A neurodegenerative disease mutation that accelerates
the clearance of apoptotic cells
Aimee W. Kaoa,b, Robin J. Eisenhuta, Lauren Herl Martensc, Ayumi Nakamuraa, Anne Huanga, Josh A. Bagleya,
Ping Zhouc, Alberto de Luisd, Lukas J. Neukomme,1, Juan Cabellod, Robert V. Farese, Jr.a,c,f, and Cynthia Kenyona,2
Departments of aBiochemistry and Biophysics and bNeurology, University of California, San Francisco, CA 94158; cGladstone Institute of Cardiovascular
Disease, San Francisco, CA 94158; dCenter for Biomedical Research of La Rioja, 26006 Logrono, Spain; eInstitute of Molecular Life Sciences, University of Zurich,
CH8057 Zurich, Switzerland; and fDepartment of Internal Medicine, University of California, San Francisco, CA 94143
Frontotemporal lobar degeneration is a progressive neurodegenerative syndrome that is the second most common cause of earlyonset dementia. Mutations in the progranulin gene are a major
cause of familial frontotemporal lobar degeneration [Baker M, et al.
(2006) Nature 442:916–919 and Cruts M, et al. (2006) Nature
442:920–924]. Although progranulin is involved in wound healing,
inflammation, and tumor growth, its role in the nervous system and
the mechanism by which insufficient levels result in neurodegeneration are poorly understood [Eriksen and Mackenzie (2008) J Neurochem 104:287–297]. We have characterized the normal function of
progranulin in the nematode Caenorhabditis elegans. We found
that mutants lacking pgrn-1 appear grossly normal, but exhibit
fewer apoptotic cell corpses during development. This reduction
in corpse number is not caused by reduced apoptosis, but instead
by more rapid clearance of dying cells. Likewise, we found that
macrophages cultured from progranulin KO mice displayed enhanced rates of apoptotic-cell phagocytosis. Although most neurodegenerative diseases are thought to be caused by the toxic effects
of aggregated proteins, our findings suggest that susceptibility to
neurodegeneration may be increased by a change in the kinetics of
programmed cell death. We propose that cells that might otherwise
recover from damage or injury are destroyed in progranulin
mutants, which in turn facilitates disease progression.
Alzheimer’s disease
| apoptosis
F
rontotemporal lobar degeneration (FTLD) comprises a group
of neurodegenerative syndromes that are characterized by behavioral changes, progressive aphasia, and/or semantic language
deficits (1). Pathologically, the syndromes are characterized by
frontal and temporal lobe atrophy and by intraneuronal deposition
of aggregated tau, TDP-43, or FUS proteins (2–4). Several genetic
mutations have been associated with the development of FTLD,
including mutations affecting MAPT, VCP, CHMP2B, FUS, and
progranulin (2, 5–11).
Progranulin mutations are present in 5% to 10% of all patients
with FTLD (11, 12). Unlike mutations affecting tau, APP, αsynuclein, and huntingtin, which result in decreased protein solubility and formation of toxic oligomers (13), FTLD-associated
progranulin mutations are not gain-of-function mutations, and
progranulin itself is not found in neuronal inclusions. Instead,
disease-associated progranulin mutations result in nonsensemediated decay of progranulin mRNA or an inability to secrete
the mutant protein (14). Individuals with these mutations exhibit
less than half the normal blood and cerebrospinal fluid levels of
progranulin (15–17). As the disease phenotype is inherited in an
autosomal-dominant fashion, progranulin mutations appear to
result in haploinsufficiency. In addition, variations in the human
progranulin gene may also increase the risk for developing ALS,
Alzheimer’s disease, and Parkinson disease (18–21).
Progranulin (also known as GRN, acrogranin, proepithelin,
granulin–epithelin precursor, and PC cell-derived growth factor)
is a highly conserved glycoprotein involved in a variety of biological processes, including embryogenesis, inflammation, and
www.pnas.org/cgi/doi/10.1073/pnas.1100650108
wound healing (22). Increased progranulin secretion is associated with more aggressive forms of breast, brain, renal, and other
tumors (22). Progranulin is secreted by many cell types including
neurons and glia, and can be cleaved into 6-kDa cysteine-rich
granulins whose functions are unclear, although they may oppose
the activity of the proprotein (23, 24). In cultured neurons and
neuronal cell lines, progranulin knockdown can adversely affect
neurite outgrowth and long-term neuronal survival under serumdeprived conditions (25, 26).
