Molecular Biology and Genetics / Moleküler Biyoloji ve Genetik
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Review Citation - WoS: 6Citation - Scopus: 8Molecular Trojan Horses for Treating Lysosomal Storage Diseases(Academic Press, 2023) Leal, Andres Felipe; Rintz, Estera; Çelik, Betül; Ago, Yasuhiko; León, Daniel; İnci, Orhan Kerim; Seyrantepe, VolkanLysosomal storage diseases (LSDs) are caused by monogenic mutations in genes encoding for proteins related to the lysosomal function. Lysosome plays critical roles in molecule degradation and cell signaling through interplay with many other cell organelles, such as mitochondria, endoplasmic reticulum, and peroxisomes. Even though several strategies (i.e., protein replacement and gene therapy) have been attempted for LSDs with promising results, there are still some challenges when hard-to-treat tissues such as bone (i.e., cartilages, ligaments, meniscus, etc.), the central nervous system (mostly neurons), and the eye (i.e., cornea, retina) are affected. Consistently, searching for novel strategies to reach those tissues remains a priority. Molecular Trojan Horses have been well-recognized as a potential alternative in several pathological scenarios for drug delivery, including LSDs. Even though molecular Trojan Horses refer to genetically engineered proteins to overcome the blood-brain barrier, such strategy can be extended to strategies able to transport and deliver drugs to specific tissues or cells using cell-penetrating peptides, monoclonal antibodies, vesicles, extracellular vesicles, and patient-derived cells. Only some of those platforms have been attempted in LSDs. In this paper, we review the most recent efforts to develop molecular Trojan Horses and discuss how this strategy could be implemented to enhance the current efficacy of strategies such as protein replacement and gene therapy in the context of LSDs. © 2023Article Citation - WoS: 4Citation - Scopus: 4Autophagic Flux Is Impaired in the Brain Tissue of Tay-Sachs Disease Mouse Model(Public Library of Science, 2023) Şengül, Tuğçe; Can, Melike; Ateş, Nurselin; Seyrantepe, VolkanTay-Sachs disease is a lethal lysosomal storage disorder caused by mutations in the HexA gene encoding the α subunit of the lysosomal β-hexosaminidase enzyme (HEXA). Abnormal GM2 ganglioside accumulation causes progressive deterioration in the central nervous system in Tay-Sachs patients. Hexa-/-mouse model failed to display abnormal phenotype. Recently, our group generated Hexa-/-Neu3-/-mouse showed severe neuropathological indications similar to Tay-Sachs patients. Despite excessive GM2 ganglioside accumulation in the brain and visceral organs, the regulation of autophagy has not been clarified yet in the Tay-Sachs disease mouse model. Therefore, we investigated distinct steps of autophagic flux using markers including LC3 and p62 in four different brain regions from the Hexa-/-Neu3-/-mice model of Tay-Sachs disease. Our data revealed accumulated autophagosomes and autophagolysosomes indicating impairment in autophagic flux in the brain. We suggest that autophagy might be a new therapeutic target for the treatment of devastating Tay-Sachs disease. © 2023 Sengul et al.Article Citation - WoS: 6Citation - Scopus: 6Analysis of Brain Lipids in the Early-Onset Tay–sachs Disease Mouse Model With the Combined Deficiency of Β-Hexosaminidase a and Neuraminidase 3(Frontiers Media S.A., 2022) Can, Melike; Şengül, Tuğçe; Akyıldız Demir, Seçil; İnci, Orhan K.; Basırlı, Hatice Hande; Seyrantepe, VolkanTay–Sachs disease is an autosomal recessively inherited lysosomal storage disease that results from loss-of-function mutations in the HEXA gene coding βhexosaminidase A. HEXA gene deficiency affects the central nervous system owing to GM2 ganglioside accumulation in lysosomes resulting in progressive neurodegeneration in patients. We recently generated a novel mice model with a combined deficiency of βhexosaminidase A and neuraminidase 3 (Hexa−/−Neu3−/−) that mimics both the neuropathological and clinical abnormalities of early-onset Tay–Sachs disease. Here, we aimed to explore the secondary