Henrieta Scholtzova

Immunization with a Nontoxic/Nonfibrillar Amyloid-β Homologous Peptide Reduces Alzheimer’s Disease-Associated Pathology in Transgenic Mice

Transgenic mice with brain amyloid-beta (Abeta) plaques immunized with aggregated Abeta1-42 have reduced cerebral amyloid burden. However, the use of Abeta1-42 in humans may not be appropriate because it crosses the blood brain barrier, forms toxic fibrils, and can seed fibril formation. We report that immunization in transgenic APP mice (Tg2576) for 7 months with a soluble nonamyloidogenic, nontoxic Abeta homologous peptide reduced cortical and hippocampal brain amyloid burden by 89% (P = 0.0002) and 81% (P = 0.0001), respectively. Concurrently, brain levels of soluble Abeta1-42 were reduced by 57% (P = 0.0019). Ramified microglia expressing interleukin-1beta associated with the Abeta plaques were absent in the immunized mice indicating reduced inflammation in these animals. These promising findings suggest that immunization with nonamyloidogenic Abeta derivatives represents a potentially safer therapeutic approach to reduce amyloid burden in Alzheimers disease, instead of using toxic Abeta fibrils.

Detection of Alzheimer's amyloid in transgenic mice using magnetic resonance microimaging.

The presence of amyloid‐β (Aβ) plaques in the brain is a hallmark pathological feature of Alzheimers disease (AD). Transgenic mice overexpressing mutant amyloid precursor protein (APP), or both mutant APP and presenilin‐1 (APP/PS1), develop Aβ plaques similar to those in AD patients, and have been proposed as animal models in which to test experimental therapeutic approaches for the clearance of Aβ. However, at present there is no in vivo whole‐brain imaging method to detect Aβ plaques in mice or men. A novel method is presented to detect Aβ plaques in the brains of transgenic mice by magnetic resonance microimaging (μMRI). This method uses Aβ1‐40 peptide, known for its high binding affinity to Aβ, magnetically labeled with either gadolinium (Gd) or monocrystalline iron oxide nanoparticles (MION). Intraarterial injection of magnetically labeled Aβ1‐40, with mannitol to transiently open the blood–brain barrier (BBB), enabled the detection of many Aβ plaques. Furthermore, the numerical density of Aβ plaques detected by μMRI and by immunohistochemistry showed excellent correlation. This approach provides an in vivo method to detect Aβ in AD transgenic mice, and suggests that diagnostic MRI methods to detect Aβ in AD patients may ultimately be feasible. Magn Reson Med 50:293–302, 2003.

Blocking the apolipoprotein E/amyloid-β interaction as a potential therapeutic approach for Alzheimer's disease

The amyloid-β (Aβ) cascade hypothesis of Alzheimers disease (AD) maintains that accumulation of Aβ peptide constitutes a critical event in the early disease pathogenesis. The direct binding between Aβ and apolipoprotein E (apoE) is an important factor implicated in both Aβ clearance and its deposition in the brains parenchyma and the walls of meningoencephalic vessels as cerebral amyloid angiopathy. With the aim of testing the effect of blocking the apoE/Aβ interaction in vivo as a potential novel therapeutic target for AD pharmacotherapy, we have developed Aβ12-28P, which is a blood-brain-barrier-permeable nontoxic, and nonfibrillogenic synthetic peptide homologous to the apoE binding site on the full-length Aβ. Aβ12-28P binds with high affinity to apoE, preventing its binding to Aβ, but has no direct effect on Aβ aggregation. Aβ12-28P shows a strong pharmacological effect in vivo. Its systemic administration resulted in a significant reduction of Aβ plaques and cerebral amyloid angiopathy burden and a reduction of the total brain level of Aβ in two AD transgenic mice models. The treatment did not affect the levels of soluble Aβ fraction or Aβ oligomers, indicating that inhibition of the apoE/Aβ interaction in vivo has a net effect of increasing Aβ clearance over deposition and at the same time does not create conditions favoring formation of toxic oligomers. Furthermore, behavioral studies demonstrated that treatment with Aβ12-28P prevents a memory deficit in transgenic animals. These findings provide evidence of another therapeutic approach for AD.

