Bid signal pathway components are
identified in the temporal cortex with
Hong Jiang, PhD
Ping He, PhD
Charles H. Adler, MD,
Holly Shill, MD
Thomas G. Beach, MD,
Rena Li, MD, PhD
Yong Shen, PhD
Objective: Parkinson disease (PD), a devastating neurodegenerative disorder, affects motor abili-
ties and cognition as well. It is not clear whether the proapoptotic protein, Bid, is involved in tumor
necrosis factor death receptor I (TNFRI)–mediated destructive signal transduction pathways such
as cell dysfunction or neurodegeneration in the temporal cortex of patients with PD.
Methods: Molecular and biochemical approaches were used to dissect mitochondrial related com-
ponents of the destructive signaling pathway in the temporal cortex from rapidly autopsied brains
(postmortem interval mean 2.6 hours). Brains from patients with PD (n ? 15) had an average age
of 81.4 years, compared to the average age of 84.36 years in age-matched control patient brains
(n ? 15).
Results: TNFRI and its adaptor protein, TRADD, were not only present in the cytoplasm of the
temporal cortex, but were significantly elevated (42.3% and 136.1%, respectively) in PD brains
compared to age-matched control brains. Bid in the PD temporal cortex could be further cleaved
into tBid in the cytosol, which is translocated into the mitochondria, where cytochrome c is then
released and caspase-3 is subsequently activated.
Conclusion: Patients with PD have an activated Bid-mediated destructive signal pathway via
TNFRI in the temporal cortex. Such deficits are pervasive, suggesting that they might contribute
to cortex degeneration as PD manifests. Neurology®2012;79:1767–1773
AD ? Alzheimer disease; BBDP ? brain and body donation program; DD ? death domain; FADD ? Fas-associated death
domain; GFAP ? glial fibrillary acid protein; NPC ? nonpathologic control; PD ? Parkinson disease; SDS-PAGE ? sodium
dodecyl sulfate polyacrylamide gel electrophoresis; SN ? substantia nigra; TNF ? tumor necrosis factor; TNFRI ? tumor
necrosis factor death receptor I; TRADD ? tumor necrosis factor death receptor I–associated death domain protein.
Parkinson disease (PD) is a progressive neurodegenerative disorder characterized by the loss of
dopaminergic neurons in the substantia nigra. Apoptosis may be involved in dopaminergic cell
death in sporadic and familial PD.1–7However, whether other brain regions undergo the
destructive signal pathways is still unknown. Cell apoptosis is regulated by a wide number of
factors, such as tumor necrosis factor (TNF) and Fas ligand. Evidence implicates neuroinflam-
mation from TNF as the fundamental piece in the pathogenesis of PD.8–12Elevated levels of
TNF in the CSF and postmortem brains of patients with PD suggest that this proinflammatory
cytokine plays an important role in the pathophysiology of the disease. Elucidation of the
mechanisms underlying neuroinflammation in PD may contribute to new and effective thera-
pies, especially for cognitive decline of patients with PD. TNF receptor subtype I (TNFRI)
contains a “death domain” (DD) that is associated with DD-containing adaptor proteins,
including TNFRI-associated DD protein (TRADD), Fas-associated DD (FADD), or receptor-
interacting protein. The detailed apoptotic pathway that may be mediated by TNFRI is still
unknown in PD, although studies have shown elevated TNFRI levels in the substantia nigra of
patients with PD.13
From the Sun Health Research Institute (H.J., P.H., H.S., T.G.B., Y.S.), Sun City, AZ; Mayo Clinic Scottsdale (C.H.A.), Scottsdale, AZ; and
Roskamp Institute (P.H., R.L., Y.S.), Sarasota, FL.
Study funding: Supported by National Institutes of Health (RO1AG025888, RO1AG032441), Alzheimer’s Association IIRG-07-59510, and
American Health Assistance Foundation (AHAF) grant G2006-118 and A2008-642.
Go to Neurology.org for full disclosures. Disclosures deemed relevant by the authors, if any, are provided at the end of this article.
Correspondence & reprint
requests to Dr. Shen:
Copyright © 2012 by AAN Enterprises, Inc.
Bid is a proapoptotic protein of the Bcl-2
family that is crucial for death receptor–
mediated apoptosis and is activated post-
translationally via caspase-8-mediated cleavage
into a truncated form, tBid.14–16In the pres-
ent study, we have identified an activated
Bid-mediated destructive pathway in the
temporal cortex of patients with PD, sug-
gesting that they might contribute to cogni-
tive decline manifestation.