The toxic insults to neurons that ultimately result in neuronal
death and neurodegeneration in humans are generally thought to
occur in a cell-autonomous fashion, although evidence is accumulating that microglia, the resident phagocytic and immune effector cells of the CNS, may be involved in neuronal killing (27).
Here, by using Caenorhabditis elegans and ex vivo macrophage
studies, we describe an unexpected role for progranulin. We find
that loss of progranulin accelerates the clearance of apoptotic cells
in both worms and mammals. Others have shown that the apoptotic cell-clearance machinery can remove cells that are dying but
not yet dead, raising the possibility that loss of progranulin causes
damaged neurons to be engulfed before they can be repaired.
Results
C. elegans PGRN-1 Protein Is Likely Secreted from Neurons and
Intestine. Many human disease proteins have conserved func-
tions in invertebrates. Because of this, and because of the many
molecular, genetic, and cell biological approaches available in
C. elegans, we chose to study the function of progranulin in the
nematode. The C. elegans progranulin gene (pgrn-1) encodes
a protein with three predicted granulin domains, compared with
the 7.5 granulin domains in human progranulin (Fig. S1). We
generated a transgenic C. elegans line expressing a fluorescent
mCherry protein that acts as a transcriptional reporter for pgrn-1
expression. In developing C. elegans larvae, mCherry protein was
present in intestinal cells and select neurons (Fig. 1 A–F and Fig.
S2) and was excluded from muscle cells in a pattern reminiscent
of human progranulin expression (23). We tracked the timing of
progranulin expression throughout development. C. elegans
embryos are laid as eggs, hatch, and proceed through four larval
stages to adulthood. The total time from embryo to adulthood
takes approximately 3 d. The mCherry transcriptional reporter
was first visible in the embryonic intestinal precursor cells at
Author contributions: A.W.K., R.V.F., and C.K. designed research; A.W.K., R.J.E., L.H.M.,
A.N., A.H., J.A.B., A.d.L., L.J.N., and J.C. performed research; A.W.K., P.Z., and L.H.M.
contributed new reagents/analytic tools; A.W.K., R.J.E., L.J.N., J.C., and C.K. analyzed data;
and A.W.K. and C.K. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
1
Present address: Department of Neurobiology, Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, MA 01605.
2
To whom correspondence should be addressed. E-mail: [email protected].
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1100650108/-/DCSupplemental.
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Contributed by Cynthia Kenyon, February 1, 2011 (sent for review November 26, 2010)
We also generated a translational reporter with an RFP fluorescent tag fused to PGRN-1. In contrast to the transcriptional
reporter, this PGRN-1::RFP fusion protein was not found in
discrete tissues. Instead, it was visible in what appeared to be
intestinal organelles, in the pseudocoelomic cavity, and in scavenging coelomocytes, suggesting that, as in mammals (14), progranulin is secreted (Fig. 1 G–J and Fig. S3 C–F).
Progranulin Loss-of-Function Mutant Has a Normal Lifespan. The
pgrn-1(tm985) allele contains a 347-bp deletion encompassing
part of the progranulin promoter, all of exon 1, and part of the
first intron (Fig. S1). Animals carrying this mutation did not
produce progranulin mRNA as assayed by quantitative RT-PCR
(Fig. S4A). Thus, pgrn-1(tm985) is likely to be a null allele. pgrn-1
mutants appeared grossly normal, and both the homozygous and
heterozygous mutants exhibited a normal lifespan (Fig. S4B and
Tables S1 and S2). Likewise, overexpression of progranulin did
not significantly alter lifespan (Fig. S4B and Table S3). However,
the homozygous mutant had approximately 20% reduction in
total number of progeny (Fig. S4C).
