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author:("Hol, elle M.")
1.  GFAP isoforms control intermediate filament network dynamics, cell morphology, and focal adhesions 
Cellular and Molecular Life Sciences  2016;73(21):4101-4120.
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.
PMCID: PMC5043008  PMID: 27141937
GFAP; Astrocytoma; FRAP; Intermediate filaments
2.  Frequency of nuclear mutant huntingtin inclusion formation in neurons and glia is cell‐type‐specific 
Glia  2016;65(1):50-61.
Huntington's disease (HD) is an autosomal dominant inherited neurodegenerative disorder that is caused by a CAG expansion in the Huntingtin (HTT) gene, leading to HTT inclusion formation in the brain. The mutant huntingtin protein (mHTT) is ubiquitously expressed and therefore nuclear inclusions could be present in all brain cells. The effects of nuclear inclusion formation have been mainly studied in neurons, while the effect on glia has been comparatively disregarded. Astrocytes, microglia, and oligodendrocytes are glial cells that are essential for normal brain function and are implicated in several neurological diseases. Here we examined the number of nuclear mHTT inclusions in both neurons and various types of glia in the two brain areas that are the most affected in HD, frontal cortex, and striatum. We compared nuclear mHTT inclusion body formation in three HD mouse models that express either full‐length HTT or an N‐terminal exon1 fragment of mHTT, and we observed nuclear inclusions in neurons, astrocytes, oligodendrocytes, and microglia. When studying the frequency of cells with nuclear inclusions in mice, we found that half of the population of neurons contained nuclear inclusions at the disease end stage, whereas the proportion of GFAP‐positive astrocytes and oligodendrocytes having a nuclear inclusion was much lower, while microglia hardly showed any nuclear inclusions. Nuclear inclusions were also present in neurons and all studied glial cell types in human patient material. This is the first report to compare nuclear mHTT inclusions in glia and neurons in different HD mouse models and HD patient brains. GLIA 2016;65:50–61
PMCID: PMC5129569  PMID: 27615381
Huntington's disease; inclusions; astrocytes; microglia; oligodendrocytes; neurons
3.  The Indispensable Roles of Microglia and Astrocytes during Brain Development 
Glia are essential for brain functioning during development and in the adult brain. Here, we discuss the various roles of both microglia and astrocytes, and their interactions during brain development. Although both cells are fundamentally different in origin and function, they often affect the same developmental processes such as neuro-/gliogenesis, angiogenesis, axonal outgrowth, synaptogenesis and synaptic pruning. Due to their important instructive roles in these processes, dysfunction of microglia or astrocytes during brain development could contribute to neurodevelopmental disorders and potentially even late-onset neuropathology. A better understanding of the origin, differentiation process and developmental functions of microglia and astrocytes will help to fully appreciate their role both in the developing as well as in the adult brain, in health and disease.
PMCID: PMC5099170  PMID: 27877121
microglia; astrocytes; brain development; glial cells; neurodevelopmental disorders
4.  Isolation of Neural Progenitor Cells From the Human Adult Subventricular Zone Based on Expression of the Cell Surface Marker CD271 
A method was developed to specifically isolate neural progenitor cells (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. This study shows that a pure population of NPCs can be isolated from the adult human subventricular zone (SVZ), which is highly instrumental for developing future therapies based on stimulating endogenous SVZ neurogenesis.
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.
PMCID: PMC3973708  PMID: 24604282
Glial fibrillary acidic protein delta; Neurodegenerative disorders; Specific isolation; Human adult neural progenitor cells; Subventricular zone
5.  Visualization of Active Glucocerebrosidase in Rodent Brain with High Spatial Resolution following In Situ Labeling with Fluorescent Activity Based Probes 
PLoS ONE  2015;10(9):e0138107.
