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Pericytes are vascular mural cells that surround capillaries of the central nervous system (CNS). They are crucial for brain development and contribute to CNS homeostasis by regulating blood–brain barrier function and cerebral blood flow. It has been suggested that pericytes are lost in Alzheimer's disease (AD), implicating this cell type in disease pathology. Here, we have employed state‐of‐the‐art stereological morphometry techniques as well as tissue clearing and two‐photon imaging to assess the distribution of pericytes in two independent cohorts of AD (n = 16 and 13) and non‐demented controls (n = 16 and 4). Stereological quantification revealed increased capillary density with a normal pericyte population in the frontal cortex of AD brains, a region with early amyloid β deposition. Two‐photon analysis of cleared frontal cortex tissue confirmed the preservation of pericytes in AD cases. These results suggest that pericyte demise is not a general hallmark of AD pathology.

Dysregulation of microglial function contributes to Alzheimer’s disease (AD) pathogenesis. Several genetic and transcriptome studies have revealed microglia specific genetic risk factors, and changes in microglia expression profiles in AD pathogenesis, viz. the human-Alzheimer’s microglia/myeloid (HAM) profile in AD patients and the disease-associated microglia profile (DAM) in AD mouse models. The transcriptional changes involve genes in immune and inflammatory pathways, and in pathways associated with Aβ clearance. Aβ oligomers have been suggested to be the initial trigger of microglia activation in AD. To study the direct response to Aβ oligomers exposure, we assessed changes in gene expression in an in vitro model for microglia, the human monocyte-derived microglial-like (MDMi) cells. We confirmed the initiation of an inflammatory profile following LPS stimulation, based on increased expression of IL1B, IL6, and TNFα. In contrast, the Aβ1-42 oligomers did not induce an inflammatory profile or a classical HAM or DAM profile. Interestingly, we observed a specific increase in the expression of metallothioneins in the Aβ1-42 oligomer treated MDMi cells. Metallothioneins are involved in metal ion regulation, protection against reactive oxygen species, and have anti-inflammatory properties. In conclusion, our data suggests that Aβ1-42 oligomers may trigger a protective response both in vitro and in vivo.

Dysregulation of microglial function contributes to Alzheimer's disease (AD) pathogenesis. Several genetic and transcriptome studies have revealed microglia specific genetic risk factors, and changes in microglia expression profiles in AD pathogenesis, viz. the human-Alzheimer's microglia/myeloid (HAM) profile in AD patients and the disease-associated microglia profile (DAM) in AD mouse models. The transcriptional changes involve genes in immune and inflammatory pathways, and in pathways associated with Aβ clearance. Aβ oligomers have been suggested to be the initial trigger of microglia activation in AD. To study the direct response to Aβ oligomers exposure, we assessed changes in gene expression in an in vitro model for microglia, the human monocyte-derived microglial-like (MDMi) cells. We confirmed the initiation of an inflammatory profile following LPS stimulation, based on increased expression of IL1B, IL6, and TNFα. In contrast, the Aβ

Gliomas are the most common primary brain tumors. Their highly invasive character and the heterogeneity of active oncogenic pathways within single tumors complicate the development of curative therapies and cause poor patient prognosis. Glioma cells express the intermediate filament protein glial fibrillary acidic protein (GFAP), and the level of its alternative splice variant GFAP-δ, relative to its canonical splice variant GFAP-α, is higher in grade IV compared with lower-grade and lower malignant glioma. In this study we show that a high GFAP-δ/α ratio induces the expression of the dual-specificity phosphatase 4 (DUSP4) in focal adhesions. By focusing on pathways up- and downstream of DUSP4 that are involved in the cell-extracellular matrix interaction, we show that a high GFAP-δ/α ratio equips glioma cells to better invade the brain. This study supports the hypothesis that glioma cells with a high GFAP-δ/α ratio are highly invasive and more malignant cells, thus making GFAP alternative splicing a potential therapeutic target.-Van Bodegraven, E. J., van Asperen, J. V., Sluijs, J. A., van Deursen, C. B. J., van Strien, M. E., Stassen, O. M. J. A., Robe, P. A. J., Hol, E. M. GFAP alternative splicing regulates glioma cell-ECM interaction in a DUSP4-dependent manner.

The glial fibrillary acidic protein (GFAP) is a type III intermediate filament (IF) protein that is highly expressed in astrocytes, neural stem cells, and in gliomas. Gliomas are a heterogeneous group of primary brain tumors that arise from glia cells or neural stem cells and rely on accurate diagnosis for prognosis and treatment strategies. GFAP is differentially expressed between glioma subtypes and, therefore, often used as a diagnostic marker. However, GFAP is highly regulated by the process of alternative splicing; many different isoforms have been identified. Differential expression of GFAP isoforms between glioma subtypes suggests that GFAP isoform-specific analyses could benefit diagnostics. In this study we report on the differential expression of a new GFAP isoform between glioma subtypes, GFAPµ. A short GFAP transcript resulting from GFAP exon 2 skipping was detected by RNA sequencing of human glioma. We show that GFAPµ mRNA is expressed in healthy brain tissue, glioma cell lines, and primary glioma cells and that it translates into a ~21 kDa GFAP protein. 21 kDa GFAP protein was detected in the IF protein fraction isolated from human spinal cord as well. We further show that induced GFAPµ expression disrupts the GFAP IF network. The characterization of this new GFAP isoform adds on to the numerous previously identified GFAP splice isoforms. It emphasizes the importance of studying the contribution of IF splice variants to specialized functions of the IF network and to glioma research.

