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Beclin 2-mediated autophagic degradation of the pathogenic proteins in neurodegenerative diseases
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Beclin 2-mediated autophagic degradation of the pathogenic proteins in neurodegenerative diseases
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Content
Beclin 2-mediated autophagic degradation of the pathogenic
proteins in neurodegenerative diseases
by
Siyao Liu
A Thesis Presented to the
FACULTY O THE USC KECK SCHOOL OF MEDICINE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirement for the Degree
MASTER OF SCIENCE
(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)
August 2021
Copyright [2021] Siyao Liu
ii
Acknowledgments
I would like to thank my thesis advisor Dr. Rongfu Wang for giving me the opportunity to
study and work in his lab. Throughout my master’s program, his patient instructions and
constructive suggestions are greatly beneficial to me.
I am very thankful to my committee members Dr. Weiming Yuan and Dr. J. H. James Ou
for their valuable time and constant encouragement. They are also the referees of my
Ph.D. application. Without their support, I will not get admitted and continue to chase my
academic dream.
I would also like to thank Dr. Yang Du and Dr. Motao Zhu, my mentors and friends, for
their continuous guidance throughout my training in this lab. When I need help, they are
always there for me.
Last but not the least, I would like to convey my heartful regards to my lab mates Dr.
Changsheng Xing, Dr. Tianhao Duan, Dr. Xin Liu, Dr. Junjun Chu, Dr. Chen Qian, and
Ms. Bingnan Yin for their help during the course of my project. I will always remember the
delightful and joyful moments we have spent in the last two years.
iii
TABLE OF CONTENTS
Acknowledgments ....................................................................................................................... ii
List of Figures ............................................................................................................................ iv
Abstract....................................................................................................................................... v
Chapter I. Introduction ................................................................................................................ 1
1.1 Neurodegeneration and proteinopathies ............................................................................................. 1
1.2 The molecular machinery of autophagy .............................................................................................. 7
1.3 Autophagy mechanism in neurodegenerative disorder ...................................................................... 8
1.4 Beclin 2 mediated non-canonical autophagy .................................................................................... 10
Chapter II. Materials and methods ............................................................................................12
2.1 Cell culture ........................................................................................................................................ 12
2.2 Antibodies and inhibitors ................................................................................................................... 12
2.3 Cell transfection ................................................................................................................................ 12
2.4 Immunoprecipitation and immunoblot analysis ................................................................................. 13
Chapter III. Results ...................................................................................................................15
3.1 Beclin 2 targets the key proteins in AD and other neurodegenerative diseases .............................. 15
3.2 Beclin 2 downregulates the key pathogenic proteins for neurodegenerative diseases .................... 16
3.3 Beclin 2 regulates the pathogenic proteins for neurodegenerative diseases through a lysosomal
degradation pathway ............................................................................................................................... 16
3.4 Determination of the key binding domains of Beclin 2 in mediating the degradation process ......... 18
Chapter IV. Discussion ..............................................................................................................20
Figures ......................................................................................................................................22
Figure 1. Beclin 2 interacts with the key molecules involved in neurodegenerative diseases................ 23
Figure 2. Beclin 2 downregulates key proteins involved in neurodegenerative diseases ....................... 25
Figure 3. Beclin 2 regulates the pathogenic proteins involved in neurodegenerative diseases through a
lysosomal degradation pathway .............................................................................................................. 27
Figure 4. Determination of the key binding domains of Beclin 2 in mediating the degradation process 28
References ...............................................................................................................................29
iv
List of Figures
Figure 1. Beclin 2 interacts with the key molecules involved in neurodegenerative diseases. 23
Figure 2. Beclin 2 downregulates key proteins involved in neurodegenerative diseases ........25
Figure 3. Beclin 2 regulates the pathogenic proteins involved in neurodegenerative diseases
through a lysosomal degradation pathway .............................................................................27
Figure 4. Determination of the key binding domains of Beclin 2 in mediating the degradation
process ..................................................................................................................................28
v
Abstract
The unprecedented increase in the number of older adults brings a higher risk of chronic
diseases in the United States and the whole world as well, such as dimentias, heart
disease, type 2 diabetes, arthritis, and cancer. Among all these diseases,
neurodegenerative disorders, such as Alzheimer's disease (AD), Parkinson's disease
(PD), and amyotrophic lateral sclerosis (ALS), are classified as one of the most severe
types, as they are mostly irreversible and incurable, lack robust early prediction, but affect
every aspects of the patient’s life. As the senior adults (65 or older) are expected to make
up nearly 25% of the population by 2060, there is an urgent need to develop new and
more effective therapeutic strategies to combat these devastating diseases.
The progression of neurodegenerative diseases is usually accompanied with the
accumulation of pathogenic proteins, such as the beta-amyloid and tau in AD, Lewy
bodies in PD, and TDP-43 in ALS. The prompt clearance of these pathogenic proteins is
critical for the prevention and early treatment of the neurodegenerative diseases.
Autophagy is a self-degradative program that removes misfolded or aggregated proteins,
damaged organelles, and intracellular pathogens, thus plays important housekeeping role
in maintaining the cellular homeostasis. In addition, autophagy protects against genome
instability and promotes cellular senescence, thus preventing chronic diseases such as
cancer, diabetes, and neurodegeneration.
