Lecture 11: Cellular Structure of the Brain 3 Flashcards
diabetic NEPHROpathy causes
main alterations induced by diabetes in axons and cytoskeletal components
NEPHROpathy
With the increase in glucose, lots of proteins get glycated so we get tubulin that gets glycated in the microtubules which disrupts the microtubules function which therefore disrupts the function of axonal transport
Actin becomes glycalted which causes an affect on the plasma membrane, actin stabilises proteins and it also is important for transport as well therefore not having actin there has a huge toll on axonal transport as well
With the neuro filaments, the structural loss of these (the stabilisers of the house framework), if they are affected by the lack of mRNA then we have structural change in the axon as well
So not many microtubules, a lot less actin microfilaments and have a decrease in axon caliber (actual axon can be shorter and potentially wider because neuro filament maintains width of axon), because there is no nice plasma membrane which would be keeping the membrane proteins in place the speed and conduction of our axons are also changing as well, nerve regeneration changes too
Being able to manage the glucose levels at a good level can have a beneficial role in preventing this illness itself
What causes AD?
Alzheimer’s disease causes neurons start to retract and they start to die, they become rem a nets of neurons called tangles, also have plaques made up of amyloid beta - these are the pathological hallmarks of Alzheimer’s disease
Beta amyloid clumps together and form plaques and they are toxic and influence all the cells around it by killing them, beta amyloid caused by protein on the plasma membrane being cleaved differently
When looking inside the neuron - microtubules, motor proteins are taking down organelles down towards the axon which is the standard procedure in axonal transport, in Alzheimer’s disease what happens is that the tau proteins which stabilised the microtubules (microtubules associated proteins) become hyper phosphorylated which causes the disintegration of microtubules and the tau forms a tangle (clumps together) and the neuron dies
Tau mediated neurodegeneration
1 - hyperphosphorylation of tau which leads to disassembly of microtubules and axonal transport insufficiency
2 - tau aggregates in axons/dendrites - congest axonal transport
3 - tau pathology is transmitted synaptically
Hyper phosphorylated tau and the disintegration of the microtubules, tau attaches and then the microtubules just falls apart, because tau is clumping together and forming these tangles it actually stops/congests axonal transport so even if that tau hasn’t hyper phosphorylated that particular microtubule the presence of these tau aggregates in the axon can also cause inhibition of axonal transport
Interestingly now what it looks like is that the tau molecule is hyper phosphorylated and it can transfer its toxicity from one cell to another and it seems to spread through the brain so the hypothesis of tau is that it moves from one cell to another and we do know that there is a standard progression of the illness in that you start in one localised area and then it spreads so tau can potentially be transferred form one cell to another
Chronological relationships among AD pathology and clinical symptoms
amyloid beta and tau have pathological changes during the preclinical stages of AD i.e. before the clinical symptoms of cognitive and functional impairment come on
tau has as much important as amyloid beta because it could be happening quite a lot earlier, research says that tau may be as equally important as amyloid beta
Summary of the cytoskeleton
Enables neuron shape
́Intracellular transport
́Organization e.g. protein location in membrane
Summary of AD and diabetic neuropathy
Disrupt protein structure – disrupt structure – disrupt function – finally clinical symptoms – maybe too late for recovery
Nissl staining
Nissl was found to be selective for RNA and so represents large stacks of rough endoplasmic reticulum
dark in the cytoplasm because it is staining rER because it is producing large amounts of proteins, lots of proteins in the soma
Cell membrane composition
Phospholipid bilayer
hydrophobic ends in middle zone
hydrophilic ends –intra/extracellular compartment
not permeable to ions
hydrophobic = non-polar hydrophilic = polar
need protein to allow function
Proteins in plasma membrane
Proteins in plasma membrane
Composition of protein (task specific)
may span the membrane, occupy part (external/internal)
a simple channel
a complex folded structure
composed of subunits (same/different)
Synaptic vesicles
not simple phospholipid spheres
contain multiple membrane associated proteins
The plasma membrane of a neuron
Neuron
́Part of a network with specific connections
́To do this is polarized – different parts
́Proteins essential for this - specificity
Also
́A neuron has a huge membrane area
́Cell soma 20μm diameter
́Epithelial cell 20μm soma – membrane area 1,256μm2 ́neuron – 20μm soma membrane area 250,000μm2
́200x greater membrane area(than an epithelial cell)
́To make these proteins – lots of RER
́Specialised vesicles
~10,000 spines per neuron
Cytoskeleton and associated proteins role
important role in axonal transport/actin anchoring role
majority of these proteins are made in the cell body and this is why axonal transport is so important as it transports proteins where they are supposed to be
Membrane proteins are essential components of neuronal structure …
are essential for neuronal function
Proteins need to be in the ‘right’ place - Need to be sitting in the right place in the membrane, need to be held by scaffolding proteins (you have your transmitting protein and then you have you scaffolding protein and then you have your actin mesh underneath which is stabilising that protein because the plasma membrane is quite fluid and labile therefore need to stabilise these proteins)
any change may result in dysfunction
membrane proteins are held in black by scaffolding proteins
Technique to identify and locate proteins
Immunohistochemistry (good to look at what is happening at a cellular level with these proteins)
IHC to visualise …
visible product on LM
Fluorescent molecule
electron dense product in TEM
using IHC we can identify multiple proteins in multiple locations -> precision of function and a range of functions
General method of IHC
protein of interest called the antigen on a rat neuron - primary antibody to the antigen, made in a ‘goat’ - secondary antibody, antibody to the species goat - visual label or fluorescent molecule to visualise
secondary antibody is specific to the goat because it is the only goat thing that is amongst the rat neurons
Two reasons for using IHC