Following the decline of neurogenesis at birth, progenitors of the subventricular zone (SVZ) remain mostly in a quiescent state in the adult human brain. The mechanisms that regulate this quiescent state are still unclear. Here, we isolate CD271

The glial fibrillary acidic protein (GFAP) is a type III intermediate filament (IF) protein that is highly expressed in astrocytes, neural stem cells, and in gliomas. Gliomas are a heterogeneous group of primary brain tumors that arise from glia cells or neural stem cells and rely on accurate diagnosis for prognosis and treatment strategies. GFAP is differentially expressed between glioma subtypes and therefore often used as a diagnostic marker. However, GFAP is highly regulated by the process of alternative splicing; many different isoforms have been identified. Differential expression of GFAP isoforms between glioma subtypes suggests that GFAP isoform-specific analyses could benefit diagnostics. In this study we report on the differential expression of a new GFAP isoform between glioma subtypes, GFAPμ. A short GFAP transcript resulting from GFAP exon 2 skipping was detected by RNA sequencing of human glioma. We show that GFAPμ mRNA is expressed in healthy brain tissue, glioma cell lines, and primary glioma cells and that it translates into a ~21 kDa GFAP protein. 21 kDa GFAP protein was detected in the IF protein fraction isolated from human spinal cord as well. We further show that induced GFAPμ expression disrupts the GFAP IF network. The characterization of this new GFAP isoform adds on to the numerous previously identified GFAP splice isoforms. It emphasizes the importance of studying the contribution of IF splice variants to specialized functions of the IF network and to glioma diagnostics.Summary statementRNA sequencing data of glioma patient material reveals the differential expression of a short GFAP splice isoform, GFAPμ, between glioma subtypes. GFAPμ translates into a 21 kDa N-terminal GFAP protein which can disrupt the GFAP intermediate filament network.

Neurogenesis continues throughout adulthood in specialized regions of the brain. One of these regions is the subventricular zone. During brain development, neurogenesis is regulated by a complex interplay of intrinsic and extrinsic cues that control stem-cell survival, renewal and cell lineage specification. Cerebrospinal fluid (CSF) is an integral part of the neurogenic niche in development as it is in direct contact with radial glial cells, and it is important in regulating proliferation and migration. Yet, the effect of CSF on neural stem cells in the subventricular zone of the adult human brain is unknown. We hypothesized a persistent stimulating effect of ventricular CSF on neural stem cells in adulthood, based on the literature, describing bulging accumulations of subventricular cells where CSF is in direct contact with the subventricular zone. Here, we show by immunohistochemistry on post-mortem adult human subventricular zone sections that neural stem cells are in close contact with CSF via protrusions through both intact and incomplete ependymal layers. We are the first to systematically quantify subventricular glial nodules denuded of ependyma and consisting of proliferating neural stem and progenitor cells, and showed that they are present from foetal age until adulthood. Neurosphere, cell motility and differentiation assays as well as analyses of RNA expression were used to assess the effects of CSF of adult humans on primary neural stem cells and a human immortalized neural stem cell line. We show that human ventricular CSF increases proliferation and decreases motility of neural stem cells. Our results also indicate that adult CSF pushes neural stem cells from a relative quiescent to a more active state and promotes neuronal over astrocytic lineage differentiation. Thus, CSF continues to stimulate neural stem cells throughout aging., De Sonnaville et al. demonstrate that subventricular glial nodules in the human subventricular zone—denuded of ependyma—consist of accumulating proliferating neural stem and progenitor cells, corroborating in vitro findings that adult cerebrospinal fluid retains the capacity to stimulate proliferation, influence motility and promote differentiation of neural stem cells., Graphical Abstract Graphical Abstract

Astrocytomas are the most common malignant brain tumours and are to date incurable. It is unclear how astrocytomas progress into higher malignant grades. The intermediate filament cytoskeleton is emerging as an important regulator of malignancy in several tumours. The majority of the astrocytomas express the intermediate filament protein Glial Fibrillary Acidic Protein (GFAP). Several GFAP splice variants have been identified and the main variants expressed in human astrocytoma are the GFAPα and GFAPδ isoforms. Here we show a significant downregulation of GFAPα in grade IV astrocytoma compared to grade II and III, resulting in an increased GFAPδ/α ratio. Mimicking this increase in GFAPδ/α ratio in astrocytoma cell lines and comparing the subsequent transcriptomic changes with the changes in the patient tumours, we have identified a set of GFAPδ/α ratio-regulated high-malignant and low-malignant genes. These genes are involved in cell proliferation and protein phosphorylation, and their expression correlated with patient survival. We additionally show that changing the ratio of GFAPδ/α, by targeting GFAP expression, affected expression of high-malignant genes. Our data imply that regulating GFAP expression and splicing are novel therapeutic targets that need to be considered as a treatment for astrocytoma.

