Loss of function of the ALS protein SigR1 leads to ER pathology associated with defective autophagy and lipid raft disturbances

Intracellular accumulations of altered, misfolded proteins in neuronal and other cells are pathological hallmarks shared by many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Mutations in several genes give rise to familial forms of ALS. Mutations in Sigma receptor 1 have been found to cause a juvenile form of ALS and frontotemporal lobar degeneration (FTLD). We recently described altered localization, abnormal modification and loss of function of SigR1 in sporadic ALS. In order to further elucidate the molecular mechanisms underlying SigR1-mediated alterations in sporadic and familial ALS, we extended our previous studies using neuronal SigR1 knockdown cell lines. We found that loss of SigR1 leads to abnormal ER morphology, mitochondrial abnormalities and impaired autophagic degradation. Consistent with these results, we found that endosomal trafficking of EGFR is impaired upon SigR1 knockdown. Furthermore, in SigR1-deficient cells the transport of vesicular stomatitis virus glycoprotein is inhibited, leading to the accumulation of this cargo protein in the Golgi apparatus. Moreover, depletion of SigR1 destabilized lipid rafts and associated calcium mobilization, confirming the crucial role of SigR1 in lipid raft and intracellular calcium homeostasis. Taken together, our results support the notion that loss of SigR1 function contributes to ALS pathology by causing abnormal ER morphology, lipid raft destabilization and defective endolysosomal pathways.ER stress causes alterations in protein quality control, autophagy, calcium imbalance and mitochondrial dysfunction.^{1, 2, 3} It is central to the pathogenesis of many neurodegenerative diseases.^{4, 5} Altered proteins are targeted by molecular chaperones for protein repair or refolding. However, failure of this first line of defense leads to abnormal aggregates of such proteins that then form ubiquitinated cellular inclusions and compromise UPS function.^{6, 7, 8, 9} Macroautophagy, the major lysosomal degradative pathway in cells, is responsible for degrading long-lived cytoplasmic constituents; it is the principal mechanism for turning over cellular organelles and protein aggregates too large to be degraded by the proteasome.^{10, 11, 12, 13} Macroautophagy, hereafter referred to as autophagy, is a multistep process, initiated primarily by sequestration of portions of the cytoplasm in double-membrane-bound vesicles to form an autophagosome. Autophagosomes along with their cargoes are then degraded upon fusing with late endosome- or lysosome-containing cathepsins, other acid hydrolases, and vacuolar [H+] ATPase.¹⁴ Even though autophagy is a selective and efficient mechanism for the degradation of misfolded and mutant proteins related to neurodegeneration, recent evidence indicates that the alterations in certain disease-related genes may actually impair autophagic activity at different levels, including accumulation of autophagic vacuoles,¹⁵ substrate recognition, lysosomal acidity and autophagosome membrane nucleation.^{16, 17}SigR1 interacts with a variety of ligands and is involved in a broad array of biological functions that have only been partially defined so far. They include regulation of neuronal survival, neuritogenesis, ion channel activity, IP3R-mediated Ca²⁺ signaling, memory and drug addiction. SigR1 is an ER chaperone that is located at the mitochondria–ER interface and is normally bound to another chaperone, BiP/GRP78.¹⁸ Upon IP₃ receptor stimulation or Ca²⁺ depletion within the ER, SigR1 dissociates from BiP and stabilizes IP₃ receptors, leading to prolonged Ca²⁺ signaling into mitochondria.¹⁸ Recently, a mutation in SigR1, E1O2Q, has been reported to cause a juvenile form of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD).¹⁹ SigR1 protein is decreased significantly in human sporadic ALS spinal cord.²⁰ In addition, functional relevance of SigR1 in ALS pathogenesis is demonstrated by SigR1 knockout mice that display motor deficits with a shorter latency period in rotarod experiments, and by another study showing that lack of SigR1 exacerbates ALS progression in SOD1 tg mice.^{21, 22} Furthermore, a recent study showed improvement in motor function and survival of motor neurons in SOD1 mice treated with a SigR1 agonist.²³ Recently, we reported altered localization and abnormal modification of SigR1 in sporadic ALS and showed that loss of SigR1 function leads to deformities in ER structure, formation of ER-derived autophagic vacuoles and induction of ER stress.²⁰ Loss of function of SigR1 also leads to aberrant calcium homeostasis and cell death.²⁰ Together, these data suggest a crucial role of SigR1 in neuronal function and survival.Autophagy has been known to be tightly linked to ER function and is decisive in neurodegeneration mediated by ER stress.^{24, 25, 26} Therefore, we hypothesized that loss of SigR1 function contributes to a vicious circle including ER stress, defective autophagy and altered calcium signaling that causes multifactorial ALS pathology. In the present study, we show that depletion of SigR1 leads to the accumulation of numerous autophagic vacuoles often filled with nondegraded autophagic substrates, and severe deformities in ER ultrastructure including loss of ER tethering. Biochemical analysis in SigR1-deficient cells revealed the accumulation of various autophagic substrates and defects in endosomal trafficking, suggesting an impairment of endolysosomal pathways. Finally, loss of SigR1 destabilized lipid rafts causing both impaired calcium mobilization and altered endosomal trafficking. Altogether, our results support the notion that loss of SigR1 contributes to ALS pathogenesis by causing abnormal ER morphology, lipid raft destabilization and defective autophagy.     

