Aging Cell (2017) 16, pp349–359
Doi: 10.1111/acel.12561
The amino acid transporter SLC36A4 regulates the amino acid
pool in retinal pigmented epithelial cells and mediates the
mechanistic target of rapamycin, complex 1 signaling
Peng Shang,1,2 Mallika Valapala,2 Rhonda Grebe,2
Stacey Hose,2 Sayan Ghosh,2 Imran A. Bhutto,2 James T. Handa,2
Gerard A. Lutty,2 Lixia Lu,1 Jun Wan,2 Jiang Qian,2
Yuri Sergeev,3 Rosa Puertollano,4 J. Samuel Zigler Jr,2
Guo-Tong Xu1,5,6 and Debasish Sinha2
1
Department of Ophthalmology of Shanghai Tenth People’s Hospital and
Laboratory of Clinical Visual Science of Tongji Eye Institute, Tongji University
School of Medicine, Shanghai, China
2
The Wilmer Eye Institute, The Johns Hopkins University School of Medicine,
Baltimore, MD, USA
3
National Eye Institute, National Institutes of Health, Bethesda, MD, USA
4
Cell Biology and Physiology Center, National Heart, Lung and Blood
Institute, National Institutes of Health, Bethesda, MD, USA
5
Translational Medical Center for Stem Cell Therapy, Shanghai East Hospital,
Tongji University School of Medicine, Shanghai, China
6
The Collaborative Innovation Center for Brain Science, Tongji University,
Shanghai, China
Summary
Aging Cell
The dry (nonneovascular) form of age-related macular degeneration (AMD), a leading cause of blindness in the elderly, has few, if
any, treatment options at present. It is characterized by early
accumulation of cellular waste products in the retinal pigmented
epithelium (RPE); rejuvenating impaired lysosome function in RPE
is a well-justified target for treatment. It is now clear that amino
acids and vacuolar-type H+-ATPase (V-ATPase) regulate the mechanistic target of rapamycin, complex 1 (mTORC1) signaling in
lysosomes. Here, we provide evidence for the first time that the
amino acid transporter SLC36A4/proton-dependent amino acid
transporter (PAT4) regulates the amino acid pool in the lysosomes
of RPE. In Cryba1 (gene encoding bA3/A1-crystallin) KO (knockout)
mice, where PAT4 and amino acid levels are increased in the RPE,
the transcription factors EB (TFEB) and E3 (TFE3) are retained in the
cytoplasm, even after 24 h of fasting. Consequently, genes in the
coordinated lysosomal expression and regulation (CLEAR) network
are not activated, and lysosomal function remains low. As these
mice age, expression of RPE65 and lecithin retinol acyltransferase
(LRAT), two vital visual cycle proteins, decreases in the RPE. A
defective visual cycle would possibly slow down the regeneration
of new photoreceptor outer segments (POS). Further, photoreceptor degeneration also becomes obvious during aging, reminiscent
of human dry AMD disease. Electron microscopy shows basal
Correspondence
Debasish Sinha, The Wilmer Eye Institute, The Johns Hopkins University School of
Medicine Baltimore, MD 21287, USA. Tel.: 410 502 2100; fax: 410-614-6728;
e-mail: Debasish@jhmi.edu
and
Guo-Tong Xu, Department of Ophthalmology of Shanghai Tenth People’s Hospital
and Laboratory of Clinical Visual Science of Tongji Eye Institute, Tongji University
School of Medicine, Shanghai 200 092, China. Tel.: 86 21 6598 6358; fax: +86-216598-6358; e-mail: gtxu@tongji.edu
Accepted for publication 24 November 2016
laminar deposits in Bruch’s membrane, a hallmark of development
of AMD. For dry AMD patients, targeting PAT4/V-ATPase in the
lysosomes of RPE cells may be an effective means of preventing or
delaying disease progression.
Key words: amino acid transporter (PAT4/SLC36A4); agerelated macular degeneration; coordinated lysosomal expression and regulation (CLEAR) network; lysosomes; mechanistic
target of rapamycin; complex 1 (mTORC1); mouse model;
retinal pigmented epithelium (RPE); photoreceptor degeneration; signal transduction; transcription factors EB (TFEB) and
E3 (TFE3); visual cycle proteins.
Introduction
Crystallins are highly abundant proteins of the lens, essential for
maintaining its transparency and refractivity. In addition to their roles as
structural elements in the lens, crystallins may also have diverse functions
in other parts of the eye (Horwitz, 2003; Piatigorsky, 2008; Zigler &
Sinha, 2015). bA3/A1-crystallin, a member of the b-crystallin subfamily
encoded by the Cryba1 gene, is also expressed in retinal pigmented
epithelial (RPE) cells and astrocytes (Parthasarathy et al., 2011).
We have demonstrated that bA3/A1-crystallin in the RPE is localized
to the lysosomal lumen, where it regulates endolysosomal acidification
by modulating V-ATPase, thereby affecting lysosomal clearance by both
phagocytosis and autophagy (Zigler et al., 2011; Valapala et al., 2014a,
b). We have shown that bA3/A1-crystallin binds to ATP6V0A1/V0-ATPase
and is involved in the mechanistic target of rapamycin, complex 1
(mTORC1) signaling pathway in RPE cells. Loss of bA3/A1-crystallin
results in decreased V-ATPase activity, elevated lysosomal pH, activation
of mTORC1, and inhibition of autophagy (Valapala et al., 2014a).
It is now recognized that an interplay between V-ATPase and amino
acids is essential in regulation of mTORC1 signaling (Zoncu et al., 2011).
