Massimo A. Hilliard

Neurites of sensory neurons innervate the skin and are embedded within it. These delicate structures are chronically exposed to mechanical strain and yet their integrity is maintained throughout life. In C. elegans, UNC-70/β-spectrin functions in synergy with the small GTPase RAB-35 within the skin to stabilize neuron-epidermal attachment against mechanical strain, and prevent movement-induced damage to mechanosensitive axons. However, the molecular pathway regulating these specialized attachments remains elusive. Here, through an unbiased genetic screen we have identified a guanine nucleotide exchange factor (GEF), AGEF-1, that impacts axonal maintenance. This molecule is known to function in endocytic-recycling and its human ortholog, BIG2, is associated with the development of the periventricular nodular heterotopia. We show that AGEF-1 functions selectively within the skin to regulate axonal integrity of mechanosensitive neurons. Mechanistically, we reveal that AGEF-1 binds to epidermal RAB-35 and regulates its activity, modulating neuron-epidermal attachment stability. Finally, we demonstrate that this GEF is highly conserved, with its human ortholog BIG2, capable of replacing AGEF-1. Together, we reveal the molecular machinery responsible for fine-tuning neuron-epidermal attachments and maintaining axonal integrity during life. Resumo Neuritos de neurônios sensoriais inervam densamente a pele e situam-se inseridos nela. Essas estruturas delicadas são cronicamente expostas a tensão mecânica, contudo sua integridade é mantida ao longo da vida. Em C. elegans, a β-espectrina UNC-70 atua em sinergia com a GTPase RAB-35 dentro da pele para estabilizar adesões neuro-epidermais contra tensão mecânica e protege axônios mecanosensitivos contra dano mediado pelo movimento. No entanto, não são conhecidos todos os componentes da maquinaria molecular que regula essas adesões. Através de uma mutagênese química, identificamos que AGEF-1, um fator de troca de nucleotídeos de guanina (GEF), impacta a manutenção axonal de neuronios mecanosensitivos. Essa molecula funciona no sistema de reciclagem endocítica e seu ortologo em humanos, BIG2, é associado ao desenvolvimento de heterotropia nodular periventricular. Aqui demostramos que essa proteína atua seletivamente na pele para essa função. Mecanisticamente, revelamos que AGEF-1 liga-se a RAB-35 epidermal regulando sua ativação e modulando a estabilidade de adesões neuro-epidermais. Por fim, demonstramos que AGEF-1 é altamente conservado, com o seu ortólogo humano BIG2 sendo capaz de substituir a molécula nematoide. Ao todo, revelamos a maquinária molecular responsável pelo ajuste delicado das adesões neuro-epidermais e da integridade axonal ao longo da vida.

Spectrins are highly conserved molecules that form a distinct membrane-associated periodic scaffold within axons to provide mechanical resilience. In Caenorhabditis elegans , UNC-70/ß-Spectrin also functions within the epidermis to maintain the integrity of sensory neurons. The precise molecular organization in this tissue and the cellular mechanisms that mediate this protection are unknown. Here, using three-dimensional structured illumination microscopy, we show that epidermal SPC-1/α-Spectrin and UNC-70/ß-Spectrin form a crescent-shaped scaffold with a periodicity of ~200 nm that embraces adjacent axons. This epidermal Spectrin scaffold is induced by developing axons and reformed during axonal regeneration, creating a “molecular imprint” of the nervous system. Disruption of this epidermal scaffold causes axonal damage, and we propose that it protects axons by restricting the endocytosis of cell adhesion molecules required for axonal-epidermal adhesion. Our findings reveal a distinct periodic Spectrin scaffold within the epidermis that is molded by the developing nervous system and protects axons from damage.

Neurites of sensory neurons densely innervate the skin and are embedded within it. These delicate structures are exposed to acute and chronic mechanical strain and yet their integrity is maintained throughout life. Although evidence suggests a neuroprotective role for the skin, the molecular pathways involved are still poorly understood. In C. elegans , the cytoskeletal molecule UNC-70/β-spectrin functions in synergy with the small GTPase RAB-35 within the skin to stabilize neuron-epidermal attachment structures against mechanical strain and prevent movement-induced damage to mechanosensitive axons. However, the full suite of molecules regulating these specialized attachments remains elusive. Here, through an unbiased forward genetic screen we have identified a guanine nucleotide exchange factor (GEF) previously associated with the endocytic-recycling machinery, AGEF-1a, that impacts axonal maintenance. We show that AGEF-1a functions selectively within the skin to regulate the integrity of the embedded axons. Mechanistically, we reveal that this effect is achieved through an interaction between AGEF-1a and epidermal RAB-35 to facilitate its activation, which in turn modulates the neuron-epidermal attachments. Finally, we demonstrate that the function of this GEF is highly conserved, with the expression of its human ortholog, BIG2 capable of replacing AGEF-1a. Together, we reveal the specific molecular machinery responsible for fine-tuning neuron-epidermal attachments and maintaining axonal integrity during life.

Oxidative stress, caused by the accumulation of reactive oxygen species (ROS), is a pathological factor in several incurable neurodegenerative conditions as well as in stroke. However, our knowledge of the genetic elements that can be manipulated to protect neurons from oxidative stress-induced cell death is still very limited. Here, using Caenorhabditis elegans as a model system, combined with the optogenetic tool KillerRed to spatially and temporally control ROS generation, we identify a previously uncharacterized gene, oxidative stress protective 1 (osp-1), that protects C. elegans neurons from oxidative damage. Using rodent and human cell cultures, we also show that the protective effect of OSP-1 extends to mammalian cells. Moreover, we demonstrate that OSP-1 functions in a strictly cell-autonomous fashion, and that it localizes to the endoplasmic reticulum (ER) where it has an ER-remodeling function. Finally, we present evidence suggesting that OSP-1 may exert its neuroprotective function by influencing autophagy. Our results point to a potential role of OSP-1 in modulating autophagy, and suggest that overactivation of this cellular process could contribute to neuronal death triggered by oxidative damage.

