Toxic Proteins in Neurodegenerative Diseases

1
Toxic Proteins in Neurodegenerative Diseases

• Neurodegenerative disorders as diverse as
  Alzheimers disease, Parkinsons disease, prion
  diseases, Huntingtons disease, frontotemporal
  dementia, and motor neuron disease all share a
  conspicuous common characteristicsabnormal
  processing, aggregation and deposition of
  misfolded protein in neuronal inclusions and
  plaques.

(Taylor et al., 2002, Science)
2
Aggregation of misfolded proteins in
microscopically visible inclusions or plaques in
various neurodegenerative diseases. (A)
Alzheimers disease. Arrowhead, intracellular
neurofibrillary tangles arrow, extracellular
amyloid plaque. (B) Fibrillar tau inclusions in
Picks disease. (C) PrPSc amyloid deposition in
prion disease. (D) Multiple Lewy bodies in a
nigral neuron in Parkinson disease. (E) Neuronal
intranuclear inclusions of mutant ataxin-3 in
Machado-Josephs disease. (F) Higher power
micrograph of nuclear inclusion of mutant
ataxin-3, demonstrating that it is distinct from
the nucleolus.
(Taylor et al., 2002, Science)
3
Alzheimers disease

• Alzheimers disease is the most common
  neurodegenerative disease causing the dementia,
  Pathologically, the disease is characterized by
  the presence of two lesions an extracellular
  plaque made up largely of the b-amyloid peptide
  (Ab), and an intracellular tangle made up largely
  of the cytoskeletal protein tau.
• The main components of extracellular plaque are
  the amyloid b-peptides, Ab40 and Ab42, which
  contain 40 and 42 amino acids, respectively, and
  appear to be toxic to neurons.
• Ab is generated from b-APP (b-amyloid precursor
  protein) by endoproteolytic processin involving
  sequential cleavages by b- and g-secretase. The
  name secretase reflects the fact that their
  substrates are liberated from the membrane and
  are then found in extracellular fluids.

4
. Proteolytic processing of
b-APP The initial cleavage of b-APP by
b-secretase generates membrane-bound carboxyl
(C)-terminal fragment (CTF-b) starting at the
Asp1 amino (N)-terminus of the Ab domain.
Subsequently, g-secretase mediates the apparently
intramembraneous cleavage of CTF-b resulting in
the liberation of Ab. In an alternative pathway,
b-APP can be cleaved by a-secretase in the center
of the Ab domain, thereby inhibiting generation
of Ab.
(Walter et al., 2001)
5
The etiology of Alzheimers disease

• Under normal cellular conditions, Ab is
  constitutively released from APP-expressing
  cells. Currently, the most compelling hypothesis
  on the etiology of Alzheimer's disease (AD)
  centers on the overproduction of b-amyloid
  peptide generated through sequential cleavages of
  the amyloid precursor protein (APP) by two
  proteases, b- and g -secretases.
• In addition to non-familial form of AD, mutation
  of two genes also cause Alzheimers disease
  inherited as an autosomal dominant disorder of
  mid-life. These genes include the amyloid
  precursor protein (APP) gene and the presenilin
  protein genes (PS1 and PS2).
• \
• Missense mutations of APP are believed to result
  in overproduction of Ab40 and Ab42 peptides.
  However, APP mutation is the rare cause of
  familial autosomal dominant AD.

6

• The g-secretase is a multimeric complex
  consisting of presenilins (PS-1 and PS-2), Aph-1,
  Pen-2, and nicastrin. PSs seem to be the key
  contributors for the active site of the enzyme.
  Thus, g-secretase activity is abolished after
  knockout of PS genes.
• Missense mutations in the presenilins is the most
  common cause of autosomal dominant AD. Presenilin
  1 missense mutations cause the earliest and most
  aggressive form of AD, commonly leading to onset
  of symptoms before the age of 50 and demise of
  the patient in his/her 60s.
• Previous studies using transfected cells and
  transgenic mice demonstrated that presenilin
  mutations causing AD lead to an overproduction of
  Ab42 peptides.
• .

