J. Steven Jacobsen

Acute γ-Secretase Inhibition Improves Contextual Fear Conditioning in the Tg2576 Mouse Model of Alzheimer's Disease

Transgenic mice (Tg2576) overexpressing the Swedish mutation of the human amyloid precursor protein display biochemical, pathological, and behavioral markers consistent with many aspects of Alzheimers disease, including impaired hippocampal function. Impaired, hippocampal-dependent, contextual fear conditioning (CFC) is observed in mice as young as 20 weeks of age. This impairment can be attenuated after treatment before training with the phosphodiesterase-4 inhibitor rolipram (0.1 mg/kg, i.p.). A rolipram-associated improvement is also observed in the littermate controls, suggesting that the effect of rolipram is independent of β-amyloid. Acute treatment before training (but not after training or before testing) with the γ-secretase inhibitor (GSI) N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine-t-butylester (DAPT), at a dose that reduces brain concentrations of β-amyloid (100 mg/kg), attenuates the impairment in 20- to 65-week-old Tg2576 mice. Importantly, DAPT had no effect on performance of control littermates. These data are supportive of a role of β-amyloid in the impairment of CFC in Tg2576 mice. Furthermore, they suggest that acute treatment with GSI may provide improved cognitive functioning as well as disease-modifying effects in Alzheimers disease.

In Vitro Phosphorylation of the Cytoplasmic Domain of the Amyloid Precursor Protein by Glycogen Synthase Kinase‐3β

Abstract: The two pathological lesions found in the brains of Alzheimers disease patients, neurofibrillary tangles and neuritic plaques, are likely to be formed through a common pathway. Neurofibrillary tangles are intracellular aggregates of paired helical filaments, the main component of which is hyperphosphorylated forms of the microtubule‐associated protein τ. Extracellular neuritic plaques and diffuse and vascular amyloid deposits are aggregates of β‐amyloid protein, a 4‐kDa protein derived from the amyloid precursor protein (APP). Using conditions in vitro under which two proline‐directed protein kinases, glycogen synthase kinase‐3β (GSK‐3β) and mitogen‐activated protein kinase (MAPK), were able to hyperphosphorylate τ, GSK‐3β but not MAPK phosphorylated recombinant APPcyt. The sole site of phosphorylation in APPcyt by GSK‐3β was determined by phosphoamino acid analysis and phosphorylation of APPcyt mutant peptides to be Thr743 (numbering as for APP770). This site was confirmed by endoproteinase Glu‐C digestion of APPcyt and peptide sequencing. The ability of GSK‐3β to phosphorylate APPcyt and τ provides a putative link between the two lesions and indicates a critical role of GSK‐3β in the pathogenesis of Alzheimers disease.

Begacestat (GSI-953): A Novel, Selective Thiophene Sulfonamide Inhibitor of Amyloid Precursor Protein γ-Secretase for the Treatment of Alzheimer's Disease

The presenilin containing γ-secretase complex is responsible for the regulated intramembraneous proteolysis of the amyloid precursor protein (APP), the Notch receptor, and a multitude of other substrates. γ-Secretase catalyzes the final step in the generation of Aβ40 and Aβ42 peptides from APP. Amyloid β-peptides (Aβ peptides) aggregate to form neurotoxic oligomers, senile plaques, and congophilic angiopathy, some of the cardinal pathologies associated with Alzheimers disease. Although inhibition of this protease acting on APP may result in potentially therapeutic reductions of neurotoxic Aβ peptides, nonselective inhibition of the enzyme may cause severe adverse events as a result of impaired Notch receptor processing. Here, we report the preclinical pharmacological profile of GSI-953 (begacestat), a novel thiophene sulfonamide γ-secretase inhibitor (GSI) that selectively inhibits cleavage of APP over Notch. This GSI inhibits Aβ production with low nanomolar potency in cellular and cell-free assays of γ-secretase function, and displaces a tritiated analog of GSI-953 from enriched γ-secretase enzyme complexes with similar potency. Cellular assays of Notch cleavage reveal that this compound is approximately 16-fold selective for the inhibition of APP cleavage. In the human APP-overexpressing Tg2576 transgenic mouse, treatment with this orally active compound results in a robust reduction in brain, plasma, and cerebral spinal fluid Aβ levels, and a reversal of contextual fear-conditioning deficits that are correlated with Aβ load. In healthy human volunteers, oral administration of a single dose of GSI-953 produces dose-dependent changes in plasma Aβ levels, confirming pharmacodynamic activity of GSI-953 in humans.

