca homeostasis. Increased levels
of misfolded -syn and toxic fibrils allow for significant membrane
leakage with neuronal lysis, closely following the release of misfolded and
modified -syn proteins into the brain parenchyma . These
-syn protofibrils inhibit tyrosine hydroxylase (TH), the rate-limiting
enzyme in dopamine synthesis, through TH phosphorylation - ultimately reducing
circulating dopamine levels (Fig. 1). Dopamine is believed to be a major
regulator of inflammation, involved in the activity, migration, differentiation,
and proliferation of immune cells involved in cognitive functions .
MPTP-exposed mice express a significant loss of TH neurons with concomitant
increase in -syn aggregation and neuronal inclusion formation [7, 45].
Semiquantitative analysis of TH- immunoreactive (IR) neurons in sections taken
from SN of MPTP injected mice reveals a ~55% reduction of TH-IR
dopaminergic neurons compared to controls. Despite the variability in the loss of
SN dopaminergic neurons, this MPTP toxin-induced PD model is still widely used to
understand the disease’s pathophysiology and to explore the neuroprotective
-Syn aggregation has also been shown to have a negative effect on
motor neurons in the SC [3, 4, 42]. Significant colocalization of TUNEL in
neuronal nuclear protein, NeuN, positive neurons highlights neuronal death in
both the dorsal and ventral SC . Axonal degradation is also similarly
prevalent in MPTP-treated mice, as identified by intense dephosphorylated
neurofilament protein (deNFP) IR immunofluorescent staining in the cervical and
lumbar SC samples of MPTP-treated mice with phosphorylation of neurofilament
proteins (NFP) in these regions . Axonal transport motor proteins are also
degraded, with decreasing concentrations of dynein and kinesin found in the
dorsal horns (DH), ventral horns (VH), and interneurons (IN) of MPTP-treated mice
. Motor protein decay is strongly correlated with PD progression as motor
proteins are responsible for synaptic neurotransmission. Studies on the
involvement of the SC in PD need to be investigated in order to provide better
quality therapeutics to patients suffering from motor function abnormalities and
Toll-like receptor 2 and 4 (TLR2 and TLR4) , innate immune system proteins
upregulated in monocytes and microglia of PD patients, recognize protofibril
forms of -syn, prompting the release of pro-inflammatory signals,
neuronal degeneration, and activation of inflammasomes (Fig. 1) [38, 40].
Additionally, in the -syn driven PD model, Adeno-associated virus(AAV)
2-SY, increased expression of major histocompatibility complex-II (MHC-II) on
resident microglia and elevated levels of macrophages and monocytes mediating T
cell infiltration is observed compared to controls . Similarly, MHC-I
expression on CD8+ T-cells is increased by AAV2-SY transduction, with MHC-I
detected outside TH expressing cells. AAV-9--syn injections in rats
also upregulate MHC-I and MHC-II molecules, increasing the presentation of
antigenic peptides to cytotoxic T-cells. AAV models, however, are limited by the
lack of typical spreading in the CNS by transduced neurons .
-Syn also interacts with CD36, an integral membrane protein. Together
with an Src family non-receptor tyrosine kinase (FYN), CD36 mediates further
-syn uptake and NOD-, LRR- and pyrin domain-containing protein 3
(NLRP3) inflammasome activation, inducing neurodegeneration in various PD models
(Fig. 1) . AAV-9-induced expression of -syn results in dopaminergic
neuron loss in the SN accompanied by forelimb akinesia, assessed by cylinder
tests. Dopaminergic neuron loss is observed in T cell competent rats expressing
-syn, but not in T cell-deficient rats .
A cross-sectional study conducted for over 20 years on peripheral blood
mononuclear cells (PBMCs) stimulated with -syn epitopes revealed the
-syn-specific T-cell response is significantly greater in the first ten
years prior to PD diagnosis compared to the 17 years following PD diagnosis .
Furthermore, the -syn-specific T-cell response is more pronounced
immediately following PD diagnosis; this gradually declines afterward .
Hence, -syn-specific T cell response screening prior to PD diagnosis
may be a viable, early detection paradigm.
3. Activated microglia and astrocytes in PD
MPTP and other neurotoxins (rotenone and 6-OHDA) induce inflammatory responses
in the brain and SC through activation of microglia and astrocytes in rodent
models. Activated microglia and astrocytes release inflammatory factors such as
cytokines, chemokines and free radicals that lead to neuronal death (i.e.,
RANTES, Cox-2, IL-1, IFN-, and TNF-) [7, 14, 19].
RANTES, also known as CCL5, and eotaxin chemokine supplementation results in the
marked loss of nigral TH-positive neurons in combination with MPTP exposure .
