Faculty Bibliography

Background: Immunomodulation is a promising therapeutic approach for Alzheimer's disease (AD); however, major drawbacks are cerebral microhemorrhages associated with increased cerebral amyloid angiopathy (CAA) and excessive inflammation. Our initial findings indicate that stimulation of TLR9 signaling with CpG oligodeoxynucleotide (ODN) is effective against CAA without inducing toxicity in AD mouse models. To further assess potential human use of CpG ODN we advanced our studies using a well-established non-human primate model of sporadic CAA, squirrel monkey (Saimiri Boliviensis). Methods: Safety and efficacy assessment studies were first performed in young squirrel monkeys (SQM). Elderly female monkeys were subcutaneously injected either with the most effective and non-toxic dosages of the class B CpG ODN containing a primate specific immunostimulatory sequence or saline. Both age groups were subjected to behavioral testing. Plasma taken during the course of treatment was analyzed to identify immune responses and AD biomarkers. Fluidigm RT-PCR was used to evaluate mRNA levels of cytokines in SQM PBMCs. Results: CpG ODN elevated the levels of various Th1/Th2 cytokines in plasma from old monkeys. Upregulation of cytokines in CpG ODN group was further confirmed by RTPCR. Pre-treatment behavioral assessment in our aged monkeys demonstrated cognitive deficits on the Inhibitory Control of Behavior and Delayed Response tests. Age effect on cognitive abilities was observed as the young group performed with overall lower session error rates compared to old animals. Post-treatment behavioral testing in our aged monkeys is ongoing. Here we report the first pyroglutamate (pE3) immunohistochemistry of aged Saimiri Boliviensis. In addition to 6E10/4G8 As-positive plaques, pyroglutamate As-positive deposits in the form of CAA and parenchymal plaques were detected. Our preliminary biomarker analyses revealed a noticeable increase in As40, As42 and AspE3 plasma levels in CpG ODN-treated group. Further longitudinal assessment of potential AD biomarkers is currently in progress. Conclusions: The presented studies represent the first trial of specifically targeting CAA in non-human primates. We hope that our research will validate this novel approach of immunomodulation as a safer method to successfully ameliorate AD related pathologies and provide critical data for potential clinical use of CpG ODN in AD patients

Background: We have previously demonstrated that immuno-intervention in AD animal models can lead to prevention of some pathology through innate immune system stimulation via TLR9 induced by CpG ODN (Scholtzova et al 2014) or the modulation of the adaptive immune system through active vaccination with the beta-sheet oligomeric form of the polymerized Bri peptide (Goni et al 2014). A challenge to therapeutic immune stimulation of old AD Tg animals, with preexisting extensive pathology, is senescence of the immune system. We have now vaccinated old 3xTg AD animals with both Abeta and tau pathology, with the pBri as a conformational antigen and CpGODNas an immune stimulator. Methods: Two groups of at least 15 months old 3xTg AD animals were inoculated four times over a period of two months with either vehicle or oligomeric pBri in Alum as previously described. Another group was inoculated from 12 to 17 month old with five doses of pBri or CpG ODN in alternate weeks as reported. Behavioral and locomotor tests were performed after the 17 month of age. The animals were then euthanized, followed by histological and biochemical analyses. Results: We show that old animals still could mount a conformational immune response that results in diminished pathology, as well as rescue of cognitive function. Both groups vaccinated with pBri and pBri-CpG ODN showed behavioral rescue when compared to age matched controls. Biochemistry and immuno-histology showed improvements of some pathological features; importantly including diminished oligomeric Abeta and tau. Conclusions: The active immunomodulation using polymerized beta-sheet oligomeric pBri can elicit a conformational antibody response even in old animals. These antibodies directed to beta-sheet conformation can retard the progression and reverse some preexisting pathology. The use of CpG ODN can help to boost the innate immune system, in senescent animals, to help establish the subsequent adaptive conformational response

Alzheimer's disease (AD) is the leading cause of dementia worldwide. Late-onset AD (LOAD), is the most common form of Alzheimer's disease, representing about >95% of cases and early-onset AD represents <5% of cases. Several risk factors have been discovered that are associated with AD, with advancing age being the most prominent. Other environmental risk factors include diabetes mellitus, level of physical activity, educational status, hypertension and head injury. The most well known genetic risk factor for LOAD is inheritance of the apolipoprotein (apo) E4 allele. Recently, rare variants of TREM2 have been reported as a significant risk factor for LOAD, comparable to inheritance of apoE4. In this review we will focus on the role(s) of TREM2 in AD as well as in other neurodegenerative disorders.

