Module 1: Cellular and Development Neurobiology Flashcards
1
Q
What is the difference between CNS and PNS Neurons?
A
Peripheral Neurons have their cell bodies outside the brain or spinal cord
Central Neurons have their cell bodies inside the brain or spinal cord
Some central neurons have axons that extend extensively into the peripheral nervous system (e.g. motor neurons). Because their CELL bodies are in the CNS they are no doubt central neurons
Some PNS neurons have axons that project into the CNS (e.g. dorsal root ganglion sensory neurones). Because their cell bodies are in the PNS, they are nevertheless peripheral neurons.
2
Q
What are the features of neurons and their diversity?
A
- Basic structural and functional unit of the nervous system
- information processing unit
- responsible for the generation and conduction of electrical signals
- communicate with one another via chemicals released at the synapse
- enormous heterogeneity
- supported by neuroglia, comprising of several different cell types
3
Q
What are the cellular features of a neuron?
A
- large nucleus
- prominent nucleolus
- abundant rough ER
- well developed Golgi
- abundant mitochondria
- highly organised cytoskeleton
- HIGHLY ORGANISED METABOLICALLY ACTIVE CELL
4
Q
What is the classification of neurons based on their morphology?
A
- Unipolar
- Multipolar
- Bipolar
- Pseudounipolar
5
Q
What are the types of neurons? (Morphology)
A
Multipolar
- Motor neuron
- Pyramidal neuron
- Purkinje cell
Bipolar
- Retinal neuron
- Olfactory neuron
Unipolar
- Touch and Pain Sensory neuron
Anaxonic neuron
- Amacrine cell
6
Q
What is the classification of neurons based on their function?
A
- Sensory neurons (afferent neurons) (e.g. in skin, ear, tongue) transmit information about the surrounding environment to the central nervous system neurons.
- Motor neurons (efferent neurons) innervate muscle and stimulate muscle contraction.
- Interneurons are CNS neurons that communicate with other CNS neurons
7
Q
What is the organisation of neuronal circuits?
A
- Divergence: a mechanism for spreading stimulation to multiple neurons or neuronal pools in the CNS.
- Convergence: a mechanism providing input to a single neuron from multiple sources
- Serial processing: neurons or pools work in a consequential manner
- Parallel processing: individual neurons or neuronal pools process information simultaneously
- Reverberation: a feedback mechanism that may be excitatory or inhibitory
8
Q
Why must the axonal compartment be kept separate from the somato-dendritic compartment?
A
- Certain proteins, complexes and cargo must be delivered to particular regions of the neuron
9
Q
What are somato-dendritic specific components?
A
- Microtubule stabilising protein: MAP2B
- All the neurotransmitter receptors, post-synaptic density (PSD) scaffolding and signalling proteins required at post-synapse
10
Q
What are some features of dendrites?
A
- major area of reception of incoming information
- spread from cell body and branch frequently
- greatly increase the surface area of a neuron
- location of branches determines the origin of incoming signals
- often covered in protrusions called spines
11
Q
What are axon specific components?
A
- Neurofilaments, only present in axons where they are important for strength
- Microtubules
- Microtubule stabilising protein tau
- Cell adhesion molecules L1 (NgCAM), TAG-1
- All the neurotransmitters, growth factor receptors, SNARE complexes etc, required at the pre-synapse
12
Q
What are some features of axons?
A
- conduct impulses away from the cell body
- emerge at the axon hillock
- usually one per cell
- may branch after leaving cell body and at target
- prominent microtubules and neurofilaments
13
Q
What are some structural features of axons?
A
- axons contain abundant intermediate filaments and microtubules
- axons can be myelinated or unmyelinated
- axonal membrane of myelinated fibre only exposed at node of Ranvier
- has cable properties to maintain constant speed of conduction
- large numbers of mitochondria required to maintain action potential
14
Q
How is the axonal membrane organised in specific domains?
A
Soma - Axon initial segment - Myelinated axon - Axon terminal
Within myelinated axon:
Juxtaparanode - paranode - node - paranode - juxtaparanode
15
Q
What are the different types of synapses?
A
- Synapses with other neurons
- Neuromuscular
- Neuroglandular
16
Q
What are some structural features of a synapse?
A
- Synaptic vesicles packaged in the Golgi and shipped by fast anterograde transport
- specialised mechanisms for association of synaptic vesicles with the plasma membrane (active zone)
- voltage gated Ca2+ channels enriched
- abundant mitochondria - around 45% of total energy consumption is required for ion pumping and synaptic transmission - sensitivity to O2 deprivation
17
Q
What is the organisation of synapses?
A
- neurons receive multiple synaptic input
- neurons use a diversity of neurotransmitters, inhibitory and excitatory
- most synapses are axo-dendritic which are usually excitatory
- axo-somatic and axo-axonic synapses are usually inhibitory or modulatory
- competing inputs are integrated in the postsynaptic neuron (neuronal integration)
- axon potential generated at the axon hillock
18
Q
What are some features of synaptic diversity?
A
- Some neurotransmitters REDUCE THE LIKELIHOOD OF ACTION POTENTIAL FIRING by the target neuron (Decrease depolarisation e.g. GABA, dopamine)
- Some neurotransmitters INCREASE THE LIKELIHOOD OF ACTION POTENTIAL FIRING by the target neuron (Increase depolarisation e.g. glutamate, acetycholine)
19
Q
What are some features of synaptic diversity? (cont)…
A
- Each synapse has multiple receptors e.g. 11 glutamate receptor proteins, 3 GABA receptors, 5 dopamine receptors, 14 5HT receptors etc
20
Q
What is the structure of Astrocytes?
A
- 40% cells in human brain
- Many thin processes around capillaries (left), synapses, surface of neurons.
- Cytoskeleton - GFAP (Glial fibrillary acidic protein), microtubules, actin.
- Expression of vimentin
- glycogen granules
- Rough ER, Golgi apparatus
- Large nucleus
- Light cytoplasm
21
Q
What may astrocytes come into contact with?
A
- Capillary endothelial cells (round BV)
- Neuronal cell bodies
- Initial segment
- Axon (at nodes)
- Dendrities
- Synapses
- Ependymal cells lining the ventricles
- Pial surface of the brain
22
Q
What are different types of astrocytes?
A
- Fibrous
- Protoplasmic
23
Q
What are functions of astrocytes?
A
Modulation of synaptic function:
synaptogenesis and synaptic pruning
- Metabolic function: production of cholesterol
- Maintenance of the BBB
- Regulation of blood flow
- Neuroprotective: release of growth factors
- Recycling of neurotransmitters
- Production of anti-inflammatory cytokines
24
Q
What are some dysfunctions of astrocytes?
A
- Increased glutamate cytotoxicity
- Increased levels of Calcium and ATP release
- Increased production of nitric oxide
- Accumulation of superoxide dismutase
- Formation of glial scar
25
Why is the BBB essential?
It is essential for controlling entry of molecules and ions from the general circulation into the nervous system
26
What forms the BBB?
- A tight association of brain capillary endothelial cells
- Astrocytic endfeet enwrap enthodelial cells providing a gateway of nutrients etc into the CNS and the removal of metabolites out of the CNS
27
How is AQP4 involved in the BBB?
- Regulation of extracellular space volume
- Potassium buffering
- CSF circulation
- interstitial fluid resorption
- metabolic waste clearance
- Ca2+ signalling
28
Astrocytes express a large array of:
- transport proteins for nutrients (e.g. glucose) and metabolites
- neurotransmitters (e.g. GABA and glutamate)
- neurotransmitter receptors
- neuronal trophic factors (e.g. GDNF, FGF, IGF)
29
How are astrocytes associated with synapses?
- Well placed to interact with released transmitters and to respond to synaptic activation
- well placed to modulate neuronal function via transmitter removal and also release
- regulation of synaptic pruning
30
How are astrocytes involved in neurotransmitter recycling?
- Astrocytic processes surrounding the synapse endocytose the unbound GABA or glutamate and converts GABA to Glu then Glu to Gln to be re-supplied to the neuron.
31
How is astrocytes involved with potassium?
- Astrocytes act as a potassium reservoir, maintaining a good supply of potassium,
but keeping it away from the extracellular space until required
- Remove potassium ions from extracellular space – essential for neuronal function, since extracellular potassium
concentration must be kept low.
32
What happens when local K+ concentrations are too high?
- Potassium is redistributed from these regions to other regions, by transport through the astrocytic network via gap junctions. Thus they play a role in “spatial buffering” of
potassium
33
What are the metabolic functions of astrocytes?
```
- Provide energy to neurons in the form
of lactate produced from glycogen
specifically in astrocytes.
Astrocytes are the only cells in the brain
that store glycogen (hence the large
glycogen granules in the cytoplasm).
Enough to last for 10s of minutes.
```
```
- Astrocytes produce cholesterol:
Brain is the most cholesterol-rich organ,
it contains about 20% of the whole
body’s cholesterol. Adult neurons
essentially rely on astrocyte for
cholesterol providing.
```
- Synthesize Apolipoprotein E
34
What constitutes the structural support of astrocytes?
```
- Radial Glia
Span the cortex radially
from the inner to outer
layers.
Are important for neuronal
migration.
```
35
What is an example of a Radial Glia?
- the Bergmann glia in the
cerebellum and the Muller
cells in the retina.
36
What common progenitor cell do fibrous astrocytes and oligodendrocytes originate from?
O-2A progenitor cell
37
What is a key factor released from type-1 astrocytes to induce O-2A cells proliferation?
