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nature research | reporting summary
October 2018
Corresponding author(s):
Viviana Gradinaru
Last updated by author(s):
Jan 26, 2020
Reporting Summary
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Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Me
thods section.
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Confirmed
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) for each experimental group/condition, given as a discrete number and unit of measurement
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ion coefficient)
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value noted
Give P values as exact values whenever suitable.
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, Pearson's
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), indicating how they were calculated
Our web collection on
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Data collection
ImageJ/FIJI 1.51, Image Lab (Bio-Rad) 6.0.1, Python 3.7.2, Zen (Zeiss) 14.0.0.0, Solis (Andor) 4.31.30022
Data analysis
Python 3.7.2 (pandas, seaborn), GraphPad Prism 6, ImageJ/FIJI 1.51, Image Lab (Bio-Rad) 6.0.1
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No datasets were generated or analysed during the current study
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nature research | reporting summary
October 2018
Life sciences study design
All studies must disclose on these points even when the disclosure is negative.
Sample size
The estimated number of animals needed to attain >90% power for each experiment was based on an expected effect size of 0.7 cal
culated
from preliminary experiments comparing wild type to
α
-synuclein overexpressing mice. Power analysis was performed using a SAS based
model for multifactorial ANOVA designs.
Data exclusions
No data points were excluded from statistical analysis. Outliers that were 1.5x outside of the 25th/75th interquartile range we
re not plotted
on graphs for presentation purposes.
Replication
Experiments involving mice were performed using 2+ cohorts initiated at different times. Attempts at replication were successfu
l.
Randomization
For given age-matched animal cohorts, mice were evenly divided between the experimental conditions.
Blinding
Behavioral analysis was performed blinded and condition revealed after analysis was completed.
Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here,
indicate whether each material,
system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the approp
riate section before selecting a response.
Materials & experimental systems
n/a
Involved in the study
Antibodies
Eukaryotic cell lines
Palaeontology
Animals and other organisms
Human research participants
Clinical data
Methods
n/a
Involved in the study
ChIP-seq
Flow cytometry
MRI-based neuroimaging
Antibodies
Antibodies used
1. Mouse IgG2a monoclonal anti-
α
-synuclein phospho (Ser129, Clone81A) (BioLegend MMS-5091, lot #B230550, Duodenum IHC
1:300, Brain IHC 1:500)
2. Rabbit polyclonal anti-
α
-synuclein phosphor (Ser129) (Abcam ab59264, lot #GR52476-60, Western 1:500)
3. Rabbit monoclonal anti-Alpha-synuclein filament antibody [MJFR-14-6-4-2] - Conformation-Specific (Abcam ab209538,
lot#GR293949-11, Dot blot 2ng/mL)
4. Rabbit polyclonal anti-Protein gene product 9.5 (PGP9.5) (Millipore AB1761-I, lot #2581358, Duodenum IHC 1:300, Nodose IHC
1:100)
5. Chicken polyclonal anti-Protein gene product 9.5 (PGP9.5) (ThermoFisher Scientific PA1-10011, lot # TI2631136, Duodenum
IHC 1:300)
6. Chicken polyclonal anti-Glial fibrillary acidic protein (GFAP) (Millipore AB5541, lot # 2728385, Duodenum IHC 1:300)
7. Goat polyclonal anti-Choline acetyltransferase (ChAT) (Millipore AB144P, lot #2620146, Brain IHC 1:500)
8. Rabbit polyclonal anti-Tyrosine hydroxylase (TH) (Millipore AB152, lot #2665965, Brain IHC 1:500)
9. Chicken polyclonal anti-Green fluorescent protein (GFP) (Aves Labs GFP-1010, lot #GFP697986, Brain IHC 1:1000)
10. Rabbit polyclonal anti-Red fluorescent protein (RFP) (Rockland 600-401-379, lot #35868, Brain IHC 1:1000)
11. Rabbit polyclonal anti-GBA1 (Abcam ab175869, lot #GR221342-17, Western 1:1000)
12. Rabbit polyclonal anti-Interleukin 6 (IL6) (Abcam ab7737, lot #GR3185165-1, Western 1:500)
13. Rabbit polyclonal anti-Iba1 (Wako 016-20001, lot # LKH4161, Western 1:1000)
14. Rabbit polyclonal anti-
β
-Tubulin (Abcam ab6046, Western 1:1000)
15. Mouse IgG2b anti-
β
-actin (Cell Signaling 3700, Western 1:1000)
16. AlexaFluor 488 Goat anti-Mouse IgG2a (ThermoFisher Scientific A-21131, IHC 1:300-1000)
17. AlexaFluor 488 Donkey anti-Mouse IgG (ThermoFisher Scientific A-21202, IHC 1:300-1000)
18. AlexaFluor 488 Donkey anti-Chicken IgY (Jackson ImmunoResearch 703-545-155, IHC 1:300-1000)
19. AlexaFluor 555 Donkey anti-Rabbit IgG (ThermoFisher Scientific A-31572, IHC 1:300-1000)
20. AlexaFluor 555 Donkey anti-Goat IgG (ThermoFisher Scientific A-21432, IHC 1:300-1000)
21. AlexaFluor 633 Goat anti-Rabbit IgG (ThermoFisher Scientific A-21071, IHC 1:300-1000)
22. AlexaFluor 633 Goat anti-Chicken IgY (ThermoFisher Scientific A-21103, IHC 1:300-1000)
23. Horseradish peroxidase (HRP)-linked Goat anti-Rabbit IgG (CellSignaling 7074, Western, dot blot 1:2000)
24. Horseradish peroxidase (HRP)-linked Goat anti-Mouse IgG (CellSignaling 7076, Western 1:2000)
25. Horseradish peroxidase (HRP)-linked Goat anti-Mouse IgG2a (Abcam ab97245, Western 1:2000)
Validation
Antibodies were only used if validated by the manufacturer via their website. Most antibodies used are standard in the field an
d
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nature research | reporting summary
October 2018
are cited in the manuscript where relevant:
1. Use of antibody for immunohistochemistry (IHC) was validated in mouse brain tissue in Adamowicz DH, et al. 2017. J.
Neurosci. 37(7):1675-1684.
