The upper scheme depicts the experimental design . Representative microphotographs show that injection of LPS primed MSCs into wounds increased the expression of CXCL6 , IL - 8 and IL - 1β . Graphs display numbers of double positive cells for h - β2M + CXCL6 , h - β2M + IL - 8 and h - β2M + IL - 1β , respectively . MSCs injected wounds served as controls . Statistical analysis was performed using unpaired t - test , values are represented as mean ± SEM , six biological replicates . To facilitate comparison , areas inside the rectangles are shown at 5x magnification in the insets . Es , eschar on wound margin ; wm , wound margin ; scale bars : 50µm .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The upper scheme depicts the experimental design. Representative microphotographs show that injection of LPS primed MSCs into wounds increased the expression of CXCL6, IL-8 and IL-1β. Graphs display numbers of double positive cells for h-β2M + CXCL6, h-β2M + IL-8 and h-β2M + IL-1β, respectively. MSCs injected wounds served as controls. Statistical analysis was performed using unpaired t-test, values are represented as mean ± SEM, six biological replicates. To facilitate comparison, areas inside the rectangles are shown at 5x magnification in the insets. Es, eschar on wound margin; wm, wound margin; scale bars: 50µm.


Results show that LPS primed MSCs injected into wounds provoke increased expression of MIP / KC in endogenous neutrophils . MIP / KC represents functional homologues of IL - 8 in mice and are known as neutrophil chemoattractant . Similar results were found for IL - 1β , which is a strong chemoattractant for neutrophil recruitment and bacterial clearance . In addition , LIX which shares 63 % amino acid sequence identity with human GCP - 2 / CXCL6 , a chemoattractant for neutrophils . The graphs display numbers of double positive cells for Ly6G MIP / KC , IL - 1β and LIX , respectively . PBS and non - primed MSCs injected wounds served as controls . To facilitate comparison , areas inside the rectangles are shown at 5x magnification in the insets . Statistical analysis was performed using one way ANOVA , values are represented as mean ± SEM , six biological replicates . Scale bars : 50µm .

O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Results show that LPS primed MSCs injected into wounds provoke increased expression of MIP/KC in endogenous neutrophils. MIP/KC represents functional homologues of IL-8 in mice and are known as neutrophil chemoattractant. Similar results were found for IL-1β, which is a strong chemoattractant for neutrophil recruitment and bacterial clearance. In addition, LIX which shares 63% amino acid sequence identity with human GCP-2/CXCL6, a chemoattractant for neutrophils. The graphs display numbers of double positive cells for Ly6G MIP/KC, IL-1β and LIX, respectively. PBS and non-primed MSCs injected wounds served as controls. To facilitate comparison, areas inside the rectangles are shown at 5x magnification in the insets. Statistical analysis was performed using one way ANOVA, values are represented as mean ± SEM, six biological replicates. Scale bars: 50µm.


Representative microphotographs of TLR4 silenced and non - silenced MSCs primed with LPS and co - cultured with neutrophils . Immunostaining was performed with an antibody detecting NETs ( DNA - histone , red ) . Of note , TLR4 silenced MSCs upon LPS priming cannot mount any adaptive response and fail to enhance NET formation ( lower row , outer right panel ) as compared to non - silenced , LPS primed MSCs co - cultured with PMA activated neutrophils ( lower row , outer left panel ) . Neutrophils and DMSO served as a negative control ( upper row , outer left panel ) , while neutrophils activated by the protein C kinase activator PMA served as a positive control depicting enhanced NET formation ( upper row , middle panel ) . Co - culture with MSCs suppressed enhanced NET formation ( upper row , outer left panel ) induced by PMA . Scale bars : 50μm .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O

Representative microphotographs of TLR4 silenced and non-silenced MSCs primed with LPS and co-cultured with neutrophils. Immunostaining was performed with an antibody detecting NETs (DNA-histone, red). Of note, TLR4 silenced MSCs upon LPS priming cannot mount any adaptive response and fail to enhance NET formation (lower row, outer right panel) as compared to non-silenced, LPS primed MSCs co-cultured with PMA activated neutrophils (lower row, outer left panel). Neutrophils and DMSO served as a negative control (upper row, outer left panel), while neutrophils activated by the protein C kinase activator PMA served as a positive control depicting enhanced NET formation (upper row, middle panel). Co-culture with MSCs suppressed enhanced NET formation (upper row, outer left panel) induced by PMA. Scale bars: 50μm.


Quantitative assessment of NET bound elastase employing a specific ELISA in identical experimental setting as described in Figure 1 . Similarly , to immunostaining , a significant reduction of NET bound elastase was observed in TLR4 silenced LPS primed MSCs when co - cultured with activated neutrophils as opposed to high NET bound elastase in non - silenced LPS primed MSCs co - cultured with activated neutrophils . Statistical analysis was performed using one way ANOVA , values are represented as mean ± SEM , three biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantitative assessment of NET bound elastase employing a specific ELISA in identical experimental setting as described in Figure 1. Similarly, to immunostaining, a significant reduction of NET bound elastase was observed in TLR4 silenced LPS primed MSCs when co-cultured with activated neutrophils as opposed to high NET bound elastase in non-silenced LPS primed MSCs co-cultured with activated neutrophils. Statistical analysis was performed using one way ANOVA, values are represented as mean ± SEM, three biological replicates.


High resolution scanning electron microscope analysis depicting enhanced NET formation ( red arrows ) expulsed from neutrophils ( blue arrows ) in the presence of LPS primed MSC co - cultured with PMA activated neutrophils ( middle row , outer left panel ) as compared to reduced NETs in non - primed MSCs co - cultured with activated neutrophils ( middle row , outer right panel ) . Of note , TLR4 silenced LPS primed MSCs failed to activate neutrophils and NETs ( lower row , outer left panel ) . TLR4 silenced and non - primed MSCs display reduced NETs in co - cultures with activated neutrophils ( lower row , outer right panel ) , suggesting that PMA activation shares TLR4 signaling components . PMA activated neutrophils alone served as positive control with highly enhanced NETs ( upper row , outer right panel ) and DMSO treated neutrophils as negative controls ( upper row , outer left panel ) . Red stars indicate neutrophil derived granules . Scale bars : 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

High resolution scanning electron microscope analysis depicting enhanced NET formation (red arrows) expulsed from neutrophils (blue arrows) in the presence of LPS primed MSC co-cultured with PMA activated neutrophils (middle row, outer left panel) as compared to reduced NETs in non-primed MSCs co-cultured with activated neutrophils (middle row, outer right panel). Of note, TLR4 silenced LPS primed MSCs failed to activate neutrophils and NETs (lower row, outer left panel). TLR4 silenced and non-primed MSCs display reduced NETs in co-cultures with activated neutrophils (lower row, outer right panel), suggesting that PMA activation shares TLR4 signaling components. PMA activated neutrophils alone served as positive control with highly enhanced NETs (upper row, outer right panel) and DMSO treated neutrophils as negative controls (upper row, outer left panel). Red stars indicate neutrophil derived granules. Scale bars: 0.1µm


Representative clinical pictures of murine wounds at 0 , 3 , 5 , 7 and 10 days after wounding . Enhanced wound healing in LPS primed MSCs group as opposed to all other groups . Statistical analysis of 20 wound areas per group at the indicated time points , expressed as percentage of the initial wound size ( day 0 ) , for PBS control , non - primed MSCs , LPS primed MSCs , non - primed scrambled siRNA - treated ( Scr ) MSCs , LPS primed Scr MSCs and LPS primed TLR4 silenced MSCs . Results are mean ± SD of five biological replicates representing 1 of 3 independent experiments . Statistical analysis was performed using one way ANOVA .

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative clinical pictures of murine wounds at 0, 3, 5, 7 and 10 days after wounding. Enhanced wound healing in LPS primed MSCs group as opposed to all other groups. Statistical analysis of 20 wound areas per group at the indicated time points, expressed as percentage of the initial wound size (day 0), for PBS control, non-primed MSCs, LPS primed MSCs, non-primed scrambled siRNA-treated (Scr) MSCs, LPS primed Scr MSCs and LPS primed TLR4 silenced MSCs. Results are mean ± SD of five biological replicates representing 1 of 3 independent experiments. Statistical analysis was performed using one way ANOVA.


Representative photomicrographs of confocal microscopy of sections from differently injected day 1 wounds stained for Ly6G + neutrophils ( green ) and F4 / 80 + macrophages ( red ) . Nuclei are stained with DAPI ( blue ) . Double staining was performed for sections of day 1 wounds injected with PBS ( control ) , non - primed MSCs , LPS primed MSCs and LPS primed TLR4 - silenced MSCs Double stained cells indicate phagocytic engulfment of neutrophils by macrophages . To facilitate comparison , areas inside the rectangles are shown at 5x magnification in the insets . Scale bar : 100µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative photomicrographs of confocal microscopy of sections from differently injected day 1 wounds stained for Ly6G+ neutrophils (green) and F4/80+ macrophages (red). Nuclei are stained with DAPI (blue). Double staining was performed for sections of day 1 wounds injected with PBS (control), non-primed MSCs, LPS primed MSCs and LPS primed TLR4-silenced MSCs Double stained cells indicate phagocytic engulfment of neutrophils by macrophages. To facilitate comparison, areas inside the rectangles are shown at 5x magnification in the insets. Scale bar: 100µm.


Quantification of Ly6G and F4 / 80 double positive cells on sections of differently injected day 1 wounds . Wounds were injected as described in ( a ) . Double positive cells were counted and statistical analysis was performed using one way ANOVA , values are represented as mean ± SEM , six biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of Ly6G and F4/80 double positive cells on sections of differently injected day 1 wounds. Wounds were injected as described in (a). Double positive cells were counted and statistical analysis was performed using one way ANOVA, values are represented as mean ± SEM, six biological replicates.


