Huntington*s disease is an autosomal dominant neuro-degenerative disorder
caused by an expanding polyglutamine repeat in the huntingtin gene. Though
huntingtin is widely expressed and is required for normal development, the
pathology of HD is restricted to the brain.
Huntingtin-associated protein-1 (HAP-1) was found by Li et al(1995) to be
enriched in brain. It*s capability of binding to huntingtin is enhanced with
the length of CAG repeats, suggesting a possible basis for the selective
brain pathology of HD.
Apopain , a human counterpart of the nematode cysteine protease death-gene
product, has a key role in proteolytic events leading to apoptosis. Goldberg
et al showed that apoptotic extract , and apopain itself ,specifically,
cleave huntingtin. The rate of cleavage increased with the length of the
polyglutamine tract , providing an explanation for the gain-of function
associated with CAG expansion.
Gutekunst et al(1995) found that huntingtin was associated with
microtrbules, indicating the mutation impairs the cytoskeletal anchoring or
transport of organelles or molecules.
Based on the above facts, we postulate that huntingtin might play a vital
role in neuronal apoptosis of Huntingtin*s disease. The presumed pathway is
as follows:
HAP1/Apopain ---* block/degrade huntingtin (related to polyglutamine tract
length) ---* lose inhibitory functions ----* apoptosis begins or accelarates
Two sets of experiments are applied to prove the hypothesis. Firstly,
induce neuronal and somatic apoptosis with TNF or Glutamate. Assay the
calcium concentration intracellularly and observe the extent of cell death
under SEM. Secondly, incorporate huntingtin gene (with variant repeat
numbers of CAG tract) into YAC vectors, transfect the vector into both
neurons and somatic cells then repeat the first experiment to see of the
overexpressed huntingtin slows down apoptosis. Thirdly, coexpress hap1 and
huntingtin (with variant repeat numbers of CAG tract) genes in both cells,
iterate the first experiments. If the result appears that huntingtin
decreases the range of apoptosis or calcium concentration, we can conclude
that huntingtin is an apopaosis inhibiting protein. And then hap1-huntingtin
coexpressing experiment shows whether HAP1 block huntingtin protein*s
inhibitory functions.
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Introduction:
Huntington*s disease is a nearly completely penetrant autosomal dominant
neurodegenerative disease that is characterized by psychiatric disorders,
demantia, and involuntary movements. The typical age of onset of HD is in
the fourth and fifth decades of life and the average duration of disease is
15 to 20 years. HD is currently incurable, and death results usually from
infectious complications of immobility. The social and emotional impact of
HD is disproportionately greater than its prevalence because of its onset in
the priming working years, the prolonged and progressive course and the
familial nature of the disease.
Molecular genetics of HD
McLaughlin et al. (1996) reported that cytoplasmic protein extracts
from several rat brain regions, including striatum and cortex (sites of
neuronal degeneration in HD), contain a 63 kD RNA-binding protein that
interacts specifically with CAG repeat sequences. They noted that the
protein RNA interactions are dependent upon the length of the CAG repeat,
and that longer repeats bind substantially more protein. McLaughlin et al.
(1996) identified 2 CAG binding proteins in human cortex and striatum, one
of 63 kD and another of 49 kD. They concluded that these data suggest
mechanisms by which RNA binding proteins may be involved in the pathological
course of trinucleotide-associated neurological diseases.
Myers et al (1989) performed molecular genetic studies in 4 offsprings of
3 different affected x affected matings for possible homozygosity. One of
the 4 was found to have a 95% likelihood of being an HD homozygote. The
individuals*s age at onset and symptoms were similar to those in affected HD
heterozygous relatives. Thus, the findings from the New England Huntington
Disease Research Center corroborated the findings of Wexler et al (1987).
Harper et al (1985) stated that the polymorphism with 4 enzymes (HindIII,
EcoRI, NciI, and BstI) applied to the G8 locus shows that over 80% of
subjects are heterozygous. They further stated the latest estimate of the
interval between the G8 and the HD loci is 5 cM. In 16 British kindreds ,
Youngman et al (1986) found 2 recombinants yielding a maximum lod score of
17.6 at theta 0.02. This is more evidence against multilocus heterogeneity
in HD.
