Meclofenamate causes loss of cellular tethering and decoupling of functional networks in glioblastoma.

BACKGROUND: Glioblastoma cells assemble to a syncytial communicating network based on tumor microtubes (TMs) as ultra-long membrane protrusions. The relationship between network architecture and transcriptional profile remains poorly investigated. Drugs that interfere with this syncytial connectivity such as meclofenamate (MFA) may be highly attractive for glioblastoma therapy. METHODS: In a human neocortical slice model using glioblastoma cell populations of different transcriptional signatures, three-dimensional tumor networks were reconstructed, and TM-based intercellular connectivity was mapped on the basis of two-photon imaging data. MFA was used to modulate morphological and functional connectivity; downstream effects of MFA treatment were investigated by RNA sequencing and fluorescence-activated cell sorting (FACS) analysis. RESULTS: TM-based network morphology strongly differed between the transcriptional cellular subtypes of glioblastoma and was dependent on axon guidance molecule expression. MFA revealed both a functional and morphological demolishment of glioblastoma network architectures which was reflected by a reduction of TM-mediated intercellular cytosolic traffic as well as a breakdown of TM length. RNA sequencing confirmed a downregulation of NCAM and axon guidance molecule signaling upon MFA treatment. Loss of glioblastoma communicating networks was accompanied by a failure in the upregulation of genes that are required for DNA repair in response to temozolomide (TMZ) treatment and culminated in profound treatment response to TMZ-mediated toxicity. CONCLUSION: The capacity of TM formation reflects transcriptional cellular heterogeneity. MFA effectively demolishes functional and morphological TM-based syncytial network architectures. These findings might pave the way to a clinical implementation of MFA as a TM-targeted therapeutic approach.

Novel Hexb-based tools for studying microglia in the CNS.

Microglia and central nervous system (CNS)-associated macrophages (CAMs), such as perivascular and meningeal macrophages, are implicated in virtually all diseases of the CNS. However, little is known about their cell-type-specific roles in the absence of suitable tools that would allow for functional discrimination between the ontogenetically closely related microglia and CAMs. To develop a new microglia gene targeting model, we first applied massively parallel single-cell analyses to compare microglia and CAM signatures during homeostasis and disease and identified hexosaminidase subunit beta (Hexb) as a stably expressed microglia core gene, whereas other microglia core genes were substantially downregulated during pathologies. Next, we generated HexbtdTomato mice to stably monitor microglia behavior in vivo. Finally, the Hexb locus was employed for tamoxifen-inducible Cre-mediated gene manipulation in microglia and for fate mapping of microglia but not CAMs. In sum, we provide valuable new genetic tools to specifically study microglia functions in the CNS.

A Subset of Skin Macrophages Contributes to the Surveillance and Regeneration of Local Nerves.

The skin comprises tissue macrophages as the most abundant resident immune cell type. Their diverse tasks including resistance against invading pathogens, attraction of bypassing immune cells from vessels, and tissue repair require dynamic specification. Here, we delineated the postnatal development of dermal macrophages and their differentiation into subsets by adapting single-cell transcriptomics, fate mapping, and imaging. Thereby we identified a phenotypically and transcriptionally distinct subset of prenatally seeded dermal macrophages that self-maintained with very low postnatal exchange by hematopoietic stem cells. These macrophages specifically interacted with sensory nerves and surveilled and trimmed the myelin sheath. Overall, resident dermal macrophages contributed to axon sprouting after mechanical injury. In summary, our data show long-lasting functional specification of macrophages in the dermis that is driven by stepwise adaptation to guiding structures and ensures codevelopment of ontogenetically distinct cells within the same compartment.

Human organotypic brain slice culture: a novel framework for environmental research in neuro-oncology.

When it comes to the human brain, models that closely mimic in vivo conditions are lacking. Living neuronal tissue is the closest representation of the in vivo human brain outside of a living person. Here, we present a method that can be used to maintain therapeutically resected healthy neuronal tissue for prolonged periods without any discernible changes in tissue vitality, evidenced by immunohistochemistry, genetic expression, and electrophysiology. This method was then used to assess glioblastoma (GBM) progression in its natural environment by microinjection of patient-derived tumor cells into cultured sections. The result closely resembles the pattern of de novo tumor growth and invasion, drug therapy response, and cytokine environment. Reactive transformation of astrocytes, as an example of the cellular nonmalignant tumor environment, can be accurately simulated with transcriptional differences similar to those of astrocytes isolated from acute GBM specimens. In a nutshell, we present a simple method to study GBM in its physiological environment, from which valuable insights can be gained. This technique can lead to further advancements in neuroscience, neuro-oncology, and pharmacotherapy.

Spinal muscular atrophy pathogenic mutations impair the axonogenic properties of axonal-survival of motor neuron.

The axonal survival of motor neuron (a-SMN) protein is a truncated isoform of SMN1, the spinal muscular atrophy (SMA) disease gene. a-SMN is selectively localized in axons and endowed with remarkable axonogenic properties. At present, the role of a-SMN in SMA is unknown. As a first step to verify a link between a-SMN and SMA, we investigated by means of over-expression experiments in neuroblastoma-spinal cord hybrid cell line (NSC34) whether SMA pathogenic mutations located in the N-terminal part of the protein affected a-SMN function. We demonstrated here that either SMN1 missense mutations or small intragenic re-arrangements located in the Tudor domain consistently altered the a-SMN capability of inducing axonal elongation in vitro. Mutated human a-SMN proteins determined in almost all NSC34 motor neurons the growth of short axons with prominent morphologic abnormalities. Our data indicate that the Tudor domain is critical in dictating a-SMN function possibly because it is an association domain for proteins involved in axon growth. They also indicate that Tudor domain mutations are functionally relevant not only for FL-SMN but also for a-SMN, raising the possibility that also a-SMN loss of function may contribute to the pathogenic steps leading to SMA.