Supplemental Figure Legends and References

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1 Targeting novel Integrative Nuclear FGFR1 Signaling by nanoparticle-mediated gene transfer stimulates neurogenesis in adult brain Ewa K. Stachowiak, Indrajit Roy, Yu-Wei Lee, Mariolina Capacchietti, John M. Aletta, Paras N. Prasad, Michal K. Stachowiak 2* Supplemental Figure Legends and References Supplemental Figure 1. (A) A sagittal view of the rodent brain showing the neurogenic subventricular zone (SVZ; red) of the lateral ventricle (LV). Neural progenitor cells from the SVZ migrate through the rostral migratory stream (RMS; red) and differentiate into neurons in the olfactory bulb (OB). The other marked structures include corpus callosum (cc), striatum (STR) and brain cortex (cortex). (B) The microscopic images show an example of FGFR1 localization in cells of the mouse SVZ. Brain sections were co-immunostained with FGFR1 C- term Ab (B2) and anti-cbp Ab (B1) a well known nuclear marker. We previously showed that CBP is a binding partner for FGFR1 1. Arrows indicate nuclei of two cells in the same focal plane. One nucleus shows high concentration of FGFR1 while in the other this concentration is not observed. This observation is consistent with our earlier report that FGFR1 is present in the cytoplasm of many SVZ cells, while some cells accumulate FGFR1 in the nucleus 1. The role of nuclear FGFR1 and its ligand in neuronal differentiation is the subject of the present investigation. Size bar: 10 µm. Supplemental Figure 2. Expression of EGFP 5 days after injection of ORMOSIL complexed with EGFP DNA into the mouse brain lateral ventricle. As observed previously 2 the fixation process necessary for preserving the brain tissue cellular structure abolished EGFP fluorescence. Therefore, the transfected cells were visualized with a rabbit antibody raised against EGFP (+ anti-rabbit CY5 IgG). The illustration shows immunostaining in subventricular zone (SVZ) and the rostral migratory stream (RMS) after intraventricular injection of the EGFP expression vector mixed with ORMOSIL nanoparticles with (A) and without (B) surface amino groups. Specificity of immunostaining was further verified as previously 2 by omitting the primary EGFP antibody 1

2 which abolished the immunofluorescence (not shown). Amino-terminated ORMOSIL produces robust EGFP expression that is focused in SVZ cells lining the LV (A1). In addition, EGFP expression was observed in cells entering the RMS (A2). Some of these cells have neuronal-like morphology and could represent maturing neurons, however the great majority appear as typical round non-differentiated SVZ/RMS cells. In sharp contrast, negligible EGFP expression is observed following transfection mediated by non-amino terminated ORMOSIL in SVZ (B1) and RMS (B2). Thus the presence of amino-groups on the surface of ORMOSIL nanoparticles is critical for achieving high transfection efficiency. This provides an additional control for ORMOSIL nanoparticle mediated transfection. We conclude that the presence of surface amino groups drastically increases the transfection efficiencies. The modified formulation of the nanoparticles which has higher content of amino groups on their surface 2, 3 produces initial expression of the transfected EGFP that is restricted to the cells in the immediate vicinity of the lateral ventricle (injection site), thus allowing us to study the subsequent migration and development of these transfected cells. To ascertain that proteins encoded by two co-transfected plasmids are co-expressed in the same cells, the ORMOSIL nanoplexes containing1.3 μg EGFP μg of FGF receptor 1 tagged with of human Myc epitope 4 (C) or EGFP alone (D) were injected intraventricularily (4 μl) and the brains were co-immunsotained for EGFP (C1, D1) and for the Myc epitope (C2, D2) (C3, D3 merged images). In the SVZ area co-expression of both proteins in the same cells was observed in brains receiving double plasmid injection. Size bar: 25 μm. Supplemental Figure 3. Transfection of nuclear FGF receptor inhibits proliferation of NS/PC cells. Fourteen days after ORMOSIL transfection of control EGFP (A) or EGFP + R1 (B) the animals were injected with BrdU (i.p.) and were perfused 2 hours later. Brain sections were immunostained for EGFP (A1,B1) or DNA-incorporated BrdU (A2,B2); A3,B3 - merged color images. B2- arrow points towards nontransfected BrdU + cells. Size bar: 40 um Supplemental Figure 4. Reduced number of nestin-expressing cells in SVZ and adjacent area following transfection of R1 when compared to control DNA. Mice received injections of ORMOSIL complexed with control DNA (A) or with EGFP+R1 (B) into the brain lateral 2