Platelet-derived growth factor (PDGF)
38
Which PDGF receptor must O-2A and oligodendroctye progenitor cells express?
PDGFaa receptor
39
Where is PDGFaR expressed?
PDGFαR is expressed in the ventral half of the spinal cord, initially only two cells wide but the cells subsequently appear to proliferate and disseminate throughout the spinal cord
40
Where do the earliest oligodendrocyte precursors in the spinal cord originate?
In a restricted area in the ventricular zone during a brief time around E14
41
Where do migrating PDGFaR+ cells originate in the embryonic brain?
In a localised germinal zone in the ventral diencephalon
42
What are markers for oligodendrocytes?
PDGFαR: Oligodendrocyte precursor
Olig2: General marker for oligodendrocyte lineage. A basic helix-loop-helix transcription factor
NG2: Expressed by OPC. A type of chondroitin sulfate proteoglycan
O4: Marker for both immature and mature oligodendrocytes. An unidentified sulfated glycolipid antigen called POA (Proligodendrocyte Antigen)
GSTn: Marker for mature oligodendrocytes. n-type Glutathione S-transferase
MAG, CNPase, MBP, CREB: Markers for myelin
43
What does Olig1 do?
Olig1 plays a minor and non-essential role in oligodendrocyte development
44
What does Olig2 do?
Olig2 is essential for the development of motor neurons and oligodendrocytic lineages
45
What happens before oligodendrocyte progenitor formation?
- Shortly after motor neuron production ceases, proneural genes Ngn1 & 2 are downregulated in the ventral neuroepithelium.
- Extinction of neurogenins from the olig2 expressing domains precedes oligodendrocyte progenitor formation
46
What are the two morphogens?
- Sonic hedgehog (SHH) produced by floor plate, ventral to dorsal gradient
- Bone morphogenetic protein 4 (BMP4) produced by roof plate, dorsal to ventral gradient
47
What is the difference between SHH and BMP4?
SHH promotes oligodendrocyte development will BMP4 inhibits
48
What genes/proteins are responsible for moving OPCs along axons
- Netrin-1 and Sema3a
| - The cells responsive to netrin 1 and those responsive to Sema3a are different cell types
49
Which protein acts as a chemoattractant and which one acts as a chemorepellent for oligodendrocytes from chick spinal cord?
a) PDGF
b) Netrin-1
a) chemoattractant
| b) chemorepellent
50
How do activated neurons promote myelination by mature oligodendrocytes?
- ATP released by activated neurons stimulates astrocytes and promotes Leukaemia Inhibitory Factor (LIF) which in turn promotes myelination by mature oligodendrocytes
- Electrical stimulation promotes LIF expression in astrocytes
51
What are the roles of oligodendrocytes?
- myelination is vital to the correct functioning of the nervous system
- correct ratio of oligodendrocytes to axons is essential during development
- dysmyelination during development usually leads to mental retardation and/or death (leukodystrophies/leukoencephalopathies)
- energy efficient and space saving
- steps in the development of oligodendrocytes are well defined
52
What does Neuregulin do?
- Promotes oligodendrocyte survival in the developing rat nerve
Neuron, Vol. 28, 81–90, 2000
53
Describe the initiation of myelination
1) Target innervation and electrical activity in axon cause the release of ATP.
2) ATP stimulates astrocytes to produce and secrete LIF (leukaemia inhibitory factor)
3) Axons can directly stimulate oligodendrocytes through cell adhesion
molecules (Neuregulin, NCAM, L1)
4) Inhibitory molecules are Downregulated (Notch, PSA-NCAM, Lingo-1)
5) Multiple axo-glial signals result in ensheathment
54
How does myelin form?
- Oligodendrocyte process contacts axon
- Leading process tucks under and extends around axon in multiple wraps
- myelin compaction
- cytoplasm filled areas are inner and outer loops
55
What does myelination do to Na+ channels?
Causes them to cluster at Nodes of Ranvier
56
What's the difference between Oligodendrocytes and Schwann cells in terms of myelination?
Myelinating Schwann cells have a 1:1 relationship with an axon segment, whereas oligodendrocytes produce multiple myelin sheaths
57
What is Caspr?
- Contactin associated protein
| - A membrane protein found in the neuronal membrane in the paranodal section of the axon in myelinated neurons
58
What is Saltatory conduction?
- The myelin sheath is like the insulation around the wire. Therefore, the local current jumps to the next unmyelinated area (node of Ranvier).
59
What are the different types of nerve fibres?
A-fibre:
- alpha (motor), beta (touch), gamma (touch), delta (heat, touch, nociception)
B-fibre (autonomic)
C-fibre (nociception, autonomic)
60
What is the Schwann cell lineage in rodent spinal nerves?
Neural crest --> Schwann cell precursor --> Immature schwann cell precursor --> Promyelin Schwann cell --> Myelin Schwann cell
Neural crest --> Schwann cell precursor --> Immature Schwann cell precursor --> Nonmyelin (Remak) Schwann cell (through radial sorting)
61
What is essential for both myelination and Remak bundles formation?
Neuregulin-1 type III
62
What are the implications of not having Neuregulin-1 receptor ErbB2/B3?
Sensory and motor neurones are not myelinated and and 80% die by E18
63
What does Neuregulin-1 type III do?
controls the decision to myelinate and extent of myelination
64
How does Neuregulin-1 type III control myelination?
- the amount of NRG on the axon detected by SCs determines how many axons they segregate.
- when NRG1 type III is overexpressed, previously unmyelinated axons become myelinated, eg sympathetic fibres.
- the axon diameter/myelin thickness relationship is encoded by the amount of axonal NRG1 type III.
65
What is Krox-20?
A transcription factor that is selectively expressed in myelinating cells
66
What is Nav1.8?
A sodium ion channel subtype localised at discrete positions along the neurites in sensory neurons and colocalises with lipid rafts
67
What are lipid rafts?
Lipid rafts are cholesterol and sphingolipid
| -enriched microdomains of the membrane
68
What is the biological role of lipid rafts?
- Caveolae and Lipid rafts have been implicated in signal transduction, and endocytosis
- Act as a platform on the membrane where proteins are sorted and functionally localised
69
What are lipid rafts' role in the nervous system?
- Proteins involved in cell adhesion and axon guidance
- Proteins involved in synaptic activity (NMDA, AMPA receptor)
- NGF and GDNF receptors
70
What is an essential feature for transmitting the chemical stimuli to cell soma of culture DRG neurons?
TTX-resistance
71
What is Charcot-Marie-Tooth Disease?
- A group of varied inherited disorders of the peripheral nervous system characterized by progressive loss of muscle tissue and touch sensation across various parts of the body
- The most commonly inherited neurological disorder, and incurable
- More than 30 genes have been reported to be linked to this disease including Peripheral myelin protein 22 (PMP22)
72
What is Guillain-Barre Syndrome?
- Infection induced nerve inflammation
- A rapid-onset muscle weakness caused by the immune system damaging the peripheral nervous system
- Caused by antibody against gangliosides, which damages myelin sheeth
73
What are causes of axonal damage?
- Disease (MS, infection, infarction, ischemia etc)
- Drug induced
- Injury
74
What is the pathophysiology and consequences of axonal damage?
- Degeneration vs Regeneration
Reinnervation
CNS vs PNS
- Genetic changes in neurons, glial cells and macrophages
75
How do axonal varicosities, spheroids and end bulbs occur?
- Focal blockages of axonal transport, which may occur preferentially at nodes of Ranvier, lead to accumulation of organelles and dis-organized cytoskeleton in axonal varicosities
- Amyloid precursor protein (APP) also ac-cumulates in these. The swellings increase in size to form axonal spheroids. The axon re-mains continuous, but as the spheroids grow, axonal transport may become increasingly impaired (APP: consequences or cause?)
- The block of axonal transport is of sufficient magnitude to trigger Wallerian degeneration (WD) of the distal axon. An end bulb remains on the proximal axon stump. End bulbs also form when axons are transected directly
76
What is the sequence of cellular events triggered by transection of a myelinated axon? (PNS)
- Local injury to axon producing dissolution of myelin sheaths and degeneration of axoplasm distally, and sealing of the tip of the proximal stump
- The glial cell tube is invaded by macrophages which breach the basal lamina. Glial cells distal to the injury proliferate. Axon sprouts emerge from the proximal stump
- Axon sprouts elongate within the glial tube and associate with the glial cells therein
- Daughter glial cells remyelinate the regrowing axon
77
What is the "Dying back" hypothesis of axon degeneration?
A focal block of axonal transport can trigger Wallerian degeneration
78
What is Wallerian degeneration?
Process of degeneration of the axon distal to a site of transection (Augustus Waller, 1850)
- Dissociation of myelin sheaths of distal axons
- Removal of the myelin and axonal debris by macrophages
- Removal of myelin-associated molecules, e.g. MAG, which
would otherwise inhibit axonal growth
- During this process, proximal axons remain intact
- Glial cells distal to the injury proliferate
- Glial cells synthesize growth factors
- Secreted growth factors attract axonal sprouts from the proximal stump
79
What is the repair process of peripheral nerve transection of skeletal muscle?
- Macrophages clear Schwann cell debris
- Proximal nerve terminals send sprouts towards proliferated/reassembles Schwann cell tubes. Misrouting can occur if two ends are well apart
- Some sprouts make it into the tubes and reinnervate the muscle
80
What is the repair process of spinal cord injury (WD)?
