The role of cytokines, astrocytes, microglia and apoptosis in Creutzfeldt-Jakob disease.

In order to investigate inflammation and apoptosis in Creutzfeldt-Jakob disease (CJD) patients, we analyzed astrocytes, microglia and apoptotic neurons in brain and IL-1beta in cerebrospinal fluid (CSF). Our results showed increased numbers of astrocytes in CJD and increased numbers of microglia and apoptotic neurons both in CJD and Alzheimer's disease (AD) as compared to controls. All these markers correlated (P < 0.001) with the severity of the neuropathological lesions. An increased IL-1beta concentration was found in AD and CJD CSF that correlated with the number of microglia and which did not change in the disease course of CJD.In conclusion, apoptotic neurons in CJD correlates to the neuropathological lesions and are probably related to the presence of inflammatory cells and cytokines which are present during the whole CJD disease process.

Cerebral amyloid angiopathy is a pathogenic lesion in Alzheimer's disease due to a novel presenilin 1 mutation.

The dense-cored plaques are considered the pathogenic type of amyloid deposition in Alzheimer's disease brains because of their predominant association with dystrophic neurites. Nevertheless, in > 90% of cases of Alzheimer's disease amyloid is also deposited in cerebral blood vessel walls (congophilic amyloid angiopathy; CAA) but its role in Alzheimer's disease pathogenesis remains enigmatic. Here, we report a family (family GB) in which early-onset Alzheimer's disease was caused by a novel presenilin 1 mutation (L282V). This was unusually severe CAA reminiscent of the Flemish amyloid precursor protein (A692G) mutation we reported previously, which causes Alzheimer's disease and/or cerebral haemorrhages. In family GB, however, the disease presented as typical progressive Alzheimer's disease in the absence of strokes or stroke-like episodes. Similarly, neuroimaging studies and neuropathological examination favoured a degenerative over a vascular dementia. Interestingly, an immunohistochemical study revealed that, similar to causing dense-cored amyloid plaques, CAA also appeared capable of instigating a strong local dystrophic and inflammatory reaction. This was suggested by the observed neuronal loss, the presence of tau- and ubiquitin-positive neurites, micro- and astrogliosis, and complement activation. Together, these data suggest that, like the dense-cored neuritic plaques, CAA might represent a pathogenic lesion that contributes significantly to the progressive neurodegeneration that occurs in Alzheimer's disease.

Detection of cytokines in human sural nerve biopsies: an immunohistochemical and molecular study.

In vitro and in vivo models have implicated numerous cytokines as major modulators of inflammation, destruction and repair in the peripheral nervous system (PNS). The in situ production of cytokines in human peripheral nerve disorders is still poorly documented. We studied the expression of interleukin (IL)-1 beta, tumor necrosis factor (TNF)-alpha, IL-6, IL-10, IL-4, IL-3 and nerve growth factor (NGF) in 35 human sural nerve biopsies using immunohistochemistry; additional reverse transcription-polymerase chain reaction and mRNA in situ hybridization were performed for IL-4 and NGF. Expression of IL-1 beta and TNF-alpha was shown in both morphologically normal nerves and various neuropathies, and macrophages appeared as their predominant source. Levels of IL-1 beta and TNF-alpha expression were significantly correlated (P < 0.01) with each other and with expression of NGF. Multiple endoneurial sources were suggested for IL-6 and IL-10 with low immunoreactivity in the vast majority of cases. Conversely, IL-4 and IL-3 expression were found in neuropathies of various etiologies and Schwann cells appeared to be a predominant source of IL-4 in double-labeling immunofluorescence studies. IL-3 immunoreactivity correlated with IL-1 beta, TNF-alpha and IL-6. In this retrospective study, no specific cytokine profile of expression could be assigned to a precise subgroup of neuropathies. This is the first report of IL-4 and IL-3 expression in human neuropathies, and it may be important given the potential role of these cytokines in modulating macrophage activity in the PNS.

