Acta Neuropathol (2007) 114:537–542 DOI 10.1007/s00401-007-0217-6

CASE REPORT

Myopathy with tubulin-reactive inclusions in two cats

G. Diane Shelton · Beverly K. Sturges · Leslie A. Lyons · D. Colette Williams · Monica Aleman · Yun Jiang · Andrew P. Mizisin

Received: 26 January 2007 / Revised: 5 March 2007 / Accepted: 6 March 2007 / Published online: 29 March 2007 © Springer-Verlag 2007

Abstract Many types of inclusions have been described Keywords Cats · · Crystalline inclusions · in human including but not limited to nemaline Tubulin rod bodies, cylindrical spirals, tubular aggregates, cytoplas- mic bodies, reducing bodies, and Wngerprint bodies, and hyaline inclusions in myoWbrillar myopathy and inclusion Introduction body . There are very few reports describing inclu- sions in spontaneously occurring myopathies in cats, and Inclusions described in human myopathies include nema- these reports are limited to nemaline rod myopathy. A line rod bodies, cylindrical spirals, tubular aggregates, cyto- myopathy with tubulin-reactive crystalline inclusions has plasmic bodies, reducing bodies, Wngerprint bodies, and recently been reported in a human patient with a clinical hyaline inclusions in myoWbrillar myopathy and inclusion presentation of and fatigue. Similarly, a myopathy body myositis [2]. These inclusions may be the primary with chronic, slowly progressive muscle has been pathologic change as in myopathies associated with nema- identiWed here in two unrelated cats. Inclusions were the line rods, tubular aggregates or cylindrical spirals [9], or only pathological change in biopsies and, may be associated with other myopathic abnormalities as in ultrastructurally, groups of crystalline structures were evi- myoWbrillar myopathy [11] and dent that had a subsarcolemmal or central location, rhom- [1]. Inclusions may be characteristic of a speciWc myopa- boid or rectangular shapes, lacked orientation, and were not thy, or may occur in various myopathies, such as the Wnd- membrane bound. The crystalline structures reacted posi- ing of tubular aggregates in disorders as diverse as periodic tively with an antibody against tubulin. This feline myopa- paralysis, congenital myasthenic syndromes, and muscle thy may be the equivalent of the human myopathy with disorders associated with , , or stiVness. tubulin-positive crystalline inclusions. There are very few reports describing inclusions in spon- taneously occurring myopathies in cats, and these reports are limited to nemaline rod myopathy. There is one report of a congenital nemaline myopathy in a family of cats [3] in which nemaline rods were the primary pathologic abnor- G. D. Shelton (&) · Y. Jiang · A. P. Mizisin Department of Pathology 0709, mality. There is also a single case report of a myopathy in a University of California, San Diego, La Jolla, cat in which rod bodies were found in conjunction with the CA, 92093-0709, USA accumulation of other proteins including desmin, dystro- e-mail: [email protected] phin and spectrin [7]. B. K. Sturges · D. C. Williams · M. Aleman A myopathy with tubulin-reactive crystalline inclusions Veterinary Medical Teaching Hospital, has been reported in a human patient with myalgia, mildly University of California, Davis, Davis, CA, USA elevated serum creatine kinase concentrations (2–5 times the reference range), and fatigue [13]. Examination of cryo- L. A. Lyons Department of Population Health and Reproduction, stat sections of muscle showed aggregates of small crystal University of California, Davis, Davis, CA, USA inclusions in the sarcoplasm of type 2 Wbers. The inclusions 123 538 Acta Neuropathol (2007) 114:537–542 were eosinophilic and stained bright red with the modiWed frightened, it would occasionally lose its balance and fall to Gomori trichrome stain, indicating a membranous compo- the Xoor, struggle and be unable to get up. At the time of nent. Ultrastructurally, the crystals were randomly oriented, presentation to the VMTH, the owner was unsure whether rhomboidal or rectangular, and not membrane bound. the signs had progressed in severity, although they did Immunohistochemical studies showed strong reactivity of seem to wax and wane. the inclusions to antibodies against tubulin. In addition, Abnormalities noted on physical examination included subsarcolemmal rhomboid or rectangular crystalline struc- signs of chronic upper respiratory infection and a body con- tures have been described in muscle from two human dition score of 3/9 indicative of mild-to-moderate general- patients with diabetic amyotrophy and neuropathy [12], and ized muscle wasting (1, extreme emaciation; 5, ideal body cystine crystals described in Wbroblasts in nephropathic weight; 9, extreme obesity). On neurological examination, cystinosis [6]. the cat had normal mentation and cranial nerve function. Recently, light microscopic, immunohistochemical, and Although it was able to walk and run without apparent diY- ultrastructural changes similar to the reported human culty, it would sit or lie down whenever possible. When sit- myopathy [13] were identiWed in muscle biopsies from two ting, it assumed a characteristic pose (Fig. 1a). The cat had cats with chronic, slowly progressive . mild cervical ventroXexion and, when supporting the head, Here we analyze the inclusions in detail and report that, in would appear to use momentum to swing and move it. In a these two cats, the sarcoplasmic crystalline inclusions also sitting or lying position, it would immediately Wnd some- react with anti-tubulin antibody. These observations point thing in the environment to rest its head on (Fig. 1b). The to the likelihood that this myopathy is the feline equivalent remainder of the neurologic exam was normal, as were a of the human myopathy. routine complete blood count, serum biochemical proWle including creatine kinase (CK) concentration, and a urinal- ysis. Electromyography, done under general anesthesia, Case descriptions showed no spontaneous activity or abnormal insertional activity. Waveform conWguration and motor and sensory Case 1 nerve conduction velocities were within normal limits for the feline peroneal nerve. Cord dorsum potentials and late A 4-year-old female spayed Devon Rex cat was donated to waves were present, and repetitive stimulation (1, 2, 3, 5, 7, the Veterinary Medical Teaching Hospital (VMTH), Uni- 10, 15, 20, 30, and 50 Hz) did not show a signiWcant decre- versity of California, Davis because of generalized weak- mental response. Biopsies of the quadriceps, cranial tibial ness and episodes of “spastic” behavior. The cat was and triceps muscles were obtained, as well as a biopsy of obtained from a breeder at 8 months of age, and was thin the peroneal nerve. and underweight. It also had a severe upper respiratory infection that was a recurrent problem. The cat had diY- Case 2 culty in swallowing soft foods and would often gag while eating. Whenever it was picked up, its legs would become A 6-year-old male neutered domestic shorthaired cat was stiV and grope wildly until Wnding something on which to presented with a 1-year history of slowly progressive weak- cling. Although able to ambulate without assistance, it was ness and generalized . Weakness began ini- much weaker than the other cats in the household. When tially in the pelvic limbs and then progressed to

