Deficiency of HtrA4 in BeWo cells downregulates angiogenesis through IL-6/JAK/STAT3 signaling.

In a previous study, we investigated the effects of high-temperature requirement factor A4 (HtrA4) deficiency on trophoblasts using the BeWo KO cell line. However, the effects of this deficiency on angiogenesis remain unclear. To explore the role of HtrA4 in angiogenesis, HUVECs were co-cultured with wild-type BeWo cells (BeWo WT), BeWo KO, and HtrA4-rescued BeWo KO (BeWo KO-HtrA4 rescue) cells. Dil staining and dextran analysis revealed that HUVECs co-cultured with BeWo KO formed tubes, but they were often disjointed compared to those co-cultured with BeWo WT, BeWo KO-HtrA4 rescue, and HUVECs controls. RT-PCR, ELISA, and western blot analysis were performed to assess angiogenesis-related factors at the mRNA and protein levels. HtrA4 deficiency inhibited IL-6 expression in trophoblasts, and the reduced secretion of IL-6 decreases VEGFA expression in HUVECs by modulating the JAK2/STAT3 signaling pathway to prevent tube formation. Moreover, rescuing HtrA4 expression restored the HUVEC tube formation ability. Interestingly, IL-6 expression was lower in supernatants with only cultured HUVECs than in co-cultured HUVECs with BeWo WT cells, but the HUVEC tube formation ability was similar. These findings suggest that the promoting angiogenesis-related signaling pathway differs between only HUVECs and co-cultured HUVECs, and that the deficiency of HtrA4 weakens the activation of the IL-6/JAK/STAT3/VEGFA signaling pathway, reducing the ability of tube formation in HUVECs. HtrA4 deficiency in trophoblasts hinders angiogenesis and may contribute to placental dysfunction.

PRSS37 deficiency leads to impaired energy metabolism in testis and sperm revealed by DIA-based quantitative proteomic analysis.

Our previous studies have reported that a putative trypsin-like serine protease, PRSS37, is exclusively expressed in testicular germ cells during late spermatogenesis and essential for sperm migration from the uterus into the oviduct and sperm-egg recognition via mediating the interaction between PDILT and ADAM3. In the present study, the global proteome profiles of wild-type (wt) and Prss37-/- mice in testis and sperm were compared employing data independent acquisition (DIA) technology. Overall, 2506 and 459 differentially expressed proteins (DEPs) were identified in Prss37-null testis and sperm, respectively, when compared to control groups. Bioinformatic analyses revealed that most of DEPs were related to energy metabolism. Of note, the DEPs associated with pathways for the catabolism such as glucose via glycolysis, fatty acids via ß-oxidation, and amino acids via oxidative deamination were significantly down-regulated. Meanwhile, the DEPs involved in the tricarboxylic acid cycle (TCA cycle) and oxidative phosphorylation (OXPHOS) were remarkably decreased. The DIA data were further confirmed by a markedly reduction of intermediate metabolites (citrate and fumarate) in TCA cycle and terminal metabolite (ATP) in OXPHOS system after disruption of PRSS37. These outcomes not only provide a more comprehensive understanding of the male fertility of energy metabolism modulated by PRSS37 but also furnish a dynamic proteomic resource for further reproductive biology studies.

PRSS50 is a testis protease responsible for proper sperm tail formation and function.

Multiple morphological abnormalities of the sperm flagella (MMAF) are a major cause of asthenoteratozoospermia. We have identified protease serine 50 (PRSS50) as having a crucial role in sperm development, because Prss50-null mice presented with impaired fertility and sperm tail abnormalities. PRSS50 could also be involved in centrosome function because these mice showed a threefold increase in acephalic sperm (head-tail junction defect), sperm with multiple heads (spermatid division defect) and sperm with multiple tails, including novel two conjoined sperm (complete or partial parts of several flagellum on the same plasma membrane). Our data support that, in the testis, as in tumorigenesis, PRSS50 activates NFκB target genes, such as the centromere protein leucine-rich repeats and WD repeat domain-containing protein 1 (LRWD1), which is required for heterochromatin maintenance. Prss50-null testes have increased IκκB, and reduced LRWD1 and histone expression. Low levels of de-repressed histone markers, such as H3K9me3, in the Prss50-null mouse testis may cause increases in post-meiosis proteins, such as AKAP4, affecting sperm formation. We provide important insights into the complex mechanisms of sperm development, the importance of testis proteases in fertility and a novel mechanism for MMAF.