Glial fibrillary acidic protein (GFAP) is the characteristic intermediate filament (IF) protein in astrocytes. Expression of its main isoforms, GFAPα and GFAPδ, varies in astrocytes and astrocytoma implying a potential regulatory role in astrocyte physiology and pathology. An IF-network is a dynamic structure and has been functionally linked to cell motility, proliferation, and morphology. There is a constant exchange of IF-proteins with the network. To study differences in the dynamic properties of GFAPα and GFAPδ, we performed fluorescence recovery after photobleaching experiments on astrocytoma cells with fluorescently tagged GFAPs. Here, we show for the first time that the exchange of GFP–GFAPδ was significantly slower than the exchange of GFP–GFAPα with the IF-network. Furthermore, a collapsed IF-network, induced by GFAPδ expression, led to a further decrease in fluorescence recovery of both GFP–GFAPα and GFP–GFAPδ. This altered IF-network also changed cell morphology and the focal adhesion size, but did not alter cell migration or proliferation. Our study provides further insight into the modulation of the dynamic properties and functional consequences of the IF-network composition. Electronic supplementary material The online version of this article (doi:10.1007/s00018-016-2239-5) contains supplementary material, which is available to authorized users.

Aicardi–Goutières syndrome (AGS) is a monogenic inflammatory encephalopathy caused by mutations in TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, ADAR1, or MDA5. Mutations in those genes affect normal RNA/DNA intracellular metabolism and detection, triggering an autoimmune response with an increase in cerebral IFN-α production by astrocytes. Microangiopathy and vascular disease also contribute to the neuropathology in AGS. In this study, we report that AGS gene silencing of TREX1, SAMHD1, RNASEH2A, and ADAR1 by short hairpin RNAs in human neural stem cell–derived astrocytes, human primary astrocytes, and brain-derived endothelial cells leads to an antiviral status of these cells compared with nontarget short hairpin RNA–treated cells. We observed a distinct activation of the IFN-stimulated gene signature with a substantial increase in the release of proinflammatory cytokines (IL-6) and chemokines (CXCL10 and CCL5). A differential impact of AGS gene silencing was noted; silencing TREX1 gave rise to the most dramatic in both cell types. Our findings fit well with the observation that patients carrying mutations in TREX1 experience an earlier onset and fatal outcome. We provide in the present study, to our knowledge for the first time, insight into how astrocytic and endothelial activation of antiviral status may differentially lead to cerebral pathology, suggesting a rational link between proinflammatory mediators and disease severity in AGS.

Neural progenitor cells (NPCs) in the subventricular zone (SVZ) hold promise for future therapy for neurodegenerative disorders, because the stimulation of adult neurogenesis could potentially restore the function of degenerating neurons and glia. To obtain more knowledge on these NPCs, we developed a method to specifically isolate NPCs from postmortem adult human brains based on the expression of the specific human adult neural stem/progenitor cell marker glial fibrillary acidic protein δ (GFAPδ). An extensive immunophenotyping analysis for cell surface markers resulted in the observation that CD271 was limited to the SVZ-derived GFAPδ-positive cells. CD271+ cells developed into neurospheres and could be differentiated into astrocytes, neurons, and oligodendrocytes. We are the first to show that a pure population of NPCs can be isolated from the adult human SVZ, which is highly instrumental for developing future therapies based on stimulating endogenous SVZ neurogenesis.

ObjectiveTo report the clinical and immunological characteristics of 22 new patients with glial fibrillar acidic protein (GFAP) autoantibodies.MethodsFrom January 2012 to March 2017, we recruited 451 patients with suspected neurological autoimmune disease at the Catholic University of Rome. Patients’ serum and cerebrospinal fluid (CSF) samples were tested for neural autoantibodies by immunohistochemistry on mouse and rat brain sections, by cell-based assays (CBA) and immunoblot. GFAP autoantibodies were detected by immunohistochemistry and their specificity confirmed by CBA using cells expressing human GFAPα and GFAPδ proteins, by immunoblot and immunohistochemistry on GFAP-/- mouse brain sections.ResultsSerum and/or CSF IgG of 22/451 (5%) patients bound to human GFAP, of which 22/22 bound to GFAPα, 14/22 to both GFAPα and GFAPδ and none to the GFAPδ isoform only. The neurological presentation was: meningoencephalomyelitis or encephalitis in 10, movement disorder (choreoathetosis or myoclonus) in 3, anti-epileptic drugs (AED)-resistant epilepsy in 3, cerebellar ataxia in 3, myelitis in 2, optic neuritis in 1 patient. Coexisting neural autoantibodies were detected in five patients. Six patients had other autoimmune diseases. Tumours were found in 3/22 patients (breast carcinoma, 1; ovarian carcinoma, 1; thymoma, 1). Nineteen patients were treated with immunotherapy and 16 patients (84%) improved. Histopathology analysis of the leptomeningeal biopsy specimen from one patient revealed a mononuclear infiltrate with macrophages and CD8+ T cells.ConclusionsGFAP autoimmunity is not rare. The clinical spectrum encompasses meningoencephalitis, myelitis, movement disorders, epilepsy and cerebellar ataxia. Coexisting neurological and systemic autoimmunity are relatively common. Immunotherapy is beneficial in most cases.

