A Cleaning Crew: The Pursuit of Autophagy in Parkinson's Disease

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A Cleaning Crew: The Pursuit of Autophagy in Parkinson’s Disease Pathik Parekh, Nishant Sharma, Anagha Gadepalli, Abhishek Shahane, Monika Sharma, and Amit Khairnar ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.9b00244 • Publication Date (Web): 06 Aug 2019 Downloaded from pubs.acs.org on August 7, 2019

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ACS Chemical Neuroscience

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+-Full Title: A Cleaning Crew: The Pursuit of Autophagy in Parkinson’s Disease

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Author names: Pathik Parekh1, Nishant Sharma1, Anagha Gadepalli1, Abhishekh Sahane1, Monika

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Sharma1, Amit Khairnar1*

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Author Affiliations:

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1

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Research (NIPER), Ahmedabad, Gandhinagar, Gujarat, India.

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*Corresponding

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Amit Khairnar, Ph. D

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Assistant Professor,

Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and

author:

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National Institute of Pharmaceutical Education and Research (NIPER), Ahmedabad,

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Palaj, Gandhinagar-382355, Gujarat, India.

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Phone: +91-9284349396

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Email: [email protected], [email protected]

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ACS Paragon Plus Environment

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Abbreviations:

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ATG – Autophagy-related genes

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Hsc70 – Heat shock cognate 70KDa protein

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CHIP - Carboxy-terminus of Hsc70-interacting protein

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AP- Autophagosome

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ER- Endoplasmic Reticulum

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ULK1- Unc-51 Like Autophagy Activating Kinase 1

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FIP200- Focal adhesion kinase family-interacting protein of 200 kDa

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VPS34 - Vacuolar protein sorting 34

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PI3KC3 - Class-III Phosphatidyl-inositol-3-kinase-catalytic subunit-3

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LC3I/II – Light chain kinase 3-I/II

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CMA- Chaperone-mediated autophagy

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MA- Macroautophagy

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ALP- Autophagy-lysosomal pathway

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PFFs – Pre-formed fibrils

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KO – Knock-out

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Rab1 – Ras superfamily of small G protein-1

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ATP13A2 – Type 5 P-type ATPase

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DLB – Dementia with Lewy-bodies

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Bcl-2 – B-cell lymphoma 2

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WT – Wild-type

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WIPI2 – WD repeat domain phosphoinositide-interacting protein 2

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MAPK/ERK – Mitogen-activated protein kinase/ Extracellular signal-regulated kinase

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mTOR- Mammalian target of Rapamycin

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CaMKK-β – Calcium/calmodulin-dependent protein kinase kinase- β

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AMPK- 5'-adenosine monophosphate-activated protein kinase

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Keap-1 – Kelch Like ECH Associated Protein-1

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GSK3ß – Glycogen synthase kinase 3- ß

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MPTP – 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine


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6-OHDA – 6-Hydroxydopamine

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TFEB – Transcription factor-EB


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Abstract:

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Parkinson’s disease (PD) is the 2ndmost common neurodegenerative disorder, neuropathologically

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depicted by the aggregation of misfolded α-synuclein (α-syn) protein and appears to be central to the

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onset and progression of PD pathology. Evidence from pioneering studies has highly advocated the

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existence of impaired autophagy pathways in the brains of Parkinson’s disease patients. Autophagy is

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an evolutionary conserved, homeostatic machinery for minimizing abnormal protein aggregates and

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for facilitating organelle turnover. Any aberration in constitutive autophagy activity results in the

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aggregation of misfolded α-syn which in turn, may further inhibit their own degradation- leading to a

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vicious cycle of neuronal death. Despite the plethora of available literature, there are still lacunas

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existing in our understanding related to the exact cellular interplay between autophagy impairment and

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α-syn accumulation mediated neurotoxicity. In this context, clearance of aggregated α-syn via

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upregulation of the autophagy-lysosomal pathway could provide a pharmacologically viable approach

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in the treatment of PD. This present review highlights about basics of autophagy, detrimental crosstalk

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of α-syn and chaperone-mediated autophagy, α-syn and macroautophagy and it also depicts the

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interaction between α-syn and novel targets: LRRK2 and mTOR, followed by the role of autophagy in

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PD from the therapeutic perspective. More importantly, it further updates the reader’s understanding

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on various newer therapeutic avenues which may provide disease modification via promoting

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clearance of toxic α-syn through activation of autophagy.

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Keywords: Parkinson’s disease, Autophagy, α-synuclein, LRRK2, Chaperone-mediated autophagy,

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Macroautophagy, Neurotoxicity

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1. Introduction:

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Parkinson’s disease (PD) is the 2nd-most common neurodegenerative disorder preceded by

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Alzheimer’s disease, affecting 1 - 2% population over the age of 65 years[1]. Typically, it is

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characterized by progressive loss of specific sub-region of dopaminergic (DAnergic) neurons in

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substantia nigra-pars compacta (SNpc) of the brain which in turn give rise to the cardinal motor

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features of PD i.e. resting tremor, bradykinesia, muscle rigidity, and postural instability[2]. This, of

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course, is an oversimplified view of the disorder. In actuality, PD patients also suffer from a

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constellation of non-motor symptoms beyond cardinal motor symptoms which includes hyposmia,

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autonomic dysfunction, depression, sleep disturbances, cognitive abnormalities and gastrointestinal

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dysfunction[3]. Today, it is well-accepted fact that non-motor symptoms occur due to extensive extra-

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nigral pathology that appears approximately 20 years before the clinical onset of PD motor

12

symptoms[4].

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At the cellular level, neuronal loss is accompanied by aggregation of misfolded α-syn within the

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intracytoplasmic inclusions known as Lewy bodies[5]. α-synuclein (α-syn) is a monomeric protein

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encoded by SNCA gene which has been found to play an important role in synaptic plasticity and

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vesicle trafficking [6, 7]. Till date, more than 22 genes have been found to be associated with the PD

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[8]. Most of the cases of PD are sporadic, only 5% are of familial PD which is associated with

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autosomal dominant or autosomal recessive mutation[9]. Indeed, identification of autosomal dominant

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mutations such as A30P, A53T, E46K, G51D, H51Q mutations as well as duplication and triplication

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in SNCA gene has markedly broadened our current understanding related to the pathogenesis of

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familial PD [6, 7]. Interestingly, it has been reported that increased levels of a toxic oligomeric form

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of α-syn (aggregated α-syn) are the pre-requisite for the severity of disease[10]. In addition, there is a

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large body of evidence suggesting increased expression of α-syn mRNA levels in case of sporadic PD

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patients in comparison to control. Overall, these observations point to a pivotal involvement of α-syn

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overexpression to cause genetic PD and, that a subtle aggregation of α-syn may be central to the

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pathogenesis of PD. As the degradation of α-syn is under the control of cellular cleaning crew that is

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macroautophagy (MA) and chaperone-mediated autophagy (CMA), any dysfunction in the removal of

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α-syn might be the one reason responsible for its aggregation. Therefore, enhancing α-syn elimination

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by autophagy induction may represent a viable therapeutic strategy for the treatment of PD and other

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synucleopathies.