Gaucher disease is characterized by lysosomal accumulation of glucosylceramide due to deficient activity of lysosomal glucocerebrosidase (GBA). In cells, glucosylceramide is also degraded outside lysosomes by the enzyme glucosylceramidase 2 (GBA2) of which inherited deficiency is associated with ataxias. The interest in GBA and glucosylceramide metabolism in the brain has grown following the notion that mutations in the GBA gene impose a risk factor for motor disorders such as α-synucleinopathies. We earlier developed a β-glucopyranosyl-configured cyclophellitol-epoxide type activity based probe (ABP) allowing in vivo and in vitro visualization of active molecules of GBA with high spatial resolution. Labeling occurs through covalent linkage of the ABP to the catalytic nucleophile residue in the enzyme pocket. Here, we describe a method to visualize active GBA molecules in rat brain slices using in vivo labeling. Brain areas related to motor control, like the basal ganglia and motor related structures in the brainstem, show a high content of active GBA. We also developed a β-glucopyranosyl cyclophellitol-aziridine ABP allowing in situ labeling of GBA2. Labeled GBA2 in brain areas can be identified and quantified upon gel electrophoresis. The distribution of active GBA2 markedly differs from that of GBA, being highest in the cerebellar cortex. The histological findings with ABP labeling were confirmed by biochemical analysis of isolated brain areas. In conclusion, ABPs offer sensitive tools to visualize active GBA and to study the distribution of GBA2 in the brain and thus may find application to establish the role of these enzymes in neurodegenerative disease conditions such as α-synucleinopathies and cerebellar ataxia.
PMCID: PMC4587854  PMID: 26418157
6.  The ubiquitin proteasome system in glia and its role in neurodegenerative diseases 
The ubiquitin proteasome system (UPS) is crucial for intracellular protein homeostasis and for degradation of aberrant and damaged proteins. The accumulation of ubiquitinated proteins is a hallmark of many neurodegenerative diseases, including amyotrophic lateral sclerosis, Alzheimer’s, Parkinson’s, and Huntington’s disease, leading to the hypothesis that proteasomal impairment is contributing to these diseases. So far, most research related to the UPS in neurodegenerative diseases has been focused on neurons, while glial cells have been largely disregarded in this respect. However, glial cells are essential for proper neuronal function and adopt a reactive phenotype in neurodegenerative diseases, thereby contributing to an inflammatory response. This process is called reactive gliosis, which in turn affects UPS function in glial cells. In many neurodegenerative diseases, mostly neurons show accumulation and aggregation of ubiquitinated proteins, suggesting that glial cells may be better equipped to maintain proper protein homeostasis. During an inflammatory reaction, the immunoproteasome is induced in glia, which may contribute to a more efficient degradation of disease-related proteins. Here we review the role of the UPS in glial cells in various neurodegenerative diseases, and we discuss how studying glial cell function might provide essential information in unraveling mechanisms of neurodegenerative diseases.
PMCID: PMC4126450  PMID: 25152710
astrocytes; microglia; oligodendrocytes; gliosis; ubiquitin proteasome system; neurodegenerative diseases
7.  Induction of a common microglia gene expression signature by aging and neurodegenerative conditions: a co-expression meta-analysis 
Microglia are tissue macrophages of the central nervous system that monitor brain homeostasis and react upon neuronal damage and stress. Aging and neurodegeneration induce a hypersensitive, pro-inflammatory phenotype, referred to as primed microglia. To determine the gene expression signature of priming, the transcriptomes of microglia in aging, Alzheimer’s disease (AD), and amyotrophic lateral sclerosis (ALS) mouse models were compared using Weighted Gene Co-expression Network Analysis (WGCNA).
A highly consistent consensus transcriptional profile of up-regulated genes was identified, which prominently differed from the acute inflammatory gene network induced by lipopolysaccharide (LPS). Where the acute inflammatory network was significantly enriched for NF-κB signaling, the primed microglia profile contained key features related to phagosome, lysosome, antigen presentation, and AD signaling. In addition, specific signatures for aging, AD, and ALS were identified.
Microglia priming induces a highly conserved transcriptional signature with aging- and disease-specific aspects.
Electronic supplementary material
The online version of this article (doi:10.1186/s40478-015-0203-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4489356  PMID: 26001565
8.  Dementia in Parkinson's Disease Correlates with α-Synuclein Pathology but Not with Cortical Astrogliosis 
Parkinson's Disease  2012;2012:420957.