Molecular misreading of the ubiquitin-B (UBB) gene results in a dinucleotide deletion in UBB mRNA. The resulting mutant protein, UBB+1, accumulates in the neuropathological hallmarks of Alzheimer disease. In vitro, UBB+1 inhibits proteasomal proteolysis, although it is also an ubiquitin fusion degradation substrate for the proteasome. Using the ligase chain reaction to detect dinucleotide deletions, we report here that UBB+1 transcripts are present in each neurodegenerative disease studied (tauo- and synucleinopathies) and even in control brain samples. In contrast to UBB+1 transcripts, UBB+1 protein accumulation in the ubiquitin-containing neuropathological hallmarks is restricted to the tauopathies such as Pick disease, frontotemporal dementia, progressive supranuclear palsy, and argyrophilic grain disease. Remarkably, UBB+1 protein is not detected in the major forms of synucleinopathies (Lewy body disease and multiple system atrophy). The neurologically intact brain can cope with UBB+1 as lentivirally delivered UBB+1 protein is rapidly degraded in rat hippocampus, whereas the K29,48R mutant of UBB+1, which is not ubiquitinated, is abundantly expressed. The finding that UBB+1 protein only accumulates in tauopathies thus implies that the ubiquitin-proteasome system is impaired specifically in this group of neurodegenerative diseases and not in synucleinopathies and that the presence of UBB+1 protein reports proteasomal dysfunction in the brain.

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.

Ubiquitin-B+1 (UBB+1) is a mutant ubiquitin that accumulates in the neurones of patients with Alzheimer's disease (AD). Here we report on the biochemical and functional differences between ubiquitin and UBB+1 and the effect of the mutant protein on neuronal cells. UBB+1 lacks the capacity to ubiquitinate, and although it is ubiquitinated itself, UBB+1 is not degraded by the ubiquitin-proteasomal system and is quite stable in neuronal cells. Overexpression of UBB+1 in neuroblastoma cells significantly induces nuclear fragmentation and cell death. Our results demonstrate that accumulation of UBB+1 in neurones is detrimental and may contribute to neuronal dysfunction in AD patients.

Dinucleotide deletions (e.g. DeltaGA, DeltaGU) are created by molecular misreading in or adjacent to GAGAG motifs of neuronal mRNAs. As a result, the reading frame shifts to the +1 frame, and so-called "+1 proteins" are subsequently synthesized. +1 Proteins have a wild-type N-terminus, but an aberrant C-terminus downstream from the site of the dinucleotide deletion. Molecular misreading was discovered in the rat vasopressin gene associated with diabetes insipidus and subsequently in human genes linked to Alzheimer's disease (AD), e.g. beta amyloid precursor protein (betaAPP) and ubiquitin-B (UBB). Furthermore, betaAPP(+1) and UBB(+1) proteins accumulate in the neuropathological hallmarks (i.e. in the tangles, neuritic plaques, and neuropil threads) of AD. As these +1 proteins were also found in elderly nondemented controls, but not in younger ones (

ARTICLE Sir, We would like to thank Hoglinger and colleagues for their interest in our recent publication in Brain (van den Berge et al ., 2011). In this study, we showed that the number of adult neural stem cells and precursors in the main neurogenic niche of the human brain, the subventricular zone, is not significantly diminished in patients with Parkinson’s disease compared to age- and sex-matched controls. In addition, we provided evidence that two different human neural stem cell lines did not respond with an increase in cell proliferation following exposure to dopamine or dopamine agonists. We also examined the number of proliferating neural stem cells and precursors in the subventricular zone of mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a mouse model for Parkinson’s disease, and these results supported our findings in the human brain. Our data, however, are inconsistent with three previous studies (Hoglinger et al ., 2004; O'Keeffe et al ., 2009; O'Sullivan et al ., 2011). In a letter to the editor, Hoglinger and colleagues argue that our data should be interpreted with caution, and they state that our anatomical definition of the region of interest, the sampling procedure, some of the immunostaining procedures, and the quantification methods were of limited precision. Here, we would like to extend on the used methodologies as described in the methods section and supplementary material of our article (van den Berge et al ., 2011) and we would like to take the opportunity to clarify the issues raised by Hoglinger and colleagues. We agree that the human subventricular zone is highly variable in width, as we have shown before (van den Berge et al ., 2010), and that anatomical matching between donors is necessary for obtaining reproducible quantitative data. In our recent publication (van den Berge et al …

