Combining germline, tissue and liquid biopsy analysis by comprehensive genomic profiling to improve the yield of actionable variants in a real-world cancer cohort.

BACKGROUND: Comprehensive next-generation sequencing is widely used for precision oncology and precision prevention approaches. We aimed to determine the yield of actionable gene variants, the capacity to uncover hereditary predisposition and liquid biopsy appropriateness instead of, or in addition to, tumor tissue analysis, in a real-world cohort of cancer patients, who may benefit the most from comprehensive genomic profiling. METHODS: Seventy-eight matched germline/tumor tissue/liquid biopsy DNA and RNA samples were profiled using the Hereditary Cancer Panel (germline) and the TruSight Oncology 500 panel (tumor tissue/cfDNA) from 23 patients consecutively enrolled at our center according to at least one of the following criteria: no available therapeutic options; long responding patients potentially fit for other therapies; rare tumor; suspected hereditary cancer; primary cancer with high metastatic potential; tumor of unknown primary origin. Variants were annotated for OncoKB and AMP/ASCO/CAP classification. RESULTS: The overall yield of actionable somatic and germline variants was 57% (13/23 patients), and 43.5%, excluding variants previously identified by somatic or germline routine testing. The accuracy of tumor/cfDNA germline-focused analysis was demonstrated by overlapping results of germline testing. Five germline variants in BRCA1, VHL, CHEK1, ATM genes would have been missed without extended genomic profiling. A previously undetected BRAF p.V600E mutation was emblematic of the clinical utility of this approach in a patient with a liver undifferentiated embryonal sarcoma responsive to BRAF/MEK inhibition. CONCLUSIONS: Our study confirms the clinical relevance of performing extended parallel tumor DNA and cfDNA testing to broaden therapeutic options, to longitudinally monitor cfDNA during patient treatment, and to uncover possible hereditary predisposition following tumor sequencing in patient care.

Assessment of pharyngeal motor function using a novel velopharyngeal squeeze maneuver and a novel endoscopic pharyngeal contraction grade scale in patients with dysphagia after radiotherapy for nasopharyngeal carcinoma.

BACKGROUND: To investigate a novel velopharyngeal squeeze maneuver (VPSM) and novel endoscopic pharyngeal contraction grade (EPCG) scale for the evaluation of pharyngeal motor function. METHODS: During endoscopic examination of 77 post-irradiated nasopharyngeal carcinoma patients and control subjects, VPSM was rated and lateral pharyngeal wall movement graded with EPCG scale during swallowing. Pharyngeal constriction ratio (PCR) measured by videofluoroscopy was used for correlation. RESULTS: VPSM and EPCG scale showed almost perfect intra-rater and inter-rater reliability (Kappa: >0.90). VPSM was present in 61% of patients suggesting good pharyngeal motor function. VPSM was predictive of EPCG scale (Wald statistic = 29.99, p < 0.001). EPCG scale also correlated strongly with PCR (r: 0.812) and was predictive for aspiration (odds ratio: 22.14 [95% CI 5.01-97.89, p < 0.001]). CONCLUSIONS: VPSM and EPCG scale are two novel tools to assess pharyngeal motor function, and both correlate well with pharyngeal contractility and aspiration.

Comfort rules for face masks among healthcare workers during COVID-19 spread.

BACKGROUND: The outbreak of Coronavirus disease 2019 (COVID-19) made imperative the use of protective devices as a source control tool. As there is no definite antiviral treatment and effective vaccine, the only efficient means of protecting and mitigating infectious contagion has been the use of personal protective equipment, especially by healthcare workers. However, masks affect the humidification process of inhaled air, possibly leading to a basal inflammatory state of the upper airways. STUDY DESIGN: This is a single-center observational study conducted at the University Hospital of Catania from April 1, 2020, to June 31, 2020. METHODS: We analyzed the role of protective masks on the elimination of upper airways complaints in healthcare workers of the University Hospital of Catania. We evaluated 277 subjects through a self-administered 17 item questionnaire based on respiratory, work performance and health-related quality of life domains. RESULTS: A higher prevalence of nasal and ocular symptoms, perceived reduced work performance, difficulty in concentrating, and sleep disorders were found. After two weeks adhering to a list of good practices that we recommended, significant reversibility of the symptoms investigated and work performance enhancement were observed. CONCLUSIONS: Despite clinical complaints related to personal protective equipment, effective amelioration through usage rules is easily obtained. Given the essential use of protective masks, healthcare workers have to adhere to appropriate work and safety prevention rules.

