Evaluation of Neuropathological Features in the SOD1-G93A Low Copy Number Transgenic Mouse Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) still depicts an incurable and devastating disease. Drug development efforts are mostly based on superoxide dismutase 1 gene (SOD1)-G93A mice that present a very strong and early phenotype, allowing only a short time window for intervention. An alternative mouse model is available, that is based on the same founder line but has a reduced SOD1-G93A copy number, resulting in a weaker and delayed phenotype. To be able to use these SOD1-G93A/low mice for drug testing, we performed a characterization of ALS-typical pathologies. All analyses were performed compared to non-transgenic (ntg) littermates of the same sex and age. In vivo analysis of SOD1-G93A/low mice was performed by weekly body weight measurements, analysis of the survival rate, and measurement of the muscle strength of 24-30 weeks old female and male SOD1-G93A/low mice. Immunofluorescent labeling of SOD1, glial fibrillary acidic protein (GFAP), and ionized calcium-binding adaptor molecule 1 (Iba1) protein was performed in the cervical, thoracic, and lumbar ventral horn of the spinal cord of 24-30 weeks old male and female SOD1-G93A/low mice. The musculus gastrocnemius of male SOD1-G93A/low mice was labeled with fluorophore-conjugated α-bungarotoxin and antibodies against phosphorylated neurofilaments. Fluorescent labeling was detected and quantified by macro-based image analysis. Although SOD1 protein levels were highly increased in both sexes and all age groups, levels strongly peaked in 30 weeks old male SOD1-G93A/low mice. Astrocytosis and activated microglia in the spinal cord ventral horn and phosphorylated neurofilaments in the motor unit of the musculus gastrocnemius progressively increased, while muscle strength progressively decreased in male SOD1-G93A/low mice. In female SOD1-G93A/low mice, only activated microglia increased progressively, while muscle strength was constantly reduced starting at 26 weeks. These differences result in a shorter survival time of male SOD1-G93A/low mice of about 3 weeks compared to female animals. The results suggest that male SOD1-G93A/low mice present a stronger pathology and are, therefore, better suitable to evaluate the efficacy of new drugs against ALS as most pathological features are developing progressively paralleled by a survival time that allows treatment to start before symptom onset.

Homozygosity of BACHD rats not only causes strong behavioral deficits in young female rats but also a reduced breeding success.

Huntington's disease is known to be a purely genetic disease based on an expansion of a CAG base triplet repeat in the coding region of the Huntingtin gene. Some years ago, researchers were able to introduce the extensive full-length gene sequence of the mutant huntingtin gene into a rodent model. The resulting BACHD rat is already well characterized for behavioral deficits. So far, all analyses in this preclinical rat model were performed in male hemizygous animals. As homozygosity of transgenic models often causes an amplification of the phenotype and female HD patients present a stronger phenotype compared to men, we established a homozygous breeding colony and tested 2 and 5 months old homozygous male and female BACHD rats in a behavioral test battery. The tests included the grip strength test, Rota Rod, elevated plus maze, passive avoidance, and Barnes maze test. Our results show strong deficits in young female homozygous BACHD rats including increased body weight, motor deficits, muscle weakness, reduced anxiety and hypoactivity, as well as learning and memory deficits. Analysis of male homozygous BACHD rats showed only weak disease symptoms, similar compared to male hemizygous BACHD rats of already published studies. Evaluation of the breeding success showed that homozygous BACHD have a reduced number of pups at the time of birth that even decreases until weaning. Our results suggest that the phenotype of homozygous male BACHD rats barely differs from already published results of hemizygous BACHD rats while female homozygous BACHD rats display strong and early alterations.

Studies for Improving a Rat Model of Alzheimer's Disease: Icv Administration of Well-Characterized ß-Amyloid 1-42 Oligomers Induce Dysfunction in Spatial Memory.

During the past 15 years, several genetically altered mouse models of human Alzheimer's disease (AD) have been developed. These costly models have greatly facilitated the evaluation of novel therapeutic approaches. Injecting synthetic ß-amyloid (Aß) 1-42 species into different parts of the brain of non-transgenic rodents frequently provided unreliable results, owing to a lack of a genuine characterization of the administered Aß aggregates. Previously, we have published a new rat AD-model in which protofibrillar-fibrillar Aß1-42 was administered into rat entorhinal cortex (Sipos 2007). In order to develop a more reliable model, we have injected well-characterized toxic soluble Aß1-42 species (oligomers, protofibrils and fibrils) intracerebroventricularly (icv) into rat brain. Studies of the distribution of fluorescent-labeled Aß1-42 in the brain showed that soluble Aß-species diffused into all parts of the rat brain. After seven days, the Aß-treated animals showed a significant decrease of spatial memory in Morris water maze test and impairment of synaptic plasticity (LTP) measured in acute hippocampal slices. The results of histological studies (decreased number of viable neurons, increased tau levels and decreased number of dendritic spines) also supported that icv administration of well-characterized toxic soluble Aß species into rat brain provides a reliable rat AD-model.