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.
PMCID: PMC3347756  PMID: 22577599
9.  Abundant kif21b is associated with accelerated progression in neurodegenerative diseases 
Kinesin family member 21b (kif21b) is one of the few multiple sclerosis (MS) risk genes with a presumed central nervous system function. Kif21b belongs to the kinesin family, proteins involved in intracellular transport of proteins and organelles. We hypothesised that kif21b is involved in the neurodegenerative component of MS and Alzheimer's (AD) disease. Post-mortem kinesin expression was assessed in 50 MS, 58 age and gender matched non-demented controls (NDC) and 50 AD. Kif21b expression was five-fold increased in AD compared to MS and NDC aged below 62 years (p = 8*10-5), three-fold between 62-72 years (p = 0.005) and not different above 72 years. No significant differences were observed between MS and NDC. In AD, kif21b expression was two-fold increased in Braak stage 6 (scoring for density of neurofibrillary tangles) compared with stage 5 (p = 0.003). In MS patients, kif21b correlated with the extent of grey matter demyelination (Spearman's rho = 0.31, p = 0.03). Abundant kif21b, defined as expression above the median, was associated with a two-fold accelerated development of the Kurtzke Expanded Disability Status Scale (EDSS) 6.0 (median time in low kif21b group 16 years vs. high kif21b 7.5 years, log-rank test p = 0.04) in MS. Given the genetic association of kif21b with MS, the results were stratified according to rs12122721[A] single nucleotide polymorphism (SNP). No association was found between kif21b expression or the time to EDSS 6 in kif21b risk SNP carriers compared to non-risk carriers. Kif21b was expressed in astrocytes in addition to neurons. Upon astrocyte activation, kif21b increased nine-fold. Abundant kif21b expression is associated with severe MS and AD pathology and with accelerated neurodegeneration independent of the kif21b risk SNP.
Electronic supplementary material
The online version of this article (doi:10.1186/s40478-014-0144-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4207309  PMID: 25274010
Brain cortex; Astrocytes; Glioma; Kinesins; EDSS
10.  Defective Glial Maturation in Vanishing White Matter Disease 
Vanishing white matter disease (VWM) is a genetic leukoencephalopathy linked to mutations in the eukaryotic translation initiation factor 2B (eIF2B). It is a disease of infants, children and adults, who experience a slowly progressive neurological deterioration with episodes of rapid clinical worsening triggered by stress and eventually leading to death. Characteristic neuropathological findings include cystic degeneration of the white matter with scarce reactive gliosis, dysmorphic astrocytes, and paucity of myelin despite an increase in oligodendrocytic density. To assess whether a defective maturation of macroglia may be responsible for the feeble gliosis and lack of myelin, we investigated the maturation status of astrocytes and oligodendrocytes in the brains of 8 VWM patients, 4 patients with other white matter disorders and 6 age-matched controls with a combination of immunocytochemistry, histochemistry, scratch-wound assays, Western blot and quantitative PCR. We observed increased proliferation and a defect in the maturation of VWM astrocytes. They show an anomalous composition of their intermediate filament network with predominance of the δ-isoform of the glial fibrillary acidic protein and an increase in the heat shock protein αB-crystallin, supporting the possibility that a deficiency in astrocyte function may contribute to the loss of white matter in VWM. We also demonstrated a significant increase in numbers of pre-myelinating oligodendrocyte progenitors in VWM, which may explain the co-existence of oligodendrocytosis and myelin paucity in the patients’ white matter.
