doi:10.

1093/jmcb/mjaa027 Journal of Molecular Cell Biology (2020), 12(10), 807–816 | 807

Published online June 3, 2020

Article
Probing the therapeutic potential of TRPC6 for
Alzheimer’s disease in live neurons from
patient-specific iPSCs
Ran Tao1,†, Rui Lu2,†, Junfeng Wang2, Shujun Zeng3,4, Ting Zhang1, Wenke Guo1,5,

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Xiaobing Zhang6, Qi Cheng3,4, Chunmei Yue1,*, Yizheng Wang7,*, and Naihe Jing1,5,*

1
State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese
Academy of Sciences, Shanghai 200031, China
2
Laboratory of Neural Signal Transduction, Institute of Neuroscience, Chinese Academy of Sciences, Shanghai 200031, China
3
Department of Neurology, Ruijin Hospital affiliated with the School of Medicine, Shanghai Jiao Tong University, Shanghai 200025, China
4
School of Public Health, Shanghai Jiao Tong University, Shanghai 200025, China
5
School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China
6
Division of Regenerative Medicine, Department of Medicine, Loma Linda University, CA 92350, USA
7
National Clinical Research Center for Aging and Medicine, Huashan Hospital, Fudan University, Shanghai 200040, China
†
These authors contributed equally to the work.
* Correspondence to: Chunmei Yue, E-mail: [email protected]; Yizheng Wang, E-mail: [email protected]; Naihe Jing, E-mail: [email protected]

Edited by Zhen-Ge Luo

The induced pluripotent stem cells (iPSCs) offer an unprecedented opportunity to model and study Alzheimer’s disease (AD) under
patient-specific genetic background. The lower expression of transient receptor potential canonical 6 (TRPC6) was associated
with AD patients, which might be involved in AD pathogenesis. However, the role of TRPC6 that played in AD process still needs
more investigation in patient-relevant neurons. In this study, the iPSCs were generated from peripheral blood cells of sporadic
AD patients and efficiently differentiated into mature cortical neurons. These sporadic AD-bearing neurons displayed higher lev-
els of AD pathological markers A and phospho-tau, but lower levels of TRPC6, than those of control neurons. Treatment of AD neu-
rons with TRPC6 protein fragment or agonist inhibited the elevation of A and phospho-tau. Our results in live AD neurons mani-
fest that the compromised expression of TRPC6 substantially contributed to A pathology of sporadic AD, suggesting that
targeting TRPC6 could help to develop novel therapeutic strategies for the treatments of AD.

Keywords: transient receptor potential canonical 6 (TRPC6), Alzheimer’s disease, patient-specific induced pluripotent stem
cells (iPSCs), cellular models, amyloid-beta (Ab)

Introduction
containing mutations associated with familial AD have been
Alzheimer’s disease (AD) is a progressive debilitating
widely used in elucidating the etiology of AD. The majority of
neurodegenerative disorder pathologically characterized by
AD cases are sporadic and lack of an appropriate model sys-
accumulation of amyloid-b peptide (Ab), aggregation of hyper-
tem. Recently, the derivation of induced pluripotent stem cells
phosphorylated tau, and neuronal loss in the brain. The mo-
(iPSCs) from somatic cells of AD patients and neural differenti-
lecular mechanisms underlying pathogenesis of AD are
ation of iPSCs offer a new strategy to model AD-relevant phe-
complicated and needed more comprehensive investigation.
notypes in live neurons with genetically identical background
In the past, animal models expressing human genes
of individual patient (Marchetto et al., 2011). The patient
Received November 25, 2019. Revised April 14, 2020. Accepted April 27, 2020. iPSC-derived neurons were demonstrated to display canonical
C The Author(s) (2020). Published by Oxford University Press on behalf of Journal
V
neuropathological features of AD (Israel et al., 2012; Kondo
of Molecular Cell Biology, IBCB, SIBS, CAS.
This is an Open Access article distributed under the terms of the Creative et al., 2013; Young et al., 2015). Then, the key challenges in
Commons Attribution Non-Commercial License (http://creativecommons.org/ the field are to explore the possible application of the result-
licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and re-
production in any medium, provided the original work is properly cited. For com-
ing iPSC-based cellular models in uncovering the molecular
mercial re-use, please contact [email protected] mechanisms of AD pathogenesis and in subsequently
808 | Tao et al.

