Episodic memory refers to the conscious recalling of a personal experience and includes information of an event and the context in which the event took place.

This function is the first to be impaired in Alzheimer's disease, a degenerative condition in which pathological changes are found initially in the medial temporal cortex and then spread in the rest of the cortex starting from post-Rolandic areas.

This study aims at examining the mechanisms that enhance memory processes, based on the information acquired by studying hypermnesic subjects. The recent discovery of subjects with an extraordinary ability to remember past events (highly above-average autobiographical memory) and the development of techniques to manipulate memory circuits in rodents provide a unique opportunity to study the mechanisms that determine the facilitation of memories.

As part of a multicenter project funded by the Ministry of Health in collaboration with La Sapienza University of Rome, the University of Perugia and the Santa Lucia Rehabilitation Center in Rome, the aspect of the project carried out at CIMeC (University of Trento) will consist in evaluating the changes induced by rTMS in patients with prodromal Alzheimer's disease, after stimulation of the regions that appear particularly active in hypermnesic subjects.

This project would offer the possibility of accessing an innovative non-invasive, and non-pharmacological treatment.

The specific objectives are:

(i) To evaluate the effectiveness of rTMS applied to hyperactive areas in hypermnesic subjects in enhancing autobiographical memories; (ii) Analyzing the neural correlates of the behavioral variations. The study will allow us to define whether it is possible to improve the recollection of autobiographical events by stimulating the circuits that are more active in hypermnesic subjects.

The results will be crucial to gain a better understanding of the mechanisms through which brain stimulation contributes to the promotion of neuroplasticity and the effects of rTMS in the prodromal stages of Alzheimer's dementia.

METHODS AND PROCEDURES:

Materials and methods of the investigation will be the following:

• repetitive Transcranial Magnetic Stimulation (rTMS)
• A general neuropsychological assessment battery
• Questionnaires and scales
• Electroencephalogram (EEG) recording
• Combined EEG recording and single-pulse TMS (TMS- EEG)

Different rTMS stimulation protocols will be applied:

1. rTMS: this protocol consists in the administration of 1600 pulses at 20 Hz, alternating 2 s of stimulation and 28 seconds of pause. rTMS will be applied over the left dorsolateral prefrontal cortex (left DLPFC). The coil will be placed at the EEG 10-20 International System position of the F3 electrode. The stimulation intensity will be equal to 100% of the motor threshold value at rest. rTMS will be delivered through a Medtronic-Magpro magnetic stimulator and a 70-mm figure-of-eight coil.
2. sham rTMS: as for the protocol that involves the application of sham/placebo stimulation, the rTMS will be administered by applying to the coil a piece of wood or plastic of about 30 mm in thickness, a distance that ensures that the magnetic pulse does not reach the cortex and built so to appear as an integral part of the apparatus.

All stimulation parameters are in accordance with the safety guidelines for the application of rTMS.

Neuropsychological and psychological evaluation:

All patients will undergo a neuropsychological assessment before starting the treatment (baseline=t0=week 1), at the end of the rTMS phase (t1=Week 4), after 3 months (t2=Week 16), and after 6 months (t3=Week 28) from the beginning.

The follow-up evaluation after the end of the treatment will allow verifying long-term effects.

A possible "practice effect" resulting from the frequent and close administration of neuropsychological tests is expected and will be considered in the data analysis, as in all experimental protocols of this type. The practice effect is a common factor for all experimental groups and does not affect the evaluation of the treatment effectiveness.

TMS-EEG data recording: the EEG will be acquired from 64 sintered Ag / AgCl electrodes placed on the scalp in accordance with the international 10-20 system through an EEG acquisition system compatible with TMS. The EEG signal will be acquired with a high-pass filter at 0.01 Hz, a low-pass filter at 1000 Hz, and a sampling frequency of 5000 Hz. The impedance of the electrodes will be kept below 5 kΩ.

The TMS-EEG co-registration will consist of the administration of 180 pulses on the target area stimulated in the application phase of the protocol (left DLPFC), with a random inter-stimulus interval of 2-3 s. rTMS single pulse will be delivered through a Magstim Rapid2 magnetic stimulator (Magstim, Whitland, UK) and a 70-mm figure-of-eight coil.

