A Rapid and Sensitive Method for Measuring N-Acetylglucosaminidase Activity in Cultured Cells

Mauri, Victor; Lotfi, Parisa; Segatori, Laura; Sardiello, Marco

doi:10.1371/journal.pone.0068060

Cited by 16 publications

(11 citation statements)

References 78 publications

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“…The ERG is a direct functional measurement of the retinal neurons which has the advantages of being non-invasive, repeatable and quantitative, and would be particularly instrumental to evaluate the effectiveness of various emerging therapeutic options for this neurodegenerative disease (see Mauri et al . for a review 30 ).…”

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confidence: 99%

Electrophysiological and Histological Characterization of Rod-Cone Retinal Degeneration and Microglia Activation in a Mouse Model of Mucopolysaccharidosis Type IIIB

Tse

Lotfi

Simons

et al. 2015

Sci Rep

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Sanfilippo syndrome Type B or Mucopolysaccharidosis IIIB (MPS IIIB) is a neurodegenerative autosomal recessive lysosomal storage disorder in which patients suffer severe vision loss from associated retinopathy. Here we sought to study the underlying retinal functional and morphological changes associated with MPS IIIB disease progression using the established model of MPS IIIB, the B6.129S6-Naglu(tm1Efn)/J mouse line. Electroretinogram (ERG) was recorded from MPS IIIB and wild-type (WT) mice at the age of 28 and 46 weeks, and retinal tissues were subsequently collected for immunohistochemistry analysis. At the 28th week, rod a- and b-wave amplitudes were significantly diminished in MPS IIIB compared to WT mice. The cone a- and b-waves of MPS IIIB mice were not significantly different from those of the control at the 28th week but were significantly diminished at the 46th week, when MPS IIIB mice showed a major loss of rods and rod bipolar cells in both central and peripheral regions and a minor loss of cones in the periphery. Activation of microglia and neovascularization were also detected in the MPS IIIB retina. The new findings that cones and rod bipolar cells also undergo degeneration, and that retinal microglia are activated, will inform future development of therapeutic strategies.

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confidence: 99%

Electrophysiological and Histological Characterization of Rod-Cone Retinal Degeneration and Microglia Activation in a Mouse Model of Mucopolysaccharidosis Type IIIB

Tse

Lotfi

Simons

et al. 2015

Sci Rep

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“…The enzyme activity of lysosomal hydrolase TPP1 was measured by adapting a single-step intact cell assay protocol as described previously 46 . Briefly, SHSY-5Y cells were plated at a density of 20,000 cells per well in a 96-well plate (Corning, Inc.) and incubated at 37 °C and 5% CO 2 overnight to achieve cell attachment.…”

Section: Methodsmentioning

confidence: 99%

NADPH oxidase promotes Parkinsonian phenotypes by impairing autophagic flux in an mTORC1-independent fashion in a cellular model of Parkinson’s disease

Pal

Bajaj

Sharma

et al. 2016

Sci Rep

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Oxidative stress and aberrant accumulation of misfolded proteins in the cytosol are key pathological features associated with Parkinson's disease (PD). NADPH oxidase (Nox2) is upregulated in the pathogenesis of PD; however, the underlying mechanism(s) of Nox2-mediated oxidative stress in PD pathogenesis are still unknown. Using a rotenone-inducible cellular model of PD, we observed that a short exposure to rotenone (0.5 μM) resulted in impaired autophagic flux through activation of a Nox2 dependent Src/PI3K/Akt axis, with a consequent disruption of a Beclin1-VPS34 interaction that was independent of mTORC1 activity. Sustained exposure to rotenone at a higher dose (10 μM) decreased mTORC1 activity; however, autophagic flux was still impaired due to dysregulation of lysosomal activity with subsequent induction of the apoptotic machinery. Cumulatively, our results highlight a complex pathogenic mechanism for PD where short-and long-term oxidative stress alters different signaling pathways, ultimately resulting in anomalous autophagic activity and disease phenotype. Inhibition of Nox2-dependent oxidative stress attenuated the impaired autophagy and cell death, highlighting the importance and therapeutic potential of these pathways for treating patients with PD.Reactive oxygen species (ROS) play pivotal roles in regulating signaling molecules, but when in excess they induce oxidative stress; which has been implicated as a key pathological factor in "sporadic" forms of Parkinson's disease (PD) and other neurodegenerative diseases. Environmental toxins, such as rotenone, have been well-established as causal agents of sporadic form of PD due to its ability to generate reactive oxygen and nitrogen species (ROS/ RNS) 1 . Previous work has shown that oxidative stress impairs autophagic flux and decreases lysosomal biogenesis and function in animal models of PD 2,3 . Up-regulation of NADPH oxidase (Nox2), a major superoxide-producing enzyme complex 4 , has recently been associated with PD pathogenesis in human patients and animal models 5 . Shacka and colleagues suggest that rotenone-induced oxidative stress impairs autophagic flux and promotes accumulation of protein aggregates in an in vitro model of PD 6 . We and others have recently demonstrated the ability of rotenone to induce oxidative stress via activation of the Nox2 complex [7][8][9] . We have also shown that enhanced Nox2-dependent ROS production drives impaired autophagic flux and lysosomal dysfunction by activation of Src/PI3K/mTORC1 pathway in a mouse model of muscle degeneration 10 . The classical paradigm of autophagy in mammalian cells involves ULK1, a key pro-autophagy adapter kinase essential to the nucleation of the autophagophore membrane. Activation of the serine/threonine kinase mTORC1 inhibits autophagy through phosphorylation of ULK1 at S757, thus subsequent inhibition of ULK1 activity 11 . The energy sensitive AMP activated protein kinase (AMPK) promotes autophagy, in an mTORC1-independent manner, by directly activating ULK1 through

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“…2-dimensional electrophoresis (2-DE) was performed with standard Alcian blue reagent method [4]. Enzyme assays were performed by using artificial fluorigenic (4-methylumbelliferyl) and chromogenic substrates [5][6][7][8][9][10][11][12][13][14].…”

Section: Methodsmentioning

confidence: 99%

Amniotic fluid glycosaminoglycans in the prenatal diagnosis of mucopolysaccharidoses - A useful biomarker

Akella

Kadali²

2016

Clinica Chimica Acta

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A Rapid and Sensitive Method for Measuring N-Acetylglucosaminidase Activity in Cultured Cells

Cited by 16 publications

References 78 publications

Electrophysiological and Histological Characterization of Rod-Cone Retinal Degeneration and Microglia Activation in a Mouse Model of Mucopolysaccharidosis Type IIIB

Electrophysiological and Histological Characterization of Rod-Cone Retinal Degeneration and Microglia Activation in a Mouse Model of Mucopolysaccharidosis Type IIIB

NADPH oxidase promotes Parkinsonian phenotypes by impairing autophagic flux in an mTORC1-independent fashion in a cellular model of Parkinson’s disease

Amniotic fluid glycosaminoglycans in the prenatal diagnosis of mucopolysaccharidoses - A useful biomarker

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