Background
Synucleinopathies are a group of neurodegenerative diseases characterized by the presence of intra-cytoplasmic amyloidogenic inclusions comprised of the protein α-synuclein (αSyn) [
1,
2]. In most of these disorders, such as Parkinson’s disease (PD) or dementia with Lewy bodies (DLB), these αSyn inclusions are predominantly neuronal [
2,
3]. The majority of pathological αSyn in these inclusions are phosphorylated at the serine 129 residue and this modification is generally used as a biological marker to monitor inclusion formation when combined with other histological assays [
3,
4].
The aberrant aggregation of αSyn to form amyloidogenic inclusions is thought to follow a prion-like mechanism involving the molecular conversion of protein monomers from their predominantly unfolded structure to a β-pleated sheet that can then polymerize into amyloid (reviewed in [
4]). As part of this prionoid mechanism, such conformationally altered forms of αSyn can readily aggregate and can subsequently be propagated between cells [
5‐
12]. Consistent with this model, both intracerebral or peripheral injections of recombinant αSyn amyloid seeds result in robust induction of αSyn pathology in αSyn transgenic mice and to a lesser extent, in wild type mice [
6,
7,
9,
13‐
15]. Modeling studies have further shown that both soluble and aggregated αSyn can be released and taken up by cells via various mechanisms [
16‐
18], providing proof of concept that pathological αSyn shares prion-type transmission properties.
The observations that innate immune activation is an invariant finding in synucleinopathies and that αSyn, by itself, can directly interact with immune cells suggest that innate immunity can potentially modify how exogenous αSyn is able to influence the onset and progression of α-synucleinopathy (reviewed in [
19,
20]). In mouse models of αSyn inclusion pathology induced by exogenous αSyn aggregates, a significant component of the αSyn pathology is retained in glial cells [
7,
21]. Glial cytoplasmic inclusions (Papp-Lantos bodies) and tuft-shaped astrocytes laden with aggregated αSyn have been reported in patients with synucleinopathies, such as PD, multiple system atrophy (MSA) and DLB [
22‐
25] as well as in transgenic mouse models of α-synucleinopathy [
26‐
28]. Mechanistically, extracellular αSyn can directly activate microglia by interacting with microglial receptors including TLR2 and TLR4 [
29‐
31]. CNS resident astrocytes as well as macrophages can endocytose αSyn via dynamin-related pathways [
16,
17], suggesting that immune pathways can potentially have disease modifying effects in α-synucleinopathies.
To investigate how innate immune activation alters seeded α-synucleinopathy in wild type mice, we used an Interleukin (IL)-6 driven somatic transgenesis model. IL-6 is a pleiotropic cytokine that plays a key role in immune regulation, hematopoiesis and acute phase reactions [
32]. Under chronic conditions, IL-6 induces an acute inflammatory condition by activating immune cells, such as macrophages, B cells, microglia and astrocytes. Using recombinant adeno-associated viruses overexpressing IL-6 in the brains of wild type mice [
33], we explored how preconditioning innate immune milieu in the CNS affects the induction of αSyn pathology following challenge with exogenous aggregated αSyn. IL-6 expression, as expected, leads to widespread astrogliosis and, surprisingly, attenuates the induction of αSyn pathology in these mice. Thus, contrary to our expectations that an inflammatory milieu might exacerbate α-synucleinopathy, we report that IL-6 induced immune preconditioning limits induction of α-synucleinopathy following injection of exogenous αSyn aggregates.
Discussion
Here we have investigated the effect of inflammatory immune activation on the induction of αSyn pathology in wild type mice following intra-hippocampal delivery of pre-fibrillized αSyn aggregates. We show that expression of IL-6 in two independent time progressive cohorts of wild type mice results in 1) widespread and massive astrogliosis, 2) attenuation of induced αSyn pathology in mice challenged with exogenous αSyn aggregates and 3) concurrent reduction in p62 and argyrophilic inclusion pathology in mice injected with αSyn. Our study is in agreement with earlier reports that αSyn can be readily endocytosed by astrocytes [
16] and this is potentially an endogenous mechanism for rebalancing proteostasis abnormalities via lysosomal degradation [
17]. Taken together, this suggests that prior activation of astroglia alters the innate immune milieu to generate a beneficial response in wild type mouse brains challenged with exogenous αSyn aggregates. However, whether IL-6 induced inflammatory activation will have a different outcome in a chronic setting, especially with regard to neurodegeneration and behavioral outcome measures, remains to be investigated.
Templated conformational alterations in intracellular αSyn and its subsequent secretion across neuroanatomic junctions has emerged as a possible mechanism of disease progression in α-synucleinopathies [
4]. However, little is known about the non-cell autonomous effects of extracellular αSyn on innate immunity in the CNS. More importantly, whether innate immune based strategies could potentially induce rapid uptake and degradation of extracellular αSyn in vivo by immune cells has not been studied. While chronic inflammation can have a detrimental role in CNS homeostasis [
41], whether transient activation of astrocytes and microglia can be harnessed as a disease modifying therapy in αSyn models of PD is still unknown.