Intracerebroventricular Cerliponase Alfa for Neuronal Ceroid Lipofuscinosis Type 2 Disease: Clinical Practice Considerations From US Clinics.

BACKGROUND: Neuronal ceroid lipofuscinosis type 2 or CLN2 disease is a rare, autosomal recessive, neurodegenerative lysosomal storage disorder caused by tripeptidyl peptidase 1 deficiency. Cerliponase alfa, a recombinant human tripeptidyl peptidase 1 enzyme, is the first and only approved treatment for CLN2 disease and the first approved enzyme replacement therapy administered via intracerebroventricular infusion. METHODS: A meeting of health care professionals from US institutions with experience in cerliponase alfa treatment of children with CLN2 disease was held in November 2018. Key common practices were identified, and later refined during the drafting of this article, that facilitate safe chronic administration of cerliponase alfa. RESULTS: Key practices include developing a multidisciplinary team of clinicians, pharmacists, and coordinators, and institution-specific processes. Infection risk may be reduced through strict aseptic techniques and minimizing connections and disconnections during infusion. The impact of intracerebroventricular device design on port needle stability during extended intracerebroventricular infusion is a critical consideration in device selection. Monitoring for central nervous system infection is performed at each patient contact, but with flexibility in the degree of monitoring. Although few institutions had experienced positive cerebrospinal fluid test results, the response to a positive cerebrospinal fluid culture should be determined on a case-by-case basis, and the intracerebroventricular device should be removed if cerebrospinal fluid infection is confirmed. CONCLUSIONS: The key common practices and flexible practices used by institutions with cerliponase alfa experience may assist other institutions in process development. Continued sharing of experiences will be essential for developing standards and patient care guidelines.

ABHD11, a new diacylglycerol lipase involved in weight gain regulation.

Obesity epidemic continues to spread and obesity rates are increasing in the world. In addition to public health effort to reduce obesity, there is a need to better understand the underlying biology to enable more effective treatment and the discovery of new pharmacological agents. Abhydrolase domain-containing protein 11 (ABHD11) is a serine hydrolase enzyme, localized in mitochondria, that can synthesize the endocannabinoid 2-arachidonoyl glycerol (2AG) in vitro. In vivo preclinical studies demonstrated that knock-out ABHD11 mice have a similar 2AG level as WT mice and exhibit a lean metabolic phenotype. Such mice resist to weight gain in Diet Induced Obesity studies (DIO) and display normal biochemical plasma parameters. Metabolic and transcriptomic analyses on serum and tissues of ABHD11 KO mice from DIO studies show a modulation in bile salts associated with reduced fat intestinal absorption. These data suggest that modulating ABHD11 signaling pathway could be of therapeutic value for the treatment of metabolic disorders.

MiniBacillus PG10 as a Convenient and Effective Production Host for Lantibiotics.

Efficient bacterial cell factories are important for the screening and characterization of potent antimicrobial peptides such as lantibiotics. Although lantibiotic production systems have been established in Lactococcus lactis and Escherichia coli, the industrial workhorse Bacillus subtilis has been left relatively unexplored as a lantibiotic production host. Therefore, we tested different B. subtilis strains for their ability to produce lantibiotic peptides by using the subtilin modification and transport enzymes derived from the natural subtilin producer B. subtilis ATCC 6633. Our study shows that although B. subtilis ATCC 6633 and 168 are able to produce various processed lantibiotic peptides, an evident advantage of using either the 8-fold protease-deficient strain WB800 or the genome-minimized B. subtilis 168 strain PG10 is the lack of extracellular serine protease activity. Consequently, leader processing of lantibiotic precursor peptides is circumvented and thus potential toxicity toward the production host is prevented. Furthermore, PG10 provides a clean secondary metabolic background and therefore appears to be the most promising B. subtilis lantibiotic production host. We demonstrate the production of various lantibiotic precursor peptides by PG10 and show different options for their in vitro activation. Our study thus provides a convenient B. subtilis-based lantibiotic production system, which facilitates the search for novel antimicrobial peptides.

Extracellular Vesicles as Drug Carriers for Enzyme Replacement Therapy to Treat CLN2 Batten Disease: Optimization of Drug Administration Routes.

