Manuel B. Graeber

Surveillance, Intervention and Cytotoxicity: Is There a Protective Role of Microglia?

The study of microglial cell biology has become the key to understanding the brains fundamental tissue reactions as well as the cellular mechanisms underlying CNS disease. This article focuses on glial-neuronal interactions with special reference to human pathology. Three important areas of brain pathology are critically reviewed: multiple sclerosis and CNS inflammation, the brain in AIDS and opportunistic infections, and neurodegenerative disorders. Although microglial cytotoxicity may cause bystander damage, e.g. in ischemia, there is little evidence to support the view that microglial activation per se is pathogenic. Results suggesting that one important normal function of microglia is to protect the integrity of the central nervous system are discussed. The concept is proposed that microglia function as a highly developed guardian to the CNS.

On the question of apoptosis in the parkinsonian substantia nigra.

Abstract Apoptosis has been postulated as a mechanism of nerve cell death in Parkinson’s disease. In the present study, the substantia nigra of 22 neuropathologically confirmed Parkinson cases and 8 control brains was studied using the in situ end-labeling (TUNEL) method. About 50% of parkinsonian brains showed a small number of TUNEL-positive glial cells in the substantia nigra, whereas no neurons showed convincing TUNEL positivity or any morphological signs of apoptosis. No correlation was observed between the number of TUNEL-positive glial cells and microglial activation. Our results fail to demonstrate apoptosis as a mechanism of cell death in Parkinson’s disease.

Synaptic stripping in the human facial nucleus

SummaryAn autopsy case of severe peripheral facial nerve paresis with disconnection of synapses from facial motor neurons is reported. A 77-year-old man presented with left-sided otitis media and subsequent development of facial nerve paresis. Three months later, the patient died of an acute gastrointestinal bleeding from a chronic duodenal ulcer. Gross inspection of the brain revealed non-stenosing arteriosclerotic vascular changes and a single small cystic lesion in the right putamen. Microscopically, marked chromatolytic changes were observed in the left facial nucleus. Immunocytochemistry for synaptophysin revealed a marked loss of afferent synaptic contacts from somatic and stem dendritic surface membranes of all chromatolytic motor neurons. Wrapping of a number of neurons by newly formed glial fibrillary acidic protein-positive astrocytic cell processes could be detected in the regenerting facial motor nucleus. In addition, expression of HLA-DR was increased on a small number of microglia and pertivascular cells. These changes were absent from the contralateral, normal-appearing facial nucleus. To our knowledge, this case provides the first evidence for disconnection of synapses following peripheral nerve lesioning in humans. Occurrence of synaptic stripping is likely to explain nuclear hyperexcitability and failure of recovery of complex fine motor movements that are commonly observed following peripheral injury to the facial nerve.

Microglia and the development of spongiform change in Creutzfeldt-Jakob disease.

Recent in vitro experiments suggest that neurotoxicity of the prion protein is dependent on the presence of microglia. We have studied 11 cases of Creutzfeldt-Jakob disease (CJD) using immunocytochemistry in combination with computerized image analysis to clarify the relationship between spongiform change and microglial activation. MHC class II-positive microglia were almost exclusively confined to cortical gray matter where the neuropil area occupied by these cells exceeded that of controls more than 350-fold. In cortical regions with a bimodal distribution of spongiform degeneration, the presence of class II-positive microglia correlated well with the presence of vacuolation in layer V, but significantly less with spongiform change in layers II and III. In areas where spongiform degeneration affected the entire depth of the cortex, activated microglia were predominantly located in the inner one-half of the cortex or were evenly distributed throughout all cortical laminae. Here, microglia exhibited atypical, tortuous cell processes and occasionally intracytoplasmic vacuoles, suggesting that microglia themselves may become a disease target. Taken together, our results provide indirect evidence against an early causative involvement of microglia in the development of spongiform change. At later stages, however, diseased microglia could produce harmful factors which mediate both astrogliosis and neuronal injury.

CR3/43, a marker for activated human microglia: application to diagnostic neuropathology

