Background
Multiple system atrophy (MSA) is a progressive, neurodegenerative disease characterized by parkinsonism resistant to dopamine therapy, ataxia, autonomic dysfunction, and pathological accumulation of α-synuclein (α-syn) [
1-
4]. MSA differs from other synucleinopathies in that α-syn accumulates not only within neurons and astrocytes, but also within oligodendrocytes in the form of glial cytoplasmic inclusions [
5]. This intracellular accumulation of toxic α-syn species leads to degeneration of oligodendroglial cells, loss of trophic support to neurons and subsequent neurodegeneration.
In recent years increasing evidence supports the notion that α-syn is primarily generated by neurons, where it aggregates and gets released to the extracellular environment [
6,
7]. Extracellular aggregated α-syn would then propagate to other neurons and glial cells in a prion-like fashion [
8,
9]. However, a recent report of MSA oligodendrocytes also expressing α-syn mRNA [
10] suggests that the origin of oligodendroglial α-syn might be both of endogenous nature and the result of propagation from neurons and/or other oligodendroglial cells. Furthermore, propagation and accumulation of α-syn within astrocytes could lead to activation of these cells and subsequent neuroinflammation [
11-
13]. Therefore, the development of therapeutic interventions/strategies for MSA and related neuropathologies has been focused on reducing α-syn accumulation, increasing α-syn clearance and/or inhibiting α-syn propagation. One of these therapeutic alternatives is immunotherapy.
To date there are no disease-modifying treatments for α-synucleinopathies. The discovery that α-syn oligomers can be secreted [
14,
15] and propagate extracellularly [
16,
17] provided a clear rationale for immunotherapy [
18]. Humoral immunization against α-syn can occur in one of two forms, active or passive immunity [
18]. Active immunization involves stimulating the immune system to produce antibodies against toxic α-syn conformations, while passive immunization involves administering anti-α-syn antibodies to the patient, which confers temporary protection against the disease. Recent preclinical studies have been successful in clearing intraneuronal α-syn aggregates and reducing neuron-to-neuron α-syn propagation by immunotherapy, focusing on stimulating or restoring the ability of the immune system to fight the disease [
18-
22]. In this sense, Phase 1 clinical trial is currently investigating the use of active immunotherapy with PD01A for Parkinson’s disease (PD), and intravenous immunoglobulins are being used in a Phase 2 clinical trial for MSA.
Recent studies suggest that active immunotherapy increases α-syn clearance and might be a viable therapy for PD, a closely related neurodegenerative disease characterized by extensive α-syn deposition in neurons [
19,
20]. AFFiRiS has developed novel active immunogens (AFFITOPEs®) that hold the promise of treating these disorders. AFFITOPEs® are short immunogenic peptides that are too short for inducing a T-cell response (autoimmunity) and do not carry the native epitope but rather a sequence that mimics the original epitope [
23,
24]. This methodology allows for the generation of long term, sustained, more specific, non-cross reacting antibody responses suitable for the treatment of synucleinopathies. The main objective of this study was to evaluate the effects vaccination with the AFFITOPE® proven most effective for PD models on reducing the MSA-like pathology in the MBP-α-syn transgenic (tg) mice [
19].
Discussion
The present study showed that immunization with the AFFITOPE® vaccine AFF 1 reduced propagation and accumulation of α-syn, demyelination, motor deficits and neurodegeneration in a tg mouse model of MSA. AFF 1 was selected for this study because it had previously demonstrated a high ability to elicit α-syn specific antibodies and to ameliorate behavioral and neurodegenerative pathology in two models of synucleinopathies [
19]. Furthermore, AFF 1 elicits the generation of antibodies that do not cross-react with other members of the synuclein family, and does so without promoting α-syn specific T-cell responses [
19]. This approach allows for the generation of long term, specific responses suitable for the treatment of synucleinopathies such as MSA.
Active and passive immunization studies have previously shown that immunization reduces the pathology in α-syn tg models of PD. Active immunization of PDGF-α-syn mice with full-length human α-syn induces the production of high affinity antibodies, together with a decrease in neuronal α-syn accumulation and neurodegeneration [
20]. Furthermore, vaccination is also effective experimentally in other mouse models of neurodegenerative diseases, by reducing the accumulation of toxic proteins aggregates such as amyloid β [
36-
39], tau [
40,
41], prion protein [
42] and huntingtin [
43,
44]. Interestingly, the antibodies produced by mice immunized with full-length human α-syn recognized epitopes within the C-terminal region of human α-syn [
20]. Passive immunization studies with antibodies against the C-terminus of α-syn further confirmed this observation [
21]. Epitope mapping of AFF 1-induced antibodies shows that the antibodies generated also recognize an area in the C-terminal region of α-syn (110–130) [
19], supporting the involvement of this part of the protein in antibody-mediated targeting and clearance. Interestingly, here we report that AFF 1 vaccination also reduced α-syn spreading from oligodendrocytes to astroglia. This finding confirms previous passive immunization studies showing that antibodies against the C-terminus of α-syn also reduced α-syn propagation [
45]. Furthermore, this is the first observation that propagation between glial cells can be halted by active immunization. Propagation of α-syn from oligodendrocytes to astroglia has been previously described in the MBP-α-syn mice [
28], and although its relevance in MSA has not been determined yet, the recent finding that oligodendrocytes do express α-syn in the MSA brain [
10] suggests that this type of propagation could be also happening in MSA. Moreover, reducing propagation of α-syn from oligodendrocytes to astroglia might also prevent neuroinflammatory responses derived from the accumulation of α-syn within astrocytes [
11-
13]. However, a more detailed analysis of neuroinflammation after immunization with AFF 1 in MBP-α-syn tg mice is needed to elucidate the protective effect of AFF 1 against inflammation in MSA.
