Induction of COX-2-PGE2 synthesis by activation of the MAPK/ERK pathway contributes to neuronal death triggered by TDP-43-depleted microglia

Neuroinflammation is a striking hallmark of amyotrophic lateral sclerosis (ALS) and other neurodegenerative disorders. Previous studies have shown the contribution of glial cells such as astrocytes in TDP-43-linked ALS. However, the role of microglia in TDP-43-mediated motor neuron degeneration remains poorly understood. In this study, we show that depletion of TDP-43 in microglia, but not in astrocytes, strikingly upregulates cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production through the activation of MAPK/ERK signaling and initiates neurotoxicity. Moreover, we find that administration of celecoxib, a specific COX-2 inhibitor, greatly diminishes the neurotoxicity triggered by TDP-43-depleted microglia. Taken together, our results reveal a previously unrecognized non-cell-autonomous mechanism in TDP-43-mediated neurodegeneration, identifying COX-2-PGE2 as the molecular events of microglia- but not astrocyte-initiated neurotoxicity and identifying celecoxib as a novel potential therapy for TDP-43-linked ALS and possibly other types of ALS.Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by the degeneration of motor neurons in the brain and spinal cord.¹ Most cases of ALS are sporadic, but 10% are familial. Familial ALS cases are associated with mutations in genes such as Cu/Zn superoxide dismutase 1 (SOD1), TAR DNA-binding protein 43 (TARDBP) and, most recently discovered, C9orf72. Currently, most available information obtained from ALS research is based on the study of SOD1, but new studies focusing on TARDBP and C9orf72 have come to the forefront of ALS research.^{1, 2} The discovery of the central role of the protein TDP-43, encoded by TARDBP, in ALS was a breakthrough in ALS research.^{3, 4, 5} Although pathogenic mutations of TDP-43 are genetically rare, abnormal TDP-43 function is thought to be associated with the majority of ALS cases.¹ TDP-43 was identified as a key component of the ubiquitin-positive inclusions in most ALS patients and also in other neurodegenerative diseases such as frontotemporal lobar degeneration,^{6, 7} Alzheimer''s disease (AD)^{8, 9} and Parkinson''s disease (PD).^{10, 11} TDP-43 is a multifunctional RNA binding protein, and loss-of-function of TDP-43 has been increasingly recognized as a key contributor in TDP-43-mediated pathogenesis.^{5, 12, 13, 14}Neuroinflammation, a striking and common hallmark involved in many neurodegenerative diseases, including ALS, is characterized by extensive activation of glial cells including microglia, astrocytes and oligodendrocytes.^{15, 16} Although numerous studies have focused on the intrinsic properties of motor neurons in ALS, a large amount of evidence showed that glial cells, such as astrocytes and microglia, could have critical roles in SOD1-mediated motor neuron degeneration and ALS progression,^{17, 18, 19, 20, 21, 22} indicating the importance of non-cell-autonomous toxicity in SOD1-mediated ALS pathogenesis.Very interestingly, a vital insight of neuroinflammation research in ALS was generated by the evidence that both the mRNA and protein levels of the pro-inflammatory enzyme cyclooxygenase-2 (COX-2) are upregulated in both transgenic mouse models and in human postmortem brain and spinal cord.^{23, 24, 25, 26, 27, 28, 29} The role of COX-2 neurotoxicity in ALS and other neurodegenerative disorders has been well explored.^{30, 31, 32} One of the key downstream products of COX-2, prostaglandin E2 (PGE2), can directly mediate COX-2 neurotoxicity both in vitro and in vivo.^{33, 34, 35, 36, 37} The levels of COX-2 expression and PGE2 production are controlled by multiple cell signaling pathways, including the mitogen-activated protein kinase (MAPK)/ERK pathway,^{38, 39, 40} and they have been found to be increased in neurodegenerative diseases including AD, PD and ALS.^{25, 28, 32, 41, 42, 43, 44, 45, 46} Importantly, COX-2 inhibitors such as celecoxib exhibited significant neuroprotective effects and prolonged survival or delayed disease onset in a SOD1-ALS transgenic mouse model through the downregulation of PGE2 release.²⁸Most recent studies have tried to elucidate the role of glial cells in neurotoxicity using TDP-43-ALS models, which are considered to be helpful for better understanding the disease mechanisms.^{47, 48, 49, 50, 51} Although the contribution of glial cells to TDP-43-mediated motor neuron degeneration is now well supported, this model does not fully suggest an astrocyte-based non-cell autonomous mechanism. For example, recent studies have shown that TDP-43-mutant astrocytes do not affect the survival of motor neurons,^{50, 51} indicating a previously unrecognized non-cell autonomous TDP-43 proteinopathy that associates with cell types other than astrocytes.Given that the role of glial cell types other than astrocytes in TDP-43-mediated neuroinflammation is still not fully understood, we aim to compare the contribution of microglia and astrocytes to neurotoxicity in a TDP-43 loss-of-function model. Here, we show that TDP-43 has a dominant role in promoting COX-2-PGE2 production through the MAPK/ERK pathway in primary cultured microglia, but not in primary cultured astrocytes. Our study suggests that overproduction of PGE2 in microglia is a novel molecular mechanism underlying neurotoxicity in TDP-43-linked ALS. Moreover, our data identify celecoxib as a new potential effective treatment of TDP-43-linked ALS and possibly other types of ALS.     

Secretory production of an FAD cofactor-containing cytosolic enzyme (sorbitol–xylitol oxidase from Streptomyces coelicolor) using the twin-arginine translocation (Tat) pathway of Corynebacterium glutamicum

