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LAMP-2 deficiency leads to hippocampal dysfunction but normal clearance of neuronal substrates of chaperone-mediated autophagy in a mouse model for Danon disease.

Rothaug M, Stroobants S, Schweizer M, Peters J, Zunke F, Allerding M, D'Hooge R, Saftig P, Blanz J - Acta Neuropathol Commun (2015)

Bottom Line: Intellectual dysfunction observed in the human disease suggests a pivotal role of LAMP-2 within brain.The absence of LAMP-2 in murine brain led to inflammation and abnormal behavior, including motor deficits and impaired learning.The absence of LAMP-2 did not apparently affect MA or steady-state levels of selected CMA substrates in brain or neuroblastoma cells under physiological and prolonged starvation conditions.

View Article: PubMed Central - PubMed

ABSTRACT
The Lysosomal Associated Membrane Protein type-2 (LAMP-2) is an abundant lysosomal membrane protein with an important role in immunity, macroautophagy (MA) and chaperone-mediated autophagy (CMA). Mutations within the Lamp2 gene cause Danon disease, an X-linked lysosomal storage disorder characterized by (cardio)myopathy and intellectual dysfunction. The pathological hallmark of this disease is an accumulation of glycogen and autophagic vacuoles in cardiac and skeletal muscle that, along with the myopathy, is also present in LAMP-2-deficient mice. Intellectual dysfunction observed in the human disease suggests a pivotal role of LAMP-2 within brain. LAMP-2A, one specific LAMP-2 isoform, was proposed to be important for the lysosomal degradation of selective proteins involved in neurodegenerative diseases such as Huntington's and Parkinson's disease. To elucidate the neuronal function of LAMP-2 we analyzed knockout mice for neuropathological changes, MA and steady-state levels of CMA substrates. The absence of LAMP-2 in murine brain led to inflammation and abnormal behavior, including motor deficits and impaired learning. The latter abnormality points to hippocampal dysfunction caused by altered lysosomal activity, distinct accumulation of p62-positive aggregates, autophagic vacuoles and lipid storage within hippocampal neurons and their presynaptic terminals. The absence of LAMP-2 did not apparently affect MA or steady-state levels of selected CMA substrates in brain or neuroblastoma cells under physiological and prolonged starvation conditions. Our data contribute to the understanding of intellectual dysfunction observed in Danon disease patients and highlight the role of LAMP-2 within the central nervous system, particularly the hippocampus.

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Lysosomal protein content and CMA substrate steady-state levels after prolonged starvation. Immunoblotting and quantitative RT-PCR of lysates from N2a cells either non-transfected (non) or stably transfected with scramble shRNA (scmbl) or shRNA targeting LAMP-2 mRNA. Cells were cultured in EBSS media for 24 hours to induce CMA. (a) Immunoblots and respective densitometric quantification showing protein levels of CMA substrates MEF2D and GAPDH (b) Immunoblots and respective densitometric quantification of LAMP-2A and (c) quantitative RT-PCR of LAMP-2A and LAMP-2B. (d) Immunoblots and respective densitometric quantification showing protein levels of LAMP-1. (e) Quantitative RT-PCR of LAMP-1. (f) Immunoblots and respective densitometric quantification showing protein levels of LIMP-2. (g) Quantitative RT-PCR of LIMP-2. (Samples were cultured in EBSS for 24 hours to induce CMA; actin was used to control loading; *p < 0.05, **p < 0.01, ***p < 0.001).
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Fig7: Lysosomal protein content and CMA substrate steady-state levels after prolonged starvation. Immunoblotting and quantitative RT-PCR of lysates from N2a cells either non-transfected (non) or stably transfected with scramble shRNA (scmbl) or shRNA targeting LAMP-2 mRNA. Cells were cultured in EBSS media for 24 hours to induce CMA. (a) Immunoblots and respective densitometric quantification showing protein levels of CMA substrates MEF2D and GAPDH (b) Immunoblots and respective densitometric quantification of LAMP-2A and (c) quantitative RT-PCR of LAMP-2A and LAMP-2B. (d) Immunoblots and respective densitometric quantification showing protein levels of LAMP-1. (e) Quantitative RT-PCR of LAMP-1. (f) Immunoblots and respective densitometric quantification showing protein levels of LIMP-2. (g) Quantitative RT-PCR of LIMP-2. (Samples were cultured in EBSS for 24 hours to induce CMA; actin was used to control loading; *p < 0.05, **p < 0.01, ***p < 0.001).

Mentions: Under these conditions we observed a significant increase in LAMP-2A expression in control cells as shown by immunoblotting (Figure 7b) and qRT-PCR (Figure 7c). Interestingly, an increase was also observed in LAMP-2B (Figure 7c) as well as two other lysosomal membrane proteins investigated namely LAMP-1 (Figure 7d/e) and LIMP-2 (Figure 7f/g).Figure 7


LAMP-2 deficiency leads to hippocampal dysfunction but normal clearance of neuronal substrates of chaperone-mediated autophagy in a mouse model for Danon disease.