Quantification of Ly6G + neutrophils and F4 / 80 + macrophages on sections of differently injected day 1 wounds . Wounds were injected as described in ( a ) . Single positive cells were counted and statistical analysis was performed using one way ANOVA , values are represented as mean ± SEM , six biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of Ly6G+ neutrophils and F4/80+ macrophages on sections of differently injected day 1 wounds. Wounds were injected as described in (a). Single positive cells were counted and statistical analysis was performed using one way ANOVA, values are represented as mean ± SEM, six biological replicates.


Representative photomicrographs of confocal microscopy of sections from differently injected day 3 wounds double - stained for TGFβ - 1 ( red ) and for F4 / 80 + macrophages ( green ) . Nuclei are stained with DAPI ( blue ) . Double staining was performed for sections of day 5 wounds injected with PBS ( control ) , non - primed MSCs , LPS primed MSCs and LPS primed TLR4 - silenced MSCs . Scale bar : 50µm . Representative photomicrographs of sections of day 5 wounds immunostained for CD31 ( indicative of endothelial cells and newly formed vessels ) and for α - SMA ( indicative of myofibroblasts differentiation ) after injection of LPS primed MSCs , non - primed MSCs , LPS primed TLR4 - silenced MSCs or PBS ( middle and lower panel ) . To facilitate comparison , areas inside the rectangles are shown at 5x magnification in the insets . Scale bars : 50µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative photomicrographs of confocal microscopy of sections from differently injected day 3 wounds double-stained for TGFβ-1 (red) and for F4/80+ macrophages (green). Nuclei are stained with DAPI (blue). Double staining was performed for sections of day 5 wounds injected with PBS (control), non-primed MSCs, LPS primed MSCs and LPS primed TLR4-silenced MSCs. Scale bar: 50µm. Representative photomicrographs of sections of day 5 wounds immunostained for CD31 (indicative of endothelial cells and newly formed vessels) and for α-SMA (indicative of myofibroblasts differentiation) after injection of LPS primed MSCs, non-primed MSCs, LPS primed TLR4-silenced MSCs or PBS (middle and lower panel). To facilitate comparison, areas inside the rectangles are shown at 5x magnification in the insets. Scale bars: 50µm.


Double positive macrophages stained for TGFβ - 1 and F4 / 80 were counted and statistical analysis was performed using one way ANOVA , values are represented as mean ± SEM , six biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Double positive macrophages stained for TGFβ-1 and F4/80 were counted and statistical analysis was performed using one way ANOVA, values are represented as mean ± SEM, six biological replicates.


Quantitative analysis of CD31 positive endothelial cells in sections of wounds injected with PBS , non - primed MSCs , LPS primed MSCs and LPS primed TLR4 silenced LPS Cell counting was performed on immunostained wound sections and statistical analysis was performed using one way ANOVA , values are represented as mean ± SEM , six biological replicates .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantitative analysis of CD31 positive endothelial cells in sections of wounds injected with PBS, non-primed MSCs, LPS primed MSCs and LPS primed TLR4 silenced LPS Cell counting was performed on immunostained wound sections and statistical analysis was performed using one way ANOVA, values are represented as mean ± SEM, six biological replicates.


Western blot analysis of lysates from day 5 wounds ( left panel ) and the corresponding densitometric analysis ( right panel ) depicts enhanced α - SMA protein expression in wounds injected with LPS primed MSCs as opposed to the respective control groups . Actin served as loading control . Statistical analysis was performed using one way ANOVA , values are represented as mean ± SEM , three biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Western blot analysis of lysates from day 5 wounds (left panel) and the corresponding densitometric analysis (right panel) depicts enhanced α-SMA protein expression in wounds injected with LPS primed MSCs as opposed to the respective control groups. Actin served as loading control. Statistical analysis was performed using one way ANOVA, values are represented as mean ± SEM, three biological replicates.


( a - d ) Onset of rod death visualized by staining for cleaved nuclear envelope protein LaminA ( a ) , cleaved caspase3 ( arrowheads , magenta and red signal , b ) and TUNEL ( arrows , brown signal , c , d ) . Blue in a and b shows nuclear DAPI staining . A retinal flat mount with a view onto the photoreceptor layer is shown in d .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a-d) Onset of rod death visualized by staining for cleaved nuclear envelope protein LaminA (a), cleaved caspase3 (arrowheads, magenta and red signal, b) and TUNEL (arrows, brown signal, c, d). Blue in a and b shows nuclear DAPI staining. A retinal flat mount with a view onto the photoreceptor layer is shown in d.


( e - h ) Progression of rod death determined by the reduction of the ONL was visualized by hematoxylin and eosin staining .

O O O O O O O O O O O O O O O O O O O O O O O O

(e-h) Progression of rod death determined by the reduction of the ONL was visualized by hematoxylin and eosin staining.


( i - q ) End phase of rod death was assessed by section analysis ( i - l ) or by retinal flat mounts ( m - q ) . In the Rho − / − mouse , the onset of rod death was around PW5 ( a ) and progressed up to PW25 ( l ) . By PW17 , the ONL was reduced to one row of cells ( h , j ) and the remaining rods died in the following 8 weeks ( j - q ) , as seen by immunofluorescence with an antibody to Gnat1 on sections of progressively older animals ( j - l ) . Retinal flat mounts showing rods visualized by immunofluorescence microscopy with an antibody Gnat1 are shown in m - q . The entire retina is shown in m , higher magnifications around the optic nerve head are shown in n and o , and the peripheral region is shown in p . No signal was seen at PW25 on flat mounts ( q ) or sections ( l ) . Age ( in PW ) is indicated in the panels . Vertical bar in a - c and e - l indicates thickness of the ONL .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(i-q) End phase of rod death was assessed by section analysis (i-l) or by retinal flat mounts (m-q). In the Rho−/− mouse, the onset of rod death was around PW5 (a) and progressed up to PW25 (l). By PW17, the ONL was reduced to one row of cells (h, j) and the remaining rods died in the following 8 weeks (j-q), as seen by immunofluorescence with an antibody to Gnat1 on sections of progressively older animals (j-l). Retinal flat mounts showing rods visualized by immunofluorescence microscopy with an antibody Gnat1 are shown in m-q. The entire retina is shown in m, higher magnifications around the optic nerve head are shown in n and o, and the peripheral region is shown in p. No signal was seen at PW25 on flat mounts (q) or sections (l). Age (in PW) is indicated in the panels. Vertical bar in a-c and e-l indicates thickness of the ONL.


( a ) qRT - PCR analysis for Opn1sw during cone degeneration .

(a) qRT-PCR analysis for Opn1sw during cone degeneration.


( b - o ) All panels show retinal flat mounts except for i and l . Green signal indicates PNA expression and red signal indicates red / green or blue opsin . The wild - type retina is shown at P35 in b - d . Red / green opsin ( b ) and PNA ( c , d ) expression were detected both dorsally and ventrally , whereas blue opsin ( c , d ) was detected only ventrally .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(b-o) All panels show retinal flat mounts except for i and l. Green signal indicates PNA expression and red signal indicates red/green or blue opsin. The wild-type retina is shown at P35 in b-d. Red/green opsin (b) and PNA (c, d) expression were detected both dorsally and ventrally, whereas blue opsin (c,d) was detected only ventrally.


( b - o ) All panels show retinal flat mounts except for i and l . Green signal indicates PNA expression and red signal indicates red / green or blue opsin . The wild - type retina is shown at P35 in b - d . Red / green opsin ( b ) and PNA ( c , d ) expression were detected both dorsally and ventrally , whereas blue opsin ( c , d ) was detected only ventrally . The Pde6b − / − mouse is analyzed in e - g and j - o . A central to peripheral gradient of PNA and shortening of cone outer segments is shown in e - g . At P20 , there were fewer elongated outer segments in the center ( e ) as compared with the periphery . Higher magnifications of a central or peripheral outer segment from e are shown in f and g , and a wild - type outer segment is shown in h ( white line marks the outer segment ) . A quantification of outer segment length at 3 weeks is shown in i ( error bars represent s . d . of 15 measurements each ) . With the shortening of outer segments during degeneration , red / green opsin was localized throughout the membrane of the cell body , and PNA , which detects an extracellular protein ( s ) , was reduced to a small dot attached to the residual outer segment ( j ) ( yellow shows red / green and PNA overlap , arrow ) . A higher magnification of a cone showing red / green at the membrane is displayed in k ( arrow ) . A cross section showing red / green in cell body is shown in l ( arrows ) ( P70 ; j - l ) . During degeneration , red / green opsin was detected mainly dorsally ( m ) , whereas PNA ( m , n ) or blue opsin ( o ) expression were not altered ( m and n , P21 ; o , P49 ) . GCL , ganglion cell layer ; INL , inner nuclear layer .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(b-o) All panels show retinal flat mounts except for i and l. Green signal indicates PNA expression and red signal indicates red/green or blue opsin. The wild-type retina is shown at P35 in b-d. Red/green opsin (b) and PNA (c, d) expression were detected both dorsally and ventrally, whereas blue opsin (c,d) was detected only ventrally. The Pde6b−/− mouse is analyzed in e-g and j-o. A central to peripheral gradient of PNA and shortening of cone outer segments is shown in e-g. At P20, there were fewer elongated outer segments in the center (e) as compared with the periphery. Higher magnifications of a central or peripheral outer segment from e are shown in f and g, and a wild-type outer segment is shown in h (white line marks the outer segment). A quantification of outer segment length at 3 weeks is shown in i (error bars represent s.d. of 15 measurements each). With the shortening of outer segments during degeneration, red/green opsin was localized throughout the membrane of the cell body, and PNA, which detects an extracellular protein(s), was reduced to a small dot attached to the residual outer segment (j) (yellow shows red/green and PNA overlap, arrow). A higher magnification of a cone showing red/green at the membrane is displayed in k (arrow). A cross section showing red/green in cell body is shown in l (arrows) (P70; j-l). During degeneration, red/ green opsin was detected mainly dorsally (m), whereas PNA (m, n) or blue opsin (o) expression were not altered (m and n, P21; o, P49). GCL, ganglion cell layer; INL, inner nuclear layer.


