Animal model for the pathogenesis of reactive amyloidosis

Reviews . . . .

Animal Model for the Pathogenesis of Reactive Amyloidosis Z. Ali-Khan, W. Li and S.L. Chan The pathogenesis of amyloidosis is not well understood. Here, Zafer All-Khan, Weihua Li and Sic L. Chan present a metazoan parasite mouse model of reactive amyloidosis, review the relationship between chronic inflammation and multiorgan AA amyloidosis and postulate how ubiquitin might fimction in the processing of serum amyloid A and in AA amyloid formation in the endosomes-lysosomes of activated murine reticuloendothetial cells. Amyloidosis describes a heterogeneous collection of systemic diseases characterized by the extracellular deposition of [3-pleated amyloid fibrils in various organs and tissues (for reviews, see Refs 1,2). At least 15 different amyloid precursor proteins form amyloid, with or without partial proteolysis1,2. All amyloid fibrils share certain physicochemical propertiesL2: fibrils 8-10 nm in thickness; congophilic and tinctorial properties; generally a high degree of insolubility under physiological conditions; and association with various proteins, including amyloid P component 1-~and glycosaminoglycans3. Ubiquitin (UB), one of the heat-shock proteins, also hinds to at least two amyloid proteins: reactive amyloid protein AA, derived from acute phase serum amyloid A4,5; amyloid A~2M derived from [32-microglobulin in chronic haemodialysis patients6; and co-localizes with amyloid A[3 derived from Alzheimer amyloid precursor protein 7. The pathophysiology of amyloidogenesis is not well understood. Although this review focuses on the biological characteristics of alveolar hydatid cyst (AHC, the larval stage of Echinococcus multilocularis), and on its ability to induce reactive AA amyloidosis in the infected hosts, it is appropriate to summarize here some aspects of AA amyloid-related human diseases. A common pathway Gajduseka has proposed a common pathogenetic pathway for all forms of amyloidosis: (1) preliminary processing of the precursor into amyloid subunits or monomer, (2) nucleation-induced configurafional change in the subunit into a cross [~-pleated configuration; and (3) formation of oligomers or polymers, which may polymerize to form amyloid fibrils. The implication is that an understanding of the pathogenetic processes involved in one chemical form of amyloid may help elucidate the underlying mechanisms for other amyloids as well. With the discovery, about 30 years ago, that certain mouse strains become amyloidotic after repeated (25 to 30) daily injections of casein, an animal model of reactive amyloidosis came into being9. This led to numerous experimental approaches to identify the stimuli Zafer Aft-Khan, Weihua Li and Sic L, Chan are at the

Department of Microbiology and Immunology. McGi;i University. 3775 University Street, Montreal, Quebec, Canada H3A 2B4.

Tel: +1 514 398 ]9]0. Fax: 4"1 SI4 398 7052, e-maih A D I [email protected]

