Faculty Bibliography

Epilepsy is a common neurological disorder affecting over 70 million people worldwide, with a high rate of pharmaco-resistance, diverse comorbidities including progressive cognitive and behavioural disorders, and increased mortality from direct (e.g. sudden unexpected death in epilepsy, accidents, drowning) or indirect effects of seizures and therapies. Extensive research with animal models and human studies provides limited insights into the mechanisms underlying seizures and epileptogenesis, and these have not translated into significant reductions in pharmaco-resistance, morbidities or mortality. To help define changes in molecular signalling networks associated with seizures in epilepsy with a broad range of aetiologies, we examined the proteome of brain samples from epilepsy and control cases. Label-free quantitative mass spectrometry was performed on the hippocampal cornu ammonis 1-3 region (CA1-3), frontal cortex and dentate gyrus microdissected from epilepsy and control cases (n = 14/group). Epilepsy cases had significant differences in the expression of 777 proteins in the hippocampal CA1 - 3 region, 296 proteins in the frontal cortex and 49 proteins in the dentate gyrus in comparison to control cases. Network analysis showed that proteins involved in protein synthesis, mitochondrial function, G-protein signalling and synaptic plasticity were particularly altered in epilepsy. While protein differences were most pronounced in the hippocampus, similar changes were observed in other brain regions indicating broad proteomic abnormalities in epilepsy. Among the most significantly altered proteins, G-protein subunit beta 1 (GNB1) was one of the most significantly decreased proteins in epilepsy in all regions studied, highlighting the importance of G-protein subunit signalling and G-protein-coupled receptors in epilepsy. Our results provide insights into common molecular mechanisms underlying epilepsy across various aetiologies, which may allow for novel targeted therapeutic strategies.

Accumulation of phosphorylated tau is a key pathological feature of Alzheimer's disease. Phosphorylated tau accumulation causes synaptic impairment, neuronal dysfunction and formation of neurofibrillary tangles. The pathological actions of phosphorylated tau are mediated by surrounding neuronal proteins; however, a comprehensive understanding of the proteins that phosphorylated tau interacts with in Alzheimer's disease is surprisingly limited. Therefore, the aim of this study was to determine the phosphorylated tau interactome. To this end, we used two complementary proteomics approaches: (i) quantitative proteomics was performed on neurofibrillary tangles microdissected from patients with advanced Alzheimer's disease; and (ii) affinity purification-mass spectrometry was used to identify which of these proteins specifically bound to phosphorylated tau. We identified 542 proteins in neurofibrillary tangles. This included the abundant detection of many proteins known to be present in neurofibrillary tangles such as tau, ubiquitin, neurofilament proteins and apolipoprotein E. Affinity purification-mass spectrometry confirmed that 75 proteins present in neurofibrillary tangles interacted with PHF1-immunoreactive phosphorylated tau. Twenty-nine of these proteins have been previously associated with phosphorylated tau, therefore validating our proteomic approach. More importantly, 34 proteins had previously been associated with total tau, but not yet linked directly to phosphorylated tau (e.g. synaptic protein VAMP2, vacuolar-ATPase subunit ATP6V0D1); therefore, we provide new evidence that they directly interact with phosphorylated tau in Alzheimer's disease. In addition, we also identified 12 novel proteins, not previously known to be physiologically or pathologically associated with tau (e.g. RNA binding protein HNRNPA1). Network analysis showed that the phosphorylated tau interactome was enriched in proteins involved in the protein ubiquitination pathway and phagosome maturation. Importantly, we were able to pinpoint specific proteins that phosphorylated tau interacts with in these pathways for the first time, therefore providing novel potential pathogenic mechanisms that can be explored in future studies. Combined, our results reveal new potential drug targets for the treatment of tauopathies and provide insight into how phosphorylated tau mediates its toxicity in Alzheimer's disease.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder that is growing in prevalence globally. It is the only major cause of death without any effective pharmacological means to treat or slow progression. Inheritance of the ε4 allele of the Apolipoprotein (APO) E gene is the strongest genetic risk factor for late-onset AD. The interaction between APOE and amyloid β (Aβ) plays a key role in AD pathogenesis. The APOE-Aβ interaction regulates Aβ aggregation and clearance and therefore directly influences the development of amyloid plaques, congophilic amyloid angiopathy and subsequent tau related pathology. Relatively few AD therapeutic approaches have directly targeted the APOE-Aβ interaction thus far. Here we review the critical role of APOE in the pathogenesis of AD and some of the most promising therapeutic approaches that focus on the APOE-Aβ interaction.

