Showing papers in "Neuropathology and Applied Neurobiology in 2003"

Capillary and arterial cerebral amyloid angiopathy in Alzheimer's disease: defining the perivascular route for the elimination of amyloid beta from the human brain.

Abstract: Accumulation of amyloid beta (Abeta) in the extracellular spaces of the cerebral cortex and in blood vessel walls as cerebral amyloid angiopathy is a characteristic of Alzheimer's disease (AD) and the ageing human brain. Studies in animals suggest that Abeta is eliminated from the brain either directly into the blood or along perivascular interstitial fluid drainage channels. The aim of the present study is to define the perivascular route for the drainage of Abeta from the human brain. Smears and paraffin sections of post-mortem cortical tissue from 17 cases of AD and from two controls were stained with thioflavin and for Abeta by immunohistochemistry. Histology and confocal microscopy showed that deposits of Abeta in the cortical parenchyma were continuous with Abeta in capillary walls but Abeta in artery walls was not in continuity with Abeta in brain parenchyma. Quantitative studies supported these observations. The results of this study suggest that when Abeta is eliminated from the extracellular spaces of the human brain by the perivascular route, it enters pericapillary spaces and from there drains along the walls of cortical arteries to leptomeningeal arteries. Factors such as overproduction of Abeta, entrapment of Abeta in drainage pathways and poor drainage of Abeta due to functional changes in ageing arteries might result in the failure of elimination of Abeta from the ageing brain and play a major role in the pathogenesis of AD. Such factors might affect therapies for AD that entail administration of anti-Abeta antibodies to eliminate Abeta from the human brain.

Dural haemorrhage in non-traumatic infant deaths: does it explain the bleeding in 'shaken baby syndrome'?

Abstract: A histological review of dura mater taken from a post-mortem series of 50 paediatric cases aged up to 5 months revealed fresh bleeding in the dura in 36/50, the bleeding ranging from small perivascular haemorrhages to extensive haemorrhage which had ruptured onto the surface of the dura Severe hypoxia had been documented clinically in 27 of the 36 cases (75%) In a similar review of three infants presenting with classical 'shaken baby syndrome', intradural haemorrhage was also found, in addition to subdural bleeding, and we believe that our findings may have relevance to the pathogenesis of some infantile subdural haemorrhage Recent work has shown that, in a proportion of infants with fatal head injury, there is little traumatic brain damage and that the significant finding is craniocervical injury, which causes respiratory abnormalities, severe global hypoxia and brain swelling, with raised intracranial pressure We propose that, in such infants, a combination of severe hypoxia, brain swelling and raised central venous pressure causes blood to leak from intracranial veins into the subdural space, and that the cause of the subdural bleeding in some cases of infant head injury is therefore not traumatic rupture of bridging veins, but a phenomenon of immaturity Hypoxia with brain swelling would also account for retinal haemorrhages, and so provide a unified hypothesis for the clinical and neuropathological findings in cases of infant head injury, without impact or considerable force being necessary

Neurotrophins and neurodegeneration.

Abstract: There is growing evidence that reduced neurotrophic support is a significant factor in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS). In this review we discuss the structure and functions of neurotrophins such as nerve growth factor, and the role of these proteins and their tyrosine kinase (Trk) receptors in the aetiology and therapy of such diseases. Neurotrophins regulate development and the maintenance of the vertebrate nervous system. In the mature nervous system they affect neuronal survival and also influence synaptic function and plasticity. The neurotrophins are able to bind to two different receptors: all bind to a common receptor p75NTR, and each also binds to one of a family of Trk receptors. By dimerization of the Trk receptors, and subsequent transphosphorylation of the intracellular kinase domain, signalling pathways are activated. We discuss here the structure and function of the neurotrophins and how they have been, or may be, used therapeutically in AD, PD, Huntington's diseases, ALS and peripheral neuropathy. Neurotrophins are central to many aspects of nervous system function. However they have not truly fulfilled their therapeutic potential in clinical trials because of the difficulties of protein delivery and pharmacokinetics in the nervous system. With the recent elucidation of the structure of the neurotrophins bound to their receptors it will now be possible, using a combination of in silico technology and novel screening techniques, to develop small molecule mimetics with much improved pharmacotherapeutic profiles.

Pathological inclusion bodies in tauopathies contain distinct complements of tau with three or four microtubule-binding repeat domains as demonstrated by new specific monoclonal antibodies.

Abstract: Pathological inclusions containing fibrillar aggregates of hyperphosphorylated tau protein are a characteristic feature in the tauopathies, which include Alzheimer's disease, frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy, corticobasal degeneration and Pick's disease Tau isoform composition and cellular and regional distribution as well as morphology of these inclusions vary in each disorder Recently, several pathological missense and exon 10 splice-donor site mutations of the tau gene were iden-tified in FTDP-17 Exon 10 codes for the second of four microtubule-binding repeat domains The splice-site mutations result in increased inclusion of exon 10 which causes a relative increase in tau isoforms containing four microtubule-binding repeat domains over those containing three repeat domains This could be a central aetiological mechanism in FTDP-17 and, perhaps, other related tauopathies We have investigated changes in the ratio and distribution of three-repeat and four-repeat tau in the different tauopathies as a basis of the phenotypic range of these disorders and the selective vulnerability of different subsets of neurones In this study, we have developed two monoclonal antibodies, RD3 and RD4 that effectively distinguish these closely related tau isoforms These new isoform-specific antibodies are useful tools for analysing tau isoform expression and distribution as well as pathological changes in the human brain

