R. M. Garruto

Low-calcium, high-aluminum diet-induced motor neuron pathology in cynomolgus monkeys

SummaryLong-term epidemiological studies indicate that environmental factors play a causative role in high-incidence amyotrophic lateral sclerosis (ALS) and parkinsonism-dementia (PD) in the western Pacific. An increased risk for disease is acquired in youth and remains for life. The low concentrations of calcium and magnesium and high levels of aluminum in the soil and drinking water, along with the relative isolation of these populations, constitute an unusual environmental feature common to all three high-incidence foci. Studies of mineral deposition in brain tissue of Guamanian ALS and PD patients, as well as of neurologically normal Guamanians with neurofibrillary degeneration, demonstrate accumulations of calcium, aluminum and silicon in neurofibrillary tangle-bearing neurons. In an attempt to duplicate the low calcium and high aluminum and manganese in soil and drinking water in these foci, we maintained juvenile cynomolgus monkeys for 41 to 46 months on a low-calcium diet with or without supplemental aluminum and manganese. Experimental animals exhibited mild calcium and aluminum deposition and degenerative changes, compatible with those of early ALS and PD, in motor neurons of the spinal cord, brain stem, substantia nigra and cerebrum. Neuropathological findings included chromatolysis, aberrant perikaryal accumulation of phosphorylated neurofilament, neurofibrillary tangles, axonal spheroids, and basophilic and hyaline-like inclusions consisting of abnormal cytoskeletal elements by electron microscopy. The magnitude and extent of these lesions far exceeded those found in normal aged monkeys.

Histochemical and X-ray microanalytical localization of aluminum in amyotrophic lateral sclerosis and parkinsonism-dementia of Guam

SummaryHistochemical staining for aluminum, using Solochrome azurine or Morin, provided a rapid, simple and reliable means of identifying areas and structures of the brain of interest for closer scrutiny by X-ray microanalysis in patients with amyotrophic lateral sclerosis and parkinsonism-dementia of Guam. Neuronal perikarya, dendritic processes, and the walls of some cerebral vessels were aluminum positive by Solochrome azurine staining. In some cases, the deposition of aluminum was rather diffuse, particularly in the white matter. Fluorescent localization of aluminum using Morin was equally sensitive and specific, but provided less morphological detail than Solochrome azurine. Confirmation of histochemical detection of aluminum was achieved by examining adjacent tissue sections using wavelength-dispersive spectrometry coupled to a computer-controlled electron beam X-ray microprobe. Although the minimum detectable limits for aluminum by these histochemical procedures are unknown, the lower detection limit of our X-ray microanalytical technique is 10–100 ppm dry weight. Solochrome and Morin staining, as verified by X-ray microanalysis, afford a useful and reliable means of surveying multiple anatomical regions for aluminum deposition in naturally occurring and experimentally induced neurodegenerative disorders.

N-butyl benzenesulfonamide : a neurotoxic plasticizer inducing a spastic myelopathy in rabbits

SummaryN-Butyl benzenesulfonamide (NBBS), a plasticizer used commercially in the polymerization of polyamide compounds, is neurotoxic. Young adult New Zealand white rabbits, inoculated repeatedly with NBBS by the intracisternal or intraperitoneal routes, developed a dose-dependent motor dysfunction characterized by limb splaying, hyperreflexia, hypertonia, gait impairment, and abnormal righting reflexes. Histopathological changes consisted of intramedullary thickening of the ventral horn axons, random neuroaxonal spheroids confined to brain stem nuclei and spinal motor neurons, and swollen dendritic processes of spinal motor neurons. Immunoreactivity to a monoclonal antibody against microtubule-associated protein-2 (MAP-2) was markedly increased in the dendrites of spinal motor neurons following thrice weekly intraperitoneal inoculations of NBBS for 4 months, whereas after 12 monthly intracisternal inoculations. MAP-2 immunoreactivity was absent or strikingly reduced in the same neuronal populations. Ultrastructurally, postsynaptic zones contained vacuoles and multilamellar bodies. These findings raise questions about the safety of NBBS to humans.