Sir-We have used the major histocompatibility complex (MHC) class I1 specific monoclonal antibody (mAb) CR3/43 (DAKO, M 775) to study microglia in routinely processed, paraffin-embedded human central nervous tissue. CR3/43 is directed against the P-chain of all products of the MHC class I1 gene subregions HLA-DR, HLA-DQ, and HLA-DP. We have investigated CR3/43 staining characteristics in a variety of disease conditions, and here report on its use as a sensitive diagnostic marker for activated human microglia. CR3/43 labelling characteristics were evaluated by using a panel of 50 autopsies and surgical cases including senile dementia of the Alzheimer type (SDAT; n = 4 ) , Parkinsons disease (n = 5). progressive supranuclear palsy (PSP; n = l), HIV-1 infection (n=15) , multiple sclerosis (MS: n= 3). acute perivenous encephalitis (n= l), subacute sclerosing panencephalitis (SSPE; n = l), toxoplasmosis (n= 1). hypoxic brain injury ( n = 2), normal ageing (n=2) , pilocytic astrocytoma (n= 5), astrocytic tumours graded WHO 11-IV (n=6), and glioblastoma (n=4). All tissue samples had been routinely fixed in unbuffered 3.7% formalin, processed through xylene or acetone and embedded into paraffin. Paraffin sections. 2 pm, were used for all stains. CR3/43 labelling was compared to the macrophage/microglia markers L-CA (DAKO M 701; 1 : l O O ) . KP1 (DAKO M 814; 1:50), and RCA-1 (biotinylated, Vector; 1 0 pg/ml). In addition, immunocytochemistry employing the class I1 specific mAb TAL.lB5 (DAKO M 7 4 6 ; 1:50), and an mAb directed against the astrocyte-specific glial fibrillary acidic protein, GFAP (DAKO M 761; 1:50), was performed. Double-labelling on serial sections was carried out for CR3/43 and L-CA and GFAP. Immunocytochemical staining results were visualized using a standard biotin-streptavidin-peroxidase detection system [3]. The CR3/43 mAb is known to label B cells. interdigitating reticulum cells, Langerhans cells and many macrophages of peripheral organs. CR3/43 binding to human microglia has been occasionally described [5, 11, 121 but not studied in a systematic manner. We found CR3/43 labelling of microglia in all pathological situations examined (Figure 1-5). Yet, morphology of the stained cells was that of activated microglial cells bearing stout cell processes rather than that of highly ramified, resting microglia [2, 10, 121. Resting. RCA-l+ microglia of histologically normal tissue, including cases without known CNS disease. were C R 3 / 4 3 ~. The microglial/macrophage nature of the CR3/43* parenchymal cells was conhmed by doublelabelling for CR3143 and the markers listed above. Interestingly, in tissue samples exhibiting no or only slight pathological change in conventional histological stains, CR3/43 immunoreactivity of activated microglia allowed detection and localization OC even subtle tissue pathology. All of the other markers tested, including GFAP, were much less sensitive in this respect. We have not found constitutive expression of MHC class I1 molecules by microglia [l], although this may be due to technical factors [8]. It should be noted, however, that reactive changes in microglial phenotype have been observed within minutes following ischaemia [9]. suggesting that microglia are the most susceptible sensors of CNS pathology [ 71. Thus, agonal and early post-mortem changes might be sufficient to induce low levels of MHC

Association of the mitochondrial tRNAA4336G mutation with Alzheimer's and Parkinson's diseases

Alzheimers and Parkinsons diseases (AD, PD) are among the most common neurodegenerative disorders in adults. Both AD and PD have a complex aetiology, and it is widely considered that genetic factors, acting independently or in concert with other genetic and/or environmental factors, modify the risk of developing them. While the apolipoprotein E (ApoE) ε4 allele represents an established risk factor for familial and sporadic late‐onset AD, it has been suggested that a common polymorphism in the α1‐antichymotrypsin gene modifies the ApoE ε4 dosage effect in AD. Moreover, it has been proposed that a mitochondrial tRNAGln sequence variant (A4336G transition) confers an increased risk for both AD and PD. This finding is of particular interest as the A4336G mutation seems to predispose to two clinically and neuropathologically distinct neurodegenerative disorders. We have examined the allelic frequencies of these putative susceptibility genes in 28 neuropathologically confirmed cases of AD, 23 cases with Lewy‐body PD and 100 age‐matched controls without clinical or histological evidence of neurodegenerative disease. The ApoE ε4 allele frequency was significantly over‐represented in AD patients vs controls (0.35 vs 0.11) but we could not find evidence for an association between the α1‐antichymotrypsin AA genotype, the ApoE ε4 allele and AD. In contrast, the mtDNAA4336G mutation was present in one of 28 AD cases and in two of 23 PD patients, whereas no mutation was found in 100 age‐matched controls (P<0.05). Our data therefore support the hypothesis that the mitochondrial A4336G mutation represents a risk factor for AD and PD.

