Endocytosis-Like Vesicle Fission Mediated by a Membrane-Expanding Molecular Machine Enables Virus Encapsulation for In Vivo Delivery.

Biological membranes are functionalized by membrane-associated protein machinery. Membrane-associated transport processes, such as endocytosis, represent a fundamental and universal function mediated by membrane-deforming protein machines, by which small biomolecules and even micrometer-size substances can be transported via encapsulation into membrane vesicles. Although synthetic molecules that induce dynamic membrane deformation have been reported, a molecular approach enabling membrane transport in which membrane deformation is coupled with substance binding and transport remains critically lacking. Here, we developed an amphiphilic molecular machine containing a photoresponsive diazocine core (AzoMEx) that localizes in a phospholipid membrane. Upon photoirradiation, AzoMEx expands the liposomal membrane to bias vesicles toward outside-in fission in the membrane deformation process. Cargo components, including micrometer-size M13 bacteriophages that interact with AzoMEx, are efficiently incorporated into the vesicles through the outside-in fission. Encapsulated M13 bacteriophages are transiently protected from the external environment and therefore retain biological activity during distribution throughout the body via the blood following administration. This research developed a molecular approach using synthetic molecular machinery for membrane functionalization to transport micrometer-size substances and objects via vesicle encapsulation. The molecular design demonstrated in this study to expand the membrane for deformation and binding to a cargo component can lead to the development of drug delivery materials and chemical tools for controlling cellular activities.

Treatment of Retinoblastoma 1-Intact Hepatocellular Carcinoma With Cyclin-Dependent Kinase 4/6 Inhibitor Combination Therapy.

BACKGROUND AND AIMS: Synthetic cyclin-dependent kinase (CDK) 4/6 inhibitors exert antitumor effects by forcing RB1 in unphosphorylated status, causing not only cell cycle arrest but also cellular senescence, apoptosis, and increased immunogenicity. These agents currently have an indication in advanced breast cancers and are in clinical trials for many other solid tumors. HCC is one of promising targets of CDK4/6 inhibitors. RB family dysfunction is often associated with the initiation of HCC; however, this is revivable, as RB family members are not frequently mutated or deleted in this malignancy. APPROACH AND RESULTS: Loss of all Rb family members in transformation related protein 53 (Trp53)-/- mouse liver resulted in liver tumor reminiscent of human HCC, and re-expression of RB1 sensitized these tumors to a CDK4/6 inhibitor, palbociclib. Introduction of an unphosphorylatable form of RB1 (RB7LP) into multiple liver tumor cell lines induced effects similar to palbociclib. By screening for compounds that enhance the efficacy of RB7LP, we identified an I kappa B kinase (IKK)ß inhibitor Bay 11-7082. Consistently, RB7LP expression and treatment with palbociclib enhanced IKKα/ß phosphorylation and NF-κB activation. Combination therapy using palbociclib with Bay 11-7082 was significantly more effective in hepatoblastoma and HCC treatment than single administration. Moreover, blockade of IKK-NF-κB or AKT pathway enhanced effects of palbociclib on RB1-intact KRAS Kirsten rat sarcoma viral oncogene homolog mutated lung and colon cancers. CONCLUSIONS: In conclusion, CDK4/6 inhibitors have a potential to treat a wide variety of RB1-intact cancers including HCC when combined with an appropriate kinase inhibitor.

Self-assembling Molecular Medicine for the Subacute Phase of Ischemic Stroke.

Ischemic stroke leads to acute neuron death and forms an injured core, triggering delayed cell death at the penumbra. The impaired brain functions after ischemic stroke are hardly recovered because of the limited regenerative properties. However, recent rodent intervention studies manipulating the extracellular environments at the subacute phase shed new light on the regenerative potency of the injured brain. This review introduces the rational design of artificial extracellular matrix (ECM) mimics using supramolecular peptidic scaffolds, which self-assemble via non-covalent bonds and form hydrogels. The facile customizability of the peptide structures allows tuning the hydrogels' physical and biochemical properties, such as charge states, hydrophobicity, cell adhesiveness, stiffness, and stimuli responses. Supramolecular peptidic materials can create safer and more economical drugs than polymer materials and cell transplantation. We also discuss the importance of activating developmental programs for the recovery at the subacute phase of ischemic stroke. Self-assembling molecular medicine mimicking the ECMs and activating developmental programs may stand as a new drug modality of regenerative medicine in various tissues.

Hydrogel-Stiffening and Non-Cell Adhesive Properties of Amphiphilic Peptides with Central Alkylene Chains.

