Journal: Cellular and Molecular Neurobiology

Article Title: Enhanced Spine Stability and Survival Lead to Increases in Dendritic Spine Density as an Early Response to Local Alpha-Synuclein Overexpression in Mouse Prefrontal Cortex

doi: 10.1007/s10571-024-01472-7

Figure Lengend Snippet: Increased dendritic spine density in response to alpha-Synuclein overexpression occurs in pSer129-negative neurons within the injection location. A Mice expressing Green fluorescent protein (GFP) in Layer V neurons (Thy1-GFP) mice were injected with control vector (AAV-mCherry) in one hemisphere and vector for α-Syn overexpression (OE): (AAV-α-Syn-IRES-mCherry) in the contralateral hemisphere for within animal comparison. B Spines from basilar dendrites 6 weeks post-injection were quantified via confocal microscopy. Sections were immunostained with antibodies against GFP and pSer129-α-Syn and dendritic spines were counted. C Basilar dendrites were analyzed in bins based on distance from the soma and grouped according to the hemisphere treatment (control or α-Syn OE). For the α-Syn OE hemisphere, cells were further divided into pSer129-α-Syn positive or negative. pSer129-α-Syn-negative dendrites had greater spine density than both mCherry-negative dendrites on the control hemisphere and pSer129-α-Syn-positive dendrites from the same hemisphere (Mixed-effects model, Main effect: treatment: F(2,63) = 3.397, p = 0.0397). Individual comparisons were corrected via Tukey: * p = 0.0106, *** p = 0.0010

Article Snippet: The Thy1-GFP mice ( n = 4) received bilateral injections with control virus into one hemisphere and α-Synuclein overexpression into the other hemisphere: Control: AAV6-CAG-mCherry-WPRE (1.7 × 10 12 gc/mL, Vector Biolabs, 1 μL, left hemisphere) and overexpression: vector carrying cDNA coding for human, wild-type α-Synuclein: AAV6-CAG-hSNCA-IRES-mCherry-WPRE (1.0 × 10 12 gc/mL, VectorBiolabs, 1 μL right hemisphere).

Techniques: Over Expression, Injection, Expressing, Plasmid Preparation, Comparison, Confocal Microscopy

Journal: bioRxiv

Article Title: Increased CD33 levels tune activation and function of induced human microglial cells through inhibition of the TREM2 pathway

doi: 10.64898/2026.01.28.701050

Figure Lengend Snippet: (A) Schematic of AAV6-mediated transduction of iMG. Cells were transduced with an MOI of 125000 at the time of plating. On day 3, cells were incubated with 2 μg/mL of pHrodo-labeled oligomerized amyloid (1-42) and imaged over a 20-hour window. (B) Protein lysates isolated from iMG 72 hours after transduction were used to quantify percent CD33 protein expression by MSD. Data are normalized to no AAV6 transduction control. (C) Confocal fluorescence images of iMG assessed for internalization of pHrodo-oligomerized amyloid beta treated as in (A). Digital phase contrast was used to visualize iMG. Images are stitched from four 20X images. Scale bar represents 400 μm. (D) Normalized intensity of internalized pHrodo-oligomerized amyloid beta in iMG transduced with or without AAV6-CD33 or AAV6-mCherry per hour. Data are normalized to no AAV6 transduction control. (E) Normalized intensity of internalized pHrodo-oligomerized amyloid beta in iMG transduced with or without AAV6-CD33 or AAV6-mCherry at endpoint. Data are normalized to no AAV6 transduction control. (F) TREM2 secretion in conditioned media was quantified from iMG 72 hours after transduction by MSD. (G)(left) Percent change in TREM2 secretion in conditioned media was quantified from HMC3 cells 48 hours after transduction with increasing AAV6-CD33 concentrations by MSD. Data are represented as a percent change relative to dose-matched AAV6-mCherry. (right) TREM2 secretion in conditioned media was quantified from HMC3 cells 48 hours after transduction of AAV6-CD33 by MSD. (MOI = 250000). (H) Protein lysates isolated from HMC3 cells 48 hours after transduction were used to quantify percent full length TREM2 protein expression by MSD. (MOI = 250000). Data represent mean ± SEM using one-way ANOVA with post-hoc Tukey’s multiple comparisons test (B), with uncorrected Fisher’s LSD (E), with post-hoc Dunnett multiple comparisons test (F, G) and one-tailed unpaired t-test (H). Data points (n-numbers) are plotted on each bar graph and scatter plot each representing an independent experimental replicate. Line plots represent three independent experimental replicates (D, G).