Dementia is a common feature in Parkinson’s disease (PD) and is considered to be the result of limbic and cortical Lewy bodies and/or Alzheimer changes. Astrogliosis may also affect the development of dementia, since it correlates well with declining cognition in Alzheimer patients. Thus, we determined whether cortical astrogliosis occurs in PD, whether it is related to dementia, and whether this is reflected by the presence of glial fibrillary acidic protein (GFAP) and vimentin in cerebrospinal fluid (CSF). We have examined these proteins by immunohistochemistry in the frontal cortex and by Western blot in CSF of cases with PD, PD with dementia (PDD), dementia with Lewy bodies (DLB) and nondemented controls. We were neither able to detect an increase in cortical astrogliosis in PD, PDD, or DLB nor could we observe a correlation between the extent of astrogliosis and the degree of dementia. The levels of GFAP and vimentin in CSF did not correlate to the extent of astrogliosis or dementia. We did confirm the previously identified positive correlation between the presence of cortical Lewy bodies and dementia in PD. In conclusion, we have shown that cortical astrogliosis is not associated with the cognitive decline in Lewy body-related dementia.

There are many indications that neurogenesis is impaired in Parkinson's disease, which might be due to a lack of dopamine in the subventricular zone. An impairment in neurogenesis may have negative consequences for the development of new therapeutic approaches in Parkinson's disease, as neural stem cells are a potential source for endogenous repair. In this study, we examined the subventricular zone of 10 patients with Parkinson's disease and 10 age-and sex-matched controls for proliferation and neural stem cell numbers. We also included five cases with incidental Lewy body disease, which showed Parkinson's disease pathology but no clinical symptoms and thus did not receive dopaminergic treatment. We quantified the neural stem cell number and proliferative capacity in the subventricular zone of these three donor groups. We found subventricular neural stem cells in each donor, with a high variation in number. We did not observe significant differences in neural stem cell number or in proliferation between the groups. Additionally, we were able to culture neural stem cells from post-mortem brain of several patients with Parkinson's disease, confirming the presence of viable neural stem cells in these brains. We have also examined the subventricular zone of a chronic, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine- induced Parkinson's disease mouse model, and again found no effect of dopaminergic denervation on precursor proliferation. Lastly, we investigated the proliferation capacity of two different human neural stem cell lines in response to dopamine. Both cell lines did not respond with a change in proliferation to treatment with dopamine agonists and an antagonist. In summary, the adult neural stem cell pool in the subventricular zone was not clearly affected in the human parkinsonian brain or a Parkinson's disease mouse model. Furthermore, we did not find evidence that dopamine has a direct effect on human neural stem cell proliferation in vitro. Thus, we conclude that the number of adult neural stem cells is probably not diminished in the parkinsonian brain and that dopamine depletion most likely has no effect on human neural stem cells. © 2011 The Author.

The 39- to 42-residue amyloid β (Aβ) peptide is deposited in extracellular fibrillar plaques in the brain of patients suffering from Alzheimer's Disease (AD). Vaccination with these peptides seems to be a promising approach to reduce the plaque load but results in a dominant antibody response directed against the N-terminus. Antibodies against the N-terminus will capture Aβ immediately after normal physiological processing of the amyloid precursor protein and therefore will also reduce the levels of non-misfolded Aβ, which might have a physiologically relevant function. Therefore, we have targeted an immune response on a conformational neo-epitope in misfolded amyloid that is formed in advance of Aβ-aggregation. A tetanus toxoid-conjugate of the 11-meric cyclic peptide Aβ(22-28)-YNGK' elicited specific antibodies in Balb/c mice. These antibodies bound strongly to the homologous cyclic peptide-bovine serum albumin conjugate, but not to the homologous linear peptide-conjugate, as detected in vitro by enzyme-linked immunosorbent assay. The antibodies also bound--although more weakly--to Aβ(1-42) oligomers as well as fibrils in this assay. Finally, the antibodies recognized Aβ deposits in AD mouse and human brain tissue as established by immunohistological staining. We propose that the cyclic peptide conjugate might provide a lead towards a vaccine that could be administered before the onset of AD symptoms. Further investigation of this hypothesis requires immunization of transgenic AD model mice.

