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PubMed2024

A Transcriptional Signature of Induced Neurons Differentiates Virologically Suppressed People Living With HIV from People Without HIV.

Neurocognitive impairment is a prevalent and important co-morbidity in virologically suppressed people living with HIV (PLWH), yet the underlying mechanisms remain elusive and treatments lacking. Here, we explored for the first time, use of participant-derived directly induced neurons (iNs) to model neuronal biology and injury in PLWH. iNs retain age- and disease-related features of the donors, providing unique opportunities to reveal novel aspects of neurological disorders. We obtained primary dermal fibroblasts from six virologically suppressed PLWH (range: 27 - 64 years, median: 53); 83% Male; 50% White) and seven matched people without HIV (PWOH) (range: 27 - 66, median: 55); 71% Male; 57% White). iNs were generated using transcription factors NGN2 and ASCL1, and validated by immunocytochemistry and single-cell-RNAseq. Transcriptomic analysis using bulk-RNAseq identified 29 significantly differentially expressed genes between iNs from PLWH and PWOH. Of these, 16 genes were downregulated and 13 upregulated in PLWH iNs. Protein-protein interaction network mapping indicates that iNs from PLWH exhibit differences in extracellular matrix organization and synaptic transmission. IFI27 was upregulated in iNs from PLWH, which complements independent post-mortem studies demonstrating elevated IFI27 expression in PLWH-derived brain tissue, indicating that iN generation reconstitutes this pathway. Finally, we observed that expression of the FOXL2NB-FOXL2-LINC01391 genome locus is reduced in iNs from PLWH and negatively correlates with neurocognitive impairment. Thus, we have identified an iN gene signature of HIV through direct reprogramming of skin fibroblasts into neurons revealing novel mechanisms of neurocognitive impairment in PLWH.

HAND↗

PubMed2001

Xebf3 is a regulator of neuronal differentiation during primary neurogenesis in Xenopus.

During primary neurogenesis in Xenopus, a cascade of helix--loop--helix (HLH) transcription factors regulates neuronal determination and differentiation. While XNeuroD functions at a late step in this cascade to regulate neuronal differentiation, the factors that carry out terminal differentiation are still unknown. We have isolated a new Xenopus member of the Ebf/Olf-1 family of HLH transcription factors, Xebf3, and provide evidence that, during primary neurogenesis, it regulates neuronal differentiation downstream of XNeuroD. In developing Xenopus embryos, Xebf3 is turned on in the three stripes of primary neurons at stage 15.5, after XNeuroD. In vitro, XEBF3 binds the EBF/OLF-1 binding site and functions as a transcriptional activator. When overexpressed, Xebf3 is able to induce ectopic neurons at neural plate stages and directly convert ectodermal cells into neurons in animal cap explants. Expression of Xebf3 can be activated by XNeuroD both in whole embryos and in animal caps, indicating that this new HLH factor might be regulated by XNeuroD. Furthermore, in animal caps, XNeuroD can activate Xebf3 in the absence of protein synthesis, suggesting that, in vitro, this regulation is direct. Similar to XNeuroD, but unlike Xebf2/Xcoe2, Xebf3 expression and function are insensitive to Delta/Notch-mediated lateral inhibition. In summary, we conclude that Xebf3 functions downstream of XNeuroD and is a regulator of neuronal differentiation in Xenopus.

Amino Acid Sequence↗

PubMed2004

Activation of REST/NRSF target genes in neural stem cells is sufficient to cause neuronal differentiation.

REST/NRSF is a transcriptional repressor that acts at the terminal stage of the neuronal differentiation pathway and blocks the transcription of several differentiation genes. REST/NRSF is generally downregulated during induction of neuronal differentiation. The recombinant transcription factor REST-VP16 binds to the same DNA binding site as does REST/NRSF but functions as an activator instead of a repressor and can directly activate the transcription of REST/NRSF target genes. However, it is not known whether REST-VP16 expression is sufficient to cause formation of functional neurons from neural stem cells (NSCs). Here we show that regulated expression of REST-VP16 in a physiologically relevant NSC line growing under cycling conditions converted the cells rapidly to the mature neuronal phenotype. Furthermore, when grown in the presence of retinoic acid, REST-VP16-expressing NSCs activated their target, as well as other differentiation genes that are not their direct target, converting them to the mature neuronal phenotype and enabling them to survive in the presence of mitotic inhibitors, which is a characteristic of mature neurons. In addition, these neuronal cells were physiologically active. These results showed that direct activation of REST/NRSF target genes in NSCs with a single transgene, REST-VP16, is sufficient to cause neuronal differentiation, and the findings suggested that direct activation of genes involved in the terminal stage of differentiation may cause neuronal differentiation of NSCs.