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A most common response to the food abstinence is activation of autophagy via Autophagy-Lysosomal

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Pathway (ALP) in which cell starts to digest its own components in order to recycle the nutrients from

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dying cells. Autophagy contributes to the maintenance of the nutritional status of the cell in the fasting

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condition and also helps in the removal or clearance of misfolded proteins or damaged organelles[11].



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Christian de Duve, the discoverer of the Lysosome gave birth to the new research field of autophagy

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and received the Nobel prize in physiology or medicine in 1974[12]. However, the research on

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autophagy didn’t receive that much attention at that time. In 1993, the laboratory of Yoshinori Ohsumi

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conducted ground-breaking experiments of genetic analyses of apg mutants in yeast and identified 15

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APG genes (Autophagy-related genes) involved in autophagy [13, 14]. From that point, research on

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autophagy was tremendously up taken and the role of autophagy in human health was explored. Along

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with it, there is unequivocal evidence that suggests impairment in the ALP in PD initiates misfolding

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of α-syn and dysfunction in protein clearance system converse to form two interconnected concepts

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related to the cellular mechanism of PD pathology. From the last decade research on PD has been

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shifted on understanding the mechanism behind the clearance of toxic α-syn via focusing on

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autophagy. So, by keeping this viewpoint in mind, this review begins with existing knowledge on the

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complex process of autophagy.

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2. Autophagy

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Autophagy is an intracellular degradation process which involves degradation of cytoplasmic contents

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including both misfolded proteins and damaged organelles inside the lysosome or vacuole (in case of

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yeast) [15]. It is followed by subsequent re-entry of breakdown products into the cytosol in order to

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promote recycling of the cellular constituents and to get energy from it and maintain the viability of

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the cells under stressful conditions [16]. Thus, the autophagy is important in two aspects: first is the

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removal of toxic waste from the cells and second is to promote the recycling of nutrients from damaged

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organelles and proteins [17]. Based on the method of delivery of cargo to the lysosome, autophagy can

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be categorized into three classes: Chaperone-mediated autophagy (CMA), Macroautophagy (MA),

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Microautophagy.

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Autophagy was considered as nonselective “in bulk” degradation process before the discovery of the

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CMA. Discovery of CMA made the first evidence that autophagy may be selective as only proteins

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having KFERQ motif (about 40% of proteins) can be selectively degraded by CMA[18]. Basically,

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any misfolded proteins like α-syn having KFERQ motif are identified by chaperone cytosolic Hsc70

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and co-chaperones Hsp70-interacting protein (CHIP), Hsp40, Hsp70-Hsp90 organizing protein (HOP)

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[19]. Once identified they form complex and then targeted to the lysosomal surface where it will bind

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to the lysosomal surface receptor, LAMP2A (Lysosome-associated membrane protein 2A). This is the

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first identified component of lysosome which is involved in the CMA[20]. LAMP2 mRNA undergoes

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alternative splicing and thus exists as three isoforms: LAMP2A, LAMP2B, LAMP2C[21] but, the

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LAMP2A is selectively involved in the autophagy [22]. All isoforms differ in their luminal, cytosolic


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and transmembrane region[23] and further gets internalized by the lysosome and promote its

2

degradation via lysosomal hydrolases.

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Macroautophagy (MA) (hereafter referred as autophagy), is preserved intracellular degradation

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pathway involves directing substrates like damaged or superfluous organelles (mitochondria,

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peroxisomes), aggregated proteins, aggregation-prone proteins, etc. towards the lysosome via double-

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membrane vesicles known as autophagosome (AP). Phagophore is cup-shaped double-membrane

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vesicle which is the perceivable herald of AP. Edges of phagophore extend and fuse with each other

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and lead to the formation of AP. Mechanism of MA has been divided into different stages: initiation,

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elongation, and maturation of AP. Noteworthy progress has been made on the identification of proteins

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that controls the biogenesis of AP. Although, how AP is built up of and what could be the source of

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the AP membrane are prime questions in this area. Many studies advocate relationship between ER

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and autophagy structures and put forth the hypothesis that membranes of AP are derived from the

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ER[24]. However, ER is not the only source, but, mitochondria, ER-mitochondrial contact sites, Golgi

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apparatus, Plasma membrane, and ER-Golgi intermediate compartment are also involved in the

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biogenesis of AP as they supply lipids to the growing isolation membrane[25] and its underlying

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molecular mechanism is still unclear. There are lot many genes as well as proteins which are involved

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in the biogenesis of AP, most of them are conserved from yeast to human. Atg1 is the autophagy-

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related gene-1 which is also known as ULK1 in mammals and reported to form complex in

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combination with Atg13 and FIP200. Further, Hosokawa et. al found that Atg101, anAtg13-binding

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protein molds tetrameric complex in association with Atg13 and FIP200 and upregulates autophagy

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towards nutrient deficiency[26]. The activity of ULK1 kinase is essential for recruitment of VPS34

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(vacuolar protein sorting 34) to the phagophore[27]. VPS34 is class-III PI3KC3 (Class-III

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Phosphatidyl-inositol-3-kinase-catalytic subunit-3) complex which is required for the formation of the

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phagophore. After completion of the initiation process, it further leads to elongation of AP, which is

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mainly controlled by the ubiquitin-like conjugation system. This system consists of Atg7 [ubiquitin-

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activating-enzyme (E1)-like] and Atg10 [ubiquitin-conjugating enzyme (E2)-like]. Both Atg7 and

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Atg10 assists in the conjugation of Atg12 with Atg5 and forms complex which ultimately forms

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another complex Atg12-Atg5 with Atg16L, an E3-ubiquitin ligase and promotes the conversion of

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LC3 to LC3I and finally LC3I to LC3II causing elongation of AP [28]. Furthermore, maturation of AP

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involves fabrication of AP throughout the cytoplasm followed by the trafficking of AP by means of

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dynein machinery on the microtubules to carry AP into the juxtaposition of the lysosome[29]. After

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fusion of AP with a lysosome, contents of AP will get degraded into free amino acid by lysosomal

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hydrolases. The primitive function of autophagy is to foster the cells against starvation or any other

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stress conditions which are preserved from yeast to human. Autophagy has a precious role in health as



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well as in several diseases. Defective autophagy may be responsible for the development of pathology

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in various neurodegenerative disorders. For example, aggregation of Tau protein and α-syn in

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Alzheimer’s disease and Parkinson’s disease respectively. Refer to the previous work for more detailed

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molecular mechanisms related to autophagy [28].

