Presently, few studies have evaluated the clinical impact of rTMS in Alzheimer's disease. Though some studies have demonstrated an improvement, there have been conflicting results, as others do not seem to demonstrate beneficial effects. Furthermore, it is the combined application of rTMS with cognitive training that could represent a real turning point in interventions aiming to slow down cognitive decline resulting from AD. Research has shown that the best way to promote the strengthening of a network is to stimulate the area while simultaneously activating the network (i.e. through cognitive training) which supports the specific function of interest.

Recently, there have been new protocols from animal model research showing that "bursts" of repetitive stimulation at a high theta frequency induce synaptic plasticity in a much shorter time period than required by standard rTMS protocols. This type of rTMS stimulation, theta-burst stimulation (TBS), is therefore even more compelling as a therapeutic intervention given that it includes the benefits previously ascribed to other rTMS protocols, but requires less administration time. Furthermore, studies conducted using both types of stimulation suggest that TBS protocols are capable of producing long term effects on cortical excitability that exceed the efficacy of those using standard rTMS protocols.

This project offers patients the possibility of accessing an innovative non-invasive, and non-pharmacological treatment. The goal is to evaluate the clinical efficacy TBS in patients diagnosed with mild cognitive decline (MCI) and AD, verifying if TBS in conjunction with cognitive training produces results better than those obtainable with only one of the two methodologies alone. Patients will be evaluated throughout the full scope of the treatment period, through clinical assessments and neuropsychological evaluations. We will examine neuroplastic changes by investigating the neural correlates underlying improvements using the multimodal imaging technique: TMS-EEG co-registration. A secondary objective will be to define the most effective stimulation protocol, verifying if TBS applied continuously (cTBS) or intermittently (iTBS) produces better behavioral outcomes. 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 efficacy of TBS combined with cognitive training.

METHODS AND PROCEDURES

The materials and methods of investigation proposed will be the following:

• Administration of rTMS in theta burst mode (TBS - intermittent and continuous)
• Administration of computerized cognitive training
• Administration of a battery of neuropsychological tests
• Administration of questionnaires and scales
• Recording of the electroencephalogram (EEG)
• Combination of EEG recording with single-pulse TMS administration (TMS-EEG)

Different TBS stimulation protocols will be applied:

1. Intermittent theta burst stimulation (iTBS): this protocol consists in the administration of 600 pulses divided into blocks of 3 pulses at 50 Hz which are applied at 5 Hz (every 200 ms), alternating 2 s of stimulation with 8 s of pause.
2. Continuous theta burst stimulation (cTBS): this protocol consists in the administration of 600 pulses divided into blocks of 3 pulses at 50 Hz that are applied at 5 Hz (every 200 ms).

In both stimulation protocols, the stimulation intensity will be equal to 80% of the motor threshold value at rest. As for the protocols that involve 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 as to appear as an integral part of the apparatus. All stimulation parameters adopted in this study are in accordance with the safety guidelines for the application of rTMS.

Cognitive rehabilitation protocol For patients assigned to the protocol including the application of cognitive training (TBS + CT; CT), the training will be administered immediately following the application of rTMS (both in the real intermittent or continuous condition, and placebo) and will last 25 minutes. Cognitive training will be administered through dedicated software that uses an individualized adaptive methodology based on the participant's performance.

The rehabilitation of memory functions, associated with the stimulation of the left DLPFC, will be focused on learning face-name associations. The face-name association training involves an acquisition phase in which patients are shown faces with an associated name and are asked to memorize these associations. The reproduction phase follows the training phase, in which the patient's task will consist in finding the face that corresponds to the associated name. Based on the patient's performance, the level of difficulty is modulated by increasing or decreasing the number of associations to be memorized and possibly, for higher difficulty levels, by adding other information to be memorized (for example, a profession).

Neuropsychological and psychological evaluation All patients will undergo a neuropsychological assessment before the start of treatment (t0), at the end of the intensive treatment phase (t1), at the end of the maintenance phase (t2), and after 3 (t3) and 5 months (t4) from the start of treatment (Figure 1).

The evaluation of the patients after some time (follow-up) from the end of the treatment will allow for the verification of long-term effects.

A possible "practice effect" resulting from the repeated and quick 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 factor common to all experimental groups and does not affect the evaluation of the efficacy of the treatment, the primary objective of the study.

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 with a sampling frequency of 5000 Hz. The impedance of the electrodes will be kept below 5 kΩ. The TMS-EEG co-registration will consist in the administration of 120 pulses on the target area stimulated in the application phase of the protocol (right DLPFC or left DLPFC) at an intensity equal to 110% of the motor threshold at rest with a random frequency between 0.2- 0.4 Hz. The analysis of the data recorded by the combination of TMS-EEG will allow an in-depth evaluation of the modulations of cortical activity induced by the different treatment protocols and, in particular, will allow the investigation of cortical excitability and inhibition, connectivity cortico-cortical and the intrinsic ability of the stimulated areas to generate oscillatory activity. This method will be able to provide a unique measure of local cortical activity and effective cortical-cortical connectivity .

The characterization and organization of brain networks will be investigated using graph theory.

