Karen M. Cullen

Characterization of the Kynurenine Pathway in Human Neurons

The kynurenine pathway is a major route of l-tryptophan catabolism producing neuroactive metabolites implicated in neurodegeneration and immune tolerance. We characterized the kynurenine pathway in human neurons and the human SK-N-SH neuroblastoma cell line and found that the kynurenine pathway enzymes were variably expressed. Picolinic carboxylase was expressed only in primary and some adult neurons but not in SK-N-SH cells. Because of this difference, SK-N-SH cells were able to produce the excitotoxin quinolinic acid, whereas human neurons produced the neuroprotectant picolinic acid. The net result of kynurenine pathway induction in human neurons is therefore predicted to result in neuroprotection, immune regulation, and tumor inhibition, whereas in SK-N-SH cells, it may result in neurotoxicity, immune tolerance, and tumor promotion. This study represents the first comprehensive characterization of the kynurenine pathway in neurons and the first description of the involvement of the kynurenine pathway as a mechanism for controlling both tumor cell neurotoxicity and persistence.

Indoleamine 2,3 dioxygenase and quinolinic acid Immunoreactivity in Alzheimer's disease hippocampus

The present immunohistochemical study provides evidence that the kynurenine pathway is up‐regulated in Alzheimers disease (AD) brain, leading to increases in the excitotoxin quinolinic acid (QUIN). We show that the regulatory enzyme of the pathway leading to QUIN synthesis, indoleamine 2,3 dioxygenase (IDO) is abundant in AD compared with controls. In AD hippocampus, both IDO‐ and QUIN‐immunoreactivity (‐IR) was detected in cortical microglia, astrocytes and neurones, with microglial and astrocytic expression of IDO and QUIN highest in the perimeter of senile plaques. QUIN‐IR was present in granular deposits within the neuronal soma of AD cortex and was also seen uniformly labelling neurofibrillary tangles. Our data imply that QUIN may be involved in the complex and multifactorial cascade leading to neuro‐degeneration in AD. These results may open a new therapeutic door for AD patients.

Topography of brain atrophy during normal aging and alzheimer's disease

The present study investigated the effect of age on total and regional brain volumes and compared age-associated changes in 20 healthy controls with those observed in 12 patients with Alzheimers disease (AD). Weights and volumes of the whole brain and cerebrum, as well as the fractional volumes of the frontal, temporal, and parieto-occipital cortices, medial temporal structures, deep brain structures, and white matter were measured. Males had larger and heavier brains than females of comparable age. A small decline in brain volume with age was found (approximately 2 ml per year), but only within the white matter. In comparison, no further loss of white matter occurred in AD; however, the cerebral cortex was significantly reduced in volume, with the greatest loss from the medial temporal structures. This loss was related to disease progression; greater proportional loss was associated with more rapid decline in older patients. This study suggests that significant brain atrophy is not a consequence of advancing age. In addition, it suggests a regional specificity of damage in AD.

Microvascular pathology in the aging human brain: Evidence that senile plaques are sites of microhaemorrhages

Amyloid-rich plaques are a feature of the aging human cerebral cortex. We have recently described another feature of aging human cortex, microhaemorrhages, identified by their content of haem, red blood cells, collagen and clotting factors, and their spatial relationship to capillaries. Here we relate microhaemorrhages to amyloid deposits. Observations were made in three groups: patients with no history of dementia, patients with Alzheimers disease (AD) and patients with Downs syndrome (DS) and dementia. Amyloid deposits and microhaemorrhages were labelled in adjacent sections, amyloid deposits with antibodies to beta-amyloid (betaA), and microhaemorrhages by Prussian blue histochemistry for haem. The densities and sizes of betaA deposits and haem-rich deposits (HRDs), and their relationship to blood vessels, were surveyed in temporal, cingulate and superior frontal cortex. Our results suggest that HRDs and betaA deposits are the same sites of pathology. Their densities in the cortex and white matter of the regions surveyed varied markedly between cases, particularly between demented and non-demented cases, but they always co-varied; where haem deposits were sparse or numerous, so were betaA deposits. Both HRDs and betaA deposits formed adjacent to or encircling small vessels, often at branch points, and a spatial proximity analysis confirmed that both were found close to or colocalising with microvessels. Both HRDs and betaA deposits were associated with blood- or vessel-derived proteins (fibrinogen, von Willebrand factor and collagen VI). Since haem is an established marker of cerebral bleeding, and amyloid is a marker of senile plaques, our results indicate that senile plaques are sites of microhaemorrhages. This colocalisation raises the very testable questions of whether microhaemorrhages are early events in plaque formation and whether therapies which stabilise cerebral microvessels can prevent the onset or slow the progress of dementias associated with plaque formation.

