Patricia F. Kao

An epigenetic blockade of cognitive functions in the neurodegenerating brain

Cognitive decline is a debilitating feature of most neurodegenerative diseases of the central nervous system, including Alzheimer’s disease. The causes leading to such impairment are only poorly understood and effective treatments are slow to emerge. Here we show that cognitive capacities in the neurodegenerating brain are constrained by an epigenetic blockade of gene transcription that is potentially reversible. This blockade is mediated by histone deacetylase 2, which is increased by Alzheimer’s-disease-related neurotoxic insults in vitro, in two mouse models of neurodegeneration and in patients with Alzheimer’s disease. Histone deacetylase 2 associates with and reduces the histone acetylation of genes important for learning and memory, which show a concomitant decrease in expression. Importantly, reversing the build-up of histone deacetylase 2 by short-hairpin-RNA-mediated knockdown unlocks the repression of these genes, reinstates structural and synaptic plasticity, and abolishes neurodegeneration-associated memory impairments. These findings advocate for the development of selective inhibitors of histone deacetylase 2 and suggest that cognitive capacities following neurodegeneration are not entirely lost, but merely impaired by this epigenetic blockade.

Differential Expression of Exosomal microRNAs in Prefrontal Cortices of Schizophrenia and Bipolar Disorder Patients

Exosomes are cellular secretory vesicles containing microRNAs (miRNAs). Once secreted, exosomes are able to attach to recipient cells and release miRNAs potentially modulating the function of the recipient cell. We hypothesized that exosomal miRNA expression in brains of patients diagnosed with schizophrenia (SZ) and bipolar disorder (BD) might differ from controls, reflecting either disease-specific or common aberrations in SZ and BD patients. The sources of the analyzed samples included McLean 66 Cohort Collection (Harvard Brain Tissue Resource Center), BrainNet Europe II (BNE, a consortium of 18 brain banks across Europe) and Boston Medical Center (BMC). Exosomal miRNAs from frozen postmortem prefrontal cortices with well-preserved RNA were isolated and submitted to profiling by Luminex FLEXMAP 3D microfluidic device. Multiple statistical analyses of microarray data suggested that certain exosomal miRNAs were differentially expressed in SZ and BD subjects in comparison to controls. RT-PCR validation confirmed that two miRNAs, miR-497 in SZ samples and miR-29c in BD samples, have significantly increased expression when compared to control samples. These results warrant future studies to evaluate the potential of exosome-derived miRNAs to serve as biomarkers of SZ and BD.

Full-length TDP-43 forms toxic amyloid oligomers that are present in frontotemporal lobar dementia-TDP patients

Proteinaceous inclusions are common hallmarks of many neurodegenerative diseases. TDP-43 proteinopathies, consisting of several neurodegenerative diseases, including frontotemporal lobar dementia (FTLD) and amyotrophic lateral sclerosis (ALS), are characterized by inclusion bodies formed by polyubiquitinated and hyperphosphorylated full-length and truncated TDP-43. The structural properties of TDP-43 aggregates and their relationship to pathogenesis are still ambiguous. Here we demonstrate that the recombinant full-length human TDP-43 forms structurally stable, spherical oligomers that share common epitopes with an anti-amyloid oligomer-specific antibody. The TDP-43 oligomers are stable, have exposed hydrophobic surfaces, exhibit reduced DNA binding capability and are neurotoxic in vitro and in vivo. Moreover, TDP-43 oligomers are capable of cross-seeding Alzheimers amyloid-β to form amyloid oligomers, demonstrating interconvertibility between the amyloid species. Such oligomers are present in the forebrain of transgenic TDP-43 mice and FTLD-TDP patients. Our results suggest that aside from filamentous aggregates, TDP-43 oligomers may play a role in TDP-43 pathogenesis.

Increased expression of TrkB and Capzb2 accompanies preserved cognitive status in early Alzheimer disease pathology.

