Integrin Linked Kinase (ILK) regulates astrocytic differentiation by NSCs.

<p>A. NSCs cultured in either control (scrambled) PA (VVIAK PA) or IKVAV PA for one day, and immunostained with activated β1integrin antibody HUTS-4 (green) or ILK (red). <i>Scale bar in HUTS-4 staining image = 10 µm, and in the ILK staining image = 20 µm.</i> B. Western blot analysis of NSCs cultured in either control PA (VVIAK PA) or IKVAV PA for one day. Note that levels of ILK are markedly elevated in the IKVAV-PA group without any change in FAK. C. β1-integrin KO and control NSCs were transfected with either a control construct or a construct expressing a constitutively active form of ILK (ILK-CA). At 7DIV the cells were immunostained for GFAP and numbers of GFAP⁺ cells were counted. (*p≤0.05) D. β1-integrin KO and control NSCs were transfected with a control construct, a construct expressing constitutively active ILK (ILK-CA), or a construct expressing kinase-dead ILK (ILK-KD) and cultured for 7DIV. RNA was extracted for qPCR measurement of GFAP mRNA graphed as a ratio compared to the control (**p≤0.004). Note that β1-integrin KO increased levels of GFAP mRNA, but this increase was blocked by expression of ILK-CA. E. Western blot analysis of GFAP expression in control NSCs and in β1-integrin KO NSCs transfected with either with a control construct or ILK-CA. Note that transfection with ILK-CA prevented the increase in GFAP expression in β1-integrin KO NSCs. F. NSCs were treated with different doses of the ILK inhibitor, Cpd22. After 6DIV RNA was extracted for qPCR quantification of GFAP. Note that GFAP expression increased in a dose dependent manner after treatment with Cpd22. H. NSCs were treated with different doses of the ILK inhibitor, Cpd22 for 3h, and protein was extracted for western blot analysis. Note that treatment with the drug reduced levels of phospho-Akt without any change in levels of AKT.</p

β1-integrin regulates astrocytic differentiation.

<p>A. NSCs were infected with retrovirus expressing GFP alone (ctrl pCLE) orβ1-integrinlacking the cytoplasmic domain (β1-integrin ΔC), cultured for 7 days on PDL-laminin and immunostained for GFAP (red) and DAPI (blue). <i>Scale bar = 20 µm</i>. B. Astrocytic differentiation (GFAP expression) increased more than 2.5 fold (**p≤0.005) in the β1- integrin ΔC group compared to control while neuronal (βIIItubulin) and oligodendroglial (O4) differentiation were unchanged. C. NSCs were cultured either on PDL-laminin or IKVAV-PA for one day and immunostained with β1- integrin (green) and DAPI (blue). Culture in IKVAV-PA dramatically increased expression of β1- integrin. <i>Scale bar = 20 µm</i>. D. Western Blot analysis shows that NSCs express higher levels of β1integrin protein when cultured for one day in IKVAV-PA compared to control (PDL-laminin). E. Real time PCR amplification plots showing that the β1integrin transcripts increase in cells cultured in IKVAV-PA compared to control (PDL-laminin). F. Quantitation of the increase in transcript levels of β1integrin in the IKVAV PA versus PDL-laminin group. (**p≤0.0007). G. KO of β1-integrin increases the number of astrocytes (GFAP – red) generated by NSCs cultured in IKVAV PA for 7 DIV. Blue = DAPI. H. NSCs were cultured in either control PA (VVIAK PA) or IKVAV PA for 7DIV, and RNA was extracted for qPCR quantification. The graph represents each lineage marker mRNA level in IKVAV PA normalized to levels in the control PA. Levels of GFAP mRNA were profoundly decreased (**p≤0.0005 in the IKVAV-PA group) without any significant change in levels of βIIItubulin of CNPase mRNAs. I. NPCs were cultured either on PDL-laminin or in IKVAV PA for 7DIV and immunostained with DAPI (blue) and several lineage markers (red). Note the absence of GFAP staining in the IKVAV PA group and the increase in the progenitor markers Sox2 and nestin. <i>Scale bar = 20 µm</i>. J. Quantification of the percentages of cells in the conditions described in (i) (*p≤0.017, **p≤0.005).</p

β1-integrin signaling in vivo regulates astrogliogenesis after spinal cord injury (SCI).

<p>A. Low magnification (10X) images of representative longitudinal sections of injured mouse spinal cords at 3 weeks post SCI, that were injected with either the IKVAV PA, scrambled KIAVV PA or vehicle injected at 24 hours post injury. Sections are stained with DAPI (blue) and GFAP (red). <i>Scale bar = 200 µm.</i> B. Representative confocal Z-stacks taken at higher magnification (20X) of the areas boxed in (a) showing reduced gliosis in the IKVAV PA injected animals at 3 weeks post SCI. <i>Scale bar = 100 µm.</i> C. High magnification (63x) confocal images of the ependymal regions stained with activated β1integrin antibody HUTS-4 (red) and DAPI (blue). Injured mouse spinal cords were injected either with IKVAV PA or vehicle 2 days post injury. <i>Scale bar = 20 µm.</i> D. Spinal cord injured rats were injected with either Tenascin-C epitope presenting PA, RGD epitope presenting PA or vehicle 2 days post injury, and monitored with BBB scoring for 12 weeks. Both PA groups showed significant improvement compared to the vehicle group.</p

β1-integrin suppresses generation of astrocytes by NSCs derived from the spinal cord.

<p>A. Ependymal NSCs in adult mouse spinal cord express β1-integrin. B. NSCs derived from spinal cord are able to differentiate into astrocytes (GFAP -red), neurons (βIIItubulin-green) and oligodendrocytes (CNPase-green). β1-integrin Knock-Out (KO) increases astrocyte differentiation without altering neuronal or oligodendroglial differentiation. <i>Scale bar = 20 µm</i>. C. Western blot analysis shows depletion of β1 integrin protein levels in β1-itg KO NSCs derived from the spinal cord. D. Quantification of total cell numbers (DAPI) in the cultures of control and β1-integrin null cells analyzed for lineage commitment (E). There was no difference in overall cell numbers (n = 12, p = 0.89). Values are means ± SEM. E. Quantification of cell numbers of different lineages shows a significant increase (**p≤0.004) in astrocytes (67% of increase) in cultures of β1 integrin null cells at 7DIV without any change in neuronal or oligodendroglial differentiation.</p

β1-integrin suppresses generation of astrocytes by NSCs derived from the SVZ.

<p>A. NSCs derived from the SVZ show a similar profile of differentiation as those derived from spinal cord. β1-integrin Knock-Out (KO) increases astrocytic differentiation (GFAP - red) without altering neuronal (βIIItubulin - green) or oligodendroglial (O4 -red) differentiation. S<i>cale bar = 20 µm</i>. B. Western blot analysis shows depletion of β1-integrin protein in the β1KO derived NSCs compared with control. C. Quantification of total cell numbers (DAPI) in the cultures of control and β1-integrin null cells analyzed for lineage commitment (D). There was no difference in overall cell numbers (n = 20, p = 0.91). Values are means ± SEM. D. Quantification of cell numbers of different lineages shows a significant increase (*p≤0.046) in astrocytes (56% increase) in the β1-integrin KO group compared with control at 7DIV without any change in neuronal or oligodendroglial differentiation. E. Quantification of mRNA levels by qPCR after 7DIV reveals a significant increase in GFAP mRNA in the β1-integrin KO group compared with control. βIIItubulin and CNPase mRNAs were unchanged.</p