healthmedicinet

Adeno-Associated Viral vector production

Recombinant AAV2/9-p.A53T-human-?-syn (AAV-h?-syn) vectors were produced by polyethylenimine
(PEI) mediated triple transfection of low passage HEK-293 T /17 cells (ATCC; cat number
CRL-11268). The AAV expression plasmid pAAV2-CMVie/hSyn-synA53T-WPRE-pA (provided
by Dr V. Baekelandt) was co-transfected with the adeno helper pAd Delta F6 plasmid
(Penn Vector Core, cat # PL-F-PVADF6) and AAV Rep Cap pAAV2/9 plasmid (Penn Vector
Core, cat # PL-T-PV008).

AAV vectors were purified as previously described 11]. Cells are harvested 72 h post transfection, resuspended in lysis buffer (150 mM
NaCl, 50 mM Tris–HCl pH 8.5) and lysed by 3 freeze-thaw cycles (37 °C/-80 °C). The
cell lysate is treated with 150units/ml Benzonase (Sigma, St Louis, MO) for 1 h at
37 °C and the crude lysate is clarified by centrifugation. Vectors are purified by
iodixanol step gradient centrifugation, and concentrated and buffer-exchanged into
Lactated Ringer’s solution (Baxter, Deerfield, IL) using vivaspin20 100 kDa cut off
concentrator (Sartorius Stedim, Goettingen, Germany).

Titrations were performed at the transcriptome core facility (Neurocentre Magendie,
INSERM U862, Bordeaux, France). The genome-containing particle (gcp) titer was determined
by quantitative real-time PCR using the Light Cycler 480 SYBR green master mix (Roche,
cat # 04887352001) with primers specific for the AAV2 ITRs (fwd 5’-GGAACCCCTAGTGATGGAGTT-3’;
rev 5’-CGGCCTCAGTGAGCGA-3’) 12] on a Light Cycler 480 instrument.

Purity assessment of vector stocks was estimated by loading 10 ?l of vector stock
on 10 % SDS acrylamide gels, total proteins were visualized using the Krypton Infrared
Protein Stain according to the manufacturer’s instructions (Life Technologies).

Animals

Experiments were performed in accordance with the European Union directive of September
22, 2010 (2010/63/EU) on the protection of animals used for scientific purposes. The
Institutional Animal Care and Use Committee of Bordeaux (CE50) approved experiments
under the license number 5012099-A (rodents) and 50120102-A (monkeys). Kinematics
experiments were performed under the guidelines established at EPFL. Local Swiss Veterinary
Offices approved all the procedures. Experiments were performed on Sprague Dawley
(53 animals), Wistar (8 animals) and Lewis rats (10 animals) with initial weight of
approximately 200 g and age of 8 weeks. Rats were ordered from Janvier Labs or Charles
River in France. Mouse experiments were performed on C57Bl/6 J (12 animals – ordered
from Charles River laboratories), SAMR1 (19 animals) and SAMP8 (21 animals). Both
SAMR1 and SAMP8 were purchased from the research animal facility of the Barcelona
Science Park (Barcelona, Spain).

Monkey experiments were performed in an Association for Assessment and Accreditation
of Laboratory Animal Care accredited facility following acceptance of study design
by the Institute of Lab Animal Science (Chinese Academy of Science, Beijing, China)
Institutional Animal Care and Use Committee. Thirteen male marmoset monkeys (Callithrix jacchus; Beijing, People’s Republic of China) were housed in 2 primate cages, allowing visual
contacts and interactions with monkeys housed inside the cage. The use of wood chip
or shredded paper litter on the cage floor is a source of environmental enrichment.
Food and water were available ad libitum and animal care was supervised daily by veterinarians
skilled in the health care and maintenance of nonhuman primates.

Rodent experiments

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine-intoxicated mice

Eight- to 10-week-old male C57BL/6 J, SAMP8 and SAMR1 mice received one intraperitoneal
injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-HCl per day (30 mg/kg
free base; Sigma) for five consecutive days. Control mice received saline injections
only. Mice were killed five months after MPTP intoxication. Four to nine mice were
used in each group.