PMCID: PMC4135437  PMID: 21157376
Astrocytes; GFAPδ; Glia maturation; Olig2 cytoplasmic translocation; Oligodendrocyte progenitor cells; Vanishing white matter
11.  Reducing hippocampal extracellular matrix reverses early memory deficits in a mouse model of Alzheimer’s disease 
Alzheimer’s disease is caused by increased production or reduced clearance of amyloid-β, which results in the formation amyloid-β plaques and triggers a cascade of downstream events leading to progressive neurodegeneration. The earliest clinical symptoms of Alzheimer’s disease, i.e., memory loss, are however poorly understood from a molecular and cellular perspective. Here we used APPswe/PS1dE9 (APP/PS1) transgenic mice to study the early pre-pathological effects of increased amyloid-β levels on hippocampal synaptic plasticity and memory. Using an unbiased proteomics approach we show that the early increase in amyloid-β levels in APP/PS1 mice at three months of age coincides with a robust and significant upregulation of several protein components of the extracellular matrix in hippocampal synaptosome preparations. This increase in extracellular matrix levels occurred well before the onset of plaque formation and was paralleled by impairments in hippocampal long-term potentiation and contextual memory. Direct injection into the hippocampus of the extracellular matrix inactivating enzyme chondroitinase ABC restored both long-term potentiation and contextual memory performance. These findings indicate an important role for the extracellular matrix in causing early memory loss in Alzheimer’s disease.
Electronic supplementary material
The online version of this article (doi:10.1186/s40478-014-0076-z) contains supplementary material, which is available to authorized users.
PMCID: PMC4149201  PMID: 24974208
Alzheimer’s disease; Hippocampus; Memory; Plasticity; Extracellular matrix; Perineuronal net
12.  Activation of endogenous neural stem cells for multiple sclerosis therapy 
Multiple sclerosis (MS) is a chronic inflammatory disorder of the central nervous system, leading to severe neurological deficits. Current MS treatment regimens, consist of immunomodulatory agents aiming to reduce the rate of relapses. However, these agents are usually insufficient to treat chronic neurological disability. A promising perspective for future therapy of MS is the regeneration of lesions with replacement of the damaged oligodendrocytes or neurons. Therapies targeting to the enhancement of endogenous remyelination, aim to promote the activation of either the parenchymal oligodendrocyte progenitor cells or the subventricular zone-derived neural stem cells (NSCs). Less studied but highly potent, is the strategy of neuronal regeneration with endogenous NSCs that although being linked to numerous limitations, is anticipated to ameliorate cognitive disability in MS. Focusing on the forebrain, this review highlights the role of NSCs in the regeneration of MS lesions.
PMCID: PMC4299409  PMID: 25653584
multiple sclerosis; neurogenesis; gliogenesis; neural stem cells; therapy
13.  GFAPδ Expression in Glia of the Developmental and Adolescent Mouse Brain 
PLoS ONE  2012;7(12):e52659.
Glial fibrillary acidic protein (GFAP) is the major intermediate filament (IF) protein in astrocytes. In the human brain, GFAP isoforms have unique expression patterns, which indicate that they play distinct functional roles. One isoform, GFAPδ, is expressed by proliferative radial glia in the developing human brain. In the adult human, GFAPδ is a marker for neural stem cells. However, it is unknown whether GFAPδ marks the same population of radial glia and astrocytes in the developing mouse brain as it does in the developing human brain. This study characterizes the expression pattern of GFAPδ throughout mouse embryogenesis and into adolescence. Gfapδ transcripts are expressed from E12, but immunohistochemistry shows GFAPδ staining only from E18. This finding suggests a translational uncoupling. GFAPδ expression increases from E18 to P5 and then decreases until its expression plateaus around P25. During development, GFAPδ is expressed by radial glia, as denoted by the co-expression of markers like vimentin and nestin. GFAPδ is also expressed in other astrocytic populations during development. A similar pattern is observed in the adolescent mouse, where GFAPδ marks both neural stem cells and mature astrocytes. Interestingly, the Gfapδ/Gfapα transcript ratio remains stable throughout development as well as in primary astrocyte and neurosphere cultures. These data suggest that all astroglia cells in the developing and adolescent mouse brain express GFAPδ, regardless of their neurogenic capabilities. GFAPδ may be an integral component of all mouse astrocytes, but it is not a specific neural stem cell marker in mice as it is in humans.
PMCID: PMC3528700  PMID: 23285135
14.  Migrating neuroblasts in the adult human brain: a stream reduced to a trickle 
Cell Research  2011;21(11):1523-1525.
