p62: a potential target for neurodegenerative disease - ACS

Subscriber access provided by Iowa State University | Library

Review

SQSTM1/p62: a potential target for neurodegenerative disease Shifan Ma, Insiya Attarwala, and Xiang-Qun (Sean) Xie ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00516 • Publication Date (Web): 18 Jan 2019 Downloaded from http://pubs.acs.org on January 19, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 59 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

ACS Chemical Neuroscience

SQSTM1/p62: a potential target for neurodegenerative disease Shifan Ma1,2, Insiya Attarwala 1,2, and Xiangqun Xie1,2,3,4,5*

2 3 4

1

5

Center, School of Pharmacy; 2NIDA National Center of Excellence for Computational Drug

6

Abuse Research; 3Drug Discovery Institute; 4ID4Pharma LLC, Bridgeville, Pennsylvania 15017;

7

5

8

Pittsburgh, Pennsylvania 15261.

Department of Pharmaceutical Sciences and Computational Chemical Genomics Screening

Departments of Computational Biology and of Structural Biology, University of Pittsburgh,

9 10 11 12

*Corresponding Author: Xiang-Qun Xie, MBA, Ph.D.

13

Professor of Pharmaceutical Sciences/Drug Discovery Institute

14

Director of CCGS and NIDA CDAR Centers

15

335 Sutherland Dr., 206 Salk Hall Pavilion

16

University of Pittsburgh

17

Pittsburgh, PA 15261, USA

18

412-383-5276 (Phone)

19

412-383-7436 (Fax)

20

Email: [email protected]

21 1 ACS Paragon Plus Environment

ACS Chemical Neuroscience 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

Abstract

2

Neurodegenerative diseases, characterized by a progressive loss of brain function, affect the lives

3

of millions of individuals worldwide. The complexity of the brain poses a challenge for scientists

4

who are trying to map the biochemical and physiological pathways in order to identify areas of

5

pathological errors. Brain samples of patients with neurodegenerative diseases have been shown

6

to contain large amounts of misfolded and abnormally aggregated proteins, resulting in

7

dysfunction in certain brain centers. Removal of these abnormal molecules is essential in

8

maintaining protein homeostasis and overall neuron health. Macroautophagy is the major route

9

by which cells achieve this. Administration of certain autophagy-enhancing compounds has been

10

shown to provide therapeutic effects for individuals with neurodegenerative conditions.

11

SQSTM1/p62 is a scaffold protein closely involved in the macroautophagy process. P62

12

functions to anchor the ubiquitinated proteins to the autophagosome membrane, promoting

13

degradation of unwanted molecules. Modulators targeting p62 to induce autophagy and promote

14

its protective pathways for aggregate protein clearance have high potential in the treatment of

15

these conditions. Additionally, causal relationships have been found between errors in regulation

16

of SQSTM1/p62 and the development of a variety of neurodegenerative disorders, including

17

Alzheimer’s, Parkinson’s, Huntington’s, Amyotrophic lateral sclerosis, and Frontotemporal lobar

18

degeneration. Furthermore, SQSTM1/p62 also serves as a signaling hub for multiple pathways

19

associated with neurodegeneration, providing a potential therapeutic target in the treatment of

20

neurodegenerative diseases. However, rational design of a p62-oriented autophagy modulator

21

that can balance the negative and positive functions of multiple domains in p62 requires further

22

efforts in the exploration of the protein structure and pathological basis.

23

Key words: SQSTM1/p62, neurodegenerative diseases, autophagy

24

2 ACS Paragon Plus Environment

Page 2 of 59

Page 3 of 59 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

1. Introduction

2

Neurodegenerative diseases result from the progressive loss of function and eventual death of

3

neurons in the central and peripheral nervous systems. Common neurodegenerative conditions

4

include Alzheimer's disease (AD), Parkinson's disease (PD), Huntington’s disease (HD),

5

Amyotrophic lateral sclerosis (ALS), Motor neuron disease, Prion Diseases, Spinocerebellar

6

Ataxia, and Spinal Muscular Atrophy 1. Millions of people worldwide are suffering from

7

neurodegenerative disease, among which at least 500,000 Americans are affected by AD, and

8

even more with PD.

9

These disorders primarily affect neurons. Unlike other cells in the body, neurons lack

10

regenerative capabilities: once damaged or injured, they cannot be repaired, renewed or replaced.

11

Onset of these conditions results in inevitable progressive neuron dysfunction leading to neuron

12

death, resulting in loss of brain function with corresponding physical and/or mental symptoms.

13

Current pharmacological treatments are only able to reduce the symptoms associated with

14

neurodegenerative diseases in an effort to improve the comfort and well-being of the patient. But

15

current methods are unable to prevent, stop, or reverse disease progression.

16

Researchers are working to understand the genetic and biochemical etiological components, in

17

addition to studying the pathogenesis of common neurodegenerative diseases. Studies show these

18

conditions are likely caused by complex interactions of several factors including genetic,

19

epigenetic, pathogenic, environmental and other unknown ones. The complexity of these

20

disorders makes it challenging to map the biochemical and physiological pathways in order to

21

better identify areas of potential treatment.



1

The SQSTM1 gene codes for P62, and was first identified by Jaekyoon Shin and his colleagues 2.

2

Also known as sequestosome-1, p62 is a scaffold protein, crucial in modulating enzyme function

3

through several domain interactions. For example, p62 promotes autophagy degradation by

4

directly binding to an autophagy biomarker, the microtubule light chain 3 (LC3) through a LC3

5

interacting region 3, 4. Combined with its ability to bind ubiquitinated proteins at the C-terminus

6

ubiquitin-binding domain5, p62 serves as an autophagy receptor in the clearance of unwanted

7

protein molecules and aggregates. In addition to ubiquitin binding domain (UBA) and LIR,

8

which play critical roles in autophagy uptake, other protein-interaction motifs, including an N-

9

terminal Phox-BEM1 domain (PB1), a ZZ-type zinc finger domain 6, and Tumor necrosis factor

10

receptor-associated factor 6 (TRAF6) binding (TBS) domain, are functional domains influential

11

in the regulation of inflammation, oxidative stress, osteoclast genesis and apoptosis 7, 8. In the

12

past decade, studies have shown that p62 is associated with several diseases including Paget’s

13

disease of bone (PDB), PD, AD9, HD 10, liver cancer 8, breast cancer 11, obesity and diabetes 12.

14

In this review, we will focus on the physiological role of p62 in neurodegenerative diseases.

15

2. Neurodegenerative Diseases and Misfolded Protein Aggregation and Clearance

16

The maintenance of protein homeostasis is essential in sustaining a viable neuronal

17

microenvironment to support neuron health and adequate function, especially under metabolic

18

stress

19

neurodegenerative diseases14. For this reason, these conditions are often referred to as

20

“proteinopathies”. As shown in Figure 1, under normal conditions, misfolded malfunctioning

21

proteins are removed by a protective mechanisms. However, impairment of these mechanisms

22

can lead to accumulation of misfolded peptides, disrupting protein homeostasis and causing

23

neuronal toxicity

13

. Protein misfolding and aggregation are hallmark signs for the most common forms of

15

. Evidence suggests that polypeptide conformational changes can lead to


Page 4 of 59

Page 5 of 59 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

instability of the misfolded intermediates due to interactions between hydrophobic regions and

2

the surrounding aqueous solution. Consequently, the polypeptide forms β-sheets to shield the

3

hydrophobic regions. Aggregation of the β-sheet oligomers can seriously disrupt the neuronal

4

environment, as in the case of β-amyloid buildup into plaques associated with Alzheimer’s

5

disease 14. The conversion of misfolded protein oligomers into insoluble fibrillary species can be

6

directly linked to cell death

7

soluble protein, an intermediate in the production of the final amyloid fibril.