- Damage occurs to spinal cord axons
- Brain macrophages (microglia) begin to clear debris and astrocytes begin to enlarge and proliferate
- Astrocytes synthesize GFAP (Glial fibrillary acidic protein) and a glial scar is formed, blocking axonal growth
81
What are proteins are upregulated by denervated Schwann cells?
- p75NTR (low affinity neurotrophin receptor)
- NGF, BDNF (growth factors)
- GFAP (glial fibrillary acidic protein)
- Glial maturation factor-b
- c-erbB2, c-erbB3 (neu receptors – responsible for Schwann cell proliferation)
- LP (regulates the morphology/adhesion of Schwann cells)
- N-CAM, L1 etc (cell adhesion molecules)
netrin-1 (responsible for growth cone and axon guidance)
- IL-1a, IL-6, TNF-a, TGF-b1 (cytokines – induce expression of LIF)
- Erythropoeitin (a neuroprotective cytokine)
- Connexin 46 (a gap-junction protein – facilitates the rapid diffusion of signalling molecules between cells downstream of the site of injury)
- MMP-2, MMP-9 (matrix metalloproteinase – remodelling of BNB)
- Pax3, c-jun (transcription factors – regulate Schwann cell de- and redifferentiation)
82
What are proteins are downregulated by denervated Schwann cells?
- MAG, MBP, P0, PMP22, periaxin (myelin-associated protein)
- Connexin 32, E-cadherin (important for maintaining the complex structural organisation)
- Krox-20 (transcription factor – regulates Schwann cell de- and redifferentiation)
83
Describe the metastatic cascade
1) Cells in primary tumour undergo Epithelial-Mesenchymal Transition (EMT) and acquire invasive properties
2) Degradation of basement membrane and ECM remodelling by proteinases facilitates tumour cell invasion
3) Tumour cells invade surrounding tissues as single cells or collectively
4) Intravasation of tumour cells into newly formed vessels within or nearby tumour
5) Tumour cells are transported through vasculature and arrest in a capillary bed where they extravasate
6) Extravasated tumor cells can stay dormant for years.
7) Eventually, some disseminated cells grow out to a secondary tumor / macrometastasis, requiring ongoing
ECM remodeling and angiogenesis
8) Cells outside their normal microenvironment undergo anoikis
(“detachment-induced apoptosis”). Anoikis could hamper metastasis at several steps of the cascade, as
indicated in the scheme.
84
What are the challenges to brain tumour treatment?
* Blood Brain Barrier (BBB)
* Resistance
* Heterogeneity
* Diffusing nature of some brain tumours
* Invasiveness of local delivery into the brain
* Lack of efficacy through the systemic circulation
* Limitations attributable to medicines themselves
85
What are obstacles to chemotherapy?
- Only lipid-soluble (lipohilic) low molecular weight drugs (<600 Da) have the
potential to cross the BBB.
- Many chemotherapy drugs often fail to treat brain tumours.
- Moreover, p-glycoprotein function as an efflux pump to pump anticancer drugs out of the cells.
- High systemic levels of a drug is often required to achieve therapeutic concentrations with the tumour, but limited by systemic toxicity.
86
Why does doxorubicin accumulate poorly in the brain when given systematically?
Low lipophilicity and high molecular weight prevent penetration across the BBB.
87
What is the most used chemotherapeutic drug to treat brain tumours?
Temolozomide (TMZ)
88
What is a solution for improving systematic chemotherapeutic drug delivery to the brain? (1/2)
Systemic Delivery to the brain:
- Osmotic opening of the BBB by intracranial infusion
of hypertonic arabinose or mannitol is mediated by dilation of the cerebral blood vessels and shrinkage of the endothelial cells, causing widening of the endothelial tight junctions.
89
What is a solution for improving systematic chemotherapeutic drug delivery to the brain? (2/2)
Local Delivery to the brain:
- Convection enhanced delivery (CED) for local delivery of chemotherapeutic drugs. Continuous injection of the drug solution via a catheter under positive pressure.
- Polymeric vesicles: Local delivery technology for chemotherapy in
the brain. GliadelR was approved by FDA in 1996, and is now in commercial use.
90
What is a Glioblastoma?
- Glioblastoma multiforme: a World Health Organization (WHO) grade IV glioma, is the most common and lethal primary malignancy of the CNS.
- Despite aggressive treatment including surgical resection, chemo- and radiotherapy, median survival time for patients is only 14.6 months.
- GBM is an incurable disease that almost invariably leads to neurological failure and death.
- Due to high degree of invasiveness, radical resection of the primary tumour mass is not curative.
- Infiltrating tumour cells remain in the surrounding brain and lead to recurrence.
- Glioma Stem Cells.
91
What is an example of novel therapeutic approaches to Glioblastoma?
Molecularly Targeted Therapies
92
Anti-angiogenic therapies: Why is the vasculature of a solid tumour an attractive target for intervention?
- Endothelial cells lining the blood vessels are directly accessible to drugs via the systemic circulation.
- It is estimated that up of 100 tumour cells are sustained by a single endothelial cell.
- Endothelial cells are genetically more stable and are therefore unlikely to acquire resistance to therapy.
- The Tumour endothelium expresses specific markers, that are absent or barely detectable in the normal quiescent blood vessels (zip codes, vascular targets).
93
What is angiogenesis?
The growth of new blood vessels
94
At what diameter must tumours grow to before they must generate their own blood supply?
2 - 3 mm
95
How do tumours form new blood vessels?
Tumors secrete a number of growth factors and proteolytic enzymes into the interstitium that act on endothelial cells and basement membranes to remodel existing vessels and stimulate the release of endothelial progenitor stem cells from the bone marrow to form new vessels.
96
What are vascular targets in angiogenic blood vessels?
Endothelial cells & Tumours:
- VEGF and VEGFR Receptors
- alphav beta3 & alphav beta5 Integrins
- MMP-2 & MMP-9
- EGFR
Perivascular cells (Pericytes):
- Aminopeptidases APA & APN
- NG2 Proteoglycan
- PDGFRs
97
What are some examples of VEGF inhibitors?
- Aflibercept
| - Bevacizumab
98
What are some examples of VEGF receptor inhibitors?
- AEE788
- Cedirnab
- Sorafenib
- Sunitinib
99
What are some examples of antiangiogenic agents which are currently in clinical trials for Adult Malignant Glioma?
- VEGF inhibitors
- VEGF receptor inhibitors
- Alternative angiogenesis pathway inhibitors
- Endothelial cell migration inhibitors
100
What are potential limitations of antiangiogenic agents and other therapeutic medicines?
- Short half-lives
- Rapid renal clearance
- High chance of non specific accumulation
- Inefficient accumulation at the diseased site
- Severe side effects at high doses
- Poor tissue and cellular membrane permeability in vivo requiring cell transduction systems when the molecular target is intracellular
- Tumour resistance due to GBM heterogeneity
101
What are key factors to take in account for chemotherapeutic drugs?
- Stability
- Half-life
- Rapid clearance
- Side effects
102
What proportion of the dose of chemotherapeutic drugs that accumulates in normal organs reach the tumour?
around 5 - 10%
103
What is the benefit of targeted drug delivery?
- Increase EFFICACY and SAFETY
- Can allow doctors to transport medicine to an
exact location in the body, and control the rate of drug release
104
What are some examples of drug delivery systems?
- Polymers
- Liposomes
- Viruses
105
What are some challenges to drug delivery systems?
* Inefficacy of delivery through the Systemic Route
* Targeting is challenging- Getting the right ratio: Vector/Drug/Targeting agent.
* Reproducibility in quality and quantity sufficient for pharmaceutical applications are challenging problems.
* Preparative conditions for the vesicles of different formulations differ markedly from one targeted particle to another.
* Safety concerns
* Cost
106
What can gene delivery be used for?
- hormonal therapy, vaccine, cytotoxic peptides and
| proteins, cytokine therapy, cancer immunotherapy.
107
What are some drawbacks to gene delivery?
- Stability
- Half-life
- Side-effects
108
What is Cancer Gene Therapy?
- the delivery of therapeutic genes to tumours such as cytotoxic genes, tumour suppressor genes, antivascular genes, anti-angiogenic genes etc…
- Gene therapy was originally intended to treat congenital diseases
- Today more than 70% of ongoing gene therapy clinical trials are designed to treat cancer
109
What are some of the different types of viruses used as gene therapy vectors?
- Retroviruses and Lentiviruses
- Adenoviruses (Ad)
- Adeno-associated viruses (AAV)
- Herpes simplex viruses (HSV)
110
Why have eukaryotic virus vectors not been successful in gene therapy?
* Undesired uptake by the liver
* Uptake by the reticuloendothelial system (RES)
* Broad tropism for normal tissues causing toxicity
* Poor penetration into Tumor tissues
* Presence of antiviral neutralizing antibodies
111
What are bacteriophages?
- Viruses that infect only bacteria
- Do not infect mammalian or plant cells
- Human are routinely exposed to bacteriophage at high levels through food and water without adverse effects
112
When was the first phage therapy application to treat dysentery?
1919
113
Why is there a rekindled interest in phage therapy?
Due to the rapid and alarming emergency of antibiotic-resistant superbugs
114
What are some examples of bacteriophage groups?
- Tailed: bacteriophage Lambda (Double-stranded DNA)
- Filamentous: bacteriophage M13 (Circular Single-stranded DNA)
880 nm long and 6.6 nm in diameter
115
What are some advantages to bacteriophages as cancer gene therapy vectors?
- Safe, administered to humans in antibiotic therapy.
- No need to ablate any native tropism.
- ligand-directed targeting is well established.
