combined active humoral and cellular immunization ...combined active humoral and cellular...
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Research Articles: Neurobiology of Disease
Combined active humoral and cellular immunization approaches for thetreatment of Synucleinopathies.
Edward Rockenstein1, Gary Ostroff2, Fusun Dikengil2, Florentina Rus2, Michael Mante1, Jazmin Florio1,
Anthony Adame1, Ivy Trinh1, Changyoun Kim1,3, Cassia Overk1, Eliezer Masliah1,3 and Robert A.
Rissman1,4
1Department of Neurosciences, University of California, San Diego, La Jolla, California 92093-0624.2University of Mass Massachusetts Medical School, Program in Molecular Medicine Worcester MA 016053Molecular Neuropathology Section, Laboratory of Neurogenetics, National Institute on Aging, National Institutesof Health, Bethesda, MD 20892, USA4Veterans Affairs San Diego Healthcare System, La Jolla, CA 92161
DOI: 10.1523/JNEUROSCI.1170-17.2017
Received: 6 April 2017
Revised: 4 December 2017
Accepted: 6 December 2017
Published: 15 December 2017
Author contributions: E.R., G.O., F.D., F.R., M.M., J.F., A.A., I.T., C.K., and E.M. performed research; G.O.,C.R.O., and E.M. wrote the paper; F.D., F.R., C.R.O., E.M., and R.A.R. analyzed data; E.M. designed research.
Conflict of Interest: The authors declare no competing financial interests.
This work was funded by NIH grants AG18840, NS044233, BX003040, AG051839, and AG10483. The fundingsources had no role in study design; in the collection, analysis and interpretation of data; in the writing of thereport; or in the decision to submit the article for publication.
Corresponding author's email: [email protected]
Cite as: J. Neurosci ; 10.1523/JNEUROSCI.1170-17.2017
Alerts: Sign up at www.jneurosci.org/cgi/alerts to receive customized email alerts when the fully formattedversion of this article is published.
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Combined active humoral and cellular immunization approaches for the 1
treatment of Synucleinopathies. 2
3
Abbreviated title: Humoral/cellular immunization for synucleinopathy 4
5
Edward Rockenstein1, Gary Ostroff2, Fusun Dikengil2, Florentina Rus2, Michael 6
Mante1, Jazmin Florio1, Anthony Adame1, Ivy Trinh1, Changyoun Kim1,3, Cassia 7
Overk1, Eliezer Masliah1,3 and Robert A. Rissman,1,4* 8
9
1Department of Neurosciences, University of California, San Diego, La Jolla, 10
California 92093-0624. 11
2University of Mass Massachusetts Medical School, Program in Molecular 12
Medicine Worcester MA 01605 13
3Molecular Neuropathology Section, Laboratory of Neurogenetics, National 14
Institute on Aging, National Institutes of Health, Bethesda, MD 20892, USA 15
4. Veterans Affairs San Diego Healthcare System, La Jolla, CA 92161 16
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*Corresponding author’s email: [email protected] 18
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Pages: 41 20
Figures: 14 21
Tables: 1 22
Number of words: Abstract (250); Introduction (644); Discussion (790) 23
2
Conflict of Interest: The authors declare no competing financial interests 24
Acknowledgements: This work was funded by NIH grants AG18840, 25
NS044233, BX003040, AG051839, and AG10483. The funding sources had no 26
role in study design; in the collection, analysis and interpretation of data; in the 27
writing of the report; or in the decision to submit the article for publication. 28
29
ABSTRACT 30
Dementia with Lewy bodies (DLB), Parkinson’s disease (PD) and Multiple 31
System Atrophy (MSA) are age-related neurodegenerative disorders 32
characterized by progressive accumulation of -synuclein ( -syn) and jointly 33
termed synucleinopathies. Currently, no disease-modifying treatments are 34
available for these disorders. Previous preclinical studies showed that active and 35
passive immunizations targeting -syn partially ameliorates behavioral deficits 36
and -syn accumulation; however, it is unknown if combining humoral and 37
cellular immunization might act synergistically to reduce inflammation and 38
improve microglial-mediated -syn clearance. Since combined delivery of an 39
antigen + rapamycin (RAP) in nanoparticles is known to induce antigen-specific 40
regulatory T (Tregs) cells, we adapted this approach to -syn using the antigen-41
presenting cell-targeting glucan microparticle (GP) vaccine delivery system. 42
PDGF- -syn tg male and female mice, , were immunized with: GP-alone; GP- -43
syn (active humoral immunization); GP+RAP; or GP+RAP/ -syn (combined 44
active humoral and Treg) and analyzed using neuropathological and biochemical 45
markers. Active immunization resulted in higher serological total IgG, IgG1 and 46
3
IgG2a anti- -syn levels. Compared to mice immunized with GP-alone or GP- -47
syn, mice vaccinated with GP+RAP or GP+RAP/ -syn displayed increased 48
numbers of CD25-, FoxP3- and CD4-positive cells in the CNS. GP- -syn or 49
GP+RAP/ -syn immunizations resulted in 30-45% reduction in -syn 50
accumulation, neuroinflammation and neurodegeneration. Mice immunized with 51
GP+RAP/ -syn further rescued neurons and reduced neuroinflammation. Levels 52
of TGFβ1 were increased with GP+RAP/ -syn immunization, while levels of 53
TNF and IL6 were reduced. We conclude that the observed effects of 54
GP+RAP/ -syn immunization support the hypothesis that cellular immunization 55
might enhance the effects of active immunotherapy for the treatment of 56
synucleinopathies. 57
58
SIGNIFICANCE STATEMENT: We show that a novel vaccination modality 59
combining antigen-presenting cell-targeting glucan particle (GP) vaccine delivery 60
system with encapsulated antigen (α-synuclein) + rapamycin (RAP) induced both 61
strong anti-α-synuclein antibody titers and regulatory T (Tregs) cells. This 62
vaccine, collectively termed GP+RAP/ -syn, is capable of triggering 63
neuroprotective Treg cell responses in synucleinopathy models, and the 64
combined vaccine is more effective than the humoral or cellular immunization 65
alone. Together, these results support the further development of this multi-66
functional vaccine approach for the treatment of synucleinopathies, such as 67
Parkinson’s disease, dementia with Lewy bodies and multiple systems atrophy. 68
69
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INTRODUCTION 70
Immunotherapy approaches for the management of neurodegenerative 71
disorders other than Alzheimer’s disease (AD) have gained considerable interest 72
(Brody and Holtzman, 2008; Valera et al., 2016). In addition, preclinical and 73
clinical trials are underway to test the safety and efficacy of antibodies for the 74
treatment of synucleinopathies (Masliah et al., 2011; Lee and Lee, 2016; Valera 75
and Masliah, 2016a) and tauopathies (Sigurdsson, 2008; Ubhi and Masliah, 76
2013; Iqbal et al., 2016). Parkinson’s disease (PD), dementia with Lewy bodies 77
(DLB), and multiple systems atrophy (MSA). These are neurodegenerative 78
disorders of the aging population pathologically characterized by progressive 79
accumulation of α-synuclein (α-syn) and clinically manifested by fluctuating 80
cognitive impairment, parkinsonism, and dysautonomia (McKeith, 2006; Wenning 81
et al., 2008). For these synucleinopathies (Dickson et al., 1999; Spillantini and 82
Goedert, 2000; Galvin et al., 2001), no disease-modifying treatments are 83
currently available. Progressive accumulation of -syn in cortical and subcortical 84
brain regions is assumed to lead to neurodegeneration by interfering with 85
synaptic, mitochondrial and lysosomal function (Wong and Krainc, 2017). 86
Clinically, PD is characterized by motor deficits including bradykinesia, tremor, 87
