Metalloprotease MP100: a synaptic protease in rat brain

Ž .Brain Research 837 1999 193–202www.elsevier.comrlocaterbres

Research report

Metalloprotease MP100: a synaptic protease in rat brain

Andrea B. Huber a,b, Christian Brosamle b, Hans Mechler a, Gerda Huber a,)ä Pharma DiÕision, Preclinical CNS Research, F. Hoffmann-La Roche, Bldg. 69r452, 4002 Basel, Switzerlandb Brain Research Institute, UniÕersity of Zurich and Swiss Federal Institute of Technology, Zurich, Switzerland¨ ¨

Accepted 1 June 1999

Abstract

Proteases are expressed widely throughout the nervous system and perform essential functions. We have earlier characterized andŽ .cloned the metalloprotease MP100, an enzyme originally described as a b-amyloid precursor protein b-APP processing candidate. In the

present study we describe the cellular and subcellular localization of MP100 in rat brain. A punctuate intracellular immunostaining incortical, hippocampal and cerebellar neurons suggests its high abundance in vesicular intracellular structures. The MP100 staining patternresembled that of the presynaptic protein synaptophysin. In gel filtration chromatography of isolated rat brain synaptosomal membranes,MP100 co-fractionated with synaptophysin and b-APP. Furthermore, pre-embedding immunoelectron microscopy of the cerebellumrevealed MP100 to be localized at synaptic sites. All together, these data might indicate a role for MP100 in functions such as proteolyticmodification of synaptic proteins. q 1999 Elsevier Science B.V. All rights reserved.

Keywords: Metalloprotease; Neuronal; Synapse; Immunohistochemistry

1. Introduction

In the nervous system, proteases of various classesperform multiple functions. Extracellular serine proteases,such as plasmin, are involved in modulating short-term

w xpotentiation 25 . Others have been shown to participate inprocesses related to synaptic plasticity and neuronal sur-

w xvival 17,34 . Activity-dependent synapse elimination in-w xvolves proteases such as thrombin 1 . Proteases of the

cathepsin family or the proteasome multicatalytic complexplay roles in lysosomal or non-lysosomal pathways of

w xintracellular protein degradation 23,27 . Also neuropeptidemetabolism is mediated by protease actions, e.g., the inac-

w xtivation of cholecystokinin by aminopeptidase A 24 . Pro-teases may even, under neurodegenerative conditions, takeon functions such as prion protein processing in Prion

w xdiseases 38 or alternative b-amyloid precursor proteinŽ . w xb-APP processing in Alzheimer’s Disease 3,13 . Rapidphysiological turnover of presynaptic b-APP has beendemonstrated in vivo in the primary visual projection

w xpathway 19 where proteolysis is thought to occur eitherin transport vesicles in the axon or at the presynaptic

) Corresponding author. Fax: q 41-688-1720; E-m ail:[email protected]

w xterminal membrane 2 . Although some b-APP is delivereduncleaved to the cell surface, most can be cleaved atseveral sites by at least three different proteases termedsecretases. No secretase has yet been clearly identified buta number of b-secretases have been postulated by in vitro

w xcleavage studies 12,16,21,29 among which the metallo-w xprotease MP100 was one candidate 30 . However, it was

recently found unable to exert true b-secretase-like func-tion in cultured cells although it might be involved as

w xsecondary exoprotease in b-APP metabolism 11 . RecentcDNA cloning demonstrated nearly complete homology of

Ž .MP100 to a puromycin-sensitive aminopeptidase PSAw x11 enriched in brain and potentially involved in

w xenkephalin cleavage 22,35 . Among the potential sub-strates b-APPs are a family of proteins which are develop-mentally regulated and b-APP , the major isoform in695

brain, shows a peak of expression during synaptogenesisw x18 . High levels of another isoform, b-APP , are onlyKPI

found in the olfactory areas in brain, where constantw xsynaptogenesis occurs 18 . Overall, b-APP immuno-

reactivity is described as patches near the plasma mem-w xbrane of neurons 33 . Higher-resolution studies have de-

scribed co-localization of b-APPs with synaptophysin atsynaptic sites and with vesicular structures in various

w xneuronal populations in human and rat neurons 31 . In thepresent study, the expression of MP100 was investigated in

0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.Ž .PII: S0006-8993 99 01693-5

( )A.B. Huber et al.rBrain Research 837 1999 193–202194

rat brain and compared to the biochemical and subcellulardistribution of synaptic marker proteins.