Enhanced clearance of Aβ in brain by sustaining the plasmin proteolysis cascade

The amyloid hypothesis states that a variety of neurotoxic β-amyloid (Aβ) species contribute to the pathogenesis of Alzheimers disease. Accordingly, a key determinant of disease onset and progression is the appropriate balance between Aβ production and clearance. Enzymes responsible for the degradation of Aβ are not well understood, and, thus far, it has not been possible to enhance Aβ catabolism by pharmacological manipulation. We provide evidence that Aβ catabolism is increased after inhibition of plasminogen activator inhibitor-1 (PAI-1) and may constitute a viable therapeutic approach for lowering brain Aβ levels. PAI-1 inhibits the activity of tissue plasminogen activator (tPA), an enzyme that cleaves plasminogen to generate plasmin, a protease that degrades Aβ oligomers and monomers. Because tPA, plasminogen and PAI-1 are expressed in the brain, we tested the hypothesis that inhibitors of PAI-1 will enhance the proteolytic clearance of brain Aβ. Our data demonstrate that PAI-1 inhibitors augment the activity of tPA and plasmin in hippocampus, significantly lower plasma and brain Aβ levels, restore long-term potentiation deficits in hippocampal slices from transgenic Aβ-producing mice, and reverse cognitive deficits in these mice.

Discovery of Begacestat, a Notch-1-Sparing γ-Secretase Inhibitor for the Treatment of Alzheimer's Disease

SAR on HTS hits 1 and 2 led to the potent, Notch-1-sparing GSI 9, which lowered brain Abeta in Tg2576 mice at 100 mg/kg po. Converting the metabolically labile methyl groups in 9 to trifluoromethyl groups afforded the more stable analogue 10, which had improved in vivo potency. Further side chain modification afforded the potent Notch-1-sparing GSI begacestat (5), which was selected for development for the treatment of Alzheimers disease.

Current concepts in therapeutic strategies targeting cognitive decline and disease modification in Alzheimer’s disease

SummaryAlzheimer’s disease is a progressive neurodegenerative disorder and the leading cause of dementia in the Western world. Postmortem, it is characterized neuropathologically by the presence of amyloid plaques, neurofibrillary tangles, and a profound gray matter loss. Neurofibrillary tangles are composed of an abnormally hyperphosphorylated intracellular protein called tau, tightly wound into paired helical filaments and thought to impact microtubule assembly and protein trafficking, resulting in the eventual demise of neuronal viability. The extracellular amyloid plaque deposits are composed of a proteinacious core of insoluble aggregated amyloid-β (Aβ) peptide and have led to the foundation of the amyloid hypothesis. This hypothesis postulates that Aβ is one of the principal causative factors of neuronal death in the brains of Alzheimer’s patients. With multiple drugs now moving through clinical development for the treatment of Alzheimer’s disease, we will review current and future treatment strategies aimed at improving both the cognitive deficits associated with the disease, as well as more novel approaches that may potentially slow or halt the deadly neurodegenerative progression of the disease.

Selective Aggregation of Endogenous β‐Amyloid Peptide and Soluble Amyloid Precursor Protein in Cerebrospinal Fluid by Zinc

Abstract: Zinc added to buffered solutions of synthetic β‐amyloid peptide (Aβ) has been reported to induce accelerated formation of insoluble aggregates. This observation suggests that zinc may play a role in the formation of senile plaques, which contain Aβ, in Alzheimers disease. To test this hypothesis under conditions more representative of the brain, we investigated the ability of zinc to induce aggregation of Aβ in freshly drawn canine CSF, which contains the same sequence as human Aβ. Aggregates were separated from CSF by ultracentrifugation before and after incubation with zinc and assayed by quantitative western blotting and ELISA. We found that zinc induced the rapid aggregation of endogenous Aβ in CSF, with an EC50 of 120–140 µM. The reaction was specific, because most (≥95%) CSF protein remained soluble under conditions where most Aβ was insoluble, as assayed by scanning densitometry of Coomassie‐stained gels. Staining of the precipitated material resulted in the visualization of punctate regions that were thioflavin positive or birefringent when stained with Congo red, suggesting the formation of amyloid‐related structures. These results suggest that zinc could play a role in amyloid deposition, because there is overlap between the regions of the brain where zinc concentrations are highest and regions with the highest amyloid content. It is surprising that zinc induced the aggregation of endogenous soluble APP at lower concentrations than required for Aβ (EC50 80 µM). The possibility that zinc‐induced aggregation of APP may precede the deposition of Aβ into plaques is discussed. Investigation of aggregation of Aβ in CSF will aid in assessing the biological relevance of other agents that have been reported to accelerate amyloid formation.

