Cdk12 maintains the integrity of adult axons by suppressing actin remodeling.

The role of cyclin-dependent kinases (CDKs) that are ubiquitously expressed in the adult nervous system remains unclear. Cdk12 is enriched in terminally differentiated neurons where its conical role in the cell cycle progression is redundant. We find that in adult neurons Cdk12 acts a negative regulator of actin formation, mitochondrial dynamics and neuronal physiology. Cdk12 maintains the size of the axon at sites proximal to the cell body through the transcription of homeostatic enzymes in the 1-carbon by folate pathway which utilize the amino acid homocysteine. Loss of Cdk12 leads to elevated homocysteine and in turn leads to uncontrolled F-actin formation and axonal swelling. Actin remodeling further induces Drp1-dependent fission of mitochondria and the breakdown of axon-soma filtration barrier allowing soma restricted cargos to enter the axon. We demonstrate that Cdk12 is also an essential gene for long-term neuronal survival and loss of this gene causes age-dependent neurodegeneration. Hyperhomocysteinemia, actin changes, and mitochondrial fragmentation are associated with several neurodegenerative conditions such as Alzheimer's disease and we provide a candidate molecular pathway to link together such pathological events.

SMAD transcription factors are altered in cell models of HD and regulate HTT expression.

Transcriptional dysregulation is observable in multiple animal and cell models of Huntington's disease, as well as in human blood and post-mortem caudate. This contributes to HD pathogenesis, although the exact mechanism by which this occurs is unknown. We therefore utilised a dynamic model in order to determine the differential effect of growth factor stimulation on gene expression, to highlight potential alterations in kinase signalling pathways that may be in part responsible for the transcriptional dysregulation observed in HD, and which may reveal new therapeutic targets. We demonstrate that cells expressing mutant huntingtin have a dysregulated transcriptional response to epidermal growth factor stimulation, and identify the transforming growth factor-beta pathway as a novel signalling pathway of interest that may regulate the expression of the Huntingtin (HTT) gene itself. The dysregulation of HTT expression may contribute to the altered transcriptional phenotype observed in HD.

Integration-Free Reprogramming of Lamina Propria Progenitor Cells.

Producing induced pluripotent stem cells (iPSCs) from human tissue for use in personalized medicine strategies or therapeutic testing is at the forefront of medicine. Therefore, identifying a source of cells to reprogram that is easily accessible via a simple noninvasive procedure is of great clinical importance. Reprogramming these cells to iPSCs through nonintegrating methods for genetic manipulation is paramount for regenerative purposes. Here, we demonstrate reprogramming of oral mucosal lamina propria progenitor cells from patients undergoing routine dental treatment. Reprogramming was performed utilizing nonintegrating plasmids containing all 6 pluripotency genes (OCT4, SOX2, KLF4, NANOG, LIN28, and cMYC). Resulting iPSCs lacked genetic integration of the vector genes and had the ability to differentiate down mesoderm, ectoderm, and endoderm lineages, demonstrating pluripotency. In conclusion, oral mucosal lamina propria progenitor cells represent a source of cells that can be obtained with minimal invasion, as they can be taken concurrently with routine treatments. The resulting integration-free iPSCs therefore have great potential for use in personalized medicine strategies.

Retinoid-independent motor neurogenesis from human embryonic stem cells reveals a medial columnar ground state.

A major challenge in neurobiology is to understand mechanisms underlying human neuronal diversification. Motor neurons (MNs) represent a diverse collection of neuronal subtypes, displaying differential vulnerability in different human neurodegenerative diseases. The ability to manipulate cell subtype diversification is critical to establish accurate, clinically relevant in vitro disease models. Retinoid signalling contributes to caudal precursor specification and subsequent MN subtype diversification. Here we investigate the necessity for retinoic acid in motor neurogenesis from human embryonic stem cells. We show that activin/nodal signalling inhibition, followed by sonic hedgehog agonist treatment, is sufficient for MN precursor specification, which occurs even in the presence of retinoid pathway antagonists. Importantly, precursors mature into HB9/ChAT-expressing functional MNs. Furthermore, retinoid-independent motor neurogenesis results in a ground state biased to caudal, medial motor columnar identities from which a greater retinoid-dependent diversity of MNs, including those of lateral motor columns, can be selectively derived in vitro.