3 ventricle and were killed 14 days later. Brain sections were immunostained with α-nestin + ALEXA558 fluorophor-conjugated secondary antibody. Cells in SVZ and RMS express nestin, a marker protein of proliferating immature neural stem/progenitor cells (NS/PC). A marked depletion of cells expressing Nestin can be observed in the rostral migratory stream (RMS) of R1-transfected mice compared to control DNA transfected mice indicating R1-induced cell differentiation. Hence, the loss of mitotic activity in the RMS in mice transfected with nuclear FGFR1 (Fig. S3) was accompanied by decreased expression of nestin. Size bar: 25 um. Supplemental Figure 5 (A-C). Nuclear R1 induces appearance of immature migrating neuroblasts in striatum, septum and cortex. Earlier, the reduction in the number of Nestin expressing cells SVZ/RMS suggested increased differentiation of the NS/PCs (supplemental Fig. 4). Using brains of the same mice we also examined whether nuclear R1 can induce early differentiation and migration of new neuroblasts towards their target brain regions. As a marker for such cells we tested Doublecortin (DCX) expression. Brain sections were immunostained with αdcx + Cy3 goat anti-rabbit 2nday antibody. In the brains of mice transfected with EGFP+R1 we found DCX expressing immature neuroblasts in periventricular brain regions striatum (A2) and, septum (B2) which were absent in control DNA-transfected brains (A1 and B1, respectively). In the brain cortex (C2) in mice transfected with nuclear R1, we observed an 2.4- fold increase in the numbers of DCX positive cells relative to that in control EGFP transfected mice (C1). Size bar: 25 um. Supplemental Figure 6. Mice were perfused 18 days after ORMOSIL transfection of EGFP + R1 Transfected cells migrate to the mouse cerebral cortex. Transfected cells marked by EGFP (A1) are found extensively in the mouse cerebral cortex. Many of these cells co-express DCX (A2) as illustrated by the merged images (A3). A similar robust appearance of transfected (EGFP + ) cells that are also DCX + is observed in HMW transfected mice (Fig. 4 A,B) in the brain cortex (cf. ORMOSIL transfection of control DNA where few transfected cells are DCX + (Fig. 3 A,C; the same experiment, parallel stained sections]. These observations emphasize the role of INFS in generation of DCX + migrating neuroblasts. Size bar: 25 um. 3

4 Supplemental Figure 7. Effects of nuclear R1 and its ligand (HMW) on the long-term retention of BrdU labeling by brain cells. Mice were injected intraperitonealy 4 times a day with 50 mg/kg of BrdU for 5 days. 24 hours after the last injection, mice were subjected to stereotaxic surgery during which the ORMOSIL-coupled DNA was injected bilaterally in the brain lateral ventricle. After an additional 18 days mice were perfused with 4% paraformaldehyde, the brain sections were processed for double-labeling immunocytochemistry. ORMOSIL transfections and brain regions shown as: (A) control DNA: SVZ and adjacent RMS; (B,C) EGFP + HMW: SVZ + RMS (B) and striatum (C). Double immunostaining for BrdU (A2-C2) with either EGFP (A1- B1) or DCX (C1) merged color images (A3-C3). Note presence of BrdU staining (B2,C2) of EGFP+ HMW transfected cells (B1,C1) and the general absence of BrdU (A2) in control DNA transfected (A1) cells; arrow points to a single EGFP + /BrdU + cell. This outcome of the longterm BrdU labeling is consistent with the results of the acute BrdU labeling experiment (two hours after BrdU injection) where the BrdU + cells were those transfected with control DNA (Fig. S3A) while the BrdU negative cells had co-transfected nuclear R1 (Fig. S3B) or HMW (not shown). Together these complementary experiments show that nuclear R1 and its HMW ligand inhibit cell proliferation (i.e., block acute BrdU labeling and prevent label dilution in pre-labeled cell). Double immunostaining for BrdU (C2) and DCX (C1) indicates that many of the BrdUlabeled cells have become the DCX-positive neuroblasts (C3) following the transfection of HMW. Size bar: 25 um. Supplemental Figure 8. Differentiation of newly generated SVZ cells into neurofilament L (NfL) expressing neurons. Mice received i.p. BrdU injections for 5 consecutive days followed by intraventricular ORMOSIL transfections of: (A) control DNA, SVZ/RMS or (B,C) EGFP+HMW, SVZ/RMS (B), RMS (C). Mice were perfused after an additional 18 days. Brain sections were triple-immunostained for EGFP (A1-C1), BrdU (A2-C2) and NfL (A3-C3); merged images (A4-C4). Note lack of EGFP coimmunostaining with BrdU or NfL in (A) and frequent triple coimmunostaining in (B). Higher magnification of the triple immunostained cortical neuron (C). 4

