A current view of mitochondria damage and the diversity of lipopigment inclusions in neuronal ceroid lipofuscinose type 2 from rectal biopsy.

Neuronal ceroid lipofuscinoses (NCLs) are a growing group of neurodegenerative storage diseases, in which specific features are sought to facilitate the creation of a universal diagnostic algorithm in the future. In our ultrastructural studies, the group of NCLs was represented by the CLN2 disease caused by a defect in the TPP1 gene encoding the enzyme tripeptidyl-peptidase 1. A 3.5-year-old girl was affected by this disease. Due to diagnostic difficulties, the spectrum of clinical, enzymatic, and genetic tests was extended to include analysis of the ultrastructure of cells from a rectal biopsy. The aim of our research was to search for pathognomonic features of CLN2 and to analyse the mitochondrial damage accompanying the disease. In the examined cells of the rectal mucosa, as expected, filamentous deposits of the curvilinear profile (CVP) type were found, which dominated quantitatively. Mixed deposits of the CVP/fingerprint profile (FPP) type were observed less frequently in the examined cells. A form of inclusions of unknown origin, not described so far in CLN2 disease, were wads of osmophilic material (WOMs). They occurred alone or co-formed mixed deposits. In addition, atypically damaged mitochondria were observed in muscularis mucosae. Their deformed cristae had contact with inclusions that looked like CVPs. Considering the confirmed role of the c subunit of the mitochondrial ATP synthase in the formation of filamentous lipopigment deposits in the group of NCLs, we suggest the possible significance of other mitochondrial proteins, such as mitochondrial contact site and cristae organizing system (MICOS), in the formation of these deposits. The presence of WOMs in the context of searching for ultrastructural pathognomonic features in CLN2 disease also requires further research.

Upregulation of shelterin and CST genes and longer telomeres are associated with unfavorable prognostic characteristics in prostate cancer.

INTRODUCTION: Search for new clinical biomarkers targets in prostate cancer (PC) is urgent. Telomeres might be one of these targets. Telomeres are the extremities of linear chromosomes, essential for genome stability and control of cell divisions. Telomere homeostasis relies on the proper functioning of shelterin and CST complexes. Telomeric dysfunction and abnormal expression of its components are reported in most cancers and are associated with PC. Despite this, there are only a few studies about the expression of the main telomere complexes and their relationship with PC progression. We aimed to evaluate the role of shelterin (POT1, TRF2, TPP1, TIN2, and RAP1) and CST (CTC1, STN1, and TEN1) genes and telomere length in the progression of PC. METHODS: We evaluated genetic alterations of shelterin and CST by bioinformatics in samples of localized (n = 499) and metastatic castration-resistant PC (n = 444). We also analyzed the expression of the genes using TCGA (localized PC n = 497 and control n = 152) and experimental approaches, with surgical specimens (localized PC n = 81 and BPH n = 10) and metastatic cell lines (LNCaP, DU145, PC3 and PNT2 as control) by real-time PCR. Real-time PCR also determined the telomere length in the same experimental samples. All acquired data were associated with clinical parameters. RESULTS: Genetic alterations are uncommon in PC, but POT1, TIN2, and TEN1 showed significantly more amplifications in the metastatic cancer. Except for CTC1 and TEN1, which are differentially expressed in localized PC samples, we did not detect an expression pattern relative to control and cell lines. Nevertheless, except for TEN1, the upregulation of all genes is associated with a worse prognosis in localized PC. We also found that increased telomere length is associated with disease aggressiveness in localized PC. CONCLUSION: The upregulation of shelterin and CST genes creates an environment that favors telomere elongation, giving selective advantages for localized PC cells to progress to more aggressive stages of the disease.

Evolution of the retinal function by flash-ERG in one child suffering from neuronal ceroid lipofuscinosis CLN2 treated with cerliponase alpha: case report.

