Re: 병든 뇌세포 제거 시스템 탐구 -- neuro-immunity와 synaptic plascicity

[문형철]

Open Access Review

Cell Clearing Systems Bridging Neuro-Immunity and Synaptic Plasticity

by 

Fiona Limanaqi

 1,†

Human Anatomy, Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Via Roma 55, 56126 Pisa (PI), Italy

2

I.R.C.C.S Neuromed, Via Atinense, 86077 Pozzilli (IS), Italy

*

Author to whom correspondence should be addressed.

†

These authors equally contributed to the present work.

Int. J. Mol. Sci. 2019, 20(9), 2197; https://doi.org/10.3390/ijms20092197

Submission received: 9 April 2019 / Revised: 29 April 2019 / Accepted: 30 April 2019 / Published: 4 May 2019

(This article belongs to the Special Issue Molecular Mechanisms of Synaptic Plasticity: Dynamic Changes in Neurons Functions)

Abstract

In recent years, functional interconnections emerged between synaptic transmission, inflammatory/immune mediators, and central nervous system (CNS) (patho)-physiology. Such interconnections rose up to a level that involves synaptic plasticity, both concerning its molecular mechanisms and the clinical outcomes related to its behavioral abnormalities. Within this context, synaptic plasticity, apart from being modulated by classic CNS molecules, is strongly affected by the immune system, and vice versa. This is not surprising, given the common molecular pathways that operate at the cross-road between the CNS and immune system. When searching for a common pathway bridging neuro-immune and synaptic dysregulations, the two major cell-clearing cell clearing systems, namely the ubiquitin proteasome system (UPS) and autophagy, take center stage. In fact, just like is happening for the turnover of key proteins involved in neurotransmitter release, antigen processing within both peripheral and CNS-resident antigen presenting cells is carried out by UPS and autophagy. Recent evidence unravelling the functional cross-talk between the cell-clearing pathways challenged the traditional concept of autophagy and UPS as independent systems. In fact, autophagy and UPS are simultaneously affected in a variety of CNS disorders where synaptic and inflammatory/immune alterations concur. In this review, we discuss the role of autophagy and UPS in bridging synaptic plasticity with neuro-immunity, while posing a special emphasis on their interactions, which may be key to defining the role of immunity in synaptic plasticity in health and disease.

Abstract

최근 몇 년 동안

시냅스 전달,

염증/면역 매개체,

중추신경계(CNS) (병리)-생리학 간에

기능적 상호 연결이 나타났습니다.

synaptic transmission,

inflammatory/immune mediators, and

central nervous system (CNS) (patho)-physiology

이러한 상호 연결은

시냅스 가소성의 분자 메커니즘과

행동 이상과 관련된 임상 결과와 관련된 수준까지 올라갔습니다.

이러한 맥락에서

시냅스 가소성은

고전적인 CNS 분자에 의해 조절되는 것 외에도

면역 체계에 의해 크게 영향을 받으며,

그 반대의 경우도 마찬가지입니다.

이는

중추신경계와 면역계 사이의 교차로에서 작동하는

공통된 분자 경로를 고려할 때

놀라운 일이 아닙니다.

신경 면역과 시냅스 조절 이상을 연결하는 공통 경로를 찾을 때

유비퀴틴 프로테아좀 시스템(UPS)과

오토파지라는

두 가지 주요 세포 청소 시스템이 중심이 됩니다.

실제로

신경전달물질 방출에 관여하는

주요 단백질의 턴오버와 마찬가지로

말초 및 중추신경계 항원 제시 세포 내 항원 처리도

UPS와 자가포식에 의해 수행됩니다.

세포 제거 경로 간의 기능적 상호 작용을 밝혀낸 최근의 증거는

오토파지와 UPS를 독립적인 시스템으로 간주하는

기존의 개념에 도전장을 던졌습니다.

실제로

시냅스 및 염증/면역 변화가 동시에 일어나는

다양한 중추신경계 질환에서

자가포식과 UPS가 동시에 영향을 받습니다.

이 리뷰에서는

시냅스 가소성과 신경 면역을 연결하는 오토파지와 UPS의 역할에 대해 논의하고,

건강과 질병에서 시냅스 가소성에서 면역의 역할을 정의하는 데

핵심이 될 수 있는

이들의 상호작용에 특히 중점을 두고자 합니다.

Keywords: 

autophagy; proteasome; immunoproteasome; mTOR; T-cells; glia; dopamine; glutamate; neuro-inflammation

1. Introduction

In recent years, unexpected connections have emerged between synaptic transmission, inflammatory/immune mediators, and brain (patho)-physiology [1,2,3]. In fact, the prevailing dogma that portrayed the nervous and immune system as two independent entities has been progressively replaced by new levels of functional connections and commonalities [4,5,6]. This interconnection rose up to a level that involves synaptic plasticity concerning both its molecular mechanisms and the clinical outcomes related to behavioral abnormalities [7,8]. Synaptic plasticity refers to those activity-dependent changes in the strength or efficacy of synaptic transmission, which occur continuously upon exposure to either positive or negative stimuli, such as learning, exercise, stress, or substance abuse, as well as the subsequent mood conditions [8].

Modifications of the neural circuits entail a variety of cellular and molecular events, encompassing neurotransmitter release; ionic activity; and metabolic, epigenetic, and transcriptional changes, which converge to shape the neuronal proteome and phenotype in an attempt to restore homeostasis [9,10,11]. The ability to re-establish and/or sustain baseline brain functions depends on a plethora of synchronized activities, which indeed involve both neuronal- and immune-related mechanisms. In this scenario, neurotransmitters and immune-related molecules adopt a common language to fine-tune brain functions [12,13,14,15]. In fact, classic immune molecules, including cytokines, major histocompatibility complex (MHC) molecules, and T-cells, are deeply involved in central nervous system (CNS) plasticity, while CNS factors, mostly neurotransmitters encompassing dopamine (DA) and glutamate (GLUT), actively participate in shaping immune functions [14].

Neuro-immune surveillance is a critical component for brain function, as circulating T-cells that recognize CNS antigens (Ags) are key in supporting the brain’s plasticity, both in health and disease [8]. The functional anatomy from which the molecular interplay between the immune system and brain matter stems, was recently identified at the level of lymphatic pathways operating in the perivascular (also known as “glymphatic”) and dural meningeal spaces [16,17,18]. Lymphatic flows foster the drainage of the brain interstitial fluid into the cerebrospinal fluid, and then back again into the bloodstream, or even directly into the secondary lymphoid organs. Functionally, this translates into a clearance of potentially threatening interstitial solutes and the drainage of CNS-derived Ag peptides to the deep cervical lymph-nodes to be captured and processed by antigen presenting cells (APCs) [19,20].

Within this context, synaptic plasticity, apart from being modulated by classic CNS molecules, is strongly affected by the immune system. This is not surprising, given the common molecular pathways that operate at the cross-road between the nervous- and immune-system. In fact, just like what is happening for the key proteins involved in neurotransmitter release [21,22], Ag processing within APCs is carried out by the two major cell-clearing machineries, ubiquitin proteasome (UPS) and autophagy [23,24,25]. In detail, UPS and autophagy operate both in the CNS and immune system, to ensure protein turnover and homeostasis. In the CNS, UPS- and autophagy-dependent protein degradation is seminal to protect neurons from potentially harmful proteins, and to modulate neurotransmitter release and synaptic plasticity [21,26,27,28].

Similarly, in the immune system, UPS and autophagy cleave endogenously- and exogenously-derived proteins to produce Ag peptides, which bind to MHC molecules class I and II [23,24,25,29]. Indeed, these pathways converge when the CNS components are cleared by immunocompetent mechanisms [24,29]. Thus, CNS-derived Ags bound to MHC-I and –II may be exposed on the plasma membrane of APCs, for presentation to CD8+ and CD4+ T-lymphocytes, respectively [29,30]. The associative binding of MHC molecules with T-cells receptors (TCR), coupled with co-stimulatory signals and the presentation of CNS-derived Ags, fosters the activation of naïve T-cells in the periphery, while mounting CNS-directed adaptive immune responses, which may produce either beneficial or detrimental effects already pertaining to the field of CNS plasticity [2,14,31,32,33]. Still, at anatomical level, the sympathetic innervation of both primary and secondary lymphoid organs provides a means of functional connection between the immune- and nervous-system [34].

1. 소개

최근 몇 년 동안

시냅스 전달, 염증/면역 매개체, 뇌(병리)-생리학 사이에

예상치 못한 연관성이 밝혀지고 있습니다[1,2,3].

실제로

신경계와 면역계를 두 개의 독립된 개체로 묘사하던 기존의 도그마는

점차 새로운 수준의 기능적 연결과 공통점으로 대체되고 있습니다[4,5,6].

이러한 상호 연결은

분자 메커니즘과 행동 이상과 관련된 임상 결과와 관련된

시냅스 가소성과 관련된 수준까지 올라갔습니다 [7,8].

시냅스 가소성이란

학습, 운동, 스트레스 또는 약물 남용과 같은 긍정적 또는 부정적 자극과

그에 따른 기분 상태에 노출될 때

지속적으로 발생하는 시냅스 전달의 강도 또는 효능의 활동 의존적 변화를 말합니다 [8].

신경 회로의 변형은

신경전달물질 방출, 이온 활동, 대사, 후성유전학 및 전사적 변화를 포함하는

다양한 세포 및 분자적 사건을 수반하며,

이러한 변화는 항상성을 회복하기 위해

신경세포 단백질체와 표현형을 형성하는 데 수렴됩니다[9,10,11].

기본 뇌 기능을 회복하거나 유지하는 능력은

신경세포와 면역 관련 메커니즘을 모두 포함하는

수많은 동기화된 활동에 달려 있습니다.

이 시나리오에서

신경전달물질과 면역 관련 분자는

뇌 기능을 미세 조정하기 위해 공통 언어를 채택합니다[12,13,14,15].

실제로

사이토카인,

주요 조직 적합성 복합체(MHC) 분자,

T세포를 포함한 대표적인 면역 분자는

중추 신경계(CNS) 가소성에 깊이 관여하며,

도파민(DA)과 글루타메이트(GLUT)를 포괄하는 신경 전달 물질인

CNS 인자는

면역 기능을 형성하는 데 적극적으로 참여합니다[14].

In fact, classic immune molecules, including

cytokines,

major histocompatibility complex (MHC) molecules, and

T-cells,

are deeply involved in central nervous system (CNS) plasticity, while CNS factors, mostly neurotransmitters encompassing dopamine (DA) and glutamate (GLUT), actively participate in shaping immune functions

신경 면역 감시 Neuro-immune surveillance 는

뇌 기능의 중요한 구성 요소로,

CNS 항원(Ag)을 인식하는 순환 T세포는

건강과 질병 모두에서 뇌의 가소성을 지원하는 데

핵심적인 역할을 합니다[8].