CLN2 Batten disease (BD) is one of a broad class of lysosomal storage disorders that is characterized by the deficiency of lysosomal enzyme, TPP1, resulting in a build-up of toxic intracellular storage material in all organs and subsequent damage. A major challenge for BD therapeutics is delivery of enzymatically active TPP1 to the brain to attenuate progressive loss of neurological functions. To accomplish this daunting task, we propose the harnessing of naturally occurring nanoparticles, extracellular vesicles (EVs). Herein, we incorporated TPP1 into EVs released by immune cells, macrophages, and examined biodistribution and therapeutic efficacy of EV-TPP1 in BD mouse model, using various routes of administration. Administration through intrathecal and intranasal routes resulted in high TPP1 accumulation in the brain, decreased neurodegeneration and neuroinflammation, and reduced aggregation of lysosomal storage material in BD mouse model, CLN2 knock-out mice. Parenteral intravenous and intraperitoneal administrations led to TPP1 delivery to peripheral organs: liver, kidney, spleen, and lungs. A combination of intrathecal and intraperitoneal EV-TPP1 injections significantly prolonged lifespan in BD mice. Overall, the optimization of treatment strategies is crucial for successful applications of EVs-based therapeutics for BD.

Loss of PRSS56 function leads to ocular angle defects and increased susceptibility to high intraocular pressure.

Glaucoma is a leading cause of blindness, affecting up to 70 million people worldwide. High intraocular pressure (IOP) is a major risk factor for glaucoma. It is well established that inefficient aqueous humor (AqH) outflow resulting from structural or functional alterations in ocular drainage tissues causes high IOP, but the genes and pathways involved are poorly understood. We previously demonstrated that mutations in the gene encoding the serine protease PRSS56 induces ocular angle closure and high IOP in mice and identified reduced ocular axial length as a potential contributing factor. Here, we show that Prss56-/- mice also exhibit an abnormal iridocorneal angle configuration characterized by a posterior shift of ocular drainage structures relative to the ciliary body and iris. Notably, we show that retina-derived PRSS56 is required between postnatal days 13 and 18 for proper iridocorneal configuration and that abnormal positioning of the ocular drainage tissues is not dependent on ocular size reduction in Prss56-/- mice. Furthermore, we demonstrate that the genetic context modulates the severity of IOP elevation in Prss56 mutant mice and describe a progressive degeneration of ocular drainage tissues that likely contributes to the exacerbation of the high IOP phenotype observed on the C3H/HeJ genetic background. Finally, we identify five rare PRSS56 variants associated with human primary congenital glaucoma, a condition characterized by abnormal development of the ocular drainage structures. Collectively, our findings point to a role for PRSS56 in the development and maintenance of ocular drainage tissues and IOP homeostasis, and provide new insights into glaucoma pathogenesis.

The Replisome Mediates A-NHEJ Repair of Telomeres Lacking POT1-TPP1 Independently of MRN Function.

Telomeres use shelterin to protect chromosome ends from activating the DNA damage sensor MRE11-RAD50-NBS1 (MRN), repressing ataxia-telangiectasia, mutated (ATM) and ATM and Rad3-related (ATR) dependent DNA damage checkpoint responses. The MRE11 nuclease is thought to be essential for the resection of the 5' C-strand to generate the microhomologies necessary for alternative non-homologous end joining (A-NHEJ) repair. In the present study, we uncover DNA damage signaling and repair pathways engaged by components of the replisome complex to repair dysfunctional telomeres. In cells lacking MRN, single-stranded telomeric overhangs devoid of POT1-TPP1 do not recruit replication protein A (RPA), ATR-interacting protein (ATRIP), and RAD 51. Rather, components of the replisome complex, including Claspin, Proliferating cell nuclear antigen (PCNA), and Downstream neighbor of SON (DONSON), initiate DNA-PKcs-mediated p-CHK1 activation and A-NHEJ repair. In addition, Claspin directly interacts with TRF2 and recruits EXO1 to newly replicated telomeres to promote 5' end resection. Our data indicate that MRN is dispensable for the repair of dysfunctional telomeres lacking POT1-TPP1 and highlight the contributions of the replisome in telomere repair.

Deletion of BCG Hip1 protease enhances dendritic cell and CD4 T cell responses.

