Joseph Ojo

Exposure to an organophosphate pesticide, individually or in combination with other Gulf War agents, impairs synaptic integrity and neuronal differentiation, and is accompanied by subtle microvascular injury in a mouse model of Gulf War agent exposure.

Gulf War illness (GWI) is a currently untreatable multi‐symptom disorder experienced by 1990–1991 Persian Gulf War (GW) veterans. The characteristic hallmarks of GWI include cognitive dysfunction, tremors, migraine, and psychological disturbances such as depression and anxiety. Meta‐analyses of epidemiological studies have consistently linked these symptomatic profiles to the combined exposure of GW agents such as organophosphate‐based and pyrethroid‐based pesticides (e.g. chlorpyrifos (CPF) and permethrin (PER) respectively) and the prophylactic use of pyridostigmine bromide (PB) as a treatment against neurotoxins. Due to the multi‐symptomatic presentation of this illness and the lack of available autopsy tissue from GWI patients, very little is currently known about the distinct early pathological profile implicated in GWI (including its influence on synaptic function and aspects of neurogenesis). In this study, we used preclinical models of GW agent exposure to investigate whether 6‐month‐old mice exposed to CPF alone, or a combined dose of CPF, PB and PER daily for 10 days, demonstrate any notable pathological changes in hippocampal, cortical (motor, piriform) or amygdalar morphometry. We report that at an acute post‐exposure time point (after 3 days), both exposures resulted in the impairment of synaptic integrity (reducing synaptophysin levels) in the CA3 hippocampal region and altered neuronal differentiation in the dentate gyrus (DG), demonstrated by a significant reduction in doublecortin positive cells. Both exposures also significantly increased astrocytic GFAP immunoreactivity in the piriform cortex, motor cortex and the basolateral amygdala and this was accompanied by an increase in (basal) brain acetylcholine (ACh) levels. There was no evidence of microglial activation or structural deterioration of principal neurons in these regions following exposure to CPF alone or in combination with PB and PER. Evidence of subtle microvascular injury was demonstrated by the reduction of platelet endothelial cell adhesion molecule (PECAM)‐1 levels in CPF+PB+PER exposed group compared to control. These data support early (subtle) neurotoxic effects on the brain following exposure to GW agents.

Neurobehavioral, neuropathological and biochemical profiles in a novel mouse model of co-morbid post-traumatic stress disorder and mild traumatic brain injury.

Co-morbid mild traumatic brain injury (mTBI) and post-traumatic stress disorder (PTSD) has become the signature disorder for returning combat veterans. The clinical heterogeneity and overlapping symptomatology of mTBI and PTSD underscore the need to develop a preclinical model that will enable the characterization of unique and overlapping features and allow discrimination between both disorders. This study details the development and implementation of a novel experimental paradigm for PTSD and combined PTSD-mTBI. The PTSD paradigm involved exposure to a danger-related predator odor under repeated restraint over a 21 day period and a physical trauma (inescapable footshock). We administered this paradigm alone, or in combination with a previously established mTBI model. We report outcomes of behavioral, pathological and biochemical profiles at an acute timepoint. PTSD animals demonstrated recall of traumatic memories, anxiety and an impaired social behavior. In both mTBI and combination groups there was a pattern of disinhibitory like behavior. mTBI abrogated both contextual fear and impairments in social behavior seen in PTSD animals. No major impairment in spatial memory was observed in any group. Examination of neuroendocrine and neuroimmune responses in plasma revealed a trend toward increase in corticosterone in PTSD and combination groups, and an apparent increase in Th1 and Th17 proinflammatory cytokine(s) in the PTSD only and mTBI only groups respectively. In the brain there were no gross neuropathological changes in any groups. We observed that mTBI on a background of repeated trauma exposure resulted in an augmentation of axonal injury and inflammatory markers, neurofilament L and ICAM-1 respectively. Our observations thus far suggest that this novel stress-trauma-related paradigm may be a useful model for investigating further the overlapping and distinct spatio-temporal and behavioral/biochemical relationship between mTBI and PTSD experienced by combat veterans.

