Transcript Histone modifications in Huntington`s Disease

Mechanisms of Disease:
histone modifications in
Huntington's disease
Nature Clinical Practice Neurology (2006) 2, 330-338
In 1872, the American physician George Huntington
wrote about an illness that he called "an heirloom
from generations away back in the dim past." and
described the disorder in his first paper "On
Chorea" at the age of 22
"In
the history of medicine, there are few instances in which
a disease has been more accurately, more graphically or
more briefly described."
*One of its earliest names was chorea, which, as in
"choreography," is the Greek word for dance. The term
chorea describes how people affected with the disorder
twist, and turn in a constant, uncontrollable dance--like
motion.
*"Hereditary chorea" emphasizes how the disease is
passed from parent to child.
*"Chronic progressive chorea" stresses how symptoms
of the disease worsen over time.
*Today, physicians commonly use the simple term
Huntington's disease (HD) to describe this highly
complex disorder that causes untold suffering for
thousands of families.
*Examining the combined medical history of several
generations of a family exhibiting similar symptoms, he
realized their conditions must be linked; he presented
his detailed and accurate definition of the disease as
his first paper. Unknowingly, described the exact
pattern of inheritance of autosomal dominant disease
years before the rediscovery of Mendelian inheritance
*During the rediscovery of Mendelian inheritance at the
turn of the 20th century, HD was used tentatively as an
example of autosomal dominant inheritance
*Huntingtin (Htt), the protein that is defective in HD, is
expressed throughout the body, principally affects the
brain.
*including the cortex, thalamus and sub thalamic
nucleus, the striatum is the most severely affected
region.
*In striatum, the medium spiny projection neurons are
preferentially targeted by the disease and interneurons
are relatively spared
*preferential loss of medium spiny neurons and sparing
of interneuron populations indicates that
neurodegeneration in HD is cell-type-specific.
*Htt is expressed in all mammalian cells.
*The highest concentrations are found in the brain and
testes
*The function of Htt in humans is unclear.
*It interacts with proteins which are involved in transcription,
cell signaling and intracellular transporting.
*In animals genetically modified to exhibit HD, several
functions of Htt have been found.
*In these animals, Htt is important for embryonic
development, as its absence is related to embryonic death.
*It also acts as an anti-apoptotic agent preventing
programmed cell death and controls the production of
brain-derived neurotrophic factor, a protein which protects
neurons and regulates their creation during neurogenesis.
*Htt also facilitates vesicular transport and synaptic
transmission and controls neuronal gene transcription.
*If the expression of Htt is increased and more Htt
produced, brain cell survival is improved and the effects of
mHtt are reduced, whereas when the expression of Htt is
reduced, the resulting characteristics are more typical of
the presence of mHtt.
* In humans the disruption of the normal gene does not
cause the disease.
*It is currently concluded that the disease is not caused by
inadequate production of Htt, but by a gain of toxic function
of mHtt.
THE HUNTINGTON'S DISEASE MUTATION
*is caused by a mutation in the IT15 gene on the short arm
of chromosome 4.
*This gene, which was
renamed HD, consists of 67 exons
that encode Htt, a 350-kD protein of 3,144 amino acids.
*mutation is an expansion of the cytosine–adenine–guanine
(CAG) trinucleotide repeat in exon 1, which codes for a pg
moiety in the Htt protein.
*Normal individuals have CAG repeat lengths of 7–34.
*CAG repeat is expanded and unstable in HD patients
*Repeat lengths of more than 40 glutamines produce HD,
and repeats of over 70 glutamines invariably cause juvenile
onset.
*When the HD mutation was eventually identified as a CAG
trinucleotide repeat expansion, HD joined a novel class of
neurodegenerative disease, the polyglutamine
diseases(PGD)
*expansion of the CAG repeat occurs within the coding
region of the gene, so the CAG repeat is translated into a
polyglutamine stretch
* PGD are all autosomal dominant or sex-linked dominant
diseases
*In both humans and transgenic mouse models, these
disorders are characterized pathologically by pg proteincontaining intracellular inclusions
*the pg moiety is strongly implicated as the portion of the
protein that makes the greatest contribution to disease
*As the disease progresses, concentration on
intellectual tasks becomes increasingly difficult.
