WP1: Set-up of the experimental platform for the study of neural circuits in BO/BA models (DIE) 

DIE will supervise the design step of the technological platform by interfacing with the other partners for feedback related to the diverse area of expertise. WP1 activities outcomes will serve as input for all other work package activities.

Task 1.1 Definition of system requirement (DIE)(M3)

Assessment of the state-of-the-art methodologies and technological solutions

Task 1.2 Reconfigurable devices for optical stimulation, activity and morphodynamic recording (calcium imaging, fluorescence, microscopy facility) (DIE)(M12)

Establishing the requirements for the design of the integrated platform with particular attention to technological solutions that allow re-configurability and minimal invasiveness.

Task 1.3 Microfluidics for the administration of chemical stimuli, for organoid culturing, and sensors integration (chip, pumps, pH and oxygen sensors) (DIE)(M12)

  • integration of all the individual parts investigated in task 1.2
  • interface with the optical sensors, to correlate molecular changes (see WP4) with functional/morphological ones. 

Task 1.4 Management software (interface with microscope, microfluidics, sensors) (DIE)(M12)

Develop a management software will be designed, with the aim to drive in a controlled modality all the parts in the ALTERA platform

WP2: Development of organoid/assembloid models (DBP)  

WP2 activities are devoted to the long-term growth of the organoids and assembloids model: definition of the protocol for the culture, coordination of the growth process of BO/BAs and the optogenetic process to control the activity of individual neurons, recapitulation of the human brain microenvironment. 

Task 2.1 Definition of the protocols for long-term culture of BO/BA and optogenetic manipulation

  • Study of the existing protocols for the organoid growth and the transition from organoid to assembloids.
  • Study of patient-specific organoids built from hiPSCs to model various neurodevelopmental, neurodegenerative, and neuropsychiatric disorders
  • Study of the protocols for optogenetic manipulation of the organoids.

Task 2.2 Organoids cultures

  • Design and implementation of optimal culturing conditions for hiPSCs to growth organoids: a direct method which involves obtaining dorsal and ventral forebrain and an indirect method which will involve the use of the STEMdiff™ Cerebral Organoid Kit (Stemcell Technologies Cat.#08570), a serum-free culture system.
  • Characterization of the organoids by cryosectioning/immunolabeling and/or RT-qPCR.

Task 2.3 Assembloids cultures

  • Pattern ventral and dorsal forebrain, after organoid will be cultured together so that they fuse and generate forebrain assembloids. This enables cortical GABAergic neurons to migrate towards the dorsal forebrain spheroid and then to synaptically integrate into networks with cortical glutamatergic neurons.
  • Perform live monitoring as well as genetic and pharmacological manipulation of cortical interneuron migration.
  • Cretae of assembloids deriving from cerebral organoids and microglial cells. Multi-lineage assembloids will allow the study of morphological and functional aspects of microglia in 3D, including the response to neuronal injury.

WP3: Machine learning methods for BO/BA (DIE)

WP3 activities are related to the design and development of machine learning algorithms for the analysis of the electro-optical outcome from BO/BAs models after stimulation. In particular, image and video analysis methodologies will be designed to perform feature extraction from fluorescence microscopy images and from calcium imaging. ML algorithms will be also exploited to create the control loop to manage passive and active tasks (see WP5 activities) in BO/BAs.  In addition, GNN will be applied to correlate heterogeneous patterns coming from different omics (membrane phenotype, proteomic, electrophysiological activities) with the performance degradation in learning and memory tasks in healthy and unhealthy subjects.  

Task 3.1 Design and development of algorithms for dynamic optical stimulation and extraction of calcium imaging responses (M1-M9, M13-M15, M21-M23)

  • Development of algorithms for the generation of stimulation patterns.
  • Development of algorithms for calcium imaging responses analysis.

Task 3.2 Data and image analysis for the characterization of BO/BA (M10-M36)

Multivariate analysis of the platform output data (sensor responses, fluorescent video sequences, and functional signals) using DL, generative and autoencoders models.

