Persistent activation of p38 mitogen-activated protein kinase in a mouse model of familial amyotrophic lateral sclerosis correlates with disease progression.

The p38 mitogen-activated protein kinase (p38MAPK) is activated via phosphorylation in neurones and glial cells by a variety of stimuli including oxidative stress, excitotoxicity, and inflammatory cytokines. Activated p38MAPK can in turn induce phosphorylation of cytoskeletal proteins and activation of cytokines and nitric oxide, thus contributing to neurodegeneration. We investigated the expression and distribution of p38MAPK in the spinal cord of transgenic mice expressing a superoxide dismutase 1 mutation (SOD1G93A), a model of familial amyotrophic lateral sclerosis (ALS). Accumulation of p38MAPK was found by immunoblotting in the spinal cord of G93A mice during the progression of disease, but no changes were detected in its mRNA levels. Immunostaining for phosphorylated p38MAPK in lumbar spinal cord sections of SOD1G93A mice at the presymptomatic and early stages of disease showed an increased labeling in motor neurones that colocalized with phosphorylated neurofilaments in vacuolized perikarya and neurites, as detected by confocal microscopy. As the disease progressed, activated p38MAPK also accumulated in hypertrophic astrocytes and reactive microglia, as demonstrated by colocalization with GFAP and CD11b immunostaining, respectively. These data suggest that activation of p38MAPK in motor neurons and then in reactive glial cells may contribute, respectively, to the development and progression of motor neuron pathology in SOD1G93A mice.

Molecular mechanisms of neuronal death in the dorsal lateral geniculate nucleus following visual cortical lesions.

We investigated the molecular mechanisms of cell death in the dorsal lateral geniculate nucleus of the rat, following suction lesion of the visual cortex at birth or in the third postnatal week, using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) technique and immunohistochemistry for caspase-3, -7, -8, and cleaved poly(ADP-ribose) polymerase. Following lesion at birth, TUNEL-positive neurons were found in the dorsal lateral geniculate nucleus between 24 h and 3 days after lesion, with a peak on the second day. Shorter survival times (12-18 h) resulted in labeling of very few neurons in dorsal lateral geniculate nucleus and of several neurons in the perilesional cortex. Activated caspase-3 was expressed from the first to the third days after lesion, whereas cleaved poly(ADP-ribose) polymerase and activated caspase-8 were expressed on the second and third day. Activated caspase-7 was expressed mainly in pretectal nuclei. Caspase-3 activation coincided with the appearance of TUNEL-positive profiles, but decreased earlier than TUNEL. In the ipsi- and contralateral cerebral cortex, all parameters were unchanged. In animals lesioned in the third week, rare apoptotic thalamic neurons were detected as TUNEL- and activated caspase-3-positive profiles 2 days after cortical ablation, and were still present 1 week after lesion.Thus, early target ablation has dramatic effects on neonatal thalamic neurons, which die following activation of caspases 3 and 8. In contrast, cortical neurons are relatively unaffected by target deprivation. Compared with early lesions, late lesions induce a limited thalamic cell death, that persists over time.

Extensive DNA fragmentation in oxyphilic cell lesions of the thyroid.

The in situ end-labeling (ISEL) method demonstrates DNA fragmentation, commonly regarded as a marker of apoptosis. We investigated by the ISEL procedure a series of 52 thyroid lesions, including 24 lesions of mitochondrion-rich oxyphilic cells, both benign and malignant, and 28 non-oxyphilic control tumors. A high percentage of nuclear ISEL staining (approximating to 100% in most cases) was observed in the vast majority of oxyphilic cells from both adenomas and carcinomas, in the absence of morphological apoptotic changes and with no immunocytochemical evidence of caspase activation. This pattern of DNA fragmentation was not observed in non-oxyphilic lesions and was confirmed in total extracted DNA. Moreover, a peculiar cytoplasmic staining was also observed in oxyphilic cells from both benign and malignant lesions, probably related to abnormal fragmentation of mitochondrial DNA. Similar staining patterns were detected in oxyphilic cell tumors of other organs (parathyroids, salivary glands, and kidneys). These findings are consistent with an extensive DNA fragmentation peculiar to oxyphilic cells, which is not directly related to apoptosis and whose origin and biological significance are presently unknown.

Cellular and transgenic models of familial prion diseases.