developing new treatment strategies for AD, such as identify- generated from the peripheral blood mononuclear cells of these
ing or validating novel therapeutic target genes or drugs that 29 participants using an episomal vector-mediated integration-
are safe and effective for AD patients. free approach as previously reported (Chou et al., 2011; Dowey
Transient receptor potential canonical 6 (TRPC6) is an impor- et al., 2012; Su et al., 2013). Immunostaining analysis showed
tant Ca2þ-permeable non-selective cation channel protein that the iPSC colonies homogeneously expressed pluripotency
(Montell et al., 2002) and plays critical roles in brain develop- markers OCT4, TRA-1-60, TRA-1-81 as well as SSEA-4 and acquired
ment (Tai et al., 2009), neuronal survival (Jia et al., 2007), and pluripotency (Supplementary Figure S1A). PCR analysis on DNAs
synaptic formation (Zhou et al., 2008). Disruption of TRPC6 ex- from multiple iPSCs at early passages did not detect exogenous
pression was reported to affect the development and function of pluripotent genes, confirming that the iPSCs were integration-free
neurons and may be involved in the etiology of autism spectrum (Supplementary Figure S1B). In addition, we observed that the
disorders (Griesi-Oliveira et al., 2015). Previously, we found that patient-specific iPSCs displayed normal karyotypes as control

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TRPC6 specifically interacted with amyloid precursor protein iPSCs (Supplementary Figure S1C) and possessed pluripotent ca-
(APP) via the second transmembrane domain (TM2), modulated pacities to form three germ layers in teratoma assay
the c-secretase cleavage of APP, and reduced Ab production (Supplementary Figure S1D).
(Wang et al., 2015). Moreover, the overexpression of TRPC6 led Then, we analyzed the neural differentiation capacity of the
to the reduction of Ab burden in the AD mice brain and improved generated iPSC lines. We found that differentiation from human
the learning abilities of AD mice (Wang et al., 2015). The follow- iPSCs to neurons was hardly initiated relying on the embryoid
ing study revealed the reduction of TRPC6 expression in periph- body (EB) formation in serum-free culture conditions, which is
eral blood of sporadic AD patients at different clinical stages (Lu suitable for human embryonic stem cell (hESC) neural differentia-
tion (Yue et al., 2015; Li et al., 2017). To promote the neural dif-
et al., 2018). Based on these observations from AD model mice
ferentiation of human iPSCs, an approach combining the EB-
and cohort studies among AD patients, we have hypothesized
based method with dual-SMAD inhibitors (TGF-b inhibitor
that TRPC6 might be involved in the progress of AD by interfering
SB431542 and BMP inhibitor dorsomorphin) (Chambers et al.,
with Ab generation. Due to the fundamental species-specific dif-
2009) was established (Figure 1A). With the treatment of two
ferences between rodents and human, the mouse models are
inhibitors at the first 6 days, the expression of OCT4 quickly de-
usually not faithfully reflective of the clinic conditions of AD
creased, while the neuroepithelia markers OCT6 and ZIC2 in-
patients, thus compromising the fidelity of assessing the thera-
creased and the neural differentiation of iPSCs initiated
peutic potential of TRPC6 for AD diagnosis and treatment. Thus,
(Figure 1B). Then the differentiating cells acquired the forebrain
it is required to investigate the role of TRPC6 in AD pathogenesis
identity by expressing anterior forebrain neuroectoderm marker
under the genetic background of patients. However, the expres-
OTX2 and anterior forebrain progenitor marker FOXG1 (Figure 1B).
sion of TRPC6 in live neurons of AD patients and its correlativity
Finally, the expression of neural progenitor markers PAX6 and
with Ab production remain largely unexplored. Hyperforin is a SOX1 as well as subsequent neuronal markers TUJ1 and MAP2
drug derived from plant (Singer et al., 1999), which can induce was indicative of the progressive fate commitment from human
the expression of TRPC6 in short term cultured human skin iPSCs to neuronal progenitors, then to neurons (Figure 1B). At
explants (Muller et al., 2008) and mouse cortex (Gibon et al., Day 50, 90% of cells in iPSC-neural differentiated cultures were
2013). However, it is still unknown whether hyperforin treatment TUJ1- or MAP2-positive neurons, showing the high efficiency of
can increase the expression of TRPC6 and then interfere with Ab our approach (Figure 1C and D). Majority of iPSC-derived TUJ1þ
generation under genetic background of AD patients. neurons were mature cortical neurons and expressed mature neu-
In this study, we generated multiple lines of sporadic AD ronal marker NEUN (75.5%) and cortical marker TBR1 (83.6%)
patient-specific iPSCs that efficiently differentiated into mature (Figure 1E and F). In addition, VGLUTþ glutamatergic neurons
AD neurons. Relative to control neurons, sporadic AD neurons (89.3%) among iPSC-derived cortical neurons were dominant
exhibited higher levels of Ab and phospho-tau but lower compared with other subtypes, such as GAD67þ GABAergic neu-
TRPC6, while increasing TRPC6 level by TRPC6 peptides or ago- rons (6.9%), THþ dopaminergic neurons (5.1%) and ChATþ cho-
nist hyperforin caused marked reductions of Ab and tau in spo- linergic neuron (not detected) (Figure 1E and F). Whole-cell patch-
radic AD neurons, providing evidences that TRPC6 inhibits Ab clamp recording revealed that most of iPSC-derived neurons fired
elevation in human AD neurons. action potentials (APs) (15/17) at Days 55–65, either repetitive
(9/17) or one APs (Figure 1G and H) and some neurons generated
spontaneous APs (Figure 1I) or expressed post-synaptic currents
Results (Figure 1J), indicating that iPSC-derived cortical neurons pos-
The patient iPSC-derived neurons exhibit AD-related sessed functional membrane properties.
phenotypes Next, the reprogramming efficiency and neural differentiation
Twelve diagnosed sporadic AD patients and 17 age-matched capacity between control and AD group were evaluated. First,
healthy controls who participated in our previous study (Lu et al., the efficiency to generate iPSCs from peripheral blood mononu-
2018) were recruited and the detailed information was summa- clear cells (PB MNCs) of AD patients vs. controls was compara-
rized in Supplementary Table S1. Altogether, 145 iPSC lines were ble (Supplementary Figure S2A). The similar expression pattern
 Probing TRPC6 in live neurons from sporadic AD iPSCs | 809

