TDP43 proteinopathy is associated with aberrant DNA methylation in human amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neurone (MN) degeneration and death. ALS can be sporadic (sALS) or familial, with a number of associated gene mutations, including C9orf72 (C9ALS). DNA methylation is an epigenetic mechanism whereby a methyl group is attached to a cytosine (5mC), resulting in gene expression repression. 5mC can be further oxidized to 5-hydroxymethylcytosine (5hmC). DNA methylation has been studied in other neurodegenerative diseases, but little work has been conducted in ALS. AIMS: To assess differences in DNA methylation in individuals with ALS and the relationship between DNA methylation and TDP43 pathology. METHODS: Post mortem tissue from controls, sALS cases and C9ALS cases were assessed by immunohistochemistry for 5mC and 5hmC in spinal cord, motor cortex and prefrontal cortex. LMNs were extracted from a subset of cases using laser capture microdissection. DNA from these underwent analysis using the MethylationEPIC array to determine which molecular processes were most affected. RESULTS: There were higher levels of 5mC and 5hmC in sALS and C9ALS in the residual lower motor neurones (LMNs) of the spinal cord. Importantly, in LMNs with TDP43 pathology there was less nuclear 5mC and 5hmC compared to the majority of residual LMNs that lacked TDP43 pathology. Enrichment analysis of the array data suggested RNA metabolism was particularly affected. CONCLUSIONS: DNA methylation is a contributory factor in ALS LMN pathology. This is not so for glia or neocortical neurones.

Review: Microglia in motor neuron disease.

Motor Neuron Disease (MND) is a fatal neurodegenerative condition, which is characterized by the selective loss of the upper and lower motor neurons. At the sites of motor neuron injury, accumulation of activated microglia, the primary immune cells of the central nervous system, is commonly observed in both human post mortem studies and animal models of MND. Microglial activation has been found to correlate with many clinical features and importantly, the speed of disease progression in humans. Both anti-inflammatory and pro-inflammatory microglial responses have been shown to influence disease progression in humans and models of MND. As such, microglia could both contribute to and protect against inflammatory mechanisms of pathogenesis in MND. While murine models have characterized the microglial response to MND, these studies have painted a complex and often contradictory picture, indicating a need for further characterization in humans. This review examines the potential role microglia play in MND in human and animal studies. Both the pro-inflammatory and anti-inflammatory responses will be addressed, throughout the course of disease, followed by the potential of microglia as a target in the development of disease-modifying treatments for MND.

Expression of p16 and p21 in the frontal association cortex of ALS/MND brains suggests neuronal cell cycle dysregulation and astrocyte senescence in early stages of the disease.

AIMS: Cellular senescence plays a role in organismal ageing and has been linked to persistent DNA damage in age-related diseases. Brain senescence has been described in astrocytes and microglia, but it is less well understood in neurones. Evidence suggests that neurones activate a senescence-like mechanism that could contribute to neurodegeneration. We aimed to determine whether a persistent DNA damage response (DDR) and senescence activation are features of motor neurone disease (amyotrophic lateral sclerosis, ALS/MND). METHODS: We examined expression of senescence (p16 and p21) and DNA damage markers (8-OHdG and γH2AX) in motor cortex (MCx), frontal association cortex (FACx) and occipital cortex (OCx) in post-mortem tissue donated by patients with ALS/MND and controls. RESULTS: Nuclear expression of p16 and p21 was detected in glial cells; double immunofluorescence for p16/p21 and glial fibrillary acidic protein (GFAP) suggested that some of these cells were GFAP+ astrocytes. p21 nuclear expression was also found in neurones. Higher levels of p16+ (glia, P = 0.028) and p21+ (glia, P = 0.003; neurones, P = 0.008) cells were found in the FACx of ALS/MND donors but not in the MCx or OCx. Expression of p16 and p21 did not correlate with 8-OHdG or γH2AX. CONCLUSIONS: Expression of p16 and p21 in glia, mainly in astrocytes, suggests senescence induction in these cells; however, neuronal p21 expression might reflect a more general mechanism of age-related cell cycle dysregulation. The significantly higher proportion of cells expressing either p16 or p21 in the FACx of ALS/MND donors could indicate senescence activation and cell cycle dysregulation in early stages of the disease.

