PAR1 inhibition suppresses the self-renewal and growth of A2B5-defined glioma progenitor cells and their derived gliomas in vivo.

Glioblastoma (GBM) remains the most common and lethal intracranial tumor. In a comparison of gene expression by A2B5-defined tumor-initiating progenitor cells (TPCs) to glial progenitor cells derived from normal adult human brain, we found that the F2R gene encoding PAR1 was differentially overexpressed by A2B5-sorted TPCs isolated from gliomas at all stages of malignant development. In this study, we asked if PAR1 is causally associated with glioma progression. Lentiviral knockdown of PAR1 inhibited the expansion and self-renewal of human GBM-derived A2B5(+) TPCs in vitro, while pharmacological inhibition of PAR 1 similarly slowed both the growth and migration of A2B5(+) TPCs in culture. In addition, PAR1 silencing potently suppressed tumor expansion in vivo, and significantly prolonged the survival of mice following intracranial transplantation of human TPCs. These data strongly suggest the importance of PAR1 to the self-renewal and tumorigenicity of A2B5-defined glioma TPCs; as such, the abrogation of PAR1-dependent signaling pathways may prove a promising strategy for gliomas.

Sustained induction of neuronal addition to the adult rat neostriatum by AAV4-delivered noggin and BDNF.

Intraventricular ependymal infection by adenoviruses expressing brain-derived neurotrophic factor (BDNF) and noggin is sufficient to induce the heterotopic recruitment of new medium spiny neurons to the adult neostriatum, from endogenous subependymal neural progenitor cells. This approach was found to slow disease progression and extend survival in an R6/2 mouse model of Huntington's disease (HD). However, the practical therapeutic value of this strategy is limited by the transient expression and immunogenicity of adenoviral vectors. In addition, it has been unclear whether sustained overexpression of BDNF and noggin would yield similarly sustained neuronal production and striatal recruitment, or whether progenitor depletion or tachyphylaxis might supervene to limit the therapeutic potential of this approach. To address these issues, we used adeno-associated virus serotype 4 (AAV4), an ependymotrophic vector that is neither immunogenic nor neurotoxic, to achieve sustained BDNF and noggin expression. Using AAV4, we found that BDNF and noggin achieved levels sufficient to initiate and maintain, for at least 4 months, ongoing neuronal addition to the neostriatum and olfactory bulb. Over this period, we noted no diminution of treatment-associated neuronal recruitment from resident progenitors. AAV4:BDNF and noggin-induced neuronal addition may thus provide a means to provide longlasting and persistent striatal neuronal replacement in conditions of striatal neuronal loss, such as HD.

Partial cure of established disease in an animal model of metachromatic leukodystrophy after intracerebral adeno-associated virus-mediated gene transfer.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by genetic deficiency of arylsulfatase A (ARSA) enzyme. Failure in catalyzing the degradation of its major substrate, sulfatide (Sulf), in oligodendrocytes and Schwann cells leads to severe demyelination in the peripheral (PNS) and central nervous system (CNS), and early death of MLD patients. The ARSA knockout mice develop a disease that resembles MLD but is milder, without significant demyelination in the PNS and CNS. We showed that adeno-associated virus serotype 5-mediated gene transfer in the brain of ARSA knockout mice reverses Sulf storage and prevents neuropathological abnormalities and neuromotor disabilities when vector injections are performed at a pre-symptomatic stage of disease. Direct injection of viral particles into the brain of ARSA knockout mice at a symptomatic stage results in sustained expression of ARSA, prevention of Sulf storage and neuropathological abnormalities. Despite these significant corrections, the treated mice continue to develop neuromotor disability. We show that more subtle biochemical abnormalities involving gangliosides and galactocerebroside are in fact not corrected.

High-yield selection and extraction of two promoter-defined phenotypes of neural stem cells from the fetal human brain.

Neural stem and precursor cells reside in the ventricular lining of the fetal forebrain, and may provide a cellular substrate for brain repair. To selectively identify and extract these cells, we infected dissociated fetal human brain cells with adenoviruses bearing the gene for green fluorescence protein (GFP), placed under the control of enhancer/promoters for two genes (nestin and musashi1) that are expressed in uncommitted neuroepithelial cells. The cells were then sorted by fluorescence-activated cell sorting (FACS) on the basis of E/nestin- or P/musashi1-driven GFP expression. Both P/musashi1:hGFP- and E/nestin:EGFP-sorted cells were multipotent: limiting dilution with clonal expansion as neurospheres, in tandem with retroviral lineage analysis and xenograft to E17 and P0-2 rat forebrain, revealed that each phenotype was able to both self-renew and co-generate neurons and glia. Thus, fluorescent genes placed under the control of early neural promoters allow neural stem cells to be specifically targeted, isolated, and substantially enriched from the fetal human brain.

