The actin cytoskeleton plays an important role in morphological changes of ameloblasts during the formation of enamel, which is indispensable for teeth to withstand wear, fracture and caries progression. This study reveals that the actin nucleator Cobl is expressed in ameloblasts of mandibular molars during amelogenesis. Cobl expression was particularly pronounced during the secretory phase of the enamel-forming cells. Cobl colocalized with actin filaments at the cell cortex. Importantly, our analyses show an influence of Cobl on both ameloblast morphology and cytoskeletal organization as well as on enamel composition. At P0, Cobl knock-out causes an increased height of ameloblasts and an increased F-actin content at the apical membrane. During the maturation phase, the F-actin density at the apical membrane was instead significantly reduced when compared to WT mice. At the same time, Cobl-deficient mice showed an increased carbon content of the enamel and an increased enamel surface of mandibular molars. These findings demonstrate a decisive influence of the actin nucleator Cobl on the actin cytoskeleton and the morphology of ameloblasts during amelogenesis. Our work thus expands the understanding of the regulation of the actin cytoskeleton during amelogenesis and helps to further elucidate the complex processes of enamel formation during tooth development.

JOURNAL/nrgr/04.03/01300535-202501000-00030/figure1/v/2024-05-14T021156Z/r/image-tiff Axonal remodeling is a critical aspect of ischemic brain repair processes and contributes to spontaneous functional recovery. Our previous in vitro study demonstrated that exosomes/small extracellular vesicles (sEVs) isolated from cerebral endothelial cells (CEC-sEVs) of ischemic brain promote axonal growth of embryonic cortical neurons and that microRNA 27a (miR-27a) is an elevated miRNA in ischemic CEC-sEVs. In the present study, we investigated whether normal CEC-sEVs engineered to enrich their levels of miR-27a (27a-sEVs) further enhance axonal growth and improve neurological outcomes after ischemic stroke when compared with treatment with non-engineered CEC-sEVs. 27a-sEVs were isolated from the conditioned medium of healthy mouse CECs transfected with a lentiviral miR-27a expression vector. Small EVs isolated from CECs transfected with a scramble vector (Scra-sEVs) were used as a control. Adult male mice were subjected to permanent middle cerebral artery occlusion and then were randomly treated with 27a-sEVs or Scra-sEVs. An array of behavior assays was used to measure neurological function. Compared with treatment of ischemic stroke with Scra-sEVs, treatment with 27a-sEVs significantly augmented axons and spines in the peri-infarct zone and in the corticospinal tract of the spinal grey matter of the denervated side, and significantly improved neurological outcomes. In vitro studies demonstrated that CEC-sEVs carrying reduced miR-27a abolished 27a-sEV-augmented axonal growth. Ultrastructural analysis revealed that 27a-sEVs systemically administered preferentially localized to the pre-synaptic active zone, while quantitative reverse transcription-polymerase chain reaction and Western Blot analysis showed elevated miR-27a, and reduced axonal inhibitory proteins Semaphorin 6A and Ras Homolog Family Member A in the peri-infarct zone. Blockage of the Clathrin-dependent endocytosis pathway substantially reduced neuronal internalization of 27a-sEVs. Our data provide evidence that 27a-sEVs have a therapeutic effect on stroke recovery by promoting axonal remodeling and improving neurological outcomes. Our findings also suggest that suppression of axonal inhibitory proteins such as Semaphorin 6A may contribute to the beneficial effect of 27a-sEVs on axonal remodeling.

