Early Diagnosis of Alzheimer's Disease

June 2020 29, June 2020 2, July 2020 

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1. Background

Alzheimer’s Disease (Alzheimer’s Disease) is the most generic form of degenerative dementia, characterized by widespread neuron destruction, a sharp decrease in Acetylcholine in patients’ brain, and the accumulation of a protein called beta-amyloid (βA) in the extracellular environment and of anomalously phosphorylated Tau protein, within neurons. Especially important in Alzheimer’s Disease are the Basal Forebrain Cholinergic neurons, we consider whose destruction the major cause of the patients’ memory loss. We know that the genetic mutations responsible for the genetic forms of Alzheimer’s Disease often interfere with the correct function of the main cleaning mean of our Central Nervous System, and the Glymphatic System. We think that this happens in the sporadic forms of Alzheimer’s Disease, also, for other reasons, which often involve the breaking of the Blood-Brain-Barrier integrity aging.

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The CNS Clearance and the Glymphatic System

The waste products, deriving from the cellular activity of neurons, are partly eliminated within the same cells by intracellular clearance mechanisms; the substances that are not eliminated inside the neurons, are expelled in the extracellular matrix and eliminated through the Glymphatic System, the principal instrument for removing extracellular waste substances in the Central Nervous System.

3. Intracellular Clearance Mechanisms

The main Alzheimer’s disease genetic alterations slow the clearance mechanisms that, within neurons, are performed by the Ubiquitin-Proteasome System or by autophagy, a process by which it delivers superfluous or potentially dangerous cytoplasmic material to lysosomes for degradation. We know three types of autophagy:

Micro autophagy, in which the cytosolic material is directly engulfed by lysosome invaginations. Chaperone-Mediated autophagy (CMA), in which chaperone proteins lead the waste to the lysosome. Macro autophagy (autophagic-lysosomal network or ALN), which involves the seizure of cytosolic material in auto-phagosomes that provide their content to lysosomes for digestion.

The laboratory findings show that in Alzheimer’s Disease, UPS, CMA, and ALN, are compromised, often because of gene alterations in APOE4, PS1, PS2, APP, PICALM, TREM2, among the main recognized genes responsible for Alzheimer cases. Naturally, the substances not removed by the endocellular mechanisms are expelled by neurons, in the extracellular environment. 

Extracellular Clearance Mechanism: Glymphatic System

The main tool for removing extracellular waste substances is Glymphatic System. The clearance of soluble proteins, waste products, and extracellular fluid excess is achieved through the convective flow of the interstitial fluid, facilitated by the presence of channels called aquaporins), located in the astrocyte membrane, which plays a crucial role in water flow regulation in and out of cells. Aquaporins facilitate the cell’s water permeability up to thirty times. The main aquaporins types, expressed in the Central Nervous System, are aquaporin 1, which is expressed by the epithelial cells of the choroid plexus, and aquaporin, astrocytes express which.

The AQP4 in astrocytes is present in their terminal processes (end-feet) that cover the encephalic vessels. Up to 50% of the surface of these feet is occupied by AQP4, and the glymphatic system is critically based on astrocytic AQP4.

Studies have shown deficiencies in this pathway to contribute to Alzheimer’s Disease, and a perivascular AQP4 reduced we associate number with Alzheimer’s Disease diagnosis and pathology.

With glymphatic system deficiency, the clearance of the β-amyloid protein is altered, and thus that of Adenosine, present in the Basal Forebrain (BF). The key factor in the Glymphatic System functioning, therefore in the pathogenesis of Alzheimer’s Disease, is sleep. Glymphatic System works up to 60% better during sleep and especially during the N3 phase of NREM (deeper sleep). Several studies have shown that limited sleep increases the level of β-amyloid and of Tau protein neurofibrillary tangles.