Activated microglia also contribute to the inflammatory cycle seen in PD through
the recruitment of and interaction with T-cells. Microglia secrete cytokines and
chemokines that recruit T-cells to the site of damage, initiating an inflammatory
response amplified by incoming immune cells . Activated microglia express
increased levels of MHC-I and MHC-II proteins, which are able to interact with
CD4+ and CD8+ T-cells, respectively . Both MHC-I and MHC-II house human
leukocyte antigen (HLA) genes that form complexes with peptide epitopes,
presenting intracellular antigens to the CD4+ and CD8+ T-cells [27, 49, 52]. Once
T-cells have migrated to the site of damage, they are able to interact with MHC
on microglia and other antigen presenting cells (APCs), leading to further T cell
infiltration and consequent inflammatory effects .
Microglia are critical components of the inflammatory cycle found in PD
pathogenesis due to their ability to be both pro- and anti-inflammatory [53, 54, 55].
Microglia are commonly classified with different phenotypes, some being pro- and
others anti-inflammatory. Pro-inflammatory microglia present high levels of
MHC-II (M1 microglia) and antigens with the capacity to prime T-cells. In
contrast, anti-inflammatory microglia express low levels of MHC-II and release
beneficial, anti-inflammatory cytokines (M2 microglia). For example, IL-4, a
cytokine released by anti-inflammatory microglia, suppresses microglial
pro-inflammatory activity and modulates neurogenesis. IL-4 is also implicated in
improving neuronal survival and initiating axonal healing following neuronal
damage as seen in PD . As PD progresses, pro-inflammatory microglia gradually
replace anti-inflammatory microglia, leading to increased inflammation and
neurodegeneration. Liposomal clodronate, a potent anti-macrophage agent,
attenuates pro-inflammatory macrophage proliferation induced by MPTP-treatment
through inhibition of Nuclear Factor Kappa B (NF-B) phosphorylation
[57, 58]. Once CD4+ T-cells infiltrate the SN/SC, microglia function as APC’s and
present nitrated -syn-derived antigens on MHC-II; this leads CD4+
T-cells to produce interferon gamma (IFN-) and tumor necrosis factor
alpha (TNF-) . Other studies support -syn’s role in
upregulating MHC-II on CNS myeloid cells, facilitating the infiltration of
IFN--producing CD4+ and CD8+ T-cells . These inflammatory markers
further contribute to the activation of microglia, and astrocytes.
Glial cells in the ventral-regions of cervical and lumbar SC samples tested for
inflammatory markers using immunofluorescent staining confirm MPTP’s role in
neurotoxicity [14, 59]. Activated microglia (ionized calcium binding adaptor
molecule 1 (Iba-1)), astrocytes (Glial Fibrillary Acidic Protein (GFAP)), and
peripheral macrophages (ED-2) in the brain and SC samples of MPTP-treated mice
support MPTP induced neurotoxicity. Deficiencies of Aquaporin-4 (AQP4), a family
of water channel proteins, in MPTP-treated cells exhibit severe PD-like symptoms
due to the hyperactivation of microglial pro-inflammatory responses . Other
inflammatory markers such as cyclooxygenase-2 (Cox-2), caspase-1, and Nitric
Oxide Synthase-2 (NOS-2) are also significantly upregulated in MPTP mouse tissue.
Studies showed in comparison to CNS samples harvested from control mice,
MPTP-treated mouse tissue show a 39% increase in Cox-2, a 22% increase in
caspase-1, and a 112% increase in NOS-2 using Western blotting . In
addition, caspase-1 expression causes -syn truncation, triggering
additional caspase-1 expression with further inflammation . The increased
presence of inflammatory markers suggests neuroinflammation initiated by glial
cell activation plays a vital role in the neurodegenerative processes of PD.
4. Oxidative stress in PD
The accumulation of -syn in the SN/SC due to microglia depletion has
been linked to mitochondrial dysfunction and the generation of reactive oxygen
species (ROS) [60, 61]. Aggregated -syn contributes to increased oxygen
free radical production by interacting with CD11b (an integrin protein found on
the surface of innate immune cells) and activation of NADPH Oxidase 2 (NOX2), a
crucial enzyme in antimicrobial host defense and immune regulation [40, 62, 63].
The increase in ROS accumulates in oxidative stress with resulting damage to
neurons in the CNS (Fig. 1).
