Chloride and the endosomal–lysosomal pathway: emerging ... · J Physiol 578.3 (2007) pp 633–640 633 PRIZE LECTURE Chloride and the endosomal–lysosomal pathway: emerging roles

J Physiol 578.3 (2007) pp 633–640 633

PR IZE LECTURE

Chloride and the endosomal–lysosomal pathway:emerging roles of CLC chloride transporters

Thomas J. Jentsch

FMP/MDC (Leibniz-Institut fur Molekulare Pharmakologie and Max-Delbruck-Zentrum fur Molekulare Medizin), Robert-Rossle Strasse 10,

D-13125 Berlin, FRG

Several members of the CLC family of Cl− channels and transporters are expressed in vesicles

of the endocytotic–lysosomal pathway, all of which are acidified by V-type proton pumps.

These CLC proteins are thought to facilitate vesicular acidification by neutralizing the electric

current of the H+-ATPase. Indeed, the disruption of ClC-5 impaired the acidification of

endosomes, and the knock-out (KO) of ClC-3 that of endosomes and synaptic vesicles. KO

mice are available for all vesicular CLCs (ClC-3 to ClC-7), and ClC-5 and ClC-7, as well as its

β-subunit Ostm1, are mutated in human disease. The associated mouse and human pathologies,

ranging from impaired endocytosis and nephrolithiasis (ClC-5) to neurodegeneration (ClC-3),

lysosomal storage disease (ClC-6, ClC-7/Ostm1) and osteopetrosis (ClC-7/Ostm1), were crucial

in identifying the physiological roles of vesicular CLCs. Whereas the intracellular localization of

ClC-6 and ClC-7/Ostm1 precluded biophysical studies, the partial expression of ClC-4 and -5

at the cell surface allowed the detection of strongly outwardly rectifying currents that depended

on anions and pH. Surprisingly, ClC-4 and ClC-5 (and probably ClC-3) do not function as Cl−

channels, but rather as electrogenic Cl−–H+ exchangers. This hints at an important role for

luminal chloride in the endosomal–lysosomal system.

(Received 9 November 2006; accepted after revision 14 November 2006; first published online 16 November 2006)

Corresponding author T. J. Jentsch: FMP/MDC, Leibniz-Institut fur Molekulare Pharmakologie and Max-Delbruck-

Zentrum fur Molekulare Medizin, Robert-Rossle Strasse 10, D-13125 Berlin, FRG. Email: [email protected]

The lumen of the vesicles of the endosomal pathway isincreasingly acidified along their way from the plasmamembrane to lysosomes. Likewise, synaptic vesicles andvesicles in the exocytotic pathway display an acidicinterior. Their low luminal pH is important for severalprocesses like receptor–ligand interactions, traffickingalong the endosomal pathway, and luminal enzymaticactivities. Although other transport processes like Na+–H+

exchangers may play a role in acidifying some vesiclepopulations, the active accumulation of protons inthese compartments is mainly accomplished by V-typeH+-ATPases, which function as ‘proton pumps’. As thesepumps are electrogenic, i.e. transport net positive charge,they would create a lumen-positive voltage in the absenceof a neutralizing current. This voltage would soon inhibitfurther acidification on energetic grounds. It has beenknown for decades that chloride needs to be present inthe medium to achieve an efficient luminal acidification

The Hodgkin-Huxley-Katz Prize Lecture was given on 5 July 2006 at The

Physiological Society Meeting at University College London.

of diverse vesicle preparations. This suggested that theneutralizing current may be mediated by vesicular chloridechannels. The molecular identity of these channels,however, remained obscure for a long time.

The molecular identification in my laboratory of ClC-0,a voltage-gated chloride channel from the electric organof the marine ray Torpedo marmorata (Jentsch et al.1990), led to the identification of all nine members ofthe mammalian CLC gene family (Jentsch et al. 2005).By their degree of homology, they can be grouped intothree branches. Similar to ClC-0, members of the firstbranch (ClC-1, -2, -Ka, -Kb) are plasma membrane Cl−

channels, whereas members of the two other branches(ClC-3, -4 and -5, and ClC-6 and -7, respectively) residepredominantly on intracellular vesicles (Fig. 1). ClC-4 andClC-5 are now known to operate as voltage-dependentCl−–H+ exchangers (Picollo & Pusch, 2005; Scheel et al.2005).