Sleep Deprivation (SD) simulates what can happen because of some factors, such as trauma, stress, and aging, which can alter the control of the endoplasmic reticulum on protein quality and lead to an “Unfolding Protein Response” (UPR), which causes the production of “misfolding” proteins, i.e. poorly aggregated and hyperphosphorylated. In SD, adenosine levels are raised (+140%) in the cholinergic BF; adenosine, a small purine molecule that makes up the central element of adenosine triphosphate (ATP), the major energy source of all our cells, including neurons.

This molecule is produced everywhere, in the Central Nervous System, but accumulates only in the Basal Forebrain where it inhibits cholinergic neurons by stimulating its A1 receptors; this induces drowsiness and reduces the waking state.

The Glymphatic system also plays a key role in the transport of extra-synaptic glutamate excess, which, if not eliminated, can cause excitotoxicity, the most notable cause of neuron loss in Alzheimer’s Disease. Some drugs (e.g. memantine) are effective (but not decisive) in Alzheimer’s Disease care.

The Glymphatic System may encounter difficulties in functioning following Traumatic Brain Injury, in Depression, following general anesthesia, in Diabetes, following Stroke and with aging. In all these cases there would be substance accumulation, both in the extracellular and in the intracellular environment.

A clearance system that does not function optimally will penalize the cells with high activity. Therefore, the cells of ARAS nuclei, which perform many functions, even the most disparate, must present a remarkable metabolism. In fact, besides the Tau and Aβ proteins in Alzheimer’s Disease, some of the most important neurotransmitters (NT) in our body, such as Serotonin, Norepinephrine, Histamine and Dopamine and above all Acetylcholine are involved (reduced in quantity) .

Orexin (OX) is an exception: the number of orexinergic neurons decreases with age, but the Orexin concentration in CSF of Alzheimer’s Disease patients is increased.

It constitutes the most vital component of a much broader system of cholinergic cells distributed throughout the Central Nervous System, from the rostral portions of the Striato until, caudally, to the spinal motor neurons. This structure regulates phenomena such as attention, learning, and memory and is implicated in the cognitive alterations present in different neurological pathologies such as Alzheimer’s Disease. BFC neurons also project towards the preoptic nuclei (VLPO and MnPO) and Tubero-mammillary (TMN) of the Hypothalamus and, through these projections, take part in wake/sleep modulation. Cholinergic neurons present many Adenosine receptors.

The cellular bodies of neurons expressing the orexin/hypocretin neuropeptides, present only in the lateral hypothalamus and in the contiguous perifornical area, provide diffuse projections towards the basal forebrain which increase the cortical Acetylcholine release. Orexin has a strong and direct excitatory effect on BFC neurons, contributes to cortical activation associated with wakefulness, more than all the other NTs that promote wakefulness and works in concert with cholinergic ones.

If the Glymphatic System does not work well, as with aging, diabetes, lack of sleep, etc., at the Basal Forebrain level, there is an increase in adenosine, which inhibits cholinergic cells. We expect acetylcholine production to decrease but the hypothalamus produces more Orexin and stimulates the remaining cholinergic cells to produce enough Acetylcholine to make Central Nervous System work properly. When the cholinergic cell number decreases too much, Alzheimer’s disease symptoms begin. Orexin determines the vigil and there is a sleep mechanism alteration with daytime sleepiness, because of the excess of Adenosine and nocturnal vigil, caused by the excess of Orexin-a. The Glymphatic System becomes less and less effective causing a further Acetylcholine deficiency in a vicious circle that leads to Alzheimer’s Disease. Adenosine, β-Amyloid, and TAU protein are not disposed of by the glymphatic system and accumulate this causes further loss of BFC, the Ach decreases further and the OX increases further: Alzheimer’s Disease. Thus, in Alzheimer’s Disease, while many neurons die and all other ARAS Neurotransmitters decrease in quantity, the OX increases, and we can use this detail for an early diagnosis. The OX increase is an especially important fact because it causes an acceleration of neurodegeneration (due to sleep loss) and is Alzheimer’s disease specific. This is the reason for which Alzheimer’s Disease is the main neurodegenerative disease, and we can use this increase to make an early diagnosis of the disease. The orexinergic “compensation” of Ach deficiency can mask the disease for years but could allow us to intervene for an early diagnosis.