In healthy individuals, ROS and reactive nitrogen species (RNS) levels are
maintained through a strict balance of production and clearance . In PD
patients, however, ROS and RNS production dramatically exceeds clearance. The
unpaired electrons in ROS react with surrounding molecules and mediate the
formation of dopamine quinones, resulting in adducts with proteins and other
biomolecules which affect structural proteins and enzyme function. For example,
dopamine quinones result in the intracellular formation of reactive peroxynitrite
in dopaminergic neurons. Peroxynitrite reacts with proteins such as
-syn, parkin, DJ-1, and PTEN Induced Kinase 1 (PINK1) through
nitratation and nitrosylation of tyrosine and cysteine moieties, respectively.
Oxidative stress is then recognized by the ataxia telangiectasia mutated kinase
(ATM), and ATM- and Rad3-related (ATR) kinase proteins, resulting in
phosphorylation of murine double minute gene 2 (MDM2), a ubiquitin-ligase that
enhances p53 degradation . This post-translational modification inactivates
MDM2 while simultaneously activating p53. In cases where DNA damage is not
corrected, p53 initiates programmed cell death as observed in dopaminergic
neurons/PC12 cells .
P53-induced pro-apoptotic genes also release cytochrome-c (cyt c), a protein
complex linked to oxidative stress, from the mitochondrial membrane. Pyroptosis,
a form of programmed cell death induced by the inflammatory caspase cascade,
generates a pro-inflammatory milieu with increased levels of IL-1 and
IL-18 cytokines . Inflammasomes and intracellular multiprotein complexes
mediate the maturation of IL-1 and IL-18 through caspase-1; these are
activated by damage-associated molecular patterns (DAMPs) and pathogen-associated
molecular patterns (PAMPs). Activation of inflammasomes and intracellular
multiprotein complexes results in a cascade of inflammatory processes
contributing to neuronal injury and rapid cell death in both the brain and SC
[38, 39]. These processes also amplify the ability of innate cells to actively
present antigens on MHCs, further directing T cell activation.
5. Disruption of blood brain barrier (BBB) in PD
The BBB is compromised by the release of inflammatory factors, i.e., cytokines,
following glial cell activation (Fig. 1) . A damaged BBB contributes to
dopaminergic neuronal death by facilitating the infiltration of cytotoxic T-cells
into the SN . With chronic inflammation, tight junctions between endothelial
cells, normally preventing T cell diffusion, allow passage of antibodies and
otherwise restricted immune cells . Furthermore, these inflamed CNS
endothelial cells upregulate the expression of adhesion molecules, such as
intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule-1
(VCAM-1), that bind and recruit circulating T-cells and monocytes. MPTP-induced
BBB disruption in mice increases the frequency of CD4+ T-cell infiltration in the
ventral midbrain following effector T (Teff) cell transfer . One study
suggests retinoic acid receptor (RAR)-related orphan nuclear receptor-t
(RORt — a specific transcriptional factor of the Th17 phenotype)
positive cells localize around disrupted BBBs in the hippocampus of rats,
indicating Th17 cells infiltrate the brain through the BBB .
The inflammatory cytokine IL-17A is also correlated with BBB leakage, as
IL-17A-KO MPTP-induced mice demonstrate significant alleviation of BBB leakage in
the SN . Furthermore, secukinumab, an FDA-approved anti-IL-17 antibody,
successfully prevents neuronal death in ex vivo cultures . IL-17
activates astrocytes, resulting in the production of multiple chemokines, which
in turn act on endothelial cells to disrupt the BBB . Lymphocyte
function-associated antigen-1 (LFA-1) is a key T cell integrin, regulating T cell
activation and migration . Blocking this LFA-1 activity reduces Treg cell
population in SN of MPTP treated mice. Additionally, blocking CD45, a
transmembrane molecule, in Tregs impaired the ability of Tregs to protect
dopaminergic neurons against MPP+ toxicity . Furthermore, a chronic
inflammatory environment is induced by Th17 infiltration through upregulation of
the pro-inflammatory and downregulation of the anti-inflammatory microglia
phenotype; this promotes local T-cell differentiation into the Th17 phenotype
6. Dysregulation of Ca homeostasis in PD
MPP+, rotenone, and 6-OHDA are mitochondrial toxins which result in: prevention
of oxidative phosphorylation, depletion of adenosine triphosphate (ATP),
mitochondrial membrane potential disruption, ROS production, and elevation of
intracellular Ca levels in the brain and SC (Fig. 2) . These
neurotoxins raise Ca levels by impairing the mitochondrial electron
transport chain (ETC), specifically through the inhibition of Complex I. MPTP
crosses the BBB and is converted to MPP+ by monoamine oxidase-B (MOA-B), an
enzyme localized in CNS astrocytes. Through passive transport, MPP+ enters the
mitochondria and inhibits Complex I of the ETC [7, 71]. Dysfunctional Complex I
depletes cellular ATP levels, leading to a partial depolarization of the cell
through reduced Sodium (Na+)/Potassium (K+) ATPase activity. In addition, the
overactivation of NMDA receptors results in excitotoxicity, raising cytosolic
Ca concentrations .