Expression of Bcl-2 and Bax proteins in relation to quality of bovine oocytes and embryos produced in vitro

The mechanisms underlying the visual assessment and selection of immature oocytes resulting in optimum embryonic development following in vitro maturation, fertilization and culture (in vitro maturation (IVM)/in vitro fertilization (IVF)/in vitro embryo culture (IVC)) are unknown. Also, the reasons for the more frequent occurrence of cytoplasmic fragmentation in in vitro produced bovine embryos, resulting in poor survival following cryopreservation and decreased pregnancy rates following embryo transfer are not clear. The objectives of this study are: (1) to investigate whether differences in the quality of immature oocytes and embryo fragmentation are associated with apoptosis; and (2) to study the pattern of Bcl-2 and Bax expression in oocytes and embryos to help elucidate their potential roles in the regulation of apoptosis during development. Bovine oocytes were obtained from slaughterhouse ovaries and divided into four grades (grades I–IV) based on their morphology. Oocytes of different grades were cultured in serum-free medium for 48 h. Embryos were produced only from grade I oocytes (highest quality) via IVM, IVF and IVC procedures. The morphological analysis of apoptosis in oocytes and embryos was carried out using propidium iodide staining and terminal deoxynucleotidyl transferase mediated dUTP nick end labeling. The expression of Bcl-2 and Bax in oocytes and embryos of different qualities and stages was determined using western blotting. The results showed that the number of morphologically abnormal oocytes with shrinkage and/or fragmentation of the ooplasm, which are typical features of apoptosis, was significantly higher in grade IV oocytes (denuded oocytes, the lowest quality) than in grade I oocytes after 48 h in vitro culture (P<0.05). DNA fragmentation, a hallmark of the biochemical changes seen in apoptotic cell death, was observed in morphologically fragmented oocytes and embryos. The expression of Bcl-2 was high in good quality oocytes and embryos, low in fragmented embryos, and hardly detectable in denuded oocytes. In contrast, the expression of Bax was found in all types of oocytes and embryos with the highest expression in the denuded oocytes. This implies that the ratio of Bcl-2 to Bax may be used to gauge the tendency of oocytes and embryos towards either survival or apoptosis. Overall, our results show that apoptosis appears to be an underlying mechanism of bovine oocyte degeneration and embryo fragmentation. Interactions between the Bcl-2 family of proteins may play a critical role in pre-implantation embryo development. These findings could have important implications for improving IVF and related techniques.     