INTRODUCTION: Neuronal ceroid lipofuscinoses (CLN) are neurodegenerative disorders among the most frequent, inherited as an autosomal recessive trait. Affected patients can present with progressive decline in cognitive and motor functions, seizures, a shortened life span and visual deficiency. CLN2 is one of the rare CLN that benefits from treatment by cerliponase alpha an enzyme replacement therapy. Preliminary results on treated animal models have shown delayed neurological signs and prolonged life span. However, cerliponase alpha did not prevent vision loss or retinal degeneration in those animal models. Cerliponase alpha has currently been delivered to a few CLN2-affected patients. We report the case of one patient suffering from CLN2 treated with intracerebroventricular infusions of cerliponase alpha 300 mg every two weeks. Evolution of his retinal function was assessed by three successive flash-ERG and flash-VEP recordings throughout his treatment over a 4-year period. RESULTS: Before treatment at the age of 4 years 5 months, patient's retinas were normal (normal fundi and normal flash-ERG). After 29 infusions at the age of 6 years 10 months, a-wave combined response was absent, while cone and flicker responses were normal. After 80 infusions at the age of 8 years 9 months, a-wave cone response was absent with b-wave peak time increased, and no combined response. COMMENTS: Despite treatment, our patient's retinas showed a progressive abnormal and inhomogeneous function. Rods function was altered first, then the scotopic system and afterward, the cones. This result differs from those recorded in animal models. The relative preservation of cone functioning for a while could not be unequivocally attributed to enzyme replacement therapy as we lack comparison with the evolution of flash-ERGs recorded in untreated subjects.

Harnessing affinity-based protein profiling to reveal a novel target of nintedanib.

Nintedanib (BIBF1120), a triple angiokinase inhibitor, was first approved for idiopathic pulmonary fibrosis (IPF) therapy and is also efficacious for lung carcinoma, and interstitial lung diseases, far beyond its inhibition of VEGFR/PDGFR/FGFR. We identified tripeptidyl-peptidase 1 (TPP1) as one of the direct targets of nintedanib employing the affinity-based protein profiling (AfBPP) technique. This may be a new mechanism for nintedanib's role different from tyrosine kinase inhibition.

Identification of tumor-associated antigens of lung cancer: SEREX combined with bioinformatics analysis.

The aim of this study is to identify novel tumor-associated antigens (TAAs) of lung cancer by using serological analysis of recombinant cDNA expression library (SEREX) and bioinformatics analysis as well as to explore their humoral immune response. SEREX and pathway enrichment analysis were used to immunoscreen TAAs of lung cancer and elaborate their function in biological pathways, respectively. Subsequently, the sera level of autoantibodies against the selected TAAs (TOP2A, TRIM37, HSP90AB1, EEF1G and TPP1) was detected by immunoserological analysis to explore the immune response of these antigens. The Gene Expression Profiling Interactive Analysis (GEPIA) and Human Protein Atlas (HPA) database were applied to explore the mRNA and protein expression level of TOP2A, TRIM37 and HSP90AB1 in tissues, respectively. Seventy positive clones were identified by SEREX which contain 63 different genes, and 35 genes of them have been reported. These 35 genes were mainly related to regulation of different transcription factor and performed enrichment in legionellosis, RNA transport, IL-17 signaling pathway via enrichment analysis. Additionally, the positive rate of autoantibodies against TOP2A, TRIM37 and HSP90AB1 in lung cancer patients were typically higher than normal control (NC; P < 0.05). Moreover, the combination of the autoantibodies against TOP2A, TRIM37 and HSP90AB1 possessed an excellent diagnostic performance with sensitivity of 84% and specificity of 60%. The mRNA expression level of TOP2A was obviously unregulated in squamous cell carcinoma (SCC) tissues and adenocarcinoma (ADC) tissues compared to normal tissues (P < 0.05). In addition, TRIM37 and HSP90AB1 also showed a significant difference between SCC and NC at the mRNA expression level (P < 0.05). This study combining comprehensive autoantibody and gene expression assays has added to the growing list of lung cancer antigens, which may aid the development of diagnostic and immunotherapeutic targets for lung cancer patients.

Neuronal Ceroid Lipofuscinosis: Potential for Targeted Therapy.