Fig. 1. Progranulin expression in the intestine and neurons begins during
development. Light (Left) and fluorescent (Right) images of animals expressing a fluorescent mCherry transcriptional reporter (Ppgrn-1::pgrn-1::polycistronic mCherry) (A–F) or a translational RFP reporter (Ppgrn-1::pgrn-1::RFP)
(G–J). (A and B) Confocal images of representative mixed-stage embryos
demonstrate that progranulin expression begins at approximately 180 min
after the first cleavage event in embryogenesis (at approximately the time
programmed cell death begins). Left embryo is at approximately 150 min of
development and displays no fluorescent signal. Center embryo is at approximately 180 min of development, during early to mid-gastrulation when intestinal precursor cells are seen clearly in the interior of the embryo. These four
intestinal precursor cells contain the mCherry fluorescent protein (dashed
line). Right: Comma-stage embryo (∼390 min) demonstrates strong mCherry
expression (See also Fig. S3 A and B). (C–F) Nomarski and fluorescent micrographs show a representative late larval (L4) animal producing PGRN-1 in the
intestine (C and D) and select neurons (E and F). (G–J) Nomarski and fluorescent micrographs of a comma-stage animal (G and H) and day 1 adult animal
(I and J) expressing a PGRN-1::RFP translational reporter show the protein
diffusing throughout the animal. Arrowheads mark a refractile corpse (G and
H) and secreted PGRN-1::RFP taken up by coelomocytes (I and J). (Scale bars: A,
B, E–J, 10 μm; C and D, 100 μm.)
midgastrulation stage approximately 180 min after the first embryonic cleavage (Fig. 1 A and B and Fig. S3 A and B). We
observed neuronal mCherry expression in L1 larvae, and neuronal and intestinal mCherry expression persisted throughout
larval development and into adulthood (Fig. 1 C–F).
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Programmed Cell Death Is Altered in pgrn-1 Mutants. C. elegans
development is characterized by an invariant pattern of cell division and cell death (28). The initiation of PGRN-1 production
and secretion coincided temporally with the first programmed
cell deaths in C. elegans (Fig. 1 A and B). Because of this temporal link between PGRN-1 production and the onset of programmed cell death, and because progranulin haploinsufficiency
has been linked to human neurodegenerative disease, we investigated whether pgrn-1 mutants exhibited defective regulation
of programmed cell death. In developing nematodes and the
adult nematode germ line, cells dying via the process of apoptosis can be visualized by Nomarski microscopy as refractile
bodies or “corpses.” We counted apoptotic corpses in embryos at
several developmental stages and found significantly fewer apoptotic bodies in pgrn-1 mutants compared with control animals
(Fig. 2A). To verify that the cell death defect was a result of
progranulin deficiency, we reintroduced the C. elegans progranulin gene and observed partial rescue of the mutant phenotype (Fig. S4D). We speculate that the incomplete rescue may
be because expression of pgrn-1 from a transgene does not fully
mimic the expression of the endogenous gene. In addition to cell
deaths that occur as part of the developmental program, apoptosis
can be triggered in the germ line by UV irradiation. We found that
pgrn-1 mutations did not affect UV-induced germ cell death
(Fig. 2B).
Reduced numbers of apoptotic bodies in embryos could be
caused by a failure of cells to die or by an increase in the rate of
apoptotic cell clearance. To distinguish these possibilities, we
used fluorescent markers that label the sisters of apoptotic cells
that survive. These fluorescent markers are known to label
“undead” cells in ced-3 caspase mutants in which cell death is
blocked. If we saw “extra” cells, this would suggest that some
cells normally programmed to die did not. First, we used an
ODR-1::RFP reporter that normally is present in AWB, AWC,
I1, and ASI neurons (29). I1 and ASI have sister cells that die
(28). The same number of ODR-1::RFP fluorescent cell bodies
were observed in pgrn-1 mutants and controls, suggesting that
cell death occurs normally in the I1 and ASI lineage (Fig. 2C).
We also used a LIN-11::GFP reporter which is seen in approximately five extra ventral cord motor neurons in strong ced-3(lf)
mutants (30). Once again, pgrn-1 mutants had similar numbers of
LIN-11::GFP fluorescent cell bodies compared with controls
(Fig. 2D). Finally, we looked for extra cells in the anterior
pharynx, where, in strong ced-3(lf) mutants, approximately 12
extra cells are found (31). Once again, pgrn-1 mutants had the
same number of cells in the anterior pharynx as control worms
Kao et al.
