Cerebrospinal Fluid Protein Biomarker Discovery in CLN3.

Syndromic CLN3-Batten is a fatal, pediatric, neurodegenerative disease caused by variants in CLN3, which encodes the endolysosomal transmembrane CLN3 protein. No approved treatment for CLN3 is currently available. The protracted and asynchronous disease presentation complicates the evaluation of potential therapies using clinical disease progression parameters. Biomarkers as surrogates to measure the progression and effect of potential therapeutics are needed. We performed proteomic discovery studies using cerebrospinal fluid (CSF) samples from 28 CLN3-affected and 32 age-similar non-CLN3 individuals. Proximal extension assay (PEA) of 1467 proteins and untargeted data-dependent mass spectrometry [MS; MassIVE FTP server (ftp://MSV000090147@massive.ucsd.edu)] were used to generate orthogonal lists of protein marker candidates. At an adjusted p-value of <0.1 and threshold CLN3/non-CLN3 fold-change ratio of 1.5, PEA identified 54 and MS identified 233 candidate biomarkers. Some of these (NEFL, CHIT1) have been previously linked with other neurologic conditions. Others (CLPS, FAM217B, QRICH2, KRT16, ZNF333) appear to be novel. Both methods identified 25 candidate biomarkers, including CHIT1, NELL1, and ISLR2 which had absolute fold-change ratios >2. NELL1 and ISLR2 regulate axonal development in neurons and are intriguing new candidates for further investigation in CLN3. In addition to identifying candidate proteins for CLN3 research, this study provides a comparison of two large-scale proteomic discovery methods in CSF.

A mouse mutant deficient in both neuronal ceroid lipofuscinosis-associated proteins CLN3 and TPP1.

Late-infantile neuronal ceroid lipofuscinosis (LINCL) and juvenile neuronal ceroid lipofuscinosis (JNCL) are inherited neurodegenerative diseases caused by mutations in the genes encoding lysosomal proteins tripeptidyl peptidase 1 (TPP1) and CLN3 protein, respectively. TPP1 is well-understood and, aided by animal models that accurately recapitulate the human disease, enzyme replacement therapy has been approved and other promising therapies are emerging. In contrast, there are no effective treatments for JNCL, partly because the function of the CLN3 protein remains unknown but also because animal models have attenuated disease and lack robust survival phenotypes. Mouse models for LINCL and JNCL, with mutations in Tpp1 and Cln3, respectively, have been thoroughly characterized but the phenotype of a double Cln3/Tpp1 mutant remains unknown. We created this double mutant and find that its phenotype is essentially indistinguishable from the single Tpp1-/- mutant in terms of survival and brain pathology. Analysis of brain proteomic changes in the single Tpp1-/- and double Cln3-/- ;Tpp1-/- mutants indicates largely overlapping sets of altered proteins and reinforces earlier studies that highlight GPNMB, LYZ2, and SERPINA3 as promising biomarker candidates in LINCL while several lysosomal proteins including SMPD1 and NPC1 appear to be altered in the Cln3-/- animals. An unexpected finding was that Tpp1 heterozygosity significantly decreased lifespan of the Cln3-/- mouse. The truncated survival of this mouse model makes it potentially useful in developing therapies for JNCL using survival as an endpoint. In addition, this model may also provide insights into CLN3 protein function and its potential functional interactions with TPP1.

Elevated levels of tripeptidyl peptidase 1 do not ameliorate pathogenesis in a mouse model of Alzheimer disease.

One potential therapeutic strategy for Alzheimer disease (AD) is to promote degradation of amyloid beta (Aß) and we previously demonstrated that the lysosomal protease tripeptidyl peptidase 1 (TPP1) can degrade Aß fibrils in vitro. In this study, we tested the hypothesis that increasing levels of TPP1 might promote degradation of Aß under physiological conditions, slowing or preventing its accumulation in the brain with subsequent therapeutic benefits. We used 2 approaches to increase TPP1 activity in the brain of J20 mice, an AD model that accumulates Aß and exhibits cognitive defects: transgenic overexpression of TPP1 in the brain and a pharmacological approach employing administration of recombinant TPP1. While we clearly observed the expected AD phenotype of the J20 mice based on pathology and measurement of behavioral and cognitive defects, we found that elevation of TPP1 activity by either experimental approach failed to have any measurable beneficial effect on disease phenotype.

A Method to Estimate the Distribution of Proteins across Multiple Compartments Using Data from Quantitative Proteomics Subcellular Fractionation Experiments.