Fig. 1 A 4-year-old female spayed Devon Rex (cat 1) with myopathy associated with crystalline inclusions in muscle Wbers. When sitting, it assumed a characteristic position (a). In a lying position, it would immediately Wnd something in the environment to rest its head on (b)

123 Acta Neuropathol (2007) 114:537–542 539 tetraparesis. General physical examination was considered sections stained with uranyl acetate and lead citrate for normal, and routine blood counts and serum chemistry electron microscopy. analyses including CK concentration were within reference ranges. With the exception of weakness and muscle atro- ImmunoXuorescence phy, the neurological examination was unremarkable with normal spinal reXexes and sensations, and normal mental After washing with 0.05 M Tris buVer solution (TBS, pH attitude and cranial nerve functions. An electromyogram 7.6) containing 0.2% Triton-X (TBST), the 8 m thick sec- was performed and revealed no abnormalities in insertional tions were blocked with 10% goat serum for 30 min. The activity or waveforms. Measurement of the tibial motor sections were then incubated with one of the following pri- nerve conduction velocity and amplitude of the compound mary antibodies in TBST containing 5% goat serum over- muscle action potential was within the reference range for night at 4°C: rabbit antibodies against desmin (1:20, cats. As a neuromuscular disease was suspected, muscle D8281, Sigma, St Louis, MO), -amyloid (1:200, 51–2700, and peripheral nerve biopsies were performed. Zymed, South San Francisco, CA), and ubiquitin (1:250, Z0458, DakoCytomation, Carpinteria, CA), mouse antibod- ies against myeloid leukemia factor 1 (MLF1, 1:100, Methods Abgent, San Diego, CA), two mouse antibodies against - tubulin (1:2000, T6074, Sigma, St Louis, MO; 1:1000, gift Histopathology and histochemistry from Dr. Donald Cleveland, University of California, San Diego), and a rat anti-tubulin antibody against the tyrosi- Immediately following collection, muscle biopsy samples nated form of -tubulin (1:2000, gift from Dr. Donald (cranial tibial, triceps, and quadriceps muscle from case 1, Cleveland). Following incubation with primary antibody, cranial tibial muscle from case 2) were Xash frozen in goat anti-rabbit Alexa-488 (1:500, Molecular Probe, isopentane pre-cooled in liquid nitrogen and the muscles Eugene, OR) or goat anti-mouse Alexa-594 (1:500, Molec- stored at ¡80°C until further processed. Sections were cut ular Probe, Eugene, OR) in TBST containing 2% goat 8-m thick in a cryostat and evaluated using a standard serum was added onto the sections and incubated for 2 h at panel of histochemical stains and enzyme reactions [4] room temperature. Finally, all sections were mounted in including hematoxylin and eosin (H&E), modiWed Gomori anti-fade mounting medium (Molecular Probe), cover- trichrome, periodic acid-SchiV, oil red O, myoWbrillar slipped and examined using Xuorescence microscopy adenosine triphosphatase (ATPase) reactions at pH 9.8, 4.6 (Olympus BX41). Control sections were performed in par- and 4.3, acid phosphatase, alkaline phosphatase, the allel without addition of the primary antibody or using non- reduced form of nicotinamide–adenine dinucleotide, succi- immune IgG. nic dehydrogenase, and the alizarin stain for calcium. Sec- tions were also evaluated for congo red reactivity using rhodamine Xuorescence. Results