The Huntington's Disease Collaborative Research Group (1993) found that a
"new" gene, designated IT15 (important transcript 15) and later called
huntingtin, which was isolated using cloned trapped exons from the target
area, contains a polymorphic trinucleotide repeat that is expanded and
unstable on HD chromosomes. A (CAG)n repeat longer than the normal range was
observed on HD chromosomes from all 75 disease families examined. The
families came from a variety of ethnic backgrounds and demonstrated a
variety of 4p16.3 haplotypes. The (CAG)n repeat appeared to be located
within the coding sequence of a predicted protein of about 348 kD that is
widely expressed but unrelated to any known gene. Thus it turned out that
the HD mutation involves an unstable DNA segment similar to those previously
observed in several disorders, including the fragile X syndrome, Kennedy
syndrome, and myotonic dystrophy.
The fact that the phenotype of HD is completely dominant suggests that the
disorder results from a gain-of-function mutation in which either the mRNA
product or the protein product of the disease allele has some new property
or is expressed inappropriately (Myers et al., 1989). (According to the
tabulation of Parrish and Nelson (1993), HD was the 21st genetic disorder of
previously unknown basic biochemical defect in which the gene was isolated
by positional cloning.
MacDonald et al. (1993) found that unlike the similar CCG repeat in the
fragile X syndrome, the expanded HD repeat shows no evidence of somatic
instability in a comparison of blood, lymphoblast, and brain DNA from the
same persons. Furthermore, 4 pairs of monozygotic HD twins displayed
identical CAG repeat lengths, suggesting that repeat size is determined in
gametogenesis. However, in contrast to the fragile X syndrome and with HD
somatic tissue, mosaicism was readily detected as a diffuse spread of repeat
lengths in DNA from HD sperm samples. Thus, the developmental timing of
repeat instability appears to differ between HD and fragile X syndrome,
indicating perhaps that the fundamental mechanisms leading to repeat
expansion are distinct. Goldberg et al. (1993) reported findings in 3
families in which a new mutation for HD had arisen. In all 3 families, a
parental intermediate allele (with expansion to 30-38 CAG repeats, greater
than that seen in the population but below the range seen in patients with
HD) had expanded in more than 1 offspring. In one of the families, 2 sibs
with the expanded CAG repeat were clinically affected with HD, thus
presenting a pseudorecessive pattern of inheritance.
Zuhlke et al. (1993) studied the length variation of the repeat in 513
non-HD chromosomes from normal individuals and HD patients; the group
comprised 23 alleles with 11 to 33 repeats. In an analysis of the
inheritance of the (CAG)n stretch, they found meiotic instability for HD
alleles, (CAG)40 to (CAG)75, with a mutation frequency of approximately 70%;
following the HD allele in 38 pedigrees during 54 meioses, they found a
ratio of stable to altered copy number of 15:39. On the other hand, in 431
meioses of normal alleles, only 2 expansions were identified. They found
that the risk of expansion during spermatogenesis was enhanced compared to
oogenesis, explaining juvenile onset by transmission from affected fathers.
No mosaicism or differences in repeat lengths were observed in the DNA from
different tissues, including brain and lymphocytes of 2 HD patients,
indicating mitotic stability of the mutation. Thus, the determination of the
repeat number in the DNA of blood lymphocytes is probably representative of
all tissues in a patient.
Andrew et al. (1994) found that 30 of 1,022 persons with HD (2.9%) did not
have an expanded CAG repeat in the disease range. They showed that most of
these individuals with normal sized alleles, namely 18, represented
misdiagnosis, sample mix-up, or clerical error. The remaining 12 patients
represented possible phenocopies for HD. In at least 4 cases, family studies
of these phenocopies excluded 4p16.3 as the region responsible for the
phenotype. Mutations in the HD gene other than CAG expansion have not been
excluded for the remaining 8 cases; however, in as many as 7 of these
patients, retrospective review of their clinical features identified
characteristics not typical for HD. Andrew et al. (1994) concluded that on
rare occasions mutations in other, as-yet-undefined genes can present with a
clinical phenotype very similar to that of HD.
