Faculty Bibliography

Transgenic mice are used increasingly to model brain amyloidosis, mimicking the pathogenic processes involved in Alzheimer's disease (AD). In this chapter, an in vivo strategy is described that has been successfully used to map amyloid-beta deposits in transgenic mouse models of AD with magnetic resonance imaging (MRI), utilizing both the endogenous contrast induced by the plaques attributed to their iron content and by selectively enhancing the signal from amyloid-beta plaques using molecular-targeting vectors labeled with MRI contrast agents. To obtain sufficient spatial resolution for effective and sensitive mouse brain imaging, magnetic fields of 7-Tesla (T) or more are required. These are higher than the 1.5-T field strength routinely used for human brain imaging. The higher magnetic fields affect contrast agent efficiency and dictate the choice of pulse sequence parameters for in vivo MRI, all addressed in this chapter. Two-dimensional (2D) multi-slice and three-dimensional (3D) MRI acquisitions are described and their advantages and limitations are discussed. The experimental setup required for mouse brain imaging is explained in detail, including anesthesia, immobilization of the mouse's head to reduce motion artifacts, and anatomical landmarks to use for the slice alignment procedure to improve image co-registration during longitudinal studies and for subsequent matching of MRI with histology.

The increased availability of transgenic mouse models for studying human diseases has shifted the focus of many laboratories from in vitro to in vivo assays. Herein, methods are described to allow investigators to obtain well-preserved mouse tissue to be stained with the standard histological dyes for amyloid, Congo Red, and Thioflavin S. These sections can as well be used for immunohistological procedures that allow detection of tissue amyloid and pre-amyloid, such as those composed of the amyloid-beta peptide, the tau protein, and the islet amyloid polypeptide.

Pathological tau protein is found in Alzheimer's disease and related tauopathies. The protein is hyperphosphorylated and/or mutated which leads to aggregation and neurotoxicity. Because cognitive functions correlate well with the degree of tau pathology, clearing these aggregates is a promising therapeutic approach. Studies pioneered by our laboratory and confirmed by others have shown that both active and passive immunizations targeting disease-related tau epitopes successfully reduce tau aggregates in vivo and slow or prevent behavioral impairments in mouse models of tauopathy. Here, we summarize recent advances in this new field

Impairment of synaptic plasticity underlies memory dysfunction in Alzheimer's disease (AD). Molecules involved in this plasticity such as PSD-95, a major postsynaptic scaffold protein at excitatory synapses, may play an important role in AD pathogenesis. We examined the distribution of PSD-95 in transgenic mice of amyloidopathy (5XFAD) and tauopathy (JNPL3) as well as in AD brains using double-labeling immunofluorescence and confocal microscopy. In wild type control mice, PSD-95 primarily labeled neuropil with distinct distribution in hippocampal apical dendrites. In 3-month-old 5XFAD mice, PSD-95 distribution was similar to that of wild type mice despite significant Abeta deposition. However, in 6-month-old 5XFAD mice, PSD-95 immunoreactivity in apical dendrites markedly decreased and prominent immunoreactivity was noted in neuronal soma in CA1 neurons. Similarly, PSD-95 immunoreactivity disappeared from apical dendrites and accumulated in neuronal soma in 14-month-old, but not in 3-month-old, JNPL3 mice. In AD brains, PSD-95 accumulated in Hirano bodies in hippocampal neurons. Our findings support the notion that either Abeta or tau can induce reduction of PSD-95 in excitatory synapses in hippocampus. Furthermore, this PSD-95 reduction is not an early event but occurs as the pathologies advance. Thus, the time-dependent PSD-95 reduction from synapses and accumulation in neuronal soma in transgenic mice and Hirano bodies in AD may mark postsynaptic degeneration that underlies long-term functional deficits.

Neurofibrillary tangles (NFTs) are one of the pathological hallmarks of Alzheimer's disease (AD) and are primarily composed of aggregates of hyperphosphorylated forms of the microtubule associated protein tau. It is likely that an imbalance of kinase and phosphatase activities leads to the abnormal phosphorylation of tau and subsequent aggregation. The wide ranging therapeutic approaches that are being developed include to inhibit tau kinases, to enhance phosphatase activity, to promote microtubule stability, and to reduce tau aggregate formation and/or enhance their clearance with small molecule drugs or by immunotherapeutic means. Most of these promising approaches are still in preclinical development whilst some have progressed to Phase II clinical trials. By pursuing these lines of study, a viable therapy for AD and related tauopathies may be obtained

Neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease, Huntington's disease (HD) or amyotrophic lateral sclerosis (ALS) are all characterized histologically by the presence of deposits of misfolded proteins, tau and amyloid, -synuclein, huntingtin or superoxide dismutase respectively. Currently these illnesses do not have any disease modifying treatment options. A novel therapeutic strategy that is being pursued is immunomodulation, which is using the body's immune system to target the self proteins that are deposited. Most of these promising approaches are still in preclinical development whilst some have progressed to Phase III clinical trials. As new insights are gained, it is hoped that these immunotherapies will be effective tools at slowing the progression of these debilitating diseases

J. Neurochem. (2011) 118, 658-667. ABSTRACT: Targeting hyperphosphorylated tau by immunotherapy is emerging as a promising approach to treat tauopathies such as Alzheimer's disease and frontotemporal dementia. We have previously reported that active tau immunization clears tau aggregates from the brain and attenuates or prevents functional impairments in two different tangle mouse models. Here, we assessed the efficacy of passive immunization with the PHF1 antibody, which targets a phospho-epitope within one of our active immunogens. Homozygous female tangle mice (JNPL3, 2-3 months) were injected intraperitoneally once per week with PHF1 or pooled mouse IgG (250 mug/125 muL; n = 10 per group) for a total of 13 injections. Their behavior was assessed at 5-6 months of age and brain tissue was subsequently harvested for analyses of treatment efficacy. The treated mice performed better than controls on the traverse beam task (p < 0.03), and had 58% less tau pathology in the dentate gyrus of the hippocampus (p = 0.02). As assessed by western blots, the antibody therapy reduced the levels of insoluble pathological tau by 14-27% (PHF1, p < 0.05; PHF1/total tau, p < 0.0001) and 34-45% (CP13 or CP13/total tau, p < 0.05). Levels of soluble tau and sarkosyl soluble tau were unchanged, compared with controls, as well as total tau levels in all the fractions. Plasma levels of PHF1 correlated inversely with tau pathology in the brainstem (p < 0.01), with a strong trend in the motor cortex (p < 0.06) as well as with insoluble total tau levels (p < 0.02), indicating that higher dose of antibodies may have a greater therapeutic effect. Significant correlation was also observed between performance on the traverse beam task and PHF1 immunoreactivity in the dentate gyrus (p < 0.05) as well as with insoluble PHF1/total tau ratio on western blots (p < 0.04). These results show that passive immunization with tau antibodies can decrease tau pathology and functional impairments in the JNPL3 model. Future studies will determine the feasibility of this approach with other monoclonals and in different tangle models in which thorough cognitive assessment can be performed

Background: Active anti-amyloid immunotherapy is a strategy developed againstAlzheimer's disease.ApproacheswithAs1-42 orK6As1-30 immunogens in an adjuvant decrease amyloid-s burden and prevent cognitive decline in transgenic mice (Asuni et al, 2006). However, clinical trials of As1-42 immunotherapy have induced side effects like encephalitis and possibly microhemorrhages (Orgogozo et al, 2003; Ferrer et al, 2004). Mouse lemurs can develop As plaques with age (Mestre-Frances et al, 2000). Such a primate modelmay be more predictive than rodents of human side effects.We studied, by magnetic resonance imaging (MRI), immunotherapies in these primates. Methods: A first cohort was used to compare K6As1-30 (n = 4; 5.8 6 0.2years) and As1-42 (n = 4; 5.96 0.2years) immunogens in alumadjuvant. Asecond cohortwas used to evaluate K6As1-30 (n=6; 4.660.2years) compared to adjuvant alone (n = 6; 4.7 6 0.3years). All the animals were followed- up by MRI (7T PharmaScan-Bruker) to evaluate neuroinflammation, microhemorrhages and other forms of iron deposition, with T2-weighted and T2*-weighted sequences (resolution = (234x234x234)Amm3). The hypointense regions from T2*-weighted images were quantified and evaluate by histology. A complementary study of age effect was performed with twenty other naive animals (1.5 to 4.9years). Results: TheT2-weighted images did not show any neuroinflammation during immunization, irrespective of the immunogen. Microhemorrhages were detected in the cerebral parenchyma at the histological analysis of the first cohort. The animals treated with K6As1-30 presented less microhemorrhages compared to those treated with As1-42 vaccine (Mann-Whitney, p < 0.05). These small microhemorrhages were not detected on the T2*-weighted images. However hypointense signal was detected onMRI and corresponded to iron deposits in the choroid plexus. Its volume increased with natural aging (r=0.60; p<0.001) and with As1-42 compared toK6As1-30 treatment (ANOVA, p<0.05).No difference was detected between K6As1-30 and adjuvant alone. Conclusions: The immunotherapies studied in the mouse lemur primate did not lead to any MRI sign of neuroinflammation. The K6As1-30 strategy appears to be safer than the As1-42 strategy as it provokes less microhemorrhages in the cerebral parenchyma and less iron deposits in the choroid plexus

