Histone acetylation in astrocytes suppresses GFAP and stimulates a reorganization of the intermediate filament network.

Glial fibrillary acidic protein (GFAP) is the main intermediate filament in astrocytes and is regulated by epigenetic mechanisms during development. We demonstrate that histone acetylation also controls GFAP expression in mature astrocytes. Inhibition of histone deacetylases (HDACs) with trichostatin A or sodium butyrate reduced GFAP expression in primary human astrocytes and astrocytoma cells. Because splicing occurs co-transcriptionally, we investigated whether histone acetylation changes the ratio between the canonical isoform GFAPα and the alternative GFAPδ splice variant. We observed that decreased transcription of GFAP enhanced alternative isoform expression, as HDAC inhibition increased the GFAPδ∶GFAPα ratio. Expression of GFAPδ was dependent on the presence and binding of splicing factors of the SR protein family. Inhibition of HDAC activity also resulted in aggregation of the GFAP network, reminiscent of our previous findings of a GFAPδ-induced network collapse. Taken together, our data demonstrate that HDAC inhibition results in changes in transcription, splicing and organization of GFAP. These data imply that a tight regulation of histone acetylation in astrocytes is essential, because dysregulation of gene expression causes the aggregation of GFAP, a hallmark of human diseases like Alexander's disease.

Silencing GFAP isoforms in astrocytoma cells disturbs laminin-dependent motility and cell adhesion.

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein expressed in astrocytes and neural stem cells. The GFAP gene is alternatively spliced, and expression of GFAP is highly regulated during development, on brain damage, and in neurodegenerative diseases. GFAPα is the canonical splice variant and is expressed in all GFAP-positive cells. In the human brain, the alternatively spliced transcript GFAPδ marks specialized astrocyte populations, such as subpial astrocytes and the neurogenic astrocytes in the human subventricular zone. We here show that shifting the GFAP isoform ratio in favor of GFAPδ in astrocytoma cells, by selectively silencing the canonical isoform GFAPα with short hairpin RNAs, induced a change in integrins, a decrease in plectin, and an increase in expression of the extracellular matrix component laminin. Together, this did not affect cell proliferation but resulted in a significantly decreased motility of astrocytoma cells. In contrast, a down-regulation of all GFAP isoforms led to less cell spreading, increased integrin expression, and a >100-fold difference in the adhesion of astrocytoma cells to laminin. In summary, isoform-specific silencing of GFAP revealed distinct roles of a specialized GFAP network in regulating the interaction of astrocytoma cells with the extracellular matrix through laminin.-Moeton, M., Kanski, R., Stassen, O. M. J. A., Sluijs, J. A., Geerts, D., van Tijn, P., Wiche, G., van Strien, M. E., Hol, E. M. Silencing GFAP isoforms in astrocytoma cells disturbs laminin dependent motility and cell adhesion.

A star is born: new insights into the mechanism of astrogenesis.

Astrocytes emerge as crucial cells for proper neuronal functioning in the developing and adult brain. Neurons and astrocytes are sequentially generated from the same pool of neural stem cells (NSCs). Tight regulation of the neuron-to-astrocyte switch is critical for (1) the generation of a balanced number of astrocytes and neurons and (2) neuronal circuit formation, since newborn astrocytes regulate synapse formation. This review focuses on signaling pathways that instruct astrogenesis, incorporating recently discovered intrinsic and extrinsic regulators. The canonical pathway of astrocytic gene expression, JAK/STAT signaling, is inhibited during neurogenesis to prevent premature astrocyte differentiation. At the onset of astrogenesis, Notch signaling induces epigenetic remodeling of astrocytic genes like glial fibrillary acidic protein to change NSC competence. In turn, astrogenesis is initiated by signals received from newborn neurons. We highlight how key molecular pathways like JAK/STAT and Notch are integrated in a complex network of environmental signals and epigenetic and transcriptional regulators to determine NSC differentiation. It is essential to understand NSC differentiation in respect to future NSC-based therapies for brain diseases, as transplanted NSCs preferentially become astrocytes. As emphasized in this review, many clues in this respect can be learned from development.

