Current Alzheimer Research - Volume 4, Issue 5, 2007

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A number of factors limit the therapeutic application of neurotrophin proteins, such as nerve growth factor (NGF) and brain-derived growth factor (BDNF), for Alzheimer's and other neurodegenerative diseases. These factors include unfavorable pharmacological properties typical of proteins and the pleiotropic effects mediated by protein-ligand interactions with p75NTR, Trk, and sortilin neurotrophin receptors. Targeted modulation of p75NTR provides a strategy for preventing degeneration without promoting TrkA-mediated deleterious effects, and targeted activation of TrkB might achieve more favorable neurotrophic effects than those achieved by concomitant activation of p75NTR and TrkB. The discovery of small molecules functioning as ligands at specific neurotrophin receptors has made possible for the first time approaches for modulating selected components of neurotrophin signaling processes for the purpose of modulating underlying Alzheimer's disease mechanisms.

NAP (NAPVSIPQ), derived from activity-dependent neuroprotective protein (ADNP) provides neuroprotection in vitro and in vivo against a wide variety of neurotoxic substances. To further understand the mechanism by which NAP provides broad neuroprotection it was essential to find NAP's binding partners. Previous results, using affinity chromatography coupled with mass spectrometry, identified tubulin, the subunit protein of microtubules, as the major NAP binding protein in neurons and glial cells. Here, following microtubule depolymerization in the presence of nocodazole, NAP treatment enhanced rapid microtubule assembly and stimulated neurite outgrowth. Nocodazole is an established inhibitor of axoplasmic transport and cell division that exerts its effect by depolymerizing microtubules. NAP shows selectivity in interacting with brain tubulin and does not affect dividing cells. This data demonstrates that NAP functions as a neuroprotectant, at least in part, through its interaction with tubulin with a resulting increase in microtubule assembly.

Confronting the efficacy of a regenerative therapeutic is the degenerative environment that is characterized by neuronal loss, physical plague and glial scar barriers and inflammation. But perhaps more fundamental from a regenerative perspective, are changes in the biochemical milieu of steroid and peptide growth factors, cytokines and neurotransmitter systems. Data from multiple levels of analysis indicate that gonadal steroid hormones and their metabolites can promote neural health whereas their decline or absence are associated with decline in neural health and increased risk of neurodegenerative disease including Alzheimer's. Among the steroids in decline, is allopregnanolone (APα), a neurosteroid metabolite of progesterone, which was found to be reduced in the serum [1,2] and plasma [3] and brain of aged vs. young subjects [4]. Further, Alzheimer disease (AD) victims showed an even further reduction in plasma and brain levels of APα relative to age-matched neurologically normal controls [1,4,5]. Our earlier work has shown that AP?? is a neurogenic agent for rodent hippocampal neural progenitors and for human neural progenitor cells derived from the cerebral cortex [6]. Our ongoing research seeks to determine the neurogenic potential of APα in the triple transgenic mouse model of Alzheimer's disease (3xTgAD) as AD related pathology progresses from imperceptible to mild to severe. Initial analyses suggest that neurogenic potential changes with age in nontransgenic mice and that the neurogenic profile differs between non-transgenic and 3xTgAD mice. Comparative analyses indicate that APα modifies neurogenesis in both nontransgenic and 3xTgAD mice. Preliminary data suggest that APα may modify Alzheimer's pathology progression. Together the data indicate that APα may maintain the regenerative ability of the brain and modify progression of AD related pathology. Challenges for efficacy of regenerative agents within a degenerative milieu are discussed.

The progressive memory loss observed in Alzheimer's disease (AD) is accompanied by an increase in the levels of amyloid-β peptide (Aβ) and a block of synaptic plasticity. Both synaptic plasticity and memory require changes in the expression of synaptic proteins such as the activity-regulated cytoskeleton-associated protein, Arc (also termed Arg3.1). Using a model of synaptic plasticity in which BDNF increases Arc expression in cultured cortical neurons, we have found that an oligomeric form of Aβ strongly inhibits the BDNF-induced increase of Arc expression. Given that Aβ oligomers are likely to be involved in the synaptic dysfunction and cognitive impairment observed in amyloid depositing mouse models, we hypothesize that inhibition of Arc induction by BDNF contributes to the synaptic and memory deficits at early stages of AD.

Traditionally, drug design programs are focused on optimizing the specificity of lead compounds against a carefully selected drug target. Disappointingly, this approach to discover a “magic bullet” drug has not met with the expected success for CNS disorders. Transcriptomics and proteomic profiling of neurodegenerative diseases have indicated that they are poly-etiological in origin and that the processes leading to neuronal death are multifactorial. An emerging concept is the design of drug ligands that modulate multiple drug targets identified for a particular disease. In this review we explore some examples of multifunctional drugs which may be useful in the treatment of neurodegenerative diseases, such as Alzheimer's and Parkinson's disease.

