Pten regulates endocytic trafficking of cell adhesion and Wnt signaling molecules to pattern the retina.

The retina is exquisitely patterned, with neuronal somata positioned at regular intervals to completely sample the visual field. Here, we show that phosphatase and tensin homolog (Pten) controls starburst amacrine cell spacing by modulating vesicular trafficking of cell adhesion molecules and Wnt proteins. Single-cell transcriptomics and double-mutant analyses revealed that Pten and Down syndrome cell adhesion molecule Dscam) are co-expressed and function additively to pattern starburst amacrine cell mosaics. Mechanistically, Pten loss accelerates the endocytic trafficking of DSCAM, FAT3, and MEGF10 off the cell membrane and into endocytic vesicles in amacrine cells. Accordingly, the vesicular proteome, a molecular signature of the cell of origin, is enriched in exocytosis, vesicle-mediated transport, and receptor internalization proteins in Pten conditional knockout (PtencKO) retinas. Wnt signaling molecules are also enriched in PtencKO retinal vesicles, and the genetic or pharmacological disruption of Wnt signaling phenocopies amacrine cell patterning defects. Pten thus controls vesicular trafficking of cell adhesion and signaling molecules to establish retinal amacrine cell mosaics.

Sensitization of cancer cells towards Cisplatin and Carboplatin by protein kinase D inhibitors through modulation of ATP7A/B (copper transport ATPases).

Drug resistance of cancer cells is a significant impediment to effective chemotherapy. One primary reason for this is copper exporters - ATPase copper transporting alpha (ATP7A) and ATPase copper transporting beta (ATP7B). These molecular pumps belong to P-type ATPases and dispose off the Platinum (Pt) based anticancer drugs from cancer cells, causing resistance in them. For the disposal of Pt-drugs, copper exporters require phosphorylation mediated by protein kinase D (PKD) for their activation and trafficking. Even though various research works are underway to overcome resistance to anticancer drugs, the role of PKD is mainly ignored. In this study, we have found a significant upregulation of ATP7A and ATP7B in cervical cancer cells (HeLa) and Liver Hepatocellular Carcinoma cells (HepG2) in the presence of Cisplatin or Carboplatin; both at transcriptional as well as translational levels. Interestingly, the expression of ATP7A and ATP7B were significantly downregulated in the presence of a PKD inhibitor (CID2011756), resulting in the reduction of PKD mediated phosphorylation of ATP7A/7B. This causes enhancement of proteasome-mediated degradation of ATP7A/7B and thereby sensitizes the cells towards Cisplatin and Carboplatin. Similarly, the treatment of Cisplatin resistant HepG2 cells with PKD inhibitor causes enhanced sensitivity towards Cisplatin drug. However, the presence of proteasome inhibitor (MG132) reversed the effect of the PKD inhibitor on the expression level of ATP7A/7B, indicating the necessity of phosphorylation for its stability. Hence, we conclude that the combinatorial usage of Cisplatin with drugs targeting PKD can be developed as an effective chemotherapeutic approach to overcome drug resistance.

Focal ablation of apical prostate cancer lesions with irreversible electroporation (IRE).

INTRODUCTION AND OBJECTIVE: To assess the safety, oncological and quality-of-life (QoL) outcomes of focal ablation of apical prostate cancer (PCa) lesions with irreversible electroporation (IRE). METHODS: Patients were included in the study if they had a PCa lesion within 3 mm of the apical capsule treated with IRE. The IRE procedure was performed in our institution by a single urologist. The QoL and functional data was collected prospectively from patients who provided consent using the Expanded Prostate Cancer Index Composite (EPIC). Oncological follow up included 3-month PSA levels, mpMRI at 6 months and transperineal biopsy at 1-year post treatment. RESULTS: A total of 50 patients had apical PCa lesions treated between February 2013 and September 2018. Median follow-up was 44 months. There were no Clavien-Dindo grade 3 events or higher. No perioperative complications were recorded. No significant difference was observed in the EPIC urinary or bowel QoL domain between baseline and 12-month post-treatment. One patient (2%) required one pad per day for urinary incontinence 12-month post-treatment. There was a small but significant decline in EPIC sexual QoL (65 at baseline and 59 at 12-month post-IRE). Of patient's potent pre-treatment, 94% remained potent after treatment. The median PSA nadir decreased by 71% (6.25-1.7 ng/mL). Only one patient (2.5%) had in-field residual disease on repeat biopsy. CONCLUSION: Focal ablation using IRE for PCa in the distal apex appears safe and feasible with acceptable early QoL and oncologic outcomes.

