Sphingomyelin-induced inhibition of the plasma membrane calcium ATPase causes neurodegeneration in type A Niemann-Pick disease.

Niemann-Pick disease type A (NPA) is a rare lysosomal storage disorder characterized by severe neurological alterations that leads to death in childhood. Loss-of-function mutations in the acid sphingomyelinase (ASM) gene cause NPA, and result in the accumulation of sphingomyelin (SM) in lysosomes and plasma membrane of neurons. Using ASM knockout (ASMko) mice as a NPA disease model, we investigated how high SM levels contribute to neural pathology in NPA. We found high levels of oxidative stress both in neurons from these mice and a NPA patient. Impaired activity of the plasma membrane calcium ATPase (PMCA) increases intracellular calcium. SM induces PMCA decreased activity, which causes oxidative stress. Incubating ASMko-cultured neurons in the histone deacetylase inhibitor, SAHA, restores PMCA activity and calcium homeostasis and, consequently, reduces the increased levels of oxidative stress. No recovery occurs when PMCA activity is pharmacologically impaired or genetically inhibited in vitro. Oral administration of SAHA prevents oxidative stress and neurodegeneration, and improves behavioral performance in ASMko mice. These results demonstrate a critical role for plasma membrane SM in neuronal calcium regulation. Thus, we identify changes in PMCA-triggered calcium homeostasis as an upstream mediator for NPA pathology. These findings can stimulate new approaches for pharmacological remediation in a disease with no current clinical treatments.

High sphingomyelin levels induce lysosomal damage and autophagy dysfunction in Niemann Pick disease type A.

Niemann Pick disease type A (NPA), which is caused by loss of function mutations in the acid sphingomyelinase (ASM) gene, is a lysosomal storage disorder leading to neurodegeneration. Yet, lysosomal dysfunction and its consequences in the disease are poorly characterized. Here we show that undegraded molecules build up in neurons of acid sphingomyelinase knockout mice and in fibroblasts from NPA patients in which autophagolysosomes accumulate. The latter is not due to alterations in autophagy initiation or autophagosome-lysosome fusion but because of inefficient autophago-lysosomal clearance. This, in turn, can be explained by lysosomal membrane permeabilization leading to cytosolic release of Cathepsin B. High sphingomyelin (SM) levels account for these effects as they can be induced in control cells on addition of the lipid and reverted on SM-lowering strategies in ASM-deficient cells. These results unveil a relevant role for SM in autophagy modulation and characterize autophagy anomalies in NPA, opening new perspectives for therapeutic interventions.

Endocytosis of NBD-sphingolipids in neurons: exclusion from degradative compartments and transport to the Golgi complex.

Sphingolipids are abundant constituents of neuronal membranes that have been implicated in intracellular signaling, neurite outgrowth and differentiation. Differential localization and trafficking of lipids to membrane domains contribute to the specialized functions. In non-neuronal cultured cell lines, plasma membrane short-chain sphingomyelin and glucosylceramide are recycled via endosomes or sorted to degradative compartments. However, depending on cell type and lipid membrane composition, short-chain glucosylceramide can also be diverted to the Golgi complex. Here, we show that NBD-labeled glucosylceramide and sphingomyelin are transported from the plasma membrane to the Golgi complex in cultured rat hippocampal neurons irrespective of the stage of neuronal differentiation. Golgi complex localization was confirmed by colocalization and Golgi disruption studies, and importantly did not result from conversion of NBD-glucosylceramide or NBD-sphingomyelin to NBD-ceramide. Double-labeling experiments with transferrin or wheat-germ agglutinin showed that NBD-sphingolipids are first internalized to early/recycling endosomes, and subsequently transported to the Golgi complex. The internalization of these two sphingolipid analogs was energy and temperature dependent, and their intracellular transport was insensitive to the NBD fluorescence quencher sodium dithionite. These results indicate that vesicles mediate the transport of internalized NBD-glucosylceramide and NBD-sphingomyelin to the Golgi complex.

Brain plasmin enhances APP alpha-cleavage and Abeta degradation and is reduced in Alzheimer's disease brains.

The proteolytic processing of amyloid precursor protein (APP) has been linked to sphingolipid-cholesterol microdomains (rafts). However, the raft proteases that may be involved in APP cleavage have not yet been identified. In this work we present evidence that the protease plasmin is restricted to rafts of cultured hippocampal neurons. We also show that plasmin increases the processing of human APP preferentially at the alpha-cleavage site, and efficiently degrades secreted amyloidogenic and non-amyloidogenic APP fragments. These results suggest that brain plasmin plays a preventive role in APP amyloidogenesis. Consistently, we show that brain tissue from Alzheimer's disease patients contains reduced levels of plasmin, implying that plasmin downregulation may cause amyloid plaque deposition accompanying sporadic Alzheimer's disease.

