Post-mortem assessment of the short and long-term effects of the trophic factor neurturin in patients with α-synucleinopathies.

Substantial interest persists for developing neurotrophic factors to treat neurodegenerative diseases. At the same time, significant progress has been made in implementing gene therapy as a means to provide long-term expression of bioactive neurotrophic factors to targeted sites in the brain. Nonetheless, to date, no double-blind clinical trial has achieved positive results on its primary endpoint despite robust benefits achieved in animal models. A major issue with advancing the field is the paucity of information regarding the expression and effects of neurotrophic factors in human neurodegenerative brain, relative to the well-characterized responses in animal models. To help fill this information void, we examined post-mortem brain tissue from four patients with nigrostriatal degeneration who had participated in clinical trials testing gene delivery of neurturin to the putamen of patients. Each had died of unrelated causes ranging from 1.5-to-3-months (2 Parkinson's disease patients), to 4+-years (1 Parkinson's disease and 1 multiple-system atrophy-parkinsonian type patient) following gene therapy. Quantitative and immunohistochemical evaluation of neurturin, alpha-synuclein, tyrosine hydroxylase (TH) and an oligodendroglia marker (Olig 2) were performed in each brain. Comparable volumes-of-expression of neurturin were seen in the putamen in all cases (~15-22%; mean=18.5%). TH-signal in the putamen was extremely sparse in the shorter-term cases. A 6-fold increase was seen in longer-term cases, but was far less than achieved in animal models of nigrostriatal degeneration with similar or even far less NRTN exposure. Less than 1% of substantia nigra (SN) neurons stained for neurturin in the shorter-term cases. A 15-fold increase was seen in the longer-term cases, but neurturin was still only detected in ~5% of nigral cells. These data provide unique insight into the functional status of advanced, chronic nigrostriatal degeneration in human brain and the response of these neurons to neurotrophic factor stimulation. They demonstrate mild but persistent expression of gene-mediated neurturin over 4-years, with an apparent, time-related amplification of its transport and biological effects, albeit quite weak, and provide unique information to help plan and design future trials.

Oncolytic virus therapy of multiple tumors in the brain requires suppression of innate and elicited antiviral responses.

The occurrence of multiple tumors in an organ heralds a rapidly fatal course. Although intravascular administration may deliver oncolytic viruses/vectors to each of these tumors, its efficiency is impeded by an antiviral activity present in complement-depleted plasma of rodents and humans. Here, this activity was shown to interact with complement in a calcium-dependent fashion, and antibody neutralization studies indicated preimmune IgM has a contributing role. Short-term exposure to cyclophosphamide (CPA) partially suppressed this activity in rodents and humans. At longer time points, cyclophosphamide also abrogated neutralizing antibody responses. Cyclophosphamide treatment of rats with large single or multiple intracerebral tumors substantially increased viral survival and propagation, leading to neoplastic regression.

Physostigmine and recent memory: effects in young and aged nonhuman primates.

The effect of physostigmine on recent memory was evaluated in young and aged rhesus monkeys. All aged monkeys had previously shown impaired memory. The performance of the young monkeys treated with physostigmine was similar to that recently reported for young humans--no effects at low doses, some improvement at a restricted range of doses, and deficits at the highest dose. Although the aged subjects also improved at the same general doses, their overall response as a group was much more variable than that of the younger subjects. The performance of some aged monkeys was impaired by low doses that did not affect young monkeys. Continued improvement was observed in some aged monkeys at the highest dose, which typically impaired young monkeys. These variable effects across aged subjects suggest that physostigmine cannot easily or reliably be used as an agent for treating geriatric cognition. Nevertheless, the differential age-related effects suggest that appropriate manipulation of the cholinergic system may eventually be developed to alleviate some of the cognitive impairments suffered by aged subjects.

The cholinergic hypothesis of geriatric memory dysfunction.

