Caitlin S. Latimer

Vitamin D prevents cognitive decline and enhances hippocampal synaptic function in aging rats

Significance Higher blood levels of vitamin D are associated with better health outcomes. Vitamin D deficiency, however, is common among the elderly. Despite targets in the brain, little is known about how vitamin D affects cognitive function. In aging rodents, we modeled human serum vitamin D levels ranging from deficient to sufficient and tested whether increasing dietary vitamin D could maintain or improve cognitive function. Treatment was initiated at middle age, when markers of aging emerge, and maintained for ∼6 mo. Compared with low- or normal-dietary vitamin D groups, only aging rats on higher vitamin D could perform a complex memory task and had blood levels considered in the optimal range. These results suggest that vitamin D may improve the likelihood of healthy cognitive aging. Vitamin D is an important calcium-regulating hormone with diverse functions in numerous tissues, including the brain. Increasing evidence suggests that vitamin D may play a role in maintaining cognitive function and that vitamin D deficiency may accelerate age-related cognitive decline. Using aging rodents, we attempted to model the range of human serum vitamin D levels, from deficient to sufficient, to test whether vitamin D could preserve or improve cognitive function with aging. For 5–6 mo, middle-aged F344 rats were fed diets containing low, medium (typical amount), or high (100, 1,000, or 10,000 international units/kg diet, respectively) vitamin D3, and hippocampal-dependent learning and memory were then tested in the Morris water maze. Rats on high vitamin D achieved the highest blood levels (in the sufficient range) and significantly outperformed low and medium groups on maze reversal, a particularly challenging task that detects more subtle changes in memory. In addition to calcium-related processes, hippocampal gene expression microarrays identified pathways pertaining to synaptic transmission, cell communication, and G protein function as being up-regulated with high vitamin D. Basal synaptic transmission also was enhanced, corroborating observed effects on gene expression and learning and memory. Our studies demonstrate a causal relationship between vitamin D status and cognitive function, and they suggest that vitamin D-mediated changes in hippocampal gene expression may improve the likelihood of successful brain aging.

Dietary vitamin D deficiency in rats from middle to old age leads to elevated tyrosine nitration and proteomics changes in levels of key proteins in brain: implications for low vitamin D-dependent age-related cognitive decline.

In addition to the well-known effects of vitamin D (VitD) in maintaining bone health, there is increasing appreciation that this vitamin may serve important roles in other organs and tissues, including the brain. Given that VitD deficiency is especially widespread among the elderly, it is important to understand how the range of serum VitD levels that mimic those found in humans (from low to high) affects the brain during aging from middle age to old age. To address this issue, 27 male F344 rats were split into three groups and fed isocaloric diets containing low (100 IU/kg food), control (1000 IU/kg food), or high (10,000 IU/kg food) VitD beginning at middle age (12 months) and continued for a period of 4-5 months. We compared the effects of these dietary VitD manipulations on oxidative and nitrosative stress measures in posterior brain cortices. The low-VitD group showed global elevation of 3-nitrotyrosine compared to control and high-VitD-treated groups. Further investigation showed that this elevation may involve dysregulation of the nuclear factor κ-light-chain enhancer of activated B cells (NF-κB) pathway and NF-κB-mediated transcription of inducible nitric oxide synthase (iNOS) as indicated by translocation of NF-κB to the nucleus and elevation of iNOS levels. Proteomics techniques were used to provide insight into potential mechanisms underlying these effects. Several brain proteins were found at significantly elevated levels in the low-VitD group compared to the control and high-VitD groups. Three of these proteins, 6-phosphofructokinase, triose phosphate isomerase, and pyruvate kinase, are involved directly in glycolysis. Two others, peroxiredoxin-3 and DJ-1/PARK7, have peroxidase activity and are found in mitochondria. Peptidyl-prolyl cis-trans isomerase A (cyclophilin A) has been shown to have multiple roles, including protein folding, regulation of protein kinases and phosphatases, immunoregulation, cell signaling, and redox status. Together, these results suggest that dietary VitD deficiency contributes to significant nitrosative stress in brain and may promote cognitive decline in middle-aged and elderly adults.

Reversal of glial and neurovascular markers of unhealthy brain aging by exercise in middle-aged female mice.