Article Snippet: AAV6-mCherry was obtained from Charles River Laboratories.

Techniques: Transduction, Incubation, Labeling, Isolation, Expressing, Control, Fluorescence, One-tailed Test

Journal: bioRxiv

Article Title: Increased CD33 levels tune activation and function of induced human microglial cells through inhibition of the TREM2 pathway

doi: 10.64898/2026.01.28.701050

Figure Lengend Snippet: (A) Schematic of AAV6-mediated transduction of iMG. Cells were transduced with an MOI of 125000 at the time of plating. On day 1, cells were transfected with a di-siRNA targeting CD33 for 48 hours. On day 3, cells were incubated with 2 μg/mL of pHrodo-labeled oligomerized amyloid (1-42) and imaged over a 20-hour window. (B) Confocal fluorescence images of iMG assessed for internalization of pHrodo-oligomerized amyloid beta treated as in (A). Digital phase contrast was used to visualize iMG. Images are stitched from four 20X images. Scale bar represents 400 μm. (C) Normalized intensity of internalized pHrodo-oligomerized amyloid beta in iMG transduced with or without AAV6-CD33 or AAV6-mCherry per hour after transfection of increasing concentrations of CD33-targeting di-siRNA per hour. Data are normalized to mock transfected no AAV6 transduction control. (D) Normalized intensity of internalized pHrodo-oligomerized amyloid beta in iMG transduced with or without AAV6-CD33 or AAV6-mCherry per hour after transfection of increasing concentrations of CD33-targeting di-siRNA at endpoint. Data are normalized to mock transfected no AAV6 transduction control. (E) Protein lysates isolated from iMG 72 hours after transduction and 48 hours after transfection were used to quantify percent CD33 protein expression by MSD. Data are normalized to mock transfected AAV6-CD33-treated iMG. (F) Linear regression of data from (D) and (E) was plotted to demonstrate the correlation between percent CD33 expression levels and oligomerized amyloid-beta uptake. Data represent mean ± SEM using one-way ANOVA with uncorrected Fisher’s LSD (D) and Dunnett’s multiple comparison test (E). Data points (numbers) are plotted on each bar graph and scatter plot, each representing an independent experimental replicate. Line plots are representative of three independent experimental replicates (C, F).

Article Snippet: AAV6-mCherry was obtained from Charles River Laboratories.

Techniques: Transduction, Transfection, Incubation, Labeling, Fluorescence, Control, Isolation, Expressing, Comparison

Journal: Cellular and Molecular Neurobiology

Article Title: Enhanced Spine Stability and Survival Lead to Increases in Dendritic Spine Density as an Early Response to Local Alpha-Synuclein Overexpression in Mouse Prefrontal Cortex

doi: 10.1007/s10571-024-01472-7

Figure Lengend Snippet: Alpha-Synuclein overexpression causes increases in dendritic spine density on individual dendrites. A 2-photon imaging was performed 3 weeks after cranial window surgery to generate 2 weeks of pre-treatment imaging of dendrites (week -1 and week 0, followed by injection of AAV-mCherry (control) or AAV-mCherry/α-Syn (α-Syn OE) immediately after the week 0 imaging session. Nine weeks of 2-photon microscopy was performed on the same pool of dendrites that were established in weeks − 1 and 0. B Representative, single plane images of control (top) and α-Syn OE (bottom) dendrites. Purple arrowheads represent new spines; red arrow is a lost spine, each identified by comparison of the full 3D stacks, not visible here. C Dendritic spine density increases in α-Syn OE animals compared with control. Each dendrite was divided by its pre-treatment density to evaluate the effect on individual dendrites. The orange and blue colored numbers represent the number of dendrites measured for each group at each week from n = 6 control, n = 8 α-Syn OE; dendrites were excluded at some weeks due to changes in the cranial window transparency or angle, equipment malfunction, and the covid pandemic. α-Syn OE led to increased dendritic spine density starting at week 5 and continuing for the duration of imaging (* p < 0.05)

Article Snippet: For Thy1-YFP mice, we used the following viral treatments: Control: AAV6-CAG-mCherry-WPRE (1.7 × 10^12 gc/mL, Vector Biolabs, unilateral); α-Syn overexpression: vector carrying cDNA coding for human, wild-type α-Synuclein: AAV6-CAG-hSNCA-WPRE (1.0 × 10 13 gc/mL, VectorBiolabs) was mixed with the mCherry virus outlined above.

