Homocysteine,Infections,Polyamines,Oxidative Metabolism,and the Pathogenesis of Dementia and Atherosclerosis

Abstract

Hyperhomocysteinemia is a risk factor for development of dementia and Alzheimer’s disease (AD), and low blood levels of folate and cobalamin are associated with hyperhomocysteinemia and AD. In elderly subjects with cognitive decline, supplementation with folate, cobalamin, and pyridoxal demonstrated reduction of cerebral atrophy in gray matter regions vulnerable to the AD process. Multiple pathogenic microbes are implicated as pathogenic factors in AD and atherosclerosis, and the deposition of amyloid-β (Aβ), phosphorylation of tau protein, neuronal injury, and apoptosis in AD are secondary to microbial infection. Glucose utilization and blood flow are reduced in AD, and these changes are accompanied by downregulation of glucose transport, Na, K-ATPase, oxidative phosphorylation, and energy consumption. Thioretinaco ozonide, the complex formed from thioretinamide, cobalamin, ozone, and oxygen is proposed to constitute the active site of oxidative phosphorylation, catalyzing synthesis of adenosine triphosphate (ATP) from nicotinamide adenine dinucleotide (NAD⁺) and phosphate. Pathogenic microbes cause synthesis of polyamines in host cells by increasing the transfer of aminopropyl groups from adenosyl methionine to putrescine, resulting in depletion of intracellular adenosyl methionine concentrations in host cells. Depletion of adenosyl methionine causes dysregulation of methionine metabolism, hyperhomocysteinemia, reduced biosynthesis of thioretinamide and thioretinaco ozonide, decreased oxidative phosphorylation, decreased production of nitric oxide and peroxynitrite, and impaired host response to infectious microbes, contributing to the pathogenesis of dementia and atherosclerosis.

Keywords

Adenosyl methionine aging atherosclerosis cystathionine synthase dementia homocysteine nitric oxide oxidative phosphorylation peroxynitrite polyamines thioretinaco ozonide

HOMOCYSTEINE AND DEMENTIA

A prospective study of 1,092 participants in the Framingham Heart Study without dementia demonstrated that those subjects with elevated plasma homocysteine levels have an increased risk of subsequent development of dementia and Alzheimer’s disease (AD) after eight years of observation [1]. Previous studies established that low blood levels of folate and cobalamin and elevated homocysteine levels are associated with clinically or histopathologically confirmed dementia of the Alzheimer type [2, 3]. An interventional study to lower homocysteine levels with B-vitamins (folic acid 0.8 mg, vitamin B6 20 mg, vitamin B12 0.5 mg) in elderly subjects with mild cognitive impairment demonstrated reduction of cerebral atrophy in gray matter regions specifically vulnerable to the AD process, including the medial temporal lobe, during a 2-year treatment protocol [4]. A recent analysis demonstrated a decreased incidence of dementia over three decades among participants in the Framingham Heart Study [5]. Although the prevalence of vascular disease risk factors (except for diabetes and obesity) associated with dementia has decreased over the period of observation, these trends do not completely explain the observed decrease in dementia [5].

INFECTIONS IN DEMENTIA AND ATHEROSCLEROSIS

Because of the failure of 413 therapeutic trials of AD, based on the amyloid cascade hypothesis, current opinion is that therapeutic intervention based on the microbial etiology of AD is more likely to be successful [6]. A recent review summarizes the evidence for the pathogenesis of AD from chronic infection by Herpes Simplex Virus, Cytomegalovirus, other Herpesviridae, Chlamydophilia pneumoniae, spirochetes, Helicobacter pylori, and various periodontal pathogens [7]. According to this view, the deposition of amyloid-β (Aβ), phosphorylation of tau protein, neuronal injury, and apoptosis are reactive processes caused by chronic microbial infection. Supporting evidence is that Aβ is an anti-microbial peptide deposited in senile plaques and neurofibrillary tangles as a normal function of the innate immune system in AD [8]. Host cell toxicity from anti-microbial peptides induces mitochondrial dysfunction by Aβ peptides by decreasing membrane fluidity [9], opening of the mitochondrial permeability transition pore [10], release of cytochrome C, induction of DNA breaks, and cleavage of poly-ADP ribose polymerase (PARP), the NAD⁺-dependent enzyme that is involved in DNA repair [11].