- Cost-effective production in bacteria & at high titers.
116
How does RGD4C/AAVP have potential for non-invasive IV treatment of brain tumours?
- Specific to brain tumours
RGD peptide binds to αVβ3 integrin overexpressed on the surface of tumour-derived endothelial cells and tumour cells in brain tumours
- Intravenous delivery to the brain
Non invasive
Diffusing tumours
- BBB Cross Ability
117
What are some novel therapeutic strategies against glioblastoma?
- Improving Conventional Therapies against
Glioblastoma
- Combination of Chemo- and radiotherapy with
Gene Therapy
118
What is a medulloblastoma?
Most common brain tumour in children
119
What are microglia?
CNS Macrophages
120
What are the three different types of receptors on microglia?
- Physical receptors e.g. CD200R & CD45
- Receptors to soluble mediators released by neurones e.g. CX3CR1 detects CX3CL1
- Neurotransmitter receptors e.g. dopamine receptors and adrenoceptors.
These monitor the health of the neuron.
121
What genes are related to early onset AD?
Amyloid related genes (APP etc)
122
What genes are related to late onset AD?
Immune function and lipid handling genes
123
What proportion of brain cells are microglia?
5-10%
124
Which area has a higher density of microglia?
e.g. Midbrain
125
What are the other myeloid cells in the CNS?
- Perivascular macrophages
- Choroid plexus macrophages
- Meningeal macrophages
126
Are microglial mesodermal or neuroectodermal?
Mesodermal
127
How are microglial unlike other resident myeloid cells?
- Derived from yolk sac myeloid cells
| - Self renewing population (doesn't need myeloid progenitor cells)
128
How can you cause microglial activation without affecting the BBB?
Facial axotomy
129
What is the normal macrophage function of microglia?
Surveillance / activation / immune response
- Mount immune response
- Attract other immune cells
- Phagocytose pathogens
- Present antigen
- Injury resolution
- Phagocytosis of apopotic cells and debris
- Tissue repair
130
What is the CNS specific function of microglia?
Dynamic interaction with synapses
131
Explain microglial synaptic pruning
- Prenatally, neurones have more synapses than in the mature brain
- They are pruned by microglia, mediated by complement
- This process continues post-natally and may contribute to pathology in later life
132
What is the difference between striatal and cerebellar microglia?
Cerebellum
- High neuronal turnover
- Microglia express phagocytic markers
Striatum
- Low neuronal turnover
- Microglial do not express phagocytic markers
133
Describe microglia's surveillance function
- Occupy own spatial territory
- Monitor extracellular environment
- Processes directly contact neurons, astrocytes and blood vessels to receive signals concerning changes in state
- Undergo rapid transformation into alerted or reactive state
- Initiate an immune response
- Support endangered neurons or interfere with potential threats to tissue integrity
134
What functions do activated microglia upregulate?
- Migration
- Cell proliferation
- Secretion of anti-inflammatory compounds and neurotrophic factors
- Secretion of proinflammatory compounds and cytotoxic factors
- Upregulation of innate immune response cell surface receptors
- Phagocytosis
- Upregulation of antigen presenting capabilities
135
Describe the rat facial axotomy model
- Motor neurons innervate muscles that control whisker movement
- Axon transection (periphery) --> rapid microgliosis in facial nerve nucleus (CNS)
- Distressed neurons release ATP which activate microglia
- Microglia proliferate
136
What happens days after axotomy?
- Axotomised perikaya is surrounded by activated microglia
- Microglia do not display phagocytic properties (depends on extent of injury)
- Microglia may released factors (e.g. TGF beta) to protect neurons and promote regeneration
- Microglia replace axosomatic terminals so microglial and neuronal membranes become opposed
- Deafferentation promotes neurons from being exposed to excitatory afferent impulses
- Facilitates exchange of signalling or trophic molecules between them
137
What happens several weeks after axotomy?
- Microglial activation subsides coinciding with successful regeneration e.g. rat regains whisker movement
- Number of microglia die due to apoptosis
138
What are the genes expressed by microglia but not peripheral macrophages?
- P2Y12 (in human), TMEM 19 (in human), GPR34, Hexb
| - Most not in human
139
Why are microglia different to peripheral macrophages?
CNS environment influences microglial phenotype
140
What does axon growth and pathway finding and synapse formation depend on?
* Extracellular guidance cues
* Growth cone receptors
* Intracellular signalling
* Cytoskeletal rearrangements
* Changes in transcription in the nucleus/local translation
141
How do axons develop?
- Once neurons reach their final position, they extend an axon to an appropriate target tissue
- The pathway may be long and circuitous, requiring axons to use molecular cues for navigation
- The distal tip of the axon, the growth cone, is morphologically specialised and enriched in receptors that detect molecular cues and stimulate intracellular signalling pathways leading to cytoskeleton arrangements and changes in gene transcription
- Intracellular signalling leads to cytoskeletal changes (actin filaments and microtubules) underlie growth cone dynamics and hence control axon growth
- Growth cone responses depend on the receptor complement and signalling history of the neuron.
- Limiting target-derived trophic factors (growth factors) stimulate survival of appropriately innervating neurons.
- Target-derived signals instruct differentiation of the growth cone into a pre-synaptic terminal. Signals may be cell-associated or soluble.
- Axon guidance signals (including growth factors) often control structural plasticity of synapses, e.g. for learning and memory.
142
What is the Wnt family?
Large family of secreted molecules that stimulate pre-synaptic differentiation by modulating microtubule dynamics in the growth cone.
143
What is neurexin/neuroligin?
Target cells expressing cell surface receptor Neuroligin stimulates pre-synaptic differentiation by binding to cell-surface Neurexin on the incoming cell.
144
The outgrowth of axons, synaptogenesis etc is activity-dependent. TRUE or FALSE?
FALSE
Once neurons form their synapses with their target cells, activity-dependent remodelling refines connections
145
What are some examples of +ve long range cues?
- growth factors such as the neurotrophins (e.g. nerve growth factor, NGF), stimulate axon outgrowth and survival of selective groups of neurons.
- Netrin, which can attract (and repel – see later) different classes of neurons.
146
What does netrin-1 and netrin-2 do?
Attracts commisural neurons in the dorsal half ventrally to the floor plate
Netrin-1 (gradually)
Netrin-2 (steeply)
147
What's the name of a transmembrane receptor for netrin expressed by commisural neurons?
Deleted in Colerectal Cancer (DCC)
148
What are some examples of neurotrophins?
NGF, BDNF, NT-3, NT 4/5
149
Which Tyrosine Receptor Kinases bind to which neurotrophins?
1.TrkA: NGF
2.TrkB: BDNF & NT4/5
•Trk C: NT3
1. All the neurotrophins also bind with lower affinity to the p75 receptor.
2. p75 is associated with inhibitory signals (e.g. myelin, see later).
150
What are some +ve short range cues for axon outgrowth?
Cell Associated (cell surface)
- NCAM
- Fyn/FGF receptor
- Cell Adhesion Molecule
- N-Cadherin
ECM-associated
- Laminin: beta 1 integrin receptor
- Cells secrete components of Basal lamina (neurons prefer Laminin > Collagen)
- Astrocytic endfeet lay down path of Laminin along Optic nerve for retinal ganglion cells to navigate to Optic Tectum
151
What are negative long range cues?
- Netrin, which can attract and repel different classes of neurons
- Semaphorins such as semaphorin III (collapsin), which stimulates growth cone collapse
- Slit, which is a soluble repellent secreted by midline cells.
- Robo, Comm
- To cross midline, Comm removes Robo receptor for Slit --> can cross midline
152
Why are trochlear motor neurons repulsed by netrin?
- They express DCC but also Unc5, and the combination changes a chemoattractive response to chemorepulsion.
(Involves modulation of cAMP levels)
153
What are Semaphorins?
Soluble chemorepulsnts that stimulate growth cone collapse
154
What are the receptors for Semaphorins?
Plexins
155
Where is Sema III expressed?
Ventral half of the spinal cord
156
Where do NT3 and NGF responsive sensory neurons terminate?
NT3 ones terminate in the ventral part
NGF ones terminates in the dorsal part
157
What do in vitro studies using Sema III transfected fibroblasts show?
- NGF responsive neurons are repulsed by Sema III, but NT3 responsive neurons are not.
- Therefore NGF-responsive neurons do not enter ventral part, while NT3-responsive neurons do.
158
What are some negative short-range cues?
- Cell associated:
1. Ephrins
2. Some of the growth-cone collapse inducing semaphorins are membrane tethered.
- ECM-associated:
•Repulsive cues are also present in the ECM, such as S-laminin, tenascin, chondroitin sulphate proteoglycan.
159
What are Ephrins?
- Membrane-associated repulsive ligands which may be either GPI-anchored (A-type) or transmembrane (B-type).
- Growth cone receptors for ephrins are the Eph Receptors
- There are two classes of Eph receptor in vertebrates, EphA & EphB receptors.
- They are tyrosine kinase receptors that activate Rho GTPases to stimulate growth cone collapse.
- Ephrin B/EphB binding stimulates bi-directional signalling (both are transmembrane).
160
What is the Growth cone composed of?
Lamellopodia (sheet-like) and Filopodia (finger-like) which constantly sample enviroment
161
What do positive guidance cues do to the growth cone?
- positive cue --> increased F-actin assembly --> decreased F-actin retrograde flow
162
What do negative guidance cues do to the growth cone?
- negative cue --> decreased F-actin assembly --> increased F-actin retrograde flow
163
What's the pathogenesis of tissue damage?
