ALS and MND Flashcards
compare and contrast the the pathology of SMA and ALS.
SMA causes muscle weakness soley in the lower motor neurons of the spine in the peripheral system.
ALS is a form o MND and will cause degenration of bot higher motor neurons in the brain and motor neruosn n the brain stem and spine.
in SMA this is a cell autonomous disease with the mtor neurons being selctve tragetted, meanwhile dysfunction in schwann cells seen here.. ALS is not, SOD1 mouse models report degenration of oligondendrocyte. There is also slectivity of the degen of certain motor neurons. Also selective sparring of some MNs neurons in ALS, sphincter, bladder, nonm-voulntary less vulnerbale and slow twitch less
vulnerable
SMA shows symetrical weakness in all systems.
what is the muscle weakness that defines motor nueron disease?
MND is defined by motor weakness that results in parysis in mre than one muscle group.
is there a gender aspect in MND-
MND risk is higher in men with 5000 being affected in th uk at any one time.
what are the 4 main types of MND? How can you differentiate between them.
Progressive bulbar palsy (PBP) effecting the bulbar nerves of the face.
Progressive lateral sclerosis (PLS) this effects on upper (central) motor neurons.
Progressive muscular atrophy (PMA) this effects only lower neurons.
Amyotrophic lateral sclerosis (ALS) this effects both upper and lower neurons.
What extent of ALS is sporadic and inherited mutations.
0% of cases are sporadic and 10% are fammilial
despite this the onset is usually the same and the symptoms are aswell. onset is between 50-70
outline the disease phenotype in ALS (what and and what is not effected, what are the unique characteristics?
effected:
- May experience weakness in the arms or legs leading to issues walking*
- Many struggle to keep their head up*
- there are problems with breathing and emotional lability*.
- Some cognitive deficits exists: mild issues with memory and concentration and language*
Rare:
there can be some aspcects of FTD that appear in aound 5% prior to motor issues. this can be linked t the overlap of the 2 diseases.
Not effected:
The 5 core senses are sparred.
Bladder and bowel movements unaffected.
eyes, heart also unnafected.
what are the 2 current approved therapeutics?
Riluzone- Anti-glutamatergic agent, attempots to stop excitotoxcity as models of ALS report reduced glutamate uptake and ssues with Astrocytes uffering of levels.
This was able to increase life span by 2-3 months but does not infleunce quality of life.
EDARAVONE- This targets oxidative stress by acting as a free-radical scavenger and thus countering the effects of ROS molecues.
It has only been shown to be effective in fast progressing subgroups and so this may have exagerated the appearance of this efficacy.
what is the mitochondrial dysfunction and oxidative stress assocciated with MND (ALS)?
There is alot of evidence of oxidative stress in MND and ALS.
- increase nitrotyrosine (a marker of stress) and nitrated magnese SOD can be seen in CSF samples, indicating increased ROS levels.
- increased marker of protein and lipid oxidation are also observed in motorneruons, macrophages and astrocytes. all cell effected in ALS.
there is eveidence of oxidative DNA damage in the ventral horn of the spine.
Mt dysfunction.
Mitochondria with abnormal morphology are found in post-mortem samples of ALS patients.
ALS motor neurons show reduced caclium buffering capacity, disrupted mitochondrial membrane pottential, and reduced activity of the ETC.
Linking back to ideas of enhanced oxidative stress. Mt are a large source of this through the production of ROS molecules and damge here will inrease this releas. ROS can act back on MT to damge the function of the MRC and the mtDNA, which correlates with INCREASED levels of MTDNA deletion observed in the spinal cord and motor cortex of ALS patients.
Mutant SOD1 a cause of familail ALS 15% showed specific localisation at mitcohondria in spinal cord.
What other cell types beyond motor neurons are implicated in MND? (3)
Astrocytes- These play a key role in the buffering of the glutamate level via uptake by EAAT2 (excitatory amino acid transporter 2) reduced glutamate uptake is observed in ALS.(disrupted function) (progressive decline in EAAT2 presentation seen in G93A model.