2. Materials and methods

2.1. Primary antibodies

The mouse monoclonal antibodies anti-synapsin, anti-Ž .synaptophysin and anti-b-APP 22C11 were purchased

from Promega and Boehringer Mannheim. The polyclonalanti-MP100 antibody was raised in rabbits against thesynthetic peptide AATEDLWESENA, affinity purified by

Ž .peptide chromatography and termed anti-MP100 p89 .Ž .Working dilutions were as follows: anti-MP100 p89

Ž . Ž .1:1000 Western blot and 1:50 immunohistochemistry ;Ž .anti-synaptophysin 0.5 mgrml Western blots and 2

Ž . Žmgrml immunohistochemistry ; anti-synapsin 1:5 im-. Ž .munohistochemistry ; 22C11 1 mgrml Western blots .

2.2. Brain tissue and MP100 preparation

Brain extracts from adult SPF rats were prepared at 48Cfrom frozen tissue stored at y708C. The tissue was rapidlyhomogenized in 5 mlrg of cold 50 mM sodium phosphate

Ž . Ž .buffer pH 7 containing 1 mM dithiothreitol DTT usinga glass homogenizer with a Teflon pistil. The homogenatewas centrifuged at 5000=g for 30 min at 48C, the super-natant was collected and recentrifuged at 100 000=g for1 h at 48C. This fraction was named crude brain extract.Protein concentrations were determined using a commer-

Ž .cial assay BioRad with bovine serum albumin as astandard. Human MP100 was expressed as described in

w xRef. 11 .

2.3. Rat brain synaptosomal membrane fractionation

Synaptosomal membranes were prepared as describedw x28 . Briefly, 30 g of rat brain tissue were homogenized in100 ml ice-cold 0.32 M sucrose in 100 mM potassium

Žphosphate, pH 7.4 containing protease inhibitors Com-.pletee, Boehringer Mannheim . The crude homogenate

was centrifuged at 25 000=g for 60 min at 48C and theŽ .resulting pellet was salt-washed 0.5 M NaCl and recen-

trifuged. The pellet was resuspended in 25 ml of 3.2 Msucrose in 100 mM potassium phosphate, pH 7.4 andaliquots of the suspension were layered on discontinuous

Žsucrose gradients. Following ultracentrifugation 100 000.=g , three bands of material could be observed at the

interface between each solution and the band enriched inw xsynaptosomal membranes 28 was recovered, diluted with

60 ml of 100 mM potassium phosphate, pH 7.4 andcentrifuged at 100 000=g for 90 min. The pellet wasresuspended in 20 mM TrisrHCl pH 7.5, 300 mM NaCl, 1

Ž .mM DTT, 2.5% 3- 3-cholamidopropyl dimethylammonio-

Ž .1-propanesulfonate CHAPS to solubilize membrane asso-ciated proteins and the suspension was diluted to 0.25%CHAPS. Non-solubilized material was removed by ultra-

Ž .centrifugation 100 000=g for 60 min at 48C and theresulting supernatant containing synaptosomal proteins wasfurther processed. The solubilized synaptosomal proteinswere concentrated by Ultrafiltration and applied to a Su-perdex 200 HR column previously equilibrated with 20mM TrisrHCl pH 7.5, 300 mM NaCl, 1 mM DTT, 0.25%

Ž .CHAPS. The fast protein liquid chromatography FPLCcolumn was run at 0.3 mlrmin and the fractions collectedwere precipitated by trichloroacetic acid and analyzed byWestern blotting.