Familial Creutzfeldt-Jakob disease in a patient carrying both a presenilin 1 missense substitution and a prion protein gene insertion.

We describe a patient who was clinically diagnosed with familial early-onset Alzheimer disease (AD) carrying both the E318G substitution in presenilin 1 (PSEN1) and an insertion of 7 octapeptide coding repeats in the prion protein gene (PRNP). Neuropathological examination revealed elongated cerebellar prion protein deposits in the absence of AD pathology. Further analysis of other family members showed that the Creutzfeldt-Jakob disease phenotype in this family was caused solely by the PRNP insertion. This observation is consistent with our previous finding that PSEN1 E318G is not causally related to AD.

Behavioral disturbances without amyloid deposits in mice overexpressing human amyloid precursor protein with Flemish (A692G) or Dutch (E693Q) mutation.

The contribution of mutations in the amyloid precursor protein (APP) gene known as Flemish (APP/A692G) and Dutch (APP/E693Q) to the pathogenesis of Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis of the Dutch type, respectively, was studied in transgenic mice that overexpress the mutant APP in brain. These transgenic mice showed the same early behavioral disturbances and defects and increased premature death as the APP/London (APP V717I), APP/Swedish (K670N, M671L), and other APP transgenic mice described previously. Pathological changes included intense glial reaction, extensive microspongiosis in the white matter, and apoptotic neurons in select areas of the brain, while amyloid deposits were absent, even in mice over 18 months of age. This contrasts with extensive amyloid deposition in APP/London transgenic mice and less pronounced amyloid deposition in APP/Swedish transgenic mice generated identically. It demonstrated, however, that the behavioral deficiencies and the pathological changes in brain resulting from an impaired neuronal function are caused directly by APP or its proteolytic derivative(s). These accelerate or impinge on the normal process of aging and amyloid deposits per se are not essential for this phenotype.

Detection of beta-A4 amyloid and its precursor protein in the muscle of a patient with juvenile neuronal ceroid lipofuscinosis (Spielmeyer-Vogt-Sjögren).

Muscle biopsy tissue from a patient affected by the juvenile form of neuronal ceroid lipofuscinosis (NCL) was studied immunohistochemically using antibodies to beta-amyloid peptide and amyloid precursor protein. Positive reaction in muscle was specifically localized to autophagic vacuoles and blood vessel walls. Increased acid phosphatase reaction suggested enhanced lysosomal activity. We hypothesize that beta-amyloid is deposited in NCL muscle by a lysosomal mechanism similar to that proposed in other disorders involving beta-amyloid.

Identification and localization of ataxin-7 in brain and retina of a patient with cerebellar ataxia type II using anti-peptide antibody.

Autosomal dominant cerebellar ataxias (ADCAs) are a complex group of neurodegenerative disorders characterized by progressive degeneration of the cerebellum, brain stem and spinal cord. The spinocerebellar ataxia type 7 (SCA7) is associated with pigmentary macular dystrophy and retinal degeneration leading to blindness caused by a CAG/polyglutamine (polyGln) expansion in the coding region of the SCA7 gene/protein. The SCA7 gene codes for ataxin-7, a protein of unknown function. To investigate its cellular and subcellular localization, we have developed a sequence-specific polyclonal antibody against the N-terminal part of the protein. Immunohistochemical analysis indicated that ataxin-7 accumulates as single nuclear inclusion (NI) in the cells of the brain and retina of a SCA7 patient but not of controls. The 1C2 antibody, directed against expanded polyGln, confirmed the aggregation of mutant ataxin-7 in these NIs. Furthermore, ubiquitin was found in these aggregates, suggesting that mutant ataxin-7 is a target for ubiquitin-dependent proteolysis, but resistant to removal. Electron microscopic studies using immunogold labeling showed that ataxin-7 immunoreactive NIs appear as dense aggregates containing a mixture of granular and filamentary structures. Together, these data confirm the presence of NIs in brain and retina of a SCA7 patient, a common characteristic of disorders caused by expanded CAG/polyGln repeats.