7
Alzheimers disease

• Amyloid cascade hypothesis----Ab deposition is
  intimately connected to the initiation of
  Alzheimer pathogenesis and that all the other
  features the tangles and the cell and synapse
  loss are secondary to this initiation.
• Previous studies using transgenic mice model of
  Alzheimers disease showed that Ab levels are
  elevated without plaque formation or nerve cell
  loss, yet learning and memory deficits are
  evident. Thus, it has been hypothesized that a
  soluble form (dimers and/or small oligomers) of
  Ab is mainly responsible for the disruption of
  neuronal signaling, particularly at an early
  stage in Alzheimers disease.

8
Alzheimers disease and cholinergic
transmisssion

• Alzheimers disease (AD) is characterized by an
  increasing loss of cognitive function and
  accompanied by various deficits in cholinergi
  neurotransmission, including the loss of
  cholinergic neurons in the basal forebrain,
  decrease in release of ACh, and decrease in
  choline acetyltransferase activity. This suggests
  that impairment of the cholinergic system may
  occur early in AD and lead to cognitive deficits
• Potential targets in AD pathology are the
  nicotinic AChRs because they are widely expressed
  throughout the CNS, they are known to participate
  in cognition, and AD patients exhibit decreased
  numbers. It was reported that Ab42 binds the a7
  and non-a7 subtypes of nicotinic AChRs.
• Ab42 inhibits the cholinergic transmission by
  directly blocking the postsynaptic nicotinic AChR
  channels. Chronic inhibition of cholinergic
  signaling by Ab42 could contribute to the
  cognitive deficits and loss of cholinergic
  function associated with Alzheimers disease.

9
Ab42 inhibits Ach-evoked whole-cell and
single-channel currents from rat hippocampal
interneurons by directly blocking the
postsynaptic nicotinic AChR channels.
(Pettit et al., J. Neurosci., vol. 21, 2001)
10
Ab42 impairs the LTP induction in the hippocampus

• Long-term potentiation (LTP) is a
  neurophysiological model of activity dependent
  changes in synaptic strength that are believed to
  underlie information storage in the hippocampus
  and neocortex.
• Ab-containing C-terminus of b-APP (CT) blocked
  the LTP induction in the intact hippocampus at a
  time when baseline transmission was not affected
  (Kim et al., J. Neurosci., 2001, 2113271333).
• Ab42 and CT facilitate the induction of long-term
  depression (LTD) in vivo. The Ab-facilitated LTD
  was blocked by the NMDA receptor antagonist
  D-(2)-2-amino-5-phosphonopentanoic acid (D-AP5).

11
The C-terminal fragment of b-amyloid precursor
protein (CT) reverses the LTP induction in the
intact hippocampus.
Ab42 facilitates the induction of LTD in the rat
hippocampus in vivo.
(Kim et al., J. Neurosci., 2001, 2113271333)
12
Molecular mechanisms of Ab-induced neuronal death

• The b-amyloid plaques activate microglia and
  monocytes, which results in production of
  neurotoxic secretory products, proinflammatory
  cytokines, and reactive oxygen species. b-amyloid
  peptides increases the production of cytokines,
  TNFa and IL1-b, through activating Src or Syk
  family tyrosine kinases. Subsequently, TNFa
  causes the apoptotic death of neurons by
  increasing the expression of iNOS and
  peroxynitrite.

Ab-stimulated microglial and monocytic
proinflammatory products cause TNFa/iNOS-dependent
neuronal apoptosis. (Combs et al., J.
Neurosci., 2001, 21, 11791188)
13
Molecular mechanisms of Ab-induced neuronal death

• The fibrillar form ofAb peptides activates c-Jun
  N-terminal kinase (JNK) is required for the
  phosphorylation and activation of the c-Jun
  transcription factor, which in turn stimulates
  the transcription of several key target genes,
  including the death inducer Fas ligand. The
  binding of Fas ligand to its receptor Fas then
  induces a cascade of events that lead to caspase
  activation and ultimately neuronal death.
• ( Morishima et al., J. Neurosci., 2001, 21,
  75517560)