Morphology and morphometry Histopathology and histochemistry

Inclusions from all muscles of cat 1 were characterized as Biopsies from the quadriceps, triceps, and cranial tibial to location, shape, and size, number per high power Weld, muscles were evaluated from cat 1 (Fig. 2) and the cranial and presence in speciWc myoWber type. Computer digitali- tibial muscle from cat 2 (not shown). Inclusions were the zation of light microscopic images in transverse planes only pathological change in muscles from both cats. Inclu- were evaluated with NIH image™ software (NIH image, sions were eosinophilic with H&E staining (Fig. 2a), bright Bethesda, MA, USA). Cross-sectional area and size in rela- red with the modiWed Gomori trichrome stain (Fig. 2b), and tion to the muscle Wbers were determined. Only areas with- restricted to type 2B Wbers (Fig. 2c,d). Vacuoles were pres- out artifacts and Wbers with distinct borders were measured. ent in <1% of muscle Wbers from the quadriceps muscle, 33% from the triceps muscle, and 10% from the cranial tib- Electron microscopy ial muscle from cat 1, and in 5% of the muscle Wbers from the cranial tibial muscle of cat 2. Other than a dark blue rim Glutaraldehyde-Wxed muscle biopsy specimens were post- around the inclusions, reactivity was negative for NADH Wxed in osmium tetroxide, and dehydrated in serial alcohol (Fig. 2e) and SDH (not shown). Light PAS positive stain- solutions and propylene oxide prior to embedding in aral- ing was present around the periphery of the inclusions dite resin. Thick sections (1 m) were stained with tolui- (Fig. 2f), but staining did not indicate the presence of gly- dine blue for light microscopy and ultrathin (60 nm) cogen, polysaccharide, or lipid (not shown) within the 123 540 Acta Neuropathol (2007) 114:537–542

Fig. 2 Fresh-frozen muscle biopsy sections from the triceps muscle of cat 1. Subsarcolemmal and central inclusions were eosinophilic with the H&E stain (a, arrows) and stained bril- liantly red with the modiWed Gomori trichrome stain (b). The ATPase reaction performed with preincubations at pH 4.5 (c) and pH 4.3 (d) localized the inclusions to type 2B Wbers. Although positive staining was present around the periphery of the inclusion (e, arrow), the inclusions were not positive for nicotinamide–adenine dinucleo- tide (e) or succinic dehydroge- nase (not shown) reactivity. Light positive staining was also evident around the periphery of the inclusions with PAS (f, non- serial section). Bar = 40 m for all Wgures

