In utero gene therapy rescues microcephaly caused by Pqbp1-hypofunction in neural stem progenitor cells.

Human mutations in PQBP1, a molecule involved in transcription and splicing, result in a reduced but architecturally normal brain. Examination of a conditional Pqbp1-knockout (cKO) mouse with microcephaly failed to reveal either abnormal centrosomes or mitotic spindles, increased neurogenesis from the neural stem progenitor cell (NSPC) pool or increased cell death in vivo. Instead, we observed an increase in the length of the cell cycle, particularly for the M phase in NSPCs. Corresponding to the developmental expression of Pqbp1, the stem cell pool in vivo was decreased at E10 and remained at a low level during neurogenesis (E15) in Pqbp1-cKO mice. The expression profiles of NSPCs derived from the cKO mouse revealed significant changes in gene groups that control the M phase, including anaphase-promoting complex genes, via aberrant transcription and RNA splicing. Exogenous Apc4, a hub protein in the network of affected genes, recovered the cell cycle, proliferation, and cell phenotypes of NSPCs caused by Pqbp1-cKO. These data reveal a mechanism of brain size control based on the simple reduction of the NSPC pool by cell cycle time elongation. Finally, we demonstrated that in utero gene therapy for Pqbp1-cKO mice by intraperitoneal injection of the PQBP1-AAV vector at E10 successfully rescued microcephaly with preserved cortical structures and improved behavioral abnormalities in Pqbp1-cKO mice, opening a new strategy for treating this intractable developmental disorder.

iNOS: a potential therapeutic target for malignant glioma.

Glioblastoma is the most aggressive adult primary brain tumor. Although progress has been made in understanding the molecular mechanisms underlying these tumors, current treatments are ineffective. Recent studies have identified iNOS as a critical regulator of glial transformation downstream of EGFRvIII/STAT3 signaling, a key oncogenic pathway in glioblastoma. STAT3 directly binds the promoter of the iNOS gene and thereby stimulates its expression. Importantly, inhibition of iNOS by genetic and pharmacological approaches impedes glial cell proliferation, invasiveness, and tumor growth in vivo. iNOS expression is also elevated in a population of human brain tumor stem cells (BTSCs), and iNOS is required for BTSC proliferation and tumorigenesis. Together, these findings suggest that development of iNOS-targeted therapies may prove valuable in the treatment of glioblastoma. Here, we review our current understanding of iNOS signaling in the regulation of glioblastoma pathogenesis and the potential mechanisms by which iNOS inhibition might suppress the malignant behavior of these devastating tumors.

SnoN: bridging neurobiology and cancer biology.

The transcriptional regulator SnoN has been the subject of growing interest due to its diverse functions in normal and pathological settings. A large body of evidence has established a fundamental role for SnoN as a modulator of signaling and responses by the transforming growth beta (TGFbeta) family of cytokines, though how SnoN regulates TGFbeta responses remains incompletely understood. In accordance with the critical and complex roles of TGFbeta in tumorigenesis and metastasis, SnoN may act as a tumor promoter or suppressor depending on the stage and type of cancer. Beyond its role in cancer, SnoN has also been implicated in the control of axon morphogenesis in postmitotic neurons in the mammalian brain. Remarkably, signaling pathways that control SnoN functions in the divergent cycling cells and postmitotic neurons appear to be conserved. Identification of novel SnoN regulatory and effector mechanisms holds the promise of advances at the interface of cancer biology and neurobiology.

Signaling pathways regulating gliomagenesis.

