Characterizing primary human microglia: A comparative study with myeloid subsets and culture models.

The biology of microglia has become subject to intense study, as they are widely recognized as crucial determinants of normal and pathologic brain functioning. While they are well studied in animal models, it is still strongly debated what specifies most accurately the phenotype and functioning of microglia in the human brain. In this study, we therefore isolated microglia from postmortem human brain tissue of corpus callosum (CC) and frontal cortex (CTX). The cells were phenotyped for a panel of typical microglia markers and genes involved in myeloid cell biology. Furthermore, their response to pro- and anti-inflammatory stimuli was assessed. The microglia were compared to key human myeloid cell subsets, including monocytes, monocyte-derived macrophages and monocyte-derived dendritic cells, and several commonly used microglial cell models. Protein and mRNA expression profiles partly differed between microglia isolated from CC and frontal cortex and were clearly distinct from other myeloid subsets. Microglia responded to both pro- (LPS or poly I:C) and anti-inflammatory (IL-4 or dexamethasone) stimuli. Interestingly, pro-inflammatory responses differed between microglia and monocyte-derived macrophages, as the former responded more strongly to poly I:C and the latter more strongly to LPS. Furthermore, we defined a large phenotypic discrepancy between primary human microglia and currently used microglial cell models and cell lines. In conclusion, we further delineated the unique and specific features that discriminate human microglia from other myeloid subsets, and we show that currently used cellular models only partly reflect the phenotype of primary human microglia. GLIA 2016;64:1857-1868.

Deep brain stimulation and the role of astrocytes.

Deep brain stimulation (DBS) has emerged as a powerful surgical therapy for the management of treatment-resistant movement disorders, epilepsy and neuropsychiatric disorders. Although DBS may be clinically effective in many cases, its mode of action is still elusive. It is unclear which neural cell types are involved in the mechanism of DBS, and how high-frequency stimulation of these cells may lead to alleviation of the clinical symptoms. Neurons have commonly been a main focus in the many theories explaining the working mechanism of DBS. Recent data, however, demonstrates that astrocytes may be active players in the DBS mechanism of action. In this review article, we will discuss the potential role of reactive and neurogenic astrocytes (neural progenitors) in DBS.

Expression patterns of glial fibrillary acidic protein (GFAP)-delta in epilepsy-associated lesional pathologies.

AIMS: Glial fibrillary acidic protein (GFAP)-delta is a novel isoform that differs in its C-terminal sequence from other GFAP isoforms. Previous studies suggest restriction of expression to the subpial layer, subventricular zone and the subgranular zone astrocytes, with an absence in pathological conditions causing reactive gliosis. GFAP-delta is speculated to have roles in regulation of astrocyte size and motility and a subpopulation of GFAP-delta-positive glia may be multipotent stem cells. The aim of this study was to investigate its expression in common causes of lesion-related refractory epilepsy. METHODS: Hippocampal sclerosis (HS), focal cortical dysplasia (FCD) type IIB, cortical tuberous sclerosis (TSC) lesions, gangliogliomas, grey matter heterotopias and hemimegalencephaly from a wide age range of patients using both surgical and post mortem tissue specimens were studied. RESULTS: GFAP-delta expression was observed in CA4 and CA1 astrocytes in HS with less frequent labelling in the granule cell layer, even where granule cell dispersion was present. No significant labelling was noted in the subiculum in HS cases or in any subfields in non-HS epilepsy cases. Balloon cells in FCDIIB and hemimegalencephaly, giant cells in TSC and the astrocytic component of gangliogliomas showed immunoreactivity, colocalizing with conventional GFAP. No neuronal expression for GFAP-delta was seen in any of the pathologies. Quantitative analysis in 10 FCDIIB and five TSC cases revealed greater numbers of GFAP-delta-positive balloon cells than conventional GFAP. There was no GFAP-delta expression within nodular heterotopia. CONCLUSIONS: GFAP-delta expression patterns in HS overall appears to mirror regional reactive gliosis. It is a useful marker for the demonstration of balloon cells in FCD and TSC, which may be relevant to their abnormal size and localization. The lack of GFAP-delta within heterotopia supports their composition from cells destined for deeper cortical layers.