면역 체계와 뇌 물질 간의 분자적 상호 작용이 발생하는 기능적 해부학은

최근 혈관 주위(일명 “림프”) 및 경막 수막 공간에서 작동하는

림프 경로 수준에서 확인되었습니다[16,17,18].

림프 흐름은

뇌 간질액이 뇌척수액으로 배출된 후

다시 혈류로,

또는 이차 림프 기관으로 직접 배출되는 것을

촉진합니다.

기능적으로 이는

잠재적으로 위협적인 간질 용질의 제거와

항원 제시 세포(APC)에 의해 포획 및 처리될 수 있도록

CNS 유래 Ag 펩타이드가 심부 경부 림프절로 배출되는 것을 의미합니다[19,20].

이러한 맥락에서

시냅스 가소성은

전통적인 CNS 분자에 의해 조절되는 것 외에도

면역 체계에 의해 크게 영향을 받습니다.

신경계와 면역계 사이의 교차로에서 작동하는

일반적인 분자 경로를 고려할 때

이는 놀라운 일이 아닙니다.

실제로

신경전달물질 방출에 관여하는 주요 단백질에서 일어나는 일과 마찬가지로[21,22],

APC 내의 Ag 처리는

두 가지 주요 세포 청소 기계인 유비퀴틴 프로테아좀(UPS)과

오토파지에 의해 수행됩니다[23,24,25].

구체적으로

UPS와 오토파지는

중추신경계와 면역계에서 모두 작동하여

단백질 회전율과 항상성을 보장합니다.

CNS에서

UPS 및 자가포식에 의존하는 단백질 분해는

잠재적으로 유해한 단백질로부터 뉴런을 보호하고

신경전달물질 방출과 시냅스 가소성을 조절하는 데

매우 중요합니다[21,26,27,28].

마찬가지로

면역계에서도

UPS와 자가포식은

내인성 및 외인성 유래 단백질을 분해하여

MHC 분자 클래스 I 및 II에 결합하는

Ag 펩타이드를 생성합니다[23,24,25,29].

실제로 이러한 경로는

CNS 성분이 면역 기능 메커니즘에 의해 제거되면 수렴합니다[24,29].

따라서

MHC-I 및 -II에 결합된 CNS 유래 Ag는

각각 CD8+ 및 CD4+ T 림프구에 제시하기 위해

APC의 혈장막에 노출될 수 있습니다 [29,30].

MHC 분자와 T세포 수용체(TCR)의 결합은

공동 자극 신호 및 CNS 유래 Ag의 제시와 결합하여

말초에서 순진한 T세포의 활성화를 촉진하는 동시에

CNS 주도 적응 면역 반응을 일으켜

이미 CNS 가소성 분야와 관련된 유익한 또는 해로운 효과를 낼 수 있습니다 [2,14,31,32,33].

https://www.nature.com/articles/s12276-020-00486-7

하지만

해부학적 수준에서 일차 및 이차 림프 기관의 교감 신경 분포는

면역계와 신경계 사이의 기능적 연결 수단을 제공합니다 [34].

In fact, catecholamine, and mostly DA released from sympathetic nerve terminals, is an active regulator of the metabolism, fate, and activity of naïve CD4+ and CD8+ T-cells [35,36]. This is achieved through the binding of DA to its cognate receptors and transporters, which are abundantly expressed on lymphoid cells. Likewise, the GLUT released in the bloodstream or within the CNS modulates T-cells activity, through binding to its cognate receptors, which are expressed on T-cells [2,37]. In this way, neurotransmitters and CNS-derived Ag presentation synergize to define the pool of immunocompetent cells that travel back and forth between the brain and periphery, to guarantee neuro–immune surveillance and synaptic plasticity. Antigen presentation and immune responses may also occur directly in the brain upon interactions between CNS circulating T-cells and glia, or even neurons [38,39,40,41,42]. Unexpectedly, recent studies showed that naïve T-cells are able to cross CNS barriers and infiltrate the brain parenchyma [38,39,40,41,42,43,44,45,46,47].

This is magnified during pro-inflammatory conditions when the glia and even neurons operate as competent APCs, as they become able to process and present Ags via MHC molecules [39,47,48]. At the same time, T-cells possess all of the machinery that is necessary for releasing and responding to neurotransmitters, just like neurons and glia do [35,37]. The existence of such a bi-directional dialogue between nerve and immune cells has now challenged the classical dichotomy between inflammatory and degenerative disorders of the CNS. In fact, defective or inappropriate communication between the immune and nervous system gives rise to a chain of events, where inflammatory/immune and synaptic alterations intermingle to produce CNS disorders, encompassing neuro-developmental, neurodegenerative, and auto-immune diseases [2,12,13,20,49].

When searching for a common pathway bridging neuro–immune and synaptic dysregulations, UPS and autophagy machineries take center stage. The dysregulations of both UPS and autophagy characterize a plethora of CNS disorders, where synaptic and neuro-inflammatory/immune alterations co-exist, such as Parkinson’s, Alzheimer’s, and Huntingtin’s diseases (PD, AD, and HD); epilepsy; ischemia; brain tumors; multiple sclerosis (MS), and psychiatric and substance-abuse disorders [6,21,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77].

The reason for such a common dysregulation of UPS and autophagy in etiologically different CNS disorders is rooted in their pleiotropic catalytic functions, which are seminal for both synaptic plasticity and neuro-immunity [30,78,79,80,81,82,83,84,85,86,87]. Despite being traditionally considered as independent systems, recent evidence has unraveled a functional cross-talk between UPS and autophagy, which occurs at both biochemical and morphological levels [73,88,89,90]. Thus, it is not surprising that autophagy and UPS share most of their substrates and functions, and they operate dynamically and coordinately in both nerve and immune cells so as to modulate neurotransmission, oxidative/inflammatory stress response, and immunity [91,92,93,94].

This is accomplished through the degradation and turnover of proteins, including those involved in endocytic and secretory pathways, transcription factors, and oxidized and/or immunogenic proteins. The present review aims to analyze those molecular interactions that are related to both UPS and autophagy, and that enable neurons and immune cells to surveil synaptic and neuro–immune activity. Apart from being well known triggers of synaptic plasticity, environmental agents such as pathogens, inflammatory cytokines, free radicals, and abnormal neurotransmitter release can profoundly affect cell-clearing systems [51,52,94,95,96,97,98,99,100]. As a proof of concept, when a dysregulation of cell-clearing systems occurs, the altered communication between the nervous and immune cells translates into maladaptive plasticity, which may underlie behavioral alterations. Given the variety of specific regulatory signals and molecules involved in the interplay between UPS and autophagy, a better understanding of their interactions is key in order to define the role of immunity in synaptic plasticity in health and disease.

실제로

교감신경 말단에서 방출되는

카테콜아민과 대부분 DA는

순진한 CD4+ 및 CD8+ T세포의 대사, 운명 및 활동을

적극적으로 조절하는 역할을 합니다[35,36].

이는 림프 세포에서 풍부하게 발현되는

동종 수용체 및 수송체에 대한 DA의 결합을 통해 이루어집니다.

마찬가지로

혈류 또는 CNS 내에서 방출되는 GLUT는

T세포에서 발현되는 유사 수용체와의 결합을 통해

T세포 활동을 조절합니다 [2,37].

이러한 방식으로

신경전달물질과 CNS 유래 Ag 제시가

시너지 효과를 발휘하여 뇌와 말초를 오가는 면역 능력 세포 풀을 정의하고

신경 면역 감시와 시냅스 가소성을 보장합니다.

항원 제시와 면역 반응은

CNS 순환 T세포와 신경교세포 또는 뉴런 간의 상호작용을 통해

뇌에서 직접 발생할 수도 있습니다[38,39,40,41,42].

예기치 않게도

최근 연구에 따르면

순진한 T세포가 CNS 장벽을 통과하여

뇌실질에 침투할 수 있는 것으로 나타났습니다 [38,39,40,41,42,43,44,45,46,47].

이는

신경교세포와 심지어 뉴런이

유능한 APC로 작동하는 전염증성 조건에서 증폭되며,

이들은 MHC 분자를 통해 Ag를 처리하고 제시할 수 있게 됩니다[39,47,48].

동시에 T세포는

뉴런과 신경교세포처럼 신경전달물질을 방출하고

이에 반응하는 데 필요한 모든 메커니즘을 갖추고 있습니다[35,37].

신경세포와 면역세포 사이에 이러한 양방향 대화가 존재한다는 사실은

이제 중추신경계의 염증성 질환과 퇴행성 질환이라는

고전적인 이분법에 도전장을 내밀었습니다.

실제로

면역계와 신경계 간의 결함 또는

부적절한 의사소통은

염증성/면역성 및 시냅스 변화가 상호 작용하여

신경 발달, 신경 퇴행성 및 자가 면역 질환을 포괄하는 CNS 장애를 일으키는

일련의 사건을 일으킵니다 [2,12,13,20,49].

신경 면역과 시냅스 조절 이상을 연결하는 공통 경로를 찾을 때

UPS와 오토파지 메커니즘이 중심이 됩니다.

UPS와 자가포식의 조절 이상은

파킨슨병, 알츠하이머병, 헌팅턴병(PD, AD, HD)과 같이

시냅스 및 신경 염증/면역 변화가 공존하는

수많은 CNS 장애의 특징입니다;

간질, 허혈, 뇌종양, 다발성 경화증(MS), 정신과 및 약물 남용 장애[6,21,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77].

병인학적으로

다른 CNS 장애에서

UPS와 자가포식의 공통적인 조절 장애가 발생하는 이유는

시냅스 가소성과 신경 면역 모두에 중요한 역할을 하는 이들의

다발성 촉매 기능에 뿌리를 두고 있습니다[30,78,79,80,81,82,83,84,85,86,87].

전통적으로 독립적인 시스템으로 간주되어 왔지만,

최근의 증거에 따르면

생화학적 및 형태학적 수준에서 발생하는

UPS와 오토파지 사이의 기능적 crosstalk가 밝혀졌습니다[73,88,89,90].

따라서

자가포식과 UPS가 대부분의 기질과 기능을 공유하며

신경세포와 면역세포 모두에서

역동적이고 조화롭게 작동하여

신경 전달, 산화/염증 스트레스 반응 및 면역을 조절한다는 것은

놀라운 일이 아닙니다[91,92,93,94].

이는

세포 내 및 분비 경로, 전사인자, 산화 및/또는

면역원성 단백질에 관여하는 단백질을 포함한

단백질의 분해와 전환을 통해 이루어집니다.

본 리뷰에서는

UPS와 자가포식과 관련이 있고

뉴런과 면역 세포가 시냅스 및 신경 면역 활동을 감시하는 데 도움이 되는

분자 상호작용을 분석하는 것을 목표로 합니다.