How Might C. elegans Progranulin Influence the Kinetics of Programmed
Cell Death? We did not observe PGRN-1 production from dying
Fig. 2. Loss of pgrn-1 causes a defect in embryonic programmed cell death.
(A) Refractile bodies representing apoptotic corpses were counted at the
comma, 1.5-, and twofold stages in control and pgrn-1(tm985) embryos
(Student t test, **P < 0.01 and ***P < 0.001; n ≥ 35 embryos per condition).
(B) Apoptotic corpses in the germ line were quantified 24 h after UV irradiation (100 J/m2) and were not statistically different (Student t test, P = 0.7;
n ≥ 15 animals per strain). (C–E) To identify potential extra cells that evade
the normal program of cell death, we expressed fluorescent markers in cells
with sisters that die. (C) Fluorescent cell bodies were counted in day-1 adult
Podr-1::RFP or pgrn-1(tm985); Podr-1::RFP animals (n ≥ 18 animals per strain).
The numbers of fluorescent cells in these two strains were similar (Student t
test, P = 0.34). (D) Fluorescent cell bodies were counted in day-1 adult Plin-11::
GFP or pgrn-1(tm985); Plin-11::GFP animals (n ≥ 20 animals per strain). These
two strains did not exhibit a statistically significant difference in GFP pattern
(Student t test, P = 0.07). (E) The number of extra cells in the anterior
pharynx of late-larval control, pgrn-1(tm985), and ced-3(n2433) animals was
quantified (n ≥ 15 animals per strain). Control and pgrn-1(tm985) were not
significantly different from one another. Error bars in A represent SEM. Error
bars in B–E represent SD (n.s., not significant).
(Fig. 2E). Taken together, our inability to find extra cells suggests
that cells that normally die still do so in pgrn-1 mutants.
Loss of pgrn-1 Accelerates the Clearance of Dying Cells. Apoptotic
corpses are visible from the time they are generated until they
are engulfed and degraded by neighboring cells. We next tested
whether the reduced cell corpse number in pgrn-1 embryos was
caused by a change in the kinetics of apoptosis: either delayed
initiation of cell death or faster engulfment and clearance of the
dying cell. To do this, we used four-dimensional Nomarski timelapse video microscopy to follow the first 13 programmed cell
deaths in the AB cell lineage (32). By using this technique, we
found striking alterations in the kinetics of cell death (Fig. 3).
The time from which a cell is born until it begins to show morphological characteristics of apoptosis (i.e., rounding of the cell,
Kao et al.
cells or their engulfing sister cells during the embryonic stage in
which this altered progression of cell death and clearance took
place (Fig. S3 A and B). In fact, the only cells that produced
PGRN-1 at this time were intestinal precursor cells. As progranulin is secreted (Fig. 1 G–J and Fig. S3 C–F), it is likely to act
cell-non autonomously—on dying cells, engulfing cells, or both—
to delay the clearance of apoptotic cells.
Studies in C. elegans have established two partially redundant
pathways in engulfing cells that converge on the small GTPase
CED-10 to promote phagocytosis of apoptotic cells. One pathway contains CED-1, 6, and 7 and DYN-1 and the other contains
CED-2, 5, and 12, MIG-2, and UNC-73 (33). Mutations affecting these pathways slow the clearance of apoptotic cells by
neighboring engulfing cells. To determine if progranulin was
acting through these engulfment pathways, we generated double
mutants between pgrn-1 and cell-engulfment mutants. As loss-offunction mutations in pgrn-1 accelerate corpse clearance, we
would expect that the number of corpses observed in double
mutants to be decreased if pgrn-1 were acting independently of
an engulfment pathway. This was seen with a mutation in abl-1,
which also accelerates corpse clearance (34). In engulfment
mutants, uncleared corpses from cells that die during embryogenesis are visible in newly hatched L1 worms. We counted
corpses in these newly hatched L1 heads and found that loss of
progranulin did not affect the number of corpses (Table S5).
Because loss of either of these partially redundant engulfment
pathways abrogated the effect of pgrn-1, WT progranulin
appears to require fully functional engulfment machinery to
exert its effects on the rate of apoptotic cell clearance.
