Investigating the role of filamin C in Belgian patients with frontotemporal dementia linked to GRN deficiency in FTLD-TDP brains

TAR DNA-binding protein 43 (TDP-43) inclusions are pathological hallmarks of patients with frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Loss of TDP-43 in zebrafish engenders a severe muscle and vascular phenotype with a concomitant elevation of filamin C (FLNC) levels, an observation confirmed in the frontal cortex of FTLD-TDP patients. Here, we aimed to further assess the contribution of FLNC to frontotemporal dementia (FTD) etiology. We conducted a mutational screening of FLNC in a cohort of 529 unrelated Belgian FTD and FTD-ALS patients, and a control cohort of 920 unrelated and age-matched individuals. Additionally we performed an in-depth characterization of FLNC expression levels in FTD patients and a murine FTD model. In total 68 missense variants were identified of which 19 (MAF C) loss-of-function mutation. Increased FLNC levels were, to a lesser extent, also identified in a FLNC p.V831I variant carrier and in FTD patients with the p.R159H mutation in valosin-containing protein (VCP). The GRN-associated increase of FLNC was confirmed in the frontal cortex of aged Grn knockout mice starting at 16-18 months of age. Combined quantitative proteomic and bioinformatic analyses of the frontal cortex of FTD patients possessing elevated FLNC levels, identified multiple altered protein factors involved in accelerated aging, neurodegeneration and synaptogenesis. Our findings further support the involvement of aberrant FLNC expression levels in FTD pathogenesis. Identification of increased FLNC levels in aged Grn mice and impaired pathways related to aging and neurodegeneration, implies a potential role for FLNC in mediating or accelerating the aging process

Alzheimer's disease: Identification of oxidative stress biomarkers in red blood cells

Background: Oxidative stress plays a significant role in the physiological aging process and in some pathological conditions, such as Alzheimer's disease (AD). Studies suggested that biomarkers of oxidative and nitrosative stress are elevated in brain of AD patients and that oxidative damage is one of the earliest events in AD. In this experimental study, we focused on protein carbonylation and S- nitrosylation, which are irreversible and reversible post-translational modifications, respectively. The aim of this project was to identify protein markers of oxidative stress as an early event in AD pathogenesis. We hypothesized first that levels of carbonylated and nitrosylated membrane proteins of red blood cell (RBC) - including spectrin -, are increased in patients with cognitive impairment (CI) compared to controls, as based on measurements made in human autopsy brain tissue. The CI group includes patients with mild cognitive impairment (MCI) and Alzheimer's disease (AD). Secondly we split the CI group in MCI and AD groups, and hypothesized that AD contains less oxidized RBC proteins than MCI, supporting the idea that oxidative stress decreases with the disease progression. Method: We analysed blood samples from a cohort of one hundred and twenty subjects aged 49 to 85 years, including 69 cognitive impairment (CI) subjects and 51 age-matched controls. The CI group was composed of MCI and AD patients. RBC membrane proteins were extracted and separated by one-dimensional electrophoresis. Fluorescent detection was then performed by using anti- dinitrophenyl (DNP) and anti-S-nitrocysteine antibodies to detect carbonylated and S-nitrosylated proteins, respectively. Then we quantified total RBC-membrane protein content and RBC-spectrin proteins, as well as their oxidation levels. Finally, we compared the levels of oxidation in CI and control groups. Further comparisons were performed in groups with/without established molecular biomarkers of AD risk, i.e. increased p-tau/Abeta1-42 ratio in cerebrospinal fluid (CSF) and APOE4 allele carriers. Two-dimensional gel-electrophoresis of selected RBC-membrane extracts was performed to test for differences in protein content and oxidation. Results: The data revealed that protein carbonylation and S-nitrosylation were significantly decreased in cases of CI, as determined by a clinical dementia rate (CDR) score compared to controls. Oxidized and nitrosylated proteins also tended to decrease significantly in the group with pathological p-tau/Abeta1-42 ratio. Finally, S-nitrosylated beta spectrin was found decreased in APOEepsilon4 carriers when compared to APOEepsilon4 non-carriers. Two-dimensional gel-electrophoresis of selected RBC-membrane extracts revealed the presence of heatshock protein 27 (hsp27) and an elongation factor 2 (EF2) in CI samples. Conclusion: The results showed that the blood of CI patients contained less oxidized proteins than controls. This discovery might suggest that oxidative stress has a role in the beginning or before the dementia development, and suggests an activation of stress response in the blood, possibly by activation of heatshock proteins. Oxidative damages in blood are not accumulated as it was found in autopsy brain tissue, but may be eliminated due to the RBC turnover and microvesiculation process, thus decreasing the levels of marker proteins. The relation between oxidative stress in blood and AD should be more studied by measuring the capacity of antioxidant mechanisms and following the progression of the oxidative protein pattern in the same patients. Such an understanding may be necessary in order to develop the diagnostic tools to evaluate the role of redox mechanisms in peripheral tissues as well as for targeting and designing antioxidant therapies