Background: Immunotherapy targeting pathological tau is emerging as a promising approach to treat tauopathies such as Alzheimer's disease (AD) and frontotemporal dementia. We have previously shown that prophylactic active or passive tau immunotherapy, starting at 2-3 months of age, clears tau pathology and improves function (Asuni A. et al., J. Neurosci. 2007, Boutajangout A. et al., J. Neurosci. 2010, Boutajangout A. et al., ICAD 2010). Here we assessed if a phospho-specific monoclonal, 4E6G7, targeting the same epitope, would be efficacious if treatment commenced at an advanced stage of tau pathology (8-9 months). Methods: htau/PS1 mice without mouse tau protein (8-9 months) were injected intraperitoneally (250 mg/125 ml) once per week with a novel phospho- specific tau monoclonal, 4E6G7 (n = 9) or pooled mouse IgG (n = 10) for a total of 13 injections. Their behavior was analyzed at 11-12 months of age and brain tissue subsequently harvested for analyses of treatment efficacy. Results: The immunized mice (n = 7-8) performed substantially better than controls (n = 6) on the Radial Arm Maze (p < 0.0001; post hoc, p < 0.01-0.001 on days 2, 3, 5-9), the Closed Field Symmetrical Maze with 35-69% fewer errors in simple, intermediate and complex tasks (p < 0.05-0.003), and the Object Recognition Task (63% time spent with a novel object vs. 46% for controls, p < 0.05). The groups did not differ in various sensorimotor tasks, indicating that the robust cognitive improvements cannot be explained by sensorimotor effects, which further strengthens our results. Interestingly, more controls died during the study (40%) than immunized mice (22%), providing an additional support for the beneficial effect of the monoclonal tau antibody. Histological and biochemical analyses are underway. Conclusions: These results indicate pronounced efficacy of passive tau immunotherapy at an advanced stage of tauopathy, which suggests that this approach may be beneficial after clinical onset of AD and other tauopathies

Background: Active anti-amyloid immunotherapy is a strategy developed against Alzheimer's disease.ApproacheswithAs1-42 orK6As1-30 immunogens in an adjuvant decrease amyloid-s burden and prevent cognitive decline in transgenic mice (Asuni et al, 2006). However, clinical trials of As1-42 immunotherapy have induced side effects like encephalitis and possibly microhemorrhages (Orgogozo et al, 2003; Ferrer et al, 2004). Mouse lemurs can develop As plaques with age (Mestre-Frances et al, 2000). Such a primate modelmay bemore predictive than rodents of human side effects.We studied, by magnetic resonance imaging (MRI), immunotherapies in these primates. Methods: A first cohort was used to compare K6As1-30 (n = 4; 5.8 +/- 0.2 years) and As1-42 (n = 4; 5.9 +/- 0.2 years) immunogens in alum adjuvant. Asecond cohortwas used to evaluateK6As1-30 (n=6; 4.660.2 years) compared to adjuvant alone (n = 6; 4.7 +/- 0.3 years). All the animals were followed- up by MRI (7T PharmaScan-Bruker) to evaluate neuroinflammation, microhemorrhages and other forms of iron deposition, with T2-weighted and T2*-weighted sequences (resolution=(234x234x234)mm³). The hypointense regions from T2*-weighted images were quantified and evaluate by histology. A complementary study of age effectwas performedwith twenty other naive animals (1.5 to 4.9 years). Results: The T2-weighted images did not show any neuroinflammation during immunization, irrespective of the immunogen. Microhemorrhages were detected in the cerebral parenchyma at the histological analysis of the first cohort. The animals treated with K6As1-30 presented less microhemorrhages compared to those treated with As1-42 vaccine (Mann-Whitney, p < 0.05). These small microhemorrhages were not detected on the T2*-weighted images. However hypointense signalwas detected on MRI and corresponded to iron deposits in the choroid plexus. Its volume increasedwith natural aging (r= 0.60; p< 0.001) and withAs1-42 compared to K6As1-30 treatment (ANOVA, p < 0.05). No difference was detected between K6As1-30 and adjuvant alone. Conclusions: The immunotherapies studied in the mouse lemur primate did not lead to any MRI sign of neuroinflammation. The K6As1-30 strategy appears to be safer than theAs1-42 strategy as it provokes less microhemorrhages in the cerebral parenchyma and less iron deposits in the choroid plexus