A spatial-temporal map of glutamatergic neurogenesis in the murine embryonic cerebellar nuclei uncovers a high degree of cellular heterogeneity.

The nuclei are the main output structures of the cerebellum. Each and every cerebellar cortical computation reaches several areas of the brain by means of cerebellar nuclei processing and integration. Nevertheless, our knowledge of these structures is still limited compared to the cerebellar cortex. Here, we present a mouse genetic inducible fate-mapping study characterizing rhombic lip-derived glutamatergic neurons of the nuclei, the most conspicuous family of long-range cerebellar efferent neurons. Glutamatergic neurons mainly occupy dorsal and lateral territories of the lateral and interposed nuclei, as well as the entire medial nucleus. In mice, they are born starting from about embryonic day 9.5, with a peak between 10.5 and 12.5, and invade the nuclei with a lateral-to-medial progression. While some markers label a heterogeneous population of neurons sharing a common location (BRN2), others appear to be lineage specific (TBR1, LMX1a, and MEIS2). A comparative analysis of TBR1 and LMX1a distributions reveals an incomplete overlap in their expression domains, in keeping with the existence of separate efferent subpopulations. Finally, some tagged glutamatergic progenitors are not labeled by any of the markers used in this study, disclosing further complexity. Taken together, our results obtained in late embryonic nuclei shed light on the heterogeneity of the excitatory neuron pool, underlying the diversity in connectivity and functions of this largely unexplored cerebellar territory. Our findings contribute to laying the groundwork for a comprehensive functional analysis of nuclear neuron subpopulations.

Ex Vivo Analysis of Cell Differentiation, Oxidative Stress, Inflammation, and DNA Damage on Cutaneous Field Cancerization.

Cutaneous field cancerization (CFC) refers to a skin region containing mutated cells' clones, predominantly arising from chronic exposure to ultraviolet radiation (UVR), which exhibits an elevated risk of developing precancerous and neoplastic lesions. Despite extensive research, many molecular aspects of CFC still need to be better understood. In this study, we conducted ex vivo assessment of cell differentiation, oxidative stress, inflammation, and DNA damage in CFC samples. We collected perilesional skin from 41 patients with skin cancer and non-photoexposed skin from 25 healthy control individuals. These biopsies were either paraffin-embedded for indirect immunofluorescence and immunohistochemistry stain or processed for proteins and mRNA extraction from the epidermidis. Our findings indicate a downregulation of p53 expression and an upregulation of Ki67 and p16 in CFC tissues. Additionally, there were alterations in keratinocyte differentiation markers, disrupted cell differentiation, increased expression of iNOS and proinflammatory cytokines IL-6 and IL-8, along with evidence of oxidative DNA damage. Collectively, our results suggest that despite its outwardly normal appearance, CFC tissue shows early signs of DNA damage, an active inflammatory state, oxidative stress, abnormal cell proliferation and differentiation.

ZFP423 regulates early patterning and multiciliogenesis in the hindbrain choroid plexus.

The choroid plexus (ChP) is a secretory tissue that produces cerebrospinal fluid (CSF) secreted into the ventricular system. It is a monolayer of secretory, multiciliated epithelial cells derived from neuroepithelial progenitors and overlying a stroma of mesenchymal cells of mesodermal origin. Zfp423, which encodes a Kruppel-type zinc-finger transcription factor essential for cerebellar development and mutated in rare cases of cerebellar vermis hypoplasia/Joubert syndrome and other ciliopathies, is expressed in the hindbrain roof plate, from which the IV ventricle ChP arises, and, later, in mesenchymal cells, which give rise to the stroma and leptomeninges. Mouse Zfp423 mutants display a marked reduction of the hindbrain ChP (hChP), which: (1) fails to express established markers of its secretory function and genes implicated in its development and maintenance (Lmx1a and Otx2); (2) shows a perturbed expression of signaling pathways previously unexplored in hChP patterning (Wnt3); and (3) displays a lack of multiciliated epithelial cells and a profound dysregulation of master genes of multiciliogenesis (Gmnc). Our results propose that Zfp423 is a master gene and one of the earliest known determinants of hChP development.