Neuronal ceroid lipofuscinosis (NCLs) is a group of inherited neurodegenerative lysosomal storage diseases that together represent the most common cause of dementia in children. Phenotypically, patients have visual impairment, cognitive and motor decline, epilepsy, and premature death. A primary challenge is to halt and/or reverse these diseases, towards which developments in potential effective therapies are encouraging. Many treatments, including enzyme replacement therapy (for CLN1 and CLN2 diseases), stem-cell therapy (for CLN1, CLN2, and CLN8 diseases), gene therapy vector (for CLN1, CLN2, CLN3, CLN5, CLN6, CLN7, CLN10, and CLN11 diseases), and pharmacological drugs (for CLN1, CLN2, CLN3, and CLN6 diseases) have been evaluated for safety and efficacy in pre-clinical and clinical studies. Currently, cerliponase alpha for CLN2 disease is the only approved therapy for NCL. Lacking is any study of potential treatments for CLN4, CLN9, CLN12, CLN13 or CLN14 diseases. This review provides an overview of genetics for each CLN disease, and we discuss the current understanding from pre-clinical and clinical study of potential therapeutics. Various therapeutic interventions have been studied in many experimental animal models. Combination of treatments may be useful to slow or even halt disease progression; however, few therapies are unlikely to even partially reverse the disease and a complete reversal is currently improbable. Early diagnosis to allow initiation of therapy, when indicated, during asymptomatic stages is more important than ever.

Tripeptidyl peptidase I promotes human endometrial epithelial cell adhesive capacity implying a role in receptivity.

The endometrium undergoes cyclic remodelling throughout the menstrual cycle in preparation for embryo implantation which occurs in a short window during the mid-secretory phase. It is during this short 'receptive window' that the endometrial luminal epithelium acquires adhesive capacity permitting blastocysts firm adhesion to the endometrium to establish pregnancy. Dysregulation in any of these steps can compromise embryo implantation resulting in implantation failure and infertility. Many factors contribute to these processes including TGF-ß, LIF, IL-11 and proteases. Tripeptidyl peptidase 1 (TPP1) is a is a lysosomal serine-type protease however the contribution of the TPP1 to receptivity is unknown. We aimed to investigate the role of TPP1 in receptivity in humans.In the current study, TPP1 was expressed in both epithelial and stromal compartments of the endometrium across the menstrual cycle. Expression was confined to the cytoplasm of luminal and glandular epithelial cells and stromal cells. Staining of mid-secretory endometrial tissues of women with normal fertility and primary unexplained infertility showed reduced immunostaining intensity of TPP1 in luminal epithelial cells of infertile tissues compared to fertile tissues. By contrast, TPP1 levels in glandular epithelial and stromal cells were comparable in both groups in the mid-secretory phase. Inhibition of TPP1 using siRNA compromised HTR8/SVneo (trophoblast cell line) spheroid adhesion on siRNA-transfected Ishikawa cells (endometrial epithelial cell line) in vitro. This impairment was associated with decreased sirtuin 1 (SIRT1), BCL2 and p53 mRNA and unaltered, CD44, CDH1, CDH2, ITGB3, VEGF A, OSTEOPONTIN, MDM2, CASP4, MCL1, MMP2, ARF6, SGK1, HOXA-10, LIF, and LIF receptor gene expression between treatment groups. siRNA knockdown of TPP1 in primary human endometrial stromal cells did not affect decidualization nor the expression of decidualization markers prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP1). Taken together, our data strongly suggests a role for TPP1 in endometrial receptivity via its effects on epithelial cell adhesion and suggests reduced levels associated with unexplained infertility may contribute to implantation failure.

Slowing late infantile Batten disease by direct brain parenchymal administration of a rh.10 adeno-associated virus expressing CLN2.