CLC proteins, whether ion channels or exchangers,function as (homo)dimers (Ludewig et al. 1996; Middletonet al. 1996), with each of the two ion translocation pathwaysentirely contained within a single subunit (Weinreich &

C© 2007 The Author. Journal compilation C© 2007 The Physiological Society DOI: 10.1113/jphysiol.2006.124719

634 T. J. Jentsch J Physiol 578.3

Jentsch, 2001). This was beautifully confirmed by thecrystal structure of bacterial CLC proteins (Dutzler et al.2002). To some degree, different CLC isoforms mayassemble to heteromers that are functional in heterologousexpression systems (Lorenz et al. 1996; Weinreich &Jentsch, 2001). So far, only two accessory and structurally

Figure 1. Subcellular localization of vesicular CLC proteinsA, proposed localization of different CLC isoforms along theendosomal–lysosomal pathway. Vesicles are progressively acidifiedfrom the neutral extracellular pH (∼7.4) to the acidic pH (∼4.5) oflysosomes. Acidification is performed by an ATP-driven proton pumpthat needs a net influx of negative charge for electroneutrality. Theneutralizing current is thought to be mediated by CLC isoforms. ClC-4and -5 mediate nCl−–H+ exchange (with an imprecisely knownstoichiometry n, which might be n = 2 as in ClC-e1) and this very likelyapplies for ClC-3 as well. ClC-6 and ClC-7/Ostm1 are also shown asantiporters, although this remains to be demonstrated. Thelocalization of ClC-4 is rather uncertain, but may also be endosomal(Suzuki et al. 2006). B, in osteoclasts attached to bone, ClC-7/Ostm1 isco-inserted with the proton pump into the highly infolded ‘ruffledborder’ that acidifies the underlying resorption lacuna.

unrelated β-subunits of CLC proteins are known: Barttinfor both ClC-K isoforms (Estevez et al. 2001) and Ostm1for ClC-7 (Lange et al. 2006).

Loss of ClC-5: impaired renal endocytosis leadsto kidney stones

Human mutations in ClCN-5 lead to low molecularweight proteinuria, urinary loss of phosphate and calcium,and frequently to kidney stones in a syndrome calledDent’s disease (Lloyd et al. 1996). ClC-5 is expressed inrenal proximal tubule (PT) cells. In these cells, ClC-5co-localizes with proton pumps in apical endosomes,suggesting that the lack of ClC-5 might impair endosomalacidification, thereby compromising the reabsorption offiltered proteins and causing proteinuria (Gunther et al.1998). Indeed, ClC-5 KO mice from two independentstrains (Piwon et al. 2000; Wang et al. 2000) lose lowmolecular weight proteins into the urine. In vivo endo-cytosis experiments revealed that the broad defect in endo-cytosis is cell-autonomous. It affects receptor-mediatedendocytosis, fluid-phase endocytosis and the retrieval ofplasma membrane proteins such as the sodium–phosphatecotransporter NaPi-2a and the Na+–H+ exchanger NHE3(Piwon et al. 2000). The abundance of the endocytoticreceptor megalin was decreased in KO PT cells, possiblypointing to a defect in recycling it back to the surface(Piwon et al. 2000). As a consequence, receptor-mediatedendocytosis is reduced more severely than fluid-phaseendocytosis. The role of ClC-5 in endosomal acidificationwas tested with suspensions of renal cortical endosomes(Piwon et al. 2000; Gunther et al. 2003) and in cellculture (Hara-Chikuma et al. 2005a). In both protocols,the disruption of ClC-5 reduced endosomal acidification.The concomitant increase in luminal Cl− concentrationwas blunted as well (Hara-Chikuma et al. 2005a).

