453

We thank Dr P. Jahrling and Dr J. B. McCormick for their critical review of this manuscript; the many people who assisted in identifying, tracking, and caring for the animals, especially Dr T. Butler, Southwest Biomedical Foundation and Dr Stephen L. Pearson, Texas Primate Center, who performed some of the necropsies; the many people who cared for and helped us with the animals, especially Ms Lynnette Brammer, Ms Bertha Farrar, Ms Vonme Stone, and Ms Patricia Behr. We also thank Dr T. Sanchez, Ms A. Conaty and Mr S. Trappier for technical advice and assistance with PCR. Our espeaal thanks is for the help and support given us by Mr Lee Aldermann of the Office of Health and Safety, CDC. The animals used in this study were used according to a protocol approved by the Animal Care and Use Committee and were maintained in accordance with the Guide for the Care and Use of Laboratory Animals and the Animal Welfare Act. The animal facilities and programmes at CDC are fully accredited by the American Association for the Accreditation of Laboratory Animal Care (AAALAC).

REFERENCES

Marburg virus disease, clinical syndrome. In: Martini GA, Siegert R, eds. Marburg virus disease. Berlin: Springer-Verlag, 1971:

1. Martini GA.

1-9. 2. World Health Organization. Ebola haemorrhagic fever in Zaire, 1976. . Report of an International Commission. Bull WHO 1978; 56: 271-93. 3. World Health Organization. Ebola haemorrhagic fever in Sudan, 1976. Report of a WHO/International Study Team. Bull WHO 1978; 56: 247-70. 4. Heymann DL, Weisfeld JS, Webb PA, Johnson KM, Cairns T, Berquist H. Ebola hemorrhagic fever: Tandala, Zaire, 1977-1978. J Infect Dis 1980; 142: 372-76. 5. CDC. Ebola virus infection in imported primates-Virginia, 1989. MMWR 1989; 38: 831-38. 6. CDC. Ebola-related filovirus infection in nonhuman primates and interim guidelines for handling non-human primates during transit and quarantine. MMWR 1990; 39: 22-30. 7. Jahrling PB, Geisbert TW, Dalgard DW, et al. Preliminary report:

SHORT REPORT Low

cerebrospinal-fluid

concentrations of soluble amyloid &bgr;-protein precursor in hereditary Alzheimer’s disease

In Alzheimer’s disease, deposits of amyloid &bgr;-protein are apparently derived from intracellular processing of a large precursor protein. We have measured concentrations of this precursor in cerebrospinal fluid (CSF) from six members of a family affected by presenile Alzheimer’s disease associated with a point mutation of the precursor gene. One gene carrier with clinical signs of the disorder had low CSF concentrations of the precursor, similar to those of three patients with sporadic Alzheimer’s disease subsequently confirmed at necropsy. Two symptom-free gene carriers had CSF precursor concentrations similar to those of non-demented controls, though the value was lower in one, who had deficits revealed on neuropsychological testing, than in the other. These findings suggest that low concentrations of soluble amyloid precursor proteins in the CSF reflect the process that results in amyloid plaque formation and vascular deposition in Alzheimer’s disease.

isolation of Ebola virus from monkeys imported to USA. Lancet 1990; 335: 502-05. 8. Miranda ME, White ME, Dayrit MM, Hayes CG, Ksiazek TG, Burans JP. Seroepidemiological study of filovirus related to Ebola in the Philippines. Lancet 1991; 337: 425-27. 9. World Health Organization. Viral haemorrhagic fever in imported monkeys. Weekly Epidemiol Rep 1992; 67: 142-43. 10. Fisher-Hoch SP, Brammer TL, Trappier SG, et al. Pathogenic potential of filoviruses: role of geographic origin of primate host and virus stain. J Infect Dis (in press). 11. CDC. Update: filovirus infection in animal handlers. MMWR 1990; 39: 221.

12.

Wulff H, Johnson KM. Immunoglobulin M and G responses measured by immunofluorescence in patients with Lassa or Marburg virus

