Pretreatment with Perlecan-Conjugated Laminin-E8 Fragment Enhances Maturation of Grafted Dopaminergic Progenitors in Parkinson’s Disease Model

The therapeutic effect of a cell replacement therapy for Parkinson's disease (PD) depends on the proper maturation of grafted dopaminergic (DA) neurons and their functional innervation in the host brain. In the brain, laminin, an extracellular matrix protein, regulates signaling pathways for the survival and development of neurons by interacting with integrins. The heparan sulfate (HS) chain binds mildly to various neurotrophic factors and regulates their intracellular signaling. Perlecan-conjugated laminin 511/521-E8 fragments (p511/p521) were designed to contain an integrin-binding site and HS chains. Here we examined the effect of treating DA progenitors with p511/p521 prior to transplantation in rodent PD models. In vitro and in vivo experiments showed that p511/p521 treatment enhanced the maturation and neurite extension of the grafted DA progenitors by activating RAS-ERK1/2 signaling. This strategy will contribute to an efficient cell replacement therapy for PD in the future

Viral delivery of L1CAM promotes axonal extensions by embryonic cerebral grafts in mouse brain

遺伝子治療によるホスト脳の環境最適化が細胞移植効果を高める --ホスト脳へのL1CAMの強制発現によるマウス胎仔脳移植片の軸索伸長促進効果--. 京都大学プレスリリース. 2023-03-24.Combining cell transplantation and gene therapy to enhance axonal outgrowth in the central nervous system. 京都大学プレスリリース. 2023-04-06.Cell replacement therapy is expected as a new and more radical treatment against brain damage. We previously reported that transplanted human cerebral organoids extend their axons along the corticospinal tract in rodent brains. The axons reached the spinal cord but were still sparse. Therefore, this study optimized the host brain environment by the adeno-associated virus (AAV)-mediated expression of axon guidance proteins in mouse brain. Among netrin-1, SEMA3, and L1CAM, only L1CAM significantly promoted the axonal extension of mouse embryonic brain tissue-derived grafts. L1CAM was also expressed by donor neurons, and this promotion was exerted in a haptotactic manner by their homophilic binding. Primary cortical neurons cocultured on L1CAM-expressing HEK-293 cells supported this mechanism. These results suggest that optimizing the host environment by the AAV-mediated expression of axon guidance molecules enhances the effect of cell replacement therapy

A comprehensive approach to characterize navigation instruments for magnetic guidance in biological systems

Abstract Achieving non-invasive spatiotemporal control over cellular functions, tissue organization, and behavior is a desirable aim for advanced therapies. Magnetic fields, due to their negligible interaction with biological matter, are promising for in vitro and in vivo applications, even in deep tissues. Particularly, the remote manipulation of paramagnetic (including superparamagnetic and ferromagnetic, all with a positive magnetic susceptibility) entities through magnetic instruments has emerged as a promising approach across various biological contexts. However, variations in the properties and descriptions of these instruments have led to a lack of reproducibility and comparability among studies. This article addresses the need for standardizing the characterization of magnetic instruments, with a specific focus on their ability to control the movement of paramagnetic objects within organisms. While it is well known that the force exerted on magnetic particles depends on the spatial variation (gradient) of the magnetic field, the magnitude of the field is often overlooked in the literature. Therefore, we comprehensively analyze and discuss both actors and propose a novel descriptor, termed ‘effective gradient’, which combines both dependencies. To illustrate the importance of both factors, we characterize different magnet systems and relate them to experiments involving superparamagnetic nanoparticles. This standardization effort aims to enhance the reproducibility and comparability of studies utilizing magnetic instruments for biological applications

Perspectives of RAS and RHEB GTPase Signaling Pathways in Regenerating Brain Neurons

Cellular activation of RAS GTPases into the GTP-binding “ON„ state is a key switch for regulating brain functions. Molecular protein structural elements of rat sarcoma (RAS) and RAS homolog protein enriched in brain (RHEB) GTPases involved in this switch are discussed including their subcellular membrane localization for triggering specific signaling pathways resulting in regulation of synaptic connectivity, axonal growth, differentiation, migration, cytoskeletal dynamics, neural protection, and apoptosis. A beneficial role of neuronal H-RAS activity is suggested from cellular and animal models of neurodegenerative diseases. Recent experiments on optogenetic regulation offer insights into the spatiotemporal aspects controlling RAS/mitogen activated protein kinase (MAPK) or phosphoinositide-3 kinase (PI3K) pathways. As optogenetic manipulation of cellular signaling in deep brain regions critically requires penetration of light through large distances of absorbing tissue, we discuss magnetic guidance of re-growing axons as a complementary approach. In Parkinson’s disease, dopaminergic neuronal cell bodies degenerate in the substantia nigra. Current human trials of stem cell-derived dopaminergic neurons must take into account the inability of neuronal axons navigating over a large distance from the grafted site into striatal target regions. Grafting dopaminergic precursor neurons directly into the degenerating substantia nigra is discussed as a novel concept aiming to guide axonal growth by activating GTPase signaling through protein-functionalized intracellular magnetic nanoparticles responding to external magnets