The proton-assisted amino acid transporter (PAT)/solute-linked carrier 36
(SLC36) family members regulate intracellular amino acid concentrations
and mTORC1 signaling in lysosomes (Taylor, 2014). Recently, SLC38A9
(solute carrier family 38, member 9) was identified as an amino acid
sensor that activates mTORC1 activity by interacting with the RagulatorRag GTPase scaffolding complex in lysosomes (Rebsamen et al., 2015;
Wang et al., 2015). Here, we show for the first time that PAT4/
SLC36A4, a member of the PAT/SLC36 family, is expressed in RPE cells
and is involved in the lysosomal dysfunction caused by loss of bA3/A1crystallin. PAT4 can mediate the amino acid-sensing mechanism that
regulates mTORC1 activation inside the cell (Heublein et al., 2010). It has
been shown that Rab12 promotes constitutive degradation of PAT4
(Matsui & Fukuda, 2013). The accumulation of PAT4 in Rab12
knockdown cells increased mTORC1 activity and decreased autophagy.
mTORC1 signaling modulates lysosomal homeostasis (Laplante &
Sabatini, 2013). As the RPE maintains the health of photoreceptors,
preserving its normal clearance functions is essential to insure functional
integrity of the neural retina (Strauss, 2005). Impaired lysosome-
ª 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use,
distribution and reproduction in any medium, provided the original work is properly cited.
349
350 PAT4 regulates the amino acid pool in RPE, P. Shang et al.
mediated clearance results in toxic accumulation of undegraded waste
products within the RPE, severely stressing these cells (Sinha et al.,
2016). Further, the accumulated toxic material may be released by the
RPE, thereby generating subretinal drusenoid deposits and drusen
(deposits in Bruch’s membrane), two signs indicative of development of
dry age-related macular degeneration (AMD), a major cause of vision
loss in the elderly (Sivaprasad et al., 2016). With the human lifespan
increasing, the management of aging-related diseases becomes more
important. There currently is no definitive treatment or prevention for
dry AMD (Buschini et al., 2015). A new treatment that targets early dry
AMD before significant vision loss would have great benefit for these
patients.
We have now generated a global knockout mouse for Cryba1.
These mice have pathological changes in the retina, mimicking some
characteristics of dry AMD. Using this model, we provide novel
evidence that the bA3/A1-crystallin/PAT4/V-ATPase complex is a
potential therapeutic target for preventing or delaying the progression
of dry AMD.
Results
bA3/A1-crystallin interacts with PAT4, and loss of bA3/A1crystallin elevates cellular amino acid concentration in RPE
cells and induces the mTORC1 pathway
We recently performed a human proteome high-throughput array
(CDI Laboratories, Inc.) and found that bA3/A1-crystallin interacts with
PAT4/SLC36A4, an amino acid transporter (Supplementary Table 1).
The PAT4/SLC36A4 family of amino acid transporters is known to
regulate intracellular amino acid concentrations and mTORC1 activity
in lysosomes. Here, we show that in a pull-down assay, bA3/A1crystallin binds to PAT4 in RPE cells from two-month-old Cryba1fl/fl
mice. Such binding did not occur in cells from Cryba1 KO mice
(Fig. 1A). Further, PAT4 RNA (from primary RPE cells in culture) and
protein (RPE from tissue) levels were determined by quantitative PCR
(QPCR) and western blot, respectively, in RPE of Cryba1fl/fl and Cryba1
KO mice after fasting. In Cryba1fl/fl (control) mice, PAT4 RNA, as well
Fig. 1 PAT4, interacting with bA3/A1crystallin, may modulate the mTORC1
signaling pathway by elevating cellular
amino acid concentration. (A) Pull-down
assay using antibody to bA3/A1-crystallin
demonstrates that PAT4 and bA3/A1crystallin interact in RPE lysates from twomonth-old Cryba1fl/fl mice, but not in
lysates from Cryba1 KO mice. (B) qPCR
analysis of PAT4 transcript showing lower
basal levels in Cryba1 KO primary RPE cells
relative to floxed control samples. Upon
serum starvation, the level decreases in RPE
cells from floxed mice, but markedly
increases in KO cells. (n = 4) (C) Western
blot showing corresponding protein levels
for PAT4 in RPE (n = 3). Quantification of
data in C is shown in D. (E) Total L-amino
acids increase in RPE of Cryba1 KO mice
after 24-h fasting in vivo. No such increase
is found in Cryba1fl/fl mice following fasting
(n = 8). Actin was used as internal control
in all blots. All data expressed as mean
SEM. *P < 0.05, **P < 0.01, ***P < 0.01.
ª 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.
PAT4 regulates the amino acid pool in RPE, P. Shang et al. 351
as protein levels, were downregulated in RPE following 24-h fasting
(Fig. 1B–D). However, in Cryba1 KO mice, both PAT4 RNA and
protein expression increased in the RPE after 24-h fasting (Fig. 1B–D).
Cryba1 KO mice appear to have a lower basal level of PAT4
expression compared with control mice. PAT4 has previously been
shown to regulate amino acid sensing inside cells (Matsui & Fukuda,
2013). Interestingly, our data indicate that the concentration of free Lamino acids after 24 h of fasting is significantly elevated in RPE cells
of Cryba1 KO mice relative to fed controls. This increase is not seen in
cells from Cryba1fl/fl following fasting (Fig. 1E).
It is known that when the cellular environment is amino acid rich,
mTORC1 is activated. When Cryba1fl/fl mice were fasted for 24 h, we found
that phosphorylated mTORC1 (Ser2448) and phosphorylated p70S6K
(T421/S424) levels decreased in RPE cells, as compared to fed mice.