8

As seen in Figure 1, misfolded and aggregated protein molecules are eliminated by three

9

different protective mechanisms, primarily based on molecule size: proteolysis, autophagy, and

10

inclusion body formation. Unfolded peptides and misfolded monomers are eliminated via

11

proteasomal degradation. The majority of misfolded oligomers, in addition to some misfolded

12

monomers, are removed via autophagic clearance. Larger oligomers and insoluble fibrils, which

13

can typically be visualized in cells, will form inclusion bodies. Ubiquitin plays a critical role in

14

all three pathways, as it marks proteins for proteolytic and autophagic degradation, and is found

15

in abundance in inclusion bodies. P62 has an ubiquitin binding domain, which can associate with

16

ubiquitin-modified proteins and shuttle them to the autophagosomal or proteasomal pathways for

17

degradation.

18

In the case of neurodegenerative conditions, mutations or posttranslational alterations lead to

19

protein misfolding. When these misfolded proteins evade degradation, they are then processed

20

into small-misfolded oligomers, at which point they become toxic to the neuronal environment.

21

Examples of these include α-synuclein, β-amyloid, and poly Q proteins, oligomers known for

22

their pathological roles in Parkinson’s, Alzheimer’s, and Huntington’s diseases, respectively.

23

The toxic oligomeric aggregates are primarily cleared via macroautophagy. Therefore, one of the

15

. The most cytotoxic molecule is considered to be the oligomeric



Page 6 of 59

1

attractive

2

neurodegenerative diseases, is to induce the removal of toxic oligomeric molecules by promoting

3

macroautophagy and the ubiquitin proteasome system (UPS) (Figure 1).

4

As shown in Figure 2, many neurodegenerative diseases share several of the hallmark misfolded

5

protein aggregates, highlighting a common underlying mechanism for neuron dysfunction 13. AD

6

is characterized by amyloid plaques comprised of Aβ and intracellular neurofibrillary tangles

7

formed by the accumulation of phosphorylated tau protein. The mutations in β-amyloid precursor

8

protein (APP) and presenlin 1, two proteins in Aβ metabolism signaling, are identified as disease

9

related gene changes. Hereditary cystatin C amyloid angiopathy (HCCAA) is a rare, but fatal

10

amyloid disease observed in young people in Iceland and caused by a mutation in cystatin C

11

gene. Cystatin C is colocalized with amyloid beta in AD and CAA. Familial Amyloidotic

12

Polyneuropathy (FAP) is typically caused by the aggregation of mutant transthyretin but is

13

sometimes due to aggregation of the wild type protein. Familial British Disease (FBD) and

14

Familial Danish dementias (FDD) are associated with mutations in the BRI2 gene, which are

15

characterized by cerebral deposition of the 34-mer British amyloid (ABri) and Danish amyloid

16

(ADan) peptides, and are accompanied by the observation of neurofibrillary tangles and neuro-

17

inflammation

18

α-Synuclein and polyubiquitinated proteins, is the central pathological feature of PD. Lewy

19

bodies have also been linked to the pathological outcomes of multiple system atrophy (MSA)

20

and other Lewy body diseases (LBD). Poly-Q expanded huntingtin, one of the various mutations

21

of the Poly-Q protein, is known to cause HD10. Other disease-associated mutations of the Poly-Q

22

protein have been identified in other neurodegenerative diseases, including Dentatorubral-

23

pallidoluysian atrophy (DRPLA), Spinal and Bulbar Muscle Atrophy (SBMA), and

therapeutic

17, 18

strategies

to

treat

proteinopathies,

including

those

found

in

. The presence of protein deposits called Lewy bodies, formed by aggregated


Page 7 of 59 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

Spinocerebellar Ataxia (SCA). TAR DNA-binding protein 43 (TDP-43) was noted as the

2

disease-causing protein for both Frontotemporal Dementia (FTLD) and ALS. Mutations in TDP-

3

43 cause familial ALS, while cytoplasmic and nuclear inclusions of TDP-43 are found in glial

4

cells and neurons for nearly all anatomical studies of sporadic ALS and FTLD. Prion diseases are

5

a family of rare progressive neurodegenerative disorders characterized by the spongiform

6

changes associated with neuronal loss and failed response to inflammation. Prion disease is

7

caused by a “prion”, which refers to a transmissible, pathogenic agent that can trigger misfolding

8

of cellular proteins called prion proteins, most of which are located in the brain. The common

9

types of Prion diseases affecting humans include Creutzfeldt-Jakob Disease (CJD), Variant 19

19

10

Creutzfeldt-Jakob Disease (vCJD), Gerstmann-Straussler-Scheinker Syndrome

11

Familial Insomnia

12

aggregates overlap the most commonly known neurodegenerative diseases, it is reasonable to

13

predict that treatment targeting the degradation of these misfolded or aggregated proteins might

14

have therapeutic effects. Macroautophagy is of importance then, as it is the major cellular

15

clearance mechanism of these toxic protein aggregates 21.

16

3. Autophagy in neurodegenerative diseases

17

Autophagy plays a crucial physiological and pathological role in the regulation of cell growth,

18

survival, and death, as well as macromolecule catabolic signaling, aging, inflammation, and

19

immunity. Either a deficiency or over-activation of autophagy will cause neuronal dysfunction,

20

an underlying condition for several brain pathologies

21

divided into several sequential steps: induction, initiation/vesicle nucleation, autophagosome

22

elongation and completion, maturation and fusion, and degradation (Figure 3). Genetic and

23

pharmacological regulations of many key players in the autophagy process are associated with

20 20

, Fatal

, and Kuru disease. In summary, since many of the misfolded protein

22

. The process of autophagy can be



Page 8 of 59

1

neurodegeneration, indicating a strong relationship between autophagy and neurodegenerative

2

diseases 22, 23.

3

3.1 Autophagosome formation and autophagic flux

4

As indicated in Figure 3, the autophagosome formation can be induced by inhibition of

5

mammalian target of rapamycin complex 1 (mTORC1) and activation of AMP-activated protein

6

kinase (AMPK), thus suggesting two independent ways that autophagy premature induction

7

could be resolved therapeutically. This will lead to the phosphorylation of serine/threonine-

8

protein kinase ULK1 and the subsequent phosphorylation of all the components in ULK1-Atg1

9

(AuTophaGy related 1) complex, including Atg 13, Atg 101, FIP200 (FAK family-interacting

10

protein of 200 kDa), ULK1, and ULK2 23. Phosphorylated serine/threonine-protein kinase ULK1

11

can also phosphorylate AMBRA in class III phosphatidylinositol 3-kinase (PI3K) CIII complex I,

12

which is composed of Phosphatidylinositol 3-kinase Vps34, Vps15, Atg14, and BCL-2-

13

interacting protein (Beclin-1), enabling the complex to relocate from the cytoskeleton to the

14

isolation membrane in the pre-autophagosome structure. In the PI3K CIII complex I, Beclin-1 is

15

negatively regulated by Bcl-2 and Bcl-X that related to ER (Endoplasmic reticulum) stress

16

Then, Vps34 in PI3K CIII complex will generate PI3K, which selectively interacts with the PI3P

17

effectors WD repeat domain phosphoinositide-interacting 1 and 2 (WIPIs), catalyzing two

18

reactions that mediate the isolation membrane elongation

19

conjunction of Atg5 and Atg12 in the presence of Atg7 and Atg10, followed by the Atg5-Atg12-

20

Atg16 complex formation

21

autophagosomes and promote the covalent interaction of Microtubule-associated proteins 1A/1B

22

light chain (LC3)-I with phosphatidylethanolamine (PE). In the process, Atg4 helps pro-LC3

23

translocate from the cell membrane to the early autophagosomal membrane, thus conjugating

23

23

22, 23

.

