TKI increases parkin ubiquitination and interaction with Beclin-1.

<p><i>In Situ</i> PLA in 20 µm thick mouse brain sections shows <b>A</b>) parkin-Beclin-1 interaction in the striatum of C57BL/6 control, <b>B</b>) parkin^−/− mice, <b>C</b>) transgenic A53T mice treated with daily I.P injection for 3 weeks of DMSO <b>D</b>) 10 mg/kg nilotinib and <b>E</b>) 5 mg/kg bosutinib (n = 5). <i>In situ</i> PLA in 20 µm thick mouse brain sections showing parkin ubiquitination via <b>F</b>) parkin-ubiquitin interaction in the striatum of C57BL/6 control, <b>G</b>) transgenic A53T mice treated with daily I.P injection for 3 weeks of DMSO <b>H</b>) 10 mg/kg nilotinib and <b>I</b>) 5 mg/kg bosutinib (n = 5). <b>J</b>) Histograms represent parkin ubiquitination in B35 rat neuroblastoma cells treated with either 10 µM nilotinib or 1 µM bosutinib and 20 µM proteasome inhibitor MG132. Parkin ubiquitination was observed with WT ubiquitin not the K0 control (n = 6), compared to recombinant E1-E2-E3 control. Asterisk indicates significantly different, means±SEM, ANOVA, Newman Keuls, n = 6) and <b>K</b>). immnunoprecipitation of cell extracts with anti-ubiquitin antibodies and WB with parkin showing ubiquitinated proteins (n = 5).</p

Parkin co-localizes with Beclin-1 in human brain but their interaction is decreased in sporadic PD.

<p>Immunostaining of paraffin embedded 20 µm thick human midbrain sections <b>A</b>) shows parkin expression in DAPI stained neurons co-labeled with GFAP. Inset is higher magnification showing parkin expression. <b>B</b>) TH⁺ neurons counterstained with DAB in serial midbrain sections of healthy human subjects. <b>C</b>) <i>In situ</i> proximity ligation assay (PLA) shows human parkin-Beclin-1 interaction in control midbrain neurons (n = 7). <b>D</b>) Parkin expression in DAPI stained neurons co-labeled with GFAP indicating active astrocytes. Inset is higher magnification showing cytosolic parkin accumulation. <b>E</b>) TH⁺ neurons counterstained with DAB in serial midbrain sections of post-mortem PD brains. <b>F)</b><i>In situ</i> PLA shows reduced human parkin-Beclin-1 interaction in midbrain neurons in PD brains (n = 9). <b>G</b>). parkin and <b>H</b>) and Beclin-1 labeling are co-localized <b>I</b>) in human midbrain sections (n = 7). <b>J</b>) shows alteration of parkin expression and <b>K</b>) Beclin-1 staining with <b>L</b>) reduced co-localization in sporadic PD brains (n = 9). <b>M</b>) graph represents stereological counting of C and F <i>in situ</i> PLA and <b>N</b>) WB analysis on 4−12% NuPage SDS gel showing beclin-1 levels relative to actin and immunoprecipitation in post-mortem human brain tissues. Asterisk indicates significantly different, means±SEM, ANOVA, Newman Keuls.</p

TKIs stimulate autophagic α-Synuclein clearance in A53T mice.

<p>ELISA measurement of human α-Synuclein in the <b>A</b>) brain and <b>B</b>) blood of 1 month old A53T mice injected I.P. with either 5 mg/kg or 1 mg/kg bosutinib once every other day for 6 weeks (n = 10). <b>C</b>). Insert shows separation of AVs and lysosomal fractions. Histograms represent subcellular fractionation of total brain lysates from A53T mice and ELISA of human α-Synuclein in AVs in 1 and 5 months old A53T brains after daily I.P. injection with 10 mg/kg nilotinib or 5 mg/kg bosutinib for 3 weeks (n = 5). <b>D</b>). ELISA showing parkin level in total brain extracts of A53T mice compared to WT and parkin^−/− in the presence of nilotinib and bosutinib (n = 10). <b>E</b>). Histograms represent subcellular fractionation of total brain lysates from A53T mice and parkin ELISA in AVs in 1 and 5 months old A53T brains after daily I.P. injection with 10 mg/kg nilotinib or 5 mg/kg bosutinib for 3 weeks (n = 5). Asterisk indicates significantly different, means±SEM, ANOVA, Newman Keuls. F) WB analysis on 4−12% NuPage SDS gel showing parkin and α-Synuclein levels relative to actin in A53T mice treated with either DMSO or bosutinib.</p