In this study, we illustrated a previously unrecognized function of Beclin 2 as an important
regulator of neurodegenerative diseases for the clearance of pathogenic proteins by
vi
direct interaction. We also identified the key domains of Beclin 2 in the degradation
process and revealed that the ATG9A-dependent removal of these pathogenic proteins
required the existing components in the autophagic pathway. Therefore, our study not
only expands the current understanding in this field, but also provides potential
therapeutic targets for the prevention and better treatment of the neurodegenerative
disease
1
Chapter I. Introduction
1.1 Neurodegeneration and proteinopathies
Neurons are the foundation of the nervous system, including the brain and spinal cord. In
general, neurons do not reproduce for self-replacement. Therefore, when the neurons are
damaged or even dead, they cannot be regenerated by the host. Neurodegenerative
diseases are incurable and debilitating conditions that result in progressive degeneration
and death of the nerve cells
[1]
. According to their clinical manifestations, the
neurodegenerative disorders can be roughly classified. The most common
representations are extrapyramidal and pyramidal dyskinesias with cognitive or
behavioral disorders. Most of the patients show mixed clinical features, yet few have
single syndrome
[2]
. Although having a variety of clinical manifestations that reflect the loss
of specific neurons and synapses in different brain regions, the neurodegenerative
diseases have some common features and mechanisms. One of such features is the
accumulation of misfolded proteins into insoluble aggregates (or tangles) in the central
nervous system (CNS) with continued loss of neurons in the lesion area
[3]
.
Common neurodegenerative diseases include Alzheimer’s disease (AD), Parkinson’s
disease (PD), amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Huntington’s
disease (HD), and multiple system atrophy (MSA). The neurodegenerative disease
usually extends over a decade, and the actual onset may be many years earlier than the
clinical manifestations. Although it has been widely investigated, the mechanisms leading
to the chronic development of these neurodegenerative disorders are still obscure
[4]
.
2
Effective treatments could come up upon a deep understanding of the cause and
mechanism of each disease.
Alzheimer’s Disease
AD is a progressive neurodegenerative disease with insidious onset. Clinically, it is
characterized by general dementia such as memory impairment, aphasia, agnosia,
impairment of visual-spatial skills, executive dysfunction, and personality and behavior
changes. Alzheimer’s disease is the sixth-leading cause of death in the United States
[5]
,
and more than 5 million Americans with an age of 65 or older are living with Alzheimer’s
dementia; and the total cost for this disease was around 227 billion USD in 2018
[6]
.
Although the etiology is still unknown, AD causes the loss of neurons and synapses in
the cerebral cortex and certain subcortical structures, leading to the shrinkage of the
temporal, parietal, and frontal cortex, as well as cingulate gyrus
[7]
. AD is characterized by
the presence of two types of neuropathological hallmarks: senile plaques (SPs) and
neurofibrillary tangles (NFTs)
[8]
. SPs are the deposit of a protein fragment called beta-
amyloid (Aβ) that builds up in the spaces between nerve cells. The Aβ peptide is
generated by the amyloid precursor protein (APP), which is a type Ⅰ transmembrane
glycoprotein. APP is first cleaved by an extracellular protease α-secretase, which
liberates a soluble protein, sAPPα. Meanwhile, the fragment is also cleaved by an aspartyl
protease known as β-secretase 1 (BACE1), generating sAPPβ and a cell-membrane
bound fragment (C99). In the cytoplasm, C99 is cleaved by an enzyme complex called γ-
secretase. The fragment between the cleavage sites of BACE1 and γ-secretase is Aβ
[9]
.
Under pathological conditions, Aβ aggregates to form oligomers, protofibrils, fibrils, and
3
eventually plaques, which is believed to be the initial event of the AD process
[10]
. The
involvement of Aβ-induced synaptic dysfunction disrupts the neural connectivity and
associates with neuron death
[11]
. Beside Aβ, tau is a cytosolic soluble microtubule-binding
protein, which is the primary constituent of NFTs. Tau protein is associated with
dystrophic neurites and the loss of synapses, as well as microgliosis and astrogliosis.
Under normal condition, tau stabilizes the microtubule and associates with the regulation
of intracellular trafficking. Before the formation of tangles, tau undergoes a series of
modifications, including hyperphosphorylation, acetylation, and glycosylation. In AD, tau
could exist as monomers, small oligomeric species, paired helical filaments (PHFs), and
straight filaments
[12]
. Large fibrils contribute to cell dysfunction by disturbing the
intracellular material transportation in neurons, causing molecular crowding and effects
on cell metabolism
[12]
. Moreover, intracellular cytosolic protein tau can also be found
extracellularly
[13]
. The prion hypothesis holds that tau aggregates escape cells of origin to
enter adjacent cells, where they induce further tau aggregation and thus propagate
pathological tau
[14]
. Recombinant tau fibrils were observed to be able to induce
aggregation of intracellular tau in cultured cells
[15]
. Besides, tau pathology correlates with
the deposition of Aβ. In the absence of Aβ, hippocampal tau aggregation is insufficient to
trigger the neurodegenerative process. The mechanism of how protein misfolding
spreads through the brain could be a key point to better understand the myriad
neurodegenerative diseases.