. The first reaction is a covalent

. This complex will translocate to the membrane of early


Page 9 of 59 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

with PE and becoming LC3-II. LC3-II can interact with p62 bodies (p62/ Next to BRCA1 gene 1

2

protein (NBR1) complex with ubiquitinated proteins and organelles) and thereby facilitate the

3

elongation and closure of autophagosomal membrane (Shown in Figure 3).

4

Finally, the autophagosome will fuse with the lysosome and its hydrolytic activity, forming an

5

autolysosome where the ubiquitinated proteins and organelle complexes are degraded.

6

Autolysosomes move along microtube tracks to fuse with lysosome. Microtube acetylation,

7

regulated by Histone deacetylase 6 (HDAC 6), is essential for fusion. Autolysosome formation

8

requires late endosome proteins, such as Ras-related protein Rab-7 (Rab7), several SNAREs, and

9

Lysosome-associated membrane glycoproteins (LAMPs) 23. For lysosomal degradation of cargos,

10

lysosomal acidification relies on vATPase, a proton channel on the lysosome membrane. “The

11

electrical gradient created is counterbalanced by parallel influx of anions mediated by chloride

12

proton antiporters. Cations, including calcium, can efflux through distinct channels or

13

transporters, including Two pore calcium channel protein 2 (TPC2) and Transient receptor

14

potential cation channel mucolipin-like protein (TRPML, mucolipin), which may also influence

15

pH

16

debris will be removed from the autolysosome (Figure 3) 23.

17

3.2 Neurodegeneration caused by abnormal gene regulation of components in autophagy

18

Many studies have reported that the Beclin-1 level is closely associated with neurodegeneration.

19

Reduced Beclin-1 level was detected in early stage AD9 and HD24. Impaired Beclin-1 expression

20

will increase Aß accumulation and mutant Huntingtin accumulation in AD

21

patients

22

aggregated proteins and improve neuron functions, thus providing protection against

23

. The cargo will be degraded in the autolysosome and the signaling factors and organelle

24

9

mice and HD

. Upregulation of Beclin-1 expression can be used to increase the clearance of



Page 10 of 59

1

neurodegeneration and prolonging the life span in AD 9, 25, HD 26, 27, PD 28, and Machado–Joseph

2

disease, a disease characterized by polyglutamine protein accumulation

3

enhanced Beclin-1 level has been reported in ALS 20, but a reduced Beclin-1 expression in ALS

4

patients has been found to increase neural protective activity against the disease 22, 29. It has been

5

reported that heterozygous deletion of Beclin1 caused earlier SOD1 aggregation, onset of

6

symptoms, motor neuron loss, and a markedly shortened survival in ALS mouse models 30.

7

Several research studies have indicated that regulating the Beclin-1 through its interaction with

8

other proteins can also alter the initiation step in autophagy and modulate aggregated protein

9

clearance in neurodegenerative disease models 22. In PD, both Parkin and PTEN induced putative

24

. Interestingly, an

31, 32

10

kinase 1 (PINK1) can interact with Beclin-1 to alter autophagy function

11

and mutant Leucine-Rich Repeat Kinase 2 (LRRK2) can also regulate the elimination of

12

damaged ubiquitinated mitochondria, thus affecting the mitophagic pathway

13

mutant SOD1 will interfere with the interaction between Beclin-1 and BCL-X, thus influencing

14

the autophagy level

15

autophagy dysfunction due to impaired vesicle sequestering 22, 34.

16

In the autophagosome elongation step, several mutations of the autophagy adaptor protein p62

17

have been identified in both familial and sporadic ALS patients. Gene expressions of p62 are

18

also related to many neurodegenerative diseases as mentioned above

19

(mHTT) expression leads to altered cargo recognition and autophagy failure. It is also reported

20

that α-Synuclein can bind to Rab1a and inhibit the interaction between Rab1a and the Atg9

21

complex, thus hampering the trafficking of autophagy vesicles

22

Atg5 and Atg7 in the central nervous system of mice will induce autophagy dysfunction and

23

spontaneous neurodegeneration, causing accumulation of aggregated proteins, extensive neuron

29

. Parkin, PINK1,

33

. For ALS, the

. VPS35, a rare mutation observed in PD patients, can also contribute to


36

35

. Mutant Huntingtin

. The genetic inactivation of

Page 11 of 59 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


37, 38

1

loss, and death of the mice

2

be modified by a polymorphism in Atg7 39.

3

In the fusion and degradation step, the proteolytic ability of lysosome depends on the luminal pH

4

in the lysosome

5

membrane, including v-ATPase proton channels, chloride proton antiporters, and calcium

6

transporters and so on. In AD, presenilin -1 can interact with v-ATPase subunit, regulating its

7

maturation and function to control the pH level in the lysosome, thereby influencing lysosome

8

function 40. Other studies showed restored lysosome function can alleviate AD-related symptoms

9

and improve neural function, which is in accordance with the above findings

41

10

ATPase ATP13A2 in the lysosomal membrane was observed in familial PD

42

11

with impaired lysosome function, altered proteolytic activity, and abnormal accumulation of

12

autophagosome and α-synuclein, indicating that lysosome dysfunction also influences the

13

occurrence of PD 43.

14

All these studies indicate a critical role of autophagy in neurodegenerative pathologies in

15

clearing toxic protein aggregates, protecting neurons against degeneration, and prolonging

16

neuronal survival.

17

3.3 Autophagy-enhancing compounds as therapeutic agents for neurodegenerative diseases

18

Since autophagy serves as an efficient approach to selectively degrade abnormal disease-related

19

proteins and damaged organelles in neurodegenerative diseases, several compounds were

20

screened and identified to enhance autophagy in specific steps and have potential therapeutic

21

efficacy to treat different neurodegenerative diseases. Here, we have summarized some of the

22

studies with compounds that can induce autophagy, promote autophagic degradation of disease

22

. Silke Metzger et al. reported that the age of onset in HD could

. The pH in the lysosome is regulated by ion channels on the lysosomal


. Mutations in

, accompanied


Page 12 of 59

1

related protein aggregates, and improve neuron function in cell and animal models (Table 1).

2

Some of the compounds have shown therapeutic efficacy in clinical trials. We will use the

3

mTOR inhibitor rapamycin, an agent widely tested in preclinical models in different

4

neurodegenerative diseases, as an example to illustrate the pharmacological effect of targeting

5

autophagy in neurodegenerative diseases.