AD accounts for 50 to 56% of cases of dementia in autopsy and clinical series, and AD combined with intracerebral vascular disease accounts for another 13 to 17% of cases of dementia [12]. Aging is the principal risk factor for AD, with the incidence doubling every 5 years after 65 years of age, affecting approximately one third of individuals over the age of 85 [12]. Atherosclerosis is also associated with hyperhomocysteinemia and aging, and blood homocysteine levels increase approximately 1 μmol/L per decade over the age of 60 [13]. Increasing evidence supports the view that microbial infections, including most of the organisms implicated in the etiology of AD, are causal in the pathogenesis of atherosclerosis [14]. The origin of vulnerable atherosclerotic plaques is attributed to obstruction of vasa vasorum by aggregates of microbes and lipoproteins, exacerbated by homocysteinylation of low-density lipoprotein (LDL), production of autoantibodies to LDL, endothelial dysfunction and impaired erythrocyte deformability, resulting in an intimal micro-abscess, the vulnerable plaque [15].

OXIDATIVE METABOLISM AND DEMENTIA

Imaging of subjects with AD using positron emission spectroscopy demonstrates progressive reduction in brain glucose metabolism and blood flow in relation to the severity of dementia [16]. These reductions follow regional synaptic loss or dysfunction, reflecting downregulation of gene expression for glucose transport, Na, K-ATPase, oxidative phosphorylation, and energy consumption in brain [16]. In the process of aging, dysfunctional mitochondria show a decreased capacity to produce ATP by oxidative phosphorylation because of diminished activities of complexes I and IV [17]. In addition to decreased electron transfer and reduced oxygen consumption in mitochondria of aged animals, a decreased membrane potential, increased oxidation products of phospholipids, proteins, and DNA, and increased size and fragility of mitochondria are also observed. In a study of hippocampal neurons in AD, the levels of mitochondrial DNA and cytochrome oxidase-1 were found to be increased, even though the number of mitochondria per neuron is decreased, and evidence of mitosis in pyramidal neurons is interpreted to indicate reactive synthesis of new mitochondria [18].

THIORETINACO OZONIDE AND OXIDATIVE PHOSPHORYLATION

Because of the discovery of failure of malignant cells to oxidize the sulfur atom of homocysteine thiolactone to sulfate [19], a series of synthetic derivatives of homocysteine thiolactone was tested for anti-neoplastic activity in mice with transplanted tumors [20]. The amide synthesized from homocysteine thiolactone and retinoic acid, thioretinamide (TR), was found to have anti-neoplastic, anti-carcinogenic, and anti-atherogenic activity in mice and rats [20, 21]. Two molecules of thioretinamide form a complex with cobalamin to form thioretinaco (TR₂Co), and the disulfonium derivative of thioretinaco, produced by reaction of thioretinaco with ozone, was proposed to catalyze ATP synthesis by the F1F0 complex of mitochondria by stereospecific binding of the phosphate groups of ATP to the disulfonium sulfur atoms of thioretinaco ozonide complexed with oxygen (TR₂CoO₃O₂ATP) [20]. The active site of oxidative phosphorylation was proposed to result from binding of nicotinamide adenine dinucleotide (NAD⁺) and inorganic phosphate (H₂PO₄^-) to form TR₂CoO₃O₂NAD⁺H₂PO₄^-, which catalyzes ATP synthesis in coordination with reduction of oxygen by electrons from electron transport complexes and production of a trans membrane potential through creation of a proton gradient [22].

ADENOSYL METHIONINE AND HYPERHOMOCYSTEINEMIA

Adenosyl methionine is the sulfonium derivative of methionine formed from the reaction of ATP and methionine [23]. Adenosyl methionine is the allosteric regulator of methionine metabolism which inhibits the activity of methylenetetrahydrofolate reductase [24] and stimulates the activity of cystathionine synthase [25]. Because of these regulatory effects, decreased intracellular concentrations of adenosyl methionine cause increased methylation of homocysteine to methionine and decreased conversion of homocysteine and serine to cystathionine, leading to hyperhomocysteinemia. A proposed scheme for adenosyl methionine synthesis from methionine requires thioretinaco ozonide and ATP [26]. The increasing plasma homocysteine levels and decreasing cellular oxidative phosphorylation observed in human aging are attributed to loss of thioretinaco ozonide from cellular membranes and decreased production of adenosyl methionine [26]. The hyperhomocysteinemia and decreased oxidative phosphorylation observed in AD [1, 16] are attributable to loss of thioretinaco ozonide from mitochondria and dysregulation of methionine metabolism because of decreased biosynthesis of adenosyl methionine [26]. The proposal for the dependence of oxidative phosphorylation upon TR₂CoO₃O₂ and NAD⁺ suggests that declining NAD⁺ and TR₂CoO₃O₂ concentrations occur because of loss of these coenzymes from mitochondrial F1F0 complexes during aging [22].