Knowledge of cellular location is key to understanding the biological function of proteins. One commonly used large-scale method to assign cellular locations is subcellular fractionation, followed by quantitative mass spectrometry to identify proteins and estimate their relative distribution among centrifugation fractions. In most of such subcellular proteomics studies, each protein is assigned to a single cellular location by comparing its distribution to those of a set of single-compartment reference proteins. However, in many cases, proteins reside in multiple compartments. To accurately determine the localization of such proteins, we previously introduced constrained proportional assignment (CPA), a method that assigns each protein a fractional residence over all reference compartments (Jadot Mol. Cell Proteomics 2017, 16(2), 194-212. 10.1074/mcp.M116.064527). In this Article, we describe the principles underlying CPA, as well as data transformations to improve accuracy of assignment of proteins and protein isoforms, and a suite of R-based programs to implement CPA and related procedures for analysis of subcellular proteomics data. We include a demonstration data set that used isobaric-labeling mass spectrometry to analyze rat liver fractions. In addition, we describe how these programs can be readily modified by users to accommodate a wide variety of experimental designs and methods for protein quantitation.

Comparative Analysis of Quantitative Mass Spectrometric Methods for Subcellular Proteomics.

Knowledge of intracellular location can provide important insights into the function of proteins and their respective organelles, and there is interest in combining classical subcellular fractionation with quantitative mass spectrometry to create global cellular maps. To evaluate mass spectrometric approaches specifically for this application, we analyzed rat liver differential centrifugation and Nycodenz density gradient subcellular fractions by tandem mass tag (TMT) isobaric labeling with reporter ion measurement at the MS2 and MS3 level and with two different label-free peak integration approaches, MS1 and data independent acquisition (DIA). TMT-MS2 provided the greatest proteome coverage, but ratio compression from contaminating background ions resulted in a narrower accurate dynamic range compared to TMT-MS3, MS1, and DIA, which were similar. Using a protein clustering approach to evaluate data quality by assignment of reference proteins to their correct compartments, all methods performed well, with isobaric labeling approaches providing the highest quality localization. Finally, TMT-MS2 gave the lowest percentage of missing quantifiable data when analyzing orthogonal fractionation methods containing overlapping proteomes. In summary, despite inaccuracies resulting from ratio compression, data obtained by TMT-MS2 assigned protein localization as well as other methods but achieved the highest proteome coverage with the lowest proportion of missing values.

Lysosomal protein thermal stability does not correlate with cellular half-life: global observations and a case study of tripeptidyl-peptidase 1.

Late-infantile neuronal ceroid lipofuscinosis (LINCL) is a neurodegenerative lysosomal storage disorder caused by mutations in the gene encoding the protease tripeptidyl-peptidase 1 (TPP1). Progression of LINCL can be slowed or halted by enzyme replacement therapy, where recombinant human TPP1 is administered to patients. In this study, we utilized protein engineering techniques to increase the stability of recombinant TPP1 with the rationale that this may lengthen its lysosomal half-life, potentially increasing the potency of the therapeutic protein. Utilizing multiple structure-based methods that have been shown to increase the stability of other proteins, we have generated and evaluated over 70 TPP1 variants. The most effective mutation, R465G, increased the melting temperature of TPP1 from 55.6°C to 64.4°C and increased its enzymatic half-life at 60°C from 5.4 min to 21.9 min. However, the intracellular half-life of R465G and all other variants tested in cultured LINCL patient-derived lymphoblasts was similar to that of WT TPP1. These results provide structure/function insights into TPP1 and indicate that improving in vitro thermal stability alone is insufficient to generate TPP1 variants with improved physiological stability. This conclusion is supported by a proteome-wide analysis that indicates that lysosomal proteins have higher melting temperatures but also higher turnover rates than proteins of other organelles. These results have implications for similar efforts where protein engineering approaches, which are frequently evaluated in vitro, may be considered for improving the physiological properties of proteins, particularly those that function in the lysosomal environment.

Analysis of Brain and Cerebrospinal Fluid from Mouse Models of the Three Major Forms of Neuronal Ceroid Lipofuscinosis Reveals Changes in the Lysosomal Proteome.