Parasitology Today, vol. 12, no. 8, 1996

that trigger amyloid formation. In 1983, we describe:d the AHC-mouse model of reactive amyloidosis10. Animal bioassay, immunocytochemcial and immunogold electron microscopy studies indicate that hepatocytederived serum amyloid A protein (SAA), the precursor of protein AA amyloid, in conjunction with UB, is targeted to endosomes-lysosomes in activated murine monocytoid cells5,11;endosomes-lysosomes have been implicated in SAA processing and AA formation (Ref. 11 and references therein). Ubiquitin (UB) is a highly conserved (8kDa) eukaryotic stress protein 12. Its expression is upregulated during inflammation and during Jarious stress-related conditions4,:z. On passive transfer, purified UB induces accelerated amyloidogenesis in the recipient mice13-15;this activity is functionally a:~alogous to amyloid-enhancing factor (AEF.16, which is believed to act as a common pathogenetic link in various types of amyloidosis and, thus, to be one of the crucial factors in amyloidogenesis~7.~s.How UB might function as AEF is still unclear. Nonetheless, the AHC mouse has provided an important clue to an interesting biological property of UB. We are now pursuing this concept in order to elucidate UBmediated mechanisms that regulate the metabolism of amyloidogenic mouse SAA (see below). Clinical aspects Reactive amyloidosis, characterized by the tissue deposition of insoluble AA, is an infrequent but a IX>" tentially serious complication of inflammation-associated diseases l~. It is associated with various chronic infectious and inflammatory diseases (tuberculosis, leprosy, osteomyelitis, familial Mediterranean fever, juvenile and adult rheumatoid arthritis), and certain neoplasms 1~. Induction of AA amyloidosis has been recognized in a number of parasitic diseases, such as leishmaniasis~9,malaria 20, filariasis2~, schistosomiasis22 and alveolar hydatid disease23~4. Ozeretskovskaya et al. 24 found signs of renal disorders in 25 of 35 alveolar hydatid disease patients and amyloid deposits in spleens, livers and kidneys of patients with the metastatic form of alveolar hydatid disease; renal failure was found to be one of the most frequent causes of death in these patients. Of the four liver and two kidney samples examined from Alaskan alveolar hydatid disease patients, we found AA deposits in two of the four liver samples23. Implicit in these reports is the strong possibility of AA amyloidosis occurring as a complicating factor in chronic bacteria[ and parasitic diseases. In a recent hydatidosis meeting he~d in China and summarized by Craig2s, alveolar hydatid disease is recognized as a major public health problem in several provinces in China. Clinical aspects of alveolar hydatid disease were not the focus of this meeting, and the unanswered question ~ a i n S aS to whether any of the Chinese alveolar hydatid d!~a ~ patients aisplayed signs ano symptoms or reactive amyloidosis. ....

c~,~,t© 19~6.k,se~rs...... u~ A,.~ht. . . . . . d 0,6~ 4~8~i~:0o

Reviews Box 1. A Schematic Presentation of the Key Events in Reactive Amyloidosis Chronic inflammation, a sequela of alveolar hydatid cyst (AHC) infection in mice, leads to the induction of two inflammation-associated acute phase and stress responses and overexpression of their respective reactants sermn amyloid A protein (SAA) and ubiquitin (UB). Published reports show the following sequence of events (see Fig. below): increased expression and release of UB by activated monocytoid cells 4 (a); increased synthesis and secretion of SAA by hepatocytes induced by activated monocytoid cell-derived interleukin (IL)-I, IL-6 and tumor necrosis factor (TNF)2 (b); and co-deposition of SAA and UB in the tissue interstitium4 (c), which, during the preamyloid phase, may lead to UB-SAA, ~mplex formation. That UB interacts avidly and selectively with murine SAA is shown in Ref. 37. Both UB and SAA as well as UB-associated AA amyloid are found in the endosomes-lysosomes (EL)S,N. We hypothesize that UB may have a processing role for the short-lived SAA at the afferent end; UB, by interacting with SAA (as proposed in Ref. 38) may alter the molecule in such a way as to make it more susceptible to phagocytosis by activated monocytoid cells and EL-mediated degradation. Sustained overproduction of SAA followed by incessant accumulation of UB-SAA complexes in the EL may lead to incomplete/abnormal SAA degradation, and thus AA fibril formation1L~.

manifestations m a y include diarrhoea, malabsorption, motility disturbances, mucosal friability and intestinal perforation 26. AA amyloidosisrelated hepatosplenomegaly and atrophic changes in the adrenal gland leading to adrenal insufficiency have also beea reported 27. Am yloidosis is clearly a progressive disease, involving possibly every organ and tissue. The biochemical processes involved in the conversion of amyloidogenic SAA into fibrillar AA amyloid are unclear and remain to be elucidated 1,2. The AHC parasite-mouse model