Microglia- as with many other neurodegenrative diases neruoinflamation is involved. this has been associated with exxcaerbating the situation by attacking MNs. This has been related to hyperactivity lnked to over release of superoxide ROS.
Oligodendrocytes. These are key to myelination of MNs for fast propagation. In the SOD1 mutant mouse model they showed that oligodendrocytes where degenrated and the expression of Monocarboxylase transporter 1 (MCT1) is reduced, this is key for the transport of the lactate to neurons whcih is part of oligodendrocytes metbolic support of neurons.
- Expression of mutant SOD1 G93A
- In microglia slectively silencing this can increase life span and survival. The same is seen with astoglia.
In BOTHciruumstances soleley expressing Micoglia or astrocytes overexpressing SOD1 G93A in WT mice or Motor neuron cell cultures respectively annot induced neuronal death. This is in line with their pottentiation of the condition.
What pathological mechanisms are indicated by genetics in MND?
Protein aggregation:
Protein aggregation as a result of dysfunction in TDP-43, FUS and SOD1 is associated both with toxic gain of funcion and loss of function.
the downstream effects could be Blockage of the proteasome (a toxic SOD function),
overload of the autophagy system (in removing blocks C9orF72 has pottential to be a GEF for small Rab GTPases giving it a role in lysosmal biogeneis and autophagy so LOF could be linke here.),
depletion of essential cellualr proteins (through accumualtion into aggreagates and RNA interfearance FUS and TDP cause aberant stress granule formation)
RNA/metabolism and processing- TDP-43 (xryptic exon inclusions and abberant protein formation) and FUS (expression of toxic form cause drosophilla degen prevented by removal of RNA bindng domains.) are RNA/DNA binding proteins adn LOF has severe effects on the processing and ths function of many proteins upstream. (C9orF72 toxicity is ascoiated to its abberant processing potentialy impact on disrupting the porcess of other Rna sequesters RNA binding proteins by repeats frming G-quadruplexs (made from G-base atypical pairing to fro G-quartets that stack to form G-quadruplex) allowing ofr atypical pairing
Axonal trasport:
this is mainly linked to SOD1 mutant role in toxic aggregation and endoplasmic stress, induces these issues.
ALS can also present with aggreations of neuroflaments which usually act as the tracks for transport.
Mice show deficits in both anterograde and retrograde transport.
Glutamate excitotoxicity. (SOD1 increases this)
We can observe increase glutamate in the CSF
Reduced Glutamate upatke stemming from astrocyte dysfunction.
MNs have a low calcium buffering capacity but a high number of calcium permeable AMPAR indicated by lack of GLUR2A subunit. this could explain selctive degen of MNs.
Also progressive decline in EAAT2 seen in the G93A mose model.
Outline the genetics linked to ALS (3 detail) (4 names)
SOD1:
*mutation in cu/zn SOD1 is seen in 15% of fammilial ALS cases.
*SOD1 has the normal role of converting superoxide to hydrogen peroxide and molecular Oxygen.
*There are 160 identified mutations all of which cause dominant inheritance Familial ALS but 1.
*this has been associated o toxic gain of fucntion playing role in:
-Mitochondrial damage
-glutamate excitotoxicity
-axonal transport disruption
-proteasome inhibition
-increase extracellular superoxide
-Endoplasmic reticulum stress.
A transgenic mouse model of the mutant SOD1 has been created.: G93A model is the gold standard with large overlaps with human condition.
-has a charceterisitc progression of disease pathology and showe behavioural signs of ALS. progressive motor neuron degen eventually led to parylysis seen in the Hind Limbs following a period of locomotor defects. 20% loss in body weight and inability to right themselves when placed on their side. degernaion is sen cocomitnalty with progressive accumulation of microgliosis and astrogliosis
- Expression of mutant SOD1 G93A
- In microglia slectively silencing this can increase life span and survival. The same is seen with astoglia.