Prion diseases are fatal neurodegenerative disorders of humans and animals, which result from the conformational conversion of a normal, cell surface glycoprotein (PrP(C)) into a pathogenic isoform (PrP(Sc)) that is the main component of infectious prions (1,2) . Familial prion diseases, which include 10% of the cases of Creutzfeldt-Jakob disease and all cases of Gerstmann-Sträussler syndrome and fatal familial insomnia, are linked in an autosomal dominant fashion to point and insertional mutations in the PrP gene on chromosome 20 (3,4). These mutations are presumed to favor spontaneous conversion of PrP to the PrPSc state. One way to experimentally model familial prion diseases is to express PrP molecules carrying disease-associated mutations in either cultured mammalian cells or transgenic mice. The authors review their own work using these two kinds of model systems, which have provided complementary information about the PrPC?PrP(Sc) conversion process, and about the pathogenic effects of mutant PrP.

Activation of the JNK/p38 pathway occurs in diseases characterized by tau protein pathology and is related to tau phosphorylation but not to apoptosis.

JNK and p38, two members of the MAP kinase family, are strongly induced by various stresses including oxidative stress and have been involved in regulation of apoptosis. As both kinases phosphorylate tau protein in vitro, we have investigated their immunohistochemical localization in a group of neurodegenerative diseases characterized by intracellular deposits of hyperphosphorylated tau. Cases included Alzheimer disease, Pick disease, progressive supranuclear palsy, corticobasal degeneration, Gerstmann-Sträussler-Scheinker disease-Indiana kindred, and frontotemporal dementia with parkinsonism linked to chromosome 17. In all tissue samples, strong immunoreactivity for both MAP kinases was found in the same neuronal or glial cells that contained tau-positive deposits. By double immunohistochemistry, JNK and p38 colocalized with tau in the inclusions. Analysis of apoptosis-related changes (DNA fragmentation, activated caspase-3) showed that the expression of JNK and p38 was unrelated to activation of an apoptotic cascade. Our data indicate that phospho-JNK and phospho-p38 are associated with hyperphosphorylated tau in a variety of abnormal tau inclusions, suggesting that these kinases may play a role in the development of degenerative diseases with tau pathology.

Defective neurogenesis in citron kinase knockout mice by altered cytokinesis and massive apoptosis.

Citron-kinase (Citron-K) has been proposed by in vitro studies as a crucial effector of Rho in regulation of cytokinesis. To further investigate in vivo its biologic functions, we have inactivated Citron-K gene in mice by homologous recombination. Citron-K-/- mice grow at slower rates, are severely ataxic, and die before adulthood as a consequence of fatal seizures. Their brains display defective neurogenesis, with depletion of specific neuronal populations. These abnormalities arise during development of the central nervous system due to altered cytokinesis and massive apoptosis. Our results indicate that Citron-K is essential for cytokinesis in vivo but only in specific neuronal precursors. Moreover, they suggest a novel molecular mechanism for a subset of human malformative syndromes of the CNS.

Carnosine-related dipeptides in neurons and glia.

Carnosine-related dipeptides have been demonstrated to occur in the nervous tissue of many vertebrates, including humans. Although several hypotheses have been formulated, to date their precise physiological role in the nervous system remains unknown. This article will review the studies on the presence and distribution of these dipeptides in the nervous system of different classes of vertebrates. It will focus on the most recent data on their cellular localization and potential functions in mammals. The studies on localization of carnosine-related dipeptides show a complex pattern of expression that involves both neuronal and glial cell types. The glial localization, widely distributed throughout the whole brain and spinal cord, includes a subset of both mature astrocytes and oligodendrocytes, whereas the neuronal localization is restricted to a particular type of neurons (the olfactory receptor neurons), and to restricted populations of putative migrating neurons and neuroblasts. There is no definitive demonstration of the function of these dipeptides in the various cell types. However, a wide array of evidence suggests that carnosine-related dipeptides could act as natural protective agents. Moreover, recent studies have suggested that, as previously postulated for the olfactory receptor neurons, in mature functional glial cells as well, carnosine-related dipeptides could be implicated in a neuromodulatory functional mechanism.

Accumulation of protease-resistant prion protein (PrP) and apoptosis of cerebellar granule cells in transgenic mice expressing a PrP insertional mutation.

We have generated lines of transgenic mice that express a mutant prion protein (PrP) containing 14 octapeptide repeats whose human homologue is associated with an inherited prion dementia. These mice develop a neurological illness with prominent ataxia at 65 or 240 days of age, depending on whether the transgene array is, respectively, homozygous or hemizygous. Starting from birth, mutant PrP is converted into a protease-resistant and detergent-insoluble form that resembles the scrapie isoform of PrP, and this form accumulates dramatically in many brain regions throughout the lifetime of the mice. As PrP accumulates, there is massive apoptosis of granule cells in the cerebellum. Our analysis provides important insights into the molecular pathogenesis of inherited prion disorders in humans.