Walking the line: a randomised trial on the effects of a short term walking programme on cognition in dementia.

BACKGROUND: Walking has proven to be beneficial for cognition in healthy sedentary older people. The aim of this study was to examine the effects of a walking intervention on cognition in older people with dementia. METHODS: 97 older nursing home residents with moderate dementia (mean age 85.4 years; 79 female participants; mean Mini-Mental State Examination 17.7) were randomly allocated to the experimental or control condition. Participants assigned to the experimental condition walked for 30 min, 5 days a week, for 6 weeks. To control for personal communication, another group received social visits in the same frequency. Neuropsychological tests were assessed at baseline, directly after the 6 week intervention and again 6 weeks later. Apolipoprotein E (ApoE) genotype was determined. RESULTS: Differences in cognition between both groups at the three assessments were calculated using a linear mixed model. Outcome measures included performance on tests that formed three domains: a memory domain, an executive function domain and a total cognition domain. Results indicate that there were no significant time x group interaction effects or any time x group x ApoE4 interaction effects. CONCLUSION: Possible explanations for the lack of a beneficial effect of the walking programme on cognition could be the level of physical activation of the intervention or the high frequency of comorbid cardiovascular disease in the present population of older people with dementia.

Frameshift mutants of beta amyloid precursor protein and ubiquitin-B in Alzheimer's and Down patients.

The cerebral cortex of Alzheimer's and Down syndrome patients is characterized by the presence of protein deposits in neurofibrillary tangles, neuritic plaques, and neuropil threads. These structures were shown to contain forms of beta amyloid precursor protein and ubiquitin-B that are aberrant (+1 proteins) in the carboxyl terminus. The +1 proteins were not found in young control patients, whereas the presence of ubiquitin-B+1 in elderly control patients may indicate early stages of neurodegeneration. The two species of +1 proteins displayed cellular colocalization, suggesting a common origin, operating at the transcriptional level or by posttranscriptional editing of RNA. This type of transcript mutation is likely an important factor in the widely occurring nonfamilial early- and late-onset forms of Alzheimer's disease.

Mutant ubiquitin found in Alzheimer's disease causes neuritic beading of mitochondria in association with neuronal degeneration.

A dinucleotide deletion in human ubiquitin (Ub) B messenger RNA leads to formation of polyubiquitin (UbB)+1, which has been implicated in neuronal cell death in Alzheimer's and other neurodegenerative diseases. Previous studies demonstrate that UbB+1 protein causes proteasome dysfunction. However, the molecular mechanism of UbB+1-mediated neuronal degeneration remains unknown. We now report that UbB+1 causes neuritic beading, impairment of mitochondrial movements, mitochondrial stress and neuronal degeneration in primary neurons. Transfection of UbB+1 induced a buildup of mitochondria in neurites and dysregulation of mitochondrial motor proteins, in particular, through detachment of P74, the dynein intermediate chain, from mitochondria and decreased mitochondria-microtubule interactions. Altered distribution of mitochondria was associated with activation of both the mitochondrial stress and p53 cell death pathways. These results support the hypothesis that neuritic clogging of mitochondria by UbB+1 triggers a cascade of events characterized by local activation of mitochondrial stress followed by global cell death. Furthermore, UbB+1 small interfering RNA efficiently blocked expression of UbB+1 protein, attenuated neuritic beading and preserved cellular morphology, suggesting a potential neuroprotective strategy for certain neurodegenerative disorders.

Protein quality control in neurodegeneration: walking the tight rope between health and disease.