Mutant ubiquitin decreases amyloid ß plaque formation in a transgenic mouse model of Alzheimer's disease.

The mutant ubiquitin UBB(+1) is a substrate as well as an inhibitor of the ubiquitin-proteasome system (UPS) and accumulates in the neuropathological hallmarks of Alzheimer's disease (AD). A role for the UPS has been suggested in the generation of amyloid ß (Aß) plaques in AD. To investigate the effect of UBB(+1) expression on amyloid pathology in vivo, we crossed UBB(+1) transgenic mice with a transgenic line expressing AD-associated mutant amyloid precursor protein (APPSwe) and mutant presenilin 1 (PS1dE9), resulting in APPPS1/UBB(+1) triple transgenic mice. In these mice, we determined the Aß levels at 3, 6, 9 and 11 months of age. Surprisingly, we found a significant decrease in Aß deposition in amyloid plaques and levels of soluble Aß(42) in APPPS1/UBB(+1) transgenic mice compared to APPPS1 mice at 6 months of age, without alterations in UBB(+1) protein levels or proteasomal chymotrypsin activity. These lowering effects of UBB(+1) on Aß deposition were transient, as this relative decrease in plaque load was not significant in APPPS1/UBB(+1) mice at 9 and 11 months of age. We also show that APPPS1/UBB(+1) mice exhibit astrogliosis, indicating that they may not be improved functionally compared to APPPS1 mice despite the Aß reduction. The molecular mechanism underlying this decrease in Aß deposition in APPPS1/UBB(+1) mice is more complex than previously assumed because UBB(+1) is also ubiquitinated at K63 opening the possibility of additional effects of UBB(+1) (e.g. kinase activation).

asb11 is a regulator of embryonic and adult regenerative myogenesis.

The specific molecular determinants that govern progenitor expansion and final compartment size in the myogenic lineage, either during gestation or during regenerative myogenesis, remain largely obscure. Recently, we retrieved d-asb11 from a zebrafish screen designed to identify gene products that are downregulated during embryogenesis upon terminal differentiation and identified it as a potential regulator of compartment size in the ectodermal lineage. A role in mesodermal derivatives remained, however, unexplored. Here we report pan-vertebrate expression of Asb11 in muscle compartments, where it highly specifically localizes to the Pax7(+) muscle satellite cell compartment. Forced expression of d-asb11 impaired terminal differentiation and caused enhanced proliferation in the myogenic progenitor compartment both in in vivo and in vitro model systems. Conversely, introduction of a germline hypomorphic mutation in the zebrafish d-asb11 gene produced premature differentiation of the muscle progenitors and delayed regenerative responses in adult injured muscle. Thus, the expression of d-asb11 is necessary for muscle progenitor expansion, whereas its downregulation marks the onset of terminal differentiation. Hence, we provide evidence that d-asb11 is a principal regulator of embryonic as well as adult regenerative myogenesis.

Impairment of the tRNA-splicing endonuclease subunit 54 (tsen54) gene causes neurological abnormalities and larval death in zebrafish models of pontocerebellar hypoplasia.