Carbohydrate oxidases are biotechnologically interesting enzymes that require a tightly or covalently bound cofactor for activity. Using the industrial workhorse Corynebacterium glutamicum as the expression host, successful secretion of a normally cytosolic FAD cofactor-containing sorbitol–xylitol oxidase from Streptomyces coelicolor was achieved by using the twin-arginine translocation (Tat) protein export machinery for protein translocation across the cytoplasmic membrane. Our results demonstrate for the first time that, also for cofactor-containing proteins, a secretory production strategy is a feasible and promising alternative to conventional intracellular expression strategies.The secretory expression of recombinant proteins can offer significant process advantages over cytosolic production strategies, since secretion into the growth medium greatly facilitates downstream processing and therefore can significantly reduce the costs of producing a desired target protein (Quax, 1997). And, in fact, the enormous secretion capacity of certain Gram-positive bacteria (e.g. various Bacillus species) has been used since many years in industry for the production of mainly host-derived secretory proteins such as proteases and amylases, resulting in amounts of more than 20 g l⁻¹ culture medium (Harwood and Cranenburg, 2008). In contrast, attempts to use Bacillus species for the secretory production of heterologous proteins have often failed or led to disappointing results, a fact that, among other reasons, could in many cases be attributed to the presence of multiple cell wall-associated and secreted proteases that rapidly degraded the heterologous target proteins (Li et al., 2004; Sarvas et al., 2004; Westers et al., 2011). Therefore, an increasing need exists to explore alternative host systems with respect to their ability to express and secrete problematic and/or complex heterologous proteins of biotechnological interest.So far, the Gram-positive bacterium Corynebacterium glutamicum has been used in industry mainly for the production of amino acids and other low-molecular weight compounds (Leuchtenberger et al., 2005; Becker and Wittmann, 2011; Litsanov et al., 2012). However, various recent reports have indicated that C. glutamicum might likewise possess a great potential as an alternative host system for the secretory expression of foreign proteins. Corynebacterium glutamicum belongs to a class of diderm Gram-positive bacteria that, besides the cytoplasmic membrane, possess an additional mycolic acid-containing outer membrane-like structure that acts as an extremely efficient permeability barrier for hydrophilic compounds (Hoffmann et al., 2008; Zuber et al., 2008). Despite this fact, an efficient secretion of various heterologous proteins into the growth medium of this microorganism has been observed (e.g. Billman-Jacobe et al., 1995; Meissner et al., 2007; Kikuchi et al., 2009; Tateno et al., 2009; Tsuchidate et al., 2011).In bacteria, two major export pathways exist for the transport of proteins across the cytoplasmic membrane that fundamentally differ with respect to the folding status of their respective substrate proteins during the actual translocation step. The general secretion (Sec) system transports its substrates in a more or less unfolded state and folding takes places on the trans side of the membrane after the actual transport event (Yuan et al., 2010; du Plessis et al., 2011). In contrast, the alternative twin-arginine translocation (Tat) system translocates its substrates in a fully folded form and therefore provides an attractive alternative for the secretory production of proteins that cannot be produced in a functional form via the Sec route (Brüser, 2007). Carbohydrate oxidases are biotechnologically interesting enzymes (van Hellemond et al., 2006) that are excluded from Sec-dependent secretion since they depend on a tightly or covalently bound cofactor for their activity and, for this reason, require that their folding and cofactor insertion has to take place in the cytosol. Because C. glutamicum has shown to be an excellent host for the Tat-dependent secretion of the cofactor-less model protein GFP (Meissner et al., 2007; Teramoto et al., 2011), we now asked whether it is likewise possible to secrete a cofactor-containing enzyme into the supernatant of C. glutamicum using the same protein export route.As a model protein, we chose the sorbitol–xylitol oxidase (SoXy) from Streptomyces coelicolor, a normally cytosolic enzyme that possesses a covalently bound FAD molecule as cofactor (Heuts et al., 2007; Forneris et al., 2008). FAD is incorporated into the apoprotein in a post-translational and self-catalytic process that only occurs if the polypeptide chain has adopted a correctly folded structure (Heuts et al., 2007; 2009). To direct SoXy into the Tat export pathway of C. glutamicum, we constructed a gene encoding a TorA–SoXy hybrid precursor in which SoXy is fused to the strictly Tat-specific signal peptide of the periplasmic Escherichia coli Tat substrate trimethylamine N-oxide reductase (TorA) (Fig. 1) which, in our previous study, has been proven to be a functional and strictly Tat-specific signal peptide also in C. glutamicum (Meissner et al., 2007). The corresponding torA–soxy gene was cloned into the expression vector pEKEx2 (Eikmanns et al., 1991) under the control of an IPTG-inducible P_tac promotor. After transformation of the resulting plasmid pTorA–SoXy into the C. glutamicum ATCC13032 wild-type strain, two independent colonies of the resulting recombinant C. glutamicum (pTorA–SoXy) strain and, as a control, a colony of a strain that contained the empty expression vector without insert [C. glutamicum (pEKEx2)] were grown in CGXII medium (Keilhauer et al., 1993) at 30°C for 16 h in the presence of 1 mM IPTG. Subsequently, the proteins present in the culture supernatants were analysed by SDS-PAGE followed by staining with Coomassie blue. As shown in Fig. 2, in the supernatants of the pTorA–SoXy-containing cells (lanes 3 and 4), a prominent protein band of approximately 44 kDa can be detected, the size of which is very similar to the calculated molecular mass (44.4 kDa) of SoXy. Since this band is completely lacking in the supernatant of the control strain (lane 2), this strongly suggests that this band corresponds to SoXy that has been secreted into the culture supernatant of C. glutamicum (pTorA–SoXy). And, in fact, this suggestion was subsequently confirmed in a direct way by MALDI-TOF mass spectrometry after extraction of the protein out of the gel followed by tryptic digestion (Schaffer et al., 2001) (data not shown).Open in a separate windowFigure 1The TorA–SoXy hybrid precursor protein. Upper part: Schematic drawing of the relevant part of the pTorA–SoXy expression vector. P_tac, IPTG-inducible tac promotor. RBS, ribosome binding site. To maintain the authentic TorA signal peptidase cleavage site, the first four amino acids of the mature TorA protein (black bar) were retained in the TorA–SoXy fusion protein. White bar: TorA signal peptide (TorA^SP); grey bar: SoXy (amino acids 2–418). Lower part: Amino acid sequence of the signal peptide and early mature region of the TorA–SoXy hybrid precursor. The twin-arginine consensus motif of the TorA signal peptide is underlined. The four amino acids derived from mature TorA are shown in italics. The signal peptidase cleavage site is indicated by an arrowhead.Open in a separate windowFigure 2Secretion of SoXy into the growth medium of C. glutamicum. Cells of C. glutamicum ATCC13032 containing the empty vector pEKEx2 and two independently transformed colonies of C. glutamicum (pTorA–SoXy) were grown overnight in 5 ml of BHI medium (Difco) at 30°C. The cells were washed once with CGXII medium (Keilhauer et al., 1993) and inoculated to an OD₆₀₀ of 0.5 in 5 ml of fresh CGXII medium containing 1 mM IPTG. After 16 h of further growth at 30°C, the supernatant fractions were prepared as described previously (Meissner et al., 2007). Samples corresponding to an equal number of cells were subjected to SDS-PAGE followed by staining with Coomassie blue. Lane 1, molecular mass marker (kDa). Lane 2, C. glutamicum (pEKEx2); lanes 3 and 4, C. glutamicum (pTorA–SoXy). The position of the secreted SoXy protein is indicated by an arrow.Next, the supernatant of C. glutamicum (pTorA–SoXy) was analysed for SoXy enzyme activity by measuring the production of H₂O₂ that is formed during the enzymatic conversion of sorbitol to fructose (Meiattini, 1983). Six hours after induction of gene expression by 1 mM IPTG, an enzymatic activity of 10.3 ± 1.6 nmol min⁻¹ ml⁻¹ could be determined in the supernatant of C. glutamicum (pTorA–SoXy). In contrast, no such activity was found in the supernatant of the control strain C. glutamicum (pEKEx2). From these results we conclude that we have succeeded in the secretion of enzymatically active and therefore FAD cofactor-containing SoXy into the culture supernatant of C. glutamicum.Finally, we examined whether the secretion of SoXy had in fact occurred via the Tat pathway of C. glutamicum. Plasmid pTorA–SoXy was used to transform C. glutamcium ATCC13032 wild type and a C. glutamicum ΔTatAC mutant strain that lacks two essential components of the Tat transport machinery and therefore does not possess a functional Tat translocase (Meissner et al., 2007). The corresponding cells were grown in BHI medium (Difco) at 30°C in the presence of 1 mM IPTG for 6 h. Subsequently, the proteins present in the cellular and the supernatant fractions of the corresponding cells were analysed by SDS-PAGE followed by Western blotting using SoXy-specific antibodies. As shown in Fig. 3, polypeptides corresponding to the unprocessed TorA–SoXy precursor and some minor smaller degradation products of it can be detected in the cellular fractions of both the wild-type and the ΔTatAC deletion strains (lanes 3 and 5). In the supernatant fraction of the Tat⁺ wild-type strain (lane 4), but not that of the ΔTatAC strain (lane 6), a polypeptide corresponding to mature SoXy is present, clearly showing that export of SoXy in the wild-type strain had occurred in a strictly Tat-dependent manner. Another noteworthy finding is the observation that hardly any mature SoXy protein accumulated in the cellular fraction of the Tat⁺ wild-type strain (lane 3), indicating that SoXy is, after its Tat-dependent translocation across the cytoplasmic membrane and processing by signal peptidase, rapidly transported out of the intermembrane space across the mycolic acid-containing outer membrane into the supernatant. However, the mechanism of how proteins cross this additional permeability barrier is completely unknown so far (Bitter et al., 2009).Open in a separate windowFigure 3Transport of TorA–SoXy occurs in a strictly Tat-dependent manner. Plasmid pTorA–SoXy was transformed into C. glutamcium ATCC13032 (Tat⁺) and a C. glutamicum ΔTatAC mutant that lacks a functional Tat translocase (Meissner et al., 2007). As a control, the empty pEKEx2 expression vector was transformed into C. glutamicum ATCC13032 (Tat⁺). The respective strains were grown overnight in 5 ml of BHI medium (Difco) at 30°C. The cells were washed once with BHI and resuspended in 20 ml of fresh BHI medium containing 1 mM IPTG. After 6 h of further growth at 30°C, the cellular (C) and supernatant (S) fractions were prepared as described previously (Meissner et al., 2007). Samples of the C and S fractions were subjected to SDS-PAGE followed by immunoblotting using anti-SoXy antibodies as indicated at the top of the figure. Lanes 1 and 2: C. glutamicum ATCC13032 (pEKEx2); lanes 3 and 4: C. glutamicum ATCC13032 (pTorA–SoXy); lanes 5 and 6: C. glutamicum ΔTatAC (pTorA–SoXy). Asterisk: TorA–SoXy precursor; arrow: secreted SoXy protein. The positions of molecular mass markers (kDa) are indicated at the left margin of the figure.To the best of our knowledge, our results represent the first documented example of the successful secretion of a normally cytosolic, cofactor-containing protein via the Tat pathway in an active form into the culture supernatant of a recombinant expression host. Our results clearly show that, also for this biotechnologically very interesting class of proteins, a secretory production strategy can be a promising alternative to conventional intracellular expression strategies. Besides for SoXy and other FAD-containing carbohydrate oxidases, for which various applications are perceived by industry such as the in situ generation of hydrogen peroxide for bleaching and disinfection performance in technical applications, their use in the food and drink industry, as well as their use in diagnostic applications and carbohydrate biosynthesis processes (Oda and Hiraga, 1998; Murooka and Yamashita, 2001; van Hellemond et al., 2006; Heuts et al., 2007), a secretory production strategy might now be an attractive option also for biotechnologically relevant enzymes that are used as biocatalysts in chemo-enzymatic syntheses and that possess cofactors other than FAD, such as pyridoxal-5′-phosphate (PLP)-dependent ω-transaminases (Mathew and Yun, 2012) or various thiamin diphosphate (TDP)-dependent enzymes (Müller et al., 2009).     

