**From Cerebrospinal Fluid Neurochemistry to Clinical Diagnosis of Alzheimer's Disease in the Era of Anti-Amyloid Treatments. Report of Four Patients**

**Ioanna Tsantzali 1,†, Fotini Boufidou 2,†, Eleni Sideri 1, Antonis Mavromatos 1, Myrto G. Papaioannou 2, Aikaterini Foska 1, Ioannis Tollos 1, Sotirios G. Paraskevas 2, Anastasios Bonakis 1, Konstantinos I. Voumvourakis 1, Georgios Tsivgoulis 1, Elisabeth Kapaki <sup>2</sup> and George P. Paraskevas 1,2,\***

	- <sup>2</sup> Neurochemistry and Biological Markers Unit, 1st Department of Neurology, School of Medicine, National and Kapodistrian University of Athens, "Eginition" Hospital, 11528 Athens, Greece; fboufidou@med.uoa.gr (F.B.); myrtop@yahoo.com (M.G.P.); sotirispar5@gmail.com (S.G.P.); ekapaki@med.uoa.gr (E.K.)

**Abstract:** Analysis of classical cerebrospinal fluid biomarkers, especially when incorporated in a classification/diagnostic system such as the AT(N), may offer a significant diagnostic tool allowing correct identification of Alzheimer's disease during life. We describe four patients with more or less atypical or mixed clinical presentation, in which the classical cerebrospinal fluid biomarkers amyloid peptide with 42 and 40 amino acids (Aβ<sup>42</sup> and Aβ40, respectively), phospho-tau (τP-181) and total tau (τT) were measured. Despite the unusual clinical presentation, the biomarker profile was compatible with Alzheimer's disease in all four patients. The measurement of classical biomarkers in the cerebrospinal fluid may be a useful tool in identifying the biochemical fingerprints of Alzheimer's disease, especially currently, due to the recent approval of the first disease-modifying treatment, allowing not only typical but also atypical cases to be enrolled in trials of such treatments.

**Keywords:** Alzheimer's disease; beta amyloid; tau protein; phospho-tau; cerebrospinal fluid; biomarkers; anti-amyloid antibodies; aducanumab

#### **1. Introduction**

Alzheimer's disease (AD), the most common cause of dementia, is a neurodegenerative disorder characterized by neuronal and synaptic loss and eventually brain atrophy, due to extracellular polymerization and the accumulation of amyloid peptide with 40 and especially 42 amino acids (Aβ<sup>40</sup> and Aβ42, respectively) in the form of amyloid plaques and intracellular polymerization of hyper-phosphorylated tau protein in the form of paired helical filaments, viewed microscopically as neurofibrillary tangles [1]. This pathophysiological/pathobiochemical process of AD starts many years before, and likely, one to three decades prior to symptom onset [2,3]. Following this long asymptomatic or "preclinical" phase of the disease [4], the symptomatic phase starts [5] initially with mild cognitive impairment (MCI) [6] and finally dementia [7]. At the symptomatic phase, the typical presentation of AD is usually of the "hippocampal amnestic-type", characterized by a deficit in episodic memory with difficulty in both free and cued recall [8]. However, in approximately 10–15% of AD patients, atypical (non-amnestic) presentations have been described [5] and this percentage may rise to 22–64% in early-onset (pre-senile) cases [9]. Such atypical presentations include primary progressive aphasia (PPA) [10], frontal dementia

**Citation:** Tsantzali, I.; Boufidou, F.; Sideri, E.; Mavromatos, A.; Papaioannou, M.G.; Foska, A.; Tollos, I.; Paraskevas, S.G.; Bonakis, A.; Voumvourakis, K.I.; et al. From Cerebrospinal Fluid Neurochemistry to Clinical Diagnosis of Alzheimer's Disease in the Era of Anti-Amyloid Treatments. Report of Four Patients. *Biomedicines* **2021**, *9*, 1376. https:// doi.org/10.3390/biomedicines9101376

Academic Editor: Lorenzo Falsetti

Received: 31 August 2021 Accepted: 30 September 2021 Published: 2 October 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

which may mimic frontotemporal degeneration [11], corticobasal syndrome (CBS) [12], and posterior cortical atrophy [13]. Furthermore, cases of AD mixed with cerebrovascular disease [14], Lewy body pathology [15], and even normal pressure hydrocephalus (NPH) [16] are not uncommon, especially in the elderly. Thus, AD is no longer viewed as synonymous with amnestic dementia [17]. It may be viewed as a biological process, irrespective of the presence (or absence) and the type and severity of symptoms at a certain time point during disease evolution and progression [18]. Then, how can we diagnose AD?

As in any aspect of medicine, the initial approach is always clinical and, clinical criteria formulated more than 35 years ago [19], may show a diagnostic accuracy > 90% when typical patients are examined in specialized centers [20]. However, in the community, in early disease, in atypical or mixed cases, and the presence of comorbidities, diagnostic accuracy may decrease substantially [21]. Thus, it has been estimated that up to 30% of patients with a clinical diagnosis of AD during life will prove to have non-AD pathology at autopsy [22] and, vice versa, for patients with a clinical presentation suggestive of a non-AD disorder, there is a 39% chance that an autopsy will prove the (co)occurrence of AD pathology [23]. The gold standard for verification of the AD diagnosis is a *postmortem* neuropathological examination. However, correct diagnosis during life is needed, since it allows a more accurate estimation of prognosis and better therapeutic decisions [24,25].

Until now, the pharmaceutical treatment of Alzheimer's disease was dependent on drugs introduced 20–25 years ago. However, on 7 June 2021, the Food and Drug Administration (FDA) in the USA, approved the anti-amyloid monoclonal antibody aducanumab, as the first disease-modifying treatment for AD in the early clinical stages (MCI, mild dementia) [26]. Aducanumab was approved under the accelerated approval pathway, which requires a long (nine years) post-marketing phase IV study to confirm the drug's cognitive benefits. Despite the intense discussion, the arguments and debates triggered, all agree that, if such a specific disease-modifying treatment is to be used the diagnosis of AD should be verified with the maximum accuracy as possible.

For in vivo diagnosis, various biomarkers have been studied during the last 25 years, including cerebrospinal fluid (CSF) biomarkers [27]. Among these, three are considered as classical or "core" biomarkers for AD [28]: Aβ42, which is decreased in AD and is inversely related to amyloid plaque burden [29]; tau protein phosphorylated to a threonine residue at position 181 (τP-181) which is increased in AD and it is considered as a marker of tangle formation [30]; total tau protein (τT) which is increased in AD and it is a nonspecific marker of neuronal and/or axonal loss [31]. The Aβ42/Aβ<sup>40</sup> ratio may be used instead of Aβ<sup>42</sup> and seems to perform diagnostically better than the latter [32]. With sensitivities and specificities approaching or exceeding 90%, CSF biomarkers offer added diagnostic value compared to clinically-based diagnosis alone [5] and they have been incorporated in newer diagnostic criteria and guidelines [5–7]. A combination of decreased Aβ<sup>42</sup> with increased τP-181 and τ<sup>T</sup> is highly specific for the presence of AD, while normal levels of all three biomarkers are highly specific for the absence of AD [33]. Increased levels of the τP-181/Aβ<sup>42</sup> ratio have also been observed to provide high specificity for the differential diagnosis of AD from other dementias [34]. More recently, the AT(N) classification system has been introduced for diagnostic classification of AD (and possibly other dementia disorders), based on biomarkers [35]. The letter A stands for markers of amyloid pathology, T for markers of tau pathology (tangle formation), and N for markers of neurodegeneration (neuronal/axonal loss). Each letter is followed by either <sup>+</sup> or <sup>−</sup>, representing the positive (abnormal) or negative (normal) result of testing, respectively. The profile ("fingerprint") of AD is either A+T+(N)+ or A+T+(N)<sup>−</sup> [18]. Profiles such as A+T−(N)<sup>−</sup> or A+T−(N)+ are compatible with Alzheimer's *pathological change* (change from normal with the acquisition of amyloid biochemistry/pathology, without or with additional non-AD pathologies), but not Alzheimer's disease (which requires *both* amyloid plaques and neurofibrillary tangles [1]) [18]. Although the AT(N) system was designed mainly for research purposes, it can be used in clinical practice, even with clinically relevant prognostic value [36] and it

may be suitable for in vivo AD verification in patients suitable for aducanumab treatment, especially during the long phase IV trial of aducanumab.

#### **2. Patients and Methods**

#### *2.1. Patients*

The four patients presented here were examined at the 2nd Department of Neurology. They had cognitive impairment with an atypical presentation, creating clinical diagnostic uncertainty, with CSF biomarkers resolving the problem by revealing the CSF "neurochemical fingerprint" of AD (otherwise, there were no specific selection criteria).

Initially, history, neurological and complete physical examination were recorded routinely. Secondary causes including thyroid disease, B12 deficiency, neurosyphilis, brain tumor, or subdural hematoma (but not normal pressure hydrocephalus) were excluded. Written informed consent was obtained for all cases. The study had the approval of the Scientific Board and Ethics Committee of "Attikon" Hospital (project identification codes of approval: A13, 7 April 2021 and 157, 16 March 2021 respectively) and was conducted according to the ethical guidelines of the 1964 Declaration of Helsinki.

#### *2.2. Neuropsychological Approach*

Following history and clinical examination, a battery of neuropsychological tests was performed. Global tests for the assessment of cognition and activities of daily living included the Addenbrooke's Cognitive Examination-Revised version (ACE-R), the Mini Mental State Examination (MMSE), and the Instrumental Activities of Daily Living (IADL), all of which have been validated in Greece [37–39]. Brief bed-side tests for memory (free and cued recall), frontal function, visuospatial skills, and possible depression included the 5-words memory test [40], the Frontal Assessment Battery (FAB) [41], the CLOX (1 and 2) [42], and the short version of the Geriatric Depression Scale (GDS) [43], respectively. Finally, as a tool for the concomitant assessment of cognitive and functional status, the Clinical Dementia Rating (CDR, both sum of boxes and overall score) was used [44].