There are multiple reports showing that various therapies can attenuate αSyn pathology in transgenic mouse models (reviewed in [
42]). However, only one previous report, using a peripherally administered monoclonal anti-αSyn antibody showed that prefibrillar αSyn aggregate induced α-synucleinopathy can be blocked in vivo [
43]. Additionally, the reported safety and preliminary efficacy of the ongoing AFFITOPE® PD01A (AFFiRis AG) trials in human patients lends support to the idea that activating the patient’s immune system to generate anti-αSyn response may be potentially beneficial in limiting the progression of αSyn pathology. Herein, we show that pro-inflammatory preconditioning significantly attenuates induction of endogenous αSyn pathology in wild type mice. Further, our data seems to suggest that in addition to potential immune scavenging of exogenous αSyn aggregates around the time of administration, IL-6 induced gliosis was instrumental in removing the secondary pathological forms of αSyn at a later timepoint. The mechanism of such time-progressive reduction remains unknown but given the present understanding, it is likely that attenuation of αSyn pathology may occur via upregulation of phago-lysosomal function of the glial cells (reviewed in [
44]). Both the autophagy-lysosomal pathway and the ubiquitin proteasome system have been shown to play crucial roles in αSyn clearance [
45]. However, there may well be additional clearance mechanisms underlying the reduction in αSyn pathology observed in our study. Our data thus provides a second and unique instance where manipulating the immune system provides a beneficial response in a model of exogenous fibril-induced αSyn pathology. There are some caveats to such immune manipulations. Given that microarray expression data suggest that aging itself skews the human brain towards a pro-inflammatory state, it is interesting to consider how those age-induced alterations might modify the spread of neurodegenerative pathology in humans [
46]. In particular, special cognizance ought to be placed on harnessing the beneficial nature of innate immune function vis-a-vis the potentially harmful outcomes of a chronic response.
Our present data as well as earlier reports show that CNS injection of exogenous αSyn aggregates leads to focal gliosis, especially discernible along the needle track [
7]. Exogenous αSyn aggregates may cause glial dysfunction followed by intracellular proteostasis resembling neuropathological changes in synucleinopathies [
7,
9]. On the other hand, astroglial activation may provide trophic support to the CNS in rodent models of PD and therefore may have beneficial neuroprotective effects overall [
47,
48]. A major function of glial cells is to scavenge debris [
49] and it is possible that facilitating clearance of extracellular αSyn may attenuate inter-neuronal transfer of αSyn and support normal homeostasis. Interestingly, αSyn is thought to be taken up by astrocytes around axon terminals in brains of PD patients [
50], which was demonstrated in cell culture using exogenous αSyn [
16]. Both astrocytes and microglia can internalize and degrade extracellular and cell-derived αSyn [
11,
17,
29,
30,
51,
52]. Two microglial receptors, TLR2 and TLR4, have been identified as possible endogenous receptors responsible for αSyn internalization; indeed, TLR4 ablation reduces αSyn clearance and exacerbates neurodegeneration [
30]. Therefore, such astrocytic and microglial scavenging and clearance mechanisms hold promise in preventing the spread of αSyn pathology during inter-cellular transfer of αSyn seeds.
Although the general dogma in many neurodegenerative disorders is that inflammatory stimuli might promote disease [
53], data in preclinical models of Alzheimer’s disease demonstrate that activating the immune system can attenuate the underlying proteinopathy [
33,
54‐
57]. We and others have demonstrated that at least early in the disease process, inflammatory activation in the CNS can have positive disease modifying effects in mouse models of Alzheimer’s disease [
33,
54,
58,
59]; similarly, in this study, we demonstrate that at least in an acute scenario, IL-6 induced innate immune activation can have a protective outcome in wild type mice following exogenous αSyn challenge. To our knowledge the current data presented here is the first to show that an inflammatory interventional strategy may also work in a mouse model of seeded synucleinopathy. However, in Lewy Body diseases, the role of individual cytokines still remains to be elucidated (reviewed in [
60]). Different inflammatory cytokines have been shown to be correlated with the disease process in mouse models of nigro-striatal degeneration as well as in Parkinsonian patients. Both IFN-γ and TNFα have been shown to be associated with neurodegeneration in PD mouse models [
61‐
63] while the role of other cytokines, such as IL-6 and IL-4 that are upregulated in PD patients [
64], have not been studied before in mouse models of synucleinopathies. In particular, whether any of these latter cytokine signaling pathways can modify induction and progression of α-synucleinopathy and therefore have translatable disease modifying outcomes is of paramount interest in therapeutics against synucleinopathies.
Acknowledgements
Not Applicable.