Maintenance of a healthy proteome is essential for cellular homeostasis and loss of proteostasis is associated with tissue dysfunction and neurodegenerative disease. The mechanisms that support proteostasis in healthy cells and how they become defective during aging or in disease states are not fully understood. Here, we investigate the transcriptional programs that are essential for neural stem and progenitor cell (NSPC) function and uncover a program of autophagy genes under the control of the transcription factor FOXO3. Using genomic approaches, we observe that FOXO3 directly binds a network of target genes in adult NSPCs that are involved in autophagy, and find that FOXO3 functionally regulates induction of autophagy in these cells. Interestingly, in the absence of FOXO activity, aggregates accumulate in NSPCs, and this effect is reversed by TOR (target of rapamycin) inhibition. Surprisingly, enhancing FOXO3 causes nucleation of protein aggregates, but does not increase their degradation. The work presented here identifies a genomic network under the direct control of a key transcriptional regulator of aging that is critical for maintaining a healthy mammalian stem cell pool to support lifelong neurogenesis.

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.

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.

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.

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.

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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.

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