Insulin responsiveness of glucose transporter 4 in 3T3-L1 cells depends on the presence of sortilin

Insulin-dependent translocation of glucose transporter 4 (Glut4) to the plasma membrane of fat and skeletal muscle cells plays the key role in postprandial clearance of blood glucose. Glut4 represents the major cell-specific component of the insulin-responsive vesicles (IRVs). It is not clear, however, whether the presence of Glut4 in the IRVs is essential for their ability to respond to insulin stimulation. We prepared two lines of 3T3-L1 cells with low and high expression of myc₇-Glut4 and studied its translocation to the plasma membrane upon insulin stimulation, using fluorescence-assisted cell sorting and cell surface biotinylation. In undifferentiated 3T3-L1 preadipocytes, translocation of myc₇-Glut4 was low regardless of its expression levels. Coexpression of sortilin increased targeting of myc₇-Glut4 to the IRVs, and its insulin responsiveness rose to the maximal levels observed in fully differentiated adipocytes. Sortilin ectopically expressed in undifferentiated cells was translocated to the plasma membrane regardless of the presence or absence of myc₇-Glut4. AS160/TBC1D4 is expressed at low levels in preadipocytes but is induced in differentiation and provides an additional mechanism for the intracellular retention and insulin-stimulated release of Glut4.Adipocytes, skeletal muscle cells, and some neurons respond to insulin stimulation by translocating intracellular glucose transporter 4 (Glut4) to the plasma membrane. In all these cells, the insulin-responsive pool of Glut4 is localized in small membrane vesicles, the insulin-responsive vesicles (IRVs; Kandror and Pilch, 2011 ; Bogan, 2012 ). The protein composition of these vesicles has been largely characterized (Kandror and Pilch, 2011 ; Bogan, 2012 ). The IRVs consist predominantly of Glut4, insulin-responsive aminopeptidase (IRAP), sortilin, low-density-lipoprotein receptor–related protein 1 (LRP1), SCAMPs, and VAMP2. Glut4, IRAP, and sortilin physically interact with each other, which might be important for the biogenesis of the IRVs (Shi and Kandror, 2007 ; Shi et al., 2008 ). In addition, the IRVs compartmentalize recycling receptors, such as the transferrin receptor and the IGF2/mannose 6-phosphate receptor, although it is not clear whether these receptors represent obligatory vesicular components or their presence in the IRVs is explained by mass action (Pilch, 2008 ), inefficient sorting, or other reasons.Deciphering of the protein composition of the IRVs is important because it is likely to explain their unique functional property: translocation to the plasma membrane in response to insulin stimulation. Even if we presume that IRV trafficking is controlled by loosely associated peripheral membrane proteins, the latter should still somehow recognize the core vesicular components that create the “biochemical individuality” of this compartment. In spite of our knowledge of the IRV protein composition, however, the identity of the protein(s) that confer insulin sensitivity to these vesicles is unknown.Insulin responsiveness of the IRVs was associated with either IRAP or Glut4. Thus it was shown that Glut4 interacted with the intracellular anchor TUG (Bogan et al., 2003 , 2012 ), whereas IRAP associated with other proteins implemented in the regulation of Glut4 translocation, such as AS160 (Larance et al., 2005 ; Peck et al., 2006 ), p115 (Hosaka et al., 2005 ), tankyrase (Yeh et al., 2007 ), and several others (reviewed in Bogan, 2012 ). Results of these studies, or at least their interpretations, are not necessarily consistent with each other, as the existence of multiple independent anchors for the IRVs is, although possible, unlikely.Ablation of the individual IRV proteins has also led to controversial data. Thus knockout of IRAP decreases total protein levels of Glut4 but does not affect its translocation in the mouse model (Keller et al., 2002 ). On the contrary, knockdown of IRAP in 3T3-L1 adipocytes has a strong inhibitory effect on translocation of Glut4 (Yeh et al., 2007 ). In yet another study, knockdown of IRAP in 3T3-L1 adipocytes did not affect insulin-stimulated translocation of Glut4 but increased its plasma membrane content under basal conditions (Jordens et al., 2010 ). By the same token, total or partial ablation of Glut4 had various effects on expression levels, intracellular localization, and translocation of IRAP (Jiang et al., 2001 ; Abel et al., 2004 ; Carvalho et al., 2004 ; Gross et al., 2004 ; Yeh et al., 2007 ). Knockdown of either sortilin or LRP1 decreased protein levels of Glut4 (Shi and Kandror, 2005 ; Jedrychowski et al., 2010 ).One model that might explain these complicated and somewhat inconsistent results is that depletion of either major integral protein of the IRVs disrupts the network of interactions between vesicular proteins and thus decreases the efficiency of protein sorting into the IRVs (Kandror and Pilch, 2011 ). Correspondingly, the remaining IRV components that cannot be faithfully compartmentalized in the vesicles are either degraded (Jiang et al., 2001 ; Keller et al., 2002 ; Abel et al., 2004 ; Carvalho et al., 2004 ; Shi and Kandror, 2005 ; Yeh et al., 2007 ; Jedrychowski et al., 2010 ) or mistargeted (Jiang et al., 2001 ; Jordens et al., 2010 ), depending on experimental conditions and types of cells used in these studies. In other words, knockdown of any major IRV component may decrease vesicle formation along with insulin responsiveness. Thus, in spite of a large body of literature, the identity of protein(s) that confer insulin responsiveness to the IRVs is unknown.Here we used a gain-of-function approach to address this question. Specifically, we attempted to “build” functional IRVs in undifferentiated 3T3-L1 preadipocytes by forced expression of the relevant proteins. Undifferentiated preadipocytes do not express Glut4 or sortilin and lack IRVs (ElJack et al., 1999 ; Shi and Kandror, 2005 ; Shi et al., 2008 ). Correspondingly, IRAP, which is expressed in these cells, shows low insulin response (Ross et al., 1998 ; Shi et al., 2008 ). We found that ectopic expression of increasing amounts of Glut4 in undifferentiated preadipocytes does not lead to its marked translocation to the plasma membrane upon insulin stimulation. On the contrary, sortilin expressed in undifferentiated preadipocytes was localized in the IRVs and was translocated to the plasma membrane in response to insulin stimulation. Moreover, upon coexpression with Glut4, sortilin dramatically increased its insulin responsiveness to the levels observed in fully differentiated adipocytes. Thus sortilin may represent the key component of the IRVs, which is responsible not only for the formation of vesicles (Shi and Kandror, 2005 ; Ariga et al., 2008 ; Hatakeyama and Kanzaki, 2011 ), but also for their insulin responsiveness. It is worth noting that sortilin levels are significantly decreased in obese and diabetic humans and mice (Kaddai et al., 2009 ). We thus suggest that sortilin may be a novel and important target in the fight against insulin resistance and diabetes.Our experiments also demonstrate that undifferentiated preadipocytes lack a mechanism for the full intracellular retention of Glut4 that can be achieved by ectopic expression of AS160/TBC1D4.     

