The value of database controls in pilot or futility studies in ALS.

OBJECTIVE: To evaluate the use and reliability of database controls in place of a placebo group in pilot or "futility" ALS trials. METHODS: We compared the rates of disease progression in the placebo arm of the clinical phase III US Insulin-like Growth Factor-I Trial (n = 90) with the rates of disease progression of 207 patients with ALS selected from 1,600 ALS database patients using the same inclusion criteria. RESULTS: The mean rates of change in the Appel ALS (AALS) score were nearly identical in the placebo group (4.70 points/month) and in the database group (4.79 points/month). In addition, there was no significant difference in the median time to achieving a 20-point progression in AALS score: 143 days for database match vs 146 days for the placebo group (log rank p = 0.88). Furthermore, in the multivariate Cox analysis, both the rate at which the disease had progressed prior to first exam (preslope) (p < 0.001) and first exam AALS total score (p = 0.01) were shown to be covariates of subsequent rate of disease progression. CONCLUSION: The similarity in disease progression between placebo arm of clinical phase III trial and matched database group suggests the value of historical databases in futility trials. However, the proposed study design requires appropriate matching of study patients with database controls. Based on our results, we suggest matching by stage of the disease and rate of clinical decline in a contemporaneous ALS population.

The HHQK domain of beta-amyloid provides a structural basis for the immunopathology of Alzheimer's disease.

The beta-amyloid peptide 1-42 (Abeta1-42), a major component of neuritic and core plaques found in Alzheimer's disease, activates microglia to kill neurons. Selective modifications of amino acids near the N terminus of Abeta showed that residues 13-16, the HHQK domain, bind to microglial cells. This same cluster of basic amino acids is also known as a domain with high binding affinity for heparan sulfate. Both Abeta binding to microglia and Abeta induction of microglial killing of neurons were sensitive to heparitinase cleavage and to competition with heparan sulfate, suggesting membrane-associated heparan sulfate mediated plaque-microglia interactions through the HHQK domain. Importantly, small peptides containing HHQK inhibited Abeta1-42 cell binding as well as plaque induction of neurotoxicity in human microglia. In vivo experiments confirmed that the HHQK peptide reduces rat brain inflammation elicited after infusion of Abeta peptides or implantation of native plaque fragments. Strategies which exploit HHQK-like agents may offer a specific therapy to block plaque-induced microgliosis and, in this way, slow the neuronal loss and dementia of Alzheimer's disease.

Prevalence and correlates of neuropsychological deficits in amyotrophic lateral sclerosis.

OBJECTIVE: To determine the prevalence and correlates of neuropsychological impairment in a large cohort (n = 146) of patients with typical, sporadic (non-familial) amyotrophic lateral sclerosis. METHODS: A battery of neuropsychological tests was administered to patients with amyotrophic lateral sclerosis who were attending a monthly outpatient clinic or who were in hospital undergoing diagnostic tests. RESULTS: Comparing individual patient's scores with relevant normative data, 35.6% of the patients displayed evidence of clinically significant impairment, performing at or below the 5th percentile on at least two of the eight neuropsychological measures. Deficits were most common in the areas of problem solving, attention/mental control, continuous visual recognition memory, word generation, and verbal free recall. Impairment was most prevalent in patients with dysarthria (48.5%), but 27.4% of non-dysarthric patients were also impaired. Impaired patients had more severe or widespread symptoms of amyotrophic lateral sclerosis than non-impaired patients, and had fewer years of education. CONCLUSION: Neither the conventional wisdom that cognition is intact in nearly all patients with amyotrophic lateral sclerosis, nor more recent suggestions that cognition is often at least mildly impaired seems to be correct. A minority of patients with amyotrophic lateral sclerosis displayed evidence of significant impairment. Dysarthria, low education, and greater severity of motor symptoms were risk factors for impairment.

Specific domains of beta-amyloid from Alzheimer plaque elicit neuron killing in human microglia.

