Current Alzheimer Research - Volume 6, Issue 5, 2009

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Recent results from high-throughput and other screening approaches reveal that small molecules can directly interact with recombinant full-length tau monomers and fibrillar tau aggregates in three distinct modes. First, in the high concentration regime (>10 micromolar), certain anionic molecules such as Congo red efficiently promote tau filament formation through a nucleation-elongation mechanism involving a dimeric nucleus and monomer-mediated elongation. These compounds are useful for modeling tau aggregation in vitro and in biological models. Second, in the low concentration regime (<1 micromolar), other ligands, including cyanine dyes, display aggregation antagonist activity. Compounds that can prevent or reverse fibrillization are candidate modifiers of disease pathology. Finally, certain compounds bind mature tau fibrils with varying affinities at multiple binding sites without modulating the aggregation reaction. For some ligands, >10-fold selectivity for tau aggregates relative to filaments composed of beta-amyloid or alpha-synuclein can be demonstrated at the level of binding affinity. Together these observations suggest that small-molecules have utility for interrogating the tau aggregation pathway, for inhibiting neuritic lesion formation, and for selective pre-mortem detection of neurofibrillary lesions through whole brain imaging.

Apolipoprotein E4 (apoE4) is the major genetic risk factor for Alzheimer's disease (AD) by an as yet to be defined mechanism. Since the structure or biophysical properties of a protein directly determines function, our approach to addressing mechanism is structure:function based. Domain interaction a structural property of apoE4 that distinguishes it from apoE3 is predicted to contribute to the association of apoE4 with AD. We developed a mouse model, the Arg-61 apoE model, which is specific for domain interaction. These mice display synaptic, functional, and cognitive deficits, demonstrating domain interaction is the causative factor. We present evidence that domain interaction results in stressed astrocytes that are dysfunctional and propose that dysfunctional astrocytes are an early player in apoE4-associated AD and that domain interaction is a potential therapeutic target.

The concept and rationale for neuroprotection are presented. Several examples of small molecule neurotrophic agents with favourable drug-like and pharmacological properties are shown. Compound efficacy in acute neurodegenerative models (optic nerve axotomy) and chronic neurodegenerative models (glaucoma, age-associated cognitive impairment, Alzheimer's Disease) are evaluated and discussed. Targeting neurotrophin receptors with ligands that activate survival pathways or inhibit death pathways is an alternative worth pursuing.

The mechanisms responsible for oxidative damage, pathological brain iron deposition and mitochondrial insufficiency in Alzheimer disease (AD) remain enigmatic. Heme oxygenase-1 (HO-1) is a 32 kDa stress protein that catabolizes heme to biliverdin, free iron and carbon monoxide. The HO-1 gene is exquisitely sensitive to oxidative stress and is induced in brain and other tissues in various models of disease and trauma. Our laboratory demonstrated that 1) HO-1 protein is significantly over-expressed in AD-affected temporal cortex and hippocampus relative to neurohistologicallynormal control preparations, 2) in cultured astrocytes, HO-1 up-regulation by transient transfection of the human ho-1 gene, or stimulation of endogenous HO-1 expression by exposure to β-amyloid, TNFα or IL-1β, promotes intracellular oxidative stress, opening of the mitochondrial permeability transition pore and accumulation of non-transferrin iron in the mitochondrial compartment, and 3) the glial iron sequestration renders co-cultured neuron-like PC12 cells prone to oxidative injury. Induction of the astroglial ho-1 gene may constitute a ‘common pathway’ leading to pathological brain iron deposition, intracellular oxidative damage and bioenergetic failure in AD and other human CNS disorders. Hypothesis: Targeted suppression of glial HO-1 hyperactivity may prove to be a rational and effective neurotherapeutic intervention in AD and related neurodegenerative disorders. To begin testing this hypothesis, studies have been initiated to determine whether systemic administration of a novel, selective and brain-permeable inhibitor of HO-1 activity ameliorates cognitive dysfunction and neuropathology in a transgenic mouse model of AD.

Agonists of the peroxisome proliferator activated receptor gamma (PPARγ) have been shown to reduce inflammatory responses in several animal models of neurological diseases and conditions and to reduce amyloid burden in transgenic mice expressing mutant forms of human amyloid precursor protein. However, the effects of activating the related receptor PPARdelta (PPARδ), which is expressed at higher levels in the brain than PPARγ, on inflammation and amyloid burden have not been explored. In this study we tested the effects of the selective PPARδ agonist GW742 in 5xFAD mice which harbor 3 mutations in amyloid precursor protein and 2 mutations in presenilin 1, develop plaques by 5-6 weeks of age, and show robust inflammation and neuronal damage. Oral delivery of GW742 significantly reduced amyloid plaque burden in the subiculum region of 3-month old male and female 5xFAD mice. GW742 also significantly reduced astrocyte activation, suggesting anti-inflammatory effects on glia cells. The changes in plaque burden were accompanied by increased expression of the amyloid degrading enzymes neprilysin and insulin degrading enzyme, while in transfected HEK293 cells, GW742 activated a neprilysin promoter driving luciferase expression. These results suggest that, as found for some PPARγ agonists, PPARδ agonists can also reduce amyloid burden likely to be mediated by effects on amyloid clearance.