시냅스 가소성의 잘 알려진 유발 요인 외에도

병원균, 염증성 사이토카인, 활성산소,

비정상적인 신경전달물질 방출과 같은 환경적 요인도

세포 청소 시스템에 큰 영향을 미칠 수 있습니다[51,52,94,95,96,97,98,99,100].

개념 증명으로,

세포 제거 시스템의 조절 장애가 발생하면

신경 세포와 면역 세포 간의 변경된 통신이 부적응 가소성으로 전환되어

행동 변화의 근간이 될 수 있습니다.

UPS와 자가포식 간의 상호작용에 관여하는

다양한 특정 조절 신호와 분자를 고려할 때,

건강과 질병에서 시냅스 가소성에서 면역의 역할을 정의하기 위해서는

이들의 상호작용을 더 잘 이해하는 것이 핵심입니다.

2. Cell Clearing Systems: Tracing the Path of the Interplay between Proteasome and Autophagy

Autophagy and UPS ensure eukaryotic cell proteostasis by clearing unfolded, misfolded, oxidized, or disordered proteins, so as to prevent their accumulation, aggregation, and spreading [60,101,102,103,104,105,106]. Besides being seminal in extreme cell conditions when cell survival is jeopardized, autophagy and UPS activities operate in baseline conditions in order to keep the turnover of proteins that naturally occur within a living cell steady. In fact, as actors of protein degradation, autophagy and UPS regulate most cell functions encompassing cell cycle and division, cell differentiation and development, endo- and exo-cytosis, and, specifically, synaptic strength and Ag processing [6,21,22,25,26,27,107,108,109,110,111]. Autophagy initiates with the formation of double-layered membrane vacuoles, named phagophores. The maturation and sealing of the phagophore leads to the formation of the autophagosome, which stains for autophagy markers such as beclin-1 (the orthologue of yeast Atg6) and LC3 (Atg8) [112,113]. The autophagosome shuttles a variety of substrates, including ubiquitinated proteins and whole organelles (e.g., mitochondria, endoplasmic reticula, ribosomes, and synaptic vesicles) to the lysosomal compartment, which is gifted with a rich enzymatic activity. The merging of the autophagosome with endosomes and lysosomes generates the catalytic organelle autophagolysosome, where the degradation and recycling of “in bulk” sequestered cytosolic cargoes occurs [112,113].

Protein tagging with ubiquitin chains, which is carried out by the UPS system, represents a sorting signal for either UPS- or autophagy-dependent protein degradation [114]. Protein ubiquitination is an ATP-dependent process that is accomplished by three enzymes, namely ubiquitin-activating (E1), ubiquitin-conjugating (E2), and ubiquitin-ligase (E3). Several proteins operate at the cross-road between UPS and autophagy, to regulate the sorting and shuttling of ubiquitinated substrates towards either system. Among these proteins, which indeed constitute a much longer list, three are worth mentioning, namely (i) Parkin, (ii) histone deacetylase 6 (HDAC6), and (iii) Sequestosome-1 (SQSTM1)/p62.

(i) Parkin is an ubiquitin-ligase enzyme (E3 ligase), which mediates protein polyubiquitination and serves as a signal for targeting misfolded proteins to the aggresome, where autophagy is recruited [115]. Parkin-dependent ubiquitination triggers the removal of the pro-apoptotic proteins BAX and BCL-2 by either UPS or autophagy, and it is seminal to induce mitophagy, that is, mitochondria-specific autophagy [116]. After ubiquitin linkage, Parkin also induces the coupling of target proteins with dynein motor complexes via the adaptor protein HDAC6 in order to facilitate their transport to the aggresome, where autophagy is recruited;

(ii) HDAC6 is a microtubule-associated histone deacetylase, which shuttles polyubiquitinated substrates along the microtubules for autophagosomal engulfment, while fostering lysosomes transport to the site of autophagy occurrence. In detail, HDAC6 binds the polyubiquitin chains [117,118] or even C-terminal regions of free ubiquitin [119] via a C-terminal zinc finger-containing domain (called BUZ domain). Then, HDAC6 binds to the microtubule-associated dynein motors to shuttle the polyubiquinated proteins to the aggresomes, while fostering the recruitment of autophagy to the aggresomes [120,121].  Again, HDAC6 participates in the fusion of autophagosomes with lysosomes for final autophagy degradation [122,123]. Remarkably, HDAC6 activity is essential for autophagy, to compensate for protein degradation and rescue cell survival when UPS is impaired [124], thus providing a functional link between autophagy and UPS.

(iii) SQSTM1/p62 is a ubiquitin-binding scaffold protein that links ubiquitinated proteins to autophagy machinery in order to enable their degradation [125]. This occurs through a direct interaction between SQSTM1/p62 and ubiquitinated proteins via a C-terminal UBA domain, and their subsequent binding to autophagy proteins such as LC3 and GABARAP family proteins. As p62 is itself degraded by autophagy, it is widely used as a marker of autophagy flux [126].

Once tagged with ubiquitin, proteins are recognized by autophagy and/or the proteasome 26S (P26S) multimeric complex, which is formed by a catalytic core (P20S) and two regulatory subunits (P19S, also known as PA700) capping the ends of P20S [127]. P19S binds the poly-ubiquitin chain and cleaves it from the substrate. In this way, the unfolded substrate enters the P20S to be degraded by the β1, β2, and β5 catalytic subunits of the P20S, which own chymotrypsin-like, trypsin-like, and caspase-like activity, respectively. Despite being traditionally considered as cytosolic catalytic machinery, UPS also associates with vesicular organelles, including precursor synaptic vesicles (SVs), Golgi-derived vesicles, mitochondria, and lysosomes [128]. Far from being a mere phenomenon of morphological co-localization, the association of UPS with vesicular structures probably underlies a sophisticated functional cooperation. In fact, vacuolar organelles may serve as a ferryboat to shuttle UPS in different cell-compartments, while the UPS handles the turnover of vesicle-associated proteins. This is in line with the recent studies characterizing a novel organelle named “autophagoproteasome”, where the autophagy and UPS markers co-localize [73,88] (Figure 1).

2. 세포 청소 시스템: 프로테아좀과 오토파지의 상호 작용 경로 추적하기

오토파지와 UPS는

펼쳐지거나 잘못 접히거나 산화되거나 무질서한 단백질을 제거하여

축적, 응집, 확산을 방지함으로써

진핵세포의 단백질 항상성을 보장합니다 [60,101,102,103,104,105,106].

세포 생존이 위태로운 극한의 세포 조건에서 중요한 역할을 하는 것 외에도,

오토파지와 UPS 활동은

살아있는 세포 내에서 자연적으로 발생하는 단백질의 회전율을 일정하게 유지하기 위해

기본 조건에서 작동합니다.

실제로

단백질 분해의 주체로서 자가포식과 UPS는

세포 주기와 분열, 세포 분화 및 발달, 세포 내 및 세포 외 세포증,

특히 시냅스 강도 및 Ag 처리를 포함한

대부분의 세포 기능을 조절합니다[6,21,22,25,26,27,107,108,109,110,111].

자가포식은

식세포라고 하는

이중막 액포의 형성으로 시작됩니다.

식세포의 성숙과 밀봉은

오토파지좀의 형성으로 이어지며,

이 오토파지좀은 베클린-1(효모 Atg6의 상동체) 및 LC3(Atg8)와 같은

오토파지 마커에 염색됩니다[112,113].

오토파지는

유비퀴틴화된 단백질과 전체 소기관(예: 미토콘드리아, 소포체, 리보솜, 시냅스 소포체)을 포함한

다양한 기질을 풍부한 효소 활성을 지닌

리소좀 구획으로 운반합니다.

오토파지솜과 엔도솜 및 리소좀의 병합은

촉매 소기관인 오토파지리소좀을 생성하여

“대량으로” 격리된 세포질화물의

분해 및 재활용을 진행합니다[112,113].

UPS 시스템에 의해 수행되는 유비퀴틴 사슬을 사용한 단백질 태깅은

UPS 또는 오토파지 의존 단백질 분해에 대한

분류 신호를 나타냅니다[114].

단백질 유비퀴틴화는

유비퀴틴 활성화(E1), 유비퀴틴 접합(E2), 유비퀴틴 리가제(E3)라는

세 가지 효소에 의해 수행되는

ATP 의존적인 과정입니다.

여러 단백질이 UPS와 오토파지 사이의 교차로에서 작동하여 유비퀴틴화된 기질이 두 시스템으로 분류되고 셔틀링되는 것을 조절합니다. 실제로 훨씬 더 긴 목록을 구성하는 이러한 단백질 중에서

(i) Parkin,

(ii) 히스톤 탈아세틸화 효소 6(HDAC6),

(iii) 시퀘스토솜-1(SQSTM1)/p62 등

세 가지를 언급할 가치가 있습니다.

 (i) Parkin,

(ii) histone deacetylase 6 (HDAC6), 

(iii) Sequestosome-1 (SQSTM1)/p62.

(i) 파킨은 유비퀴틴 리가제 효소(E3 리가제)로서

단백질 다중유비퀴틴화를 매개하고

잘못 접힌 단백질을 어그레좀으로 표적화하여

오토파지가 모집되는 신호로 작용합니다[115].

파킨 의존적 유비퀴틴화는

UPS 또는 오토파지에 의해

세포사멸 단백질인 BAX와 BCL-2의 제거를 촉발하며,

미토파지, 즉 미토콘드리아 특이적 오토파지를 유도하는 데 중요한 역할을 합니다 [116].

유비퀴틴 연결 후,

파킨은 또한 표적 단백질과 다이나인 모터 복합체와의 결합을

어댑터 단백질 HDAC6을 통해 유도하여

오토파지가 모집되는 어그레좀으로의 수송을 용이하게 합니다;

(ii) HDAC6는

미세소관 관련 히스톤 탈아세틸화 효소로,

미세소관을 따라 폴리유비퀴틴화된 기질을 셔틀링하여

오토파지를 포식하는 동시에

리소좀이 오토파지 발생 부위로 운반되도록 촉진합니다.

구체적으로

HDAC6는

폴리유비퀴틴 사슬[117,118] 또는

유리 유비퀴틴의 C-말단 영역[119]에 C-말단 아연 핑거 함유 도메인(BUZ 도메인이라고 함)을 통해

결합합니다.

그런 다음

HDAC6는

미세소관 관련 다이인 모터에 결합하여

폴리유비퀴틴화된 단백질을 응집체로 이동시키는 동시에

응집체에 대한 오토파지의 모집을 촉진합니다[120,121].

다시 말하지만,

HDAC6는 최종 자가포식 분해를 위해

오토파지와 리소좀의 융합에 참여합니다 [122,123].

놀랍게도

HDAC6 활성은 자가포식에 필수적이며,

UPS가 손상되었을 때 단백질 분해를 보상하고

세포 생존을 구출하는 데 필수적이므로[124]

자가포식과 UPS 사이의 기능적 연결 고리를 제공합니다.