Alzheimer's disease (AD) is found to have striking brain inflammation characterized by clusters of reactive microglia that surround senile plaques. A recent study has shown that microglia placed in contact with isolated plaque fragments release neurotoxins. To explore further this process of immunoactivation in AD, we fractionated plaque proteins and tested for the ability to stimulate microglia. Three plaque-derived fractions, each containing full-length native A beta 1-40 or A beta 1-42 peptides, elicited neurotoxin release from microglia. Screening of various synthetic peptides (A beta 1-16, A beta 1-28, A beta 12-28, A beta 25-35, A beta 17-43, A beta 1-40, and A beta 1-42) confirmed that microglia killed neurons only after exposure to nanomolar concentrations of human A beta 1-40 or human A beta 1-42, whereas the rodent A beta 1-40 (5Arg-->Gly, 10Tyr-->Phe 13His-->Arg) was not active. These findings suggested that specific portions of human A beta were necessary for microglia-plaque interactions. When coupled to microspheres, N-terminal portions of human A beta (A beta 1-16, A beta 1-28, A beta 12-28) provided anchoring sites for microglial adherence whereas C-terminal regions did not. Although itself not toxic, the 10-16 domain of human A beta was necessary for both microglial binding and activation. Peptide blockade of microglia-plaque interactions that occur in AD might prevent the immune-driven injury to neurons.

Morphology and toxicity of Abeta-(1-42) dimer derived from neuritic and vascular amyloid deposits of Alzheimer's disease.

In the course of analyzing the chemical composition of Alzheimer's disease neuritic and vascular amyloid, we have purified stable dimeric and trimeric components of Abeta peptides. These peptides (molecular mass 9.0 and 13.5 kDa) were separated by size exclusion chromatography in the presence of 80% formic acid or 5 guanidine thiocyanate, pH 7.4. The average ratio of monomers, dimers, and trimers was 55:30:15, respectively. Similar structures were produced over time upon incubation of synthetic Abeta-(1-42) at pH 7.4. The stability of these oligomeric forms was also demonstrated by Western blot and mass spectrometry. Atomic force microscopy and electron microscopy rotary shadowing revealed that the monomers polymerized into 8-10-nm filaments, whereas the dimers generated prolate ellipsoids measuring 3-4 nm in diameter. The pathogenic effects of the dimeric Abeta-(1-40/42) were tested in cultures of rat hippocampal neuron glia cells. Only in the presence of microglia did the dimer elicit neuronal killing. It is possible that these potentially pathogenic Abeta-(1-40/42) dimers and trimers from Alzheimer's disease amyloid represent the soluble oligomers of Abeta recently described in Alzheimer's disease brains (Kuo, Y.-M., Emmerling, M. R., Vigo-Pelfrey, C., Kasunic, T. C., Kirkpatrick, J. B., Murdoch, G. H., Ball, M. J., and Roher, A. E. (1996) J. Biol. Chem., 271, 4077-4081).

Senile plaques stimulate microglia to release a neurotoxin found in Alzheimer brain.

Senile plaques found in the brains of patients with Alzheimer's disease (AD) are surrounded by clusters of reactive microglia. Isolated human microglia placed in contact with plaques in vitro are activated to release a factor which is toxic to neurons. This same neurotoxin is found in AD brain tissue and causes damage to pyramidal neurons in vivo when infused into rat hippocampus. Highest concentrations of the neurotoxin are in those brain structures most burdened by reactive microglia, suggesting that plaque-activated cells contribute to the neuronal damage and impaired cognition seen in patients with Alzheimer's dementia.

Natural history of amyotrophic lateral sclerosis in a database population. Validation of a scoring system and a model for survival prediction.

Over 1200 patients with motor neuron disease have been carefully diagnosed, followed, and included in a detailed database delineating characteristics of the disease. Of these patients, 831 were identified as exhibiting typical, sporadic amyotrophic lateral sclerosis (ALS). The progression of the disease in these patients has been followed with our scoring system, and the ALS score was verified as a significant covariate of survival. Age at first symptom, delay from first symptom to entering ALS clinic, and rate of change of respiratory function were also identified as significant covariates of survival. These measures, applied to the Cox proportional hazards model, were used to develop a mathematical model for prediction of survival time in ALS, which proved highly accurate for the 80% of patients at intermediate risk. For those patients, a second model was developed which accurately predicts, after an initial period of observation, the time over which ALS patients will decline a set number of points in total ALS score. Such validation permits initial trials for drug therapies in ALS by comparison of relatively small groups of treated patients to this historical control group, based on the model of predicted time to a particular decrement in total ALS score.

Biological studies of a putative avian muscle-derived neurotrophic factor that prevents naturally occurring motoneuron death in vivo.

