Istvan Sohar

A mutation in the ovine cathepsin D gene causes a congenital lysosomal storage disease with profound neurodegeneration

The neuronal ceroid lipofuscinoses (NCLs) constitute a group of neurodegenerative storage diseases characterized by progressive psychomotor retardation, blindness and premature death. Pathologically, there is accumulation of autofluorescent material in lysosome‐derived organelles in a variety of cell types, but neurons in the central nervous system appear to be selectively affected and undergo progressive death. In this report we show that a novel form of NCL, congenital ovine NCL, is caused by a deficiency in the lysosomal aspartyl proteinase cathepsin D. A single nucleotide mutation in the cathepsin D gene results in conversion of an active site aspartate to asparagine, leading to production of an enzymatically inactive but stable protein. This results in severe cerebrocortical atrophy and early death, providing strong evidence for an important role of cathepsin D in neuronal development and/or homeostasis.

Quantitation of oxidative damage to tissue proteins

Active oxygen species are thought to be involved in many physiological and pathological processes and are known to oxidatively modify DNA, lipids and proteins. One such modification is the addition of carbonyl groups to amino acid residues in proteins. The number of carbonyl groups on proteins can be quantitated spectrophotometrically using 2,4-dinitrophenylhydrazine (DNPH). The DNPH assay described in the literature was found to be unreliable in samples containing high amounts of chromophore (e.g. hemoglobin, myoglobin, retinoids). By using an HCl-acetone wash, hemes from the chromophores could be extracted, enabling the determination of carbonyl content to be made even in highly colored tissue extracts. Residual DNPH, which was also found to interfere with the assay, was removed by additional washes with trichloroacetic acid and ethanol-ethylacetate. These improvements are known to remove lipids, do not lengthen the time required to do the assay, permit quantification of carbonyl content in 1-4 mg protein from a variety of tissue types and provide a sensitive and reliable method for assessing oxidative damage to tissue proteins.

Rat brain contains high levels of mannose-6-phosphorylated glycoproteins including lysosomal enzymes and palmitoyl-protein thioesterase, an enzyme implicated in infantile neuronal lipofuscinosis.

Mannose 6-phosphate (Man-6-P) is a posttranslational carbohydrate modification typical of newly synthesized acid hydrolases that signals targeting from the Golgi apparatus to the lysosome via Man-6-P receptors (MPRs). Using iodinated cation independent MPR as a probe in a Western blot assay, we surveyed levels of Man-6-P glycoproteins in a number of different rat tissues. Considerable variation was observed with respect to total amounts and types of Man-6-P glycoproteins in the different tissues. Brain contained 2-8-fold more Man-6-P glycoproteins than other tissues, with relative abundance being brain ≫ testis ≈ heart > lung ≈ kidney ≈ ovary ≈ spleen > skeletal muscle ≈ liver ≈ serum. Analysis of 16 different lysosomal enzyme activities revealed that brain contains lower activities than other tissues which suggested that decreased removal of Man-6-P results in increased levels of Man-6-P glycoproteins. This was directly demonstrated by comparing activities of phosphorylated lysosomal enzymes, purified by immobilized MPR affinity chromatography, with total activities. The phosphorylated forms accounted for a considerable proportion of the MPR-targeted activities measured in brain (on average, 36.2%) but very little in lung, kidney, and liver (on average, 5.5, 2.3, and 0.7%, respectively). Man-6-P glycoproteins were also isolated from rat brain by MPR affinity chromatography on a preparative scale. Of the 18 bands resolvable by SDS-polyacrylamide gel electrophoresis, seven bands were NH2-terminally sequenced and identified as the known lysosomal enzymes cathepsin L, cathepsin A, cathepsin D, α-galactosidase A, arylsulfatase A, and α-iduronidase. One of the major Man-6-P glycoproteins was identified as palmitoyl protein thioesterase, which was not previously thought to be lysosomal. This finding raises important questions about the cellular location and function of palmitoyl protein thioesterase, mutations in which result in the neurodegenerative disorder, infantile neuronal ceroid lipofuscinosis.

Identification and Validation of Mannose 6-Phosphate Glycoproteins in Human Plasma Reveal a Wide Range of Lysosomal and Non-lysosomal Proteins

Acid hydrolase activities are normally confined within the cell to the lysosome, a membrane-delimited cytoplasmic organelle primarily responsible for the degradation of macromolecules. However, lysosomal proteins are also present in human plasma, and a proportion of these retain mannose 6-phosphate (Man-6-P), a modification on N-linked glycans that is recognized by Man-6-P receptors (MPRs) that normally direct the targeting of these proteins to the lysosome. In this study, we purified the Man-6-P glycoforms of proteins from human plasma by affinity chromatography on immobilized MPRs and characterized this subproteome by two-dimensional gel electrophoresis and by tandem mass spectrometry. As expected, we identified many known and potential candidate lysosomal proteins. In addition, we also identified a number of abundant classical plasma proteins that were retained even after two consecutive rounds of affinity purification. Given their abundance in plasma, we initially considered these proteins to be likely contaminants, but a mass spectrometric study of Man-6-phosphorylation sites using MPR-purified glycopeptides revealed that some proportion of these classical plasma proteins contained the Man-6-P modification. We propose that these glycoproteins are phosphorylated at low levels by the lysosomal enzyme phosphotransferase, but their high abundance results in detection of Man-6-P glycoforms in plasma. These results may provide useful insights into the molecular processes underlying Man-6-phosphorylation and highlight circumstances under which the presence of Man-6-P may not be indicative of lysosomal function. In addition, characterization of the plasma Man-6-P glycoproteome should facilitate development of mass spectrometry-based tools for the diagnosis of lysosomal storage diseases and for investigating the involvement of Man-6-P-containing glycoproteins in more widespread human diseases and their potential utility as biomarkers.

