Ruth F. Itzhaki

A micro-biuret method for estimating proteins

Abstract A rapid and sensitive method for estimating proteins using an alkaline copper sulfate reagent is described. The method is reasonably nonspecific for type of protein and can be used for solutions containing DNA even at concentrations of the latter as high as 0.7 mg/ml in the final reaction mixture. In the presence of DNA, 0.15 to 3.0 mg of protein can be estimated and in the absence of DNA, as little as 0.075 mg. The effects are described of various salts, of perchloric acid, and of urea on the estimation. Measurements on several dipeptides showed that proline peptides do not form ultraviolet-absorbing complexes with copper whereas prolyl peptides do form such complexes. This explains the finding that gelatin (which has a high proportion of proline and of hydroxyproline residues) is less reactive with copper than are the other proteins studied here.

Herpes simplex virus type 1 in brain and risk of Alzheimer's disease

BACKGROUND The apolipoprotein E epsilon 4 (APOE-epsilon 4) allele is a risk factor for Alzheimers disease (AD), but it is neither essential nor sufficient for development of the disease. Other factors-genetic or environmental-must therefore have a role. By means of a PCR we have detected herpes simplex virus type 1 (HSV1) in latent form in brains of elderly people with and without AD. We have postulated that limited reactivation of the virus causes more damage in AD patients than in elderly people without AD because of a difference in the hosts. We now report the APOE genotypes of AD patients and non-AD sufferers with and without HSV1 in brain. METHODS DNA was extracted from 84 samples of brain from 46 AD patients (39 temporal lobe, 39 frontal lobe, three hippocampus) and from 75 samples of brain from 44 non-AD elderly people (33 temporal lobe, 36 frontal lobe, six hippocampus). PCR amplification was used to detect HSV1 thymidine kinase gene and the host APOE gene. FINDINGS By multiple logistic regression, the APOE-epsilon 4 allele frequency was significantly higher in the patients positive for HSV1 in brain than in the HSV1-negative AD group, the HSV1-positive non-AD group, or the HSV1-negative non-AD group (52.8% vs 10.0%, 3.6%, and 6.3%, respectively). The odds ratio for APOE-epsilon 4 in the HSV1-positive AD group compared with HSV1-negative non-AD group was 16.8 (95% CI 3.61-77.8) and in the HSV1-negative AD group, 1.67 (0.21-13.4). We also compared APOE genotypes of 40 people who had recurrent cold sores and 33 non-sufferers; the APOE-epsilon 4 allele frequencies were 36% and 9%, respectively (p < 0.0001). INTERPRETATION These findings suggest that the combination of HSV1 in brain and carriage of an APOE-epsilon 4 allele is a strong risk factor for AD, whereas either of these features alone does not increase the risk of AD. The findings in people with cold sores support our hypothesis that APOE-epsilon 4 and HSV1 together are damaging in the nervous system.

Herpes simplex virus type 1 DNA is located within Alzheimer's disease amyloid plaques.

The brains of Alzheimers disease sufferers are characterized by amyloid plaques and neurofibrillary tangles. However, the cause(s) of these features and those of the disease are unknown, in sporadic cases. We previously showed that herpes simplex virus type 1 is a strong risk factor for Alzheimers disease when in the brains of possessors of the type 4 allele of the apolipoprotein E gene (APOE‐ε4), and that β‐amyloid, the main component of plaques, accumulates in herpes simplex virus type 1‐infected cell cultures and mouse brain. The present study aimed to elucidate the relationship of the virus to plaques by determining their proximity in human brain sections. We used in situ polymerase chain reaction to detect herpes simplex virus type 1 DNA, and immunohistochemistry or thioflavin S staining to detect amyloid plaques. We discovered a striking localization of herpes simplex virus type 1 DNA within plaques: in Alzheimers disease brains, 90% of the plaques contained the viral DNA and 72% of the DNA was associated with plaques; in aged normal brains, which contain amyloid plaques at a lower frequency, 80% of plaques contained herpes simplex virus type 1 DNA but only 24% of the viral DNA was plaque‐associated (p < 0.001). We suggest that this is because in aged normal individuals, there is a lesser production and/or greater removal of β‐amyloid (Aβ), so that less of the viral DNA is seen to be associated with Aβ in the brain. Our present data, together with our finding of Aβ accumulation in herpes simplex virus type 1‐infected cells and mouse brain, suggest that this virus is a major cause of amyloid plaques and hence probably a significant aetiological factor in Alzheimers disease. They point to the usage of antiviral agents to treat the disease and possibly of vaccination to prevent it. Copyright

The significance of environmental factors in the etiology of Alzheimer's disease

The proposition that environmental agents, such as diet, aluminum, and viruses, are as important as genetic factors in the etiology of Alzheimers disease (AD) was advanced by the authors at the Challenging Views of Alzheimers Disease meeting held in Cincinnati on July 28 and 29, 2001. Diet, dietary fat, and to a lesser extent, total energy (caloric intake), were found to be significant risk factors for the development of AD in a dozen countries, while fish consumption was found to be a significant risk reduction factor. An acid-forming diet, such as one high in dietary fat or total energy, can lead to increased serum and brain concentrations of aluminum and transition metal ions, which are implicated in oxidative stress potentially leading to the neurological damage characteristic of AD. Many of the risk factors for AD, such as cholesterol and fat, and risk reduction factors, such as whole grain cereals and vegetables, are shared with ischemic heart disease. Aluminum may cause neurological damage and a number of studies have linked aluminum to an increased risk for developing AD. The evidence for viral agents playing a role in AD is the strong association between the presence of HSV1 in brain and carriage of an apoE-epsilon4 allele in the case of AD patients but not of controls; statistical analysis shows the association is causal. Diet, aluminum, and viral infections may increase the prevalence of AD by eliciting inflammation, which may cause the neurological damage that results in AD.