Late infantile Batten disease (CLN2 disease) is an autosomal recessive, neurodegenerative lysosomal storage disease caused by mutations in the CLN2 gene encoding tripeptidyl peptidase 1 (TPP1). We tested intraparenchymal delivery of AAVrh.10hCLN2, a nonhuman serotype rh.10 adeno-associated virus vector encoding human CLN2, in a nonrandomized trial consisting of two arms assessed over 18 months: AAVrh.10hCLN2-treated cohort of 8 children with mild to moderate disease and an untreated, Weill Cornell natural history cohort consisting of 12 children. The treated cohort was also compared to an untreated European natural history cohort of CLN2 disease. The vector was administered through six burr holes directly to 12 sites in the brain without immunosuppression. In an additional safety assessment under a separate protocol, five children with severe CLN2 disease were treated with AAVrh.10hCLN2. The therapy was associated with a variety of expected adverse events, none causing long-term disability. Induction of systemic anti-AAVrh.10 immunity was mild. After therapy, the treated cohort had a 1.3- to 2.6-fold increase in cerebral spinal fluid TPP1. There was a slower loss of gray matter volume in four of seven children by MRI and a 42.4 and 47.5% reduction in the rate of decline of motor and language function, compared to Weill Cornell natural history cohort (P < 0.04) and European natural history cohort (P < 0.0001), respectively. Intraparenchymal brain administration of AAVrh.10hCLN2 slowed the progression of disease in children with CLN2 disease. However, improvements in vector design and delivery strategies will be necessary to halt disease progression using gene therapy.

Identification of three predictors of gastric cancer progression and prognosis.

Abnormal gene expression is an established cause of gastric cancer (GC) initiation and progression. In this study, we aimed to identify several key genes that could be used to effectively predict progression and prognosis in patients with GC. The Cancer Genome Atlas and the Gene Expression Omnibus database were used to identify candidate genes. Fourteen genes were found to associate highly with progress, metastasis, and survival of GC. Five of these genes were overexpressed in tumor tissue compared to adjacent normal tissue. This was confirmed by reverse transcription-polymerase chain reaction and western blotting for myosin-Va (MYO5A), phospholipid transfer protein (PLTP), and tripeptidyl peptidase 1 (TPP1), while the CCK8 assay was used to show that these three genes promote GC cell proliferation. In summary, we demonstrate that MYO5A, PLTP, and TPP1 expression may be suitable markers for the progression and prognosis of GC.

FDA orphan drug designations for lysosomal storage disorders - a cross-sectional analysis.

PURPOSE: To provide a quantitative clinical-regulatory insight into the status of FDA orphan drug designations for compounds intended to treat lysosomal storage disorders (LSDs). METHODS: Assessment of the drug pipeline through analysis of the FDA database for orphan drug designations with descriptive and comparative statistics. RESULTS: Between 1983 and 2019, 124 orphan drug designations were granted by the FDA for compounds intended to treat 28 lysosomal storage diseases. Orphan drug designations focused on Gaucher disease (N = 16), Pompe disease (N = 16), Fabry disease (N = 10), MPS II (N = 10), MPS I (N = 9), and MPS IIIA (N = 9), and included enzyme replacement therapies, gene therapies, and small molecules, and others. Twenty-three orphan drugs were approved for the treatment of 11 LSDs. Gaucher disease (N = 6), cystinosis (N = 5), Pompe disease (N = 3), and Fabry disease (N = 2) had multiple approvals, CLN2, LAL-D, MPS I, II, IVA, VI, and VII one approval each. This is an increase of nine more approved drugs and four more treatable LSDs (CLN2, MPS VII, LAL-D, and MPS IVA) since 2013. Mean time between orphan drug designation and FDA approval was 89.7 SD 55.00 (range 8-203, N = 23) months. CONCLUSIONS: The drug development pipeline for LSDs is growing and evolving, with increased focus on diverse small-molecule targets and gene therapy. CLN2 was the first and only LSD with an approved therapy directly targeted to the brain. Newly approved products included "me-too"-enzymes and innovative compounds such as the first pharmacological chaperone for the treatment of Fabry disease.

Mechanisms Inspired Targeting Peptides.