( b ) Summary of rod and cone death kinetics . Time is indicated in postnatal days or weeks . ( c ) Immunofluorescence microscopy on retinal flat mounts showing the ventral reduction of red / green opsin expression found in the four mutants during cone degeneration . Strain and time , in postnatal days , are indicated below each image ( for higher magnification of wild type , see Fig . 2b ) .

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(b) Summary of rod and cone death kinetics. Time is indicated in postnatal days or weeks. (c) Immunofluorescence microscopy on retinal flat mounts showing the ventral reduction of red/green opsin expression found in the four mutants during cone degeneration. Strain and time, in postnatal days, are indicated below each image (for higher magnification of wild type, see Fig. 2b).


( a - c ) P - mTOR levels in wild - type retinae . Dorsal ( top ) enrichment of P - mTOR ( a ) . Higher magnifications of the dorsal and ventral regions are shown to the right , with P - mTOR in red and cone segments in green , as detected by PNA . Dorsal retinal sections stained for P - mTOR ( red ) and PNA ( b ) or antibody to β - galactosidase ( green , c ) . Higher magnification ( insets ) images suggest that the P - mTOR signal is located in the lower part of the outer segment ( OS ) . IS , inner segment .

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(a-c) P-mTOR levels in wild-type retinae. Dorsal (top) enrichment of P-mTOR (a). Higher magnifications of the dorsal and ventral regions are shown to the right, with P-mTOR in red and cone segments in green, as detected by PNA. Dorsal retinal sections stained for P-mTOR (red) and PNA (b) or antibody to β-galactosidase (green, c). Higher magnification (insets) images suggest that the P-mTOR signal is located in the lower part of the outer segment (OS). IS, inner segment.


( d - g ) Rapamycin treatment of wild - type mice led to downregulation of red / green opsin ventrally ( e ) but not dorsally ( red , d ) . Ventral blue opsin ( red , f ) remained unaffected , as did PNA ( d - g ) ( green ) and P - mTOR itself ( red , g ) .

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(d-g) Rapamycin treatment of wild-type mice led to downregulation of red/green opsin ventrally (e) but not dorsally (red, d). Ventral blue opsin (red, f) remained unaffected, as did PNA (d-g) (green) and P-mTOR itself (red, g).


( h - m ) Reduced levels of dorsal P - mTOR during photoreceptor degeneration ( red ) . The wild - type control is shown in h and the Pde6b mutant is shown in i and j . Reduction started during rod death at P15 ( i ) as the outer segments ( green , PNA ) detached from the retinal pigmented epithelium . By P30 , only a few cones ( green signal : α - β - galactosidase ) showed high levels of P - mTOR ( red ) ( j ) . ( k - m ) Similar reduction was seen in dorsal cones of the other three mutants ( cones marked in green by PNA ; Pde6g − / − is shown at P35 in k ; Rho − / − is shown at PW20 in l ; P23H is shown at PW70 in m ) . All panels show immunofluorescence microscopy on retinal flat mounts ( photoreceptor side up ) with the exception of b , c and g , which show retinal sections . Blue indicates DAPI .

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(h-m) Reduced levels of dorsal P-mTOR during photoreceptor degeneration (red). The wild-type control is shown in h and the Pde6b mutant is shown in i and j. Reduction started during rod death at P15 (i) as the outer segments (green, PNA) detached from the retinal pigmented epithelium. By P30, only a few cones (green signal: α-β-galactosidase) showed high levels of P-mTOR (red) (j). (k-m) Similar reduction was seen in dorsal cones of the other three mutants (cones marked in green by PNA; Pde6g−/− is shown at P35 in k; Rho−/− is shown at PW20 in l; P23H is shown at PW70 in m). All panels show immunofluorescence microscopy on retinal flat mounts (photoreceptor side up) with the exception of b, c and g, which show retinal sections. Blue indicates DAPI.


All panels show immunofluorescent staining . Blue shows nuclear DAPI staining and green shows cones marked with PNA . ( a - f ) Staining for HIF - 1α ( red signal ) . ( a ) Wild type ( PW10 ) stained for HIF - 1α ( inset shows higher magnification ) . ( b , c ) Cross sections in wild type stained for HIF - 1α ( PW10 ) . DAPI overlap is shown in c . ( d - f ) During cone degeneration in Pde6b − / − ( PW10 ) mice , we found increased levels of HIF - 1α in cones ( inset in d shows higher magnification with DAPI overlap ) . Cross sections indicated that the increase of Hif - 1α occurred mainly in cones ( arrows point to cones that are located in the top layer of the inner nuclear layer at this stage ; e ) . DAPI overlap is shown in f .

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All panels show immunofluorescent staining. Blue shows nuclear DAPI staining and green shows cones marked with PNA. (a-f) Staining for HIF-1α (red signal). (a) Wild type (PW10) stained for HIF-1α (inset shows higher magnification). (b, c) Cross sections in wild type stained for HIF-1α (PW10). DAPI overlap is shown in c. (d-f) During cone degeneration in Pde6b−/− (PW10) mice, we found increased levels of HIF-1α in cones (inset in d shows higher magnification with DAPI overlap). Cross sections indicated that the increase of Hif-1α occurred mainly in cones (arrows point to cones that are located in the top layer of the inner nuclear layer at this stage; e). DAPI overlap is shown in f.


( g ) GLUT1 expression in wild type ( PW10 ) ( red ) . Most of the signal in between the cones reflects expression in rods . ( h - j ) Increased expression of GLUT1 in cones during degeneration seen in flat mounts ( h ) and sections ( i , j ) . PNA overlap of j is shown in i . Retinal flat mounts are shown in a , d , g and h . Retinal sections are shown in b , c , e , f , i and j .

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(g) GLUT1 expression in wild type (PW10) (red). Most of the signal in between the cones reflects expression in rods. (h-j) Increased expression of GLUT1 in cones during degeneration seen in flat mounts (h) and sections (i, j). PNA overlap of j is shown in i. Retinal flat mounts are shown in a, d, g and h. Retinal sections are shown in b, c, e, f, i and j.


( a - c ) Immunofluorescence microscopy on retinal flat mounts ( LAMP - 2 is shown in green , red / green opsin in red and DAPI in blue ) . Insets show enlarged cells ( arrow ) . A wild - type retinae at PW5 showing a lysosome ( small green dots ) with normal LAMP - 2 distribution is imaged in a . Weak red / green opsin signal was detected at the level of the photoreceptornuclei , as it was mainly found in the outer segments in the wild type . Enlarged lysosomes ( dots ) resulting from an accumulation of LAMP - 2 at the lysosomal membrane were seen specifically in cones from Pde6b − / − micePW5 ( b ) . Confocal section of same field as in b taken at the level of the inner nuclear layer showed LAMP - 2 levels similar to those in wild type ( c ) .

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(a-c) Immunofluorescence microscopy on retinal flat mounts (LAMP-2 is shown in green, red/green opsin in red and DAPI in blue). Insets show enlarged cells (arrow). A wild-type retinae at PW5 showing a lysosome (small green dots) with normal LAMP-2 distribution is imaged in a. Weak red/green opsin signal was detected at the level of the photoreceptornuclei, as it was mainly found in the outer segments in the wild type. Enlarged lysosomes (dots) resulting from an accumulation of LAMP-2 at the lysosomal membrane were seen specifically in cones from Pde6b−/− micePW5 (b). Confocal section of same field as in b taken at the level of the inner nuclear layer showed LAMP-2 levels similar to those in wild type (c).


( d ) qRT - PCR for the three different LAMP - 2 splice forms showing the relative concentration and the ratios between the Pde6b − / − mutant and wild type . Error bars represent s . d . of three measurements .

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(d) qRT-PCR for the three different LAMP-2 splice forms showing the relative concentration and the ratios between the Pde6b−/− mutant and wild type. Error bars represent s.d. of three measurements.


( a - c ) Retinal flat mounts of Pde6b − / − mutants at PW7 stained for LacZ to detect cones ( see Methods and Supplementary Fig . 9 ) . An example of untreated control is shown in a , an example of a mouse injected with streptozotocin is shown in b and an example of a mouse injected daily with insulin is shown in c . ( d ) Quantification of cone survival after four weeks of treatment . Data represents an average of at least eight retinae . The y axis represents the percentage of the cone surface area versus the surface area of entire retina ( see Supplementary Figs . 8 and 10 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a-c) Retinal flat mounts of Pde6b−/− mutants at PW7 stained for LacZ to detect cones (see Methods and Supplementary Fig. 9). An example of untreated control is shown in a, an example of a mouse injected with streptozotocin is shown in b and an example of a mouse injected daily with insulin is shown in c. (d) Quantification of cone survival after four weeks of treatment. Data represents an average of at least eight retinae. The y axis represents the percentage of the cone surface area versus the surface area of entire retina (see Supplementary Figs. 8 and 10).


( e , f ) Measurements of blood glucose levels ( e ) and body weight ( f ) performed weekly over the time span of the experiment .

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(e, f) Measurements of blood glucose levels (e) and body weight (f) performed weekly over the time span of the experiment.


( g , h ) Immunofluorescence microscopy staining on retinal flat mounts for HIF - 1α ( red ) and PNA ( green ) in untreated control Pde6b − / − ( g ) and Pde6b − / − mice treated for 4 weeks with insulin ( h ) . DAPI is shown in blue . Error bars in d - f represent s . d .

