Journal: bioRxiv

Article Title: Neuregulin-1 protects against respiratory viral induced mortality

doi: 10.1101/2023.05.10.540232

Figure Lengend Snippet: ( A ) CD11c + cells are increased in the lungs of atopic mice. Flow cytometry of lung cell suspension showing frequency (left) and cell number (right) at day 3 post inoculation (PI) high dose SeV ( B ) Adoptive transfer of CD11c + cells isolated from NA or atopic mice into naïve mice 24 h before inoculation with high dose SeV delays but does not prevent mortality; n=4 per group. ( C ) Transcriptomic (RNAseq) comparison between FACS isolated lung CD11c + cells from atopic and NA mice identifies several disparately expressed gene products, including Nrg1 (n=4 per group). Selected gene products shown. ( D ) NRG1 is markedly increased in atopic mouse lung (“tissue”), BAL, and supernatant from 1 x10 6 atopic lung CD11c + cells (“CD11c sup”) cultured for 24 h. ( E ) The scRNA (10x Platform) tSNE coordinates show CD14 + monocytes and dendritic cell (“DC”) sub-populations expressing NRG1 in peripheral blood cells of healthy human donors.*p<0.05, **p<0.01, ****p<0.0001

Article Snippet: Cell culture basal media was supplemented with recombinant human NRG1 alpha (cat#: NBP2-35093, Novus Bio) for hBEC or with mouse NRG1 for mTEC on day five and three before and at the time of infection with 4000 pfu of rgRSV or SeV-GFP.

Techniques: Flow Cytometry, Suspension, Adoptive Transfer Assay, Isolation, Comparison, Cell Culture, Expressing

Journal: bioRxiv

Article Title: Neuregulin-1 protects against respiratory viral induced mortality

doi: 10.1101/2023.05.10.540232

Figure Lengend Snippet: ( A ) NRG1(1ng to 1000ng) i.n. (in 30µL) given daily to naïve mice for 5d before inoculation with high dose SeV reduces viral mortality; n=4 per group (1ng, 10ng 1000ng & PBS), n=8 per group (100 ng and 500 ng). ( B ) Ratio of EBD in the BAL to that in the lung shows reduced EBD in NRG1 treated mice on day 8 PI SeV. n≥8 per group, median ± IQR shown, Mann-Whitney U test.

Article Snippet: Cell culture basal media was supplemented with recombinant human NRG1 alpha (cat#: NBP2-35093, Novus Bio) for hBEC or with mouse NRG1 for mTEC on day five and three before and at the time of infection with 4000 pfu of rgRSV or SeV-GFP.

Techniques: MANN-WHITNEY

Journal: bioRxiv

Article Title: Neuregulin-1 protects against respiratory viral induced mortality

doi: 10.1101/2023.05.10.540232

Figure Lengend Snippet: ( A ) Adding NRG1 to hBEC inoculated with rgRSV (left) and mTEC inoculated with GFP-SeV (right) reduces spread of infection. Representative images shown. ( B ) Quantification of (A) for rgRSV and hBEC and ( C ) for GFP-SeV and mTEC. GFP positive cells quantified by ImageJ. Representative images from ≥3 separate experiments. *p<0.05, **p<0.01. ( D ) Transcriptomic analysis of hBEC cultures treated with NRG1 (100 ng) on the basolateral side of the Transwell for 5 days and inoculated with RSV (4000 pfu). RNA was isolated 48 h PI RSV and qRT-PCR performed using a custom Prime PCR array plate: (i) Transcripts in which NRG1 treatment reduced gene expression from that seen in RSV infected cells. (ii) Transcripts with low level expression that show small but significant change in expression relative to naïve control with NRG1 alone or genes significantly increased with RSV but whose expression levels were not affected by NRG1. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n=3.

Article Snippet: Cell culture basal media was supplemented with recombinant human NRG1 alpha (cat#: NBP2-35093, Novus Bio) for hBEC or with mouse NRG1 for mTEC on day five and three before and at the time of infection with 4000 pfu of rgRSV or SeV-GFP.