A subpopulation of glial fibrillary acidic protein (GFAP)-expressing cells located along the length of the lateral ventricles in the subventricular zone (SVZ) have been identified as the multipotent neural stem cells of the adult mammalian brain. We have previously found that, in the adult human brain, a splice variant of GFAP, termed GFAPdelta, was expressed specifically in these cells. To investigate whether GFAPdelta is also present in the precursors of SVZ astrocytes during development and whether GFAPdelta could play a role in the developmental process, we analyzed GFAPdelta expression in the normal developing human cortex and in the cortex of foetuses with the migration disorder lissencephaly type II. We demonstrated for the first time that GFAPdelta is specifically expressed in radial glia and SVZ neural progenitors during human brain development. Expression of GFAPdelta in radial glia starts at around 13 weeks of pregnancy and disappears before birth. GFAPdelta is continuously expressed in the SVZ progenitors at later gestational ages and in the postnatal brain. Co-localization with Ki67 proved that these GFAPdelta-expressing cells are able to proliferate. Furthermore, we showed that the expression pattern of GFAPdelta was disturbed in lissencephaly type II. Overall, these results suggest that the adult SVZ is indeed a remnant of the foetal SVZ, which develops from radial glia. Furthermore, we provide evidence that GFAPdelta can distinguish resting astrocytes from proliferating SVZ progenitors.

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

The glial fibrillary acidic protein (GFAP) gene is alternatively spliced to give GFAP-alpha, the most abundant isoform, and seven other differentially expressed transcripts including GFAP-delta. GFAP-delta has an altered C-terminal domain that renders it incapable of self-assembly in vitro. When titrated with GFAP-alpha, assembly was restored providing GFAP-delta levels were kept low (approximately 10%). In a range of immortalized and transformed astrocyte derived cell lines and human spinal cord, we show that GFAP-delta is naturally part of the endogenous intermediate filaments, although levels were low (approximately 10%). This suggests that GFAP filaments can naturally accommodate a small proportion of assembly-compromised partners. Indeed, two other assembly-compromised GFAP constructs, namely enhanced green fluorescent protein (eGFP)-tagged GFAP and the Alexander disease-causing GFAP mutant, R416W GFAP both showed similar in vitro assembly characteristics to GFAP-delta and could also be incorporated into endogenous filament networks in transfected cells, providing expression levels were kept low. Another common feature was the increased association of alphaB-crystallin with the intermediate filament fraction of transfected cells. These studies suggest that the major physiological role of the assembly-compromised GFAP-delta splice variant is as a modulator of the GFAP filament surface, effecting changes in both protein- and filament-filament associations as well as Jnk phosphorylation.

Tyrosine hydroxylase (TH), the first and limiting enzyme for catecholamine synthesis, has been identified immunohistochemically (IHC) in human neurosecretory neurons where it is found to colocalize with vasopressin (AVP) or oxytocin. TH expression shows striking interindividual variability and appears to depend on neuronal activation. Since GTP cyclohydrolase I (GCHI), the first enzyme for tetrahydrobiopterin synthesis, the essential cofactor of TH, and aromatic L-amino acid decarboxylase (AADC) have so far not been detected in neurosecretory neurons, the functional role of TH in catecholamine synthesis is still questionable. Our purpose was to investigate in postmortem hypothalamus whether GCHI and AADC mRNAs are co-expressed with TH in human AVP-synthesizing neurons. Total RNA was extracted from laser microdissected TH-IHC-identified neurons as well as from dissected parts of the dorsolateral supraoptic nucleus (dl-SON) of 12 control subjects, i.e. without known neurological, psychiatric or endocrinological illness. GCHI, AADC and TH mRNA expression was determined by real-time PCR. Our results showed that GCHI mRNA is co-expressed with TH in almost all cases that had a considerable number of TH-immunoreactive (TH-IR) neurosecretory neurons. A positive correlation was found between TH-immunohistochemical intensity and the presence of GCHI mRNA. AADC mRNA expression was detected only in microdissected areas of dl-SON in 2 cases that showed an increased number of TH-IR neurons. The co-expression of GCHI with TH indicates that TH is indeed active in human neurosecretory neurons. The apparent limited expression of AADC indicates that dopamine might be produced in human neurosecretory neurons under activation of the hypothalamoneurohypophyseal system, although the possibility that L-dopa is the final product cannot be excluded.

Down syndrome (DS) patients suffer from mental retardation, but also display enhanced beta-APP production and develop cortical amyloid plaques at an early age. As beta-APP and Notch are both processed by gamma-secretase, we analyzed expression of the Notch signaling pathway in the adult DS brain and in a model system for DS, human trisomy 21 fibroblasts by quantitative PCR. In adult DS cortex we found that Notch1, Dll1 and Hes1 expression is up-regulated. Moreover, DS fibroblasts and Alzheimer disease cortex also show overexpression of Notch1 and Dll1, indicating that enhanced beta-APP processing found in both DS and AD could be instrumental in these changes. Using pull-down studies we could demonstrate interaction of APP with Notch1, suggesting that these transmembrane proteins form heterodimers, but independent of gamma-secretase. We could demonstrate binding of the intracellular domain of Notch1 to the APP adaptor protein Fe65. Furthermore, activated Notch1 can trans-activate an APP target gene, Kai1, and vice versa, activated APP can trans-activate the classical Notch target gene Hes1. These data suggest that Notch expression is activated in Down syndrome, possibly through cross-talk with APP signaling. This interaction might affect brain development, since the Notch pathway plays a pivotal role in neuron-glia differentiation.

