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openwetware class on amyloid

Animal models

• "hemizygous" is when an animal has 1 copy of an inserted gene (e.g., PDAPP+/-); "heterozygous" is when 1 copy of a normal gene is knocked out (e.g., apoE-/+)
• PDAPP:
  • deposition at 7-8 months
  • homos more variable than hemis (?)
• APPsw:
  • 90% a-beta40 (higher than humans or PDAPP mice)

Name: PDAPP

• Promoter: platelet-derived growth factor (PDGF)-b
• Mutations: APP (V717F)
• Where: mostly CNS neurons
• Origin:
• Who: Indiana family
• hAPP levels: 10x higher than endogenous mouse APP
• Initial age of A-beta deposition: 6-9
• Fibrillar? Only ~10% of A-beta
• Where: hippocampus, cortex, corpus callosum, some cerebral arterioles
• Other (gliosis, CAA, etc): gliosis, a little CAA
• Dystrophy? yes
• Cell death? no
• Synaptic density? decreased
• Electrophysiology? abnormal
• Anatomy? abnormal
• Behavioral deficits / age? Yes, with plaque deposition

Name: Tg2576, APPsw

• Promoter: hamster prion protein
• Mutations: double mutation (K670N, M671L) in APP695
• Where:
• Origin:
• Who: Swedish family
• hAPP levels: 5-6x higher than endogenous mouse APP
• Initial age of A-beta deposition: 9-12
• Fibrillar:
• Where:
• Other (gliosis, CAA, etc): gliosis, wCAA
• Dystrophy? yes
• Cell death? no
• Synaptic density? decreased
• Electrophysiology? abnormal
• Anatomy?
• Behavioral deficits / age? Water maze 6 months (just prior to plaque deposition)… normal at 3 months (?). circular maze spatial learning deficits have been reported as early as 3 months of age for female, but not male, APPsw mice (King et al., 1999), although no gender effects were reported for the water maze task (Westerman et al., 2002; Hartman et al, 2006).

Name: APP23

• Promoter: murine Thy-1 (neuron specific)
• Mutations: overexpress human APP751 with Swedish double mutation (K670N, M671L) and London mutation (V717I)
• Where:
• Origin:
• Who: Swedish, London families
• hAPP levels: 7x higher than endogenous mouse APP
• Initial age of A-beta deposition: 6 months (cortex)
• Fibrillar: majority of even initial plaques
• Where: cortex, hippocampus
• Other (gliosis, CAA, etc): hyperphosphorylated Tau, gliosis, CAA, micro/macro hemorrhages
• Dystrophy? yes
• Cell death? 25% of CA1
• Synaptic density? decreased
• Electrophysiology? abnormal
• Anatomy?
• Behavioral deficits / age? Water maze (some yes, some no)

Name: PSAPP

• Promoter: murine Thy-1 (neuron specific)
• Mutations: APPsw (double mutation (K670N, M671L) in APP695) x PS1 mutation (M146L)
• Where:
• Origin:
• Who: Swedish, London families
• hAPP levels: 7x higher than endogenous mouse APP
• Initial age of A-beta deposition: majority A-beta42 (41% more than APPsw mice), fibrillar plaques in cortex at 3 months, to hippocampus by 6-8 months
• Fibrillar:
• Where: cortex, hippocampus
• Other (gliosis, CAA, etc): hyperphosphorylated Tau, gliosis, CAA, micro/macro hemorrhages
• Dystrophy? yes
• Cell death? 25% of CA1
• Synaptic density? decreased
• Electrophysiology? abnormal
• Anatomy?
• Behavioral deficits / age? Evidence for late (15-17 month) deficits (???)