Neurological aspects of osteopetrosis

Abstract: The osteopetroses are caused by reduced activity of osteoclasts which results in defective remodelling of bone and increased bone density. They range from a devastating neurometabolic disease, through severe malignant infantile osteopetrosis (OP) to two more benign conditions principally affecting adults [autosomal dominant OP (ADO I and II)]. In many patients the disease is caused by defects in either the proton pump [the a3 subunit of vacuolar-type H(+)-ATPase, encoded by the gene variously termed ATP6i or TCIRG1] or the ClC-7 chloride channel (ClCN7 gene). These pumps are responsible for acidifying the bone surface beneath the osteoclast. Although generally thought of as bone diseases, the most serious consequences of the osteopetroses are seen in the nervous system. Cranial nerves, blood vessels and the spinal cord are compressed by either gradual occlusion or lack of growth of skull foramina. Most patients with OP have some degree of optic atrophy and many children with severe forms of autosomal recessive OP are rendered blind; optic decompression is frequently attempted to prevent the latter. Auditory, facial and trigeminal nerves may also be affected, and hydrocephalus can develop. Stenosis of both arterial supply (internal carotid and vertebral arteries) and venous drainage may occur. The least understood form of the disease is neuronopathic OP [OP and infantile neuroaxonal dystrophy, MIM (Mendelian inheritance in man) 600329] which causes rapid neurodegeneration and death within the first year. Although characterized by the finding of widespread axonal spheroids and accumulation of ceroid lipofuscin, the biochemical basis of this disease remains unknown. The neurological complications of this disease and other variants are presented in the context of the latest classification of the disease.

Protein aggregation in motor neurone disorders

Abstract: Toxicity associated with abnormal protein folding and protein aggregation are major hypotheses for neurodegeneration. This article comparatively reviews the experimental and human tissue-based evidence for the involvement of such mechanisms in neuronal death associated with the motor system disorders of X-linked spinobulbar muscular atrophy (SBMA; Kennedy's disease) and amyotrophic lateral sclerosis (ALS), especially disease related to mutations in the superoxide dismutase (SOD1) gene. Evidence from transgenic mouse, Drosophila and cell culture models of SBMA, in common with other trinucleotide repeat expansion disorders, show protein aggregation of the mutated androgen receptor, and intraneuronal accumulation of aggregated protein, to be obligate mechanisms. Strong experimental data link these phenomena with downstream biochemical events involving gene transcription pathways (CREB-binding protein) and interactions with protein chaperone systems. Manipulations of these pathways are already established in experimental systems of trinucleotide repeat disorders as potential beneficial targets for therapeutic activity. In contrast, the evidence for the role of protein aggregation in models of SOD1-linked familial ALS is less clear-cut. Several classes of intraneuronal inclusion body have been described, some of which are invariably present. However, the lack of understanding of the biochemical basis of the most frequent inclusion in sporadic ALS, the ubiquitinated inclusion, has hampered research. The toxicity associated with expression of mutant SOD1 has been intensively studied however. Abnormal protein aggregation and folding is the only one of the four major hypotheses for the mechanism of neuronal degeneration in this disorder currently under investigation (the others comprise oxidative stress, axonal transport and cytoskeletal dysfunctions, and glutamatergic excitotoxicity). Whilst hyaline inclusions, which are strongly immunoreactive to SOD1, are linked to degeneration in SOD1 mutant mouse models, the evidence from human tissue is less consistent and convincing. A role for mutant SOD1 aggregation in the mitochondrial dysfunction associated with ALS, and in potentially toxic interactions with heat shock proteins, both leading to apoptosis, are supported by some experimental data. Direct in vitro data on mutant SOD1 show evidence for spontaneous oligomerization, but the role of such oligomers remains to be elucidated, and therapeutic strategies are less well developed for this familial variant of ALS.

APOE epsilon 4 influences the pathological phenotype of Alzheimer's disease by favouring cerebrovascular over parenchymal accumulation of A beta protein.

Abstract: The relative amounts of amyloid beta-protein (A beta) in cerebral blood vessels and parenchyma vary considerably amongst patients with Alzheimer's disease (AD). Although several mechanisms have been proposed to explain this variability, the underlying genetic and environmental determinants are still unclear, as are the functional consequences. Polymorphisms in APOE, the gene for apolipoprotein E (ApoE), influence the risk of developing AD and of deposition of A beta within the brain. We examined the relationship between the APOE genotype and the relative extent of accumulation of A beta as plaques within the cerebral parenchyma and in cortical blood vessels in the form of cerebral amyloid angiopathy (CAA), in autopsy brain tissue from 125 AD cases and from 53 elderly, neurologically normal controls of which 19 had CAA without other neuropathological features of AD. In the AD cases, we also assessed whether the severity of CAA was related to the age of onset and duration of dementia, risk factors for atherosclerotic vascular disease, and histologically demonstrable cerebral infarcts or foci of haemorrhage. The APOE genotype was determined by a standard polymerase chain reaction-based method. Paraffin sections of frontal, temporal and parietal lobes were immunolabelled for A beta and the parenchymal A beta load (total A beta minus vessel-associated A beta) was quantified by computer-assisted image analysis. CAA severity was scored for cortical and leptomeningeal vessels. The relevant clinical data were obtained from the database of the South West Brain Bank. In AD, we found the severity of CAA to be strongly associated with the number of epsilon 4 alleles (P < 0.0001) but the parenchymal A beta load to be independent of APOE genotype. Cases with severe CAA had a lower parenchymal A beta load than had those with moderate CAA (P = 0.003). Neither the severity of CAA nor the parenchymal A beta load correlated with age of onset, duration of disease or age at death, and the severity of CAA also did not correlate with the presence of cerebral infarcts or foci of haemorrhage. These findings indicate that possession of the APOE epsilon 4 allele favours vascular over parenchymal accumulation of A beta in AD. This may influence the pathogenesis of neurodegeneration in epsilon 4-associated AD.

