Luke Esposito

Antiamyloidogenic and Neuroprotective Functions of Cathepsin B: Implications for Alzheimer's Disease

Alzheimers disease (AD) may result from the accumulation of amyloid-beta (Abeta) peptides in the brain. The cysteine protease cathepsin B (CatB) is associated with amyloid plaques in AD brains and has been suspected to increase Abeta production. Here, we demonstrate that CatB actually reduces levels of Abeta peptides, especially the aggregation-prone species Abeta1-42, through proteolytic cleavage. Genetic inactivation of CatB in mice with neuronal expression of familial AD-mutant human amyloid precursor protein (hAPP) increased the relative abundance of Abeta1-42, worsening plaque deposition and other AD-related pathologies. Lentivirus-mediated expression of CatB in aged hAPP mice reduced preexisting amyloid deposits, even thioflavin S-positive plaques. Under cell-free conditions, CatB effectively cleaved Abeta1-42, generating C-terminally truncated Abeta peptides that are less amyloidogenic. Thus, CatB likely fulfills antiamyloidogenic and neuroprotective functions. Insufficient CatB activity might promote AD; increasing CatB activity could counteract the neuropathology of this disease.

Deficiency in neuronal TGF-β signaling promotes neurodegeneration and Alzheimer's pathology

Alzheimers disease (AD) is characterized by progressive neurodegeneration and cerebral accumulation of the beta-amyloid peptide (Abeta), but it is unknown what makes neurons susceptible to degeneration. We report that the TGF-beta type II receptor (TbetaRII) is mainly expressed by neurons, and that TbetaRII levels are reduced in human AD brain and correlate with pathological hallmarks of the disease. Reducing neuronal TGF-beta signaling in mice resulted in age-dependent neurodegeneration and promoted Abeta accumulation and dendritic loss in a mouse model of AD. In cultured cells, reduced TGF-beta signaling caused neuronal degeneration and resulted in increased levels of secreted Abeta and beta-secretase-cleaved soluble amyloid precursor protein. These results show that reduced neuronal TGF-beta signaling increases age-dependent neurodegeneration and AD-like disease in vivo. Increasing neuronal TGF-beta signaling may thus reduce neurodegeneration and be beneficial in AD.

Reduction in mitochondrial superoxide dismutase modulates Alzheimer's disease-like pathology and accelerates the onset of behavioral changes in human amyloid precursor protein transgenic mice.

Alzheimers disease (AD) is associated with accumulations of amyloid-β (Aβ) peptides, oxidative damage, mitochondrial dysfunction, neurodegeneration, and dementia. The mitochondrial antioxidant manganese superoxide dismutase-2 (Sod2) might protect against these alterations. To test this hypothesis, we inactivated one Sod2 allele (Sod2+/−) in human amyloid precursor protein (hAPP) transgenic mice, reducing Sod2 activity to ∼50% of that in Sod2 wild-type (Sod2+/+) mice. A reduction in Sod2 activity did not obviously impair mice without hAPP/Aβ expression. In hAPP mice, however, it accelerated the onset of behavioral alterations and of deficits in prepulse inhibition of acoustic startle, a measure of sensorimotor gating. In these mice, it also worsened hAPP/Aβ-dependent depletion of microtubule-associated protein 2, a marker of neuronal dendrites. Sod2 reduction decreased amyloid plaques in the brain parenchyma but promoted the development of cerebrovascular amyloidosis, gliosis, and plaque-independent neuritic dystrophy. Sod2 reduction also increased the DNA binding activity of the transcription factor nuclear factor κB. These results suggest that Sod2 protects the aging brain against hAPP/Aβ-induced impairments. Whereas reductions in Sod2 would be expected to trigger or exacerbate neuronal and vascular pathology in AD, increasing Sod2 activity might be of therapeutic benefit.