Intrathecal tripeptidyl-peptidase 1 reduces lysosomal storage in a canine model of late infantile neuronal ceroid lipofuscinosis

Late infantile neuronal ceroid lipofuscinosis (LINCL) is caused by mutations in the gene encoding tripeptidyl-peptidase 1 (TPP1). LINCL patients accumulate lysosomal storage materials in the CNS accompanied by neurodegeneration, blindness, and functional decline. Dachshunds homozygous for a null mutation in the TPP1 gene recapitulate many symptoms of the human disease. The objectives of this study were to determine whether intrathecal (IT) TPP1 treatment attenuates storage accumulation and functional decline in TPP1-/- Dachshunds and to characterize the CNS distribution of TPP1 activity. TPP1 was administered to one TPP1-/- and one homozygous wild-type (WT) dog. An additional TPP1-/- and WT dog received vehicle. Four IT administrations of 32 mg TPP1 formulated in 2.3 mL of artificial cerebrospinal fluid (aCSF) or vehicle were administered monthly via the cerebellomedullary cistern from four to seven months of age. Functional decline was assessed by physical and neurological examinations, electrophysiology, and T-maze performance. Neural tissues were collected 48 h after the fourth administration and analyzed for TPP1 activity and autofluorescent storage material. TPP1 was distributed at greater than WT levels in many areas of the CNS of the TPP1-/- dog administered TPP1. The amount of autofluorescent storage was decreased in this dog relative to the vehicle-treated affected control. No improvement in overall function was observed in this dog compared to the vehicle-treated TPP1-/- littermate control. These results demonstrate for the first time in a large animal model of LINCL widespread delivery of biochemically active TPP1 to the brain after IT administration along with a decrease in lysosomal storage material. Further studies with this model will be necessary to optimize the dosing route and regimen to attenuate functional decline.

Crystal Structure and Autoactivation Pathway of the Precursor Form of Human Tripeptidyl-peptidase 1, the Enzyme Deficient in Late Infantile Ceroid Lipofuscinosis

Late infantile neuronal ceroid lipofuscinosis is a fatal childhood neurological disorder caused by a deficiency in the lysosomal protease tripeptidyl-peptidase 1 (TPP1). TPP1 represents the only known mammalian member of the S53 family of serine proteases, a group characterized by a subtilisin-like fold, a Ser-Glu-Asp catalytic triad, and an acidic pH optimum. TPP1 is synthesized as an inactive proenzyme (pro-TPP1) that is proteolytically processed into the active enzyme after exposure to low pH in vitro or targeting to the lysosome in vivo. In this study, we describe an endoglycosidase H-deglycosylated form of TPP1 containing four Asn-linked N-acetylglucosamines that is indistinguishable from fully glycosylated TPP1 in terms of autocatalytic processing of the proform and enzymatic properties of the mature protease. The crystal structure of deglycosylated pro-TPP1 was determined at 1.85 Å resolution. A large 151-residue C-shaped prodomain makes extensive contacts as it wraps around the surface of the catalytic domain with the two domains connected by a 24-residue flexible linker that passes through the substrate-binding groove. The proenzyme structure reveals suboptimal catalytic triad geometry with its propiece linker partially blocking the substrate-binding site, which together serve to prevent premature activation of the protease. Finally, we have identified numerous processing intermediates and propose a structural model that explains the pathway for TPP1 activation in vitro. These data provide new insights into TPP1 function and represent a valuable resource for constructing improved TPP1 variants for treatment of late infantile neuronal ceroid lipofuscinosis.

Residual levels of tripeptidyl-peptidase I activity dramatically ameliorate disease in late-infantile neuronal ceroid lipofuscinosis.