6

Inhibition of mTOR by rapamycin enhances autophagy in the early stage of AD, improving

7

cognitive function, correlating with reduced levels of amyloid-β and tau phosphorylation as well

8

as delayed formations of plaques and tangles

9

has a protective function against neurodegeneration in fly and mouse HD models, inducing the

44-46

. D. Rubinsztein et al. reported that rapamycin

47, 48

10

clearance of mHTT associated with motor activity

11

neurons against degeneration and death in both in vitro and in vivo HD models, accompanied by

12

an ameliorated amount of Lewy bodies and α-synuclein in the brain

13

autophagy in both FTLD-U

14

autophagy alleviated the FTLD symptoms in the mice models

15

degeneration in ALS models

16

pathology via apoptosis, oxidative stress, and other mechanisms in SOD1G93A mice, according to

17

the severe mitochondrial impairment, higher apoptosis regulator Bax levels, and greater caspase-

18

3 activation 51.

19

4. Role and biomarker of p62/SQSTM1 in autophagy, and degradation of

20

ubiquitinated proteins

21

Ubiquitin-enriched misfolded protein inclusions represent an invariant characteristic for almost

22

all the neurodegenerative diseases 14. P62 serves as a protein adaptor for ubiquitinated substrates

50

51

. Rapamycin administration also protects

and SOD mutated mice models

51

50

49

. Rapamycin increases

. The rapamycin-induced

, but augments the motor

. It was indicated that rapamycin might exacerbate the ALS


Page 13 of 59 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

and selective macroautophagy 52. As shown in Figure 4, three domains contribute to the role of

2

p62 to shuttle ubiquitinated proteins to autophagosome for degradation, including C-terminal

3

UBA domain, LC3B-interaction region domain and N-terminal Phox-BEM1 (PB1) domain

4

Misfolded proteins first self-aggregate, are bound to chaperone proteins, and are then

5

ubiquitinated by UPS enzymes. The mono- or poly-ubiquitinated proteins then recruit p62 via its

6

C-terminal ubiquitin-associated

7

UBA domain binds to both mono- and poly-ubiquitinated proteins, with a preference for the K63

8

ubiquitinated proteins. Tanji et al. reported that K63-linked polyubiquitin is the most stable

9

enhancer for protein inclusions formation by increasing the protein accumulation and facilitating

10

the formation of intracellular inclusion bodies under normal conditions. Under pathological

11

conditions, co-cultured with tau and SOD1 mutation, K63 promotes the accumulation of tau and

12

the formation of SOD1-contained inclusion bodies. K63-linked polyubiquitin, acts as a partner

13

with p62 to enhance autophagic clearance of protein inclusions linked to common

14

neurodegenerative diseases

15

with K63 ubiquitinating E3 ligases (TRAF6) 55-57.

16

As shown in Figure 4, ubiquitinated protein/p62 aggregates continue growing larger and become

17

p62 bodies, which are then transported to a phagophore-forming location to form an

18

autophagosome. The formation, growth, and transportation of p62 bodies cannot be achieved

19

without the assistance of N-terminal PB1 domain in p62. The formation of p62 bodies relies on

20

p62 dimerization and further oligomerization. PB1 domain is indispensable and responsible for

21

p62 dimerization; any mutation or variants in this domain will hamper the p62 dimerization.

22

Both p62 homo-dimerization and heter-dimerization with NBR1, both of which are important for

23

the formation of p62 bodies, are regulated by the PB1 domain. Although the underlying

54

53

52

.

domain, leading to p62-promoted protein aggregation. The

. P62 may regulate K63-linked polyubiquitination via interaction



Page 14 of 59

1

mechanism is still unknown, the transportation of p62 bodies to autophagosome formation

2

location is also dependent on the p62-PB1 mediated homo-dimerization and heter-dimerization 21,

3

52, 56, 57

4

There are several biological processes related to p62. These processes could be measured by

5

their correlated biomarkers, such as LC3B and Beclin 1, biomarkers for macroautophagy. These

6

autophagy biomarkers might also be used as clinical prognostic biomarker, which can provide

7

information on the likely outcome of diseases on patients and help identify patients for the

8

specific treatment group. For instance, LC3B is one of the best and most commonly used

9

autophagy markers in multiple in vitro assays. The expression levels of LC3B protein have also

10

been examined as a useful biomarker by immunohistochemistry (IHC) for the selection of many

11

cancer patients treated with chloroquine (CQ) and other lysosomal inhibitors. The treatment of

12

lysosomal inhibitors causes the accumulation of p62 due to the blockage of p62 degradation in

13

autolysosomes. In addition to LC3B, other proteins, such as LC3A, Beclin 1, ULK1, and VPS34

14

are also used as autophagy biomarkers to monitor the autophagosome formation and autophagy

15

flux. They might be applied as potential clinical prognostic biomarker for many cancers as well.

16

The Beclin 1-VPS34 complex is a central coordinator for autophagy downstream

17

was identified as a potential prognostic biomarker with favorable outcomes for patients with lung

18

cancer, breast cancer, lymphoma and gastric cancer 59. Additionally, p62 itself is long served as a

19

biomarker for autophagy flux. Accumulation of p62 protein measured by immunofluorescence

20

and Western blot is usually considered an indication of autophagy inhibition 58.

21

LC3 interaction region is another critical domain in p62 that plays a role in autophagy. LC3

22

protein is the key component in autophagy that is necessary for autophagosome elongation and

23

closure as depicted in Figure 4. LC3 protein is cleaved by Atg4 protease to expose its C terminal

.


58

. Beclin-1

Page 15 of 59 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

Gly residue, and then conjugates to a phosphatidylethanolamine (PE) group to get its active form

2

LC3-II. LC3-II directly binds to the autophagosomal membrane and p62, and links them together

3

to form an autophagosome with ubiquitinated protein-p62 aggregates. P62 interacts with LC3-II

4

via its LIR domain (AA332-343). It has been suggested that the LIR domain also binds to key

5

mitophagy components BCL2 Interacting Protein 3 (BNIP3) and BCL2/adenovirus E1B 19 kDa

6

protein-interacting protein 3-like 23 60, 61, as well as the autophagy-dependent cell death mediator

7

Tumor protein p53-inducible nuclear protein 1 (TP53INP1)

8

oligomer constituted of about 11 amino acids shared by ten proteins, including p62. The

9

interaction between p62 and LC3-II anchors the p62 bodies onto LC3-II-containing

10

autophagosomal membrane, which then continues its maturation and elongation in the autophagy

11

process. Based on the selective interaction between LC3-II and p62, the autophagosome will

12

only contain the ubiquitinated protein aggregates with p62/NBR1, and go through a selective

13

autophagic degradation with lysosome fusion. Therefore, p62 and LC3-II are indispensable

14

regulators for misfolded protein clearance through selective autophagy. Knocking down of LC3

15

proteins has been shown to induce the accumulation of p62/NBR1-ubiquitinated proteins in the

16

cell plasma. Other studies have shown that ubiquitinated unfolded proteins or organelles could

17

interact with p62/NBR1, then attach to LC3-II in the autophagosomal membrane, to trigger

18

specific autophagic elimination

19

that shuttles the ubiquitinated proteins to autophagic degradation 56.

