Developing a Gene Therapy for the Treatment of Autosomal Dominant Alzheimer's Disease.

Autosomal dominant Alzheimer's disease (ADAD) is a rare early-onset form of Alzheimer's disease, caused by dominant mutations in one of three genes: presenilin 1, presenilin 2, and amyloid ß precursor protein (APP). Mutations in the presenilin 1 gene (PSEN1) account for the majority of cases, and individuals who inherit a single-mutant PSEN1 allele go on to develop early-onset dementia, ultimately leading to death. The presenilin 1 protein (PS1) is the catalytic subunit of the γ-secretase protease, a tetrameric protease responsible for cleavage of numerous transmembrane proteins, including Notch and the APP. Inclusion of a mutant PS1 subunit in the γ-secretase complex leads to a loss of enzyme function and a preferential reduction of shorter forms of Aß peptides over longer forms, an established biomarker of ADAD progression in human patients. In this study, we describe the development of a gene therapy vector expressing a wild-type (WT) copy of human PSEN1 to ameliorate the loss of function associated with PSEN1 mutations. We have carried out studies in mouse models using a recombinant AAV9 vector to deliver the PSEN1 gene directly into the central nervous system (CNS) and shown that we can normalize γ-secretase function and slow neurodegeneration in both PSEN1 conditional knockout and PSEN1 mutant knockin models. We have also carried out biodistribution studies in nonhuman primates (NHPs) and demonstrated the ability to achieve broad PS1 protein expression throughout the cortex and the hippocampus, two regions known to be critically involved in ADAD progression. These studies demonstrate preclinical proof of concept that expression of a WT human PSEN1 gene in cells harboring a dominant PSEN1 mutation can correct the γ-secretase dysfunction. In addition, direct administration of the recombinant AAV9 into the NHP brain can achieve broad expression at levels predicted to provide efficacy in the clinic.

Dopamine transporter (DAT) inhibitors alleviate specific parkinsonian deficits in monkeys: association with DAT occupancy in vivo.

Viable dopamine neurons in Parkinson's disease express the dopamine transporter (DAT) and release dopamine (DA). We postulated that potent DAT inhibitors, with low affinity for the serotonin transporter (SERT), may elevate endogenously released extracellular dopamine levels to provide therapeutic benefit. The therapeutic potential of eight DAT inhibitors was investigated in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated cynomolgus monkeys (Macaca fascicularis), with efficacy correlated with DAT occupancy as determined by positron emission tomography imaging in striatum. Four potent DAT inhibitors, with relatively high norepinephrine transporter, but low SERT affinities, that occupied the DAT improved activity in parkinsonian monkeys, whereas three high-affinity DAT inhibitors with low DAT occupancy did not. 2beta-Carbomethoxy-3alpha-(3,4-dichlorophenyl)-7beta-hydroxy-8-methyl-8-azabicyclo[3.2.1.]octane (O-1163) occupied the DAT but had short-lived pharmacological effects. The benztropine analog difluoropine increased general activity, improved posture, reduced body freeze, and produced sleep disturbances at high doses. (1R)-2beta-(1-Propanoyl)-3alpha-(4-fluorophenyl)tropane (O-1369) alleviated parkinsonian signs in advanced parkinsonian monkeys, by increasing general activity, improving posture, reducing body freeze, and sedation, but not significantly reducing bradykinesia or increasing locomotor activity. In comparison with the D(2)-D(3) DA receptor agonist quinelorane, O-1369 elicited oral/facial dyskinesias, whereas quinelorane did not improve posture or reduce balance and promoted stereotypy. In conclusion, DAT inhibitors with therapeutic potential combine high DAT affinity in vitro and high DAT occupancy of brain striatum in vivo with enduring day-time effects that do not extend into the nighttime. Advanced parkinsonian monkeys (80% DAT loss) respond more effectively to DAT inhibitors than mild parkinsonian monkeys (46% DAT loss). The therapeutic potential of dopamine transport inhibitors for Parkinson's disease warrants preclinical investigation.

Partial reversal of phencyclidine-induced impairment of prepulse inhibition by secretin.