Tau pathology in the olfactory bulb correlates with Braak stage, Lewy body pathology and apolipoprotein epsilon4.

Abstract: Olfactory dysfunction increases with disease severity in Alzheimer's disease (AD), is early and independent of disease severity in Parkinson's disease (PD), but is absent in progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). Previous histopathologic studies of olfactory bulbs in AD have shown neurofibrillary tangles (NFTs) and senile plaques while Lewy bodies (LBs) have been described in PD. Little is known about olfactory bulb pathology in PSP and CBD. Tau and alpha-synuclein pathology was assessed with immunohistochemistry in olfactory bulbs of AD (N=15), Lewy body disease (LBD; N=10), LBD with concurrent AD (AD/LBD; N=19), PSP (N=27), CBD (N=3) and cases with no significant neurodegenerative pathology (NSP; N=15). The Braak NFT stage, counts of senile plaques and NFT in cortical and hippocampal sections, and counts of LBs in amygdala and cortical sections were recorded for each case. Apolipoprotein E (APOE) genotypes were determined on DNA prepared from frozen brain tissue. All AD and AD/LBD cases and nine of 10 LBD cases had tau pathology in the anterior olfactory nucleus (AON), but it was uncommon in PSP (9/27), CBD (0/3) and NSP (5/15). Multiple linear regression analysis demonstrated that tau pathology in the AON correlated with Braak stage (P<0.001), cortical LB counts (P<0.001), as well as APOE epsilon4. Tau pathology is common in the olfactory bulb of AD and LBD but is minimal or absent in PSP and CBD. It correlates with APOE epsilon4, severity of tau pathology in the brain and surprisingly with cortical and amygdala LBs, suggesting a possible synergistic effect between tau and synuclein in the AON in cases with both pathologic processes.

Caspase‐dependent and caspase‐independent signalling of apoptosis in the penumbra following middle cerebral artery occlusion in the adult rat

Abstract: Transient focal ischaemia by middle cerebral artery occlusion (MCAO) may produce cell death, but the mechanisms leading to cell death differ in the infarct core and in the penumbra, the immediate zone surrounding the infarct core. In the present study, transient focal ischaemia to adult rats was produced by intraluminal occlusion of the middle cerebral artery for 1 h followed by 0 h (n=6), 1 h (n=10), 4 h (n=8), 6 h (n=2) and 12 h (n=3) of reperfusion. The present model of ischaemia causes a large cortico-striatal infarct extending through the mediolateral cortex and dorsolateral striatum at 12 h. The expression and subcellular distribution of several proteins involved in apoptosis have been examined in the penumbra and in the infarct core by using combined methods of immunohistochemistry, cell subfractionation and Western blotting. Transient focal ischaemia by MCAO results in activation of complex signal pathways for cell death in the penumbra. Increased expression of Bcl-2 and Bax, but not of Bcl-x, occurs in the penumbra at the time when Bax translocates from the cytosol to the mitochondria, cytochrome c is released to the cytoplasm and active caspase-3 is expressed. Bax translocation, cytochrome c release and active caspase-3 are observed at 4 h, but not at 1 h, following reperfusion, and together indicate activation of the caspase-dependent pathway of apoptosis in the penumbra. In contrast, reduced Bax expression but not Bax translocation and cytochrome c release occurs in the infarct core, thus suggesting apoptosis signals restricted to the penumbra. In addition, increased expression of an apoptosis-inducing factor in the cytoplasm and nuclei of selected cells shows, for the first time, activation of the caspase-independent mitochondrial pathway in the penumbra following transient focal ischaemia and reperfusion.

Tau immunohistochemistry in acute brain injury

Abstract: Epidemiological studies have identified a history of head injury as a risk factor for Alzheimer's disease. However, the neuropathological mechanism underlying this relationship is as yet unclear. Neuronal cytoskeletal changes in the form of neurofibrillary tangles and neuropil threads have recently been demonstrated in young men who had sustained repetitive head injury and subsequently died in their 20s. In addition, recent experimental studies have found accumulation of tau within neuronal somata and damaged axons following diffuse brain injury. We hypothesized that tau-immunoreactive tangles may be present in the brains of patients who died after a single acute blunt head injury. A total of 45 cases of fatal head injury were immunostained for tau. They comprised nine groups (n=5 for each group) separated by age (0-19 years, 20-50 years, 50+ years) and survival time (<24 h, 24 h-1 week, 1 week-1 month) and were compared with age-matched controls. Subtle alterations in tau immunoreactivity, for example, in oligodendrocytes, were present in some head injury cases but not controls. However, neurofibrillary tangles did not appear more prevalent after traumatic brain injury (TBI) when compared with age-matched controls. Although alterations in tau immunoreactivity may occur which warrant further study, neurofibrillary tangles were not more prevalent after a single fatal episode of TBI.

