Pip5k1γ regulates axon formation by limiting Rap1 activity.

During their differentiation, neurons establish a highly polarized morphology by forming axons and dendrites. Cortical and hippocampal neurons initially extend several short neurites that all have the potential to become an axon. One of these neurites is then selected as the axon by a combination of positive and negative feedback signals that promote axon formation and prevent the remaining neurites from developing into axons. Here, we show that Pip5k1γ is required for the formation of a single axon as a negative feedback signal that regulates C3G and Rap1 through the generation of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). Impairing the function of Pip5k1γ results in a hyper-activation of the Fyn/C3G/Rap1 pathway, which induces the formation of supernumerary axons. Application of a hyper-osmotic shock to modulate membrane tension has a similar effect, increasing Rap1 activity and inducing the formation of supernumerary axons. In both cases, the induction of supernumerary axons can be reverted by expressing constitutively active Pip5k. Our results show that PI(4,5)P2-dependent membrane properties limit the activity of C3G and Rap1 to ensure the extension of a single axon.

Optical Clearing and Labeling for Light-sheet Fluorescence Microscopy in Large-scale Human Brain Imaging.

Despite the numerous clearing techniques that emerged in the last decade, processing postmortem human brains remains a challenging task due to its dimensions and complexity, which make imaging with micrometer resolution particularly difficult. This paper presents a protocol to perform the reconstruction of volumetric portions of the human brain by simultaneously processing tens of sections with the SHORT (SWITCH - H2O2 - Antigen Retrieval - 2,2'-thiodiethanol [TDE]) tissue transformation protocol, which enables clearing, labeling, and sequential imaging of the samples with light-sheet fluorescence microscopy (LSFM). SHORT provides rapid tissue clearing and homogeneous multi-labeling of thick slices with several neuronal markers, enabling the identification of different neuronal subpopulations in both white and grey matter. After clearing, the slices are imaged via LSFM with micrometer resolution and in multiple channels simultaneously for a rapid 3D reconstruction. By combining SHORT with LSFM analysis within a routinely high-throughput protocol, it is possible to obtain the 3D cytoarchitecture reconstruction of large volumetric areas at high resolution in a short time, thus enabling comprehensive structural characterization of the human brain.

The balance of mitochondrial fission and fusion in cortical axons depends on the kinases SadA and SadB.

Neurons are highly polarized cells that display characteristic differences in the organization of their organelles in axons and dendrites. The kinases SadA and SadB (SadA/B) promote the formation of distinct axonal and dendritic extensions during the development of cortical and hippocampal neurons. Here, we show that SadA/B are required for the specific dynamics of axonal mitochondria. Ankyrin B (AnkB) stimulates the activity of SadA/B that function as regulators of mitochondrial dynamics through the phosphorylation of tau. Suppression of SadA/B or AnkB in cortical neurons induces the elongation of mitochondria by disrupting the balance of fission and fusion. SadA/B-deficient neurons show an accumulation of hyper-fused mitochondria and activation of the integrated stress response (ISR). The normal dynamics of axonal mitochondria could be restored by mild actin destabilization. Thus, the elongation after loss of SadA/B results from an excessive stabilization of actin filaments and reduction of Drp1 recruitment to mitochondria.

On the role of vesicle transport in neurite growth: Modeling and experiments.

The processes that determine the establishment of the complex morphology of neurons during development are still poorly understood. Here, we focus on the question how a difference in the length of neurites affects vesicle transport. We performed live imaging experiments and present a lattice-based model to gain a deeper theoretical understanding of intracellular transport in neurons. After a motivation and appropriate scaling of the model we present numerical simulations showing that initial differences in neurite length result in phenomena of biological relevance, i.e. a positive feedback that enhances transport into the longer neurite and oscillation of vesicles concentrations that can be interpreted as cycles of extension and retraction observed in experiments. Thus, our model is a first step towards a better understanding of the interplay between the transport of vesicles and the spatial organization of cells.

Igf1R/InsR function is required for axon extension and corpus callosum formation.

