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DOI: 10.1126/science.1209870, 1133 (2011);334 Science
, et al.Artur CzuprynAmeliorate Obesity in db/db MiceTransplanted Hypothalamic Neurons Restore Leptin Signaling and
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more primed than those from chemoresistant can-cers and chemoresistant normal tissues. Priminglevels were similar among typically chemoresist-ant cancers and chemoresistant normal tissues,which may explain why we are unable to findconventional chemotherapy regimens of toler-able toxicity to successfully treat these cancers.The chemosensitive hematologic tissues, includ-ing murine bone marrow, thymus, and spleen,and human bone marrow and peripheral bloodmononuclear cells are the most highly primednormal tissues tested. Prior work has demon-strated that these are also the most radiosensitivetissues in mice and the tissues most sensitiveto p53 activation (30, 31), suggesting that theapoptotic death observed after these insults mayalso be regulated by the mitochondrial presetmeasured by BH3 profiling.
Cytotoxic chemotherapy has been the main-stay of clinical cancer therapy for the past 60years despite an incomplete understanding of whyit works—why it is more toxic to some cancercells than others and, most importantly, why it ismore toxic to tumors than to normal tissues. Herewe show that differential mitochondrial primingcorrelates with, and may be a determinant of,differential chemosensitivity. One implication ofthese results is that agents that selectively in-crease priming in cancer cells, even if they do notcause cell death by themselves, might enhancethe response of tumors to conventional chemo-therapy (Fig. 3, D and E). A tool such as BH3profiling, which can detect changes in priming,might be useful in identifying such agents.
References and Notes1. H. E. Skipper, Cancer 28, 1479 (1971).2. F. Bosch et al., Clin. Cancer Res. 14, 155 (2008).3. R. Marcus et al., Blood 105, 1417 (2005).4. S. Noguchi, Cancer Sci. 97, 813 (2006).5. J. L. Pasieka, Curr. Opin. Oncol. 15, 78 (2003).6. J. L. Hurwitz, F. McCoy, P. Scullin, D. A. Fennell,
Oncologist 14, 986 (2009).7. N. N. Danial, S. J. Korsmeyer, Cell 116, 205 (2004).8. M. C. Wei et al., Science 292, 727 (2001).9. M. F. van Delft, D. P. Smith, M. H. Lahoud, D. C. Huang,
J. M. Adams, Cell Death Differ. 17, 821 (2010).10. J. E. Chipuk, T. Moldoveanu, F. Llambi, M. J. Parsons,
D. R. Green, Mol. Cell 37, 299 (2010).11. M. Certo et al., Cancer Cell 9, 351 (2006).12. J. Deng et al., Cancer Cell 12, 171 (2007).13. J. A. Ryan, J. K. Brunelle, A. Letai, Proc. Natl. Acad.
Sci. U.S.A. 107, 12895 (2010).14. J. K. Brunelle, J. Ryan, D. Yecies, J. T. Opferman, A. Letai,
J. Cell Biol. 187, 429 (2009).15. V. Del Gaizo Moore et al., J. Clin. Invest. 117, 112
(2007).16. L. Chen et al., Mol. Cell 17, 393 (2005).17. T. Kuwana et al., Mol. Cell 17, 525 (2005).18. K. C. Anderson, Semin. Hematol. 42 S3 (2005).19. D. Chauhan et al., Oncogene 15, 837 (1997).20. T. Hideshima et al., Cancer Res. 61, 3071 (2001).21. D. Chauhan et al., Blood 115, 834 (2010).22. B. G. Durie et al.; International Myeloma Working Group,
Leukemia 20, 1467 (2006).23. A. Burnett, M. Wetzler, B. Löwenberg, J. Clin. Oncol. 29,
487 (2011).24. C. H. Pui, W. E. Evans, N. Engl. J. Med. 354, 166
(2006).25. T. Oltersdorf et al., Nature 435, 677 (2005).26. F. J. Kubben et al., Gut 35, 530 (1994).27. R. van Gent et al., Cancer Res. 68, 10137 (2008).28. G. D. Weinstein, J. L. McCullough, P. Ross, J. Invest.
Dermatol. 82, 623 (1984).29. G. I. Evan, K. H. Vousden, Nature 411, 342 (2001).30. A. V. Gudkov, E. A. Komarova, Nat. Rev. Cancer 3, 117
(2003).
