Beneficial effects of lysosome-modulating and other pharmacological and nanocarrier agents on amyloid-beta-treated cells.

The progression of Alzheimer's disease (AD) is accompanied by disturbances of the endosome/lysosome (EL) system and there is accumulation of peptides of the AD-associated amyloid beta (Abeta) type in EL vesicles of affected neurons. EL modulating agents partially ameliorate the Abeta-mediated cell abnormalities. However, no extensive studies on the potential pharmaceutical applications of combinations of such agents and their synergistic effects have been performed. This study shows the beneficial anti-amyloid effects of several combinations of lysosomal modulators and other pharmacological and new nanobiotechnological agents. Some agents potentiated each other's action and some of them facilitated the anti-amyloid actions of memantine, a modifier of Ca2+-permeable channels involved in AD and one of the few drugs used for treatment of AD. Another compound used in nanobiotechnology ameliorated as a nanocarrier the beneficial effects of some of these potential pharmaceutical agents. They may be considered as additional drugs to improve the efficacy of the therapeutic approaches for AD and related neurodegenerative disorders.

The cation channel mucolipin-1 is a bifunctional protein that facilitates membrane remodeling via its serine lipase domain.

Phospholipase modulators have been shown to affect the topology of lipid bilayers and the formation of tubulo-vesicular structures, but the specific endogenous phospholipases involved have yet to be identified. Here we show that TRPML1 (MLN1), a Ca(2+)-permeable channel, contributes to membrane remodeling through a serine lipase consensus domain, and thus represents a novel type of bifunctional protein. Remarkably, this serine lipase active site determines the ability of MLN1 to generate tubulo-vesicular extensions in mucolipin-1-expressing oocytes, human fibroblasts and model membrane vesicles. Our demonstration that MLN1 is involved in membrane remodeling and the formation of extensions suggests that it may play a role in the formation of cellular processes linked to the late endosome/lysosome (LE/L) pathway. MLN1 is absent or mutated in patients with mucolipidosis IV (MLIV), a lysosomal disorder with devastating neurological and other consequences. This study provides potential insight into the pathophysiology of MLIV.

Calcium-sensing receptor stimulates secretion of an interferon-gamma-induced monokine (CXCL10) and monocyte chemoattractant protein-3 in immortalized GnRH neurons.

Biology of GnRH neurons is critically dependent on extracellular Ca(2+) (Ca(2+) (o)). We evaluated differences in gene expression patterns with low and high Ca(2+) (o) in an immortalized GnRH neuron line, GT1-7 cells. Mouse global oligonucleotide microarray was used to evaluate transcriptional differences among the genes regulated by elevated Ca(2+) (o). Our result identified two interferon-gamma (IFNgamma)-inducible chemokines, CXCL9 and CXCL10, and a beta chemokine, monocyte chemoattractant protein-3 (MCP-3/CCL7), being up-regulated in GT1-7 cells treated with high Ca(2+) (o) (3.0 mM) compared with low Ca(2+) (o) (0.5 mM). Up-regulation of these mRNAs by elevated Ca(2+) (o) was confirmed by quantitative PCR. Elevated Ca(2+) (o) stimulated secretion of CXCL10 and MCP-3 but not CXCL9 in GT1-7 cells, and this effect was mediated by an extracellular calcium-sensing receptor (CaR) as the dominant negative CaR attenuated secretion of CXCL10 and MCP-3. CXCL10 and MCP-3 were localized in mouse GnRH neurons in the preoptic hypothalamus. Suppression of K(+) channels (BK channels) with 25 nM charybdotoxin inhibited high-Ca(2+) (o)-stimulated CXCL10 release. Accordingly, CaR activation by a specific CaR agonist, NPS-467, resulted in the activation of a Ca(2+)-activated K(+) channel in these cells. CaR-mediated MCP-3 secretion involves the PI3 kinase pathway in GT1-7 cells. MCP-3 stimulated chemotaxis of astrocytes treated with transforming growth factor-beta (TGFbeta). With TGFbeta-treated astrocytes, we next observed that conditioned medium from GT1-7 cells treated with high Ca(2+) promoted chemotaxis of astrocytes, and this effect was attenuated by a neutralizing antibody to MCP-3. These results implicate CaR as an important regulator of GnRH neuron function in vivo by stimulating secretion of heretofore unsuspected cytokines, i.e., CXCL10 and MCP-3.

Calcium receptor stimulates chemotaxis and secretion of MCP-1 in GnRH neurons in vitro: potential impact on reduced GnRH neuron population in CaR-null mice.

The factors controlling the migration of mammalian gonadotropin-releasing hormone (GnRH) neurons from the nasal placode to the hypothalamus are not well understood. We studied whether the extracellular calcium-sensing receptor (CaR) promotes migration/chemotaxis of GnRH neurons. We demonstrated expression of CaR in GnRH neurons in the murine basal forebrain and in two GnRH neuronal cell lines: GT1-7 (hypothalamus derived) and GN11 (olfactory bulb derived). Elevated extracellular Ca(2+) concentrations promoted chemotaxis of both cell types, with a greater effect in GN11 cells. This effect was CaR mediated, as, in both cell types, overexpression of a dominant-negative CaR attenuated high Ca(2+)-stimulated chemotaxis. We also demonstrated expression of a beta-chemokine, monocyte chemoattractant protein-1 (MCP-1), and its receptor, CC motif receptor-2 (CCR2), in the hypothalamic GnRH neurons as well as in GT1-7 and GN11 cells. Exogenous MCP-1 stimulated chemotaxis of both cell lines in a dose-dependent fashion; the effect was greater in GN11 than in GT1-7 cells, consistent with the higher CCR2 mRNA levels in GN11 cells. Activating the CaR stimulated MCP-1 secretion in GT1-7 but not in GN11 cells. MCP-1 secreted in response to CaR stimulation is biologically active, as conditioned medium from GT1-7 cells treated with high Ca(2+) promoted chemotaxis of GN11 cells, and this effect was partially attenuated by a neutralizing antibody to MCP-1. Finally, in the preoptic area of anterior hypothalamus, the number of GnRH neurons was approximately 27% lower in CaR-null mice than in mice expressing the CaR gene. We conclude that the CaR may be a novel regulator of GnRH neuronal migration likely involving, in part, MCP-1.

