Ken-ichi Fujimoto

Triple Transduction with Adeno-Associated Virus Vectors Expressing Tyrosine Hydroxylase, Aromatic-L-Amino-Acid Decarboxylase, and GTP Cyclohydrolase I for Gene Therapy of Parkinson's Disease

Parkinsons disease (PD), a neurological disease suited to gene therapy, is biochemically characterized by a severe decrease in the dopamine content of the striatum. One current strategy for gene therapy of PD involves local production of dopamine in the striatum achieved by inducing the expression of enzymes involved in the biosynthetic pathway for dopamine. We previously showed that the coexpression of tyrosine hydroxylase (TH) and aromatic-L-amino-acid decarboxylase (AADC), using two separate adeno-associated virus (AAV) vectors, resulted in more effective dopamine production and more remarkable behavioral recovery in 6-hydroxydopamine-lesioned parkinsonian rats, compared with the expression of TH alone. Not only levels of TH and AADC but also levels of tetrahydrobiopterin (BH4), a cofactor of TH, and GTP cyclohydrolase I (GCH), a rate-limiting enzymes for BH4 biosynthesis, are reduced in parkinsonian striatum. In the present study, we investigated whether transduction with separate AAV vectors expressing TH, AADC, and GCH was effective for gene therapy of PD. In vitro experiments showed that triple transduction with AAV-TH, AAV-AADC, and AAV-GCH resulted in greater dopamine production than double transduction with AAV-TH and AAV-AADC in 293 cells. Furthermore, triple transduction enhanced BH4 and dopamine production in denervated striatum of parkinsonian rats and improved the rotational behavior of the rats more efficiently than did double transduction. Behavioral recovery persisted for at least 12 months after stereotaxic intrastriatal injection. These results suggest that GCH, in addition to TH and AADC, is important for effective gene therapy of PD.

A Phase I Study of Aromatic l-Amino Acid Decarboxylase Gene Therapy for Parkinson's Disease

Gene transfer of dopamine-synthesizing enzymes into the striatal neurons has led to behavioral recovery in animal models of Parkinsons disease (PD). We evaluated the safety, tolerability, and potential efficacy of adeno-associated virus (AAV) vector-mediated gene delivery of aromatic L-amino acid decarboxylase (AADC) into the putamen of PD patients. Six PD patients were evaluated at baseline and at 6 months, using multiple measures, including the Unified Parkinsons Disease Rating Scale (UPDRS), motor state diaries, and positron emission tomography (PET) with 6-[(18)F]fluoro-L-m-tyrosine (FMT), a tracer for AADC. The short-duration response to levodopa was measured in three patients. The procedure was well tolerated. Six months after surgery, motor functions in the OFF-medication state improved an average of 46% based on the UPDRS scores, without apparent changes in the short-duration response to levodopa. PET revealed a 56% increase in FMT activity, which persisted up to 96 weeks. Our findings provide class IV evidence regarding the safety and efficacy of AADC gene therapy and warrant further evaluation in a randomized, controlled, phase 2 setting.Gene transfer of dopamine-synthesizing enzymes into the striatal neurons has led to behavioral recovery in animal models of Parkinsons disease (PD). We evaluated the safety, tolerability, and potential efficacy of adeno-associated virus (AAV) vector-mediated gene delivery of aromatic L-amino acid decarboxylase (AADC) into the putamen of PD patients. Six PD patients were evaluated at baseline and at 6 months, using multiple measures, including the Unified Parkinsons Disease Rating Scale (UPDRS), motor state diaries, and positron emission tomography (PET) with 6-[18F]fluoro-L-m-tyrosine (FMT), a tracer for AADC. The short-duration response to levodopa was measured in three patients. The procedure was well tolerated. Six months after surgery, motor functions in the OFF-medication state improved an average of 46% based on the UPDRS scores, without apparent changes in the short-duration response to levodopa. PET revealed a 56% increase in FMT activity, which persisted up to 96 weeks. Our findings provide class IV evidence regarding the safety and efficacy of AADC gene therapy and warrant further evaluation in a randomized, controlled, phase 2 setting.

Intramuscular injection of AAV-GDNF results in sustained expression of transgenic GDNF, and its delivery to spinal motoneurons by retrograde transport

Adeno-associated virus (AAV) vector has been developed as an attractive gene delivery system with proven safety. Glial cell line-derived neurotrophic factor (GDNF) is proposed to be a promising therapeutic agent for amyotrophic lateral sclerosis (ALS) and other motor neuron diseases. The purpose of this report was to investigate transgenic GDNF expression at different time points post AAV mediated GDNF intramuscular delivery. An AAV vector was constructed to encode a recombinant fusion of GDNF tagged with a FLAG sequence at the C-terminal (AAV-GDNF) to distinguish it from its endogenous counterpart. A single intramuscular injection of AAV-GDNF led to substantial expression of transgenic GDNF which remained for at least 10 months in transduced gastrocnemius muscle. This transgenic GDNF was distributed in a large number of myofibers, mainly in the vicinity of the sarcolemma and predominantly concentrated at the sites of neuromuscular junctions (NMJs). Furthermore, transgenic GDNF, but not beta-galactosidase expressed as a control, was detected in the motoneurons that projected axons to the injected muscles, thus, indicating retrograde axonal transportation of the transgenic GDNF. This study provides a basis for a strategy of intramuscular AAV-GDNF delivery to protect motoneurons as a possible means of ALS treatment.

Dropped head in Parkinson's disease.

