Faculty Bibliography

INTRODUCTION/BACKGROUND:In autism spectrum disorder, lack of coherence and of complex information processing, and narrowly focused interests and repetitive behaviors are considered a sign of long-range underconnectivity and short-range overconnectivity. The goal of this morphometric study of five anatomically and functionally different segments of the corpus callosum (CC) was to establish patterns of differences between long-range interhemispheric connections in nine neurotypical and nine autistic subjects. RESULTS:Electron microscopy revealed a significant reduction in average axon diameter and axon cross-sectional area in autistic subjects, and reduction in CC segment-specific diversification of connections of functionally different cortical regions. The study shows an increase in the percentage of small diameter axons (< 0.651 μm) and a decrease in the percentage of axons with large diameter (> 1.051 μm). The total number of small-diameter axons is reduced in segment I and III by 43% on average. The number of medium- and large-diameter axons is reduced in all five CC segments by an average of 49 and 72%, respectively. CONCLUSIONS:The detected pattern of pathology suggests a failure of mechanisms controlling guidance of axons during development leading to axonal deficit, and failure of mechanisms controlling axon structure. A reduction in axon diameter may affect the velocity and volume of signal transmission, and distort functional specialization of CC segments. Significant deficits in axon number and reduction in axon size in all five CC segments appear to be substantial components of brain connectome integrity distortion which may contribute to the autism phenotype.

Structural and functional elements of biological systems are highly conserved across vertebrates. Many neurological and psychiatric conditions affect both humans and animals. A cross-species approach to the study of brain and behaviour can advance our understanding of human disorders via the identification of unrecognized natural models of spontaneous disorders, thus revealing novel factors that increase vulnerability or resilience, and via the assessment of potential therapies. Moreover, diagnostic and therapeutic advances in human neurology and psychiatry can often be adapted for veterinary patients. However, clinical and research collaborations between physicians and veterinarians remain limited, leaving this wealth of comparative information largely untapped. Here, we review pain, cognitive decline syndromes, epilepsy, anxiety and compulsions, autoimmune and infectious encephalitides and mismatch disorders across a range of animal species, looking for novel insights with translational potential. This comparative perspective can help generate novel hypotheses, expand and improve clinical trials and identify natural animal models of disease resistance and vulnerability.

Fragile X syndrome (FXS), caused by lack of fragile X mental retardation protein (FMRP), is associated with a high prevalence of autism. The deficit of FMRP reported in idiopathic autism suggests a mechanistic overlap between FXS and autism. The overall goal of this study is to detect neuropathological commonalities of FMRP deficits in the brains of people with idiopathic autism and with syndromic autism caused by dup15q11.2-q13 (dup15). This study tests the hypothesis based on our preliminary data that both idiopathic and syndromic autism are associated with brain region-specific deficits of neuronal FMRP and structural changes of the affected neurons. This immunocytochemical study revealed neuronal FMRP deficits and shrinkage of deficient neurons in the cerebral cortex, subcortical structures, and cerebellum in subjects with idiopathic and dup(15)/autism. Neuronal FMRP deficit coexists with surprising infiltration of the brains of autistic children and adults with FMRP-positive astrocytes known to be typical only for the fetal and short postnatal periods. In the examined autistic subjects, these astrocytes selectively infiltrate the border between white and gray matter in the cerebral and cerebellar cortex, the molecular layer of the cortex, part of the amygdala and thalamus, central cerebellar white matter, and dentate nucleus. Astrocyte pathology results in an additional local loss of FMRP in neurons and their shrinkage. Neuronal deficit of FMRP and shrinkage of affected neurons in structures free of FMRP-positive astrocytes and regions infiltrated with FMRP-expressing astrocytes appear to reflect mechanistic, neuropathological, and functional commonalities of FMRP abnormalities in FXS and autism spectrum disorder.

