A Neurodegenerative Disease Mutation That Accelerates the Clearance of Apoptotic Cells

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

Contributed by Cynthia Kenyon, February 1, 2011 (sent for review November 26, 2010) Frontotemporal lobar degeneration is a progressive neurodegen- wound healing (22). Increased progranulin secretion is associ- erative syndrome that is the second most common cause of early- ated with more aggressive forms of breast, brain, renal, and other onset dementia. Mutations in the progranulin gene are a major tumors (22). Progranulin is secreted by many cell types including cause of familial frontotemporal lobar degeneration [Baker M, et al. neurons and glia, and can be cleaved into 6-kDa cysteine-rich (2006) Nature 442:916–919 and Cruts M, et al. (2006) Nature granulins whose functions are unclear, although they may oppose 442:920–924]. Although progranulin is involved in wound healing, the activity of the proprotein (23, 24). In cultured neurons and inﬂammation, and tumor growth, its role in the nervous system and neuronal cell lines, progranulin knockdown can adversely affect the mechanism by which insufﬁcient levels result in neurodegener- neurite outgrowth and long-term neuronal survival under serum- ation are poorly understood [Eriksen and Mackenzie (2008) J Neuro- deprived conditions (25, 26). chem 104:287–297]. We have characterized the normal function of The toxic insults to neurons that ultimately result in neuronal progranulin in the nematode Caenorhabditis elegans. We found death and neurodegeneration in humans are generally thought to that mutants lacking pgrn-1 appear grossly normal, but exhibit occur in a cell-autonomous fashion, although evidence is accu- fewer apoptotic cell corpses during development. This reduction mulating that microglia, the resident phagocytic and immune ef- in corpse number is not caused by reduced apoptosis, but instead fector cells of the CNS, may be involved in neuronal killing (27).

by more rapid clearance of dying cells. Likewise, we found that Here, by using Caenorhabditis elegans and ex vivo macrophage NEUROSCIENCE macrophages cultured from progranulin KO mice displayed en- studies, we describe an unexpected role for progranulin. We find hanced rates of apoptotic-cell phagocytosis. Although most neuro- that loss of progranulin accelerates the clearance of apoptotic cells degenerative diseases are thought to be caused by the toxic effects in both worms and mammals. Others have shown that the apo- of aggregated proteins, our findings suggest that susceptibility to ptotic cell-clearance machinery can remove cells that are dying but neurodegeneration may be increased by a change in the kinetics of not yet dead, raising the possibility that loss of progranulin causes programmed cell death. We propose that cells that might otherwise damaged neurons to be engulfed before they can be repaired. recover from damage or injury are destroyed in progranulin mutants, which in turn facilitates disease progression. Results C. elegans PGRN-1 Protein Is Likely Secreted from Neurons and Alzheimer’s disease | apoptosis Intestine. Many human disease proteins have conserved functions in invertebrates. Because of this, and because of the many rontotemporal lobar degeneration (FTLD) comprises a group molecular, genetic, and cell biological approaches available in Fof neurodegenerative syndromes that are characterized by be- C. elegans, we chose to study the function of progranulin in the havioral changes, progressive aphasia, and/or semantic language nematode. The C. elegans progranulin gene (pgrn-1) encodes deficits (1). Pathologically, the syndromes are characterized by a protein with three predicted granulin domains, compared with frontal and temporal lobe atrophy and by intraneuronal deposition the 7.5 granulin domains in human progranulin (Fig. S1). We of aggregated tau, TDP-43, or FUS proteins (2–4). Several genetic generated a transgenic C. elegans line expressing a fluorescent mutations have been associated with the development of FTLD, mCherry protein that acts as a transcriptional reporter for pgrn-1 including mutations affecting MAPT, VCP, CHMP2B, FUS,and expression. In developing C. elegans larvae, mCherry protein was progranulin (2, 5–11). present in intestinal cells and select neurons (Fig. 1 A–F and Fig. Progranulin mutations are present in 5% to 10% of all patients S2) and was excluded from muscle cells in a pattern reminiscent with FTLD (11, 12). Unlike mutations affecting tau, APP, α- of human progranulin expression (23). We tracked the timing of synuclein, and huntingtin, which result in decreased protein sol- progranulin expression throughout development. C. elegans ubility and formation of toxic oligomers (13), FTLD-associated embryos are laid as eggs, hatch, and proceed through four larval progranulin mutations are not gain-of-function mutations, and stages to adulthood. The total time from embryo to adulthood progranulin itself is not found in neuronal inclusions. Instead, takes approximately 3 d. The mCherry transcriptional reporter fi disease-associated progranulin mutations result in nonsense- was rst visible in the embryonic intestinal precursor cells at mediated 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 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. progranulin (15 17). As the disease phenotype is inherited in an contributed new reagents/analytic tools; A.W.K., R.J.E., L.J.N., J.C., and C.K. analyzed data; autosomal-dominant fashion, progranulin mutations appear to and A.W.K. and C.K. wrote the paper. result in haploinsufficiency. In addition, variations in the human The authors declare no conflict of interest. progranulin gene may also increase the risk for developing ALS, Freely available online through the PNAS open access option. ’ – Alzheimer s disease, and Parkinson disease (18 21). 