*the disease may begin with uncontrolled movements in
the fingers, feet, face,….
*The disease can reach the point where speech is
slurred and vital functions, such as swallowing, eating,
speaking, and especially walking, continue to decline.
Some individuals cannot recognize other family
members
*The most common causes of death are infection (most
often pneumonia), injuries related to a fall, or other
complications
*Huntington's disease has autosomal dominant
inheritance, meaning that an affected individual
typically inherits one copy of the gene with an
expanded trinucleotide repeat (the mutant allele) from
an affected parent.
*In this type of inheritance pattern, each offspring of an
affected individual has a 50% risk of inheriting the
mutant allele and therefore being affected with the
disorder . This probability is sex-independent.
Is there any age limit…………………..
*Some individuals develop symptoms of HD when they
are very young--before age 20.--"early-onset" or
"juvenile" HD
*and death often follows within 10 years.
*A few individuals develop HD after age 55.
*Diagnosis in these people can be very difficult.
* The symptoms of HD may be masked by other health
problems, or the person may not display the severity of
symptoms seen in individuals with HD of earlier onset.
A baby History
*To verify the link between the number of CAG repeats
in the HD gene and the age at onset of symptoms,
scientists studied a boy who developed HD symptoms
at the age of two, one of the youngest and most severe
cases ever recorded.
*They found that he had the largest number of CAG
repeats of anyone studied so far--nearly 100.
* The boy's case was central to the identification of the
HD gene and at the same time helped confirm that
juveniles with HD have the longest segments of CAG
repeats, the only proven correlation between repeat
length and age at onset.
Heart Breaking or Interesting……?
*The great American folk singer and composer Woody
Guthrie died on October 3, 1967, after suffering from
HD for 13 years.
* He had been misdiagnosed, considered an alcoholic,
and shuttled in and out of mental institutions and
hospitals for years before being properly diagnosed.
*His case, sadly, is not extraordinary, although the
diagnosis can be made easily by experienced
neurologists
*The physician may ask the individual to undergo a brain
imaging test. Computed tomography (CT) and magnetic
resonance imaging (MRI) provide excellent images of brain
structures with little if any discomfort.
*Those with HD may show shrinkage of some parts of the
brain—particularly two areas known as the caudate nuclei
and putamen—and enlargement of fluid-filled cavities within
the brain called ventricles. These changes do not definitely
indicate HD,
*When used in conjunction with a family history and record
of clinical symptoms, however, CT can be an important
diagnostic tool.
*Another technology for brain imaging includes positron
emission tomography (PET,) which is important in HD
research efforts but is not often needed for diagnosis.
*The most prominent early effects are in a part of the
basal ganglia called the neostriatum, which is
composed of the caudate nucleus and putamen.
*Other areas affected include the substantia nigra,
layers of cerebral cortex, the hippocampus, purkinje
cells in the cerebellum, lateral tuberal nuclei of the
hypothalamus
*reducing in size as they lose cells Striatal spiny
neurons
*The basal ganglia—the part of the brain most
prominently affected by HD—play a key role in
movement and behavior control.
*current theories propose that they are part of the
Area of the brain damaged
by Huntington's disease –
striatum (shown in purple)
*The basal ganglia ordinarily inhibit a large number of
circuits that generate specific movements.
* To initiate a particular movement, the cerebral cortex
sends a signal to the basal ganglia that causes the
inhibition to be released.
*Damage to the basal ganglia can cause the release or
reinstatement of the inhibitions to be erratic and
uncontrolled, which results in an uncontrolled
movement
*The accumulating damage to this area causes the
characteristic erratic movements associated with HD.
* Coronal section from a MR brain scan of a patient with HD showing
atrophy of the heads of the caudate nuclei, enlargement of the
frontal horns of the lateral ventricles
How is Huntington's disease diagnosed?
