MAIN POINTS
- L-DOPA-induced dyskinesia produces a decrease of the NG2-glia density
in the striatum, an area critical to Parkinson’s disease
pathophysiology LID development.
- The antidyskinetic therapy with doxycycline revealed a robust increase
of the NG2-glia in the dorsomedial, dorso- and ventrolateral striatum
- NG2-glia has a negative correlation with LID score contrasting to GFAP
and OX-42 immunopositive cells.
Abbreviations - 6-OHDA: 6-hydroxydopamine; AIMs: axial, limb
and orofacial abnormal involuntary movements; ALO: global AIMs Score
including all AIM categories: axial, forelimb, and orofacial; DOXY:
doxycycline; GFAP: glial fibrillary acidic protein from astrocyte;
L-DOPA: 3,4-dihydroxyphenyl-L-alanine; LID: L-DOPA-induced dyskinesia;
NG2-glia: Nerve/glial antigen 2 glial cells; OX-42: CD11b/c equivalent
protein of microglia; PD: Parkinson’s disease; SNc: substantia nigra
compacta; TH: tyrosine hydroxylase.
INTRODUCTION (742 words)
Parkinson’s disease (PD) is a neurodegenerative disorder, triggered by
the progressive loss of dopamine-producing neurons in the substantia
nigra compacta (SNc) region of the basal ganglia (Vaillancourt and
Lehericy, 2018). The disease was considered mainly by motor
symptomatology, including resting tremor, bradykinesia, limb rigidity,
and defects in gait and balance (Obeso et al., 2018). Currently,
symptomatic therapies are available, mainly the dopamine replacement
therapy with levodopa (L-DOPA: l-3,4-dihydroxyphenylalanine). L-DOPA
improves motor symptoms, but long-term L-DOPA use leads to the gradual
development of side effects such as on-off fluctuations, abnormal
involuntary movements, and hallucinations (Obeso et al., 2000; Dauer and
Przedborski, 2003). The abnormal involuntary movements termed
L-DOPA-induced dyskinesia (LID), are among the main challenges in
treating PD since they limit the L-DOPA effectiveness.
To date, LID cause is not entirely understood (Fahn et al., 2004; Olanow
et al., 2004; Jenner, 2008). Several studies aimed to find alternative
therapies that reduce LID (Cenci et al., 2020). It was recently
demonstrated the presence of an inflammatory reaction in the brains of
patients exhibiting a history of dyskinesia (Del-Bel et al., 2016; Carta
et al., 2017; Junior et al., 2020). In the PD post mortem brain
patients, the basal ganglia is marked by angiogenesis, vascular
endothelial growth factor up-regulation, and altered brain blood barrier
properties (Ohlin et al., 2011, 2012; Janelidze et al., 2015; Lerner et
al., 2017). Pre-clinical studies showed evidence of an inflammatory
environment in the dopamine depleted striatum, with sustained activation
of astrocytes and microglia and the recruitment of immune elements
contributing to the pathophysiology of LID (Picconi et al., 2002;
Robelet et al., 2004; Meissner et al., 2006; Buck and Ferger, 2010;
Bortolanza et al., 2015a; Del-Bel et al et al., 2016; Guerra et al.,
2019). Supporting the hypothesis, the striatum of lesioned rats long
after the microinjection of the neurotoxin 6-hydroxydopamine (6-OHDA),
receiving L-DOPA treatment, has been revealed a sustained
neuroinflammatory reaction (Spinnewyn et al., 2011; Aron-Badin et al.,
2013; Muñoz et al., 2014; Bortolanza et al., 2015a and b; Ramirez-Garcia
et al., 2016; Teema et al., al., 2016; Boi et al., 2019). Targeting
neuroinflammation may be a strategy to limit LID (Del Bel et al., 2016;
Dos-Santos-Pereira et al., 2016; Carta et al., 2017; Junior et al.,
2020).
A population of dividing glial
progenitors, called NG2-glia, considered the fourth glial type in the
adult central nervous system, has been identified throughout adult brain
parenchyma (Dimou et al. 2008; Richardson et al., 2011). This population
express markers typically found in oligodendrocyte precursor cells
during development such as chondroitin sulfate proteoglycan (Levine et
al., 1998; Peters, 2004; Nishiyama et al., 2009). It has been reported
their potential to generate a wide variety of cell types besides
oligodendrocytes, including astrocytes and neurons (Belachew et al.,
2003; Aguirre and Gallo, 2004; Aguirre et al., 2010; Baracskay et al.,
2007), although the latter is debated (Dimou et al., 2008; Nishiyama et
al., 2009; Zhu et al., 2011; Richardson et al., 2011). Rodent NG2 glia
receives direct synaptic inputs from glutamatergic and GABAergic
neurons, a feature that is unique among glial cells (Bergles, et al.,
2000; Lin & Bergles, 2004). Furthest, it was demonstrated that neuronal
activity promotes the recruitment and differentiation of NG2 cells in
the adult brain, which could contribute to neural plasticity (Gibson et
al., 2014).
