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23 Cards in this Set
- Front
- Back
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1) Describe PrP indels. s8 |
1) -> genetic prion diseases are linked to point mutations and insertions in the PRNP gene encoding PrPc on chromosome 20 -> Point mutations mainly clustered in the protein's C terminus, leading to amino acid substitutions or protein truncations -> The insertions consist of additional copies of an octapeptide repeat in the N-terminal region, which normally contains one nonapeptide and four octapeptides -> Mutant PrP is thought to mis-fold and aggregate spontaneously, eventually acquiring PrPsc structure -> position 129 and 178 indels seem to be most important esp in combinaiton; can be inherited |
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1) What are some diseases that different types of PRNP mutations associated with 2) Describe impact of region 129 s10 |
1) -> CJD, FFI, GSS, PrP-cerebral amyloid angiopathy, PrP systemic amyloidosis 2) Disease phenotype also influenced by PRNP polymorphic codon 129, where met or val can be encoded |
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1) What are the exact mutations in FFI and CJD, and what are the associated phenotypes? s11 |
1) -> If have Met at 129 and another mutation e.g. aspartic acid to asparagine at 178, this is FFI (severe sleep disorders and autonomic dysfunction). -> If have Val at 129 and same mutation as before at 178, have Creutzfeldt-Jakob Disease (global cortical dementia and motor abnormalities) |
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1) Describe the histopathology of FFI and CJD. s12 |
1) FFI: Prions aggregate in thalamus CJD: Large vacuoles found throughout cortex Both see big holes ^ |
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1) Describe the PG14 mouse model, the pathway of prion pathology and what it leads to. 2) What is the hypothesis that was created based on results of the PG14 mice experiments? s13 |
1) PG14 mouse: extra nine-octapeptide repeat insertion associated with GSS (inherited) and also a moPrP mutation homologous to the human CJD178. -> Prions tend to be accumulating in the ER. -> Intracellular accumulation of these PrP mutants impair the secretory transport of the voltage-gated calcium channel (VGCC), resulting in inefficient targeting of the VGCC complex to presynaptic terminals -> leads to inefficient glutamatergic neurotransmission in cerebellar granuole neurons and abnormal motor behaviour in transgenic mice. 2) ER retention of mutant PrP causes motor disease by altering the secretory trafficking of calcium channels for synaptic activity. |
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1) Describe the inheritance pattern and the pathophysiology of FFI (Fatal Familial Insomnia)
s15 |
1) -> Autosomal dominant -> Spongiform in nature, lethal within 1-1.5 years -> Hallucinations, paranoia, psychoses, rapid weight loss -> Autonomic functions affected -> Relatively late onset (40s-50s) -> cant sleep |
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1) Describe the basic physiology of sleep 2) compare sleep patterns through life s16, 17 |
1) -> Sleep debt dependent -> Circadian Rhythm (blue light) dependent -> Mammals, suprachiasmatic nucleus (SCN) and other groups of neurons within hypothalamus involved -> role debatable 2) Newborns have random cycles, get entrained later and build sleep cycle towards adulthood |
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1) What is the sleep pattern of Narcolepsy, and what are associated conditions? When would symptoms occur? s18 |
1) Excessive daytime sleepiness and prolonged drowsiness. Opposite sleep cycle than normal.
-> When go to sleep with narcolepsy, also get cataplexy: sudden loss of skeletal muscle tone, often due to emotional reactions. -> Sleep paralysis (abnormal REM and EEG) -> Get hypnagogic hallucinations (Vivid dreaming episodes; from suddenly falling in and out of sleep) -> Loss of memory in some cases -> ages 10 - 25 |
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1) What is one of the best animal model of studying narcolepsy? 2) What system is the neurotransmitter in the hypothalamus that are important for feeding behaviour and what were they found to be associated with? s19 |
1) Various dog species, have spontaneous narcolepsy 2) Stimulate orexin/hypocretin system, control feeding behaviour. When knocked out, showed narcolepsy. Reduced numbers of hypocretin neurons in individuals with narcolepsy. |
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1) What was found in the dog models; and what was found in narcoleptic people? s23, 26 |
1) They did positional cloning on narcoleptic dogs and found that had mutations in the hypocretin2 receptor (Hcrtr) -> GCPR -> In people, they found lack of hypocretin/orexin containing neurons within the hypothalamus lateral wall, less connections to rest of the brain, which is why they fall sleep |
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1) What is hypocretin (same as orexin), what does it act like, where can it be found and what is its risk ratio to narcolepsy ? s25 |
1) Neuropeptide produced by a small number of neurons found in the posterior/lateral wall of hypothalamus -> 2 forms: Hypocretin 1 and hypocretin 2 (Orexin A and Orexin B) -> Acts much like a neurotransmitter and is found in presynaptic vesicles -> Extremely high relative risk ratios associated with narcolepsy -> neurons destroyed in auto immune fashion to produce narcolepsy |
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1) What is saporin and what was seen in the saporin experiment s27 |