1Present address: Department of Neurobiology, Howard Hughes Medical Institute, Uni- Progranulin (also known as GRN, acrogranin, proepithelin, versity of Massachusetts Medical School, Worcester, MA 01605. granulin–epithelin precursor, and PC cell-derived growth factor) 2To whom correspondence should be addressed. E-mail: [email protected]. is a highly conserved glycoprotein involved in a variety of bi- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ological processes, including embryogenesis, inflammation, and 1073/pnas.1100650108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1100650108 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 We also generated a translational reporter with an RFP ﬂuo- rescent 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 scav- enging 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).

Programmed Cell Death Is Altered in pgrn-1 Mutants. C. elegans development is characterized by an invariant pattern of cell di- vision 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 in- vestigated 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 Fig. 1. Progranulin expression in the intestine and neurons begins during mimic the expression of the endogenous gene. In addition to cell development. Light (Left) and fluorescent (Right) images of animals express- deaths that occur as part of the developmental program, apoptosis fl ing a uorescent mCherry transcriptional reporter (Ppgrn-1::pgrn-1::poly- can be triggered in the germ line by UV irradiation. We found that cistronic mCherry)(A–F) or a translational RFP reporter (Ppgrn-1::pgrn-1::RFP) – pgrn-1 mutations did not affect UV-induced germ cell death (G J). (A and B) Confocal images of representative mixed-stage embryos (Fig. 2B). demonstrate that progranulin expression begins at approximately 180 min after the first cleavage event in embryogenesis (at approximately the time Reduced numbers of apoptotic bodies in embryos could be programmed cell death begins). Left embryo is at approximately 150 min of caused by a failure of cells to die or by an increase in the rate of development and displays no fluorescent signal. Center embryo is at approx- apoptotic cell clearance. To distinguish these possibilities, we imately 180 min of development, during early to mid-gastrulation when in- used fluorescent markers that label the sisters of apoptotic cells testinal precursor cells are seen clearly in the interior of the embryo. These four that survive. These fluorescent markers are known to label intestinal precursor cells contain the mCherry fluorescent protein (dashed “undead” cells in ced-3 caspase mutants in which cell death is ∼ line). Right: Comma-stage embryo ( 390 min) demonstrates strong mCherry blocked. If we saw “extra” cells, this would suggest that some 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 cells normally programmed to die did not. First, we used an intestine (C and D) and select neurons (E and F). (G–J) Nomarski and fluores- ODR-1::RFP reporter that normally is present in AWB, AWC, cent micrographs of a comma-stage animal (G and H) and day 1 adult animal I1, and ASI neurons (29). I1 and ASI have sister cells that die (I and J) expressing a PGRN-1::RFP translational reporter show the protein (28). The same number of ODR-1::RFP fluorescent cell bodies diffusing throughout the animal. Arrowheads mark a refractile corpse (G and were observed in pgrn-1 mutants and controls, suggesting that H) and secreted PGRN-1::RFP taken up by coelomocytes (I and J). (Scale bars: A, cell death occurs normally in the I1 and ASI lineage (Fig. 2C). B, E–J,10μm; C and D, 100 μm.) 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 midgastrulation stage approximately 180 min after the first em- LIN-11::GFP fluorescent cell bodies compared with controls bryonic cleavage (Fig. 1 A and B and Fig. S3 A and B). We (Fig. 2D). Finally, we looked for extra cells in the anterior observed neuronal mCherry expression in L1 larvae, and neu- pharynx, where, in strong ced-3(lf) mutants, approximately 12 ronal and intestinal mCherry expression persisted throughout extra cells are found (31). Once again, pgrn-1 mutants had the larval development and into adulthood (Fig. 1 C–F). same number of cells in the anterior pharynx as control worms

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1100650108 Kao et al. Downloaded by guest on September 28, 2021 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 distributedinaGaussianmanner.Inpgrn-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). Al- though 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.