PMCID: PMC3364722  PMID: 21691300
15.  GFAP Isoforms in Adult Mouse Brain with a Focus on Neurogenic Astrocytes and Reactive Astrogliosis in Mouse Models of Alzheimer Disease 
PLoS ONE  2012;7(8):e42823.
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.
PMCID: PMC3418292  PMID: 22912745
16.  A Cyclic Undecamer Peptide Mimics a Turn in Folded Alzheimer Amyloid β and Elicits Antibodies against Oligomeric and Fibrillar Amyloid and Plaques 
PLoS ONE  2011;6(4):e19110.
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.
PMCID: PMC3079747  PMID: 21526148
17.  Subventricular Zone Neural Progenitors from Rapid Brain Autopsies of Elderly Subjects with and without Neurodegenerative Disease 
In mice and young adult humans, the subventricular zone (SVZ) contains multipotent, dividing astrocytes, some of which, when cultured, produce neurospheres that differentiate into neurons and glia. It is unknown whether the SVZ of very old humans has this capacity. Here, we report that neural stem/progenitor cells can also be cultured from rapid autopsy samples of SVZ from elderly human subjects, including patients with age-related neurologic disorders. Histological sections of SVZ from these cases showed a GFAP-positive ribbon of astrocytes similar to the astrocyte ribbon in human periventricular white matter biopsies that is reported to be a rich source of neural progenitors. Cultures of the SVZ contained (1) neurospheres with a core of Musashi-1-, nestin-, and nucleostemin-immunopositive cells, as well as more differentiated GFAP-positive astrocytes; (2) SMI-311-, MAP2a/b-, and β-tubulin (III)-positive neurons; and (3) galactocerebroside-positive oligodendrocytes. Neurospheres continued to generate differentiated progeny for months after primary culturing, in some cases nearly two years post initial plating. Patch clamp studies of differentiated SVZ cells expressing neuron-specific antigens revealed voltage-dependent, tetrodotoxin-sensitive, inward Na+ currents and voltage-dependent, delayed, slowly inactivating K+ currents, electrophysiologic characteristics of neurons. A subpopulation of these cells also exhibited responses consistent with the kinetics and pharmacology of the h current. However, while these cells displayed some aspects of neuronal function, they remained immature, as they did not fire action potentials. These studies suggest that human neural progenitor activity may remain viable throughout much of the life span, even in the face of severe neurodegenerative disease.
PMCID: PMC2757160  PMID: 19425077
neural stem cells; neural precursors; neurospheres; neuronal differentiation; Alzheimer’s disease
18.  Specific Human Astrocyte Subtype Revealed by Affinity Purified GFAP+1 Antibody; Unpurified Serum Cross-Reacts with Neurofilament-L in Alzheimer 
PLoS ONE  2009;4(11):e7663.
The human GFAP splice variants GFAPΔ164 and GFAPΔexon6 both result in a GFAP protein isoform with a unique out-of-frame carboxy-terminus that can be detected by the GFAP+1 antibody. We previously reported that GFAP+1 was expressed in astrocytes and in degenerating neurons in Alzheimer's disease brains. In this study we aimed at further investigating the neuronal GFAP+1 expression and we started by affinity purifying the GFAP+1 antibody. The purified antibody resulted in a loss of neuronal GFAP+1 signal, although other antibodies directed against the amino- and carboxy-terminus of GFAPα still revealed GFAP-immunopositive neurons, as described before. With an in-depth analysis of a western blot, followed by mass spectrometry we discovered that the previously detected neuronal GFAP+1 expression was due to cross-reactivity of the antibody with neurofilament-L (NF-L). This was confirmed by double-label fluorescent immunohistochemistry and western blotting with the unpurified GFAP+1 antibody and an antibody against NF-L. Our data imply that NF-L can accumulate in some tangle-like structures in Alzheimer brains. More importantly, the purified GFAP+1 antibody clearly revealed a specific subtype of astrocytes in the adult human brain. These large astrocytes are present throughout the brain, e.g., along the subventricular zone, in the hippocampus, in the striatum and in the spinal cord of controls, Alzheimer, and Parkinson patients. The presence of a specific GFAP-isoform suggests a specialized function of these astrocytes.