Neprilysin Overexpression Inhibits Plaque Formation But Fails to Reduce Pathogenic Aβ Oligomers and Associated Cognitive Deficits in Human Amyloid Precursor Protein Transgenic Mice

The accumulation of amyloid-β (Aβ) peptides in the brain of patients with Alzheimers disease (AD) may arise from an imbalance between Aβ production and clearance. Overexpression of the Aβ-degrading enzyme neprilysin in brains of human amyloid precursor protein (hAPP) transgenic mice decreases overall Aβ levels and amyloid plaque burdens. Because AD-related synaptic and cognitive deficits appear to be more closely related to Aβ oligomers than to plaques, it is important to determine whether increased neprilysin activity also diminishes the levels of pathogenic Aβ oligomers and related neuronal deficits in vivo. To address this question, we crossed hAPP transgenic mice with neprilysin transgenic mice and analyzed their offspring. Neprilysin overexpression reduced soluble Aβ levels by 50% and effectively prevented early Aβ deposition in the neocortex and hippocampus. However, it did not reduce levels of Aβ trimers and Aβ*56 or improve deficits in spatial learning and memory. The differential effect of neprilysin on plaques and oligomers suggests that neprilysin-dependent degradation of Aβ affects plaques more than oligomers and that these structures may form through distinct assembly mechanisms. Neprilysins inability to prevent learning and memory deficits in hAPP mice may be related to its inability to reduce pathogenic Aβ oligomers. Reduction of Aβ oligomers will likely be required for anti-Aβ treatments to improve cognitive functions.

Early induction of oxidative stress in mouse model of Alzheimer disease with reduced mitochondrial superoxide dismutase activity

While oxidative stress has been linked to Alzheimers disease, the underlying pathophysiological relationship is unclear. To examine this relationship, we induced oxidative stress through the genetic ablation of one copy of mitochondrial antioxidant superoxide dismutase 2 (Sod2) allele in mutant human amyloid precursor protein (hAPP) transgenic mice. The brains of young (5–7 months of age) and old (25–30 months of age) mice with the four genotypes, wild-type (Sod2+/+), hemizygous Sod2 (Sod2+/−), hAPP/wild-type (Sod2+/+), and hAPP/hemizygous (Sod2+/−) were examined to assess levels of oxidative stress markers 4-hydroxy-2-nonenal and heme oxygenase-1. Sod2 reduction in young hAPP mice resulted in significantly increased oxidative stress in the pyramidal neurons of the hippocampus. Interestingly, while differences resulting from hAPP expression or Sod2 reduction were not apparent in the neurons in old mice, oxidative stress was increased in astrocytes in old, but not young hAPP mice with either Sod2+/+ or Sod2+/−. Our study shows the specific changes in oxidative stress and the causal relationship with the pathological progression of these mice. These results suggest that the early neuronal susceptibility to oxidative stress in the hAPP/Sod2+/− mice may contribute to the pathological and behavioral changes seen in this animal model.

Intracellularly generated amyloid‐β peptide counteracts the antiapoptotic function of its precursor protein and primes proapoptotic pathways for activation by other insults in neuroblastoma cells

Most mutations in amyloid precursor proteins (APPs) linked to early onset familial Alzheimers disease (FAD) increase the production of amyloid‐β peptides ending at residue 42 (Aβ42), which are released from APP by β‐ and γ‐secretase cleavage. Stably transfected cells expressing wild‐type human APP (APPWT) were more resistant to apoptosis‐inducing treatments than cells expressing FAD‐mutant human APP (APPFAD). Preventing Aβ42 production with an M596I mutation (β–), which blocks β‐secretase cleavage of APP, or by treatment with a γ‐secretase inhibitor increased the resistance of APPFAD‐expressing cells to apoptosis. Exposing hAPPFAD/β– cells to exogenous Aβ42 or conditioned medium from Aβ42‐producing APPFAD cells did not diminish their resistance to apoptosis. Preventing APP from entering the distal secretory pathway, where most Aβ peptides are generated, by retaining APP in the endoplasmic reticulum (ER)/intermediate compartment (IC) increased the resistance of APPFAD‐expressing cells to apoptosis and did not alter the resistance of APPWT‐expressing cells. p53‐mediated gene transactivation after apoptosis‐inducing treatments was much stronger in APPFAD cells than in hAPPWT or hAPPFAD/β– cells. In contrast, upon induction of ER stress, cells expressing APPFAD, hAPPFAD/β–, or APPWT had comparable levels of glucose‐regulated protein‐78 mRNA, an unfolded protein response indicator. We conclude that Aβ, especially intracellular Aβ, counteracts the antiapoptotic function of its precursor protein and predisposes cells to p53‐mediated, and possibly other, proapoptotic pathways.