*The doctor will also ask about recent intellectual or
emotional problems, which may be indications of HD,
and will test the person's hearing, eye movements,
strength, coordination, involuntary movements
(chorea), sensation, reflexes, balance, movement, and
mental status, and will probably order a number of
laboratory tests as well.
*People with HD commonly have impairments in the
way the eye follows or fixes on a moving target
*The discovery of the HD gene in 1993 resulted in a
direct genetic test to make or confirm a diagnosis of
HD in an individual who is exhibiting HD-like
symptoms.
* Using a blood sample, the genetic test analyzes DNA
for the HD mutation by counting the number of repeats
in the HD gene region.
A model for HD pathogenesis :pathogenic
mechanisms
*HD pathogenesis begins with altered conformation of
the protein containing the expanded pg repeat.
* Proteolysis generates a fragment that leads to toxicity
through several pathways.
* Nuclear importation may lead to altered gene
transcription with a detrimental effect on cell survival.
*Inclusions also form in the nucleus, but may not be a
major cause of cell death.
*Huntington fragments may interfere with mitochondrial
energy metabolism, either directly, or more likely
indirectly, perhaps via altered gene transcription.
*Micro aggregation of the fragment may lead to
caspase activation and the consequent initiation of cell
death pathways.
*Fragments may be transported into neuritis, interfering
with cytoskeleton function.
*Autophagy has emerged as a mechanism of HD
pathogenesis mutant Htt accumulation activates the
endosomal– lysosomal system and contributes to Htt
proteolysis and autophagic cell death.
*Inhibition of mTOR (mammalian target of rapamycin;
also known as FRAP) activates autophagy and
attenuates Htt-induced toxicity, indicating that
autophagy might in fact be helpful as a clearance
mechanism
*There are multiple cellular changes through which the
toxic function of mHtt may manifest and produce the
HD pathology.
* During the biological process of posttranslational
modification of mHtt, cleavage of the protein can leave
behind shorter fragments constituted of parts of the pg
expansion.
*The polar nature of glutamine causes interactions with
other proteins when it is overabundant in Htt proteins.
*Thus, the Htt molecule strands will form hydrogen
bonds with one another, forming a protein aggregate
rather than folding into functional proteins.
*Over time, the aggregates accumulate, ultimately interfering
with neuron function because these fragments can then
misfold and coalesce, in a process called protein aggregation,
to form inclusion bodies within cells.
*Neuronal inclusions run indirect interference.
*The excess protein aggregates clump together at axons and
dendrites in neurons which mechanically stops the
transmission of neurotransmitters because vesicles (filled with
neurotransmitters) can no longer move through the
cytoskeleton.
*Ultimately, over time, less and less neurotransmitters are
available for release in signaling other neurons as the
neuronal inclusions grow.
*Inclusion bodies have been found in both the cell nucleus and
cytoplasm.
A microscope image
of Medium spiny
neurons (yellow) with
nuclear inclusions
(orange), which
occur as part of the
disease process
TRANSCRIPTIONAL DYSREGULATION IN
HD
*transcriptional dysregulation is an important underlying
mechanism in HD pathogenesis.
*Pg
repeats in the N-terminal region of Htt protein gives
it structural similarities to known transcription factors
and repeat expansion leads to aberrant cleavage by
caspases
*The cleaved fragments gain access to the nucleus and
form nuclear aggregates that might disrupt
transcription
*interacts with numerous transcription factors including
CREB-binding protein (CBP), TATA-binding protein
*Transcriptional dysregulation has been proposed to
have an important role in the pathology of HD
*control of eukaryotic gene expression depends on the
modification of histone proteins associated with specific
genes, with histone acetylation playing a crucial role.
*Acetylation of histones at specific residues increases
gene transcription; conversely, histone deacetylation
represses transcription.
Recent studies in numerous HD models have
demonstrated a potential therapeutic role for histone
deacetylase (HDAC) inhibitors in the treatment of
polyglutamine diseases.