Alzheimer inverted question marks disease (AD) is the most common cause of dementia, and currently, there is no effective treatment. The major neuropathological lesions in AD are accumulation of amyloid beta (Abeta) as amyloid plaques and congophilic amyloid angiopathy, as well as aggregated tau in the form of neurofibrillary tangles (NFTs). In addition, inflammation and microglia/macrophage function play an important role in AD pathogenesis. We have hypothesized that stimulation of the innate immune system via Toll-like receptor 9 (TLR9) agonists, such as type B CpG oligodeoxynucleotides (ODNs), might be an effective way to ameliorate AD related pathology. We have previously shown in the Tg2576 AD model that CpG ODN can reduce amyloid deposition and prevent cognitive deficits. In the present study, we used the 3xTg-AD mice with both Ass and tau related pathology. The mice were divided into 2 groups treated from 7 to 20 months of age, prior to onset of pathology and from 11 to 18 months of age, when pathology is already present. We demonstrated that immunomodulatory treatment with CpG ODN reduces both Ass and tau pathologies, as well as levels of toxic oligomers, in the absence of any apparent inflammatory toxicity, in both animal groups. This pathology reduction is associated with a cognitive rescue in the 3xTg-AD mice. Our data indicates that modulation of microglial function via TLR9 stimulation is effective at ameliorating all the cardinal AD related pathologies in an AD mouse model mice suggesting such an approach would have a greater chance of achieving clinical efficacy.

Inheritance of the apolipoprotein E4 (apoE4) genotype has been identified as the major genetic risk factor for late-onset Alzheimer's disease (AD). Studies have shown that the binding between apoE and amyloid-beta (Abeta) peptides occurs at residues 244-272 of apoE and residues 12-28 of Abeta. ApoE4 has been implicated in promoting Abeta deposition and impairing clearance of Abeta. We hypothesized that blocking the apoE/Abeta interaction would serve as an effective new approach to AD therapy. We have previously shown that treatment with Abeta12-28P can reduce amyloid plaques in APP/PS1 transgenic (Tg) mice and vascular amyloid in TgSwDI mice with congophilic amyloid angiopathy (CAA). In the present study, we investigated whether the Abeta12-28P elicits a therapeutic effect on tau-related pathology in addition to amyloid pathology using old triple transgenic Alzheimer's disease mice (3xTg, with PS1M146V , APPS we and tauP30 IL transgenes) with established pathology from the ages of 21 to 26 months. We show that treatment with Abeta12-28P substantially reduces tau pathology both immunohistochemically and biochemically, as well as reducing the amyloid burden and suppressing the activation of astrocytes and microglia. These affects correlate with a behavioral amelioration in the treated Tg mice

Background: Immunomodulation has shown great promise as an Alzheimer's disease (AD) therapy but major limitations must be overcome, such as the need for effectively reducing cerebral amyloid angiopathy (CAA), without associated hemorrhages. CAA is a common feature in AD and cognitively normal elderly individuals. Our initial findings indicate that stimulation of innate immunity with CpG ODN appears to be an effective means of reducing vascular amyloid without inducing toxicity in AD mouse models. We are currently testing our approach in a well characterized nonhuman primate model of sporadic CAA, squirrel monkey (Saimiri Boliviensis), which share numerous biological similarities with humans. Methods: Varying doses of the class B CpG ODN preparation containing the primate specific immunostimulatory sequence were administered in young monkeys by a subcutaneous route (s.c.). The most effective and non-toxic dosage described in young animals was selected for our long term studies in the older animals (with expected CAA deposits). During the treatment, primates were closely monitored for signs of toxicity. The peripheral cytokine responses were determined in PBMC supernatants and plasma from control and treated monkeys at selected time points. In addition, the performances on cognitive tests are being compared between our aged and young squirrel monkeys. Results: We have shown that a TLR9 agonist prevented shortterm memory deficits in Tg-SwDI mice, a model with extensive CAA. CpG ODN treatment led to a reduction of CAA in the absence of microhemorrhages and increased inflammation. Transgenic models are ideal for initial screening of a potential therapy but prior to clinical trials it is imperative to perform studies in non-human primates, which are a more biologically proximate model to humans. Short duration safety and efficacy assessment studies were first performed in young monkeys. Characterization of immune responses and evaluation of cognitive function and working memory in our monkeys is curr!

