Journal of Neuropathology & Experimental Neurology Advance Access published September 26, 2016
J Neuropathol Exp Neurol
Vol. 0, No. 0, 2016, pp. 1–10
doi: 10.1093/jnen/nlw087
ORIGINAL ARTICLE
Shared and Distinct Patterns of Oligodendroglial Response in
a-Synucleinopathies and Tauopathies
Zdenek Rohan, MD, PhD, Ivan Milenkovic, MD, PhD, Mirjam I. Lutz, MSc,
Radoslav Matej, MD, PhD, and Gabor G. Kovacs, MD, PhD
Abstract
Pathological protein deposits in oligodendroglia are common but
variable features of various neurodegenerative conditions. To evalu-
ate oligodendrocyte response in neurodegenerative diseases (NDDs)
with different extents of oligodendroglial protein deposition we per-
formed immunostaining for tubulin polymerization-promoting pro-
tein p25a (TPPP/p25a), a-synuclein (a-syn), phospho-tau, ubiquitin,
myelin basic protein, and the microglial marker HLA-DR. We inves-
tigated cases of multiple system atrophy ([MSA] n ¼ 10), Lewy
body disease ([LBD] n ¼ 10), globular glial tauopathy ([GGT]
n ¼ 7) and progressive supranuclear palsy ([PSP] n ¼ 10). Loss of
nuclear TPPP/p25a immunoreactivity correlated significantly with
the degree of microglial reaction and loss of myelin basic prtein den-
sity as a marker of tract degeneration. This was more prominent in
MSA and GGT, which, together with enlarged cytoplasmic TPPP/
p25a immunoreactivity and inclusion burden allowed these disor-
ders to be grouped as predominant oligodendroglial proteinopathies.
However, distinct features, ie more colocalization of a-syn than tau
with TPPP/p25a, more obvious loss of oligodendrocyte density in
MSA, but more prominent association of tau protein inclusions in
GGT to loss of nuclear TPPP/p25a immunoreactivity, were also rec-
ognized. In addition, we observed previously underappreciated oli-
godendroglial a-synuclein pathology in the pallidothalamic tract in
LBD. Our study demonstrates common and distinct aspects of oligo-
dendroglial involvement in the pathogenesis of diverse NDDs.
From the Department of Pathology and Molecular Medicine, Thomayer Hos-
pital, Prague, Czech Republic (ZR, RM), Institute of Neurology, Medical
University of Vienna, Vienna, Austria (IM, MIL, GGK), and Institute of
Pathology of the First Faculty of Medicine of Charles University in
Prague and General Teaching Hospital, Prague, Czech Republic (RM)
Send correspondence to: Gabor G. Kovacs, MD, PhD, Institute of Neurology,
AKH 4J, W€ahringer Gürtel 18-20, A-1097 Vienna, Austria. E-mail:
gabor.kovacs@meduniwien.ac.at
Disclosure/conflict of interest
The authors have no duality or conflicts of interest to declare.
Funding
The study was supported by projects PRVOUK-P26 and 27/LF1/1 from
Charles University in Prague, project OPPK No CZ.2.16/3.1.00/24509,
grant P303/12/1791 of the Grant Agency of the Czech Republic and proj-
ect GAUK 113115 from Grant Agency of Charles University in Prague.
ZR was supported by AVASTipendium for Human Brain from Czech
Alzheimer Foundation-Avast project.
C 2016 American Association of Neuropathologists, Inc. All rights reserved.
V 1
Key Words: Globular glial tauopathy, Lewy body disease, Multiple
system atrophy, Oligodendroglia, Progressive supranuclear palsy,
TPPP/p25a.
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INTRODUCTION
Oligodendrocytes are increasingly recognized as impor-
tant cellular components of the pathogenesis of neurodegener-
ative diseases (NDDs). Apart from their myelinating
functions, oligodendrocytes play essential roles in maintaining
axonal and neuronal metabolism (1–3). In addition, perineuro-
nal oligodendrocytes, provide metabolic support through their
direct contact with neurons (4). Accordingly, dysfunction of
this glioneuronal network may lead to complex pathogenic
events. Importantly, oligodendrocytes show specific reactions
to various CNS diseases (5, 6). Primary dysfunction of oligo-
dendrocytes may lead to neuronal damage and death, e.g. in
the pathogenesis of motor neuron disease (MND) and other
diseases (7–11). Oligodendroglial pathology is underappreci-
ated in the most common NDDs such as Alzheimer disease
and Lewy body diseases (LBDs), including Parkinson disease
and dementia with Lewy bodies. In contrast, oligodendroglial
abnormalities are a major component of the pathology of mul-
tiple system atrophy (MSA) (12), and a recently recognized
entity designated as globular glial tauopathy (GGT) (13).