Calpain activation resulting in mitochondrial damage,
neuroinflammation, and neurodegeneration. Calpain, a neutral protease, is
activated in the presence of elevated intracellular Ca levels. Calpain
activation leads to the cleavage of various mitochondrial components, resulting
in dysfunctional mitochondrial activity. When the ETC is disrupted by calpain,
ATP production is reduced. The resulting increase in Ca levels causes
further calpain activation, ROS production, and neuronal death as a result of
microglial activation. Calpain activation results in decreased local microglial
populations, disrupting the normal functions of autophagy and lysosome clearance
by microglia. Dysfunctional microglia allow for the accumulation and aggregation
of -syn, resulting in pro-inflammatory responses. Calpain inhibition,
however, allows normal microglial autophagy and lysosome clearance, preventing
the accumulation of -syn. (Figure created with BioRender.com).
Maintaining Ca homeostasis is vital for regulating many signaling
pathways and biological systems in the body . Among other roles, Ca
serves as a physiological messenger for transport across the plasma membrane and
enzymatic activation . Ca is unique from other signaling ions, such as
Na+ and K+, as the concentration is 20,000-fold lower in the cytoplasm compared
to the extracellular space (significantly less than the ~10- to
30-fold difference for Na+ and K+ ion concentrations). As a result of the extreme
concentration gradient, Ca acts as a potent intracellular signaling ion,
responding rapidly to changes in extracellular and intracellular environments
. Thus, controlling Ca movement enables a wide range of physiological
functions through the activation and inhibition of Ca-dependent signals.
Irregular Ca homeostasis, particularly elevated intracellular Ca
concentration levels, is implicated in the development of PD through the
activation of calpain (Fig. 2), a neutral protease that mediates neuronal death
through activation of inflammatory T-cells and microglia [7, 72]. Studies showed
exposure to MPP+ for over 24 hours causes a significant rise in intracellular
Ca levels which is four times greater in MPP+ treated hybrid rodent VSC
4.1 motor neuron cells compared to controls .
7. Calpain activation leading to mitochondrial dysfunction and
oxidative stress in PD
MPP+-induced increases in Ca significantly upregulate calpain and
caspase-3 activity. Calpain activation contributes to the dysfunction of
mitochondria by cleaving essential components of the organelle (Fig. 2). For
example, -calpain cleaves the Na/Ca exchanger responsible for
decreasing cytosolic Ca concentrations in normal mitochondria, leading to
elevated intracellular Ca levels and release of apoptosis-inducing factors
. Calpain-10, an atypical calpain, similarly cleaves Complex I of the ETC and
ATP Synthase in mitochondria . Other mitochondrial proteins affected by
calpain activation include Bcl-2-associated X protein (Bax-2), an apoptotic
membrane protein, and B-cell lymphoma 2 (Bcl-2), a survival membrane protein.
Bax-2 contributes to apoptotic neuronal death in the brain and SC whereas Bcl-2
protects neurons from cell death. The ratio of Bax-2 to Bcl-2 is significantly
increased after calpain activation, mediating apoptosis in neuronal cells.
Quantitative analysis of cervical SC samples showed apoptotic Bax proteins are
upregulated in MPTP mice . Thus, a cycle is perpetuated where mitochondrial
dysfunction leads to calpain activation, which in turn furthers mitochondrial
dysfunction with resulting ROS production and ultimate cell death (Fig. 2).
The activation of calpain by MPTP is confirmed in multiple studies. Brain
samples of MPTP-treated mice show an MPTP-induced increase in 80 kDa and active
76 kDa forms of m-calpain compared to controls. Furthermore, enhanced formation
of 145 kDa calpain-specific spectrin breakdown product (SBDP) is observed in the
SN of MPTP-treated mice at a significantly greater level compared to control
samples. SBDP is known to colocalize in TH neurons of MPTP-treated mice .
Calpain-specific SBDP are associated with acute neuronal damage and act as
biomarkers for neurodegenerative diseases such as PD. Thus, a marked increase of
SBDP in the SN of MPTP-injected mice suggests calpain plays a crucial role in the
progression of dopaminergic neuronal death in PD .