Expression of Epsin3 and its interaction with Notch signalling in oral epithelial dysplasia and oral squamous cell carcinoma.

BACKGROUND: Most oral squamous cell carcinoma patients present with late-stage disease. Early detection of the disease is considered to be the most effective way of improving patient outcomes. Several biomarkers have been identified as indicators of oral cancer development and progression; however, none have been translated into clinical practice. In this study, we have investigated the role of Epsin3, an endocytic adaptor protein, and Notch1, a transmembrane signalling protein, in oral carcinogenesis with a view to explore their potential as biomarkers. METHODS: Oral cancer cell lines and a normal oral keratinocyte cell line were used together with tissue samples of normal oral mucosa (n = 21), oral epithelial dysplasia (n = 74) and early stage (Stages I and II) oral squamous cell carcinoma (n = 31). Immunocytochemical staining, immunoblotting and real-time quantitative polymerase chain reaction (PCR) were performed to assess protein as well as gene expression levels. RESULTS: The expression levels of Epsin3 and Notch1 mRNA and protein are variable across different oral squamous cell carcinoma derived cell lines. Epsin3 was upregulated in oral epithelial dysplasia and oral squamous cell carcinoma tissues compared with normal epithelium. Overexpression of Epsin3 resulted in a significant reduction of Notch1 expression in oral squamous cell carcinoma. Notch1 was generally downregulated in the dysplasia and oral squamous cell carcinoma samples. CONCLUSION: Epsin3 is upregulated in oral epithelial dysplasia and oral squamous cell carcinoma and has the potential to be used as a biomarker for oral epithelial dysplasia. Notch signalling is downregulated in oral squamous cell carcinoma, possibly through an Epsin3-induced de-activation pathway.

HYPERREFLECTIVE BAND IN THE GANGLION CELL LAYER IN RETINITIS PIGMENTOSA.

PURPOSE: To describe a sign that takes the form of a continuous hyperreflective band within the thickness of the ganglion cell layer (GCL), thus dubbed the "hyperreflective ganglion cell layer band" (HGB), which the authors detected in a fraction of patients affected by retinitis pigmentosa (RP). METHODS: Retrospective, cross-sectional, observational study. Optical coherence tomography (OCT) images of patients with RP examined between May 2015 and June 2021 were retrospectively reviewed for the presence of HGB, epiretinal membrane (ERM), macular hole, and cystoid macular edema (CME). The ellipsoid zone (EZ) width was also measured. A subgroup of patients underwent microperimetry in the central 2°, 4°, and 10°. RESULTS: One hundred and fifty-four eyes from 77 subjects were included in the study. The HGB was present in 39 (25.3%) eyes with RP. Mean best-corrected visual acuity (BCVA) was 0.39 ± 0.05 logMAR (approximately 20/50 Snellen equivalent) and 0.18 ± 0.03 logMAR (approximately 20/32 Snellen equivalent) in eyes with and without HGB, respectively ( P < 0.001). The two groups did not differ regarding EZ width; mean 2°, 4°, and 10° retinal sensitivity; and prevalence of CME, ERM, and macular hole. The multivariable analysis showed the presence of HGB to be a predictor of poorer BCVA ( P < 0.001). CONCLUSION: HGB is an OCT finding detectable in approximately a quarter of eyes with RP and is associated with a poorer visual function. In the discussion, the authors speculate about possible morphogenetic scenarios to explain this observation.

Tetracyclines in COVID-19 patients quarantined at home: Literature evidence supporting real-world data from a multicenter observational study targeting inflammatory and infectious dermatoses.

