RESUMEN
Neurodevelopmental disorders are often caused by chromosomal microdeletions comprising numerous contiguous genes. A subset of neurofibromatosis type 1 (NF1) patients with severe developmental delays and intellectual disability harbors such a microdeletion event on chromosome 17q11.2, involving the NF1 gene and flanking regions (NF1 total gene deletion [NF1-TGD]). Using patient-derived human induced pluripotent stem cell (hiPSC)-forebrain cerebral organoids (hCOs), we identify both neural stem cell (NSC) proliferation and neuronal maturation abnormalities in NF1-TGD hCOs. While increased NSC proliferation results from decreased NF1/RAS regulation, the neuronal differentiation, survival, and maturation defects are caused by reduced cytokine receptor-like factor 3 (CRLF3) expression and impaired RhoA signaling. Furthermore, we demonstrate a higher autistic trait burden in NF1 patients harboring a deleterious germline mutation in the CRLF3 gene (c.1166T>C, p.Leu389Pro). Collectively, these findings identify a causative gene within the NF1-TGD locus responsible for hCO neuronal abnormalities and autism in children with NF1.
Asunto(s)
Cerebro/patología , Deleción Cromosómica , Cromosomas Humanos Par 17/genética , Células Madre Pluripotentes Inducidas/patología , Modelos Biológicos , Neurogénesis/genética , Organoides/patología , Receptores de Citocinas/metabolismo , Trastorno Autístico/genética , Línea Celular , Proliferación Celular , Dendritas/metabolismo , Dendritas/patología , Activación Enzimática , Eliminación de Gen , Genes de Neurofibromatosis 1 , Humanos , Mutación/genética , Transducción de Señal , Proteínas ras/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
BACKGROUND: The function of synaptotagmins (syt) in Ca2+-dependent transmitter release has been attributed primarily to Ca2+-dependent isoforms such as syt I. Recently, syt IV, an inducible Ca2+-independent isoform has been implicated in transmitter release. We postulated that the effects of syt IV on transmitter release are dependent on the expression of syt I. RESULTS: To test this, we increased syt IV expression in PC12 cells by either upregulation with forskolin treatment or overexpression with transfection. Two separately generated stable PC12 cell lines with syt I expression abolished by RNAi targeting were used and compared to control cells. We measured catecholamine release from single vesicles by amperometry and neuropeptide Y release from populations of cells by an immunoassay. In syt I targeted cells with forskolin-induced syt IV upregulation, amperometry measurements showed a reduction in the number of release events and the total amount of transmitter molecules released per cell. In cells with syt IV overexpressed, similar amperometry results were obtained, except that the rate of expansion for full fusion was slowed. Neuropeptide Y (NPY) release from syt I knockdown cells was decreased, and overexpression of syt IV did not rescue this effect. CONCLUSIONS: These data support an inhibitory effect of syt IV on release of vesicles and their transmitter content. The effect became more pronounced when syt I expression was abolished.
Asunto(s)
Neurotransmisores/metabolismo , Sinaptotagmina I/biosíntesis , Sinaptotagminas/biosíntesis , Animales , Western Blotting , Calcio/metabolismo , Catecolaminas/metabolismo , Colforsina/farmacología , Electroquímica , Humanos , Inmunohistoquímica , Cinética , Ratones , Microscopía Electrónica , Neuropéptido Y/metabolismo , Células PC12 , Interferencia de ARN , ARN Interferente Pequeño/biosíntesis , ARN Interferente Pequeño/genética , Ratas , Sinaptotagmina I/genética , Sinaptotagminas/fisiología , Transfección , Regulación hacia Arriba/fisiologíaRESUMEN
Neurofibromatosis type 1 (NF1) is a common neurodevelopmental disorder caused by a spectrum of distinct germline NF1 gene mutations, traditionally viewed as equivalent loss-of-function alleles. To specifically address the issue of mutational equivalency in a disease with considerable clinical heterogeneity, we engineered seven isogenic human induced pluripotent stem cell lines, each with a different NF1 patient NF1 mutation, to identify potential differential effects of NF1 mutations on human central nervous system cells and tissues. Although all mutations increased proliferation and RAS activity in 2D neural progenitor cells (NPCs) and astrocytes, we observed striking differences between NF1 mutations on 2D NPC dopamine levels, and 3D NPC proliferation, apoptosis, and neuronal differentiation in developing cerebral organoids. Together, these findings demonstrate differential effects of NF1 gene mutations at the cellular and tissue levels, suggesting that the germline NF1 gene mutation is one factor that underlies clinical variability.
