Re: 글루탐산 흥분성 독성 .. 성상교세포, 미세아교세포에서 재흡수 장애.. 치매, 파킨슨 치료...nature

[문형철]

글루타민 흥분성 독성은 neuronal cell death를 유발함.

어떻게?

흥분성 물질 글루탐산 재흡수 장애 --> 글루탐산 과잉

mitochondria permeability transition --> secondary 흥분성 독성

--> calcium buffering 손상

--> ROS 생성

Critical Review

Glutamate-induced excitotoxicity in Parkinson's disease: The role of glial cells

Author links open overlay panelL. Iovino a, M.E. Tremblay b, L. Civiero a c

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https://doi.org/10.1016/j.jphs.2020.07.011Get rights and content

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Abstract

Glutamate is the major excitatory neurotransmitter in the central nervous system. Glutamate transmission efficiency depends on the correct functionality and expression‎ of a plethora of receptors and transporters, located both on neurons and glial cells. Of note, glutamate reuptake by dedicated transporters prevents its accumulation at the synapse as well as non-physiological spillover. Indeed, extracellular glutamate increase causes aberrant synaptic signaling leading to neuronal excitotoxicity and death. Moreover, extrasynaptic glutamate diffusion is strongly associated with glia reaction and neuroinflammation. Glutamate-induced excitotoxicity is mainly linked to an impaired ability of glial cells to reuptake and respond to glutamate, then this is considered a common hallmark in many neurodegenerative diseases, including Parkinson's disease (PD). In this review, we discuss the function of astrocytes and microglia in glutamate homeostasis, focusing on how glial dysfunction causes glutamate-induced excitotoxicity leading to neurodegeneration in PD.

글루타메이트는 

중추신경계의 주요 흥분성 신경전달물질입니다. 

글루타메이트 전달 효율은 

뉴런과 신경교세포에 위치한 

수많은 수용체와 수송체의 올바른 기능과 발현에 따라 달라집니다. 

주목할 점은 

전용 수송체에 의한 글루타메이트 재흡수는 

시냅스에 축적되는 것을 방지하고 

비생리적인 파급을 방지한다는 것입니다. 

실제로 

세포 외 글루타메이트 증가는 

비정상적인 시냅스 신호를 유발하여 

신경세포 흥분 독성과 사멸로 이어집니다. 

또한 

시냅스 외 글루타메이트 확산은 

신경아교세포 반응 및 신경염증과 밀접한 관련이 있습니다. 

글루타메이트에 의한 흥분 독성은 

주로 신경교 세포의 글루타메이트 재흡수 및 반응 능력 장애와 관련이 있으며, 

이는 파킨슨병(PD)을 포함한 많은 신경 퇴행성 질환의 공통적인 특징으로 간주됩니다. 

이 리뷰에서는 

글루타메이트 항상성에서 

성상교세포와 미세아교세포의 기능에 대해 논의하고, 

신경교세포 기능 장애가 

어떻게 글루타메이트에 의한 흥분성 독성을 유발하여 

PD의 신경 퇴행을 유발하는지에 중점을 두고 설명합니다.

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Keywords

Parkinson's disease

Glutamate-induced excitotoxicity

Inflammation

Astrocytes

Microglia

Abbreviations

Introduction

In the central nervous system (CNS), the balance between excitatory and inhibitory neuronal connections is crucial for maintaining proper function. The majority of excitatory signals are mediated by glutamate, which is the predominant neurotransmitter in the mammalian CNS.1 Glutamatergic neurotransmission is responsible for many cognitive, motor, sensory and autonomic activities.2, 3, 4 As a consequence, keeping extracellular glutamate levels in a physiological range is crucial to ensure proper neuronal transmission and viability. Indeed, impaired glutamate homeostasis has important neuropathological consequences and has been associated to several neurological or neurodegenerative disorders (NDs) such as epilepsy, multiple sclerosis, Alzheimer's disease (AD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD).5

At the excitatory synapse, glutamate is continuously released and recaptured through the well described ‘glutamate–glutamine cycle’.6,7 Glutamate is released by neurons into the synaptic cleft where it binds to both ionotropic and metabotropic receptors to transmit an excitatory message. It has been estimated that, following action potential, glutamate concentration in the synaptic cleft exceeds 1 mM for <10 ms.8,9 Afterwards, glutamate is rapidly removed from the cleft and its concentration returns to nanomolar levels, thanks to high-affinity transporters expressed both on neurons and glial cells (Fig. 1).6,10 In astrocytes, the enzyme glutamine synthetase (GS) converts glutamate into inert glutamine which, in turn, can be promptly released and taken up by neurons as a precursor for glutamate synthesis.6,7 Neurons are able to uptake glutamate11,12 and this strategy has been described as a way to replenish the presynaptic glutamate stores, bypassing the glutamate–glutamine cycle.11 Glutamate uptake occurs via five specialized glutamate transporters, defined as excitatory amino acid transporters (EAATs) 1 to 5 6,10. Two of these, the EAAT1 and EAAT2 (respectively named GLAST and GLT-1 in rodent brain) are considered, in terms of function and distribution, to be the most represented glutamate transporters in the CNS.13,14 In more detail, EAAT1 is mainly expressed on astrocytes, while EAAT2, that is responsible for 90% of the brain glutamate reuptake, is highly expressed on astrocytes, but also on neurons11,15 (Fig. 1). Moreover, EAAT1 and EAAT2 are present in oligodendrocytes,16,17 while microglia, which do not express glutamate transporters at steady-state, start to synthetize EAATs when they become reactive18, 19, 20 (Fig. 1). Overall, the expression‎ pattern of glutamate transporters on glial cells implies a crucial role in the maintenance of glutamate homeostasis both in health and disease.

소개

중추신경계(CNS)에서는

흥분성 신경세포 연결과

억제성 신경세포 연결 사이의 균형이 적절한 기능을 유지하는 데 매우 중요합니다.

대부분의 흥분성 신호는

포유류 CNS의 주요 신경 전달 물질인

글루타메이트에 의해 매개됩니다.1

글루타메이트 신경 전달은

많은 인지, 운동, 감각 및 자율 활동을 담당합니다.2, 3, 4

따라서

세포 외 글루타메이트 수준을 생리적 범위로 유지하는 것이

적절한 신경 전달과 생존을 보장하는 데 중요합니다.

실제로

글루타메이트 항상성 장애는

중요한 신경 병리학적인 결과를 초래하며

간질, 다발성 경화증, 알츠하이머병(AD), 헌팅턴병(HD), 근위축성 측색 경화증(ALS), 파킨슨병(PD)과 같은

여러 신경학적 또는 신경 퇴행성 장애(ND)와 연관되어 있습니다.5

흥분성 시냅스에서

글루타메이트는

잘 설명된 '글루타메이트-글루타민 주기'를 통해

지속적으로 방출되고 재포획됩니다.6,7

성상교세포는 알츠하이머병(AD)의 복잡한 세포 병리에 기여합니다. 뉴런과 성상 세포는 신경 전달을 유지하는 데 필수적인 글루타메이트/GABA-글루타민 주기로 통칭되는 신경 전달 물질 재활용을 통해 긴밀하게 협력하여 기능합니다. 신경전달물질 재활용은 성상교세포 에너지 대사와 밀접한 관련이 있습니다. 알츠하이머병이 진행되는 동안 성상교세포는 광범위한 대사 리모델링을 거치며, 이는 글루타메이트/GABA-글루타민 주기에 큰 영향을 미칠 수 있습니다. 신경전달물질 재활용과 관련된 성상교세포 기능 및 대사 변화의 결과는 아직 밝혀지지 않았습니다. 알츠하이머병에서 성상교세포의 대사 변화는 뉴런의 대사 지원을 박탈하여 시냅스 기능 장애와 신경 퇴화에 기여합니다. 또한 글루타민 합성 및 시냅스 신경전달물질 흡수를 포함한 글루타메이트/GABA-글루타민 주기의 여러 성상교세포 특이적 구성 요소는 AD에서 교란됩니다. AD의 맥락에서 복잡한 성상교세포 생물학을 통합하는 것은 질병의 근본적인 메커니즘을 이해하는 데 필수적이며, 성상교세포 대사를 회복하는 것은 AD의 임상적 진행을 억제하거나 심지어 되돌릴 수 있는 접근 방식이 될 수 있습니다.