Exebryl-1: A novel small molecule currently in human clinical trials as a disease-modifying drug for the treatment of Alzheimer's disease

injections of vehicle, CHF5074 (10 or 30 or 100 mg/kg) or DAPT (100 mg/ kg) 24 and 3 hours before the training session. Wild-type animals received vehicle, CHF5074 (100 mg/kg) or DAPT (100 mg/kg). Freezing was expressed as a percentage of time in each portion of the test in which the animal remained immobile (at least 95% of his body for at least 500 msec) and analyzed using two-way ANOVA (with transgene and treatments ad fixed factors) followed by the Holm-Sidak’s comparison procedure vs the vehicle-treated groups. Results: Compared to vehicle-treated wild-type mice, vehicle-treated transgenic animals had significantly lower freezing to the context (59.1 6 3.9% vs 77.5 6 2.6%, p < 0.001). CHF5074 showed a bell-shaped dose-response curve with a non-significant increase of the contextual freezing with the 10 mg/kg dose (66.1 6 3.6%, p 1⁄4 0.128), a significant improvement with 30 mg/kg (71.5 6 3.9%, p 1⁄4 0.008) and no effects with the highest dose of 100 mg/kg (58.1 6 4.6%). Compared to transgenic controls, DAPT 100 mg/kg had not effects on contextual freezing (55.6 6 5.8%). In wild-type mice, neither CHF5074 (100 mg/kg) nor DAPT (100 mg/kg) had effects on behavior compared to vehicle-treated animals. No significant effects of drug treatments were observed in hippocampal-independent cue conditioning. Conclusions: These data show that acute treatment with CHF5074 improves hippocampal-dependent memory in a transgenic mouse model of AD and support its development as potentially disease-modifying agent of AD.

Identification of novel small molecules as tau protein aggregation inhibitors

Background:A key neuropathological characteristic shared by several neurodegenerative disorders including Alzheimer’s disease (AD) is the misfolding and aberrant aggregation of the microtubule-associated protein, tau. The accumulation of tau into neurofibrillary tangles is concomitant with the severity of dementia in AD. Thus, targeting tau aggregation has emerged as a promising therapeutic strategy for slowing the progression of dementia in AD. ProteoTech, Inc. has developed several in vitro screening technologies and cellular and animal models in order to identify novel and effective inhibitors of amyloidogenesis, in general. From structure activity relationship studies we have designed and synthesized a class of novel small molecules, which have been found to be potent inhibitors of tau aggregation. Methods:A high throughput screen was developed by incubating a purified tau protein 4-repeat domain with heparin in vitro. Tau aggregated to form Thioflavin S (ThioS)-positive, b-sheet-containing paired helical filaments. More than 100 of ProteoTech’s novel small molecules were screened for their ability to inhibit/disrupt tau fibrils. Inhibitory potencies were evaluated by several independent in vitromethodologies, including ThioS fluorometry, circular dichroism (CD) spectroscopy, and negative stain electron microscopy (EM). Select small molecules were further tested for their ability to disrupt intracellular tau aggregation in cellular models byWestern blot analysis for detergent-insoluble tau aggregates and by florescent staining for aggregated tau. Results: A number of ProteoTech’s novel small molecules caused a dose-dependent inhibition of tau aggregation and disruption of preformed tau fibrils. Ten lead small molecule compounds were identified by ThioS assays with low IC50s ranging from 1-15 mM. These lead compounds also dose-dependently inhibited tau protein ß-sheet formation (determined by CD analysis), and tau protein fibrillogenesis (visualized by EM). In cell culture experiments, some of the lead compounds also reduced levels of tau oligomers by Western and dot blot analyses. Conclusions: ProteoTech has identified ten lead compounds that show robust efficacy against tau aggregation using independent in vitro and cell-based assays. These lead compounds are promising novel small molecule candidate therapeutics for treatment of AD and other tauopathies, and will be further evaluated in relevant transgenic mouse models of tauopathy.