20

5. Role of p62 in UPS in neurodegenerative diseases

21

The UPS is responsible for the degradation of misfolded proteins via the 26S proteasome and

22

serves as a cellular quality control system. UPS protein clearance has two steps, covalent linkage

23

of polyubiquitin chains to target proteins, and degradation of the ubiquitinated proteins by 26S

57, 63

62

. The LIR domain is a short

. All these studies demonstrated that p62 is a cargo protein



1

proteasome complex and releasing free and reusable ubiquitin. Three enzymes take part in

2

ubiquitination of substrates and formation of a bond between the C-terminus of ubiquitin and

3

substrate lysine residue (K6, K11, K27, K33, K29, K48, and K63). “Ubiquitin-activating enzyme

4

(E1) forms a thiol ester with the carboxyl group of Gly76, activating the C terminus of ubiquitin.

5

The activated ubiquitin molecule is carried by ubiquitin-conjugating enzyme (E2) and transferred

6

to the substrate lysine residue by ubiquitin-ligases (E3)” 63

7

As discussed above, the UBA domain in p62 can bind to ubiquitinated proteins, especially those

8

possessing K63-linked polyubiquitin chains. N-terminus of p62 can directly interact with 26S

9

proteasome subunit component Rpn10 and Rpt164. Therefore, p62 can shuttle the

10

polyubiquitinated protein via UBA domain to 26S proteasome via its N-terminus for degradation.

11

In addition, as mentioned above, the PB1 domain of p62 can regulate p62 self-interaction and

12

heter-interaction with PB1 domains of other proteins. It has also been reported that PB1 domain

13

can assume an ubiquitin fold, which might be favorable for p62 N-terminal interaction with the

14

proteasomal subunit64. It has also been reported that p62 associates with polyubiquitinated tau

15

and targets it for proteasomal degradation, supporting the shuttling role of p62 in proteasome-

16

mediated protein degradation 65. A similar role for p62 has also been reported for TrkA shuttling

17

to the proteasome 66.

18

Proteasome inhibitor epoxomicin treatment can enhance levels of ubiquitinated protein and p62

19

in SK-N-SH cells 67. P62 overexpression does not affect proteasome catalytic activity, but delays

20

UPS substrate delivery to the proteasome's proteases, leading to their accumulation 68. Moreover,

21

p62 overexpression along with pharmacological inhibition of UPS and/or autophagy does not

22

further increase ubiquitin aggregates. It was also observed that soluble p62 co-localized with

23

proteasomes and p62 aggregates contained inactive proteasomes, ubiquitinated proteins, and 16 ACS Paragon Plus Environment

Page 16 of 59

Page 17 of 59 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


67

1

autophagosomes with epoxomicin treatment

2

critical role with shuttling ubiquitinated proteins for proteasome degradation.

3

Similar with macroautophagy, UPS, as another intracellular protein clearance system, is also

4

involved in the pathology of neurodegenerative diseases. One possible assessment of UPS

5

function in neurodegenerative diseases is to measure the endpoint proteolytic activity of the

6

proteasome.

7

different cell lines, animal models and brain tissues. The results have not been consistent. Some

8

studies have shown reduced proteasome activities in neurodegenerative diseases, including

9

Parkinson’s disease 40, 69-72, Alzheimer’s disease 73-77, ALS78, Huntington’s disease79, 80, and prion

. These studies further suggest that p62 plays a

Many studies have measured proteasome activities with purified proteasome,

10

diseases81.

11

neurodegenerative disease models81-85. This increase might be due to an induction of

12

immunoproteasomes in inflammatory response86, or could be an adaptive response to the

13

augmented load of misfolded proteins that occurs in neurodegeneration. 87

14

Given tremendous amount of proteins are involved in UPS, they might be also associated with

15

neurodegenerative diseases. For example, mutations in the vcp gene, which encodes valsolin-

16

containing protein (a ubiquitin binding protein involved in UPS trafficking), were reported to be

17

related to FTLD.

18

(UBQLN1), which encodes another shuttling protein that delivers ubiquitinated protein to the

19

proteasome, increase the risk of AD. The deubiquitinating enzyme ubiquitin carboxyl-terminal

20

hydrolase isozyme L1 gene (UCHL1) removes ubiquitin from proteins and is known to stabilize

21

monomer ubiquitin and facilitates E3 ligase in UPS. A certain mutation in UCHL1 has been

22

reported to inhibit the proteasomal degradation of α-synuclein, which makes individuals more

Some studies reported an unchanged or even increased proteasome activity in

A genetic study implied that certain variants in the ubiquilin 1 gene



88

Page 18 of 59

1

susceptible to PD

2

and restores synaptic functions in AD mouse models 89.

3

Parkin, an E3 ligase involved in UPS, is associated with the pathology of both AD and PD, and

4

is one of the major components found in lewy bodies and lewy neurites, the major pathological

5

features of PD

6

impaired LTP and behavioral abnormalities in APP/PS1 mouse model.

7

Hsp70-interacting protein (CHIP) is another important E3 ligase in UPS and has been implicated

8

in AD, HD, and PD pathogenesis. CHIP and heat shock protein 70 (HSP70) are upregulated in

9

the brain of AD mouse model

90

. On the other hand, UCHL1 overexpression is found to improve memory

. Parkin overexpression has also been reported to decrease Aβ and restore

94

91-93

. C-terminus of

. CHIP and HSP70 can induce the ubiquitination and degradation

10

of p-tau. In addition, overexpression of CHIP or HSP70 reduces steady-state Aβ levels and

11

promotes Aβ degradation in AD models

12

synuclein

13

Furthermore, CHIP can suppress the aggregation of polyglutamate proteins and reduce their

14

neuronal toxicity in HD models; while knockdown of CHIP exacerbates the HD process in a

15

transgenic model 99. 93

16

6. Sequestosome 1/p62: A multi-domain protein functioning as a signaling hub

17

As shown in Figure 5, Sequestosome 1/p62, originally identified in 1996 by Joung, functions as

18

an intracellular signal modulator in multiple signaling pathways. P62 is a multifunctional protein

19

rich of protein-interaction domains, including an N-terminal PB1 domain, ZZ-type zinc finger

20

domain, nuclear localization signal (NLS), TRAF6 binding domain (TBS), export motif (NES),

21

LC3-interacting region 3, Kelch-like ECH-associated protein 1 (Keap1)-interacting region (KIR),

22

and a C-terminal UBA domain, consistent with its role as a signaling hub 100. P62 protein acts as

97

95

. Moreover, CHIP can ubiquitinate LRRK2

96

and α-

, promote their degradation, and improve the ligase activity of Parkin in PD models 98.


Page 19 of 59 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

a signaling hub for different signaling pathways related to neuron disease, as shown in Figure 6

2

13, 101

3

Among its multiple motifs, p62 has an N-terminal PB1 domain, which is a protein-protein

4

interaction module present in many other signaling molecules, such as atypical protein kinases

5

Cs (αPKCs) and Mitogen-activated protein kinase kinase kinase 3 (MEKK3). These proteins and

6

p62 can bind to each other and themselves through their PB1 domains 7, 102. First, p62 interacts

7

with itself and aggregates via PB1 domain, facilitating its homo-oligomerization and cellular

8

function103. Additionally, heter-oligomerization can also occur with p62 and other PB1 domain-

9

containing proteins, including αPKCs, Dual specificity mitogen-activated protein kinase kinase 5

10

(MEK5), Mitogen-activated protein kinase 3 (MAPK3), MEKK3 and Rpt1, which have critical

11

roles in different signaling pathways that modulate adipogenesis, angiogenesis, neuron survival,

12

cardiovascular pathologenesis, as well as osteoclastogenesis 104.