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The idea that membrane of lysosome undergoes invagination to confine cytosolic components to

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degrade them was put forth in early days and the process is commonly known as microautophagy. The

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phrase microautophagy is reserved for removal of damaged or misfolded proteins and superfluous

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organelles that are directly engulfed by lysosomes or vacuoles in case of yeast. In early years it was

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known that microautophagy is not selective but it was proven that cargo can be distinguished in case

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of microautophagy also and it gave genesis to new terms like micromitophagy, microlipophagy,

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micropexophagy. As the link between microautophagy and α-syn is not clearly established, discussing

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microautophagy is beyond the scope of this article.

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3. α-syn and cleaning crew: single-player and multiple cross-talks

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3.1.α-syn and CMA: Detrimental cross-talk

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The cross-talk between α-syn and CMA has been of great interest and witnessed to have detrimental

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outcomes from the last several years. It has been observed monomers and dimers of α-syn can be easily

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degraded by CMA however, α-syn oligomers can’t be degraded by CMA.[30]. Both in vivo and in

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vitro studies have found the increased levels of LAMP2A and decreased levels of α-syn aggregation

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which supports the notion that proper functioning of CMA is required to mitigate neurotoxicity

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imparted by α-syn aggregation.[31-33]. Thus, impairment of CMA leads to the formation of α-syn

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oligomers and development of α-syn pathology[32, 34]. Not only α-syn oligomers but several other

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factors like a mutation in LRRK2 (leucine-rich repeat kinase 2) and VPS35 (vacuolar protein sorting-

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associated protein 35) and post-translational modifications (PTMs) also impair CMA and thus leads to

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α-syn aggregation. Mutations in the leucine-rich repeat kinase 2 (LRRK2) including G2019S mutant

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inhibited the translocation complex at the lysosome membrane and blocked CMA[35]. PTMs like

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nitration and oxidation of α-syn promotes mild impairment of CMA. Nevertheless, phosphorylation of

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α-syn and/or dopamine-induced oligomerization of α-syn completely impairs the CMA[30].

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Interestingly, it has been reported that activity of VPS35 in DAnergic neurons is essential for

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endosome-to-Golgi retrieval of LAMP2A, a receptor of CMA that is critical for degradation of α-syn

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and deficiency as well as mutation of VPS35 lead to impairment of LAMP2A and subsequently α-syn

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aggregation[36]. Impairment of CMA by overexpression of WT α-syn or A53T-α-syn induces

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translocation of Myocyte enhancer factor 2D (MEF2D) which is one of the neuronal survival markers


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from the nucleus to the cytosol and induces neuronal death[37]. Apart from in-vivo studies, clinical

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studies with post-mortem brains of PD patients also suggested impairment of CMA which can be seen

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from decreased levels of LAMP2A and Hsc70 in the SN and amygdala, which was correlated with the

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α-syn aggregation [38]. Moreover, mRNA levels of LAMP2 isoforms and protein expression of

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LAMP2B, 2C were not altered significantly in the initial stages of PD compared to control. However,

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protein expression of LAMP2A was significantly up-regulated that has been linked with α-syn

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aggregation suggesting impairment of CMA in PD[38].

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Recently research is focused on the role of microRNA (miRNA) in CMA in PD. miRNAs are the

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conserved non-protein coding sequence that emerged as a key post-transcriptional regulator of gene

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expression. In general, it has been observed that miRNAs promote degradation of α-syn either directly

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targeting α-syn or indirectly via activation of CMA (Zhao L et al., 2019). miRNA-133b is found to be

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involved in the degradation of α-syn through CMA. Kim et. al reported a significant decrease in the

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level of miRNA-133b in PD patients which is normally present in the DAnergic neurons of the

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midbrain of healthy person [39]. While most of the other miRNAs (miRNA-320A) were found to

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inhibit the degradation of α-syn and thus it alters the normal state of protein to aggregated state[40].

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Alvarez-Erviti, L and group recently with the help of luciferase reporter assay in SHSY5Y cells

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reported that increased levels of, miRNA-21, miRNA-224, miRNA-373, miRNA-379 caused a

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reduction in levels of LAMP2A whereas increased levels of miRNA-26b, miRNA-106a, miRNA-301b

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reduced levels of Hsc70 which was correlated with an increase in the level of α-syn. [41]. Recently,

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it was reported that miRNA-7 promotes clearance of α-syn aggregates and pre-formed fibrils (PFFs)

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via activation of CMA [42]. Recent studies reported that inhibition of glucocerebrosidase (GCase), a

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lysosomal hydrolase lead to α-syn aggregation via blockade of CMA [38]. However, the role of GCase

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in PD is not only limited to the blockage of CMA. Recently, Gegg et. al has described that inhibition

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of GCase may alleviate ER stress, mitochondrial dysfunction and neuroinflammation, thus lead to the

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induction of PD pathology[43]. As we have discussed crosstalk between α-syn and CMA and its

26

detrimental consequences. Now it is quite clear that α-syn aggregation impairs the CMA and vice

27

versa. As inhibition of CMA shifts total burden of clearance of α-syn on MA, for this reason, we will

28

discuss how α-syn interacts with the MA. Please refer Fig.1 for diagrammatic representation.

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Figure 1: Illustration representing crosstalk in between α-syn and chaperone-mediated

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autophagy (CMA): Under pathological conditions:1) A30P/A53T-mutant α-syn binds with

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LAMP2A with the high affinity 2) mutated LRRK2 inhibits translocation complex at lysosome surface

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3) post-translational modifications (PTMs) like nitration and oxidation as well as phosphorylated α-

16

syn directly inhibits CMA 4) deficiency of VPS35 impairs retrieval of LAMP2A while miRNAs

17

21,224,373,379 directly inhibits LAMP2A 5) Exposure of dopamine to WT-α-syn promotes

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aggregation of α-syn which on turn inhibits LAMP2A 6)miRNA 26b,301b,106a reduces levels of

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Hsc70 and blocks downstream cascade. Mechanisms 1-6 ultimately impairs CMA and leads to α-syn

20

aggregation. Under physiological conditions: 7) Monomeric α-syn binds with chaperone cytosolic

21

Hsc70 and forms complex which further targeted to LAMP2A, further internalized into the lysosome

22

where α-syn interacts with the lysosomal hydrolase, cathepsin-D and ultimately undergoes breakdown.