Healthy brain aging and cognitive function are promoted by exercise. The benefits of exercise are attributed to several mechanisms, many which highlight its neuroprotective role via actions that enhance neurogenesis, neuronal morphology and/or neurotrophin release. However, the brain is also composed of glial and vascular elements, and comparatively less is known regarding the effects of exercise on these components in the aging brain. Here, we show that aerobic exercise at mid-age decreased markers of unhealthy brain aging including astrocyte hypertrophy, a hallmark of brain aging. Middle-aged female mice were assigned to a sedentary group or provided a running wheel for six weeks. Exercise decreased hippocampal astrocyte and myelin markers of aging but increased VEGF, a marker of angiogenesis. Brain vascular casts revealed exercise-induced structural modifications associated with improved endothelial function in the periphery. Our results suggest that age-related astrocyte hypertrophy/reactivity and myelin dysregulation are aggravated by a sedentary lifestyle and accompanying reductions in vascular function. However, these effects appear reversible with exercise initiated at mid-age. As this period of the lifespan coincides with the appearance of multiple markers of brain aging, including initial signs of cognitive decline, it may represent a window of opportunity for intervention as the brain appears to still possess significant vascular plasticity. These results may also have particular implications for aging females who are more susceptible than males to certain risk factors which contribute to vascular aging.

Obesity and diabetes cause cognitive dysfunction in the absence of accelerated β-amyloid deposition in a novel murine model of mixed or vascular dementia

Mid-life obesity and type 2 diabetes mellitus (T2DM) confer a modest, increased risk for Alzheimer’s disease (AD), though the underlying mechanisms are unknown. We have created a novel mouse model that recapitulates features of T2DM and AD by crossing morbidly obese and diabetic db/db mice with APPΔNL/ΔNLx PS1P264L/P264L knock-in mice. These mice (db/AD) retain many features of the parental lines (e.g. extreme obesity, diabetes, and parenchymal deposition of β-amyloid (Aβ)). The combination of the two diseases led to additional pathologies-perhaps most striking of which was the presence of severe cerebrovascular pathology, including aneurysms and small strokes. Cortical Aβ deposition was not significantly increased in the diabetic mice, though overall expression of presenilin was elevated. Surprisingly, Aβ was not deposited in the vasculature or removed to the plasma, and there was no stimulation of activity or expression of major Aβ-clearing enzymes (neprilysin, insulin degrading enzyme, or endothelin-converting enzyme). The db/AD mice displayed marked cognitive impairment in the Morris Water Maze, compared to either db/db or APPΔNLx PS1P264L mice. We conclude that the diabetes and/or obesity in these mice leads to a destabilization of the vasculature, leading to strokes and that this, in turn, leads to a profound cognitive impairment and that this is unlikely to be directly dependent on Aβ deposition. This model of mixed or vascular dementia provides an exciting new avenue of research into the mechanisms underlying the obesity-related risk for age-related dementia, and will provide a useful tool for the future development of therapeutics.

Flow cytometry analysis of synaptosomes from post-mortem human brain reveals changes specific to Lewy Body and Alzheimer’s Disease

Synaptic dysfunction is thought to have an important role in the pathophysiology of neurodegenerative diseases, such as Alzheimer’s disease (AD) and Lewy body disease (LBD). To improve our understanding of synaptic alterations in health and disease, we investigated synaptosomes prepared from post-mortem human cerebral cortex, putamen (PT), and two regions of the caudate nucleus, dorso-lateral (DL) and ventro-medial (VM), regions commonly affected in AD and LBD. We observed that the fraction of synaptosomal particles with reactivity for dopamine transporter (DAT) was significantly reduced in the PT and VM caudate of patients with neuropathological diagnosis of LBD. As expected, these differences also were reflected in direct measurements of dopamine (DA) and its metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), in caudate and PT of LBD patients. The fraction of synaptosomal particles positive for amyloid β (Aβ) was significantly increased in frontal cortical samples of patients with the neuropathological diagnosis of severe AD, and was positively correlated with disease progression. We also prepared synaptosomes from the striatum of mice with severe loss of DA neurons (Slc6a3-DTR mice) and wild-type littermate controls. We observed markedly reduced levels of DAT-positive synaptosomes in Slc6a3-DTR mice following exposure to diphtheria toxin (DT). Striatal levels of DA and DOPAC in Slc6a3-DTR mice also were reduced significantly following DT exposure. We conclude that flow cytometric analysis of synaptosomes prepared from human or mouse brain provides an opportunity to study expression of pathology-associated proteins and also the specific loss of dopaminergic nerve terminals. Hence, we believe it is a valid method to detect pathological changes at the level of the synapse in LBD as well as AD.