Treatments are emerging for the neuronal ceroid lipofuscinoses (NCLs), a group of similar but genetically distinct lysosomal storage diseases. Clinical ratings scales measure long-term disease progression and response to treatment but clinically useful biomarkers have yet to be identified in these diseases. We have conducted proteomic analyses of brain and cerebrospinal fluid (CSF) from mouse models of the most frequently diagnosed NCL diseases: CLN1 (infantile NCL), CLN2 (classical late infantile NCL) and CLN3 (juvenile NCL). Samples were obtained at different stages of disease progression and proteins quantified using isobaric labeling. In total, 8303 and 4905 proteins were identified from brain and CSF, respectively. We also conduced label-free analyses of brain proteins that contained the mannose 6-phosphate lysosomal targeting modification. In general, we detect few changes at presymptomatic timepoints but later in disease, we detect multiple proteins whose expression is significantly altered in both brain and CSF of CLN1 and CLN2 animals. Many of these proteins are lysosomal in origin or are markers of neuroinflammation, potentially providing clues to underlying pathogenesis and providing promising candidates for further validation.

Inducible transgenic expression of tripeptidyl peptidase 1 in a mouse model of late-infantile neuronal ceroid lipofuscinosis.

Late-infantile neuronal ceroid lipofuscinosis is a fatal neurodegenerative disease of children caused by mutations resulting in loss of activity of the lysosomal protease, tripeptidyl peptidase 1 (TPP1). While Tpp1-targeted mouse models of LINCL exist, the goal of this study was to create a transgenic mouse with inducible TPP1 to benchmark treatment approaches, evaluate efficacy of treatment at different stages of disease, and to provide insights into the pathobiology of disease. A construct containing a loxP-flanked stop cassette inserted between the chicken-actin promoter and a sequence encoding murine TPP1 (TgLSL-TPP1) was integrated into the ROSA26 locus in mice by homologous recombination. Tested in both transfected CHO cells and in transgenic mice, the TgLSL-TPP1 did not express TPP1 until cre-mediated removal of the LSL cassette, which resulted in supraphysiological levels of TPP1 activity. We tested four cre/ERT2 transgenes to allow tamoxifen-inducible removal of the LSL cassette and subsequent TPP1 expression at any stage of disease. However, two of the cre/ERT2 driver transgenes had significant cre activity in the absence of tamoxifen, while cre-mediated recombination could not be induced by tamoxifen by two others. These results highlight potential problems with the use of cre/ERT2 transgenes in applications that are sensitive to low levels of basal cre expression. However, the germline-recombined mouse transgenic that constitutively overexpresses TPP1 will allow long-term evaluation of overexposure to the enzyme and in cell culture, the inducible transgene may be a useful tool in biomarker discovery projects.

Proteomic Analysis of Brain and Cerebrospinal Fluid from the Three Major Forms of Neuronal Ceroid Lipofuscinosis Reveals Potential Biomarkers.

Clinical trials have been conducted for the neuronal ceroid lipofuscinoses (NCLs), a group of neurodegenerative lysosomal diseases that primarily affect children. Whereas clinical rating systems will evaluate long-term efficacy, biomarkers to measure short-term response to treatment would be extremely valuable. To identify candidate biomarkers, we analyzed autopsy brain and matching CSF samples from controls and three genetically distinct NCLs due to deficiencies in palmitoyl protein thioesterase 1 (CLN1 disease), tripeptidyl peptidase 1 (CLN2 disease), and CLN3 protein (CLN3 disease). Proteomic and biochemical methods were used to analyze lysosomal proteins, and, in general, we find that changes in protein expression compared with control were most similar between CLN2 disease and CLN3 disease. This is consistent with previous observations of biochemical similarities between these diseases. We also conducted unbiased proteomic analyses of CSF and brain using isobaric labeling/quantitative mass spectrometry. Significant alterations in protein expression were identified in each NCL, including reduced STXBP1 in CLN1 disease brain. Given the confounding variable of post-mortem changes, additional validation is required, but this study provides a useful starting set of candidate NCL biomarkers for further evaluation.

Using whole-exome sequencing to investigate the genetic bases of lysosomal storage diseases of unknown etiology.

