Characterization of the Stability and Bio-functionality of Tethered Proteins on Bioengineered Scaffolds: Implications for stem cell biology and tissue repair

Background: Tethering proteins onto bioengineered scaffolds enables longterm delivery, however protein stability, release kinetics, and functionality over time remains unknown. Results: Tethered proteins remain stable and functional for several months, capable of activating intracellular signaling pathways and influencing cell fate. Conclusion: Tethered proteins are stable and functional long-term. Significance: Such knowledge may have implications for promoting tissue repair

Characterization of the Stability and Bio-functionality of Tethered Proteins on Bioengineered Scaffolds IMPLICATIONS FOR STEM CELL BIOLOGY AND TISSUE REPAIR

Various engineering applications have been utilized to deliver molecules and compounds in both innate and biological settings. In the context of biological applications, the timely delivery of molecules can be critical for cellular and organ function. As such, previous studies have demonstrated the superiority of long-term protein delivery, by way of protein tethering onto bioengineered scaffolds, compared with conventional delivery of soluble protein in vitro and in vivo. Despite such benefits little knowledge exists regarding the stability, release kinetics, longevity, activation of intracellular pathway, and functionality of these proteins over time. By way of example, here we examined the stability, degradation and functionality of a protein, glial-derived neurotrophic factor (GDNF), which is known to influence neuronal survival, differentiation, and neurite morphogenesis. Enzyme-linked immunosorbent assays (ELISA) revealed that GDNF, covalently tethered onto polycaprolactone (PCL) electrospun nanofibrous scaffolds, remained present on the scaffold surface for 120 days, with no evidence of protein leaching or degradation. The tethered GDNF protein remained functional and capable of activating downstream signaling cascades, as revealed by its capacity to phosphorylate intracellular Erk in a neural cell line. Furthermore, immobilization of GDNF protein promoted cell survival and differentiation in culture at both 3 and 7 days, further validating prolonged functionality of the protein, well beyond the minutes to hours timeframe observed for soluble proteins under the same culture conditions. This study provides important evidence of the stability and functionality kinetics of tethered molecule

Mutant TDP-43 deregulates AMPK activation by PP2A in ALS models.

Bioenergetic abnormalities and metabolic dysfunction occur in amyotrophic lateral sclerosis (ALS) patients and genetic mouse models. However, whether metabolic dysfunction occurs early in ALS pathophysiology linked to different ALS genes remains unclear. Here, we investigated AMP-activated protein kinase (AMPK) activation, which is a key enzyme induced by energy depletion and metabolic stress, in neuronal cells and mouse models expressing mutant superoxide dismutase 1 (SOD1) or TAR DNA binding protein 43 (TDP-43) linked to ALS. AMPK phosphorylation was sharply increased in spinal cords of transgenic SOD1G93A mice at disease onset and accumulated in cytoplasmic granules in motor neurons, but not in presymptomatic mice. AMPK phosphorylation also occurred in peripheral tissues, liver and kidney, in SOD1G93A mice at disease onset, demonstrating that AMPK activation occurs late and is not restricted to motor neurons. Conversely, AMPK activity was drastically diminished in spinal cords and brains of presymptomatic and symptomatic transgenic TDP-43A315T mice and motor neuronal cells expressing different TDP-43 mutants. We show that mutant TDP-43 induction of the AMPK phosphatase, protein phosphatase 2A (PP2A), is associated with AMPK inactivation in these ALS models. Furthermore, PP2A inhibition by okadaic acid reversed AMPK inactivation by mutant TDP-43 in neuronal cells. Our results suggest that mutant SOD1 and TDP-43 exert contrasting effects on AMPK activation which may reflect key differences in energy metabolism and neurodegeneration in spinal cords of SOD1G93A and TDP-43A315T mice. While AMPK activation in motor neurons correlates with progression in mutant SOD1-mediated disease, AMPK inactivation mediated by PP2A is associated with mutant TDP-43-linked ALS

AMPK activation is diminished in spinal cords and brains of mutant TDP-43 mice from pre-symptomatic age.