글루타메이트는

뉴런에 의해 시냅스 틈새로 방출되어

이온성 및 대사성 수용체와 결합하여 흥분성 메시지를 전달합니다.

활동 전위 후 시냅스 틈새의 글루타메이트 농도는

10밀리초 미만 동안 1밀리미터를 초과하는 것으로 추정됩니다.8,9

그 후,

글루타메이트는

뉴런과 신경교세포 모두에서 발현되는

고친화성 수송체 덕분에 틈새에서 빠르게 제거되고

그 농도는 나노몰 수준으로 돌아갑니다(그림 1).6,10

성상교세포에서

글루타민 합성효소(GS)는

글루타메이트를 불활성 글루타민으로 전환하고,

이는 글루타민 합성의 전구체로서 뉴런에서 즉시 방출되어 흡수될 수 있습니다.6,7

뉴런은

글루타메이트를 흡수할 수 있으며11,12

이 전략은 글루타메이트-글루타민 순환을 우회하여

시냅스 전 글루타메이트 저장소를 보충하는 방법으로 설명되었습니다.11

글루타메이트 흡수는

흥분성 아미노산 수송체 (EAAT) 1 ~ 5 6,10 로 정의되는

5개의 특수 글루타메이트 수송체를 통해 이루어집니다.

이 중 EAAT1과 EAAT2(설치류 뇌에서는 각각 GLAST와 GLT-1로 명명)는 기능 및 분포 측면에서 CNS에서 가장 대표적인 글루타메이트 수송체로 간주됩니다.13,14 더 자세히 살펴보면, EAAT1은 주로 성상세포에서 발현되며 뇌 글루타메이트 재흡수의 90%를 담당하는 EAAT2는 성상세포뿐만 아니라 신경세포에서도 많이 발현됩니다11,15 (그림 1). 또한, EAAT1과 EAAT2는 희소돌기아교세포에 존재하며16,17 정상 상태에서는 글루타메이트 수송체를 발현하지 않는 미세아교세포는 반응성이 되면 EAAT를 합성하기 시작합니다18, 19, 20 (그림 1). 전반적으로 신경교세포에서 글루타메이트 수송체의 발현 패턴은 건강과 질병 모두에서 글루타메이트 항상성 유지에 중요한 역할을 한다는 것을 의미합니다.

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Fig. 1. Expression‎ pattern of glutamate receptors and transporters in brain cells. Transcriptomic data showing the expression‎ of mouse (A) and human (B) genes in astrocytes, neurons, as well as microglia/macrophages.176,177 Mouse and human astrocytes n = 12 and n = 6 cells, human and mouse neurons n = 1 and n = 2 cells, human and mouse microglia n = 4 and n = 2 cells. The majority of genes involved in glutamate handling are considerably expressed in astrocytes and neurons but not in microglia. Abbreviation: RPKM, reads per kilobase of transcript per million mapped reads.

Glutamate accumulation at the synapse over physiological range is toxic and triggers apoptotic cell death due to Ca2+ overload upon overstimulation of glutamate receptors.5 Glutamate-induced excitotoxicity is considered a common hallmark of many NDs, including PD, and has been linked to alterations in the expression‎ of glutamate transporters and receptors possibly in relation to inflammatory processes. Indeed, neuronal death but also glutamate spillover from the synaptic area can contribute to neuroinflammation.21 Neuroinflammation is a complex phenomenon during which microglia and astrocytes become reactive and start to secrete excessive amounts of proinflammatory cytokines as well as chemokines.22 Moreover, inflammation in the periphery is transmitted to the brain through changes in the integrity of the blood brain barrier.23 Both peripheral and central inflammation are observed in NDs.24,25 Of note, chemokines impact on glutamate homeostasis both downregulating EAATs expression‎ and upregulating glutamate receptors, thus potentiating glutamatergic neurotransmission and excitotoxicity.21

PD is the second most common neurodegenerative disorder that affects movements as well as cognition. It is characterized by i) the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) that project to the dorsal striatum, ii) a massive accumulation of aggregated α-synuclein (α-syn) as the main component of Lewy bodies and Lewy neurites and iii) extended neuroinflammation.26 PD mainly occurs as sporadic condition, but it is now confirmed that a substantial proportion of risk to develop the pathology is driven by genetic variants.27 Moreover, highly-penetrant rare mutations in SNCA, LRRK2, VPS35, PRKN, PINK1 and DJ-1 have been linked to familiar forms of PD.28 Despite PD being generally described as a DA disease, it is now accepted that alterations of glutamatergic neurotransmission play a major role in PD progression.29 Indeed, glutamate intervenes at crucial points of fronto-basal circuits involved in the modulation of voluntary movements (Fig. 2A). The striatum circuitry is mainly composed of inhibitory spiny projection neurons (SPNs) that receive DA innervation from SNpc and glutamatergic inputs from the cerebral cortex (CTX) and thalamus.29,30 SPNs are divided into two subpopulations: the striatonigral neurons are connected to the substantia nigra pars reticulata (SNr), while the striatopallidal neurons project to neurons of the internal segment of the globus pallidus (GPi) directly (direct pathway) or communicate with the external segment of the globus pallidus (GPe) and the subthalamic nucleus (STN) through glutamate release (indirect pathway).29,30 In addition, a direct glutamatergic connection exists between the STN and the SNpc.29,30 The coordinated activity of the direct and indirect pathways becomes impaired in PD due to the progressive loss of nigral DA neurons.29 A decreased stimulation of the DA receptors (D1R) affects the direct pathway leading to a reduced inhibition of the output signals while the lack of DA receptor (D2R)-mediated stimulation of the indirect pathway results in a disinhibition of the STN causing a glutamatergic overstimulation of the output signals29 (Fig. 2B). Overall, unbalance of the direct–indirect pathways prevents the activation of motor thalamic nuclei, which results in a reduced stimulation of motor areas in the frontal cortex and hence in a decreased capacity to perform voluntary movements.29 Of note, high-frequency stimulation of cortico-striatal connectivity or inhibition of glutamate transporters can increase glutamate striatal levels in mice. This promotes glutamate spillover that can modulate/suppress DA evoked release by activating the group I metabotropic glutamate receptors (mGluR) 1 in SNpc DA terminals.31

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Fig. 2. Glutamate handling at the striatum and the role of glial cells. Striatal events are detailed in healthy condition with a focus on astrocytes and microglia functions (A). Impact of astrocytes and microglia loss or gain-of-functions in glutamate uptake, spillover and inflammation resulting in exacerbated neurodegeneration (B). Fronto-basal circuits involved in the modulation of voluntary movements and impaired connectivity caused by DA degeneration in PD (grey box).

