American Journal of Pathology, Vol 139, No. 3, September 1991 Copyright © American Association of Pathologists

Rapid Communication Amyloid Precursor Protein and Ubiquitin Immunoreactivity in Dystrophic Axons Is Not Unique to Alzheimer's Disease E. Cochran, B. Bacci,* Y. Chen, A. Patton, P. Gambetti, and L. Autilio-Gambetti From the Division ofNeuropathology, Institute of Pathology, Case Western Reserve University, Cleveland, Ohio, and the Department ofNeurobiology of Aging,* FIDIA Research

Laboratories, Albana Terme, Italy A distinctive feature ofAlzbeimer's disease (AD) is the presence of dystrophic neurites that immunoreact with antibodies to amyloid precursor protein (APP) and ubiquitin (Ub). The authors examined dystrophic axons (DA) present in other chronic conditions such as familial infantile neuroaxonal dystrophy (INAD), aging, cysticfibrosis, and biliary obstruction as well as in conditions of shorter duration such as

human immunodeficiency virus (HIV) leucoencephalopathy, infarction and radiation therapy to determine whether APP and Ub immunoreactivity was unique to the DA of AD. A large number of DA immunoreacted with antibodies to theA4, C- and N-terminal regions of APP as well as to Ub. Ub and APP immunoreactivities oftent but not always, colocalized 'Acute" DA generally reacted more intensely and in larger number with antibodies to APP than to Ub, whereas the reverse was true for "chronic" DA Structureless DA immunostained diffusely. In DA with cores or granules, the Ub immunoreaction was occasionally limited to these structures, whereas reaction with antibodies to APP was more diffuse. In view of the contention that impairment ofproteolysis is the common pathogenetic step in the formation of DA, Ub immunoreactivity in all DA may indicate a vicarious attempt to degrade accumulated components through an activation of the Ub systems The role of APP in the formation of DA remains to be determined (Am J Pathol 1991, 139:485-489)

Axonal dystrophy is a frequent alteration in human and animal pathology.1 It is characterized by focal enlargement of the terminal region of the axon, which is variably

filled with membranous profiles, lysosomes, and mitochondria.1'2 It is a distinctive feature of a group of familial diseases, the neuroaxonal dystrophies, but dystrophic axons (DA) are also associated with a variety of conditions such as aging, cystic fibrosis, diabetes1' 3'4 and can be induced experimentally with toxic and deficiency conditions.1' 56 Reactive axonal enlargements, morphologicalIly similar to DA, also form around infarcts of the white matter in axons abutting the infarcted area,' in areas exposed to radio- and/or chemotherapy8 and are often present in human immune deficiency virus (HIV) leukoencephalopathy.9'10 For simplicity, we will refer to all these reactive axonal enlargements as DA. Attention has been recently drawn to the presence of dystrophic neurites, neuronal processes inclusive of axons and dendrites, in Alzheimer's disease (AD). These neurites are associated with amyloid plaques and immunoreact with antibodies to amyloid precursor protein (APP) and to ubiquitin (Ub).11 To determine whether APP and Ub immunoreactivities are an exclusive characteristic of DA associated with amyloid plaques of AD or are shared by DA of different origin, we carried out an immunohistochemical study of DA in other pathologic conditions including familial infantile neuroaxonal dystrophy (INAD), aging, HIV leukoencephalopathy, cystic fibrosis, biliary obstruction, infarction, and radiation therapy. Part of this study has been reported.12

Materials and Methods Tissue Procurement and Processing Tissue from cases whose primary disease had been confirmed at autopsy was obtained from a) medulla of a 25Supported in part by NIA Merit Award AG08155 and NIH NS14509. B. Bacci was supported by FIDIA Research Laboratories. Accepted for publication July 9, 1991. Address reprint requests to Dr. L. Autilio-Gambetti, Division of Neuropathology, Institute of Pathology, Case Western Reserve University, 2085 Adelbert Rd, Cleveland, OH 44106.