rigidity, and postural instability (Jankovic, 2008), along with behavioral 88
alterations, cognitive impairment, sleep disorders, olfactory deficits, and 89
gastrointestinal dysfunction (Savica et al., 2013). 90
Several experimental strategies were directed at reducing -syn levels, 91
including decreasing the expression of -syn with anti-sense oligonucleotides 92
5
(Murphy et al., 2000) or miRNA (Junn et al., 2009); decreasing -syn aggregation 93
with small molecules (Wrasidlo et al., 2016), increasing the clearance of -syn 94
via autophagy (Sarkar and Rubinsztein, 2008) and preventing the seeding and 95
prion-like spreading of -syn (Lashuel et al., 2013; Valera and Masliah, 2016b). 96
Immunotherapeutic approaches targeting -syn have the advantage of 97
addressing several of these mechanisms. For example, active and passive 98
vaccination (Bae et al., 2012; Lindstrom et al., 2014) prevented 99
neurodegeneration and reduced -syn accumulation by promoting clearance via 100
autophagy (Masliah et al., 2005; Masliah et al., 2011; Mandler et al., 2014) and 101
microglial cells (Mandler et al., 2015). Using this mouse model, we showed that 102
active immunization against -syn triggered a strong antibody response with 103
antibodies trafficking into the CNS (Masliah et al., 2005; Mandler et al., 2015). 104
Likewise, modulation of T-cell trafficking with a copolymer has been shown to 105
reduce dopaminergic deficits and inflammation (Laurie et al., 2007). Similarly, 106
passive immunization with monoclonal antibodies recognizing the N- 107
(Shahaduzzaman et al., 2015) and C-terminus of -syn ameliorated behavioral 108
deficits, reduced neurodegeneration and -syn accumulation in neurons (Masliah 109
et al., 2011) as well as in glial cells (Bae et al., 2012) by reducing the cell-to-cell 110
transmission of -syn (Bae et al., 2012; Valera and Masliah, 2013; Games et al., 111
2014) and prion-like propagation (Tran et al., 2014). 112
Recent preclinical studies have shown that these active, passive, and 113
cellular immunization approaches are safe and some of them are currently under 114
clinical development. However, it is unknown if combining humoral and cellular 115
6
immunization might synergistically reduce inflammation and improve microglial-116
mediated -syn clearance. Therefore, we investigated if combining active cellular 117
and humoral immunization was more effective at ameliorating the deficits 118
associated in models of synucleinopathy than either cellular or humoral 119
immunization alone. Rapamycin has been used as part of a combination 120
immunotherapy in other diseases, including tuberculosis and influenza 121
(Jagannath and Bakhru, 2012; Keating et al., 2013), and recent clinical trials 122
(Mannick et al., 2014; Xie et al., 2016). It has been recently reported that the co-123
delivery of an antigen + rapamycin (RAP) in nanoparticles induced antigen-124
specific T regulatory (iTreg) cells (Maldonado et al., 2015). We adapted this 125
immunization strategy to -syn using the antigen-presenting cell targeting glucan 126
particle (GP) vaccine delivery system to test the hypothesis that combining 127
humoral and immunosuppressive cellular immunization would synergize to 128
enhance -syn clearance, reduce inflammation and neuropathological 129
symptoms. For this purpose, -syn tg mice were immunized with the GP vaccine 130
complex and analyzed by neuropathological and immunological markers. We 131
show that the combined vaccination generating iTreg cells might enhance the 132
effects of active immunotherapy for the treatment of synucleinopathies. 133
134
EXPERIMENTAL PROCEDURES 135
Preparation of the GP vaccine and in vitro testing in the LC3-GFP cell line 136
It has been shown that the combined delivery of an antigen + RAP in 137
nanoparticles induced antigen-specific Tregs cells(Maldonado et al., 2015) We 138
7
adapted this vaccination approach to -syn using the antigen-presenting cell 139
targeting glucan particle (GP) vaccine delivery system (Figure 1A). For this 140
purpose the GP vaccines were prepared as follows: 1) GP-alone (200 μg GP with 141
10 μg Ovalbumin + 25 μg mouse serum albumin (MSA, carrier protein); 2) GP- -142
syn (200 μg GP with 10 μg human -syn + 25 μg MSA); 3) GP encapsulated 143
RAP alone (200 μg GP containing 10 μg RAP + 10 μg Ovalbumin + 25 μg MSA) 144
and 4) GP co-encapsulated RAP/ -syn (200 μg GP containing 10 μg RAP + 10 145
μg human -syn + 25 μg MSA) using the antigen-adjuvant co-loading methods 146
as previously described (Huang et al., 2009; Huang et al., 2010; Hurtgen et al., 147
2012; Cole et al., 2013; Huang et al., 2013; Specht et al., 2015). 148
An immortalized C57/BL6 macrophage cell line expressing microtubule-149
associated protein 1A/1B-light chain 3 fused to GFP (LC3-GFP) was used to 150
demonstrate that the RAP encapsulated in the GPs was delivered and induced 151
autophagy (Hartman and Kornfeld, 2011). For this purpose, LC3-GFP 152
macrophage cells were plated on glass coverslips, incubated overnight with the 153
following treatments: a) saline alone (control 1); b) free RAP (10 μg/ml); c) GP 154
alone) at 10 particles/cell (control 2); d) GP+RAP (10 μg/ml); e) GP+RAP (1 155
μg/ml) and f) GP+RAP (0.1 μg/ml) and used fluorescent microscopy to the next 156
day to determine the formation of LC3-GFP punctae. Fluorescent puncta (white 157
arrows) indicate autophagy induction. Free rapamycin (10 μg/ml) stimulates 158
autophagy over saline and empty GP controls. GP encapsulated rapamycin 159
efficiently stimulates autophagy between 0.1-10 μg/ml) 160
161
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Mouse model and immunotherapy 162
For these experiments, we utilized 6-month-old (male and female) mice 163
over-expressing human -syn under the PDGFβ promoter (PDGFβ -syn, Line 164
D) (Masliah et al., 2000) and age-matched (male and female) non-tg mouse 165
littermates. The Line D model was selected because these mice develop 166
behavioral deficits (Amschl et al., 2013), axonal pathology, neurodegeneration, 167
neuroinflammation, and accumulate -syn in the neocortex and limbic system 168
mimicking DLB-like pathology (Lee et al., 2010). Moreover, this model has been 169
previously used for proof of concept studies of active and passive vaccination 170
and shown that the antibodies penetrate the CNS, recognize -syn in aggregates 171
in neurons and reduce the accumulation of -syn in the brain (Masliah et al., 172
2005; Masliah et al., 2011). These studies have provided important preclinical 173
data that have informed the advancement of immunotherapeutics to early-stage 174
clinical trials in patients with PD. 175
A total of n=30 (n=6 non-tg and n=24 tg) age-matched and gender-176
balanced mice were utilized for this study. For each group n=6 non-tg and n=6 tg 177
mice were treated with the following vaccines: a) GP-alone (control 1); b) GP- -178
syn (active immunization); c) GP+RAP (control 2) and d) GP+RAP/ -syn 179
(combined active and Treg vaccine). Mice were 6 m/o at the start of the 180
treatment, each mouse was injected with 0.1ml (intra-peritoneal; IP) every other 181
week for 4 weeks and sacrificed 4 weeks after the last injection (at the age 182
between 7.5-8 m/o). This timeline has previously been shown to produce 183
antibody titers levels sufficient to interfere with α-syn (Masliah et al., 2005). All 184
9
experiments described were performed according to NIH guidelines for animal 185
use, approved by the animal use and care committee of the University of 186
California San Diego (UCSD), were randomized and had power calculations 187
performed. 188