Maturation of the axonal plasma membrane requires upregulation of sphingomyelin synthesis and formation of protein-lipid complexes.

Neuronal maturation is a gradual process; first axons and dendrites are established as distinct morphological entities; next the different intracellular organization of these processes occurs; and finally the specialized plasma membrane domains of these two compartments are formed. Only when this has been accomplished does proper neuronal function take place. In this work we present evidence that the correct distribution of a class of axonal membrane proteins requires a mechanism which involves formation of protein-lipid (sphingomyelin/cholesterol) detergent-insoluble complexes (DIGs). Using biochemistry and immunofluorescence microscopy we now show that in developing neurons the randomly distributed Thy-1 does not interact with lipids into DIGs (in fully developed neurons the formation of such complexes is essential for the correct axonal targeting of this protein). Using lipid mass spectrometry and thin layer chromatography we show that the DIG lipid missing in the developing neurons is sphingomyelin, but not cholesterol or glucosylceramide. Finally, by increasing the intracellular levels of sphingomyelin in the young neurons the formation of Thy-1/DIGs was induced and, consistent with a role in sorting, proper axonal distribution was facilitated. These results emphasize the role of sphingomyelin in axonal, and therefore, neuronal maturation.

Tau glycation is involved in aggregation of the protein but not in the formation of filaments.

A tau peptide, peptide 2R, with capacity for self assembly into filaments was used as a model to test the role of glycation on tau assembly or aggregation. Our results indicate that glycation of that peptide facilitates dimer formation but not assembly into filaments. However, glycation of tau results in the bundling of the tau filaments formed by glycosaminoglycan-induced polymerisation. These results suggest a role of glycation in the formation of covalent links among pre-formed filaments but not in the assembly of those filaments.

Neuronal polarity: essential role of protein-lipid complexes in axonal sorting.

The viral glycoprotein hemagglutinin (HA) and the endogenous glycosylphosphatidylinositol-anchored protein Thy-1 are efficiently targeted to the axonal surface of fully polarized hippocampal neurons in culture. Here we have shown that in these cells HA and Thy-1 interact with sphingolipid-cholesterol rafts and are included in detergent-insoluble glycolipid-enriched complexes. Axonal HA and Thy-1, but not two dendritic membrane proteins, resisted extraction to detergents at 4 degrees C. Both HA and Thy-1 became detergent-soluble in neurons with reduced levels of cholesterol or sphingolipids. Missorting of the axonal Thy-1 but not of a dendritic membrane protein occurred in sphingolipid-deprived cells. These results indicate that neurons sort a subset of axolemmal proteins by a mechanism that requires the formation of protein-lipid rafts. The involvement of rafts in axonal membrane sorting may explain the neurological deficits observed in patients with certain types of Niemann-Pick disease.

The in vitro formation of recombinant tau polymers: effect of phosphorylation and glycation.

Tau Isolated from paired helical filaments, aberrant structures that appear in Alzheimer disease (AD) patients' brains, show at least two posttranslational modifications: phosphorylation (Grundke-Iqbal et al., 1986; Ihara et al., 1986) and glycation (Ledesma et al., 1994; Yan et al., 1994). To test whether these modifications could affect the capacity of tau to self-aggregate, recombinant tau was phosphorylated and glycated, and its capacity to form polymers analyzed. Our results indicate that on phosphorylation and glycation, the capacity of tau to form aggregates increases, and that glycation of tau could stabilize the assembled polymers and could facilitate formation of bundles from these polymers.

The role of the Maillard reaction in other pathologies: Alzheimer's disease.

Many approaches have and are being undertaken to treat Alzheimer's disease but, as yet, no therapy is available with any established efficacy. Given the heterogeneity of the aetiological factors involved in Alzheimer's disease and the difficulties encountered in the clinical diagnosis, the lack of pharmacological success is not surprising. Furthermore, the lack of an adequate animal model of Alzheimer's disease has delayed the development of novel therapeutic strategies. At present, and with the exception of the rarer forms of familial Alzheimer's disease, the need remains to treat the symptoms rather than the causes of the disease, primarily because the pathogenesis of Alzheimer's disease is still unknown. The evidence for the role of glycation and advanced glycation end-products (AGEs) in the formation of neurofibrillary tangles and neuritic plaques, the characteristic histopathological lesions of Alzheimer's disease, is briefly reviewed. While the role of glycation in the pathogenesis of Alzheimer's disease is not yet unequivocally proven, it is the only single protein modification that would explain the formation of both the characteristic histopathological lesions first described by Alois Alzheimer in 1907. With our improved understanding of the molecular basis for the clinical symptoms of dementia, it is hoped that the aetiological causes will afford more suitable targets for therapeutic intervention. In this respect it is interesting to note that the anti-inflammatory compounds indomethacin and acetylsalicylic acid, both inhibitors of the Maillard reaction, have been reported to have therapeutic potential and the nootropic agent tenilsetam inhibits protein cross-linking by AGEs.