Besides the aforementioned pathological structural changes, a number of AD studies
exhibited the important association of apolipoprotein E (APOE), which is involved in the
age of onset and the risk of AD
[16]
. As the major risk factor that determines the late-onset
4
Alzheimer’s disease (LOAD), APOE gene locates on chromosome 12 and contains three
allelic variants named ε2, ε3, and ε4. Carriers of ε4 are most likely to develop AD, while
ε2 is related to a decreased risk
[17]
. In peripheral tissues, APOE also mediates cholesterol
metabolism. In CNS, APOE is mainly produced by astrocytes and transports cholesterol
to neurons via APOE receptors
[18]
. Clinical samples show that Aβ deposition in the form
of SPs is more abundant in APOE4 carriers than noncarriers, and increasing researches
show that the effect of APOE4 on AD is executed by inhibiting the Aβ clearance while
promoting the Aβ aggregation. Besides, some studies implicate that APOE affects tau
pathology in an isoform-specific manner
[19]
. Similarly, the genome-wide association
studies highlight TREM2 in AD development. Commonly considered as a receptor of the
innate immune system, TREM2 is expressed by astrocytes in the brain. And the variants
of TREM2 have been found to increase the risk of LOAD. Consistently, the increased
expression of TREM2 has been observed in AD patients and in mouse models of Aβ and
tau pathology
[20, 21]
. Moreover, recent report reveals that TREM2 impacts microglia
metabolism during AD development by sustaining mTOR activation and suppressing
autophagy
[22]
.
Parkinson’s disease
After AD, PD is the second most common neurodegenerative disorder
[23]
, a progressive
neuron system disease that affects movement. Its symptoms include shaking, tremors,
bradykinesia, and walking difficulty. PD is characterized by the loss of dopaminergic
neurons from substantia nigra and the presence of intracellular structures called Lewy
bodies (LBs). And the α-synuclein (SNCA) is considered as an important factor in the
5
onset of PD. Under physiological conditions, SNCA associates with the regulation of
dopamine release and transport; and in non-dopaminergic neurons, it diminishes
caspase-3 and provides neuroprotective function
[24]
. LBs in PD are similar to Aβ in AD,
and the major component of LBs is aggregated SNCA. Recent works also show that
aggregated SNCA can spread seed between cells and interconnection regions of the
brain
[25]
. In addition, mutations in a number of related genes, such as Parkin RBR E3
ubiquitin-protein ligase (PARK2), Parkinson disease protein 7 (PARK7), and PTEN-
induced putative kinase (PINK1), have been identified as causes of PD
[26]
. PARK7 has
multiple functions; it works as an antioxidant, regulates transcription, prevents the
aggregation of SNCA, and is involved in the proteolytic pathway
[27]
. PINK1 is a
mitochondrial serine/threonine-protein kinase, which is thought to protect cells from
stress-induced mitochondrial dysfunction
[28]
. PINK1 accumulates on the surface of
damaged mitochondria, regulates PARK2 translocation, and recruits other factors to the
impaired mitochondria. PARK2 is an E3 ubiquitin ligase with an amino-terminal ubiquitin-
like (Ubl) domain and a carboxyl-terminal ubiquitin ligase domain. PARK2 ubiquitinates
outer mitochondrial membrane proteins and triggers selective autophagy, which is known
as mitophagy
[29]
. Healthy neurons efficiently eradicate impaired mitochondria by
mitophagy to maintain the cellular homeostasis. Excessive mitochondria stress may
cause both neurodegeneration and neuroinflammation. Increasing evidence shows the
implication between mitochondrial dysfunction and the pathogenesis of PD, and
PINK1/PARK2-mediated mitophagy is an attractive target for the therapeutics of this
disease
[30]
.
Amyotrophic lateral sclerosis
6
ALS is a progressive nervous system disease characterized by gradual deterioration and
death of motor neurons in the brain and spinal cord, causing the failure of muscle control.
The underlying mechanisms of neurodegeneration in ALS are still not fully clarified. This
disease is thought to have sporadic connections with both environmental and genetic
risks. Myriad cellular and molecular processes are involved, including mitochondrial
dysfunction, axonal transport, toxic protein aggregation, impaired protein degradation,
etc
[31]
. Abnormal aggregation of transactive response DNA binding protein 43 (TDP-43),
as the major component of neuronal inclusions in ALS, is the most common pathological
feature for this disease. TDP43 is generally localized in the nucleus and regulates gene
transcription, however, misfolded TDP43 forms aggregates in the cytoplasm. The
mislocalization leads to the loss of their transcriptional function in the nuclear, and toxic
TDP43 deposition segregates the essential cellular components
[32]
. Superoxide
dismutase 1 (SOD1) is commonly present in both cytosol and mitochondria
intermembrane to control the level of oxidizing species, and is found to be critical for
repressing respiration and directing energy metabolism. However, mutated SOD1 in
family ALS and wild-type (WT) SOD1 in sporadic ALS exert their detrimental effects by
the formation of protein aggregates that accumulate in mitochondria and disturb different
cellular processes
[33]
. Evidence shows that SOD1 and TDP43 aggregates are capable of
spreading through the neuroanatomical pathway through a self-perpetuating or prion-like
mechanism which is shared in many other diseases
[34]
.
In most neurodegenerative disorders, specific proteins have been found to misfold and
aggregate to disturb intracellular and extracellular physiology processes. A majority of
aggregates can form seeds and propagate themselves to other cells and tissues for the
7
instigation and progression of the diseases. Targeting and clearance of these protein
aggregates highlight potential therapeutic strategies to better treat these
neurodegenerative diseases.