Techniques: Over Expression, Imaging, Injection, Microscopy, Comparison

Journal: Cellular and Molecular Neurobiology

Article Title: Enhanced Spine Stability and Survival Lead to Increases in Dendritic Spine Density as an Early Response to Local Alpha-Synuclein Overexpression in Mouse Prefrontal Cortex

doi: 10.1007/s10571-024-01472-7

Figure Lengend Snippet: Experimental design for overexpression of human alpha-Synuclein in the mouse PFC. A We placed round cranial windows over the putative imaging site. AAV injections were performed after two imaging sessions, through a pre-drilled hole plugged with silicon. Injection sites for mice in the study are shown as yellow circles, with the lines (blue = control, green = α-Syn OE) representing the angle of injection. The colored boxes (blue = control, green = α-Syn OE) represent the imaging sites of apical tufts. B Representative live image from a highly transduced area using 2-photon. C – D Example of the localized spread of transduction in a Thy1-YFP coronal section, showing AAV-induced mCherry expression in a section from control ( C ) and α-Syn OE ( D ), localized to the mPFC (M2, cingulate) region. E Overexpression of human-α-Syn leads to robust phosphorylation at Ser129 in treated mice, including in soma ( F ) and some dendrites ( G ). E ; Tiled confocal image, scale bar 100um. F single confocal slice, 40 × objective, scale bar 10um. G Max projection of 3-confocal slices, 0.33um step size

Article Snippet: For Thy1-YFP mice, we used the following viral treatments: Control: AAV6-CAG-mCherry-WPRE (1.7 × 10^12 gc/mL, Vector Biolabs, unilateral); α-Syn overexpression: vector carrying cDNA coding for human, wild-type α-Synuclein: AAV6-CAG-hSNCA-WPRE (1.0 × 10 13 gc/mL, VectorBiolabs) was mixed with the mCherry virus outlined above.

Techniques: Over Expression, Imaging, Injection, Transduction, Expressing

Journal: Cellular and Molecular Neurobiology

Article Title: Enhanced Spine Stability and Survival Lead to Increases in Dendritic Spine Density as an Early Response to Local Alpha-Synuclein Overexpression in Mouse Prefrontal Cortex

doi: 10.1007/s10571-024-01472-7

Figure Lengend Snippet: Increased dendritic spine density in response to alpha-Synuclein overexpression occurs in pSer129-negative neurons within the injection location. A Mice expressing Green fluorescent protein (GFP) in Layer V neurons (Thy1-GFP) mice were injected with control vector (AAV-mCherry) in one hemisphere and vector for α-Syn overexpression (OE): (AAV-α-Syn-IRES-mCherry) in the contralateral hemisphere for within animal comparison. B Spines from basilar dendrites 6 weeks post-injection were quantified via confocal microscopy. Sections were immunostained with antibodies against GFP and pSer129-α-Syn and dendritic spines were counted. C Basilar dendrites were analyzed in bins based on distance from the soma and grouped according to the hemisphere treatment (control or α-Syn OE). For the α-Syn OE hemisphere, cells were further divided into pSer129-α-Syn positive or negative. pSer129-α-Syn-negative dendrites had greater spine density than both mCherry-negative dendrites on the control hemisphere and pSer129-α-Syn-positive dendrites from the same hemisphere (Mixed-effects model, Main effect: treatment: F(2,63) = 3.397, p = 0.0397). Individual comparisons were corrected via Tukey: * p = 0.0106, *** p = 0.0010

Article Snippet: For Thy1-YFP mice, we used the following viral treatments: Control: AAV6-CAG-mCherry-WPRE (1.7 × 10^12 gc/mL, Vector Biolabs, unilateral); α-Syn overexpression: vector carrying cDNA coding for human, wild-type α-Synuclein: AAV6-CAG-hSNCA-WPRE (1.0 × 10 13 gc/mL, VectorBiolabs) was mixed with the mCherry virus outlined above.

Techniques: Over Expression, Injection, Expressing, Plasmid Preparation, Comparison, Confocal Microscopy