Indeed, MSA is also considered to be a primary oligodendro-
gliopathy (14). Other NDDs often present with oligodendrog-
lial inclusions, such as the tauopathies progressive supranu-
clear palsy (PSP), corticobasal degeneration, argyrophilic
grain disease (15), or disorders in the spectrum of frontotem-
poral lobar degeneration (FTLD) with TDP-43 pathology (10,
16). Studies on the oligodendroglial response in NDDs have
been hindered by a lack of reliable immunohistochemical
markers of differentiated myelinating oligodendrocytes that
can be used on formalin-fixed and paraffin-embedded tissues.
The tubulin polymerization protein (TPPP) p25a (TPPP/p25a)
(17), also known as p25a (18), has been shown to be expressed
in the nucleus, cytoplasm, and cellular processes of differenti-
ated myelinating oligodendrocytes (19, 20). Using anti-TPPP/
p25a antibodies, the oligodendroglial response has been stud-
ied in multiple sclerosis (MS) (9), temporal lobe epilepsy (11),
MSA (17, 21, 22), and MND (10). TPPP/p25a has been shown
to be present in a-synuclein inclusions in MSA and in some
Rohan et al
cases of MND with TDP-43 pathology (10, 17). Indeed, in
MSA, TPPP/p25a is expressed in the enlarged oligodendrog-
lial cytoplasm and its accumulation has also been suggested to
be the first step in a-synuclein aggregation and inclusion for-
mation (18, 21–23). Moreover, re-localization of TPPP/p25a
immunoreactivity from the nucleus and cellular processes to
the perinuclear cytoplasm has also been described (21, 23). In
MS, increases in TPPP/p25a-immunoreactive (IR) oligoden-
drocytes and accumulation of TPPP/p25a leads to enlarge-
ment of cytoplasm volume that is reminiscent of that seen in
MSA but without evidence for inclusion body formation (9).
This process was observed mostly in the peri-plaque white
matter and was interpreted as an oligodendroglial reaction to a
chronic injury (9). These results emphasize the role of TPPP/
p25a in modulating or promoting an oligodendroglial
response in neurodegenerative conditions. However, observa-
tions are lacking on TPPP/p25a immunoreactivity in tauopa-
thies with oligodendroglial tau pathology and also in the
a-synucleinopathy LBD, in which oligodendroglial a-synu-
clein pathology is considered to be much less significant than
in MSA.
Based on these observations, in the present study we
compared the oligodendroglial response in different NDDs
with oligodendroglial a-synuclein or tau deposits and evalu-
ated its relation to white matter pathology.
MATERIALS AND METHODS
Patient Samples
A total of 37 diseased and 10 control cases were in-
cluded in the study. The neuropathologically confirmed diag-
noses were MSA (n ¼ 10), LBD (n ¼ 10; 6 cases Braak stage
6 and 2 each of Braak stages 4 and 5), GGT (n ¼ 7), and PSP
(n ¼ 10). Control cases (n ¼ 10) were neurologically healthy
individuals without neuropathological evidence for NDDs, in-
flammatory, or vascular diseases. The study was approved by
the ethical committee of the Medical University of Vienna
(Nr. 396/2011).
Immunohistochemistry
Formalin fixed, paraffin-embedded tissue blocks from
the investigated cases were evaluated. The following mono-
clonal (mouse) antibodies were used for immunohistochemis-
try: anti-TPPP/p25a (1:2000; [9]), anti-ubiquitin (Ubi;
1:50000, Millipore, Temecula, CA), anti-tau AT8 (pS202/
pT205, 1:200, Pierce Biotechnology, Rockford, IL), anti-
HLA-DR (1:100, Dako, Glostrup, Denmark), and anti-a-synu-
clein (a-syn; 1:2000, clone 5G4, Roboscreen, Leipzig,
Germany, which is specific for disease-associated forms;
[24]). In addition, polyclonal anti-myelin basic protein
([MBP] rabbit, 1:200; Dako) was also used. A DAKO
EnVision detection kit, peroxidase/DAB, rabbit/mouse (Dako)
was used for visualization of antibody reactions. Double
immunolabeling was performed using monoclonal anti-a-syn-
uclein (5G4), -tau (AT8), and polyclonal TPPP/p25a (1:1000;
[25]). The fluorescence-labeled secondary antibodies were
Alexa Fluor (AF) 555 donkey anti-mouse IgG (1:200, Molecu-
lar Probes, Inc., Eugene, OR) and AF 488 goat anti-rabbit
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(1:200, Molecular Probes, Inc.). The following combinations
were used: polyclonal TPPP/p25a (AF 555)/monoclonal 5G4
or AT8 (AF 488). We evaluated double immunofluorescent la-
beling with a Zeiss LSM 510 confocal laser microscope.