NEUROPROTECTIVE STRATEGIES FOLLOWING EXPERIMENTAL TRAUMATIC BRAIN INJURY: LIPID PEROXIDATION-DERIVED ALDEHYDE SCAVENGING AND INHIBITION OF MITOCHONDRIAL PERMEABILITY TRANSITION

Traumatic brain injury (TBI) represents a significant health crisis. To date there are no FDA-approved pharmacotherapies available to prevent the neurologic deficits caused by TBI. Following TBI, dysfunctional mitochondria generate reactive oxygen and nitrogen species, initiating lipid peroxidation (LP) and the formation of LP-derived neurotoxic aldehydes, which bind mitochondrial proteins, exacerbating dysfunction and opening of the mitochondrial permeability pore (mPTP), resulting in extrusion of mitochondrial sequestered calcium into the cytosol, and initiating a downstream cascade of calpain activation, spectrin degradation, neurodegeneration and neurologic impairment. As central mediators of the TBI secondary injury cascade, mitochondria and LP-derived neurotoxic aldehydes make promising therapeutic targets. In fact, Cyclosporine A (CsA), an FDA-approved immunosuppressant capable of inhibiting mPTP has been shown to be neuroprotective in experimental TBI. Additionally, phenelzine (PZ), an FDA-approved non-selective irreversible monoamine oxidase inhibitor (MAOI) class antidepressant has also been shown to be neuroprotective in experimental TBI due to the presence of a hydrazine (-NH-NH2) moiety allowing for the scavenging of LP-derived neurotoxic aldehydes. The overall goal of this dissertation is to further examine the neuroprotective effects of the mPTP inhibitor, CsA, and the LP-derived neurotoxic aldehyde scavenger, PZ, using a severe controlled cortical impact injury (CCI) model in 3-month old male Sprague-Dawley rats. First, the effects of CsA on cortical synaptic and non-synaptic mitochondria, two heterogeneous populations, are examined. Our results indicate that compared to non-synaptic mitochondria, synaptic mitochondria sustain greater damage 24h following CCI and are protected to a greater degree by CsA. Second, the neuroprotective effects of a novel 72h continuous subcutaneous infusion of CsA combined with PZ are compared to monotherapy. Following CCI, our results indicate that individually both CsA and PZ attenuate modification of mitochondrial proteins by LP-derived neurotoxic aldehydes, PZ is able to maintain mitochondrial respiratory control ratio and cytoskeletal integrity, but together, PZ and CsA, are unable to improve and in some cases negate monotherapy neuroprotective effects. Finally, the effects of PZ (MAOI, aldehyde scavenger), pargyline (PG, MAOI, non-aldehyde scavenger) and hydralazine (HZ, non-MAOI, aldehyde scavenger) are compared. Our results indicate that PZ, PG, and HZ are unable to improve CCI-induced deficits to learning and memory as measured by Morris water maze (post-CCI D3-7). Of concern, PZ animals lost a significant amount of weight compared to all other group, possibly due to MAOI effects. In fact, in uninjured cortical tissue, PZ administration leads to a significant increase in norepinephrine and serotonin. Additionally, although PZ, PG, and HZ did not lead to a statistically significant improvement in cortical tissue sparing 8 days following CCI, the HZ group saw a 10% improvement over vehicle. Overall, these results indicate that pharmacotherapies which improve mitochondrial function and decrease lipid peroxidation should continue to be pursued as neuroprotective approaches to TBI. However, further pursuit of LP-derived aldehyde scavengers for clinical use in TBI may require the development of hydrazine (-NH-NH2)-compounds which lack additional confounding mechanisms of action