The discovery of the AHCinfected m o u s e model of reactive amyloidosis was accidental. Mice infected intraperitoneally with 50AHC, at 12weeks post-infection, demonstrated atrophic changes in AHC-infectedmouse the thymus, lymphadenopathy, plasmacytosis in the paracortical Acute/chronic inflammation areas and hyaline eosinophilic areas Activated monocytoidceils in the spleen 1°,29,3°.The hyaline areas were subsequently identified to be J ~ "*.,t-, amyloid deposits of AA typet0,31. a G . . . . . lized st . . . . . . . p . . . . ~ ~ ~ I L - 6 / Since 1983, we have studied amyloid susceptibility in several m o u s e strains (C57L, C57BL/6, CBA, A/J, ]Ubiquitin. (UB)I BALB/c, C3H/HeSn and C3H/HeJ), the effects of graded A H C doses Increased cellular expression b Acute phase response (10, 20, 50,100 and 250 AHC) on the amyloid induction period and s o m e Extracellular release 1 aspects of AA pathogenesis 4,5,1t,32. NNN~ [ Serum amyloid A (SAA)] Experimental A H C infection in Increased synthesis and secretion mice triggers a p r o m p t influx of inflammatory cells at the inoculation / site 33. This is mediated by highly C Depositionof UB-SAA in extracellulartissue matrix phlogistic AHC-elaborated products ~. Despite heavy leukocyte infiltration into their matrix, A H C s SAA-UBinteraction grow as vascularized tumour-like masses in various soft organs, with Interna izationof SAA-UB 1 complexand endosomalthe potential to metastasize35. The lysosomal (EL)degradationby m0hucyt0idcells / infected mice appear not to control Overloading of EL with SAA-UB complexes the proliferation or metastasis of AbnormalSAA clearance Normalclearance- SAA degradation A H C effectively, nor are they able to abrogate the infection 3~,32,35. GenIncomplete SAA degradation eralized lymphadenopathy, anemia, Formationof UB-associatedamyloidin EL splenomegaly (up to fivefold), Exoeytosis and/or release efUB boundamyloidextraeellulady peripheral leukocytosis, loss of subcutaneous and retroperitoneal fat, and depression of cell-mediated Ara.yloid AA-related complications also occur in immunity correspond with the increasing AHCpati:~nts with rheumatoid arthritis 26-28. As in h u m a n parasite biomass in mice -~0,32.36.In short, the proliferatalveolar hydatid diseasea4, chronic AA-related nephing AHC acts as a potent amyloidogen in mice. This ritis is the principal cause of death in rheumatoid leads to the induction of two inflammation-related arthritis patients (5.8% male and 12.8% female) 27. acute phase and stress responses and the upreguReports based on autopsy samples indicate that u p to lation of their expressed reactants SAA and UB, 50% of patients with reactive amyloidosis develop respectively (Box 1)4. The mechanisms responsible amyloid lymphadenopathy2S. Gastrointestinal (GI) for the triggering of UB and SAA responses in the amyloidosis has a m u c h higher incidence (up to 98%) A H C - m o u s e appear to be analogous to those in patients with systemic amyloidosis and the clinical described previously 1,2,4,m. ~

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Parasitology Today, voL 12, no 8, i ?96

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Fig. I. (left) Congo red-stained sections of spleens at one week (a) and 12weeks (b) post-infection: amyloid was detected using fluorescence microscopy (rhodamine filter); note amyloid deposits in perifollicular sinus (PS) walls (arrow) and its extension into the pulp (a); and, at 12 weeks post-infection, amyloid has obliterated the PS, extended into the follicle and the pulp (b) and has also involved the follicular arterioles (arrows). Scale b~rs = 20 p.m (a), 12 Itm (b). Fig. 2. (middle) Detection of hepatic amyloid deposits by Congo red stain using fluorescence microscopy (rhodamine filter) (a,b) and immunohistochemically (c) in C57BU6 mice at one week (a) and 12weeks (b.c) post-infection. Liver section was reacted with monospecific rabbit anti-mouse AA amyloid IgG and the bound antibody was detected with biotinylated donkey anti-rabbit IgG and avidin-peroxidase complex 4. Note light (a) and heavy (b) fluorescent or immunopositive (c) amyloid deposits involving the sinusoids (arrowheads) and the portal vessels (arrow). Scale bars = 12t~m (a), 20p.m (b). 71~m (c). Fig. 3. (right) Detection of gastric (a,b) a,',d renal (c) amyloid deposits by Congo red stain (a) using fluorescence microscopy (rhodamine filter) and immunohistochemically (b.c) (as described in Fig. 2c) in C57BL/6 mice at four weeks (a), 12weeks (b), and eight weeks (c) post-infection. Note perivascular and connective tissue amyloid deposits forming a network in the gastric mucosa (a,b) and expanded renal tubules and immunopositive amyloid deposits in kidney glomeruli and interstitially in the medulla (c). Scale bars = 7 p.m (a)o7 i~m (b), 20 p.m (c).