Techniques: Infection, Isolation, Quantitative RT-PCR, Gene Expression, Expressing, Control

Journal: Glia

Article Title: BACE1 Processing of NRG1 Type III Produces a Myelin-Inducing Signal but Is Not Essential for the Stimulation of Myelination

doi: 10.1002/glia.21255

Figure Lengend Snippet: Proteases serve as positive and negative regulators of myelination. ( A ) Pharmacological BACE1 inhibition blocks myelination in DRG dissociated explant cultures, whereas inhibition of ADAMs promotes myelination. Fluorescent imunostaining for MBP (green). Numbers of MBP-positive segments (mean ± SEM) after 14 days in vitro (DMSO, ADAMs inh n = 3 cultures; BACE1 inh n = 2 cultures; 2 coverslips/treatment; * P < 0.05, ** P < 0.01). Scale bar, 200 μm. ( B ) Axonal association of Schwann cells is not impaired upon BACE1 or ADAMs inhibition in DRG neuron dissociated explant cultures. Immunostaining for axons (Tuj1; red) and counterstaining for nuclei (DAPI; blue) after 9 d of protease inhibitor treatment. Representative axon bundles used for quantification are delineated. Numbers of axon-associated Schwann cells (per bundle length in μm) are displayed as percentage ± SEM relative to DMSO treated controls ( n = 2 cultures; 3 coverslips/condition; 3 bundles/coverslip; P = 0.38, ** P < 0.01). Scale bar, 20 μm. ( C ) Cleavage of full-length (HA-NRG1 FL ), but not “processed” NRG1 type III (HA-NRG1 GIEF ) by BACE1. Western blot of transfected HEK293T cell lysates probed with an anti-HA antibody. Expression of BACE1 (+B) strongly reduced the level of full-length HA-NRG1 FL (black arrowheads) and resulted in the accumulation of a ∼75 kD N-terminal processing product (white arrowhead), which corresponds in size to HA-NRG1 GIEF . HA-NRG1 FL processing by BACE1 was blocked by BACE1 inhibitor treatment (+B/Bi). Expression of BACE1 had no visible impact on HA-NRG1 GIEF levels. Note that “base line” processing of HA-NRG1 FL (+Bi) was not affected by BACE1 treatment. ( D ) BACE1 cleaves HA-NRG1 FL in the juxtamembrane “stalk” region. Western blot of transfected HEK293T cell lysates probed with an antibody against the C-terminal peptide sequence “GIEF”. Note that the C-terminal in ‘GIEF’ epitope only accumulates in HA-NRG1 FL lysates when BACE1 was coexpressed (+B). HA-NRG1 GIEF lysates served as a positive control. The membrane was reprobed for tubulin as a loading control. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: The following primary antibodies were used: mouse anti-HA tag (1:250; Covance), rabbit anti-carboxy terminus of neuregulin1 (1:100; Santa Cruz sc-348), rabbit anti-neurofilament 200 (1:200; Sigma), rabbit anti-peripherin (1:500; Chemicon), mouse anti-class III β-tubulin (1:250; Covance), mouse anti-NeuN (1:100; Chemicon), rabbit anti-parvalbumin (1:200, Swant), rabbit anti-Olig2 (1:100; gift from John Alberta), and rabbit anti-Necl1 (1:1000; gift from Elior Peles).

Techniques: Inhibition, In Vitro, Immunostaining, Protease Inhibitor, Western Blot, Transfection, Expressing, Sequencing, Positive Control, Membrane, Control

Journal: Glia

Article Title: BACE1 Processing of NRG1 Type III Produces a Myelin-Inducing Signal but Is Not Essential for the Stimulation of Myelination

doi: 10.1002/glia.21255

Figure Lengend Snippet: BACE1 cleavage in the “stalk” region produces a myelination-inducing NRG1 type III variant. ( A ) Domain structure of HA-NRG1 FL and HA-NRG1 GIEF . BACE1 cleavage of HA-NRG1 FL is indicated by scissors. Protein domains: EGF-like domain (green), cystein-rich domain (purple/black), transmembrane domain (brown), HA epitope tags (red). EC: extracellular; IC: intracellular. ( B ) HA-NRG1 FL and HA-NRG1 GIEF induce heterodimerization of ErbB2 and ErbB3 receptors in a two-cell split-TEV assay as measured by a ∼5 fold increase in luciferase reporter activity compared with controls. Results are displayed as relative luminescence units (RLUs) ±SD ( n = 6; *** P < 0.001). ( C ) HA-NRG1 FL and HA-NRG1 GIEF stimulate myelination in DRG neuron-Schwann cell co-cultures. Immunostaining for MBP (red) demonstrates a dramatic increase in the number of myelin segments upon lentiviral expression of HA-NRG1 FL or HA-NRG1 GIEF in sensory neurons compared with noninfected control cultures (ctrl) 7 days after myelination induction. Scale bar, 50 μm. ( D ) Sensory neurons prepared from NRG1 type III null mutants (CRD-NRG1−/−) are not myelinated 14 days after induction of myelination. Myelination is restored in NRG1 type III-deficient axons after lentiviral overexpression of HA-NRG1 FL or HA-NRG1 GIEF as demonstrated by immunostaining for MBP (red). Neurofilament (NF) staining (green) reveals the normal maintenance of sensory axons in mutant cultures. Scale bar, 50 μm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: The following primary antibodies were used: mouse anti-HA tag (1:250; Covance), rabbit anti-carboxy terminus of neuregulin1 (1:100; Santa Cruz sc-348), rabbit anti-neurofilament 200 (1:200; Sigma), rabbit anti-peripherin (1:500; Chemicon), mouse anti-class III β-tubulin (1:250; Covance), mouse anti-NeuN (1:100; Chemicon), rabbit anti-parvalbumin (1:200, Swant), rabbit anti-Olig2 (1:100; gift from John Alberta), and rabbit anti-Necl1 (1:1000; gift from Elior Peles).