Early Diagnosis Model:

Of course, we could take a cerebrospinal fluid sample and dose Orexin, but this method is risky and painful for the patient’s health, therefore unsuitable for large numbers of patients.

Hanazawa T and Kamijo Y have administered Suvorexant to four Alzheimer’s Disease patients, all four patients with nocturnal delirium successfully fell asleep rapidly, suggesting that resolving delirium may be related to the effects of Suvorexant on sleep dysregulation. In all four cases, Suvorexant drastically resolved delirium symptoms and improved their sleep. The nocturnal delirium recurred at once following the discontinuation of Suvorexant. It thus suggested the effect of Suvorexant on nocturnal delirium to be reproducible. The medical history of these patients showed a progressive decline in cognitive function, neuroimaging results including computed tomography of the brain, cognitive tests, and laboratory data all satisfied the DSM-5 criteria for Alzheimer’s Disease with an elevated level of evidence. The administration of Suvorexant to manage nocturnal delirium, in several elderly patients with dementia other than Alzheimer’s Disease, had no effects at all. Then the administration of Suvorexant allows us a differential diagnosis between Alzheimer’s Disease and other similar neurodegenerative diseases. We know that the orexin, besides being important for the maintenance of wakefulness, is fundamental for the stabilization of the wakefulness-sleep switch ; and we also know that nocturnal delirium depends on Acetylcholine deficiency and that by administering an anticholinergic we can cause hallucinations and delirium (Atum M, 2020), while, with the administering an acetylcholinesterase inhibitor, which increases the amount of Acetylcholine, we can stop these hallucinations and delirium. So, if we administer a Dual Orexin Receptor Antagonist (DORA) and the delirium ceases it means that this patient has Ach deficiency and, of course, excess of Orexina. DORA eliminates the excess of Orexin-a and the patient sleeps: hallucinations and delirium are due to the complex: too much Orexin-a, that does not make the patient sleep and little Acetylcholine, which, during insomnia, causes delirium. During sleep (NREM sleep) it is normal, however, that there is little Acetylcholine. If we administer a DORA to an awake patient, the effects of Orexin will be zero and, if that patient is an asymptomatic Mild Cognitive Impairment, the Ach will show its real levels, low. We can administer a DORA to the suspect patient and perform an instrumental check: e.g., a Functional Magnetic Resonance Imaging (fMR). If the BFC does not have enough Acetylcholine, its O ₂ consumption will be significantly reduced and we will see it in the neuroimaging that will present signs of impaired hippocampus function and of other CC areas, particularly related to cholinergic innervations. In case of doubt, we can perform the same analysis, after a few times, without DORA and compare the two results. In the Alzheimer’s Disease patient, the DORA administration will increase both the amount of total sleep and the NREM. REM sleep, instead, will decrease, both in quantity (time) both, above all, in quality, due to the inability of BFC cells to support it, proportionally to the gravity of the situation, with the disappearance of posterior dominant alpha rhythm and the diffuse slowing in EEG, specifically a reduction of power in the alpha (8 - 15 Hz) and beta (16 - 31 Hz) bands and an increase in the theta (4 - 8 Hz) and delta (0.5 - 4 Hz) bands. This because the BFC system, which is impaired in Alzheimer’s disease, is more crucial for the activation of REM sleep EEG than it is for wakefulness 110 - 120. The phenomena related to sleep, in Alzheimer’s Disease, are early and present for the entire duration of the disease. We could make a first Polysomnographic (PSG) check on the “suspect patient”, evaluate the various parameters, and above all the quantity and quality of the REM. Perform a second PSG after DORA administration to the patient and rechecking the values obtained, especially the REM, again. If we administer DORA during the waking state and subject the patient to Alzheimer’s Disease tests (Mini-Mental State Examination, Clock Drawing Test, etc.) its performance will be poor, like those of a patient frankly Alzheimer’s Disease or MCI, Finally, to be sure of the diagnosis we can make more invasive examinations (e.g., Cerebrospinal Fluid control).