8. Invasion of inflammatory T-cells in PD
Calpain also influences the infiltration of T-cells as a consequence of calpain
induced microglial activation . Pro-inflammatory microglia, activated by
calpain induced increase in Ca, express increased levels of MHC antigens
and pro-inflammatory cytokines as opposed to beneficial neurotrophic factors such
as Insulin-like growth factor-1 (IGF-1) and GDNF [29, 73, 74]. Antigenic
peptide-loaded MHC molecules, MHC-I and MHC-II, bind to the surface of
professional antigen-presenting cells (PAPCs), recognized by either CD4+ or CD8+
T-cells, respectively . CD4+ and CD8+ T-cells, but not B-cells or natural
killer (NK) cells, are subsequently activated with subsequent cellular death in
PD patients . Some studies, however, demonstrate significantly higher levels
of NK cells and B-cells in PD patients, suggesting these cells may also serve as
biomarkers of PD .
The peripheral population of T-cells is significantly increased in PD patients,
supporting T cell trafficking into the CNS [76, 77]. Th1 and Th17 cell peripheral
blood levels measured in in vitro analyses in human PD samples show a
significant increase in the frequency of peripheral blood Th1 and Th17 cell
concentrations . Although CD8+ T cell concentrations are consistently higher
in MPTP-treated mice and PD patients, removal of CD8+ T cell subsets in CD8a–/–
mice has not been shown to mitigate MPTP injury. Furthermore, AAV2-SYN treated
mice lacking CD8+ T-cells exhibit myeloid inflammatory responses, similar to wild
type (WT) mice; thus, CD8+ T-cells may not directly cause the
-syn-driven myeloid response. Moreover, Th1 deficiency in mice leads to
significant neuroprotection from MPTP insult, suggesting CD4+ T-cells are
primarily responsible for mediating the adaptive immune response .
AAV2-SYN-treated CD4-/- mice exhibit significantly less activation of myeloid
cells, microglia and monocytes, compared to AAV2-SYN-treated WT mice and AAV2-GFP
(control)-treated CD4-/- mice. These results suggest CD4+ T-cells are crucial
mediators of the pro-inflammatory myeloid response to -syn expression
in the SN and SC . Thus, CD4+ T-cells not only drive the activated myeloid
response to -syn expression but are also critical to neurodegeneration,
as evidenced by decreased TH+ neuronal loss in AAV2-SYN-treated CD4-/- mice.
Infiltration of CD4+ and CD8+ T-cells is significantly elevated in the SN and SC
of MPTP-treated mice [21, 27]. CD4+ T cell toxicity depends on the Fas/Fas Ligand
(FasL) pathway . FasL, a membrane-bound ligand, exhibits significantly
decreased affinity for the receptor Fas when mutated. Mice with mutated FasL are
characterized by a minor reduction in nigral dopaminergic neurons following MPTP
treatment, suggesting CD4+ T-cell-mediated dopaminergic neuronal death relies on
functional FasL . In comparison, despite elevated IFN- levels
detected in MPTP-treated mice and post-mortem PD patients, deletion of
IFN- in mice does not significantly alter dopaminergic neuron death.
Studies demonstrated mice reconstituted with IFN--deficient phenotypes
and those lacking CD8+ T-cells are not protected - supporting the theory of
T-cell mediated dopaminergic toxicity by CD4+ T-cells and the FasL pathway .
Investigations into Calpain’s role as a mediator of the T cell mediated toxicity
in PD should be conducted to better understand calpain’s potential as a
therapeutic target for PD.
9. Anti-inflammatory T-cells neuroprotective role in PD
Treg cells are thought to play an essential role in preventing the onset of PD
through migration to the site of injury and interaction with local CNS glia .
Activation of glial cells in the CNS causes an inflammatory cascade, ultimately
signaling Treg cells to migrate to the SN/SC and perform their immune functions.
This process may alter the toxic, reactive microglia phenotype (M1 type) to a
non-toxic M2 type. Additionally, the process may drive astrocytes to produce
brain-derived neurotrophic factor (BDNF) and GDNF to provide trophic support to
SN neurons . Similarly, treatment with granulocyte-macrophage
colony-stimulating factor (GM-CSF), such as Sargramostim, before
MPTP-intoxication increases Treg cell concentrations in a dose-dependent manner.
Diminished neuroinflammatory responses and improved motor function are
consequently observed [66, 81]. For this reason, Treg cells confer a significant
neuroprotective effect via various modes of action including downregulation of
pro-inflammatory cytokines, upregulation of anti-inflammatory cytokines,
attenuation of Th1-/Th17- driven inflammation, and interaction with local
In addition, Treg cells provide nigrostriatal protection via cell-cell contact
with dopaminergic neurons through the CD47-Signal Regulatory Protein
(CD47-SIRP) receptor interaction, triggering Ras-related C3 botulinum
toxin substrate 1 (Rac1)/Protein kinase B (Akt) signaling in vitro .