How Might C. elegans Progranulin Influence the Kinetics of Programmed NEUROSCIENCE Cell Death? We did not observe PGRN-1 production from dying 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 Fig. 2. Loss of pgrn-1 causes a defect in embryonic programmed cell death. cell-non autonomously—on dying cells, engulfing cells, or both— (A) Refractile bodies representing apoptotic corpses were counted at the to delay the clearance of apoptotic cells. comma, 1.5-, and twofold stages in control and pgrn-1(tm985) embryos < < ≥ Studies in C. elegans have established two partially redundant (Student t test, **P 0.01 and ***P 0.001; n 35 embryos per condition). pathways in engulfing cells that converge on the small GTPase (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; CED-10 to promote phagocytosis of apoptotic cells. One path- n ≥ 15 animals per strain). (C–E) To identify potential extra cells that evade way contains CED-1, 6, and 7 and DYN-1 and the other contains the normal program of cell death, we expressed fluorescent markers in cells CED-2, 5, and 12, MIG-2, and UNC-73 (33). Mutations affect- with sisters that die. (C) Fluorescent cell bodies were counted in day-1 adult ing these pathways slow the clearance of apoptotic cells by ≥ Podr-1::RFP or pgrn-1(tm985); Podr-1::RFP animals (n 18 animals per strain). neighboring engulfing cells. To determine if progranulin was The numbers of fluorescent cells in these two strains were similar (Student t acting through these engulfment pathways, we generated double test, P = 0.34). (D) Fluorescent cell bodies were counted in day-1 adult Plin-11:: mutants between pgrn-1 and cell-engulfment mutants. As loss-of- GFP or pgrn-1(tm985); Plin-11::GFP animals (n ≥ 20 animals per strain). These fi function mutations in pgrn-1 accelerate corpse clearance, we two strains did not exhibit a statistically signi cant difference in GFP pattern would expect that the number of corpses observed in double (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 mutants to be decreased if pgrn-1 were acting independently of quantified (n ≥ 15 animals per strain). Control and pgrn-1(tm985) were not an engulfment pathway. This was seen with a mutation in abl-1, significantly different from one another. Error bars in A represent SEM. Error which also accelerates corpse clearance (34). In engulfment bars in B–E represent SD (n.s., not significant). 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 (Fig. 2E). Taken together, our inability to find extra cells suggests progranulin did not affect the number of corpses (Table S5). that cells that normally die still do so in pgrn-1 mutants. Because loss of either of these partially redundant engulfment pathways abrogated the effect of pgrn-1, WT progranulin Loss of pgrn-1 Accelerates the Clearance of Dying Cells. Apoptotic appears to require fully functional engulfment machinery to corpses are visible from the time they are generated until they exert its effects on the rate of apoptotic cell clearance. are engulfed and degraded by neighboring cells. We next tested whether the reduced cell corpse number in pgrn-1 embryos was Macrophages from Progranulin KO Mice Display Enhanced Phago- caused by a change in the kinetics of apoptosis: either delayed cytosis. Many biological processes discovered in C. elegans are initiation of cell death or faster engulfment and clearance of the conserved in mammals. To determine whether this might be the dying cell. To do this, we used four-dimensional Nomarski time- case with progranulin’s effect on the kinetics of apoptotic cell lapse video microscopy to follow the first 13 programmed cell engulfment, we asked whether peritoneal macrophages from deaths in the AB cell lineage (32). By using this technique, we progranulin-deficient animals differed from WT in their rates of found striking alterations in the kinetics of cell death (Fig. 3). phagocytosis. Macrophages are involved in the clearance of ap- The time from which a cell is born until it begins to show mor- optotic cells (35), express progranulin (36), and can cross the phological characteristics of apoptosis (i.e., rounding of the cell, blood–brain barrier to become indistinguishable from resident