Involvement of precerebellar nuclei in multiple system atrophy

Abstract: In this semiquantitative study based on 26 post-mortem cases, we describe the involvement of precerebellar nuclei in multiple system atrophy (MSA), a progressive degenerative disorder of the human central nervous system characterized by abnormal, argyrophilic and alpha-synuclein immunopositive intracellular inclusions within selectively vulnerable oligodendrocytes and nerve cells. The Campbell-Switzer silver-pyridine technique with alpha-synuclein immunoreactions using 100-microm thick sections is recommended over more conventional methods, thereby permitting visualization of the pertinent lesions in greater detail and facilitating post-mortem diagnosis of MSA specimens. Affected oligodendrocytes occur in specific fibre tracts and grey matters, with most pathology being observed in projections from the precerebellar nuclei to the cerebellum (ponto-cerebellar, olivo-cerebellar, reticulo-cerebellar tracts) and in descending/ascending fibre tracts of the motor system (cortico-pontine, cortico-bulbar, cortico-spinal, spino-reticular, spino-olivary, spino-cerebellar tracts). Three types of abnormal intraneuronal aggregations occur: (i) a loosely woven network within the cell nucleus; (ii) a latticework accumulating in peripheral portions of the cell body; and (iii) irregularly outlined patches of compact, intensely argyrophilic material usually located within deposits of lipofuscin granules. Counter-staining for the presence of extraneuronal lipofuscin can aid neuropathologists in the recognition of lost existent neurones in MSA. Neurones with inclusion bodies occur in the inferior olivary nuclear complex, lateral reticular nucleus, external cuneate nucleus, conterminal nucleus, interfascicular nucleus, nucleus of Roller, dorsal paramedian reticular nucleus, subventricular nucleus, arcuate nucleus, pontobulbar body and pontine grey. The lateral reticular nucleus and accessory nuclei of the inferior olive sustain the most damage and reveal prominent neuronal loss, followed by the pontobulbar body and arcuate nucleus. The uniformly bilateral damage and, in some cases, even obliteration of the nuclei studied, supply additional evidence for the pathoanatomical substrata of the cerebellar dysfunction that reportedly emerges in the clinical course of MSA.

Lymphatic cerebrospinal fluid absorption pathways in neonatal sheep revealed by subarachnoid injection of Microfil.

Abstract: There is mounting evidence that a significant portion of cerebrospinal fluid drainage is associated with transport along cranial and spinal nerves with absorption taking place into lymphatic vessels external to the central nervous system. To characterize these pathways further, yellow Microfil was infused into the cisterna magna of 2-7-day-old lambs post mortem to perfuse either the cranial or spinal subarachnoid compartments. In some animals, blue Microfil was perfused into the carotid arteries simultaneously. Microfil was observed in lymphatic networks in the nasal mucosa, covering the hard and soft palate, conchae, nasal septum, the ethmoid labyrinth and the lateral walls of the nasal cavity. Many of these lymphatics drained into vessels located on the lateroposterior wall of the nasopharynx and from this location drained to the retropharyngeal lymph nodes. Additionally, lymphatics containing Microfil penetrated the lateral wall of the nasal cavity and joined with superficial lymphatic ducts travelling towards the submandibular and preauricular lymph nodes. In two cases, lymphatic vessels were observed anastomosing with deep veins in the retropharyngeal area. Microfil was also distributed within the nerve trunks of cranial and spinal nerves. The contrast agent was located in longitudinal channels within the endoneurial space and lymphatics containing Microfil were observed emerging from the mesoneurium. In summary, Microfil distribution patterns in neonatal lambs illustrated the important role that cranial and spinal nerves play in linking the subarachnoid compartment with extracranial lymphatics.

Sudden and unexpected death in epilepsy (SUDEP): evidence of acute neuronal injury using HSP‐70 and c‐Jun immunohistochemistry

Abstract: Post-mortem and neuropathological examination in sudden and unexpected death in epilepsy (SUDEP) shows no specific lesions and the exact cause and mechanism of death in these cases remains undetermined. There is clinical evidence to support the fact that SUDEP is a seizure-mediated event, and patients with poorly controlled seizures are at higher risk. We aimed to identify any evidence of acute neuronal injury in SUDEP cases at post-mortem to support that a recent seizure had occurred. We analysed the distribution and frequency of heat shock protein (HSP)-70 and c-Jun immunopositive neurones in the hippocampus in 18 SUDEP cases and 22 control cases, both markers being nonspecific but early and reliable indicators of acute neuronal injury. Post-mortem control groups included patients with epilepsy with cause of death other than SUDEP (including status epilepticus and accidental death), and patients with sudden cardiac death without an epilepsy history. An additional surgical control group included patients with refractory epilepsy and hippocampal sclerosis who had undergone temporal lobectomy. Semiquantitative analysis of the distribution of HSP-70 staining showed significantly more SUDEP cases with positively labelled neurones in hippocampal subfields compared to epilepsy and cardiac post-mortem controls (P < 0.001) but not compared to the epilepsy surgical controls (P = 0.4). No significant difference in immunostaining patterns between groups was seen in the parahippocampal gyrus with HSP-70 or with c-Jun in either the hippocampus or parahippocampal gyrus regions. The detection of HSP-70 positive neurones in the hippocampus in SUDEP is supportive of ante-mortem neuronal injury including a recent seizure prior to death.

Frontotemporal and motor neurone degeneration with neurofilament inclusion bodies: additional evidence for overlap between FTD and ALS

Abstract: We present the case of a patient who had clinical frontal lobe dementia without apparent motor neurone disease (MND), with pathologic findings not typical of any single currently classified frontotemporal degeneration (FTD). At autopsy, the brain had frontal and temporal atrophy with neuronal loss, gliosis, and superficial spongiosis, typical of all FTDs. There were at least three different morphologic types of intracytoplasmic neuronal inclusions in a variety of brain and brainstem regions, including the hippocampal dentate gyrus and pyramidal neurones, the neocortex (in particular, the motor cortex), basal ganglia, thalamus, subthalamic nucleus, basis pontis, and inferior olivary nuclei. Inclusions had the morphologies of Pick-like bodies, pleomorphic inclusions, and hyaline conglomerate (HC)-like inclusions. None of these were positive with tau immunostains. Pick-like bodies in the dentate gyrus were labelled with ubiquitin. The pleomorphic inclusions in the neocortex and dentate gyrus and the HC-like inclusions in the motor and parietal cortex were strongly positive with immunostains for neurofilament. We discuss the differential diagnosis and compare this case with those disorders to which it is most similar. In particular, we compare the unique neurofilament-positive inclusions to the inclusions of FTD-MND, to Pick bodies, and to the basophilic and HC inclusions that are occasionally seen in amytrophic lateral sclerosis (ALS). Although FTD-MND may be found in ALS, the findings in this case may have additional implications for a link between FTD and ALS.