Numerous viruses use specialized surface molecules called fusogens to enter host cells. Many of these viruses, including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), can infect the brain and are associated with severe neurological symptoms through poorly understood mechanisms. We show that SARS-CoV-2 infection induces fusion between neurons and between neurons and glia in mouse and human brain organoids. We reveal that this is caused by the viral fusogen, as it is fully mimicked by the expression of the SARS-CoV-2 spike (S) protein or the unrelated fusogen p15 from the baboon orthoreovirus. We demonstrate that neuronal fusion is a progressive event, leads to the formation of multicellular syncytia, and causes the spread of large molecules and organelles. Last, using Ca ²⁺ imaging, we show that fusion severely compromises neuronal activity. These results provide mechanistic insights into how SARS-CoV-2 and other viruses affect the nervous system, alter its function, and cause neuropathology.

Axonal fusion is a neuronal repair mechanism that results in the reconnection of severed axon fragments, leading to the restoration of cytoplasmic continuity and neuronal function. While synaptic vesicle recycling has been linked to axonal regeneration, its role in axonal fusion remains unknown. Dynamin proteins are large GTPases that hydrolyze lipid-binding membranes to carry out clathrin-mediated synaptic vesicle recycling. Here, we show that the Caenorhabditis elegans dynamin protein DYN-1 is a key component of the axonal fusion machinery. Animals carrying a temperature-sensitive allele of dyn-1(ky51) displayed wild-type levels of axonal fusion at the permissive temperature (15°C) but presented strongly reduced levels at the restrictive temperature (25°C). Furthermore, the average length of regrowth was significantly diminished in dyn-1(ky51) animals at the restrictive temperature. The expression of wild-type DYN-1 cell-autonomously into dyn-1(ky51) mutant animals rescued both the axonal fusion and regrowth defects. Furthermore, DYN-1 was not required prior to axonal injury, suggesting that it functions specifically after injury to control axonal fusion. Finally, using epistatic analyses and superresolution imaging, we demonstrate that DYN-1 regulates the levels of the fusogen protein EFF-1 post-injury to mediate axonal fusion. Together, these results establish DYN-1 as a novel regulator of axonal fusion.

Spectrins are highly conserved molecules that form a distinct membrane associated periodic scaffold within axons thought to provide mechanical resilience. In C. elegans , UNC-70/ß-Spectrin also functions outside the nervous system, within the epidermis, to maintain the integrity of sensory neurons. The precise molecular organization and cellular mechanisms that mediate this protection are unknown. Here, using 3D-structured illumination microscopy, we show that within the epidermis spectrins form a crescent-shaped scaffold with a periodicity of ∼ 200 nm that embraces adjacent axons. This epidermal spectrin scaffold is induced by developing axons and reformed during axonal regeneration, and creates a “molecular imprint” of the adjacent nervous system. Disruption of this epidermal scaffold causes widespread axonal damage in sensory and motor neurons. These findings reveal the existence of a distinct and periodic spectrin framework within the epidermis that is molded by the developing nervous system and is necessary to protect it from mechanical damage. Teaser: Spectrin forms a periodic scaffold in the epidermis that protects axons from damage.

Axonal fusion is an efficient means of repair following axonal transection, whereby the regenerating axon fuses with its own separated axonal fragment to restore neuronal function. Despite being described over 50 years ago, its molecular mechanisms remain poorly understood. Here, we demonstrate that the Caenorhabditis elegans metalloprotease ADM-4, an ortholog of human ADAM17, is essential for axonal fusion. We reveal that animals lacking ADM-4 cannot repair their axons by fusion, and that ADM-4 has a cell-autonomous function within injured neurons, localizing at the tip of regrowing axon and fusion sites. We demonstrate that ADM-4 overexpression enhances fusion to levels higher than wild type, and that the metalloprotease and phosphatidylserine-binding domains are essential for its function. Last, we show that ADM-4 interacts with and stabilizes the fusogen EFF-1 to allow membranes to merge. Our results uncover a key role for ADM-4 in axonal fusion, exposing a molecular target for axonal repair.

Numerous enveloped viruses use specialized surface molecules called fusogens to enter host cells ¹ . During virus replication, these fusogens decorate the host cells membrane enabling them the ability to fuse with neighboring cells, forming syncytia that the viruses use to propagate while evading the immune system. Many of these viruses, including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), infect the brain and may cause serious neurological symptoms through mechanisms which remain poorly understood ^2–4 . Here we show that expression of either the SARS-CoV-2 spike (S) protein or p15 protein from the baboon orthoreovirus is sufficient to induce fusion between interconnected neurons, as well as between neurons and glial cells. This phenomenon is observed across species, from nematodes to mammals, including human embryonic stem cells-derived neurons and brain organoids. We show that fusion events are progressive, can occur between distant neurites, and lead to the formation of multicellular syncytia. Finally, we reveal that in addition to intracellular molecules, fusion events allow diffusion and movement of large organelles such as mitochondria between fused neurons. Our results provide important mechanistic insights into how SARS-CoV-2 and other viruses could affect the nervous system circuitries causing neurological symptoms.