We recently identified Secernin-1 (SCRN1) as a novel amyloid plaque associated protein using localized proteomics. Immunohistochemistry studies confirmed that SCRN1 was present in plaque-associated dystrophic neurites and also revealed distinct and abundant co-localization with neurofibrillary tangles (NFTs). Little is known about the physiological function of SCRN1 and its role in Alzheimer's disease (AD) and other neurodegenerative diseases has not been studied. Therefore, we performed a comprehensive study of SCRN1 distribution in neurodegenerative diseases. Immunohistochemistry was used to map SCRN1 accumulation throughout the progression of AD in a cohort of 58 patients with a range of NFT pathology (Abundant NFT, n = 21; Moderate NFT, n = 22; Low/No NFT, n = 15), who were clinically diagnosed as having AD, mild cognitive impairment or normal cognition. SCRN1 accumulation was also examined in two cases with both Frontotemporal Lobar Degeneration (FTLD)-Tau and AD-related neuropathology, cases of Down Syndrome (DS) with AD (n = 5), one case of hereditary cerebral hemorrhage with amyloidosis - Dutch type (HCHWA-D) and other non-AD tauopathies including: primary age-related tauopathy (PART, [n = 5]), Corticobasal Degeneration (CBD, [n = 5]), Progressive Supranuclear Palsy (PSP, [n = 5]) and Pick's disease (PiD, [n = 4]). Immunohistochemistry showed that SCRN1 was a neuronal protein that abundantly accumulated in NFTs and plaque-associated dystrophic neurites throughout the progression of AD. Quantification of SCRN1 immunohistochemistry confirmed that SCRN1 preferentially accumulated in NFTs in comparison to surrounding non-tangle containing neurons at both early and late stages of AD. Similar results were observed in DS with AD and PART. However, SCRN1 did not co-localize with phosphorylated tau inclusions in CBD, PSP or PiD. Co-immunoprecipitation revealed that SCRN1 interacted with phosphorylated tau in human AD brain tissue. Together, these results suggest that SCRN1 is uniquely associated with tau pathology in AD, DS and PART. As such, SCRN1 has potential as a novel therapeutic target and could serve as a useful biomarker to distinguish AD from other tauopathies.

There are growing genetic, transcriptomic and proteomic data pointing to the complexity of Alzheimer's disease (AD) pathogenesis. Unbiased "omics" approaches are essential for the future development of effective AD research, which will need to be combined and personalized, given that multiple distinct pathways can drive AD pathology. It is essential to gain a better understanding of the AD pathogenesis subtype variety and to develop several distinct therapeutic approaches tailored to address this diversity, as well as the common presence of mixed pathologies. These nonmutually exclusive therapeutic approaches include the targeting of multiple toxic oligomeric species concurrently, targeting the apolipoprotein E/amyloid β interaction and the modulation of innate immunity, as well as more "out of the box" ideas such as targeting infectious agents that may play a role in AD.

INTRODUCTION/BACKGROUND:Alzheimer's disease (AD) is a devastating neurodegenerative disease characterized by protein aggregates of amyloid β (Aβ) and tau. These proteins have normal physiological functions, but in AD they undergo a conformational change and aggregate as toxic oligomeric and fibrillar species with a high β-sheet content. Areas covered: Active and passive immunotherapeutic approaches are among the most attractive methods for targeting misfolded Aβ and tau. Promising preclinical testing of various immunotherapeutic approaches have yet to translate to cognitive benefits in human clinical trials. Knowledge gained from these past failures has led to the development of second generation Aβ active immunotherapies, anti-Aβ monoclonal antibodies targeting a wide array of Aβ conformations, and to a number of immunotherapies targeting pathological tau. This review covers the more recent advances in vaccine development for AD from 2016 to present. Expert commentary: Due to the complex pathophysiology of AD, greatest clinical efficacy will most likely be achieved by concurrently targeting the most toxic forms of both Aβ and tau.

BACKGROUND:Oligomeric forms of amyloid-β (Aβ) and tau are increasing being recognized as key toxins in the pathogenesis of Alzheimer's disease (AD). METHODS:We developed a novel monoclonal antibody (mAb), GW-23B7, that recognizes β-sheet secondary structure on pathological oligomers of neurodegenerative diseases. RESULTS:The pentameric immunoglobulin M kappa chain (IgMκp) we developed specifically distinguishes intra- and extracellular pathology in human AD brains. Purified GW-23B7 showed a dissociation constant in the nanomolar range for oligomeric Aβ and did not bind monomeric Aβ. In enzyme-linked immunosorbent assays, it recognized oligomeric forms of both Aβ and hyperphosphorylated tau. Aged triple-transgenic AD mice with both Aβ and tau pathology infused intraperitoneally for 2 months showed IgMκp in the soluble brain homogenate, peaking at 24 h postinoculation. Treated mice exhibited significant cognitive rescue on radial arm maze testing compared with vehicle control-infused mice. Immunohistochemically, treatment resulted in a significant decrease of extracellular pathology. Biochemically, treatment resulted in significant reductions of oligomeric forms of Aβ and tau. CONCLUSIONS:These results suggest that GW-23B7, an anti-β-sheet conformational mAb humanized for clinical trials, may be an effective therapeutic agent for human AD.

Here, we describe a new method that allows localized proteomics of amyloid plaques and neurofibrillary tangles (NFTs), which are the two pathological hallmarks of Alzheimer's disease (AD). Amyloid plaques and NFTs are visualized using immunohistochemistry and microdissected from archived, formalin-fixed paraffin-embedded (FFPE) human tissue samples using laser-capture microdissection. The majority of human tissue specimens are FFPE; hence the use of this type of tissue is a particular advantage of this technique. Microdissected tissue samples are solubilized with formic acid and deparaffinized, reduced, alkylated, proteolytically digested, and desalted. The resulting protein content of plaques and NFTs is determined using label-free quantitative LC-MS. This results in the unbiased and simultaneous quantification of ~900 proteins in plaques and ~500 proteins in NFTs. This approach permits downstream pathway and network analysis, hence providing a comprehensive overview of pathological protein accumulation found in neuropathological features in AD.