2.4. Gel electrophoresis and Western-blotting

Ž .Sodium-dodecyl sulphate SDS –polyacrylamide gelŽ .electrophoresis PAGE was performed according to stan-w xdard methods 15 using either 7.5% SDS–PAGE in a

Ž .mini-gel system Bio-Rad or 4–20% SDS–PAGEŽ .NOVEX . Electrotransfer of proteins from gels to nitro-

Ž .cellulose membranes Amersham was performed in aŽ .cooled Trans-Blot mini cell Bio-Rad according to Ref.

w x36 , except that 0.1 grl SDS was included in the transferbuffer. After blocking non-specific binding sites by immer-sion in 50 grl skimmed milk powder in Tris-buffered

Ž .saline TBS containing 0.5 mlrl Tween20 for 1 h, themembranes were incubated overnight at 48C or for 2 h atroom temperature with the appropriate primary antibody.Bound antibodies were detected using alkaline phos-

Žphatase-conjugated secondary antibodies Dakopatts or. Ž .Bio-Rad and a corresponding substrate kit Bio-Rad or

Ž .peroxidase-conjugated secondary antibodies DakopattsŽ .with an ECL substrate kit Amersham . For p89 preabsorp-

Ž .tion experiments the anti-MP100 p89 was incubated with0.2 mgrml of p89 peptide at 48C overnight and then usedlike the non-preabsorbed antibody for Western-blotting.

2.5. Immunohistochemistry

Ž .Adult rats RoRo SPF of 150 to 200 g b.wt. weredeeply anaesthetized by intraperitoneal injection of pento-

Ž .barbital 40 mgrkg . The animals were transcardially per-Ž .fused with 200 ml saline 0.9% NaCl followed by 250 ml

fixative containing 4% paraformaldehyde in 0.4 M phos-Ž .phate buffer pH 7.4 . The brains were rapidly dissected

out and postfixed in the same fixative at 48C overnight.Ž .Sagittal vibratome sections 40 mm were cut; the cerebel-

lum was embedded in 2% agar in phosphate-bufferedŽ .saline PBS for better stability before cutting. Before

incubation of the tissue sections with the primary antibodyovernight at 48C, non-specific binding sites were blocked

Ž .with 3% fetal calf serum FCS in PBS during 30 min atroom temperature. After primary antibody incubation, thesections were incubated for 2 h with secondary biotinyl-

( )A.B. Huber et al.rBrain Research 837 1999 193–202 195

Ž .ated antibody Dako and then with streptavidine-biotinyl-Ž .ated horseradish complex Amersham for 1 h. Between

each step, the sections were washed 4= with PBS contain-ing 0.05% Tween20. The sections were finally stainedwith 4-chloro-1-naphthol, mounted onto slides with 50%glycerol in PBS containing 0.2% sodium azide and photo-graphed using a Zeiss Axiophot microscope.

2.6. Electron microscopy

Animals were deeply anesthetized and perfused tran-scardially with a Ringer solution containing 100.000 IUheparin and 0.25% NaNO followed by 4% formaldehyde2

in 0.1 M phosphate buffer pH 7.4 containing 5% sucrose.Cerebella were dissected, embedded in a glutaraldehyde-polymerized protein matrix, 50 mm sagittal sections werecut on a vibratome and processed for immunohistochemi-cal staining in a semifree-floating way as described earlierw x8 . Briefly, sections were washed in TBS pH 8, 3= for 10min, then incubated with either MP100 immune or the

corresponding pre-immune serum overnight at 48C. Afterwashing 2= for 15 min in TBS and incubation with a

Ž .biotinylated secondary anti-rabbit antibody Vector di-luted 1:200 for 2 h at RT, sections were washed 2= for 15min in TBS, incubated with a horseradish peroxidase–

Ž .avidin complex ABC standard, Vector washed again 2=

for 10 min in TBS, rinsed in 50 mM Tris buffer, andreacted with 0.0015% diaminobenzidine and 0.004% H O2 2

in the presence of 0.4% ammonium nickel sulfate. Sectionswere then examined at low power magnification, dissectedfurther and transferred to 0.1-M cacodylate buffer. Sec-tions selected for electron microscopy were osmicated for

Ž .1 h 1% OsO4 in 0.1 PB , washed again in cacodylatebuffer, dehydrated through an alcohol series ending in two100% propylene oxide steps and flat embedded in Epon

Ž .araldite. Ultrathin sagittal sections 70–90 nm were takenoff the cerebellar molecular layer, contrasted withReynold’s lead citrate and observed under a Zeiss trans-mission electron microscope.