BACKGROUND: Secretin is a "gut-brain" peptide whose neural function is as yet poorly understood. Several clinical studies have reported modestly increased social interaction in autistic children following intravenous secretin administration. Very recently secretin also was administered to schizophrenic patients and found to increase social interaction in some individuals. METHODS: In light of this finding, we assessed the ability of secretin to reverse phencyclidine- (PCP) induced impairment in prepulse inhibition (PPI), a leading animal model of sensorimotor gating deficits in schizophrenia. RESULTS: Similar to atypical antipsychotics, secretin (1, 3, 10, 30, and 100 microg/kg) partially and dose-dependently reversed the PCP-induced deficit in PPI without significantly affecting baseline startle when administered intraperitoneally (IP) 10 minutes following IP administration of PCP (3 mg/kg). CONCLUSIONS: This finding may be relevant to observations of antipsychotic efficacy of secretin in schizophrenic patients as well as our previous report that systemically administered secretin is capable of modulating conditioned fear, even at quite low doses.

Age-related and regional differences in secretin and secretin receptor mRNA levels in the rat brain.

In the present study expression levels of secretin and secretin receptor mRNAs in several brain regions of rats ranging in age from postnatal days 7 to 60 were investigated by quantitative real-time PCR. Expression of secretin and secretin receptor was detected in the central amygdala, hippocampus, area postrema, nucleus of the tractus solitary and cerebellum. The cerebellum expressed secretin receptor at significantly higher levels than that found in other brain regions within all the ages examined. In contrast, secretin mRNA was significantly higher in the nucleus of the tractus solitary than in the other four brain regions examined in postnatal day-21, -30 and -60 rats. Within most brain regions, both secretin and secretin receptor mRNAs were more abundant in postnatal day-7 and -14 rats as compared to postnatal day-21, -30 and -60 rats. Thus, secretin and its receptor are widely expressed in rat brain and the expression of both genes is developmentally regulated during the first few weeks following birth.

Secretin depolarizes nucleus tractus solitarius neurons through activation of a nonselective cationic conductance.

The recent suggestion that secretin may be useful in treating autism and schizophrenia has begun to focus attention on the mechanisms underlying this gut-brain peptide's actions in the central nervous system (CNS). In vitro autoradiographic localization of (125)I-secretin binding sites in rat brain shows the highest binding density in the nucleus tractus solitarius (NTS). Recent evidence suggests that intravenous infusion of secretin causes fos activation in NTS, a relay station playing important roles in the central regulation of autonomic functions. In this study, whole cell patch-clamp recordings were obtained from 127 NTS neurons in rat medullary slices. The mean resting membrane potential of these neurons was -54.7 +/- 0.3 mV, the mean input resistance was 3.7 +/- 0.2 GOmega, and the action potential amplitude of these neurons was always >70 mV. Current-clamp studies showed that bath application of secretin depolarized the majority (80.8%; 42/52) of NTS neurons tested, whereas the remaining cells were either unaffected (17.3%; 9/52) or hyperpolarized (1.9%; 1/52). These depolarizing effects were maintained in the presence of 5 microM TTX and found to be concentration dependent from 10(-12) to 10(-7) M. Using voltage-clamp techniques, we also identified modulatory actions of secretin on specific ion channels. Our results demonstrate that while secretin is without effect on net whole cell potassium currents, it activates a nonselective cationic conductance (NSCC). These results show that NTS neurons are activated by secretin as a consequence of activation of a NSCC and support the emerging view that secretin can act as a neuropeptide within the CNS.

Inhibition of fear potentiated startle in rats following peripheral administration of secretin.

RATIONALE: Previous results indicate that peripheral administration of secretin leads to robust Fos protein expression in the central nucleus of the rat amygdala. The implications of this observation on rat brain function, if any, remain unclear. OBJECTIVES: We examined the effect of systemic secretin administration on the expression of fear-potentiated startle in rats, a behavioral response known to require an intact, functional central nucleus of the amygdala. METHODS: Rats were trained to associate a neutral light conditioned stimulus (CS) with footshock, a fear-inducing unconditioned stimulus (US). Twenty-four hours later, rats were administered secretin or vehicle and were tested immediately for their startle response to a loud noise in the presence or absence of the light. RESULTS: Within a dose range relevant to its clinical use in autistic children, secretin dose-dependently decreased the magnitude of fear-potentiated startle in rats. CONCLUSIONS: This investigation provides additional evidence that systemically administered secretin can influence a neural network implicated in the acquisition and expression of emotional behaviors, including fear and anxiety.

Non-amine-based dopamine transporter (reuptake) inhibitors retain properties of amine-based progenitors.

