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(Rcccivcd 24 February. I t.u~2) (Revised. rccci,,ed I Jura:. I(>)u2) (Acccplcd 1() Jtmc, 1~.)2) Key word~: Am?,,otrophic lateral sclerosis: T h r o m b o s p o n d i n ; E x t r a c c l l u l a r matrix: B a s c n l c n t m e m b r a n e

Summao In an a t t e m p t to o b t a i n a biological m a r k e r for the e n i g m a t i c a n d fatal n c u r o h ) g i c d i s o r d e r , a m y o l r o p h i c lateral sclerosis (AI.S). scvei-al l a b o r a t o r i e s have e x p l o r e d a l t e r a t i o n s in wtrious e x t r a c e l l u l a r mnlrix c o m p o n c n l s in b o t h skeletal muscle and skin. W c have s t u d i c d the d i s t r i b u t i o n of f i b r o n e c t i n , himinin, h c p a r a n sulfatc p r o t c o g l y c a n ( I I S P G ) a n d collagen iypcs 1, 111 a n d IV. ahing x,¢iih lhc platelcl ~ - g r a n u l c g l y c o p r o t e i n , t h r o m b o s p ( m d i n (TSP). by i m m u n o f h l o r c s c e n c c in frozcn s e c i i o n s e l musclc from

control dcncrwtting conditions and ALS paticnts. In ALS and control muscle, types I ;.u)d Ili collagen wcrc localizcd 1o the cndomysium and the pcrimysium. Typc IV collagen and laminin prccisely dclineaicd each muscle fibcr (cndomysium t)r bascmcnl membrane) but did not slain thc pcrimysium. Wc found no marked quantilativc or qualitativc diffcrcnccs in the distributiotl of collagen types 1. 111 and IV, laminin, fibroncctin or I tSPG in AI~S p a t i c n t s compared to controls, t lowcvcr, when polyclomll antisct-a for TSP was used wc found a markcd increase in the deposition of this multi-domain glycoprotcin in AI,S patients' muscle comparcd to control muscle. Quantitative analysis of soluble extracts Irtml control and AI,S patients' muscle hy ELISA also indicated that TSP was incrcased in ALS. TSP is released from platclcl +~-gninulcs in response to thrombin stitnulati(m. TSP clcwtlion implics coagulation activity via the extravascular thrombolytic system in ALS and may corrclatc with rcgcncratiim. ()thor sludics havc indicated dccreascd circuhiting protcasc inhibitors and incrcascd scrine protcases in this disorder.

Introduction T h e c x t r a c e l l u l a r matrix ( E C M ) plays an i m p o r t a n t role in thc function and d e v e l o p m e n t of all tissues ( L c b l o n d and I n o u c 1989). Collagens, laminin, fib r o n c c t i n a n d p r o t e o g [ y c a n s are the m a j o r E C M comp o n e n t s of connective tissue. In a d d i t i o n , the last several years haxe w i t n e s s e d thc c h a r a c t e r i z a t i o n of o t h e r E C M g l y c o p r o t e i n s from various tissue sources. Bit)c h e m i c a l and u l t r a s t r u c t u r a l studies have d e m o n s t r a t e d the p r e s e n c e ()f t h e s e c o m p o n e n t s in the E C M of

I tS"c~cnI addre.s.s: Department el Neurosurgcry, MD Ande)son (':inter ('elliot, 15 I0 1h)Icoml~c Ave. l louston, TX 77030. USA. ('orreg~mt('m~' to: Bar)5, W. Feslol'f, MD, Ncurobiology Research Lab (151). Department of Vclcrans Affairs Medical Center. 4N01 Litlwood Boulevard. Kansas City, M() 64128. USA. Tel.: (NI6) 861 4700 cxl. 3551): ]ax (81(',) N61 Ill0.

