Faculty Bibliography

Alzheimer's disease (AD) is the most common form of dementia worldwide. It is characterized by an imbalance between the production and clearance of amyloid beta (Abeta) and tau proteins. In AD these normal proteins accumulate, leading to aggregation and a conformational change forming oligomeric and fibrillary species with a high beta-sheet content. Active and passive immunotherapeutic approaches result in dramatic reduction of Abeta pathology in AD animal models. However, there is much more limited evidence in human studies of significant clinical benefits from these strategies and it is becoming apparent that they may only be effective very early in AD. Vaccination targeting only tau pathology has shown benefits in some mouse studies but human studies are limited. Greater therapeutic efficacy for the next generation of vaccine approaches will likely benefit from specifically targeting the most toxic species of Abeta and tau, ideally simultaneously.

BACKGROUND: Peripheral surgical trauma may incite neuroinflammation that leads to neuronal dysfunction associated with both depression and cognitive deficits. In a previous study, we found that adult mice developed neuroinflammation and short-term working memory dysfunction in a delayed, transient manner after splenectomy that was ameliorated by the cyclooxygenase-2 inhibitor meloxicam. We tested the hypothesis that splenectomy in mice would also cause anhedonia, the diminished response to pleasure or rewarding stimuli that is a hallmark of depression, and that treatment with meloxicam would be ameliorative. METHODS: After Institutional Animal Care and Use Committee approval, Swiss-Webster mice underwent sucrose preference training before being randomized into groups on day 0, when they had either splenectomy and anesthesia or anesthesia alone. Within each group, half were randomized to receive intraperitoneal saline at 24 hours, while the other half received intraperitoneal meloxicam at 24 hours. Sucrose preference ratios were determined on days 1, 5, 9, and 14. Additional mice were randomized into groups for brain histochemistry. Specimens were stained for glial fibrillary acidic protein (GFAP), a marker of astrocytes, and CD45, a protein tyrosine phosphatase that identifies microglial activation. RESULTS: On day 5, mice receiving splenectomy and saline demonstrated diminished sucrose preference, which was not seen in mice receiving splenectomy and meloxicam. Semiquantitative analysis of histological slides taken from splenectomized mice treated with meloxicam revealed reduced microglial-based neuroinflammation and reactive astrocytosis compared to mice receiving saline. CONCLUSION: Splenectomy in mice is associated with neuroinflammation and anhedonia, as evidenced by reactive microgliosis, astrocytosis, and behavioral changes. Postsurgical treatment with meloxicam attenuates both neuroinflammation and anhedonia. These findings suggest that cyclooxygenase-2-dependent mechanisms may play a role in the development of postoperative mood disorders, possibly via modulation of peripheral effects on neuroinflammation.

Aggregation of amyloid beta-peptide (Abeta) is implicated in the pathology of Alzheimer's disease (AD), with the soluble, Abeta oligomeric species thought to be the critical pathological species. Identification and characterization of intermediate species formed during the aggregation process is crucial to the understanding of the mechanisms by which oligomeric species mediate neuronal toxicity and following disease progression. Probing these species proved to be extremely challenging, as evident by the lack of reliable sensors, due to their heterogeneous and transient nature. We describe here an oligomer-specific fluorescent chemical probe, BoDipy-Oligomer (BD-Oligo), developed through the use of the diversity-oriented fluorescent library approach (DOFLA) and high-content, imaging-based screening. This probe enables dynamic oligomer monitoring during fibrillogenesis in vitro and shows in vivo Abeta oligomers staining possibility in the AD mice model.

The vast majority of human tissue specimens are formalin-fixed, paraffin embedded (FFPE) archival samples, making this type of tissue a potential gold mine for medical research. It is now accepted that proteomics can be done using FFPE tissue and can generate similar results as snap-frozen tissue. However, the current methodology requires a large amount of starting protein, limiting the questions that can be answered in these types of proteomics studies and making cell-type specific proteomics studies difficult. Cell-type specific proteomics has the potential to greatly enhance understanding of cell functioning in both normal and disease states. Therefore, here we describe a new method that allows localized proteomics on individual cell populations isolated from FFPE tissue sections using laser capture microdissection. To demonstrate this technique we microdissected neurons from archived tissue blocks of the temporal cortex from patients with Alzheimer's disease. Using this method we identified over 400 proteins in microdissected neurons; on average 78% that were neuronal and 50% that were associated with Alzheimer's disease. Therefore, this technique is able to provide accurate and meaningful data and has great potential for any future study that wishes to perform localized proteomics using very small amounts of archived FFPE tissue.