Glial levels determine severity of white matter disease in Alzheimer's disease: a neuropathological study of glial changes.

Abstract: The morphological components of cerebral white matter disease (WMD) were studied in 17 cases of clinically diagnosed dementia and neuropathologically verified Alzheimer’s disease (AD) with concomitant WMD. The distribution of grey and white matter changes was evaluated and overall as well as regional severity was graded. Total glial numbers in frontal white matter were counted using a light microscope. Oligodendrocyte and astrocyte quantities as well as astrocytic reactivity were assessed from frontal and parietal lobe white matter using a computer assisted morphometric method. The AD‐WMD group was compared with 10 nondemented age-matched controls. Astrocyte/oligodendrocyte ratio (AOR) was calculated, total glial counts and AOR were compared with severity of WMD, and Alzheimer encephalopathy grade was subjectively assessed. Astrocytic numbers, AOR and astrocytic reactivity proved to be significantly higher in the demented group, whereas oligodendrocytic and total glial counts were significantly lower. Furthermore, AOR proved to be positively correlated with severity of WMD, whereas no association was found with Alzheimer encephalopathy. We conclude that WMD in dementia, for example, of the type seen in AD, can easily be detected, measured and graded quantitatively, with AOR being a significant indicator of the severity of changes. This could serve as a tool for differentiating white matter pathologies in dementia and may be the basis for recognition of the mildest white matter changes with new imaging methods, and enable potential clinical intervention.

The pathology and pathogenesis of retinal vasculitis

Abstract: Retinal vasculitis is a rare, but potentially blinding intraocular inflammatory condition with diverse aetiology. Although commonly idiopathic, it has a strong association with systemic inflammatory diseases known to involve other areas of the central nervous system, most notably Behcet's disease, sarcoidosis, systemic lupus erythematosis and multiple sclerosis. This article describes the clinicopathologic features of retinal vasculitis and its visually damaging sequelae, reviewing available human histopathologic studies and work with experimental models to discuss the pathogenesis and immunopathology. Evidence indicates that noninfective retinal vasculitis is an autoimmune condition that may be induced by antecedent infection with microbes cross-reacting with putative autoantigens, influenced by genetic susceptibility of both HLA associations and cytokine polymorphisms. The growing understanding of the cellular mechanisms involved in the effector immune response is already providing a rationale for more specific therapeutic approaches.

CDKN2A, CDKN2B and p14ARF are frequently and differentially methylated in ependymal tumours.

Abstract: Ependymal tumours are histologically and clinically varied lesions. Numerical abnormalities of chromosome 9 are frequently associated with these tumours. Nevertheless, the three important tumour suppressor genes located in this chromosome, CDKN2A, CDKN2B and p14 ARF, have not been reported to be commonly altered in them. We studied promoter methylation of these genes, an important mechanism associated with gene silencing in a series of 152 ependymal tumours of WHO grades I to III. Methylation status of the CDKN2A, CDKN2B and p14 ARF promoters was assessed by methylation-specific polymerase chain reaction and the genetic results were correlated to clinicopathological features. We observed promoter methylation for CDKN2A in 21% (26/123) of tumours, for CDKN2B in 32% (23/71) and p14 ARF in 21% (23/108). For all three genes, posterior fossa ependymomas were less frequently methylated in paediatric patients than in adults. For CDKN2B, extracranial tumours were more frequently methylated than intracranial ones. For CDKN2B and p14 ARF, methylation was more frequent in low-grade tumours; the reverse was observed for CDKN2A. CDKN2A, CDKN2B and p14 ARF promoters were methylated in 21-32% of the tumours. Frequencies of methylation varied according to clinicopathological features. This suggests a role for these genes in ependymoma tumorigenesis.

Apoptotic mitochondrial pathway in neurones and astrocytes after neonatal hypoxia‐ischaemia in the rat brain

Abstract: Neuronal apoptosis plays an essential role in early brain development and contributes to secondary neuronal loss after acute ischaemia. Recent studies have provided evidence that caspase-3 is an important downstream event after hypoxia-ischaemia in the immature brain, but a minor event in the adult brain. Our investigations have focused on cell populations that expressed apoptotic effectors in the enzymatic death pathway including cytochrome c, caspase-9 and caspase-3. Expression, activation and cellular localization of these proteins were studied using cleavage of fluorogenic substrate and immunohistochemistry in neonatal rat brain after unilateral focal ischaemia. Caspase-3 enzyme activity was elevated in brain homogenate between 6 and 48 h after reperfusion. This activation was preceded by that of caspase-9, between 3 and 24 h. Apoptotic cell death was finally accomplished by poly-ADP-ribose polymerase cleavage, an endogenous caspase-3 substrate. In addition, immunodetection demonstrated that cytochrome c and activated caspase-9 and caspase-3 were expressed not only in the neurones, the primarily affected cells, but also within the astrocytes, which constituted a dense network delineating the infarct. These results suggested that glial injury may promote the formation of cystic lesions such as those observed clinically in the newborn brain.

Chemokine receptors on infiltrating leucocytes in inflammatory pathologies of the central nervous system (CNS).