T-cells harvested from MPTP-treated mice show decreased Treg cell populations
compared to control mice (3.6% vs. 1.1%, respectively) . In PD patients,
anti-inflammatory Th2 cell phenotypes of CD4+ T-cells, are measured at lower
concentrations compared to Th1 cell phenotypes . However, Th1 and Th17 cells
demonstrate suppressed pathogenic function after Treg cell administration in PD
models [21, 69].
Treg cell administration results in reduction of pro-inflammatory cytokines
(IL-17, IL-22, IFN-, TNF-, and IL-1) in the SN of
MPTP-treated mice. Studies demonstrate anti-TNF- neutralizing
antibodies in combination with Treg cell administration significantly reduce Th1
cell populations [6, 82]. Additionally, polyclonal Treg cells activated by
anti-CD3 antibodies upregulate anti-inflammatory cytokines, IL-10 and
transforming growth factor- (TGF-), in the ventral midbrains of
MPTP-treated mice, significantly attenuating neuroinflammation and inhibiting
microglial activation . Increases in anti-inflammatory cytokines that mediate
inflammatory cell suppression by conversion of adenosine monophosphate (AMP) to
adenosine (IL-10, IL-13, Granzyme B (GZMB), and 5’- Nucleotidase Ecto (NT5E)) are
observed in cells enriched with Treg markers CD4+, CD25+,
and forkhead box P3 (FOXP3+) .
Treg cells, specifically CD4+ and CD25+ T-cells expressing Foxp3, function by
secreting TGF- and IL-10 to suppress immune cell activation . CD4+
and CD25+ Treg cells harvested from spleen cells of C57BL/6 mice, when activated
by anti-CD3 and anti-CD28 antibodies, significantly attenuate dopaminergic neuron
loss in comparison to MPTP-treated mice. As a result, dopamine content in the
striatum of Treg cell-treated mice is considerably higher . Furthermore,
vasoactive intestinal peptide (VIP)-induced Treg cells, from the transfer of
pooled splenocytes of N-4YSyn-immunized donors and VIP-treated donors, are able
to overcome the toxic environment produced by Th17 amplified
N--syn-mediated nigrostriatal degeneration. In vitro data
suggests VIP modulated N-4YSyn CD4+ T-cells favor the Treg cell phenotype over
the Th17 cell phenotype. Co-cultures of T-cells from N-4YSyn immunized-mice with
T-cells from VIP-injected mice demonstrate preferential production of the Treg
phenotype rather than that of Th17 . Investigations into Treg cell activity
from calpain activation should be conducted to better understand the connection
and therapeutic potential.
10. Dopamine receptors (DR) and CD4+ T-cells in PD
-Syn is ubiquitously expressed throughout the body; however, as
previously discussed, elevated calpain levels have been shown to damage
-syn proteins. Harmful -syn aggregation results in LB
formation in the SN/SC but also leads to dysfunctional TH enzymes in the CNS with
decreased dopamine synthesis . Dopamine is essential for motor control,
executive function, motivation and arousal etc. but also modulates the function
of immune effector cells through DRs [44, 84]. It is hypothesized different DRs
may play an important role in the recruitment of -syn associated T
cells to the brain through a positive feedback loop during inflammatory responses
initiated by calpain activation . -Syn reactive T cells in the
periphery, when activated by aggregated -syn, migrate to the CNS due to
the disruption of the BBB . BBB disruption allows -syn reactive T
cells to infiltrate the CNS and migrate to areas containing high levels of
-syn, i.e., the SN and recently discovered in the SC . Once in the
SN/SC, T cells interact with -syn and undergo differentiation into
various CD4+ T phenotypes; these release various cytokines/chemokines, leading to
a cycle of neuroinflammation and neuronal loss [24, 33].
Although the expression of different DRs on various CD4+ T cells is poorly
understood, it is clear different DRs play an important role in PD pathogenesis.
In healthy individuals, dopaminergic signaling relies on low-affinity DRs, DRD1
and DRD2, that exhibit anti-inflammatory effects. Dopamine interaction with these
low-affinity receptors decreases cytokine production, regulates inflammasome
activity, and suppress inflammation [86, 87]. In PD patients, however, the use of
high-affinity DRs, such as DRD3 and DRD5, is favored in dopaminergic signaling
(Fig. 3). These high-affinity DRs, specifically DRD3, upregulate the migration of
inflammatory Th1 and Th17 cell phenotypes . Activation of these receptors on
Treg cells also suppresses anti-inflammatory responses.