TKI clears α-Synuclein and protects TH⁺ neurons in a parkin-dependent manner.

<p>Immunostaining of 20 µm thick mouse brain sections showing human α-Synuclein counterstained with DAB in the SN of lentiviral injected <b>A</b>) LacZ WT mice treated with DMSO for 3 weeks, <b>B</b>) LacZ WT treated with 5 mg/kg bosutinib, <b>C</b>) human WT α-Synuclein in WT mice treated with DMSO, and <b>D</b>) human WT α-Synuclein in WT mice treated with 5 mg/kg bosutinib (n = 10). TH⁺ labeling counterstained with DAB in the SN of lentiviral injected <b>E</b>) LacZ WT mice treated with DMSO for 3 weeks, <b>F</b>) LacZ WT treated with 5 mg/kg bosutinib, <b>G</b>) human WT α-Synuclein in WT mice treated with DMSO, and <b>H</b>) human WT α-Synuclein in WT mice treated with 5 mg/kg bosutinib (n = 10). Human α-Synuclein in the SN of parkin^−/− mice counterstained with DAB injected with <b>I)</b> LacZ treated with DMSO, or human α-Synuclein treated with <b>J</b>) DMSO, <b>K</b>) 5 mg/kg bosutinib and <b>L</b>) 10 mg/kg nilotinib for 3 weeks (n = 10). Staining of TH⁺ neurons in the SN of parkin^−/− mice counterstained with DAB injected with <b>M</b>) LacZ treated with DMSO <b>O</b>) is higher magnification, or lentiviral α-Synuclein treated with <b>N</b>) DMSO, <b>R</b>) is a higher magnification <b>O</b>) 5 mg/kg bosutinib, <b>S</b>) is a higher magnification and <b>P</b>) 10 mg/kg nilotinib, <b>T</b>) is a higher magnification (n = 10). <b>U</b>) graph represents stereological counting and <b>V</b>) RT-PCR showing equal amount of α-Synuclein mRNA relative to GADPH in both parkin^−/− and WT mice with and without TKIs (n = 10). Asterisk indicates significantly different, means±SEM, ANOVA, Newman Keuls.</p

Parkin deletion increases α-Synuclein secretion from the brain into the blood.

<p>Histograms represent ELISA in WT and parkin^−/− mice stereotaxically injected with lentiviral human α-Synuclein or LacZ showing α-Synuclein levels in <b>A</b>) brain lysates and <b>B</b>) total blood (n = 10) in mice treated with DMSO, 10 mg/kg nilotinib or 5 mg/kg bosutinib for 3 weeks. <b>C</b>). Histograms represent subcellular fractionation of midbrain lysates and ELISA of human α-Synuclein in AVs of WT and parkin^−/− mice after daily I.P. injection with DMSO, 10 mg/kg nilotinib or 5 mg/kg bosutinib for 3 weeks (n = 5). <b>D</b>) WB on 4−12% SDS-NuPAGE gel showing parkin (1^st blot), α-Synuclein (2^nd blot) Beclin-1 (3^rd blot) and LC3-I/LC3-II (4^th blot) relative to actin (5^th blot). <b>E</b>). Histograms represent dopamine and HVA levels in total brain lysates in WT and parkin^−/− mice injected with lentiviral α-Synuclein or LacZ and treated with daily I.P. of DMSO, 10 mg/kg nilotinib or 5 mg/kg bosutinib for 3 weeks (n = 10). Asterisk indicates significantly different, means±SEM, ANOVA, Newman Keuls, p<0.05.</p

Schematic representation of MMP-dependent ICAM-5 signaling at the synapse.

