Classical late-infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal neurodegenerative disease whose defective gene has remained elusive. A molecular basis for LINCL was determined with an approach applicable to other lysosomal storage diseases. When the mannose 6-phosphate modification of newly synthesized lysosomal enzymes was used as an affinity marker, a single protein was identified that is absent in LINCL. Sequence comparisons suggest that this protein is a pepstatin-insensitive lysosomal peptidase, and a corresponding enzymatic activity was deficient in LINCL autopsy specimens. Mutations in the gene encoding this protein were identified in LINCL patients but not in normal controls.

The late-infantile form of neuronal ceroid lipofuscinosis (LINCL) is a progressive and ultimately fatal neurodegenerative disease of childhood. The defective gene in this hereditary disorder, CLN2, encodes a recently identified lysosomal pepstatin-insensitive acid protease. To better understand the molecular pathology of LINCL, we conducted a genetic survey of CLN2 in 74 LINCL families. In 14 patients, CLN2 protease activities were normal and no mutations were identified, suggesting other forms of NCL. Both pathogenic alleles were identified in 57 of the other 60 LINCL families studied. In total, 24 mutations were associated with LINCL, comprising six splice-junction mutations, 11 missense mutations, 3 nonsense mutations, 3 small deletions, and 1 single-nucleotide insertion. Two mutations were particularly common: an intronic G-->C transversion in the invariant AG of a 3' splice junction, found in 38 of 115 alleles, and a C-->T transition in 32 of 115 alleles, which prematurely terminates translation at amino acid 208 of 563. An Arg-->His substitution was identified, which was associated with a late age at onset and protracted clinical phenotype, in a number of other patients originally diagnosed with juvenile NCL.

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Autism is a neurodevelopmental disability characterized by deficits in verbal communications, impairments in social interactions, and repetitive behaviors. Several studies have indicated strong involvement of multigenic components in the etiology of autism. Linkage analyses and candidate gene search approaches so far have not identified any reliable susceptibility genes. We are using a proteomic approach to identify protein abnormalities due to aberrant gene expression in autopsied autism brains. In four of eight autism brains, we have found an increase in polarity (more acidic) of glyoxalase I (Glo1) by two-dimensional gel electrophoresis. To identify the molecular change resulting in the shift of Glo1 polarity, we undertook sequencing of GLO1 gene. Direct sequencing of GLO1 gene/mRNA in these brains, has identified a single nucleotide polymorphism (SNP), C419A. The SNP causes an Ala111Glu change in the protein sequence. Population genetics of GLO1 C419A SNP studied in autism (71 samples) and normal and neurological controls (49 samples) showed significantly higher frequency for the A419 (allele frequency 0.6 in autism and 0.4 in controls, one-tailed Fisher's test P < 0.0079). Biochemical measurements have revealed a 38% decrease in Glo1 enzyme activity in autism brains (one-tailed t-test P < 0.026). Western blot analysis has also shown accumulation of advanced glycation end products (AGE's) in autism brains. These data suggest that homozygosity for A419 GLO1 resulting in Glu111 is a predisposing factor in the etiology of autism.

The name Batten disease (or neuronal ceroid lipofuscinosis ) is used to unify the spectrum of clinical and pathological conditions covered by the names infantile, late infantile, juvenile, and adult variants with their respective eponyms. The past was largely devoted to clinical diagnosis. The present is devoted to specific diagnostic tests. The future will be devoted to prevention and treatment. Treatment may consist of specific drug treatment, enzyme replacement, or gene replacement. Early diagnosis is important in order to provide genetic counseling and to establish family support for those patients who have a protracted, progressive disabling and ultimate fatal course.

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A lysosomal pepstatin-insensitive proteinase (CLN2p) deficiency is the underlying defect in the classical late-infantile neuronal ceroid lipofuscinosis (LINCL, CLN2). The natural substrates for CLN2p and the causative factors for the neurodegeneration in this disorder are still not understood. We have now purified the CLN2p from bovine brain to apparent homogeneity. The proteinase has a molecular mass of 46 kDa and an aminoterminal sequence, L-H-L-G-V-T-P-S-V-I-R-K, that is identical to the human enzyme. Peptide: N-glycosidase F and endoglycosidase H treatment of the CLN2p reduced its molecular mass to 39.5 and 40.5 kDa, respectively, suggesting the presence of as many as five N-glycosylated residues. The CLN2p activity was not affected by common protease inhibitors, and thiol reagents, metal chelators, and divalent metal ions had no significant effect on the proteolytic activity of the CLN2p. Among the naturally occurring neuropeptides, angiotensin II, substance P, and ␤-amyloid were substrates for the CLN2p, whereas angiotensin I, Leu-enkephalin, and ␥-endorphin were not. Peptide cleavage sites indicated that the CLN2p is a tripeptidyl peptidase that cleaves peptides having free amino-termini. Synthetic amino-and carboxyl-terminal peptides from the subunit c sequence, which is the major storage material in LINCL, are hydrolyzed by the CLN2p, suggesting that the subunit c may be one of the natural substrates for this proteinase and its accumulation in LINCL is the direct result of the proteinase deficiency. Key Words: Late-infantile neuronal ceroid lipofuscinosis-Batten disease -Pepstatin-insensitive -Tripeptidyl peptidase I-Acid proteinase -␤-AmyloidSubstance P-Angiotensin II.

Mannose 6-phosphate (Man-6-P) is a carbohydrate modification that is generated on newly synthesized lysosomal proteins. This modification is specifically recognized by two Man-6-P receptors that direct the vesicular transport of the lysosomal enzymes from the Golgi to a prelysosomal compartment. The Man-6-P is rapidly removed in the lysosome of most cell types; however, in neurons the Man-6-P modification persists. In this study we have examined the spectrum of Man-6-P-containing glycoproteins in brain specimens from patients with different neuronal ceroid lipofuscinoses (NCLs), which are progressive neurodegenerative disorders with established links to defects in lysosomal catabolism. We find characteristic alterations in the Man-6-P glycoproteins in specimens from late-infantile (LINCL), juvenile (JNCL) and adult (ANCL) patients. Man-6-P glycoproteins in LINCL patients were similar to controls, with the exception that the band corresponding to CLN2, a recently identified lysosomal enzyme whose deficiency results in this disease, was absent. In an ANCL patient, two Man-6-P glycoproteins were elevated in comparison with normal controls, suggesting that this disease also results from a perturbation in lysosomal hydrolysis. In JNCL, total levels of Man-6-P glycoproteins were 7-fold those of controls. In general this was reflected by increased lysosomal enzyme activities in JNCL but three Man-6-P glycoproteins were elevated to an even greater degree. These are CLN2 and the unidentified proteins that are also highly elevated in the ANCL.