Through research
work……
*R6 mice were created by inserting exon 1 of the
human HD gene containing 150 pg repeats, under the
control of the human HD promoter, into the mouse
genome.
*Alterations of neurotransmitter receptor protein levels,
as well as of mRNA levels, have been reported in the
R6/2-before the onset of abnormal symptoms
*In very early-grade HD cases there are profound
reductions in the expression levels of cannabinoid
CB1, dopamine D2 and adenosine A2a receptors.
*screening of mRNA levels, using DNA microarrays, in
the brains of R6/2 mice confirmed the finding that
specific genes are downregulated in this HD mouse
*It affects neuronal disfunction,neurotransmitter signaling…..
*Gene expression studies have also been carried out in
other HD mouse models.
*The HD-N171-82Q transgenic mouse model expresses a
COMPLEMENTARY DNA that encodes a 171-amino-acid
N-terminal fragment of Htt exon 1 containing 82 CAG
repeats.
*The changes in gene expression in HD-N171-82Q mice are
similar to those observed in R6/2 mice
*changes are most pronounced in the striatum, with the
motor cortex affected to a lesser degree, and the
cerebellum showing the fewest gene changes.
*Tm models expressing full-length versions of Htt, such as
the YAC72 mice, demonstrate fewer gene changes,
indicating that increasing the length of Htt reduces the
severity of pg-induced gene changes
*Recently, researchers have shown that mutant
versions of N-terminal Htt can dissociate components
of the transcriptional complex on gene promoters
*transcriptional dysregulation is likely to be an important
mechanism in the pathogenesis not only of HD, but
also of other pg disorders
HISTONE MODIFICATIONS
*The N-terminal tails of the core histones (H2A, H2B,
H3 and H4) are strongly basic---contain specific aminoacid residues ---sites for several post-translational
modifications, including acetylation, methylation,
phosphorylation
*acetylation of lys residues corresponds to
transcriptionally active chromatin, whereas methylation
of lys and arg residues leads to transcriptional
repression.
*acetylation of lys 9 and 14 on histone H3 correlates
with active chromatin and leads to transcription,
whereas methylation of lys 9 on histone H3 is a marker
Known modifications of human histone H3.
*Histone acetylation and deacetylation are modulated
by the interplay between histone acetyltransferases
(HATs) and HDACs
* HAT activity leads to increases in gene transcription
by creating a more open conformation of chromatin,
whereas HDACs remove acetyl groups, leading to
gene repression through condensation of chromatin
*Abnormalities of histone acetylation have been
associated with a number of human cancers like
leukemia non-Hodgkin's lymphoma
*In human gastrointestinal cancers, histone acetylation
is globally reduced
*Acetylation of core histone proteins by the activity of HAT
proteins or treatment with HDAC inhibitors ---more open
conformation of chromatin---transcriptionally active state.
Removal of acetyl groups by HDAC leads ----repressed
chromatin----- transcriptional repression. Mutant Htt expression -- repressed chromatin acetylation of histones
Modification
Histone
Residue
Effects on transcription
K, lysine; R, arginine; S, serine; T, threonine.
Acetylation
H2A
H2B
H3
H4
H3
Methylation
H4
Phosphorylation
Ubiquitination
H2A
H2AX
H3
H4
H2A
H2B
K5
K5, K12, K15, K20
K4, K14, K18, K23,
K27
K9
K5, K12
K8, K16
Activation
Activation
Activation
Histone deposition
Histone deposition
Activation
K4, K79
K9, K27
R17
K36
R3
K20
Euchromatin
Silencing
Activation
Elongation
Activation
Silencing
S1, T119
S139
T3, S10, T11, S28
S1
K119
K120
Mitosis
DNA repair
Mitosis
Mitosis
Silencing
Activation
*HDAC inhibitors increase acetylation of histones,
thereby increasing transcription of genes that have
been silenced.