PMCID: PMC2766629  PMID: 19888461
19.  Glial Fibrillary Acidic Protein Filaments Can Tolerate the Incorporation of Assembly-compromised GFAP-δ, but with Consequences for Filament Organization and αB-Crystallin Association 
Molecular Biology of the Cell  2008;19(10):4521-4533.
The glial fibrillary acidic protein (GFAP) gene is alternatively spliced to give GFAP-α, the most abundant isoform, and seven other differentially expressed transcripts including GFAP-δ. GFAP-δ has an altered C-terminal domain that renders it incapable of self-assembly in vitro. When titrated with GFAP-α, assembly was restored providing GFAP-δ levels were kept low (∼10%). In a range of immortalized and transformed astrocyte derived cell lines and human spinal cord, we show that GFAP-δ is naturally part of the endogenous intermediate filaments, although levels were low (∼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-δ 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 αB-crystallin with the intermediate filament fraction of transfected cells. These studies suggest that the major physiological role of the assembly-compromised GFAP-δ 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.
PMCID: PMC2555932  PMID: 18685083
20.  Polyglutamine Expansion Accelerates the Dynamics of Ataxin-1 and Does Not Result in Aggregate Formation 
PLoS ONE  2008;3(1):e1503.
Polyglutamine expansion disorders are caused by an expansion of the polyglutamine (polyQ) tract in the disease related protein, leading to severe neurodegeneration. All polyQ disorders are hallmarked by the presence of intracellular aggregates containing the expanded protein in affected neurons. The polyQ disorder SpinoCerebellar Ataxia 1 (SCA1) is caused by a polyQ-expansion in the ataxin-1 protein, which is thought to lead to nuclear aggregates.
Methodology/Principal Findings
Using advanced live cell fluorescence microscopy and a filter retardation assay we show that nuclear accumulations formed by polyQ-expanded ataxin-1 do not resemble aggregates of other polyQ-expanded proteins. Instead of being static, insoluble aggregates, nuclear accumulations formed by the polyQ-expanded ataxin-1 showed enhanced intracellular kinetics as compared to wild-type ataxin-1. During mitosis, ataxin-1 accumulations redistributed equally among daughter cells, in contrast to polyQ aggregates. Interestingly, polyQ expansion did not affect the nuclear-cytoplasmic shuttling of ataxin-1 as proposed before.
These results indicate that polyQ expansion does not necessarily lead to aggregate formation, and that the enhanced kinetics may affect the nuclear function of ataxin-1. The unexpected findings for a polyQ-expanded protein and their consequences for ongoing SCA1 research are discussed.
PMCID: PMC2200796  PMID: 18231590
21.  hUPF2 Silencing Identifies Physiologic Substrates of Mammalian Nonsense-Mediated mRNA Decay†  
Molecular and Cellular Biology  2006;26(4):1272-1287.
Nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic surveillance pathway that selectively degrades aberrant mRNAs with premature termination codons (PTCs). Although a small number of cases exist in mammals, where NMD controls levels of physiologic PTC transcripts, it is still unclear whether the engagement of NMD in posttranscriptional control of gene expression is a more prevalent phenomenon. To identify physiologic NMD substrates and to study how NMD silencing affects the overall dynamics of a cell, we stably down-regulated hUPF2, the human homolog of the yeast NMD factor UPF2, by RNA interference. As expected, hUPF2-silenced HeLa cells were impaired in their ability to recognize ectopically expressed aberrant PTC transcripts. Surprisingly, hUPF2 silencing did not affect cell growth and viability but clearly diminished phosphorylation of hUPF1, suggesting a role of hUPF2 in modulating NMD activity through phosphorylation of hUPF1. Genome-wide DNA microarray expression profiling identified 37 novel up-regulated and 57 down-regulated transcripts in hUPF2-silenced cells. About 60% of the up-regulated mRNAs carry typical NMD motifs. Hence, NMD is important not only for maintaining the transcriptome integrity by removing nonfunctional and aberrant PTC-bearing transcripts but also for posttranscriptional control of selected physiologic transcripts with NMD features.
