Supplementary Methods
MALDI-MSI of tryptic peptides
For MALDI-MSI of tryptic peptides, the brain hemispheres from both genotypes and disease stages (n= 24)
were coronally sectioned at 12 µm thickness (at the level of hippocampus and thalamic regions, between 2.46 to -3,00 mm relative to the bregma and interaural between 1.34 to 0.8 mm), using a Leica CM3050
cryostat (Leica Microsystems GmbH, Wetzlar, Germany) and thaw-mounted on conductive slides (Indium tin
oxide -coated slides, ITO from Bruker Daltonics, Bremen, Germany) using a minimum amount of TissueTek
O.C.T. To avoid systematic experimental errors, the sections from different genotypes but the same
advanced stage time-points were mounted on ITO slides. Sectioned tissues were fixed briefly in cold
acetone and dried for 5 minutes in a speed-vac, prior to washing. In order to fix tissue and remove excessive
salts, the cryosections were washed in 70% EtOH for 45 seconds followed by two 30 seconds washes in
96% EtOH, as described (Mainini et al. 2015). Sections were then dried in a speed-vac for 10 minutes and
dlipidized for 30 seconds with chloroform. Tryptic digestion was performed through the ImagePrep
automated system. Trypsin (200μl) was prepared at a concentration of 100 ng/μL, in a 10 mM NH4HCO3
buffer dissolved in HPLC grade water: acetonitrile (9:1 ratio), and loaded into the ImagePrep™ (Bruker
Daltonics, Bremen, Germany) holder. ImagePrep method for tryptic digestion consisted of 30 cycles of 1.25
second spraying, followed by 45 second of drying. An extra cycle of 60 seconds of drying was performed
after deposition. After trypsin digestion the tissue was incubated at 37°C for 180 minutes in a humidity
chamber moisturized with 50% MetOH. Matrix was sprayed over ITO-coated glass slides at 7 mg/mL
concentration of CHCA in 50% Acetonitrile/ 1% TFA. The sprayed tissue sections were analyzed using a
MALDI-TOF/TOF mass spectrometer (UltrafleXtreme, Bruker Daltonics) equipped with SmartBeam II
Nd:YAG/355 nm laser operating in reflectron, positive mode at 2 kHz with a laser focus down to 70 µm
diameter using the ‘large’ setting. Spectra were acquired in the range of m/z 700-3500 kDa. Prior to image
analysis, each MALDI plate was externally calibrated using a standard protein mix (Peptide Calibration
Standard II, m/z range ~1-3000 Da, Bruker Daltonics). For MSI data acquisition, 1000 shots were summed
per array position with a spatial resolution of 150 µm x 150 µm. Preparation of sequence, acquisition and
visualization of MSI data was accomplished with FlexControl™ (version 3.4) and FlexImaging™ softwares
(version 3.0, Bruker Daltonics, Bremen, Germany). Spectra pre-processing was achieved with Matlab. All
spectra have been resampled to 10.000 samples with linearly increasing spacing. Savitzky Golay smoothing
was applied three times (once with width of 10 samples and degree of polynomial equal to 1, followed by
smoothing with width of 4 samples and degree of polynomial 1). This smoothing procedure was implemented
in order to remove the high frequency spikes which were present in the raw spectra and remained if a higher
order polynomial was applied. The baseline was removed using cubic spline interpolation, with a window
size of 20 samples. For subsequent peak detection, we utilized Pearson correlation to estimate the
consistency of peaks among samples and set a threshold to avoid inclusion of peaks with too low intensity
which may correspond to noise. Pearson correlation coefficient was calculated for every pair of masses
generating a 10.000 x 10.000 matrix. We defined the correlation threshold as the mean of this matrix plus 5
standard deviations from the mean. The intensity threshold was set as a median of the spectrum plus half
(0.5) standard deviation.
Supplemental Figures legends
Supplemental Figure S1. Analysis of the differentially expressed proteins in the Ppt1-/- thalamus at the
symptomatic stage. Proteins that were differentially expressed in the Ppt1-/- thalamus were linked using
Ingenuity pathways (IPA) algorithms. Network 1 linked 35 molecules including 17 differentially expressed
proteins while the second network linked 35 proteins including 10 differentially expressed ones. The
networks with the highest IPA scores are portrayed. Protein families’ annotation: rhomb- enzyme, trianglekinase, horizontal oval- transcriptional activator, vertical oval- transmembrane receptor, pentagontransporter, circle- other. Green coloring- proteins with down-regulated expression, in red- proteins with upregulated expression. The different shades in coloring portray changes in protein expression from less to
more down-(up)-regulated. For details see Online Resource 7, Supplemental Table S7.