infections. Bull WHO 1979; 57: 631-35. 13. Gear J S, Cassel GA, Gear AJ, et al. Outbreak of Marburg virus disease in Johannesburg. BMJ 1975; 4: 489-93. 14. Fisher-Hoch SP, Platt GS, Neild GH, et al. Pathophysiology of shock and hemorrhage in a fulminating viral infection (Ebola). J Infect Dis 1985; 152: 887-94. 15. CDC. Update: evidence of filovirus infection in an animal caretaker in research/service facility. MMWR 1990; 39: 296-97. 16. Tomori O, Fabiyi A, Sorungbe A, Smith A, McCormick JB. Viral hemorrhagic fever antibodies in Nigerian populations. Am J Trop Med Hyg 1988; 38: 407-10. 17. Johnson BK, Gitau LG, Gichogo A, et al. Marburg, Ebola and Rift Valley Fever virus antibodies in East African primates. Trans R Soc Trop Med Hyg 1982; 76: 307-10. 18. Sanchez A, Kiley MP, Klenk HD, Feldmann H. Sequence analysis of the Marburg virus nucleoprotein gene: comparison to Ebola virus and other non-segmented negative-strand RNA viruses. J Gen Virol 1992; 73: 347-57. 19. Stegall MD, Chabot J, Weber C, Reemtsma K, Hardy MA. Pancreatic islet transplantation in cynomolgus monkeys. Initial studies and evidence that cyclosporine impairs glucose tolerance in normal monkeys. Transplantation 1989; 48: 944-50. 20. Henrickson RV, Maul DH, Osborn KG, et al. Epidemic of acquired immunodeficiency in rhesus monkeys. Lancet 1983; ii: 388-90.

Alzheimer’s disease is characterised by amyloid p-protein deposition in senile plaques and in cerebral vessels; the protein is apparently derived from intracellular processing of a large precursor protein.! Extracellular processing of the precursor seems to yield non-amyloidogenic soluble proteins. Concentrations of non-amyloidogenic amyloid p-protein precursor in the cerebrospinal fluid (CSF) are three to four times lower among severely demented patients diagnosed as having probable Alzheimer’s disease than among non-demented controls or demented patients without Alzheimer’s disease.2 Patients with the rare autosomal dominant disorder hereditary cerebral haemorrhage with amyloidosis-Dutch type, who carry a point mutation that leads to a glutamine-for-glutamate substitution within the p-protein domain of the precursor proteinalso have low CSF concentrations of the precursor amyloid-(3.4 We have analysed CSF from members of a family affected by presenile hereditary Alzheimer’s disease. Affected members have a point mutation at codon 717 in exon 17 (numbered by the 770 transcript of amyloid 0-protein precursor) resulting in a single aminoacid substitution (phenylalanine for valine) in the transmembrane domain of the precursor.5,6 The mean onset of clinical disease in this family is in the early 40s .6 CSF was collected from six members of the family (aged 31-45 years) by sterile lumbar puncture. CSF samples were immediately placed on dry ice and stored at -70°C. Three individuals were shown by direct sequencing of PCR-amplified genomic DNA to carry the point mutation,6 but at the time of the study only one of these patients had clinical signs of Alzheimer’s disease (subject M3; aged 44 years). One of the two symptom-free gene carriers (Ml; aged 35 years) had good intellectual abilities with no deficits in memory or cognition, and one (M2; aged 39 years) had impairment of verbal and visual recent memory, information processing, and conceptual reasoning, revealed only by neuropsychological testing. CSF samples were also obtained from three non-demented unrelated controls (aged 59-90 years) and from three patients

454

1-Soluble amyloid concentrations in CSF.

Fig

0-protein

precursor

(A(3PP)

Cl -C3 =non -demented controls; C4*-C6*= unaffected family members, M1-M3=carriers of point mutation leading to familial Alzheimer’s disease confirmed AD.

(AD);

AD1-AD3 = patients

with

necropsy

(53-74 years) diagnosed clinically as probably having Alzheimer’s disease, which was confirmed at necropsy. Concentrations of amyloid 0-protein precursor in CSF were measured by investigators unaware of the source of CSF with an enzyme-linked immunosorbent assays that uses a mouse monoclonal antibody to human protease nexin-2 (mAbp2-1); the antibody cross-reacts with all secreted isoforms of human amyloid 0-protein precursor. The sensitivity of the antibody means that as little as 5 III CSF is needed. Immunoblotting with mAbp2-1 was done for some subjects.2

Family member M3, who had clinical signs of Alzheimer’s disease, had low CSF concentrations of amyloid p-protein with necropsyconfirmed Alzheimer’s 1). Precursor concentrations in the other two gene carriers (Ml and M2) did not differ significantly (p < 0-05, Student’s t test) from those of the controls or the other family members who were not gene carriers (C4-C6; aged 31-42 years). However, the concentration in subject M2 (with deficits detectable on neuropsychological testing) was lower than that of the younger unaffected gene carrier Ml. Immunoblotting results were consistent with the quantitative measurements; subjects M3 and AD3 had virtually undetectable amyloid p-protein precursor, whereas all the other subjects tested (C3-C6, Ml, M2) had highly visible bands at the appropriate molecular mass (fig 2). Our findings suggest that CSF concentrations of soluble amyloid-(3-protein precursor reflect the disease process that results in neuritic plaque formation and vascular deposition of amyloid in the central nervous system of patients with familial Alzheimer’s disease associated with this mutation. Affected members of this family from previous generations showed clinical signs at ages 40-45 and died aged 48-58; the disease duration is typically only 7 years.6 The fmding that symptom-free gene carriers had normal CSF concentrations precursor, similar to those of