The impairment of proximal tubular endocytosis mightbe explained by a reduced electrical shunt for end-osomal proton pumps, but how can one explain thekidney stones observed in many, but not all patientswith Dent’s disease? A unifying, experimentally supportedhypothesis (Piwon et al. 2000) links these symptoms tothe primary defect in endocytosis (Fig. 2). The smallpeptide hormone parathyroid hormone (PTH) passes theglomerular filter into the primary urine and is normallyendocytosed by PT cells (Fig. 2A). The lack of ClC-5severely impairs PTH endocytosis, leading to higherthan normal PTH levels in the lumen of the nephroneven when the blood concentration of PT is normal.This leads to an excessive stimulation of apical PTHreceptors in the late PT, triggering the endocytic removalof the phosphate transporter NaPi-2a from the plasmamembrane and thereby causing hosphaturia (Piwon et al.2000) (Fig. 2B). The increased stimulation of apical PTHreceptors in the PT also augments the transcription of

C© 2007 The Author. Journal compilation C© 2007 The Physiological Society

J Physiol 578.3 Endosomal CLC chloride transport 635

1α-25(OH)-VitD3-hydroxylase (Gunther et al. 2003), amitochondrial enzyme that converts the inactive precursor25(OH)-VitD3 into the active form 1,25-(OH)2-VitD3.This active hormone, which is slightly increased in theserum of patients with Dent’s disease (Scheinman, 1998),is expected to stimulate the intestinal resorption of Ca2+,which then must be eliminated by the kidney. On the otherhand, the main supply of 25(OH)-VitD3 to the activatinghydroxylase occurs through apical, megalin-dependentendocytosis, which is severely impaired in the absence

Figure 2. Model for renal pathology in Dent’s disease (due to a loss of ClC-5)The small peptide PTH (parathyroid hormone) and various forms of vitamin D (VitD; bound to their bindingprotein) pass the glomerular filter into the early proximal tubule (A). PTH and VitD-binding protein complexesare normally endocytosed by proximal tubular cells after binding to megalin. PTH is degraded in lysosomes,whereas 1,25-VitD reaches VitD receptors that regulate the transcription of nuclear target genes (not shown) and25-VitD is metabolized by mitochondrial enzymes: 1α-hydroxylase activates the precursor to the active hormone1,25(OH)2-vitaminD3 (1,25-VitD), whereas the 24-hydroxylase inactivates VitD. The endocytotic uptake of PTH andVitD is severely impaired in the absence of ClC-5 (A and B; indicated by red minus symbols). As a consequence,their concentration increases in the lumen of later nephron segments compared to wild-type (A–C). This leads toan enhanced stimulation of apical PTH receptors (B; green plus symbols), causing the endocytosis and degradationof the sodium–phosphate cotransporter NaPi-2a (leading to phosphaturia) and increasing α-hydroxylase whiledecreasing 24-hydroxylase levels. Similar changes in hydroxylase levels also result from the impaired apicalendocytosis of VitD, which acts on the transcription of the respective genes (not shown). The alteredhydroxylase activities tend to produce more active hormone (1,25-VitD). Such an increase, however, is counteractedby a decrease in precursor availability due to its defective endocytotic uptake. Thus, depending on other factors,the amount of 1,25-VitD released into the blood may be decreased or increased (B). C, in contrast to proximaltubules, in which VitD is taken up primarily by apical endocytosis of its binding protein and is hence decreasedin ClC-5 KO cells, it enters distal tubular cells mainly by diffusion of the free hormone. The increased luminalconcentration of 1,25-VitD in distal segments of Clcn5− nephrons enhances the transcription of distal VitD targetgenes like that of the ion channel TRPV5 (Maritzen et al. 2006).

of ClC-5. The outcome of these opposing mechanisms(increase in hydroxylase activity versus decreasedprecursor availability; Fig. 2B) determines whether thereis an increase in serum 1,25-(OH)2-VitD3 and hencehypercalciuria and kidney stones. This model therebycan account for the clinical variability of Dent’sdisease.

This hypothesis has recently been put to moretests (Maritzen et al. 2006). Expression profiling andquantitative PCR indicated that VitD target genes are



down-regulated in proximal tubules, but up-regulated inmore distal nephron segments of Clcn5−/− mice. Thisfinding is consistent with the decreased endocytotic accessof VitD3 to PT cells and its increased concentration inthe lumen of later segments (Fig. 2C). KO mice alsoshowed increased transcription of distal tubular targetgenes of retinoic acid, pointing to a more generalizedimportance of changes in hormone concentration in thenephron lumen in pathological states (Maritzen et al.2006).