However, phosphorylated mTORC1 (Ser2448) and phosphorylated p70S6K
(T421/S424) were observed at consistently higher levels in cells from Cryba1
KO mice, even after fasting, suggesting activation of mTORC1 (Fig. 2A–C).
It is also true that amino acid-sensing interactions are required for
proper nucleotide loading of the Rag GTPases, recruitment of mTORC1
to the lysosome, and the subsequent activation of mTORC1 (Zoncu
et al., 2011). The Rag GTPases reside on the lysosome and modulate
amino acid import. They exist as obligate heterodimers (RagA or RagB
with RagC or RagD) and interact with Ragulator (LAMTOR1-5). The
Ragulator-Rag multiprotein complex is a critical component in the
shuttling of mTORC1 to late endosomes/lysosomes. The protein levels of
Ragulator and Rag GTPases, as indicated by western analysis of the
mTORC1 signaling intermediates (LAMTOR2, LAMTOR3, RagA, and
RagB), were higher in Cryba1 KO mice than in floxed control mice, but
only RagA was statistically significantly higher (Fig. 2D,E). After 24 h of
fasting, the levels of LAMTOR2, LAMTOR3, and RagB were statistically
significantly higher in cells from KO mice relative to floxed controls.
Loss of bA3/A1-crystallin in RPE cells affects TFEB/TFE3
phosphorylation as well as expression of CLEAR network
genes
mTORC1 modulates the stress-induced transcription factor EB (TFEB) to
regulate a group of genes known as the coordinated lysosomal
expression and regulation (CLEAR) network, which maintain normal
lysosomal function. Amino acids can regulate TFEB through mTORC1.
Fig. 2 Increased mTORC1 signaling intermediates and persistent activation of mTORC1 signaling pathway in RPE of Cryba1 KO mice even after fasting. (A) Representative
western blot of p-mTOR (Ser2448) and p70S6K (Thr421/Ser424) in RPE lysates from fed and 24-h-fasted Cryba1fl/fl or Cryba1 KO mice (n = 4). (B) and (C) show
densitometric quantification for p-mTOR and p-70S6K, respectively. (D) Representative western blots for LAMTOR2, LAMTOR3, RagA, and RagB expression and (E)
densitometric quantification of the western blot data for RPE lysates from fed and 24-h-fasted Cryba1fl/fl or Cryba1 KO mice (n = 5). Actin was used as internal control in all
blots. Values were normalized to that of fed Cryba1fl/fl samples which relative expression was as 1. All data are expressed as mean SEM. *P < 0.05, **P < 0.01.
ª 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.
352 PAT4 regulates the amino acid pool in RPE, P. Shang et al.
TFEB, when phosphorylated by mTORC1, is retained in the cytoplasm;
when not phosphorylated, it translocates to the nucleus and activates
CLEAR genes, thereby stimulating lysosomal biogenesis and function
(Settembre et al., 2012; Martina et al., 2012; Roczniak-Ferguson et al.,
2012). Our data suggest that even when autophagy is induced in the RPE
by fasting in vivo, the absence of bA3/A1-crystallin causes TFEB to remain
in the cytosol, thereby preventing activation of CLEAR genes (Fig. 3A,B).
Under normal physiological conditions, western analyses detected TFEB in
both the cytoplasm and nucleus of RPE cells of Cryba1fl/fl mice, with an
increased proportion in the nucleus after fasting (Fig. 3A). In Cryba1 KO
mouse RPE, TFEB was predominantly cytoplasmic, with no indication of
movement into the nucleus after fasting (Fig. 3A). Quantitative real-time
PCR showed that expression levels of lysosomal genes in the CLEAR
network were significantly lower in RPE of Cryba1 KO mice than in controls
(Fig. 3B). TFE3, similar to TFEB, is also involved in nutrient sensing and
maintenance of cellular homeostasis. TFE3 accumulates in the nucleus
upon nutrient deprivation, but is retained in the cytosol when phosphorylated by mTORC1 (Martina et al., 2014). In Cryba1fl/fl control mice,
phosphorylated TFE3 decreased in RPE after 24-h fasting, indicating the
accumulation of TFE3 in the nucleus. In contrast, the level of phosphorylated TFE3 was not reduced in the RPE of fasted Cryba1 KO mice like
Cryba1fl/fl control mice, even though the phosphorylated TFE3 level in RPE
of KO mice is much lower than that in RPE of control mice (Fig. 3C,D).
We further investigated the expression of cathepsin D (CTSD), a
CLEAR network gene with significantly decreased expression in Cryba1
KO RPE relative to control. Interestingly, while CTSD expression increased
significantly with age in RPE cells from Cryba1fl/fl mice (Fig. 3E,F,G), in
Cryba1 KO cells, the overall CTSD expression level was lower at both
ages relative to controls (Fig. 3E,F,G). Further, CTSD immunolabeling
suggests that the capacity of intracellular degradation in the RPE of
Cryba1 KO mice is considerably less than in control mice (Fig. 3H). Our
transmission electron microscopy (TEM) data from 20-month-old KO
mice show accumulation of undegraded material in the RPE (Fig. 3I) as
compared to control. Numerous lipidated vacuoles (Fig. 3I, middle and
right panels) and, most importantly, greater accumulation of autolysosomes (Fig. 3I, right panel) result from impaired lysosome-mediated
degradation and recycling. We also found that levels of p62, a receptor
for cargo destined to be degraded by autophagy, were higher in
10-month-old Cryba1 KO RPE cells than in controls (Fig. 3J,K).