#### *2.3. Neuroimaging*

A routine 1.5 or 3T brain magnetic resonance imaging (MRI) scan was the preferred method of neuroimaging, including 3D T1W sequences, suitable for assessing cortical and central atrophy, including medial temporal atrophy, according to a visual scale [45]. The Evans index and callosal angle were also calculated as appropriate [46]. Alternatively, a brain computerized (CT) scan was obtained in cases with MRI contraindication (orthopedic prostheses).

#### *2.4. Lumbar Puncture and CSF Biomarker Measurements*

A lumbar puncture was performed using a standard, 21–22G, Quincke-type needle, at the L4–L5 interspace, at 9–12 a.m. according to widely accepted recommendations on standardized operative procedures for CSF biomarkers [47]. In brief, CSF was collected in six polypropylene tubes. The first and second tubes (1 mL each) were used for routine CSF cytology and biochemistry, respectively. The third tube (2 mL) was used for oligoclonal bands and IgG index determinations. The following two tubes (5 mL each) were used for biomarker determinations. The last tube (~2 mL) was used for syphilis serology or other tests according to clinical indications. All CSF samples had <500 red blood cells/μL.

The two tubes intended for CSF biomarker analysis were immediately centrifuged (2000× *g* 15 min), aliquoted in polypropylene tubes (1 mL each), and finally stored at −80 ◦C. Aliquots were thawed only once, just before analysis, which was performed within three months of storage.

Classical CSF biomarkers (Aβ42, Aβ40, τP-181, and τT) were measured in a Euroimmun Analyzer I (Euroimmun, Lübeck, Germany), in duplicate, with a double sandwich enzymelinked immunosorbent assay (ELISA) by commercially available kits (EUROIMMUN Beta-Amyloid (1-42) ELISA, EUROIMMUN Beta-Amyloid (1-40) ELISA, EUROIMMUN

pTau(181) ELISA, and EUROIMMUN Total-Tau ELISA, respectively), according to the manufacturer's instructions and by the use of 4-parameter logistic curves as described elsewhere [48]. All procedures were performed under a stable temperature (21 ± 2 ◦C) and quality control samples (both in-house and provided by the manufacturer) were used in each run. The inter- and intra-assay coefficients of variation were both <7% for all biomarkers. CSF biomarkers were considered normal according to cut-off values of the Neurochemistry and Biological Markers Unit (Aβ<sup>42</sup> > 480–500 pg/mL, Aβ42/Aβ<sup>40</sup> > 0.09, τP-181 < 60 pg/mL, τ<sup>T</sup> < 400 pg/mL).

The CSF AD profile ("fingerprint") was defined as decreased Aβ<sup>42</sup> or decreased Aβ42/Aβ<sup>40</sup> and increased τP-181, and thus, compatible with the A+T+(N)+ or A+T+(N)<sup>−</sup> profiles of the AT(N) classification system [18], according to Figure 1.

**Figure 1.** Biomarker levels in the CSF and interpretation of results for clinical purposes in our departments according to the AT(N) classification system, using the classical CSF biomarkers and structural imaging (MRI or CT) [18]. \* Abnormal have decreased levels (positive result). ‡ Abnormal have increased levels (positive result). § Abnormal have increased CSF levels or atrophy in structural neuroimaging (positive result). Negative results indicate normal findings. AD: Alzheimer's disease.

#### **3. Results**

The demographic, clinical, neuropsychological, and CSF neurochemical data of the four patients are summarized in Table 1.



ACE-R: Addenbrooke's Cognitive Examination-Revised, MMSE: Mini Mental State Examination, IADL: Instrumental Activities of Daily Living, FAB: Frontal Assessment Battery, GDS: Geriatric Depression Scale, CDR: Clinical Dementia Rating, PPA: Primary Progressive Aphasia, NPH: Normal Pressure Hydrocephalus, CBS: Corticobasal Syndrome, VCI: Vascular Cognitive Impairment, NA: not available. ↓ Decreased levels, ↑ increased levels, ? diagnostic uncertainty remains.

#### *3.1. Patient 1*

A seventy-six-year-old female was examined due to four years of "memory problems". She increasingly had to keep memos and frequently repeated the same questions. According to the results of the neuropsychological testing, she had incipient dementia, with a profile more compatible with a frontal or frontal-subcortical syndrome (decreased attention and concentration and executive function) rather than the typical hippocampal amnestic syndrome (Table 1). Neuroimaging showed frontal–frontoparietal atrophy and asymmetric hippocampal atrophy (Figure 2a). Biomarker assessment showed decreased Aβ<sup>42</sup> and Aβ42/Aβ<sup>40</sup> ratio and increased both τP-181 and τT, compatible with AD.

**Figure 2.** (**a**) T1 Magnetic Resonance Imaging (MRI) sequences of patient 1. Frontal (mainly), frontoparietal, perisylvian, and left hippocampal (grade 3) atrophy is observed. (**b**) T1 MRI sequences of patient 2. Atrophy in the left posterior perisylvian and parietal area is observed with preservation of the hippocampus. (**c**) Computerized tomography (CT) scan of patient 3. Some degree of frontal and parietal atrophy is seen. The white matter shows decreased density consistent with subcortical small vessel disease, in addition to periventricular caps. The parietal convexity is tight, the callosal angle is 84.4◦ and the Evans index has been calculated to 0.36. (**d**) CT scan of patient 4. Frontal (mainly) and parietal asymmetric atrophy are observed. Although the parietal convexity is not tight, the callosal angle is 88.4◦ and the Evans index has been calculated to 0.38. Decreased density of the white matter at centrum semiovale is noted, consistent with small vessel disease, with additional periventricular caps.

#### *3.2. Patient 2*

This seventy-six-year-old female suffered gradually progressive difficulty in speech for three years. Upon examination, she had a perfect understanding of language, but during spontaneous speech she made many pauses in an effort to "recall" the appropriate word. Upon naming testing, anomic (word-finding) difficulty was obvious, with object knowledge and single-word comprehension completely spared. Phonological errors were frequent and sentence repetition was severely affected. The motor and grammatical aspects of speech were normal. No difficulty in other cognitive domains was reported and decreased scores in neuropsychological testing were attributed mainly to the language (aphasic) disorder. She had no other significant difficulty in activities of daily living except in communication due to the aphasic disorder, which was compatible with Primary Progressive Aphasia (PPA) of the logopenic-type [49]. Atrophy was predominant in the left perisylvian and parietal areas (Figure 2b). Biomarker analysis revealed normal Aβ<sup>42</sup> with reduced Aβ42/Aβ<sup>40</sup> ratio, together with increased τP-181 and τT, compatible with AD.

#### *3.3. Patient 3*

An eighty-one-year-old male developed a gradually progressive cognitive decline during the last four years. He had apathy, social withdrawal difficulty in performing complex tasks, mental "slowness", and reduced attention. The previous year, progressive gait difficulty was noticed, with slow and short steps, sometimes a "magnetic" gait, and occasional falls with one fracture. The previous month, urinary urgency and sometimes incontinence was added into the clinical picture. Neuropsychological testing revealed moderate-stage dementia showing a mixed profile, including significant frontal, amnestic, and visuoconstructive components. Neuroimaging revealed an increased Evans index, acute callosal angle, tight convexity and periventricular caps, suggestive of normal pressure hydrocephalus [46], but cerebral small vessel disease was also evident (Figure 2c). Consistently with the suspicion of normal pressure hydrocephalus, a spinal taping test (removal of 40 mL of CSF) resulted in a significant improvement of gait and cognition. However, CSF biomarkers analysis revealed decreased Aβ<sup>42</sup> and increased τP-181 and τT, compatible with the additional presence of AD.

#### *3.4. Patient 4*

This eighty-three-year-old female developed gradually progressive gait difficulty with slow and short steps, postural instability, and frequent falls during the last three years and was unresponsive to L-dopa treatment. In addition, apathy, mental "slowness" and reduced attention were reported. In the previous year, urinary incontinence was noted. Upon clinical examination, she was practically bed-ridden, with asymmetric parkinsonism, including limb bradykinesia and rigidity more evident in the left limbs, while pyramidal signs were additionally present, more evident in the left limbs. Frequent myoclonic jerks were observed in the upper limbs, especially the left. Cortical sensory loss and sensory neglect were present in the right limbs. Primitive reflexes (especially grasping) were also present. Neuropsychological testing revealed moderate-stage dementia showing a mixed profile, including significant frontal, amnestic and visuoconstructive components, while significant upper limb apraxia was present. The patient met clinical criteria for corticobasal syndrome [50]. Despite some degree of asymmetrical atrophy, neuroimaging revealed an increased Evans index, acute callosal angle, and periventricular caps, suggestive of normal pressure hydrocephalus [46], while some degree of cerebral small vessel disease was also evident (Figure 2d). The spinal taping test (removal of 40 mL of CSF) resulted in a significant improvement of cognition, but there was no change in gait. Analysis of CSF biomarkers showed reduced Aβ42/Aβ<sup>40</sup> ratio, together with increased τP-181 and τT, compatible with the presence of AD.