accumulation of lipids in the brain of Hexa−/ −Neu3−/− mice.Article Citation - WoS: 5Citation - Scopus: 5Sialidase Neu4 Deficiency Is Associated With Neuroinflammation in Mice(Springer, 2021) Timur, Zehra Kevser; İnci, Orhan Kerim; Akyıldız Demir, Seçil; Seyrantepe, VolkanSialidases catalyze the removal of sialic acid residues from glycoproteins, oligosaccharides, and sialylated glycolipids. Sialidase Neu4 is in the lysosome and has broad substrate specificity. Previously generated Neu4-/- mice were viable, fertile and lacked gross morphological abnormalities, but displayed a marked vacuolization and lysosomal storage in lung and spleen cells. In addition, we showed that there is an increased level of GD1a ganglioside and a markedly decreased level of GM1 ganglioside in the brain of Neu4-/- mice. In this study, we further explored whether sialidase Neu4 deficiency causes neuroinflammation. We demostrated that elevated level of GD1a and GT1b is associated with an increased level of LAMP1-positive lysosomal vesicles and Tunel-positive neurons correlated with alterations in the expression of cytokines and chemokines in adult Neu4-/- mice. Astrogliosis and microgliosis were also significantly enhanced in the hippocampus, and cerebellum. These changes in brain immunity were accompanied by motor impairment in these mice. Our results indicate that sialidase Neu4 is a novel mediator of an inflammatory response in the mouse brain due to the altered catabolism of gangliosides.Article Citation - WoS: 37Citation - Scopus: 40Gm2 Ganglioside Accumulation Causes Neuroinflammation and Behavioral Alterations in a Mouse Model of Early Onset Tay-Sachs Disease(BioMed Central Ltd., 2020) Akyıldız Demir, Seçil; Timur, Zehra Kevser; Ateş, Nurselin; Martinez, Luis Alarcon; Seyrantepe, VolkanBackground Tay-Sachs disease (TSD), a type of GM2-gangliosidosis, is a progressive neurodegenerative lysosomal storage disorder caused by mutations in the alpha subunit of the lysosomal beta-hexosaminidase enzyme. This disease is characterized by excessive accumulation of GM2 ganglioside, predominantly in the central nervous system. Although Tay-Sachs patients appear normal at birth, the progressive accumulation of undegraded GM2 gangliosides in neurons leads to death. Recently, an early onset Tay-Sachs disease mouse model, with genotypeHexa-/-Neu3-/-, was generated. Progressive accumulation of GM2 led to premature death of the double KO mice. Importantly, this double-deficient mouse model displays typical features of Tay-Sachs patients, such as cytoplasmic vacuolization of nerve cells, deterioration of Purkinje cells, neuronal death, deceleration in movement, ataxia, and tremors. GM2-gangliosidosis is characterized by acute neurodegeneration preceded by activated microglia expansion, macrophage, and astrocyte activation, along with the production of inflammatory mediators. However, the mechanism of disease progression inHexa-/-Neu3-/-mice, relevant to neuroinflammation is poorly understood. Method In this study, we investigated the onset and progression of neuroinflammatory changes in the cortex, cerebellum, and retina ofHexa-/-Neu3-/-mice and control littermates by using a combination of molecular genetics and immunochemical procedures. Results We found elevated levels of pro-inflammatory cytokine and chemokine transcripts, such as Ccl2, Ccl3, Ccl4, and Cxcl10 and also extensive microglial and astrocyte activation and proliferation, accompanied by peripheral blood mononuclear cell infiltration in the vicinity of neurons and oligodendrocytes. Behavioral tests demonstrated a high level of anxiety, and age-dependent loss in both spatial learning and fear memory inHexa-/-Neu3-/-mice compared with that in the controls. Conclusion Altogether, our data suggest thatHexa-/-Neu3-/-mice display a phenotype similar to Tay-Sachs patients suffering from chronic neuroinflammation triggered by GM2 accumulation. Furthermore, our work contributes to better understanding of the neuropathology in a mouse model of early onset Tay-Sachs disease.Article Characterization of the Human Sialidase Neu4 Gene Promoter(TÜBİTAK - Türkiye Bilimsel ve Teknolojik Araştırma Kurumu, 2014) Seyrantepe, Volkan; Delman, MuratThere are 4 different sialidases that have been described in humans: lysosomal (Neu1), cytoplasmic (Neu2), plasma membrane (Neu3), and lysosomal/mitochondrial (Neu4). Previously, we have shown that Neu4 has a broad substrate specificity and is active against glyco-conjugates, including GM2 ganglioside, at the acidic pH of 3.2. An overexpression of Neu4 in transfected neuroglia cells from a Tay-Sachs patient shows a clearance of accumulated GM2, indicating the biological importance of Neu4. In this paper, we aimed to characterize a minimal promoter region of the human Neu4 gene in order to understand the molecular mechanism regulating its expression. We cloned 7 different DNA fragments from the human Neu4 promoter region into luciferase expression vectors for a reporter assay and also performed an electrophoretic mobility shift assay to demonstrate the binding of transcription factors. We demonstrated that -187 bp upstream of the Neu4 gene is a minimal promoter region for controlling transcription from the human Neu4 gene. The electrophoretic mobility shift assay showed that the minimal promoter region recruits a c-myc transcription factor, which might be responsible for regulation of Neu4 gene transcription. The data we obtained might be useful to discover small molecules, which control selective high expression of the human Neu4 gene, resulting in the normal morphological phenotype in the lysosomes of Tay-Sachs patients.Article Citation - WoS: 2Citation - Scopus: 2Identification of Cytoplasmic Sialidase Neu2-Associated Proteins by Lc-ms/Ms(Türk Biyokimya Derneği, 2019) Akyıldız Demir, Seçil; Seyrantepe, VolkanBackground: Cytoplasmic sialidase (NEU2) plays an active role in removing sialic acids from oligosaccharides, gly-copeptides, and gangliosides in mammalian cells. NEU2 is involved in various cellular events, including cancer metabolism, neuronal and myoblast differentiation, proliferation, and hypertrophy. However, NEU2-interacting protein(s) within the cell have not been identified yet. Objective: The aim of this study is to investigate NEU2 interacting proteins using two-step affinity purification (TAP) strategy combined with mass spectrometry analysis. Methods: In this study, NEU2 gene was cloned into the pCTAP expression vector and transiently transfected to COS-7 cells by using PEI. The most efficient expression time of NEU2- tag protein was determined by real-time PCR and Western blot analysis. NEU2-interacting protein(s) were investigated by using TAP strategy combined with two different mass spectrometry experiment; LC-MS/MS and MALDI TOF/TOF. Results: Here, mass spectrometry analysis showed four proteins; a-actin, beta-actin, calmodulin and histone H1.2 proteins are associated with NEU2. The interactions between NEU2 and actin filaments were verified by Western blot analysis and immunofluorescence analysis. Conclusions: Our study suggests that association of NEU2 with actin filaments and other protein(s) could be important for understanding the biological role of NEU2 in mammalian cells.Article Citation - WoS: 28Citation - Scopus: 31Lysosomal Cathepsin a Plays a Significant Role in the Processing of Endogenous Bioactive Peptides(Frontiers Media S.A., 2016) Timur, Zehra Kevser; Akyıldız Demir, Seçil; Seyrantepe, VolkanLysosomal serine carboxypeptidase Cathepsin A (CTSA) is a multifunctional enzyme with distinct protective and catalytic function. CTSA present in the lysosomal multienzyme complex to facilitate the correct lysosomal routing, stability and activation of with beta-galactosidase and alpha-neuraminidase. Beside CTSA has role in inactivation of bioactive peptides including bradykinin, substances P, oxytocin, angiotensin I and endothelin-I by cleavage of 1 or 2 amino acid(s) from C-terminal ends. In this study, we aimed to elucidate the regulatory role of CTSA on bioactive peptides in knock-in mice model of CTSA(S190A). We investigated the level of bradykinin, substances P, oxytocin, angiotensin I and endothelin-I in the kidney, liver, lung, brain and serum