Lysosomes are membrane-bound, acidic eukaryotic cellular organelles that play important roles in the degradation of macromolecules. Mutations that cause the loss of lysosomal protein function can lead to a group of disorders categorized as the lysosomal storage diseases (LSDs). Suspicion of LSD is frequently based on clinical and pathologic findings, but in some cases, the underlying genetic and biochemical defects remain unknown. Here, we performed whole-exome sequencing (WES) on 14 suspected LSD cases to evaluate the feasibility of using WES for identifying causal mutations. By examining 2,157 candidate genes potentially associated with lysosomal function, we identified eight variants in five genes as candidate disease-causing variants in four individuals. These included both known and novel mutations. Variants were corroborated by targeted sequencing and, when possible, functional assays. In addition, we identified nonsense mutations in two individuals in genes that are not known to have lysosomal function. However, mutations in these genes could have resulted in phenotypes that were diagnosed as LSDs. This study demonstrates that WES can be used to identify causal mutations in suspected LSD cases. We also demonstrate cases where a confounding clinical phenotype may potentially reflect more than one lysosomal protein defect.

A Basic ApoE-Based Peptide Mediator to Deliver Proteins across the Blood-Brain Barrier: Long-Term Efficacy, Toxicity, and Mechanism.

We have investigated delivery of protein therapeutics from the bloodstream into the brain using a mouse model of late-infantile neuronal ceroid lipofuscinosis (LINCL), a lysosomal disease due to deficiencies in tripeptidyl peptidase 1 (TPP1). Supraphysiological levels of TPP1 are delivered to the mouse brain by acute intravenous injection when co-administered with K16ApoE, a peptide that in trans mediates passage across the blood-brain barrier (BBB). Chronic treatment of LINCL mice with TPP1 and K16ApoE extended the lifespan from 126 to >294 days, diminished pathology, and slowed locomotor dysfunction. K16ApoE enhanced uptake of a fixable biotin tracer by brain endothelial cells in a dose-dependent manner, suggesting that its mechanism involves stimulation of endocytosis. Pharmacokinetic experiments indicated that K16ApoE functions without disrupting the BBB, with minimal effects on overall clearance or uptake by the liver and kidney. K16ApoE has a narrow therapeutic index, with toxicity manifested as lethargy and/or death in mice. To address this, we evaluated variant peptides but found that efficacy and toxicity are associated, suggesting that desired and adverse effects are mechanistically related. Toxicity currently precludes direct clinical application of peptide-mediated delivery in its present form but it remains a useful approach to proof-of-principle studies for biologic therapies to the brain in animal models.

Chronic Enzyme Replacement to the Brain of a Late Infantile Neuronal Ceroid Lipofuscinosis Mouse Has Differential Effects on Phenotypes of Disease.

Late infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal inherited neurodegenerative disease caused by loss of lysosomal protease tripeptidyl peptidase 1 (TPP1). We have investigated the effects of chronic intrathecal (IT) administration using enzyme replacement therapy (ERT) to the brain of an LINCL mouse model, in which locomotor function declines dramatically prior to early death. Median lifespan was significantly extended from 126 days to >259 days when chronic IT treatment was initiated before the onset of disease. While treated animals lived longer and showed little sign of locomotor dysfunction as measured by stride length, some or all (depending on regimen) still died prematurely. One explanation is that cerebrospinal fluid (CSF)-mediated delivery may not deliver TPP1 to all brain regions. Morphological studies support this, showing delivery of TPP1 to ventral, but not deeper and dorsal regions. When IT treatment is initiated in severely affected LINCL mice, lifespan was extended modestly in most but dramatically extended in approximately one-third of the cohort. Treatment improved locomotor function in these severely compromised animals after it had declined to the point at which animals normally die. This indicates that some pathology in LINCL is reversible and does not simply reflect neuronal death.

Accounting for Protein Subcellular Localization: A Compartmental Map of the Rat Liver Proteome.

Accurate knowledge of the intracellular location of proteins is important for numerous areas of biomedical research including assessing fidelity of putative protein-protein interactions, modeling cellular processes at a system-wide level and investigating metabolic and disease pathways. Many proteins have not been localized, or have been incompletely localized, partly because most studies do not account for entire subcellular distribution. Thus, proteins are frequently assigned to one organelle whereas a significant fraction may reside elsewhere. As a step toward a comprehensive cellular map, we used subcellular fractionation with classic balance sheet analysis and isobaric labeling/quantitative mass spectrometry to assign locations to >6000 rat liver proteins. We provide quantitative data and error estimates describing the distribution of each protein among the eight major cellular compartments: nucleus, mitochondria, lysosomes, peroxisomes, endoplasmic reticulum, Golgi, plasma membrane and cytosol. Accounting for total intracellular distribution improves quality of organelle assignments and assigns proteins with multiple locations. Protein assignments and supporting data are available online through the Prolocate website (http://prolocate.cabm.rutgers.edu). As an example of the utility of this data set, we have used organelle assignments to help analyze whole exome sequencing data from an infant dying at 6 months of age from a suspected neurodegenerative lysosomal storage disorder of unknown etiology. Sequencing data was prioritized using lists of lysosomal proteins comprising well-established residents of this organelle as well as novel candidates identified in this study. The latter included copper transporter 1, encoded by SLC31A1, which we localized to both the plasma membrane and lysosome. The patient harbors two predicted loss of function mutations in SLC31A1, suggesting that this may represent a heretofore undescribed recessive lysosomal storage disease gene.