In BOTHciruumstances soleley expressing Micoglia or astrocytes overexpressing SOD1 G93A in WT mice or Motor neuron cell cultures respectively annot induced neuronal death. This is in line with their pottentiation of the condition. - Simply over expressing wild type SOD1 in mice does not cause MN pathology showing this is a gain of function.
- Trangenic SOD1 mouse models show degenration of oligodendrocytes and the reduced expression of monocarboxylase transporter 1. Lathough in MND it is defined by the degenration of motor neurosn it is not the only area effected and this is also seen. MCT1 are key to providing metabolic support to neurons also.
- Studies have shown that SOD1 can show a prion like spread of pathlogy. reports on their structure show a structyure densein Beta pleated sheets, a structure which fascilitates promiscuous bindin and is believed to allow for permissive templating.
- A study showed CSF from a SOD1 mouse model could induce WT SOD1 to form amylid like fibrils. suggesting in may be involved the the sporead ot toxicity in SOD1 mutant ALS.
TDP-43 AND FUS: TDP-43 pathology is not seen in FUS-ALS or SOD-ALS bus it seen in the majority of cases. FUS is very rare.
*these proteins are involved in the processing of RNA>
-involved in the transcriptional levels of nuclear RNA
with roles in splicing, polyadenyaltion and long intron stabilisation.
-miRNA processing roles, key to the stabilisation of 3’ UTR binding. (UTR binding in in TDP FUS is not selctive.)
-They also play roles in the protein translation and stress granule formation
- when mutated they form cytoplasmic inclusions (TDP-43 also forma extracellular inclusions in Type A-C FTD-TDP)(aggreagates).
- this leads to a depletion from the nucleus and a LOF of roles in splcing and transcription of RNA.
- They also get a toxic gain of cytoplasmic function in altering RNA transport
- abberant stress granule formation
- The hyperphosphorylation and ubiqutination of TDP-43 has been reported in inclusions. an was solely recovered in damaged areas such as the Hippocampus, spinal cord and frontal cortex.
- Note- work in drosophilla has reported: LOF of TDP-43 has been related to motor abnormalities in larvae and decreased boutons at NMJ. Over expression of human trangene in drosophila also led to reduced boutons and dendritic branchhing. So both linked to toxic dysfcuntion of synaptic trasnmission that cause neurodegenration. Synaptic dysfunction is one of he early pathologies of ALS. skewed to wards LOF as theyre removed from fucntional location.
Ling et al 2015- Showed that CRE=ER controlled KO of TDP-43 led to loss of splicing of cryptic EXONs tat led to disrupted MRNA translation and the formation of several abberant porteins in mouse embryonic stem cell. Restoring splicing of these expns was able to prevent cell death in these lines.,
Fus- same functions as TDP-43. Expressing Human ALS causing tragenes in drosophilla results in brain, eye and Motor neuron degeneration. This can be stopped by mutating the FUS domains fasciliatting RNA bidning. This suggests that its toxicity stems fom abberant binding and aggregate formation in the cytoplasm.
VCP also seen here think of RITSON 2010 (FTD plan.)
C9orF72- this is the core link between FTD and ALS, being a large genetic contributor in both cases.
- The GGGGCC repeats can be translated in either direction in any reading frame producing 5 different transcripts. (GP,GA,GR,PR,PA)
- it is predminatly asscoiated with FTD-TDP type 1 and 2 pathology, and thus unsuprisingly, behvaioural and non-fluent phenotypes, alongside, psychosis, MND.
- Was discovered in the FTD-MND phenotype.
- the notmal function of the disease is unknown, allthough some reaserc has pointed towards a role in LYSOSOMAL biogenesis and autophagy by interacting with GEF, serine threonine kinases Like TBK1 (autophagy) and RAB GTPases)
- Mutations are the results of a hexanucleotide repeat expansion, from around 22 repeats of 100s -1000 IN SOME pathogenic cases. This is a GGGGCC repeat
The are 3 mechanisms by which they believe this could be toxic.