Adenoviral brain-derived neurotrophic factor induces both neostriatal and olfactory neuronal recruitment from endogenous progenitor cells in the adult forebrain.

Neural progenitor cells persist throughout the adult forebrain subependyma, and neurons generated from them respond to brain-derived neurotrophic factor (BDNF) with enhanced maturation and survival. To induce neurogenesis from endogenous progenitors, we overexpressed BDNF in the adult ventricular zone by transducing the forebrain ependyma to constitutively express BDNF. We constructed a bicistronic adenovirus bearing BDNF under cytomegalovirus (CMV) control, and humanized green fluorescent protein (hGFP) under internal ribosomal entry site (IRES) control. This AdCMV:BDNF:IRES:hGFP (AdBDNF) was injected into the lateral ventricles of adult rats, who were treated for 18 d thereafter with the mitotic marker bromodeoxyuridine (BrdU). Three weeks after injection, BDNF averaged 1 microg/gm in the CSF of AdBDNF-injected animals but was undetectable in control CSF. In situ hybridization demonstrated BDNF and GFP mRNA expression restricted to the ventricular wall. In AdBDNF-injected rats, the olfactory bulb exhibited a >2.4-fold increase in the number of BrdU(+)-betaIII-tubulin(+) neurons, confirmed by confocal imaging, relative to AdNull (AdCMV:hGFP) controls. Importantly, AdBDNF-associated neuronal recruitment to the neostriatum was also noted, with the treatment-induced addition of BrdU(+)-NeuN(+)-betaIII-tubulin(+) neurons to the caudate putamen. Many of these cells also expressed glutamic acid decarboxylase, cabindin-D28, and DARPP-32 (dopamine and cAMP-regulated phosphoprotein of 32 kDa), markers of medium spiny neurons of the neostriatum. These newly generated neurons survived at least 5-8 weeks after viral induction. Thus, a single injection of adenoviral BDNF substantially augmented the recruitment of new neurons into both neurogenic and non-neurogenic sites in the adult rat brain. The intraventricular delivery of, and ependymal infection by, viral vectors encoding neurotrophic agents may be a feasible strategy for inducing neurogenesis from resident progenitor cells in the adult brain.

Behavioral and electrophysiological study of phonological priming between bisyllabic spoken words.

Phonological priming between bisyllabic (CV.CVC) spoken items was examined using both behavioral (reaction times, RTs) and electrophysiological (event-related potentials, ERPs) measures. Word and pseudoword targets were preceded by pseudoword primes. Different types of final phonological overlap between prime and target were compared. Critical pairs shared the last syllable, the rime or the coda, while unrelated pairs were used as controls. Participants performed a target shadowing task in Experiment 1 and a delayed lexical decision task in Experiment 2. RTs were measured in the first experiment and ERPs were recorded in the second experiment. The RT experiment was carried out under two presentation conditions. In Condition 1 both primes and targets were presented auditorily, while in Condition 2 the primes were presented visually and the targets auditorily. Priming effects were found in the unimodal condition only. RTs were fastest for syllable overlap, intermediate for rime overlap, and slowest for coda overlap and controls that did not differ from one another. ERPs were recorded under unimodal auditory presentation. ERP results showed that the amplitude of the auditory N400 component was smallest for syllable overlap, intermediate for rime overlap, and largest for coda overlap and controls that did not differ from one another. In both experiments, the priming effects were larger for word than for pseudoword targets. These results are best explained by the combined influences of nonlexical and lexical processes, and a comparison of the reported effects with those found in monosyllables suggests the involvement of rime and syllable representations.

In vitro neurogenesis by progenitor cells isolated from the adult human hippocampus.