Tetracyclines (TetraC) are widely used in dermatology for both inflammatory and infectious dermatoses; recently both in vivo and in vitro studies started to suggest also a potential antiviral effect. During COVID-19 outbreak, several dermatological patients contracted SARS-CoV-2 experiencing only mild symptoms, but no protocol were approved. A multicenter prospective observational study that enrolled COVID-19 patients visited with teledermatology and undergoing TetraC was performed. About 38 adult outpatients (M/F: 20/18, age 42.6 years [21-67]) were enrolled. During the TetraC treatment, symptoms resolved in all patients within 10 days. Remarkably, ageusia and anosmia disappeared in the first week of TetraC treatment. TetraC seem a promising drug to treat COVID-19 outpatients with mild symptoms.

Zfp423/ZNF423 regulates cell cycle progression, the mode of cell division and the DNA-damage response in Purkinje neuron progenitors.

The Zfp423/ZNF423 gene encodes a 30-zinc-finger transcription factor involved in key developmental pathways. Although null Zfp423 mutants develop cerebellar malformations, the underlying mechanism remains unknown. ZNF423 mutations are associated with Joubert Syndrome, a ciliopathy causing cerebellar vermis hypoplasia and ataxia. ZNF423 participates in the DNA-damage response (DDR), raising questions regarding its role as a regulator of neural progenitor cell cycle progression in cerebellar development. To characterize in vivo the function of ZFP423 in neurogenesis, we analyzed allelic murine mutants in which distinct functional domains are deleted. One deletion impairs mitotic spindle orientation, leading to premature cell cycle exit and Purkinje cell (PC) progenitor pool deletion. The other deletion impairs PC differentiation. In both mutants, cell cycle progression is remarkably delayed and DDR markers are upregulated in cerebellar ventricular zone progenitors. Our in vivo evidence sheds light on the domain-specific roles played by ZFP423 in different aspects of PC progenitor development, and at the same time strengthens the emerging notion that an impaired DDR may be a key factor in the pathogenesis of JS and other ciliopathies.

Clinical Implications of Acquired BRAF Inhibitors Resistance in Melanoma.

Understanding the role of mitogen-activated protein kinase (MAPK) pathway-activating mutations in the development and progression of melanoma and their possible use as therapeutic targets has substantially changed the management of this neoplasm, which, until a few years ago, was burdened by severe mortality. However, the presence of numerous intrinsic and extrinsic mechanisms of resistance to BRAF inhibitors compromises the treatment responses' effectiveness and durability. The strategy of overcoming these resistances by combination therapy has proved successful, with the additional benefit of reducing side effects derived from paradoxical activation of the MAPK pathway. Furthermore, the use of other highly specific inhibitors, intermittent dosing schedules and the association of combination therapy with immune checkpoint inhibitors are promising new therapeutic strategies. However, numerous issues related to dose, tolerability and administration sequence still need to be clarified, as is to be expected from currently ongoing trials. In this review, we describe the clinical results of using BRAF inhibitors in advanced melanoma, with a keen interest in strategies aimed at overcoming resistance.

Dynamic Expression and New Functions of Early B Cell Factor 2 in Cerebellar Development.

The collier/Olf1/EBF family genes encode helix-loop-helix transcription factors (TFs) highly conserved in evolution, initially characterized for their roles in the immune system and in various aspects of neural development. The Early B cell Factor 2 (Ebf2) gene plays an important role in the establishment of cerebellar cortical topography and in Purkinje cell (PC) subtype specification. In the adult cerebellum, Ebf2 is expressed in zebrin II (ZII)-negative PCs, where it suppresses the ZII+ molecular phenotype. However, it is not clear whether Ebf2 is restricted to a PC subset from the onset of its expression or is initially distributed in all PCs and silenced only later in the prospective ZII+ subtype. Moreover, the dynamic distribution and role of Ebf2 in the differentiation of other cerebellar cells remain unclarified. In this paper, by genetic fate mapping, we determine that Ebf2 mRNA is initially found in all PC progenitors, suggesting that unidentified upstream factors silence its expression before completion of embryogenesis. Moreover we show Ebf2 activation in an early born subset of granule cell (GC) precursors homing in the anterior lobe. Conversely, Ebf2 transcription is repressed in other cerebellar cortex interneurons. Last, we show that, although Ebf2 only labels the medial cerebellar nuclei (CN) in the adult cerebellum, the gene is expressed prenatally in projection neurons of all CN. Importantly, in Ebf2 nulls, fastigial nuclei are severely hypocellular, mirroring the defective development of anterior lobe PCs. Our findings further clarify the roles of this terminal selector gene in cerebellar development.