Medical terminations of pregnancy: a viable source of tissue for cell replacement therapy for neurodegenerative disorders.

"Proof-of-principle" that cell replacement therapy works for neurodegeneration has been reported, but only using donor cells collected from fetal brain tissue obtained from surgical terminations of pregnancy. Surgical terminations of pregnancy represent an increasingly limited supply of donor cells due to the tendency towards performing medical termination in much of Europe. This imposes a severe constraint on further experimental and clinical cell transplantation research. Therefore, we explore here the feasibility of using medical termination tissue as a donor source. Products of conception were retrieved from surgical terminations over the last 7 years and from medical terminations over the last 2.5 years. The number of collections that yielded fetal tissue, viable brain tissue, and identifiable brain regions (ganglionic eminence, ventral mesencephalon, and neocortex) were recorded. We studied cell viability, cell physiological properties, and differentiation potential both in vitro and following transplantation into the central nervous system of rodent models of neurodegenerative disease. Within equivalent periods, we were able to collect substantially greater numbers of fetal remains from medical than from surgical terminations of pregnancy, and the medical terminations yielded a much higher proportion of identifiable and dissectible brain tissue. Furthermore, we demonstrate that harvested cells retain the capacity to differentiate into neurons with characteristics appropriate to the region from which they are dissected. We show that, contrary to widespread assumption, medical termination of pregnancy-derived fetal brain cells represent a feasible and more readily available source of human fetal tissue for experimental cell transplantation with the potential for use in future clinical trials in human neurodegenerative disease.

Carbon monoxide is a rapid modulator of recombinant and native P2X(2) ligand-gated ion channels.

BACKGROUND AND PURPOSE: Carbon monoxide (CO) is a potent modulator of a wide variety of physiological processes, including sensory signal transduction. Many afferent sensory pathways are dependent upon purinergic neurotransmission, but direct modulation of the P2X purinoceptors by this important, endogenously produced gas has never been investigated. EXPERIMENTAL APPROACH: Whole-cell patch-clamp experiments were used to measure ATP-elicited currents in human embryonic kidney 293 cells heterologously expressing P2X(2), P2X(3), P2X(2/3) and P2X(4) receptors and in rat pheochromocytoma (PC12) cells known to express native P2X(2) receptors. Modulation was investigated using solutions containing CO gas and the CO donor molecule, tricarbonyldichlororuthenium (II) dimer (CORM-2). KEY RESULTS: CO was a potent and selective modulator of native P2X(2) receptors, and these effects were mimicked by a CO donor (CORM-2). Neither pre-incubation with 8-bromoguanosine-3',5'-cyclomonophosphate nor 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (a potent blocker of soluble guanylyl cyclase) affected the ability of the CO donor to enhance the ATP-evoked P2X(2) currents. The CO donor caused a small, but significant inhibition of currents evoked by P2X(2/3) and P2X(4) receptors, but was without effect on P2X(3) receptors. CONCLUSIONS AND IMPLICATIONS: These data provided an explanation for how CO might regulate sensory neuronal traffic in physiological reflexes such as systemic oxygen sensing but also showed that CO could be used as a selective pharmacological tool to assess the involvement of homomeric P2X(2) receptors in physiological systems.

Mice with very low expression of the vesicular monoamine transporter 2 gene survive into adulthood: potential mouse model for parkinsonism.