Examined Areas
For each disease, a representative brain area with white
matter a-synuclein and tau oligodendroglial pathology was
selected as follows: 1) MSA, white matter from cerebellar
hemispheres; 2) LBD, white matter tract corresponding to the
pallidothalamic tract in the thalamo-subthalamic area; 3)
GGT, frontal subcortical white matter, and 4) PSP, the internal
capsule at the basal ganglia (striatum). In each of the controls,
cerebellar white matter, the pallidothalamic tract, frontal white
matter, and the internal capsule were selected as reference
areas. It should be noted that oligodendroglial a-synuclein in-
clusions have een seen in the mesencephalon and brainstem of
LBD patients (26). For the purpose of this study, we selected
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white matter areas with a consistent occurrence of a-syn oligo-
dendroglial inclusions (GCIs). Therefore, the first step was to
systematically evaluate the 10 LBD cases. Unexpectedly,
more voluminous oligodendroglial a-syn immunoreactivity in
the pallidothalamic tract (27) was observed compared with
that seen in the substantia nigra (Fig. 1). To confirm this find-
ing, a further 13 cases were evaluated [6 cases Braak 2 and 3
and 7 cases Braak 4–6 (28)]. The finding appeared to be con-
firmed in Braak stages above 3. Because the substantia nigra
showed variable amounts of thin oligodendroglial cytoplasmic
a-synuclein immunoreactivity, we included the consistently
involved pallidothalamic tract for studying the oligodendrog-
lial response in LBD.
Slide Scanning
Stained slides were scanned using a Hamamatsu Slide
Scanner, and from the digital slides 2 different fields were se-
lected. These fields represented 1) areas with the most and the
least prominent a-syn pathology in MSA; 2) immediately sub-
cortical and central subcortical white matter of the temporal
lobe in GGT; 3) the pallidothalamic tract in LBD; and 4) the
internal capsule in PSP cases, respectively. All fields were
viewed at 40 magnification. Based on the exact location of
the fields examined for tau and a-syn pathology, all other sec-
tions stained for TPPP/p25a, Ubi, MBP, and HLA-DR were
selected to represent images of corresponding areas in consec-
utive 4-mm-thick sections.
Immunohistochemistry Assessment
Tau-, a-syn-, and Ubi-IR GCIs were manually counted
from the scanned images by one observer (Z.R.). Oligodendro-
glial inclusions were defined as filamentous, coiled, globose,
flame-like, or dot-like immunoreactivity in the cytoplasm,
which was immediately adjacent to the nucleus of an oligo-
dendrocyte. Double-immunolabeling for TPPP/p25 confirmed
that these morphologies represent oligodendroglial inclusions.
Furthermore, we evaluated white matter areas that did not ex-
hibit astroglial tau pathology (eg white matter tau positive
thorn-shaped astrocytes). TPPP/p25a-IR was evaluated as the
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FIGURE 1. Topographical localization of oligodendroglial a-synuclein pathology in the pallidothalamic pathway in LBD.
(A) Myelin basic protein immunostaining of thalamus and subthalamic area at the level of nucleus ruber. Dotted line represents
the pallidothalamic pathway; dashed line delimits subthalamic nucleus; solid line is in the area of substantia nigra; (asterisk) is in
the area of a “comb system” that contains fibers of striatonigral pathway. (B, C) a-Synuclein immunostain with part of
pallidothalamic tract (black rectangle, B) magnified in (C), where numerous a-synuclein oligodendroglial inclusions and IR
threads are clearly visible. Scale bars: A, B ¼ 5 mm; C ¼ 240 mm.

total number of cells with immunostained nuclei or cytoplasm,
or both. In addition, the density of TPPP/p25a-IR oligoden-
drocytes was assessed in respective areas as the overall num-
ber of TPPP/p25a-IR cells (cytoplasmic and/or nuclear) per
total of four 40 fields of the scanned image (total area ¼ 4 
0.107 mm2 ¼ 0.428 mm2). MBP- and HLA-DR-IR was evalu-
ated using densitometry analysis as described in our previous
study (10). Loss of MBP- and increase of HLA-DR-IR were
considered to be surrogate markers of tissue damage for the
purpose of this study. For the evaluation of colocalizations, us-
ing the 40 objective, we made 5 photos each from 5 non-
overlapping areas in 3 representative cases of each disease
group. We counted the total number of a-syn or tau positive
oligodendroglial inclusions and also those showing colocaliza-
tion with TPPP/p25a (yellow colored). Pooled numbers from
each disease group were used to calculate percentages and en-
tered statistical analysis.
Statistical Analysis
Cases included in the study (diseased n ¼ 37; controls
n ¼ 10) were grouped according to neuropathological diagno-
sis (ie MSA, LBD, GGT, or PSP) (Table). In addition to
comparative analysis of different diseases, based on the obser-
vation that MSA and GGT cases are predominantly character-
ized by oligodendroglial inclusion body pathology, we
grouped them together as predominantly oligodendroglial pro-
teinopathies ([POP] n ¼ 17). PSP and LBD were grouped to-
gether as predominantly non-oligodendroglial proteinopathies
([NOP] n ¼ 20), ie diseases that show less oligodendroglial
protein pathology.