*promote growth arrest by inducing the expression of
tumor suppressor genes, and are commonly used as
anticancer
*Recent studies in yeast, cell culture, Drosophila and
mouse models of pg disease indicate that HDAC
inhibitors might be useful as therapeutic agents in HD
HISTONE DEACETYLASE INHIBITORS IN HUNTINGTON'S
DISEASE
*Many Htt-interacting proteins possess histone-modifying activity
*CBP contains an acetyltransferase domain and is a coactivator
at a number of promoters
*overexpression of CBP decreases pg-induced cell death
*Htt exon 1 with 51 glutamines containing the polyproline domain
directly binds to the acetyltransferase domain of CBP and
p300/CBP-associated factor
*expression of Httex1p 20Q or Httex1p 103Q in PC12 cells
causes a global hypoacetylation of histones, an effect that is
reversed by the presence of the HDAC inhibitors sodium
butyrate, trichostatin A (TSA) and suberoylanilide hydroxamic
acid (SAHA)
Huntington's
disease model
Histone deacetylase inhibitor
3-NP, 3-nitropropionic acid; Httex1p 20Q, huntingtin exon 1
with 20 glutamines containing the polyproline domain; TSA,
trichostatin A; SAHA, suberoylanilide hydroxamic acid.
Httex1p 20Q and
Httex1p 103Q PC12
cells
Transgenic
Drosophila (Httex1p
93Q)
R6/2 transgenic
mice
R6/2 transgenic
mice
HD-N171-82Q
transgenic mice
Sodium butyrate, TSA, SAHA
Sodium butyrate, SAHA
Sodium butyrate
SAHA
Phenylbutyrate
*in transgenic Drosophila that express Httex1p 93Q, a
decrease in neuron degeneration and early adult death
are observed if the flies have been reared on SAHA or
sodium butyrate
*These results indicated that reduced acetyltransferase
activity might be an important component of pg
pathogenesis in vivo, and paved the way for HDAC
inhibitor studies in transgenic mouse models of HD.
*Then investigated the effects of HDAC inhibitors in
R6/2 mice.
*Sodium butyrate treatment significantly prolonged the
survival of R6/2 mice.
* sodium butyrate and SAHA improved performance on
a ROTAROD TEST, and were neuroprotective
* there was a decrease in gross brain atrophy and
ventricular enlargement in the sodium-butyrate-treated
mice,and a reduction in neuronal atrophy in the
striatum of SAHA-treated mice.
*There was no effect on Htt aggregates
*In both studies, administration of HDAC inhibitors
corrected global hypoacetylation of histones.
*In sodium-butyrate-treated R6/2 mice, there was an
increase in Sp1 acetylation, but no change in basal
levels of Sp1 was detected.
*Sodium butyrate also provided protection against 3nitropropionic-acid (3-NP)-induced striatal damage in
R6/2 mice.
*3-NP is a mitochondrial toxin that specifically targets
complex II of the electron transport chain.
*Striatal administration of 3-NP produces neuronal damage
similar to that observed in the brains of individuals with HD,
and 3-NP striatal lesioning has been used as a toxin model
of HD
*Microarray analysis of sodium-butyrate-treated R6/2
striatum demonstrates a selective change in gene
expression, but there was no uniform correction of genes
downregulated by mutant Htt.
*the HDAC inhibitor phenylbutyrate after the onset of
symptoms in another transgenic mouse model, HD-N17182Q.
*Phenylbutyrate administered to HD-N171-82Q mice at 75
days of age increased survival, and decreased striatal
*There was no effect on weight loss or Htt aggregate
formation.
*Histone H3 and H4 acetylation was increased in the
striatum following phenylbutyrate treatment, and there
was a decrease in methylation of histone H3
*Microarray analysis of the phenylbutyrate-treated HDN171-82Q striatum demonstrated that some genes
were upregulated and others were downregulated.
* phenylbutyrate did not improve the expression of
mutant-Htt-downregulated genes,
*it is promising that transgenic mice showed an overall
improvement in their condition, given that treatment
began after the onset of symptoms.