Neurofibrillary tangles of Guamanian amyotrophic lateral sclerosis, parkinsonism-dementia and neurologically normal Guamanians contain a 4-to 4.5-kilodalton protein which is immunoreactive to anti-amyloid β/A4-protein antibodies

SummaryNeurofibrillary tangles (NFT), one of the neurodegenerative features of Alzheimers disease, Downs syndrome and normal aging, is a constant, widespread neuropathological finding in Guamanian amyotrophic lateral sclerosis (ALS), parkinsonism-dementia (PD) and in neurologically normal Guamanians, dying of causes other than ALS and PD. NFT in brain tissue sections of patients with Guamanian ALS and PD were immunoreactive to antibodies directed against a 43-amino acid synthetic peptide homologous to amyloid β/A4-protein (anti-SP43) associated with Alzheimers disease. NFT extracted from frozen brain tissues of Guamanian patients with ALS and PD and from tissues of neurologically normal Guamanians were congophilic and birefringent. By negative-stain electron microscopy, NFT preparations contained bundles and/or isolated single, straight, unpaired filaments in Guamanian AlS and occasionally pairing of filaments in neurologically normal Guamanians, measuring 5–20 nm in diameter. Formic acid digestion of NFT preparations, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion high-pressure liquid chromatography, showed a protein with an apparent molecular mass of 4-to 4.5-kDa, which by Western blot analysis was immunoreactive to anti-SP43. Immunoabsorption of purified NFT or SP43 with anti-SP43 abolished immunostaining. Our study corroborate previous data that amyloid β/A4-protein is present in NFT in Guamanian PD. Furthermore, our data indicate that amyloid β/A4-protein is present in NFT in brain tissues of patients with Guamanian ALS and in neurologically normal Gumananians, suggesting a common mechanism of amyloidogenesis with NFT formation in Alzheimers disease and normal brain aging.

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.

High prevalence of human T-lymphotropic virus type I infection in isolated populations of the Western Pacific region confirmed by Western immunoblot

High prevalences of antibodies against human T‐lymphotropic virus type I (HTLV‐I), as confirmed by Western immunoblot, were found in several remote indigenous populations of the Solomon Islands and Vanuatu and in some isolated populations of New Guinea that had no contact with Japanese or Africans and little contact with Caucasians prior to our bleedings. By contrast, zero or very low prevalences of HTLV‐I infection were found in Guamanians and Carolinians, despite more than 30 years of intense contact with the Japanese. A total of 1,601 sera, collected between 1963 and 1981 from 21 population groups in the Western Pacific, was tested by enzyme‐linked immunosorbent assay (ELISA) for IgG antibodies to HTLV‐I. By ELISA, prevalences of antibodies against HTLV‐I ranged from zero to 50%. Seropositivity could be confirmed in only 12.5% of 48 ELISA‐positive sera selected for testing by Western immunoblot. However, the confirmed HTLV‐I seroprevalences in some Melanesian populations were still as high as those found in HTLV‐I‐endemic regions, such as southwestern Japan and the Caribbean basin. HTLV‐I prevalences were similar among males and females, and acquisition of antibodies increased with age. Our data indicate that infections with HTLV‐I or a related retrovirus have been widespread in the southwestern Pacific for over 25 year in populations with minimal outside contact, while some populations which had extensive Japanese contact have no evidence of infection. Furthermore, based on the high frequency of indeterminate Western immunoblots, we conclude that in Melanesia this may represent either incomplete specific reactivity to HTLV‐I or the existence of an antigenic variant of HTLV‐I, distinct from prototype Japanese, American, and European HTLV‐I strains.