As the major genetic risk factor for Alzheimer's disease, the apolipoprotein (apo) E4 isoform is a promising therapeutic target. ApoE4 likely contributes to Alzheimer's disease pathology by interacting with multiple factors through various pathways. Interactions with the amyloid β peptide and the amyloid cascade, for example, may lead to cognitive decline and neurodegeneration. Alternatively, apoE4 might act independently of the amyloid β peptide. Our working hypothesis is that apoE has isoform-specific effects on neuronal repair and remodeling. One or more injurious agents could result in neuronal damage, requiring neuronal repair or remodeling. The injurious agents (or “second hits”) may be genetic, metabolic, or environmental. Potential therapeutic strategies include changing the structure of apoE4 to be more apoE3-like, inhibiting the protease that cleaves apoE4 into toxic fragments, and protecting mitochondria from apoE4 toxicity. Structural features that distinguish apoE4 and apoE3 determine their functional differences and hold the key to understanding how apoE4 is involved in Alzheimer's disease.

While much of the focus on Alzheimer's disease therapeutics has been directed at beta-amyloid peptide or at cholinergic synaptic transmission, recent data suggest that targeting signal transduction by the amyloid precursor protein (APP) itself may be an alternative approach with significant potential [1]. Here we discuss the possibility that APPmediated signal transduction, downstream from amyloid-beta peptide production itself, may be an appropriate therapeutic target in Alzheimer's disease.

The pathological aggregation of tau into paired helical filaments is a hallmark of several neurodegenerative diseases, including Alzheimer's disease. We have generated cell models of tau aggregation in order to study mechanisms involving abnormal changes of tau. In the cell models the repeat domain of tau (tauRD) and some of its variants are expressed in a regulated fashion, e.g. the 4-repeat domain of tau with the wild-type sequence, the repeat domain with the ΔK280 mutation (“pro-aggregation mutant”), or the repeat domain with additional proline mutations (“anti-aggregation mutant”). The aggregation of tauRD is toxic to the cells, but aggregation and toxicity can be prevented by low molecular weight compounds identified by a screen for inhibitors. Thus the cell models are suitable for developing aggregation inhibitor drugs and testing their cellular roles.

Cyclin-dependent kinase 5 (cdk5) is a member of the serine-threonine kinase family of cyclin-dependent kinases. This family is known for its role in the cell cycle, but cdk5 differs due to its interaction with activators p35 or p39, both abundant in post-mitotic neurons. Cdk5 is not known to have a role in cell cycle regulation at all, but is known to be an important modulator of neuronal activity. Cdk5 has been an attractive target for CNS diseases for a number of years. Among its attractions is the possibility that inhibitors will prevent the pathological phosphorylation of tau and neurofibrillary pathology in both Alzheimer's disease and tauopathies. More recently, there has been evidence that cdk5 is involved in the processing of pain and therefore inhibitors would also have potential therapeutic value for acute pain. Several classes of potent chemical inhibitors for cdk5 have been identified but most are competitive with the ATP binding site, resulting in a lack of specificity among the other cyclin-dependent kinases as well as other ATP-dependent kinases. We are working to discover specific inhibitors that might disrupt the interaction of tau and cdk5 at sites other than the ATP binding site. We are screening our compound library of 110,000 compounds using the full length tau as a substrate and will separate ATP competitive from non-competitive binders. In addition, we are taking a computational approach with virtual screening to identify non-ATP-competitive binders. These two approaches may lead to the discovery of sitespecific inhibitors for tau and cdk5 interactions rather than competitive inhibitors for ATP binding. The hope is that non- ATP competitive compounds will more likely be selective and will be better therapeutics.

Background: Increasing basic science and clinical evidence implicates inflammatory processes and resulting glial activation in the pathogenesis of Alzheimer's Disease. Excess TNF-alpha, a cytokine with pleotropic effects in the CNS, has been suggested to be involved in the pathogenesis of AD. In addition to its pro-inflammatory effects, TNF-alpha affects synaptic transmission; and glutamate, NMDA, and amyloid pathways. More specifically, TNF-alpha, produced by glia, has been shown to affect both synaptic strength and to mediate synaptic scaling, a homeostatic mechanism important to the control of neural networks. A recently published small, open-label pilot study suggested that inhibition of the inflammatory cytokine TNF-alpha utilizing the perispinal administration of etanercept may lead to sustained cognitive improvement for six months in patients with mild, moderate, and severe Alzheimer’s disease. Results: Continued open-label clinical experience with this new treatment modality, now for more than two years, suggests that weekly maintenance treatment with perispinal etanercept may have a sustained positive effect. In addition, rapid clinical improvement, within minutes of dosing, has been observed on a repeated basis in multiple patients. Discussion: It is hypothesized that perispinal administration of etanercept may enable rapid delivery to the CNS via the cerebrospinal venous system, resulting in improvement in synaptic mechanisms which have been dysregulated by excess TNF-alpha. TNF-alpha modulation in Alzheimer's disease may also act by influencing glutamate, NMDA, amyloid and other inflammatory pathways. Methods of perispinal administration, as described in the pilot study, may prove useful for delivering other therapeutics, particularly large molecules, to the CNS. Further study in randomized, placebo-controlled clinical trials and in basic science studies is merited.