Neurogenesis persists in the adult mammalian hippocampus. To identify and isolate neuronal progenitor cells of the adult human hippocampus, we transfected ventricular zone-free dissociates of surgically-excised dentate gyrus with DNA encoding humanized green fluorescent protein (hGFP), placed under the control of either the nestin enhancer (E/nestin) or the Talpha1 tubulin promoter (P/Talpha1), two regulatory regions that direct transcription in neural progenitor cells. The resultant P/Talpha1:hGFP+ and E/nestin:enhanced (E)GFP+ cells expressed betaIII-tubulin or microtubule-associated protein-2; many incorporated bromodeoxyuridine, indicating their genesis in vitro. Using fluorescence-activated cell sorting, the E/nestin:EGFP+ and P/Talpha1:hGFP+ cells were isolated to near purity, and matured antigenically and physiologically as neurons. Thus, the adult human hippocampus contains mitotically competent neuronal progenitors that can be selectively extracted. The isolation of these cells may provide a cellular substrate for re-populating the damaged or degenerated adult hippocampus.

Promoter-based isolation and fluorescence-activated sorting of mitotic neuronal progenitor cells from the adult mammalian ependymal/subependymal zone.

Neuronal precursor cells are widespread in the subependyma of the forebrain ventricular lining, and may provide a cellular substrate for brain repair. We have previously identified and isolated them from fetal brain, by sorting forebrain cells transfected with plasmid DNA encoding the gene for green fluorescent protein (hGFP), driven by the early neuronal promoter for Talpha1 tubulin (P/Talpha1). Fetal neuronal precursors were thereby identified and harvested with both a high degree of enrichment, and a virtual abolition of glial contaminants. We have now extended this approach to include the isolation and purification of neuronal progenitors from the adult brain. Dissociates of the lateral ventricular wall, that included the combined ependymal/subependymal zone, were obtained from 3-month-old adult rats. These cells were cultured and transfected with P/Talpha1:hGFP plasmid DNA. Two days later, the cells were redissociated, sorted on the basis of Talpha1-driven GFP expression, and replated. The majority of these cells expressed the early neuronal proteins Hu and TuJ1/betaIII-tubulin upon FACS; within the week thereafter, most matured as morphologically-evident neurons, that coexpressed betaIII-tubulin and MAP-2. Fewer than 5% expressed astrocytic markers, compared to over half of the cells in matched samples that were either not sorted, or sorted after transfection with a plasmid bearing the nonfluorescent lacZ gene under the control of P/Talpha1 tubulin. Thus, the use of a fluorescent transgene under the control of an early neuron-selective promoter permits the enrichment of neuronal progenitor cells from the adult rat brain, in a form that may allow their heterologous implantation.

Promoter-targeted selection and isolation of neural progenitor cells from the adult human ventricular zone.

Adult humans, like their nonhuman mammalian counterparts, harbor persistent neural progenitor cells in the forebrain ventricular lining. In the absence of adequate surface markers, however, these cells have proven difficult to isolate for study. We have previously identified and selected neural progenitor cells from both the fetal and adult rodent ventricular zone (VZ), by sorting forebrain cells transfected with plasmid DNA encoding the gene for green fluorescent protein driven by the early neuronal promoter for Talpha1 tubulin (P/Talpha1:hGFP). We have now extended this approach by purifying both P/Talpha1:hGFP tubulin-defined neuronal progenitors, as well as potentially less committed E/nestin:hGFP-defined neural progenitor cells, from the adult human VZ. The ventricular wall of the temporal horn of the lateral ventricle was dissected from temporal lobes obtained from four adult patients undergoing therapeutic lobectomy. These samples were dissociated, and the cultured cells transduced with either P/Talpha1:hGFP or E/nestin:EGFP plasmid DNA. A week later, the cells were redissociated, selected via fluorescence-activated cell sorting (FACS) on the basis of neural promoter-driven GFP expression, and replated. The majority of these cells expressed the early neuronal protein betaIII-tubulin upon FACS; within the week thereafter, most matured as morphologically evident neurons that coexpressed betaIII-tubulin and microtubule-associated protein (MAP)-2. Many of these neurons had incorporated bromodeoxyuridine in vitro in the days before FACS, indicating their mitogenesis in vitro. Thus, the use of fluorescent transgenes under the control of early neural promoters permits the enrichment of neuronal progenitor cells from the adult human ventricular zone. The specific acquisition, in both purity and number, of residual neural progenitor cells from the adult human brain may now permit hitherto unfeasible studies of both their biology and practical application.

From print to meaning: an electrophysiological investigation of the role of phonology in accessing word meaning.