Human glial fibrillary acidic protein-delta (GFAP-delta) is a GFAP protein isoform that is encoded by an alternative splice variant of the GFAP-gene. As a result, GFAP-delta protein differs from the predominant splice form, GFAP-alpha, by its C-terminal protein sequence. In this study, we show that GFAP-delta protein is not expressed by all GFAP-expressing astrocytes but specifically by a subpopulation located in the subpial zone of the cerebral cortex, the subgranular zone of the hippocampus, and, most intensely, by a ribbon of astrocytes following the ependymal layer of the cerebral ventricles. Therefore, at least in the sub ventricular zone (SVZ), GFAP-delta specifically marks the population of astrocytes that contain the neural stem cells in the adult human brain. Interestingly, the SVZ astrocytes actively splice GFAP-delta transcripts, in contrast to astrocytes adjacent to this layer. Furthermore, we show that GFAP-delta protein, unlike GFAP-alpha, is not upregulated in astrogliosis. Our data therefore indicate a different functional role for GFAP-delta in astrocyte physiology. Finally, transfection studies showed that GFAP-delta protein expression has a negative effect on GFAP filament formation, and therefore could be important for modulating intermediate filament cytoskeletal properties, possibly facilitating astrocyte motility. Further studies on GFAP-delta and the cells that express it are important for gaining insights into its function during differentiation, migration and during health and disease.

Frame-shifted amyloid precursor protein (APP(+1)), which has a truncated out-of-frame C-terminus, accumulates in the neuropathological hallmarks of patients with Alzheimer's disease pathology. To study a possible involvement of APP(+1) in the pathogenesis of Alzheimer's disease, we expressed APP695 and APP(+1) in the HEK293 cell-line and studied whether the processing of APP695 was affected. APP(+1) is a secretory protein, but high expression of APP695 and APP(+1) results in the formation of intracellular aggregate-like structures containing both proteins and Fe65, an adaptor protein that interacts with APP695. APP(+1) is shown to interact with APP695, suggesting that these structures consist of functional protein complexes. Such an interaction can also be anticipated in post-mortem brains of young Down's syndrome patients without any sign of neuropathology. Here we observed APP(+1) immunoreactivity in beaded fibres. Additional support for functional consequences on the processing of APP695 comes from a 1.4-fold increase in levels of secreted amyloid beta40 in cells co-expressing APP695 and APP(+1), although APP(+1) itself does not contain the amyloid beta sequence. Taken together, these data show that co-expression of APP695 and APP(+1) affects the processing of APP695 in a pro-amyloidogenic way and this could gradually contribute to Alzheimer's disease pathology, as has been implicated in Down's syndrome patients.

Glial fibrillary acidic protein (GFAP) is considered to be a highly specific marker for glia. Here, we report on the expression of GFAP in neurons in the human hippocampus. Intriguingly, this neuronal GFAP is coded by out-of-frame splice variants and its expression is associated with Alzheimer pathology. We identified three novel GFAP splice forms: Delta 135 nt, Delta exon 6 and Delta 164 nt. Neuronal GFAP is mainly observed in the pyramidal neurons of the hippocampus of Alzheimer and Down syndrome patients and aged controls, but not in neurons of patients suffering from hippocampal sclerosis. Apparently, the hippocampal neurons in patients with Alzheimer's disease pathology are capable of expressing glia-specific genes.

Molecular misreading of the beta-amyloid precursor protein (APP) gene generates mRNA with dinucleotide deletions in GAGAG motifs. The resulting truncated and partly frameshifted APP protein ( APP(+1)) accumulates in the dystrophic neurites and the neurofibrillary tangles in the cortex and hippocampus of Alzheimer patients. In contrast, we show here that neuronal cells transfected with APP(+1) proficiently secreted APP(+1). Because various secretory APP isoforms are present in cerebrospinal fluid (CSF), this study aimed to determine whether APP(+1) is also a secretory protein that can be detected in CSF. Post-mortem CSF was obtained at autopsy from 50 non-demented controls and 122 Alzheimer patients; all subjects were staged for neuropathology (Braak score). Unexpectedly, we found that the APP(+1) level in the CSF of non-demented controls was much higher (1.75 ng/ml) than in the CSF of Alzheimer patients (0.51 ng/ml) ( p

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 GFAP alpha and the alternative GFAP delta splice variant. We observed that decreased transcription of GFAP enhanced alternative isoform expression, as HDAC inhibition increased the GFAP delta: GFAP alpha ratio. Expression of GFAP delta 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 GFAP delta-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.

