Long-term consequences of human alpha-synuclein overexpression in the primate ventral midbrain.

Overexpression of human alpha-synuclein (alpha-syn) using recombinant adeno-associated viral (rAAV) vectors provides a novel tool to study neurodegenerative processes seen in Parkinson's disease and other synucleinopathies. We used a pseudotyped rAAV2/5 vector to express human wild-type (wt) alpha-syn, A53T mutated alpha-syn, or the green fluorescent protein (GFP) in the primate ventral midbrain. Twenty-four adult common marmosets (Callithrix jacchus) were followed with regular behavioural tests for 1 year after transduction. alpha-Syn overexpression affected motor behaviour such that all animals remained asymptomatic for at least 9 weeks, then motor bias comprising head position bias and full body rotations were seen in wt-alpha-syn expressing animals between 15 and 27 weeks; in the later phase, the animals overexpressing the A53T alpha -syn, in particular, showed a gradual worsening of motor performance, with increased motor coordination errors. Histological analysis from animals overexpressing either the wt or A53T alpha -syn showed prominent degeneration of dopaminergic fibres in the striatum. In the ventral midbrain, however, the dopaminergic neurodegeneration was more prominent in the A53T group than in the WT group suggesting differential toxicity of these two proteins in the primate brain. The surviving cell bodies and their processes in the substantia nigra were stained by antibodies to the pathological form of alpha-syn that is phosphorylated at Ser position 129. Moreover, we found, for the first time, ubiquitin containing aggregates after overexpression of alpha-syn in the primate midbrain. There was also a variable loss of oligodendroglial cells in the cerebral peduncle. These histological and behavioural data suggest that this model provides unique opportunities to study progressive neurodegeneration in the dopaminergic system and deposition of alpha-syn and ubiquitin similar to that seen in Parkinson's disease, and to test novel therapeutic targets for neuroprotective strategies.

Neglect of memory after dopaminergic lesions in monkeys.

Crossed unilateral dopaminergic lesions of the nigrostriatal bundle and unilateral inferotemporal cortex ablations (DA x IT lesions) in marmoset monkeys produced impaired retention of object discriminations first learnt before, or after, the DA lesion but no impairment on new learning of the same type of task. Retention testing of a pre-operatively learned task was given after new learning of a different task so impairment cannot be attributed to improvement with practice or spontaneous recovery. We argue that the DA lesion produces a form of intentional neglect, a defect of volition, which is the mnemonic counterpart of the volitional neglect of directional hypokinesia, which animals with this lesion also exhibit. The DA lesion was unilateral (for welfare reasons) so the information to be retrieved had to be confined to that hemisphere by the use of an IT ablation in the other hemisphere. Unilateral DA lesion compromises the competence of ipsilateral fronto-striatal interactions and our results parallel those found in monkeys with crossed IT x frontal lesions that are impaired on complex tasks requiring effortful implementation of a cognitive strategy but are not impaired on discrimination learning. Parkinsonian patients with sub-total but bilateral DA loss may lack 'top-down' conative mechanisms as well as 'top-down' movement initiation mechanisms. They may fail to initiate retrieval strategies, although they may not exhibit retrograde amnesia under test conditions that provoke retrieval. Failure to self-initiate retrieval of relevant knowledge may contribute to the paucity of cognitive style and loss of executive skills exhibited by some patients with Parkinson's disease.

Further analysis of the effects of immunotoxic lesions of the basal nucleus of Meynert reveals substantial impairment on visual discrimination learning in monkeys.

In this paper we undertake a combined analysis of several studies in which marmoset monkeys received immunotoxic lesions of the cortical cholinergic projections from the basal nucleus of Meynert (NBM) bilaterally and/or in combination with immunotoxic lesions of other parts of the cholinergic system or ablations of the target inferotemporal neocortical area. Analysis of the mean learning scores across all visual discriminations learning tasks for each lesion combination revealed highly significant impairments where the NBM was lesioned bilaterally or where an NBM lesion in one hemisphere was crossed with an inferotemporal cortical ablation in the other hemisphere. This demonstrates that the cholinergic projection from the NBM to the major target area of neocortex involved in visual discrimination learning, i.e. the inferotemporal cortex, makes an important contribution to the perceptuo-mnemonic processes necessary for this type of learning. A new study demonstrates a significant effect of a subtotal bilateral cholinergic lesion confined to the NBM on a concurrent object-reward association task using black objects which is perceptually and mnemonically demanding. These results do not preclude the possibility that cholinergic projections from the NBM to other parts of the neocortex make a contribution to other cortical functions which are not mnemonic. It is well established that lesions of the cholinergic projection from the diagonal band of Broca disrupts the mnemonic functions of the hippocampus. The results described here suggest that degeneration of the cholinergic projections in Alzheimer's disease and other dementias will contribute to the loss of those mnemonic functions which are dependent on the neocortex.