Kao et al. PNAS Early Edition | 3of6 Downloaded by guest on September 28, 2021 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 Discussion +/+ peritoneal macrophages from WT (i.e., ) and progranulin- We have identified a function for progranulin in modulating the KO littermates in heat-inactivated serum for several days and kinetics of programmed cell death that may be relevant for then exposed them to one of several targets. First, we exposed neuron loss in neurodegenerative diseases. We showed that lack them to FITC-labeled polystyrene beads. We found that, com- of progranulin caused apoptotic cells to be cleared more rapidly pared with WT macrophages, progranulin-KO macrophages in C. elegans. This type of change in the kinetics of programmed engulfed latex beads more rapidly (Fig. 4A). We then tested cell death would have been difficult to observe in mammals. substrates likely internalized via receptor-mediated endocytosis, However, we have extended this finding to mouse macrophages, yeast cell wall β-glucan particles, and, most significantly, apo- which also exhibit enhanced engulfment of latex beads, yeast cell − − ptotic thymocytes. Compared with Pgrn+/+ controls, Pgrn / wall, and, importantly, apoptotic cells. A defect in programmed macrophages had increased levels of phagocytosis of yeast cell cell death kinetics could gradually contribute to neuronal loss wall particles and apoptotic thymocytes (Fig. 4 B–D). Together, over the lifetime of human progranulin-mutation carriers. A these data suggest that, as in C. elegans, loss of progranulin neurodegenerative phenotype was not observed in C. elegans, but influences phagocytosis by macrophages, including engulfment this may be because, unlike vertebrates, adult worms do not of apoptotic cells. 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).

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1100650108 Kao et al. Downloaded by guest on September 28, 2021 Apoptosis was once thought to be an irreversible process, such addition, our findings suggest an additional mechanism by which that, when programmed cell death had been initiated, death of progranulin overexpression by tumor cells may promote tumor a cell was inevitable. Recent studies have found, however, that growth; namely, by inhibiting the activity of surveillance phag- even after a cell initiates the program of cell death, the process ocytes and promoting tumor immune evasion. can be reversed (30, 32, 38, 39). Additionally, the process of Although this model to explain the effect of progranulin de- apoptosis is not entirely cell autonomous, as engulfing cells ac- ficiency on development of FTLD remains speculative, several tively promote cell death (30, 32). Because lack of progranulin in groups have recently implicated abnormal microglial activity in C. elegans causes apoptotic cells to be cleared more quickly than FTLD and related diseases. While our studies were in progress, if progranulin is present, one normal function of progranulin progranulin-deficient mice were shown to exhibit an exaggerated appears to be to regulate the rate of cell clearance. Others have inflammatory response after infection (43) as well as an age- shown that expression of human progranulin in an immortalized dependent increased accumulation and activation of microglia mouse neuronal cell line can decrease the percentage of cells (43–45). Our findings with cultured macrophages would be undergoing apoptosis after serum deprivation even in the ab- consistent with this type of enhanced microglial activity. More- sence of any “engulfing” cells (26). Our results with the use of over, they suggest that progranulin loss alters the activity of cultured primary macrophages now also implicate the engulfing engulfing cells even in the absence of progranulin-deficient cell in regulating the kinetics of cell engulfment and death in neurons. Enhanced microglial action has also been observed in response to progranulin. It is not clear how progranulin acts a mouse model of Alzheimer’s disease. Here, microglia were biochemically to influence the rate of cell engulfment. Our shown to increase in number and migration velocity around studies in worms suggest that progranulin acts through the nor- neurons just before their elimination (46). Finally, in a mouse mal cell engulfment machinery to affect the rate of apoptotic cell model of ALS, a neurodegenerative disorder linked to FTLD clearance, as the slower engulfment that occurs in the absence of by overlapping genetic and pathological changes, expression of this machinery is not affected by loss of progranulin. In mam- mutant SOD1 protein in microglia and not neurons is the mals, progranulin can activate specific kinases such as MAPK major determinant of disease progression (47, 48). These and and AKT, and it interacts with sortilin, a membrane