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(g, h) Immunofluorescence microscopy staining on retinal flat mounts for HIF-1α (red) and PNA (green) in untreated control Pde6b−/− (g) and Pde6b−/− mice treated for 4 weeks with insulin (h). DAPI is shown in blue. Error bars in d-f represent s.d.


( H ) UMAP maps color - coded for transcript counts of genes enriched in subsets of the analyzed cells . The scale bars represent color coded sequencing depth - corrected log transformed transcript counts .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) UMAP maps color-coded for transcript counts of genes enriched in subsets of the analyzed cells. The scale bars represent color coded sequencing depth-corrected log transformed transcript counts.


( H ) Representative immunofluorescence images of Iba1 ( green ) and APOE ( red ) on cortical sections from SPF and GF mice . Nuclei were stained with DAPI ( blue ) . Scale bar : 50 μm . White arrowheads indicate APOE + / Iba1 + microglia and arrows show APOE - / Iba1 + microglia . Quantification of APOE + / Iba1 + microglia . Each symbol represents one mouse . At least three slides were examined per individual mouse . Data are presented as mean ± s . e . m . Significant differences were determined by unpaired t - test and marked with asterisks ( * P < 0 . 05 ) . Data are representative of two independent experiments .

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(H) Representative immunofluorescence images of Iba1 (green) and APOE (red) on cortical sections from SPF and GF mice. Nuclei were stained with DAPI (blue). Scale bar: 50 μm. White arrowheads indicate APOE+/Iba1+ microglia and arrows show APOE-/Iba1+ microglia. Quantification of APOE+/Iba1+ microglia. Each symbol represents one mouse. At least three slides were examined per individual mouse. Data are presented as mean ± s.e.m. Significant differences were determined by unpaired t-test and marked with asterisks (*P < 0.05). Data are representative of two independent experiments.


( H ) Representative Iba1 + immunohistochemistry in the choroid plexus of SPF , GF , ABX - treated , ASF , recolonized ASF , sDMDMm2 , TLR2 , 3 , 4 , 7 , 9 - deficient , SCFA - treated GF mice or FFAR2 - deficient mice . Scale bar = 50 µm . ( I - O ) Quantification of stromal CPMΦ . Each symbol represents one mouse . Three to four sections per mouse were examined . Means ± s . e . m . are indicated . Significant differences were evaluated by unpaired t - test or one - way ANOVA followed by Tukey ' s post - hoc comparison test and marked with asterisks ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Data are representative of at least two independent experiments .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Representative Iba1+ immunohistochemistry in the choroid plexus of SPF, GF, ABX-treated, ASF, recolonized ASF, sDMDMm2, TLR2,3,4,7,9-deficient, SCFA-treated GF mice or FFAR2-deficient mice. Scale bar = 50 µm. (I-O) Quantification of stromal CPMΦ. Each symbol represents one mouse. Three to four sections per mouse were examined. Means ± s.e.m. are indicated. Significant differences were evaluated by unpaired t-test or one-way ANOVA followed by Tukey's post-hoc comparison test and marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). Data are representative of at least two independent experiments.


( H ) Representative Iba1 + immunohistochemistry in the perivascular space of SPF , GF , ABX - treated , ASF , recolonized ASF , sDMDMm2 , TLR2 , 3 , 4 , 7 , 9 - deficient , SCFA - treated GF mice or FFAR2 - deficient mice . Arrows indicate Iba1 + perivascular macrophages . Scale bar = 50 µm . ( I - O ) Quantification thereof . Each symbol represents one mouse . Three to four sections per mouse were examined . Means ± s . e . m . are indicated . No significant differences were determined by unpaired t - test or one - way ANOVA followed by Tukey ' s post - hoc comparison test . Data are representative of at least two independent experiments .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Representative Iba1+ immunohistochemistry in the perivascular space of SPF, GF, ABX-treated, ASF, recolonized ASF, sDMDMm2, TLR2,3,4,7,9-deficient, SCFA-treated GF mice or FFAR2-deficient mice. Arrows indicate Iba1+ perivascular macrophages. Scale bar = 50 µm. (I-O) Quantification thereof. Each symbol represents one mouse. Three to four sections per mouse were examined. Means ± s.e.m. are indicated. No significant differences were determined by unpaired t-test or one-way ANOVA followed by Tukey's post-hoc comparison test. Data are representative of at least two independent experiments.


( H ) Representative Iba1 + immunohistochemistry in the leptomeninges of SPF , GF , ABX - treated , ASF , recolonized ASF , sDMDMm2 , TLR2 , 3 , 4 , 7 , 9 - deficient , SCFA - treated GF mice or FFAR2 - deficient mice . Arrows indicate Iba1 + meningeal macrophages . Scale bar = 50 μm . ( I - O ) Quantification thereof . Each symbol represents one mouse . Three to four sections per mouse were examined . Means ± s . e . m . are indicated . No significant differences were determined by unpaired t - test or one - way ANOVA followed by Tukey ' s post - hoc comparison test . Data are representative of at least two independent experiments .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Representative Iba1+ immunohistochemistry in the leptomeninges of SPF, GF, ABX-treated, ASF, recolonized ASF, sDMDMm2, TLR2,3,4,7,9-deficient, SCFA-treated GF mice or FFAR2-deficient mice. Arrows indicate Iba1+ meningeal macrophages. Scale bar = 50 μm. (I-O) Quantification thereof. Each symbol represents one mouse. Three to four sections per mouse were examined. Means ± s.e.m. are indicated. No significant differences were determined by unpaired t-test or one-way ANOVA followed by Tukey's post-hoc comparison test. Data are representative of at least two independent experiments.


( A ) Iba1 immunohistochemistry depicting cortical microglia , meningeal macrophages , perivascular macrophages and choroid plexus macrophages 4 days after i . c . challenge with LCMV or vehicle controls . Arrows indicate Iba1 + meningeal or perivascular macrophages , respectively . ( B ) Quantification of Iba1 + microglia , choroid plexus macrophages , meningeal macrophages and perivascular macrophages . Each symbol represents one mouse . Three to four sections per mouse were examined . Significant differences were evaluated by two - way ANOVA followed by Tukey ' s post - hoc comparison test and marked with asterisks ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Data are representative of two independent experiments .

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(A) Iba1 immunohistochemistry depicting cortical microglia, meningeal macrophages, perivascular macrophages and choroid plexus macrophages 4 days after i.c. challenge with LCMV or vehicle controls. Arrows indicate Iba1+ meningeal or perivascular macrophages, respectively. (B) Quantification of Iba1+ microglia, choroid plexus macrophages, meningeal macrophages and perivascular macrophages. Each symbol represents one mouse. Three to four sections per mouse were examined. Significant differences were evaluated by two-way ANOVA followed by Tukey's post-hoc comparison test and marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). Data are representative of two independent experiments.


( C , D ) Representative immunofluorescence images ( left ) of ( C ) Iba1 ( green ) and KI67 ( red ) or ( D ) CD74 ( red ) on cortical sections from SPF and GF mice . Nuclei were stained with DAPI ( blue ) . Scale bars : 25 μm . White arrows indicate KI67 + / Iba1 + or CD74 + / Iba1 + macrophages and arrowheads show KI67 - / Iba1 + or CD74 - / Iba1 + macrophages . Quantification thereof ( right ) . Each symbol represents one mouse . At least three slides were examined per individual mouse . Data are presented as mean ± s . e . m . Significant differences were evaluated by two - way ANOVA followed by Tukey ' s post - hoc comparison test and marked with asterisks ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Data are representative of two independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C, D) Representative immunofluorescence images (left) of (C) Iba1 (green) and KI67 (red) or (D) CD74 (red) on cortical sections from SPF and GF mice. Nuclei were stained with DAPI (blue). Scale bars: 25 μm. White arrows indicate KI67+/Iba1+ or CD74+/Iba1+ macrophages and arrowheads show KI67-/Iba1+ or CD74-/Iba1+ macrophages. Quantification thereof (right). Each symbol represents one mouse. At least three slides were examined per individual mouse. Data are presented as mean ± s.e.m. Significant differences were evaluated by two-way ANOVA followed by Tukey's post-hoc comparison test and marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). Data are representative of two independent experiments.


( A - C ) Representative immunofluorescence images visualizing ( A ) cortical mMΦ , ( B ) cpMΦ located in the lateral ventricle and ( C ) hippocampal pvMΦ from SPF ( left ) , GF ( middle ) and ABX - treated 5xFAD mice ( right ) . Iba1 ( green ) and Aβ ( 6E10 , red ) . Nuclei were stained with DAPI ( blue ) . White arrowheads indicate Iba1 + Aβ + MΦ and white arrows show Iba1 + Aβ - MΦ . Scale bar : 50 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-C) Representative immunofluorescence images visualizing (A) cortical mMΦ, (B) cpMΦ located in the lateral ventricle and (C) hippocampal pvMΦ from SPF (left), GF (middle) and ABX-treated 5xFAD mice (right). Iba1 (green) and Aβ (6E10, red). Nuclei were stained with DAPI (blue). White arrowheads indicate Iba1+Aβ+ MΦ and white arrows show Iba1+Aβ- MΦ. Scale bar: 50 μm.


( D ) Quantification of the number of 6E10 + Aβ - plaques per mm2 . Each symbol represents one mouse . Means ± s . e . m . are indicated . Significant differences were evaluated by one - way ANOVA followed by Tukey ' s post - hoc comparison test and marked with asterisks ( * * * P < 0 . 001 ) . Data are representative of four independent experiments .

