Mark P. Burns, PhD - US grants

[unreadable] DESCRIPTION (provided by applicant): Abstract Brain trauma in humans may be a significant risk factor in developing Alzheimer's disease. A number of studies have shown the presence of A-beta plaques in postmortem brains of single-incidence brain trauma victims. A-beta is neurotoxic peptide that forms the plaques that are the major pathology in Alzheimer's disease. The production and accumulation of A-beta may be responsible for neuronal cell loss that occurs in the hours and days following brain trauma. However, why the production of A-beta increases following brain trauma is not yet known. By using animal models of brain trauma we can elucidate mechanisms involved in A-beta production, as well as understanding pathways involved in neuronal cell death following brain trauma. A-beta is formed when a transmembrane precursor protein is cleaved. Cholesterol is an integral membrane lipid and is found in high concentrations in the brain. In vitro, when cellular cholesterol levels increase, cleavage of the precursor protein to A-beta also increases. A decrease in cholesterol levels causes a decrease in A-beta formation. These results have been replicated with cholesterol lowering drugs in vivo. In humans, taking cholesterol lowering drugs may reduce the incidence of Alzheimer's disease. Traumatic brain trauma in rodents causes a chain of cell death, starting in the region of direct impact. As the cells die and degrade, their constituent parts are recycled. We hypothesize that this leads to a transient increase in cholesterol levels in cells immediately surrounding the injured area, and this increase is responsible for the acute increase in A-beta levels following brain trauma. We aim to test this hypothesis by 1) Measuring cholesterol-related regulatory proteins and the A-beta response following brain trauma in rodents and 2) attempting to intervene and inhibit these changes by using an LXR agonist (TO-901317) and a statin (lovastatin) prior to and following injury. We believe that TO-901317 will prevent cells surrounding the injured area from accepting cholesterol from damaged neurons, and that this will prevent the TBI-induced A-beta spike. Statins have previously been shown to be beneficial in cognitive recovery and lesion size following TBI, therefore we will examine the effects of lovastatin on both the cholesterol response to TBI, and its anti-inflammatory effects in an attempt to elucidate a possible mechanism of action. These studies aim to understand how A-beta is produced, and why brain injury can lead to production of the neurotoxin, A-beta Project Relevance This research aims to investigate how cholesterol homeostasis is altered following traumatic brain injury (TBI) in mice, and if this change in cholesterol is responsible for the increased production of Abeta that has been reported in this model. As CNS cholesterol is independent of fluctuations in peripheral cholesterol, it is difficult to examine the effects of cholesterol in the CNS independent of effects in the periphery. Using TBI as a model of dysfunctional intracerebral cholesterol homeostasis is a novel approach to examine how changes in CNS cholesterol alone can alter Abeta levels. Furthermore, the data we provide in this project will help determine the course of events following TBI, and the pathways identified may help guide future drug development studies. As such this project has multidisciplinary benefits and is relevant to the fields of CNS cholesterol, Alzheimer's disease and brain injury. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Traumatic brain injury (TBI) is a degenerative process, with an initial primary injury which causes immediate mechanical cell death. This injury also induces biochemical and cellular changes that contribute to continuing neuronal damage and death over time. This continuing damage is known as secondary injury, and multiple apoptotic and inflammatory pathways are activated as part of this process. One of the neurotoxic elements produced following TBI is the Alzheimer's disease-related protein A[unreadable]. A[unreadable] deposits, similar to those in Alzheimer's disease, are seen within 24 hours after exposure to TBI. A[unreadable] is produced following sequential cleavage of the amyloid precursor protein (APP) by [unreadable]- and ?-secretase. We have recently reported that A[unreadable] and the APP secretases are elevated in non-transgenic mice following TBI, with protein levels peaking at 3 days post-trauma. We found that immediate treatment with a ?- secretase inhibitor (DAPT) can completely block the learning deficits following TBI, and reduce brain lesion volume by 70%. Thus, we conclude that ?- secretase is a promising target for treatment of TBI. In order to fully exploit this new target, a key set of experiments have been designed. Firstly, the therapeutic window for APP secretase inhibition will be calculated. By narrowing the treatment window (both the start and end points of treatment), we can determine the time at which APP secretases are initiating secondary injury, and determine how long treatment should be maintained. This data will help us identify where in the sequence of secondary injury that APP secretases are important, and help to establish a therapeutic strategy for this class of inhibitors. Secondly, it is unclear from our data what the downstream target of APP secretase inhibitors are. Aim 2 of this application examines APP and A[unreadable] as primary downstream targets of ?-secretase following trauma. While between them [unreadable]- and ?-secretase have multiple downstream targets, there are a limited set of proteins that are cleaved by both [unreadable]- and ?-secretase. Given the excess of data suggesting that A[unreadable] can impair blood flow, induce inflammation and cause apoptosis - all hallmarks of secondary injury - APP/A[unreadable] is the most apparent of these targets. The specific aims are designed to enhance our understanding of ?- secretase inhibitors as a treatment for TBI, and to determine if the continuing cell death following TBI is mediated through APP processing. PUBLIC HEALTH RELEVANCE: This application aims to establish the role of the Alzheimer's disease peptide A[unreadable] after traumatic brain injury (TBI). A[unreadable] levels rapidly increase after TBI in the days following injury. We have previously shown that blocking [unreadable]- and ?- secretase activity after TBI can reduce hippocampal cell death and prevent learning impairments after trauma. Both [unreadable]- and ?- secretase inhibitors are currently in clinical trials for Alzheimer's disease, making these drugs a novel therapeutic strategy for TBI. This application will enhance our understanding of APP-secretase inhibitors as a treatment for TBI, and determine if the continuing cell death following TBI is mediated through cleavage of APP or production of the A[unreadable] peptide.