31. I. Ringshausen, C. C. O’Shea, A. J. Finch, L. B. Swigart,G. I. Evan, Cancer Cell 10, 501 (2006).
Acknowledgment: We thank I. Galinsky, J. Feather,R. Berkowitz, N. Horowitz, C. Feltmate, M. Muto, E. Kantoff,K. Daniels, and C. Doyle for assistance with human tumorsample procurement; K.-K. Wong and T. Mitchison forcritical review; and K. Stevenson for help with biostatisticalanalysis. We thank Abbott Laboratories for providing ABT-737.We gratefully acknowledge funding from the MultipleMyeloma Research Foundation (T.N.C.), American CancerSociety Postdoctoral Fellowship 121360-PF-11-256-01-TBG(K.A.S.), Women’s Cancers Program at the Dana-FarberCancer Institute (K.A.S.), Gabrielle’s Angel Foundation forCancer Research (T.-T.V.), FLAMES Fund of the Pan-MassChallenge, and NIH grants RO1CA129974, P01CA068484,and P01CA139980. A.L. is a Leukemia and LymphomaSociety Scholar. A.L. is a cofounder of and a paidadvisor to Eutropics Pharmaceuticals, which haspurchased a license for BH3 profiling from Dana-FarberCancer Institute. P.R. is a paid advisor to MillenniumPharmaceuticals, Celgene Corporation, NovartisPharmaceuticals, Johnson & Johnson, and Bristol MyersSquibb. T.N.C. and J.A.R. are paid consultants to EutropicsPharmaceuticals. R.D. is a paid consultant for NovartisPharmaceuticals and Forma Therapeutics. C.S.M. is a paidconsultant for Millennium, Celgene, Novartis, Bristol-MyersSquibb, Merck & Co., Kosan Pharmaceuticals, Pharmion,Centocor, and Amnis Therapeutics. The authors (A.L.) andDana-Farber Cancer Institute have applied for a patentrelating to the use of BH3 profiling in predictingchemosensitivity.
Supporting Online Materialwww.sciencemag.org/cgi/content/full/science.1206727/DC1Materials and MethodsFigs. S1 to S14Tables S1 to S5References (32–35)
8 April 2011; accepted 7 October 2011Published online 27 October 2011;10.1126/science.1206727
Transplanted Hypothalamic NeuronsRestore Leptin Signaling andAmeliorate Obesity in db/db MiceArtur Czupryn,1,2,3* Yu-Dong Zhou,4*† Xi Chen,5* David McNay,5‡ Matthew P. Anderson,4§∥Jeffrey S. Flier,5,6§∥ Jeffrey D. Macklis1,2,6§∥
Evolutionarily old and conserved homeostatic systems in the brain, including the hypothalamus,are organized into nuclear structures of heterogeneous and diverse neuron populations. Toinvestigate whether such circuits can be functionally reconstituted by synaptic integration ofsimilarly diverse populations of neurons, we generated physically chimeric hypothalami bymicrotransplanting small numbers of embryonic enhanced green fluorescent protein–expressing,leptin-responsive hypothalamic cells into hypothalami of postnatal leptin receptor–deficient(db/db) mice that develop morbid obesity. Donor neurons differentiated and integrated as fourdistinct hypothalamic neuron subtypes, formed functional excitatory and inhibitory synapses,partially restored leptin responsiveness, and ameliorated hyperglycemia and obesity in db/db mice.These experiments serve as a proof of concept that transplanted neurons can functionallyreconstitute complex neuronal circuitry in the mammalian brain.
To functionally repair complex circuitry inthe central nervous system (CNS), newlyincorporated neurons should be electrophys-
iologically and functionally integrated, whethertransplanted or derived from endogenous progen-itors. Newly integrated neurons might optimally
be a single neuronal subtype in some “point-to-point” sensorimotor CNS systems, or instead adiverse and nucleus-specific population of neu-ronal subtypes for some critical, homeostatic sys-tems organized into distributed nuclear structures.These systems often signal polymodally via neu-
ropeptides and are regulated by paracrine andendocrine mechanisms via multiple neuronalsubtypes operating in parallel. One such criticalhomeostatic system is that of leptin signaling inthe hypothalamus, regulating energy balance,glucose, food intake, and body weight.