METHODS Standard protocol approvals, registra-
tions, and patient consents. All subjects or their legally au-
thorized representatives sign an informed consent to research
and autopsy; the research has been approved by the institutional
review board. Brain tissue was obtained from the Banner Sun
Health Research Institute brain and body donation program
(BBDP), Sun City, AZ.17
Acquisition of samples. The research subjects were recruited
primarily from the Sun City retirement communities located in
the northwest metropolitan region of Phoenix, AZ. These com-
munities have a population of approximately 900,000, an aver-
age age of 72 years, and a minimum age of 55 years. The BBDP
is unique for its rapid autopsy program (median postmortem
interval is 2.8 hours), allowing the provision of unusually high-
quality brain tissue. The study subjects consisted of 15 non-
pathologic control (NPC) subjects (at age of 78 to 90 years) and
15 patients with PD (at age of 72 to 90 years, disease duration of
14.4 ? 8.4 years). The subject profiles are presented in table 1.
Clinical diagnosis and pathologic confirmation. Hu-
man subjects were specifically chosen with no dementia and PD
in the temporal cortex who might be showing early changes but
not be end-stage as might be seen in individuals with dementia.
The criteria for Alzheimer disease (AD) are defined by NIA–
Reagan “intermediate or high”18and pathologic Consortium to
Establish a Registry for Alzheimer’s Disease neuritic plaque den-
sity19as well as Braak staging.20The detail information is shown
in table e-1 on the Neurology®Web site at www.neurology.org.
PD diagnosis was determined when there were at least 2 of the
cardinal clinical signs of PD as well as pathologic evidence of
Lewy bodies and pigmented neuron loss in the substantia nigra
(SN). Scores for brain regions are based on Dementia with Lewy
Body Consortium density estimates21and Unified Staging Sys-
tem for Lewy body disorders.22The detail information is shown
in table e-2. Patients who met pathologic criteria for PD are
levodopa responsive. NPCs were selected based on the absence of
a clinical history of parkinsonism or dementia and the absence of
Lewy bodies on neuropathologic examination.
Brain tissue preparation. Protein was extracted from ho-
mogenates of frozen tissue from the gray matter of the temporal
cortex in 15 PD cases and 15 controls. Approximately 500 mg of
tissue was homogenized in phosphate-buffered saline containing
1% Triton X-100 and 0.1% sodium dodecyl sulfate (SDS) (pH
7.0) plus proteinase inhibitors, and it was centrifuged at 14,000
rpm for 30 minutes. The protein concentration in the superna-
tant was measured with a Bio-Rad protein assay kit. A total of 50
?g of protein was mixed with 1:1 of sample buffer (25 mM Tris,
250 mM Glycine, pH 7.0) and loaded on a 12% SDS polyacryl-
amide gel electrophoresis (SDS-PAGE) gel. The protein was
then transferred to a PDVF membrane.
ELISA. The content of TNFRI in the lysed extracts was deter-
mined using a commercially available ELISA kit for human
TNFRI (R&D System). Dilution curve homogenate samples of
the brain were parallel to the standard dilution curve. The assay
followed the manufacturer’s recommended procedures. The ab-
sorbance was measured at 450 nm with an automatic wavelength
correction of 540 nm. The experiments were repeated 3 times.