The astrocytomas represent the most common primary tumors of the brain. Despite efforts to improve the treatment of astrocytomas, these tumors and in particular the high-grade astrocytoma termed glioblastoma multiforme still carry a poor prognosis. In recent years, there has been an intensive effort to gain an understanding of the cellular and molecular mechanisms that contribute to the pathogenesis of astrocytomas as a first step toward the development of better treatments for these devastating tumors. Here, we will review our current understanding of the signaling pathways that underlie glial transformation. Studies of astrocytomas have led to the identification of two major groups of signaling proteins whose abnormalities contribute to gliomagenesis: the cell cycle pathways and the growth factor-regulated signaling pathways. Among the cell cycle proteins, the p16-cdk4-pRb and ARF-MDM2-p53 cell cycle arrest pathways play a prominent role in glial transformation. In addition, deregulation of polypeptide growth factors acting via receptor tyrosine kinases (RTKs) and of intracellular signals, including the lipid phosphatase PTEN, that regulate cellular responses to RTKs plays a critical role in gliomagenesis. In addition to the identification of the signaling proteins targeted in glial transformation, the cell-of-origin of astrocytomas has been investigated. Genetic modeling of astrocytomas in mice suggests that neuroepithelial precursor cells represent preferred cellular substrates of gliomas or that either astrocytes or precursor cells constitute potential cells-of-origin of astrocytomas. During normal brain development, neuroepithelial precursor cells, including neural stem cells, differentiate into astrocytes. As the mechanisms that control gliogenesis during normal brain development become better understood, it will be important to determine if deregulation of these mechanisms might contribute to the pathogenesis of astrocytomas. The elucidation of the molecular underpinnings of astrocytomas holds the promise of improved treatment options for patients with these devastating brain tumors.

Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and -independent mechanisms.

A mechanism by which the Ras-mitogen-activated protein kinase (MAPK) signaling pathway mediates growth factor-dependent cell survival was characterized. The MAPK-activated kinases, the Rsks, catalyzed the phosphorylation of the pro-apoptotic protein BAD at serine 112 both in vitro and in vivo. The Rsk-induced phosphorylation of BAD at serine 112 suppressed BAD-mediated apoptosis in neurons. Rsks also are known to phosphorylate the transcription factor CREB (cAMP response element-binding protein) at serine 133. Activated CREB promoted cell survival, and inhibition of CREB phosphorylation at serine 133 triggered apoptosis. These findings suggest that the MAPK signaling pathway promotes cell survival by a dual mechanism comprising the posttranslational modification and inactivation of a component of the cell death machinery and the increased transcription of pro-survival genes.

Neuronal activity-dependent cell survival mediated by transcription factor MEF2.

During mammalian development, electrical activity promotes the calcium-dependent survival of neurons that have made appropriate synaptic connections. However, the mechanisms by which calcium mediates neuronal survival during development are not well characterized. A transcription-dependent mechanism was identified by which calcium influx into neurons promoted cell survival. The transcription factor MEF2 was selectively expressed in newly generated postmitotic neurons and was required for the survival of these neurons. Calcium influx into cerebellar granule neurons led to activation of p38 mitogen-activated protein kinase-dependent phosphorylation and activation of MEF2. Once activated, MEF2 regulated neuronal survival by stimulating MEF2-dependent gene transcription. These findings demonstrate that MEF2 is a calcium-regulated transcription factor and define a function for MEF2 during nervous system development that is distinct from previously well-characterized functions of MEF2 during muscle differentiation.

Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor.

Survival factors can suppress apoptosis in a transcription-independent manner by activating the serine/ threonine kinase Akt, which then phosphorylates and inactivates components of the apoptotic machinery, including BAD and Caspase 9. In this study, we demonstrate that Akt also regulates the activity of FKHRL1, a member of the Forkhead family of transcription factors. In the presence of survival factors, Akt phosphorylates FKHRL1, leading to FKHRL1's association with 14-3-3 proteins and FKHRL1's retention in the cytoplasm. Survival factor withdrawal leads to FKHRL1 dephosphorylation, nuclear translocation, and target gene activation. Within the nucleus, FKHRL1 triggers apoptosis most likely by inducing the expression of genes that are critical for cell death, such as the Fas ligand gene.

Neurotrophin regulation of gene expression.

The neurotrophins comprise a family of secreted proteins that elicit profound responses in cells of the developing and mature vertebrate nervous system including the regulation of neuronal survival and differentiation. The molecular mechanisms by which the neurotrophins exert their effects have been the subject of intense investigation. The neurotrophins elicit responses in neurons via members of the Trk family of receptors and the p75 neurotrophin receptor. Once activated, neurotrophin receptors trigger a large number of biochemical events that propagate the neurotrophin signal from the plasma membrane to the interior of the cell. An important target of the neurotrophin-induced signaling pathways is the nucleus, where neurotrophin-induced signals are coupled to alterations in gene expression. These neurotrophin-induced changes in gene expression are critical for many of the phenotypic effects of neurotrophins including the regulation of neuronal survival and differentiation.