Cystatin C Plays a Sex-Dependent Detrimental Role in Experimental Autoimmune Encephalomyelitis.

The cysteine protease inhibitor Cystatin C (CST3) is highly expressed in the brains of multiple sclerosis (MS) patients and C57BL/6J mice with experimental autoimmune encephalomyelitis (EAE; a model of MS), but its roles in the diseases are unknown. Here, we show that CST3 plays a detrimental function in myelin oligodendrocyte glycoprotein 35-55 (MOG35-55)-induced EAE but only in female animals. Female Cst3 null mice display significantly lower clinical signs of disease compared to wild-type (WT) littermates. This difference is associated with reduced interleukin-6 production and lower expression of key proteins (CD80, CD86, major histocompatibility complex [MHC] II, LC3A/B) involved in antigen processing, presentation, and co-stimulation in antigen-presenting cells (APCs). In contrast, male WT and Cst3-/- mice and cells show no differences in EAE signs or APC function. Further, the sex-dependent effect of CST3 in EAE is sensitive to gonadal hormones. Altogether, we have shown that CST3 has a sex-dependent role in MOG35-55-induced EAE.

The Emerging Role of Extracellular Vesicles in the Glioma Microenvironment: Biogenesis and Clinical Relevance.

Gliomas are a diverse group of brain tumors comprised of malignant cells ('tumor' cells) and non-malignant 'normal' cells, including neural (neurons, glia), inflammatory (microglia, macrophage) and vascular cells. Tumor heterogeneity arises in part because, within the glioma mass, both 'tumor' and 'normal' cells secrete factors that form a unique microenvironment to influence tumor progression. Extracellular vesicles (EVs) are critical mediators of intercellular communication between immediate cellular neighbors and distantly located cells in healthy tissues/organs and in tumors, including gliomas. EVs mediate cell-cell signaling as carriers of nucleic acid, lipid and protein cargo, and their content is unique to cell types and physiological states. EVs secreted by non-malignant neural cells have important physiological roles in the healthy brain, which can be altered or co-opted to promote tumor progression and metastasis, acting in combination with glioma-secreted EVs. The cell-type specificity of EV content means that 'vesiculome' data can potentially be used to trace the cell of origin. EVs may also serve as biomarkers to be exploited for disease diagnosis and to assess therapeutic progress. In this review, we discuss how EVs mediate intercellular communication in glioma, and their potential role as biomarkers and readouts of a therapeutic response.

Ascl1 is required to specify a subset of ventromedial hypothalamic neurons.

Despite clear physiological roles, the ventromedial hypothalamus (VMH) developmental programs are poorly understood. Here, we asked whether the proneural gene achaete-scute homolog 1 (Ascl1) contributes to VMH development. Ascl1 transcripts were detected in embryonic day (E) 10.5 to postnatal day 0 VMH neural progenitors. The elimination of Ascl1 reduced the number of VMH neurons at E12.5 and E15.5, particularly within the VMH-central (VMHC) and -dorsomedial (VMHDM) subdomains, and resulted in a VMH cell fate change from glutamatergic to GABAergic. We observed a loss of Neurog3 expression in Ascl1-/- hypothalamic progenitors and an upregulation of Neurog3 when Ascl1 was overexpressed. We also demonstrated a glutamatergic to GABAergic fate switch in Neurog3-null mutant mice, suggesting that Ascl1 might act via Neurog3 to drive VMH cell fate decisions. We also showed a concomitant increase in expression of the central GABAergic fate determinant Dlx1/2 in the Ascl1-null hypothalamus. However, Ascl1 was not sufficient to induce an ectopic VMH fate when overexpressed outside the normal window of competency. Combined, Ascl1 is required but not sufficient to specify the neurotransmitter identity of VMH neurons, acting in a transcriptional cascade with Neurog3.

