Journal: The Journal of Physiology

Article Title: New perspectives on amyotrophic lateral sclerosis: the role of glial cells at the neuromuscular junction

doi: 10.1113/JP270213

Figure Lengend Snippet: In normal conditions (left panel), the presynaptic nerve terminal (green), the postsynaptic muscle fibre (pink) and the PSCs (blue) regulate synaptic functions in a coordinated fashion. Synaptic transmission is induced when an action potential reaches the presynaptic nerve terminal, activating voltage‐dependent calcium channels. This activation will trigger a rapid calcium entry into the nerve terminal, and induce synaptic vesicle exocytosis and subsequent neurotransmitter release into the synaptic cleft. ACh will be co‐released with ATP. ACh will bind to nAChRs on the muscle fibre and to mAChRs on PSCs. The binding of ACh to the nAChRs will depolarize the muscle fibre, which can result in the opening of voltage‐dependent sodium channels and subsequent muscle contraction (myosin and actin movement). The binding of ACh to mAChRs will trigger an increase in intracellular Ca2+ via the activation of the IP3 receptors (IP3Rs) of the ER. ATP released during synaptic activity will be detected by PSCs via P2Y G‐protein‐coupled receptors. They, too, will trigger an increase of intra‐PSC Ca2+ concentration by releasing Ca2+ from IP3‐driven internal stores. In return, PSC detection of neurotransmission will regulate synaptic activity by acting on presynaptic adenosine receptors (A1/A2ARs). Interactions between MN nerve terminals and PSCs can also occur via different pathways that influence NMJ stability and repair. For example, PSCs express receptors such as ErbBs that, if activated, can influence NMJ structure. PSCs can also release TGF‐β1 and agrin, and synthesize MMPs. TGF‐β1 will promote NMJ formation and stability, while agrin, which can be cleaved by MMP, will act on the LRP4 (low density lipoprotein receptor‐related protein 4)–MuSK complex to influence NMJ stability. Also, note the presence of mitochondria and wt SOD1 in all three synaptic elements. In ALS pathological conditions (right panel), these different PSC signalling pathways can be altered to promote NMJ denervation (D). Over‐activation of PSC muscarinic pathway leads to greater intracellular Ca2+ responses, alters gene expression and, hence, influences NMJ repair. Furthermore, the activation of the ErbB pathway can be implicated in alterations in PSC position and morphology as well as synaptic loss. Finally, the PSC agrin/MuSK pathway can be altered such that MMP release by PSCs can be upregulated and released agrin can be reduced, leading to NMJ instability. Boxes: hypothesis and proposed mechanisms. Dotted lines: pathways that are yet to be confirmed. Line thickness: relative increase or decrease of the pathway in comparison to the normal condition. LRP4, low density lipoprotein receptor‐related protein 4; nAChR, nicotinic acetylcholine receptor; SOD1, superoxide dismutase 1.

Article Snippet: In symptomatic SOD1 G93A mice, where NMJ denervation is already ongoing, data reveal at least two distinct groups of NMJs: one having the same synaptic properties as their wild‐type littermates and the other group exhibiting reduced synaptic strength (Rocha et al . 2013 ).

Techniques: Transmission Assay, Activation Assay, Binding Assay, Activity Assay, Concentration Assay, Gene Expression, Comparison

Journal: ASN NEURO

Article Title: SOD1 G93A Astrocyte-Derived Extracellular Vesicles Induce Motor Neuron Death by a miRNA-155-5p-Mediated Mechanism

doi: 10.1177/17590914231197527

Figure Lengend Snippet: List of Primer Sequences of Primers Employed for Real-Time RT-PCR.

Article Snippet: Male hemizygous NTac: SD-Tg (SOD1 G93A ) L26H rats (RRID: IMSR_TAC:2148) were obtained from Taconic (Hudson, NY, USA) and were mated with outbred Sprague–Dawley background.