Ab upregulates Fas ligand expression.
Ab activates c-Jun and JNK in cortical neurons.
14

• Prion Diseases
• Prions are defined as small proteinaceous
  infectious particles that resist inactivation by
  procedures which modify nucleic acids (Prusiner,
  S.B. , 1982. Science, 216, pp134)
• Scrapie, bovine spongiform encephalopathy (BSE),
  and human CreutzfeldtJakob disease (CJD) are
  examples of prion diseases or transmissible
  spongiform encephalopathies (TSEs). These
  diseases result in fatal neurodegenerative
  conditions. The main hallmarks of prion diseases
  are neuronal cell death, vacuolation of neuropil,
  and astrogliosis.
• The genetic code of the prion protein (PrPc) was
  identified only after the isolation of an
  abnormal isoform (PrPSc) from brains of mice that
  were infected with the disease scrapie (Prusiner,
  S.B. , 1982. Science, 216, pp134). The one
  consistent pathological feature of the prion
  diseases is the accumulation of amyloid material
  that is immunopositive for prion protein (PrP),
  which is encoded by a single gene on the short
  arm of chromosome 20.

15
Normal PrP (PrPC) and disease-causing PrP (PrPSc)

• Substantial evidence now supports the contention
  that prions consist of an abnormal isoform of
  PrP. Structural analysis indicates that normal
  cellular PrP (designated PrPC) is a soluble
  protein rich in a helix with little b-pleated
  sheet content.
• PrP extracted from the brains of affected
  individuals (designated PrPSc) is highly
  aggregated and detergent insoluble. PrPSc is less
  rich in a helix and has a greater content of
  b-pleated sheet.
• Amino acid sequences of PrPC and PrPSc are
  identical. They differ only in their
  three-dimensional conformation.

The primary structure of the mouse prion protein.
This protein is anchored to the cell membrane by
a glycosylphosphatidylinositol (GPI) anchor. The
signal peptide for entry into the endoplasmic
reticulum and the GPI signal peptide are cleaved
before the protein reaches the cell surface.
(Brown, TINS, 2001)
16
Prion diseases are thought to propagate by a
high-fidelity process in which PrPSc
self-replicates by templating the conformational
rearrangement of endogenous PrPC

• The PrP is believed to fluctuate between a native
  state (PrPC) and a series of additional
  conformations, one or a set of which may
  self-associate to produce a stable supramolecular
  structure composed of misfolded PrP monomers.
  Thus, PrPSc may serve as a template that promotes
  the conversion of PrPC to PrPSc.
• Initiation of a pathogenic self-propagating
  conversion reaction may be induced by exposure to
  a seed of b-sheetrich PrP after prion
  inoculation, thus accounting for
  transmissibility.
• The conversion reaction may also depend on an
  additional, species-specific factor termed
  protein X. Alternatively, aggregation and
  deposition of PrPSc may be a consequence of a
  rare, stochastic conformational change leading to
  sporadic cases. Hereditary prion disease is
  likely a consequence of a pathogenic mutation tha
  predisposes PrPC to the PrPSc structure.

17
Possible mechanism for prion propagation. Largely
a-helical PrPC proceed via an unfolded state (A)
into a largely b-sheet form, b-PrP (B). b-PRP is
prone to aggregation. Prion replication may
require PrPSc as a critical seed. Further
recruitment of b-PRP and unfolded PrP then
occurs as an essentially irreversible process
(Collinge, Annu. Rev. Neurosci., 2001, 24,
519-550).
18
Physiological functions of normal prion protein

• PrPc is a glycoprotein expressed on the surface
  of many cells including neurons and binds copper.
  PrPc expression has been shown both
  presynaptically and postsynaptically. PrPc
  knockout mice that were studied
  electrophysiologically were found to have altered
  GABA-type inhibitory currents and also altered
  LTP.
• There is also evidence for differences in the
  responses to stress-inducing agents, such as
  exogenous copper and hydrogen peroxide. At the
  cellular level, PrPc-deficient cells are less
  viable in culture compared with wild-type cells
  and are more susceptible to oxidative damage and
  toxicity caused by agents such as copper and
  cytosine arabinoside. In addition, astrocytes
  show changes in their ability to take up
  glutamate. Therefore, at all levels
  PrPc-deficient mice show a clear phenotype
  indicating that they are more sensitive to
  various kinds of stresses. These data imply that
  PrPc has a neuroprotective effect.