Neuropathological characterization of a novel TANK binding kinase (TBK1) gene loss of function mutation associated with amyotrophic lateral sclerosis.

AIMS: Mutations in TANK binding kinase gene (TBK1) are causative in amyotrophic lateral sclerosis (ALS), however correlations between clinical features and TBK1 mutations have not been fully elucidated. We aimed to identify and compare TBK1 mutations to clinical features in a cohort of ALS patients from Northern England. METHODS: TBK1 mutations were analysed in 290 ALS cases. Immunohistochemistry was performed in brain and spinal cord of one case with a novel in-frame deletion. RESULTS: Seven TBK1 variants were identified, including one novel in-frame deletion (p.85delIle). In silico analysis and literature suggested four variants were pathogenic, and three were variants of uncertain significance or benign. Post-mortem immunohistochemistry established an individual with the novel in-frame deletion had classical ALS and Type B FTLD-TDP pathology, with no changes in TBK1 staining or interferon regulatory factor IRF3. CONCLUSIONS: TBK1 mutations were present in 1.38% of our cohort, and screening showed no clear genotype-phenotype associations compared to other genetic and sporadic ALS cases. TBK1 immunohistochemistry was consistent with previously published literature and we are the first to show no differential expression of interferon regulatory factor IRF3, a downstream effector of TBK1 in the immune pathway, in the TBK1-mutant tissue, compared to controls.

Age-associated changes in the blood-brain barrier: comparative studies in human and mouse.

AIMS: While vascular pathology is a common feature of a range of neurodegenerative diseases, we hypothesized that vascular changes occur in association with normal ageing. Therefore, we aimed to characterize age-associated changes in the blood-brain barrier (BBB) in human and mouse cohorts. METHODS: Immunohistochemistry and Evans blue assays were used to characterize BBB dysfunction (tight junction protein expression and serum plasma protein accumulation), vascular pathology (pericyte loss and vascular density) and glial pathology (astrocyte and microglial density) in ageing neurological control human prefrontal cortex (a total of 23 cases from 5 age groups representing the spectrum of young adult to old age: 20-30 years, 31-45 years, 46-60 years, 61-75 years and 75+) and C57BL/6 mice (3 months, 12 months, 18 months and 24 months, n = 5/6 per group). RESULTS: Quantification of the tight junction protein ZO-1 within the cortex and cerebellum of the mouse cohort showed a significant trend to both increased number (cortex P < 0.001, cerebellum P < 0.001) and length (cortex P < 0.001, cerebellum P < 0.001) of junctional breaks associated with increasing age. GFAP expression significantly correlated with ageing in the mice (P = 0.037). In the human cohort, assessment of human protein accumulation (albumin, fibrinogen and human IgG) demonstrated cells morphologically resembling clasmatodendritic astrocytes, indicative of BBB dysfunction. Semiquantitative assessment of astrogliosis in the cortex expression revealed an association with age (P = 0.003), while no age-associated changes in microglial pathology, microvascular density or pericyte coverage were detected. CONCLUSIONS: This study demonstrates BBB dysfunction in normal brain ageing, both in human and mouse cohorts.

Review: Neuropathology and behavioural features of transgenic murine models of Alzheimer's disease.

Our understanding of the underlying biology of Alzheimer's disease (AD) has been steadily progressing; however, this is yet to translate into a successful treatment in humans. The use of transgenic mouse models has helped to develop our understanding of AD, not only in terms of disease pathology, but also with the associated cognitive impairments typical of AD. Plaques and neurofibrillary tangles are often among the last pathological changes in AD mouse models, after neuronal loss and gliosis. There is a general consensus that successful treatments need to be applied before the onset of these pathologies and associated cognitive symptoms. This review discusses the different types of AD mouse models in terms of the temporal progression of the disease, how well they replicate the pathological changes seen in human AD and their cognitive defects. We provide a critical assessment of the behavioural tests used with AD mice to assess cognitive changes and decline, and discuss how successfully they correlate with cognitive impairments in humans with AD. This information is an important tool for AD researchers when deciding on appropriate mouse models, and when selecting measures to assess behavioural and cognitive change.