INFECTIONS, POLYAMINE BIOSYNTHESIS, NITRIC OXIDE, AND PEROXYNITRITE

Pathogenic microbes, as observed in brain in AD and in atherosclerotic arterial plaques, synthesize polyamines that are necessary for a broad range of functions, including genetic translation, genetic regulation, resistance to stress, cell proliferation, and differentiation [27]. Polyamines are synthesized in cells infected with viruses [28] and a wide variety of microorganisms [29]. In a recent study, Chlamydia trachomatis, the most common agent of sexually transmitted disease, was found to inhibit cellular nitric oxide (NO) synthesis in cultured human mesenchymal stem cells by stimulating polyamine synthesis [30]. Infection by C. trachomatis produced downregulation of inducible NO synthase (iNOS) and upregulation of ornithine decarboxylase, which is the rate-limiting enzyme in the polyamine biosynthetic pathway. No studies of polyamine biosynthesis and downregulation of NO by Chlamydia pneumoniae, spirochetes, viruses, periodontal pathogens, and other microbes implicated in AD and atherosclerosis have been reported. NO has powerful anti-microbial activity because of formation of peroxynitrite (OONOO^-) from superoxide (O₂^-) [31]. Large quantities of NO are produced during infections caused by pathogens, including bacteria, viruses, parasites, and fungi, and peroxynitrite has bactericidal activity which aids in the cytotoxic action of macrophages and neutrophils by inducing nitrative stress and formation of 3-nitro tyrosine [32]. Both reactive oxygen intermediates and reactive nitrogen intermediates are delivered to phagosomes of neutrophils and macrophages to mediate anti-microbial activity against Chlamydia pneumoniae, Mycoplasma pneumoniae, cytomegalovirus, Staphylococcus aureus, and other pathogenic microbes implicated in the pathogenesis of AD and atherosclerosis [7 , 33].

Increased susceptibility to infectious microbes may occur in aging because of reduced concentrations of thioretinaco ozonide within cellular membranes, resulting in decreased production of nitric oxide, superoxide, and peroxynitrite, potentially explaining the exponential increase in incidence of dementia and atherosclerosis with aging. Recent results demonstrate a role for cystathionine synthase in intracellular NO biosynthesis because of the ability of the heme co-factor of cystathionine synthase to reduce nitrite and generate NO [34]. Studies of the kinetics of nitrite formation and peroxynitrite formation by ferrous heme implicate cystathionine synthase as a previously unrecognized source of NO and peroxynitrite [35].

The synthesis of the polyamine spermidine is accomplished by transfer of the aminopropyl group of adenosyl methionine to the amino group of putrescine (di-amino butane) [29]. This transfer reaction is catalyzed by S-adenosylmethionine decarboxylase and spermidine synthase (putrescine aminopropyltransferase). Thus microbial infection by a wide variety of microorganisms, including viruses, bacteria, protozoans, and fungi, causes a depletion of adenosyl methionine within host cells because of increased synthesis of polyamines. Decreased adenosyl methionine within infected host cells causes dysregulation of methionine metabolism because of decreased allosteric inhibition of methylenetetrahydrofolate reductase and decreased allosteric activation of cystathionine synthase, resulting in excess production of homocysteine, explaining the hyperhomocysteinemia observed in AD and atherosclerosis[1, 13].

INFECTIONS, OXIDATIVE PHOSPHORYLATION, THIORETINACO OZONIDE, AND AGING

Infectious microbes may deplete host cells of the active site of oxidative phosphorylation, TR₂CoO₃O₂NAD⁺H₂PO₄^-, because of utilization of this complex for oxidative metabolism by these chronic intracellular pathogens. Depletion of adenosyl methionine from infected host cells by polyamine synthesis [29] will inhibit biosynthesis of thioretinamide because of decreased allosteric activation of cystathionine synthase [25], decreased heme oxygenase activity, resulting in reduced conversion of retinol to retinoic acid by superoxide, and reduced reaction of retinoic acid with homocysteine thiolactone to form thioretinamide [36]. Support for this proposal is the recent observation of reduced concentrations of the cobalamin co-enzymes, methyl-cobalamin and adenosyl-cobalamin in human brain tissue in aging, autism, and schizophrenia [37]. This study supports the concept of a decreased concentration of thioretinaco ozonide concentration within mitochondrial and cellular membranes as an important factor in the process of aging [26]. In addition, young males with schizophrenia have been observed to have hyperhomocysteinemia [38] and impaired glutathione synthesis, associated with oxidative stress from genetic and functional factors [39].