This study investigated the role and time-course of phonology in accessing meaning during silent reading. In Experiment 1, the homophone effect was replicated in a semantic categorization task in French. When deciding whether a stimulus belonged to a semantic category (FOOD), subjects made more errors to homophones (MEET) than to orthographic controls (MELT). In Experiment 2, event-related brain potentials (ERPs) were used to study the online development of this effect. If access to meaning was mediated by phonology, smaller N400 components should be obtained to homophones than to orthographic controls. The ERP data exhibited a full-blown N400 component to homophones that did not differ from the N400 to controls. No differences between homophones and controls were found before the N400. After the N400, however, homophones differed from controls, with ERPs to homophones being similar to those of correct category exemplars. The results suggest that the final selection of a word's meaning does not depend on its phonological form. This result is incompatible with a strong phonological view according to which the only way to meaning is via a word's phonology.

Identification, isolation, and promoter-defined separation of mitotic oligodendrocyte progenitor cells from the adult human subcortical white matter.

Previous studies have suggested the persistence of oligodendrocyte progenitor cells in the adult mammalian subcortical white matter. To identify oligodendrocyte progenitors in the adult human subcortical white matter, we transfected dissociates of capsular white matter with plasmid DNA bearing the gene for green fluorescence protein (hGFP), placed under the control of the human early promoter (P2) for the oligodendrocytic protein cyclic nucleotide phosphodiesterase (P/hCNP2). Within 4 d after transfection with P/hCNP2:hGFP, a discrete population of small, bipolar cells were noted to express GFP. These cells were A2B5-positive (A2B5(+)), incorporated bromodeoxyuridine in vitro, and constituted <0.5% of all cells. Using fluorescence-activated cell sorting (FACS), the P/hCNP2-driven GFP(+) cells were then isolated and enriched to near-purity. In the weeks after FACS, most P/hCNP2:hGFP-sorted cells matured as morphologically and antigenically characteristic oligodendrocytes. Thus, the human subcortical white matter harbors mitotically competent progenitor cells, which give rise primarily to oligodendrocytes in vitro. By using fluorescent transgenes of GFP expressed under the control of an early oligodendrocytic promoter, these oligodendrocyte progenitor cells may be extracted and purified from adult human white matter in sufficient numbers for implantation and cell-based therapy.

Neurogenesis during caudal spinal cord regeneration in adult newts.

After tail amputation in urodele amphibians, dramatic changes appear in the spinal cord rostral to the amputation level. Transection induces a proliferation response in cells lining the ependymal canal, giving rise to an ependymal tube in which neurogenesis occurs. Using the thymidine analog bromodeoxyuridine (BrdU) in short- and long-term labeling of cells undergoing DNA synthesis (S phase of the cell cycle), specific cell markers, and cell cultures, we show that neurons derive from the proliferative ependymal layer of the ependymal tube.

Neural crest-like cells originate from the spinal cord during tail regeneration in adult amphibian urodeles.

Using in vitro cell-marking experiments and transplantation in tail regenerates, we have recently shown (Benraiss et al., 1996) that clonal cells derived from adult newt spinal cord (SC) cultures could find suitable cues in blastemal mesenchyme to enable them to differentiate into melanocytes or Schwann cells. This led to the question of whether neural crest-like cell derivatives might emerge from the ependymal tube as tail regeneration proceeded. To address this question we used the biolistic method for in situ transfection of caudal SC cells. These cells were transfected with an alkaline phosphatase marker gene. The potentialities of transfected cell derivatives in tail regenerates were analyzed using histochemistry or immunohistochemistry. As early as eight days after transfection, labeled cells were detected in the regenerating SC and around its "terminal vesicle" (TV). Two to four weeks following transfection, most of the labeled cell derivatives could be identified either by dark granules as melanocytes or by galactocerebroside staining as Schwann cells. Electron microscopic investigations revealed the incompletely organized cytoarchitecture of the TV, suggesting that an exit of cells was possible at this level, at least from its "open" dorsal part. Furthermore, the localization of ciliated cells in the blastemal mesenchyme, especially around the TV, supported this view by suggesting that they might be ependymal cells detached from it. Our findings therefore led us to believe that in the newt, during tail regeneration, neural crest-like cells emerging from the TV could participate in the formation of the peripheral nervous system, especially by providing Schwann cells and melanocytes.

Correlations between standard auditory evoked potentials and symptomatology in a group of 50 schizophrenic patients.