Review: Astrocytes in Alzheimer's disease and other age-associated dementias: a supporting player with a central role.

Astrocytes have essential roles in the central nervous system and are also implicated in the pathogenesis of neurodegenerative disease. Forming non-overlapping domains, astrocytes are highly complex cells. Immunohistochemistry to a variety of proteins can be used to study astrocytes in tissue, labelling different cellular components and sub-populations, including glial fibrillary acidic protein, ALDH1L1, CD44, NDRG2 and amino acid transporters, but none of these labels the entire astrocyte population. Increasing heterogeneity is recognized in the astrocyte population, a complexity that is relevant both to their normal function and pathogenic roles. They are involved in neuronal support, as active components of the tripartite synapse and in cell interactions within the neurovascular unit (NVU), where they are essential for blood-brain barrier maintenance and neurovascular coupling. Astrocytes change with age, and their responses may modulate the cellular effects of neurodegenerative pathologies, which alone do not explain all of the variance in statistical models of neurodegenerative dementias. Astrocytes respond to both the neurofibrillary tangles and plaques of Alzheimer's disease, to hyperphosphorylated tau and Aß, eliciting an effect which may be neuroprotective or deleterious. Not only astrocyte hypertrophy, in the form of gliosis, occurs, but also astrocyte injury and atrophy. Loss of normal astrocyte functions may contribute to reduced support for neurones and dysfunction of the NVU. Understanding how astrocytes contribute to dementia requires an understanding of the underlying heterogeneity of astrocyte populations, and the complexity of their responses to pathology. Enhancing the supportive and neuroprotective components of the astrocyte response has potential translational applications in therapeutic approaches to dementia.

Calcium dysregulation in relation to Alzheimer-type pathology in the ageing brain.

AIMS: Calcium dyshomeostasis is implicated in the pathogenesis of several neurodegenerative disorders including Alzheimer's disease. However, much of the previous research has focused on changes in neuronal calcium signalling. In a recent microarray study we identified dysregulation of several key signalling pathways including the Ca(2+) signalling pathway in astrocytes as Alzheimer-type pathology developed. In this study we sought to determine the expression of calpain-10 and calcium/calmodulin-dependent kinase alpha (CamKIIα) in relation to Alzheimer-type pathology in a population-based study. METHODS: Using post mortem temporal cortex samples derived from the Medical Research Council Cognitive Function and Ageing Study (MRC-CFAS) ageing brain cohort we examined calpain-10 and CamKIIα gene and protein expression using quantitative polymerase chain reaction and immunohistochemistry. RESULTS: We demonstrate that astrocytic expression of calpain-10 is up-regulated, and CamKIIα down-regulated with increasing Braak stage. Using immunohistochemistry we confirm protein expression of calpain-10 in astrocytes throughout the temporal cortex and demonstrate that calpain-10 immunoreactivity is correlated with both local and global measures of Alzheimer-type pathology. In addition, we identify a subpopulation of calpain-10 immunoreactive interlaminar astrocytes that extend processes deep into the cortex. CamKIIα is predominantly neuronal in localization and is associated with the presence of diffuse plaques in the ageing brain. DISCUSSION: Dysregulated expression of key calcium signalling molecules occurs with progression of Alzheimer-type pathology in the ageing brain, highlighting the need for further functional studies of astrocytic calcium signalling with respect to disease progression.

Expression microdissection isolation of enriched cell populations from archival brain tissue.