5 In conclusion, when triple immunostaining for EGFP, BrdU and the mature neuronal marker neurofilament L (NfL) was employed, cells expressing all three antigens in mice transfected with HMW are observed. Examples of such cells in SVZ-striatum region and an enlarged triple stained neuron are shown. No such cells were observed in control DNA transfected mice. Thus, activation of INFS promotes differentiation of newly generated SVZ cells into neurofilament L (NfL) expressing neurons (see footnote). Size bar (A,B): 25 um, (C): 15 um. Supplemental Figure 9. INFS stimulates DCX and tyrosine hydroxylase (TH) expression by PC12 cells. Cultured PC 12 cells were transfected with pcdna3.1 (control) or HMW or R1 expressing plasmids (transfection of cells was verified using EGFP plasmid, not shown) and were analyzed 5days after transfection. Some EGFP-transfected cultures were incubated with nerve growth factor (NGF, 50 ng/ml) 1 day after transfection for an additional 4 days after which the cells were fixed and stained with TH (blue) or DCX (red) antibodies. (A1,A2) control DNA, (B1,B2) control DNA + NGF, (C1,C2) HMW, (D1,D2) R1. In control nonstimulated cells (- NGF), the TH or DCX immunoreactivity was weak. However, both were markedly increased by treatment with NGF. The effects of NGF on DCX and TH expression were mimicked by transfection of HMW or R1. The experiment was repeated 3 times with similar results. Transfection efficiency evaluated by cotransfection with EGFP was similar for all experiment conditions (> 50%). Quantitative analysis of confocal images collected in the linear range of the fluorescent signal using Image J 4 confirmed that stimulation of INFS by DNA transfection or treatment with NGF increased intensity of immunofluorescence 4-10 fold (not shown). Thus, the overexpression of nuclear R1 or FGF-2 ligand is sufficient to induce neuronal differentiation in the absence of additional differentiation promoting stimulation (NGF). Footnote: One interesting question raised by this work is whether nuclear FGF2/FGFR1, in addition to promoting NS/PC differentiation, regulates the maturation process of newly generated neuroblasts. Further analyses of multiple time points after transfection and specifically the analysis of the ratio of new-born transfected neuroblasts (BrdU + /EGFP + /DCX + ) to new-born transfected neurons (BrdU/EGFP/NfL) will address this question. Progenitors transfected with nuclear FGF-2/FGFR1 can determine if the increased entry of NS/PC to the neuronal pathway (DCX-positive cells) is accompanied by accelerated formation of mature neurons or whether generation of mature neurons is increased simply reflecting the increased generation of DCX-positive cells. 5

6 References: 1. X. Fang, E. K. Stachowiak, S. M. Dunham-Ems, I. Klejbor and M. K. Stachowiak, J Biol Chem, 2005, 280, D. J. Bharali, I. Klejbor, E. K. Stachowiak, P. Dutta, I. Roy, N. Kaur, E. J. Bergey, P. N. Prasad and M. K. Stachowiak, Proc Natl Acad Sci U S A, 2005, 102, I. Klejbor, E. K. Stachowiak, D. J. Bharali, I. Roy, I. Spodnik, J. Morys, E. J. Bergey, P. N. Prasad and M. K. Stachowiak, J Neurosci Methods, 2007, 165, H. Peng, J. Myers, X. Fang, E. K. Stachowiak, P. A. Maher, G. G. Martins, G. Popescu, R. Berezney and M. K. Stachowiak, J Neurochem, 2002, 81,

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