Publisher Summary Familial Alzheimer's disease (FAD) represents about 40% of the total Alzheimer's disease (AD) cases. Most of these FAD cases ( 35% of all AD patients) do not inherit AD as an autosomaldominant trait. Although these patients have at least one other relative in the first degree suffering from the disease, the genetic factor causing AD in these cases is not known. Families with an autosomal-dominant inheritance pattern of AD, account for only 5% of the total number of AD patients. In a subset of these families, missense mutations in the genes for β-amyloid precursor protein (β-APP), presenilin-1, and -2 underlie the AD pathogenesis. The non-familial or sporadic form of AD comprises approximately 60% of the total AD cases. Aging is probably an important factor in the AD etiology. The central nervous system (CNS), however, displays a high degree of plasticity, such that initial or minor damage to the CNS will not directly lead to neuropathology and can be compensated for. On the other side, the aging CNS is very vulnerable, because it is not capable to compensate for lost neurons that are especially prominent in a number of areas. Thus, during life-time irreversible cellular damage caused by somatic mutations, oxidative stress, or synapse loss accumulate in the post-mitotic neurons. Especially in AD most of these neurons do not die, but appear to become less active and show shrinkage. This chapter proposes that transcript mutations occurring in neuronal genes might be one of these unknown aging factors and might be a part of a general mechanism that could contribute to the neuropathogenesis in the majority of the AD cases, apart from the autosomal dominant forms. Recently this process was designated as molecular misreading.

24 p. Glial fibrillary acidic protein (GFAP) is the main astrocytic intermediate filament (IF). GFAP splice isoforms show differential expression patterns in the human brain. GFAPδ is preferentially expressed by neurogenic astrocytes in the subventricular zone (SVZ), whereas GFAP+1 is found in a subset of astrocytes throughout the brain. In addition, the expression of these isoforms in human brain material of epilepsy, Alzheimer and glioma patients has been reported. Here, for the first time, we present a comprehensive study of GFAP isoform expression in both wild-type and Alzheimer Disease (AD) mouse models. In cortex, cerebellum, and striatum of wild-type mice, transcripts for Gfap-α, Gfap-β, Gfap-γ, Gfap-δ, Gfap-κ, and a newly identified isoform Gfap-ζ, were detected. Their relative expression levels were similar in all regions studied. GFAPα showed a widespread expression whilst GFAPδ distribution was prominent in the SVZ, rostral migratory stream (RMS), neurogenic astrocytes of the subgranular zone (SGZ), and subpial astrocytes. In contrast to the human SVZ, we could not establish an unambiguous GFAPδ localization in proliferating cells of the mouse SVZ. In APPswePS1dE9 and 3xTgAD mice, plaque-associated reactive astrocytes had increased transcript levels of all detectable GFAP isoforms and low levels of a new GFAP isoform, Gfap-ΔEx7. Reactive astrocytes in AD mice showed enhanced GFAPα and GFAPδ immunolabeling, less frequently increased vimentin and nestin, but no GFAPκ or GFAP+1 staining. In conclusion, GFAPδ protein is present in SVZ, RMS, and neurogenic astrocytes of the SGZ, but also outside neurogenic niches. Furthermore, differential GFAP isoform expression is not linked with aging or reactive gliosis. This evidence points to the conclusion that differential regulation of GFAP isoforms is not involved in the reorganization of the IF network in reactive gliosis or in neurogenesis in the mouse brain. This work was supported by the Internationale Stichting Alzheimer Onderzoek (ISAO; M.O. [#08504]) and the Netherlands Organization for Scientific Research (NWO; VICI grant to E.M.H. [865.09.003]). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

In Alzheimer's disease (AD), amyloid plaques are surrounded by reactive astrocytes with an increased expression of intermediate filaments including glial fibrillary acidic protein (GFAP). Different GFAP isoforms have been identified that are differentially expressed by specific subpopulations of astrocytes and that impose different properties to the intermediate filament network. We studied transcript levels and protein expression patterns of all known GFAP isoforms in human hippocampal AD tissue at different stages of the disease. Ten different transcripts for GFAP isoforms were detected at different abundancies. Transcript levels of most isoforms increased with AD progression. GFAPδ-immunopositive astrocytes were observed in subgranular zone, hilus, and stratum–lacunosum–moleculare. GFAPδ-positive cells also stained for GFAPα. In AD donors, astrocytes near plaques displayed increased staining of both GFAPα and GFAPδ. The reading-frame–shifted isoform, GFAP+1, staining was confined to a subset of astrocytes with long processes, and their number increased in the course of AD. In conclusion, the various GFAP isoforms show differential transcript levels and are upregulated in a concerted manner in AD. The GFAP+1 isoform defines a unique subset of astrocytes, with numbers increasing with AD progression. These data indicate the need for future exploration of underlying mechanisms concerning the functions of GFAPδ and GFAP+1 isoforms in astrocytes and their possible role in AD pathology.