The loss of SNpc DA terminals in the striatum was proposed to be the first trigger for neurodegeneration, occurring as a ‘dying-back’ process that affects the neuronal cell body at later stages.32 But, what is the contribution of altered glutamate homeostasis to the DA neurodegeneration in the striatum? Are astrocytes and microglia involved by a loss or gain-of-function in glutamate-induced excitotoxicity and how do their activities coordinate together? In this review, we extensively discuss the evidence of impaired glutamatergic neurotransmission and glutamate clearance in genetic and idiopathic forms of PD, as well as the state of art concerning the role of glial cells in glutamate-mediated neuroinflammation, analyzing also these cells as a potential and attractive target for PD cure.

Evidence of impaired glutamate homeostasis in PD

Several clinical results obtained using magnetic resonance imaging (MRI), positron emission tomography (PET) and single photon emission computed tomography (SPET) have revealed slight alterations in glutamate content in the brain of PD patients that indicate increased glutamate neurotransmission.33, 34, 35 Of note, a two-fold increase in glutamate level (~70 μmol/L) was also measured in the plasma from PD patients compared to controls (~30 μmol/L), reflecting increases in cerebral glutamatergic activity.36,37 Whether glutamate alteration causes or exacerbates neurodegeneration in PD patients is still unclear. One possibility is that, with the progression of the disease, the sustained glutamatergic excitation of the SNpc neurons caused by STN disinhibition can further promote DA neuronal loss. However, several studies conducted in both acute and genetic in vitro and in vivo models of PD suggest that i) glutamate dyshomeostasis might induce DA neuronal death and ii) both nigral and striatal glutamatergic imbalance are involved in PD. The marked alterations of glutamate homeostasis and glutamatergic neurotransmission is detailed below and summarized in Table 1.

Table 1. Reported glutamatergic defects in PD models.

PD modelsGlutamatergic phenotypeReferences

Acute models	MPTP-lesioned mice	<< GLT-1 protein and mRNA expression‎ in primary astrocytes and in the striatum	37,38
	6-0HDA-lesioned rats	<< GLT-1 and GLAST protein in the striatum but not in the substantia nigra	39
Genetic models	SNCA mice	<< GLT -1 and GLAST protein in A53T astrocytes from brainstem	48
		>> Release of glutamate in forebrain synaptoneurosomes of ASOTg mice	50
	PARK7 KO mice	<< GLT-1 but not of GLAST in primary cortical neurons and in whole brain	52
	PINK1 KO rats	<< Basal striatal glutamate neurotransmission	58
		>> glutamate striatal levels upon potassium stimulation	58
	PRNK rat neurons	<< Cell surface distribution of AMPA and NMDA receptors	59
	LRRK2 mice	>> EPSCs of SNPs in acute slices from Lrrk2 KO pups >> EPSCs of cortical neurons from Lrrk2 G2019S mice >> EPSCs of SNPs in acute slices from Lrrk2 G2019S adult mice	65,68 69,70
		= EPSCs of SNPs in ≥6 months KI mice	72
Genetic manipulation of EAAT	Astrocytic downregulation of GLT-1 in the SNpc induces PD phenotypes in terms of progressive motor deficits and nigral DA neuronal death in mice		74

Acute exposure to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-OHDA) neurotoxins is largely used to investigate biochemical and cellular dysfunctions in PD cellular and animal models.38 Of note, in vivo microdialysis experiments demonstrated an increase of basal extracellular glutamate levels by two-fold in the striatum of 1 month 6-OHDA-injected rats and MPTP-injected mice.39,40 Also, a three-fold increase in glutamate extracellular concentration has been reported in the SNpc of MPTP-treated mice.41 In the same study, the authors demonstrated that the affinity of glutamate transporters for their substrate was increased by MPTP treatment, without changing the rate of transport.41 In primary astrocytes, Zhang et al. reported that treatment with MPTP reduces cellular viability, glutamate uptake and membrane expression‎ of GLT-1 protein.42 Specifically, glial cells biotransform MPTP into MPP+ which activates the release of the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB).42 Consequently, the activation of NF-κB stimulates c-Jun N-terminal kinase (JNK)/c-Jun signaling and negatively modulates GLT-1 expression‎ thus promoting cell death.42 In the same report, they have shown that ceftriaxone, a β-lactame antibiotic, restores astrocytic viability, GLT-1 protein and mRNA expression‎, and promotes glutamate reuptake by suppressing the NF-κB/JNK/c-Jun signaling pathway in primary astrocytes.42 More recently, the same group obtained similar data in vivo in mice intraperitoneally-injected with MPTP.43 The animal model displays typical behavioral and histopathological deficits of PD coupled with downregulation of GLT-1 protein and mRNA levels, extracellular glutamate accumulation, excitotoxicity, as well as astrocytic and microglial reactivity.43 The decrease in GLT-1 levels has been attributed to the increased ubiquitination of the transporter by the neuronal precursor cell expressed developmentally down-regulated 4–2 (Nedd4-2), a member of the ubiquitin ligase enzymes E3 43. The increased Nedd4-2-mediated GLT-1 ubiquitination leads to GLT-1 internalization from the plasma membrane and lysosomal degradation.43 Indeed, in vivo knockdown of Nedd4-2 ameliorates locomotor phenotypes, reverts DA neuron degeneration and attenuates the inflammatory activation of astrocytes and microglia suggesting that glutamate dyshomeostasis might be upstream of DA neurodegeneration.43 Similar to MPTP, the injection of 6-OHDA in rat striatum causes a downregulation of GLT-1 and GLAST expression‎ in the striatum, but not in the SNpc of 6-OHDA animals.44 Again, treatment with ceftriaxone reduces striatal DA degeneration and the locomotor defects of 6-OHDA-injected rats.45 The authors suggested that the antibiotic stimulates astrocytic GLT-1 expression‎, thus preventing overactivation of glutamatergic ionotropic receptors, i.e. N-methyl-d-aspartate (NMDA) receptors, and the subsequent Ca2+ overload in DA neurons. Recently, a novel molecular mechanism underlying GLT-1 downregulation in PD has been proposed by Wu et al. In MPP+-treated astrocytes and MPTP-injected mice, the authors have identified miR-543–3p as a suppressor of GLT-1 protein and mRNA expression‎, and hence of its transport activity.46 Furthermore, miR-543–3p inhibition was shown to increase GLT-1 expression‎ and function thus relieving dyskinesia in the PD model.46 A similar regulatory mechanism but mediated by miR-342–3p has been proposed by the same group.47 Furthermore, intranigral application of glutamate receptor antagonists, like antagonists of group I metabotropic receptors, that comprise mGluR1 and mGluR5, appears to be efficient in preventing DA neurodegeneration in a 6-OHDA rat model.48 Also, intrastriatal injection on MK-801, an antagonist of NMDA ionotropic receptors, prevented the Levodopa (l-Dopa)-induced increase in glutamate in 6-OHDA-lesioned rats.49 Taking together these findings indicate that intervening with both nigral and striatal glutamate handling could block or slow DA neurodegeneration in PD.