A Synthetic Peptide Blocking the Apolipoprotein E/β-Amyloid Binding Mitigates β-Amyloid Toxicity and Fibril Formation in Vitro and Reduces β-Amyloid Plaques in Transgenic Mice

Alzheimer’s disease (AD) is associated with accumulation of β-amyloid (Aβ). A major genetic risk factor for sporadic AD is inheritance of the apolipoprotein (apo) E4 allele. ApoE can act as a pathological chaperone of Aβ, promoting its conformational transformation from soluble Aβ into toxic aggregates. We determined if blocking the apoE/Aβ interaction reduces Aβ load in transgenic (Tg) AD mice. The binding site of apoE on Aβ corresponds to residues 12 to 28. To block binding, we synthesized a peptide containing these residues, but substituted valine at position 18 to proline (Aβ12–28P). This changed the peptide’s properties, making it non-fibrillogenic and non-toxic. Aβ12–28P competitively blocks binding of full-length Aβ to apoE (IC50 = 36.7 nmol). Furthermore, Aβ12–28P reduces Aβ fibrillogenesis in the presence of apoE, and Aβ/apoE toxicity in cell culture. Aβ12–28P is blood-brain barrier-permeable and in AD Tg mice inhibits Aβ deposition. Tg mice treated with Aβ12–28P for 1 month had a 63.3% reduction in Aβ load in the cortex (P = 0.0043) and a 59.5% (P = 0.0087) reduction in the hippocampus comparing to age-matched control Tg mice. Antibodies against Aβ were not detected in sera of treated mice; therefore the observed therapeutic effect of Aβ12–28P cannot be attributed to an antibody clearance response. Our experiments demonstrate that compounds blocking the interaction between Aβ and its pathological chaperones may be beneficial for treatment of β-amyloid deposition in AD.

Anti-prion antibodies for prophylaxis following prion exposure in mice

Prion disease is characterized by a conformational change of the normal form of the prion protein (PrP(C)) to the scrapie-associated form (PrP(Sc)). Since the emergence of new variant Creutzfeldt-Jakob disease a potentially large human population is at risk for developing prion disease. Currently, no effective treatment or form of post-exposure prophylaxis is available for prion disease. We recently showed that active immunization with recombinant PrP prolongs the incubation period of scrapie. Here we show that anti-PrP antibodies following prion exposure are effective at increasing the incubation period of the infection. Stimulation of the immune system is an important therapeutic target for the prion diseases, as well as for other neurodegenerative illnesses characterized by abnormal protein conformation.

Induction of Toll-Like Receptor 9 Signaling as a Method for Ameliorating Alzheimer's Disease-Related Pathology

The pathogenesis of Alzheimers disease (AD) is thought to be related to the accumulation of amyloid β (Aβ) in amyloid deposits and toxic oligomeric species. Immunomodulation is emerging as an effective means of shifting the equilibrium from Aβ accumulation to clearance; however, excessive cell mediated inflammation and cerebral microhemorrhages are two forms of toxicity which can occur with this approach. Vaccination studies have so far mainly targeted the adaptive immune system. In the present study, we have stimulated the innate immune system via the Toll-like receptor 9 (TLR9) with cytosine-guanosine-containing DNA oligodeoxynucleotides in Tg2576 AD model transgenic mice. This treatment produced a 66% and 80% reduction in the cortical (p = 0.0001) and vascular (p = 0.0039) amyloid burden, respectively, compared with nontreated AD mice. This was in association with significant reductions in Aβ42, Aβ40, and Aβ oligomer levels. We also show that treated Tg mice performed similarly to wild-type mice on a radial arm maze. Our data suggest that stimulation of innate immunity via TLR9 is highly effective at reducing the parenchymal and vascular amyloid burden, along with Aβ oligomers, without apparent toxicity.

Links between the pathology of Alzheimer's disease and vascular dementia.

The major neuropathological lesions defining Alzheimers disease (AD) include neurofibrillary tangles and amyloid plaques, which are mainly composed of abnormally phosphorylated tau and amyloid-β (Aβ), respectively. Numerous neuropathological and neuroimaging studies indicate that at least one-third of AD cases are complicated by some degree of vascular pathology, whereas in a similar proportion of patients clinically diagnosed with vascular dementia, AD pathology is also present. Many classical vascular risk factors such as hypertension, diabetes mellitus, and hypercholesterolemia have recently been shown also to increase the risk of AD. Growing evidence suggests that vascular pathology lowers the threshold for the clinical presentation of dementia at a given level of AD-related pathology and potentially directly promotes AD lesions such as Aβ plaques. Cerebral ischemia chronically up-regulates expression of the amyloid precursor protein (APP), which is the precursor to the amyloid β peptide and damages the blood–brain barrier (BBB), affecting Aβ peptide clearance from the brain. Recognition of the importance of these vascular risk factors for AD-related dementia and their treatment will be beneficial not only for preventing cardiac, cerebral, and peripheral complications of vascular disease, but also will likely have a direct impact on the occurrence of sporadic AD in older subjects. In this paper, we review some of the links between vascular risk factors and AD pathology and present data on the direct effect of ischemia on cognitive function and Aβ deposition in a mouse model of AD.