First studies on active vaccination against α-syn were performed using full-length human α-syn in the mThy1-α-syn tg model of dementia with Lewy bodies [
20]. In this model, active vaccination with human α-syn decreased accumulation of aggregated α-syn in neuronal cell bodies and synapses and reduced neurodegeneration. Antibodies produced by immunized mice recognized abnormal α-syn associated with the neuronal membrane and promoted the degradation of α-syn via lysosomal pathways [
20,
46]. In the present study we did not observe α-syn or antibody reactivity within neurons, this result suggesting that antibodies elicited by AFF 1 do not recognize murine α-syn, which is expressed predominantly by neurons, and that neurons do not play a significant role in clearing extracellular human α-syn in the MBP-α-syn tg model of MSA. Furthermore, AFF 1 has been previously assessed in an immunization protocol of two different tg mouse models of synucleinopathies, the PDGF-α-syn and the mThy1-α-syn tg mice [
19]. Vaccination with AFF 1 in those animals resulted in high antibody titers that crossed into the CNS and recognized α-syn aggregates, similar to the results obtained in MBP-α-syn tg mice. Interestingly in those models clearance of α-syn also involved activation of microglia and increased anti-inflammatory cytokine expression [
19]. In this sense and similarly to what we observed in the MBP-α-syn tg mice, AFF 1 also induced an increase the levels of anti-inflammatory cytokines such as IL-1Ra in the PDGF-α-syn tg mouse model of PD [
19]. These results combined suggest that the clearance mechanism of extracellular antibody-antigen complexes is probably common to all synucleinopathies. The mechanism through which the antibodies are internalized is not completely understood, but might include interaction with Fcγ receptors [
27] and endolysosomal trafficking for autophagy degradation [
20]. However, we also observed internalization of antibodies in α-syn-expressing oligodendrocytes in the MBP-α-syn tg mice, suggesting that these cells can also uptake antibody-antigen complexes or that their membrane integrity is compromised and antibodies can access intracellular α-syn deposits. Finally, other clearance mechanisms such as clearing α-syn into the venous system or via other glial mechanisms have not been evaluated and therefore cannot be ruled out.
Also of interest is the fact that immunization with AFF 1 prevented myelin loss and reduced motor deficits in the MBP-α-syn tg mice. MBP-α-syn tg animals show significant myelin pallor in white matter tracts [
32], similar to the demyelination observed in the MSA brain [
47]. The protective effect of immunotherapy on myelination is probably a consequence of the reduction in α-syn accumulation within oligodendrocytes and/or the reduction in α-syn propagation. Moreover, a direct protective effect of the treatment on myelin integrity cannot be excluded either. Interestingly, this protective effect on myelination has never been reported in active immunization studies for synucleinopathies, and it is a very good indicator of the effects of the AFFITOPE® on the preservation of the brain structure in MSA.
Finally, it is important to consider that MBP-α-syn tg animals over-express human α-syn directly in oligodendrocytes, therefore not showing α-syn propagation from neurons to oligodendrocytes, which it has been reported to occur in MSA patients [
48-
50]. In this case, AFF 1-induced antibodies might also be beneficial in halting α-syn transmission from neurons [
19], which is likely an early event in MSA progression. Moreover, as AFF 1-induced antibodies are specific to human α-syn, the question remains to if they could interact and block the physiological activity of this protein. In this sense, it must be noted that in healthy individuals α-syn should not be present in the extracellular space or bound to the plasma membrane, where the interaction with the antibodies might take place. However, in MSA patients AFF 1-induced antibodies could interact with extracellular or membrane-bound oligomeric α-syn, which are key features of the disease pathology [
51]. Therefore, binding of AFF 1-induced antibodies to intracellular, physiological human α-syn is not expected. In this regard, AFF 1 is currently being studied in clinical phase I studies in patients with early MSA.
Acknowledgements
We thank Andrea Achleitner, Martina-Anna Gschirtz, Michael Hierzer, Beate Pilz, Martina Trefil and Christina Wöss for their contribution in conducting the experiments. This work was funded by the National Institutes of Health (NIH) grants NS044233, AG18440, NS047303, AG022074 and NS057096. Additional funding was provided by Austrian Science promotion agency (FFG) grants 813335, 817969, 821453 and by the Michael J. Fox foundation for PD research (MJFF) grant: AFFITOPE® based immunotherapeutic strategies for Parkinson’s disease.
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Competing interests
The authors MM, HW, SS, RS, AS, WS and FM are employees of AFFiRiS, the company that commercialize the AFFITOPEs® described in the manuscript. The author FM is co-founder of AFFiRiS. The authors EV, ER, MM, CP, AA and EM declare that they have no competing interests.
Authors’ contributions
MM, EV, ER, FM and EM conceived the study and participated in its design. MM, EV, MM, HW, CP, AA, SS, RS, AS and WS performed the experiments. MM, EV and EM wrote the paper. All authors read and approved the final manuscript.