Immunoreactivity of presenilin-1 and tau in Alzheimer's disease brain.

Mutations in the presenilin-1 gene (PS-1) on chromosome 14 are causative for early-onset familial Alzheimer's disease (AD). In order to study the localization of PS-1 in human brain, a polyclonal antibody, SB63, against a N-terminal epitope of PS-1 (25VRSQNDNRERQEHND40), was raised in rabbits and characterized. Immunolabeling with SB63 of formalin-fixed sections of hippocampus from cases of PS-1-linked AD (PS-1 I143T (AD/A), G384A (AD/B)), sporadic AD, and controls showed a predominant neuronal staining pattern with a stronger immunoreactivity in pyramidal neurons. Staining was mainly granular and localized in the neuronal cell body as well as in neuronal processes. In AD some dystrophic neurites surrounding the amyloid plaques were stained, but no immunoreactivity was observed in the amyloid core. Although PS-1 was present in tangle bearing neurons, colocalization of PS-1 and tau could not be detected using immunofluorescence double labeling. Our data indicate that the pattern of PS-1 immunoreactivity in the hippocampus does not substantially differ between PS-1-linked AD, sporadic AD, and controls.

Discordant light microscopic, electron microscopic, and in vitro contracture study findings in a family with central core disease.

We report a family that was referred to our laboratory after a fatal malignant hyperthermia (MH) accident during general anesthesia. Postmortem study of different muscles of the proband pointed retrospectively to the presence of central core disease (CCD). Of the 8 family members investigated by histology and in vitro contracture testing (IVCT) 5 were found to be MH-susceptible. Neurological examination was completely normal. Histologically, these 5 patients showed a highly variable proportion (6-89%) of cores in type 1 fibers on light microscopy. In 3 patients definite central cores were found, in 1 patient multicore disease was diagnosed, and 1 patients presented with a mixed central/paracentral form. Electron microscopy could detect cores in only 4 out of 5 patients. These results demonstrate the difficulty to diagnose central or multicore disease and suggest that mixed forms within the same family may occur. The one histologically dubious patient in this family shows that the most sensitive test for the diagnosis of this myopathy might be the IVCT.

Microtubule-associated protein tau epitopes are present in fiber lesions in diverse muscle disorders.

The microtubule-associated protein tau is a major cytoskeletal protein involved in the neurofibrillary tangles of Alzheimer's disease. Although tau is predominantly a neuronal protein, it has been demonstrated in glia and other nonneuronal cells. We describe the presence of microtubule-associated protein tau epitopes in various muscle fiber lesions in oculopharyngeal and Becker muscular dystrophy, dermatomyositis, central core disease, neurogenic atrophy, and in the recovery phase of an attack of malignant hyperthermia. Western blot demonstrated a 100- to 110-kd tau-immunoreactive protein probably corresponding to 'big tau' as described in peripheral nerves. Tau immunoreactivity in muscle fiber lesions usually co-localized with tubulin, although electron microscopy failed to show an increase in microtubules. Tau and tubulin reactivity also correlated with the presence of desmin and vimentin epitopes. Possible explanations for the presence of tau are briefly discussed.

The abnormal phosphorylation of tau protein at Ser-202 in Alzheimer disease recapitulates phosphorylation during development.

Tau is a neuronal phosphoprotein whose expression is developmentally regulated. A single tau isoform is expressed in fetal human brain but six isoforms are expressed in adult brain, with the fetal isoform corresponding to the shortest of the adult isoforms. Phosphorylation of tau is also developmentally regulated, as fetal tau is phosphorylated at more sites than adult tau. In Alzheimer disease, the six adult tau isoforms become abnormally phosphorylated and form the paired helical filament, the major fibrous component of the characteristic neurofibrillary lesions. We show here that Ser-202 (in the numbering of the longest human brain tau isoform) is a phosphorylation site that distinguishes fetal from adult tau and we identify it as one of the abnormal phosphorylation sites in Alzheimer disease. The abnormal phosphorylation of tau at Ser-202 in Alzheimer disease thus recapitulates normal phosphorylation during development.