Techniques: Sequencing

Journal: ASN NEURO

Article Title: SOD1 G93A Astrocyte-Derived Extracellular Vesicles Induce Motor Neuron Death by a miRNA-155-5p-Mediated Mechanism

doi: 10.1177/17590914231197527

Figure Lengend Snippet: Morphological characterization and size distribution of extracellular vesicles present in astrocyte CM. (a) TEM micrographs (negative staining) from extracellular vesicles isolated from astrocyte CM. Bars: 100 nm. (b) Frequency distribution of EV diameter as a percentage of the total number of vesicles from the three independent experiments. (c) Representative western blot of the exosomes from non-Tg or SOD1 G93A astrocyte CM using antibodies against TSG101, flotillin-1, and human SOD1.

Article Snippet: Male hemizygous NTac: SD-Tg (SOD1 G93A ) L26H rats (RRID: IMSR_TAC:2148) were obtained from Taconic (Hudson, NY, USA) and were mated with outbred Sprague–Dawley background.

Techniques: Negative Staining, Isolation, Western Blot

Journal: ASN NEURO

Article Title: SOD1 G93A Astrocyte-Derived Extracellular Vesicles Induce Motor Neuron Death by a miRNA-155-5p-Mediated Mechanism

doi: 10.1177/17590914231197527

Figure Lengend Snippet: Human SOD1 G93A mRNA is undetectable in exosomes of SOD1 G93A astrocytes. (a) Electropherogram results of total RNA extracted with TRIzol. (b) SOD1 G93A mRNA expression in non-Tg and SOD1 G93A astrocytes and astrocyte-derived exosomes, as assessed by RT-PCR.

Article Snippet: Male hemizygous NTac: SD-Tg (SOD1 G93A ) L26H rats (RRID: IMSR_TAC:2148) were obtained from Taconic (Hudson, NY, USA) and were mated with outbred Sprague–Dawley background.

Techniques: Expressing, Derivative Assay, Reverse Transcription Polymerase Chain Reaction

Journal: ASN NEURO

Article Title: SOD1 G93A Astrocyte-Derived Extracellular Vesicles Induce Motor Neuron Death by a miRNA-155-5p-Mediated Mechanism

doi: 10.1177/17590914231197527

Figure Lengend Snippet: SOD1 G93A astrocyte-derived exosomes reduce MN survival, neurite length, and neurite branching. (a) Representative images of primary cultures of MNs treated with GDNF (1 ng/ml, CTL) or with GDNF and exosomes from non-Tg or SOD1 G93A astrocytes. MNs were immunostained with beta III tubulin, scale bar = 250 μm. (b) MN survival after treatment with GDNF and following the addition of the indicated CM (non-Tg and SOD1 G93A ), exosomes (Exo; non-Tg and SOD1 G93A ) or CM depleted of exosomes (Exo free CM; non-Tg and SOD1 G93A ). Data are expressed as a percentage of survival after GDNF addition (100%, upper dotted line) compared to MN survival with no addition of trophic factor (lower dotted line). Unpaired t -tests were carried out, and the levels of significance were represented as * p < .05, ** p < .01. (c) and (d) Longest neurite length and total neurite length plot. MNs were treated with GDNF (control) and with GDNF and exosomes from nontransgenic and SOD1 G93A astrocytes (Exo; non-Tg and SOD1 G93A ). At least seven neurites per treatment were quantified with the Fiji program. Data are expressed as the mean ± SEM from five to six independent experiments (GDNF, Exo; non-Tg and SOD1 G93A ). Unpaired t -tests were performed, and significance levels were denoted as unpaired t -tests, * p < .05. (e) Representative images of Sholl graphs of MNs treated with exosomes. Mean ± SEM from at least three independent experiments. A two-way ANOVA followed by Fisher's LSD test for multiple comparisons was performed. p < .05 was used to determine statistical significance. (f) Intersections at 150 μm from the soma. An unpaired t -test was used to analyze the significant branching of neurites at 150 μm. An unpaired t -test was executed, and the significance levels were indicated as follows: * p < .05. Experiments performed with non-Tg astrocyte fractions are depicted in blue, and those from SOD1 G93A ones are in red.

Article Snippet: Male hemizygous NTac: SD-Tg (SOD1 G93A ) L26H rats (RRID: IMSR_TAC:2148) were obtained from Taconic (Hudson, NY, USA) and were mated with outbred Sprague–Dawley background.