Macrophages from Progranulin KO Mice Display Enhanced Phagocytosis. Many biological processes discovered in C. elegans are
conserved in mammals. To determine whether this might be the
case with progranulin’s effect on the kinetics of apoptotic cell
engulfment, we asked whether peritoneal macrophages from
progranulin-deficient animals differed from WT in their rates of
phagocytosis. Macrophages are involved in the clearance of apoptotic cells (35), express progranulin (36), and can cross the
blood–brain barrier to become indistinguishable from resident
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cytoplasmic condensation, nuclear swelling) is referred to as the
“time to die.” The time from which initial morphological signs of
apoptosis are seen until the time at which the dying cell is
completely eliminated and disappears is referred to as the “time
to clearance” (Fig. 3A). We observed a striking change in the
mean time to clearance, which was decreased by nearly half in
pgrn-1 mutants (harmonic mean of 5.7 min in pgrn-1 mutants vs.
10.7 min in WT; P < 0.0036; Fig. 3B and Table S4) This indicates
that, when the cell death program has been initiated, corpse
clearance is accelerated in pgrn-1 mutants.
We also measured the time to die in WT and pgrn-1 mutant
animals. In WT animals, the time to die for apoptotic cells is
distributed in a Gaussian manner. In pgrn-1 mutants, the distribution was skewed and no longer Gaussian (P < 0.0001).
However, the mean time to die of the two strains was not
significantly different (harmonic mean: control, 20.2 min vs.
pgrn-1 mutants, 21.7 min; Fig. 3 C and D and Table S4). Many
mutations have been described that change programmed cell
death specification or slow the process of cell corpse engulfment; however, this is a mutation that accelerates the process
of cell corpse clearance (i.e., time to clearance) and narrows
the time window of cell death initiation (i.e., time to die). Although the effect of narrowed initiation time on number of
apoptotic corpses is unclear, more rapid clearance can explain
the reduced apoptotic cell corpse numbers observed during
embryonic development.
Fig. 3. Loss of progranulin accelerates the clearance of apoptotic cells in C. elegans. With the use of four-dimensional
Nomarski time-lapse video microscopy, the time to clearance
and time to die were determined for early deaths in the AB
lineage in control animals and pgrn-1(tm985) mutants (n = 3
embryos per strain). (A) Diagram depicting approximate
relative timing of events in time to die and time to clearance.
(B) Results for time to clearance are shown in a scatterplot in
which each circle represents a single cell-death observation.
Harmonic means were 10.7 min for control and 5.7 min for
pgrn-1–mutant animals (Mann–Whitney t test, P = 0.0036).
(C) Results for time to die are depicted in a scatterplot in
which each circle represents a single cell-death observation.
Mean times to die were 20.2 min for control animals and 21.7
min for pgrn-1 mutants (harmonic means, Mann–Whitney
t test, P = 0.3). (D) Frequency distribution histogram for time
to die. The distribution of deaths in control animals was
Gaussian, whereas in pgrn-1 mutants it was not (D’Agostino–
Pearson omnibus normality test, P = 0.05 and P < 0.0001,
respectively).
microglia, the phagocytic cells of the CNS (37). We cultured
peritoneal macrophages from WT (i.e., +/+) and progranulinKO littermates in heat-inactivated serum for several days and
then exposed them to one of several targets. First, we exposed
them to FITC-labeled polystyrene beads. We found that, compared with WT macrophages, progranulin-KO macrophages
engulfed latex beads more rapidly (Fig. 4A). We then tested
substrates likely internalized via receptor-mediated endocytosis,
yeast cell wall β-glucan particles, and, most significantly, apoptotic thymocytes. Compared with Pgrn+/+ controls, Pgrn−/−
macrophages had increased levels of phagocytosis of yeast cell
wall particles and apoptotic thymocytes (Fig. 4 B–D). Together,
these data suggest that, as in C. elegans, loss of progranulin
influences phagocytosis by macrophages, including engulfment
of apoptotic cells.
Discussion
We have identified a function for progranulin in modulating the
kinetics of programmed cell death that may be relevant for
neuron loss in neurodegenerative diseases. We showed that lack
of progranulin caused apoptotic cells to be cleared more rapidly
in C. elegans. This type of change in the kinetics of programmed
cell death would have been difficult to observe in mammals.