Biochemical, electrophysiological, and pharmacological evidence supporting a role for cholinergic dysfunction in age-related memory disturbances is critically reviewed. An attempt has been made to identify pseudoissues, resolve certain controversies, and clarify misconceptions that have occurred in the literature. Significant cholinergic dysfunctions occur in the aged and demented central nervous system, relationships between these changes and loss of memory exist, similar memory deficits can be artificially induced by blocking cholinergic mechanisms in young subjects, and under certain tightly controlled conditions reliable memory improvements in aged subjects can be achieved after cholinergic stimulation. Conventional attempts to reduce memory impairments in clinical trials hav not been therapeutically successful, however. Possible explanations for these disappointments are given and directions for future laboratory and clinical studies are suggested.

Intravenous RMP-7 selectively increases uptake of carboplatin into rat brain tumors.

Rats implanted with RG-2 gliomas were administered i.v. RMP-7 and [14C]carboplatin. Changes in the permeability of the blood-brain barrier to carboplatin were determined using quantitative autoradiography. i.v. infusions of RMP-7 induced an increase in the permeability of the vascular barrier within the tumor to carboplatin. Additionally, permeability of brain tissue proximal to, but clearly outside the tumor mass, was also increased. Progressively less uptake of [14C]carboplatin was observed as distance from the tumor border increased. The increases in permeability induced by RMP-7 occurred in a dose-related fashion. No increase in carboplatin level was observed in several nonbrain tissues, including sciatic nerve, retina, heart, lung, liver, kidney, and spleen. Finally, the permeabilizing effects of RMP-7 were shown to occur independent of histaminergic or hypotensive mechanisms. These data provide additional insight into the permeabilizing effects and mechanism of RMP-7 and offer additional support for the therapeutic utility of this novel compound as an adjunctive treatment for human gliomas.

Behavioral efficacy of posttraumatic calpain inhibition is not accompanied by reduced spectrin proteolysis, cortical lesion, or apoptosis.

Administration of the selective calpain inhibitor AK295 has been shown to attenuate motor and cognitive dysfunction after brain trauma in rats. To explore mechanisms underlying the behavioral efficacy of posttraumatic calpain inhibition, we investigated histologic consequences of AK295 administration. Anesthetized Sprague-Dawley rats received lateral fluid percussion brain injury of moderate severity (2.2 to 2.4 atm) or served as uninjured controls. At 15 minutes after injury, animals were randomly assigned to receive a 48-hour infusion of either 2 mmol/L AK295 (120 to 140 mg/kg) or saline via the carotid artery. At 48 hours and 1 week after injury, spectrin fragments generated specifically by calpain were detected by Western blotting and immunohistochemistry, respectively, in saline-treated, brain-injured animals. Interestingly, equivalent spectrin breakdown was observed in AK295-treated animals when cortical and hippocampal regions were evaluated. Similarly, administration of the calpain inhibitor did not attenuate cortical lesion size or the numbers of apoptotic cells in the cortex, subcortical white matter, or hippocampus, as verified by terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick-end labeling and morphology, at 48 hours after injury. These data suggest that an overt reduction in spectrin proteolysis, cortical lesion, or apoptotic cell death at 48 hours or 1 week is not required for behavioral improvements associated with calpain inhibition by AK295 after experimental brain injury in rats.

Time-related neuronal changes following middle cerebral artery occlusion: implications for therapeutic intervention and the role of calpain.

Changes in neocortex and striatum were characterized over time following focal ischemia to the brain. Rats were subjected to permanent middle cerebral artery occlusion (MCA-O) and sacrificed 1, 3, 6, 12, or 24 h later. The affected tissue was processed for tetrazolium chloride (TTC) and cresyl violet staining, as well as for Western blots to detect calpain-induced spectrin proteolysis. Significant changes in cell size and spectrin breakdown occurred within the first hour of occlusion, with further, dramatic changes in these two early markers continuing over time. Initial evidence of cell loss was noted at 1 h postocclusion in the striatum and at 3 h in the neocortex. However, even in the center of the most affected portion of the neocortex, the majority of cells appeared to be intact through 6 h. By this time, a significant TTC-defined infarct also emerged. These quantitative data indicate that calpain-induced proteolysis occurs very soon after the ischemic insult, is correlated with earliest changes in cell hypotrophy, and precedes or occurs in tandem with evidence of significant cell loss. They also demonstrate that, while some cell loss occurs earlier than previously believed, the majority of cells remains morphologically intact well beyond what is typically thought to be the window of opportunity for intervention. The results thus raise the question of how long after the ischemic event pharmaceutic intervention might be employed to salvage substantial numbers of neurons.