Peptides, as a large group of molecules, are composed of amino acid residues and can be divided into linear or cyclic peptides according to the structure. Over 13,000 molecules of natural peptides have been found and many of them have been well studied. In artificial peptide libraries, the number of peptide diversity could be up to 1 × 1013. Peptides have more complex structures and higher affinity to target proteins comparing with small molecular compounds. Recently, the development of targeting cancer immune checkpoint (CIP) inhibitors is having a very important role in tumor therapy. Peptides targeting ligands or receptors in CIP have been designed based on three-dimensional structures of target proteins or directly selected by random peptide libraries in biological display systems. Most of these targeting peptides work as inhibitors of protein-protein interaction and improve CD8+ cytotoxic T-lymphocyte (CTL) activation in the tumor microenvironment, for example, PKHB1, Ar5Y4 and TPP1. Peptides could be designed to regulate CIP protein degradation in vivo, such as PD-LYSO and PD-PALM. Besides its use in developing therapeutic drugs for targeting CIP, targeting peptides could be used in drug's targeted delivery and diagnosis in tumor immune therapy.

Impact of Gln94Glu mutation on the structure and function of protection of telomere 1, a cause of cutaneous familial melanoma.

Protection of telomere 1 (POT1) is a key component of shelterin complex, essential for maintaining telomere length and its regulation. It consists of N-terminal domain (residues 1-299), which interacts with telomeric ssDNA, and the C-terminal domain (residues 320-634) that binds to the tripeptidyl-peptidase I (TPP1). A large number of naturally occurring mutations in the POT1 gene are associated with glioma, cardiac angiosarcoma and cutaneous familial melanoma (FM). In particular, Q94E mutation disrupts the interaction of POT1 with telomeric DNA which subsequently enhances telomere uncapping and elongation and promotes the development of cutaneous familial melanoma. To understand the underlying mechanism of familial melanoma developed by Q94E-mutation, we have performed extensive structure analysis of WT and mutant protein followed by molecular dynamics simulations. Q94E mutation causes a dramatic change in the structure and stability of POT1 protein. A considerable decrease in the flexibility, fluctuation and solvent accessibility of Q94E was observed in comparison to the WT, indicating overall destabilization of protein. Essential dynamics and Anisotropic Network Mode analysis have quantified a significant change in direction and magnitude of conformational motion in Q94E mutant compared to WT. A significant loss of frustration due to Q94E mutation was also observed. Our findings indicate the loss of protein stability and dynamics of POT1 protein by Q94E mutation may be associated with the familial melanoma. AbbreviationsANManisotropic network modeEDessential dynamicsFMfamilial melanomaMDmolecular dynamicsPOT1protection of telomere 1Rgradius of gyrationRMSDroot-mean-square deviationRMSFroot-mean-square fluctuationsSASAsolvent accessible surface areaSIFTsorting Intolerant from TolerantTPP1tripeptidyl-peptidase IWTwild typeCommunicated by Ramaswamy H. Sarma.

Telomere erosion in Sjögren's syndrome: A multi-tissue comparative analysis.

BACKGROUND: Acinar progenitor cells within salivary glands have decreased regenerative capacity and exhibit shorter telomeres in primary Sjögren's syndrome (pSS) patients. We investigated whether DNA of saliva, PBMCs, and labial salivary gland (LSG) biopsy tissue have shorter telomeres in pSS compared to controls. mRNA expression of genes associated with pSS pathogenesis (ETS1, LEF1, and MMP9), telomere DNA damage response (ATM), senescence (CDKN2A), telomerase inhibition (IFN-y, TGFß1), and the shelterin complex (TPP1, POT1) were assessed in LSG tissue by qRT-PCR to examine potential defects in telomere maintenance. METHODS: Relative telomere length in DNA of saliva, PBMCs, and LSGs from non-pSS sicca and pSS patients was measured using qPCR. Saliva DNA telomere length was further compared to healthy controls. Expression of genes affecting telomere maintenance was analyzed in LSGs using qRT-PCR. RESULTS: Primary Sjögren's syndrome patients have shorter telomeres in saliva DNA (n = 21) than healthy controls (n = 27) (P = .0035). ATM mRNA expression was higher in pSS LSG tissue (n = 16) vs non-pSS sicca patients (n = 13) (P = .0283) and strongly correlated with LEF1, TPP1, and POT1 (P < .01, r > 0.6). CONCLUSIONS: Patients with pSS exhibited significant telomere erosion in saliva DNA. Overexpression of ATM in LSGs could represent a compensatory response to telomere shortening. The role of LEF1 in telomere erosion remains to be elucidated.