Ž .Fig. 1. Characterization of the MP100 antibody. Anti-MP100 p89 strongly reacts with active soluble human MP100 over-expressed in HEK293 cells asŽ .well as with native rat brain MP100 arrow and smaller breakdown products. Crude soluble cell or tissue extracts were separated on 7.5% PAGE,

Ž . Ž .electro-blotted and immunoprobed on nitrocellulose membranes with anti-MP100 p89 or p89 preabsorbed anti-MP100 p89 . Molecular masses of standardŽ .proteins MS-St. are indicated.


3. Results

3.1. Expression of MP100 in brain and synaptic membranefractions

The expression of the protease MP100 in rat brain wasstudied using the monospecific affinity purified anti-

Ž .MP100rp89 antibody Fig. 1 . The antibody strongly re-acted with active soluble human MP100 expressed in

Ž .HEK293 cells arrow . The same size protein is immunola-beled in soluble rat brain extracts and also HEK293 cellsexpress basal levels of MP100. MP100 is prone to degra-dation as can be seen by the presence of smaller break-down products, which vary between preparations. This isparticularly critical in multi-step preparations such as brainsynaptosomes where multiple lower molecular weight frag-

Ž .ments appear Fig. 2 . In rat synaptosomal membranesŽ .active MP100 Fig. 2, arrowhead co-fractionates with

Ž .synaptophysin and b-APP e.g., lanes 12, 13 while manyof the lower size fragments are spread throughout allsynaptophysin fractions. Large immunoreactive MP100

Ž .forms 120–200 kDa are also seen together with b-APPŽ .and synaptophysin lanes 10–12 and could represent

membrane-bound enzyme precursor.

3.2. MP100 distribution in the cerebral cortex

In the cerebral cortex, as in all other rat brain regionsexamined, large numbers of immunoreactive neurons wereseen. These MP100-immunostained cells were localized in

Ž .all layers of the cortex Fig. 3A . Pyramidal cells werelabeled in a patchy manner revealing vesicle-like structures

Ž .beneath the cell surface of perikarya arrows in Fig. 3Band extending into dendrites, possibly representing secre-tory organelles as they are mostly located close to the cellsurface as well as occasionally as fine dots in the cyto-plasm. In the neuropil, the staining pattern for MP100 was

Ž .very punctate Fig. 3E and the cerebral white matter wasŽ .unstained not shown .

3.3. MP100 in the hippocampus and dentate gyrus

A large number of immunoreactive neurons were foundwithin all regions of the hippocampus in all sections

Žexamined. Granule cells of the dentate gyrus Fig. 4C and.D and pyramidal cells of layers CA3 and CA1 of the

Ž .hippocampus B and E showed equally strong immuno-reactivity. MP100-immunolabeled patchy structures withinthe perikarya, dendrites and along the surface of pyramidal

Ž .and granule cells were seen arrows in Fig. 4B and D . Thecharacteristic punctuate distribution of MP100-immunola-beled structures observed in the pyramidal cells of thecortex appeared also in the cells of the hippocampus.Immunoreactive pyramidal neurons with perinuclear im-munostaining were found in all parts of the hippocampus.In the granular cell layer also other cells showed a re-

Fig. 2. MP100 in synaptic membranes. In synaptic membranes, MP100Ž . Ž . Ž .A co-fractionates with both synaptophysin B and b-APPs C whereby

Ž .the active MP100 form arrowhead co-purifies best with b-APPs. Alsohigh-molecular weight MP100-immunoreactive forms occur in synapto-

Ž .somes A, lanes 10–12 . Rat brain synaptosomal membrane fractionsafter Superdex 200 HR chromatography were run on 4–20% SDS–PAGE

Ž . Ž . Ž .and probed by Western-blots, A with anti-MP100 p89 , B with anti-Ž .synaptophysin and C with anti-b-APP.

stricted punctuate immunoreactive structure in their cyto-plasm.