(iii) SQSTM1/p62는 유비퀴틴 결합 스캐폴드 단백질로,

유비퀴틴화된 단백질을

오토파지 기계에 연결하여 분해할 수 있도록 합니다[125].

이는 C-말단 UBA 도메인을 통해 SQSTM1/p62와 유비퀴틴화된 단백질 간의 직접적인 상호 작용과 LC3 및 GABARAP 계열 단백질과 같은 오토파지 단백질과의 후속 결합을 통해 발생합니다.

p62는 그 자체가

오토파지에 의해 분해되기 때문에

오토파지 플럭스의 마커로 널리 사용됩니다[126].

유비퀴틴으로 태그가 부착된 단백질은

자가포식 및/또는 프로테아좀 26S(P26S) 다중 복합체에 의해 인식되며,

이는 촉매 코어(P20S)와 2개의 조절 서브유닛(P19S, PA700이라고도 함)이 P20S의 끝을 감싸는 형태로 구성됩니다[127].

P19S는 폴리유비퀴틴 사슬을 결합하여 기질에서 절단합니다.

이러한 방식으로 펼쳐진 기질은 P20S로 들어가 각각 키모트립신 유사, 트립신 유사, 카스파제 유사 활성을 갖는 P20S의 β1, β2, β5 촉매 서브유닛에 의해 분해됩니다. 전통적으로 세포질 촉매 기계로 간주되어 왔지만 UPS는 전구체 시냅스 소포(SV), 골지 유래 소포, 미토콘드리아 및 리소좀을 포함한 소포체 소기관과도 연관되어 있습니다[128].

단순한 형태학적 공동 위치화 현상이라기보다는 소포 구조와 UPS의 연관성은 아마도 정교한 기능적 협력의 기반이 될 것입니다. 실제로 액포 소기관은 다른 세포 구획에서 UPS를 셔틀하는 페리선 역할을 하고, UPS는 소포 관련 단백질의 턴오버를 처리하는 역할을 할 수 있습니다. 이는 오토파지와 UPS 마커가 함께 위치하는 “오토파지 프로테아좀”이라는 새로운 세포소기관의 특징을 규명한 최근 연구 결과와 일치합니다[73,88](그림 1).

The formation of this specific vacuolar compartment is hindered by the administration of the neurotoxic abused drug methamphetamine (meth), while its rescue via the inhibition of the mammalian target of rapamycin (mTOR) correlates with cell protection and survival [73]. This is line with studies indicating the mTOR pathway as a common modulator of both UPS- and autophagy-dependent protein degradation [89]. These findings configure mTOR inhibition as a potential strategy to synergistically enhance autophagy and UPS-dependent protein degradation. mTOR is a ubiquitously expressed serine-threonine kinase, which senses and integrates several environmental and intracellular cues to orchestrate major processes, such as cell growth and metabolism [55,75,129]. mTOR has been widely implicated in synaptic plasticity, inflammation, and immunity, although this was merely related to the role in protein synthesis. In the last decades, mTOR has been posed at the center stage on a variety of cell functions, mostly related to autophagy and UPS. The emerging mechanisms linking mTOR with autophagy and UPS unravel a close interdependency between the cell-clearing systems. In detail, the duration and amplitude of the autophagy response depends on the stability of the serine/threonine kinase ULK1/Atg1, which, in turn, is coordinately regulated by UPS and mTOR [130]. ULK1 acts at multiple steps of autophagy initiation and response, in part by phosphorylating autophagy proteins, including Atg13, Beclin 1, and Atg9 [131]. mTOR activation inhibits ULK1 kinase activity (and thus autophagy initiation) via phosphorylation, and also coordinates ULK1 de novo protein synthesis [130,132].

In this context, UPS behaves as a sentinel in sensing and regulating mTOR/ULK1-dependent autophagy. In fact, during the early stages of autophagy, UPS mediates the K63-linked polyubiquitination of ULK1 via the AMBRA1–TRAF6 (E3 ligase) complex to maintain its stability, self-association, and kinase activity [133]. Conversely, during prolonged nutrient starvation, UPS targets ULK1 for degradation, following Cullin/KLHL20-dependent K48-linked polyubiquitination, thus providing a feedback control of the autophagy response [134]. In turn, autophagy may control UPS efficacy and activity through the degradation of inactive UPS subunits, which are shuttled to autophagosomes, a phenomenon known as “proteophagy” [135,136,137]. This may explain the intriguing effects that are observed on autophagy upon UPS inhibition, and vice versa, while remarking on the importance of autophagy-UPS cross-talk in cell homeostasis. In fact, the inhibition of either autophagy or UPS alone may produce detrimental effects to cell survival [138,139,140,141,142,143], which are bound to impaired protein turnover by both cell-clearing systems. For instance, autophagy inhibition leads to the accumulation of ubiquitinated substrates by affecting UPS either upstream, or at the level of its catalytic activity [144,145]. Conversely, UPS inhibition may induce an enhancement of autophagy as an early compensatory response to cope with protein overload and grant cell-survival [146,147,148].

Such an effect turns out to be only transitory, as UPS dysfunction at later stages impedes mitophagy and decreases the levels of essential autophagy proteins, such as Atg9 and LC3B [93]. This is not surprising, as UPS is essential for endo–lysosome membrane fusion [149,150], which, in turn, is involved in the late steps of autophagy. In fact, UPS modulates the activity of Rab GTPases (GTP-bound Ras proteins in the brain), which are involved in all cell-trafficking mechanisms, including autophagy-dependent endocytosis and autophagy membrane fusion [150,151,152,153]. These findings indicate that the synergistic and compensatory functional interplay between autophagy and UPS needs to be taken into account in experimental approaches modulating either systems alone. On the one hand, this may lead to confounding outcomes when assessing the effects of autophagy and UPS alone; on the other, it calls for investigating the potential strategies that can simultaneously rescue the defects of autophagy and UPS. In keeping with this, it is worth of mentioning that UPS exists as two alternative isoforms, the standard 26S proteasome and the immuno-proteasome (SP and IP, respectively). It is remarkable that the mTOR pathway also modulates the switch between these alternative subtypes of UPS, which evolution has preserved in order to optimize different tasks according to specific cell demands [154,155,156,157]. In fact, SP is ubiquitously expressed in all eukaryotic cells, and it is generally enhanced by mTOR inhibition, while IP is an alternative, cytokine-inducible form that is downregulated by mTOR inhibition. Despite overlapping in structure and functions, these alternative UPS isoforms differ in catalytic subunits and substrate specificity [83,84,85,101,102,103]. In fact, the IP operates constitutively in all immune-related cells, including professional APCs (e.g., dendritic cells—DC) and lymphocytes, and thus, it is mostly involved in potentiating innate and adaptive immunity [158].

이 특정 액포 구획의 형성은 신경독성 남용 약물인 메탐페타민(meth)의 투여에 의해 방해받는 반면, 포유류 표적 라파마이신(mTOR)의 억제를 통한 구조는 세포 보호 및 생존과 상관관계가 있습니다[73]. 이는 UPS 및 오토파지 의존성 단백질 분해의 공통 조절자로서 mTOR 경로를 나타내는 연구 결과와 일치합니다[89]. 이러한 연구 결과는 mTOR 억제를 자가포식 및 UPS 의존적 단백질 분해를 시너지적으로 향상시키는 잠재적 전략으로 구성합니다. mTOR는 유비쿼터스적으로 발현되는 세린-트레오닌 키나제로서, 여러 환경 및 세포 내 신호를 감지하고 통합하여 세포 성장 및 대사와 같은 주요 과정을 조율합니다 [55,75,129]. mTOR는 단백질 합성에서의 역할과 관련이 있지만 시냅스 가소성, 염증 및 면역에 널리 관여해왔습니다. 지난 수십 년 동안 mTOR는 주로 자가포식 및 UPS와 관련된 다양한 세포 기능의 중심에 서게 되었습니다. mTOR와 오토파지 및 UPS를 연결하는 새로운 메커니즘은 세포 청소 시스템 간의 긴밀한 상호 의존성을 밝혀냈습니다. 구체적으로, 자가포식 반응의 지속 시간과 진폭은 세린/트레오닌 키나아제 ULK1/Atg1의 안정성에 따라 달라지며, 이는 차례로 UPS와 mTOR에 의해 조정됩니다 [130]. ULK1은 자가포식 개시 및 반응의 여러 단계에서 작용하며, 부분적으로는 Atg13, Beclin 1, Atg9 등 자가포식 단백질을 인산화하여 작용합니다 [131]. mTOR 활성화는 인산화를 통해 ULK1 키나제 활성(따라서 자가포식 개시)을 억제하고 ULK1 신규 단백질 합성도 조율합니다 [130,132].

이러한 맥락에서 UPS는 mTOR/ULK1 의존성 자가포식을 감지하고 조절하는 감시자 역할을 합니다. 실제로 자가포식의 초기 단계에서 UPS는 안정성, 자기 결합 및 키나아제 활성을 유지하기 위해 AMBRA1-TRAF6(E3 리가제) 복합체를 통해 ULK1의 K63-연결 폴리유비퀴틴화를 매개합니다[133]. 반대로, 장기간 영양 결핍이 지속되는 동안 UPS는 Cullin/KLHL20 의존성 K48 연결 폴리유비퀴틴화에 따라 ULK1을 표적으로 삼아 분해하여 자가포식 반응의 피드백 제어를 제공합니다 [134]. 결과적으로 자가포식은 “프로테오파지”로 알려진 현상인 오토파지솜으로 셔틀되는 비활성 UPS 서브유닛의 분해를 통해 UPS 효능과 활동을 제어할 수 있습니다[135,136,137]. 이는 UPS 억제 시 오토파지에서 관찰되는 흥미로운 효과를 설명할 수 있으며, 세포 항상성에서 오토파지-UPS 누화의 중요성을 상기시켜 줍니다. 실제로 자가포식 또는 UPS 중 하나만 억제하면 세포 생존에 해로운 영향을 미칠 수 있으며[138,139,140,141,142,143], 이는 두 세포 청소 시스템에 의한 단백질 턴오버 장애와 관련이 있습니다. 예를 들어, 자가포식 억제는 UPS의 업스트림 또는 촉매 활성 수준에 영향을 미쳐 유비퀴틴화된 기질의 축적을 초래합니다[144,145]. 반대로, UPS 억제는 단백질 과부하에 대처하고 세포 생존을 위한 초기 보상 반응으로서 자가포식의 강화를 유도할 수 있습니다 [146,147,148].

