Motor neuron disease 2 Flashcards
latest experimental research, disease mechanisms, treatments and new candidate therapies
What are the pathological proteins found in ALS inclusions?
Proteins such as TDP-43 and SOD1 are commonly observed in ALS inclusions
These proteins are associated with neurodegeneration.
how can we study ALS
- post-mortem tissue (brain SC)
- clinical imaging (e.g. PET)
- patient blood/CSF
What is the limitation of using post-mortem tissue for ALS research?
It provides a snapshot in time of the end-stage disease, limiting insights into early neurodegeneration
Most neurons of interest may already be dead.
What are the two main types of model systems used to study ALS?
In vivo and in vitro systems
In vivo refers to whole organisms, while in vitro refers to cell cultures.
in vitro disease models
- immortalised cell lines
- Human iPSC-derived neurons
- Primary cell cultures from animals
what are Human iPSC-derived neurons
Human induced pluripotent stem cell (iPSC) derived neurons
- take skin samples and turn them into pluipotent stem cells that can turn into cell type we are interested in
What are immortalized cells?
Cells that have been manipulated to divide indefinitely for experimentation
Examples include certain epithelial cell lines.
What is a major advantage of using primary cell cultures from animals?
They provide cells that are more similar to in vivo conditions
However, they can only be used once, requiring more animals for additional experiments.
Which animal models are commonly used in in vivo ALS research?
Rodents (mice and rats), fruit flies (Drosophila), zebrafish, and C. elegans
advantage of in vivo disease models
These models help elucidate genetic and cellular mechanisms as are similar to humans
What are transgenic models of ALS?
Models where genes have been altered to study ALS mechanisms
- Knockout
OR
- Knock-in
What does a knockout model entail?
Deleting a gene to study its normal function or effects on ALS pathology
This helps to understand loss of function in genes like SOD1.
What is a knock-in model?
Introducing a specific mutation into the genome to study its effects
An example is the SOD1 G93A mutation linked to familial ALS.
What is the significance of the C9orf72 gene in ALS?
It involves a long repeat expansion affecting protein expression and RNA processing (toxic RNA and reduce C9 protein and increased DPRs)
This gene is associated with both ALS and frontotemporal dementia.
What types of assays are used to quantify motor function in ALS mouse models?
Behavioral tests such as the Rotorod test
These tests measure balance and coordination in mice.
True or False: In vitro models can provide longitudinal information about ALS progression.
False
In vitro models are not suitable for tracking disease progression over time.
Fill in the blank: The study of ALS mechanisms is primarily focused on the __________.
[underlying causes of neurodegeneration]
What is the purpose of using high-throughput screens in ALS research?
To quickly test numerous compounds or genetic modifications for potential treatments
This is often done in simpler models before moving to more complex organisms.
What is the main focus of the discussion on ALS?
The complexity of ALS and various cellular processes involved
The discussion highlights that multiple factors contribute to neuroregeneration in ALS, not just one cellular process.
What are the three specific processes discussed in relation to ALS?
- Glutamate-excited toxicity
- Axonal transport
- Microglial function and inflammation
what is glutamate
Glutamate is the most common excitatory neurotransmitter in the CSF.
What is glutamate in the context of ALS?
An excitatory neurotransmitter that can cause excitotoxicity if not controlled
What are the three types of receptors that glutamate can bind to?
- AMPA receptors
- NMDA receptors
- Kainate receptors
all cause an influx of calcium across the membrane into the cell
What role do EAATs play in synaptic transmission?
They reuptake glutamate from the synaptic cleft to maintain control over synaptic transmission
EAATs are excitatory amino acid transporters found on neurons and astrocytes.
What happens if there is too much glutamate in the system?
Excess calcium influx can lead - activation of signalling cascades
- damage to mambrane and DNA
- mitochondira release toxic free radicals
- apoptosis (capase cleavage)
What evidence suggests that glutamate is not well controlled in ALS?
evidence for glutamate ecxitotoxicity in ALS
- Increased glutamate levels in patients’ CSF compared to healthy controls
- Reduced expression of gluatame transporters (EAAT2) in ALS post-mortem tissue for motor cortex and spinal cord
in cell cultures what is evidence for excitotoxicity (not ALS specific)
- cultured motor neurons more vulnerable compared to other neuronal subtypes
- patient CSF toxic to cultured motor neurons
- rescued motor neuron cell cultures by glutamate receptor antagonists
evidence for glutamate excitotoxicity in mice for ALS
- EAAT2 knockout makes motor impairements worse and reduced survival in mSOD1 mice
overexpression for EAAT2 gene can improe symptoms
What is the only approved ALS treatment in the UK and what does it do?
- Riluzole
- It reduces glutamate transmission
only modest theraputic benefit
What is axonal transport?
The transport of molecules and organelles up and down the axon
This process is crucial for delivering proteins and other materials from the cell body to various parts of the neuron.
what modulates axonal transport
- microtubules that act as train tracks
- powered by ATP
What motor proteins are involved in axonal transport?
- Kinesins (anterograde transport)
- Dyneins (retrograde transport)
walk along microtubules
anterograde transport
away from cell body- mostly controled by kinesins
retrograde transport
towards cell body
- mostly controled by dyneins
What patient evidence indicates impaired axonal transport in ALS?
- Mutations in kinase proteins linked to ALS (KIR1A,KIR5A)
- Abnormal accumulation of cargo in ALS patient tissues (vesicles, mitochondria, lysosomes etc.)
what evidence in mice and cells indicates impaired axonal transport in ALS?