Most neurodegenerative disorders are characterised by deposits of aggregated proteins that are readily visualised by light microscopy. Although the presence of such a bulky structure inside the cell or in the extracellular space is likely not to be healthy, over recent years the idea has emerged that these end-stage aggregates are a relatively safe way to deposit harmful aberrant proteins. Protein quality control is a multi-level security system to safeguard cells from aberrant proteins and is therefore a protective response. However, protein quality control may turn destructive in case of impairment of protein quality control for example by aging or because of overflow of the quality control systems due to prolonged exposure. In many cases the medicine is worse than the cause and the "protective" response of the cell to aggregates kills the cell, rather than the aggregate itself. Here we review the role of protein quality control in neurodegeneration and aim to distinguish protective and destructive responses to aggregates in order to find targets for therapeutic intervention.

Role of von Willebrand factor and ADAMTS-13 in early brain injury after experimental subarachnoid hemorrhage.

Essentials von Willebrand Factor (VWF) and ADAMTS13 may affect early injury after subarachnoid hemorrhage (SAH). Early brain injury was assessed in VWF-/- , ADAMTS13-/- and recombinant (r) ADAMTS13 treated mice. VWF-/- and rADAMTS13 treated mice had less brain injury than ADAMTS13-/- and wild-type mice. Early administration of rADAMTS13 may improve outcome after SAH by reducing early brain injury. SUMMARY: Background Early brain injury is an important determinant of poor functional outcome and case fatality after aneurysmal subarachnoid hemorrhage (SAH), and is associated with early platelet aggregation. No treatment exists for early brain injury after SAH. We investigated whether von Willebrand factor (VWF) is involved in the pathogenesis of early brain injury, and whether ultra-early treatment with recombinant ADAMTS-13 (rADAMTS-13) reduces early brain injury after experimental SAH. Methods Experimental SAH in mice was induced by prechiasmatic injection of non-anticoagulated blood from a littermate. The following experimental SAH groups were investigated: C57BL/6J control (n = 21), VWF-/- (n = 25), ADAMTS-13-/- (n = 23), and C57BL/6J treated with rADAMTS-13 (n = 26). Mice were killed at 2 h after SAH. Primary outcome measures were microglial activation (IBA-1 surface area) and neuronal injury (number of cleaved caspase-3-positive neurons). Results As compared with controls, microglial activation was decreased in VWF-/- mice (mean difference of - 20.0%, 95% confidence interval [CI] - 4.0% to - 38.6%), increased in ADAMTS-13-/- mice (mean difference of + 34.0%, 95% CI 16.2-51.7%), and decreased in rADAMTS-13-treated mice (mean difference of - 22.1%, 95% CI - 3.4% to - 39.1%). As compared with controls (185 neurons, interquartile range [IQR] 133-353), neuronal injury in the cerebral cortex was decreased in VWF-/- mice (63 neurons, IQR 25-78), not changed in ADAMTS-13-/- mice (53 neurons, IQR 26-221), and reduced in rADAMTS-13-treated mice (45 neurons, IQR 9-115). Conclusions Our findings suggest that VWF is involved in the pathogenesis of early brain injury, and support the further study of rADAMTS-13 as a treatment option for early brain injury after SAH.

Genetic vulnerability to DUSP22 promoter hypermethylation is involved in the relation between in utero famine exposure and schizophrenia.

Epigenetic changes may account for the doubled risk to develop schizophrenia in individuals exposed to famine in utero. We therefore investigated DNA methylation in a unique sample of patients and healthy individuals conceived during the great famine in China. Subsequently, we examined two case-control samples without famine exposure in whole blood and brain tissue. To shed light on the causality of the relation between famine exposure and DNA methylation, we exposed human fibroblasts to nutritional deprivation. In the famine-exposed schizophrenia patients, we found significant hypermethylation of the dual specificity phosphatase 22 (DUSP22) gene promoter (Chr6:291687-293285) (N = 153, p = 0.01). In this sample, DUSP22 methylation was also significantly higher in patients independent of famine exposure (p = 0.025), suggesting that hypermethylation of DUSP22 is also more generally involved in schizophrenia risk. Similarly, DUSP22 methylation was also higher in two separate case-control samples not exposed to famine using DNA from whole blood (N = 64, p = 0.03) and postmortem brains (N = 214, p = 0.007). DUSP22 methylation showed strong genetic regulation across chromosomes by a region on chromosome 16 which was consistent with new 3D genome interaction data. The presence of a direct link between famine and DUSP22 transcription was supported by data from cultured human fibroblasts that showed increased methylation (p = 0.048) and expression (p = 0.019) in response to nutritional deprivation (N = 10). These results highlight an epigenetic locus that is genetically regulated across chromosomes and that is involved in the response to early-life exposure to famine and that is relevant for a major psychiatric disorder.

