Ikuro Wakayama

Advanced Glycation End Products in Alzheimer’s Disease and Other Neurodegenerative Diseases

Advanced glycation end products (AGEs) have been implicated in the chronic complications of diabetes mellitus and have been reported to play an important role in the pathogenesis of Alzheimers disease. In this study, we examined the immunohistochemical localization of AGEs, amyloid beta protein (A beta), apolipoprotein E (ApoE), and tau protein in senile plaques, neurofibrillary tangles (NFTs), and cerebral amyloid angiopathy (CAA) in Alzheimers disease and other neurodegenerative diseases (progressive supranuclear palsy, Picks disease, and Guamanian amyotrophic lateral sclerosis/Parkinsonism-dementia complex). In most senile plaques (including diffuse plaques) and CAA from Alzheimers brains, AGE and ApoE were observed together. However, approximately 5% of plaques were AGE positive but A beta negative, and the vessels without CAA often showed AGE immunoreactivity. In Alzheimers disease, AGEs were mainly present in intracellular NFTs, whereas ApoE was mainly present in extracellular NFTs. Picks bodies in Picks disease and granulovacuolar degeneration in various neurodegenerative diseases were also AGE positive. In non-Alzheimer neurodegenerative diseases, senile plaques and NFTs showed similar findings to those in Alzheimers disease. These results suggest that AGE may contribute to eventual neuronal dysfunction and death as an important factor in the progression of various neurodegenerative diseases, including Alzheimers disease.

Relationship of microglia and scrapie amyloid-immunoreactive plaques in kuru, Creutzfeldt-Jakob disease and Gerstmann-Sträussler syndrome.

Kuru, Creutzfeldt-Jakob disease (CJD) and Gerstmann-Sträussler syndrome (GSS) are transmissible dementias affecting humans characterized neuropathologically by intraneuronal vacuolation, spongiform change, astrocytic hypertrophy and hyperplasia and the variable presence of amyloid plaques. It has been suggested that microglia are amyloid-forming cells, which play an essential role in amyloid plaque formation. To study the relationship between microglia and amyloid plaques in kuru, CJD and GSS, cerebellar tissues were examined by the double-immunostaining technique using anti-ferritin antibodies as the microglial marker and anti-scrapie amyloid antibody as plaque marker. Ferritin-immunoreactive microglia were observed interdigitating with and among unicentric, multicentric and diffuse types of scrapie amyloid-immunoreactive plaques and were found to a lesser extent in the neuropil. In kuru and CJD, scrapie amyloid-immunoreactive plaques were predominantly unicentric and were observed in the granular layer. In kuru, 53% of the amyloid plaques were associated with microglia, whereas only 30% of plaques in CJD were. In contrast, scrapie-amyloid-immunoreactive plaques in GSS were of the multicentric type, predominantly observed in the molecular layer, and 90% of these plaques were associated with microglia. Our data indicate that microglia are frequently associated with scrapie amyloid-immunoreactive plaques in GSS, less commonly in kuru and to a much lesser extent in CJD, suggesting that microglia may play a variable but important role in the formation of plaques in the transmissible spongiform encephalopathies.

Comparative study of chronic aluminum-induced neurofilamentous aggregates with intracytoplasmic inclusions of amyotrophic lateral sclerosis

Abstract Amyotrophic lateral sclerosis (ALS) is characterized neuropathologically by chromatolysis, Bunina bodies, hyaline inclusions, skein-like inclusions and axonal spheroids. Aluminum, a known neurotoxin, is the cause of dialysis encephalopathy and is considered to be a causative agent in high incidence foci of ALS in the western Pacific. We have developed an experimental model of motor neuron degeneration in New Zealand white rabbits using chronic low-dose intracisternal administration of aluminum and compared the clinical and neuropathological changes to those of human ALS. Aluminum-inoculated rabbits developed progressive hyperreflexia, hypertonia, limb splaying, gait impairment, muscle wasting, hindlimb paralysis and impaired tonic immobility responses without overt encephalopathic features over a 14-month period. Examination of spinal cords from these animals demonstrated the frequent occurrence and progressive development of anterior horn cell lesions that included small, round, argentophilic perikaryal inclusions similar to hyaline inclusions seen in human ALS. Other inclusions were more condensed and eosinophilic, while still others had neurofibrillary tangle-like morphologies. Axonal spheroids and neuritic thickenings were also prominent and were identical to those seen in human ALS. We believe that the similar and progressive development of neuropathological changes observed in the chronic aluminum-intoxication model, compared to human ALS, warrants further study to aid in understanding the cellular and molecular mechanisms of human motor neuron disease.

