Double labelling of NG2-glia and astrocytes or microglia
in the striatum of 6-OHDA-lesioned parkinsonian rats treated with L-DOPA
and/or doxycycline
We performed a double immunofluorescence reaction to determine the
presence and/or co-localization of the astroglial protein GFAP, the
microglial protein OX-42 and the NG2-antigen in the striatum from
6-OHDA-injected animals and those presenting LID. The yellow
pseudo-color for these structures visible in microscopic images
identified the superimposition of NG2-glia immunoreactivity (green) on
either GFAP or OX-42 positive immunoreactivity (red) cell body/process
and vice versa .
Similar to Bortolanza et al., (2015), the dopamine-depleted striatum of
rats under L-DOPA treatment (6OHDA+Vehicle+L-DOPA ,
p<0.001, Table 1) had a prominent GFAP and OX42
immunoreactivity (6OHDA+Vehicle+L-DOPA , p<0.05, Table
1). Doxycycline administered before L-DOPA reduced LID and also GFAP and
OX42 immunoreactivity in the depleted striatum
(6OHDA+DOXY+L-DOPA , p<0.001, Table 1).
High resolution in single plane image showed an extensive
interdigitation of the processes deriving from astrocytes/microglia and
NG2-glia. The fine processes of NG2 (green) and astrocytes (red) or
microglia (red), characteristically shown as irregular shaped dots,
closely associated in tiny points (yellow) of all subdivisions of
striatum (Fig. 4, 5A-D). There was no effect of the lesion or the
treatment in the measured parameters of the microglia and astrocytes
markers.
DISCUSSION (1.002 words)
Our data revealed (i) a slight increase in the immunoreactivity of
NG2-glia in the lesioned dorso- and ventrolateral striatum 36 days after
the 6-OHDA-microinjection; (ii) a robust decrease of NG2-glia
immunoreactivity in the dorsolateral, dorso- and ventromedial striatum
of rats presenting LID with NG2-glia density negatively correlated with
LID score; (iii) a robust increase of NG2-glia density in the
dorsomedial, dorso- and ventrolateral striatum following doxycycline
antidyskinetic therapy, in contrast to a decrease in the GFAP and OX-42
immunoreactivity. The morphometric analysis of the striatum of the
animals expressing LID revealed (iv) an increase in the NG2-glia
indicators of activation in the dorso- and ventromedial striatum with a
decrease in the dorsolateral one. The aforementioned results revealed:
(v) a further increase in the activation cells parameters only in the
dorsolateral striatum, associated to doxycycline antidyskinetic effect;
(vi) a decrease in the NG2-glia indicators of activation in the dorso-
and ventromedial striatum. The no-lesion animals and the only 6-OHDA
lesioned rats receiving vehicle or doxycycline exhibited: (vii) a
NG2-glia morphological features of a typical resting cell; (viii) a
NG2-glia immunoreactivity decrease in the dorsal and ventromedial
striatum. The results provide evidence of the dynamic involvement of
NG2-glia in the dyskinesia induced by L-DOPA chronic treatment of
parkinsonian rats.
One of the remarkable findings of the present study is that NG2-glia
expression decreases in the dorsal striatum of animals’ disclosing LID.
The dorsal striatum receives dense dopaminergic innervation from the SNc
and the lateral portion of the ventral tegmental area (Haber et al.,
2000; Ikemoto, 2007; Lerner et al., 2015), strong projections from
somatomotor cortical areas and lateral thalamic motor nuclei (Smith, et
al., 2004). Independently, controlled information flows from the SNc to
the dorsolateral and dorsomedial striatum (Lerner et al., 2015). The
dorsolateral striatum presents a sensorimotor role, controlling
voluntary movement severely compromised in PD (Flaherty and Graybiel,
1994).
The doxycycline antidyskinestic action increased NG2-glia cell density,
with activated phenotype. In this study, we did not measure L-DOPA
plasmatic concentrations. Therefore, a potential doxycycline
interference on L-DOPA gastrointestinal absorption cannot be ruled out.
However, this is an unlikely possibility because previous research from
our group (Bortolanza et al., 2020) provide evidences of no drug
interference with the positive motor effects of L-DOPA and the
anti-dyskinetic effects of doxycycline were still present when L-DOPA
was administered subcutaneously. The large majority of NG2-glia in the
adult brain is maintained in a quiescent state under physiological
conditions (Hughes et al., 2013). Phenotypically, NG2 glia provides a
stereotypic reaction with increased NG2 expression, retraction of cell
processes, cell body swelling, cell proliferation, and migration toward
the lesion site to almost all kind of injury, independently of the
extent of myelin loss (Bedner et al., 2020; Jin et al., 2018; Valny et
al., 2018). A neuromodulatory mechanism has been proposed to be related
to NG2-glia reactivity (Levine et al., 2016). A co-culture of primary
NG2-glia with damage hippocampal slices activated NG2-glia, produced
neurotrophic factors and immunomodulatory action by creating
anti-inflammatory cytokines as interleukin-10 and transforming growth
factor (TGF) β2 (Sypecka and Sarnowska, 2014).