Animals↗

PubMed2004

Acid-sensing ion channel 2 contributes a major component to acid-evoked excitatory responses in spiral ganglion neurons and plays a role in noise susceptibility of mice.

Ion channels in the degenerin-epithelial sodium channel (DEG-ENaC) family perform diverse functions, including mechanosensation. Here we explored the role of the vertebrate DEG-ENaC protein, acid-sensing ion channel 2 (ASIC2), in auditory transduction. Contributions of ASIC2 to hearing were examined by comparing hearing threshold and noise sensitivity of wild-type and ASIC2 null mice. ASIC2 null mice showed no significant hearing loss, indicating that the ASIC2 was not directly involved in the mechanotransduction of the mammalian cochlea. However, we found that (1) ASIC2 was present in the spiral ganglion (SG) neurons in the adult cochlea and that externally applied protons induced amiloride-sensitive sodium currents and action potentials in SG neurons in vitro, (2) proton-induced responses were greatly reduced in SG neurons obtained from ASIC2 null mice, indicating that activations of ASIC2 contributed a major portion of the proton-induced excitatory response in SG neurons, and (3) ASIC2 null mice were considerably more resistant to noise-induced temporary, but not permanent, threshold shifts. Together, these data suggest that ASIC2 contributes to suprathreshold functions of the cochlea. The presence of ASIC2 in SG neurons could provide sensors to directly convert local acidosis to excitatory responses, therefore offering a cellular mechanism linking hearing losses caused by many enigmatic causes (e.g., ischemia or inflammation of the inner ear) to excitotoxicity.

Acid Sensing Ion Channels↗

PubMed2000

Effect of oral CDP-choline on plasma choline and uridine levels in humans.

Twelve mildly hypertensive but otherwise normal fasting subjects received each of four treatments in random order: CDP-choline (citicoline; 500, 2000, and 4000 mg) or a placebo orally at 8:00 a.m. on four different treatment days. Eleven plasma samples from each subject, obtained just prior to treatment (8:00 a.m.) and 1-12 hr thereafter, were assayed for choline, cytidine, and uridine. Fasting terminated at noon with consumption of a light lunch that contained about 100 mg choline. Plasma choline exhibited dose-related increases in peak values and areas under the curves (AUCs), remaining significantly elevated, after each of the three doses, for 5, 8, and 10 hr, respectively. Plasma uridine was elevated significantly for 5-6 hr after all three doses, increasing by as much as 70-90% after the 500 mg dose, and by 100-120% after the 2000 mg dose. No further increase was noted when the dose was raised from 2000 to 4000 mg. Plasma cytidine was not reliably detectable, since it was less than twice blank, or less than 100 nM, at all of the doses. Uridine is known to enter the brain and to be converted to UTP; moreover, we found that uridine was converted directly to CTP in neuron-derived PC-12 cells. Hence, it seems likely that the circulating substrates through which oral citicoline increases membrane phosphatide synthesis in the brains of humans involve uridine and choline, and not cytidine and choline as in rats.