Task 3.3 Machine learning methods for training BO/BA to solve specific tasks (M10-M36)

  • Development of algorithm to support the cognitive tasks, such as feedback in the active learning paradigm, signalling to control the “protagonist’s motion” in the BO/BA player video-game, analysis of the output responses.
  • Reproducibility tests will be performed to investigate the performance of the brain computing over time and in relation to the biological heterogeneity
  • Fault tolerance analysis

Task 3.4 Correlation networks for the study of BO/BA (M13-M36)

  • Correlation of the functional neurotransmitter receptor and the voltage-operated channels in the host (oocyte) of the cell membrane injected (BO cell membrane)  with the A) electro-optical activities of the organoid in the original situ (DIE) and B) with the learning capability of the BO/BA.
  • Graph Neural Networks will be used as model in addition to data reduction approaches (PCA, t-SNE).

Task 3.5 Definition and development of countermeasure strategies to mitigate performance degradation in diseased BO/BA (M13-M36)

Define a methodology to study the effect of drug (treatment) over the cognitive tasks performance, by comparing learning performance of control and diseased BO/BA.

WP4: Molecular phenomics analyses of BO/BA models using micro-transplantation of cellular membranes into Xenopus oocytes (SR)

WP4 will deal with the micro-transplantation of cellular membranes from the BO/BA into the Xenopus oocytes. This procedure will be fundamental to conduct molecular phenomics analysis of the BO/BA models and to provide the whole analysis (WP3) with multivariate characteristics of the BO/BA status. After micro-transplantation, electrophysiological studies will be conducted through voltage clamps methodology in order to measure excitatory and inhibitory neurotransmission in BO/BAs providing a level of maturation and/or of drug response in presence of treatment. Phenotypic changes induced by new senotherapeutic drugs will be also evaluated in aged or diseased BO/BA models.

Task 4.1 Development of dedicated procedures of BO/BA cellular membranes into Xenopus oocytes

  • Cellular membranes are extracted from organoids as described in the literature.
  • Through well-established procedures, cells treated properly are injected in the Xenopus oocytes cytoplasm.

Task 4.2 Electrophysiological studies

From 12 to 48 hr after injection, membrane currents will be recorded from voltage clamped placed in a recording chamber and perfused continuously. This approach will allow a functional study of GABAergic and glutamatergic neurotransmission in the organoids, and of their state of functional maturation after the different treatments.

Task 4.3 Analyses of the molecular and functional phenotype of the BO/BA aging and neurodegenerative models

  • Analysis of the protein phenotype of healthy and diseased BO/BA models.
  • Phenotypic changes induced by new seno-therapeutic drugs will be also evaluated.

WP5: Experimental tests for the study of the learning and memorization capabilities in BO/BA (M1-M36) (DIE, DMS2)

WP5 activities will be related to the design and implementation of the experimental tests for the study of learning and memorization tasks in healthy and in diseased BO/BAs to verify the feasibility of using assembloids models as processor are validated through the WP5 work. In particular, the BO/BA will be connected to an external control software developed in the project to study the possibility to train the OA to perform some tasks, using learning strategies such as reinforcement learning or habituation.

Task 5.1 Protocols for stimulation and recording of the neural activity (M1-M9) 

  • Design a method to identify regions for optical stimulation (through optogenetic, Task 2.1) and regions for electrical activity recording (through calcium imaging). 
  • Sensitivity analysis and Monte Carlo simulations for fault tolerance test.

Task 5.2 Cognitive tests and experiments for input-response evaluation (DMS2) (M4-M27)

  • Perform a passive learning  task where the BO/BA will be initially fed by repeated input stimuli followed by a very different set of stimuli.
  • Study the ability of the BO/BA to habituate to the stimuli and react to a diverse input after habituation convergence. (see Fig. 1, bottom-right).
  • Perform an active learning task, where the BO/BA will be tested against its capacity to learn by reward.
  • Implement a video-game platform in which the protagonist’s motion is controlled by the output of the ‘BO/BA player’.

Task 5.3 Effects of the cooperative activity in BA models (M16-M27)

Understand and quantify differences between single organoids and assembloids (and related subtypes) either in the electrical activities and in the morphodynamic response.

Task 5.4 Application of tests in diseased OA (M22-M36)

Apply tests developed in Task 5.2 to the diseased BO/BA developed in WP6. Results of healthy and diseased input/output response model will be compared for a better insight of the disease mechanisms. 