Targeting the ERK Signaling Pathway in Melanoma.

The discovery of the role of the RAS/RAF/MEK/ERK pathway in melanomagenesis and its progression have opened a new era in the treatment of this tumor. Vemurafenib was the first specific kinase inhibitor approved for therapy of advanced melanomas harboring BRAF-activating mutations, followed by dabrafenib and encorafenib. However, despite the excellent results of first-generation kinase inhibitors in terms of response rate, the average duration of the response was short, due to the onset of genetic and epigenetic resistance mechanisms. The combination therapy with MEK inhibitors is an excellent strategy to circumvent drug resistance, with the additional advantage of reducing side effects due to the paradoxical reactivation of the MAPK pathway. The recent development of RAS and extracellular signal-related kinases (ERK) inhibitors promises to add new players for the ultimate suppression of this signaling pathway and the control of pathway-related drug resistance. In this review, we analyze the pharmacological, preclinical, and clinical trial data of the various MAPK pathway inhibitors, with a keen interest for their clinical applicability in the management of advanced melanoma.

A novel neuroferritinopathy mouse model (FTL 498InsTC) shows progressive brain iron dysregulation, morphological signs of early neurodegeneration and motor coordination deficits.

Neuroferritinopathy is a rare genetic disease with a dominant autosomal transmission caused by mutations of the ferritin light chain gene (FTL). It belongs to Neurodegeneration with Brain Iron Accumulation, a group of disorders where iron dysregulation is tightly associated with neurodegeneration. We studied the 498-499InsTC mutation which causes the substitution of the last 9 amino acids and an elongation of extra 16 amino acids at the C-terminus of L-ferritin peptide. An analysis with cyclic voltammetry on the purified protein showed that this structural modification severely reduces the ability of the protein to store iron. In order to analyze the impact of the mutation in vivo, we generated mouse models for the some pathogenic human FTL gene in FVB and C57BL/6J strains. Transgenic mice in the FVB background showed high accumulation of the mutated ferritin in brain where it correlated with increased iron deposition with age, as scored by magnetic resonance imaging. Notably, the accumulation of iron-ferritin bodies was accompanied by signs of oxidative damage. In the C57BL/6 background, both the expression of the mutant ferritin and the iron levels were lower than in the FVB strain. Nevertheless, also these mice showed oxidative alterations in the brain. Furthermore, post-natal hippocampal neurons obtained from these mice experienced a marked increased cell death in response to chronic iron overload and/or acute oxidative stress, in comparison to wild-type neurons. Ultrastructural analyses revealed an accumulation of lipofuscin granules associated with iron deposits, particularly enriched in the cerebellum and striatum of our transgenic mice. Finally, experimental subjects were tested throughout development and aging at 2-, 8- and 18-months for behavioral phenotype. Rotarod test revealed a progressive impaired motor coordination building up with age, FTL mutant old mice showing a shorter latency to fall from the apparatus, according to higher accumulation of iron aggregates in the striatum. Our data show that our 498-499InsTC mouse models recapitulate early pathological and clinical traits of the human neuroferritinopathy, thus providing a valuable model for the study of the disease. Finally, we propose a mechanistic model of lipofuscine formation that can account for the etiopathogenesis of human neuroferritinopathy.

Selective high-level expression of epsin 3 in gastric parietal cells, where it is localized at endocytic sites of apical canaliculi.