Novel mutations of mitochondrial complex I in pathologically proven Parkinson disease

ABSTRACT Complete sequence analysis of all mitochondrial complex I genes was performed in 22 cases of neuropathologically confirmed idiopathic Parkinson disease (PD). DNA from the substantia nigra was used as a template for polymerase chain reaction-based genomic sequencing. Seven novel mutations causing the exchange of amino acids were detected in subunit genes ND1 (3992 C/T, 4024 A/G), ND4 (11253 T/C, 12084 C/T), ND5 (13711 G/A, 13768 T/C), and ND6 (14582 T/C). In addition, five known missense mutations affecting the ND1 (3335 T/C, 3338 T/C), ND2 (5460 G/A), ND3 (10398 A/G), and ND5 (13966 A/G) genes as well as three secondary LHON mutations (4216 T/C, 4917 A/G, 13708 G/A) were found in the PD group. Among the novel mutations, the 11253 T/C transition which changes a conserved isoleucine residue into threonine is most likely to be of functional relevance. Furthermore, 43 synonymous polymorphisms were detected in PD brains, including 20 novel sequence variants. Haplogroup analysis revealed that most unique missense mutations were found in PD cases belonging to the Dc haplogroup. Our data are in line with the view that PD is not a single disease entity but comprises a genetically heterogeneous group of disorders. The results of our study further suggest that 90% or more of all idiopathic PD cases are not due to sequence variation of mitochondrial complex I, but that mitochondrial mutations may play a pathogenic role in a subset of PD patients.

Use of neuropathological tissue for molecular genetic studies: parameters affecting DNA extraction and polymerase chain reaction

Nuclear and mitochondrial DNA were extracted from gray matter of human cerebral cortex which had either been formalin-fixed and embedded into paraffin or stored in formalin for up to 26 years. Extraction conditions were optimized for proteinase K digestion, i.e., enzyme concentration, digestion temperature and incubation time. Using the polymerase chain reaction (PCR), DNA was successfully amplified from archival material and sequenced employing a direct nonradioactive cycle sequencing protocol. In general, tissue embedded into paraffin following brief fixation in formalin gave good quantitative results, i.e., up to 1 μg DNA/mg tissue were extracted. This yield was at least one order of magnitude higher than that obtained with tissue stored in formalin. However, paraffin-embedded neuropathological material was found to contain an as-yet-unidentified PCR inhibitor, and a deleterious effect of long-term fixation in unbuffered low-grade formalin was clearly detectable. Importantly, both paraffin-embedded tissue blocks and human brain that had been stored in formalin for many years yielded DNA sufficient for qualitative analysis. The implications of these findings for the use of neuropathological material in molecular genetic studies are discussed.

MITOCHONDRIAL NADH DEHYDROGENASE AND CYP2D6 GENOTYPES IN LEWY-BODY PARKINSONISM

The cause of nerve‐cell death in sporadic Parkinsons disease remains unknown. Although environmental factors have been traditionally implicated in the etiology of Parkinsons disease, recent studies strongly suggest that there is a genetic contribution to this multifactorial disorder. We studied archival brain tissue from clinically and neuropathologically verified cases of Parkinsons disease, using nonradioactive cycle sequencing and restriction enzymatic analysis of polymerase chain reaction products. Twenty‐one Parkinsonian brains with brain stem Lewy‐bodies and 77 control brains were genotyped at two mitochondrial loci previously implicated in the etiology of neurodegenerative disease. In addition, genotyping was performed for two alleles of the debrisoquine 4‐hydroxylase gene (CYP2D6). A heteroplasmic mtDNAG5460A missense mutation in the ND2 subunit gene of NADH dehydrogenase was three times more frequent in Parkinson cases (4/21) compared to controls (5/77). A homoplasmic mtDNAA4336G transition which alters the mitochondrial tRNAGln gene product was found in one Parkinson case. Frequencies of the CYP2D6G1934A and CYP2D6C2938T alleles were not significantly different between Parkinson cases and controls. Two Parkinsonian brains with high degrees of heteroplasmy for the ND2G5460A mutation and one CYP2D6C2938T homozygous case showed very high numbers of Lewy‐bodies in the substantia nigra. The results of this study are in line with the concept that different genetic loci may be involved in Parkinsons disease susceptibility. They provide a hint that the ND25460 mutation, in combination with other factors, could play a role in disease pathogenesis in a subset of patients.

Long-lasting perivascular accumulation of major histocompatibility complex class II-positive lipophages in the spinal cord of stroke patients: possible relevance for the immune privilege of the brain

Abstract Six cases of middle cerebral artery occlusion are presented in which the cellular changes accompanying descending degeneration of the lateral corticospinal tract were studied at different time points (5 days–10 years) following the insult. Microglia and perivascular cells were found to ingest large amounts of myelin degradation products, while expressing high levels of major histocompatibility complex (MHC) class II molecules. Activation of perivascular macrophages, as indicated by increased class II expression, lasted for many years and appeared to follow down-regulation of both phagocytic activity and class II expression on parenchymal microglia. TUNEL labeling was absent from both microglia and perivascular cells at all time points investigated. Indirect evidence is presented that microglia may transfer myelin degradation products to the perivascular space. Perivascular cells which express MHC class II molecules constitutively do not appear to leave the perivascular compartment in large numbers and could release myelin degradation products into the cerebrospinal fluid. The possible immunological consequences of these findings are discussed with respect to their possible relevance for antigen presentation and autoimmune central nervous system disease.