Compelling evidence demonstrate an aberrant glutamate reuptake in several genetic animal models of PD. Mutations in and duplication or triplication of the SNCA gene, that encodes for the protein α-syn, were found to be associated with PD onset in human.50, 51, 52 In a mouse model overexpressing the human PD-related A53T α-syn mutation selectively in astrocytes, it was described that A53T α-syn mutation results in a decreased expression‎ level of functional GLT-1 and GLAST in the brainstem.53 The authors hypothesized (but not demonstrated) that reduced expression‎ of the two glutamate transporters could induce glutamate-induced excitotoxicity.53 Furthermore, extended astrogliosis has been reported in the cerebral cortex, striatum, brainstem and spinal cord; microgliosis was observed in the brainstem, cerebellum, SNpc, but not in the cortex of this animal model.53 Also, DA neuron degeneration has been found in the SNpc and in the ventral tegmental area of the same model.53 A clear indication that aggregated α-syn (both fibrillar and oligomeric) might directly influence glutamatergic transmission comes from electrophysiology measurements.54 In a study carried out on SH-SY5Y neuroblastoma cells and hippocampal mouse slices, Diógenes et al. have shown that exposure to α-syn oligomers, but not monomers or fibrils, increases basal synaptic transmission through NMDA receptor activation, triggering an enhanced contribution of glutamatergic Ca2+-permeable (α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) AMPA ionotropic receptors.54 They demonstrated that hippocampal brain slices treated with α-syn oligomers do not respond with further potentiation to a theta-burst stimulation, leading to impaired long-term potentiation (LTP).54 This study provided new insights into a possible mechanism trough which α-syn could exert its toxicity in PD. Finally, a direct stimulation of glutamate release by both monomeric and fibrillar α-syn has been reported using synaptoneurosomes, an in vitro model that mimics functional synapses.55 Moreover, α-syn increases Ca2+ depolarization-dependent presynaptic glutamate release as demonstrated using forebrain synaptoneurosomes from transgenic mice overexpressing the human α-syn protein throughout the brain (ASOTg).55 Overall, these data suggest that α-syn can exert its toxicity by impacting glutamate clearance and transmission both in neurons and astrocytes.

Mutations in PARK7 gene, that encodes for Protein/Nucleic Acid Deglycase (DJ-1) protein, are linked to early onset, familial forms of PD.56 Downregulated levels of the glutamate transporter GLT-1 have been recognized in Park7 knockout (KO) and mutant (M26I, E64D, and L166P) astrocytes.57 DJ-1 regulates the assembly of lipid rafts, highly organized membrane microdomains that are involved in membrane receptor trafficking, endocytosis, and signal transduction.58 At the membrane, GLT-1 assembles in the lipid rafts where it undergoes recycling.59 In DJ-1 KO models, downregulation of the functional GLT-1, but not GLAST, correlates with a decreased level of two proteins crucially involved in lipid rafts assembly, flotillin-1 and caveolin-1, pointing to DJ-1 as a regulator of GLT-1 cellular localization.57 Interestingly, the overexpression‎ of wild-type but not DJ-1 familial mutants (M26I and L166P) in a DJ-1-null background reverts the decrease in glutamate uptake and GLT-1 downregulation.57 DJ-1 protein is also involved in cellular protection against oxidative stress, especially reactive oxygen species (ROS) damage.56 In addition to its association with neuroinflammation, cellular aging and degeneration, ROS production can reduce the uptake of glutamate.60,61 Therefore, DJ-1 loss-of-function might intervene in PD pathology by inducing ROS-derived glutamate-induced excitoxicity. As a vicious circle, dysfunctional glutamate homeostasis could increase glutamate extracellular content which in turn would stimulate increases in Ca2+ levels at the mitochondria, eliciting overproduction of ROS, causing oxidative stress and its deleterious outcomes.

Mutations in PINK1 (encoding for PINK1, PTEN-induced kinase 1) and PRKN (encoding for Parkin, E3-ubiquitin ligase) have been identified in familial recessive forms of PD.62 Both PINK1 and Parkin are mitochondrial proteins involved in mitophagy, a cellular process that mediates mitochondrial quality control.62 Age-dependent abnormalities in striatal glutamatergic transmission have been found in one study carried out in Pink1 KO rats.63 Creed et al. applied in vivo microdialysis to measure both basal and potassium-induced release of different neurotransmitters and their metabolites in the striatum of awake versus freely moving rats.63 Pink1 KO rats displayed a significant decrease of basal glutamate release between 4 and 12 months of age.63 However, upon potassium stimulation, Pink1 KO rats showed an age-dependent increase in striatal glutamate levels.63 Instead, no significant changes in both basal and evoked potassium-induced release of glutamate were reported for Parkin KO rats compared to controls and over time with age by the authors.63 However, Zhu et al. revealed that overexpressing four Parkin forms commonly associated with PD (T240M, R275W, R334C, G430D) in hippocampal neurons from Parkin-null background rats impairs glutamatergic synaptic function.64 Specifically, Parkin mutations disrupted glutamatergic synaptic transmission and plasticity by altering NMDA and AMPA cell-surface levels and, consequently, the receptor-mediated currents thus impairing LTP.64 Moreover, they showed that Parkin regulates NMDA receptor trafficking through its direct ubiquitination while all Parkin mutants display a non-efficient receptor ubiquitination.64 In the same study, PD pathological mutations in Parkin impacted the ability to regulate AMPA receptor trafficking by preventing the binding and retention of the postsynaptic scaffold Homer1.64 Overall, this paper points to a major impact of Parkin pathological mutations in the biology of glutamate receptors. While neurons only were studied so far, the pathological implication of mutated Parkin in glial cells, especially astrocytes and microglia, remains to be investigated. In addition, the impaired glutamate homeostasis observed in the whole brain of Pink1 KO mice raises the intriguing possibility that glial cells, possibly acting together, are responsible for the phenotype in whole brain and neurons.

The majority of inherited forms of PD are caused by mutations in the PARK8 gene encoding for Leucine-rich repeat kinase 2 (LRRK2).65 Mutations reside in the GTPase or kinase domains of LRRK2.65 Among them, the pathogenic G2019S mutation is located in the kinase domain of the protein and is the most frequent variant identified in familial forms of PD.66,67 The G2019S mutation induces a significant increase in LRRK2 kinase activity both in vitro and in vivo 68,69. In the brain, LRRK2 is most highly expressed in striatal SPNs where it plays an important role in establishing and shaping the activity of corticostriatal circuits.70 Indications of a physiological role of LRRK2 in modulating synaptic activity come from many studies carried out silencing Lrrk2 expression‎ in cortical neurons or in SPNs of Lrrk2 KO mice. In 2011, Piccoli et al. demonstrated that Lrrk2 silencing in primary cortical neurons affects evoked postsynaptic currents and induces a redistribution of presynaptic vesicles, alteration of recycling dynamics, and increase of vesicle kinetics.71 Furthermore, they found that LRRK2 protein interacts with several proteins involved in vesicular recycling, such as adaptor proteins 1 and 2, α-actinin 2, the clathrin coat assembly protein AP180, synapsin 1, VAMP2, SNAP25, dynamin 1 and synaptophysin.71,72 Moreover, Parisiadou et al. revealed that Lrrk2 KO in mice have altered dendritic spine number and morphology on the SPNs, contributing to an abnormal synaptogenesis during development.73 To determine if these alterations correlate with changes in SPNs synaptic transmission, they measured glutamatergic spontaneous excitatory postsynaptic currents (sEPSC) in striatal slices from Lrrk2 KO pups, revealing that EPSCs frequency is significantly reduced.73 They finally demonstrated that Lrrk2 negatively modulates protein kinase A (PKA) activity during synaptogenesis and in response to D1 activation.73 The absence of Lrrk2 promoted the synaptic translocation of PKA and increased PKA-mediated phosphorylation of AMPA glutamate receptor GluR1, resulting in abnormal synaptogenesis and transmission in the developing SPNs.73 Furthermore, PKA-dependent phosphorylation of GluR1 was aberrantly enhanced in the striatum of P21 and 18-month-old Lrrk2 KO mice after treatment with a D1 agonist.73