Dysfunctional dopamine signaling resulting in neuroinflammation
and dopaminergic neuron loss. In healthy individuals, dopamine signaling is
controlled through low affinity DRs (DRD1 and DRD2). DRD1 and DRD2 serve a
neuroprotective role and lead to anti-inflammatory responses. In PD individuals,
however, decreased dopamine levels prompt activation of high affinity DRs (DRD3
and DRD5) on Treg cells, leading to pro-inflammatory responses. DRD3 and DRD5 on
Treg cells interact with dopamine and signal T cell differentiation into Th1 and
Th17 cell phenotypes. Treg cell interaction with ICAM and LFA-1 also causes
stimulation of the Th17 and Th1 cell phenotypes. These Th cells release
pro-inflammatory cytokines and chemokines (IFN-, TNF-, IL-17,
IL-22, etc.), resulting in a pro-inflammatory cascade, neuroinflammation, and
dopaminergic neuron loss. Treatment with Pramipexole has been shown to inhibit
these high affinity DRs with consequent anti-inflammatory effects. (Figure
created with BioRender.com).
The Th17 phenotype significantly contributes to dopaminergic neuron death
through interaction with LFA-1 and ICAM; Th17 secretion of pro-inflammatory
cytokines IL-17, IL-8, IL-21, IL-22, IL-26, TNF-, GM-CSF, and
IFN- also promotes neuronal death [24, 67, 88]. These cytokines support
Th17 cell survival, creating an environment in the brain conducive to chronic
neuronal damage . Th17 T-cells thus appear to possess a greater capacity to
exacerbate dopaminergic neurodegeneration than Th1 T-cells - identifying the Th17
cell phenotype as the primary driver of neuroinflammation .
Furthermore, DRD3 expression favors CD4+ T cell differentiation into the Th1
phenotype rather than the Th17 phenotype . DRD3 is expressed on CD4+ T-cells
involved in Th1 differentiation, but not in Treg or Th17 cell differentiation.
CD4+ T-cells of DRD3 deficient mice demonstrate significant neuroprotection from
MPTP treatment, suggesting the Th1 cell phenotype is a major contributor to
neuroinflammation . Likewise, systemic administration of DRD3-antagonists in
MPTP-treated mice significantly attenuates nigrostriatal neurodegeneration and
motor impairment caused by CD4+ T cell-mediated inflammation (as characterized by
microgliosis reduction) [24, 32]. Thus, medications blocking the DRD3 receptor,
such as pramipexole, demonstrate a neuroprotective effect which may provide
clinical benefits in PD patients (Fig. 3) . However, these medications also
downregulate dopamine transporter (DAT), a membrane-spanning protein responsible
for dopamine reuptake from the synapse, essential for PD patients .
Peripheral T-cells in PD patients demonstrate a significant reduction of DAT
immunoreactivity, suggesting the peripheral dopaminergic system participates in
PD pathogenesis .
11. Calpain-inhibitors (calpeptin, SJA6017, SNJ-1945) as novel
treatment options for PD
Calpeptin, an inhibitor of calpain, has been evaluated as a potential treatment
option in PD animal models . Treatment with calpeptin in MPTP-treated mice
leads to a marked reduction in TUNEL and NeuN expression within cervical and
lumbar SC samples, providing protection for dorsal and ventral root ganglion
neurons . -Syn aggregation is also reduced in both SN and SC of
MPTP-treated mice, as demonstrated by immunofluorescent staining using
-syn/TH and NeuN antibodies, respectively . In addition,
pre-treatment with calpeptin protects SC neurons from MPTP-induced toxicity by
preventing neuronal degeneration and axonal alterations, thereby improving gait
dynamics and restoring motor functionality in MPTP-treated mice .
Bax, an apoptotic protein responsible for neuronal death, is inhibited in the
CNS after calpeptin treatment, whereas Bcl-2, is observed in elevated
concentrations post-calpeptin treatment. A marked decrease in deNFP IR levels is
also detected after calpeptin treatment in the cervical and lumbar SC,
demonstrating axonal preservation. Moreover, Treg cell populations exhibit
significant growth after calpeptin treatment, resulting in suppression of
microglial pro-inflammatory responses and Th1/Th17 cell functions .
SJA6017, a cell-permeable calpain inhibitor, is also an effective
neuroprotective agent against MPP+ induced damage in spinal motoneurons .
This calpain inhibitor provides significant cytoprotection in VSC 4.1 motoneurons
after MPP+ induced damage. In addition, SJA6017 attenuates the MPP+ induced rise
in intracellular Ca, reduces SBDP levels, and diminishes ROS elevated
after MPP+ exposure. Motor proteins dynein and kinesin also show partial
improvement after SJA6017 treatment.