Lack of ClC-3, ClC-6 or ClC-7 causesneurodegeneration

Surprisingly, the disruption in mice of ClC-3, -6, or-7 led to a neurodegeneration in the central nervoussystem (Stobrawa et al. 2001; Kasper et al. 2005; Poetet al. 2006). The severity and the morphological andbiochemical characteristics of the degeneration differedsignificantly between the genotypes. In mice lacking ClC-6or ClC-7, neurons displayed intracellular, electron-densedeposits that stained for lysosomal marker proteins and thesubunit c of ATP-synthase, a protein typically accumulatedin a subset of human lysosomal storage disease calledneuronal ceroid lipofuscinosis (NCL). Storage occurredthroughout neuronal cell bodies in ClC-7 KO mice (Kasperet al. 2005), whereas it accumulated specifically in initialaxon segments of mice lacking ClC-6 (Poet et al. 2006)(Fig. 3). In comparison, no severe intraneuronal storagewas observed in ClC-3 KO mice (Stobrawa et al. 2001;Kasper et al. 2005), although Clcn3−/− mice were reportedto display NCL-like features (Yoshikawa et al. 2002). Theneuronal cell loss was severe in mice lacking ClC-3 orClC-7, leading to a complete loss of the hippocampus inadult Clcn3−/− mice (Stobrawa et al. 2001). These lattermice, however, survived quite happily for more than ayear, whereas Clcn7−/− mice died after about 2 monthseven if their osteopetrosis (see below) was cured (Kasper

A B

nuc

nuc

Figure 3. Lysosomal storage in neurons ofmice lacking ClC-7 or ClC-6Electron-dense lysosomal storage material(indicated by arrows) is present throughout thecytoplasm of Clcn7−/− neurons (A), whereas itaccumulates exclusively in initial axonsegments of Clcn6−/− neurons (B). Scale bars,1.5 μm in A and 0.4 μm in B. The plasmamembrane of the neuron in B is highlighted bya dashed line. nuc, nucleus. Modified imagesfrom Kasper et al. (2005) and Poet et al.(2006).

et al. 2005). Neuronal cell loss was nearly absent inClC-6 KO mice, which showed nearly normal life spanand only mild neurological abnormalities that includeda reduced sensitivity to pain (Poet et al. 2006). Whereasthe combination of severe osteopetrosis with lysosomalstorage disease may also occur in human patients homo-zygous for severe CLCN7 mutations, it is currentlyunclear whether human mutations in CLCN3 or CLCN6may cause phenotypes similar to ClC-3 and ClC-6 KOmice.

The lysosomal pathologies observed with a disruption ofeither ClC-6 or ClC-7 raise the question of the subcellularlocalization of these CLC proteins (Fig. 1A). Immuno-cytochemistry and subcellular fractionation demonstratedthe presence of ClC-7 in lysosomes and late endosomes(Kornak et al. 2001; Kasper et al. 2005). Staining forClC-7 and the late endosomal–lysosomal protein lamp-1overlapped almost completely, whereas there was only apartial overlap with ClC-6 (Poet et al. 2006). ClC-6 andClC-3 may both be expressed predominantly on late endo-somes (Stobrawa et al. 2001; Poet et al. 2006), a conclusionalso supported by localizing epitope-tagged CLCs intransfected cells (Suzuki et al. 2006). A pre-lysosomallocalization of ClC-3 and -6 is further suggested by thepartial shift of both proteins, but not of ClC-4, intolysosomal fractions in Clcn7−/− mice (Poet et al. 2006).In neuronal cells, ClC-3 is additionally expressed onsynaptic vesicles (Stobrawa et al. 2001). The subcellularlocalization of ClC-6 and -7 suggests that the lysosomalstorage in the respective KO mice is a consequence ofimpaired late endosomal or lysosomal function. As theneurodegeneration of Clcn3−/− mice does not display thetypical features of a lysosomal storage disease (Stobrawaet al. 2001; Kasper et al. 2005), it may be caused bya different mechanism. For instance, abnormal intra-cellular trafficking as in ClC-5 KO mice might change thesubcellular localization of various proteins in a mannerthat is detrimental for the cell.