Regulation of gliogenesis in the central nervous system by the JAK-STAT signaling pathway.

A mechanism by which members of the ciliary neurotrophic factor (CNTF)-leukemia inhibitory factor cytokine family regulate gliogenesis in the developing mammalian central nervous system was characterized. Activation of the CNTF receptor promoted differentiation of cerebral cortical precursor cells into astrocytes and inhibited differentiation of cortical precursors along a neuronal lineage. Although CNTF stimulated both the Janus kinase-signal transducer and activator of transcription (JAK-STAT) and Ras-mitogen-activated protein kinase signaling pathways in cortical precursor cells, the JAK-STAT signaling pathway selectively enhanced differentiation of these precursors along a glial lineage. These findings suggest that cytokine activation of the JAK-STAT signaling pathway may be a mechanism by which cell fate is controlled during mammalian development.

Interleukin 2 signaling involves the phosphorylation of Stat proteins.

One of the most important cytokines involved in immune response regulation is interleukin 2 (IL-2), a potent activator of the proliferation and function of T lymphocytes and natural killer cells. The mechanisms by which the effects of IL-2 are propagated within cells are not understood. While the binding of IL-2 to its receptor was recently shown to lead to the activation of two kinases, Jak-1 and Jak-3, subsequent steps in the signaling pathway to the nucleus that lead to the activation of specific genes had not been characterized. Since many cytokines that activate Jak kinases also lead to the tyrosine phosphorylation and activation of members of the Stat family of transcription factors, the ability of IL-2 to trigger Stat phosphorylation was examined. Exposure of activated human T lymphocytes or of a natural killer cell line (NKL) to IL-2 leads to the phosphorylation of Stat1 alpha, Stat1 beta, and Stat3, as well as of two Stat-related proteins, p94 and p95. p94 and p95 share homology with Stat1 at the phosphorylation site and in the Src homology 2 (SH2) domain, but otherwise are immunologically distinct from Stat1. These Stat proteins were found to translocate to the nucleus and to bind to a specific DNA sequence. These findings suggest a mechanism by which IL-2 binding to its receptor may activate specific genes involved in immune cell function.

Serine 133-phosphorylated CREB induces transcription via a cooperative mechanism that may confer specificity to neurotrophin signals.

A mechanism has been characterized by which the transcription factor CREB regulates neurotrophin-induced gene expression. Whereas CREB can mediate calcium- or cyclic AMP-induced c-fos transcription independently of other promoter-bound transcription factors, CREB mediates NGF induction of c-fos transcription via a novel mechanism that appears to require a cooperative interaction with another transcription factor, the serum response factor. A similar transcriptional mechanism may explain how neurotrophins and growth factors induce distinct subsets of delayed response genes. Neurotrophins induce the phosphorylation of CREB at a key regulatory site, Serine 133, with prolonged kinetics that are distinct from the transient kinetics of CREB phosphorylation elicited by growth factors. These results indicate that CREB is a versatile transcription factor that activates transcription via distinct mechanisms in a stimulus-specific manner. In addition, by selectively activating delayed response genes, CREB may confer specificity to neurotrophin signals that promote the survival and differentiation of neurons.

L-type voltage-sensitive Ca2+ channel activation regulates c-fos transcription at multiple levels.

A mechanism by which voltage-sensitive Ca2+ channel (VSCC) activation triggers c-fos transcription has been characterized. Ca2+ influx through VSCCs stimulates phosphorylation of the transcription factor cAMP response element-binding protein (CREB) on serine 133 leading to an increase in the formation of transcription complexes that can elongate through a transcription pause site within the c-fos gene. Ca(2+)-stimulated CREB serine 133 phosphorylation is mediated by a Ca(2+)-activated kinase and is not dependent on the cAMP-dependent protein kinase (PKA). While necessary for c-fos transcriptional induction following VSCC opening, CREB serine 133 phosphorylation is not sufficient for transcriptional activation. A second, PKA-dependent event is required. Following induction, c-fos transcription is rapidly down-regulated. Dephosphorylation of CREB serine 133 parallels and likely mediates the transcriptional shut-off event. These results suggest that the phosphorylation and dephosphorylation of CREB controls its ability to regulate transcription in membrane-depolarized cells and that multiple pathways contribute to Ca(2+)-activated gene expression.