Techniques: Variant Assay, Luciferase, Activity Assay, Immunostaining, Expressing, Control, Over Expression, Staining, Mutagenesis

Journal: Glia

Article Title: BACE1 Processing of NRG1 Type III Produces a Myelin-Inducing Signal but Is Not Essential for the Stimulation of Myelination

doi: 10.1002/glia.21255

Figure Lengend Snippet: Neuronal overexpression of epitope-tagged NRG1 type III-variants in transgenic mice. ( A ) Schematic drawing of the Thy1.2 cassette used for neuronal expression of HA-NRG1 FL and HA-NRG1 GIEF cDNAs (tg). XhoI, restriction site for subcloning; NotI/PvuI, restriction sites for cassette release; exons Ia, Ib, II, IV, and locations of RT-PCR primers used in ( B ) are indicated. (B) HA-NRG1 FL and HA-NRG1 GIEF transgenes are expressed in spinal cord at the onset of peripheral myelination. Transgene-specific RT-PCR of spinal cord cDNAs from transgenic and wildtype (wt) mice at P1. Amplification of β-actin was used as a positive control. ( C ) HA-NRG1 FL and HA-NRG1 GIEF proteins are expressed in the spinal cord of transgenic mice. Western blot of spinal cord protein lysates (40 μg) at 2 months of age probed with an anti-HA antibody. Arrowheads mark full-length (∼ 140 kD) and processed HA-NRG1 type III (∼ 70 kD) as well as a lower molecular weight protein band (∼ 45 kD), which most likely results from further NRG1 type III processing. ( D ) Spinal cord motor neurons and sensory neurons express HA-NRG1 FL and HA-NRG1 GIEF proteins at their plasma membrane. Confocal images of spinal cord and DRG cross sections after immunostaining for the HA-epitope, neurofilament 200 (NF), and peripherin (Prph). White line marks the border between gray and white matter. Scale bars, 50 μm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: The following primary antibodies were used: mouse anti-HA tag (1:250; Covance), rabbit anti-carboxy terminus of neuregulin1 (1:100; Santa Cruz sc-348), rabbit anti-neurofilament 200 (1:200; Sigma), rabbit anti-peripherin (1:500; Chemicon), mouse anti-class III β-tubulin (1:250; Covance), mouse anti-NeuN (1:100; Chemicon), rabbit anti-parvalbumin (1:200, Swant), rabbit anti-Olig2 (1:100; gift from John Alberta), and rabbit anti-Necl1 (1:1000; gift from Elior Peles).

Techniques: Over Expression, Transgenic Assay, Expressing, Subcloning, Reverse Transcription Polymerase Chain Reaction, Amplification, Positive Control, Western Blot, Molecular Weight, Clinical Proteomics, Membrane, Immunostaining

Journal: Glia

Article Title: BACE1 Processing of NRG1 Type III Produces a Myelin-Inducing Signal but Is Not Essential for the Stimulation of Myelination

