NAP (davunetide) rescues neuronal dysfunction in a Drosophila model of tauopathy.

Alzheimer's disease (AD) is a devastating neurodegenerative disease causing irreversible cognitive decline in the elderly. There is no disease-modifying therapy for this condition and the mechanisms underpinning neuronal dysfunction and neurodegeneration are unclear. Compromised cytoskeletal integrity within neurons is reported in AD. This is believed to result from loss-of-function of the microtubule-associated protein tau, which becomes hyper-phosphorylated and deposits into neurofibrillary tangles in AD. We have developed a Drosophila model of tauopathy in which abnormal human tau mediates neuronal dysfunction characterised by microtubule destabilisation, axonal transport disruption, synaptic defects and behavioural impairments. Here we show that a microtubule-stabilising drug, NAPVSIPQ (NAP), prevents as well as reverses these phenotypes even after they have become established. Moreover, it does not alter abnormal tau levels indicating that it by-passes toxic tau altogether. Thus, microtubule stabilisation is a disease-modifying therapeutic strategy protecting against tau-mediated neuronal dysfunction, which holds great promise for tauopathies like AD.

Increased throughput assays of locomotor dysfunction in Drosophila larvae.

Larval locomotion is a sensitive readout of a range of nervous system deficits in Drosophila, and has been utilised to quantify modulation of the disease phenotype in models of human disease. Single larvae are typically analysed in series using manual quantification of parameters such as contraction rate, or grouped together and studied en-masse. Here, we describe the development of tests for the analysis of several spatially isolated third instar larvae in parallel. We rapidly quantify larval turning rate and velocity during wandering behaviour in a 4 plate assay. In a second test, larvae are recorded as they race along five parallel lanes towards a yeast stimulus. This allows increased throughput analysis of comparative genotypes simultaneously, video archiving, and detection of exacerbation or rescue of defective locomotion in a Drosophila model of tauopathy, as we demonstrate genetically and through delivery of candidate therapeutic chemicals in fly food. The tests are well-suited for rapid comparison of locomotion capability in Drosophila mutants or candidate modulation screens in Drosophila models of human disease.

Characterization of xenobiotic-induced hepatocellular enzyme induction in rats: anticipated thyroid effects and unique pituitary gland findings.

During routine safety evaluation of RO2910, a non-nucleoside reverse transcriptase inhibitor for HIV infection, histopathology findings concurrent with robust hepatocellular induction occurred in multiple organs, including a unique, albeit related, finding in the pituitary gland. For fourteen days, male and female rats were administered, by oral gavage vehicle, 100, 300, or 1000 mg/kg/day of RO2910. Treated groups had elevated serum thyroid-stimulating hormone and decreased total thyroxine, and hypertrophy in the liver, thyroid gland, and pituitary pars distalis. These were considered consequences of hepatocellular induction and often were dose dependent and more pronounced in males than in females. Hepatocellular centrilobular hypertrophy corresponded with increased expression of cytochrome P450s 2B1/2, 3A1, and 3A2 and UGT 2B1. Bilateral thyroid follicular cell hypertrophy occurred concurrent to increased mitotic activity and sometimes colloid depletion, which were attributed to changes in thyroid hormone levels. Males had hypertrophy of thyroid-stimulating hormone-producing cells (thyrotrophs) in the pituitary pars distalis. All findings were consistent with the well-established adaptive physiologic response of rodents to xenobiotic-induced hepatocellular microsomal enzyme induction. Although the effects on the pituitary gland following hepatic enzyme induction-mediated hypothyroidism have not been reported previously, other models of stress and thyroid depletion leading to pituitary stimulation support such a shared pathogenesis.

Spontaneous cardiomyopathy in cynomolgus monkeys (Macaca fascicularis).

A previously undescribed spontaneous cardiomyopathy was identified by routine light microscopic examination of the heart from four clinically healthy purpose-bred cynomolgus monkeys that ranged from four to nine years of age and included 2 males and 2 females. Special stains of Sirius red, Masson's trichrome, and Mallory's phosphotungstic acid hematoxylin (PTAH); and immunohistochemistry using anti-CD68, troponin-I, and desmin antibodies were used to facilitate lesion characterization and assess cardiomyocyte viability. Microscopically, the apical to mid-ventricular myocardium to subendocardium had foci of cardiomyocyte disarray with cytoplasmic pallor to stippling and karyomegaly, vacuolization of the perimyseal connective tissue, a meshwork of fibrous tissue that concentrated around medium-sized blood vessels and dissected between or less often replaced affected cardiomyocytes; and a minimal, predominantly macrophage infiltrate. The disrupted cardiomyocytes were immunoreactive to desmin and troponin-I antibodies and had a normal cross-striation pattern by PTAH, indicating the chronic cardiomyopathy was not associated with active cardiomyocyte damage. The consistent distribution and morphology of the cardiomyopathy suggested a common etiology and pathogenesis. The features were reminiscent of chronic catecholamine-induced experimental cardiomyopathy and stress cardiomyopathy in monkeys and humans, respectively. This report documents another spontaneous heart lesion in clinically healthy monkeys for consideration during interpretation of toxicology studies.