Administration, Oral↗

PubMed1998

Structure and promoter analysis of Math3 gene, a mouse homolog of Drosophila proneural gene atonal. Neural-specific expression by dual promoter elements.

ath3, a vertebrate basic helix-loop-helix gene homologous to Drosophila proneural gene atonal, can directly convert non-neural cells into neurons with the anterior features. In the mouse, ath3 expression initially occurs widely in the developing nervous system and then gradually becomes restricted to the neural retina. Here, we characterized the genomic organization and promoter activity of mouse ath3 (Math3). Math3 gene consists of two exons separated by an 8-kilobase intron, and the whole protein-coding region is located in the second exon. Transcription starts at two sites, which are 75 nucleotides apart from each other, and there is no typical TATA box in the upstream region of either start site. Transient transfection analysis showed that the 5'-region of Math3 can direct efficient expression in neuroblastoma cells but not in glioma or fibroblast cells. Deletion studies revealed that the proximal 193-base pair region, which contains the downstream transcription initiation site but not the upstream site, is essential for the Math3 promoter activity and can direct efficient expression in neuroblastoma cells. In contrast, retrovirus-mediated promoter analysis demonstrated that a region further upstream is additionally necessary for retinal expression. These results indicate that Math3 promoter contains two essential regulatory regions, the proximal 193-base pair region, which confers efficient neural-specific expression, and a region further upstream, required for retinal expression.

Animals↗

PubMed1992

Techniques for converting Golgi precipitate in CNS neurons into stable electron microscopic markers.

Direct electron microscopy of nervous tissue stained with the Golgi impregnation method is unsatisfactory because the cytoplasm of the cell bodies and processes of the impregnated neurons are completely filled with a compact precipitate of electron dense silver chromate. This precipitate entirely obscures the cytological details of the impregnated neurons. Because of its solidity and instability in aqueous solutions, the silver chromate is also a source of inconvenience during the preparation of the ultrathin sections. This review summarizes methods that have been developed with the aim of replacing the Golgi precipitate in CNS neurons with a more convenient electron dense material--for example, heavy metal salts or metallic particles. Conversion of the precipitate into a stable electron dense marker is done before the material is embedded for electron microscopy. The methods include lead, gold, and bromide substitution, treatment with ammonia, direct chemical reduction into metallic silver, and photoreduction of the silver chromate into silver through irradiation with ultraviolet light.

Aminophenols↗

PubMed1992

Mutations in the channel domain of a neuronal nicotinic receptor convert ion selectivity from cationic to anionic.

Introduction by site-directed mutagenesis of three amino acids from the MII segment of glycine or gamma-aminobutyric acid (GABAA) receptors into the MII segment of alpha 7 nicotinic receptor was sufficient to convert a cation-selective channel into an anion-selective channel gated by acetylcholine. A critical mutation was the insertion of an uncharged residue at the amino-terminal end of MII, stressing the importance of protein geometrical constraints on ion selectivity.

Acetylcholine↗

PubMed2004

Conversion of myoblasts to physiologically active neuronal phenotype.

Repressor element 1 (RE1)-silencing transcription factor (REST)/neuron-restrictive silencer factor (NRSF) can repress several terminal neuronal differentiation genes by binding to a specific DNA sequence (RE1/neuron-restrictive silencer element [NRSE]) present in their regulatory regions. REST-VP16 binds to the same RE1/NRSE, but activates these REST/NRSF target genes. However, it is unclear whether REST-VP16 expression is sufficient to cause formation of functional neurons either from neural stem cells or from heterologous stem cells. Here we show that the expression of REST-VP16 in myoblasts grown under muscle differentiation conditions blocked entry into the muscle differentiation pathway, countered endogenous REST/NRSF-dependent repression, activated the REST/NRSF target genes, and, surprisingly, activated other neuronal differentiation genes and converted the myoblasts to a physiologically active neuronal phenotype. Furthermore, in vitro differentiated neurons produced by REST-VP16-expressing myoblasts, when injected into mouse brain, survived, incorporated into the normal brain, and did not form tumors. This is the first instance in which myoblasts were converted to a neuronal phenotype. Our results suggest that direct activation of REST/NRSF target genes with a single transgene, REST-VP16, is sufficient to activate other terminal neuronal differentiation genes and to override the muscle differentiation pathways, and they suggest that this approach provides an efficient way of triggering neuronal differentiation in myoblasts and possibly other stem cells.

Animals↗

PubMed1999

Endothelin converting enzyme-1-, endothelin-1-, and endothelin-3-like immunoreactivity in the rat brain.