- SOD1 and TARDBP mutatant mice and cells have axonal transport defects
- cargoes fluorescently labelled and tracked move more slowly or not at all in ALS models
What did the study by Fumigale et al. investigate?
The motility of mitochondria in iPSC-derived neurons from ALS patients in fluorescent live-imaging
- found sig. reduction in C9-carrying in ALS neurons
was this caused by C9orf72 dipeptides ?
continuing on from Fumigale et al.,
What was the outcome of treating healthy motor neurons with synthetic DPR peptides (dipeptides)?
poly-GR and poly-PR peptides caused a significant reduction in mitochondrial motility
The effects were dose-dependent, indicating that higher concentrations resulted in worse outcomes.
C9orf72 expansion impairement in axonal transport
C9orf72 dipeptides bind to several axonal trnasport proteins, no longer free to do their job resulting in defects
- C9orf72 expansion impairs axonal transport
Fill in the blank: Excess glutamate can lead to _______.
excitotoxicity
True or False: Riluzole is known to significantly increase survival in ALS patients.
False
What are microtubules’ role in axonal transport?
They act as tracks for motor proteins to transport cargo along the axon
Microtubules are composed of tubulin and are a key part of the cytoskeleton.
What happens to protein transport when the EAAT2 gene is knocked out in mice?
Motor impairments worsen and survival is reduced
This indicates the importance of EAAT2 in maintaining neuronal health.
What does the term ‘excitotoxicity’ refer to?
Cellular damage caused by excessive stimulation by excitatory neurotransmitters like glutamate
What are the two types of transport in axonal transport?
- Anterograde transport
- Retrograde transport
What does a reduction in mitochondrial motility indicate in ALS?
Impaired axonal transport and potential cellular dysfunction
What are GR and PR in the context of patient cell lines?
Proteins that form insoluble aggregates in the cytoplasm
GR stands for GGG repeat, and PR stands for poly-Glycine-Arginine repeat.
What unexpected behavior was observed in kinase family proteins in patient cell lines?
They formed cytoplasmic inclusions co-localizing with DPR
This was not seen in control cell lines.
What is co-immunoprecipitation used for?
To determine if proteins are physically interacting with each other.
How does co-immunoprecipitation work?
Beads coated with an antibody bind to a protein of interest, capturing it and any interacting proteins.
what did co-immunoprecipitation show for C9orf72
- GR and PR interact with multiple components of machinery and hence why we get axonal impairment
Neuroinflammation in ALS
- common in all neurodegenerative diseases
- microgliosis in affected brain regions
- increased pro-inflammatory cytokines in CSF
What is the role of microglia in neurodegenerative diseases?
They show excessive activation and increased inflammatory cytokines.
microgli in ALS
more amoeboid shape
What is SOD-1 mutant mouse used for in research?
To study microglial activation in ALS
- increased activation in ALS
What does shawl analysis measure?
The morphology and processes of microglia.
What shape do activated microglia take?
Amoeboid shape with fewer processes.
True or False: Neuroinflammation is always harmful in ALS.
False.
What genetic evidence suggests microglia have an important role in ALS?
Mutations in microglial proteins are identified as risk factors for ALS.
e.g. mSOD1 microglia
what is
what is one potential cause of neuroinflammation
NLRP3 inflammasome
What is the NLRP3 inflammasome?
A multi-protein complex that form cytoplasm that responds to stimuli in immune cells e.g. tissue damage, pathogens, particulates that need clearing
what does inflammasome activation produce
- caspase-1 activation
- cleaves pro-interleukin-1B (pro-IL-1B) and IL-18
- these are inflammatory cytokine that can damage motor neurons
NLRP3 inhibition
rescues cogntion in Alzheimer’s rodents
suggests inflammasome not passive but directly contributes to neuronal dysfunction and degeneration
What effect does GR have on IL-1 beta release?
It significantly increases IL-1 beta release from macrophages.
(causes cleavage of pro-IL-1B)
GR activates NLRP3
What happens to IL-1 beta release when an NLRP3 inhibitor is applied?
The release is blocked.
rtells us il-1b is mediated by NLP3 inflammasone
What does immunofluorescence staining reveal when GR is applied in hippocampal mice slices?
Formation of inflammasome complexes in cells
can visualise inflammasomes using antibody against ASC - GR does activate inflammasome
What does the presence of ASC indicate?
Formation of inflammasomes.
What is the significance of knocking out NLRP3 in GR mice?
It rescues some ALS phenotypes - as NLPR3 contributes to neurodegeneration symptoms
highlights inflammasone as potential theraputic target
What potential therapeutic target is highlighted for ALS?
The NLRP3 inflammasome.
What does ALS stand for?
Amyotrophic Lateral Sclerosis
ALS is a progressive neurodegenerative disease affecting nerve cells in the brain and spinal cord.
What does the speaker imply about the current understanding of ALS?
It is not fully understood, and there are still unknown factors
The speaker mentions that not everything about ALS has been discovered yet.
What kind of mechanisms did the speaker mention discussing?
Three different mechanisms
The speaker acknowledges the complexity of explaining ALS mechanisms.
Fill in the blank: Autoimmune conditions involve the immune system targeting _______.
the body’s own cells
The speaker explains the nature of autoimmune responses.
What does the speaker suggest about the complexity of autoimmune responses?
They are more targeted and involve antibodies
The speaker highlights the specificity of autoimmune mechanisms.