Cryba1 deprivation leads to age-dependent defects in
architecture of RPE cells
We next asked whether molecular dysregulation of normal lysosomal
function in Cryba1 KO RPE cells has an effect on RPE structure and,
most importantly, whether waste products accumulate in the Cryba1
KO RPE cells. We observed abnormalities in the cellular architecture of
Cryba1 KO RPE by TEM (Fig. 4). Large vacuoles, not seen in Cryba1fl/fl
cells (Fig. 4C), and increased numbers of melanosomes (Fig. 4B) were
observed in RPE cells from two-month-old Cryba1 knockout mice, as
compared to controls (Fig. 4A). These abnormalities became more
severe as the animals aged. Cellular debris enclosed in vacuoles was
not efficiently digested (Fig. 4E). In some areas, the Cryba1 KO RPE
cells began to lose basal infoldings and showed intracytoplasmic
disruption (Fig. 4F). Such abnormalities were not seen in floxed
controls (Fig. 4D). In Cryba1 KO animals, melanosomes were sometimes found to move from RPE cells into the photoreceptor out
segments (POS). Large basal laminar deposits could be seen in Cryba1
KO RPE (Fig. 4G–I) and were also found above Bruch’s membrane
(Fig. 4F).
The visual cycle is impaired in aging Cryba1 knockout mice
One of the critical functions of RPE cells is the recycling of retinoids that
are essential for the visual cycle. RPE65 and lecithin retinol acyltransferase (LRAT) are key enzymes in converting all-trans-retinal to 11-cisretinal (Redmond et al., 1998; Jin et al., 2007). RPE flat mounts from
Cryba1fl/fl and Cryba1 KO mice were stained with the high-affinity
filamentous actin probe, phalloidin, and with RPE65 antibody. Phalloidin
staining demonstrated differences in both the size and shape of RPE cells
in Cryba1 KO mice (Fig. 5A). A large number of RPE cells in Cryba1 KO
mice gradually lose their regular hexagonal shape (arrow heads) and
exhibit reduced staining for RPE65 (arrows) as they age (Fig. 5A). Both
RPE flat mounts and western blots showed a gradual loss of RPE65 in
Cryba1 KO mice as a function of aging. LRAT was also reduced in the
Cryba1 KO mice by 9 months of age compared with age-matched
Cryba1fl/fl mice (Fig. 5B,C).
Photoreceptor OS dysfunction due to abnormal lysosomalmediated clearance of RPE cells from Cryba1 KO mice
Several elegant studies have suggested that a symbiotic relationship
between photoreceptors and RPE cells is necessary for maintaining the
proper health of the neural retina (Sparrow et al., 2010). As the normal
functioning of the RPE is compromised in Cryba1 KO mice, we evaluated
their POS. Immunofluorescence studies for rhodopsin show stronger
staining in Cryba1 KO retinas in cryosections from 20-month-old mice
compared with those of Cryba1fl/fl, but the staining was more diffuse
(first panel in Fig. 6A). These data are consistent with staining caused by
shed photoreceptor OS, which were not engulfed by RPE cells, but
Fig. 3 Predominantly cytosolic TFEB and abnormal levels of phosphorylated TFE3 in RPE cells from fasted and fed Cryba1 KO mice. (A) Western blot showing increased levels
of TFEB in Cryba1fl/fl nuclear extracts following fasting. TFEB is predominantly localized in the cytosol (C) in cells from normally fed Cryba1fl/fl (control) mice, but after fasting
the nuclear (N) proportion increases. In cells from Cryba1 KO mice, significantly lower nuclear levels of TFEB were observed in both fasted and fed conditions. (B) Cryba1fl/fl
and Cryba1 KO cells were subjected to qPCR analysis using Taqman probes for some CLEAR (coordinated lysosomal expression and regulation) genes after fasting. Cryba1
KO cells showed a reduction in the transcript levels for lysosomal hydrolases: CTSD (55%), CTSB (75%); lysosomal acidification: ATP6V0A1 (56%); lysosomal membrane
proteins: LAMP1 (15%), LAMP2 (40%), and MCOLIN1 (35%); and gene related to autophagy: BECN1 (51%), UVRAG (35%). The graph shows mean SD from triplicate
experiments, representative of at least three independent experiments. (two-tailed t-test) (C) Western blot for p-TFE3 in RPE isolated from Cryba1fl/fl and Cryba1 KO mice.
The data indicate decreased p-TFE3 in controls, but increased p-TFE3 in KO RPE after fasting. (n = 4) Quantification of C is shown in D. (E) Western blot showing cathepsin D
(both precursor and immature forms) expression in the RPE from four-month-old (n = 6) and 10-month-old (n = 3) Cryba1 KO mice vs. floxed controls. F and G show
densitometric quantification for data in E. (H) CTSD immunostaining (red) and DAPI staining (blue) on RPE flat mounts from Cryba1fl/fl and Cryba1 KO mice at 2 and
10 months of age (n = 3). There are fewer fluorescent puncta in Cryba1 KO RPE cells, than in Cryba1fl/fl RPE at both ages. Scale bars=10 lm. (I) TEM of RPE in 20-month-old
Cryba1fl/fl mouse showing photoreceptor outer segments (POS) and RPE (left panel). Unlike Cryba1 KO mouse, TEM (center panel) showed numerous vacuoles with possible
accumulation of lipid-like droplets (arrow). TEM also showed many autolysosomes, some with incomplete degradation (arrow) retained in 20-month-old Cryba1 KO mouse
(right panel). (J) Western blot and quantification (K) of protein level of SQSTM1 (p62) in RPE of 10-month-old Cryba1 KO mice relative to control mice. (n = 3) *P < 0.05,
**P < 0.01.
ª 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.