7-Dehydrocholesterol enhances ultraviolet A-induced oxidative stress in keratinocytes: roles of NADPH oxidase, mitochondria, and lipid rafts

Long wavelength solar UVA radiation stimulates formation of reactive oxygen species (ROS) and prostaglandin E(2) (PGE(2)), which are involved in skin photosensitivity and tumor promotion. High levels of 7-dehydrocholesterol (7-DHC), the precursor to cholesterol, cause exaggerated photosensitivity to UVA in patients with Smith-Lemli-Opitz syndrome (SLOS). Partially replacing cholesterol with 7-DHC in keratinocytes rapidly (<5 min) increased UVA-induced ROS, intracellular calcium, phospholipase A(2) activity, PGE(2), and NADPH oxidase activity. UVA-induced ROS and PGE(2) production were inhibited in these cells by depleting the Nox1 subunit of NADPH oxidase using siRNA or using a mitochondrial radical quencher, MitoQ. Partial replacement of cholesterol with 7-DHC also disrupted membrane lipid raft domains, although depletion of cholesterol, which also disrupts lipid rafts, did not affect UVA-induced increases in ROS and PGE(2). Phospholipid liposomes containing 7-DHC were more rapidly oxidized by a free radical mechanism than those containing cholesterol. These results indicate that 7-DHC enhances rapid UVA-induced ROS and PGE(2) formation by enhancing free radical-mediated membrane lipid oxidation and suggests that this mechanism might underlie the UVA photosensitivity in SLOS.     

1-D coordination chains of Cu(hfac)2 and Mn(hfac)2 bridged by phenyl-nitronylnitroxide derivatives

Two alternating 1-D metal-radical linear [L:Cu(hfac)₂]_n and zig-zag [L:Mn(hfac)₂]_n chains (where L = 4-trimethylsilylethynyl-1-(4,4,5,5-tetramethyl-3-oxylimidazoline-1-oxide)benzene) and hfac = hexafluoroacetylacetonate) are described and characterized by X-ray diffraction of their crystals. Bulk magnetic measurements of L:Cu(hfac)₂ indicated a ferromagnetic interaction with J = 6 cm⁻¹ and L:Mn(hfac)₂ yielded ferrimagnetic interactions with J = −95 cm⁻¹. For the latter, a strong increase of their magnetic moment at lowest temperatures was observed only at very low static magnetic field, while for H_dc > 0.05 T saturation effect led to a downward slope after reaching a maximum.     

Microbial diversity in coastal sediments during pre- and post-tsunami periods in the south east coast of India

Sediment samples were collected from 12 beaches affected by the 2004 Asian Tsunami in the south-east coast of India between Vanagiri and Nagoor. The objective of the present study is to delineate the microbial diversity in pre- and post-tsunami disaster coastal sediments. The collected marine sediments indicate that the overall microbial diversity is higher in the pre-tsunami sediments. The increase in pathogenic bacteria and fungal species after the tsunami is obscured due to inundation and backwashing of seawater along the coast. The reduction of other microbial diversity after the tsunami is attributed that the coastal and shelf sediments play an important role in the demineralization of organic matter, which supports the growth of microbes. The continuous exchange of ocean water and backwashing of coastal sediments by the tsunami wave probably reduced the pathogenic bacterial diversity in the sediments.     

Fungi hijack a ubiquitous plant apoplastic endoglucanase to release a ROS scavenging β-glucan decasaccharide to subvert immune responses

Plant pathogenic and beneficial fungi have evolved several strategies to evade immunity and cope with host-derived hydrolytic enzymes and oxidative stress in the apoplast, the extracellular space of plant tissues. Fungal hyphae are surrounded by an inner insoluble cell wall layer and an outer soluble extracellular polysaccharide (EPS) matrix. Here, we show by proteomics and glycomics that these two layers have distinct protein and carbohydrate signatures, and hence likely have different biological functions. The barley (Hordeum vulgare) β-1,3-endoglucanase HvBGLUII, which belongs to the widely distributed apoplastic glycoside hydrolase 17 family (GH17), releases a conserved β-1,3;1,6-glucan decasaccharide (β-GD) from the EPS matrices of fungi with different lifestyles and taxonomic positions. This low molecular weight β-GD does not activate plant immunity, is resilient to further enzymatic hydrolysis by β-1,3-endoglucanases due to the presence of three β-1,6-linked glucose branches and can scavenge reactive oxygen species. Exogenous application of β-GD leads to enhanced fungal colonization in barley, confirming its role in the fungal counter-defensive strategy to subvert host immunity. Our data highlight the hitherto undescribed capacity of this often-overlooked EPS matrix from plant-associated fungi to act as an outer protective barrier important for fungal accommodation within the hostile environment at the apoplastic plant–microbe interface.

