β-Amyloid (Aβ) peptides are thought to be critically involved in the etiology of Alzheimer's disease (AD). The aspartyl protease β-site amyloid precursor protein cleaving enzyme 1 (BACE1) is required for the production of Aβ, and BACE1 inhibition is thus an attractive target for the treatment of AD. We show that verubecestat (MK-8931) is a potent, selective, structurally unique BACE1 inhibitor that reduced plasma, cerebrospinal fluid (CSF), and brain concentrations of Aβ40, Aβ42, and sAPPβ (a direct product of BACE1 enzymatic activity) after acute and chronic administration to rats and monkeys. Chronic treatment of rats and monkeys with verubecestat achieved exposures >40-fold higher than those being tested in clinical trials in AD patients yet did not elicit many of the adverse effects previously attributed to BACE inhibition, such as reduced nerve myelination, neurodegeneration, altered glucose homeostasis, or hepatotoxicity. Fur hypopigmentation was observed in rabbits and mice but not in monkeys. Single and multiple doses were generally well tolerated and produced reductions in Aβ40, Aβ42, and sAPPβ in the CSF of both healthy human subjects and AD patients. The human data were fit to an amyloid pathway model that provided insight into the Aβ pools affected by BACE1 inhibition and guided the choice of doses for subsequent clinical trials.

Glial cell line-derived neurotrophic factor (GDNF) is able to protect dopaminergic neurons against various insults and constitutes therefore a promising candidate for the treatment of Parkinson's disease. Lentiviral vectors that infect quiescent neuronal cells may allow the localized delivery of GDNF, thus avoiding potential side effects related to the activation of other brain structures. To test this hypothesis in a setting ensuring both maximal biosafety and optimal transgene expression, a self-inactivating (SIN) lentiviral vector was modified by insertion of the posttranscriptional regulatory element of the woodchuck hepatitis virus, and particles were produced with a multiply attenuated packaging system. After a single injection of 2 microl of a lacZ-expressing vector (SIN-W-LacZ) in the substantia nigra of adult rats, an average of 40.1 +/- 6.0% of the tyrosine hydroxylase (TH)-positive neurons were transduced as compared with 5.0 +/- 2.1% with the first-generation lentiviral vector. Moreover, the SIN-W vector expressing GDNF under the control of the mouse phosphoglycerate kinase 1 (PGK) promoter was able to protect nigral dopaminergic neurons after medial forebrain bundle axotomy. Expression of hGDNF in the nanogram range was detected in extracts of mesencephalon of animals injected with an SIN-W-PGK-GDNF vector, whereas it was undetectable in animals injected with a control vector. Lentiviral vectors with enhanced expression and safety features further establish the potential use of these vectors for the local delivery of bioactive molecules into defined structures of the central nervous system.

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Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor which has been purified on the basis of its ability to promote the survival of dopaminergic neurons in vitro. GDNF has subsequently been cloned and its sequence shown to be distantly related to transforming growth factor-beta (TGF-beta). To identify GDNF expressing cells in the adult rat brain, in situ hybridization using a digoxygenin (DIG)-labelled riboprobe has been performed. Our results show that GDNF mRNA is mainly expressed in neurons and that its synthesis is not restricted to dopaminergic areas. It is widely expressed in the cortex, the hippocampus, the striatum, the substantia nigra, the thalamus, the cerebellum and the spinal cord. Neuronal GDNF expression varies among brain regions as determined by the intensity of the in situ signal. Double labelling of the substantia nigra using tyrosine hydroxylase immunohistochemistry, associated with GDNF in situ hybridization, show that the majority of dopaminergic neurons express GDNF. The widespread expression of GDNF throughout the adult brain suggests that its administration in Parkinson's disease should be restricted to the altered structures, in order to avoid possible deleterious side effects.

Parkinson's disease (PD) is characterized by the progressive loss of the substantia nigra (SN) dopaminergic neurons projecting to the striatum. Neurotrophic factors may have the potential to prevent or slow down the degenerative process occurring in PD. To that end, we examined whether low amounts of glial cell line-derived neurotrophic factor (GDNF) continuously released from polymer-encapsulated genetically engineered cells are able to prevent the loss of tyrosine hydroxylase immunoreactivity (TH-IR) in SN neurons and ameliorate the amphetamine-induced rotational asymmetry in rats that have been subjected to a unilateral medial forebrain bundle (MFB) axotomy. Baby hamster kidney (BHK) cells transfected with the cDNA for GDNF were encapsulated in a polymer fiber and implanted unilaterally at a location lateral to the MFB and rostral to the SN. ELISA assays before implantation show that the capsules release approximately 5 ng of GDNF/capsule per day. One week later, the MFB was axotomized unilaterally ipsilateral to the capsule placement. Seven days later, the animals were tested for amphetamine-induced rotational asymmetry and killed. The striatum was excised and analyzed either for catecholamine content or TH-IR, while the SN was immunostained for the presence of TH-IR. GDNF did not prevent the loss of dopamine in the striatum. However, GDNF significantly rescued TH-IR neurons in the SN pars compacta. Furthermore, GDNF also significantly reduced the number of turns per minute ipsilateral to the lesion under the influence of amphetamine. Improvement of rotational behavior in the absence of dopaminergic striatal reinnervation may reflect neuronal plasticity in the SN, as suggested by the dendritic sprouting observed in animals receiving GDNF. These results illustrate that the continuous release of low levels of GDNF close to the SN is capable of protecting the nigral dopaminergic neurons from an axotomy-induced lesion and significantly improving pharmacological rotational behavior by a mechanism other than dopaminergic striatal reinnervation.

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