CSB332 Lecture 4 Notes

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Cell and Systems Biology
Francis Bambico

CSB332 Lecture 4 Slide 7 - Narcoleptic dog o Orexin, also called hypocretin, is the neuropeptide that is affected  Released by the hypothalamus  Controls arousal and wakefulness  Mutations in orexin receptors results in narcolepsy o Narcolepsy is associated with deficits in the release of orexin or hypocretin  Low levels of orexin - Dad carrying daughter to safety o Endorphins are associated with the ability to modulate pain  Family of opioid neurotransmitters (e.g., enkephalin)  Similar to exogenous substances (e.g., morphine, vicodin, oxycontin) o Enhanced release of substance P in the spinal cord or in the trigeminal areas of the brain stem is associated with increased pain Slide 8 - Enkephalin is released by the substantia gelatinosa interneurons and inhibits the transmission of signals mediating pain o Co-transmission of glutamate and substance P  Glutamate and substance P are released into the synapse by DRG sensory neurons  Activates glutamate receptors and neurokinin receptors in the dorsal horn ascending commissural interneuron  Relays pain information into the thalamus  Stimulates secondary sensory neurons (or third order sensory neurons) in the thalamus into the somatosensory cortex for final pain processing o To prevent that, you may want to stimulate the substantia gelatinosa interneurons  When stimulated, substantia gelatinosa interneurons release enkephalins  Enkephalins binds opioid receptors that are expressed in the axon terminals  inhibits release of substance P and glutamate  prevents the further transmission of pain o There are two fiber types that compose the axon fibers  A-delta fibers are myelinated  C-type fibers are unmyelinated • Interacting with substantia gelatinosa interneurons o What conditions allow for the overstimulation of substantia gelatinosa interneurons? Under emergency type events. Other types of neurotransmitters are stimulated and are able to activate the substantia gelatinosa interneurons. The substantia gelatinosa interneurons contain many receptors (e.g., serotonin receptors, dopamine receptors, glutamate receptors). The picture on slide 7 is associated with norepinephrine rush. There is an emergency type of situation, the locus coeruleus gets over activated and releases norepinephrine (associated with alertness, wakefulness, readiness to move), norepinephrine neurons synapse with substantia gelatinosa, norepinephrine activate receptors of substantia gelatinosa interneurons and stimulate the substantia gelatinosa interneurons, increases the release of substance P, inhibits pain sensations. o There is a type of interneuron that crosses the other side of the spinal cord before sending their axons into the thalamus and the somatosensory cortex. These axons compose a pathway called the spinothalamic tract. - The spinal cord is composed of gray matter. The gray matter is a butterfly-shaped structure that is composed of cell bodies. There is a dorsal horn and a ventral horn of the gray matter. o The dorsal horn gray matter contains the [Lissauer’s] tract that is composed of axons that are being projected by the sensory neurons. The sensory neurons are afferent neurons that relay information from the external world into the CNS, and synapse with interneurons in the spinal cord. o There is another type of spinal neurons called lower motor neurons in the ventral horn of the spinal cord. These are efferent neurons because they project their axons from the CNS into the periphery (e.g., the muscles). These neurons are responsible for innervating the muscles in response to a brain process. Slide 9 - How can the axons find their way to the proper synapse? Slide 10 - In a developing brain nervous system, the end terminals of the axon would look like a stump-like structure, called the axon growth cone. The axon growth cone is located in the terminal of a growing axon that allows the axon to navigate through the environment. - Filopodia contains aggregates of microtubules, F-actin, and G-actins. o Microtubules terminate at the central core of the axon growth cone. There are spike-like structures emanating from the central core called filopodia. There is a thin membrane sheet of tissue called lamellipodium surrounding the filopodia. - Both filopodia and lamellipodium are important for navigation into the extracellular environment. Slide 12 - This is a two-photon image. Filopodia are composed of F-actin and its monomeric counterpart, G-actin. G-actin is the monomer. When G-actin monomers aggregate, they form F-actin. Slide 13 - Stationary phase o Microtubules are located in the central core of the growth cone. Actin filaments are located anterior to the microtubules. The depolymerisation and polymerization process of G-actin and F-actin is cyclic. - Protrusive growth o The actin filament is immobilized by attachment to a substrate. The immobilization leads to the build-up of G-actin in the anterior part of the anterior filament, allowing for the extension of the actin filaments. o The actin filaments are extended forward, so the microtubules can readily move forward via myosin motor proteins. This allows for the forward movement of the axon growth cone. Slide 14 - How can the axon growth cone find its post-synaptic target? o For the growth cones to be guided to their respective destinations and targets, molecules need to guide the axon growth cones to respective targets. o E.g., The guidance of growth cones of commissural interneurons in the developing spinal cord.  Presynaptic axon growth has to be in physical contact with postsynaptic cell. CAM requires the physical contact of the presynaptic growth cone and the postsynaptic cell.  Some molecules are secreted, called extracellular matrix adhesion molecules. The presynaptic growth cone should still be in close contact with the postsynaptic cell. For these to be able to guide the growth cone, they have to be released within a small area. The movement that is produced is very minimal.  The interactions produced by CAM and ECM only allow very minimal movement of the growth cone (less than 100 microns). CAM can interact with the same species of molecules (e.g., DCC can interact with DCC, TAG-1 can interact with TAG-1, DCC can interact with TAG-1).  