Hemodynamics regulate spatiotemporal artery muscularization in the developing circle of Willis.

Vascular smooth muscle cells (VSMCs) envelop vertebrate brain arteries, playing a crucial role in regulating cerebral blood flow and neurovascular coupling. The dedifferentiation of VSMCs is implicated in cerebrovascular diseases and neurodegeneration. Despite its importance, the process of VSMC differentiation on brain arteries during development remains inadequately characterized. Understanding this process could aid in reprogramming and regenerating differentiated VSMCs in cerebrovascular diseases. In this study, we investigated VSMC differentiation on the zebrafish circle of Willis (CoW), comprising major arteries that supply blood to the vertebrate brain. We observed that the arterial expression of CoW endothelial cells (ECs) occurs after their migration from the cranial venous plexus to form CoW arteries. Subsequently, acta2+ VSMCs differentiate from pdgfrb+ mural cell progenitors upon recruitment to CoW arteries. The progression of VSMC differentiation exhibits a spatiotemporal pattern, advancing from anterior to posterior CoW arteries. Analysis of blood flow suggests that earlier VSMC differentiation in anterior CoW arteries correlates with higher red blood cell velocity wall shear stress. Furthermore, pulsatile blood flow is required for differentiation of human brain pdgfrb+ mural cells into VSMCs as well as VSMC differentiation on zebrafish CoW arteries. Consistently, the flow-responsive transcription factor klf2a is activated in ECs of CoW arteries prior to VSMC differentiation, and klf2a knockdown delays VSMC differentiation on anterior CoW arteries. In summary, our findings highlight the role of blood flow activation of endothelial klf2a as a mechanism regulating the initial VSMC differentiation on vertebrate brain arteries.

Interference with SRF expression in skeletal muscles reduces peripheral nerve regeneration in mice.

Traumatic injury of peripheral nerves typically also damages nerve surrounding tissue including muscles. Hence, molecular and cellular interactions of neighboring damaged tissues might be decisive for successful axonal regeneration of injured nerves. So far, the contribution of muscles and muscle-derived molecules to peripheral nerve regeneration has only poorly been studied. Herein, we conditionally ablated SRF (serum response factor), an important myofiber transcription factor, in skeletal muscles of mice. Subsequently, the impact of this myofiber-restricted SRF deletion on peripheral nerve regeneration, i.e. facial nerve injury was analyzed. Quantification of facial nerve regeneration by retrograde tracer transport, inspection of neuromuscular junctions (NMJs) and recovery of whisker movement revealed reduced axonal regeneration upon muscle specific Srf deletion. In contrast, responses in brainstem facial motor neuron cell bodies such as regeneration-associated gene (RAG) induction of Atf3, synaptic stripping and neuroinflammation were not overly affected by SRF deficiency. Mechanistically, SRF in myofibers appears to stimulate nerve regeneration through regulation of muscular satellite cell (SC) proliferation. In summary, our data suggest a role of muscle cells and SRF expression within muscles for regeneration of injured peripheral nerves.

The FTLD Risk Factor TMEM106B Regulates the Transport of Lysosomes at the Axon Initial Segment of Motoneurons.

Genetic variations in TMEM106B, coding for a lysosomal membrane protein, affect frontotemporal lobar degeneration (FTLD) in GRN- (coding for progranulin) and C9orf72-expansion carriers and might play a role in aging. To determine the physiological function of TMEM106B, we generated TMEM106B-deficient mice. These mice develop proximal axonal swellings caused by drastically enlarged LAMP1-positive vacuoles, increased retrograde axonal transport of lysosomes, and accumulation of lipofuscin and autophagosomes. Giant vacuoles specifically accumulate at the distal end and within the axon initial segment, but not in peripheral nerves or at axon terminals, resulting in an impaired facial-nerve-dependent motor performance. These data implicate TMEM106B in mediating the axonal transport of LAMP1-positive organelles in motoneurons and axonal sorting at the initial segment. Our data provide mechanistic insight into how TMEM106B affects lysosomal proteolysis and degradative capacity in neurons.

Three-Dimensional In vivo Magnetic Resonance Imaging (MRI) of Mouse Facial Nerve Regeneration.