Live axonal transport disruption by mutant huntingtin fragments in Drosophila motor neuron axons.

Huntington's Disease is a neurodegenerative condition caused by a polyglutamine expansion in the huntingtin (Htt) protein, which aggregates and also causes neuronal dysfunction. Pathogenic N-terminal htt fragments perturb axonal transport in vitro. To determine whether this occurs in vivo and to elucidate how transport is affected, we expressed htt exon 1 with either pathogenic (HttEx1Q93) or non-pathogenic (HttEx1Q20) polyglutamine tracts in Drosophila. We found that HttEx1Q93 expression causes axonal accumulation of GFP-tagged fast axonal transport vesicles in vivo and leads to aggregates within larval motor neuron axons. Time-lapse video microscopy, shows that vesicle velocity is unchanged in HttEx1Q93-axons compared to HttEx1Q20-axons, but vesicle stalling occurs to a greater extent. Whilst HttEx1Q93 expression did not affect locomotor behaviour, external heat stress unveiled a locomotion deficit in HttEx1Q93 larvae. Therefore vesicle transport abnormalities amidst axonal htt aggregation places a cumulative burden upon normal neuronal function under stressful conditions.

Overexpression of tau results in defective synaptic transmission in Drosophila neuromuscular junctions.

Synaptic dysfunction is believed to be an early pathological change in neurodegenerative diseases and may cause the earliest clinical symptoms. We have used Drosophila to model a tauopathy in order to analyse the earliest neuronal and synaptic dysfunction. Our work has shown that overexpression of human tau (0N3R) in larval motor neurons causes a disruption of axonal transport and a morphological and functional disruption of NMJs (neuromuscular junctions). Tau-expressing NMJs are smaller with an abnormal structure. Despite abnormal morphology, tau-expressing NMJs retain synaptotagmin expression and can form active zones. Tau-expressing NMJs are functionally abnormal and exhibit disrupted vesicle cycling and synaptic transmission. At low-frequency stimulation (1 Hz), ESPs (evoked synaptic potentials) produced by tau-expressing motor neurons were indistinguishable from wild-type; however, following high-frequency stimulation (50 Hz), ESPs from tau-expressing NMJs were significantly decreased in amplitude. To investigate the mechanism underlying the change in ESPs, we analysed the relative numbers and distribution of mitochondria. This revealed that motor neurons expressing tau had a significant reduction in the number of detectable mitochondria in the pre-synaptic terminal. Our results demonstrate that tau overexpression results in synaptic dysfunction, associated with a reduced complement of functional mitochondria. These findings suggest that disruption of axonal transport and synaptic transmission may be key components of the pathogenic mechanism that underlie neuronal dysfunction in the early stages of tauopathies.

Susceptibility of rats to corneal lesions after injectable anesthesia.

Corneal injury is not a commonly reported side effect after injectable or inhalation anesthesia in rats, but a number of surgery studies at our facility resulted in a high incidence of these injuries. To explore the potential association of various anesthetic protocols with the development of corneal lesions in rats, we retrospectively evaluated clinical records and sections of eyes from 215 male and 187 female Wistar rats used in eight intravenous infusion toxicology studies. None of the studied compounds was associated with eye toxicity. For placement of jugular vein vascular access ports, rats were anesthetized with enflurane, isoflurane, ketamine-xylazine, or Hypnorm-midazolam. Histologically, corneal changes were scored from 0 to 4 in light of degree of mineralization, leukocytic infiltrates, neovascularization, fibrosis, and ulceration. Prestudy (postsurgical) ophthalmic examination findings of corneal opacities were correlated with mineralization of the anterior limiting membrane and corneal ulceration. Corneal lesions were more severe in animals anesthetized with ketamine-xylazine, and minimal changes occurred after anesthesia with either enflurane or isoflurane. The results of further analysis suggest that corneal lesions can be observed within 24 h after injectable anesthetic administration and are not reversible. The severity of corneal changes was reduced when ketamine-xylazine anesthesia was reversed with yohimbine. Compared with Sprague-Dawley and Lewis rats, Wistar, Long-Evans, and Fischer 344 rats had increased incidence and severity of corneal lesions after anesthesia with ketamine-xylazine, suggesting that these three strains are at increased risk for developing postanesthetic corneal lesions with this regimen.

