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.

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.

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.

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.

A mouse model of classical late-infantile neuronal ceroid lipofuscinosis based on targeted disruption of the CLN2 gene results in a loss of tripeptidyl-peptidase I activity and progressive neurodegeneration.

Mutations in the CLN2 gene, which encodes a lysosomal serine protease, tripeptidyl-peptidase I (TPP I), result in an autosomal recessive neurodegenerative disease of children, classical late-infantile neuronal ceroid lipofuscinosis (cLINCL). cLINCL is inevitably fatal, and there currently exists no cure or effective treatment. In this report, we provide the characterization of the first CLN2-targeted mouse model for cLINCL. CLN2-targeted mice were fertile and apparently healthy at birth despite an absence of detectable TPP I activity. At approximately 7 weeks of age, neurological deficiencies became evident with the onset of a tremor that became progressively more severe and was eventually accompanied by ataxia. Lifespan of the affected mice was greatly reduced (median survival, 138 d), and extensive neuronal pathology was observed including a prominent accumulation of cytoplasmic storage material within the lysosomal-endosomal compartment, a loss of cerebellar Purkinje cells, and widespread axonal degeneration. The CLN2-targeted mouse therefore recapitulates much of the pathology and clinical features of cLINCL and represents an animal model that should provide clues to the normal cellular function of TPP I and the pathogenic processes that underlie neuronal death in its absence. In addition, the CLN2-targeted mouse also represents a valuable model for the evaluation of different therapeutic strategies.

Genetic evidence for nonredundant functional cooperativity between NPC1 and NPC2 in lipid transport.

Niemann-Pick C (NPC) disease is a fatal neurodegenerative disorder characterized by a lysosomal accumulation of cholesterol and other lipids within the cells of patients. Clinically identical forms of NPC disease are caused by defects in either of two different proteins: NPC1, a lysosomal-endosomal transmembrane protein and NPC2, a soluble lysosomal protein with cholesterol binding properties. Although it is clear that NPC1 and NPC2 are required for the egress of lipids from the lysosome, the precise roles of these proteins in this process is unknown. To gain insight into the normal function of NPC2 and to investigate its interactions, if any, with NPC1, we have generated a murine NPC2 hypomorph that expresses 0-4% residual protein in different tissues and have examined its phenotype in the presence and absence of NPC1. The phenotypes of NPC1 and NPC2 single mutants and an NPC1;NPC2 double mutant are similar or identical in terms of disease onset and progression, pathology, neuronal storage, and biochemistry of lipid accumulation. These findings provide genetic evidence that the NPC1 and NPC2 proteins function in concert to facilitate the intracellular transport of lipids from the lysosome to other cellular sites.