Intervertebral disc and endplate cells response to IL-1ß inflammatory cell priming and identification of molecular targets of tissue degeneration.

Inflammation represents an important factor leading to metabolic imbalance within the intervertebral disc (IVD), conducive to degenerative changes. Therefore, a thorough knowledge of the IVD and endplate (EP) cell behaviour in such pathological environments is essential when designing regenerative therapeutic strategies. The present study aimed at assessing the molecular response of the IVD constitutive nucleus pulposus (NPCs)-, annulus fibrosus (AFCs)- and endplate (EPCs)-derived cells to interleukin (IL)-1ß treatment, through large-scale, high-throughput microarray and protein analysis, identifying the differentially expressed genes and released proteins. Overall, the inflammatory stimulus downregulated stemness genes while upregulating pro-inflammatory, pro-angiogenic and catabolic genes, including matrix metalloproteases, which were not balanced by a concomitant upregulation of their inhibitors. Upregulation of anti-inflammatory and anabolic tumour necrosis factor inducible gene 6 protein (TNFAIP6), of IL-1 receptor antagonist (IL-1Ra) (at gene and protein levels) and of trophic insulin-like growth factor 1 (IGF1) was also observed in all cell types; IGF1 particularly in AFCs. An overall inhibitory effect of tumour necrosis factor alpha (TNFα) signal was observed in all cell types; however, EPCs showed the strongest anti-inflammatory behaviour. AFCs and EPCs shared the ability to limit the activation of the signalling mediated by specific chemokines. AFCs showed a slightly senescent attitude, with a downregulation of genes related to DNA repair or pro-mitosis. Results allowed for the identification of specific molecular targets in IVD and EP cells that respond to an inflammatory environment. Such targets can be either silenced (when pathological targets) or stimulated to counteract the inflammation.

Intervertebral disc and endplate cell characterisation highlights annulus fibrosus cells as the most promising for tissue-specific disc degeneration therapy.

Degenerative processes of the intervertebral disc (IVD) and cartilaginous endplate lead to chronic spine pathologies. Several studies speculated on the intrinsic regenerative capacity of degenerated IVD related to the presence of local mesenchymal progenitors. However, a complete characterisation of the resident IVD cell populations, particularly that isolated from the endplate, is lacking. The purpose of the present study was to characterise the gene expression profiles of human nucleus pulposus (NPCs), annulus fibrosus (AFCs) and endplate (EPCs) cells, setting the basis for future studies aimed at identifying the most promising cells for regenerative purposes. Cells isolated from NP, AF and EP were analysed after in vitro expansion for their stemness ability, immunophenotype and gene profiles by large-scale microarray analysis. The three cell populations shared a similar clonogenic, adipogenic and osteogenic potential, as well as an immunophenotype with a pattern resembling that of mesenchymal stem cells. NPCs maintained the greatest chondrogenic potential and shared with EPCs the loss of proliferation capability during expansion. The largest number of selectively highly expressed stemness, chondrogenic/tissue-specific and surface genes was found in AFCs, thus representing the most promising source of tissue-specific expanded cells for the treatment of IVD degeneration.

Resistin is associated with overall survival in non-small cell lung cancer patients during nivolumab treatment.

PURPOSE: Since the role of resistin was evaluated only in patients with non-small cell lung cancer (NSCLC) not treated with immunotherapy, we aimed to evaluate levels of resistin during immunotherapy (nivolumab) and its prognostic role with regard to OS. METHODS/PATIENTS: From a cohort of 78 patients with advanced NSCLC enrolled in a prospective study at Ospedale Policlinico San Martino in Genoa (Italy), 43 patients have been considered for this sub-analysis because of the availability of samples. Before and during nivolumab administration, clinical information and blood samples were collected and resistin, matrix metalloproteinase (MMP)-8, MMP-9, and myeloperoxidase were evaluated by enzyme-linked immunosorbent assay (ELISA). RESULTS: Median age was 71 with a prevalence of males and former smokers. Median resistin levels presented a peak at cycle 2 and then dropped down until the last cycle. Resistin correlated with all neutrophil degranulation products at cycle 1 (except for MMP-9) and at cycle 2 as well as with white blood cells and neutrophils. By a ROC curve analysis, a resistin value at cycle 2 of 19 ng/mL was tested as the best cut-off point for OS. Kaplan-Meier analysis demonstrated that patients above the resistin cut-off experienced a reduced OS (median OS 242.5 vs. 470 days, p = 0.0073), as confirmed by Cox proportional hazards regression analysis. CONCLUSIONS: Resistin levels > 19 ng/mL at the time of the second cycle of nivolumab treatment independently predict a reduced OS in patients with advanced NSCLC.

