Micellar nanocomplexes for biomagnetic delivery of intracellular proteins to dictate axon formation 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.
Section snippets
Materials and antibodies
Glycol chitosan (250 kDa molecular weight, degree of deacetylation > 60%), 5β-cholanic acid, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), and N-Hydroxy-Succinimide (NHS) were purchased from Sigma-Aldrich (St. Louis, MO). Monoreactive hydroxysuccinimide ester of Cyanine 5.5 (Cy5.5-NHS) was obtained from Lumiprobe (Hallandale Beach, FL). Biotin-4-fluorescein (B4F) was obtained from Biotium (Hayward, CA). Superparamagnetic iron oxide nanoparticles (nano-screenMAG-ARA,
Physicochemical characteristics of LKB1-MNP-chitosan nanocomplexes (LKB1-NCs)
We describe the design and fabrication (Fig. 1) of biomagnetic micellar LKB1-nanocomplexes (LKB1-NCs), assembled using a naturally-derived polymer, chitosan, that serves two crucial purposes. First, chitosan should allow rapid delivery of the NCs into the cell [39], [40] (see Fig. 3), and enable their escape from endosomal entrapment and lysosomal degradation [41], [42], [43] (Supplementary Fig. S3). Nanoparticle capture within the endosomal-lysosomal pathway as a consequence of endocytosis is
Discussion
Fundamental events during embryonic neuronal development as neurogenesis, migration, directed formation of axon and dendrite, and synaptogenesis, are regulated by protein activity in a highly spatio-temporal manner. Our study aims to manipulate an early event in neuronal development, where neuronal cells polarize to create morphologically and functionally distinct compartments of axon and dendrite, a critical architecture that allows the directed connectivity and information flow in the brain.
Conclusions
We here report the development of a micellar biomagnetic system that can be used to restrict the activity of LKB1, a critical intracellular kinase for neuronal polarization, in a spatio-temporal manner, to regulate axon development, in cultured neurons and in the live rodent embryonic brain in utero. LKB1 was complexed with amphiphilic chitosan micelles and MNPs, yielding a stable engineered nanocomplex that preserved kinase-activity. Our findings showed that the nanocomplexes were rapidly
Acknowledgements
This project was supported by a grant from NIH, NS084111 (M. Shelly), and the SUNY M&AM Network of Excellence, 71036 (Y. Meng). Research was carried out in part at the Center for Functional Nanomaterials, Brookhaven National Laboratory, which is supported by the U.S. Department of Energy, Office of Basic Energy Sciences, under Contract No. DE-SC0012704. We thank J. Cathcart for help with the endocytic routing studies.
References (68)
- et al.
New sights into the molecular mechanisms specifying neuronal polarity in vivo
Curr. Opin. Neurobiol.
(2008) - et al.
Molecular motors in neurons: transport mechanisms and roles in brain function, development, and disease
Neuron
(2010) - et al.
The role of the cytoskeleton during neuronal polarization
Curr. Opin. Neurobiol.
(2008) - et al.
Hippocampal neuronal polarity specified by spatially localized mPar3/mPar6 and PI 3-kinase activity
Cell
(2003) - et al.
GSK-3 beta regulates phosphorylation of CRMP-2 and neuronal polarity
Cell
(2005) - et al.
Both the establishment and the maintenance of neuronal polarity require active mechanisms: critical roles of GSK-3 beta and its upstream regulators
Cell
(2005) - et al.
A change in the selective translocation of the Kinesin-1 motor domain marks the initial specification of the axon
Neuron
(2006) - et al.
Phosphorylation of E3 ligase Smurf1 switches its substrate preference in support of axon development
Neuron
(2011) - et al.
Robust neuronal symmetry breaking by Ras-triggered local positive feedback
Curr. Biol.
(2008) - et al.
LKB1 and SAD kinases define a pathway required for the polarization of cortical neurons
Cell
(2007)
LKB1/STRAD promotes axon initiation during neuronal polarization
Cell
Light activation as a method of regulating and studying gene expression
Curr. Opin. Chem. Biol.
Cell polarity in eggs and epithelia: parallels and diversity
Cell
Efficient gene transfer into the embryonic mouse brain using in vivo electroporation
Dev. Biol.
Potential use of tight junction modulators to reversibly open membranous barriers and improve drug delivery
Biochim. Biophys. Acta-Biomembr.
Progress in developing cationic vectors for non-viral systemic gene therapy against cancer
Biomaterials
Self-assembled nanoparticles based on glycol chitosan bearing 5 beta-cholanic acid for RGD peptide delivery
J. Control. Release
Effect of 0.2 T static magnetic field on human neurons: remodeling and inhibition of signal transduction without genome instability
Neurosci. Lett.
Complete polarization of single intestinal epithelial cells upon activation of LKB1 by STRAD
Cell
Accumulation of iron oxide nanoparticles by cultured primary neurons
Neurochem. Int.
Neuronal polarity: from extracellular signals to intracellular mechanisms
Nat. Rev. Neurosci.
The establishment of polarity by hippocampal-neurons in culture
J. Neurosci.
Experimentally induced alteration in the polarity of developing neurons
Nature
Establishment of axon-dendrite polarity in developing neurons
Annu. Rev. Neurosci.
Shootin1: a protein involved in the organization of an asymmetric signal for neuronal polarization
J. Cell Biol.
Centrosome localization determines neuronal polarity
Nature
Asymmetric membrane ganglioside sialidase activity specifies axonal fate
Nat. Neurosci.
CRMP-2 induces axons in cultured hippocampal neurons
Nat. Neurosci.
The sequential activity of the GTPases Rap1B and Cdc42 determines neuronal polarity
Nat. Neurosci.
Microtubule affinity-regulating kinase 2 functions downstream of the PAR-3/PAR-6/atypical PKC complex in regulating hippocampal neuronal polarity
Proc. Natl. Acad. Sci. U. S. A.
Local and long-range reciprocal regulation of cAMP and cGMP in axon/dendrite formation
Science
Self-amplifying autocrine actions of BDNF in axon development
Proc. Natl. Acad. Sci. U. S. A.
Mammalian SAD kinases are required for neuronal polarization
Science
Non-hyperpolarizing GABA(B) receptor activation regulates neuronal migration and neurite growth and specification by cAMP/LKB1
Nat. Commun.
Cited by (10)
Biohybrid nanointerfaces for neuromodulation
2024, Nano TodayEngineering magnetic nanoparticles for repairing nerve injuries
2020, Advances in Nanostructured Materials and Nanopatterning Technologies: Applications for Healthcare, Environmental and Energy