#### **4. Discussion**

In the present study, we present four cognitively impaired patients with clinical presentations creating diagnostic uncertainty. The first patient was at the transition from MCI to mild dementia and, while she complained of memory problems, the total delayed recall (including memory cues) was normal, which is considered not compatible with the hippocampal amnestic disorder (typically expected in AD), but more compatible with a frontal–subcortical-type of memory decline. Despite a senile onset of disease and a presumably higher probability for AD, this is estimated to be no more than ~70% in such cases with early-stage disease and non-typical presentation [21,22], with other pathologies entering in the differential diagnosis. In the second patient, the clinical profile was compatible with PPA of the logopenic-type, which is due to AD in approximately 50– 80% of patients [10,51]. However, it should be not considered synonymous with AD [49], since, in ~25%, it is caused by one of the frontotemporal pathologies [51].

Thus, in both patients 1 and 2, there was still a significant chance (at the level of 25–30%) that a non-AD pathology may be the cause of the cognitive decline. Since both patients had MMSE > 20, making them eligible for aducanumab treatment, it is necessary to increase the diagnostic certainty from 70–75% to as high as possible, in order to initiate such a specific, expensive, and with potentially serious complications, treatment. In both patients, the CSF biomarker results, according to the AT(N) classification system [18], were compatible with the presence of AD.

In patients 3 and 4, the case was quite different since they were mixed cases of dementia. Patient 3 had typical clinical and imaging characteristics of normal pressure hydrocephalus and the positive taping test was consistent with this notion. Normal-pressure hydrocephalus may occur alone, but in three-quarters of cases, AD and/or cerebrovascular disease (usual of the small vessel-type) may be additionally present [52]. In the additional presence of AD, a shunting operation may offer some degree of gait improvement, which may positively affect the quality of life [53]; however, cognitive improvement may be modest [53] and the overall improvement is traditionally thought to be moderate at best and short-lived [54]. Thus, the possible co-occurrence of AD should be known prior to the selection of optimal treatment (or treatment combinations). In patient 3, the whole picture was compatible with NPH and concomitant small vessel disease, both of which may contribute to the clinical picture. However, CSF biomarkers revealed a third significant component in this patient's dementia, that of AD.

Patient 4 was the most intriguing. She had a mixed movement and cognitive disorder, with a clinical picture typical of corticobasal *syndrome*, while neuroimaging revealed a normal pressure hydrocephalus-like picture and some degree of small vessel disease. A taping test resulted in the improvement of cognition only, but not of gait, probably because the corticobasal component of the motor disability was already severe enough to oppose any improvement. The corticobasal *syndrome* is not a disease, but a clinical picture that can be due to many neurodegenerative diseases, the most common being corticobasal *degeneration* which belongs to the 4-repeat tauopathies [50]. However, it can be caused by AD, Lewy body pathology, progressive supranuclear palsy, and even Creutzfeldt–Jakob disease [12], with AD accounting for a significant percentage of cases with corticobasal *syndrome* [55]. CSF biomarker analysis in patient 4 revealed that AD was indeed the underlying cause. Normal-pressure hydrocephalus was probably present as well (hence the cognitive improvement following the taping test), however, it was superimposed on AD.

Classical CSF biomarkers are useful in identifying the AD biochemical fingerprint in typical and atypical AD cases [27,28]. Their diagnostic performance has been validated in autopsy-proven cases [56]. They have been proven useful in cases with primary progressive aphasia [51], corticobasal syndrome [57], and cases of AD mixed with Lewy body pathology [58] or cerebrovascular disease [14,59]. They can identify the concomitant presence of AD in cases with normal pressure hydrocephalus [60,61], and possibly predict a

worse neurosurgical prognosis [62], although recent data suggest that they may predict the opposite [16].

When incorporated in the AT(N) classification system, CSF biomarkers may be used effectively not only in research but also in clinical practice [36,63]. It should be noted that in patients 2 and 4, CSF levels of Aβ<sup>42</sup> were normal. However, the Aβ42/Aβ<sup>40</sup> ratio was abnormally reduced in both, allowing the diagnosis of AD. Despite some concerns about the interchangeability between Aβ<sup>42</sup> and the Aβ42/Aβ<sup>40</sup> ratio in the AT(N) system [64], the ratio shows better diagnostic accuracy compared to Aβ<sup>42</sup> alone [32,65], correlates better with amyloid imaging by positron emission tomography [32], and its better diagnostic performance has been confirmed in pathologically proven cases [32].

There are some limitations in classical CSF biomarker determination. Preanalytical factors, including CSF sampling and storage, may affect test results and internationally accepted guidelines have been formulated for this reason [47]. International quality control programs and projects have been organized, in order to identify and control for confounding factors, improve the methodologies used, optimize analytical performance, and harmonize the levels of biomarkers [66–68]. However, there is still a significant intraand inter-laboratory variability [67,69] and each laboratory should have its own cut-off values [28]. Discordant biomarker results have been observed in different reference laboratories, especially for Aβ<sup>42</sup> [70]. Diagnostically gray zones also exist and, when added to the possible measurement error, they may lead to a variability of ±25% [70]. Normal levels of all three CSF classical biomarkers may be observed in normal aging, but also in psychiatric disorders which may present with cognitive complaints, sometimes entering in the differential diagnosis of frontotemporal dementia. Furthermore, the classical CSF biomarkers cannot identify additional neurodegenerative pathologies, which are not rare in older patients with AD [71]. Finally, determination of CSF biomarkers requires a lumbar puncture which is a cause of concern and anxiety in many patients and caregivers, and it cannot be easily repeated for frequent follow-up.

Other molecules are under intense investigation in an effort to optimize the differential diagnostic value of the classic biomarkers and identify possible additional neurodegenerative pathologies. They include markers of neuroinflammation such as the triggering receptor expressed on myeloid cells 2 (TREM2), progranulin, and chitinase-3-like protein-1 (YKL-40), markers of synaptic dysfunction such as neurogranin, and markers of neuronal injury such as neurofilament light (NfL) and visinin-like protein 1 (VILIP-1), while miR-NAs could also be helpful [72–77]. Oligomeric forms of Aβ<sup>42</sup> [78], α-synuclein [79], and TAR DNA-Binding Protein 43 (TDP43) [80] are emerging biomarkers, but work must still be carried out to achieve adequate diagnostic performance. Especially for α-synuclein, which has been traditionally considered as a marker of synuclein pathology, results are conflicting [79], partially due to the effect of preanalytical and analytical factors, including differences in a-synuclein species detected by different methods [81]. Recent evidence suggests that α-, and also β- and γ-synuclein, may be effective markers of AD rather than synucleinopathy [82]. Both α- and β-synuclein may be early markers of AD, even in non-demented elder subjects [83,84], while the ratio of total tau/α-synuclein may serve as a marker of tau phosphorylation, even allowing patients with the A−T+(N+) profile to re-enter the AD diagnostic group [85]. Blood-based classical [86,87] and exosomal [88] biomarkers may prove helpful, especially for frequent monitoring of the biochemical effects of anti-amyloid antibodies. The AT(N) system is flexible and may expand to an ATX(N) form, incorporating such new or evolving biomarkers of AD-related or additional non-AD pathologies [89].

#### **5. Conclusions**

Biomarkers are not stand-alone tools and should always be interpreted along with clinical, neuropsychological, and imaging data. Keeping this in mind, analysis of classical CSF biomarkers, especially when incorporated in a classification/diagnostic system such as the AT(N), may offer a significant diagnostic tool [90,91], with both added [92] and prognostic [36] value, allowing the correct identification of AD during life, especially in cases with atypical or mixed presentations [93]. This is always important for correct therapeutic decisions, and it is of paramount importance currently, due to the recent approval of aducanumab as a disease-modifying treatment. Whether atypical cases are going to have the same benefit (from classical or newer treatments) as the typical ones, remains to be elucidated.

**Author Contributions:** Conceptualization, I.T. (Ioanna Tsantzali), F.B., E.K. and G.P.P.; methodology, I.T. (Ioanna Tsantzali), F.B., E.S., G.T., E.K. and G.P.P.; formal analysis, I.T. (Ioanna Tsantzali), F.B., E.S., A.M., M.G.P., A.F., I.T. (Ioannis Tollos), S.G.P., A.B., K.I.V., G.T., E.K. and G.P.P.; investigation, I.T. (Ioanna Tsantzali), F.B., E.S., A.M., M.G.P., A.F., I.T. (Ioannis Tollos), S.G.P., A.B., K.I.V., G.T., E.K. and G.P.P.; data curation, I.T. (Ioanna Tsantzali), F.B., E.S. and G.P.P.; writing—original draft preparation, I.T. (Ioanna Tsantzali), F.B., and G.P.P.; writing—review and editing, A.M., M.G.P., A.F., I.T. (Ioannis Tollos), S.G.P., A.B., K.I.V., G.T. and E.K.; visualization, G.T., E.K. and G.P.P.; supervision, K.I.V., G.T., E.K. and G.P.P.; project administration, G.T., E.K. and G.P.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** E.K. has received research funding from ELPEN Pharmaceutical Co. Inc. and NUTRICIA.

**Institutional Review Board Statement:** The study had the approval of the Bioethics Committee and the Scientific Board of "Attikon" Hospital (approval numbers 157/16-03-2021 and A13/07-04-2021, respectively). It was performed according to the 1964 Declaration of Helsinki.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study and/or next of kin caregivers (depending on the severity of cognitive impairment).

**Data Availability Statement:** The data presented in this study are available upon request from the corresponding author. The data are not publicly available due to privacy restrictions.

**Acknowledgments:** We would like to thank the patients and their caregivers for their participation. We would also like to thank the laboratory technician Olga Petropoulou for her valuable assistance in sample handling and biomarker measurements.