Background: Alzheimer's Disease (AD) is the most common of the conformational neurodegenerative disorders (NDD), while prion diseases are the most transmissible NDD. No highly effective treatment is currently available for any NDD. Previously, we have shown that mutated Ab peptides soluble and devoid of T-cell epitopes, produced a response which correlates with amelioration of AD pathology in animal models. Our lab has demonstrated that active immunization with antigens mimicking abnormal conformations such as polymerized ABri (pABri), could elicit antibody responses to oligomerized Ab and conformational aberrant hyperphosphorylated tau (ptau).We have now tested a combined vaccination using pBri and polymerized mutated Ab (pAb) in 3xTg mice and human PrP-Tg animals. Methods: We produced pBri and pAb1-30K18K19, which by electron microscopy formed oligomer-like structures. The pBri alone was used as an immunogen in HuPrPTg animals and in conjunction with pAb1-30KK in AD 3xTg mice using alum as an adjuvant. At the age of 15-16 months the AD mice were subjected to behavioral testing followed by histological and biochemical analysis. The HuPrPTg were assessed for the presence of antibodies against PrPRes. Results: 3xTg mice immunized animals showed no difference in locomotor activity compared to controls but a significant behavioral rescue by radial arm maze testing. The animals produced significant titers of IgM and IgG against Ab42 oligomers. Histological analysis showed a significant reduction of both amyloid and tau pathology. Biochemically, Ab42 and soluble ptau were significantly reduced. In HuPrPTg animals a systemic IgM and IgG response against PrPRes was observed, similar to the systemic response obtained in our prior successful mucosal PrP vaccinations. Conclusions: pBri conformational vaccination can be used to elicit antibodies that recognize multiple pathological proteins, including PrPRes, oligomer Ab and ptau. In 3x Tg mice with both Ab and tau pathology which were vaccinated with the combined pABri+pAb1-30KK, each of these pathologies were reduced histologically and biochemically, in association with a behavioral rescue. Such a combined approach targeting both pathological Ab and tau is more likely to be efficacious in AD patients

Background: Alzheimer's disease (AD) is the most common cause of dementia and has a major societal and economic impact. Immunomodulation has shown great promise as an AD therapy, even though the initial clinical trial was associated with severe adverse effects in a minority of patients. Our research group postulated stimulation of the innate immune system, via the Toll-like receptor 9 (TLR9), as a possible alternative method for ameliorating AD pathology, without associated toxicity. A significant concern with immunotherapy is clearance of vascular amyloid and associated microhemorrhages. This is an important issue, since cerebral amyloid angiopathy (CAA) is a common feature in AD and cognitively normal elderly individuals. We tested the efficacy of TLR9 signaling stimulation for reducing parenchymal and vascular amyloid, as well as tau related pathology. Methods: We utilized TLR9 agonist type B CpG oligonucleotides (ODNs) to stimulate innate immunity in 3xTg-AD mice, which develop both plaque and tangle pathology, and in Tg-SwDI mice with extensive CAA. Animals were divided into 2 study groups treated prior to or after the onset of AD pathology. Results: After treatment the mice were behaviorally tested. No statistical differences were observed between the groups in any of the locomotor parameters measured. CpG ODN treatment improved working memory in 3xTg-AD mice as indicated by radial arm maze testing. TLR9 stimulation was also effective at improving short-term memory in Tg-SwDI mice as evidenced by novel-object recognition testing. In 3xTg-AD mice TLR9 signaling reduced both amyloid deposits and hyperphosphorylated tau pathology. Semiquantitative analysis of hippocampal CA1 neurons revealed reduction in AT8 and PHF1 immunoreactivity in CpG ODN-treated 3xTg-AD mice. The reduction of plaque and tangle pathology was paralleled by an overall reduction in the numbers of activated microglia. There were no group differences in the levels of CNS astrocytosis; hence there was no evidence of encephalitis in the brains of treated mice. Further histological, biochemical analyses and characterization of immune responses are ongoing. Conclusions: Overall, stimulation of the TLR9 and thus innate immunity with CpG ODN (currently used in clinical trials for a variety of other diseases) represents a novel immunotherapeutic approach for AD