Invited for the cover of this issue is the group of Takahiro Muraoka at Tokyo University of Agriculture and Technology and collaborators. The image depicts nanofiber formation of an amphiphilic peptide with a central alkylene chain that shows non-cell adhesive properties. Read the full text of the article at 10.1002/chem.202100739.

Hydrogel-Stiffening and Non-Cell Adhesive Properties of Amphiphilic Peptides with Central Alkylene Chains.

Amphiphilic peptides bearing terminal alkyl tails form supramolecular nanofibers that are increasingly used as biomaterials with multiple functionalities. Insertion of alkylene chains in peptides can be designed as another type of amphiphilic peptide, yet the influence of the internal alkylene chains on self-assembly and biological properties remains poorly defined. Unlike the terminal alkyl tails, the internal alkylene chains can affect not only the hydrophobicity but also the flexibility and packing of the peptides. Herein, we demonstrate the supramolecular and biological effects of the central alkylene chain length inserted in a peptide. Insertion of the alkylene chain at the center of the peptide allowed for strengthened ß-sheet hydrogen bonds and modulation of the packing order, and consequently the amphiphilic peptide bearing C2 alkylene chain formed a hydrogel with the highest stiffness. Interestingly, the amphiphilic peptides bearing internal alkylene chains longer than C2 showed a diminished cell-adhesive property. This study offers a novel molecular design to tune mechanical and biological properties of peptide materials.

Dnmt1-dependent Chk1 pathway suppression is protective against neuron division.

Neuronal differentiation and cell-cycle exit are tightly coordinated, even in pathological situations. When pathological neurons re-enter the cell cycle and progress through the S phase, they undergo cell death instead of division. However, the mechanisms underlying mitotic resistance are mostly unknown. Here, we have found that acute inactivation of retinoblastoma (Rb) family proteins (Rb, p107 and p130) in mouse postmitotic neurons leads to cell death after S-phase progression. Checkpoint kinase 1 (Chk1) pathway activation during the S phase prevented the cell death, and allowed the division of cortical neurons that had undergone acute Rb family inactivation, oxygen-glucose deprivation (OGD) or in vivo hypoxia-ischemia. During neurogenesis, cortical neurons became protected from S-phase Chk1 pathway activation by the DNA methyltransferase Dnmt1, and underwent cell death after S-phase progression. Our results indicate that Chk1 pathway activation overrides mitotic safeguards and uncouples neuronal differentiation from mitotic resistance.

Glycine Substitution Effects on the Supramolecular Morphology and Rigidity of Cell-Adhesive Amphiphilic Peptides.

Self-assembling peptides that are capable of adopting ß-sheet structures can generate nanofibers that lead to hydrogel formation. Herein, to tune the supramolecular morphologies, mechanical properties, and stimuli responses of the hydrogels, we investigated glycine substitution in a ß-sheet-forming amphiphilic peptide. Glycine substitution generally enhances conformational flexibility. Indeed, glycine substitution in an amphiphilic peptide weakened the hydrogels or even inhibited the gelation. However, unexpectedly, glycine substitution at the center of the peptide molecule significantly enhanced the hydrogel stiffness. The central glycine substitution affected the molecular packing and led to twisted ß-sheet structures and to nanofiber bundling, which likely led to the stiffened hydrogel. Importantly, the supramolecular structures were accurately predicted by molecular dynamics simulations, demonstrating the helpfulness of these techniques for the identification of self-assembling peptides. The hydrogel formed by the amphiphilic peptide with the central glycine substitution had cell adhesive function, and showed a reversible thermal gel-to-sol transition. Thus, glycine substitution is effective in modulating self-assembling structures, rheological properties, and dynamics of biofunctional self-assembling peptides.

Near-Infrared Optogenetic Genome Engineering Based on Photon-Upconversion Hydrogels.

Photon upconversion (UC) from near-infrared (NIR) light to visible light has enabled optogenetic manipulations in deep tissues. However, materials for NIR optogenetics have been limited to inorganic UC nanoparticles. Herein, NIR-light-triggered optogenetics using biocompatible, organic TTA-UC hydrogels is reported. To achieve triplet sensitization even in highly viscous hydrogel matrices, a NIR-absorbing complex is covalently linked with energy-pooling acceptor chromophores, which significantly elongates the donor triplet lifetime. The donor and acceptor are solubilized in hydrogels formed from biocompatible Pluronic F127 micelles, and heat treatment endows the excited triplets in the hydrogel with remarkable oxygen tolerance. Combined with photoactivatable Cre recombinase technology, NIR-light stimulation successfully performs genome engineering resulting in the formation of dendritic-spine-like structures of hippocampal neurons.