A series of in vivo studies have been carried out using the chick embryo to address several critical questions concerning the biological, and to a lesser extent, the biochemical characteristics of a putative avian muscle-derived trophic agent that promotes motoneuron survival in vivo. A partially purified fraction of muscle extract was shown to be heat and trypsin sensitive and rescued motoneurons from naturally occurring cell death in a dose-dependent fashion. Muscle extract had no effect on mitotic activity in the spinal cord and did not alter cell number when administered either before or after the normal cell death period. The survival promoting activity in the muscle extract appears to be developmentally regulated. Treatment with muscle extract during the cell death period did not permanently rescue motoneurons. The motoneuron survival-promoting activity found in skeletal muscle was not present in extracts from a variety of other tissues, including liver, kidney, lung, heart, and smooth muscle. Survival activity was also found in extracts from fetal mouse, rat, and human skeletal muscle. Conditioned medium derived from avian myotube cultures also prevented motoneuron death when administered in vivo to chick embryos. Treatment of embryos in ovo with muscle extract had no effect on several properties of developing muscles. With the exception of cranial motoneurons, treatment with muscle extract did not promote the survival of several other populations of neurons in the central and peripheral nervous system that also exhibit naturally occurring cell death. Initial biochemical characterization suggests that the activity in skeletal muscle is an acidic protein between 10 and 30 kD. Examination of a number of previously characterized growth and trophic agents in our in vivo assay have identified several molecules that promote motoneuron survival to one degree or another. These include S100 beta, brain-derived neurotrophic factor (BDNF), neurotrophin 4/5 (NT-4/5), ciliary neurotrophic factor (CNTF), transforming growth factor beta (TGF beta), platelet-derived growth factor-AB (PDGF-AB), leukemia inhibitory factor (CDF/LIF), and insulin-like growth factors I and II (IGF). By contrast, the following agents were ineffective: nerve growth factor (NGF), neurotrophin-3 (NT3), epidermal growth factor (EGF), acidic and basic fibroblast growth factors (aFGF, bFGF), and the heparin-binding growth-associated molecule (HB-GAM).(ABSTRACT TRUNCATED AT 400 WORDS)

Nigral damage and dopaminergic hypofunction in mesencephalon-immunized guinea pigs.

To support a potential role for immune mechanisms in the destruction of substantia nigra (SN) neurons, guinea pigs were immunized with bovine mesencephalon containing SN neurons. After immunization no clinical signs of basal ganglia dysfunction appeared. However, pathological examination revealed evidence of neuronal damage in the SN in 8 of 17 guinea pigs immunized with bovine mesencephalon. No nigral pathology was noted in animals immunized with spinal cord gray matter or Freund's adjuvant alone. Accompanying the SN damage in mesencephalon-immunized guinea pigs was a 25% decrease in tyrosine hydroxylase activity in the SN and a 27% decrease in dopamine content in the striatum. Deposits of IgG were detected by immunohistochemical techniques in sections of SN from mesencephalon-immunized guinea pigs and in sections of human SN after exposure to serum from mesencephalon-immunized guinea pigs. These data document the antigenicity of SN and suggest the possibility that immune mechanisms can contribute to basal ganglia pathology.

Ontogeny of high- and low-affinity nerve growth factor receptors in the lumbar spinal cord of the developing chick embryo.

The binding of 125I-labeled nerve growth factor-beta (NGF) to soluble extracts of intact or dissociated embryonic chick lumbar cords was used to investigate the kinetic properties and to quantify the levels of NGF receptors (NGFRs) in the developing chick between Embryonic Day 6 (E6) and E10. Both high-affinity (type I; Kd = 7.4 x 10(-11) M) and low-affinity (type II; Kd = 2.4 x 10(-9) M) NGFRs were detected by Scatchard analysis of 125I-NGF binding to E6 spinal cord extracts. A total of 4 x 10(9) type I and 5 x 10(10) type II receptors/cord were found in extracts of E6 cords. As development progressed there was a decline of both types of NGFRs; however, the decline of type I receptors occurred more rapidly than that of type II. Between E6 and E8 greater than 90% of the type I but only 25% of the type II receptors were lost. These relative rates of loss were maintained over the next week of development, with type I receptors no longer detectable by E12, and type II receptors reduced to 0.025% of their E6 numbers by E15. Analyses of NGFR levels in subpopulations of E6 and E8 lumbar cord cells, prepared by metrizamide density gradient centrifugation, showed that during this period there is an enrichment of both types of NGFRs in the motoneuron-containing subpopulation, relative to other cell populations. The loss of NGFRs does not appear to be influenced by those peripheral-trophic interactions which control other aspects of motoneuron development: curarization of the embryos between E6 and E9 increased motoneuron number in E10 embryos by 30%, but did not significantly affect the loss of NGFRs. These results provide the first quantitative evidence that type I and type II NGFRs are differentially regulated in the spinal cord during embryonic development and raise the possibility that distinct cellular mechanisms may govern their expression.