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(D) Quantification of the number of 6E10+ Aβ-plaques per mm2. Each symbol represents one mouse. Means ± s.e.m. are indicated. Significant differences were evaluated by one-way ANOVA followed by Tukey's post-hoc comparison test and marked with asterisks (***P < 0.001). Data are representative of four independent experiments.


( E - G ) Quantification of Iba1 + ( E ) mMΦ , ( F ) cpMΦ and ( G ) pvMΦ per mm2 . Each symbol represents one mouse . At least three slides were examined per individual mouse . Data are presented as mean ± s . e . m . Significant differences were determined by two - way ANOVA followed by Tukey ' s post - hoc comparison test and marked with asterisks ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Data are representative of two independent experiments .

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(E-G) Quantification of Iba1+ (E) mMΦ, (F) cpMΦ and (G) pvMΦ per mm2. Each symbol represents one mouse. At least three slides were examined per individual mouse. Data are presented as mean ± s.e.m. Significant differences were determined by two-way ANOVA followed by Tukey's post-hoc comparison test and marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). Data are representative of two independent experiments.


( H - J ) Percentage of Iba1 + Aβ + ( H ) cortical mMΦ , ( I ) cpMΦ and ( J ) hippocampal pvMΦ of SPF , GF and ABX - treated 5xFAD mice . Means ± s . e . m . are indicated . Significant differences were evaluated by one - way ANOVA followed by Tukey ' s post - hoc comparison test and marked with asterisks ( * P < 0 . 05 ) . Data are representative of two independent experiments .

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(H-J) Percentage of Iba1+Aβ+ (H) cortical mMΦ, (I) cpMΦ and (J) hippocampal pvMΦ of SPF, GF and ABX-treated 5xFAD mice. Means ± s.e.m. are indicated. Significant differences were evaluated by one-way ANOVA followed by Tukey's post-hoc comparison test and marked with asterisks (*P < 0.05). Data are representative of two independent experiments.


GSC neurospheres were dissociated to single cell suspension and transfected with either miR - 10b inhibitor ( labeled " miR - 10b - i " ) or non - targeting control oligonucleotide , or treated with Lipofectamine 2000 only ( Mock ) . Statistical significance of the difference was determined by Student ' s t test , with p - values < 0 . 001 indicated by three asterisks . Numbers of replicates and exact P - values are included in Appendix Table S4 . A . Cell viability was monitored at day 5 after transfection as described in Materials and Methods .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

GSC neurospheres were dissociated to single cell suspension and transfected with either miR-10b inhibitor (labeled "miR-10b-i") or non-targeting control oligonucleotide, or treated with Lipofectamine 2000 only (Mock). Statistical significance of the difference was determined by Student's t test, with p-values < 0.001 indicated by three asterisks. Numbers of replicates and exact P-values are included in Appendix Table S4.A. Cell viability was monitored at day 5 after transfection as described in Materials and Methods.


B . The number and size of GSC colonies were monitored at day 5 after transfection .

B. The number and size of GSC colonies were monitored at day 5 after transfection.


C . MiR - 10b inhibition induces cleavage of caspases 3 and 7 in GSC , as determined by Western blot analysis at day 5 after transfection with the inhibitor . The signals were quantified using ImageJ and normalized to beta - actin .

C. MiR-10b inhibition induces cleavage of caspases 3 and 7 in GSC, as determined by Western blot analysis at day 5 after transfection with the inhibitor. The signals were quantified using ImageJ and normalized to beta-actin.


D . Flow Cytometry analysis of Annexin V and 7 - AAD staining of GSC GBM8 at day 5 after miR - 10b inhibition .

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D. Flow Cytometry analysis of Annexin V and 7-AAD staining of GSC GBM8 at day 5 after miR-10b inhibition.


Three types of GSC ( GBM4 , GBM6 , and GBM8 ) were transfected with miR - 10bASO , and gene expression was analyzed 24 hours later by the Affymetrix microarrays . The heatmaps colors intensity demonstrate altered expression of the genes ( up - or down - regulated relative to the mock treated samples ) with the fold change > 1 . 2 and p < 0 . 05 in at least two out of the three GSC cultures . A . The genes associated with " cell cycle " bioterm have been selected using Ingenuity Pathway Analysis . The treatment with the miR - 10b inhibitor is indicated as " miR - 10b - i " .

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Three types of GSC (GBM4, GBM6, and GBM8) were transfected with miR-10bASO, and gene expression was analyzed 24 hours later by the Affymetrix microarrays. The heatmaps colors intensity demonstrate altered expression of the genes (up- or down-regulated relative to the mock treated samples) with the fold change > 1.2 and p < 0.05 in at least two out of the three GSC cultures.A. The genes associated with "cell cycle" bioterm have been selected using Ingenuity Pathway Analysis. The treatment with the miR-10b inhibitor is indicated as "miR-10b-i".


B . The genes associated with " RNA splicing " bioterm have been selected using Gene Ontology ( GO ) . The treatment with the miR - 10b inhibitor is indicated as " miR - 10b - i " . Arrows depict the genes selected as candidate direct targets for further study .

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B. The genes associated with "RNA splicing" bioterm have been selected using Gene Ontology (GO). The treatment with the miR-10b inhibitor is indicated as "miR-10b-i". Arrows depict the genes selected as candidate direct targets for further study.


C . miR - 10b binding motifs are enriched in 5 ' UTRs of the genes up - regulated by miR - 10b ASO . The graph shows the probability that enrichment of the miR - 10b motifs in mRNAs up - regulated vs . unchanged ( p < 0 . 05 ) by anti - miR - 10b does not occur by chance .

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C. miR-10b binding motifs are enriched in 5'UTRs of the genes up-regulated by miR-10b ASO. The graph shows the probability that enrichment of the miR-10b motifs in mRNAs up-regulated vs. unchanged (p<0.05) by anti-miR-10b does not occur by chance.


D . The miR - 10b octamer motifs ' composition of the 5 ' UTRs was compared between transcripts up - and down - regulated on the microarrays . The relative frequencies of various miR - 10b binding motifs are shown , indicating that mostly miR - 10b 3 ' - end binding motifs are enriched in the up - regulated mRNAs .

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D. The miR-10b octamer motifs' composition of the 5'UTRs was compared between transcripts up- and down-regulated on the microarrays. The relative frequencies of various miR-10b binding motifs are shown, indicating that mostly miR-10b 3'-end binding motifs are enriched in the up-regulated mRNAs.


Figure 3 . Expression analysis of splicing factor mRNAs in various GBM datasets . A . The genes encoding splicing factors down - regulated by miR - 10b are expressed at lower levels in various GBM datasets relative to their expression in normal brain tissues . B . In contrast , many splicing factors up - regulated by miR - 10b are overexpressed in the GBM datasets . Six high - content GBMmicroarray datasets from the Oncomine resource ( https : / / www . oncomine . org / resource / main . html ) , including TCGA _ BrainGBM ( 2 ) , Bredel Brain2 ( 31 ) , Lee Brain ( 32 ) , Liang Brain ( 33 ) , Murat Brain ( 34 ) and Sun Brain ( 35 ) , that collectively contain information for 858 GBM and 52 control samples , have been utilized for the analysis . The data is presented as log2 fold change between GBM and normal brain tissues .

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Figure 3.Expression analysis of splicing factor mRNAs in various GBM datasets.A. The genes encoding splicing factors down-regulated by miR-10b are expressed at lower levels in various GBM datasets relative to their expression in normal brain tissues.B. In contrast, many splicing factors up-regulated by miR-10b are overexpressed in the GBM datasets.Six high-content GBMmicroarray datasets from the Oncomine resource (https://www.oncomine.org/resource/main.html), including TCGA_BrainGBM (2), Bredel Brain2 (31), Lee Brain (32), Liang Brain (33), Murat Brain (34) and Sun Brain (35), that collectively contain information for 858 GBM and 52 control samples, have been utilized for the analysis. The data is presented as log2 fold change between GBM and normal brain tissues.


B . qRT - PCR analysis validates that mRNA of MBNL1 - 3 , SART3 , RSRC1 and other splicing factors are de - repressed by miR - 10b ASO in different GSC and GBM cell lines . mRNA expression levels were normalized to GAPDH expression .

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B. qRT-PCR analysis validates that mRNA of MBNL1-3, SART3, RSRC1 and other splicing factors are de-repressed by miR-10b ASO in different GSC and GBM cell lines. mRNA expression levels were normalized to GAPDH expression.


C . Regulation of representative splicing - related proteins by miR - 10b mimic in GSC , as demonstrated by Western blot analysis . The signals were quantified using ImageJ and normalized to beta - actin . The ratios between miR - 10b mimic expressing and control samples are indicated .

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C. Regulation of representative splicing-related proteins by miR-10b mimic in GSC, as demonstrated by Western blot analysis. The signals were quantified using ImageJ and normalized to beta-actin. The ratios between miR-10b mimic expressing and control samples are indicated.


D . miR - 10b mimic regulates 5 ' UTR Luciferase reporter containing a single miR - 10b complementary site .

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D. miR-10b mimic regulates 5'UTR Luciferase reporter containing a single miR-10b complementary site.


E . miR - 10b mimic regulates 5 ' UTR Luciferase reporters of some splicing factors genes bearing wild type ( WT ) , but not mutated ( Mut ) miR - 10b binding sites . Statistical significance of the differences was determined by Student ' s t test , with p - values < 0 . 05 indicated by asterisks , p < 0 . 01 by two asterisks , and p < 0 . 001 by three asterisks . Numbers of replicates and exact P - values are included in Appendix Table S4 .

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E. miR-10b mimic regulates 5'UTR Luciferase reporters of some splicing factors genes bearing wild type (WT), but not mutated (Mut) miR-10b binding sites. Statistical significance of the differences was determined by Student's t test, with p-values < 0.05 indicated by asterisks, p < 0.01 by two asterisks, and p< 0.001 by three asterisks. Numbers of replicates and exact P-values are included in Appendix Table S4.


