key: cord-0015305-z320ddrv authors: Fessel, Jeffrey title: A vaccine to prevent initial loss of cognition and eventual Alzheimer's disease in elderly persons date: 2021-02-05 journal: Alzheimers Dement (N Y) DOI: 10.1002/trc2.12126 sha: 0a1c28b7719cca7dc78b95631fb4e01f16e1bfda doc_id: 15305 cord_uid: z320ddrv Prevention is better than cure and prevention of Alzheimer's disease (AD) may be possible. In elderly persons who are cognitively normal, synaptic hypometabolism as shown by reduced cerebral uptake of fluorodeoxyglucose ((18)F‐FDG), provides a premonitory signal of potential, future loss of cognition if those individuals also have present evidence of amyloid deposition seen in the Pittsburgh compound B positron emission tomography (PIB‐PET) scan for amyloid. Those are the persons who should be targeted if one aims to prevent AD. The synaptic hypometabolism implies that the brain's availability of adenosine triphosphate (ATP) is inadequate for performance of all required synaptic functions. This review first describes the basis for asserting that reduced cerebral uptake of (18)F‐FDG accurately reflects synaptic hypometabolism; second, explains the basis for asserting that hypometabolism implies inadequate ATP; third, shows that amyloid beta (Aβ) itself, Aβ modified by pyroglutamate to become a molecule termed pE(3)Aβ, and cyclophilin‐D, in concert are the main contributors to inadequate synaptic ATP and that, therefore, reducing all of their levels would neutralize their combined effect and correct the hypometabolism. pE(3)Aβ is more neurotoxic than unmodified Aβ; and cyclophilin D inhibits ATP synthase and reduces ATP formation. Finally, this review describes an mRNA self‐replicating vaccine that will raise brain levels of ATP by reducing Aβ, pyroglutamate‐modified Aβ, and cyclophilin‐D, and thereby—in cognitively normal elderly persons who have synaptic hypometabolism—prevent initiation of the process that terminates in AD. early use of anti-inflammatory drugs; and it is widely recognized that treatment for human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) should begin immediately when the diagnosis is made. The concept by Hardy and Higgins, 1 and Selkoe 2 that the primary cause of Alzheimer's disease (AD) is cerebral amyloid or its oligomers, was based upon rational analysis of the facts known at the time. The most likely reason why, subsequently, so many trials based upon the amyloid hypothesis have failed either to reverse or halt AD, is because once the causal process is in motion it may be unstoppable due to the known multiplicity of abnormalities of the brain in AD, many of which are also present at the earlier stage of mild cognitive impairment (MCI). On the other hand, it may be possible to prevent AD before it has even commenced, which is the time when there is not yet cognitive loss but when amyloid is detectable by Pittsburgh compound B positron emission tomography (PIB-PET) scan and when synaptic metabolism as shown by fluorodeoxyglucose PET ( 18 F-FDG-PET) scan is reduced. 3 That condition has been termed pre-clinical AD. 4 Because synapses account for a large portion of the brain's energy use, an immediate question is whether preventing this reduction of neuronal adenosine triphosphate (ATP) might prevent the start of cognitive loss in predisposed persons. This article will first describe the basis for asserting the presence of synaptic hypometabolism before cognitive loss has appeared. Second, it will describe the basis for asserting that hypometabolism, as shown by low uptake of 18 F-FDG, implies inadequate synaptic ATP. Third, it will show that amyloid beta (Aβ) itself, pyroglutamate-modified Aβ, and cyclophilin-D are, in concert, the main contributors to inadequate synaptic ATP and, therefore, that reducing their input would neutralize their combined effect and correct that hypometabolism. Finally, it will describe a self-replicating, mRNA vaccine that will reduce the levels of Aβ, pyroglutamate-modified Aβ, and cyclophilin-D, thereby preventing the process that terminates in AD. Among nine proteins in cerebrospinal fluid (CSF) that participate in core synaptic processes, Lleó et al. found that six were reduced in preclinical AD. 5 Lim et al. caused stress to cholinergic neurotransmission by administering scopolamine to 63 persons aged 55 to 75, who had subjective memory complaints but normal neuropsychological test results. 6 Aβ PET imaging showed that 15 subjects were Aβ +ve and therefore had preclinical AD and 48 were Aβ -ve. At Dienel pointed out that evidence for glucose being the obligatory fuel for brain metabolism comes from insulin-induced hypoglycemia, in which the cognitive status progressively changes to lethargy, stupor, and coma as plasma glucose level falls. 