Effect of atorvastatin on Aß1-42 -induced alteration of SESN2, SIRT1, LC3II and TPP1 protein expressions in neuronal cell cultures.

OBJECTIVES: Sestrins (SESNs) and sirtuins (SIRTs) are antioxidant and antiapoptotic genes and crucial mediators for lysosomal autophagy regulation that play a pivotal role in the Alzheimer's disease (AD). Recently, statins have been linked to the reduced prevalence of AD in statin-prescribed populations yet molecular basis for the neuroprotective action of statins is still under debate. METHODS: This study was undertaken whether Aß-induced changes of SESN2 and SIRT1 protein expression, autophagy marker LC3II and lysosomal enzyme TPP1 affected by atorvastatin (Western blot) and its possible role in Aß neurotoxicity (ELISA). KEY FINDINGS/RESULTS: We showed that SESN2 and LC3II expressions were elevated, whereas SIRT1 and TPP1 expressions were decreased in the Aß1-42 -exposed human neuroblastoma cells (SH-SY5Y). Co-administration of atorvastatin with Aß1-42 compensates SESN2 increase and recovers SIRT1 decline by reducing oxidative stress, decreasing SESN2 expression and increasing SIRT1 expression by its neuroprotective action. Atorvastatin induced LC3II but not TPP1 level in the Aß1-42 -exposed cells suggested that atorvastatin is effective in the formation of autophagosome but not on the expression of the specific lysosomal enzyme TPP1. DISCUSSION AND CONCLUSION: Together, these results indicate that atorvastatin induced SESN2, SIRT1 and LC3II levels play a protective role against Aß1-42 neurotoxicity.

Long telomeres and cancer risk: the price of cellular immortality.

The distribution of telomere length in humans is broad, but it has finite upper and lower boundaries. Growing evidence shows that there are disease processes that are caused by both short and long telomere length extremes. The genetic basis of these short and long telomere syndromes may be linked to mutations in the same genes, such as the telomerase reverse transcriptase (TERT), but through differential effects on telomere length. Short telomere syndromes have a predominant degenerative phenotype marked by organ failure that most commonly manifests as pulmonary fibrosis and are associated with a relatively low cancer incidence. In contrast, insights from studies of cancer-prone families as well as genome-wide association studies (GWAS) have identified both rare and common variants that lengthen telomeres as being strongly associated with cancer risk. We have hypothesized that these cancers represent a long telomere syndrome that is associated with a high penetrance of cutaneous melanoma and chronic lymphocytic leukemia. In this Review, we will synthesize the clinical and human genetic observations with data from mouse models to define the role of telomeres in cancer etiology and biology.

The lysosomal aminopeptidase tripeptidyl peptidase 1 displays increased activity in malignant pancreatic cysts.

Incidental detection of pancreatic cysts has increased dramatically over the last decade, but risk stratification and clinical management remain a challenge. Mucinous cysts are precursor lesions to pancreatic cancer, however, the majority are indolent. Current diagnostics cannot identify mucinous cysts that harbor cancer or reliably differentiate these lesions from nonmucinous cysts, which present minimal risk of malignant progression. We previously determined that activity of two aspartyl proteases was increased in mucinous cysts. Using a global protease activity profiling technology, termed multiplex substrate profiling by mass spectrometry (MSP-MS), we now show that aminopeptidase activity is also elevated in mucinous cysts. The serine aminopeptidase, tripeptidyl peptidase 1 (TPP1), was detected by proteomic analysis of cyst fluid samples and quantitation using targeted MS demonstrated that this protease was significantly more abundant in mucinous cysts. In a cohort of 110 cyst fluid samples, TPP1 activity was increased more than 3-fold in mucinous cysts relative to nonmucinous cysts. Moreover, TPP1 activity is primarily associated with mucinous cysts that harbor high-grade dysplasia or invasive carcinoma. Although only 59% accurate for differentiating these lesions, measurement of TPP1 activity may improve early detection and treatment of high-risk pancreatic cysts when used in conjunction with other promising biomarkers.