BACKGROUND: Laser capture microdissection (LCM) is an established technique for the procurement of enriched cell populations that can undergo further downstream analysis, although it does have limitations. Expression microdissection (xMD) is a new technique that begins to address these pitfalls, such as operator dependence and contamination. NEW METHOD: xMD utilises immunohistochemistry in conjunction with a chromogen to isolate specific cell types by extending the fundamental principles of LCM to create an operator-independent method for the procurement of specific CNS cell types. RESULTS: We report how xMD enables the isolation of specific cell populations, namely neurones and astrocytes, from rat formalin fixed-paraffin embedded (FFPE) tissue. Subsequent reverse transcriptase-polymerase chain reaction (RT-PCR) analysis confirms the enrichment of these specific populations. RIN values after xMD indicate samples are sufficient to carry out further analysis. COMPARISON WITH EXISTING METHOD: xMD offers a rapid method of isolating specific CNS cell types without the need for identification by an operator, reducing the amount of unintentional contamination caused by operator error, whilst also significantly reducing the time required by the current basic LCM technique. CONCLUSIONS: xMD is a superior method for the procurement of enriched cell populations from post-mortem tissue, which can be utilised to create transcriptome profiles, aiding our understanding of the contribution of these cells to a range of neurological diseases. xMD also addresses the issues associated with LCM, such as reliance on an operator to identify target cells, which can cause contamination, as well as addressing the time consuming nature of LCM.

Quantitative assessment of AMPA receptor mRNA in human spinal motor neurons isolated by laser capture microdissection.

Disturbance of glutamate neurotransmission may contribute to the motor neuron injury seen in amyotrophic lateral sclerosis. Previous studies have suggested that human spinal motor neurons express a specific profile of the AMPA subtype of glutamate receptor with low mRNA expression for the GluR2 AMPA receptor subunit but other studies have contested this finding. The present study uses laser capture microdissection to isolate specifically identified neurons coupled with quantitative RT-PCR to demonstrate that the level of expression of the GluR2 subunit is lower in spinal motor neurons than in dorsal horn neurons from the same spinal cord region. Thus, it is likely that human spinal motor neurons express a proportion of Ca2+-permeable AMPA receptors which may contribute to the selective vulnerability of these cells in amyotrophic lateral sclerosis.

Allelic variations of the human histamine H2 receptor gene.

The gene encoding the human histamine H2 receptor (H2R) has previously been cloned and sequenced from gastric cDNA. Following PCR amplification of a fragment of the H2R gene from total human DNA, three single nucleotide base substitutions were observed and confirmed when compared with the previously published sequence. One of these base changes introduces an additional TaqI restriction endonuclease site in the coding portion of the gene. PCR amplification of human H2R gene fragments followed by cleavage with TaqI demonstrated the existence of allelic variation of the human H2R gene.

The relative importance of premortem acidosis and postmortem interval for human brain gene expression studies: selective mRNA vulnerability and comparison with their encoded proteins.

To help account for the variable quality and quantity of RNA in human brain, we have studied the effect of premortem (agonal state) and postmortem factors on the detection of poly(A)+mRNA and eight mRNAs. For comparison, the influence of the same factors upon gene products encoded by the mRNAs was studied immunocytochemically or by receptor autoradiography. Brain pH declined with increasing age at death and was related to agonal state severity, but was independent of postmortem interval and the histological presence of hypoxic changes. By linear regression, pH was significantly associated with the abundance of several of the RNAs, but not with poly(A)+mRNA, immunoreactivities, or binding site densities. Postmortem interval had a limited influence upon mRNA and protein products. Freezer storage time showed no effect. Parallel rat brain studies showed no relationship between postmortem interval (0-48 h) and amounts of total RNA, poly(A)+RNA, or two individual mRNAs; however, RNA content was reduced by 40% at 96 h after death. pH is superior to clinical assessments of agonal state or mode of death in predicting mRNA preservation. It provides a simple means to improve human brain gene expression studies. pH is stable after death and during freezer storage and can be measured either in cerebrospinal fluid or in homogenised tissue.