n u m e r o u s tissues including skeletal muscle ( S c h u b e r t and L a C o r b i e r e 1980: M i l l e r ct al. lgt,)l). O n e of these is t h r o m b o s p o n d i n (TSP), a trimcric g l y c o p r o t e i n that is synthesized, s e c r e t e d and incorpor a t e d into the E C M by a variety of cclls including e n d o t h e l i a l , s m o o t h musclc cells and fibroblasts ( R a u g i et al. 1982: M o s h e r et al. 198;2). Like o t h e r E C M c o m p o n e n t s , T S P is also m u l t i - d o m a i n , i n t e r a c t i n g with h c p a r i n (Coligan and Slayter 1984), f i b r o n c c t i n (l,ahav et al. 1t,)82), fibrinogen (Dixit ct al., 1~184), p l a s m i n o g e n (Silverstcin et al. 1985), types I-V collagcn ( M u m b y et al., 1984; Galvin et al., lt)87), and h c p a r a n sulfate p r o t c o g l y c a n s ( H S P G s ; S a g e and Bornstein lt)91 ). Extensive p r o l i f e r a t i o n of connective tissue c h a r a c terizes several n c u r o m u s c u l a r d i s e a s e s of gcnetic origin. l n m T u n o t l u o r e s c e n c c studies c a r r i c d out in cascs of l ) u c h e n n e m u s c u l a r dystrophy, polymyositis and various n e u r o m u s c u l a r d i s e a s e s have d e m o n s t r a t e d notablc i n c r c a s e s in collagen types I and III (1)uancc et

100 al. 1980; Foidart et al. 1981). Alterations of ECM macromotecules lead to changes in composition, and, eventually, disruption of the basal lamina, thereby disturbing the critical relationship between nerve and muscle. In this regard, it has also been reported that increases in collagen types I and III are found in some, but not all, patients with amyotrophic lateral sclerosis (ALS) (Peltonen et al. 1982). The distribution of other skeletal muscle matrix components (laminin, fibronectin and HSPG) has not been studied in ALS patients. Only preliminary information is available on the content and distribution of TSP in human skeletal muscle (Wight et al. 1985). However, a recent study of direct muscle trauma in rats showed dramatic increase in extracellular accumulation of TSP during the regenerative phase (Watkins et al. 1990). Of interest to remodeling situations, TSP undergoes a peculiar biphasic pattern of distribution in muscle during development (O'Shea and Dixit, 1988; Hanta'/et al. 1991). In order to gain a better understanding of the distribution of these ECM components in ALS muscle, we carried out a comparative immunohistochemical study of the patterns for fibronectin, laminin, HSPG and types I, 111 and IV collagen in ALS, and disease control muscle. We also employed antisera to platelet a-granule-derived TSP to estimate the levels of this glycoprotein and its proteolytic fragments in ALS and control muscle extracts. A preliminary report of this work has appeared in abstract form (Rao et al. 1987).

Materials and methods

Kansas or the Neuromuscular Diseasc Center of the Kansas City Department of Veterans Affairs Medical Centers. Four fulfilled the criteria tot probable "classical" ALS (El Escorial WFN Working Group Meeting, June, 1990) and were biopsied within 12 months of onset. Controls all had a dcnervating condition: one (B) was considered normal by histochemical criteria and, clinically, had a mild chronic inflammatory demyelinating polyneuropathy (CIDP); another had mild denervation (D) due to mixed motor-sensory neuropathy, a third had severe CIDP (1F) and the last (2F) had clinically progressive muscular atrophy (PMA) who responded clinically to chelation. They were evenly matched for age and all were males, as were the ALS patients. After informed consent was obtained, an open muscle biopsy was performed and 200-400 mg of muscle tissue (either biceps or quadriceps) was removed. The specimens were rapidly frozen and mounted in OCT compound and cooled in liquid nitrogen. Transverse sections 8 /~m thick were cut in a cryostat and later air-dried on microscope slides.

Antibodies Polyclonal rabbit antibodies against collagen types 1, III, IV and V, laminin and fibronectin were kindly provided by Dr. Hynda Kleinmann (NIDR, NIH). Rabbit antibodies against HSPG were generously provided by Drs. John Hassall (NIDR, NIH) and Magnus H66k (University of Alabama at Birmingham) Rabbit potyclonal anti-TSP was donated by Dr. George Tuszynski (Lankenau Research Center, Philadelphia, PA). The specificity of these antibodies was characterized by Western blotting and shown to be monospecific.