Compelling evidence of an association between LRRK2 and glutamate in PD pathogenesis derives from studies carried out on mice harboring the common G2019S mutation. The majority of LRRK2 animal models display nearly absent or subtle DA neurodegeneration suggesting that glutamate dyshomeostasis is an early event in LRRK2-PD. An in vitro study by Beccano-Kelly et al. comparing primary cortical neuronal cultures derived from Lrrk2 KO and G2019S knock-in (KI) mice revealed that glutamate release is augmented in KI versus KO mice, as demonstrated by a >35% increase in sEPSC.74 An independent study also revealed a four-fold increase in SPN sEPSC frequency at postnatal day 21 in KI mice and demonstrated that the application of LRRK2 kinase inhibitors normalized the electrical activity, suggesting that excessive neural activity in KI SPNs is LRRK2 kinase dependent.70 Accordingly, Volta et al. demonstrated that sEPSC frequency, but not amplitude, is significantly changed in SNP acute striatal slices of aged 1–3 months KI mice compared to control.75 These specific increases in glutamate release frequency were not maintained in older animals (>12 months).75 From a behavioral point of view, young mice also manifested exploratory hyperactivity in cylinder exploration test.75 In addition to early glutamatergic changes, also DA neurotransmission increases in SPNs from young (3 months) KI mice and decreases with aging (>12 months).75 Of note, Lrrk2 G2019S-linked alterations in young adult mice are followed by hypodopaminergia, as well as mitochondrial and tau pathology.76 A similar description of hyperactive phenotype in KI mice, starting at 3 months and continuing during aging, has been reported by Tozzi et al.77 In agreement with Volta's results, no change in sEPSC was observed in SPNs of acute slices from 6 months old KI mice.77 Moreover, the authors focused on DA receptors since they are primarily involved in the modulation of striatal synaptic transmission.77 In order to analyze the effect of D2 receptor stimulation on striatal SPNs, they recorded sEPSC of KI SPNs in the presence of quinpirole, a D2 receptor agonist. Interestingly, they found that quinpirole does not modify sEPSC amplitude or frequency in wild-type animals.77 By contrast, quinpirole significantly reduced sEPSC frequency without affecting amplitude in KI mice, suggesting a presynaptic modulation of this drug on striatal neurotransmission.77 The authors eval‎uated the role of endocannabinoids (eCB) to explain the inhibitory presynaptic effects of quinpirole.77 eCB are released into the extracellular space upon intracellular Ca2+ changes in SPNs and can bind CB1 inhibitory receptors on presynaptic glutamatergic neurons, thus promoting glutamate release inhibition.77 In this line, they found that application of CB1 inhibitors prevented quinpirole-induce inhibitory responses.77 At a first glance, these findings suggest that LRRK2 G2019S mutation profoundly impact glutamatergic neurotransmission by acting on neurons. However, LRRK2 is expressed at comparable levels also in astrocytes where it is emerging to play a functional role. Of note, Xiong et al. developed human TH promoter-controlled tetracycline-sensitive LRRK2 G2019S transgenic mice and showed using these mice that the mutation leads to an age- and kinase-dependent cell-autonomous neurodegeneration of DA neurons with a concomitant increase of reactive, glial fibrillary acidic protein (GFAP) positive astrocytes in the striatum.78 Additional studies are required to shed light on possible implication of mutated LRRK2 in astrocytes and glia-mediated glutamate clearance.

Finally, recent observations clearly associate aberrant glutamate homeostasis with PD pathological manifestations. Indeed, exclusive downregulation of astrocytic GLT-1 in the SNpc, using adeno-associated viral vectors, induced PD phenotypes in terms of progressive motor deficits and nigral DA neuronal death in mice.79 As reported for the acute and genetic PD models described above, the authors detected reactive astrocytes and microglia in the SNpc upon GLT-1 knockdown.79 Furthermore, RNA sequencing analysis revealed altered gene expression‎ patterns following GLT-1 knockdown in the SNpc.79 Specifically, the results revealed disrupted Ca2+ signaling pathways that might be associated with apoptotic DA neuronal death in the SNpc.79

Concluding, increasing evidence highlight defects in glutamate handling in PD and robust data from genetics and pharmacology suggest that recovering aberrant glutamate homeostasis might be key to stop and revert neurodegenerative phenotypes.

The role of glial cells in PD pathology

Astrocytes and microglia are highly specialized glial cells of the CNS6,21 (Fig. 2A). Astrocytes maintain neuronal vitality, supporting neurons in their metabolic and synaptic functions.6 They possess a multitude of receptors to respond to neurotransmitters, including glutamate.6 As a consequence, astrocytes promptly release “gliotransmitters” such as ATP, d-serine, GABA and glutamate that can modulate neuronal activity.6,80 On one hand, as mentioned, they clear up the extrasynaptic space via the reuptake of glutamate as well as other neurotransmitters.6 On the other hand, astrocytes control glutamate extra-synaptic diffusion thus protecting from glutamate spillover.9,21 Finally, they cooperate with microglia in maintaining the innate immune response of the CNS to trauma, injury or disease.81 Microglia, the well-recognized resident immune cells, are specialized in coordinating immune response and protecting the CNS parenchyma from disease.82,83 In normal physiological conditions, microglia are present in a “surveillance” phenotype, in which they appear highly ramified, with their dynamic processes continuously monitoring and maintaining the CNS homeostatic status.21,82,83 By secreting neurotrophic factors such as nerve growth factor (NGF) and fibroblast growth factor (FGF), microglia help to maintain neuronal cell survival as well as circuit formation, integrity and plasticity.84 Also, ramified microglia extend processes, contact synapses and play a functional dynamic role in synaptic plasticity using different mechanisms such as phagocytosis of synaptic elements.84 Additionally, when an immune response recruits microglia, astrocytes surround the area and create a barrier to prevent the spread of toxic signals into the surrounding healthy CNS tissue.85

Accumulating evidence suggest that the “activation” or reactivity of astrocytes and microglia to challenges, referred as astrogliosis and microgliosis, play a pivotal role in both PD initiation and progression86 (Fig. 2B). Immunohistochemical studies, labeling GFAP, a marker of reactive astrocytes, revealed the presence of astrogliosis in α-synucleinopathies87 and α-syn-positive inclusions in astrocytes of idiopathic PD patients in different brain regions, including the striatum and cerebral cortex.87, 88, 89 Furthermore, in vivo PET screening studies using [11C](R)-PK11195, a radiotracer for pro-inflammatory glial cell activity (mainly used to label microglia, but also associated with astrocytic reactivity), revealed increased binding in the pons, basal ganglia and frontal and temporal cortical regions of PD patients.90,91 The presence of reactive microglia, positively stained for the major histocompatibility complex class II antigen, tumor necrosis factor- α (TNF-α) and interleukin (IL)-6 has also been found in the SN and putamen of idiopathic PD patients.92