**Conflicts of Interest:** G.P.P. receives fees from Biogen International as a consultant of the advisory board. All other authors: none. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**


## *Review* **Shared Molecular Mechanisms among Alzheimer's Disease, Neurovascular Unit Dysfunction and Vascular Risk Factors: A Narrative Review**

**Lorenzo Falsetti 1,\*, Giovanna Viticchi 2, Vincenzo Zaccone 1, Emanuele Guerrieri 3, Gianluca Moroncini 4, Simona Luzzi <sup>2</sup> and Mauro Silvestrini <sup>2</sup>**

	- e.guerrieri93@gmail.com

**Abstract:** Alzheimer's disease (AD) is the most common type of dementia, affecting 24 million individuals. Clinical and epidemiological studies have found several links between vascular risk factors (VRF), neurovascular unit dysfunction (NVUd), blood-brain barrier breakdown (BBBb) and AD onset and progression in adulthood, suggesting a pathogenetic continuum between AD and vascular dementia. Shared pathways between AD, VRF, and NVUd/BBB have also been found at the molecular level, underlining the strength of this association. The present paper reviewed the literature describing commonly shared molecular pathways between adult-onset AD, VRF, and NVUd/BBBb. Current evidence suggests that VRF and NVUd/BBBb are involved in AD neurovascular and neurodegenerative pathology and share several molecular pathways. This is strongly supportive of the hypothesis that the presence of VRF can at least facilitate AD onset and progression through several mechanisms, including NVUd/BBBb. Moreover, vascular disease and several comorbidities may have a cumulative effect on VRF and worsen the clinical manifestations of AD. Early detection and correction of VRF and vascular disease by improving NVUd/BBBd could be a potential target to reduce the overall incidence and delay cognitive impairment in AD.

**Keywords:** Alzheimer's disease; vascular risk factors; hypertension; type 2 diabetes mellitus; dyslipidemia; cigarette smoking

#### **1. Introduction**

Alzheimer's disease (AD) is the most common neurodegenerative dementia, affecting two-thirds of individuals with cognitive decline worldwide [1]. Its main pathological features are represented by neuroinflammation, extracellular amyloid-β (Aβ) peptide deposition, intracellular neurofibrillary tangles, tau protein degeneration, and neural loss with progressive deterioration of cognitive function [2–4]. Considering a doubling in 20 years, the prevalence of AD will reach 130 million people in 2050, with the greatest increase expected in the poorest countries [5]. Due to its high prevalence, several studies are focusing on reliable serum biomarkers to accurately diagnose AD [3,4].

The neuropathology of AD is characterised by structural and physiological changes that may involve different brain areas. This variability contributes to a certain heterogeneity in the final clinical manifestations in AD patients, each of whom may exhibit a variable association of different neuropsychological deficits. In fact, while the classic cognitive profile of AD is mainly characterised by episodic memory deficits due to the impairment of the temporal lobe, several recent studies have shown that in relation to the different brain

**Citation:** Falsetti, L.; Viticchi, G.; Zaccone, V.; Guerrieri, E.; Moroncini, G.; Luzzi, S.; Silvestrini, M. Shared Molecular Mechanisms among Alzheimer's Disease, Neurovascular Unit Dysfunction and Vascular Risk Factors: A Narrative Review. *Biomedicines* **2022**, *10*, 439. https:// doi.org/10.3390/ biomedicines 10020439

Academic Editor: Kuen-Jer Tsai

Received: 3 January 2022 Accepted: 12 February 2022 Published: 14 February 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

171

areas predominantly involved, different clinical pictures may be present. For example, alterations in the medial prefrontal cortex are associated with impaired retrieval and extinction memories, whereas impairment of emotional and executive processing, similarly to the psychiatric population, reflects a probable impairment of the lateral orbitofrontal cortex or the inferior frontal gyrus [6–8]. It is worth underlining that multiple pathways are implicated in the onset and progression of neurodegeneration and specific functions, such as emotional control, are often impaired in dementia. Indeed, impairment of the prefrontal cortex causes in AD patients an impairment in emotion processing that impacts on action and motor control [7].

The AD pathophysiology is considered largely heterogeneous and characterized by both neurodegeneration—characterized by aberrant, misfolded and aggregated Aβ [9] and hyperphosphorylated tau proteins [10]—and vascular disease, with a common involvement of large [11–14] and small brain vessels. Other neurotoxic elements could also play a relevant role: oxidative stress with reactive oxygen species overproduction, mitochondrial dysfunction or metal accumulation have been extensively studied in the last years [15].

Recently a great attention was put on the interaction between neuronal (neurons and glia) and vascular tissues (endothelial cells, pericytes, and adventitial cells) that are functionally organized in the neurovascular unit (NVU). The NVU is responsible for socalled neurovascular coupling, an organized vascular response to specific neuronal stimuli aimed at modifying regional cerebral blood flow and neuronal metabolic activity [16]. The blood-brain barrier (BBB) is a part of the NVU that controls the transfer of molecules and pathogens to and from brain tissue by adopting a specific transport system in brain endothelial cells [17]. The BBB transports brain metabolic waste products from the brain interstitial fluid to the bloodstream. Thus, BBB represents the most important site of metabolic homeostasis in the central nervous system [17]. Neurovascular coupling is typically deranged in several pathological conditions such as hypertension [18], acute ischemic stroke [19] and AD [20], suggesting a potential role of NVU dysfunction (NVUd) in the progression of cognitive impairment. Of note, neurovascular coupling is strongly influenced by VRF [21–26] and atherosclerotic vascular pathology [14,27], as synthesized in Figure 1.

**Figure 1.** Known interactions between vascular and neurodegenerative factors in AD.

Postmortem studies emphasize an important role of vascular pathology in a large percentage of AD subjects. As first observed in the Nun study [6], the presence of neuropathologic findings of vascular lesions in AD subjects was associated with a history of

worse cognitive performances. In addition, other studies have confirmed the presence of atherosclerosis of large and small vessels in AD [12]. These alterations, which could affect NVU activities, may be expressed by pathologic adaptations of cerebrovascular responsiveness to hypercapnia [26,28]. A dysfunction of the BBB has been associated with oxidative stress [29], advanced glycation end products (AGEs) and their receptor (RAGE) [30,31] and increased production of proinflammatory cytokines [32]. A blood-brain barrier breakdown (BBBb) could also contribute to AD onset and progression.

This review focuses on the main and most common vascular risk factors that can be easily detected, monitored, and addressed in common clinical practice, their impact on AD neurovascular and neurodegenerative pathology and the potential links between VRF and AD at a molecular level.

#### **2. Research Strategy**

The review team identified first the MeSH major terms to explore the association between AD, VRF, NVUd and BBB breakdown and performed the literature search in PubMed/Medline and Web of Science for case reports, reviews, and original research articles from 1 January 1991, to 1 December 2021. We used MeSH major terms and considered: "Alzheimer's disease" [MeSH], "Adult-onset diabetes mellitus" [MeSH], "Hypertension" [MeSH], "Dyslipidemia" [MeSH], "Cigarette smoking" [MeSH], "Neurovascular coupling" [MeSH], "Blood brain barrier" [MeSH] and "Neurovascular abnormalities" [MeSH] alone or in combination. The review team favored the inclusion of articles from the last 10 years to give up-to-date information, although they did not exclude older highly referenced reports. The reference lists of articles identified by this search strategy were also reviewed, and the working group selected relevant references. We chose to consider this time frame and all types of articles to obtain a comprehensive overview of this topic.

#### **3. Discussion of the Results of the Research Strategy**

According to the pre-specified research strategy, the review team selected 156 unique papers regarding the clinical, epidemiological and molecular relationships between AD, NVUd, BBBb, type 2 diabetes mellitus (T2DM), hypertension, smoking and dyslipidemia.

#### *3.1. Type 2 Diabetes Mellitus*

#### 3.1.1. The Clinical and Epidemiological Link between AD and T2DM

Both AD and T2DM prevalence are progressively increasing, especially among elderly patients [1,33]. Among adults, 1 in 11 suffers from diabetes mellitus and 90% of cases are T2DM, which is a chronic, multi-organ disease characterised by a high burden of co-morbidities and a low quality of life [34]. While aging itself is the strongest risk factor for both AD [35] and T2DM [36], emerging epidemiological data suggest that T2DM and other VRFs may contribute to the pathogenesis of AD directly, in association, or as cofactors [37–39]. AD and T2DM are epidemiologically associated as AD patients appear more vulnerable to T2DM [40], and individuals with T2DM show an increased risk of dementia, including AD [41].

The Rotterdam Study confirmed that the presence of T2DM increases the risk of AD [42], and that this association is stronger in patients with a long history of T2DM. This epidemiological relationship has been confirmed in other cohorts [43]. However, despite epidemiological studies suggesting T2DM as a potential risk factor for AD, the demonstration of a complete overlap between the two diseases is lacking. Some authors have associated this effect to different insulin resistance in target organs. Cerebral hyperglycemia [44] in the absence of clinically evident T2DM has been positively associated with accelerated cognitive impairment, even adjusting for other risk factors, including age and macrovascular disease. In addition, patients with AD often exhibit both insulin resistance and insulin insufficiency even when not affected by T2DM. These observations have led to the current concept of AD as a special form of T2DM, defined by several authors as "type 3 diabetes mellitus" (T3DM) [45–47]. T3DM refers to insulin/insulin-like growth factor (IGF) deficiency and insulin/IGF resistance in brain tissue [46].

#### 3.1.2. The Role of Insulin Signalling

Insulin/IGF and their receptors are widely expressed in the cortex, hippocampus, and hypothalamus of the human brain. Several investigations support the hypothesis that cognitive impairment in AD might be, at least in part, mediated by insulin resistance and deficiency of the insulin/IGF cascade in the brain [48–50]. These mechanisms activate multiple intracellular signalling pathways ensuing in intrinsic tyrosine kinases (iTK) activation starting with ligand binding to cell surface receptors, followed by iTK autophosphorylation and activation [51,52]. iTK phosphorylate IRS molecules [53–55], which transmit signals downstream by activating the extracellular/mitogen-activated signal-related kinase (ERK/MAPK) and PI3K pathways and inhibit glycogen synthase kinase 3beta (GSK3). PI3K/AKT/mTOR cascade activation leads to synaptic formation, increased neuronal cell survival [56], regional vasodilation, and regulation of cerebrovascular reactivity in the neurovascular unit [57].