<p>In figure 6 we show a hypothetical model in which MMPs are rapidly released from preformed peri-synaptic stores to cleave ICAM-5 (green and lavender) at a membrane proximal site. The released N terminal fragment can bind unengaged integrins (red ovals) to stimulate intracellular signaling cascades leading to increased phosphorylation and membrane insertion of GluA1 subunits. Following ectodomain shedding, the C terminal fragment of ICAM-5 could undergo additional processing followed by internalization and degradation. It is worth noting that following MMP or ADAM mediated shedding, select CAMs are further processed by intramembranous proteolysis. ICDs thus generated may be degraded or, in some cases, influence gene transcription.</p

Soluble ICAM-5 affects an increase in GluA1 surface staining along dendrites in particular.

<p>Figure 4 shows data from live cell surface staining for GluA1 in control and ICAM-5 treated hippocampal neurons at 14 and 21 DIV. Indicated cultures were treated with 2.5 µg/ml soluble ICAM-5 and surface staining performed 1 hour later. Representative images are shown in A and C, while quantitative data is shown in B and D. The mean and standard error for percent control values were 100 +/- 8.8, n=21 for the DIV 14 control group; 187.5 +/- 16.4, n=16 for the DIV 14 ICAM-5 group; 100 +/- 5.8, n=25 for the DIV 21 control group; and 146.3 +/- 7.9, n=25 for the DIV 21 ICAM-5 group. Differences in GluA1 staining between control and ICAM-5 treated cultures are significant at <i>p</i>< 0.01 (*) at both 14 and 21 DIV.</p

The ICAM-5 ectodomain stimulates an increase in surface levels of the glutamate receptor subunit GluA1.

<p>Rat hippocampal neurons were unstimulated (control) or stimulated for 60 min. with 1 µg/ml of the ICAM-5 ectodomain (R & D Systems). Surface proteins were then biotinylated, and biotinylated proteins pulled down to be analyzed by Western blot. As can be appreciated, ICAM-5 was associated with an increase in surface GluA1 (A). Blots from separate experiments are shown. Densitometric analysis showing the fold increase in GluA1 band intensity in ICAM-5 versus control treated cultures in shown in (B). The mean and standard error for the fold increase from 6 replicate experiments is shown, and the difference between control and ICAM-5 groups is significant at <i>p</i> < 0.1 (*<i>p</i>=0.05). A representative blot for GluA2 in surface protein preparations is shown in (C), and densitometric analysis showing the fold change in GluA2 band intensity from 3 replicate experiments follows in (D). The mean and standard error for the fold change from 3 replicate experiments is shown, and the difference between control and ICAM-5 groups is not significant (<i>p</i>= 0.6).</p

The ICAM-5 ectodomain affects an increase in mini excitatory post synaptic current (mEPSC) frequency.

<p>Stimulation of rat hippocampal neurons with 1 µg/ml ICAM-5 ectodomain (60 min. pretreatment) is associated with an increase in mEPSC frequency. In these experiments, 1,468 events from 11 control cells and 2353 events from 16 ICAM-5 stimulated cells were evaluated using standard techniques [74]. Representative tracings are shown in (A) while the average mEPSC frequency is shown in (B) and amplitude in (C). The difference between mEPSC frequency in control and ICAM treated neurons was significant (*<i>p</i> < 0.05, Student’s <i>t</i> test).</p

Phosphorylation of GluA1 at serine-845 is increased by soluble ICAM-5.

<p>Rat hippocampal neurons were unstimulated (control) or treated for 60 min. with 1 µg/ml of soluble ICAM-5 and lysates tested by Western blot for phospho-serine 845 GluA1. A representative blot is shown in (A) while densitometric analysis of blots from 5 experiments using distinct cultures is shown in (B). The mean and standard error for the fold increase is shown, and the fold increase is significant at <i>p</i> < 0.1 (*<i>p</i>= 0.06).</p