B . 2 ' - O - MOE - POmiR - 10b inhibitor ( miR - 10b - i ) or non - targeting control ( 1 μg of each ) formulated with InVivo - jet PEI were injected intratumorally at days 20 and 25 after cells implantation . The efficacy of miR - 10b inhibition was assessed by qRT - PCR analysis of the resected tumors , with miR - 10b expression levels normalized to miR - 125b .

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B. 2'-O-MOE-POmiR-10b inhibitor (miR-10b-i) or non-targeting control (1 μg of each) formulated with InVivo-jet PEI were injected intratumorally at days 20 and 25 after cells implantation. The efficacy of miR-10b inhibition was assessed by qRT-PCR analysis of the resected tumors, with miR-10b expression levels normalized to miR-125b.


C . qRT - PCR analysis demonstrates that miR - 10b inhibition in orthotopic GBM8 leads do de - repression of its mRNA targets . mRNA expression levels were normalized to GAPDH .

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C. qRT-PCR analysis demonstrates that miR-10b inhibition in orthotopic GBM8 leads do de-repression of its mRNA targets. mRNA expression levels were normalized to GAPDH.


D . Inverse correlation between miR - 10b levels and expression of its mRNA targets in resected GBM8 tumors .

D. Inverse correlation between miR-10b levels and expression of its mRNA targets in resected GBM8 tumors.


E . Inhibition of miR - 10b markedly reduces tumor burden . The left panels illustrate tumor imaging in representative animals at day 29 . The bars represent average signal ratios for each group at day 29 ( after treatment ) to day 20 ( at the beginning of treatment ) . N = 7 animals per group at treatment initiation .

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E. Inhibition of miR-10b markedly reduces tumor burden. The left panels illustrate tumor imaging in representative animals at day 29. The bars represent average signal ratios for each group at day 29 (after treatment) to day 20 (at the beginning of treatment). N = 7 animals per group at treatment initiation.


F . Growth curves of individual tumors , based on the ratios of bioluminescence signals to the baseline signals at day 5 .

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F. Growth curves of individual tumors, based on the ratios of bioluminescence signals to the baseline signals at day 5.


G . Each mouse was sacrificed when the tumor - generated signal reached 1 . 5x107 photons / sec , and Kaplan - Meier survival plots were built retrospectively .

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G. Each mouse was sacrificed when the tumor-generated signal reached 1.5x107 photons/sec, and Kaplan-Meier survival plots were built retrospectively.


A . Intravenously injected Cy5 - labeled 2 ' - O - MOE - PSoligonucleotide is distributed to intracranialGBM8tumor . In the normal brain , the signal is observed in blood vessels but not within brain parenchyma . " T " - tumor , " B " - brain tissue . Each image is representative of three mice analyzed .

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A. Intravenously injected Cy5-labeled 2'-O-MOE-PSoligonucleotide is distributed to intracranialGBM8tumor. In the normal brain, the signal is observed in blood vessels but not within brain parenchyma. "T" - tumor, "B" - brain tissue. Each image is representative of three mice analyzed.


B - D . Systemic inhibition of miR - 10b markedly reduces tumor burden . Uncomplexed 2 ' - O - MOE - PSmiR - 10b inhibitor ( miR - 10b - i ) or non - targeting control of the same chemistry were injected at 80 mg / kg through the tail vein at the days indicated by arrows . B . The left panels illustrate tumorimages of representative animals at day 34 , and average signals ( photons / sec ) are indicated below the images . The bars represent average signal ratios for each group at day 34 , relative to day

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B-D. Systemic inhibition of miR-10b markedly reduces tumor burden. Uncomplexed 2'-O-MOE-PSmiR-10b inhibitor (miR-10b-i) or non-targeting control of the same chemistry were injected at 80 mg/kg through the tail vein at the days indicated by arrows.B. The left panels illustrate tumorimages of representative animals at day 34, and average signals (photons/sec) are indicated below the images. The bars represent average signal ratios for each group at day 34, relative to day 6


B - D . Systemic inhibition of miR - 10b markedly reduces tumor burden . Uncomplexed 2 ' - O - MOE - PSmiR - 10b inhibitor ( miR - 10b - i ) or non - targeting control of the same chemistry were injected at 80 mg / kg through the tail vein at the days indicated by arrows . B . The left panels illustrate tumorimages of representative animals at day 34 , and average signals ( photons / sec ) are indicated below the images . The bars represent average signal ratios for each group at day 34 , relative to day 6 . C . Each mouse was sacrificed when the tumor - generated signal reached 5X107 photons / sec , and Kaplan - Meier survival plots were built retrospectively . D . Growth curves of individual tumors based on the ratios of bioluminescence signals to the baseline signals at day 6 .

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B-D. Systemic inhibition of miR-10b markedly reduces tumor burden. Uncomplexed 2'-O-MOE-PSmiR-10b inhibitor (miR-10b-i) or non-targeting control of the same chemistry were injected at 80 mg/kg through the tail vein at the days indicated by arrows.B. The left panels illustrate tumorimages of representative animals at day 34, and average signals (photons/sec) are indicated below the images. The bars represent average signal ratios for each group at day 34, relative to day 6.C. Each mouse was sacrificed when the tumor-generated signal reached 5X107 photons/sec, and Kaplan-Meier survival plots were built retrospectively.D. Growth curves of individual tumors based on the ratios of bioluminescence signals to the baseline signals at day 6.


E . The efficacy of miR - 10b inhibition in intracranial tumors was assessed by qRT - PCR analysis of the resected tumor tissues , with miR - 10b expression levels normalized to miR - 125b .

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E. The efficacy of miR-10b inhibition in intracranial tumors was assessed by qRT-PCR analysis of the resected tumor tissues, with miR-10b expression levels normalized to miR-125b.


F . qRT - PCR analysis demonstrates that miR - 10b targets were de - repressed in orthotopic GBM8 upon systemic administration of the miR - 10b inhibitor . Seven tumors per condition , and two specimens per tumor have been analyzed . mRNA expression levels were normalized to GAPDH .

F. qRT-PCR analysis demonstrates that miR-10b targets were de-repressed in orthotopic GBM8 upon systemic administration of the miR-10b inhibitor. Seven tumors per condition, and two specimens per tumor have been analyzed. mRNA expression levels were normalized to GAPDH.


G . Inverse correlation between miR - 10b levels and expression of its mRNA targets in resected GBM8 tumors .

G. Inverse correlation between miR-10b levels and expression of its mRNA targets in resected GBM8 tumors.


A . Uptake of the uncomplexed Cy5 - labeled 2 ' - O - MOE - PSoligonucleotide ( 80 mg / kg injected via the tail vein ) by normal extracranial tissues was examined by fluorescence microscopy24 h after injections .

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A. Uptake of the uncomplexed Cy5-labeled 2'-O-MOE-PSoligonucleotide (80 mg/kg injected via the tail vein) by normal extracranial tissues was examined by fluorescence microscopy24 h after injections.


B - D . Systemic treatment of intracranialGBM8tumors with uncomplexed 2 ' - O - MOE - PSmiR - 10b inhibitor ( miR - 10b - i ) or non - targeting control oligonucleotide ( at 80 mg / kg ) was not associated with toxic effects . B . No significant difference in average mice weight was observed between the anti - miR - 10b and control treatment groups . C . No significant difference in average organs ' weight was observed between the anti - miR - 10b and control treatment groups . D . No significant difference in tissue histology using Hematoxylin and Eosin staining was observed between the anti - miR - 10b and control treatment groups .

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B-D. Systemic treatment of intracranialGBM8tumors with uncomplexed 2'-O-MOE-PSmiR-10b inhibitor (miR-10b-i) or non-targeting control oligonucleotide (at 80 mg/kg) was not associated with toxic effects.B. No significant difference in average mice weight was observed between the anti-miR-10b and control treatment groups.C. No significant difference in average organs' weight was observed between the anti-miR-10b and control treatment groups.D. No significant difference in tissue histology using Hematoxylin and Eosin staining was observed between the anti-miR-10b and control treatment groups.


A . Continuous osmotic delivery of miR - 10b inhibitor markedly reduces tumor burden . The osmotic pumps , loaded with lipid nanoparticles formulated with 2 ' - O - MOE - POmiR - 10b inhibitor or non - targeting control infused 2 μg of the oligonucleotides per day introtumorally , over 13 days . Tumors growth was monitored by the WBI , and the left panels illustrate tumor imaging of representative animals at the end of treatment . The bars represent average signal ratios for each group at day 13 ( end of treatment ) , relative to day 2 ( treatment initiation ) .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Continuous osmotic delivery of miR-10b inhibitor markedly reduces tumor burden. The osmotic pumps, loaded with lipid nanoparticles formulated with 2'-O-MOE-POmiR-10b inhibitor or non-targeting control infused 2 μg of the oligonucleotides per day introtumorally, over 13 days. Tumors growth was monitored by the WBI, and the left panels illustrate tumor imaging of representative animals at the end of treatment. The bars represent average signal ratios for each group at day 13 (end of treatment), relative to day 2 (treatment initiation).


B . Representative immunostaining of tumors for PCNA proliferation marker .

B. Representative immunostaining of tumors for PCNA proliferation marker.


C . Representative immunostaining of the tumors for KI67 proliferation marker .

C. Representative immunostaining of the tumors for KI67 proliferation marker.


D . Representative tumor immunostaining for cleaved Caspase 3 as a marker of apoptosis .

D. Representative tumor immunostaining for cleaved Caspase 3 as a marker of apoptosis.


E . Tumor cell invasion was examined by fluorescence microscopy for mCherry - positive cells migrating through the tumor border .