Mild topographical memory impairment following crossed unilateral lesions of the mediodorsal thalamic nucleus and the inferotemporal cortex.

Monkeys with crossed unilateral lesions of the dorsomedial thalamus and contralateral ablations of the inferotemporal cortex were mildly impaired on acquisition and retention of visual conditional tasks requiring the integration of information about objects and their positions in space. They were not impaired on other conditional and nonconditional tasks. This impairment pattern resembles, qualitatively, that found following crossed unilateral lesions of the anterior thalamus and the inferotemporal cortex or bilateral lesions of the anterior and mediodorsal thalamic nuclei. Although the flow of visual information from the inferotemporal cortex through the hippocampal-fornix-anterior thalamic circuit plays a major part in memory for objects in places, the flow of information between inferotemporal cortex and mediodorsal thalamus, possibly by means of the frontal cortex, also makes some contribution.

Continuous low-level glial cell line-derived neurotrophic factor delivery using recombinant adeno-associated viral vectors provides neuroprotection and induces behavioral recovery in a primate model of Parkinson's disease.

The therapeutic potential of glial cell line-derived neurotrophic factor (GDNF) for Parkinson's disease is likely to depend on sustained delivery of the appropriate amount to the target areas. Recombinant adeno-associated viral vectors (rAAVs) expressing GDNF may be a suitable delivery system for this purpose. The aim of this study was to define a sustained level of GDNF that does not affect the function of the normal dopamine (DA) neurons but does provide anatomical and behavioral protection against an intrastriatal 6-hydroxydopamine (6-OHDA) lesion in the common marmoset. We found that unilateral intrastriatal injection of rAAV resulting in the expression of high levels of GDNF (14 ng/mg of tissue) in the striatum induced a substantial bilateral increase in tyrosine hydroxylase protein levels and activity as well as in DA turnover. Expression of low levels of GDNF (0.04 ng/mg of tissue), on the other hand, produced only minimal effects on DA synthesis and only on the injected side. In addition, the low level of GDNF provided approximately 85% protection of the nigral DA neurons and their projections to the striatum in the 6-OHDA-lesioned hemisphere. Furthermore, the anatomical protection was accompanied by a complete attenuation of sensorimotor neglect, head position bias, and amphetamine-induced rotation. We conclude that when delivered continuously, a low level of GDNF in the striatum (approximately threefold above baseline) is sufficient to provide optimal functional outcome.

Analysis of infant carrying in large, well-established family groups of captive marmosets (Callithrix jacchus).

To assess the pattern of infant carrying across time and family members, we counted which animals in 13 well-established family groups of captive-bred marmosets (Callithrix jacchus) carried neonates during the first 8 weeks of life. The neonates were carried almost continuously for the first 3 weeks and then spent progressively more time independently. The mother did most of the carrying for the first 2 weeks, her contribution rising from day 1 to day 3 and declining thereafter. The contribution of the father was high on day 1, declined during the first week, and then rose to a peak in the fourth week. The contribution of the siblings rose sharply during the first week and declined thereafter. There was no overall difference in amount of infant carrying by each parent. The contribution of each sibling was small although in these large families the total contribution by siblings was large. These data may differ from previous observations because the breeding pairs were very well established, the families were large, and all except the youngest animals were very experienced in rearing and carrying. These data emphasise the group-dynamic nature of infant carrying in a primate species.

Recombinant adeno-associated viral vector (rAAV) delivery of GDNF provides protection against 6-OHDA lesion in the common marmoset monkey (Callithrix jacchus).

Glial cell line-derived neurotrophic factor (GDNF) has shown potential as a treatment for Parkinson's disease. Recombinant adeno-associated viral vectors expressing the GDNF protein (rAAV-GDNF) have been used in rodent models of Parkinson's disease to promote functional regeneration after 6-OHDA lesions of the nigrostriatal system. The goal of the present study was to assess the anatomical and functional efficacy of rAAV-GDNF in the common marmoset monkey (Callithrix jacchus). rAAV-GDNF was injected into the striatum and substantia nigra 4 weeks prior to a unilateral 6-OHDA lesion of the nigrostriatal bundle. Forty percent of the dopamine cells in the lesioned substantia nigra of the rAAV-GDNF-treated monkeys survived, compared with 21% in the untreated monkeys. Fine dopaminergic fibres were observed microscopically in the injected striatum of some rAAV-GDNF-treated monkeys, suggesting that rAAV-GDNF treatment may have prevented, at least in part, the loss of dopaminergic innervation of the striatum. Protection of dopamine cells and striatal fibre innervation was associated with amelioration of the lesion-induced behavioural deficits. rAAV-GDNF-treated monkeys showed partial or complete protection not only in the amphetamine and apomorphine rotation but also in head position and the parkinsonian disability rating scale. Therefore, our study provides evidence for the behavioural and anatomical efficacy of GDNF delivered via an rAAV vector as a possible treatment for Parkinson's disease.