1Department of Stem Cell and Regenerative Biology, andHarvard StemCell Institute, HarvardUniversity, Cambridge,MA02138, USA. 2MGH-HMS Center for Nervous System Repair,Departments of Neurology and Neurosurgery, and Program inNeuroscience, Harvard Medical School; Nayef Al-Rodhan Lab-oratories, Massachusetts General Hospital, Boston, MA 02114,USA. 3Nencki Institute of Experimental Biology, Department ofMolecular and Cellular Neurobiology,Warsaw, 02-093, Poland.4Departments of Neurology and Pathology, and Program inNeuroscience, HarvardMedical School; Center for Life Sciences,Beth Israel Deaconess Medical Center, Boston, MA 02215, USA.5Beth Israel Deaconess Medical Center, Division of Endocrinol-ogy, Diabetes, and Metabolism, and Harvard Medical School,Boston, MA 02215, USA. 6Harvard Medical School, Boston, MA02215, USA.
*These authors contributed equally to this work.†Present address: Department of Neurobiology, ZhejiangUniversity School of Medicine, Hangzhou, Zhejiang 310058,China.‡Present address: Institute of Biological and EnvironmentalSciences, University of Aberdeen, Aberdeen AB24 2TZ, Scot-land, UK.§These authors contributed equally to this work.||To whom correspondence should be addressed. E-mail:[email protected] (J.D.M.); [email protected] (J.S.F.); [email protected] (M.P.A.)
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The adipocyte-derived hormone leptin signalsthrough a variety of hypothalamic neuropeptide-producing neurons; defective leptin signaling inthe CNS due to gene mutations in either leptin(ob/ob) or its receptor (db/db) results in severeobesity and diabetes in both humans and rodents(1, 2). Deletion or rescue of leptin receptors spe-cifically in the CNS is sufficient to fully repro-duce or rescue, respectively, the obesity and diabeticphenotypes (3, 4). Recent studies link enhancedadult neurogenesis in the hypothalamus to per-sistent body-weight reduction (5, 6); a substantialnumber of these newborn neurons were leptin-responsive. Leptin receptors are highly expressedby diverse populations of hypothalamic neurons;these include anorexigenic proopiomelanocortin(POMC) neurons and orexigenic neuropeptideY (NPY) neurons. Cell type–specific deletion ofleptin receptors selectively in POMC neurons orin both POMC and NPY neurons only partiallyreplicates the phenotype of db/db mice (7). Thisdemonstrates that additional neuronal populationsin the hypothalamus or other brain regions arerequired for this modulatory system to functioncorrectly and suggests that local hypothalamicrestoration of leptin receptor signaling by diverseneuron subtypes might lead to partial phenotypicrescue.
To investigate whether partial circuit recon-stitution with the diverse hypothalamic neuronalpopulations involved in leptin signaling mightsubstantially reverse energy balance defects indb/db mice, we generated physical hypothalamicchimeras by microtransplantation of small num-bers of dissociated immature, wild-type, enhancedgreen fluorescent protein (eGFP)–expressing,leptin-responsive, hypothalamic cells (~1.5 × 104
per hypothalamus) into medial hypothalamus ofpostnatal day 0.5 (P0.5) to P5.5 db/dbmice underhigh-resolution ultrasound guidance (8, 9) (Fig.1A). Donor cells were developmentally appro-priate E13.5 immature neurons and progenitors,poised to develop into leptin-responsive hypotha-lamic neurons, able to integrate in circuitry (10–18)[fig. S1 and supporting online material (SOM)text 1].
Twenty weeks after microtransplantation, clus-ters of eGFP+ cells were located primarily in thearcuate nucleus (ARC), ventromedial hypotha-lamic nuclei (VMH), dorsomedial hypothalamicnuclei (DMH), and lateral hypothalamic nuclei(LH; not shown) (Figs. 1, B and C, and fig. S2).Most displayed typical polarized hypothalamicneuron morphology (fig. S3). Fewer eGFP+ cellswere astroglia (figs. S3 and S4). Double-labelingimmunocytochemistry revealed that 45 T 3%of eGFP+ cells expressed the mature neuronalmarker NeuN, and 4 T 4% expressed the matureastroglial marker S100b (fig. S3). Transplantedcells differentiated into several neuronal subtypesthat could be distinguished using specific mark-ers: pan neuronal HuC/D; g-aminobutyric acid(GABA); GABAergic neuron calbindin; and do-paminergic synthetic tyrosine hydroxylase (TH)(fig. S5). Notably, some of the newly incorpo-
rated eGFP+ cells expressed neuronal subtypemarkers linked to regulation of energy balance.For example, 7 T 5% of transplanted eGFP+ cellsin representative sections developed and maturedas POMC neurons (identified by immunocyto-chemistry directed against the POMC cleavageproduct b-endorphin) (Fig. 1D). Fewer eGFP+
cells differentiated and survived as NPY neurons(Fig. 1E). These morphologic and neurochemicalresults demonstrate that transplanted eGFP+ cellsmature and survive in recipient brains for at least5 months and develop into multiple distinct neu-ron types that normally reside in the hypothalamus.