Supplemental Figure S2. Interaction network linking differentially expressed proteins at the
symptomatic stage. Differentially expressed proteins in the thalamus of the Ppt1-/- mice were connected
using multiple interaction data and filtered for Gene Ontology biological process participation. Using these
criteria 88 proteins were connected. A) The regulation of vesicle-mediated transport (P= 6.58E-9) linked 14
nodes of the network including 5 differentially expressed ones and PPT1. Inset presents a 3-node functional
module formed by PPT1 interacting partner Atp5b/ATP5B (Lyly et al. 2008; Scifo et al. 2015a; Scifo et al.
2015b), Atpa1/ATPA1 and Atpaf1/ATPAF1. The down-regulation of Stx1a/STX1A and Vamp2/VAMP2 axis
(Kim et al. 2008) is confirmed in this study and depicted in the upper inset. B) Synaptic vesicle transport (P =
6.03E-7) encompassing 8 molecules is depicted. The functional attributes of the network nodes including
being parts of PPT1, CLN3/CLN5 interactomes or lipofuscin and myelin proteomes, and those validated by
other methods are portrayed (for details see Online Resource 2 and 5, Supplemental Tables S2 and S5).
Supplemental
Figure
S3.
Immunohistochemical
analysis
of
glial
acidic
fibrillary
protein
immunoreactivity in the thalamus. The immunoreactivity with Gfap/GFAP- specific antibodies was studied
at symptomatic and advanced stages, respectively. A strong up-regulation of immunoreactivity specifically in
GFAP-positive astrocytes of Ppt1-/- thalamus can be observed. Scale bars- 200 µm and 50 µm respectively.
Supplemental Figure S4. Representation of Signaling by Rho Family GTPases pathway affected in
CLN1 deficiency. A) The dysregulation of this signaling pathway at the symptomatic and advanced stages
in the Ppt1-/- thalamus is depicted. Differentially up-regulated proteins are shown in magenta while downregulated are illustrated in light green. The dysregulated genes associated with this pathway, which were
previously determined by microarray profiling (Jalanko et al. 2005; von Schantz et al. 2008) are also listed.
B) Transcriptomic analysis in fibroblasts derived from two CLN1 disease patients, with missense mutation
p.(Leu222Pro; patient 1) (Simonati et al. 2009) and single adenine insertion p.(Met57Asnfs*45; patient 2)
(Santorelli et al. 1998) validated the down-regulation of the same pathway (respective Z scores= -1.941 with
15 molecules assigned and Z= -2.324 with 16 molecules assigned).
Supplemental Figure S5. Down-regulation of myelin sheath components at the advanced stage. Three
proteins linked to ensheathment of axons, namely claudin 11 (Cldn11/CLDN11), proteolipid 1 (Plp1/PLP1)
and myelin basic protein (MbP/MBP) are strongly down-regulated in their expression in the Ppt1-/- thalamus
at the advanced stage (upper panel). The down-regulated protein CLDN11 forms tight junction complex (P=
E-28), together with other claudins (CLDNs), tetraspanins (TSPANs) and integrin beta 1 (ITGB1), as
predicted by various interaction data (depicted in the lower panel).
Supplemental Figure S6. Depiction of overall average MALDI-MSI spectra measured in wild-type and
Ppt1-/- brain. The MALDI-MSI was performed at the pre-symptomatic, symptomatic and advanced stages,
respectively. Over 900 peaks were detected based on the peak detection algorithm implemented in Matlab.
The spectra were recorded in positive, average mode on UltrafleXtreme spectrometer. Lateral resolution
150µm x 150µm. The details of MSI measurements (see Online Resource 15, Supplemental Material and
Methods).
Supplemental Figure S7. Myelin basic proteins detected in wild-type and Ppt1-/- brain. A) Two different
isoforms of Mbp detected in LC-MSE experiments at the advanced stage. The brain tissue slices were
digested with trypsin, and the digests from selected brain regions, pinpointed by immunohistochemistry
(Figure 8E) were resolved by LC-MSE. The sequences of Mbp peptides detected in these experiments are
underlined. Phosphorylated Tyrosine (T) measured in the LC-MSE experiments (Supplemental Table S13) is
shown in orange. Boxed sequences-peptides detected in MALDI-MSI experiments. In magenta- MbP peptide
(HGFLPR, m/z 726.67) measured in MALDI-MSI experiments of tryptic peptides on tissues, shown in B). The
Mbp peptides were measured in MALDI-MSI experiments in reflectron, positive mode on UltrafleXtreme
spectrometer. For details of the procedure see Supplemental Material and Methods. The mean of peak
intensity is indicated. Relative intensity: dark blue- 0% intensity, red 100% intensity. The maximum peak
intensity in each image was set at 100%. T- Total ion current normalization (TIC).
Supplemental Figure S8. Differentially expressed m/z at the advanced stage. The m/z with differential
expression in the Ppt1-/- brain at the advanced stage measured in MALDI-MSI experiments. 24 m/z with
differential expression were detected at this stage, pointed by the arrows. The spectra were recorded in
positive, average mode on UltrafleXtreme spectrometer. The means of each differential m/z are shown. The
details of differential m/z peak statistics are provided in Online Resource 12, Supplemental Table S12.
Supplemental references
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