Analysis of large-scale whole exome sequencing data to determine the prevalence of genetically-distinct forms of neuronal ceroid lipofuscinosis.

The neuronal ceroid lipofuscinoses (NCLs) are a group of fatal, mostly recessive neurodegenerative lysosomal storage diseases. While clinically similar, they are genetically distinct and result from mutations in at least twelve different genes. Estimates of NCL incidence range from 0.6 to 14 per 100,000 live births but vary widely between populations and are influenced by whether patients are classified based upon clinical or genetic criteria. We investigated mutations in twelve NCL genes in ~61,000 individuals represented in the Exome Aggregation Consortium (ExAC) whole exome sequencing database. Variants were extracted from ExAC and pathogenic alleles were differentiated from neutral polymorphisms using annotated variant databases and missense mutation prediction tools. Carrier frequency was dependent on ethnicity, with the highest (1/75) observed for PPT1 in the Finnish. When data are adjusted for ethnic diversity within the USA, PPT1, TPP1 and CLN3 carrier frequencies were found to be the highest of the NCLs, each at ~1/500. Carrier frequencies calculated from ExAC correlated well with incidence estimated from numbers of living NCL patients in the US. In addition, the analysis identified numerous variants that are annotated as pathogenic in public repositories but have a predicted frequency that is not consistent with patient studies. These variants appear to be neutral polymorphisms that are reported as pathogenic without validation. Based upon literature reports, such alleles may be annotated in public databases as pathogenic and this propagates errors that can have clinical consequences.

Potential pitfalls and solutions for use of fluorescent fusion proteins to study the lysosome.

Use of fusion protein tags to investigate lysosomal proteins can be complicated by the acidic, protease-rich environment of the lysosome. Potential artifacts include degradation or release of the tag and acid quenching of fluorescence. Tagging can also affect protein folding, glycosylation and/or trafficking. To specifically investigate the use of fluorescent tags to reveal lysosomal localization, we tested mCherry derivatives as C-terminal tags for Niemann-Pick disease type C protein 2 (NPC2), a luminal lysosomal protein. Full-length mCherry was released from the NPC2 chimera while deletion of the 11 N-terminal residues of mCherry generated a cleavage-resistant (cr) fluorescent variant. Insertion of proline linkers between NPC2 and crmCherry had little effect while Gly-Ser linkers promoted cleavage. The NPC2-crmCherry fusion was targeted to the lysosome and restored function in NPC2-deficient cells. Fusion of crmCherry to known and candidate lysosomal proteins revealed that the linkers had different effects on lysosomal localization. Direct fusion of crmCherry impaired mannose 6-phosphorylation and lysosomal targeting of the lysosomal protease tripeptidyl peptidase I (TPP1), while insertion of linkers corrected the defects. Molecular modeling suggested structural bases for the effects of different linkers on NPC2 and TPP1 fusion proteins. While mCherry fusion proteins can be useful tools for studying the lysosome and related organelles, our findings underscore the potential artifacts associated with such applications.

Effective intravenous therapy for neurodegenerative disease with a therapeutic enzyme and a peptide that mediates delivery to the brain.

The blood-brain barrier (BBB) presents a major challenge to effective treatment of neurological disorders, including lysosomal storage diseases (LSDs), which frequently present with life-shortening and untreatable neurodegeneration. There is considerable interest in methods for intravenous delivery of lysosomal proteins across the BBB but for the most part, levels achievable in the brain of mouse models are modest and increased lifespan remains to be demonstrated. In this study, we have investigated delivery across the BBB using a mouse model of late-infantile neuronal ceroid lipofuscinosis (LINCL), a neurodegenerative LSD caused by loss of tripeptidyl peptidase I (TPP1). We have achieved supraphysiological levels of TPP1 throughout the brain of LINCL mice by intravenous (IV) coadministration of recombinant TPP1 with a 36-residue peptide that contains polylysine and a low-density lipoprotein receptor binding sequence from apolipoprotein E. Importantly, IV administration of TPP1 with the peptide significantly reduces brain lysosomal storage, increases lifespan and improves neurological function. This simple "mix and inject" method is immediately applicable towards evaluation of enzyme replacement therapy to the brain in preclinical models and further exploration of its clinical potential is warranted.