Sensorimotor deficits in a unilateral intrastriatal 6-OHDA partial lesion model of Parkinson's disease in marmoset monkeys.

Animal studies investigating the efficacy of neurotrophic factors as treatments for Parkinson's disease (PD) ideally require partial dopamine (DA) lesion models. The intrastriatal 6-hydroxydopamine (6-OHDA) lesion model may be suitable for this purpose. Although this model has been well characterized in rodents, it has not previously been used in monkeys. The goal of the present study was to characterize the behavioral effects of unilateral injections of 6-OHDA in the basal ganglia of common marmoset monkeys (Callithrix jacchus). Cell counts from tyrosine hydroxylase immunochemistry 5 months postlesion revealed DA cell loss in the substantia nigra on the lesioned side to approximately 46% of relative to the unlesioned side. 6-OHDA lesioned monkeys showed a variety of behavioral deficits. Apomorphine induced rotation and simple sensorimotor measures (head position bias and PD disability rating score) were most affected by the lesion. The largest deficits were seen at 1 or 2 weeks postsurgery but had recovered by week 10. 6-OHDA lesioned monkeys took longer to complete a more complex sensorimotor staircase task. At 3.5 months postlesion, 6-OHDA monkeys also showed deficits on an object retrieval task designed to measure sensorimotor planning and skilled hand use. alpha-Methyl-p-tyrosine, a tyrosine hydroxylase inhibitor, reinstated those deficits which had undergone recovery in the lesioned animals and also exacerbated the deficits on the staircase task. This model has potential in assessing treatments for PD aimed at curtailing disease progression such as continuous delivery of neurotrophic factors.

Functional and histological evidence for the protective effect of NXY-059 in a primate model of stroke when given 4 hours after occlusion.

BACKGROUND AND PURPOSE: NXY-059 has substantial protective effects when administered immediately after the onset of ischemia in a primate model of stroke. This study examined the efficacy of this drug when administered 4 hours after onset, a more clinically relevant time point. METHODS: Before surgery, marmosets were trained and tested on a number of neurological tests, which assessed general neurological function, motor ability, and spatial awareness. Four hours after permanent middle cerebral artery occlusion (pMCAO), marmosets received a bolus of saline (n=13) or NXY-059 (n=13), and osmotic minipumps were implanted, providing 48-hour saline or drug (85 micromol/kg per hour) infusion. The monkeys were retested 3 and 10 weeks after surgery. Finally, infarct size was evaluated with histological analysis. RESULTS: The unbound plasma NXY-059 concentration was 200+/-9 micromol/L after 24-hour infusion, a concentration well tolerated in stroke patients. Drug treatment ameliorated the long-term motor impairment produced by pMCAO; the marmosets were better at using their contralesional, stroke-affected arm than controls at both 3 and 10 weeks. Saline-treated animals had a debilitating spatial neglect at 3 weeks with residual signs evident at 10 weeks. NXY-059 treatment substantially attenuated neglect at 3 weeks, with no deficit being seen at 10 weeks. NXY-059 reduced the overall infarct size by 28% (saline, 324+/-46 mm3; NXY-059, 234+/-30 mm3) with protection to the cortex, white matter, and subcortical structures. CONCLUSIONS: NXY-059 is an effective neuroprotective agent when administered 4 hours after pMCAO in a primate species, attenuating both motor and spatial neglect. The compound also substantially lessened the volume of cerebral damage.

What would T. H. Huxley have made of prion diseases?

T. H. Huxley was "Darwin's bulldog," and took the offensive in championing the cause of evolution against skeptical scientists and outraged theologians. As such, he took part in one of the great "paradigm shifts" of biology, at the end of the nineteenth century. Huxley was a rigorous scientist and wrote important articles on scientific method, as well as publishing extensively on a wide range of subjects in natural history. In the second half of the twentieth century, the "prion hypothesis" was put forward to explain the pathogenesis of a curious group of diseases known as the transmissible spongiform encephalopathies. This also involved a "paradigm shift" because the prion hypothesis postulated that biologically relevant information could be enciphered in protein conformation (rather than encoded in nucleic acid base sequences), and could be transmitted from one molecule to another, thereby causing infectious disease. This article examines a few of Huxley's remarks to speculate on how he might have responded to the scientific debate about prion disease had he lived a century later.

Comparison of the neuroprotective effect of clomethiazole, AR-R15896AR and NXY-059 in a primate model of stroke using histological and behavioural measures.