An aspect of Alzheimer neuropathology after suicide transport damage.

Concentrations of APP-like immunoreactivity have been determined by western blotting in a soluble fraction and two membrane fractions of brain cortex from demented patients (14 with Alzheimer's disease and 8 with other diagnoses). The concentration of APP in the soluble fraction correlated with the number of pyramidal neurones but not astrocytes or indices of interneurones. Experimental lesions in rats and quantitative autoradiography were used to investigate the cellular localisation of receptors. Lesions were produced by intrastriatal or intracortical injections of volkensin to destroy corticofugal and corticortical pyramidal neurons respectively. Volkensin treatment caused significant loss of pyramidal neurones which was accompanied by reduced binding to muscarinic cholinergic m1 receptors. [3H] 8-OH-DPAT (serotonin 1A receptors) binding was reduced only following intrastriatal volkensin. Results from the human and rat investigations are discussed in terms of the biology of cortical pyramidal neurones and drugs for the treatment of Alzheimer's disease.

Isolation of enriched glial populations from post-mortem human CNS material by immuno-laser capture microdissection.

Isolating individual populations of cells from post-mortem (PM) central nervous system (CNS) tissue for transcriptomic analysis will provide important insights into the pathogenesis of neurodegenerative diseases. To date, research on individual CNS cell populations has been hindered by the availability of suitable PM material, unreliable sample preparation and difficulties obtaining individual cell populations. In this paper we report how rapid immunohistochemistry combined with laser capture microdissection (immuno-LCM) enables the isolation of specific cell populations from PM CNS tissue, thereby enabling the RNA profile of these individual cell types to be investigated. Specifically, we detail methods for isolating enriched glial populations (astrocytes, oligodendrocytes and microglia) and confirm this cell enrichment by polymerase chain reaction (PCR). In addition, the study details the numbers of each glial population required to obtain 50ng RNA, a suitable amount of starting material required to carry out microarray analysis that potentially may identify alterations of cell-specific genes and pathways associated with a range of neurodegenerative disorders.

Clinical manifestations of trisomy 5q.

A patient with a small deletion of the short arm and a partial duplication of the long arm of chromosome 5 is described. The main clinical features include craniofacial dysmorphism, growth failure, developmental retardation, and congenital heart defect. The mother and male sib each carried an inv(5) (p15.3q35) but were phenotypically normal. The possible clinical manifestations of partial duplication of the long arm of chromosome 5 are discussed with a review of previous published reports.

MAL mRNA is induced during the differentiation of human embryonal carcinoma cells into neurons and is also localised within specific regions of the human brain.

We have found that the MAL gene, which encodes a membrane proteolipid expressed during the late stages of T-lymphocyte maturation, is also activated during neuronal differentiation of NTERA2 human embryonal carcinoma cells following induction with retinoic acid. An RT-PCR fragment with a sequence identical to MAL was found amongst 30 cloned DNA fragments corresponding to genes putatively activated during NTERA2 differentiation and isolated using a differential display PCR screen. PCR and Northern blot analysis with a cloned MAL cDNA as a probe confirmed that MAL is not expressed by undifferentiated NTERA2 EC cells, but is expressed, predominantly as a 1.1 kb transcript, within 7 days of retinoic acid-induced differentiation and later in the post-mitotic neurons arising in such cultures. MAL was not expressed in the non-neuronal lineages induced by treatment of NTERA2 cells with the gratuitous inducer hexamethylene bisacetamide. Analysis of cDNA libraries constructed from EC cells, purified neurons and a sub-population of non-neuronal cells (ME311+), confirmed that expression of the MAL gene is activated in the neural lineage of NTERA2 differentiation. Using in situ hybridisation we found that MAL is expressed in the human CNS and especially in grey matter of the cerebral cortex, with less in the cerebellum and the amygdala and little or none in subcortical white matter. In contrast to reports concerning the expression pattern of a rat MAL homologue, MAL was expressed in the human brain predominantly in cell bodies which include neurons, correlating with in vitro data from the NTERA2 line.

