SLAS Discovery - Special Issue on Advances in MALDI Mass Spectrometry for Drug Discovery - Volume 22 Issue 10 - December 2017 - 1224

1224

SLAS Discovery 22(10)

GD/ 4L/PS-NA

Control

5mm

LacCer d18:1/18:0
4L/PS-NA

pmole/mg tissue

200

4L/PS-NA
Control

150
100
50

OB
5.5

CO
2.7

MID
7.0

BS
3.3

CB
12.2 Fold change

m/z 968.570
LacCer d18:1/22:0

LacCer d18:1/20:0

Control

25
8

pmole/mg tissue

4L/PS-NA
Control

6
4
2
0

CO
2.0

MID
6.0

BS
4.6

CB
12.1 Fold change

4L/PS-NA
Control

20
15
10
5
0

OB
3.6

60%

5mm
m/z 940.510

m/z 928.618

LacCer d18:1/22:0 +Na
GD
Control

LacCer d18:1/20:0 +Na
GD
Control

LacCer d18:1/18:0 +K

pmole/mg tissue

OB
2.8

CO
2.7

MID
2.9

BS
2.8

CB
11.9 Fold change

Figure 4. Spatial distribution of LacCer species using MALDI FTICR IMS and quantitation of LacCers by LC-ESI-MS/MS. (A) H&E
stain of 4L/PS-NA and control brain sections analyzed in MALDI FTICR IMS. (B) 4L/PS-NA mice brains had increased LacCer signal
intensity compared with the control, preferentially in the CB. (C) Quantitative analysis of LacCer species by LC-ESI-MS/MS confirms
the difference in signal intensity shown as fold changes of LacCers levels in 4L/PS-NA over the control. n = 2 mice. All intensity values
were scaled at 0%-60%, as indicated by the color bar on the far right.

features of GSL storage diseases, including GD.24,29 It is
also possible that accumulated LacCer may also be a contributing factor to the loss of Purkinje cells in CB observed
in 4L/PS-NA mice and GD type 3 patients.3,16
Saposin B has an in vitro activation effect on sialidase
for the degradation of ganglioside GM3.30 Since the 4L/
PS-NA mice have 45% lower levels of saposin B than the
control mice, the effect of the sialidase on GM3 degradation, along with gangliosides GM1 and GM2, was assessed
(Suppl. Fig. S3). Among the detected gangliosides, the GM
with a C18 fatty acyl chain length was the predominant species identified by LC-ESI-MS/MS. MALDI IMS data
depict GM1 d18:1/18:0 distribution more in the OB than in
other areas, whereas GM3 d18:1/16:0 and d18:1/18:0 accumulations were different across all four regions, which was
consistent with LC-ESI-MS/MS results (Suppl. Fig. S3).
One of the potential issues for imaging gangliosides in tissue using MALDI MS is that sialic acid residues can be
dissociated in the ionization process,31 although this effect
is less when using MALDI FTICR IMS. Discrepancies
were observed in the identification and quantification of
some of these complex GSLs. Saposin B also activates arylsulfatase A, the major enzyme responsible for the degradation of sulfatides.30 Analyses of sulfatide levels by

LC-ESI-MS/MS showed slight increases in the 4L/PS-NA
brain and were more abundant in the MID and BS regions
(Suppl. Fig. S4).32 MALDI FTICR IMS and LC-ESI-MS/
MS also identified increased sulfatide d18:1/24:1 in the
MID and BS regions of the 4L/PS-NA brain.

GD Mice Brain Pathology Associated with GSL
Accumulation
Brain pathology was assessed by immunohistochemistry
using an anti-GFAP astrocyte marker, anti-CD68-activated
microglial/macrophage marker, or p62 autophagy marker
(Fig. 5). Strong GFAP signals were detected predominantly
in the thalamus within MID, BS, and CB regions of the 4L/
PS-NA brain compared with the CO and OB of control mice
and likely demonstrate astrogliosis indicative of inflammation (Fig. 5B,C). CD68-positive cells represent active
microglials/macrophages, which were detected uniformly
across the brain regions of 4L/PS-NA mice, signifying
ubiquitous brain inflammation (Fig. 5B,C). p62 is an
autophagosome marker, which, in comparison with the control mice, was elevated in the OB, MID, thalamus, and BS
regions of the 4L/PS-NA mouse brains, suggesting enhanced
autophagy occurring in these selected regions. Regional

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