ENDOTHELIAL DYSFUNCTION, HOMOCYSTEINE, AND ENDOPLASMIC RETICULUM STRESS

Endothelial dysfunction, one of the earliest manifestations of atherogenesis, is promoted by hyperhomocysteinemia [13, 40]. In human endothelial cells homocysteine induces apoptosis through activation of the unfolded protein response, signaled by the endoplasmic reticulum kinase IRE-1 [41]. Induction of endoplasmic reticulum stress by homocysteine causes dysregulation of the pathways for cholesterol and triglyceride biosynthesis, causing fat deposition in liver [42]. The unfolded protein response is clearly established as a factor in the atherogenic effect of hyperhomocysteinemia in production of human and model atherosclerotic plaques, because of a response to endoplasmic reticulum stress, resulting in apoptosis [43]. Herp is an endoplasmic reticulum protein encoded by the HERPUD-1 (homocysteine-inducible, endoplasmic stress inducible, ubiquitin-like domain member 1) gene, which is induced by homocysteine, facilitates endoplasmic stress, is expressed in neurons and glial cells including astrocytes, and is deposited in the Lewy bodies of neurons and in substantia nigra glial cells in Parkinson’s disease [44].

NEUROTOXICITY, MISFOLDED PROTEIN RESPONSE, AND NEURODEGENERATION

Exposure to the neurotoxic amino acid, β-methylamino alanine (BMAA), which is produced by cyanobacteria, is implicated in the etiology of amyotrophic lateral sclerosis (ALS)/Parkinsonism dementia complex in Chamorro patients from Guam [45]. Subsequently BMAA was detected in patients with sporadic AD and ALS from North America, using a validated fluorescent high performance liquid chromatography (HPLC) method with tandem mass spectroscopy for confirmation of BMAA identification [46]. Mis-incorporation of BMAA into human proteins in place of L-serine was found to cause protein misfolding and aggregation in cell cultures [47]. Using a mouse model of the sticky mutation, which is characterized by follicular dystrophy, hair loss, cerebellar Purkinje cell loss, and ataxia, amissense mutation of the alanyl-tRNA synthetase gene was found to result in low levels of mischarged tRNA molecules, producing misfolded proteins and cell death associated with neurodegeneration [48]. Experimental administration of the neurotoxin BMAA to vervets produces an animal model of AD, characterized by neurofibrillary tangles and amyloid deposits in the brain [49].

ADENOSINE MONOPHOSPHATE KINASE, HOMOCYSTEINE, AND HEPATIC STEATOSIS

Adenosine monophosphate-activated kinase (AMPK) is a metabolic master switch, which controls the metabolic pathways of hepatic ketogenesis, cholesterol biosynthesis, lipogenesis, triglyceride synthesis, adipocyte lipolysis, and fatty acid oxidation by phosphorylation of key enzyme proteins [50]. AMPK is activated by an increased intracellular ratio of AMP to ATP, stimulating oxidative phosphorylation and increased ATP synthesis by mitochondria [51]. In a study of cultured adipocytes, homocysteine was demonstrated to suppress lipolysis by activating the AMPK pathway, resulting in elevation of intracellular triglycerides [52]. In a related study, homocysteine was demonstrated to increase resistin production from adipose tissue in mice with hyperhomocysteinemia and from cultured adipocytes [53]. These studies implicate activation of AMPK in the production of fatty liver in subjects with homocystinuria and in subjects with type 2 diabetes [13].

HOMOCYSTEINE, EXCITOTOXICITY, AND NEURODEGENERATION

Homocysteine is an excitatory neurotransmitter that binds to the N-methyl D-aspartate (NMDA) receptor and leads to oxidative stress, cytoplasmic calcium influx, apoptosis, and endothelial dysfunction [40]. Homocysteine sulfinic acid, an oxidized derivative of homocysteine, is a potent excitatory neurotransmitter, which stimulates glucose uptake through the calcium-dependent AMPK-p38 MAPK-protein kinase C pathway in muscle cells [54]. The Aβ₄₂ oligomers that are present in neuritic plaques in AD activate the calmodulin-dependent protein kinase kinase (CAMKK2)-AMPK kinase pathway through phosphorylation of tau protein [55]. Over activation of CAMKK2 or AMPK induces dendritic spine loss in hippocampal neurons of transgenic mice for human amyloid-β protein precursor [55].