Classical late-infantile neuronal ceroid lipofuscinosis (LINCL) is a hereditary neurodegenerative disease of childhood that is caused by mutations in the gene (CLN2) encoding the lysosomal protease tripeptidyl-peptidase I (TPPI). LINCL is fatal and there is no treatment of demonstrated efficacy in affected children but preclinical studies with AAV-mediated gene therapy have demonstrated promise in a mouse model. Here, we have generated mouse CLN2-mutants that express different amounts of TPPI activity to benchmark levels required for therapeutic benefits. Approximately 3% of normal TPPI activity in brain delayed disease onset and doubled lifespan to a median of approximately 9 months compared to mice expressing approximately 0.2% of normal levels. Expression of 6% of normal TPPI activity dramatically attenuated disease, with a median lifespan of approximately 20 months which approaches that of unaffected mice. While the lifespan of this hypomorph is shortened, disease is late-onset, less severe and progresses slowly compared to mice expressing lower TPPI levels. For gene therapy and other approaches that restore enzyme activity, these results suggest that 6% of normal TPPI activity throughout the CNS of affected individuals will provide a significant therapeutic benefit but higher levels will be required to cure this disease.

Large-volume Intrathecal Enzyme Delivery Increases Survival of a Mouse Model of Late Infantile Neuronal Ceroid Lipofuscinosis

Late infantile neuronal ceroid lipofuscinosis (LINCL) is a progressive neurodegenerative lysosomal storage disorder caused by mutations in TPP1, the gene encoding the lysosomal protease tripeptidyl-peptidase (TPP1). LINCL primarily affects children, is fatal and there is no effective treatment. Administration of recombinant protein has proved effective in treatment of visceral manifestations of other lysosomal storage disorders but to date, only marginal improvement in survival has been obtained for neurological diseases. In this study, we have developed and optimized a large-volume intrathecal administration strategy to deliver therapeutic amounts of TPP1 to the central nervous system (CNS) of a mouse model of LINCL. To determine the efficacy of treatment, we have monitored survival as the primary endpoint and demonstrate that an acute treatment regimen (three consecutive daily doses started at 4 weeks of age) increases median lifespan of the LINCL mice from 16 (vehicle treated) to 23 weeks (enzyme treated). Consistent with the increase in life-span, we also observed significant reversal of pathology and improvement in neurological phenotype. These results provide a strong basis for both clinical investigation of large-volume/high-dose delivery of TPP1 to the brain via the cerebrospinal fluid (CSF) and extension of this approach towards other neurological lysosomal storage diseases.

Determination of the Substrate Specificity of Tripeptidyl-peptidase I Using Combinatorial Peptide Libraries and Development of Improved Fluorogenic Substrates

Classical late-infantile neuronal ceroid lipofuscinosis is a fatal neurodegenerative disease caused by mutations in CLN2, the gene encoding the lysosomal protease tripeptidyl-peptidase I (TPP I). The natural substrates for TPP I and the pathophysiological processes associated with lysosomal storage and disease progression are not well understood. Detailed characterization of TPP I substrate specificity should provide insights into these issues and also aid in the development of improved clinical and biochemical assays. To this end, we constructed fluorogenic and standard combinatorial peptide libraries and analyzed them using fluorescence and mass spectrometry-based activity assays. The fluorogenic group 7-amino-4-carbamoylmethylcoumarin was incorporated into a series of 7-amino-4-carbamoylmethylcoumarin tripeptide libraries using a design strategy that allowed systematic evaluation of the P1, P2, and P3 positions. TPP I digestion of these substrates liberates the fluorescence group and results in a large increase in fluorescence that can be used to calculate kinetic parameters and to derive the substrate specificity constant kcat/KM. In addition, we implemented a mass spectrometry-based assay to measure the hydrolysis of individual peptides in peptide pools and thus expand the scope of the analysis. Nonfluorogenic tetrapeptide and pentapeptide libraries were synthesized and analyzed to evaluate P1′ and P2′ residues. Together, this analysis allowed us to predict the relative specificity of TPP I toward a wide range of potential biological substrates. In addition, we evaluated a variety of new fluorogenic peptides with a P3 Arg residue, and we demonstrated their superiority compared with the widely used substrate Ala-Ala-Phe-AMC for selectively measuring TPP I activity in biological specimens.

Systemic administration of tripeptidyl peptidase I in a mouse model of late infantile neuronal ceroid lipofuscinosis: effect of glycan modification.

Late-infantile neuronal ceroid lipofuscinosis (LINCL) is a recessive genetic disease of childhood caused by deficiencies in the lysosomal protease tripeptidyl peptidase I (TPP1). Disease is characterized by progressive and extensive neuronal death. One hurdle towards development of enzyme replacement therapy is delivery of TPP1 to the brain. In this study, we evaluated the effect of modifying N-linked glycans on recombinant human TPP1 on its pharmacokinetic properties after administration via tail vein injection to a mouse model of LINCL. Unmodified TPP1 exhibited a dose-dependent serum half-life of 12 min (0.12 mg) to 45 min (2 mg). Deglycosylation or modification using sodium metaperiodate oxidation and reduction with sodium borohydride increased the circulatory half-life but did not improve targeting to the brain compared to unmodified TPP1. Analysis of liver, brain, spleen, kidney and lung demonstrated that for all preparations, >95% of the recovered activity was in the liver. Interestingly, administration of a single 2 mg dose (80 mg/kg) of unmodified TPP1 resulted in ∼10% of wild-type activity in brain. This suggests that systemic administration of unmodified recombinant enzyme merits further exploration as a potential therapy for LINCL.