We have created a transgenic mouse with a hypomorphic allele of the vesicular monoamine transporter 2 (Vmat2) gene by gene targeting. These mice (KA1) have profound changes in monoamine metabolism and function and survive into adulthood. Specifically, these animals express very low levels of VMAT2, an endogenous protein which sequesters monoamines intracellularly into vesicles, a process that, in addition to being important in normal transmission, may also act to keep intracellular levels of the monoamine neurotransmitters below potentially toxic thresholds. Homozygous mice show large reductions in brain tissue monoamines, motor impairments, enhanced sensitivity to dopamine agonism, and changes in the chemical neuroanatomy of the striatum that are consistent with alterations in the balance of the striatonigral (direct) and striatopallidal (indirect) pathways. The VMAT2-deficient KA1 mice are also more vulnerable to the neurotoxic effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in terms of nigral dopamine cell death. We suggest that the mice may be of value in examining, long term, the insidious damaging consequences of abnormal intracellular handling of monoamines. On the basis of our current findings, the mice are likely to prove of immediate interest to aspects of the symptomatology of parkinsonism. They may also, however, be of use in probing other aspects of monoaminergic function and dysfunction in the brain, the latter making important contributions to the pathogenesis of schizophrenia and addiction.

Conditional ablation of neurones in transgenic mice.

Conditional targeted ablation of specific cell populations in living transgenic animals is a very powerful strategy to determine cell functions in vivo. This approach would be of particular value to study the functions of distinct neuronal populations; however, the transgene of choice for conditional cell ablation studies in mice, the herpes simplex virus thymidine kinase gene, cannot be used to ablate neurones as its principal mode of action relies on cell proliferation. Here we report that expression of the E.coli nitroreductase gene (Ntr) and metabolism of the prodrug CB1954 (5-aziridin-1-yl-2-4-dinitrobenzamide) to its cytotoxic derivative can be used to conditionally and acutely ablate specific neuronal populations in vivo. As proof of principal, we have ablated olfactory and vomeronasal receptor neurones by expressing Ntr under the control of the olfactory marker protein (OMP) gene promoter. We demonstrate that following CB1954 administration, olfactory and vomeronasal receptor neurones expressing the transgene were selectively eliminated from the olfactory epithelium (OE), and projections to the olfactory bulb (OB) were lost. The functional efficacy of cell ablation was demonstrated using a highly sensitive behavioural test to show that ablated mice had lost the olfactory ability to discriminate distinct odors and were consequently rendered anosmic. Targeted expression of Ntr to specific neuronal populations using conventional transgenes, as described here, or by "knock-in" gene targeting using embryonic stem cells may be of significant value to address the functions of distinct neuronal populations in vivo.

Inducible ablation of astrocytes shows that these cells are required for neuronal survival in the adult brain.

To study the function of astrocytes in the adult brain, we have targeted the expression of E. coli nitroreductase (NTR) to the astrocytes of transgenic mice under the control of the GFAP promoter. The astrocytes expressing NTR were selectively ablated after administration of the prodrug CB1954, resulting in motor discoordination. Histological examination showed that the region most affected in the brain was the cerebellum, in which the Bergmann glia were eliminated and the granular neurons had degenerated. Specific effects were also noted on the dendrites of the Purkinje cells, and the junction between these neurons and granular layer was disrupted. Astrocyte ablation was associated with a dramatic decrease in the expression of glutamate transporters, which may account for the degeneration of granular neurons since the excitotoxic effects of glutamate result in a similar phenotype. These results provide the first evidence that astrocytes are important for the survival of neurons in the adult brain in vivo.

GAP-43 N-terminal translocation signal targets beta-galactosidase to developing axons in a pan-neuronal transgenic mouse line.

Tracing neural connectivity is important in understanding the intricacy of the nervous system as this represents the functional unit throughout the system. Here, we provide evidence that beta-galactosidase (beta-gal) linked to the N-terminal axonal translocation signal of GAP-43 provides a reproducible and versatile reporter system for analyzing the developing nervous system in vivo. When expressed by the GAP-43 promoter in transgenic mice, the fusion protein is detected equally within the developing axons of the peripheral and the central nervous systems, directly reflecting the promoter activity.