Tissue processing 189
Mice were anesthetized with chloral hydrate and terminal blood sampling 190
was performed. Blood was saved in EDTA treated tubes and spun for 191
subsequent serological analysis. Then brains were removed and divided sagittal, 192
the left hemibrain was post-fixed in phosphate-buffered 4% paraformaldehyde 193
(pH 7.4) at 4°C for 48 hr and sectioned at 40 μm with a Vibratome (Leica, 194
Germany) for immunocytochemical analysis, while the right hemibrain was snap 195
frozen and stored at -70°C for subsequent fractionation and immunoblot analysis. 196
197
Serological studies 198
Control and vaccinated mouse sera were diluted as indicated in PBS +0.05% 199
Tween 20 (wash buffer) and analyzed by ELISA. Human recombinant alpha-200
synuclein (10 ng/well) was bound to Costar 3695-96 well microtiter plates in 201
Binding Buffer (eBioscience), washed three times with wash buffer, blocked with 202
Blocking Buffer (eBioscience), washed 3x with wash buffer and diluted sera were 203
added in triplicate. After 2 hr incubation at room temperature wells were washed 204
3x with wash buffer and secondary antibodies were added: total IgG (Goat anti-205
mouse IgG-HRP; Life Technologies-A16072; dilution 1:500), IgG1 (Goat anti-206
mouse IgG1-HRP; SBiotech-1071-05; dilution 1:5000), and IgG2a (Goat anti-207
10
mouse IgG2a-HRP; SBiotech-1081-05; dilution 1:5000) for 1 hr at room 208
temperature. After washing the plate was developed with TMB Substrate 209
Solution (eBioscience 88-7324-88), stopped with 2N sulfuric acid and 210
absorbance measured at OD405. Standard concentration curves of primary anti-211
alpha-synuclein antibodies: A) total IgG and B) IgG1 (alpha Synuclein 212
Monoclonal Antibody Syn 211, Invitrogen, 0-500 ng/ml), and C) IgG2a (alpha 213
Synuclein Monoclonal Antibody Syn 202, Invitrogen, 0-500 ng/ml) were detected 214
with the isotype-specific-HRP conjugated antibodies as described above and 215
used to determine α-syn antibody sera concentrations. 216
Tissue fractionation and western blot analysis 217
Briefly, as previously described, the frozen cortex and hippocampus were 218
homogenized and separated by ultracentrifugation into cytosolic and membrane 219
fractions (Tofaris et al., 2003). For immunoblot analysis, 20 μg of protein per lane 220
were loaded into 4-12% Bis-Tris SDS-PAGE gels and blotted onto polyvinylidene 221
fluoride (PVDF) membranes. To determine the effects of vaccination on -syn 222
levels, blotted samples from treated -syn tg mice were probed with an antibody 223
against full length (FL)- -syn (monoclonal, SYN-1, 1:1000, BD Biosciences) 224
(Masliah et al., 2011) and an antibody against human -syn (monoclonal, 225
SYN211, 1:2000, Millipore). To evaluate the effects of the combined vaccine of 226
immunological markers the blots were probed with antibodies against CD25 227
(monoclonal, 1:1000, MBL); TGFβ1 (polyclonal, 1:2500, Abcam); CD68 228
(monoclonal, 1:1000, Abcam), TNFα (polyclonal, 1:2000, Abcam) and IL6 229
(polyclonal, 1:1000, Santa Cruz). 230
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Incubation with primary antibodies was followed by species-appropriate 231
incubation with secondary antibodies tagged with horseradish peroxidase 232
(1:5000, Santa Cruz Biotechnology, Santa Cruz, CA), visualization with 233
enhanced chemiluminescence and analysis with a Versadoc XL imaging 234
apparatus (BioRad, Hercules, CA). Analysis of -actin (Sigma) levels was used 235
as a loading control. 236
237
Immunocytochemical studies of -syn, markers of neurodegeneration, 238
inflammation and immune response 239
Analysis of -syn accumulation was performed using vibratome-cut free-240
floating coronal brain sections (40 μm). Brain sections were incubated overnight 241
at 4°C with a monoclonal antibody against total -syn (1:500, SYN-1, BD 242
Systems) (Masliah et al., 2000; Games et al., 2013) in the presence or absence 243
of proteinase K (10 μg/ ml for 15 min) or non-immune IgG controls (background 244
levels), phosphor Ser129 -syn (rabbit monoclonal, Abcam, 1:250), a neuronal 245
marker (NeuN, mouse monoclonal, Millipore 1:2500), an astroglial cell marker 246
(glial fibrillary acidic protein [GFAP], mouse monoclonal, Millipore 1:2500), a 247
dopaminergic neuron marker (TH, mouse monoclonal, Millipore, 1:1,000), a 248
microglial cell marker (Iba1; goat polyclonal, Abcam, 1:5000), a Treg marker 249
(CD25, monoclonal, 1:250, MBL), a B cell marker (CD20, mouse monoclonal, 250
Millipore, 1:100) a marker of learning and memory (Arc, rabbit polyclonal Santa 251
Cruz, 1:500), an anti-CD68 (monoclonal, 1:100, Abcam), an anti-FOXP3 252
(monoclonal, 1:250, Abcam), a T cell marker (CD4) (monoclonal, 1:250, Abcam), 253
12
or anti-TGFβ1 (polyclonal, 1:500, Abcam) followed by incubations with secondary 254
antibodies biotinylated (1:100, Vector Laboratories, Inc., Burlingame, CA), Avidin 255
D-HRP (1:200, ABC Elite, Vector) and detection with 3,3’-diaminobenzidine 256
(Masliah et al., 2011). All sections were processed simultaneously under the 257
same conditions and experiments were performed in triplicate in order to assess 258
the reproducibility of results. 259
Sections were analyzed with an Olympus BX41 digital video microscope 260
equipped with an Optronics magna fire SP camera, LucisTM DHP unique 261
contrast enhancement software and Image Pro ExpressTM for live 262
image acquisition and processing at 630X magnification. For each section 4 263
areas of interest (1024 x 1024 pixels) within the neocortex and hippocampus, 264
were analyzed in real time using the Image-Pro Plus program. -Syn and GFAP 265
levels of immunoreactivity were expressed as corrected optical density (arbitrary 266
units). Sections labeled with Iba-1 were analyzed utilizing the Image-Pro Plus 267
program (Media Cybernetics, Silver Spring, MD) (10 digital images per section at 268
400 × magnification) and analyzed in order to estimate the average number of 269
immuno-labeled cells per unit area (100 μm2). Sections with LC3-GFP positive 270
punctae were counted semiautomatically with a macro available in Image 271
J, as previously described (Suberbielle et al., 2015). Sections were scanned 272
with a digital Olympus bright field digital microscope (BX41). Briefly as 273
previously described (Overk et al., 2014), the numbers of NeuN-immunoreactive 274
neurons in the hippocampus were estimated utilizing unbiased stereological 275
methods. The CA3 region of the hippocampus was outlined using an Olympus 276
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BX51 microscope running StereoInvestigator 8.21.1 software (Micro-BrightField, 277
Colchester, VT) (grid sizes 150 × 150 μm and the counting frames were 30 × 30 278
μm). The average coefficient of error for each region was below 0.1. Sections 279
were analyzed using a 100 × 1.4 PlanApo oil-immersion objective. The average 280
mounted tissue thickness allowed for 2 μm top and bottom guard-zones and a 281
5 μm high disector. 282
283
Double immunolabeling and confocal microscopy 284
To evaluate the effects of immunization on -syn clearance via immune 285
cells, briefly as previously described (Bae et al., 2012) sections were double 286
labeled with antibodies against -syn (Millipore, 1:500) detected with Tyramide 287
Signal Amplification™-Direct (Red) system and microglial markers (1:2500, 288
Wako) detected with FITC. All sections were processed simultaneously under the 289
same conditions and experiments were performed in triplicate in order to assess 290
the reproducibility of results. Sections were imaged with a Zeiss 63X (N.A. 1.4) 291
objective on an Axiovert 35 microscope (Zeiss) with an attached MRC1024 292