To further classify the diverse neuronal subtypesof the newly incorporated neurons by theirdistinct electrophysiological properties, and todetermine whether they functionally integrateinto recipient hypothalamic circuitry,weperformedwhole-cell patch-clamp recordings of eGFP+ cellsin acute brain slices from the hypothalamus ofrecipient db/dbmice, using reportedmethods (19).We identified four different neuronal subtypeswith distinct electrophysiological properties (Figs.1, F to I, and table S1). The first and most fre-quent subtype, nonrebound regular-spiking neurons(NRRS) (n = 17) (Fig. 1F), showed an adapting,
Fig. 1. Transplanted E13.5 eGFP+ hypothalamic cells mature into appropriate hypothalamic neuronsubtypes. (A) Ultrasound-guided microtransplantation of dissociated E13.5 hypothalamic cells into earlypostnatal db/db mouse hypothalamus in sagittal view. The ~100-mm-diameter pulled glass micropipette(MP) appears on the ultrasonogram much larger than its real size due to ultrasound shadow. (B)Schematic of a coronal brain section magnified in C. (C) Location and survival of transplanted eGFP+ cellsin the representative recipient hypothalamus 20 weeks after microtransplantation. (D and E) Representativeconfocal three-dimensional (3D) reconstructions of donor-derived eGFP+ neurons in the VMH of db/dbmice 20 weeks after microtransplantation, showing (D) a POMC neuron identified by the cleavageproduct b-endorphin and (E) a NPY neuron. For each confocal reconstruction, the x-z plane is shown atthe bottom and the y-z plane is shown at the right. (F to I) Four distinct electrophysiological donor-derivedeGFP+ neuron subtypes. (F) The firing behavior of a representative newly integrated eGFP+ donor-derivedNRRS neuron. “Type A” and “type C” neurons can be distinguished by the absence or presence,respectively, of delayed firing (bottom traces). Note the delayed firing in “type C” neurons (arrow) whenhyperpolarizing current is withdrawn. (G) Representative donor-derived eGFP+ RRS neuron. An apparentvoltage sag (dashed line) and a single rebound action potential were typical. (H) Representative donor-derived eGFP+ BS neuron. A low-threshold calcium spike and cluster of rebound action potentials weretypical in response to hyperpolarizing current injection. (I) Representative donor-derived eGFP+ FS neuron.A high firing frequency and absent firing adaptation are two typical characteristics of this neuron type. DS,dorsum sellae (bone); ST, sella turcica (bone); AC, anterior commissure; S, skin and fur on the head; 3V, thirdventricle. Asterisks demarcate the rostrocaudal extent of the transplantation tracks. Scale bars in (A), 1 mm;in (C), 100 mm; in (D) to (I), 10 mm.
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regular firing response to depolarizing currentinjections, with no voltage sag and no reboundaction potentials fired after a strong hyperpolariz-ing current injection. These properties are typicalof “type A” and “type C” neurons found in ARC(20) and VMH (21) and type 1 neurons found inLH (22). The second subtype, rebound regular-spiking neurons (RRS) (n = 7) (Fig. 1G), displayedboth an adapting firing response to depolarizingcurrent injections, and a depolarizing sag duringhyperpolarizing current injections (table S1) fol-lowed by a single rebound action potential, sug-gesting the presence of hyperpolarization-activatedcation currents (Ih). This is typical of hypotha-lamic POMC neurons (23) and type 2 neurons ofLH (22). The third subtype, burst-spiking neu-rons (BS) (n = 7) (Fig. 1H), displayed a clusterof rebound sodium action potentials overlying aslower calcium spike in response to hyperpolar-izing current injections, indicating the presenceof a low threshold T-type Ca2+ conductance. Thisfiring behavior is similar to that of type B neu-rons found in bothARC (20) andVMH (21). Thefourth subtype, fast-spiking neurons (FS) (n = 5)(Fig. 1I), displayed nonadapting, high-frequencyfiring of narrow sodium spikes (table S1) withlong depolarizing current injections. These prop-erties are typical of fast-spiking GABAergic neu-rons and the type 3 neurons in LH (22). These
results indicate that transplanted immature wild-type hypothalamic neurons develop and matureelectrophysiologically into a specific and diverseset of neuronal subtypes typical of the normal hy-pothalamic neuron populations (tables S1 and S2).