L-type voltage-sensitive calcium channel activation stimulates gene expression by a serum response factor-dependent pathway.

A mechanism by which calcium-induced signals are transduced to the nucleus to activate transcription of the c-fos proto-oncogene has been characterized. The serum response element (SRE), a region of the c-fos gene which controls growth factor-induced transcription, is now shown to mediate c-fos transcription in response to activation of L-type voltage-sensitive calcium channels. Calcium-dependent transcriptional activation through the SRE is mediated by the serum response factor (SRF). Membrane depolarization induces phosphorylation of SRF at Ser-103, an event shown to enhance the ability of SRF to bind the SRE. Ca(2+)-induced SRF phosphorylation occurs via a pathway that may involve Ca2+/calmodulin-dependent kinases.

Nerve growth factor activates a Ras-dependent protein kinase that stimulates c-fos transcription via phosphorylation of CREB.

A mechanism by which the nerve growth factor (NGF) signal is transduced to the nucleus to induce gene expression has been characterized. An NGF-inducible, Ras-dependent protein kinase has been identified that catalyzes the phosphorylation of the cyclic AMP response element-binding protein (CREB) at Ser-133. Phosphorylation of Ser-133 stimulates the ability of CREB to activate transcription in NGF-treated cells. These findings suggest that CREB has a more widespread function than previously believed and functions in the nucleus as a general mediator of growth factor responses.

Characterization of a pathway for ciliary neurotrophic factor signaling to the nucleus.

Components of a signaling pathway that couples the ciliary neurotrophic factor (CNTF) receptor to induction of transcription were identified. CNTF stimulated the tyrosine phosphorylation of p91, a protein implicated in interferon signaling pathways, and of two proteins that are distinct but related to p91. Tyrosine-phosphorylated p91 translocated to the nucleus, where p91 and p91-related proteins bound to a DNA sequence found in promoters of genes responsive to CNTF. This DNA sequence, when inserted upstream of a reporter gene, conferred a transcriptional response to CNTF. A pathway that transduces interferon signals may therefore have a more general function in the propagation of responses to certain neurotrophic factors.

Metachromatic leukodystrophy: multiple nonfunctional and pseudodeficiency alleles in a pedigree: problems with diagnosis and counseling.

Metachromatic leukodystrophy is due to deficient activity of arylsulfatase A, an enzyme important in myelin catabolism. The deficiency can be caused by different point mutations in the gene coding for arylsulfatase A (nonfunctional alleles). In addition, certain mutations result in low levels of enzyme activity detectable with artificial substrates in vitro but no clinical dysfunction (pseudodeficiency alleles). The described family has various combinations of normal, nonfunctional, and pseudodeficiency alleles that presented diagnostic and counseling dilemmas which were resolved at the genomic level. We find no evidence that compound heterozygote individuals have subclinical involvement of the nervous system. We report the clinical, pathological, electrophysiological, imaging, biochemical, and genetic data of this family and discuss the difficulties in analyzing such pedigrees.

Magnetic resonance imaging in the diagnosis of dominantly inherited cerebello-olivary atrophy: a clinicopathologic study.

To facilitate the study of cerebellar degenerative disorders, improved clinical diagnosis is needed. Cerebello-olivary atrophy is pathologically distinct, but until now its diagnosis has been thought to require postmortem examination. This condition was considered as a possible diagnosis in two patients from different families with dominantly inherited ataxia. The affected members of each family demonstrated a stereotyped, progressive, "pure" cerebellar syndrome, which began with gait ataxia followed years later by dysarthria and limb ataxia. The autopsy findings for the first patient's father revealed paleocerebellar and olivary atrophy, characteristic of cerebello-olivary atrophy. Magnetic resonance imaging (MRI) of the brain of both patients revealed medullary, vermian and, to a lesser extent, cerebellar hemispheric atrophy but a normal pons. Dominantly inherited cerebello-olivary atrophy was diagnosed in both patients. Characteristic clinical and MRI features thus permit a confident clinical diagnosis of dominantly inherited cerebello-olivary atrophy. Recognition of this entity during life should advance the classification of cerebellar degenerative disorders.