A transgenic model of a familial prion disease.

We have generated lines of transgenic mice that express a mutant prion protein containing 14 octapeptide repeats whose human homologue is associated with an inherited prion dementia. These mice develop an ataxic illness that begins at 65 days of age when the transgene array is homozygous, and results in death by 115-138 days. Starting from birth, mutant PrP is converted into a protease-resistant and detergent-insoluble form that resembles PrP(Sc), and this form accumulates dramatically in many brain regions throughout the lifetime of the mice. As PrP accumulates, there is massive apoptosis of cerebellar granule cells, as well as astrocytosis and deposition of PrP in a punctate pattern. These results establish a new transgenic animal model of an inherited human prion disease, and provide important insights into the molecular pathogenesis of these disorders.

A cell cycle alteration precedes apoptosis of granule cell precursors in the weaver mouse cerebellum.

A missense mutation in the gene coding for the G-protein-activated inwardly rectifying potassium (GIRK) channel, GIRK2, is responsible for apoptosis in the external germinal layer (EGL) of the cerebellum and a nonapoptotic death of midbrain dopaminergic neurons in the weaver (wv) mouse. Failure of axonogenesis and migration are considered to be the primary consequences of GIRK2 channel malfunction in the cerebellum. We investigated whether a disruption of the cell cycle precedes the failure of migration and axonogenesis and leads to massive apoptosis. To this end, immunohistochemistry and immunoblotting for PCNA, Cdk4, cyclin D, cyclin A, and the Cdk inhibitor p27/kip1, as well as in situ end-labeling for apoptotic DNA fragmentation, were applied to cerebella of P7-P21+/+, wv/+, and wv/wv mice. In +/+ and wv/+ mice, the expression of cell cycle proteins was limited to the outer, premigratory zone of the EGL. Antibodies to p27, a marker of cell differentiation, gave a reverse staining pattern. Due to migration delay, patches of p27-positive cells persisted in the outer EGL in P21 wv/+ mice. On the contrary, marked cell cycle up-regulation and absence of p27 occurred throughout the EGL at all ages in wv/wv mice, indicating an inability to switch off the cell cycle. Mitotic index evaluation showed that cell cycle activation was unrelated to proliferative events. Cell cycle proteins were not expressed in the substantia nigra, suggesting that nonapoptotic death of mature dopaminergic neurons is not preceded by abortive cell cycle re-entry. Our data show that abnormalities of the cell cycle in wv/wv cerebellum represent a major and early consequence of GIRK2 channel malfunction and may strongly influence the susceptibility of EGL cells to apoptosis. These observations may help in understanding the pathogenesis of human neurological channelopathies.

S-100beta protein is upregulated in astrocytes and motor neurons in the spinal cord of patients with amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive motor neuron loss and astrogliosis. We studied the immunohistochemical expression of S-100beta, a calcium-binding protein with both neurotrophic and neurotoxic activities, in the spinal cord of patients with ALS. Adjacent sections were processed with an in situ end-labeling technique for the demonstration of apoptosis-related DNA fragmentation. In controls, low expression of S-100beta was found in astrocytes but not motor neurons. Compared to controls, S-100beta was overexpressed in ALS. Most stained cells were reactive astrocytes, but a minority of motor neurons was also labeled. Neuronal labeling was unrelated to the presence of signs of atrophy/degeneration. S-100beta expression was also unrelated to neuronal or glial apoptosis. S-100beta upregulation in ALS spinal cord suggests that the protein might be involved in cellular defense mechanisms against oxidative stress.

Isochromosome 17q is a constant finding in medulloblastoma. An interphase cytogenetic study on tissue sections.

Isochromosome 17q (i[17q]) is the most frequent chromosomal abnormality in medulloblastoma, occurring in 30-60% of cases by karyotype analysis. In the present study i[17q] was demonstrated in routinely processed tissue sections of 20 medulloblastomas by in situ hybridization (ISH), using a chromosome 17 centromeric alpha satellite DNA probe. All medulloblastomas showed the i[17q] specific signal, i.e. two hybridization spots slightly apart from each other. The specific hybridization signal was not observed in ependymomas, cerebellar astrocytomas, haemangioblastomas, supratentorial neuroblastomas and ependymoblastomas. The constant finding of i[17q] in medulloblastoma depends on the much higher number of nuclei which can be analysed by ISH compared with cytogenetic techniques. Molecular data on medulloblastoma are consistent with the present results. The number of cells with i[17q] in medulloblastoma cases ranged from 3% to 9%; these figures are underestimated because of nuclear truncation in tissue sections. The percentage was not linked to patients' age, location of tumour, MIB-1 labelling index and histological type (classical vs desmoplastic). The present results indicate that i[17q] is a key event in the pathogenesis of medulloblastoma, and suggest a genetic difference between medulloblastoma and other primitive neuroectodermal tumours.

c-Jun, JNK/SAPK kinases and transcription factor NF-kappa B are selectively activated in astrocytes, but not motor neurons, in amyotrophic lateral sclerosis.