lntraperitoneal infection of mice with 10AHC is non-amyloidogenic 32. With 50AHC infection, the amyloid induction period is approximately six weeks post-infection"~.32; this period is reduced to six days with 250AHC 32, indicating a dose-dependent amplification of the mediators involved in AA amyloidogenesis. Indeed, both a higher inflammatory response and elevated levels of SAA are seen in mice infected with 250AHC 4.~4.32. The crucial changes in the preamyloidotic phase include expansion of the reticuloendothelial cells (Kupffer cells and monocytoid cells in the splenic perifollicular areas) 3°.32.3~, upregulation and redistribution of UB in the reticuloendothelial cells4,5, co-deposition of SAA and UB in the splenic perifoUicular areas followed by co-localization of SAA and UB to endosomes-lysosomes in the reticuloendothelial cells 4.-~.ll. This preferential colocalization indicates a SAA-processing role for UB, although more detailed studies are required to understand the functional implication of this phenomenon. This phase is followed by active amyloid formation and its tissue deposition; UB-associated AA are found in the endosomes-lysosomes, as well as extraceltularly 4,5,n,3~. The intriguing possibility is that UBassociated AA, formed in the endosomes-lysosomes, might then be excytosed, as proposed previously N. Since activated monocytoid cells do not appear to phagocytose native amyloid fibrils, the sequence of Parasitology Today. rot. 12. no. 8. 1996

events described above is in agreement with the formation of AA in the endosomes-lysosomes (Ref. 11 and references therein). The primary targets of amyloid deposition are the marginal zone sinuses in the spleen (Fig. la), portal and central veins and sinus walls in the liver (Fig. 2a), and both the mucosal and submucosal blood vessels in the GI tract (stomach, duodenum, jejunum, ileum, large intestine) (Figs 3a, 4a). Progressive amyloid deposition between six days and 12weeks post-infection significantly distorts the splenic architecture (Fig. lb); AA-related daanges in the liver include hepatocyte chord atrophy and disruption of the trabeculae including the portal and centrilobular vessels (Fig. 2, b and c). Similar obliterative changes occur in the kidneys, which include glomeruli, mesangium, renal blood vessels and the renal papilla 1°,29(Fig. 3c). As stated above, AA deposition in the GI tract starts as early as six clays post-infection in the 250 AHC-infected mice. By 12 weeks post-infection, AArelated atrophic changes in the villus core (Fig. 4, b and c) lead to epithelial cell degeneration and protrusion of the interstitial collagen (Fig. 4b); the Peyer's patch blood vessels also show amyloid angi0pathy (Fig. 5). Collectively, these findings in the AHC-mice

Reviews

.., Fig. 4. Congo red-stained sections from C57BL/6 mice of ileum at one week (a, oblique sections), t2weeks (b, transverse section) and one week (c, oblique sections of villi) post-infection. Amyloid was detected by fluorescence microscopy (rhodamine filter). Note amyloid deposit perivascularly in the rnucosa and subrnucosa (arrows) and in the lamina propria (arrowhead) (a), amyloid in the villus core. sloughed epithelium (arrowhead) (b), and exposed amyloidotic interstitial collagen at the villus tip (arrows) (b), and encasement of capillaries and lacteals by amyloid (c). Scale bars =7p.rn (a), 7txm (b), 3txm (c).

~o red-stained turn showing a Peyer's patch J6 mouse at post-infection. microscopy liter) was used ascular am),loid )ws). Also note /Ioid deposition li (arrowheads). Izm.

