Hossein A. Ghanbari

Ectopic localization of phosphorylated histone H3 in Alzheimer's disease: A mitotic catastrophe?

Despite their terminally differentiated status, vulnerable neurons in Alzheimers disease (AD) display evidence of cell cycle activation, suggesting that mitotic dysfunction may be important in disease pathogenesis. To further delineate the role of mitotic processes in disease pathogenesis, we investigated phosphorylated histone H3, a key component involved in chromosome compaction during cell division. Consistent with an activation of the mitotic machinery, we found an increase in phosphorylated histone H3 in hippocampal neurons in AD. However, rather than within the nucleus as in actively dividing cells, activated phosphorylated histone H3 in AD is restricted to the neuronal cytoplasm despite activation of the mitotic machinery. Therefore, the aberrant cytoplasmic localization of phosphorylated histone H3 indicates a mitotic catastrophe that leads to neuronal dysfunction and neurodegeneration in AD.

Characterization of the AD7C-NTP cDNA expression in Alzheimer's disease and measurement of a 41-kD protein in cerebrospinal fluid.

We have isolated a novel Alu sequence-containing cDNA, designated AD7c-NTP, that is expressed in neurons, and overexpressed in brains with Alzheimers disease (AD). The 1,442-nucleotide AD7c-NTP cDNA encodes an approximately 41-kD protein. Expression of AD7c-NTP was confirmed by nucleic acid sequencing of reverse transcriptase PCR products isolated from brain. AD7c-NTP cDNA probes hybridized with 1. 4 kB mRNA transcripts by Northern blot analysis, and monoclonal antibodies generated with the recombinant protein were immunoreactive with approximately 41-45-kD and approximately 18-21-kD molecules by Western blot analysis. In situ hybridization and immunostaining studies localized AD7c-NTP gene expression in neurons. Using a quantitative enzyme-linked sandwich immunoassay (Ghanbari, K., I. Beheshti, and H. Ghanbari, manuscript submitted for publication) constructed with antibodies to the recombinant protein, AD7c-NTP levels were measured under code in 323 clinical and postmortem cerebrospinal fluid (CSF) samples from AD, age-matched control, Parkinsons disease, and neurological disease control patients. The molecular mass of the AD7c-NTP detected in CSF was approximately 41 kD. In postmortem CSF, the mean concentration of AD7c-NTP in cases of definite AD (9.2+/-8.2 ng/ml) was higher than in the aged control group (1.6+/-0.9; P < 0.0001). In CSF samples from individuals with early possible or probable AD, the mean concentration of AD7c-NTP (4.6+/-3.4) was also elevated relative to the levels in CSF from age-matched (1.2+/-0.7) and neurological disease (1.0+/-0.9) controls, and ambulatory patients with Parkinsons disease (1.8+/-1.1) (all P < 0.001). CSF levels of AD7c-NTP were correlated with Blessed dementia scale scores (r = 0. 66; P = 0.0001) rather than age (r = -0.06; P > 0.1). In vitro studies demonstrated that overexpression of AD7c-NTP in transfected neuronal cells promotes neuritic sprouting and cell death, the two principal neuroanatomical lesions correlated with dementia in AD. The results suggest that abnormal AD7c-NTP expression is associated with AD neurodegeneration, and during the early stages of disease, CSF levels correlate with the severity of dementia.

Oxidative damage in the olfactory system in Alzheimer's disease

Increased oxidative damage is a prominent and early feature of vulnerable neurons in Alzheimers disease (AD). However, while damage to proteins, sugars, lipids, nucleic acids and organelles such as lysosomes, mitochondria, and endoplasmic reticulum are evident, the source of increased reactive oxygen species has not been determined. Furthermore, a major limitation in further determining the source, as well as finding a means to arrest damage, is the paucity of cellular models directly homologous to AD since the vulnerable neurons of the brain in AD cannot be studied in vitro. Here, we examined the olfactory epithelium in situ to see if neurons there exhibit a similar pathological oxidative balance to vulnerable neurons in AD. In biopsy specimens, (eight AD and three controls) we found that neurons, and also the surrounding epithelial cells, show an increase in oxidative damage for a subset of the markers increased in the brain of cases of AD. Lipid peroxidation and heme oxygenase-1, a stress response protein, were increased, while nucleic acid or protein oxidation, demonstrated in vulnerable neurons in AD, were not increased. These findings highlight the systemic nature of oxidative abnormalities in AD, but that different cell types may express this abnormality by a different array of oxidative stress markers, supporting the potential for using olfactory neurons or other cells derived from AD patients in culture to understand the mechanistic basis for increased oxidative damage in AD and as a model to screen compounds for therapeutic intervention.