Epsin is a ubiquitin-binding endocytic adaptor, which is highly concentrated at clathrin-coated pits and coordinates acquisition of bilayer curvature with coat recruitment and cargo selection. Epsin is encoded by three distinct genes in mammals. Epsin 1 and 2 have broad tissue distribution with high-level expression in the brain. In contrast, epsin 3 was reported to be expressed primarily in immature keratinocytes. Here, we show that epsin 3 is selectively expressed at high levels in the stomach (including the majority of gastric cancers), where it is concentrated in parietal cells. In these cells, epsin 3 is enriched and colocalized with clathrin around apical canaliculi, the sites that control acidification of the stomach lumen via the exo-endocytosis of vesicles containing the H/K ATPase. Deletion of the epsin 3 gene in mice did not result in obvious pathological phenotypes in either the stomach or other organs, possibly because of overlapping functions of the other two epsins. However, levels of EHD1 and EHD2, two membrane tubulating proteins with a role in endocytic recycling, were elevated in epsin 3 knock-out stomachs, pointing to a functional interplay of epsin 3 with EHD proteins in the endocytic pathway of parietal cells. We suggest that epsin 3 cooperates with other bilayer binding proteins with curvature sensing/generating properties in the specialized traffic and membrane remodeling processes typical of gastric parietal cells.

Neuronal models of TDP-43 proteinopathy display reduced axonal translation, increased oxidative stress, and defective exocytosis.

Amyotrophic lateral sclerosis (ALS) is a progressive, lethal neurodegenerative disease mostly affecting people around 50-60 years of age. TDP-43, an RNA-binding protein involved in pre-mRNA splicing and controlling mRNA stability and translation, forms neuronal cytoplasmic inclusions in an overwhelming majority of ALS patients, a phenomenon referred to as TDP-43 proteinopathy. These cytoplasmic aggregates disrupt mRNA transport and localization. The axon, like dendrites, is a site of mRNA translation, permitting the local synthesis of selected proteins. This is especially relevant in upper and lower motor neurons, whose axon spans long distances, likely accentuating their susceptibility to ALS-related noxae. In this work we have generated and characterized two cellular models, consisting of virtually pure populations of primary mouse cortical neurons expressing a human TDP-43 fusion protein, wt or carrying an ALS mutation. Both forms facilitate cytoplasmic aggregate formation, unlike the corresponding native proteins, giving rise to bona fide primary culture models of TDP-43 proteinopathy. Neurons expressing TDP-43 fusion proteins exhibit a global impairment in axonal protein synthesis, an increase in oxidative stress, and defects in presynaptic function and electrical activity. These changes correlate with deregulation of axonal levels of polysome-engaged mRNAs playing relevant roles in the same processes. Our data support the emerging notion that deregulation of mRNA metabolism and of axonal mRNA transport may trigger the dying-back neuropathy that initiates motor neuron degeneration in ALS.

Embryonic arrest at midgestation and disruption of Notch signaling produced by the absence of both epsin 1 and epsin 2 in mice.

Epsins are endocytic adaptors with putative functions in general aspects of clathrin-mediated endocytosis as well as in the internalization of specific membrane proteins. We have now tested the role of the ubiquitously expressed epsin genes, Epn1 and Epn2, by a genetic approach in mice. While either gene is dispensable for life, their combined inactivation results in embryonic lethality at E9.5-E10, i.e., at the beginning of organogenesis. Consistent with studies in Drosophila, where epsin endocytic function was linked to Notch activation, developmental defects observed in epsin 1/2 double knockout (DKO) embryos recapitulated those produced by a global impairment of Notch signaling. Accordingly, expression of Notch primary target genes was severely reduced in DKO embryos. However, housekeeping forms of clathrin-mediated endocytosis were not impaired in cells derived from these embryos. These findings support a role of epsin as a specialized endocytic adaptor, with a critical role in the activation of Notch signaling in mammals.

Multiple Basal Cell Carcinomas in Immunocompetent Patients.