The Excitotoxin Quinolinic Acid Induces Tau Phosphorylation in Human Neurons

Some of the tryptophan catabolites produced through the kynurenine pathway (KP), and more particularly the excitotoxin quinolinic acid (QA), are likely to play a role in the pathogenesis of Alzheimers disease (AD). We have previously shown that the KP is over activated in AD brain and that QA accumulates in amyloid plaques and within dystrophic neurons. We hypothesized that QA in pathophysiological concentrations affects tau phosphorylation. Using immunohistochemistry, we found that QA is co-localized with hyperphosphorylated tau (HPT) within cortical neurons in AD brain. We then investigated in vitro the effects of QA at various pathophysiological concentrations on tau phosphorylation in primary cultures of human neurons. Using western blot, we found that QA treatment increased the phosphorylation of tau at serine 199/202, threonine 231 and serine 396/404 in a dose dependent manner. Increased accumulation of phosphorylated tau was also confirmed by immunocytochemistry. This increase in tau phosphorylation was paralleled by a substantial decrease in the total protein phosphatase activity. A substantial decrease in PP2A expression and modest decrease in PP1 expression were observed in neuronal cultures treated with QA. These data clearly demonstrate that QA can induce tau phosphorylation at residues present in the PHF in the AD brain. To induce tau phosphorylation, QA appears to act through NMDA receptor activation similar to other agonists, glutamate and NMDA. The QA effect was abrogated by the NMDA receptor antagonist memantine. Using PCR arrays, we found that QA significantly induces 10 genes in human neurons all known to be associated with AD pathology. Of these 10 genes, 6 belong to pathways involved in tau phosphorylation and 4 of them in neuroprotection. Altogether these results indicate a likely role of QA in the AD pathology through promotion of tau phosphorylation. Understanding the mechanism of the neurotoxic effects of QA is essential in developing novel therapeutic strategies for AD.

Practical considerations for the use of the optical disector in estimating neuronal number

The aim of this study was to establish methodological variability in the estimation of the total number of neurones using the optical disector. Variations in the 3 dimensions of the disector probe were analysed under uniform sampling conditions in 50-microns-thick frozen sections of the human mediodorsal thalamic nucleus. There was no significant difference between the estimated neuronal number using samples of variable height (fractionator vs. non-fractionator sampling). In addition, different methods of volume calculation (individual sample vs. an average) did not significantly change the estimated total neuronal number. Large variations in the estimated total neuronal number occurred when the x and y dimensions of the disector probe were altered. In this study, accurate and reproducible estimates were achieved when the disector probe was large enough to have a probability of sampling at least 2 cells per frame. We conclude that the variables in the x-y plane (the disector frame size as well as the sample interval) significantly contribute to differences in the estimated total neuronal number. Several practical measurements to estimate this probability and enhance experimental design are discussed.

Pericapillary haem-rich deposits: evidence for microhaemorrhages in aging human cerebral cortex

In this Post mortem study, we examined haem-rich deposits (HRDs) in patients with and without dementia, using a histochemical label (Prussian blue) to show haem, autofluorescence to detect red blood cells (RBCs), and immunohistochemistry for clotting-related factors and collagen IV. The patients studied had no clinical or Post mortem evidence of macrovascular stroke. To allow examination of the spatial relationships between HRDs and the microvasculature, we cut 45-µm sections. Haem-rich deposits were small (< 200 µm diameter). They were rare in younger (< 50 years) patients but were more common in older (> 70 years) patients, particularly in cerebral cortex, and were most abundant in cases with senile plaques. Wherever HRDs appeared they were perivascular and appeared to form around capillaries or small arterioles. Using a software package (Proxan) developed to outline vessels and HRDs, and to analyse the distances between them, a tight spatial correlation between HRDs and capillaries was shown. In addition, HRDs were rich in von Willebrand factor (vWF), fibrinogen, collagen IV and RBCs. These observations suggest that HRDs are the residua of capillary bleeds (microhaemorrhages), and that microhaemorrhages are a common feature of the aging cerebral cortex, particularly where plaque pathology is present.

Expression of tryptophan 2,3-dioxygenase and production of kynurenine pathway metabolites in triple transgenic mice and human Alzheimer's disease brain.