Dendrite defects and loss are early cellular alterations observed across neurodegenerative diseases that play a role in early disease pathogenesis. Dendrite degeneration can be modeled by expressing pathogenic polyglutamine disease transgenes in Drosophila neurons in vivo. Here, we show that we can protect against dendrite loss in neurons modeling neurodegenerative polyglutamine diseases through injury to a single primary dendrite branch. We find that this neuroprotection is specific to injury-induced activation of dendrite regeneration: neither injury to the axon nor injury just to surrounding tissues induces this response. We show that the mechanism of this regenerative response is stabilization of the actin (but not microtubule) cytoskeleton. We also demonstrate that this regenerative response may extend to other neurodegenerative diseases. Together, we provide evidence that activating dendrite regeneration pathways has the potential to slow-or even reverse-dendrite loss in neurodegenerative disease.

The coordinated action of a plethora of factors is required for the organization and dynamics of membranous structures critically underlying the development and function of cells, organs, and organisms. The evolutionary acquisition of additional amino acid motifs allows for expansion and/or specification of protein functions. We identify a thus far unrecognized motif specific for chordata EHBP1 proteins and demonstrate that this motif is critically required for interaction with syndapin I, an F-BAR domain-containing, membrane-shaping protein predominantly expressed in neurons. Gain-of-function and loss-of-function studies in rat primary hippocampal neurons (of mixed sexes) unraveled that EHBP1 has an important role in neuromorphogenesis. Surprisingly, our analyses uncovered that this newly identified function of EHBP1 did not require the domain responsible for Rab GTPase binding but was strictly dependent on EHBP1's syndapin I binding interface and on the presence of syndapin I in the developing neurons. These findings were underscored by temporally and spatially remarkable overlapping dynamics of EHBP1 and syndapin I at nascent dendritic branch sites. In addition, rescue experiments demonstrated the necessity of two additional EHBP1 domains for dendritic arborization, the C2 and CH domains. Importantly, the additionally uncovered critical involvement of the actin nucleator Cobl in EHBP1 functions suggested that not only static association with F-actin via EHBP1's CH domain is important for dendritic arbor formation but also actin nucleation. Syndapin interactions organize ternary protein complexes composed of EHBP1, syndapin I, and Cobl, and our functional data show that only together these factors give rise to proper cell shape during neuronal development.

Compensatory plastic changes in the remaining intact brain regions are supposedly involved in functional recovery following stroke. Previously, a compensatory increase in cortical activation occurred in the ventral premotor cortex (PMv), which contributed to the recovery of dexterous hand movement in a macaque model of unilateral internal capsular infarcts. Herein, we investigated the structural plastic changes underlying functional changes together with voxel-based morphometry (VBM) analysis of magnetic resonance imaging data and immunohistochemical analysis using SMI-32 antibody in a macaque model. Unilateral internal capsular infarcts were pharmacologically induced in 5 macaques, and another 5 macaques were used as intact controls for immunohistochemical analysis. Three months post infarcts, we observed significant increases in the gray matter volume (GMV) and the dendritic arborization of layer V pyramidal neurons in the contralesional rostral PMv (F5) as well as the primary motor cortex (M1). The histological analysis revealed shrinkage of neuronal soma and dendrites in the ipsilesional M1 and several premotor cortices, despite not always detecting GMV reduction by VBM analysis. In conclusion, compensatory structural changes occur in the contralesional F5 and M1 during motor recovery following internal capsular infarcts, and the dendritic growth of pyramidal neurons is partially correlated with GMV increase.

Axon regeneration in response to injury has been documented in many animals over several hundred years. In contrast, how neurons respond to dendrite injury has been examined only in the last decade. So far, dendrite regeneration after injury has been documented in invertebrate model systems, but has not been assayed in a vertebrate. In this study, we use zebrafish motor neurons to track neurons after dendrite injury. We address two major gaps in our knowledge of dendrite regeneration: 1) whether post-synaptic dendrites can regenerate and 2) whether vertebrate dendrites can regenerate. We find that motor neurons survive laser microsurgery to remove one or all dendrites. Outgrowth of new dendrites typically initiated one to three days after injury, and a new, stable dendrite arbor was in place by five days after injury. We conclude that zebrafish motor neurons have the capacity to regenerate a new dendrite arbor.