R.W.H. Verwer

Toll‐like receptor 3 on adult human astrocytes triggers production of neuroprotective mediators

Toll‐like receptors (TLRs) are innate immunity receptors that are expressed on a wide range of cell types, including CNS glial cells. In general, TLR engagement by specific sets of microbial ligands triggers production of pro‐inflammatory factors and enhances antigen‐presenting cell functions. The functional roles of TLR in the CNS, however, are still poorly understood. While adult human astrocytes in culture dominantly express TLR4, they display a strikingly strong and selective induction of TLR3 when activated by pro‐inflammatory cytokines, TLR3 or TLR4 agonists, or oxidative stress. Gene profiling analysis of the astrocyte response to either TLR3 or TLR4 activation revealed that TLR3, but not TLR4, induces expression of a range of neuroprotective mediators and several other molecules that regulate cellular growth, differentiation, and migration. Also, TLR3 triggered enhanced production of anti‐inflammatory cytokines including interleukin‐9 (IL‐9), IL‐10, and IL‐11 and downregulation of the p40 subunit of IL‐12 and IL‐23. The collective TLR3‐induced products were found in functional assays to inhibit astrocyte growth, promote human endothelial cell growth, and importantly, to enhance neuronal survival in organotypic human brain slice cultures. Together, our data indicate that TLR3 is induced on human astrocytes upon inflammation and when activated, mediates a comprehensive neuroprotective response rather than a polarized pro‐inflammatory reaction.

The vasopressin and oxytocin neurons in the human supraoptic and paraventricular nucleus; changes with aging and in senile dementia

The neuropeptides vasopressin (AVP) and oxytocin (OXT) are supposed to be involved not only in peripheral functions (e.g. diuresis, labour and lactation) but also in central processes that are frequently disturbed during aging and senile dementia (e.g. fluid and electrolyte homeostasis and cognitive functions). A concomitant decrease in activity of the hypothalamo-neurohypophyseal system (HNS) with aging has been postulated in the literature, but has not yet been established. In order to investigate possible age-related changes in the human HNS, immunocytochemically identified AVP and OXT neurons in the paraventricular and supraoptic nucleus (PVN and SON) were analysed morphometrically in subjects from 10 to 93 years of age, including patients with senile dementia of the Alzheimer type (SDAT). Cell size was used as a parameter for peptide production. Mean profile area of OXT cells did not show any significant changes with increasing age. Mean profile area of AVP cells, however, showed an initial decrease up to the sixth decade of life, after which a gradual increase was observed. Size of AVP and OXT cell nuclei did not change significantly with aging. Observations in brains from patients with SDAT were within the range for their age group. The present results do not support degeneration or diminished function of the HNS in senescence or SDAT, as generally presumed in the literature, but suggest an activation of AVP cells after 80 years of age. The activation of AVP cells in senescence is in accordance with previous findings in the aged Wistar rat.

Cells in human postmortem brain tissue slices remain alive for several weeks in culture

Animal models for human neurological and psychiatric diseases only partially mimic the underlying pathogenic processes. Therefore, we investigated the potential use of cultured postmortem brain tissue from adult neurological patients and controls. The present study shows that human brain tissue slices obtained by autopsy within 8 h after death can be maintained in vitro for extended periods (up to 78 days) and can be manipulated experimentally. We report for the first time that 1) neurons and glia in such cultures could be induced to express the reporter gene LacZ after transduction with adeno‐associated viral vectors and 2) cytochrome oxidase activity could be enhanced by the addition of pyruvate to the medium. These slice cultures offer new opportunities to study the cellular and molecular mechanisms of neurological and psychiatric diseases and new therapeutic strategies.— Verwer, R. W. H., Hermens, W. T. J. M. C. Dijkhuizen, P. A., ter Brake, O., Baker, R. E., Salehi, A., Sluiter, A. A., Kok, M. J. M., Müller, L. J., Verhaagen, J., Swaab, D. F. Cells in human postmortem brain tissue slices remain alive for several weeks in culture. FASEB J. 16, 54–60 (2002)

Activation of vasopressin neurons in the human supraoptic and paraventricular nucleus in senescence and senile dementia

A recent study has shown that vasopressin (AVP) cells in the human supraoptic (SON) and paraventricular (PVN) nuclei increase in size after 60 years of age, suggesting that AVP production is increased in senescence. In the present study, the same brain material was used for the determination of nucleolar size in immunocytochemically identified AVP and oxytocin (OXT) neurons as an additional parameter for peptide production. A strong correlation was found between nucleolar size and cell size, both in AVP and OXT neurons. Nucleolar size of AVP but not of OXT neurons increased significantly in senescence. Observations in brains from patients with senile dementia of the Alzheimer type (SDAT) were commensurate with their ages. These results strongly support the hypothesis that AVP neurons in the SON and PVN are activated in old age.

Reduced neuronal activity and reactivation in Alzheimer's disease.