Techniques: Derivative Assay, Control

Journal: ASN NEURO

Article Title: SOD1 G93A Astrocyte-Derived Extracellular Vesicles Induce Motor Neuron Death by a miRNA-155-5p-Mediated Mechanism

doi: 10.1177/17590914231197527

Figure Lengend Snippet: miR-155-5p and miR-582-3p show different expression levels in SOD1 G93A and non-Tg astrocyte exosomes. (a) and (c) Relative expression levels of miR-155-5p and miR-582-3p in astrocytes. (b) and (d) Relative expression levels of miR-155-5p and miR-582-3p in astrocyte exosomes. qRT-PCR quantification was performed with the ΔΔCt method using non-Tg samples as negative control and GAPDH mRNA as an endogenous reference control. Data are the mean ± SEM from at least three independent experiments. Unpaired t -tests were performed, and significance levels were denoted as * p < .05, ** p < .01, *** p < .005.

Article Snippet: Male hemizygous NTac: SD-Tg (SOD1 G93A ) L26H rats (RRID: IMSR_TAC:2148) were obtained from Taconic (Hudson, NY, USA) and were mated with outbred Sprague–Dawley background.

Techniques: Expressing, Quantitative RT-PCR, Negative Control, Control

Journal: ASN NEURO

Article Title: SOD1 G93A Astrocyte-Derived Extracellular Vesicles Induce Motor Neuron Death by a miRNA-155-5p-Mediated Mechanism

doi: 10.1177/17590914231197527

Figure Lengend Snippet: Inhibition of miR-155-5p rescue MN death induced by SOD1 G93A astrocyte exosomes. MN survival 24 h after incubation with SOD1 G93A (red) or non-Tg (blue) exosomes plus the miR155-5p mimic or inhibitor as indicated. Data show the percentage of MNs compared to control (MNs treated with non-Tg astrocyte exosomes). 24 h primary MN cultures were treated with non-Tg CM depleted of exosomes from non-Tg and SOD1 G93A astrocytes (Exo free CM; non-Tg, and SOD1 G93A , respectively), or with exosomes of non-Tg and SOD1 G93A astrocytes (Exo; non-Tg and SOD1 G93A , respectively), cotransfected with miR-155-5p mimetics (Exo miR; non-Tg or SOD1 G93A ) or miR-155-5p inhibitors (α-miR; non-Tg and SOD1 G93A ). Mean ± SEM from at least three independent experiments. One-way ANOVA results: * p < .05, from non-Tg treated control. (b) Representative images of Sholl graphs of MNs treated with exosomes from SOD1 G93A astrocytes (Exo-SOD1 G93A ) in the absence or presence of miR-155-5p inhibitors (Exo-SOD1 G93A -αmiR). Mean ± SEM from at least three independent experiments. A two-way ANOVA followed by Fisher's LSD test for multiple comparisons was performed. p < .05 was employed to determine statistical significance. (c) Intersections at 150 μm from the soma. An unpaired t -test was used to analyze the significant branching of neurites at 150 μm. Experiments performed with Exo-SOD1 G93A astrocytes are depicted in red, and those from Exo-SOD1 G93A -αmiR ones are in green. An unpaired t -test was conducted, and the results were denoted as * p < .05, ** p < .01, and *** p < .005.

Article Snippet: Male hemizygous NTac: SD-Tg (SOD1 G93A ) L26H rats (RRID: IMSR_TAC:2148) were obtained from Taconic (Hudson, NY, USA) and were mated with outbred Sprague–Dawley background.

Techniques: Inhibition, Incubation, Control

Journal: Glia

Article Title: Identification of a chronic non-neurodegenerative microglia activation state in a mouse model of peroxisomal β-oxidation deficiency

doi: 10.1002/glia.22831

Figure Lengend Snippet: (A-B) After transcriptomics analyses of Mfp2−/− microglia (A) and SOD1G93A microglia (B) (data from GEO GSE43366) the expression of genes associated with the Lysosome (mmu04142) KEGG pathway were plotted. The percentage of genes of this pathway differentially expressed in both mouse models is shown. (C) Heatmap for enriched functions of SOD1G93A microglia as compared to Mfp2−/− microglia (Ingenuity analysis). Enrichment is depicted as activated (orange), not enriched (white) or deactivated (blue). Red arrows point to functions related to phagocytosis, green arrows to growth and proliferation. (D-F) Staining of the lysosomal marker Cathepsin D (green) in visual cortex of control (D) and Mfp2−/− mice (E) and in spinal cord of endstage SOD1G93A mice (F). (G-I) Co-staining of the microglial marker Iba1 (red) and the lysosomal marker LAMP2/MAC3 (green) in brainstem of control (G) and Mfp2−/− mice (H) and in spinal cord of endstage SOD1G93A mice (I). White arrowheads point to LAMP2+ microglia. (J-L) Staining for nitrosylated proteins (green) in visual cortex of control (J) and Mfp2−/− mice (K) and in spinal cord of endstage SOD1G93A mice (L). Nuclei are stained blue with DAPI.