Chronic repetitive mild traumatic brain injury results in reduced cerebral blood flow, axonal injury, gliosis, and increased T-Tau and Tau oligomers

Exposure to repetitive mild traumatic brain injury (mTBI) is a risk factor for chronic traumatic encephalopathy, which is characterized by patchy deposition of hyperphosphorylated tau aggregates in neurons and astrocytes at the depths of cortical sulci. We developed an mTBI paradigm to explore effects of repetitive concussive-type injury over several months in mice with a human tau genetic background (hTau). Two injuries were induced in the hTau mice weekly over a period of 3 or 4 months and the effects were compared with those in noninjured sham animals. Behavioral and in vivo measures and detailed neuropathological assessments were conducted 6 months after the first injury. Our data confirm impairment in cerebral blood flow and white matter damage. This was accompanied by a 2-fold increase in total tau levels and mild increases in tau oligomers/conformers and pTau (Thr231) species in brain gray matter. There was no evidence of neurofibrillary/astroglial tangles, neuropil threads, or perivascular foci of tau immunoreactivity. There were neurobehavioral deficits (ie, disinhibition and impaired cognitive performance) in the mTBI animals. These data support the relevance of this new mTBI injury model for studying the consequences of chronic repetitive mTBI in humans, and the role of tau in TBI.

Ultrastructural Changes in the White and Gray Matter of Mice at Chronic Time Points After Repeated Concussive Head Injury.

Abstract Mild traumatic brain injury is a risk factor for neurodegenerative disease. We recently developed a model of repetitive concussive injury in mice that we have extensively characterized from 24 hours to 24 months after injury. Animals show evidence of progressive spatial memory deficits, thinning of the corpus callosum, axonal injury, and neuroglial activation. Here, we extended our neuropathologic characterization to the ultrastructural level in both a qualitative and a quantitative study. We focused on chronic (3 and 6 months) postinjury time points when the earliest stages of degenerative secondary changes were previously observed. In both C57BL/6 and hTau mice, we found white matter damage typified by axonal degeneration, microglial phagocytosis, and increased neuroglial cell density. In the cerebral cortex, we observed evidence of synaptic degeneration, dark neurons, altered dendritic microfilaments, subtle changes to the microvasculature, a mild augmentation of age-related features such as lipofuscin deposition, and electron-dense inclusions in microglial and perivascular cells. The majority of these ultrastructural features seemed to be more prominent at 3 versus 6 months after injury. Similar patterns were observed in C57BL/6 and hTau mice. These findings further support the relevance of our concussive injury model to the consequences of repetitive mild traumatic brain injury in humans.

Lifelong behavioral and neuropathological consequences of repetitive mild traumatic brain injury

Exposure to repetitive concussion, or mild traumatic brain injury (mTBI), has been linked with increased risk of long‐term neurodegenerative changes, specifically chronic traumatic encephalopathy (CTE). To date, preclinical studies largely have focused on the immediate aftermath of mTBI, with no literature on the lifelong consequences of mTBI in these models. This study provides the first account of lifelong neurobehavioral and histological consequences of repetitive mTBI providing unique insight into the constellation of evolving and ongoing pathologies with late survival.

Chronic White Matter Degeneration, But No Tau Pathology at One-Year Post-Repetitive Mild Traumatic Brain Injury in a Tau Transgenic Model

Tau pathology associated with chronic traumatic encephalopathy has been documented in the brains of individuals with a history of repetitive mild traumatic brain injury (r-mTBI). At this stage, the pathobiological role of tau in r-mTBI has not been extensively explored in appropriate pre-clinical models. Here, we describe the acute and chronic behavioral and histopathological effects of single and repetitive mild TBI (five injuries given at 48 h intervals) in young adult (3 months old) hTau mice that express all six isoforms of hTau on a null murine tau background. Animals exposed to r-mTBI showed impaired visuospatial learning in the Barnes maze test that progressively worsened from two weeks to 12 months post-injury, which was also accompanied by significant deficits in visuospatial memory consolidation at 12 months post-injury. In contrast, only marginal changes were observed in visuospatial learning at six and 12 months after single mTBI. Histopathological analyses revealed that hTau mice developed axonal injury, thinning of the corpus callosum, microgliosis and astrogliosis in the white matter at acute and chronic time points after injury. Tau immunohistochemistry and enzyme-linked immunosorbent assay data suggest, however, only transient, injury-dependent increases in phosphorylated tau in the cerebral cortex beneath the impact site and in the CA1/CA3 subregion of the hippocampus after single or r-mTBI. This study implicates white matter degeneration as a prominent feature of survival from mTBI, while the role of tau pathology in the neuropathological sequelae of TBI remains elusive.