Rapid progression of a walking disability in a 5-year-old boy with a CLN6 mutation.

INTRODUCTION: Neuronal ceroid lipofuscinoses (NCLs; CLN) are mainly autosomal recessive neurodegenerative disorders characterized by the accumulation of autofluorescent lipopigments in neuronal and other cells. Symptoms include visual disabilities, motor decline, and epilepsy. Causative genes are CLN1, CLN2, CLN3, CLN5, CLN6, CLN7, CLN8, CLN10, CLN11, CLN12, CLN13, and CLN14. We present the fourth Japanese case with a CLN6 mutation. CASE PRESENTATION: At 3 years of age, our patient became clumsy and fell down easily. He developed focal seizures with impaired consciousness and was started on carbamazepine. He showed ataxic walking and dysarthria with increased deep tendon reflexes. Interictal electroencephalogram revealed slow waves in the left temporal and occipital areas. Brain magnetic resonance imaging showed cerebellar atrophy and ventriculomegaly. In optical coherence tomography (OCT), the inner layer of the retina was thick and highly reflective. Exome sequencing revealed a known homozygous mutation, C.794_976del, p. (Ser265del) in CLN6. DISCUSSION: A total of 130 cases of NCL with CLN6 mutations have been reported globally, of which only four were from Japan including the current patient. The deletion of serine at position 265 has been reported in six cases. Ser265 is located in a region of short repeated sequences that is susceptible to mutation. Clinical trials of gene therapy using adeno-associated virus serotype 9 have started for NCL6, making early diagnosis crucial. OCT examination might be helpful in achieving a diagnosis.

TPP1 Delivery to Lysosomes with Extracellular Vesicles and their Enhanced Brain Distribution in the Animal Model of Batten Disease.

Extracellular vesicles (EVs) are promising natural nanocarriers for delivery of various types of therapeutics. Earlier engineered EV-based formulations for neurodegenerative diseases and cancer are reported. Herein, the use of macrophage-derived EVs for brain delivery of a soluble lysosomal enzyme tripeptidyl peptidase-1, TPP1, to treat a lysosomal storage disorder, Neuronal Ceroid Lipofuscinoses 2 (CLN2) or Batten disease, is investigated. TPP1 is loaded into EVs using two methods: i) transfection of parental EV-producing macrophages with TPP1-encoding plasmid DNA (pDNA) or ii) incorporation therapeutic protein TPP1 into naive empty EVs. For the former approach, EVs released by pretransfected macrophages contain the active enzyme and TPP1-encoding pDNA. To achieve high loading efficiency by the latter approach, sonication or permeabilization of EV membranes with saponin is utilized. Both methods provide proficient incorporation of functional TPP1 into EVs (EV-TPP1). EVs significantly increase stability of TPP1 against protease degradation and provide efficient TPP1 delivery to target cells in in vitro model of CLN2. The majority of EV-TPP1 (≈70%) is delivered to target organelles, lysosomes. Finally, a robust brain accumulation of EV carriers and increased lifespan is recorded in late-infantile neuronal ceroid lipofuscinosis (LINCL) mouse model following intraperitoneal administration of EV-TPP1.

Trehalose induces autophagy via lysosomal-mediated TFEB activation in models of motoneuron degeneration.