The role of CEA, CYFRA21-1 and NSE in monitoring tumor response to Nivolumab in advanced non-small cell lung cancer (NSCLC) patients.

BACKGROUND: CEA, CYFRA21-1 and NSE are tumor markers used for monitoring the response to chemotherapy in advanced adenocarcinoma, squamous cell carcinoma and small-cell lung cancer, respectively. Their role in cancer immunotherapy needs to be elucidated. METHODS: Patients with advanced non-small cell lung cancer (NSCLC) were treated with nivolumab 3 mg/kg every 2 weeks within the Italian Nivolumab Expanded Access Program. Blood samples were collected at baseline, at each cycle up to cycle 5 and then every two cycles until patient's withdrawn from the study. All patients underwent a CT-scan after every 4 cycles of treatment and responses were classified according to RECIST 1.1. The biomarkers serum levels were measured with a chemiluminescent microparticle immunoassay for CEA and with an immuno radiometric assay for CYFRA21-1 and NSE. The markers values at baseline and after 4 cycles were used to analyze the relationship between their variation over baseline and the tumor response, evaluated as disease control rate (DCR: CR + PR + SD), and survival (PFS and OS). RESULTS: A total of 70 patients were evaluable for the analysis. Overall, a disease control was obtained in 24 patients (35.8%, 4 PR + 20 SD). After 4 cycles of nivolumab a CEA or CYFRA21-1 reduction ≥ 20% over the baseline was significantly associated with DCR (CEA, p = 0.021; CYFRA21-1, p < 0.001), PFS (CEA, p = 0.028; CYFRA21-1, p < 0.001) and OS (CEA, p = 0.026; CYFRA21-1, p = 0.019). Multivariate analysis confirmed the ability of CYFRA21-1 reduction ≥ 20% to predict DCR (p = 0.002) and PFS (p < 0.001). CONCLUSION: The reduction in serum level of CYFRA21-1 or CEA might be a reliable biomarker to predict immunotherapy efficacy in NSCLC patients. NSE was not significant for monitoring the efficacy of nivolumab.

Vinflunine for the treatment of non-small cell lung cancer.

INTRODUCTION: Vinflunine belongs to the class of vinca alkaloids and acts by disrupting the microtubule dynamics during cell cycle; this agent is currently available for previously treated advanced transitional cell carcinoma in Europe. The aim of this invited review is to evaluate the potential role of vinflunine for the treatment of non-small cell lung cancer (NSCLC). Areas covered: The potential role of vinflunine in NSCLC is discussed on the basis of the available data, including full papers and meeting abstracts. Relevant preclinical studies describing the pharmacological properties of vinflunine are also included. The review also summarizes clinical studies, including phase I trials involving NSCLC among other tumors as well as phase II/III trials specifically addressing this malignancy. Additionally, the safety profile and the current regulatory status of vinflunine is discussed. Expert opinion: Vinflunine is active as single agent and as part of platinum-based combinations in NSCLC. It results non-inferior to docetaxel in a randomized phase III trial including previously treated NSCLC patients; additionally, its safety profile is generally considered manageable. Ultimately, further studies are needed to confirm the role of vinflunine in NSCLC, in consideration of the evolving evidence regarding targeted therapies and immune check-point inhibitors.

Sequential use of vinorelbine followed by gefitinib enhances the antitumor effect in NSCLC cell lines poorly responsive to reversible EGFR tyrosine kinase inhibitors.