To compare the mean ranks of the variables, the Mann-
Whitney U test was used with a significance level at p < 0.05
and, when significant, followed by effect size (r) calculations
using the formula (r ¼ z冑N), with r < 0.3 considered small, r
¼ 0.3–0.5 as medium, and r > 0.5 as a large effect (29). A
Spearman rank-order correlation test was performed to assess
the relationship between variables; p < 0.05 was considered
significant.
RESULTS
TPPP/p25a Cytoplasmic Immunoreactivity
Pattern Differs between Disease Groups
In all examined cases, we observed TPPP/p25a-IR in
both the cytoplasm and nuclei of oligodendrocytes as well as a
population of oligodendrocytes that had TPPP/p25a-positive
cytoplasm and TPPP/p25a-negative nuclei. In MSA, the cyto-
plasmic TPPP/p25a-IR pattern was often dense, round to oval,
and flame-shaped, which was in addition to strong diffuse cy-
toplasmic IR (Fig. 2A). In GGT, the TPPP/p25a-IR pattern
was similar to that of MSA; however, in some cases we also
observed oligodendrocytes with markedly enlarged
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FIGURE 2. Comparison of TPPP/p25a immunoreactivity pattern with a-synuclein and tau oligodendroglial inclusions.
Immunostaining for TPPP/p25a (A–D), a-synuclein (E, G) and tau AT8 (F, H), in MSA (A, E), GGT (B, F), LBD (C, G), and PSP
(D, H). Compare the oligodendrocytes with loss of nuclear TPPP/p25a immunoreactivity and enlarged TPPP/p25a-IR cytoplasm
(white arrowheads) to oligodendrocytes with TPPP/p25a-IR nucleus and barely visible or rather pale (“empty”) cytoplasm (black
arrowheads). Scale bar in A ¼ 25 mm.

cytoplasmic volume with diffuse and fine granular TPPP/
p25a-IR (Fig. 2B). In LBD and PSP, the TPPP/p25a oligoden-
droglial cytoplasmic IR was more similar to that of the con-
trols, although a few enlarged TPPP/p25a-IR cells were noted
in some LBD cases (Fig. 2C, D).
In MSA, we found variable numbers of mostly flame-
shaped, bullet-shape or globose a-syn-IR GCIs (Fig. 2E) that
often resembled cytoplasmic TPPP/p25a-IR (Fig. 2A). In
GGT, less affected regions showed less globular, dense, and
more finely granular tau-IR GCIs. In severely affected areas,
numerous well-formed globular tau-IR inclusions were ob-
served (Fig. 2F). In LBD, in the pallidothalamic tract, we saw
various, partly coiled-body type, slightly more voluminous ol-
igodendroglial inclusions as well as neuropil thread- and dot-
like IR (Fig. 2G). In PSP, oligodendroglial cytoplasmic tau-IR
inclusions were mostly of the coiled-body type (Fig. 2H).
The IR pattern of TPPP/p25a myelin staining was dif-
ferent among individual disease groups. Instead of a rather
dense, diffuse, and filamentous IR as seen in controls, we ob-
served a more fragmented pattern of TPPP/p25a IR in MSA
and some GGT cases (Fig. 3A, B). LBD and PSP were similar
to controls (Fig. 3C, D). This disruption of TPPP/p25a myelin
background stain was paralleled by a similar MBP-IR pattern
(Fig. 3E, F), whereas in LBD and PSP cases, the fragmentation
of the MBP-IR was not obvious (Fig. 3G, H). However, in
areas with fewer inclusions, the myelin TPPP/p25a- and MBP
IR pattern was indistinguishable from that in controls (Fig. 3I–
P). According to densitometry, loss of MBP-IR was
pronounced in POP cases (MSA plus GGT), vs controls
(p ¼ 0.018, r ¼ 0.4). This was not significant when we com-
pared the pooled group of LBD and PSP (NOP cases) with
representative controls (p ¼ 0.211). On the other hand, when
compared with respective controls, we found a significant loss
of MBP-IR in LBD cases in the area of the pallidothalamic
tract (p ¼ 0.004, r ¼ 0.7), as well as in MSA (p ¼ 0.035, r ¼
0.5), although not in GGT (cases with variable number of in-
clusions in the examined region included) and PSP (p ¼ 0.417
and 0.211, respectively).
Double immunolabeling revealed that the protein inclu-
sions are associated with oligodendrocytes in the white matter
(Fig. 4). Significantly higher numbers of cells showed redistribu-
tion of TPPP/p25a into a-syn inclusions in both a-synucleinopa-
thies pooled when compared with that of tau-IR inclusions and
TPPP/p25a in tauopathies (GGT and PSP) (p < 0.001; r ¼ 0.6).