Dendritic cells (DCs) play a key role in the generation of CD4 T cell responses to pathogens. Mycobacterium tuberculosis (Mtb) harbors immune evasion mechanisms that impair DC responses and prevent optimal CD4 T cell immunity. The vaccine strain Mycobacterium bovis Bacille Calmette-Guérin (BCG) shares many of the immune evasion proteins utilized by Mtb, but the role of these proteins in DC and T cell responses elicited by BCG is poorly understood. We previously reported that the Mtb serine protease, Hip1, promotes sub-optimal DC responses during infection. Here, we tested the hypothesis that BCG Hip1 modulates DC functions and prevents optimal antigen-specific CD4 T cell responses that limit the immunogenicity of BCG. We generated a strain of BCG lacking hip1 (BCGΔhip1) and show that it has superior capacity to induce DC maturation and cytokine production compared with the parental BCG. Furthermore, BCGΔhip1-infected DCs were more effective at driving the production of IFN-γ and IL-17 from antigen-specific CD4 T cells in vitro. Mucosal transfer of BCGΔhip1-infected DCs into mouse lungs induced robust CD4 T cell activation in vivo and generated antigen-specific polyfunctional CD4 T cell responses in the lungs. Importantly, BCGΔhip1-infected DCs enhanced control of pulmonary bacterial burden following Mtb aerosol challenge compared with the transfer of BCG-infected DCs. These results reveal that BCG employs Hip1 to impair DC activation, leading to attenuated lung CD4 T cell responses with limited capacity to control Mtb burden after challenge.

Glial cells are functionally impaired in juvenile neuronal ceroid lipofuscinosis and detrimental to neurons.

The neuronal ceroid lipofuscinoses (NCLs or Batten disease) are a group of inherited, fatal neurodegenerative disorders of childhood. In these disorders, glial (microglial and astrocyte) activation typically occurs early in disease progression and predicts where neuron loss subsequently occurs. We have found that in the most common juvenile form of NCL (CLN3 disease or JNCL) this glial response is less pronounced in both mouse models and human autopsy material, with the morphological transformation of both astrocytes and microglia severely attenuated or delayed. To investigate their properties, we isolated glia and neurons from Cln3-deficient mice and studied their basic biology in culture. Upon stimulation, both Cln3-deficient astrocytes and microglia also showed an attenuated ability to transform morphologically, and an altered protein secretion profile. These defects were more pronounced in astrocytes, including the reduced secretion of a range of neuroprotective factors, mitogens, chemokines and cytokines, in addition to impaired calcium signalling and glutamate clearance. Cln3-deficient neurons also displayed an abnormal organization of their neurites. Most importantly, using a co-culture system, Cln3-deficient astrocytes and microglia had a negative impact on the survival and morphology of both Cln3-deficient and wildtype neurons, but these effects were largely reversed by growing mutant neurons with healthy glia. These data provide evidence that CLN3 disease astrocytes are functionally compromised. Together with microglia, they may play an active role in neuron loss in this disorder and can be considered as potential targets for therapeutic interventions.

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.

AAV gene transfer delays disease onset in a TPP1-deficient canine model of the late infantile form of Batten disease.

The most common form of the childhood neurodegenerative disease late infantile neuronal ceroid lipofuscinosis (also called Batten disease) is caused by deficiency of the soluble lysosomal enzyme tripeptidyl peptidase 1 (TPP1) resulting from mutations in the TPP1 gene. We tested whether TPP1 gene transfer to the ependyma, the epithelial lining of the brain ventricular system, in TPP1-deficient dogs would be therapeutically beneficial. A one-time administration of recombinant adeno-associated virus (rAAV) expressing canine TPP1 (rAAV.caTPP1) resulted in high expression of TPP1 predominantly in ependymal cells and secretion of the enzyme into the cerebrospinal fluid leading to clinical benefit. Diseased dogs treated with rAAV.caTPP1 showed delays in onset of clinical signs and disease progression, protection from cognitive decline, and extension of life span. By immunostaining and enzyme assay, recombinant protein was evident throughout the brain and spinal cord, with correction of the neuropathology characteristic of the disease. This study in a naturally occurring canine model of TPP1 deficiency highlights the utility of AAV transduction of ventricular lining cells to accomplish stable secretion of recombinant protein for broad distribution in the central nervous system and therapeutic benefit.

Shrimp serine proteinase homologues PmMasSPH-1 and -2 play a role in the activation of the prophenoloxidase system.