Immunoprecipitation. For the immunoprecipitation of
TRADD, 500 ?g of brain extracts in a 300 ?L volume contain-
ing 20 ?g anti-TRADD polyclonal antibody (catalog: ab18914,
Abcam) was incubated at 4°C overnight. A total of 20 ?L of
Protein-G agarose (Pierce) was then mixed with the samples and
incubated with orbitary rotation at 4°C for 4 hours. The beads
were collected and resuspended in 50 ?L of SDS-PAGE loading
dye and heated at 95°C for 5 minutes. A total of 10 ?L of each
sample was separated by 12% SDS-PAGE gel and transferred to
Immunoblotting. Western blots were probed with the follow-
ing antibodies: anti-TRADD monoclonal antibody (1:500, cata-
log: sc-46653, Santa Cruz); anti-Bid polyclonal antibody (1:
5,000, catalog: AF846, R&D Systems); anti-cytochrome c
monoclonal antibody (1:5,000, clone: 7H8.2C12, catalog:
ab13575, Abcam); and anti-caspase-3 polyclonal antibody (1:
5,000, catalog: sc-7148, Santa Cruz). Blots were also probed
with anti-?-actin monoclonal antibody (1:5,000, catalog:
#1978, Sigma) as a loading control. For mitochondria samples,
the blots were performed with anti-Cox IV monoclonal antibody
(1:5,000, clone: 20E8C12, catalog: ab14744, Abcam) as a mito-
chondrial loading control. The protein levels were normalized to
?-actin or Cox IV (for mitochondria samples) and indicated in
Immunohistochemistry. The immunohistostaining was per-
formed as described previously.23,24Fresh tissues of the temporal
cortex were postfixed with 4% paraformaldehyde and sectioned
to 30 ?m. Pretreatment of sections was performed with 0.3%
triton X-100 and blocked with 10% goat serum for 30 minutes.
Primary antibodies were incubated with polyclonal rabbit anti-
Bid (1:400, clone: Y8, catalog: ab32060, Abcam), mouse anti-
NeuN (MAB377, Millipore, Billerica, MA), monoclonal mouse
anti–glial fibrillary acid protein (GFAP, 1:5,000, clone: SMI22,
catalog: SMI-22R, Covance), mouse anti-human CD45 (1:400,
clone: F10-89-4, catalog: MCA87, Serotec) overnight at 4°C.
Secondary antibodies of goat against mouse or rabbit immuno-
globulin G with fluorescence 488 or 568 (1:1,000; Invitrogen,
Carlsbad, CA) were incubated for 30 minutes. The images were
taken with BX63 microscope (Olympus, Tokyo, Japan).
Mitochondria isolation. Mitochondria were isolated from
approximately 100 mg of gray matter tissue from the temporal
cortex using a mitochondria isolation kit (BioChain Institute
Table 1Human subject profiles from the patients with PD and
size, n Age, y
onset age, y
1584.26 ? 1.26 7/8NA2.85 ? 0.48
1581.40 ? 1.30 5/1067 ? 2.942.47 ? 0.72
Abbreviations: NA ? not applicable; NPC ? nonpathologic control; PD ? Parkinson disease.
Neurology 79October 23, 2012
Inc, Hayward, CA). The isolation procedure followed the man-
ufacturer’s recommended procedures. Protein concentration was
measured with Bio-Rad protein assay kit. Samples for separation
on a 12% SDS-PAGE gel were prepared as described in the
Caspase-3 activity assay. Caspase-3 activity was determined
using a caspase-3 activity kit according to the manufacturer’s
protocol (Calbiochem). Briefly, the colorimetric assay was con-
ducted with an equal amount of brain lysate combined with the
fluorescent labeled DEVD substrate in a 96-well plate. The plate
was incubated at 37°C for 2 hours and the fluorescence was
measured at 400 nm and 505 nm. The amount of luminescence
expressed as relative light units reflects caspase activity. These
assays were performed in triplicate.
Statistical analysis. Data were presented as mean ? SEM.
Differences in means were determined by an unpaired t test.
A probability value of p ? 0.05 was considered statistically
RESULTS TNFRI signaling is elevated in PD brains.
Quantification of TNFRI protein levels in the tem-
poral cortex of PD and age-matched NPC brains
were determined by ELISA. As shown in figure 1A,
the levels of TNFRI in PD brains (2.22 ? 0.24 ng/
mg) was higher (p ? 0.05) than that in NPC brains
(1.56 ? 0.12 ng/mg). The activation of TNFRI re-
cruited several adaptor proteins containing DDs. We
examined the expression of TRADD, a molecule in-
volved in a TNFRI-mediated destructive signal
transduction pathway, in the temporal cortex using
immunoprecipitation. We observed a band of
TRADD around 34 kD (figure 1B). Density assay
showed increased TRADD levels in PD brains (fig-
ure 1C, p ? 0.01).
Bid expression in neurons and activated astrocytes as
well as cleaved Bid translocation to the mitochondria
in PD brains. To verify whether Bid expression is
activated and in which cell type in brains, immuno-
staining was performed with the brain sections from
the temporal cortex. We found Bid expression in
most of the neurons (NeuN, a neuron marker, figure
2A) and some activated astrocytes (GFAP, an astro-
cyte marker, figure 2B) but not in activated microglia
(CD45, an activated microglial marker, figure 2C).