At 5, 10, and 50 M MPP+ concentrations, pre-treatment with 10 M
SJA6017 effectively increases cell viability and provides resistance to MPP+
induced toxicity . However, at 100 M and 200 M MPP+
concentrations, higher concentrations (100 M) of SJA6017 are required.
Pre-treatment with 100 M of SJA6017 prevents the loss of kinesin and
dynein after MPP+ exposure. With ROS production significantly attenuated, cells
pre-treated with SJA6017 show a 50% increase in survival rate compared to
controls with MPP+ induced toxicity . MPP+ concentrations of 5, 10, and 50
M also cause a significant increase in calpain and caspase-3 expression
compared with controls and SJA6017 pretreated cells. In addition, SJA6017
pre-treated cells show reduced SBDP formation with decreased calpain,
pro-caspase-3, and active caspase-3 levels after MPP+ exposure. Resting membrane
potentials in VSC 4.1 cells are also reduced after MPP+ exposure compared to
controls, but pre-treatment with SJA6017 maintains standard cellular membrane
potentials, restoring electrophysiological functionality .
Calpeptin substantially attenuates MPP+ and rotenone-induced toxicity; however,
calpeptin’s application is constrained due to its limited water solubility. Thus,
SNJ-1945, a water-soluble calpain inhibitor , may serve as a better treatment
option for neuroprotection in PD patients. SNJ-1945 pre-treatment alone, at its
highest concentration of 250 M, shows no overt effects on SH-SY5Y-DA and
SH-SY5Y-ChAT cells; however, SNJ-1945 is significantly protective at lower
concentrations from the effects of MPP+ and rotenone. SNJ-1945 pre-treatment at
concentrations of 50, 100 or 250 M, (dependent on the dosage of MPP+ or
rotenone) also attenuates the neurotoxicant-induced inflammatory mediators Cox-2,
caspase-1, and p10. Furthermore, calpain activity is diminished after SNJ-1945
pre-treatment, as marked by decreased levels of the 145 kDa calpain-specific SBDP
band induced following rotenone and MPP+ exposure .
The role of neuroinflammation in the SN and SC in PD pathogenesis has been
elucidated further with recent studies. Calpain, a calcium-activated neutral
protease (upregulated after MPTP, rotenone, and 6-OHDA injections) contributes to
microglia/astrocyte activation, resulting in the release of various cytokines and
chemokines which promote infiltration of toxic T-cells and pro-inflammatory
responses. Glial cell activation also causes oxidative stress from ROS/RNS and
damage to the BBB, allowing for CNS parenchymal inflammatory progression.
Moreover, calpain plays a significant role in the deletion and cleavage of
-syn, which aggregates into harmful LBs with resulting oxidative stress
and impaired mitochondrial function. -Syn aggregation also inhibits an
essential enzyme in dopamine synthesis (TH), resulting in depleted dopamine
levels and altered DR signaling with resulting infiltration of T-cells and
ultimately dopaminergic neuron loss.
In addition, calpain activation related to Ca homeostasis dysregulation
leads to elevated levels of intracellular Ca, further activating calpain.
Thus, calpain inhibitors are being investigated as potential treatment options to
prevent neurodegeneration and dopaminergic neuron loss. Calpeptin, SJA6017, and
SNJ-1945 are effective calpain inhibitors, mitigating the neuroinflammation and
neurodegeneration induced from MPTP, rotenone, and 6-OHDA. These calpain
inhibitors effectively decreases calpain levels, reduce inflammation, and protect
neuronal viability in both the SN and SC. Further research involving these
calpain inhibitors will be required to determine clinical efficacy and safety
AG designed and wrote the manuscript. HMM designed, re-wrote, revised, and
edited the manuscript. HHM also designed the figures. AG and HMM contributed
equally. VZ edited the figures and manuscript. DCS and NLB edited the manuscript.
AH conceived, designed, and edited the manuscript. All authors reviewed and
approved the final version of the manuscript.
Ethics approval and consent to participate
We thank Denise Matzelle for her technical assistance.
This work was supported in part by funding from the National Institutes of
Health (1R21 NS118393-01). This work was also supported in part by
funding from the Veterans Administration (1IOBX001262, 1I01 BX004269).
Conflict of interest
The authors declare no conflict of interest.
AH is serving as one of the Editorial Board members of this journal. We
declare that AH had no involvement in the peer review of
this article and has no access to information regarding its
peer review. Full responsibility for the editorial process for
this article was delegated to GP.