Compounds and reagents OCT compound was obtained from Ames (Ames, IA). N-ethylmaleimide (NEM), phenylmethylsulfonyl fluoride (PMSF), benzamidine p-nitrophenyl phosphate disodium and goat anti-rabbit IgG conjugated to alkaline phosphatase were from Sigma Chemical Co. (St. Louis, MO). Aprotinin (Trasylol ®) was a generous gift of Dr. F. Schumann (Bayer AG, Wuppertal, FRG). e-Aminocaproic acid (EACA; Amicar ~) was a gift of Lederle Labs (Pearl River, NY). Fluorescein isothiocyanate-tagged streptavidin and biotinylated goat IgG against rabbit or mouse IgG was purchased from Amersham Corp. (Arlington Heights, IL). Mowiol 4,88 was purchased from Hoechst (Frankfurt, FRG). Nunc lmmulon-2 microtiter plates were obtained from Dynatech Labs (Chantilly, VA). BCA reagent was obtained from Pierce Chemical (Rockford, IL). Nitrocellulose was purchased from Scheicher and Schuell (Keane, NH).

lmmunocytochemistry The antibodies for these proteins were diluted with phosphate-buffered saline (PBS) and the sections incubated for 60 rain at room temperature. The sections were rinsed for 45 rain in PBS and then incubated with the specific rabbit or mouse IgG for the respective ECM antigen for 60 min at room temperature. After washing in PBS sections were then incubated with biotinylated goat anti-rabbit or anti-mouse IgG and then, after rinsing, with FITC-tagged streptavidin at room temperature for 60 rain. The sections were then rinsed in PBS followed by double quartz-distilled water and mounted in Mowiol mounting medium under cover slips (Heimer and Taylor 1974). Sections were observed using a Leitz Ultraphot microscope equipped with Pl6em narrow band FITC filters and the photomicrographs made with a Nikon UFX camera using Kodak Tri-X 400 ASA film.

Muscle biopsy Eight patients were recruited from either the Muscular Dystrophy Association Clinic of the University of

Muscle extracts Small amounts of muscle tissue (100-200 mg) were homogenized in a Brinkmann Polytron in buffer (1 : 10,

1(tl w / v : Ili(I m M l'ris-('l, 2 m M [ + I ) T A . p H 7.4) cont:iin+ ing ~l c o c k t M l o l l~roic;.tsc i n h i b i t o r s : H E M , b c t l Z a m i dine, e-lilllillt/Ciipl+oJC itcid ( E A C A ) . iiIld aprotJnhl, all :il 1 m M and p h c n y h l l o t h y l s u l f o n y l f l u o r i d e ( P M S F t , I).~ raM+

I sl`/S,,I "l'hc ct+ncuntr'or which s u r r o u n d s each muscle fiber) arid p o r i m y s i u m (the thicker connective tissue matrix s u r r o u n d i n g muscle fihcr b u n d l e s in which blood vcsscls and ncrvc t,a.igs ~nc located). In our

stud), anti-lilmmcctin, anti-type 1 nnd Ill colhigcn antibodius (l:ig. 1) all stained cndon1vsitl111 allCl pcrinlySitllll contirnling " "" " our previous rcsulls (thintai cl al. 1985). Thc fluorescence intcnsil\ ~ll the pcrimysiUlll was the strongest with anti-type 1 c~lhlgcn ;ullibodv. D e s p i t e tl slight increase of the l l u o r o s c c n c c intcnsJly duc to fibronoctJn in some of the AI.S p a t i e n t nnisclc biopsies, thcrc was rio significant d i f f e r e n c e holy t a capilI:.it-\, cxpansioi+i thesis is the lack o l itlct-ca',,u ii+i cLipillar) prt}lil¢,s idmtltifiud in both [amiriin ~tilct cl+lla~ut+ typu I V itnnlunoflut+rusccncL' IFi+,,. 2. 3 ) a n d t I + I S A dtita { I;thim I). Somu of the clovatcd T+t > dutcctcd in ,,\1 .S i'~atiunl:,, lnu,'-;clc i~ in the lt}I-ill of ;t dcgractcd Ir'nlint+7cn. and Ul{)killa:,,¢-P/\ diructl\ l',Il~..I. ('ell P,i~l.. I l l 7 : 2 7 2 7 274S. Pclluncn, I . N1),I!yI{L P,.. l ~ h m c n . /:. :lnc[ MyllyliL \ . V . lickS2) ('h:.illgC