BACKGROUND: The onset of multiple BCCs is a relatively common condition, not only among patients undergoing chronic treatment with immunosuppressant drugs, but also in the general population, although specific risk factors for immunocompetent patients have not been identified. A putative role of somatic mutations in the hedgehog pathway should be considered. METHODS: This study is a retrospective observation of all patients diagnosed and surgically treated for BCCs during 5 years at our Dermatological Division. For these patients, we evaluated clinical and histopathological characteristics and data about possible risk factors for BCC. RESULTS: Five-hundred and six patients affected by multiple BCCs, accounting for the 24.2% of the entire sample, have been identified. In these patients, the total number of BCCs was 1516, ranging from 2 to 11. Subjects affected by multiple BCCs were more frequently males, with an older age at diagnosis; multiple BCCs developed mainly on the trunk and were often represented by a nodular histotype. The multivariate analysis highlighted that male gender, older age, nodular BCC, or face involvement at the first diagnosis are risk factors for the development of multiple BCCs. CONCLUSIONS: The frequency of multiple BCCs even among the non-immunocompromised population underlines the need to subject patients to a close surveillance program, to allow early diagnosis and treatment of additional cancers.

The dual phosphatase activity of synaptojanin1 is required for both efficient synaptic vesicle endocytosis and reavailability at nerve terminals.

Phosphoinositides have been implicated in synaptic vesicle recycling largely based on studies of enzymes that regulate phosphoinositide synthesis and hydrolysis. One such enzyme is synaptojanin1, a multifunctional protein conserved from yeast to humans, which contains two phosphoinositol phosphatase domains and a proline-rich domain. Genetic ablation of synaptojanin1 leads to pleiotropic defects in presynaptic function, including accumulation of free clathrin-coated vesicles and delayed vesicle reavailability, implicating this enzyme in postendocytic uncoating of vesicles. To further elucidate the role of synaptojanin1 at nerve terminals, we performed quantitative synaptic vesicle recycling assays in synj1(-/-) neurons. Our studies show that synaptojanin1 is also required for normal vesicle endocytosis. Defects in both endocytosis and postendocytic vesicle reavailability can be fully restored upon reintroduction of synaptojanin1. However, expression of synaptojanin1 with mutations abolishing catalytic activity of each phosphatase domain reveals that the dual action of both domains is required for normal synaptic vesicle internalization and reavailability.

Cell- and stimulus-dependent heterogeneity of synaptic vesicle endocytic recycling mechanisms revealed by studies of dynamin 1-null neurons.

Mice lacking expression of dynamin 1, a GTPase implicated in the fission reaction of synaptic vesicle endocytosis, fail to thrive and exhibit severe activity-dependent endocytic defects at their synapses. Here, we have used electron tomography to investigate the massive increase in clathrin-coated pit abundance that is selectively observed at a subset of synapses in dynamin 1 KO primary neuron cultures under conditions of spontaneous network activity. This increase, leading to branched tubular plasma membrane invaginations capped by clathrin-coated buds, occurs selectively at inhibitory synapses. A similar massive increase of clathrin-coated profiles (in this case, of clathrin-coated vesicles) is observed at inhibitory synapses of neurons that lack expression of synaptojanin 1, a phosphoinositide phosphatase involved in clathrin-coated vesicle uncoating. Thus, although excitatory synapses are largely spared under these conditions, inhibitory synapses are uniquely sensitive to perturbation of endocytic proteins, probably as a result of their higher levels of tonic activity leading to a buildup of clathrin-coated intermediates in these synapses. In contrast, the predominant endocytic structures observed at the majority of dynamin 1 KO synapses after acute stimulation are endosome-like intermediates that originate by a dynamin 1-independent form of endocytosis. These findings reveal a striking heterogeneity in the mode of synaptic vesicle recycling in different synapses and functional states.

The Italian law on body donation: A position paper of the Italian College of Anatomists.