DESCRIPTION (provided by applicant): After traumatic brain injury (TBI) the human APOE-?4 (APOE4) gene polymorphism is associated with increased mortality, increased coma time, poor prognosis, and an increased risk of late-onset Alzheimer's disease (AD). The APOE4 gene is found in 27% of the US population, and as such affects an estimated 459,000 TBI cases each year. It is not known how APOE4 genotype negatively impacts outcome after TBI, or if genotype-specific treatments are required to improve prognosis. TBI causes the accumulation and deposition of a neurotoxic peptide called amyloid-ß (Aß). Approximately 30% of all fatal TBI cases present with Aß plaques, however the deposition of Aß is dependent on the APOE genotype of the patient. Only 10% of non-APOE4 brains have Aß plaques after injury, while 35% of heterozygous APOE4 brains, and 100% of homozygous APOE4 brains, develop Aß plaques. The APOE gene encodes for the apolipoprotein E (apoE) protein, which was recently shown to facilitate the enzymatic degradation of Aß. These data suggest that individuals carrying the APOE4 genotype are unable to clear the excess Aß that is produced as a result of TBI. Accumulation of excess Aß is known to cause neuronal apoptosis and trigger neuroinflammation. We have recently shown that preventing Aß production, or enhancing Aß clearance, can ameliorate secondary injury and prevent cognitive and motor deficits caused by experimental TBI in mice. Here we will study the role of apoE isoforms in Aß clearance after TBI. We are testing the hypothesis that apoE is instrumental in Aß degradation after TBI, but the apoE4 isoform is dysfunctional at this process. We believe that the accumulation of Aß in APOE4 mice leads to increased cell death and poorer functional and cognitive outcome after injury. We will test this hypothesis in our Specific Aims: Aim 1) Determine the role of apoE in Aß clearance after TBI Aim 2) Determine the effect of APOE genotype on Aß clearance after TBI Aim 3) Test if the poorer prognosis after TBI in APOE4 carriers is due to prolonged Aß accumulation These data will allow us to determine the mechanism by which Aß accumulates aggressively in APOE4 patients after TBI, and the functional consequences of that Aß accumulation.

PROJECT SUMMARY The rationale for this proposal is that synaptic integrity is essential for neuronal function. Disruptions to synaptic integrity are downstream of common TBI pathophysiological events including neuronal cell death, axonal injury, p-tau aggregation, and neuroinflammation. The ability to detect discrete changes in synaptic integrity ? regardless of the upstream mechanism of synapse loss ? will allow clinicians to track the ability of clinical interventions to improve chronic outcome in patients. The objective of this proposal is to use measures of synaptic integrity to predict chronic behavioral outcomes, and to validate Hcorr as a non-invasive marker of synaptic integrity for use in TBI.