1.2 The molecular machinery of autophagy
Autophagy is an ancient life phenomenon existing universally in eukaryotes. Autophagy
was first observed by Ashford and Porten in human hepatocytes through electron
microscopy in the 1960s, yet it was not until the 1990s that autophagy-related (ATG)
genes were identified in yeast
[35]
. The results of these studies have incredibly broadened
our knowledge of the mechanism and function of autophagy. Autophagy is a programmed
intracellular degradation mechanism that involves the lysosomes to clear cytoplasmic
components to achieve cellular metabolic needs and the renewal of certain organelles,
thereby maintaining the cellular homeostasis
[36]
. According to the different pathways of
substrates entering the lysosome, autophagy can be divided into three classes: (1)
macroautophagy, in which cytoplasmic cargo is transported into the lysosome by forming
a vesicle to degrade the aged or damaged proteins and organelles. (2) microautophagy,
in which small components are internalized by lysosome directly. (3) chaperon-mediated
autophagy (CMA), in which substrate proteins enter the lysosome through a multimeric
translocation complex with the absence of membrane recognition
[37]
.
The research on macroautophagy is the most in-depth, and it is considered as the main
type of autophagy. Upon autophagy stimulation, the cytoplasm will be wrapped in a
double-membrane structure called phagocytes that are formed de novo. The edges of the
phagophore then curve and fuse to form a closed double-membrane vacuole, called
8
autophagosome. Consequently, the inner membrane and luminal constituents of the
autolysosome are degraded by lysosomal acid
[38]
. The operation and regulation of
autophagy is a very sophisticated process. Despite the rapid advances of autophagy
research, the knowledge of some mechanisms is still under conundrum.
1.3 Autophagy mechanism in neurodegenerative disorder
Autophagy is a critical biological pathway that functions to promote cell health and
longevity. The housekeeping functions of autophagy include the eradications of defective
proteins and organelles, misfolded protein aggregates, and intracellular pathogens
[39]
.
Mounting evidence reveals that the abnormal alteration in autophagy occurs in many
human diseases, as autophagy participates in many crucial cellular pathways and yet
plays an important role in disease conditions. Since the presence of abnormal proteins is
recognized as a prevailing subject in all neurodegenerative disorders
[40]
, the stimulation
of autophagy becomes a promising therapeutic strategy to promote the degradation of
these abnormal intracellular components
[41]
.
Recent studies further reveal that the degradation of the disease-related proteins highly
depends on autophagy. TREM2-deficient mice with AD-like pathology have increased
autophagic vesicles and damaged mTOR activation ability
[22]
. Up-regulation of the
endosomal-lysosome is found in the early stage in AD, which might be related to the
processing of APP secretion
[42]
. The excessive autophagy processes, including lysosome
proliferation and increased expression of lysosomal enzyme, are imperative for the
clearance of misfolded protein aggregates and toxic proteins in the early stage of AD. On
the contrary, the fusion of mature autophagic vacuoles (AVs) and lysosome is impaired
9
during the pathological progress, resulting in the insufficient removal of enzymes that
associated with generation of Aβ and other intracellular components
[43]
. Similarly, the
autophagy-lysosomal pathway (ALP) is also damaged during AD development and
causes the accumulation of toxic tau. Pharmacological inhibition on mTOR activation
promotes the autophagy clearance of tau and relieves the phenotype in AD patient-
derived neurons
[44]
. In PD, SNCA can be degraded through both autophagy and
proteasome pathways
[45]
. However, the fibrillar forms of proteins influence the protease
activation by blocking the main enzyme functions due to the large structure. Moreover,
soluble SNCA can be degraded by CMA, but pathogenic SNCA is poorly removed by
CMA, as it blinds the CMA receptor at the lysosomal surface. This process also blocks
the clearance of other molecules and worsens the disease progression
[46]
. The increased
autophagosome marker and decreased lysosomal marker have been reported in PD
patients with LBs, further indicating the presence of dysfunctional ALP. Toxin-induced
cells and PD animal model both show the increases of AVs after the treatment of
mesenchymal stem cells (MSCs), which generate different cytotropic factors that exert
neuroprotective functions with enhanced AVs formation. MSCs treatment also reduces
SNCA and improves cellular viability
[47]
. The correlation between enhanced autophagy
and SNCA implies that autophagy plays an essential role in maintaining SNCA
homeostasis, therefore, the modulation of autophagy pathways could be applied in PD
treatment. Likewise, in ALS, the accumulation of TDP43 concurs with the inhibition of
proteasome and autophagy pathway
[48]
. After the treatment of autophagy stimulators,
neuron cells have accelerated TDP43 clearance and reduced mislocalization.
10
Furthermore, autophagy improves the survival of human iPSC-derived astrocytes and
neurons from ALS patients
[49]
.
Conclusively, these convincing evidences support the scheme that insufficient cellular
control system is the basis causation of neurodegenerative disorders, which is related to
the impaired self-cleaning system. Activation of the autophagy process can be utilized as
an effective therapeutic approach for the clearance of abnormal aggregates and toxic
structures.