LSCM (laser scanning confocal microscope) system (BioRad) (Masliah et al., 293
2000). Image analysis using Image J was utilized as previously described 294
(Marxreiter et al., 2013) to determine the percent of immune cells displaying -295
syn immunoreactivity. 296
297
Experimental Design and Statistical Analysis All experiments were done 298
blind-coded and in triplicate. Values in the figures are expressed as means ± 299
14
SEM. To determine the statistical significance, values were compared using one-300
way analysis of variance (ANOVA) with Dunnett’s post-hoc test as indicated in 301
each figure legend. Adjusted p-values are reported for specific comparisons in 302
the Results section. Each analysis had 25 degrees of freedom with the 303
exception of the PK-α-syn and pSer-α-syn experiments (Figure 6) which had 20 304
degrees of freedom. The differences were considered to be significant if the p-305
value was less than 0.05. 306
307
RESULTS 308
309
The GP vaccine is active in vitro and elicits serological and Treg responses in 310
vivo 311
To verify the activity of the GP vaccine in vitro, macrophages expressing 312
LC3-GFP were treated with GP-alone or GP+RAP (0.1 to 10 μg/ml). Under 313
baseline conditions, macrophages treated with saline (Figure 1B) or GP-alone 314
(Figure 1C) displayed only a few LC3-GFP positive punctae. In contrast, 315
treatment with RAP (10 μg/ml) alone resulted in a considerable increase in 316
punctae per cell compared to GP-alone (Figure 1D, p-value = 0.0005), likewise, 317
treatment with the GP+RAP resulted in a dose-dependent increases in LC3-GFP 318
positive grains (Figure 1E-HH) with both 1 μg/mL (p-value = 0.0116) and 10 319
μg/mL (p-value = 0.0001) having significant increases compared to GP-alone.e 320
Next, we analyzed the titers of total IgG, IgG1 and IgG2a anti- -syn antibody 321
generation in the sera by ELISA. For the present study, analysis by ELISA 322
15
showed that vaccination with GP- -syn or GP+RAP/ -syn resulted in high-level 323
titers of total IgG, IgG1, and IgG2a compared to GP-alone negative control 324
(Figure 2, Table 1). These studies support the notion that the GP vaccine is 325
active and promotes the generation of high levels of anti- -syn antibodies. 326
For all experiments we decided to utilize the Line D mice (PDGFβ- -syn 327
tg) (Masliah et al., 2000; Amschl et al., 2013) because these animals mimic some 328
pathological and functional aspects of DLB including the accumulation of -syn in 329
the neocortex and hippocampus and degeneration in layers 5-6 of the neocortex 330
and CA3 of the hippocampus (Amschl et al., 2013). 331
Next, we evaluated the effects of the GP vaccine on T and B cells in the 332
CNS by immunostaining sections from the mice with antibodies against CD4 (T 333
cells), CD25 (Treg), FOXP3 (Treg) and 20CD20 (B cells). In the non-tg and -syn 334
tg mice treated with GP, only rare CD4 positive cells were detected (Figure 3A, 335
B). In mice treated with GP- -syn some scattered CD4 positive cells were found 336
(Figure 3A, B). in contrast mice treated with GP+RAP (p-value = 0.0001) or 337
GP+RAP/ -syn (p-value = 0.0001) displayed abundant CD4 positive cells 338
scattered in the neuropil or in proximity with blood vessels compared to GP-alone 339
treated non-tg mice (Figure 3A, B). Similarly, mice treated with GP-alone or GP-340
-syn showed some scattered CD25 (Figure 3C, D) and FOXP3 positive cells 341
(Figure 3E, F), while mice immunized with GP+RAP(p-value = 0.0001) or 342
GP+RAP/ -syn (p-value = 0.0001) displayed abundant CD25 (Figure 3C, D p-343
value = 0.0001 for both comparisons) and FOXP3 positive Treg cells (Figure 3E, 344
F; p-value = 0.0001for both comparisons). With CD20 only a few positive cells 345
16
were observed, with no differences between non-tg and tg mice or among groups 346
(Figure 3G, H). Consistent with the immunocytochemical analysis, western blot 347
showed that levels of CD25 and FOXP3 were increased in the brains of mice 348
vaccinated with GP+RAP (p-value = 0.0002 and 0.0001, respectively) and 349
GP+RAP/ -syn (p-value = 0.0001 and 0.0001, respectively) compared to α-syn 350
tg animals treated with GP alone- (Figure 4A-C). In summary, these data show 351
that the GP+RAP/ -syn vaccine elicited a potent antibody response and elicited 352
Treg cells (CD25 and FOXP3) in the CNS of tg mice. 353
354
The GP vaccine reduces the accumulation of -syn in the brains of tg mice and 355
ameliorates neurodegeneration. 356
As expected, in the non-tg mice stained with an antibody against total -357
syn, which also recognizes endogenous murine α-syn, immunoreactivity was 358
detected only in the neuropil but no staining was seen in the neuronal cell bodies 359
(Figure 5A). In contrast, in the tg mice there was increased -syn accumulation in 360
the neuropil and cell bodies in the neocortex and hippocampus (Figure 5A). 361
Compared to GP-alone or GP+RAP, tg mice vaccinated with GP- -syn (p = 362
0.0001; p = 0.0004, respectively) or GP+RAP/ -syn (p < 0.0001; p < 0.0001, 363
respectively) displayed a significant reduction in -syn accumulation in the 364
neuropil and neuronal cell bodies in the neocortex (Figure 5A, B). Moreover, 365
GP+RAP/α-syn significantly decreased α-syn accumulation compared to GP-α-366
syn treatment (Figure 5A, B; p-value = 0.0352). Likewise, the hippocampus 367
displayed a significant reduction in -syn accumulation in the neuropil and 368
17
neuronal cell bodies (Figure 5A, C) when comparing tg mice vaccinated with GP-369
alone or GP+RAP to tg mice vaccinated with GP- -syn (p-value = 0.0003; p = 370
0.0009, respectively) or GP+RAP/ -syn (p-value = 0.0001; p-value = 0.0001, 371
respectively). 372
To evaluate the effects of the GP vaccine on aggregated -syn species, 373
immunocytochemical analysis was performed in sections pretreated with PK or 374
with an antibody against pSer129- -syn. In the non-tg mice no -syn 375
immunostaining was detected in the neocortex and hippocampus with PK 376
treatment (Figure 6A-C) or with anti-pSer129 (Figure 6D-F). Tg mice treated with 377
GP-alone or GP+RAP displayed high levels of PK-resistant α-syn (Figure 6A-B) 378
in the neocortex. In contrast, tg mice vaccinated with GP+RAP/ -syn showed a 379
significant reduction in PK-resistant α-syn accumulation (Figure 6A-B) compared 380
to GP-alone (p-value = 0.0001), and GP+RAP (p-value = 0.0001), and a trend 381
towards reduction compared to GP-α-syn (p-value = 0.0683). A similar reduction 382
in PK-resistant α-syn accumulation was seen in the hippocampus (Figure 6A, C) 383
with GP+RAP/α-syn treatment compared to GP-alone (p-value = 0.0003), GP-α-384
syn (p-value = 0.0369) and GP+RAP (p-value = 0.0369). pSer129- -syn 385
immunoreactivity (Figure 6) was also increased in neurons in the deep layers of 386
the neocortex (Figure 6A) and CA3 of the hippocampus (Figure 6D). Additionally, 387
pSer129 -syn (Figure 6D) accumulation in the neocortex (Figure 6E) was 388
significantly reduced with GP+RAP/α-syn treatment compared to GP-alone (p-389
value = 0.0051) or GP+RAP (p-value = 0.0001). GP-α-syn also significantly 390
reduced pSer α-syn accumulation compared to GP+RAP (p-value = 0.0013). In 391
18
the hippocampus (Figure 6F) GP-α-syn and GP+RAP/α-syn treatments 392
significantly reduced pSer-α-syn compared to GP-alone (p-value = 0.0001 and 393
0.0001, respectively) and GP+RAP (0.0002 and 0.0001, respectively). Consistent 394
with these results, western blot analysis showed that levels of insoluble -syn 395
were reduced in the brains of tg mice vaccinated with GP- -syn or GP+RAP/ -396