2. Use of antibody for Western blot was validated by Abcam using brain extracts from transgenic mice overexpressing human
alpha-synuclein at a dilution of 1:1000.
3. Use of antibody for dot blot was validated by Abcam via Professor Paul Henning Jensen against recombinant alpha-synuclein
filaments with 2.2 ng/mL of antibody.
4. Use of antibody for IHC was validated by Millipore using cultured rat cortical cells at a dilution of 1:100.
5. Use of antibody for IHC was validated by ThermoFisher in cortical neuron-glia co-cultures at a dilution of 1:2000.
6. Use of antibody for IHC was validated by Millipore using cultured rat neonatal forebrain cells at a dilution of 1:500 and is
evaluated by Western blot on mouse brain lysates.
7. Use of antibody for IHC was validated by Millipore via D. Anandh, K Shobha and Dr. Bindu M Kutty in rat neocortex and is
evaluated by Western blot on mouse brain lysates.
8. Use of antibody for IHC was validated by Millipore in mouse primary neural cultures at a dilution of 1:1000 and is evaluated
by
Western blot on PC12 cell lysates.
9. Use of antibody was validated by Western blot analysis (1:5000 dilution) and IHC (1:500 dilution) using transgenic mice
expressing the GFP gene product.
10. Use of antibody for IHC was validated by Rockland by staining retina tissue from DsRed transgenic mouse at a dilution of
1:10,000 and by Western Blot for recombinant RFP protein.
11. Use of antibody for Western blot was validated by Abcam using mouse lung lysate at a dilution of 1:1000.
12. Use of antibody for Western blot was validated by Abcam at a dilution of 1:20 and in Cao W, et al. 2018. Front Immunol.
9:647.
13. Use of antibody for Western blot was validated by Wako via Sanagi,T., Ichinohe, N., and Kohsaka, S using purified Iba1
protein and rat microglia at a dilution of 1:1000.
14. Use of antibody as a loading control for Western blot was validated by Abcam using HeLa cell lysate, A431 cell lysate, MCF7
cell lysate, and 293 cell lysate at a dilution of 1:500.
15. Use of antibody as a loading control for Western blot was validated by Cell Signaling using various cell types at a dilutio
n of
1:1000.
16. Use of antibody for secondary detection in IHC was validated by ThermoFisher in HeLa cells at a concentration of 1
μ
g/mL.
17. Use of antibody for secondary detection in IHC was validated by ThermoFisher in MCF-7 cells at a concentration of 0.2
μ
g/
mL.
18. Use of antibody for secondary detection in IHC was validated in Durand de-Cuttoli R, et al. 2018. Elife. pii: e37487 at a
dilution of 1:1000.
19. Use of antibody for secondary detection in IHC was validated by ThermoFisher in HeLa cells at a concentration of 4
μ
g/mL.
20. Use of antibody for secondary detection in IHC was validated in Sauvegarde C, et al. 2016. PLoS One. 11(10):e0165898 at a
dilution of 1:500.
21. Use of antibody for secondary detection in IHC was validated by ThermoFisher in HepG2 cells at a concentration of 4
μ
g/mL.
22. Use of antibody for secondary detection in IHC was validated in Chen Z, et al. 2013. J Neurosci. 33(19):8336-51 at a diluti
on
of 1:2000.
23. Use of antibody for secondary detection in Western blots was validated in Shaw JH, et al. 2018. Front Cell Infect Microbiol
.
8:415 at a dilution of 1:1000.
24. Use of antibody for secondary detection in Western blots was validated in Jeong JH, et al. 2018. PLoS Biol. 16(4):e2004399
at
a dilution of 1:3000.
25. Use of antibody for secondary detection in Western blots was validated by Abcam at dilutions of 1:2000-1:20000.
Eukaryotic cell lines
Policy information about
cell lines
Cell line source(s)
HEK293T (ATCC, CRL 3216; for virus production)
Authentication
Cell line was authenticated by production of AAV9-CAG-mNeonGreen virus, which was validated in cell culture
Mycoplasma contamination
The cell line was tested negative for mycoplasma
Commonly misidentified lines
(See
ICLAC
register)
No commonly misidentified cell lines were used
Animals and other organisms
Policy information about
studies involving animals
;
ARRIVE guidelines
recommended for reporting animal research
Laboratory animals
C57BL/6N mice (males, 8-10 weeks old or 16 month old); Thy1-
α
-synuclein overexpressing mice (males, 6 month or 12 month
old; generated by crossing female BDF1/Thy1-
α
-synuclein mice heterozygous for transgene to wild type male BDF1)
Wild animals
Study did not use wild animals
Field-collected samples
Study did not use field-collected samples
Ethics oversight
Care and experimental manipulation of animals were in accordance with the National Institutes of Health Guide for the Care and
Use of Laboratory Animals and approved by the Caltech Institutional Animal Care and Use Committee.
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nature research | reporting summary
October 2018
Note that full information on the approval of the study protocol must also be provided in the manuscript.