Oxidative damage in cultured human olfactory neurons from Alzheimer's disease patients

Oxidative abnormalities precede clinical and pathological manifestations of Alzheimers disease and are the earliest pathological changes reported in the disease. The olfactory pathways and mucosa also display the pathological features associated with Alzheimers disease in the brain. Olfactory neurons are unique because they can undergo neurogenesis and are able to be readily maintained in cell culture. In this study, we examined neuronal cell cultures derived from olfactory mucosa of Alzheimers disease and control patients for oxidative stress responses. Levels of lipid peroxidation (hydroxynonenal), Nɛ‐(carboxymethyl)lysine (glycoxidative and lipid peroxidation), and oxidative stress response (heme oxygenase‐1) were measured immunocytochemically. We found increased levels for all the oxidative stress markers examined in Alzheimers disease neurons as compared to controls. Interestingly, in one case of Alzheimers disease, we found hydroxynonenal adducts accumulated in cytoplasmic lysosome‐like structures in about 20% of neurons cultured, but not in neurons from control patients. These lysosome‐like structures are found in about 100% of the vulnerable neurons in brains of cases of Alzheimers disease. This study suggests that manifestations of oxidative imbalance in Alzheimers disease extend to cultured olfactory neurons. Primary culture of human olfactory neurons will be useful in understanding the mechanism of oxidative damage in Alzheimers disease and can even be utilized in developing therapeutic strategies.

Neuronal CDK7 in hippocampus is related to aging and Alzheimer disease

Despite their supposedly terminally-differentiated quiescent status, many neurons in Alzheimer disease display an ectopic re-expression of cell-cycle related proteins. In the highly regulated process of cell cycle, cyclin-dependent kinase 7 (Cdk7) plays a crucial role as a Cdk-activating kinase and activates all of the major Cdk-cyclin substrates. In this study, we demonstrate that Cdk7 immunoreactivity is significantly elevated in susceptible hippocampal neurons of Alzheimer disease patients in comparison with age-matched controls. Notably, the expression of Cdk7 is age-dependent, with decreased levels between the ages of 54 and 65 years and after the age of 78. While the Cdk7 levels in Alzheimer disease patients are higher than controls within each age group, the difference is greatest between ages 54-65 where disease susceptibility and/or progression is likely more related to genetic factors.

Neuronal polo-like kinase in Alzheimer disease indicates cell cycle changes

Neurons of adults apparently lack the components necessary to complete the cell division process. Therefore, in Alzheimer disease, the increased expression of cell cycle-related proteins in degenerating neurons likely leads to an interrupted mitotic process associated with cytoskeletal abnormalities and, ultimately, neuronal degeneration. In this study, to further delineate the role of mitotic processes in the pathogenesis of Alzheimer disease, we undertook a study of polo-like kinase (Plk), a protein that plays a crucial role in the cell cycle. Our results show disease-related increases in Plk in susceptible hippocampal and cortical neurons in comparison to young or age-matched controls. An increase in neuronal Plk further implicates aberrations in cell cycle control in the pathogenesis of Alzheimer disease and provides a novel mechanistic basis for therapeutic intervention.

Biochemical assay for AD7C-NTP in urine as an Alzheimer's disease marker.

A reliable and specific immunoassay has been developed to detect and measure AD7C‐NTP, a biochemical marker for Alzheimers disease, in urine. The urine samples are first processed by centrifugation and ultrafiltration to fractionate and concentrate AD7C‐NTP. The urinary AD7C‐NTP has the same molecular weight as AD7C‐NTP in brain and cerebro‐spinal fluid by size exclusion chromatography. It has also retained the binding properties to the monoclonal and polyclonal antibodies developed against recombinantly produced AD7C‐NTP. This assay is an enzyme linked sandwich immunoassay (ELSIA) using 96 well microtiter plates. The plate surface is coated with a monoclonal antibody (N3I4) which has a high affinity and specificity for AD7C‐NTP, capturing it effectively from the samples. The detection was achieved using a polyclonal antibody (ADRI). The utility of the assay has been demonstrated using urine specimens from Alzheimers disease (AD) patients and non‐Alzheimers controls. Urinary AD7C‐NTP in the AD group (2.5 ng/mL, n=66) was significantly higher than the non‐AD group (0.8 ng/mL, n=134).Using 1.5 ng/mL as cut off, in this patient population, specificity and sensitivity of urinary AD7C‐NTP were comparable to CSF AD7C‐NTP. J. Clin. Lab. Anal. 12:285–288, 1998.