Once reactive, astrocytes and microglia change their morphology and acquire novel functions, producing and responding to anti-/pro-inflammatory cytokines and chemokines, which control, maintain and enhance the inflammatory condition.93, 94, 95 Cytokines such as TNF-α and the chemokine ligands (CCL) 2 and 3 were shown to mediate microglial communication with astrocytes in mouse primary cultures, leading to astrocytic reactivity, for instance after exposure to the neurotoxin manganese.96 In addition, microglia interact with astrocytes in rat primary cultures via ATP signaling onto astrocytic P2Y1, leading to neuroprotection after exposure to the neurotoxin methylmercury.97 In acute mouse hippocampal slices, microglia-derived ATP was previously shown to stimulate astrocytic P2Y1 at steady-state, which resulted in an increased frequency of EPSCs via a mGluR5-dependent mechanism.98 Depending on the context, reactive microglia can become deramified and ameboid, exacerbate their phagocytosis, exert anti-inflammatory and neuroprotective actions, or secrete pro-inflammatory cytokines and chemokines, such as inducible nitric oxide synthase (iNOS), ROS, reactive nitrogen species and CCL2 that attract peripheral monocytes into the brain and propagate the inflammatory state.21,99,100 Furthermore, cytokines released by reactive microglia can activate receptor-mediated proapoptotic pathways on DA neurons, induce nitric oxide (NO) generation and increase the production of ROS, which lead to DNA damage, protein disruption and lipid peroxidation.100 The latter causes increased prostaglandin E2 production by microglia resulting in direct toxicity to the DA neurons.100 Increased levels of IL-6, IL-1β, TNF- α, and their receptors have been observed in both SN and striatum in a mouse model of acute PD.101, 102, 103 In fact, application of the antibiotic minocycline, which blocks the production of IL-1 β, iNOS and nicotinamide adenine dinucleotide phosphate hydrogen (NADPH)-oxidase, was shown to be neuroprotective in acute MPTP- and 6-OHDA PD models.101,104, 105, 106 It has also been reported that pro-inflammatory microglia gradually outnumber the anti-inflammatory microglia in an MPTP-induced PD model.100,107,108 Indeed, intraperitoneal injection of MPTP in mice increases the expression‎ of IL-1 β, TNF-α and other inflammatory cytokines in vitro within nigral microglial cells and this phenotype is associated with progressive DA degeneration.100,107,108

Astrocytes can acquire different reactive phenotypes. In analogy with microglia, reactive astrocytes can be considered harmful to the CNS, since they produce proinflammatory factors, such as TNF-α, IL-1β, and ROS.109 On the other side, in their reactive state astrocytes can be trophic and neuroprotective, by secreting neurotrophic factors like NGF, brain-derived neurotrophic factor (BDNF) and increasing the expression‎ of glutamate reuptake systems.109 Recently, Liddelow et al. reported the robust expression‎ of neurotoxic reactive astrocytes in PD human brain.94 Using in situ hybridization and immunohistochemical experiments on human brain slices from idiopathic PD patients, they found in the SN of PD patients a strong colocalization between GFAP and component complement 3 protein (C3), two markers of astrocytes with neurotoxic behaviors.94 In the same study, they described that proinflammatory reactive microglia, secreting IL-1α, TNFα, and complement protein 1q (C1q), promote the astrocytic shift from neuroprotective to neurotoxic.94 A recent study, carried out on the α-syn preformed fibril (α-syn PFF) model of sporadic PD indicates that α-syn fibrils induce microglial proinflammatory phenotype that, in turn, stimulates an astrocyte neurotoxic phenotype.110 Similarly, astrocytes exposure to a α-syn PFF microglial conditioned medium results in a shift toward a toxic phenotype as suggested by the robust expression‎ of GFAP and C3.106

Finally, a clear indication that astrocytes and microglia contribute to PD pathology comes from genetics. As we have already discussed, PD genes are expressed in astrocytes and microglia, and compelling evidence indicate that astrocytes and microglia harboring PD gene mutations display loss-of neuroprotective as well as gain of novel toxic functions possibly interfering with glutamate homeostasis or signaling.86,111,112

Glia-mediated handling of glutamate

Both astrocytes and microglia contribute to mediating glutamatergic transmission (Fig. 2A). As mentioned, astrocytes are responsible for most of the glutamate reuptake via their expression‎ of EAAT1 and EAAT2 and they can sense glutamate extracellular content since they express AMPA and NMDA as well as group II mGluR3 and group I mGluR5.6 The transformation into GFAP-positive, neurotoxic astrocytes can negatively impact on their ability to reuptake glutamate as identified in HD and ALS mouse models.113, 114, 115 Vacuolization and astrogliosis are associated with a focal loss of the GLT-1 glutamate transporter. On this ground, as discussed above, astrogliosis and toxic reactive astrocytes have been observed also in PD brain, suggesting a similar reduction in glutamate buffering activity (Fig. 2B). Furthermore, it is worth noting that other mechanisms could be brought into action in non-physiological glutamate release. Na+ gradient is indispensable for the inward direction of glutamate uptake and perturbation of Na+ dynamics has been associated to many neurological disorders, including PD.116,117 As a consequence, a reverse outward transport of glutamate could cause extracellular glutamate accumulation.

On the other hand, microglia in their surveillance state do not intervene in glutamate reuptake since they do not express glutamate transporters at rest (Fig. 2A).21 However, microglia can start to synthetize de novo glutamate transporters when they become reactive, probably in response to the reduced ability of astrocytes to reuptake glutamate18,20,118 (Fig. 2B). Indeed, an increased microglial expression‎ of EAAT1 has been found in cerebral cortex and striatum of human Creutzfeldt-Jakob disease as well as fatal familial insomnia.118 Also, EAAT1 has been found to be upregulated in the brain of human immunodeficiency virus (HIV)-Infected patients.20 Moreover, microglial GLAST and GLT-1 expression‎ was found to increase in a rat model of cortical impact injury, while increases of EAAT2 and GS, which converts glutamate into glutamine, were observed in macaques infected with the simian immunodeficiency virus (SIV), an established animal model of HIV infection.19

Microglia also express glutamate receptors, both ionotropic and metabotropic, and can release glutamate.21,119,120 Microglia express AMPA-type GluR1–GluR4 and kainate receptors, and were shown to respond to an NMDA agonist in the cortex of neonatal rats, indicating their display of functional NMDA receptors.121,122 Also, microglia express members of all three groups of metabotropic glutamate receptors. Cultured rat microglia express group II mGluR2 and mGluR3 and group III mGluR4, mGluR6 and mGluR8, but not mGluR7.119,120,123,124 In addition, microglia produce glutaminase, which generates glutamate from glutamine, and release glutamate through connexin 32 (Cx32) hemichannels. This mechanism was shown to cause microglial release of glutamate, leading to excitotoxic damage to neurons and dendrites in hippocampal culture from a mouse model of Rett syndrome.125 In pathological contexts, other important non-vesicular mechanisms are involved in glutamate release by microglia21 (Fig. 2B). The cysteine-glutamate transporters (also named cysteine–glutamate exchange system or xCs) is a Na+-independent amino acid transporter that intakes one extracellular cystine extruding one glutamate.1,5,21,126,127 Of note, it has been shown that xCs are activated during inflammation (see next paragraph for details).