Mucosal vaccination delays or prevents prion infection via an oral route

In recent years major outbreaks of prion disease linked to oral exposure of the prion agent have occurred in animal and human populations. These disorders are associated with a conformational change of a normal protein, PrP(C) (prion protein cellular), to a toxic and infectious form, PrP(Sc) (prion protein scrapie). None of the prionoses currently have an effective treatment. A limited number of active immunization approaches have been shown to slightly prolong the incubation period of prion infection. Active immunization in wild-type animals is hampered by auto-tolerance to PrP and potential toxicity. Here we report that mucosal vaccination with an attenuated Salmonella vaccine strain expressing the mouse PrP, is effective at overcoming tolerance to PrP and leads to a significant delay or prevention of prion disease in mice later exposed orally to the 139A scrapie strain. This mucosal vaccine induced gut anti-PrP immunoglobulin (Ig)A and systemic anti-PrP IgG. No toxicity was evident with this vaccination approach. This promising finding suggests that mucosal vaccination may be a useful method for overcoming tolerance to PrP and preventing prion infection among animal and potentially human populations at risk.

Vaccination of Alzheimer’s model mice with Aβ derivative in alum adjuvant reduces Aβ burden without microhemorrhages

Immunotherapy holds great promise for Alzheimers disease (AD) and other conformational disorders but certain adverse reactions need to be overcome. The meningoencephalitis observed in the first AD vaccination trial was likely related to excessive cell‐mediated immunity caused by the immunogen, amyloid‐β (Aβ) 1–42, and the adjuvant, QS−21. To avoid this toxicity, we have been using Aβ derivatives in alum adjuvant that promotes humoral immunity. Other potential side effects of immunotherapy are increased vascular amyloid and associated microhemorrhages that may be related to rapid clearance of parenchymal amyloid. Here, we determined if our immunization strategy was associated with this form of toxicity, and if the therapeutic effect was age‐dependent. Tg2576 mice and wild‐type littermates were immunized from 11 or 19 months and their behaviour evaluated prior to killing at 24 months. Subsequently, plaque‐ and vascular‐Aβ burden, Aβ levels and associated pathology was assessed. The therapy started at the cusp of amyloidosis reduced cortical Aβ deposit burden by 31% and Aβ levels by 30–37%, which was associated with cognitive improvements. In contrast, treatment from 19 months, when pathology is well established, was not immunogenic and therefore did not reduce Aβ burden or improve cognition. Significantly, the immunotherapy in the 11–24 months treatment group, that reduced Aβ burden, did not increase cerebral bleeding or vascular Aβ deposits in contrast to several Aβ antibody studies. These findings indicate that our approach age‐dependently improves cognition and reduces Aβ burden when used with an adjuvant suitable for humans, without increasing vascular Aβ deposits or microhemorrhages.

Memantine leads to behavioral improvement and amyloid reduction in Alzheimer's-disease-model transgenic mice shown as by micromagnetic resonance imaging.

Memantine, an N‐methyl‐D‐aspartate (NMDA) receptor antagonist, has been shown to improve learning and memory in several preclinical models of Alzheimers disease (AD). Memantine has also been shown to reduce the levels of amyloid β (Aβ) peptides in human neuroblastoma cells as well as to inhibit Aβ oligomer‐induced synaptic loss. In this study, we assessed whether NMDA receptor inhibition by memantine in transgenic mice expressing human amyloid‐beta precursor protein (APP) and presenilin 1 (PS1) is associated with cognitive benefit and amyloid burden reduction by using object recognition, micromagnetic resonance imaging (μMRI), and histology. APP/PS1 Tg mice were treated either with memantine or with vehicle for a period of 4 months starting at 3 months of age. After treatment, the mice were subjected to an object recognition test and analyzed by ex vivo μMRI, and histological examination of amyloid burden. μMRI was performed following injection with gadolinium‐DTPA‐Aβ1–40. We found that memantine‐treated Tg mice performed the same as wild‐type control mice, whereas the performance of vehicle‐treated Tg mice was significantly impaired (P = 0.0081, one‐way ANOVA). Compared with vehicle‐treated animals, memantine‐treated Tg mice had a reduced plaque burden, as determined both histologically and by μMRI. This reduction in amyloid burden correlates with an improvement in cognitive performance. Thus, our findings provide further evidence of the potential role of NMDA receptor antagonists in ameliorating AD‐related pathology. In addition, our study shows, for the first time, the utility of μMRI in conjunction with gadolinium‐labeled Aβ labeling agents to monitor the therapeutic response to amyloid‐reducing agents.