ALZHEIMER’S DISEASE, THIORETINACO OZONIDE, AND CANCER

In a study of 1,278 participants in the Framingham Heart Study, survivors of cancer were found to have a 33% decreased risk of developing AD, compared with participants without cancer, and participants with probable AD had a decreased risk of incident cancer, confirming the results of previous studies [56]. These authors considered polymorphisms of p53, the tumor suppressor gene, or Pin-1, a protein which is necessary for cell division and control of protein folding, as possible explanations of this observation. Another possible explanation of this observation is the upregulation of oxidative phosphorylation of impaired neurons which propagates to neighboring cells, promoting cell death in AD [57]. These authors point to the increased glycolysis in cancer cells as a metabolic factor that may explain the observation of an inverse association of AD and cancer. A possible molecular explanation of this inverse association is the depletion of thioretinaco ozonide from malignant cells, leading to aerobic glycolysis, because of proliferation of a clone of cells with loss of the heme oxygenase function of cystathionine synthase and consequent deficient synthesis of thioretinamide, thioretinaco, and thioretinaco ozonide [36]. In AD the depletion of thioretinaco ozonide and consequent impaired oxidative phosphorylation [16] is attributable to decreased biosynthesis of adenosyl methionine because of increased polyamine synthesis by infectious microbes, impairing biosynthesis of thioretinamide by cystathionine synthase and reducing production of thioretinaco ozonide from thioretinamide and cobalamin [29, 36]. In addition, oncogenic viruses may suppress cystathionine synthase function produced by depletion of intracellular adenosyl methionine because of increased polyamine synthesis, allowing a clone of cells with loss of the heme oxygenase function of cystathionine synthase to proliferate. According to this view, carcinogenesis by viruses or carcinogenic chemicals may inhibit oxidative metabolism, reducing the risk of AD because competition for thioretinaco ozonide biosynthesis may suppress the metabolic activity and viability of infectious microbes involved in the pathogenesis of AD.

DETECTION, PREVENTION, AND TREATMENT OF ALZHEIMER’S DISEASE

Early detection of subjects at risk for AD may be accomplished by the finding of mild cognitive impairment through abnormal Mini-Mental State Examination (MMSE) scores, computed tomography or magnetic resonance imaging scans of medial temporal lobe thickness, cerebrospinal fluid Aβ₄₀, Aβ₄₂, or tau protein, plasma homocysteine, C-reactive protein, and ocular biomarkers [4, 58]. Identification of pathogenic microbes by sero-positivity, positive culture, or other methods will guide the choice of antibiotic, vaccination, or other anti-microbial strategy [7, 59]. Treatment of the metabolic alterations induced by pathogenic microbes in AD and atherosclerosis, including hyperhomocysteinemia, increased polyamine synthesis, and impaired oxidative metabolism from depletion of thioretinaco ozonide, may be addressed by a proposed protocol of thioretinamide, vitamin B complex vitamins, including methyl-cobalamin, methyl-folate, pyridoxal phosphate, and nicotinamide riboside, ascorbate, co-enzyme Q10, adenosyl methionine, menoquinone, amygdalin, vitamin D3, pancreatic enzymes, cod liver oil, and dietary improvement to eliminate processed foods and to prevent subclinical protein energy malnutrition [13, 60]. In addition, meticulous oral hygiene, consumption of dietary monolaurin and other nutrients with anti-microbial activity, consumption of adequate dietary protein, and avoidance of neurotoxins from foods or environmental contaminants may also retard the progression of mild cognitive impairment to dementia [61, 62]. The efficacy of this proposed protocol requires validation by a properly designed clinical trial.

CONCLUSION

The metabolic abnormalities caused by infectious microbes in dementia and atherosclerosis affect homocysteine metabolism, oxidative phosphorylation, and biosynthesis of polyamines, leading to neurodegeneration and atherosclerotic arterial plaques. These abnormalities consist of decreased concentrations of thioretinaco ozonide, adenosyl methionine, and reduced allosteric activation of cystathionine synthase in host cells. These metabolic changes impair the host response to infectious microbes because of impaired production of nitric oxide and peroxynitrite in macrophages. A proposed strategy for prevention and treatment of dementia and atherosclerosis consists of early detection of cognitive decline, dietary improvement to eliminate highly processed foods, adequate dietary protein to prevent subclinical protein energy malnutrition, dietary consumption of anti-microbial nutrients, meticulous oral hygiene, and a homocysteine-lowering protocol consisting of thioretinamide, B vitamins, coenzyme Q10, ascorbate, adenosyl methionine, menoquinone, amygdalin, vitamin D3, cod liver oil, and pancreatic enzymes.

DISCLOSURE STATEMENT

The author’s disclosure is available online (http://j-alz.com/manuscript-disclosures/16-0549r1).

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