9 Administration of glucose rapidly reverses these behavioral consequences and, of other tested compounds, only mannose and maltose are effective, whereas many others, for example, glycerol, ethanol, lactate, pyruvate, glyceraldehyde, fumarate, acetate, β-hydroxybutyrate, and galactose, are ineffective. Although adult brain tissue can metabolize most of those substrates, blood-brain barrier (BBB) transport capacity and their normal blood concentrations are insufficient to deliver enough of these alternative oxidative fuels to the brain to support cognitive activities. Calculations show that metabolism of ≈1 molecule of glucose produces ≈28 molecules of ATP by the oxidative pathways per synapse and ≈2 molecules of ATP by glycolysis. 9 Synaptic activity accounts for the majority of energy expenditure by the brain, and ATP formation in the brain is primarily from oxidative phosphorylation of glucose, 10 so when cerebral glucose metabolism is reduced there is also reduced ATP available. The cerebral metabolic rate for glucose (CMR glc ) is assessed by means of 18 F/fluoro-2-deoxy-D-glucose ( 18 F-FDG) PET scanning. 11 18 F-FDG enters the cell by the same membrane transport mechanism as glucose. After penetration of the cellular membrane via glucose transporters, both 18 F-FDG and glucose are phosphorylated by hexokinase but unlike glucose-6-phosphate, 18 The term "synaptic hypometabolism" refers to a low CMR glc as shown by reduced uptake of the 18 shown a reduced baseline CMR glc in frontal and temporal lobes with normal aging, and that hypometabolism of glucose in the entorhinal cortex predicted an MCI diagnosis 3 years later with 83% sensitivity and 85% specificity. 13, 14 The above studies show cognitive loss as occurring soonest in those individuals with cerebral hypometabolism; note, however, that deliberately raising blood glucose levels to prevent cognitive loss would be inappropriate because many studies have demonstrated that prediabetic levels of blood glucose are themselves associated with either current or future cognitive impairment (see Roriz-Filho et al. 15 for citations). The importance of Aβ unmodified by pyroglutamate, particularly its soluble oligomers, is widely recognized as an important player in the pathogenesis of AD. As is discussed elsewhere and not expanded upon in this essay, unmodified Aβ is essential but insufficient for that pathogenesis. 16 Modification of Aβ by pyroglutamate (pE), produces much-reduced levels of ATP as well as greatly aggravated neurotoxicity. Each of these actions will be explained below but first is an explanation of how pyroglutamate modifies Aβ. The modification occurs because, mediated by the enzyme glutaminyl cyclase, pyroglutamic acid truncates and caps the Aβ oligomeric peptide at position #3 of its N-terminus sequence. That position is occupied by glutamate (E), and pyroglutamate (p) attaches to E, forming a molecule that is written as pE (3)Aβ (sometimes written as pGlu3-Aβ, or AβpGlu3, or Aβ N3(pE) ; whatever its description in the various cited reports, it will, for uniformity, be written here as pE (3) Their In brief, the reduction of pE(3)Aβ levels would be beneficial for elderly individuals who are at risk of future loss of cognition. Increased reactive oxygen species (ROS) in AD, which would produce lipid peroxidation, has been known for many years. 34 37 Thus, reducing cyclophilin D negated its deleterious consequence of excessively raising Ca 2+ . Cyclophilin D also binds ANT, which is another way whereby cyclophilin D reduces ATP. 38 Giorgio et al. showed that binding to the F 0 F 1 -ATP synthase and impairing its enzymatic activity is yet another mechanism by which cyclophilin D decreases the synthesis of ATP. 39 ATP synthase is organized into supramolecular units called synthasomes that increase the efficiency of ATP production, and Beutner et al. showed that deletion of cyclophilin D was advantageous, by increasing synthasome assembly in various tissues including the brain. 40 In an AD mouse model, Du et al. showed that abrogation of cyclophilin D resulted in persistent, life-long protection against Aβ toxicity thereby suggesting that blockade of cyclophilin D may be of benefit for AD treatment. 