13

Among them, the interaction between p62 and αPKCs is associated with the activation of the

14

nuclear factor NF-kappa-B (NF-κB), which is the downstream of cell stimulation by interleukin

15

1 (IL-1)

16

Nerve growth factor (NGF), to promote neuron survival and to trigger inflammation

17

downstream signaling cascades will be elevated or suppressed based on the up- or down-

18

regulation of p62 protein expression level. It was reported that knocking out p62 would reduce

19

the aPKC activity, thus increasing MAPK, RAC-alpha serine/threonine-protein kinase (AKT),

20

and Mitogen-activated protein kinase 8-10 (JNK) signaling

21

leading to Aβ pathology and inflammation in neurodegenerative disease. The p62 PB1 domain

22

also binds to MEKK3, thus activating NF-κB signaling

23

kinase-3 beta (GSK3β) activity was also enhanced in the p62 knock out mice, leading to tau

.

105

, (Tumor necrosis factor receptor superfamily member 11A) RANK ligand

102

109


106

107, 108

, or

. The

, which have detrimental effects

. In addition, glycogen synthase


110, 111

Page 20 of 59

1

hyperphosphorylation

2

p62 protein activated MAPK signaling, leading to insulin resistance, impaired plasma glucose

3

levels, and obesity

4

reported that the interaction between p62 and MAPK3 will promote adipogenesis 114.

5

Next, p62 has a zinc finger 106 domain, which interacts with receptor interaction protein (RIP) to

6

regulate NF-κB signaling and inflammation and necroptosis pathways in conjunction with

7

atypical PKC 5, 115-117. In addition, p62 possesses a newly reported region located between the ZZ

8

and TB domain that interacts with mTOR regulator Raptor

9

component for mTORC1 complex. P62 is necessary for mTORC1 activation in response to the

112

. Normally, p62 will suppress the activity of MAPK, knocking out

insulin resistance has been observed in AD brain samples

118

113

. It is also

, which makes p62 an integral

118

10

uptake of amino acids and the subsequent mTORC1 recruitment to lysosomes

11

their collaborators have discovered P62 ZZ domain specific inhibitors and identified them as

12

potential treatment for multiple myeloma and Huntington’s disease

13

have shown effect to modulate autophagy and proteolysis 123-125.

14

Moreover, p62 interacts with TRAF6, a lysine 63 E3 ubiquitin ligase, via its central the TRAF6

15

binding site (TBS) to regulate several signaling pathways that related to neurodegenerative

16

diseases

17

followed by K63 polyubiquitination of TRAF6, which leads to the activation of NF-κB 101, 105, 107.

18

In 2000, Sanz et al. reported that p62 can interact selectively with TRAF6, thereby activating the

19

NF-κB activation in response to IL-1

20

kinase A (TrkA), leading to NF-κB activation

21

that p62 binds to TrkA but not p75, whereas TRAF6 binds to p75 but not TrkA. They

22

demonstrated that an interaction between p62 and TRAF6 could act as a bridge to link p75 and

23

TrkA signaling, and a high affinity-binding site for NGF. They also suggested that p62 serves as

101, 105, 126

3, 119-122

. Xie lab and

. These inhibitors

. The interaction of p62 and TRAF6 induces p62-TRAF6 oligomerization,

105

. NGF interacts with both p75 and Tropomyosin receptor 127

. In 2001, Wooten and his colleagues showed


Page 21 of 59 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

a scaffold protein for the activation of NF-κB signaling by NGF, which mediates neuron survival

2

and differentiation responses

3

polyubiquitination, and this polyubiquitination of TrkA was reduced in p75 knock out mice.

4

Both mutations in ubiquitin (K63R) and an absence of TRAF6 will abolish the

5

polyubiquitination 128. Moreover, blocking the TBS domain in p62 and mutating the K485 in Trk

6

A with arginine will also eliminate the polyubiquitination of TrkA, and the following NGF

7

activated NF-κB signaling. This work indicated that polyubiquitination serves a role as a

8

common signaling platform for the complex formation and receptor internalization 128.

9

In 2005, Wooten showed that p62 facilitated the polyubiquitination of TRAF6

127

. Geetha and his coworkers found that NGF stimulated TrkA

129

. This

10

polyubiquitination will be inhibited or blocked by mutation or deletion of either the PB1, or

11

UBA, or TBS domain in p62. NGF stimulates the TRAF5 polyubiquitination and p62-TRAF6-

12

IKK-PKC complex formation, which are suppressed by the blocker of p62-TRAF6 interaction

13

129

14

ubiquitination, complex formation with TRAF6-P62-P75

15

impairment on Trk A tyrosine phosphorylation, ubiquitination, and downstream signaling in AD

16

patient brain hippocampus samples compared with the control. A possible explanation is the

17

nitrotyrosylation of TrkA is increased in the AD hippocampus, which might in turn reduce the

18

TrkA that undergoes ubiquitination and phosphorylation

19

reduced production of matrix metalloproteinase-7 (MMP-7) in AD hippocampus samples, which

20

cleaves proNGF, resulting in an accumulation of proNGF and an attenuated level of active NGF

21

130

22

and neuron death. Further analysis showed Aß and AD not only lessened the ubiquitination and

23

phosphorylation of Trk A, but also Trk A regulated downstream signaling, such as NF-κB, p38-

. C Zheng et al. reported that in PC12 cells, Aß impaired the Trk A phosphorylation, 130

. They also observed similar

55, 130

. Additionally, they reported a

. The accumulated proNGF will activate the p75 (not with TrkA), thereby inducing apoptosis



55, 101, 130

Page 22 of 59

1

MAPK, PI3K-AKT pathways

2

A/p75 induced neurotrophin signaling caused by lack of p62 or p75 have been linked to

3

cholinergic dysfunction in AD 131.

4

Furthermore, the LIR domain and C-terminal UBA domain enable p62 to function as an adaptor

5

between autophagy and ubiquitinated proteins. P62 binds to ubiquitin and ubiquitinated proteins

6

via its UBA domain, and then traffics the ubiquitinated protein complex to autophagosome

7

through LIR interaction with LC3. The detailed description was discussed in the above

8

macroautophagy section.

9

Additionally, p62 has a KIR domain that directly binds to Keap1, and interferes with the Keap-

10

nuclear factor erythroid 2-related factor 2 (Nrf2) interaction, activating the Nrf2 mediated

11

Reactive oxygen species (ROS) elimination. Keap1 is an adaptor for Cullin-3 ubiquitin ligase

12

that senses oxidative stress and binds to Nrf2. Nrf2, a basic leucine zipper protein, is responsible

13

for a series of antioxidant proteins and detoxifies enzymes, which protect cells against oxidative

14

damage triggered by injury and inflammation

15

for Keap1-Nrf2 interaction, thus inhibiting the interaction between Keap1 and Nrf2, leading to

16

the stabilization of Nrf2 and transcriptional hyperactivation of Nrf2 target gene 132, 133. Given that

17

p62 is closely involved in selective autophagy, the p62-Keap1-Nrf2 axis is also linked to

18

selective autophagy by some post-translational modification like phosphorylation and

19

ubiquitination 134. Moreover, since p62 is degraded through autophagy, the lack or deficiency of

20

autophagy in hepatocellular carcinoma cells or liver disease patients will result in the p62

21

accumulation, thereby evoking persistent activation of Nrf2

22

stimulates the p62 protein expression, which then produces a positive feedback loop between

23

Nrf2 activation and p62 protein expression. These studies showed that p62 served as a bridge to

. Furthermore, studies showed that deregulation of Trk

132

. Keap1 binds to KIR at the same binding site


135, 136

. On the other hand, Nrf2

Page 23 of 59 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

link the selective autophagy and ubiquitination system to the oxidative stress response system

2

and redox regulation

3

health. Kanninen showed that the AD symptoms in transgenic AD mice would be improved by

4

elevated Nrf2 expression138-140.

