Lanny J. Haverkamp

Morphology and Toxicity of Aβ-(1-42) Dimer Derived from Neuritic and Vascular Amyloid Deposits of Alzheimer's Disease

In the course of analyzing the chemical composition of Alzheimers disease neuritic and vascular amyloid, we have purified stable dimeric and trimeric components of Aβ 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 M 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 Aβ-(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 Aβ-(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 Aβ-(1-40/42) dimers and trimers from Alzheimers disease amyloid represent the soluble oligomers of Aβ recently described in Alzheimers 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).

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 patients 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.

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

Senile plaques found in the brains of patients with Alzheimers 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 Alzheimers dementia.

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

The β-amyloid peptide 1–42 (Aβ1–42), 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 Aβ 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 Aβ binding to microglia and Aβ 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 Aβ1–42 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 Aβ 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.

Incidence and characteristics of spinal decompression surgery after the onset of symptoms of amyotrophic lateral sclerosis

OBJECTIVE:The high incidence of spondylosis in patients at the mean age of onset (55.7 yr) of amyotrophic lateral sclerosis (ALS) can make recognition of ALS as a cause of weakness difficult. METHODS:To assess the impact of this diagnostic dilemma on neurosurgical practice, we performed a retrospective analysis of a database of more than 1500 patients with motor neuron disease. RESULTS:Of 1131 patients with typical, sporadic ALS, 47 (4.2%) underwent decompressive spinal surgery after the onset of retrospectively recognized symptoms of ALS. Among 55 operations in 47 ALS patients, 86% yielded no improvement, 9% produced minor improvement, and only 5% provided significant benefit. Cervical decompression was performed in 56%, lumbar in 42%, and thoracic in 2%. Foot drop was a symptom prompting surgery in 11 patients, and in 10, this finding was subsequently attributed solely to ALS. No differences between ALS patients who underwent spinal decompression and other ALS patients were noted regarding age at symptom onset, severity of impairment at time of diagnosis, or rate of disease progression. Not surprisingly, patients who had spinal surgery tended to have a longer interval between retrospectively recognized symptom onset and diagnosis of ALS. CONCLUSION:A small but significant number of patients with unrecognized ALS undergo spinal surgery that in retrospect may be inappropriate. The possibility of ALS must be considered in the evaluation of patients with weakness even in the presence of radiographic evidence of spondylosis and nerve root or spinal cord impingement.

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.

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.

Muscle-derived trophic factors influencing cholinergic neurons in vitro and in vivo

Publisher Summary This chapter review the effects of two different factors derived from muscle which enhance cholinergic properties of neurons in vitro. In the absence of muscle extract the development of choline acetyltransferase (CAT) in tissue culture follows a biphasic time course in which there is an initial decrease in CAT activity followed by a period of steadily increasing activity. The addition of muscle extract to the cultures prevents the initial decline and stimulates the subsequent development of CAT activity. A factor (cholinergic development factor, CDF) in the muscle extract which produces such effects has been purified and partially characterized. While CDF was being purified from rat skeletal muscle, attempts were made to purify neurotrophic activity from autopsied human skeletal muscle. Chick ciliary neurons were used to assay the enhancement of acetylcholine synthesis, CAT. Although neurotrophic factors may exhibit a range of specific behaviors in vitro such as neuron survival or enhancement of cholinergic activity, for complete validation such factors must be shown to be capable of influencing neuronal survival in vivo. An in vivo effect could eliminate a trivial explanation of the in vitro phenomena, i.e. that purified extracts may provide essential components missing or perturbed in the tissue culture environment, or that they act in a manner normally inoperative in vivo.

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.

Design using historical controls

Over the last 20 years, our clinic has evaluated and followed over 2100 patients, recording clinical data for each patient, and assessing the extent of dysfunction and rates of progression. This historical database provides a unique benchmark for carrying out ‘futility design’ experiments as described by Dr. Palesch, to assess whether therapies are sufficiently promising to warrant intensive investigation. This futility design could weed out ineffective drugs that probably should not proceed to Phase III studies, and permits a concentrated effort on drugs that have an increased likelihood of demonstrating efficacy. More than 1,000 patients have had two or more examinations, for a total of 6,000 examinations. Seventyfive percent of the patients had probable or definite ALS, and 9% were familial. Approximately 25% of patients presented with bulbar onset. The higher the age of onset, the more likely that patients will present with bulbar compromise. Females are more likely than males to present with bulbar compromise. In general, recent additions to our database have substantiated our prior publication, with one exception: we now agree that bulbar onset is associated with shorter survival at all ages, including both older and younger patients. The major question to be addressed in this discussion is how a historical database can be used most effectively in drug trials with the futility design. What are the relative strengths and limitations of such an approach? When evaluating the plotted clinical progression, we noted that many patients display a small lag before the course becomes linear. If you look at the design (Figure 1), the time from first symptom to a screening examination is the preslope. Next, we have from screening to baseline and then the 0–3 month prediction, which is very similar to when preslope is used. If you look at the pre-slope predicted versus the actual progression, there is very poor correlation. Similarly, if you compare the screening-to-baseline predicted to actual progression, there is also poor correlation. The best correlation with subsequent progression was found by using sequential examination scores from the first three months after the baseline examination. That gives us some confidence that it may be possible to use our database to weed out those drugs that are likely to be ineffective. We have used our database in validation studies as well as in determining potential efficacy of drugs to proceed to Phase III studies. First, to validate the database, we took all the placebo data from the Myotrophin (IGF-1) trial and looked at it in relation to our database. There were 90 placebo patients in the American trial, and there clearly was a statistical difference in slope of 25–26%. However, the placebos, intriguingly, matched our database when selecting patients with the same criteria (Figure 2, top). The same was done for riluzole. We had 199 patients who were on riluzole for at least six months, and we matched them against 459 patients who were not on riluzole, not on IGF, nor in any of our trials. Interestingly, we found no statistical difference in rate of progression (Figure 2, bottom). These have been the arguments that favor the use of a historical database, with some examples. I would like to highlight a point raised by Dr. Nigel Leigh which is that we have been very successful in our clinics – so successful that you might not be able to use a historical database. That is a reasonable concern; if you examine the data from 1983–87, compared to 1997–2001, the more-recent patients survived longer. Realizing that the introduction of newer medications and the wider use of supportive therapies may have contributed to improved patient survival, we excluded patients who were in the riluzole study and other patients who had been on riluzole or on myotrophin. Moreover, we tried to account for possible effects from BiPap respiratory assistance. However, we still found an improvement. Indeed, when comparing outcomes from the earlier time periods, 1983–87 and 1990–94, there was already an improvement in survival (Figure 3). In addition to survival time, improvement was also seen in the rates of disease progression; the patients from 1997–2001 changed less per month than the patients in the 1983–87 group. This improvement in survival and rate of progression raises a difficulty if we go back and use old database patients for the historical control, even for rejecting futility. This bias is not usually mentioned when the use of a historical database is being considered. Nevertheless, if looking for a substantial change, say 30%, comparison of efficacy of a tested compound to a historical database may still be a useful and meaningful first step, and may represent an appropriate ‘futility design’.