Protein accumulation leads to CNS effects in Alzheimer's disease, frontotemporal dementia, and other agerelated disorders. Common mechanisms may contribute to the progressive pathology in the different protein accumulation disorders, and synergistic toxicity between dissimilar protein structures may also be involved. Among several avenues being pursued to reduce proteins prone to oligomerization and/or aggregation, a lysosomal avenue has been described that regulates the lysosomal system's broad clearance capability. Lysosomes are the primary site for protein clearance, to remove old and misfolded proteins and maintain cellular homeostasis. Small-molecule lysosomal modulators trigger a feedback response in vitro and in vivo, resulting in marked up-regulation of cathepsins and other lysosomal enzymes without any indications of synaptic pathology, behavioral abnormalities, or major organ malfunctions. For the characterization and screening of lysosomal modulatory drugs, the hippocampal slice model of protein accumulation has proved very useful. The model exhibits experimentally-induced phosphorylated tau species, paired helical filament deposits, ubiquitinated inclusions, and protein oligomers, thus providing a valuable tool to study the associated sequelae underlying progressive cellular and synaptic compromise. In the absence of modulatory drugs, the protein accumulation events lead to microtubule destabilization, transport failure, and synaptic decline. When lysosomal modulators are administered to slices with pre-existing deposits, protein accumulations are reduced causing normalization of tau chemistry, restoration of tubulin structures and tubulin-binding proteins, and recovery of synaptic composition. Thus, positive modulators of the lysosomal system represent first-in-class drugs, providing a suitable strategy to enhance protein clearance, promote synaptic health, and slow the progression of proteinopathies.

Immunotherapies targeting the amyloid-β (Aβ) peptide in Alzheimer's disease (AD) have consistently been effective in mouse studies and shown promise in clinical trials, although some setbacks have occurred. First, encephalitis was observed in a small subset of patients. More recent autopsy data from a few subjects suggests that clearance of Aβ plaques may not halt cognitive deterioration once impairments are evident, emphasizing the need for other more effective approaches at that stage of the disease. Another important target in AD is the neurofibrillary tangles and its precursors, composed primarily of hyperphosphorylated tau proteins, which correlate well with the degree of dementia. As Aβ and tau pathologies are likely synergistic, targeting both together may be more effective, and perhaps essential as early diagnosis prior to cognitive decline is currently unavailable. Also, Aβ immunotherapy results in a very limited indirect clearance of tau aggregates, showing the importance of developing a separate therapy that directly targets pathological tau. Our findings in two tangle mouse models indicate that active immunization targeting an AD phospho-tau epitope reduces aggregated tau in the brain and prevents/ slows progression of the tangle-related behavioral phenotype, including cognitive impairment. These antibodies enter the brain and bind to pathological tau within neurons although the therapeutic effect may at least in part be due to clearance of extracellular tau that may have biological effects. We are currently clarifying the mechanism of these promising findings, determining its epitope specificity as well as assessing the feasibility of this approach for clinical trials.

The aggregation of the microtubule-associated protein tau into paired-helical filaments is the defining characteristic of the tauopathies. It has become apparent that the hyperphosphorylation of tau likely plays a role in the aggregation process and thus strategies to reduce tau phosphorylation are generating wide interest. The O-GlcNAc posttranslational modification of tau has been shown to be reciprocal to its phosphorylation; increasing O-GlcNAc leads to reductions in tau phosphorylation. In this mini-review, we highlight the use of chemical compounds as a means of understanding the reciprocal nature of tau phosphorylation and tau O-GlcNAcylation and highlight some recent progress in this area.

AL-108 is the intranasal formulation of NAP (a peptide of eight amino acids, NAPVSIPQ). Phase IIa clinical results have recently shown that AL-108 has a positive impact on memory function in patients with amnestic mild cognitive impairment (aMCI), a precursor to Alzheimer's disease (AD). The clinical development of AL-108 has been based on extensive studies showing pre-clinical efficacy for NAP. NAP has demonstrated potent neuroprotective activity in vitro and in vivo. Its mechanism of action is thought to center on the modulation of microtubule stability in the face of outside damage. Such an effect on structures of such central importance in a broad range of cellular functions is thought to explain NAP's activity in wide ranging models of cellular damage and neurodegeneration. The following article reviews NAP's discovery and pharmacological characterization that has led to clinical development of a novel tangle-directed drug candidate.