There is increasing evidence that oxidative damage plays a major role in amyotrophic lateral sclerosis (ALS), but how it contributes to motor neuron degeneration and astrocytic gliosis, two pathologic hallmarks of the disease, is unknown. A few studies have suggested that ALS motor neurons die via apoptosis and show upregulation of c-jun, an immediate early gene that is necessary for neuronal apoptosis. In order to elucidate the mechanisms of cell damage induced by oxidant stress, we have studied in ALS and control spinal cord the immunohistochemical expression of c-Jun, of JNK/SAPK, a kinase that activates c-Jun following various types of stress, and of NF-kappa B, a transcription factor that is induced by oxidant stress and has prominent neuroprotective functions. An in situ end-labeling assay was performed for detecting apoptotic cells. We show that (a) the JNK/SAPK-c-Jun pathway is dramatically overexpressed in ALS spinal cord; (b) the strongest activation occurs in astrocytes, while motor neurons show unusually low expression of the pathway; (c) increased JNK/SAPK expression in glial cells is accompanied by NF-kappa B activation, indicating the presence of a protective response to oxidant sress, which is deficient in motor neurons; (d) activation of JNK/SAPK, c-Jun and NF-kappa B is unrelated to apoptotic cell death. These results support the view that astrocytes are directly involved in the pathologic process of ALS, and might explain the selective vulnerability of motor neurons by their relative lack of antioxidant defenses.

Diverse cell death pathways result from a single missense mutation in weaver mouse.

Neuronal death affects selectively granule cell precursors of the cerebellum and the dopaminergic neurons of midbrain in the weaver mutant mouse. The weaver phenotype is associated with a missense mutation in the gene coding for the GIRK2 potassium channel, which results in chronic depolarization. Using DNA gel electrophoresis, electron microscopy (EM), the in situ end-labeling (ISEL) technique at the light and EM level, and immunohistochemistry for apoptosis-related proteins c-Jun and proliferating cell nuclear antigen (PCNA), we have investigated the mechanisms of cell death in cerebellum and substantia nigra. Between postnatal day P1 and P21, in the external germinal layer of the cerebellum, most degenerating granule cell precursors were found to aggregate to form clusters. Degenerating cells exhibited strong nuclear staining for ISEL, c-Jun, and PCNA and had a typical apoptotic morphology by EM. Increased c-Jun and ISEL staining were also occasionally seen in Purkinje cells. Between P14 and P21, when dopaminergic neurons start to degenerate, staining for ISEL, c-Jun, and PCNA in weaver substantia nigra was the same as in controls. By EM, however, we found only in weaver mice numerous dopaminergic cells that showed extensive vacuolar and autophagic changes of cytoplasm, preservation of membrane and organelle integrity, and absence of chromatin condensation or DNA fragmentation by EM-ISEL. The combination of vacuolar and autophagic changes identifies a novel type of non-necrotic, nonapoptotic cell death. After biochemical analysis of DNA, a clear-cut laddering, suggestive of oligonucleosomal fragmentation, was present in samples from weaver cerebellum. Cell death diversity appears to be influenced by specific features of target cells. These findings may be relevant for understanding the mechanisms of cell death in neurodegenerative diseases.

Role of apoptosis in the prognosis of oligodendrogliomas.

Prognostic factors in oligodendrogliomas are not well defined, even considering the labeling index of proliferation markers. As in other neuroepithelial tumors, the difficulty in calculating cell loss may contribute to this uncertainty. Proliferation markers Ki-67/MIB.1 and PCNA, mitoses, apoptotic nuclei, p53 and bcl-2 expression were investigated in 98 oligodendrogliomas. Apoptosis was assessed by the aspect of nuclei, by in situ end-labeling (ISEL) technique and by c-Jun immunohistochemical demonstration. The Bcl-2 also was immunohistochemically studied for its anti-apoptotic role. Mitotic index (MI), labeling index (LI) for MIB.1 and PCNA and apoptotic index (AI) were calculated and compared among themselves and with histology and survival. It was found that AI correlated with MI (p = 0.001) and was significantly higher in anaplastic than in classic oligodendrogliomas (p = 0.001). Apoptosis occurred only slightly more frequently in cases with high LIs for proliferation markers (MIB.1 and PCNA) (p = non-significant) and it was definitely higher in p53-positive cases (p = 0.008). It did not correlate with bcl-2 which was poorly expressed in oligodendrogliomas, with the exception of cells with astrocytic features. Apoptotic index correlated very weakly with survival (p = 0.05); therefore, it cannot be considered a highly reliable prognostic factor in oligodendrogliomas.