1.4 Beclin 2 mediated non-canonical autophagy
The essential autophagy protein Beclin 1 is well-established for its role in the regulation
of autophagy and oncogenic suppression by interacting with several cofactors
[50]
.
Recently, an uncharacterized mammalian-specific gene that shared 57% sequence
identity with Beclin 1 was clarified, named Beclin 2
[51]
. The analysis of mouse mRNA level
of Beclin 2 shows that it can be expressed in multiple organs and tissues, including the
brain, lung, placenta, thymus, uterus, stomach, and testis. Beclin 2 protein expression is
also detected in human brain. Further studies show that Beclin 2 interacts with multiple
known Beclin 1-interacting proteins, and shares similar functions in controlling
autophagy
[51]
. Moreover, Beclin 2 interacts with G-protein-couple protein (GPCR)
associated sorting protein 1 (GASP1), and functions in degrading GPCR in a lysosome-
dependent pathway. A followed study also shows that Beclin 2 controls the oncogenesis
induced by Kaposi’s sarcoma-associated herpesvirus (KSHV) by enhancing the
degradation of viral-GPCR (vGPCR) via an endolysosome trafficking route
[52]
. However,
11
the detailed mechanism of how Beclin 2 performs a lysosomal-dependent function, which
is also independent of the traditional autophagy machinery, remains unknown.
Recent studies reveal that Beclin 2 regulates inflammation by inhibiting ERK1/2 signaling,
and promotes MEKK3 and TAK1 by a non-canonical autophagic process, which is
ATG16L/LC3B/Beclin 1 independent
[53]
. Mechanistically, Beclin 2 interacts with MEKK3
and ATG9A through the activation of ULK1 complex and forms Beclin 2-ATG9A-MEKK3
complex on ATG9A+ vesicle. Beclin 2 further interacts with STX5 and STX6 to promote
the fusion of ATG9A+ vesicle and phagophores, then engages in the elongation of
autophagosome, followed by the fusion with lysosome for subsequent degradation
[53]
.
These findings elucidate the important role of Beclin 2 in the negative regulation of innate
immune signaling through an ATG9A-dependent autophagic pathway. As the autophagy
has been implicated in diverse tissue homeostasis and renovation in both health and
disease conditions
[38]
, whether and how Beclin 2 participates in normal cellular regulation
and pathogenesis of human diseases remain incomprehensible.
This study illustrates that Beclin 2 is responsible for the removal of pathogenic proteins
that are involved in neurodegenerative diseases, mainly through a non-canonical
autophagic pathway. These findings indicate that an alternative autophagy process might
contribute to the cellular metabolism and play an important neuroprotective function.
12
Chapter II. Materials and methods
2.1 Cell culture
HEK293T cell line from human embryonic kidney was used for the in vitro experiments.
HEK293T cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Gibco),
supplemented with 10% Fetal Bovine Serum (FBS) (Gibco) and 1% penicillin-
streptomycin (P/S) (Gibco). Cell stock was removed from the -80 ℃ freezer, thawed in a
37 ℃ water bath, and seeded in T75 flasks. Cells were incubated in a 37 ℃ incubator
and passaged when the confluency reached 80%-90%.
2.2 Antibodies and inhibitors
Primary antibodies for western blotting: anti-HA-Peroxidase (#12013819001, Sigma-
Aldrich), and anti-Flag-Peroxidase (#A8592, Sigma-Aldrich).
Second antibodies for western blotting: anti ‐mouse IgG ‐HRP (#7076, Cell Signaling
Technology), and anti ‐rabbit IgG ‐HRP (#7074, Cell Signaling Technology).
Drugs: Bafilomycin A1 (#54645S, Cell Signaling Technology).
2.3 Cell transfection
HEK293T cells were seeded in 500 μL complete DMEM in 24 well-plates (3-4× 10
5
cells/well). After attached, cells were transfected using Lipofectamine 2000 (Invitrogen).
For immunoprecipitation, cells were co-transfected with the expression vectors of both
HA- and Flag-tagged SOD1, APP, GAK, PARK2, PARK7, SUPT4H1, SUPT5H, TDP43,
13
GAK, TREM 2, PINK, and SNCA, alone with empty pcDNA3.1 vector (EV) HA- or Flag-
tagged Beclin2. For immunoblot analysis, cells were transfected with Flag-tagged APP,
GAK, PARK2, PARK7, SUPT5H, TDP43, and GAK, HA-tagged PINK and TREM 2, alone
with EV or increasing amount of HA-tagged or Flag-tagged Beclin 2. Cells were collected
for analysis 24 hours after the transfection.
2.4 Immunoprecipitation and immunoblot analysis
Aspirate the cell culture medium, add precooled low salt lysis buffer (for 1 liter: 3.79 g
glycine, 10 g sodium dodecyl sulfate (SDS), then adjust PH to 2.0), place cell culture
plates on the rotor at 4 ℃ for 30 minutes for cell lysis. The supernatant was obtained by
centrifuging the cell lysates at 13000 g for 10 minutes. For immunoprecipitation, incubate
200 μL cell lysates with anti-Flag agarose gel and anti-HA agarose gel, wash the samples
with the low salt lysis buffer, and place samples at 4 ℃ overnight. Beads were then
washed 5 times with low salt lysis buffer, followed by the elution with 20 μL 3×SDS loading
buffer. For immunoblot analysis, add 6 μL 5×SDS loading buffer in 15 μL cell lysates.