Insights Into the Role and Potential of Schwann Cells for Peripheral Nerve Repair From Studies of Development and Injury.

Peripheral nerve injuries arising from trauma or disease can lead to sensory and motor deficits and neuropathic pain. Despite the purported ability of the peripheral nerve to self-repair, lifelong disability is common. New molecular and cellular insights have begun to reveal why the peripheral nerve has limited repair capacity. The peripheral nerve is primarily comprised of axons and Schwann cells, the supporting glial cells that produce myelin to facilitate the rapid conduction of electrical impulses. Schwann cells are required for successful nerve regeneration; they partially "de-differentiate" in response to injury, re-initiating the expression of developmental genes that support nerve repair. However, Schwann cell dysfunction, which occurs in chronic nerve injury, disease, and aging, limits their capacity to support endogenous repair, worsening patient outcomes. Cell replacement-based therapeutic approaches using exogenous Schwann cells could be curative, but not all Schwann cells have a "repair" phenotype, defined as the ability to promote axonal growth, maintain a proliferative phenotype, and remyelinate axons. Two cell replacement strategies are being championed for peripheral nerve repair: prospective isolation of "repair" Schwann cells for autologous cell transplants, which is hampered by supply challenges, and directed differentiation of pluripotent stem cells or lineage conversion of accessible somatic cells to induced Schwann cells, with the potential of "unlimited" supply. All approaches require a solid understanding of the molecular mechanisms guiding Schwann cell development and the repair phenotype, which we review herein. Together these studies provide essential context for current efforts to design glial cell-based therapies for peripheral nerve regeneration.

Plag1 and Plagl2 have overlapping and distinct functions in telencephalic development.

The Plag gene family has three members; Plagl1/Zac1, which is a tumor suppressor gene, and Plag1 and Plagl2, which are proto-oncogenes. All three genes are known to be expressed in embryonic neural progenitors, and Zac1 regulates proliferation, neuronal differentiation and migration in the developing neocortex. Here we examined the functions of Plag1 and Plagl2 in neocortical development. We first attempted, and were unable to generate, E12.5 Plag1;Plagl2 double mutants, indicating that at least one Plag1 or Plagl2 gene copy is required for embryonic survival. We therefore focused on single mutants, revealing a telencephalic patterning defect in E12.5 Plagl2 mutants and a proliferation/differentiation defect in Plag1 mutant neocortices. Specifically, the ventral pallium, a dorsal telencephalic territory, expands into the ventral telencephalon in Plagl2 mutants. In contrast, Plag1 mutants develop normal regional territories, but neocortical progenitors proliferate less and instead produce more neurons. Finally, in gain-of-function studies, both Plag1 and Plagl2 reduce neurogenesis and increase BrdU-uptake, indicative of enhanced proliferation, but while Plagl2 effects on proliferation are more immediate, Plag1 effects are delayed. Taken together, we found that the Plag proto-oncogenes genes are essential regulators of neocortical development and although Plag1 and Plagl2 functions are similar, they do not entirely overlap. This article has an associated First Person interview with the first author of the paper.

A non-canonical role for the proneural gene Neurog1 as a negative regulator of neocortical neurogenesis.

Neural progenitors undergo temporal identity transitions to sequentially generate the neuronal and glial cells that make up the mature brain. Proneural genes have well-characterised roles in promoting neural cell differentiation and subtype specification, but they also regulate the timing of identity transitions through poorly understood mechanisms. Here, we investigated how the highly related proneural genes Neurog1 and Neurog2 interact to control the timing of neocortical neurogenesis. We found that Neurog1 acts in an atypical fashion as it is required to suppress rather than promote neuronal differentiation in early corticogenesis. In Neurog1-/- neocortices, early born neurons differentiate in excess, whereas, in vitro, Neurog1-/- progenitors have a decreased propensity to proliferate and form neurospheres. Instead, Neurog1-/- progenitors preferentially generate neurons, a phenotype restricted to the Neurog2+ progenitor pool. Mechanistically, Neurog1 and Neurog2 heterodimerise, and while Neurog1 and Neurog2 individually promote neurogenesis, misexpression together blocks this effect. Finally, Neurog1 is also required to induce the expression of neurogenic factors (Dll1 and Hes5) and to repress the expression of neuronal differentiation genes (Fezf2 and Neurod6). Neurog1 thus employs different mechanisms to temper the pace of early neocortical neurogenesis.

