Faculty Bibliography

Background: Anti-amyloid immunotherapy reduces Ab plaques and prevents cognitive decline in transgenic mouse models of Alzheimer's disease (AD) (Asuni et al, 2006). Nevertheless, in humans, a strategy based on Ab1- 42 peptide induced encephalomyelitis and possibly microhemorrhages (Orgogozo et al, 2003; Ferrer et al, 2004). These outcomes were not expected from studies in rodents. Mouse lemur, as a primate model may be more predictive of human side effects. A small proportion of old animals develop spontaneously Ab plaques (Mestre-Franc-es et al, 2000). Thus this primate models prodromal AD stages, and we used it to evaluate side effects of immunotherapies. Methods: Animals were treated with K6Ab1-30 (n = 4; 5.860.2 years) or Ab1-42 (n = 4; 5.960.2 years) immunogens in alum adjuvant. They were followed-up for 9 months with biochemistry (anti-Ab40 antibodies and Ab40 levels in the plasma), as well as T2-weighted (T2w) and T2-weighted (T2w) MRI (7T PharmaScan-Bruker; resolutions =(234x234x234)mm3. Histological analyses was performed to evaluate amyloidosis, neuroinflammation, and iron deposits/microhemorrhages. Age-associated occurrences of hypointense signals were evaluated in twenty other naive animals (1.5 to 4.9 years). Results: In this particular study, the animals responded mainly to the Ab1-42 immunogen, which differs from our prior study with this Ab derivative (Trouche et al, 2009). This treatment induced an immune response and increased Ab levels in plasma. No severe neuroinflammation were observed (either on MRI or histology). Compared to K6Ab1-30 vaccine, Ab1-42 immunogen increased microhemorrhages (Mann Whitney test U=1, P<0.05) and the size of hypointense signal corresponding to iron deposits in the choroid plexus (CP) (F(2,5)= 4.627; P<0.05). The study in naive lemurs showed that iron accumulates in the CP with normal aging (r=0.60; P<0.001). Hence, immunotherapy with Ab1-42 immunogen accelerated this age-associated phenomenon. Conclusions: Ab-immunization can lead to side effects such as microhemorrhages in a primate model of normal aging or prodromal stage of AD with minimal extracellular Ab deposition. Also, iron accumulation in the CP is a potential side effect of Ab-immunization. This should be taken into account in future evaluations of clinical trials with AD patients

Background: We previously reported human catalytic autoantibodies to amyloid b peptide (Ab). We hypothesize that recognition of electrophilic amyloid epitopes by nucleophilic autoantibodies is an innate immune function that is recruited for catalytic clearance of amyloid deposits associated with aging and Alzheimer's disease (AD). Methods: Ab cleavage was measured by HPLC, acid precipitation, mass spectroscopy or electrophoresis. Electrophilic Ab (E-Ab) was prepared by carbonylation with the lipid peroxidation end products 4-hydroxynonenal (HNE)/malonaldehyde (MDA) or phosphonate diester insertion. Covalent immune complexes were analyzed by SDS-electrophoresis. Ab1-42 aggregates were identified by antibody or Thioflavin-T staining. Results: IgM from healthy human sera, the first antibody class produced during B cell differentiation, catalyzed Ab cleavage at rates superior to IgGs. Preferential Ab cleavage by IgMs was also observed for antibodies from the sera and cerebrospinal fluid from patients with AD. Two Ab cleaving antibody fragments were isolated from a phage library, a heterodimeric V L -V L construct (2E6) and a single domain V L construct (5D3). Treatment with antibody 2E6 induced disappearance of oligomeric and fibrillar Ab. Intracranial antibody injection in Ab-overexpressing transgenic mice cleared the Ab plaques. Traditional antibodies bind antigens at complementarity determining regions (CDRs). The Ab cleaving antibodies contained CDRs with no or minimum mutations acquired by antigen-driven diversification. Deleting the CDRs did not attenuate Ab cleavage by antibody 2E6 but the catalytic activity was lost by replacing the framework regions (FRs) with corresponding FRs from a non-catalytic antibody. The FRs are evolutionarily conserved segments important for innate recognition of B cell superantigens without requirement for adaptive immune processes. From protease inhibitor and epitope mapping studies, the catalytic mechanism entails noncovalent binding at the Ab C terminus followed by nucleophilic peptide bond cleavage. Antibody 2E6 reacted covalently with an electrophilic phosphonate-containing Ab analog and the naturally-occurring Ab-HNE/Ab-MDA analogs (E-Ab). Monoclonal murine antibodies (MAbs) that cleaved Ab at low substrate concentrations were identified by immunization with non-electrophilic Ab. A subset of MAbs induced by immunization with E-Ab cleaved Ab robustly. Conclusions: Amplification of the innate noncovalent recognition and catalytic functions of antibodies driven by age/ disease-associated Ab accumulation can remove toxic amyloid deposits