Asunto(s)
Encéfalo/patología , Genes de Neurofibromatosis 1 , Células Madre Pluripotentes Inducidas/patología , Mutación/genética , Neuronas/patología , Organoides/patología , Animales , Apoptosis , Astrocitos/patología , Diferenciación Celular , Línea Celular , Proliferación Celular , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones Mutantes , Neurogénesis , Neuronas/metabolismo , Proteínas ras/metabolismoRESUMEN
Calcein, a fluorescent fluid phase marker, has been used to track and visualize cellular processes such as synaptic vesicle fusion. It is also the fluorophore for live cells in the commonly used Live/Dead viability assay. In pilot studies designed to determine fusion pore open size and vesicle movement in secretory cells, imaging analysis revealed that calcein reduced the number of vesicles released from the cells when stimulated with nicotine. Using amperometry to detect individual vesicle release events, we show that when calcein is present in the media, the number of vesicles that fuse with the cellular membrane is reduced when cells are stimulated with either nicotine or high K+. Experimentally, amperometric electrodes are not undergoing fouling in the presence of calcein. We hypothesized that calcein, when activated by light, releases reactive oxygen species that cause a reduction in secreted vesicles. We show that when calcein is protected from light during experimentation, little to no reduction of vesicle secretion occurred. Therefore, photoactivated calcein can cause deleterious results for measurements of cellular processes, likely to be the result of release of reactive oxygen species.
Asunto(s)
Catecolaminas/metabolismo , Células Cromafines/metabolismo , Exocitosis/fisiología , Fluoresceínas/metabolismo , Animales , Membrana Celular/metabolismo , Colorantes Fluorescentes , Luz , Ratas , Vesículas Secretoras/metabolismoRESUMEN
Peripheral nerve injuries present challenges to regeneration. Currently, the gold standard for nerve repair is an autograft that results in another region of the body suffering nerve damage. Previously, bioactive borate glass (BBG) has been studied in clinical trials to treat patients with non-healing wounds, and we have reported that BBG is conducive for soft tissue repair. BBG provides structural support, degrades in a non-cytotoxic manner, and can be chemically doped. Here, we tested a wide range of chemical compounds that are reported to have neuroprotective characteristics to promote regeneration of peripheral neurons after traumatic injury. We hypothesized that chemical dopants added in trace amounts to BBG would improve neuronal survival and neurite outgrowth from dorsal root ganglion (DRG) explants. We measured neurite outgrowth from whole DRG explants, and survival rates of dissociated neurons and support cells that comprise the DRG. Results show that chemically doped BBGs have differentially variable effects on neuronal survival and outgrowth, with iron, gallium, and zinc improving outgrowth of neurons, and iodine causing the most detriment to neurons. Because chemically doped BBGs support increased nerve regrowth and survival, they show promise for use in peripheral nerve regeneration.
Asunto(s)
Boratos/química , Ganglios Espinales/metabolismo , Vidrio/química , Regeneración Nerviosa , Neuritas/metabolismo , Traumatismos de los Nervios Periféricos , Andamios del Tejido/química , Animales , Bovinos , Células Cultivadas , Ganglios Espinales/patología , Neuritas/patología , Traumatismos de los Nervios Periféricos/metabolismo , Traumatismos de los Nervios Periféricos/patología , Traumatismos de los Nervios Periféricos/terapiaRESUMEN
Electrical and chemical stimulation have been studied as potent mechanisms of enhancing nerve regeneration and wound healing. However, it remains unclear how electrical stimuli affect nerve growth, particularly in the presence of neurotrophic factors. The objective of this study was to explore (1) the effect of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) supplementation to support neurite outgrowth in a 3D scaffold, and (2) the effect of brief, low voltage, electrical stimulation (ES) on neurite outgrowth prior to neurotrophin supplementation. Dissociated E11 chick dorsal root ganglia (DRG) were seeded within a 1.5 mg/mL type-I collagen scaffold. For neurotrophin treatments, scaffolds were incubated for 24 h in culture media containing NGF (10 ng/mL) or BDNF (200 ng/mL), or both. For ES groups, scaffolds containing neurons were stimulated for 10 min at 8-10 V/m DC, then incubated for 24 h with neurotrophin. Fixed and labeled neurons were imaged to measure neurite growth and directionality. BDNF supplementation was not as effective as NGF at supporting DRG neurite outgrowth. ES prior to NGF supplementation improved DRG neurite outgrowth compared to NGF alone. This combination of brief ES with NGF treatment was the most effective treatment compared to NGF or BDNF alone. Brief ES had no impact on neurite directionality in the 3D scaffolds. These results demonstrate that ES improves neurite outgrowth in the presence of neurotrophins, and could provide a potential therapeutic approach to improve nerve regeneration when coupled with neurotrophin treatment.