PD preclinical studies evidenced NG2-glia as a protector factor against
neuroinflammation and dopaminergic neuron apoptosis (Kitamura et al.,
2010; Zhang et al., 2019). The NG2-glia ablated mouse brain exhibited
increases in pro-inflammatory response subsequent exposure to endotoxin
lipopolysaccharide (Zhang et al., 2019). An enriched environment with
physical activity induces increased numbers of newborn NG2-positive and
GFAP-positive cells in the adult SN and improves motor behavior function
in the 6-OHDA rat model of PD (Steiner et al., 2006; Kirby et al.,
2019). There are however, contradictory observations. Nakano et al.
(2017) and Zhang et al. (2019) found that NG2-glia ablation exacerbated
dopaminergic neuronal cell loss in a mouse PD model, induced
neurodegeneration, microglia activation, and neuroinflammation in the
adult hippocampus. In the post-mortem brain specimens from patients with
Alzheimer’s disease, there is a reduction in NG2-glia immunoreactivity,
which is negatively correlated to microglial immunoreactivity (Nielsen
et al., 2013), but associated with the amyloid-β plaques (Fiedorowicz et
al., 2008; Zhang et al., 2019). As shown in amyotrophic lateral
sclerosis models NG2-glia has been observed either to limit central
nervous system damage or to actively contribute to neuroinflammation or
neurotoxicity (Kang et al., 2013).
Consistent with the notion that NG2-glia, microglia, and astrocytes are
distinct glial cells subpopulations NG2/GFAP or NG2/OX42 staining was
not once found to be co‐localized on the same cell. Noteworthy, the
number of NG2/GFAP or NG2/OX42 staining superimposition points was not
altered by the factors lesion or treatment. An interaction was
demonstrated between NG2 glia and astrocytes (Hamilton et al., 2010; Xu
et al., 2014), microglia (Nishiyama et al., 1997), and neurons
(Maldonado and Angulo, 2015). This anatomical relationship is a basis
for the coupling and functional communication between the glial
subtypes. Further, NG2-glia is a source of neurotrophic factors in the
central nervous system (Bankston et al., 2013). Liu and Aguzzi (2020)
demonstrated that NG2-glia and microglia interaction played pivotal
roles in regulating microglia states in the adult mouse brain in
Alzheimer model disease. Astrocytes signal to NG2-glia via the release
of ATP and glutamate that evokes a Ca2+ rise in NG2-glia (Hamilton et
al., 2010). Considering that the TGFβ pathway is essential for normal
microglia development (Butovsky et al., 2014; Krasemann et al., 2017),
TGFβ pathway suppression may be associated with microglia dysfunction.
Whether the NG2-glia function contributes to the disturbing microglia
and astrocyte signature in the dorsal striatum of rats presenting LID is
not determined yet.
A limitation of the present study was the performance only in male rats
to avoid gender influence in the results. It is important to highlight
that the analysis of dyskinesia induced by L-DOPA was originally
standardized in female rats (Cenci et al., 1999).
As aforementioned, there is an association between damaged dopamine
neurons, L-DOPA-treatment, and dysregulated inflammation (Bortolanza et
al., 2015b; Del-Bel et al., 2016; Mulas et al., 2016; Teema et al.,
2016). The hypothesis of NG2-glia decrease inflammatory reaction is
compatible with the observation of the NG2-glia immunoreactivity
decrease in LID, and the increase in the microglia/astrocytes. The
implications of this discovery are far-reaching and needs more
investigation.
SIGNIFICANCE: Our findings provide the first description of the
distribution and morphological changes of NG2-glia in the striatum of
parkinsonian rats presenting LID and the effect of doxycycline therapy.
They support an inverse link between the transformation of NG2 glia to
the reactive form and microglial/astrocyte activation/recruitment in a
specific brain region, directly enrolled in PD and the dyskinesia
manifestation. These data could reflect the possibility that NG2-glia
cells promote striatal plasticity as a form of dyskinesia recovery.
Besides, this study may also contribute to establishing NG2-glia as a
novel therapeutic target for LID and so, we believe that our findings
are of great interest to the neuroscience and medical community.
ACKNOWLEDGMENTS The authors would like to thank Célia A.
da-Silva, Sara Saltareli, and Vitor Castania for their technical
assistance.
CONFLICT OF INTEREST EADB is listed as co‐inventor on a U.S.
patent application covering doxycycline and related compounds (Patent
No. EP18306400.5. PCT: 1054). All other co‐authors declare that no
conflict of interest exists.
AUTHOR CONTRIBUTIONS GCN, MB, AB, GCLL performed the
experiments. EDB, LLM, GCN, MB AB contributed to the experimental
design. EDB, LLM contributed with resources. GCN, MB, AB, EDB, LLM wrote
and edited the manuscript. GCN, MB performed data acquisition and
analysis. All authors have approved the final manuscript.
DATA AVAILABILITY STATEMENT: The data that support the findings
of this study are available from the corresponding author upon
reasonable request.
FUNDING STATEMENT: The study was supported by the São Paulo
State Foundation for the Support of Research (FAPESP, Brazil; Grants
2014/25029-4 and 2017/24304-0). EDB is a recipient of grants from the
National Council for Scientific and Technological Development (CNPq,
Brazil). EDB is a CNPq research fellow. MB was recipient of FAPESP,
Brazil, fellowship 2016-06602-0). GCN was recipient of FAPESP, Brazil,
fellowships 2015/03053-3 and 2018/05146-7 and PNPD CNPQ,
88882.317597/2019-01. LLM: Research Grants from the MICINN
(PID2019-105218RB-I00).