doi: 10.1002/glia.21255

Figure Lengend Snippet: Expression of the epitope-tagged NRG1 type III-variants in the brains of transgenic mice. ( A ) HA-NRG1 GIEF expression in the cortex exclusively localizes to neurons. Confocal images of cortical layer V immunostained for the HA-epitope and cell type-specific markers. HA-NRG1 GIEF accumulates in a subset of NeuN-positive cortical neurons but is absent from parvalbumin (Parv)-expressing interneurons and Olig2-positive oligodendrocytes. Scale bars, 25 μm. ( B ) Confocal image of a HA epitope-positive neocortical projection neuron from a HA-NRG1 GIEF transgenic mouse at 4 months of age. HA-NRG1 GIEF is expressed in the soma, axon (arrowheads), and proximal dendrites (left panel), as well as dendritic spines (right panel; arrowheads). Scale bars, 10 μm. ( C ) Electron micrograph of a cortical synapse (postsynapse is to the lower right) after immunogold labeling using anti HA- (black arrowhead, 10 nm gold) and anti-PSD95 antibodies (white arrowhead, 15 nm gold). The HA-NRG1 FL transgene preferentially localizes to the PSD95-positive postsynaptic compartment. Scale bar, 100 nm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: The following primary antibodies were used: mouse anti-HA tag (1:250; Covance), rabbit anti-carboxy terminus of neuregulin1 (1:100; Santa Cruz sc-348), rabbit anti-neurofilament 200 (1:200; Sigma), rabbit anti-peripherin (1:500; Chemicon), mouse anti-class III β-tubulin (1:250; Covance), mouse anti-NeuN (1:100; Chemicon), rabbit anti-parvalbumin (1:200, Swant), rabbit anti-Olig2 (1:100; gift from John Alberta), and rabbit anti-Necl1 (1:1000; gift from Elior Peles).

Techniques: Expressing, Transgenic Assay, Labeling

Journal: Glia

Article Title: BACE1 Processing of NRG1 Type III Produces a Myelin-Inducing Signal but Is Not Essential for the Stimulation of Myelination

doi: 10.1002/glia.21255

Figure Lengend Snippet: Limited axonal transport of NRG1 type III. ( A ) (left panel) Endogenous full-length NRG1 (140 kD) and a C-terminal processing product (∼ 60 kD; arrowheads) are detected with an antibody against the C-terminus of NRG1 (αNRG1 C-term) in wt spinal cord protein lysates at P12, whereas corresponding sciatic nerve expression is virtually absent. (right panel) Endogenous NRG1 accumulates on the plasma membrane of spinal cord motor neurons after immunostaining with the αNRG1 C-term antibody. Nuclei are counterstained with DAPI (blue). Corresponding NRG1 immunoreactivity is absent from the sciatic nerve. Sciatic nerve axons are marked by tubulin (Tuj1; red). ( B,C ) (left panels) When probed with an anti HA-antibody (αHA), HA-NRG1 type III (140 kD) and two N-terminal processing products (∼ 70 kD and ∼ 45 kD; arrowheads) are present in spinal cord protein lysates from HA-NRG1 FL transgenic mice at P12, but barely detectable in sciatic nerve. Similarly, HA-NRG1 GIEF (∼ 70 kD) and a smaller protein species (∼ 45 kD; arrowheads) are prominently expressed in spinal cord lysates from HA-NRG1 GIEF mice, whereas sciatic nerve expression is low. (right panels) Immunostaining for axons (neurofilament 200, NF; red) and the HA-epitope (green) reveals localization of HA-NRG1 FL and HA-NRG1 GIEF at the surface of individual sciatic nerve axons (insets). Scale bars, 10 μm. ( D ) Confocal images of an individual sciatic nerve axon in cross section from an adult HA-NRG1 GIEF transgenic mouse. Immunostaining for neurofilament 200 (NF; red) and the HA tag (green) reveals expression of HA-NRG1 GIEF in vesicle-like structures (white arrowheads) and on the axonal surface (empty arrowheads). Scale bar, 2.5μm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: The following primary antibodies were used: mouse anti-HA tag (1:250; Covance), rabbit anti-carboxy terminus of neuregulin1 (1:100; Santa Cruz sc-348), rabbit anti-neurofilament 200 (1:200; Sigma), rabbit anti-peripherin (1:500; Chemicon), mouse anti-class III β-tubulin (1:250; Covance), mouse anti-NeuN (1:100; Chemicon), rabbit anti-parvalbumin (1:200, Swant), rabbit anti-Olig2 (1:100; gift from John Alberta), and rabbit anti-Necl1 (1:1000; gift from Elior Peles).

Techniques: Expressing, Clinical Proteomics, Membrane, Immunostaining, Transgenic Assay

Journal: Glia

Article Title: BACE1 Processing of NRG1 Type III Produces a Myelin-Inducing Signal but Is Not Essential for the Stimulation of Myelination