Abstract Brain-derived neurotrophic factor (BDNF) and its receptor tyrosine kinase B (TrkB) may influence brain reserve, the ability of the brain to tolerate pathological changes without significant decline in function. Here, we explore whether a specifically vulnerable population of human neurons shows a compensatory response to the neuropathological changes of Alzheimer disease (AD) and whether that response depends on an upregulation of the BDNF pathway. We observed increased neuronal TrkB expression associated with early-stage AD pathology (Braak and Braak stages I–II) in hippocampal CA1 region samples from cognitively intact Framingham Heart Study subjects (n = 5) when compared with cognitively intact individuals with no neurofibrillary tangles (n = 4). Because BDNF/TrkB signaling affects memory formation and retention through modification of the actin cytoskeleton, we examined the expression of actin capping protein &bgr;2 (Capzb2), a marker of actin cytoskeleton reorganization. Capzb2 expression was also significantly increased in CA1 hippocampal neurons of cognitively intact subjects with early-stage AD pathology. Our data suggest that increased expression of TrkB and Capzb2 accompanies adequate brain reserve in the initial stages of AD pathology. In subsequent stages of AD, the higher levels of TrkB and Capzb2 expression achieved may not be sufficient to prevent cognitive decline.

Modulators of Cytoskeletal Reorganization in CA1 Hippocampal Neurons Show Increased Expression in Patients at Mid-Stage Alzheimer's Disease

During the progression of Alzheimers disease (AD), hippocampal neurons undergo cytoskeletal reorganization, resulting in degenerative as well as regenerative changes. As neurofibrillary tangles form and dystrophic neurites appear, sprouting neuronal processes with growth cones emerge. Actin and tubulin are indispensable for normal neurite development and regenerative responses to injury and neurodegenerative stimuli. We have previously shown that actin capping protein beta2 subunit, Capzb2, binds tubulin and, in the presence of tau, affects microtubule polymerization necessary for neurite outgrowth and normal growth cone morphology. Accordingly, Capzb2 silencing in hippocampal neurons resulted in short, dystrophic neurites, seen in neurodegenerative diseases including AD. Here we demonstrate the statistically significant increase in the Capzb2 expression in the postmortem hippocampi in persons at mid-stage, Braak and Braak stage (BB) III-IV, non-familial AD in comparison to controls. The dynamics of Capzb2 expression in progressive AD stages cannot be attributed to reactive astrocytosis. Moreover, the increased expression of Capzb2 mRNA in CA1 pyramidal neurons in AD BB III-IV is accompanied by an increased mRNA expression of brain derived neurotrophic factor (BDNF) receptor tyrosine kinase B (TrkB), mediator of synaptic plasticity in hippocampal neurons. Thus, the up-regulation of Capzb2 and TrkB may reflect cytoskeletal reorganization and/or regenerative response occurring in hippocampal CA1 neurons at a specific stage of AD progression.

Detection of TDP-43 Oligomers in Frontotemporal Lobar Degeneration–TDP

The proteinaceous inclusions in TDP‐43 proteinopathies such as frontotemporal lobar degeneration (FTLD)‐TDP are made of high–molecular‐weight aggregates of TDP‐43. These aggregates have not been classified as amyloids, as prior amyloid staining results were not conclusive. Here we used a specific TDP‐43 amyloid oligomer antibody called TDP‐O to determine the presence and abundance of TDP‐43 oligomers among different subtypes of FTLD‐TDP as well as in hippocampal sclerosis (HS), which represents a non‐FTLD pathology with TDP‐43 inclusions.

Myelin basic protein associates with AβPP, Aβ1-42, and amyloid plaques in cortex of Alzheimer's disease brain.

The goal of this study was to show that myelin and axons in cortical gray matter are damaged in Alzheimers disease (AD) brain. Superior temporal gyrus gray matter of AD patients (9 male, 14 female) was compared to cognitively normal controls (8 male, 7 female). Myelin basic protein (MBP) and a degraded myelin basic protein complex (dMBP) were quantified by Western blot. Brain sections were immunostained for MBP, dMBP, axonal neurofilament protein (NF), autophagy marker microtubule-associated proteins 1A/B light chain 3B precursor (LC3B), amyloid-β protein precursor (AβPP), and amyloid markers amyloid β1-42 (Aβ1-42) and FSB. Co-immunoprecipitation and mass spectroscopy evaluated interaction of AβPP/Aβ1-42 with MBP/dMBP. Evidence of axonal injury in AD cortex included appearance of AβPP in NF stained axons, and NF at margins of amyloid plaques. Evidence of myelin injury in AD cortex included (1) increased dMBP in AD gray matter compared to control (p < 0.001); (2) dMBP in AD neurons; and (3) increased LC3B that co-localized with MBP. Evidence of interaction of AβPP/Aβ1-42 with myelin or axonal components included (1) greater binding of dMBP with AβPP in AD brain; (2) MBP at the margins of amyloid plaques; (3) dMBP co-localized with Aβ1-42 in the core of amyloid plaques in AD brains; and (4) interactions between Aβ1-42 and MBP/dMBP by co-immunoprecipitation and mass spectrometry. We conclude that damaged axons may be a source of AβPP. dMBP, MBP, and NF associate with amyloid plaques and dMBP associates with AβPP and Aβ1-42. These molecules could be involved in formation of amyloid plaques.