Transgenics identify distal 5'- and 3'-sequences specifying gonadotropin-releasing hormone expression in adult mice.

GnRH neurons play a critical role in regulating gonadotropin secretion, but their scattered distribution has prevented detailed understanding of their molecular and cellular properties in vivo. Using GnRH promoter-driven transgenics we have examined here the role of 5'- and 3'-murine GnRH sequences in specifying GnRH expression in the adult mouse. Transgenic mice bearing a lacZ construct incorporating 5.5 kb of 5'-, all the introns and exons, and 3.5 kb of 3'-murine GnRH sequence were found to express beta-galactosidase (betagal) immunoreactivity in approximately 85% of all GnRH neurons. Deletion of GnRH sequence 3' to exon II had no effect upon transgene expression in the GnRH population (89%) but resulted in the appearance of ectopic betagal immunoreactivity in several regions of the brain. The production of additional mice in which 5'-elements were deleted to leave only -2.1 kb of sequence resulted in an approximately 40% reduction in the number of GnRH neurons expressing betagal. Mice in which further deletion of 400 bp allowed only -1.7 kb of 5'-sequence to remain exhibited a complete absence of betagal immunoreactivity within GnRH and other neurons. These results suggest that elements 3' to exon II of the GnRH gene have little role in enabling GnRH expression within the GnRH phenotype but, instead, are particularly important in repressing the GnRH gene in non-GnRH neurons. In contrast, elements located between -2.1 and -1.7 kb of distal 5'-sequence appear to be critical for the in vivo activation of GnRH expression within GnRH neurons in the adult brain.

Promoter transgenics reveal multiple gonadotropin-releasing hormone-I-expressing cell populations of different embryological origin in mouse brain.

Gonadotropin-releasing hormone-I (GnRH-I) is thought to be expressed by a single, highly spatially restricted group of neurons, which originate in the olfactory placode and migrate through the nose into the medial septum and hypothalamus from where they control fertility. Transgenic mice bearing a 13.5 kb GnRH-I-lacZ reporter construct were derived and found to express high levels of beta-galactosidase mRNA and protein within the septohypothalamic GnRH neurons in a correct temporal and spatial manner. Unexpectedly, low levels of beta-galactosidase were also present in three further populations of cells within the lateral septum, bed nucleus of the stria terminalis, and tectum. Analysis of wild-type mice with three different GnRH-I antibodies revealed distinct and transient patterns of GnRH-I peptide expression during development in all three of these populations revealed by transgenics. The synthesis of GnRH by cells of the lateral septum was the most persistent and remained until the third postnatal week. Embryonic "small eye" Pax-6 null mice, which fail to develop an olfactory placode, were also examined and shown to have equivalent populations of GnRH-I-immunoreactive cells in the lateral septum, tectum, and bed nucleus of the stria terminalis but none of the migrating cells that form the septohypothalamic GnRH population. These results prove that so-called "ectopic" expression in promoter transgenic lines can reflect authentic developmental patterns of gene expression. They further provide the first demonstration in mammalian brain that multiple neuronal populations of different embryological origin express GnRH-I peptide during embryonic and postnatal development.

Transgenic mice ubiquitously expressing human placental alkaline phosphatase (PLAP): an additional reporter gene for use in tandem with beta-galactosidase (lacZ).

A fundamental keystone of developmental biology has been the growing use of reporter genes in model transgenic systems. Their use has greatly facilitated investigations of cell lineage and cell fate in addition to aiding experiments aimed at determining patterns of gene expression, gene interaction and gene regulation. Through construction of transgenic mice, ubiquitously expressing human placental alkaline phosphatase (PLAP), we demonstrate the suitability of PLAP as a reporter gene for use in conjunction with, or as an alternative to, beta-galactosidase (lacZ). Our findings demonstrate that over-expression of PLAP has no adverse effects on mouse development or viability, despite a widespread pattern of expression. This technology provides a simple yet effective mechanism based on eukaryotic reporter gene technology to facilitate the identification of transgenic cells within complex in vivo systems.

