01. Gene therapy Boulikas.pdf - Gene therapy & Molecular Biology

A. Etiology and mechanisms of destruction
of neurons in PD
First described by James Parkinson in 1817 this
neurodegenerative disorder is characterized by resting
tremor, postural instability and bradykinesia (slow
movement); surviving neurons display intracytoplasmic
inclusions known as Lewy bodies. PD symptoms ensue
when the pars compacta region of the substantia nigra
(black substance) at the base of the brain loses neurons
that normally issue motion-controlling signals (dopamine)
to the striatum (divided into caudate nucleus and
putamen). The death of neurons is believed to be caused
by oxygen free radical damage; brain contains unusually
low levels of antioxidants. This damage might be caused
by a decline in the activity of the mitochondrial complex I.
PD can be induced in experimental animals by
selective destruction of the dopaminergic neurons of the
substantia nigra by the neurotoxic drug 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine
(MPTP) through
inhibition of complex I of the mitochondrial respiratory
chain (see Polymeropoulos et al, 1996 and the references
cited therein). MPTP was found as an impurity in heroin
and explained some earlier observations of addicts who
became almost completely immobile after making use of
the drug, a symptom characteristic of severe PD. MPTP
crosses the brain-blood barrier and is converted by
mitochondrial monoamine oxidase B into a reactive
molecule that inhibits the complex I enzyme resulting in
energy deficit and increase in free radicals in the cell
(reviewed by Youdim and Riederer, 1997).
There is substantial evidence which implicates immune
mechanisms in the destruction of neurons. The substantia
nigra of Parkinson’s patients contains active microglia
which, after stimulation by cytokines, could produce the
free radical nitric oxide which can penetrate the cell
membrane of vicinal neurons, inhibit the complex I
mitochondrial enzyme and activate signal transduction
pathways. Furthermore, NO with superoxide, emitted by
hyperactive microglia, can free iron ions from intracellular
stores which can oxidize dopamine into neuromelanin, a
molecule that acts as an oxidant when complexed with
transition metals. These oxidative stress mechanisms
could trigger apoptosis in neurons. Excessive release of
the neurotransmitter glutamate (known to occur in stroke)
into the striatum and substantia nigra could induce a
similar cascade of NO and free radical damage. These
mechanisms suggest that excessive stressful conditions in
predisposed individuals might precipitate the onset of PD
symptoms.
B. Drug treatment of PD
The first medicament in the mid-1900s included
extracts of the deadly nightshade plant which inhibited the
activity of acetylcholine in the striatum; acetylcholine
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overexcites striatal neurons that projected to higher motor
regions of the brain, an effect normally counteracted by
dopamine. Later in 1960s L-DOPA, which is converted
into dopamine, proved valuable for treatment of PD
patients; dopamine itself cannot cross the blood-brain
barrier (a network of specialized blood vessels that control
which substances are allowed to pass from the blood into
the central nervous system). Drugs that mimic the actions
of dopamine (agonists) have also been used. Selegiline
(also called deprenyl), an inhibitor of monoamine oxidase
B, the enzyme that breaks down dopamine in the
astrocytes and microglia, is of therapeutic potential
(reviewed by Youdim and Riederer, 1997).
Amantadine is used to block the effects of glutamate in
substantia nigra. Antioxidants able to cross the brain-blood
barrier could have a protective effect on the destruction of
neurons. Unfortunately, the first indications show that
vitamin E in the low doses tested, which can cross to some
extent the brain-blood barrier, is ineffective; however, the
effect of higher doses of vitamin E need to be investigated.
The efficacy of glial-derived neurotrophic factor (GDNF)
injected into the brain of PD patients is in trials.
Rasagiline, which could activate neuronal growth factors
in the brain is under investigation on humans. Also in
clinical trials are strategies of direct implantation of
dopamine-producing cells into the brain of patients
(Youdim and Riederer, 1997).
Ex vivo and in vivo gene therapy strategies for PD
have a promising future (see below).
C. Candidate genes for PD
A susceptibility gene for PD has been mapped to
chromosome 4q21-q23 by genotyping genomic DNA from
a large family in Contursi in the Salemo province of
Southern Italy where 60 individuals out of 592 members
are affected by PD at an average age of 46. A total of 140
genetic markers were typed in the pedigree and only those
associated with this chromosomal region were altered
showing recombination events in PD patients in this
family; this type of recombination does not involve
expansions of the CAG trinucleotide repeat
(Polymeropoulos et al, 1996).
The neurologic abnormalities associated with PD were
thought to result from a severe reduction in L-DOPA as a
consequence of degeneration of dopaminergic neurons of
the nigrostriatal pathway. L-DOPA is synthesized from
tyrosine by the enzyme tyrosine hydroxylase (TH); L-
DOPA is then converted into the neurotransmitter
dopamine by a decarboxylase. Besides TH, other genes
whose malfunction has been linked to PD include
glutathione peroxidase, a brain-derived neurotrophic
factor, catalase, amyloid precursor protein, Cu/Zn
superoxide dismutase, and debrisoquine 4-hydroxylase;
however, none of these candidate genes are found in the
4q21-q23 region to be linked as etiologic agents of PD;