In the first session, the stimulation hot spot, coil orientation, and TMS intensity will be decided using the rt-TEP software. Specifically, individual stimulation sites (over left DLPFC) will be determined based on the assessed amplitude, morphology, and topography of the average EEG response, following the software developer indications (based on 20-trial average EEG responses to TMS, the investigators will look for a peak-to-peak amplitude near Vpp > 10 µV, in the early 10-50 ms components in the EEG channels located underneath the TMS coil, avoiding the muscle artifacts as much as possible by rotating the coil). During all TMS-EEG recordings, a masking sound, based on the TMS Adaptable Auditory Control -TAAC, will be played via earphones in order to avoid auditory EEG responses evoked by the TMS coil discharge.

The analysis of the data recorded by the combined TMS-EEG will allow an in-depth evaluation of the modulation of cortical activity induced by the different stimulation protocols (real or sham rTMS) and, in particular, will allow investigating cortical excitability and inhibition, connectivity cortico-cortical and the intrinsic ability of the stimulated areas to generate oscillatory activity. This method will provide a unique measure of local cortical activity and effective cortical-cortical connectivity.

STATISTICAL ANALYSIS:

The variables that will be considered for the analysis of neuropsychological and neurophysiological data are:

1. rTMS stimulation effect over time [Prior to treatment (baseline=t0=week 1), at the end of the rTMS phase (t1=Week 4), 3 months post-treatment (t2=Week 16)]
2. type of stimulation protocol (real or sham rTMS); The experimental design will be both "within-subjects" within each variable of interest (for example, investigating the difference between t1 and baseline to evaluate the effect of rTMS stimulation), and "between subjects" regarding the data between the two stimulation protocols (for example, investigating the difference between real and sham rTMS stimulation).

Correlation analyzes will be performed to identify which neuroplastic changes are linked to improved behavioral performance.

Calculation of sample size:

The primary outcome for the calculation of the sample size was defined as the effect of the rTMS protocol compared to rTMS placebo, on the score achieved at the end of the treatment.

Considering an alpha value of 0.5 - 0,8 and a power of 80% - 95%, the investigators estimate that the number of patients to be recruited should be 14 - 16 patients per group, increased to 20 per group to take into account a possible dropout rate of 20% (pAD participants real stimulation vs. sham stimulation).

Techniques provided for data processing: behavioral and neurophysiological data will be analyzed by analysis of variance (ANOVA) and posthoc comparisons (t-test, contrast analysis).

Statistical processing software: Data processing will be performed using Matlab R2020b (The MathWorks, Massachusetts, USA) and in-house scripts based on functions of the open-source toolbox EEGLAB (https://sccn.ucsd.edu/eeglab/), SPSS and/or Statistica software.

Ethical Considerations and Assessment of the Risk/Benefit Ratio:

Expected benefits: Based on the assumptions of the present project, participants who will receive real rTMS should show a clinical response, based on the primary endpoints reported above, superior to the participants assigned to the sham rTMS protocol. The study also should provide indirect scientific/cognitive benefits, in terms of advancing knowledge on the development of treatments with proven efficacy and on the mechanisms underlying Alzheimer's dementia.

Potential Risks:

The risks are represented by the use of electro-medical devices, that, however, have EC authorization for use with patients. In addition, all the appropriate safety measures will be put in place as indicated by the international scientific community for studies with brain stimulation. Although no adverse events are expected, when the international guidelines for the safe administration of TMS are followed, researchers involved in the project are able to cope with any side effects of stimulation. The stimulation parameters take into account the clinical goals and safety of the participants. Regarding EEG procedures, skin redness immediately under the electrodes is possible, following abrasion from the application of the electroconductive gel.

All the procedures foreseen by the research will be carried out by taking care of the patient, adopting all the necessary measures so that no critical issues related to stress or fatigue arise.

Risk/Benefit Ratio:

In the proposed study, the risk/benefit ratio is in favor of benefit, in terms of increased knowledge and expected direct benefit for the participants.