patients disease (fig

_

-

n,

of soluble precursor suggests that before clinical onset of the disease, at least in the pathways that would be measured by this assay, the precursor is normally processed and secreted. Alternatively, low CSF precursor concentrations may be associated with the disease process only after widespread amyloid deposition and neuronal loss. Low concentrations of soluble precursor proteins in CSF have been seen in other early-onset familial amyloidoses with known single aminoacid substitutions.3,9 In hereditary cerebral haemorrhage with amyloidosis (Icelandic type), a single aminoacid substitution in cystatin C leads to cerebral amyloid angiopathy with early-onset dementia and recurrent cerebral haemorrhages.8,9 The haemorrhages apparently result from the extensive deposition of amyloidotic fragments of cystatin C within the cerebral vessels. In the similar Dutch-type syndromeamyloidotic fragments of amyloid p-protein precursor are deposited within the walls of the cerebral vessels, resulting in recurrent haemorrhage and stroke.1O These findings and ours support the hypothesis that substantial reductions in concentrations of soluble amyloid precursor proteins in the CSF may indicate cerebral amyloid deposition in vessel walls or plaques resulting from excessive hydrolysis of precursor proteins produced by mutant genes, abnormal processing, or both. It remains to be seen, from a larger series of patients with both familial and sporadic Alzheimer’s disease, whether the measurement of amyloid 0-protein precursor will become a generally applicable diagnostic test for the disorder. Part of this work G.

was

supported by a public health service grant (P30 AG

10133) to B.

REFERENCES 1. Golde TE, Estus S, Younkin LH, et al. Processing of the amyloid protein precursor to potentially amyloidogenic derivatives. Science 1992; 255: 728-30. 2. Van Nostrand WE, Wagner SL, Shankle WR, et al. Decreased levels of soluble amyloid. &bgr;-protein precursor in cerebrospinal fluid of live Alzheimer disease patients. Proc Natl Acad Sci USA 1992; 89: 2251-55. 3. Levy E, Carman MD, Fernandez-Madrid IJ, et al. Mutation of the Alzheimer’s disease amyloid gene in hereditary cerebral hemorrhage, Dutch-type. Science 1990; 248: 1124-26. 4. Van Nostrand WE, Wagner SL, Haan J, Bakker E, Roos RAC. Alzheimer’s disease with hereditary cerebral hemorrhage with amyloidosis Dutch type share a decrease in cerebrospinal fluid levels of amyloid &bgr;-protein precursor. Ann Neural (in press). 5. Ghetti B, Murrell J, Benson MD, et al. Spectrum of amyloid &bgr;-protein immunoreactivity in hereditary Alzheimer disease with a guanine to thymine missense change at position 1924 of the APP gene. Brain Res 1992; 571: 133-39. 6. Murrell J, Farlow M, Ghetti B, Benson MD. A mutation in the amyloid precursor protein associated with hereditary Alzheimer’s disease. Science 1991; 254: 97-99. 7. Van Nostrand WE, Wagner SL, Suzuki M, et al. Protease nexin II, a potent anti-chymoptrypsin, shows identity to amyloid &bgr;-protein precursor. Nature 1989; 341: 546-49. 8. Grubb A, Jensson O, Gudmundsson G, Arnason A, Lofberg H, Malm J. Abnormal metabolism of &ggr;-trace alkaline microprotein. N Engl J Med 1984; 311: 1547-49. 9. Ghiso J, Jenssen O, Frangione B. Amyloid fibrils in hereditary cerebral hemorrhage with amyloidosis in Icelandic type is a variant of &ggr;-trace basic protein (cystatin C). Proc Natl Acad Sci USA 1986; 83: 2974-78. 10. van Duinen SG, Castano EM, Prelli F, et al. Hereditary cerebral hemorrhage with amyloidosis in patients of Dutch origin is related to Alzheimer’s disease. Proc Natl Acad Sci USA 1987; 84: 5991-94.

ADDRESSES

Departments of Neurology (M. Farlow, MD), Pathology (B Ghetti, MD) and Medicine (M. D Benson, MD), Indiana University School of Medicine, Indianapolis; Microbiology and Molecular Genetics, University of California, Irvine (W. E. van Nostrand, PhD); and Salk Institute Biotechnology/ Industrial Associates (SIBIA), La Jolla, California, USA (J. S Farrow, BS, S. L Wagner, PhD). Correspondence to Dr S. L. Wagner, Salk Institute Biotechnology/Industrial Associates, Inc, 505 Coast Boulevard

Fig 2-lmmunoblot analysis.

South, La Jolla, California 92037-4641, USA.