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Figure 1 The efficient differentiation from patient-specific iPSCs into neurons displaying pathological features associated with AD.
(A) Schematic representation of the approach used to direct the differentiation from iPSCs into neurons. (B) Gene expression heatmap for
marker genes during the neural differentiation of human iPSCs. The value indicates the fold change that is relative to GAPDH and normal-
ized to the highest value. Red and white colors represent higher and lower gene expression levels, respectively. (C) Immunocytochemistry
analysis of the expression levels of neuronal markers TUJ1 (a) and MAP2 (b) in iPSC-derived neurons at Day 50. (D) Quantification of the
results shown in C. n ¼ 3. (E) Immunocytochemistry analysis of the expression levels of mature neuronal marker NEUN (a), cortical neuronal
marker TBR1 (b), glutamatergic neuronal marker VGLUT (c), GABAergic neuronal marker GAD67 (d), dopaminergic neuronal marker TH (e), and
810 | Tao et al.

of representative marker genes during neural differentiation quantitative real-time polymerase chain reaction (qRT-PCR)
measured showed the similar neural differentiation process be- analysis was performed in the control and AD iPSC lines same as
tween AD and control iPSCs (Figure 1B; Supplementary Figure those used for the measurements of Ab and tau. The mRNA level
S2B). Consequently, both control and AD iPSC lines showed of TRPC6 in sporadic AD iPSCs was much lower than that in con-
preference to give rise to cortical neurons with similar effi- trols (Figure 2A). During the neural differentiation, the mRNA lev-
ciency (Figure 1C–F; Supplementary Figure S2C and D). Total els of TRPC6 kept elevating from Day 34 to Day 50 in a linear
protein levels measured at Day 50 were slightly but not signifi- manner with the maturation of neurons in both control and spo-
cantly lower in AD neurons than those in controls radic AD neurons. However, the expression of TRPC6 in sporadic
(Supplementary Figure S2E). The levels of apoptosis-associated AD neurons was markedly lower from Day 34 and kept lower at
markers, PARP (a substrate of activated Caspase-3), cleaved all the following time points measured relative to controls, indic-
PARP, and Caspase-3, were similar between AD and control ative of the compromised expression of TRPC6 in sporadic AD