Background: Manipulation of the immune system is becoming increasingly important for the potential treatment of Alzheimer's disease (AD). However, problems in the initial human clinical trials have included toxicity from excessive cell mediated immunity, hemorrhages and limited cognitive benefits. Our research group postulated stimulation of innate immunity, via the Toll-like receptors (TLRs), as an alternative approach to overcome these side effects. Our prior work in prion disease, suggested TLR9 to be an attractive candidate for AD prevention and therapy. We sought to determine the effectiveness of TLR9 stimulation on both amyloid and tau related pathology. Various CpG DNA drugs that are TLR9 agonist are safe in humans and rodents. Methods: We utilized type B CpG oligodeoxynucleotides (ODNs) to stimulate innate immunity in 3xTg-AD mice, which develop both plaque and tangle pathology. The mice were divided into 2 study groups treated from 11 to 17 months and from 7 to 20 months of age. After the treatment, the mice were subjected to behavioral testing. In addition, we tested the effect of the TLR9 agonist CpG ODN on microglial As42 uptake and degradation in culture. Results: The transgenic mice were less active than their mu/d-littermates, but no significant differences between Tg groups were found in any of the locomotor parameters. CpG ODN treatment led to working memory improvement in both study groups of 3xTg- AD mice as indicated by radial arm maze testing. As immunostaining showed a 58% reduction in hippocampal amyloid burden (p=0.009) in the group treated from 11 to 17 months and 48% reduction (p=0.003) in the group treated from 7 to 20 months compared to vehicle-treated Tg animals. Biochemical analyses and evaluation of the impact of our approach on tau related pathology are ongoing. In vitro results indicate that TLR9 activation enhances microglial uptake and degradation of aggregated As42. Whether TLR9 agonist can boost peripheral macrophages to clear As is being further explored in our studies using RAW 264.7 and differentiated human THP-1 macrophages cells. Conclusions: Immunomodulation targeting TLR9 signaling represents a novel immunotherapeutic approach for AD. The current results provide essential information prior to any clinical use of CpG ODN

Many neurodegenerative diseases are characterized by the conformational change of normal self-proteins into amyloidogenic, pathological conformers, which share structural properties such as high beta-sheet content and resistance to degradation. The most common is Alzheimer's disease (AD) where the normal soluble amyloid beta (sAbeta) peptide is converted into highly toxic oligomeric Abeta and fibrillar Abeta that deposits as neuritic plaques and congophilic angiopathy. Currently, there is no highly effective treatment for AD, but immunotherapy is emerging as a potential disease modifying intervention. A major problem with most active and passive immunization approaches for AD is that both the normal sAbeta and pathogenic forms are equally targeted with the potential of autoimmune inflammation. In order to avoid this pitfall, we have developed a novel immunomodulatory method that specifically targets the pathological conformations, by immunizing with polymerized British amyloidosis (pABri) related peptide which has no sequence homology to Abeta or other human proteins. We show that the pABri peptide through conformational mimicry induces a humoral immune response not only to the toxic Abeta in APP/PS1 AD transgenic mice but also to paired helical filaments as shown on AD human tissue samples. Treated APP/PS1 mice had a cognitive benefit compared to controls (p<0.0001), associated with a reduction in the amyloid burden (p = 0.0001) and Abeta40/42 levels, as well as reduced Abeta oligomer levels. This type of immunomodulation has the potential to be a universal beta-sheet disrupter, which could be useful for the prevention or treatment of a wide range of neurodegenerative diseases