GSK-3beta inhibition reverses axonal transport defects and behavioural phenotypes in Drosophila.

The tauopathies are a group of disorders characterised by aggregation of the microtubule-associated protein tau and include Alzheimer's disease (AD) and the fronto-temporal dementias (FTD). We have used Drosophila to analyse how tau abnormalities cause neurodegeneration. By selectively co-expressing wild-type human tau (0N3R isoform) and a GFP vesicle marker in motorneurons, we examined the consequences of tau overexpression on axonal transport in vivo. The results show that overexpression of tau disrupts axonal transport causing vesicle aggregation and this is associated with loss of locomotor function. All these effects occur without neuron death. Co-expression of constitutively active glycogen-synthase kinase-3beta (GSK-3beta) enhances and two GSK-3beta inhibitors, lithium and AR-A014418, reverse both the axon transport and locomotor phenotypes, suggesting that the pathological effects of tau are phosphorylation dependent. These data show that tau abnormalities significantly disrupt neuronal function, in a phosphorylation-dependent manner, before the classical pathological hallmarks are evident and also suggest that the inhibition of GSK-3beta might have potential therapeutic benefits in tauopathies.

Differentiating spontaneous from drug-induced vascular injury in the dog.

When vascular injury is observed in dogs used in preclinical toxicology studies, careful evaluation of the lesions is warranted, especially when differentiating drug-induced vascular changes from spontaneous findings, such as idiopathic canine polyarteritis. The clinical signs as well as the nature and distribution of lesions can often be distinguishing, as is the case with vasoactive drugs, including vasodilators and/or positive inotropes (hydralazine, minoxidil, endothelin receptor antagonists, and phosphodiesterase III inhibitors). For most types of vasodilator-induced vascular injury, the lesion is often restricted to coronary arteries, whereas in idiopathic canine polyarteritis, arterial lesions not only involve coronary arteries, but also medium to small arteries of other organs. In addition, the nature of the changes in vessels yields important clues. Medial and adventitial hemorrhage is generally associated with vasodilator-induced arterial lesion, whereas hemorrhage is generally absent in idiopathic polyarteritis. Although idiopathic canine polyarteritis can generally be differentiated from vasoactive-induced vascular injury in dogs, there are increasing incidences of this type of polyarteritis in dogs receiving any 1 of a number of unrelated classes of compounds, suggestive of an exacerbation of the spontaneous disease. Therefore, in order to differentiate drug-induced injury from idiopathic canine polyarteritis, it is critical that examination of the vascular pathology be conducted with good understanding of clinical, pharmacological, and mechanistic data associated with the drug.

Presenilin 1 independently regulates beta-catenin stability and transcriptional activity.

Presenilin 1 (PS1) regulates beta-catenin stability; however, published data regarding the direction of the effect are contradictory. We examined the effects of wild-type and mutant forms of PS1 on the membrane, cytoplasmic, nuclear, and signaling pools of endogenous and exogenous beta-catenin by immunofluorescence microscopy, subcellular fractionation, and in a transcription assay. We found that PS1 destabilizes the cytoplasmic and nuclear pools of beta-catenin when stabilized by Wnt or Dvl but not when stabilized at lower levels of the Wnt pathway. The PS1 mutants examined were less able to reduce the stability of beta-catenin. PS1 also inhibited the transcriptional activity of endogenous beta-catenin, and the PS1 mutants were again less inhibitory at the level of Dvl but showed a different pattern of inhibition toward transcription below Dvl. The transcriptional activity of exogenously expressed wild-type beta-catenin and two mutants, DeltaN89beta-catenin and DeltaSTbeta-catenin, were also inhibited by wild-type and mutant PS1. We conclude that PS1 negatively regulates the stability and transcriptional activity of beta-catenin at different levels in the Wnt pathway, that the effect on transcriptional activity appears to be independent of the GSK-3beta mediated degradation of beta-catenin, and that mutations in PS1 differentially affect the stability and transcriptional activity of beta-catenin.

Dishevelled regulates the metabolism of amyloid precursor protein via protein kinase C/mitogen-activated protein kinase and c-Jun terminal kinase.