후기 단계의 UPS 기능 장애는 미토파지를 방해하고 Atg9 및 LC3B와 같은 필수 자가포식 단백질의 수준을 감소시키기 때문에 이러한 효과는 일시적인 것으로 밝혀졌습니다 [93]. UPS는 리소좀 막 융합에 필수적이며[149,150], 이는 다시 오토파지의 후기 단계에 관여하기 때문에 이는 놀라운 일이 아닙니다. 실제로 UPS는 자가포식 의존성 소포체화 및 자가포식 막 융합을 포함한 모든 세포 이동 메커니즘에 관여하는 Rab GTPase(뇌의 GTP 결합 Ras 단백질)의 활성을 조절합니다[150,151,152,153]. 이러한 연구 결과는 오토파지와 UPS 간의 시너지 및 보상 기능적 상호 작용을 두 시스템만을 조절하는 실험적 접근 방식에서 고려할 필요가 있음을 시사합니다. 한편으로 이는 오토파지와 UPS의 효과를 단독으로 평가할 때 혼란스러운 결과를 초래할 수 있으며, 다른 한편으로는 오토파지와 UPS의 결함을 동시에 해결할 수 있는 잠재적 전략을 조사할 것을 요구합니다. 이와 관련하여 UPS는 표준 26S 프로테아좀과 면역 프로테아좀(각각 SP 및 IP)이라는 두 가지 대체 이소형으로 존재한다는 점을 언급할 가치가 있습니다. mTOR 경로가 특정 세포의 요구에 따라 다양한 작업을 최적화하기 위해 진화가 보존해 온 이러한 대체 아형 UPS 간의 전환을 조절한다는 점은 주목할 만합니다[154,155,156,157]. 실제로 SP는 모든 진핵세포에서 유비쿼터스적으로 발현되며 일반적으로 mTOR 억제를 통해 강화되는 반면, IP는 mTOR 억제를 통해 하향 조절되는 사이토카인 유도 가능한 대체 형태입니다. 구조와 기능이 겹치지만 이러한 대체 UPS 동형체는 촉매 서브유닛과 기질 특이성이 다릅니다[83,84,85,101,102,103]. 실제로 IP는 전문 APC(예: 수지상세포-DC)와 림프구를 포함한 모든 면역 관련 세포에서 구성적으로 작동하므로 선천성 및 적응성 면역 강화에 주로 관여합니다[158].

Figure 1. A schematic overview of the cell-clearing systems, with a focus on the interplay between autophagy and proteasome. The cartoon offers a rough schematization of the autophagy pathway, starting from the phagophore biogenesis staining for LC3 and Beclin1, which engulfs cytoplasmic portions containing insoluble aggregates, ubiquitinated substrates, and damaged organelles. The phagophore membrane seals to from the autophagosome, which then fuses with the late endosomes to generate the amphisome. This latter fuses with lysosomes to generate the autophagosome (staining for LC3, Beclin1, Rab24, VPS34, and p62) where cargo degradation occurs. At the same time, ubiquitinated substrates can be degraded by the 26S ubiquitin proteasome (UPS), which is formed by the regulatory subunits 19s and the catalytic subunits 20s. Signals such as p62 contribute to sort ubiquitinated proteins for either UPS or autophagy degradation. At the same time, p62 may serve as a signal to promote the merging of UPS and autophagy into a single organelle, “autophagoproteasome”, where potentiated cell-clearance may take place. Alternatively, the autophagy-dependent degradation of inactive UPS subunits may occur within this compartment, a phenomenon that is named “proteophagy”. Dotted arrows indicate the formation of insoluble aggregates from misfolded proteins and their ubiquitination (red), the shuttling of substrates to the phagophore (blue), and the fusion of endosomes and lysosomes with autophagy vacuoles (blue). Solid blue arrows indicate the progression of the autophagy machinery, the shuttling of ubiquitinated substrates to the UPS, and the shuttling of the UPS within autophagosomes.

Remarkably, persistent oxidative/inflammatory stress may concomitantly affect autophagy flux and IP–SP switch, either in the immune periphery or within the CNS. This is expected to alter the clearing capacity and/or substrate specificity of the cell-clearing systems, while triggering a cascade of molecular events that synergize to produce synaptic dysfunctions/toxicity, along with a loss of auto-immune tolerance up to the development of CNS-directed inflammatory and auto-immune reactions. In the present manuscript, we discuss the role of autophagy, SP, and IP at the level of classic neuronal and immunological synapses, while posing a special emphasis on their effects at the level of hybrid junctions, which establish “neuro-immunological synapses” between immune and nerve cells. This is critical to comprehend those autophagy and UPS-dependent mechanisms that finely tune T-cells populations that migrate to the CNS. Again, the role of autophagy and UPS is seminal to disclose those molecular events, which induce neurons and glia to behave as competent APCs, and, as such, become possible targets for auto-immune damage.

그림 1. 자가포식과 프로테아좀의 상호 작용에 초점을 맞춘 세포 청소 시스템의 개략적인 개요.

이 만화는 불용성 응집체, 유비퀴틴화된 기질, 손상된 소기관을 포함하는 세포질 부분을 포식하는 LC3 및 Beclin1의 식세포 생체 생성 염색에서 시작하여 자가포식 경로의 대략적인 도식을 보여줍니다. 식세포막은 오토파지솜으로부터 봉인된 다음 후기 엔도솜과 융합하여 앰피솜을 생성합니다. 이 후자는 리소좀과 융합하여 화물 분해가 일어나는 오토파지좀(LC3, Beclin1, Rab24, VPS34 및 p62 염색)을 생성합니다. 동시에, 유비퀴틴화된 기질은 조절 서브유닛 19와 촉매 서브유닛 20에 의해 형성되는 26S 유비퀴틴 프로테아좀(UPS)에 의해 분해될 수 있습니다. p62와 같은 신호는 UPS 또는 오토파지 분해를 위해 유비퀴틴화된 단백질을 분류하는 데 기여합니다. 동시에, p62는 UPS와 자가포식이 하나의 세포소기관인 “자가포식 프로테아좀”으로 합쳐지는 것을 촉진하는 신호로 작용하여 강화된 세포 청소가 일어날 수 있습니다. 또는 이 구획 내에서 “프로테오파지”라는 이름의 비활성 UPS 하위 유닛의 자가포식 의존적 분해가 발생할 수 있습니다. 점선 화살표는 잘못 접힌 단백질에서 불용성 응집체의 형성 및 그 유비퀴틴화(빨간색), 기질이 식세포로 이동(파란색), 엔도솜과 리소좀이 자가포식 액포와 융합(파란색)함을 나타냅니다. 파란색 실선 화살표는 오토파지 기계의 진행, 유비퀴틴화된 기질과 UPS의 셔틀링, 오토파지솜 내에서 UPS의 셔틀링을 나타냅니다.
놀랍게도, 지속적인 산화/염증 스트레스는 면역 말초 또는 CNS 내에서 자가포식 플럭스와 IP-SP 스위치에 동시에 영향을 미칠 수 있습니다. 이는 세포 제거 시스템의 제거 능력 및/또는 기질 특이성을 변화시키는 동시에 시냅스 기능 장애/독성을 생성하기 위해 상승 작용을 하는 일련의 분자 사건을 촉발하고, 자가 면역 내성의 상실과 함께 CNS 주도 염증 및 자가 면역 반응의 발달까지 유발할 것으로 예상됩니다. 본 원고에서는 고전적인 신경 및 면역 시냅스 수준에서 자가포식, SP, IP의 역할을 논의하는 한편, 면역세포와 신경세포 사이에 “신경 면역 시냅스”를 형성하는 하이브리드 접합 수준에서의 영향에 대해 특별히 강조하고 있습니다. 이는 CNS로 이동하는 T세포 집단을 미세하게 조정하는 자가포식 및 UPS 의존 메커니즘을 이해하는 데 매우 중요합니다. 다시 말하지만, 자가포식과 UPS의 역할은 뉴런과 신경교세포가 유능한 APC로 행동하도록 유도하여 자가면역 손상의 표적이 될 수 있는 분자적 사건을 밝히는 데 매우 중요합니다.

3. Autophagy and Proteasome Tune Synaptic Plasticity by Modulating Neurotransmission and Immunity

Autophagy and SP are constitutively expressed in neurons either in the cell body, nucleus, or synapses, where they modulate synaptic plasticity by surveilling oxidative stress, gene transcription, and neurotransmitter release [22,159,160,161,162,163,164,165,166,167,168,169,170,171]. Autophagy and SP operate at various sub-cellular levels in both pre- and post-synaptic sites, and in detail, they are the following:

(i) intersect with secretory pathways to modulate SV trafficking, as well as the size and number of SV pools [21,128,161,172,173];

(ii) degrade protein isoforms and presynaptic chaperone proteins such as alpha-synuclein, beta amyloid, and tau [62,65,108,174,175], which, when altered in either amount or conformation, can drive synaptic dysfunctions [176,177,178,179];

(iii) modulate the rate and duration of neurotransmitter release (including DA and GLUT) by degrading whole SVs (in the case of autophagy), as well as soluble Nsf attachment protein receptor (SNARE) and accessory proteins, which are involved in SV exo-/endo-cytosis [21,22,152,161,162,166,167,168,180,181,182,183];

(iv) foster the internalization and degradation of DA and GLUT receptors, which are coupled with downstream intracellular cascades driving metabolic, transcriptional, and epigenetic changes within neurons [11,98,184,185,186,187].

As such, autophagy and SP are deeply involved in those mechanisms driving synaptic plasticity, such as long term-potentiation and -depression, which are directly related to neuronal and behavioral phenotypes. In fact, the inhibition of either SP or autophagy in experimental models produces profound alterations in neurotransmitter release and the expression‎ of neurotransmitter receptors [31,161,173,181,182,183]. Reiterated stimuli that alter neurotransmitter activity are seminal to induce maladaptive changes in synaptic strength and connectivity, which translate into long-lasting psychomotor changes. In the last decades, experimental evidence has accumulated, suggesting that early synaptic alterations may represent a major event fostering neuronal degeneration [2,188,189].

This is best exemplified by the mechanisms of action of abused drugs such as meth, which produces psychiatric alterations including addiction and psychoses, and even neurotoxicity affecting the DA terminals, DA cell bodies, and post-synaptic neurons of the DA circuitry within the striatum, iso-cortex, and limbic brain areas [11,190,191,192]. This occurs through the joined contribution of epigenetic events and protein alterations (oxidation, aggregation, and spreading) arising from abnormal DA release, the abnormal pulsatile stimulation of DA receptors, and also the increased responsiveness of neurons to GLUT and GLUT exitotoxicity. In fact, abnormal levels of DA and abnormal stimulation of DA receptors play a key role in GLUT excitotoxicity, which stands for the over-activation of specific types of GLUT receptors, resulting in neuronal death, tissue damage, and loss of brain function, as it occurs both during meth toxicity and in various neurological diseases [2,193].