PAT4 regulates the amino acid pool in RPE, P. Shang et al. 353
ª 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.
354 PAT4 regulates the amino acid pool in RPE, P. Shang et al.
A
B
C
D
E
F
G
H
I
Fig. 4 Ultrastructure of RPE in two-month-old and 20-month-old Cryba1 knockout mice and age-matched Cryba1fl/fl mice by transmission electron microscopy. POS
(photoreceptor outer segment), AM (apical microvilli), BL (basal lamina), BM (Bruch’s membrane), CC (choriocapillaris). (A) RPE of a two-month-old Cryba1fl/fl mouse showing
normal phenotype. (B) RPE of a two-month-old Cryba1 KO mouse showing increased melanosomes (arrows). (C) Vacuoles appear in RPE of two-month-old KO mice
(arrows). (D) RPE of a 20-month-old Cryba1fl/fl mouse. (E) RPE of a 20-month-old Cryba1 KO mice with much bigger vacuoles containing undigested cellular debris (arrows)
and highly disrupted basal infoldings. (F, G) Asterisks indicate basal laminar deposits in KO RPE cells. Arrow heads in F indicate a layer of possible basal laminar deposit
between Bruch’s membrane and RPE. Inset in F is magnified area in the lower right frame of F, showing a thin part of Bruch’s membrane. (H, I) Melanosomes (arrows) from
20-month-old KO RPE move into the POS layer.
accumulated between the photoreceptors and the RPE. This was
confirmed by TEM and 1D4 (POS marker) staining (Fig. 6B). M-opsin
and peanut agglutinin lectin (PNAL), cone photoreceptor markers, were
almost undetectable in some regions of the Cryba1 KO retina (middle
two panels in Fig. 6A). Furthermore, the thickness of the outer nuclear
layer was significantly reduced in the regions with loss of M-opsin
and PNAL (bottom panel in Fig. 6A), indicating degeneration of
photoreceptors in 20-month-old Cryba1 KO mice. TEM also
showed degenerating POS in Cryba1 KO compared with floxed
controls.
ª 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.
PAT4 regulates the amino acid pool in RPE, P. Shang et al. 355
Fig. 5 Impaired visual cycle in Cryba1 KO
mice. (A) RPE65 (retinal pigment
epithelium-specific 65 kDa protein, red)
and phalloidin (green) immunofluorescent
staining of Cryba1fl/fl and Cryba1 KO mice
RPE flat mounts. With increasing age, RPE
cells in Cryba1 KO mice gradually lose their
regular hexagonal shape (arrow heads and
have less RPE65 (arrows). Scale
bar = 20 lm. (B, C) Western blots and
densitometric quantification show that as
Cryba1 KO mice age, there is significant
loss of RPE65 and LRAT (Lecithin retinol
acetyltransferase) in Cryba1 KO mice by
9 months.
Discussion
Many age-related diseases, including dry AMD, severely impair quality of
life in the elderly. We and others have postulated that abnormal
lysosome function in RPE cells could ultimately contribute to dry AMD
(Guha et al., 2014; Sinha et al., 2016). In fact, the efficiency of
lysosomes in degrading cellular components declines over time, an effect
linked both to aging and the development of age-related diseases
(Cuervo & Dice, 2000). Therefore, we have generated unique mouse
models to evaluate defective lysosomal degradative function in the RPE
and its possible role in AMD.
RPE cells are postmitotic, have high metabolic activity, and are among
the most active phagocytic cells in the body (Strauss, 2005). For efficient
cellular clearance in the RPE, normal lysosome function through both
phagocytosis and autophagy is key (Boya & Codogno, 2013). Lysosomes
are no longer regarded as simply a heterogeneous collection of
degradative organelles, but also as a platform for signaling pathways
(Puertollano, 2014). Our previous work confirmed that bA3/A1-crystallin
regulates mTORC1 signaling, possibly by modulating the assembly/
disassembly of the proton pump, V-ATPase (Valapala et al., 2014a).
mTORC1 is a platform for a major signaling axis within lysosomes that
supports normal lysosomal function and thus cellular homeostasis
(Settembre et al., 2013).
It is now clear that amino acids and V-ATPase are absolutely essential
for mTORC1 signaling. Two ubiquitously expressed members of the PAT
family, PAT1/SLC36A1 and PAT4/SLC36A4, have been shown to affect
mTORC1 activity (Heublein et al., 2010; Matsui & Fukuda, 2013; Fan
et al., 2016; Goberdhan et al., 2016). We made the unexpected
observation that bA3/A1-crystallin binds to PAT4. This is the first report
showing PAT4 expression in the eye. It is known that when the cellular
environment is amino acid rich, mTORC1 is active and inhibits
autophagy. Our data provide evidence that when bA3/A1-crystallin is
lacking, fasting upregulates PAT4, cellular amino acid levels increase and
mTORC1 is activated. Moreover, Rag GTPases, supported by the
ª 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.