To investigate whether these newly incorpo-rated eGFP+ neurons are functionally integratedinto the recipient db/db hypothalamic circuitry, weexamined whether synapses are formed betweennative and donor eGFP+ neurons. We first usedimmunocytochemical analysis of synaptophysin(a presynaptic marker) for synaptic localization.Synaptophysin immunopositive puncta developedon the somas and processes of eGFP+ neurons(Fig. 2, A and B). NPY-positive neurons had ex-tensive contacts with the transplanted eGFP+ cells(fig. S6). We then examined functional synaptictransmission by assessing postsynaptic currentsin the transplanted neurons. Notably, all recordedtransplanted neurons that fired action potentialsalso displayed spontaneous inhibitory (sIPSC)and spontaneous excitatory (sEPSC) postsynapticcurrents under voltage-clamp conditions (Fig. 2Cand fig. S7). The four neuronal subtypes showedsimilar mean sEPSC and sIPSC amplitudes andfrequencies (fig. S7). Electron microscopy (EM),combined with anti-GFP immunogold labeling tounequivocally identify transplanted neurons, fur-ther confirmed that synapses form between native
and donor-derived eGFP+ neurons (fig. S8). Thepresence of synaptophysin immunopositive punc-ta and spontaneous synaptic events indicate thateGFP+ neurons receive synaptic inputs in thechimeric hypothalamus. To investigate whethereGFP+ donor neurons form functional synapseswith native neurons, we performed dual whole-cell patch-clamp recordings of eGFP+ and nativeneurons. Fast synaptic connections from eGFP+
to native neurons were observed (n = 3) (Fig. 2D).Firing of different eGFP+ neurons (green tracings)elicited either an EPSP (upper trace) or IPSP (lowertrace) in adjacent native neurons (Fig. 2D).Together,the immunofluorescence labeling, electrophysio-logical, and ultrastructural results strongly indicatethat incorporated eGFP+ neurons form synapticconnections with native hypothalamic circuitry foras long as 5 months after microtransplantation.
To determine whether the newly integratedeGFP+ neurons were responsive to leptin, we ex-amined a known leptin-activated signaling path-way by immunocytochemistry, and we measuredthe electrophysiological response to leptin in theseneurons. The phosphorylation and nuclear trans-location of signal transducer and activator of tran-scription 3 (STAT3) is a well-characterized leptinsignaling response (24–26). After intraperitonealadministration of leptin to hypothalamically chimer-ic db/db mice, 8 T 3% of eGFP+ neurons exhibited
Fig. 2. Transplanted eGFP+ hypothalamic neurons form excitatory and in-hibitory synaptic connections and response to the energy state signals leptin,glucose, and insulin. (A and B) Donor-derived eGFP+ neurons in db/db re-cipients bear extensive appositions with synaptophysin-positive puncta (red).(B) Higher-magnification confocal image of the boxed region of a dendrite inthe merged confocal image in (A), showing synaptophysin-positive punctaindicating synaptic contacts. (C) Spontaneous EPSCs and IPSCs were observedin eGFP+ neurons. (D) Representative dual whole-cell recording showing thatstimulation of donor-derived eGFP+ neurons (green traces) elicits EPSPs (upper)or IPSPs (lower) in native neurons (black traces). (E) Representative confocal
3D reconstruction of donor-derived eGFP+ neuron in the VMH of db/db mice20 weeks after microtransplantation with leptin-induced signaling indicatedby phosphorylated STAT3 in response to leptin administration. (Bottom) x-zplane; (right) y-z plane. (F) Representative voltage tracing from a leptin-depolarized eGFP+ neuron. Leptin depolarized the membrane potential andsubstantially increased the firing rate; insulin had the opposite effect. (G) Rep-resentative voltage tracing from a leptin-hyperpolarized eGFP+ neuron. Bothleptin and insulin hyperpolarized the membrane potential and substantiallydecreased the firing rate; low glucose had the opposite effect. Scale bars in (A),8 mm; in (B), 2 mm; in (E), 10 mm.