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3.2.α-syn and macroautophagy: Deliberating crosstalk

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Several studies enlightened involvement of oligomeric/fibrillar form of α-syn in the pathogenesis of

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PD. It is evident that MA is involved in the degradation of the mainly oligomeric or fibrillar form of

27

α-syn. Thus, it becomes foremost to understand how MA is intricated in the clearance of aggregated

28

α-syn. Most importantly both in vitro and in vivo studies suggests that clearance of mutated α-syn is

29

mainly governed by Beclin-1, which is an important component of the MA pathway[44]. Beclin-1 is

30

the autophagic protein which is encoded by the BECN1 gene, which interacts with Bcl-2 and plays an

31

important role in the regulation of autophagy. Till date, various activators, as well as inhibitors, have

32

been identified related to macroautophagy (MA) which are involved in the degradation of α-syn.

33

Besides, the clearance of α-syn is found to be dependent on the type of activators/inhibitors used, few

34

studies suggested MA activator rapamycin and Trehalose selectively targeted E46K mutated α-syn


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through inhibition of JNK1/Bcl-2 pathway[45]. Eventually, the blockade in this pathway leads to

2

accumulation of oligomeric or fibrillar of α-syn [46]. However, it is still ambiguous whether α-syn is

3

accumulated only due to impairment of clearance of α-syn due to inhibition of MA or not. As exposure

4

of PFFs of α-syn to HEK293 induced α-syn accumulation and finally lead to impairment of MA [47].

5

Exhaustively studies suggest clearance of α-syn via MA relies on its exact conformational state like

6

whether α-syn is in the monomeric, oligomeric or fibrillar state. Ahmed et al developed a transgenic

7

knock out (KO) mice lacking Atg -7, an important autophagic protein that is responsible for the

8

formation of AP. The same study ascertained that KO of Atg-7 leads to impairment in the autophagy,

9

age-related DAnergic neuronal loss in striatum, α-syn accumulation and up-regulation of LRRK-2

10

proteins [48, 49] Not only this but also PTMs in α-syn like sumoylation and phosphorylation are

11

critical factor for its clearance by MA[50-52].

12

As the final destination of cargo in the MA is the lysosomes, so straightway AP containing cargo (here,

13

α-syn) enters into the lysosomes and once inside the lysosomes, it is mostly degraded by lysosomal

14

hydrolase specifically cathepsin-D (CTSD)[53]. The role of CTSD in the degradation of α-syn has

15

been confirmed by a study where they have used CTSD KO mice which showed increased

16

accumulation of oligomeric α-syn [54]. Furthermore, mislocalization of CTSD has been observed due

17

to a mutation in VPS35 which also promoted to the accumulation of α-syn[55]. Intriguingly, a mutation

18

in VPS35 has been already linked to the development of familial PD [56]. Not only CTSD but, VPS35

19

mutation also induces mislocalization of Atg9 and hampers the biogenesis of AP which can cause a

20

decrease in α-syn clearance. [57]. Apart from this, studies with mammalian cells found α-syn

21

accumulation also inhibited Rab1 protein, involved in the localization of Atg9 and ultimately inhibits

22

AP biogenesis[58].PARK9, a lysosomal gene was found to be mutated in autosomal PD which encodes

23

for Type 5 P-type ATPase (ATP13A2) protein which is involved in exosomal biogenesis. Kong SM

24

has reported reduced levels of α-syn aggregation due to elevated expression of ATP13A2 which

25

induced externalization of α-syn through exosomes[59]. Thus, the above study supports the transfer of

26

α-syn from dying neurons to healthy neurons might be occurring through the exosomal secretion of α-

27

syn [60]. Furthermore, it has been evident by the observation that expression of ATP13A2 was found

28

to be elevated in surviving DAnergic neurons of SNpc in sporadic PD suggesting that secreted α-syn

29

through exosomes might be taken up by surviving neurons. [59, 61]. Distinctive studies reported

30

increased secretion of α-syn either mediated by exosomes[62, 63]or by exophagy[64] upon the

31

inhibition of MA or lysosomal dysfunction. Above studies suggest debilitating equilibrium exists

32

between α-syn aggregation and its secretion which is mainly mediated by MA leads to the development

33

of PD pathology.



Page 12 of 37

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Multitudinous studies described an increased number of AP in the brains of PD patients[65]. Similarly,

2

increased levels of LC3-II, a marker of AP biogenesis has been observed in PD and DLB patients[66,

3

67]. It is clear from the above studies thatα-syn accumulation might be one reason for increased AP

4

formation. However, it might be possible that lysosomal dysfunction dampens the clearance of α-syn

5

aggregates. High mobility group box 1 (HMGB1) has been identified as a novel target to promote

6

clearance of α-syn via activation of autophagy. Inversely, the α-syn aggregation has also been found

7

to disturb binding of HMGB1 to Beclin1 and promotes binding of Beclin1 to Bcl-2 which ultimately

8

impairs the autophagic process[68, 69].

9

Taken together all studies advocates that α-syn may act as a single-player and multiple roles in the

10

process of modulation of autophagy which relies on the levels of α-syn, the conformation of α-syn

11

whether it is in monomeric form or oligomeric, species whether it is WT or mutated α-syn or cellular

12

environment. We have summarized that instead of the presence of conflicting evidence, there is no

13

doubt about the existence of robust crosstalk in between α-syn and autophagy pathways (MA) and thus

14

may enable the development of therapeutics. Refer Fig.2 for diagrammatic representation.

15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Figure 2: Illustration representing the crosstalk in between α-syn and macroautophagy: A)

31

Under physiological conditions: 1) α-syn overexpression/mutation 2) A53T α-syn 3) post-

32

translational modifications like SUMOylation and phosphorylation 4) monomeric α-syn can be

33

effectively cleared out from the cells as it upregulates MA. Under pathological conditions: 1)

34

Deletion of Atg-7 2) E46K α-syn inhibits MA via inhibition of JNK1/Bcl-2 pathway 3) Exposure of


Page 13 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

pre-formed fibrils of α-syn inhibits MA as it promotes aggregation of WT-α-syn4)α-syn aggregation

2

disturbs binding of HMGB1 to Beclin1 and promotes binding of Beclin1 to Bcl-2 which ultimately

3

impairs the MA 5) α-syn aggregation also inhibits the MA via inhibition of Rab1, protein regulates

4

localization of Atg9 required for biogenesis of AP 6) VPS35 mutation also inhibits AP biogenesis via

5

inhibition of Rab1 7) VPS35 mutation also promotes aggregation of α-syn via mislocalization of

6

cathepsin-D, a lysosomal hydrolase involved in degradation of α-syn and ultimately leads to

7

aggregation of α-syn.

8 9

3.3. α-syn and LRRK2: Partners of crime????