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year-old subject with acquired biliary obstruction secondary to liver metastasis; b) medulla of a 39-year-old subject with cystic fibrosis; c) medulla of a 94-year-old subject with atrial fibrillation, hemiparesis and multi-infarct dementia; d) various regions of the brain stem white matter from two cases of HIV leukoencephalopathy of 35 and 38 years of age; e) the cervico medullatory junction and upper cervical spinal cord from a 8-year-old familial case of INAD (provided by Dr. D. Cowen of Columbia Presbyterian Medical Center (case 1 of reference 13); f) periventricular white matter of the centrum ovale from a 66-year-old subject with rectal carcinoma and multiple cerebral infarcts due to embolization; g) pons from a 16year-old subject with a brain stem glioma which had undergone radiation therapy.

Immunohistochemistry Paraffin sections of the aforementioned tissues, fixed in 10% unbuffered formalin or methacarn (methanol:chloroform:acetic acid, 3:2:1), were immunostained using the peroxidase-antiperoxidase (PAP) method.14 The following antibodies and dilutions were used: antiserum to Ub (1:800)15; antibodies to various APP regions, numbered according to Kang et al16: monoclonal antibody 22C1 1 (1:100)17 obtained from Boehringer Mannheim Biochimica) that recognizes an epitope located within residues 60 and 10018; antisera to residues 676-695 (1:1000),20 681-695 (1:100),21 and 597-624, in the A4 region, (1:300)22 Sections were treated with formic acid for 10 minutes before incubation with antiserum to the A4 region. Absorption of these antibodies with the corresponding peptides abolished immunostaining. For double immunostaining, reaction of the first antibody was detected by the alkaline phosphatase-antialkaline-phosphatase procedure22 with Fast Red as chromogen and photographed; sections were then incubated with the other antibody and reaction visualized with the PAP procedure14 using diaminobenzidine as cosubstrate.

Results DA were present in tissues from all the cases and displayed morphologic features previously reported (Figure 1).910132-27 DA associated with chronic conditions such as INAD, aging, and cystic fibrosis were often larger, displayed regions that appeared denser and more granular than the remaining content of the DA and formed either cores, targetlike or granular patterns (Figure 1A). In contrast, DA associated with conditions of

shorter duration, such as HIV encephalopathy, infarct and radiation were often structureless (Figure 1 B). A large number of DA immunoreacted with the antiserum to Ub and with at least two antibodies to APP (Table 1, Figure 2). In the larger DA present in the chronic conditions, the immunoreaction with all antibodies was often unevenly distributed, and was more intense in a central core or ring or formed a punctate pattern (Figure 2A, B). This pattern of immunostaining resembled that observed with conventional histologic procedures (Figure 1). On the contrary, immunostaining of small DA was generally uniform (Figure 2C). The immunostaining pattern with antibodies to APP appeared to be similar regardless of whether the antibody recognized the A4, the N- or the Cterminal region of APP (not shown). Differences in the intensity of reaction with antibodies to APP (Table I) could be due to variations in the length of fixation. When the immunostaining patterns of APP and Ub antibodies were compared after double immunostaining, several differences were observed: a) in the structured DA, the immunoreactivity of the two sets of antibodies often but not always colocalized (Figure 3A-D). When there was no colocalization, APP reactivity was often diffuse in the whole DA but more marked in certain regions that were the only ones immunostained by the Ub antiserum (Figure 3A, B). In other instances Ub immunoreactivity was present in regions unstained by antibodies to APP (Figure 3C, D); b) immunoreactivity to APP was generally stronger and present in a larger number of recent than old, structured DA (Figure 3E). The converse held true for Ub immunoreactivity (Figure 3B, D).

Discussion The current study shows that immunoreactivity with antibodies to APP and Ub is not an exclusive feature of the DA associated with amyloid plaques of Alzheimer disease but is shared by DA which form in a variety of other conditions. It also indicates that the presence of APP in DA does not necessarily lead to the formation of amyloid plaques. The finding of immunoreactivity with antibodies to various regions of APP, including the amyloidogenic A4 region, suggest that the entire APP is present in DA. Antibodies to A4 have been reported not to recognize intracellular APP due to unaccessible epitopes in this transmembrane region. The reactivity of DA with antiserum to A4 may indicate partial processing of APP and unmasking of A4 epitopes. DA are believed to form because of an impairment of the axonal transport.1' 28 The DA forming at the site of axonal transection, such as those associated with infarct, trauma, radiation, and HIV leucoencephalopathy, might