Thick bones, grey hair and lysosomal storage:identification of a new ClC-7 β-subunit

The immediately obvious phenotype of Clcn7−/− mice,however, is not lysosomal storage, but rather a severeosteopetrosis that leads to skeletal deformities, lack oftooth eruption, and impaired growth (Kornak et al.2001). The bone marrow cavity is replaced by calcifiedbone. ClC-7 is highly expressed in osteoclasts, where itcan be inserted into the ‘ruffled border’ together withthe vacuolar proton pump (Fig. 1B). This specializedmembrane secretes acid into the resorption lacuna thatdirectly faces the calcified bone and that may be regardedas an ‘extracellular lysosome’. The acidic pH in thisextracellular compartment is necessary both for thechemical dissolution of inorganic bone and for the activityof secreted lysosomal enzymes that degrade organic bonematerial. Clcn7−/− osteoclasts still attached to ivory(a bone surrogate) in vitro, but did neither acidifya resorption lacuna nor degrade bone like wild-type(Kornak et al. 2001). As their ‘ruffled border’ was under-developed, ClC-7 might contribute to the exocytoticbuild-up of this membrane. Our mouse model suggestedthat ClC-7 might also play a role in human osteopetrosis.Indeed, CLCN7 was mutated on both alleles in patientswith malignant infantile osteopetrosis (Kornak et al.2001), and heterozygous missense mutations of CLCN7underlie the less severe osteopetrosis of the dominantAlbers-Schonberg disease (Cleiren et al. 2001). Mutationsin the a3 subunit of the H+-ATPase, a subunit highly

Figure 4. ClC-5 is a Cl−–H+ exchanger, whereas ClC-0 is a Cl− channelHEK cells transfected with ClC-5 (A) or ClC-0 (B) were clamped to different voltages (lower traces) using thegramicidin-perforated patch clamp technique to minimize the equilibration of their internal pH (pHi) with thepatch pipette. The middle traces shows clamp currents, while the upper panels show pHi (measured using aratiometric pH-sensitive dye). When ClC-5-transfected cells were clamped to voltages more positive than +30 mV,pHi increased, indicating an exit of H+ in exchange for Cl− entry. Consistent with the steep outward rectificationof ClC-5 currents (Friedrich et al. 1999), the rate of intracellular alkalinization increased steeply with inside-positivevoltage. By contrast, a similar voltage-clamp protocol did not change the pHi of cells expressing the Torpedochannel ClC-0 (B). Panels taken from Scheel et al. (2005).

expressed in osteoclasts, may also underlie severe infantileosteopetrosis (Frattini et al. 2000; Kornak et al. 2000), againhighlighting the importance of having an acidic resorptionlacuna.

Grey lethal mice, a spontaneous, severely osteopetroticmouse mutant, resemble Clcn7−/− mice not only intheir severe osteopetrosis, but also in having grey furin an agouti background. We therefore searched for apossible link between ClC-7 and Ostm1, the proteinencoded by the grey lethal gene (Chalhoub et al.2003). Ostm1 is a type I membrane protein with aheavily glycosylated amino-terminal portion and a shortcytoplasmic tail (Lange et al. 2006). It co-localizeswith ClC-7 in lysosomes and in the ruffled border ofosteoclasts. Ostm1 needs ClC-7 to travel to lysosomes,whereas ClC-7 reaches lysosomes also without Ostm1.ClC-7 could be co-immunoprecipitated with Ostm1 andvice versa, identifying Ostm1 as a novel β-subunit ofClC-7 (Lange et al. 2006). Importantly, the stabilityof either protein depends on the coexpression withits partner. The pathology observed upon a loss ofOstm1 may be entirely due to the ensuing instabilityof the ClC-7 chloride transporter. Indeed, grey lethalmice do not only resemble Clcn7−/− mice in theirosteopetrosis, but also display similar lysosomal storagedisease (Lange et al. 2006). The grey hair of eithermouse model is not yet fully understood, but may berelated to a dysfunction of melanosomes, lysosome-relatedorganelles.



Lysosomal storage, but normal lysosomal pH:a clue for Cl−–H+ exchange?

The subcellular localization of ClC-6 and ClC-7 indicatedthat the lysosomal storage disease observed upontheir loss might be a consequence of impaired lateendosomal–lysosomal function. Because ClC-5 facilitatesthe acidification of renal endosomes (Gunther et al.2003), and as ClC-3 plays a similar role in endo-somes (Hara-Chikuma et al. 2005b) and synaptic vesicles(Stobrawa et al. 2001), we expected Clcn6−/− and Clcn7−/−

lysosomes to be more alkaline. However, steady-statelysosomal pH was unchanged in either mouse model(Kasper et al. 2005; Poet et al. 2006). Several hypothesesmay reconcile these findings with the assumed role ofvesicular CLCs: even if ClC-6 and -7 would accountfor the bulk of lysosomal membrane conductance, othersmaller conductances remaining after their eliminationmay suffice to neutralize proton pump currents overthe long run, leading to identical lysosomal pH underour experimental conditions where we had chased apH-sensitive dye into lysosomes overnight (Kasper et al.2005; Poet et al. 2006). The observed pathology might bedue to a slower rate of acidification on the way to lysosomes(fitting to the likely late endosomal localization of ClC-6),or might hint at an important role of lysosomal chloride.