Neurons likely to use endothelin as a neurotransmitter/neurohormone were mapped in the rat brain using polyclonal antibodies directed against endothelin-converting enzyme-1, endothelin-1, and endothelin-3. Anti-endothelin-converting enzyme-1 antibodies produced the most robust staining, permitting the best visualization of the distribution and morphology of neurons. Labeled neurons were found in the dorsal thalamic nuclei and reticular thalamic nuclei, medial preoptic area, pontine nucleus, and locus coeruleus. Localization of endothelin-converting enzyme-like immunoreactivity in the locus coeruleus and in the reticular nucleus of the thalamus suggests that endothelin is co-localized with norepinephrine and GABA, respectively. Additionally, endothelin-converting enzyme-like immunoreactivity was found in the globus pallidus, septal nuclei, and in both the vertical and horizontal limbs of the nucleus of the diagonal band of Broca, and the ventrolateral area of the caudate-putamen. Strong endothelin-converting enzyme-like immunoreactivity was found in a continuous band of pyramidal neurons throughout the neocortex primarily in layer V, extending into the cingulate gyrus and piriform cortex. Motor nuclei, including oculomotor, facial, and trigeminal nuclei, were also endothelin-converting enzyme-immunoreactive. In the cerebellum, Purkinje cells were stained. Non-neuronal cells such as oligodendroglia, microglia, and astrocytes generally were not endothelin-converting enzyme-immunoreactive, although astrocytes were rarely stained. Endothelin-converting enzyme-, endothelin-1-, and endothelin-3-like immunoreactivities were generally found co-existing in given nuclei. The diversity of neurons immunostained for endothelin suggests multiple roles of endothelin in the CNS.

Animals↗

PubMed1997

Chaperone-supervised conversion of prion protein to its protease-resistant form.

Transmissible spongiform encephalopathies (TSEs) are lethal, infectious disorders of the mammalian nervous system. A TSE hallmark is the conversion of the cellular protein PrPC to disease-associated PrPSc (named for scrapie, the first known TSE). PrPC is protease-sensitive, monomeric, detergent soluble, and primarily alpha-helical; PrPSc is protease-resistant, polymerized, detergent insoluble, and rich in beta-sheet. The "protein-only" hypothesis posits that PrPSc is the infectious TSE agent that directly converts host-encoded PrPC to fresh PrPSc, harming neurons and creating new agents of infection. To gain insight on the conformational transitions of PrP, we tested the ability of several protein chaperones, which supervise the conformational transitions of proteins in diverse ways, to affect conversion of PrPC to its protease-resistant state. None affected conversion in the absence of pre-existing PrPSc. In its presence, only two, GroEL and Hsp104 (heat shock protein 104), significantly affected conversion. Both promoted it, but the reaction characteristics of conversions with the two chaperones were distinct. In contrast, chemical chaperones inhibited conversion. Our findings provide new mechanistic insights into nature of PrP conversions, and provide a new set of tools for studying the process underlying TSE pathogenesis.

Adenosine Triphosphate↗

PubMed2006

Effect of steroid milieu on gonadotropin-releasing hormone-1 neuron firing pattern and luteinizing hormone levels in male mice.

GnRH neuronal function is regulated by gonadal hormone feedback. In males, testosterone can act directly or be converted to either dihydrotestosterone (DHT) or estradiol (E2). We examined central steroid feedback by recording firing of green fluorescent protein (GFP)-identified GnRH neurons in brain slices from male mice that were intact, castrated, or castrated and treated with implants containing DHT, E2, or E2 + DHT. Castration increased LH levels. DHT or E2 alone partially suppressed LH, whereas E2 + DHT reduced LH to intact levels. Despite the inhibitory actions on LH, the combination of E2 + DHT increased GnRH neuron activity relative to other treatments, reflected in mean firing rate, amplitude of peaks in firing rate, and area under the curve of firing rate vs. time. Cluster8 was used to identify peaks in firing activity that may be correlated with hormone release. Castration increased the frequency of peaks in firing rate. Treatment with DHT failed to reduce frequency of these peaks. In contrast, treatment with E2 reduced peak frequency to intact levels. The frequency of peaks in firing rate was intermediate in animals treated with E2 + DHT, perhaps suggesting the activating effects of this combination partially counteracts the inhibitory actions of E2. These data indicate that E2 mediates central negative feedback in males primarily by affecting the pattern of GnRH neuron activity, and that androgens combined with estrogens have a central activating effect on GnRH neurons. The negative feedback induced by E2 + DHT to restore LH to intact levels may mask an excitatory central effect of this combination.