Postmortem studies pointed out that, in brain samples from AD patients, insulin and insulin receptor expression were severely impaired and their levels were inversely proportional to the extent of neurodegeneration [45,58] along with impairment of insulin receptor binding capacity and reduced expression of insulin, IGF-1, IGF2 mRNA and their receptors, with a reduction in the cytosolic level of PI3K p85a and p110a subunits [59]. This was consensual with a tau protein reduction, regulated by insulin/IGF-1.

Decreased choline acetyltransferase (ChAT) expression, typically described in AD, is associated with reduced ChAT colocalization with the insulin/IGF-1 receptor, confirming that neuronal expression of tau and ChAT is regulated by insulin/IGF-1 in the human brain [46,47]. Reduced insulin and poor insulin receptor sensitivity contribute to decreased acetylcholine (ACh), further elucidating a possible biochemical link between diabetes and AD [58]. Thus, insulin resistance and deficiency in the brain could explain, at least in part, the alterations observed in AD, such as cytoskeletal collapse, retraction of neurites, synaptic disconnection, loss of neuronal plasticity, and deficiencies in ACh production. Moreover, T2DM is known to be one of the most important factors for accelerated atherosclerosis [60], and these observations suggest that cerebral vessel atherosclerosis could be another potential link between the two diseases, as confirmed by clinical studies [14].

#### 3.1.3. Shared Molecular Mechanisms between AD and T2DM

Primary biological responses to insulin/IGF include increase in cell growth, survival, energy metabolism and cholinergic gene expression, and inhibition of oxidative stress and of apoptosis. These signalling pathways are activated in different cell types and tissues capable of expressing insulin/IGF receptor. Thus they are virtually universal [55,61–63]. Several authors enlightened different abnormalities in IRS-1 phosphorylation (IRS-1p) in AD brains [64]. IRS-1p on tyrosine residues is needed for insulin-stimulated responses, whereas IRS-1p on serine residues was associated to an insulin reduced response, which was consistent with insulin resistance [65].

This pathway modulates the expression of Aβ precursor protein (APP), kinesin, Abelson helper integration site-1 (AHI-1), huntingtin-associated protein-1 (HAP-1), and tau, which are all involved in the neuropathology of AD. Furthermore, neuronal and oligodendroglial cell survival and function are fully linked to the integrity of the insulin/IGF-1 pathway [46,47,49]. Impairment of these metabolic pathways leads to deficits in energy metabolism resulting in increased oxidative stress, mitochondrial dysfunction, activation of proinflammatory cytokines and APP expression. Consequently, reduced expression of neuronal and oligodendroglial specific genes and increased expression of astrocytic and microglial inflammatory genes in AD have been attributed to progressive brain insulin/IGF deficiency and resistance.

Microglial and astrocytic APP mRNA levels are increased in the early stages of neurodegeneration in AD [66]. Microglia activation promotes APP gene expression, cleavage and accumulation. Impairment of insulin/IGF signalling leads to oxidative stress and mitochondrial dysfunction that induces APP gene expression and cleavage, thus resulting in neurotoxicity due to APP-A accumulation. Tau gene expression and tau protein phosphorylation are specifically mediated by this signalling cascade [67,68].

#### 3.1.4. The Role of AGE/RAGE System in AD

Glycosylation is a non-reversible and non-enzymatic reaction that occurs between proteins and glucose and eventually leads to the production of AGEs [69], which is especially observed in subjects affected by complicated T2DM [70]. The presence of AGEs marginally affects cell survival, but can significantly alter neuronal metabolism and thus brain function in several neurodegenerative disorders, such as AD [71,72]. In addition, AGEs can directly induce oxidative stress and promote the release of proinflammatory cytokines, thereby worsening cognitive dysfunction in AD [73]. AGEs and RAGEs colocalize with Aβ, senile plaques, and neurofibrillary tangles. Specifically, the interaction between RAGE and Aβ activates neuroinflammatory signalling pathways, causes the release of reactive oxygen species, and ultimately induces neuronal and mitochondrial dysfunction [31].

However, one large study observed a lack of longitudinal association between AGE-RAGE system dysfunction and dementia, suggesting a potential short-term association or reverse causality [74], thus supporting the need for further studies to explore this association.

#### *3.2. Hypertension*

#### 3.2.1. The Clinical and Epidemiological Link between AD and Hypertension

Aging is an important risk factor for hypertension, representing one of the epidemiological links between AD and VRF. However, the clinical association between hypertension and AD seems weaker than that with T2DM. Some studies have described this potential association [75–77] while others failed to demonstrate any link [78,79]. Papers underlining this epidemiological link have longer follow-up times [75–77]. The Rotterdam study pointed out that hypertension preceded the onset of AD by nine years [75], while in the Honolulu-Asia Aging Study, a temporal relationship of 20–26 years was observed [77].

On the other hand, some studies have shown that low blood pressure is also associated with incident dementia, and that blood pressure drops in the preclinical stages of AD, during AD, and consistently with advanced cognitive impairment [80]. Recently, a U-shaped relationship between hypertension and cognition has been confirmed, especially among the elderly [81]. Several authors have suggested that this effect might be mediated by neurodegeneration of brain structures involved in the central regulation of blood pressure (hypothalamus, amygdala, insular cortex, medial prefrontal cortex, locus coeruleus, parabrachial nucleus, pons and medulla oblongata). This hypothesis is supported by studies underlining a direct positive relationship between neuron number in pons or medulla and blood pressure in AD [82]. Further, brain atrophy has been correlated with lower blood pressure in elderly patients, regardless of dementia [83]. In addition, lower blood pressure can result in greater neuronal damage by worsening regional cerebral blood flow regulation by generating local hypoperfusion [84].

Thus, middle-aged hypertension acts as a risk factor for AD before its onset, whereas low blood pressure in the elderly should be interpreted as a consequence of neural loss, especially in advanced AD. Hypertension is known to induce cerebral vascular changes and vascular dementia. Animal models have shown that high blood pressure can also lead to AD-like neuropathology [85–87], with accumulation and deposition of Aβ.

#### 3.2.2. Shared Molecular Mechanisms between AD and Hypertension

While the "classical" amyloid hypothesis suggested a cytotoxic accumulation of Aβ in the brain tissue of AD patients due to its overproduction [88], more recent evidence has shown that Aβ accumulation might be more related to an altered clearance of this molecule from the BBB due to NVU dysfunction [89].

One of the pathways linking hypertension to AD is RAGE, which modulates Aβ clearance in the BBB. Its expression is critically increased in endothelial cells and at the level of the AD brain neurovascular unit [90]. Furthermore, in experimental models, its expression is upregulated in cerebral vessels of the cortex and hippocampus after exposure to a hypertensive condition [86]. RAGE acts as a scavenger receptor for Aβ. In the BBB it mediates the passage of Aβ from the blood to the brain. It also stimulates Aβ production [91] and induces tau hyperphosphorylation [92] by activating the GSK-3 cascade. The angiotensin-II type 1 receptor suggests another link between AD and hypertension. In hypertensive subjects, activation of this receptor increased RAGE mRNA expression, suggesting a link between activation of the renin-angiotensin axis and AD progression [93]. RAGE also responds to AGEs, which are elevated in AD, especially in patients with T2DM, and this represents another possible link between VRF and AD.

The other molecular mechanism linking AD to hypertension is low-density lipoprotein receptor-related protein-1 (LRP-1). Most cell types in the neurovascular unit express LRP-1, which is able to maintain the BBB integrity and transport Aβ from the brain to the blood vessels, in a direction opposite to that by RAGE. LRP-1 acts primarily by releasing Aβ from the brain, but its soluble form (sLRP-1) can bind Aβ and remove it from the circulation, reducing its bioavailability. Interestingly, the expression of LRP-1 in endothelial cells of the neurovascular unit is reduced with aging and its activity is mediated by ApoE [94]. In murine models of hypertension, RAGE expression is increased, whereas LRP-1 expression is unchanged, suggesting increased Aβ influx that is not adequately counteracted by increased efflux [95]. Furthermore, the presence of oxidative stress, as commonly observed in association with the presence of VRF, decreases sLRP-1 activity and increases serum levels of Aβ, which negatively correlates with cognition [96].

Finally, hypertensive patients often show increased serum levels of several markers of endothelial damage, such as soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), and endothelin-1 (ET-1), which might be implicated in the dysregulation of cerebrovascular reactivity in AD and other neurodegenerative diseases by promoting vasoconstriction [97–99].

#### *3.3. Dyslipidaemia*

3.3.1. The Clinical and Epidemiological Link between AD and Dyslipidaemia

Dyslipidaemias are a heterogeneous group of diseases defined as disorders of lipid metabolism that lead, alone or in association with other VRFs, to cerebral and systemic atherosclerosis. The current management of dyslipidaemias is closely dependent on the presence and extent of other VRFs, and current guidelines suggest treatment according to the patient's overall cardiovascular risk, as assessed by formal scores [100]. The systematic assessment and proper management of cardiovascular risk is leading to a progressive reduction in the incidence of atherosclerosis. However, this disease remains one of the leading causes of mortality and morbidity worldwide [100]. The link between dyslipidaemia and AD has been described at several levels. Epidemiological evidence suggests an association between high serum cholesterol levels and AD, with a potential role for lipids in modulating AD expression. Total cholesterol serum levels appear to be independently associated with increased AD prevalence, with a potential modulation of the effect by ApoE genotype [101]. Similar to hypertension, increased serum total cholesterol in middle age also appears to be strongly associated with the risk of AD, with a 3-fold increase in the likelihood of development, independent of ApoE genotype [102]. High levels of LDL cholesterol (LDL-C) correlate with lower global cognition in the absence of clinical dementia [103], and with more rapid cognitive decline in individuals who will develop AD [104]. Some authors underlined a paradoxically protective effect of increased serum cholesterol levels from dementia in late life [105], underlining the detrimental role of dyslipidaemia in younger subjects [106]. In addition, intracranial and extracranial atherosclerosis, one of the major consequences of inadequately treated dyslipidaemia, is significantly associated with the risk of AD onset and progression [14,107].