Pontocerebellar hypoplasia (PCH) represents a group (PCH1-6) of neurodegenerative autosomal recessive disorders characterized by hypoplasia and/or atrophy of the cerebellum, hypoplasia of the ventral pons, progressive microcephaly and variable neocortical atrophy. The majority of PCH2 and PCH4 cases are caused by mutations in the TSEN54 gene; one of the four subunits comprising the tRNA-splicing endonuclease (TSEN) complex. We hypothesized that TSEN54 mutations act through a loss of function mechanism. At 8 weeks of gestation, human TSEN54 is expressed ubiquitously in the brain, yet strong expression is seen within the telencephalon and metencephalon. Comparable expression patterns for tsen54 are observed in zebrafish embryos. Morpholino (MO) knockdown of tsen54 in zebrafish embryos results in loss of structural definition in the brain. This phenotype was partially rescued by co-injecting the MO with human TSEN54 mRNA. A developmental patterning defect was not associated with tsen54 knockdown; however, an increase in cell death within the brain was observed, thus bearing resemblance to PCH pathophysiology. Additionally, N-methyl-N-nitrosourea mutant zebrafish homozygous for a tsen54 premature stop-codon mutation die within 9 days post-fertilization. To determine whether a common disease pathway exists between TSEN54 and other PCH-related genes, we also monitored the effects of mitochondrial arginyl-tRNA synthetase (rars2; PCH1 and PCH6) knockdown in zebrafish. Comparable brain phenotypes were observed following the inhibition of both genes. These data strongly support the hypothesis that TSEN54 mutations cause PCH through a loss of function mechanism. Also we suggest that a common disease pathway may exist between TSEN54- and RARS2-related PCH, which may involve a tRNA processing-related mechanism.

Alzheimer-associated mutant ubiquitin impairs spatial reference memory.

UBB(+1) is a mutant ubiquitin which accumulates in the hallmarks of tauopathies, including Alzheimer's disease. Transgenic mice expressing high levels of neuronal UBB(+1) exhibit moderately decreased proteasome activity and spatial reference memory deficits at 9months of age. In the present study, we characterized the behavioral phenotype of male UBB(+1) transgenic mice at different ages. We show that UBB(+1) transgenic mice displayed an age-related functional decline similar to wild-type littermates, without gross neurological abnormalities or alterations in procedural motor-learning and motor coordination. At 15months of age, a transgene-specific spatial learning deficit was dependent on the period of training in the Morris watermaze. This deficit could be eliminated after additional training. We conclude that the previously reported spatial reference memory deficits of UBB(+1) transgenic mice persist during aging. In addition, our results demonstrate that the subtle defect in spatial reference memory formation, caused by a decrease in forebrain proteasome activity, is a persistent defect and not a structural defect.

Presenilin mouse and zebrafish models for dementia: focus on neurogenesis.

Autosomal dominant mutations in the presenilin gene PSEN cause familial Alzheimer's disease (AD), a neurological disorder pathologically characterized by intraneuronal accumulation and extracellular deposition of amyloid-ß in plaques and intraneuronal, hyperphosphorylated tau aggregation in neurofibrillary tangles. Presenilins (PS/PSENs) are part of the proteolytic γ-secretase complex, which cleaves substrate proteins within the membrane. Cleavage of the amyloid precursor protein (APP) by γ-secretase releases amyloid-ß peptides. Besides its role in the processing of APP and other transmembrane proteins, presenilin plays an important role in neural progenitor cell maintenance and neurogenesis. In this review, we discuss the role of presenilin in relation to neurogenesis and neurodegeneration and review the currently available presenilin animal models. In addition to established mouse models, zebrafish are emerging as an attractive vertebrate model organism to study the role of presenilin during the development of the nervous system and in neurodegenerative disorders involving presenilin. Zebrafish is a suitable model organism for large-scale drug screening, making this a valuable model to identify novel therapeutic targets for AD.

Low levels of mutant ubiquitin are degraded by the proteasome in vivo.

The ubiquitin-proteasome system fulfills a pivotal role in regulating intracellular protein turnover. Impairment of this system is implicated in the pathogenesis of neurodegenerative diseases characterized by ubiquitin- containing proteinaceous deposits. UBB(+1), a mutant ubiquitin, is one of the proteins accumulating in the neuropathological hallmarks of tauopathies, including Alzheimer's disease, and polyglutamine diseases. In vitro, UBB(+1) properties shift from a proteasomal ubiquitin-fusion degradation substrate at low expression levels to a proteasome inhibitor at high expression levels. Here we report on a novel transgenic mouse line (line 6663) expressing low levels of neuronal UBB(+1). In these mice, UBB(+1) protein is scarcely detectable in the neuronal cell population. Accumulation of UBB(+1) commences only after intracranial infusion of the proteasome inhibitors lactacystin or MG262, showing that, at these low expression levels, the UBB(+1) protein is a substrate for proteasomal degradation in vivo. In addition, accumulation of the protein serves as a reporter for proteasome inhibition. These findings strengthen our proposition that, in healthy brain, UBB(+1) is continuously degraded and disease-related UBB(+1) accumulation serves as an endogenous marker for proteasomal dysfunction. This novel transgenic line can give more insight into the intrinsic properties of UBB(+1) and its role in neurodegenerative disease.

