Appunti e schemi lezioni Molecular pharmacology e approfondimenti libro

DEVELOPMENTAL STUDIES.

RIGENERATIVE MEDICINE.

Hybrid Enhanced Regenerative Medicine System (HERMES) is an Eu project evolved by Decimo’s group in

order to develop brain organoid to generate functional hippocampal organoid to repair lesion tissue caused

by epilepsy. Epilepsy alters the hippocampus and the aim of the study was to generate the hippocampal

tissue starting from mouse’s neuro stem cells.

HERMES pursues the long-term vision of healing disabling brain disorders by means of brain tissue transplants

→ Establishment of biohybrid neuronics (neural electronics), that is the symbiotic integration of

bioengineered brain tissue, neuromorphic microelectronics and artificial intelligence.

The objectives are:

1. Generation of functional bioengineered hippocampal tissue

2. Integration of neuromorphic engineering and AI with bioengineered hippocampal tissue to yield a

functional intelligent biohybrid construct

3. Repair epileptic networks by means of the intelligent biohybrid construct.

There wasn’t a protocol available. They use mouse ES cells (embryonic stem cells).

Development of mouse Neural Stem Cells (NSCs)-derived organoids:

1- Isolation of embryonic neuronal stem cells from pregnant mouse. They isolate tissue from meninges,

SVZ (subventricular zone) and hippocampus.

[extract embryo from pregnant mouse- extract tissue from brain of embrio].

They dissociate tissue by enzymatic molecules and have a single cells suspension.

2- NSCs culture (neural stem cells) and seeding.

Intrinsical property of stem cells to generate an aggregate and form an organoid.

3- Organoid formation.

There are three phases in organoid formation: the expansion phase with high concentration of

growth factors, the induction phase with low concentration of GF and the differentiation phase with

differentiation factor. 44 Bacci Anna – University of Verona

There is a low variability, an high efficacy and the consistent development, all of these improve the

reproducibility of the method.

They characterised the NSC by transcriptomic and immunofluorescence analysis to confirm stem cells

phenotype (stem cells marker such as vimentin).

They analyse the metabolic phenotype of the cells. During the differentiation the neural stem cells

undergo a swith on the metabolic profile. NSCs use glycolysis in order to produce energy while during

the differentiation the cells use an oxidative phosphorylation that allow to produce energy in a more

efficient way. This switch on metabolic process can be observed in vivo but also in vitro. In fact they look

at the proteins related to oxidative phosphorylation pathway (OxPhos). The maturation process is paired

to an increased mithocondrial content.

Excitatory and inhibitory synapses.

Proper brain development and function require stringent balance between excitatory and inhibitory

synapses formation during neural circuit assembly. Imbalances of E/I function are related to several

neurological diseases including epilepsy.

There is an initial overproduction of excitatory synapses. Then an active-dependent elimination of

excitatory synapses until the synaptic density is almost stable. The inhibitory synapses refine the amount

of excitatory synapses.

We see that the organoids are able to mimic the same biological processes that occur in vivo.

Calcium imaging assay.

It’s the capability of the cells inside the organoids to be functional. They perform calcium imaging assay

by using fluorescent dye that can binf the calcium. Calcium ions is important to generate the calcium

potential. We have the presence of calcium oscillations, that means that the cells inside the organoid are

acrive. We demonstrate the activation of the cells taking advantages of synaptic tracing. The synaptic

tracing is based on viral neuronal tracing, that is the use of a virus to trace neural pathways, providing a

self-replicating tracer.

It exploits the ability of some neurotropic viruses to travel across neuronal pathways and to function as

self-amplifying markers.

Rabies virus is the only viral tracer that is entirely specific, as it propagates exclusively between

connected neurons by strictly unidirectional (retrograde) trans-neuronal transfer, allowing the

identification of neuronal connections.

Cellular composition.

They have different cellular types inside the organoids. Remember that there are two different methods,

the unguided and the guided one.

So they modify the first protocol (that use the same basic growth factors, unguided method) and use

specific GFs in order to prompt the hippocampal phenotype. They use the WNT3a morphogenic factors

that induces organoid with CA3 hippocampal identity. There is a gradient of wnt3a that is low at CA1

level, medium at CA3 level and DG level (Dentate gyrus).

In the region in which is developed the epilepsy they use low concentration of growth factors in order to

prompt CA3 phenotype.

DISEASE MODELLING.

They try to mimic a pathological scenario in the brain organoid. The syndrome is the Allan-Herndon-Dudley

syndrome (AHDS), a severe neurodevelopmental disorder, caused by the absence of a gene of transporter

hormone thyroid T3. This condition affects male children. It causes mental retardation, delay in

neurodevelopment. 45 Bacci Anna – University of Verona

T3 hormone is important in metabolic process of the cells, for their differentiation, migration and also

synapsis establishment. It regulates the neural cells commitment by inducing a switch between neuronal cells

and associated cells. The absence of T3 lead to a hypothyroid condition that impair the neuronal

development.