Load all samples on the 10% SDS-Polyacrylamide gels after boiling at 95 ℃ for 5 minutes.
The protein marker (#1610395, Bio-Rad) was loaded to indicate the protein sizes. Run
the gel at 80 Volts for 30 minutes, then change the Volt to 120V and run for another 70
minutes. Activate the Polyvinylidene Fluoride (PVDF) membrane in methanol for 1 minute
before transferring the protein samples onto it, then run the protein transfer program at
100 volts for 2 hours on ice. The sample-loaded membranes were blocked in 5% non-fat
milk/TBST for 1 hour at room temperature, incubated with appropriate antibodies, and
14
developed with Immobilon Western chemiluminescent HRP substrate (Millipore) for
protein detection.
15
Chapter III. Results
3.1 Beclin 2 targets the key proteins in AD and other neurodegenerative
diseases
Our previous examination of Beclin 2 expression has indicated that this gene could be
widely expressed in multiple mouse organs and tissues, including the brain, at least on
mRNA level
[53]
. Human Beclin 2 protein expression has also been detected in human fetal
and adult brain samples
[51]
. As an essential regulator of the autophagy pathway, Beclin 2
targets MEKK3 and TAK1, and promotes their degradation
[54]
. Since autophagy has been
linked to several neurodegenerative diseases, we aim to figure out whether Beclin 2 may
function in regulating the nerve cell metabolism and the neurodegenerative diseases.
We first co-transfected 293T cells with GFP-Tau and Flag-Beclin 2. After 24 hours, cells
were collected for co ‐immunoprecipitation (co ‐IP) and immunoblot analyses. We showed
that Beclin 2 could interact with Tau, suggesting that Beclin 2 signaling may be linked to
the AD development (Figure 1A). To explore the possibility that Beclin 2 could directly
target key pathogenic proteins in AD and other neurodegenerative diseases, we further
performed co-IP analyses in the 293T cells expressing Beclin 2 and other potential target
proteins in AD, PD, and ALS. We found that Beclin 2 could interact with APP, APOE,
TREM2, PINK1, PARK2, PARK7, SOD1, GAK, and SUPT5H (Figures 1B and 1C),
besides Tau protein. These data suggest the potential association of Beclin 2 signaling
with multiple neurodegenerative diseases.
16
3.2 Beclin 2 downregulates the key pathogenic proteins for
neurodegenerative diseases
We next sought to determine whether Beclin 2 could target the key molecules involved in
neurodegenerative diseases for protein degradation. We co-transfected 293T cells with
Tau, TuaE14 (A pseudohyperphosphorylated mutant carrying 14 (serine/threonine to
glutamate) mutations), APP, APOE, TREM2, PINK1, PARK2, PARK7, SOD1, GAK,
SUPT5H, and TDP43 expression vectors, along with EV or increasing amount of Beclin
2. Our results showed that the protein levels of Tau, TauE14, APP, APOE, TREM2, and
PARK7 were considerably reduced with the increase of Beclin 2 expression, while GAK
protein level was mildly decreased (Figure 2A). In contrast, we did not observe the protein
level changes for PINK1, PARK2, SOD1, and SUPT5H (Figure 2B). Next, we detected
whether the endogenous protein levels could be affected by Beclin 2. The 293T cells were
transfected with an increasing amount of Beclin 2. By using relative antibodies, the
western blotting results showed decreased protein amounts of endogenous Tau, PINK1,
PARK7, and APOE in 293T cells (Figure 2C). Therefore, our data suggest that Beclin 2
could promote the degradation of these important pathogenic proteins, thus may
ameliorate the development of relevant neurodegenerative diseases.
3.3 Beclin 2 regulates the pathogenic proteins for neurodegenerative
diseases through a lysosomal degradation pathway
After we identified the significance of Beclin 2 in regulating the aforementioned
pathogenic proteins, we further investigated the underlying mechanisms. Since Beclin 2-
mediated non-canonical degradation requires the lysosome for conducting its function,
17
we used bafilomycin A (BafA) to inhibit the autophagy-dependent degradation. BafA has
been reported to inhibit the acidification of the lysosome and impair the lysosomal
protease activity, and block the fusion between autophagosome and lysosome as well.
We added 500 nM BafA into the cell culture medium for a treatment of 6 hours, and found
that the Beclin 2 mediated degradations of Tau, Tau E14, APOE, TREM2, TDP43, APP,
and PARK7 can be significantly suppressed by this autophagy inhibitor (Figure 3A), thus
indicating that the autophagic pathway is required for the protein degradation.
Furthermore, Beclin 2 mediated autophagy does not require the hierarchical action of
ATG proteins, but rather a set of ATG proteins that are critical for a pre-existing double-
membrane structure for autophagosome formation. ATG9A is the key to bridging the
degraded proteins and Beclin 2. Thus, we utilized ATG9A knock out 293T cells to validate
whether the degradation of these pathogenic proteins is similarly through the ATG9A-
dependent pathway. We found that ATG9A ablation rescued the degradation of Tau and
TauE14 by Beclin 2 signaling (Figure 3B). These results indicate that Beclin 2 degrades
the neurodegenerative disease-associated proteins in an ATG9A-dependent manner.