NG2-glia reacts to many types of injury or pathological conditions
changing their morphology and proliferation rate. Additionally, NG2-glia
responds to inflammatory cues, exhibiting a behaviour remarkably similar
to microglial cells (Kang et al., 2013; Wang et al., 2017). NG2-glia
surveys their microenvironment through constant filopodia extension
changing their morphology (Nishiyama et al., 1997; Martín-López et al.,
2013; Bribian et al., 2018; Okada et al., 2018). Zhang et al. (2019)
described a downregulation of NG2-glia expression in the SNc of PD
patient brain compared with healthy subjects. Kitamura et al. (2010)
detected activated NG2-positive cells in the nigral 6-OHDA-injected
model but not in the striatum. These findings suggest the dysfunction of
NG2 glia in the PD brain and provide a compelling rationale for
developing new studies.
Here we analyzed the NG2-glia response in the striatum of parkinsonian
rats expressing LID using immunoconfocal morphometry,
immunohistochemistry and immunoblotting. We examined NG2-glia
distribution in the lesioned striatum, the cells’ phenotypic
characteristics, and the association with astrocytes and microglia
cells. Because molecules capable of modulating glial cells’ activation
were effective in resolving LID, we determine the antidyskinetic effect
of doxycycline (6-Deoxy-5-hydroxytetracycline, Bortolanza et al., 2020),
in NG2 expression and NG2-glia activation.
METHODS (2.243 words)
Subjects Adult, male Wistar rats (n=64, 250–300 g, aged 9–11
weeks) were used in this study. Animals were housed in groups of three
per cage, maintained at a temperature of 22–25 °C, on a 12-h light/dark
cycle, and with food and water (autoclaved tap water) available ad
libitum. Studies regarding sex differences, which may produce biological
variables, were not investigated in this study. The experiments were
performed in compliance with the recommendations of the US National
Institutes of Health Guide for Care and Use of Laboratory Animals and
were approved by the Institutional Animal Care and Use Committee at the
University of Sao Paulo (Approval Number: 2017-0014-02). All efforts of
the researchers minimized the animal suffering and the number of animals
used.
Drugs The dose regimen and route of administration of drugs
were based on previously published studies (Cenci et al., 1998; Gomes et
al., 2008; Lazzarini et al., 2013; Padovan-Neto et al., 2015). L-DOPA
(L-3,4-Dihydroxyphenylalanine methyl ester hydrochloride; 20 mg/kg
orally- Prolopa dispersive, Hoffman-LaRoche, Rio de Janeiro, RJ,
Brazil), plus benserazide–HCl (5 mg/kg) were dissolved in water.
Doxycycline (40 mg/kg i. p., Sigma-Aldrich, St. Louis, MO, USA) was
dissolved in saline and administered 30 min before L-DOPA. All drugs and
their respective vehicle (VEH) were freshly prepared before use and
injected in a volume of 1mL/kg.
Parkinsonian Lesion Microinjection of 6-OHDA was delivered into
the medial forebrain bundle as previously described (Gomes and Del-Bel,
2003; Gomes et al., 2008; Padovan-Neto et al., 2009). Animals were
anaesthetized with 2,2,2-tribromoethanol (Sigma-Aldrich, St. Louis, MO,
USA) (250 mg kg-1, i.p.) and fixed into the stereotaxic apparatus for
performing the surgery (David Kopf, model USA, 9:57). Stereotaxic
coordinates were (from bregma in mm: AP = -4.3; LL = -1.6; DV = -8.3)
were based on Paxinos & Watson, (2007). Rats received microinjection of
6-OHDA in a volume of 2μl into the left medial forebrain bundle (6-OHDA
- 2.5 µg µl-1 in 0.9% NaCl supplemented with 0.02% ascorbic acid, 1 μL
min-1). After the microinjection, the cannula was left in place for two
additional minutes to prevent the injected solution’s reflux. At the end
of the surgical procedure, the animals were kept warm by a 60W light
bulb until full recovery from anaesthesia. The dopamine lesion was
confirmed by analysis of tyrosine hydroxylase immunoreactivity (TH-ir)
as described before (Padovan-Neto et al., 2015) in the striatum and SNc
(Fig. 1).