Dual spatio-temporal regulation of axon growth and microtubule dynamics by RhoA signaling pathways.

RhoA plays a crucial role in neuronal polarization, where its action restraining axon outgrowth has been thoroughly studied. We now report that RhoA has not only inhibitory but also a stimulatory effect on axon development depending on when and where exerts its action and the downstream effectors involved. In cultured hippocampal neurons, FRET imaging revealed that RhoA activity selectively localizes in growth cones of undifferentiated neurites, while in developing axons it displays a biphasic pattern, being low in nascent axons and high in elongating ones. RhoA-Rho kinase (ROCK) signaling prevents axon initiation but has no effect on elongation, while formin inhibition reduces axon extension without significantly altering initial outgrowth. Besides, RhoA-mDia promotes axon elongation by stimulating growth cone microtubule stability and assembly, as opposed to RhoA-ROCK that restrains growth cone microtubule assembly and protrusion.

Neuroprotective Effect of a Pharmaceutical Extract of Cannabis with High Content on CBD Against Rotenone in Primary Cerebellar Granule Cell Cultures and the Relevance of Formulations.

Introduction: Preclinical research supports the benefits of pharmaceutical cannabis-based extracts for treating different medical conditions (e.g., epilepsy); however, their neuroprotective potential has not been widely investigated. Materials and Methods: Using primary cultures of cerebellar granule cells, we evaluated the neuroprotective activity of Epifractan (EPI), a cannabis-based medicinal extract containing a high level of cannabidiol (CBD), components like terpenoids and flavonoids, trace levels of Δ9-tetrahydrocannabinol, and the acid form of CBD. We determined the ability of EPI to counteract the rotenone-induced neurotoxicity by analyzing cell viability and morphology of neurons and astrocytes by immunocytochemical assays. The effect of EPI was compared with XALEX, a plant-derived and highly purified CBD formulation (XAL), and pure CBD crystals (CBD). Results: The results revealed that EPI induced a significant reduction in the rotenone-induced neurotoxicity in a wide range of concentrations without causing neurotoxicity per se. EPI showed a similar effect to XAL suggesting that no additive or synergistic interactions between individual substances present in EPI occurred. In contrast, CBD did show a different profile to EPI and XAL because a neurotoxic effect per se was observed at higher concentrations assayed. Medium-chain triglyceride oil used in EPI formulation could explain this difference. Conclusion: Our data support a neuroprotective effect of EPI that may provide neuroprotection in different neurodegenerative processes. The results highlight the role of CBD as the active component of EPI but also support the need for an appropriate formulation to dilute pharmaceutical cannabis-based products that could be critical to avoid neurotoxicity at very high doses.

Extracellular matrix stiffness negatively affects axon elongation, growth cone area and F-actin levels in a collagen type I 3D culture.

Three dimensional (3D) in vitro neuronal cultures can better reproduce physiologically relevant phenotypes compared to 2D-cultures, because in vivo neurons reside in a 3D microenvironment. Interest in neuronal 3D cultures is emerging, with special attention to the mechanical forces that regulate axon elongation and sprouting in three dimensions. Type I collagen (Col-I) is a native substrate since it is present in the extracellular matrix and hence emulates an in vivo environment to study axon growth. The impact of its mechanical properties needs to be further investigated. Here, we generated Col-I 3D matrices of different mechanical stiffness and evaluated axon growth in three dimensions. Superior cervical ganglion (SCG) explants from neonatal rats were cultured in soft and stiff Col-I 3D matrices and neurite outgrowth was assessed by measuring: maximum neuritic extent; neuritic halo area and fasciculation. Axonal cytoskeletal proteins were examined. Axon elongation in stiff Col-I 3D matrices was reduced (31%) following 24 h in culture compared to soft matrices. In stiff matrices, neurites fasciculated and formed less dense halos. Consistently, almost no F-actin rich growth cones were recognized, and F-actin staining was strongly reduced in the axonal compartment. This study shows that stiffness negatively affects 3D neurite outgrowth and adds insights on the cytoskeletal responses upon mechanic interactions of axons with a 3D environment. Our data will serve to facilitate the development of model systems that are mechanically well-behaved but still mimic key physiologic properties observed in vivo.