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neurons, indicating that the genetic differences between AD neurons (Figure 2B). In addition, western blotting analysis was
and controls did not entail evident apoptosis in AD-bearing performed to measure the protein levels of TRPC6, TRPC5 (an-
neurons up to Day 50 (Supplementary Figure S2F and G). Thus, other TRPC family member), APP, and PS1 in sporadic AD and
from the PB MNCs of AD patients and controls, we generated control neurons at Days 34, 42, and 50, respectively (Figure 2C).
faithfully reprogrammed iPSC lines with similar capacity and ef- The quantitative analysis revealed that the levels of these pro-
ficiency of neural differentiation, which allowed us to compare teins in both sporadic AD and control neurons increased in a
the Ab secretion and tau level between control and AD iPSC- time-dependent manner (Figure 2D). However, TRPC6, but not
derived neurons in a reliable and consistent way. other proteins, in sporadic AD neurons was markedly lower than
To detect the AD-relevant phenotypes in sporadic AD neu- in controls, displaying similar expression pattern as TRPC6
rons, iPSC lines of three AD patients and three controls were mRNAs (Figure 2D). Furthermore, the mRNA levels of key factors
simultaneously subjected to neural differentiation. Enzyme- involved in APP processing, including a-secretase ADAM10 and
linked immunosorbent assay (ELISA) was performed to mea- TACE, b-secretase BACE1, as well as c-secretase subunits PS1,
sure the levels of Ab in medium and tau in cell lysates of PEN2, nicastrin, and APH-1 in AD neurons, were comparable with
neurons derived from each iPSC line at Days 34, 42, and 50, those in control neurons (Supplementary Figure S3), indicating
respectively. Both the secreted Ab (Ab42 and Ab40) and intra- that sporadic AD neurons with lower TRPC6 displayed relatively
cellular tau (total tau and phospho-tau) increased at a time- normal transcriptional levels of key factors involved in APP proc-
dependent manner in AD and control neurons (Figure 1K and essing. Thus, we confirmed that the expression of TRPC6 at both
L). The levels of Ab42, Ab40, total tau, and phospho-tau were mRNA and protein levels in sporadic AD neurons was compro-
similar to controls at Day 34, but became markedly higher than mised. In addition, we observed that during the neural differenti-
controls from Day 42 in AD neurons and maintained higher ation, the sporadic AD neurons exhibiting compromised
thereafter (Figure 1K and L). expression of TRPC6 displayed evidently higher Ab and tau
Collectively, the sporadic AD patient-specific iPSCs efficiently (Figure 1K and L). It is interesting to note that the detectable re-
differentiated into mature cortical glutamatergic neurons that duced expression of TRPC6 in AD neurons occurred at Day 34,
display pathological features associated with human AD at which is earlier than the elevation of Ab and tau in AD neurons,
later differentiation days, suggesting that the iPSC-derived spo- which begins from Day 42. Thus, the consistent compromised
radic AD neurons could serve as a cellular platform to study the expression of TRPC6 might be an early molecular event during
pathogenesis of AD. AD progression, which was even prior to abnormal Ab accumula-
tion or tauopathy, suggesting that TRPC6 might be a potentially
therapeutic target gene of AD.
Compromised expression of TRPC6 in patient iPSC-derived
sporadic AD neurons
Since we have observed that TRPC6 is protective against pa- The expression of TRPC6 inhibits the elevation of Ab and tau in
thology of AD in mouse model, we next investigate this gene by live AD neurons
assessing the expression of TRPC6 and the correlativity between To assess the therapeutic potential of TRPC6, we next ex-
TRPC6 and Ab in the patient iPSC-based cellular platform. The plored whether the rescue of TRPC6 expression could alleviate

cholinergic neuronal marker ChAT (f) in human iPSC-derived neurons at Day 50. (F) Quantification of the results shown in E. n ¼ 3.
(G) Representative traces of no AP (upper), one AP (middle), and repetitive AP (lower) from human iPSC-derived neurons at Days 55–65.
(H) Percentages of human iPSC-derived neurons firing different types of AP presented in G. (I) Representative traces of spontaneous AP
recorded in human iPSC-derived neurons at Days 55–65. (J) Representative traces showing spontaneous PSCs received by human iPSC-
derived neurons at Days 55–65. (K and L) The levels of Ab42 and Ab40 (K) as well as total tau and phosphorylated tau (L) in control or AD
iPSC-derived neurons at Days 34, 42, and 50. Data are normalized to total protein of whole-cell lysates. n ¼ 4. Scale bar, 50 lm. Data are
represented as mean 6 SD. *P < 0.05, **P < 0.01, and ***P < 0.001.
 Probing TRPC6 in live neurons from sporadic AD iPSCs | 811