Impact of age on acute post-TBI neuropathology in mice expressing humanized tau: A chronic effects of neurotrauma consortium study

ABSTRACT Objectives: We hypothesized that polypathology is more severe in older than younger mice during the acute phase following repetitive mild traumatic brain injury (r-mTBI). Methods: Young and aged male and female mice transgenic for human tau (hTau) were exposed to r-mTBI or a sham procedure. Twenty-four hours post-last injury, mouse brain tissue was immunostained for alterations in astrogliosis, microgliosis, tau pathology, and axonal injury. Results: Quantitative analysis revealed a greater percent distribution of glial fibrillary acid protein and Iba-1 reactivity in the brains of all mice exposed to r-mTBI compared to sham controls. With respect to axonal injury, the number of amyloid precursor protein-positive profiles was increased in young vs aged mice post r-mTBI. An increase in tau immunoreactivity was found in young and aged injured male hTau mice. Conclusions: We report the first evidence in our model that r-mTBI precipitates a complex sequelae of events in aged vs young hTau mice at an acute time point, typified by an increase in phosphorylated tau and astroglisosis, and a diminished microgliosis response and axonal injury in aged mice. These findings suggest differential age-dependent effects in TBI pathobiology.

APOE4 GENOTYPE-DEPENDENT DEFICITS IN DHA CONTAINING PHOSPHOLIPIDS AND DHA TRANSPORTERS IN THE CEREBROVASCULATURE OF ALZHEIMER'S DISEASE PATIENTS

Background:The contribution of apolipoprotein E (APOE) E4 to the cerebrovascular dysfunction in Alzheimer’s disease (AD) is characterized by the presence of severe cerebral amyloid angiopathy, increased blood-brain-barrier (BBB) breakdown, reduced cerebral vascularization, and basement membrane thinning in E4 carriers compared to non-carriers. It has also been proposed that the diminished capacity of the apoE4 protein to transport essential polyunsaturated fatty acids (PUFAs) that are required for the structural and functional maintenance and vascular integrity of the brain also contribute to AD pathogenesis. However, it remains to be determined if there are changes in the profiles of phospholipids (PL) and in the expression of lipid transporters within the brain vasculature in relation to the E4 allele and AD diagnosis.Methods:We performed liquid chromatography/mass spectrometry based lipidomic analysis of the cerebrovascular and parenchymal fractions from autopsied human brain tissue of pathologically confirmed AD cases and controls stratified by APOE genotype. In order to determine if there were changes in the expression of lipid transporters in relation to the APOE E4 allele, we performed antibody based examination of the major facilitator superfamily domain containing 2A (mfsd2a) protein in the cerebrovasculature from these subjects. Results:Total phosphatidylcholine (PC) was significantly lower in the cerebrovascular fractions of AD patients compared to controls. While docosahexaenoic acid (DHA) containing PL species were lower in heterozygous E4 AD patients compared to E4 controls in both the cerebrovascular and parenchymal fractions, an ether PC species containing arachidonic acid (AA) was elevated within the cerebrovasculature of E4 carriers relative to non-carriers and was highest among E4 AD specimens compared to E4 controls. We also observed an APOE E4 dependent difference in mfsd2a expression. Among AD patients, E4 homozygotes had lower expression of mfsd2a than E4 heterozygotes and non-carriers. Conclusions: These findings demonstrate that deficiencies in DHA within the brains of APOE E4 carriers may, in part, be due to lower expression of mfsd2a. Thus, targeting this transport mechanism may improve the bioavailability of DHA to the brain of APOE E4 individuals providing a novel approach to the treatment of AD.