- Neuronal and glial cell damage or death
- Inflammation
- Ischemia/hemorrhage for stroke or trauma
164
What are the goals of Regenerative Neuroscience?
- Inhibit the scar formation
- Inhibit the axon regeneration inhibitory signaling
- Promote pro-regenerative pathways
- Replace cell loss
165
What are some mechanisms of post-injury circuitry remodelling?
- Regeneration of transected axons
| - Compensatory sprouting from preserved axons
166
How do neurotrophins cause local cytoskeletal changes?
- Neurotrophin --> Trk --> Dimerization --> Intracellular signalling via Rho GTPases --> Local cytoskeletal changes
- NGF-Trk complex endocytosed and retrogradely transported to cell body --> cascade --> stimulate neuronal survival
167
What is a locus?
The physical location of a gene
168
What is an allele?
One of 2 forms of a gene
169
What is a phenotype?
Expression of a diploid genotype
170
What is additive inheritance?
Intermediate expression of alleles
| Phenotype halfway
171
What is the "epigenetic landscape" model of Waddington (1957)?
Genetics can determine neurodevelopmental landscape but trajectory of development (ball) can be influenced by epigenetic factors and random development fluctuations
172
What is an example of disorders of cortical organisation?
Polymicrogyria
173
Underline the mechanism of Polymicrogyria
ARX or TUBB2B mutation --> Many microscopic gyri --> Polymicrogyria (associated with epilepsy and LD)
174
What are examples of disorders of proliferation?
- Microecephaly (germline mutations)
- Hemimegalencephaly (somatic/new mutation)
- Focal Cortical Dysplasia (FCD): somatic mutation in mTOR --> abnormally shaped neurons --> abnormal electrical firing --> epilepsy
175
What are the main molecular pathways and mechanisms underpinning regenerative failure?
- Extrinsic (inhibitory) signals
| - Intrinsic signals (promote regeneration)
176
How do extrinsic signals cause regenerative failure?
All inhibitory pathways cause RhoA --> ROCK --> LIMK --> Actin depolymerisation --> Growth cone collapse
- Astrocytes secrete CSPG --> PTP-sigma receptor/Nogo receptor --> Activate RhoA --> Growth cone collapse - Tx: Dissolve CSPGs
- Injury to Oligs --> Myelin proteins exposed (MAG, OMgp, Nogo) --> Activate RhoA --> Growth cone collapse - Tx: Nogo-R antagonist
- Ephrin/Semaphorn --> RhoA --> Growth cone collapse
177
How do intrinsic signals promote regeneration?
- Neurotrophin --> Trk Receptor --> cAMP --> PKA phosphorylates CREB --> activates pro-regenerative genes. Tx: promote TrkR signalling
- Injury --> Inflammation --> IL-6 --> Gp130-Jak2 dimer --> Phosphorylates STAT3 --> activates pro-regenerative genes, Tx: Increase IL-6?
- mTOR role in protein synthesis, Tx: increase mTOR
178
How do histone deactylase inhibitors work?
- Dephosphorylation of HDAC3 --> decreases activity of HDAC3 (in PNS not CNS)
- HDAC3 inhibition --> increased accessibility of TF to pro-regenerative genes
179
How does PTEN deletion promote regeneration?
- PTEN is an inhibitor of mTOR
180
What are examples of disorders of migration?
- Mutation in Filamin-1 --> Stuck in ventricles/unable to migrate out of ventricular zone --> Periventricular heterotopia
- Mutation in Doublecortin (X-linked):
- Males --> Lissencephaly (smooth cortical surface)
- Females --> Subcortical band heterotopia (SCBH, halfway)
181
What is the common variant common disease hypothesis?
- Genetic variant confers small risk but it is so common (>5%) that disease is common
182
What is the rare variant common disease hypothesis?
- Rare variant (<1%) or de novo mutation leads to protein disruption --> disease
- Mutation in different genes may lead to same disease via different mechanism
183
What is genetic heterogeneity of epilepsy?
Different genes may cause same epilepsy
184
What is phenotypic heterogenity of epilepsy?
Mutation in same gene may cause different types of epilepsy and different drug responses
185
What are the different genes involved in mendellian epilepsy?
- Ion channel subset genes
- Voltage gated (Sodium and Potassium)
- Ligand gated (Nicotonic and GABA)
- Non-ion channel genes
- LGI1 (lateral temporal lobe epilepsy
- GLUT1 (absences and other generalised epilepsies
- PCDH19 (female limited epilepsy)
- DEPDC5 ('variable foci' - frontal, temporal)
186
How many protein disrupting mutations does everyone receive and why do they not cause disease in healthy people?
- 1-4
| - Mutations fall in more tolerant genes in healthy people
187
What is the most important epilepsy gene?
SCN1A (sodium ion channel)
188
What epilepsy genes are involved in synaptogenesis?
PCDH7 and VRK2
189
Which gene regulatory network is associated with human cognition and ND a disease?
M3
190
What are the main causes of neurological impairment?
- Poor neuronal intrinsic regenerative capacity
- Presence of growth inhibitory environment
- Cell loss- imperfect generation of new cells
191
How does Taxol enhance DC axonal regeneration?
- Stabilises microtubules (decreased growth cone collapse, decreased glial cell motility --> decreased glial scar)
- Anti-cancer drug
- Decreased lesion core, Axons reach lesion + into lesion, decreased foot slips (motor coordination) - Hella F, 2011
192
How does Nogo Ab cause axonal regeneration?
- Increased sprouting, Axon grows past lesion (Liebscher, 2005), decreased growth cone collapse
- Phase 1 Clinical Trials shown safe in SCI
193
How does Chrondroitinase cause axonal regeneration?
- Axons grows past lesion, decreased foot slips (Bradbury, 2002)
- Dissolves CSPGs
- Inhibits glial scar
- Delivered by lentivirus
194
How do cAMP analogues/Phosphodiesterase inhibitors work?
- Act as cAMP/Increase cAMP levels
| - Stimulates growth cone remodelling
195
What does Sox11 overexpression do?
- Decreased net reaction of injured DRG axons
| - Increased growth of CST axons, Wang, 2015
196
How do RhoA inhibitors cause axonal regeneration?
Lesioned axons approach lesion (partially go around it)
197
What does Nogo receptor KO do?
Makes axons extend past lesion
198
How does modifying epigenetics cause axonal regeneration?
Relaxes histones to allow TF to access pro-regenerative genes
199
How does PCAF cause regeneration in PNS?
- PCAF promotes histone acetylation
- Increases accesibility
- Increases regeneration
(Doesn't occur in CNS)
200
How does PNS injury trigger regeneration?
- NGF/MEK/ERK retrogradely transported --> activate PCAF --> Histone acetylation--> activate pro-regenerative genes
- Not in CNS
201
What does PCAF overexpression do?
- Axons growth past lesion site
| - Astrocytes support developing axons - Puttagunta, 2014
202
How can electrical stimulation cause axonal regeneration?
ES below lesion site in spinal cord (dura mater) enabled muscle contraction - 1st SCI patient - Harkema S, 2011
203
How can optogenetics cause regeneration?
- Increases serotinin above lesion
| - Enhances neuronal activity
204
How can cell/tissue transplantation cause regeneration?
Pre-differentiated stem cells injected into spinal cord, new synapses, less foot slips, decreased transection score, Lu K, 2001
205
What are different types of pain?
- Somatic pain (nociceptive pain due to injury, sharp/burning
- Visceral pain (nociceptive pain from organs, dull
- Neuropathic pain (non-nociceptive, structural damage, to nerve units, pain in absence of injury)
206
What's the difference between nociception and pain?
- Nociception = sensing damage
| - Pain = experience with the brain
207
What is acute pain?
- Sudden, self-limiting < 6 months
208
What is chronic pain?
Sudden or gradual, periods of remission/exacerbation > 6 months
209
What are the different types of sensory neurons?
- Aβ fibres: Large diameter (>50µm), heavily myelinated --> Touch (fastest)
- A𝛿 fibres: Medium diameter (25 - 50µm), thinly myelinated --> Acute pain
- C fibres: Small diameter (20 - 25µm), unmyelinated --> chronic pain
210
What areas of brain are involved in pain?
- SI, SII, Anterior Insula, Cingulate gyrus
211
What is the Specificity Theory of Pain?
Intensity of pain is directly related to amount of associated injury (Descartes)
212
What is the Gate Theory of Pain?
- When the gates are open, pain can get through more or less easily and pain can be intense
- When the gates close, pain messages are prevented from reaching the brain and may not even be experienced
213
How do bradykinins work?
- Binds to Bradykinin receptor (GPCR)
- PLA2 activation
- Converts Arachidonic acid to Prostanoids
214
What are prostanoids?
- Pain mediators synthesised from AA by COX, released by nociceptive neurons (pain) and immune cells (inflammation)
- Pro-inflammatory cytokines (TNF-alpha) induce COX-2
- NSAIDs/Aspirin blocks COX --> decreased PGE2 (painkiller)
215
What is Nerve Growth Factor's involvement in pain?
- NGF binds to TrkA --> increased peripheral sensitivity of nociceptive neurons, Na+ channels are upregulated
- NGF antibody is a painkiller
- TrkA is selectively expressed on unmyelinated nociceptive sensory neurons (C-fibres)
216
What is glutamate's role in pain?
- Tissue Damage --> sensory neuron releases glutamate --> post-synaptic Na+ influx --> AP --> Pain signal to CNS
217
What is GABA's role in pain?