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Figure 2 The TRPC6 expression in sporadic AD neurons derived from patient-specific iPSCs. (A) The mRNA levels of TRPC6 in AD and control
iPSCs derived from PB MNCs. n ¼ 3. (B) The mRNA levels of TRPC6 in iPSC-derived AD or control neurons at differentiation Days 34, 42,
and 50. n ¼ 8. (C) The protein levels of TRPC6, TRPC5, APP, and PS1 in iPSC-derived AD or control neurons at Days 34, 42, and 50.
(D) Quantification of the results shown in C. n ¼ 6. Data are represented as mean 6 SD. *P < 0.05 and **P < 0.01.

the AD-relevant phenotypes in sporadic AD neurons. We previ- hyperforin resulted in an increase of TRPC6 protein in a dose-
ously found that the TRPC6 inhibits APP processing via its TM2 dependent manner and 1 lM or higher hyperforin markedly in-
domain and mutations in TM2 (TM2-mut) abolished the inhibi- duced the protein expression level of TRPC6 (Figure 4A). More im-
tory effect of TRPC6 in HEK293 cells (Wang et al., 2015). Then, portantly, co-immunoprecipitation assay showed that more
the TM2 fused with transmembrane peptide TAT was added to TRPC6 proteins bound to APP due to hyperforin treatment, which
neurons from sporadic AD and control iPSCs at Day 48. Two might further modulate the APP processing (Figure 4B). We also
days later, the secretion of both Ab42 and Ab40 decreased in a consistently observed that hyperforin treatment resulted in a re-
dose-dependent manner in sporadic AD or control neurons duction of secreted Ab42 or Ab40 in a dose-dependent manner
(Figure 3A). In addition, TM2 induced marked decrease of total and 1 lM or higher hyperforin caused evident decrease of Ab in
tau and phospho-tau (Figure 3B). The treatment with TAT only both AD and control neurons, and the sporadic AD neurons were
or TM2-mut had no effect on Ab secretion or tau expression in more sensitive to hyperforin than controls (Figure 4C;
either AD or control neurons (Figure 3C and D). These results in- Supplementary Table S2). Similar inhibitory effects on phospho-
dicate that TRPC6 can inhibit the elevation of Ab and phospho- tau and total tau by hyperforin were observed in both AD and con-
tau via TM2 in sporadic AD neurons. trol neurons (Figure 4D; Supplementary Table S2).
Hyperforin is a drug, which was reported to increase TRPC6 ex- Together, these results reveal that increasing TRPC6 by TM2
pression (Muller et al., 2008; Gibon et al., 2013). We confirmed peptide and hyperforin inhibits the elevation of Ab and tau in
that treatment with hyperforin increased the expression of TRPC6 sporadic AD neurons, suggesting that targeting TRPC6 might
at both mRNA and protein levels in HEK293 cells (Supplementary help to develop novel therapeutic strategies for the treatment
Figure S4A and B). Exposure of sporadic AD or control neurons to of AD.
812 | Tao et al.

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Figure 3 The decrease of Ab and tau due to the treatment of TRPC6 peptide fragments in sporadic AD neurons. (A and B) The secretion of
Ab42 and Ab40 (A) as well as the intracellular tau and phospho-tau (B) in AD and control neurons exposed to second transmembrane of
TRPC6 fused with transmembrane peptide TAT (TM2) at different concentrations. (C and D) The secretion of Ab42 and Ab40 (C) as well as
the intracellular tau and phospho-tau (D) in AD and control neurons exposed to TAT, TM2, and mutated TM2 (TM2-MUT). Data are normal-
ized to total protein of whole-cell lysates and represented as mean 6 SD. n ¼ 3. *P < 0.05, **P < 0.01, and ***P < 0.001.