PMCID: PMC1367210  PMID: 16449641
22.  Regulation of stearoyl-CoA desaturase-1 after central and peripheral nerve lesions 
BMC Neuroscience  2004;5:15.
Interruption of mature axons activates a cascade of events in neuronal cell bodies which leads to various outcomes from functional regeneration in the PNS to the failure of any significant regeneration in the CNS. One factor which seems to play an important role in the molecular programs after axotomy is the stearoyl Coenzyme A-desaturase-1 (SCD-1). This enzyme is needed for the conversion of stearate into oleate. Beside its role in membrane synthesis, oleate could act as a neurotrophic factor, involved in signal transduction pathways via activation of protein kinases C.
In situ hybridization and immunohistochemistry demonstrated a strong up-regulation of SCD at mRNA and protein level in regenerating neurons of the rat facial nucleus whereas non-regenerating Clarke's and Red nucleus neurons did not show an induction of this gene.
This differential expression points to a functionally significant role for the SCD-1 in the process of regeneration.
PMCID: PMC411035  PMID: 15099403
23.  Identification of regeneration-associated genes after central and peripheral nerve injury in the adult rat 
BMC Neuroscience  2003;4:8.
It is well known that neurons of the peripheral nervous system have the capacity to regenerate a severed axon leading to functional recovery, whereas neurons of the central nervous system do not regenerate successfully after injury. The underlying molecular programs initiated by axotomized peripheral and central nervous system neurons are not yet fully understood.
To gain insight into the molecular mechanisms underlying the process of regeneration in the nervous system, differential display polymerase chain reaction has been used to identify differentially expressed genes following axotomy of peripheral and central nerve fibers. For this purpose, axotomy induced changes of regenerating facial nucleus neurons, and non-regenerating red nucleus and Clarke's nucleus neurons have been analyzed in an intra-animal side-to-side comparison. One hundred and thirty five gene fragments have been isolated, of which 69 correspond to known genes encoding for a number of different functional classes of proteins such as transcription factors, signaling molecules, homeobox-genes, receptors and proteins involved in metabolism. Sixty gene fragments correspond to genomic mouse sequences without known function. In situ-hybridization has been used to confirm differential expression and to analyze the cellular localization of these gene fragments. Twenty one genes (~15%) have been demonstrated to be differentially expressed.
The detailed analysis of differentially expressed genes in different lesion paradigms provides new insights into the molecular mechanisms underlying the process of regeneration and may lead to the identification of genes which play key roles in functional repair of central nervous tissues.
PMCID: PMC161801  PMID: 12756057
24.  Mutant ubiquitin found in neurodegenerative disorders is a ubiquitin fusion degradation substrate that blocks proteasomal degradation 
The Journal of Cell Biology  2002;157(3):417-427.
Loss of neurons in neurodegenerative diseases is usually preceded by the accumulation of protein deposits that contain components of the ubiquitin/proteasome system. Affected neurons in Alzheimer's disease often accumulate UBB+1, a mutant ubiquitin carrying a 19–amino acid C-terminal extension generated by a transcriptional dinucleotide deletion. Here we show that UBB+1 is a potent inhibitor of ubiquitin-dependent proteolysis in neuronal cells, and that this inhibitory activity correlates with induction of cell cycle arrest. Surprisingly, UBB+1 is recognized as a ubiquitin fusion degradation (UFD) proteasome substrate and ubiquitinated at Lys29 and Lys48. Full blockade of proteolysis requires both ubiquitination sites. Moreover, the inhibitory effect was enhanced by the introduction of multiple UFD signals. Our findings suggest that the inhibitory activity of UBB+1 may be an important determinant of neurotoxicity and contribute to an environment that favors the accumulation of misfolded proteins.
PMCID: PMC2173284  PMID: 11980917
proteasome; neurodegeneration; aggregate; tauopathies; polyglutamine disorders

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