Tau protein from Alzheimer's disease patients is glycated at its tubulin-binding domain.

Glycated residues of tau protein from paired helical filaments isolated from the brains of Alzheimer's disease patients were localized by doing a proteolytic cleavage of the protein, fractionation of the resulting peptides, and identification of those peptides using specific antibodies. The most suitable residues for glycation, lysines, present at the tubulin-binding motif of tau protein, seem to be preferentially modified compared with those lysines present at other regions. Among these modified lysines, those located in the sequence comprising residues 318-336 (in the largest human tau isoform) were found to be glycated, as determined by the reaction with an antibody that recognizes a glycated peptide containing this sequence. Because those lysines are present in a tubulin binding motif of tau protein, its modification could result in a decrease in the interaction of tau with tubulin.

Isolation of a phosphorylated soluble tau fraction from Alzheimer's disease brain.

Modified forms of tau proteins are major components of the paired helical filaments (PHFs) present in Alzheimer brains. In this study, tau from cytosolic samples obtained from normal and Alzheimer disease brains were fractionated by iron-chelated affinity chromatography (ICAC) to discriminate between isoforms phosphorylated to different extents using an stepwise pH gradient. Immunoblot analysis of the different fractions using antibody Tau-1 (recognizing an unphosphorylated epitope in tau and in PHF-tau after dephosphorylation) and antibody SMI 31 (recognizing a phosphorylated epitope in PHF-tau) have been carried out. Phosphorylated tau species (Tau 1-nonreactive and SMI 31-reactive) are only isolated from the Alzheimer samples at pH = 8.5. These tau species although having other Ser/Thr-Pro motifs susceptible of phosphorylation by proline-directed protein kinases are not further phosphorylated in vitro by MAP2 kinase whereas the fraction isolated at pH 7.0, which contains underphosphorylated tau species, is phosphorylated. Thus, soluble tau species phosphorylated both at the sites constituting the Tau-1 and the SMI 31 epitopes are present in Alzheimer but not in normal brain cytosol and can be isolated by ICAC. These modifications may be a prerequisite for PHF formation.

Analysis of microtubule-associated protein tau glycation in paired helical filaments.

Alzheimer's disease is typified by the characteristic histopathological lesions of neurofibrillar plaques and tangles. The latter are composed of paired helical filaments (PHFs), the major components of which are modified forms of the microtubule-associated protein tau. The exact nature of these modifications remains unknown, although the presence of hyperphosphorylated tau in PHFs argues strongly that phosphorylation is one of the modifications that result in the polymerization of tau into PHFs. However, hyperphosphorylation alone is insufficient to explain the formation of PHFs. In an attempt to characterize other post-translational modifications of PHF-tau, we have analyzed its glycation. A fraction of PHF-tau seems to be glycated in vivo, whereas soluble tau from either Alzheimer's disease or non-demented human brain is not glycated at all. Purified tau from bovine brain can be efficiently glycated in vitro. Tau glycation is accompanied by a decrease in the tau binding to tubulin. These results support the view that glycation may be one of the modifications hampering the binding of tau to tubulin in Alzheimer's disease, thus facilitating tau aggregation into PHFs.

Dephosphorylation of tau protein from Alzheimer's disease patients.

Tau protein-prepared post-mortem from brains of Alzheimer's disease patients was treated with protein phosphatase 1 catalytic subunit, 2A catalytic subunit, and 2B (calcineurin). Dephosphorylation was monitored by immunoblotting with two monoclonal antibodies, TAU-1 and SMI31, which recognize in tau the dephospho- and phospho-states, respectively, of proline-directed protein kinase phosphorylation sites. Out of the three enzymes tested, protein phosphatase 2A was only effective in dephosphorylating tau at these Alzheimer-type epitopes.

Implication of brain cdc2 and MAP2 kinases in the phosphorylation of tau protein in Alzheimer's disease.

Brain tau protein is phosphorylated in vitro by cdc2 and MAP2 kinases, obtained through immunoaffinity purification from rat brain extracts. The phosphorylation sites are located on the tau molecule both upstream and downstream of the tubulin-binding motifs. A synthetic peptide comprising residues 194-213 of the tau sequence, which contains the epitope recognized by the monoclonal antibody tau-1, is also efficiently phosphorylated in vitro by cdc2 and MAP2 kinases. Phosphorylation of this peptide markedly reduces its interaction with the antibody tau-1, as it has been described for tau protein in Alzheimer's disease. Both cdc2 and MAP2 kinases are present in brain extracts obtained from Alzheimer's disease patients. Interestingly, the level of cdc2 kinase may be increased in patient brains as compared with non-demented controls. These results suggest a role for cdc2 and MAP2 kinases in phosphorylating tau protein at the tau-1 epitope in Alzheimer's disease.