A Longitudinal Investigation of Blood Neurofilament Light Chain Levels in Chronic Cocaine Users

The identification of a blood marker of brain pathology that is sensitive to substance-induced neurotoxicity and dynamically responds to longitudinal changes in substance intake would substantially improve clinical monitoring in the field of substance use and addiction. Here, we explored the hypothesis that plasma levels of neurofilament light chain (NfL), a promising marker of neuroaxonal pathology, are elevated in chronic cocaine users and longitudinally associated with changes in cocaine use. Plasma NfL levels were determined using single molecule array (SIMOA) technology at baseline and at a 4-month follow-up. Substance use was subjectively assessed with an extensive interview and objectively measured via toxicological analysis of urine and 4-month hair samples. In a generalized linear model corrected for sex, age, and body mass index, NfL plasma levels were elevated in cocaine users (n=35) compared to stimulant-naïve healthy controls (n=35). A positive correlation between cocaine hair concentration and NfL levels was also found. Changes in cocaine hair concentration (group analysis of increasers vs. decreasers) over the 4-month interval predicted NfL levels at follow-up, indicating a rise in NfL with increased cocaine use and a reduction with decreased use. No associations between use or change of use of other substances (including the neurotoxic cocaine adulterant levamisole) and NfL levels were found. Our findings demonstrate that NfL is a sensitive marker for assessing cocaine-related neuroaxonal pathology, supporting the utility of blood NfL analysis in addiction research but also suggesting the detailed assessment of substance use in neurological studies and diagnostics

Effect of space conditions on neuronal plasticity and connectivity

Looking for opportunities to explore new frontiers and developing new technologies have always been in the nature of mankind. In 1957, the first rocket in space opened a new era for space traveling towards other planets. Concomitantly, a wide range of concerns related to human health risks that could occur during spaceflight was raised. Up to now, a large number of experiments has been performed to determine the biological effects of space conditions on human health, in order to develop appropriate countermeasures. However, extensive investigations still need to be performed before considering long-term spaceflight towards other planets such as Mars. Since the first human space flight, it has been observed that in weightlessness conditions, equilibrium sense organs can send misleading inputs to the central nervous system which is forced to develop new strategies and adapt to adequately translate these messages. Furthermore, cosmic radiations are known to induce oxidative stress as well as genomic damages. In this thesis, we studied concomitant microgravity and radiation exposures as models for space conditions and developed various methods to analyse their specific and combined effects on in vitro neuronal network models. In vitro primary neuronal network cultures were established and exposed to simulated space conditions to investigate neuronal network remodelling (plasticity and connectivity) as well as genomic damage/repair dynamics. This work was performed to address questions on neuronal network disorders occurring during spaceflights and, in the future, to develop strategies against these effects