syn compared to animals treated with GP-alone (p-value = 0.0394 and 0.0001, 397
respectively) or GP+RAP (Figure 4A, D; p-value = 0.0001 and 0.0001, 398
respectively). 399
Next, we investigated the effects of the GP vaccine at ameliorating the 400
neurodegenerative phenotype in the tg mice. We have previously shown that in 401
aged -syn tg mice there is a loss of neurons in deeper layers of the neocortex 402
and CA3 of the hippocampus (Overk et al., 2014). Consistent with these reports 403
compared to vehicle-treated non-tg mice, the -syn tg mice treated with GP-404
alone (p-value = 0.0001), GP-α-syn (p-value = 0.0024), and GP+RAP (p-value = 405
0.0001) showed a 25-30% reduction in the estimated number of NeuN positive 406
neurons in layers 5-6 of the neocortex (Figure 7A, B). In the CA3 pyramidal 407
neurons in the hippocampus (Figure 7A, C) there was a 45-50% decrease in the 408
estimated number of NeuN positive neurons with GP-alone (p-value = 0.0001), 409
GP-α-syn (p-value = 0.0091), or GP+RAP (p-value = 0.0001) treatment 410
compared to non-tg mice. In contrast, treatment with GP+RAP/ -syn ameliorated 411
the loss of neurons in the neocortex (Figure 7A, B; p-value = 0.0001) and 412
hippocampus (Figure 7A, C; p-value = 0.0001) compared to vehicle-treated α-syn 413
tg mice. In summary, these studies showed that the GP+RAP/ -syn vaccine 414
19
reduced the accumulation of -syn and neurodegeneration in the -syn tg mice 415
to a similar if not greater extent than GP- -syn. 416
Effects of the GP vaccine on markers of functional recovery from 417
neurodegeneration in the brains of tg mice. 418
We used neuronal activation (Arc) and the dopaminergic system (TH) as 419
biomarkers, which are impacted in this mouse model of α-synucleinopathies to 420
assess functional recovery in the α-syn tg mice. Compared to non-tg mice, the 421
-syn tg mice treated with GP-alone showed decreased neuronal activation in 422
the neocortex (Figure 8A, B; p-values = 0.0001). Treatment with GP+α-syn or 423
GP+RAP/α-syn significantly improved the neuronal activation in α-syn tg mice 424
compared to GP-alone in the neocortex (Figure 8A, B; p-values = 0.0386 and 425
0.0001, respectively), with GP+RAP/α-syn improving neuronal activation to a 426
greater extent than GP+α-syn treatment (Figure 8A, B; p-value = 0.0002). 427
Similarly, in the hippocampus, α-syn tg mice treated with GP-alone had a 428
significant decrease in the Arc biomarker of neuronal activation in α-syn tg mice 429
compared to non-tg mice (Figure 8C, D; p-value = 0.0006). Although treatment 430
with GP+α-syn had no significant effect (Figure 8C, D; p-value = 0.1351), 431
treatment with GP+RAP/α-syn significantly improved neuronal activation in α-syn 432
tg mice compared to GP-alone (Figure 8C, D; p-value = 0.0003). We also 433
evaluated the functional consequences of these treatments on the dopaminergic 434
system in α-syn tg mice. In the striatum, α-syn tg mice treated with GP-alone 435
showed a significant decrease in TH-immunoreactivity compared to non-tg mice 436
(Figure 9A, B; p-value = 0.0003). Treatment with GP-α-syn or GP+RAP/α-syn 437
20
significantly increased TH-immunoreactivity in the striatum of α-syn tg mice 438
compared to GP-alone (Figure 9A, B; p-values = 0.0013 and 0.0001, 439
respectively). However, there were no significant differences in TH-440
immunoreactivity between any of the groups in the s. nigra (Figure 9C, D). 441
Effects of the GP vaccine on immunomodulatory cytokines in the brains of tg 442
mice. 443
Previous studies have shown that in aged -syn tg mice there are neuro-444
inflammatory changes associated with microgliosis (Iba1), astrogliosis (GFAP) 445
and increased IL6 and TNFα expression (Bae et al., 2012). Given that we have 446
shown that the GP+RAP/ -syn vaccine, in addition to eliciting an anti- -syn 447
antibody response, also induces CD25/FOXP3 cells in the CNS, we 448
hypothesized that Treg signaling with glial cells might further modulate 449
neuroinflammation. 450
Compared to non-tg mice, the -syn tg mice treated with GP-alone, GP-α-451
syn, or GP+RAP showed increased numbers of microglial cells in the neocortex 452
(Figure 10A, B; p-values = 0.0001, 0.0001, and 0.0006, respectively), as well as 453
in the hippocampus of GP-alone treated α-syn tg mice (Figure 10C, D; p-values = 454
0.0001). Moreover, in the neocortex, there was a significant increase in the 455
number of activated microglia in α-syn tg mice treated with GP-alone compared 456
to non-tg mice (Figure 10E, F; p-value = 0.0007), which was significantly reduced 457
by treatment only with GP+RAP/α-syn (Figure 10E, F; p-value = 0.0001). 458
Similarly, the number of activated microglia was also increased in the 459
hippocampus in α-syn tg mice treated with GP-alone compared to non-tg mice 460
21
(Figure 10G, H; p-value = 0.0001). The number of activated microglia was 461
significantly reduced with GP+RAP/α-syn treatment (Figure 10G, H; p-value = 462
0.0001). Further investigation revealed a significant increase in the colocalization 463
between α-syn and microglia in α-syn tg mice treated with GP-alone compared to 464
non-tg mice (Figure 11; p-value =0.0001). Treatment with either GP+α-syn or 465
GP+RAP/α-syn significantly increased the percent of colocalization between 466
microglia and α-syn suggesting increased clearance of α-syn with these 467
treatments (Figure 11; p-values = 0.0001 and 0.0001, respectively). Likewise, 468
when compared to non-tg mice, the -syn tg mice treated with GP-alone 469
displayed increased astrogliosis in the in the neocortex (Figure 12A, B; p-value = 470
0.0001) and hippocampus (Figure 12C, D; p-value = 0.0001)). Astrocytosis 471
(Figure 12) was similarly decreased with GP- -syn and GP+RAP/ -syn 472
treatment both in the neocortex (Figure 12 A, B; p-value = 0.0020 and 0.0001, 473
respectively) and in the hippocampus (Figure 12 C, D; p-value = 0.0024 and 474
0.0001, respectively). Interestingly, GP+RAP/ -syn treatment resulted in a 475
greater reduction in microgliosis in both the neocortex and hippocampus (Figure 476
10B, D; p-value = 0.0002 and 0.0081, respectively) and astrocytosis (Figure 12B, 477
D; p-value = 0.0134 and 0.0016, respectively) compared to GP- -syn treatment. 478
Next, we analyzed the effects of the GP vaccination on levels of pro-479
inflammatory cytokines IL6, TNFα, and IL8. Compared to non-tg mice, the -syn 480
tg mice treated with GP-alone displayed increased levels of IL6 (Figure 13A, B; 481
p-value = 0.0001) and TNFα immunoreactivity (Figure 313C, D, p-value = 482
0.0001). Treatment with GP- -syn and GP+RAP/ -syn resulted in decreased IL6 483
22
(Figure 13A, B; p-value = 0.0384 and 0.0010, respectively) and TNFα (Figure 484
13C, D; p-value = 0.0006 and 0.0001) compared to α-syn tg mice treated with 485
GP-alone. No significant differences or effects of the GP vaccination were 486
detected among the groups when analyzing IL8 levels of immunoreactivity 487
(Figure 13 E, F). Consistent with these results, immunoblot analysis showed that 488
GP-α-syn, GP+RAP, and GP+RAP/α-syn treatment also led to a reduction in 489
TNFα compared to GP-alone-treated α-syn tg mice (Figure 4A, E; p-value for 490
each comparison = 0.0001) and IL6 (Figure 4 A, F; p-value for each comparison 491
= 0.0001). 492
Finally, in order to begin to understand the mechanisms through which the 493
GP vaccine might reduce inflammatory responses levels of immunomodulatory 494
cytokines, TGFβ1 and IL2 were analyzed. Levels of TGFβ1 were similar between 495
the non-tg and the -syn tg mice treated with GP-alone or GP- -syn (Figure 13G, 496