Muscle apoptosis in humans occurs in normal and denervated muscle, but not in myotonic dystrophy, dystrophinopathies or inflammatory disease.

Recent data suggest that death of muscle cells during development and in selected pathological conditions occurs via apoptosis. We investigated the occurrence of apoptosis in normal and pathological human skeletal muscle, using in situ end-labeling (ISEL) to detect DNA fragmentation, and immunohistochemistry for the expression of tissue transglutaminase and interleukin-1beta-converting enzyme (ICE)-like proteases. In normal subjects, apoptotic myonuclei were occasionally observed as evidence of normal tissue turnover. Myonuclear apoptosis due to a deficit of trophic support from nerve cells also occurred in spinal muscular atrophies. No apoptosis of muscle cells was found in dystrophinopathies, myotonic dystrophy and inflammatory myopathies, suggesting that death of myofibers in those conditions is not due to activation of a gene-directed program of death. In dystrophinopathies and inflammatory myopathies, apoptosis was found in interstitial mononuclear cells, as a likely mechanism of clearance of the inflammatory infiltrates.

RAGE and amyloid-beta peptide neurotoxicity in Alzheimer's disease.

Amyloid-beta peptide is central to the pathology of Alzheimer's disease, because it is neurotoxic--directly by inducing oxidant stress, and indirectly by activating microglia. A specific cell-surface acceptor site that could focus its effects on target cells has been postulated but not identified. Here we present evidence that the 'receptor for advanced glycation end products' (RAGE) is such a receptor, and that it mediates effects of the peptide on neurons and microglia. Increased expressing of RAGE in Alzheimer's disease brain indicates that it is relevant to the pathogenesis of neuronal dysfunction and death.

Reactive astrogliosis of the spinal cord in amyotrophic lateral sclerosis.

Many observations have been carried out on astrogliosis in the cerebral cortex in amyotrophic lateral sclerosis (ALS), whereas little attention has been paid to astrogliosis in the spinal cord. Twenty autopsy cases of sporadic, common form of ALS have been studied. Spinal cords have been examined at the cervical, thoracic and lumbar levels by histological methods and immunohistochemistry for GFAP, Vimentin, Tau-protein, Neurofilaments, PCNA. A gliosis was found in the ventral horns, in dorsal horns and at the transition between gray matter and anterior and lateral funiculi, especially close to laminae VII, VI and V as being due to secondary gliosis. The findings cannot be interpreted on the only basis of the substitutive role of reacting glia. The proposed pathogenetic mechanisms of ALS are evaluated as possible responsible stimuli; the coincidence of the distribution of reactive astrocytes with the entering points of the corticospinal tracts into the gray matter is considered of primary importance. Of special interest are reactive astrocytes at the transition between laminae VII, VI and V and the lateral funiculus, where dystrophic neurites are known to concentrate.

Bcl-2 distribution in neuroepithelial tumors: an immunohistochemical study.

Bcl-2 proto-oncogene prevents apoptosis in many conditions. First detected in lymphomas, it has been also described in non-lymphoid tissues. The immunohistochemical distribution of bcl-2 protein in 100 neuroepithelial tumors is presented. Bcl-2 was positive in some neurons of normal nervous tissue, in reactive astrocytes and variably in all neuroepithelial tumor. The reaction product was either diffuse or granular, due to bcl-21 protein localization on cytoplasmic, nuclear and mitochondrial membranes. The positivity was high in medulloblastomas and in astrocytic tumors. In the latter, the strongest staining was found in cells retaining the astrocytic aspect. Oligodendroglial cells were minimally stained. No correlation of bcl-2 staining with survival was found in each tumor type. The interpretation of the results is based on the one side on the constitutive role played by bcl-2 in the nervous tissue and its neoplastic derivatives. On the other side, in tumors bcl-2 acts by preventing tumor cells from undergoing apoptosis. BCl-2 expression in brain tumors, therefore, receives a dual interpretation. For this reason and for the lacking of correlation with survival, bcl-2 expression cannot be regarded as a prognostic factor.