Immune involvement in the pathogenesis of schizophrenia: a meta-analysis on postmortem brain studies.

Although the precise pathogenesis of schizophrenia is unknown, genetic, biomarker and imaging studies suggest involvement of the immune system. In this study, we performed a systematic review and meta-analysis of studies investigating factors related to the immune system in postmortem brains of schizophrenia patients and healthy controls. Forty-one studies were included, reporting on 783 patients and 762 controls. We divided these studies into those investigating histological alterations of cellular composition and those assessing molecular parameters; meta-analyses were performed on both categories. Our pooled estimate on cellular level showed a significant increase in the density of microglia (P=0.0028) in the brains of schizophrenia patients compared with controls, albeit with substantial heterogeneity between studies. Meta-regression on brain regions demonstrated this increase was most consistently observed in the temporal cortex. Densities of macroglia (astrocytes and oligodendrocytes) did not differ significantly between schizophrenia patients and healthy controls. The results of postmortem histology are paralleled on the molecular level, where we observed an overall increase in expression of proinflammatory genes on transcript and protein level (P=0.0052) in patients, while anti-inflammatory gene expression levels were not different between schizophrenia and controls. The results of this meta-analysis strengthen the hypothesis that components of the immune system are involved in the pathogenesis of schizophrenia.

Mutations in RNA: a first example of molecular misreading in Alzheimer's disease.

In the past decade, considerable progress has been made in the understanding of the neurodegenerative changes that occur in Alzheimer's disease (AD). Knowledge about this disease is based mainly on studies of inherited forms of AD, although most cases of AD are of the non-familial type. Recently, a novel type of mutation in 'vulnerable' dinucleotide repeats in messenger RNA was discovered in AD patients: in this type of mutation a mutated transcript is produced from a correct DNA sequence, a process that we call 'molecular misreading'. The resulting mutated '+1 proteins' are prominent neuropathological hallmarks of AD and they are present in most elderly non-demented people also. This suggests that the dinucleotide deletions in transcripts could be one of the earliest events in the neuropathogenesis of AD and an important factor in normal aging.

Frameshift proteins in Alzheimer's disease and in other conformational disorders: time for the ubiquitin-proteasome system.

Neuronal homeostasis requires a constant balance between biosynthetic and catabolic processes. Eukaryotic cells primarily use two distinct mechanisms for degradation: the proteasome and autophagy of aggregates by the lysosomes. We focused on the ubiquitin-proteasome system (UPS) and discovered a frameshift protein for ubiquitin (UBB+1), that accumulates in the neuritic plaques and tangles in patients with Alzheimer's disease (AD). UBB+1, unable to tag proteins to be degraded, has been shown to be a substrate for ubiquitination and subsequent proteasomal degradation. If UBB+1 is accumulated, it inhibits the proteasome, which may result in neuronal death. We showed that UB+1 is also present in other tauopathies (e.g. Pick's disease) and in several polyglutamine diseases, but remarkably not in synucleinopathies (e.g. Parkinson's disease). Accumulation of UBB+1-being a reporter for proteasomal dysfunctioning- thus differentiates between these conformational diseases. The accumulation of UBB+1 causes a dysfunctional UPS in these multifactorial neurodegenerative diseases. Novel transgenic mouse models and large-scale expression profiling and functional analyses of enzymes of the UPS compounds - enabling us to identify the targets of the UPS in these conformational diseases - may now pave the way for intervention and treatment of AD.

