Journal: Scientific Reports

Article Title: Live Neuron High-Content Screening Reveals Synaptotoxic Activity in Alzheimer Mouse Model Homogenates

doi: 10.1038/s41598-020-60118-y

Figure Lengend Snippet: Validation of PSD95-mVenus postsynaptic, VAMP2-mRFP presynaptic, and colocalized puncta. The majority of anti-VAMP2 and anti-Synapsin1 stained puncta were colocalized with endogenous VAMP2-mRFP puncta. Similarly, the majority of the antibody stained anti-PSD95 and anti-Homer2 puncta were colocalized with endogenous PSD95-mVenus puncta. ( a–c ) Presynaptic VAMP2-mRPF in singly transgenic mice was validated with immunocytochemistry stained ( a ) anti-VAMP2 and ( b ) anti-Syn1, ( c ) 92.1 ± 4.8% of stained VAMP2 puncta and 80.8 ± 3.9% of stained Syn1 puncta was colocalized with endogenous VAMP2-mRFP; ( d–f ) Postsynaptic PSD95-mVenus expression in singly transgenic mice was validated with immunocytochemistry stained ( d ) anti-PSD95 and ( e ) anti-Homer2, ( f ) 107.5 ± 4.5% of stained PSD95 puncta and 113.7 ± 6.2% of stained Homer2 puncta was colocalized with endogenous PSD95-mVenus (mean ± SD%, n = 3).

Article Snippet: All cells were blocked with 5% bovine serum albumin and 0.2% Triton X-100 (Sigma-Aldrich) in PBS for 1 hour at room temperature, and then incubated with anti-Synaptobrevin 2, anti-Synapsin1, anti-PSD95, or anti-Homer2 (Synaptic Systems) antibodies at 1:500 in blocking solution overnight at 4 °C, subsequently washed in 0.1% Triton X-100 in PBS three times.

Techniques: Staining, Transgenic Assay, Immunocytochemistry, Expressing

Journal: PLoS Genetics

Article Title: HOMER2, a Stereociliary Scaffolding Protein, Is Essential for Normal Hearing in Humans and Mice

doi: 10.1371/journal.pgen.1005137

Figure Lengend Snippet: (A) The pedigree of a five-generation family segregating progressive ADNSHL. DNA samples were obtained for 9 unaffected and 10 affected individuals. The partially filled symbol represents a two-year-old female who carries the HOMER2 mutation but in whom a formal ABR has not been performed. Individuals who underwent TGE+MPS using either the deafness panel (OtoSCOPE) or WES are marked with an O or W, respectively. Genotypes of participating family members are shown below each symbol in single letter amino acid nomenclature: P for Proline and R for Arginine. ( B) Age-related typical audiograms (ARTA). Binaural mean air conduction thresholds (dB HL) are presented for the ages 10–70 years. Hearing levels ranged from 0 to 120 dB depending on age and frequency; the annual threshold deterioration (ATD) was 1.2–1.6 dB/year at all frequencies. ( C) Representative chromatograms from wild-type and mutant sequences. ( D) Diagram of HOMER2 structure; the amino acid numbering indicates the beginning and end of the EVH1 and CC domains. The CC includes the CDC42 binding domain (CBD), Leucine Zipper-A (LZA) and Leucine Zipper-B (LZB). The position of the p.Arg185Pro mutation is shown in red.

Article Snippet: Following infiltration using 0.3% Triton X-100 and blocking with 5% normal goat serum, we incubated the tissues in rabbit HOMER2 polyclonal primary antibody (NB100-98712, Novus Biologicals, Littleton, CO) diluted 1:1000 in PBS overnight at 4°C.

Techniques: Mutagenesis, Binding Assay

Journal: PLoS Genetics

Article Title: HOMER2, a Stereociliary Scaffolding Protein, Is Essential for Normal Hearing in Humans and Mice

doi: 10.1371/journal.pgen.1005137

Figure Lengend Snippet: (A ) Staining with F-actin shows three rows of OHCs and one row of IHCs in the cochlea. ( B ) Homer2 staining in the OHCs and IHCs shows localization to stereocilia. ( C ) Merged pictures showing co-localization of Homer2 with F-actin in HC stereocilia. ( D ) Zoomed view of OHCs shows pronounced localization of Homer2 to the tips of stereocilia. Scale bar represents 10μm.

Article Snippet: Following infiltration using 0.3% Triton X-100 and blocking with 5% normal goat serum, we incubated the tissues in rabbit HOMER2 polyclonal primary antibody (NB100-98712, Novus Biologicals, Littleton, CO) diluted 1:1000 in PBS overnight at 4°C.

Techniques: Staining

Journal: PLoS Genetics

Article Title: HOMER2, a Stereociliary Scaffolding Protein, Is Essential for Normal Hearing in Humans and Mice