Modest proteasomal inhibition by aberrant ubiquitin exacerbates aggregate formation in a Huntington disease mouse model.

UBB(+1), a mutant form of ubiquitin, is both a substrate and an inhibitor of the proteasome which accumulates in the neuropathological hallmarks of Huntington disease (HD). In vitro, expression of UBB(+1) and mutant huntingtin synergistically increase aggregate formation and polyglutamine induced cell death. We generated a UBB(+1) transgenic mouse line expressing UBB(+1) within the neurons of the striatum. In these mice lentiviral driven expression of expanded huntingtin constructs in the striatum results in a significant increase in neuronal inclusion formation. Although UBB(+1) transgenic mice show neither a decreased lifespan nor apparent neuronal loss, they appear to be more vulnerable to toxic insults like expanded polyglutamine proteins due to a modest proteasome inhibition. These findings underscore the relevance of an efficient ubiquitin-proteasome system in HD.

Intermediate filament transcription in astrocytes is repressed by proteasome inhibition.

Increased expression of the astrocytic intermediate filament protein glial fibrillary acidic protein (GFAP) is a characteristic of astrogliosis. This process occurs in the brain during aging and neurodegeneration and coincides with impairment of the ubiquitin proteasome system. Inhibition of the proteasome impairs protein degradation; therefore, we hypothesized that the increase in GFAP may be the result of impaired proteasomal activity in astrocytes. We investigated the effect of proteasome inhibitors on GFAP expression and other intermediate filament proteins in human astrocytoma cells and in a rat brain model for astrogliosis. Extensive quantitative RT-PCR, immunocytochemistry, and Western blot analysis resulted unexpectedly in a strong decrease of GFAP mRNA to <4% of control levels [Control (DMSO) 100+/-19.2%; proteasome inhibitor (epoxomicin) 3.5+/-1.3%, n=8; P < or = 0.001] and a loss of GFAP protein in astrocytes in vitro. We show that the proteasome alters GFAP promoter activity, possibly mediated by transcription factors as demonstrated by a GFAP promoter-luciferase assay and RT(2) Profiler PCR array for human transcription factors. Most important, we demonstrate that proteasome inhibitors also reduce GFAP and vimentin expression in a rat model for induced astrogliosis in vivo. Therefore, proteasome inhibitors could serve as a potential therapy to modulate astrogliosis associated with CNS injuries and disease.

Long-term proteasome dysfunction in the mouse brain by expression of aberrant ubiquitin.

Many neurodegenerative diseases are characterized by deposits of ubiquitinated and aberrant proteins, suggesting a failure of the ubiquitin-proteasome system (UPS). The aberrant ubiquitin UBB(+1) is one of the ubiquitinated proteins accumulating in tauopathies such as Alzheimer's disease (AD) and polyglutamine diseases such as Huntington's disease. We have generated UBB(+1) transgenic mouse lines with post-natal neuronal expression of UBB(+1), resulting in increased levels of ubiquitinated proteins in the cortex. Moreover, by proteomic analysis, we identified expression changes in proteins involved in energy metabolism or organization of the cytoskeleton. These changes show a striking resemblance to the proteomic profiles of both AD brain and several AD mouse models. Moreover, UBB(+1) transgenic mice show a deficit in contextual memory in both water maze and fear conditioning paradigms. Although UBB(+1) partially inhibits the UPS in the cortex, these mice do not have an overt neurological phenotype. These mouse models do not replicate the full spectrum of AD-related changes, yet provide a tool to understand how the UPS is involved in AD pathological changes and in memory formation.