Alzheimer's disease (AD) is a disorder of two pathologies: amyloid plaques, the core of which is a peptide derived from the amyloid precursor protein (APP), and neurofibrillary tangles composed of highly phosphorylated tau. Protein kinase C (PKC) is known to increase non-amyloidogenic alpha-secretase cleavage of APP, producing secreted APP (sAPPalpha), and glycogen synthase kinase (GSK)-3beta is known to increase tau phosphorylation. Both PKC and GSK-3beta are components of the wnt signaling cascade. Here we demonstrate that overexpression of another member of this pathway, dishevelled (dvl-1), increases sAPPalpha production. The dishevelled action on APP is mediated via both c-jun terminal kinase (JNK) and protein kinase C (PKC)/mitogen-activated protein (MAP) kinase but not via p38 MAP kinase. These data position dvl-1 upstream of both PKC and JNK, thereby explaining the previously observed dual signaling action of dvl-1. Furthermore, we show that human dvl-1 and wnt-1 also reduce the phosphorylation of tau by GSK-3beta. Therefore, both APP metabolism and tau phosphorylation are potentially linked through wnt signaling.

Deafferentation of the hippocampus results in the induction of AT8 positive 'granules' in the rat.

Hyperphosphorylated tau is a pathological hallmark of Alzheimer's disease, but the mechanisms that lead to its formation are poorly understood. To investigate what effect deafferentation of the hippocampus has on the phosphorylation state of tau, we lesioned the entorhinal cortex in rats and looked for hyperphosphorylated tau in the hippocampus at various days post lesioning. After 7 and 21 days, small AT8-positive 'granules' appeared in the molecular layer of the dentate gyrus on the lesioned side. No such staining was seen in the animals injected with saline. This study shows that deafferentation leads to induction of hyperphosphorylated tau. The AT8 positive 'granules' seen resemble the argyrophilic grains that characterize Argyrophilic Grain disease suggesting that lesioning the perforant pathway may serve as a useful model for inducing argyrophilic grains in vivo.

Induction of hyperphosphorylated tau in living slices of rat hippocampal formation and subsequent detection using an ELISA.

Although hyperphosphorylated tau is an established feature of Alzheimer's Disease, its role in the disease process is poorly understood, partly because of lack of suitable animal models. We describe the use of living slices of rat hippocampal formation to study tau phosphorylation. Using the AT8 antibody in an ELISA, phosphorylated tau was detected in freshly frozen slices and it increased significantly in slices that were incubated in an electrophysiological recording chamber; the amount detected was greatest when the homogenisation buffer contained phosphatase and kinase inhibitors. The phosphorylated tau content of the slices increased significantly after exposure to the phosphatase 1 and 2A inhibitor okadaic acid (OA) - 1.5 microM. Electrophysiological recordings confirmed that slices were alive and that OA had no acute toxic effect. In control slices phosphorylated tau, detected immunohistochemically, was mainly in the somatodendritic compartment of neurones; in OA treated slices, there was an apparent decrease in somatodendritic AT8 staining and an increase in neuropil staining. Our system enables the induction of hyperphosphorylated tau within living slices, in an experimental environment that can be used to study the biological consequences of such a change, and may therefore help further our understanding of the significance of hyperphosphorylated tau in Alzheimer's Disease.

Heparin injection into the adult rat hippocampus induces seizures in the absence of macroscopic abnormalities.

The pathological hallmarks of Alzheimer's disease include neurofibrillary tangles, neuropil threads and neuritic plaques. Neurofibrillary tangles and neuropil threads are comprised of paired helical filaments which are themselves composed of a hyperphosphorylated form of the microtubule-associated protein tau. Neuritic plaques are extracellular deposits of aggregated beta amyloid associated with neurites containing hyperphosphorylated tau. The mechanisms by which the neurofibrillary tangles and neuritic plaques develop in Alzhemier's disease are not clear but it is hypothesized that sulphated glycosaminoglycans are important in their formation. This impression is based on the finding that the glycosaminoglycan, heparan sulphate, is found associated with neurofibrillary tangles, neuritic plaques and neuropil threads while dermatan sulphate, chondroitin sulphate and keratan sulphate immunoreactivity is found around neuritic plaques in brains of Alzheimer's disease patients. Furthermore, in vitro studies demonstrate that sulphated glycosaminoglycans such as heparan sulphate and the closely related molecule heparin interact with tau and potentiate its phosphorylation by a number of serine/threonine kinases, reduce its ability to bind to microtubules and induce paired helical filament formation, all properties associated with tau isolated from Alzheimer's disease brain. Thus, we were interested to learn whether intracerebral injection of the sulphated glycosaminoglycan heparin would give rise to alterations in the cytoskeletal protein tau in the rat brain. Although no cytoskeletal changes were observed, to our considerable surprise we found that the intrahippocampal injection of heparin gave rise to seizures. We have investigated this unexpected effect further in vivo and by using in vitro electrophysiological techniques.