5

In addition to signaling pathways mentioned above, p62 protein also interacts with other proteins

6

and regulates other signaling pathways influential to brain function. For instance, p62 binds to

7

ubiquitinated Dishevelled protein (Dvl2) and mediates its autophagic clearance, so that p62 can

8

inhibit the Wnt signaling, which is known to play a role in AD pathogenesis by its ability to

9

control neuron development and maintain synaptic function in the brain

137

. Maintenance of homeostasis of p62 protein levels is crucial for neuron

141

. Moreover, two

10

nuclear localization systems (NLS1 and NLS2) and one nuclear export system (NES) were

11

identified in the structure of p62 protein. They are involved in the nucleo-cytoplasmic shuttling

12

for p62 and other scaffold proteins. P62 contains two PEST regions rich in proline (P), glutamic

13

acid (E), serine (S) and threonine (T), which serve as proteolytic signals for rapid degradation 142.

14

Furthermore, scientists reported that p62 could induce the intracellular aggregation and

15

autophagic clearance of cyclic AMP phosphodiesterase-4A4 (PDE4A4), eliminating the

16

PDE4A4 from their functional sites and thereby augmenting cyclic adenosine monophosphate

17

(cAMP) signaling. So the reduced p62 protein expression will attenuate the cAMP signaling,

18

which plays an important role in the mediation of memory and synaptic plasticity. 143-146

19

7. Genetic regulations of p62/SQSTM1 with neurodegenerative diseases

20

Dysregulations of p62 levels and functions were observed in several neurodegenerative diseases.

21

For example, studies in p62 knockout mice have clearly demonstrated that the lack of p62

22

protein could result in neuropathological lesions including the increase of hyperphosphorylated



Page 24 of 59

1

tau and neurofibrillary tangles, anxiety, depression, loss of working memory and reduced serum

2

brain-derived neurotrophic factor levels and neurodegeneration

3

a detectable increase in accumulation of insoluble Lys63 ubiquitinated proteins in the neuron of

4

p62 knockout mice 149i.

5

7.1 Regulation of p62 in ALS, FTLD, and ALS/FTLD

6

Amyotrophic lateral sclerosis is a progressive, invariable fatal neurological disease, which is

7

characterized by the gradual degeneration and depletion of motor neurons in both the brain and

8

spinal cord that are responsible for controlling voluntary muscle movement. Eventually, all

9

voluntary muscles will be affected, and the patient will lose control of muscle movement and

10

strength. Fatality in ALS patients is due to the loss of function of the respiratory muscles. The

11

formations of ubiquitin-immunoreactive inclusions are widely detected in the brain of ALS

12

patients. In 1991, Okamoto K et al. examined the brains of 27 amyotrophic lateral sclerosis

13

patients and 50 controls

14

hippocampal granular cell layer and entorhinal cortex in seven of the ALS patients. Then in 2004,

15

Nakano T and his colleagues tested p62 immunoreactivity in ubiquitin-positive inclusions in five

16

ALS-D patients and confirmed that the ubiquitin-immunoreactive inclusions are also p62

17

positive

18

inclusions and in preventing neuronal death in the ALS degenerative disease process.

19

Concurrently, mutations in SQSTM1 encoding p62 protein were screened in a large cohort of

20

both familial and sporadic ALS patients. Fecto et al. examined SQSTM1 mutations in a cohort of

21

546 patients 152. They identified 10 novel SQSTM1 mutations in 15 patients. Teyssou and his/her

22

colleagues sequenced SQSTM1 in 74 sporadic ALS cases and 90 familial ALS French patients

151

150

105, 147, 148

. Wooten et al. reported

. They found new ubiquitin-positive intra-neuronal inclusions in the

. This suggested that p62 and ubiquitin could play a common role in formation of


Page 25 of 59 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

153

2

Paget’s disease patients. They also identified two new missense mutations in sporadic ALS cases,

3

which were not identified in the control group. Further analysis revealed increased p62 levels in

4

the spinal cord and large p62 inclusions in motor neurons. A study from UK confirmed the

5

presence of the p(Pro392Leu) SQSTM1 mutation in UBA region in both familiar ALS and PDB,

6

indicating a common pathogenic pathway that converges on protein homeostasis 154. Jozsef et al.

7

reported that accumulation of p62 was can increase formation of polyubiquitinated proteins and

8

mutant SOD1 aggregates in G93A mouse spinal cord affected cells

9

recognize and bind to an ALS associated SOD1 isoform through its SOD1 mutant interaction

10

region (SMIR), indicating that p62 could link mutant SOD1 to autophagy and proteasome

11

pathways in both ubiquitin-dependent and independent mechanisms 156. Their findings suggested

12

a direct genetic role for p62 in ALS pathogenesis and a potential therapeutic use for ALS.

13

Absence of SQSTM1/p62 was found in children-onset neurodegenerative disease patients

14

fibroblast, and has been linked to defects in mitophagy owing to compromised autophagosome

15

formation following mitochondrial depolarization

16

has been evaluated in zebrafish model of ALS/FTLD, where p62 knockdown led to abnormal

17

motor behavior and shortening of motor neurons, which can be rescued by rapamycin

18

administration 157, 158.

19

Frontotemporal degeneration (FTD) is the most common cause of dementia under the age of 65

20

years and the third most common cause of neurodegenerative dementia, after AD and PD 159, 160.

21

The syndrome is also called frontotemporal lobar degeneration (FTLD). However, FTLD refers

22

to a larger group of disorders FTD being one of its subgroups. The other subgroups of FTLD are

23

progressive nonfluent aphasia (PFNA), and semantic dementia

. They confirmed two mutations in the UBA domain that were previously reported in ALS and

19

155

. They found p62 can

. The effect SQSTM1/p62 loss-of-function


61 161

. Neuronal cytoplasmic


Page 26 of 59

162

1

inclusions are pathological hallmarks in several neurodegenerative diseases

2

cytoplasmic inclusions composed of either the 43-kDa transactive response-DNA-binding

3

protein (TDP-43) or tau are characterized in almost 90% of FTLD

4

colleagues immunoprecipitated p62 in the cerebral cortex from FTLD patients with TDP-43 and

5

found that p62 co-immunoprecipitated several proteins, including TDP-43

6

involved in the degradation of 35kDa TDP-43, a truncated isoform of TDP-43

7

degradation and dysregulation of TDP-43 might play a causative role in ALS and FTLD

8

Mutations distributed throughout the p62 might affect the interaction between p62 and TDP-43,

9

and may participate in the pathogenesis of TDP-43 proteinopathy 54, 56.

