Faculty Bibliography

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: 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: Tau pathology is involved in multiple neurodegenerative disorders, for example in Alzheimer's disease, Parkinson's disease, and Frontotemporal dementia (FTD). Tau is a neuronal protein that binds microtubules and is thought to be involved in the stabilization of microtubules. Over 50 different mutations within the MAPT gene, the gene encoding for Tau, have been associated with inherited FTD. FTD is thought to involve deficits in the communication between neurons and in the mechanisms neuronal adaptation to experience, synaptic plasticity. The role of Tau in mechanisms of synaptic plasticity is not well-understood. To address this gap in the field, we have investigated synaptic plasticity and behavior in P301L mice, a mouse model for tau pathology that over-expresses human Tau protein carrying an inherited human mutation. Methods: Long-lasting forms of plasticity, late-phase long term potentiation (L-LTP) were examined in P301L (JNPL3) mice and age-matched controls by measuring field excitatory postsynaptic potentiation (fEPSP) in the CA1 hippocampal region after high frequency electrophysiological stimulation. Two behaviors associated with GABAergic function were assayed, prepulse inhibition of startle response (PPI) and susceptibility to epileptic seizures. GABAergic interneurons were stained using two markers; paravalbumin and somatostatin. Results: By examining long-lasting forms of plasticity in aged (>18 months old) in hippocampal brain slices we found surprisingly enhanced L-LTP in P301L mice compared to age-matched controls. The enhanced L-LTP in P301L slices was rescued by treatment with a GABA agonist, Zolpidem. These results suggest a loss of GABAergic neurons in P301L mice. Next we examined PPI and susceptibility to epileptic seizures in P301L and control mice. We found an altered PPI response and differences in epileptic seizures grades. Finally, we stained GABAergic interneurons in the hippocampus using two markers that identify GABAergic cell types showing a decrease in GABAergic neurons in the hippocampal CA1 region. Conclusions: Our results suggest that GABAergic interneurons are more vulnerable to molecular lesions caused by pathological Tau, which may result in the selective loss of hippocampal GABAergic interneurons. The molecular mechanisms involved in this specific GABAergic loss remains to be resolved, but may help to explain the pathophysiological symptoms of diseases like FTD, which involve altered Tau function

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

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

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

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.

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: 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