Animals↗

PubMed1996

Targeting toxins to neural antigens and receptors.

Immunotoxins have been used to study the targeting of biologically active substances at neurons in vivo and to make experimental neural lesions. OX7-saporin, directed against Thy 1, destroys any neuron. 192 IgG-saporin, directed against the 'low affinity' neurotrophin receptor (p 75NTR), selectively destroys neurons expressing this receptor (sympathetic, sensory, cholinergic basal forebrain, cerebellar Purkinje). Anti-D beta H-saporin, directed against dopamine beta-hydroxylase, the enzyme that converts dopamine to norepinephrine, selectively destroys noradrenergic neurons (sympathetic, CNS). These agents show that several types of neural antigens may prove useful in treating pain, and anti-D beta H-saporin may be active against pheochromocytoma or neuroblastoma.

Animals↗

PubMed1991

Activation of N-methyl-d-aspartate Receptors Induces Endogenous Rhythmic Bursting Activities in Nucleus Tractus Solitarii Neurons: An Intracellular Study on Adult Rat Brainstem Slices.

A brainstem slice preparation and intracellular recording techniques were used to examine the effects of N-methyl-d-aspartate (NMDA) application on neurons within the swallowing area of the nucleus tractus solitarii (NTS). According to their cellular properties, NTS neurons were classified into type I and type II neurons. The most striking difference was the occurrence of delayed excitation in type I but not in type II neurons, when they were depolarized from membrane potentials more negative than -60 mV. Bath application of NMDA (30 - 60 microM) elicited depolarization and triggered stable repetitive firing in all the NTS neurons but one. During the NMDA-induced depolarization, hyperpolarization below -60 mV elicited, in some type I neurons, a rhythmic bursting pattern. The duration of the bursts (300 - 1000 ms) and their frequency (0.5 - 2 Hz) depended on the membrane potential. With hyperpolarizations below -75 mV, rhythmic bursting was converted into rhythmic single discharges, a pattern elicited directly in the other type I neurons. In all cases, rhythmic patterns were superimposed on cyclic depolarizations of the membrane potential characterized by an initial ramp-shaped phase. In type II neurons, rhythmic bursting discharges, superimposed on rhythmic oscillations of the membrane potential, were also obtained upon hyperpolarization during the NMDA-induced depolarization. In all type I neurons tested, NMDA-induced cyclic ramp-shaped depolarizations continued after addition of tetrodotoxin to the medium. Rhythmic bursting was not elicited by bath application of kainate (10 - 20 microM). Application of d-2-amino-5-phosphonovalerate (50 microM) blocked NMDA-induced depolarizations without modifying those elicited by kainate, which were selectively depressed by 6-cyano-7-nitroquinoxaline-2,3-dione (10 microM). Moreover, removal of Mg2+ from the medium suppressed NMDA-induced cyclic depolarizations. Results demonstrate that both NMDA and non-NMDA receptors are present in NTS neurons and that selective activation of NMDA receptors induced rhythmic bursting and/or rhythmic single discharges. Rhythmic patterns were not driven by synaptic mechanisms but originated from endogenous properties of NTS neurons activated by NMDA. Thus, NTS neurons can be considered as conditional pacemakers. According to the location of the neurons, the conditional properties shown in these in vitro experiments might be involved in vivo in the generation of rhythmic motor activities set up at the NTS level, such as swallowing.

Journal Article↗

PubMed1990

Retinoids increase perinatal spinal cord neuronal survival and astroglial differentiation.