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STAT3 phosphorylation (Fig. 2E), comparedwith 0% in the recipient (eGFP–) db/db hypo-thalamic neurons lacking leptin receptors. Wefurther investigated the effects of leptin on actionpotential firing and membrane potential in 17eGFP+ transplanted neurons and in endogenousneurons in hypothalamically chimeric db/db mice.Leptin (100 nM) depolarized the membrane po-tential (4.5 T 1.2 mV) (n = 4) and increased thespontaneous firing rate (96.0 T 51.6%) (n = 4) in 4of the 17 neurons tested (Fig. 2F and table S3).This response is typical of hypothalamic POMCneurons (27). Consistent with this conclusion, 3of 4 leptin-depolarized neurons also displayedthe RRS firing behavior typical of POMC neu-rons. In contrast, leptin hyperpolarized the mem-brane potential (–3.1 T 0.6 mV) (n = 10) anddecreased the spontaneous firing rate (–62.3 T6.4%) (n = 10) in the majority of NRRS and BSneurons in the ARC, VMH, and LH, as well asin FS neurons in the mammillary nucleus (n =10) (Fig. 2G and table S3). This inhibitory re-sponse to leptin is typical of NPY neurons (28).Only 3 of the 17 eGFP+ neurons were insensitiveto leptin. In contrast, all recordings from neigh-boring eGFP– neurons in the same hypothalam-ically chimeric db/db brain slices displayed noresponse to leptin (n = 3) (fig. S9). These resultsindicate that newly incorporated eGFP+ neuronsreconstitute the typical diversity of excitatory andinhibitory leptin responses found in the normalhypothalamus.
We next investigated whether chimericallyincorporated and synaptically integrated neuronsexhibit electrophysiological responses to glu-cose and insulin typical of neurons in these hypo-thalamic nuclei. Hypothalamic neuronswith leptinresponses typically display distinct matching in-sulin and glucose responses (27). Like nativeneurons, all leptin-depolarized eGFP+ donor-derived neurons examined were hyperpolarizedin response to 100 nM insulin (Fig. 2F and tableS3) (n = 3) (29). In response to reduction of glu-cose from 10 mM to 5 mM, two of four leptin-depolarized eGFP+ neurons exhibited membranehyperpolarization and decreased their firing rates(table S3), typical of normal leptin-depolarizedhypothalamic neurons (23, 29, 30). Two of threeleptin-hyperpolarized neurons also hyperpolarizedin response to insulin (Fig. 2G and table S3),whereas one neuron was strongly excited by insu-lin (table S3) (29). Of seven leptin-hyperpolarizedeGFP+ neuronswith glucose reduction, four weredepolarized (Fig. 2G and table S3), normal forhypothalamic neurons (31). These results indicatethat newly integrated neurons exhibit responses toglucose, insulin, and leptin typical of native neu-rons of the medial hypothalamus.