41 The same group of investigators showed that in mice with streptozotocin-induced diabetes, blockade of the F 1 F 0 ATP synthasecyclophilin D interaction by cyclophilin D ablation, protected against decrease of ATP synthesis; and, further, that the absence of cyclophilin D alleviated deficits in synaptic plasticity, learning, and memory in the diabetic mice. 42 In brief, reduction of cyclophilin D would lead to increase in ATP formation and benefit persons with synaptic hypometabolism. The ultimate cause of synaptic hypometabolism is reduced availability of ATP, largely due to its reduced production by mitochondria, and its regulation involves both Ca 2+ and Mg 2+ . For more extensive details than provided in this synopsis, in which oversimplification of the many complexities is intended to assist understandability, the interested reader is referred to recent reviews. [43] [44] [45] This section will briefly discuss the synthesis of ATP and its role as a store of energy; the concept behind the motive force of protons; the uptake and removal from mitochondria of Ca 2+ ; the mitochondrial permeability transition (MPT); the MPTP; and the crucial role of cyclophilin D. Within the intermembrane space, addition of a third phosphate group to ADP adds the energy that is stored as ATP. This addition requires complex V (ATP synthase) and complexes I, II, and IV (cytochrome c oxidase) of the electron transport chain (ETC), that transport protons in the form of H + across the inner mitochondrial membrane (which is almost impermeable but has a variety of ion channels and transporters). In the matrix, the oxidation of NADH produces the H + (the proton), and the ETC transports the H + into the intermembrane space, which now becomes electrically positive; and the matrix, having lost its H + is now electrically negative. That translocation of protons by complexes 1, 111, and 1V, from the mitochondrial matrix into the intermembrane space, creates the force to drive ATP synthesis by ATP synthase. The energy of this concentration gradient is the so-called proton motive force (ΔΨ). 42, 46 ΔΨ is regulated by the Ca 2+ concentration in the mitochondrial matrix, where the net level of Ca 2+ is the difference between Ca 2+ uptake and its extrusion: uptake is via the mitochondrial Ca 2+ uniporter (MCU) transporting Ca 2+ into the mitochondrial matrix: the main extrusion pathway is the Na + /Ca 2+ exchanger pumping Ca 2+ from the matrix, back into the intermembrane space. Wescott et al. described experiments on isolated mitochondria that elucidated the main mechanisms involved in ATP formation, for which the three critical components are ADP (the substrate), Ca 2+ (the regulator), and ΔΨ (the energy source). 45 By using isolated mitochondria and controlling the levels of Ca 2+ , Wescott et al. showed that Ca 2+ regulates mitochondrial use of carbohydrates and amino acids in the tricarboxylic acid cycle, and through this regulation, increased Ca 2+ powered by ΔΨ, augments ATP production. If, however, the Ca 2+ concentration in the mitochondrial matrix becomes excessive, Ca 2+ may then cause the MPTP to swell and open; when that happens, the inner mitochondrial membrane can no longer maintain a barrier to protons, leading to dissipation of the proton motive force, that is, ΔΨ m , and now there is uncoupling of oxidative phosphorylation and reduced formation of ATP. 47, 48 Further, extrusion of excessive Ca 2+ requires ATP and causes its further depletion. 49 Those high levels of Ca 2+ may be prevented by dantrolene, discussed in detail in Only the briefest synopsis of the possible methodology is provided here: for further details the reader is referred to Pardi et al. 51 and Zhang et al. 52 mRNA has the capacity for self-replication, which means that susceptible persons may be vaccinated just once and the effect should be long-lasting because a self-replicating vaccine induces longterm antibody production, which is an important asset in the present context. An additional reason to use an mRNA-based vaccine is that a single vaccine can incorporate several antigens and thereby minimize the number of oral drugs that might be required to achieve the same therapeutic objective. The basic principle of mRNA vaccines is to use the host cell's transcription system to produce target antigens that stimulate adaptive immunity. Traditional mRNA-based vaccines contain only the target antigen gene and cannot replicate themselves. Self-amplifying mRNA vaccines encode the engineered genome of an RNA virus; one commonly used for this purpose is a modified vaccinia virus Ankara, which is an attenuated poxviral vector that is widely used to develop vaccines because of (1) its absence of harmful effects in humans, (2) its excellent safety record, (3) its capacity to express multiple antigens, and (4) its ability to express antigen-specific humoral and cellular immunity. 