Clinical-pathologic correlations in vascular cognitive impairment and dementia

The most common causes of cognitive impairment and dementia are Alzheimers disease (AD) and vascular brain injury (VBI), either independently, in combination, or in conjunction with other neurodegenerative disorders. The contribution of VBI to cognitive impairment and dementia, particularly in the context of AD pathology, has been examined extensively yet remains difficult to characterize due to conflicting results. Describing the relative contribution and mechanisms of VBI in dementia is important because of the profound impact of dementia on individuals, caregivers, families, and society, particularly the stability of health care systems with the rapidly increasing age of our population. Here we discuss relationships between pathologic processes of VBI and clinical expression of dementia, specific subtypes of VBI including microvascular brain injury, and what is currently known regarding contributions of VBI to the development and pathogenesis of the dementia syndrome. This article is part of a Special Issue entitled: Vascular Contributions to Cognitive Impairment and Dementia edited by M. Paul Murphy, Roderick A. Corriveau and Donna M. Wilcock.

Resistance to Alzheimer Disease Neuropathologic Changes and Apparent Cognitive Resilience in the Nun and Honolulu-Asia Aging Studies

Two population-based studies key to advancing knowledge of brain aging are the Honolulu-Asia Aging Study (HAAS) and the Nun Study. Harmonization of their neuropathologic data allows cross comparison, with findings common to both studies likely generalizable, while distinct observations may point to aging brain changes that are dependent on sex, ethnicity, environment, or lifestyle factors. Here, we expanded the neuropathologic evaluation of these 2 studies using revised NIA-Alzheimers Association guidelines and compared directly the neuropathologic features of resistance and apparent cognitive resilience. There were significant differences in prevalence of Alzheimer disease neuropathologic change, small vessel vascular brain injury, and Lewy body disease between these 2 studies, suggesting that sex, ethnicity, and lifestyle factors may significantly influence resistance to developing brain injury with age. In contrast, hippocampal sclerosis prevalence was very similar, but skewed to poorer cognitive performance, suggesting that hippocampal sclerosis could act sequentially with other diseases to impair cognitive function. Strikingly, despite these observed differences, the proportion of individuals resistant to all 4 diseases of brain or displaying apparent cognitive resilience was virtually identical between HAAS and Nun Study participants. Future in vivo validation of these results awaits comprehensive biomarkers of these 4 brain diseases.

First confirmed case of chronic traumatic encephalopathy in a professional bull rider