Both forms of Bid, full length Bid (?22 kD) and
tBid (?14 kD), were detected in temporal cortex
brain lysate and mitochondria extractions. The levels
of full-length Bid in the cytosol were increased in PD
brains compared to NPC brains. A dramatically in-
creased level of tBid was detected in all PD brains,
but only existed in a few NPC brains (figure 2, D and
E). In the mitochondria, there was an increase in
full-length Bid expression exclusively in PD group
compared to NPC brains. Similar results were ob-
served in tBid levels (figure 2, F and G); tBid was not
found in mitochondria extractions from NPC brain
Cytochrome c is released from the mitochondria in PD
brains. The mitochondria from the temporal cortex
were isolated. Western blot was performed and re-
sults showed that levels of cytoplasmic cytochrome c
were increased in PD brain samples after normaliza-
tion to ?-actin level (figure 3, A and B, p ? 0.01).
However, the levels of cytochrome c in mitochondria
were decreased after normalization to Cox IV level
(figure 3, C and D, p ? 0.01), suggesting that the
translocation of tBid to mitochondria causes a signif-
icant cytochrome c release from the mitochondria.
Figure 1 Tumor necrosis factor death receptor I (TNFRI) signaling is
increased in Parkinson disease (PD) brains
(A) ELISA showed that TNFRI levels are elevated in the temporal cortex of PD brains com-
pared to the control brains. (B) Representative immunoprecipitation of tumor necrosis fac-
tor death receptor I–associated death domain protein (TRADD) was presented in the
temporal cortex from parkinsonian brains and controls. (C) Spot density analysis of TRADD
was normalized to corresponding ?-actin level (ap ? 0.05;bp ? 0.01) compared to non-
pathologic control (NPC).
Neurology 79October 23, 2012
Figure 2 Bid is expressed in neurons and astrocytes, and cleaved Bid is located to the mitochondria in
Parkinson disease (PD) brains
body CD45. Bars: 20 ?m. (D) A full-length Bid around 22 kD and the cleaved Bid (tBid) around 14 kD were detected in cytoplasmic
Neurology 79 October 23, 2012
Activity of caspase-3 is increased in PD brains. To
address the particular role of caspases in TNFRI-
mediated destructive signal transduction pathway,
we used both Western blot and procaspase-3 activity
assay. Three bands were detected using Western blot,
1 fragment of procaspase-3 (32 kD), and 2 cleaved
forms of proteins (21 kD and 12 kD). There was an
increase of procaspase-3 and its cleaved fragments in
the temporal cortex of PD brains compared to NPC
brains (figure 4A). Procaspase-3 activity was detected
and we found an increased caspase activity in PD
brains compared to NPC brains, which is in agree-
ment with the Western blot results (figure 4B).
No changes are found in Bid signal components be-
tween controls without dementia and preclinical AD
among the patients with PD. According to the current
criteria for a pathologic diagnosis of preclinical AD,
in the 15 cases of patients with PD listed in table e-1,
6 cases of patients with PD accompany the cortical
changes of preclinical AD (moderate/frequent neu-
ritic plaques and Braak III/IV of neurofibrillary tan-
gles). To clarify the possible influences of preclinical
AD-like changes in patients with PD, the values of
each protein band density of Western blotting were
measured. Results showed no significant differences
in the expression levels of TNFRI, TRADD, Bid,
tBid, cytochrome c, and procaspase 3 and cleaved
caspase 3 between no dementia and brain preclinical
AD changes among all patients with PD.
DISCUSSION Available evidence suggests that pa-
tients with PD exhibit neurodegeneration not only in
the dopaminergic neurons in SN, but also other
brain regions including the cortex. The mechanisms
underlying the neurodegenerative process remain
elusive. In the present study, we found that the cleav-
age of Bid and its truncated tBid translocation to
the mitochondria are implicated in the TNFRI-
mediated signaling pathway in the temporal cortex of
PD brains. These findings might provide a mecha-
nistic link to how the death receptor leads to the
activation of Bid, the dysfunction of mitochondria,
and the consequent activation of caspases in the cor-
tex regions of PD brains (figure e-1).
