Micellar nanocomplexes for biomagnetic delivery of intracellular proteins to dictate axon formation during neuronal development

Abstract

During mammalian embryonic development, neurons polarize to create distinct cellular compartments of axon and dendrite that inherently differ in form and function, providing the foundation for directional signaling in the nervous system. Polarization results from spatio-temporal segregation of specific proteins' activities to discrete regions of the neuron to dictate axonal vs. dendritic fate. We aim to manipulate axon formation by directed subcellular localization of crucial intracellular protein function. Here we report critical steps toward the development of a nanotechnology for localized subcellular introduction and retention of an intracellular kinase, LKB1, crucial regulator of axon formation. This nanotechnology will spatially manipulate LKB1-linked biomagnetic nanocomplexes (LKB1-NCs) in developing rodent neurons in culture and in vivo. We created a supramolecular assembly for LKB1 rapid neuronal uptake and prolonged cytoplasmic stability. LKB1-NCs retained kinase activity and phosphorylated downstream targets. NCs were successfully delivered to cultured embryonic hippocampal neurons, and were stable in the cytoplasm for 2 days, sufficient time for axon formation. Importantly, LKB1-NCs promoted axon formation in these neurons, representing unique proof of concept for the sufficiency of intracellular protein function in dictating a central developmental event. Lastly, we established NC delivery into cortical progenitors in live rat embryonic brain in utero. Our nanotechnology provides a viable platform for spatial manipulation of intracellular protein-activity, to dictate central events during neuronal development.

Introduction

During mammalian embryonic development, neuronal cells polarize to create morphologically and functionally distinct compartments of axon and dendrite. Axons and dendrites inherently differ in molecular composition of their cytoplasm, cytoskeleton, and plasma membrane, in morphology and function, to underlie the directed information flow in the mature brain [1], [2], [3], [4], [5], [6], [7]. During neuronal development, the polarized architecture of axon and dendrite arises from highly regulated spatial segregation of specific proteins' activities to discrete regions of the cell, to respectively dictate the axonal or dendritic fate [1], [2], [3], [4], [5], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26]. Aberrations in the localization of these proteins' activity lead to defective neuron polarization, and underlie developmental neuropathologies including intellectual and motor disabilities, epilepsy and autism spectrum disorders. The ability to exert spatio-temporal control on intracellular protein activity would allow directed regulation of neuronal polarization, and would lead to the generation of new protein-based therapeutic approaches for the repair of neuropathologies that result from break-down of polarity.

Current methodologies for spatio-temporal manipulation of protein function predominantly use light-controlled activation [27], [28], [29], [30]. Such approaches were applied to short-term (minutes) localized control of cytoskeleton dynamics by small GTPases, resulting in directed cell protrusions and movement [27] in cultured fibroblasts. However, light-controlled protein activation does not enable its long-term spatial confinement, as the protein can diffuse or actively relocate following activation. Furthermore, molecular-genetics techniques only allow whole-cell up- or down-regulation but not spatio-temporal manipulation of protein function. Thus, the ability for spatial restriction of intracellular protein activity to dictate a complex and long-term developmental process as axon formation, which lasts for hours and days, is currently missing. We sought to develop a biomagnetic-based nanotechnology that would deliver and restrict the activity of critical intracellular proteins in a spatio-temporal manner to control axon formation. In this study we report the essential steps toward the development of this nanotechnology. We engineered biomagnetic micellar nanoparticles for the purpose of subcellular delivery of a crucial intracellular protein, the kinase LKB1, in developing neurons in culture and the rodent embryonic brain, to dictate axon formation.

Much of the current understanding of the molecular events underlying axon initiation is based on studies using dissociated rodent embryonic hippocampal neurons in culture [1], [2], [3], [4], [5]. These neurons undergo polarization - from a cell exhibiting several morphologically similar neurites within hours of plating, to a mature neuron with a single axon and multiple dendrites within days. Studies from these cultured neurons implicated several signaling determinants in axon formation, including the six Par (partitioning-defective) proteins (Par-1 through Par-6) [8], [31]. Recent studies [20], [22], [23], [25] have established the mammalian kinase LKB1, Par-4 counterpart, as a crucial upstream regulator of axon formation. We and others have shown that down-regulation of LKB1 in cultured hippocampal neurons greatly reduced axon formation [22], [23], [25]. Moreover, deletion of LKB1 in cortical progenitors in the rodent embryonic brain, resulted in striking absence of axon formation in neurons throughout cortical layers in vivo [22], [23], [25]. Furthermore, our previous findings underscore the critical importance for spatial restriction of LKB1-activity in the undifferentiated neurite to promote its axon development, in vitro and in vivo [20], [25]. Localized LKB1-activity in a single neurite may initiate axon development via phosphorylation and activation of the down-stream effectors SAD [22], [23] and MARK [17], [32], [33], [34], [35] kinases, and their downstream actions on local regulation of the cytoskeleton. SAD-A and SAD-B were shown, in an LKB1-dependent manner, to phosphorylate the axonal microtubule binding protein Tau [22], [23], which regulates local microtubule (MT) organization that is required for axon formation.

Together, these findings suggest that localized LKB1-activity might serve an instructive signal for axon formation from a single undifferentiated neurite. However, direct demonstration that localized LKB1 activity indeed instructs axon formation, is currently absent. Our overall objective is to develop a biomagnetic-based nanotechnology to control intracellular protein activity, the LKB1 kinase, in a spatio-temporal manner, with the specific goal of locally activating the cell-signaling for axon formation, sustained over a period of 48 h, in culture and in the embryonic brain in vivo. We will direct localized subcellular introduction and retention of LKB1 into single undifferentiated neuritic processes, by spatially manipulating magnetic nanoparticles (MNPs) complexed with LKB1, using an externally applied magnetic field, to dictate the axon fate of these neuritic processes. Continuous localized LKB1-activity delivered in this manner will manipulate axon formation in developing rodent neurons in culture and the embryonic brain.

Here we report the critical steps toward the development of this nanotechnology. First, we created a supramolecular micellar assembly for effectively complexing LKB1 with MNPs to allow its rapid neuronal uptake and prolonged cytoplasmic availability and stability. We show that LKB1 linked to the nanocomplex retained its kinase activity and phosphorylated downstream targets. The nanocomplexes were successfully delivered to cultured embryonic hippocampal neurons and did not affect their growth or development. Importantly, delivery of LKB1-linked nanocomplexes promoted axon formation in these cultured neurons. Lastly, we established a nanotechnology for micellar delivery in cortical progenitor cells in the live rat embryonic brain in utero.

Together, here we report the generation of a multifunctional nanocomplex and show that it is indeed a suitable platform for the localized delivery of LKB1 into an undifferentiated neurite to dictate axonal fate. We emphasize two particularly unique findings reported in this study. First, the demonstration of the promotion of axon formation in cultured hippocampal neurons upon delivery of a critical intracellular protein, the kinase LKB1, represents an important proof of concept for the sufficiency of intracellular protein function in dictating a central developmental event as axon formation, over a physiological time scale (2–3 days). Second, the methodology for the intracellular micelle delivery into the live rodent embryonic brain in utero, with the purpose of manipulating neuronal development, represents a unique approach that was established in this study. These findings offer a framework for our overall goal of directing localization of intracellular protein-activity during crucial events of neuronal development.