Three experimental neuroprotective agents (clomethiazole, AR-R15896AR and NXY-059) have recently been tested in a primate model of acute ischaemic stroke. As the experimental techniques used in all three studies were similar and the compounds were administered at clinically relevant doses, a comparative analysis of the functional benefits of these drug-treatments has now been performed. Furthermore a more detailed histological analysis of the neuroprotection afforded by the drugs has also been made. NXY-059 produced almost twice the degree of neuroprotection than that seen following clomethiazole or AR-R15896AR. Protection by NXY-059 was seen in measurements of damage to cortex and white matter. Clomethiazole and AR-R15896AR provided less protection of cortex and white matter than NXY-059. Conspicuously, AR-R15896AR was without effect in sub-cortical regions. NXY-059 was the only compound to produce a major, statistically significant improvement in the motor deficit induced by the stroke. All three drugs also reduced the degree of spatial neglect 3 weeks after pMCAO, and 10 weeks later only NXY-059 still provided significant additional functional benefit to the spontaneous improvement seen in stroked control animals not receiving treatment. The overview of the behavioural effects and these new histological findings suggest that NXY-059 was by far the most effective neuroprotective agent of the three examined.

Unilateral hippocampal and inferotemporal cortex lesions in opposite hemispheres impair learning of single-pair visual discriminations as well as visuovisual conditional tasks in monkeys.

Monkeys with unilateral ablations of the inferotemporal (IT) cortex were not impaired on learning or retention of single-pair object discriminations or visuovisual conditional tasks. Addition of an excitotoxic hippocampal lesion to the hemisphere opposite to the IT ablation impaired retention and acquisition of single-pair object discriminations and visuovisual conditional tasks. Histology revealed no areas of bilaterally symmetrical damage. Previous experiments have shown that bilateral excitotoxic hippocampal lesions do not impair single-pair object discriminations although they do produce a substantial impairment on visuovisual conditional tasks. Bilateral IT ablations produce impairment on single-pair object discrimination tasks. It is argued that the hippocampus in the hemisphere with the IT ablation is deprived of feed-forward visual input and that this, in addition to the contralateral hippocampal lesion, accounts for the impairment on the visuovisual conditional tasks. It is also argued that feed-back projections from the hippocampus to the IT cortex influence the learning of single-pair object discriminations. This influence may be difficult to demonstrate by the addition of hippocampal lesions to IT lesions because of the substantial effect of the IT lesion alone. It may be difficult to demonstrate by bilateral hippocampal lesions alone since the effect may be below that which generates an observable impairment. Nonetheless, an effect may be seen when a hippocampal lesion is made in monkeys with some IT damage, as in this experiment, as well as by the general observation that large lesions of the temporal lobes produce larger perceptuo-mnemonic impairments than lesions confined to the hippocampus or temporal neocortex in monkeys and man.

Assessment of cognitive and motor deficits in a marmoset model of stroke.

The Stroke Therapy Academic Industry Roundtable noted the need for standardized, well-accepted primate models of stroke to help develop both neuroprotective and restorative therapies. One primate model has been developed using the marmoset, a small New World species of monkey, in which long-term functional deficits can be assessed. The surgery and postoperative care of the animals is described, as well as the behavioral tests used to quantify the postoperative disability. The types of deficits seen are illustrated by reference to some of the findings with neuroprotective treatments. Nevertheless, the long-term nature and consistency of the motor deficits make this model ideal for assessing the worth of restorative therapies.

Learning impairments in monkeys with combined but not separate excitotoxic lesions of the anterior and mediodorsal thalamic nuclei.

Clinical studies in humans and experiments in macaques suggest that damage to the anterior and the mediodorsal thalamus can induce a moderate amnesia, but a more dense impairment may result from substantial damage within the temporal lobes or their subcortical connections. Lesions of the anterior thalamus in macaques produce impairments which resemble those seen after lesions of the fornix-mamillary pathway, which carries projections from the hippocampus to the anterior thalamus, while lesions of the mediodorsal thalamus, which receives inputs from frontal and temporal cortex, produce moderate impairments on a wider range of memory tasks. In the present study, we have made bilateral excitotoxic lesions of either the anterior or the mediodorsal thalamus, or both, in marmoset monkeys. Monkeys with lesions of both thalamic nuclei were severely impaired on retention and new learning of examples of the visuospatial conditional task, a task which is specifically impaired by lesions of the fornix or hippocampus. They were not impaired on performance of a visuovisual conditional task on which monkeys with hippocampal lesions are impaired, nor were they impaired on any visual discrimination task, including the concurrent discrimination task on which monkeys with temporal neocortical ablations are impaired. Monkeys with separate lesions of either the anterior or the mediodorsal thalamus were not impaired on any of these tasks. These results suggest that the mediodorsal thalamus and the anterior thalamus are both involved in processing the output of the hippocampal-fornix-thalamic circuit. Dense amnesia may result from damage to circuits additional to the temporal lobe efferents to either the anterior or the mediodorsal nuclei.