PHOSPHOLIPID DISRUPTION IN THE BRAIN LINKS MOUSE MODELS OF REPETITIVE MILD TRAUMATIC BRAIN INJURY AND ALZHEIMER’S DISEASE

normal, mild cognitive impairment or dementia). SNAP is also a complement to the new National Institute on Aging–Alzheimer Association (NIA-AA) research criteria of preclinical AD. We aimed to explore the biomarkers in different clinical status with SNAP. Methods:We used the baseline data from Alzheimer’s Disease Neuroimaging Initiative 1 (ADNI1) and collected the individuals with SNAP (clinically normal, mild cognitive impairment or dementia). The criteria were decreased CSF Ab1-42 (<192pg/ml) and elevated CSF Tau (total Tau>93pg/ml, or phosphorylated Tau>23pg/ml). The data of biomarkers from CSF (e.g. Ab, Tau, a-synuclein, sAPPb, neurogranin, b-secretase, neurofilament, isoprostane, multiplex proteomics) and plasma (e.g. Ab, Tau, neurofilament) were extracted. Mann-Whitney U tests were carried out between the groups of different clinical status, and P<0.05 was regarded as statistical significance. Results: A total of 31 SNAP individuals were selected from ADNI1 cohort, with 18 clinically normal, 9 MCI and 4 dementia. APOE4 alleles were only 8%. In comparison with clinically normal, CSF CD40 (Mann-Whitney U test, Z1⁄42.024, P 1⁄40.046) and fibrinogen (Mann-Whitney U test, Z1⁄42.239, P 1⁄40.025) significantly changed in MCI, as well as CSF isoprostane (ISO8PGF2A) was elevated in dementia. In comparison with clinically normal, CSF isoprostane (ISO8PGF2A) (Mann-Whitney U test, Z1⁄4-2.315, P1⁄40.020) was elevated in dementia. Conclusions: APOE4 is less common in SNAP. Only elevated ISO8PGF2A is observed in dementia in comparison with clinically normal and MCI, which is not found between clinically normal and MCI. Large-sample is required to confirm our findings and seek better biomarkers to differentiate the clinical status of SNAP. As the previous opinion “SNAP individuals are stable over time”, biomarkers signature suggests SNAP may be normal aging rather than pathological changes, in which brain atrophy and cognitive decline progress slowly.

APOE-E4 DEPENDENT DEFICITS IN DHA PHOSPHOLIPIDS AND TRANSPORTERS IN ALZHEIMER’S DISEASE PATIENTS WITH SEVERE CEREBRAL AMYLOID ANGIOPATHY

Background: The contribution of apolipoprotein E (APOE) e4 to Alzheimer’s disease (AD) pathology is characterized by the presence of severe cerebral amyloid angiopathy (CAA), increased blood-brain-barrier (BBB) breakdown and reduced cerebral vascularization in e4 carriers. It has been proposed that the diminished capacity of the apoE4 protein to transport docosahexaenoic acid (DHA), an essential fatty acid that is required for the structural and functional maintenance and vascular integrity of the brain also contributes to AD pathogenesis. However, it remains to be determined if there are changes in brain DHA content of phospholipids (PL) and in DHA transporters in relation to the e4 allele and AD diagnosis and brain cerebrovascular amyloid pathology. Methods:We performed liquid chromatography/mass spectrometry based PL analysis of the cerebrovascular and parenchymal fractions from autopsied human brain tissue of pathologically confirmed AD cases and controls. We performed antibody-based examination of the major facilitator superfamily domain containing 2A (mfsd2a) protein in the cerebrovasculature from these subjects. Results: In the cerebrovascular and parenchymal fractions, DHA containing PL species were lower in e4 carriers with AD cases compared to control ε4 carriers. We observed an APOE e4 dependent decreases in mfsd2a expression in the brain cerebrovasculature. The mfsd2a expression was lower in e4 carriers compared to non-carriers. Stratification of DHA containing PL by CAA showed that these PL levels were reduced in e4 positive AD patients with severe CAA. Conclusions: These findings demonstrate that brain DHA deficiencies in e4 carriers may be due to reduced mfsd2a expression and partly associated with CAA. Thus, targeting this transport mechanism may improve the bioavailability of DHA to the brain of e4 carriers who are at risk of developing AD.