H). However, treatment with GP+RAP (p-value = 0.0001 for each comparison) 497
and GP+RAP/ -syn (p-value = 0.0001 for each comparison) resulted in 498
increased levels of TGFβ1 immunoreactivity in the tg mice (Figure 13G, H) 499
compared to both non-tg and tg mice treated with GP-alone. No significant 500
effects of the GP vaccination or with GP+α-syn were detected among the groups 501
when analyzing levels of IL2 immunoreactivity (Figure 13I, J). Consistent with 502
these results, immunoblot analysis showed that treatment with GP+RAP and 503
GP+RAP/α-syn similarly resulted in increased levels of TGFβ1 in the α-syn tg 504
mice compared to treatment with GP-alone in either the non-tg or α-syn tg mice 505
(Figure 4A, G; p-value = 0.0001 for each comparison). 506
23
Together, these results suggest that the GP vaccines containing RAP 507
triggered increased expression of TGFβ1, which in turn promoted retention of 508
Treg cells in the CNS that might regulate and hamper microglial and astroglial 509
pro-inflammatory responses and decrease expression of IL6 and TNF (Figure 510
14). This, in combination with the production of anti- -syn antibodies, resulted in 511
a potentially more effective vaccine compared to GP- -syn alone (Figure 14). 512
513
DISCUSSION 514
The present study shows that vaccination with GP-+RAP/α-syn can elicit 515
the production of high titers of anti- -syn antibodies in combination with an anti-516
inflammatory cellular response mediated by TGFβ1-produced by iTreg (CD25 517
and FOXP3 +) cells in the CNS. We found that these effects were associated 518
with greater attenuation of microglial and astroglial neuro-inflammatory 519
responses, as well as a complete amelioration of neuronal loss in an -syn tg 520
mouse model of DLB when compared to GP- -syn alone. Moreover, vaccination 521
with GP+RAP/ -syn resulted in reduced accumulation of -syn and microglia-522
mediated clearance of -syn comparable to the effects observed when 523
vaccinating tg mice with GP- -syn alone. 524
For these experiments we chose to utilize the PDGF- -syn tg mice 525
(Masliah et al., 2000) because these animals display a widespread accumulation 526
of -syn in neuronal cells in the deeper layers of the neocortex and hippocampus 527
(CA2-3) mimicking some aspects of DLB (Amschl et al., 2013; Adamowicz et al., 528
2017). We have also shown in independent studies that these mice have high 529
24
titers of anti- -syn antibody upon vaccination with -syn peptides with trafficking 530
of antibodies into the CNS and target engagement of the -syn aggregates in the 531
neocortex and limbic system (Masliah et al., 2005; Mandler et al., 2014). 532
In our previous studies, mice were vaccinated with recombinant -syn or 533
with peptides mimicking the antigenicity of -syn that resulted in the production of 534
antibodies that recognized the c-terminus of -syn and partially blocked neuro-535
inflammation and neurodegeneration (Masliah et al., 2005; Mandler et al., 2014). 536
The difference between our previous studies and the present study is that we 537
now immunized with GP+RAP/ -syn, which triggered a robust anti- -syn 538
humoral response and promoted the formation of iTreg cells in the CNS. In 539
combination, these iTreg cells were more effective at modulating neuro-540
inflammatory responses and ameliorating the neurodegenerative pathology in the 541
neocortex and hippocampus, than upon immunization with the antigen alone. 542
Regulatory T cells are potent suppressors, playing important roles in 543
autoimmunity and transplantation tolerance (Fu et al., 2004), which can be 544
classified as induced (iTreg) or natural Treg . In the CNS Tregs have been 545
studied mostly in the context of auto-immunity (eg: multiple sclerosis) and 546
persistent viral infection in the CNS (eg: HIV-1) (Reuter et al., 2012), but less is 547
known as to their role in neurodegeneration and neuro-therapeutics. For 548
example, recent studies have shown that manipulation of Tregs can be utilized to 549
regulate virus-specific CD8+ effector T cells and virus persistence in the brain 550
(Reuter et al., 2012). It has been reported that Tregs induce neuroprotective 551
immune responses in murine models of stroke, amyotrophic lateral sclerosis and 552
25
HIV-1 associated neurocognitive disorders (Reynolds et al., 2009) 553
Moreover, previous studies have shown that iTregs can be 554
neuroprotective in a neurotoxin-induced model of PD (Reynolds et al., 2007) by 555
modulating Th17 responses (Reynolds et al., 2010). For those experiments, 556
adoptive transfer of T cells was performed by administering copolymer-1 to mice 557
challenged with the nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-558
tetrahydropyridine (MPTP) (Reynolds et al., 2010). iTregs were found to mediate 559
neuroprotection in the MPTP challenged mice by reducing microglial responses 560
to stimuli, including aggregated, nitrated -syn. In addition, iTreg-mediated 561
neuroprotection was functional upon removal of iTregs from culture prior to 562
stimulation (Reynolds et al., 2010). 563
It has been recently reported that the co-delivery of an antigen plus RAP 564
in nanoparticles induced Treg cells (Maldonado et al., 2015). We adapted this 565
immunization strategy to -syn using the antigen-presenting cell GP vaccine 566
delivery system to test the hypothesis that combining humoral and 567
immunosuppressive cellular immunization would synergize to enhance -syn 568
clearance, and reduce inflammation and neuropathological symptoms. The 569
mechanisms through which the GP+RAP/ -syn vaccine might work probably 570
involve several mechanisms (Figure 14). We propose that, in addition to the 571
effects of the antibodies, the GP+RAP vaccine might trigger iTreg cells to 572
produce TGFβ1 which in turn might mediate the conversion of microglial cells 573
into a neuroprotective state (Figure 14). Recent reports suggest a role for TGFβ1 574
in the generation of iTregs from CD4 + CD25 - precursors, and recent studies 575
26
have shown that TGFβ1 triggers FOXP3 expression in CD4 + CD25 - precursors, 576
and these FOXP3+ cells act like conventional Tregs. The generation of FOXP3+ 577
iTregs requires stimulation of the T-cell receptor, the IL-2R and the TGFβ1 578
receptors (Fu et al., 2004). In this context, it is worth mentioning that while 579
vaccination with both GP+RAP or GP+RAP/ -syn induced a Treg cell response, 580
only the GP+RAP/ -syn vaccine combination was effective in reducing -syn 581
accumulation, neurodegenerative, and inflammatory pathology. In summary, we 582
have shown that a novel vaccination modality based on GP-RAP/α-syn is 583
capable of triggering both neuroprotective humoral and iTreg cell responses in 584
mouse models of synucleinopathy and that the combined vaccine is more 585
effective than either humoral or cellular immunization alone. Together, these 586
results support the further development of this multi-functional vaccine approach 587
for the treatment of synucleinopathies such as PD, DLB, and MSA. 588
589
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802
Figure Legends 803
804
Figure 1. Diagrammatic representation of the preparation of the vaccine 805
and in vitro verification of GP+rapamycin to induce autophagy in the LC3-806
GFP macrophage cell line. (A) Schematic representation of the glucan (GP) 807
vaccine delivery system loaded with α-syn and designated as GP-α-syn (left), 808
which could also be co-loaded with rapamycin (RAP) and designated as GP + 809
RAP/α-syn (right). (B) Macrophages expressing LC3-GFP were treated with 810
vehicle (saline) or (C) GP-alone displayed few LC3-GFP positive punctae 811