Article Snippet: Frozen sections of spinal cord of endstage SOD1 G93A mice were kindly provided by Prof. L. Van Den Bosch (VIB and KU Leuven, Belgium).

Techniques: Expressing, Staining, Marker, Control

Journal: The Journal of Neuroscience

Article Title: Microarray Analysis of the Cellular Pathways Involved in the Adaptation to and Progression of Motor Neuron Injury in the SOD1 G93A Mouse Model of Familial ALS

doi: 10.1523/JNEUROSCI.1470-07.2007

Figure Lengend Snippet: Genes significantly altered at 60 d in motorneurons expressing the G93A SOD1

Article Snippet: Murine SOD1 G93A transgenic mouse model. Transgenic mice B6SJL-Tg (SOD1-G93A) 1 Gur/J high copy number were backcrossed onto C57BL/6J Ola-Hsd (Harlan UK, Bicester, UK) for >20 generations to eliminate the SJL dysferlin mutation ( Bittner et al., 1999 ) and to achieve a homogeneous genetic background more suitable for microarray studies.

Techniques: Expressing, Membrane, Sequencing, Activity Assay, Binding Assay, Protein Binding, Translocation Assay, Ubiquitin Proteomics, Virus

Journal: The Journal of Neuroscience

Article Title: Microarray Analysis of the Cellular Pathways Involved in the Adaptation to and Progression of Motor Neuron Injury in the SOD1 G93A Mouse Model of Familial ALS

doi: 10.1523/JNEUROSCI.1470-07.2007

Figure Lengend Snippet: Q-PCR results. A, Significant upregulation of necdin at 60 d (p = 0.04), which is downregulated at the late stage of the disease (p = 0.003). B, Consistent upregulation of cyclin I throughout the pathology (60 d, p = 0.05; 90 d, p = 0.1; 120 d, p = 0.03). C, Consistent downregulation of plexin domain containing 1 (60 d, p = 0.02; 90 d, p = 0.003; 120 d, p = 0.001). Error bars indicate SD. Tg, Transgenic; N-Tg, nontransgenic. *p ≤ 0.05; **p ≤ 0.01.

Article Snippet: Murine SOD1 G93A transgenic mouse model. Transgenic mice B6SJL-Tg (SOD1-G93A) 1 Gur/J high copy number were backcrossed onto C57BL/6J Ola-Hsd (Harlan UK, Bicester, UK) for >20 generations to eliminate the SJL dysferlin mutation ( Bittner et al., 1999 ) and to achieve a homogeneous genetic background more suitable for microarray studies.

Techniques: Transgenic Assay

Journal: The Journal of Neuroscience

Article Title: Microarray Analysis of the Cellular Pathways Involved in the Adaptation to and Progression of Motor Neuron Injury in the SOD1 G93A Mouse Model of Familial ALS

doi: 10.1523/JNEUROSCI.1470-07.2007

Figure Lengend Snippet: Genes significanlty altered at 90 d in motorneurons expressing the G93A SOD1

Article Snippet: Murine SOD1 G93A transgenic mouse model. Transgenic mice B6SJL-Tg (SOD1-G93A) 1 Gur/J high copy number were backcrossed onto C57BL/6J Ola-Hsd (Harlan UK, Bicester, UK) for >20 generations to eliminate the SJL dysferlin mutation ( Bittner et al., 1999 ) and to achieve a homogeneous genetic background more suitable for microarray studies.