Postischemic administration of AK275, a calpain inhibitor, provides substantial protection against focal ischemic brain damage.

Experiments were conducted to determine whether a potent, reversible calpain inhibitor could reduce the cortical ischemic brain damage associated with focal ischemia in the rat. AK275 (Z-Leu-Abu-CONH-CH2CH3), the active isomer of the diastereomeric mixture, CX275, was employed in conjunction with a novel method of perfusing drug directly onto the infarcted cortical surface. This protocol reduced or eliminated numerous, nonspecific pharmacokinetic, hemodynamic, and other potentially confounding variables that might complicate interpretation of any drug effect. Focal ischemia was induced using a variation of the middle cerebral artery occlusion method. These studies demonstrated a reliable and robust neuroprotective effect of AK275 over the concentration range of 10 to 200 microM (perfused supracortically at 4 microliters/h for 21 h). Moreover, a 75% reduction in infarct volume was observed when initiation of drug treatment was delayed for 3 h postocclusion. Our data further support an important role of calpain in ischemia-induced neuropathology and suggest that calpain inhibitors may provide a unique and potentially powerful means of treating stroke and other ischemic brain incidents.

The need for common perspectives in the development and use of animal models for age-related cognitive and neurodegenerative disorders.

Research toward the development and use of animals models of age-related neurodegeneration and memory loss has accelerated rapidly during the past decade. However, the degree to which various investigators have been able to integrate their findings has not kept pace. Given the apparent complexity of neurodegenerative and cognitive phenomena, it seems essential that more effective dialogue between investigators with diverse backgrounds and approaches be achieved. Such a dialogue would likely reduce certain confusion and misunderstanding that currently exist in this field. It might also be expected that the dialogue would lead to the development of a working framework for continued growth and integration of novel findings and ideas. This paper attempts to promote a dialogue by identifying and discussing certain broad issues that produce unnecessary confusion in the common pursuit of using animals to understand various aspects of complex human neurodegenerative diseases. Also, some perspectives are suggested that may facilitate discussion and comparison of different animal models, independent of the paradigms and species used.

Tetrahydroaminoacridine, 3,4 diaminopyridine and physostigmine: direct comparison of effects on memory in aged primates.

The effects of tetrahydroaminoacridine (THA), 3,4 diaminopyridine (3,4 DAP) and physostigmine were evaluated for their ability to reduce memory impairments in aged, test-sophisticated cebus monkeys (18 to 26 years old). Several doses of each drug were tested (PO) in each of ten different monkeys, allowing for direct and extensive comparison of each drug's efficacy in this model. The results of this comparative test revealed several potentially interesting findings: (1) all drugs produced improvement in a portion of the monkeys tested; (2) as in many past tests with aged monkeys and humans, wide variations in most effective dose, per subject, were observed; (3) different monkeys responded more effectively to one drug than another; and (4) under these tightly controlled conditions, physostigmine produced the most reliable and robust effects (p less than 0.005), in more monkeys, than did either THA (p less than 0.05) or 3,4 DAP (p less than 0.10).

Lack of efficacy of clonidine on memory in aged cebus monkeys.

Using a dosing paradigm and an automated behavioral testing procedure designed to maximize potential drug efficacy, we evaluated the ability of clonidine to improve performance of aged monkeys on a trial-specific memory task. These results indicated that neither acutely administered (0.0025-0.04 mg/kg) or subchronically administered (0.01-0.06 mg/kg for 3 consecutive days) clonidine improved the performance of aged monkeys. These data are discussed in view of previously reported, conflicting data, as well as their implication regarding probable neurochemical factors involved in age-associated memory impairment and approaches to future palliative treatments.

Memory deficits in aged cebus monkeys and facilitation with central cholinomimetics.