number of the soft organs (kidney, pancreas and adrenal glands) do not become amyloidotic until 4-6 weeks postqnfection, even in the 250 AHCinfected mice. We have no objective explanation for this phenomenon. Figure 6, a and b illustrate amyloidrelated changes in the pancreas and adrenal glands, respectively. Initially AA appears as focal deposits at the adrenal cortico-medullary junction. • With time, the fucal deposits coa-lesce to form a band and extend peripherally along the connective tissue. This eventually involves the cortical blood capillaries and disrupts the cortical cell masses in the zona fasciculata (Fig. 6a). In contrast, the pancreatic involvement is less severe; the islets of Langerhans are spared although AA deposits are present in the pancreatic interlobular septum, in most of the large and medium-sized blood capillaries and in the connective tissue around the pancreatic excretory ducts (Fig. 6b). Studies on reactive amyloidosis in normal and immunocompromised mice infected with Sdlistosotna japonicure have shown a link between immunological reactivity and amyloidogenesis; the T-cell-deprived mice were most susceptible to amyloidosis and demonstrated relatively severe amyloid-related obliterative changes in the tissues4°. These observations are in some ways compatible with our findings in the AHCinfected mice 3°,36. Biological features of reactive amyioldosls In rheumatoid arthritis patients, recurrent inflammation coincides with an increase in SAA concentration; these are believed to be the principal predisposing factors to AA amyloidosis1,2. Previous studies in mice and, to a limited extent, in humans have established that inflammatory macrophage-derived cytokines interleukin 1 (IL-1), 1L-6 and tumor necrosis factor (TNF) enhance the production of SAA by the hepatoeytes and are thus critical in the regulation of SAA gene expressionL Biochemical analysis of purified proteins SAA and AA has confirmed that the N-terminal two-thirds of human SAA (or SAAz in mice) form AA amyloid 2. Thus, in humans and in experimental animals, the predisposing factors and the precursor-product relationship between SAA and AA are similar. What is unclear, however, is the role of certain accessory factors involved Parasitoiogy Today, voL 12, no. 8. 1996

Reviews .....

in A A amyloidogenesis. UB, recently identified to be functionally analogous to AEF, is one of them ~ 5 . it is k n o w n to be involved in ATP-dependent proteolysis of a b n o r m a l or short-lived n o r m a l regulatory proteins 12. Stress-related conditions, including chronic inflammation (as s h o w n in the AHC-mice), p r o f o u n d l y affect the cellular expression of UB 5,7,m4. A l t h o u g h the biological characteristics of UB expression and its functional relationship to AEF activity h a v e been explored in the A H C - m o u s e model, the crucial question is: h o w does UB w o r k as AEF? Box 1 provides clues as to h o w this m i g h t occur. The u n d e r l y i n g a s s u m p t i o n is that convergence of t w o inflammation-induced biological p h e n o m ena, the acute p h a s e a n d stress responses and interaction between their expressed reactants, SAA a n d UB, respectively, m a y lead to A A a m y l o i d o g e n e s i s ~4. Conclusions A central t h e m e of the current research in a m y l o i d osis ( i n c l u d i n g A l z h e i m e r ' s disease) is to define the m o l e c u l a r m e c h a n i s m s i n v o l v e d in the p r o c e s s i n g / c o n v e r s i o n of ' n o r m a l ' soluble a m y l o i d precursor proteins into i n s o l u b l e a m y l o i d fibrils. R e g a r d l e s s of the k n o w n b i o c h e m i c a l d i v e r s i t y of v a r i o u s a m y l o i d prec u r s o r p r o t e i n s L2, it s e e m s likely that a m y l o i d form a t i o n m a y b e g o v e r n e d b y one s i n g l e m e c h a n i s m : a n a b n o r m a l or defective clearance of the p a r e n t protein. O u r w o r k i n g h y p o t h e s i s (Box 1) p r e d i c t s that spont a n e o u s interaction b e t w e e n UB a n d SAA, l e a d i n g to U B - S A A c o m p l e x f o r m a t i o n in the tissue interstitium, m a y c o n s t i t u t e the p r i m a r y p h y s i o l o g i c a l m e c h a n i s m for the n o r m a l clearance of e l e v a t e d levels (up to 1000-fold) 2 of short-lived SAA (half-life 90 min) 2 d u r i n g the acute phase. UB is k n o w n to exert a 'chaotropic' effect on the s u b s t r a t e protein -~s. This m a y r e n d e r the interstitially d e p o s i t e d S A A m o r e s u s c e p t i b l e to nonspecific reticuloendothelial cell-med:ated p h a g o c y t o s i s f o l l o w e d b y their e n d o s o m e s - l y s o s o m e s - m e d i a t e d degradation. E v i d e n c e s u g g e s t s that A A a m y i o i d o s i s , e v e n in r h e u m a t o i d arthritis patients, is a rare condition; not e v e r y i n f l a m m a t o r y e p i s o d e results in A A a m y l o i d o s i s t,2. H o w e v e r , as a secondary, consequence, incessant o v e r l o a d i n g of the e n d o s o m e s - l y s o s o m e s w i t h UB-associated SAA d u r i n g chronic inflamm a t i o n m a y lead to i n c o m p l e t e SAA d e g r a d a t i o n a n d f o r m a t i o n of U B - b o u n d A A fibrils in the e n d o s o m e l y s o s o m e 5,u,~'~ (Box 1). As o t h e r p h a s e s of this prop o s e d p a t h w a y are e l u c i d a t e d , it will b e possible to g a i n i n s i g h t into d e s i g n i n g t h e r a p e u t i c strategies for the m a n a g e m e n t of reactive a m y l o i d o s i s . The A H C is clearly a potent a m y l o i d o g e n , a n d the AHC-infected m o u s e a p p e a r s to be a potentially useful m o d e l for investigations, in viva, on the pathogenesis of amyloidosis. With 2 5 0 A H C infection, the e m y l o i d induction period is shortened to six days. It is approxim a t e l y 3-4 w e e k s in the casein-treated m o u s e 2.~ a n d 9-30 m o n t h s in the a g e i n g m o u s e model4L Thus, both the m o l e c u l a r p a t h o g e n e s i s of a m y l o i d fibril formation a n d the evolution of A A amyloid-related pathological c h a n g e s in the affected o r g a n s can b e s t u d i e d in the AHC-infected mice w i t h i n a r e a s o n a b l y short time.