356 PAT4 regulates the amino acid pool in RPE, P. Shang et al.
Fig. 6 Photoreceptor outer segment dysfunction due to abnormal lysosomal-mediated clearance of RPE cells from Cryba1 KO mice. (A) antibodies to rhodopsin (labels rod
photoreceptors—green), M-opsin (labels cones—red), and DAPI (labels nuclei—blue) were used to stain retinal cross sections. Representative data of immunofluorescent
staining of photoreceptors: rhodopsin, M-opsin, peanut agglutinin lectin (PNAL) in 20-month-old Cryba1 KO and age-matched Cryba1fl/fl retina. DAPI staining showed the
number of nuclei and the thickness of outer nuclear layer. ONL (outer nuclear layer), INL (inner nuclear layer), scale bar = 20 lm. (n = 2) (B) Transmission electron
microscopy (scale bar = 2 lm) and 1D4 (a marker for shed outer segments) immunostaining (scale bar = 20 lm) showing accumulation of photoreceptor outer segment
tips in the subretinal layer in 20-month-old Cryba1 KO mice (arrows), but not in the age-matched Cryba1fl/fl mice. (C) Three-dimensional modeling is shown, depicting
structures of PAT-4 (magenta), bA3-crystallin (green), and V-ATPase (Cyan) forming a complex, obtained by Hex Protein Docking.
Ragulator complex (Bar-Peled et al., 2012), modulate amino acid import
by lysosomes. Our data clearly show abnormal regulation of Rag GTPases
and mTORC1 activity after fasting in Cryba1 KO RPE cells.
Alternatively, amino acid sensing might occur by an ‘inside-out’
mechanism with direct coupling between intralysosomal amino acids
and activation of mTORC1 leading to subsequent conformational
change in the lysosomal V-ATPase (Zoncu et al., 2011). A direct
interaction between V-ATPase and the Rag GEF (guanine nucleotideexchange factor) complex was observed. Interestingly, amino acids
weaken the interaction between Ragulator and the V-ATPase V1
domain, but have no effect on the interaction with the V0 domain.
These amino acid-sensitive interactions were shown to be essential for
proper nucleotide loading of the Rag GTPases, recruitment of mTORC1
to the lysosome, and subsequent activation of mTORC1 (Zoncu et al.,
2011). However, we have previously shown (Valapala et al., 2014a) that
bA3/A1-crystallin binds only to the V-ATPase V0 domain, which is
responsible for carrying out proton translocation to the endolysosomal
compartments and is critical for pH-dependent processes (Forgac, 2007;
Breton & Brown, 2013). It is highly likely that bA3/A1-crystallin is an
upstream regulator of both V-ATPase and PAT4 and is essential for
mTORC1 signaling in RPE cells.
The cellular demand for amino acids is cell and tissue specific. In fact,
a direct role for amino acids in mTOR signaling is supported by the welldocumented observation that treatment of cells with protein translation
inhibitors (such as cycloheximide), which contribute to increased
intracellular concentration of amino acids, can activate mTORC1 and
inhibit autophagy even under nutrient deprivation (Beugnet et al., 2003;
Watanabe-Asano et al., 2014). This is reminiscent of the situation in our
Cryba1 KO mice, suggesting that for normal mTORC1 signaling and
thereby maintenance of lysosomal homeostasis in RPE cells, it is
important to have a functional bA3/A1-crystallin-PAT4-V-ATPase complex.
Amino acids also control lysosomal homeostasis through the regulation of TFEB. TFEB localization and inactivation in lysosomes are
dependent upon the activation of Rag GTPases and mTORC1 (Martina
et al., 2012; Settembre et al., 2012; Martina & Puertollano, 2013). TFEB
binds to the cytosolic chaperone protein 14-3-3 when phosphorylated
and is retained in the cytoplasm; when not phosphorylated, it translocates to the nucleus and activates CLEAR genes (Settembre et al., 2011;
Martina et al., 2012; Roczniak-Ferguson et al., 2012). Cells adapt to
diminished amino acid levels by increasing the lysosomal and autophagic
compartments in order to maintain a critical level of metabolites. In
ª 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.
PAT4 regulates the amino acid pool in RPE, P. Shang et al. 357
Cryba1 KO RPE cells, the amino acid levels are high, and TFEB is retained
in the cytoplasm, even after fasting. In contrast, in RPE cells from control
Cryba1fl/fl mice, TFEB translocates to the nucleus and activates CLEAR
genes after fasting.
Cathepsin D (CTSD), a CLEAR network gene product involved in
phagocytic and autophagic degradation in the RPE (Rakoczy et al., 1997;
Valapala et al., 2014a), is significantly decreased in Cryba1 KO RPE,
relative to control. Previously, we found that the absence of bA3/A1crystallin decreased CTSD activity in both astrocytes and RPE cells;
however, overexpression of bA3/A1-crystallin in the KO cells restored
normal activity (Valapala et al., 2013, 2014a). We also found that p62, a
receptor for cargo destined to be degraded by autophagy, was higher in
10-month-old Cryba1 KO RPE cells than in controls; this difference was
not found in younger mice. p62 contributes to both amino acid sensing
and the regulation of autophagy. It has been shown that p62 is required
for maximal mTORC1 activity in response to amino acids. p62 is
postulated to promote recruitment of mTORC1 to lysosomes via its
interaction with raptor (Duran et al., 2011). Taken together, our data
indicate that normal lysosomal function is significantly perturbed in
Cryba1 KO RPE cells.
Interestingly, Spatacsin KO mice, which progressively lose cortical
motor neurons and Purkinje cells, also have defective lysosomal function
(Varga et al., 2015). Spatacsin is essential for the reformation of
lysosomes from autolysosomes in vivo. The loss of lysosomes in these
mice preceded neuronal degeneration, a situation analogous to the
AMD-like phenotype in the Cryba1 KO mouse. In Cryba1 KO mice, the
RPE also loses expression of two vital visual cycle proteins, RPE65 and
LRAT. A defective visual cycle would slow down the regeneration of new
POS. As these mice age, photoreceptor degeneration also becomes
obvious, reminiscent of human dry AMD disease.