Melanization mediated by the prophenoloxidase (proPO) activating system is a rapid immune response used by invertebrates against intruding pathogens. Several masquerade-like and serine proteinase homologues (SPHs) have been demonstrated to play an essential role in proPO activation in insects and crustaceans. In a previous study, we characterized the masquerade-like SPH, PmMasSPH1, in the black tiger shrimp Penaeus monodon as a multifunctional immune protein based on its recognition and antimicrobial activity against the Gram-negative bacteria Vibrio harveyi. In the present study, we identify a novel SPH, known as PmMasSPH2, composed of an N-terminal clip domain and a C-terminal SP-like domain that share high similarity to those of other insect and crustacean SPHs. We demonstrate that gene silencing of PmMasSPH1 and PmMasSPH2 significantly reduces PO activity, resulting in a high number of V. harveyi in the hemolymph. Interestingly, knockdown of PmMasSPH1 suppressed not only its gene transcript but also other immune-related genes in the proPO system (e.g., PmPPAE2) and antimicrobial peptides (e.g., PenmonPEN3, PenmonPEN5, crustinPm1 and Crus-likePm). The PmMasSPH1 and PmMasSPH2 also show binding activity to peptidoglycan (PGN) of Gram-positive bacteria. Using a yeast two-hybrid analysis and co-immunoprecipitation, we demonstrate that PmMasSPH1 specifically interacted with the final proteinase of the proPO cascade, PmPPAE2. Furthermore, the presence of both PmMasSPH1 and PmPPAE2 enhances PGN-induced PO activity in vitro. Taken together, these results suggest the importance of PmMasSPHs in the activation of the shrimp proPO system.

Enzyme replacement therapy delays pupillary light reflex deficits in a canine model of late infantile neuronal ceroid lipofuscinosis.

Late-infantile neuronal ceroid lipofuscinosis (CLN2 disease) is a hereditary neurological disorder characterized by progressive retinal degeneration and vision loss, cognitive and motor decline, seizures, and pronounced brain atrophy. This fatal pediatric disease is caused by mutations in the CLN2 gene which encodes the lysosomal enzyme tripeptidyl peptidase-1 (TPP1). Utilizing a TPP1-/- Dachshund model of CLN2 disease, studies were conducted to assess the effects of TPP1 enzyme replacement administered directly to the CNS on disease progression. Recombinant human TPP1 (rhTPP1) or artificial cerebrospinal fluid vehicle was administered to CLN2-affected dogs via infusion into the CSF. Untreated and vehicle treated affected dogs exhibited progressive declines in pupillary light reflexes (PLRs) and electroretinographic (ERG) responses to light stimuli. Studies were undertaken to determine whether CSF administration of rhTPP1 alters progression of the PLR and ERG deficits in the canine model. rhTPP1 administration did not inhibit the decline in ERG responses, as rhTPP1 treated, vehicle treated, and untreated dogs all exhibited similar progressive and profound declines in ERG amplitudes. However, in some of the dogs treated with rhTPP1 there were substantial delays in the appearance and progression of PLR deficits compared with untreated or vehicle treated affected dogs. These findings indicate that CSF administration of TPP1 can attenuate functional impairment of neural pathways involved in mediating the PLR but does not prevent loss of retinal responses detectable with ERG.

Streptococcus pneumoniae serine protease HtrA, but not SFP or PrtA, is a major virulence factor in pneumonia.

Streptococcus (S.) pneumoniae is a common causative pathogen in pneumonia. Serine protease orthologs expressed by a variety of bacteria have been found of importance for virulence. Previous studies have identified two serine proteases in S. pneumoniae, HtrA (high-temperature requirement A) and PrtA (cell wall-associated serine protease A), that contributed to virulence in models of pneumonia and intraperitoneal infection respectively. We here sought to identify additional S. pneumoniae serine proteases and determine their role in virulence. The S. pneumoniae D39 genome contains five putative serine proteases, of which HtrA, Subtilase Family Protein (SFP) and PrtA were selected for insertional mutagenesis because they are predicted to be secreted and surface exposed. Mutant D39 strains lacking serine proteases were constructed by in-frame insertion deletion mutagenesis. Pneumonia was induced by intranasal infection of mice with wild-type or mutant D39. After high dose infection, only D39ΔhtrA showed reduced virulence, as reflected by strongly reduced bacterial loads, diminished dissemination and decreased lung inflammation. D39ΔprtA induced significantly less lung inflammation together with smaller infiltrated lung surface, but without influencing bacterial loads. After low dose infection, D39ΔhtrA again showed strongly reduced bacterial loads; notably, pneumococcal burdens were also modestly lower in lungs after infection with D39Δsfp. These data confirm the important role for HtrA in S. pneumoniae virulence. PrtA contributes to lung damage in high dose pneumonia; it does not however contribute to bacterial outgrowth in pneumococcal pneumonia. SFP may facilitate S. pneumoniae growth after low dose infection.