To assess whether the donor-derived neuronshad a functional effect on body weight, we com-pared db/db recipients of the hypothalamic neu-ron transplants with four relevant control groups(SOM text 2). Db/db mice with hypothalamicneuron transplants displayed significantly reducedbody weight compared to nonoperated or sham-
operated db/db mice (Fig. 3A), intermediate be-tween db/db and control nonobese mice. Shamexperiments, in which cell-free medium was in-jected into the same hypothalamic location ofdb/db mice, revealed no effect on body weightcompared with db/db nonoperated littermates(Fig. 3A and fig. S10). Similarly, microtransplan-tation of E13.5 wild-type hypothalamic eGFP+
cells into hypothalami of control nonobese micedid not result in body weight differences com-pared with nonoperated, nonobese littermates(Fig. 3A). To further investigate the specificity ofthe effect of transplanted immature hypothalamicneurons and progenitors, we microtransplantedimmature neurons from the neocortex isolatedduring an equivalent developmental stage (E16.5to E18.5) (11, 12, 32) to control for potential non-specific incorporation of non–leptin-responsive,nonhypothalamic neurons (SOM text 3). Twentyweeks after transplantation, almost all survivingneocortical donor cells were S100b positive gliathat did not fire action potentials (fig. S11). Theseresults with neocortical donor cells confirm pre-vious results in the neuronal transplantation lit-
erature that the specific sources and subtypes ofdonor neurons are critical for successful neuronalcircuit integration and survival (11). The bodyweight of db/db mice transplanted with corticalcells was unaltered (fig. S10A), reinforcing thespecific ability of E13.5 hypothalamic donor neu-rons and progenitors to reconstitute functionalhypothalamic circuitry. Taken together, these re-sults demonstrate that leptin-responsive donorneurons derived from wild-type E13.5 hypothal-amus are able to ameliorate obesity in hypotha-lamically chimeric db/db mice after functionalintegration and partial circuit reconstitution. Thisdecrease in body weight is equivalent in magni-tude to the increase observed when leptin recep-tors are selectively deleted from the ventromedialhypothalamic nucleus (33), reinforcing the phys-iological level of this repair.
To further investigate tissue-specific contri-butions to body-weight reduction in transplanteddb/db mice, postmortem body composition wasassessed using dual-energy x-ray absorptiometry(DEXA). There was a significant decrease in fatmass (Fig. 3B) but not in lean mass (fig. S12A).
Fig. 3. Transplanted E13.5 wild-type immature hypothalamic neurons reduce body weight, fat mass,serum leptin, and glucose levels in leptin receptor–deficient db/db mice. (A) Hypothalamically chimericdb/db mice (db/db mice microtransplanted with E13.5 hypothalamic cells into the medial hypothalamus)have significantly lower body-weight gain than nonoperated db/db mice or sham-operated db/db mice(F2,361 = 120.75; chimeric db/db versus db/db nonoperated mice, P < 0.001; chimeric db/db versus db/dbsham-operated mice, P < 0.001). Microtransplantation of E13.5 hypothalamic cells into nonobese controlmice did not change body weight compared with nonobese, nonoperated littermates. (B) Analysis of bodycomposition using DEXA demonstrates significant fat mass reduction in transplanted compared withnontransplanted obese (db/db) mice. (C and D) Analysis of blood parameters demonstrates statisticallysignificant reductions in (C) serum leptin levels at 13 weeks of age and (D) blood glucose at 9 and 13 weeksof age. *P < 0.05; **P < 0.01; ***P < 0.001. Data reported as mean T SEM.
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Of note, food intake was not significantly alteredin these mice, suggesting that changes in fat masswere likely due to increased energy expenditure(fig. S12B). Consistent with the body-weight re-duction, serum leptin levels were also significant-ly reduced (Fig. 3C).
The db/db mutant mice display a charac-teristic dysregulation of blood glucose and seruminsulin levels, and we studied whether the hypo-thalamic cell transplantation corrected thesemetabolic abnormalties. In comparison with con-trols, hypothalamically chimeric db/db mice dis-played a large and significant reduction of bloodglucose levels at both 9 and 13 weeks after trans-plantation (Fig. 3D), consistent with the knowncentral role of hypothalamic leptin receptors inperipheral glucose regulation (34). Interestingly,this rescue of peripheral glucose homeostasisoccurred despite the lack of difference in seruminsulin relative to controls (fig. S12C).
These results demonstrate that transplantednewborn hypothalamic neurons and progenitorsof appropriate developmental stage can chimeri-cally integrate into hypothalamic circuitry as func-tional neurons with subtype diversity typical ofthe normal hypothalamus, and that this relative-ly small number of functionally integrated donorneurons can ameliorate obesity, hyperleptinemia,and hyperglycemia in db/db mice. At least twopossible networkmechanismsmight explain howthe relatively few integrated neurons partially res-cue the db/db phenotype. Rescue might be solelydue to the new neurons themselves. Alternatively,the new, cell-autonomously leptin-responsive neu-rons might modulate their firing in response tosystemic leptin levels, secondarily regulating oth-er neurons in hypothalamic circuitry. Thus, trans-planted neurons might act as leptin sensors whoseoutput is then transmitted by endogenous, hypo-
thalamic circuitry still unresponsive to direct leptinactivation. Irrespective of the underlying mech-anism, these experiments demonstrate that synap-tic integration by leptin-responsive donor neuronscan impart an organism-level rescue of metabolicdefects, thereby providing a proof of concept forcell-mediated repair of a neuronal circuit control-ling a complex phenotype.