Endocannabinoids modulate neuroglial phenotype and proteotoxic stress

ABSTRACT Neuronal survival in neurodegenerative diseases and brain damage is closely related to the cell populations of the environment and in particular to glial cells. Astrocytes, microglia and oligodendrocytes oversee brain homeostasis providing the intrinsic brain defence system. Damage to brain cells triggers a condition generally referred to as reactive gliosis, which includes astrogliosis and activation of microglia. Neuroglia is also thoroughly involved in pathogenesis of many chronic neurological disorders and in neurodegeneration. Endocannabinoids modulating the behaviour of microglia and astrocytes might act as possible targets for therapeutic intervention. Recent studies have indicated that endocannabinoid levels and metabolic enzymes change during the progression of Alzheimer's disease (AD) and that the inhibition of fatty acid amide hydrolase (FAAH), the main catabolic enzyme of anandamide (AEA), has beneficial effects in mice with AD. The aim of this study was to determine whether URB597, a FAAH inhibitor, targets microglia polarization by altering the cytoskeleton reorganization induced by amyloid-β peptide (Aβ) in BV-2 microglial cells. Evaluation of actin cytoskeleton showed that Aβ treatment increased the surface area of BV-2 cells, which acquired a flat and polygonal morphology. Although URB597 did not affect cell morphology only, it partially rescued the control phenotype in BV-2 cells incubated with the combined treatment. Rho family proteins have a critical role in the plasticity of the actin cytoskeleton, influencing morphological changes, migration and phagocytic activity of cells. We observed an increase of Rho protein activation in Aβ samples and a decrease in samples treated with URB597 alone or in combination with Aβ compared to controls, while an increase of Cdc42 protein activation was observed in all samples with respect to control. Aβ induced the migration of BV-2 cells up to 2 h after stimulation. We also found that by reducing Rho protein activity, URB597 was able to reduce the migration rate. URB597 also increased the number of BV-2 cells performing phagocytosis. Taken together, these data suggest that an increase of anandamide (AEA), due to FAAH inhibition, may induce cytoskeleton reorganization, regulating phagocytosis and cell migration processes, and promote microglial polarization towards an anti-inflammatory phenotype. As most research worldwide has focused on neurons, there is a dearth of protocols to generate glial cells and to produce co-culture systems for biomedical research. The aim of this project has also been the generation of co-culture with neurons, astrocytes and microglia cells and the subsequent characterization of the resulting model, evaluating interspecies differences through the generation of co-cultures with murine microglia. We focused our interest on the repair functions during brain injury and on the interactions between microglia and astrocytes. The protective effect of astrocytes and microglia against neuronal cells in the presence of inflammatory and pro-apoptotic processes was investigated. Human astrocytes and human microglia cells were activated with TNF-, IL-1 and IFN- to evaluate the inflammatory response. The results showed an increase of inflammatory cytokines gene expression such as IL-6 and IL-8 in both cell lines examined. The astrocytes activation by TNF-, or by conditioned medium (CM) of activated microglia cells was confirmed by NF-kB nuclearization. Therefore, the arise of inflammatory process in astrocyte cells is driven not only by TNF- induction, but also by a synergic effect due to microglia activation. Neuroinflammation, oxidative stress, and progressive degeneration of specific brain regions is also driven by proteasomal impairment, promoting protein accumulations. Since LUHMES neurons are quite susceptible cells to proteotoxic stress and amino acid starvation, we investigated whether murine microglia and human astrocytes exerted a protective effect also when the cell lines were treated with URB597. The obtained data demonstrated that the astrocytes through the glutathione (GSH) release, were able to attenuate neuronal proteotoxic stress in LUHMES cells. URB597 contributed to GSH anti-oxidant effects modulating GSH metabolism. The overall data demonstrated that neuroglial cells play a pivotal role on neuronal protection from noxious stimuli

Chronic Stress Effects on Prefrontal Cortical Structure and Function

Stressful life events have been implicated clinically in the pathogenesis of major depression, but the neural substrates that may account for this observation remain poorly understood. Attentional impairments symptomatic of depression are associated with structural and functional abnormalities in the prefrontal cortex. In three parallel rodent and human neuroimaging studies, this project assessed the effects of chronic stress on prefrontal cortical structure and function and the behavioral correlates of these changes. The first study used fMRI to elucidate the precise computational contributions of frontoparietal circuitry to attentional control in human subjects, using a task that could be adapted for rats. The results confirmed that the contributions of dorsolateral frontoparietal areas to visual attentional shifts could be dissociated from the regulatory influences of more ventrolateral areas on stimulus/response mappings, in a manner consistent with studies in animal models. They also indicated that anterior cingulate and posterior parietal cortex may act in concert to detect dissociable forms of information processing conflicts and signal to dorsolateral prefrontal cortex the need for increased attentional control. Stress-induced alterations in these regions and in the connections between them may therefore contribute to attentional impairments. The second study tested this hypothesis in rats by examining whether chronic stress effects on medial prefrontal (mPFC) and orbitofrontal (OFC) dendritic morphology underlie impairments in the behaviors that they subserve. Chronic stress induced a selective impairment in attentional control and a corresponding retraction of apical dendritic arbors in mPFC. By contrast, stress did not adversely affect reversal learning or OFC dendritic arborization. These results suggest that prefrontal dendritic remodeling may underlie the attentional deficits that are symptomatic of stress-related mental illness. The third study was designed to extend these findings to human subjects, using the techniques developed in Study 1. Accordingly, chronic stress predicted selective attentional impairments and alterations in prefrontal functional coupling that were reversible after four weeks. Together, these studies outline in broad strokes a mechanistic model by which chronic stress may predispose susceptible persons to the attentional impairments that are characteristic of major depression. Future studies will assess the roles of serotonin and neurotrophins in mediating these changes

