SLAS Discovery - Special Issue on Advances in MALDI Mass Spectrometry for Drug Discovery - Volume 22 Issue 10 - December 2017 - 1226

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SLAS Discovery 22(10)

Table 1. Immunohistochemistry Analyses of 4L/PS-NA Mice Brain.

GFAP
CD68
p62
APP
αSyn

Olfactory Bulb

Cortex

Thalamus

Midbrain

Cerebellum

Brain Stem

±
+++
+
+
++

+
+++
-
+
+++

+++
+++
++
+++
++

+++
+++
++
+++
++

+++
+++
-
+++
++

+++
+++
++
+++
++

The signal levels of GFAP, CD68, and p62 in each brain region are from the data in Figure 5. APP and αSyn staining data are from previous studies.29,33

distributions of these pathological events are summarized in
Table 1. Enhanced amyloid precursor protein (APP), which
is associated with Alzheimer's disease, and αSyn accumulation, a pathological marker of Parkinson's disease, are
included and reflect data from 21-week-old 4L/PS-NA mice
brains determined previously.29,33

Discussion
HPLC MS/MS is the most widely used method for the
quantification of GSL species in cells or tissues. Although
this approach sufficiently quantifies the level of individual
glycolipid species, without laborious tissue dissections, it is
unable to offer localization information of an analyte of
interest in relation to the tissue pathology.16 MALDI IMS
workflows provide analyte localization information and
have been used previously to image GSL species in GD tissues; however, GD brains have never been assessed.34 To
gain spatial and quantitative information on GSL accumulation in CNS-involved GD brains, a feasibility study using
both MALDI FTICR IMS and LC-ESI-MS/MS was utilized. Simultaneous analysis found the overall level of
GluCer species to be almost four-fold higher in the brain of
a mouse model of GD versus the control. Differential
GluCer species distribution detected by MALDI FTICR
IMS could then be correlated to brain disease pathology
using immunohistochemistry. Several GluCer species had
higher ion intensities in GD brains than in the control,
which may be related to their precursors, ceramides that are
produced by ceramide synthases (CerSs).35 For example,
six CerSs have been identified with different tissue expressions, which, except for CerS3, express in the CNS.35 Each
CerS has a preference for a defined fatty acid; for example,
CerS1 synthesizes Cer d18:1/18:0.36 CerS2 has specificity
for Cer d18:1/22 and C24, and CerS2 mRNA expression is
associated with white matter prevalent in BS and MID.37
Our data for GluCer d18:1/18:0, GluCer d18:1/22, and C24
distribution in the 4L/PS-NA brain are consistent with the
data for CerS1 and CerS2 mRNA distribution in the brain
seen previously, suggesting that spatial changes in GluCer
species in 4L/PS-NA brains may be associated with CerS1
and CerS2 expression and affect regional cellular functions
underlying GD-associated brain disease.

Sulfatides, LacCers, and gangliosides were also assessed
by both platforms. MALDI FTICR IMS identified differential signal intensities of these GSL species in the 4L/PS-NA
brain, which was confirmed by LC-ESI-MS/MS. However,
slight discrepancies were observed for gangliosides that
might be due to the potential disassociation of sialic acid
residues in the MALDI ionization process.31 Elevation of
the gangliosides has been reported previously in the liver
and spleen of GD patients, and thus was not a surprising
finding. However, immunohistochemistry revealed regional
neuropathology (inflammation, autophagy, or defective
protein degradation) in these regions, suggesting that the
lipids potentiate distinct pathologies.38 For example, wholebrain microglial activation determined by positive CD68
signals correlated predominantly with GluCer d18:1/18:0
accumulations across all brain regions. Since brain inflammation caused by lipid accumulation is universal in GSL
storage diseases,39-41 it is likely that the accumulation of
LacCers, gangoliosides, and sulfatides found in 4L/PS-NA
brain leads to brain microglial activation. In the 4L/PS-NA
brain, the thalamus and MID appear to be the most affected
by defective protein degradation (APP and αSyn) and
autophagy (p62). In addition to GluCer d18:1/18:0, longchain GluCer species (d18:1/24:0) were distributed in this
region as well, compared with other regions. It has recently
been demonstrated that reduction of GCase substrate accumulation alleviates αSyn aggregation,11,15 and there is evidence that APP aggregation is associated with GluCer in
4L/PS-NA brains.29 The cells in this region appear to be
more vulnerable to the insults from those lipids, resulting in
defective autophagosome and lysosomal functions of proper
degradation of APP and αSyn. In addition, increased
LacCers specifically in the CB (Fig. 4) might have led to
the degeneration of Purkinje cells observed in 4L/PS-NA
mice.24
Identification of accumulating GSL species with brain
colocalization information as described herein is essential
for understanding the pathological pathways of brain diseases. Using a combined LC-ESI-MS/MS and MALDI
FTICR IMS approach helped us to define regional GSL
species distribution in the context of brain pathology in a
quick and robust manner, and has the potential to offer
greater insight in future studies assessing a larger sample
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