PROJECT SUMMARY (ABSTRACT) An athletic career filled with head impacts (HI) predisposes athletes to chronic traumatic encephalopathy (CTE), a neurodegenerative disease characterized by behavioral deficits, cognitive impairment, and p-tau pathology in the sulcal depths where the brain is most susceptible to stress and strain. A burning question in the CTE field is to the role of p-tau in behavioral impairments: is it causative, downstream, or incidental? In Alzheimer's disease and other tauopathies considerable p- tau accumulation and neuronal death is required before strong behavioral changes are detected, and it remains debatable if the low levels of p-tau found in Stage I and II CTE are causative factors of the behavioral changes reported in these same athletes. As such, exploration of the causes of behavioral deficits beyond the protein aggregation spectrum is required. Repeat traumatic brain injury (TBI) rodent models are widely used to study CTE. Overall, this work has had partial success ? almost all groups report chronic cognitive deficits and neurobehavioral abnormalities. In contrast, immense difficulty remains recapitulating the chronic neuropathology of CTE in rodents, with one major reason being the lisencephalic brain of the rodent. TBI-induced acute accumulations of p-tau and amyloid-? are possible, but these require axonal injury to generate. Repeat HI models without axonal injury do exist, and do not have either an acute or chronic increase in p-tau or amyloid-?. Precisely because of these limitations, repeat HI mice without axonal injury are excellent models to study the tau-independent effects of repetitive injury. In this proposal we use a HI mouse model with high frequency impacts (HI-HF) that does not present with structural damage outside of the optic tract: with no axonal injury, no neuronal cell death, no neuroinflammation, and no p-tau accumulation. We will focus on hippocampal neurons to study their physiological response to HI-HF. The long-term goal of this project is to understand how repeat HI disrupts physiological brain function, why these changes persist after HI exposure has stopped, and to identify molecular targets to reverse these changes. In this proposal we are testing the hypothesis that HI-HF causes chronic synaptic adaptation. The purpose of these adaptations is to reduce calcium influx into neurons in response to HI, but the consequences include chronic behavioral deficits including cognitive impairment. !

SUMMARY/ABSTRACT Traumatic Brain Injury (TBI) is a major health problem in the US. It is estimated that over 3 million people live with disabilities caused by TBI with many suffering from disorders including depression or anxiety. The goal of this research proposal is to elucidate how TBI affects the function of peripheral systems and alters the microbiome and the resultant impact on TBI-induced affective disorders. Our preliminary data indicate that CCI causes a rapid shift in microbiota diversity within 24h of TBI, including a dramatic change in the diversity of the psychoactive Lactobacillus family. Based on our data, we are testing the hypothesis that changes to commensal gut microbiota after TBI modulates brain inflammation and drives the development of affective disorder phenotypes in mice. Using 16S rRNA gene sequencing analysis, we will look for differentially abundant taxa, comparing sham to TBI mice. We will also examine if these changes are altered by injury severity, and we will study the impact of sex on the outcome. To test our hypothesis, we will: 1) identify differentially abundant bacteria before and after mild and severe control cortical impact by 16S and metagenomics analysis; 2) perform rescue experiments with probiotics containing TBI-impacted bacteria to study the progression of neuroinflammation process, neuronal death, and neurobehavioral abnormalities; and 3) compare the recovery of germ-free mice exposed to TBI following either sham or TBI mouse fecal transplants. This project will help to further elucidate the gut-brain axis cross-talk and clarify the role of the microbiome on recovery from TBI. Specifically, this project will explore if fecal transplants alter brain function and represent a potential novel therapeutic avenue for TBI. In summary, this research is based on the scientific premise that bacterial within the gut and bacterial diversity can impact behavior, and that brain injury can disrupt diversity in a healthy gut microbiome. !

PROJECT SUMMARY (ABSTRACT) An athletic career filled with head impacts (HI) predisposes athletes to memory impairments and is a risk factor for dementias including Alzheimer?s disease. While proteinopathy is clearly linked to brain dysfunction in the late stages of these disease, memory problems are also a common symptom in reported cases with early-stage disease ? and even those cases with no neurodegenerative disease pathology. To understand the nature of the chronic memory impairments caused by HI, we are focused on the physiological changes that occur in the brain after exposure to a high frequency of non-damaging HI (HFHI), and comparing our results to a single severe single TBI. Our preliminary data shows that exposing mice to HFHI causes an adaptive response in excitatory synapses. These adaptations occur at the cost of normal brain function, with widespread and prolonged impairments in learning and memory. This synaptic dampening is permanent, and does not spontaneously reverse. In this proposal we aim to determine if we can reverse chronic synaptic dysfunction in the injured brain, and recall a forgotten memory. In this proposal we will test the hypothesis that HFHI disrupts synaptic transmission within the memory circuits of the hippocampus, and that activation of engram neurons can override these cognitive deficits and reanimate a forgotten memory. These data will allow us to test if it will be possible to treat cognitive impairments by targeting the synapse in different types of TBI. This will have profound implications for the millions of people living with cognitive and behavioral dysfunction after head impact, and could be harnessed to reduce the number of TBI patients that progress to develop Alzheimer?s disease each year.