MRI (magnetic resonance imaging) is an indispensable tool in the diagnosis of centrals nervous system (CNS) disorders such as spinal cord injury and multiple sclerosis (MS). In contrast, diagnosis of peripheral nerve injuries largely depends on clinical and electrophysiological parameters. Thus, currently MRI is not regularly used which in part is due to small nerve calibers and isointensity with surrounding tissue such as muscles. In this study we performed translational MRI research in mice to establish a novel MRI protocol visualizing intact and injured peripheral nerves in a non-invasive manner without contrast agents. With this protocol we were able to image even very small nerves and nerve branches such as the mouse facial nerve (diameter 100-300 µm) at highest spatial resolution. Analysis was performed in the same animal in a longitudinal study spanning 3 weeks after injury. Nerve injury caused hyperintense signal in T2-weighted images and an increase in nerve size of the proximal and distal nerve stumps were observed. Further hyperintense signal was observed in a bulb-like structure in the lesion site, which correlated histologically with the production of fibrotic tissue and immune cell infiltration. The longitudinal MR representation of the facial nerve lesions correlated well with physiological recovery of nerve function by quantifying whisker movement. In summary, we provide a novel protocol in rodents allowing for non-invasive, non-contrast agent enhanced, high-resolution MR imaging of small peripheral nerves longitudinally over several weeks. This protocol might further help to establish MRI as an important diagnostic and post-surgery follow-up tool to monitor peripheral nerve injuries in humans.

Functional and Molecular Characterization of a Novel Traumatic Peripheral Nerve-Muscle Injury Model.

Traumatic injuries to human peripheral nerves are frequently associated with damage to nerve surrounding tissues including muscles and blood vessels. Currently, most rodent models of peripheral nerve injuries (e.g., facial or sciatic nerve) employ surgical nerve transection with scissors or scalpels. However, such an isolated surgical nerve injury only mildly damages neighboring tissues and weakly activates an immune response. In order to provide a rodent nerve injury model accounting for such nerve-associated tissue damage and immune cell activation, we developed a drop tower-based facial nerve trauma model in mice. We compare nerve regeneration in this novel peripheral nerve trauma model with the established surgical nerve injury along several parameters. These include gene expression, histological and functional facial motoneuron (FMN) regeneration, facial nerve degeneration, immune cell activation and muscle damage. Regeneration-associated genes (RAGs; e.g., Atf3) were strongly induced in FMNs subjected to traumatic and surgical injury. Regeneration of FMNs and functional recovery of whisker movement were faster in traumatic versus complete surgical injury, thus cutting down experimentation time. Wallerian degeneration of distal nerve stumps was readily observed in this novel trauma injury model. Importantly, drop tower-inflicted facial nerve injury resulted in muscle damage, activation of muscle satellite cell markers (PAX7) and pronounced infiltration of immune cells to the injury site only in this model but not upon surgical nerve transection. Thus, we provide a novel rodent PNS trauma model that can be easily adopted to other PNS nerves such as the sciatic nerve. Since this nerve trauma model replicates multiple tissue damage frequently encountered in clinical routine, it will be well suited to identify molecular and cellular mechanisms of PNS nerve repair in wild-type and genetically modified rodents.

Atf3 mutant mice show reduced axon regeneration and impaired regeneration-associated gene induction after peripheral nerve injury.

Axon injury in the peripheral nervous system (PNS) induces a regeneration-associated gene (RAG) response. Atf3 (activating transcription factor 3) is such a RAG and ATF3's transcriptional activity might induce 'effector' RAGs (e.g. small proline rich protein 1a (Sprr1a), Galanin (Gal), growth-associated protein 43 (Gap43)) facilitating peripheral axon regeneration. We provide a first analysis of Atf3 mouse mutants in peripheral nerve regeneration. In Atf3 mutant mice, facial nerve regeneration and neurite outgrowth of adult ATF3-deficient primary dorsal root ganglia neurons was decreased. Using genome-wide transcriptomics, we identified a neuropeptide-encoding RAG cluster (vasoactive intestinal peptide (Vip), Ngf, Grp, Gal, Pacap) regulated by ATF3. Exogenous administration of neuropeptides enhanced neurite growth of Atf3 mutant mice suggesting that these molecules might be effector RAGs of ATF3's pro-regenerative function. In addition to the induction of growth-promoting molecules, we present data that ATF3 suppresses growth-inhibiting molecules such as chemokine (C-C motif) ligand 2. In summary, we show a pro-regenerative ATF3 function during PNS nerve regeneration involving transcriptional activation of a neuropeptide-encoding RAG cluster. ATF3 is a general injury-inducible factor, therefore ATF3-mediated mechanisms identified herein might apply to other cell and injury types.