Skeletal muscle proteins rescue motor neurons from cell death in vivo.

Because embryonic neurons are more sensitive to the effects of target derived factors than adult neurons, the developing nervous system provides a sensitive model for investigating the in vivo actions of target-derived growth factors. We have used the developing chick embryo to document that skeletal muscle contains substances that selectively enhance the in vivo survival of motor neurons. We have also shown that a single purified skeletal muscle protein (CDF) is capable of rescuing motor neurons during the period of naturally occurring cell death. The rescue of motor neurons in vivo by CDF is consistent with the hypothesis that distinct neurotrophic factors exist which regulate the timing and extent of the naturally occurring death of specific populations of neurons. The effects of CDF appear to be specific for cholinergic somatic motor neurons, since the survival of other types of spinal cord neurons, which also exhibit cell loss during the treatment period, was not affected by CDF treatment. In contrast, treatment of the embryos with extracts of tissues not innervated by motor neurons, or with NGF or bFGF, does not affect motor neuron survival. Thus the ability to rescue motor neurons during the period of cell death appears to be a distinct property of CDF and provides indirect evidence that this molecule may play a role in the survival and development of motor neurons. The role of neurotrophic factor involvement in the pathophysiology of degenerative diseases such as ALS remains entirely speculative. However, the demonstration that such factors affect the neuronal subtypes at risk in these diseases provides experimental support for this possibility.(ABSTRACT TRUNCATED AT 250 WORDS)

The regulation of motoneuron survival and differentiation by putative muscle-derived neurotrophic agents: neuromuscular activity and innervation.

The chronic blockade of neuromuscular activity is known to promote the survival of developing motoneurons in vivo in the chick, mouse and rat embryo. Increased survival in this situation may reflect an activity-dependent mechanism for the regulation of trophic factor production by target cells. To test this notion, we have examined motoneuron survival in vivo and choline acetyltransferase (ChAT) development in vitro following treatment of chick embryos and rat spinal cord cultures with partially purified skeletal muscle extracts derived from normally active, chronically paralyzed and aneural embryos, and from denervated postnatal chickens. Extracts from active and paralyzed chick embryos were equally effective in promoting motoneuron survival and ChAT activity. Aneural embryonic muscle extracts were slightly less effective in promoting motoneurons survival in vivo, but were not significantly different from control extracts in the in vitro ChAT assay. Denervated postnatal muscle extracts, however, were more effective in enhancing both motoneuron survival and ChAT activity. These data indicate that: (1) the promotion of motoneuron survival in vivo by activity blockade may not be mediated by an up-regulation of trophic factor synthesis in target cells; (2) postnatal innervation may regulate the production of putative muscle-derived neurotrophic factors; and (3) the synthesis or availability of trophic agents may be regulated differently in embryonic and postnatal muscle.

Activity blockade at the neuromuscular junction: effects on spinal cord choline acetyltransferase during and after the period of naturally occurring cell death.

Treatment of developing chick embryos with curare during the period of naturally occurring cell death is known to ameliorate the motoneuron loss which occurs at this time. Such treatment was found likewise to lead to an increase in levels of lumbar spinal cord choline acetyltransferase (ChAT), indicating that the levels of this enzyme may act as a marker for motoneuron number. Treatment after the cell death period also increased lumbar ChAT activity, suggesting that the neurotrophic effects resulting from curare treatment may also include the stimulation of motoneuron cholinergic differentiation.

A functional framework for neurotrophic activities.

In attempts to understand the diverse roles which trophic activities exert in neuronal development and maintenance, it is useful to define some framework from within which to view their function. An example of such an outline is presented, sufficiently broad to avoid excluding by definition agents with clearly neurotrophic effect.

Reduction of naturally occurring motoneuron death in vivo by a target-derived neurotrophic factor.

Treatment of chick embryos in ovo with crude and partially purified extracts from embryonic hindlimbs (days 8 to 9) during the normal cell death period (days 5 to 10) rescues a significant number of motoneurons from degeneration. The survival activity of partially purified extract was dose-dependent and developmentally regulated. The survival of sensory, sympathetic, parasympathetic, and a population of cholinergic sympathetic preganglionic neurons was unaffected by treatment with hindlimb extract. The massive motoneuron death that occurs after early target (hindlimb) removal was partially ameliorated by daily treatment with the hindlimb extract. These results indicate that a target-derived neurotrophic factor is involved in the regulation of motoneuron survival in vivo.