Both autophagy and UPS are severely affected by meth administration [70,71,72,73], while the mTOR inhibitor rapamycin prevents both the behavioral and neurotoxic effects of meth by rescuing autophagy and UPS [73,194]. This is in line with several studies showing that the genetic or pharmacological occlusion of autophagy and UPS leads to the accumulation of ubiquitinated protein-aggregates and recapitulates neurodegeneration [138,139,140,141,142,143,195]. As a support to these findings, SP and autophagy dysfunctions occur in human brain disorders characterized by early synaptic dysfunctions, which precede protein aggregation [6,21,75,196,197,198]. On the other hand, mTOR inhibition, which is supposed to restore both autophagy and UPS activity, ameliorates early psychomotor and cognitive behavioral alterations by recuing neurotransmission defects and by restoring proteostasis in a variety of CNS disorders, both in humans and experimental models [75,175,194,199,200,201,202].

3. 오토파지와 프로테아좀은 신경전달과 면역을 조절하여 시냅스 가소성을 조정합니다.

오토파지와 SP는 세포체, 핵 또는 시냅스에서 뉴런에서 구성적으로 발현되며 산화 스트레스, 유전자 전사 및 신경 전달 물질 방출을 감시하여 시냅스 가소성을 조절합니다 [22,159,160,161,162,163,164,165,166,167,168,169,170,171]. 오토파지와 SP는 시냅스 전후 부위에서 다양한 하위 세포 수준에서 작동하며, 세부적으로 살펴보면 다음과 같습니다:

(i) SV 트래피킹과 SV 풀의 크기와 수를 조절하기 위해 분비 경로와 교차합니다[21,128,161,172,173];
(ii) 알파-시누클레인, 베타 아밀로이드, 타우[62,65,108,174,175]와 같은 단백질 이소형 및 시냅스 전 샤페론 단백질을 분해하며, 그 양이나 형태가 변경되면 시냅스 기능 장애를 유발할 수 있습니다[176,177,178,179];
(iii) 전체 SV(자가포식의 경우)는 물론, SV 세포외/내 세포증식에 관여하는 가용성 Nsf 부착 단백질 수용체(SNARE) 및 보조 단백질[21,22,152,161,162,166,167,168,180,181,182,183]을 분해하여 신경 전달 물질 방출 속도와 기간(DA 및 GLUT 포함)을 조절합니다[21,22,152,161,162,166,167,168,183];
(iv) 뉴런 내에서 대사, 전사, 후성유전학적 변화를 주도하는 세포 내 캐스케이드와 결합된 DA 및 GLUT 수용체의 내재화 및 분해를 촉진합니다[11,98,184,185,186,187].

따라서 오토파지와 SP는 신경세포 및 행동 표현형과 직접적으로 관련된 장기 강화 및 억제와 같은 시냅스 가소성을 유도하는 메커니즘에 깊이 관여합니다. 실제로 실험 모델에서 SP 또는 오토파지를 억제하면 신경전달물질 방출과 신경전달물질 수용체의 발현에 중대한 변화가 생깁니다[31,161,173,181,182,183]. 신경전달물질 활동을 변화시키는 반복적인 자극은 시냅스 강도와 연결성에 부적응적인 변화를 유도하여 오래 지속되는 정신운동 변화를 유발하는 데 매우 중요합니다. 지난 수십 년 동안 실험적 증거가 축적되어 초기 시냅스 변화가 신경세포의 퇴행을 촉진하는 주요 사건일 수 있음을 시사합니다[2,188,189].

이는 중독과 정신병을 포함한 정신과적 변화를 일으키는 필로폰과 같은 남용 약물의 작용 메커니즘과 선조체, 등피질 및 변연계 뇌 영역 내 DA 회로의 DA 단자, DA 세포체 및 시냅스 후 뉴런에 영향을 미치는 신경독성에서 가장 잘 드러납니다[11,190,191,192]. 이는 비정상적인 DA 방출, DA 수용체의 비정상적인 박동성 자극, GLUT 및 GLUT 출구 독성에 대한 뉴런의 반응성 증가로 인해 발생하는 후성유전학적 사건과 단백질 변화(산화, 응집 및 확산)의 결합된 기여를 통해 발생합니다. 실제로 비정상적인 DA 수치와 DA 수용체의 비정상적인 자극은 특정 유형의 GLUT 수용체가 과도하게 활성화되어 신경세포 사멸, 조직 손상 및 뇌 기능 상실을 초래하는 GLUT 흥분 독성에서 중요한 역할을 하며, 이는 메스 독성 및 다양한 신경 질환에서 모두 발생합니다[2,193].

오토파지와 UPS는 모두 필로폰 투여에 의해 심각하게 영향을 받는 반면[70,71,72,73], mTOR 억제제 라파마이신은 오토파지와 UPS를 구조함으로써 필로폰의 행동 및 신경 독성 효과를 모두 예방합니다[73,194]. 이는 자가포식 및 UPS의 유전적 또는 약리학적인 폐색이 유비퀴틴화된 단백질 응집체의 축적을 초래하고 신경 퇴화를 요약한다는 여러 연구 결과와 일치합니다[138,139,140,141,142,143,195]. 이러한 연구 결과를 뒷받침하듯, 단백질 응집에 앞서 초기 시냅스 기능 장애를 특징으로 하는 인간 뇌 질환에서 SP 및 오토파지 기능 장애가 발생합니다[6,21,75,196,197,198]. 반면, 오토파지와 UPS 활동을 모두 회복시키는 것으로 알려진 mTOR 억제는 인간과 실험 모델 모두에서 신경 전달 결함을 회복하고 다양한 CNS 장애에서 단백질 정체 상태를 회복시킴으로써 초기 정신 운동 및 인지 행동 변화를 개선합니다 [75,175,194,199,200,201,202].

3.1. Autophagy- and Proteasome-Dependent Neurotransmission Linking Immune-Cells’ Activity and Synaptic Plasticity

As modulators of neurotransmitter release, autophagy and SP also modulate CNS-directed immune responses by operating at the level of the neuro-immunological synapse, which may be established between the sympathetic nerve terminals and T-cells within lymphoid organs [12]. Remarkably, both SP and autophagy modulate the release of DA [161,162,181,182,183], which besides being crucial for brain functions such as movement, cognition, attention, memory, and reward [203], also orchestrates the differentiation, maturation, selection, trafficking, and migration of T-lymphocytes [34,35,36,204,205,206]. In fact, T-cells express G-coupled D1-like (D1 and D5) and D2-like (D2, D3, and D4) DA receptors, and just like it occurs for the neurons, the magnitude and duration of the DA release is key to trigger the specific metabolic and intracellular cascades, switching T-cells phenotype and function [35,36,206]. As thoroughly revised elsewhere, depending on the DA concentration and the pattern of stimulation of specific DA receptors, naïve T-cells may be induced to differentiate into either memory, regulatory, or effector cells, including CD4+ T helper (Th) 1, 2, or 17, and CD8+ cytotoxic T-lymphocyte (CTL) phenotype [34,35,36,206]. In this context, the autophagy- and SP-dependent surveillance of the DA release at the level of the neuro–immunological synapse is expected to guarantee the physiological stimulation of the DA-receptors placed on the T-cells, and control the neuro–immune activity (Figure 2). The circulation of T-lymphocytes in the brain occurs physiologically, since the early development, and persists during adulthood, to guarantee synaptic plasticity [8,207,208]. For instance, both CD4+ and CD8+ T-cells are essential for spinogenesis and GLUT synaptic function in the hippocampus [209]. In addition, CD8+ T cells regulate the hippocampal volume by promoting neurogenesis [210]. Intriguingly, peripheral and brain-infiltrating T-cells, besides regulating GLUT synaptic transmission and plasticity, are regulated themselves by GLUT [2,37]. Both autophagy and UPS modulate, and are in turn modulated by GLUT transmission [95,211,212,213]. GLUT is a major excitatory neurotransmitter, which besides being critical for the brain’s development and function, participates in tuning the T-cells activity. In fact, ionotropic and metabotropic GLUT receptors are differently expressed among resting and activated T-cells, as well as in different T-cells subtypes [2,37]. At low physiological concentrations, GLUT promotes T-cell adhesion, migration, proliferation, and protection of activated T-cells from Ag-induced apoptotic cell death. Yet, depending on the abnormalities concerning either the GLUT concentration, stimulation of specific GLUT receptors, or the presence of other converging stimuli (such as inflammatory cytokines or other neurotransmitters), GLUT may profoundly affect T-cells activity, thus playing an active role in immune diseases [2,37]. Remarkably, brain infiltrating T-cells were shown to respond to GLUT by activating a neuroprotective pathway, thus providing a potential feedback regulatory mechanism to limit GLUT excitotoxic damage in the CNS [214]. A loss of GLUT-mediated responsiveness of T-cells has been described in MS [215]. Furthermore, various alterations in CNS-circulating T-lymphocyte populations are described in both classic and autoimmune degenerative disorders, such as PD, AD, and MS [216]. Emerging evidence also indicates an association between early inflammatory mechanisms underlying neurodegeneration, and synaptic alterations involving abnormal levels of DA and/or GLUT, as well as the deregulation of their receptors on T-cells [2,37,217,218,219]. In line with this, specific modulators of DA and/or GLUT activity, may have beneficial effects, not only in classic neurodegenerative diseases, but also in auto-immune CNS disorders such as MS [2,35,217].

Figure 2. Autophagy and proteasome modulate immune activity by surveilling dopamine (DA) release. The standard proteasome (SP) and autophagy blunt DA release by degrading entire synaptic vesicles (SVs), as well as soluble Nsf attachment protein receptor (SNARE)- and SNARE associated-proteins, which foster synaptic vesicle exocytosis. In fact, they both prevent the rapid recycling of SV proteins back to the plasma membrane, which would otherwise lead to a further round of exocytosis. In this way, SP- and autophagy-dependent amount and duration of DA released at the level of the neuro-immunological synapse surveils the stimulation of DA receptors expressed on T-cells. This is seminal to modulate the differentiation of T-cells toward cytotoxic-, regulatory-, or helper-T-cells, as well as T-cell migration in periphery. For instance, the abnormal stimulation of D1-like receptors increases cyclic adenosine monophosphate (cAMP) levels to inhibit cytotoxic CD8+ cells; it impairs the differentiation and activity of T-regulatory cells, while inducing polarization of naive CD4+ cells toward the Th17 phenotype. On the other hand, the stimulation of D2-like receptors induces the differentiation of CD8+ cells into cytotoxic T-lymphocytes, induces the polarization of naive CD4+ cells toward the Th1 phenotype, and controls T-cell migration and adhesion. Dotted blue arrows indicate the progression of the SV cycle. Solid blue arrows indicate the targeting and shuttling of SNARE proteins to the UPS and autophagy. Solid red arrows indicate the increase/decrease in SNAREs degradation/recycling and exocytosis rate. Dotted red lines indicate the induction (arrows) or inhibition (lines) of naïve T-cells differentiation towards various phenotypes following abnormal stimulation of DA receptors.