Both MSA and LBD showed more prominent colocalization of
TPPP/p25a- and a-syn-IR inclusions when compared separately
to GGT and PSP (p < 0.002; r ¼ 0.4–0.7 for all except LBD/
GGT comparison where p ¼ 0.044; r ¼ 0.4). The percent of
colocalization did not differ significantly between a-synucleino-
pathies (MSA and LBD; p ¼ 0.66), but was significantly differ-
ent between tauopathies (GGT > PSP; p < 0.001; r ¼ 0.6).
Excessive Loss of TPPP/p25a Nuclear Staining
Characterizes Tract Degeneration
In the next step we focused only on nuclear TPPP/p25a-
IR. In all diseased cases pooled together, we observed that loss
of nuclear TPPP/p25a-IR was more evident than in control

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FIGURE 3. Patterns of white matter changes in the examined diseases.Disintegration of TPPP/p25a immunoreactivity of the
myelin in MSA (A) and GGT (B) with its relative preservation in LBD (C) and PSP (D) vs controls (I–L). There are analogous
changes in the myelin basic protein immunoreactivity pattern in MSA (E) and GGT (F) as compared with LBD (G), PSP (H) and
controls (M–P). Scale bar in A 5 100 mm.

cases (p < 0.001, r ¼ 0.6). When correlated with markers of
tissue damage, ie loss of MBP-IR and increased HLA-DR den-
sity, the number of oligodendroglia without TPPP/p25a nu-
clear IR was negatively correlated with MBP density in all dis-
eased cases (R ¼ 0.455, p ¼ 0.007) and positively correlated
with HLA-DR density (R ¼ 0.394, p ¼ 0.023).
In both MSA and GGT we found a positive correlation
between the number of TPPP/p25a-negative nuclei and
HLA-DR density (R ¼ 0.850, p ¼ 0.004 and R ¼ 0.899, p ¼
0.015, respectively), which was paralleled by an inverse cor-
relation between HLA expression and the number of TPPP/
p25a-IR nuclei (R ¼ 0.895, p ¼ 0.001 and R ¼ 0.829, p
¼ 0.015, respectively). However, these correlations were
not significant in either LBD or PSP. Moreover, we found a
strong trend for correlation of decreased MBP density and
increased number of TPPP/p25a-negative nuclei in MSA

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FIGURE 4. Double-immunolabeling for TPPP/p25a, a-synuclein and tau. Immunostaining for TPPP/p25a (A, E, I, M), a-
synuclein (B, F) and phospho-tau AT8 (J, N), nuclear staining (C, G, K, O) and merged images (D, H, L, P) in MSA (A–D), LBD
(E–H), GGT (I–L), and PSP (M–P). Weakly stained oligodendroglial nuclei are marked with white arrowheads in K. Scale bar in
A ¼ 15 mm for A-H; I–P ¼ 25 mm.

(R ¼ 0.58, p ¼ 0.07) and GGT (R ¼ 0.51, p ¼ 0.1). More-
over, in the pooled POP group, MBP density negatively and
HLA-DR density positively correlated with loss of nuclear
TPPP/p25a-IR (R ¼ 0.561, p ¼ 0.024 and R ¼ 0.785,
p ¼ 0.001, respectively) and this was not seen in the NOP
group (R ¼ 0.248, p ¼ 0.322). This indicated that the loss
of TPPP/p25a nuclear staining could be considered a marker
of tract degeneration.
We then compared different disease groups and their
representative controls. Loss of TPPP/p25a nuclear staining
was more pronounced in the POP group compared with the
NOP group (p < 0.001; r ¼ 0.6) and their respective controls
(p ¼ 0.001; r ¼ 0.8 and p ¼ 0.022, r ¼ 0.4, respectively). In a-
synucleinopathies the loss of TPPP/p25a nuclear IR was
marked in MSA relative to LBD (p < 0.001; r ¼ 0.8). In tauo-
pathies we did not find any difference in the loss of

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FIGURE 5. Bar chart of relative proportion of TPPP/p25a-
negative nuclei to all TPPP/p25a-IR oligodendrocytes in
examined diseases and compared with controls. GGT,
globular glial tauopathy; LBD, Lewy body disease; MSA,
multiple system atrophy; PSP, progressive supranuclear palsy;
SE: *p < 0.05.
TPPP/p25a nuclear stain between GGT and PSP (p ¼ 0.328).
In addition, there was no difference between MSA and GGT
(p ¼ 0.093) or PSP and LBD (p ¼ 0.094). However, the num-
ber of TPPP/p25a-negative nuclei differed significantly be-
tween controls and MSA and GGT (p < 0.001; r ¼ 0.8 and
p ¼ 0.005; r ¼ 0.7, respectively) and also between controls
and PSP (p ¼ 0.045; r ¼ 0.5), but did not differ significantly
between controls and LBD (p ¼ 0.2) (Fig. 5). It must be noted
that the examined diseases, in particular GGT, contained cases
with low and high numbers of protein inclusions. Correlation
statistics indicated that in GGT the number of TPPP/p25a-
negative nuclei correlated with the number of tau-IR oligoden-
droglial inclusions (R ¼ 0.841, p ¼ 0.036); however, this was
not the case for PSP (R ¼ 0.577, p ¼ 0.104). In a-synucleino-
pathies, the number of a-syn-IR GCIs did not correlate with
the loss of TPPP/p25a nuclear IR in MSA (R ¼ 0.079,
p ¼ 0.829) or LBD (R ¼ 0.403, p ¼ 0.282).