Involvement of Onuf's nucleus in amyotrophic lateral sclerosis

The Onufrowicz (Onufs) nuclei from eight amyotrophic lateral sclerosis (ALS) cases and nine neurological control cases were studied histologically and morphometrically. To clarify the factors relating to the involvement of the Onufs nucleus in ALS, we correlated the relationships among the age at death, clinical duration, morphometric findings for Onufs neurons, and neuronal numbers in the posteroposterolateral (PPL) nuclei in the ALS cases with those in neurological controls. Intracytoplasmic inclusions such as Bunina bodies, ubiquitin-reactive inclusions, and conglomerate inclusion were found in the Onufs neurons in ALS, but not in the controls. The total number of Onufs neurons in the ALS cases was not decreased, but that of normal-appearing neurons was decreased while that of atrophic neurons was increased. Significantly decreased perikaryal, nuclear and nucleolar areas and decreased perikaryal (P)/nuclear and P/nucleolar area ratios of Onufs neurons were found in ALS, not only in the atrophic neurons but also in the normal-appearing neurons, compared with the controls. The shrinkage in Onufs neurons of ALS was different from that seen in the ageing process or in the axonal reactions of controls with atonic bladder. In ALS, the morphometric findings for the Onufs neurons showed no correlation with age at death, clinical duration, or number of PPL neurons. Our results indicate that in ALS Onufs nucleus is principally vulnerable to the ALS process, although the degree of degeneration differs from that seen in other motor neurons. The involvement of Onufs nucleus might be slowed due to factors specific to this nucleus, including the biochemical and autonomic properties of the nucleus; nevertheless, it is histologically classified as part of the somatic cell column.

Kii ALS dementia

Epidemiological surveys in the foci of ALS of the Kii Peninsula of Japan started in the early 1960s. Continuous surveys conducted for decades revealed that there have been two foci in the Kii Peninsula: one in Kozagawa in the southern part, and the other in Hobara in the south‐east. Clinically, ALS patients of the Kii foci occasionally showed parkinsonian features or dementia that have not been reported in the sporadic form of ALS. Neuropathologically, numerous NFT that are identical to those of Alzheimers disease were observed in the cerebral cortex and in the brainstem nuclei. To elucidate the etiopathogenesis of this unique form of ALS, an analysis was conducted of the environment in the focus areas and of the specimens from the patients with ALS. It was hypothesized that the long exposure of these environments to low calcium and magnesium, and an excess of aluminum and manganese in the drinking water and the soil, might lead to the deposition of some trace elements in the CNS, eventually causing neuronal degeneration and death.

Ultrastructural neuropathology of chronic wasting disease in captive mule deer.

SummaryChronic wasting disease (CWD), a progressive and uniformly fatal neurological disorder, is characterized neuropathologically by intraneuronal vacuolation, spongiform change of the neuropil and astrocytic hyperplasia and hypertrophy. Ultrastructural neuropathological findings consist of (1) extensive vacuolation in neuronal processes, within myelin sheaths, formed by splitting at the major dense lines or within axons; (2) dystrophic neurites (dendrites, axonal preterminals and myelinated axons containing degenerating mitochondria and pleomorphic, electron-dense inclusion bodies); (3) prominent astrocytic gliosis; (4) amyloid plaques; and (5) giant neuronal autophagic vacuoles. Other findings include activated macrophages and occasional spheroidal structures containing densely packed fibrillar material of unknown origin, abundant structures suggestive of degenerating microtubules entrapped in filamentous masses, vacuoles and myelin figures. Similar findings have been previously observed in scrapie-infected hamsters and Creutzfeldt-Jakob disease (CJD)-infected mice, bovine spongiform encephalopathy, and CJD indicating that CWD in captive mule deer belongs to the subacute spongiform encephalopathies (transmissible brain amyloidoses).

Dose-Dependent Selective Suppression of Light (NFL) and Medium (NFM) but Not Heavy (NFH) Molecular Weight Neurofilament mRNA Levels in Acute Aluminum Neurotoxicity