Commissural interneuron axon fibres need to travel further. After it crosses the midline, the commissural axon travels hundreds of millimeters in order to go to its target in the thalamus. CAM and ECM are not sufficient to guide the growth cone to travel far distances to the thalamus. It has to interact with another set of molecules called diffusible factors or chemotactic molecules, which are released by glial cells. - Cell adhesion molecules (CAM) o Membrane-bound glycoproteins o Located on the membrane of the developing axon growth cone o Allow slight movement of the growth cone (e.g., few hundreds of micrometers)  Not sufficient to take the growth cone to the thalamus - Extracellular matrix adhesion molecules (ECM) o Secreted glycoproteins o Secreted by glial cells - Chemotactic molecules o Diffusible gradients o Released by glial cells and neurons o Interact with CAMs that are bound on the axon growth cone o Growth cone is able to navigate long distances by computing the concentration gradient of the diffusible factors Slide 15 - The growth cone from the dorsal region has to traverse down to the ventral area of the spinal cord. Netrin-1 (a chemotactic molecule) is released by floor plate cells (e.g., immature glial cells) located in the ventral area. Netrin-1 is the main midline attractant molecule, which attracts the growth cone to travel down into the ventral area close to the midline. The receptor for netrin-1 is DCC (a CAM that is expressed on the membrane of a developing axon). The growth cone is able to compute the concentration gradient produced by the diffusion of netrin-1via DCC to travel down the midline of the spinal cord into the ventral area. - It has to maintain its adherence to the floor plate glial cells and be directed to cross the midline. TAG-1 (a CAM that is expressed in the membrane of the developing axon growth cone) and NrCAM (a CAM that is expressed in the membrane glial cells) will bind each other once the growth cone gets in physical contact with the floor plate cells. They have to be in contact with each other before they can bind. The interaction of TAG-1 and NrCAM propel the growth cone to cross the midline. Once the growth cone crosses the midline, then the expression of TAG-1 in the growth cone is genetically suppressed, then the synthesis of Robo (a CAM expressed on the axon growth cone membrane) is induced. - Robo interacts with Slit (a chemorepulsive molecule that is released by the floor plate glial cells). Slit interacts with the receptor Robo, which would repel the axon growth cone and drive away the axon growth cone to move laterally. Then the growth cone needs to synapse with neurons in the thalamus. The growth cone needs to travel anteriorly. There is another set of signaling molecules that are involved (e.g., Wnt, Fz, Shh, Bmp). - Motor neuron cell bodies are located in the ventral horn. The axons have to leave the spinal cord, and it shouldn’t get attracted by molecules to the midline. A combination of the signals released by the floor plate, Slit and netrin-1, and the receptor, unc5, repel the axons growth cones of the motor neurons out of the spinal cord. Slide 16 - The explant is grown in laminin. Laminin is applied in the medium, which is an ECM. Laminin receptors are located in the growth cone. The culture is filled with laminin, and laminin binds its corresponding receptors that are expressed in the cell membrane of the growth cones, called integrins. The binding allows for minimal movement and navigating. Slide 17 - The same explant or neuron is plated with diffusible factors (e.g., netrin, slit). Slide 18 - When the presynaptic growth cone approaches the target postsynaptic cell, then there is a courtship. The courtship involves many other adhesion molecules and signaling molecules. The synapse formation takes place when the presynaptic growth cone approaches the postsynaptic cell. For example, the axons of the commissural interneurons reach the thalamus and approach the postsynaptic thalamic neurons. - This is an illustration depicting synaptogenesis in the NMJ. o (A)  A primitive motor neuron, which originates in the ventral horn of the spinal cord, and via interaction with unc5, netrin, and slit gradients, the growth cones of the motor neurons are repelled and pushed away from the spinal cord. The axons are pushed away from the spinal cord and motor neural axons synapse with muscle fibres.  The MN slow approaches the muscle fibre. When it approaches the muscle fibre, the synapse would have to be forged by a molecule that is released in the presynaptic axonal growth cone and another molecule that has to be released from the muscle fibre. o (B)  When the MN approaches the muscle fibre, it releases a specific ECM molecule called agrin. Agrin travels to the extracellular fluid and binds to a receptor that is already present in the membrane of the muscle fibre. The receptor for agrin is called LRP. When LRP is activated, it will change the conformation of another receptor called MuSK. MuSK is a tyrosine kinase. Tyrosine kinase is an enzyme that is able to cleave ATP and transfer a phosphate group of ATP to another protein. MuSK is activated and is able to transfer a phosphate group from ATP (in the cytosol) to itself. MuSK autophosphorylates in response to its activated by LRP-4. MuSK phosphorylates two other proteins within the muscle fibre: RATL and two other intracellular tyrosine kinases (Src and Fyn). RATL phosphorylates rapsyn. Src and Fyn phosphorylates cholinergic receptors.  For the postsynaptic cell to be well developed, it has to assemble specializations. What are the specializations of a mature postsynaptic cell (e.g., to be a postsynaptic target for a presynaptic axon)? • Receptors have to aggregate or cluster in the area close to the presynaptic terminal. The receptors have to cluster in a small area that is opposite to the presynaptic axon. o RATL mobilize rapsyn proteins, which cluster cholinergic receptors in a small region of the muscle fibre that is opposite to the presynaptic axon terminal. • Must contain GPCR • Must contain intracellular proteins that are responsible for the intracellular cascade associated with the activation of the receptors • Must contain enzymes that will break up the neurotransmitters (e.g., acetylcholinesterase is synthesized in the postsynaptic fibre and found in the synapse)  The signal involved in initiation postsynaptic specialization is agrin.  The presynaptic axon growth cone is not fully developed, so it also has to be developed. • Must have a molecule that would transduce biochemical changes within the presynaptic axon to initiate the development of
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