inclusions. The alizarin stain was negative for calciWc encompassed from 3 to 34% of the entire myoWber in both deposits within muscle Wbers or calcium within the inclu- the triceps and cranial tibial muscles. The cross sectional sions. The inclusions were not congophilic in either case area of the central inclusions ranged from 35 to 600 2 (not shown). (200 § 133 2; mean § SD), and from 332 to 4,463 2 (1,918 § 2,227 2) for the subsarcolemmal inclusions. Morphology and morphometry Electron microscopy With few exceptions, myoWbers containing inclusions retained a normal polygonal shape. In cross sections of Ultrastructurally, groups of crystalline structures were from muscle biopsies from cat 1, a total of 134 inclusions were both cats, had a subsarcolemmal or central location, rhom- observed in the triceps muscle with 112 and 22 located in boid or rectangular shapes, and lacked orientation (Fig. 3a, central and subsarcolemmal areas, respectively. Thirty- b). The crystals were not membrane bound. EM showed eight inclusions were observed in the cranial tibial muscle that they had a compact granular substructure with no dis- with 27 located centrally and 11 under the sarcolemma, cernible periodicity. Glycogen granules were scattered while the quadriceps muscle contained only one centrally between crystal structures (Fig. 3c, d). located inclusion. The number of inclusions per myoWber varied from one to three, with the majority containing a sin- Immunohistochemistry gle inclusion. In a transverse plane, inclusions were pre- dominantly oval in shape, while elongated in the Using a monoclonal antibody against tubulin (Sigma longitudinal plane. Central inclusions encompassed from 1 T6074), positive staining of inclusions was present in mus- to 3% of myoWber area for the triceps muscle, <1 to 3% for cles from both cats (Fig. 4). Cross-reactivity of the other the cranial tibial muscle, and <1% for the quadriceps mus- mouse and rat -tubulin antibodies with cat muscle was not cle in the transverse plane. The subsarcolemmal inclusions found at the dilutions used. The staining intensity in cat 2

123 Acta Neuropathol (2007) 114:537–542 541

Fig. 3 Electron micrographs of muscles from cat 1 (a, c) and cat 2 (b, d) demonstrate groups of crystalline structures having a rhomboid or rectangular shape and not membrane bound. The low power micrograph from cat 1 (a) shows a central inclusion and from cat 2 (b) subsarcolemmal inclusions. At higher power, glycogen granules and membranous material were scattered between crystal structures in both cats (c, d). Bar = 2.22 m for a, 1.82 m for b, 0.31 m for c, and 0.41 m for d

Fig. 4 Fresh frozen muscle biopsy sections were incubated with a monoclonal antibody against -tubulin (Sigma T6074) and evaluated by Xuorescence microscopy. Positive staining of inclusions was present in muscles from both cat 1 (a) and cat 2 (b). Bar = 55 m