Activation of volume-sensitive outwardly rectifying chloride channel by ROS contributes to ER stress and cardiac contractile dysfunction: involvement of CHOP through Wnt

Endoplasmic reticulum (ER) stress occurring in stringent conditions is critically involved in cardiomyocytes apoptosis and cardiac contractile dysfunction (CCD). However, the molecular machinery that mediates cardiac ER stress and subsequent cell death remains to be fully deciphered, which will hopefully provide novel therapeutic targets for these disorders. Here, we establish tunicamycin-induced model of cardiomyocyte ER stress, which effectively mimicks pathological stimuli to trigger CCD. Tunicamycin activates volume-sensitive outward rectifying Cl⁻ currents. Blockade of the volume-sensitive outwardly rectifying (VSOR) Cl⁻ channel by 4,4''-diisothiocya-natostilbene-2,2''-disulfonic acid (DIDS), a non-selective Cl⁻ channel blocker, and 4-(2-butyl-6,7-dichlor-2-cyclopentyl-indan-1-on-5-yl) oxybutyric acid (DCPIB), a selective VSOR Cl⁻ channel blocker, improves cardiac contractility, which correlates with suppressed ER stress through inhibiting the canonical GRP78/eIF2α/ATF4 and XBP1 pathways, and promotes survival of cardiomyocytes by inverting tunicamycin-induced decrease of Wnt through the CHOP pathway. VSOR activation of tunicamycin-treated cardiomyocytes is attributed to increased intracellular levels of reactive oxygen species (ROS). Our study demonstrates a pivotal role of ROS/VSOR in mediating ER stress and functional impairment of cardiomyocytes via the CHOP-Wnt pathway, and suggests the therapeutic values of VSOR Cl⁻ channel blockers against ER stress-associated cardiac anomalies.The endoplasmic reticulum (ER) is characterized as an organelle that participates in the folding of membrane and secretory proteins.^1,2 Efficient functioning of the endoplasmic reticulum is important for cell function and survival. Perturbations of ER homeostasis by energy deprivation and glucose,³ viral infections⁴ and accumulation of misfolded and/or unfolded proteins² interfere with ER function, leading to a state of ER stress.^{5, 6, 7} A cohort of chemicals, for example, tunicamycin and thapsigargin, also trigger ER stress.^{8, 9, 10} Thapsigargin disrupts the calcium storage of ER by blocking calcium reuptake into the ER lumen, thus by depleting calcium from the organelle.¹¹ In particular, tunicamycin is a highly specific ER stress inducer by inhibiting N-linked glycosylation of protein, representing a well-documented method to artificially elicit unfolded protein response.⁸ In response to ER stress, ER chaperones such as glucose-regulated protein 78 kDa (GRP78) and glucose-regulated protein 94 kDa (GRP94) are upregulated to facilitate the recovery of unfolded or misfolded proteins.¹² ER stress may act as a defense mechanism against external insults; however, prolonged and/or severe ER stress may ultimately trigger apoptosis.⁸ The C/EBP homologous protein (CHOP) has been defined as a pivotal mediator of cell death signaling in ER stress.^{13, 14} Accumulating evidence has demonstrated that ER stress-induced cell death is an essential step in the pathogenesis of a wide variety of cardiovascular diseases such as ischemia reperfusion heart diseases,¹⁵ atherosclerosis,^{5, 16, 17, 18} myocardial infarction,¹⁹ hypertension^{20, 21} and heart failure.^{8, 22, 23} Inhibiting ER stress has great therapeutic values for cardiac anomalies. However, the precise mechanism involved in ER stress-induced cardiovascular diseases has not been well identified, which impedes the translation of our understanding of ER stress-induced cardiovascular anomalies into effective therapeutic strategies. Apoptosis induction requires persistent cell shrinkage, named apoptotic volume decrease (AVD).^{24, 25, 26, 27} It is an early prerequisite for the activation of caspases.²⁴ In various types of cells including cardiomyocytes, AVD process is accomplished by the activation of volume-sensitive outwardly rectifying (VSOR) Cl⁻ channel and is concomitant with the egress of water from the cells undergoing mitochondrion-initiated or death receptor-induced apoptosis.^{25, 28, 29, 30} Although inhibition of VSOR Cl⁻ channel by DIDS (4,4''-diisothiocyanatostilbene-2,2''-disulphonic acid) and DCPIB (4-(2-butyl-6,7- dichlor-2-cyclopentyl-indan-1-on-5-yl) oxybutyric acid) blocked AVD and rescued cardiomyocytes from mitochondrial and death receptor pathway-induced apoptosis,^{31, 32} it remains largely unknown concerning the role of VSOR Cl⁻ channel and how it is regulated in ER stress-induced apoptotic cardiomyocyte death.Emerging evidence indicates that Wnt signal pathways are found to be anti-apoptotic in the cardiovascular diseases,^{33, 34, 35} regulating crucial aspects of cardiovascular biology. However, up to now, its activity in ER stress-induced apoptosis and in the process of AVD in cardiomyocytes remains elusive.In the present study, we probed the role of VSOR Cl⁻ channel in ER stress-induced apoptosis of cardiomyocytes, which intimately correlates with cardiac contractile dysfunction (CCD). We hypothesized that VSOR Cl⁻ channel controls the process of AVD occurring concomitantly with ER stress-induced apoptosis of cardiomyocytes. To test this hypothesis, we investigated VSOR Cl⁻ currents in cardiomyocytes treated with the ER stress inducer tunicamycin. The pathophysiological role of VSOR Cl⁻ channel and the potential signaling mechanisms in the development of ER stress-induced apoptosis in CCD were also dissected.     

Protection by natural IgG: a sweet partnership with soluble lectins does the trick!

