Protocol for labeling and fixation of intact lysosomes with esterified amino acid analogs to assess lysosomal expansion in living eukaryotic cells

Scerra, Gianluca; Caporaso, Maria Gabriella; Renna, Maurizio; D’Agostino, Massimo

doi:10.1016/j.xpro.2021.100916

Cited by 6 publications

(4 citation statements)

References 10 publications

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“…Immunofluorescence staining was performed as previously reported. − Briefly, cells grown on glass coverslips were washed with PBS and fixed in 3.7% formaldehyde at room temperature for 30 min. After fixation, cells were washed with PBS and permeabilized by incubation in blocking buffer (PBS containing 1% BSA, 0.01% sodium azide, and 0.02% saponin) for 20 min at room temperature.…”

Section: Biological Evaluationmentioning

confidence: 99%

N-Substituted l-Iminosugars for the Treatment of Sanfilippo Type B Syndrome

et al. 2023

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Sanfilippo syndrome comprises a group of four genetic diseases due to the lack or decreased activity of enzymes involved in heparan sulfate (HS) catabolism. HS accumulation in lysosomes and other cellular compartments results in tissue and organ dysfunctions, leading to a wide range of clinical symptoms including severe neurodegeneration. To date, no approved treatments for Sanfilippo disease exist. Here, we report the ability of N-substituted l-iminosugars to significantly reduce substrate storage and lysosomal dysfunctions in Sanfilippo fibroblasts and in a neuronal cellular model of Sanfilippo B subtype. Particularly, we found that they increase the levels of defective α-N-acetylglucosaminidase and correct its proper sorting toward the lysosomal compartment. Furthermore, l-iminosugars reduce HS accumulation by downregulating protein levels of exostosin glycosyltransferases. These results highlight an interesting pharmacological potential of these glycomimetics in Sanfilippo syndrome, paving the way for the development of novel therapeutic approaches for the treatment of such incurable disease.

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Section: Biological Evaluationmentioning

confidence: 99%

N-Substituted l-Iminosugars for the Treatment of Sanfilippo Type B Syndrome

et al. 2023

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“…Cell morphology was evaluated with phase contrast images taken at 20x with the EVOS FLc system (Life Technologies). Visible vacuoles (>0.4 um) were measured using ImageJ software [33]. Ratiometric measurement of lysosomal pH was carried with the dye Oregon Green 488-dextran as previously described [21].…”

Section: Cell Culture and Imagingmentioning

confidence: 99%

The chloride antiporter CLCN7 is a modifier of lysosome dysfunction in FIG4 and VAC14 mutants

et al. 2023

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The phosphatase FIG 4 and the scaffold protein VAC14 function in the biosynthesis of PI(3,5)P2, a signaling lipid that inhibits the lysosomal chloride transporter ClC-7. Loss-of-function mutations of FIG 4 and VAC14 reduce PI(3,5)P2 and result in lysosomal disorders characterized by accumulation of enlarged lysosomes and neurodegeneration. Similarly, a gain of function mutation of CLCN7 encoding ClC-7 also results in enlarged lysosomes. We therefore tested the ability of reduced CLCN7 expression to compensate for loss of FIG 4 or VAC14. Knock-out of CLCN7 corrected lysosomal swelling and partially corrected lysosomal hyperacidification in FIG 4 null cell cultures. Knockout of the related transporter CLCN6 (ClC-6) in FIG 4 null cells did not affect the lysosome phenotype. In the Fig 4 null mouse, reduction of ClC-7 by expression of the dominant negative CLCN7 variant p.Gly215Arg improved growth and neurological function and increased lifespan by 20%. These observations demonstrate a role for the CLCN7 chloride transporter in pathogenesis of FIG 4 and VAC14 disorders. Reduction of CLCN7 provides a new target for treatment of FIG 4 and VAC14 deficiencies that lack specific therapies, such as Charcot-Marie-Tooth Type 4J and Yunis-Varón syndrome.

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“…Then, cells were permeabilized with 0.2% Triton X-100 for 5 min and blocked for 30 min in PBS containing 2% FBS and 0.2% bovine serum albumin (BSA). For Lamp-1 immunolabeling, cells were fixed with 100% ice-cold methanol for 5 min for preserving lysosomal morphology as recently shown (Scerra et al, 2021); then blocked for 30 min in PBS containing 2% FBS and 0.2% BSA. Primary antibodies were detected with Alexa Fluor-conjugated secondary antibodies.…”

Section: Fluorescence Microscopymentioning

confidence: 99%

Down Syndrome Fetal Fibroblasts Display Alterations of Endosomal Trafficking Possibly due to SYNJ1 Overexpression

et al. 2022

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Endosomal trafficking is essential for cellular homeostasis. At the crossroads of distinct intracellular pathways, the endolysosomal system is crucial to maintain critical functions and adapt to the environment. Alterations of endosomal compartments were observed in cells from adult individuals with Down syndrome (DS), suggesting that the dysfunction of the endosomal pathway may contribute to the pathogenesis of DS. However, the nature and the degree of impairment, as well as the timing of onset, remain elusive. Here, by applying imaging and biochemical approaches, we demonstrate that the structure and dynamics of early endosomes are altered in DS cells. Furthermore, we found that recycling trafficking is markedly compromised in these cells. Remarkably, our results in 18–20 week-old human fetal fibroblasts indicate that alterations in the endolysosomal pathway are already present early in development. In addition, we show that overexpression of the polyphosphoinositide phosphatase synaptojanin 1 (Synj1) recapitulates the alterations observed in DS cells, suggesting a role for this lipid phosphatase in the pathogenesis of DS, likely already early in disease development. Overall, these data strengthen the link between the endolysosomal pathway and DS, highlighting a dangerous liaison among Synj1, endosomal trafficking and DS.

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Protocol for labeling and fixation of intact lysosomes with esterified amino acid analogs to assess lysosomal expansion in living eukaryotic cells

Cited by 6 publications

References 10 publications

N-Substituted l-Iminosugars for the Treatment of Sanfilippo Type B Syndrome

N-Substituted l-Iminosugars for the Treatment of Sanfilippo Type B Syndrome

The chloride antiporter CLCN7 is a modifier of lysosome dysfunction in FIG4 and VAC14 mutants

Down Syndrome Fetal Fibroblasts Display Alterations of Endosomal Trafficking Possibly due to SYNJ1 Overexpression

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