After making a diagnosis, as early as possible, we must first try to investigate the plausible causes: ageing, genetics, diabetes, depression, stroke, etc. and try to intervene on these. We must try to restore the optimal functioning of the Glymphatic System by acting on the lifestyle, especially regarding the quantity and quality of sleep. Using drugs such as Suvorexant itself which, by eliminating the effect of OX excess will improve sleep and, therefore the function of the Glymphatic system, and some antihistamines such as Pitolisant, an H3 receptor agonist/inverse antagonist of histamine, which has been shown to be effective in Alzheimer’s Disease, improving sleep. We can improve the action of Ach with cholinesterase inhibitors (if Ach increases, less Ox will be produced and sleep will improve. All this will improve the clearance made by the Glymphatic System which will reduce the amount of Aβ, Tau etc. taking care, in fact, of the causes of Alzheimer’s Disease. The patient can keep himself constantly under control by monitoring his sleep.

The pathogenetic Alzheimer’s Disease model we presented is quite simple and shared by many other authors: the cleaning system (Glymphatic System) in our Central Nervous System does not work properly and the waste accumulates. There is a substantial loss of neurons, especially of cholinergic ones, while the Orexin production increases. Despite many similarities, the increase in Orexin is not present in other neurodegenerative diseases. We can show the decrease of Acetylcholine by eliminating the excess of Orexina with specific drugs and make an early diagnosis, even many years before the symptoms of this disease, Alzheimer’s, appear.

We strongly believe in the pathophysiological model we propose because it explains many characteristics of this disease, but if it were wrong, the system for early diagnosis, that we have devised would work, anyway. The administration of Suvorexant, in asymptomatic patients, allows us an early diagnosis, a differential diagnosis, and a more targeted therapy, both with Suvorexant itself and with cholinesterase inhibitor drugs. This model of early diagnosis is not invasive; it is quite simple, quick and to our knowledge; there are no better ones.

 

Table of Abbreviations
A1	Adenosine 1	NREM	Movement for Non-Rapid Eyes
Ach	Acetylcholine	NT	Neurotransmitter
AD	Alzheimer’s Disease	OX	Orexin
AGP	Aquaporin	PB	Parabrachial Area
ALN	Autophagic-Lysosomal Network	PICALM	Clathrin Assembly Protein Binding the Inositol with Phosphatidyl
APOE	Apolipoprotein E	PPT	Peduncolo-Pontine del Tegmentum
APP	Amyloid Precursor Protein	PS	Presenilin
ARAS	Ascending Reticular Activation System	P2X7	Purinoceptor 7
ATP	Adenosine Triphosphate	REM	Rapid Eyes Movement
βA	Beta Amyloid	SCN	Suprachiasmatic Nucleus
BF	Basal Forebrain	SD	Sleep Deprivation
BFC	Basal Forebrain Cholinergic neurons	SLD	Sub Laterodorsal Core
Ca	Calcium	TMN	Tuber-Mammillary Nuclei
CC	Cerebral Cortex	TREM	Trigger of Receptors Expressed on Myeloid cells
CMA	Chaperone-Mediated Autophagy	UPR	Explainable Protein Response
CNS	Central Nervous System	UPS	Ubiquitin-Proteasome System
CSF	CerebroSpinal Fluid	VLPO	Pre-Optic Ventro-Lateral Nucleus
DA	Dopamine	5-ht	Serotonin
DORA	Dual Orexin Receptor Antagonist	KirNB	Inward Rectifier K + Channel of the Basal Nucleus
DR	Dorsal Raphe	LC	Locus Coeruleus
EEG	Electroencephalogram	LDT	Latero-Dorsal Nuclei of the Tegmentum
GABA	Aminobutyric Acid Gamma	LGN	Lateral Geniculate Nucleus
GIRK	Potassium Channels Coupled with Inward Radical Proteins	MnPO	Median Preoptic
GS	Glymphatic System	NA	Norepinephrine
H	Histamine
K	Potassium