Preclinical studies have suggested that combining cytotoxic agents with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) to treat EGFR-mutated tumors may increase their inhibitory effect depending on the order of drug administration. The antitumor efficacy of different treatment sequences using vinorelbine (VNB) and gefitinib (GEF) was investigated both in vitro and in vivo in non-small cell lung cancer (NSCLC) cell lines with the rationale of potentially translating these findings into the clinical setting. The EGFR-wild-type A549 and the EGFR-mutated (exon 21 L858R/exon 20 T790M) H1975 cell lines were treated as follows: GEF followed by VNB, VNB followed by GEF and the two drugs applied individually or concurrently. Results in vitro demonstrated that the sequence of VNB followed by GEF was significantly more active than single-agent treatments. The expression of activated EGFR and its downstream pathway genes indicated that the increased cytotoxic effect of the VNB and GEF treatment sequence was accompanied by inhibition of EGFR, AKT and ERK1/2. Moreover, the increased inhibition of tumor growth after treatment with VNB followed by GEF was also confirmed in CD1-nude mice that were xenotransplanted with H1975 cells (p < 0.0001). This effect was paralleled by a corresponding decrease in cancer glucose consumption, as assessed by micro-positron emission tomography scans (p < 0.05). These preclinical findings in NSCLC cell lines, which are poorly responsive to EGFR-TKIs, demonstrated that the sequential treatment of VNB followed by GEF induced a significant antitumor effect, which supports the translation of this treatment schedule into a clinical setting.

Role of microRNAs in malignant mesothelioma.

Malignant mesothelioma (MM) is an aggressive tumor, mainly derived from the pleura, which is predominantly associated with exposure to asbestos fibers. The prognosis of MM patients is particularly severe, with a median survival of approximately 9-12 months and latency between exposure and diagnosis ranging from 20-50 years (median 30 years). Emerging evidence has demonstrated that tumor aggressiveness is associated with genome and gene expression abnormalities; therefore, several studies have recently focused on the role of microRNAs (miRNAs) in MM tumorigenesis. miRNAs are small non-protein coding single-stranded RNAs (17-22 nucleotides) involved in numerous cellular processes that negatively regulate gene expression by modulating the expression of downstream target genes. miRNAs are often deregulated in cancer; in particular, the differential miRNA expression profiles of MM cells compared to unaffected mesothelial cells have suggested potential roles of miRNAs as either oncogenes or tumor suppressor genes in MM oncogenesis. In this review, the mechanism of MM carcinogenesis was evaluated through the analysis of the published miRNA expression data. The roles of miRNAs as diagnostic biomarkers and prognostic factors for potential therapeutic strategies will be presented and discussed.

Genomic aberrations in normal appearing mucosa fields distal from oral potentially malignant lesions.

OBJECTIVES: Oral fields of visually normal and non-dysplastic mucosa (ODFs) may represent the precursors of oral potentially malignant lesions (OPMLs). Aim of the study was to provide new evidence for the concept of the "field carcinogenesis" model by comparing the ODF and OPML genomic aberration profiles obtained by high resolution DNA flow cytometry (hr DNA-FCM) and array-Comparative Genomic Hybridization (a-CGH). A second aim was to investigate if specific CGH aberrations were associated with DNA aneuploidy. METHODS: Nineteen patients with single OPMLs were recruited for the study. In parallel with obtaining samples of OPML tissue from 11 leukoplakias without dysplasia (nd-OPMLs) and 8 with dysplasia (d-OPMLs), we also obtained samples from distant ODFs. DNA aneuploid nuclei detected by hr DNA-FCM were physically separated, based on DNA content, from the DNA diploid components with a DNA-FCM-Sorter. These relatively pure subpopulations of epithelial nuclei were then submitted to DNA extraction and a-CGH for a genome-wide analysis of DNA copy number aberrations (CNAs). RESULTS: The frequencies of DNA aneuploidy (DI ≠ 1) among ODFs and OPMLs were respectively 5.3% and 32%. The DI aneuploid values of ODFs and nd-OPMLs were all near-diploid (DI ≠ 1 and DI ≤ 1.4), while for d-OPMLs were high-aneuploid (DI > 1.4) in 40% of the cases. CNA averages were 1.9 in ODFs and 6.5 in OPMLs. The gain of the chromosomal region 20q13.33-qter was observed in 37% of both ODFs and corresponding OPMLs. Additional common regions included 7p22.2-pter, 11p15.5-pter and 16p13.3-pter where gains were observed. Furthermore, gains of 20q13.31-q13.33 and of 5p13.33-pter and loss of 9p21.3 were detected at high frequency (respectively, at 62.5%, 50% and 50%) only in d-OPMLs. In particular, loss at 9p21.3, gain at 5p13.33-pter and gain of 20q13.31-q13.33 were associated with DNA aneuploidy (p = 0.00004; p = 0.0005; p = 0.01). CONCLUSIONS: ODFs and OPMLs showed common CNAs in specific chromosomal regions suggesting that they may represent early events of the natural history of oral carcinogenesis according to the field effect cancerization and may contribute to the ODF-OPML transition. In addition, loss at 9p21.3 and gains at 5p13.33-pter and 20q13.31-q13.33 may contribute to DNA aneuploidization.