Acknowledgements This work was supported by a grant from the Medical Research Council of Canada (MA-11426).

Parasitology Today, vol. 12, ~o. 8, 1996

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28 Kaiserlung, E. and Krober, S. (1994) Lymphatic amyl0id~is, previously unrecognized form o f amyloid deposition in gen~ eralized amyl0idosis. Histot~#hology 24, 21~Z21

Reviews 20 All-Khan, Z., Jothy, S. and Siboo, R. (19821 Amyloldosis in experimental murine alveolar hydatldosis. Trans. R. Soc. Trop. Med. Hyg. 76,1690-1710 30 Ali-Khan,Z. (19781Cellular changes in the lymphorelicular tissues of C571d! mice infecled with Echhtococcus nndlilocularis cyst. hmnunola~y 34,831-839 31 AIkarmi, T,O., All-Khan, Z. and Zarkadas, C.G. (1986) Characterization of amyloid protein from mice infected with alveolar hydafid cyst: Isolation, purification and amino acid composition. Exp. MoL Palhal.45, t42-159 32 Du, T. and All-Khan, Z. (1990) Pathogenesls ef secondary amyloidosis in alveolar hydatid cyst-mouse model: Histopathology ;rod immuno/enzyme-histochemical analysis of splenic marginal zone cells during amyloidogenesis. ]. EaTs. Patht;L71, 313-335 33 Treves, S. and Ali-Khal~, Z. (19841 Characterlz.~tion of the inflammatory cells in progressing tumor-like al~, olar hydatidcysts: 1. Kinetics and composition of inflammatory infiltrate. Trop. Med. ParasitoL35,183-188 34 Alkarmi, T. and Ali-Khal,, Z. (19891 Phloglstic and chemotactic activities of alveolar hydiltid cyst antigen. I. Par,l.,itoL75, 711-719

35 All-Khan, Z. et al. (1983) Cytolytic events and the possible rod of germinal cells in metaslatls i~ chronic alveolar hydalidosis. Ann. Trop.Mat. i;arasilol.77, 4.97-312 36 All-Khan, Z. (1978) Echinococc'~t.~ tnultilocularis: cell-mediated immune response in early and chronic alveolar routine hydafidosis. Exp. Parasitol.46, ! 57-165 37 Chan, S.L., Bell, A.W. and All-Khan, Z. (19951Mouse SAAI and SAAz bind avidly to head "denatured" ublqultln. Amyh~id:hit. J. Exp. Clin. ha,est. 2, 257-264 38 Wenzel, T. and Baumeister, W. (lq93) Thermoplasma acidophilium proteosomes degrade partially unfolded and ubiquilinassociated proteins. FEBS Lett. 326, 215-218 39 Chronopoulos, S. et aL (19951 Colocalizalion of ubiquitin and serum amyloid A and ubiquitin-bound AA in the endosomeslysosomes: A double immunogold electron microscopy study. Atnyh~ht: hit. ]. Exp. C1i11.Invest. 2,191-194 40 Luty, A.J.F.,Mackenzie, C.D. and Matoney, N.A. (1987)Secondary amylrfid0sis in normal and immunocompromised raice infected with Schi;tosomajapanicum. Br. ]. Exp. Palhol.68, 839--845 41 Thnng, P.I. (1957) The relation between amyloid and aging in comparative pathology Gerontologla l, 234--25,1