Besides the effects in T-cells within the lymphoid organs, DA and GLUT release may also modulate the activity of immune cells, including glia and T-cells, directly in the CNS. In line with this, a number of studies pointed to the unexpected ability of naïve CD4+ and CD8+ T-cells to infiltrate the brain parenchyma [38,39,40,41,42,43,44,45,46,47]. This occurs mostly during pro-inflammatory conditions, which enhance naïve T-cell recruitment in the CNS, while fostering T-cells activation and phenotypic commitment once they encounter activated glial cells exposing MHC-bound Ags. Thus, the effects of DA and GLUT on brain-infiltrating naïve T-cells may synergize with local Ag presentation in order to dictate the activation or suppression of T-cells directly in the CNS. However, as a general consensus view, only peripherally activated T-cells can migrate into the brain. If they encounter a CNS-resident APCs exposing the cognate Ag, T-cells become re-activated and recruit their effector machineries to produce cytotoxicity or cytokine release. In this scenario, the effects of DA and GLUT focus mostly on the glial cells, which behave as CNS-resident immune cells. Inflammatory cytokines (such as those released by brain infiltrating T-cells) synergize with neurotransmitters to induce the activation of the glial cells, which encompass the morphological changes, increased proliferation rate, and ability to operate as APCs [9,220,221]. Nonetheless, because of an intimate functional association with the synapses, the glia is deeply involved in synaptic plasticity [9]. Experimental studies suggest that activated microglia are responsible for the synaptic alterations observed in a variety of neurological disorders [2,222]. Both microglia and astrocytes express many different neurotransmitter receptors (including DA and GLUT receptors), which, when stimulated, foster the release of soluble factors acting, in turn, on neurons to alter neurotransmitter release, neurotransmitter receptor activation, and synaptic efficacy [2,9]. Besides neurotransmitters such as DA and GLUT, these include mediators of constitutive immunity such as tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), and interleukin 1 beta (IL-1β); pentraxins; and growth factors such as brain-derived neurotrophic factor (BDNF), which altogether influence synaptic activity, mostly by enhancing the long-term potentiation of excitatory transmission [2,9,77,81,223]. Once again, in this context, autophagy and UPS configure as actors in the communication between T-cells, glia, and neurons, as they (i) surveil neurotransmitter release, which is important for glial, lymphocytic, and neuronal activity; (ii) determine the metabolism and activity of the glia and lymphocytes and the subsequent production and release of soluble factors (paragraph 3.2); and (iii) process Ag peptides, which are presented to T-cells by DCs, glial cells, or neurons (paragraph 3.3).

3.2. Cell Clearing System in the Metabolism and Fate of Immune Cells

Similar to what occurs in the neurons, the activity of immune cells largely depends on UPS and autophagy. The multitude of metabolic changes that occur upon glial and T-cells activation is tightly intermingled with autophagy and UPS activity. As detailed in the previous paragraph, both UPS and autophagy regulate DA and GLUT release, and are also involved in the turnover of DA and GLUT receptors, thus influencing the metabolic cascades, which participate in T-cells and glia activation. Moreover, both autophagy and UPS modulate the turnover of inflammatory-related transcription factors such as nuclear factor k beta (NF-κB), which, in turn, fosters the production of cytokines by glia or T-cells [25,224,225,226]. Within T-cells, autophagy and UPS directly govern the metabolic cascades, which dictate T-cells differentiation, function, and activity [6,158,226]. Again, such an overlapping task may be bound to mTOR activity, which is deeply involved in T-cell metabolism [227]. Moreover, the co-existence of UPS and autophagy degradation pathways enables APCs (including peripheral DCs and glia) to present either endogenous or exogenous Ag peptides, which is key for determining the T-cells state [30]. While autophagy operates constitutively in all immune cells, the UPS exists mostly as IP, which is an alternative, cytokine-inducible isoform of the SP possessing enhanced chymotrypsin-like activity and peculiar structural features, compared with SP [228,229,230,231,232]. In fact, within IP, β1, β2, and β5 subunits of the SP-20S catalytic core are replaced with β1i, β2i, and β5i. Among these, β1i possesses a chymotrypsin-like activity contrarily to the standard counterpart possessing a caspase-like activity. Thus, compared with SP, IP produces, more efficiently, Ag peptides with C-terminal hydrophobic amino acids, which are suitable for binding the groove of MHC class I molecules [228,229,230,231,232]. The major task of IP is to process either endogenous or exogenous proteins, and generate Ag peptides, which are first complexed to MHC-I in the endoplasmic reticulum, and then exposed on the plasma membrane of APCs for either direct or cross-presentation to CD8+ T-lymphocytes. Thus, the generation of defined T-cell epitopes and the expression‎ of MHC-I molecules largely depend on the IP activity [6,35,228,229,230,231,232]. Similar to IP, autophagy is key in adaptive immunity, though it is mostly implicated in the MHC-II restricted presentation of exogenously-derived Ag to CD4+ T cells [23,30]. Nonetheless, a few reports demonstrated that autophagy can also process and load endogenous (viral) peptides to MHC-I [233]. Remarkably, autophagy is implicated in MHC class I molecules internalization and degradation, thus influencing MHC-I stability at the plasma membrane of APCs, and subsequent CD8+ T-cell responses [234,235]. In fact, autophagy inactivation within APCs occludes the surface internalization of MHC-I molecules, leading to an increased Ag presentation and enhanced CD8+ T cell responses against viral peptides, both in vitro and in vivo [233]. Thus, autophagy fosters MHC II-restricted Ag presentation, while controlling MHC-I expression‎ [233,234,235]. Autophagy also provides an alternative pathway to the direct IP- and MHC-I-dependent Ag presentation pathway [236]. In this case, Ags normally targeted to autophagy and exposed by MHC-II can also be loaded to MHC-I in recycling endosomes, which is seminal to trigger adaptive immune response upon viral infections. In the immune periphery, autophagy- and UPS-dependent Ag processing is also seminal for T-cells thymic selection. In the thymus, specialized forms of IP operate together with SP and autophagy to finely-tune T-cell proliferation, along with positive and negative T-cell selection [228,237]. In this way, UPS (SP and IP) and autophagy coordinately guarantee immune-tolerance and define the pool of immunocompetent T-cells, which are released in the bloodstream to reach secondary lymphoid organs, and subsequently, the brain.

4. Autophagy and Proteasome Linking Altered Immunity and Synaptic Plasticity with Neurodegeneration

In neurons and glial cells, autophagy and SP operate constitutively, while the IP is generally induced by the pro-inflammatory cytokines IFNγ and TNFα, and by oxidative stress [85,158,229]. These challenging conditions contribute to disassemble SP for the sake of IP induction, which is likely to cope with the protein overload, as it is endowed with an enhanced catalytic activity [238,239]. Remarkably, the IP cleaves both microbial- and oxidized/aggregated-proteins to produce immunogenic peptides, which are exposed on glial and neuronal MHC-I for presentation to CD8+ T cells. In fact, IP is able to degrade aggregation-prone proteins such as alpha-synuclein and beta amyloid, which are conventionally degraded by SP and autophagy, although some debate still exists concerning the degradation rate and efficacy of IP compared with SP [238,240,241]. In any case, the IP-dependent degradation of aggregation-prone proteins produces Ag peptides, which activate adaptive immunity [6,238]. This provides an oxidation-linked explanation for the baseline activity of UPS in neuro-immune surveillance [85,238]. IP recruitment may serve as a compensatory pro-survival mechanism, allowing cells to quickly expand the peptides repertoire and aid immune defense in a challenged organism. This is supported by the fact that IP also operates in baseline conditions in neurons and glia, which indeed express low amounts of IP and MHC-I, even in the absence of cytokine stimulation [238,242,243]. In line with this, MHC-I-selective expression‎ within the neurons and glia throughout the brain and spinal cord extends well beyond a classic antigen-presenting role. In fact, the MHC-I neuronal expression‎ is key in early neuronal development, axonal regeneration, synaptic plasticity, reward, and memory [243,244,245,246]. Nonetheless, IP induction is a tightly regulated and transient response, as cells must rapidly switch back to SP once the IP function is no longer required [247]. Abnormal IP expression‎ and the subsequent MHC-I-dependent Ag presentation enhances the APC-like behavior of neurons, and, as such, increases their susceptibility to CD8+ auto-immune attack. In fact, a dramatic increase in the amount of IP is bound to an abnormal auto-immune response in a variety of CNS disorders [6]. As recently reviewed, IP is significantly and constantly up-regulated in the glia and neurons, both in patients and experimental models of classic and auto-immune neurodegenerative disorders [6]. Nonetheless, the functional role of IP induction differs between auto-immune compared with classic neurodegenerative disorders. In neurodegenerative disorders such as PD, AD, and HD, the upregulation of IP occurs as a compensatory response to cope with inflammatory conditions that develop during proteinopathy, when SP is downregulated [6,238,248,249]. In fact, general UPS inhibitors targeting both SP and IP produce a detrimental effect, which recapitulates neurodegeneration, while selective IP inhibitors have only limited beneficial effects in the models of neurodegenerative disorders [6]. On the other hand, in auto-immune disorders, including MS and experimental autoimmune encephalomyelitis (EAE), IP inhibitors significantly ameliorate neurological and inflammatory disease scores [6,250]. There is also evidence indicating that a combination of autophagy and IP inhibitors may be an effective strategy against EAE [251]. In keeping with this, a number of studies reported that exposure to cytokines, such as IFN-γ, also up-regulates autophagy to promote the activation of innate and adaptive auto-immunity [252]. In this context, autophagy has been suggested to represent a tolerance-avoidance mechanism, being strongly recruited during CD4+ T-cells activation [253]. Instead, autophagy inhibition induces a long-lasting state of hypo-responsiveness within T-cells [253]. In vivo, autophagy inhibition during Ag priming induces T-cell energy, and decreases the severity of disease in EAE [253]. On the other hand, studies in humans showed that autophagy activity is not increased in neither the peripheral nor brain-circulating CD4+ T cells of MS patients compared with controls, despite having increased Atg5 gene and protein levels [254]. Other studies centered on the role of microglia-related inflammation suggest that autophagy induction via mTOR inhibition contributes to reducing both demyelination and inflammation in EAE [255]. As far as it concerns neurodegenerative disorders, autophagy induction seems to play a beneficial effect in counteracting acute and chronic inflammation [256]. For instance, in an in vitro model of PD, TNF-α was shown to impair autophagy flux in microglia, while fostering microglia polarization towards the pro-inflammatory phenotype M1 [257]. The inhibition of autophagy consistently aggravates M1 polarization induced by TNF-α, and remarkably, autophagy inhibition alone is sufficient to trigger microglia activation toward M1 status, along with producing neurotoxicity [257]. Conversely, the upregulation of autophagy via serum deprivation or pharmacologic activators (rapamycin and resveratrol) promotes microglia polarization toward the M2 phenotype, thus fostering inflammation resolution and preventing neurotoxicity [257]. Again, enhancing autophagy in the microglia in an in vitro model of AD promotes the degradation of the phagocytosed fibrils of amyloid beta, along with restraining the inflammasome activation and pro-inflammatory cytokine release [258]. This is in line with findings indicating that impaired autophagy in microglia associates with synaptic defects, and with the subsequent psychiatric alterations observed in experimental models [76,77]. Again, the disruption of autophagy within neurons occurs following infection-induced microglial activation, which results in neurodegeneration [52]. This is in line with the plethora of studies pointing at autophagy dysfunction in neurodegenerative disorders, such as AD, PD, and HD. In these disorders, a progressive dysfunction of autophagy within the CNS is reminiscent of that reported for SP. In a scenario where the autophagy–UPS interplay appears critical, it is worth of considering some overlapping molecular mechanisms that may operate in various CNS disorders to foster neuro-inflammation and maladaptive synaptic plasticity through IP induction and concomitant SP-autophagy downregulation. Remarkably, autophagy and UPS activities are influenced by the same intracellular cascades that are triggered by the DA receptors expressed on the neurons and glia. In fact, signaling pathways placed downstream to plasma membrane D1-like and D2-like DA receptors converge on the mTORC1 pathway [96], which, in turn, may either suppress or enhance the baseline SP/IP and autophagy activities, depending on the pattern of stimulation of the specific DA receptors. Thus, a feedback loop is established between DA signaling and mTOR-dependent cell-clearing systems in neurons, glia, or even in T-cells. The intrinsic oxidative potential of DA, along with the abnormal stimulation of DA receptors, are primary candidates fostering protein oxidation, inflammation, impairment of autophagy flux, SP disassembly, and the subsequent IP upregulation [48,71,96,97,99,259,260] (Figure 3). This is supported by the effects of exogenously administered DA precursors in enhancing neuronal Ag presentation via MHC-I, and the subsequent activation of CTLs [48], which, in fact, is a major task of the IP.