Asunto(s)
Colágeno/química , Ganglios Espinales/metabolismo , Neuritas/metabolismo , Andamios del Tejido/química , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Células Cultivadas , Embrión de Pollo , Estimulación Eléctrica , Ganglios Espinales/citología , Factor de Crecimiento Nervioso/metabolismo , Factores de Crecimiento Nervioso/metabolismoRESUMEN
The primary goal of this research was to characterize the effect of laminin on three-dimensional (3D) neurite growth. Gels were formed using type I collagen at concentrations of 0.4-2.0 mg mL(-1) supplemented with laminin at concentrations of 0, 1, 10, or 100 µg mL(-1). When imaged with confocal microscopy, laminin was shown to follow the collagen fibers; however, the addition of laminin had minimal effect on the stiffness of the scaffolds at any concentration of collagen. Individual neurons dissociated from E9 chick dorsal root ganglia were cultured in the gels for 24 h, and neurite lengths were measured. For collagen gels without laminin, a typical bimodal response of neurite outgrowth was observed, with increased growth at lower concentrations of collagen gel. However, alteration of the chemical nature of the collagen gel by the laminin additive shifted, or completely mitigated, the bimodal neurite growth response seen in gels without laminin. Expression of integrin subunits, α1, α3, α6 and ß1, were confirmed by PCR and immunolabeling in the 3D scaffolds. These results provide insight into the interplay between mechanical and chemical environment to support neurite outgrowth in 3D. Understanding the relative impact of environmental factors on 3D nerve growth may improve biomaterial design for nerve cell regeneration.
Asunto(s)
Colágeno/administración & dosificación , Laminina/administración & dosificación , Neuritas/fisiología , Animales , Embrión de Pollo , Pollos , Colágeno/química , Femenino , Ganglios Espinales/citología , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/crecimiento & desarrollo , Geles , Laminina/química , Neuritas/efectos de los fármacos , RatasRESUMEN
In sympathetic neurons, it is well-established that the neurotransmitters, norepinephrine (NE), neuropeptide Y (NPY), and ATP are differentially coreleased from the same neurons. In this study, we determined whether synaptotagmin (syt) I, the primary Ca(2+) sensor for regulated release, could function as the protein that differentially regulates release of these neurotransmitters. Plasmid-based RNA interference was used to specifically and stably silence expression of syt I in a model secretory cell line. Whereas stimulated release of NPY and purines was abolished, stimulated catecholamine (CA) release was only reduced by approximately 50%. Although expression levels of tyrosine hydroxylase, the rate-limiting enzyme in the dopamine synthesis pathway, was unaffected, expression of the vesicular monoamine transporter 1 was reduced by 50%. To evaluate whether NPY and CAs are found within the same vesicles and whether syt I is found localized to each of these NPY- and CA-containing vesicles, we used immunocytochemistry to determine that syt I colocalized with large dense core vesicles, with NPY, and with CAs. Furthermore, both CAs and NPY colocalized with one another and with large dense core vesicles. Electron micrographs show that large dense core vesicles are synthesized and available for release in cells that lack syt I. These results are consistent with syt I regulating differential release of transmitters.
Asunto(s)
Dopamina/metabolismo , Neuropéptido Y/metabolismo , Norepinefrina/metabolismo , Vesículas Sinápticas/metabolismo , Sinaptotagmina I/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Expresión Génica , Inmunohistoquímica , Microscopía Electrónica de Transmisión , Células PC12 , Interferencia de ARN , Ratas , Vesículas Sinápticas/ultraestructura , Sinaptotagmina I/genética , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Synaptotagmin (syt) I is a Ca2+-binding protein that is well accepted as a major sensor for Ca2+-regulated release of transmitter. However, controversy remains as to whether syt I is the only protein that can function in this role and whether the remaining syt family members also function as Ca2+ sensors. In this study, we generated a PC12 cell line that continuously expresses a short hairpin RNA (shRNA) to silence expression of syt I by RNA interference. Immunoblot and immunocytochemistry experiments demonstrate that expression of syt I was specifically silenced in cells that stably integrate the shRNA-syt I compared with control cells stably transfected with the empty shRNA vector. The other predominantly expressed syt isoform, syt IX, was not affected, nor was the expression of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins when syt I levels were knocked down. Resting Ca2+ and stimulated Ca2+ influx imaged with fura-2 were not altered in syt I knockdown cells. However, evoked release of catecholamine detected by carbon fiber amperometry and HPLC was significantly reduced, although not abolished. Human syt I rescued the release events in the syt I knockdown cells. The reduction of stimulated catecholamine release in the syt I knockdown cells strongly suggests that although syt I is clearly involved in catecholamine release, it is not the only protein to regulate stimulated release in PC12 cells, and another protein likely has a role as a Ca2+ sensor for regulated release of transmitter.