T. Yamada

Apoptosis related antigen, LeY and nick-end labeling are positive in spinal motor neurons in amyotrophic lateral sclerosis

Expression of LeY, a difucosylated type 2 chain determinant, has been previously identified as a characteristic of cells undergoing apoptosis. Immunohistochemistry using an antibody for LeY, as well as nick-end labeling for the detection of DNA breaks, was done on cervical spinal cord sections from ten patients with amyotrophic lateral sclerosis (ALS) and nine patients who had died from other causes. LeY-positive immunoreactivity was seen in the motor neurons of seven ALS cases, but in none of the other cases. Nickend labeling was also positive in four ALS cases. Double staining of motor neurons by anti-LeY antibody and nick-end labeling was shwon in these cases. Other LeY-positive structures, such as reactive astrocytes and fatladen microglia/macrophages in the lateral and anterior columns, were negative for nick-end labeling. These results suggest that the mechanism of cell death in the spinal motor neurons of ALS may be apoptosis.

Loss of insulin receptor immunoreactivity from the substantia nigra pars compacta neurons in Parkinson's disease

Immunohistochemistry using both a newly developed polyclonal, and a commercially available monoclonal, anti-insulin receptor antibody was done on the midbrain from cases of idiopathic Parkinsons disease (PD), Alzheimers disease, amyotrophic lateral sclerosis, vascular parkinsonism and non-neurological controls. Both antibodies gave indentical patterns of neuronal staining. The neurons of the oculomotor nucleus were immunopositive in all the brains. However, the neurons in the pars compacta of the substantia nigra, paranigral nucleus, parabrachial pigmental nucleus, tegmental pedunculopontine nucleus, supratrocheal nucleus, cuneiform nucleus, subcuneiform nucleus and lemniscus medialis, which were positive in other diseases and in non-neurological controls, were not stained by these antibodies in PD brains. These results suggest that, in PD, a dysfunction of the insulin/insulin receptor system may precede death of the dopaminergic neurons.

Insulin receptor mRNA in the substantia nigra in Parkinson's disease

Neurotrophic effects resulting from the insulin/insulin receptor system have been recognized as important in determining the etiological basis of neurodegenerative disorders. In Parkinsons disease, selective neuronal loss in the substantia nigra is accompanied by decreased immunoreactivity of the insulin receptor as determined using immunohistochemical studies. We performed semiquantitative mRNA analysis by reverse transcription-polymerase chain reaction (RT-PCR) using specific primers for human insulin receptor exon 22, which encodes a region of the beta subunit of the receptor serving as a tyrosine kinase domain. The relative levels of mRNA in the substantia nigra from Parkinsons brain tissues showed a marked depression compared with those of normal controls. Further investigations are needed to decide whether this is a primary, disease-specific alteration of gene expression or merely a secondary process.

White matter microglia produce membrane-type matrix metalloprotease, an activator of gelatinase A, in human brain tissues

Membrane-type matrix metalloprotease (MT-MMP) is an activator of gelatinase A (MMP-2), which has previously been found in carcinoma cells. We examined non-neurological and Alzheimers disease brain tissues for MT-MMP by immunohistochemistry and in situ hybridization. The anti-MT-MMP antibodies gave positive staining of brain microglial cells in all the brain tissues. Positively stained microglia were found only in the white matter. The cells producing MT-MMP protein were also shown to be white matter microglia. These results provide further evidence that activated gelatinase A, which may be a processing enzyme for degradation of β-amyloid protein, may be produced in white matter microglia.

Immunohistochemistry with antibodies to hepatocyte growth factor and its receptor protein (c-MET) in human brain tissues.

Hepatocyte growth factor (HGF) is a potent mitogen for mature hepatocytes, and also has multifunctional effects on some other cells in various organs. The human c-Met proto-oncogene product has recently been identified as its high-affinity receptor. We examined HGF-like and c-Met protein-like immunoreactivities in the brains of neurologically normal, lacunar stroke and Alzheimer disease (AD) cases. The HGF antibody stained only round cells in the capillaries and astrocytes in the white matter. Positive staining with the antibody to c-Met protein was seen in microglia, predominantly in the white matter. The possibility of interactions between astrocytes and microglia through HGF and its receptor is suggested.