Impaired striatal dopamine release in homozygous Vps35 D620N knock-in mice.

Point mutations in the vacuolar protein sorting 35 gene (VPS35) have been associated with an autosomal dominant form of late-onset Parkinson disease (PARK17), but there has been considerable debate over whether it is caused by a loss- or gain-of-function mechanism and over the intracellular target site of neurotoxicity. To investigate the pathogenesis of PARK17 in vivo, we generated Vps35 D620N knock-in (KI) mice, expressing the homologous mutant protein with endogenous patterns of expression, simultaneously with Vps35 deletion 1 (Del1) mice, which carry 1bp deletion in the exon15 of Vps35, by CRISPR/Cas9-mediated genome engineering. Neither homozygous nor heterozygous Vps35 D620N KI mice suffered from premature death or developed clear neurodegeneration up to 70 weeks of age. Vps35 Del1 allele appeared to be a null or at least severely hypomorphic allele and homozygous Vps35 Del1 showed early embryonic lethality. Heterozygous crossings between Del1 and D620N knock-in mice revealed that the D620N/Del1 compound heterozygous mice, but not heterozygous Del1 mice, suffered from survival disadvantage. In vivo microdialysis showed that DA release evoked by 120 mM potassium chloride was significantly reduced in the caudate putamen of adult homozygous Vps35 D620N KI mice. Taken together, these results suggest that Vps35 D620N allele is a partial-loss-of-function allele and that such a genetic predisposition and age-related alterations in the nigrostriatal dopamine system cooperatively influence the pathogenesis of PARK17.

Brg1 coordinates multiple processes during retinogenesis and is a tumor suppressor in retinoblastoma.

Retinal development requires precise temporal and spatial coordination of cell cycle exit, cell fate specification, cell migration and differentiation. When this process is disrupted, retinoblastoma, a developmental tumor of the retina, can form. Epigenetic modulators are central to precisely coordinating developmental events, and many epigenetic processes have been implicated in cancer. Studying epigenetic mechanisms in development is challenging because they often regulate multiple cellular processes; therefore, elucidating the primary molecular mechanisms involved can be difficult. Here we explore the role of Brg1 (Smarca4) in retinal development and retinoblastoma in mice using molecular and cellular approaches. Brg1 was found to regulate retinal size by controlling cell cycle length, cell cycle exit and cell survival during development. Brg1 was not required for cell fate specification but was required for photoreceptor differentiation and cell adhesion/polarity programs that contribute to proper retinal lamination during development. The combination of defective cell differentiation and lamination led to retinal degeneration in Brg1-deficient retinae. Despite the hypocellularity, premature cell cycle exit, increased cell death and extended cell cycle length, retinal progenitor cells persisted in Brg1-deficient retinae, making them more susceptible to retinoblastoma. ChIP-Seq analysis suggests that Brg1 might regulate gene expression through multiple mechanisms.

Cortical excitatory neurons become protected from cell division during neurogenesis in an Rb family-dependent manner.

Cell cycle dysregulation leads to abnormal proliferation and cell death in a context-specific manner. Cell cycle progression driven via the Rb pathway forces neurons to undergo S-phase, resulting in cell death associated with the progression of neuronal degeneration. Nevertheless, some Rb- and Rb family (Rb, p107 and p130)-deficient differentiating neurons can proliferate and form tumors. Here, we found in mouse that differentiating cerebral cortical excitatory neurons underwent S-phase progression but not cell division after acute Rb family inactivation in differentiating neurons. However, the differentiating neurons underwent cell division and proliferated when Rb family members were inactivated in cortical progenitors. Differentiating neurons generated from Rb(-/-); p107(-/-); p130(-/-) (Rb-TKO) progenitors, but not acutely inactivated Rb-TKO differentiating neurons, activated the DNA double-strand break (DSB) repair pathway without increasing trimethylation at lysine 20 of histone H4 (H4K20), which has a role in protection against DNA damage. The activation of the DSB repair pathway was essential for the cell division of Rb-TKO differentiating neurons. These results suggest that newly born cortical neurons from progenitors become epigenetically protected from DNA damage and cell division in an Rb family-dependent manner.

Coordination of proliferation and neuronal differentiation by the retinoblastoma protein family.