Cloning of the mouse organic cation transporter 2 gene, Slc22a2, from an enhancer-trap transgene integration locus.

A novel mouse gene, associated with the enhancer-trap mutation TKZ736, has been cloned and sequenced. It encodes a polyspecific transmembrane transporter with 12 putative transmembrane domains, that shares significant homology with the mouse organic cation transporter 1 (Oct1/Slc22a1) called Lx1. Like Oct1/Slc22a1/Lx1, this gene maps to the proximal part of Chromosome (Chr) 17, but shows a different expression pattern from Oct1/Slc22a1/Lx1. The gene identified here is predominantly expressed in the kidney and ureter, but no expression is detectable in liver. Sequence comparisons suggest that this novel gene most likely represents the mouse homolog of the rat organic cation transporter 2 gene. The genomic DNA flanking the 3' transgene integration site in the enhancer-trap mutation TKZ736 encodes the second exon of the Oct2/Slc22a2 gene.

Parental allele-specific chromatin configuration in a boundary-imprinting-control element upstream of the mouse H19 gene.

The mouse H19 gene is expressed from the maternal chromosome exclusively. A 2-kb region at 2 to 4 kb upstream of H19 is paternally methylated throughout development, and these sequences are necessary for the imprinted expression of both H19 and the 5'-neighboring Igf2 gene. In particular, on the maternal chromosome this element appears to insulate the Igf2 gene from enhancers located downstream of H19. We analyzed the chromatin organization of this element by assaying its sensitivity to nucleases in nuclei. Six DNase I hypersensitive sites (HS sites) were detected on the unmethylated maternal chromosome exclusively, the two most prominent of which mapped 2.25 and 2.75 kb 5' to the H19 transcription initiation site. Five of the maternal HS sites were present in expressing and nonexpressing tissues and in embryonic stem (ES) cells. They seem, therefore, to reflect the maternal origin of the chromosome rather than the expression of H19. A sixth maternal HS site, at 3.45 kb upstream of H19, was detected in ES cells only. The nucleosomal organization of this element was analyzed in tissues and ES cells by micrococcal nuclease digestion. Specifically on the maternal chromosome, an unusual and strong banding pattern was obtained, suggestive of a nonnucleosomal organization. From our studies, it appears that the unusual chromatin organization with the presence of HS sites (maternal chromosome) and DNA methylation (paternal chromosome) in this element are mutually exclusive and reflect alternate epigenetic states. In addition, our data suggest that nonhistone proteins are associated with the maternal chromosome and that these might be involved in its boundary function.

Parental chromosome-specific chromatin conformation in the imprinted U2af1-rs1 gene in the mouse.

The imprinted U2af1-rs1 gene on mouse chromosome 11 is expressed exclusively from the paternal allele. We found that U2af1-rs1 resides in a chromosomal domain that displays marked differences in chromatin conformation and DNA methylation between the parental chromosomes. Chromatin conformation was assayed in brain and liver, in fetuses, and in embryonic stem cells by sensitivity to nucleases in nuclei. In all these tissues, the unmethylated paternal chromosome is sensitive to DNase-I and MspI and has two DNase-I hypersensitive sites in the 5'-untranslated region. In brain and in differentiated stem cells, which display high levels of U2af1-rs1 expression, a paternal DNase-I hypersensitive site is also readily apparent in the promoter region. On the maternal chromosome, in contrast, the entire U2af1-rs1 gene and its promoter are highly resistant to DNase-I and MspI in all tissues analyzed and are fully methylated. No differential MNase sensitivity was detected in this imprinted domain. The parental chromosome-specific DNA methylation and chromatin conformation were also present in parthenogenetic and androgenetic cells and in tissues from animals maternally or paternally disomic for chromosome 11. This demonstrates that these parental chromosome-specific epigenotypes are independently established and maintained and provides no evidence for interallelic trans-sensing and counting mechanisms in U2af1-rs1.