Genome and transcriptome analysis of neuroblastoma advanced diagnosis from innovative therapies.

Neuroblastoma is an extracranial solid tumor which occurs in infants and young children and accounts for 8% of pediatric cancers. It origins from neural crest cells of the sympathetic nervous system. Disease-free survival ranges from 95% for localized tumors to 30% for metastatic disease in children over 1 year of age and patients' outcome depends on dissemination and tissue histology. Despite the most recent therapies, the overall survival for high risk patients is still low and the outcome is invariably fatal. Improvement of neuroblastoma treatment is one of the highest priorities in pediatric oncology and a major challenge to clinicians and researchers. Understanding the biology and genetics of pediatric malignancies will be the key to identify molecular targets for innovative treatments as well as to individual management of disease. The success of human genome project and recent advances in technology have provided new tools to investigate cancer cells and to discover new tumor-associated genes. High-throughput efforts include array-based comparative genomic hybridization, single-nucleotide polymorphism arrays and expression microarrays. Here we present an overview on the most recent advances in wide-genome analysis of neuroblastoma. We also focus on the potential clinical application of genome and transcriptome information to the diagnosis, prognosis and neuroblastoma therapy.

Insulin resistance predicts endothelial dysfunction and cardiovascular risk in HIV-infected persons on long-term highly active antiretroviral therapy.

OBJECTIVE: Cardiovascular disease risk among persons with HIV is likely multifactorial, thus testing a variety of available noninvasive vascular ultrasound and other surrogate tests may yield differing results. To address this issue, we assessed multiple metabolic and clinical predictors of endothelial function and carotid intima-media thickness in HIV-infected subjects and compared results with HIV-negative controls. DESIGN: Prospective, cross-sectional study of 50 HIV-infected, healthy adults on stable highly active antiretroviral therapy matched to 50 HIV-negative controls by age, sex, race, and body mass index. METHODS: Flow-mediated vasodilation of the brachial artery, carotid intima-media thickness, dual energy X-ray absorptiometry (HIV-infected subjects), and fasting insulin, lipids, and oral glucose tolerance tests were performed. Results were compared between HIV-infected and control groups. RESULTS: Fifty percent of subjects were African-American with 34% women. Among HIV-infected, mean CD4 cell count was 547 cells/microl; 90% had HIV RNA less than 50 copies/ml. There were no significant differences between HIV-infected and control subjects with regard to brachial artery flow-mediated vasodilation or carotid intima-media thickness. In multivariate analyses of the HIV cohort, independent predictors of endothelial dysfunction (lower flow-mediated vasodilation) were increasing insulin resistance, greater alcohol consumption, and higher baseline brachial artery diameter (P < 0.05); predictors of increased carotid intima-media thickness were hypertension, higher trunk/limb fat ratio, and insulin resistance (P < 0.05). CONCLUSION: In this HIV cohort on modern highly active antiretroviral therapy with well controlled HIV, there were no significant differences with regard to preclinical markers of cardiovascular disease. Insulin resistance was a strong predictor of impaired brachial artery flow-mediated vasodilation and increased carotid intima-media thickness, and may be an important cardiovascular disease risk factor in the HIV population.

Polarization of caveolins and caveolae during migration of immortalized neurons.