protein that other studies implicate microglial function in the pathogenesis NTR can complex with pro-NGF and p75 to activate proapoptotic of neurodegenerative disease. Aslossofprogranulinchanges signals (40–42). It would be interesting to ask, by using mutants, macrophage engulfment characteristics, and reduced pro- whether progranulin acts through any of these proteins to in- granulin is linked to FTLD, our findings suggest a potentially fluence the rate of cell engulfment. new mechanism by which phagocytic cells may contribute to NEUROSCIENCE Based on these findings, we propose a model in which in- neuron loss and neurodegeneration. Nonetheless, the direct adequate progranulin levels may promote neurodegeneration by effect of reduced progranulin on microglia in vivo remains to disrupting the kinetics of programmed cell death (Fig. 5). Under be demonstrated in mouse models of progranulin deficiency. conditions in which WT levels of progranulin are produced, As polymorphisms in the human progranulin gene are asso- a cell that undergoes a sublethal insult has adequate time to ciated with increased risk of ALS, Alzheimer’s disease, and repair itself and survive. If insufficient progranulin is present, the Parkinson disease (18–21), our findings here could have rate at which an injured cell is recognized and/or engulfed by implications on the development of neurodegenerative disease phagocytic cells is accelerated, and the damaged cell has less more generally. A change in the regulation of programmed time to recover. Such changes in the kinetics of cell death could cell death kinetics represents an attractive and potentially cause a shift in the dynamic equilibrium toward survival or death important hypothesis to explain how mutations in the pro- of individual cells that could ultimately result in cumulative granulin gene may result in neurodegeneration and suggests neuronal loss. Likewise, precocious clearance could conceivably future avenues for investigation in the pathogenesis of neuro- prevent cells damaged by toxic neurodegenerative proteins from degenerative diseases. recovering. This model could help explain how identical mutations within a family can manifest in disease with differing phe- Materials and Methods notypes and ages of onset, as extent and location of neuronal Strains. C. elegans were cultured at 20 °C according to standard procedures injury could vary dramatically from individual to individual. In (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 situa- tion, the balance of events may tip toward increased cell loss after what would otherwise be a nonlethal insult.

Kao et al. PNAS Early Edition | 5of6 Downloaded by guest on September 28, 2021 Tokyo Women’s Medical University (Tokyo, Japan) and out-crossed four ACKNOWLEDGMENTS. We thank the Caenorhabditis Genetics Center at the times to our laboratory’s N2 strain. Other published strains were obtained University of Minnesota and the Mitani and Ashrafi laboratories for strains fi from the Caenorhabditis Genetics Center at the University of Minnesota. SI and reagents. We thank Kaveh Ashra , Shai Shaham, Michael Hengartner, Bruce Miller, Suneil Koliwad, Laura Mitic, Kenyon laboratory members, and Materials and Methods provides additional strain information. members of the Consortium for Frontotemporal Dementia Research (CFR) for helpful advice. A.W.K. was supported by a Hillblom Foundation Postdoc- Assays. Somatic cell and germ line corpses were visualized directly on a Zeiss toral Fellowship and by the CFR. L.H.M. and R.V.F. were supported by CFR Axioplan 2 microscope fitted with Nomarski optics as described previously (28). and by National Institutes of Health (NIH) Grant P50 AG023501 and NIH/ C. elegans embryos, larvae, and adults were mounted on 2% agarose pads in National Center for Research Resources Grant C06 RR018928 for animal fa- M9 with 2.5 mM levamisole. Apoptotic corpses were counted in a blinded cilities. 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 fashion. To measure the kinetics of cell corpse formation and clearance, four- was supported by NIH Grants K08 NS59604 (to A.W.K.) and R01 AG11816 (to dimensional time-lapse Nomarski microscopy was performed as described C.K.). C.K. is an American Cancer Society Professor and director of the Uni- previously (50). Results are shown in scatterplots and histogram form. SI versity of California, San Francisco, Hillblom Foundation for the Biology Materials and Methods provides additional details on phagocytosis assays. of Aging.