Inter- and Intracellular Effects of Traumatic Axonal Injury

Mild Traumatic Brain Injuries (mTBIs) are non-penetrating brain injuries that do not result in gross pathological lesions, yet they may cause a spectrum of cognitive and behavioral deficits. mTBI has been placed in the spotlight because of increased awareness of blast induced and sports-related concussions, but the underlying pathophysiological mechanisms are poorly understood. Several studies have implicated neuronal membrane poration and ion channel dysfunction as the primary mechanism of injury. We hypothesized that injury forces utilize mechanically-sensitive, transmembrane integrin proteins, which are coupled to the neuronal cytoskeleton (CSK) and distribute injury forces within the intracellular space, disrupting CSK organization and reducing intercellular neuronal functionality. To test this, magnetic beads were coated with adhesive protein, allowing them to bind to integrins in the neuronal membrane in vitro. To apply forces to the neurons via the bound beads, we built custom magnetic tweezers and demonstrated that focal adhesions (FACs) formed at the site of bead binding. We showed that the beads were coupled to the CSK via integrins by measuring the disparate adhesion of the soma and neurite to their underlying substrate. The soma also required more force to detach than neurites, correlating with the FAC density between each neuronal microcompartment and substrate. We then utilized the magnetic tweezers to test whether beads bound to integrins injured neurons more than beads that bound to neurons nonspecifically. Integrin-bound beads injured neurons more often and the injury was characterized by the formation of focal swellings along axons, reminiscent of Diffuse Axonal Injury. While integrin-bound beads initiated swellings throughout neurons, beads bound nonspecifically only caused local injury where beads were attached to neurons. To demonstrate the electrical dysfunction of integrin-mediated injury forces, we adapted Magnetic Twisting Cytometry to simultaneously apply injury forces to beads bound to multiple cells within neuronal networks in vitro. The formation of focal swellings resulted in reduced axonal electrical activity and decreased coordinated network activity. These data demonstrate that the mechanical insult associated with mTBI is propagated into neurons via integrins, initiating maladaptive CSK remodeling that is linked to impaired electrical function, providing novel insight into the underlying mechanisms of mTBI.Engineering and Applied Science

EFFECTS OF ESTRADIOL AND SELECTIVE ESTROGEN RECEPTOR AGONISTS ON BIOCHEMICAL ENDPOINTS IN THE BRAIN: A COMPARISON BETWEEN TRANSITIONAL AND SURGICAL MENOPAUSE RAT MODELS

Women spend the last third of their lifetime postmenopause, with cessation of ovarian activity and diminished production of systemic estrogen. The loss of ovarian estrogen production is correlated with age-related cognitive decline, dementia and a shift in brain metabolism from glucose to ketone bodies. Estrogen treatment may prevent or reverse these changes. Our goal is to understand how the loss of ovarian function and estrogen replacement therapy affect metabolism and function of brain regions that are involved in cognitive functions. In this study, we evaluated the differences between surgical (ovariectomized, OVX) to transitional (ovatotoxin-treated, 4-vinylcyclohexene diepoxide, VCD) menopause rat models in comparison to normally cycling rats and with agonists treatments for the selective activation of estrogen receptor α (ERα), ERβ and G-protein coupled estrogen receptor 1 (GPER-1). Our endpoints were chosen to represent pivotal targets in major metabolic, energy, cytoskletal and neurological pathways which are modulated by estrogens with corresponding effects on neuronal or cognitive functions. Overall these studies established a highly reliable method to relatively quantify proteins in brain homogenates, a direct comparison of metabolic endpoints between OVX, VCD and cycling animals as well as the effects of ER agonists between OVX and VCD rats in two time points of continuous treatment (1- and 6- weeks) and three brain regions: hippocampus (HPC), frontal cortex (FCX) and striatum (STR). These studies illustrates that type and onset of menopause have versatile impact on brain metabolism, glucose utilization, cytoskeletal and cholinergic endpoints. We also demonstrated the differences in response to selective activation of different estrogen receptors and provided an insight into changes that occur in across duration of treatment, menopause models and treatments with selective estrogen receptor agonists. These can serve as a pre-clinical map for the development of more selective estrogen replacement therapy for postmenopausal women