One of the earliest steps during the development of the nervous system is the establishment of neuronal polarity and the formation of an axon. The intrinsic mechanisms that promote axon formation have been extensively analyzed. However, much less is known about the extrinsic signals that initiate axon formation. One of the candidates for these signals is Insulin-like growth factor 1 (Igf1) that acts through the Igf1 (Igf1R) and insulin receptors (InsR). Since Igf1R and InsR may act redundantly we analyzed conditional cortex-specific knockout mice that are deficient for both Igf1r and Insr to determine if they regulate the development of the cortex and the formation of axons in vivo. Our results show that Igf1R/InsR function is required for the normal development of the embryonic hippocampus and cingulate cortex while the lateral cortex does not show apparent defects in the Igf1r;Insr knockout. In the cingulate cortex, the number of intermediate progenitors and deep layer neurons is reduced and the corpus callosum is absent at E17. However, cortical organization and axon formation are not impaired in knockout embryos. In culture, cortical and hippocampal neurons from Igf1r;Insr knockout embryos extend an axon but the length of this axon is severely reduced. Our results indicate that Igf1R/InsR function is required for brain development in a region-specific manner and promotes axon growth but is not essential for neuronal polarization and migration in the developing brain.

Curcumin Analogue C1 Promotes Hex and Gal Recruitment to the Plasma Membrane via mTORC1-Independent TFEB Activation.

The monocarbonyl analogue of curcumin (1E,4E)-1,5-Bis(2-methoxyphenyl)penta-1,4-dien-3-one (C1) has been used as a specific activator of the master gene transcription factor EB (TFEB) to correlate the activation of this nuclear factor with the increased activity of lysosomal glycohydrolases and their recruitment to the cell surface. The presence of active lysosomal glycohydrolases associated with the lipid microdomains has been extensively demonstrated, and their role in glycosphingolipid (GSL) remodeling in both physiological and pathological conditions, such as neurodegenerative disorders, has been suggested. Here, we demonstrate that Jurkat cell stimulation elicits TFEB nuclear translocation and an increase of both the expression of hexosaminidase subunit beta (HEXB), hexosaminidase subunit alpha (HEXA), and galactosidase beta 1 (GLB1) genes, and the recruitment of ß-hexosaminidase (Hex, EC 3.2.1.52) and ß-galactosidase (Gal, EC 3.2.1.23) on lipid microdomains. Treatment of Jurkat cells with the curcumin analogue C1 also resulted in an increase of both lysosomal glycohydrolase activity and their targeting to the cell surface. Similar effects of C1 on lysosomal glycohydrolase expression and their recruitment to lipid microdomains was observed by treating the SH-SY5Y neuroblastoma cell line; the effects of C1 treatment were abolished by TFEB silencing. Together, these results clearly demonstrate the existence of a direct link between TFEB nuclear translocation and the transport of Hex and Gal from lysosomes to the plasma membrane.

TFEB activation restores migration ability to Tsc1-deficient adult neural stem/progenitor cells.

Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disorder caused by mutations in either of two genes, TSC1 or TSC2, resulting in the constitutive activation of the mammalian target of rapamycin complex 1 (mTORC1). mTOR inhibitors are now considered the treatment of choice for TSC disease. A major pathological feature of TSC is the development of subependymal giant cell astrocytomas (SEGAs) in the brain. Nowadays, it is thought that SEGAs could be a consequence of aberrant aggregation and migration of neural stem/progenitor cells (NSPCs). Therefore, reactivation of cell migration of NSPCs might be the crucial step for the treatment of patients. In order to identify potential in vitro targets activating migration, we generated Tsc1-deficient NSPCs. These cells summarize most of the biochemical and morphological characteristics of TSC neural cells, such as the mTORC1 activation, the formation of abnormally enlarged astrocytes-like cells, the reduction of autophagy flux and the impairment of cell migration. Moreover, nuclear translocation, namely activation of the transcription factor EB (TFEB) was markedly impaired. Herein, we show that compounds such as everolimus, ionomycin and curcumin, which directly or indirectly stimulate TFEB nuclear translocation, restore Tsc1-deficient NSPC migration. Our data suggest that reduction of TFEB activation, caused by mTORC1 hyperactivation, contributes to the migration deficit characterizing Tsc1-deficient NSPCs. The present work highlights TFEB as a druggable protein target for SEGAs therapy, which can be additionally or alternatively exploited for the mTORC1-directed inhibitory approach.