EMBO J 32: 2905–2919 10.1038/emboj.2013.199; published online September032013Some B cells of the adaptive immune system secrete polyreactive immunoglobulin G (IgG) in the absence of immunization or infection. Owing to its limited affinity and specificity, this natural IgG is thought to play a modest protective role. In this issue, a report reveals that natural IgG binds to microbes following their opsonization by ficolin and mannan-binding lectin (MBL), two carbohydrate receptors of the innate immune system. The interaction of natural IgG with ficolins and MBL protects against pathogenic bacteria via a complement-independent mechanism that involves IgG receptor FcγRI expressing macrophages. Thus, natural IgG enhances immunity by adopting a defensive strategy that crossovers the conventional boundaries between innate and adaptive microbial recognition systems.The adaptive immune system generates protective somatically recombined antibodies through a T cell-dependent (TD) pathway that involves follicular B cells. After recognizing antigen through the B-cell receptor (BCR), follicular B cells establish a cognate interaction with CD4⁺ T follicular helper (T_FH) cells and thereafter either rapidly differentiate into short-lived IgM-secreting plasmablasts or enter the germinal centre (GC) of lymphoid follicles to complete class switch recombination (CSR) and somatic hypermutation (SHM) (Victora and Nussenzweig, 2012). CSR from IgM to IgG, IgA and IgE generates antibodies with novel effector functions, whereas SHM provides the structural correlate for the induction of affinity maturation (Victora and Nussenzweig, 2012). Eventually, this canonical TD pathway generates long-lived bone marrow plasma cells and circulating memory B cells that produce protective class-switched antibodies capable to recognize specific antigens with high affinity (Victora and Nussenzweig, 2012).In addition to post-immune monoreactive antibodies, B cells produce pre-immune polyreactive antibodies in the absence of conventional antigenic stimulation (Ehrenstein and Notley, 2010). These natural antibodies form a vast and stable repertoire that recognizes both non-protein and protein antigens with low affinity (Ehrenstein and Notley, 2010). Natural antibodies usually emerge from a T cell-independent (TI) pathway that involves innate-like B-1 and marginal zone (MZ) B cells. These are extrafollicular B-cell subsets that rapidly differentiate into short-lived antibody-secreting plasmablasts after detecting highly conserved microbial and autologus antigens through polyreactive BCRs and nonspecific germline-encoded pattern recognition receptors (Pone et al, 2012; Cerutti et al, 2013).The most studied natural antibody is IgM, a pentameric complement-activating molecule with high avidity but low affinity for antigen (Ehrenstein and Notley, 2010). In addition to promoting the initial clearance of intruding microbes, natural IgM regulates tissue homeostasis, immunological tolerance and tumour surveillance (Ochsenbein et al, 1999; Zhou et al, 2007; Ehrenstein and Notley, 2010). Besides secreting IgM, B-1 and MZ B cells produce IgG and IgA after receiving CSR-inducing signals from dendritic cells (DCs), macrophages and neutrophils of the innate immune system (Cohen and Norins, 1966; Cerutti et al, 2013). In humans, certain natural IgG and IgA are moderately mutated and show some specificity, which may reflect the ability of human MZ B cells to undergo SHM (Cerutti et al, 2013). Yet, natural IgG and IgA are generally perceived as functionally quiescent.In this issue, Panda et al show that natural IgG bound to a broad spectrum of bacteria with high affinity by cooperating with ficolin and MBL (Panda et al, 2013), two ancestral soluble lectins of the innate immune system (Holmskov et al, 2003). This binding involved some degree of specificity, because it required the presence of ficolin or MBL on the microbial surface as well as lower pH and decreased calcium concentration in the extracellular environment as a result of infection or inflammation (see Figure 1).Open in a separate windowFigure 1Ficolins and MBL are produced by hepatocytes and various cells of the innate immune system and opsonize bacteria after recognizing conserved carbohydrates. Low pH and calcium concentrations present under infection-inflammation conditions promote the interaction of ficolin or MBL with natural IgG on the surface of bacteria. The resulting immunocomplex is efficiently phagocytosed by macrophages through FcγR1 independently of the complement protein C3, leading to the clearance of bacteria.Ficolins and MBL are soluble pattern recognition receptors that opsonize microbes after binding to glycoconjugates through distinct carbohydrate recognition domain (CRD) structures (Holmskov et al, 2003). While ficolins use a fibrinogen domain, MBL and other members of the collectin family use a C-type lectin domain attached to a collagen-like region (Holmskov et al, 2003). Similar to pentraxins, ficolins and MBL are released by innate effector cells and hepatocytes, and thus may have served as ancestral antibody-like molecules prior to the inception of the adaptive immune system (Holmskov et al, 2003; Bottazzi et al, 2010). Of note, MBL and the MBL-like complement protein C1q are recruited by natural IgM to mediate complement-dependent clearance of autologous apoptotic cells and microbes (Holmskov et al, 2003; Ehrenstein and Notley, 2010). Panda et al found that a similar lectin-dependent co-optation strategy enhances the protective properties of natural IgG (Panda et al, 2013).By using bacteria and the bacterial glycan N-acetylglicosamine, Panda et al show that natural IgG isolated from human serum or T cell-deficient mice interacted with the fibrinogen domain of microbe-associated ficolins (Panda et al, 2013). The resulting immunocomplex was phagocytosed by macrophages via the IgG receptor FcγRI in a complement-independent manner (Panda et al, 2013). The additional involvement of MBL was demonstrated by experiments showing that natural IgG retained some bacteria-binding activity in the absence of ficolins (Panda et al, 2013).Surface plasmon resonance provided some clues regarding the molecular requirements of the ficolin–IgG interaction (Panda et al, 2013), but the conformational changes required by ficolin to interact with natural IgG remain to be addressed. In particular, it is unclear what segment of the effector Fc domain of natural IgG binds to ficolins and whether Fc-associated glycans are involved in this binding. Specific glycans have been recently shown to mitigate the inflammatory properties of IgG emerging from TI responses (Hess et al, 2013) and this process could implicate ficolins and MBL. Moreover, it would be important to elucidate whether and how the antigen-binding Fab portion of natural IgG regulates its interaction with ficolins and MBL.The in vivo protective role of natural IgG was elegantly demonstrated by showing that reconstitution of IgG-deficient mice lacking the CSR-enzyme activation-induced cytidine deaminase with natural IgG from T cell-insufficient animals enhanced resistance to pathogenic Pseudomonas aeruginosa (Panda et al, 2013). This protective effect was associated with reduced production of proinflammatory cytokines, occurred independently of the complement protein C3 and was impaired by peptides capable to inhibit the binding of natural IgG to ficolin (Panda et al, 2013). Additional in vivo studies will be needed to determine whether natural IgG exerts protective activity in mice lacking ficolin, MBL or FcγRI, and to ascertain whether these molecules also enhance the protective properties of canonical or natural IgG and IgA released by bone marrow plasma cells and mucosal plasma cells, respectively.In conclusion, the findings by Panda et al show that natural IgG adopts ‘crossover'' defensive strategies that blur the conventional boundaries between the innate and adaptive immune systems. The sophisticated integration of somatically recombined and germline-encoded antigen recognition systems described in this new study shall stimulate immunologists to further explore the often underestimated protective virtues of our vast natural antibody repertoire. This effort may lead to the development of novel therapies against infections.     

Proteomic Analysis of Dynein-Interacting Proteins in Amyotrophic Lateral Sclerosis Synaptosomes Reveals Alterations in the RNA-Binding Protein Staufen1

Synapse disruption takes place in many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). However, the mechanistic understanding of this process is still limited. We set out to study a possible role for dynein in synapse integrity. Cytoplasmic dynein is a multisubunit intracellular molecule responsible for diverse cellular functions, including long-distance transport of vesicles, organelles, and signaling factors toward the cell center. A less well-characterized role dynein may play is the spatial clustering and anchoring of various factors including mRNAs in distinct cellular domains such as the neuronal synapse. Here, in order to gain insight into dynein functions in synapse integrity and disruption, we performed a screen for novel dynein interactors at the synapse. Dynein immunoprecipitation from synaptic fractions of the ALS model mSOD1^G93A and wild-type controls, followed by mass spectrometry analysis on synaptic fractions of the ALS model mSOD1^G93A and wild-type controls, was performed. Using advanced network analysis, we identified Staufen1, an RNA-binding protein required for the transport and localization of neuronal RNAs, as a major mediator of dynein interactions via its interaction with protein phosphatase 1–beta (PP1B). Both in vitro and in vivo validation assays demonstrate the interactions of Staufen1 and PP1B with dynein, and their colocalization with synaptic markers was altered as a result of two separate ALS-linked mutations: mSOD1^G93A and TDP43^A315T. Taken together, we suggest a model in which dynein''s interaction with Staufen1 regulates mRNA localization along the axon and the synapses, and alterations in this process may correlate with synapse disruption and ALS toxicity.Amyotrophic lateral sclerosis (ALS)¹ is an adult-onset progressive neurodegenerative disease that targets both upper and lower motor neurons via an unknown mechanism, leading to paralysis and eventually death. Pathological changes affecting synapses in both the primary motor cortex and the peripheral neuromuscular junctions (NMJs) are considered an early occurrence in ALS, often preceding the degeneration of the axons and clinical symptomatic onset (1). Although synapse disruption is common to many neurodegenerative diseases and the molecular mechanisms underlying synapse stabilization and maintenance are of keen interest, the exact mechanisms governing synapse disruption have yet to be understood.Both upper and lower motor neurons are highly polarized cells, with axons that are several orders of magnitude longer than the diameter of their cell bodies. To survive and maintain proper function, these neurons depend on active intracellular transport (2). The molecular motor kinesin drives transport from the cell body to the nerve periphery, supplying proteins, lipids, RNAs, and other essential materials to the synapse. The dynein/dynactin protein complex drives retrograde transport, moving damaged proteins for degradation, as well as critical signaling molecules such as neurotrophins, to the cell body (3). Dynein is a pleiotropic cellular motor, whose function in numerous cellular pathways may be regulated by specific interactions with different binding partners (4, 5). In addition to its canonical role as a motor protein, dynein has been shown to have an anchoring role as well. For example, the interaction of dynein with microtubule binding nuclear mitotic apparatus protein (NuMA)-protein coupled receptor 1 (LGN) allows dynein to be cortically anchored in order to function in the spindle-positioning process during cell division (4, 6). In neurons, dynein interacts with the neuronal adhesion molecule neural-cell-adhesion-molecule-180, which leads to the specific recruitment of dynein to the cell cortex for synapse stabilization (7). Another example, best characterized in the oocyte, is mRNA anchoring at specific cellular locations (8). Thus, dynein can serve as a motor conducting long-distance signaling, as well as an anchoring agent at distinct domains like the synapse. The switch between dynein''s different capacities may be regulated by its phosphorylation state, which may be mediated by protein phosphatase 1 (PP1) (9, 10).Transport deficits are common in many neurodegenerative disorders (3, 11, 12). In the ALS mouse model SOD1^G93A, transport dysfunction can be observed as early as at the embryonic stage (13). Although mutations in dynein or its activator dynactin were demonstrated to lead to synapse disruption and neurodegeneration (14–16), the effect of the mutations in slowing down dynein-mediated transport is not sufficient to create the harsh neurodegeneration observed in ALS (17, 18), suggesting an additional mechanism. One possibility is a switch in the nature of the retrogradely transported cargo from survival signals to stress signals (19). Hence, a change in the composition of dynein complexes may underlie neurodegenerative and synapse elimination mechanisms.General proteomic screens of protein complexes at the synapse have presented high complexity of both protein composition and signaling network architecture (20–23). Proteomics following immunoprecipitation of receptors such as α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate from synaptosomes reveal large protein complexes, up to 3000 kDa that can incorporate up to 185 proteins (21, 24). Notably, many of these proteins are involved in localized protein synthesis (25).Interestingly, dynein was found to be one of the proteins identified from synaptosome proteomics (26), suggesting that dynein plays a role in maintaining synaptic function. Although synapse disruption is one of the early events occurring in many neurodegenerative diseases, the identity of dynein complexes in the synapse and molecular mechanisms of synapse protection are still largely unknown.Here, we sought to characterize synaptic dynein complexes using a differential proteomic screen of the SOD1^G93A mouse model for ALS. The SOD1^G93A mouse model is the most studied model for ALS, manifesting many ALS phenotypes, including upper and lower motoneuron degeneration, synaptic disruption, and alterations in dynein functions. Here, we purified synaptosomes from brains of SOD1^G93A and control mice, followed by dynein-intermediate chain immunoprecipitation and mass spectrometry analysis to identify changes in dynein interactors. We further utilized the Advanced Network Analysis Tool (ANAT) (27) to predict potential pathways connecting dynein to the immunoprecipitated proteins in the ALS model and control mice. Our results demonstrate distinct populations of dynein-interacting proteins in ALS and in control mice, in addition to several common interactors. In both networks, the RNA-binding protein Staufen1 appeared as a predicted central node linking dynein to PP1B, a component of the catalytic subunit of PP1. In vitro and in vivo validation of the interaction and synaptic colocalization of both Staufen1 and PP1B with dynein, together with altered localization caused by ALS-linked mutations, suggest a role for dynein in the localization of Staufen1 ribonucleoproteins (RNPs) in neurodegenerative diseases such as ALS.     