Extending the mannose 6-phosphate glycoproteome by high resolution/accuracy mass spectrometry analysis of control and acid phosphatase 5-deficient mice.

In mammals, most newly synthesized lumenal lysosomal proteins are delivered to the lysosome by the mannose 6-phosphate (Man6P) targeting pathway. Man6P -containing proteins can be affinity-purified and characterized using proteomic approaches, and such studies have led to the discovery of new lysosomal proteins and associated human disease genes. One limitation to this approach is that in most cell types the Man6P modification is rapidly removed by acid phosphatase 5 (ACP5) after proteins are targeted to the lysosome, and thus, some lysosomal proteins may escape detection. In this study, we have extended the analysis of the lysosomal proteome using high resolution/accuracy mass spectrometry to identify and quantify proteins in a combined analysis of control and ACP5-deficient mice. To identify Man6P glycoproteins with limited tissue distribution, we analyzed multiple tissues and used statistical approaches to identify proteins that are purified with high specificity. In addition to 68 known Man6P glycoproteins, 165 other murine proteins were identified that may contain Man6P and may thus represent novel lysosomal residents. For four of these lysosomal candidates, (lactoperoxidase, phospholipase D family member 3, ribonuclease 6, and serum amyloid P component), we demonstrate lysosomal residence based on the colocalization of fluorescent fusion proteins with a lysosomal marker.

Proteomic analysis of mouse models of Niemann-Pick C disease reveals alterations in the steady-state levels of lysosomal proteins within the brain.

Niemann-Pick C disease (NPC) is a neurodegenerative lysosomal disorder characterized by storage of cholesterol and other lipids caused by defects in NPC1, a transmembrane protein involved in cholesterol export from the lysosome, or NPC2, an intralysosomal cholesterol transport protein. Alterations in lysosomal activities have been implicated in NPC pathogenesis therefore the aim of this study was to conduct a proteomic analysis of lysosomal proteins in mice deficient in either NPC1 or NPC2 to identify secondary changes that might be associated with disease. Lysosomal proteins containing the specific mannose 6-phosphate modification were purified from wild-type and Npc1(-/-) and Npc2(-/-) mutant mouse brains at different stages of disease progression and identified by bottom-up LC-MS/MS and quantified by spectral counting. Levels of a number of lysosomal proteins involved in lipid catabolism including prosaposin and the two subunits of ß-hexosaminidase were increased in both forms of NPC, possibly representing a compensatory cellular response to the accumulation of glycosphingolipids. Several other lysosomal proteins were significantly altered, including proteases and glycosidases. Changes in lysosomal protein levels corresponded with similar alterations in activities and transcript levels. Understanding the rationale for such changes may provide insights into the pathophysiology of NPC.

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.

Loss of Niemann-Pick C1 or C2 protein results in similar biochemical changes suggesting that these proteins function in a common lysosomal pathway.

Niemann-Pick Type C (NPC) disease is a lysosomal storage disorder characterized by accumulation of unesterified cholesterol and other lipids in the endolysosomal system. NPC disease results from a defect in either of two distinct cholesterol-binding proteins: a transmembrane protein, NPC1, and a small soluble protein, NPC2. NPC1 and NPC2 are thought to function closely in the export of lysosomal cholesterol with both proteins binding cholesterol in vitro but they may have unrelated lysosomal roles. To investigate this possibility, we compared biochemical consequences of the loss of either protein. Analyses of lysosome-enriched subcellular fractions from brain and liver revealed similar decreases in buoyant densities of lysosomes from NPC1 or NPC2 deficient mice compared to controls. The subcellular distribution of both proteins was similar and paralleled a lysosomal marker. In liver, absence of either NPC1 or NPC2 resulted in similar alterations in the carbohydrate processing of the lysosomal protease, tripeptidyl peptidase I. These results highlight biochemical alterations in the lysosomal system of the NPC-mutant mice that appear secondary to lipid storage. In addition, the similarity in biochemical phenotypes resulting from either NPC1 or NPC2 deficiency supports models in which the function of these two proteins within lysosomes are linked closely.