According to the classification of a consensus paper, this protocol is part of class 2 studies, which identify studies with indirect benefits and moderate risks: these are studies with patients where the clinical benefit is speculative, but from which important data could come for the development of effective treatments.

Ethical Considerations:

At the end of the study, patients will not be informed of the treatment protocol to which they have been assigned but about the overall results of the study, receiving a report containing a summary of the results achieved by the project.

Informed Consent:

Participation in the study is on a voluntary basis: each subject will obtain explicit information regarding the nature of the project and will have to sign a written consent before they can be included. Participants can withdraw their consent to participate at any time, without any consequences and without providing explanation.

Data storage and processing:

The data will be protected and anonymized according to the current procedures. All identification data will be encrypted within the database and the subjects will be identified by a code. However, the nature of the study makes it necessary to maintain the data regarding the identification of the participants because of the follow-up evaluations. Access to the database containing the collected data and the results will be restricted to the researchers involved in the project. Sensitive data and all paper data will be kept under lock and key at CERiN (TMS-EEG laboratory). The research manager will also be responsible for the appropriate conservation of these data. As this study involves experimental data, the experimental data will later be published and shared as aggregated data with national and international scientific communities.

Inclusion Criteria:

• right-handed
• meet inclusion criteria related to TMS (attached in the protocol)
• to be able to provide information regarding their cognitive and functional skills, or have a caregiver available who is able to provide the patient information necessary for participation in the study and who is present when signing the patient's informed consent.

pAD Patient Inclusion Criteria:

• Clinical Dementia Rating Scale <2;
• Diagnosis of prodromal Alzheimer's disease (amnesic MCI) according to the diagnostic criteria;
• Neurodegeneration biomarker for AD (FDG-PET or CSF), diagnosis confirmed by clinical follow-up;
• Montreal Cognitive Assessment (MoCA) test overall score within the normal limits (equivalent score of 1);
• Absence of severe vascular distress; Patients will be selected through clinical evaluation (battery of neuropsychological tests at the Neurocognitive Rehabilitation Center - CeRiN)

Exclusion Criteria:

• Patients who are unable to perform the tasks required by the experimental procedure;
• History and/or evidence of any other central nervous system disorder that could be interpreted as a cause of dementia such as structural or developmental abnormality, infectious epilepsy, degenerative or inflammatory/demyelinating diseases of the central nervous system such as Parkinson's disease;
• History of significant psychiatric disease which, in the investigator's judgment, could interfere with study participation.
• History of alcohol or other substance abuse, according to DSM-V criteria, if this could be a contributing factor to dementia;
• Presence of cardiac pacemakers, electronic prostheses, bio-stimulators, metal inserts, or electrodes implanted in the brain or skull, or spine;
• Inability to read and /or understand the written information;
• Dermatitis, eczema, extensive scars on the scalp

Absolute exclusion criteria (criteria for TMS)

• presence of cardiac pacemakers, artificial heart valves and/or bio- stimulators;
• presence of hearing aids located in the middle ear;
• presence of metal inserts on the head and shoulders;

Individual participant's data be available?

Yes, after deidentification.

What data will be shared?

Individual participant's data that underlie the results reported in the article, after deidentification.

Which other documents will be available?

The study protocol.

When will data be available?

Data will be made available with the article publication, and the period for sharing this data will have no foreseen end date.

With whom will the data be shared?

The data will be shared with Investigators whose proposed use of the data has been approved by an independent review committee, for research purposes only.

For what types of analyses will the data be shared?

The only data that will be shared will be those that contribute to achieve the research aims of the proposal, previously approved by an independent ethical review committee.

Data will be made available with the article publication, and the time frame for sharing this data will have no foreseen end-date.

Proposals for data sharing should be directed to costanza.papagno@unitn.it following the ethical committee approval. Data requestors need to sign a data access agreement to gain access. The sharing of the data will be contingent on the approval of an independent ethics committee and the relevance of the requested data as it pertains to the research question.

Triple (Participant, Care Provider, Outcomes Assessor) We will implement a randomized, non-pharmacological study, with a double-blind certified medical device (neither the patient nor the clinician / researcher who will carry out the evaluations will be aware of the group to which the patient has been assigned).