Phosphorylation of the C Terminus of RHD3 Has a Critical Role in Homotypic ER Membrane Fusion in Arabidopsis

The endoplasmic reticulum (ER) consists of dynamically changing tubules and cisternae. In animals and yeast, homotypic ER membrane fusion is mediated by fusogens (atlastin and Sey1p, respectively) that are membrane-associated dynamin-like GTPases. In Arabidopsis (Arabidopsis thaliana), another dynamin-like GTPase, ROOT HAIR DEFECTIVE3 (RHD3), has been proposed as an ER membrane fusogen, but direct evidence is lacking. Here, we show that RHD3 has an ER membrane fusion activity that is enhanced by phosphorylation of its C terminus. The ER network was RHD3-dependently reconstituted from the cytosol and microsome fraction of tobacco (Nicotiana tabacum) cultured cells by exogenously adding GTP, ATP, and F-actin. We next established an in vitro assay system of ER tubule formation with Arabidopsis ER vesicles, in which addition of GTP caused ER sac formation from the ER vesicles. Subsequent application of a shearing force to this system triggered the formation of tubules from the ER sacs in an RHD-dependent manner. Unexpectedly, in the absence of a shearing force, Ser/Thr kinase treatment triggered RHD3-dependent tubule formation. Mass spectrometry showed that RHD3 was phosphorylated at multiple Ser and Thr residues in the C terminus. An antibody against the RHD3 C-terminal peptide abolished kinase-triggered tubule formation. When the Ser cluster was deleted or when the Ser residues were replaced with Ala residues, kinase treatment had no effect on tubule formation. Kinase treatment induced the oligomerization of RHD3. Neither phosphorylation-dependent modulation of membrane fusion nor oligomerization has been reported for atlastin or Sey1p. Taken together, we propose that phosphorylation-stimulated oligomerization of RHD3 enhances ER membrane fusion to form the ER network.In eukaryotic cells, the endoplasmic reticulum (ER) is the organelle with the largest membrane area. The ER consists of an elaborate network of interconnected membrane tubules and cisternae that is continually moving and being remodeled (Friedman and Voeltz, 2011). In plant cells, ER movement and remodeling is primarily driven by the actin-myosin XI cytoskeleton (Sparkes et al., 2009; Ueda et al., 2010; Yokota et al., 2011; Griffing et al., 2014) and secondarily by the microtubule cytoskeleton (Hamada et al., 2014). Several factors involved in creating the ER architecture have been also identified (Anwar et al., 2012; Chen et al., 2012; Goyal and Blackstone, 2013; Sackmann, 2014; Stefano et al., 2014a; Westrate et al., 2015). Among them, ER membrane-bound GTPases, animal atlastins and yeast Sey1p (Synthetic Enhancement of Yop1), function as ER fusogens to form the interconnected tubular network (Hu et al., 2009; Orso et al., 2009; Anwar et al., 2012). Atlastin molecules on the two opposed membranes have been proposed to transiently dimerize to attract the two membranes to each other (Bian et al., 2011; Byrnes and Sondermann, 2011; Morin-Leisk et al., 2011; Moss et al., 2011; Lin et al., 2012; Byrnes et al., 2013). Closely attracted lipid bilayers are supposed to be destabilized by an amphipathic helical domain at the atlastin C terminus to facilitate membrane fusion (Bian et al., 2011; Liu et al., 2012; Faust et al., 2015). Knockdown of atlastins leads to fragmentation of the ER and unbranched ER tubules, while overexpression of atlastins enhances ER membrane fusion, which enlarges the ER profiles (Hu et al., 2009; Orso et al., 2009).An Arabidopsis (Arabidopsis thaliana) protein, ROOT HAIR DEFECTIVE3 (RHD3), has been proposed as a fusogen because (1) when it is disrupted, the ER network is modified into large cable-like strands of poorly branched membranes (Zheng et al., 2004; Chen et al., 2011; Stefano et al., 2012), (2) it shares sequence similarity with the above-mentioned fusogen Sey1p (Hu et al., 2009), and (3) it has structural similarity to atlastin and Sey1p, with a functional GTPase domain at the N-terminal cytosolic domain (Stefano et al., 2012) followed by two transmembrane domains and a cytosolic tail. RHD3 has a longer cytosolic C-terminal tail than do atlastin and Sey1p (Stefano and Brandizzi, 2014). It contains not only an amphipathic region but also a Ser/Thr-rich C terminus.Arabidopsis has two RHD3 isoforms called RHD3-Like 1 and RHD3-Like 2. Fluorescently tagged RHD3 and RHD3-Like 2 localize to the ER (Chen et al., 2011; Stefano et al., 2012; Lee et al., 2013). RHD3 and the two RHD3-Like proteins likely have redundant roles in ER membrane fusion (Zhang et al., 2013). Overexpression of either RHD3 or RHD3-Like 2 with a defective GTPase domain phenocopies the aberrant ER morphology in rhd3-deficient mutants (Chen et al., 2011; Lee et al., 2013).In this study, we show that the Ser/Thr-rich C terminus enhances ER membrane fusion following phosphorylation of its C terminus. We propose a model in which phosphorylation and oligomerization of RHD3 is required for efficient ER membrane fusion. Our findings clarify the mechanisms that regulate RHD3 and consequently the homeostasis of membrane fusion in the ER.     