During CNS development neurons undergo directional migration to achieve their adult localizations. To study neuronal migration, we used a model cell line of immortalized murine neurons (gonadotropin-releasing hormone expressing neurons; GN11), enriched with caveolins and caveolae invaginations that show in vitro chemotaxis upon serum exposure. Cholesterol depletion with methyl-beta-cyclodextrin induced the loss of caveolae and the inhibition of chemotaxis, thus suggesting that GN11 migration depends upon the structural integrity of caveolae. Polarization of proteins and organelles is a hallmark of cell migration. Accordingly, GN11 cells transmigrating through filter pores exhibited a polarized distribution of caveolin-1 isoform (cav-1) in the leading processes. In contrast, during two-dimensional migration cav-1 and caveolae polarized at the trailing edge. As caveolae are enriched with signaling molecules, we suggest that asymmetrical localization of caveolae may spatially orient GN11 neurons to incoming migratory signals thereby transducing them into directional migration.

Analysis of SNAP-25 immunoreactivity in hippocampal inhibitory neurons during development in culture and in situ.

Synaptosomal associated protein of 25 kDa (SNAP-25) is a component of the soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor (SNARE) complex which plays a central role in synaptic vesicle exocytosis. We have previously demonstrated that adult rat hippocampal GABAergic synapses, both in culture and in brain, are virtually devoid of SNAP-25 immunoreactivity and are less sensitive to the action of botulinum toxin type A, which cleaves this SNARE protein [Neuron 41 (2004) 599]. In the present study, we extend our findings to the adult mouse hippocampus and we also provide demonstration that hippocampal inhibitory synapses lacking SNAP-25 labeling belong to parvalbumin-, calretinin- and cholecystokinin-positive interneurons. A partial colocalization between SNAP-25 and glutamic acid decarboxylase is instead detectable in developing mouse hippocampus at P0 and, at a lesser extent, at P5. In rat embryonic hippocampal cultures at early developmental stages, SNAP-25 immunoreactivity is detectable in a percentage of GABAergic neurons, which progressively reduces with time in culture. Consistent with the presence of the substrate, botulinum toxin type A is partially effective in inhibiting synaptic vesicle recycling in immature GABAergic neurons. Since SNAP-25, beside its role as a SNARE protein, is involved in additional processes, such as neurite outgrowth and regulation of calcium dynamics, the presence of higher levels of the protein at specific stages of neuronal differentiation may have implications for the construction and for the functional properties of brain circuits.

Chronic blockade of glutamate receptors enhances presynaptic release and downregulates the interaction between synaptophysin-synaptobrevin-vesicle-associated membrane protein 2.

During development of neuronal circuits, presynaptic and postsynaptic functions are adjusted in concert, to optimize interneuronal signaling. We have investigated whether activation of glutamate receptors affects presynaptic function during synapse formation, when constitutive synaptic vesicle recycling is downregulated. Using primary cultures of hippocampal neurons as a model system, we have found that chronic exposure to both NMDA and non-NMDA glutamate receptor blockers during synaptogenesis produces an increase in miniature EPSC (mEPSC) frequency, with no significant changes in mEPSC amplitude or in the number of synapses. Enhanced synaptic vesicle recycling, selectively in glutamatergic nerve terminals, was confirmed by the increased uptake of antibodies directed against the lumenal domain of synaptotagmin. No increased uptake was detected in neuronal cultures grown in the chronic presence of TTX, speaking against an indirect effect caused by decreased electrical activity. Enhanced mEPSC frequency correlated with a reduction of synaptophysin-synaptobrevin-vesicle-associated membrane protein 2 (VAMP2) complexes detectable by immunoprecipitation. Intracellular perfusion with a peptide that inhibits the binding of synaptophysin to synaptobrevin-VAMP2 induced a remarkable increase of mEPSC frequency in control but not in glutamate receptor blocker-treated neurons. These findings suggest that activation of glutamate receptors plays a role in the downregulation of the basal rate of synaptic vesicle recycling that accompanies synapse formation. They also suggest that one of the mechanisms through which this downregulation is achieved is an increased interaction of synaptophysin with synaptobrevin-VAMP2.

A common exocytotic mechanism mediates axonal and dendritic outgrowth.