1. Neary D, et al. (1998) Frontotemporal lobar degeneration: A consensus on clinical 26. Ryan CL, et al. (2009) Progranulin is expressed within motor neurons and promotes diagnostic criteria. Neurology 51:1546–1554. neuronal cell survival. BMC Neurosci 10:130. 2. Hutton M, et al. (1998) Association of missense and 5′-splice-site mutations in tau with 27. Ilieva H, Polymenidou M, Cleveland DW (2009) Non-cell autonomous toxicity in the inherited dementia FTDP-17. Nature 393:702–705. neurodegenerative disorders: ALS and beyond. J Cell Biol 187:761–772. 3. Neumann M, et al. (2006) Ubiquitinated TDP-43 in frontotemporal lobar 28. Sulston JE, Schierenberg E, White JG, Thomson JN (1983) The embryonic cell lineage degeneration and amyotrophic lateral sclerosis. Science 314:130–133. of the nematode Caenorhabditis elegans. Dev Biol 100:64–119. 4. Mackenzie IR, et al. (2010) Nomenclature and nosology for neuropathologic subtypes 29. L’Etoile ND, Bargmann CI (2000) Olfaction and odor discrimination are mediated by of frontotemporal lobar degeneration: an update. Acta Neuropathol 119:1–4. the C. elegans guanylyl cyclase ODR-1. Neuron 25:575–586. 5. Watts GD, et al. (2004) Inclusion body myopathy associated with Paget disease of 30. Reddien PW, Cameron S, Horvitz HR (2001) Phagocytosis promotes programmed cell bone and frontotemporal dementia is caused by mutant valosin-containing protein. death in C. elegans. Nature 412:198–202. Nat Genet 36:377–381. 31. Shaham S, Reddien PW, Davies B, Horvitz HR (1999) Mutational analysis of the 6. Skibinski G, et al. (2005) Mutations in the endosomal ESCRTIII-complex subunit Caenorhabditis elegans cell-death gene ced-3. Genetics 153:1655–1671. CHMP2B in frontotemporal dementia. Nat Genet 37:806–808. 32. Hoeppner DJ, Hengartner MO, Schnabel R (2001) Engulfment genes cooperate with 7. Kwiatkowski TJ, Jr., et al. (2009) Mutations in the FUS/TLS gene on chromosome 16 ced-3 to promote cell death in Caenorhabditis elegans. Nature 412:202–206. cause familial amyotrophic lateral sclerosis. Science 323:1205–1208. 33. Kinchen JM, et al. (2005) Two pathways converge at CED-10 to mediate actin 8. Vance C, et al. (2009) Mutations in FUS, an RNA processing protein, cause familial rearrangement and corpse removal in C. elegans. Nature 434:93–99. amyotrophic lateral sclerosis type 6. Science 323:1208–1211. 34. Hurwitz ME, et al. (2009) Abl kinase inhibits the engulfment of apoptotic [corrected] 9. Baker M, et al. (2006) Mutations in progranulin cause tau-negative frontotemporal cells in Caenorhabditis elegans. PLoS Biol 7:e99. dementia linked to chromosome 17. Nature 442:916–919. 35. Hu B, Sonstein J, Christensen PJ, Punturieri A, Curtis JL (2000) Deficient in vitro and in 10. Cruts M, et al. (2006) Null mutations in progranulin cause ubiquitin-positive vivo phagocytosis of apoptotic T cells by resident murine alveolar macrophages. frontotemporal dementia linked to chromosome 17q21. Nature 442:920–924. J Immunol 165:2124–2133. 11. Gass J, et al. (2006) Mutations in progranulin are a major cause of ubiquitin-positive 36. He Z, Ong CH, Halper J, Bateman A (2003) Progranulin is a mediator of the wound frontotemporal lobar degeneration. Hum Mol Genet 15:2988–3001. response. Nat Med 9:225–229. 12. Le Ber I, et al.; French Research Network on FTD/FTD-MND (2007) Progranulin null muta- 37. Lucin KM, Wyss-Coray T (2009) Immune activation in brain aging and tions in both sporadic and familial frontotemporal dementia. Hum Mutat 28:846–855. neurodegeneration: too much or too little? Neuron 64:110–122. 13. Kayed R, et al. (2003) Common structure of soluble amyloid oligomers implies 38. Albeck JG, et al. (2008) Quantitative analysis of pathways controlling extrinsic common mechanism of pathogenesis. Science 300:486–489. apoptosis in single cells. Mol Cell 30:11–25. 