Abstract: Haematogenous leucocytes enter the central nervous system (CNS) during diverse disorders of varied aetiologies. Understanding the trafficking cues that mediate CNS leucocyte infiltration might promote the development of flexible and selective means to modulate inflammation to achieve clinical benefit. The trafficking machinery of leucocytes has been elucidated during the past decade and consists of cell-surface adhesion molecules, chemoattractant cytokines (chemokines) and their receptors. Recent work in our laboratory characterized chemokine receptors found on T lymphocytes and monocytes in brain sections from subjects with one pathological subtype of multiple sclerosis (MS), an immune-mediated inflammatory demyelinating disease. In these tissues, the types 1 and 5 CC chemokine receptors (CCR1 and CCR5) were detected on perivascular monocytic cells whereas only CCR5 was present on parenchymal macrophages. The type 3 CXC chemokine receptor (CXCR3) was present on virtually all CD3-positive T cells. In the current study, we evaluated the expression of these receptors on the infiltrating cells present in cases of other inflammatory CNS disorders including those of dysimmune, infectious, neoplastic, and vascular aetiology. Perivascular and parenchymal monocytic cells expressed CCR1 in all cases and CXCR3 was consistently present on a substantial proportion of CD3+ T cells. The occurrence of CCR5 on parenchymal macrophages was much less uniform across the varied disorders. These data implicate CCR1 in monocyte infiltration of the CNS and are consistent with reports of studies in CCR1-deficient mice. CXCR3 is also likely to play a role in accumulation of T cells in the inflamed CNS. By contrast, our findings suggest that regulation of CCR5 on phagocytic macrophages may be contingent on the lesion environment.

Comparative analysis of mRNA levels in the frontal cortex and the hippocampus in the basal state and in response to experimental brain injury.

Abstract: Damage to the frontal cortex and to the hippocampus, both in terms of cell loss and neuronal dysfunction, is thought to underlie many of the neurological and behavioural consequences of traumatic brain injury (TBI). Several studies have indicated that the hippocampus is particularly susceptible to central nervous system insults, whereas the frontal cortex possesses relatively higher capacities for regeneration and plasticity. It has been postulated that dissimilarities in the gene expression profiles in these structures, both in the normal and the postinjury states, may underlie these differences. In order to explore this issue, mRNA samples taken from the frontal cortex and the hippocampus of uninjured animals were subjected to high-density microarray analysis. The analysis indicated that the mRNA levels of 65 genes were differentially expressed between these two brain regions. Among these, genes involved in intracellular signalling, neurotransmitter release, and genes encoding for channels and receptors were identified. Samples taken from animals injured using controlled cortical impact (a model of TBI) showed altered mRNA levels for 341 frontal cortex genes 24 h following injury. These genes can be broadly classified into one of 12 functional classes: cell cycle, metabolism, reactive oxygen metabolism, inflammation, receptors, channels and transporters, signal transduction, cytoskeleton, membrane proteins, neuropeptides, growth factors, and proteins involved in transcription/translation. The expression profile of these genes is compared to the expression profile of 241 genes in the hippocampus 24 h following cortical impact injury as previously reported by our laboratory. In addition to genes previously reported in the literature, this study found several genes that have not been associated with TBI. The functional implications of changes in the expression of some of these genes are discussed.

Accumulation of flotillin-1 in tangle-bearing neurones of Alzheimer's disease.

Abstract: The protein flotillin-1 is associated with the 'lipid rafts', that is, membrane microdomains that are enriched in cholesterol and sphingolipids. We compared flotillin-1 immunoreactivity in the hippocampus, amygdala and isocortex (Brodmann area 22) of six controls and 13 Alzheimer's disease (AD) cases (10 sporadic and three familial). A diffuse labelling of the neuropil was observed in most of the samples. The intensity of this labelling was not correlated with the density of neurofibrillary tangles (NFT) or of senile plaques. Some neuronal cell bodies were diffusely labelled in patients as in controls. Immunostained granular bodies were found in the cell body of a few neurones. The density of neuronal profiles containing large granular bodies (diameter > or =2 microm) was significantly higher in AD cases and was correlated with the density of NFTs in the three regions that were studied. Sections stained by double immunofluorescence methods and examined with confocal microscopy suggested that flotillin-1 accumulated most often in tangle-bearing neurones (76% of flotillin-1-positive neurones contained a NFT). Flotillin-1 immunoreactivity, even when found in a tangle-bearing neurone, was not colocalized with tau protein indicating that the two proteins were not in close contact and probably in different subcellular compartments. Flotillin-1-positive granular bodies were also found in neurones containing Pin1-positive vesicles but were not colocalized with them. Flotillin-1 immunoreactivity was colocalized with cathepsin D, a lysosomal marker. These data indicate that flotillin-1, a marker of rafts, accumulates in lysosomes of tangle-bearing neurones in the course of AD.

Anatomically based guidelines for systematic investigation of the central somatosensory system and their application to a spinocerebellar ataxia type 2 (SCA2) patient

Abstract: Dysfunctions of the somatosensory system are among the clinical signs that characterize a variety of polyglutamine or CAG-repeat diseases. Deficits within this system may hinder the perception of potential threats, be detrimental to somatomotor functions, and result in uncoordinated movements, ataxia, and falls. Despite the considerable clinical relevance of such deficits, however, no systematic pathoanatomical studies of the central somatosensory system in polyglutamine diseases are currently available. The present paper has two goals: (1) recommendation of an economical tissue sampling method and optimized histological processing of this tissue to allow rapid and reliable evaluation of the structural integrity of all known relay stations and interconnecting fibre tracts within this complex system, and (2) the proposal of guidelines for a rapid and detailed pathoanatomical investigative procedure of the human central somatosensory system. In so doing, we draw on the current state of neuroanatomic research and apply the methods and guidelines proposed here to a 25-year-old female patient with spinocerebellar ataxia type 2 (SCA2). The use of 100 microm serial sections through the SCA2 patient's central somatosensory components showed that obvious neuronal loss occurred in nearly all of the relay stations of this system (Clarke's column; cuneate, external cuneate and gracile nuclei; spinal, principal and mesencephalic trigeminal nuclei; ventral posterior lateral and ventral posterior medial nuclei of the thalamus), whereas the majority of interconnecting fibre tracts (dorsal spinocerebellar tract; cuneate and gracile fascicles; medial lemniscus; spinal trigeminal tract, trigeminal nerve and mesencephalic trigeminal tract) displayed signs of atrophy accompanied by demyelinization. These pathological findings suffice to explain the patient's impaired senses of vibration, position and temperature. Moreover, together with the lesions seen in the motor cerebellothalamocortical feedback loop (pontine nuclei, deep cerebellar nuclei and cerebellar cortex, ventral lateral nucleus of the thalamus), they also account for the somatomotor deficits that were observed in the young woman (gait, stance, and limb ataxia, falls, and impaired writing). In proposing these new guidelines, we hope to enable others to study the hitherto unknown morphological counterparts of somatosensory dysfunctions in additional CAG-repeat disease patients.