Rothaug M, Stroobants S, Schweizer M, Peters J, Zunke F, Allerding M, D'Hooge R, Saftig P, Blanz J - Acta Neuropathol Commun (2015)

Lysosomal protein content and CMA substrate steady-state levels after prolonged starvation. Immunoblotting and quantitative RT-PCR of lysates from N2a cells either non-transfected (non) or stably transfected with scramble shRNA (scmbl) or shRNA targeting LAMP-2 mRNA. Cells were cultured in EBSS media for 24 hours to induce CMA. (a) Immunoblots and respective densitometric quantification showing protein levels of CMA substrates MEF2D and GAPDH (b) Immunoblots and respective densitometric quantification of LAMP-2A and (c) quantitative RT-PCR of LAMP-2A and LAMP-2B. (d) Immunoblots and respective densitometric quantification showing protein levels of LAMP-1. (e) Quantitative RT-PCR of LAMP-1. (f) Immunoblots and respective densitometric quantification showing protein levels of LIMP-2. (g) Quantitative RT-PCR of LIMP-2. (Samples were cultured in EBSS for 24 hours to induce CMA; actin was used to control loading; *p < 0.05, **p < 0.01, ***p < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4359523&req=5

Fig7: Lysosomal protein content and CMA substrate steady-state levels after prolonged starvation. Immunoblotting and quantitative RT-PCR of lysates from N2a cells either non-transfected (non) or stably transfected with scramble shRNA (scmbl) or shRNA targeting LAMP-2 mRNA. Cells were cultured in EBSS media for 24 hours to induce CMA. (a) Immunoblots and respective densitometric quantification showing protein levels of CMA substrates MEF2D and GAPDH (b) Immunoblots and respective densitometric quantification of LAMP-2A and (c) quantitative RT-PCR of LAMP-2A and LAMP-2B. (d) Immunoblots and respective densitometric quantification showing protein levels of LAMP-1. (e) Quantitative RT-PCR of LAMP-1. (f) Immunoblots and respective densitometric quantification showing protein levels of LIMP-2. (g) Quantitative RT-PCR of LIMP-2. (Samples were cultured in EBSS for 24 hours to induce CMA; actin was used to control loading; *p < 0.05, **p < 0.01, ***p < 0.001).
Mentions: Under these conditions we observed a significant increase in LAMP-2A expression in control cells as shown by immunoblotting (Figure 7b) and qRT-PCR (Figure 7c). Interestingly, an increase was also observed in LAMP-2B (Figure 7c) as well as two other lysosomal membrane proteins investigated namely LAMP-1 (Figure 7d/e) and LIMP-2 (Figure 7f/g).Figure 7

Bottom Line: Intellectual dysfunction observed in the human disease suggests a pivotal role of LAMP-2 within brain.The absence of LAMP-2 in murine brain led to inflammation and abnormal behavior, including motor deficits and impaired learning.The absence of LAMP-2 did not apparently affect MA or steady-state levels of selected CMA substrates in brain or neuroblastoma cells under physiological and prolonged starvation conditions.

View Article: PubMed Central - PubMed

ABSTRACT
The Lysosomal Associated Membrane Protein type-2 (LAMP-2) is an abundant lysosomal membrane protein with an important role in immunity, macroautophagy (MA) and chaperone-mediated autophagy (CMA). Mutations within the Lamp2 gene cause Danon disease, an X-linked lysosomal storage disorder characterized by (cardio)myopathy and intellectual dysfunction. The pathological hallmark of this disease is an accumulation of glycogen and autophagic vacuoles in cardiac and skeletal muscle that, along with the myopathy, is also present in LAMP-2-deficient mice. Intellectual dysfunction observed in the human disease suggests a pivotal role of LAMP-2 within brain. LAMP-2A, one specific LAMP-2 isoform, was proposed to be important for the lysosomal degradation of selective proteins involved in neurodegenerative diseases such as Huntington's and Parkinson's disease. To elucidate the neuronal function of LAMP-2 we analyzed knockout mice for neuropathological changes, MA and steady-state levels of CMA substrates. The absence of LAMP-2 in murine brain led to inflammation and abnormal behavior, including motor deficits and impaired learning. The latter abnormality points to hippocampal dysfunction caused by altered lysosomal activity, distinct accumulation of p62-positive aggregates, autophagic vacuoles and lipid storage within hippocampal neurons and their presynaptic terminals. The absence of LAMP-2 did not apparently affect MA or steady-state levels of selected CMA substrates in brain or neuroblastoma cells under physiological and prolonged starvation conditions. Our data contribute to the understanding of intellectual dysfunction observed in Danon disease patients and highlight the role of LAMP-2 within the central nervous system, particularly the hippocampus.

Show MeSH
Related in: MedlinePlus