A very relevant recent discovery is that ClC-4 and ClC-5,just like the E. coli protein ClC-e1 (Accardi & Miller, 2004),function as electrogenic Cl−–H+ exchangers rather thanbeing Cl− channels (Picollo & Pusch, 2005; Scheel et al.2005). While residing mainly in intracellular vesicles, aminor portion of ClC-4 and ClC-5 reaches the plasmamembrane, at least upon heterologous expression. Thislocalization allowed biophysical studies that showed thateither protein mediates anion currents which decrease withextracellular (and, by extension, luminal) acidification(Friedrich et al. 1999). These currents displayed a Cl− > I−

conductance sequence, as found in other CLC proteins.These currents, as it has emerged now, reflect an electro-genic exchange of Cl− for H+ (Fig. 4). Whereas thestoichiometry of the ion exchange could not be determinedprecisely for ClC-4 and -5 (Picollo & Pusch, 2005; Scheelet al. 2005), it is 2Cl− for 1H+ in the bacterial ClC-e1(Accardi & Miller, 2004). Mutating a key ‘gating’ glutamatein ClC-e1, ClC-4 or ClC-5 uncouples the Cl− conductancefrom H+ countertransport and abolishes the rectificationof ClC-4 and -5 currents (Friedrich et al. 1999; Accardi& Miller, 2004; Picollo & Pusch, 2005; Scheel et al. 2005).It is unknown whether ClC-6 and ClC-7 also functionas Cl−–H+ exchangers, but the presence of anotherglutamate typically found in CLC exchangers, but notchannels (Accardi et al. 2005), suggests that they do. Severalmutually incompatible channel functions were assignedto ClC-3 (e.g. Duan et al. 1997; Wang et al. 2006), butit is very likely that ClC-3 rather functions as a Cl−–H+

antiport as well. ClC-3, -4, and -5 share the same highdegree of sequence homology (including the two criticalglutamate residues), and Weinman’s group reported ClC-3currents that were nearly identical to those of ClC-4 and -5(Li et al. 2000). Importantly, these currents were affectedsimilarly by mutating the key ‘gating’ glutamate (Friedrichet al. 1999; Li et al. 2002). Unfortunately, the low currentsobtained with ClC-3 did not allow direct tests for Cl−–H+

exchange activity (Picollo & Pusch, 2005).At first glance, it seems counterproductive to use

Cl−–H+ exchangers rather than Cl− channels tocompensate proton pump currents, since a portion ofthe pumped protons will leave the vesicles via the trans-porter. Nonetheless, electrogenic Cl−–H+ exchangers willfacilitate acidification, albeit at the expense of consumingmore metabolic energy. The most important differencemay be the direct coupling of Cl− gradients to H+

gradients. H+-ATPases and Cl− channels operating inparallel will accumulate Cl− during active acidification,but the antiport will keep vesicular Cl− high understeady-state conditions as long as the inside-acidic pHgradient is maintained. In indirect support for this notion,atClCa, an anion–H+ exchanger from Arabidopsis thaliana,has recently been shown to use the pH gradient of theplant vacuole to accumulate nitrate in that organelle (DeAngeli et al. 2006). Hence, although the pH of Clcn6−/−

and Clcn7−/− lysosomes was normal (Kasper et al.2005; Poet et al. 2006), their luminal Cl− concentrationmight be changed. This altered Cl− concentration mightcontribute to the lysosomal storage disease of Clcn6−/− andClcn7−/− mice. Our work may foreshadow an importantrole of vesicular chloride that has not been recognizedpreviously.

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Acknowledgements

I would like to express my gratitude to the talented and

enthusiastic coworkers of my lab whose work I have presented

in this review. Our research was supported by the Deutsche

Forschungsgemeinschaft, the European Union, the BMBF

(NGFN2) program, the Prix Louis-Jeantet de Medecine and the

Ernst Jung-Preis fur Medizin.


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