Assessment of Oligodendrocyte Density
The densities of oligodendrocytes with any type of
TPPP/p25a IR did not differ between diseased cases and re-
spective controls. We interpreted this observation to mean that
the clinical-neuropathological heterogeneity of the included
diseased cases with different numbers of inclusions (ie repre-
senting both early and late stage diseases). Therefore, we per-
formed the Spearman correlation test to define whether the oli-
godendrocyte density correlated with markers of tissue
damage (including MBP and HLA-DR density and load of a-
syn-, tau-, and Ubi-IR inclusions). Indeed, but in MSA cases
Oligodendroglia in a-Synucleinopathies and Tauopathies
only, we found a negative correlation between TPPP/p25a-IR
oligodendrocyte density and HLA-DR density (R ¼ 0.8;
p ¼ 0.01).
Inclusion Burden in Examined Diseases
To assess the significance of the inclusion burden in the
evaluated diseases, we counted the Ubi, a-syn, and tau-IR in-
clusions in the previously mentioned selected areas. Using the
Mann-Whitney U test we found that the total number of Ubi-
IR inclusions did not significantly differ between MSA and
GGT (ie POP group; p ¼ 0.315), nor did it differ between
LBD and PSP (ie NOP group; p ¼ 0.631). However, in MSA,
the number of both a-syn- and Ubi-IR inclusions was higher
than in LBD (p ¼ 0.011, r ¼ 0.5 for a-syn and p ¼ 0.002, r ¼
0.7 for Ubi) and, similarly, the number of tau- and Ubi-IR in-
clusions was higher in GGT than in PSP (p ¼ 0.033, r ¼ 0.5
for tau and p ¼ 0.005, r ¼ 0.7 for Ubi).
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DISCUSSION
In this study, we systematically evaluated oligodendrog-
lial pathology in a-synucleinopathies and tauopathies and pro-
vide the following four novel observations: 1) We identified
the loss of nuclear TPPP/p25a-IR as a general marker of tract
degeneration. 2) Oligodendroglial dysfunction in NDDs is
characterized by loss of nuclear TPPP/25a-IR, increased den-
sity of oligodendroglial inclusion bodies, enlargement of the
oligodendroglial cytoplasm with redistribution of TPPP/p25a-
IR to the cytoplasm and inclusions, disintegration of MBP-
and TPPP/p25a-IR of the myelin along with increased density
of HLA-DR and, finally, a gradual decrease in the overall
number of TPPP/p25a-IR oligodendrocytes. Therefore, in the
appropriate context, these features can be regarded as criteria
for significant oligodendroglial degeneration and dysfunction.
3) Our study revealed that MSA and the white matter predomi-
nant form of GGT fulfilled most of the points listed above,
thus supporting the idea that GGT is a morphological tau-
equivalent of a-synucleinopathy in MSA, which has already
been suggested in a comprehensive study on one subtype
(white matter predominant) of GGT (30). 4) Moreover, we
present previously underappreciated oligodendroglial pathol-
ogy in progressed LBD corresponding to the degeneration of
the pallidothalamic tract where a-syn- and Ubi-IR GCIs and
white matter thread IR were consistently found in all exam-
ined LBD cases with Braak stage above 3.
We propose that NDDs (which fulfill most criteria of oli-
godendroglial dysfunction and are characterized by extensive
oligodendroglial pathology with less neuronal and astroglial in-
clusion pathology) should be defined as POPs. In our study,
MSA and 1 type of GGT (13, 30) fulfilled this requirement, in
contrast to NOPs such as LBD or PSP (which were evaluated
in this study) or other diseases such as argyrophilic grain dis-
ease or FTLD-TDP including MND. MSA has been clinically
and neuropathologically recognized for decades (31), whereas
GGT is still regarded as rather rare with variable clinical symp-
tomatology and relatively few cases described to date (13).
Loss of nuclear TPPP/p25a-IR and TPPP/p25a re-
localization from oligodendroglial cellular processes to the
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FIGURE 6. Overview of oligodendroglial response in tauopathies and a-synucleinopathies. Oligodendrocytes react to different
pathogenic mechanisms with loss of nuclear immunoreactivity for TPPP/p25a or enlarged cytoplasmic volume with or without
the development of protein inclusions. Protein inclusions can show intense colocalization with TPPP/p25a or only be related to
TPPP/p25a IR oligodendrocytes without significant colocalization. Our observations in MSA, LBDs, GGT, and PSP reveal that
various combinations of TPPP/p25 a and a-synuclein immunoreactivity can be seen in MSA, followed by the tauopathy GGT.