14. Eriksen JL, Mackenzie IR (2008) Progranulin: Normal function and role in 39. Rehm M, Huber HJ, Dussmann H, Prehn JH (2006) Systems analysis of effector caspase neurodegeneration. J Neurochem 104:287–297. activation and its control by X-linked inhibitor of apoptosis protein. EMBO J 25: 15. Ghidoni R, Benussi L, Glionna M, Franzoni M, Binetti G (2008) Low plasma progranulin 4338–4349. levels predict progranulin mutations in frontotemporal lobar degeneration. 40. Zanocco-Marani T, et al. (1999) Biological activities and signaling pathways of the Neurology 71:1235–1239. granulin/epithelin precursor. Cancer Res 59:5331–5340. 16. Finch N, et al. (2009) Plasma progranulin levels predict progranulin mutation status in 41. Nykjaer A, et al. (2004) Sortilin is essential for proNGF-induced neuronal cell death. frontotemporal dementia patients and asymptomatic family members. Brain 132: Nature 427:843–848. 583–591. 42. Hu F, et al. (2010) Sortilin-mediated endocytosis determines levels of the 17. Sleegers K, et al. (2009) Serum biomarker for progranulin-associated frontotemporal frontotemporal dementia protein, progranulin. Neuron 68:654–667. lobar degeneration. Ann Neurol 65:603–609. 43. Yin F, et al. (2010) Exaggerated inflammation, impaired host defense, and 18. Brouwers N, et al. (2007) Alzheimer and Parkinson diagnoses in progranulin null neuropathology in progranulin-deficient mice. J Exp Med 207:117–128. mutation carriers in an extended founder family. Arch Neurol 64:1436–1446. 44. Ahmed Z, et al. (2010) Accelerated lipofuscinosis and ubiquitination in granulin 19. Brouwers N, et al. (2008) Genetic variability in progranulin contributes to risk for knockout mice suggests a role for progranulin in successful aging. Am J Pathol 177: clinically diagnosed Alzheimer disease. Neurology 71:656–664. 311–324. 20. Sleegers K, et al. (2008) Progranulin genetic variability contributes to amyotrophic 45. Yin F, et al. (2010) Behavioral deficits and progressive neuropathology in progranulin- lateral sclerosis. Neurology 71:253–259. deficient mice: A mouse model of frontotemporal dementia. FASEB J, 10.1096/fj.10- 21. Viswanathan J, et al. (2009) An association study between granulin gene 161471. polymorphisms and Alzheimer’s disease in Finnish population. Am J Med Genet B 46. Fuhrmann M, et al. (2010) Microglial Cx3cr1 knockout prevents neuron loss in Neuropsychiatr Genet 150B:747–750. a mouse model of Alzheimer’s disease. Nat Neurosci 13:411–413. 22. Bateman A, Bennett HP (2009) The granulin gene family: From cancer to dementia. 47. Clement AM, et al. (2003) Wild-type nonneuronal cells extend survival of SOD1 Bioessays 31:1245–1254. mutant motor neurons in ALS mice. Science 302:113–117. 23. Daniel R, He Z, Carmichael KP, Halper J, Bateman A (2000) Cellular localization of 48. Boillée S, et al. (2006) Onset and progression in inherited ALS determined by motor gene expression for progranulin. J Histochem Cytochem 48:999–1009. neurons and microglia. Science 312:1389–1392. 24. Irwin D, Lippa CF, Rosso A (2009) Progranulin (PGRN) expression in ALS: An 49. Brenner S (1974) The genetics of Caenorhabditis elegans. Genetics 77:71–94. immunohistochemical study. J Neurol Sci 276:9–13. 50. Schnabel R, Hutter H, Moerman D, Schnabel H (1997) Assessing normal 25. Van Damme P, et al. (2008) Progranulin functions as a neurotrophic factor to regulate embryogenesis in Caenorhabditis elegans using a 4D microscope: Variability of neurite outgrowth and enhance neuronal survival. J Cell Biol 181:37–41. development and regional specification. Dev Biol 184:234–265.

6of6 | www.pnas.org/cgi/doi/10.1073/pnas.1100650108 Kao et al. Downloaded by guest on September 28, 2021