WP6: Disease modelling (DMS1)

This WP is devoted to the development of the aging and disease model and to the protocols to correctly manage tissue samples from unhealthy patients (see Ethical Committee approval). WP6 will provide fundamental support to all WPs.

Task 6.1 Definition and measurement protocol of BO/BA aging and neurodegenerative modelling

  • Compare different aging protocols, using specific molecules (doxorubricin) or using senescence-associated β-galactosidase (SA-β-gal) staining.
  • Study an Alzheimer’s disease induction procedure  by treating BO/BA with beta amyloid fragment 1-42.
  • Assess beta-amyloid plaque formation through microscopy examination.

Task 6.2 Validation of models with standard techniques

  • Examine oxidative stress and inflammation to assess the molecular mechanisms underlying aging and neurodegeneration. Intracellular and mitochondrial ROS production will be measured using cytofluorimetric analysis.
  • Levels of pro-inflammatory and anti-inflammatory cytokines will be evaluated using a commercial inflammatory array.
  • Approximately 1300 proteins will be examined using a proteome array to identify potential novel molecules involved in the studied disorders.

Task 6.3 Pharmacological tests for reducing the degradation effects for learning and memorization

  • A novel mix of polyphenols with established antioxidant and anti-inflammatory properties, will be used to prevent and/or reduce aging and neurodegeneration.
  • Analyses described in Task 6.2 will be conducted to assess the potential beneficial effects of the mix.

WP7: Project Dissemination (DMS2)

WP7 will be led by DMS2. WP7 activities devoted to the project results dissemination and sharing with the Scientific Community, are articulated into five tasks, Task7.1-Task7.5 and detailed below.

Task 7.1. Dissemination, exploitation, and communication plan. Lead: DMS2. [M01-M36]

  • Development of a plan for the Dissemination, Exploitation and Communication Activities – DEC Plan
  • An ALTERA logo has been already created to promote the visual identity of the project (see Fig.1)

Task 7.2 ALTERA website and social media.  Lead: DIE. [M01-M36].

  • ALTERA’s website will be created by M04 to allow open access to information about the project to anyone with an interest.
  • An account of the project on the main social media.

Task 7.3 Publications. Lead: DIE. [M06-M36]

  • Manage journal and conference publications starting from the second year (4+4) and a few blogs.
  • The PI will guarantee open access to all peer-reviewed scientific publications related to the results obtained within the project and will undertake to deposit the data necessary to validate the results presented in the deposited scientific publications.

Task 7.4 Exploitation activities. Lead: DMS2. [M04-M36]

  • Early identification of potentially exploitable results.
  • Ensure IPR (Intellectual property rights) protection (patent)
  • Evaluate future key markets opportunities and of future funding agencies or potential investors.

Task 7.5 Preparation of final symposium

Organize a workshop highlighting results and implications for policy and practice.

WP8: Project coordination and ethics Lead: DIE. [M06-M36].

This WP will ensure overall coordination of the partner’s activities, internal sharing of the relevant information especially towards personnel acquired for the project and their traineeship. Moreover, the WP will be devoted to assure the quality of the project results and timely delivery of deliverables and milestones scheduled. The WP8 activities, leaded by DIE, will be divided into four tasks, Task8.1-Task8.4 that are listed in Table VIII.

Task 8.1 Scientific, administrative, legal and financial coordination. Lead: DIE. [M01-M36].

A Data Management Plan will be defined at the beginning of the project to properly manage the data and exploit the project results following FAIR and Open science principles. 

Task 8.2 Internal meetings. Lead: DIE. [M01-M36]

Kick-off meeting [M1] followed by yearly project meetings [M16,  M36] will be regularly held online (Teams, Zoom Skype).

Task 8.3. Manage reporting to the Italian Minister of University. Lead: DIE [M01-M36]

Redaction of project reports [M12, M24, M36] including ethics reports when needed.

Task 8.4. Ethics. Lead: DMS1. [M01-M36]

Ensure research ethics and integrity guidance, monitoring, and reporting and guarantee that ALTERA project will be performed with the highest standards of research ethics and integrity, in compliance with EU, national and international regulations and legislations throughout the overall duration of the project also under the guidance of the Host Institution Ethical Committee at the PTV Hospital.