Mitochondrial DNA mutations in Alzheimer's disease.

Several reports have indicated that point mutations of the mitochondrial DNA (mtDNA) contribute to the pathogenesis of Alzheimer's disease (AD). However, other groups have failed to find similar associations between these mutations and AD. A recent report described a set of mutations in the mtDNA encoded cytochrome oxidase genes which may account for 20% of all AD cases. We screened brain tissue from 65 AD patients for each of these previously reported mtDNA mutations but were unable to find an increased incidence of any of them in our AD sample. However, one patient with a mutation in the APP gene did harbour a novel mtDNA mutation (G to C at position 5705 in the tRNAAsn gene) that might have contributed to the very early onset of dementia in this individual. The role of mtDNA mutations in the pathogenesis of AD remains unclear, but they do not appear to be primary causes but may contribute to the onset of the disease.

Selective loss of cholinergic receptors following unilateral intracortical injection of volkensin.

Experimental lesions and quantitative autoradiography were used to investigate the cellular distribution of neurotransmitter receptors in rats. Lesions were produced by intracortical injections of either volkensin or ricin. However, only the former is retrogradely transported and volkensin treatment causes significant loss of contralateral cortical pyramidal neurones. Binding of [3H]pirenzepine (muscarinic M1 receptors) and [3H]nicotine was reduced in contralateral cortex in volkensin compared with ricin and/or control (uninjected) animals. However, binding of [3H]8-hydroxy-2-(n-dipropylamino)tetralin (5-HT1A receptors), [3H]ketanserin (5-HT2A receptors), and [3H]1,3-dipropylcyclopentylxanthine (adenosine A1 receptors) was unchanged. The most likely explanation for these results is that M1 and nicotinic receptors are present in large numbers on those pyramidal neurones that are lost. The results are discussed in terms of the biology of cortical pyramidal neurones, drugs for Alzheimer's disease, and novel ligands for improving human brain scanning techniques.

Neuronal degeneration by suicide transport following injection of volkensin into rat cerebral cortex.

We have examined the time course of neurodegeneration in subcortical nuclei and other cortical areas known to project to the rat parietal cortex, following unilateral injection of the suicide transport agent, volkensin, into the cortex of one side. Degenerating neurons, visualized by Gallyas silver staining were most prominent 21 days after injection. At this time darkly staining neurons were present in nuclei and areas known to project to the injected cortical area but not in other sites. Affected subcortical nuclei included the ipsilateral ventral thalamus and intralaminar nuclei, the basal nucleus of Meynert and claustrum of the same side, and the dorsal median raphé nucleus of both sides. Within the cortex degenerating pyramidal neurons were visible in the contralateral parietal cortex and in the frontal cortex of the same side. The distribution of degenerating cells is in agreement with the conclusion that only neurons projecting to the injection site were affected. The time course of the appearance of the degeneration and its distribution are in keeping with axonal transport rather than spread by diffusion of the toxin. Neuronal counts in Nissl-stained sections of the contralateral SMI confirmed significant neuronal loss 28 days after injection. In situ hybridization studies using an oligonucleotide probe directed against GAD mRNA and counts of GAD mRNA-positive neurons in the contralateral cortex confirmed that this population of cortical interneurons, which do not project to the injection site, were unaffected.

Trophoblast glycoprotein recognised by monoclonal antibody 5T4 maps to human chromosome 6q14-q15.

Human X rodent hybrids were stained by indirect immunofluorescence with 5T4, a murine monoclonal antibody that recognises a 72 kdalton glycoprotein expressed by human trophoblasts and a very restricted range of adult tissues; they were analysed by flow cytometry. Concordance analysis supported by segregation data allowed assignment of the gene controlling glycoprotein expression (M6P1) to chromosome 6. Similar analysis with translocation hybrids gave a regional assignment to 6q14-q15. M6P1 is distinct from NT5, coding for 5' nucleotidase, which maps to the same region.