Note that in MSA and GGT the cytoplasmic volume is also increased showing an oval appearance, whereas cytoplasmic TPPP/
p25 is usually not enlarged in LBD and PSP. The most distinctive and typical TPPP/p25a and a-synuclein or tau IR pattern is
shown in the left with decreasing frequency of patterns to the right.

perinuclear cytoplasm has been observed in other studies that
examined MSA cases (21, 23). Ota et al (21) suggested that
TPPP/p25a re-localization from the nucleus might not neces-
sarily be a general feature of oligodendroglial distress. More-
over, the authors divided oligodendrocytes into 6 types based
on combinations of TPPP/p25a- and a-syn nuclear and cyto-
plasmic IR and concluded that nuclear loss and cytoplasmic in-
crease of TPPP/p25a-IR may precede a-syn accumulation and
thus could signal oligodendroglial distress, which could lead to
inclusion formation (21). This is also supported by the results of
our study and, moreover, we also suggest that this event may be
specific to or at least enhanced in POPs since we showed that
loss of nuclear TPPP/p25a-IR is more pronounced in POPs
(MSA and GGT) vs NOPs (LBD and PSP). In addition, in
MSA and GGT the loss of nuclear TPPP/p25a-IR correlated
with markers of white matter tissue damage. Interestingly, only
in GGT and not in MSA, LBD or PSP we observed positive cor-
relation between the number of tau-IR GCIs and loss of nuclear
TPPP/p25a-IR. This finding suggests a possible toxic role of
tau protein deposits in chronically injured oligodendrocytes. On
the other hand, in MSA not all a-syn IR inclusions might lead
to oligodendrocyte dysfunction or there are different stages of
inclusion development with different effects.
Both MSA and GGT, when compared with other studied
diseases, often presented with TPPP/p25a-IR that resembled
the shape of a-syn- and tau-IR GCIs. Moreover, mainly in
GGT, we observed rather diffuse and finely granular TPPP/
p25a-IR in the expanded volume of oligodendroglial cyto-
plasm compared with the rather dense, inclusion-body-like ap-
pearance of TPPP/p25a-IR in MSA (Fig. 2A, B). A similar
TPPP/p25a-IR morphology has been described in primary
progressive MS, although, it lacks any protein accumulations
or inclusions in oligodendrocytes (9). Therefore, an increase
in cytoplasmic TPPP/p25a-IR alone, without accumulation of
protein inclusions, may represent a nonspecific chronic reac-
tion to oligodendrocyte injury (Fig. 6). During a further patho-
genic step, a specific interaction develops between TPPP/
p25a and neurodegeneration-related proteins leading to inclu-
sion formation. Although many elements of the primary crite-
ria of oligodendrocyte dysfunction are seen in MS, even a
long-standing disease course does not initiate formation of
pathogenic protein inclusions (9).
Our results show that various combinations of nuclear
and cytoplasmic TPPP/p25a associate with protein inclusions,
either showing or lacking colocalization (Fig. 6). TPPP/p25a
often colocalizes with a-syn-IR inclusions in MSA and LBD

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J Neuropathol Exp Neurol • Volume 0, Number 0, Month 2016
and also with tau-IR inclusions in GGT, but less in PSP. The
observation of tau-TPPP/p25a colocalization in oligodendro-
cytes with tau-IR inclusions in GGT also expands the possible
role of TPPP/p25a in the formation of oligodendroglial
inclusion bodies (22), as seen in MSA and also in TDP-43 pro-
teinopathies with MND (FTLD-MND/ALS) (10). The mor-
phological differences of TPPP/p25a immunolabeling and the
higher number of colocalized inclusions in MSA may thus
suggest that the pathophysiological mechanisms related to
TPPP/p25a pathology (i.e. mainly changes in its compartmen-
talization within oligodendrocytes) is probably different be-
tween MSA and GGT, ie TPPP/p25a is more linked to a-syn
than to tau pathology (Fig. 6). This is supported by our obser-
vation that in LBD we also see frequent colocalization, and
also by other studies focusing on MSA and non-GGT type
tauopathies (17, 21, 32). Importantly, in MSA and PSP, oligo-
dendrocyte precursor cells do not harbor inclusions and they
may be able to potentially compensate for the progressive loss
of oligodendrocytes in MSA (33).
TPPP/p25a is also recognized as a constituent of myelin
and we described changes in myelin TPPP/p25a-IR. More-
over, MBP-IR pattern changes have been described in MSA
(34). Here, we confirmed these results and also described simi-
lar changes in the TPPP/p25a-IR myelin pattern in MSA. We
also extended this finding to more progressed cases of GGT,
in which disintegration and overall loss of TPPP/p25a-and
MBP-IR was clearly apparent. In general, we found a signifi-
cant loss of MBP-IR in POPs compared with their respective
controls.