Outgrowth of the dendrites and the axon is the basis of the establishment of the neuronal shape, and it requires addition of new membrane to both growing processes. It is not yet clear whether one or two exocytotic pathways are responsible for the respective outgrowth of axons and dendrites. We have previously shown that tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) defines a novel network of tubulovesicular structures present both at the leading edge of elongating dendrites and axons of immature hippocampal neurons developing in primary culture and that TI-VAMP is an essential protein for neurite outgrowth in PC12 cells. Here we show that the expression of the N-terminal domain of TI-VAMP inhibits the outgrowth of both dendrites and axons in neurons in primary culture. This effect is more prominent at the earliest stages of the development of neurons in vitro. Expression of the N-terminal domain deleted form of TI-VAMP has the opposite effect. This constitutively active form of TI-VAMP localizes as the endogenous protein, particularly concentrating at the leading edge of growing axons. Our results suggest that a common exocytotic mechanism that relies on TI-VAMP mediates both axonal and dendritic outgrowth in developing neurons.

Different localizations and functions of L-type and N-type calcium channels during development of hippocampal neurons.

Using immunocytochemical assays and patch-clamp and calcium-imaging recordings, we demonstrate that L-type and N-type calcium channels have distinct patterns of expression and distribution and play different functional roles during hippocampal neuron differentiation. L-type channels, which support the depolarization-induced calcium influx in neurons from the very early developmental stages, are functionally restricted to the somatodendritic compartment throughout neuronal development and play a crucial role in supporting neurite outgrowth at early developmental stages. N-type channels, which start contributing at later neuronal differentiation stages (3-4 DIV), are also functionally expressed in the axons of immature neurons. At this developmental stage preceding synaptogenesis, N-type (but not L-type) channels are involved in controlling synaptic vesicle recycling. It is only at later developmental stages (10-12 DIV), when the neurons have established a clear axodendritic polarity and form synaptic contacts, that N-type channels are progressively excluded from the axon. Electrophysiological recordings of single neurons growing in microislands revealed that synaptic maturation coincides with a progressive increase in N-type channels in the somatodendritic region and a progressive decrease in the N-type channels supporting glutamate release from the presynaptic terminal. These results indicate that L-type and N-type calcium channels undergo dynamic, developmentally regulated rearrangements in regional distribution and function and also suggest that different mechanisms may be involved in the sorting and/or stabilization of these two types of channels in different plasma membrane domains during neuronal differentiation.

Subcellular localization of tetanus neurotoxin-insensitive vesicle-associated membrane protein (VAMP)/VAMP7 in neuronal cells: evidence for a novel membrane compartment.

The clostridial neurotoxin-insensitive soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptors, tetanus neurotoxin-insensitive (TI)-vesicle-associated membrane protein (VAMP)/VAMP7, SNAP23, and syntaxin 3 have recently been implicated in transport of exocytotic vesicles to the apical plasma membrane of epithelial cells. This pathway had been shown previously to be insensitive to tetanus neurotoxin and botulinum neurotoxin F. TI-VAMP/VAMP7 is also a good candidate to be implicated in an exocytotic pathway involved in neurite outgrowth because tetanus neurotoxin does not inhibit this process in conditions in which it abolishes neurotransmitter release. We have now found that TI-VAMP/VAMP7 has a widespread distribution in the adult rat brain in which its localization strikingly differs from that of nerve terminal markers. TI-VAMP/VAMP7 does not enrich in synaptic vesicles nor in large dense-core granules but is associated with light membranes. In hippocampal neurons developing in vitro, TI-VAMP/VAMP7 localizes to vesicles in the axonal and dendritic outgrowths and concentrates into the leading edge of the growth cone, a region devoid of synaptobrevin 2, before synaptogenesis. After the onset of synaptogenesis, TI-VAMP/VAMP7 is found predominantly in the somatodendritic domain. In PC12 cells, TI-VAMP/VAMP7 does not colocalize with synaptobrevin 2, chromogranin B, or several markers of endocytic compartments. At the electron microscopic level, TI-VAMP/VAMP7 is mainly associated with tubules and vesicles. Altogether, these results suggest that TI-VAMP/VAMP7 defines a novel membrane compartment in neurite outgrowths and in the somatodendritic domain.

Synaptogenesis in hippocampal cultures.

Hippocampal cultures offer unique advantages for the study of neuronal development and synaptogenesis. Studies performed on this model enabled dissection of the temporal sequence of events which lead to the differentiation of pre- and postsynaptic compartments.