19

• There is increasing evidence that the normal
  prion protein binds copper and the resulting
  complex possesses anti-oxidant activity, and that
  this might have an important role in regulating
  the synaptic transmission.

.
(Brown, 2001, 24, pp85)
PrPSc could induce neurodegeration by causing
the loss of PrPc functions.
20
Cross-Linking Cellular Prion Protein Triggers
NeuronalApoptosis in Vivo (Solforosi et al.,
Science, 2004, 303, pp1514)

• In the absence of PrPC, PrPSc does not produce
  neurotoxic effects, suggesting that PrPC itself
  may participate directly in the prion
  neurodegenerative cascade.
• A recent study by Solforosi et al showed that
  cross-linking PrPC in vivo with specific
  monoclonal antibodies was found to trigger rapid
  and extensive apoptosis in hippocampal and
  cerebellar neurons. These findings suggest that
  PrPC functions in the control of neuronal
  survival and provides a model to explore whether
  cross-linking of PrPC by oligomeric PrPSc can
  promote neuronal loss during prion infection.

21
PrPC-specific antibody triggers neuronal
apoptosis. (DAPI)-labeled neuronal cell nuclei
(left panels), TUNEL staining of apoptotic cells
(center panels), and a merge of these two images
(right panels). (A) Control anti-HIV-1 envelope
glycoprotein gp120 IgG b12 (B) Monoclonal
anti-PrPC IgG P, (C) Monoclonal anti-PrPC IgG D13.
PrPC-specific antibodies mediate neuron death in
the hippocampus. (B) and (E) Control anti- HIV-1
envelope glycoprotein gp120 IgG b12 (C)
Monoclonal anti-PrPC IgG P (F) Monoclonal
anti-PrPC IgG D13
22
Polyglutamine neurodegenerative disorders

• The expansion of an unstable CAG repeat within
  the coding region of gene, is the major cause of
  hereditary neurodegenerative diseases. Since the
  CAG repeat encodes a polyglutamine tract in the
  respective proteins, these neurological disorders
  are characterized as polyglutamine diseases. Up
  to now, at least nine polyglutamine diseases have
  been identified, including Huntington disease
  (HD), spinobulbar muscular atropy (SBMA),
  dentatorubral pallidoluysian atrophy (DRPLA) and
  six forms of spinocerebellar ataxias.

  Molecular characteristics of
polyglutamine diseases __________________________
________________________________ Disease
Protein Normal CAG (n)
Pathological CAG (n) Huntingtons disease
Huntingtin 6-35
38-180 SBMA Androgen
receptor 9-36
38-65 DRPLA Atropin-1
6-36
49-88 SCA1 Ataxin-1
6-44
39-82 SCA2 Ataxin-2
15-31
34-64 SCA3 Ataxin-3
12-41
62-84 SCA6 a1A Ca channel
4-18 21-33 SCA7
Ataxin-7
4-35 37-306 SCA17
TATA-binding protein 29-42
47-55 ______________________
_____________________________________________
23
Mutant huntingtin activates the apoptotic pathway

• Huntingtons disease (HD) is a devastating
  inherited before neurological symptoms and
  neurodegeneration neurodegenerative disease
  characterized by chorea, personality changes,
  dementia, and early death.
• HD leads to significant cell death of the
  enkephalin-containing medium spiny neurons within
  the striatum, and to a lesser extent within the
  cortex.
• It has been reported that mutant huntingtin acts
  within the nucleus and induces the apoptotic
  death of striatal neurons (Saudou et al., Cell,
  95, 1998,5566). Another study suggests that
  expression of mutant huntingtin leads to the
  recruitment of caspase-8 to the polyglutamine
  aggregates, resulting in the activation of
  apoptotic pathway (Sanchez et al., Neuron, 1999,
  22, 623-633).