1) A toxin that disrupts the ER and the transport from ER to Golgi. When targeted the neuron containing neurons orexin, this disrupted the sleep cycle. |
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1) What were the results of characterizing the prions in western for FFI and what antibodies were used? s30 |
1) The mutant PrP was largely insoluble and Protease K resistant. -> The size of both the FFI and CJD were 19 kDa, which is smaller than normal due to deglycosylation. This is same as observed in patients. -> 12B2 and Sha31 antibodies were used |
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1) What were observed in the the transgenic mice that expressed the mutant gene twice the wild type FFI gene? What was seen in EEG hypnograms? s31 |
1) Found to develop a fatal neurological syndrome with motor and cognitive deficits. Circadian organization of sleep and motor activity was lost, nocturnal activity lost. -> EEG hypnogram showed that the mutant mice fell directly from wakefulness to REM sleep, instead of going through non-REM first. |
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1) Describe the experiments in which the mice were kept awake and what characteristics were observed? s33 |
1) Kept awake for 6 hours, then allowed to sleep freely for 18 hours. REM and NREM sleep were measured. As REM was already affected, there was little lose, also little compensation (sleep rebound). -> EEG activity found to be altered after deprivation; observed unique burst of high voltage polyphasic complexes of neuron spindles, similar to those in CJD. |
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1) What conditions did the mice develop later in life? What were the result of the Rotarod essay? s34 |
1) -> Progressive neurological disease -> developed ataxia (lose of bodily movement), with abnormal flexed posture of hind legs, kyphosis (spine curvature), foot clasp reflex -> Mice later lost weight and were unable to feed themselves, died a little earlier than normal -> FFI mice performed on Rotarod assay (rotating bar that mice run on) well until 50-70 days up until 90 days, and when reaching 100 ish days, they started showing progressive motor dysfunction. Even worse in homozygous mice. |
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1) How was cognitive function was tested in the FFI mice and how did they account for motor dysfunction? s36 |
1) Found alterations in long-term recognition and spatial working memory in Tg FFI mice. -> Tested in novel objection recognition, and eight-arm radial maze (reward in different paths of maze) -> Mice were impaired in long term memory, as shown by lower discrimination index in the tasks, and higher errors than the Non-Tg mice. -> Allowed for more movement freedom to account for motor dysfunction. |
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1) What are the brain areas that are affected and when in the FFI lifetime? s37 |
1) Not much difference in 80 days. In Tg mice older than 400 days, whole brain volume was 12% smaller largely in thalamic and cerebral volume. |
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1) What did they observed in immunohistochemical detection of PrP using monoclonal antibody 12B2 after PK digestion s38 |
1) -> Overall staining is significantly less. The cerebral cortex, hippocampus, thalamus, and molecular layer of cerebellum showed aberrant misfolding of the PrP. Overall prion deposition was diffuse, not clumped. striplike staining in fimbria, and more dot-like in the mesencephalic trigeminal nucleus. overall idea: Tg mice see lots of expression of the prion in the 12B2 antibody. |
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1) What were observed in EM? s 39,40 |
1) -> increase in abundance of autophagosome, and the associated lysozomes -> higher protein turnover. -> structural changes in neocortex, hippocampus, thalamus, cerebellum. -> Lipofuscin (lysosomal marker) residual bodies in a thalamic neuron (suggest that the neurons are degenerating, neurons that are going under oxidative stress). Lots in older neurons. -> The golgi bodies look unusual, onion-like appearance instead of stack. Confirmed by 3D tomography, showing concentric isolated Golgi cisternae and invaginations of medial golgi cisternae. -> overall: possibility of something wrong with ER to Golgi transport and golgi are transport. also mice might have issues with transport of transport and degradation machinery. |
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1) What happened when they tried to test infectivity? s41 |
1) Expected Intracerebral inoculation of brain homogenates from FFI and CJD induced prion disease in experimental animals to gain spontaneous mutation but when inoculated in Tg mice that overexpressed moPrP at 8x normal levels and are highly sensitive to prions, none developed neurological dysfunction. |
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1) What is PMCA and what results did they get? s43 |
1) -> Serial protein misfolding cyclic amplication. Do serial dilutions, should see misfolded prions in all the levels. Take already misfolded protein in there to make the reaction faster -> but saw no change in this experiemnt |
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1) So what were the final conclusions? s44 |
1) -> They achieved expected phenotype and pathology of FFI, and saw same ER retention issue; but no evidence of transmissibility. -> But showed that Tg mice were useful for testing potential therapies, and useful for comparing Tg FFI and Tg CJD to compare molecular mechanisms responsible for the phenotypic heterogeneity with the polymorphic mutation variants. -> Could be a new prion model; not really sure. |