Cebus monkeys of 3 different age groups were trained to perform an automated behavioral task (delayed response), intended to measure recent memory ability. In in initial study, the aged monkeys (18 years and older) exhibit prprogressively greater performance impairments (relative to young monkeys) as they were required to remember the location of a visual stimulus for increasingly longer durations (0 to 20 sec). This deficits replicated previously published results from aged Rhesus monkeys and appeared similar to the primary memory deficits reported in elderly humans and demented patients. In subsequent studies, the effects of three different cholinomimetics were evaluated for their ability to improve the aged monkey's performance on this task. Each monkey was tested under several acute doses of the cholinergic precursor, choline, the anticholinesterase, physostigmine, and the cholinergic muscarinic receptor agonist, arecoline. The results revealed clear differences in the ability of these drugs to improve performance on this task. Choline exerted no apparent effects in the aged monkeys at any dose tested. Physostigmine clearly enhanced performance in certain aged monkeys, but the optimal dose varied dramatically between subjects, replicating previously published results with aged Rhesus monkeys and humans. Arecoline produced clear improvement within a restricted dose range, with little variation in optimal dose between subjects. In addition to demonstrating differences in the effects of different cholinomimetics on memory performance in aged primates, these data also suggest a possible rationale for future investigations. Assuming that each of these drugs primarily affected cholinergic function in the manner conventionally attributed, these data suggest that, within the cholinergic system, the more directly one stimulates the receptor, the more one might expect robust and consistent effects on memory performance in aged subjects.

Neuropeptide effects on memory in aged monkeys.

The effects of several neuropeptides were evaluated using a non-human primate model of age-related memory impairments. Several doses of ACTH4-10, lysine vasopressin, arginine vasopressin, oxytocin and somatostatin were each tested in several aged monkeys. Because data from a large number of non-drug control sessions was collected before, during and after this study, it was possible to define the normal range of control performance for each monkey and statistically determine whether a change in performance under any single dose of drug reflected a significant change from the particular monkey's normal baseline performance. Although none of the neuropeptides produced consistent group effects, evaluations of individual subjects against their own baseline performance revealed reliable changes at certain doses. Arginine vasopressin appeared to produce the best overall effects with three of the five monkeys exhibiting reliable changes in performance from baseline. These same three monkeys also responded positively to the lysine form. Oxytocin impaired memory in three of the six aged monkeys tested over a wide range of doses. Three of six aged monkeys performed better under ACTH4-10 compared to baseline; however, in two of these cases only a single dose was effective. The performance of only one subject was improved under somatostatin, and this was at a single dose only. The data reported here provide evidence for neuropeptides producing behavioral improvement in non-human primates using an appetitive task, eliminating a popular criticism that the data in this literature has depended too heavily on the testing of rodents in shock-motivated tasks. Additionally, the improvements observed in this study involve a behavior that it naturally impaired by age and one which has many operational similarities and some empirical relevance to measures of recent memory in humans. However, these positive findings must be tempered by the lack of robust effects and high individual variation observed.

Localization of cellular changes within multimodal sensory regions in aged monkey brain: possible implications for age-related cognitive loss.

Young (4 to 7 years) and aged (18 to 28 years) rhesus monkeys were sacrificed and various neuromorphometric analyses performed to determine age differences in gross topography, cell population and patterns of cellular degeneration. Two brain regions implicated for their role in age-related cognitive disturbances, the hippocampus and the gyri bordering the principal sulcus in the frontal cortex were selected for these comparisons. Reliable morphometric differences between age groups were observed in both neural areas. One significant difference observed in the hippocampus was a reduced mean depth of the pyramidal layer of the CA-1 zone in the aged monkeys. Also, the mean number of neurons per transverse section in the CA-1 zone of the pyramidal layer was significantly less in the aged monkeys, and in certain instances cell gaps were observed in this region. In the lateral principal gyrus of the frontal cortex, the number of neurons in full-depth "cores" was lower in the old monkeys, glial count was higher and the ratio of neurons to neuroglia, therefore, lower in the older monkeys. Further, the mean area of the principal gyri, measured planimetrically from the apex of the medial gyrus to that of the lateral gyrus was significantly smaller in the aged monkeys. These findings indicate that significant age-dependent cellular differences occur in brain areas thought to be functionally involved in the particular cognitive behaviors most severely impaired in aged monkeys. These differences in brain morphology may, therefore, help provide some leads into the types of neurological changes contributing to the severe cognitive disorders suffered by the elderly.

