The intracellular deposition of amyloid-␤ (A␤) peptides has been described in the brains of both Alzheimer's disease (AD) patients and animal models. A correlation between the intracellular amyloid burden and neurodegeneration has recently been reported in a triple-transgenic AD (3xTg-AD) murine model. In the present study, we assessed the effect of scFv-h3D6, an anti-A␤ single-chain variable fragment (scFv) derived from the antibody bapineuzumab, on amyloid pathology in 5-month-old 3xTg-AD female mice, focusing on intracellular A␤ clearance, neuronal survival, and functional abilities. We also examined neuroinflammation and the histology of peripheral organ samples to detect any adverse effects. A single intraperitoneal injection of scFv-h3D6 dramatically reduced intracellular A␤ burden in the deep layers of the cerebral cortex, pyramidal cells layer of the hippocampus, and basolateral amygdalar nucleus. The treatment prevented neuronal loss in the hippocampus and amygdala, while neither astrogliosis nor microgliosis was induced. Instead, an increase in the size of the white pulp after the treatment indicated that the spleen could be involved in the clearance mechanism. Although the treatment did not ameliorate behavioral and psychological symptoms of dementia-like symptoms, the results of cognitive testing pointed to a noticeable improvement in spatial memory. These findings indicated that the mechanism underlying the therapeutic effect of scFv-h3D6 was the clearance of intracellular A␤, with subsequent prevention of neuronal loss and amelioration of cognitive disabilities. The treatment was safe in terms of neuroinflammation and kidney and liver function, whereas some effects on the spleen were observed.

The success of the targeting of amyloid-β (Aβ) oligomers through immunotherapy in Alzheimer’s disease (AD) mouse models has not been translated into the clinics. The use of single-chain variable fragments (scFvs) has been proposed to prevent the potential severe effects of full-length mAbs by precluding crystallizable fraction-mediated microglia activation. The efficacy of scFv-h3D6, a bapineuzumab-derived anti-Aβ scFv, has been extensively proven. In this work, we compared scFv-h3D6-EL, an elongated variant of the scFv-h3D6, with its original version to assess whether its characteristic higher thermodynamic stability improved its pharmacokinetic parameters. Although scFv-h3D6-EL had a longer half-life than its original version, its absorption from the peritoneal cavity into the systemic compartment was lower than that of the original version. Moreover, we attempted to determine the mechanism underlying the protective effect of scFv-h3D6. We found that scFv-h3D6 showed compartmental distribution and more interestingly crossed the blood–brain barrier. In the brain, scFv-h3D6 was engulfed by glial cells or internalized by Aβ peptide-containing neurons in the early phase post-injection, and was colocalized with the Aβ peptide almost exclusively in glial cells in the late phase post-injection. Aβ peptide levels in the brain decreased simultaneously with an increase in scFv-h3D6 levels. This observation in addition to the increased tumor necrosis factor-α levels in the late phase post-injection suggested that the engulfment of Aβ peptide/scFv-h3D6 complex extruded from large neurons by phagocytic cells was the mechanism underlying Aβ peptide withdrawal. The mechanism of action of scFv-h3D6 demonstrates the effectivity of Aβ-immunotherapy and lays the background for other studies focused on the finding of a treatment for AD.

The main histopathological hallmarks of Alzheimer's disease (AD) are the extracellular deposition of neuritic amyloid plaques, composed of amyloid-β (Aβ) peptide, and the intracellular accumulation of neurofibrillary tangles, composed of hyperphosphorylated tau. Both traits are emulated in the 3xTg-AD mouse model. Because the relevance of this model in the bibliography and the main role of Aβ in neuronal impairment, here we have detailed the brain Aβ/AβPP distribution to subsequently quantify cellular density and intracellular burden for specific neuronal populations in the early stages of the disease. 6E10 immunoreactivity was evident in the deep layers of the cerebral cortex, in the pyramidal cell layer of the hippocampus, in the basolateral amygdala nucleus, and in the deep cerebellar nuclei macroneurons; whereas the specific neuronal populations with decreased cellular density were the large pyramidal neurons from the layers V-VI in the cerebral cortex, the pyramidal neurons from the CA2-3 region in the hippocampus, and the large neurons from the basolateral nucleus in the amygdala, apart from the already reported deep cerebellar nuclei. Interestingly, we found a strong correlation between intracellular Aβ/AβPP burden and cellular density in all these populations. In addition, behavioral testing showed the functional consequences of such a neuronal depletion. Concretely, anxious-like behavior is manifested in the corner and open-field tests, as well as cognitive functions shown to be impaired in the novel object recognition test and Morris water maze paradigm. To our knowledge, this is the first deep characterization of the specific neuronal populations affected in the 3xTg-AD mouse model.

We have optimised an indirect immunoperoxidase technique demonstrating bromodeoxyuridine (BrdU) incorporation into dividing cells for cerebellar tissue sections of four-day-old rats injected with this marker. This permits confident identification of granule-cell precursors engaged in DNA synthesis in the external granular layer of the developing cerebellum. Preservation of BrdU immunoreactivity is attained using methanol/acetic acid fixation and different pretreatments before immunostaining, while unlabeled nuclei can be recognized clearly after Feulgen or hematoxylin counterstaining. We established conditions to ensure satisfactory BrdU uptake without affecting cell-cycle progression during the postlabeling time period. The dose of BrdU employed provides saturation S-phase labeling from at least 1 h after BrdU delivery. Various kinetic parameters and phase durations have been determined in experiments involving a single injection or cumulative labeling sequences, and the cycle time was calculated based on two models of generative behavior: steady-state and exponential growth. The working hypothesis of steady-state kinetics can be adopted successfully if the existence of neuroblasts with different proliferation rates is taken into account.

Clinical symptoms of Alzheimer’s Disease (AD) include behavioral alterations and cognitive impairment. These functional phenotypes early occur in triple-transgenic (3xTg-AD) mice. Specifically, behavioral alterations are first detected when mice are at around 2.5 months old and cognitive impairment in between 3- and 5-month-old mice. In this work, the effect of chronic Aβ-immunotherapy on behavioral and cognitive abilities was tested by monthly administering the antibody fragment scFv-h3D6 to 3xTg-AD female mice from 5 to 9 months of age. An untreated group was used as a reference, as well as to attain some information on the effect of training during the longitudinal study. Behavioral and psychological symptoms of dementia (BPSD)-like symptoms were already evident in 5-month-old mice, in the form of neophobia and anxious-like behavior. The exploratory activity decreased over the longitudinal study, not only for 3xTgAD mice but also for the corresponding non-transgenic mice (NTg). Learning abilities of 3xTg-AD mice were not seriously compromised but an impairment in long-term spatial memory was evident at 5 months of age. Interestingly, scFv-h3D6-treatment affected the cognitive impairment displayed by 5-month-old 3xTg-AD mice. It is worth noting that training also reduced cognitive impairment of 3xTg-AD mice over the longitudinal study, suggesting that to properly quantify the isolated therapeutic potential of any drug on cognition using this model it is convenient to perform a prompt, age-matched study rather than a longitudinal study. In addition, a combination of both training and Aβ-immunotherapy could constitute a possible approach to treat Alzheimer’s disease.