Discussion in iPSC-derived live AD neurons are consistent with our previ-

In this study, we report the derivation of patient-specific spo- ous findings in mouse models and AD patients, suggesting that
radic AD iPSCs and the efficient differentiation of iPSCs into AD TRPC6 might be a potentially therapeutic target for AD.
neurons. Relative to controls, the sporadic AD neurons exhib- The differentiation process from AD iPSCs to neurons recapit-
ited severer neuropathological phenotypes associated with AD ulates aspects of neural developmental and degenerative pro-
and compromised expression of TRPC6. More importantly, we cesses. The efficient neural differentiation of iPSCs is critical to
observed that the increase of TRPC6 inhibits the elevation of generate the patient iPSC-based AD cellular models. However,
both Ab and tau in sporadic AD neurons. Together, these the previous studies reported that human iPSCs showed poor
results indicated that TRPC6 is involved in the pathogenesis of efficiency in neural commitment, delayed differentiation and
AD by interfering with the generation of Ab, even tau. Our data line variations during neural differentiation (Hu et al., 2010;
 Probing TRPC6 in live neurons from sporadic AD iPSCs | 813

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Figure 4 The decrease of Ab and tau due to the treatment of TRPC6 agonist in sporadic AD neurons. (A) Western blotting analysis of TRPC6
expression in AD-1 and Control-1 neurons exposed to hyperforin at different concentrations (top) and quantification (bottom). (B) The im-
munoprecipitation analysis of whole-cell lysates from AD and control neurons treated with DMSO or hyperforin precipitated by anti-APP an-
tibody and immunoblotted by anti-TRPC6 antibody. (C and D) The secretion of Ab42 and Ab40 (C) as well as the intracellular tau and
phospho-tau (D) in AD and control neurons exposed to hyperforin at different concentrations. Data are normalized to total protein of
whole-cell lysates and represented as mean 6 SD. n ¼ 3. *P < 0.05, **P < 0.01, and ***P < 0.001.

Kim et al., 2010; Chambers et al., 2012). Consistently, we differentiation of human ESCs (Chambers et al., 2009), was
found that the serum free, floating EB approaches for human reported to be able to promote neural commitment (Kim et al.,
ESC neural differentiation cannot sufficiently induce neural dif- 2010). In this study, we combined the EB formation and dual-
ferentiation of human iPSCs (data not shown). The dual-SMAD SMAD inhibitors to establish a neural differentiation method
inhibition (TGF-b inhibitor SB431542 and BMP inhibitor dorso- for human iPSCs. This approach efficiently directed both spo-
morphin) strategy, which was widely used in monolayer neural radic AD and control iPSCs into TUJ1þ (92.3%) or MAP2þ
814 | Tao et al.

(87.7%) neurons (Figure 1C and D) and the high efficiency was regulated protein kinases and transcription factors via activation
consistent among independent iPSC lines (Supplementary of TRPC6 channels, effect downstream targets like BDNF/TrkB
Figure S2C). In addition, majority of iPSC-derived neurons was pathway in neurons (Gibon et al., 2013; Thiel and Rossler,
mature and functional cortex neurons (Figure 1E–J) and these 2017). Hyperforin also has been proposed to functions as a pro-
AD neurons from sporadic AD iPSCs recapitulated canonical AD tonophore and causes cytosolic acidification in a TRPC6-
features including increased Ab secretion and tau phosphoryla- independent manner (Sell et al., 2014). In addition to the mech-
tion (Figure 1K and L) as previously reported in familial AD anism illustrated in our previous study (Wang et al., 2015; Lu
iPSCs (Israel et al., 2012; Kondo et al., 2013; Young et al., et al., 2018), we deduced that hyperforin might also modulate
2015). Collectively, these results suggested that our system Ab and tau levels by activating TRPC6 channel-associated path-
based on efficient neural differentiation of sporadic AD-specific ways. Thus, the hyperforin treatment illustrates the potential to
iPSCs could serve as a cellular platform to provide new insights increase TRPC6 expression by drugs in AD patients and the de-

