LAMP-2 deficiency leads to hippocampal dysfunction but normal clearance of neuronal substrates of chaperone-mediated autophagy in a mouse model for Danon disease.
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.
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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Mentions: LAMP-2A is reported to be the rate-limiting factor in CMA [14,15]. Such proteins that are degraded via CMA include those associated with neurodegenerative diseases such as MEF2D, Htt as well as the glycolytic enzyme GAPDH . Thus, we assessed a potential accumulation of these substrates, due to the lack of the CMA receptor LAMP-2A, through analysis of brain lysates from knockout animals as well as in vitro after stable knockdown of LAMP-2 in murine neuroblastoma cells (N2a). Absence of LAMP-2 expression in cortex and hippocampus (Figure 6a) did not affect the steady-state levels of MEF2D (Figure 6b), GAPDH (Figure 6c) or Htt (Figure 6d, Additional file 6a). Expression levels of Htt were also found to be unchanged in striatum (Figure 6d) which is the primary site of neuronal damage in Huntington`s disease. Similarly, stable knockdown of LAMP-2 in N2a cells using shRNA directed against LAMP-2 mRNA significantly reduced protein levels of LAMP-2 (Figure 6e) but not LAMP-1 (Additional file 6b) and did not alter steady-state levels of MEF2D (Figure 6f), GAPDH (Figure 6g) or Htt (Figure 6h). Furthermore, an accumulation of MEF2D within the cytosol, which has been reported after blockage of CMA by LAMP-2 silencing , was not revealed neither in LAMP-2-deficient primary neurons (Additional file 6c) nor in brain sections (Additional file 6d). The typical nuclear localization of this protein was observed independent of the expression of LAMP-2.Figure 6