Macroautophagy/autophagy, a defense mechanism against aberrant stresses, in neurons counteracts aggregate-prone misfolded protein toxicity. Autophagy induction might be beneficial in neurodegenerative diseases (NDs). The natural compound trehalose promotes autophagy via TFEB (transcription factor EB), ameliorating disease phenotype in multiple ND models, but its mechanism is still obscure. We demonstrated that trehalose regulates autophagy by inducing rapid and transient lysosomal enlargement and membrane permeabilization (LMP). This effect correlated with the calcium-dependent phosphatase PPP3/calcineurin activation, TFEB dephosphorylation and nuclear translocation. Trehalose upregulated genes for the TFEB target and regulator Ppargc1a, lysosomal hydrolases and membrane proteins (Ctsb, Gla, Lamp2a, Mcoln1, Tpp1) and several autophagy-related components (Becn1, Atg10, Atg12, Sqstm1/p62, Map1lc3b, Hspb8 and Bag3) mostly in a PPP3- and TFEB-dependent manner. TFEB silencing counteracted the trehalose pro-degradative activity on misfolded protein causative of motoneuron diseases. Similar effects were exerted by trehalase-resistant trehalose analogs, melibiose and lactulose. Thus, limited lysosomal damage might induce autophagy, perhaps as a compensatory mechanism, a process that is beneficial to counteract neurodegeneration. Abbreviations: ALS: amyotrophic lateral sclerosis; AR: androgen receptor; ATG: autophagy related; AV: autophagic vacuole; BAG3: BCL2-associated athanogene 3; BECN1: beclin 1, autophagy related; CASA: chaperone-assisted selective autophagy; CTSB: cathepsin b; DAPI: 4',6-diamidino-2-phenylindole; DMEM: Dulbecco's modified Eagle's medium; EGFP: enhanced green fluorescent protein; fALS, familial amyotrophic lateral sclerosis; FRA: filter retardation assay; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GLA: galactosidase, alpha; HD: Huntington disease; hIPSCs: human induced pluripotent stem cells; HSPA8: heat shock protein A8; HSPB8: heat shock protein B8; IF: immunofluorescence analysis; LAMP1: lysosomal-associated membrane protein 1; LAMP2A: lysosomal-associated membrane protein 2A; LGALS3: lectin, galactose binding, soluble 3; LLOMe: L-leucyl-L-leucine methyl ester; LMP: lysosomal membrane permeabilization; Lys: lysosomes; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MCOLN1: mucolipin 1; mRNA: messenger RNA; MTOR: mechanistic target of rapamycin kinase; NDs: neurodegenerative diseases; NSC34: neuroblastoma x spinal cord 34; PBS: phosphate-buffered saline; PD: Parkinson disease; polyQ: polyglutamine; PPARGC1A: peroxisome proliferative activated receptor, gamma, coactivator 1 alpha; PPP3CB: protein phosphatase 3, catalytic subunit, beta isoform; RT-qPCR: real-time quantitative polymerase chain reaction; SBMA: spinal and bulbar muscular atrophy; SCAs: spinocerebellar ataxias; siRNA: small interfering RNA; SLC2A8: solute carrier family 2, (facilitated glucose transporter), member 8; smNPCs: small molecules neural progenitors cells; SOD1: superoxide dismutase 1; SQSTM1/p62: sequestosome 1; STED: stimulated emission depletion; STUB1: STIP1 homology and U-box containing protein 1; TARDBP/TDP-43: TAR DNA binding protein; TFEB: transcription factor EB; TPP1: tripeptidyl peptidase I; TREH: trehalase (brush-border membrane glycoprotein); WB: western blotting; ZKSCAN3: zinc finger with KRAB and SCAN domains 3.

Administration of a Nucleoside Analog Promotes Cancer Cell Death in a Telomerase-Dependent Manner.

Telomerase, the end-replication enzyme, is reactivated in malignant cancers to drive cellular immortality. While this distinction makes telomerase an attractive target for anti-cancer therapies, most approaches for inhibiting its activity have been clinically ineffective. As opposed to inhibiting telomerase, we use its activity to selectively promote cytotoxicity in cancer cells. We show that several nucleotide analogs, including 5-fluoro-2'-deoxyuridine (5-FdU) triphosphate, are effectively incorporated by telomerase into a telomere DNA product. Administration of 5-FdU results in an increased number of telomere-induced foci, impedes binding of telomere proteins, activates the ATR-related DNA-damage response, and promotes cell death in a telomerase-dependent manner. Collectively, our data indicate that telomerase activity can be exploited as a putative anti-cancer strategy.