Name: TgCRND8

• Promoter: prion
• Mutations: APP695 w/ Swedish (double mutation (K670N, M671L) in APP695) and Indiana (V717F)
• Where:
• Origin:
• Who: Swedish, Indiana families
• hAPP levels in brain: 5x higher than endogenous mouse APP
• Initial age of A-beta deposition: fibrillar deposits by 3 months
• Fibrillar:
• Where:
• Other (gliosis, CAA, etc): gliosis
• Dystrophy? yes
• Cell death?
• Synaptic density?
• Electrophysiology?
• Anatomy?
• Behavioral deficits / age? Water maze deficits by 11 weeks that worsens with age

Name: J20

• Promoter: PDGF (only difference between J20 and TgCRND8 mice)
• Mutations: APP695 w/ Swedish (double mutation (K670N, M671L) in APP695) and Indiana (V717F)
• Where:
• Origin:
• Who: Swedish, Indiana families
• hAPP levels in brain: 5x higher than endogenous mouse APP
• Initial age of A-beta deposition: 5-7 months in cortex and hippocampus
• Fibrillar:
• Where:
• Other (gliosis, CAA, etc):
• Dystrophy?
• Cell death?
• Synaptic density? Decreased even before amyloid deposition
• Electrophysiology?
• Anatomy?
• Behavioral deficits / age? Water maze probe deficits by 7 months

Name: APPDutch

• Promoter: neuron specific Thy-1
• Mutations: human APP w/ E693Q mutation (leads to higher levels of A-beta40 than 42 and CAA)
• Where:
• Origin:
• Who:
• hAPP levels in brain:
• Initial age of A-beta deposition: predominantly CAA
• Fibrillar:
• Where:
• Other (gliosis, CAA, etc):
• Dystrophy?
• Cell death?
• Synaptic density?
• Electrophysiology?
• Anatomy?
• Behavioral deficits / age?

Name: APPDutch x PS1

• Promoter: neuron specific Thy-1 (APPDutch… PS1 promoter?)
• Mutations: human APP w/ E693Q mutation (leads to higher levels of A-beta40 than 42) and human PS1 w/ G348A mutation (leads to higher levels of A-beta42 than 40)
• Where:
• Origin:
• Who:
• hAPP levels in brain:
• Initial age of A-beta deposition: cortex, hippocampus (less CAA than APPDutch mice)
• Fibrillar:
• Where:
• Other (gliosis, CAA, etc):
• Dystrophy?
• Cell death?
• Synaptic density?
• Electrophysiology?
• Anatomy?
• Behavioral deficits / age?

Name: Tg-SwDI

• Promoter: neuron specific Thy-1 (APPDutch… PS1 promoter?)
• Mutations: human APP770 w/ Swedish (K670N, M671L), Dutch (E693Q), and Iowa (D694N – leads to CAA) mutations
• Where:
• Origin:
• Who:
• hAPP levels in brain: A-beta40:42 ratio is 10:1
• Initial age of A-beta deposition: prominent fibrillar amyloid in the vasculature, parenchymal plaques by 3 months (almost exclusively diffuse)
• Fibrillar:
• Where:
• Other (gliosis, CAA, etc):
• Dystrophy?
• Cell death?
• Synaptic density?
• Electrophysiology?
• Anatomy?
• Behavioral deficits / age?

Name: APPsw x Arctic

• Promoter:
• Mutations: human APP770 w/ Swedish (K670N, M671L) and Arctic (E693G – very amyloidogenic)
• Where:
• Origin:
• Who:
• hAPP levels in brain:
• Initial age of A-beta deposition: intraneuronal A-beta aggregation (not fibrillar) before parenchymal deposition
• Fibrillar:
• Where:
• Other (gliosis, CAA, etc):
• Dystrophy?
• Cell death?
• Synaptic density?
• Electrophysiology?
• Anatomy?
• Behavioral deficits / age?

Name: BRI-Ab40 and BRI-Ab42

• Promoter:
• Mutations: 243 amino acids of BRI protein followed by a sequence of either (mutant?) A-beta40 or A-beta42…
• Where:
• Origin:
• Who:
• hAPP levels in brain: none
• Initial age of A-beta deposition:
• Fibrillar:
• Where:
• Other (gliosis, CAA, etc):
• Dystrophy?
• Cell death?
• Synaptic density?
• Electrophysiology?
• Anatomy?
• Behavioral deficits / age?