Two reasons why we use this technique…
Firstly is because protein of interest could be tucked in betweeen spines and dendrites etc and so you do not want a large complex trying to find it because it could be quite hard to get to, secondly is because as we do this we can actually amplify the signal so you can add more labels to it or make the fluorescent molecule bigger and bigger (something might only be in a small concentration but by doing this you can see it better)
IHC in action - ankG
scaffolding protein - specific for scaffolding protein that is found on the proximal axons also shown between the nodes
proximal axon
nodes
IHC in action - Caspr
transmembrane protein
member of neurexin family
cell adhesion
paranoidal protein
IHC in action - MAP2
microtubule associated protein
specific for dendrites
absent in most astrocytes
IHC in action - PO
myelin protein
not in the nodes of ranvier at all
Protein location helps understand
function
many membrane proteins …
local or regional
concentrated or diffuse
Chemical synapse concept
the synapse is a complex membrane specialisation = membrane proteins
Chemical synapse - presynaptic
specialised membrane - active zones with neurotransmitter release
Chemical synapse - cleft
matrix fibrous proteins
Chemical synapse - postsynaptic
specialised membrane - receptors for neurotransmitters
Major requirement for the soma to make proteins for the synapse … presynaptic
- Calcium channels
- Docking proteins
- Transporter proteins
- Reuptake transporter
- Specific vesicle proteins
Major requirement for the soma to make proteins for the synapse …postsynaptic
Voltage gated channels
transmitter gated ion channels
Synapse proteins must remain in the
correct location
NMDA receptor and PSD-95
́NMDA receptor- transmitter gated ion channel
Complex protein - post synaptic density
́PSD-95 - scaffold protein, under the plasma membrane for the clustering of receptors, ion channels etc.
post synaptic density have the NMDA receptor which is a protein molecule and then there is a scaffold protein which is PSD-95 and this stabiliser is connected onto the actin skeleton via another protein
Complex neuronal shapes need to
maintain shape
stay connected to the next neuron
- essential at the synaptic cleft
Cleft protein
Trans-synaptic proteins
Mind the gap (MTG) - binding protein - holds synapse together, parallel, lined correctly to function correctly
Secreted presynaptically
cell adhesion molecules - interact with molecules on surface of adjacent cell
Staining nissl substance =
rER - synthesis of proteins - protein is packaged and moved to location required
the neuron makes so many proteins
The same proteins are required in different parts of the neuron therefore
there are different isoforms such as potassium channels
Basic organisation of synaptic cargo transport in the axon
Motor proteins carrying the mitochondria to where it needs to go and mitochondria is important for energy which is why we need lots of it there and also need the transport of protein such as all of the receptors that are important for neurotransmission, so what happens here is that the mitochondria is transported along the microtubule by a specific motor protein and then at the neck of the spine since the microtubule doesn’t continue any further and the organelle is transferred on to the actin cytoskeleton and then taken to the place where it is required also happens in the presynaptic terminal (both), in the presynaptic terminal the microtubules are also carrying the proteins of interest required from the soma and carrying it to where it is needed at the site, the think is is that it is a long way from the soma to the dendrites and it is also a long way from the soma to the axon so there is actually a reserve somewhere so that it is not dependent on the soma to make all the proteins and now we know that there is local protein synthesis at the synapses
Local protein synthesis
local synthesis of proteins at the postsynaptic membrane, local command to change the synapses very quickly - want a new protein - need to transport mRNA to polyribosomes
Local protein synthesis at synapses, have protein of interest that is transported by motor proteins along microtubule and then hops onto actin skeleton and gets delivered to the synapse but what we have found out now is that it is not just proteins or vesicles being transported we also know that mRNA can be transported as well
mRNA is bound to an mRNA binding protein which is really called a repressed mRNA, repressed mRNA gets attached onto to the motor protein like other organelles, repressed mRNA is taken out via kinesin motor and hops onto the actin network and attaches to a myosin motor protein and is transported to where it is and then this rna is just sitting there waiting to be used and when it is required repressed rna can be translated to form a protein
Polyribosomes at postsynaptic membrane because
of local protein synthesis, these are needed to translate the RNA into proteins
Flurescence in situ hybridisation (FISH)
FISH - complimentary sequence to the sequence of interest, fluorescent molecule so that you can detect the fluorescent mRNA in our cells
Fluorescence in situ hybridization (FISH) is a laboratory technique for detecting and locating a specific DNA sequence on a chromosome.
molecule beacon - quince so that it only emits light at a specific point
Molecular beacons are hairpin-shaped molecules with an internally quenched fluorophore whose fluorescence is restored when they bind to a target nucleic acid sequence. This is a novel non-radioactive method for detecting specific sequences of nucleic acids.
Why local protein synthesis? in relation to the anatomy of a neuron
Neuron is very big in comparison to an epithelial cell, every micrometer has 4 synapses, lots of requirement and need for local protein synthesis just by the number of synapses input and output, the morphological complexity of our neurons and the volume of the neurons and their capacity for plasticity (ability to change) are the reasons we have local protein synthesis, need it as well as axonal transport because it takes some time to get to the soma to the axon, local protein synthesis is about getting local processing of information within minutes rather than hours and days, local protein synthesis as well as axonal transport
local protein synthesis at dendritic spines, presynaptic terminals and axons, growth cones
______ different proteins detected at synapses
2500
The unique structures and function of neurons
Importance of membrane proteins in structure/function ́Variability of proteins
́Huge protein demand – RER dominant soma - proteins ́Proteins can be changed
́You can visualize location of proteins and RNA
́Also local protein synthesis – synapse for rapid response