.p _

Figure 1. Dystrophic axons (DA) associated uith conditions of long and short duration. A: DA in infantile neuroaxonal dystrophty (INAD) general/lp larger and often contain targetlike or granular structures. B: DA in an area of uwhite-matter spongiosis of human immunodeficiency v-irus (HIV) encephalopathty are ofteni structureless. H&E, 400.

are

X

Figure 2. DA in all the conditions examined immunoreact with antibodies to amlloid precursor protein (APP) and to ubiquitin (Ub). The structured DA shou regions qf intense immunoreaction that are likelly to correspond to the targetlike and granular structures revealed by histological stains. A, B: INAD; C: HIVencepbalopathy; A: Antiserum to APP residues 597-624, B, C: Antiserum to Ub. PAPprocedure, X400.

Figure 3. Double immunostaining shous frequent but not constant colocalization of APP and Ub immunoreactivities. In structured DA, such as those found in INAD (A-D), APP (red) and Ub (brown) immunoreactivities often colocalize (A,B). Occasionally, intense Ub immunostaining isfocal u'bereas imnmunostaining by APP antibodies is either diffuse or lacks altogetherffrom Ub-reactive structures (C, D). DA present in conditions of short duration, such as those in an area surrounding an acute infarct (E), generally react more intensily with antibodies to APP than to Ub. A, C: Mlonclonal antibody to APP residues 60-100 (APAAP, red); B, D: Same sections as (A) and (C) after immunostaining uwith the antiserum to Ub (PAP, brou'n). E: Double immunostained as mentioned earlier. A, B: x 660; C, D: x 730; E: x 400.

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Table 1. Immunostaining of DystrophicAxons Antibodies

Dystrophic axons

Chronic INAD CF Aging BO Acute Tumor Infarct HIV

Ub

APP60-100 APP597624 APP676-695 ++ ++

++

++

++

++

++

++

++

+

+++

+

++

+++

+++

+ ++

+ +

+++ +++ +++

+++ ++ +++ + ++ +++

in Alzheimer's disease APP accumulating in DA contributes to the formation of amyloid deposits and why only DA of Alzheimer's disease are associated with extracellular amyloid deposits, although all DA appear to contain an excess of APP, remain to be determined.

Acknowledgments The authors thank Drs. B. Frangione, T. Ishii, and D. Selkoe for providing antibodies, and Dr. M. Fiori for his continuous support.

CF

= cystic fibrosis; BO = biliary obstruction. + + + = reaction strong in most DA; + + = reaction strong in some DA and weaker in others; + = reaction weaker with only occasionally strong staining of DA; + = reaction weak.

result from the accumulation of transported structures at the proximal stump of the transected axon.28 DA associated with aging or metabolic conditions, such as INAD, cystic fibrosis and BPAU intoxication are believed to be caused by an impairment of the "turn around" mechanism of the axonal transport, the process at the axon terminal that converts membranous structures conveyed distally by the fast anterograde component of the axonal transport into retrogradely transported organelles.1 28 The recent findings that administration of protease inhibitors consistently lead to the formation of DA28 30 and that familial cases of INAD are associated with a deficiency of the lysosomal enzyme acetyl galactosaminidase,31 have lead to the suggestion that proteases and glycosidases are necessary for the conversion of anterogradely transported membranous structures into retrogradely transported organelles and to the removal of these organelles from the axon tip.28.31 Interestingly, treatment of cells in culture with protease inhibitors results in accumulation of APP and Ub conjugates in lysosomes.32'33 Our present data indicate that the Ub system is activated in DA regardless of the cause leading to their formation. If impairment of proteolysis is indeed a common step in the formation of DA, participation of the Ub system may represent an attempt to eliminate components of membranous structures that have not "turned around" and were not retrogradely transported. The segregation of the Ub and, to a lesser extent, the APP reactivity into selective regions of the chronic DA raises the possibility that these are areas of intense proteolytic activity and may be rich in secondary lysosomes, proteasomes, or related structures. The role of APP in the formation of DA is unclear. APP has been shown to be a membrane protein that in neurons undergoes axonal transport with the fast component.3" Since membranes that probably originate from the smooth endoplasmic reticulum (1.2 and Bacci et al, unpublished) are the main component of the DA, it is unclear if the presence of APP in DA is selective or is secondary to the accumulation of membranes. Whether

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