*The simplest mechanism is that administration of
HDAC inhibitors corrects the downregulation of specific
genes caused by mutant Htt.
* An alternative possibility is that the benefits of HDAC
inhibitors derive from global increased gene
expression.
*Thus HDAC inhibitors might be beneficial in other
neurodegenerative conditions beyond HD.
* HDAC inhibitors have also shown promise in
transgenic mouse models of amyotrophic lateral
sclerosis, schizophrenia and ischemia
*A molecular definition of the mechanisms that underlie
transcriptional dysregulation is required.
*But there remains the issue of whether histones that
are specifically associated with those genes are
hypoacetylated
*Using the molecularly specific technique of chromatin
immunoprecipitation (ChIP), they recently
demonstrated that hypoacetylation of histones is
associated with downregulated genes, whereas
histones associated with genes that are expressed at a
normal level are acetylated to the same degree as in
wild-type mice
HD model
Httex1p 20Q
,Httex1p 103Q PC12
cells
Transgenic
Drosophila (Httex1p
93Q)
HDAC inhibitor
Effect
Sodium butyrate,
TSA, SAHA
Reversed histone hypoacetylation
Sodium butyrate,
SAHA
Decreased rhabdomere degeneration
R6/2 transgenic
mice
Sodium butyrate
Increased survival,Neuroprotection,Motor
improvement,Reversed histone
hypoacetylation,Increased Sp1 acetylation
Protection against 3-NP toxicity
R6/2 transgenic
mice
SAHA
Motor improvement,Neuroprotection
Reversed histone hypoacetylation
Phenylbutyrate
Increased surviva,lNeuroprotection,Increased
histone acetylation,Decreased histone
methylation
HD-N171-82Q
transgenic mice
*There are three classes of HDACs:
1. class I and class II HDACs have been classified on
the basis of sequence similarities
2. class III HDACs are a group of NAD-dependent
deacetylase enzymes related to the yeast Sir2
protein.
*The role of each individual HDAC in regulating gene
expression is not known.
* there are tissue-specific and cell-type-specific
differences in HDAC expression, localization and
targets---might have different effects in different
biological systems.
BUT……………………………..
*HDAC inhibitors are toxic and can induce cell cycle
arrest by increasing the transcription of p21 and p53
* so it will be important to target individual HDACs to
minimize cytotoxicity.
* Other side effects are associated with HDAC inhibitor
therapy;
1. for example, prolonged TSA therapy enhances
chromosomal instability, leading to defective
centromeres and abnormal chromosomal
segregation.
2. HDAC inhibitors can promote tumor development in
some cases.
vital to determine the toxicity of these
*HD remains a fatal untreatable disease.
* Recent advances in understanding the
underlying pathologic mechanisms, have
provided hints to the development of effective
therapies.
*recent evidence points strongly to
transcriptional dysregulation as an important
mechanism.
*The alteration of gene expression is likely to
occur as a consequence of abnormal histone
*The use of HDAC inhibitors and other therapies that
target gene transcription is an exciting development in
the field of HD therapeutics.
*There are strong indications that HDAC inhibitors
might be of therapeutic benefit in HD, but their precise
mechanism of action has yet to be determined.
A ray hope still
exits…………………………..
Tackling the dreadful HD
*There is no cure for Huntington's disease,
*The goal of treatment is to slow down the course of the
disease and help the person function for as long and
as comfortably as possible.
*Dopamine blockers may help reduce abnormal
behaviors and movements.
*Drugs such as haloperidol, tetrabenazine, and
amantadine are used to try to control extra movements.
*There has been some evidence to suggest that coenzyme Q10 may also help slow down the course of
the disease.
*As the disease progresses, patients will need
assistance and supervision. They may eventually need
Mental exercise will avoid Deadly Huntington's disease
Leading scientist, Professor Martin Delatycki has said that,
people with an inherited tendency of the syndrome are the
most possible victim of such diseases. He as advised that
intellectual drills such as Sudoku, riddles etc can be helpful in
postponement of the syndrome.