THE NUCLEUS BASALIS OF MEYNERT IN PARKINSONISM–DEMENTIA OF GUAM: A MORPHOMETRIC STUDY

The nucleus basalis of Meynert (nbM) was studied morphometrically in three Guamanians with parkinsonism–dementia (PD) and in two Guamanian and two non–Guamanian controls. Paraffin–embedded blocks of the nbM were serially sectioned (20 μm thick) at increments of 200 μm so that a total of 24 sections (eight each from the anterior, intermediate and posterior sectors of the nbM) were studied. The mean cell density was determined for each sector and the diameter of 50 neurons, randomly chosen in the region of apparent maximal density, was calculated. A decrease of the mean cell density, due to the loss of neurons with diameters larger than 20 μm, was found in the PD cases compared to the controls. Two PD patients exhibited striking neuronal loss (65–95%) with predominant involvement of the intermediate and posterior sectors, while the third case showed only minimal neuronal loss in these sectors (15–40%). In both Guamanian and non–Guamanian controls large neurons (diameters ≥ 20 μm) exceeded small neurons while the reverse was true in all sectors of the nbM for the PD cases. These data, while confirming a previous study reporting neuronal loss in the nbM of PD patients, underline the importance of detailed morphometric analysis of the different sectors of the nbM to recognize those patients in whom lesions are not uniformly distributed.

N-butylbenzenesulphonamide, a novel neurotoxic plasticising agent

A typical result of this procedure is shown in fig 1. In this experiment, the exon-12-containing region of the human phenylalanine hydroxylase gene was amplified from the Guthrie cards of four phenylketonuria patients from Leningrad. This region of the phenylalanine hydroxylase gene contains the most prevalent caucasian phenylketonuria mutation (R408W).5 The primers used in this amplification were identical to those described by DiLella et al. This experiment demonstrates amplification of a single band of appropriate size (245 bp). The sequence identity of the amplification product was confirmed by slot-blot hybridisation analysis using allele-specific oligonucleotides as described by DiLella et al (fig 2).6 The primary advantage of this technique over other methods is the significant increase in the number of potential mutations which can be determined from a single card. Based on the size of the

Human T-lymphotropic virus type I: A retrovirus causing chronic myeloneuropathies in tropical and temperate climates

Human T‐cell lymphotropic virus type I (HTLV‐I), the first human retrovirus to be isolated, is the cause of endemic tropical spastic paraparesis (TSP). Originally, this chronic neurological disorder was described as a disease seen among blacks of low socioeconomic status living in tropical countries, and thus for many decades TSP remained a little known curiosity outside the endemic regions. The link between HTLV‐I infection and TSP was made fortuitously, when antibodies to HTLV‐I were found in serum and cerebrospinal fluid of TSP patients in Jamaica, Colombia, and Martinique. Soon thereafter a similar disorder, designated HTLV‐I associated myelopathy (HAM), was reported from southern Japan. This broadened the geographic and ethnic boundaries of this chronic myelopathy and the disease has now been reported in multiple ethnic groups from more than 40 countries, in both tropical and temperate regions. The name TSP/HAM is now used to include all patients (regardless of race or country of origin) who have HTLV‐I‐positive endemic TSP or HAM.

Ultrastructural analysis of neurofibrillary tangles of Alzheimer's disease using computerized digital processing

SummaryThe ultrastructure of neurofibrillary tangles of Zlzheimers disease was analyzed by computerized digital processing of electron micrographs. Processing of the electron micrographs consists of four steps: digitizing the electron micrograph, Fourier transformation, noise filtering and inverse Fourier transformation and Laplacian operation. In the present study, we have confirmed that neurofibrillary tangles are composed of a pair of helical filaments (PHF), which appear characteristically as an unbranched rigid structure. The periodicity of PHF is 78nm on the diffractogram. The dimensions of PHF obtained by our analysis, although basically similar to those described earlier by other investigators using conventional techniques, more precisely defines its structural conformation. We have also demonstrated that the spatial relationship of two filaments appears symmetrical after two-way tilting of the specimen about the axis of rotation. Our observations emphasize the importance of digital image processing as an effective tool for structural analytical research in biology and medicine.