Altogether, this work illustrated a previously unrecognized function of Beclin 2 as an
important regulator of neurodegenerative diseases for the clearance of pathogenic
proteins by direct interaction. We also identified the key domains of Beclin 2 in the
degradation process and revealed that the ATG9A-dependent removal of these
pathogenic proteins required the existing components in the autophagic pathway.
Therefore, our study not only expands the current understanding in this field, but also
provides potential therapeutic targets for the prevention and better treatment of the
neurodegenerative diseases.
18
3.4 Determination of the key binding domains of Beclin 2 in mediating
the degradation process
Different structural domains of Beclin 1 have been identified to have close interaction with
various elements that are involved in autophagy, tumor suppression, and programmed
cell death
[55-57]
. Similar to Beclin 1, Beclin 2 contains 4 structural domains: an N-terminus
domain (N), a BH3 domain, a central coiled-coil domain (CCD), and a C-terminal
evolutionarily conserved domain (ECD)
[51]
. Given that Beclin 2 act as an autophagy
regulator and mediate the degradation of its interacted proteins, we aim to distinguish
which domains of Beclin 2 are involved in the interaction with the essential elements for
protein degradation process. Mechanistically, Beclin 2 recruited the degraded proteins
through ATG9A, and form a complex (Beclin 2-ATG9A-MEKK3) on ATG9A+ vesicles.
Beclin 2 further interacts with STX5 and STX6 to promote the fusion of ATG9A+ vesicle
to phagophore and the elongation of autophagosome
[53]
. To further identify the key
domains of Beclin 2, we constructed HA-tagged full-length (FL) Beclin 2, ΔN, CC-ECD,
ECD, and ΔECD expression plasmids. We co-transfected those plasmids with ATG9A,
STX5, and STX6. After harvesting the cells, Flag antibody was used in the pull-down
assay to detect the interactions. Co-IP results showed all these 3 proteins (ATG9A, STX5,
and STX6) could interact with FL, ΔN, and CC-ECD, but not with ECD or ΔECD (Figure
4), suggesting that CC-ECD domain is an indispensable component for Beclin 2 to
regulate the degradation pathway. Importantly, we also analyzed the interaction of Beclin
2 with Tau, and the CC-ECD domain is also the minimum fragment for Tau binding (Figure
4). Since the protein removal through autophagy is highly selective
[58]
, the CC-ECD
19
domain might play a critical role in special cargo recognition of Beclin 2 and the target
proteins.
20
Chapter IV. Discussion
In this study, we first investigated the interactions between the key autophagy protein
Beclin 2 and the pathogenic proteins involved in AD, PD, and ALS, and identified several
pathogenic proteins that might be regulated by this protein. Beclin 2 is a relatively novelty
autophagy gene identified in 2013. It has been reported that homozygous deletion Beclin
2 leads to significantly impaired autophagy signaling
[51]
, indicating the importance of
Beclin 2 in the metabolism of nervous system. Thus, we further studied whether Beclin 2
could mediate the degradation of these important target proteins, and showed that the
protein levels of Tau, TauE14, APP, APOE, TREM2, and PARK7 were markedly reduced
by the presence of Beclin 2.
Preliminary mechanistical studies elucidated that Beclin 2 controlled the stability of
MEKK3 and TAK1 through an ATG9A-dependent, but ATG616L/LC3/Beclin 1-
independent, autophagic pathway. Thus, further experiments have been performed to
explore the specific process of Beclin 2 in degrading those proteins. With the treatment
of autophagy inhibitor BafA1, we confirmed that Beclin 2 degraded the key proteins in
neurodegenerative diseases through an autophagy process. Meanwhile, the Beclin 2-
mediated degradation of Tau and TauE14 was also inhibited by the ablation of ATG9A,
which is a crucial element in this non-canonical autophagic pathway. These data further
demonstrated that the degradation of these pathogenic proteins by Beclin 2 may go
through a unique selective autophagy pathway, with tightly regulated specific cargo
recognition by the autophagy machinery.
Previous reports have showed that the distinct functional domains of Beclin 1 could
associate various factors in the governance of critical signaling pathways other than
21
autophagy
[59]
. Moreover, the conformational integrity is also the key for Beclin 1 to
execute the complete functions
[57]
. However, similar studies have not been performed for
Beclin 2. To clarify the potential functional domain of Beclin 2 that is responsible for taking
part in the autophagy process, the Beclin 2 FL, ΔN, CC-ECD, ECD, and ΔECD expression
plasmids were generated for the detection of the interaction with key elements in Beclin
2-mediated autophagy. CC-ECD domain was identified to have robust binding affinity to
ATG9A, STX5, and STX6 as key autophagy elements, as well as Tau protein for its
degradation. Therefore, our domain results promote a deeper understanding of Beclin 2-
associated intracellular physiological processes.
Current researches on neurodegeneration therapeutics have proposed several
candidates
[60-63]
, including the autophagy modulators
[44, 64, 65]
, and the upregulation of
autophagy is associated with beneficial effects. Our study describes Beclin 2 as involved
in the regulation of pathogenic protein levels in the nervous system, and reveals the novel
critical roles of Beclin 2-mediated autophagy in controlling the health and diseases in the
nervous system, thus provides potential therapeutic targets for the treatment of the
neurodegenerative diseases.
22
Figures
23
Figure 1. Beclin 2 interacts with the key molecules involved in neurodegenerative
diseases.