Ž . Ž . Ž . Ž . Ž .Fig. 3. MP100 expression in rat cerebral cortex. A and B show frontal cortex in low and high magnification. In C , D and E , a comparison ofsynaptophysin, synapsin and MP100, respectively, is shown in the cortex. All three proteins revealed similar punctate staining in the neuropil, highlighted

Ž . Ž . Ž . Ž . Ž .with arrows. In addition, MP100 immunoreactivity was found in cytoplasm of pyramidal cells. Bar in A 70 mm; in B 15 mm; in C , D and E 30mm.

3.4. MP100 in the cerebellum

The cerebellum exhibited a very characteristic distribu-tion of immunoreactive perikarya. Of the four major celltypes within the laminated portion of the cerebellum, the

Purkinje cells showed particularly marked MP100immunoreactivity. Again, no homogeneous cytoplasmiclabeling was observed but immunostaining of structuresclose beneath the cell surface of Purkinje cells was foundŽ .Fig. 5B, arrows . No staining within the white matter was


Fig. 4. MP100 expression in rat hippocampus. This figure shows the neuronal distribution of MP100 labeling in rat hippocampus. Prominent intracellularŽ . Ž . Ž . Ž . Ž . Ž .punctate staining arrows can be seen in pyramidal and granule cells. A and B , hippocampal area CA1; C and D , dentate gyrus; E area CA3. Bar

Ž . Ž . Ž . Ž . Ž .in A , C 70 mm; in B , D 30 mm; and in E 15 mm.

Ž .observed Fig. 5A whereas cellular structures both in thegranular and molecular layers were stained. In the latter,

parallel fiber axons terminating on Purkinje cell dendriteswere labeled. Electronmicroscopy confirms MP100 im-


Fig. 5. MP100 localization in rat cerebellum. MP100 is present in the molecular, Purkinje cell and granule cell layers as shown in panel A. HigherŽ .magnifications inset from A, shown in B reveal MP100 immunoreactivity in small, dot-like structures, especially dense around Purkinje cell somata

Ž . Ž .arrowheads , but also in the molecular and granule cell layer. Immunoelectronmicroscopy confirms that MP100 is localized at synaptic contacts C .Ž . Ž .Electron dense reaction product is present both in the presynaptic terminal arrows and at the postsynaptic density asterisks . No such staining was

Ž . Ž . Ž . Ž . Ž .observed when the tissue reacted with the pre-immune serum D . Bars in A 70 mm, B 10 mm, C and D 0.5 mm.

Ž .munoreactivity at synaptic contacts Fig. 5C . Electrondense reaction product could be identified in the presynap-

Ž .tic bouton arrows as well as under the postsynapticŽ .density asterisks . No such staining was observed when

Ž .the tissue reacted with the pre-immune serum Fig. 5D .No conclusive vesicle-associated staining was observed byelectronmicroscopy in the cell bodies of large neurons,possibly due to a suboptimal ultrastructural preservationduring the pre-embedding immunohistochemical proce-dure.

3.5. MP100 distribution in comparison with synaptic pro-teins

As described above, the distribution of MP100 wasmost pronounced as intracellular patches in pyramidal cellsof the cortex and hippocampus, in granule cells of thedentate gyrus, in Purkinje cells of the cerebellum and aspunctuate staining in the neuropil. To investigate whetherthe punctate MP100 pattern of distribution resemblessynaptic structures, immunostaining of MP100 was com-


ŽFig. 6. MP100, synapsin and synaptophysin in hippocampus. Hippocampal layer CA3 stained for synapsin, MP100 and synaptophysin A, B and C,.respectively . All three proteins show punctuate immunoreactivity that was most similar between MP100 and synaptophysin. Bar 30 mm.

pared to synapsin and synaptophysin, which are synapticmarkers in brain and periphery. Synapsin and synapto-physin showed the most prominent punctate labeling of the

Ž .neuropil in the cortex Fig. 3C and D and hippocampusŽ .Fig. 6A and C . As expected, neuronal cell bodies werehardly stained with neither synapsin nor with synapto-physin since the bulk of these proteins is located atsynapses. The distribution pattern of MP100 in adjacent

Ž . Ž .sections of cortex Fig. 3E and hippocampus Fig. 6Bshared some similarity with the pattern particularly ob-served for synaptophysin. Also in fractionation of synapticmembrane proteins by gel filtration chromatographyMP100 co-purified with the peak of the synaptophysin

Ž .protein Fig. 2A,B ; synaptic b-APP was also found en-Ž .riched in these fractions C .