24
Wild-type Htt
Mutant Htt
Wild-type Htt
Mutant Htt
Mutant huntingtin-induced apoptosis is prevented
by antiapoptotic compounds and neurotrophic
factors.
25
The huntingtin interacting protein (HIP-1) is
involved in mutant huntingtin-induced apoptosis

• The huntingtin interact ingprotein (HIP-1) was
  identified by its altered interaction with mutant
  huntingtin and acts as a proapoptotic protein.
  HIP-1 contains a death effector domains (DEDs )
  and activates caspase-3. pathway of apoptosis in
  HIP-1-induced cell death (Hackam, et al., J.
  Biol. Chem., 2000, 275, pp. 4129941308).
• Co-expression of a normal huntingtin fragment
  capable of binding HIP-1 significantly reduced
  cell death. The affinity of Hip-1 for Htt is
  reduced by the presence of expanded
  poly-glutamine, resulting in elevated free Hip-1,
  which forms heterodimers with Hippi. Hip-1/Hippi
  complexes are able to bind and activate caspase-8
  and thus induce apoptosis (Gervais et al., Nat
  Cell Biol 2002, 495-105).

26
HIP-1 induces cell death by activating caspase-3.
Coexpression of wild-type huntingtin reduced
HIP-1-induced cell death
Wild-type Htt Mutant Htt
27
Mutant huntingtin expressed in nucleus could
induce neuronal death and dysfunction by causing
transcriptional dysregulation.

• Mutant polyglutamine-expanded huntingtin has been
  shown to interact with short glutamine stretches
  present in many transcription factors including
  p53, Sp1 and CBP, sequestering them away from
  their targets. For example, transcriptional
  coactivator CREB-binding protein (CBP) binds
  specifically to huntingtin in an expanded
  polyglutamine-dependent manner.
• Mutant huntingtin represses CBP-mediated
  transcription (Nucifora et al., Science, 2000,
  291, 2423-2427). This finding suggests that
  mutant huntingtin causes aberrant transcriptional
  regulation through its interaction with cellular
  transcription factors, which may result in
  neuronal dysfunction and cell death in HD.

28
Control mouse HD mouse
CBP is present in huntingtin nuclear inclusions
of HD transgenic mouse.
Wild-type Htt
Primary rat cortical neurons were transfected
with Gal4-CBP luciferase reporter and cDNA of
wild-type or mutant huntingtin. Note that mutant
huntingtin decreases CBP-mediated transcription.
Mutant Htt
29
Mutant huntingtin-induced neuronal dysfunction
partially mediates the the manifestation of
clinical symptoms.

• In mouse model of Huntingtons disease, mice
  manifested behavioral and motor dysfunction
  without a severe neuronal cell death in the
  striatum. Therefore, the pathogenesis of
  Huntingtons disease can be divided into two
  steps, an early phase of neuronal dysfunction and
  a later phase of eventual neuronal death. Both
  phases of pathological events are responsible for
  the manifestation of clinical symptoms.
• Previous studies using HD transgenic mice
  suggested that alteration of morphology and
  synaptic transmission in striatal and neocortical
  neurons lead to neuronal dysfunction and motor
  deficits (Laforet et al., J. Neurosci., 2001,
  21(23)91129123).

30
Striatum Neocortex
Striatal neurons from HD mice have significantly
more dendrites with endings that curved back
toward the soma (J-dendrites) and/or had sharp
bends (wavy dendrites) compared with WT mice ( A
and B). Cortical WT neuron has smooth dendrites
and extension of the apical dendrite to the pial
surface (D). A cortical pyramidal neuron from an
HD mouse with a disoriented apical dendrite (E).
NMDA response
EPSP
In striatal neurons of HD mice, the amplitude of
EPSP is slightly decreased, and NMDA response is
potentiated.