INTRODUCTION: Autism is diagnosed in numerous genetic and genomic developmental disorders associated with an overlap in high-risk genes and loci that underlie intellectual disability (ID) and epilepsy. The aim of this stereological study of neuronal soma volume in 25 brain structures and their subdivisions in eight individuals 9 to 26 years of age who were diagnosed with chromosome 15q11.2-13.1 duplication syndrome [dup(15)], autism, ID and epilepsy; eight age-matched subjects diagnosed with autism of unknown etiology (idiopathic autism) and seven control individuals was to establish whether defects of neuronal soma growth are a common denominator of developmental pathology in idiopathic and syndromic autism and how genetic modifications alter the trajectory of neuronal soma growth in dup(15) autism. RESULTS: Application of the Nucleator software to estimate neuronal size revealed significant neuronal soma volume deficits in 11 of 25 structures and their subregions (44 %) in subjects diagnosed with dup(15) autism, including consistent neuronal soma volume deficits in the limbic system (sectors CA2, 3 and 4 in Ammon's horn, the second and third layers of the entorhinal cortex and in the amygdala), as well as in the thalamus, nucleus accumbens, external globus pallidus, and Ch3 nucleus in the magnocellular basal complex, and in the inferior olive in the brainstem. The second feature distinguishing dup(15) autism was persistent neuronal soma deficits in adolescents and young adults, whereas in idiopathic autism, neuronal volume deficit is most prominent in 4- to 8-year-old children but affects only a few brain regions in older subjects. CONCLUSIONS: This study demonstrates that alterations in the trajectory of neuronal growth throughout the lifespan are a core pathological features of idiopathic and syndromic autism. However, dup(15) causes persistent neuronal volume deficits in adolescence and adulthood, with prominent neuronal growth deficits in all major compartments of the limbic system. The more severe neuronal nuclear and cytoplasic volume deficits in syndromic autism found in this study and the more severe focal developmental defects in the limbic system in dup(15) previously reported in this cohort may contribute to the high prevalence of early onset intractable epilepsy and sudden unexpected death in epilepsy.

Accumulation of toxic protein aggregates-amyloid-beta (Abeta) plaques and hyperphosphorylated tau tangles-is the pathological hallmark of Alzheimer disease (AD). Abeta accumulation has been hypothesized to result from an imbalance between Abeta production and clearance; indeed, Abeta clearance seems to be impaired in both early and late forms of AD. To develop efficient strategies to slow down or halt AD, it is critical to understand how Abeta is cleared from the brain. Extracellular Abeta deposits can be removed from the brain by various clearance systems, most importantly, transport across the blood-brain barrier. Findings from the past few years suggest that astroglial-mediated interstitial fluid (ISF) bulk flow, known as the glymphatic system, might contribute to a larger portion of extracellular Abeta (eAbeta) clearance than previously thought. The meningeal lymphatic vessels, discovered in 2015, might provide another clearance route. Because these clearance systems act together to drive eAbeta from the brain, any alteration to their function could contribute to AD. An understanding of Abeta clearance might provide strategies to reduce excess Abeta deposits and delay, or even prevent, disease onset. In this Review, we describe the clearance systems of the brain as they relate to proteins implicated in AD pathology, with the main focus on Abeta.

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: It has been increasingly recognized that the pathogenesis of many neurodegenerative diseases is related to the accumulation of diverse proteins in aggregated/oligomeric forms. The pathological conformers can spread to different areas of the brain via a "prion-like" conversion mechanism mediated by the mobile b-sheet oligomeric structure of each particular peptide or protein. Previously we have characterized conformational monoclonal antibodies that react to both oligomers of Abeta and tau in AD, as well as to prion disease proteins. We have nowdetermined their binding specificity and capacity to be extended to synthetic oligomers of alpha-synuclein and to pathological intracellular structures present in Lewy body containing neurons of Parkinson's disease (PD) subjects. Methods: Recombinant alpha-synuclein was produced and characterized in monomeric, oligomeric and fibrillar forms by electron microscopy and circular dichroism. Histological specimens of formalin fixed brains from human AD and PD confirmed cases were used for reaction with three anti-conformational mAbs IgM previously described. The mAbs that reacted to oligomeric Abeta and tau and showed high affinity, specific binding by surface plasmon resonance, and/or were shown to reverse AD pathology after infusion in old 3xTg AD animal models were used for immunohistochemical detection on human PD brain specimens and detection of different alpha-synuclein conformers. Results: By SDS-PAGE the mAbs IgM showed specificity for oligomeric forms of polymerized alpha-synuclein but not to the monomeric forms. The mAbs showed specific intraneuronal reactivity around the Lewy bodies in human brains from confirmed cases of PD. Conclusions: Conformational monoclonal antibodies that are well characterized to react against pathological conformers in AD human brains and that can produce amelioration of existing AD pathology in AD animal models can also recognize oligomeric forms of alpha-synuclein and intraneuronal structures associated with Lewy bodies. Monoclonal antibodies that are specific for pathology associated conformations are good candidates to be used as immunotherapeutical agents alone or in combination with other approaches in many neurodegenerative diseases including Parkinson's disease