#### 3.3.2. Shared Molecular Mechanisms between AD and Dyslipidaemia

Increased serum cholesterol levels are presumed to induce neuronal apoptosis, oxidative stress, and tau hyperphosphorylation [108]. Brain lipid composition appears to be directly involved in APP processing and Aβ production: increased endosomal cholesterol levels appear to unbalance APP processing, thereby promoting the amyloid-genic pathway [109,110]. A cholesterol-rich membrane might also alter the activity of membrane secretases, thus inducing Aβ production [111]. Furthermore, dyslipidemia is thought to be associated with BBB disruption, which is commonly observed in AD [112]. Animal models, particularly low-density lipoprotein receptor (LDL-R) knock-out mice, confirm these observations: dyslipidemia increases the severity of cognitive dysfunction, especially learning and memory, and Aβ-associated neurotoxicity [113].

Recent studies have emphasized a genetic overlap between AD, C-reactive protein, and plasma lipids [114]. Genome-wide association studies emphasize a strong association between dyslipidemia and AD in several genes. ApoE genotype has been confirmed central to this interaction [115]. ApoE is the most abundant apolipoprotein in the human brain whose role is to transport lipids and facilitate brain homeostasis by removing debris from the interstitial fluid of the brain by interacting with endothelial cells, the basement membrane and glia [116]. In AD, APOE promotes Aβ clearance. The efficiency of Aβ clearance through the BBB depends on the activity of transport proteins such as APOE and APOJ, and receptors such as LRP-1 and RAGE. In particular, it has been observed that APOE ε2 and APOE ε3 genotypes bind with high affinity with LRP-1, whereas APOE ε4 binds with LDL-R [117]. The lack of interaction between APOE ε4 and LRP-1 has been associated with reduced cyclophilin A (CypA) inhibition leading to a proinflammatory state and BBB breakdown [118]. This effect appears to be mediated in pericytes by an NFB-dependent matrix metalloproteinase 9 (MMP-9), which disrupts endothelial tight junctions [117,119]. In addition, CypA has been associated with systemic atherosclerosis [117].

Other single nucleotide polymorphisms of genes implicated in lipid metabolism, such as CLU and ABCA7, have been associated with AD, underscoring a strong link between lipid homeostasis and cognitive function [120]. Of note, several genes implicated in the modulation of inflammation, such as CR1, HLA-DRB5 and TREM, have also been identified as associated with AD [120].

#### *3.4. Cigarette Smoking*

#### 3.4.1. The Clinical and Epidemiological Link between AD and Cigarette Smoking

Some cross-sectional studies, supported by the tobacco industry, reported a lower AD prevalence among smokers [121]. However, when analysing incident cases and controlling for tobacco industry affiliation [122], it was observed that smoking consistently increased the risk for AD and cognitive decline [123]. This increased risk was found in both APOE ε4 allele carriers [124] and non-carriers [125]. Particularly, mid-life smoking was associated to an increased AD risk [126]. Smoking habit shows its detrimental effects in cognition at different levels. Compared to non-smokers, middle-aged, active smokers showed poorer neurocognitive performances in executive domains (processing speed, learning and memory). Such cognitive dysfunctions were associated with a reduced volume and thickness in hippocampal, cortical, and subcortical areas, reduced neuronal and BBB integrity and neurobiological alterations like those found in early-stage AD, with a dose-dependent effect. Elderly, active-smoking subjects showed worse executive functions, processing speed, learning and memory, a greater cortical atrophy and lower grey matter density in specific brain areas when compared to non-smokers. Former smokers showed intermediate abnormalities between smokers and non-smokers. Patients with chronic obstructive pulmonary disease (COPD), which is commonly caused by smoking, often show worse cognitive performances [127] that seem to be partially preserved by long-term oxygen therapy [128]. A midlife COPD diagnosis is associated to an increased risk of a later-life cognitive deterioration [129]. However, COPD and lung function impairment seem to affect only marginally incident AD [130].

#### 3.4.2. Shared Molecular Mechanisms between AD and Cigarette Smoking

The only potential neuroprotective effect of smoking on the brain relies on the finding that nicotine showed neuroprotective activity against glutamate toxicity via α4 and α7 subunits, which can inhibit the neuronal apoptosis process similarly to therapeutic acetylcholinesterase inhibitors [131]. Cigarette smoking, however, has been associated to a downregulation of nicotinic acetylcholine receptors (nAChrs) subunit α7 expression on astrocytes [132] with a reduction of the neuroprotection offered. Furthermore, nicotine strongly affects brain endothelial function, since brain endothelium expresses several nicotinic receptor subunits (α3, α5, α7, β2 and β3) [133]. Nicotine increases BBB permeability by reducing tight junctions expression [134]. The detrimental effects of nicotine on tight junctions' permeability are worsened by oxidative stress and hypoxia. Moreover, nicotine downregulates NOTCH-4 expression in brain endothelial cells: a reduced NOTCH-4 expression is also associated to BBB breakdown [133]. Chronic cigarette smoking has been associated to an increased Aβ deposition and amyloid burden, tau phosphorylation, neuroinflammation with microglial activation, and plaque formation in a dose-dependent manner [135]. On the other side, it has been demonstrated that different central nervous system cells express nicotinic subunits (α3, α4, α5, α6, α7, β2, β4) in the context of nAChrs with a wide variability of expression within different areas of the brain.

Smoking attitude increases oxidative stress by unbalancing the production of reactive oxygen/nitrogen species and their reduction by natural antioxidants [136,137]. Notably, oxidative damage acts on nucleic acids, proteins and lipid membranes of NVU cells [136,137]. Oxidative stress induces cytokine-mediated activation of inflammation in the NVU, thus inducing neuronal cell death and BBBb.

There is a narrow link between neurodegenerative diseases, as AD, and chronic lung pathologies, as COPD [138]. Chronic brain hypoxia, which is commonly observed in advanced COPD and worsened by the occurrence of significant carotid atherosclerosis, seems to worsen cognition by increasing Aβ deposition and tau hyperphosphorylation [139,140]. Moreover, chronic hypoxia acts on VMSCs by downregulating LRP-1 [140], favouring a hypercontractile, non Aβ-clearing phenotype [141]. Moreover, chronic hypoxia has other detrimental effects on cognition: it activates microglia inducing a proinflammatory state that downregulates Aβ receptors and induces a BBB breakdown [140].

#### *3.5. Association between VRF and NVU Dysfunction in AD*

Neurovascular imbalance is sufficient to initiate neuronal damage and induce accumulation of Aβ. VRF aggregation leads to atherosclerosis, and both factors can induce NVU dysfunction and BBB disruption. These two alterations are associated with increased entry and defective clearance of neurotoxic compounds into brain tissues and reduced energy metabolites and oxygen delivery to activated areas of the brain resulting in neuronal damage. Furthermore, by regulating small vessel blood flow, neurovascular coupling aims to reduce local thrombosis by balancing pro-thrombotic and anti-thrombotic pathways. Its alteration is associated with increased vascular damage. Different combinations of VRF have been associated with cognitive impairment [26,39], especially in the presence of altered cerebrovascular reactivity [142]. Large vessels atherosclerosis is the most prominent effect of a long-term VRF combination, has been associated with an imbalance in cerebrovascular reactivity and more rapid cognitive deterioration [27,143].

At the cellular level, vascular muscle smooth cells (VMSCs) in AD exhibit a "hypercontractile phenotype," which appears to be critically involved in the dysregulation of local cerebral blood flow by inducing chronic hypoxia and hypoperfusion that facilitates neural loss [141]. In addition, AD-VMSCs exhibit impaired capacity to clear Aβ, facilitating cerebral amyloid angiopathy, which in turn leads to impaired cerebral hemodynamic adaptability [144].

BBB disruption appears to be associated with increased production and reduced clearance of Aβ, which promotes the accumulation of amyloidogenic molecules, typically present in advanced AD. BBB dysfunction is favoured by genetic traits, such as APOE ε4. APOE ε4 carriers show accelerated pericyte degeneration due to activation of the

CypA MMP-9 pathway, which is associated with BBB dysfunction with tight junctions and alteration of core proteins [145]. In addition, APOE ε4 carriers often show impaired cerebrovascular reactivity that could affect cerebral perfusion [38,146,147].

#### **4. Conclusions**

This narrative review aimed to focus on the major vascular risk factors that may contribute to AD genesis and progression (as shown in Figure 2). The reviewed literature highlights correlations between VRF, NVU dysfunction, BBB breakdown and AD onset and progression. Older and emerging data suggest data suggest the urgent need for increased attention on VRF detection, monitoring, and correction in all the ageing populations in order to reduce the burden of cognitive deterioration. In addition, these observations suggest especially in elderly patients—that a global assessment should be carried out, considering 'classical' VRF and their aggregation [26]. Moreover, special attention should be paid to various pathological conditions, which are particularly frequent in elderly people such as extracranial and intracranial atherosclerosis [107,148], atrial fibrillation [38,149], chronic lung [129] and kidney [150] disease that could have a detrimental effect on cognition. The strongest link between AD and VRF can be observed in the presence of NVUd and BBB breakdown. In these conditions most of the molecular alterations have been observed. However, although several authors underlined this correlation, less is known on neurovascular unit and blood-brain barrier function after intensive correction of VRF, especially at a molecular level. The current treatment strategy for AD progression has currently focused mainly on correcting neurodegenerative aspects, by also using Aβ-directed monoclonal antibodies [151]. In prospective, especially among elderly, multicomorbid patients with AD, a comprehensive, multi-target approach could be comprehensive not only of an early and intensive VRF correction, but also a of a personalized management of comorbidities in later life to reduce the risk of AD onset and to contain the progression of cognitive impairment also at a vascular level.