Figure 3. The effects of abnormal DA release and stimulation of DA receptors on proteasome and autophagy. An abnormal amount of intracellular DA may foster the loss of compartmentalized physiological oxidative deamination of DA, which readily undergoes auto-oxidation to produce toxic quinones and highly reactive chemical species such as reactive oxygen species (ROS). In turn, these react with sulfhydryl groups and promote the structural modifications of proteins, lipids, and nucleic acids within the DA axon terminals and surrounding compartments. Structural modifications of proteins translate into the formation of insoluble aggregates overwhelming both the SP and autophagy degradative potential. At the same, the abnormal release of DA produces an abnormal stimulation of post-synaptic DA receptors, which are coupled with intracellular cascades such as protein kinase A and C (PKA/PKC). The non-canonical activation of these cascades promotes the hyper-phosphorylation and activation of glutamate (GLUT) receptors and ion channels, which foster GLUT hyper-responsivity and Ca2+ uptake converging in the increase of oxidative stress. Again, the intracellular cascades placed downstream of the DA receptors (mostly D1-like) converge on activating the mammalian target of rapamycin (mTOR) pathway, thus promoting SP and autophagy downregulation. Red arrows in bold indicate increased levels. Plain red arrows indicate the formation of DA-derived toxic quinones and oxidized/misfolded proteins up to insoluble aggregates, and the progression of the various metabolic cascades that arise from abnormal stimulation of DA receptors.

As a consequence of the autophagy and SP dysregulation, indigested misfolded or oxidized substrates may perpetuate inflammation through the release of danger-associated molecular pattern molecules (DAMPs) (Figure 4). In fact, DAMPs activate NF-κB and the inflammasome to release cytokines such as IFNγ, along with spreading misfolded proteins, advanced glycation end-products (AGEs) and free radicals, which all converge to induce the upregulation of IP within neighboring cells via autocrine or paracrine mechanisms. DAMPs may also stimulate toll-like 4 receptor (TLR4) to impair both SP and autophagy [75]. In this scenario, impaired SP and autophagy can neither digest potentially harmful DAMPs, nor restrain the release of DA and GLUT, which may add on glia activation and the release of pro-inflammatory signals recruiting T-cells within the CNS. In this way, IP upregulation leads to an overproduction of neuronal and glial antigens co-expressed with MHC-I molecules to prime cytotoxic CD8+ T cell response (Figure 4). At the same time, autophagy cannot efficiently provide for the internalization of MHC-I molecules or the degradation of damaged proteins and organelles, which fuels inflammation and immune activation. Thus, alterations of autophagy and UPS may explain why a variety of CNS disorders feature concomitant alterations in neurotransmitter activity, oxidative-inflammatory stress, and inappropriate immune response, which synergize to alter synaptic plasticity and damage neurons.

Figure 4. Molecular mechanisms bridging neuro-inflammation, immunoproteasome (IP) induction and autophagy-SP dysfunction in the central nervous system (CNS). (1) As a consequence of autophagy and SP dysregulation, indigested misfolded or oxidized substrates may perpetuate inflammation through the release of danger-associated molecular pattern molecules (DAMPs), such as advanced glycation end-products (AGEs), lipopolysaccharides (LPS), and ROS. While DAMPs activate NF-κB and the inflammasome to release cytokines such as interferon gamma (IFNγ), the spreading of indigested misfolded proteins, AGEs, and free radicals in the extracellular space occurs. (2) All of these factors converge to induce an upregulation of IP within the neighboring cells via autocrine or paracrine mechanisms. DAMPs may also stimulate AGE receptors, IFN receptors, and toll-like 4 receptor (TLR4), to converge on molecular pathways such as mTOR, which, in turn, induce IP upregulation and SP-autophagy downregulation. In this scenario, IP upregulation leads to an overproduction of CNS self-antigens co-expressed with major histocompatibility complex (MHC)-I molecules to prime cytotoxic CD8+ T cell response. At the same time, autophagy cannot efficiently provide for the internalization of MHC-I molecules or the degradation of damaged proteins and organelles. (3) Within glial cells, the same DAMPs and cytokines that foster glial activation may contribute to the impairment of autophagy and SP, while up-regulating IP, which is able to process and cross-present phagocytosed proteins via MHC-II for the activation of CD4+ T cells. (4) In this way, IP upregulation, in an attempt to compensate for SP-autophagy downregulation, may fuel inflammation and auto-immune activation to promote altered synaptic plasticity and neuronal damage. Red arrows in bold indicate decreased/increased levels/activity of SP, IP, autophagy, MHC-I, MHC-II. Plain red arrows indicate intra-cellular signaling cascades. Dotted red arrows indicate the extracellular release of DAMPs, cytokines, and their binding to cognate receptors in neighbor cells or phagocytosis by glial cells. Dotted black arrows indicate the shuttling of substrates towards UPS or autophagy, the formation of Ag peptides deriving from UPS cleavage, and the progression of MHC-I-Ag complex from the ER and endosomes to the plasma membrane. The red frame indicates the final effect produced by CD4+ and CD8+ T-cells activation on neurons and glia.

5. Conclusions and Future Directions

The evidence reviewed here suggests that autophagy and UPS are key mediators of synaptic plasticity, being placed at the cross-road between neurotransmission and immune activity. Nonetheless, we are just scratching the surface of the intricate molecular mechanisms that translate autophagy and UPS alterations into specific CNS disorders. Further experimental studies are needed to dissect the correlation between autophagy and UPS status, disease-specificity, and disease-stage. In fact, different effects on autophagy and UPS may occur in auto-immune compared with classic neurodegenerative disorders, because of the different etiologies between these CNS diseases. Moreover, the interdependency between autophagy and UPS may lead to confounding outcomes when assessing the effects of specific compounds, which indeed modulate both systems rather than autophagy or UPS individually. In addition, there are several factors that may contribute to yielding controversial results on autophagy and UPS. One of these may be the biased interpretation of the autophagy or UPS status. In fact, most studies assess autophagy by measuring the amount of LC3B or the number of autophagosomes, which does not necessarily reflect an increased autophagy capacity. Rather, it may reflect a progressive downregulation of autophagy flux due to the impaired fusion of LC3-positive autophagosomes with lysosomes. Again, most studies detect UPS status through antibodies that recognize alpha subunits, which do not allow for distinguishing between the SP/IP ratio. Likewise, measuring the overall UPS catalytic activity does not allow for dissecting whether the contribution derives from SP or IP. In any case, dysregulations of both autophagy and UPS appear as a common signature in a variety of CNS disorders, where synaptic alterations synergize with inflammatory/immune reactions. This calls for further studies aimed at investigating the effects of additional compounds, which can synergistically modulate both UPS (IP and SP) and autophagy, in an attempt to find preventive and/or therapeutic strategies against early synaptic alterations. In keeping with this, mTOR modulators remain, to date, the best candidates for acting on autophagy, IP, and SP. Beyond the gold-standard mTOR inhibitor rapamycin, several phytochemicals, which recently gained increasing interest in CNS disorders due to their adaptogenic, anti-oxidant, and anti-inflammatory effects, act indeed as mTOR modulators. Testing the effects of these compounds on autophagy and SP–IP may disclose a potential correlation between their beneficial effects and cell-clearing pathways.

Author Contributions

Original draft preparation, writing, review and art work, F.L. and F.B.; review, editing, and art work, C.L.B., and L.R.; conceptualization, P.S. and A.F.; supervision, F.F., F.L. and F.B equally contributed to the present work.

Funding

This work was funded by Ministero Della Salute (Ricerca Corrente 2019).

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

AD	Alzheimer’s disease
Ag	Antigen
APC	Antigen presenting cell
BDNF	Brain derived neurotrophic factor
CNS	Central nervous system
CTL	Cytotoxic T-lymphocyte
DA	Dopamine
DAMPs	Danger-associated molecular pattern molecules
DC	Dendritic cell
EAE	Experimental autoimmune encephalomyelitis
GLUT	Glutamate
HD	Huntingtin’s disease
HDAC6	Histone deacetylase 6
IFNγ	Interferon gamma
IL-1β	Interleukin 1 beta
IP	Immunoproteasome
Meth	Methamphetamine
MHC	Major histocompatibility complex
MS	Multiple sclerosis
mTOR	Mammalian target of rapamycin
NF-κB	Nuclear factor K beta
PD	Parkinson’s disease
Rab GTPase	Gtp bound ras proteins in brain
SNARE	Soluble Nsf attachment protein receptor
SP	Standard proteasome
SQSTM1	Sequestosome-1
SV	Synaptic vesicle
TCR	T-cells receptor
TLR4	Toll-like receptor 4
TNFα	Tumor necrosis factor alpha
UPS	Ubiquitin proteasome

References