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into AD, especially sporadic AD, in live neurons identical to velopment of TRPC6-based therapies for the treatment of AD.
patient-genetic background. The live AD neurons derived from patient-specific iPSCs pro-
Then, we detected a novel candidate target gene, TRPC6, for vide a platform to reveal the potential of TRPC6 as a target gene
AD in this patient iPSC-based cellular platform. Our previous of AD, supporting our previous conclusions in AD mouse models
studies performed in AD mouse revealed that TRPC6 interfered and AD patients and suggesting an alternative functional assay
with Ab generation and in AD patients showed the reduction of for the identification and validation of candidate targets for the
TRPC6 mRNA levels in the peripheral blood, suggesting that therapeutic intervention and treatment of AD.
TRPC6 might be involved in the progress of AD (Wang et al.,
2015; Lu et al., 2018). Then, it is crucial to know whether re-
duced expression of TRPC6 could induce AD-associated pheno- Materials and methods
types in the patient brain. To address this issue, it can take Participants
decades by longitudinal follow-up of TRPC6 expression in AD This use of human adult peripheral blood was approved by the
patients over disease progression. Then, the iPSC-derived neu-
Research Ethics Committees of all the participating hospitals and
rons from patients provide an alternative opportunity to explore
institutes and conducted according to the principles of the
the correlativity between TRPC6 and AD-relevant phenotypes in
Declaration of Helsinki. The iPSCs were generated from 12 AD
short term. Here, we compared the expression patterns of
patients and 17 age-matched healthy controls recruited in
TRPC6 vs. Ab as well as tau in live sporadic AD neurons derived
Shanghai, China (Supplementary Table S1). Written informed con-
from patient-specific iPSCs and found that sporadic AD neurons
sent was obtained from all participants or their primary care-
with compromised expression of TRPC6 displayed evident in-
givers. Diagnosis of AD was based on the criteria of International
crease of Ab secretion and tauopathy (Figures 1K, L and 2B–D).
Classification of Diseases (ICD-10) and National Institute of
This observation is of particular interesting and suggested that,
Neurological and Communicative Disorders and Stroke-
at least in a subgroup of population, the individuals with lower
Alzheimer’s Disease and Related Disorders Association (NINCDS-
TRPC6 might be predisposed to develop abnormal Ab secretion
or tau accumulation during AD progression. Thus, these results ADRDA) and performed in our previous study (Lu et al., 2018).
from live sporadic AD neurons provide evidences to further
support our hypothesis that TRPC6 might be a potential target
in early stage of AD pathogenesis. The generation of iPSCs from human peripheral blood
Finally, we tested the therapeutic potential of TRPC6 by modu- mononuclear cells
lating its expression levels in iPSC-derived AD neurons. We dem- The generation of integration-free iPSCs from PB MNCs was
onstrated that treatment with TRCP6 peptide caused the performed according to a previously reported protocol (Dowey
decrease of Ab and tau in AD neurons (Figure 3), which is consis- et al., 2012). PB MNCs were isolated from 3–8 ml peripheral
tent with the inhibitory effects of TRPC6 on Ab in cell-free in vitro blood of AD patients or controls by density-based centrifugal
assay, HEK293-APP cell, rat primary neuron, and AD mouse in separation using Ficoll-Paque Premium (GE healthcare). The
our previous studies (Wang et al., 2015; Lu et al., 2018). proliferative erythroblasts from PB MNCs were specifically
Hyperforin, the agonist of TRPC6 and a clinical drug, was enriched after an 8–10-day expansion with defined MNC me-
reported to reduce the accumulation of Ab and alleviate the cog- dium as previously described (Chou et al., 2011). Cell numbers
nitive symptoms associated with AD in mouse models were counted every 2 days. Two million of PB MNCs in expo-
(Dinamarca et al., 2006; Cerpa et al., 2010; Inestrosa et al., nential phase were nucleofected with a mixture of 4 lg
2011) , but the mechanism underlying remained largely unclear. EBNA1/OriP-based episomal vectors EV SFFV-OCT4-2A-SOX2
Our results indicated that the sporadic AD neurons were sensi- (pEV-OS), 4 lg EV SFFV-MYC-2A-KLF4 (pEV-MK), and 2 lg EV
tive to hyperforin and hyperforin exposure resulted in a marked SFFV-BCL-XL (kind gifts from Xiao-bing Zhang, Loma Linda
increase in the expression of TRPC6 and evident decreases in University, USA) using Amaxa Nucleofector II (Lonza human
the production of Ab and tau (Figure 4), which is consistent with CD34þ cell nucleofector Kit, program ‘T-016’). Two days later,
the observations in AD mouse. It was reported that hyperforin transfected MNCs were plated onto mouse embryonic
 Probing TRPC6 in live neurons from sporadic AD iPSCs | 815