Surgical procedures

All the interventions were performed under full general anesthesia with isoflurane
in oxygen-enriched air (1-2 %). After surgery, rodents were placed in an incubator
for optimized recovery from anesthesia. Rats were injected either bilaterally (kinematic
recordings,) or unilaterally (immunohistochemical investigation and basic motor behavior
(spontaneous locomotor activity and stepping test)) in the SNpc with the AAV-h?-syn
(2 ?l – 7.0 ×10
¹²
vg/ml) or the control AAV-GFP (7.0 ×10
¹²
vg/ml – solely for kinematic recordings). Under isoflurane anesthesia, rats were placed
in a stereotaxic frame (Kopf Instruments) and received two bilateral intranigral injections
(Anteroposterior: ?4.9 and ?5.4; Mediolateral: ± 2.2 and?±?2; Dorsoventral: ?7.8,
in mm from bregma) of either vector, as previously described 13]. Mice were injected unilaterally with the AAV-h?-syn (120 nl – 7.0 ×10
¹²
vg/ml) in the right SNpc (coordinates from Bregma: AP?=??2.9, L?=??1.3, DV?=??4.5).
Viruses were injected with a glass pipette at 0.4 ?l.min
^?1
and the pipette was left in place for 4 min after injection to avoid leakage.

Kinematic Recordings

All procedures used have been detailed previously 14]. Briefly, whole-body kinematics were recorded using the high-speed motion capture
system Vicon (Vicon Motion Systems, UK), combining 12 infrared cameras (200 Hz). 4 mm
reflective markers were attached bilaterally overlying the iliac crest, the greater
trochanter (hip), the lateral condyle (knee), the malleolus (ankle), and the base
of the metatarsal phalageal joint (MTP). 3D position of the markers was reconstructed
offline using Vicon Nexus software (1.8). The body was modeled as an interconnected
chain of rigid segments, and joint angles were generated accordingly. For subsequent
kinematic analysis, only hindlimb and parameters related to the trunk were analyzed.
Parameters (Additional file 1: Table S1) describing gait timing, joint kinematics, limb endpoint trajectory, and
trunk stability were computed for each gait cycle using custom written MATLAB scripts
and according to methods described previously 14].

Behavioral Tasks

Spontaneous locomotor actimetry

Spontaneous locomotor activity was evaluated in cages similar to home cages (35x25x25
cm) flanked with photobeam to allow computerized counting of horizontal beam breaks
(protocol adapted from 15]. After a 3 h habituation session, each subsequent recording sessions (i.e., Bsl,
4, 8, 12, 16 weeks) lasted 3 h.

Stepping test

Forelimb akinesia was assessed using stepping 13], 16]. Briefly, animals were gently held and conducted over a 90 cm distance to allow forehand
followed by backhand steps count. Left and right limb performances were successively
evaluated over 2 daily sessions on 3 consecutive days. The average number of left/right
forehand steps was averaged over the 6 sessions for each time-point.

Kinematic recordings

Rats were handled and trained during one week prior to surgery in order to accustom
them to the two locomotor tasks. After AAV-h?-syn injection surgery animals were trained
2–3 times per week and monitored visually to assess whether PD-like symptoms had developed.
Out of 8 rats initially injected 6 displayed observable bradykinesia and were thus
included in the functional analysis. Symptoms emerged at different timepoints for
the individual rats (8–16 weeks post injection) and the recordings used for analysis
are from these chronic timepoints. Due to improved functional performance with training
animals were only trained once a week after symptoms had emerged. Overground and ladder
locomotion were recorded on different days. One rat was not included in the ladder
analysis since not enough functional steps could be recorded from this animal in this
task. AAV-h?-syn rats were compared to a set of healthy rats that were extensively
trained to perform both locomotor tasks.

Overground locomotion was tested on a 15 cm wide and 120 cm long, elevated runway.
10–15 steps per side were analyzed per rat. A total of 89 kinematic parameters were
computed and included in the subsequent PC analysis for this task (Additional file
1: Table S1).

Crossing of an elevated horizontal ladder (rung spacing: 5.2 +/? 0.3 cm) was tested
in alternation with overground walking. 10–15 steps per side were analyzed per rat.
Quantification of hit, slip or miss paw placement was assessed from slow motion videos
acquired at 100 Hz.

Principal Component Analysis

Behavioral data was analyzed by PC analysis 17]. Not all the steps are depicted in the figures.

Step 1: Continuous locomotion is recorded using a high-resolution kinematic system.