Structural Correlates of Antibodies Associated with Acute Reversal of Amyloid β-related Behavioral Deficits in a Mouse Model of Alzheimer Disease

Immunotherapy targeting of amyloid β (Aβ) peptide in transgenic mouse models of Alzheimer disease (AD) has been widely demonstrated to resolve amyloid deposition as well as associated neuronal, glial, and inflammatory pathologies. These successes have provided the basis for ongoing clinical trials of immunotherapy for treatment of AD in humans. Acute as well as chronic Aβ-targeted immunotherapy has also been demonstrated to reverse Aβ-related behavioral deficits assessing memory in AD transgenic mouse models. We observe that three antibodies targeting the same linear epitope of Aβ, Aβ3–7, differ in their ability to reverse contextual fear deficits in Tg2576 mice in an acute testing paradigm. Reversal of contextual fear deficit by the antibodies does not correlate with in vitro recognition of Aβ in a consistent or correlative manner. To better define differences in antigen recognition at the atomic level, we determined crystal structures of Fab fragments in complex with Aβ. The conformation of the Aβ peptide recognized by all three antibodies was highly related and is also remarkably similar to that observed in independently reported Aβ:antibody crystal structures. Sequence and structural differences between the antibodies, particularly in CDR3 of the heavy chain variable region, are proposed to account for differing in vivo properties of the antibodies under study. These findings provide a structural basis for immunotherapeutic strategies targeting Aβ species postulated to underlie cognitive deficits in AD.

Evaluation of Cathepsins D and G and EC 3.4.24.15 as Candidate β‐Secretase Proteases Using Peptide and Amyloid Precursor Protein Substrates

Abstract: No single protease has emerged that possesses all the expected properties for β‐secretase, including brain localization, appropriate peptide cleavage specificity, and the ability to cleave amyloid precursor protein exactly at the amino‐terminus of β‐amyloid peptide. We have isolated and purified a brain‐derived activity that cleaves the synthetic peptide substrate SEVKMDAEF between methionine and aspartate residues, as required to generate the amino‐terminus of β‐amyloid peptide. Its molecular size of 55–60 kDa and inhibitory profile indicate that we have purified the metalloprotease EC 3.4.24.15. We have compared the sequence specificity of EC 3.4.24.15, cathepsin D, and cathepsin G for their ability to cleave the model peptide SEVKMDAEF or related peptides that contain substitutions reported to modulate β‐amyloid peptide production. We have also tested the ability of these enzymes to form carboxy‐terminal fragments from full‐length, membrane‐embedded amyloid precursor protein substrate or amyloid precursor protein that contains the Swedish KM → NL mutation. The correct cleavage was tested with an antibody specific for the free amino‐terminus of β‐amyloid peptide. Our results exclude EC 3.4.24.15 as a candidate β‐secretase. Although cathepsin G cleaves the model peptide correctly, it displays poor ability to cleave the Swedish KM → NL peptide and does not generate carboxy‐terminal fragments that are immunoreactive with amino‐terminal‐specific antiserum. Cathepsin D does not cleave the model peptide or show specificity for wild‐type amyloid precursor protein; however, it cleaves the Swedish “NL peptide” and “NL precursor” substrates appropriately. Our results suggest that cathepsin D could act as β‐secretase in the Swedish type of familial Alzheimers disease and demonstrate the importance of using full‐length substrate to verify the sequence specificity of candidate proteases.

Altered binding of mutated presenilin with cytoskeleton-interacting proteins1

The majority of familial Alzheimers disease (AD) cases are linked to mutations on presenilin 1 and 2 genes (PS1 and PS2). The normal function of the proteins and the mechanisms underlying early‐onset AD are currently unknown. To address this, we screened an expression library for proteins that bind differentially to the wild‐type PS1 and mutant in the large cytoplasmic loop (PS1L). Thus we isolated the C‐terminal tail of the 170 kDa cytoplasmic linker protein (CLIP‐170) and Reed–Sternberg cells of Hodgkins disease‐expressed intermediate filament‐associated protein (Restin), cytoplasmic proteins linking vesicles to the cytoskeleton. PS1L binding to CLIP‐170/restin requires Ca2+. Treating cells with thapsigargin or ionomycin increased the mutated PS1 in CLIP‐170 immunoprecipitates. Further, PS1 and CLIP‐170 co‐localize in transfected cells and neuronal cultures.