54

. For FTLD,

. Kunikazu Tanji and his

54

. P62 can be 54

. The 160

.

10

In the last two decades, increasing evidence implied that ALS and FTLD share some clinical,

11

neuropathological and genetic features. A subtype of disease with both ALS and FTLD features

12

is classified as ALS-FTLD. Therefore, we mentioned the regulation of p62 in these two diseases

13

together in one section. FTLD signs can be seen in patients primarily diagnosed with ALS,

14

implying clinical overlap among these two disorders. It is reported up to 40-50% of ALS patients

15

have FTLD dysfunctions, accessed as frontal dysfunctions or language impairment

16

Additionally, some reports claimed ~50% of FTLD cases developed with ALS signs

17

subclinical motor neuron degeneration, while others reported about 10% FTD cases with ALS

18

syndromes. Probably these discrepancies are caused by different disease classification as well as

19

statistic evaluation methods

20

screening have contributed tremendously in elucidating the pathology and genetic variability

21

associated with FTD and ALS. Evidence of shared molecular pathological features was also

22

reported to explain the clinical overlaps between FTD and ALS, such as the ubiquitin-positive

23

neuronal inclusions, its associated TDP-43 aggregate proteinopathy 166-170, and tau pathology

166

163, 164

164, 165

.

, as

. In the last decade pathological investigations and genetic


Page 27 of 59 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

Some papers have uncovered the genetic overlap between FTLD and ALS. The most common

2

one is the C9orf72 repeat hexanucleotide expansion coding, which occurs in 10% of all patients.

3

Al-Sarraj and King et al. identified a new distinctive pathological subtype of C9orf72-

4

ALS/FTLD, which is characterized by p62 positive, phosphorylated TDP-43 (p-TDP-43)

5

negative cytoplasmic and nuclear inclusions

6

ubiquitinated protein in a distinctive inclusion, other than p-TDP-43 inclusion, which is the core

7

disease inclusion

8

(Gly-Ala), poly-(Gly-Pro), and poly-(Gly-Arg) repeat proteins translated from the GGGGCC

9

repeat expansion gene

160

160

. They found that p62 bound to an unknown

. Then, Mori et al. found that most of these core inclusions contain poly-

171

. The genetic mutations in ALS/FTLD and the dominant pathology in 171

10

C9orf72-ALS/FTLD patients are connected directly by this finding

11

reported a FTLD patient with mixed p62, TDP-43 and tau pathology 56, 172.

12

P62 is an excellent label to detect the ubiquitinated cytoplasmic inclusion bodies that are

13

important factors in neurodegenerative disease

14

LIR of p62, lead to defective recognition and reductive binding to LC3 and failure in

15

autophagosome formation and aggregated protein degradation. This phenotype was also

16

ameliorated by rapamycin administration, implicating a potential pathogenic role for autophagy

17

in SQSTM1-ALS

18

G351A, disrupt Keap1 binding, leaving Keap1 to interact with Nrf2, which inactivates the Nrf2

19

signaling related with oxidative stress response. This suggests that SQSTM1 mutations in KIR

20

reduce Nrf2 signaling activation and partially contribute to etiology of ALS-FTLD through a

21

mechanism of regulation of oxidative response genes 174.

22

P62-mediated degradation of SOD1 isoform and TDP-43 via autophagy may provide a

23

therapeutic option to treat a subtype of ALS, FTLD, and ALS/FTLD patients. The mTOR kinase

173

162

. Later, King et al. also

. ALS-FTLD associated L341V mutation in

. Two ALS-FTLD associated KIR mutations of SQSTM1/p62, P348L and



Page 28 of 59

1

inhibitor rapamycin enhances the autophagy in both FTLD-U 50 and SOD1 mutated mice models

2

51

3

augments the motor degeneration in ALS models

4

cognition and memory as well as decreased motor and neuronal function loss in FTLD mice

5

models treated with rapamycin, and three additional autophagy enhancers (spermidine,

6

carbamazepine, and tamoxifen)

7

motor neuron degeneration, shortening the life span of ALS mice accompanied with severe

8

mitochondrial impairment, higher Bax levels and greater caspase-3 activation

9

that rapamycin might exacerbate the ALS pathology via apoptosis, oxidative stress, and other

. The rapamycin-induced autophagy alleviates the FTLD symptoms in the mice models

50

51

51

50

, but

. Wang et al. observed the improvement in

. However, autophagy enhancer rapamycin accelerated the

51

. It is suggested

10

mechanisms in SOD1G93A mice

11

independent autophagy enhancer trehalose could also delay the motor degeneration process and

12

prolong the neuron survival via the activation of autophagy 175, 176.

13

7.2 Regulation of P62 in Parkinson’s disease (PD)

14

Parkinson’s disease (PD) is the second most common neurodegenerative disorder, following

15

Alzheimer’s disease, and the most common neurodegenerative motion disease. It is a progressive

16

disorder in the central nervous system (CNS) that affects movement. Tremors are the most well

17

documented sign of early stage PD; then progressive development of shaking, muscle rigidity,

18

slowed movements, and difficulty walking occurs. Cognitive and mental problems are common

19

in the advanced stages of PD. The cause of the disease is still unknown, but it is suggested to be

20

associated with a combination of genetic, epigenetic, and environmental factors. Intracellular

21

accumulation of Lewy bodies and Lewy neuritis, consists of aggregated proteins, such as α-

22

synuclein, parkin, and ubiquitinated proteins is one of major pathological factors of PD

23

Variants in gene encoding α-synuclein

. Additionally, other researchers suggested that the mTOR-

177, 178

93

.

present in autosomal dominant familial PD, while 28

ACS Paragon Plus Environment

Page 29 of 59 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


179, 180

181

182

183

1

mutations in genes encoding parkin

2

autosomal recessive familial PD

3

disease-causing genes identified for familial PD. Parkin is an E3 ubiquitin ligase, and PINK1 is a

4

mitochondria-targeted serine-threonine kinase. PINK1 and Parkin work together to maintain

5

mitochondrial integrity

6

of mitochondria by autophagy. Scientists reported that p62/SQSTM1 is indispensable for Parkin-

7

induced mitochondrial clustering

8

Parkin and P62 also has an important role in autophagy, loss of which in dopaminergic neurons

9

was recently demonstrated to cause Lewy pathology and motor dysfunction in aged mice 186. It is

10

also implicated that the dysregulation of parkin/p62 axis may involve in and proteasomal

11

degradation and selective vulnerability of neuronal cells during the onset of PD pathogenesis187.

12

It is reported that the interaction between p62 and LRRK2 regulates LRRK2 toxic biology in PD

13

models188. Mutations in LRRK2 lead to autosomal recessive familial PD. P62 physically

14

interacts with LRRK2 as a selective autophagic receptor, and modulate antophagic clearance of

15

LRRK2. In turns, LRRK2 indirectly regulate the phosphorylation of p62 and subsequent

16

interaction between phosphorylated p62 and Keap1

17

in hypothalamic paraventricular nucleus and the paraventricular nucleus in patients with PD and

18

Lewy body disease190. The phosphorylated α-synuclein positive Lewy body was co-localized

19

with p62, but the p62 bodies did not necessarily co-localize with phosphorylated α-synuclein.

20

The average size of p62 bodies was larger in PD patients compared to control (p

p62: a potential target for neurodegenerative disease - ACS

Recommend Documents