Concluding, both astrocytes and microglia are supplied by a plethora of glutamate transporters and receptors, as well as enzymes, confirm‎ing that they are key players in glutamate signaling, clearance and release (Fig. 1). Therefore, any loss-of neuroprotective or gain of neurotoxic phenotypes by astrocytes and microglia involving dysregulation of their glutamate handling may contribute to glutamate-mediated toxicity in PD.

Inflammation: cause or consequence of glutamate alteration in PD?

As discussed inflammation and glutamate-induced excitotoxicity are two crucial processes involved in the onset and progression of PD, with a major contribution of glial cells.25,60 However, it is still unclear whether inflammation is a cause or a consequence of glutamate dyshomeostasis. In PD, pathological mutations interfere per se with the biology of glutamate transporters and receptors supporting the idea that glutamate dyshomeostatsis may be the first or at least an early event. On this line, acute genetic manipulation of GLT-1 in SNpc astrocytes resulted in PD pathological and motor phenotypes in mice that were associated with progressing microgliosis and astrogliosis.79 Indeed, glutamate accumulation and spillover overstimulate both neuronal and glial receptors. Coherently, transcriptomic data reveal that astrocytic GLT-1 ablation in mouse brain results in dysfunction of innate and adaptive immune pathways.128 However, compelling evidence suggest that microglial reactivity contributes to non-physiologic glutamate release and promotes aberrant extrasynaptic signaling through ionotropic and metabotropic glutamate receptors.21

It is worth noting that some pathological PD-mutations are directly implicated in brain inflammation processes. For instance, LRRK2 that is highly expressed in astrocytes and microglia has been functionally linked to pathways pertinent to immune cell function, such as cytokine release, autophagy and phagocytosis.129 Abolishing LRRK2 expression‎ in BV-2 microglial-like cells130 or mouse primary microglia131 results in an higher migration capacity, while mouse primary microglia from KI mice have diminished ADP-induced migration.132 In rats carrying the G2019S LRRK2 mutation, elevated neuroinflammation has been reported using PET imaging with the [11C]PBR28 radioligand and immunohistochemistry.133 Increased levels of reactive GFAP positive astrocytes were also found in a rat model carrying the G2019S mutation.78

As discussed above, microglia can increase on one hand glutamate release during inflammation by expressing the glutaminase enzyme, which further potentiates glutamatergic signaling.21,134 On the other hand, reactive microglia can contribute to promoting glutamate-induced excitotoxicity in PD via the release of immune molecules21,134 (Fig. 2B). This concept has been recently reviewed by Blaylock and defined as “Immunoexcitotoxicity”, to indicate the mechanism by which exacerbated levels of pro-inflammatory cytokines produced by microglia and astrocytes can contribute to neurodegeneration, with a particular focus on PD pathology.134 It has been shown that TNF-α released by reactive microglia, binding to its receptors TNFR1, enhances the trafficking of Ca2+ permeable AMPA receptors at synapses.134, 135, 136 Similarly, IL-1β enhances synaptic expression‎ of NMDA receptors in the hippocampus of rats.134,137 Furthermore, TNF-α can further contribute to glutamate toxicity inhibiting glutamate reuptake via NF-kB activation in rat organotypic hippocampal slices.138 Moreover, high levels of TNF-α were measured in the striatum and cerebrospinal fluid of PD patients,139 while TNF-immunoreactive microglial cells were found in the SNpc of PD versus control subjects.140

Cysteine intake allows for the synthesis of glutathione (GSH), the most abundant anti-oxidant molecule in the brain.126 In PD, it has been shown that xCs release excessive extracellular glutamate.126,141 Glutamate can bind the xCs leading to their toxic paralysis that results in GSH depletion and cell death.21 This process has been referred as ‘oxidative glutamate toxicity’, in contrast to the excitotoxic glutamate toxicity resulting from overstimulation of ionotropic receptors.5,21,126 Of note, inflammatory molecules such as TNF and lipopolysaccharide induce microglial expression‎ of xCs.21,142, 143, 144 As a result, microglia become capable of releasing glutamate, thus propagating excitotoxicity.21,142, 143, 144 Moreover, IL1-β induces the expression‎ of xCs transport on astrocytes via NF-kB signaling.145 Increases in xCs expression‎ have been observed by Western blot analysis in the striatum of acute-PD rat and mouse models146,147 as well as in genetic mouse models of AD.5,126 Intriguingly, a similar mechanism of action has been recently described in primary cultured microglia stimulated with α-syn aggregates.148 In this study, the authors found that α-syn aggregates stimulate microglial reactivity via binding to both Toll-like receptor 2 and purinergic P2X7 receptors.148 Then, the activation of an intracellular signaling cascade that involves phosphoinositide 3-kinase (PI3K) and NADPH oxidase promotes microglial glutamate release.148 The inhibition of the xCs prevented the release of glutamate induced by aggregated α-syn, clearly indicating that xCs is a final effector of this process.148 Furthermore, DA, via the activation of D1 dopamine receptors and the inhibition PI3K, induces a complete suppression of glutamate release by primary cultured microglia.148 As a consequence, the authors concluded that the deficit in DA that characterizes PD may further aggravate this process in a vicious circle mechanism.148

Glutamate that accumulates at the synapse binds glutamatergic receptors located on microglia and can stimulate or inhibit their production and release of cytokines such as TNF-α21(Fig. 2B). In fact, activation of kainate and NMDA receptors enhances microglial release of TNF-α, IL-1 and NO, while AMPA receptor activation inhibits TNF-α release.119 Moreover, group I mGluR5 activation reduces TNF-α -production149 while, activation of group II mGlu2 and mGlu3 receptors potentiates microglial neurotoxicity123 and increased TNF-α levels.123,150 Conversely, the activation of group III metabotropic receptors has a neuroprotective role.124 Antagonists of metabotropic receptors were revealed to attenuate neurodegeneration in MPTP rat and monkey models.151,152

As previously mentioned, astrocytes acquire in PD a neurotoxic phenotype, which has been proposed to contribute to neurodegeneration. Neurotoxic astrocytes release, as the pro-inflammatory activated microglia, pro-inflammatory cytokines.94 The assumption of this proinflammatory phenotype also reduces the physiological ability of astrocytes to preserve brain integrity. As already discussed, neurotoxic reactive astrocytes in HD and ALS models displayed a reduced expression‎ level of glutamate transporters.113,114 This lead us to speculate that the reduction in glutamate transporter levels observed in both genetic and acute PD models could result from the presence of toxic astrocytes instead of neuroprotective ones, and that astrocytes in PD are mainly implicated with the course of pathology through the loss of their important physiological function. The exacerbation of microglial glutamate reuptake in this context would be insufficient to slow down or halt the disease progression and in turn, could further compromise the also important physiological functions of these resident immune cells.

Concluding, it is clear that reactive glial cells promotes an irreversible cascade of events that involve inflammation, glutamate-induced excitotoxicity and that, only as a final step, culminate with neurodegeneration. In this context, new therapeutic strategies aimed to control gliosis or to restore physiological glial cell functions could allow to treat, stop and/or prevent PD.