The Paraflagellar Rod of Kinetoplastida: Solved and Unsolved Questions R Bastin, K.R. M a t t h e w s The flagellu111 of ahnost t~ery melnber of the Ki,etoplastida COlltains, 11ext to its canonical "nine-plus-two' aXOllClne strltcture , a unique, conlplex and highly organized lattice-like structure calh,d the paraflagellar rod o1" paraxia; ,'od. Here, Philippe Bastin, Keith Mattheu$ and Keith Gull sulnntarize the latest findings Oll its strltcture, the nature # its p,'ottin components and their corresponding genes. They also consider the possible functians of this intriguing organelle. The paraflagellar rod (PFR) was first identified in trypanosomes by Keith Vickerman in 1962 (Ref. 1) and several subsequent studies have precisely defined its organization2-% The PFR is a complex and highly organized lattice-like structure (Fig. 1) that runs adjacent to the axoneme throughout its length, except in the region within the flagellar pocket. However, in kinetoplastids with large flagellar pockets (such as Blastocrithidia), the PFR is present alongside the axoneme from a position that is within the outer half of the pocket 7. The PFR is present in all members of the Kinetoplastida studied so far, with the exception of some monogenetic parasites (see Box 1). All life cycle stages of kinetoplastids express the PFR, with the notable exception of Trypanosonla cruzi and Leishmania s p p in their amastigote stage where the remnant flagellum, with a very restricted axoneme, is confined to ti~e flagellar pocket. A structure possibly related to the PFR of kinetoplastids has been identified in only two other types of organisms: the Euglenoids and the dinoflagellates (reviewed in Ref. 8). However, the PFR is an eniDnatic structure. !Is high-order organization and restricted ,ovoiutionary presence argue for a Philippe Bastin, Keith R. Matthews and Keith Gull are at the University of Manchester,School of Biological Sciences,2.205 Stopford Building, Oxford Road, Manchester, UK M 13 9PT. Tel: +44 161 2755108, Fax: +44 161 2755082t e-mail: [email protected] ~02

a n d K. G u l l

specific function in these organisms. Despite this, s o m e 30 years after its discovery, we are just starting to achieve a description, at the molecular level, of its composition and morphogenesis. ~itructure and m o r p h o g e n e s i s A cross-secUon of the flagellum of the procyclic form of Trypanosoma brucei is s h o w n in Fig. la and clearly reveals the substructure of the PFR. Three regions (proximal, distal and intermediate, relative to the axoneme) can be identified, and their structure has been clarified by extensive w o r k on Phytonlonas and Herpetomonas species h. The proxintal region is formed by t w o electron-dense 'plates' which are composed of t w b types of filaments: thin (7-10 n m diameter) and thick (25 n m diameter). Similar values have been m e a s u r e d for T. brucei 3,9 and for Crithidia fasciculata Io. At high magnification, the thick filaments appeared to be c o m p o s e d of two, closely adjacent thin filaments. T~e thick and thin filam e n t s intersect with an angle of 100 °, explaining the lattice-like structure of the rod w h e n seen on longitudinal sections (Fig. lb). The m o s t proximal plate is alw a y s connected by filaments to the axoneme t h r o u g h the g r o u p of microtubule doublets four to seven (Fig. la). In longitudinal sections, the connection appears as an alternation of simple structures it-like) and of V- or Y-like structures (40-50 n m long) with a 50-60 n m Periodicity",". Using a combination of quickfreezing and deep-etching techniques, Hemphill et al. u s h o w e d that these junctions are m o r e complex and have a fleur-de-lis-like aspect. This association between the PFR and axoneme is very tight and is not disrupted by non-ionic detergent, hypotonic shock or high-salt treatment, b u t it is trypsin sensitive 7,m,12. The distal region of the PFR is larger and is composed of u p to 11 electron-dense plates, comprising

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P,,,,.J~,tcdogyToday, vol. 12, no, 8, 1996