The phosphatase inhibitor okadaic acid induces a phosphorylated paired helical filament tau epitope in human LA-N-5 neuroblastoma cells.

Recently, a mitogen activated protein kinase has been implicated in the generation of a phosphorylated paired helical filament (PHF) epitope recognized by the monoclonal antibody AT8. This epitope consists of phosphorylated serines 199 and/or 202 of the human microtubule associated protein tau. Theoretically, aside from abnormal kinase activity, inhibition of phosphatase activity could also be involved in the abnormal phosphorylation status of the microtubule associated protein tau. To investigate this, we incubated LA-N-5 neuroblastoma cells with okadaic acid, a specific inhibitor of phosphatase 2A. We found that incubating neuroblastoma cells with okadaic acid induces the abnormally phosphorylated AT8 epitope. The effect of okadaic acid is time and dose dependent and is reversible. Our findings suggest that phosphatase activity is important in the regulation of the phosphorylation state of tau. Phosphatases may act directly on tau or may influence the activity of mitogen activated protein kinase. Incubation of LA-N-5 neuroblastoma cells with okadaic acid provides a cellular model in which the generation of a well-defined PHF-tau epitope can be investigated.

Rimmed vacuoles of inclusion body myositis and oculopharyngeal muscular dystrophy contain amyloid precursor protein and lysosomal markers.

Rimmed vacuoles are small areas of focal destruction of muscle fibres, found in inclusion body myositis, oculopharyngeal muscular dystrophy and other muscle disorders. They are known to contain amyloid proteins, probably of beta-amyloid type. We examined rimmed vacuoles immunohistochemically in 12 patients with inclusion body myositis and two patients with oculopharyngeal muscular dystrophy with antibodies to beta-amyloid precursor protein and cathepsin B and D. We found evidence for the presence of all these markers in rimmed vacuoles. These results confirm the presence of beta-amyloid in rimmed vacuoles, and provide additional support for the hypotheses that rimmed vacuoles are of lysosomal origin and that lysosomes are probably important in the metabolism of amyloid precursor protein.

Duchenne muscular dystrophy immunohistochemistry of foetal muscles.

Between 1989 and 1991, we realized 36 prenatal DNA diagnoses in 23 families at risk for Duchenne muscular dystrophy (DMD). The families were previously analyzed using multiplex polymerase chain reaction (PCR) analysis, Southern blot hybridization with cDNA markers, pulse field gel electrophoresis (PFGE) and linkage studies with polymorphic DNA markers. Eighteen male foetuses were examined and seven were found to be affected. Immunohistochemistry (IHC) of muscle tissue was realized after abortion in four foetuses aged 12 to 22 weeks. Three age-matched controls were used. Poly- and monoclonal antibodies against different epitopes of dystrophin were used. A polyclonal spectrin antibody was utilized to check for membrane integrity. DNA analysis of chorion villi had demonstrated an out of frame deletion in all four foetuses, later confirmed on abortion material. The different epitopes of dystrophin were absent on immunohistochemical sections while they were present in control cases. Spectrin was present in patients as well as in controls. IHC can be used to confirm the results of DNA diagnosis of DMD in foetuses.

Immunocytochemical detection of the growth-associated protein B-50 by newly characterized monoclonal antibodies in human brain and muscle.