In this report we demonstrate that retinol and retinoic acid (RA) increase the survival and morphological differentiation of rat spinal cord neurons in vitro. Micromolar amounts of retinol and RA increased the number of surviving neurons by 2- to 3-fold and affected neuritic density resulting in increased secondary and tertiary processes compared to untreated sister cultures. A marked morphological differentiation of the astrocyte population in conjunction with an antiproliferative effect in the presence of retinoids were apparent. These trophic effects occurred mainly after 5 days in vitro, a time that corresponds to the time of birth in vivo. Retinoic acid exerted a direct trophic effect on spinal cord neurons in the absence of glial cells while retinol lost its effectiveness. Metabolic labeling suggested that retinol is converted to the biologically active RA within astrocytes but not in neurons. Taken together, our results have demonstrated direct trophic effects of RA on spinal cord neurons and have suggested another role for astrocytes in the maintenance of normal neural physiology by regulating RA concentrations through the oxidation of retinol.

Animals↗

PubMed1979

Modifications induced by neonatal steroids in reproductive organs and behavior of male rats.

Male rats injected on Day 3 neonatally with .01, .1, 1, 10, 100, or 1,000 micrograms of estradiol benzoate (EB), 10,000 microgram of testosterone propionate (TP), or sesame oil were subsequently examined for testicular, penile, and accessory organ development. Sexual behavior was evaluated during therapy with fluoxymesterone (FM) and then with TP. Estradiol benzoate in dosages greater than 1.0 micrograms delayed testicular descent, reduced the size and hormone responsiveness of reproductive organs, and decreased sexual behavior in a dose-dependent manner. The 10,000-microgram dosage of neonatal TP delayed testicular descent and reduced sexual behavior to levels near those of the 10--100 micrograms EB groups, but it produced no significant penile or accessory organ changes. Neither reduced peripheral organ development nor inhibited neonatal testicular secretions fully explain reductions in male behavior following large dosages of neonatal TP. Neonatal androgen may reduce the responsiveness of central nervous system neurons governing male sexual behavior after being converted to estrogen or by directly altering steroid receptor systems.

Animals↗

PubMed2002

Effect of tumor necrosis factor-alpha converting enzyme (TACE) and metalloprotease inhibitor on amyloid precursor protein metabolism in human neurons.

Tumor necrosis factor-alpha (TNF-alpha) is implicated in inflammatory processes and much effort is being directed at inhibiting the release of TNF-alpha for treatment of inflammatory conditions. In this context, the drug CP-661,631 has been developed to inhibit the TNF-alpha converting enzyme (TACE). However, TACE is also implicated in amyloid precursor protein secretion. Amyloid precursor protein (APP) undergoes constitutive and regulated secretion by alpha-secretase endoproteolytic cleavage within the amyloid beta peptide (Abeta) domain. Alternative cleavage at the N- and C-terminus of the Abeta domain by beta- and gamma-secretases results in the production of Abeta. In many cellular and in vivo animal models, increased secretion of APP results in a concomitant decrease in the production of Abeta suggesting that the two pathways are intricately linked. However, in human primary neuron cultures, increased APP secretion is not associated with a decrease in total Abeta production. To determine if the use of CP-661,631 may enhance amyloidogenic processing in human brain, we have assessed the effect of CP-661,631 on APP metabolism in primary cultures of human neurons. Our results show that CP-661,631 effectively prevents regulated APP secretion but does not increase total Abeta levels in human primary neuron cultures.

ADAM Proteins↗

PubMed2006

Female snake sex pheromone induces membrane responses in vomeronasal sensory neurons of male snakes.

The vomeronasal organ (VNO) is important for activating accessory olfactory pathways that are involved in sexually dimorphic mating behavior. The VNO of male garter snakes is critically important for detection of, and response to, female sex pheromones. In the present study, under voltage-clamp conditions, male snake VNO neurons were stimulated with female sexual attractiveness pheromone. Thirty-nine of 139 neurons exhibited inward current responses (reversal potential: -10.6 +/- 2.8 mV). The amplitude of the inward current was dose dependent, and the relationship could be fitted by the Hill equation. Under current-clamp conditions, application of pheromone produced membrane depolarizing responses and increases in firing frequency. These results suggest that the female pheromone directly affects male snake VNO neurons and results in opening of ion channels, thereby converting the pheromone signal to an electrical signal. The response to female pheromone is sexually dimorphic, that is, the pheromone does not evoke responses in VNO neurons of female snakes. An associated finding of the present study is that the female sex pheromone, which is insoluble in aqueous solutions, became soluble in the presence of Harderian gland homogenate.

Animals↗