Phosphorylated tau epitope of Alzheimer's disease is coupled to axon development in the avian central nervous system

The monoclonal antibody PHF-1 recognizes phosphorylated tau isoforms present in paired helical filaments of Alzheimers disease. We have found that PHF-1 immunoreactivity is present in chick brain, which expresses three major PHF-1-reactive proteins at the same molecular weights seen in humans. The developmental pattern of expression suggests a functional role in differentiation, rather than in programmed nerve cell death. Expression of PHF-1 immunoreactivity in developing retina was highly cell selective, showing robust staining of ganglion cells, the only long-axon neuron of the seven major retina cell types. The majority of ganglion cells were PHF-1 positive. The developmental window of expression extended at least from E6 through P0, well outside the period of embryonic ganglion cell death. Mature cells did not show PHF-1 immunoreactivity. In the embryo, staining was particularly robust in ganglion cell axons (optic fiber layer), and association of PHF-1 reactivity with axonal tracts also was seen in developing forebrain. PHF-1 polarization occurred at ages when staining with polyclonal anti-tau did not show axonal selectivity. Similarly, in cell cultures, PHF-1 immunoreactivity became localized to single neurites, but polyclonal anti-tau did not. These results indicate that, rather than being associated with cell degeneration, PHF-1 immunoreactivity in the developing nervous system is associated with early stages of axon formation, both in vivo and in vitro. Therefore, expression of PHF-1 immunoreactive proteins in Alzheimers disease suggests that paired helical filament formation might be triggered by mechanisms related to axon regeneration.

Aberrant expression of metabotropic glutamate receptor 2 in the vulnerable neurons of Alzheimer’s disease

Selective neuronal dysfunction and degeneration are defining features of Alzheimer’s disease (AD). While the exact mechanism(s) contributing to this selective neuronal vulnerability remains to be elucidated, we hypothesized that the differential expression of metabotropic glutamate receptors (mGluRs) may play a key role in this process since the various mGluR groups differentially regulate neuronal cell death and survival. In the present study, we focused on the metabotropic glutamate receptor 2 (mGluR2), a subtype of group II mGluRs. The mGluR2 is expressed at low levels in pyramidal neurons in age-matched control cases, whereas we found a strikingly increased mGluR2 expression in AD, in a pattern that mirrored both the regional and cellular subtype of neuronal vulnerability to degeneration and neurofibrillary alterations. Immunoblot analysis confirmed the significant increase in the level of mGluR2 in AD compared with age-matched controls. Agonists for group II mGluRs activate extracellular receptor kinase (ERK), a kinase that is chronically activated in vulnerable neurons of AD. ERK is able to phosphorylate tau protein, so the up-regulation of mGluR2 in vulnerable neurons may represent the upstream mediator of abnormal tau phosphorylation in AD. Immunocytochemical examination revealed considerable overlap between mGluR2 and neurofibrillary alterations. Thus, it is likely that mGluR2 represents a novel therapeutic target for AD.

Differential Regulation of Glutamate Receptors in Alzheimer’s Disease

Selective neurodegeneration is a prominent feature in Alzheimer’s disease; however, the mechanism of neuronal death is still unclear. Nonetheless, the topographical distribution of different types of receptors is thought to contribute to the regional selective nature of neuronal degeneration. Specifically, since glutamatergic transmission is severely altered by the early degeneration of cortico-cortical connections and hippocampal projections in Alzheimer’s disease, we suspect that glutamate receptors may play a new role in the pathophysiology of disease. Here we review the salient aspects of glutamate receptor expression in Alzheimer’s disease and how their differential regulation can contribute to the selective neurodegeneration seen in the disease. Additionally, we assess the potential therapeutic value of glutamate receptors as a target for drug intervention in Alzheimer’s disease.