Introduction
Parkinson disease (PD) is the most common neurodegenerative disorder after Alzheimer disease (AD) and is neuropathologically characterized by nigrostriatal dopaminergic degeneration and the presence of aggregated and misfolded alpha-synuclein (aSyn). In clinical examination, PD patients show motor deficits such as bradykinesia, rigidity, tremor and postural instability, which reflect the degeneration of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) and impairment of DA neurotransmission to basal ganglia motor circuits. The accompanying heavy burden of non-motor symptoms such as autonomic failure, daytime sleepiness, cognitive deficits, or even psychiatric alterations indicates the involvement of other CNS neurotransmitter systems [
1].
As the main neuropathologic culprit, aSyn has been identified in humans and was found to be located in aSyn-immunopositive Lewy neurites and Lewy bodies. Several human postmortem studies have demonstrated that not only the nigrostriatal dopaminergic system is affected in PD but that—depending on the (prodromal) disease stage—also Lewy pathology can be early found in the peripheral autonomic nervous system, including neurons of the enteric plexus of the gastrointestinal tract, paravertebral autonomic ganglia and sympathetic nerve fibers in the adrenal gland and heart and in cutaneous nerves. In subsequent disease stages, the medulla, pons, midbrain, diencephalon, basal forebrain, amygdala, olfactory bulb, limbic cortex and finally higher order association cortices can be involved, too [
1‐
3].
Thus, the extent of aSyn pathology is not a stable state but seems to progress in a prion-like cell-to-cell spreading to continuously involve further neuronal and non-neuronal cells—a disease propagation process that is also discussed for other neurodegenerative diseases in a similar way [
4]. Importantly, aSyn-associated neurodegeneration is accompanied by neuroinflammatory features, which highlights the role of aSyn for the immune system and non-neuronal cells [
5,
6]. As much as this spread of the disease in the CNS proves its aggressive character, there are also possibilities for new therapeutic approaches. A very obvious one is to interfere with disease dissemination and eliminate excess or toxic compounds of aSyn that are located extracellularly and therefore are readily accessible to therapeutic approaches.
In this review, we will describe the properties of aSyn and its pathophysiologic implications, in particular regarding its activation of cellular neuroinflammatory cascades. Thus, a clear rationale opens up to counteract this vicious circle by controlling the spread of disease with the help of immunologic, antibody-based technologies. The potential of these active and passive immunotherapies is presented from the fundamental scientific side as well as from recent human clinical data. Current knowledge is critically evaluated with the goal to provide a timely review on immunotherapies for PD for both the clinician and basic scientist. This article is based on previously conducted studies and does not contain any studies with human participants or animals performed by any of the authors.
Alpha-Synuclein and its Role in the Pathogenesis of PD
Alpha-synuclein is a soluble and cytoplasmic protein of 140-amino-acid (aa) length consisting of three domains: an amphipathic N-terminal region (1–65 aa), a non-amyloid-β component (NAC) region (66–95 aa) and a C-terminal domain (96–140 aa) [
7]. In the CNS, aSyn is predominately expressed in neurons of the thalamus, basal ganglia and substantia nigra [
8] but also in the peripheral nervous system in blood cells and in platelets [
9,
10].
In human neuropathology, aggregated aSyn represents the main component of so-called Lewy bodies and Lewy neurites, which are intraneuronal filamentous inclusions and are located in perikarya or neurites, respectively, of degenerating DA neurons in PD patients [
8,
11,
12]. In familial Parkinson disease, rare human genetic alterations occur mostly as point mutations in aSyn (A30P, E46 K, H50Q, G51D, A53E and A53T) or as gene duplications and triplications that cause autosomal-recessive or -dominant forms of PD [
13].
The function of aSyn in neurons is only incompletely understood, but it is known to play an important role in neurotransmitter release, synaptic plasticity and synaptic vesicle recycling [
7,
14,
15]. aSyn is stored in presynaptic nerve terminals and mediates synaptic vesicle fusion with soluble NSF attachment protein (SNARE) complex assemblies and via direct interaction with syntaxin-1, SNAP-25 and vesicle-associated membrane protein 2 (VAMP2)/synaptobrevin-2 [
15‐
17]. This enables synaptic vesicle trafficking from the ready releasable pool to the presynaptic active zone where the content of the synaptic vesicles is secreted. In case of aSyn accumulation, the neurotransmitter release, vesicle trafficking, recycling and SNARE complex stability are all affected and thus severely impair synaptic function of dopaminergic neurons [
16‐
19].