Here, we provide a direct link between amino acid availability and
mTORC1 signaling during aging in our mouse model. If lysosomemediated clearance is perturbed during aging, it could have important
implications for age-related disorders, such as AMD. The ability to sense
and appropriately respond to cellular stresses, such as amino acid
depletion, is commonly diminished during aging. This could have direct
consequences for the onset and progression of aging-related diseases.
For AMD patients, targeting the bA3/A1-crystallin/PAT4/V-ATPase complex (Fig. 6C) in the RPE may be an effective means of preventing or
delaying the progression of the disease.
Autophagy induction
Autophagy was induced in Cryba1fl/fl and KO mice by withholding
food for 24 h, but with no restriction on water availability. For in vitro
starvation, primary cultures of RPE cells from Cryba1fl/fl and KO mice
were maintained in growth medium lacking serum and glutamine for
24 h.
Antibodies
The following antibodies were used in this study: bA3/A1-crystallin
antibody (described previously Zigler et al., 2011), Slc36a4 antibody–
N-terminal (Aviva System Biology, San Diego, CA, ARP44114-P050),
Actin (Sigma, St. Louis, MO, A2066), PhosphoPlus-p70 S6 Kinase
(Thr389, Thr421/Ser424) Antibody Kit (Cell Signaling, Danver, MA,
#9430), Phospho-mTOR (Ser2448) (Cell Signaling, #5536), Rag and
LAMTOR Antibody Sampler Kit (Cell Signaling, #8665), TFEB (Bethyl
Laboratories, Montgomery, MA, A303-673A), Lamin A/B (Santa Cruz,
sc-6215), Tubulin (MBL, PM054-7Y), Phospho-TFE3 (as described in
Martina et al., 2016), CTSD (Gift from Dr. Ralph Nixon, NYU School of
Medicine), SQSTM1/P62 (Abcam, Cambridge, MA, ab91526), Rhodopsin, M-opsin, and PNAL (three gifts from Dr. Donald Zack, Wilmer Eye
Institute, The Johns Hopkins University School of Medicine), 1D4 (gift
from Dr. Krzysztof Palczewski, Case Western University), RPE65 (gift
from Dr. T. Michael Redmond, NEI, NIH), and LRAT (Santa Cruz, Dallas,
TX, sc-99015).
Co-immunoprecipitation
Pierce Co-Immunoprecipitation Kit (Thermo Scientific, Waltham, MA,
#26149) was used to carry out the immunoprecipitation studies.
Briefly, RPE-choroid preparations from seven mice of each genotype
were sonicated in IP Lysis/Wash Buffer (provided in the kit) plus 1%
protease inhibitors (Sigma). The total lysates were processed with the
kit according to the instructions. Seventy micrograms of lysates of
each genotype were immunoprecipitated with 10ug immobilized bA3/
A1-crystallin antibody at 4°C overnight. Normal rabbit IgG (Santa
Cruz, sc-2027) was the negative control. Samples from elution were
loaded for SDS-PAGE analysis. Fifteen micrograms of RPE-choroid
lysates of each genotype were loaded as the input for the SDS-PAGE
analysis.
Experimental procedures
Protein extraction and western blot analysis
Cryba1 global knockout mice
bA3/A1-crystallin conditional knockout (Cryba1 cKO) mice were generated as previously described (Valapala et al., 2014a,b). The controls used
in this study are Cryba1fl/fl mice. It is known that germline deletion of
floxed alleles may occur when floxed mice are maintained for multiple
generations with the Best1-Cre allele, creating a global knockout of the
floxed gene. We used this as a strategy to generate Cryba1 complete
knockout (KO) mice. All animal studies were conducted in accordance
with the Guide for the Care and Use of Animals (National Academy
Press) and were approved by the Animal Care and Use Committee of
Johns Hopkins University.
Isolation and culture of mouse primary RPE cells
Mouse RPE cells were isolated and cultured as previously described
(Valapala et al., 2014a).
RPE-choroid preparations from freshly dissected mice were sonicated in
RIPA lysis buffer (Millipore, Billerica, MA, 20-188) plus 1% protease and
phosphatase inhibitors (Sigma). Samples were incubated on ice for
20 min and centrifuged at 13 000 g for 20 min. The supernatants were
mixed with 4X protein sample buffer (Invitrogen, Carlsbad, CA) plus 5%
2-mercaptoethanol (Sigma) and heated at 100°C for 10 min to
denature. Samples were loaded into a 4–12% Bis-Tris Nu-PAGE gel
(Invitrogen) and run with MES buffer (Novex, Waltham, MA). Proteins
were transferred to nitrocellulose membranes which were then blocked
in 5% skim milk (Invitrogen) or 5% BSA (Sigma, for phosphorylated
proteins). The membranes were incubated with primary antibody
overnight followed by horseradish peroxidase-conjugated secondary
antibodies for 1 h at room temperature. Blots were developed by
chemiluminescence (ECL) methodology. Densitometric analysis was
carried out using QUANTITY ONE software (Bio-Rad Laboratories, Hercules,
CA).
ª 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.
358 PAT4 regulates the amino acid pool in RPE, P. Shang et al.
Human proteome high-throughput array
The human proteome microarray 2.0 analysis was performed as a paid
service from CDI NextGen Proteomics, MD, USA. For hit identification,
we first obtained the ratio of median value of the foreground to the
median of the surrounding background for each protein probe on the
microarray, followed by the normalization by the median value of all
neighboring probes within a 9 9 9 window size. Then, we compared
normalized value of each probe to the distribution of noise signals to
obtain a Z-score representing the significance of the probe binding signal
different from random noises we chose. The cutoff of Z-score was 6 in
this study. The protein was determined as a hit only if its Z-score was
above the cutoff for all triplicates.
