Lecture 11: Cellular Structure of the Brain 3

Question 1

diabetic NEPHROpathy causes

Answer 1

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

Question 2

What causes AD?

Answer 2

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

Question 3

Tau mediated neurodegeneration

Answer 3

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

Question 4

Chronological relationships among AD pathology and clinical symptoms

Answer 4

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

Question 5

Summary of the cytoskeleton

Answer 5

Enables neuron shape
́Intracellular transport
́Organization e.g. protein location in membrane

Question 6

Summary of AD and diabetic neuropathy

Answer 6

Disrupt protein structure – disrupt structure – disrupt function – finally clinical symptoms – maybe too late for recovery

Question 7

Nissl staining

Answer 7

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

Question 8

Cell membrane composition

Answer 8

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

Question 9

Proteins in plasma membrane

Answer 9

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)

Question 10

Synaptic vesicles

Answer 10

not simple phospholipid spheres

contain multiple membrane associated proteins

Question 11

The plasma membrane of a neuron

Answer 11

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

Question 12

Cytoskeleton and associated proteins role

Answer 12

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

Question 13

Membrane proteins are essential components of neuronal structure …

Answer 13

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

Question 14

Technique to identify and locate proteins

Answer 14

Immunohistochemistry (good to look at what is happening at a cellular level with these proteins)

Question 15

IHC to visualise …

Answer 15

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

Question 16

General method of IHC

Answer 16

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

Question 17

Two reasons for using IHC

Answer 17

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)

Question 18

IHC in action - ankG

Answer 18

scaffolding protein - specific for scaffolding protein that is found on the proximal axons also shown between the nodes
proximal axon
nodes

Question 19

IHC in action - Caspr

Answer 19

transmembrane protein
member of neurexin family
cell adhesion
paranoidal protein

Question 20

IHC in action - MAP2

Answer 20

microtubule associated protein
specific for dendrites
absent in most astrocytes

Question 21

IHC in action - PO

Answer 21

myelin protein

not in the nodes of ranvier at all

Question 22

Protein location helps understand

Answer 22

function

Question 23

many membrane proteins …

Answer 23

local or regional

concentrated or diffuse

Question 24

Chemical synapse concept

Answer 24

the synapse is a complex membrane specialisation = membrane proteins

Question 25

Chemical synapse - presynaptic

Answer 25

specialised membrane - active zones with neurotransmitter release

Question 26

Chemical synapse - cleft

Answer 26

matrix fibrous proteins

Question 27

Chemical synapse - postsynaptic

Answer 27

specialised membrane - receptors for neurotransmitters

Question 28

Major requirement for the soma to make proteins for the synapse … presynaptic

Answer 28

• Calcium channels
• Docking proteins
• Transporter proteins
• Reuptake transporter
• Specific vesicle proteins

Question 29

Major requirement for the soma to make proteins for the synapse …postsynaptic

Answer 29

Voltage gated channels

transmitter gated ion channels

Question 30

Synapse proteins must remain in the

Answer 30

correct location

Question 31

NMDA receptor and PSD-95

Answer 31

́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

Question 32

Complex neuronal shapes need to

Answer 32

maintain shape
stay connected to the next neuron
- essential at the synaptic cleft

Question 33

Cleft protein

Answer 33

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

Question 34

Staining nissl substance =

Answer 34

rER - synthesis of proteins - protein is packaged and moved to location required

the neuron makes so many proteins

Question 35

The same proteins are required in different parts of the neuron therefore

Answer 35

there are different isoforms such as potassium channels

Question 36

Basic organisation of synaptic cargo transport in the axon

Answer 36

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

Question 37

Local protein synthesis

Answer 37

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

Question 38

Polyribosomes at postsynaptic membrane because

Answer 38

of local protein synthesis, these are needed to translate the RNA into proteins

Question 39

Flurescence in situ hybridisation (FISH)

Answer 39

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.

Question 40

Why local protein synthesis? in relation to the anatomy of a neuron

Answer 40

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

Question 41

______ different proteins detected at synapses

Answer 41

2500

Question 42

The unique structures and function of neurons

Answer 42

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