However, we have extended this finding to mouse macrophages,
which also exhibit enhanced engulfment of latex beads, yeast cell
wall, and, importantly, apoptotic cells. A defect in programmed
cell death kinetics could gradually contribute to neuronal loss
over the lifetime of human progranulin-mutation carriers. A
neurodegenerative phenotype was not observed in C. elegans, but
this may be because, unlike vertebrates, adult worms do not
exhibit programmed cell death in somatic tissues.
Fig. 4. Loss of progranulin accelerates
phagocytosis in mouse macrophages.
Peritoneal macrophages were cultured
from Pgrn+/+ and Pgrn−/− animals and
incubated with FITC-labeled polystyrene
beads (A), yeast cell-wall particles (zymosan) (B), or pHrodo-labeled apoptotic
thymocytes (C and D) in the presence of
10% heat-inactivated FBS. (A) The average number of beads engulfed by each
macrophage after 90 min incubation was
determined (n ≥ 100 cells per condition).
Error bars represent SD (Student t test,
***P < 0.0001). (B) Average increase in
zymosan uptake by colorimetric assay
after 90 min of incubation. Error bars
represent SEM of two independent
experiments (Student t test, *P < 0.05).
(C and D) The average number of apoptotic thymocytes engulfed by each
macrophage after 90 min of incubation
was determined. Representative fields
are shown in C (with pHrodo-labeled apoptotic thymocytes in red and CD-11b-FITC–labeled macrophage membranes in green) and average counts shown in D
(n ≥ 100 cells per condition). Error bars represent SEM (Student t test, ***P < 0.0001).
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Kao et al.
addition, our findings suggest an additional mechanism by which
progranulin overexpression by tumor cells may promote tumor
growth; namely, by inhibiting the activity of surveillance phagocytes and promoting tumor immune evasion.
Although this model to explain the effect of progranulin deficiency on development of FTLD remains speculative, several
groups have recently implicated abnormal microglial activity in
FTLD and related diseases. While our studies were in progress,
progranulin-deficient mice were shown to exhibit an exaggerated
inflammatory response after infection (43) as well as an agedependent increased accumulation and activation of microglia
(43–45). Our findings with cultured macrophages would be
consistent with this type of enhanced microglial activity. Moreover, they suggest that progranulin loss alters the activity of
engulfing cells even in the absence of progranulin-deficient
neurons. Enhanced microglial action has also been observed in
a mouse model of Alzheimer’s disease. Here, microglia were
shown to increase in number and migration velocity around
neurons just before their elimination (46). Finally, in a mouse
model of ALS, a neurodegenerative disorder linked to FTLD
by overlapping genetic and pathological changes, expression of
mutant SOD1 protein in microglia and not neurons is the
major determinant of disease progression (47, 48). These and
other studies implicate microglial function in the pathogenesis
of neurodegenerative disease. As loss of progranulin changes
macrophage engulfment characteristics, and reduced progranulin is linked to FTLD, our findings suggest a potentially
new mechanism by which phagocytic cells may contribute to
neuron loss and neurodegeneration. Nonetheless, the direct
effect of reduced progranulin on microglia in vivo remains to
be demonstrated in mouse models of progranulin deficiency.
As polymorphisms in the human progranulin gene are associated with increased risk of ALS, Alzheimer’s disease, and
Parkinson disease (18–21), our findings here could have
implications on the development of neurodegenerative disease
more generally. A change in the regulation of programmed
cell death kinetics represents an attractive and potentially
important hypothesis to explain how mutations in the progranulin gene may result in neurodegeneration and suggests
future avenues for investigation in the pathogenesis of neurodegenerative diseases.
Materials and Methods
Strains. C. elegans were cultured at 20 °C according to standard procedures
(49). The pgrn-1(tm985) strain was provided by the Mitani Laboratory at the
Fig. 5. Speculative model for
how decreased progranulin levels may result in premature cell
death. In normal animals, a cell
experiencing a sublethal insult
has sufficient time to recover
because progranulin slows the
rate of engulfment. In an animal
with insufficient progranulin,
a cell experiencing a sublethal
insult has inadequate time to
repair itself before engulfment
by phagocytic cells. In this situation, the balance of events may
tip toward increased cell loss after what would otherwise be
a nonlethal insult.