Quantitative expansion microscopy for the characterization of the spectrin periodic skeleton of axons using fluorescence microscopy.

Fluorescent nanoscopy approaches have been used to characterize the periodic organization of actin, spectrin and associated proteins in neuronal axons and dendrites. This membrane-associated periodic skeleton (MPS) is conserved across animals, suggesting it is a fundamental component of neuronal extensions. The nanoscale architecture of the arrangement (190 nm) is below the resolution limit of conventional fluorescent microscopy. Fluorescent nanoscopy, on the other hand, requires costly equipment and special analysis routines, which remain inaccessible to most research groups. This report aims to resolve this issue by using protein-retention expansion microscopy (pro-ExM) to reveal the MPS of axons. ExM uses reagents and equipment that are readily accessible in most neurobiology laboratories. We first explore means to accurately estimate the expansion factors of protein structures within cells. We then describe the protocol that produces an expanded specimen that can be examined with any fluorescent microscopy allowing quantitative nanoscale characterization of the MPS. We validate ExM results by direct comparison to stimulated emission depletion (STED) nanoscopy. We conclude that ExM facilitates three-dimensional, multicolor and quantitative characterization of the MPS using accessible reagents and conventional fluorescent microscopes.

Author Correction: Remodeling of the Actin/Spectrin Membrane-associated Periodic Skeleton, Growth Cone Collapse and F-Actin Decrease during Axonal Degeneration.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Remodeling of the Actin/Spectrin Membrane-associated Periodic Skeleton, Growth Cone Collapse and F-Actin Decrease during Axonal Degeneration.

Axonal degeneration occurs in the developing nervous system for the appropriate establishment of mature circuits, and is also a hallmark of diverse neurodegenerative diseases. Despite recent interest in the field, little is known about the changes (and possible role) of the cytoskeleton during axonal degeneration. We studied the actin cytoskeleton in an in vitro model of developmental pruning induced by trophic factor withdrawal (TFW). We found that F-actin decrease and growth cone collapse (GCC) occur early after TFW; however, treatments that prevent axonal fragmentation failed to prevent GCC, suggesting independent pathways. Using super-resolution (STED) microscopy we found that the axonal actin/spectrin membrane-associated periodic skeleton (MPS) abundance and organization drop shortly after deprivation, remaining low until fragmentation. Fragmented axons lack MPS (while maintaining microtubules) and acute pharmacological treatments that stabilize actin filaments prevent MPS loss and protect from axonal fragmentation, suggesting that MPS destruction is required for axon fragmentation to proceed.

Reinnervation of rat endometrium in the anterior eye chamber model of experimental endometriosis: Old methods for new questions.

Endometriosis is a benign estrogen-dependent chronic gynecological disease characterized by the presence of endometrial-like tissue outside the uterine cavity. In both women and experimental endometriotic rats, endometriosis lesions endow autonomic and sensory nerves, which are thought to contribute to the disease-associated pain. Some evidence indicates that the reinnervation of lesions is regulated by factors produced by the endometrial tissue as well as by environmental factors from the peritoneum. In this study, we examined the reinnervation of the rat endometrial tissue in an ectopic environment different from the peritoneum employing the anterior eye chamber model of experimental endometriosis. At 3 and 6weeks following transplantation, endometrial grafts retained many histological features of the eutopic tissue. Both sympathetic and sensory nerves reinnervated endometrial grafts and distributed in the stroma-like tissue, around blood vessels and in close proximity to the glands and lining epithelium. Sympathetic innervation was more robust than sensory innervation. No significant topographical relationship between sympathetic nerves and macrophages was observed. These results suggest that the rat endometrium possesses intrinsic neuritogenic capacities and can be reinnervated by sympathetic and sensory nerves in ectopic sites different from the peritoneum.