Publisher Summary Alzheimers disease (AD) is a multifactorial disease in which APOE-ɛ4 and age are important risk factors. In addition, various mutations and even viral infections, such as herpex simplex may play a role. AD is characterized histopathologically by the presence of large numbers of neuritic plaques (NPs) and cytoskeletal changes that are present as pretangles after staining— that is, by Alz-50 or AT8, or as neurofibrillary tangles (NFT) after silver staining. NFT are present in the cell bodies of affected neurons, while the same cytoskeletal changes are called neuropil threads when present outside neuritic plaques or dystrophic neurites when they are the neuritic components of neuritic plaques (NPs). Dystrophic neurites or neuropil threads are short, thickened, curly, coiled, or sometimes hooked fibres. To a lesser degree, NPs and cytoskeletal changes can also be observed in aged, nondemented control subjects. This chapter provides evidence in favor of the hypotheses that (1) the neuropathological Alzheimer changes cannot all be explained by a cascade starting with amyloid (β/A4) deposits as suggested example by Selkoe, but that (2) the neuropathological hallmarks of Alzheimers disease are basically independent phenomena, and that (3) cell death in Alzheimers disease is not a major generally occurring phenomenon, but is restricted to a few brain areas. A long struggle has been going on about the question what is more important for the development of dementia: cytoskeletal changes or amyloid. The answer is probably neither. As it is argued in this chapter, (4) reduced neuronal activity is most probably one of the major characteristics of AD and may underlie the clinical symptoms of dementia. Experiments are discussed that indicate that it is attractive to direct therapeutic strategies towards restimulation of neuronal metabolism and repair mechanisms in order to improve cognitive and behavioral symptoms of AD.

Therapeutic Strategies for Alzheimer Disease Focus on Neuronal Reactivation of Metabolically Impaired Neurons

Based on several lines of evidence, it has been hypothesized that decreased neuronal metabolic rate may precede cognitive impairment, contributing to neuronal atrophy as well as reduced neuronal function in Alzheimer disease (AD). Additionally, studies have shown that stimulation of neurons through different mechanisms may protect those cells from the deleterious effects of aging and AD, a phenomenon we paraphrased as “use it or lose it.” Therefore, it is attractive to direct the development of therapeutic strategies toward stimulation of metabolic rate/neuronal activity to improve cognition and other symptoms in AD. A number of pharmacological and nonpharmacological approaches discussed here support the concept that stimulation of the brain has beneficial effects and may, to a certain degree, restore several aspects of cognition and other central functions. For instance, the circadian system, which controls the sleep/wake cycle, may be stimulated in AD patients by exposing them to more light or transcutaneous nerve stimulation. We will also discuss a procedure that has been developed to culture human postmortem brain tissue, which allows testing of the efficacy of putative stimulatory compounds.

Decreased hippocampal metabolic activity in Alzheimer patients is not reflected in the immunoreactivity of cytochrome oxidase subunits.

In the present study we have compared histochemically determined cytochrome oxidase activity with the levels of immunocytochemically stained cytochrome oxidase subunits (CO II and CO IV) and ATP synthase in the human hippocampus in relation with Alzheimers disease. Cytochrome oxidase activity was significantly reduced in all hippocampal areas of Alzheimer patients. The protein levels of subunits II and IV were not different between control subjects and Alzheimer patients. Additionally, it was observed that the active cytochrome oxidase is evenly distributed over both cell bodies and neuropil, while a relatively large pool of inactive enzyme or precursors is limited to the neuronal somata. Further, in Alzheimer patients the CO IV immunoreactivity decreased with age, whereas in control subjects it increased with age. Our results suggest that the assembly of cytochrome oxidase or the processing of its subunits may be impaired.

Post-mortem brain tissue cultures from elderly control subjects and patients with a neurodegenerative disease

Aging may be viewed as a progressive loss of normal biological function. Due to complex genetic and environmental interactions, the sequence of functional impairment shows a high degree of individual variability. In humans life style and health care have an additional influence on the aging process. To study aging and age-related disorders of the human nervous system, brain tissue that has undergone aging and pathological alterations can provide valuable study material. Recently, we have shown that adult human postmortem brain tissue can be cultured and experimentally manipulated. This approach permits the study of cellular aspects of human neuronal aging and neurodegenerative processes and complements those existing research methods such as in vivo imaging (MRI, PET, etc.) and fixed or frozen postmortem brain tissue examination.

Tissue cultures from adult human postmortem subcortical brain areas.

Animal models used to study human aging and neurodegeneration do not display all symptoms of these processes as they are found in humans. Recently, we have shown that many cells in neocortical slices from adult human postmortem brain may survive for extensive periods in vitro. Such cultures may enable us to study age and disease related processes directly in human brain tissue. Here, we present observations on subcortical brain tissue.

Increased Metabolic Activity in Nucleus Basalis of Meynert Neurons in Elderly Individuals With Mild Cognitive Impairment as Indicated by the Size of the Golgi Apparatus

In this study, we examined the metabolic activity of nucleus basalis of Meynert (NBM) neurons in individuals clinically diagnosed with no cognitive impairment (NCI, n = 8), mild cognitive impairment (MCI, n = 9), and subjects with moderate Alzheimer disease (AD, n = 7). We used Golgi apparatus (GA) size as a measure of neuronal metabolic activity. Subjects with MCI showed increased NBM metabolic activity; they had significantly more neurons with larger GA size as compared with NCI and AD subjects. In contrast, more NBM neurons with extremely small GA sizes, indicating reduced metabolic activity, were seen in AD. When these cases were classified according to their AD pathology (Braak I-II, III-IV, or V-VI), Braak III-IV subjects showed significantly increased GA sizes, comparable with the increase in clinically diagnosed MCI, whereas in Braak V-VI, GA sizes were dramatically reduced. Of all MCI and NCI subjects with similar Braak III-IV pathology, the MCI subjects again had significantly larger GA sizes. The larger NBM neuronal GA size seen in MCI suggests increased metabolic activity, associated with both the clinical progression from NCI to MCI, and with the early stages of AD pathology.