“A propensity to bend the trunk forward” and “the chin is now almost immovably bent down upon the sternum” were described by James Parkinson in patients with Parkinson’s disease (PD). The term “dropped head” was first reported in “Gerlier disease” in Switzerland and ‘kubisagari’ in Japan and since then also reported in myositis, myopathy, myasthenia gravis, amyotrophic lateral sclerosis, neuropathy, and hypothyroidism. Disproportionate antecollis occurs in about half cases of multiple system atrophy (MSA) and is considered dystonic in nature. Dropped head is considered rare in PD, both in advanced and early stages of PD. However, it is known to progress subacutely over a period of several days. In my experience, dropped head is relatively common in PD. The mechanism of dropped head in PD is either dystonia of flexor neck muscles or weakness of extensor neck muscles. The response of dropped head to various anti-parkinsonian medications is rather inconsistent. Levodopa is reported to induce amelioration in some patients while dopamine agonists can cause deterioration. Muscle afferent block with lidocaine and ethanol is reported to be effective, while the effect of botulinum toxin injection into the affected muscles is limited. The effect of stereotaxic neurosurgery on dropped head is controversial. Early diagnosis and prompt treatment is necessary to prevent muscle damage associated with longterm overstretch of extensor neck muscles.

Impaired acquisition, preserved retention and retrieval of avoidance behavior after destruction of pedunculopontine nucleus areas in the rat

Abstract Bilateral lesions of the pedunculopontine nucleus area produced by injection of ibotenic acid in rats resulted in severe impairment of acquisition of both one-trial passive avoidance and two-way shuttle-box active avoidance, while retention and retrieval of acquired avoidance behaviors were perfectly preserved and neither short-term nor long-term buffer of memory was affected.

Prevention of dopaminergic neuron death by adeno-associated virus vector-mediated GDNF gene transfer in rat mesencephalic cells in vitro.

Glial cell line-derived neurotrophic factor (GDNF) is known as a potent neurotrophic factor for dopaminergic neurons. Since adeno-associated virus (AAV) vector is a suitable vehicle for gene transfer into neurons, rat E14 mesencephalic cells were transduced with an AAV vector expressing GDNF. When compared with mock transduction, a larger number of dopaminergic neurons survived in AAV-GDNF-transduced cultures (234% and 325% of controls at 1 and 2 weeks, respectively; P < 0.01). Furthermore, the dopaminergic neurons in the latter cultures grew more prominent neurites than those in the former. These findings suggest that AAV vector-mediated GDNF gene transfer may prevent dopaminergic neuron death, and is therefore a logical approach for the treatment of Parkinsons disease.

Temporal trends and geographic clusters of mortality from amyotrophic lateral sclerosis in Japan, 1995-2004.

The present study examined temporal trends and geographic clustering of amyotrophic lateral sclerosis (ALS) mortality in Japan, during 1995-2004, using vital statistics based on death certificates. ALS was usually diagnosed by neurologists according to clinical guidelines that complied with the El Escorial Criteria. The underlying cause of death for ALS was coded as G12.2A. Regression analysis was used to examine temporal trends. Spatial scan statistic was used to detect any area of elevated risk as a cluster. A total of 12,173 (6864 male and 5309 female) ALS deaths were reported. Annual crude mortality rate per 100,000 population was 1.07 (1.26 for males and 0.89 for females) in 2004. Although the overall temporal trend was stable, the trend increased in the 70+ years age group (p for trend, <0.001 in males and <0.05 in females), while it declined in the under 70 years age group (p for trend, <0.01 for both sexes). Male preponderance and M/F ratio remained nearly constant over time. Three clusters were detected: two (p<0.005 in males and p<0.05 in females) in northeast and one (p<0.05 in males) in west-central Japan. Further research is needed to clarify contributing factors for the observed trends and clusters in ALS mortality.

Impairment of active avoidance produced after destruction of pedunculopontine nucleus areas in the rat

Pedunculopontine nucleus areas were lesioned bilaterally in the rat by local injection of kainic acid. When rats so treated were tested for active avoidance behavior in shuttle boxes we found that their avoidance acquisition was completely abolished, whereas their intertrial locomotor activity and escape behavior were not affected. In the lesioned rats, choline acetyltransferase activity in the medial thalamus and substantia nigra decreased, but not in the lateral thalamus, hippocampus, or cerebral cortex. These findings suggest that the cholinergic pedunculopontine-medial thalamus projection plays an important role in memory acquisition in the rat.

Neuronal mechanism underlying dystonia induced by bicuculline injection into the putamen of the cat

Microinjection of bicuculline (Bic) into the unilateral putamen (Put) of the cat resulted in periodic occurrence of contralateral movement of the head (dystonia). Corresponding to this dystonic movement, repetitive spikes of the field potential occurring in the Put of the injected side and strong inhibition of the extracellularly recorded unit discharges in the neurons of the substantia nigra pars reticulata (SNr) of the same side were observed.

Gene therapy of Parkinson's disease using Adeno-Associated Virus (AAV) vectors

Parkinsons disease (PD) is characterized by the progressive loss of the dopaminergic neurons in the substantia nigra and a severe decrease in dopamine in the striatum. A promising approach to the gene therapy of PD is intrastriatal expression of dopamine-synthesizing enzymes [tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC)]. The most appropriate gene-delivery vehicles for neurons are adeno-associated virus (AAV) vectors, which are derived from non-pathogenic virus. Therefore, TH and AADC genes were introduced into the striatum in the lesioned side using separate AAV vectors in parkinsonian rats, and the coexpression of TH and AADC resulted in better behavioral recovery compared with TH alone. Another strategy for gene therapy of PD is the protection of dopaminergic neurons in the substantia nigra using an AAV vector containing a glial cell line-derived neurotrophic factor (GDNF) gene. Combination of dopamine-supplement gene therapy and GDNF gene therapy would be a logical approach to the treatment of PD.