Protein quality control in Alzheimer's disease by the ubiquitin proteasome system.

The ubiquitin proteasome system (UPS) is the major protein quality control system in eukaryotic cells. Many neurodegenerative diseases are characterized by aggregates and inclusions of aberrant proteins, implying a sub-optimal functioning or defective UPS. The last few years have seen increasing evidence for the involvement of the UPS in neurodegenerative disorders, including Alzheimer's disease (AD). Notably, decreases in proteasome activity were detected in several cortical areas in AD patients. In addition, proteins that accumulate in the classical hallmarks of AD were linked to UPS function. This review specifically discusses the involvement of the UPS in AD pathogenesis. First, a detailed overview of the UPS is presented, after which AD pathology and its relation to the UPS is discussed.

Protein quality control in Alzheimer's disease: a fatal saviour.

Aggregation of Abeta plays a key role in the pathogenesis of Alzheimer's disease. Although the highly structured Abeta aggregates (fibrils) have long been thought to be the toxic form of Abeta, recent evidence suggests that smaller, soluble intermediates in Abeta aggregation are the real culprit. Because these oligomeric aggregates are already formed in the secretory pathway, this raises another issue: Is intra- or extracellular Abeta involved in the pathogenic cascade? Because aggregated proteins are very toxic, cells have developed quality control responses to deal with such proteins. A prime site for quality culum. Here, aberrant proteins are recognized and can be targeted for degradation to the cytosolic quality control system. In addition, there is accumulating evidence for quality control in other subcellular compartments in the cell. All quality control mechanisms are initially protective, but will become destructive after prolonged accumulation of aggregated proteins. This is enhanced by decreased efficiency of these systems during aging and therefore, these responses may play an important role in the pathogenesis of Alzheimer's disease. In this review, we will discuss the role of protein quality control in the neurotoxicity of Abeta.

Mutant ubiquitin expressed in Alzheimer's disease causes neuronal death.

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.

Nimodipine protects cultured spinal cord neurones from depolarization-induced inhibition of neurite outgrowth.

In the nervous system calcium ions play a crucial role in the regulation of growth cone motility, cell migration and neurite outgrowth. High intracellular Ca2+ concentrations severely disturb Ca(2+)-regulated processes and may lead to neuronal death. We studied whether the Ca(2+)-antagonist nimodipine could prevent inhibition of neurite outgrowth which occurs in depolarized cultures of rat foetal spinal neurones. Spinal cord slices were depolarized in culture with 50 mM K+. Nimodipine (0.01-10 microM) was added before depolarization. After 5 and 7 days the effect of treatment was determined by: (a) blind scoring of neurite outgrowth under phase contrast; and (b) measuring neurofilament (NF) protein with an ELISA. Neurite outgrowth was markedly decreased after depolarization, but was restored to control values by nimodipine (0.1 microM). Depolarization also led to a decrease in total NF content (18%). The NF content of depolarized slices incubated with 0.1 microM nimodipine was the same as in the controls. Thus, depolarization-induced Ca2+ entry into spinal neurones inhibits neurite outgrowth from spinal neurones. Low concentrations of nimodipine prevented this inhibition. As nimodipine had no effect on neurite outgrowth in control cultures, we conclude that nimodipine does not act as a neurotrophic factor but rather as a neuroprotective agent.

+1 Proteins and aging.

Molecular misreading is an expression used to describe errors in RNA that lead to the translation of mutated proteins. We have shown that dinucleotide deletions (delta GA, delta GU) are introduced in simple sequence repeats (e.g. GAGAG) of mRNA. If the resulting mutant transcripts escape RNA quality control systems, they are translated into +1 proteins. If functional domains are located downstream of the frameshift site, the result will be a protein with either a partial or complete loss of function. A clear example is ubiquitin(+1) (UBB(+1)), which has lost its capacity to ubiquitinate, i.e. tagging proteins destined for proteasomal degradation. This is an important step in regulating the degradation of misfolded proteins and transcription factors. In fact, UBB(+1) seems to block the proteasome. UBB(+1) and other proteins accumulate in the neuropathological hallmarks of Alzheimer's disease (AD), which suggests a causal relationship. We have hypothesized that quality control mechanisms for both transcripts and proteins work less efficiently during aging. In this manner +1 proteins may become manifest and contribute to age-related diseases.