Perspectives of RAS and RHEB GTPase signaling pathways in regenerating brain neurons

Cellular activation of RAS GTPases into the GTP-binding “ON” state is a key switch for regulating brain functions. Molecular protein structural elements of rat sarcoma (RAS) and RAS homolog protein enriched in brain (RHEB) GTPases involved in this switch are discussed including their subcellular membrane localization for triggering specific signaling pathways resulting in regulation of synaptic connectivity, axonal growth, differentiation, migration, cytoskeletal dynamics, neural protection, and apoptosis. A beneficial role of neuronal H-RAS activity is suggested from cellular and animal models of neurodegenerative diseases. Recent experiments on optogenetic regulation offer insights into the spatiotemporal aspects controlling RAS/mitogen activated protein kinase (MAPK) or phosphoinositide-3 kinase (PI3K) pathways. As optogenetic manipulation of cellular signaling in deep brain regions critically requires penetration of light through large distances of absorbing tissue, we discuss magnetic guidance of re-growing axons as a complementary approach. In Parkinson’s disease, dopaminergic neuronal cell bodies degenerate in the substantia nigra. Current human trials of stem cell-derived dopaminergic neurons must take into account the inability of neuronal axons navigating over a large distance from the grafted site into striatal target regions. Grafting dopaminergic precursor neurons directly into the degenerating substantia nigra is discussed as a novel concept aiming to guide axonal growth by activating GTPase signaling through protein-functionalized intracellular magnetic nanoparticles responding to external magnets

Novel Tools towards Magnetic Guidance of Neurite Growth: (I) Guidance of Magnetic Nanoparticles into Neurite Extensions of Induced Human Neurons and In Vitro Functionalization with RAS Regulating Proteins

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Magnetic spatiotemporal control of SOS1 coupled nanoparticles for guided neurite growth in dopaminergic single cells

The axon regeneration of neurons in the brain can be enhanced by activating intracellular signaling pathways such as those triggered by the membrane-anchored Rat sarcoma (RAS) proto-oncogene. Here we demonstrate the induction of neurite growth by expressing tagged permanently active Harvey-RAS protein or the RAS-activating catalytic domain of the guanine nucleotide exchange factor (SOS1cat), in secondary dopaminergic cells. Due to the tag, the expressed fusion protein is captured by functionalized magnetic nanoparticles in the cytoplasm of the cell. We use magnetic tips for remote translocation of the SOS1cat-loaded magnetic nanoparticles from the cytoplasm towards the inner face of the plasma membrane where the endogenous Harvey-RAS protein is located. Furthermore, we show the magnetic transport of SOS1cat-bound nanoparticles from the cytoplasm into the neurite until they accumulate at its tip on a time scale of minutes. In order to scale-up from single cells, we show the cytoplasmic delivery of the magnetic nanoparticles into large numbers of cells without changing the cellular response to nerve growth factor. These results will serve as an initial step to develop tools for refining cell replacement therapies based on grafted human induced dopaminergic neurons loaded with functionalized magnetic nanoparticles in Parkinson model systems

Novel tools towards magnetic guidance of neurite growth

Parkinson’s disease (PD) is a neurodegenerative disease associated with loss or dysfunction of dopaminergic neurons located in the substantia nigra (SN), and there is no cure available. An emerging new approach for treatment is to transplant human induced dopaminergic neurons directly into the denervated striatal brain target region. Unfortunately, neurons grafted into the substantia nigra are unable to grow axons into the striatum and thus do not allow recovery of the original connectivity. Towards overcoming this general limitation in guided neuronal regeneration, we develop here magnetic nanoparticles functionalized with proteins involved in the regulation of axonal growth. We show covalent binding of constitutive active human rat sarcoma (RAS) proteins or RAS guanine nucleotide exchange factor catalytic domain of son of sevenless (SOS) by fluorescence correlation spectroscopy and multiangle light scattering as well as the characterization of exchange factor activity. Human dopaminergic neurons were differentiated from neural precursor cells and characterized by electrophysiological and immune histochemical methods. Furthermore, we demonstrate magnetic translocation of cytoplasmic $\gamma-Fe_{2}O_{3}@SiO_{2}$ core-shell nanoparticles into the neurite extensions of induced human neurons. Altogether, we developed tools towards remote control of directed neurite growth in human dopaminergic neurons. These results may have relevance for future therapeutic approaches of cell replacement therapy in Parkinson’s disease