Tau phosphorylation and kinase activation in familial tauopathy linked to deln296 mutation.

Abstract: Tau phosphorylation has been examined by immunohistochemistry in the brain of a patient affected with familial tauopathy with progressive supranuclear palsy-like phenotype linked to the delN296 mutation in the tau gene. Phospho-specific tau antibodies Thr181, Ser202, Ser214, Ser396 and Ser422, and antibodies to glycogen synthase kinase-3alpha/beta (GSK-3alpha/beta) and to phosphorylated (P) mitogen-activated protein kinase/extracellular signal-regulated kinases (MAPK/ERK), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), p38 kinase (p38) and GSK-3betaSer9 have been used to gain understanding of the identification of phosphorylation sites, as well as of the specific kinases that regulate tau phosphorylation at those specific sites, in a familial tauopathy. The neuropathological examination disclosed atrophy of the right precentral gyrus and the brainstem. Neurone loss and gliosis were observed in the substantia nigra, several nuclei of the brainstem and diencephalon. Hyper-phosphorylated tau accumulated in neurones with neurofibrillary tangles and in neurones with pretangles in the substantia nigra, locus ceruleus, peri-aqueductal grey matter, reticular formation, motor nuclei of the brainstem, and thalamus, amygdala and hippocampus. tau-immunoreactive astrocytes and, particularly, oligodendrocytes with coiled bodies were widespread in the brainstem, diencephalons, cerebral white matter and cerebral cortex. Increased expression of MAPK/ERK-P, SAPK/JNK-P, p-38-P and GSK-3beta-P was observed in select subpopulations of neurones with neurofibrillary tangles and in neurones with pretangles. MAPK/ERK-P, SAPK/JNK-P, p38-P and GSK-3beta-P were also expressed in tau-containing astrocytes and in oligodendrocytes with coiled bodies. These findings show, for the first time, activation of precise kinases that regulate tau phosphorylation at specific sites in familial tauopathy.

Comparison of the neuropathological characteristics of bovine spongiform encephalopathy (BSE) and variant Creutzfeldt–Jakob disease (vCJD) in mice

Abstract: Bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob disease (vCJD) belong to a group of diseases called the transmissible spongiform encephalopathies (TSEs). Transmission studies in inbred mice (strain typing) provided overwhelming evidence that vCJD arose from BSE. In this study, we compare the patterns of neuropathology in a panel of three inbred mouse strains (RIII, C 5 7BL and VM) and one cross (C 5 7BL x VM) infected with either vCJD or BSE. For each mouse strain, patterns of abnormal prion protein (PrP res ) deposition, astrocytosis and vacuolation were similar in the vCJD- and BSE-challenged mice. Prion protein (PrP)-positive plaques were prominent in the VM and C 5 7BL x VM mice in addition to diffuse PrP res accumulation, whereas only diffuse PrP res labelling was observed in the RIII and C57BL mice. The hippocampus was targeted in all mouse strains, as was the cochlear nucleus in the medulla, both showing consistent severe vacuolation and heavy PrP res deposition. Although the targeting of PrP res was similar in the BSE- and vCJD-infected brains, the amount and intensity of PrP res observed in the brains treated with formic acid during fixation was reduced considerably. The distribution of astrocytosis was similar to the targeting of PrP res deposition in the brain, although some differences were observed in the hippocampi of mice challenged with vCJD. We conclude that there are no significant differences in the targeting of neuropathological changes observed in the BSE- and vCJD-infected mice, consistent with the previous evidence of a link between BSE and vCJD.

Misrouted olfactory fibres and ectopic olfactory glomeruli in normal humans and in Parkinson and Alzheimer patients.

Abstract: The presence and distribution of misrouted (MR) olfactory projection fibres were studied in the olfactory bulbs of control human brains and in the brains of people who had suffered from Parkinson's and Alzheimer's disease. It appeared that MR fibres, that is, fibres that terminate deep into the glomerular layer, are a common phenomenon in the bulbs of aged people. In all but one of the bulbs studied, MR fibres are present. The amount of MR fibres is not related to age. With a few exceptions, the MR fibres are restricted to the external plexiform layer (EPL). Only in Parkinson bulbs the MR fibres occasionally form glomerulus-like structures. These pseudo glomeruli are located in the EPL. It is concluded that MR olfactory projection fibres are a normal phenomenon in the human olfactory bulb. In nonhuman mammalians, MR fibres have only been observed in foetal and neonatal olfactory bulbs. Possibly, the age-related loss of mitral cells, which are the natural synaptic targets for the olfactory projection fibres, may play a role in the aberrant behaviour of the MR olfactory receptor cell axons. The ectopic glomerulus-like structures in Parkinson bulbs share some characteristics with normal rat glomeruli that are not observed in normal human olfactory glomeruli. This may refer to possible changes in the genetic content of olfactory structures in Parkinson patients.