(arrows). (D) Macrophages treated with RAP (10 μg/ml) and (E-G) increasing 812
concentrations of GP+RAP (0.1, 1.0, and 10 μg/ml) showed dose-dependent 813
increases in LC3-GFP positive grains. (H) Quantification of the number of LC3-814
33
GFP-positive grains per cell. *P-value < 0.05 using ANOVA followed by 815
Dunnett’s post-hoc test compared to control-treated cells. Scale bar = 15 μm. 816
817
Figure 2. Antibody titers in response to GP-α-syn+/- RAP vaccination in tg 818
mice. α-Syn tg (Line D) mice and non-tg littermates were injected biweekly IP for 819
4 weeks and sacrificed 4 weeks after the last injection (7.5-8 m/o). Total IgG was 820
increased in (A) GP-α-syn and (B) GP+RAP/α-syn-immunized mice (blue lines, 821
respectively) compared to their negative controls (red lines, respectively. 822
Similarly, (C, D) IgG1 and (E, F) IgG2a serum levels were increased for GP-α-823
syn (C and E blue lines, respectively) and GP+RAP/α-syn (D and F blue lines, 824
respectively) vaccinated mice compared to their negative controls (red lines). N= 825
6 per treatment group. Blue lines represent vaccination with GP+RAP/α-syn or 826
GP-α-syn while the red lines represent the respective controls with GP alone. 827
828
Figure 3. Effects of combined GP+RAP/α-syn vaccination on trafficking of T 829
and B cells in the brains of α-syn tg mice. Brain sections from α-syn tg mice 830
immunized with GP, GP-α-syn, GP+RAP, or GP+RAP/α-syn and non-tg control 831
mice immunized with GP were analyzed immunohistochemically for markers of T 832
cells (CD4), Treg (CD25, FOXP3) and B cells (CD20). There was a significant 833
increase in the numbers (A, B) of the CD4 immunoreactive T-cells, (C, D) the 834
CD25 and (E, F) the FOXP3 markers of Treg cells in α-syn tg mice treated with 835
either GP+RAP or GP+RAP/α-syn compared to non-tg mice treated with GP-836
alone. (G, H) There was no change in the CD20 marker for B cells across any of 837
34
the treatment groups. P-value < 0.05 using ANOVA followed by Dunnett’s post-838
hoc test comparing (&) GP+RAP/α-syn or (#) GP+RAP treatment to all other 839
groups. N=6 mice/group. Scale bar = 25 μm. 840
841
Figure 4. Verification of effects of combined GP+RAP/α-syn vaccination on 842
α-synuclein and immune activation markers by immunoblot. Brain 843
homogenates from α-syn tg mice immunized with GP, GP-α-syn, GP+RAP, or 844
GP+RAP/α-syn and non-tg control mice immunized with GP were fractioned and 845
analyzed by Western blot. (A) Representative western blot images and 846
quantitative analysis for (B) CD25, (C) FOXP3, (D) α-syn, (E) TNFα, (F) IL6, and 847
(G) TGFβ1 normalized to actin. N=6 mice/group. P-value < 0.05 using ANOVA 848
followed by Dunnett’s post-hoc test comparing each of the other groups with (*) 849
GP-alone treated tg mice; (&) GP+RAP/α-syn, or (#) GP+RAP treatment. 850
851
Figure 5. Effects of combined vaccination with GP+RAP/α-syn on levels of 852
total α-synuclein in the brains of α-syn tg mice. Immunohistochemical 853
analysis of brain sections from α-syn tg mice immunized with GP, GP-α-syn, 854
GP+RAP, or GP+RAP/α-syn and non-tg control mice immunized with GP was 855
performed using an antibody against total α-syn. (A) Representative 856
photomicrographs and quantitative analysis of α-syn immunoreactivity in the (B) 857
neocortex and (C) hippocampus. Compared to non-tg mice immunized with GP 858
alone, the α-syn tg mice treated with GP alone there is an extensive 859
accumulation of α-syn in the neocortex and hippocampus. Vaccination with GP-860
35
α-syn and GP+RAP/α-syn significantly reduced the accumulation of α-syn. P-861
value < 0.05 using ANOVA followed by Dunnett’s post-hoc test comparing each 862
of the other groups with (&) GP+RAP/α-syn or (#) GP-α-syn treatment. 863
N=6/group. Overview scale bar = 250 μm; higher magnification scale bars = 25 864
μm. 865
866
Figure 6. Effects of combined vaccination with GP+RAP/α-syn on levels of 867
PK-resistant and phosphorylated- α-syn in the brains of α-syn tg mice. 868
Immunocytochemical analysis of brain sections pretreated with PK (followed by 869
immunostaining with an antibody against total α-syn) or with an antibody against 870
pSer129-α-syn in α-syn tg mice immunized with GP, GP-α-syn, GP+RAP, or 871
GP+RAP/α-syn and non-tg control mice immunized with GP. (A) Representative 872
photomicrographs of PK-resistant -syn immunoreactivity in the frontal cortex 873
and hippocampus of immunized -syn tg mice. No immunoreactivity was 874
detected in non-tg mice, while in GP alone treated mice there was an 875
accumulation of PK-resistant α-syn; treatment with GP-α-syn or GP+RAP/α-syn 876
reduced levels of PK-resistant α-syn immunostaining. (B) Computer-aided 877
quantitation of the optical density of PK-resistant -syn immunoreactivity in the 878
(B) frontal cortex and (C) hippocampus. (D) Representative photomicrographs of 879
frontal cortex and CA3 hippocampal sections pretreated with anti-pSer129-α-syn 880
from immunized -syn tg mice and non-tg control mice. No immunoreactivity was 881
detected in non-tg mice, while in GP alone treated tg mice there was an 882
accumulation of pSer129-α-syn; treatment with GP-α-syn or GP+RAP/α-syn 883
36
reduced levels of pSer129-α-syn immunostaining. (E) Computer-aided 884
quantitation of -syn immunoreactivity using optical density from sections 885
pretreated with anti-pSer129 α-syn in the frontal cortex and (F) hippocampus. 886
Magnification scale bar = 10 μm. P-value < 0.05 using ANOVA followed by 887
Dunnett’s post-hoc test comparing each of the three α-syn tg groups with (&) 888
GP+RAP/α-syn or (#) GP-α-syn treatment. N=6/group. 889
890
Figure 7. Effects of combined vaccination with GP+RAP/α-syn on 891
neurodegeneration in the brains of α-syn tg mice. Immunohistochemical 892
analysis of neocortex and CA3 hippocampal brain sections from α-syn tg mice 893
immunized with GP, GP-α-syn, GP+RAP, or GP+RAP/α-syn and non-tg control 894
mice immunized with GP. Neuronal cells were visualized with an antibody 895
against NeuN. (A) Representative low and high magnification photomicrographs 896
and (B) computer-aided analysis of neocortex and (C) CA3 hippocampus 897
immunostained with the neuronal antibody marker NeuN. Compared to non-tg 898
GP alone, in the α-syn tg mice treated with GP alone had a decreased number of 899
NeuN-positive cells in the neocortex and CA3 of hippocampus; treatment with 900
GP-α-syn partially prevented the loss, while GP+RAP/α-syn treatment fully 901
protected the mice against neuronal loss. Low magnification scale bar = 250 μm; 902
high magnification scale bar = 50 μm. P-value < 0.05 using ANOVA followed by 903
Dunnett’s post-hoc test comparing each of the other groups with (*) GP-alone 904
treated non-tg mice; (&) GP+RAP/α-syn, or (#) GP-α-syn treatment. N=6/group. 905
906
37
Figure 8. Effects of combined GP+RAP/α-syn vaccination on markers of 907
neuronal activation. Immunocytochemical analysis of neocortex and CA3 908
hippocampal brain sections from α-syn tg mice immunized with GP, GP-α-syn, 909
GP+RAP, or GP+RAP/α-syn and non-tg control mice immunized with GP. 910
Activated neuronal cells were visualized with an antibody against Arc. (A) 911
Representative photomicrographs at low and high magnification from neocortex 912
and (B) quantitative analysis of neuronal activation. (C) Representative 913
photomicrographs at low and high magnification from hippocampus and (D) 914
quantitative analysis of neuronal activation. Compared to non-tg GP alone, in the 915
α-syn tg mice treated with GP-alone, there was a decrease in Arc-916