53 The vaccine contains the virus's non-structural protein gene, and its structural protein gene is replaced by the mRNA(s) of interest. The resulting proteins are antigens expressed at a high level due to the amplification effect of the RNA template, and these replicons cannot produce infectious virions because the gene for the structural protein of the virus has been deleted. Cells replicate, including neurons and glia, and the daughter cells maintain the parental molecular biology. A self-amplifying mRNA vaccine would produce a longlasting effect and should protect the individual for many years from synaptic dysfunction, whereas a traditional vaccine would produce an effect of far shorter duration. Another major advantage of mRNA vaccines is that by targeting multiple antigens they minimize the number of oral drugs needed. The initial trials should be in AD-model rodents. The vaccine should be produced with the collaboration of a vaccinologist. Only after an animal trial has shown that the vaccine produces a significant rise in available ATP would a trial in humans be contemplated. The primary goal of a human trial would be to demonstrate that the vaccine raises ATP levels and prevents cognitive loss in elderly persons who have not yet lost cognition. Accrual of subjects would require that they are age 70 to 75 years, and have health adequate to allow follow-up of >5 years; uncontrolled diabetes and hypertension would be among many exclusions, so accrual to the trial would be slow. There are several concerns about administering a vaccine to healthy persons in the present context. 3. Cytokine release is of major concern because the report of a "cytokine storm" that developed in six healthy human volunteers administered an anti-CD28 super-agonistic monoclonal antibody, during a phase I clinical trial. 55 There are premonitory signs of a cytokine storm and preventative action is possible. 4. Next, there is a risk that a vaccine might provoke auto-immune diseases. "Non-self" molecules such as pE (3)Aβ can induce the production of antibodies that subsequently react with epitopes of "self" origin and cause autoimmune disease. If the proposed vaccine were subjected to validation by clinical trial, a family history or personal history of autoimmune disease should be an exclusionary criterion. Another risk is antibody-dependent cellular cytotoxicity (ADCC), an immune mechanism whereby an effector cell of the immune system lyses a target cell, whose membrane-surface antigens have been bound by specific antibodies that were induced by the vaccine. This issue as well as antibody-dependent enhancement (ADE), is reviewed in detail by Arvin et al. 56 Almost always it occurs when an intact virus is part of the vaccine, because antibodies present at the time of an infection may increase the severity of an illness: this is not the case in the present context and with the proposed vaccine. Arvin et al. also warned that the effector function of antibodies is species specific, so that results in animals may not apply to humans; and further, that protective and non-protective antibodies may be elicited by different forms of the same protein, which reminds us of the various lengths of Aβ that may be present in AD. 6. Infectious complications such as the occurrence of progressive multifocal leukoencephalopathy, which is caused by the JC virus, was seen in three multiple sclerosis patients treated with natalizumab, are risks that are related to ADE and ADCC and to the presence of immune suppression. 57 Because prior exposure to the JC virus is very frequent in the general population, this potential possibility cannot be discounted but it was rare, even in patients with immune suppression due to infection by HIV: a history of infection by HIV should be an exclusionary criterion for potential recipients of a vaccine to prevent ATP reduction. 7. It is also worth pointing to the age-related attenuation of the immune system, because the subjects exposed to the vaccine will usually be over age 70 years. In brief: although the proposed vaccine might theoretically minimize ATP depletion and benefit synaptic function, risks must be acknowledged and, if possible, minimized. The various risks of vaccination should be considered if animal experiments suggest contemplating human trials. None of these risks to human subjects will actualize unless trials occur in human subjects. Synaptic hypometabolism in cognitively normal elderly persons provides a premonitory signal of potential, future loss of cognition. 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A vaccine to prevent initial loss of cognition and eventual Alzheimer's disease in elderly persons No funding from public or private sources was received. There are no conflicts of interest.