Without exception, therapeutic and addictive drugs that produce their primary effects by blocking monoamine transporters in brain contain an amine nitrogen in their structure. This fundamental canon of drug design was based on a prevailing premise that an amine nitrogen is required to mimic the structures of monoamine neurotransmitters and other natural products. Non-amines, a novel class of compounds that contain no amine nitrogen, block monoamine transporters in the nM range and display markedly high selectivity for monoamine transporters, but not for receptors. Non-amines retain the spectrum of biochemical and pharmacological properties characteristic of amine-bearing counterparts. These novel drugs compel a revision of current concepts of drug-monoamine transporter complex formation and open avenues for discovery of a new generation of therapeutic drugs.

Differential transport of a secretin analog across the blood-brain and blood-cerebrospinal fluid barriers of the mouse.

Secretin is a gastrointestinal peptide belonging to the vasoactive intestinal peptide (VIP)/glucagon/pituitary adenylate cyclase-activating polypeptide (PACAP) family recently suggested to have therapeutic effects in autism. A direct effect on brain would require secretin to cross the blood-brain barrier (BBB), an ability other members of the VIP/PACAP family have. Herein, we examined whether a secretin analog (SA) radioactively labeled with (131)I (I-SA) could cross the BBB of 4-week-old mice. We found I-SA was rapidly cleared from serum with fragments not precipitating with acid appearing in brain and serum. Levels of radioactivity were corrected to reflect only intact I-SA as estimated by acid precipitation. After i.v. injection, I-SA was taken up by brain at a modest rate of 0.9 to 1.5 microl/g-mm. Capillary depletion, brain perfusion, and high-performance liquid chromatography were used to confirm the passage of intact I-SA across the BBB. I-SA entered every brain region, with the highest uptake into the hypothalamus and cerebrospinal fluid (CSF). Unlabeled SA (10 microg/mouse) did not inhibit uptake by brain but did inhibit clearance from blood and uptake by the CSF, colon, kidney, and liver. The decreased clearance of I-SA from blood increased the percentage of the i.v. injected dose taken up per brain (%Inj/g) from about 0.118 to 0.295%Inj/g. In conclusion, SA crosses the vascular barrier by a nonsaturable process and the choroid plexus by a saturable process in amounts that for other members of its family produce central nervous system (CNS) effects. This passage provides a pathway through which peripherally administered SA could affect the CNS.

Alteration of glutamate receptors in the striatum of dyskinetic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated monkeys following dopamine agonist treatment.

The effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced nigrostriatal lesion and dopaminomimetic treatment on parameters of glutamatergic activity within the basal ganglia of monkeys were studied in relation with the development of dyskinesias. Drug-naive controls, saline-treated MPTP monkeys, as well as MPTP monkeys treated with either a long-acting D2 agonist (cabergoline) or a D1 agonist (SKF-82958) given by intermittent injections or continuous infusion, were included in this study. 3H-L-glutamate, 3H-alpha-amino-3-hydroxy-5-methylisoxasole-4-propionate (AMPA), 3H-glycine, 3H-CGP39653 (an N-methyl-D-aspartate, NMDA, antagonist selective for NR1/NR2A assembly) and 3H-Ro 25-6981 (an NMDA antagonist selective for NR1/NR2B assembly), specific binding to glutamate receptors, the expression of the NR1 subunit of NMDA receptors and glutamate, glutamine and glycine concentrations were studied by autoradiography, in situ hybridization and high-performance liquid chromatography (HPLC), respectively. Pulsatile SKF-82958 and cabergoline treatment relieved parkinsonian symptoms, whereas animals continuously treated with SKF-82958 remained akinetic. Pulsatile SKF-82958 induced dyskinesias in two of the three animals tested, whereas cabergoline did not. MPTP induced no significant changes of striatal specific binding of the radioligands used, NR1 mRNA expression and amino acid concentrations. In the putamen, pulsatile SKF-82958 treatment was associated with decreased content of glycine and glutamate, whereas only glycine was decreased in cabergoline-treated monkeys. Cabergoline and continuous administration of SKF-82958 led to lower levels of NR1 mRNA in the caudate in comparison to pulsatile SKF-82958 administration. The development of dyskinesias following a D1 agonist treatment was associated with an upregulation of 3H-glutamate [+49%], 3H-AMPA [+38%], 3H-CGP39653 [+ 111%], 3H-glycine [+ 26%, nonsignificant] and 3H-Ro 25-6981 [+ 33%] specific binding in the striatum in comparison to nondyskinetic MPTP monkeys. Our data suggest that supersensitivity to glutamatergic input in the striatum might play a role in the pathogenesis of dopaminomimetic-induced dyskinesias and further support the therapeutic potential of glutamate antagonists in Parkinson's disease.