Journal: bioRxiv

Article Title: Enhanced legumain activity links progranulin deficiency to TDP-43 pathology in frontotemporal lobar degeneration

doi: 10.1101/2024.01.16.575687

Figure Lengend Snippet: a , Representative immunoblots for total LGMN and human LGMN (hLGMN) in total brain homogenate (NP40) of Grn KO mice, three months p.i. and age-matched non-injected controls, GAPDH verified equal loading. LGMN expression is quantified and normalized to Grn ko (n=3 mice per condition, mean ± s.d) b , In vitro LGMN activity assay of total brain homogenate of AAV-injected Grn ko mice and age-matched non-injected controls (n=6 mice per condition, mean ± s.d). c , Representative immunoblot of TDP-43 in the RIPA soluble fraction of total brain lysates and pTDP-43 in the urea-soluble fraction, GAPDH verified equal loading. For quantification of TDP-43 processing in the RIPA fraction, the signal of indicated TDP-43 fragments was normalized to the total TDP-43 signal (n=6 mice per condition, mean ± s.d., multiple t-tests with FDR correction). pTDP-43 accumulation in the urea fraction was quantified and normalized to Grn ko (n=6 mice per condition, mean ± s.d.), d , Representative images show assessment of hind-limb clasping phenotype in hLGMN overexpressing mice and age-matched non-injected Grn ko mice (n=6 mice per condition). The clasping phenotype was quantified (n=6 mice per condition, mean ± s.e.m.). e , Longitudinal rotarod performance of Grn ko mice overexpressing hLGMN and age-matched controls (n=6 for Grn ko + hLGMN, n=6 for Grn ko control mice, mean ± s.e.m., multiple t-tests with FDR correction). f , Plasma NfL levels were measured before injection,1.5 months p.i and terminally. (n=6, mean ± s.d.).

Article Snippet: The AAV-PhP.eB-CAG- hLGMN vector used in this study was produced under research-grade conditions at SignaGen by triple transfection of adherent HEK293T cells.

Techniques: Western Blot, Injection, Expressing, In Vitro, Activity Assay

Journal: bioRxiv

Article Title: Enhanced legumain activity links progranulin deficiency to TDP-43 pathology in frontotemporal lobar degeneration

doi: 10.1101/2024.01.16.575687

Figure Lengend Snippet: a , Immunofluorescence staining of sagittal brain sections 3 months post tail vein injection of AAV-PHP.eB.CAG-hLGMN (DAPI in blue, hLGMN in cyan, n=3 mice, scale bar = 1500 μm). b , Immunofluorescence staining for neuronal marker MAP2 (magenta), microglial marker IBA1 (orange) or astrocytic marker GFAP (yellow) and DAPI (blue). White arrowheads show co-localization of hLGMN with astrocytes, green arrowheads co-localization of hLGMN with microglia. (n=3 mice, scale bar = 20 μm).

Article Snippet: The AAV-PhP.eB-CAG- hLGMN vector used in this study was produced under research-grade conditions at SignaGen by triple transfection of adherent HEK293T cells.

Techniques: Immunofluorescence, Staining, Injection, Marker

Journal: Nature Communications

Article Title: Defects in hair cells disrupt the development of auditory peripheral circuitry

doi: 10.1038/s41467-024-55275-x

Figure Lengend Snippet: a Diagram of spiral ganglion neuron (SGN) innervation patterns of the cochlear sensory epithelium in adult mice. b Experimental strategy for viral labeling with an adeno-associated virus (AAV) encoding enhanced green fluorescent protein (EGFP) under the control of the human synapsin (hSyn) promoter (AAV9.hSyn.EGFP). c Mid-cochlear whole-mounts from P21 Tmie +/- and Tmie -/- mice after high titer viral injection. Tissue was immunolabeled for MYO7A (magenta) to visualize inner hair cells (IHCs) and outer hair cells (OHCs) and EGFP (green) to visualize projections of infected neurons; scale bar: 10 μm. d P21 mid-cochlear whole mounts after low titer viral injection. Yellow arrow: SGN projecting past IHC; white arrow: SGN with abnormal branching; arrowhead: SGN with no projection to IHC; scale bar: 10 μm. e Representative images of P11 mid-cochlear whole mounts after low titer viral injection; arrow: single SGN projecting to several IHCs; arrowheads: single SGN projecting both towards the apex and base in OHC region; Scale bar: 10 μm. f Quantified results at P11 ( +/-: IHC contact only- 82%; abnormal projection- 18%; n = 111 neurons/3 mice; -/-: IHC contact only- 34%; abnormal projection- 66%; n = 168 neurons/3 mice). g Quantified results at P21 ( +/-: IHC contact only- 97%; abnormal projection- 3% n = 92 neurons/5 mice; -/-: IHC contact only- 48%; abnormal projection- 52%; n = 22 neurons/5 mice).

Article Snippet: Control AAV (PHP.eB.CAG.EGFP, 1.14 × 10 13 GC/mL was purchased from SignaGen (SL116010).

Techniques: Labeling, Virus, Control, Injection, Immunolabeling, Infection

Journal: Nature Communications

Article Title: Defects in hair cells disrupt the development of auditory peripheral circuitry

doi: 10.1038/s41467-024-55275-x

Figure Lengend Snippet: a Diagram of experimental strategy. Adeno-associated viruses (AAV) were used to drive enhanced green fluorescent protein (EGFP) or TMIE and EGFP expression in hair cells of Tmie -/- mice. b Auditory brainstem response thresholds for click and tone stimulation in Tmie +/- (non-injected, black, n = 11) and Tmie -/- mice injected with AAV.PHP.eB.EGFP (green, n = 11) or AAV.PHP.eB.TMIE::EGFP (blue, n = 15). c Representative apex, middle, and base regions of whole mounts of cochleae at P21 following AAV.PHP.eB.TMIE::EGFP injection at P1. Tissue was stained for EGFP (green) and DAPI (magenta). Scale bar, 20 μm. d Quantification of AAV.PHP.eB.TMIE::EGFP transduction in cochlear inner hair cells (IHCs, hollow points) and outer hair cells (OHCs, filled points) as a function of distance from apex ( n = 5 Tmie -/- mice). e Representative apical-cochlear whole mounts at P21 immunolabeled for EGFP and NGFR; scale bars, left and middle panels: 10 μm, right panels: 5 μm. f NGFR + terminals near IHCs/100 μm in P21 Tmie -/- mice infected with AAV.PHP.eB.EGFP ( n = 3 mice) or AAV.PHP.eB.TMIE::EGFP ( n = 5 mice) (apex: two-sided Welch’s t test: p = 0.0003, t = 8.21, df = 5.30; middle: two-sided Welch’s t test: p = 0.034, t = 8.24, df = 3.07; base: two-sided Welch’s t test: p = 0.0426, t = 3.27, df = 3.19). g NGFR + branches to OHCs/100 μm in P21 Tmie -/- mice infected with AAV.PHP.eB.EGFP or AAV.PHP.eB.TMIE::EGFP (apex: two-sided Welch’s t test: p = 2.024*10 −4 , t = 9.56, df = 5.945; middle: two-sided Welch’s t test: p = 0.0058, t = 6.76, df = 3.12; base: two-sided Welch’s t test: p = 0.0123, t = 7.79, df = 2.19). Values are mean ± SEM.

Article Snippet: Control AAV (PHP.eB.CAG.EGFP, 1.14 × 10 13 GC/mL was purchased from SignaGen (SL116010).

Techniques: Expressing, Injection, Staining, Transduction, Immunolabeling, Infection