Herpes simplex virus infection causes cellular β-amyloid accumulation and secretase upregulation

It is uncertain whether environmental factors contribute to the formation of senile plaques and neurofibrillary tangles, the abnormal features that define the Alzheimers disease (AD) brain. We previously proposed that herpes simplex virus type 1 (HSV1) is a strong risk factor for AD when it is present in the brains of people who possess the type 4 allele of the apolipoprotein E gene (APOE-epsilon4); however a direct biochemical link between viral infection and the development of the AD pathological features has never previously been examined. Here we show that infection of cultured neuronal and glial cells with HSV1 leads to a dramatic increase in the intracellular levels of beta-amyloid (Abeta) 1-40 and 1-42, whilst levels of amyloid precursor protein (APP) in cells decrease. Similarly, Abeta1-42 deposits are present in mouse brain after HSV1 infection. In the cultured cells the mechanism involves increased Abeta production, rather than merely greater retention of cellular Abeta, as levels of beta-site APP-cleaving enzyme (BACE-1) and of nicastrin, a component of gamma-secretase, both increase in HSV1-infected cells. These novel data show that HSV1 can directly contribute to the development of senile plaques.

Alzheimer's disease and infection: Do infectious agents contribute to progression of Alzheimer's disease?

Infection with several important pathogens could constitute risk factors for cognitive impairment, dementia, and Alzheimers disease (AD) in particular. This review summarizes the data related to infectious agents that appear to have a relationship with AD. Infections with herpes simplex virus type 1, picornavirus, Borna disease virus, Chlamydia pneumoniae, Helicobacter pylori, and spirochete were reported to contribute to the pathophysiology of AD or to cognitive changes. Based on these reports, it may be hypothesized that central nervous system or systemic infections may contribute to the pathogenesis or pathophysiology of AD, and chronic infection with several pathogens should be considered a risk factor for sporadic AD. If this hypothesis holds true, early intervention against infection may delay or even prevent the future development of AD.

Microbes and Alzheimer's Disease

We are researchers and clinicians working on Alzheimer’s disease (AD) or related topics, and we write to express our concern that one particular aspect of the disease has been neglected, even thoug ...

Herpes simplex virus type 1 in Alzheimer's disease: the enemy within.

Alzheimers disease is a modern scourge and is likely to become increasingly so in the future, with increasing longevity. The disease has been investigated for over one hundred years yet its causes and that of the neuropathological characteristics seen in AD brain are still completely unknown. Evidence for a major causative role of a common virus, herpes simplex virus type 1 (HSV1), acting in combination with a genetic factor - the type 4 allele of the apolipoprotein gene, a known susceptibility factor - is presented here. The characteristics of the virus, some of which make it an especially likely candidate for this role, are described, as are the many precedents for the action of a genetic factor modulating outcome of infection. Various possible ways in which HSV1 might lead to development of AD, such as its up-regulation of various enzymes and in particular certain kinases, its effect on the cell cycle, on autophagy, and its inflammatory and oxidative effects are also discussed. It is concluded that there is strong evidence that the virus is indeed a major factor in AD and therefore there is a strong case for appropriate treatment, and possibly for prevention in the future.

Colorimetric method for estimating polylysine and polyarginine.

Abstract A rapid and sensitive method is described for estimating polylysine and polyarginine. The method involves the stoichiometric precipitation of these polyamino acids by excess of the anionic dye methyl orange, followed by spectrophotometric determination of unbound dye. 10 μg or less of polylysine or polyarginine can thus be estimated. The effects of various salts on the estimation are described.

Alzheimer's disease-specific tau phosphorylation is induced by herpes simplex virus type 1.

Neurofibrillary tangles are one of the main neuropathological features of Alzheimers disease (AD) and are composed of abnormally phosphorylated forms of a microtubule-associated protein called tau. What causes this abnormal phosphorylation is unknown. Our previous studies have implicated herpes simplex virus type 1 (HSV1) as an etiological agent in AD, and so we investigated whether infection with this virus induces AD-like tau phosphorylation. Here we demonstrate that HSV1 causes tau phosphorylation at several sites, including serine 202, threonine 212, serine 214, serine 396 and serine 404. In addition, we have elucidated the mechanism involved by showing that the virus induces glycogen synthase kinase 3beta and protein kinase A, the enzymes that cause phosphorylation at these sites. Our data clearly reveal the importance of HSV1 in AD-type tau phosphorylation, and support the case that the virus is a cause of the disease. Together with our previous data, our results point to the use of antiviral agents to slow the progression of the disease.