They use the same organoids protocol, but they modify the protocol to resemble the pathological scenario.

The cells have the receptor of T3 hormone, but they remove the T3 from the medium. The removal of the T3

from the medium has an effect. The maturation of the cells is delayed.

The organoids without the T3 hormone have a downregulation for the genes that are important for neuron

differentiation, neuron action potential and myelination and neuron development. But there is a up-

regulation for genes related to the maintenance of stem cells and the regulation of proliferation.

The pathological models maintain a higher stem cells phenotype.

They confirm the delay in maturation by looking at a specific marker, β3 tubulin and also astrocytic marker

GFAP. Its’ important because in this pathological scenario there is an imbalance between the astrocytic cells

and neurons (that normally is 1:1). The absence of T3 can influence also the commitment of stem cells.

By administrating a drug called NR (nicotinamide riboside), that is a precursor of NAD+ and act as a booster

of mitochondrial metabolism. If the T3 is involved in neurons development delay, by boosting the metabolism

of the cells we can restore the pathological condition.

They perform immunofluorescence analysis and see that after drug administration there is a drastically

decrease in proliferative activity of the cells (the cells have a less stem cells potential) and we can see this

because the vimentin marker is less expressed.

LESSON 15 – 05/12/2023

9 ANTI-INFLAMMATORY DRUGS

The anti-inflammatory drugs are divided into two classes:

1- FANS (NSAID: Non-Steroidal Anti-inflammatory Drugs)

2- Corticosteroids

The aim of these drugs is to decrease the inflammatory response, that can be summerized into these

syntoms:

 Rubor: redness (hyperemia)

 Tumor: tissue swelling

 Calor: heat (local hypothermia, fever)

 Dolor: burning pain

 Function laesa: loss of function or functional impairment

INFLAMMATION is part of the complex biological response of body tissues to harmful stimuli, such as

pathogens, damaged cells and is a protective response involving immune cells, blood vessels, and molecular

mediators.

➢ →

Vessel modification arteriole and venule dilatation, expansion of capillary bed and increase in

blood flow.

➢ Immune cell extravasation (from vessels to damaged tissue)

➢ Immune cell migration and response to chemotactic stimuli.

46 Bacci Anna – University of Verona

Increased permeability of the blood vessels results in an exudation (leakage) of plasma proteins (and fibrin)

and fluid into the tissue (edema), which manifests itself as swelling (tumor).

The nomenclature used to describe inflammation in different tissues employs the tissue name and the suffix

“-itis”.

The acute inflammation involves:

➢ →

Alteration of vascular caliber vasodilation leads to increased blood flow (20-60 μm)

➢ →

Changes of microvasculature increased permeability for plasma proteins and cells

The loss of proteins from plasma leads to edema due to reduce osmotic pressure in the vasculature

and to increase it in the interstitium.

➢ →

Emigration of leukocytes from microcirculation leukocyte activation leads to elimination of

offending agend.

Neutrophils are commonly the first inflammatory cells (6-24 h) recruited to a site of inflammation.

The extravasation of leukocytes is a coordinated event of margination, rolling, adhesion,

transmigration (diapedesis) and migration.

1- In the first phase, leukocytes interact with the endothelium (tethering) and roll on it; this event is

mediated by the interaction between selectins and mucins.

2- During rolling, leukocytes come in contact with chemotactic factors, adsorbed on the endothelium

surface, through specific receptors belonging to the large family of G‐protein‐coupled receptors.

Interaction between these receptors and their appropriate ligands triggers the inside‐out

intracellular signals, leading to the third step responsible for leukocyte migration.

3- The third stage consists in a type of adhesion defined as “firm” that is mediated by the interaction

between integrins of the β1 or β2 subfamilies and their ligands expressed on the endothelial cell

surface, such as ICAM‐1/ICAM‐2 and VCAM‐1. Firm adhesion depends on a strengthening

(activation) of the adhesive capacity of integrins induced in step 2.

Integrin activation includes two distinct modalities: (i) conformational modification of the

heterodimer, resulting in increased affinity and (ii) induction of lateral mobility of the heterodimer,

resulting in cluster formation and valency increase. The valency corresponds to the density of integrin

heterodimers per area of plasma membrane involved in cell adhesion.

4- Only after firm adhesion to the vascular endothelium, leukocytes can pass through the inter-

endothelial junctions and migrate into the interstitium.

Normally the flowing blood erythrocytes are confined to a central axial column, displacing leukocytes towards

the wall of vessels. As blood flows slows in inflammation more white cells assume peripheral position along

the endothelial surface. This process of leukocyte accumulation is called MARGINATION.

The leukocytes follow towards the site of injury in the tissue along a chemical gradient of chemo-attractants

in a process called chemotaxis. Chemo-attractants could be exogenous (bacteri