We previously reported that TNFRI levels are el-
evated in the cortex from AD brains.25Here, we
found an elevated expression of TNFRI and its adap-
tor protein, TRADD, in the temporal cortex of par-
kinsonian brains. All of these suggest a possible
association of death receptors with cell death in neu-
rodegenerative disorders. A likely interaction partner
for TRADD may be FADD, which can then interact
with procaspase-8. Activation of procaspase-8 through
self-cleavage leads to a series of downstream events, in-
Bid is a unique BH3-only molecule of Bcl-2 fam-
ily,15which is largely present in cytosol and is in-
volved in the cellular destructive process mediated by
TNFRI death receptor. A truncated fragment tBid is
generated from a proteolytic digestion at Asp-59, a
site with a conserved caspase-8-cleavage sequence.28
Upon the stimulation of a particular death signal,
tBid translocates to the mitochondria and induces
cytochrome c release.14,29–31This is similar to a previ-
Figure 3 Cytochrome c is released from mitochondria in Parkinson disease
(A) Western blot showed cytochrome c release in cytoplasmic fractions of samples from the
temporal cortex. (B) Spot density analysis showed increased levels of cytoplasmic cyto-
chrome c release in PD brain samples (ap ? 0.01). (C) Cytochrome c was detected in the
mitochondrial fraction from temporal cortex using Cox IV as the mitochondrial loading con-
trol. (D) Spot density analysis showed decreased levels of cytochrome c in the mitochondria
from PD brain samples (bp ? 0.01). NPC ? nonpathologic control.
Neurology 79October 23, 2012
ous report showing that cells depleted of Bid by im-
munoprecipitation are no longer able to induce
cytochrome c release.32Bid-induced cytochrome c re-
lease is mediated by 2 different mechanisms. One is
responsible for the initial release of free cytochrome
c in the intermembrane space through a Bak-
dependent mechanism. The other causes a dramatic
remodeling of mitochondrial cristae, which mobilize
cytochrome c storage. Effects of tBid on the mito-
chondria may not be limited to the induction of cy-
tochrome c release, but may include other changes in
mitochondrial physiology such as mitochondrial re-
distribution and loss of mitochondrial ??m in the
This portion of cytochrome c constitutes the ma-
jority (85%) of total release.34The released cyto-
chrome c then binds with Apaf-1 to form the
apoptosome, a multimeric Apaf-1 and cytochrome c
complex. Only the caspase-9 binding to the apopto-
some is able to efficiently cleave and activate down-
stream executioner caspases such as caspase-3.16,35
These executioner caspases will then cleave intracel-
lular substrates, leading to chromatin condensation
and DNA fragmentation. As shown in figure e-1,
TNFRI can directly activate caspase-3 through
caspase-8. This might be due to the amplification of
caspase activation and the involvement of additional
Our finding demonstrated that Bid is largely acti-
vated in the neurons of the temporal cortex from
patients with PD as well as the activation of the en-
tire TNFRI-Bid death signaling pathway in the cor-
tex relevant to nonmotor PD. The results are similar
to the observation of TNFRI-engaged hepatocyte apo-
ptosis in which Bid is required for TNF?- and Fas-
induced apoptosis through a mitochondrial pathway.34
Our data might in part explain the possibly emerging
cognitive decline associated with cortical pathogenesis
Dr. Jiang and Dr. He performed biochemical and immunochemical ex-
periments, participated in data analyses, and wrote the initial manuscript
draft. Dr. Adler and Dr. Shill made clinical diagnosis. Dr. Beach made
pathologic evaluation. Dr. Shen and Dr. Li initiated the project and de-
signed the experiments, and participated in data analyses and overviewed
The samples are from the Brain and Tissue Bank, Sun Health Research
Institute. The BBDP is supported by the National Institute of Neurolog-
ical Disorders and Stroke (U24NS072026 National Brain and Tissue Re-
source for PD and Related Disorders), the National Institute on Aging
(P30AG19610 Arizona AD Core Center), the Arizona Department of
Health Services (contract 211002, Arizona Alzheimer’s Research Cen-
ter), the Arizona Biomedical Research Commission (contracts 4001,
0011, 05-901, and 1001 to the Arizona PD Consortium), and the
Michael J. Fox Foundation for Parkinson’s Research.
The authors report no disclosures relevant to the manuscript. Go to
Neurology.org for full disclosures.
Received October 20, 2011. Accepted in final form May 8, 2012.
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