Dihydropyridine Derivatives Modulate Heat Shock Responses and have a Neuroprotective Effect in a Transgenic Mouse Model of Alzheimer's Disease.

Heat shock proteins (Hsps) have chaperone activity and play a pivotal role in the homeostasis of proteins by preventing misfolding, by clearing aggregated and damaged proteins from cells, and by maintaining proteins in an active state. Alzheimer's disease (AD) is thought to be caused by amyloid-ß peptide that triggers tau hyperphosphorylation, which is neurotoxic. Although proteostasis capacity declines with age and facilitates the manifestation of neurodegenerative diseases such as AD, the upregulation of chaperones improves prognosis. Our research goal is to identify potent Hsp co-inducers that enhance protein homeostasis for the treatment of AD, especially 1,4-dihydropyridine derivatives optimized for their ability to modulate cellular stress responses. Based on favorable toxicological data and Hsp co-inducing activity, LA1011 was selected for the in vivo analysis of its neuroprotective effect in the APPxPS1 mouse model of AD. Here, we report that 6 months of LA1011 administration effectively improved the spatial learning and memory functions in wild type mice and eliminated neurodegeneration in double mutant mice. Furthermore, Hsp co-inducer therapy preserves the number of neurons, increases dendritic spine density, and reduces tau pathology and amyloid plaque formation in transgenic AD mice. In conclusion, the Hsp co-inducer LA1011 is neuroprotective and therefore is a potential pharmaceutical candidate for the therapy of neurodegenerative diseases, particularly AD.

Intranasal delivery of human beta-amyloid peptide in rats: effective brain targeting.

(1) Intranasal administration is a non-invasive and effective way for the delivery of drugs to brain that circumvents the blood-brain barrier. The aims of the study were to test a nasal delivery system for human beta-amyloid (A beta) peptides, to measure the delivery of the peptides to brain regions, and to test their biological activity in rats. (2) A beta(1-42), in the form of a mixture of oligomers, protofibrils, and fibrils was dissolved in a nasal formulation containing hydrophobic, hydrophylic, and mucoadhesive components. The peptide solution was administered intranasally to rats as a single dose or in repeated doses. (3) Nasally injected A beta labeled with the blue fluorescent dye amino-methyl coumarinyl acetic acid (AMCA) could be detected by fluorescent microscopy in the olfactory bulb and frontal cortex. The concentration of the peptide was quantified by fluorescent spectroscopy, and a significant amount of AMCA-A beta peptide could be detected in the olfactory bulb. Unlabeled A beta also reached the olfactory bulb and frontal cortex of rats as evidenced by intense immunostaining. (4) In behavioral experiments, nasal A beta treatment did not affect anxiety levels (open-field test) and short-term memory (Y-maze test), but significantly impaired long-term spatial memory in the Morris water maze. The treatments did not result in A beta immunization. (5) The tested intranasal delivery system could successfully target a bioactive peptide into the central nervous system and provides a basis for developing a non-invasive and cost effective, new model to study amyloid-induced dysfunctions in the brain.

Controlled in situ preparation of A beta(1-42) oligomers from the isopeptide "iso-A beta(1-42)", physicochemical and biological characterization.

Beta-amyloid (A beta) peptides play a crucial role in the pathology of the neurodegeneration in Alzheimer's disease (AD). Biological experiments (both in vitro and animal model studies of AD) require synthetic A beta peptides of standard quality, aggregation grade, neurotoxicity and water solubility. The synthesis of A beta peptides has been difficult, owing to their hydrophobic character, poor solubility and high tendency for aggregation. Recently an isopeptide precursor (iso-A beta(1-42)) was synthesized by Fmoc-chemistry and transformed at neutral pH to A beta(1-42) by O-->N acyl migration in a short period of time. We prepared the same precursor peptide using Boc-chemistry and studied the transformation to A beta(1-42) by acyl migration. The peptide conformation and aggregation processes were studied by several methods (circular dichroism, atomic force and transmission electron microscopy, dynamic light scattering). The biological activity of the synthetic A beta(1-42) was measured by ex vivo (long-term potentiation studies in rat hippocampal slices) and in vivo experiments (spatial learning of rats). It was proven that O-->N acyl migration of the precursor isopeptide results in a water soluble oligomeric mixture of neurotoxic A beta(1-42). These oligomers are formed in situ just before the biological experiments and their aggregation grade could be standardized.