Lysosomal exocytosis is impaired in mucolipidosis type IV.

Mucolipidosis type IV (MLIV) is an autosomal recessive disease characterized by severe neurological impairment, ophthalmologic defects, and gastric dysfunction. MLIV cells have a deficiency in the late endosomal/lysosomal (LEL) pathway that results in the buildup of lysosomal inclusions. Using a Xenopus oocyte expression system, we previously showed that mucolipin-1 (MLN1), the protein encoded by the MCOLN1 gene is a Ca2+ -permeable non-selective cation channel that is transiently modulated by elevations in intracellular Ca2+. We further showed that MLN1 is translocated to the plasma membrane during lysosomal exocytosis. In this study we show that lysosomal exocytosis is impaired in fibroblasts from MLIV patients, indicating that MLN1 plays an active role in this process. Further, we show that transfection with wild type MLN1 cDNA rescues exocytosis, suggesting the possibility of treatments based on the restoration of this crucial cellular function.

Functional links between mucolipin-1 and Ca2+-dependent membrane trafficking in mucolipidosis IV.

Most of the membrane trafficking phenomena including those involving the interactions between endosomes and lysosomes are regulated by changes in intracellular Ca2+ (Cai). These processes are disturbed in some types of mucolipidoses and other lysosomal storage disorders, such as mucolipidosis IV (MLIV), a neurological disorder that usually presents during the first year of life with blindness, cognitive impairment, and psychomotor delays. It is caused by mutations in MCOLN1, the gene encoding mucolipin-1 (MLN1), which we have recently established to represent a Ca2+-permeable cation channel that is transiently modulated by changes in Cai. The cells of MLIV patients contain enlarged lysosomes that are likely associated with abnormal sorting and trafficking of these and related organelles. We studied fibroblasts from MLIV patients and found disturbed Ca2+ signaling and large acidic organelles such as late endosomes and lysosomes (LEL) with altered cellular localization in these cells. The fusion between LEL vesicles in these cells was defective. This is a Ca2+-dependent process related to signaling pathways involved in regulation of Ca2+ homeostasis and trafficking. The MLN1 channels could play a key role in Ca2+ release from LEL vesicles, which triggers the fusion and trafficking of these organelles. The characterization of this MLN1-mediated Ca2+-dependent process should provide new insights into the pathophysiological mechanisms that lead to the development of MLIV and other mucolipidoses associated with similar disturbances in membrane trafficking.

Native polycystin 2 functions as a plasma membrane Ca2+-permeable cation channel in renal epithelia.

Mutations in polycystin 2 (PC2), a Ca(2+)-permeable cation channel, cause autosomal dominant polycystic kidney disease. Whether PC2 functions in the endoplasmic reticulum (ER) or in the plasma membrane has been controversial. Here we generated and characterized a polyclonal antibody against PC2, determined the subcellular localization of both endogenous and transfected PC2 by immunohistochemistry and biotinylation of cell surface proteins, and assessed PC2 channel properties with electrophysiology. Endogenous PC2 was found in the plasma membrane and the primary cilium of mouse inner medullar collecting duct (IMCD) cells and Madin-Darby canine kidney (MDCK) cells, whereas heterologously expressed PC2 showed a predominant ER localization. Patch-clamping of IMCD cells expressing endogenous or heterologous PC2 confirmed the presence of the channel on the plasma membrane. Treatment with chaperone-like factors facilitated the translocation of the PC2 channel to the plasma membrane from intracellular pools. The unitary conductances, channel kinetics, and other characteristics of both endogenously and heterologously expressed PC2 were similar to those described in our previous study in Xenopus laevis oocytes. These results show that PC2 functions as a plasma membrane channel in renal epithelia and suggest that PC2 contributes to Ca(2+) entry and transport of other cations in defined nephron segments in vivo.

Identification and characterization of the single channel function of human mucolipin-1 implicated in mucolipidosis type IV, a disorder affecting the lysosomal pathway.

Mucolipin-1 (MLN1) is a membrane protein with homology to the transient receptor potential channels and other non-selective cation channels. It is encoded by the MCOLN1 gene, which is mutated in patients with mucolipidosis type IV (MLIV), an autosomal recessive disease that is characterized by severe abnormalities in neurological development as well as by ophthalmologic defects. At the cellular level, MLIV is associated with abnormal lysosomal sorting and trafficking. Here we identify the channel function of human MLN1 and characterize its properties. MLN1 represents a novel Ca(2+)-permeable channel that is transiently modulated by changes in [Ca(2+)]. It is also permeable to Na(+) and K(+). Large unitary conductances were measured in the presence of these cations. With its Ca(2+) permeability and modulation by [Ca(2+)], MLN1 could play a major role in Ca(2+) transport regulating lysosomal exocytosis and potentially other phenomena related to the trafficking of late endosomes and lysosomes.