Developmental discord among markers for cholinergic differentiation: in vitro time courses for early expression and responses to skeletal muscle extract.

The effects of skeletal muscle extract on the development of CAT, ACh synthesis, high affinity choline uptake, and AChE activities were studied in dissociated ventral spinal cord cultures prepared from 14-day gestational rat embryos. In the absence of muscle extract, the development of CAT and AChE follow biphasic time courses in which they show initial declines followed by periods of steadily increasing activity. In contrast, ACh synthesis and high affinity choline uptake both gradually increase throughout the entire culture period. The presence of muscle extract both prevents the initial decline of CAT and AChE as well as stimulates the rates of development of all four cholinergic markers; however, the degrees and time courses of stimulation differ markedly. The effects of muscle extract on the kinetic and pharmacological properties of ACh synthesis and choline uptake in rat ventral cord cultures were also investigated. Cells treated with muscle extract for 2 days express both high affinity (Km = 1.6 microM) and low affinity (Km = 22 microM) choline uptake mechanisms. Control cells, on the other hand, express only low affinity uptake at this stage but develop a high affinity uptake mechanism by Day 7. During this time both ACh synthesis and high affinity choline uptake become increasingly sensitive to inhibition by hemicholinium-3. These results demonstrate that skeletal muscle factors enhance the development of cholinergic properties in embryonic spinal cord cultures. However, differences in sensitivity to muscle extract concentration, time courses of development, and degrees of stimulation suggest that these changes may involve distinct cellular mechanisms which are differentially affected by skeletal muscle factors.

Neurotrophic interactions in the development of spinal cord motoneurons.

The final number of spinal cord motoneurons is attained by a two-step process involving the proliferation of precursor cells and the loss by cell death of a proportion (approximately 50%) of the post-mitotic neurons. Although the mechanisms responsible for the proliferation of stereotyped numbers of motoneurons are not understood, considerable evidence from in vitro as well as in vivo studies indicates that the second step in attaining population size (cell death) is controlled by the interaction of motoneurons with both their efferent targets and their afferent inputs. Target influences on motoneuron survival are thought to be regulated by muscular activity and by competition for limited amounts of neurotrophic factors derived from striated skeletal muscles. However, evidence that such putative neurotrophic factors actually modulate motoneuron survival in vivo has been lacking. Using crude and partially purified extracts from embryonic hindlimbs (Days 8-9) we have found that the treatment of chick embryos in ovo with these agents during the normal cell death period (Days 5-10) rescues a significant number of motoneurons from degeneration. Kidney or lung extracts and heat-inactivated hindlimb extracts were ineffective. The survival-inducing activity of partially purified extract was dose dependent and developmentally regulated. The survival of sensory, sympathetic and a population of cholinergic sympathetic preganglionic neurons was unaffected by treatment with hindlimb extract. The massive motoneuron death that occurs after early target (hindlimb) removal was partially ameliorated by daily treatment with the hindlimb extract. Survival-inducing activity of the extract is lost after trypsin treatment. Taken collectively these results indicate that a target-derived protein or polypeptide neurotrophic factor is involved in the regulation of motoneuron survival in vivo.

Behavioral development in the absence of neural activity: effects of chronic immobilization on amphibian embryos.

Embryos of Xenopus laevis and Ambystoma mexicanum were continually immobilized from premotile stages of development to stages at which normally reared embryos were swimming well. Immobilization was achieved through exposure to solutions of chloretone, lidocaine, or alpha-bungarotoxin. At a number of stages after recovery from the drugs, spontaneous and stimulated behaviors were extensively quantified. Immobilization of Ambystoma embryos resulted in temporary defects in musculoskeletal development. In contrast, treated Xenopus embryos could not be distinguished from controls by simple visual observation within minutes to hours after removal from the drug solutions. Quantifications of behavior revealed, however, a transient period of 24-48 hr during which treated embryos exhibited consistently reduced measures of stimulated swimming, while showing an increase in frequency of spontaneous movements. Detailed behavioral testing could detect no permanent effects of chronic immobilization in either species after this initial period of recovery. The results are discussed in reference to the classic works of Harrison (1904), Carmichael (1926, 1927), and Matthews and Detwiler (1926).