Standard auditory evoked potentials (AEP) were recorded in 50 schizophrenic patients and 47 normal controls. All patients were rated on the Brief Psychiatric Rating Scale (BPRS), the Scale for the Assessment of Negative Symptoms (SANS), the Scale for the Assessment of Positive Symptoms (SAPS), and the Positive and Negative Syndrome Scale (PANSS), and were classified in three groups (positive-type [n = 10], negative-type [n = 23]and mixed-type [n = 17]patients) according to the normative criteria suggested by Kay. The mean latencies of AEP components (N1, P2, N2) and mean peak-to-peak amplitudes (N1P2, P2N2) did not correlate with age, duration of illness, length of hospitalisation or neuroleptic dosage. The evoked response did not differ between the three groups of patients (positive, negative and mixed). There was only a trend (P = 0.075) to a longer N1 latency in the negative-type group and a shorter one in the positive-type group than in the mixed-type and the control groups. The latency of N1 component correlated significantly with negative symptoms of schizophrenia (SANS scores). This correlation was related to the severity of a depressive dimension of the disorder reflected by the "depressive factor" of BPRS or "affective flattening" and "avolition" subscales of SANS.

Clonal cell cultures from adult spinal cord of the amphibian urodele Pleurodeles waltl to study the identity and potentialities of cells during tail regeneration.

The urodele amphibians are nearly the only adult vertebrates able to regenerate their missing or amputated tail. The most striking feature of this model lies in the ability of the spinal cord (SC) to differentiate, within the regenerating tail, a new ependymal tube from which the SC and the peripheral nervous system originate. A fundamental question is whether, in response to tail excision, the ependymoglia of the old SC stump behaves as an embryonic neuroepithelium. To evaluate this possibility, cell lines from primary cell cultures of adult SC were established for the first time in newts, and two cell clones, immunochemically characterized as ependymoglial cell populations, could be obtained. To analyze the potentialities of these clonal cells, after transplantation in tail regenerates, cell-marking experiments, using either in vitro transfection with lacZ gene or the lineage tracer lysinated rhodamine dextran (LRD), were performed. One to 2 weeks postimplantation, most of labeled derivatives were identified as melanocytes. Interestingly, labeled cells were also seen integrated in the ependymoglia of the regenerating SC. Two to 6 weeks after implantation in young regenerates, we also observed LRD-labeled elongated cells close to nerves or myofibers which were unambiguously identified as Schwann cells by galactocerebroside staining. Taken together, these findings showed that clonal cells derived from adult newt SC cultures could largely find, in regenerate mesenchyme, suitable environmental conditions to differentiate into melanocytes or Schwann cells. Because these two cells types arise from neural crest cells during embryo-genesis, this supports the interesting view that multipotent cells are still present in the SC of adult urodeles.

Tenascin expression in developing, adult and regenerating caudal spinal cord in the urodele amphibians.

Tenascin (Tn) protein and transcripts were analyzed in developing, adult and regenerating caudal spinal cord (SC) of Pleurodeles waltl. A polyclonal antibody (PAb) against Xenopus Tn and a newt Tn cDNA probe were used. In Western blots, anti-Tn PAb recognized Tn polypeptides of 200-220 kDa in tail regenerate extracts, but also the homolog of Tn/Cytotactin/J1 in brain and SC of adult newt. Immunofluorescence studies showed some reactivity around ependymoglial cells and strong labeling in the nervous tracts, in the developing as well as in the regenerating SC or adult SC. Immunogold electron microscopy revealed the presence of Tn throughout the ependymoglial cells, particularly near and along the plasma membrane of radial processes surrounding axons, especially growth cones. Tn could be more precisely found within rough endoplasmic reticulum and Golgi structures, or again in the surrounding extracellular space. This suggested that Tn was at least produced by radial glial profiles forming axonal compartments in which axons grew. Using the DNA probe for Tn, expression of Tn mRNA was also examined by Northern blot and RNAase protection analyses and by in situ hybridization, respectively. The levels of transcripts, barely detectable in adult tail, increased in regenerates from 3 days through 4-8 weeks post-amputation. In situ Tn mRNA were mainly localized in the mesenchyme, especially at the epithelial-mesenchymal interface, and in the developing cartilage, at the early regeneration stages, whereas high amounts of transcripts were seen not only at these stages, but also later, in the regenerating SC. Our main results supported the view that, in the caudal SC of newts, Tn, synthesized by radial ependymoglial cells, was similarly expressed during regeneration as well as larval development, and exhibited a sustained high accumulation level in the adult SC. On the basis of the multifunctional properties of Tn, the putative roles played by Tn as a substrate for neuronal pathfinding and boundary shaping were discussed.