Once neurons enter the post-mitotic G0 phase during central nervous system (CNS) development, they lose their proliferative potential. When neurons re-enter the cell cycle during pathological situations such as neurodegeneration, they undergo cell death after S phase progression. Thus, the regulatory networks that drive cell proliferation and maintain neuronal differentiation are highly coordinated. In this review, the coordination of cell cycle control and neuronal differentiation during development are discussed, focusing on regulation by the Rb family of tumor suppressors (including p107 and p130), and the Cip/Kip family of cyclin dependent kinase (Cdk) inhibitors. Based on recent findings suggesting roles for these families in regulating neurogenesis and neuronal differentiation, I propose that the Rb family is essential for daughter cells of neuronal progenitors to enter the post-mitotic G0 phase without affecting the initiation of neuronal differentiation in most cases, while the Cip/Kip family regulates the timing of neuronal progenitor cell cycle exit and the initiation of neuronal differentiation at least in the progenitor cells of the cerebral cortex and the retina. Rb's lack of involvement in regulating the initiation of neuronal differentiation may explain why Rb family-deficient retinoblastomas characteristically exhibit neuronal features.

Amphiphilic peptide-tagged N-cadherin forms radial glial-like fibers that enhance neuronal migration in injured brain and promote sensorimotor recovery.

The mammalian brain has very limited ability to regenerate lost neurons and recover function after injury. Promoting the migration of young neurons (neuroblasts) derived from endogenous neural stem cells using biomaterials is a new and promising approach to aid recovery of the brain after injury. However, the delivery of sufficient neuroblasts to distant injured sites is a major challenge because of the limited number of scaffold cells that are available to guide neuroblast migration. To address this issue, we have developed an amphiphilic peptide [(RADA)3-(RADG)] (mRADA)-tagged N-cadherin extracellular domain (Ncad-mRADA), which can remain in mRADA hydrogels and be injected into deep brain tissue to facilitate neuroblast migration. Migrating neuroblasts directly contacted the fiber-like Ncad-mRADA hydrogel and efficiently migrated toward an injured site in the striatum, a deep brain area. Furthermore, application of Ncad-mRADA to neonatal cortical brain injury efficiently promoted neuronal regeneration and functional recovery. These results demonstrate that self-assembling Ncad-mRADA peptides mimic both the function and structure of endogenous scaffold cells and provide a novel strategy for regenerative therapy.

Efficient protein incorporation and release by a jigsaw-shaped self-assembling peptide hydrogel for injured brain regeneration.

During injured tissue regeneration, the extracellular matrix plays a key role in controlling and coordinating various cellular events by binding and releasing secreted proteins in addition to promoting cell adhesion. Herein, we develop a cell-adhesive fiber-forming peptide that mimics the jigsaw-shaped hydrophobic surface in the dovetail-packing motif of glycophorin A as an artificial extracellular matrix for regenerative therapy. We show that the jigsaw-shaped self-assembling peptide forms several-micrometer-long supramolecular nanofibers through a helix-to-strand transition to afford a hydrogel under physiological conditions and disperses homogeneously in the hydrogel. The molecular- and macro-scale supramolecular properties of the jigsaw-shaped self-assembling peptide hydrogel allow efficient incorporation and sustained release of vascular endothelial growth factor, and demonstrate cell transplantation-free regenerative therapeutic effects in a subacute-chronic phase mouse stroke model. This research highlights a therapeutic strategy for injured tissue regeneration using the jigsaw-shaped self-assembling peptide supramolecular hydrogel.

Sequence-Dependent Bioactivity and Self-Assembling Properties of RGD-Containing Amphiphilic Peptides as Extracellular Scaffolds.

Cell adhesion is a fundamental biological process involved in a wide range of cellular and biological activity. Integrin-ligand binding is largely responsible for cell adhesion with an extracellular matrix, and the RGD sequence is an epitope in ligand proteins such as fibronectin. The extracellular matrix consists of fibrous proteins with embedded ligands for integrins. Such a biological architecture has been reconstructed for biochemical, pharmaceutical, and biomaterial studies using artificial supramolecular systems to reproduce cell adhesion functionality, and fiber-forming self-assembling peptides containing RGD are one such promising material for this purpose. In this study, using RADA16 as a model fiber-forming peptide, a series of RGD-containing variants have been synthesized by the replacement of one alanine with glycine at different positions, in which all the variants consist of identical amino acid components. The position of the RGD unit influenced the supramolecular self-assembly of the amphiphilic peptide to inhibit ß-sheet formation (A6G) or twist the molecular alignment in ß-sheet-type assemblies (A10G and A14G). Furthermore, A10G and A14G formed assembled nanofibers, which afforded hydrogels with higher viscoelasticities than other RGD-containing variants. In contrast to A10G and A14G, which exhibit substantial cell adhesion functionality, the cell adhesion efficiencies of the other RGD-containing variants were significantly reduced. This suggests that the higher order structure could strongly influence the cell adhesion functionality of RGD-containing supramolecular nanofibers.