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A. Palmer, L. Pasquina, K. Quinn, A. Wheeler, andP. Davis (J.D.M. laboratory), and H. Yin (J.S.F. laboratory)for excellent technical assistance. We thank O.K. Ronnekleivat Oregon Health and Science University for providingantibody to b-endorphin. We thank M. McKee of the MGHMicroscopy Core for excellent EM assistance. This studywas partially supported by NIH grant NS41590 (to J.D.M.),with additional infrastructure supported by NS45523 andNS49553 (to J.D.M.), and with additional support fromthe Jane and Lee Seidman Fund for Central NervousSystem Research and the Emily and Robert PearlsteinFund for Nervous System Repair (to J.D.M.), grantDKR37-28082 (to J.S.F.), the Picower Foundation (toJ.S.F. and M.P.A.), National Institute of NeurologicalDisorders and Stroke (NINDS) grants NS057444,NS054674, and NS070295 (to M.P.A.), and the NancyLurie Marks Family Foundation (to M.P.A.). A.C. waspartially supported by Ministerstwa Nauki i SzkolnictwaWyższego grant N303 298437 and by the Foundationfor Polish Science.
Supporting Online Materialwww.sciencemag.org/cgi/content/full/334/6059/1133/DC1Materials and MethodsSOM TextFigs. S1 to S12Tables S1 to S3Reference (35)
30 September 2010; accepted 14 October 201110.1126/science.1209870
Polarization of PAR Proteinsby Advective Triggeringof a Pattern-Forming SystemNathan W. Goehring,1 Philipp Khuc Trong,2,1* Justin S. Bois,2,1† Debanjan Chowdhury,2‡Ernesto M. Nicola,2§ Anthony A. Hyman,1 Stephan W. Grill2,1∥
In the Caenorhabditis elegans zygote, a conserved network of partitioning-defective (PAR) polarityproteins segregates into an anterior and a posterior domain, facilitated by flows of the corticalactomyosin meshwork. The physical mechanisms by which stable asymmetric PAR distributions arisefrom transient cortical flows remain unclear. We present evidence that PAR polarity arises fromcoupling of advective transport by the flowing cell cortex to a multistable PAR reaction-diffusionsystem. By inducing transient PAR segregation, advection serves as a mechanical trigger for theformation of a PAR pattern within an otherwise stably unpolarized system. We suggest that passiveadvective transport in an active and flowing material may be a general mechanism formechanochemical pattern formation in developmental systems.
Developmental form emerges from cou-pling of pattern-forming biochemical net-works with mechanical processes (1–3).
In Caenorhabditis elegans zygotes, transient flowsof a thin film of a mechanically active actomy-osin cell cortex instruct the patterning of a con-
served cell polarity pathway consisting of twogroups of partitioning-defective (PAR) proteinsthat mutually exclude one another from the cellmembrane (4–14). Initially, anterior PARs (aPARs:PAR-3, PAR-6, and atypical protein kinase C)cover the entire cell membrane. During polariza-tion, flows of cortical actomyosin oriented awayfrom the posterior-localized centrosome induces
1Max Planck Institute of Molecular Cell Biology and Genetics(MPI-CBG), 01307 Dresden, Germany. 2Max Planck Institutefor the Physics of Complex Systems (MPI-PKS), 01187 Dresden,Germany.
*Present address: Department of Physics, and Departmentof Applied Mathematics and Theoretical Physics, Universityof Cambridge, Cambridge CB3 OHE, UK.†Present address: Department of Chemistry and Biochemistry,University of California, Los Angeles, Los Angeles, CA 90095, USA.‡Present address: Department of Physics, Harvard University,Cambridge, MA 02138, USA.§Present address: Institute for Cross-Disciplinary Physics andComplex Systems (IFISC), Consejo Superior de InvestigacionesCientificas–Universitat de les Illes Balears, E-07122 Palma deMallorca, Spain.||To whom correspondence should be addressed. E-mail:[email protected]
www.sciencemag.org SCIENCE VOL 334 25 NOVEMBER 2011 1137
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