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.     

Changes in the Spinal Cord Proteome of an Amyotrophic Lateral Sclerosis Murine Model Determined by Differential In-gel Electrophoresis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by loss of motor neurons resulting in progressive paralysis. To date, more than 140 different mutations in the gene encoding CuZn-superoxide dismutase (SOD1) have been associated with ALS. Several transgenic murine models exist in which various mutant SOD1s are expressed. We used DIGE to analyze the changes in the spinal cord proteome induced by expression of the unstable SOD1 truncation mutant G127insTGGG (G127X) in mice. Unlike mutants used in most other models, G127X lacks SOD activity and is present at low levels, thus reducing the risk of overexpression artifacts. The mice were analyzed at their peak body weights just before onset of symptoms. Variable importance plot analysis showed that 420 of 1,800 detected protein spots contributed significantly to the differences between the groups. By MALDI-TOF MS analysis, 54 differentially regulated proteins were identified. One spot was found to be a covalently linked mutant SOD1 dimer, apparently analogous to SOD1-immunoreactive bands migrating at double the molecular weight of SOD1 monomers previously detected in humans and mice carrying mutant SOD1s and in sporadic ALS cases. Analyses of affected functional pathways and the subcellular representation of alterations suggest that the toxicity exerted by mutant SODs induces oxidative stress and affects mitochondria, cellular assembly/organization, and protein degradation.Amyotrophic lateral sclerosis (ALS)¹ is a devastating neurodegenerative disease characterized by loss of motor neurons in the motor cortex, the brainstem, and the spinal cord. This results in progressive muscular atrophy, and the patients usually succumb to respiratory failure within a few years. About 10% of ALS cases are familial (1), and in some patients the disease is linked to mutations in the CuZn-superoxide dismutase (SOD1) gene (2). SOD1 is a ubiquitously expressed antioxidant enzyme. Overall about 6% of all cases with ALS show SOD1 mutations, and more than 140 such mutations have been identified (3). The mutations confer a cytotoxic gain of function of unknown character to the enzyme (4, 5). ALS caused by mutant SOD1s shows the same spectrum of disease phenotypes as is seen in sporadic cases lacking such mutations. This suggests that the pathogenesis of ALS induced by SOD1 mutations should show significant similarities with that in sporadic disease, e.g. similar pathogenic protein alterations.ALS has been modeled in mice via transgenic overexpression of mutant SOD1s (5–8). To cause disease within the short lifespan of a mouse, the mutant SOD1s have to be expressed at rates around 25-fold higher than the rate of expression of the endogenous murine enzyme (9). Mostly structurally stable mutants have been used, resulting in up to 10-fold increases in SOD activity and 20-fold increases in SOD1 protein levels in the CNS that may cause overexpression artifacts. Overloading of mitochondria with mutant SOD1s and vacuolization have been observed in such models (10).To explore ALS pathogenesis, we studied changes in the proteome of spinal cords of SOD1 transgenic mice using DIGE. To reduce the risk of overexpression artifacts, we used mice that express the unstable human SOD1 (hSOD1) truncation mutant G127insTGGG (G127X) (7). These mice develop an aggressive form of the disease, which is of short duration, despite the fact that the mutant lacks SOD activity and the fact that the level of G127X hSOD1 protein is less than half that of the endogenous murine SOD1. The mice were studied at their peak body weights just before development of paralytic symptoms. Here we present the identity of and discuss the possible significance of 53 proteins found to be differentially regulated in ALS transgenic mice.     

Gut microbiota of healthy Canadian infants: profiles by mode of delivery and infant diet at 4 months

Background:

The gut microbiota is essential to human health throughout life, yet the acquisition and development of this microbial community during infancy remains poorly understood. Meanwhile, there is increasing concern over rising rates of cesarean delivery and insufficient exclusive breastfeeding of infants in developed countries. In this article, we characterize the gut microbiota of healthy Canadian infants and describe the influence of cesarean delivery and formula feeding.

Methods:

We included a subset of 24 term infants from the Canadian Healthy Infant Longitudinal Development (CHILD) birth cohort. Mode of delivery was obtained from medical records, and mothers were asked to report on infant diet and medication use. Fecal samples were collected at 4 months of age, and we characterized the microbiota composition using high-throughput DNA sequencing.

Results:

We observed high variability in the profiles of fecal microbiota among the infants. The profiles were generally dominated by Actinobacteria (mainly the genus Bifidobacterium) and Firmicutes (with diverse representation from numerous genera). Compared with breastfed infants, formula-fed infants had increased richness of species, with overrepresentation of Clostridium difficile. Escherichia–Shigella and Bacteroides species were underrepresented in infants born by cesarean delivery. Infants born by elective cesarean delivery had particularly low bacterial richness and diversity.

Interpretation:

These findings advance our understanding of the gut microbiota in healthy infants. They also provide new evidence for the effects of delivery mode and infant diet as determinants of this essential microbial community in early life.The human body harbours trillions of microbes, known collectively as the “human microbiome.” By far the highest density of commensal bacteria is found in the digestive tract, where resident microbes outnumber host cells by at least 10 to 1. Gut bacteria play a fundamental role in human health by promoting intestinal homeostasis, stimulating development of the immune system, providing protection against pathogens, and contributing to the processing of nutrients and harvesting of energy.^1,2 The disruption of the gut microbiota has been linked to an increasing number of diseases, including inflammatory bowel disease, necrotizing enterocolitis, diabetes, obesity, cancer, allergies and asthma.¹ Despite this evidence and a growing appreciation for the integral role of the gut microbiota in lifelong health, relatively little is known about the acquisition and development of this complex microbial community during infancy.³Two of the best-studied determinants of the gut microbiota during infancy are mode of delivery and exposure to breast milk.^4,5 Cesarean delivery perturbs normal colonization of the infant gut by preventing exposure to maternal microbes, whereas breastfeeding promotes a “healthy” gut microbiota by providing selective metabolic substrates for beneficial bacteria.^3,5 Despite recommendations from the World Health Organization,⁶ the rate of cesarean delivery has continued to rise in developed countries and rates of breastfeeding decrease substantially within the first few months of life.^7,8 In Canada, more than 1 in 4 newborns are born by cesarean delivery, and less than 15% of infants are exclusively breastfed for the recommended duration of 6 months.^9,10 In some parts of the world, elective cesarean deliveries are performed by maternal request, often because of apprehension about pain during childbirth, and sometimes for patient–physician convenience.¹¹The potential long-term consequences of decisions regarding mode of delivery and infant diet are not to be underestimated. Infants born by cesarean delivery are at increased risk of asthma, obesity and type 1 diabetes,¹² whereas breastfeeding is variably protective against these and other disorders.¹³ These long-term health consequences may be partially attributable to disruption of the gut microbiota.^12,14Historically, the gut microbiota has been studied with the use of culture-based methodologies to examine individual organisms. However, up to 80% of intestinal microbes cannot be grown in culture.^3,15 New technology using culture-independent DNA sequencing enables comprehensive detection of intestinal microbes and permits simultaneous characterization of entire microbial communities. Multinational consortia have been established to characterize the “normal” adult microbiome using these exciting new methods;¹⁶ however, these methods have been underused in infant studies. Because early colonization may have long-lasting effects on health, infant studies are vital.^3,4 Among the few studies of infant gut microbiota using DNA sequencing, most were conducted in restricted populations, such as infants delivered vaginally,¹⁷ infants born by cesarean delivery who were formula-fed¹⁸ or preterm infants with necrotizing enterocolitis.¹⁹Thus, the gut microbiota is essential to human health, yet the acquisition and development of this microbial community during infancy remains poorly understood.³ In the current study, we address this gap in knowledge using new sequencing technology and detailed exposure assessments²⁰ of healthy Canadian infants selected from a national birth cohort to provide representative, comprehensive profiles of gut microbiota according to mode of delivery and infant diet.     