<p>Immunoblot analysis of phosphorylated (pAMPK) and total AMPK levels in <b>A</b>, spinal cord and <b>E</b>, brain of pre-symptomatic (P60) and symptomatic (P90) transgenic TDP-43^A315T and age-matched WT mice. Quantification of pAMPK/AMPK ratio level in <b>B</b>, spinal cord and <b>F</b>, brain from immunoblots normalised to WT mice.Immunoblot analysis of <b>C</b>, PP2A, PP2C, PP1 and PPM1E with<b>E</b>, quantification of PP2A level from immunoblots for spinal cord. Data represent mean ± SEM, <i>n</i> = 4-5 mice per group, *<i>p</i><0.05 and ***<i>p</i><0.001 compared to WT mice using unpaired t-test.</p

AMPK activation is diminished in NSC-34 cells stably expressing mutant TDP-43 by a PP2A-dependent mechanism.

<p><b>A</b>, Immunoblot analysis of phosphorylated (pAMPK) and total AMPK in NSC-34 cells stably transfected with human wild-type (WT) or mutant D169G, A315T, Q331K and M337V TDP-43. <b>B</b>, Quantification of pAMPK/AMPK ratio level from immunoblots normalised to WT TDP-43 expressing cells. Data represent mean ± SEM, <i>n</i> = 3 experiments, *<i>p</i><0.05 and **<i>p</i><0.01 compared to WT using one-way ANOVAwith Tukey's post-hoc test. <b>A</b>, PP2A immunoblot analysis and <b>C</b>, quantification of PP2A level from immunoblots. <b>D</b>,Immunocytochemical analysis of cells expressing WT TDP-43 shows cytoplasmic pAMPK granules which are reduced in mutant TDP-43 expressing cells. <b>E</b>, Immunoblot analysis of pAMPK and total AMPK levels in TDP-43 stable cells treated with 0-1 µM okadaic acid (OA) for 30 min.</p

AICAR stimulates activation of AMPK in NSC-34 cells.

<p><b>A</b>, Immunoblot analysis of phosphorylated (pAMPK) and total AMPK levels in cells treated with 0–2 mM AICAR for 2 hr. <b>B</b>, Immunocytochemical analysis of untreated cells shows predominantly cytoplasmic pAMPK. Cells treated with AICAR revealdose-dependent and increased cytoplasmic pAMPK immunoreactivity. <b>C</b>,Survival analysis of cells treated with 0–2 mM AICAR for 2 hr. Data represent mean ± SEM, <i>n</i> = 3 experiments. None of the AICAR treatment groups were significantly different to untreated controls using one-way ANOVAwith Tukey's post-hoc test.</p

AMPK activation in NSC-34 cells stably expressing normal or mutant SOD1.

<p><b>A</b>, Immunoblot analysis of phosphorylated (pAMPK) and total AMPK in NSC-34 cells stably transfected with human wild-type (WT) or mutant A4V, G37R, G85R or G93A SOD1 reveals similar levels of AMPK activation. <b>B</b>,Quantification of pAMPK/AMPK ratio level in cells from immunoblots normalised to WT SOD1 expressing cells. Data represent mean ± SEM, <i>n</i> = 3 experiments.<b>A</b>, PP2A immunoblot analysis and <b>C</b>, quantification of PP2A level from immunoblots.<b>D</b>,Immunocytochemical analysis of cells expressing WT or mutant SOD1 shows similar cytoplasmic localisation of pAMPK.</p

AMPK activation is increased in spinal cords of symptomatic mutant SOD1 mice, but not pre-symptomatic mice.

<p>Immunoblot analysis of phosphorylated (pAMPK) and total AMPK levels in <b>A</b> spinal cord,<b>C</b> brain,<b>E</b> liver and <b>G</b> kidney of pre-symptomatic (P60) and symptomatic (P90) transgenic SOD1^G93A and age-matched wild-type (WT) mice. Quantification of pAMPK/AMPK ratio level in <b>B</b> spinal cord, <b>D</b> brain, <b>F</b> liver and <b>H</b> kidney from immunoblots normalised to WT mice. Immunoblot analysis of <b>A</b>, PP2A and <b>I</b>, quantification of PP2A level from immunoblots for spinal cord. Data represent mean ± SEM, <i>n</i> = 4–5 mice per group, *<i>p</i><0.05 and **<i>p</i><0.01 compared to WT mice using unpaired t-test.</p