In Italy, recent legislation (Law No. 10/2020) has tuned regulations concerning the donation of one's postmortem body and tissues for study, training, and scientific research purposes. This study discusses several specific issues to optimise the applicability and effectiveness of such an important, novel regulatory setting. Critical issues arise concerning the learners, the type of training and teaching activities that can be planned, the position of academic anatomy institutes, the role of family members in the donation process, the time frame of the donation process, the eligibility of partial donation, or the simultaneous donation of organs and tissues to patients awaiting transplantation. In particular, a universal time limit for donations (i.e., one year) makes it impossible to plan the long-term use of specific body parts, which could be effectively preserved for the advanced teaching and training of medical students and surgeons. The abovementioned conditions lead to the limited use of corpses, thus resulting in the inefficiency of the whole system of body donation. Overall, the donors' scope for the donation of their body could be best honoured by a more flexible and tuneable approach that can be used on a case-by-case basis. Furthermore, it is deemed necessary to closely monitor the events scheduled for corpses in public nonacademic institutions or private enterprises. This paper presents useful insights from Italian anatomists with the hope of providing inspiration for drafting the regulations. In conclusion, this paper focuses on the critical issues derived from the recently introduced Italian law on the donation and use of the body after death and provides suggestions to lawmakers for future implementations.

Dynamic interaction of amphiphysin with N-WASP regulates actin assembly.

Amphiphysin 1, an endocytic adaptor concentrated at synapses that couples clathrin-mediated endocytosis to dynamin-dependent fission, was also shown to have a regulatory role in actin dynamics. Here, we report that amphiphysin 1 interacts with N-WASP and stimulates N-WASP- and Arp2/3-dependent actin polymerization. Both the Src homology 3 and the N-BAR domains are required for this stimulation. Acidic liposome-triggered, N-WASP-dependent actin polymerization is strongly impaired in brain cytosol of amphiphysin 1 knock-out mice. FRET-FLIM analysis of Sertoli cells, where endogenously expressed amphiphysin 1 co-localizes with N-WASP in peripheral ruffles, confirmed the association between the two proteins in vivo. This association undergoes regulation and is enhanced by stimulating phosphatidylserine receptors on the cell surface with phosphatidylserine-containing liposomes that trigger ruffle formation. These results indicate that actin regulation is a key function of amphiphysin 1 and that such function cooperates with the endocytic adaptor role and membrane shaping/curvature sensing properties of the protein during the endocytic reaction.

Normal human macula densa morphology and cell turnover: A histological, ultrastructural, and immunohistochemical investigation.

The aim was to analyze the morphology of normal human macula densa (MD), evaluate the cells that may be responsible for its turnover, and collect quantitative data. Of four samples of normal human renal tissue, two were embedded in resin to measure the longitudinal extension and examine the ultrastructure of the MD, the other two were embedded in paraffin to study apoptosis and cell proliferation. The MD is composed of a monolayer tissue about 40 µm long, which includes 35-40 cells arranged in overlapping rows. Ultrastructurally, MD cells show two polarized portions: an apical end, with sensory features, and a basolateral aspect, with paracrine function. MD cells are connected apically by tight junctions, with/without adherens junctions, which form a barrier between the distal tubule lumen and the interstitium. Cells in degeneration, often associated with macrophages, and undifferentiated cells were found in the MD and adjacent distal tubule. A filamentous mat previously described in proximal tubule scattered tubular cells (STCs) was detected in the basal cytoplasm in undifferentiated cells. The tissue was consistently negative for the proliferation marker Ki67 and for the apoptotic markers caspase-3 and caspase-9. This work confirms our earlier morphological findings and provides new data: (a) MD cells display both apical adherens and tight junctions, the latter forming a tubulo-mesangial barrier; (b) the MD is a monolayer made up of about 40 cells arranged in rows; (c) the simultaneous presence of degenerating (8-13%) and undifferentiated (4-13%) cells reminiscent of STCs suggests a non-negligible cell turnover.