Techniques: Expressing, Variant Assay, Activity Assay, Membrane

Journal: The Journal of Neuroscience

Article Title: Microarray Analysis of the Cellular Pathways Involved in the Adaptation to and Progression of Motor Neuron Injury in the SOD1 G93A Mouse Model of Familial ALS

doi: 10.1523/JNEUROSCI.1470-07.2007

Figure Lengend Snippet: Genes significantly altered at 120 d in motorneurons expressing the G93A SOD1

Article Snippet: Murine SOD1 G93A transgenic mouse model. Transgenic mice B6SJL-Tg (SOD1-G93A) 1 Gur/J high copy number were backcrossed onto C57BL/6J Ola-Hsd (Harlan UK, Bicester, UK) for >20 generations to eliminate the SJL dysferlin mutation ( Bittner et al., 1999 ) and to achieve a homogeneous genetic background more suitable for microarray studies.

Techniques: Expressing, Binding Assay, Sequencing, Activity Assay, Protein Binding, Transformation Assay, Ubiquitin Proteomics, Activation Assay, Membrane

Journal: The Journal of Neuroscience

Article Title: Microarray Analysis of the Cellular Pathways Involved in the Adaptation to and Progression of Motor Neuron Injury in the SOD1 G93A Mouse Model of Familial ALS

doi: 10.1523/JNEUROSCI.1470-07.2007

Figure Lengend Snippet: A, Motor neurons isolated from G93A mice at 60 d show upregulation in several classes of genes delineating what is likely to be happening in these cells under stress. There is upregulation of the transcriptional machinery, along with upregulation of translation-related ribosomal and folding proteins, as the motor neuron attempts to compensate for the ongoing cellular stress. All these mechanisms require ATP, provoking a massive increase in the work load of mitochondria, leading to upregulation of carbohydrate metabolism and respiratory chain activity, which in turn causes increased ROS production. The observed imbalance among the subunits forming the ATP synthase complex, shown by downregulation of the δ subunit, will generate additional oxidative stress, with consequent production of oxidized proteins. These are then ubiquitinated and targeted for proteasomal degradation. B, Over time, the accumulation of damaged proteins and ROS is likely to cause a general collapse in cellular functioning, leading to downregulation of the compensatory pathways previously activated and leaving the cell with decreased energy and protein turnover. The cell increases protein degradation functions, with activation of the lysosomal machinery. Production and secretion of some subunits of the complement cascade are important signals of cellular stress for neighboring cells. The final abortive attempt at survival comes through activation of the cell cycle, with upregulation of cyclin L1 (involved in the transition from the quiescent state, G0, to the first phase of the cell cycle, G1) and cyclins D2 and E2 (involved in the progression of the cell cycle through G1 phase). The upregulation of cyclin I suggests that motor neurons are trying to exercise negative control on the transition between the G1 and S phase to prevent the abnormal progression through the cell cycle. Atf4, Activating transcription factor 4; Atp5a1, ATP synthase F1 complex α1 subunit; Atp5d, ATP synthase F1 complex δ subunit; Cct4, chaperonin subunit 4; Ctsz, cathepsin-Z; Eef1, eukaryotic translation elongation factor 1; Eif3, eukaryotic translation initiation factor 3; Hsp, heat shock protein; Lyz, lysozyme; Lzp-s, P-lysozyme structural; Mdh1, malate dehydrogenase 1; Ndn, necdin; Psmc6, proteasome 26S; Rpl, ribosomal protein L; Sdha, succinate dehydrogenase complex subunit A; Taf9, transcription activator factor 9; Tcerg1, transcription elongation regulator 1 (CA150); Tgfb1i4, transforming growth factor β1-induced transcript 4; Ube1c, ubiquitin-activating enzyme E1C; Uble1b, ubiquitin-like activating enzyme E1B; Usp36, ubiquitin-specific preotease 36; H+, hydrogen ion; TCA, tricarboxylic acid.

Article Snippet: Murine SOD1 G93A transgenic mouse model. Transgenic mice B6SJL-Tg (SOD1-G93A) 1 Gur/J high copy number were backcrossed onto C57BL/6J Ola-Hsd (Harlan UK, Bicester, UK) for >20 generations to eliminate the SJL dysferlin mutation ( Bittner et al., 1999 ) and to achieve a homogeneous genetic background more suitable for microarray studies.

Techniques: Isolation, Activity Assay, Activation Assay, Negative Control, Ubiquitin Proteomics