Background: Immunotherapy targeting hyperphosphorylated tau is a promising prospect to mitigate the neurodegenerative effects of tauopathies. Assessing the effectiveness of such immunotherapies often involves sacrifice of the animal. However, Manganese-Enhanced Magnetic Resonance Imaging (MEMRI) permits the longitudinal study of neuronal function with minimal risk to the animal. We hypothesize that tract-tracing MEMRI in a mouse model of tau pathology should enable non-invasive monitoring of various tau targeting therapies aimed at improving neuronal integrity. Methods: Twenty-five homozygous JNPL3 tangle transgenic mice underwent MEMRI at 6 months of age. Thirteen of the mice received tau immunotherapy with Tau379-408[P-Ser396,404] in alum adjuvant from 3 months of age, and twelve controls received an adjuvant alone. Imaging studies were performed on a 7-T micro-MRI. Mice were imaged pre-injection, then injected in one nostril with a solution of 2.5 M MnCl 2, under isoflurane anesthesia. Image sets were acquired at 1, 4, 8, 12, 24, 36 and 48 hours, and finally at 7 days (Fig 1). The datasets were processed using ImageJ. Normalized measurements for each mouse were plotted and fitted to a tract tracing bolus model using MATLAB. Fitting enabled the estimation of the timing (Pt) and intensity (Pv) of the bolus peak of Mn, and maximal slope of uptake (Sv). Results: Asignificant increase in maximal slope of manganese uptake, Sv, was observed in the mitral cell layer (35%, P <.005) and glomerular layer (36%, P <0.02) in treated JNPL3 mice compared to identical controls. There was also a significant increase in bolus peak value, Pv, in the mitral layer in the treated group (7%, P = 0.02). Furthermore, in the immunized mice, there was a strong trend for a decrease in the time to peak value, Pt (-9%P = 0.10), in the mitral cell layer, compared to the controls. Conclusions: Utilizing MEMRI's non-invasive, longitudinal measurements from 1 hour to 7 days, allowed us to detect substantial improvements in neuronal transport following tau immunotherapy. We are analyzing tau pathology in olfactory sections from these mice to assess the correlation of these benefits with clearance of tau lesions, which we have shown previously to occur with this treatment

In the last couple of decades, substantial progress has been made in the development of transgenic mouse models developing amyloid-beta deposits and/or neurofibrillary tangles. These mouse models of Alzheimer's disease and related disorders provide an excellent tool for investigating etiology, pathogenic mechanisms, and potential treatments. An essential component of their characterization is a detailed behavioral assessment, which clarifies the functional consequences of these pathologies. We have selected and refined a series of cognitive and sensorimotor tasks that are ideal for studying these models and the efficacy of various treatments.

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.

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.

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

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

Recent studies have shown that immunotherapy clears amyloid beta (Abeta) plaques and reduces Abeta levels in mouse models of Alzheimer's disease (AD), as well as in AD patients. Tangle pathology is also relevant for the neurodegeneration in AD, and our studies have shown that active immunization with an AD related phospho-tau peptide reduces aggregated tau within the brain and slows the progression of tauopathy-induced behavioral impairments. Thus, clearance of neurofibrillary tangles and/or their precursors may reduce synaptic and neuronal loss associated with AD and other tauopathies. So far the mechanisms involved in antibody-mediated clearance of tau pathology are yet to be elucidated. In this study we have used a mouse brain slice model to examine the uptake and localization of FITC labeled anti-tau antibodies. Confocal microscopy analysis showed that the FITC labeled anti-tau antibody co-stained with phosphorylated tau, had a perinuclear appearance and co-localized with markers of the endosomal/lysosomal pathway. Additionally, tau and FITC-IgG were found together in an enriched lysosome fraction. In summary, antibody-mediated clearance of intracellular tau aggregates appears to occur via the lysosomal pathway