10

Leucine-rich repeat kinase 2 (LRRK2) is one of the most important genetic contributors to the

11

development of PD. Several mutations in LRRK2 has been reported in patients of PD suggests LRRK2

12

undergoes mutation in familial PD. Specifically, mutations in the coding region (G2019S, R1441G,

13

R1441H, R1441C, Y1699C, I2020T) or polymorphism in coding, as well as non-coding region, are a

14

major cause of autosomal dominant PD or sporadic PD respectively[70, 71]. Continuing further, these

15

mutations can be identified due to the presence of pleomorphic characteristic in patients of mutant

16

LRRK2, which generally absents in the patients of sporadic PD[72]. However, it is merely impossible

17

to differentiate patients of LRRK2-associated PD from sporadic PD. LRRK2 has been reported to have

18

multiple roles ranging from autophagy, mitochondrial maintenance, immune system-related activity,

19

protein synthesis, etc. Out of which, the role of LRRK2 in the context of autophagy has been widely

20

studied. LRRK2 is a large, multi-domain protein having both GTPase and kinase activity. LRRK2

21

regulates the process of autophagy through its kinase activity. In that direction, Manzoni et. al studied

22

the role of LRRK2 in autophagy by taking primary fibroblasts from individuals having mutations in

23

the functional domain of LRRK2 and found that LRRK2-mutation impaired autophagy that is evident

24

from the altered ratio of LC3-II/I, p62 and WIPI2 positive puncta, an autophagy marker [73].

25

Interestingly, LRRK2 KO mice showed increased accumulation of α-syn, as the deficiency of LRRK2

26

impairs protein degradation pathways [74]. Moreover, inhibition of LRRK2 was associated with the

27

impaired degradation of AP, as it facilitated downregulation of Rab7, a major component involved in

28

AP degradation [75]. However, this defective degradation of AP may be due to impaired microtubule

29

mediated trafficking of AP towards the lysosome[76]suggests an evident role of LRRK2 and its kinase

30

activity in the process of autophagy [77].

31

Most recurrent mutation involved in LRRK2 that has been seen in 40% of familial PD patients is

32

G2019S, moreover, mutation I2020T also has been reported in the kinase domain of LRRK2. G2019S-

33

LRRK2 upregulates MA via activation of mitogen-activated protein kinase/ extracellular signal-



Page 14 of 37

1

regulated protein kinase (MAPK/ERK) pathway[78] while in another study mutant-LRRK2 (R1441G,

2

G2019S, Y1699C-LRRK2) has been shown to downregulate MA via inactivation of mTOR[73].

3

LRRK2 has been studied from so many times by using different approaches like knockdown,

4

overexpression of wild-type (WT) or mutant LRRK2 in the different model systems. Firstly

5

overexpression of WT-LRRK2 has been reported to activate MA via activation of CaMKK-β/AMPK

6

(calcium/calmodulin-dependent protein kinase kinase-β/ 5’-Adenosine monophosphate-activated

7

protein kinase) pathway[79]. However, overexpression of G2019S-LRRK2 has been associated with

8

reduced degradation capacity as it reduces lysosomal PH and increased expression of ATP13A2 a gene

9

associated with Parkinsonian syndrome[80]. In addition, Park et. al reported interplay between

10

LRRK2 and p62 and observed that LRRK2 promotes phosphorylation of p62 which weakens the

11

interaction of p62 with keap1 and ultimately upregulates autophagy[81]. Recently, it has been reported

12

that G2019S-LRRK2 impairs autophagy as it mediates phosphorylation of leucyl-tRNΑ-synthetase,

13

the substrate of LRRK2 [82].

14

Above studies suggests the involvement of link between LRRK2 and autophagy in PD. However, the

15

link between α-syn and LRRK2 and how α-syn aggregation affects LRRK2 has been poorly

16

understood. But it has been answered by a study using brains of PD patients carrying LRRK2 mutation,

17

showed the presence of α-syn positive Lewy bodies (LBs) suggests that α-syn aggregation is in part

18

under the control of LRRK2[70] and overexpression of the same in the A53T mutated mice has been

19

reported to enhance cytotoxicity mediated by α-syn aggregation. Inhibition of LRRK2 has also been

20

reported to protect the DAnergic neurons from α-syn mediated toxicity[83]. But, the inhibition

21

mediated by the α-syn aggregation has been shown to be well tolerated and can protect the

22

degeneration of DAnergic neurons for a time period not more than 4-week[84]. Nuclear factor

23

erythroid 2-related factor (Nrf2), a well-known anti-oxidant gene, has been identified as a novel target

24

that mitigates toxicity induced by α-syn and LRRK2 by promoting neuronal protein homeostasis in a

25

time-dependent manner[85]. Nrf2 enhances the accumulation of LRRK2 in the LBs and thus it

26

decreases the levels of mutant LRRK2 in other places of neurons[85]. LRRK2 has been reported to be

27

involved in the propagation of α-syn mediated by phosphorylation of RAB35 and the same has been

28

confirmed by LRRK2 inhibitor resulted in decreased α-syn aggregation[86]. Taken together all studies

29

suggest that LRRK2 is a negative regulator of autophagy and thereby promotes α-syn aggregation,

30

imparts neurotoxicity and provides indications that α-syn and LRRK2 might act as partners of crime.

31 32 33


Page 15 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

4. The interplay between α-syn and mTOR: Double-edged sword

2

The etiology and pathogenesis behind the PD still have to be uncovered. From so many years, it has

3

been considered in the literature that many signaling mechanisms including mTOR get disturbed in

4

case of PD[87]. Mutant forms of α-syn have been found in familial PD patients [23], as well as oxidized

5

forms of α-syn [40] have been observed in patients of sporadic PD and DLB which are shown to block

6

autophagy by deregulation of mTOR. Indeed, most of the neurotoxins like rotenone, 6-OHDA, MPTP

7

exert its neurotoxicity by arresting the activation of mTOR in PD [88, 89]. On that account, compelling

8

evidence is in support of the neuroprotective role of mTOR activation, in a various model of PD. In

9

that accordance, a separate study done by Choi et al. revealed that phylogenetically conserved Oxi-α

10

(novel activator of mTOR) rescues oxidative stress-induced death of DAnergic neurons by inhibiting

11

autophagy and subsequent accumulation of autophagic vacuoles [90]. Above these studies are in

12

support of the neuroprotective role of mTOR activation.

13

Howbeit, transcriptome analysis in the SN of post mortem brains of well-characterized PD patients

14

suggested increased levels of mTOR throughout the progression of PD pathology that advocates the

15

neurotoxic role of mTOR activation[91]. Likewise, administration of L-DOPA facilitates dopamine

16

D1 receptor-mediated activation of mTOR selectively in the GABAergic medium spiny neurons of the

17

striatum, associated with the development of dyskinesia in the mouse model and is abrogated by

18

mTOR inhibitor rapamycin.[92].Intriguingly, these findings collectively raise the possibility that

19

inhibition of mTOR might able to provide the neuroprotection via activation of autophagy.