doi: 10.1002/glia.21255

Figure Lengend Snippet: HA-NRG1 GIEF promotes hypermyelination in the PNS and CNS of transgenic mice. ( A ) Sciatic nerve hypermyelination in HA-NRG1 FL and HA-NRG1 GIEF transgenic mice. Methylenblue-Azure II staining for myelin on semithin sciatic nerve cross sections at 2 months of age. Scale bar, 10 μm. ( B ) Quantification of myelin sheath thickness by g -ratio analysis. HA-NRG1 FL and HA-NRG1 GIEF transgenic mice exhibit significantly thicker myelin compared with wt (* P < 0.05). Bars represent mean g -ratios ± SEM ( n = 3; >100 axons/mouse). ( C ) The number of myelinated and nonmyelinated Schwann cells is not changed in HA-NRG1 GIEF transgenic mice compared with wt at 2 months of age ( n = 3; mSchwann cells P = 0.08; nmSchwann cells P = 0.18). Error bars, ±SEM. ( D ) The total number of myelinated axons in sciatic nerves from HA-NRG1 GIEF transgenic mice at 2 months of age is unaltered ( n = 3 each; P = 0.29). Error bars, ±SEM. ( E ) Electron micrographs of the corpus callosum at 2 months of age. Scatter plot (upper panel) displays g -ratios as a function of axon diameter. The average g -ratio (lower panel) in HA-NRG1 GIEF transgenic mice is significantly reduced when compared with wt (wt, n = 3; HA-NRG1 GIEF n = 4; * P < 0.05). Error bars, ±SEM. Scale bar, 0.25 μm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: The following primary antibodies were used: mouse anti-HA tag (1:250; Covance), rabbit anti-carboxy terminus of neuregulin1 (1:100; Santa Cruz sc-348), rabbit anti-neurofilament 200 (1:200; Sigma), rabbit anti-peripherin (1:500; Chemicon), mouse anti-class III β-tubulin (1:250; Covance), mouse anti-NeuN (1:100; Chemicon), rabbit anti-parvalbumin (1:200, Swant), rabbit anti-Olig2 (1:100; gift from John Alberta), and rabbit anti-Necl1 (1:1000; gift from Elior Peles).

Techniques: Transgenic Assay, Staining

Journal: Glia

Article Title: BACE1 Processing of NRG1 Type III Produces a Myelin-Inducing Signal but Is Not Essential for the Stimulation of Myelination

doi: 10.1002/glia.21255

Figure Lengend Snippet: The myelination-promoting competence of NRG1 type III is not compromised in the absence of BACE1. ( A ) Semithin sciatic nerve cross sections stained for myelin at 2 months of age. Sciatic nerves from BACE1−/− mutants are severely hypomyelinated and HA-NRG1 FL transgenic nerves are hypermyelinated compared with wt. Expression of the HA-NRG1 FL transgene in a BACE1 null background restores myelination. Scale bar, 10 μm. ( B ) Quantification of myelin sheath thickness by g-ratio analysis. HA-NRG1 FL overexpression in BACE1−/− mutants significantly increases myelin sheath thickness compared with BACE1−/− mutants (** P < 0.01) and even wt (** P < 0.01). Myelin sheath thickness in HA-NRG1 FL *BACE1−/− mutants is not significantly reduced compared with HA-NRG1 FL transgenic mice ( P = 0.35). ( n = 3 per genotype, >100 axons/mouse). The scatter plot (lower panel) illustrates that HA-NRG1 FL expression in BACE1−/− mice rescues the myelination deficit of BACE1 mutants across all axon diameters. ( C ) Myelinating Schwann cells (left panel) are increased and nonmyelinating Schwann cells (right panel) decreased in adult BACE1−/− mutants ( n = 4) compared with wt ( n = 3) at 2 months of age (mean±SEM; * P < 0.05; ** P < 0.01) ( D ) “Stalk” cleavage of NRG1 type III is reduced but not abolished in the absence of BACE1. Western blot of spinal cord protein lysates from 2 months old mice probed with an anti-HA antibody. Processed NRG1 type III (∼70 kD) is reduced, whereas full-length NRG1 type III (∼140 kD) accumulates in HA-NRG1 FL *BACE1−/− compound mutants compared with HA-NRG1 FL transgenic mice. Membrane was reprobed for tubulin as a loading control. Densitometric quantification demonstrates a significant increase in the relative signal ratio (∼140 kD/∼70 kD protein bands relative to tubulin) in HA-NRG1 FL *BACE1−/− mice ± SEM; * P < 0.05). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: The following primary antibodies were used: mouse anti-HA tag (1:250; Covance), rabbit anti-carboxy terminus of neuregulin1 (1:100; Santa Cruz sc-348), rabbit anti-neurofilament 200 (1:200; Sigma), rabbit anti-peripherin (1:500; Chemicon), mouse anti-class III β-tubulin (1:250; Covance), mouse anti-NeuN (1:100; Chemicon), rabbit anti-parvalbumin (1:200, Swant), rabbit anti-Olig2 (1:100; gift from John Alberta), and rabbit anti-Necl1 (1:1000; gift from Elior Peles).

Techniques: Staining, Transgenic Assay, Expressing, Over Expression, Western Blot, Membrane, Control