Transactivation of Igf2 in a mouse model of Beckwith-Wiedemann syndrome.

The gene IGF2, which encodes a fetal insulin-like growth factor, is imprinted, so only one of two parental copies of the gene is expressed. The altered expression of IGF2 has been implicated in Beckwith-Wiedemann syndrome, a human fetal overgrowth syndrome, which is characterized by overgrowth of several organs and an increased risk of developing childhood tumours. We have introduced Igf2 transgenes into the mouse genome by using embryonic stem cells, which leads to transactivation of the endogenous Igf2 gene. The consequent overexpression of Igf2 results in most of the symptoms of Beckwith-Wiedemann syndrome, including prenatal overgrowth, polyhydramnios, fetal and neonatal lethality, disproportionate organ overgrowth including tongue enlargement, and skeletal abnormalities. These phenotypes establish Igf2 overexpression as a key determinant of Beckwith-Wiedemann syndrome.

Distribution of parthenogenetic cells in the mouse brain and their influence on brain development and behavior.

A systematic analysis of parthenogenetic (PG) cell fate within the central nervous system (CNS) was made throughout fetal development and neonatal and adult life. Chimeras were made between PG embryos carrying a ubiquitously expressed lacZ transgene and normal fertilized embryos. After detailed histological analysis, we find that the developmental potential of PG cells is spatially restricted to certain parts of the brain. PG cells are prevalent in telencephalic structures and are largely excluded from diencephalic structures, especially the hypothalamus. These spatial restrictions are established early in development. Behavioral studies with chimeras identified an increase in male aggression when the proportion of PG cells in the brain was high. These studies demonstrate that imprinted genes play key roles in development of the CNS and may be involved in behavior.

Chromatin structure and imprinting: developmental control of DNase-I sensitivity in the mouse insulin-like growth factor 2 gene.

The insulin-like growth factor 2 (Igf2) gene on distal mouse chromosome 7 is expressed predominantly from the paternal allele. In previous studies we identified two regions of paternal allele-specific methylation; one at approximately 3 kb upstream of promoter 1, and a second in the 3', coding portion of the gene. The 3' region is methylated in an expressing tissue (fetal liver), whereas in a non-expressing tissue (fetal brain), it is not methylated. By contrast, in the 5' region, the paternal allele is highly methylated in all tissues. Here, we have studied another characteristic of chromatin, namely, sensitivity to DNase-I and have focused our developmental analysis on the two differentially methylated regions of Igf2. In the upstream region, four clustered DNase-I hypersensitive sites (HSS) were detected in embryonic stem (ES) cells and in midgestation embryos, but not in neonatal liver or brain. In promoter 1 (P1), at approximately 0.3 kb upstream of exon 1, we detected a tissue-specific HSS that was present in neonatal liver, in which P1 is active, but was absent in ES cells, the embryo, and in neonatal brain. No DNase-I HSS were detected in the 3' differentially methylated region of Igf2. In all these regions, we did not detect differences in DNase-I sensitivity between the parental chromosomes. These results establish major developmental and tissue-specific control of chromatin in the Igf2 locus. The presence of the HSS upstream of Igf2 precedes transcriptional activation of the Igf2 gene and may be indicative of a promoter for another transcript that is transcribed in the opposite direction. The HSS in P1 is largely liver-specific; this promoter therefore is differently regulated than the more general fetal promoters P2 and P3. Whereas methylation can be allele-specific, presumably reflecting the gene imprint, the nuclease sensitivity, as detected by our assay, is not. These results, taken together with previous observations, reveal developmental and tissue-specific complexity in the expression of the parental imprint at the level of chromatin and transcription. We propose that epigenetic features of tissue-specific control and of the control of allelic expression are intricately linked.