A zebrafish model of CLN2 disease is deficient in tripeptidyl peptidase 1 and displays progressive neurodegeneration accompanied by a reduction in proliferation.

Tripeptidyl peptidase 1 (TPP1) deficiency causes CLN2 disease, late infantile (or classic late infantile neuronal ceroid lipofuscinosis), a paediatric neurodegenerative disease of autosomal recessive inheritance. Patients suffer from blindness, ataxia, epilepsy and cognitive defects, with MRI indicating widespread brain atrophy, and profound neuron loss is evident within the retina and brain. Currently there are no effective therapies for this disease, which causes premature death in adolescence. Zebrafish have been successfully used to model a range of neurological and behavioural abnormalities. The aim of this study was to characterize the pathological and functional consequences of Tpp1 deficiency in zebrafish and to correlate these with human CLN2 disease, thereby providing a platform for drug discovery. Our data show that homozygous tpp1(sa0011) mutant (tpp1(sa0011)(-/-)) zebrafish display a severe, progressive, early onset neurodegenerative phenotype, characterized by a significantly small retina, a small head and curved body. The mutant zebrafish have significantly reduced median survival with death occurring 5 days post-fertilization. As in human patients with CLN2 disease, mutant zebrafish display storage of subunit c of mitochondrial ATP-synthase, hypertrophic lysosomes as well as localized apoptotic cell death in the retina, optic tectum and cerebellum. Further neuropathological phenotypes of these mutants provide novel insights into mechanisms of pathogenesis in CLN2 disease. Secondary neurogenesis in the retina, optic tectum and cerebellum is impaired and axon tracts within the spinal cord, optic nerve and the posterior commissure are disorganized, with the optic nerve failing to reach its target. This severe neurodegenerative phenotype eventually results in functional motor impairment, but this is preceded by a phase of hyperactivity that is consistent with seizures. Importantly, both of these locomotion phenotypes can be assayed in an automated manner suitable for high-throughput studies. Our study provides proof-of-principle that tpp1(sa0011)(-/-) mutants can utilize the advantages of zebrafish for understanding pathogenesis and drug discovery in CLN2 disease and other epilepsies.

Different molecular mechanisms involved in spontaneous and oxidative stress-induced mitochondrial fragmentation in tripeptidyl peptidase-1 (TPP-1)-deficient fibroblasts.

NCLs (neuronal ceroid lipofuscinoses) form a group of eight inherited autosomal recessive diseases characterized by the intralysosomal accumulation of autofluorescent pigments, called ceroids. Recent data suggest that the pathogenesis of NCL is associated with the appearance of fragmented mitochondria with altered functions. However, even if an impairement in the autophagic pathway has often been evoked, the molecular mechanisms leading to mitochondrial fragmentation in response to a lysosomal dysfunction are still poorly understood. In this study, we show that fibroblasts that are deficient for the TPP-1 (tripeptidyl peptidase-1), a lysosomal hydrolase encoded by the gene mutated in the LINCL (late infantile NCL, CLN2 form) also exhibit a fragmented mitochondrial network. This morphological alteration is accompanied by an increase in the expression of the protein BNIP3 (Bcl2/adenovirus E1B 19 kDa interacting protein 3) as well as a decrease in the abundance of mitofusins 1 and 2, two proteins involved in mitochondrial fusion. Using RNAi (RNA interference) and quantitative analysis of the mitochondrial morphology, we show that the inhibition of BNIP3 expression does not result in an increase in the reticulation of the mitochondrial population in LINCL cells. However, this protein seems to play a key role in cell response to mitochondrial oxidative stress as it sensitizes mitochondria to antimycin A-induced fragmentation. To our knowledge, our results bring the first evidence of a mechanism that links TPP-1 deficiency and oxidative stress-induced changes in mitochondrial morphology.