Research Plan
Rationale
Based on three facts below we postulate that huntingtin is crucial in
pathological apoptosis in HD:
(1) The length of polyglutamine tract determines the age of onset and the
severity of symptoms.
(2) HD is caused by neuronal apoptosis within CNS.
(3) HAP1,apopain and GAPD increase affinity for huntingtin with the length
of polyglutamine tract. Among these proteins, apopain specifically cleaves
huntingtin and the rate of cleavage increases as the polyglutamine tract
extends.
(4) Huntingtin is particularly associated with microtubules. While taxol, a
promoting chemical for tubulin polymerization, can cause apoptosis.
Consequently we presume that huntingtin might inhibit apoptosis or at
least slow down the progress of it. There exist molecules that trigger
apoptosis inside or outside cells especially when cells are aging.
Huntingtin may play a role in aging neurons to prevent onset of apoptosis.
Once huntingtin acquires abnormal length of polyglutamine tract, certain
kinds of intracellular proteins such as HAP1 or apopain would soon bind to
it and block the inhibitory functions. Apoptosis begins as a result.
The experiment design is as follows:
(1) TNF/Glu stimulates normal neurons/somatic cells * apoptosis? Ca2+
concentration?
TNF/Glu stimulates neurons/somatic cells with overexpressed huntingtins
with variable lengths of polyglutamine repeats * survive?Ca2+
concentration?
(2) HAP1/huntintin-specific Ab microinject into neurons/somatic cells *
TNF/Glu treatment * apoptosis? Ca2+ concentration?
Material and Method
Material: fresh rat brain neurons
Method:
I. Apoptosis observation: SEM
II. Assaing Ca2+: Fluorescent Calcium indicator - fura-2 fluorescence
excitation spectrum
Titration: 5ml 100mM KCl + 10mM K-MOPS + 10mM K2H2EGTA + 1uM fura-2 *
adjusting pH to 7.20 * recording the spectrum * discard 0.5ml * replace with
100mM KCl + 10mM K-MOPS + 10mM K2CaEGTA + 1uM fura-2 * readjust pH to 7.20 *
record the spectrum * 9mM K2H2EGTA + 1mM K2CaEGTA * record * iterate to
reach n mM CaEGTA + (10-n) mM EGTA ,n = 2~10, discarding 5.0/(11-n) ml and
replace with equal volume of 10mM K2CaEGTA, 1uM fura-2 stock * after n = 10
has been reached to give a free Ca2+ between 10^-4 and 10^-5 M addition of
1mM CaCl2 has no further effect on the spectrum.
Calculation: [Ca2+] = f Kd (R-Rmin)/(Rmax-R)
III.HAP1 and Huntingtin expression:
Clone hap1 and huntingtin genes into YAC and then express them in
neurons
Possible results and deductions
1. Huntingtin blocks apoptosis and HAP1/Apopain counteract against
huntingtin * huntingtin is an inhibitor of neuronal apoptosis and
HAP1/Apopain act as inducers of apoptosis by hindering huntintin*s
funtions.
2. Huntinting blocks apoptosis but HAP1/Apopain don*t affect the effect
* huntingtin is the inhibitor of apoptosis but HAP1/Apopain are not
included.*HAP1/Apopain are either unrelated to the pathology of HD
or are engaged in pathways other than apoptosis.
Further experiments:
(1)Knock out apopain or HAP1 gene to see if HD symptoms are
alleviated.
(2) Continuously assay quantities of HAP1/apopain and measure relative
quantities of either proteins binding to huntinting in the progress of
apoptosis. Compare the data to relative ion concentration changes(Ca2+, Na+,
H+, Cl-..etc) and second messanger concentrations.
3.Huntingtin does not block apoptosis * huntingtin is involved in other
pathways
Further experiments
(1) Assay huntingtin*s interaction with telomerase
(2) Assay the amount of huntingtin in patients or long-repeat carriers at
different ages to understand the relative quantity of huntingtin over
time.
(3) Try to trigger expression of huntingtin gene with known transcription
factors.
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