Step 2: Custom-written MATLAB scripts are applied to reconstructed kinematic data
in order to compute basic parameters and highly elaborated variables that provide
a comprehensive quantification of gait features. All variables computed are specified
in Additional file 1: Table S1. Approximately 10–15 steps were extracted per rat and experimental condition.

Step 3: The matrix combining all values of variables from all rats and steps was then
subjected to a PC analysis. For this purpose, we used the correlation method, which
adjusts the mean of the data to 0 and the SD to 1. This method allows the comparison
of variables with disparate values (large versus small), and/or different variances.
The result of the PC analysis is a new set of synthetic uncorrelated variables, i.e.,
the PCs, which each explains the maximum possible amount of variance.

Step 4: The new coordinates of gait patterns along each PC, termed PC scores, are
extracted for each rat. PC scores are used to represent gait patterns in the “denoised”
PC space to visualize differences between mice and experimental conditions.

Step 5: PC scores are averaged for each experimental condition and represented in
histogram plots to identify the type of information differentiated along each PC axis.

Step 6: Each PC is a linear combination of the original parameters with appropriate
weights, which are termed “factor loadings.” The values of factor loadings range from
?1 to 1, and correspond to correlations between original parameters and a given PC.

Step 7: Factor loadings with a high value (|factor loading|??0.5) are extracted,
color-coded based on their correlation value, and regrouped into functional clusters
based on the type of gait control aspects they refer to. This process leads to an
objective extraction of the most relevant behavioral parameters to account for the
effects of a specific experimental condition (Additional file 2: Figure S6).

Step 8: To provide a more classical representation of the observed effects, relevant
parameters representative of functional clusters are extracted and represented in
histogram plots.

Primate experiments

Surgery

Eight two-years-old (young group) and five six-years-old (old group) common marmosets
(Callithrix jacchus) were unilaterally injected with 4 ?L AAV-h?-syn in 2 points of the SNpc following
electrophysiological recordings (AP: +4 and +3.3 mm, L: +3.7 and +3.5 mm, D: +15 mm
from interaural line). Recording system: 16 channel wireless system, Multichannel
Systems, Reutlingen, BW, Germany). Animals were anaesthetized with atropine SO
₄
at 0.04 mg/kg, i.m. prior to preparation for surgery. At least 10 min later, the animals
were anaesthetized with ketamine HCl at 10 mg/kg, IM. Following stereotaxic injections,
viral expression was allowed for the next eleven weeks.

Post-mortem processing

At the end of different experiments, animals were killed by sodium pentobarbital overdose
(150 mg/kg, i.v.), and perfused transcardiacally with 0.9 % saline solution (containing
1 % heparin) followed by 4 % PFA performed in accordance with accepted European Veterinary
Medical Association guidelines. Brains were removed quickly after death and post-fixed
overnight in the same fixative, then cryoprotected in PBS containing 20 % sucrose
and frozen by immersion in a cold isopentane bath (?45 °C), before being stored at
?80 °C until sectioning. Brains were sectioned in a Leica CM3050S cryostat (Leica
Microsystems, Wetzlar, Germany) at ?20 °C. Brains were cut at 50 ?m-thick sections
and both striatal and SNpc levels were collected.

Immunohistochemistry

Extent of lesion

To assess the integrity of the nigrostriatal pathway, tyrosine hydroxylase (TH) immunohistochemistry
was performed on striatal and SN free-floating sections (Additional file 3: Table S2). On the rat experiment, dopamine transporter (DAT) immunochemistry on
striatal sections was also done. Briefly, striatal sections from three representative
levels (anterior, medial and posterior) were incubated with a mouse monoclonal antibody
raised against human TH (Millipore, MAB318, 1/5 000) or rabbit polyclonal antibody
raised against DAT (18], 1/5 000) for 72 h at room temperature. The staining was revealed by a specific peroxidase
EnVisionâ„¢ system (mouse or rabbit HRP EnVisonâ„¢ kit DAB+ DAKO, K4007 or K4011) followed
by DAB visualization. Midbrain sections containing the SNpc were processed for tyrosine
hydroxylase. Serial Free-floating sections were incubated in mouse monoclonal TH antibody
(Millipore, MAB318, 1/5 000) for one night at room temperature and revealed by an
anti-mouse peroxidase EnVisionâ„¢ system followed by DAB staining. SN free floating
sections are mounted on gelatinized slides, counterstain with 0.1 % cresyl violet
solution, dehydrated and coversliped while striatal sections are mounted on gelatinized
slides and coversliped only.