- If in too much pain, Brain's central descending inhibitory pathways release GABA --> Cl- influx --> Hyperpolarisation --> decreased AP
218
What is a TRP channel?
- Non-selective cation channel (Na+, Ca2+)
- Gated by Capsaicin, Anandamide, High temperature, Low pH
- Only expressed in nociceptive sensory neurons
- TRPV1 KO mice have impaired detection of painful heat stimuli
- TRPV1 antagonists block pain (but temperature fluctuates)
219
What are the different types of ATP-gated channels?
- P2X (ligand-gated ionotropic channel)
- P2X3
- P2X4
- P2Y (GPCR)
220
What is the P2X3 channel?
- Non-selective cation channel (Na+, Ca2+)
| - P2X3 KO mice have deficiency in detecting painful stimuli
221
What is the P2X4 channel?
- Expressed on Microglia, causes Microglia to release BDNF --> decreased KCC2 expression in neurons --> increased intra [Cl-]
- Tactile allodynia: increased sensitivity so that even light touch causes pain
- ATP release --> P2X4 on Microglia --> release BDNF --> decreased KCC2 neuron expression (Cl- efflux pump) --> increased intra [Cl-]
- GABA release --> Cl- moves down conc. grad - [intra] > [extra] --> depolarisation (GABA is now excitatory) --> Pain (AP)
- Brain senses more pain --> Increased GABA release --> vicious cycle
222
What are calcium channels' involvement in pain?
- 4 subunits (alpha1, beta, lambda, alpha2beta), alpha1 chaperones alpha2beta to membrane (blocked by CCB) | 4th TM domain is +ve (voltage sensor)
- Ion selective filter: EEEE
- Cav2.2 KO mice --> decreased response to neuropathic pain
223
What Na+ channels are involved in pain?
- Nav1.7, 1.8, 1.9
224
What is the ion selective filter of Na+ channels?
- D+E+K-A- pore loops (Aspartate, Glutamate, Lysine, Alanine) hang off within pore
- Point mutation: DEKA --> DEEA (Ca2+ permeable): not viable
225
Describe the inactivation gate.
- IFN (Isoleucin, Phenylalanine, Methionine) tripeptide can move to block Na+ entry
226
What is Tetrodotoxin?
- A Na+ blocker (except: Nav1.5, 1.8, 1.9)
- Hear and Pain pathways unaffected but resp failure
- Serine in Nav1.8 conveys TTX resistance
- Ser --> Phe mutation increases TTX sensitivity
227
Describe the voltage sensor of sodium channels
- Every 3rd position of the 4th TM segement has a +ve charge (alpha-helices)
228
What are the sliding helix and paddle models?
- Sliding helix: Depolarisation --> extracellular more -ve --> alpha-helix twists 30 degrees upwards --> channels open
- Paddle model: jumps
229
Describe the Nav1.7
- Quick to open, quick to close, long refractory period, found at sensory nerve endings
230
What are some mutations of Nav1.7
- Inability to feel pain (Loss of function mutation, STOP codon truncation)
- Paroxysmal extreme pain disorder (gain of function, dysfunctional inactivation gate, constant Na+ in/pain signal
- Inherited Erythromelalgia (gain of function mutation, L858H mutations --> prolonged openings, voltage current left-shift)
231
Describe Nav1.8
- Slower to close, minimal refractory period, located at axons - KEY SUBTYPE IN PAIN PATHWAY
- PGE2/Bradykinin enhances TTX-resistant Na+ current (lower threshold)
- Nav1.8 exclusively expressed in nociceptive small diameter C fibre sensory neurons
- Nav1.8 KO do not feel pain
- Gain of function mutation --> Painful neuropathy
232
What is the current hypothesis of AP propagation in unmyelinated sensory neurons
- Na+ channels (e.g. Nav1.8) clusters correlated with Lipid rafts (cholesterol-enriched micro domains
- Depolarisation spreads but low magnitude due to lack of myelinaion but reaches cluster before potential decays and drops sub-threshold
- AP fires and continues
- However MbCD destroys lipid rafts --> AP cannot propagate
- Nav1.9: leaky, persistent current, sets RMP
- Nav1.9 KO mice have normal responsiveness to thermal and mechanical stimuli - may not be important in pain?
233
Which voltage gated Na+ channels are TTX sensitive?
- Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav.16, Nav1.7
234
Which voltage gated Na+channels are not TTX sensitive?
- Nav1.5, Nav1.8, Nav1.9
235
Which Na+ channel activates at around -70mV and produces a persistent current that is likely to play a role in setting resting membrane potential?
Nav1.9
236
Which Na+ channel inactivates slowly at negative membrane potential and sets threshold for generation of action potentials?
Nav1.7
237
What does Nav1.8 do?
Re-primes rapidly and contributes to continuous firing activity during sustained depolarisations
238
What happens to Nav1.9 KO mice?
- Nothing
| - Normal responsiveness to mechanical and thermal stimuli
239
What happens to Nav1.9 KO mice?
Reduced response to mechanical stimuli but not thermal stimuli
240
What are some examples of mutations of skeletal muscle sodium channel, Nav1.4?
- Mutations linked to myotona congenital, paramyotona congenita
- Hyperkalaemic periodic paralysis
- Normo- and hypokalaemic periodic paralysis
- Myasthenic syndrome
241
What are some examples of mutations of cardiac sodium channel, Nav1.5?
- Long QT syndrome
| - Brugada syndrome
242
What are some examples of mutations of brain sodium channel, Nav1.1 and 1.2?
- Generalised seizures with febrile seizures plus
| - Severe myoclonic epilepsy in infancy
243
What is paroxysmal extreme pain disorder?
- Previously known as familial rectal pain, is an autosomal dominant paroxysmal disorder of pain and autonomic dysfunction. The distinctive features of this disorder are paroxysmal episodes of burning pain in the rectal, ocular, and mandibular areas accompanied by autonomic manifestations such as skin flushing.
244
Why is Nav1.8 a key molecule in the pain pathway?
- In vitro, tetrodotoxin (TTX)-resistant Na+ current are enhanced by inflammation mediators such as PGE2
- In vivo, there is an enhancement of TTX-resistant Na+ current density after peripheral inflammation
- NaV1.8 is expressed exclusively in nociceptive small diameter C fibre sensory neurons, and TTX-resistant
- NaV1.8 knock-out mice have diminished mechanosensation and delayed inflammatory pain thresholds
245
What is p11?
- A novel subunit for Nav1.8
- A member of the family of S-100 related calcium-binding proteins
- p11 is localized at the cytoplasmic surface of the plasma membrane, and p11 plays a role in membrane trafficking events such as exocytosis, endocytosis and cell-cell adhesion
- has been identified as an interactor for NaV1.8 by Yeast two-hybrid screening system. p11 controls the translocation of NaV1.8 to the plasma membrane
246
What do p22 KO mice show?
- marked deficits in mechanically and thermally-evoked spinal cord neuronal activity
247
What do NSAIDs do?
- Prevent prostaglandin biosynthesis by inhibiting cyclo-oxygenase (COX), the crucial enzyme in the initial synthesis of prostaglandins
248
What does COX-1 do?
- Makes prostaglandins vital for protecting the stomach through mucus production, and maintenance of renal blood flow
249
What does COX-2 do?
- The inducible form that mediates the pain of inflammation by sensitising peripheral nociceptors
250
What NSAID causes irreversible COX inactivation?
Aspirin
251
What NSAID causes reversible competitive COX inactivation?
Ibuprofen and Mefenamic acid
252
What drug causes reversible non-competitive COX inactivation?
- Paracetamol
| - No anti-inflammatory effect
253
What are examples of COX-2 inhibitors?
- Celebrex
| - Vioxx (withdrawn)
254
Which endogenous ligands regulate nociception?
- Enkephalins
| - Endorphins
255
What is the postulated mechanism of Gabapentin?
- to reduce calcium currents by binding a2beta subunit of voltage gated calcium channel, and prevent translocation of a1 subunit to the plasma membrane
256
What signs and symptoms can brain tumours present with?
- Headache
- Weakness
- Clumsiness
- Difficulty walking
- Seizures
257
What signs and symptoms can brain tumours in children present with?
- Delayed puberty
- Irritability OR Rest too much
- Seizures
258
What are some risk factors and genetic dispositions for brain tumours?
- Mutations may cause certain syndrome (e.g. Neurofibromatosis, NF2, Ependymoma, Meningioma)
- Environmental factors - Ionising radiation (?mobile phones)
259
What is the aim of imaging modalities?
- Assess tumour type
- Assess response to treatment
- Guide Resection
- See recurrences
260
What does CT do?
- Identify tumour, size, quick, mass effect, oedema +/- constrast
261
What does MRI do?
- Identify boundaries, +/- contrast, features of tumour
262
What does MR Spectroscopy do?
- Looks at the spectra of different tumour markers
263
What does PET scan do?
- Looks at metabolic activity beyound tumour margin
264
What are the 3 types of neurosurgery treatment?
- Stereotactic biopsy: inoperable tumour, Requires stereotactic frame, Deep tumours, Aids Dx not Tx
- Open biopsy: Inoperable but approachable tumours, Only surface of brain
- Craniotomy: Debulking (as much as possible)
265
What are examples of post-operative treatments?
- Convectional fractionated radiotherapy
- Chemotherapy (TMZ)
- Gamma knife
- Proton beam
- Steroids (usually pre-op)
- Anti-angiogenic factors (Avastin)
266
Why are brain tumours really difficult to treat?