Neurog2 and Ascl1 together regulate a postmitotic derepression circuit to govern laminar fate specification in the murine neocortex.

A derepression mode of cell-fate specification involving the transcriptional repressors Tbr1, Fezf2, Satb2, and Ctip2 operates in neocortical projection neurons to specify six layer identities in sequence. Less well understood is how laminar fate transitions are regulated in cortical progenitors. The proneural genes Neurog2 and Ascl1 cooperate in progenitors to control the temporal switch from neurogenesis to gliogenesis. Here we asked whether these proneural genes also regulate laminar fate transitions. Several defects were observed in the derepression circuit in Neurog2-/-;Ascl1-/- mutants: an inability to repress expression of Tbr1 (a deep layer VI marker) during upper-layer neurogenesis, a loss of Fezf2+/Ctip2+ layer V neurons, and precocious differentiation of normally late-born, Satb2+ layer II-IV neurons. Conversely, in stable gain-of-function transgenics, Neurog2 promoted differentiative divisions and extended the period of Tbr1+/Ctip2+ deep-layer neurogenesis while reducing Satb2+ upper-layer neurogenesis. Similarly, acute misexpression of Neurog2 in early cortical progenitors promoted Tbr1 expression, whereas both Neurog2 and Ascl1 induced Ctip2. However, Neurog2 was unable to influence the derepression circuit when misexpressed in late cortical progenitors, and Ascl1 repressed only Satb2. Nevertheless, neurons derived from late misexpression of Neurog2 and, to a lesser extent, Ascl1, extended aberrant subcortical axon projections characteristic of early-born neurons. Finally, Neurog2 and Ascl1 altered the expression of Ikaros and Foxg1, known temporal regulators. Proneural genes thus act in a context-dependent fashion as early determinants, promoting deep-layer neurogenesis in early cortical progenitors via input into the derepression circuit while also influencing other temporal regulators.

Temporal Analysis of Gene Expression in the Murine Schwann Cell Lineage and the Acutely Injured Postnatal Nerve.

Schwann cells (SCs) arise from neural crest cells (NCCs) that first give rise to SC precursors (SCPs), followed by immature SCs, pro-myelinating SCs, and finally, non-myelinating or myelinating SCs. After nerve injury, mature SCs 'de-differentiate', downregulating their myelination program while transiently re-activating early glial lineage genes. To better understand molecular parallels between developing and de-differentiated SCs, we characterized the expression profiles of a panel of 12 transcription factors from the onset of NCC migration through postnatal stages, as well as after acute nerve injury. Using Sox10 as a pan-glial marker in co-expression studies, the earliest transcription factors expressed in E9.0 Sox10+ NCCs were Sox9, Pax3, AP2α and Nfatc4. E10.5 Sox10+ NCCs coalescing in the dorsal root ganglia differed slightly, expressing Sox9, Pax3, AP2α and Etv5. E12.5 SCPs continued to express Sox10, Sox9, AP2α and Pax3, as well as initiating Sox2 and Egr1 expression. E14.5 immature SCs were similar to SCPs, except that they lost Pax3 expression. By E18.5, AP2α, Sox2 and Egr1 expression was turned off in the nerve, while Jun, Oct6 and Yy1 expression was initiated in pro-myelinating Sox9+/Sox10+ SCs. Early postnatal and adult SCs continued to express Sox9, Jun, Oct6 and Yy1 and initiated Nfatc4 and Egr2 expression. Notably, at all stages, expression of each marker was observed only in a subset of Sox10+ SCs, highlighting the heterogeneity of the SC pool. Following acute nerve injury, Egr1, Jun, Oct6, and Sox2 expression was upregulated, Egr2 expression was downregulated, while Sox9, Yy1, and Nfatc4 expression was maintained at similar frequencies. Notably, de-differentiated SCs in the injured nerve did not display a transcription factor profile corresponding to a specific stage in the SC lineage. Taken together, we demonstrate that uninjured and injured SCs are heterogeneous and distinct from one another, and de-differentiation recapitulates transcriptional aspects of several different embryonic stages.