doi: 10.1371/journal.pgen.1005137

Figure Lengend Snippet: (A-D) Auditory brainstem responses (ABR) to broad band clicks (A) and tone bursts (B-D) at 8, 16, and 32 kHz in P14, P28 and P56 Homer2 -/- , Homer2 +/- and WT mice. (A) Raised mean ABR thresholds (dB SPL) were detected as early as P14 in Homer2 -/- mice and continued to increase with age. (B) At P14 Homer2 -/- mice had severe-to-profound hearing loss at high frequencies (16 kHz and 32 kHz) whereas hearing thresholds in their WT and Homer2 +/- littermates were in the normal range. (C) At P28 Homer2 -/- mice had hearing loss in the lower frequencies. (D) P56 Homer2 -/- mice had profound hearing loss across all tested frequencies (click, 8 kHz, 16 kHz and 32 kHz). (E-G) DPOAE levels. (E) In P14 Homer2 -/- mice, DPOAE amplitudes are significantly lower in the high frequencies (16, 22.6, and 32.0 kHz) as compared to their WT and Homer2 +/- littermates. (F) In P28 Homer2 -/- mice, DPOAE amplitudes are lower in nearly all frequencies (5.7, 8, 11.3, 16, 22.6, and 32.0 kHz). (G) In P56 Homer2 -/- mice, DPOAEs deteriorate across all frequencies consistent with profound hearing loss. (H) Representative Alexa-Fluor-488-phalloidin immunofluorescence shows no obvious hair cell death in cochleae in P56 Homer2 -/- (n = 5), Homer2 +/- (n = 4) and WT (n = 3) mice. Mean ABR thresholds and DPOAE amplitudes: Homer2 -/- mice are shown in red; Homer2 +/- in blue; and WT mice in green. The number of ears tested is shown in parentheses. P-values were calculated with One-way ANOVA and post-hoc T-test analysis. Asterisks indicate statistical significance: *P<0.05, **P<0.005, ***P<0.0005. Error bars represent SEM. Scale bar represents 10μm.

Article Snippet: Following infiltration using 0.3% Triton X-100 and blocking with 5% normal goat serum, we incubated the tissues in rabbit HOMER2 polyclonal primary antibody (NB100-98712, Novus Biologicals, Littleton, CO) diluted 1:1000 in PBS overnight at 4°C.

Techniques: Immunofluorescence

Journal: Nature neuroscience

Article Title: Synaptic depression via mGluR1 positive allosteric modulation suppresses cue-induced cocaine craving

doi: 10.1038/nn.3590

Figure Lengend Snippet: (a) Co-immunoprecipitation (co-IP) experiments assessing the physical associations between mGluR1 and Homer proteins on withdrawal day (WD) 14, 25 or 48 from extended-access cocaine (coc) or saline (sal) self-administration (see timeline in ). No significant changes in association between mGluR1 and Homer proteins were observed at any of the 3 withdrawal time-points. ( b ) In the case of mGluR5, no change in association with Homer proteins was observed at the two earlier withdrawal time-points (WD14 and WD25). However, a significant decrease in association between mGluR5 and both Homer isoforms was found on WD48 in animals that previously self-administered cocaine. Data are expressed as percent of Saline group (± s.e.m.) at each time-point (n values are provided within each bar). **p=0.01 (Homer1bc) and *p=0.04 (Homer2) versus respective Saline groups. Full-length blots are presented in .

Article Snippet: Validation is provided on the supplier’s website for all antibodies utilized, as well as on Antibodypedia for Homer1bc (Santa Cruz) and Homer2 (Abnova).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Saline

Journal: Nature neuroscience

Article Title: Synaptic depression via mGluR1 positive allosteric modulation suppresses cue-induced cocaine craving

doi: 10.1038/nn.3590

Figure Lengend Snippet: ( a ) Timeline (SA, self-administration). To assess the effects of Homer overexpression on incubation of cocaine craving, viruses (AAV-GFP, Homer1c or Homer2) were injected into the NAc core at the same time that animals underwent jugular catheterization surgery, allowing Homer expression to peak during early withdrawal. ( b ) Staining for the HA tag on cDNA-Homer1c verified localized overexpression in the NAc core (20X magnification; image taken ~3 months after virus injection; scale bar, 100μm; ac, anterior commissure). ( c ) Following ~1 week of recovery from surgeries, animals self-administered cocaine (0.5 mg/kg) for 6 h/day for 10 days. No difference in cocaine intake (average infusions obtained each day ± s.e.m.) was observed between the 3 groups. ( d ) All animals showed an increase in cue-induced cocaine-seeking on WD48 compared to WD1 (i.e., incubation), regardless of which virus infusion they received. ***p<0.001, **p=0.01, WD1 versus WD48 (ANOVA followed by least significant difference post-hoc comparisons); GFP, n=10 rats; Homer1c, n=8 rats; Homer2, n=10 rats.

Article Snippet: Validation is provided on the supplier’s website for all antibodies utilized, as well as on Antibodypedia for Homer1bc (Santa Cruz) and Homer2 (Abnova).

Techniques: Over Expression, Incubation, Injection, Expressing, Staining, Virus

Journal: Molecular Brain

Article Title: Optimization of protocols for pre-embedding immunogold electron microscopy of neurons in cell cultures and brains

doi: 10.1186/s13041-021-00799-2

Figure Lengend Snippet: Comparison on structural preservation upon different fixation conditions and subsequent treatments. Images show synaptic profiles from 3 week-old dissociated hippocampal cultures without ( a , b ) or with ( c , d ) immunogold labeling. Clusters of synaptic vesicles (SV in a ) and the postsynaptic density (PSD, edges of which marked with arrows in a ) are characteristics of glutamatergic asymmetric synapses. The fixation treatments were 4% PF in PBS for 45 min ( a ), 4% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.4 for 30 min ( b ), 4% PF in PBS for 30 min and labeled for CaMKII ( c ), a cytosolic protein in neurons , and 2% PF in PBS for 15 min and labeled for IRSp53 ( d ), an actin-associated protein enriched at the PSD . All fixations were carried out at room temperature. Membranes were poorly preserved in ( d ) due to the lower concentration of PF and the shorter fixation time. Scale bars = 100 nm

Article Snippet: Homer 2 , PSD , Rabbit pAb , Synaptic Systems.

Techniques: Preserving, Labeling, Concentration Assay