E. Tumor cell invasion was examined by fluorescence microscopy for mCherry-positive cells migrating through the tumor border.


F . Quantitative immunostainings analysis indicates that proliferation and apoptosis markers are affected by anti - miR - 10b treatment . No significant effect on invasion of intracranial GBM8 was observed .

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F. Quantitative immunostainings analysis indicates that proliferation and apoptosis markers are affected by anti-miR-10b treatment. No significant effect on invasion of intracranial GBM8 was observed.


A , B . miR - 10b inhibition decreases GL261 cellviability . Cellviability was measured at days 3 - 7 after transfection with miR - 10b inhibitor , non - targeting control , or Lipofectamine 2000 alone ( mock ) . A . Phase contrast photographs of GL261 cultures at day 6 post - transfection . B . Growth curves of cultured GL261 cells , based on the viability assay .

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A, B. miR-10b inhibition decreases GL261 cellviability. Cellviability was measured at days 3-7 after transfection with miR-10b inhibitor, non-targeting control, or Lipofectamine 2000 alone (mock).A. Phase contrast photographs of GL261 cultures at day 6 post-transfection.B. Growth curves of cultured GL261 cells, based on the viability assay.


C . InVivo - jet PEI - formulated 2 ' - O - MOE - PS / PO miR - 10b inhibitor or non - targeting control were infused to orthotopic GL261 tumors by osmotic pumps , starting at day 6 after cell implantation . The uptake of ASOs was confirmed by IHC for PS - containing oligonucleotides ( green ) . GL261 tumor cells expressing M - Cherry are red .

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C. InVivo-jet PEI - formulated 2'-O-MOE-PS/PO miR-10b inhibitor or non-targeting control were infused to orthotopic GL261 tumors by osmotic pumps, starting at day 6 after cell implantation. The uptake of ASOs was confirmed by IHC for PS-containing oligonucleotides (green). GL261 tumor cells expressing M-Cherry are red.


D . Osmotic delivery of miR - 10b inhibitor markedly reduces GL261tumorgrowth in immunocompetent Black 6 Albino mice . Micephotographs show tumor imaging in representative animals at day 3 after pump implantation , and average signals in photons per second are indicated . Tumorgrowth rates were calculated as ratios of the signals at day 3 of the treatment to day 1 prior to initiation of the treatment .

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D. Osmotic delivery of miR-10b inhibitor markedly reduces GL261tumorgrowth in immunocompetent Black 6 Albino mice. Micephotographs show tumor imaging in representative animals at day 3 after pump implantation, and average signals in photons per second are indicated. Tumorgrowth rates were calculated as ratios of the signals at day 3 of the treatment to day 1 prior to initiation of the treatment.


F . qRT - PCR analysis demonstrates that miR - 10b target p21 was de - repressed in GL261 tumors upon miR - 10b inhibition . mRNA expression levels were normalized to GAPDH .

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F. qRT-PCR analysis demonstrates that miR-10b target p21 was de-repressed in GL261 tumors upon miR-10b inhibition. mRNA expression levels were normalized to GAPDH.


Western blot analysis of total brain lysates of wild type , Tmem106b - / - , Grn - / - and Grn - / - / Tmem106b - / - mice using antibody 6F2 to detect TMEM106B . ( 4 . 5 - month - old mice with the given genotype ; n = 3 biological replicates per genotype ) .

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Western blot analysis of total brain lysates of wild type, Tmem106b-/-, Grn-/- and Grn-/-/Tmem106b-/- mice using antibody 6F2 to detect TMEM106B. (4.5-month-old mice with the given genotype; n = 3 biological replicates per genotype).


Hind limb clasping test wild type , Tmem106b - / - , Grn - / - , Grn + / - / Tmem106b - / - and Grn - / - / Tmem106b - / - mice . Representative pictures are shown of 3 mice analyzed for each genotype at 3 months of age .

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Hind limb clasping test wild type, Tmem106b-/-, Grn-/-, Grn+/-/Tmem106b-/- and Grn-/-/Tmem106b-/- mice. Representative pictures are shown of 3 mice analyzed for each genotype at 3 months of age.


Rotarod performance of wild type , Tmem106b - / - , Grn - / - , Grn + / - / Tmem106b - / - and double knockout animals at 3 to 4 months of age . Number of animals used for analysis , 10 wild type , 12 Tmem106b - / - , 5 Grn - / - , 7 Grn + / - / Tmem106b - / - , 3 Grn - / - / Tmem106b - / - mice .

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Rotarod performance of wild type, Tmem106b-/-, Grn-/-, Grn+/- /Tmem106b-/- and double knockout animals at 3 to 4 months of age. Number of animals used for analysis, 10 wild type, 12 Tmem106b-/-, 5 Grn-/-, 7 Grn+/-/Tmem106b-/-, 3 Grn-/-/Tmem106b-/- mice.


Representative picture of a 4 . 5 - month - old double knockout ( Grn - / - / Tmem106b - / - ) mouse after manual flip .

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Representative picture of a 4.5-month-old double knockout (Grn-/-/Tmem106b-/- ) mouse after manual flip.


Volcano plot of differentially expressed brain mRNAs of 4 . 5 - month - old mice ( n = 3 biological replicates per genotype ) detected by the Neuropathology panel of NanoString . Expression changes were only taken into consideration when the extent of change was above 20 % as indicated by vertical lines for all volcano plots . Gliosis and Myelination related genes are highlighted if changes are above 20 % . Fold changes are displayed after log2 transformation . ( A ) Comparison of differentially expressed brain mRNAs from Tmem106b - / - and wild type mice . From 680 detected genes 21 are significantly upregulated , while 10 are significantly reduced .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Volcano plot of differentially expressed brain mRNAs of 4.5-month-old mice (n = 3 biological replicates per genotype) detected by the Neuropathology panel of NanoString. Expression changes were only taken into consideration when the extent of change was above 20% as indicated by vertical lines for all volcano plots. Gliosis and Myelination related genes are highlighted if changes are above 20%. Fold changes are displayed after log2 transformation. (A) Comparison of differentially expressed brain mRNAs from Tmem106b-/- and wild type mice. From 680 detected genes 21 are significantly upregulated, while 10 are significantly reduced.


Volcano plot of differentially expressed brain mRNAs of 4 . 5 - month - old mice ( n = 3 biological replicates per genotype ) detected by the Neuropathology panel of NanoString . Expression changes were only taken into consideration when the extent of change was above 20 % as indicated by vertical lines for all volcano plots . Gliosis and Myelination related genes are highlighted if changes are above 20 % . Fold changes are displayed after log2 transformation . ( B ) Comparison of Grn - / - and wild type mice . From 680 detected genes 12 are significantly upregulated , while 6 are significantly reduced .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Volcano plot of differentially expressed brain mRNAs of 4.5-month-old mice (n = 3 biological replicates per genotype) detected by the Neuropathology panel of NanoString. Expression changes were only taken into consideration when the extent of change was above 20% as indicated by vertical lines for all volcano plots. Gliosis and Myelination related genes are highlighted if changes are above 20%. Fold changes are displayed after log2 transformation. (B) Comparison of Grn-/- and wild type mice. From 680 detected genes 12 are significantly upregulated, while 6 are significantly reduced.


Volcano plot of differentially expressed brain mRNAs of 4 . 5 - month - old mice ( n = 3 biological replicates per genotype ) detected by the Neuropathology panel of NanoString . Expression changes were only taken into consideration when the extent of change was above 20 % as indicated by vertical lines for all volcano plots . Gliosis and Myelination related genes are highlighted if changes are above 20 % . Fold changes are displayed after log2 transformation . ( C ) Volcano plot for Grn - / - / Tmem106b - / - and wild type mice . From 680 detected genes 79 are significantly upregulated , while 24 are significantly reduced .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Volcano plot of differentially expressed brain mRNAs of 4.5-month-old mice (n = 3 biological replicates per genotype) detected by the Neuropathology panel of NanoString. Expression changes were only taken into consideration when the extent of change was above 20% as indicated by vertical lines for all volcano plots. Gliosis and Myelination related genes are highlighted if changes are above 20%. Fold changes are displayed after log2 transformation. (C) Volcano plot for Grn-/-/Tmem106b-/- and wild type mice. From 680 detected genes 79 are significantly upregulated, while 24 are significantly reduced.


D . Display of differently expressed genes overlapping between the analyzed genotypes from ( A - C ) . Overlap was considered if a gene was significantly changed in the respective mouse models with more than 20 % change in at least one mouse model . Note that 9 genes are significantly altered in all 3 genotypes in comparison to the wild type . 7 genes are exclusively overlapping between the Grn - / - and the Grn - / - / Tmem106b - / - mice . Tmem106b - / - and Grn - / - / Tmem106b - / - mice share 15 significantly altered genes . Tmem106b - / - and Grn - / - mice share one significant altered gene . Data represent the mean ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Display of differently expressed genes overlapping between the analyzed genotypes from (A-C). Overlap was considered if a gene was significantly changed in the respective mouse models with more than 20% change in at least one mouse model. Note that 9 genes are significantly altered in all 3 genotypes in comparison to the wild type. 7 genes are exclusively overlapping between the Grn-/- and the Grn-/-/Tmem106b-/- mice. Tmem106b-/- and Grn-/-/Tmem106b-/- mice share 15 significantly altered genes. Tmem106b-/- and Grn-/- mice share one significant altered gene. Data represent the mean ± SD.


Analysis of brain mRNA expression of 4 . 5 - month - old mice ( 5 wild type 4 Tmem106b - / - mice ) detected by the Neuroinflammation panel by NanoString . Expression changes were only taken into consideration when the extent of change was above 20 % as indicated by vertical lines for in all volcano plots . Gliosis related genes are highlighted if changes are above 20 % . Fold changes are displayed after log2 transformation . ( A ) Comparison of differentially expressed mRNA of Tmem106b - / - mice in comparison to wild type mice . From 622 detected genes 21 are significantly upregulated , while 12 are significantly reduced .