**Figure 2.** Shared molecular mechanisms linking vascular risk factors, vascular pathology, APOE genotype, neurovascular unit dysfunction, blood-brain barrier dysfunction and Alzheimer's disease onset and progression. Legend: AGE: advanced glycation end products; AT-II: angiotensin receptor 2; CypA: cyclophilin A; HyperC-VMSCs: hyper-contractile phenotype vascular muscular smoot cells; LRP-1: low-density lipoprotein receptor-related protein-1; nAChrs: nicotinic acetylcholine receptors subunit α7; IGF1: insulin growth factor; RAGE: advanced glycation end products receptor; sICAM1: soluble intercellular adhesion molecule-1; sVCAM1: soluble vascular cell adhesion molecule 1.

#### **5. Future Directions**

Midlife VRF correction by drugs [152] or physical activity [153,154] has been associated to a reduction of incident dementia, especially AD, and cognitive deterioration in later life. Antihypertensive drugs have already been shown to reduce both the risk and progression of cognitive decline [155]. Oral antidiabetics and insulin seem able to reduce cognitive impairment in AD [156]. Statin use is not associated with an increased risk of cognitive impairment, and some small observational studies seem to associate this treatment with a potentially favourable role in the setting of AD [157]. Long-term oxygen therapy, also, seems to improve cognition hypoxemic patients affected by AD [128]. Recently, 5-phosphodiesterase inhibitors, such as sildenafil, have been shown to improve neurovascular and neurometabolic function in AD [158,159], and are currently under investigation as repurposed drugs for AD treatment by improving NVU function [160]. Analyses of small groups of subjects show that the correction of extracranial carotid stenosis could be associated to an improvement of NVU dysfunction and a reduction of cognitive decline [161]. However, all these observations are largely based on retrospective or nonrandomized prospective cohort studies. Larger, robust and long-term trials are required to assess the role of neurometabolic and neurovascular treatment to prevent AD onset and progression. At the present time, in conjunction with the evaluation of the possible benefits of the most modern therapies as Aβ directed treatment or brain stimulation techniques, it could be useful to pay attention to the potential role of carotid surgery or drugs that improve neurovascular and neurometabolic balance [162–164].

**Author Contributions:** Conceptualization: L.F., G.V., S.L. and M.S.; methodology: L.F. and G.V.; validation: L.F., G.V., V.Z., E.G., S.L., M.S.; investigation, L.F., G.V., V.Z., E.G.; writing- original draft preparation: L.F., G.V., V.Z., E.G., G.M., S.L., M.S.; writing—review and editing: L.F., G.V., V.Z., E.G.; supervision: G.M., S.L., M.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Opinion* **Plasma Phospho-Tau-181 as a Diagnostic Aid in Alzheimer's Disease**

**Ioanna Tsantzali †, Aikaterini Foska †, Eleni Sideri, Evdokia Routsi, Effrosyni Tsomaka, Dimitrios K. Kitsos, Christina Zompola, Anastasios Bonakis, Sotirios Giannopoulos, Konstantinos I. Voumvourakis, Georgios Tsivgoulis and George P. Paraskevas \***

> 2nd Department of Neurology, School of Medicine, National and Kapodistrian University of Athens, "Attikon" General University Hospital, 12462 Athens, Greece; docjo1989@gmail.com (I.T.); dkfoska@gmail.com (A.F.); elenisideri1985@gmail.com (E.S.); evd.routsi@hotmail.com (E.R.); tsomaka@gmail.com (E.T.); dkitsos@icloud.com (D.K.K.); chriszompola@yahoo.gr (C.Z.); bonakistasos@med.uoa.gr (A.B.); sgiannop@uoi.gr (S.G.); cvoumvou@otenet.gr (K.I.V.); tsivgoulisgiorg@yahoo.gr (G.T.)

**\*** Correspondence: geoprskvs44@gmail.com; Tel.: +30-2105832466

† These authors contributed equally to this work.

**Abstract:** Cerebrospinal fluid (CSF) biomarkers remain the gold standard for fluid-biomarker-based diagnosis of Alzheimer's disease (AD) during life. Plasma biomarkers avoid lumbar puncture and allow repeated sampling. Changes of plasma phospho-tau-181 in AD are of comparable magnitude and seem to parallel the changes in CSF, may occur in preclinical or predementia stages of the disease, and may differentiate AD from other causes of dementia with adequate accuracy. Plasma phospho-tau-181 may offer a useful alternative to CSF phospho-tau determination, but work still has to be done concerning the optimal method of determination with the highest combination of sensitivity and specificity and cost-effect parameters.

**Keywords:** Alzheimer's disease; cerebrospinal fluid; plasma; biomarkers; phospho-tau

#### **1. Introduction**

Cerebrospinal fluid (CSF) levels of amyloid peptide β with 42 amino acids (Aβ42), tau protein phosphorylated at a threonine residue at position 181 (τP-181) and total tau protein (τT) constitute the three established (classical) biomarkers for Alzheimer's disease (AD) [1]. They have been studied extensively during the last two decades and, with estimated sensitivities and specificities approaching or exceeding 90%, they have been incorporated in diagnostic criteria [2] and recommendations [3]. More recently, they have been considered as core features for the definition of AD as an in vivo biological process [4], regardless of the presence or absence of symptoms and their type or severity (mild cognitive impairment or dementia). They have proven to be useful as diagnostic tools for the diagnostic work-up of dementia [5–8] and some movement disorders [9,10] during life. Additional candidate CSF biomarkers, including α-synuclein [11,12] and the transactive response DNA binding protein-43 (TDP-43) [13], are being thoroughly investigated, but work still has to be done before they become established biomarkers.

Over the last few years, blood-based biomarkers for AD, especially the classical Aβ42, τP-181 and τT, have received much attention [14,15]. It has been observed that plasma biomarkers show changes almost simultaneously with CSF biomarkers, following similar trajectories [16]. Although the range of changes for plasma Aβ<sup>42</sup> and τ<sup>T</sup> is lower compared to CSF changes, it is similar for τP-181 [16]. Thus, the later could serve as a surrogate biomarker for AD.

**Citation:** Tsantzali, I.; Foska, A.; Sideri, E.; Routsi, E.; Tsomaka, E.; Kitsos, D.K.; Zompola, C.; Bonakis, A.; Giannopoulos, S.; Voumvourakis, K.I.; et al. Plasma Phospho-Tau-181 as a Diagnostic Aid in Alzheimer's Disease. *Biomedicines* **2022**, *10*, 1879. https://doi.org/10.3390/ biomedicines10081879

Academic Editor: Lorenzo Falsetti

Received: 26 April 2022 Accepted: 27 July 2022 Published: 3 August 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

187

#### **2. Why Plasma Biomarkers? Blood vs. CSF Sampling**

Since the CSF is in close contact with extracellular/interstitial fluid, it is expected to reflect the biochemical changes occurring within the central nervous system with adequate accuracy and thus, it may be preferable to blood [17]. However, CSF sampling requires lumbar puncture (LP). It is a routine procedure in neurological wards, well-tolerated, with a very low incidence of complications, the most frequent being post-LP headache [18]. The use of atraumatic needles reduces the likelihood of headache [18] and, in dementia patients, a headache incidence of <4.5% has been repeatedly reported [19] even with the use of Quincke-type needles [20].

Despite the above, LP is a relatively (minimally) invasive procedure, rarely performed by non-neurologists, requiring hospitalization in some countries or institutions, and it is a source of concern or anxiety for some patients or relatives. Furthermore, the amount of CSF collected is not unlimited. On the other hand, blood sampling is a non-invasive, much more easy-to-perform and acceptable procedure, has no complications, requires no hospitalization, and it can be performed in outpatient wards or in the community, permitting the collection of a larger sample volume which, in turn, facilitates biochemical determination of a wider spectrum of analytes, whilst repeated venipuncture (if necessary for equivocal or conflicting results, for additional biochemical assessments or for follow-up) is far more easy and acceptable than repeated LP.

#### **3. Plasma** τ**P-181 and Alzheimer's Disease**

Plasma τP-181 levels significantly correlate with the cerebrospinal fluid levels [16] and with the Aβ and τ protein load in the cerebral parenchyma, according to studies using Positron Emission Tomography-scan [21] (Table 1). Plasma τP-181 levels are 3.5-fold increased in patients with AD as compared to controls, and this change is greater than the one of any other plasma biomarker [16,21–23]. In asymptomatic individuals and in patients with mild cognitive impairment, increased plasma τP-181 levels predict future transition to Alzheimer's dementia [23], indicating that τP-181 levels may become abnormal during the pre-dementia or even the presymptomatic stage of AD.

From the clinical point of view, plasma τP-181 levels may show a significant diagnostic value, in order to discriminate Alzheimer's disease from other neurodegenerative disorders, with an area under the curve (AUC) reaching 0.94–0.98 [23]. This discriminative value may prove useful for the differential diagnosis of AD from frontotemporal dementia [24], with an AUC at the level of 0.88 [22]. For the discrimination from vascular dementia AUC reaches 0.92, for the discrimination from progressive supranuclear palsy and corticobasal degeneration, AUC reaches 0.88, and for the discrimination from Parkinson disease or multiple system atrophy, AUC may reach 0.82 [25]. Furthermore, plasma τP-181 may identify an additional AD pathology in patients with Lewy body diseases [26]. Based on the above, the diagnostic value of plasma τP-181 may approach that of CSF τP-181 [25], introducing the former as a promising surrogate biomarker for AD.