Targeting glial cells to moderate glutamate-induced toxicity

PD is the second most common ND affecting more than 6 million people worldwide and is currently without a cure.153 Most of the current treatments are only able to manage few symptoms, such as motor ones, but do not prevent neurodegeneration and cognitive decline. Many of the therapeutic strategies aim to restore the function of DA neurons and are effective only within a narrow temporal window.154 Prolonged treatment with l-Dopa is often accompanied by several side effects such as dyskinesia that limit its use.155 There is therefore an urgent need to find innovative and more efficient treatments. Given the crucial role of glutamate-induced excitotoxicity in PD due to neuronal apoptosis induced via Ca2+ overload in cells, therapeutic strategies aiming to improve of all those mechanisms that promote glutamate homeostasis (i.e. potentiation of the expression‎ of EAATs or inhibition of glutamate receptors) could be promising for the development of a PD cure.15,156

Glutamate transporters are also emerging as promising therapeutic targets.15 As mentioned above, the downregulation of glutamate transporters is a common characteristic of NDs associated with cognitive deficits, and has been commonly found in PD. As already discussed, ceftriaxone treatment reverts the PD phenotype in MPTP and 6-OHDA models, by inducing GLT-1 expression‎.42,45 Also, the finding that MPTP injection in the striatum decreased GLT-1 levels via Nedd4-2-mediated aberrant ubiquitination of the transporter, while promoting its lysosomal degradation, revealed that targeting Nedd4-2 could be beneficial to restore glutamate physiological levels.43 More recently, it has been shown that treatment with ginsenoside Rb1, the main active ingredient of Panax ginseng, by promoting the expression‎ of GLT-1, ameliorates motor deficits, prevents DA neurodegeneration and suppresses α-syn expression‎ and astrogliosis in a MPTP mouse model.157 Recent findings also point to vesicular glutamate transporter type 2 (VgluT2) as neuroprotective.158 A novel report indicates that selective deletion of VgluT2 in DA neurons of conditional VgluT2-KO mice abolishes glutamate release from DA neurons and reduced their expression‎ of BDNF and tyrosine receptor kinase B (TrkB), exacerbating the pathological effects of exposure to the MPTP.158 Viral rescue of VgluT2 expression‎ in DA neurons restored BDNF/TrkB expression‎ as well as attenuated MPTP-induced DA neurodegeneration and locomotor impairment.158 On this line, therapeutic strategies aimed to restore the physiological content of glutamate transporters could provide a great opportunity to modulate glutamate-induced excitotoxicity and to prevent excitotoxicity upstream. An extensive review on the potential therapeutic role of glutamate transporters in NDs has been recently published.15

Glutamate spillover induces the hyperactivation of glutamate receptors. Several studies propose to inhibit ionotropic glutamate receptors, in particular AMPA and NMDA, considering their major role in inducing excitotoxicity.159 Antagonists of NMDA ionotropic glutamatergic receptors have been shown to prevent the locomotor behavioral phenotypes in many NDs, using for instance 6-OHDA rats160 and MPTP-lesioned monkeys,161,162 indicating that NMDA receptor blockade is efficacious in chronic models of PD. Also, combined with l-Dopa, AMPA receptor antagonists were able to improve the ability of l-Dopa to reverse motor deficits in SNc-lesioned rats and primates.163, 164, 165 Nevertheless, the clinical application of glutamate ionotropic receptor antagonist drugs reveals no successful therapeutic outcomes because of their adverse side effects like psychosis, hallucinations, and impaired learning, likely due to the effects of altering glutamatergic signaling in healthy areas of the brain.166 A recent research points to kainate glutamate receptors as a target to prevent DA neuron degeneration in a MPTP mouse model of PD.167 On the other hand, metabotropic receptors seem to be more attractive therapeutic targets since they are not directly involved in excitotoxicity, but intervene in modulating glutamate activity.168 Antagonists of these receptors are emerging as promising, without important side effects.168 The activation of mGluR4 has been shown to prevent neuronal loss and inflammation resulting from microglial reactivity.169 Furthermore, since mGluR4 are expressed on the terminals of GABAergic striatopallidal axons, their activation or potentiation has been shown to reduce excessive GABA release from these terminals and induces anti-PD effects.170 On the other side, activation of mGluR2 has been recently found to prevent dyskinesia and behavioral alteration in a marmoset MPTP model of PD.171

Accumulated evidence suggest that anti-inflammatory drugs could have potential protective effects on DA neurons loss in PD animal models.172 Other studies revealed a protective effect of non-aspirin non-steroidal anti-inflammatory drug use in PD patients, coherently with the presence of an extensive neuroinflammatory state in PD pathogenesis.173 More recently, a novel attractive mechanism to inhibit neuroinflammation through regulating microglial reactivity was proposed using a MPTP mouse model of PD.174 In this study, the authors identified Src, a non-receptor tyrosine kinase, and its pathway signaling as an attractive target to modulate neuroinflammation by demonstrating that Src regulates microgliosis in vitro and in vivo.174

The link between glutamate-induced excitotoxicity, inflammation and gliosis has received more attention. On the basis of the novel discoveries concerning the role of astrocytes and microglia in PD onset and progression, novel strategies have been proposed to promote glial beneficial phenotypes. Glial cell reactivity often results in “loss-of-function” changes and promotes pathogenesis in vivo. Recent findings in a MPTP mouse model of PD demonstrated that treatment with the secreted protein prokineticin-2 (PK2) promotes neuroprotective astrocytic phenotypes and increases expression‎ of glutamate transporters (i.e. GLAST), thus promoting glutamate reuptake and homeostasis.175 Also, PK2-induced astrocytic reactivity leads to an increase in antioxidant and anti-inflammatory proteins, along with decreased inflammatory factors in both primary astrocytes and in the striatum.175 On the basis of these results, the authors proposed that PK2 treatment could be a potential novel therapeutic strategy to prevent glutamate-induced excitotoxicity and inflammation not only in PD, but also in other related chronic NDs.

Despite all the novel discoveries suggesting that the modulation of astrogliosis and microgliosis could be a promising therapeutic target for PD, to date no therapies are in use to modulate glial activity in PD.

Conclusions

Impaired glutamate homeostasis in the striatum is emerging as a key feature of PD pathology. In the last decades, many studies have increased the current knowledge on the biology of glial cells and growing evidence indicates that astrocytes and microglia are active and crucial players in brain glutamate handling. On the other hand, increasing data strongly reveal that gain- or loss-of-function of these glial cells lead to important neuropathological consequences that markedly impact on brain functionality and behavior. On this line, recent findings point to the involvement of reactive astrocytes and microglia in several events that promote the onset and progression of PD, such as glutamate-induced excitotoxicity and neuroinflammation, opening a novel and thrilling field of investigation that needs to be pursued. Considering the absence of efficient therapeutic strategies for PD cure, and the increasing need to find treatments that are not only symptomatic, astrocytes and microglia are emerging as valuable candidates in this scenario. As discussed in the review, some appealing pharmacological targets might be identified by studying in depth the role of glial cells in glutamate homeostasis at the synapse. Future studies aimed to identify the basic molecular mechanisms that link the glutamate-induced excitotoxicity and inflammation processes to PD onset and progression, need to receive attention, since they could help to bridge the gap between research discoveries and clinical applications.

Contributions

LC and LI conceived and wrote the manuscript. MET contributed to writing and revising the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by UniPD (STARs 2019: Suppoting TAlents in ReSearch) and the Italian Ministry of Health (GR-2016-02363461) to LC. LI is a post-doctoral fellow supported by the Department of Biology, UniPD.

Declaration of competing interest

The Authors have no conflicts of interest to declare.

Acknowledgements

University of Padova to support LC as assistant professor and Canada Research Chair (Tier II) in Neurobiology of Aging and Cognition to MET.

References

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