Analysis of brain mRNA expression of 4.5-month-old mice (5 wild type 4 Tmem106b-/- mice) detected by the Neuroinflammation panel by NanoString. Expression changes were only taken into consideration when the extent of change was above 20% as indicated by vertical lines for in all volcano plots. Gliosis related genes are highlighted if changes are above 20%. Fold changes are displayed after log2 transformation. (A) Comparison of differentially expressed mRNA of Tmem106b-/- mice in comparison to wild type mice. From 622 detected genes 21 are significantly upregulated, while 12 are significantly reduced.


Analysis of brain mRNA expression of 4 . 5 - month - old mice ( 5 wild type , 4 Grn - / mice ) detected by the Neuroinflammation panel by NanoString . Expression changes were only taken into consideration when the extent of change was above 20 % as indicated by vertical lines for in all volcano plots . Gliosis related genes are highlighted if changes are above 20 % . Fold changes are displayed after log2 transformation . ( B ) Comparison of differentially expressed mRNA in Grn - / - and wild type mice . From 622 detected genes 32 are significantly upregulated , while only 1 is significantly reduced .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Analysis of brain mRNA expression of 4.5-month-old mice (5 wild type, 4 Grn-/ mice) detected by the Neuroinflammation panel by NanoString. Expression changes were only taken into consideration when the extent of change was above 20% as indicated by vertical lines for in all volcano plots. Gliosis related genes are highlighted if changes are above 20%. Fold changes are displayed after log2 transformation. (B) Comparison of differentially expressed mRNA in Grn-/- and wild type mice. From 622 detected genes 32 are significantly upregulated, while only 1 is significantly reduced.


Analysis of brain mRNA expression of 4 . 5 - month - old mice ( 5 wild type 5 Grn - / - / Tmem106b - / - mice ) detected by the Neuroinflammation panel by NanoString . Expression changes were only taken into consideration when the extent of change was above 20 % as indicated by vertical lines for in all volcano plots . Gliosis related genes are highlighted if changes are above 20 % . Fold changes are displayed after log2 transformation . ( C ) Volcano plot of differentially expressed brain mRNAs from Grn - / - / Tmem106b - / - mice in comparison to wild type mice . From 622 detected genes 110 are significantly upregulated , while 16 are significantly reduced .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Analysis of brain mRNA expression of 4.5-month-old mice (5 wild type 5 Grn-/-/Tmem106b-/- mice) detected by the Neuroinflammation panel by NanoString. Expression changes were only taken into consideration when the extent of change was above 20% as indicated by vertical lines for in all volcano plots. Gliosis related genes are highlighted if changes are above 20%. Fold changes are displayed after log2 transformation. (C) Volcano plot of differentially expressed brain mRNAs from Grn-/-/Tmem106b-/- mice in comparison to wild type mice. From 622 detected genes 110 are significantly upregulated, while 16 are significantly reduced.


D . Display of differently expressed genes overlapping between the analyzed genotypes from ( A - C ) . Overlap was considered if a gene was significantly changed in the respective mouse models with more than 20 % change in at least one mouse model . Note that 19 genes are significantly altered in all 3 genotypes in comparison to the wild type . 17 genes are exclusively overlapping between the Grn - / - and the Grn - / - / Tmem106b - / - mice . Tmem106b - / - and Grn - / - / Tmem106b - / - mice share 17 significantly altered genes . Data represent the mean ± SD .

D. Display of differently expressed genes overlapping between the analyzed genotypes from (A-C). Overlap was considered if a gene was significantly changed in the respective mouse models with more than 20% change in at least one mouse model. Note that 19 genes are significantly altered in all 3 genotypes in comparison to the wild type. 17 genes are exclusively overlapping between the Grn-/- and the Grn-/-/Tmem106b-/- mice. Tmem106b-/- and Grn-/-/Tmem106b-/- mice share 17 significantly altered genes. Data represent the mean ± SD.


Western blot analysis of GFAP and IBA1 in total brain lysates from 4 . 5 - month - old mice with the given genotype . Quantification of A . Protein expression was normalized to levels in wild type animals . Data represent the mean ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Western blot analysis of GFAP and IBA1 in total brain lysates from 4.5-month-old mice with the given genotype. Quantification of A. Protein expression was normalized to levels in wild type animals. Data represent the mean ± SD.


Expression of GFAP in sagittal brain sections . Representative images of indicated brain regions ( CTX = Cortex ; TH = Thalamus ; CBX = Cerebellum ; MY = Medulla ) . Expression of IBA1 in sagittal brain sections . Representative images of indicated brain regions ( CTX = Cortex ; TH = Thalamus ; CBX = Cerebellum ; MY = Medulla ) . Scale bar indicates 100 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Expression of GFAP in sagittal brain sections. Representative images of indicated brain regions (CTX = Cortex; TH = Thalamus; CBX = Cerebellum; MY = Medulla). Expression of IBA1 in sagittal brain sections. Representative images of indicated brain regions (CTX = Cortex; TH = Thalamus; CBX = Cerebellum; MY = Medulla). Scale bar indicates 100 µm.


In vivo 18F - GE180 TSPO μPET imaging . Two female Grn - / - / Tmem106b - / - and five female wild type mice underwent longitudinal TSPO μPET at 2 . 0 and 3 . 5 months of age . All analyses were performed by PMOD ( V3 . 5 , PMOD technologies ) . Normalization of injected activity was performed by the previously validated myocardium correction method .

In vivo 18F-GE180 TSPO μPET imaging. Two female Grn-/-/Tmem106b-/- and five female wild type mice underwent longitudinal TSPO μPET at 2.0 and 3.5 months of age. All analyses were performed by PMOD (V3.5, PMOD technologies). Normalization of injected activity was performed by the previously validated myocardium correction method.


Western blot analysis of CD68 , CLEC7A , TREM2 and ApoE in total brain lysates from 4 . 5 - month - old mice with the given genotype . Asterisk indicate unspecific band . ( n = 3 biological replicates per genotype ) . Quantification of A . Protein expression was normalized to levels in wild type animals .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Western blot analysis of CD68, CLEC7A, TREM2 and ApoE in total brain lysates from 4.5-month-old mice with the given genotype. Asterisk indicate unspecific band. (n = 3 biological replicates per genotype). Quantification of A. Protein expression was normalized to levels in wild type animals.


Expression of CD68 and the microglial marker IBA1 in sagittal brain sections . Representative images of indicated brain regions ( TH = Thalamus ) . Expression of CLEC7A and the microglial marker IBA1 in sagittal brain sections . Representative images of indicated brain regions ( MY = medulla ) . Expression of TREM2 and the microglial marker IBA1 in sagittal brain sections . Representative images of indicated brain regions ( MY = medulla ) . Scale bar indicates 25 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Expression of CD68 and the microglial marker IBA1 in sagittal brain sections. Representative images of indicated brain regions (TH = Thalamus). Expression of CLEC7A and the microglial marker IBA1 in sagittal brain sections. Representative images of indicated brain regions (MY = medulla). Expression of TREM2 and the microglial marker IBA1 in sagittal brain sections. Representative images of indicated brain regions (MY = medulla). Scale bar indicates 25 µm.


Western blot analysis of cathepsin ( Cat ) D , B , and L in total brain lysates from 4 . 5 - month - old mice with the indicated genotype . Different maturation variants are indicated ( p = preform , sc = single chain , hc = heavy chain ) ( n = 3 biological replicates per genotype ) . Quantification of proteolytically generated variants of cathepsin D , B , and L normalized to levels in wild type animals from ( A ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Western blot analysis of cathepsin (Cat) D, B, and L in total brain lysates from 4.5-month-old mice with the indicated genotype. Different maturation variants are indicated (p = preform, sc = single chain, hc = heavy chain) (n = 3 biological replicates per genotype). Quantification of proteolytically generated variants of cathepsin D, B, and L normalized to levels in wild type animals from (A).


Catalytic activity of cathepsin D , B , and L . Total brain lysates from 4 . 5 - month - old mice were incubated with quenched fluorogenic substrate for Cat D , Cat B and Cat L . The linear increase of fluorescence signal was measured and then normalized to wildtype . Data represent the mean normalized activity ± SD . ( n = 3 biological replicates per genotype ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Catalytic activity of cathepsin D, B, and L. Total brain lysates from 4.5-month-old mice were incubated with quenched fluorogenic substrate for Cat D, Cat B and Cat L. The linear increase of fluorescence signal was measured and then normalized to wildtype. Data represent the mean normalized activity ± SD. (n = 3 biological replicates per genotype).


Immunofluorescence analysis of lipofuscin ( red ) and IBA1 ( green ) in sagittal brain sections . Representative images of the Thalamus ( TH ) . Scale bar indicates 25 µm .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunofluorescence analysis of lipofuscin (red) and IBA1 (green) in sagittal brain sections. Representative images of the Thalamus (TH). Scale bar indicates 25 µm.


Western blot analysis of ubiquitin and the autophagy marker LC3 in RIPA , and p62 in RIPA and urea total brain lysates from 4 . 5 - month - old mice with the indicated genotype . Calnexin used as a loading control . ( n = 3 biological replicates per genotype ) . Quantification of B . Protein expression was normalized to levels in wild type animals . Data represent the mean ± SD .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Western blot analysis of ubiquitin and the autophagy marker LC3 in RIPA, and p62 in RIPA and urea total brain lysates from 4.5-month-old mice with the indicated genotype. Calnexin used as a loading control. (n = 3 biological replicates per genotype). Quantification of B. Protein expression was normalized to levels in wild type animals. Data represent the mean ± SD.