Biochemical Characterization of a Lysosomal Protease Deficient in Classical Late Infantile Neuronal Ceroid Lipofuscinosis (LINCL) and Development of an Enzyme-Based Assay for Diagnosis and Exclusion of LINCL in Human Specimens and Animal Models Istvan Sohar, David E. Sleat, *Michel Jadot, and Peter Lobel Center for Advanced Biotechnology and Medicine; Department of Pharmacology, Robert Wood Johnson Medical School–University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey, U.S.A.; and *Laboratory of Physiological Chemistry, Faculte ´s Universitaires Notre-Dame de la Paix, Namur, Belgium Abstract: Classical late-infantile neuronal ceroid lipofus- cinosis (LINCL), a progressive and fatal neurodegenera- tive disease of childhood, results from mutations in a gene (CLN2) that encodes a protein with significant se- quence similarity to prokaryotic pepstatin-insensitive acid proteases. We have developed a sensitive protease activity assay that allows biochemical characterization of the CLN2 gene product in various human biological sam- ples, including solid tissues (brain and chorionic villi), blood (buffy coat leukocytes, platelets, granulocytes, and mononuclear cells), and cultured cells (lymphoblasts, fi- broblasts, and amniocytes). The enzyme has a pH opti- mum of 3.5 and is rapidly inactivated at neutral pH. A survey of fibroblasts and lymphoblasts demonstrated that lack of activity was associated with LINCL arising from mutations in the CLN2 gene but not other neuronal ceroid lipofuscinoses (NCLs), including the CLN6 variant LINCL, classical infantile NCL, classical juvenile NCL, and adult NCL (Kufs’ disease). A study conducted using blood samples collected from classical LINCL families whose affliction was confirmed by genetic analysis indi- cates that the assay can distinguish homozygotes, het- erozygotes, and normal controls and thus is useful for diagnosis and carrier testing. Analysis of archival speci- mens indicates that several specimens previously classi- fied as LINCL have enzyme activity and thus disease is unlikely to arise from mutations in CLN2. Conversely, a specimen previously classified as juvenile NCL lacks pepinase activity and is associated with mutations in CLN2. In addition, several animals with NCL-like neuro- degenerative symptoms [mutant strains of mice (nclf and mnd ), English setter, border collie, and Tibetan terrier dogs, sheep, and cattle] were found to contain enzyme activity and are thus unlikely to represent models for classical LINCL. Subcellular fractionation experiments in- dicate that the CLN2 protein is located in lysosomes, which is consistent with its acidic pH optimum for activity and the presence of mannose 6-phosphate. Taken to- gether, these findings indicate that LINCL represents a lysosomal storage disorder that is characterized by the absence of a specific protease activity. Key Words: Neuronal ceroid lipofuscinosis—Batten disease — Lysosomal storage disease —Proteinosis—Enzyme- based diagnosis. J. Neurochem. 73, 700 –711 (1999). The neuronal ceroid lipofuscinoses (NCLs) are a het- erogeneous group of related progressive neurodegenera- tive diseases (reviewed by Goebel and Sharp, 1998). To date, five childhood forms have been identified by clin- icopathological criteria and/or molecular basis: infantile NCL (INCL; OMIM entry 600722, where the defective gene is CLN1), classical late-infantile NCL (LINCL; OMIM 204500, CLN2), classical juvenile NCL (JNCL; OMIM 204200, CLN3), and two variant late-infantile forms (OMIM 256731, CLN5; and OMIM 601780, CLN6). All forms are characterized by visual failure, increasingly severe epileptic seizures, and mental dete- rioration, and all eventually prove fatal. LINCL has traditionally been differentiated from other forms of NCL by age of onset of symptoms and cellular morphology. Typically, onset of visual problems, sei- zures, and dementia occurs between 2 and 4 years of age, and death occurs between 8 and 15 years old. Electron microscopy of skin biopsy samples reveals cellular in- Resubmitted manuscript received March 3, 1999; accepted March 18, 1999. Address correspondence and reprint requests to Dr. P. Lobel at Center for Advanced Biotechnology and Medicine, 679 Hoes Lane, Piscataway, NJ 08854-5638, U.S.A. Abbreviations used: DCI, 3,4-dichloroisocoumarin; FITC, fluores- cein isothiocyanate; INCL, infantile neuronal ceroid lipofuscinosis; JNCL, juvenile neuronal ceroid lipofuscinosis; LINCL, late infantile neuronal ceroid lipofuscinosis; NCL, neuronal ceroid lipofuscinosis; pepinase, pepstatin-insensitive protease; TCA, trichloroacetic acid. 700 Journal of Neurochemistry Lippincott Williams & Wilkins, Inc., Philadelphia © 1999 International Society for Neurochemistry