(Fig. 4b), however, was stronger than in cat 1 (Fig. 4a), organelles, and chromosomes [8]. Microtubules, which which may be indicative of diVering amounts of tubulin in have intrinsic dynamic properties, are essential for myotube the structure of the inclusions. Positive staining of inclu- formation and allow the cells to respond mechanically to sions was not observed using antibodies against desmin, - the environment. The function of tubulin and MTs in skele- amyloid, ubiquitin, and MLF1 in either cat (not shown). tal muscle after diVerentiation is still unknown. To date, of the many known varieties of inclusions, only nemaline rod bodies have been described in cats [3, 7]. As Discussion far as we know, this is the Wrst description of crystalline inclusions in myoWbers in this species. Similar to the clini- Crystalline inclusions, similar in structure and composition cal course in the human patient with myopathy associated to those in a previously reported human myopathy [13], with sarcoplasmic crystalline inclusions [13], both cats in were found within muscle Wbers from two cats with this report had a chronic, slowly progressive course of clin- chronic, progressive muscle weakness. This Wnding demon- ical signs. Although myalgia was reported in the human strates that these crystalline inclusions are not speciWc to patient, it was not described by the cat owners or noted on humans but occur across species lines. Similar to the physical examination. However, muscle pain can be diY- human patient [13], the crystalline inclusions stained posi- cult to evaluate in animals. Neurologic examinations sup- tively with an antibody against tubulin. Tubulin is a hetero- ported a myopathy rather than neuropathy, as spinal dimer of - and -tubulin, and is a major component of the reXexes were normal. Electromyography, measurement of microtubules (MTs) involved in movements of Xagella, motor nerve conduction velocity, and amplitude of the 123 542 Acta Neuropathol (2007) 114:537–542 compound muscle action potential was normal in both Acknowledgments The authors thank Dr. Steven Skinner, Oregon cases, and the serum CK concentration was not elevated. Veterinary Specialty Clinic, for referral of cat 2, and Dr. Eva Engvall for critical review of the manuscript. At the light microscopic level, the inclusions stained bright red with the modiWed Gomori trichrome stain, also similar to that of the human patient [13]. Other structures, References such as tubular aggregates, cylindrical spirals and nemaline W rods, also stain bright red with the modi ed trichrome stain, 1. Askanas V, Engel WK (2002) Inclusion-body myositis and myop- but can be further diVerentiated from the crystalline inclu- athies: diVerent etiologies, possibly similar pathogenic mecha- sions ultrastructurally. Although similar subsarcolemmal nisms. Curr Opin Neurol 15:525–531 rhomboid or rectangular crystalline structures have been 2. Banker BQ, Engel AG (2004) Basic reactions of muscle. In: Engel AG, Franzini-Armstrong C (eds) Myology. McGraw-Hill, New described in muscle from human patients with diabetic York, pp 691–747 amyotrophy and neuropathy [12] and nephropathic cystino- 3. Cooper BJ, DeLahunta A, Gallagher EA, Valentine BA (1986) sis [6], neither cat had clinically apparent diabetes mellitus Nemaline myopathy of cats. Muscle Nerve 9:618–625 or signs of cystinosis at the time of diagnosis. Further, pero- 4. Dubowitz V (1985) Histological and histochemical stains and reactions. In: Dubowitz V (ed) Muscle biopsy. A practical ap- neal nerve biopsies from both cats were normal. With the proach. Balliere Tindall, London, pp 19–40 exception of muscle weakness, no other clinical abnormali- 5. Engel AG, Banker BQ (2004). Ultrastructural changes in diseased ties were detected in either cat. muscle. In: Engel AG, Franzini-Armstrong C (eds) Myology. Accumulation of protein crystals in muscle is rare [5] McGraw-Hill, New York, pp 749–887 6. Gahl WA, Dalakas M, Charnas L, Chen KT, Pezeshkpour GH, Ku- and, in the case of tubulin reactive crystals, is thought to be wabara T, Davis SL, Chesney RW, Fink J, Hutchison HT (1988) a consequence of a defect in tubulin synthesis, processing, Myopathy and cystine storage in muscles in a patient with nephro- or degradation [13]. Similar to the Wndings in this study, pathic cystinosis. N Engl J Med 319:1461–1464 crystals appear mainly in type 2 Wbers. As shown by our 7. Kube SA, Vernau KM, LeCouteur RA, Mizisin AP, Shelton GD  (2006) Congenital myopathy with abundant nemaline rods in a cat. immunohistochemical stainings, -tubulin is a structural Neuromusc Disord 16:188–191 component of the inclusions. Staining for other proteins 8. Makowski L (1995) Taxol found on tubulin. Nature 375:361–362 such as desmin, -amyloid, and ubiquitin was negative. 9. North K (2004). Congenital myopathies. In: Engel AG, Franzini- Congo red positivity was also not obvious. Staining for Armstrong C (eds) Myology. McGraw-Hill, New York, pp 1473– 1533 MLF1, a protein highly expressed in skeletal muscle [15], 10. Sakurai T, Fujita Y, Ohto E, Oguro A, Atomi Y (2005) The de- was also negative. crease of the cytoskeleton tubulin follows the decrease of the asso- Although the function(s) of tubulin and microtubules in ciating molecular chaperone alpha B-crystallin in unloaded soleus V muscle atrophy without stretch. FASEB J 19:1199–1201 skeletal muscle after di erentiation are still not understood, W W 11. Selcen D, Ohno K, Engel AG (2004) Myo brillar myopathy: clin- recent studies have suggested a signi cant contribution of ical, morphological, and genetic studies in 63 patients. Brain tubulin and microtubules to muscle plasticity and remodel- 127:439–451 ing of cellular architecture [10]. Recently, a role for alpha 12. ShaWq SA, Milhorat AT, Gorycki MA (1968) Crystals in muscle W B-crystallin was recognized during microtubule reorganiza- bers in patients with diabetic amyotrophy and neuropathy. Neu- rology 18:785–790 tion in muscle. Tubulin and microtubules were revealed to 13. Vu TH, Hays AP, Tanji K, Younger D, Gundersen GG, Eastwood be one of the substrates of alpha B-crystallin, and their A, Braun CW, DiMauro S, Bonilla E (2001) Myopathy with tubu- interactions may be key mechanisms for muscle adaptation, lin-reactive crystalline inclusions. 57:149–152 protection from atrophy, and muscle diVerentiation [10]. 14. Xi JH, Bai F, McGaha R, Andley UP (2006). Alpha-crystallin expression aVects microtubule assembly and prevents their aggre- Further, alpha B-crystallin expression has recently been gation. FASEB J 20:846–857 shown to prevent microtubule aggregation in lens epithelial 15. Yoneda-Kato N, Look AT, Kirstein MN, Valentine MB, Raimondi cells [14]. The cat may provide a spontaneously occurring SC, Cohen KJ, Carroll AJ, Morris SW (1996) The animal model for investigation of the interactions of alpha t(3;5)(q25.1;q34) of myelodysplastic syndrome and acute myeloid leukemia produces a novel fusion gene, NPM-MLF1. Oncogene B-crystallin and tubulin in myopathies associated with 12:265–275 tubulin-positive crystalline inclusions.

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