Synapse reorganization—a new partnership revealed

Changes in synaptic function require both qualitative and quantitative reorganization of the synaptic components. Ca²⁺ plays a central role in this process, but the mechanism has not been fully elucidated. Zhang et al report a novel mechanism whereby Ca²⁺/calmodulin (CaM) regulates the stability of the postsynaptic scaffold. Ca²⁺/CaM interacts with PSD-95, a core protein in the postsynaptic density (PSD) that supports synaptic signaling and structural components. Ca²⁺/CaM interferes with the palmitoylation of PSD-95, resulting in the dissociation of PSD-95 from the postsynaptic membrane. This process may explain the reduction of surface glutamate receptor observed during synaptic depression and homeostatic regulation of the synaptic response after prolonged neuronal activity.The adaptation of synaptic strength is a fundamental process for learning and memory. This form of synaptic plasticity is triggered by influx of Ca²⁺ from NMDA-type glutamate receptor (NMDAR), leading to the synaptic insertion or removal of AMPA-type glutamate receptor (AMPAR) and determines the strength of synaptic transmission. This process is mediated by the gross reorganization of the postsynaptic composition in a qualitative and quantitative fashion (Bosch et al, 2014). Importantly, the number of synaptic AMPAR is regulated by the number and affinity of postsynaptic ‘slots’, a hypothetical receptor binding site within a synapse, which is regulated during synaptic plasticity processes.PSD-95, a scaffolding protein at excitatory synapses, has been considered as a major candidate for the slot. It interacts with AMPAR through the TARP/stargazin protein family and modulates the synaptic localization of the receptor as well as the strength of synaptic transmission (El-Husseini et al, 2000). In turn, the localization of PSD-95 at the synapse is regulated by a constant cycle of palmitoylation by protein palmitoyl acyltransferases (PAT) at cysteines 3 and 5, which is required for efficient synaptic targeting of the protein, and depalmitoylation by palmitoyl protein thioesterases (PPT) (El-Husseini et al, 2002; Noritake et al, 2009). Activation of glutamate receptors increases depalmitoylated PSD-95 and releases it from the postsynaptic site (El-Husseini et al, 2002; Sturgill et al, 2009), whereas blockage of neuronal activity by TTX increases palmitoylated PSD-95 and targets it to the synapse (Noritake et al, 2009). However, it remains unclear how neuronal activity controls the palmitoylation/depalmitoylation cycle and the subsequent trafficking of PSD-95 to and from the synapse.In this issue of The EMBO Journal, using a combination of structural biological, biochemical, and cell biological approaches, Zhang et al (2014) revealed a novel mechanism by which Ca²⁺ regulates the synaptic localization of PSD-95. The authors found that Ca²⁺ complexed to CaM can bind PSD-95 within the first 13 residues—the exact location where palmitoylation takes place. Ca²⁺/CaM binds preferentially to unmodified PSD-95, thereby blocking the accessibility of the PAT to the palmitoylation sites. In contrast, Ca²⁺/CaM does not bind to palmitoylated PSD-95, and therefore, palmitoylated PSD-95 is subject to depalymitoylation by PPT. Overall, the net effect of Ca²⁺/CaM is a reduction of PSD-95 bound to the synaptic membrane (Fig​(Fig1).1). Consistent with this, a PSD-95 mutant that was unable to bind Ca²⁺/CaM does not leave the synapse following glutamate/glycine treatment. Furthermore, the mutant PSD-95 showed an increased in synaptic accumulation, indicating that the treatment also increases palmitoylation activity but it is normally dominated by the depalmitoylating action of Ca²⁺/CaM binding (Noritake et al, 2009).Open in a separate windowFigure 1Calcium influx-induced release of PSD-95 and glutamate receptor from the synapseAt a synapse, cysteine residues of PSD-95 (C3 and C5) are under a continuous cycle of palmitoylation by protein palmitoyl acyltransferase (PAT), and depalmitoylation by palmitoyl protein thioesterase (PPT). A. Palmitoylated PSD-95 associates with the synaptic membrane and CDKL5 and serves as a ‘slot’ for AMPAR at the synapse through the interaction with TARP/stargazin. B. Upon glutamate stimulation, Ca²⁺ influx through NMDARs induces binding of Ca²⁺/CaM to PSD-95. Ca²⁺/CaM blocks the accessibility of PAT, thereby facilitating the depalmitoylation of PSD-95, which subsequently allows PSD-95 to dissociate from the synaptic membrane and CDKL5. C. The dissociation of PSD-95 from the synaptic membrane reduces the number of available ‘slots’ for AMPAR on the postsynaptic membrane, leading to a reduction of AMPAR-mediated synaptic transmission.The beauty of the work by Zhang et al (2014) is that the structure fully explains the biology. However, several important questions remain. Above all, it is still unclear when this mechanism would operate. Generally, the palmitoylation/depalmitoylation cycle is considered to be in the order of minutes. Therefore, for Ca²⁺/CaM to effectively reduce the palmitoylation of PSD-95, a prolonged influx of Ca²⁺ is required. In contrast, the rise in intracellular Ca²⁺ induced by a single excitatory postsynaptic current (EPSC) or dendritic action potential is in the order of milliseconds to seconds. A slow repetitive stimulation protocol, such as that used to induce long-term depression (LTD) (1 Hz, 15 min), may be effective in increasing Ca²⁺/CaM for a sufficiently long period of time. Homeostatic scaling induced by a prolonged increase in network activity (for example, via the pharmacological blockade of inhibitory synaptic transmission) may also be a possible mechanism for the Ca²⁺/CaM-mediated removal of synaptic PSD-95. In contrast, stimulation used to induce long-term potentiation (LTP) (a brief high-frequency stimulation such as 100 Hz, 1 s) may not be effective. Indeed, Bosch et al (2014) found that the induction of LTP at single dendritic spines in hippocampal CA1 pyramidal neurons does not decrease or increase PSD-95 during the first hour after induction even if the dendritic spine enlarges during this period.In this context, it is important to understand what impact Ca²⁺/CaM-mediated removal of synaptic PSD-95 has on synaptic transmission. If PSD-95 is indeed the slot for AMPAR, the Ca²⁺/CaM-mediated removal of synaptic PSD-95 is expected to reduce the synaptic transmission. The stimulation protocol used here by Zhang et al (bath application of glutamate/glycine) is similar to previously reported approaches to induce ‘chemical’ LTD and hence can provide an explanation for the decrease in PSD-95 from the synapse for 10–15 min after stimulation. The PSD-95 mutant that is unable to bind Ca²⁺/CaM will be useful to further analyze the link between the observed PSD-95-Ca²⁺/CaM interaction and synaptic plasticity.PSD-95 is also known to interact with the cyclin-dependent protein kinase-like kinase 5 (CDKL5) at the first 19 residues in a palmitoylation-dependent manner (Zhu et al, 2013). As expected, Ca²⁺/CaM binding also regulates CDKL5 association with PSD-95. The treatment of neurons with NMDA reduces the palmitoylation of PSD-95 and, concomitantly, the association with CDKL5. Mutations of CDKL5 and netrin-G1 gene have been reported in patients with an atypical form of Rett syndrome. Netrin-G1 ligand (NGL1) has been identified as an interaction partner and substrate of CDKL5 (Ricciardi et al, 2012). CDKL5 phosphorylates NGL1, and this phosphorylation stabilizes the interaction of NGL1 with PSD-95. Given that the Ca²⁺ signal only lasts a few milliseconds to seconds, it is important to investigate the spatiotemporal interaction between CaM, PSD-95, CDKL5, and NGL1 in dendritic spines during physiological and pathological conditions. In addition, CDKL5 can function as an upstream modulator of Rac signaling during development (Chen et al, 2010). Together with the fact that Rac also plays an important role in long-term potentiation, the PSD-95/CDKL5 complex might regulate Rac activity in the vicinity of the PSD.Other neuronal proteins including AMPAR subunit GluR1/2, glutamate receptor interacting protein (GRIP), G-protein-coupled receptors, δ-catenin, and small and trimeric G-proteins can also undergo palmitoylation, suggesting that this process plays an essential role in subcellular targeting and that these proteins can be regulated by activity (Kang et al, 2008). The question of whether Ca²⁺/CaM can regulate the palmitoylation of these proteins remains. Interestingly, the Ca²⁺/CaM interaction site of PSD-95 does not conform to a canonical IQ-motif, a CaM binding motif found in many other proteins (Zhang et al, 2014). Therefore, a bioinformatic approach is not possible at this point in time. This opens further directions of research.