Background: We have previously demonstrated the feasibility of eliciting a unique antibody response independent of the primary and tertiary structure of proteins/peptides and specifically recognizing generic secondary beta-sheet structure dominant on misfolded proteins of oligomeric form; a hallmark of toxic prion-like "infectious" pathology in neurodegenerative diseases and specifically in the dual Abeta and tau Alzheimer's disease (AD) (Goni et al 2013 J. Neuroinflammation). As a putative antigen we have used a small peptide with a non-self primary structure, only beta-sheet secondary structure and no tertiary structure do to its size. The highly polymerized peptide (pBri) preserved a stable 90% beta-sheet secondary structure without any other outstanding epitope that could be recognized by an immune system. Young animals of AD models APP/PS1, 3xTg and SwDI as well as wild type animals, all developed polyclonal antibodies IgM and IgG that recognized oligomers of Abeta and tau and significantly ameliorated pathology. CD-1 animals were used to produced monoclonal antibodies (mAbs) that proved unequivocally the recognition was not to a sequence dependent epitope but to a generic beta-sheet secondary structure of pathologic oligomers but not fibrils or native proteins (Goni et al 2017 Scientific reports). The mAbs were characterized as specific anti-beta-sheet secondary structure monoclonal antibodies (AbetaComAbs). Either the stable IgM or the engineered IgG AbetaComAbs could reverse pathology and produce cognitive rescue in 3xTg animals (Goni et al 2018, Alz Res Therapy; Herline et al 2018 Alz Res Therapy).
Objective(s): To demonstrate that small engineered peptides with similar beta-sheet dominant structure but different primary sequences would elicit similar anti-beta-sheet responses and ameliorate pathology on 3xTg animals. To demonstrate that old 3xTg animals with already flourished pathology would develop the same type of anti-beta-sheet polyclonal IgM and IgG response and compared to AbetaComAbs infused animals produced comparable cognitive rescue and reduced oligomeric pathology. Finally, to demonstrate the specific reaction to the secondary structure but not the primary/tertiary structure does not produce antibody dependent cerebral microhemorrhages in an old SwDI CAA model of AD.
Method(s): Groups of 8 to 12 male and female 16 months old 3xTg AD and SwDI animals were inoculated i.p. weekly for 9 weeks with either 100 mug of the stable IgMk GW-23B7 or the derived TWF9 IgG2ak AbetaComAbs in 100 muL of sterile saline or with 100 muL of vehicle alone. Other groups of young 3xTg were inoculated with several tailored made mutations of the original pBri; whereas groups of 17 months old 3xTg were inoculated with the highly polymerized original pBri. The pBri immunizations and the AbetaComAbs were repeated in aged 16 month old SwDI animals prone to develop cerebral amyloid angiopathy due to the nature of the model, exacerbated by eventual reaction to Abeta amyloid epitopes existing in vessels of the brain. All animals were subjected to radial arm maze, locomotor tests or Barnes maze after the treatment and before sacrifice. Histochemical and biochemical analysis were performed on brains from treated and control animals.
Result(s): No adverse reactions were demonstrated during any one of the different treatments. Old animals inoculated with the pBri produced a fare anti-beta-sheet response; however, lower than in young animals. There was a significant cognitive rescue compared to controls and a significant reduction of both Abeta and tau oligomers. The mutated peptides were not as efficient to elicit anti-beta-sheet polyclonal response but still produce a significant amelioration on cognitive decline close to the results obtained by the original pBri. All SwDI animals either pBri immunized or AbetaComAbs infused -stable IgMk or IgG2aK- did not show any signs of vascular complication. In all cases the main biochemical improvement was the significant decrease on the number of oligomers of both Abeta and tau as assessed by specific blots of solubilized brain extractions, ELISAs and MSD measurements. Immunohistochemically the extracellular Abeta in plaques was significantly decreased whereas the intracellular PHF-tau remained fairly the same.
Conclusion(s): The development of a specific antibody response -polyclonal or monoclonal- to strictly the secondary structure of a protein or peptide frozen in a stable beta-sheet state is successfully achieved by the immunization with a highly polymerized, beta-sheet only, immunogen with no other visible epitope. That specific response is completely independent of the primary structure of pathological conformers; thus, it interferes only with oligomeric pathologic conformers that show the dominant beta-sheet secondary structure. Either treatment can produce , in various degrees, amelioration of AD pathology or evident cognitive rescue depending on the time of start of treatment. The mechanism is likely related to reductions of the levels of soluble oligomeric forms of Abeta and Tau; the species most closely linked to cognitive deficits in AD patients and the prionlike propagation. These results are extremely encouraging for the further testing of potential combinations of immunizations and AbetaComAbs in clinical trials with disease modifying potential and minimal risk of autoimmune complications