To assess the role of the kynurenine pathway in the pathology of Alzheimers disease (AD), the expression and localization of key components of the kynurenine pathway including the key regulatory enzyme tryptophan 2,3 dioxygenase (TDO), and the metabolites tryptophan, kynurenine, kynurenic acid, quinolinic acid and picolinic acid were assessed in different brain regions of triple transgenic AD mice. The expression and cell distribution of TDO and quinolinic acid, and their co-localization with neurofibrillary tangles and senile β amyloid deposition were also determined in hippocampal sections from human AD brains. The expression of TDO mRNA was significantly increased in the cerebellum of AD mouse brain. Immunohistochemistry demonstrated that the density of TDO immuno-positive cells was significantly higher in the AD mice. The production of the excitotoxin quinolinic acid strongly increased in the hippocampus in a progressive and age-dependent manner in AD mice. Significantly higher TDO and indoleamine 2,3 dioxygenase 1 immunoreactivity was observed in the hippocampus of AD patients. Furthermore, TDO co-localizes with quinolinic acid, neurofibrillary tangles-tau and amyloid deposits in the hippocampus of AD. These results show that the kynurenine pathway is over-activated in AD mice. This is the first report demonstrating that TDO is highly expressed in the brains of AD mice and in AD patients, suggesting that TDO-mediated activation of the kynurenine pathway could be involved in neurofibrillary tangles formation and associated with senile plaque. Our study adds to the evidence that the kynurenine pathway may play important roles in the neurodegenerative processes of AD.

Activated actin-depolymerizing factor/cofilin sequesters phosphorylated microtubule-associated protein during the assembly of alzheimer-like neuritic cytoskeletal striations.

In Alzheimers disease (AD), rod-like cofilin aggregates (cofilin–actin rods) and thread-like inclusions containing phosphorylated microtubule-associated protein (pMAP) tau form in the brain (neuropil threads), and the extent of their presence correlates with cognitive decline and disease progression. The assembly mechanism of these respective pathological lesions and the relationship between them is poorly understood, yet vital to understanding the causes of sporadic AD. We demonstrate that, during mitochondrial inhibition, activated actin-depolymerizing factor (ADF)/cofilin assemble into rods along processes of cultured primary neurons that recruit pMAP/tau and mimic neuropil threads. Fluorescence resonance energy transfer analysis revealed colocalization of cofilin-GFP (green fluorescent protein) and pMAP in rods, suggesting their close proximity within a cytoskeletal inclusion complex. The relationship between pMAP and cofilin–actin rods was further investigated using actin-modifying drugs and small interfering RNA knockdown of ADF/cofilin in primary neurons. The results suggest that activation of ADF/cofilin and generation of cofilin–actin rods is required for the subsequent recruitment of pMAP into the inclusions. Additionally, we were able to induce the formation of pMAP-positive ADF/cofilin rods by exposing cells to exogenous amyloid-β (Aβ) peptides. These results reveal a common pathway for pMAP and cofilin accumulation in neuronal processes. The requirement of activated ADF/cofilin for the sequestration of pMAP suggests that neuropil thread structures in the AD brain may be initiated by elevated cofilin activation and F-actin bundling that can be caused by oxidative stress, mitochondrial dysfunction, or Aβ peptides, all suspected initiators of synaptic loss and neurodegeneration in AD.

Neurofibrillary degeneration and cell loss in the nucleus basalis in comparison to cortical Alzheimer pathology

Neurofibrillary tangle staging was compared in the nucleus basalis and cerebral cortex of Alzheimers disease patients with and without Lewy body disease. In pure Alzheimers disease, cholinergic nucleus basalis cell number, as determined from counts in serial forebrain sections, was 22-60% of control mean, with the majority of residual cells containing tangles. A comparison between control cell number and the combined number of tangles plus tangle-free neurons in pure Alzheimers disease suggests that the majority of nucleus basalis neurons were lost through neurofibrillary degeneration. The staging of neurofibrillary degeneration in the nucleus basalis was discordant with cortical changes as some controls had more extensive tangle formation in the nucleus basalis than in the cerebral cortex. Patients having both Alzheimers disease and Lewy body pathology had few or no tangles in the nucleus basalis despite greater loss of neurons than purely demented patients. The presence of concomitant pathology had a greater effect on nucleus basalis tangle burden than did cortical disease stage, suggesting dichotomous disease processes in the cerebral cortex and forebrain.