To be able to evaluate progressive changes of oligo-
dendrocytes, we included less severely affected cases.
Accordingly, our finding of relatively stable oligodendro-
cyte density in diseased cases and controls may be attribut-
able to this methodological aspect of the study. However,
this finding may suggest dysfunction of TPPP/p25a-IR oli-
godendrocytes in myelin formation without their significant
loss, a concept recently proposed by Ettle et al (34) in MSA
cases. The finding of a negative correlation between HLA-
DR IR and oligodendrocyte density in MSA cases may sug-
gest a possible microglial reaction to damage to myelin or
oligodendroglial processes or somata. Taken together with
our observations of profound disintegration of the white
matter in advanced MSA and abnormal TPPP/p25a re-
localization in oligodendrocytes, we speculate that micro-
glia probably contribute to ongoing toxic or protective
mechanisms (5). However this was not a consistent feature
in the examined GGT cases.
Another novel observation of this study was the consis-
tent finding of bulky a-syn-IR oligodendroglial cytoplasmic
inclusions in the subthalamic area corresponding to the pallid-
othalamic tract in Braak 3 and higher stages of LBD.
Moreover, we found that in this region, a-syn GCIs tended to
colocalize with TPPP/p25a in oligodendroglial cytoplasm in a
similar way to that seen in MSA and GGT, and also, that their
morphology was reminiscent of a-syn GCIs in MSA. Even
though a-syn-IR GCIs can be variably found in the mesen-
cephalon in LBD cases, they are not a prominent feature of
LBDs (26). Indeed, apart from a-syn-IR GCIs in the pallido-
thalamic tract, we only found scattered, thin, coiled-body-type
Oligodendroglia in a-Synucleinopathies and Tauopathies
TABLE. Case characteristics.
Disease Males Females Mean age (years) Age range (years)
MSA 5 5 65.0 46–76
LBD 8 2 77.3 61–90
PSP 5 5 77.2 59–87
GGT 3 4 73.0 63–81
Controls 5 5 65.5 51–78
GGT, globular glial tauopathy; LBD, Lewy body disease; MSA, multiple system at-
rophy; PSP, progressive supranuclear palsy.
a-syn-IR GCIs in the substantia nigra. Selective oligodendrog-
lial degeneration in the pallidothalamic tract could thus be
involved in motor and working memory alterations such as
inability to initiate movement, which corresponds to exces-
sive GABA-mediated suppression of the thalamus by the in-
ternal pallidum (27). However, the clinical relevance of this
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selective anatomical involvement merits further studies.
Lenticular and thalamic fascicles are very subtle and reliable
placement of tractographic seeds in subthalamic nucleus is
extremely difficult on 3 Tesla MRI scanner. Qualitative as-
sessment of this pathway using higher field strength (ie 7
Tesla) MRI could improve understanding of the role of this
pathway in Parkinson disease. Moreover, we found the den-
sity of MBP in the pallidothalamic tract was decreased in
LBD cases compared with controls. However, the decrease
in MBP density was not significantly associated with either
number of GCIs or the loss of nuclear TPPP/p25a-IR. Strik-
ingly, MBP loss was also significantly associated with the
presence of Ubi-IR GCIs in the pallidothalamic tract of LBD
cases. Even though the oligodendroglial pathology in the
pallidothalamic tract seems to be distinct in LBD compared
with other anatomical regions, not all features of the above
mentioned criteria allow this alteration to be characterized
as a primary oligodendrocyte dysfunction disorder such as
that seen in MSA.
In conclusion, 1) loss of nuclear TPPP/p25a is a marker
of tract degeneration in neurodegenerative conditions; 2) GGT
and MSA share common features such as loss of nuclear
TPPP/p25a-IR, enlargement of cytoplasmic TPPP/p25a-IR,
increased inclusion burden when compared with other dis-
eases of their respective proteinopathy groups, together with
signs of white matter damage; however, 3) GGT and MSA
show distinct features, such as more colocalization of a-syn
than tau with TPPP/p25a, more obvious loss of oligodendro-
cyte density in MSA, but more prominent association of tau
protein inclusions in GGT to loss of nuclear TPPP/p25a-IR,
altogether suggesting different role of TPPP/p25a in the path-
ogenesis of a-synucleinopathies and tauopathies; and finally,
4) we observed a significant oligodendroglial a-syn inclusion
pathology in the thalamic fascicle in LBD, which may be
linked to the motor symptoms associated with Braak 3 and
higher stages of LBDs.
ACKNOWLEDGMENT
The authors wish to thank Thomas Secrest, MSc. for re-
visions on the English version of this article. The authors
9
Rohan et al
wish to thank Jiri Keller, MD, PhD (Na Homolce Hospital,
Prague, Czech Republic) for consultation of MRI imaging of
pallidothalamic tract.
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