Human ?-syn expression

Human ?-syn expression levels were revealed in the striatum (anterior, median and
posterior part) and in the SNpc by immunohistochemistry (Additional file 3: Table S2). The selected sections of the striatum, and serial free-floating sections
of the SN were incubated with a mouse monoclonal antibody raised against human ?-syn
(clone syn211 Thermo Scientific, MS1572, 1:1000) for one night at room temperature
and revealed by an anti-mouse peroxidase EnVisionâ„¢ system (DAKO, K4007) followed by
DAB incubation. Phosphorylated synuclein immunochemistry was investigated in the substantia
nigra in mouse and marmoset experiments. Briefly, serial sections of the SN were incubated
for 3 nights at room temperature in a rabbit anti p-Syn (Abcam AB51253 – 1/500) and
revealed with the rabbit HRP EnVision polymere systemâ„¢ followed by DAB as substrate.
Free-floating sections were mounted on gelatinized slides, dehydrated and coverslipped
before further analysis.

Fluorescence co-localization

Co-localization of Ubiquitin and TH with ?-syn was revealed by a triple fluorescence
immunohistochemistry within the SNpc of sham or AAV-h?-syn injected rat at 16 week
post-surgery. All sections were individually identified to perform staining with all
sections in the same conditions. After a blocking step in 5 % fat milk, TH was first
revealed with a sheep anti TH (Abcam ?1/1000) for one night followed by a donkey anti-sheep
Alexa 488 (1/500-1 h). The staining was briefly observed under microscope before to
continue with a simultaneous incubation of a mouse anti-?-syn and rabbit anti-Ubiquitin
both diluted at 1/1000 in PBS. Then, the ?-syn signal is revealed first by incubate
sections in a Alexa Cys5 goat anti mouse for 1 h at RT and the double staining TH-
?-syn was observed before the last staining with an anti-rabbit HRP EnVisionâ„¢ system
(DAKO, K4011) followed by an 549 Dylight anti HRP diluted at 1/1000 for 1 h. Free-floating
sections were mounted on slides and coverslipped with fluoromountâ„¢ aqueous mounting
media (Sigma, F4680).

Image analysis

Grey level TH quantification in the striatum

TH level in the striatum is quantified by densitometry. Sections are scanned in an
Epson expression 10000XL high-resolution scanner and images are used in ImageJ software
to compare the grey level in the delineated striatum for each animal.

Stereological assessment of TH positive cells in the SN

TH-positive SN cells were counted by stereology blind with regard to the experimental
condition using a Leica DM6000B motorized microscope coupled with the Mercator Pro
software (ExploraNova, La Rochelle, France).The SN was delineated for each section
and probes for stereological counting were applied to the map obtained (sampling and
size of probes, see Additional file 4: Table S3). Each TH-positive cell with its nucleus included in the probe or intersecting
any of the acceptance lines was counted. The optical fractionator method was finally
used to estimate the total number of TH-positive cells in the SN of each animal.

?-syn and DAT surface quantification in the STR

?-syn expression and DAT staining were quantified in the striatum on high-resolution
images from NanoZoomer 2.0 HT (BIC facility). Each NDPI image obtained was used in
Mercator Pro software (Explora Nova, La Rochelle, France) to quantify the representative
surface of ?-syn or DAT staining within the striatum. Briefly, striata were delineated
and a color threshold was applied for each image using the same parameters allowing
comparison between experimental groups. The surface detected was then compared to
the whole striatum surface in order to obtain a percentage of stained structure.

?-syn and p-?-syn volume quantification in the SN

In order to quantify the expression of ?-syn or p-?-syn in the whole SN, a semi-stereological
assessment was used for each animal. As for the striatal ?-syn quantification, all
slides with serial sections of the whole SNpc were scanned in a NanoZoomer 2.0 HT
(BIC facility). First, NDPI images obtained were analyzed in Mercator Pro software
(Explora Nova, La Rochelle, France) to quantify the representative surface of the
staining in each SN section using a color threshold. Then, surfaces detected were
reported to the sampling scheme (Additional file 4: Table S3) to assess the representative volume of ?-syn or p-?-syn expression for
each SN using the Cavalieri method. Finally the percentage of SN volume stained for
?-syn was used to compare the effect of each treatment.