Immunohistochemistry using antibodies to α-interferon and its induced protein, MxA, in Alzheimer's and Parkinson's disease brain tissues

The localization of alpha-interferon (alpha-IFN) and its induced protein, MxA, was examined in human brain tissues from neurologically normal, Alzheimers disease (AD) and Parkinsons disease (PD) cases. In all cases, a few neurons in the superficial cortical layers and microglial cells in the white matter were stained with the antibody to alpha-IFN. In AD brains, white matter microglia were intensely labeled for alpha-IFN and reactive microglia, such as those on senile plaques, were strongly positive for MxA protein. In PD, Lewy bodies in the substantia nigra were positive for MxA, but there was no staining for alpha-IFN in that region. These results suggest that increased expression of alpha-IFN in the white matter microglia and appearance of MxA protein in reactive microglia contribute to Alzheimer pathology. The staining of some Lewy bodies for MxA may be indicative of a viral infection or other unknown factor.

Detection of benzodiazepine receptor occupancy in the human brain by positron emission tomography

Benzodiazepine receptor occupancy in the brain following oral administration of clonazepam (CZP) with a dose of 30 μg/kg in six healthy young men and a further dose of 50 μg/kg in one of the subjects was estimated by carbon-11 labeled Ro15-1788 and positron emission tomography (PET). The effects of CZP on the latency of auditory event-related potentials (P300) were also studied. Overall brain 11C uptake was depressed and the % inhibition of 11C uptake in the gray matter of the brain at 30 min after [11C]Ro15-1788 injection was 15.3–23.5% (mean, n=6) following 30 μg/kg CZP when compared with that in the control experiment without any previous treatment. The 11C uptake in the cerebral cortex in the subject who received both doses decreased in a dose-related manner after 30 μg/kg and 50 μg/kg CZP. The P300 latency was prolonged significantly by 30 μg/kg CZP [31.6±16.3 ms (mean±SD, n=6), P<0.05]. The P300 latency in the same subject was prolonged in a dose-related manner by 30 μg/kg and 50 μg/kg CZP. The technique using [11C]Ro15-1788 and PET permits comparison of the pharmacological effects with the percentage of receptor sites which benzodiazepines occupy in the human brain. P300 also seems to be useful to investigate the pharmacological effects of benzodiazepines.

Selective localization of gelatinase A, an enzyme degrading β-amyloid protein, in white matter microglia and in Schwann cells

Gelatinase A is an enzyme capable of cleaving soluble β-amyloid protein (βAP), and may function as an α-secretase to produce secretory forms of amyloid precursor protein. We examined gelatinase A immunoreactivity in the brains and posterior roots of neurologically normal, lacunar stroke, Alzheimer disease (AD), amyotrophic lateral sclerosis, progressive supranuclear palsy and myasthenia gravis cases. The gelatinase A antibody stained only microglial cells in the white matter in all the brain tissues. In AD brain, the reactive microglia located in the center of classical senile plaques, as well as in other microglial cells in the gray matter, showed no immunoreactivity. Gelatinase A in white matter microglial cells may play a role in preventing local deposition of βAP. In the posterior root, Schwann cells had positive immunoreactivity. As with other metalloproteases, gelatinase A in Schwann cells may play an antiproliferative role.

Increased concentration of C4d complement protein in CSF in amyotrophic lateral sclerosis.

Plasma and CSF concentrations of C4d and the circulating immune complex to C1q were measured in 27 patients with amyotrophic lateral sclerosis (ALS) or cervical spondylosis. There was no significant difference among groups in plasma C4d or in plasma or CSF concentrations of the circulating immune complex to C1q. The ALS group, however, had a significantly higher CSF concentration of C4d than the group with cervical spondylosis, as well as a higher C4d index (CSF to plasma C4d ratio x serum to CSF albumin ratio). These results suggest that augmented complement activation in the CNS occurs in ALS. Increased CSF concentration of C4d or raised C4d index may serve as a basis for differentiating ALS from cervical spondylosis.

Pontine lesion in opsoclonus-myoclonus syndrome shown by MRI.

Two patients with opsoclonus-myoclonus syndrome are reported whose magnetic resonance imaging (MRI) showed brain stem lesions. Both patients developed the opsoclonus-myoclonus syndrome after an upper respiratory illness. One case had visual hallucinations during the course of illness and MRI revealed a focal lesion in the pons involving the junction of basis and tegmentum. MRI of the second case showed a focal lesion at the upper pontine tegmentum.