- Brain surrounding tumour or away from tumour can also be affected which is picked up by scan
267
What are measures of outcome for treating brain tumours?
- Mortality
- Morbidity
- Recurrence
268
What is the difference between intra-axial and extra-axial tumours?
- Intra-axial = located inside CNS e.g. astrocytomas
| - Extra-axial = located outside CNS e.g. meningiomas
269
What is the general grading system?
- Grade I - Long term survival/cure
- Grade II - Cause death in more than 5 years
- Grade III - Cause death within 5 years
- Grade IV - Death within 6 months to 1 year
270
What are the types of tumours that can produce tumours?
- Astrocytomas
- Oligodendrogliomas
- Ependymomas
271
What are the different grades of astroyctomas?
- Pilocytic (Grade I, benign, well-defined cyst with nodule and enhancing walls, do not spread, surgery curative)
- Diffuse (Grade II, invade surrounding tissue, grow slowly, undefined margins)
- Anaplastic (Grade III, aggressive treatment required)
- Glioblastoma Multiforme (Grade IV, primary or secondary, most aggressive brain tumour, increased mitoses, increased neoangiogenesis, cysts)
272
What are oligodendrogliomas?
- Tumours derived from oligodendrocytes
- Cerebral hemisphere (50% frontal lobe)
- Chemosensitive
- Better prognosis than Astrocytoma
- Boiled egg features
273
What are the different types of ependymomas?
- Subependymomas (Grade I, near ventricles, slow-growing)
- Myxopapillary ependymomas (Grade I, Cauda equine causing SC compression, may metastasise despite G1)
- Ependymomas (most common, next to ventricles)
- Anaplastic ependymomas (fast growing)
274
What are features of medulloblastomas?
- Arise from neural stem cells
- Typically in children
- Cerebellum
- May metastasize
- Fast-growing
- Grade IV
- Brainstem often involved, Chemotherapy, High morbidity (20%)
275
What are some features of meningiomas?
- 1/3 of all brain tumours
- Usually adults
- Well-defined round mass
- Progressive Sx
- Surgery curative
276
What are stem cells?
A cell that can differentiate from an unspecified cell to a specialised cell
277
What is differentiation?
Give rise to specialised types
278
What is self-renewal?
When a cell divides to generate daughter cell(s) equivalent to the mother cell
279
What is cell potency?
The range of commitment options available to a cell
- Totipotent
- Pluripotent
- Multipotent
280
What is Totipotent?
A Totipotent cell has the capacity to form an organism
281
What is pluripotent?
Able to for, all the body's cell lineages, including germ cells
- Ectoderm
- Mesoderm
- Endoderm
- Germ cells
282
What is Multipotent?
Can form multiple cell types that constitute an entire tissue or organ
283
What are the embryonic germ layers?
- Ectoderm: Neural lineages, skin cells etc
- Mesoderm: Bone, muscle, blood cells etc
- Endoderm: Liver, pancreas, lungs etc
284
What are some stem cell types?
- Pluripotent Stem Cells
- Somatic (Adult) Stem Cells
- Cancer Stem Cells
285
What are some types of human pluripotent stem cells?
- Embryonic stem cells (ESCs)
| - Induced pluripotent stem cells (iPSCs)
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PSCs exist in vivo, TRUE or FALSE?
FALSE
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What's unique about PSC's self-renewal?
It's unlimited
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What regulates PSC self-renewal?
- Intrinsic Factors
| - Extrinsic Factors
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What are the intrinsic factors of PSC Renewal?
- Oct-4, Sox2, Nanog
| - These transcription factors form a complex and promote expression of pluripotent cells
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What do the extrinsic factors do?
They regulate the intrinsic factors and block differentiation into the cell lineages
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What are sources of adult/somatic stem cells?
- Hair
- Blood
- Intestines
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What are some features of somatic stem cells?
- Limited self-renewal capacity
- Niche-dependent self-renewal
- Capable of life-long self-renewal
- Multipotent lineage commitment
- Lower plasticity (potency)
- No teratoma formation
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What is the stem cell niche?
- The microenvironment surrounds and nurtures stem cells and enables them to maintain tissue homeostasis
- Difficult to replicate in vitro
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What are some types of Multipotent somatic stem cells?
- HSC: all cell types in the blood
- NSC: neuron, astrocytes and oligodendrocytes
- ISC: enterocytes, Paneth cells, Goblet cells etc
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What are the differences between PSC and Somatic Stem Cells?
PSC
- Derived/generated in vitro, not exist in vivo
- Self-renewal: indefinite in vitro, regulated by extrinsic and intrinsic factors
- Differentiation: pluripotency
SSC
- Exist in vivo; limited growth in vitro.
- Self-renewal: life-time in vivo, very limited in vitro, niche-dependent regulation.
- Differentiation: multipotency
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What is the outline of neural differentiation from PSCs?
Undifferentiated hESCs --> Neural Initiation --> Neural tube and rosette --> Neuronal progenitor --> Neural progenitor/stem cells
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What inhibits PSCs from becoming neural stem cells?
BMP
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What inhibits BMP in neural differentiation?
Noggin
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Why does most neural disease modelling uses pluripotent stem cells?
It is difficult to obtain somatic neural stem cells
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What are the advantages of somatic stem cells for cell therapy?
Autologous, low risk of tumour growth, less ethical issue
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What are the limitations of somatic stem cells for cell therapy?
Limited growth and accessibility, limited differentiation potency
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What are advantages of hESCs/hiPSCs and cell therapy?
- Can be expanded to large amounts
| - Capable of generation of all cell types
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What are the challenges of hESCs/hiPSCs?
- differentiation of special cell types
- integration and survival
- immune rejection
- tumorigenesis
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Why is there some debate about which stage to implant cells?
- Too early in differentiation and the environment may not be appropriate but too late and they may not be able to integrate and survive
- Cells are currently transplanted as, for example neural progenitors or dopaminergic neuron precursors rather than ESCs or DA Neurons
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How to avoid tumorigenesis in hESCs?
- Generation of iPSCs without changing genomic DNA
Using small molecules, RNAs and proteins
- Optimal hiPSC culture conditions
Maintain genomic stability
- Eliminating undifferentiated hPSC before transplantation
Removing undifferentiated hPSC by FACS sorting, etc.
- Genetic modification of hPSC to prevent tumour formation.
Toxic ablation of tumour cells.
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Which germ layer is the CNS derived from?
Ectoderm
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At what time point does the neural plate form?
Around 3 weeks post fertilisation
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what does the neural plate tube form from?
Neural plate (basic CNS)
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What do the neural crest cells form?
PNS
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Describe the brain patterning at around 5-8 weeks
- After closure of rostral neuropore, three primary brain vesicles
Prosencephalon (Forebrain)
Mesencephalon (Midbrain)
Rhombencephalon (Hindbrain)
- Next, five secondary brain vesicles
Telencephalon
Diencephalon
Mesencephalon (midbrain)
Metencephalon
Myelencephalon
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How is the Neuron + Glia fate choice regulated
- Temporally and/or spatially regulated by supply/expression
Neuronal or glial growth factors
Transcription factors
- Competing use of shared components in signalling pathways
- Epigenetic regulation of neuronal/glial differentiation
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What does neurogenin do?
| Neural differentiation
- Promotes neurogenesis and inhibits glial differentiation by independent mechanisms
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What is a key factor in astrocyte differentiation?
DNA Methylation
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Where are NSCs found in the adult brain?
- The subventricular zone (SVZ) of the lateral ventricles
| - The subgranular zone (SGZ) of the hippocampal dentate gyrus
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What have transplantation studies suggested about the environment?
- Environment determines NSC fate
- NSC from SGZ to SVZ --> olfactory bulb neurons
- NSC from SVZ to SGZ --> new neurons in the dentate granule cell layer
- NSC to non-germinal regions --> failure to generate new neurons
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What is in the NSC Niche?
- Cellular elements
Astrocytes
Endothelial cells
Ependymal cells
```
- Molecular signals
VEGF
FGF
EGF
BDNF
SHH
```
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What transcription factors are used for iPSCs?
- Oct4
- Sox2
- cMyc
- KIf4
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What are the conditions for modern mammalian cell culture cells?
- Humid
- 37 degrees Celsius
- 5% CO2 for pH
- phenol red so pH can be gauged readily at a glance
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What are different types of cellular models?
- Primary: derived directly from the animal tissue
- Secondary: formed after sub-culturing the primary culture
- Immortalised cell lines
- Organotypic cultures/tissue explants
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What are immortalised cell lines?
A population of cells from a multicellular organisms which would normally not proliferate indefinitely but due to mutation can keep undergoing division
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What are the different methods for generating immortalised cell lines?
- Isolation from a naturally occurring cancer
- Introduction of a mutation that deregulates the cell cycle
- Fusion of two cells (hybridoma)
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What are some examples of neuronal cell lines?
- PC-12 Neuroblastoma rat adrenal medulla
- SH-SY5Y Neuroblastoma cells (dopaminergic characteristics)
- F11 - somatic hybrid of a rat - embryonic DRG and mouse Neuroblastoma cell
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What are the advantages of neuronal cell lines?
- Very easy manipulation
- Outgrowth assay
- Fast intrepretation of the results
- Clear distinction of cellular subtype
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What are the limitations of neuronal cell lines?
- Non-physiological growth
- Neuronal differentiation requires use of neurotrophic growth-factors and removal of serum
- Not a multi-cellular environment
- No tissue around cells
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What is sub-culturing?