-LOF th natural function is unkown but losing this could be toxic.
*KD studies have shown that this will inhibit the INDUCTION of autophagy that correlated with increased P62 AND TDP inclusions. pottentially a link to increased TDP.
*Ideas arre supported by SHI et al (2018) that show in patient derived and Crispr/CAS9 KO IPSC there is reduced endocytosis and vesicular trafficking.
*However= in CRISPR.CAS9 KO MOUSE MODELS, non precipitate the disease (ALS or FTD) suggesting that the haploinsuffieiciency alone cause the disease
ALthough, it could still contriute to excaerbate the condition through idsrupted lysomal fucntion e.t.c.
- RNA toxicty- The formation of snese and antisense RNA FOCI have been repored [primarily in the frontal cortices of ALS and FTD patients. Commonly these are foun inside NEURONAL NUCLEI. The Repeats form loop called G-quadruplexs that allow for atypical pairing, this allows for the binding of RNA-binding like TDP=43 AND FUS dirupting their function in RNA processing and trappingproteins in the nucleus. These can trap RNA in nucleus
- PROTEIN TOXICITY - the long repeat can cause an abberant processing of RNA called REPEAT ASSOCIATED NON-ATG translation (RAN-translation). This forms toxic DIPEPTIDE REPEAT PROTEINS, which are aggregation prone and interfere with RNA processing.
MIZIELINSKA et al 2014
-Support from study in FTD studies in drosophilla.
-expressed pure RNA and RNA-only forms in models. They showed via FISH that both could form RNA foci, but in IMMUNOBLOTTING they showed Pure RNA ALONE could cause DPRs.
-Using eye degeneration as a marker of neurodegen> Those soley expressing GGGGCC repeat expanded RNA (no protein) did not cause neurodegen. Whilst models only expressing pure RNA did cause degeneration suggeststing dipeptide repeats mediates toxicity.
- To see what was the cause of this they expressed a PROTEIN ONLY model differentiating between 2 codons in the REPEATS the sample those RICH IN ARGININE poly GLYCINE-ARGININES AND PROLINE ARGININES VS poly Glycine and proline alanines. They showed those
ARGININE RICH REPEATS DPRs alone caused increased eye degeneration.
Hence the arginine rich DPRs would seem to be the cause of C9orF72 neurotoxicity.
However, in posrt-mortem study the pattern of TDP pathology much btter tracks neruodegenration and is NOT common seen with DPR inclusios.
Given the links of DPRs to neruodegen in mouse models, it is likely DPR acts upstream fo TDP expalining its better correlation with DEGEN in psot-mortem studies which tend to represent end stages of the disease.
However, Knockin mouse models of C9orf72do not show TDP iclusions. despite observing motor, Cognitive defects with degeneration.
The downstream effects of C9orF72 DPRs have been implicated partiucuarly in issues with Nucleocytoplasmic transport.
- Zhang et al showed that the expression of poly GRs and PRs was able to induce increased stress granule formation that drove NCT disruption.
- He equally showed using ASOs to sequester C9orf72 could reduce NCT defects and TDP mislocalisation.
What MNs are damaged and Which are sparred in MND? what does this say about motor neurodegenration here.
Those associated with voluntary movement are vulnerable and those innervating fas twitch fibres
Those innervating slow twtich fibres are resistant aswell as occulomotor and innervators of the Urethra and Sphincter.
what is thought to underpin phenotypic variance in ALS?
Phenotypic varinace in ALS is thought to be mediated by the co-experinece of different genetic factors and environemntal factors. E.g C9orF72 with TDP-43 AND fus.
what environmental factors can contribute to ALS
HEAD INJURY
EXPOSURE TO HEAVY METALS
HIGH LEVELS OF EXERCISE, RELATED TO HEAD INJURY
POOR DIET AND LOW VITAMIN E INTAKE