Misc

• a-beta is a short peptide (protein fragment) derived from larger APP protein
• "diffuse" a-beta is in an alpha-helix configuration, "amyloid" is in a beta-sheet configuration
• APP molecules have one end embedded in the neuronal membrane
• 2 proteases, beta and gamma, cut a-beta from APP
• a-beta production may be part of the normal signaling pathway
• the membrane ends of a-beta are composed of water repelling lipids that cling to each other, forming small soluble assemblies that can interfere with LTP
• these assemblies form fibers in vitro that are toxic to cultured neurons
• APP and presenilin (part of gamma secretase complex) mutations result in increased a-beta / a-beta42
  • chromosome 21, APP, Down
• apoE "brings together" a-beta peptides into assemblies/filaments
  • apoE (and apoJ) are high density lipoproteins
  • produced in liver, glia, neurons
  • changes a-beta to beta-sheet (amyloid) conformation in vitro

apoE-/- mice get diffuse, but not amyloid, a-beta deposition

• apoJ is the 2nd most abundant lipoprotein
  • apoJ-/- mice get fibrillar plaques, but no neuritic dystrophy
    • amyloid does not appear to be toxic, so apoJ facilitates a-beta's neurotoxicity?
• these extracellular aggregates can initiate intracellular cascades, including kinases that add phosphates onto tau proteins (twisting them and thus killing the cell)
• tau mutations can have similar effects
• thus, a-beta is the initiator of AD, and tangled tau filaments are a general event leading to cell death

Names / discoveries

• 1984 George Glenner / Caine Wong characterized a-beta peptide
• early 90s Peter Lansbury - abeta can form fibers in vitro
• 1991 John Hardy discoved APP mutations in FAD
• then Dennis Selkoe / Steve Younkin - APP mutations result in more Ab
• 1995 Peter St George-Hyslop found Presenilin 1 & 2 mutations
• 1998 Bart de Strooper - reduced gamma secretase cleavage of APP in presenilin1-/- mice
• then Michael Wolfe - aspartyl protease inhibitors block gamma secretase cleavage of APP
• 1999 Dale Schenk / Elan - active Ab immunization prevented / reduced plaques in APP tg mice
• then Eddie Koo / Todd Golde found Flurizan, which prevents formation of fibrillar Ab (?)
• then Cindy Lemere - active immunization w/ Ab fragments can stimulate antibody-producing B cells w/o triggering encephalitis-causing T cells
• then Mark Tuszynski - skin cells with NGF genes injected into brains of AD patients

Alzheimer's stats

• 1 in 10 over 65
• 1 in 2 over 85
• Slowing progress by 5 years 1/2s prevalence

• Brain cells fire in patterns - Pinker

Alzheimer's symptoms

age of onset

• early onset familial
• spontaneous

Neuropathology

• A-beta40/42 ratio:

high -> parenchymal deposition

low -> vascular deposition

• Cholesterol:

statin drugs lower cholesterol and risk for AD

increase membrane fluidity???

increases processing of APP by alpha-secretase

reducing INTRACELLULAR cholesterol inhibits Abeta formation

impairs vascular perfusion

high dietary cholesterol increases Abeta deposition

BUT dietary cholesterol barely crosses the BBB

brain cholesterol is mainly in the myelin

• Low-density lipoproteins (LDLs)

more vulnerable to free radical damage

damaged LDL may clump together

• DHA

comprises 17% of brain’s total fatty acids

gray matter is 30-40% DHA

reduces risk of AD

decreases Abeta deposition

improves vascular health & regional cerebral blood volume (rCBV)

• Phospholipids

omega 3 fatty acids (e.g. DHA) - increase membrane fluidity

omega 6 fatty acids (e.g. arachadonic acid) - decrease membrane fluidity ???

• Altered levels of phospholipid and cholesterol in the membrane can modulate the activity of membrane-bound enzymes (e.g., the secretases)

beta & gamma secretase require cholesterol-rich “lipid rafts” within the membrane

alpha-secretase requires a cholesterol-poor / phospholipid-rich(???) / MORE FLUID membrane

Risk factors

• Risk factors:

apoe4

TBI

hypoperfusion

high cholesterol / carb diet

• apoE4 genotype (cholesterol transporter)

risk factor for:

AD

synergistic with injury

hypercholesterol

vascular dementia

less of a risk for hemorrhagic stroke?