- Referred to as splitting or passaging cells
- contact inhibition prevents them from continuing to divide (they become quiescent)
- if left they become senescent
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What is the typical protocol to harvest adherent cells?
- Cells are washed with PBS (Phosphate buffered saline)
- PBS containing trypsin and EDTA is the most common solution used – “trypsinisation”
- The proteolytic enzyme trypsin gently cleaves the proteins holding cells together, helped with gentle agitation.
- EDTA chelates calcium and magnesium ions to inhibit cadherin- and integrin-dependent cell-cell adhesion
- After 5-10 mins, the cells should be detached
Trypsin then needs to be neutralised (or it will digest the cells away)
- Can be neutralised by a trypsin inhibitor, but more routinely by flooding the plate with protein solution, e.g. equal volume of 10% FCS solution
- Cells are then separated from the trypsin solution by centrifugation
- The unwanted solution is removed from the cell pellet and discarded
- The pellet can be resuspended in a suitable growth medium.
- Cell counting in a haemocytometer gives an indication of cell density.
- Cells can be plated into fresh plates/wells at a desired density.
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What are some neuronal primary cell cultures?
- E16-18 or P0 cortical neurons
- Adult DRG
- P7 cerebellar granule neurons
- E16-18/P0 Hippocampal
- P0 Retinal Ganglion Cell
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What are the advantages of neuronal primary cell cultures?
- Very easy manipulation
- Outgrowth assay
- Fast intrepretation of the results
- Clear distinction of cellular subtype
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What are the limitations of neuronal primary cell cultures?
- Partly multi-cellular environment
| - No tissue around cells
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What are ways of purify primary cell cultures?
- Fluoresence-activated cell sorting (FACS)
- Use of antibodies/dyes/genetic to sort cell populations
- Immunopanning
- Use of cell specific antibodies to sort out cell population
- Magnetic cell sorting
- Use of antibodies and magnetic tagging to sort out population
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What are permissive and inhibitive substrates of neurite outgrowth assays
Permissive substrates
- PLL/PDL
- Laminin
Inhibitory substrates
- Myelin
- CSPGs
- MAG
- Stripe assay
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Describe the Scratch assay
- Mechanical injury
- Neurons are allowed to grow axons in vitro
- Injured and regrowth is measured
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What do microfluidic chambers do?
- Make it possible to fluidically isolate cell bodies from axons
- The device contains two chambers connected by small microgrooves
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Describe neuronal activity in culture
- Primary cells and some secondary cells are electrically active and form synapses in culture
- Calcium dyes or genetically encoded calcium indicators (GCaMP) can investigate neuronal signalling and excitability
- Calicum ions can alter gene expression and transmitter release from synaptic vesicles
- Calcium imaging visualise calcium signalling from neurons
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Describe high-throughout assays
- Can screen genes or small molecule compounds
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Describe TUNEL assay
- Double-strand nicks are symptomatic of DNA of cells undergoing apoptosis
- Addition of DNA Polymerase I together with fluorescently tagged nucleotides allows nicked DNA labelling
- Nuclei of cells undergoing apoptosis show up clearly on the microscope
- Staining total nuclei e.g. using propidium iodide, the proportion of cells undergoing apoptosis can be determined
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Describe assaying for proliferation
- Adding labelled nucleotides to culture medium which will be incorporated into the chromosomes of dividing cells
- Able to measure the proportion of cells that have entered the S phase within a given culture period
- Labelled using tritiated thymidine (radioactive)
- bromodeoxyuridine (BrdU- can be detected using anti-BrdU antibody)
- Cell nuclei are stained blue using fluoresent dye DAPI
- Red nucleus has incorporated fluorophore-tagged nucleotides because it has undergone S-phase during the period of the assay.
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Assaying for cell numbers
- Colorimetric assay: Addition of a tetrazolium salt (MTT or MTS) that changes colour from bright yellow to brown when reduced by electrons from the electron transport chain in respiring cells.
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What are examples of Organotypic cultures?
- DRG
- Brain
- Hippocampus
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What are the advantages of Organotypic cultures?
- Easy manipulation
- Fast interpretation of results
- Synapses intact
- Mimic the in vivo settings
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What are the disadvantages of Organotypic cultures?
- Cannot discern easily among cell types with biochemistry
- Outgrowth assay difficult
- Lack of vasculature as compared to in vivo
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What are some applications of organotypic cultures?
- Pharmacological manipulations
- Molecular biology
- Gene or protein delivery
- Gene silencing
- Use of transgenic ex vivo samples
- Electrophysiology
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What's involved in a neurite outgrowth assay?
- Permissive substrates
PLL/PDL
Laminin
- Inhibitory substrates
Myelin
CSPGs
MAG
- Stripe assay
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What is involved in a scratch assay?
- Mechanical injury – scratch assay
Neurons are allowed to grow axons in vitro
Then injured and regrow is measured
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What are microfluidic chambers?
- Microfluidic chambers make it possible to fluidically isolate cell bodies from axons
- The device consists of two chambers connected by small microgrooves
- By keeping the volumes in the wells on one side of the device higher than the other side, creates hydrostatic pressure, fluidically isolating each compartment
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What are the use of microfluidic chambers?
- Axotomy
- Electrophysiology
- Co-cultures
- Small molecules
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How do calcium ions affect neurons in culture?
- Calcium ions act as intracellular signals that can elicit responses such as altered gene expression and neurotransmitter release from synaptic vesicles
- Calcium imaging takes advantage of intracellular calcium flux to directly visualize calcium signalling in living neurons
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Describe high-throughout assays
- In vitro transfection is simple to do although often inefficient and cytotoxic
Chemical
Electroporation
- High-throughput assays can be used to screen genes or small-molecule compounds
- In vivo transduction can be more successful but is technical, invasive and requires large quantities of virus - expensive
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Describe TUNEL assay
- Assaying for apoptosis
- Double-strand nicks are symptomatic of DNA of cells undergoing apoptosis.
- Addition of DNA Polymerase I together with fluorescently-tagged nucleotides allows labelling of nicked DNA.
- The nuclei of cells undergoing apoptosis shows up clearly under the fluorescence microscope.
- By staining total nuclei, eg. Using propidium iodide, the proportion of cells undergoing apoptosis can be determined
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Describe DNA synthesis assays
- If labelled nucleotides are added to the culture medium they will incorporate into the chromosomes of dividing cells.
- This provides a means of quantifying the proportion of cells that have entered S phase within a given culture period.
- The most common nucleotide labelling is either tritiated thymidine, which can be detected because it is radioactive, or bromodeoxyuridine (BrdU) which can be detected by immunocytochemistry using a specific anti-BrdU antibody.
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Describe a coliorimetric assay
- Colorimetric assay: Addition of a tetrazolium salt (MTT or MTS) that changes colour from bright yellow to brown when reduced by electrons from the electron transport chain in respiring cells.
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Compare monoclonal and polyclonal antibodies (Immunocytochemistry)
Monoclonal
- More expensive
- Take longer to produce
- Very consistent batch-to-batch
Polyclonal
- Quicker and Easier to produce
- Tends to have more non-specific reactivity
- Can have very different avidity/affinity batch-to-batch
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How do we use antibodies to see cells or their components?
- Link antibody to an enzyme such as alkaline phosphatase
- When substrate is added, a coloured reaction product marks the presence of an antigen
- Link antibody to fluorophores which can be detected by a flurescent microscope
- Most common fluorophores are TRITC (Tetramethylrhodamine) and FITC (Fluorescein isothiocyanate)
- Newer Cy3 and Cy5 and Alexa probes are becoming more popular.
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What are secondary antibodies?
- Antibodies that will recognise the Fc region of the primary antibodies
- The most versatile way of doing fluorescence immunostaining is to use fluorescently-conjugated species specific secondary antibodies that recognise the –Fc portion of the primary antibody, rather than directly coupling each primary antibody to a fluorophore.
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How can you amplify signals in immunostaining?
- Using biotinylated secondary antibodies and then fluorophore-coupled streptavidin which has multiple binding sites for biotin
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What is fixing?
- Cross-linking proteins at the cell surface
| - Cells die and are fixed at the moment before the fixing chemical is added
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What are commonly used fixatives for immunostaining?
- Paraformaldehyde
- Formaldehyde
- Glutaraldehyde
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Describe Paraffin sectioning
- Must heat and process through xylenes and alcohols – ruins some antigens
- Not good for long term storage
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Describe Frozen sectioning
- Can be stored longer in -80 deg C
- Sucrose treated
- Better survival of many antigens
- Cutting more challenging
- Morphology may not be as good
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What are two types of antigen retrieval
- Heat-Induced Epitope Retrieval
| - Proteolytic-Induced Epitope Retrieval
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What is Heat-Induced Epitope Retrieval?
- Microwave/steamer/pressure cooker ~ 20 minutes, slow cool
- Citrate buffer 6.0
- Tris-EDTA 9.0
- EDTA 8.0
- Must determine for each new antibody/antigen target
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What is Proteolytic-Induced Epitope Retrieval?
- Proteinase K
- Trypsin
- Pepsin
- Destroys some epitopes
- Bad for morphology
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What is permeablisation?
- Making small holes in the membrane, allowing free access to the cell interior
- Methanol
- Triton-X100 or Tween (detergent, 0.3%)
- Cells would die on permeablisation so only cell surface antigens can be stained live
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What is co-immunostaining?
- Primary antibodies for each antigen from different animals or same animal (different isotopes e.g. IgM and IgG)
- Secondary antibodies to each primary antibody must be coupled to a different fluorophore