Time-course phosphorylation of the mitogen activated protein (MAP) kinase group of signalling proteins and related molecules following middle cerebral artery occlusion (MCAO) in rats

Abstract: Recovery from the debilitating effects of ischaemic stroke is variable and unpredictable. To maximize patient recovery, a greater understanding of the molecular mechanisms involved in regulating both apoptosis and the repair processes affecting neuronal protection, particularly in the penumbra region, is desirable. We have previously shown, in human subjects, the increased expression of several growth factors soon after stroke, together with appearance of tyrosine phosphorylated proteins, in particular mitogen activated protein (MAP) kinase (ERK1/2). In this paper, we demonstrate a relatively short-lasting ( 4 days. Evidence suggests that short-term activation of these proteins may be detrimental to neuronal survival, while their transient nature makes them unlikely to support angiogenesis, revascularization and reperfusion over a period of days and weeks. On the other hand, short-medium-term up-regulation of neuronal p-JNK, p-c-Jun, p-Stat-1 and p-p38 might be a factor in the regulation of apoptosis. Therapeutic manipulation of phosphorylation/activation of these and other important signalling intermediates might form the basis of an appropriate treatment to maximize revascularization and neuronal protection after ischaemic stroke.

AMOG/β2 and glioma invasion: does loss of AMOG make tumour cells run amok?

Abstract: The beta2 subunit of Na,K-ATPase, initially described as adhesion molecule on glia (AMOG), has been shown to mediate neurone-astrocyte adhesion as well as neural cell migration in vitro. We have investigated the expression of AMOG/beta2 in human gliomas and its effect on glioma cell adhesion and migration. Compared to normal astrocytes of human brain, AMOG/beta2 expression levels of neoplastic astrocytes were down-regulated in biopsy specimens and inversely related to the grade of malignancy. One rat and four human glioma cell lines showed complete loss of AMOG. To investigate the function of AMOG/beta2, its expression was re-established by transfecting an expression plasmid into AMOG/beta2-negative C6 rat glioma cells. In vitro assays revealed increased adhesion and decreased migration on matrigel of AMOG/beta2-positive cells as compared to their AMOG/beta2-negative counterparts. We conclude that increasing loss of AMOG/beta2 during malignant progression parallels and may underlie the extensive invasion pattern of malignant gliomas.

Relationship of Nef-positive and GFAP-reactive astrocytes to drug use in early and late HIV infection.

Abstract: Reactive astrocytosis is a well-documented feature of HIV encephalitis (HIVE), but it is unclear whether restricted infection of astrocytes contributes to this phenomenon. In addition, the part played by reactive and/or infected astrocytes in AIDS-related dementia is not fully understood. In this study of patients at different stages of the human immunodeficiency virus (HIV) infection, who had been treated at most with one antiretroviral drug, reactive astrocytes were identified by immunopositivity for glial fibrillary acidic protein (GFAP) and infected astrocytes by positivity for HIV Nef protein. Results were compared for drug-using AIDS patients with (n=9) and without (n=7) HIVE, for presymptomatic HIV-positive drug users (n=12) and for control HIV-negative subjects (n=20), including a group who used drugs (n=10). GFAP-reactive astrocytes in both grey and white matter were significantly more numerous in HIVE subjects than in each of the other groups but did not correlate with viral load. Nef-positive astrocytes were confined to HIVE cases and to white matter, but were numerous in only one subject who was treatment-naive. Nef-positive microglia were identified in all HIVE cases and in occasional AIDS and presymptomatic subjects who did not have HIVE. The results suggest that astrocytes may form an additional viral reservoir in late HIV infection and may contribute to HIVE. However, the number of GFAP-positive astrocytes was neither increased in pre-AIDS nor in drug abuse, in contrast with microglia which we have shown previously to be up-regulated in both states.

Mitochondrial dysfunction in myofibrillar myopathy.

Abstract: J. Reimann, W. S. Kunz, S. Vielhaber, K. Kappes-Horn and R. Schroder (2003) Neuropathology and Applied Neurobiology 29, 45–51 Mitochondrial dysfunction in myofibrillar myopathy ‘Myofibrillar myopathy’ defines a myopathic condition with focal myofibrillar destruction and accumulation of degraded myofibrillar elements. Despite the fact that a number of mutations in different genes as well as cytotoxic agents lead to the disease, abnormal accumulation of desmin is a typical, common feature. Pathological changes of mitochondrial morphology and function have been observed in animal models with intermediate filament pathology. Therefore, in the present study we tested for mitochondrial pathology in skeletal muscle of five patients with the pathohistological diagnosis of myofibrillar myopathy. Screening for large-scale mtDNA deletions and the frequent MERRF (myoclonic epilepsy; ragged red fibres) and MELAS (mitochondrial encephalomyopathy; lactic acidosis; stroke) point mutations was negative in all patients. Histologically, all muscle biopsies showed nonspecific abnormalities of the oxidative/mitochondrial enzyme stainings (histochemistry for reduced nicotinamide adenine dinucleotide, succinic dehydrogenase, cytochrome c oxidase), only one of them had ragged red fibres and a significant number of cytochrome c oxidase-negative fibres. Upon biochemical investigation, four of our patients showed pathologically low respiratory chain complex I activities. Only one of our patients had a pathologically low complex IV activity, while the measurements of the others were within low normal range. The single patient with pathological values for both complex I and IV was the one with the clear histological hallmarks (ragged red and cytochrome c oxidase-negative fibres) of mitochondrial pathology. She also was the only patient with clinical signs hinting at a mitochondrial disorder. Together with data from observations in desmin- and plectin-deficient mice, our results support the view that desmin intermediate filament pathology in these cases is closely linked to mitochondrial dysfunction in skeletal muscle.