A β-1,3;1,6-glucan decasaccharide released from the fungal matrix by an apoplastic host hydrolase contributes to plant immune suppression and fungal accommodation.

IN A NUTSHELL Background: Plants secrete various hydrolytic enzymes into the apoplastic space to protect themselves against invading microbes. Some of these enzymes target the fungal cell wall polymer chitin. This enzymatic attack leads to the release of chitin oligomers, which can be perceived by the plant immune system, informing the plant to activate its defense machinery. However, chitin accounts for only a small part of most fungal cell walls. Recent studies have highlighted a largely uncharacterized, β-glucan-rich extracellular polysaccharide matrix (EPS) surrounding the cell wall of various plant-colonizing fungi. Question: This EPS matrix is made of glucose and abundantly produced during colonization. As its secretion into the extracellular environment is costly for the fungus, we explored how this EPS matrix affects plant immunity and fungal colonization. Findings: We demonstrated that EPS matrices from a symbiotic and pathogenic plant-colonizing fungus are distinct from the nonsoluble fungal cell walls with respect to their protein and carbohydrate composition. Enzymatic digests revealed that a secreted plant hydrolase from barley (HvBGLUII) acts on these EPS matrices and releases a highly branched β-glucan decasaccharide (β-GD) fragment. This fragment is not perceived by the plant immune system but instead detoxifies reactive oxygen species produced by the plant host as a defense mechanism and contributes to host colonization. We thus have shown that the outermost fungal EPS layer represents a protective shield against oxidative stress. Next steps: The diversity of linkage types and branching patterns of β-glucans not only accounts for their different biochemical properties, but also makes them important messengers for the plant, potentially encoding specific information on the approaching fungal invader. Future studies should aim to identify other plant hydrolases and the elusive glucan receptors, to disentangle the contribution of β-glucans to the communication between plant hosts and fungi.     

Plant species diversity and tree population structure of a humid tropical forest in Tamil Nadu, India

Vegetation structure and species composition of tropical ecosystems were studied through nine transects at Veerapuli and Kalamalai reserve forests in the Western Ghats of Tamil Nadu, India. Species diversity, dominance, species richness and evenness indices of plant communities and also population structure of woody plants were enumerated. A total of 244 species (183 genera and 76 families) were recorded. Species richness (number of species) were 82,142 and 96 species per 0.3 ha respectively for the study areas of low-elevation forest (LEF), mid-elevation forest (MEF) and high elevation forest (HEF). Species diversity indices were greater in MEF compared to the other two forests except juveniles. In contrast, greater dominance value indices were recorded in LEF than other forests. Density and basal area of the MEF were twice greater than the LEF, while HEF showed greater tree density and low basal area when compared to LEF. The stem density and species richness (number of species) decreased with increased size classes of trees observed in the present study indicated good regeneration status. Population structure of juveniles and seedlings also reflects good regeneration status. Terminalia paniculata (IVI of 99.9) and Hopea parviflora (IVI of 103.8) were dominant tree species respectively in LEF and MEF whereas in HEF Agrostistachys meeboldii (63.65), Cullenia excelsa (63.67) and Drypetes oblongifolia (39.67) share the dominance. Past damage (anthropogenic perturbation) may be one of the reasons for single species dominance in LEF and MEF. Occurrence of alien species such as Eupatorium odoratum and Ageratum conyzoides also indicated the past disturbance in LEF. The variations in plant diversity and population structure are largely due to anthropogenic perturbation and other abiotic factors.