Neurodegenerative disease models

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Neurodegenerative disease models
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Neurodegeneration models in C.elegans

• The development of transgenic models, has been
  intensively used for investigating
  neurodegenerative disorders such as Alzheimer's
  disease, Parkinson's disease, Huntington's
  disease and tauopathies.
• In some of these cases, the underlying genes do
  not have readily recognizable orthologues in C.
  elegans.
• Nevertheless, conserved responses or interactions
  can often be detected.

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Neurodegeneration models in C.elegans
Kaletta et al. Nature Reviews Drug Discovery
advance online publication published online 21
April 2006 doi10.1038/nrd2031
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Alzheimers disease (AD)
The g-secretase protein quartet, and its roles in brain development and Alzheimer's disease. Presenilin-1, nicastrin, APH-1 and PEN-2 form a functional g-secretase complex, located in the plasma membrane and endoplasmic reticulum (ER) of neurons. The complex cleaves Notch (left) to generate a fragment (NICD) that moves to the nucleus and regulates the expression of genes involved in brain development and adult neuronal plasticity. The complex also helps in generating the amyloid b-peptide (Ab centre). This involves an initial cleavage of the amyloid precursor protein (APP) by an enzyme called BACE (or b-secretase). The g-secretase then liberates Ab, as well as an APP cytoplasmic fragment, which may move to the nucleus and regulate gene expression. Mutations in presenilin-1 that cause early-onset Alzheimer's disease enhance g-secretase activity and Ab production, and also perturb the ER calcium balance. Consequent neuronal degeneration may result from membrane-associated oxidative stress, induced by aggregating forms of Ab (which create Ab plaques), and by the perturbed calcium balance. Mattson, M. 2003 Nature
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Alzheimers disease model

• The Aß1-42 fragment is the main neurotoxic
  peptide generated from APP
• Invertebrate APP-like genes do not include the
  region encoding the neurotoxic Aß1-42 fragment
  and, therefore, direct disease modelling based on
  endogenous APP cleavage is not possible.
• The most developed invertebrate model of Aß1-42
  toxicity involves intracellular expression of a
  human Aß1-42 fragment in the C. elegans bodywall
  muscle, leading to adult-onset progressive
  paralysis and shortened lifespan
• In the nematode model, Aß1-42 forms cytoplasmic ß
  -amyloid with some properties similar to those
  found in diseased human brains

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Model of age-related protection against
proteotoxicity
Cohen et al. 2006

• DAF-2 represses two downstream pathways
  transcription factor HSF-1 and transcription
  factor DAF-16.
• Both provide protection against proteotoxicity
  of the amyloid 42 peptide, an aggregation-prone
  peptide that can spontaneously form small toxic
  aggregates.
• The default pathway, regulated by HSF-1,
  identifies and breaks apart toxic aggregates.
• When the HSF-1 machinery is overloaded, however,
  a molecular apparatus regulated by DAF-16 leads
  to formation of less toxic high-molecular-weight
  aggregates.

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Parkinsons disease

• PD is the most common neurodegenerative movement
  disorder.
• Approximately 1 of the population older than 65
  years suffers from this slowly progressive
  neurodegenerative disease 95 of PD cases are
  sporadic.
• The symptoms of PD are caused by selective and
  progressive degeneration of pigmented
  dopaminergic (DA) neurons in the substantia nigra
  pars compacta.
• Current treatments, such as administration of
  L-DOPA to produce dopamine, are only symptomatic
  and do not stop or delay the progressive loss of
  neurons.
• In fact, some studies have suggested that
  oxidative injury via dopamine may lead to further
  neuronal damage.
• 7 known genetic loci.
• PARK1 is mutation in the gene for a-synuclein
  (synaptic vesicle fusion).
• PARK2 is mutation in parkin, the ubiquitin ligase
  for a-synuclein.
• The a-synuclein protein is also seen in
  Alzheimers plaques.

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C. elegans Parkinsons disease models

• C. elegans has 302 neurons of which 8 are
  dopaminergic
• Expression of a human gene encoding the
  PD-associated protein, alpha-synuclein, in C.
  elegans neurons results in dosage and
  age-dependent neurodegeneration
• ?-synuclein-induced dopaminergic
  neurodegeneration could be rescued in these
  animals by torsinA, a protein with molecular
  chaperone activity

http//www.jove.com/index/details.stp?ID835
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Phenotypes in a worm model of human PD
postural and movement deficits
loss of neurons
Lakso et al., 2003
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up-regulated mitochondrial and proteasomal genes
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C. elegans Parkinsons disease models

• Impact of 6-hydroxydopamine (6-OHDA) on dopamine
  (DA) neurons in C. elegans
• intact green-fluorescing DA neurons in an
  untreated worm
• similar worm three days after exposure to 6-OHDA
• The partial loss of green fluorescence is
  indicative of progressive death of the dopamine
  neurons - due to destructive reactive oxygen
  species produced by 6-OHDA

Nass et al., 2002 PNAS
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• 6-OHDA has been found in brain and urine samples
  of PD patients
• DA transporter (DAT-1) in C. elegans is
  essentially the same, structurally and
  genetically, as the one found in mammals
• Blocking DAT-1 function through drugs or by
  genetic disruption eliminates 6-OHDA-sensitivity
  in the worms
• These results suggest that the toxic effect of
  6-OHDA occurs after it enters the DA neuron.