fibroblasts (MEFs) and maintained with hESC medium supple- resolved in DMSO. TAT: GRKKRRQRRRC; TM2: TSCFSWMEM
mented with 10 ng/ml bFGF (Pufei) and 0.25 mM NaB (Sigma). LIISWVIGMIWAGRKKRRQRRRC; TM2-MUT: TSCFSWMEMLIISDDIG
The iPSC colonies were picked up 2–3 weeks after transduc- MDDAGRKKRRQRRRC. Hyperforin and peptides were supple-
tion and maintained on MEF with hESC medium. For each indi- mented into neuronal cultures at differentiation Day 48 and
vidual, six iPSC lines from different mono-colonies were treated for 48 h.
established. The iPSC lines from three sporadic AD patients
(designated as AD-1, AD-2, AD-3) and three controls (desig-
nated as Control-1, Control-2, Control-3) were used for the sub- Statistics
sequent neural differentiation and analysis, respectively For qRT-PCR analysis, GAPDH was used as a reference gene
(Supplementary Table S1). Two or three iPSC lines of each indi- and the relative levels of target genes were calculated by the
vidual were used. All reagents were purchased from Life comparative Ct method (Schmittgen and Livak, 2008) . For

Downloaded from https://academic.oup.com/jmcb/article/12/10/807/5850867 by guest on 22 September 2022

Technology if not otherwise specified. ELISA analysis, data sets are normalized to total protein of
whole-cell lysates and are relative to the mean of control. For
western blotting analysis, data sets are normalized to the ex-
The Neural differentiation of human iPSCs pression of tubulin and are relative to the mean of control. All
To direct the differentiation of human iPSCs into neurons, statistical analyses were performed using GraphPad Prism.
the feeder cells were removed from iPSC cultures. Then, the Sample size (n) values were provided in the relevant text, fig-
iPSCs were dissociated into clumps to form EBs. The EBs ures, and figure legends. Data of each sample were calculated
were cultured in hESC medium without bFGF for 4 days and from 23 technical replications. The statistical analyses were
then with N2 medium for another 2 days. During EB stage, obtained from three independent experiments. All data were
both media were supplemented with 10 lM SB431542 presented as mean 6 SD. Two-tailed Student’s t-test was
(Selleck) and 2 lM dorsomorphin (Sigma). At Day 6, EBs used for two groups and one-way ANOVA with Newman–Keuls
were transferred into matrigel-coated culture plates and at- post hoc test for more than two groups. The P < 0.05 was con-
tached to form rosettes maintaining in N2 medium and then sidered as statistically significant. For all, *P < 0.05,
N2B27 medium from Day 8. At Day 16, the rosettes were **P < 0.01, and ***P < 0.001.
digested and cultured in Petri-dish for another 10 days to al-
low the formation of neural spheres in N2B27 medium. At
Day 26, neural spheres were digested into single cells Supplementary material
and reseeded on matrigel-coated dishes in B27 medium Supplementary material is available at Journal of Molecular
(Sigma) for neuronal differentiation and maturation. Cell Biology online.
Neuronal culture medium was refreshed every 2 days. All
reagents were purchased from Life Technology if not other-
wise specified. Acknowledgements
We are grateful to T. Li and D.M. Lai at Shanghai Jiao Tong
University School of Medicine for their support in karyotype
ELISA assays for the measurement of Ab40, Ab42, total tau, and analysis.
phospho-tau
To measure Ab40 and Ab42, the culture medium of iPSC-
derived neurons was collected in polypropylene tubes at differen- Funding
tiation Day 34, 42, or 50 and stored at 80 C, while the neuronal This work was supported in part by the ‘Strategic Priority
lysates were collected for protein concentration analysis. Levels Research Program’ of the Chinese Academy of Sciences
of Ab40 and Ab42 were assayed by Ab40 Human ELISA Kit (XDA16020501 and XDA16020404), National Key Basic Research
(Invitrogen, KHB3482) and Ab42 Human Ultrasensitive ELISA Kit and Development Program of China (2018YFA0108000,
(Invitrogen, KHB3544), following the manufacturer’s instructions, 2018YFA0107200, 2017YFA0102700, 2015CB964500, and
respectively. To measure the intracellular total tau and phospho- 2014CB964804), and the National Natural Science Foundation of
tau, whole-cell lysates of iPSC-derived neurons were extracted us- China (81671224, 81830034, 31800854, 31661143042,
ing cell extraction buffer were assayed by Total Tau Human ELISA 91519314, 31630043, 31571513, and 31430058).
Kit (Invitrogen, KHB0042) and TAU [pT231] Phospho-ELISA Kit,
Conflict of interest: none declared.
Human (Invitrogen, KHB8051). All experiments were repeated
twice with duplicate samples for each iPSC line.

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