We inoculated 5- to 6-week old New Zealand white rabbits intracisternally with either 100, 250, 500, 750, or 1000 micrograms of AlCl3 or 0.9% NaCl and correlated the extent of cervical motor neuron neurofilamentous inclusion formation at 48 h postinoculation with alterations in neurofilament (NF) mRNA levels. RNA was isolated from cervical spinal cord by the guanidine isothiocyanate method and individual RNA samples were normalized for poly(A+) content. Northern blot analysis was performed with cDNA probes for light (NFL), medium (NFM), and heavy (NFH) neurofilament subunit protein or with oligonucleotide probes for alpha-tubulin or actin. No significant alteration in the levels of alpha-tubulin, actin, or NFH mRNA were observed, regardless of the aluminum dose. In contrast, dose-dependent reductions in NFL and NFM mRNA levels occurred in direct proportion to the extent of neurofilamentous inclusion formation. While inoculums of NaCl or 100 or 250 micrograms AlCl3 induced neither inclusion formation or alterations in mRNA levels, both inclusion formation and reductions in the levels of NFL and NFM mRNA occurred thereafter, becoming maximal with inoculums of 1000 micrograms AlCl3. These experiments indicate that intracisternally administered AlCl3 acutely suppresses NFL and NFM mRNA levels without affecting those of NFH. This pattern is in distinct contrast to the uniform reductions of all NF mRNA transcript levels during neurogenesis or following axotomy, indicating a specific effect of aluminum upon steady-state levels of NF mRNA that correlates with the induction of neurofilamentous aggregates.

Bunina body formation in amyotrophic lateral sclerosis: a morphometric-statistical and trace element study featuring aluminum.

Clinico-environmental and pathological variables were obtained from 10 patients with amyotrophic lateral sclerosis using particle-induced X-ray emission spectrometry (PIXE) and morphometric-statistical analysis. Statistical analysis identified a model that maximally predicts the Bb% (frequency of Bunina bodies) from a selected set, four variables: (1) nucleolar index, (2) magnesium (Mg) content, (3) aluminum (Al) content, and (4) duration of illness. Among them, only the Al content proved important. To determine their chemical nature, electron energy loss spectrometry (EELS) was applied at the ultrastructural level; it revealed that within the motor neuron, Al strongly binds to the Bunina body as well as rough endoplasmic reticulum (rER), and lesser strongly to mitochondria and lipofuscin granule. Thus, it is chemically similar to the rER, providing preferential binding sites to aluminum. The Bunina bodies may be an end-product of the nucleic acid dysmetabolism at rER caused by Al along with Mg depletion.

Immunocytochemical and ultrastructural evidence of dendritic degeneration in motor neurons of aluminum-intoxicated rabbits

SummaryUsing immunocytochemical and ultrastructural methods, we observed extensive and characteristic dendritic changes in motor neurons of rabbits inoculated intracisternally with aluminum phosphate. Anti-microtubule-associated protein 2 immunostaining revealed markedly reduced immunoreactivity in motor neuron dendrites and a reduced number of dendritic trees in aluminum phosphate-intoxicated rabbits. These dendritic changes were confirmed at the ultrastructural level; neurofilamentous accumulations, membranous inclusions and disrupted microtubules were common features of motor neuron dendrites, but less prominent in motor neuron axons. These observations suggest that dendrites are characteristically involved in aluminum intoxication in addition to the widely reported accumulation of phosphorylated neurofilament in perikarya and axons.

Neuronal degeneration in amyotrophic lateral sclerosis is mediated by a possible mechanism different from classical apoptosis

In order to determine the potential role of apoptosis in the degeneration in amyotrophic lateral sclerosis (ALS), we correlated the morphological characteristics of the remaining neurons in the spinal cord with the results of in situ DNA fragmentation detected by terminal transferase‐mediated deoxyuridinetriphosphate (dUTP)‐biotin nick‐end labeling (TUNEL) in 17 cases of ALS, five age‐matched control cases, six aged control cases, and three disease control cases with spinal cord lesions. The remaining neurons were classified into five categories: normal appearing, chromatolytic, degenerative, atrophic, and inclusion laden. We measured the areas of the per©karya, the nuclei, and the nucleoli of the neurons in these categories. The areas of perikarya, nuclei, and nucleoli in the postero‐posterolateral neurons of the ALS cases were significantly decreased compared with those of the age‐matched controls. The nucleolar and nuclear areas of even normal‐appearing neurons from the ALS cases were significantly decreased compared with those from the age‐matched controls, suggesting that nuclear and nucleolar shrinkage in normal‐appearing neurons in ALS is an early change in this disease. However, the frequency of TUNEL‐positive neurons in the normal‐appearing, degenerative, atrophic, or inclusion‐laden neurons in the spinal cord of the ALS cases was not significantly elevated compared with those in the age‐matched, aged, or disease controls. No apoptotic bodies were detected by electron microscopy in the ALS spinal cords examined. Our results suggest that neuronal degeneration in ALS involves a certain mechanism different from classical apoptosis. We hypothesize that classical apoptosis may be a terminal event of neuronal degeneration in ALS, even though classical apoptosis has a role in the disease processes of ALS.