20

Interestingly, feeding mTOR inhibitor rapamycin to adult Drosophila produces longevity in

21

Drosophila life span previously exposed to paraquat and the underlying mechanism is the activation

22

of autophagy via inhibition of mTOR [93]. Further, MANF a mesencephalic astrocyte-derived

23

neurotrophic factor showed its neuroprotective and neurorestorative effect selectively to the DAnergic

24

neurons against 6-OHDA-induced neurotoxicity in SH-SY5Y cells at even very low concentration.

25

This potential mechanism was partly executed through the downregulation of p-AMPK and

26

mTOR[94]The research focused on traditional Chinese medicine, Loganin suggested that Loganin

27

protects zebrafish from MPTP-induced neurotoxicity via inhibition of PI3K/Akt/mTOR signaling

28

mechanism[95].

29

Additionally, Pérez-Revuelta, et al. delineate the direct relationship between mTOR, α-syn

30

aggregation and autophagy by using SH-SY5Y and HeLa cells and found that activation of mTOR

31

fosters aggregation of α-syn via inhibition of protein phosphatase 2A (PP2A) [96]. PP2A is an enzyme

32

belongs to a family of phosphatase which promotes phosphorylation of Beclin-1 and aids in autophagy.

33

In value addition, wang et. al perceived that moxibustion lowers the expression of p-mTOR and

34

p70S6K leads to significant improvement in behavioral performance, TH immunoreactivity, and levels



Page 16 of 37

1

of LC3-II as well as decreased immunoreactivity towards α-syn in SN of rotenone-induced PD rat

2

model[97].MANF facilitates the autophagy-mediated clearance of misfolded α-syn prevents DA

3

neuronal degeneration in human α-synuclein-induced C. elegans PD model. This protective effect was

4

found to be mitigated by silencing 26 autophagy-related genes using RNAi [98]. An explorative study

5

by using uric acid on PC12 cells overexpressing A53T mutant SNCA have found to decrease

6

aggregation of α-syn. This effect might be related to downregulation of mTOR along with upregulation

7

of LC3II and p62. By treatment with 3-BDO (3-benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran-2-

8

(3H)-one), mTOR activator this effect was found to be reversed [99]. Taken into consideration that

9

interplay between α-syn and mTOR might be threatening and has detrimental consequences. However,

10

it still remains debatable whether activation of mTOR is neurotoxic or neuroprotective. Taken together

11

these lines of evidence suggest that mTOR acts as a double-edged sword which may play a beneficial

12

or detrimental role in PD.

13 14

5. Targeting Autophagy-Lysosomal Pathway (ALP): Implications of Promising targets and

15

therapeutic intervention

16

Pioneering studies have been done to explore the influence of different therapeutic agents on the

17

autophagy-lysosomal pathway (ALP) by using different models of PD. The succeeding section will

18

emphasize on the modulation of autophagy by various therapeutics in the clinical and preclinical

19

studies. These therapeutics have been reported to modulate autophagy by mTOR-dependent pathway,

20

mTOR-independent pathway, and other mechanisms. Key targets for all of these pathways are different

21

i.e. in case of the mTOR-dependent pathway, key targets are mTOR, AMPK, Beclin-1while for

22

mTOR-independent pathway key target is inositol monophosphatase (IMPase). Recent studies on the

23

c-Abl kinase, GSK3ß and Nrf2have significantly gained interest as potential targets.

24

Starting with the mTOR-dependent pathway; Ganoderma Lucidum extract (GLE)(by restored AMPK,

25

mTOR, ULK1 and normalized LC3-II/LC3-I ratio)[100], Resveratrol (by activation go AMPK via

26

SIRT1 which enhances autophagy)[101]and Rosuvastatin[102] fosters autophagy by virtue of

27

activation of AMPK. Drugs like Rapamycin, Moxibustion and Loganin were found to alleviate

28

neurotoxicity in PD based animal models via inhibition of mTOR[92, 95, 97]. Interestingly, Metformin

29

was found to exert neuroprotective effect by using both the above approaches (activation of AMPK

30

and inhibition of mTOR) which resulted in the activation of autophagy. This was reflected by

31

downregulated levels of p-ser129, α-syn and mTOR along with upregulated AMPK and LC3-II

32

levels[96, 103, 104]. Taking into account the activation of Beclin-1, Prolyloligopeptidase (PREP)and

33

its inhibitor KYP-2047was found to ameliorate PD condition through activation of autophagy via


Page 17 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

activation of Beclin-1[105]. Similar effects using caffeine was found to mitigate α-syn induced defects

2

in the autophagy via activation of Beclin-1[42].

3

Up till now, several therapeutics have been developed to target mTOR-independent pathway focusing

4

on Inositol-monophosphatase (IMPase), responsible for cleavage of inositol-monophosphate (IMP) to

5

inositol (Ins) which ultimately downregulates autophagy. in-vitro and in-vivo studies carried out by

6

using SH-SY5Y, PC12 cells and MPTP-induced mouse model of PD respectively suggests that lithium

7

chloride and valproic acid promotes autophagy via inhibition of IMPase as it is evident from decreased

8

levels of IMPase and increased levels of LC3-II [106-108].

9

Recently, other targets have emerged which plays an important role in the modulation of autophagy

10

i.e. c-Abl, GSK3ß, Nrf2. Among them, c-Abl is activated non-receptor tyrosine kinase Abelson,

11

promotes α-syn aggregation via activation of GSK3ß, in turn, causes phosphorylation of α-syn. In

12

2018, Ren and group demonstrated the role of c-Abl-GSK3β Signalling in MPP+-Induced ALP

13

dysfunction. Study results revealed that inhibition of c-Abl by STI-571 rescued the function of ALP

14

through facilitating the nuclear translocation of TFEB and diminished the phosphorylation of GSK3β

15

at Tyr216 which was previously enhanced under MPP+ treatment in SN4741 cells and in primary

16

midbrain neurons ultimately imparted protection against MPP+-induced neuronal cell death [109].