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Since the discovery that mutations of alpha-synuclein (AS) gene are responsible for rare forms of familiar Parkinson's disease this synaptic protein attracted increased interest. AS is the main constituent of Lewy bodies. In spite the physiological function is still unclear there is an ongoing discussion if over-expression is already dangerous, or if toxicity is subjected to oligomers, protofibrilles or mature aggregates. The fact that the central hydrophobic part of AS is a constituent of amyloid plaques in Alzheimer patients and the finding that a majority of AD patients have Lewy bodies and Lewy neurites in specific brain areas, raised our interest in the possible contribution of AS to pathogenesis of AD. Betasynuclein (ßS) a protein of the same gene family seems to be a naturally occurring anti aggregatory factor preventing AS aggregation in vitro and in vivo. The N-terminal amino acid sequence 1 to 15 is responsible for this effect. Based on this finding we synthesized a peptide library with different sequence variations. Several of these peptides displayed distinct neuroprotective activity in tissue culture models of neurodegeneration induced by oxidative stress or Aß1-42. In spite these peptides have a short half-life, in vivo significant reduction in brain plaque load and improvement of behavioral deficits was demonstrated in an APP-tg mouse model after intranasal treatment for 2 months. KEGV, the shortest sequence was also active after intraperitoneal application. Neuroprotective data in tissue cultures and results from transgenic mice are some how in conflict because in vitro effects can not be explained by the antiaggregatory potential, but most likely by interaction of ßS derivates with anti-apoptotic PI3/Akt cell signaling or interference with anti-oxidative pathways (JNK/JIB). The possibility that such ßS derived peptidomimetics might act as neuroprotectants and at the same time prevent protein missfolding suggests possible therapeutic usefulness in different neurodegenerative disorders.

Amyloid β-derived diffusible ligands (ADDLs) comprise the neurotoxic subset of soluble Aβ1-42 oligomers, now widely considered to be the molecular cause of memory malfunction and neurodegeneration in Alzheimer's disease (AD). We have developed a screening cascade which identifies small molecule modulators of ADDL-mediated neurotoxicity. The primary screen involves a fluorescence resonance energy transfer (FRET)-based assay which selects inhibitors of Aβ1-42 oligomer assembly. The identified hits were further characterized by assessing their ability to inhibit the assembly and binding of ADDLs to cultures of primary hippocampal neurons. This approach has led to the identification of a number of small molecules which inhibit ADDL assembly and their subsequent binding to neurons. Here we describe our small molecule discovery efforts to identify ADDL assembly blocker and ADDL binding inhibitors, and to transform validated hits into pre-clinical lead compounds.

Pathophysiologic hypotheses for Alzheimer's disease (AD) are centered on the role of the amyloid plaque Aβ peptide and the mechanism of its derivation from the amyloid precursor protein (APP). As part of the disease process, an aberrant axonal sprouting response is known to occur near Aβ deposits. A Nogo to Nogo-66 receptor (NgR) pathway contributes to determining the ability of adult CNS axons to extend after traumatic injuries. Here, we consider the potential role of NgR mechanisms in AD. Both Nogo and NgR are mislocalized in AD brain samples. APP physically associates with the NgR. Overexpression of NgR decreases Aβ production in neuroblastoma culture, and targeted disruption of NgR expression increases transgenic mouse brain Aβ levels, plaque deposition, and dystrophic neurites. Infusion of a soluble NgR fragment reduces Aβ levels, amyloid plaque deposits, and dystrophic neurites in a mouse transgenic AD model. Changes in NgR level produce parallel changes in secreted APP and AB, implicating NgR as a blocker of secretase processing of APP. The NgR provides a novel site for modifying the course of AD and highlights the role of axonal dysfunction in the disease.