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:Alzheimer's disease (AD) is characterized by physiologically endogenous proteins amyloid beta (Aβ) and tau undergoing a conformational change and accumulating as soluble oligomers and insoluble aggregates. Tau and Aβ soluble oligomers, which contain extensive β-sheet secondary structure, are thought to be the most toxic forms. The objective of this study was to determine the ability of TWF9, an anti-β-sheet conformation antibody (aβComAb), to selectively recognize pathological Aβ and phosphorylated tau in AD human tissue compared with cognitively normal age-matched controls and to improve the performance of old 3xTg-AD mice with advanced pathology in behavioral testing after acute treatment with TWF9. METHODS:In this study, we used immunohistochemistry, immunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) to characterize TWF9 specificity. We further assessed cognitive performance in old (18-22 months) 3xTg-AD mice using both a Barnes maze and novel object recognition after intraperitoneal administration of TWF9 (4 mg/kg) biweekly for 2 weeks before the start of behavioral testing. Injections continued for the duration of the behavioral testing, which lasted 2 weeks. RESULTS:Histological analysis of TWF9 in formalin-fixed paraffin-embedded human control and AD (ABC score: A3B3C3) brain tissue revealed preferential cytoplasmic immunoreactivity in neurons in the AD tissue compared with controls (p < 0.05). Furthermore, ELISA using oligomeric and monomeric Aβ showed a preferential affinity for oligomeric Aβ. Immunoprecipitation studies showed that TWF9 extracted both phosphorylated tau (p < 0.01) and Aβ (p < 0.01) from fresh frozen brain tissues. Results show that treated old 3xTg-AD mice have an enhanced novel object recognition memory (p < 0.01) and Barnes maze performance (p = 0.05) compared with control animals. Overall plaque burden, neurofibrillary tangles, microgliosis, and astrocytosis remained unchanged. Soluble phosphorylated tau was significantly reduced in TWF9-treated mice (p < 0.05), and there was a trend for a reduction in soluble Aβ levels in the brain homogenates of female 3xTg-AD mice (p = 0.06). CONCLUSIONS:This study shows that acute treatment with an aβComAb can effectively improve performance in behavioral testing without reduction of amyloid plaque burden, and that peripherally administered IgG can affect levels of pathological species in the brain.

Mounting evidence suggests that mitochondrial dysfunction plays a causal role in the etiology and progression of Alzheimer's disease (AD). We recently showed that the carbonic anhydrase inhibitor (CAI) methazolamide (MTZ) prevents amyloid β (Aβ)-mediated onset of apoptosis in the mouse brain. In this study, we used MTZ and, for the first time, the analog CAI acetazolamide (ATZ) in neuronal and cerebral vascular cells challenged with Aβ, to clarify their protective effects and mitochondrial molecular mechanism of action. The CAIs selectively inhibited mitochondrial dysfunction pathways induced by Aβ, without affecting metabolic function. ATZ was effective at concentrations 10 times lower than MTZ. Both MTZ and ATZ prevented mitochondrial membrane depolarization and H₂ O₂ generation, with no effects on intracellular pH or ATP production. Importantly, the drugs did not primarily affect calcium homeostasis. This work suggests a new role for carbonic anhydrases (CAs) in the Aβ-induced mitochondrial toxicity associated with AD and cerebral amyloid angiopathy (CAA), and paves the way to AD clinical trials for CAIs, FDA-approved drugs with a well-known profile of brain delivery.

Traumatic brain injury (TBI) and Alzheimer's disease (AD) are devastating neurological disorders, whose complex relationship is not completely understood. Cerebrovascular pathology, a key element in both conditions, could represent a mechanistic link between Aβ/tau deposition after TBI and the development of post concussive syndrome, dementia and chronic traumatic encephalopathy (CTE). In addition to debilitating acute effects, TBI-induced neurovascular injuries accelerate amyloid β (Aβ) production and perivascular accumulation, arterial stiffness, tau hyperphosphorylation and tau/Aβ-induced blood brain barrier damage, giving rise to a deleterious feed-forward loop. We postulate that TBI can initiate cerebrovascular pathology, which is causally involved in the development of multiple forms of neurodegeneration including AD-like dementias. In this review, we will explore how novel biomarkers, animal and human studies with a focus on cerebrovascular dysfunction are contributing to the understanding of the consequences of TBI on the development of AD-like pathology.

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.