Under physiologic conditions, wild-type aSyn is a natively soluble unfolded monomer, which binds to curved membranes such as synaptic vesicles and regulates the above-mentioned SNARE-complex chaperoning function. Under pathologic circumstances with changes in pH level or oxidative stress, aSyn can convert to insoluble and aggregated forms that are enriched in β-sheets and assemble into oligomeric and fibrillar structures. These forms of aSyn have a strong pathogenic potential and may harm several physiologic functions of the cell [
15,
20,
21].
Importantly, aSyn can be released from intracellular compartments to the extracellular space. From there it may propagate to other neuronal or glial cells in which it elicits pathogenic cascades that impair their cellular function. Concerning these aSyn propagation mechanisms, three main principles are known. First, in healthy neurons a non-classical ER-/Golgi-independent protein export pathway can be used whereby aSyn can be directly integrated into secretory vesicles and subsequently released by exocytosis. Second, aSyn vesicles can be translocated into early endosomes and then be released to the extracellular space through recycling endosomes. As a third alternative, early endosomal aSyn can be incorporated to intraluminal vesicles that are part of the so-called multivesicular bodies (MVB). These can undergo a degradative process after fusion with lysosomes or directly secrete by fusion with the plasma membrane [
22]. Several in vitro and in vivo animal studies have shown that especially oligomeric aSyn directly confers toxic effects to surrounding cells, whether they are neuronal or non-neuronal glial cells.
Concerning direct effects of aggregated aSyn onto neuronal cells, several in vitro studies have been performed. Primary hippocampal neuronal cells readily internalized exogenous preformed fibrils of artificial recombinant aSyn if provided in the cell culture medium. In the cytoplasm, these exogenous fibrils recruited endogenous aSyn and induced pathologic misfolded aSyn, which was phosphorylated, ubiquitinated and aggregated to insoluble fibrils. Similarly to human PD, a LN-like pathology developed, first located in axons, and was then propagated to form LB-like inclusions in the cell perikarya [
23,
24]. The axonal protein accumulation was preceded by axonal transport alterations with an initial predominant anterograde direction of aSyn, being similar to the amyloid-beta
1-42 form but different from the processing of huntingtin protein fragments [
25]. Accumulating pathologic aSyn then led to selective decreases in synaptic proteins, progressive impairments in neuronal excitability and connectivity and finally neuronal death. Interestingly, endogenous aSyn was sufficient for the formation of these aggregates, and overexpression of wild-type or mutant aSyn was not necessarily required. A frequently observed uptake mechanism of aSyn could be attributed to absorptive-mediated endocytosis [
23,
24]. More recent findings have shown that aSyn uptake is in part also cell surface receptor mediated utilizing the protein product of lymphocyte-activation gene 3 (LAG3) [
26], which is expressed not only by neuronal cells but also microglia [
27]. The underlying mechanism here seems to be a temporary inhibition of the NMDA receptor making it obvious that aSyn-mediated impairment of cellular function is not only restricted to dopaminergic neurons [
28].
To evaluate direct aSyn toxicity in vivo, a route of administration in animals has been the direct injection of various aggregated seeds into the substantia nigra of wild-type rats. This resulted in a progressive motor impairment and cell death with fibrils being the major toxic strain. Here, ribbons caused a histopathologic phenotype similar to human PD neuropathology [
29]. These findings indicate that distinct aSyn strains display differential seeding capacities. Interestingly, aSyn assemblies also crossed the blood-brain barrier and distributed to the central nervous system after intravenous injection [
29]. In another study, Luk et al. used wild-type mice that were unilaterally injected with recombinant aSyn into the dorsal striatum. At different time points, an increasing spread of Lewy bodies was observed, and although aSyn was only injected into one hemisphere, after several days Lewy bodies were also detected on the contralateral hemisphere and in diverse brain regions including the hippocampus [
30]. Viral-mediated overexpression of wild-type and transgenic aSyn injected into the substantia nigra in mice severely damaged nigral dopaminergic cells and its axons. Importantly, also the intrinsic regenerative capacity of dopaminergic neurons is significantly impaired if dopamine neurons suffer from aSyn overload [
31].