There is increased concern regarding the prevalence of chronic traumatic encephalopathy [5] following repeated head impact exposure in a variety of sports [2, 4, 6, 7] and the military [3] but the existence in other populations is unknown. We present the first-confirmed case of a professional bull rider with CTE. Following sustainment of at least 15 head injuries over a 10-year period confirmed by collateral sources, records review, and ante-mortem imaging studies, the majority witnessed and documented on videotape, he committed suicide. Unique to this case, we performed imaging-guided brain tissue sampling for neuropathological investigation. This approach may provide for more focused tissue sampling that is sensitive and flexible to the heterogeneity of brain injury complementing standard neuropathological evaluation strategies. Past medical history identified first-diagnosed concussion at age 16 with confirmed loss of consciousness (LOC) and additional exposures approximately biennially until age 21 when he sustained five head injuries over the course of a 12-month period. Each incident involved LOC for minutes followed by disorientation, confusion, ocular disturbance including photophobia, and periods of anterograde amnesia lasting hours to days. The decedent was evaluated by onsite medical personnel and/or admitted to a hospital for observation with radiographic CT examination, noted as unremarkable each time. At age 23, he sustained a blow to the head after being stepped on by a rearing bull crushing his helmet with LOC for 1 h, meeting criteria for moderate brain injury [8]. Initial Glasgow Coma Scale (GCS) at hospital presentation was 10, and remained 10 for 24 h before returning to 14 out of 15 by day 2. CT evaluation again was negative for pathoanatomical brain injury lesions. MRI evaluation completed 3 months later identified multiple regions of hemorrhagic foci bilaterally in the frontal lobes, right temporal lobe, left hippocampus, and left brainstem, consistent with microhemorrhage following shear injury meeting radiographic criteria for diffuse axonal injury [1]. Following these exposures, the decedent was noted to have post-traumatic headache, memory loss, concentration problems, attentional dysfunction, mood lability, disinhibition, diplopia, photophobia, phonophobia, vestibular dysfunction, insomnia, irritability, explosivity, depression, anxiety, dysarthria with mild aphasia, difficulty with mental flexibility and planning, motor slowing, exaggerated somatic concern, hostility, and conceptual disorganization. Family members described a very bright, jovial, and affable young man who was conscientious and loving but in the last 6–9 months of life rapidly deteriorated, becoming reclusive and hypervigilant with paralyzing panic attacks, and displaying significant behavioral changes characterized by erratic and impulsive decisions until his death at 25. Following consent for brain donation, familial consent was provided for review of medical records, clinical CT, and MRI scans collected on the decedent prior to death in accordance with regulations. The decedent’s fixed brain was examined for gross findings and then imaged ex vivo with high-resolution MRI for co-registration to the ante-mortem scans. This allowed imaged-guided tissue sectioning for pathoanatomic lesions visible on the ante-mortem MRI that may be grossly unremarkable. Standard sampling was performed to evaluate for traumatic brain injury, CTE [5], and other neurodegenerative processes. Sampled regions included bilateral superior and middle frontal gyri, orbitofrontal cortex, superior and middle temporal gyri, anterior temporal lobes, inferior parietal lobules, hippocampi and entorhinal cortex at two levels, amygdalae, thalami, * Christine L. Mac Donald cmacd@uw.edu

Human Striatal Dopaminergic and Regional Serotonergic Synaptic Degeneration with Lewy Body Disease and Inheritance of APOE ε4

Cognitive impairment in older individuals is a complex trait that in population-based studies most commonly derives from an individually varying mixture of Alzheimer disease, Lewy body disease, and vascular brain injury. We investigated the molecular composition of synaptic particles from three sources: consecutive rapid autopsy brains from the Adult Changes in Thought Study, a population-based cohort; four aged nonhuman primate brains optimally processed for molecular investigation; and targeted replacement transgenic mice homozygous for APOE ε4. Our major goal was to characterize the molecular composition of human synaptic particles in regions of striatum and prefrontal cortex. We performed flow cytometry to measure six markers of synaptic subtypes, as well as amyloid β 42 and paired helical filament tau. Our results showed selective degeneration of dopaminergic terminals throughout the striatum in individuals with Lewy body disease, and serotonergic degeneration in human ventromedial caudate nucleus from individuals with an APOE ε4 allele. Similar results were seen in mouse caudate nucleus homozygous for APOE ε4 via targeted replacement. Together, extension of these clinical, pathologic, and genetic associations from tissue to the synaptic compartment of cerebral cortex and striatum strongly supports our approach for accurately observing the molecular composition of human synapses by flow cytometry.

Flow Cytometric Evaluation of Crude Synaptosome Preparation as a Way to Study Synaptic Alteration in Neurodegenerative Diseases

Neurodegenerative diseases, the most common among them Alzheimers disease (AD) and Lewy body disease (LBD), are a group of progressive incurable illnesses. In both AD and LBD, abundant evidence points to the synapse as the critical and early focus of pathological changes. Here we present a method for the isolation and flow cytometric analysis of synaptosomes prepared from postmortem human brain tissue, which we also applied to animal models, including mice and nonhuman primates. The use of flow cytometry for analysis allows for relatively fast and efficient examination of thousands of synaptosome particles in a matter of minutes, and also makes it possible to use crude, rather than purified, synaptosomal preparation, thus conserving tissue resources. We have applied this method to study synaptic alteration in several brain regions in human research participants and animal models.