Microtubule-Associated Type II Protein Kinase A Is Important for Neurite Elongation

<div><p>Neuritogenesis is a process through which neurons generate their widespread axon and dendrites. The microtubule cytoskeleton plays crucial roles throughout neuritogenesis. Our previous study indicated that the amount of type II protein kinase A (PKA) on microtubules significantly increased upon neuronal differentiation and neuritogenesis. While the overall pool of PKA has been shown to participate in various neuronal processes, the function of microtubule-associated PKA during neuritogenesis remains largely unknown. First, we showed that PKA localized to microtubule-based region in different neurons. Since PKA is essential for various cellular functions, globally inhibiting PKA activity will causes a wide variety of phenotypes in neurons. To examine the function of microtubule-associated PKA without changing the total PKA level, we utilized the neuron-specific PKA anchoring protein MAP2. Overexpressing the dominant negative MAP2 construct that binds to type II PKA but cannot bind to the microtubule cytoskeleton in dissociated hippocampal neurons removed PKA from microtubules and resulted in compromised neurite elongation. In addition, we demonstrated that the association of PKA with microtubules can also enhance cell protrusion using the non-neuronal P19 cells. Overexpressing a MAP2 deletion construct which does not target PKA to the microtubule cytoskeleton caused non-neuronal cells to generate shorter cell protrusions than control cells overexpressing wild-type MAP2 that anchors PKA to microtubules. Finally, we demonstrated that the ability of microtubule-associated PKA to promote protrusion elongation was independent of MAP2 phosphorylation. This suggests other proteins in close proximity to the microtubule cytoskeleton are involved in this process.</p> </div

MAP2c-independent removal of PKA from microtubules resulted in shorter neurites in dissociated hippocampal neurons.

<p>(A) E18 mouse hippocampal neurons were transfected with plasmid overexpressing EGFP (left column) or PKABD-EGFP (right column) immediately after dissociation, incubated for 48 hours, and permeabilized with triton X-100 before formaldehyde fixation. Fixed neurons were stained with antibody against β-III-tubulin (top column) and antibody against PKA-RIIβ subunit (bottom). All scale bars represent 20 µm. (B) Quantification of microtubule-associated PKA in neurites. Error bars represent SD. All PKA signals were normalized to the PKA signal in untransfected neurons. (C) E18 mouse hippocampal neurons were transfected with plasmid overexpressing EGFP or PKABD-EGFP immediately after dissociation and fixed after 48 hours. All images show signal from EGFP and all scale bars represent 50 µm. Quantification of total neurite length per neuron (D), average neurite length (E), and average neurite number per neuron (F) in transfected 2DIV hippocampal neurons. * p < 0.05, two-tailed Student’s t-test. Error bars represent SEM from 3 independent repeats. More than 190 neurons were analyzed for each constructs.</p

MAP2c-independent PKA recruitment onto microtubules resulted in longer neurite-like cell protrusions in non-neuronal cells.

<p>(A) Image of P19 cells transfected with plasmid overexpressing PKABD-Tub1 (left) or EGFP-Tub1 (right) constructs for 20 hours, incubated with 1 µM of latrunculin-A and 0.5 µM of taxol for 4 hours, and formaldehyde fixed. All scale bars represent 20 µm. Quantification of average protrusion length (B) or average protrusion number per cell (C). ** p < 0.01, two-tailed Student’s t-test. Error bars represent SEM from three independent repeats. More than 120 cells were analyzed for each constructs.</p

Removing PKA from microtubules using MAP2c constructs resulted in shorter neurites in dissociated hippocampal neurons.

<p>(A) E18 mouse hippocampal neurons were transfected with plasmid overexpressing MAP2c-ΔRII-EEE-EGFP (left column) or MAP2c-EEE-EGFP (right column) immediately after dissociation, incubated for 48 hours, and permeabilized with triton X-100 before formaldehyde fixation. Fixed neurons were stained with antibody against β-III-tubulin (top column) and antibody against PKA-RIIβ subunit (bottom). All scale bars equal 20 µm. (B) Quantification of microtubule-associated PKA in neurites. Error bars represent SD. All PKA signals were normalized to the PKA signal in untransfected neurons. (C) E18 mouse hippocampal neurons were transfected with plasmid overexpressing MAP2c-EEE-EGFP, MAP2c-ΔRII-EEE-EGFP, or EGFP immediately after dissociation and fixed after 48 hours. All images show signal from EGFP and were inverted to improve the visual presentation. All scale bars represent 50 µm. Quantification of total neurite length per neuron (D), average neurite length (E), and average neurite number per neuron (F) in transfected 2DIV hippocampal neurons. * p < 0.05, ** p < 0.01, one way ANOVA followed by Tukey’s post-hoc analysis. Error bars represent SEM from 3 independent repeats. More than 190 neurons were analyzed for each constructs.</p

Recruiting PKA to MAP2c-stabilized microtubules resulted in longer neurite-like cell protrusions in non-neuronal cells.

<p>(A) Images of non-neuronal P19 cells transfected with plasmid overexpressing MAP2c-EGFP (left) or MAP2c-ΔRII-EGFP (right) for 20 hours, incubated with 1 µM of latrunculin-A for 4 hours, and fixed with formaldehyde. All images show signal from EGFP and all scale bars represent 20 µm. Quantification of average protrusion length (B) and average protrusion number per cell (C). * p < 0.05, two-tailed Student’s t-test. Error bars represent SEM from three independent repeats. More than 300 cells were analyzed for each constructs.</p

Photocontrollable Probe Spatiotemporally Induces Neurotoxic Fibrillar Aggregates and Impairs Nucleocytoplasmic Trafficking

The abnormal assembly of misfolded proteins into neurotoxic aggregates is the hallmark associated with neurodegenerative diseases. Herein, we establish a photocontrollable platform to trigger amyloidogenesis to recapitulate the pathogenesis of amyotrophic lateral sclerosis (ALS) by applying a chemically engineered probe as a “switch” in live cells. This probe is composed of an amyloidogenic peptide from TDP-43, a photolabile linker, a polycationic sequence both to mask amyloidogenicity and for cell penetration, and a fluorophore for visualization. The photocontrollable probe can self-assemble into a spherical vesicle but rapidly develops massive nanofibrils with amyloid properties upon photoactivation. The photoinduced <i>in vitro</i> fibrillization process is characterized by biophysical techniques. In cellular experiments, this cell-penetrable vesicle was retained in the cytoplasm, seeded the mislocalized endogenous TDP-43 into aggregates upon irradiation, and consequently initiated apoptosis. In addition, this photocontrollable vesicle interfered with nucleocytoplasmic protein transport and triggered cortical neuron degeneration. Our developed strategy provides <i>in vitro</i> and <i>in vivo</i> spatiotemporal control of neurotoxic fibrillar aggregate formation, which can be readily applied in the studies of protein misfolding, aggregation-induced protein mislocalization, and amyloid-induced pathogenesis in different diseases