Spatiotemporally Dependent Vascularization Is Differently Utilized among Neural Progenitor Subtypes during Neocortical Development.

To facilitate efficient oxygen and nutrient delivery, blood vessels in the brain form three-dimensional patterns. However, little is known about how blood vessels develop stereographically in the neocortex and how they control the expansion and differentiation of neural progenitors during neocortical development. We show that highly vascularized and avascular regions are strictly controlled in a spatially and temporally restricted manner and are associated with distinct cell populations. Dividing basal progenitors and oligodendrocyte precursors preferentially contact honeycomb vessels, but dividing apical progenitors are localized in avascular regions without Flt1-positive endothelial cells but directly contact with sprouting neovascular tip cells. Therefore, not all blood vessels are associated equally with neural progenitors. Furthermore, a disruption of normal vascular patterning can induce abnormalities in neural development, whereas the impaired features of neural progenitors influenced angiogenesis patterning. These results indicate that close association between the nervous and vascular systems is essential for neocortex assembly.

Radial Glial Fibers Promote Neuronal Migration and Functional Recovery after Neonatal Brain Injury.

Radial glia (RG) are embryonic neural stem cells (NSCs) that produce neuroblasts and provide fibers that act as a scaffold for neuroblast migration during embryonic development. Although they normally disappear soon after birth, here we found that RG fibers can persist in injured neonatal mouse brains and act as a scaffold for postnatal ventricular-subventricular zone (V-SVZ)-derived neuroblasts that migrate to the lesion site. This injury-induced maintenance of RG fibers has a limited time window during post-natal development and promotes directional saltatory movement of neuroblasts via N-cadherin-mediated cell-cell contacts that promote RhoA activation. Transplanting an N-cadherin-containing scaffold into injured neonatal brains likewise promotes migration and maturation of V-SVZ-derived neuroblasts, leading to functional improvements in impaired gait behaviors. Together these results suggest that RG fibers enable postnatal V-SVZ-derived neuroblasts to migrate toward sites of injury, thereby enhancing neuronal regeneration and functional recovery from neonatal brain injuries.

Expression profiles of Insulin-like growth factor binding protein-like 1 in the developing mouse forebrain.

Insulin-like growth factor binding protein-like 1 (IGFBPL-1) has a domain structure that resembles one in the insulin-like growth factor binding proteins (IGFBPs), which is a domain known to be bound by insulin-like growth factors (IGFs) with high affinity and to thereby affect cell growth and metabolism. However, the distribution and functions of IGFBPL-1 in the brain are not known. Thus, to clarify IGFBPL-1 expression profiles during development, we have investigated the expression patterns of IGFBPL-1 mRNA in developing mouse brains using in situ hybridization, focusing especially on the forebrain. On embryonic day (E)12, IGFBPL-1mRNA expressions were localized in the preplate of the neopallium, dorsal thalamus, the differentiating field of the epithalamus, and the hippocampus. On E14, strong expression was recognized in the subventricular zone (SVZ) and intermediate zone (IMZ) in the neopallium, hippocampus, the differentiating field of the epithalamus, and the dorsal thalamus. E14 expressions were generally the strongest among all the stages examined and signals decreased thereafter. At E16 and E18, IGFBPL-1mRNA was strongly detected in the hippocampus and the SVZ of the neopallium. In postnatal stages, mRNA was found only in the granule cell layer of the dentate gyrus (DG). Notably, at P20, expression was restricted to the deep fraction of the granule cell layer, the location of late-born DG neurons.

Affinity-Immobilization of VEGF on Laminin Porous Sponge Enhances Angiogenesis in the Ischemic Brain.

Ischemic brain stroke is caused by blood flow interruption, leading to focal ischemia, neuron death, and motor, sensory, and/or cognitive dysfunctions. Angiogenesis, neovascularization from existing blood vessel, is essential for tissue growth and repair. Proangiogenic therapy for stroke is promising for preventing excess neuron death and improving functional recovery. Vascular endothelial growth factor (VEGF) is a critical factor for angiogenesis by promoting the proliferation, the survival, and the migration of endothelial cells. Here, angiogenic biomaterials to support injured brain regeneration are developed. Porous laminin (LN)-rich sponge (LN-sponge), on which histidine-tagged VEGF (VEGF-Histag) is immobilized via affinity interaction is developed. In an in vivo mouse stroke model, transplanting VEGF-Histag-LN-sponge produces remarkably stronger angiogenic activity than transplanting LN-sponge with soluble VEGF. The findings indicate that using affinity interactions to immobilize VEGF is a practical approach for developing angiogenic biomaterials for regenerating the injured brain.