APP

• Transmembrane protein encoded on chromosome 21
• Isoforms range from 365-770 amino acids long
• in brain, most is 695-770 long
• 2 extracellular domains - E1 & E2
• APP found IN vesicles (intracellular, but inside of vesicle is released extracellularly)
• Alpha-secretase
• TACE
• ADAM-10
• Beta-secretase
• BACE1
• memapsin-2
• transmembrane - extracellular domain does cutting
• Gamma-secretase
• composed of presenilin
• also cleaves (?) notch receptor???
• As this cleavage occurs at the Lys16-Leu17 bond within the amyloid beta domain, it prevents deposition of intact amyloidogenic peptide.
• PS-1 is an integral membrane protein expressed in neurons and is localized primarily in the endoplasmic reticulum (ER). PS-1 mutations may promote neuronal degeneration by altering the processing of the beta-amyloid precursor protein (APP) and/or by engaging apoptotic pathways. Alternative processing of APP in AD may increase production of neurotoxic amyloid beta-peptide (Abeta) and reduce production of the neuroprotective alpha-secretase-derived form of APP (sAPPalpha).
• 30+ mutations have been identified that increase A_beta production

some APP (chromosome 21)

some presenilin 1 (chromosome 14) 50% OF FAD

some presenilin 2 (chromosome 1)

• • APP, PS1, and PS2 mutations all increase a-beta production by 1.5-3x
• Active Abeta42 immunization induced encephalitis
• Plaques:

abeta

Apoe

Zinc / iron / copper traces

chelating can break up plaques

into toxic oligomers????

Hemes - can cause heme deficiency

(hemes + Abeta complexes function as a peroxidase, causing damage to muscarnic receptors

polyphenols can ameliorate this effect)

• Abeta:

C-terminus (from membrane) is water-repelling lipid that clings together

in plaques, the 40-42 (n-terminus) end is on the outside of the plaques

Abeta is generated IN the cell - NOT at cell surface???

gets degraded by Lysosomes

OR

gets secreted by a “recycling endosome”

release via synaptic activity ala Cirrito?

casein kinase 1 - control bace & gamma sec?

• A-beta:

APP snipped by enzymes

Normally 39-43 amino acids long

the 2 extras in 42 cause protein to fold abnormally and clump together (oligomers)

• • it is the ends cleaved from within the membrane that stick together

• Beta-secretase (extracellular)

APPsol_beta

membrane-bound beta C-terminus fragment (CTF) [contains what will become Abeta]

• C-terminus fragment cleaved in membrane by gamma secretase

Abeta1-40 OR Abeta1-42 (WHY ONE OR THE OTHER)

has to do whether gamma-sec is associated with endoplasmic reticulum OR golgi apparatus???

gamma CTF - aka AICD - APP Intracellular Cellular Domain???

different isoforms?

goes to nucleus & affects calcium signaling

gamma-secretase inhibitors could interact

all other fragments end up extracellular (?)

• OR
• alpha secretase (extracellular - ~12 amino acids closer to membrane than beta)

APPsol_alpha (longer than APPsol_beta)

membrane-bound alpha C-terminus fragment (CTF) [contains what will become P3]

• C-terminus fragment cleaved in membrane by gamma secretase

P3 = ~Abeta1-32ish (fragment between gamma & alpha)

gamma CTF - aka AICD - APP Intracellular Cellular Domain???

different isoforms?

goes to nucleus & affects calcium signaling

gamma-secretase inhibitors could interact

all other fragments end up extracellular (?)

• ASHE 2007

APP is cleaved at alpha, beta, gamma, and epsilon-secretase sites:

releasing:

amino-terminal, internal, and carboxy-terminal polypeptides:

AICD (app intracellular domain / carboxy-terminal fragment epsilon (CTF-e)

alpha-sec: soluble APP alpha (sAPPa) - neuroprotectant/enhancer)

BACE1 cleavage, followed by y- and e- release Ab and AICD

• M1 and M3 muscarnic ACh receptors stimulates alpha-sectretase

• Type 2 Diabetes:

high blood glucose from reduced pancreatic insulin production

Type 3???

reduced brain insulin production

Therapeutics

• PJ:

reduced Abeta levels

increased alpha?

decreased beta?

altered gamma (less 42)?

• Ellagic acid and punicalagin are BACE inhibitors

Antioxidant

Anti-inflammatory

Improves lipid profile (lowers LDL, raises HDL)

Improves vasculature measures

• Eli Lilly - gamma secretase inhibitors (phase 1 trials)
• Elan - passive immunization (phase 2 trials)
• Neurochem - Alzhemed... blocks heparin from forming Ab plaques from soluble Ab (phase 3 trials)