miR-149 and miR-29c as candidates for bipolar disorder biomarkers

Bipolar disorder (BD) is a common, recurring psychiatric illness with unknown pathogenesis. Recent studies suggest that microRNA (miRNA) levels in brains of BD patients are significantly altered, and these changes may offer insight into BD pathology or etiology. Previously, we observed significant alterations of miR‐29c levels in extracellular vesicles (EVs) extracted from prefrontal cortex (Brodmann area 9, BA9) of BD patients. In this study, we show that EVs extracted from the anterior cingulate cortex (BA24), a crucial area for modulating emotional expression and affect, have increased levels of miR‐149 in BD patients compared to controls. Because miR‐149 has been shown to inhibit glial proliferation, increased miR‐149 expression in BA24‐derived EVs is consistent with the previously reported reduced glial cell numbers in BA24 of patients diagnosed with either familial BD or familial major depressive disorder. qPCR analysis of laser‐microdissected neuronal and glial cells from BA24 cortical samples of BD patients verified that the glial, but not neuronal, population exhibits significantly increased miR‐149 expression. Finally, we report altered expression of both miR‐149 and miR‐29c in EVs extracted from brains of Flinders Sensitive Line rats, a well‐validated animal model exhibiting depressive‐like behaviors and glial (astrocytic) dysfunction. These findings warrant future investigations into the potential of using EV miRNA signatures as biomarkers to further enhance the biological definition of BD.

Deregulated microRNA expression in biospecimens from patients diagnosed with schizophrenia and bipolar disorder as a disease biomarker

The biological markers for schizophrenia (SZ) and bipolar disorder (BD) would represent a precious tool in evaluating the risk for the development of these common neuropsychiatric diseases and, possibly, in the prevention of either disease episodes and/or treatment efficiency monitoring. Since both SZ and BD are diseases with a significant genetic component, the research over the last decades has focused on the genes with altered function in the central nervous system (CNS) of individuals suffering from these illnesses. Recently, however, small non-coding RNA molecules (microRNAs, miRNAs, miRs) were shown to regulate the expression of human CNS genes involved in cell processes and functions negatively affected in neuropsychiatric disorders, including synaptic development and maturation, learning and memory. Differentially expressed sets of miRNAs have been reported in the tissues of SZ and BD patients in comparison to controls suggesting the emergence of a novel class of potential biomarkers. Here we review the reports on the changes in miRNA expression in postmortem brain tissue and peripheral blood in SZ and BD. We also evaluate the potential of miRNA packaged in exosomes, signaling vesicles released by neurons and glia, to contribute to the disaggregation of the molecular machinery underlying mental disorders and provide clinically useful biomarkers.

Capzb2 protein expression in the brains of patients diagnosed with Alzheimer’s disease and Huntington’s disease

The silencing of actin capping protein ß2, Capzb2, by RNAi in developing cultured neurons results in short, dystrophic neurites reminiscent of cytoskeletal changes seen in diverse neurodegenerative diseases, including Alzheimer’s disease (AD) and Huntington’s disease (HD). Actin and tubulin are two major cytoskeletal proteins indispensable for normal neurite development and regenerative responses to injury and neurodegenerative stimuli. We have previously shown that Capzb2 binds tubulin and, in the presence of microtubule-associated protein tau, affects microtubule polymerization necessary for neurite outgrowth and normal growth cone morphology. Accordingly, Capzb2 silencing in hippocampal neurons results in short neurites with abnormal growth cones. Decreased neurite length is found in both AD and HD. In the first step towards uncovering the possible role of Capzb2 in these diseases, we studied Capzb2 protein expression in the postmortem brains of AD and HD patients. To determine whether disease-specific changes in Capzb2 protein accompany the progression of neurodegeneration, we performed Western Blot analysis of prefrontal cortices (PFC) and hippocampi (HPC) in AD patients and of PFC and heads of caudate nuclei (HCN) in HD patients. Our results show disease- and areaspecific dynamics in the levels of Capzb2 protein expression in the progressive stages of AD and HD.