Extracellular aSyn has a strong impact on glial cells and there can elicit a variety of immunologic responses. Glial cells are by far the largest cell population, accounting for more than 50% of all cells in the brain, and thus comprise about 1000 billion cells [
32]. They differentiate in the CNS, where they are collectively referred to as neuroglia, consisting of astroglia, microglia and oligodendroglia.
Astroglia account for by far the largest share of glial cells and perform many tasks essential to the survival of neurons. They ensure structural and metabolic cerebral homeostasis, regulate synaptic transmission, water transport and blood flow, and myelination and produce neurotrophic molecules such as GDNF [
33]. Experiments with human ESC-derived astrocytes have demonstrated that these cells actively transferred aggregated aSyn to healthy astrocytes via direct contact and tunneling nanotubes in a propagating manner instead of degrading them [
34]. The intracellularly accumulated aSyn affected the lysosomal machinery and induced mitochondrial damage. This elicited further internal proinflammatory responses and caused cellular death [
22,
35]. The aSyn-induced impairments of astrocyte function could be alleviated by an application of oligomer-selective anti-aSyn antibodies [
36].
Microglial cells represent the innate immune system of the CNS. Precursor cells migrate into the CNS already during the prenatal phase. Microglia account for approximately 10–20% of the total glia population [
37]. The physiological function of CNS-resident microglia involves the maintenance of homeostasis through ongoing monitoring of the CNS environment and, if necessary, phagocytosis or pinocytosis of degradation products or cell debris [
38]. Microglial cells have been shown to be able to directly engulf aSyn attempting to clear it from the extracellular space. This response was strongly dependent on its aggregation state [
39]. Distinct oligomeric forms but not monomers or fibrils of aSyn interacted with microglial toll-like receptor 2 (TLR2) and activated the TLR2 signaling pathway [
40] leading to increased production of neurotoxic ROS [
41].
To reduce increased levels of harmfully aggregated extracellular but also endogenous aSyn, a clearing process of the protein has to be established. This can be achieved with immunotherapies using vaccination strategies or antibodies directed against aSyn [
42]. If extracellular levels of aSyn decrease, the spread to other neuronal and non-neuronal cells such as astroglia or microglia will be reduced and ultimately prevent pathologic inflammatory activation [
42,
43]. Other aSyn degradative processes are mediated through ubiquitination, lysosomal degradation by macroautophagy or chaperone-mediated autophagy (CMA) [
7]. In the next section, we will present the current state of research in the first PD immunotherapies, which all have the goal of a causal disease modification in humans. Findings from animal studies and recent human clinical trials will be compared.
Future Perspectives and Conclusions
Through detailed studies on patients, but also in animal models of PD, key neurodegenerative pathomechanisms of this disease have been decrypted. In addition to the damage to neuronal cells, it is now obvious that also glial cells are affected and that a strong neuroimmunologic interaction takes place with aSyn being the central player. From today’s perspective, a modulation of this cross-cell disease propagation in PD appears possible by “targeting” of aSyn.
Very elegant neuroimmunologic tools have been developed to inhibit disease spread by extracellular aSyn. The elimination of aSyn by targeted antibody-based technologies with active or passive immunization therefore seems very pragmatic. Certainly, technologic challenges, such as overcoming the blood-brain barrier or the targeted elimination of only aggregated aSyn, still need to be optimized. However, we are well on the way to providing answers to these questions as several human clinical trials with ambitious expectations are currently underway. The simultaneous application of antibody-based therapies in atypical Parkinson’s syndromes such as PSP or also Alzheimer’s disease will in any case result in important basic insights. This will, it is our firm conviction, enable new technologic advances in the immunotherapy for PD and bring about new powerful options to modify disease progression.