Molecular misreading: a new type of transcript mutation expressed during aging.

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 (<51 years), molecular misreading in nondividing cells might act as a factor that only becomes manifest at an advanced age. Frameshift mutations (UBB(+1)) and pretangle staining (Alz-50 and MC1) seem to occur independently of each other during early stages of AD. We recently detected +1 proteins, not only in proliferating cells present in non-neuronal tissues such as the liver and epididymis, but also in neuroblastoma cell lines. These observations suggest that molecular misreading is a general source of transcript errors that are involved in cellular derangements in various age-related pathologies.

Regulation of the LIM-type homeobox gene islet-1 during neuronal regeneration.

Peripheral nerve lesion leads to prominent changes in gene expression in the injured neurons, a process co-ordinated by transcription factors. During development the transcription factor islet-1 plays an important role in differentiation and axogenesis. In axotomized adult neurons a process of axonal regrowth and re-establishment of the neuronal function has to be activated. Thus, we studied changes in the expression of islet-1 after axotomy, under the assumption that frequently developmentally regulated factors are reactivated during neuronal regeneration. We investigated the regulation of islet-1 expression with (i) semi-quantitative reverse transcription polymerase chain reaction and (ii) confocal microscopy in combination with quantitative image analysis. Islet-1 expression was suprisingly down-regulated in motoneurons and sensory neurons of adult rats after axotomy. A maximal reduction in the expression level was reached between day 3 and 7 after nerve lesion, a period of extensive axonal sprouting. Islet-1 expression attained control level at day 42 after lesion, a time-point at which target reinnervation takes place. The decreased expression of islet-1 during axonal regeneration is in contrast to the high levels of islet-1 expression during axogenesis in the developing nervous system. Thus, the proposed role of islet-1 in axonal target finding during axogenesis could not be confirmed in the adult rat. The observed down-regulation of islet-1 rather suggests that the activation of downstream genes important for the embryonic pattern of axonal path finding is suppressed. Moreover, in the adult nervous system islet-1 might be one of the transcription factors regulating the expression of proteins significant for the physiological intact neuronal phenotype.

Molecular misreading. A new type of transcript mutation in gerontology.

Molecular misreading is a novel process that causes mutations in neuronal transcripts. It is defined as the inaccurate conversion of genomic information from DNA into nonsense transcripts and the subsequent translation into mutant proteins. As a result of dinucleotide deletions (delta GA, delta GU, delta CU) in and around GAGAG motifs in mRNA the reading frame shifts to the +1 frame, and subsequently the so-called +1 proteins are synthetized. +1 Proteins have a wild-type NH2 terminus and from the site of the dinucleotide deletion onwards an aberrant, nonfunctional COOH terminus. Molecular misreading was found in the rat vasopressin gene associated with diabetes insipidus and in the human genes linked to Alzheimer's disease (AD), that is, beta-amyloid precursor protein (beta APP) and ubiquitin-B (UBB). Moreover, beta APP+1 and UBB+1 proteins accumulate in the neuropathological hallmarks of AD. Inasmuch as these +1 proteins were also found in elderly, nondemented control patients, but not in younger ones (< 72 years), molecular misreading may act as a factor that becomes manifest in aged people. A hotspot for dinucleotide deletions is GAGAG motifs. Because statistically an average of 2.1 GAGAG motifs per gene can be expected, other genes expressed in other tissues may undergo molecular misreading as well. Indeed, we recently detected +1 proteins in proliferating cells present in tissues such as the liver, epididymis, parotid gland, and neuroblastoma cell lines. Therefore, molecular misreading can be regarded as a general biological source of transcript errors that may be involved in cellular derangements in numerous age-related pathologic conditions apart from Alzheimer's disease.