17

Further continuing with GSK3ß, lithium chloride and 6-Bio, direct inhibitors of GSK3ß has been

18

shown to decrease α-syn aggregation as it inhibits GSK3ß and also activates autophagy that can be

19

seen from increased levels of LC3-II [110, 111]. Using Adeno-associated virus serotype 1/2

20

(AAV1/2)-based rat model of PD, Qing et al highlighted the ability of autophagy enhancer Trehalose

21

and suggested that Trehalose at the concentration of 2% prevents/reverse the α-syn aggregation via

22

activation of autophagy as it increases levels of LC3-II [112]. Completely different, Geraniol has been

23

reported to activate autophagy via activation of ATG genes (ATG5, 7, 12) directly in SK-N-SH cells

24

exposed to rotenone [113]. While ITC-3, Naringin, Curcumin, Safranal, Tanshinone-IIa, Pinostrobin

25

has been reported to promote activate Nrf2, an anti-oxidant enzyme and counteracts neurotoxicity in

26

different models of PD as shown in Table 1. Taking together all the studies suggests that AMPK-

27

mTOR-Beclin1 axis, c-Abl kinase, GSK3β, and Nrf2 might serve as potential therapeutic targets to

28

counteract neurotoxicity induced by α-syn due to their autophagic potential. Refer Fig.3 for

29

diagrammatic representation.

30 31 32 33 34



Page 18 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Figure 3: Illustration representing molecular mechanisms regulating the process of autophagy

20

and therapeutic agents acting on different targets for promoting autophagy via activation of

21

autophagy pathway:

22

Molecular mechanisms regulating the process of autophagy: A) Amino acid depletion as well as

23

low amount of energy levels can be detected by AMPK through LKB1, activates TSC1/2, can lead to

24

inhibition of mTORC1 facilitates autophagy via activation of Beclin1 and ULK1 and also promotes

25

dephosphorylation of TFEB leads to migrate into the nucleus and promotes biogenesis of lysosomes

26

and AP via promoting transcription of ATG genes and lysosomal genes B) Insulin and growth

27

hormones acts on growth hormone receptors and thereby activates class-I PI3K, can activates Akt and

28

thus activates mTORC1 and inhibits autophagy C) Cellular stress leads to activation of p53 which

29

activates PTEN leads to inhibition of PI3K and activates autophagy D) ROS generation due to stress

30

promotes oligomerization of monomeric α-syn which phosphorylates NRF2 and promotes

31

ubiquitination followed by degradation of NRF2 leads to enhanced levels of ROS, triggers

32

inflammatory cascade and inhibits autophagy E) GSK3β also inhibits the autophagy via

33

phosphorylation of Nrf2.


Page 19 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

Therapeutic agents acting on different targets for promoting autophagy via activation of

2

autophagy pathway: Therapeutic agents upregulates autophagy via 1) activation of AMPK

3

2)Activation of Beclin-1 3) Inhibition of mTOR 4) Enhancing biogenesis of lysosome via

4

dephosphorylation of TFEB 5) Inhibition of c-Abl involved in activation of GSK3β and thereby

5

inhibits degradation of Nrf2 6) Directly inhibiting GSK3β 7) Activation of Nrf2.

6

Table 1: List of therapeutic agents: modulation of the autophagy pathway Agent

Target

Effect

Animal/

Model

cell line

Induction

Reference

(dose) mTOR-dependent Pathway Ganoderma

AMPK

Improved

locomotor C57BL/6J

lucidum

performance,

extract (GLE)

TH+-neurones in SNpc, increased

(400 mg/kg/d) Ganoderma

increased mice levels

[100]

(20mg/kg)

of

AMPK, mTOR, ULK1 AMPK

Increased

cell Neuro-2a

lucidum

viability,increased

extract (GLE)

expression

of

MPTP (1nM)

[100]

Rotenone

[102]

cells

AMPK,

mTOR, ULK1

(800μg/ml) Rosuvastatin

MPTP

AMPK

Upregulated Beclin-1,

(40μM)

AMPK, SH-SY5Y

LC3-II,

cell cells

viability, increased α-syn

(200nM)

clearance Metformin (50mg/kg)

AMPK

Improved coordination, TH+-neurons,

motor Wildtype elevated and LC3-II, C1k1+/-

AMPK expression

mutant mice


MPTP (25mg/kg)

[103]


Metformin

AMPK

Page 20 of 37

Increased levels of LC3- Murine

MPP+

II,

(1000μM)

AMPK,

while DAnergic

[103]

decreased expression of MN9D p62 Metformin

AMPK

(100mg/kg)

cells

Attenuated induced

MPTP- AMPK

loss

positive

of

TH- KO mice

MPTP

[104]

(30mg/kg)

neurons,

Restored

DOPAC:DA

ratio, Decreased MPTPinduced gliosis

Metformin

AMPK

(1.5,2 mM) Metformin

Reduced levels of p- SH-SY5Y ser129 α-syn

AMPK

(5g/kg)

α-syn

[96]

cells

Reduced levels of p- C57BL/6J

[96]

ser129 α-syn in mouse mice brain

Resveratrol

AMPK/

Increased

oxidative Cultured

SIRT1

capacity of mitochondria, skin

mediated

cellular

model

ATP

content, fibroblasts

reduced oxidative stress KYP-2047 (PREP inhibitor) (5mg/kg) Glycyrrhizic acid (500μM)

Beclin-1 Reduced

levels

Increased levels of DA,

KO of

PARKIN [105]

syn transgenic mice

Beclin-1 Increased cell viability, SH-SY5Y LC3-II/I, Beclin-1

[101]

of A30P Α-

oligomeric α-syn,

HVA in the striatum

siRNA

cells

6-OHDA

Corticosterone (50μM)


and [114]

Page 21 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Caffeine (1g/L Beclin-1 Reduced in

drinking

p129-α-syn, C57BL/6J

apoptosis,

water)

activation,

microglial mice

A53T

α-syn [42]

(3.25μl)

astrogliosis,

increased LC3II/I ratio, Beclin-1 expression MANF

mTOR/

Increased

mTOR, SH-SY5Y

AMPK

decreased

AMPK, cells

6-OHDA

[94]

decreased apoptosis Loganin

mTOR

Suppression

of PC12 cells MPTP

[95]

PI3K/Akt/mTOR signaling,

decreased

MPTP-induced neurotoxicity Rapamycin

mTORC Decreased levels of p- α-syn

and analogues

1

ser129 α-syn

[96]

overexpre ssing SHSY5Y cells

Rapamycin

mTORC Alleviated

and analogues

1

cell

Rescued

death, SH-SY5Y

rotenone

[92]

L-DOPA cells

induced dyskinesia Rapamycin

mTORC Alleviated cell death

α-syn

and analogues

1

transgenic

[115]

rats Moxibustion

mTOR

Suppresses

Sprague-

mTOR/p70S6K,

Dawley

Activated

autophagy,

rats

increased α-syn clearance


Rotenone (2mg/ml, s.c.)