The growth-associated protein B-50 also termed GAP-43, F1, pp46, P-57 and neuromodulin is a nervous tissue-specific protein kinase C (PKC) substrate that is considered to play a major role in neurite formation, regeneration, and neuroplasticity. We describe the isolation of seven mouse monoclonal antibodies (Mabs) directed against B-50. The Mabs are produced against the bovine B-50, selected by ELISA for cross-reactivity with its human counterpart, and evaluated on Western blots in comparison with the well-characterized affinity-purified rabbit polyclonal antibodies to rat-B-50. The Western blots show that the Mabs NM1, NM4, and NM6 recognize specifically the B-50 of bovine, human, and rat brain extract and the purified PKC phosphorylated and unphosphorylated rat B-50 isoforms. The Mabs NM2 and NM3 cross-react with bovine B-50 immunoreactive c-kinase substrate (BICKS), a protein sharing a 17 amino acid sequence homology with B-50. Two Mabs are useful for the detection of B-50 immunoreactivity in formalin-fixed human and rat brain tissues. In human specimen of the hippocampus, a characteristic neuropil distribution of B-50 is detected by the Mabs. In human muscle, Mabs reveal B-50 in nerve bundles and in axons at motor end plates. Thus, these Mabs are useful in investigating the function and localization of the B-50 protein.

Affinity purification of human tau proteins and the construction of a sensitive sandwich enzyme-linked immunosorbent assay for human tau detection.

Immunoaffinity chromatography with a monoclonal antibody produced against bovine tau protein was used to purify tau proteins from human brain. Fifty grams of brain tissue yielded approximately 2 mg of pure tau proteins. The affinity-purified human tau was used to produce a high-titered rabbit anti-human tau serum. The monoclonal anti-tau antibody and the polyclonal rabbit anti-tau serum were then used to construct a sandwich enzyme-linked immunosorbent assay for detection of human tau proteins, with a sensitivity of 1 ng/ml.

Becker-type muscular dystrophy. Report of a family with one postmortem study.

Becker-type muscular dystrophy (BMD) is reported in two brothers. In one of the patients, the molecular demonstration of an in-frame deletion of exons 45, 46 and 47 has confirmed the clinical and pathological diagnosis of BMD. The autopsy of the other patient revealed mild neuronal losses in the anterior horns at C8, lumbar and sacral levels of the spinal cord. Mild neuronal losses in the spinal cord may explain the mixed type of neurogenic-myogenic features in the skeletal muscles of adult BMD patients.

Specific monoclonal antibodies against normal microtubule-associated protein-2 (MAP2) epitopes present in Alzheimer pathological structures do not recognize paired helical filaments.

We have developed monoclonal antibodies that detect normal microtubule-associated protein-2 (MAP2) epitopes in routinely fixed, paraffin-embedded tissue. The somatodendritic distribution of MAP2 in bovine and human nervous tissue was confirmed with several of these antibodies. Furthermore, some of these antibodies immunohistochemically labeled certain pathological structures in Alzheimer brain, especially neurites in senile plaques. Electron microscopic observations, however, indicate that these MAP2 epitopes are not located in the Alzheimer paired helical filaments themselves, but in amorphous granular structures coexistent with them. While the pathological nature of these structures is undetermined, they may represent artefactual modifications of normal cytoskeletal components.

Monoclonal antibodies with selective specificity for Alzheimer Tau are directed against phosphatase-sensitive epitopes.

A modified form of the microtubule-associated protein Tau is the major component of the paired helical filaments (PHF) found in Alzheimer's disease. The characterization of these posttranslational Tau modifications is hindered by the lack of sufficient PHF-Tau-specific markers. Here we describe several monoclonal antibodies, prepared by immunization with PHF, two of which showed a selective specificity for PHF-Tau without cross-reactivity with normal Tau. Epitope recognition by these two monoclonals was sensitive to alkaline phosphatase treatment. In Western blotting these monoclonal antibodies reacted specifically with the abnormally phosphorylated epitopes on Alzheimer's disease-associated PHF-Tau. One of the new antibodies can be used for the construction of a sandwich enzyme-linked immunosorbent assay for the specific detection of PHF-Tau without cross-reactivity to normal Tau proteins.