4. Discussion

Several metalloproteases are described in brain, whichare either biochemically similar to matrix metalloproteasesor are intracellular calcium-dependent metalloproteases. Of

these, some were found most active in soluble brain frac-tions whereas others occur in particulate fractions of adult

w xforebrain 26 . The metalloprotease MP100 was first iden-tified from soluble human brain extracts as a potential

w xb-secretase involved in the processing of b-APP 30while no other brain substrates are known to date. Itsaction is, however, not likely as a true b-secretase in vivow x11 , but could be as secondary protease involved in acomplex metabolism of b-APPs. The MP100 sequence

w xwas recently found to be highly homologous to PSA 11which can exist both as soluble as well as membrane-asso-ciated forms, the latter of which are restricted to brain and

w xmay be involved in enkephalin cleavage 5,22 . In thepresent study, MP100 expression was investigated in therat brain. Immunoblot analysis of crude brain extract re-vealed the presence of the 100 kDa active MP100 protein.The biochemical co-purification of MP100, synaptophysinand b-APP in synaptosomal fractions suggests that MP100is, similar to b-APPs, present at synapses, possibly withinvesicular organelles. In addition to the active form, thereare larger and smaller forms present in the isolated synap-


tosome preparations. The fast migrating ones could repre-sent degradation products indicative for a turnover ofMP100 while the larger ones might be inactivemembrane-associated enzyme precursors that become acti-vated by proteolytic removal of a pro-peptide, as it is

w xknown for many metalloproteases 20 . Light microscopyrevealed MP100 staining in vesicular-like compartments,possibly late Golgi or endosome structures in large neu-rons. On the ultrastructural level it was impossible toobserve electron dense reaction product associated withvesicular organelles probably due to non-optimal tissuepreservation. In the neuropil where the majority of synapsesare located a punctate staining pattern was observed thatclosely resembled that of well characterized synaptic pro-teins such as synapsin and, in particular, synaptophysinw x4,37 . Punctate synaptophysin staining corresponds to itspresence at synapses as shown by electron microscopy of

w xhippocampal cultures 7 . Electron microscopy in this studyshowed MP100 to be located both in the pre- and post-syn-apse. Also b-APPs are neuronal proteins highly abundant

w xat synapses 31 and they associate with certain classes ofmembrane-bound organelles, particularly with Golgi,

w xclathrin-coated vesicles or late endosomes 6 . The local-ization of MP100 in brain cells was evaluated in thecontext of intracellular pathways by which membrane-bound organelles move through the cells. Little is knownabout the trafficking events in neurons with their highlypolar structures and long processes. The absence of aribosomal machinery in the axon requires delivery ofproteins over long distances rather than local synthesis.b-APPs are usually delivered to the cell surface throughthe constitutive secretory pathway, and they were shown toundergo fast axonal transport to synaptic sites where they

w xare localized 14,31 and might perform basic functionsw x9,10 . A further indication for MP100 localization atsynapses comes from its punctate staining patter in theneuropil. Such sites are assumed to represent the majorityof cortical synapses, most of which are axo-axonal oraxo-dendritic terminals and such staining is characteristic

w xof proteins associated with synapses 4,37 . Further func-tional studies are required to determine the true substratesof MP100 in vivo and establish a possible involvement inmodulation of synaptic function.

Acknowledgements

The authors would like to thank Marcus Frank for helpwith the EM work and John Kemp and Grayson Richardsfor critical reading of the manuscript.

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Metalloprotease MP100: a synaptic protease in rat brain