Statistical Analysis:

The variables that will be considered for the analysis of clinical, neuropsychological and neurophysiological data are: a) treatment effect over time (t0, t1, t2, t3, t4); b) type of treatment protocol (combination of TBS and cognitive training, isolated application of TBS, isolated application of cognitive training); c) type of stimulation protocol (cTBS, iTBS) and d) clinical group (AD or MCI). 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 intensive treatment), and "between subjects" regarding the data between the different treatment protocols (for example, investigating the difference between combination of TBS and cognitive training and isolated application of TBS, to evaluate which protocol produces the greatest benefits), between different stimulation protocols (for example, investigating the difference between cTBS and iTBS to evaluate which protocol produces greater benefits) and between clinical conditions (to assess whether the same treatment leads to differences in the achieved benefit between the two groups of patients, AD and MCI).

Calculation of sample size:

The primary outcome for the calculation of the sample size was defined as the effect of the cTBS protocol and the iTBS protocol (both in combination with cognitive training) compared to the treatment involving the combination of cognitive training with TBS placebo, and the one which involves only the TBS protocols applied in isolation, on the MMSE score achieved at the end of the treatment. Based on the results of a previous rTMS study on a sample of AD patients (Ahmed et al. 2012), we estimate that at the end of our treatment there will be an improvement in the MMSE score of at least 3 points (SD of change = 2.95) for protocols that involve the combination of cognitive training and real TBS, and of 0.2 points (SD of change = 2.7) for the treatment that involves the combination of cognitive training and placebo TBS.Considering an alpha value of 0.05 and a power of 0.80, we estimate that the number of patients to be recruited should be 16 patients per group, increased to 20 per group to take into account a possible dropout rate of 20%.

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

Statistical processing software Data processing will be performed with BrainVision Analyzer, SPSS and/or Statistica software.

Ethical Considerations and Assessment of the Risk/Benefit Ratio:

Expected benefits Based on the assumptions of the present project, patients who will receive the treatment that involves the combined application of rTMS and cognitive training should show a clinical response, based on the primary endpoints reported above, better than the patients assigned to the protocols in which rTMS and cognitive training are applied in isolation. The research also provides 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 equipment, however, all of which have EC authorization for use with patients. For this protocol, all appropriate safety measures will be put in place for studies with brain stimulation as indicated by the international scientific community. Although, following the international guidelines for the safe administration of TMS no adverse events are expected, it should be noted that the environment in which the research will take place and the personnel involved are able to cope with any side effects of stimulation. The stimulation parameters chosen take into account the clinical goals and safety of the participants. With regard to EEG procedures, redness of the skin 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 paying particular attention to the patient involved, adopting all the necessary measures so that no critical issues related to stress or fatigue arise.

Risk/Benefit Ratio:

It is believed that in the proposed study program, 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 will be informed 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.

Data storage and processing:

The data will be protected and anonymized according to the procedures in force. All data regarding identification will be encrypted within the database and the subjects will be identified only with a code. However, the nature of the study makes it necessary to preserve the data regarding the identification of the participants because the project provides for follow-up evaluations. Access to the database containing the collected data and the results will be restricted to the researchers involved with the project. Sensitive data and all paper data will be kept under lock and key at the various facilities. 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 with national and international scientific communities.

Inclusion Criteria:

General Inclusion Criteria (must be met for both AD and MCI group):

• right-handed
• normal or corrected to normal vision through lenses
• meet inclusion criteria related to TMS
• 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.

AD Patient Inclusion Criteria:

• Mini Mental State Examination (MMSE) score ≥ 16;
• Stable intake of cholinesterase inhibitors for at least 3 months before the start of the protocol

MCI Patient Inclusion Criteria:

• Diagnosis of mild cognitive impairment
• Mini Mental State Examination (MMSE) score ≥ 24;

Patients will be selected through clinical evaluation (battery of neuropsychological tests at the Neurocognitive Rehabilitation Center (CeRiN) and, in accordance with the APSS, a CSF and PET examination will be performed as well as a further finalized neuropsychological evaluation for research.

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, epilepsy, infectious disease, degenerative or inflammatory/demyelinating diseases of the central nervous system such as Parkinson's disease or Fronto-temporal dementia
• 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, or recent or previous history of drug abuse if this could be a contributing factor to dementia
• Ongoing treatments with drugs that contain / intake of the following substances: imipramine, amitriptyline, doxepin, nortriptyline, maprotiline, chlorpromazine, clozapine, foscarnet, ganciclovir, ritonavir, amphetamines, cocaine, (MDMA, ecstasy), phencyclidine (PCP, angel dust), gamma-hydroxybutyrate acid (GHB), theophylline
• Presence of cardiac pacemakers, electronic prostheses, bio-stimulators, metal inserts or electrodes implanted in the brain or skull or spine.

Absolute exclusion criteria (Criteria for TMS), which in detail are:

• presence of cardiac pace-makers, 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 data be available (including data dictionaries) after de-identification.

The data in particular that will be shared are individual participant data that underlie the results reported in the published article, after de-identification (text, tables, figures, and appendices).

The data will be available immediately following article publication, and the time frame for sharing this data will have no foreseen end-date.

The data will be shared with Investigators whose proposed use of the data has been approved by an independent review committee ("learned intermediary") identified for this purpose, for research purposes only.

The only data will be shared will be those which aid in achieving the research aims of the approved proposal, previously approved by the above mentioned independent ethical review committee.

Proposals for data sharing should be directed to carlo.miniussi@unitn.it following ethical committee approval. To gain access, data requestors will need to sign a data access agreement. The sharing of the data will be contingent on the above-mentioned criteria, namely, the approval of an independent ethics committee and the relevance of the requested data as it pertains to the research question.

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).