A) The 293T cells were transfected with GFP-Tau along with empty vector or HA-Beclin 2.
After 24 h, cells were harvested. Cell lysates were immunoprecipitated using anti-Flag
followed by immunoblots using the indicated antibodies.
B) The 293T cells were transfected with HA-tagged SOD1, APOE, PARK7, NIPA1, SUPT5H,
TDP43, GAK, TREM2, SNCA, PINK1, PARK7 along with empty vector or HA-Beclin 2.
After 24 h, cells were harvested. Cell lysates were immunoprecipitated using anti-Flag
followed by immunoblots using the indicated antibodies.
24
C) The 293T cells were transfected with switched tagged of Beclin 2 and neurodegenerative
related genes, followed by anti-Flag pull down.
25
Figure 2. Beclin 2 downregulates key proteins involved in neurodegenerative diseases
A) 293T cells were transfected with Flag-tagged Tau (50 ng), TauE14 (50 ng), APP (400 ng),
APOE (800 ng), TDP43 (100 ng), PARK7 (200 ng), GAK (100 ng), HA-tagged TREM 2
(400 ng), and EV or increasing dose of HA-tagged Beclin 2 (0, 200, 500 ng) respectively.
24 h post ‐transfection, cells were harvested and used to perform immunoblot analysis with
the indicated antibodies.
B) 293T cells were transfected with Flag-tagged SUPT5H (100 ng), SOD1 (100 ng), PARK2
(400 ng), HA-Tagged PINK1 (800 ng), and EV or increasing dose of HA-tagged Beclin 2
(0, 200, 500 ng) respectively. 24 h post ‐transfection, cells were harvested and used to
perform immunoblot analysis with the indicated antibodies.
C) 293T cells were transfected with EV or increasing dose of none-tagged Beclin 2. 24 h
post ‐transfection, cells were harvested and used to perform immunoblot analysis with the
indicated antibodies.
26
27
Figure 3. Beclin 2 regulates the pathogenic proteins involved in neurodegenerative
diseases through a lysosomal degradation pathway
A) 293T cells were transfected with Flag-tagged Tau, TauE14, APOE, TDP43, APP, and
PARK7, and HA-tagged TREM2, together with EV or HA- or Flag-tagged Beclin 2 plasmids,
followed by 6h treatment with Bafilomycin A1 (500 nM). 24 h post ‐transfection, cells were
harvested and used to perform immunoblot analysis with the indicated antibodies.
B) 293T cells and ATG9A-KO 293T cell clones were transfected with Flag-tagged Tau,
TauE14, together with EV or increasing dose of HA-tagged Beclin 2 plasmids, 24 h post ‐
transfection, cells were harvested and used to perform immunoblot analysis with the
indicated antibodies.
28
Figure 4. Determination of the key binding domains of Beclin 2 in mediating the
degradation process
The 293T cells were transfected with HA-tagged Beclin 2 FL, ΔN, CC-ECD, ECD, ΔECD along
with empty vector or Flag-tagged STX5, STX6, ATG9A, and Tau. After 24 h, cells were harvested.
Cell lysates were immunoprecipitated using anti-Flag followed by immunoblots using the indicated
antibodies.
29
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Abstract (if available)
Abstract
The unprecedented increase in the number of older adults brings a higher risk of chronic diseases in the United States and the whole world as well, such as dementias, heart disease, type 2 diabetes, arthritis, and cancer. Among all these diseases, neurodegenerative disorders, such as Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS), are classified as one of the most severe types, as they are mostly irreversible and incurable, lack robust early prediction, but affect every aspects of the patient’s life. As the senior adults (65 or older) are expected to make up nearly 25% of the population by 2060, there is an urgent need to develop new and more effective therapeutic strategies to combat these devastating diseases. ❧ The progression of neurodegenerative diseases is usually accompanied with the accumulation of pathogenic proteins, such as the beta-amyloid and tau in AD, Lewy bodies in PD, and TDP-43 in ALS. The prompt clearance of these pathogenic proteins is critical for the prevention and early treatment of the neurodegenerative diseases. ❧ Autophagy is a self-degradative program that removes misfolded or aggregated proteins, damaged organelles, and intracellular pathogens, thus plays important housekeeping role in maintaining the cellular homeostasis. In addition, autophagy protects against genome instability and promotes cellular senescence, thus preventing chronic diseases such as cancer, diabetes, and neurodegeneration. ❧ In this study, we illustrated a previously unrecognized function of Beclin 2 as an important regulator of neurodegenerative diseases for the clearance of pathogenic proteins by direct interaction. We also identified the key domains of Beclin 2 in the degradation process and revealed that the ATG9A-dependent removal of these pathogenic proteins required the existing components in the autophagic pathway. Therefore, our study not only expands the current understanding in this field, but also provides potential therapeutic targets for the prevention and better treatment of the neurodegenerative disease.
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Asset Metadata
Creator
Liu, Siyao
(author)
Core Title
Beclin 2-mediated autophagic degradation of the pathogenic proteins in neurodegenerative diseases
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Molecular Microbiology and Immunology
Degree Conferral Date
2021-08
Publication Date
07/28/2021
Defense Date
06/04/2021
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Ou, Jing-Hsiung (
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), Wang, Rongfu (
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), Yuan, Weiming (
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