Effects of aging and dementia upon recent visuospatial memory.

In two experiments, a total of 20 young normals, 29 elderly normals, and 76 elderly demented subjects were administered a computerized delayed visuospatial recall task. Subjects were instructed to remember which room of a 25-room house had a light on in the window. A choice reaction time task was interposed during the delay interval (0-120 seconds) between stimulus presentation and recall. The test was designed to be (1) face valid-relevant to the subjects' everyday lives, (2) sensitive and specific to the cognitive decline associated with senile dementia of the Alzheimer's type (SDAT), and (3) comparable to animal memory tests. Immediate recall of the spatial location of a single stimulus was found to be deficient in severely demented subjects only, and all groups exhibited a decline in recall accuracy with increasing delay intervals. This decline in recall accuracy was greatest in severely demented subjects, smaller in less demented subjects, still smaller in aged normals, and smallest in young normals. No significant forgetting of spatial location occurred between 30 and 120 seconds after stimulus presentation. Increasing stimulus number decreased recall accuracy in all groups and the elderly and elderly demented subjects were more sensitive to the increase in stimulus load than the young normals. Choice reaction time also proved sensitive to age and severity of dementia. Correlation analyses demonstrated that delayed spatial recall (as well as choice reaction time) is highly correlated with clinically evaluated global cognitive status, as well as with tests of verbal recall.(ABSTRACT TRUNCATED AT 250 WORDS)

Psychomotor performance in the senescent rodent: reduction of deficits via striatal dopamine receptor up-regulation.

In order to determine the relationship between striatal dopamine (DA) receptor density and psychomotor performance in senescent animals, two experiments were carried out. In the first, the age-related motor deficits were characterized using a battery of four psychomotor tests (rod walking, wire hanging, inclined screen, plank walking). These tests were administered to three groups of male Fischer rats (mature, 6-8 months; middle aged, 12-18 months; and senescent, 25 months) and performance measured. Age-related differences were observed on all the tasks, with the oldest animals showing the poorest performance. These animals were then used in a second experiment in which one-half of the group of animals from each age was administered 1.86 mg/kg/day of haloperidol for 14 days (via surgically implanted Alza Minipumps. Control groups of animals from each age were given pumps which contained only the vehicle (HCl diluted with distilled water, pH = 2.9). Following the 14 day drug administration, the pumps were surgically removed and 3 days later all the groups were retested on the psychomotor tests. Stereotypy (to 0.5 mg/kg of apomorphine, sniffing, licking, grooming and cage crossings) was also re-examined. Results show that haloperidol-treated animals from all three age groups display greater response times (i.e., better performance) than vehicle-treated animals on the battery of four motor tests and, the haloperidol-treated old animals exhibit more sniffing and grooming than the vehicle-treated old animals. Parallel increases in [3H]spiperone binding seen in all haloperidol-treated groups suggest a relationship between increases in the density of striatal DA receptors and improvement in motor performance.

Age-related alterations in the rodent brain cholinergic system and behavior.

Pre- and postsynaptic cholinergic markers were studied in various brain regions of mice and rats aged 6 to 30 months in an attempt to determine whether alterations in this transmitter system occur during the normal aging process. Reliable decreases in cholinergic receptor binding and choline acetyltransferase (CAT) activity were found in the cerebral cortex and corpus striatum. These alterations in the cholinergic system were typically more consistent and robust than changes involving glutamic acid decarboxylase, and enzyme marker for GABA neurons. No statistically significant changes in any markers were found in the hippocampus of either species. Significant age-related changes in retention of passive avoidance learning and locomotor activity were also observed in these same animals. These findings demonstrate that changes in the cholinergic system occur naturally in aged mice and rats and that both the loss of cholinergic receptors and decrease in cat activity may contribute to the motor and mental impairments that often accompany old age.