0724370_m1), UVRAG (Mm00724370_m1), Beclin-1 (Mm01265461_
m1), and Mucolipin-1 (Mm00522549_m1). Actin B (Mm00607939_s1)
was used as a loading control. All data were analyzed with the ABI 7500
Real Time PCR system using the DATA ASSIST Software (Applied Biosystems), and the graphs were plotted using MICROSOFT EXCEL, Redmond, WA.
Molecular modeling
The possible interaction between PAT-4, bA3-crystallin, and V-ATPase
was tested using an interactive protein docking and molecular superposition program HEX PROTEIN DOCKING, version 6.3 (http://hex.loria.fr/ma
nual63/hex_manual.pdf).
Statistical analysis
Nuclei and cytoplasmic fraction
Nuclear and cytoplasmic fractions were isolated from extract of RPEchoroid preparations from six-month-old Cryba1fl/fl or Cryba1 KO mice
using the NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit
(Thermo Fisher Scientific, Waltham, MA, #78833) according to the
manufacturer’s protocol.
Statistical analysis was performed using MICROSOFT EXCEL. Graphs were
plotted using ORIGIN8 software or MICROSOFT EXCEL. The P-values were
determined by two-tailed Student’s t-test in at least three biological
replicate experiments. Significance was defined as *P < 0.05. Results are
presented as mean SEM.
Acknowledgments
Quantification of the intracellular L-amino-acid concentration
RPE-choroid complexes were homogenized in L-amino acid assay buffer
from the L-Amino Acid Quantification Kit (Sigma, MAK002). Lysates
were prepared as described above and then analyzed according to the
manufacturer’s instructions to determine their intracellular L-amino-acid
concentration. The value for each sample was normalized by total
protein concentration.
Immunofluorescence of RPE flat mount and cryosections
Fresh eyes were enucleated and fixed in 2% paraformaldehyde (PFA) for
10 min and then the anterior parts including cornea, lens, and attached
iris pigmented epithelium were removed. The resulting posterior
eyecups were fixed in 2% PFA for 1 h at room temperature either for
cryosections or RPE flat mount. For cryosections, the eyecups were
dehydrated through gradient sucrose solutions and embedded in OCT.
For RPE flat mounts, retinas were then removed after the eyecup was
quartered like a petaloid structure. The resulting eyecup was further cut
radially into eight pieces from the optic nerve head to the periphery.
Immunostaining on flat mount and cryosections was performed as
described previously (Zigler et al., 2011). Stained RPE-choroid sheets
with sclera were mounted on a microscope slide with RPE layer up.
Images were acquired by Zeiss LSM 710 confocal workstation.
We thank Dr. Morton Goldberg for critical reading and discussions
regarding this manuscript. We thank Drs. Ralph Nixon (CTSD), Donald
Zack (Rhodopsin, M-opsin and PNAL), Krzysztof Palczewski (1D4), and T.
Michael Redmond (RPE65) for the antibodies. DS is a guest professor at
Tongji University School of Medicine, Shanghai, China, and a recipient of
the Carolyn K. McGillvray Memorial Award for Macular Degeneration
Research from BrightFocus Foundation, the Sybil B. Harrington Special
Scholar Award for Macular Degeneration from Research to Prevent
Blindness. JTH is the Robert Bond Welch Professor.
Funding
This study was funded by an unrestricted grant to the Wilmer Eye
Institute from the Research to Prevent Blindness, National Eye Institute:
EY019037-S (DS), EY019044 (JTH), EY14005 (JTH), EY01765 (Wilmer
Imaging Core), and the National High Technology Research and
Development Program of China (2013CB967501, 2015CB964601,
2013CB967101), Shanghai East Hospital (ZJ2014-2D-002), and Tongji
Eye Institute (TEI-201403001).
Conflict of interest
None declared.
Transmission electron microscopy
Author contributions
Samples were prepared and transmission electron microscopy was
performed as previously described (Zigler et al., 2011).
DS designed the study and assisted with the generation of Cryba1
knockout (KO) mice and data analysis. G-TX participated in the study
design and analyzed data. SZ generated the Cryba1 KO mice and
analyzed data. PS conducted the majority of experiments. MV, SG, and
RP were involved with TFEB, TFE3, and CLEAR gene network studies.
TEM experiments were conducted by RG and analyzed data with JTH,
IAB, and JL. SH constructed the figures. JW and JQ analyzed the human
proteome high-throughput array data. LL assisted with the morphological studies and analyzed data. YS did the molecular modeling. DS, SZ,
SH, and PS wrote the paper. All authors have approved the final
manuscript.
RNA isolation, cDNA synthesis, and qRT–PCR
RNA isolation, cDNA synthesis, and qRT–PCR were performed as
described previously (Valapala et al., 2014a). PCR amplification was
performed using the 7500 PCR Fast Real-Time System (Applied
Biosystems, Carlsbad, CA, USA) and custom-made TaqMan probes for
LAMP1 (Mm00495262_m1), LAMP2A (Mm00495274_m1), cathepsin B
(Mm01310506_m1), cathepsin D (Mm00515586_m1), V-ATPase (Mm0-
ª 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.
PAT4 regulates the amino acid pool in RPE, P. Shang et al. 359
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Supporting Information
Additional Supporting Information may be found online in the supporting
information tab for this article.
Table S1 Heatmap of Z-scores of 78 protein hits identified for all triplicates
(Rep1/2/3) from 14 693 human proteins on the microarray. The hits were
sorted by their mean value of Z-scores.
ª 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.