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: Histological analysis of the CNS of 12-month-old LAMP-2-deficient mice revealed wide-spread astrogliosis within all brain regions, including the hippocampus, as illustrated by immunohistochemistry (Figure 1a) and immunoblotting (Figure 1b) with an antibody directed against the Glial Fibrillary Acidic Protein (GFAP). Mild microgliosis (Figure 1c and Additional file 1a), as depicted by immunohistochemical staining using an antibody specific for the microglia resident protein macrosialin/CD68, was observed throughout LAMP-2-deficient brains. In the subiculum of the hippocampus and pons, microgliosis was most prominent. Here, microglia assumed a morphology indicative of their activation (Figure 1c, left and middle panels). Toluidine blue stained sections of LAMP-2-deficient brains, revealed darkly stained degenerating neurons within the hippocampus, particularly the subiculum and the CA3 region (Additional file 1b). However, no indication of apoptosis was evident, as highlighted by Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) (Additional file 1c) and lack of caspase-3 cleavage (Additional file 1d). Using an antibody (Abl93, Development Studies Hybridoma Bank) that recognizes all LAMP-2 isoforms (pan LAMP-2), LAMP-2 was shown to be expressed in both primary cultured neurons (Figure 2a) and astrocytes (Figure 2b). Immunoblot analysis of different brain regions, obtained from wild-type mice, revealed ubiquitous expression of LAMP-2 within the CNS. Quantification of LAMP-2 immunoblot levels relative to cortex revealed the highest expression of LAMP-2 in the cerebellum and the lowest in the hippocampus (Figure 2c). Expression of both LAMP-2A and LAMP-2B isoforms in the hippocampus was also evident by quantitative (q) RT-PCR (Figure 2d/e). LAMP-2A appeared to be the most abundant isoform expressed in brain (Figure 2d) whereas transcripts of LAMP-2C, an isoform implicated in the uptake of RNA and DNA  was not detectable in brain using semi-quantitative PCR (data not shown). Immunohistochemistry of brain sections showed localized enriched expression of LAMP-2 in neurons especially in pyramidal neurons of the hippocampus (Figure 2f, upper panels and Additional file 2a, upper panels) and in the pons (Figure 2f, middle panels and Additional file 2a, lower panels). Despite its similar distribution, we did not observe a compensatory upregulation of LAMP-1 in LAMP-2-deficient brain as illustrated by LAMP-1 immunoblot (Additional file 2b) and immunohistological (Additional file 2c) analysis. From our data we conclude that LAMP-2 is an abundant protein of neuronal tissue and that loss of its expression causes neuroinflammation in mice.Figure 1