Profound effects of combining choline and piracetam on memory enhancement and cholinergic function in aged rats.

In an attempt to gain some insight into possible approaches to reducing age-related memory disturbances, aged Fischer 344 rats were administered either vehicle, choline, piracetam or a combination of choline or piracetam. Animals in each group were tested behaviorally for retention of a one trial passive avoidance task, and biochemically to determine changes in choline and acetylcholine levels in hippocampus, cortex and striatum. Previous research has shown that rats of this strain suffer severe age-related deficits on this passive avoidance task and that memory disturbances are at least partially responsible. Those subjects given only choline (100 mg/kg) did not differ on the behavioral task from control animals administered vehicle. Rats given piracetam (100 mg/kg) performed slightly better than control rats (p less than 0.05), but rats given the piracetam/choline combination (100 mg/kg of each) exhibited retention scores several times better than those given piracetam alone. In a second study, it was shown that twice the dose of piracetam (200 mg/kg) or choline (200 mg/kg) alone, still did not enhance retention nearly as well as when piracetam and choline (100 mg/kg of each) were administered together. Further, repeated administration (1 week) of the piracetam/choline combination was superior to acute injections. Regional determinations of choline and acetylcholine revealed interesting differences between treatments and brain area. Although choline administration raised choline content about 50% in striatum and cortex, changes in acetylcholine levels were much more subtle (only 6-10%). No significant changes following choline administration were observed in the hippocampus. However, piracetam alone markedly increased choline content in hippocampus (88%) and tended to decrease acetylcholine levels (19%). No measurable changes in striatum or cortex were observed following piracetam administration. The combination of choline and piracetam did not potentiate the effects seen with either drug alone, and in certain cases the effects were much less pronounced under the drug combination. These data are discussed as they relate to possible effects of choline and piracetam on cholinergic transmission and other neuronal function, and how these effects may reduce specific memory disturbances in aged subjects. The results of these studies demonstrate that the effects of combining choline and piracetam are quite different than those obtained with either drug alone and support the notion that in order to achieve substantial efficacy in aged subjects it may be necessary to reduce multiple, interactive neurochemical dysfunctions in the brain, or affect activity in more than one parameter of a deficient metabolic pathway.

Presynaptic cholinergic mechanisms in brain of aged rats with memory impairments.

Presynaptic cholinergic mechanisms were investigated in various brain regions of aged Fisher 344 rats with documented 24 hr retention deficits measured in a single-trial passive avoidance tasks. Sodium-dependent high affinity choline uptake was found to be decreased by 22% in hippocampus of 23-26 month old animals as compared to 6 month old controls. Prior depolarization of hippocampal or cortical synaptosomes with K+ resulted in stimulation of choline uptake which was similar in aged rats and young controls. No age-related differences were observed either in hippocampal, cortical, striatal acetylcholine or choline concentrations, or in the activity of choline acetyltransferase in hippocampus. Synthesis of acetylcholine in hippocampal and cortical slices under basal conditions, as well as under K+-stimulated concentrations, did not differ in the two age groups examined. These neurochemical findings are consistent with an age-related decrease in hippocampal cholinergic neuronal activity without an actual loss in cholinergic neuron number. It is further suggested that this reduction in cholinergic neuronal activity may be related to the deficit in cognitive performance observed in aged Fisher rats.

Behavioral effects of phosphatidylserine in the aged Fischer 344 rat: amelioration of passive avoidance deficits without changes in psychomotor task performance.

A series of studies was conducted to evaluate the effects of phosphatidylserine (PS) in aged Fischer 344 rats. No effects were observed in any of four psychomotor tasks in which aged rats normally show deficits, nor on measures of shock sensitivity. However, significant dose-related effects on retention of passive avoidance were observed when PS was given both 30 min prior to training and retention. Further, in a second experiment similar positive effects were observed when PS was given only 30 min prior to training, as well as only 5 min following training. These results suggest that one effect of PS may include an ability to enhance neural events involved in the encoding or consolidation of new information into memory.