[97]


2-

TFEB

Page 22 of 37

Promotes clearance of α- Transfecte

hydroxypropyl

syn

via

-β-

autophagy

cyclodextrin

activation

[116]

of d Human H4 neuroglio ma

cells

for α-syn

mTOR-independent Pathway Lithium

Inositol

chloride

monoph osphatas

Rotenone (200nM)

SH-SY5Y

Increased cell [106]

cells

viability, reduced

e

mitochondrial dysfunction, increased ration of LC3II/I

Lithium

Inositol

chloride

monoph osphatas

Rotenone (200nM)

SH-SY5Y

Increased cell [106]

cells

viability, reduced

e

mitochondrial dysfunction, increased ration of LC3II/I

Valproic

Inositol

(20mic/ml)

monoph

acid Lithium

and osphatas

MPTP (30mg/kg)

C57BL/6J

Rescues

mice

DAnergic

e

neuronal degeneration,

carbonate

decreased loss

(10mic/ml)

of


DOPAC

[107]

Page 23 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


after exposure of MPTP Lithium

Inositol

----------

A53T

& Promotes

[108]

monoph

A30P

clearance

osphatas

mutant α- mutant α-syn,

e

syn

of

expressing PC12 cells Other Mechanisms STI-571 (5μM)

Activate MPP+

SN4741

Reduced

d

cells

expression

nonrece

(200μM)

[109] of

c-Abl and p-

ptor

GSK3ß,

tyrosine

restored

kinase

Autophagy-

Abelson

lysosomal

(c-Abl)

pathway

& GSK3ß Geraniol (60nM)

ATG

Rotenone (100nM)

genes

SK-N-SH

Enhanced cell [113]

cells

viability, reduced ROS, restored MMP, reduced

ER-

stress Lithium

GSK3β

Methamphetamine

PC12 cells Increased cell [110]

chloride Or

viability,

GSK3β

siRNA



Trehalose (2- -------

AAV A53T- α-syn

Rat

5%)

Page 24 of 37

Increased

[112]

levels of LC3II,

reduced

levels of α-syn aggregates 6-Bio

GSK3β

MPP+

(50μM)

SH-SY5Y

Reduced levels [111]

cells

of

p-GSK3β,

enhanced LC3II, activation of autophagy, increased

the

clearance of αsyn 6-Bio

GSK3β

(5mg/kg)

MPTP (20mg/kg)

C57BL/6J

Enhanced

[111]

mice

autophagy, increased

the

clearance of αsyn, improved motor functions ITC-3 (30mg/kg)

Nrf2

MPTP (20mg/kg)

C57BL/6J

Increased

mice

expression nuclear

[117] of

Nrf2-

dependent oxidative enzymes, decreased activation

of

microglia and TNF-α, improved


Page 25 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


motor functions ITC-3

Nrf2

MPP+/BH4

(1μM)

BV-2

Suppressed

[117]

microglial

pro-

cells

inflammatory cytokines, Increased expression nuclear

of

Nrf2,

HO-1, NQO1, GCL gene Curcumin

Nrf2

(100mg/kg)

Rotenone

Lewis rats

(1ml/kg/day)

Decreased

[118]

ROS,

MDA

levels

while

increased GSH, Nrf2 Safranal

Nrf2

(50μg/ml)

Rotenone (100nM)

Primary

Suppressed

[119]

dopaminer apoptosis and gic cells

ROS generation induced

by

rotenone, inhibited Keap1

and

activated Nrf2, HO-1, NQO1, GCLc Naringin

Nrf2

3-nitropropionic acid

PC12 cells Increased cell [120] viability, increased antioxidant enzymes,



Page 26 of 37

decreased apoptosis, restored MMP Tanshinone-

Nrf2

IIA (20 μg/ml)

6-OHDA (100μM)

SH-SY5Y

Decreased

Cells

apoptosis,

[121]

increased expression

of

Antioxidant related enzyme (ARE)regulated gene expression, increased nuclear Nrf2 Pinostrobin

Nrf2

(125μM)

MPTP

Zebrafish

Rescued loss of [122] dopaminergic

(360μM)

neurons, improved locomotion,

Pinostrobin (25μM)

Nrf2

MPP+ (1.5mM)

SH-SY5Y

Improved cell [122]

cells

viability, increased levels

of

nuclear Nrf2 1 2

6. Concluding Remarks and Future directions:

3

Recent studies reinforce that the turnover of α-syn is the main touchstone of PD as it manifests

4

aggregation of α-syn as part of its pathology. A lot of advances have been done to understand the

5

pathophysiology of PD but still, we do not have a complete depiction regarding the pathophysiology

6

of PD. Many studies have been done which strongly advocates the role of autophagy in PD but still it

7

is quite ambiguous whether autophagy is neuroprotective or not due to its complex nature. In the


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1

closure, visionary studies employing autophagy promoting agents catered decisive insight regarding

2

the role of autophagy and the importance of autophagy activation in PD. Different agents which may

3

found to promote clearance of toxic α-syn by targeting the autophagy-lysosomal pathway, GSK3β,

4

IMPase, c-Abl, mTOR, etc might be suitable therapeutic agents to halt the progression of PD

5

pathology. Howbeit, the clinical application of autophagy promoting agents is defined because of their

6

non-selectivity and property of double-edged sword, hitherto they are not the primary choice in the

7

treatment of PD. Therefore, advancement and deep understanding of molecular mechanisms of

8

autophagy in PD is the pre-requisite and it may lead to the development of agents which may promote

9

the clearance of toxic α-syn thereby combating PD pathology. As the field of autophagy has gained

10

focus in recent years and as it is growing expeditiously, updates may be expected soon.

11 12

7. Conflict of Interests:

13

The authors declare no conflict of interest

14 15

8. Funding Sources:

16

This supplement was supported by the seed fund of the National Institute of Pharmaceutical Education

17

and Research (NIPER), Ahmedabad and Ministry of Chemical and Fertilizers, Government of India.

18 19

9. Author contribution:

20 21 22

PP, NS and AK contribute to conception and design. PP did a rigorous literature search. PP, NS, AG, AS, MS and AK participated in revising the manuscript critically for important intelligence part. PP, NS, AK wrote the manuscript. AK supervised the project.

23 24 25 26 27 28 29 30 31 32 33 34



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Table of Contents Graphic (TOC)

29



llustration representing crosstalk in between α-syn and chaperone-mediated autophagy (CMA)


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Illustration representing the crosstalk in between α-syn and macroautophagy 414x254mm (300 x 300 DPI)



Illustration representing molecular mechanisms regulating the process of autophagy and therapeutic agents acting on different targets for promoting autophagy via activation of autophagy pathway 303x193mm (300 x 300 DPI)


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309x174mm (150 x 150 DPI)


A Cleaning Crew: The Pursuit of Autophagy in Parkinson's Disease

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