from CTSA(S190A) mouse model at 3- and 6-months of age. Our results suggest CTSA selectively contributes to processing of bioactive peptides in different tissues from CTSA(S190A) mice compared to age matched WT mice.Article Citation - WoS: 54Citation - Scopus: 58Murine Sialidase Neu3 Facilitates Gm2 Degradation and Bypass in Mouse Model of Tay-Sachs Disease(Elsevier, 2018) Seyrantepe, Volkan; Akyıldız Demir, Seçil; Timur, Zehra Kevser; Von Gerichten, Johanna; Marsching, Christian; Erdemli, Esra; Öztaş, Emin; Takahashi, Kohta; Yamaguchi, Kazunori; Ateş, Nurselin; Dönmez Demir, Buket; Dalkara, Turgay; Erich, Katrin; Hopf, Carsten; Sandhoff, Roger; Miyagi, TaekoTay-Sachs disease is a severe lysosomal storage disorder caused by mutations in Hexa, the gene that encodes for the α subunit of lysosomal β-hexosaminidase A (HEXA), which converts GM2 to GM3 ganglioside. Unexpectedly, Hexa−/− mice have a normal lifespan and show no obvious neurological impairment until at least one year of age. These mice catabolize stored GM2 ganglioside using sialidase(s) to remove sialic acid and form the glycolipid GA2, which is further processed by β-hexosaminidase B. Therefore, the presence of the sialidase (s) allows the consequences of the Hexa defect to be bypassed. To determine if the sialidase NEU3 contributes to GM2 ganglioside degradation, we generated a mouse model with combined deficiencies of HEXA and NEU3. The Hexa−/− Neu3−/− mice were healthy at birth, but died at 1.5 to 4.5 months of age. Thin-layer chromatography and mass spectrometric analysis of the brains of Hexa−/− Neu3−/− mice revealed the abnormal accumulation of GM2 ganglioside. Histological and immunohistochemical analysis demonstrated cytoplasmic vacuolation in the neurons. Electron microscopic examination of the brain, kidneys and testes revealed pleomorphic inclusions of many small vesicles and complex lamellar structures. The Hexa−/− Neu3−/− mice exhibited progressive neurodegeneration with neuronal loss, Purkinje cell depletion, and astrogliosis. Slow movement, ataxia, and tremors were the prominent neurological abnormalities observed in these mice. Furthermore, radiographs revealed abnormalities in the skeletal bones of the Hexa−/− Neu3−/− mice. Thus, the Hexa−/− Neu3−/− mice mimic the neuropathological and clinical abnormalities of the classical early-onset Tay-Sachs patients, and provide a suitable model for the future pre-clinical testing of potential treatments for this condition.Article Citation - Scopus: 8The Second Case of Saposin a Deficiency and Altered Autophagy(Springer Verlag, 2018) Köse, Melis; Akyıldız Demir, Seçil; Akıncı, Gülçin; Eraslan, Cenk; Yılmaz, Ünsal; Ceylaner, Serdar; Sözmen Yıldırım, Eser; Seyrantepe, VolkanKrabbe disease is a lysosomal storage disease caused by galactosylceramidase deficiency, resulting in neurodegeneration with a rapid clinical downhill course within the first months of life in the classic infantile form. This process may be triggered by the accumulation of galactosylceramide (GalCer) in nervous tissues. Both the enzyme galactosylceramidase and its in vivo activator molecule, saposin A, are essential during GalCer degradation. A clinical manifestation almost identical to Krabbe disease is observed when, instead of the galactosylceramidase protein, the saposin A molecule is defective. Saposin A results from posttranslational processing of the precursor molecule, prosaposin, encoded by the PSAP gene. Clinical and neuroimaging findings in a 7-month-old child strongly suggested Krabbe disease, but this condition was excluded by enzymatic and genetic testing. However, at whole exome sequencing, the previously undescribed homozygous, obviously pathogenic PSAP gene NM_002778.3: c.209T>G(p.Val70Gly) variant was determined in the saposin A domain of the PSAP gene. Fibroblast studies showed GalCer accumulation and the activation of autophagy for the first time in a case of human saposin A deficiency. Our patient represents the second known case in the literature and provides new information concerning the pathophysiology of saposin A deficiency and its intralysosomal effects.