γ-Secretase is responsible for the final cut of the amyloid β-peptide (Aβ) precursor (APP) to produce the Aβ peptide implicated the pathogenesis of Alzheimer's disease (AD). Thus, this protease is a top target for the development of AD therapeutics. γ-Secretase is a complex of four different integral membrane proteins, with the multi-pass presenilin being the catalytic component of a novel intramembrane-cleaving aspartyl protease. γ-Secretase cleaves other substrates besides APP, the most notorious being the Notch receptor that is required for many cell differentiation events. Because proteolysis of Notch by γ-secretase is essential for Notch signaling, interference with this process by γ-secretase inhibitors can cause severe toxicities. Thus, the potential of &ggr-secretase as therapeutic target likely depends on the ability to selectively inhibit Aβ production without hindering Notch proteolysis. The discovery of compounds capable of such allosteric modulation of the protease activity has revived γ-secretase as an attractive target. Structural modification of these &ggr- secretase modulators through medicinal chemistry should lead to in vivo active agents suitable for clinical trials.

Drug discovery is a complex and costly endeavor, requiring multidisciplinary know-how, interdisciplinary collaboration, tenacity, and a bit of luck. For these reasons, the search for new chemical agents to treat human disease has traditionally been undertaken only within the walls of industry. While the pharmaceutical industry is often successful where it focuses its attention, it generally focuses only on those areas that are allowed by corporate financial realities. Sadly, this means that diseases effecting small populations of patients may go untreated. As a society we should not be content with this situation and must make a priority of the development of new models that will allow and encourage drug discovery in disease areas that are neglected by pharma. In this presentation, I will describe one such model that has been established to find treatments for neurodegenerative diseases.

The M1 muscarinic receptor (M1 mAChR), preserved in Alzheimer's disease (AD), is a pivotal target that links major hallmarks of AD, e.g. cholinergic deficiency, cognitive dysfunctions, β-amyloid (Aβ) and tau pathologies. Some muscarinic agonists, while effective in AD, had limited clinical value due to adverse effects and lack of M1 selectivity. The M1 selective muscarinic agonists AF102B [Cevimeline], AF150(S) and AF267B - i) elevated αAPPs, decreased Aβ levels and tau hyperphosphorylation, and blocked Aβ-induced neurotoxicity, in vitro, via M1 mAChR-modulation of kinases (e.g. PKC, MAPK and GSK3β); ii) restored cognitive deficits, cholinergic markers, and decreased tau hyperphosphorylation in relevant models with a wide safety margin. AF267B decreased brain Aβ levels in hypercholesterolemic rabbits and decreased CSF Aβ42 in rabbits and removed vascular Aβ42 deposition from cortex in cholinotoxin-treated rabbits. In 3x transgenic-AD mice that recapitulate the major pathologies and cognitive deficits of AD, chronic AF267B treatment rescued cognitive deficits and decreased Aβ42 and tau pathologies in the cortex and hippocampus (not amygdala), via M1 mAChR-activation of ADAM17/TACE and decreased BACE1 steady state levels and inhibition of GSK3β, extending findings from above. Conclusions: A comprehensive therapy should target all AD hallmarks, regardless of the culprit(s) responsible for the disease. In this context, AF267B is the 1st reported low MW CNS-penetrable mono-therapy that meets this challenge. Clinical trials will determine if AF267B may become an important therapy in AD.

The receptor for advanced glycation end products (RAGE) binds amyloid peptides with high affinity. Soluble RAGE-like peptides and Aβ-like peptides occur in relatively high concentrations in the circulation of individuals with Alzheimer's disease. Protein complexes with epitopes for both Aβ and RAGE are also present. At physiological concentrations, forms of Aβ have different, but relatively low potencies as cytotoxicants in neural cells in culture. The purpose of this study was to determine whether a synthetic peptide complex composed of Aβ1-42 and RAGE23-54, a conserved Nterminal fragment of RAGE, exhibited increased cytotoxicity in comparison with the constituent peptides. Western analysis indicated that Aβ1-42 and RAGE23-54 remained primarily in their original low molecular weight states (4-6 kDa) during the maintenance of the individual peptides (37 ° C) in water from 1 to 4 weeks. In contrast, over the same maintenance periods the combined Aβ1-42 and RAGE23-54 peptides shifted to higher molecular weight complexes (up to 80-120 kDa). Protein complexes of similar molecular weights with epitopes for Aβ and RAGE antibodies were identified in human plasma. Incubation of differentiated PC-12 cells with 10-100 μM Aβ1-42 or with RAGE23-54 resulted in concentration-dependent decreases in cell viability. The cytotoxicity of each peptide was slightly enhanced by the progressive maintenance of Aβ1- 42 and RAGE23-54 in water over 3 weeks prior to the assay. Under the same conditions, the Aβ1-42 - RAGE23-54 complex became significantly more cytotoxic. These results suggest that the formation of soluble Aβ-RAGE complexes in Alzheimer's disease could represent a mechanism for enhancing the neurotoxicity of amyloid peptides.