6-hydroxydopamine
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Huntington disease (HD)

• HD affects medium sized spiny neurons
• Uncontrollable growth of dendrites causes neurons
  to malfunction
• Parts of the brain affects include the cortex and
  basal ganglia, particular the caudate nucleus and
  the striatum
• Symptoms Involuntary chorei-form (dance-like)
  movements, psychological change, dementia
• Treatment aimed at symptoms, drugs that are
  helpful at one stage may not work at another
  stage
• Death usually occurs 15 to 20 years after onset
  of symptoms
• People die of complications to HD not HD itself -
  choking, infections, heart failure, pneumonia

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HD is a monogenic disease

• The disease is associated with increases in the
  length of a CAG triplet repeat present in a gene
  called 'huntingtin' (IT15) located on chromosome
  4p16.
• Wild-type huntingtin has a role in membrane
  trafficking in the cytoplasm and is also involved
  in microtubule-based axonal transport.
• The range of CAG repeat numbers is 9 to 37 in
  normal individuals and 37 to 86 in HD patients.

Huntingtin (Htt) domain model indicating the
location of the polyQ repeat, proline-rich domain
(PRD) and HEAT repeats. Selected interaction
partners are listed for the polyQ and PRD region
as well as the N-terminal region, including the
first HEAT repeat section
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• Mutant huntingtin binds to synaptic vesicles
  with higher affinity than does wild-type
  huntingtin and inhibits the uptake of glutamate
  by synaptic vesicles, suggesting that it might be
  able to affect synaptic homeostasis directly.
• Aggregates containing mutant huntingtin are
  usually located in the nucleus and cytoplasm, but
  they can also appear in the axon and nerve
  terminals.

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1. Immunoblot showing anti-GFP immunoreaction in C.
   elegans protein extracts using 34-day-old
   animals expressing different lengths of
   GFPHttpolyQ proteins
2. GFP fluorescence micrographs of young adult (34
   days old) C. elegans expressing different lengths
   of GFPpolyQ fusion proteins. Note that GFP
   fluorescence is mainly localized to the body wall
   muscle cells. Also, note that more compact foci
   form with increasing number of polyQ repeats
   expressed

Wang, H. et al. 2006 Hum. Mol. Genet.
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• C) Higher magnification showing the body wall of
  young adult C. elegans expressing different
  lengths of GFPpolyQ fusion proteins
• D) Motility assay measured as body bends per
  minute in wild-type and various transgenic lines
  of adult C. elegans.
• Note that the rate of movement decreases with
  increasing length of polyQ repeats.

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Ion-channel-mediated neurotoxicity

• If neurons are deprived of oxygen and the ATP
  stores drop, excess excitatory neurotransmitter
  glutamate is released into the synapse.
• Glutamate transporters, which usually rapidly
  clear synaptic glutamate, also fail to function
  appropriately in these conditions, leading to
  hyperactivation of glutamate-gated ion channels,
  excess ion influx, neuronal swelling and death.
• In C. elegans, a mutant acetylcholine receptor
  Ca2 channel DEG-3(d), and hyperactivated mutant
  variants of the degenerin Na channels cause
  neuronal swelling and death
• Transgenic expression of constitutively active
  GTPase-defective heterotrimeric G protein G?s can
  also induce necrotic-like neuronal death
• Because G proteins are known to influence
  ion-channel activity, G?s might induce neuronal
  degeneration by ion-channel hyperactivation

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• Hyperactivated ion channels, such as the MEC-4(d)
  Na channel, the DEG-3(d) Ca2 channel and
  possibly other channels that are affected if G?s
  is constitutively activated, conduct excess ions
  into neurons.
• This might induce the secondary release of
  calcium through ryanodine receptor (UNC-68) or
  InsP3R receptor (ITR-1) from ER stores, where it
  is bound by the ER chaperone proteins
  calreticulin and calnexin

• If intracellular calcium levels rise, specific
  calpain and aspartyl proteases are activated and
  the cell ultimately undergoes a necrotic death.
• In mammalian models of ischaemia and excitotoxic
  cell death, as well as in human neurodegenerative
  disease, activation of Ca2-activated calpain
  proteases is crucial for necrotic demise