Wnt signaling in Alzheimer's disease: up or down, that is the question.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder, neuropathologically characterized by amyloid-beta (Abeta) plaques and hyperphosphorylated tau accumulation. AD occurs sporadically (SAD), or is caused by hereditary missense mutations in the amyloid precursor protein (APP) or presenilin-1 and -2 (PSEN1 and PSEN2) genes, leading to early-onset familial AD (FAD). Accumulating evidence points towards a role for altered Wnt/beta-catenin-dependent signaling in the etiology of both forms of AD. Presenilins are involved in modulating beta-catenin stability; therefore FAD-linked PSEN-mediated effects can deregulate the Wnt pathway. Genetic variations in the low-density lipoprotein receptor-related protein 6 and apolipoprotein E in AD have been associated with reduced Wnt signaling. In addition, tau phosphorylation is mediated by glycogen synthase kinase-3 (GSK-3), a key antagonist of the Wnt pathway. In this review, we discuss Wnt/beta-catenin signaling in both SAD and FAD, and recapitulate which of its aberrant functions may be critical for (F)AD pathogenesis. We discuss the intriguing possibility that Abeta toxicity may downregulate the Wnt/beta-catenin pathway, thereby upregulating GSK-3 and consequent tau hyperphosphorylation, linking Abeta and tangle pathology. The currently available evidence implies that disruption of tightly regulated Wnt signaling may constitute a key pathological event in AD. In this context, drug targets aimed at rescuing Wnt signaling may prove to be a constructive therapeutic strategy for AD.

The neuronal ubiquitin-proteasome system: murine models and their neurological phenotype.

The ubiquitin-proteasome system (UPS) is the main intracellular pathway for regulated protein turnover. This system is of vital importance for maintaining cellular homeostasis and is essential for neuronal functioning. It is therefore not surprising that impairment of this system is implicated in the pathogenesis of a variety of diseases, including neurological disorders, which are pathologically characterized by the presence of ubiquitin-positive protein aggregates. A direct correlation between intact neuronal functioning and the UPS is exemplified by a range of transgenic mouse models wherein mutations in components of the UPS lead to a neurodegenerative or neurological phenotype. These models have been proven useful in determining the role of the UPS in the nervous system in health and disease. Furthermore, recently developed in vivo models harboring reporter systems to measure UPS activity could also substantially contribute to understanding the effect of neurodegeneration on UPS function. The role of the UPS in neurodegeneration in vivo is reviewed by discussing the currently available murine models showing a neurological phenotype induced by genetic manipulation of the UPS.

Dose-dependent inhibition of proteasome activity by a mutant ubiquitin associated with neurodegenerative disease.

The ubiquitin-proteasome system is the main regulated intracellular proteolytic pathway. Increasing evidence implicates impairment of this system in the pathogenesis of diseases with ubiquitin-positive pathology. A mutant ubiquitin, UBB(+1), accumulates in the pathological hallmarks of tauopathies, including Alzheimer's disease, polyglutamine diseases, liver disease and muscle disease and serves as an endogenous reporter for proteasomal dysfunction in these diseases. UBB(+1) is a substrate for proteasomal degradation, however it can also inhibit the proteasome. Here, we show that UBB(+1) properties shift from substrate to inhibitor in a dose-dependent manner in cell culture using an inducible UBB(+1) expression system. At low expression levels, UBB(+1) was efficiently degraded by the proteasome. At high levels, the proteasome failed to degrade UBB(+1), causing its accumulation, which subsequently induced a reversible functional impairment of the ubiquitin-proteasome system. Also in brain slice cultures, UBB(+1) accumulation and concomitant proteasome inhibition was only induced at high expression levels. Our findings show that by varying UBB(+1) expression levels, the dual proteasome substrate and inhibitory properties can be optimally used to serve as a research tool to study the ubiquitin-proteasome system and to further elucidate the role of aberrations of this pathway in disease.