Plasma levels of τP-181 may also have prognostic value, since they may predict cortical brain atrophy in AD [27], AD pathology at least 8 years prior to pathologic diagnosis [28] and progression to AD dementia even in presymptomatic subjects [29–31]. Indeed, longitudinal changes in plasma levels seem to correlate with the progression of the AD neurodegenerative process [32–35]. Recently, it has been suggested that τP-217 may perform better than τP-181 [31,32,36].

**Table 1.** The major conclusions of the latest studies concerning the role of plasma τP-181 in the diagnosis of Alzheimer's disease.


#### **4. Comparison with Other Plasma Biomarkers**

#### *4.1. Beta Amyloid Levels*

Shin et al. [37] had observed a statistically significant decrease of Aβ<sup>42</sup> in the plasma of patients with Alzheimer's disease, without alteration of Aβ<sup>40</sup> as compared to the control group. However, the Aβ42/Aβ<sup>40</sup> ratio made this difference even more conspicuous. Likewise, Janelidze et al. [38] observed a significant reduction of Aβ<sup>42</sup> and Aβ42/Aβ<sup>40</sup> in plasma, without change of Aβ<sup>40</sup> levels. The findings of two other studies [39,40] were headed towards the same direction, showing statistically significant differences; however, the Aβ42/Aβ<sup>40</sup> ratio (although greater than Aβ<sup>42</sup> level alone) showed a moderate capacity to separate sporadic presenile Alzheimer's disease cases from normal individuals, with an area under the Receiver Operating Characteristics curve reaching 0.76 and a sensitivity and specificity that did not exceed 70% [39], due to an adequate amount of overlapping values between Alzheimer's disease and other groups [38,40]. Nonetheless, through the use of more developed and precise detection techniques (including multiplexed, densely aligned sensor array), the Aβ42/Aβ<sup>40</sup> ratio may have the potential to reach a more compensatory capacity to separate Alzheimer's disease from the control group with an area under the curve 0.925 and a sensitivity and specificity that accedes to 90% [41].

The plasma Aβ42/Aβ<sup>40</sup> ratio seems to predict the amount of cerebral amyloid burden, irrespective of the presence of cognitive deterioration [40,42,43], a fact that could be useful for the early (pre-symptomatic) diagnosis of Alzheimer's disease and the incorporation of pre-symptomatic patients in research for new medications. An abnormal Aβ42/Aβ<sup>40</sup> ratio recognizes the presence of amyloid in cerebral parenchyma with an area under the curve reaching 0.88, and increasing to 0.94 with *APOE4* addition, whilst it recognizes the presence of increased cerebrospinal fluid levels of τP-181 with an area under the curve reaching 0.85 [44]. In addition to these, diminished levels of Aβ<sup>42</sup> are associated with decreased hippocampal volume and a higher risk of Alzheimer's disease occurrence [45].

Not all studies are in agreement with the above data; Feinkohl et al. did not conclude to a statistically significant difference between Aβ42, Aβ<sup>40</sup> and Aβ42/Aβ<sup>40</sup> in the plasma of AD patients [46], while two other studies have found an increased plasma Aβ<sup>42</sup> level as compared to the control group [47,48]. Most of the above studies use more advanced methodologies, like highly sensitive immunoassays, mass spectrometry, Simoa (single molecule array), Luminex xMAP®, *η*´ IMR (immunomagnetic reduction). The use of those techniques is associated to a higher cost, regarding that the low-cost technical infrastructure of Enzyme-linked Immunosorbent Assay (ELISA), which is used for the measurement of classical cerebrospinal fluid biomarkers, cannot generally be reclaimed in the measurement of plasma biomarkers.

#### *4.2. Total Tau Levels and Other Biomarkers*

Despite some initial indications of reduction [49], the level of τ<sup>T</sup> is elevated in the plasma of Alzheimer's disease patients, although not significantly correlated to the cerebrospinal fluid level [50,51]. Nevertheless, an elevation of total tau protein has been observed in other disorders, including frontotemporal dementia [52], thus limiting the

specificity of this biomarker, whose determination demands a Single Molecule Array (Simoa) assay.

Neurofilament light chain (NFL) level is another indicator of axonal damage that presents a significant increase in the plasma of Alzheimer's disease patients [53] and in other neurodegenerative disorders; therefore, it consists of another sensitive but not specific biomarker [15].

The plasma level of α-synuclein, which is increased in Parkinson's disease patients [54], would be considered as a suitable biomarker for the separation between Alzheimer's disease and Lewy body synucleinopathies. However, there are several restrictions that require further research to estimate the diagnostic value of this biomarker [15]; those restrictions are mainly related to the nature of the molecule under determination (monomer or oligomeric protein, total, phosphorylated) and other pre-analytical factors.

#### **5. Some Preanalytical Aspects**

As with CSF collection and handling, pre-analytical aspects in plasma biomarkers determination (including τP-181) may be extremely important for diagnostic accuracy. It seems that K2- or K3-EDTA is the preferable anticoagulant for blood collection [55]. Centrifugation should be performed within <1 h after blood collection (preferably < 30 min), followed by aliquoting in tubes filled to >75% of their volume and storage at −80 ◦C within 1 h from sampling [56–60]. Polypropylene should be the material of collecting and storage tubes. Those techniques and preanalytical protocols have been established by numerous study groups, including the Alzheimer's Biomarkers Standardization Initiative. The conditions and temporal limits under which the blood sample is centrifuged and stored may affect the levels of tau protein and β-amyloid in the sample under test. Other anticoagulants, such as Li-heparin or Na-citrate, can dramatically reduce the levels of tau protein compared to K3-EDTA. In addition, a reduction in β-amyloid levels in a plasma sample separated after 6 hours compared to a freshly separated sample has been noted. Finally, the sequalae of freeze/thaw cycles are shown to minimally affect the levels of plasma biomarkers. It is therefore important that a sample is obtained, separated, and stored under conditions that do not affect the quality of results [56–58].

#### **6. New Disease-Modifying Treatments and Plasma** τ**P-181**

Among the various disease modifying treatments tested for AD, the monoclonal antibody aducanumab has been recently approved by the Food and Drug Administration in the USA (accelerated approval pathway) [60], but not by the EMA, while other monoclonal antibodies are currently under clinical trials. Although these antibodies act by removing brain parenchymal amyloid, they also lead to a decrease of CSF phospho-tau [61]. The latter may be used to monitor the biochemical treatment effect, although there is not necessarily a correlation between the efficacy of the drug and modification of the CSF biomarker levels. Plasma phospho-tau may prove a good alternative, allowing frequent biochemical follow up, more convenient to the patient compared to repeated lumbar punctures and less costly compared to repeated positron emission tomography for amyloid load. Indeed, new data from aducanumab trials indicate a significant decrease of plasma τP-181 following treatment [62,63].

Furthermore, since disease-modifying treatments may be more effective at early stages of the disease, the diagnosis of AD during the preclinical stages by blood (and not CSF) sampling could open new perspectives in wide population screening.

#### **7. Emerging Plasma** τ**P-271**

The phosphorylation of tau proteins can emerge at multiple sites. Recent studies have shown an increased capacity of another phospho-tau protein, τP-271, to discriminate patients between Alzheimer's disease and other dementias. Studies on CSF levels of τP-271 have shown to accurately discriminate amyloid-PET-positive from amyloid-PET-negative patients. Those promising findings have led to studies involving the accuracy of plasma levels of τP-271 in early diagnosis of AD, alone or compared to τP-181. Further studies are needed to determine the possible applications of this new biomarker and its contingent superiority upon τP-181 [32,36,64].

#### **8. Conclusions**

It seems that plasma levels of τP-181 may prove helpful (and probably better than other blood-based biomarkers) in AD diagnosis, and prediction of progression. The additional combined use of other plasma biomarkers may not offer advantage over τP-181 alone. Furthermore, it may prove a useful tool for frequent biochemical follow-up of patients under disease-modifying treatments. Despite the above encouraging data, plasma biomarkers including τP-181 cannot be considered as established biomarkers yet. There are still questions concerning the optimal method of determination, and some recent studies raise doubts about the diagnostic help of τP-181, which may be lower compared to the value of other plasma biomarkers such as the combination of Aβ<sup>42</sup> and neurofilament light chain (NFL). Still, much work has to be done, including extensive real-world studies, testing various combinations of plasma biomarkers and cost-effect analyses.

**Author Contributions:** Conceptualization, I.T., E.S., G.T. and G.P.P.; critical review of the literature, I.T., A.F., E.S., E.R., E.T., D.K.K., C.Z., A.B., S.G., K.I.V., G.T. and G.P.P.; original draft preparation, I.T., A.F., E.S., E.R., E.T., D.K.K., C.Z. and G.P.P.; manuscript review and editing: I.T., A.F., E.S., E.R., E.T., D.K.K., C.Z., A.B., S.G., K.I.V., G.T. and G.P.P.; supervision, A.B., S.G., K.I.V., G.T. and G.P.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** G.P.P. has received fees from Biogen International as a consultant of advisory board. All other authors none.

#### **References**


MDPI St. Alban-Anlage 66 4052 Basel Switzerland www.mdpi.com

*Biomedicines* Editorial Office E-mail: biomedicines@mdpi.com www.mdpi.com/journal/biomedicines

Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Academic Open Access Publishing

www.mdpi.com ISBN 978-3-0365-8439-3