Kao et al.
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Apoptosis was once thought to be an irreversible process, such
that, when programmed cell death had been initiated, death of
a cell was inevitable. Recent studies have found, however, that
even after a cell initiates the program of cell death, the process
can be reversed (30, 32, 38, 39). Additionally, the process of
apoptosis is not entirely cell autonomous, as engulfing cells actively promote cell death (30, 32). Because lack of progranulin in
C. elegans causes apoptotic cells to be cleared more quickly than
if progranulin is present, one normal function of progranulin
appears to be to regulate the rate of cell clearance. Others have
shown that expression of human progranulin in an immortalized
mouse neuronal cell line can decrease the percentage of cells
undergoing apoptosis after serum deprivation even in the absence of any “engulfing” cells (26). Our results with the use of
cultured primary macrophages now also implicate the engulfing
cell in regulating the kinetics of cell engulfment and death in
response to progranulin. It is not clear how progranulin acts
biochemically to influence the rate of cell engulfment. Our
studies in worms suggest that progranulin acts through the normal cell engulfment machinery to affect the rate of apoptotic cell
clearance, as the slower engulfment that occurs in the absence of
this machinery is not affected by loss of progranulin. In mammals, progranulin can activate specific kinases such as MAPK
and AKT, and it interacts with sortilin, a membrane protein that
can complex with pro-NGF and p75NTR to activate proapoptotic
signals (40–42). It would be interesting to ask, by using mutants,
whether progranulin acts through any of these proteins to influence the rate of cell engulfment.
Based on these findings, we propose a model in which inadequate progranulin levels may promote neurodegeneration by
disrupting the kinetics of programmed cell death (Fig. 5). Under
conditions in which WT levels of progranulin are produced,
a cell that undergoes a sublethal insult has adequate time to
repair itself and survive. If insufficient progranulin is present, the
rate at which an injured cell is recognized and/or engulfed by
phagocytic cells is accelerated, and the damaged cell has less
time to recover. Such changes in the kinetics of cell death could
cause a shift in the dynamic equilibrium toward survival or death
of individual cells that could ultimately result in cumulative
neuronal loss. Likewise, precocious clearance could conceivably
prevent cells damaged by toxic neurodegenerative proteins from
recovering. This model could help explain how identical mutations within a family can manifest in disease with differing phenotypes and ages of onset, as extent and location of neuronal
injury could vary dramatically from individual to individual. In
Assays. Somatic cell and germ line corpses were visualized directly on a Zeiss
Axioplan 2 microscope fitted with Nomarski optics as described previously (28).
C. elegans embryos, larvae, and adults were mounted on 2% agarose pads in
M9 with 2.5 mM levamisole. Apoptotic corpses were counted in a blinded
fashion. To measure the kinetics of cell corpse formation and clearance, fourdimensional time-lapse Nomarski microscopy was performed as described
previously (50). Results are shown in scatterplots and histogram form. SI
Materials and Methods provides additional details on phagocytosis assays.
ACKNOWLEDGMENTS. We thank the Caenorhabditis Genetics Center at the
University of Minnesota and the Mitani and Ashrafi laboratories for strains
and reagents. We thank Kaveh Ashrafi, Shai Shaham, Michael Hengartner,
Bruce Miller, Suneil Koliwad, Laura Mitic, Kenyon laboratory members, and
members of the Consortium for Frontotemporal Dementia Research (CFR)
for helpful advice. A.W.K. was supported by a Hillblom Foundation Postdoctoral Fellowship and by the CFR. L.H.M. and R.V.F. were supported by CFR
and by National Institutes of Health (NIH) Grant P50 AG023501 and NIH/
National Center for Research Resources Grant C06 RR018928 for animal facilities. A.d.L. and J.C. were supported by the Riojasalud Foundation and by
Spanish Ministry of Science and Innovation Grant BFU2010-21794. This work
was supported by NIH Grants K08 NS59604 (to A.W.K.) and R01 AG11816 (to
C.K.). C.K. is an American Cancer Society Professor and director of the University of California, San Francisco, Hillblom Foundation for the Biology
of Aging.
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