immunoreactivity in the neocortex and CA3 of hippocampus; treatment with GP-917
α-syn partially increased neuronal activation, while GP+RAP/α-syn was more 918
effective. P-value < 0.05 using ANOVA followed by Dunnett’s post-hoc test 919
comparing each of the other groups with (*) GP-alone treated tg mice or (&) 920
GP+RAP/α-syn. N = 6 mice per group. Lower magnification scale bar = 50 μm; 921
detail scale bar = 10 μm. 922
923
Figure 9. Effects of combined GP+RAP/α-syn vaccination on the 924
dopaminergic system in the brains of α-syn tg mice. Immunocytochemical 925
analysis of striatum and s. nigra brain sections from α-syn tg mice immunized 926
with GP, GP-α-syn, GP+RAP, or GP+RAP/α-syn and non-tg control mice 927
immunized with GP. Dopaminergic cells were visualized with an antibody against 928
TH. (A) Representative photomicrographs at low and high magnification from 929
38
striatum and (B) quantitative analysis of the corrected optical density of TH-930
positive cells. (C) Representative photomicrographs at low and high 931
magnification from the s. nigra and (D) quantitative analysis of the optical density 932
of TH-positive cells. Compared to non-tg GP alone, in the α-syn tg mice treated 933
with GP-alone, there was a decrease in TH-immunoreactivity in the striatum. 934
Treatment with GP+α-syn and GP+RAP/α-syn significantly increased TH 935
immunoreactivity compared in GP-alone in α-syn tg mice. There were no 936
significant changes in TH-immunoreactivity in the s. nigra. P-value < 0.05 using 937
ANOVA followed by Dunnett’s post-hoc test comparing each of the other groups 938
with (*) GP-alone treated tg mice. N = 6 mice per group. Lower magnification 939
scale bar = 50 μm; detail scale bar = 10 μm. 940
941
Figure 10. Effects of combined GP+RAP/α-syn vaccination on microglial 942
cell markers in the brains of α-syn tg mice. Immunocytochemical analysis of 943
neocortex and CA3 hippocampal brain sections from α-syn tg mice immunized 944
with GP, GP-α-syn, GP+RAP, or GP+RAP/α-syn and non-tg control mice 945
immunized with GP. Microglial cells were visualized with an antibody against 946
Iba1. (A) Representative photomicrographs at low and high magnification from 947
neocortex and (B) quantitative analysis of the number of immunoreactive 948
microglia per 0.1 mm2. (C) Representative photomicrographs at low and high 949
magnification from hippocampus and (D) quantitative analysis of the number of 950
immunoreactive microglia per 0.1 mm2. Compared to non-tg GP alone, in the α-951
syn tg mice treated with GP-alone, there were increased numbers of Iba1+ cells 952
39
in the neocortex and CA3 of hippocampus; treatment with GP-α-syn and 953
GP+RAP partially reduced the microglial cell number, while GP+RAP/α-syn was 954
more effective. (E) Representative photomicrographs at low and high 955
magnification from neocortex and (F) quantitative analysis of the number of 956
CD68-immunoreactive cells per 0.1 mm2. (G) Representative photomicrographs 957
at low and high magnification from hippocampus and (H) quantitative analysis of 958
the number of CD68-immunoreactive cells per 0.1 mm2. Low magnification scale 959
bar = 50 μm; high magnification scale bar = 10 μm. P-value < 0.05 using ANOVA 960
followed by Dunnett’s post-hoc test comparing each of the other groups with (*) 961
GP-alone treated tg mice or (&) GP+RAP/α-syn.N = 6 mice per group. 962
963
Figure 11. Effects of combined vaccination with GP+RAP/α-syn on 964
microglial cell clearance of α-syn in the brains of α-syn tg mice. Confocal 965
analysis of neocortex brain sections from α-syn tg mice immunized with GP, GP-966
α-syn, GP+RAP, or GP+RAP/α-syn and non-tg control mice immunized with GP. 967
(A) Representative images of the colocalization and (B) quantification of α-syn 968
(green channel) and microglial cells, which were visualized with an antibody 969
against Iba-1 (red channel). The percent of colocalization of α-syn and microglia 970
was significantly increased in GP-alone in the α-syn tg mice compared to the 971
non-tg mice. In the α-syn tg mice, treatment with GP+α-syn and GP+RAP/α-syn 972
significantly increased the amount of colocalization between α-syn and microglia 973
compared to GP-alone. P-value < 0.05 using ANOVA followed by Dunnett’s 974
post-hoc test comparing (*) GP-alone in α-syn tg mice. N = 6 mice per group. 975
40
Scale bar in merged image = 15 μm; scale bar in detail = 5 μm. 976
977
Figure 12. Effects of combined vaccination with GP+RAP/α-syn on 978
astroglial cells in the brains of α-syn tg mice. Immunohistochemical analysis 979
of neocortex and CA3 hippocampal brain sections from α-syn tg mice immunized 980
with GP, GP-α-syn, GP+RAP, or GP+RAP/α-syn and non-tg control mice 981
immunized with GP. Astroglial cells were visualized with an antibody against 982
GFAP. (A) Representative photomicrographs at low and high magnification from 983
neocortex and (B) quantitative analysis of the number of immunoreactive 984
microglia per 0.1 mm2. (A) Representative photomicrographs at low and high 985
magnification from hippocampus and (B) quantitative analysis of the number of 986
immunoreactive astroglia per 0.1 mm2. Compared to non-tg GP alone, in the α-987
syn tg mice treated with GP-alone, there was an increase in astrogliosis in the 988
neocortex and CA3 of the hippocampus, treatment with GP-α-syn and GP/RAP 989
partially re-established the astroglial cell immunostaining, while GP+RAP/α-syn 990
was more effective. Low magnification scale bar = 25 μm; high magnification 991
scale bar = 10 μm. P-value < 0.05 using ANOVA followed by Dunnett’s post-hoc 992
test comparing each of the other groups with (*) GP-alone treated non-tg mice, 993
(&) GP+RAP/α-syn, or (#) GP-α-syn treatment. N = 6 mice per group. 994
995
Figure 13. Effects of combined vaccination with GP+RAP/α-syn on 996
cytokines in the brains of α-syn tg mice. Immunohistochemical analysis of 997
neocortex brain sections from α-syn tg mice immunized with GP, GP-α-syn, 998
41
GP+RAP, or GP+RAP/α-syn and non-tg control mice immunized with GP. Pro-999
inflammatory cytokine immunoreactivity was visualized with antibodies against 1000
IL6, TNFα, IL8 and the immunomodulator cytokine levels visualized with 1001
antibodies against TGFβ1 and IL2. (A) Representative photomicrographs and 1002
quantification of (B) IL6, (C) TNFα, (D) IL8, (E) TGFβ1, and (F) IL2 1003
immunoreactivity. Scale bar = 25 μm. P-value < 0.05 using ANOVA followed by 1004
Dunnett’s post-hoc test comparing each of the other groups with (*) GP-alone 1005
treated non-tg mice, (&) GP+RAP/α-syn, or (#) GP-α-syn treatment. N = 6 mice 1006
per group. 1007
1008
Figure 14. Diagrammatic representation of the potential mechanisms 1009
through which combined active and cellular vaccine with GP+RAP/α-syn 1010
might work. We propose that vaccination with GP+RAP/α-syn elicits both 1011
humoral and cellular responses that are neuroprotective. The GP+RAP/α-syn is 1012
internalized by antigen presenting cells (APCs) which in turn cross-talk with 1013
plasma cells and Treg cells to produce respectively anti-α-syn antibodies and 1014
immunomodulatory cytokines (TGFβ1); that in turn switch microglia into a 1015
neuroprotective phenotype. 1016
1017
1
Table 1. Antibody titers in response to combined syn vaccination in tg mice
*Note: Mouse serum was diluted 1: 10,000 for the total IgG and IgG1 analysis and 1: 1,000 for IgG2a.
Mouse serum Total IgG (ng/ml) (average +/- SD)
IgG1 (ng/ml) (average +/- SD)
IgG2a (ng/ml) (average +/- SD)
Negative control 0 0 0 GP α-syn vaccine 2530.4 +/- 881.5 5524 +/- 1707.4 3750 +/-1896.1
GP α-syn + rapamycin vaccine 3965.1 +/- 461.5 4479 +/- 342 3926 +/- 1531.6